Compositions and methods for modulating neural activity

Abstract

Provided herein are, inter alia, methods and compositions for transfecting a brain cell (e.g., neuron). The methods for transfecting a brain cell provided herein including embodiments thereof include, for example, transfecting the brain cell with a viral vector encoding a potassium-selective light-sensitive protein (e.g., kalium channelrhodopsin). Also provided herein are, inter alia, methods of modulating a neural network of brain cells (e.g., neurons) by activating a potassium-selective channelrhodopsin with light. The methods and compositions provided herein are, inter alia, useful for treating neurological or psychiatric diseases or disorders (e.g., epilepsies, Parkinson's disease, Alzheimer's, etc.).

Description

PATENT Attorney Docket No. 048536-799001WOCOMPOSITIONS AND METHODS FOR MODULATING NEURAL ACTIVITYRELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 703,869, filed on October 4, 2024, the contents of which are incorporated herein by reference for all purposes, including all text, tables and drawings.BACKGROUND

[0002] Seizures are made up of the coordinated activity of networks of neurons. It follows that control of neurons in the pathologic circuits of epilepsy could allow for control of the disease. In non-human disease models of epilepsy, optogenetics has been effective at stopping seizure-like activity by increasing inhibitory tone or decreasing excitation. However, this has not been shown in human brain tissue. Many of the genetic means for achieving channelrhodopsin expression in non-human models are not possible in humans, and vector-mediated methods are susceptible to species-specific tropism that may affect translational potential. There is currently no platform for testing the effects of these potentially disease-modifying tools on network activity in human brain tissue. The methods and compositions provided herein, inter alia, address these and other problems in the art.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0003] This invention was made with government support under MH130700 awarded by the National Institutes of Health. The government has certain rights in the invention.BRIEF SUMMARY

[0004] In another aspect is provided a method of treating a neurological or psychiatric disease or disorder in a subject in need thereof, the method including exposing a portion of a neural network of brain cell neurons within the subject to light thereby modulating the activity of the neural network of brain cell neurons, wherein the neural network of brain cell neurons is associated with the neurological or psychiatric disease or disorder, wherein the portion of the neural network of brain cell neurons includes a population of transduced brain cell neurons, andPATENTAttorney Docket No. 048536-799001WO wherein each transduced brain cell neuron within the population of transduced brain cell neurons includes a potassium-selective channelrhodopsin protein.

[0005] In an aspect is provided a method of transfecting a brain cell, the method including contacting the brain cell with an adeno-associated virus (AAV) viral vector including a nucleotide sequence encoding a potassium-selective channelrhodopsin protein.

[0006] In another aspect is provided a method of inhibiting neural activity of a transduced brain cell neuron, the method including exposing the transduced brain cell neuron to light thereby inhibiting neural activity of the transduced brain cell neuron, wherein transduced brain cell neuron includes an exogenous potassium-selective channelrhodopsin protein.

[0007] In another aspect is provided a method of modulating activity of a neural network of brain cell neurons, the method including exposing a portion of the neural network of brain cell neurons with light thereby modulating the activity of the neural network of brain cell neurons, wherein the portion of the neural network of brain cell neurons includes a population of transduced brain cell neurons, wherein each transduced brain cell neuron within the population of transduced brain cell neurons includes a potassium-selective channelrhodopsin protein.

[0008] In another aspect is provided an adeno-associated virus (AAV) viral vector including a nucleotide sequence encoding a potassium-selective channelrhodopsin protein, wherein the AAV viral vector is an AAV9 viral vector, an AAV-PHP.eB viral vector, an AAV.CAP-Mac viral vector, an AAV9-X1. 1 viral vector, or an AAV5 viral vector.

[0009] In another aspect is provided a brain cell including an adeno-associated virus (AAV) viral vector including a nucleotide sequence encoding a potassium-selective channelrhodopsin protein, wherein the AAV viral vector is an AAV9 viral vector, an AAV-PHP.eB viral vector, an AAV.CAP-Mac viral vector, an AAV9-X1.1 viral vector, or an AAV5 viral vector.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGS. 1A-1D show high-density microelectrode array recordings of human hippocampal slices. FIG. 1A: Workflow of hippocampal slice recordings of high-density microelectrode arrays (HD-MEAs). Hippocampus specimens were collected on postoperative day 0 (PODO = day of surgery), sliced, and plated on cell-culture inserts. Transduction of viral vectors was done on the same day as surgery. After 5-8 days in culture, slices were plated onPATENTAttorney Docket No. 048536-799001WOHD-MEAs for recording experiments. After HD-MEA recordings, slices were stained for NeuN and eYFP (FIG. IB) as markers of neurons and opsin-transduction respectively (slice 10F). FIG. 1C: Left, insets of areas shown in FIG. 1C correlating with the granule cell layer (GCL) of the dentate gyrus. Right, area of CAI showing areas of pyramidal cell (PYR) morphology. Scale bars in microns. FIG. ID: Top, raster plot of unit activity with average unit firing rate (Hz) overlaid in red, demonstrating a sample of rhythmic bursting activity after adding kainic acid to a hippocampal slice (slice 10F). Bottom, local field potential from select electrodes from recording above, showing discrete increases in LFP frequency bands strongest in the theta range.

[0011] FIGS. 2A-2D show optogenetic inhibition of human hippocampal activity. FIG. 2A: Stacked raster plot of single unit activity from a hippocampal slice expressing HcKCRl. On the y-axis are 5 trials (tl - 15) of continuous 10 s illumination, stacked to line up the phase immediately preceding illumination (pre), the light-ON phase (shaded area), and the 10 s following the end of illumination (OFF). The average firing rate of all units overlaid (red). FIG. 2B: Heat maps of the electrode arrays showing 5-rms spike activity across the electrode recording surface during the 10 s bins shown in FIG. 3 A (both FIGS. 3 A and 3B show data recorded from slice 3C). Black represents HcKCRl -eYFP staining. Similar stacked raster plot with firing rate overlay were generated from a single slice recorded in low magnesium (0-mg) media, follow ed by recordings after addition of KA to the slice. Optogenetic inhibition in low magnesium both in the presence and absence of kainic acid decreased firing rates. Non-random coherence in the theta frequency band was observed. A non-random phase distribution of theta coherence in subsets of the burst of coordinated activity in slices was also observed. Recordings from slices in the presence of a GABAergic blockade with bicuculline demonstrated that optogenetic inhibition could decrease the firing activity. FIG. 2C: Schematic of closed-loop, responsive optogenetic illumination. FIG. 2D: Example of using closed-loop optogenetic silencing of bicuculline-provoked activity.

[0012] FIGS. 3A-3C show waveform clustering analysis. FIG. 3A: Average waveform of each cluster as sorted based on HD-MEA extracellular unit activity recorded from all units in slices named in Table 2. FIG. 3B: Average trough to peak (top) and full width half maximum (bottom, FWHM) features for each of the waveform clusters. FIG. 3C: Overlay of all waveforms analyzed. Units from the recorded slice were projected spatially onto the MEA recording area and overlaid onto histology of the slice, to demonstrate whether the unit hadPATENTAttorney Docket No. 048536-799001WO granule cell layer (GCL) anatomic correlation. *Clusters significantly associated with areas of GCL coverage.

[0013] FIGS. 4A-4D shows opto-response clustering. FIG. 4A: A UMAP was generated showing units clustered through an HDBSCAN pipeline of a 20 s period of time correlating with 10 s of time prior to light-ON and 10 s during light-ON. Average firing rates for units in each cluster were calculated over the course of the analyzed 20 s period. FIG. 4B: Firing rate (left) and standard deviation of firing rate (right) of each cluster showing the distributions during light- OFF and light-ON periods. All clusters except cluster 5 showed significant differences between light-ON and light-OFF conditions. Histology showing HcKCRl-eYFP expression in white. Scale bars represent 500 um. *Significant differences in firing rate or firing rate standard deviation between light-ON and light-OFF conditions. FIGS. 4C-4D: Overlay of opto-response cluster identities with the units they were associated with on in recording areas covering 2 distinct hippocampal regions known to have with disparate cell types, the granule cell layer of the dentate gyrus (GCL) (FIG. 4C, slice 10F) and Cornu Ammonis (CA) (FIG. 4D, slice 3C). Scale bars = 500 microns. Cluster 3 was found to be positively correlated with GCL waveform clusters (Table 6). White immunohistochemistry demonstrates HcKCRl-eYFP expression.

[0014] FIGS. 5A-5I show immunohistochemistry overlay of hippocampal slices transduced with AAV9-CaMKIIa-HcKCRl-eYFP (FIGS 5A-5F) or AAV9-CaMKIIa-ChR2-eYFP (FIGS. 5G-5I). The immunostaining shows the neurons that express the HcKCRl -eYFP or ChR2-eYFP construct. FIGS. 5A-5C: Examples of slices where predominately CA regions were recorded. Slice 3C (FIG. 5A), slice 8D (FIG. 5B), and slice 12G (FIG. 5C). FIGS. 5D-5F: Examples of slices where predominately granule cell layer (GCL) of the dentate gyrus areas were recorded. Slice 9E (FIG. 5D), slice 10F (FIG. 5E), and slice 11G (FIG. 5F). Scale bars all represent 500 microns. FIGS. 57G-5I: Slice 4C, transduced with AAV9-CaMKIIa-ChR2-eYFP.

[0015] FIGS. 6A-6C show exemplary data from a patch clamp recording of HcKCRl -positive neuron. FIG. 6A: DIC microscopic image of a neuron in a hippocampal slice transduced with AAV9-CaMKIIa-HcKCRl-eYFP. FIG. 6B: Fluorescence of the same cell as in FIG. 6A, confirming viral transduction. FIG. 6C: Voltage clamp recording of the cell above showing hyperpolarizing currents activated with brief pulses of 540 nm light illumination, represented by rectangles above the hyperpolarizing currents elicited by illumination.PATENTAttorney Docket No. 048536-799001WO

[0016] FIGS. 7A-7D show Raster plots of simulated activity of an in silico model of granule and basket cells of the human hippocampus. Each black dot represents a single-unit firing event, so dark areas represent clusters of high firing rates consistent with epileptiform activity. FIG. 7A: Examples of spontaneous seizure-like events in presence of no light-responsive cells. A simulated “light-on” phase is indicated in shaded rectangles. FIGS. 7B-7D: Raster plots of the simulation data with 10% (FIG. 7B), 25% (FIG. 7C) and 50% (FIG. 7D) of excitatory granule cells harboring an inhibitory, light-sensitive channel.

[0017] FIGS. 8A-8L show optogenetic inhibition in physiologic and bicuculline media. FIGS. 8A-8C: Stacked raster plots with average firing rate plot overlaid for multiple trials of 10 s of light illumination (shaded rectangle from 10-20 s on stacked raster plots)(FIG. 8A), and paired comparisons of firing rate (FIG. 8B) and spike amplitude (FIG. 8C) for the 10 s prior to illumination (pre), during illumination (light ON) and following illumination (OFF). FIG. 8A: hippocampal slice 3C expressing HcKCRl, illuminated with 10 s of continuous 530 nm LED light. On the y-axis are 5 trials (tl - 15) of continuous 10 s illumination, stacked align the light- ON phase (shaded box). FIG. 8B: Average firing rate (Hz) of all recorded units in slice 3C during the 10-seconds prior to illumination (Pre), during illumination (light-ON) and following illumination (OFF). FIG. 8C: Average spike amplitude (uV) of all recorded units in slice 3C. FIGS. 8D-8F: Same parameters as shown in FIGS. 8A-8C are shown for slice 5C, recorded with bicuculline. FIGS. 8G-8I: Same parameters for slice 7D, recorded with bicuculline. FIGS. 8J- 8L: Same parameters for slice 8D, recorded with bicuculline. See Table 7 for p-values for each comparison. Similar experiments were performed on slices in low magnesium (0 mg) media with and without kainic acid.

[0018] FIGS. 9A-9D show' optogenetic stimulation in physiological media. FIG. 9A: Stacked raster plots with average unit firing rate overlaid for slice 4C, showing ChR2-mediated increases in firing rate during light-ON conditions. FIG. 9B: Intensity sweep showing unit response to increasing powers of LED illumination. FIG. 9C: Unit values from all trials for average firing rate and amplitude during pre, light-ON and post conditions are displayed in dot plots. Firing rate comparisons for N = 30 units recorded (p-values for pre vs light-ON: 2.43E-08; light-ON vs post: 1.72E-07; pre vs post: 0.0075). FIG. 9D: Spike amplitude comparisons for the same slice (p-values for pre vs light-ON: 0.1615; light-ON vs post: 0.0093; pre vs post: 0.3381).PATENTAttorney Docket No. 048536-799001WO

[0019] FIG. 10 shows the fraction of units with decreased firing rates during light-ON conditions in 8 slices used for optogenetic experiments. Each black dot represents a separate slice. The y-axis is the % of units in each slice that had a firing rate reduction with a magnitude greater than 25% (top), 50% (second from top), 75% (third from top) and 90% (bottom). The solid line is a linear regression line.

[0020] FIG. 11 shows mean relative firing rate calculated as the % difference between the mean firing rate the 10 s prior to light-ON conditions and the mean firing rate during light-ON conditions. Slices in physiologic or bicuculline treated media (Physio+Bic) are grouped together (slices 3C, 5C, 7D, 8D), low magnesium (Omg) media (slices 9E, 10F, 11G, 12G), and low magnesium with kainic acid (Omg+KA) media (slices 9E, 10F, 11G, 12G). Whiskers in box plot represent maximum and minimum values, middle bar represents the mean. Physio+Bic vs Omg (p=0.0095), Physio+Bic vs Omg+KA (p=0.0053), Omg vs Omg+KA (p=0.20). Raw. unadjusted p-values reported for comparisons made with unpaired t-tests with Welch's correction. *P<0.05 after adjusting for multiple comparisons.DETAILED DESCRIPTIONDEFINITIONS

[0021] While various embodiments and aspects of the present invention are show n and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0022] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0023] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.PATENTAttorney Docket No. 048536-799001WO

[0024] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed„ J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0025] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo" or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA. e.g. mRNA, siRNA. miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0026] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide throughPATENTAttorney Docket No. 048536-799001WO covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0027] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e g., phosphoramidate. phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH. Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g.. to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the intemucleotide linkages in DNA are phosphodiester, phosphodi ester derivatives, or a combination of both.

[0028] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acidPATENTAttorney Docket No. 048536-799001WO residues that does not function as an inhibitory' nucleic acid when contacted with a cell or organism.

[0029] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term ‘’polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology' searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0030] The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary' nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary' sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0031] As descnbed herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary' to eachPATENTAttorney Docket No. 048536-799001WO other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

[0032] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxy proline, y-carboxy glutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms "non-naturally occurring amino acid'’ and ‘‘unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0033] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0034] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.PATENTAttorney Docket No. 048536-799001WO

[0035] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N- terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0036] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0037] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an aminoPATENTAttorney Docket No. 048536-799001WO acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0038] The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I). Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)(see. e.g., Creighton, Proteins (1984)).

[0039] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can accountPATENTAttorney Docket No. 048536-799001WO for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0040] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0041] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of. e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in w hich a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology' alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443. by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology7(1995 supplement)).

[0042] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity7are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly availablePATENTAttorney Docket No. 048536-799001WO through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0043] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0044] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically crossPATENTAttorney Docket No. 048536-799001WO reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0045] The phrase "specifically (or selectively) binds to" when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified proteins bind to a particular protein at least two times the background and more typically more than 10 to 100 times background.

[0046] For specific proteins described herein, the named protein includes any of the protein’s naturally occurring forms, variants or homologs that maintain the protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100. 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference, homolog or functional fragment thereof.

[0047] The terms “CaMKIIa” and “CaMKIIa'’ are used interchangeably herein according to their plain ordinary meaning and includes any of the recombinant or naturally-occurring forms of the Ca2+ / calmodulin-dependent protein kinase II alpha protein, or variants or homologs thereof that maintains CaMKIIa activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to CaMKIIa). In some aspects, the variants or homologs have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous amino acid portion) compared to a naturally-PATENTAttorney Docket No. 048536-799001WO occurring CaMKIIa protein. In embodiments, the CaMKIIa protein is substantially identical to the protein identified by the UniProt reference number Q00168 or a variant or homolog having substantial identity thereto. In embodiments, the CaMKIIa protein is encoded by a CAMK2A gene.

[0048] The term “synapsin 1” or “Synl” is used herein according to its plain ordinary meaning and includes any of the recombinant or naturally-occurring forms of the synapsin 1 protein, or variants or homologs thereof that maintains Synl activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to Synl). In some aspects, the variants or homologs have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 25, 50, 100, 150, 200, 250, 300. 350, 400, 450, or 500 continuous amino acid portion) compared to a naturally-occurring Synl protein. In embodiments, the Synl protein is substantially identical to the protein identified by the UniProt reference number Pl 7600 or a variant or homolog having substantial identity thereto. In embodiments, the Synl protein is encoded by a SYN1 gene.

[0049] The term ‘‘vesicular glutamate transporter- 1 ” or “VGLUT1 ” is used herein according to its plain ordinary meaning and includes any of the recombinant or naturally-occurring forms of the vesicular glutamate transporter 1 protein, or variants or homologs thereof that maintains VGLUT1 activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to VGLUT1). In some aspects, the variants or homologs have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous amino acid portion) compared to a naturally-occurring VGLUT1 protein. In embodiments, the VGLUT1 protein is substantially identical to the protein identified by the UniProt reference number Q9P2U7 or a variant or homolog having substantial identity thereto. In embodiments, the VGLUT1 protein is encoded by an SLC17A7 gene.

[0050] The term “c-Fos” or “FOS” is used herein according to its plain ordinary meaning and includes any of the recombinant or naturally-occurring of the protein c-Fos, or variants or homologs thereof that maintains c-Fos activity (e.g. within at least 50%, 60%, 70%, 80%, 90%,PATENTAttorney Docket No. 048536-799001WO95%, 96%, 97%, 98%, 99%, or 100% activity compared to c-Fos). In some aspects, the variants or homologs have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous amino acid portion) compared to a naturally- occurring c-Fos protein. In embodiments, the c-Fos protein is substantially identical to the protein identified by the UniProt reference number P01100 or a variant or homolog having substantial identity thereto. In embodiments, the c-Fos protein is encoded by a FOS gene.

[0051] The term “phosphate-activated glutaminase” or “PAG” is used herein according to its plain ordinary meaning and includes any of the recombinant or naturally-occurring of the protein phosphate-activated glutaminase, also known as glutaminase or glutamine aminohydrolase, or variants or homologs thereof that maintains PAG activity (e g. within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to PAG). In some aspects, the variants or homologs have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 25, 50, 100. 150, 200, 250, 300. 350, 400, 450, or 500 continuous amino acid portion) compared to a naturally-occurring PAG protein. In embodiments, the PAG protein is substantially identical to the protein identified by the UniProt reference number 094925 or a variant or homolog having substantial identity7thereto. In embodiments, the PAG protein is encoded by a GLS1 gene. In embodiments, the PAG protein is substantially identical to the protein identified by the UniProt reference number Q9UI32 or a variant or homolog having substantial identity thereto. In embodiments, the PAG protein is encoded by a GLS2 gene.

[0052] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as w ell as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.PATENTAttorney Docket No. 048536-799001WO

[0053] The terms "plasmid", "vector" or "expression vector" refer to a nucleic acid molecule that encodes for genes, regulatory' elements necessary for the expression of genes, proteins, and / or recombinant proteins (e.g., potassium-selective channel rhodopsin protein). Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, the gene and the regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory' elements are encoded by separate plasmids.

[0054] The terms “virus” or “virus particle” are used herein according to their plain ordinary meaning within Virology and refer to a virion including the viral genome (e.g. DNA, RNA, single strand, double strand), viral capsid and associated proteins, and in the case of enveloped viruses (e.g. herpesvirus, poxvirus), an envelope including lipids and optionally components of host cell membranes, and / or viral proteins.

[0055] The term “viral vector” is used herein according to its plain ordinary' meaning and refers to a virion designed to deliver exogenous genetic material (e.g.. a nucleic acid or a transgene) into a cell. In embodiments, the viral vector is replication defective. In embodiments, the viral vector is an adeno-associated virus (AAV) viral vector. In embodiments, the viral vector includes a promoter.

[0056] The term “adeno-associated virus” or “adeno-associated viral vector” is used herein according to its plain ordinary meaning and refers to a replication defective, nonenveloped virus. In embodiments, the adeno-associated virus (AAV) is useful as a viral vector for delivering an exogenous nucleic acid encoding a protein (e.g., a channelrhodopsin protein) into a cell (e g., brain cell). In embodiments, the AAV an AAV viral vector. In embodiments, the AAV viral vector is a AAV9 viral vector, an AAV-PHP.eB viral vector, an AAV. CAP -Mac viral vector, an AAV9-X1. 1 viral vector, or an AAV5 viral vector. In embodiments, the AAV an AAV viral vector. In embodiments, the AAV viral vector is a AAV9 viral vector. In embodiments, the AAV viral vector is n AAV-PHP.eB viral vector. In embodiments, the AAV viral vector is an AAV.CAP-Mac viral vector. In embodiments, the AAV viral vector is an AAV9-X1.1 viral vector. In embodiments, the AAV viral vector is an AAV5 viral vector. In embodiments, the AAV viral vector includes a nucleotide sequence encoding a potassium-selective channelrhodopsin protein. In embodiments, the AAV viral vector further includes a promoter.PATENTAttorney Docket No. 048536-799001WO

[0057] The term “promoter” is used herein according to its plain ordinary' meaning and refers to a nucleic acid sequence which proteins bind to initiate transcription of a downstream nucleic acid sequence. In embodiments, the promoter controls expression of a downstream nucleic acid and / or gene. In embodiments, the promoter is a Calcium / calmodulin-dependent protein kinase II alpha (CaMKIIa) promoter, a synapsin 1 (Synl) promoter, a phosphate-activated glutaminase (PAG) promoter, a vesicular glutamate transporter-1 (VGLUT1) promoter, or a protein c-Fos (FOS) promoter.

[0058] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non-viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms "transfection" or "transduction" also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8: 1-4 and Prochiantz (2007) Nat. Methods 4: 119-20.

[0059] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzy mes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide specifically reactive with aPATENTAttorney Docket No. 048536-799001WO target peptide. Any appropriate method known in the art for conjugating a peptide to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0060] When the label or detectable moiety is a radioactive metal or paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding to these ions. The long tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which the metals or ions may be added for binding. Examples of chelating groups that may be used according to the disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), DOT A, NOTA, NET A, TETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups. The chelate is normally linked to the PSMA antibody or functional antibody fragment by a group, which enables the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies and carriers described herein. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as22?Ra for RAIT may be used. In certain embodiments, chelating moieties may be used to attach a PET imaging agent, such as an A1-18F complex, to a targeting molecule for use in PET analysis.

[0061] A "cell" as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.PATENTAttorney Docket No. 048536-799001WO

[0062] The term “brain cell” is used herein according to its plain ordinary meaning and refers to a cell that forms part of the functional tissue of an organism’s brain. In embodiments, the brain cell is a neuron or a glial cell. In embodiments, the brain cell is a neuron. In embodiments, the brain cell is a glial cell.

[0063] The term “neuron” or “neural cell” is used herein according to its plain ordinary’ meaning and refers to a polarized, electrically excitable brain cell. In embodiments, the neuron is capable of electrochemical signaling. In embodiments, the electrochemical signaling includes secretion of a neurotransmitter. In embodiments, the secreted neurotransmitter binds or interacts with a protein on the surface of another neuron. In embodiments, the neuron is an excitatory neuron, an inhibitory' neuron, or a neuromodulatory neuron. In embodiments, the neuron is an excitatory neuron. In embodiments, the neuron is an inhibitory’ neuron. In embodiments, the neuron is a neuromodulatory neuron.

[0064] The term “excitatory neuron” is used herein according to its plain ordinary meaning and refers to a neuron that stimulates or increases the activity of a second (e.g.. receiving or postsynaptic) neuron that receives the electrochemical signal (e.g., neurotransmitter) from the excitatory neuron. In embodiments, the excitatory’ neuron includes the neurotransmitter glutamate. In embodiments, the excitatory neuron secretes glutamate. In embodiments, the excitatory neuron is a glutamatergic neuron.

[0065] The term “neuromodulatory neuron” is used herein according to its plain ordinary meaning and refers to a neuron that alters activity (e.g., increases or decreases) of a second (e.g., receiving or postsynaptic) neuron that receives the electrochemical signal (e.g., neurotransmitter) from the neuromodulatory neuron. In embodiments, the neuromodulatory neuron includes dopamine, serotonin, acetylcholine, histamine, norepinephrine (noradrenaline), nitric oxide, or a neuropeptide. In embodiments, the neuromodulatory neuron secretes dopamine, serotonin, acetylcholine, histamine, norepinephrine, nitric oxide, or a neuropeptide. In embodiments, the neuromodulatory neuron is a dopaminergic neuron, a serotonergic neuron, an acetylcholinergic neuron, a histaminergic neuron, or a noradrenergic neuron. In embodiments, the neuromodulatory neuron secretes dopamine and is a dopaminergic neuron. In embodiments, the neuromodulatory^ neuron secretes serotonin and is a serotonergic neuron. In embodiments, the neuromodulatory’ neuron secretes acety lcholine and is an acetylcholinergic neuron. InPATENTAttorney Docket No. 048536-799001WO embodiments, the neuromodulatory neuron secretes histamine and is a histaminergic neuron. In embodiments, the neuromodulatory neuron secretes norepinephrine and is a noradrenergic neuron.

[0066] The term “neural network” is used herein according to its plain ordinary meaning and refers to a group of interconnected neurons that send signals to one another via electrochemical signaling (e.g., secretion of a neurotransmitter). In embodiments, neurons within the neural network transmit electrochemical signals across synapses to one another.

[0067] The term “transduced brain cell neuron” is used herein according to its plain ordinary meaning and refers to a neuron which has been transfected with an exogenous nucleic acid molecule, an exogenous protein, or an exogenous expression vector. In embodiments, the transduced brain cell neuron expresses an exogenous protein (e.g. potassium-selective channelrhodopsin protein).

[0068] The term “optogenetic” is used herein according to its plain ordinary' meaning and refers to a biological technique that controls the activity of a target cell with light. In embodiments, the activity of the target cell is modulated (e.g., increased or decreased) by activating light-sensitive biomolecule expressed on or in the target cell with light. In embodiments, the light-sensitive biomolecule is an ion channel, an ion pump, or an enzyme. In embodiments, the light-sensitive biomolecule is a light-sensitive ion channel. In embodiments, the light-sensitive ion channel is a potassium-selective channelrhodopsin protein.

[0069] The term “potassium-selective channelrhodopsin protein” is used herein according to its plain ordinary meaning and refers to a light-gated ion channel that preferentially conducts potassium ions through its pore based on the electrochemical gradient of potassium across a membrane. In embodiments, the potassium-selective channel rhodopsin is transmembrane ion channel in the cell membrane of a neuron. In embodiments, the potassium-selective channelrhodopsin has a closed configuration when not exposed to light. In embodiments, the closed configuration of the potassium-selective channelrhodopsin includes a closed ion channel pore. In embodiments, the closed ion channel pore prevents conduction of potassium ions cross the membrane. In embodiments, the potassium-selective channelrhodopsin protein is activated by light. In embodiments, the activated potassium-selective channelrhodopsin protein includes a conformational change in the ion channel. In embodiments, the activated potassium-selectivePATENTAttorney Docket No. 048536-799001WO channelrhodopsin changes from a closed configuration to an open configuration. In embodiments, the open configuration includes an open ion channel pore. In embodiments, the open configuration allows conduction of potassium ions across the membrane based on the electrochemical gradient of potassium across the membrane. In embodiments, an activated potassium-selective channelrhodopsin increases the intracellular potassium concentration of a neuron. In embodiments, the increased intracellular potassium concentration of a neuron decreases the membrane potential of the neuron (e.g., hyperpolarizes the neuron). The term “membrane potential7’ or “Vm” is used herein according to its plain ordinary meaning and refers to the difference in electric potential between the interior and exterior of a cell. In embodiments, the activated potassium-selective channelrhodopsin protein decreases the membrane potential of the neuron. In embodiments, the decreased membrane potential of the neuron decreases (e.g. inhibits) the activity of the neuron. In embodiments, an activated potassium-selective channelrhodopsin inhibits a neuron. In embodiments, the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl), a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2), a light-responsive inward rectifier potassium channel Kir2.1. a Wobblia lunata channelrhodopsin (WiChR), a K+-augmented light-gated ion channel 1 (KALI-1), or a K+-augmented light-gated ion channel 2 (KALI-2). The terms Wobblia lunata channelrhodopsin and Wobblia lunata inwardly rectifiying channelrhodopsin are used interchangeably herein. Potassium-selective channelrhodopsin proteins are well known in the art. See, e.g., Govorunova et al., Nat Neurosci. 2022, vol. 25, no. 7; and Vierock et al., Sci Adv, 2022. vol. 8, no. 49, each of which is incorporated in its entirety and for all purposes.

[0070] The term “electrochemical gradient” is used herein according to its plain ordinary meaning and refers to the electrochemical potential for an ion across a membrane. In embodiments, the electrochemical gradient includes the chemical gradient and the electrical gradient. In embodiments, the chemical gradient is the difference in solute concentration of the ion across a membrane. In embodiments, the electrical gradient is the difference in electrical charge across a membrane.

[0071] The term “modulation,” “modulate,” “modulating,” “modulator” and the like in reference to a cell activity (e.g., neuron activity or neural activity) means affecting (e.g. increasing or decreasing) the activity or function of the cell (e g., neuron) relative to the activity or function of the cell in the absence of the modulator (e.g., optogenetic light exposure). InPATENTAttorney Docket No. 048536-799001WO embodiments, optogenetic light exposure modulates (e.g., inhibits or increases) the activity’ of a target cell (e.g.. neuron) through a direct interaction (e.g. optogenetic light activation of an inhibitory channelrhodopsin protein). In embodiments, optogenetic light exposure modulates (e.g., inhibits or increases) the activity of a target cell (e.g., neuron) through an indirect interaction (e.g. optogenetic light activation of an upstream cell that stimulates and / or inhibits the target cell).

[0072] As defined herein, the term “activation,” “activate,” “activating,” “activator,” and the like in reference to a cell activity (e.g.. neuron activity or neural activity) means positively affecting (e.g. increasing) the activity or function of the cell (e.g., neuron) relative to the activity or function of the cell in the absence of the activator (e.g., optogenetic light exposure). In embodiments, activation refers to an increase in the activity7of a particular cell (e.g., neuron) or population of cells (e.g., neural network). The terms may reference activation, or activating, sensitizing, or up-regulating cellular activity, signal transduction or enzymatic activity of a cell decreased in a disease. Thus, activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up- regulating cellular activity, signal transduction or enzy matic activity7of a cell (e.g., neuron) associated with a disease (e.g., a protein which is decreased in a disease relative to a nondiseased control). Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity7of a cell (e.g., neuron). In embodiments, activation refers to an increase of activity of a target cell (e g., neuron) from an indirect interaction (e.g. optogenetic light activation of an upstream cell that inhibits the target cell).

[0073] As defined herein, the term “inhibition,” “inhibit,” “inhibiting,” and the like in reference to a cell activity (e.g., neuron activity or neural activity) means negatively affecting (e.g. decreasing) the activity or function of the cell (e.g., neuron) relative to the activity7or function of the cell in the absence of the inhibitory stimulus (e.g., optogenetic light exposure). In embodiments inhibition refers to reduction of a disease or symptoms of disease. The terms mayreference inhibition, or inhibiting, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating cellular activity, signal transduction or enzy matic activity7of a cell decreased in a disease. Thus, inhibition may include, at least in part, partially or totally blocking stimulation, decreasing.PATENTAttorney Docket No. 048536-799001WO preventing, or delaying activation, or inactivating, desensitizing, or down-regulating cellular activity, signal transduction or enzy matic activity' of a cell (e.g., neuron) associated with a disease (e.g., a protein which is decreased in a disease relative to a non-diseased control). In embodiments, inhibition refers to a reduction in the activity of a particular cell (e.g., neuron) or population of cells (e.g., neural network). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity of a cell. In embodiments, inhibition refers to a reduction of activity of a cell (e.g., neuron) resulting from a direct interaction (e.g. optogenetic light activation of an inhibitory' channelrhodopsin protein). In embodiments, inhibition refers to a reduction of activity' of a target cell (e.g., neuron) from an indirect interaction (e.g. optogenetic light activation of an upstream cell that stimulates the target cell). In embodiments, activation of an inhibitory channelrhodopsin protein (e.g., a potassiumselective channelrhodopsin protein) inhibits the cell (e.g. neuron) or the population of cells (e.g., neural network) which expresses the inhibitory' channelrhodopsin protein. In embodiments, optogenetic light activation of an inhibitory' channelrhodopsin protein (e.g., a potassium-selective channelrhodopsin protein) inhibits the cell (e.g. neuron) or the population of cells (e.g.. neural network) which expresses the inhibitory channelrhodopsin protein.

[0074] The terms “neurological disease” and “neurological disorder” are used interchangeably herein according to their plain ordinary' meaning and refer to a disease or condition in which the function of a subject’s nervous system, brain, and / or neurons become impaired. In embodiments, the neurological disease may be comorbid with a psychiatric disorder. In embodiments, the neurological disease is an epilepsy, Parkinson’s disease. Alzheimer’s disease, essential tremor, depression, chronic pain, or schizophrenia.

[0075] The terms “psychiatric disease” and “psychiatric disorder” are used interchangeably herein according to their plain ordinary' meaning and refer to a condition in which the subject’s cognition, emotional regulation, or behavior is impaired or disrupted. In embodiments, the psychiatric disorder may be comorbid with a neurological disease.

[0076] The term "recombinant" when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by' the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid orPATENTAttorney Docket No. 048536-799001WO protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0077] The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0078] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is ty pically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g.. a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e g., a fusion protein).

[0079] The term "exogenous" refers to a molecule or substance (e.g. , a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the cell or organism it is expressed by. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to, or originates within, a given cell or organism.

[0080] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g.. ELISA, Western blotting, flow cytometry. immunofluorescence, immunohistochemistry, etc. ).PATENTAttorney Docket No. 048536-799001WO

[0081] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluidjoint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0082] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., halflife) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell ty pes, specific bodily fluids, specific tissues, etc).

[0083] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.PATENTAttorney Docket No. 048536-799001WO

[0084] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.METHODS

[0085] Provided herein are, inter alia, methods and compositions for transfecting a brain cell (e.g., neuron). The methods and compositions for transfecting a brain cell provided herein including embodiments thereof include, for example, transfecting the brain cell with a viral vector including a nucleic acid encoding a potassium-selective light-sensitive protein (e.g., kalium channelrhodopsin). Also provided herein are, inter alia, methods of modulating a neural network of brain cells (e.g., neurons) by activating a potassium-selective channelrhodopsin with light. The methods of modulating a neural network of brain cells are, inter alia, useful for treating neurological or psychiatric diseases or disorders (e g., epilepsies, Parkinson’s disease, Alzheimer’s, etc.). The methods and compositions described herein demonstrate the utility of optogenetics for therapeutic effects. For example, the methods described herein demonstrate that inhibitory channelrhodopsin proteins (e.g.. a potassium-selective channelrhodopsin) are effective for decreasing the activity of a neural network, even when only a portion of the neurons express the inhibitor}7channelrhodopsin protein or are inhibited by light exposure. In addition, the methods described herein demonstrate that brain cells in human neural tissue (e.g., neurons) can be efficiently transfected with a viral vector (e g., AAV 9) which encodes an inhibitory channelrhodopsin (e.g., a potassium-selective channelrhodopsin). The methods described herein further demonstrate that activity of human neurons can be modulated (e.g., inhibited) by exposing an inhibitory channelrhodopsin (e.g., a potassium-selective channelrhodopsin) to light. The methods and compositions provided herein demonstrate that optogenetics can be used to modulate (e.g., inhibit) aberrant or neuropathological activity of a human neural network.

[0086] Thus, in an aspect is provided a method of treating a neurological or psychiatric disease or disorder in a subject in need thereof, the method including exposing a portion of a neural network of brain cell neurons within the subject to light thereby modulating the activity7of the neural network of brain cell neurons, wherein the neural network of brain cell neurons is associated with the neurological or psychiatric disease or disorder, wherein the portion of thePATENTAttorney Docket No. 048536-799001WO neural network of brain cell neurons includes a population of transduced brain cell neurons, and wherein each transduced brain cell neuron within the population of transduced brain cell neurons includes a potassium-selective channelrhodopsin protein.

[0087] In another aspect is provided a method of transfecting a brain cell, the method including contacting the brain cell with an adeno-associated virus (AAV) viral vector including a nucleotide sequence encoding a potassium-selective channelrhodopsin protein.

[0088] In another aspect is provided a method of modulating activity of a neural network of brain cell neurons, the method including exposing a portion of the neural network of brain cell neurons with light thereby modulating the activity of the neural network of brain cell neurons, wherein the portion of the neural network of brain cell neurons includes a population of transduced brain cell neurons, wherein each transduced brain cell neuron within the population of transduced brain cell neurons includes a potassium-selective channelrhodopsin protein.

[0089] In embodiments, the modulating of activity of the neural network is a change in activity of the neural network during the light exposure relative to the activity of the neural network in the absence of the light exposure. In embodiments, the modulating of activity of the neural network is decreasing the activity of the neural network. In embodiments, the decreasing the activity of the neural network during the light exposure is relative to the activity' of the neural network in the absence of the light exposure. In embodiments, the light activates the potassiumselective channelrhodopsin protein in each transduced brain cell neuron. In embodiments, the activated potassium-selective channel rhodopsin increases the intracellular concentration of potassium ions in each transduced brain cell neuron, thereby hyperpolarizing each transduced brain cell neuron. In embodiments, the activated potassium-selective channelrhodopsin protein decreases the activity of each transduced brain cell neuron. In embodiments, the light activates a plurality of potassium-selective channelrhodopsin proteins in a plurality’ of transduced brain cell neurons in the neural network of brain cell neurons. In embodiments, the plurality of activated potassium-selective channelrhodopsin proteins in the plurality of transduced brain cell neurons hyperpolarizes the plurality of transduced brain cell neurons. In embodiments, the hyperpolarized plurality of transduced brain cell neurons decreases the activity of the neural network. In embodiments, the plurality’ of activated potassium-selective channelrhodopsin proteins in the plurality of transduced brain cell neurons decreases the activity’ of the plurality’ ofPATENTAttorney Docket No. 048536-799001WO transduced brain cell neurons. In embodiments, the decreased activity of the plurality of transduced brain cell neurons decreases the activity' of the neural network.

[0090] In embodiments, the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl), a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2), a light-responsive inward rectifier potassium channel Kir2. 1 , or a Wobblia lunata channelrhodopsin (WiChR), a K+-augmented light-gated ion channel 1 (KALI- 1), a K+-augmented light-gated ion channel 2 (KALI-2). In embodiments, the potassiumselective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl). In embodiments, the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2). In embodiments, the potassium-selective channelrhodopsin protein is a light-responsive inward rectifier potassium channel Kir2.1. In embodiments, the potassium-selective channelrhodopsin protein is a Wobblia lunata channelrhodopsin (WiChR). In embodiments, the potassium-selective channelrhodopsin protein is a K+-augmented light-gated ion channel 1 (KALI-1). In embodiments, the potassiumselective channelrhodopsin protein is a K+-augmented light-gated ion channel 2 (KALI-2).

[0091] In embodiments, the wavelength of the light is between about 260 nm and about 770 nm. In embodiments, the wavelength of the light is between about 265 nm and about 770 nm. In embodiments, the wavelength of the light is between about 270 nm and about 770 nm. In embodiments, the wavelength of the light is between about 275 nm and about 770 nm. In embodiments, the wavelength of the light is between about 280 nm and about 770 nm. In embodiments, the w avelength of the light is betw een about 285 nm and about 770 nm. In embodiments, the wavelength of the light is between about 290 nm and about 770 nm. In embodiments, the wavelength of the light is between about 295 nm and about 770 nm. In embodiments, the wavelength of the light is between about 300 nm and about 770 nm. In embodiments, the w avelength of the light is betw een about 305 nm and about 770 nm. In embodiments, the wavelength of the light is between about 310 nm and about 770 nm. In embodiments, the wavelength of the light is between about 315 nm and about 770 nm. In embodiments, the wavelength of the light is between about 320 nm and about 770 nm. In embodiments, the wavelength of the light is between about 325 nm and about 770 nm. In embodiments, the w avelength of the light is betw een about 330 nm and about 770 nm. In embodiments, the wavelength of the light is between about 335 nm and about 770 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between about 340 nm and about 770 nm. In embodiments, the wavelength of the light is between about 345 nm and about 770 nm. In embodiments, the wavelength of the light is between about 350 nm and about 770 nm. In embodiments, the wavelength of the light is between about 355 nm and about 770 nm. In embodiments, the wavelength of the light is between about 360 nm and about 770 nm. In embodiments, the wavelength of the light is between about 365 nm and about 770 nm. In embodiments, the wavelength of the light is between about 370 nm and about 770 nm. In embodiments, the wavelength of the light is between about 375 nm and about 770 nm. In embodiments, the wavelength of the light is between about 380 nm and about 770 nm. In embodiments, the wavelength of the light is between about 385 nm and about 770 nm. In embodiments, the wavelength of the light is between about 390 nm and about 770 nm. In embodiments, the wavelength of the light is between about 395 nm and about 770 nm. In embodiments, the wavelength of the light is between about 400 nm and about 770 nm. In embodiments, the wavelength of the light is between about 405 nm and about 770 nm. In embodiments, the wavelength of the light is between about 410 nm and about 770 nm. In embodiments, the wavelength of the light is between about 415 nm and about 770 nm. In embodiments, the wavelength of the light is between about 420 nm and about 770 nm. In embodiments, the wavelength of the light is between about 425 nm and about 770 nm. In embodiments, the wavelength of the light is between about 430 nm and about 770 nm. In embodiments, the wavelength of the light is between about 435 nm and about 770 nm. In embodiments, the wavelength of the light is between about 440 nm and about 770 nm. In embodiments, the wavelength of the light is between about 445 nm and about 770 nm. In embodiments, the wavelength of the light is between about 450 nm and about 770 nm. In embodiments, the wavelength of the light is between about 455 nm and about 770 nm. In embodiments, the wavelength of the light is between about 460 nm and about 770 nm. In embodiments, the wavelength of the light is between about 465 nm and about 770 nm. In embodiments, the wavelength of the light is between about 470 nm and about 770 nm. In embodiments, the wavelength of the light is between about 475 nm and about 770 nm. In embodiments, the wavelength of the light is between about 480 nm and about 770 nm. In embodiments, the wavelength of the light is between about 485 nm and about 770 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between about 490 nm and about 770 nm. In embodiments, the wavelength of the light is between about 495 nm and about 770 nm.

[0092] In embodiments, the wavelength of the light is between about 500 nm and about 770 nm. In embodiments, the wavelength of the light is between about 505 nm and about 770 nm. In embodiments, the wavelength of the light is between about 510 nm and about 770 nm. In embodiments, the wavelength of the light is between about 515 nm and about 770 nm. In embodiments, the wavelength of the light is between about 520 nm and about 770 nm. In embodiments, the wavelength of the light is between about 525 nm and about 770 nm. In embodiments, the wavelength of the light is between about 530 nm and about 770 nm. In embodiments, the wavelength of the light is between about 535 nm and about 770 nm. In embodiments, the wavelength of the light is between about 540 nm and about 770 nm. In embodiments, the wavelength of the light is between about 545 nm and about 770 nm. In embodiments, the wavelength of the light is between about 550 nm and about 770 nm. In embodiments, the wavelength of the light is between about 555 nm and about 770 nm. In embodiments, the wavelength of the light is between about 560 nm and about 770 nm. In embodiments, the wavelength of the light is between about 565 nm and about 770 nm. In embodiments, the wavelength of the light is between about 570 nm and about 770 nm. In embodiments, the wavelength of the light is between about 575 nm and about 770 nm. In embodiments, the wavelength of the light is between about 580 nm and about 770 nm. In embodiments, the wavelength of the light is between about 585 nm and about 770 nm. In embodiments, the wavelength of the light is between about 590 nm and about 770 nm. In embodiments, the wavelength of the light is between about 595 nm and about 770 nm. In embodiments, the wavelength of the light is between about 600 nm and about 770 nm. In embodiments, the wavelength of the light is between about 605 nm and about 770 nm. In embodiments, the wavelength of the light is between about 610 nm and about 770 nm. In embodiments, the wavelength of the light is between about 615 nm and about 770 nm. In embodiments, the wavelength of the light is between about 620 nm and about 770 nm. In embodiments, the wavelength of the light is between about 625 nm and about 770 nm. In embodiments, the wavelength of the light is between about 630 nm and about 770 nm. In embodiments, the wavelength of the light is between about 635 nm and about 770 nm. In embodiments, the wavelength of the light is between about 640 nm and about 770 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between about 645 nm and about 770 nm. In embodiments, the wavelength of the light is between about 650 nm and about 770 nm. In embodiments, the wavelength of the light is between about 655 nm and about 770 nm. In embodiments, the wavelength of the light is between about 660 nm and about 770 nm. In embodiments, the wavelength of the light is between about 665 nm and about 770 nm. In embodiments, the wavelength of the light is between about 670 nm and about 770 nm. In embodiments, the wavelength of the light is between about 675 nm and about 770 nm. In embodiments, the wavelength of the light is between about 680 nm and about 770 nm. In embodiments, the wavelength of the light is between about 685 nm and about 770 nm. In embodiments, the wavelength of the light is between about 690 nm and about 770 nm. In embodiments, the wavelength of the light is between about 695 nm and about 770 nm. In embodiments, the wavelength of the light is between about 705 nm and about 770 nm. In embodiments, the wavelength of the light is between about 710 nm and about 770 nm. In embodiments, the wavelength of the light is between about 715 nm and about 770 nm. In embodiments, the wavelength of the light is between about 720 nm and about 770 nm. In embodiments, the wavelength of the light is between about 725 nm and about 770 nm. In embodiments, the wavelength of the light is between about 730 nm and about 770 nm. In embodiments, the wavelength of the light is between about 735 nm and about 770 nm. In embodiments, the wavelength of the light is between about 740 nm and about 770 nm. In embodiments, the wavelength of the light is between about 745 nm and about 770 nm. In embodiments, the wavelength of the light is between about 750 nm and about 770 nm. In embodiments, the wavelength of the light is between about 755 nm and about 770 nm. In embodiments, the wavelength of the light is between about 760 nm and about 770 nm. In embodiments, the wavelength of the light is between about 765 nm and about 770 nm.

[0093] In embodiments, the wavelength of the light is between about 260 nm and about 765 nm. In embodiments, the wavelength of the light is between about 260 nm and about 760 nm. In embodiments, the wavelength of the light is between about 260 nm and about 755 nm. In embodiments, the wavelength of the light is between about 260 nm and about 750 nm. In embodiments, the wavelength of the light is between about 260 nm and about 745 nm. In embodiments, the wavelength of the light is between about 260 nm and about 740 nm. In embodiments, the wavelength of the light is between about 260 nm and about 735 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between about 260 nm and about 730 nm. In embodiments, the wavelength of the light is between about 260 nm and about 725 nm. In embodiments, the wavelength of the light is between about 260 nm and about 720 nm. In embodiments, the wavelength of the light is between about 260 nm and about 715 nm. In embodiments, the wavelength of the light is between about 260 nm and about 710 nm. In embodiments, the wavelength of the light is between about 260 nm and about 705 nm. In embodiments, the wavelength of the light is between about 260 nm and about 700 nm. In embodiments, the wavelength of the light is between about 260 nm and about 695 nm. In embodiments, the wavelength of the light is between about 260 nm and about 690 nm. In embodiments, the wavelength of the light is between about 260 nm and about 685 nm. In embodiments, the wavelength of the light is between about 260 nm and about 680 nm. In embodiments, the wavelength of the light is between about 260 nm and about 675 nm. In embodiments, the wavelength of the light is between about 260 nm and about 670 nm. In embodiments, the wavelength of the light is between about 260 nm and about 665 nm. In embodiments, the wavelength of the light is between about 260 nm and about 660 nm. In embodiments, the wavelength of the light is between about 260 nm and about 655 nm. In embodiments, the wavelength of the light is between about 260 nm and about 650 nm. In embodiments, the wavelength of the light is between about 260 nm and about 645 nm. In embodiments, the wavelength of the light is between about 260 nm and about 640 nm. In embodiments, the wavelength of the light is between about 260 nm and about 635 nm. In embodiments, the wavelength of the light is between about 260 nm and about 630 nm. In embodiments, the wavelength of the light is between about 260 nm and about 625 nm. In embodiments, the wavelength of the light is between about 260 nm and about 620 nm. In embodiments, the wavelength of the light is between about 260 nm and about 615 nm. In embodiments, the wavelength of the light is between about 260 nm and about 610 nm. In embodiments, the wavelength of the light is between about 260 nm and about 605 nm. In embodiments, the wavelength of the light is between about 260 nm and about 600 nm. In embodiments, the wavelength of the light is between about 260 nm and about 595 nm. In embodiments, the wavelength of the light is between about 260 nm and about 590 nm. In embodiments, the wavelength of the light is between about 260 nm and about 585 nm. In embodiments. the wavelength of the light is between about 260 nm and about 580 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between about 260 nm and about 575 nm. In embodiments, the wavelength of the light is between about 260 nm and about 570 nm. In embodiments, the wavelength of the light is between about 260 nm and about 565 nm. In embodiments, the wavelength of the light is between about 260 nm and about 560 nm. In embodiments, the wavelength of the light is between about 260 nm and about 555 nm. In embodiments, the wavelength of the light is between about 260 nm and about 550 nm. In embodiments, the wavelength of the light is between about 260 nm and about 545 nm. In embodiments, the wavelength of the light is between about 260 nm and about 540 nm. In embodiments, the wavelength of the light is between about 260 nm and about 535 nm. In embodiments, the wavelength of the light is between about 260 nm and about 530 nm. In embodiments, the wavelength of the light is between about 260 nm and about 525 nm. In embodiments, the wavelength of the light is between about 260 nm and about 520 nm. In embodiments, the wavelength of the light is between about 260 nm and about 515 nm. In embodiments, the wavelength of the light is between about 260 nm and about 510 nm. In embodiments, the wavelength of the light is between about 260 nm and about 505 nm. In embodiments, the wavelength of the light is between about 260 nm and about 500 nm.

[0094] In embodiments, the wavelength of the light is between about 260 nm and about 495 nm. In embodiments, the wavelength of the light is between about 260 nm and about 490 nm. In embodiments, the wavelength of the light is between about 260 nm and about 485 nm. In embodiments, the wavelength of the light is between about 260 nm and about 480 nm. In embodiments, the wavelength of the light is between about 260 nm and about 475 nm. In embodiments, the wavelength of the light is between about 260 nm and about 470 nm. In embodiments, the wavelength of the light is between about 260 nm and about 465 nm. In embodiments, the wavelength of the light is between about 260 nm and about 460 nm. In embodiments, the wavelength of the light is between about 260 nm and about 455 nm. In embodiments, the wavelength of the light is between about 260 nm and about 450 nm. In embodiments, the wavelength of the light is between about 260 nm and about 445 nm. In embodiments, the wavelength of the light is between about 260 nm and about 440 nm. In embodiments, the wavelength of the light is between about 260 nm and about 435 nm. In embodiments, the wavelength of the light is between about 260 nm and about 430 nm. In embodiments, the wavelength of the light is between about 260 nm and about 425 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between about 260 nm and about 420 nm. In embodiments, the wavelength of the light is between about 260 nm and about 415 nm. In embodiments, the wavelength of the light is between about 260 nm and about 410 nm. In embodiments, the wavelength of the light is between about 260 nm and about 405 nm. In embodiments, the wavelength of the light is between about 260 nm and about 400 nm. In embodiments, the wavelength of the light is between about 260 nm and about 395 nm. In embodiments, the wavelength of the light is between about 260 nm and about 390 nm. In embodiments, the wavelength of the light is between about 260 nm and about 385 nm. In embodiments, the wavelength of the light is between about 260 nm and about 380 nm. In embodiments, the wavelength of the light is between about 260 nm and about 375 nm. In embodiments, the wavelength of the light is between about 260 nm and about 370 nm. In embodiments, the wavelength of the light is between about 260 nm and about 365 nm. In embodiments, the wavelength of the light is between about 260 nm and about 360 nm. In embodiments, the wavelength of the light is between about 260 nm and about 355 nm. In embodiments, the wavelength of the light is between about 260 nm and about 350 nm. In embodiments, the wavelength of the light is between about 260 nm and about 345 nm. In embodiments, the wavelength of the light is between about 260 nm and about 340 nm. In embodiments, the wavelength of the light is between about 260 nm and about 335 nm. In embodiments, the wavelength of the light is between about 260 nm and about 330 nm. In embodiments, the wavelength of the light is between about 260 nm and about 325 nm. In embodiments, the wavelength of the light is between about 260 nm and about 320 nm. In embodiments, the wavelength of the light is between about 260 nm and about 315 nm. In embodiments, the wavelength of the light is between about 260 nm and about 310 nm. In embodiments, the wavelength of the light is between about 260 nm and about 305 nm. In embodiments, the wavelength of the light is between about 260 nm and about 300 nm. In embodiments, the wavelength of the light is between about 260 nm and about 295 nm. In embodiments, the wavelength of the light is between about 260 nm and about 290 nm. In embodiments, the wavelength of the light is between about 260 nm and about 285 nm. In embodiments, the wavelength of the light is between about 260 nm and about 280 nm. In embodiments, the wavelength of the light is between about 260 nm and about 275 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between about 260 nm and about 270 nm. In embodiments, the wavelength of the light is between about 260 nm and about 265 nm.

[0095] In embodiments, the wavelength of the light is between 260 nm and 770 nm. In embodiments, the wavelength of the light is between 265 nm and 770 nm. In embodiments, the wavelength of the light is between 270 nm and 770 nm. In embodiments, the wavelength of the light is between 275 nm and 770 nm. In embodiments, the wavelength of the light is between 280 nm and 770 nm. In embodiments, the wavelength of the light is between 285 nm and 770 nm. In embodiments, the wavelength of the light is between 290 nm and 770 nm. In embodiments, the wavelength of the light is between 295 nm and 770 nm. In embodiments, the wavelength of the light is between 300 nm and 770 nm. In embodiments, the wavelength of the light is between 305 nm and 770 nm. In embodiments, the wavelength of the light is between 310 nm and 770 nm. In embodiments, the wavelength of the light is between 315 nm and 770 nm. In embodiments, the wavelength of the light is between 320 nm and 770 nm. In embodiments, the wavelength of the light is between 325 nm and 770 nm. In embodiments, the wavelength of the light is between 330 nm and 770 nm. In embodiments, the wavelength of the light is between 335 nm and 770 nm. In embodiments, the wavelength of the light is between 340 nm and 770 nm. In embodiments, the wavelength of the light is between 345 nm and 770 nm. In embodiments, the wavelength of the light is between 350 nm and 770 nm. In embodiments, the wavelength of the light is between 355 nm and 770 nm. In embodiments, the wavelength of the light is between 360 nm and 770 nm. In embodiments, the wavelength of the light is between 365 nm and 770 nm. In embodiments, the wavelength of the light is between 370 nm and 770 nm. In embodiments, the wavelength of the light is between 375 nm and 770 nm. In embodiments, the wavelength of the light is between 380 nm and 770 nm. In embodiments, the wavelength of the light is between 385 nm and 770 nm. In embodiments, the wavelength of the light is between 390 nm and 770 nm. In embodiments, the wavelength of the light is between 395 nm and 770 nm. In embodiments, the wavelength of the light is between 400 nm and 770 nm. In embodiments, the wavelength of the light is between 405 nm and 770 nm. In embodiments, the wavelength of the light is between 410 nm and 770 nm. In embodiments, the wavelength of the light is between 415 nm and 770 nm. In embodiments, the wavelength of the light is between 420 nm and 770 nm. In embodiments, the wavelength of the light is between 425 nm and 770 nm. In embodiments, the wavelength of the light is betweenPATENTAttorney Docket No. 048536-799001WO430 nm and 770 nm. In embodiments, the wavelength of the light is between 435 nm and 770 nm. In embodiments, the wavelength of the light is between 440 nm and 770 nm. In embodiments, the wavelength of the light is between 445 nm and 770 nm. In embodiments, the wavelength of the light is between 450 nm and 770 nm. In embodiments, the wavelength of the light is between 455 nm and 770 nm. In embodiments, the wavelength of the light is between 460 nm and 770 nm. In embodiments, the wavelength of the light is between 465 nm and 770 nm. In embodiments, the wavelength of the light is between 470 nm and 770 nm. In embodiments, the wavelength of the light is between 475 nm and 770 nm. In embodiments, the w avelength of the light is betw een 480 nm and 770 nm. In embodiments, the wavelength of the light is between 485 nm and 770 nm. In embodiments, the wavelength of the light is between 490 nm and 770 nm. In embodiments, the wavelength of the light is between 495 nm and 770 nm.

[0096] In embodiments, the wavelength of the light is between 500 nm and 770 nm. In embodiments, the wavelength of the light is between 505 nm and 770 nm. In embodiments, the w avelength of the light is betw een 510 nm and 770 nm. In embodiments, the w avelength of the light is betw een 515 nm and 770 nm. In embodiments, the w avelength of the light is betw een 520 nm and 770 nm. In embodiments, the wavelength of the light is between 525 nm and 770 nm. In embodiments, the w avelength of the light is betw een 530 nm and 770 nm. In embodiments, the wavelength of the light is between 535 nm and 770 nm. In embodiments, the wavelength of the light is betw een 540 nm and 770 nm. In embodiments, the w avelength of the light is between 545 nm and 770 nm. In embodiments, the wavelength of the light is between 550 nm and 770 nm. In embodiments, the wavelength of the light is between 555 nm and 770 nm. In embodiments, the w avelength of the light is between 560 nm and 770 nm. In embodiments, the wavelength of the light is between 565 nm and 770 nm. In embodiments, the wavelength of the light is between 570 nm and 770 nm. In embodiments, the wavelength of the light is between 575 nm and 770 nm. In embodiments, the wavelength of the light is between 580 nm and 770 nm. In embodiments, the wavelength of the light is between 585 nm and 770 nm. In embodiments, the w avelength of the light is betw een 590 nm and 770 nm. In embodiments, the w avelength of the light is betw een 595 nm and 770 nm. In embodiments, the wavelength of the light is between 600 nm and 770 nm. In embodiments, the wavelength of the light is between 605 nm and 770 nm. In embodiments, the wavelength of the light is betweenPATENTAttorney Docket No. 048536-799001WO610 nm and 770 nm. In embodiments, the wavelength of the light is between 615 nm and 770 nm. In embodiments, the wavelength of the light is between 620 nm and 770 nm. In embodiments, the wavelength of the light is between 625 nm and 770 nm. In embodiments, the wavelength of the light is between 630 nm and 770 nm. In embodiments, the wavelength of the light is between 635 nm and 770 nm. In embodiments, the wavelength of the light is between 640 nm and 770 nm. In embodiments, the wavelength of the light is between 645 nm and 770 nm. In embodiments, the wavelength of the light is between 650 nm and 770 nm. In embodiments, the wavelength of the light is between 655 nm and 770 nm. In embodiments, the w avelength of the light is betw een 660 nm and 770 nm. In embodiments, the wavelength of the light is between 665 nm and 770 nm. In embodiments, the wavelength of the light is between 670 nm and 770 nm. In embodiments, the wavelength of the light is between 675 nm and 770 nm. In embodiments, the wavelength of the light is between 680 nm and 770 nm. In embodiments, the wavelength of the light is between 685 nm and 770 nm. In embodiments, the w avelength of the light is betw een 690 nm and 770 nm. In embodiments, the w avelength of the light is betw een 695 nm and 770 nm. In embodiments, the w avelength of the light is betw een 705 nm and 770 nm. In embodiments, the wavelength of the light is between 710 nm and 770 nm. In embodiments, the wavelength of the light is betw een 715 nm and 770 nm. In embodiments, the wavelength of the light is between 720 nm and 770 nm. In embodiments, the w avelength of the light is betw een 725 nm and 770 nm. In embodiments, the w avelength of the light is betw een 730 nm and 770 nm. In embodiments, the w avelength of the light is betw een 735 nm and 770 nm. In embodiments, the wavelength of the light is between 740 nm and 770 nm. In embodiments, the w avelength of the light is betw een 745 nm and 770 nm. In embodiments, the wavelength of the light is between 750 nm and 770 nm. In embodiments, the wavelength of the light is between 755 nm and 770 nm. In embodiments, the wavelength of the light is between 760 nm and 770 nm. In embodiments, the wavelength of the light is between 765 nm and 770 nm.

[0097] In embodiments, the wavelength of the light is between 260 nm and 765 nm. In embodiments, the wavelength of the light is between 260 nm and 760 nm. In embodiments, the wavelength of the light is between 260 nm and 755 nm. In embodiments, the wavelength of the light is between 260 nm and 750 nm. In embodiments, the wavelength of the light is between 260 nm and 745 nm. In embodiments, the wavelength of the light is between 260 nm and 740PATENTAttorney Docket No. 048536-799001WO nm. In embodiments, the wavelength of the light is between 260 nm and 735 nm. In embodiments, the wavelength of the light is between 260 nm and 730 nm. In embodiments, the wavelength of the light is between 260 nm and 725 nm. In embodiments, the wavelength of the light is between 260 nm and 720 nm. In embodiments, the wavelength of the light is between 260 nm and 715 nm. In embodiments, the wavelength of the light is between 260 nm and 710 nm. In embodiments, the wavelength of the light is between 260 nm and 705 nm. In embodiments, the wavelength of the light is between 260 nm and 700 nm. In embodiments, the wavelength of the light is between 260 nm and 695 nm. In embodiments, the wavelength of the light is between 260 nm and 690 nm. In embodiments, the wavelength of the light is between 260 nm and 685 nm. In embodiments, the wavelength of the light is between 260 nm and 680 nm. In embodiments, the wavelength of the light is between 260 nm and 675 nm. In embodiments, the wavelength of the light is between 260 nm and 670 nm. In embodiments, the wavelength of the light is between 260 nm and 665 nm. In embodiments, the wavelength of the light is between 260 nm and 660 nm. In embodiments, the wavelength of the light is between 260 nm and 655 nm. In embodiments, the wavelength of the light is between 260 nm and 650 nm. In embodiments, the wavelength of the light is between 260 nm and 645 nm. In embodiments, the wavelength of the light is between 260 nm and 640 nm. In embodiments, the wavelength of the light is between 260 nm and 635 nm. In embodiments, the wavelength of the light is between 260 nm and 630 nm. In embodiments, the wavelength of the light is between 260 nm and 625 nm. In embodiments, the wavelength of the light is between 260 nm and 620 nm. In embodiments, the wavelength of the light is between 260 nm and 615 nm. In embodiments, the wavelength of the light is between 260 nm and 610 nm. In embodiments, the wavelength of the light is between 260 nm and 605 nm. In embodiments, the wavelength of the light is between 260 nm and 600 nm. In embodiments, the wavelength of the light is between 260 nm and 595 nm. In embodiments, the wavelength of the light is between 260 nm and 590 nm. In embodiments, the wavelength of the light is between 260 nm and 585 nm. In embodiments, the wavelength of the light is between 260 nm and 580 nm. In embodiments, the wavelength of the light is between 260 nm and 575 nm. In embodiments, the wavelength of the light is between 260 nm and 570 nm. In embodiments, the wavelength of the light is between 260 nm and 565 nm. In embodiments, the wavelength of the light is between 260 nm and 560 nm. In embodiments, the wavelength of the light is between 260 nm and 555 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between 260 nm and 550 nm. In embodiments, the wavelength of the light is between 260 nm and 545 nm. In embodiments, the wavelength of the light is between 260 nm and 540 nm. In embodiments, the wavelength of the light is between 260 nm and 535 nm. In embodiments, the wavelength of the light is between 260 nm and 530 nm. In embodiments, the wavelength of the light is between 260 nm and 525 nm. In embodiments, the wavelength of the light is between 260 nm and 520 nm. In embodiments, the wavelength of the light is between 260 nm and 515 nm. In embodiments, the wavelength of the light is between 260 nm and 510 nm. In embodiments, the wavelength of the light is between 260 nm and 505 nm. In embodiments, the wavelength of the light is between 260 nm and 500 nm.

[0098] In embodiments, the wavelength of the light is between 260 nm and 495 nm. In embodiments, the wavelength of the light is between 260 nm and 490 nm. In embodiments, the wavelength of the light is between 260 nm and 485 nm. In embodiments, the wavelength of the light is between 260 nm and 480 nm. In embodiments, the wavelength of the light is between 260 nm and 475 nm. In embodiments, the wavelength of the light is between 260 nm and 470 nm. In embodiments, the wavelength of the light is between 260 nm and 465 nm. In embodiments, the wavelength of the light is between 260 nm and 460 nm. In embodiments, the wavelength of the light is between 260 nm and 455 nm. In embodiments, the wavelength of the light is between 260 nm and 450 nm. In embodiments, the wavelength of the light is between 260 nm and 445 nm. In embodiments, the wavelength of the light is between 260 nm and 440 nm. In embodiments, the wavelength of the light is between 260 nm and 435 nm. In embodiments, the wavelength of the light is between 260 nm and 430 nm. In embodiments, the wavelength of the light is between 260 nm and 425 nm. In embodiments, the wavelength of the light is between 260 nm and 420 nm. In embodiments, the wavelength of the light is between 260 nm and 415 nm. In embodiments, the wavelength of the light is between 260 nm and 410 nm. In embodiments, the wavelength of the light is between 260 nm and 405 nm. In embodiments, the wavelength of the light is between 260 nm and 400 nm. In embodiments, the wavelength of the light is between 260 nm and 395 nm. In embodiments, the wavelength of the light is between 260 nm and 390 nm. In embodiments, the wavelength of the light is between 260 nm and 385 nm. In embodiments, the wavelength of the light is between 260 nm and 380 nm. In embodiments, the wavelength of the light is between 260 nm and 375 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is between 260 nm and 370 nm. In embodiments, the wavelength of the light is between 260 nm and 365 nm. In embodiments, the wavelength of the light is between 260 nm and 360 nm. In embodiments, the wavelength of the light is between 260 nm and 355 nm. In embodiments, the wavelength of the light is between 260 nm and 350 nm. In embodiments, the wavelength of the light is between 260 nm and 345 nm. In embodiments, the wavelength of the light is between 260 nm and 340 nm. In embodiments, the wavelength of the light is between 260 nm and 335 nm. In embodiments, the wavelength of the light is between 260 nm and 330 nm. In embodiments, the wavelength of the light is between 260 nm and 325 nm. In embodiments, the wavelength of the light is between 260 nm and 320 nm. In embodiments, the wavelength of the light is between 260 nm and 315 nm. In embodiments, the wavelength of the light is between 260 nm and 310 nm. In embodiments, the wavelength of the light is between 260 nm and 305 nm. In embodiments, the wavelength of the light is between 260 nm and 300 nm. In embodiments, the wavelength of the light is between 260 nm and 295 nm. In embodiments, the wavelength of the light is between 260 nm and 290 nm. In embodiments, the wavelength of the light is between 260 nm and 285 nm. In embodiments, the wavelength of the light is between 260 nm and 280 nm. In embodiments, the wavelength of the light is between 260 nm and 275 nm. In embodiments, the wavelength of the light is between 260 nm and 270 nm. In embodiments, the wavelength of the light is between 260 nm and 265 nm.

[0099] In embodiments, the wavelength of the light is about 260 nm. In embodiments, the wavelength of the light is about 265 nm. In embodiments, the wavelength of the light is about 270 nm. In embodiments, the wavelength of the light is about 275 nm. In embodiments, the wavelength of the light is about 280 nm. In embodiments, the wavelength of the light is about 285 nm. In embodiments, the wavelength of the light is about 290 nm. In embodiments, the wavelength of the light is about 295 nm. In embodiments, the wavelength of the light is about 300 nm. In embodiments, the wavelength of the light is about 305 nm. In embodiments, the wavelength of the light is about 310 nm. In embodiments, the wavelength of the light is about 315 nm. In embodiments, the wavelength of the light is about 320 nm. In embodiments, the wavelength of the light is about 325 nm. In embodiments, the wavelength of the light is about 330 nm. In embodiments, the wavelength of the light is about 335 nm. In embodiments, the wavelength of the light is about 340 nm. In embodiments, the wavelength of the light is aboutPATENTAttorney Docket No. 048536-799001WO345 nm. In embodiments, the wavelength of the light is about 350 nm. In embodiments, the wavelength of the light is about 355 nm. In embodiments, the wavelength of the light is about 360 nm. In embodiments, the wavelength of the light is about 365 nm. In embodiments, the wavelength of the light is about 370 nm. In embodiments, the wavelength of the light is about 375 nm. In embodiments, the wavelength of the light is about 380 nm. In embodiments, the wavelength of the light is about 385 nm. In embodiments, the wavelength of the light is about 390 nm. In embodiments, the wavelength of the light is about 395 nm. In embodiments, the wavelength of the light is about 400 nm. In embodiments, the wavelength of the light is about 405 nm. In embodiments, the wavelength of the light is about 410 nm. In embodiments, the wavelength of the light is about 415 nm. In embodiments, the wavelength of the light is about 420 nm. In embodiments, the wavelength of the light is about 425 nm. In embodiments, the wavelength of the light is about 430 nm. In embodiments, the wavelength of the light is about 435 nm. In embodiments, the wavelength of the light is about 440 nm. In embodiments, the wavelength of the light is about 445 nm. In embodiments, the wavelength of the light is about 450 nm. In embodiments, the wavelength of the light is about 455 nm. In embodiments, the wavelength of the light is about 460 nm. In embodiments, the wavelength of the light is about 465 nm. In embodiments, the wavelength of the light is about 470 nm. In embodiments, the wavelength of the light is about 475 nm. In embodiments, the wavelength of the light is about 480 nm. In embodiments, the wavelength of the light is about 485 nm. In embodiments, the wavelength of the light is about 490 nm. In embodiments, the wavelength of the light is about 495 nm.

[0100] In embodiments, the wavelength of the light is about 500 nm. In embodiments, the wavelength of the light is about 505 nm. In embodiments, the wavelength of the light is about 510 nm. In embodiments, the wavelength of the light is about 515 nm. In embodiments, the wavelength of the light is about 520 nm. In embodiments, the wavelength of the light is about 525 nm. In embodiments, the wavelength of the light is about 530 nm. In embodiments, the wavelength of the light is about 535 nm. In embodiments, the wavelength of the light is about 540 nm. In embodiments, the wavelength of the light is about 545 nm. In embodiments, the wavelength of the light is about 550 nm. In embodiments, the wavelength of the light is about 555 nm. In embodiments, the wavelength of the light is about 560 nm. In embodiments, the wavelength of the light is about 565 nm. In embodiments, the wavelength of the light is aboutPATENT Attorney Docket No. 048536-799001WO570 nm. In embodiments, the wavelength of the light is about 575 nm. In embodiments, the wavelength of the light is about 580 nm. In embodiments, the wavelength of the light is about 585 nm. In embodiments, the wavelength of the light is about 590 nm. In embodiments, the wavelength of the light is about 595 nm. In embodiments, the wavelength of the light is about 600 nm. In embodiments, the wavelength of the light is about 605 nm. In embodiments, the wavelength of the light is about 610 nm. In embodiments, the wavelength of the light is about 615 nm. In embodiments, the wavelength of the light is about 620 nm. In embodiments, the wavelength of the light is about 625 nm. In embodiments, the wavelength of the light is about 630 nm. In embodiments, the wavelength of the light is about 635 nm. In embodiments, the wavelength of the light is about 640 nm. In embodiments, the wavelength of the light is about 645 nm. In embodiments, the wavelength of the light is about 650 nm. In embodiments, the wavelength of the light is about 655 nm. In embodiments, the wavelength of the light is about 660 nm. In embodiments, the wavelength of the light is about 665 nm. In embodiments, the wavelength of the light is about 670 nm. In embodiments, the wavelength of the light is about 675 nm. In embodiments, the wavelength of the light is about 680 nm. In embodiments, the wavelength of the light is about 685 nm. In embodiments, the wavelength of the light is about 690 nm. In embodiments, the wavelength of the light is about 695 nm. In embodiments, the wavelength of the light is about 700 nm. In embodiments, the wavelength of the light is about 705 nm. In embodiments, the wavelength of the light is about 710 nm. In embodiments, the wavelength of the light is about 715 nm. In embodiments, the wavelength of the light is about 720 nm. In embodiments, the wavelength of the light is about 725 nm. In embodiments, the wavelength of the light is about 730 nm. In embodiments, the wavelength of the light is about 735 nm. In embodiments, the wavelength of the light is about 740 nm. In embodiments, the wavelength of the light is about 745 nm. In embodiments, the wavelength of the light is about 750 nm. In embodiments, the wavelength of the light is about 755 nm. In embodiments, the wavelength of the light is about 760 nm. In embodiments, the wavelength of the light is about 765 nm. In embodiments, the wavelength of the light is about 770 nm.

[0101] In embodiments, the wavelength of the light is 260 nm. In embodiments, the wavelength of the light is 265 nm. In embodiments, the wavelength of the light is 270 nm. In embodiments, the wavelength of the light is 275 nm. In embodiments, the wavelength of the light is 280 nm. In embodiments, the wavelength of the light is 285 nm. In embodiments, thePATENTAttorney Docket No. 048536-799001WO wavelength of the light is 290 nm. In embodiments, the wavelength of the light is 295 nm. In embodiments, the wavelength of the light is 300 nm. In embodiments, the wavelength of the light is 305 nm. In embodiments, the wavelength of the light is 310 nm. In embodiments, the wavelength of the light is 315 nm. In embodiments, the wavelength of the light is 320 nm. In embodiments, the wavelength of the light is 325 nm. In embodiments, the wavelength of the light is 330 nm. In embodiments, the wavelength of the light is 335 nm. In embodiments, the wavelength of the light is 340 nm. In embodiments, the wavelength of the light is 345 nm. In embodiments, the wavelength of the light is 350 nm. In embodiments, the wavelength of the light is 355 nm. In embodiments, the wavelength of the light is 360 nm. In embodiments, the wavelength of the light is 365 nm. In embodiments, the wavelength of the light is 370 nm. In embodiments, the wavelength of the light is 375 nm. In embodiments, the wavelength of the light is 380 nm. In embodiments, the wavelength of the light is 385 nm. In embodiments, the wavelength of the light is 390 nm. In embodiments, the wavelength of the light is 395 nm. In embodiments, the wavelength of the light is 400 nm. In embodiments, the wavelength of the light is 405 nm. In embodiments, the wavelength of the light is 410 nm. In embodiments, the wavelength of the light is 415 nm. In embodiments, the wavelength of the light is 420 nm. In embodiments, the wavelength of the light is 425 nm. In embodiments, the wavelength of the light is 430 nm. In embodiments, the wavelength of the light is 435 nm. In embodiments, the wavelength of the light is 440 nm. In embodiments, the wavelength of the light is 445 nm. In embodiments, the wavelength of the light is 450 nm. In embodiments, the wavelength of the light is 455 nm. In embodiments, the wavelength of the light is 460 nm. In embodiments, the wavelength of the light is 465 nm. In embodiments, the wavelength of the light is 470 nm. In embodiments, the wavelength of the light is 475 nm. In embodiments, the wavelength of the light is 480 nm. In embodiments, the wavelength of the light is 485 nm. In embodiments, the wavelength of the light is 490 nm. In embodiments, the wavelength of the light is 495 nm. In embodiments, the wavelength of the light is 500 nm. In embodiments, the wavelength of the light is 505 nm. In embodiments, the wavelength of the light is 510 nm. In embodiments, the wavelength of the light is 515 nm. In embodiments, the wavelength of the light is 520 nm. In embodiments, the wavelength of the light is 525 nm. In embodiments, the wavelength of the light is 530 nm. In embodiments, the wavelength of the light is 535 nm. In embodiments, the wavelength of the light is 540 nm. In embodiments, the wavelength of the light is 545 nm. InPATENTAttorney Docket No. 048536-799001WO embodiments, the wavelength of the light is 550 nm. In embodiments, the wavelength of the light is 555 nm. In embodiments, the wavelength of the light is 560 nm. In embodiments, the wavelength of the light is 565 nm. In embodiments, the wavelength of the light is 570 nm. In embodiments, the wavelength of the light is 575 nm. In embodiments, the wavelength of the light is 580 nm. In embodiments, the wavelength of the light is 585 nm. In embodiments, the wavelength of the light is 590 nm. In embodiments, the wavelength of the light is 595 nm. In embodiments, the wavelength of the light is 600 nm.

[0102] In embodiments, the wavelength of the light is 500 nm. In embodiments, the wavelength of the light is 505 nm. In embodiments, the wavelength of the light is 510 nm. In embodiments, the wavelength of the light is 515 nm. In embodiments, the wavelength of the light is 520 nm. In embodiments, the wavelength of the light is 525 nm. In embodiments, the wavelength of the light is 530 nm. In embodiments, the wavelength of the light is 535 nm. In embodiments, the wavelength of the light is 540 nm. In embodiments, the wavelength of the light is 545 nm. In embodiments, the wavelength of the light is 550 nm. In embodiments, the wavelength of the light is 555 nm. In embodiments, the wavelength of the light is 560 nm. In embodiments, the wavelength of the light is 565 nm. In embodiments, the wavelength of the light is 570 nm. In embodiments, the wavelength of the light is 575 nm. In embodiments, the wavelength of the light is 580 nm. In embodiments, the wavelength of the light is 585 nm. In embodiments, the wavelength of the light is 590 nm. In embodiments, the wavelength of the light is 595 nm. In embodiments, the wavelength of the light is 600 nm. In embodiments, the wavelength of the light is 605 nm. In embodiments, the wavelength of the light is 610 nm. In embodiments, the wavelength of the light is 615 nm. In embodiments, the wavelength of the light is 620 nm. In embodiments, the wavelength of the light is 625 nm. In embodiments, the wavelength of the light is 630 nm. In embodiments, the wavelength of the light is 635 nm. In embodiments, the wavelength of the light is 640 nm. In embodiments, the wavelength of the light is 645 nm. In embodiments, the wavelength of the light is 650 nm. In embodiments, the wavelength of the light is 655 nm. In embodiments, the wavelength of the light is 660 nm. In embodiments, the wavelength of the light is 665 nm. In embodiments, the wavelength of the light is 670 nm. In embodiments, the wavelength of the light is 675 nm. In embodiments, the wavelength of the light is 680 nm. In embodiments, the wavelength of the light is 685 nm. In embodiments, the wavelength of the light is 690 nm. In embodiments, the wavelength of thePATENTAttorney Docket No. 048536-799001WO light is 695 nm. In embodiments, the wavelength of the light is 700 nm. In embodiments, the wavelength of the light is 705 nm. In embodiments, the wavelength of the light is 710 nm. In embodiments, the wavelength of the light is 715 nm. In embodiments, the wavelength of the light is 720 nm. In embodiments, the wavelength of the light is 725 nm. In embodiments, the wavelength of the light is 730 nm. In embodiments, the wavelength of the light is 735 nm. In embodiments, the wavelength of the light is 740 nm. In embodiments, the wavelength of the light is 745 nm. In embodiments, the wavelength of the light is 750 nm. In embodiments, the wavelength of the light is 755 nm. In embodiments, the wavelength of the light is 760 nm. In embodiments, the wavelength of the light is 765 nm. In embodiments, the wavelength of the light is 770 nm.

[0103] In embodiments, the w avelength of light is about 590 nm and the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl). In embodiments, the wavelength of light is 590 nm and the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl). In embodiments, the wav elength of light is about 490 nm and the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2). In embodiments, the wavelength of light is 490 nm and the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2). In embodiments, the wavelength of light is about 470 nm and the potassium-selective channelrhodopsin protein is a light-responsive inward rectifier potassium channel Kir2.1. In embodiments, the wavelength of light is 470 nm and the potassium-selective channelrhodopsin protein is a light-responsive inward rectifier potassium channel Kir2.1. In embodiments, the w avelength of light is about 470 nm and the potassium-selective channelrhodopsin protein is a Wobblia lunata channelrhodopsin (WiChR). In embodiments, the wavelength of light is 470 nm and the potassium-selective channelrhodopsin protein is a Wobblia lunata channelrhodopsin (WiChR). In embodiments, the wavelength of light is about 590 nm and the potassium-selective channelrhodopsin protein is a K+-augmented light-gated ion channel 1 (KALI-1). In embodiments, the wavelength of light is 590 nm and the potassium-selective channelrhodopsin protein is a K+-augmented light-gated ion channel 1 (KALI-1). In embodiments, the wavelength of light is about 590 nm and the potassium-selective channelrhodopsin protein is a K+- augmented light-gated ion channel 2 (KALI-2). In embodiments, the wavelength of light is 590PATENTAttorney Docket No. 048536-799001WO nm and the potassium-selective channelrhodopsin protein is a K+-augmented light-gated ion channel 2 (KALI-2).

[0104] In embodiments, the AAV vector is an AAV9 vector, an AAV -PHP. eB vector, an AAV.CAP-Mac vector, an AAV9-X1. 1 vector, or an AAV5 vector. In embodiments, the AAV vector is an AAV9 vector. In embodiments, the AAV vector is an AAV -PHP. eB vector. In embodiments, the AAV vector is an AAV.CAP-Mac vector. In embodiments, the AAV vector is an AAV9-X1.1 vector. In embodiments, the AAV vector is an AAV5 vector.

[0105] In embodiments, the AAV viral vector further includes a Calcium / calmodulin- dependent protein kinase II alpha (CaMKIIa) promoter, a synapsin 1 (Synl) promoter, a phosphate-activated glutaminase (PAG) promoter, a vesicular glutamate transporter-1 (VGLUT1) promoter, or a protein c-Fos (FOS) promoter. In embodiments, the AAV viral vector further includes a Calcium / calmodulin-dependent protein kinase II alpha (CaMKIIa) promoter. In embodiments, the AAV viral vector further includes a synapsin 1 (Synl) promoter. In embodiments, the AAV viral vector further includes a phosphate-activated glutaminase (PAG) promoter. In embodiments, the AAV viral vector further includes a vesicular glutamate transporter-1 (VGLUT1) promoter. In embodiments, the AAV viral vector further includes a protein c-Fos (FOS) promoter.

[0106] In embodiments, the brain cell is a neuron. In embodiments, the brain cell is a glutamatergic neuron.

[0107] In another aspect is provided a method of inhibiting neural activity of a transduced brain cell neuron, the method including exposing the transduced brain cell neuron to light thereby inhibiting neural activity of the transduced brain cell neuron, wherein transduced brain cell neuron includes an exogenous potassium-selective channelrhodopsin protein.

[0108] In embodiments, the intensity’ of the light exposure is from about 20 mW / mm2to about 40 mW / mm2In embodiments, the intensity of the light exposure is from about 21 mW / mm2to about 40 mW / mm2In embodiments, the intensity of the light exposure is from about 22 mW / mm2to about 40 mW / mm2In embodiments, the intensity’ of the light exposure is from about 23 mW / mm2to about 40 mW / mm2. In embodiments, the intensity of the light exposure is from about 24 mW / mm2to about 40 mW / mm2. In embodiments, the intensity of the lightPATENTAttorney Docket No. 048536-799001WO exposure is from about 25 mW / mm2to about 40 mW / mm2. In embodiments, the intensify of the light exposure is from about 26 mW / mm2to about 40 mW / mm2In embodiments, the intensify of the light exposure is from about 27 mW / mm2to about 40 mW / mm2. In embodiments, the intensify of the light exposure is from about 28 mW / mm2to about 40 mW / mm2. In embodiments, the intensity of the light exposure is from about 29 mW / mm2to about 40 mW / mm2. In embodiments, the intensify of the light exposure is from about 30 mW / mm2to about 40 mW / mm2In embodiments, the intensify of the light exposure is from about 31 mW / mm2to about 40 mW / mm2In embodiments, the intensify of the light exposure is from about 32 mW / mm2to about 40 mW / mm2In embodiments, the intensify of the light exposure is from about 33 mW / mm2to about 40 mW / mm2. In embodiments, the intensify' of the light exposure is from about 34 mW / mm2to about 40 mW / mm2. In embodiments, the intensify of the light exposure is from about 35 mW / mm2to about 40 mW / mm2In embodiments, the intensity of the light exposure is from about 36 mW / mm2to about 40 mW / mm2. In embodiments, the intensify of the light exposure is from about 37 mW / mm2to about 40 mW / mm2. In embodiments, the intensify of the light exposure is from about 38 mW / mm2to about 40 mW / mm2. In embodiments, the intensify of the light exposure is from about 39 mW / mm2to about 40 mW / mm2.

[0109] In embodiments, the intensify' of the light exposure is from about 20 mW / mm2to about 39 mW / mm2In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 38 mW / mm2. In embodiments, the intensify' of the light exposure is from about 20 mW / mm2to about 37 mW / mm2In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 36 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 35 mW / mm2. In embodiments, the intensify' of the light exposure is from about 20 mW / mm2to about 34 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 33 mW / mm2In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 32 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 31 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 30 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 29 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 28 mW / mm2. In embodiments, the intensity of the light exposure is fromPATENTAttorney Docket No. 048536-799001WO about 20 mW / mm2to about 27 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 26 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 25 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 24 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 23 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 22 mW / mm2. In embodiments, the intensify of the light exposure is from about 20 mW / mm2to about 21 mW / mm2.

[0110] In embodiments, the intensify' of the light exposure is from 20 mW / mm2to 40 mW / mm2. In embodiments, the intensify' of the light exposure is from 21 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 22 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 23 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 24 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 25 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 26 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 27 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 28 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 29 mW / mm2to 40 mW / mm2. In embodiments, the intensify’ of the light exposure is from 30 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 31 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 32 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 33 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 34 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 35 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 36 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 37 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 38 mW / mm2to 40 mW / mm2. In embodiments, the intensify of the light exposure is from 39 mW / mm2to 40 mW / mm2.[OHl] In embodiments, the intensify of the light exposure is from 20 mW / mm2to 39 mW / mm2. In embodiments, the intensify of the light exposure is from 20 mW / mm2to 38PATENTAttorney Docket No. 048536-799001WO mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 37 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 36 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 35 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 34 mW / mm2. In embodiments, the intensity’ of the light exposure is from 20 mW / mm2to 33 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 32 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 31 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 30 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 29 mW / mm2. In embodiments, the intensity’ of the light exposure is from 20 mW / mm2to 28 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 27 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 26 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 25 mW / mm2. In embodiments, the intensity’ of the light exposure is from 20 mW / mm2to 24 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 23 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 22 mW / mm2. In embodiments, the intensity of the light exposure is from 20 mW / mm2to 21 mW / mm2.

[0112] In embodiments, the intensity' of the light exposure is from about 26.2 mW / mm2to about 35.8 mW / mm2. In embodiments, the intensity’ of the light exposure is from 26.2 mW / mm2to 35.8 mW / mm2

[0113] In embodiments, the intensity’ of the light exposure is about 26.2 mW / mm2In embodiments, the intensity of the light exposure is about 26.3 mW / mm2. In embodiments, the intensity of the light exposure is about 26.4 mW / mm2In embodiments, the intensity of the light exposure is about 26.5 mW / mm2In embodiments, the intensity of the light exposure is about 26.6 mW / mm2In embodiments, the intensity of the light exposure is about 26.7 mW / mm2In embodiments, the intensity of the light exposure is about 26.8 mW / mm2. In embodiments, the intensity of the light exposure is about 26.9 mW / mm2

[0114] In embodiments, the intensity' of the light exposure is 26.2 mW / mm2. In embodiments, the intensity’ of the light exposure is 26.3 mW / mm2. In embodiments, the intensity’ of the lightPATENTAttorney Docket No. 048536-799001WO exposure is 26.4 mW / mm2. In embodiments, the intensity of the light exposure is 26.5 mW / mm2. In embodiments, the intensity of the light exposure is 26.6 mW / mm2In embodiments, the intensity of the light exposure is 26.7 mW / mm2. In embodiments, the intensity of the light exposure is 26.8 mW / mm2. In embodiments, the intensity of the light exposure is 26.9 mW / mm2.

[0115] In embodiments, the intensity' of the light exposure is about 27.0 mW / mm2In embodiments, the intensity of the light exposure is about 27. 1 mW / mm2. In embodiments, the intensity of the light exposure is about 27.2 mW / mm2. In embodiments, the intensity of the light exposure is about 27.3 mW / mm2In embodiments, the intensity of the light exposure is about27.4 mW / mm2In embodiments, the intensity of the light exposure is about 27.5 mW / mm2In embodiments, the intensity of the light exposure is about 27.6 mW / mm2. In embodiments, the intensity of the light exposure is about 27.7 mW / mm2. In embodiments, the intensity of the light exposure is about 27.8 mW / mm2In embodiments, the intensity of the light exposure is about27.9 mW / mm2.

[0116] In embodiments, the intensity' of the light exposure is 27.0 mW / mm2. In embodiments, the intensity of the light exposure is 27. 1 mW / mm2. In embodiments, the intensity of the light exposure is 27.2 mW / mm2. In embodiments, the intensity of the light exposure is 27.3 mW / mm2. In embodiments, the intensity of the light exposure is 27.4 mW / mm2In embodiments, the intensity of the light exposure is 27.5 mW / mm2In embodiments, the intensity of the light exposure is 27.6 mW / mm2In embodiments, the intensity of the light exposure is 27.7 mW / mm2In embodiments, the intensity of the light exposure is 27.8 mW / mm2In embodiments, the intensity of the light exposure is 27.9 mW / mm2.

[0117] In embodiments, the intensity of the light exposure is about 28.0 mW / mm2. In embodiments, the intensity of the light exposure is about 28. 1 mW / mm2. In embodiments, the intensity of the light exposure is about 28.2 mW / mm2In embodiments, the intensity of the light exposure is about 28.3 mW / mm2In embodiments, the intensity of the light exposure is about28.4 mW / mm2In embodiments, the intensity of the light exposure is about 28. 5 mW / mm2. In embodiments, the intensity of the light exposure is about 28.6 mW / mm2. In embodiments, the intensity of the light exposure is about 28.7 mW / mm2In embodiments, the intensity of the light exposure is about 28.8 mW / mm2In embodiments, the intensity of the light exposure is about28.9 mW / mm2.PATENTAttorney Docket No. 048536-799001WO

[0118] In embodiments, the intensity of the light exposure is 28.0 mW / mm2. In embodiments, the intensity of the light exposure is 28. 1 mW / mm2In embodiments, the intensity' of the light exposure is 28.2 mW / mm2. In embodiments, the intensity of the light exposure is 28.3 mW / mm2. In embodiments, the intensity of the light exposure is 28.4 mW / mm2. In embodiments, the intensity of the light exposure is 28. 5 mW / mm2In embodiments, the intensity of the light exposure is 28.6 mW / mm2. In embodiments, the intensity' of the light exposure is 28.7 mW / mm2. In embodiments, the intensity of the light exposure is 28.8 mW / mm2In embodiments, the intensity of the light exposure is 28.9 mW / mm2

[0119] In embodiments, the intensity' of the light exposure is about 29.0 mW / mm2. In embodiments, the intensity of the light exposure is about 29. 1 mW / mm2. In embodiments, the intensity of the light exposure is about 29.2 mW / mm2. In embodiments, the intensity of the light exposure is about 29.3 mW / mm2In embodiments, the intensity of the light exposure is about29.4 mW / mm2In embodiments, the intensity of the light exposure is about 29.5 mW / mm2. In embodiments, the intensity of the light exposure is about 29.6 mW / mm2. In embodiments, the intensity of the light exposure is about 29.7 mW / mm2. In embodiments, the intensity of the light exposure is about 29.8 mW / mm2In embodiments, the intensity of the light exposure is about 29.9 mW / mm2.

[0120] In embodiments, the intensity’ of the light exposure is 29.0 mW / mm2. In embodiments, the intensity of the light exposure is 29. 1 mW / mm2In embodiments, the intensity of the light exposure is 29.2 mW / mm2. In embodiments, the intensity of the light exposure is 29.3 mW / mm2. In embodiments, the intensity of the light exposure is 29.4 mW / mm2In embodiments, the intensity of the light exposure is 29.5 mW / mm2. In embodiments, the intensity of the light exposure is 29.6 mW / mm2In embodiments, the intensity of the light exposure is 29.7 mW / mm2In embodiments, the intensity of the light exposure is 29.8 mW / mm2In embodiments, the intensity of the light exposure is 29.9 mW / mm2

[0121] In embodiments, the intensity of the light exposure is about 30.0 mW / mm2In embodiments, the intensity of the light exposure is about 30.1 mW / mm2. In embodiments, the intensity of the light exposure is about 30.2 mW / mm2In embodiments, the intensity of the light exposure is about 30.3 mW / mm2In embodiments, the intensity of the light exposure is about30.4 mW / mm2. In embodiments, the intensity of the light exposure is about 30.5 mW / mm2. InPATENTAttorney Docket No. 048536-799001WO embodiments, the intensity of the light exposure is about 30.6 mW / mm2. In embodiments, the intensity' of the light exposure is about 30.7 mW / mm2In embodiments, the intensity of the light exposure is about 30.8 mW / mm2In embodiments, the intensity of the light exposure is about30.9 mW / mm2.

[0122] In embodiments, the intensity' of the light exposure is 30.0 mW / mm2. In embodiments, the intensity of the light exposure is 30. 1 mW / mm2In embodiments, the intensity of the light exposure is 30.2 mW / mm2In embodiments, the intensity of the light exposure is 30.3 mW / mm2In embodiments, the intensity of the light exposure is 30.4 mW / mm2In embodiments, the intensity of the light exposure is 30.5 mW / mm2In embodiments, the intensity of the light exposure is 30.6 mW / mm2. In embodiments, the intensity of the light exposure is 30.7 mW / mm2In embodiments, the intensity' of the light exposure is 30.8 mW / mm2In embodiments, the intensity of the light exposure is 30.9 mW / mm2

[0123] In embodiments, the intensity’ of the light exposure is about 31.0 mW / mm2. In embodiments, the intensity of the light exposure is about 31.1 mW / mm2. In embodiments, the intensity of the light exposure is about 31.2 mW / mm2In embodiments, the intensity of the light exposure is about 31.3 mW / mm2In embodiments, the intensity of the light exposure is about 31.4 mW / mm2In embodiments, the intensity of the light exposure is about 31.5 mW / mm2In embodiments, the intensity of the light exposure is about 31.6 mW / mm2. In embodiments, the intensity of the light exposure is about 31.7 mW / mm2In embodiments, the intensity of the light exposure is about 31.8 mW / mm2In embodiments, the intensity' of the light exposure is about31.9 mW / mm2.

[0124] In embodiments, the intensity' of the light exposure is 31.0 mW / mm2. In embodiments, the intensity of the light exposure is 31. 1 mW / mm2. In embodiments, the intensity of the light exposure is 31.2 mW / mm2In embodiments, the intensity of the light exposure is 31.3 mW / mm2In embodiments, the intensity of the light exposure is 31.4 mW / mm2In embodiments, the intensity' of the light exposure is 31.5 mW / mm2. In embodiments, the intensity' of the light exposure is 31.6 mW / mm2. In embodiments, the intensity of the light exposure is 31.7 mW / mm2. In embodiments, the intensity of the light exposure is 31.8 mW / mm2In embodiments, the intensity of the light exposure is 31.9 mW / mm2PATENTAttorney Docket No. 048536-799001WO

[0125] In embodiments, the intensity of the light exposure is about 32.0 mW / mm2. In embodiments, the intensity of the light exposure is about 32. 1 mW / mm2. In embodiments, the intensity of the light exposure is about 32.2 mW / mm2. In embodiments, the intensity of the light exposure is about 32.3 mW / mm2In embodiments, the intensity of the light exposure is about32.4 mW / mm2In embodiments, the intensity of the light exposure is about 32.5 mW / mm2In embodiments, the intensity of the light exposure is about 32.6 mW / mm2. In embodiments, the intensity of the light exposure is about 32.7 mW / mm2In embodiments, the intensity of the light exposure is about 32.8 mW / mm2. In embodiments, the intensity of the light exposure is about32.9 mW / mm2

[0126] In embodiments, the intensity' of the light exposure is 32.0 mW / mm2. In embodiments, the intensity of the light exposure is 32. 1 mW / mm2. In embodiments, the intensity' of the light exposure is 32.2 mW / mm2In embodiments, the intensity of the light exposure is 32.3 mW / mm2In embodiments, the intensity of the light exposure is 32.4 mW / mm2. In embodiments, the intensity of the light exposure is 32.5 mW / mm2In embodiments, the intensity of the light exposure is 32.6 mW / mm2. In embodiments, the intensity of the light exposure is 32.7 mW / mm2. In embodiments, the intensity of the light exposure is 32.8 mW / mm2In embodiments, the intensity of the light exposure is 32.9 mW / mm2.

[0127] In embodiments, the intensity’ of the light exposure is about 33.0 mW / mm2. In embodiments, the intensity of the light exposure is about 33.1 mW / mm2. In embodiments, the intensity of the light exposure is about 33.2 mW / mm2. In embodiments, the intensity of the light exposure is about 33.3 mW / mm2In embodiments, the intensity of the light exposure is about33.4 mW / mm2In embodiments, the intensity of the light exposure is about 33.5 mW / mm2. In embodiments, the intensity of the light exposure is about 33.6 mW / mm2. In embodiments, the intensity of the light exposure is about 33.7 mW / mm2In embodiments, the intensity of the light exposure is about 33.8 mW / mm2In embodiments, the intensity of the light exposure is about33.9 mW / mm2

[0128] In embodiments, the intensity of the light exposure is 33.0 mW / mm2. In embodiments, the intensity of the light exposure is 33.1 mW / mm2In embodiments, the intensity of the light exposure is 33.2 mW / mm2. In embodiments, the intensity of the light exposure is 33.3 mW / mm2In embodiments, the intensity of the light exposure is 33.4 mW / mm2In embodiments, thePATENTAttorney Docket No. 048536-799001WO intensity of the light exposure is 33.5 mW / mm2. In embodiments, the intensity of the light exposure is 33.6 mW / mm2In embodiments, the intensity of the light exposure is 33.7 mW / mm2In embodiments, the intensity of the light exposure is 33.8 mW / mm2In embodiments, the intensity of the light exposure is 33.9 mW / mm2

[0129] In embodiments, the intensity' of the light exposure is about 34.0 mW / mm2In embodiments, the intensity of the light exposure is about 34. 1 mW / mm2. In embodiments, the intensity of the light exposure is about 34.2 mW / mm2. In embodiments, the intensity of the light exposure is about 34.3 mW / mm2In embodiments, the intensity of the light exposure is about34.4 mW / mm2In embodiments, the intensity of the light exposure is about 34.5 mW / mm2In embodiments, the intensity of the light exposure is about 34.6 mW / mm2. In embodiments, the intensity of the light exposure is about 34.7 mW / mm2. In embodiments, the intensity of the light exposure is about 34.8 mW / mm2In embodiments, the intensity of the light exposure is about 34.9 mW / mm2.

[0130] In embodiments, the intensity' of the light exposure is 34.0 mW / mm2. In embodiments, the intensity of the light exposure is 34. 1 mW / mm2In embodiments, the intensity of the light exposure is 34.2 mW / mm2. In embodiments, the intensity of the light exposure is 34.3 mW / mm2. In embodiments, the intensity of the light exposure is 34.4 mW / mm2In embodiments, the intensity of the light exposure is 34.5 mW / mm2In embodiments, the intensity of the light exposure is 34.6 mW / mm2In embodiments, the intensity of the light exposure is 34.7 mW / mm2In embodiments, the intensity of the light exposure is 34.8 mW / mm2In embodiments, the intensity of the light exposure is 34.9 mW / mm2.

[0131] In embodiments, the intensity of the light exposure is about 35.0 mW / mm2. In embodiments, the intensity of the light exposure is about 35. 1 mW / mm2. In embodiments, the intensity of the light exposure is about 35.2 mW / mm2. In embodiments, the intensity of the light exposure is about 35.3 mW / mm2In embodiments, the intensity of the light exposure is about35.4 mW / mm2. In embodiments, the intensity of the light exposure is about 35.5 mW / mm2In embodiments, the intensity of the light exposure is about 35.6 mW / mm2. In embodiments, the intensity of the light exposure is about 35.7 mW / mm2In embodiments, the intensity of the light exposure is about 35.8 mW / mm2PATENTAttorney Docket No. 048536-799001WO

[0132] In embodiments, the intensity of the light exposure is 35.0 mW / mm2. In embodiments, the intensity of the light exposure is 35. 1 mW / mm2In embodiments, the intensity' of the light exposure is 35.2 mW / mm2. In embodiments, the intensity of the light exposure is 35.3 mW / mm2. In embodiments, the intensity of the light exposure is 35.4 mW / mm2. In embodiments, the intensity of the light exposure is 35.5 mW / mm2In embodiments, the intensity of the light exposure is 35.6 mW / mm2. In embodiments, the intensity' of the light exposure is 35.7 mW / mm2. In embodiments, the intensity of the light exposure is 35.8 mW / mm2

[0133] In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 20 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 30 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 40 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 50 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 60 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 70 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 80 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 90 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 100 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 200 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 300 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 400 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 500 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 600 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 700 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 800 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 900 milliseconds (ms) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 1 second (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 2 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 3 seconds (s) to about 20 seconds (s). In embodiments, the duration of the lightPATENTAttorney Docket No. 048536-799001WO exposure is about 4 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 5 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 6 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 7 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 8 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 9 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 10 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 11 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 12 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 13 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 14 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 15 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 16 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 17 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 18 seconds (s) to about 20 seconds (s). In embodiments, the duration of the light exposure is about 19 seconds (s) to about 20 seconds (s).

[0134] In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 19 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 18 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 17 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 16 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 15 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 14 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 13 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 12 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 11 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 10 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 9 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 8 seconds (s). In embodiments, the duration of the lightPATENTAttorney Docket No. 048536-799001WO exposure is about 10 milliseconds (ms) to about 7 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 6 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 5 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 4 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 3 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 2 seconds (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 1 second (s). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 900 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 800 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 700 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 600 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 500 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 400 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 300 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 200 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 100 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 90 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 80 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 70 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 60 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 50 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 40 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 30 milliseconds (ms). In embodiments, the duration of the light exposure is about 10 milliseconds (ms) to about 20 milliseconds (ms).

[0135] In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 20 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 30 milliseconds (ms) to 20PATENTAttorney Docket No. 048536-799001WO seconds (s). In embodiments, the duration of the light exposure is 40 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 50 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 60 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 70 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 80 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 90 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 100 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 200 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 300 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 400 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 500 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 600 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 700 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 800 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 900 milliseconds (ms) to 20 seconds (s). In embodiments, the duration of the light exposure is 1 second (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 2 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 3 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 4 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 5 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 6 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 7 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 8 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 9 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 10 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 1 1 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 12 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 13 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 14 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 15 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 16 seconds (s) to 20 seconds (s). InPATENTAttorney Docket No. 048536-799001WO embodiments, the duration of the light exposure is 17 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 18 seconds (s) to 20 seconds (s). In embodiments, the duration of the light exposure is 19 seconds (s) to 20 seconds (s).

[0136] In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 19 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 18 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 17 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 16 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 15 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 14 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 13 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 12 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 11 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 10 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 9 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 8 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 7 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 6 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 5 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 4 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 3 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 2 seconds (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 1 second (s). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 900 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 800 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 700 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 600 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 500 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 400 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 300 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 200 milliseconds (ms). In embodiments, thePATENTAttorney Docket No. 048536-799001WO duration of the light exposure is 10 milliseconds (ms) to 100 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 90 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 80 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 70 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 60 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 50 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 40 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 30 milliseconds (ms). In embodiments, the duration of the light exposure is 10 milliseconds (ms) to 20 milliseconds (ms).

[0137] In embodiments, the duration of the light exposure is about 50 milliseconds (ms) to about 10 seconds (s). In embodiments, the duration of the light exposure is 50 milliseconds (ms) to 10 seconds (s).

[0138] In embodiments, the duration of the light exposure is about 10 milliseconds (ms). In embodiments, the duration of the light exposure is about 20 milliseconds (ms). In embodiments, the duration of the light exposure is about 30 milliseconds (ms). In embodiments, the duration of the light exposure is about 40 milliseconds (ms). In embodiments, the duration of the light exposure is about 50 milliseconds (ms). In embodiments, the duration of the light exposure is about 60 milliseconds (ms). In embodiments, the duration of the light exposure is about 70 milliseconds (ms). In embodiments, the duration of the light exposure is about 80 milliseconds (ms). In embodiments, the duration of the light exposure is about 90 milliseconds (ms). In embodiments, the duration of the light exposure is about 100 milliseconds (ms). In embodiments, the duration of the light exposure is about 200 milliseconds (ms). In embodiments, the duration of the light exposure is about 300 milliseconds (ms). In embodiments, the duration of the light exposure is about 400 milliseconds (ms). In embodiments, the duration of the light exposure is about 500 milliseconds (ms). In embodiments, the duration of the light exposure is about 600 milliseconds (ms). In embodiments, the duration of the light exposure is about 700 milliseconds (ms). In embodiments, the duration of the light exposure is about 800 milliseconds (ms). In embodiments, the duration of the light exposure is about 900 milliseconds (ms). In embodiments, the duration of the light exposure is about 1 second (s). In embodiments, the duration of the light exposure is about 2 seconds (s). In embodiments, the duration of the light exposure is about 3PATENTAttorney Docket No. 048536-799001WO seconds (s). In embodiments, the duration of the light exposure is about 4 seconds (s). In embodiments, the duration of the light exposure is about 5 seconds (s). In embodiments, the duration of the light exposure is about 6 seconds (s). In embodiments, the duration of the light exposure is about 7 seconds (s). In embodiments, the duration of the light exposure is about 8 seconds (s). In embodiments, the duration of the light exposure is about 9 seconds (s). In embodiments, the duration of the light exposure is about 10 seconds (s). In embodiments, the duration of the light exposure is about 11 seconds (s). In embodiments, the duration of the light exposure is about 12 seconds (s). In embodiments, the duration of the light exposure is about 13 seconds (s). In embodiments, the duration of the light exposure is about 14 seconds (s). In embodiments, the duration of the light exposure is about 15 seconds (s). In embodiments, the duration of the light exposure is about 16 seconds (s). In embodiments, the duration of the light exposure is about 17 seconds (s). In embodiments, the duration of the light exposure is about 18 seconds (s). In embodiments, the duration of the light exposure is about 19 seconds (s). In embodiments, the duration of the light exposure is about 20 seconds (s).

[0139] In embodiments, the duration of the light exposure is 10 milliseconds (ms). In embodiments, the duration of the light exposure is 20 milliseconds (ms). In embodiments, the duration of the light exposure is 30 milliseconds (ms). In embodiments, the duration of the light exposure is 40 milliseconds (ms). In embodiments, the duration of the light exposure is 50 milliseconds (ms). In embodiments, the duration of the light exposure is 60 milliseconds (ms). In embodiments, the duration of the light exposure is 70 milliseconds (ms). In embodiments, the duration of the light exposure is 80 milliseconds (ms). In embodiments, the duration of the light exposure is 90 milliseconds (ms). In embodiments, the duration of the light exposure is 100 milliseconds (ms). In embodiments, the duration of the light exposure is 200 milliseconds (ms). In embodiments, the duration of the light exposure is 300 milliseconds (ms). In embodiments, the duration of the light exposure is 400 milliseconds (ms). In embodiments, the duration of the light exposure is 500 milliseconds (ms). In embodiments, the duration of the light exposure is 600 milliseconds (ms). In embodiments, the duration of the light exposure is 700 milliseconds (ms). In embodiments, the duration of the light exposure is 800 milliseconds (ms). In embodiments, the duration of the light exposure is 900 milliseconds (ms). In embodiments, the duration of the light exposure is 1 second (s). In embodiments, the duration of the light exposure is 2 seconds (s). In embodiments, the duration of the light exposure is 3 seconds (s). InPATENTAttorney Docket No. 048536-799001WO embodiments, the duration of the light exposure is 4 seconds (s). In embodiments, the duration of the light exposure is 5 seconds (s). In embodiments, the duration of the light exposure is 6 seconds (s). In embodiments, the duration of the light exposure is 7 seconds (s). In embodiments, the duration of the light exposure is 8 seconds (s). In embodiments, the duration of the light exposure is 9 seconds (s). In embodiments, the duration of the light exposure is 10 seconds (s). In embodiments, the duration of the light exposure is 11 seconds (s). In embodiments, the duration of the light exposure is 12 seconds (s). In embodiments, the duration of the light exposure is 13 seconds (s). In embodiments, the duration of the light exposure is 14 seconds (s). In embodiments, the duration of the light exposure is 15 seconds (s). In embodiments, the duration of the light exposure is 16 seconds (s). In embodiments, the duration of the light exposure is 17 seconds (s). In embodiments, the duration of the light exposure is 18 seconds (s). In embodiments, the duration of the light exposure is 19 seconds (s). In embodiments, the duration of the light exposure is 20 seconds (s).

[0140] In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 20 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 30 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 40 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 50 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 60 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 70 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 80 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 90 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 100 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 200 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 300 milliseconds (ms) to about 20 seconds (s). InPATENTAttorney Docket No. 048536-799001WO embodiments, the transduced brain cell neuron is exposed to light at an interval of about 400 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 500 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 600 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 700 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 800 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 900 milliseconds (ms) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 1 second (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 2 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 3 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 4 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 5 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 6 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 7 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 8 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 9 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 11 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 12 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 13 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 14 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 15 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 16 seconds (s) to about 20 seconds (s). InPATENTAttorney Docket No. 048536-799001WO embodiments, the transduced brain cell neuron is exposed to light at an interval of about 17 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 18 seconds (s) to about 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 19 seconds (s) to about 20 seconds (s).

[0141] In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 19 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 18 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 17 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 16 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 15 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 1 milliseconds (ms) to about 14 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 13 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 12 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 11 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 10 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 9 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 8 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 7 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 6 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 5 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 4 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 3 seconds (s). In embodiments, the transduced brain cell neuron isPATENTAttorney Docket No. 048536-799001WO exposed to light at an interval of about 10 milliseconds (ms) to about 2 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 1 second (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 900 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 800 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 700 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 600 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 500 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 400 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 300 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 200 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 100 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 90 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 80 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 70 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 60 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 50 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 40 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 30 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms) to about 20 milliseconds (ms).

[0142] In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposedPATENTAttorney Docket No. 048536-799001WO to light at an interval of 20 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 30 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 40 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 50 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 60 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 70 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 80 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 90 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 100 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 200 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 300 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 400 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 500 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 600 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 700 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 800 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 900 milliseconds (ms) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 1 second (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 2 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 3 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 4 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 5 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 6 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 7 seconds (s) to 20 seconds (s). In embodiments, the transducedPATENTAttorney Docket No. 048536-799001WO brain cell neuron is exposed to light at an interval of 8 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 9 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 11 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 12 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 13 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 14 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 15 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 16 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 17 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 18 seconds (s) to 20 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 19 seconds (s) to 20 seconds (s).

[0143] In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 19 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 18 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 17 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 16 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 15 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 14 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 13 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 12 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 11 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 10 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 9 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 8 seconds (s). InPATENTAttorney Docket No. 048536-799001WO embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 7 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 6 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 5 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 4 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 3 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 2 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 1 second (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 900 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 800 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 700 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 600 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 500 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 400 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 300 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 200 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 100 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 90 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 80 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 70 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 60 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 50 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 40 milliseconds (ms). In embodiments, the transduced brain cell neuron isPATENTAttorney Docket No. 048536-799001WO exposed to light at an interval of 10 milliseconds (ms) to 30 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms) to 20 milliseconds (ms).

[0144] In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 50 milliseconds (ms) to about 10 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 50 milliseconds (ms) to 10 seconds (s).

[0145] In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 20 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 30 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 40 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 50 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 60 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 70 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 80 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 90 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 100 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 200 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 300 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 400 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 500 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 600 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 700 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 800 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 900 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 1 second (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 2 seconds (s). In embodiments, the transducedPATENTAttorney Docket No. 048536-799001WO brain cell neuron is exposed to light at an interval of about 3 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 4 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 5 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 6 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 7 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 8 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 9 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 10 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 11 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 12 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 13 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 14 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 15 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 16 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 17 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 18 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 19 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of about 20 seconds (s).

[0146] In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 20 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 30 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 40 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 50 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 60 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 70 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 80 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 90 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed toPATENTAttorney Docket No. 048536-799001WO light at an interval of 100 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 200 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 300 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 400 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 500 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 600 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 700 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 800 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 900 milliseconds (ms). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 1 second (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 2 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 3 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 4 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 5 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 6 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 7 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 8 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 9 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 10 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 11 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 12 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 13 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 14 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 15 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 16 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 17 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 18 seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 19PATENTAttorney Docket No. 048536-799001WO seconds (s). In embodiments, the transduced brain cell neuron is exposed to light at an interval of 20 seconds (s).

[0147] In embodiments, the method includes exposing a plurality of the transduced brain cell neurons with the light thereby inhibiting neural activity of the plurality transduced brain cell neurons.

[0148] In embodiments, about 5% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 6% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 7% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 8% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 9% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 10% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 15% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 20% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 25% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 30% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 35% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 40% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 45% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 46% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 47% to about 50% of the plurality7of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 48% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 49% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure.

[0149] In embodiments, about 5% to about 49% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 48% of thePATENTAttorney Docket No. 048536-799001WO plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 47% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 46% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 45% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 40% of the plurality' of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 35% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 30% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 25% of the plurality' of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 20% of the plurality' of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 15% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 10% of the plurality7of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 9% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 8% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 7% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 5% to about 6% of the plurality of transduced brain cell neurons are inhibited by the light exposure.

[0150] In embodiments, 5% to 50% of the plurality of transduced brain cell neurons are inhibited by' the light exposure. In embodiments, 6% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 7% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 8% to 50% of the plurality7of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 9% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 10% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 15% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 20% to 50% of the plurality7of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 25% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. InPATENTAttorney Docket No. 048536-799001WO embodiments, 30% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 35% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 40% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 45% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 46% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 47% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 48% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 49% to 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure.

[0151] In embodiments, 5% to 49% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 48% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 47% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 46% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 45% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 40% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 35% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 30% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 25% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 20% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 15% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 10% of the plurality7of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 9% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 8% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 7% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 5% to 6% of the plurality of transduced brain cell neurons are inhibited by the light exposure.PATENTAttorney Docket No. 048536-799001WO

[0152] In embodiments, about 5% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 6% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 7% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 8% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 9% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 10% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 15% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 20% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 25% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 30% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 35% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 40% of the plurality7of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 45% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 46% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 47% of the plurality7of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 48% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 49% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure.

[0153] In embodiments, 5% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 6% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 7% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 8% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 9% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 10% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 15% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 20% of the plurality of transduced brain cell neurons are inhibited by the lightPATENTAttorney Docket No. 048536-799001WO exposure. In embodiments, 25% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 30% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 35% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 40% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 45% of the plurality' of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 46% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 47% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 48% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 49% of the plurality of transduced brain cell neurons are inhibited by the light exposure. In embodiments, 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure.

[0154] In embodiments, the neural network of brain cell neurons includes a population of transduced brain cell neurons. In embodiments, the population of transduced brain cell neurons is a population of glutamatergic neurons. In embodiments, a transduced brain cell neuron includes a potassium-selective channelrhodopsin protein. In embodiments, each transduced brain cell neuron within the population of transduced brain cell neurons includes a potassium-selective channelrhodopsin protein.

[0155] In embodiments, the population of transduced brain cell neurons is from about 5% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 6% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 7% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 8% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 9% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 10% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 15% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 20% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 25% toPATENTAttorney Docket No. 048536-799001WO about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 30% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 35% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 40% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 45% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 46% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 47% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 48% to about 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 49% to about 50% of the neural network of brain cell neurons.

[0156] In embodiments, the population of transduced brain cell neurons is from about 5% to about 49% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 48% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 47% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 46% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 45% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 40% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 35% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 30% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 25% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 20% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 15% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 10% of the neural network of brain cellPATENTAttorney Docket No. 048536-799001WO neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 9% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 8% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 7% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from about 5% to about 6% of the neural network of brain cell neurons.

[0157] In embodiments, the population of transduced brain cell neurons is from 5% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 6% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 7% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 8% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 9% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 10% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 15% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 20% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 25% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 30% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 35% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 40% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 45% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 46% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 47% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 48% to 50% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 49% to 50% of the neural network of brain cell neurons.PATENTAttorney Docket No. 048536-799001WO

[0158] In embodiments, the population of transduced brain cell neurons is from 5% to 49% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 48% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 47% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 46% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 45% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 40% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 35% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 30% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 25% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 20% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 15% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 10% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 9% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 8% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 7% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is from 5% to 6% of the neural network of brain cell neurons.

[0159] In embodiments, the population of transduced brain cell neurons is 5% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 6% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 7% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 8% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 9% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 10% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 15% of the neural network of brain cell neurons. In embodiments, the population ofPATENTAttorney Docket No. 048536-799001WO transduced brain cell neurons is 20% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 25% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 30% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 35% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 40% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 45% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 46% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 47% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 48% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 49% of the neural network of brain cell neurons. In embodiments, the population of transduced brain cell neurons is 50% of the neural network of brain cell neurons.

[0160] In embodiments, the neurological or psychiatric disease or disorder is an epilepsy, Parkinson’s disease, Alzheimer’s disease, essential tremor, depression, chronic pain, or schizophrenia. In embodiments, the neurological or psychiatric disease or disorder is an epilepsy. In embodiments, the neurological or psychiatric disease or disorder is Parkinson’s disease. In embodiments, the neurological or psychiatric disease or disorder is Alzheimer’s disease. In embodiments, the neurological or psychiatric disease or disorder is essential tremor. In embodiments, the neurological or psychiatric disease or disorder is depression. In embodiments, the neurological or psychiatric disease or disorder is chronic pain. In embodiments, the neurological or psychiatric disease or disorder is schizophrenia.

[0161] In embodiments, the neurological disease or disorder is an epilepsy, Parkinson’s disease, Alzheimer’s disease, essential tremor, depression, chronic pain, or schizophrenia. In embodiments, the neurological disease or disorder is an epilepsy. In embodiments, the neurological disease or disorder is Parkinson’s disease. In embodiments, the neurological disease or disorder is Alzheimer’s disease. In embodiments, the neurological disease or disorder is essential tremor. In embodiments, the neurological disease or disorder is depression. In embodiments, the neurological disease or disorder is chronic pain. In embodiments, the neurological disease or disorder is schizophrenia.PATENTAttorney Docket No. 048536-799001WOVIRAL VECTOR COMPOSITIONS

[0162] The methods provided herein include, inter alia, adeno-associated virus (AAV) viral vectors useful for transfecting a brain cell (e.g., neuron). Thus, in an aspect is provided an adeno- associated virus (AAV) viral vector including a nucleotide sequence encoding a potassiumselective channelrhodopsin protein, wherein the AAV viral vector is an AAV9 viral vector, an AAV -PHP. eB viral vector, an AAV. CAP -Mac viral vector, an AAV9-X1.1 viral vector, or an AAV5 viral vector.

[0163] In embodiments, the AAV viral vector is an AAV9 viral vector. In embodiments, the AAV viral vector is an AAV -PHP. eB viral vector. In embodiments, the AAV viral vector is an AAV.CAP-Mac viral vector. In embodiments, the AAV viral vector is an AAV9-X1.1 viral vector. In embodiments, the AAV viral vector is an AAV5 viral vector.

[0164] In embodiments, the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl), a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2), a light-responsive inward rectifier potassium channel Kir2. 1, or a Wobblia lunata channelrhodopsin (WiChR), or a K+-augmented light-gated ion channel 1 (KALI-1), or a K+-augmented light-gated ion channel 2 (KALL2). In embodiments, the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl). In embodiments, the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2). In embodiments, the potassium-selective channelrhodopsin protein is a light-responsive inward rectifier potassium channel Kir2.1. In embodiments, the potassium-selective channelrhodopsin protein is a Wobblia lunata channelrhodopsin (WiChR). In embodiments, the potassium-selective channelrhodopsin protein is a K+-augmented light-gated ion channel 1 (KALI-1). In embodiments, the potassiumselective channelrhodopsin protein is a K+-augmented light-gated ion channel 2 (KALI-2).CELLULAR COMPOSITIONS

[0165] The methods provided herein are, inter alia, capable of generating a transduced brain cell (e.g., neuron). The transduced brain cells (e.g., neurons) provided herein are, inter alia, useful for modulating a neural network of brain cells. Thus, In an aspect is provided a brain cell including an adeno-associated virus (AAV) viral vector including a nucleotide sequence encoding a potassium-selective channelrhodopsin protein, wherein the AAV viral vector is anPATENTAttorney Docket No. 048536-799001WOAAV9 viral vector, an AAV-PHP.eB viral vector, an AAV. CAP -Mac viral vector, an AAV9- XI. 1 viral vector, or an AAV5 viral vector.

[0166] In embodiments, the AAV viral vector is an AAV9 viral vector. In embodiments, the AAV viral vector is an AAV-PHP.eB viral vector. In embodiments, the AAV viral vector is an AAV.CAP-Mac viral vector. In embodiments, the AAV viral vector is an AAV9-X1. 1 viral vector. In embodiments, the AAV viral vector is an AAV5 viral vector.

[0167] In embodiments, the brain cell is a glutamatergic neuron.

[0168] In embodiments, the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl), a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2), a light-responsive inward rectifier potassium channel Kir2. 1, a Wobblia lunata channelrhodopsin (WiChR), a K+-augmented light-gated ion channel 1 (KALI- 1), or a K+-augmented light-gated ion channel 2 (KALI-2). In embodiments, the potassiumselective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl). In embodiments, the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2). In embodiments, the potassium-selective channelrhodopsin protein is a light-responsive inward rectifier potassium channel Kir2.1. In embodiments, the potassium-selective channelrhodopsin protein is a Wobblia lunata channelrhodopsin (WiChR). In embodiments, the potassium-selective channelrhodopsin protein is a K+-augmented light-gated ion channel 1 (KALI-1). In embodiments, the potassiumselective channelrhodopsin protein is a K+-augmented light-gated ion channel 2 (KALI-2).EXAMPLESExample 1

[0169] Epilepsy is a disease of aberrant neuronal activity that results from an imbalance of excitation and inhibition1'3. Francis Crick famously envisioned the potential of using light to precisely control neuronal populations in the human brain4'6. Since their discovery, light- responsive channelrhodopsins7have become a cornerstone of modem neuroscience studies8,9, and optogenetic interventions have shown tremendous promise in non-human disease models of epilepsy10,11, but whether such tools will translate into human neural circuits, and under what conditions, remains unclear.PATENTAttorney Docket No. 048536-799001WO

[0170] Although direct application of optogenetics in the human brain is not currently possible, it has been translated for use in human retina12 13, and the use of optogenetics in research on human brain tissue has the potential to uncover key parameters for evaluating the potential efficacy of alternative therapeutic strategies. Development of any such therapies involving vector deliver}7of activity modulating genes would benefit from data in a directly relevant model, namely human brain tissue.

[0171] Human brain tissue slices have been used to probe mechanisms of epilepsy for decades14 17, and single neuron patch clamping data exists showing cells transduced with an activating channelrhodopsin in human brain slice can be depolarized with light18. However, optogenetics has never been used to modulate network activity in a human brain slice model. Activation of inhibitor}7GABAergic neurons10,19and silencing of glutamatergic neurons20,21have both shown promise in animal models of epilepsy. Still, neither of these approaches have been demonstrated in human brain slice.

[0172] Despite meaningful insights into human hippocampal physiology, the vectors, genetic models and optogenetic techniques available for circuit manipulation in non-human models have far outpaced those developed for human brain slice models21'26. Species-specific differences present a significant barrier to developing new therapeutic modalities27,28since cross-species applicability can be unpredictable. A model for evaluating AAVs in human brain slice models of human disease may provide a valuable method to screen for translational potential.

[0173] Tools for delivering genetic payloads to specific neuronal cell types in a modelagnostic way are advancing rapidly29'33, and offer therapeutic potential32,34,35. For example, if AAV vectors can be combined with enhancers or promoters that selectively promote gene expression in a molecularly defined subset of cells, this would allow investigation of how optogenetic control of specific cell types could impact network activity, providing a roadmap to developing interventions that modulate epileptiform activity22,36'38.

[0174] Here, we sought to validate a platform that addresses the unmet need to evaluate optogenetic interventions on network activity in human brain tissue. To this end, we evaluated the network effects of AAV -mediated optogenetic activity modulation in a human brain slice model of epileptiform activity. From the view of epilepsy as an imbalance of excitation andPATENTAttorney Docket No. 048536-799001WO inhibition1, we chose to target excitability in excitatory neurons. We found that inhibition of a subset of glutamatergic cells can limit epileptiform activity' in human hippocampal network.Example 2: Establishing human hippocampus slice culture and gene delivery into glutamatergic neurons

[0175] To model human epileptiform activity, we established human organotypic tissue slices from hippocampus tissue (FIG. 1A) donated to research by patients with drug-refractory epilepsy. Tissues were obtained from patients both with and without hippocampal sclerosis as defined by ILAE criteria39(Table 1). Resected tissue was sliced to 300 pm and cultured on cell inserts at the air-liquid interface in serum-free media as previously described16,36. Slices were transduced (see Methods in Example 11) with AAV9 carrying a HcKCRl40transgene driven by a CaMKIIa promoter and a fluorescent tag (eYFP). Fluorescent reporter expression was observed in live imaging with an epi-fluorescent microscope by day 4-6 (transduced on day 0) and confirmed by immunostaining (FIGS. IB and 5A-5I). Reporter expression w as enriched in neurons of the granule cell layer and the dentate gyrus as well as pyramidal cells (FIGS. IB and 5A-5I). The proportion of neurons expressing eYFP in areas of high transduction ranged from 10 - 54% (Table 2). This variability may be the result of focal micropipette injection. Slices that did not show detectable eYFP were excluded from the analysis of transduction rates (see methods). Together, these results indicate that we established organotypic tissue slice cultures of the human hippocampus and reproducibly deliver genetic payloads into hippocampal glutamatergic neurons using a combination of AAV9 and CaMKIIa promoter driven transcript.Example 3: Optogenetic control of human hippocampal network

[0176] HcKCRl encodes a kalium channelrhodopsin, a potassium-selective, light-sensitive ion channel that hyperpolarizes the neuronal membrane, thus reducing the probability’ of spiking when activated by 530 nm light40,41. Unlike a more traditional Channelrhodopsin-2 (ChR2), which encodes a sodium selective ion channel, inhibitory channelrhodopsin has never been applied to human tissue. To confirm that HcKCRl illumination drives membrane hyperpolarization, we conducted intracellular voltage clamp recordings. Consistent with our expectations, HcKCRl activation results in hyperpolarizing currents in human hippocampal neurons (FIGS. 6A-6C). To assess w hether AAV-based transduction of human neurons with optogenetic constructs is specific to HcKCRl. we transduced additional slices with an excitatoryPATENTAttorney Docket No. 048536-799001WO channelrhodopsin (AAV9-CaMKIIa-ChR2-eYFP) packaged in the same viral vector. In this case, we observed similar extent of expression, suggesting that our methods are not limited to HcKCRl (FIGS. 5A-5I). Together, these results demonstrate the feasibility of applying inhibitor}' optogenetics to human neurons.Example 4: Computational modeling of glutamatergic neuron inhibition

[0177] Relative rarity of human hippocampal tissue samples and the limited viability' of slices can impede optimization of any step in an experimental protocol requiring direct testing of human tissue. Computational modeling of cellular circuits is free from these limitations. We conducted in silico modeling of hippocampal epileptiform activity' based on prior electrophysiological data42to computationally predict the degree to which HcKCRl activation might dampen epileptiform activity' across a range of transduction rates in human neurons,. The model contained dentate gyrus granule cells providing excitatory input and basket cells providing inhibitory input. In the model, the light-sensitive channel was modelled after the single cell characterization of HcKCRl40. We varied the proportion of granule cells expressing this channel to model the potential effects of variability' in expression levels (FIGS. 7A-7D). In silico epileptiform activity could be dampened with optogenetic control of less than 50% of the granule cell population (FIGS. 7B-7D). Even at the lower limits of effective expression, the length and intensity of seizure-like events was experimentally controllable in our model (FIGS. 7B-7D). This analysis demonstrated that even incomplete delivery of HcKCRl into glutamatergic hippocampal neurons may be sufficient to modulate network-level epileptiform activity.Example 5: HD-MEA integration with optogenetic activation

[0178] To test the effects of optically activating channelrhodopsins in our slice model, we integrated the HD-MEA recording system with fiber-coupled LED drivers to illuminate organotypic slices plated on the recording array. The associated hardware was small enough to be contained entirely in a tissue culture incubator. While closed in the incubator, the system was controllable from external computers. Custom software was created to allow for real-time analysis of spike event data, allowing for observation of electrophysiological activity and the impact of optogenetic perturbations with minimal delay. The system allowed software control of light intensity, duration, frequency, and off-time.PATENTAttorney Docket No. 048536-799001WOExample 6: Optical HcKCRl-mediated inhibition of network activity

[0179] HcKCRl activation suppressed activity in human hippocampal neurons across all experimental conditions of spontaneous and provoked activity (FIGS. 2A-2D and Table 3). Illumination with 530 nm light40resulted in rapid silencing of hippocampal activity across all tissue slices (FIGS. 2A-2D and 8A-8L). Constant illumination for 10 seconds reduced spontaneous firing rates (FIG. 2A). Changes in spike amplitude were less consistent (FIGS. 8A- 8L). The reduction in neuronal spiking was consistent and evident in raw data.

[0180] Intensity scans across the full range of light intensity produced by the LED driver were carried out to determine the dynamic range of the control of neuronal firing rates as a function of illumination power. The intensity threshold was ty pically between (26.2 mW / mm2- 31.2 mW / mm2), correlating to 40-50% of the possible output from our drivers. For experiments, we utilized one step above the minimum threshold (i.e., 50 - 60% intensity, translating to 31.2 - 35.8 mW / mm2).

[0181] Decreases in neuronal firing rates during HcKCRl optogenetic activation were obtained from n=8 ex vivo hippocampal slices (Table 3 and FIGS. 8A-8L). We additionally carried out recordings in a hippocampal slice transduced with an identical AAV vector driving ChR2 expression (AAV9-CaMKIIa-ChR2-eYFP), which demonstrated an increase in unit activity with 470 nm light illumination (FIGS. 9A-9D). This result confirmed the specificity of light-mediated change in neuronal activity is to the channelrhodopsin-specific physiology instead of an artifact of light illumination.

[0182] AAV-mediated expression of HcKCRl was sufficient to enable optogenetic modulation of network-wide spontaneous activity despite incomplete transduction (Table 3). Spontaneous activity arises from networks perched in a balanced regime that is neither prone to silence nor saturation43. As a result, relatively small increases in inhibitory tone at a cellular level may be insufficient to offset the imbalance necessary' to generate epileptiform activity' at a network level.Example 7: Optogenetic suppression in provoked hyperexcitability

[0183] Next, we tested HcKCRl-mediated optical inhibition in the context of hyperexci table conditions. Under all these conditions, HcKCRl activation reduced firing rates in large portions of units recorded (Table 3).PATENTAttorney Docket No. 048536-799001WO

[0184] We used a magnesium-free (0-mg) media, a long-standing approach used to increase neuronal excitability and induce epileptiform activity' in hippocampal preparations14,44’45. Clear, spontaneous bursts of activity were observed 2 / 4 slices bathed with 0-mg media. These bursts of activity were <ls in duration and several minutes apart, reminiscent of interictal epileptiform spikes46. Overall firing rates of slices were reduced in slices bathed in 0-mg media during periods without spike activity'. However, spike events were not completely absent during optical inhibition two separate slices. The spikes were far enough apart in time to prevent strong conclusions about the effects of optical inhibition on these events (FIG. 10).

[0185] Addition of kainic acid (KA) has been used in various epilepsy models22,47'49Based on other groups reporting effectiveness of combining multiple pro-convulsant pharmacologic interventions to induce epileptiform activity'22,50, we tested whether adding kainate to 0-mg bathed slices would lead to epileptiform activity. The addition of KA resulted in acute elevations in firing rate, and in 2 / 4 slices recorded with KA, rhythmic bursts of activity occurred. Slices exhibiting this rhythmic bursting of unit activity also showed increased coherence across most frequency power bands, most clearly in the theta band. Repeated 10 s periods of illumination clearly showed reduced firing rates during light-ON conditions, but a causal effect of illumination on the coordinated bursts is less clear in the absence of more comprehensive characterization of the typical effects and expected frequency of rhythmic bursting with addition of KA.

[0186] To explore this platform’s ability' to perform closed-loop illumination, we used a GABAAR blockade via the application of bicuculline to elevate firing rates above their baseline levels. We utilized the reproducible increases in firing rate with GABA R blockade to detect and disrupt elevations in network firing We used automated, real-time spike event detection to trigger LED illumination by crossing a firing rate threshold. The closed-loop illumination software is activated following bicuculline application, and firing rates surpassing the threshold triggers 10-seconds of continuous illumination. This dramatically reduced the firing of recorded units. This was replicated in n=3 HcKCRl -expressing slices derived from n=2 patients. These results demonstrate a potential benefit of optogenetic interventions in epilepsy: modulation of brain activity' restricted to precisely to time and anatomy in which pathologic elevations in neuronal firing occur.PATENTAttorney Docket No. 048536-799001WO

[0187] In these three distinct methods of provoking a hyperexcitable state (0-mg, 0-mg+KA and GABAAR blockade). HcKCRl activation in a subset of glutamatergic CaMKIIa-expressing neurons reduced network firing rates. Fraction of neurons transduced in a recording area did not completely explain different fractions of neurons exhibiting reduced firing rates in light-ON conditions (Table 3 and FIG. 10). The magnitude of firing rate reductions in 0-mg and 0- mg+KA conditions was less than that observed in physiologic media and GABAergic blockade (FIG. 11) While several variables may contribute to this, HcKCRl -mediated inhibition of presynaptic CaMKIIa-expressing neurons may be partially offset by post-synaptic glutamatergic receptor activation in 0-mg and 0-mg+KA conditions. Whether HcKCRl activation of CaMKIIa-expressing neurons effectively interrupt provoked coherence seen in 0-mg + KA conditions will require additional experiments to thoroughly characterize the effects of the rhythmic burst activity induced with 0-mg + KA.Example 8: Propagation of epileptiform activity

[0188] Spatially resolved recordings of neural activity enabled by the HD-MEA allow us to analyze signal propagation patterns over the hippocampal areas (e.g., dentate gyrus) recorded during epileptiform spikes and optogenetic activation of HcKCRl. First, we examined increases in single unit firing rates during the 0-mg+KA spike events that make up the rhythmic bursting in the slice with the densest coverage of the GCL. Plotting the units with relative increases of >3 standard deviations above their baseline firing before the spike event indicated a progression from the hilar aspect of the GCL to the outer aspect. Due to the theta phase coherence observed at a network level during these bursts of activity , we examined the spatial changes in phase coherence over time during these rhythmic bursts. Plotting the relative in the theta band during these bursts of activity revealed a clear propagation of theta waves from the hilar aspect to the outer aspect of the GCL. A comparable pattern of theta propagation was also observed in the rhythmic bursting during 0-mg+KA in a separate slice, also with GCL coverage, from a different patient.

[0189] In one slice there was no overlap of these rhythmic bursts during light-ON conditions, while in the other slice these bursts did indeed have some overlap with the light-ON conditions. This variability demonstrated that reductions in overall firing rate, or reductions in CaMKIIa+PATENTAttorney Docket No. 048536-799001WO neuronal firing, may not always be sufficient for disrupting network coherence characteristic of epileptic circuits.

[0190] The traveling wave phenotype we observed has never been visualized at this level of resolution. Such a granular picture of network synchronization may offer a proxy for epileptiform activity that has been unmeasurable without HD-MEA technology. These traveling waves of theta coherence during 0-mg+KA may offer a new target for evaluating the efficacy of experimental interventions.Example 9: Waveform and opto-response clustering

[0191] Reduced unit firing with HcKCRl activation was not uniform across recorded units (FIGS. 2A-2D and 8A-8L), ranging from complete silencing to more subtle or no apparent firing rate changes in other units. Given that AAV9 transduction was not uniform across the tissue, this may partially explain such a spectrum of response, but there was not a clear correlation between transduction rate and fractions of units with reduced firing (FIG. 10). However, cell type is another variable that could affect a unit’s response to HcKCRl activation even in the presence of similar transduction. To investigate whether response parameters of the units are uniform40’31, we investigated whether unit cell-type differences could be inferred from the HD-MEA extracellular recordings.

[0192] We applied a waveform clustering method52,53(see Methods in Example 12) to group units based on features extracted from the extracellular action-potential waveform (FIGS. 3A- 3C). This analysis yielded 12 separate clusters (FIGS. 3A-3C). To validate whether the distribution of putative cell types identified by waveform clustering is consistent with anatomy, we aligned recording array electrodes with histology to approximate the anatomic slice area from which each electrode is records. If waveform clusters correlate with putative cell types, then recordings over an anatomic area with a relatively homogenous cell type should be enriched for waveform clusters underrepresented in anatomic areas without such cells. We analyzed waveform clustering in areas of the dentate gyrus granule cell layer (GCL) versus recordings without granule cell layer representation. We compared the distribution of unit waveform cluster in 3 slices with clear GCL coverage to 6 slices recorded where active recording electrodes did not overlap with GCL. Five waveform clusters were significantly overrepresented in the GCL recordings (FIGS. 3A-3C and Table 5).PATENTAttorney Docket No. 048536-799001WO

[0193] Next, to characterize the heterogeneity of unit responses to optogenetic activation, we used an HDBSCAN-based clustering technique54,55(see Methods in Example 12). We analyzed peri-stimulus histograms of neuronal firing rate spanning time segments before and during optogenetic activation. We found 5 distinct opto-response clusters (FIGs. 4A), 4 of which showed statistically significant reductions in mean firing rate and firing rate distribution with optogenetic activation (FIG. 4B).

[0194] Since our waveform clustering indicated that 5 of the waveform clusters were associated with recordings of the GCL, we treated these waveform clusters as a single category when testing whether opto-response clusters were associated with any putative cell types from waveform clustering. There was indeed a significant association between the GCL waveform cluster and the opto-response cluster 3 (Table 6), in addition to associations between several other waveform and opto-response clusters. While this is far from definitive for stipulating cell type to be the only or most prominent factor in neuronal response to HcKCRl activation, it is consistent with the hypothesis that response to optogenetic activation may differ between cell types. Moreover, it suggests the need for further clarification of how optogenetic interventions in different cell-types may affect those cell-ty pes and networks differently.Example 10

[0195] We describe a novel approach for using human organotypic brain tissue as an experimental research platform for optimizing approaches to modulate neural activity- at the network level in human hippocampus. Building on advances in culture techniques for maintaining viable human organoty pic brain slices ex vivo, we evaluated the effectiveness of AAV -mediated delivery of inhibitory channelrhodopsins expressed in a subset of glutamatergic neurons to exert network level effects in hyperexcitable conditions. By integrating HD-MEA recordings with a custom system for optogenetic control, our experiments demonstrate a robust platform for high resolution analysis of network activity and cell type specific optogenetic manipulation in human hippocampus.

[0196] Optogenetics enables high spatiotemporal control over neural activity. Targeting local circuits has been shown to decrease epileptiform activity in non-human model systems10,11,20,2136, but application of optogenetics to human neurons has been limited. Two studies have used patchclamp recordings from single neurons to show excitatory optogenetic control of human neuronsPATENTAttorney Docket No. 048536-799001WO at a single cell level18,36. Hyperpolarizing, potassium-specific, kalium channelrhodopsins are a new tool in the optogenetic armamentarium40,41. Their inhibiting effects may be relevant to diseases of neuronal hyperactivity, such as epilepsy. To the best of our knowledge, our study represents the first demonstration that optogenetic inhibition of human neurons can be used to modulate network-wide neuronal activity in human hippocampal slices.

[0197] The effects of optogenetic inhibition of glutamatergic CaMKIIa+ neurons on network firing rates were more pronounced in the conditions of physiologic media and GABAAR blockade compared to low magnesium or kainic acid (FIG. 11). Notably, the latter two conditions promote glutamate receptor activation. Optogenetic firing rate reductions in physiologic media and GABAAR blockade may be due to combined changes from HcKCRl- mediated inhibition of excitatory neurons, as well as indirect reductions in activity' of their downstream neurons. In contrast, if low magnesium17and kainic acid57promote direct activation of glutamate receptors, the downstream effects of reduced CaMKIIa+ HcKCRl-expressing neurons may be less pronounced. This may also explain some persistence of epileptiform bursts during slice illumination with kainic acid media.

[0198] In our preparations, adding kainic acid to slices in low magnesium media resulted in rhythmic bursting events, consistent with prior studies22. This bursting activity appeared to be less common during optogenetic activation of HcKCRl activation, with none occurring during light-ON conditions in slice 11G and less common, but still present during 3 of 10 trials in slice 10F. Incomplete silencing is not explained by transduction rate in these slices (FIG. 10). This may' also be related to the increased exogenous glutamatergic activation by kainic acid conditions discussed above. Further characterization of the rhythmic bursting observed is needed to understand effective methods for targeting this electrophysiologic phenotype. Interestingly, the observed results show remarkable similarity to in silico modeling (FIGS. 7A-7D), where coordinated activity’ was reduced in magnitude and frequency (but not completely extinguished) compared to control conditions.

[0199] Our in silico model is based on a recently described characterization of human hippocampal granule cell recordings.42Since one limitation of human brain slice experiments is the scarcity of tissue specimens, this model allowed us to predict that even relatively low rates of transduction and inhibition of glutamatergic neurons could have network-wide effects withoutPATENTAttorney Docket No. 048536-799001WO extensive tissue use. We envision that experimental design studies involving human tissue could involve an iterative framework involving biophysical models to predict cell type and network effects of perturbations, followed by experimentally testing and refinement. Advances in biophysical modeling of brain networks could thus significantly reduce overall needs for human tissue material42,58'61.

[0200] One notable aspect of the observed rhythmic bursting activity was the strong LFP coherence in the theta frequency band. Waves of coherence in the hippocampus, particularly in theta62, have been described in both human63'65and non-human66'68hippocampal preparations. HD-MEA recordings of this density have not been reported in the human hippocampus. This high level of resolution allowed for visualization waves of theta phase-coherence across the GCL of the dentate gyms. This wave of propagation across the GCL is consistent with prior w ork showing that granule cells of the dentate gyrus receive a large amount of excitatory input, that is often phase-locked with theta frequency LFP69. Given evidence suggesting epileptiform discharges in the human brain are waves arising from an epileptogenic focus70, better characterization of such wave propagation may have significance for elucidating epileptic circuits. These data demonstrate the utility of the HD-MEA platform used here to characterize novel network synchronization patterns as a potential target for therapeutic intervention.

[0201] The ability to record dozens to hundreds of neurons simultaneously is one of the advantages of the HD-MEA recording platform described here. Such high-throughput methods may be effective for hypothesis generation about the neuronal ensemble and cell-type responses that can be validated with patch clamp or patch-seq71based experiments. We used clustering techniques to leverage these advantages of the platform described here. We probed whether differences in neuronal cell type might contribute to differences in optogenetic response in our data, since many factors can affect neuronal responses, even to the same ligand72. The waveform clustering method distinguishes putative neuronal cell types based on extracellular spikewaveform features (FIGS. 3A-3C)52-5. To test whether the clustering results were reasonable, we used the anatomic localization of our slice recordings. The granule cell layer is composed mostly of granule cell neurons, which are not typically found elsewhere in the hippocampus73'76. Comparing the distribution of waveform clusters from recordings over the GCL to recordings not over the GCL, 5 clusters were significantly associated with localization over the GCL (FIGS. 3A-3C), suggesting these may represent GCL neurons.PATENTAttorney Docket No. 048536-799001WO

[0202] We used an unsupervised clustering (HDBSCAN54,55’77’78) approach to distinguish patterns of neuronal response to optogenetic HcKCRl activation that may not be captured by firing rate averages. Rather than simply clustering into responders and non-responders to slice illumination, this approach yielded 5 clusters (FIGS. 4A-4D). The GCL waveform clusters were overrepresented in opto-response cluster 3 (Table 6), which had on average a high baseline firing rate and modest reduction during slice illumination (FIGS. 4A-4B). This suggested that cell-type — in this case granule cell identity — may play a role in optogenetic response to HcKCRl activation, although activity-state at the time of slice-illumination cannot be ruled out based on these analyses40. Single nucleus sequencing data indicates that granule cells typically express CamKIIa at high levels compared to other hippocampal neurons79, such that promoter gene expression does not completely explain this modest reduction in firing rate seen during HcKCRl activation in putative GCL neurons.

[0203] The present work utilizes hippocampi resected en bloc from patients with drug-resistant temporal lobe epilepsy. Clinically, these hippocampi have been determined to be likely or definitively involved in generating seizure activity'. By this definition, the hippocampi recorded may have physiologic differences from what might be considered normal physiology or connectivity in non-epileptic human tissue. While this caveat should be noted when making biological conclusions about hippocampal function, it should also be noted that the intended target for new epilepsy therapies is epileptogenic hippocampi. This makes the preparation presented here an optimal model for pathologic insights, but perhaps limited in regard to normal hippocampal physiology.

[0204] Emerging strategies to develop AAVs with improved fidelity and robustness30harness methods for screening synthetic libraries of capsid proteins, as well as enhancer elements29,30’80-82AAV gene delivery studies have thus far focused on visualizing transduced neurons and describing their relevance with regard to normal CNS physiology32’80 83. Such investigations would benefit from characterizing how these tools can be utilized in pathologic circuits. Ex vivo cultures of human brain slices may both accelerate the development of such tools and serve as a platform for investigating the circuit-level effects of AAV-mediated, gain-of-function gene delivery. The techniques described herein allow' for a layer of functional screening of AAVs for gene delivery by combining validation of human brain tissue expression with the potential to measure electrophysiologic activity and circuit-level effects.PATENTAttorney Docket No. 048536-799001WO

[0205] The field of surgical treatment of epilepsy is evolving at a rapid pace. While surgical resection is the gold standard for drug-refractory focal epilepsy84, responsive neurostimulation is another increasingly common treatment for select patients85. Responsive neurostimulation involves running electrical current though neural tissue using stereotactically implanted electrodes. Neurostimulation techniques do confer clinical reductions in seizure frequency86, but the mechanism by which they modulate seizures is a topic of ongoing research87. Therapeutic translation of optogenetics for epilepsy is still theoretical88. The chronic effects and toxicityprofiles of optogenetics in human hippocampal neurons require further study89. However, permanent implantation of electrodes and power-sources into patients with refractory epilepsy and other functional neurologic diseases is now routinely practiced90'97. Since such treatments are well-received by patients and clinicians, our working hypothesis is that the invasiveness of implanting a light guide and battery will not be the rate-limiting factor in developing therapeutic optogenetics. The lack of models for testing therapeutics in human tissue, on the other hand, may well impede progress on the many experimental fronts that would be necessary for moving in a translational direction88.

[0206] In conclusion, primary human tissue can serve as an experimental platform for optimizing and testing strategies to modulate network activity with the long-term goal of derisking potential therapeutic interventions. Emerging technologies could enable rapid and scalable genetic access into cell types that could be tested in such ex vivo preparations. Here we describe a scenario whereby AAV-mediated delivery7of inhibitory7channelrhodopsin into a subset of glutamatergic neurons can be used to demonstrate network-wide activity modulation in human hippocampus. These techniques were previously relegated to animal modeling experiments. This study highlights the potential for human brain tissue method to serve as a viable model for illuminating human brain neurophysiology7.Example 11: Methods

[0207] Tissue preparation and culture for high-density MEA recordings

[0208] Human hippocampal specimens were obtained from patients undergoing temporal lobectomy with hippocampectomy for refractory epilepsy with University of California-San Francisco IRB approval. Tissue was transported in artificial cerebrospinal fluid (aCSF) bubbled with carbogen, then sliced into 300 pM sections and recovered sequentially in HEPES bufferedPATENTAttorney Docket No. 048536-799001WO aCSF and culture media. The slices are plated on cell-culture inserts to allow for long-term culture at the air-liquid interface. Viral transduction was performed on the day of plating with glass micro-pipets. Slices were subsequently plated on HD-MEAs with minimal culture media. For optogenetic recordings, a custom, open-source hardware platform was used with off-the- shelf optoelectronic equipment and 3D-printed components.

[0209] Human tissue samples were collected from select neurosurgical cases at UCSF with signed patient consent and approval from the UCSF Institutional Review Board.

[0210] Tissue transport and preparation were adapted from Ting et al.36Briefly, tissue was collected in the operating room and put in sterile artificial cerebrospinal fluid (aCSF) of the composition (in mM): 92 NMDG, 2.5 KCL 1.25 NaH2PO4, 30 NaHCO3, 20 4-(2- hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaC12 4H2O and 10 MgSO4-7H2O. Before collection, the pH of the aCSF was titrated to 7.3-7.4 with hydrochloric acid, and the osmolality was 300-305 mOsmoles / Kg. The solution was pre-chilled to 2-4°C and thoroughly bubbled with carbogen (95% 02 / 5% CO2) gas before collection.

[0211] Tissue was cut into 300 pM slices submerged in carbogen-bubbled aCSF using a vibratome. Slices w ere briefly recovered in aCSF warmed to 33 °C before plating on cell culture inserts at the air-liquid interface and incubating at 37 °C in 5% CO2 incubators.

[0212] Tissue was cultured and recorded in a media formulation adapted from prior studies16,36, referred to herein as physiologic media. 15% BME, 15% DMEM / F12 + Glutamax, 0.65% % PBS, 3% H2O, 0.91 mM CaC12, 1.96 mM KCl, 0.81 mM MgSO4, 81.90 mM NaCl, 0.84 mM NaH2PO4, 14.0 mM NaHC03, 0.5 pM ZnSO4, 0.01 pM CuSO4, 0.63 mM d-Glucose, 0.14 mM Sodium Pyruvate, 0.58 mM Ascorbic acid, 1.0 mM Oxalo-acetic acid, 4.0 mM Sodium lactate. 0.57 mM Citric acid. 4 mM Sodium lactate, 0.27% BSA, 0.001 pM 17-b-Estradiol. 2.28 pM a-Tocopherol, 2.10 pM a-Tocopherol-acetate, 0.06 pM Corticosterone, 5 mM Creatine, 1 mM Sodium b-hydroxybutyrate, 5 mM Mannitol, 0.2 mM Phosphate creatine, 0.5% N-2 supplement, 1250 U / mL Heparin, 0.01 mM ATP, 0.41 pM Insulin, 0.0003 pM Tri-iodo-I- thyronine sodium, 0.08 pM Superoxide dismutase (SOD). 0.05 mM Glycerol (lOx), 0.5 mM Glutamax, 1 pM MnCL, 1 pM g- Amino-butyric acid, 3 pM Glutathione, 8 pM Taurine.PATENTAttorney Docket No. 048536-799001WO

[0213] Plating slices on HD-MEA

[0214] To plate tissue plated on MaxWell high-density MEAs, MEAs were incubated with Matrigel for 1 hour, and subsequently filled with culture / recording media described above. Slices were floated into the MEA wells and the media was then aspirated slowly such that the slice descended onto the recording surface. The slice was kept in minimal media for the 2 hours during which recordings were carried out. For GABAAR blockade, bicuculline dissolved in recording media to a concentration of 2 pM was dripped directly on the tissue slice. For Low magnesium media, the physiologic media was made without any magnesium (MgSCL) added. For Kainic acid experiments, 100 nM kainic acid was dripped was directly on to the slice.

[0215] Examples are shown of large areas of dense activity recorded in FIG. 2 and overlaid on FIGS. 4C-4D. Slices that did not show electrophysiologic evidence of unit activity after plating were excluded from electrophysiologic analyses.

[0216] Inclusion and Exclusion Criteria

[0217] All slices included in electrophysiologic analyses were from adult patients with refractory epilepsy and were cultured for 4-8 days prior to recordings. Acute recordings were excluded from waveform clustering analyses. Any slice from which unit activity was not recorded after plating on HD-MEAs was not included in electrophysiologic analyses.

[0218] For transparency, no slices showing HD-MEA data that were transduced with HcKCRl were excluded from analyses. These slices are shown in FIGS. 8A-8L. Waveform clustering analyses used data from all slices in Table 2. Opto-response clustering analyses included all slices that were transduced with HcKCRl and yielded unit activity, listed in Table 3, and shown in FIGS. 8A-8L

[0219] Immunohistochemistry

[0220] Immunohistochemistry for NeuN and eYFP described herein was performed with the following antibodies:

[0221] NeuN: guinea pig anti-NeuN, Millipore, ABN90, dilution 1 : 1000. lot#4077530

[0222] eYFP: Chicken anti-GFP antibody. Aves, GFP-1020, dilution 1: 1000. lot#GFP3717982PATENT Attorney Docket No. 048536-799001WO

[0223] AAVs

[0224] AAV9-CaMKIIa-HcKCRl-eYFP: titer 5.00E+13 GC / mLCompany: PackGene. Lot# 12109TPurity analyzed according to PackGene company protocols by SDS-PAGE finding no other significant bands.Endotoxin assay analyzed by Quantitative LAL assay with <10EU / ml

[0225] AAV9-CaMKIIa-ChR2-eYFP: titer 2.40E+13 GC / mLCompany: Addgene. Lot# vl 13177Purified by lodixanol gradient ultracentrifugation. Purity of AAV preparation were assayed according to Addgene company protocols by comparing the relative stoichiometric ratios of the viral capsid proteins VP1, VP2 and VP3. Samples of viral preparations were subjected to polyacrylamide gel electrophoresis (PAGE) followed by silver staining or SYPRO Red staining and the molecular weight and relative intensity of the viral capsid proteins were analyzed.Endotoxin assay analyzed by Quantitative LAL assay with <5 EU / ml

[0226] Design of optogenetic system

[0227] The purpose of the designed hardware platform is to trigger an optogenetic stimulation pulse and observe the response of neurons on a high-density electrode array. The optogenetic system was designed to be easy to replicate and does not require custom PCBs or soldering. The bill of materials with pricing is available in Table 4. Lid and insert from are described in detail in Example 12.

[0228] To run an experiment, the user must specify a calibration CSV file, which contains data on an LED's power output (see Section Optogenetic power density measurement and calculation). The user also connects to the Arduino over a USB serial port. Afterward, the program creates a stimulation log CSV file into which the stimulation events with their parameters are recorded. Now, the user can construct their own stimulation sequences and use several helper functions with configurable stimulation pattern templates.PATENTAttorney Docket No. 048536-799001WO

[0229] Activity detection algorithm and closed-loop optical intervention

[0230] We implemented an algorithm that monitors spike activity' in real-time, using data from the HD-MEA (FIGS. 2A-2D). This algorithm is meant to demonstrate a therapeutic use case scenario. When a pre-defined spike-threshold is detected, the algorithm triggers illumination of the slice. Spike activity7was measured by counting the total number of 5-rms spikes that occurred in 10 seconds across all the electrodes on the HD-MEA.

[0231] Experimentally, this threshold was to be 2 to 4 times above the average baseline 5-rms spike activity for 1 minute prior to application of bicuculline. After the drug was applied, activity' quickly surpassed this threshold. After the threshold is surpassed for a 10-second window, the algorithm triggers 10 seconds of illumination. Afterward, the algorithm goes back to monitoring activity7. This algorithm is intentionally simple in nature, to provide a proof of concept that user- defined signals can be detected in real-time using HD-MEA interface with this this ex vivo model, in a closed-loop process.

[0232] Optogenetic power density measurement and calculation

[0233] Power output for the optical fiber was measured with Thorlabs meter PM100D with sensor S130C. The power density (mW / mm ) values of the light presented here were calculated by dividing the measured power by the cross-sectional area of the fiber, using this formula:For multimode fiber with a 0200 pm core: Beam radius = r =200^m= 100 m = 0.100 mm Average power = PMgSurface power densitywhere P„, is measured power and r is ' the fiber core diameter. For additional information of how this data was collected, see optogenetic power density measurement and calculation in Example 12.

[0234] Data Acquisition

[0235] Neuronal activity7was sampled simultaneously7at 20 kHz from multiple electrodes on the HD-MEA for both action potential and local field potential bandwidth. During thePATENTAttorney Docket No. 048536-799001WO experiment, an activity7scan assay using MaxLab Interface across all 26,400 electrodes was performed first, then a maximum of 1,024 available channels were manually selected to record from the most active areas spread across the MEA. Closed-loop seizure detection and optical intervention started after the assay from a custom Python script. After the experiment, raw activity data and optical stimulation timestamps were saved to an hdf5 file on local memory. The optical light protocol was written into a separate CSV logging file pairing with the raw data (see section Design of Optogenetic System).

[0236] Spike Sorting

[0237] Kilosort298was used for sorting the raw data into single unit activity. Since the high- density MEA can record one neuron from tens of channels, it is common that the spikes from many neurons overlap in time for a single channel. The template matching and clustering algorithm in Kilosort2 can distinguish spikes between different neurons based on their waveform and assign them to individual clusters. Raw data were bandpass filtered with 300 - 6000 Hz and sorted in Kilosort2 with a voltage detection threshold of 6 RMS over the baseline. Spike sorting was performed on the Pacific Research Platform computing cluster with an NVIDIA GeForce GTX 1080 Ti GPU. For spike sorting, both manual and auto-curation techniques were applied. Kilosort2’s result was manually curated using Phy GUI99by experienced electrophysiology researchers. The quality metrics100for saving high-quality units are based on the shape of the spike waveform, firing rate, and interspike interval distribution. Details and quality metrics are available in Example 12 under Spike Sorting Curation.

[0238] Activity heatmaps

[0239] Activity heatmaps in FIG. ID show the spatial distribution of significant voltage events. The MaxWell software computes a moving root-mean-square (rms) value for each electrode, and we used this to determine events that crossed a threshold set at 5x the rms value of each respective electrode. A 2D spatial grid of the counts per second was constructed, then a 2D Gaussian blur with radius was used to enable better visual continuity. Each position in the grid was divided by 2n2 so the values displayed would correspond to events per second, making it so the gaussian blur doesn't significantly alter the values of each data point. These values were then plotted as the activity heatmaps, where warmer colors represent areas of higher event frequency, and darker colors denote regions with less activity.PATENTAttorney Docket No. 048536-799001WO

[0240] Statistics

[0241] Firing rate and amplitude

[0242] To compare Firing rate and amplitude (FIGS. 8A-8L and 9A-9D) across multiple trials for the groups ‘"pre'’, “light-ON,” and “‘OFF” we used the Fisher's Combined Probability Test101,102with the fisher_combined_pvalue() function in R. This was applied with the inputs of each individual trial’s p-value from a paired t-test using the paired_t_test () function in R, with Bonferroni correction for multiple comparisons p. adjust with method= "Bonferroni”. The N for each individual trial comparison was calculated from the number of units recorded in each trial. This was done in RStudio 2022.07. 1 Build 554.

[0243] Mean relative firing rate reduction

[0244] In FIG. 11 we compared differences in magnitude of firing rate reduction from optogenetic activation between conditions exogenously increasing glutamatergic tone (low magnesium with and without kainic acid) vs those without exogenous glutamatergic tone (physiologic media with and without bicuculline). To do this, we calculated mean firing rates of all units during the 10 s prior to light-ON and 10 s during hght-ON. Slice were grouped in the following fashion: for the physiologic media with and without bicuculline (Physio + Bic) 1 slice with physiologic media only and 3 slices after addition of bicuculline. For the low magnesium (0-mg), 4 slices recorded with low magnesium media. For the low magnesium plus kainic acid (0-mg+KA), the same 4 slices recorded for the 0-mg condition, using the recordings after administration of kainic acid. For each slice mean firing rate was calculated for the 10 s prior to light-ON and 10 s during hght-ON, and the difference between the firing rates for each slice w as expressed as a % change from 10 s prior to light-ON to 10 s during hght-ON (e.g., if the mean firing rate prior to hght-ON was 10 Hz and the mean firing rate during light-ON was 7.5 Hz, this would be as 25% change in firing rate). Kruskal-Wallis test was applied to the 3 groups and Welch’s t-tests used for individual comparisons.

[0245] Waveform cluster association with GCL

[0246] To assess the association between identified w aveform clusters (FIGS. 3A-3C) and their anatomical localization within the granule cell layer (GCL) the dentate gyrus, we used R (Version 2022.07.1, Build 554). Given the binary nature of our dependent variable (presence vs.PATENTAttorney Docket No. 048536-799001WO absence of a unit within GCL), we used logistic regression analysis. This choice was predicated on the need to understand the extent to which belonging to a specific cluster could predict the likelihood of a neuronal unit being located in the GCL, with clusters serving as categorical predictor variables in the model.

[0247] Prior to analysis, neuronal unit data were rigorously cleaned and formatted. Each unit was classified into one of 12 clusters based on waveform features, with this classification serving as independent variables in our subsequent logistic regression model. The dependent variable was binary, denoting the anatomical attribution of each unit to either the GCL (1) or other hippocampal regions (0). Logistic regression was then performed using the glm function in R, specifying a binomial family to accommodate our binary dependent variable. The model included one intercept and individual coefficients for each cluster, allowing us to estimate the odds ratio of GCL localization for units in each cluster relative to the baseline condition (absence from GCL).

[0248] To address potential Type I errors due to multiple comparisons — given the 12 clusters analyzed — we implemented a p-value adjustment using the Benjamini-Hochberg method. Adjusted Confidence intervals and p-values were calculated and reported in Table 5.

[0249] Waveform cluster association with opto response clusters

[0250] To examine the association between neuronal unit classifications based on waveform cluster identity (FIGS. 3A-3C) and opto-response cluster identity (FIGS. 4A-4D), we employed a two-step statistical approach. Initially, a Chi-square test of independence was applied to a contingency table representing the distribution of units across the predetermined cluster identities (RStudio function: chisq.test). This test assesses the null hypothesis that waveform and opto- response cluster identities are independent of each other across the entire dataset. Given the categorical nature of our data, where each unit is classified into discrete clusters based on spike waveform morphology and optogenetic response, the Chi-square test provides a suitable framework for detecting overall patterns of association between these two classification methods. However, the Chi-square test alone does not indicate which specific cluster associations contribute to the overall significance. Thus, we conducted a post-hoc analysis using standardized residuals, which in this context function analogously to z-scores. For each cell in the contingency table, the standardized residual calculates the number of standard deviations the observedPATENT Attorney Docket No. 048536-799001WO frequency is from the expected frequency, under the assumption of independence. These residuals thus provide a measure of the magnitude and direction of deviation for each cluster association, enabling us to identify specific pairs of waveforms and opto-response clusters that are associated more or less frequently than would be expected by chance alone. Standardized residuals are computed as follows:O—E Standardized Residual = (v— -J?E=-) ’Where O represents the observed frequency in each cell, and E denotes the expected frequency, calculated based on the marginal totals under the assumption of independence. We applied a Bonferroni correction to control for the family-wise error rate. This correction adjusts the significance threshold by dividing the conventional alpha level (0.05) by the number of comparisons made. A comparison is considered statistically significant if its absolute standardized residual exceeds the critical value derived from the Bonferroni-adjusted alpha of 0.05.

[0251] Kullback-Liebler divergence and phase coherence (KLD)

[0252] To represent phase coherence events in time of the Local Field we first constructed an analytic (complex-valued) time series using the Hilbert transform extension. We then extracted the instantaneous phase distributions at each instant and measured the divergence between these and a uniform null hypothesis using the Kullback-Liebler divergence (KLD). Let / be a binning of the interval [0, 2TT] . Furthermore, let Px(t) be the probability distribution of the phase angle values at time t, and let Qxbe the uniform distribution with respect to / . Our Entropy-based metric of coherence across time is defined as

[0253] By inspection of the polar histograms, it was determined that in many cases the nature of the distribution deviated considerably from a von Mises distribution, making the p-value a misleading measure of coherence. In the field of mathematical statistics, the KLD serves as a statistical distance, quantifying the discrepancy between a given probability distribution P and aPATENTAttorney Docket No. 048536-799001WO reference probability distribution Q. making it a better indicator of phase coherence when the reference Q is chosen to be uniform.

[0254] Simulation Prototype

[0255] The dynamical simulation of a hippocampal network which was used as a proof of concept is a simplified version of a previously published in silico model of the human dentate gyrus100which has been used to study disease progression in epilepsy42. Simulated kalium channelrhodopsin channels40were added to a variable fraction of cells within this model in order to probe the efficacy of optogenetic feedback in disturbing seizure-like events. For further details, see "Simulation” directory in github.com / braingeneers / AndrewsGengVoitiukEtAl_SourceCode

[0256] Theta Waveform Videos

[0257] The theta waveform videos were recorded to depict how theta phase signals in local field potential propagate through the tissue over time. The video was comprised of two animated figures. The left figure, explained further below, displayed theta signals across all recording electrodes. Theta activity was aligned in time with the neural firing rate, displayed in the right figure. This was done to convey how theta activity was modulated during neural bursts. The moving vertical line in the firing rate figure illustrated the exact timing of the theta waves to the left. The videos moved at a pace that is ten times slower than real time. This was done to better understand the nuances in theta activity during bursts.

[0258] The theta wave activity for each electrode was calculated using a standard neuroscience protocol. The raw data for an electrode was normalized and then had a low band pass filter applied to it. Then a second bandpass filter was applied to select activity within the theta frequencies, between 4-8 hz.

[0259] To express the evolution in theta activity across the roughly thousand recording electrodes of the HD MEA, we used colors and circles of vary ing sizes. The center of each circle is the position of a single recording electrode. The color of the circle displays whether the theta activity for that particular electrode is in a positive or negative phase (i.e.. whether the wave is positive or negative at that point in time). The size of the circle displays the relative amplitude ofPATENTAttorney Docket No. 048536-799001WO the theta wave (i. e.. the magnitude of the wave’s value at that time). We observed at times of regular neural activity the theta waves were roughly randomly distributed, but during epileptiform bursts, there were strongly coherent propagations of synchronized theta waves.

[0260] REFERENCES

[0261] 1 Staley, K. Molecular mechanisms of epilepsy. Nature neuroscience 18, 367 (2015).

[0262] 2 Staley, K. J. & Dudek, F. E. Interictal spikes and epileptogenesis. Epilepsy Currents 6, 199-202 (2006).

[0263] 3 Paz, J. T. & Huguenard, J. R. Microcircuits and their interactions in epilepsy: is the focus out of focus? Nature neuroscience 18, 351-359 (2015).

[0264] 4 Crick. F. H. Thinking about the brain. Scientific American 241, 219-233 (1979).

[0265] 5 Boyden, E. S. Optogenetics and the future of neuroscience. Nature neuroscience18, 1200-1201 (2015).

[0266] 6 Lin, S.-C., Deisseroth, K. & Henderson, J. M. Optogenetics: background and concepts for neurosurgery. Neurosurgery 69, 1-3 (2011).

[0267] 7 Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecondtimescale, genetically targeted optical control of neural activity. Nature neuroscience 8, 1263- 1268 (2005).

[0268] 8 Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154-165 (2010).

[0269] 9 Zhang, F. et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nature protocols 5, 439-456 (2010).

[0270] 10 Krook-Magnuson, E.. Armstrong, C., Oijala, M. & Soltesz, I. On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy. Nature communications 4, 1-8 (2013).

[0271] 11 Krook-Magnuson, E. et al. In vivo evaluation of the dentate gate theory in epilepsy. The Journal of physiology 593, 2379-2388 (2015).PATENTAttorney Docket No. 048536-799001WO

[0272] 12 Fradot, M. et al. Gene therapy in ophthalmology: validation on cultured retinal cells and explants from postmortem human eyes. Human gene therapy 22, 587-593 (2011).

[0273] 13 Sahel, J.-A. et al. Partial recovery' of visual function in a blind patient after optogenetic therapy. Nature medicine 27, 1223-1229 (2021).

[0274] 14 Avoli. M. et al. Epileptiform activity induced by low extracellular magnesium in the human cortex maintained in vitro. Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology’ Society: 30, 589-596 (1991).

[0275] 15 Masukawa, L. M., Higashima, M., Kim, J. H. & Spencer, D. D. Epileptiform discharges evoked in hippocampal brain slices from epileptic patients. Brain research 493, 168- 174 (1989).

[0276] 16 Cohen, I., Navarro, V., Clemenceau, S., Baulac, M. & Miles, R. On the origin of interictal activity in human temporal lobe epilepsy in vitro. Science 298, 1418-1421 (2002).

[0277] 17 Kohling, R. & Avoli, M. Methodological approaches to exploring epileptic disorders in the human brain in vitro. Journal of neuroscience methods 155, 1-19 (2006).

[0278] 18 Andersson, M. et al. Optogenetic control of human neurons in organotypic brain cultures. Scientific Reports 6, 1-5 (2016).

[0279] 19 Ledri. M.. Andersson, M., Wickham. J. & Kokaia. M. Optogenetics for controlling seizure circuits for translational approaches. Neurobiology of Disease, 106234 (2023).

[0280] 20 Wykes, R. C. et al. Optogenetic and potassium channel gene therapy in a rodent model of focal neocortical epilepsy. Science translational medicine 4. 161ral52-161ral52 (2012).

[0281] 21 Wykes, R. C., Kullmann, D. M., Pavlov, I. & Magloire, V. Optogenetic approaches to treat epilepsy. Journal of neuroscience methods 260, 215-220 (2016).

[0282] 22 Jones, R. S., da Silva, A. B., Whittaker, R. G., Woodhall, G. L. & Cunningham, M. O. Human brain slices for epilepsy research: Pitfalls, solutions and future challenges. Journal of neuroscience methods 260, 221-232 (2016).PATENTAttorney Docket No. 048536-799001WO

[0283] 23 Grone, B. P. & Baraban, S. C. Animal models in epilepsy research: legacies and new directions. Nature neuroscience 18, 339-343 (2015).

[0284] 24 Bosque, J. R. et al. Molecular tools for the characterization of seizure susceptibility in genetic rodent models of epilepsy. Epilepsy & Behavior 121. 106594 (2021).

[0285] 25 Levesque. M., Avoli. M. & Bernard. C. Animal models of temporal lobe epilepsy following systemic chemoconvulsant administration. Journal of neuroscience methods 260, 45- 52 (2016).

[0286] 26 Armstrong, C., Krook-Magnuson, E., Oijala, M. & Soltesz, I. Closed-loop optogenetic intervention in mice. Nature protocols 8, 1475-1493 (2013).

[0287] 27 Matsuzaki, Y. et al. Intravenous administration of the adeno-associated virus- PHP. B capsid fails to upregulate transduction efficiency in the marmoset brain. Neuroscience letters 665, 182-188 (2018).

[0288] 28 Hordeaux, J. et al. The neurotropic properties of AAV-PHP. B are limited to C57BL / 6J mice. Molecular Therapy 26, 664-668 (2018).

[0289] 29 Zhu, D. et al. Optimal trade-off control in machine learning-based library design, with application to adeno-associated virus (AAV) for gene therapy. bioRxiv. 2021.2011. 2002.467003 (2021).

[0290] 30 Zhu, D., Schieferecke, A. J., Lopez, P. A. & Schaffer, D. V. Adeno-Associated Virus Vector for Central Nervous System Gene Therapy. Trends in Molecular Medicine (2021).

[0291] 31 Chen, X. et al. Engineered AAVs for non-invasive gene delivery' to rodent and non-human primate nervous systems. Neuron 110. 2242-2257. e2246 (2022).

[0292] 32 Goertsen, D. et al. AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery' in mouse and marmoset. Nature neuroscience 25, 106-115 (2022).

[0293] 33 Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery' to the central and peripheral nervous systems. Nature neuroscience 20, 1172-1179 (2017).PATENTAttorney Docket No. 048536-799001WO

[0294] 34 Deverman, B. E., Ravina, B. M., Bankiewicz, K. S., Paul, S. M. & Sah, D. W. Gene therapy for neurological disorders: progress and prospects. Nature Reviews Drug Discovery 17, 641-659 (2018).

[0295] 35 Andrews, M. R. Gene therapy in the CNS — one size does not fit all. Gene Therapy 28, 393-395 (2021).

[0296] 36 Ting, J. T. et al. A robust ex vivo experimental platform for molecular-genetic dissection of adult human neocortical cell types and circuits. Scientific reports 8, 8407 (2018).

[0297] 37 Le Duigou, C. et al. Imaging pathological activities of human brain tissue in organotypic culture. Journal of neuroscience methods 298, 33-44 (2018).

[0298] 38 Schwarz, N. et al. Long-term adult human brain slice cultures as a model system to study human CNS circuitry and disease. Elife 8, e48417 (2019).

[0299] 39 Bliimcke, I. et al. International consensus classification of hippocampal sclerosis in temporal lobe epilepsy: a Task Force report from the ILAE Commission on Diagnostic Methods. Epilepsia 54, 1315-1329 (2013).

[0300] 40 Govorunova, E. G. et al. Kalium channelrhodopsins are natural light-gated potassium channels that mediate optogenetic inhibition. Nature Neuroscience 25. 967-974 (2022).

[0301] 41 Vierock, J. et al. WiChR, a highly potassium-selective channelrhodopsin for low- light one-and two-photon inhibition of excitable cells. Science Advances 8, eadd7729 (2022).

[0302] 42 Buchin, A. et al. Multi-modal characterization and simulation of human epileptic circuitry. Cell reports 41, 111873 (2022).

[0303] 43 Ma, Z., Turrigiano, G. G., Wessel, R. & Hengen. K. B. Cortical circuit dynamics are homeostatically tuned to criticality in vivo. Neuron 104, 655-664. e654 (2019).

[0304] 44 Mele, M. et al. Transient incubation of cultured hippocampal neurons in the absence of magnesium induces rhythmic and synchronized epileptiform-like activity. Scientific reports 11, 11374 (2021).PATENTAttorney Docket No. 048536-799001WO

[0305] 45 Mody, I., Lambert, J. & Heinemann, U. Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices. Journal of neurophysiology 57 , 869-888 (1987).

[0306] 46 Keller, C. J. et al. Heterogeneous neuronal firing patterns during interictal epileptiform discharges in the human cortex. Brain 133, 1668-1681 (2010).

[0307] 47 Levesque, M. & Avoli, M. The kainic acid model of temporal lobe epilepsy. Neuroscience & Biobehavioral Reviews 37, 2887-2899 (2013).

[0308] 48 Vezzani, A. et al. Interleukin- ip immunoreactivity and microglia are enhanced in the rat hippocampus by focal kainate application: functional evidence for enhancement of electrographic seizures. Journal of Neuroscience 19, 5054-5065 (1999).

[0309] 49 Jimenez-Mateos, E. M. et al. Silencing microRNA-134 produces neuroprotective and prolonged seizure-suppressive effects. Nature medicine 18, 1087-1094 (2012).

[0310] 50 Huberfeld, G. et al. Glutamatergic pre-ictal discharges emerge at the transition to seizure in human epilepsy. Nature neuroscience 14, 627-634 (2011).

[0311] 51 Herman, A. M., Huang. L., Murphey, D. K., Garcia, I. & Arenkiel, B. R. Cell type-specific and time-dependent light exposure contribute to silencing in neurons expressing Channelrhodopsin-2. elife 3, e01481 (2014).

[0312] 52 Lee, E. K. et al. Non-linear dimensionality reduction on extracellular waveforms reveals cell type diversity in premotor cortex. Elife 10, e67490 (2021).

[0313] 53 Lee, K., Carr, N., Perliss, A. & Chandrasekaran, C. WaveMAP for identity ing putative cell types from in vivo electrophysiology. STAR protocols 4, 102320 (2023).

[0314] 54 Masuda, F. K, Aery’ Jones, E. A., Sun, Y. & Giocomo, L. M. Ketamine evoked disruption of entorhinal and hippocampal spatial maps. Nature Communications 14, 6285 (2023).

[0315] 55 Mclnnes, L. & Healy, J. in 2017 IEEE international conference on data mining workshops (ICDMW). 33-42 (IEEE).PATENTAttorney Docket No. 048536-799001WO

[0316] 56 Tonnesen, J., Sorensen, A. T., Deisseroth, K., Lundberg, C. & Kokaia, M.Optogenetic control of epileptiform activity. Proceedings of the National Academy of Sciences 106, 12162-12167 (2009).

[0317] 57 Castillo, P. E., Malenka, R. C. & Nicoll, R. A. Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons. Nature 388, 182-186 (1997).

[0318] 58 Savtchenko, L. P. et al. Disentangling astroglial physiology with a realistic cell model in silico. Nature communications 9, 3554 (2018).

[0319] 59 Ecker, A. et al. Data-driven integration of hippocampal CAI synaptic physiology in silico. Hippocampus 30, 1129-1145 (2020).

[0320] 60 Chiang, C. C., Shivacharan, R. S., Wei, X.. Gonzalez-Reyes, L. E. & Durand, D. M. Slow periodic activity in the longitudinal hippocampal slice can self-propagate non- synaptically by a mechanism consistent with ephaptic coupling. The Journal of physiology 597, 249-269 (2019).

[0321] 61 Schmalz, J., Quinarez, R. V., Kothare, M. V. & Kumar, G. Controlling neocortical epileptic seizures using forced temporal spike-time stimulation: an in silico computational study. Frontiers in Computational Neuroscience 17, 1084080 (2023).

[0322] 62 Buzsaki, G. Theta oscillations in the hippocampus. Neuron 33, 325-340 (2002).

[0323] 63 Kleen, J. K. et al. Bidirectional propagation of low frequency oscillations over the human hippocampal surface. Nature communications 12, 2764 (2021).

[0324] 64 Zhang, H. & Jacobs, J. Traveling theta waves in the human hippocampus. Journal of Neuroscience 35, 12477-12487 (2015).

[0325] 65 Goyal, A. et al. Functionally distinct high and low theta oscillations in the human hippocampus. Nature communications 11, 2469 (2020).

[0326] 66 Lubenov, E. V. & Siapas, A. G. Hippocampal theta oscillations are travelling waves. Nature 459, 534 (2009).

[0327] 67 Goutagny, R., Jackson, J. & Williams, S. Self-generated theta oscillations in the hippocampus. Nature neuroscience 12, 1491-1493 (2009).PATENTAttorney Docket No. 048536-799001WO

[0328] 68 Etter, G., van der Veldt, S., Choi, J. & Williams, S. Optogenetic frequency scrambling of hippocampal theta oscillations dissociates working memory retrieval from hippocampal spatiotemporal codes. Nature Communications 14, 410 (2023).

[0329] 69 Pemia-Andrade, A. J. & Jonas. P. Theta-gamma-modulated synaptic currents in hippocampal granule cells in vivo define a mechanism for network oscillations. Neuron 81, MO- 152 (2014).

[0330] 70 Diamond, J. M. et al. Interictal discharges in the human brain are travelling waves arising from an epileptogenic source. Brain 146, 1903-1915 (2023).

[0331] 71 Cadwell, C. R. et al. Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq. Nature biotechnology 34. 199-203 (2016).

[0332] 72 Rahmati, N. et al. Unique actions of GABA arising from cytoplasmic chloride microdomains. Journal of Neuroscience 41, 4957-4975 (2021).

[0333] 73 Yao, Z. et al. A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation. Cell 184, 3222-3241. e3226 (2021).

[0334] 74 Mott, D. D.. Turner, D. A., Okazaki, M. M. & Lewis, D. V. Interneurons of the dentate-hilus border of the rat dentate gyrus: morphological and electrophysiological heterogeneity7. Journal of Neuroscience 17, 3990-4005 (1997).

[0335] 75 Jaffe, A. E. et al. Profiling gene expression in the human dentate gyrus granule cell layer reveals insights into schizophrenia and its genetic risk. Nature neuroscience 23, 510- 519 (2020).

[0336] 76 Murphy, B. L. et al. Heterogeneous integration of adult-generated granule cells into the epileptic brain. Journal of Neuroscience 31. 105-117 (2011).

[0337] 77 Grossberger, L., Battaglia, F. P. & Vinck, M. Unsupervised clustering of temporal patterns in high-dimensional neuronal ensembles using a novel dissimilarity measure. PLoS computational biology 14, el 006283 (2018).

[0338] 78 Caldwell, D. et al. Signal recovery from stimulation artifacts in intracranial recordings with dictionary learning. Journal of neural engineering 17, 026023 (2020).PATENT Attorney Docket No. 048536-799001WO

[0339] 79 Ayhan, F. et al. Resolving cellular and molecular diversity along the hippocampal anterior-to-posterior axis in humans. Neuron 109, 2091-2105. e2096 (2021).

[0340] 80 Mich, J. K. et al. Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex. Cell Rep 34, 108754 (2021). doi.org:10.1016 / i.celrep.2021. 108754

[0341] 81 Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204-209 (2016). doi.org:10.1038 / nbt.3440

[0342] 82 Dimidschstein, J. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat Neurosci 19. 1743-1749 (2016). doi.org: 10 1038 / nn,4430

[0343] 83 Chuapoco, M. R. et al. Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain. Nature Nanotechnology, 1-11 (2023).

[0344] 84 Wiebe, S., Blume, W. T., Girvin, J. P. & Eliasziw, M. A randomized, controlled trial of surgery for temporal -lobe epilepsy. New England Journal of Medicine 345, 311-318 (2001).

[0345] 85 Ma, B. B. & Rao, V. R. Responsive neurostimulation: candidates and considerations. Epilepsy & Behavior 88, 388-395 (2018).

[0346] 86 Razavi, B. et cd. Real-world experience with direct brain-responsive neurostimulation for focal onset seizures. Epilepsia 61, 1749-1757 (2020).

[0347] 87 Khambhati, A. N., Shafi, A., Rao, V. R. & Chang, E. F. Long-term brain network reorganization predicts responsive neurostimulation outcomes for focal epilepsy. Science Translational Medicine 13, eabf6588 (2021).

[0348] 88 Bansal, A., Shikha, S. & Zhang, Y. Towards translational optogenetics. Nature Biomedical Engineering, 1-21 (2022).

[0349] 89 Priya, R. et al. Activity regulates cell death within cortical interneurons through a calcineurin-dependent mechanism. Cell reports 22, 1695-1709 (2018).PATENTAttorney Docket No. 048536-799001WO

[0350] 90 Nair, D. R. et al. Nine-year prospective efficacy and safety of brain-responsive neurostimulation for focal epilepsy. Neurology’ 95, el244-el256 (2020).

[0351] 91 Ellens, N. R., Elisevich, K.. Burdette, D. E. & Patra, S. E. A comparison of vagal nerve stimulation and responsive neurostimulation for the treatment of medically refractory complex partial epilepsy. Stereotactic and functional neurosurgery 96, 259-263 (2018).

[0352] 92 Haneef, Z. & Skrehot, H. C. Neurostimulation in generalized epilepsy: A systematic review and meta-analysis. Epilepsia 64, 811-820 (2023).

[0353] 93 Skrehot, H. C., Englot, D. J. & Haneef, Z. Neuro-stimulation in focal epilepsy: A systematic review and meta-analysis. Epilepsy & Behavior 142, 109182 (2023).

[0354] 94 Ma, B. B. et al. Responsive neurostimulation for regional neocortical epilepsy. Epilepsia 61, 96-106 (2020).

[0355] 95 Panov, F. et al. Safety of responsive neurostimulation in pediatric patients with medically refractory epilepsy. Journal of Neurosurgery: Pediatrics 26, 525-532 (2020).

[0356] 96 Ryvlin, P. & Jehi, L. E. Neuromodulation for refractory epilepsy. Epilepsy Currents 22, 11-17 (2022).

[0357] 97 Ryvlin, P., Rheims, S., Hirsch, L. J., Sokolov, A. & Jehi, L. Neuromodulation in epilepsy: state-of-the-art approved therapies. The Lancet Neurology 20. 1038-1047 (2021).

[0358] 98 Pachitariu, M., Steinmetz, N., Kadir, S., Carandini, M. & Kenneth D, H. Kilosort: realtime spike-sorting for extracellular electrophysiology with hundreds of channels. BioRxiv, 061481 (2016).

[0359] 99 Rossant, C. et al. Spike sorting for large, dense electrode arrays. Nature neuroscience 19, 634-641 (2016).

[0360] 100 Siegle, J. H. et al. Survey of spiking in the mouse visual system reveals functional hierarchy. Nature 592, 86-92 (2021).

[0361] 101 Fisher, R. Statistical Methods for Research Workers. 4th edn, (Oliver and Boyd, London, 1932).PATENTAttorney Docket No. 048536-799001WO

[0362] 102 Hu, X., Zhang, W., Zhang, S., Ma, S. & Li, Q. Group-combined P-values with applications to genetic association studies. Bioinformatics 32, 2737-2743 (2016).Example 12: Extended Methods

[0363] Design of ontogenetic system

[0364] The purpose of the designed hardware platform is to trigger an optogenetic stimulation pulse and observe the response of neurons on a high-density electrode array. The optogenetic system was designed to be easy to replicate and does not require custom PCBs or soldering. Assembly instructions are detailed below. The bill of materials with pricing is available in Table 4. Lid and insert are described in detail below.

[0365] Assembly guide for optoelectronic components: First the DAC, HDMI breakout board, two pairs of black and red jumper wires, and the screw terminal to BNC connector were assembled. The VCC (+) and GND (-) pins on the power connector were connected to VCC and GND on the DAC. For the HDMI breakout board, the black (GND) wire was connected to pin 10, and the red (Signal) wire was connected to pin 2. The printed Arduino mount and HDMI cable holde...

Claims

PATENTAtorney Docket No. 048536-799001WOWHAT IS CLAIMED IS:

1. A method of treating a neurological or psychiatric disease or disorder in a subject in need thereof, the method comprises exposing a portion of a neural network of brain cell neurons within the subject to light thereby modulating the activity of the neural network of brain cell neurons, wherein the neural network of brain cell neurons is associated with the neurological or psychiatric disease or disorder, wherein the portion of the neural network of brain cell neurons comprises a population of transduced brain cell neurons, and wherein each transduced brain cell neuron within the population of transduced brain cell neurons comprises a potassium-selective channelrhodopsin protein.

2. The method of claim 1, wherein the neurological or psychiatric disease or disorder is an epilepsy, Parkinson’s disease, Alzheimer’s disease, essential tremor, depression, chronic pain, or schizophrenia.

3. The method of claim 1, wherein the potassium-selective channelrhodopsin is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl), a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2), a light-responsive inward rectifier potassium channel Kir2.1, a Wobblia hinata channelrhodopsin (WiChR), a K+-augmented light-gated ion channel 1 (KALI-1). or a K+-augmented light-gated ion channel 2 (KALI-2).

4. The method of claim 1, wherein the wavelength of the light is between about 260 nm and about 770 nm.

5. The method of claim 1, wherein the population of transduced brain cell neurons is a population of glutamatergic neurons.

6. The method of claim 1. wherein the intensity of the light exposure is from about 26.2 mW / mm2to about 35.8 mW / mm27. The method of claim 1, wherein the duration of the light exposure is about 50 milliseconds (ms) to about 10 seconds (s).PATENTAtorney Docket No. 048536-799001WO8. The method of claim 1, wherein the portion of the neural network of brain cell neurons is exposed to light at an interval of about 50 milliseconds (ms) to about 10 seconds (s).

9. The method of claim 1, wherein the population of transduced brain cell neurons is from about 5% to about 50% of the neural network of brain cell neurons.

10. A method of transfecting a brain celt the method comprising contacting the brain cell with an adeno-associated virus (AAV) viral vector comprising a nucleotide sequence encoding a potassium-selective channelrhodopsin protein.

11. The method of claim 10, wherein the potassium-selective channelrhodopsin protein is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl), a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2). a light- responsive inward rectifier potassium channel Kir2. 1, a Wobblia lunata channelrhodopsin (WiChR), a K+-augmented light-gated ion channel 1 (KALI-1), or a K+-augmented light-gated ion channel 2 (KALI-2).

12. The method of claim 10, wherein the AAV viral vector is an AAV9 viral vector, an AAV-PHP.eB viral vector, an AAV.CAP-Mac viral vector, an AAV9-X1. 1 viral vector, or an AAV5 viral vector.

13. The method of claim 10, wherein the AAV viral vector further comprises a Calcium / calmodulin-dependent protein kinase II alpha (CaMKIIa) promoter, a synapsin 1 (Synl) promoter, a phosphate-activated glutaminase (PAG) promoter, a vesicular glutamate transporter-1 (VGLUT1) promoter, or a protein c-Fos (FOS) promoter.

14. The method of claim 10, wherein the brain cell is a glutamatergic neuron.

15. A method of inhibiting neural activity of a transduced brain cell neuron, the method comprising exposing the transduced brain cell neuron to light thereby inhibiting neural activity of the transduced brain cell neuron, wherein transduced brain cell neuron comprises an exogenous potassium-selective channelrhodopsin protein.

16. The method of claim 15, wherein the wavelength of the light is between about 260 nm and about 770 nm.PATENTAttorney Docket No. 048536-799001WO17. The method of claim 15, wherein the intensity of the light exposure is from about 26.2 mW / mm2to about 35.8 mW / mm2.

18. The method of claim 15, wherein the duration of the light exposure is about 50 milliseconds (ms) to about 10 seconds (s).

19. The method of claim 15, wherein the transduced brain cell neuron is exposed to light at an interval of about 50 milliseconds (ms) to about 10 seconds (s).

20. The method of claim 15, wherein the method comprises exposing a plurality of the transduced brain cell neurons with the light thereby inhibiting neural activity of the plurality transduced brain cell neurons.

21. The method of claim 15, wherein about 5% to about 50% of the plurality of transduced brain cell neurons are inhibited by the light exposure.

22. A method of modulating activity of a neural network of brain cell neurons, the method comprising exposing a portion of the neural network of brain cell neurons with light thereby modulating the activity of the neural network of brain cell neurons, wherein the portion of the neural network of brain cell neurons comprises a population of transduced brain cell neurons, wherein each transduced brain cell neuron within the population of transduced brain cell neurons comprises a potassium-selective channelrhodopsin protein.

23. The method of claim 22, wherein the potassium-selective channelrhodopsin is a Hyphochytrium catenoides kalium channelrhodopsin 1 (HcKCRl), a Hyphochytrium catenoides kalium channelrhodopsin 2 (HcKCR2), a light-responsive inward rectifier potassium channel Kir2.1, a Wobblia lunata channelrhodopsin (WiChR) , a K - augmented light-gated ion channel 1 (KALI-1), or a K+-augmented light-gated ion channel 2 (KALI-2).

24. The method of claim 22, wherein the wavelength of the light is between about 260 nm and about 770 nm.

25. The method of claim 22, wherein population of transduced brain cell neurons is a population of glutamatergic neurons.PATENTAtorney Docket No. 048536-799001WO26. The method of claim 22, wherein the modulating of activity of the neural network is decreasing the activity of the neural network.

27. The method of claim 22, wherein the intensity of the light exposure is from about 26.2 mW / mm2to about 35.8 mW / mm2.

28. The method of claim 22, wherein the duration of the light exposure is about 50 milliseconds (ms) to about 10 seconds (s).

29. The method of claim 22, wherein the portion of the neural network of brain cell neurons is exposed to light at an interval of about 50 milliseconds (ms) to about 10 seconds (s).

30. The method of claim 22, wherein the population of transduced brain cell neurons is from about 5% to about 50% of the neural network of brain cell neurons.

31. An adeno-associated virus (AAV) viral vector comprising a nucleotide sequence encoding a potassium-selective channelrhodopsin protein, wherein the AAV viral vector is an AAV9 viral vector, an AAV-PHP.eB viral vector, an AAV.CAP-Mac viral vector, an AAV9-X1.1 viral vector, or an AAV5 viral vector.

32. A brain cell comprising an adeno-associated virus (AAV) viral vector comprising a nucleotide sequence encoding a potassium-selective channelrhodopsin protein, wherein the AAV viral vector is an AAV9 viral vector, an AAV-PHP.eB viral vector, an AAV.CAP-Mac viral vector, an AAV9-X1.1 viral vector, or an AAV5 viral vector.

Images