Thalamic input drive to orbitofrontal cortex is mediated by GABA and is frequency dependent across the brain

Abstract

Provided herein are methods and systems for modulating the temporal patterns of neuronal activity in the brain. The methods of the present disclosure can include using optogenetics to stimulate one or more of thalamocortical projections in the brain, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO, in conjunction with fMRI of different regions of the brain to directly visualize the global impact of afferent and efferent connections of the VLO, and to characterize how different temporal patterns of activity in the VLO circuit affect brain dynamics by driving its inputs and outputs at different frequencies.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to the filing date of U.S. Provisional Patent Application Serial No. 62 / 905557, filed September 25, 2019, pursuant to 35 U.S. SC §119(e); the disclosure of which is incorporated herein by reference.

[0003] Statement on Federally Funded Research

[0004] This invention was completed with government support under contracts AG047666, MH114227, NS087159, and NS091461 granted by the National Institutes of Health in the United States. The government holds certain rights to this invention.

[0005] introduction

[0006] The orbitofrontal cortex (OFC) is associated with a variety of cognitive and emotional functions. The ventrolateral orbitofrontal cortex (VLO), one of the five parts within the OFC, prominently supports many of these functions. Thalamic input to the VLO plays a crucial role in modulating perceived pain levels during noxious stimuli and supports goal-oriented behavior by issuing predictive cues and anticipating outcomes. The VLO is associated with spatial navigation and attention, depression, memory formation, and risk assessment. Cortical input also allows the VLO to integrate information relevant to different processes. These connections, along with extensive efferent projections, suggest that the VLO may act as a global hub, modulating activity within brain circuits. Despite evidence of a global role for the VLO in brain function, the circuital mechanisms by which it achieves this effect have not been directly investigated.

[0007] To better understand how VLO supports different behavioral processes, technical methods are needed that can control the circuit elements of an individual while visualizing whole-brain responses.

[0008] Overview

[0009] This article provides methods and systems for modulating temporal patterns of neuronal activity in the brain. The methods disclosed herein may include using optogenetics to stimulate one or more of the following: thalamic cortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the infracentral thalamic nucleus, and cell bodies in the ventrolateral orbitofrontal cortex (VLO); combining this with functional magnetic resonance imaging (fMRI) of different brain regions to directly visualize the global effects of afferent and efferent connections in the VLO; and characterizing how different temporal patterns of activity in the VLO circuit affect brain dynamics by driving its inputs and outputs at different frequencies.

[0010] Brief description of the attached figures

[0011] FIGS. 1A-1GOptogenetic fMRI revealed strong but distinct responses in VLO to thalamic cortex stimulation at 10 Hz and 40 Hz. FIG. 1A Experimental design for viral injection and thalamic cortex stimulation. FIG. 1B A schematic diagram of 23 coronal sections obtained in an optogenetic fMRI (ofMRI) experiment. FIG. 1C The design matrix of the block design stimulus paradigm. FIGS. 1D-1E ) at 10Hz FIG. 1D ) and 40Hz ( FIG. 1E Group-level activation maps during hypothalamic cortex stimulation (N = 11 animals; p < 0.05, FWE corrected). In these and all other activation maps, white triangles indicate the site of stimulation; warm colors indicate positive t-scores; cool colors indicate negative t-scores; image numbers correspond to treads. FIG. 1B The slice shown. FIGS. 1F-1G From the same side ( FIG. 1F ) and the opposite side ( FIG. 1G Single-cycle fMRI time series of segmented regions of the cortex. The horizontal blue line represents the stimulation period. The error bar represents the mean ± standard error of the mean in the animals (N=11). See also Figure 8-12.

[0012] FIGS. 2A-2C The results showed that frequency scanning experiments revealed a shift in induced activity patterns between low and high stimulation frequencies. FIG. 2A Group-level activation map during thalamic cortex stimulation at frequencies of 5 to 40 Hz in VLO (N = 7 animals; p < 0.005, uncorrected). FIG. 2B Quantification of significantly regulated brain volume in the ipsilateral hemisphere. Values ​​represent the voxel fractions significantly regulated in the group-level activation map for each ROI. FIG. 2C The average single-cycle time series illustrates the frequency-dependent shift from negative to positive responses in the sensory, motor, and cingulate cortex. The horizontal blue line represents the stimulation period.

[0013] FIGS. 3A-3D The stimulation of cell bodies in the VLO or thalamus did not induce a wide range of negative fMRI signals. FIGS. 3A-3B At 10Hz ( FIG. 3A ) and 40Hz ( FIG. 3B Group-level activation map during cell body stimulation in VLO (N=5 animals; p<0.05, FWE corrected). FIGS. 3C-3D At 10Hz ( FIG. 3C ) and 40Hz ( FIG. 3D Group-level activation map of responses induced during stimulation of cell bodies in the subcentral nucleus of the thalamus (N=5 animals; p<0.05, FWE corrected).

[0014] FIGS. 4A-4O Electrophysiology confirms frequency-dependent fMRI signal. FIG. 4A Single-unit recording schematic in the VLO at the stimulation site. FIG. 4B 10 Hz and 40 Hz stimulation drives strong positive fMRI signal at the stimulation site. FIG. 4C Peri-event time histograms of a representative unit in the VLO evoked during 10 Hz and 40 Hz stimulation (p = 1.2 x 10 -7 and 7.6 x 10 -10 , respectively). Error bars represent mean ± standard error of the mean across trials. FIG. 4D Quantification of significant changes in firing rate between recording units. INC: increase, DEC: decrease, N / C: no change. FIG. 4E Histogram of stimulation-evoked changes in firing rate in the VLO (n.s. not significant; p = 0.38). FIG. 4F Single-unit recording schematic in the contralateral VLO (cVLO). FIG. 4G 10 Hz stimulation drives strong negative fMRI signal in the cVLO, which largely disappears during 40 Hz stimulation. FIG. 4H Peri-event time histograms from a representative unit in the cVLO. Firing rate decreases during 10 Hz stimulation (p = 4.6 x 10 -13 ) but does not change during 40 Hz stimulation (p = 0.42). FIG. 4I Quantification of significant changes in firing rate of recorded units in the cVLO. FIG. 4J Histogram of stimulation-evoked changes in firing rate in the cVLO (p = 4.5 x 10 -17 ). (K) Single-unit recording schematic in the ipsilateral motor cortex (iMtr). FIG. 4L 10 Hz thalamocortical stimulation drives a negative fMRI response in the iMtr, while 40 Hz stimulation drives a positive fMRI response. FIG. 4M Peri-event time histograms from a representative unit in the iMtr, which is suppressed during 10 Hz stimulation (p = 3.4 x 10 -4 ) but is excited during 40 Hz stimulation (p = 2.6 x 10 -6 ). FIG. 4N Quantification of significant changes in firing rate of recorded units in the iMtr. FIG. 4O Histogram of stimulation-evoked changes in firing rate in the iMtr (p = 3.9 x 10 -29 ). See also FIG. 12A -12D.

[0015] FIGS. 5A-5GThe display of remote cortical inhibition driven by low-frequency thalamocortical stimulation is mediated by GABA. FIG. 5A . Single unit recording in cVLO and infusion schematic during 10 Hz thalamocortical stimulation. FIG. 5B . Microphotograph of a cannula electrode used to deliver saline and BMI. FIG. 5C . Quantification of significant changes in firing rate during stimulation before and after saline or BMI bolus. FIG. 5D . Histogram of stimulation-evoked changes in firing rate before and after a single saline or BMI bolus (p = 0.07 and 1.9 x 10 -16 , respectively). FIG. 5E . Quantification of changes in baseline firing rate after BMI infusion. Error bars represent mean ± standard error of the mean across trials for each unit and are color-coded according to whether the baseline firing rate of the unit increased or decreased significantly. The thick black line represents the mean ± standard error of the mean across units. FIG. 5F . Timeline of stimulation-evoked changes averaged across all recorded units during the 20 trials before and after each bolus. Shaded regions represent one standard deviation. Values reflect the percent signal change in firing rate during the 20 seconds of stimulation for each trial relative to the pre-stimulation period of the previous 20 seconds. FIG. 5G . Peri-event time histogram from a representative unit in the cVLO. Firing rate decreased during 10 Hz stimulation before saline (p = 2.0 x 10 -6 and 1.3 x 10 -4 ) and BMI (p = 1.9 x 10 -7 ) infusions. After the BMI infusion, 10 Hz stimulation no longer resulted in a significant change in firing rate (p = 0.63). Error bars represent mean ± standard error of the mean across trials.

[0016] FIGS. 6A-6E It is shown that pharmacological inactivation of the zona incerta reduces remote cortical inhibition driven by low-frequency thalamocortical stimulation. FIG. 6A . Schematic of infusion of lidocaine in the zona incerta during 10 Hz thalamocortical stimulation and single unit recording in the cVLO. FIG. 6B . Quantification of significant changes in firing rate evoked by stimulation at baseline and after infusion of saline or lidocaine. FIG. 6C . Timeline of stimulation-evoked changes in firing rate averaged across units that did not have a significant decrease in firing rate after the lidocaine infusion. Shaded regions represent one standard deviation. Values reflect the percent signal change in firing rate during the 20 seconds of stimulation for each trial relative to the pre-stimulation period of the previous 20 seconds. FIG. 6D. Left, histograms of changes in stimulus-evoked cVLO firing rate at baseline, after saline infusion, and after lidocaine infusion. Right, corresponding group means with 95% confidence intervals and post-hoc ANOVA comparisons (***p < 0.001). FIG. 6E . Representative peri-stimulus time histogram of a cell in the contralateral VLO. During 10 Hz stimulation before and after unrimband saline perfusion, firing rate decreased (p = 3.9 x 10 -5 and 1.4 x 10 -3 , respectively). After lidocaine infusion, the cell no longer exhibited a significant change in firing rate (p = 0.31). Error bars represent mean ± standard error of the mean across trials. See also FIGS. 14A-14I .

[0017] FIGS. 7A-7H Demonstrates that optical silencing of the unrimband eliminates long-range cortical inhibition driven by low-frequency thalamocortical stimulation. FIG. 7A . Schematic of single-unit recordings in the cVLO and unrimband (ZI) during 10 Hz thalamocortical stimulation and simultaneous silencing of the ZI with eNpHR. FIG. 7B . Stimulation paradigm used to assess the role of the unrimband in mediating widespread inhibition. FIG. 7C . Quantification of the significant change in ZI firing rate evoked by 10 Hz thalamocortical stimulation with and without eNpHR activation. FIG. 7D . Quantification of the significant change in cVLO firing rate evoked by 10 Hz thalamocortical stimulation with and without unrimband silencing. FIGS. 7E-7F . Histograms of stimulus-evoked changes in ZI FIG. 7E ) and cVLO FIG. 7F ) firing rate (p = 2.2 x 10 -9 and 3.8 x 10 -7 , respectively). FIGS. 7G-7H . Peri-stimulus time histograms of representative cells in the ZI FIG. 7G ) and cVLO FIG. 7H ). Firing rate of ZI cells increased during 10 Hz thalamocortical stimulation (p = 6.1 x 10 -5 ), but decreased when paired with eNpHR activation (p = 8.6 x 10 -4 ). Firing rate of cVLO cells decreased during 10 Hz thalamocortical stimulation (p = 0.030), but did not change when paired with eNpHR activation (p = 0.23). See also FIGS. 14A-14I .

[0018] FIGS. 8A-8D Demonstrates that stimulation targets thalamocortical projections genetically and spatially in the ventrolateral subregion of the VLO; related to FIGS. 1A-1G . FIG. 8AConfocal imaging at the injection site confirmed the expression of ChR2-EYFP in the cell bodies of thalamic neurons (white arrows). 29% of cells in the bulk injection area were identified as ChR2-EYFP positive (N = 2 animals, 343 cells). FIGS. 8B-8C Confocal in VLO ( FIG. 8B ) and fluorescence ( FIG. 8C Imaging confirmed the presence of ChR2-EYFP positive neuronal processes. No ChR2-EYFP positive cell bodies were observed, confirming that stimulation was limited to thalamic cortical projections. OLF: olfactory bulb. Note that the secondary antibody emitted in the red channel was used to amplify the endogenous EYFP signal. This signal was mapped to the green channel to maintain consistency with standard EYFP visualization. FIG. 8D Representative T2-weighted MRI scans used to confirm the location of stimulation in the cortex. Arrows indicate the light transmission location at the tip of the fiber optic implant (left, coronal; right, sagittal).

[0019] FIGS. 9A-9D The fMRI activation driven by thalamic cortex stimulation showed high consistency between the scan and the subject; with FIGS. 1A-1G Related. FIG. 9A Single-scan activation maps of the response to 40Hz thalamic cortex stimulation in representative animals (p<0.001, uncorrected). Each scan represents approximately 7 minutes of acquisition within the same session. White triangles indicate stimulation sites. Image numbers correspond to... FIG. 1B The coronal section shown. FIG. 9B The mean fMRI time series measured at the stimulation (LPFC) and ipsilateral thalamus sites demonstrate a high degree of consistency in the responses evoked in repeated trials. The time series are from ( FIG. 9A The same scan as shown in the image. FIG. 9C .right FIGS. 1A-1G The activation maps of the 11 animals reported in the study responded to the 40Hz stimulus (p<0.001, uncorrected). FIG. 9D Mean fMRI time series measured at the ipsilateral LPFC and thalamus in each animal demonstrate high reproducibility among subjects. The time series were derived from... FIG. 9C The same scan as shown.

[0020] FIGS. 10A-10E Quantitative, ROI-based characterization of fMRI responses induced during thalamic cortex stimulation; compared with FIGS. 1A-1G Related. FIG. 10A Whole-brain fMRI activation was segmented based on anatomical regions of interest (ROIs) to enable quantitative analysis of spatiotemporal characteristics. The segmented ROIs were superimposed as color regions on average structural MRI images. FIG. 10B and 10DQuantification of the amount of modulated voxels in the ipsilateral ( FIG. 10B ) and contralateral ( FIG. 10D ) regions of interest during 10 Hz and 40 Hz stimulation. Ipsilateral volume significantly increased during 40 Hz stimulation, while contralateral volume significantly increased during 10 Hz stimulation (*p<0.05, **p<0.005, ***p<0.001). Red lines represent values from individual animals. Black lines represent the mean. FIG. 10C and 10E Quantification of the sum fMRI values in the ipsilateral ( FIG. 10C ) and contralateral ( FIG. 10E ) regions of interest, significantly different from zero, marked with asterisks. The three ipsilateral regions sensory, motor and cingulate cortex transitioned from a significant negative response at 10 Hz to a significant positive response at 40 Hz Contralateral sum fMRI values were significantly negative during 10 Hz stimulation, but not significantly different from zero during 40 Hz stimulation. Values with error bars represent mean ± standard error of the mean.

[0021] FIGS. 11A-11D show that the frequency-dependent effect of thalamocortical projection stimulation is preserved when the pulse width (PW) is kept constant; related to FIGS. 1A-1G (A) Activation maps from a representative animal in the VLO during 10 Hz and 40 Hz thalamocortical stimulation using a constant pulse width of 3 ms (p<0.001, uncorrected). White triangle on slice 6 indicates approximate location of stimulation. Warm colors indicate positive t-scores, while cold colors indicate negative t-scores. Image numbers correspond to coronal slices shown in FIG. 1B (B) Quantification of total fMRI modulation volume in ipsilateral and contralateral cortex (N=4 animals). Thin gray lines correspond to individual animals. Black lines represent the mean. Values are summed over the cortical ROIs. (C) Quantification of the sum fMRI values of the ipsilateral ROIs. Error bars represent mean ± standard error of the mean in the animals. (D) Time series from the ipsilateral and contralateral somatosensory cortex. Thin lines represent individual animals. Thick lines represent the mean.

[0022] FIG. 12A show that animal-specific electrophysiological results reflect the frequency-dependent trends reported in the main text; related to FIGS. 4A-4O Each column represents a different animal in which single unit recordings were made for the stimulation site in the VLO ( FIG. 12A ), contralateral VLO ( FIG. 12B ), or ipsilateral motor cortex ( FIG. 12C ).

[0023] FIGS. 13A-13H show that stimulus-evoked activity in the thalamic reticular nucleus (TRN) is greater during 40 Hz thalamocortical stimulation than during 10 Hz stimulation; related toFIGS. 5A-5G Related. FIG. 13A and 13E During VLO thalamic cortical stimulation, ipsilateral ( FIG. 13A ) and the opposite side ( FIG. 13E A diagram illustrating the location of a single cell record in a TRN. FIG. 13B Quantification of significant changes in firing frequency in the ipsilateral TRN. During 40 Hz stimulation, the firing frequency of more cells increased significantly. INC: increase, DEC: decrease, N / C: no change. FIG. 13C Histograms of changes in firing frequency induced by stimulation in the ipsilateral TRN during 10Hz and 40Hz stimulation (p = 5.3 x 10⁻⁶). -12 ). ( FIG. 13D The ring event time histogram from a representative unit in the ipsilateral TRN showed a significant increase in firing frequency during 40 Hz stimulation (p = 1.6 x 10⁻⁶). -4 However, there was no change during the 10Hz stimulus period (p = 0.39; not significant in ns). The error bar represents the mean ± standard error of the mean in the experiment. FIG. 13F Quantification of significant changes in contralateral TRN firing frequency. Activity preferentially decreased during 10 Hz stimulation. FIG. 13G Histograms of changes in firing frequency induced by stimulation in the contralateral TRN during 10Hz and 40Hz stimulation (p = 6.6 x 10⁻⁶). -31 ). ( FIG. 13H The histogram of ring event times from representative units in the contralateral TRN showed a significant decrease in firing frequency during 10 Hz stimulation (p = 7.7 x 10⁻⁶). -8 However, the firing frequency increased significantly during 40Hz stimulation (p = 0.020).

[0024] FIGS. 14A-14I Shows details of the method for undefined targeting and control; with FIGS. 6A-6E and FIGS. 7A-7E Related. FIGS. 14A-14C Stereotactic targeting precisely locates the zone of indeterminate position (ZI). FIG. 14A To assess the accuracy of stereotactic targeting in the zone of indeterminacy, bilateral implants were inserted into another group of animals at target coordinates [-3.96 mm AP, ±2.75 mm ML, -7.20 mm DV] (N=9). The resulting implantation locations were determined by MRI. Individual implants, indicated by red circles in the schematic diagram, were located directly above or inside the zone of indeterminacy [mean location: -3.92 mm AP, ±2.79 mm ML, -7.21 mm DV]. FIG. 14B) High-resolution ex vivo MRI scans confirmed correct placement of infusion cannula in the zona incerta during lidocaine hydrochloride experiments. Area outlines covering the zona incerta and underlying white matter tracts are overlaid for clarity. Fast low angle shot (FLASH) MRI sequence parameters: 0.1 x 0.1 x 0.08 mm 3 Spatial resolution, 280 x 280 matrix size, 12.9 ms TR, 4.9 ms TE, 170 slices, 30° flip angle. FIG. 14C ) Electrophysiological signals recorded at the targeted coordinates in the ZI during eNpHR experiments (high-pass filtered, 300 Hz cutoff frequency, 4-pole Bessel filter). Neurons at the targeted coordinates responded to contralateral 4s cycles but not to ipsilateral antenna stimulation, consistent with the known receptive field properties of the zona incerta. Bottom trace shows an expanded version of the contralateral antenna stimulation trial. FIGS. 14D-14G ) Histological and functional confirmation of halorhodopsin expression in the zona incerta. (D) mCherry expression in the zona incerta confirms expression of eNpHR-mCherry. FIG. 14E ) Recordings in the zona incerta during continuous 589 nm light delivery to confirm functional halorhodopsin expression. FIG. 14F ) Peri-stimulus time histogram from a representative unit in the zona incerta showing a significant decrease in firing rate (p = 1.3 x 10 -5 ) Significant decrease in firing rate was observed in all recorded units (N = 35 units, 20 trials). FIG. 14G ) Histogram of eNpHR-driven changes in firing rate in all recorded units in the zona incerta (N = 35 units). The mean change in firing rate was -30% ± 15% standard deviation. FIG. 14H ) Recordings in the contralateral VLO during continuous inhibition of the zona incerta to investigate any tonic effects of the ZI on cortex. FIG. 14I ) When the zona incerta was inhibited with halorhodopsin, most units recorded in the contralateral VLO (92%) showed no significant change. 8% showed a significant increase in activity.

[0025] FIG. 15 Amino acid sequences of depolarizing light-activated polypeptides and derivatives thereof (SEQ ID NOs: 1-20) useful in the present methods according to embodiments of the present disclosure are shown.

[0026] FIG. 16 Amino acid sequences of hyperpolarizing light-activated polypeptides and derivatives thereof (SEQ ID NOs: 21-51) useful in the present methods according to embodiments of the present disclosure are shown.

[0027] Definitions

[0028] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified; for example, disulfide- formed, glycosylated, lipidated, acetylated, phosphorylated, or otherwise derivatized, for example, with a labeling component. The term "amino acid," as used herein, refers to naturally occurring and / or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0029] The term "genetic modification" refers to a permanent or transient genetic change induced in a cell after introduction of a heterologous nucleic acid (e.g., extracellular nucleic acid) into the cell. The genetic alteration ("modification") can be achieved by incorporation of the heterologous nucleic acid into the genome of the host cell, or by transient or stable maintenance of the heterologous nucleic acid as an extrachromosomal element. When the cell is a eukaryotic cell, permanent genetic alteration can be achieved by introduction of the nucleic acid into the genome of the cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.

[0030] A "plurality" contains at least 2 members. In certain instances, a plurality can have at least 10, at least 100, at least 1000, at least 10,000, at least 100,000, at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , or more members.

[0031] As used herein, "substantially" can be used to modify any quantitative representation that is permissible to vary without causing a change in the basic function intended to be engendered by the quantitative representation.

[0032] As used herein, an "individual" can be any animal suitable for use with the methods and techniques described herein, where in some instances the individual can be a vertebrate, including mammals, birds, reptiles, amphibians, and the like. The individual can be any suitable mammal, such as a human, a mouse, a rat, a cat, a dog, a pig, a horse, a cow, a monkey, a non-human primate, and the like.

[0033] As used herein, a "set" can include one or more than one element.

[0034] As used herein, "functional" can be used to describe a process that is physiologically relevant, i.e., a process that is related to performing a process that normally occurs in a living organism. The process can be a measurement phenomenon that represents or directly or indirectly reads out a potential physiologically relevant process.

[0035] As used herein, “connectivity” can refer to a structural and / or functional relationship between two different entities, such as a cell (including a neuron), a tissue region (e.g., a brain region), a tissue, an organ, and the like. Functional connectivity between two regions of the brain can be achieved through direct and / or indirect structural connectivity (e.g., synaptic connections) between the two regions.

[0036] As used herein, “neural activity” can refer to electrical activity of a neuron (e.g., changes in membrane potential of a neuron), as well as indirect measures of electrical activity of one or more neurons. Thus, neural activity can refer to changes in field potentials, changes in intracellular ion concentrations (e.g., intracellular calcium concentration), and magnetic resonance changes caused by electrical activity of neurons, as measured by, e.g., cerebral blood volume (CBV) in functional magnetic resonance imaging.

[0037] As used herein, “dynamic” can be used to describe a process that changes over a temporal dimension.

[0038] As used herein, “quantitative” refers to a numerical attribute defined by or related to a quantity value, or a system (e.g., a brain circuit) whose output varies with different input patterns.

[0039] As used herein, “qualitative” can refer to a property that is not defined by a quantity value of a numerical quantity. For example, a qualitative determination can include a determination of a yes / no or on / off result.

[0040] In certain aspects, the term “modulate” refers to increase, decrease, or inhibit. In some cases, “modulation” can be measured using an appropriate in vitro assay, cellular assay, in vivo assay, or behavioral assay. In some cases, an increase or decrease is 10% or more relative to a reference, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, up to 100% relative to a reference. For example, an increase or decrease can be 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 50-fold or more, or 100-fold or more relative to a reference.

[0041] Before further description of the application, it should be understood that the application is not limited to the particular embodiments described, as such can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the application will only be limited by the appended claims.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the application.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0044] It must be noted that, as used herein and in the appended claims, the singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a neuron" includes more than one such neuron, reference to "a light-activated polypeptide" includes reference to one or more light-activated polypeptides and equivalents thereof known to one of ordinary skill in the art, and so forth. It must also be noted that the claims can be drafted to exclude any optional element. Thus, for example, the claims can be drafted to exclude any optional element by using "unique", "only", or "exactly" along with the claiming of the elements in the claims.

[0045] It will be appreciated that, for clarity and for reasons of convenience and federal regulation, certain features of the application that are, for brevity, described in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the application, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any appropriate subcombination. All combinations of the embodiments pertaining to the application are specifically embraced by the application and are disclosed herein just as if each and every combination was individually and explicitly disclosed herein. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the application and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0046] The publications discussed herein are provided only for their public disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to precede such publications by prior invention. Furthermore, the publication dates provided may differ from the actual publication dates that may require independent verification.

[0047] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any method described may be performed in the order of the events described or in any other logically possible order.

[0048] Although apparatuses and methods have been or will be described for the purposes of grammatical fluency and functional interpretation, it should be clearly understood that, unless pursuant to 35 U.SC §112, they should not be construed as necessarily being limited in any way by constructing “means” or “steps”, but rather should be given the full meaning and scope of equivalents as defined under the doctrine of judicial equivalents, and where the claims are expressly stated under 35 U.SC §112, they should conform to all legal equivalents under 35 U.SC §112. Detailed Implementation

[0049] This article provides methods and systems for modulating temporal patterns of neuronal activity in the brain. The methods disclosed herein may include using optogenetics to stimulate one or more of the following: thalamic cortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the subcentral thalamic nucleus, and cell bodies in the VLO (vegetative lobe loop); combining this with fMRI of different brain regions to directly visualize the global effects of the afferent and efferent connections of the VLO; and characterizing how different temporal activity patterns in the VLO circuit affect brain dynamics by driving its inputs and outputs at different frequencies.

[0050] method

[0051] As described above, methods are provided for modulating the temporal pattern of neuronal activity in an individual's brain. In some cases, the methods modulate neuronal activity in one or more brain regions or the whole brain. In some cases, the methods modulate the spatial extent of neuronal activation or inhibition in one or more brain regions or the whole brain. In some cases, the methods modulate the inhibitory or excitatory effect of input from one or more brain regions on one or more downstream brain regions. Aspects of the methods can include visualizing and / or measuring neuronal activity, e.g., the temporal and / or spatial pattern of neuronal activity, in one or more brain regions or the whole brain in response to stimulation of one or more brain regions. The methods of the present disclosure can use any number of combinations of suitable neuronal stimulation and neuronal activity measurement protocols as desired to determine functional connectivity between different brain regions. Suitable protocols include electrophysiology; optogenetic modulation of neural activity; electroencephalography (EEG) recording; functional imaging; and behavioral analysis. One or more parameters of the neuronal stimulation protocol, e.g., light pulse frequency, can be varied. One or more parameters can be varied to modulate neuronal activity as described herein. The neuronal stimulation and neuronal activity measurement protocols can be applied to the whole brain. The neuronal stimulation and neuronal activity measurement protocols can be applied to one or more brain regions. In some cases, the whole brain includes ipsilateral and contralateral regions of the brain.

[0052] As described above, the methods can include any number of combinations of neuronal stimulation and neuronal activity measurement protocols. Some protocols, e.g., fMRI, provide non-invasive, whole-brain measurements of neural activity. Some protocols, e.g., electrophysiology, provide fast measurements of neural activity with cellular resolution and fast control of neural activity with cellular resolution. Some protocols, e.g., optogenetics, provide spatially-localized and temporally-defined control of action potential firing in defined groups of neurons. Appropriate combinations of assays can be used to dissect functional brain circuits. In some cases, the combinations include: optogenetics and fMRI; optogenetics and electrophysiology; optogenetics and EEG; optogenetics and behavioral analysis. Any other suitable combination can also be used, e.g., EEG and behavioral analysis; fMRI and electrophysiology; electrophysiology and behavioral analysis; etc.

[0053] The methods disclosed herein are suitable for revealing causal links between different brain regions in a single living individual (e.g., a single mouse or rat, a single human, a single non-human primate) by using different combinations of one or more than one neuronal stimulation and activity measurement protocol as described above. In some cases, the methods determine the underlying circuit mechanisms by which one or more than one brain region controls neural activity throughout the brain. Thus, in some embodiments, one or more than one combination of the following is used to determine brain functional circuits in a single animal: optogenetics and fMRI; optogenetics and electrophysiology; optogenetics and EEG; and optogenetics and behavioral analysis. In some cases, all of the following are used to determine brain functional circuits in a single animal: optogenetics and fMRI; optogenetics and electrophysiology; optogenetics and EEG; and optogenetics and behavioral analysis. The order in which the different measurements are performed on a single animal can be any suitable order. In some cases, the combination of measurements is performed in the following order: optogenetics and fMRI; optogenetics and EEG / optogenetics and behavioral analysis; and optogenetics and electrophysiology, where "optogenetics and EEG" and "optogenetics and behavioral analysis" can be performed in any order. Other combinations of protocols can be performed at any suitable time point before or after a protocol that involves optogenetics.

[0054] Aspects of the disclosure can include methods of modulating the temporal pattern of neuronal activity in an individual's brain using a combination of optogenetic stimulation of defined neuronal populations in one or more than one brain region of the individual, and measuring the response at the whole-brain level by scanning the brain with fMRI to modulate neuronal activity after stimulation. Embodiments of the methods can include modulating the temporal pattern of neuronal activity in an individual's brain using a combination of optogenetic stimulation of defined neuronal populations in one or more than one of the VLO and thalamus of the individual, and measuring the response at the whole-brain level by scanning the brain with fMRI to modulate neuronal activity after stimulation.

[0055] The brain regions of interest in the present methods (for optogenetic stimulation and / or measuring neural activity) can vary and can be any suitable region. In certain embodiments, the brain region is an anatomically and / or functionally defined region of the brain. For example, the first region of the brain and the second region of the brain irradiated by the light pulses as described herein can be anatomically distinct regions of the brain. In some cases where the brain is a mammalian brain, the brain region of interest is selected from at least a portion of the thalamus (including the centromedial thalamus), the sensory cortex (including the somatosensory cortex), the zona incerta (ZI), the ventral tegmental area (VTA), the prefrontal cortex (PFC), the nucleus accumbens (NAc), the amygdala (BLA), the substantia nigra, the ventral pallidum, the pallidum, the dorsal striatum, the ventral striatum, the subthalamic nucleus, the hippocampus, the dentate gyrus, the cingulate, the entorhinal cortex, the olfactory cortex, the primary motor cortex, and the cerebellum. In some cases, the different brain regions (e.g., the first and second brain regions) are separated by at least one or more, e.g., 2 or more, 3 or more, 4 or more, 5 or more, including 7 or more synapse connections, and separated by at least 15 or less, e.g., 12 or less, 10 or less, 8 or less, including 6 or less synapse connections. In some embodiments, the different brain regions are separated by at least 1 to 15 synapse connections, e.g., 1 to 12 synapse connections, 1 to 10 synapse connections, 2 to 8 synapse connections, including 3 to 6 synapse connections.

[0056] The neurons of interest present in the brain region can be any suitable type of neuron. In some cases, the neurons are inhibitory neurons or excitatory neurons. In some cases, the neurons are sensory neurons, interneurons, or motor neurons. In some cases, the neurons are, but are not limited to, dopaminergic, cholinergic, GABAergic, glutamatergic, or peptidergic neurons.

[0057] In some cases, the methods of the present disclosure comprise stimulating the VLO of the brain. In some cases, the methods of the present disclosure comprise stimulating the thalamocortical projection of the brain. In some cases, the methods of the present disclosure comprise stimulating thalamic relay neurons of the brain. In some cases, the methods of the present disclosure comprise stimulating the corticofugal neurons of the brain. In some cases, the methods of the present disclosure comprise stimulating the cell bodies in the centrolateral subnucleus of the thalamus of the brain. In some cases, the methods of the present disclosure comprise stimulating the cell bodies in the VLO of the brain. In some cases, stimulating the VLO of the brain results in a positive measured fMRI signal at the VLO of the brain.

[0058] In embodiments of methods of practicing the application, the method can comprise, for example, i) stimulating one or more of thalamocortical projections in the brain, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the ventrolateral orbital frontal cortex (VLO) with a pulse of light from an optical light source, wherein the neuronal cell bodies of one or more of the VLO and the thalamus of the individual express a light-activated polypeptide; ii) measuring functional magnetic resonance imaging (fMRI) signals of the whole brain, wherein the measuring occurs during the stimulating, wherein a positive measured fMRI signal correlates with an increase in neuronal activity following the stimulating, wherein a negative measured fMRI signal correlates with a decrease in neuronal activity following the stimulating.

[0059] Stimulation

[0060] Neurons in one or more brain regions subjected to optogenetic stimulation can be modified to include a light-activated polypeptide. The modification can be made by administering, e.g., injecting, a light-activated polypeptide to the one or more brain regions. Thus, neurons in the VLO and / or the thalamus can be modified to include a light-activated polypeptide, e.g., a light-activated ion channel, wherein the light-activated polypeptide is configured to modulate, e.g., depolarize or hyperpolarize, the activity of one or more neurons upon stimulation of one or more of thalamocortical projections in the brain, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO with an appropriate wavelength, amount of illumination, and intensity. In some cases, the method comprises expressing a light-activated polypeptide in neurons of the thalamus. In some cases, the method comprises expressing a light-activated polypeptide in neurons of the centrolateral nucleus of the thalamus. In some cases, the method comprises expressing a light-activated polypeptide in neurons of the VLO. In some cases, the method comprises expressing a light-activated polypeptide in layer I and / or layer III neurons. In some cases, the light-activated polypeptide expressed in layer I and / or layer III neurons of the VLO is from neurons located in the centrolateral nucleus of the thalamus. For example, the centrolateral nucleus neurons expressing the light-activated polypeptide send projections to the VLO. In some cases, the light-activated polypeptide is a depolarizing light-activated polypeptide. In some cases, the light-activated polypeptide is a hyperpolarizing light-activated polypeptide. In some embodiments, neurons in the centrolateral nucleus are modulated by stimulating cell bodies in the centrolateral nucleus. In some embodiments, neurons in the centrolateral nucleus are modulated by stimulating projecting cell bodies in the VLO.

[0061] In some cases, the methods of the present disclosure include genetically modifying neurons of the VLO and / or thalamus, e.g., by infecting with a virus a DNA construct containing a nucleotide sequence encoding a light-activated polypeptide and any other suitable regulatory elements, to express the light-activated polypeptide. In some cases, the methods include administering a light-activated polypeptide to the centrolateral nucleus of the thalamus. As further described herein, any suitable light-activated polypeptide can be used. In some cases, the methods of the present disclosure include a first light-activated polypeptide and a second light-activated polypeptide. In some cases, the first light-activated polypeptide is a depolarizing light-activated polypeptide. In some cases, the second light-activated polypeptide is a hyperpolarizing light-activated polypeptide. In some cases, the methods of the present disclosure include administering the first and second light-activated polypeptides to the same region of the brain. In some cases, the methods of the present disclosure include administering the first and second light-activated polypeptides to different regions of the brain. Suitable light-activated polypeptides are described in U.S. Patent Publication No. 2018 / 0360343 Al, which is incorporated by reference herein in its entirety.

[0062] Aspects of the present methods can include administering a second light-activated polypeptide. In some cases, the second light-activated polypeptide is administered to a zona incerta (ZI) region of the brain. In some cases, the second light-activated polypeptide is a depolarizing light-activated polypeptide. In some cases, the second light-activated polypeptide is a hyperpolarizing light-activated polypeptide. In some cases, the methods of the present disclosure include stimulating a ZI region of the brain, e.g., when the second light-activated polypeptide is expressed in neurons of the ZI. The ZI region can be stimulated simultaneously during stimulation of other brain regions and / or performance of electrophysiological recordings. The ZI region can be stimulated simultaneously with stimulation of thalamocortical projections. The ZI region can be stimulated with light pulses having any of the frequencies described herein.

[0063] Neurons of suitable regions of the brain whose activity is to be light-modulated can be modified to express light-activated polypeptides using convenient methods. In some cases, neurons of a brain region are genetically modified to express a light-activated polypeptide. In some cases, the neurons can be genetically modified using a viral vector, e.g., an adeno-associated viral vector, that includes a nucleic acid having a nucleotide sequence encoding a light-activated polypeptide. The viral vector can include any suitable control elements (e.g., promoters, enhancers, recombination sites, etc.) to control expression of the light-activated polypeptide according to cell type, timing, presence of an inducer, etc.

[0064] Suitable neuron-specific control sequences include, but are not limited to, the neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Patent No. 6,649,811, U.S. Patent No. 5387742); the aromatic amino acid decarboxylase (AADC) promoter; the neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); the synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); the thy-1 promoter (see, e.g., Chen et al., (1987) Cell 51 :7-19; and Llewellyn et al., (2010) Nat. Med. 16: 1161); the serotonin receptor promoter (see, e.g., GenBank S62283); the tyrosine hydroxylase promoter (TH) (see, e.g., Nucl. Acids. Res. 15:2363-2384 (1987) and Neuron 6:583-594 (1991)); the GnRH promoter (see, e.g., Radovick et al., Proc. Natl. Acad. Sci. USA 88:3402-3406 (1991)); the L7 promoter (see, e.g., Oberdick et al., Science 248:223-226 (1990)); the DNMT promoter (see, e.g., Bartge et al., Proc. Natl. Acad. Sci. USA 85:3648-3652 (1988)); the enkephalin promoter (see, e.g., Comb et al., EMBO J. 17:3793-3805 (1988)); the myelin basic protein (MBP) promoter; the CMV enhancer / platelet-derived growth factor-beta promoter (see, e.g., Liu et al., (2004) Gene Therapy 11 :52-60); the motor neuron-specific gene Hb9 promoter (see, e.g., U.S. Patent No. 7632679; and Lee et al., (2004) Development 131 :3295-3306); and the Ca 2+ )-calmodulin-dependent protein kinase II (CaMKIIa) promoter a subunit (see, e.g., Mayford et al., (1996) Proc. Natl. Acad. Sci. USA 93: 13250). Other suitable promoters include the elongation factor (EF) 1a and the dopamine transporter (DAT) promoters.

[0065] In some cases, cell type-specific expression of a light-activated polypeptide can be achieved using a recombination system, such as Cre-Lox recombination, Flp-FRT recombination, and the like. Cell type-specific expression of genes using recombination has been described, e.g., in Fenno et al., Nat Methods. 2014 Jul; 11(7):763; and Gompf et al., Front Behav Neurosci. 2015 Jul 2;9: 152, which are incorporated by reference herein in their entireties.

[0066] Light stimulation can be used to illuminate one or more brain regions containing a light-activated polypeptide. Light stimulation can be used to activate one or more light-activated polypeptides. Light stimulation for activating a light-activated polypeptide can include one or more light pulses. A light pulse can be characterized by, e.g., frequency, pulse width, duty cycle, wavelength, intensity, and the like. In some cases, light stimulation includes two or more groups of different light pulses, where each group of light pulses is characterized by a different temporal pattern of light pulses. The temporal pattern can be characterized by any suitable parameter, including but not limited to frequency, period (i.e., total duration of the light stimulation), pulse width, duty cycle, and the like. Optogenetic stimulation can be performed using any suitable method. Suitable methods are described, e.g., in U.S. Patent No. 8,834,546, which is incorporated by reference herein in its entirety.

[0067] Changes in the properties of a group of light pulses can be reflected in differences in the activity of illuminated neurons. In some cases, an increase in the frequency of light pulses can result in an increase in the frequency of action potential firing in illuminated neurons when the neurons are depolarized by activation of a light-activated polypeptide. In some embodiments, the frequency of action potential firing in illuminated neurons is quantitatively proportional to an increase in the frequency of light pulses. In some cases, a linear increase in the frequency of light pulses can result in a linear increase or a non-linear but monotonic increase in the frequency of action potential firing in illuminated neurons. In some cases, the stimulation can manifest as a downregulation of neuronal activity, e.g., hyperpolarization of neurons. In some cases, an increase in the frequency of light pulses can result in a decrease in the frequency of action potential firing in illuminated neurons when the neurons are hyperpolarized by activation of a light-activated polypeptide. Aspects of the present disclosure can include stimulating or illuminating a first region of the brain with a first group of light pulses and a second group of light pulses having different temporal patterns, where neurons in the first region can generate action potentials induced by the first group and / or the second group of light pulses, or suppress action potentials following the first group and / or the second group of light pulses.

[0068] In some cases, the light stimulus comprises one or more than one, two or more than two, three or more than three, four or more than four, five or more than five, six or more than six, seven or more than seven, eight or more than eight, nine or more than nine, or ten or more than ten groups of light pulses, where the groups of light pulses are characterized by having different values of a parameter, e.g., different frequencies of the light pulses. Where the groups of light pulses have different frequencies, the duty cycles can be the same, or can be different. In some cases, the groups of light pulses of different frequencies have the same pulse width. In other cases, the groups of light pulses of different frequencies have different pulse widths.

[0069] The groups of light pulses can have any suitable frequency. In some cases, a group of light pulses comprises a single light pulse that lasts for the duration of the light stimulus. In some cases, a group of light pulses has a frequency of 0.1 Hz or higher, e.g., 0.5 Hz or higher, 1 Hz or higher, 5 Hz or higher, 10 Hz or higher, 20 Hz or higher, 30 Hz or higher, 40 Hz or higher, including 50 Hz or higher, or 60 Hz or higher, or 70 Hz or higher, or 80 Hz or higher, or 90 Hz or higher, or 100 Hz or higher, and a frequency of 100,000 Hz or lower, e.g., 10,000 Hz or lower, 1000 Hz or lower, 500 Hz or lower, 400 Hz or lower, 300 Hz or lower, 200 Hz or lower, including 100 Hz or lower. In some cases, a group of light pulses has a frequency of 0.1 Hz to 100,000 Hz, e.g., 1 Hz to 10,000 Hz, 1 Hz to 1000 Hz, including 5 Hz to 500 Hz, or 10 Hz to 100 Hz. In some embodiments, the frequency of the light pulses is 5 Hz to 40 Hz.

[0070] The light pulses of the present methods can have any suitable pulse width. In some cases, the pulse width is 0.1 ms or greater, e.g., 0.5 ms or greater, 1 ms or greater, 3 ms or greater, 5 ms or greater, 7.5 ms or greater, 10 ms or greater, including 15 ms or greater, or 20 ms or greater, or 25 ms or greater, or 30 ms or greater, or 35 ms or greater, or 40 ms or greater, or 45 ms or greater, or 50 ms or greater, and is 500 ms or less, e.g., 100 ms or less, 90 ms or less, 80 ms or less, 70 ms or less, 60 ms or less, 50 ms or less, 45 ms or less, 40 ms or less, 35 ms or less, 30 ms or less, 25 ms or less, including 20 ms or less. In some embodiments, the pulse width is 0.1 ms to 500 ms, e.g., 0.5 ms to 100 ms, 1 ms to 80 ms, including 1 ms to 60 ms, or 1 ms to 50 ms, or 1 ms to 30 ms.

[0071] The duty cycle of the pulses of the present methods can be any suitable duty cycle. In some cases, the duty cycle is 1% or greater, e.g., 5% or greater, 10% or greater, 15% or greater, 20% or greater, including 25% or greater, or 30% or greater, or 35% or greater, or 40% or greater, or 45% or greater, or 50% or greater, and can be 80% or less, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 65% or less, 50% or less, 45% or less, including 40% or less, or 35% or less, or 30% or less. In some embodiments, the duty cycle is 1% to 80%, e.g., 5% to 70%, 5% to 60%, including 10% to 50% or 10% to 40%.

[0072] The average power of the light pulses of the method, which is measured at the tip of the optical fiber that delivers the light pulses to the brain region, can be any suitable power. In some cases, the power is 0.1 mW or greater than 0.1 mW, e.g., 0.5 mW or greater than 0.5 mW, 1 mW or greater than 1 mW, 1.5 mW or greater than 1.5 mW, including 2 mW or greater than 2 mW, or 2.5 mW or greater than 2.5 mW, or 3 mW or greater than 3 mW, or 3.5 mW or greater than 3.5 mW, or 4 mW or greater than 4 mW, or 4.5 mW or greater than 4.5 mW, or 5 mW or greater than 5 mW, and can be 1000 mW or less than 1000 mW, e.g., 500 mW or less than 500 mW, 250 mW or less than 250 mW, 100 mW or less than 100 mW, 50 mW or less than 50 mW, 40 mW or less than 40 mW, 30 mW or less than 30 mW, 20 mW or less than 20 mW, 15 mW or less than 15 mW, including 10 mW or less than 10 mW, or 5 mW or less than 5 mW. In some embodiments, the power is 0.1 mW to 1000 mW, e.g., 0.5 mW to 100 mW, 0.5 mW to 50 mW, 1 mW to 20 mW, including 1 mW to 10 mW or 1 mW to 5 mW.

[0073] The wavelength and intensity of the light pulses of the method can vary and can depend on the activation wavelength of the light-activated polypeptide, the optical transparency of the brain region, the desired volume of the brain to be irradiated, etc.

[0074] The volume of the brain region irradiated by the light pulses can be any suitable volume. In some cases, the irradiated volume is 0.001 mm 3 or greater than 0.001 mm 3 , e.g., 0.005 mm 3 or greater than 0.005 mm 3 , 0.001 mm 3 or greater than 0.001 mm 3 , 0.005 mm 3 or greater than 0.005 mm 3 , 0.01 mm 3 or greater than 0.01 mm 3 , 0.05 mm 3 or greater than 0.05 mm 3 , including 0.1 mm 3 or greater than 0.1 mm 3 , and is 100 mm 3 or less than 100 mm 3 , e.g., 50 mm 3 or less than 50 mm 3 , 20 mm 3 or less than 20 mm 3 , 10 mm3 or less than 10 mm 3 , 5 mm 3 or less than 5 mm 3 , 1 mm 3 or less than 1 mm 3 , including 0.1 mm 3 or less than 0.1 mm 3 In some cases, the irradiated volume is 0.001 mm 3 to 100 mm 3 , for example 0.005 mm 3 to 20 mm 3 , 0.01 mm 3 to 10 mm 3 , 0.01 mm 3 to 5 mm 3 , including 0.05 mm 3 to 1 mm 3 .

[0075] In some cases, the methods of the present disclosure include reversibly inserting an optical light source, such as an optical fiber, into the VLO of the individual. In some cases, the optical light source is implanted. In some cases, the optical light source, such as an optical fiber, is reversibly inserted and / or implanted into the VLO. In some cases, the optical light source is removable. In some cases, the brain region having neurons comprising a light-activated polypeptide is stimulated or irradiated using an optical light source comprising one or more optical fibers. In some cases, the optical fiber is coupled to a laser source. The optical fiber can be configured in any suitable manner to direct light emitted from a suitable light source, such as a laser or light-emitting diode (LED) light source, to the brain region.

[0076] Aspects of the present disclosure also include methods of modulating pain in an individual. In some cases, the methods include i) stimulating one or more of thalamocortical projections, thalamic relay neurons, corticofugal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain of the individual with one or more light pulses, wherein neuronal cell bodies in one or more of the VLO and the thalamus of the individual express a light-activated polypeptide, and wherein the stimulation modulates pain in the individual. Modulating pain in the individual can include, for example, modulating neuronal activity in response to noxious stimuli, or modulating neuronal activity associated with aversive or painful sensations in the orbitofrontal cortex of the brain.

[0077] In some cases, stimulating one or more of thalamocortical projections, thalamic relay neurons, corticofugal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with a first set of light pulses inhibits neuronal activity in response to noxious stimuli. Noxious stimuli can include chemical, thermal, and / or mechanical stimuli. In some cases, noxious stimuli include, for example, heat, one or more chemicals, and radiation.

[0078] In some cases, stimulating one or more of thalamocortical projections, thalamic relay neurons, corticopetal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with a first set of light pulses inhibits neuronal activity associated with aversive or painful sensation in the orbitofrontal cortex of the brain.

[0079] In some cases, stimulating one or more of thalamocortical projections, thalamic relay neurons, corticopetal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with a second set of light pulses activates neuronal activity associated with aversive or painful sensation in the orbitofrontal cortex of the brain.

[0080] Response to stimulation

[0081] The response of the whole brain to stimulation by different sets of light pulses can be measured by any suitable brain imaging or measurement of neuronal activity protocol, such as fMRI. Comparison of the response of each region of the brain can indicate functional connectivity between neurons stimulated by light stimulation by one or more of the thalamocortical projections, thalamic relay neurons, corticopetal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO and other regions of the brain, such as downstream of their projection sites. In some cases, quantitative changes in light pulses can result in changes in fMRI cerebral blood volume (CBV) signal (e.g., measuring positive or negative CBV response as a function of light pulse frequency).

[0082] In some cases, the methods of the present disclosure include measuring fMRI signal of the whole brain during stimulation of one or more of thalamocortical projections, thalamic relay neurons, corticopetal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain. In some cases, the fMRI signal is measured in ipsilateral regions, including the left hemisphere of the brain, including the medial prefrontal cortex, lateral prefrontal cortex, motor cortex, cingulate cortex, sensory cortex, insular cortex, striatum, and thalamus. In some cases, the methods include measuring fMRI signal in contralateral regions of the brain, including the right hemisphere of the brain, including the medial prefrontal cortex, lateral prefrontal cortex, motor cortex, cingulate cortex, sensory cortex, insular cortex, striatum, and thalamus. In some cases, measuring fMRI signal includes measuring cerebral blood volume.

[0083] In certain embodiments, fMRI can be used to indirectly measure neuronal activity in one or more regions of the brain. For example, fMRI can be used to indirectly measure neuronal activity in different regions of the brain before, during, or after, for example, using fiber optic stimulation or illumination, stimulating or illuminating a first region of the brain with a first set of light pulses and a second set of light pulses having different temporal patterns, where neurons in the first region can generate action potentials induced by the first set and / or the second set of light pulses, or suppress action potentials after the first set and / or the second set of light pulses. In some cases, an increase in neural activity in a brain region induced by a set of light pulses, e.g., the first set of light pulses, as provided herein can be correlated with a measured fMRI signal. In addition, a decrease in neural activity in a brain region caused by a set of light pulses, e.g., the second set of light pulses, as provided herein can also be correlated with a measured fMRI signal. In some cases, a negative measured fMRI signal is correlated with a decrease in neuronal activity in one or more brain regions caused by a set of light pulses. In some cases, a positive measured fMRI signal is correlated with an increase in neuronal activity in one or more brain regions caused by a set of light pulses. In some cases, a negative measured fMRI signal is correlated with a decrease in neuronal activity following stimulation of one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies of the VLO. In some cases, a positive measured fMRI signal is correlated with an increase in neuronal activity following stimulation of one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies of the VLO.

[0084] The response to stimulation measured, for example, by fMRI, can depend on the frequency of the light pulses and / or the set of neurons or brain regions illuminated. For example, the frequency of the light pulses can determine whether the fMRI signal in one or more brain regions is positive or negative. In certain cases, the light pulses are delivered at a frequency that produces a negative measured fMRI signal. In some cases, the light pulses are delivered at a frequency that produces a positive measured fMRI signal. In some cases, stimulating a first brain region with light pulses produces a negative fMRI signal in one or more downstream brain regions, e.g., brain regions that receive input from the first brain region. In some cases, stimulating a first brain region with light pulses produces a positive fMRI signal in one or more downstream brain regions.

[0085] In some cases, the frequency of the light pulses is 5 Hz or higher. In some cases, stimulating the thalamocortical projection with pulses at a frequency of 5 Hz or higher results in a negative measured fMRI signal. In some cases, the negative measured fMRI signal is in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain. In some cases, stimulating the thalamocortical projection with pulses at a frequency of 5 Hz or higher results in a negative measured fMRI in the contralateral region of the brain. In some cases, the negative measured fMRI signal is associated with a decrease in neuronal activity in the contralateral region of the brain. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 5 Hz or higher suppresses neuronal activity in the ipsilateral thalamus of the brain.

[0086] In some cases, the frequency of the light pulses is 10 Hz or higher. In some cases, stimulating the thalamocortical projection with pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal. In some cases, the negative measured fMRI signal is in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain. In some cases, stimulating the thalamocortical projection with pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal. In some cases, the negative measured fMRI signal is in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain. In some cases, the negative measured fMRI signal is associated with a decrease in neuronal activity in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal in the contralateral region of the brain. In some cases, the negative measured fMRI signal is associated with a decrease in neuronal activity in the contralateral region of the brain. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal in the cortex, contralateral striatum, and contralateral thalamus of the brain.

[0087] In some cases, the frequency of the light pulses is 5 Hz to 20 Hz. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 5 Hz to 20 Hz results in a negative measured fMRI signal in the contralateral region of the brain. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 5 Hz to 20 Hz suppresses neuronal activity in the contralateral region of the brain. In some cases, the contralateral region includes the prefrontal cortex of the brain. In some cases, the negative measured fMRI signal is associated with a decrease in neuronal activity in the contralateral region of the brain. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 5 Hz or higher, 10 Hz or higher, 15 Hz or higher, or 20 Hz suppresses neuronal activity in the contralateral region of the brain.

[0088] In some cases, the frequency of the light pulses is 20 Hz to 40 Hz. In some cases, stimulating the thalamocortical projections with pulses having a frequency of 20 Hz to 40 Hz results in a positive measured fMRI signal. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in a homotopic region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in the homotopic thalamus of the brain. In some cases, stimulating the thalamocortical projections with light pulses having a frequency of 20 Hz or higher, 25 Hz or higher, 30 Hz or higher, 35 Hz or higher, or 40 Hz or higher results in a positive measured fMRI signal in a homotopic region of the brain that is associated with an increase in neuronal activity in the homotopic region of the brain. In some cases, stimulating the thalamocortical projections with light pulses having a frequency of 20 Hz to 40 Hz activates neuronal activity in the homotopic thalamus of the brain. In some cases, stimulating the thalamocortical projections with light pulses having a frequency of 25 Hz or higher results in a negative measured fMRI signal in a heterotopic region of the brain.

[0089] In some cases, the frequency of the light pulses is 40 Hz or higher. In some cases, stimulating the thalamocortical projections with pulses having a frequency of 40 Hz or higher results in a positive measured fMRI signal. In some cases, stimulating the thalamocortical projections with light pulses having a frequency of 40 Hz or higher results in a positive fMRI signal in a homotopic region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in a homotopic region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in the homotopic thalamus of the brain. In some cases, stimulating the thalamocortical projections with light pulses having a frequency of 40 Hz or higher results in a positive fMRI signal in the homotopic thalamus, homotopic striatum, and homotopic cortex of the brain.

[0090] In some cases, the frequency of the light pulses is 5 Hz to 40 Hz. In some cases, stimulating cell bodies in the VLO with light pulses having a frequency of 5 Hz to 40 Hz results in a positive measured fMRI signal in a homotopic region of the brain. In some cases, stimulating cell bodies in the VLO with light pulses having a frequency of 5 Hz or higher, 10 Hz or higher, 15 Hz or higher, 20 Hz or higher, 25 Hz or higher, 30 Hz or higher, 35 Hz or higher, or 40 Hz or higher results in a positive measured fMRI signal in a homotopic region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in the homotopic thalamus of the brain. In some cases, stimulating cell bodies with light pulses having a frequency of 40 Hz or higher increases neuronal activity in the homotopic thalamus of the brain.

[0091] In some cases, the frequency of the light pulses is 5 Hz to 40 Hz. In some cases, stimulating the cell bodies of the centromedian nucleus of the thalamus results in a positive measured fMRI signal in the ipsilateral thalamus of the brain. In some cases, stimulating the cell bodies of the centromedian nucleus of the thalamus with light pulses at a frequency of 5 Hz or higher, 10 Hz or higher, 15 Hz or higher, 20 Hz or higher, 25 Hz or higher, 30 Hz or higher, 35 Hz or higher, or 40 Hz or higher results in a positive measured fMRI signal in the ipsilateral region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in the ipsilateral thalamus of the brain.

[0092] In some cases, the frequency of the light pulses is 5 Hz to 10 Hz. In some cases, stimulating the thalamic cortical projections with light pulses at a frequency of 5 Hz to 10 Hz decreases brain activity in the ipsilateral region of the brain. In some cases, stimulating with light pulses at a frequency of 5 Hz to 10 Hz suppresses neuronal activity in the ipsilateral thalamus of the brain.

[0093] Aspects of the method include performing electrophysiological recordings to detect the frequency of neuronal firing in one or more brain regions associated with the measured fMRI signal. In some cases, the electrophysiological recordings detect neuronal activity associated with a positive or negative fMRI signal. In some cases, a positive fMRI signal can reflect an increase in the frequency of neuronal firing. In some cases, a negative fMRI signal can reflect a decrease in the frequency of neuronal firing. In some cases, the electrophysiological recordings are performed at the stimulation site. In some cases, the electrophysiological recordings are performed at a site downstream of one or more brain regions subjected to stimulation in the brain. In some cases, the electrophysiological recordings are performed at a site associated with the fMRI signal, e.g., a positive or negative fMRI signal. In some cases, the electrophysiological recordings are used to detect the firing frequency of one or more brain regions during or after stimulation at one or more frequencies. In some cases, the electrophysiological recordings are performed in the VLO. In some cases, the electrophysiological recordings are performed in the ipsilateral region of the brain. In some cases, the electrophysiological recordings are performed in the contralateral region of the brain. In some cases, the electrophysiological recordings are performed in the thalamic reticular nucleus. In some cases, the electrophysiological recordings are performed in the contralateral reticular nucleus. In some cases, the increase or decrease in the frequency of neuronal firing in one or more brain regions can be modulated by varying the frequency of the light pulses used for stimulation. The electrophysiology can include single electrode, multi-electrode, and / or field potential recordings.

[0094] In some cases, the method comprises electrophysiological recording in one or more brain regions comprising the ipsilateral VLO of the brain. In some cases, a positive measured fMRI signal is associated with an increase in neuronal firing frequency recorded in the ipsilateral VLO. In some cases, a negative measured fMRI signal is associated with a decrease in neuronal firing frequency recorded in the ipsilateral VLO. In some cases, the one or more brain regions are the ipsilateral motor cortex. In some cases, stimulation with light pulses at a frequency of 10 Hz or higher results in a decrease in neuronal firing frequency in the ipsilateral motor cortex. In some cases, stimulation with light pulses at a frequency of 40 Hz or higher results in an increase in neuronal firing frequency in the ipsilateral motor cortex.

[0095] In some cases, the method comprises electrophysiological recording in one or more brain regions comprising the contralateral VLO of the brain. In some cases, a negative measured fMRI signal is associated with a decrease in neuronal firing frequency in the contralateral VLO. In some cases, stimulation of the contralateral VLO, a negative measured fMRI signal is associated with a decrease in neuronal firing frequency in the contralateral VLO. In some cases, stimulation of the contralateral VLO with light pulses at a frequency of 10 Hz or higher results in a decrease in neuronal firing frequency in the contralateral VLO. In some cases, stimulation with light pulses at a frequency of 40 Hz or higher results in an increase in neuronal firing frequency in the contralateral VLO.

[0096] Systems

[0097] Aspects of the disclosure include systems for performing the methods of the disclosure to modulate the temporal patterns of neuronal activity in the brain of an individual. In some cases, the system modulates neuronal activity in one or more brain regions or the whole brain. In some cases, the system modulates the spatial extent of neuronal activation or inhibition in one or more brain regions or the whole brain. In some cases, the system modulates the inhibitory or excitatory effects of input from one or more brain regions on one or more downstream brain regions. Aspects of the system can include subsystems or devices for visualizing and / or measuring the temporal and / or spatial patterns of neuronal activity in one or more brain regions or the whole brain in response to stimulation of one or more brain regions. The systems of the disclosure can use any number of combinations of suitable subsystems, devices, or apparatuses for stimulating neurons and measuring neuronal activity as needed to determine functional connections between different brain regions. Suitable subsystems, devices, or apparatuses include those for performing electrophysiological recordings; optogenetic modulation of neural activity; electroencephalogram (EEG) recordings; functional imaging. In some cases, the whole brain comprises ipsilateral and contralateral regions of the brain.

[0098] The brain regions of interest in the present system (for optogenetic stimulation and / or measurement of neural activity) can vary and can be any suitable region. In certain embodiments, the brain region is an anatomically and / or functionally defined region of the brain. For example, the first region of the brain illuminated by the light pulses and the second region of the brain as described herein can be anatomically distinct regions of the brain. In some cases where the brain is a mammalian brain, the brain region of interest is selected from at least a portion of the thalamus (including the centromedial thalamus), the sensory cortex (including the somatosensory cortex), the zona incerta (ZI), the ventral tegmental area (VTA), the prefrontal cortex (PFC), the nucleus accumbens (NAc), the amygdala (BLA), the substantia nigra, the ventral pallidum, the pallidum, the dorsal striatum, the ventral striatum, the subthalamic nucleus, the hippocampus, the dentate gyrus, the cingulate, the entorhinal cortex, the olfactory cortex, the primary motor cortex, and the cerebellum. In some cases, the different brain regions (e.g., the first and second brain regions) are separated by at least one or more, e.g., 2 or more, 3 or more, 4 or more, 5 or more, including 7 or more synapse connections, and are separated by at least 15 or less, e.g., 12 or less, 10 or less, 8 or less, including 6 or less synapse connections. In some embodiments, the different brain regions are separated by at least 1 to 15 synapse connections, e.g., 1 to 12 synapse connections, 1 to 10 synapse connections, 2 to 8 synapse connections, including 3 to 6 synapse connections.

[0099] The neurons of interest present in the brain region can be any suitable type of neuron. In some cases, the neurons are inhibitory neurons or excitatory neurons. In some cases, the neurons are sensory neurons, interneurons, or motor neurons. In some cases, the neurons are, but are not limited to, dopaminergic, cholinergic, GABAergic, glutamatergic, or peptidergic neurons.

[0100] In some cases, the system of the present disclosure includes a light source for stimulating the VLO of the brain. In some cases, the thalamocortical projections of the brain are stimulated. In some cases, the thalamic relay neurons of the brain are stimulated. In certain cases, the cortical projection neurons of the brain are stimulated. In some cases, the cell bodies in the centrolateral subnucleus of the thalamus of the brain are stimulated. In some cases, the cell bodies in the VLO of the brain are stimulated. In some cases, the stimulation of the VLO of the brain results in a measured fMRI signal that is positive at the VLO of the brain.

[0101] In embodiments of the method of implementation, the system of the present disclosure can include, for example, i) a light source configured to stimulate one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain of the individual with pulses of light, wherein a light-responsive opsin polypeptide is expressed in cell bodies in one or more of the VLO and the thalamus of the brain; ii) an fMRI device configured to scan the whole brain during the stimulation to produce an fMRI signal; wherein a positive measured fMRI signal correlates with an increase in neuronal activity following the stimulation, wherein a negative measured fMRI signal correlates with a decrease in neuronal activity following the stimulation. Embodiments of the system can also include an electrophysiological recording device to record and detect the frequency of neuronal firing in one or more brain regions correlated with the measured fMRI signal.

[0102] As described above, aspects of the present disclosure include systems to modulate the temporal pattern of neuronal activity in the brain of an individual using a combination of optogenetic stimulation of defined neuronal groups in one or more of the VLO and the thalamus of the individual, and an fMRI device to measure the whole-brain level of response by scanning the brain with fMRI to modulate neuronal activity following the stimulation. Thus, neurons in the VLO and / or the thalamus can be modified to contain a light-activated polypeptide, such as a light-activated ion channel, wherein the light-activated polypeptide is configured to modulate the activity of, for example, depolarize or hyperpolarize neurons following stimulation of one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with light of an appropriate wavelength, illumination volume, and intensity. In some cases, neurons of the thalamus express a light-activated polypeptide. In some cases, neurons of the centrolateral nucleus of the thalamus express a light-activated polypeptide. In some cases, neurons of the VLO express a light-activated polypeptide. In some cases, the VLO neurons expressing a light-activated polypeptide are layer I and / or layer III neurons of the VLO. In some cases, the light-activated polypeptide expressed in layer I and / or layer III neurons of the VLO is from a neuron located in the centrolateral nucleus of the thalamus. For example, the centrolateral nucleus neurons expressing a light-activated polypeptide send projections to the VLO. In some cases, the light-activated polypeptide is a depolarizing light-activated polypeptide. In some cases, the light-activated polypeptide is a hyperpolarizing light-activated polypeptide. In some embodiments, neurons in the centrolateral nucleus are modulated by stimulating cell bodies in the centrolateral nucleus. In some embodiments, neurons in the centrolateral nucleus are modulated by stimulating cell bodies in the projections in the VLO.

[0103] In some cases, neurons of the VLO and / or thalamus are genetically modified, for example by viral infection of a DNA construct containing a nucleotide sequence encoding a light-activated polypeptide and any other appropriate regulatory elements, to express a light-activated polypeptide. As further described herein, any suitable light-activated polypeptide can be used. In some cases, the methods of the present disclosure include a first light-activated polypeptide and a second light-activated polypeptide. In some cases, the first light-activated polypeptide is a depolarizing light-activated polypeptide. In some cases, the second light-activated polypeptide is a hyperpolarizing light-activated polypeptide. In some cases, the methods of the present disclosure include administering the first and second light-activated polypeptides in the same region of the brain. In some cases, the methods of the present disclosure include administering the first and second light-activated polypeptides in different regions of the brain. Suitable light-activated polypeptides are described in U.S. Patent Publication No. 2018 / 0360343 Al, which is hereby incorporated by reference in its entirety.

[0104] The system can include an optical light source. The optical light source can be operably coupled to an illumination unit, which includes one or more light sources, such as light emitting diodes (LEDs) and / or laser sources, which can be configured to emit light at suitable wavelengths. Having multiple light sources can allow a user to control the illumination pattern, such as the timing of light pulses, of each light source independently of one another. The illumination unit can also include any other suitable optical components to direct, focus, and otherwise control the light produced by the light sources. Suitable optical components include, but are not limited to, lenses, barrel lenses, collimators, dichroic mirrors, filters, gratings, and the like. Thus, in certain embodiments, the illumination unit can be configured to project a light stimulus comprising light pulses of a number of wavelengths. The controller can be in communication with the illumination unit to control the timing, duration, and / or wavelength of the light pulses produced by the illumination unit. The system can also include a power source.

[0105] The light source of the system of the present disclosure can include any suitable light source. In some cases, the light source is an LED, an array of LEDs, or a laser. The light source can emit light having a wavelength in the infrared range, the near infrared range, the visible range, and / or the ultraviolet range. The light source can emit light having a wavelength of about 350 nm or greater, such as about 380 nm or greater, about 410 nm or greater, about 440 nm or greater, about 470 nm or greater, about 500 nm or greater, about 560 nm or greater, about 594 nm or greater, about 600 nm or greater, about 620 nm or greater, about 650 nm or greater, about 680 nm or greater, about 700 nm or greater, about 750 nm or greater, about 800 nm or greater, including about 900 nm or greater, and can emit light having a wavelength of about 2000 nm or less, such as about 1500 nm or less, 1000 nm or less, 800 nm or less, 700 nm or less, 650 nm or less, including 620 nm or less, or 600 nm or less. In some cases, the light source can emit light having a wavelength of about 350 nm to about 2000 nm, such as about 410 nm to about 2000 nm, about 440 nm to about 1000 nm, about 440 nm to about 800 nm, including about 440 nm to about 620 nm. The light source can be configured to produce a continuous wave, quasi-continuous wave, or pulsed wave light beam. In certain embodiments, the laser light source is a gas laser, a solid-state laser, a dye laser, a semiconductor laser (e.g., diode laser), or a fiber laser.

[0106] The number of wavelengths produced by the light source can be any suitable number of wavelengths. In some cases, the light source produces light having 1 or more than 1 different wavelength, such as 2 or more than 2, 3 or more than 3, including 4 or more than 4, or 5 or more than 5, or 6 or more than 6, or 7 or more than 7, or 8 or more than 8, or 9 or more than 9, or 10 or more than 10 different wavelengths, as well as producing light having 10 or fewer different wavelengths, such as 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, including 5 or fewer different wavelengths. In some embodiments, the light source produces light having 1 to 10 different wavelengths, such as 1 to 8, 2 to 6, 2 to 5, including 2 to 4 different wavelengths.

[0107] In some cases, the system of the present disclosure includes an optical light source that can be reversibly inserted into the brain of an individual, for example, into the VLO of the individual. In some cases, the optical light source is implanted. In some cases, the optical light source is removable. In some cases, a brain region having neurons comprising a light-activated polypeptide is stimulated or illuminated using an optical light source comprising one or more optical fibers. In some cases, the optical fiber is coupled with a laser source. The optical fiber can be configured in any suitable manner to direct light emitted from a suitable light source, for example, a laser or light-emitting diode (LED) light source, to a brain region.

[0108] In some cases, the optical light source can be reversibly inserted into one or more regions of the brain of an individual. In some cases, the optical light source can be reversibly inserted into the VLO of an individual. In certain embodiments, the optical light source can be implanted into a region of the brain. In some cases, the optical light source is configured to deliver light to a target tissue structure after being implanted at a location proximal to the target tissue structure. In certain embodiments, the optical light source can be implanted at a dorsal location in the VLO of the brain.

[0109] In some cases, the optical light source is an optical fiber. The optical fiber can be any suitable optical fiber. In some cases, the optical fiber is a multi-mode optical fiber. In some cases, the multi-mode optical fiber supports more than one mode of propagation. For example, the multi-mode optical fiber can be configured to carry light in a range of wavelengths, where each wavelength of light propagates at a different speed. The optical fiber can include a core of defined core diameter, where light from the light source passes through the core. The optical fiber can have any suitable core diameter. In some cases, the core diameter of the optical fiber is 10 pm or greater than 10 pm, for example, 20 pm or greater than 20 pm, 30 pm or greater than 30 pm, 40 pm or greater than 40 pm, 50 pm or greater than 50 pm, 60 pm or greater than 60 pm, including 80 pm or greater than 80 pm, and 1000 pm or less than 1000 pm, for example, 500 pm or less than 500 pm, 200 pm or less than 200 pm, 100 pm or less than 100 pm, including 70 pm or less than 70 pm. In some embodiments, the core diameter of the optical fiber is 10 pm to 1000 pm, for example, 20 pm to 500 pm, 30 pm to 200 pm, including 40 pm to 100 pm.

[0110] In some cases, the system includes multiple optical light sources, e.g., multiple optical fibers. In some cases, the multiple optical fibers can each be reversibly inserted into different brain regions. In some cases, the multiple optical fibers can each be implanted into different brain regions. Each optical fiber can deliver light pulses having the same or different parameters, e.g., frequency, wavelength, pulse width, intensity, etc. The number of optical fibers used in the present system can vary and can be any suitable number. In some cases, the number of optical fibers used to excite and image a targeted tissue, e.g., different regions of the brain, is 1 or more than 1, e.g., 2 or more than 2, 3 or more than 3, 4 or more than 4, 5 or more than 5, 6 or more than 6, 7 or more than 7, including 10 or more than 10, and 100 or less than 100, e.g., 80 or less than 80, 60 or less than 60, 40 or less than 40, 20 or less than 20, 15 or less than 15, 10 or less than 10, 8 or less than 8, 7 or less than 7, 6 or less than 6, including 5 or less than 5. In certain embodiments, the number of optical fibers is between 1 and 100, e.g., between 2 and 60, between 3 and 40, between 4 and 20, including between 4 and 10.

[0111] In certain cases, a cladding surrounds at least a portion of the optical fiber core. For example, the cladding can substantially surround the entire outer circumferential surface of the optical fiber. In some cases, the cladding is not present at the end of the optical fiber, e.g., the end of the optical fiber that receives light from the light source, and the opposite end of the optical fiber that transmits light to neurons of the targeted region of the brain. The cladding can be any suitable type of cladding. In some cases, the cladding has a lower refractive index than the core of the optical fiber. Suitable materials for the cladding include, but are not limited to, plastics, resins, etc., and combinations thereof.

[0112] In some cases, the optical fiber includes an outer coating. The outer coating can be disposed on the surface of the cladding. The coating can substantially surround the entire outer circumferential surface of the optical fiber. In some cases, the coating is not present at the end of the optical fiber, e.g., the end of the optical fiber that receives light from the light source, and the opposite end of the optical fiber that transmits light to neurons of the targeted region of the brain. The coating can be a biocompatible coating. A biocompatible coating includes a coating that does not significantly react with tissues, fluids, or other substances present in the subject into which the optical fiber is inserted. In some cases, the biocompatible coating is composed of a material that is inert (i.e., substantially non-reactive) with respect to the surrounding environment in which the optical fiber is used.

[0113] An optical fiber tip implanted or reversibly inserted into a target region of the brain can have any suitable configuration suitable for illuminating a brain region with light stimulation delivered through the optical fiber. In some cases, the optical fiber is removably inserted and / or implanted into the VLO. In some cases, the optical fiber includes an attachment device located at or near the distal end of the optical fiber, where the distal end of the optical fiber corresponds to the tip inserted into the subject. In some cases, the attachment device is configured to connect to the optical fiber and facilitate attachment of the optical fiber to the subject, such as the skull of the subject. Any suitable attachment device can be used. In some cases, the attachment device includes a ferrule, such as a metal, ceramic, or plastic ferrule. The ferrule can have any suitable size to hold and attach the optical fiber. In some cases, the ferrule has a diameter of 0.5 mm to 3 mm, such as 0.75 mm to 2.5 mm, or 1 mm to 2 mm.

[0114] In certain embodiments, the methods of the present disclosure can be performed using any suitable electronic components to control and / or coordinate various optical components for illuminating a brain region. The optical components (e.g., light sources, optical fibers, lenses, objectives, back mirrors, etc.) can be controlled by a controller, such as to coordinate the light sources to illuminate a brain region with light pulses. The controller can include a driver for the light sources to control one or more parameters related to the light pulses, such as but not limited to the frequency, pulse width, duty cycle, wavelength, intensity, etc. of the light pulses. The controller can communicate with components of the light sources (e.g., collimators, gratings, filter wheels, movable mirrors, lenses, etc.).

[0115] A computing unit (e.g., a computer) can be used in the methods and systems of the present disclosure to control and / or coordinate the light stimulation by one or more controllers, and to analyze data from fMRI scans of a brain region. The computing unit can include any suitable components to analyze the measured fMRI images. Thus, the computing unit can include one or more of: a processor; a non-transitory computer-readable memory, such as a computer-readable medium; an input device, such as a keyboard, mouse, touchscreen, etc.; an output device, such as a monitor, screen, speaker, etc.; a network interface, such as a wired or wireless network interface; and the like.

[0116] Optical light sources for activating light-activated polypeptides can include light pulses characterized by, for example, frequency, pulse width, duty cycle, wavelength, intensity, etc. In some cases, the light stimulation includes two or more groups of different light pulses, where each group of light pulses is characterized by a different temporal pattern of light pulses. The temporal pattern can be characterized by any suitable parameter, including but not limited to frequency, period (i.e., total duration of the light stimulation), pulse width, duty cycle, etc.

[0117] Changes in the characteristics of the group of light pulses can be reflected in differences in the activity of the illuminated neuron. In some cases, an increase in the frequency of the light pulses can result in an increase in the firing frequency of action potentials in the illuminated neuron when the neuron is depolarized by activation of the light-activatable polypeptide. In some embodiments, the firing frequency of action potentials in the illuminated neuron is quantitatively proportional to the increase in the frequency of the light pulses. In some cases, a linear increase in the frequency of the light pulses can result in a linear increase or a non-linear monotonic increase in the firing frequency of action potentials in the illuminated neuron. In some cases, the stimulation can manifest as a downregulation of the activity of the neuron, e.g., hyperpolarization of the neuron. In some cases, an increase in the frequency of the light pulses can result in a decrease in the firing frequency of action potentials in the illuminated neuron when the neuron is hyperpolarized by activation of the light-activatable polypeptide.

[0118] In some cases, the optical stimulation comprises one or more than one, two or more than two, three or more than three, four or more than four, five or more than five, six or more than six, seven or more than seven, eight or more than eight, nine or more than nine, or ten or more than ten groups of light pulses, where the groups of light pulses are characterized by having different values of a parameter, e.g., different frequencies of the light pulses. In cases where the groups of light pulses have different frequencies, the duty cycles can be the same, or can be different. In some cases, the groups of light pulses of different frequencies have the same pulse width. In other cases, the groups of light pulses of different frequencies have different pulse widths.

[0119] The group of light pulses can have any suitable frequency. In some cases, the group of light pulses comprises a single light pulse that lasts for the entire duration of the light stimulus. In some cases, the group of light pulses has a frequency of 0.1 Hz or higher, such as 0.5 Hz or higher, 1 Hz or higher, 5 Hz or higher, 10 Hz or higher, 20 Hz or higher, 30 Hz or higher, 40 Hz or higher, including 50 Hz or higher, or 60 Hz or higher, or 70 Hz or higher, or 80 Hz or higher, or 90 Hz or higher, or 100 Hz or higher, and a frequency of 100,000 Hz or lower, such as 10,000 Hz or lower, 1000 Hz or lower, 500 Hz or lower, 400 Hz or lower, 300 Hz or lower, 200 Hz or lower, including 100 Hz or lower. In some cases, the group of light pulses has a frequency of 0.1 Hz to 100,000 Hz, such as 1 Hz to 10,000 Hz, 1 Hz to 1000 Hz, including 5 Hz to 500 Hz, or 10 Hz to 100 Hz. In some embodiments, the light pulses have a frequency of 5 Hz to 40 Hz.

[0120] The light pulses of the present system can have any suitable pulse width. In some cases, the pulse width is 0.1 ms or greater, such as 0.5 ms or greater, 1 ms or greater, 3 ms or greater, 5 ms or greater, 7.5 ms or greater, 10 ms or greater, including 15 ms or greater, or 20 ms or greater, or 25 ms or greater, or 30 ms or greater, or 35 ms or greater, or 40 ms or greater, or 45 ms or greater, or 50 ms or greater, and 500 ms or less, such as 100 ms or less, 90 ms or less, 80 ms or less, 70 ms or less, 60 ms or less, 50 ms or less, 45 ms or less, 40 ms or less, 35 ms or less, 30 ms or less, 25 ms or less, including 20 ms or less. In some embodiments, the pulse width is 0.1 ms to 500 ms, such as 0.5 ms to 100 ms, 1 ms to 80 ms, including 1 ms to 60 ms, or 1 ms to 50 ms, or 1 ms to 30 ms.

[0121] The duty cycle of the pulses of the present system can be any suitable duty cycle. In some cases, the duty cycle is 1% or greater than 1%, such as 5% or greater than 5%, 10% or greater than 10%, 15% or greater than 15%, 20% or greater than 20%, including 25% or greater than 25%, or 30% or greater than 30%, or 35% or greater than 35%, or 40% or greater than 40%, or 45% or greater than 45%, or 50% or greater than 50%, and can be 80% or less than 80%, such as 75% or less than 75%, 70% or less than 70%, 65% or less than 65%, 60% or less than 60%, 65% or less than 65%, 50% or less than 50%, 45% or less than 45%, including 40% or less than 40%, or 35% or less than 35%, or 30% or less than 30%. In certain embodiments, the duty cycle is 1% to 80%, such as 5% to 70%, 5% to 60%, including 10% to 50% or 10% to 40%.

[0122] The average power of the light pulses of the present system, when measured at the tip of the optical fiber that delivers the light pulses to the brain region, can be any suitable power. In some cases, the power is 0.1 mW or greater than 0.1 mW, such as 0.5 mW or greater than 0.5 mW, 1 mW or greater than 1 mW, 1.5 mW or greater than 1.5 mW, including 2 mW or greater than 2 mW, or 2.5 mW or greater than 2.5 mW, or 3 mW or greater than 3 mW, or 3.5 mW or greater than 3.5 mW, or 4 mW or greater than 4 mW, or 4.5 mW or greater than 4.5 mW, or 5 mW or greater than 5 mW, and can be 1000 mW or less than 1000 mW, such as 500 mW or less than 500 mW, 250 mW or less than 250 mW, 100 mW or less than 100 mW, 50 mW or less than 50 mW, 40 mW or less than 40 mW, 30 mW or less than 30 mW, 20 mW or less than 20 mW, 15 mW or less than 15 mW, including 10 mW or less than 10 mW, or 5 mW or less than 5 mW. In some embodiments, the power is 0.1 mW to 1000 mW, such as 0.5 mW to 100 mW, 0.5 mW to 50 mW, 1 mW to 20 mW, including 1 mW to 10 mW or 1 mW to 5 mW.

[0123] The wavelength and intensity of the light pulses of the present system can vary and can depend on the activation wavelength of the light-activated polypeptide, the optical transparency of the brain region, the desired volume of the brain to be illuminated, etc.

[0124] The volume of the brain region illuminated by the light pulses can be any suitable volume. In some cases, the volume of illumination is 0.001 mm 3 or greater than 0.001 mm 3 , such as 0.005 mm 3or greater than 0.005 mm 3 , 0.001 mm 3 or greater than 0.001 mm 3 , 0.005 mm 3 or greater than 0.005 mm 3 , 0.01 mm 3 or greater than 0.01 mm 3 , 0.05 mm 3 or greater than 0.05 mm 3 , including 0.1 mm 3 or greater than 0.1 mm 3 , and 100 mm 3 or less than 100 mm 3 , for example, 50 mm 3 or less than 50 mm 3 , 20 mm 3 or less than 20 mm 3 , 10 mm 3 or less than 10 mm 3 , 5 mm 3 or less than 5 mm 3 , 1 mm 3 or less than 1 mm 3 , including 0.1 mm 3 or less than 0.1 mm 3 . In some cases, the illumination volume is 0.001 mm 3 to 100 mm 3 , for example, 0.005 mm 3 to 20 mm 3 , 0.01 mm 3 to 10 mm 3 , 0.01 mm 3 to 5 mm 3 , including 0.05 mm 3 to 1 mm 3 .

[0125] Aspects of the system include a second light-activated polypeptide expressed in neurons of one or more brain regions of interest. In some cases, the second light-activated polypeptide is administered to the zona incerta (ZI) region of the brain. In some cases, the second light-activated polypeptide is a depolarizing light-activated polypeptide. In some cases, the second light-activated polypeptide is a hyperpolarizing light-activated polypeptide. In some cases, the system of the disclosure includes stimulating the ZI region of the brain with an optical light source, for example, when the second light-activated polypeptide is expressed in neurons of the ZI.

[0126] Responses to different sets of light pulses can be measured by any suitable brain imaging or neuronal activity measurement system for the whole brain, e.g., fMRI, and comparison of responses in each region can indicate functional connectivity between neurons stimulated by light stimulation of one or more of the thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO, and other regions downstream of the thalamic brain regions, e.g., projection sites. In some cases, quantitative changes in light pulses can result in a change in sign of fMRI CBV (e.g., measuring a positive or negative CBV response as a function of light pulse frequency).

[0127] In some cases, systems of the present disclosure include fMRI devices for measuring fMRI signals in the whole brain during stimulation of one or more of the thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO. In some cases, fMRI signals are measured in ipsilateral regions, which include the left hemisphere of the brain, including the medial prefrontal cortex, lateral prefrontal cortex, motor cortex, cingulate cortex, sensory cortex, insular cortex, striatum, and thalamus. In some cases, systems include fMRI devices for measuring fMRI signals in the contralateral regions of the brain, which include the right hemisphere of the brain, including the medial prefrontal cortex, lateral prefrontal cortex, motor cortex, cingulate cortex, sensory cortex, insular cortex, striatum, and thalamus.

[0128] In certain embodiments, an fMRI device can be used to indirectly measure neuronal activity in one or more regions of the brain. For example, fMRI can be used to indirectly measure neuronal activity in different regions of the brain before, during, or after stimulating or illuminating a first region of the brain with a first set of light pulses and a second set of light pulses, e.g., using an optical light source, where neurons in the first region can generate action potentials induced by the first set and / or the second set of light pulses, or suppress action potentials following the first set and / or the second set of light pulses. In some cases, an increase in neuronal activity in a brain region as provided herein induced by a set of light pulses, e.g., the first set of light pulses, can be correlated with a measured fMRI signal. In addition, a decrease in neuronal activity in a brain region as provided herein caused by a set of light pulses, e.g., the second set of light pulses, can also be correlated with a measured fMRI signal. In some cases, a negative measured fMRI signal is correlated with a decrease in neuronal activity in one or more brain regions caused by a set of light pulses. In some cases, a positive measured fMRI signal is correlated with an increase in neuronal activity in one or more brain regions caused by a set of light pulses. In some cases, a negative measured fMRI signal is correlated with a decrease in neuronal activity following stimulation of one or more of thalamocortical projections, thalamic relay neurons, corticofugal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies of the VLO. In some cases, a positive measured fMRI signal is correlated with an increase in neuronal activity following stimulation of one or more of thalamocortical projections, thalamic relay neurons, corticofugal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies of the VLO.

[0129] fMRI can be performed using any suitable method. Suitable methods are described, e.g., in U.S. Patent No. 8,834,546 and U.S. Patent Publication No. 2013 / 0144153 Al, which are hereby incorporated by reference in their entirety. Functional magnetic resonance imaging (fMRI) allows visualization of areas of brain activity with high spatial resolution (millimeters) according to tasks performed by a subject within a scanner. Functional imaging can include fMRI, as well as any functional imaging protocol using genetically encoded indicators (e.g., calcium indicators, voltage indicators, etc.). fMRI can be performed in any suitable static magnetic field (e.g., > 1 Tesla) and any suitable accompanying dynamic spatially varying magnetic field. In some cases, the fMRI signal is representative of CBV in one or more regions of the brain. Suitable fMRI methods and devices are further described, e.g., in Glover. Neurosurg Clin N Am. (2011) 22(2): 133-139 and Chow et al., World J Radiol. (2017) 9(1): 5-9, the disclosures of which are hereby incorporated by reference in their entirety.

[0130] The response to a stimulus, measured, e.g., by fMRI, can depend on the frequency of the light pulses and / or the set of neurons or brain regions to be illuminated. For example, the frequency of the light pulses can determine whether the fMRI signal in one or more brain regions is positive or negative. In some cases, the light pulses are delivered at a frequency that results in a negative measured fMRI signal. In some cases, the light pulses are delivered at a frequency that results in a positive measured fMRI signal. In some cases, stimulating a first brain region with light pulses results in a negative fMRI signal in one or more downstream brain regions, e.g., brain regions that receive input from the first brain region. In some cases, stimulating a first brain region with light pulses results in a positive fMRI signal in one or more downstream brain regions.

[0131] In some cases, the frequency of the light pulses is 5 Hz or higher. In some cases, stimulating the thalamocortical projections with pulses at a frequency of 5 Hz or higher results in a negative measured fMRI signal. In some cases, the negative measured fMRI signal is in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain. In some cases, stimulating the thalamocortical projections with pulses at a frequency of 5 Hz or higher results in a negative measured fMRI in the contralateral region of the brain. In some cases, the negative measured fMRI signal is associated with a decrease in neuronal activity in the contralateral region of the brain. In some cases, stimulating the thalamocortical projections with light pulses at a frequency of 5 Hz or higher suppresses neuronal activity in the ipsilateral thalamus of the brain.

[0132] In some cases, the frequency of the light pulses is 10 Hz or higher. In some cases, stimulating the thalamocortical projections with pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal. In some cases, the negative measured fMRI signal is in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain. In some cases, stimulating the thalamocortical projections with pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal. In some cases, the negative measured fMRI signal is in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain. In some cases, the negative measured fMRI signal is associated with a decrease in neuronal activity in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain. In some cases, stimulating the thalamocortical projections with light pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal in the contralateral region of the brain. In some cases, the negative measured fMRI signal is associated with a decrease in neuronal activity in the contralateral region of the brain. In some cases, stimulating the thalamocortical projections with light pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal in the cerebral cortex, contralateral striatum, and contralateral thalamus.

[0133] In some cases, the frequency of the light pulses is 5 Hz to 20 Hz. In some cases, stimulation of the thalamic cortex projection with light pulses at frequencies of 5 Hz to 20 Hz results in negative measured fMRI signals in the contralateral brain region. In some cases, stimulation of the thalamic cortex projection with light pulses at frequencies of 5 Hz to 20 Hz inhibits neuronal activity in the contralateral brain region. In some cases, the contralateral region includes the prefrontal cortex of the brain. In some cases, negative measured fMRI signals are associated with reduced neuronal activity in the contralateral brain region. In some cases, stimulation of the thalamic cortex projection with light pulses at frequencies ranging from 5 Hz or higher, 10 Hz or higher, 15 Hz or higher, or 20 Hz inhibits neuronal activity in the contralateral brain region.

[0134] In some cases, the frequency of the light pulses is 20 Hz to 40 Hz. In some cases, stimulation of the thalamic cortex projection with pulses at frequencies of 20 Hz to 40 Hz results in positive measured fMRI signals. In some cases, positive measured fMRI signals are associated with increased neuronal activity in the ipsilateral region of the brain. In some cases, positive measured fMRI signals are associated with increased neuronal activity in the ipsilateral thalamus. In some cases, stimulation of the thalamic cortex projection with light pulses at frequencies of 20 Hz or higher, 25 Hz or higher, 30 Hz or higher, 35 Hz or higher, or 40 Hz or higher results in increased neuronal activity in the ipsilateral region of the brain, which is associated with positive measured fMRI signals in the ipsilateral region. In some cases, stimulation of the thalamic cortex projection with light pulses at frequencies of 20 Hz to 40 Hz activates neuronal activity in the ipsilateral thalamus. In some cases, stimulation of the thalamic cortex projection with light pulses in the range of 25 Hz or higher results in negative measured fMRI signals in the contralateral region of the brain.

[0135] In some cases, the frequency of the light pulses is 40 Hz or higher. In some cases, stimulating the thalamocortical projection with pulses at a frequency of 40 Hz or higher results in a positive measured fMRI signal. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 40 Hz or higher results in a positive fMRI signal in the ipsilateral region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in the ipsilateral region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in the ipsilateral thalamus of the brain. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 40 Hz or higher results in a positive fMRI signal in the ipsilateral thalamus, ipsilateral striatum, and ipsilateral cortex of the brain. In some cases, the frequency of the light pulses is 5 Hz to 40 Hz. In some cases, stimulating cell bodies in the VLO with light pulses at a frequency of 5 Hz to 40 Hz results in a positive measured fMRI signal in the ipsilateral region of the brain. In some cases, stimulating cell bodies in the VLO with light pulses having a frequency of 5 Hz or higher, 10 Hz or higher, 15 Hz or higher, 20 Hz or higher, 25 Hz or higher, 30 Hz or higher, 35 Hz or higher, or 40 Hz or higher results in a positive measured fMRI signal in the ipsilateral region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in the ipsilateral thalamus of the brain.

[0136] In some cases, the frequency of the light pulses ranges from 5 Hz to 40 Hz. In some cases, stimulating cell bodies of the centrolateral nucleus of the thalamus results in a positive measured fMRI signal in the ipsilateral thalamus of the brain. In some cases, stimulating cell bodies of the centrolateral nucleus of the thalamus with light pulses at a frequency ranging from 5 Hz or greater, 10 Hz or greater, 15 Hz or greater, 20 Hz or greater, 25 Hz or greater, 30 Hz or greater, 35 Hz or greater, or 40 Hz or greater results in a positive measured fMRI signal in the ipsilateral region of the brain. In some cases, the positive measured fMRI signal is associated with an increase in neuronal activity in the ipsilateral thalamus of the brain.

[0137] In some cases, the frequency of the light pulses is 5 Hz to 10 Hz. In some cases, stimulating the thalamocortical projection with light pulses at a frequency of 5 Hz to 10 Hz decreases brain activity in the ipsilateral region of the brain. In some cases, stimulating with light pulses at a frequency of 5 Hz to 10 Hz suppresses neuronal activity in the ipsilateral thalamus of the brain.

[0138] Aspects of the system include electrophysiological recording devices to record and detect the firing rate of neurons in one or more brain regions that correlate with measured fMRI signals. Electrophysiology can include single electrode, multi-electrode, and / or field potential recordings. In some cases, the one or more brain regions include the ipsilateral VLO of the brain. In some cases, a positive measured fMRI signal correlates with an increase in the firing rate of neurons recorded in the ipsilateral VLO. In some cases, a negative measured fMRI signal correlates with a decrease in the firing rate of neurons recorded in the ipsilateral VLO. In some cases, light pulse stimulation at 10 Hz or greater than 10 Hz frequency results in a decrease in the firing rate of neurons in the ipsilateral motor cortex. In some cases, light pulse stimulation at 40 Hz or greater than 40 Hz frequency results in an increase in the firing rate of neurons in the ipsilateral motor cortex.

[0139] In some cases, the one or more brain regions include the contralateral VLO of the brain. In some cases, a negative measured fMRI signal correlates with a decrease in the firing rate of neurons in the contralateral VLO. In some cases, stimulating the contralateral VLO, a negative measured fMRI signal correlates with a decrease in the firing rate of neurons in the contralateral VLO. In some cases, light pulse stimulation at 10 Hz or greater than 10 Hz frequency of the contralateral VLO results in a decrease in the firing rate of neurons in the contralateral VLO. In some cases, stimulation with light pulses having a frequency of 40 Hz or greater than 40 Hz results in an increase in the firing rate of neurons in the contralateral VLO.

[0140] Electrophysiological recordings can be performed using any suitable protocol and apparatus. In some cases, electrophysiological recordings include intracellular recordings. In certain cases, performing recordings includes inserting a microelectrode inside a neuron. In some cases, performing recordings includes placing a microelectrode on the surface of the cell membrane of a neuron. In some cases, performing recordings uses methods and equipment for performing patch-clamp electrophysiology and any variants, including, for example, whole-cell, inside-out, outside-out, perforated, loose patch-clamp methods. In some cases, recordings are performed using a voltage-clamp method. In some cases, recordings are performed using a current-clamp method. In some cases, recordings include extracellular recordings that can detect changes in ion concentrations in extracellular fluid or in a group of neurons. Electrophysiology can include single electrode, multiple electrode, and / or field potential recordings. In some cases, the electrode is a glass micropipette. In some cases, recordings are performed using multiple electrodes, such as a microelectrode array. Exemplary methods and equipment for performing electrophysiological recordings are described, for example, in U.S. Patent Publication Nos. 2013 / 0225963; 2017 / 0138926; and 2005 / 0231186, the disclosures of which are incorporated by reference herein in their entireties. Exemplary electrode techniques for neuronal recordings are described in Hong et al., Nat Rev Neurosci (2019) 20(6):330-345, the disclosure of which is incorporated by reference herein in its entirety. Light-induced modulation of neural activity can include any suitable optogenetic method, as described herein. In some cases, electrophysiological recordings include single unit recordings.

[0141] Aspects of the present disclosure also include systems for modulating pain in an individual. In some cases, the system comprises i) an optical light source configured to stimulate one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain of the individual with one or more light pulses, wherein neuronal cell bodies in one or more of the VLO and the thalamus of the individual express a light-activated polypeptide, and wherein the stimulation modulates pain in the individual.

[0142] In some cases, stimulating one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with a first set of light pulses suppresses neuronal activity that responds to noxious stimuli.

[0143] In some cases, stimulating one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with a first set of light pulses suppresses neuronal activity associated with aversive or painful sensations in the orbitofrontal cortex of the brain.

[0144] In some cases, stimulating one or more of thalamocortical projections in the brain, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO with the second set of light pulses activates neuronal activity associated with aversive or painful sensations in the orbitofrontal cortex of the brain.

[0145] In some cases, stimulating one or more of thalamocortical projections in the brain, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO with the second set of light pulses activates neuronal activity associated with aversive or painful sensations in the orbitofrontal cortex of the brain.

[0146] Light-activated polypeptides used in methods and systems

[0147] As described above, aspects of the present methods and systems include a plurality of brain regions containing, for example, neurons that express light-activated polypeptides. The light-activated polypeptides can be light-activated ion channels or light-activated ion pumps. When the polypeptides are illuminated with light of an activating wavelength, the light-activated ion channel polypeptides are adapted to allow one or more ions to pass through the plasma membrane of the neuron. The light-activated proteins can feature ion pump proteins that facilitate the passage of small numbers of ions through the plasma membrane with the aid of photons of light, or ion channel proteins that allow ion flow to pass freely through the plasma membrane when the channel is open. In some embodiments, the light-activated polypeptides depolarize the neuron when activated by light of an activating wavelength. Suitable light-activated polypeptides for depolarization are not limited to those shown in FIG. 15 In some embodiments, the light-activated polypeptides hyperpolarize the neuron when activated by light of an activating wavelength. Suitable light-activated polypeptides for hyperpolarization are not limited to those shown in FIG. 16

[0148] In some embodiments, the light-activated polypeptides are activated by blue light. In some embodiments, the light-activated polypeptides are activated by green light. In some embodiments, the light-activated polypeptides are activated by yellow light. In some embodiments, the light-activated polypeptides are activated by orange light. In some embodiments, the light-activated polypeptides are activated by red light.

[0149] In some embodiments, the light-activated polypeptides expressed in the cells can be fused to one or more amino acid sequence motifs selected from the group consisting of a signal peptide, an endoplasmic reticulum (ER) export signal, a membrane trafficking signal, and / or an N-terminal Golgi export signal. The one or more amino acid sequence motifs that enhance trafficking of the light-activated protein to the plasma membrane of a mammalian cell can be fused to the N-terminus, the C-terminus, or both the N- and C-termini of the light-activated polypeptide. In some cases, the one or more amino acid sequence motifs that enhance trafficking of the light-activated polypeptide to the plasma membrane of a mammalian cell are fused internally within the light-activated polypeptide. Optionally, the light-activated polypeptide and the one or more amino acid sequence motifs can be separated by a linker.​

[0150] In some embodiments, the light-activated polypeptide can be modified by the addition of a trafficking signal (ts) that enhances transport of the protein to the plasma membrane of a cell. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). A trafficking sequence suitable for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)).

[0151] The trafficking sequence can have a length of about 10 amino acids to about 50 amino acids, for example, about 10 amino acids to about 20 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 40 amino acids, or about 40 amino acids to about 50 amino acids.

[0152] ER export sequences suitable for use with the light-activated polypeptides include, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53); VLGSL (SEQ ID NO: 54); and the like); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); and the like. The ER export sequence can have a length of about 5 amino acids to about 25 amino acids, for example, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, or about 20 amino acids to about 25 amino acids.

[0153] Suitable signal sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, one of: 1) a signal peptide of hChR2 (e.g., MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO: 59)); 2) a signal peptide of the β2 subunit of the neuronal nicotinic acetylcholine receptor (e.g., MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO: 60)); 3) a signal sequence of the nicotinic acetylcholine receptor (e.g., MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO: 64)); 4) a signal sequence of the nicotinic acetylcholine receptor (e.g., MRGTPLLLVVSLFSLLQD (SEQ ID NO: 61)).

[0154] The signal sequence can have a length of about 10 amino acids to about 50 amino acids, for example, about 10 amino acids to about 20 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 40 amino acids, or about 40 amino acids to about 50 amino acids.

[0155] In some embodiments, the signal peptide sequence in the protein can be deleted or replaced by a signal peptide sequence from a different protein.

[0156] Examples of light-activated polypeptides are described, for example, in PCT Application No. PCT / US2011 / 028893, which is hereby incorporated by reference in its entirety. Representative light-activated polypeptides useful in the present disclosure are further described below.

[0157] Depolarizing light-activated polypeptide

[0158] ChR

[0159] In some aspects, the depolarizing light-activated polypeptide is derived from Chlamydomonas reinhardtii, wherein the polypeptide is capable of transporting cations across a cell membrane when the cell is illuminated by light. In another embodiment, the light-activated polypeptide comprises an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 1. Light used to activate the light-activated cation channel protein derived from Chlamydomonas reinhardtii can have a wavelength of about 460 nm to about 495 nm or can have a wavelength of about 480 nm. Further, light pulses at a temporal frequency of about 100 Hz can be used to activate the light-activated protein. In some embodiments, activation of the light-activated cation channel derived from Chlamydomonas reinhardtii with light pulses having a temporal frequency of about 100 Hz can result in depolarization of neurons expressing the light-activated cation channel. The light-activated cation channel protein can additionally comprise substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the light-activated cation channel protein to modulate the plasma membrane polarization state of a cell. Further, the light-activated cation channel protein can comprise one or more than one conservative amino acid substitution and / or one or more than one non-conservative amino acid substitution. The light-activated proton pump protein comprising substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane.

[0160] In some embodiments, the light-activated cation channel comprises a T159C substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light- activated cation channel comprises a L132C substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light-activated cation channel comprises an E123T substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light-activated cation channel comprises an E123A substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light-activated cation channel comprises a T159C substitution and an E123T substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light-activated cation channel comprises a T159C substitution and an E123A substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light-activated cation channel comprises a T159C substitution, a L132C substitution, and an E123T substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light-activated cation channel comprises a T159C substitution, a L132C substitution, and an E123A substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light-activated cation channel comprises a L132C substitution and an E123T substitution to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the light-activated cation channel comprises a L132C substitution and an E123A substitution to the amino acid sequence set forth in SEQ ID NO: 1.

[0161] In some embodiments, the ChR2 protein comprises at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the neuron to the plasma membrane selected from a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the ChR2 protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the ChR2 protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the ChR2 protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the ChR2 protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any one of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also comprise a fluorescent protein such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0162] In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0163] In certain embodiments, the ChR2 protein can have an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 2.

[0164] In other embodiments, the light-activated polypeptide is a step function opsin (SFO) or a stabilized step function opsin (SSFO), which can have specific amino acid substitutions at key positions in the retinal binding pocket of the protein. In some embodiments, the SFO protein can have a mutation at amino acid residue C128 of SEQ ID NO: 1. In other embodiments, the SFO protein has a C128A mutation in SEQ ID NO: 1. In other embodiments, the SFO protein has a C128S mutation in SEQ ID NO: 1. In another embodiment, the SFO protein has a C128T mutation in SEQ ID NO: 1.

[0165] In some embodiments, the SSFO protein can have a mutation at amino acid residue D156 of SEQ ID NO: 1. In other embodiments, the SSFO protein can have mutations at both amino acid residues C128 and D156 of SEQ ID NO: 1. In one embodiment, the SSFO protein has a C128S and D156A mutation in SEQ ID NO: 1. In another embodiment, the SSFO protein can comprise an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 1; and includes an alanine, serine, or threonine at amino acid 128; and includes an alanine at amino acid 156. In another embodiment, the SSFO protein can comprise a C128T mutation in SEQ ID NO: 1. In some embodiments, the SSFO protein includes a C128T and D156A mutation in SEQ ID NO: 1.

[0166] In some embodiments, the SFO or SSFO proteins provided herein are capable of mediating a depolarizing current in a cell when the cell is illuminated with blue light. In other embodiments, the light can have a wavelength of about 445 nm. Further, in some embodiments, due to the extended stability of SFO and SSFO photocurrents, the light can be delivered as a single light pulse or as spaced light pulses. In some embodiments, activation of the SFO or SSFO proteins with a single light pulse or spaced light pulses can result in the depolarization of neurons expressing the SFO or SSFO proteins. In some embodiments, the disclosed step-function opsin and stable step-function opsin each can have specific properties and characteristics for depolarizing the membrane of a neuronal cell in response to light.

[0167] Further disclosures related to SFO or SSFO proteins can be found in International Patent Application Publication No. WO 2010 / 056970, the disclosure of which is hereby incorporated by reference in its entirety.

[0168] In some cases, the ChR2-based SFO or SSFO comprises a membrane trafficking signal and / or an ER export signal. In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to, for example, the amino acid sequence of the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0169] In certain embodiments, the SSFO protein comprises an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 4.

[0170] Volvox carteri light-activated polypeptide

[0171] In some embodiments, a suitable light-activated polypeptide is a cation channel derived from Volvox (VChRl) that is activated by illumination with light having a wavelength of about 500 nm to about 600 nm, e.g., about 525 nm to about 550 nm, e.g., 545 nm. In some embodiments, the light-activated ion channel protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 5. The light-activated ion channel protein can additionally comprise substitutions, deletions, and / or insertions into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the light-activated ion channel protein to modulate the polarization state of the plasma membrane. Furthermore, the light-activated ion channel protein can comprise one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions. The light-activated ion channel protein comprising substitutions, deletions, and / or insertions into the native amino acid sequence suitably retains the ability to transport ions across the plasma membrane of a neuronal cell in response to light.

[0172] In some cases, the VChRl light-activated cation channel protein comprises a core amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 5, and at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport to the mammalian cell plasma membrane selected from the group consisting of a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the light-activated proton ion channel comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the light-activated ion channel protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the light-activated ion channel protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the light-activated ion channel protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be about any one of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0173] In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0174] In certain embodiments, the VChRl protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 6.

[0175] VChRl -based step-function opsin and stable step-function opsin

[0176] In other embodiments, the light-activated polypeptide is a VChRl -based SFO or SSFO. In some embodiments, the SFO protein can have a mutation at amino acid residue C123 of SEQ ID NO: 5. In other embodiments, the SFO protein has a C123A mutation in SEQ ID NO: 5. In other embodiments, the SFO protein has a C123S mutation in SEQ ID NO: 5. In another embodiment, the SFO protein has a C123T mutation in SEQ ID NO: 5.

[0177] In some embodiments, the SFO protein can have a mutation at amino acid residue D151 of SEQ ID NO: 5. In other embodiments, the SFO protein can have mutations at amino acid residues C123 and D151 of SEQ ID NO: 5. In one embodiment, the SFO protein has C123S and D151A mutations in SEQ ID NO: 5.

[0178] In some embodiments, the SFO or SSFO protein is capable of mediating a depolarizing current in a cell when the cell is illuminated with blue light. In some embodiments, the light has a wavelength of about 560 nm. Additionally, in some embodiments, the light is delivered as a single light pulse or as spaced light pulses due to the extended stability of the SFO and SSFO photocurrents. In some embodiments, activation of the SFO or SSFO protein with a single pulse or spaced light pulses can result in the depolarization of a neuron expressing the SFO or SSFO protein. In some embodiments, the disclosed step function opsin and stable step function opsin each can have specific properties and characteristics for depolarizing the membrane of a neuronal cell in response to light.

[0179] In some cases, the VChRl-based SFO or SSFO comprises a membrane trafficking signal and / or an ER export signal. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0180] C1V1 chimeric cation channel

[0181] In other embodiments, the light-activated cation channel protein is a C1V1 chimeric protein derived from VChR1 protein from Volvox carteri and ChR1 protein from Chlamydomonas reinhardtii, wherein the protein comprises the amino acid sequence of VChR1 having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChR1; is responsive to light; and is capable of mediating a depolarizing current in a cell when the cell is illuminated by light. In some embodiments, the C1V1 protein further comprises a replacement within the intracellular loop domain between the second and third transmembrane helices of the chimeric light-responsive protein, wherein at least a portion of the intracellular loop domain is replaced by a corresponding portion from ChR1. In another embodiment, a portion of the intracellular loop domain of the C1V1 chimeric protein can be replaced by a corresponding portion of ChR1 that extends to amino acid residue A145 of ChR1. In other embodiments, the C1V1 chimeric protein further comprises a replacement within the third transmembrane helix of the chimeric light-responsive protein, wherein at least a portion of the third transmembrane helix is replaced by a corresponding sequence of ChR1. In another embodiment, a portion of the intracellular loop domain of the C1V1 chimeric protein can be replaced by a corresponding portion of ChR1 that extends to amino acid residue W163 of ChR1. In other embodiments, the C1V1 chimeric protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 7.

[0182] In some embodiments, the C1V1 protein mediates a depolarizing current in a cell when the cell is illuminated with green light. In some embodiments, the light has a wavelength of between about 540 nm to about 560 nm. In some embodiments, the light can have a wavelength of about 542 nm. In some embodiments, the C1V1 chimeric protein is not capable of mediating a depolarizing current in a cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein is not capable of mediating a depolarizing current in a cell when the cell is illuminated with light having a wavelength of about 405 nm. Further, in some embodiments, light pulses having a temporal frequency of about 100 Hz can be used to activate the C1V1 protein.

[0183] In some cases, the C1V1 polypeptide comprises a membrane trafficking signal and / or an ER export signal. In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0184] In certain embodiments, the C1V1 protein comprises an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 8.

[0185] C1V1 variants

[0186] In some aspects, suitable light-activated polypeptides comprise a substituted or mutated amino acid sequence, wherein the mutated polypeptide retains the characteristic light-activated properties of the precursor C1V1 chimeric polypeptide, but can also have altered properties in certain specific aspects. For example, the mutated light-activated C1V1 chimeric proteins described herein can exhibit increased expression levels both within animal cells and on the animal cell plasma membrane; altered reactivity when exposed to different wavelengths of light, particularly red light; and / or a combination of characteristics whereby the chimeric C1V1 polypeptide has the properties of low desensitization, fast inactivation, low violet light activation to minimize cross-activation with other light-activated cation channels, and / or strong expression in animal cells.

[0187] Thus, suitable light-activated proteins include C1V1 chimeric light-activated proteins that can have specific amino acid substitutions at key positions throughout the retinal binding region of the VChRl portion of the chimeric polypeptide. In some embodiments, the C1V1 protein comprises an amino acid substitution at amino acid residue E122 of SEQ ID NO: 7. In some embodiments, the C1V1 protein comprises a substitution at amino acid residue E162 of SEQ ID NO: 7. In other embodiments, the C1V1 protein comprises substitutions at amino acid residues E162 and E122 of SEQ ID NO: 7.

[0188] In some aspects, the C1V1-E122 mutant chimeric protein is capable of mediating a depolarizing current in a cell when the cell is illuminated with light. In some embodiments, the light is green light. In other embodiments, the light has a wavelength of about 540 nm to about 560 nm. In some embodiments, the light has a wavelength of about 546 nm. In other embodiments, the C1V1-E122 mutant chimeric protein mediates a depolarizing current in a cell when the cell is illuminated with red light. In some embodiments, the red light has a wavelength of about 630 nm. In some embodiments, the C1V1-E122 mutant chimeric protein does not mediate a depolarizing current in a cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein does not mediate a depolarizing current in a cell when the cell is illuminated with light having a wavelength of about 405 nm. Further, in some embodiments, light pulses having a temporal frequency of about 100 Hz can be used to activate the C1V1-E122 mutant chimeric protein. In some embodiments, activation of the C1V1-E122 mutant chimeric protein with light pulses at a frequency of 100 Hz can result in depolarization of neurons expressing the C1V1-E122 mutant chimeric protein.

[0189] In other aspects, the C1V1-E162 mutant chimeric protein is capable of mediating a depolarizing current in a cell when the cell is illuminated with light. In some embodiments, the light can be green light. In other embodiments, the light can have a wavelength of about 535 nm to about 540 nm. In some embodiments, the light can have a wavelength of about 542 nm. In other embodiments, the light can have a wavelength of about 530 nm. In some embodiments, the C1V1-E162 mutant chimeric protein does not mediate a depolarizing current in a cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein does not mediate a depolarizing current in a cell when the cell is illuminated with light having a wavelength of about 405 nm. Further, in some embodiments, light pulses having a temporal frequency of about 100 Hz can be used to activate the C1V1-E162 mutant chimeric protein. In some embodiments, activation of the C1V1-E162 mutant chimeric protein with light pulses at a frequency of 100 Hz can result in depolarization-induced synaptic depletion of neurons expressing the C1V1-E162 mutant chimeric protein.

[0190] In other aspects, the C1V1-E122 / E162 mutant chimeric protein is capable of mediating a depolarizing current in a cell when the cell is illuminated with light. In some embodiments, the light can be green light. In other embodiments, the light can have a wavelength of about 540 nm to about 560 nm. In some embodiments, the light can have a wavelength of about 546 nm. In some embodiments, the C1V1-E122 / E162 mutant chimeric protein does not mediate a depolarizing current in a cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein does not mediate a depolarizing current in a cell when the cell is illuminated with light having a wavelength of about 405 nm. In some embodiments, the C1V1-E122 / E162 mutant chimeric protein can exhibit little activation upon exposure to violet light relative to a C1V1 chimeric protein lacking a mutation at E122 / E162 or relative to other light-activated cation channel proteins. Further, in some embodiments, light pulses having a temporal frequency of about 100 Hz can be used to activate the C1V1-E122 / E162 mutant chimeric protein. In some embodiments, activation of the C1V1-E122 / E162 mutant chimeric protein with light pulses having a frequency of 100 Hz can cause depolarization-induced synaptic depletion of neurons expressing the C1V1-E122 / E162 mutant chimeric protein.

[0191] In some cases, the C1V1 variant polypeptide comprises a membrane trafficking signal and / or an ER export signal. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0192] C1C2 chimeric cation channel

[0193] In other embodiments, the light-activated cation channel protein is a C1C2 chimeric protein derived from ChRl and ChR2 proteins of Chlamydomonas reinhardtii, wherein the protein is responsive to light and is capable of mediating a depolarizing current in a cell when the cell is illuminated with light. In another embodiment, the light-activated polypeptide comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 9. The light-activated cation channel protein can additionally comprise substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the light-activated cation channel protein to modulate the plasma membrane polarization state of a cell. In addition, the light-activated cation channel protein comprises one or more than one conservative amino acid substitution and / or one or more than one non-conservative amino acid substitution. The light-activated proton pump protein comprising substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane.

[0194] In some embodiments, the C1C2 protein comprises at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the protein to the plasma membrane, selected from the group consisting of a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the C1C2 protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the C1C2 protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the C1C2 protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the C1C2 protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0195] In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0196] In certain embodiments, the C1C2 protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 10.

[0197] ReaChR

[0198] In some aspects, the depolarizing light-activated polypeptide is a red-shifted variant of a depolarizing light-activated polypeptide derived from Chlamydomonas reinhardtii; such light-activated polypeptide is referred to herein as a "ReaChR polypeptide" or "ReaChR protein" or "ReaChR." In another embodiment, the light-activated polypeptide comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 11. The light used to activate the ReaChR polypeptide can have a wavelength of about 590 to about 630 nm or can have a wavelength of about 610 nm. The ReaChR protein can additionally comprise a substitution, deletion, and / or insertion introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the light-activated cation channel protein to modulate the polarization state of the cytoplasmic membrane. Furthermore, the ReaChR protein can comprise one or more than one conservative amino acid substitution and / or one or more than one non-conservative amino acid substitution. A ReaChR containing a substitution, deletion, and / or insertion introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane.

[0199] In some embodiments, the ReaChR protein comprises at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the neuron to the plasma membrane selected from a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the ReaChR protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the ReaChR protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the ReaChR protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the ReaChR protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also include a fluorescent protein such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0200] In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0201] In certain embodiments, the ReaChR protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence set forth in SEQ ID NO: 12.

[0202] SdChR

[0203] In some aspects, the depolarizing light-activated polypeptide is a SdChR polypeptide derived from Scherffelia dubia, wherein the SdChR polypeptide is capable of transporting cations across a cell membrane when the cell is illuminated with light. In some cases, the SdChR polypeptide comprises an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 13. The light used to activate the SdChR polypeptide can have a wavelength of about 440 to about 490 nm, or can have a wavelength of about 460 nm. The SdChR protein can additionally comprise substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the SdChR protein to modulate the polarization state of the plasma membrane of a cell. In some cases, the SdChR protein comprises one or more than one conservative amino acid substitution and / or one or more than one non-conservative amino acid substitution. The SdChR protein containing substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane.

[0204] In some embodiments, the SdChR protein comprises at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the neuron to the plasma membrane selected from a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the SdChR protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the SdChR protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the SdChR protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the SdChR protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0205] In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0206] In certain embodiments, the SdChR protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence set forth in SEQ ID NO: 14.

[0207] CnChR1

[0208] In some aspects, the depolarizing light-activated polypeptide can be CnChRl derived from Chlamydomonas noctigama, where the CnChRl polypeptide is capable of transporting cations across a cell membrane upon irradiation of the cell with light. In some cases, the CnChRl polypeptide comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 15. The light used to activate the CnChRl polypeptide can have a wavelength of about 560 to about 630 nm, or can have a wavelength of about 600 nm. The CnChRl protein can additionally comprise a substitution, deletion, and / or insertion introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the CnChRl protein to modulate the polarization state of the plasma membrane of a cell. In some cases, the CnChRl protein comprises one or more than one conservative amino acid substitution and / or one or more than one non-conservative amino acid substitution. The CnChRl protein containing a substitution, deletion, and / or insertion introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane.

[0209] In some embodiments, the CnChRl protein comprises at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the neuron to the plasma membrane selected from a signal peptide, an ER export signal, an ER export signal, and a membrane trafficking signal. In some embodiments, the CnChRl protein includes an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the CnChRl protein includes an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the CnChRl protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the CnChRl protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also include a fluorescent protein such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0210] In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifying potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of, for example, the trafficking sequence of the human inward rectifying potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0211] In certain embodiments, the CnChRl protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence set forth in SEQ ID NO: 16.

[0212] CsChrimson

[0213] In other embodiments, the light-activated cation channel protein is a CsChR protein derived from Chloromonas subdivisa and a CnChRl protein from C. noctigama, wherein the N- terminus of the protein comprises the amino acid sequence of residues 1-73 of CsChR followed by residues 79-350 of the amino acid sequence of CnChRl; is responsive to light; and is capable of mediating a depolarizing current in a cell when the cell is illuminated with light. In another embodiment, the CsChrimson polypeptide comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 17. The CsChrimson protein can additionally comprise substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the CsChrimson protein to modulate the plasma membrane polarization state of a cell. Furthermore, the CsChrimson protein can comprise one or more than one conservative amino acid substitution and / or one or more than one non-conservative amino acid substitution. The CsChrimson protein comprising substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane.

[0214] In some embodiments, the CsChrimson protein comprises at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the protein to the plasma membrane of a neuron, selected from the group consisting of a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the CsChrimson protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the CsChrimson protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the CsChrimson protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the CsChrimson protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0215] In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifying potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of, for example, the trafficking sequence of the human inward rectifying potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0216] In certain embodiments, the CsChrimson protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence set forth in SEQ ID NO: 18.

[0217] ShChR1

[0218] In some aspects, the depolarizing light-activated polypeptide can be, for example, ShChRl derived from Stigeoclonium helveticum, wherein the ShChRl polypeptide is capable of transporting cations across a cell membrane upon irradiation of the cell with light. In some cases, the ShChRl polypeptide comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 19. Light used to activate the ShChRl protein derived from Stigeoclonium helveticum can have a wavelength of about 480 nm to about 510 nm, or can have a wavelength of about 500 nm. The ShChRl protein can additionally comprise a substitution, deletion, and / or insertion introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the ShChRl protein to modulate the polarization state of the plasma membrane. Furthermore, the ShChRl protein can comprise one or more than one conservative amino acid substitution and / or one or more than one non-conservative amino acid substitution. The ShChRl protein comprising a substitution, deletion, and / or insertion introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane.

[0219] In some embodiments, the ShChRl protein comprises at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the neuron to the plasma membrane selected from a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the ShChRl protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the ShChRl protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the ShChRl protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the ShChRl protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0220] In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0221] In certain embodiments, the ShChRl protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence set forth in SEQ ID NO: 20.

[0222] Other suitable depolarizing light-activated polypeptides are described, for example, in Klapoetke et al., Nat Methods 2014 11 :338.

[0223] Hyperpolarizing light-activated polypeptide

[0224] Arch

[0225] In some embodiments, a suitable light-activated polypeptide is an Arch proton pump (e.g., a proton pump derived from Halorubrum sodomense) that can transport one or more protons across the plasma membrane of a cell when the cell is illuminated with light. The light can have a wavelength of about 530 to about 595 nm, or can have a wavelength of about 560 nm. In some embodiments, the Arch protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 21. The Arch protein can additionally have substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, to increase or decrease sensitivity to a particular wavelength of light, and / or to increase or decrease the ability of the Arch protein to transport ions across the plasma membrane of a neuron. In addition, the Arch protein can comprise one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions. The Arch protein comprising substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to transport ions across the plasma membrane of a neuron in response to light.

[0226] In some embodiments, the Arch protein comprises at least one (e.g., one, two, three, or more) amino acid sequence motif that enhances transport of the neuron to the plasma membrane selected from a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the Arch protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the Arch protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the Arch protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the Arch protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can further comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0227] In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifying potassium channel Kir2.1. In other embodiments, the trafficking signal can include the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can include an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifying potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0228] In certain embodiments, the Arch protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 22.

[0229] ArchT

[0230] In some embodiments, a suitable light-activated protein is an ArchT proton pump (e.g., a proton pump derived from Halorubrum sp. TP009) that can transport one or more protons across the plasma membrane of a cell when the cell is illuminated with light. The light can have a wavelength of about 530 nm to about 595 nm, or can have a wavelength of about 560 nm. In some embodiments, the ArchT protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 23 (ArchT). The ArchT protein can additionally comprise substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the ArchT protein to transport ions across the plasma membrane of a neuron. Moreover, the ArchT protein can comprise one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions. The ArchT protein containing substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to transport ions across the plasma membrane of a neuron in response to light.

[0231] In some cases, the ArchT polypeptide comprises a membrane trafficking signal and / or an ER export signal. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0232] In certain embodiments, the ArchT protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 24.

[0233] GtR3

[0234] In some embodiments, the light-activated polypeptide is responsive to blue light and is a proton pump protein derived from Guillardia theta, wherein the proton pump protein is capable of mediating a hyperpolarizing current in a cell when the cell is illuminated with blue light; such a protein is referred to herein as a "GtR3 protein" or "GtR3 polypeptide." The light can have a wavelength of about 450 nm to about 495 nm, or can have a wavelength of about 490 nm. In some embodiments, the GtR3 protein comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 25 (GtR3). The GtR3 protein can additionally comprise substitutions, deletions, and / or insertions into the native amino acid sequence to increase or decrease sensitivity to light, to increase or decrease sensitivity to a particular wavelength of light, and / or to increase or decrease the ability of the GtR3 protein to modulate the plasma membrane polarization state of a cell. Furthermore, the GtR3 protein can comprise one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions. The GtR3 protein containing substitutions, deletions and / or insertions into the native amino acid sequence suitably retains the ability to hyperpolarize the plasma membrane of a neuronal cell in response to light.

[0235] In some cases, the GtR3 protein comprises a core amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 25, and at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the mammalian cell to the plasma membrane selected from the group consisting of a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the GtR3 protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the GtR3 protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the light-activated proton pump protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the GtR3 protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the signal peptide comprises the amino acid sequence MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO: 59). In some embodiments, the first 19 amino acids are replaced with MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO: 59). In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be about any one of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The GtR3 protein can also comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0236] In some embodiments, the trafficking signal is derived from the amino acid sequence of human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of, for example, the trafficking sequence of human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0237] In certain embodiments, the GtR3 protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 26.

[0238] Oxy

[0239] In some embodiments, the light-activated protein is an Oxyrrhis marina (Oxy) proton pump that can transport one or more protons across the plasma membrane of a cell when the cell is illuminated with light. The light can have a wavelength of about 500 nm to about 560 nm, or can have a wavelength of about 530 nm. In some embodiments, the Oxy protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 27. The Oxy protein can additionally comprise substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the Oxy protein to transport ions across the plasma membrane of a neuron. Furthermore, the Oxy protein can comprise one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions. The Oxy protein comprising substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to transport ions across the plasma membrane of a neuron in response to light.

[0240] In some embodiments, the Oxy protein comprises at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport to the plasma membrane of a neuron selected from a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the Oxy protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the Oxy protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the Oxy protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the Oxy protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The Oxy protein can also comprise a fluorescent protein such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0241] In some cases, the Oxy polypeptide comprises a membrane trafficking signal and / or an ER export signal. In some embodiments, the trafficking signal can be derived from the amino acid sequence of human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0242] In certain embodiments, the Oxy protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 28.

[0243] Mac

[0244] In some embodiments, the light-activated proton pump protein (referred to herein as a "Mac protein") is responsive to light and is derived from Leptosphaeria maculans, wherein the Mac proton pump protein is capable of pumping protons across a cell membrane when the cell is illuminated with light of 520 nm to 560 nm. The light can have a wavelength of about 520 nm to about 560 nm. In some cases, the Mac protein comprises an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 30 (Mac; Mac 3.0). The Mac protein can additionally comprise substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the Mac protein to modulate the plasma membrane polarization state of a cell. Moreover, the Mac protein can comprise one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions. The Mac protein comprising substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to pump protons through the plasma membrane of a neuronal cell in response to light.

[0245] In other aspects, the Mac protein comprises a core amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 29, and at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the mammalian cell to the plasma membrane selected from the group consisting of a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the Mac protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the Mac protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the Mac protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the Mac protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can comprise any one of a length of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids. The Mac protein can also include a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0246] In some cases, the Mac polypeptide includes a membrane trafficking signal and / or an ER export signal. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences for use can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0247] Further disclosure relating to light-activated proton pump proteins can be found in International Patent Application No. PCT / US2011 / 028893, the disclosure of which is hereby incorporated by reference in its entirety.

[0248] NpHR

[0249] In certain cases, a suitable light-activated chloride pump protein is from Natronomonas pharaonis; this protein is referred to herein as an "NpHR protein" or "NpHR polypeptide." In some embodiments, an NpHR protein can respond to amber light as well as red light, and can mediate a hyperpolarizing current in a neuron when the NpHR protein is illuminated with amber light or red light. The wavelength of light that can activate the NpHR protein can be about 580 nm to 630 nm. In some embodiments, the light can have a wavelength of about 589 nm, or the light can have a wavelength greater than about 630 nm (e.g., less than about 740 nm). In another embodiment, the light has a wavelength of about 630 nm. In some embodiments, the NpHR protein can hyperpolarize a neural membrane for at least about 90 minutes when exposed to a continuous light pulse. In some embodiments, the NpHR protein comprises an amino acid sequence that is at least about 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 31. In addition, the NpHR protein can comprise substitutions, deletions, and / or insertions into the native amino acid sequence to increase or decrease sensitivity to light, to increase or decrease sensitivity to a particular wavelength of light, and / or to increase or decrease the ability of the NpHR protein to modulate the polarization state of the plasma membrane. In some embodiments, the NpHR protein comprises one or more conservative amino acid substitutions. In some embodiments, the NpHR protein comprises one or more non-conservative amino acid substitutions. The NpHR protein comprising substitutions, deletions, and / or insertions into the native amino acid sequence suitably retains the ability to hyperpolarize the plasma membrane of a neuronal cell in response to light.

[0250] In some cases, the NpHR protein comprises a core amino acid sequence that is at least about 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 31, and an endoplasmic reticulum (ER) export signal. The ER export signal can be fused to the C-terminus of the core amino acid sequence, can be fused to the N-terminus of the core amino acid sequence. In some embodiments, the ER export signal is linked to the core amino acid sequence by a linker. The linker can be any one of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker can also comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal comprises the amino acid sequence FXYENE (SEQ ID NO: 57), where X can be any amino acid. In another embodiment, the ER export signal comprises the amino acid sequence VXXSL, where X can be any amino acid. In some embodiments, the ER export signal comprises the amino acid sequence FCYENEV (SEQ ID NO: 58).

[0251] Suitable endoplasmic reticulum (ER) export sequences for use include, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); and the like); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), such as FCYENEV (SEQ ID NO: 58); and the like. The ER export sequence can be about 5 amino acids to about 25 amino acids in length, such as about 5 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, or about 20 amino acids to about 25 amino acids.

[0252] In other aspects, the NpHR protein comprises a core amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 31, and a trafficking signal (e.g., which can enhance transport of the NpHR protein to the plasma membrane). The trafficking signal can be fused to the C-terminus of the core amino acid sequence, or can be fused to the N-terminus of the core amino acid sequence. In some embodiments, the trafficking signal can be connected to the core amino acid sequence by a linker, which can be about any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The NpHR protein can also include a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward-rectifying potassium channel Kir2.1. In other embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56).

[0253] In some aspects, the NpHR protein comprises a core amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 31, and at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the mammalian cell to the plasma membrane selected from the group consisting of an ER export signal, a signal peptide, and a membrane trafficking signal. In some embodiments, the NpHR protein includes an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be about any one of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The NpHR protein can further include a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal can be more C-terminal than the trafficking signal. In other embodiments, the trafficking signal is more C-terminal than the ER export signal. In some embodiments, the signal peptide includes the amino acid sequence MTETLPPVTESAVALQAE (SEQ ID NO: 62). In another embodiment, the NpHR protein includes an amino acid sequence having at least 95% identity to SEQ ID NO: 31. In another embodiment, the NpHR protein includes an amino acid sequence having at least 95% identity to SEQ ID NO: 31.

[0254] Further, in other aspects, the NpHR protein comprises a core amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 31, wherein the N-terminal signal peptide of SEQ ID NO: 31 is deleted or replaced. In some embodiments, other signal peptides (e.g., signal peptides from other opsin proteins) can be used. The light-activated protein can also comprise an ER transport signal and / or a membrane trafficking signal as described herein.

[0255] In some embodiments, the light-activated protein is a NpHR protein comprising an amino acid sequence that is at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 31. In some embodiments, the NpHR protein further comprises an endoplasmic reticulum (ER) export signal and / or a membrane trafficking signal. For example, the NpHR protein comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 31 and an endoplasmic reticulum (ER) export signal. In some embodiments, the amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 31 is linked to the ER export signal by a linker. In some embodiments, the ER export signal comprises the amino acid sequence FXYENE (SEQ ID NO: 57), where X can be any amino acid. In another embodiment, the ER export signal comprises the amino acid sequence VXXSL, where X can be any amino acid. In some embodiments, the ER export signal comprises the amino acid sequence FCYENEV (SEQ ID NO: 58). In some embodiments, the NpHR protein comprises an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 31, an ER export signal, and a membrane trafficking signal. In other embodiments, the NpHR protein comprises, from N-terminus to C-terminus, an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 31, an ER export signal, and a membrane trafficking signal. In other embodiments, the NpHR protein comprises, from N-terminus to C-terminus, an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 31, a membrane trafficking signal, and an ER export signal. In some embodiments, the membrane trafficking signal is derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the membrane trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). In some embodiments, the membrane trafficking signal is linked to the amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 31 by a linker. In some embodiments, the membrane trafficking signal is linked to the ER export signal by a linker. The length of the linker can be any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids. The linker can also comprise a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the light-activated protein further comprises an N-terminal signal peptide.

[0256] Further disclosures related to light-activated chloride pump proteins can be found in U.S. Patent Application Publication Nos. 2009 / 0093403 and 2010 / 0145418 and International Patent Application No. PCT / US2011 / 028893, the disclosures of each of which are hereby incorporated by reference in their entirety.

[0257] Dunaliella salina light-activated polypeptide

[0258] In some embodiments, a suitable light-activated ion channel protein is, for example, a DsChR protein derived from Dunaliella salina, wherein the ion channel protein is capable of mediating a hyperpolarizing current in a cell when the cell is illuminated with light. The light can have a wavelength of about 470 nm to about 510 nm, or can have a wavelength of about 490 nm. In some embodiments, the DsChR protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 34. The DsChR protein can additionally comprise substitutions, deletions, and / or insertions introduced into the native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to a particular wavelength of light, and / or increase or decrease the ability of the DsChR protein to modulate the plasma membrane polarization state of a cell. Furthermore, the DsChR protein can comprise one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions. The DsChR protein comprising substitutions, deletions, and / or insertions introduced into the native amino acid sequence suitably retains the ability to transport ions across the plasma membrane of a neuronal cell in response to light.

[0259] In some cases, the DsChR protein comprises a core amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 34; and at least one (e.g., one, two, three, or more than three) amino acid sequence motif that enhances transport of the mammalian cell to the plasma membrane selected from the group consisting of a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the DsChR protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the DsChR protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some embodiments, the DsChR protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the DsChR protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C-terminal ER export signal and the C-terminal trafficking signal are connected by a linker. The linker can be any one of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The DsChR protein can also include a fluorescent protein, such as, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the ER export signal is more C-terminal than the trafficking signal. In some embodiments, the trafficking signal is more C-terminal than the ER export signal.

[0260] In some cases, the DsChR polypeptide comprises a membrane trafficking signal and / or an ER export signal. In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Suitable trafficking sequences can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of, for example, the trafficking sequence of the human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export signal is, for example, VXXSL (where X is any amino acid; SEQ ID NO: 52) (e.g., VKESL (SEQ ID NO: 53), VLGSL (SEQ ID NO: 54); etc.); NANSFCYENEVALTSK (SEQ ID NO: 55); FXYENE (SEQ ID NO: 57) (where X is any amino acid), for example, FCYENEV (SEQ ID NO: 58); etc.

[0261] In certain embodiments, the DsChR protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 35.

[0262] C1C2-based anion channel polypeptides

[0263] In some embodiments, the light-activated anion channel polypeptide is a C1C2 protein. In some embodiments, the C1C2 polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the amino acid sequence of the C1C2 protein is modified by introducing one or more than one of the following mutations to the amino acid sequence: T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K, and / or N297Q. In some embodiments, the C1C2 protein comprises the amino acid sequence of the C1C2 protein having all 9 of the above-listed amino acid substitutions, such that the amino acid sequence of the C1C2 polypeptide is as set forth in SEQ ID NO: 36.

[0264] In some embodiments, the C1C2 polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 36; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K, and / or N297Q relative to the amino acid sequence of C1C2 (SEQ ID NO: 36). In some embodiments, the C1C2 polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 36; and includes T98S, E129S, E140S, E162S, and T285N substitutions relative to the amino acid sequence of C1C2. In some embodiments, the C1C2 polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 36; and includes V156K, H173R, V281K, and N297Q substitutions relative to the amino acid sequence of C1C2.

[0265] In some embodiments, the C1C2 polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 36; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S98, S129, S140, S162, K156, R173, N285, K281, and Q297, wherein the amino acid numbering is as set forth in SEQ ID NO: 36. In some embodiments, the C1C2 polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 36; and includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297, wherein the amino acid numbering is as set forth in SEQ ID NO: 36. In any of these embodiments, the C1C2 polypeptide can comprise a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the C1C2 polypeptide can comprise an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the C1C2 polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). Thus, in certain embodiments, the C1C2 protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 36.

[0266] In some embodiments, a C1C2 polypeptide is based on the amino acid sequence of protein C1C2 (SEQ ID NO: 36), wherein the amino acid sequence has been modified by replacing the first 50 N-terminal amino acids of C1C2 with amino acids 1-11 from protein ChR2 (MDYGGALSAVG) (SEQ ID NO: 63). In some embodiments, a suitable light-activated anion channel polypeptide is referred to as “ibC1C2” and comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 40; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258, wherein the amino acid numbering is as set forth in SEQ ID NO: 40. In some embodiments, a suitable light-activated anion channel polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 40; and includes S59, S90, S101, S123, K117, R134, N246, K242, and Q258, wherein the amino acid numbering is as set forth in SEQ ID NO: 40. In some embodiments, a suitable light-activated anion channel polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 40. In any of these embodiments, a suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, a suitable anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, a suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). Thus, in certain embodiments, an ibC1C2 protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 40.

[0267] In some embodiments, a suitable light-activated anion channel polypeptide is based on the amino acid sequence of protein C1C2 (SEQ ID NO: 36), wherein the cysteine amino acid residue at position 167 has been replaced with a threonine residue. In some embodiments, a suitable light-activated anion channel polypeptide, e.g., SwiChR CT In some embodiments, a suitable light-activated anion channel polypeptide comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 38; and comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and includes T167. In some embodiments, a suitable light-activated anion channel polypeptide comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 38; includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and includes T167, where the amino acid numbering is as set forth in SEQ ID NO: 38. In some embodiments, a light-activated anion channel polypeptide comprises the amino acid sequence provided in SEQ ID NO: 38. In some of these embodiments, the light-activated polypeptide exhibits prolonged photocurrent stability. In some embodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 63). In any of these embodiments, a suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, a suitable anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, a suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0268] In some embodiments, a suitable light-activated anion channel polypeptide is based on the amino acid sequence of protein C1C2 (SEQ ID NO: 36), wherein the cysteine amino acid residue at position 167 has been replaced with a threonine residue. In some embodiments, a suitable light-activated anion channel polypeptide, e.g., SwiChRCA an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 38; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and includes A167, wherein the amino acid numbering is as set forth in SEQ ID NO: 38. In some embodiments, a suitable light-activated anion channel polypeptide comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 38; includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and includes A167, wherein the amino acid numbering is as set forth in SEQ ID NO: 38. In some embodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 63). In any of these embodiments, the suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the anion channel polypeptide of interest includes an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the anion channel polypeptide of interest comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0269] In some embodiments, a suitable light-activated anion channel polypeptide is based on the amino acid sequence of protein C1C2, wherein the cysteine amino acid residue at position 167 has been replaced with a serine residue. In some embodiments, a suitable light-activated anion channel polypeptide SwiChR CSIn some embodiments, a suitable light-activated anion channel polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 38; includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and includes S167, where the amino acid numbering is as set forth in SEQ ID NO: 38. In some embodiments, a suitable light-activated anion channel polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 38; includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and includes S167, where the amino acid numbering is as set forth in SEQ ID NO: 38. In some embodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 63). In any of these embodiments, a suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, a target anion channel polypeptide includes an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, a target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0270] In certain embodiments, a SwiChR protein comprises an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in SEQ ID NO: 39.

[0271] In some embodiments, a suitable light-activated anion channel polypeptide SwiChR comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 38; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S98, S129, S140, S162, K156, R173, N285, K281, and Q297; includes N195 or A195; and includes A167, where the amino acid numbering is as set forth in SEQ ID NO: 38. In some embodiments, a suitable light-activated anion channel polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 38; includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297; includes A167; and includes N195 or A195, where the amino acid numbering is as set forth in SEQ ID NO: 38. In some embodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 63). In any of these embodiments, the subject anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the subject anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the subject anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0272] In some embodiments, a suitable light-activated anion channel polypeptide is based on the amino acid sequence of protein C1C2 with one or more than one modification described above, wherein the aspartic acid amino acid residue at original position 195 has been replaced with an alanine residue. In some embodiments where the first 50 N-terminal amino acids of the protein are replaced with amino acids 1-11 from protein ChR2, the aspartic acid amino acid residue at position 156 (which corresponds to original position 195 of the C1C2 amino acid sequence set forth in SEQ ID NO: 36) is replaced with an alanine residue.

[0273] In some embodiments, a suitable hyperpolarizing light-activated polypeptide is based on the amino acid sequence of protein C1C2 with one or more of the above-mentioned modifications, wherein the aspartic acid amino acid residue at original position 195 has been replaced with an asparagine residue. In some embodiments, wherein the first 50 N-terminal amino acids of the protein are replaced with amino acids 1-11 from protein ChR2, the aspartic acid amino acid residue at position 156 (which corresponds to original position 195 of the C1C2 amino acid sequence set forth in SEQ ID NO: 36) is replaced with an asparagine residue.

[0274] In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 40; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258; and includes A128, T128, or S128, where the amino acid numbering is as set forth in SEQ ID NO: 40. In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 40; includes S59, S90, S101, S123, K117, R134, N246, K242, and Q258; and includes A128, T128, or S128, where the amino acid numbering is as set forth in SEQ ID NO: 40. In any of these embodiments, the subject anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the suitable anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the suitable anion channel polypeptide includes a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0275] ChR2-based anion channel proteins

[0276] In some embodiments, a suitable hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the protein ChR2. The amino acid sequence of ChR2 is set forth in SEQ ID NO:42. In some embodiments, the amino acid sequence of the ChR2 protein has been modified by introducing one or more than one of the following mutations: A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N, and / or N258Q. In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises the amino acid sequence of the protein ChR2 with all 9 or more of the listed amino acid substitutions, such that the amino acid sequence of the polypeptide is provided in SEQ ID NO:42 (iChR2).

[0277] In some embodiments, a suitable light-activated anion channel polypeptide iChR2 comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:42; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N, and / or N258Q relative to the amino acid sequence of ChR2 (SEQ ID NO: 1).

[0278] In some embodiments, a suitable light-activated polypeptide ("iChR2") comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 42; including 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, K242, N246, and Q258, where the amino acid numbering is as set forth in SEQ ID NO: 42. In some embodiments, an iChR2 polypeptide comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 42; including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of S59, S90, S101, S123, K117, R134, K242, N246, Q258, and either of N156 or A156, and either of T128, A128, or S128, where the amino acid numbering is as set forth in SEQ ID NO: 42. In some embodiments, an iChR2 polypeptide comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 42; and includes S59, S90, S101, S123, K117, R134, K242, N246, and Q258, where the amino acid numbering is as set forth in SEQ ID NO: 42. In any of these embodiments, the iChR2 polypeptide can comprise a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the iChR2 polypeptide can comprise an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the iChR2 polypeptide can comprise a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).Accordingly, in certain embodiments, the iChR2 protein comprises an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 43.

[0279] C1V1 -based anion channel polypeptides

[0280] In some embodiments, a suitable hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the protein C1V1. The amino acid sequence of C1V1 is set forth in SEQ ID NO: 44. In some embodiments, the amino acid sequence of the C1V1 protein has been modified by introducing one or more than one of the following mutations into the amino acid sequence: T98S, E129S, E140S, E162S, V156K, H173R, A285N, P281K, and / or N297Q. In some embodiments, the hyperpolarizing light-activated polypeptide comprises the amino acid sequence of the protein C1V1 with all 9 of the above-listed amino acid substitutions, such that the amino acid sequence of the polypeptide is provided in SEQ ID NO: 44.

[0281] In some embodiments, a suitable light-activated anion channel polypeptide iC1V1 comprises an amino acid sequence that is at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 44; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from T98S, E129S, E140S, E162S, V156K, H173R, A285N, P281K, and / or N297Q relative to the amino acid sequence of C1V1 (SEQ ID NO: 7).

[0282] In some embodiments, a suitable light-activated anion channel polypeptide iC1V1 comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 44; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S98, S129, S140, S162, K156, R173, N285, K281, and Q297, where the amino acid numbering is as set forth in SEQ ID NO: 44. In some embodiments, a suitable light-activated anion channel polypeptide (referred to as “iC1V1”) comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID: 44; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S98, S129, S140, S162, K156, R173, N285, K281, and Q297, and includes N195, where the amino acid numbering is as set forth in SEQ ID NO: 44. In some embodiments, a suitable light-activated anion channel polypeptide comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 44; and includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297, where the amino acid numbering is as set forth in SEQ ID NO: 44. In any of these embodiments, the suitable anion channel polypeptide includes a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the anion channel polypeptide of interest includes an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).Accordingly, in certain embodiments, the iClVl protein can have an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 45.

[0283] In some embodiments, a suitable hyperpolarizing light-activated polypeptide is based on the amino acid sequence of protein C1V1 (SEQ ID NO: 7), wherein the amino acid sequence has been modified by replacing the first 50 N-terminal amino acids of C1V1 with amino acids 1-11 from protein ChR2 (MDYGGALSAVG) (SEQ ID NO: 63). In some embodiments, a suitable hyperpolarizing light-activated polypeptide ibClV1 comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258, wherein the amino acid numbering is as set forth in SEQ ID NO: 46. In some embodiments, a suitable hyperpolarizing light-activated polypeptide (termed “ibC1V1”) comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258, and includes N156, wherein the amino acid numbering is as set forth in SEQ ID NO: 46. In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; and includes S59, S90, S101, S123, K117, R134, N246, K242, and Q258, wherein the amino acid numbering is as set forth in SEQ ID NO: 46. In some embodiments, a suitable light-activated anion channel polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 46. In any of these embodiments, a suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, a suitable anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). Thus, in certain embodiments, the ibClVl protein comprises an amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 47.

[0284] In some embodiments, a suitable hyperpolarizing light-activated polypeptide is based on the amino acid sequence of protein C1V1 (SEQ ID NO: 7), wherein the cysteine amino acid residue at position 167 has been replaced with a threonine residue. In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and includes T167. In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 44; includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and includes T167, S167, or A167, where the amino acid numbering is as set forth in SEQ ID NO: 44. In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297; includes T167, S167, or A167; and includes A195 or N195, where the amino acid numbering is as set forth in SEQ ID NO: 46. In some embodiments, the first 50 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 63). In any of these embodiments, a suitable hyperpolarizing light-activated polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).In any of these embodiments, a suitable hyperpolarizing light-activated polypeptide includes a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0285] In some embodiments, a suitable hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the C1V1 protein with one or more than one modification described above, wherein the aspartic acid amino acid residue at original position 195 has been replaced by an alanine residue. In some embodiments, wherein the first 50 N-terminal amino acids of the protein are replaced by amino acids 1-11 from the protein ChR2, the aspartic acid amino acid residue at position 156, which corresponds to the original position 195 of the C1V1 amino acid sequence set forth in SEQ ID NO: 7, is replaced by an alanine residue.

[0286] In some embodiments, a suitable hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the protein C1V1 with one or more than one modification described above, wherein the aspartic acid amino acid residue at original position 195 has been replaced by an asparagine residue. In some embodiments, wherein the first 50 N-terminal amino acids of the protein are replaced by amino acids 1-11 from the protein ChR2, the aspartic acid amino acid residue at position 156, which corresponds to the original position 195 of the C1V1 amino acid sequence set forth in SEQ ID NO: 7, is replaced by an asparagine residue.

[0287] In some embodiments, a suitable hyperpolarizing light-activated polypeptide ibClVl comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258; and includes T128, A128, or S128, where the amino acid numbering is as set forth in SEQ ID NO: 46. In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; includes S59, S90, S101, S123, K117, R134, N246, K242, and Q258; and includes T128, A128, or S128, where the amino acid numbering is as set forth in SEQ ID NO: 46. In any of these embodiments, a suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, a suitable anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, a suitable anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0288] In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258; includes T128, A128, or S128; and includes A156 or N156, wherein the amino acid numbering is as set forth in SEQ ID NO: 46. In some embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; includes S59, S90, S101, S123, K117, R134, N246, K242, and Q258; includes T128, A128, or S128; and includes A156 or N156, wherein the amino acid numbering is as set forth in SEQ ID NO: 46. In any of these embodiments, a suitable hyperpolarizing light-activated polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, a suitable hyperpolarizing light-activated polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the target anion channel polypeptide includes a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0289] ReaChR-based anion channel polypeptides

[0290] In some embodiments, the targeted hyperpolarization photoactivated peptide is based on the amino acid sequence of the protein ReaChR. The amino acid sequence of ReaChR is listed in SEQ ID NO: 11. In some embodiments, the amino acid sequence of the ReaChR protein has been modified by introducing one or more of the following mutations into the amino acid sequence: T99S, E130S, E141S, E163S, V157K, H174R, A286N, P282K, and / or N298Q. In some embodiments, the targeted hyperpolarization photoactivated peptide comprises the amino acid sequence of the protein ReaChR having all nine or more of the listed amino acid substitutions, such that the amino acid sequence of the peptide is provided in SEQ ID NO: 48.

[0291] In some embodiments, the target photoactivated anion channel polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 48; and the amino acid sequence relative to ReaChR (SEQ ID NO: 11) includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from T99S, E130S, E141S, E163S, V157K, H174R, A286N, P282K, and / or N298Q.

[0292] In some embodiments, the target light-activated anion channel polypeptide iReaChR comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:48; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S99, S130, S141, S163, K157, R174, N286, K281, and Q298, where the amino acid numbering is as set forth in SEQ ID NO:48. In some embodiments, the target light-activated anion channel polypeptide comprises an amino acid sequence that has at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:48; includes S99, S130, S141, S163, K157, R174, N286, K281, and Q298, where the amino acid numbering is as set forth in SEQ ID NO:48. In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:56)). In any of these embodiments, the target anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO:58)). In any of these embodiments, the target anion channel polypeptide includes a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:58)). Thus, in certain embodiments, the iReaChR protein comprises an amino acid sequence that has at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence set forth in SEQ ID NO:49.

[0293] In some embodiments, the target light-activated anion channel polypeptide iReaChR comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 48; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S99, S130, S141, S163, K157, R174, N286, K281, and Q298, and includes N196, where the amino acid numbering is as set forth in SEQ ID NO: 48. In some embodiments, the target light-activated anion channel polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 48; includes S99, S130, S141, S163, K157, R174, N286, K281, and Q298, and includes N196, where the amino acid numbering is as set forth in SEQ ID NO: 48. In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the target anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0294] In some embodiments, the target hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the protein ReaChR (SEQ ID NO: 11), wherein the amino acid sequence has been modified by replacing the first 51 N-terminal amino acids of ReaChR with amino acids 1-11 (MDYGGALSAVG) from the protein ChR2 (SEQ ID NO: 63). In some embodiments, the target hyperpolarizing light-activated polypeptide ibReaChR comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 50; including 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258, wherein the amino acid numbering is as set forth in SEQ ID NO: 50. In some embodiments, the target hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence set forth in SEQ ID NO: 50; and including S59, S90, S101, S123, K117, R134, N246, K242, and Q258, wherein the amino acid numbering is as set forth in SEQ ID NO: 50. In some embodiments, the target light-activated anion channel polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 50. In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the target anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). Thus, in certain embodiments, the ibReaChR protein can have an amino acid sequence having at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence set forth in SEQ ID NO: 51.

[0295] In some embodiments, the target hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the protein ReaChR (SEQ ID NO: 11), wherein the amino acid sequence has been modified by replacing the first 51 N-terminal amino acids of ReaChR with amino acids 1-11 (MDYGGALSAVG) from the protein ChR2 (SEQ ID NO: 63). In some embodiments, the target hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258, and includes N156, wherein the amino acid numbering is as set forth in SEQ ID NO: 11. In some embodiments, the target hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; includes S59, S90, S101, S123, K117, R134, N246, K242, and Q258, and includes N156, wherein the amino acid numbering is as set forth in SEQ ID NO: 11. In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the target anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0296] In some embodiments, the target hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the protein ReaChR (SEQ ID NO: 11), in which the cysteine amino acid residue at position 168 has been replaced with a threonine residue. In some embodiments, the target hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S99, S130, S141, S163, K157, R174, N286, K281, and Q298; and includes T168, S168, or A168. In some embodiments, the target hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; includes S99, S130, S141, S163, K157, R174, N286, K281, and Q298; and includes T168, S168, or A168, where the amino acid numbering is as set forth in SEQ ID NO: 11. In some embodiments, the first 51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 63). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the target anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0297] In some embodiments, the target hyperpolarizing light-activated polypeptide iReaChR comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 48; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S99, S130, S141, S163, K157, R174, N286, K281, and Q298; includes A196 or N196; and includes T168, S168, or A168, where the amino acid numbering is as set forth in SEQ ID NO: 48. In some embodiments, the target hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 48; includes S99, S130, S141, S163, K157, R174, N286, K281, and Q298; includes A196 or N196; and includes T168, S168, or A168, where the amino acid numbering is as set forth in SEQ ID NO: 48. In some embodiments, the first 51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO: 63). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the target anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0298] In some embodiments, the target hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the protein ReaChR with one or more of the above-mentioned modifications, wherein the aspartic acid amino acid residue at the original position 196 has been replaced with an alanine residue. In some embodiments, wherein the first 51 N-terminal amino acids of the protein are replaced with amino acids 1-11 from the protein ChR2, the aspartic acid amino acid residue at position 156, which corresponds to the original position 196 of the ReaChR amino acid sequence set forth in SEQ ID NO: 11, is replaced with an alanine residue.

[0299] In some embodiments, the target hyperpolarizing light-activated polypeptide is based on the amino acid sequence of the protein ReaChR with one or more of the above-mentioned modifications, wherein the aspartic acid amino acid residue at original position 196 has been replaced with an asparagine residue. In some embodiments, wherein the first 51 N-terminal amino acids of the protein are replaced with amino acids 1-11 from the protein ChR2, the aspartic acid amino acid residue at position 156 (which corresponds to the original position 196 of the ReaChR amino acid sequence set forth in SEQ ID NO: 11) is replaced with an asparagine residue.

[0300] In some embodiments, the target hyperpolarizing light-activated polypeptide ibReaChR comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 50; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258; and includes T128, S128, or A128, where the amino acid numbering is as set forth in SEQ ID NO: 50. In some embodiments, the target hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% amino acid sequence %, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 50; includes S59, S90, S101, S123, K117, R134, N246, K242, and Q258; and includes T128, where the amino acid numbering is as set forth in SEQ ID NO: 50. In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the target anion channel polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0301] In some embodiments, the target hyperpolarizing light-activated polypeptide ibReaChR comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 50; includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of S59, S90, S101, S123, K117, R134, N246, K242, and Q258; includes T128, S128, or A128; and includes A156 or N156, where the amino acid numbering is as set forth in SEQ ID NO: 50. In some embodiments, the target hyperpolarizing light-activated polypeptide comprises an amino acid sequence having at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 50; includes S59, S90, S101, S123, K117, R134, N246, K242, and Q258; includes T128, S128, or A128; and includes A156 or N156, where the amino acid numbering is as set forth in SEQ ID NO: 50. In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any of these embodiments, the target anion channel polypeptide includes an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)). In any of these embodiments, the target anion channel polypeptide comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 58)).

[0302] Utility

[0303] The methods of the present disclosure have a variety of utilities. As described above, the methods of the present disclosure can be used to modulate the temporal pattern of neuronal activity in one or more regions of the brain using fMRI, optogenetics, and / or electrophysiological recordings. In some cases, the present methods can provide a way to identify new roles of anatomically and / or functionally defined neurons in functional circuits.

[0304] In some cases, the present methods determine the specific circuit mechanisms by which VLOs control global brain neural activity. Thalamic inputs to VLOs play a key role in modulating the perceived pain level during noxious stimulation and support goal-directed behavior by sending predictive cues and anticipating outcomes.

[0305] In certain embodiments, the present methods provide for selective activation of specific populations of neurons at different temporal frequencies by the combination of selective expression of a light-activated polypeptide and selective illumination of a brain region, where the number of neurons activated at each frequency remains substantially the same. Thus, the effect of an increase in the frequency of light pulses that activate a first region on the response of a second region of functional connectivity of the brain can be attributed primarily to the change in frequency, and not other factors, e.g., recruiting more neurons in a frequency-dependent manner.

[0306] The methods of the present invention can also be used to probe deep brain stimulation (DBS) of brain regions such as the centromedian thalamus, insula, cingulate, subthalamic nucleus (STN), globus pallidus interna (GPI), zona incerta (ZI), and the like, which can be used to treat a variety of neurological disorders, such as pain, depression, addiction, Alzheimer’s disease, attention deficit disorder, autism, anhedonia, cerebral palsy, bipolar depression, unipolar depression, epilepsy, generalized anxiety disorder, acute head trauma, hedonism, obesity, obsessive compulsive disorder (OCD), acute pain, chronic pain, Parkinson’s disease, persistent vegetative state, phobia, post-traumatic stress disorder, post-stroke rehabilitation / regeneration, post-head trauma, social anxiety disorder, Tourette’s syndrome, hemorrhagic stroke, and ischemic stroke. In some cases, the methods of the present invention can provide a way to probe the effects of individual stimulation parameters, such as light pulse frequency or pulse width, of specific populations of neurons on global brain dynamics as well as cell-level functional circuits.

[0307] Examples of non-limiting aspects of the present disclosure

[0308] 1. A method for modulating the temporal pattern of neuronal activity in the brain of an individual, the method comprising:

[0309] i) stimulating one or more of thalamocortical projections in the brain, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral subthalamic nucleus, and cell bodies in the ventrolateral orbitofrontal cortex (VLO) with light pulses from an optical light source, wherein neuronal cell bodies in one or more of the VLO and thalamus of the individual express a light-activated polypeptide; and

[0310] ii) measuring functional magnetic resonance imaging (fMRI) signals of the global brain, wherein the measuring occurs during the stimulating,

[0311] wherein positive measured fMRI signals correlate with an increase in neuronal activity following stimulation and wherein negative measured fMRI signals correlate with a decrease in neuronal activity following stimulation.

[0312] 2. The method of aspect 1, wherein whole brain comprises ipsilateral regions and contralateral regions of the brain.

[0313] 3. The method of aspect 2, wherein the ipsilateral regions comprise the left hemisphere of the brain, including the medial prefrontal cortex, lateral prefrontal cortex, motor cortex, cingulate cortex, sensory cortex, insular cortex, striatum, and thalamus.

[0314] 4. The method of aspect 2, wherein the contralateral regions comprise the right hemisphere of the brain, including the medial prefrontal cortex, lateral prefrontal cortex, motor cortex, cingulate cortex, sensory cortex, insular cortex, striatum, and thalamus.

[0315] 5. The method of aspect 1, wherein the frequency of the light pulses is 5 Hz to 40 Hz.

[0316] 6. The method of aspect 5, wherein the frequency of the light pulses is 10 Hz.

[0317] 7. The method of aspect 5, wherein the frequency of the light pulses is 40 Hz.

[0318] 8. The method of aspect 1, wherein stimulating the thalamocortical projections with light pulses at a frequency of 10 Hz or higher results in negative fMRI signals in the sensory, motor, and cingulate cortex of the ipsilateral regions of the brain.

[0319] 9. The method of aspect 1, wherein stimulating the thalamocortical projections with light pulses at a frequency of 5 Hz or higher results in negative fMRI signals in the sensory, motor, and cingulate cortex of the ipsilateral regions of the brain.

[0320] 10. The method of aspect 1, wherein stimulating the thalamocortical projections with light pulses at a frequency of 40 Hz or higher results in positive measured fMRI signals.

[0321] 11. The method of aspect 1, wherein stimulating the thalamocortical projections with light pulses at a frequency of 5 Hz or higher results in negative measured fMRI signals in the contralateral regions of the brain.

[0322] 12. The method of aspect 1, wherein stimulating the thalamocortical projections with light pulses at a frequency of 10 Hz or higher results in negative measured fMRI signals in the contralateral regions of the brain.

[0323] 13. The method of aspect 1, wherein stimulating cell bodies in the VLO with light pulses at a frequency of 5 Hz to 40 Hz results in positive measured fMRI signals in ipsilateral regions of the brain.

[0324] 14. The method of aspect 1, wherein the light-activated polypeptide is expressed in neurons of the central subnucleus of the thalamus.

[0325] 15. The method of aspect 1, wherein the light-activated polypeptide is expressed in I- and III-layer neurons of the VLO of the brain.

[0326] 16. The method of aspect 1, wherein the method further comprises reversibly inserting an optical light source in the VLO of the individual.

[0327] 17. The method of aspect 1, wherein the method further comprises stimulating the VLO of the brain.

[0328] 18. The method of aspect 17, wherein stimulating the VLO of the brain results in positive measured fMRI signals at the VLO of the brain.

[0329] 19. The method of aspect 1, wherein stimulating the thalamocortical projections with light pulses at a frequency of 5 Hz to 20 Hz results in negative measured fMRI signals in contralateral regions of the brain including the prefrontal cortex.

[0330] 20. The method of aspect 1, wherein stimulating cell bodies with light pulses at a frequency of 40 Hz or higher increases neuronal activity in ipsilateral thalamus of the brain.

[0331] 21. The method of aspect 1, wherein stimulating the thalamocortical projections with light pulses at a frequency of 20 Hz to 40 Hz activates neuronal activity in ipsilateral thalamus of the brain.

[0332] 22. The method of aspect 1, wherein stimulating cell bodies of the central subnucleus of the thalamus results in positive measured fMRI signals in ipsilateral thalamus of the brain.

[0333] 23. The method of aspect 1, wherein stimulating the thalamocortical projections with light pulses at a frequency of 5 Hz or higher suppresses neuronal activity in ipsilateral thalamus of the brain.

[0334] 24. The method of aspect 1, wherein measuring fMRI signals comprises measuring cerebral blood volume (CBV).

[0335] 25. The method of aspect 1, wherein the method further comprises administering a second light-activated polypeptide.

[0336] 26. The method of aspect 25, wherein the second light-activated polypeptide is administered to a zona incerta (ZI) region of the brain.

[0337] 27. The method of aspect 1, wherein the method further comprises performing electrophysiological recordings to detect firing frequency of neurons in one or more brain regions correlated with the measured fMRI signal.

[0338] 28. The method of aspect 27, wherein the one or more brain regions comprise ipsilateral VLO of the brain.

[0339] 29. The method of aspect 28, wherein a positive measured fMRI signal correlates with increased firing frequency of neurons in the ipsilateral VLO.

[0340] 30. The method of aspect 27, wherein the one or more brain regions comprise contralateral VLO.

[0341] 31. The method of aspect 30, wherein a negative measured fMRI signal correlates with decreased firing frequency of neurons in the contralateral VLO.

[0342] 32. The method of aspect 31, wherein stimulation with light pulses at a frequency of 10 Hz or higher results in decreased firing frequency of neurons in the contralateral VLO.

[0343] 33. The method of aspect 27, wherein the one or more brain regions is ipsilateral motor cortex.

[0344] 34. The method of aspect 33, wherein stimulation with light pulses at a frequency of 10 Hz or higher results in decreased firing frequency of neurons in the ipsilateral motor cortex.

[0345] 35. The method of aspect 33, wherein stimulation with light pulses at a frequency of 40 Hz or higher results in increased firing frequency of neurons in the ipsilateral motor cortex.

[0346] 36. A method of modulating pain in an individual, the method comprising:

[0347] stimulating one or more of thalamocortical projections, thalamic relay neurons, corticofugal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the ventrolateral orbitofrontal cortex (VLO) in the brain of the individual with one or more light pulses, wherein neuronal cell bodies in one or more of the VLO and the thalamus of the individual express a light-activated polypeptide, wherein the stimulation modulates pain in the individual.

[0348] 37. The method of aspect 36, wherein stimulating one or more of thalamocortical projections, thalamic relay neurons, corticofugal neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with a first set of light pulses suppresses neuronal activity in response to noxious stimuli.

[0349] 38. The method of aspect 36, wherein stimulating one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with the first set of light pulses inhibits neuronal activity associated with aversive or painful sensation in the orbitofrontal cortex of the brain.

[0350] 39. The method of aspect 36, wherein stimulating one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain with the second set of light pulses activates neuronal activity associated with aversive or painful sensation in the orbitofrontal cortex of the brain.

[0351] 40. A system for modulating temporal patterns of neuronal activity in the brain of an individual, the system comprising:

[0352] i) a light source configured to stimulate one or more of thalamocortical projections, thalamic relay neurons, cortical projection neurons, cell bodies in the centrolateral nucleus of the thalamus, and cell bodies in the VLO in the brain of the individual with light pulses, wherein light-responsive opsin polypeptides are expressed in cell bodies of one or more of the ventrolateral orbitofrontal cortex (VLO) and the thalamus of the brain; and

[0353] ii) a functional magnetic resonance imaging (fMRI) device configured to scan the whole brain during the stimulation to produce fMRI signals;

[0354] wherein a positive measured fMRI signal is associated with an increase in neuronal activity after the stimulation, wherein a negative measured fMRI signal is associated with a decrease in neuronal activity after the stimulation.

[0355] 41. The system of aspect 40, wherein the whole brain comprises ipsilateral and contralateral regions of the brain.

[0356] 42. The system of aspect 41, wherein the ipsilateral region comprises the left hemisphere of the brain, including the medial prefrontal cortex, lateral prefrontal cortex, motor cortex, cingulate cortex, sensory cortex, insular cortex, striatum, and thalamus.

[0357] 43. The system of aspect 41, wherein the contralateral region comprises the right hemisphere of the brain, including the medial prefrontal cortex, lateral prefrontal cortex, motor cortex, cingulate cortex, sensory cortex, insular cortex, striatum, and thalamus.

[0358] 44. The system of aspect 40, wherein the frequency of the light pulses is 5 Hz to 40 Hz.

[0359] 45. The system of aspect 44, wherein the frequency of the light pulses is 10 Hz or higher.

[0360] 46. The system of aspect 44, wherein the frequency of the light pulses is 40 Hz or higher.

[0361] 47. The system of aspect 40, wherein stimulating the thalamocortical projections with light pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal in the sensory, motor, and cingulate cortex of the ipsilateral region of the brain.

[0362] 48. The system of aspect 40, wherein the light source is reversibly inserted in the VLO of the individual.

[0363] 49. The system of aspect 40, wherein stimulating the thalamocortical projections with light pulses at a frequency of 40 Hz or higher results in a positive measured fMRI signal.

[0364] 50. The system of aspect 40, wherein stimulating the thalamocortical projections with light pulses at a frequency of 10 Hz or higher results in a negative measured fMRI signal in the contralateral region of the brain.

[0365] 51. The system of aspect 40, wherein stimulating cell bodies in the VLO with light pulses at a frequency of 5 Hz to 40 Hz results in a positive measured fMRI signal in the ipsilateral region of the brain.

[0366] 52. The system of aspect 40, wherein the light activation is expressed in layer I and layer III neurons of the VLO of the brain.

[0367] 53. The system of aspect 40, wherein the implantable light source is implanted in a dorsal location of the VLO of the brain.

[0368] 54. The system of aspect 40, wherein stimulating with light pulses at a frequency of 5 Hz to 10 Hz suppresses neuronal activity of the ipsilateral thalamus of the brain.

[0369] 55. The system of aspect 40, wherein the fMRI signal comprises cerebral blood volume (CBV).

[0370] 56. The system of aspect 40, wherein the system further comprises a second light-activated polypeptide expressed in neurons of the limbic area of the brain.

[0371] 57. The system of aspect 40, wherein the system further comprises an electrophysiological recording device configured to detect firing frequency of neurons in one or more brain regions related to the measured fMRI signal.

[0372] 58. The system of aspect 57, wherein the one or more brain regions comprise the ipsilateral VLO of the brain.

[0373] 59. The system of aspect 58, wherein the positive fMRI signal is associated with an increase in firing frequency of neurons in the ipsilateral VLO of the brain.

[0374] Embodiments

[0375] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of what the inventors regard as their application, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations can be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid; kb, kilobase(s); bp, base pair(s); nt, nucleotide; i.m., intramuscularly; i.p., intraperitoneally; s.c., subcutaneously; and the like.

[0376] Example 1: Thalamic input drive to orbitofrontal cortex is mediated by GABA and undetermined band-mediated whole-brain frequency-dependent suppression Results

[0377] Applying optogenetic fMRI to the various elements that drive the VLO circuit, while visualizing the whole-brain response. Surprisingly, driving excitatory thalamocortical projections to the VLO at low frequencies (5-10 Hz) elicited a widespread, bilateral reduction in brain activity that spanned multiple cortical and subcortical structures. This pattern was unique to thalamocortical projections, and was not elicited by direct stimulation of either the VLO or the thalamus. High-frequency stimulation (25-40 Hz) of thalamocortical projections elicited a markedly different but still far-reaching response in the form of widespread ipsilateral activation. Importantly, the reduction in brain activity elicited by low-frequency thalamocortical input was mediated by GABA and zona incerta activity. These findings identify a specific circuit mechanism by which the VLO can control neural activity throughout the brain.

[0378] While evidence suggests that the VLO has a global role in brain function, the circuit mechanisms by which it achieves this influence have not been directly studied. To better understand how the VLO supports different behavioral processes, a technical approach that can control individual circuit elements and visualize the whole-brain response is needed. Optogenetic fMRI (ofMRI), the combination of optogenetic stimulation with whole-brain functional magnetic resonance imaging, was applied to directly visualize the global impact of the VLO’s afferent and efferent connections. Aspects of the present disclosure investigated how different temporal patterns of activity in the VLO circuit, by driving its inputs and outputs at different frequencies, influence brain dynamics.

[0379] FIG. 1A

[0380] The effects of thalamocortical projections on the VLO were first investigated by stimulating the thalamic terminals here. Adeno-associated viruses carrying the ChR2-EYFP excitatory opsin were injected into the centrolateral nucleus of the thalamus ( FIG. 8A ). To achieve targeted transfection, the CaMKIIa promoter was used, which is expressed in the thalamus mainly in excitatory relay neurons (Smith, 2008). This resulted in strong membrane-bound expression of ChR2 at the injection site ( FIGS. 8B-8C ). Ex vivo histology confirmed strong expression of ChR2 in layers I and III of the VLO ( FIG. 1A ), which is consistent with the known termination pattern of the injected nucleus (Krettek and Price, 1977). Stimulation of ChR2-positive terminals in the cortex was achieved by implanting optical fibers in the VLO ( FIG. 1B ).

[0381] Optogenetic fMRI experiments were performed to visualize the dynamic, whole-brain response to thalamocortical stimulation at different frequencies. Light pulses were delivered at a frequency of 10 Hz or 40 Hz. Imaging was performed on 23 coronal slices ( FIG. 1C ). Standard general linear model (GLM) statistical techniques were used to identify voxels that were significantly modulated during stimulation ( FIGS. 9A-9D ). Responses were highly consistent across scans and subjects ( FIG. 1D ).

[0382] Frequency control of thalamocortical stimulation in the VLO modulates both hemispheres

[0383] fMRI activation maps show that the stimulation frequency is a key parameter in determining the spatial extent of ipsilateral and contralateral modulation ( FIG. 10A , 1E). Both stimulation frequencies produced strong positive responses in the VLO stimulation site as well as the ipsilateral thalamus and striatum. The 10 Hz stimulation drove a measured negative response in both hemispheres, which spanned the cortex, the contralateral striatum, and the contralateral thalamus. The 40 Hz stimulation elicited strong positive activation throughout the ipsilateral cortex, but the contralateral hemisphere was largely unmodulated. Only a tiny negative response was observed in the prefrontal cortex and striatum.

[0384] To quantify the response patterns, the number of significantly modulated voxels was calculated in anatomically defined regions of interest (ROIs; FIG. 10B ). In the ipsilateral hemisphere, the number of modulated voxels increased between 10 Hz and 40 Hz for all segmented regions of the cortex and striatum ( FIG. 10D ; p < 0.05, N = 11 animals). In contrast, in the contralateral hemisphere, the amount of modulated volume decreased between 10 Hz and 40 Hz ( FIGS. 12A-12B). These results indicate that the firing frequency of thalamic input to the VLO determines the spatial extent of downstream modulation. The ipsilateral hemisphere modulates the most during 40 Hz stimulation, while the contralateral hemisphere modulates the most during 10 Hz stimulation. The same frequency-dependent trends were observed when a constant pulse width was used in control experiments FIG. 10C

[0385] Frequency control of induced response polarity of thalamic cortical stimulation in the VLO

[0386] The temporal dynamics of whole-brain responses were then examined. The quantitative measure of each ROI response polarity was calculated as the sum of its mean fMRI time series (∑fMRI). In the ipsilateral hemisphere, sensory, cingulate, and motor cortex exhibited a significant negative response during 10 Hz stimulation FIG. 1F ; p < 0.05, N = 11 animals). These same regions, including the striatum, LPFC, and MPFC, exhibited a significant positive response during 40 Hz stimulation. Visualization of the time series of the whole ipsilateral cortex largely corroborated the ∑fMRI measure FIG. 10E ; p < 0.05, N = 11 animals). These same regions, including the striatum, LPFC, and MPFC, exhibited a significant positive response during 40 Hz stimulation. Visualization of the time series of the whole ipsilateral cortex largely corroborated the ∑fMRI measure

[0387] The quantitative measure of ∑fMRI in the contralateral hemisphere differed greatly from those observed in the ipsilateral hemisphere. Cortex and striatum exhibited a significant negative response during 10 Hz stimulation FIG. 1G ; p < 0.05, N = 11 animals), but the ∑fMRI values were not significantly different from zero during 40 Hz stimulation. Visualization of the time series of the whole contralateral cortex corroborated the sharp drop in activity here during 10 Hz stimulation FIG. 12C ; p < 0.05, N = 11 animals). These data illustrate that thalamic input to the VLO produces a broad, hemispheric effect by suppressing remote activity in a frequency-dependent manner. Importantly, this effect was preserved when the pulse width was held constant in control experiments FIG. 2A -12D), which confirms that stimulation frequency is the primary factor determining the polarity of stimulation-induced responses.

[0388] Frequency sweep experiments reveal region-dependent shifts in response patterns

[0389] To explore how the frequency-dependent changes manifest, a second series of imaging sessions was performed on a subset of the animals reported above (N = 7). Stimulation was performed at 5 Hz intervals over a frequency range of 5 Hz to 40 Hz. The resulting activation maps are shown in FIG. 2B ​Mid. Stimulation at all frequencies elicited positive responses at the stimulation site. The negative responses observed during 10 Hz stimulation were observed across the entire contralateral hemisphere at frequencies from 5 Hz up to 20 Hz. At 25 Hz and above, the negative responses in the contralateral hemisphere were generally limited to the prefrontal cortex. Interestingly, widespread activation of the ipsilateral cortex also began to emerge at 25 Hz. To quantify this effect, the percentage of voxels with significant modulation at each frequency was examined FIG. 2C ). Several regions exhibited a large increase in the volume of positive modulation between 20 Hz and 25 Hz, suggesting that a threshold for widespread prefrontal activation had been reached. The volume of negative modulation was also larger during 5 Hz stimulation compared to 10 Hz stimulation, suggesting that the circuit mechanisms responsible for the negative signal have an even stronger effect at lower frequencies. Notably, the transition from negative to positive responses observed in sensory, motor, and cingulate cortex occurred between 10 Hz and 15 Hz stimulation. Time series extracted from these ROIs confirmed this trend FIGS. 3A-3B ).

[0390] Thalamic-cortical projections to the VLO uniquely drive widespread negative fMRI signals

[0391] Thalamic-cortical projections to the VLO represent only one neuronal element in the perturbed circuit. To better understand the origin of the fMRI responses, pyramidal neurons in the VLO were also stimulated. Neither 10 Hz nor 40 Hz stimulation of cell bodies in the VLO drove negative fMRI responses in any region FIGS. 3C-3D ). Similar to stimulating the thalamic-cortical projections, 40 Hz stimulation of cell bodies drove ipsilateral thalamic activation. However, the widespread cortical activation observed during stimulation of the thalamic-cortical projections did not occur. These data suggest that direct activation of the VLO does not elicit the same frequency-dependent or widespread inhibitory effects induced by thalamic input to this region. Cell bodies in the central subnucleus of the thalamus, which project heavily to the VLO, were then stimulated. As shown in the group-level activation map FIGS. 1A-1G ), driving these relay neurons at 10 Hz and 40 Hz elicited strong responses in the VLO but could not elicit negative fMRI responses in any region. Thus, direct stimulation of projections to the VLO elicits a completely different response than stimulating the cell bodies where the projections terminate.

[0392] Neuronal basis of whole-brain, frequency-dependent fMRI signals

[0393] Several classes of frequency-dependent fMRI responses were observed throughout the brain during stimulation of thalamic input to the VLO FIG. 4A ). To investigate how these dynamics relate to neuronal activity, a series of in vivo electrophysiological experiments were performed. Extracellular recordings were first made at the stimulation site in the ipsilateral VLO FIG. 4B), where fMRI responses were positive during both 10 Hz and 40 Hz stimulation FIG. 4C Ring event time histograms from representative units show that these signal changes are associated with corresponding increases in spiking ( FIG. 4D ). Over half of all recorded units were modulated by stimulation at either frequency ( FIG. 12A and FIG. 4E ; 60% and 54% during 10 Hz and 40 Hz stimulation, respectively; N = 151 units, 5 animals, 10 trials per frequency). Furthermore, the firing rate of nearly all modulated units was significantly increased (99% and 96%, respectively). The median change in firing rate was not significantly different between the two frequencies ( FIG. 4F ). These results confirm that the positive fMRI signals observed at the stimulation site reflect underlying increases in neuronal activity.

[0394] It was then investigated whether the negative fMRI signals observed throughout the contralateral cortex during 10 Hz stimulation reflect underlying decreases in neuronal activity, and whether this modulation is suppressed at higher stimulation frequencies as suggested by fMRI. Extracellular recordings were made in the contralateral VLO (cVLO; FIG. 4G ) where negative fMRI signals were observed during 10 Hz stimulation, but little modulation was observed during 40 Hz stimulation ( FIG. 4H ). Ring event time histograms from representative units in the cVLO confirm that this pattern is observed at the level of single unit activity ( FIG. 4I ). Of all recorded units, 95% exhibited a significant decrease in firing rate during 10 Hz stimulation ( FIG. 12B and FIG. 4J ; N = 55 units, 2 animals, 20 trials per frequency). During 40 Hz stimulation, 78% of recorded units exhibited no significant change, and only 18% of activity exhibited a significant decrease. The median change in firing rate was significantly different between the two frequencies ( FIG. 4K ). These results confirm that low frequency thalamic input to the VLO preferentially drives decreases in spiking in the contralateral cortex.

[0395] Finally, it was examined whether the frequency-dependent switch in fMRI polarity observed in the ipsilateral cortex is associated with corresponding changes in neuronal activity. Recordings were then made in the ipsilateral motor cortex ( FIG. 4L ) where negative fMRI signals were observed during 10 Hz stimulation, but positive signals were evoked during 40 Hz stimulation ( FIG. 4M ). Ring event time histograms from representative units show that this behavior is consistent with underlying spiking dynamics ( FIG. 4N). Of all recorded units, 46% showed a significant decrease in firing rate during 10 Hz stimulation FIG. 12C and FIG. 4O ; N = 99 units, 4 animals, 20 trials per frequency) and the rest did not show a significant change. During 40 Hz stimulation, 82% of recorded units showed a significant increase in firing rate and none showed a significant decrease. The median change in firing rate was significantly different between the two frequencies FIG. 13A ). These data confirm that the frequency-dependent shift in cortical response polarity measured with fMRI reflects underlying spiking activity.

[0396] Mechanisms of evoked cortical activity reduction

[0397] Having established that negative fMRI signals in cortex reflect a reduction in neuronal spiking, the next step was to determine the mechanism of this frequency-dependent response. The thalamic reticular nucleus (TRN) was hypothesized to play a role as it directly inhibits thalamic nuclei and can suppress excitatory input to cortex (Lewis et al., 2015, eLife 4, e08760; Pinault, 2004, Brain Res Rev 46, 1-3). To examine whether activity in the TRN was related to the reduction in cortical firing, extracellular recordings were made in the TRN during 10 Hz and 40 Hz thalamocortical stimulation FIG. 13B ). Given the frequency dependence of the evoked reduction in cortical activity, it was expected that 10 Hz stimulation would evoke the strongest response in the TRN. However, the percentage of units showing a significant increase in firing rate more than doubled between 10 Hz and 40 Hz stimulation FIGS. 13C-13D ; 25% and 69% respectively; N = 123 units, 2 animals, 10-20 trials per frequency). The median change in firing rate of recorded units was significantly different between the two frequencies, with 40 Hz stimulation driving a greater change FIG. 13E To investigate whether a transhemispheric pathway was involved, recordings were made in the contralateral reticular nucleus FIG. 13F ). During 10 Hz stimulation, 54% of units here were modulated, of which 98% showed a significant decrease in firing rate FIGS. 13G-13H , N = 199 recorded units, 20 trials per frequency). During 40 Hz stimulation, 13% of units were modulated, of which 80% showed a significant increase in firing rate. The median change in firing rate of all recorded units was significantly different between the two frequencies, with 40 Hz stimulation again driving a greater change FIG. 5A . These data suggest that inhibition of thalamus by the TRN is not the primary cause of the reduction in cortical activity observed during low frequency thalamocortical stimulation.

[0398] It was then hypothesized that the reduction in cortical activity reflected direct GABA-mediated inhibition. The dependence of this response on downstream GABA release was tested by comparing changes in firing rate before and after microinfusion of bevantolol (BMI). Although BMI can exhibit mixed pharmacological actions (e.g., block of calcium-activated potassium channels), it is a potent antagonist of GABA A Single-unit recordings were made in cVLO, where negative fMRI signals were observed during 10 Hz thalamocortical stimulation ( FIG. 5B ). To ensure that any changes associated with BMI infusion were due to its pharmacological action, a sterile saline infusion was first performed. To inject BMI and saline, a pair of cannulae was attached directly to the recording electrode above the electrical contacts ( FIG. 5C ).

[0399] Consistent with expectations, the effect of saline infusion on cortical inhibition evoked by 10 Hz stimulation was negligible. Of the 49 units recorded in cVLO, 94% showed a significant decrease in firing rate during 10 Hz stimulation before and after saline infusion ( FIG. 5D ; N = 2 animals, 20 trials per condition). Furthermore, saline infusion did not significantly affect the median change in firing rate evoked by 10 Hz stimulation ( FIG. 5C ). In contrast, infusion of BMI completely eliminated the stimulus-evoked inhibition ( FIG. 5D ). Before BMI infusion, 86% of the units showed a significant decrease in firing rate evoked by 10 Hz stimulation. After BMI infusion, the recorded units either were not modulated by the stimulus (96%) or showed a significant increase in firing rate (4%). The median change in firing rate evoked by the stimulus was also significantly different ( FIG. 5E ). Importantly, the baseline firing rate in cVLO did not change after BMI infusion in most units ( FIG. 5F ). Those that did show a difference were roughly split between an increase (27%) and a decrease (18%). Furthermore, the mean baseline firing rate after BMI infusion was not significantly different across all units (p = 0.66). These analyses suggest that the effect of the drug is related to the stimulus-evoked GABA release and is not a general decrease in local inhibitory behavior. The reduction in stimulus-evoked inhibition took effect immediately after BMI infusion and persisted for the subsequent 20 trials ( FIG. 5G ). Peri-stimulus time histograms from a representative unit illustrate how the stimulus-evoked decrease in firing rate was observed after saline infusion but abolished after BMI infusion ( FIG. 6A ). These results indicate that widespread cortical inhibition driven by low-frequency thalamic input to VLO is mediated by the release of GABA at remote sites downstream of the excitatory terminal.

[0400] Cortical inhibition driven by thalamic input to VLO is mediated by zona incerta

[0401] Having established that GABA drives the reduction in cortical activity, we determined the potential source of the inhibitory neurotransmitter and investigated the role of the zona incerta (ZI). In addition to receiving a stimulating projection from VLO and sparse input collaterals (Kuramoto et al., 2017, The Journal of comparative neurology 525, 3821-3839; Shammah-Lagnado et al., 1985, Neuroscience 15, 109-134), ZI sends GABAergic projections to the entire neocortex bilaterally (Lin et al., 1990, Science 248, 1553-1556) and has previously been shown to mediate the reduction in cortical firing frequency caused by 10 Hz thalamic stimulation (Liu et al., 2015, eLife 4, e09215). To examine whether this region mediates the widespread inhibition observed throughout the forebrain, we inactivated its activity during simultaneous 10 Hz thalamic cortical stimulation and cVLO recording.

[0402] ZI activity was first inactivated by incertal infusion of the sodium channel blocker lidocaine hydrochloride (Liu et al., 2015, eLife 4, e09215). FIGS. 14A-14B and FIG. 6B Incertal saline infusion did not affect the remote inhibition driven by 10 Hz stimulation. The firing frequency of at least 98% of units recorded in cVLO exhibited a significant reduction during stimulation before and after saline infusion ( FIG. 6B ; N = 62 units, 20 trials per condition). In contrast, after inactivation of the zona incerta with lidocaine, only 72% of units were inhibited during 10 Hz stimulation ( FIG. 6D ). The median change in firing frequency evoked by stimulation also differed significantly between the three conditions ( FIG. 6C , p = 1.1 x 10 -12 , χ 2 = 55.0, 185 degrees of freedom). Post-hoc tests confirmed that the change evoked by stimulation after saline infusion did not differ from the baseline value (p = 0.17), whereas the change evoked by stimulation after lidocaine infusion differed from both the baseline and the value after saline (p = 9.6 x 10 -10 and 3.0 x 10 -7 respectively). The reduction in evoked inhibition took effect immediately after lidocaine infusion and persisted for the subsequent twenty trials ( FIG. 6E ). Peri-stimulus time histograms from representative units show that, in a subset of units recorded, incertal infusion of lidocaine, but not saline, completely abolished the remote cortical inhibition driven by 10 Hz stimulation (FIG. 14D .

[0403] To confirm the role of the ZI in mediating remote cortical inhibition, similar experiments were performed using the inhibitory opsin eNpHR. In addition to normal ChR2-EYFP injections into the thalamus, an adeno-associated virus carrying eNpHR-mCherry under the control of the pan-neuronal hSyn promoter was injected into the zona incerta ( FIGS. 14E-14G ). This potently inhibited zona incerta activity ( FIG. 7A ). To assess the role of the ZI in remote inhibition, single unit recordings were made in the cVLO and zona incerta during 10 Hz stimulation, with and without simultaneous activation of eNpHR ( FIG. 7B ). These stimulation paradigms were alternated to ensure that differences between them were attributable to eNpHR activation ( FIG. 14C ). The light pole placement in the zona incerta was verified by confirming that the ZI had the known property (Nicolelis et al., 1992, Brain Res 577, 134-141) of the recorded population responding to contralateral whisker stimulation ( FIG. 7C ).

[0404] Of the 26 units recorded in the zona incerta, 65% exhibited a significant increase in firing rate during 10 Hz thalamocortical stimulation ( FIG. 7C , 7G). Thus, the zona incerta was recruited during the stimulation paradigm that drives widespread inhibition. Simultaneous activation of eNpHR during thalamocortical stimulation disrupted this recruitment. In 96% of recorded units, the firing rate during 10 Hz stimulation was actually lower than the pre-sti...

Claims

1. A system for modulating temporal patterns of neuronal activity in an individual's brain using fMRI, optogenetics, and / or electrophysiological recordings, the system comprising: i) A light source configured to stimulate one or more of the following in the brain of an individual: thalamic cortical projection, thalamic relay neurons, cortical projection neurons, cell bodies in the subcentral nucleus of the thalamus, and cell bodies in the VLO, wherein a light-responsive opsin polypeptide is expressed in one or more of the following cell bodies in the ventrolateral orbitofrontal cortex (VLO) and the thalamus. and ii) A functional magnetic resonance imaging (fMRI) device configured to scan the whole brain during stimulation to generate fMRI signals, wherein the fMRI signals include cerebral blood volume (CBV); iii) A computing unit used to control and / or coordinate light stimulation via one or more controllers and to analyze data from fMRI scans of brain regions; The percentage signal change calculated from the original fMRI signal is inverted. Positive fMRI signal measurements were associated with increased neuronal activity after stimulation, while negative fMRI signal measurements were associated with decreased neuronal activity after stimulation. The polarity of the response in regions of interest (ROIs) throughout the whole brain was calculated by summing the average values ​​of the time series of fMRI signals associated with the ROIs, and The time series mean is calculated based on voxel orientation using fMRI signals as a percentage of the modulated response relative to the baseline period collected before light source stimulation.

2. The system of claim 1, wherein the whole brain includes the ipsilateral and contralateral regions of the brain.

3. The system according to claim 2, wherein the ipsilateral region comprises the left hemisphere of the brain, including the medial prefrontal cortex, the lateral prefrontal cortex, the motor cortex, the cingulate cortex, the sensory cortex, the insular cortex, the striatum, and the thalamus.

4. The system of claim 2, wherein the contralateral region comprises the right hemisphere of the brain, including the medial prefrontal cortex, the lateral prefrontal cortex, the motor cortex, the cingulate cortex, the sensory cortex, the insular cortex, the striatum, and the thalamus.

5. The system according to claim 1, wherein the frequency of the optical pulse is from 5 Hz to 40 Hz.

6. The system of claim 1, wherein the frequency of the optical pulse is 10 Hz or higher.

7. The system of claim 1, wherein the frequency of the optical pulse is 40 Hz or higher.

8. The system of claim 1, wherein stimulation of the thalamic cortex projection with light pulses of 10 Hz or higher results in negative fMRI signals measured in the ipsilateral sensory, motor, and cingulate cortex of the brain.

9. The system of claim 1, wherein the optical light source is reversibly inserted into the individual VLO.

10. The system of claim 1, wherein stimulation of the thalamic cortex projection with light pulses at a frequency of 40 Hz or higher results in a positive measured fMRI signal.

11. The system of claim 1, wherein stimulation of the thalamic cortex projection with light pulses of 10 Hz or higher results in a negative measured fMRI signal in the contralateral area of ​​the brain.

12. The system of claim 1, wherein stimulation of cell bodies in the VLO with light pulses of frequency from 5 Hz to 40 Hz results in a positive measured fMRI signal in the ipsilateral region of the brain.

13. The system of claim 1, wherein the photoactivated polypeptide is expressed in neurons of layer I and layer III of the VLO in the brain.

14. The system of claim 1, wherein the implantable light source is implanted at the dorsal position of the VLO in the brain.

15. The system of claim 1, wherein light pulses at a frequency of 5 Hz to 10 Hz are used to stimulate and inhibit neuronal activity in the ipsilateral thalamus of the brain.

16. The system of claim 1, wherein the system further comprises a second photoactivated polypeptide expressed in neurons in the undefined zone region of the brain.

17. The system of claim 1, wherein the system further comprises an electrophysiological recording device configured to detect the firing frequency of neurons in one or more brain regions associated with the measured fMRI signal.

18. The system of claim 17, wherein one or more brain regions include the ipsilateral VLO of the brain.

19. The system of claim 18, wherein a positive fMRI signal is associated with an increased firing frequency of neurons in the ipsilateral VLO of the brain.

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