Systems and methods for preventing, mitigating and / or treating dementia

Abstract

To provide systems and methods for preventing, mitigating and / or treating dementia in a subject.SOLUTION: The present disclosure provides systems and methods for at least one of preventing, reducing and treating a level of or change in at least one of amyloid-β (Aβ) peptide, C-terminal fragment-β (β-CTF), β-secretase (BACE1), γ-secretase, neuroinflammation, and / or dementia (e.g., Alzheimer's disease or age-related decline) in a subject by inducing synchronized gamma oscillations in the brain of the subject using a stimulus-emitting device to emit a stimulus at a frequency (e.g., about 40 Hz) that synchronously activates in vivo a specific cell type (e.g., fast-spiking parvalbumin (FS-PV) immunoreactive interneurons) and / or brain region (e.g., a sensory cortex and / or hippocampus) of the subject.SELECTED DRAWING: Figure 16

Description

[Technical Field]

[0001] Government Support Statement This invention was made with government support under Grant No. RF1 AG047661 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Application No. 62 / 259,187, entitled "System and Methods for Preventing, Mitigating, and / or Treating Dementia," filed November 24, 2015, the disclosure of which is incorporated herein by reference in its entirety.

[0003] The present disclosure relates generally to systems and methods for preventing, alleviating, and / or treating dementia in a subject. More particularly, the present disclosure relates to systems and methods for inducing synchronized gamma oscillations in at least one brain region of a subject. [Background technology]

[0004] Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a decline in memory, orientation, and reasoning. It is the most common form of dementia worldwide, affecting approximately one in eight people over the age of 65, and is the sixth leading cause of death in the United States. The number of people with this progressive neurodegenerative disease is estimated to increase by 40% over the next decade.

[0005] Histopathologically, AD can be characterized by the accumulation of amyloid plaques containing amyloid beta (Aβ) peptides and neurofibrillary tangles (NFTs) composed of tau protein. Aβ peptides are proteins of 36–43 amino acids, the normal physiological function of which remains unknown. Aβ peptides are formed by sequential proteolytic cleavage of amyloid precursor protein (APP) by beta-secretase 1 (BACE1) and gamma-secretase. C-terminal fragment β (β-CTF) is an APP derivative produced during the amyloidogenic cleavage of APP by BACE1 and is therefore another indicator of Aβ peptide production. Under normal conditions, soluble Aβ peptides are produced and secreted by neurons and subsequently cleared from the brain via the cerebrospinal fluid (CSF) pathway. However, in AD subjects, Aβ peptides appear to aggregate into higher-order species, forming soluble oligomers and insoluble plaques in a concentration-dependent manner. This aggregation can lead to a number of neurotoxic events, including disruption of brain metabolism, neuroinflammation, reduced functional connectivity, synaptic and neuronal loss, and / or the formation of NFTs.

[0006] A fundamental relationship between Aβ concentrations and neuronal activity has been demonstrated. First, treatment of organotypic hippocampal slices prepared from transgenic (Tg) mice overexpressing APP with tetrodotoxin reduced neuronal activity and subsequently reduced Aβ levels. Subsequently, the opposite effect, i.e., increased neuronal activity, was observed upon treatment with picrotoxin. Dynamic regulation of Aβ peptide concentrations and eventual plaque deposition in vivo has also been demonstrated using neuronal activity. In human AD patients, neural imaging shows that the most severe plaque deposition can coincide with the most consistently active brain regions, known as the "default mode network."

[0007] To date, there is no cure for AD, and treatment options do not inhibit the pathological progression of AD, are primarily palliative, and / or may have multiple troublesome side effects. For example: Preventive and / or therapeutic strategies targeting Aβ peptides and / or their precursors (e.g., Aβ immunotherapy and inhibition of beta- and gamma-secretase) have been shown in clinical trials to be harmful and / or ineffective in reducing AD pathology. Clinical trials using amyloid beta vaccines (e.g., bapineuzumab) have failed due to a lack of effect on cognitive function. Gamma secretase inhibitors (e.g., semagacestat) have failed in clinical trials due to worsening cognitive impairment in subjects. Even existing drugs such as acetylcholinesterase inhibitors (e.g., donepezil and rivastigmine) and N-methyl-D-aspartate (NMDA) receptor antagonists (e.g., memantine) have only shown mild effects on cognitive function. Summary of the Invention

[0008] Key microscopic pathological features of AD include the presence of amyloid plaques, NFTs, and widespread neuronal loss. This accumulation of neuronal damage over time leads to dysfunction of macroscopic circuits in the brain, particularly inducing gamma power deficits during memory and concentration tasks. These gamma oscillations (e.g., approximately 20 Hz to approximately 100 Hz, approximately 20 Hz to approximately 80 Hz, or approximately 20 Hz to approximately 50 Hz) primarily originate from and are regulated by fast-spiking parvalbumin (FS-PV) interneurons.

[0009] In one aspect, the present disclosure provides devices, methods, and systems for preventing, alleviating, and / or treating dementia in a subject, comprising inducing synchronized gamma oscillations in at least one brain region of the subject. In some embodiments, the dementia is AD, vascular dementia, frontotemporal dementia, dementia with Lewy bodies, and / or age-related cognitive decline. The subject may be a human or an animal.

[0010] In some embodiments, the synchronized gamma oscillations have a frequency of about 20 Hz to about 50 Hz, e.g., about 40 Hz. The synchronized gamma oscillations can be induced in a cell-type-specific manner. For example, the oscillations can be coordinated with synchronous activation of FS-PV interneurons. The synchronized gamma oscillations can be induced in a brain region-specific manner. For example, the oscillations can be coordinated with synchronous activation in at least one of the hippocampal region and the sensory cortex region.

[0011] In one embodiment, a method for preventing, alleviating, and / or treating dementia in a subject includes controlling a stimulus-emitting device to emit a stimulus, and exposing the subject to the stimulus and / or administering the stimulus to the subject, thereby inducing synchronized gamma oscillations in vivo in at least one brain region of the subject. The stimulus may have a frequency of about 35 Hz to about 45 Hz, for example, about 40 Hz. The stimulus-emitting device may be a tactile device, a light-emitting device, and / or a sound-emitting device. For example, the light-emitting device may be a fiber optic device. The period of exposure of the subject to the stimulus and / or the period of administration of the stimulus to the subject may be about 1 hour. The exposure of the subject to the stimulus and / or the administration of the stimulus to the subject may be repeated over a certain period of time. For example, the exposure of the subject to the stimulus and / or the administration of the stimulus to the subject may be repeated at least once a day over the period of time. The period can include, but is not limited to, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 1 month (or longer, e.g., once daily for the life of the subject).

[0012] In one embodiment, a method for reducing the level (e.g., amount or proportion) of Aβ peptide in at least one brain region of a subject includes inducing synchronized gamma oscillations in at least one brain region of the subject. The Aβ peptide may include one or more isoforms of Aβ peptide (e.g., isoform Aβ 1-40 , isoform Aβ 1-42 , and / or isoform Aβ 1-43), soluble Aβ peptide, and / or insoluble Aβ peptide.

[0013] In some embodiments, the synchronized gamma oscillations reduce the production of Aβ peptides in the at least one brain region of the subject, for example, by reducing the level (e.g., amount or proportion) of C-terminal fragments (CTFs) and / or N-terminal fragments (NTFs) of APP in the at least one brain region of the subject. The synchronized gamma oscillations may reduce the cleavage of APP into CTFs and NTFs by BACE1 and / or gamma-secretase in the at least one brain region of the subject. The synchronized gamma oscillations may reduce the level (e.g., number or proportion) of endosomes in the at least one brain region of the subject. For example, the endosomes may be positive for early endosomal antigen 1 (EEA1) and / or a Ras-related protein encoded by the RAB5A gene (Rab5). In some embodiments, the synchronized gamma oscillations promote the clearance of Aβ peptides in the at least one brain region of the subject. The synchronized gamma oscillations may increase the uptake of Aβ peptides by microglia in the at least one brain region of the subject.

[0014] In one embodiment, a method for increasing the level (e.g., number or percentage) of microglial cells in at least one brain region of a subject, morphological changes of microglial cells consistent with a neuroprotective state, and / or activity of the microglial cells comprises inducing synchronized gamma oscillations in at least one brain region of the subject. The synchronized gamma oscillations may upregulate at least one differentially expressed gene, such as Nr4a1, Arc, Npas4, Cd68, B2m, Bsr2, Icam1, Lyz2, Irf7, Spp1, Csf1r, and / or Csf2ra, which are involved in microglial activity in the at least one brain region of the subject. The morphological changes of microglial cells consistent with the neuroprotective state may include an increase in cell body size and / or a decrease in process length.

[0015] In one embodiment, a method for reducing the level (e.g., amount or rate) of Aβ peptide in a subject's hippocampus includes optogenetically stimulating FS-PV interneurons in the hippocampus with multiple light pulses, wherein the FS-PV interneurons express an optogenetic actuator, thereby entraining in vivo synchronous gamma oscillations measured by local field potentials in excitatory neurons (e.g., FS-PV interneurons), which reduce the level of Aβ peptide in the hippocampus. The light pulses may have a pulse frequency of about 40 pulses / second. Each light pulse may have a duration of about 1 millisecond. At least one light pulse may have a wavelength of about 473 nm. The optogenetic actuator may include channelrhodopsin, halorhodopsin, and / or archerhodopsin. For example, the optogenetic actuator may be channelrhodopsin 2 (ChR2).

[0016] In one aspect, a method for reducing the level (e.g., amount or proportion) of soluble and / or insoluble Aβ peptide in the visual cortex of a subject includes stimulating the subject with a plurality of light pulses at a pulse frequency of about 40 pulses / second, thereby inducing synchronized gamma oscillations in the visual cortex in vivo, which reduces the level of soluble and / or insoluble Aβ peptide in the visual cortex.

[0017] In one embodiment, a method for reducing the level (e.g., amount or rate) of tau phosphorylation in the visual cortex of a subject includes stimulating the subject with a plurality of light pulses at a pulse frequency of about 40 pulses / second, thereby inducing synchronized gamma oscillations in vivo in the visual cortex, which reduces tau phosphorylation in the visual cortex.

[0018] In one aspect, a method for reducing the level (e.g., amount or proportion) of Aβ peptide in the hippocampus and / or auditory cortex of a subject includes stimulating the subject with a plurality of sound pulses at a pulse frequency of about 40 pulses / second, thereby inducing synchronized gamma oscillations in vivo in at least one of the hippocampus and auditory cortex, which induces synchronous gamma oscillations in at least one of the hippocampus and auditory cortex. On the one hand, it reduces the levels of Aβ peptides.

[0019] In one embodiment, a system for preventing, reducing, and / or treating levels (e.g., amount or rate) or changes in Aβ peptide, neuroinflammation, and / or cognitive function in a subject includes a stimulus-emitting device for in vivo synchronous activation of brain regions of the subject, at least one memory for storing stimulus parameters and processor execution instructions, and at least one processor communicably connected to the stimulus-emitting device and the at least one memory. Upon execution of the processor's execution instructions, the at least one processor controls the stimulus-emitting device to emit stimuli according to the stimulus parameters, including a frequency at which the brain regions are synchronously activated, thereby preventing, reducing, and / or treating Aβ peptide, neuroinflammation, and / or dementia in the subject. The frequency may be about 35 Hz to about 45 Hz, e.g., about 40 Hz. The in vivo synchronous activation may be regulated by an enzyme and / or may occur in a specific cell type, e.g., immunoreactive FS-PV interneurons. The enzyme can include an optogenetic activator, a microbial opsin, ChR2, and / or the vector AAV-DIO-ChR2-EYFP.

[0020] In one embodiment, a system for preventing, reducing, and / or treating levels (e.g., amount or rate) or changes in Aβ peptide, neuroinflammation, and / or cognitive function in a subject includes an optical occlusion device for reducing ambient light to at least one eye of the subject and / or a noise cancellation device for reducing ambient noise to at least one ear of the subject. The optical occlusion device may include a light-emitting unit for emitting light stimuli to the at least one eye for in vivo synchronous activation of at least one of the subject's visual cortex and hippocampus. The noise cancellation device may include a speaker unit for emitting sound stimuli to the at least one ear for in vivo synchronous activation of at least one of the subject's auditory cortex and hippocampus. The system also includes at least one memory for storing processor execution instructions, and at least one processor communicably connected to the optical occlusion device and / or the noise cancellation device and the at least one memory. Upon execution of the processor's executable instructions, the at least one processor may control the light occlusion device such that the light emitting unit emits the light stimulus at a frequency that synchronously activates the at least one of the visual cortex and the hippocampus. Alternatively, or in addition, the at least one processor may control the noise cancellation device such that the speaker unit operates the sound stimulus at a frequency that synchronously activates the at least one of the auditory cortex and the hippocampus.

[0021] In one aspect, a method for improving cognitive function in a subject includes controlling at least one electroacoustic transducer to convert an electrical audio signal into a corresponding sound stimulus. In some embodiments, the sound stimulus includes a click train having a click frequency of about 35 clicks / second to about 45 clicks / second. The method further includes exposing the subject to the sound stimulus and / or administering the stimulus to the subject to induce synchronized gamma oscillations in at least one brain region of the subject, where the synchronized gamma oscillations result in improved cognitive function in the subject. The cognitive function may include recognition, discrimination, and / or spatial memory.

[0022] In one aspect, a method for preventing, reducing, and / or treating levels (e.g., amount or rate) or changes in Aβ peptides, neuroinflammation, and / or cognitive function in a subject includes controlling at least one electroacoustic transducer to convert an electrical audio signal into a corresponding sound stimulus, i.e., a sound stimulus such as a click train having a click frequency of about 35 clicks / second to about 45 clicks / second, and exposing the subject to the sound stimulus and / or administering the stimulus to the subject to induce synchronous gamma oscillations in at least one brain region of the subject. wherein the synchronized gamma oscillations result in the prevention, reduction, and / or treatment of levels of Aβ peptide, neuroinflammation, and / or dementia in the subject.

[0023] The Aβ peptide may include one or more isoforms of Aβ peptide (e.g., isoform Aβ 1-40 , isoform Aβ 1-42 , and / or isoform Aβ 1-43 The synchronized gamma oscillations may include soluble Aβ peptides, soluble Aβ peptides, and / or insoluble Aβ peptides. The synchronized gamma oscillations may prevent, reduce, and / or treat levels of Aβ peptides, neuroinflammation, and / or dementia in the subject by increasing the number of microglial cells in at least one brain region of the subject and / or increasing the uptake of Aβ peptides by microglial cells in the at least one brain region. The at least one brain region may include the auditory cortex and / or the hippocampus.

[0024] The click frequency may be about 40 clicks / second. Each click in the click train may have a duration of about 1 millisecond. Each click in the click train may have a frequency of about 10 Hz to about 100 kHz, about 12 Hz to about 28 kHz, about 20 Hz to about 20 kHz, and / or about 2 kHz to about 5 kHz. Each click in the click train may have a sound pressure level of about 0 dB to about 85 dB, about 30 dB to about 70 dB, and about 60 dB to about 65 dB.

[0025] The at least one electroacoustic transducer may include at least one headphone, in which case the method may include using the at least one headphone around, on, and / or in at least one ear of the subject and directing a sound stimulus to the at least one ear of the subject. The method may also include reducing ambient noise using passive noise blocking and / or active noise cancellation.

[0026] In one embodiment, a system for preventing, reducing, and / or treating levels (e.g., amount or rate) or changes in Aβ peptides, neuroinflammation, and / or cognitive function in a subject includes at least one electro-acoustic transducer for converting electrical audio signals into corresponding sound stimuli, i.e., sound stimuli such as a click train having a click frequency of about 35 clicks / second to about 45 clicks / second, at least one memory device for storing the electrical audio signals and processor instructions, and at least one processor communicatively connected to the at least one electro-acoustic transducer and the at least one memory device. Upon execution of the processor instructions, the at least one processor controls the electro-acoustic transducer to output sound stimuli to at least one ear of the subject and induce synchronized gamma oscillations in at least one brain region of the subject, which results in the prevention, reduction, and / or treatment of levels (e.g., amount or rate) of Aβ peptides, neuroinflammation, and / or dementia in the subject.

[0027] The system may be stationary or portable. When the at least one electro-acoustic transducer comprises at least one headphone for wearing around, on, and / or in at least one ear of the subject, directing sound stimuli to the at least one ear of the subject and reducing ambient noise, the system may further comprise a headphone interface for transmitting the electrical audio signal to the at least one headphone. Alternatively, or in addition, the system may comprise a neuroimaging device for monitoring function of the at least one brain region of the subject before, during, and / or after output of the sound stimuli.

[0028] In one aspect, a method for preventing, alleviating, and / or treating dementia in a subject comprises providing a device for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0029] In one embodiment, the blood levels of glucocorticoids involved in stress response in a subject A method for maintaining and / or reducing (e.g., amount of) includes providing a device that induces synchronized gamma oscillations in at least one brain region of the subject.

[0030] In one aspect, a method for preventing and / or reducing anxiety in a subject comprises providing a device for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0031] In one aspect, a method for maintaining and / or strengthening memory associations comprises providing a device for inducing synchronized gamma oscillations in at least one brain region of the subject, wherein the memory associations can be based on spatial memory.

[0032] In one aspect, a method for maintaining and / or enhancing cognitive flexibility comprises providing a device for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0033] In one embodiment, a method for maintaining and / or reducing changes in anatomy and / or morphology in at least one brain region of a subject includes providing a device for inducing synchronized gamma oscillations in at least one brain region of the subject, where the anatomy and / or morphology can include brain weight, lateral ventricle size, cortical thickness, neuronal layer thickness, and / or vascular diameter, and the at least one brain region can include the visual cortex, somatosensory cortex, and / or insular cortex of the subject.

[0034] In one embodiment, a method for maintaining and / or reducing alterations in the number of neurons, the quality of DNA in the neurons, and / or the density of synaptic puncta in at least one brain region of a subject includes providing a device for inducing synchronized gamma oscillations in at least one brain region of the subject, which may include the visual cortex, the somatosensory cortex, the insular cortex, and / or the hippocampus.

[0035] In one aspect, a device for inducing synchronized gamma oscillations in at least one brain region of a subject can prevent, reduce, and / or treat dementia and / or anxiety in the subject, maintain and / or enhance memory associative and / or cognitive flexibility in the subject, and / or maintain and / or reduce anatomic, morphological, cellular, and molecular changes in at least one brain region of the subject.

[0036] It should be understood that all combinations of the foregoing concepts, and additional concepts discussed in more detail below, provided that such concepts are not mutually inconsistent, are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of subject matter recited in the claims appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be understood that terms explicitly used herein, which may also appear in any disclosure incorporated by reference, should be given the meaning most consistent with the particular concepts disclosed herein.

[0037] Other systems, processes, and features will become apparent to one with skill in the art upon examination of the following figures and detailed description, and it is intended that all such additional systems, processes, and features be included within this description, be within the scope of the invention, and be protected by the accompanying claims.

[0038] Those skilled in the art will appreciate that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The drawings are not necessarily to scale, and in some instances, various aspects of the inventive subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate an understanding of different features. In the drawings, like reference numbers generally, for example, refer to like features (eg, functionally like and / or structurally like elements). [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 is a schematic diagram showing a mouse running through a virtual linear maze on a spherical treadmill, according to some embodiments. [Figure 2] 2A and 2B are electrical traces showing theta oscillations and sharp wave ripples (SWRs) recorded from hippocampal CA1, according to some embodiments. [Figure 3] 3A and 3B are plots showing the mean and standard deviation of normalized power spectra and normalized power spectral densities during theta cycles of 3-month-old Tg 5XFAD and wild-type (WT) mice, according to some embodiments. [Figure 4] 4A and 4B are spectrograms showing SWRs of WT and 5XFAD mice, according to some embodiments. [Figure 5] 5A-5C are plots showing the distribution of instantaneous gamma frequencies during SWR, according to some embodiments. [Figure 6]Figure 6A is a series of graphs showing Z-scored gamma power as a function of time from the peak of an SWR in 5XFAD and WT mice, according to some embodiments. Figure 6B is a plot showing the cumulative distribution of gamma power during an SWR in 5XFAD and WT mice, according to some embodiments. Figures 6C and 6D are plots showing the cumulative distribution of Z-scored gamma power during 100 ms around the peak of an SWR in WT and 5XFAD mice, according to some embodiments. Figure 6E is a plot showing the cumulative distribution of gamma power during large SWRs in 5XFAD and WT mice, according to some embodiments. [Figure 7] 7A is a plot showing spike rate as a function of gamma oscillation phase, and FIG. 7B is a plot showing the depth of modulation of spiking during an SWR, according to some embodiments. 7C and 7D are plots showing spike rate in hippocampal CA1 during an SWR as a function of gamma oscillation phase, according to some embodiments. 7E is a plot showing spike rate as a function of gamma oscillation phase, and FIG. 7F is a plot showing the depth of modulation of spiking during a large SWR, according to some embodiments. [Figure 8] 8A and 8B are plots showing the percent SWR per non-theta cycle for each animal and all animals combined in 5XFAD and WT animals, according to some embodiments. [Figure 9] FIG. 1 is a schematic diagram showing a viral vector for modulating the activation of specific cell types in the brain of a subject, according to some embodiments. [Figure 10] 10A and 10B are schematic diagrams illustrating signal delivery to the CA1 region of the hippocampus of a subject, according to some embodiments. [Figure 11] 1 is an immunofluorescence image showing immunostaining of neural tissue in a subject with ChR2 and DAPI, according to some embodiments. [Figure 12]Figure 12A is a series of immunofluorescence images showing ChR2-EYFP expressed in PV+ interneurons, according to some embodiments. Figure 12B is a series of immunofluorescence images showing immunohistochemistry with anti-EYFP and anti-PV antibodies, according to some embodiments. [Figure 13] 13A and 13B include a schematic diagram of a study of FS-PV interneurons, electrical traces of local field potentials, and power spectral densities, according to some embodiments. [Figure 14] 14A and 14B include a raw electrical trace after the onset of a 1 ms laser pulse, a trace after filtering for spikes after optogenetic stimulation, and a plot of spike probability, according to some embodiments. [Figure 15] 15A is a histogram showing the difference in firing rate between 40 Hz and random stimulation periods, according to some embodiments, and FIG. 15B is a bar graph showing multi-unit firing rates per 40 Hz, random, and no-stimulus period for each animal, according to some embodiments. [Figure 16] 16A and 16B are electrical traces and power spectral density plots showing frequency-specific increases in local field potential power upon stimulation of specific cell types in the CA1 region of the hippocampus of a subject, respectively, according to some embodiments. [Figure 17] 17A and 17B are bar graphs showing relative Aβ1-40 and Aβ1-42 levels in 5XFAD / PV-Cre CA1 by one-way analysis of variance, according to some embodiments. [Figure 18] 18A and 18B are bar graphs showing relative Aβ1-40 and Aβ1-42 levels in 5XFAD / αCamKII-Cre CA1 by one-way ANOVA, according to some embodiments. [Figure 19]Figure 19A is a series of images showing immunohistochemistry with anti-Aβ and anti-EEA1 antibodies in the CA1 region of the hippocampus, according to some embodiments. Figure 19B is a series of bar graphs showing relative immunoreactivity of Aβ normalized to EYFP, according to some embodiments. [Figure 20] Figure 20A is a series of immunofluorescence images showing immunohistochemistry with anti-Aβ antibodies in the hippocampal CA1 region of 5XFAD / PV-Cre mice, according to some embodiments. Figure 20B is a bar graph showing relative Aβ immunoreactivity normalized to EYFP, according to some embodiments. [Figure 21] Figure 21A is a representative Western blot showing levels of APP (CT695), APP NTF (A8967), APP CTF (CT695), and β-actin (A5316) (loading control) in CA1, according to some embodiments. Figure 21B is a bar graph showing relative (normalized to actin) immunoreactivity of APP CTF at 40 Hz relative to EYFP and random conditions, according to some embodiments. Figure 21C is a series of Western blots showing levels of full-length APP2106 (CT695), APP CTF2108 (CT695), and β-actin2112 (A5316, loading control) in CA1, according to some embodiments. [Figure 22] Figure 22A is a bar graph showing the relative (normalized to actin) immunoreactivity of APP NTF in EYFP and random conditions at 40 Hz, according to some embodiments. Figure 22B is a bar graph showing the relative (normalized to actin) immunoreactivity of full-length APP in EYFP, random, and 40 Hz conditions, according to some embodiments. [Figure 23] 1 is a series of immunofluorescence images showing immunohistochemistry with anti-Rab5 (ADI-KAp-GP006-E), according to some embodiments. [Figure 24]FIG. 24A is a bar graph showing relative EEA1 immunoreactivity normalized to EYFP, and FIG. 24B is a bar graph showing relative Rab5 intensity levels in CA1 from 5XFAD / PV-Cre under EYFP, 40 Hz, and random stimulation conditions according to some embodiments. [Figure 25] Figure 25A is a bar graph showing levels of Aβ peptide isoform Aβ1-40 after different types of stimulation in the CA1 region of the hippocampus of a subject, according to some embodiments. Figure 25B is a bar graph showing a decrease in Aβ peptide isoform Aβ1-42 after stimulation of specific cell types in the CA1 region of the hippocampus of a subject with gamma oscillations, according to some embodiments. Figure 25C is a series of images showing a decrease in levels of CTF (e.g., β-CTF) and an increase in levels of full-length APP (normalized to actin) after stimulation of specific cell types in the CA1 region of the hippocampus of a subject with gamma oscillations, according to some embodiments. [Figure 26] 26A-26B are immunofluorescence images showing endosome levels (based on EEA1 levels) following stimulation of different types of CA1 region of the hippocampus of a subject, according to some embodiments. [Figure 27] 6C is a bar graph showing mean intensity values ​​(normalized to FAD) for the immunofluorescence images of FIGS. 6A-6B after different types of stimulation of the CA1 region of the hippocampus of a subject, according to some embodiments. [Figure 28] 1 is a heatmap showing differentially expressed genes determined by whole-transcriptome ribonucleic acid sequencing (RNA-seq) of the mouse hippocampal CA1 region with and without 40 Hz stimulation, according to some embodiments. [Figure 29] FIG. 10 is a box plot showing FPKM values ​​of up- and down-regulated genes in EYFP and 40 Hz conditions according to some embodiments. [Figure 30] 1 is a pie chart showing cell type-specific expression patterns of identified upregulated genes following 40 Hz stimulation, according to some embodiments. [Figure 31]1 is a bar graph showing RT-qPCR validation of specific gene targets in an RNA-seq dataset, according to some embodiments. [Figure 32] 32A and 32B are plots showing the power spectral density of local field potentials recoded over the brain while viewing a 40 Hz light flash, according to some embodiments. [Figure 33] 1 is a bar graph showing RT-qPCR validation of specific gene targets in an RNA-seq dataset, according to some embodiments. [Figure 34] 1 is a series of immunofluorescence images showing immunohistochemistry with anti-Iba1 (019-19741) and anti-Aβ (12F4) antibodies in the hippocampal CA1 region of 5XFAD / PV-Cre mice under EYFP, 40 Hz, and random stimulation conditions, according to some embodiments. [Figure 35] Figure 35A is a bar graph showing the number of microglia in EYFP and 40 Hz conditions, according to some embodiments. Figure 35B is a bar graph showing the diameter of microglial cell bodies normalized to EYFP in EYFP, 40 Hz, and random stimulation conditions, according to some embodiments. Figure 35C is a bar graph showing the average length of microglial primary processes normalized to EYFP in EYFP, 40 Hz, and random stimulation conditions, according to some embodiments. Figure 35D is a bar graph showing the percentage of Iba1-positive (microglial) cell bodies that are also Aβ-positive in EYFP and 40 Hz stimulation conditions, according to some embodiments. [Figure 36] 35 is a series of 3D renderings formed by merging the immunofluorescence images of FIG. 34 in accordance with some embodiments. [Figure 37] Figure 37A is a series of immunofluorescence images showing Hoechst immunohistochemistry in the hippocampal CA1 region of 5XFAD / PV-Cre, according to some embodiments. Figure 37B is a bar graph showing estimated CA1 thickness in 5XFAD / PV-Cre under EYFP and 40 Hz stimulation conditions, according to some embodiments. [Figure 38]Figure 38A is a heatmap showing differentially expressed genes (DEGs) determined by genome-wide RNA-seq in hippocampal CA1 upon 40 Hz FS-PV+ stimulation or control stimulation, according to some embodiments. Figure 38B shows the overlap between DEGs upregulated in the treatment conditions of Figure 38A, according to some embodiments. [Figure 39] FIG. 38B is a bar graph showing RT-qPCR validation of specific gene targets in the RNA-seq dataset of FIG. 38A according to some embodiments. [Figure 40] 38B is a plot showing the biological processes associated with the upregulated genes of FIG. 38A, according to some embodiments. [Figure 41] FIG. 38B is a plot showing the biological processes associated with the down-regulated genes of FIG. 38A, according to some embodiments. [Figure 42] Figure 42A is a series of immunofluorescence images showing levels of Iba1 after different types of stimulation of the CA1 region of a subject's hippocampus, according to some embodiments. Figure 42B is a bar graph showing mean intensity values ​​for the immunofluorescence images of Figure 42A, according to some embodiments. [Figure 43] Figure 43A is a schematic diagram showing a mouse exposed to a flashing light stimulus, according to some embodiments. Figure 43B includes plots of local field potential traces and power spectral densities in the visual cortex before and during a 40 Hz light flash, according to some embodiments. Figures 43C-43F are plots showing power spectral densities of local field potentials in the visual cortex, according to some embodiments. [Figure 44] Figure 44A is a series of histograms showing spike rates in the visual cortex as a function of time for four cycles of 40 Hz light flashes and an equivalent period for random light flashes, according to some embodiments. Figure 44B is a series of electrical traces of local field potentials recorded over the brain during exposure to light flashes, according to some embodiments. [Figure 45]Figure 45A is a histogram showing the difference in firing rate between 40 Hz light flashes and random light flashes, according to some embodiments. Figure 45B is a plot showing multi-unit firing rate in the visual cortex, according to some embodiments. [Figure 46] Figure 46A is a schematic diagram showing an experimental paradigm according to some embodiments. Figures 46B-46C are plots further showing changes in baseline levels of Aβ peptide isoforms Aβ1-40 and Aβ1-42, respectively, following the experimental paradigm of Figure 46A according to some embodiments. [Figure 47] 47A and 47B are bar graphs showing changes in baseline levels of Aβ1-40 and Aβ1-42, respectively, in the visual cortex of 5XFAD, according to some embodiments. [Figure 48] Figure 48A is a bar graph showing changes in baseline levels of Aβ1-40 and Aβ1-42 in the barrel cortex of 5XFAD under dark and 40 Hz flashing conditions, according to some embodiments. Figure 48B is a bar graph showing changes in baseline levels of Aβ1-40 and Aβ1-42 in the visual cortex of APP / PS1 under dark and 40 Hz flashing conditions, according to some embodiments. Figure 48C is a bar graph showing changes in baseline levels of Aβ1-40 and Aβ1-42 in the visual cortex of WT under dark and 40 Hz flashing conditions, according to some embodiments. [Figure 49] 10 is a series of immunofluorescence images showing immunohistochemistry with anti-Iba1 (019-19741) and anti-Aβ (12F4) antibodies in the visual cortex of 5XFAD under dark and 40 Hz flash conditions, according to some embodiments. [Figure 50]Figure 50A is a bar graph showing the number of Iba1-positive cells (microglia), according to some embodiments. Figure 50B is a bar graph showing the diameter of microglial cell bodies normalized to controls under dark and 40 Hz flashing conditions, according to some embodiments. Figure 50C is a bar graph showing the average length of microglial primary processes normalized to controls under dark and 40 Hz flashing conditions, according to some embodiments. Figure 50D is a bar graph showing the percentage of microglia that are also Aβ-positive under dark and 40 Hz flashing conditions, according to some embodiments. [Figure 51] A series of 3D renderings (from immunofluorescence images) of Iba+ microglia under dark and 40 Hz flashing conditions from a 100 μm tissue section that was CLARITY-processed according to some embodiments. CLARITY is a method for making brain tissue transparent using, for example, an acrylamide-based hydrogel that is configured within and bound to the tissue. [Figure 52] Figure 52A is a flow chart showing a method for isolating microglia from the visual cortex using fluorescence-activated cell sorting (FACS), according to some embodiments. Figure 52B is a bar graph showing levels of Aβ1-40 in microglia isolated from the visual cortex of 3-month-old 5XFAD and WT control animals using the method of Figure 52A, according to some embodiments. [Figure 53] Figure 53A is a series of immunofluorescence images showing immunohistochemistry with an SVP38 antibody to detect synaptophysin in the visual cortex of 3-month-old 5XFAD mice under dark and 40 Hz flashing conditions, according to some embodiments. Figure 53B is a bar graph showing relative SVP38 intensity levels in the visual cortex of 5XFAD mice after dark and 40 Hz flashing conditions, according to some embodiments. [Figure 54] 54A and 54B are bar graphs showing the reduction of Aβ peptide isoform Aβ1-42 following stimulation of a subject's visual cortex with gamma oscillations, according to some embodiments, and the levels of Aβ peptide isoform Aβ1-42 following stimulation of a subject's visual cortex with gamma oscillations, and further 24 hours after stimulation, according to some embodiments. [Figure 55] Figure 55A includes electrical traces and power spectral density plots of local field potentials in the hippocampus before and during 40 Hz light flashing, according to some embodiments. Figure 55B is a series of histograms of spike rate in the hippocampus as a function of time for four cycles of 40 Hz light flashing and an equivalent period for random light flashing, respectively, according to some embodiments. [Figure 56] Figure 56A is a histogram showing the difference in firing rate between 40 Hz light flashes and random light flashes, according to some embodiments. Figure 56B is a plot showing multi-unit firing rate in CA1 during 40 Hz light flashes, according to some embodiments. [Figure 57] Figure 57A is a bar graph showing relative Aβ1-40 levels in the visual cortex of 5XFAD, according to some embodiments. Figure 57B is a bar graph showing relative Aβ1-42 levels in the visual cortex of 5XFAD, according to some embodiments. [Figure 58] Figure 58A is a bar graph showing relative Aβ1-40 levels in the visual cortex of 5XFAD with recovery after a 40 Hz light flash, according to some embodiments. Figure 58B is a bar graph showing relative Aβ1-42 levels in the visual cortex of 5XFAD with recovery after a 40 Hz light flash, according to some embodiments. [Figure 59] Figure 59A is a schematic diagram showing a test according to some embodiments. Figure 59B is a bar graph showing relative Aβ1-42 levels in the visual cortex of 6-month-old 5XFAD mice after 7 days in the dark for 1 hour / day or at 40 Hz flashing, according to some embodiments. Figure 59C is a bar graph showing relative Aβ1-40 levels in the visual cortex of 6-month-old 5XFAD mice after 7 days in the dark for 1 hour / day or at 40 Hz flashing, according to some embodiments. [Figure 60]Figure 60A is a series of immunofluorescence images showing immunohistochemistry with an anti-Aβ antibody in the visual cortex of a 6-month-old 5XFAD mouse after 7 days in the dark for 1 hour / day or 40 Hz flashing, according to some embodiments. Figure 60B is a bar graph showing the number of Aβ-positive plaque deposits in the visual cortex of a 6-month-old 5XFAD mouse after 7 days in the dark for 1 hour / day or 40 Hz flashing, according to some embodiments. Figure 60C is a bar graph showing the area of ​​Aβ-positive plaques in the visual cortex of a 6-month-old 5XFAD mouse after 7 days in the dark for 1 hour / day or 40 Hz flashing, according to some embodiments. [Figure 61] Figure 61A is a series of immunofluorescence images showing immunohistochemistry with anti-phospho-Tau (S202) and anti-MAP2 antibodies in 4-month-old P301S mice after 7 days in the dark for 1 hour / day or 40 Hz flashing, according to some embodiments. Figure 61B is a bar graph showing relative phospho-Tau (pTau) (S202) intensity levels in the visual cortex of P301S mice after 7 days in the dark for 1 hour / day and 40 Hz flashing, according to some embodiments. Figure 61C is a bar graph showing relative MAP2 intensity levels in the visual cortex of P301S mice after 7 days in the dark for 1 hour / day and 40 Hz flashing, according to some embodiments. [Figure 62] Figure 62A is a series of immunofluorescence images showing immunohistochemistry with anti-pTau6202 (S404) antibody in 4-month-old P301S mice after 7 days under 1 hour / day darkness and 40 Hz flashing conditions, according to some embodiments. Figure 62B is a bar graph showing relative anti-pTau (S400 / T403 / S404) fluorescence intensity levels in the visual cortex of P301S mice after 7 days under 1 hour / day darkness and 40 Hz flashing conditions, according to some embodiments. [Figure 63]Figure 63A is a series of immunofluorescence images showing immunohistochemistry with anti-pTau6302 (S396) antibody in 4-month-old P301S mice after 7 days under 1 hour / day darkness and 40 Hz flashing conditions, according to some embodiments. Figure 63B is a bar graph showing relative pTau (S396) fluorescence intensity levels in the visual cortex of P301S mice after 7 days under 1 hour / day darkness and 40 Hz flashing conditions, according to some embodiments. [Figure 64] 1 is a series of immunofluorescence images showing immunohistochemistry with anti-Iba1 antibody in 4-month-old P301S mice after 7 days under 1 hour / day dark and 40 Hz flashing conditions, according to some embodiments. [Figure 65] Figure 65A is a bar graph showing the number of microglia after 7 days in the dark for 1 hour / day and flashing at 40 Hz, according to some embodiments. Figure 65B is a bar graph showing the diameter of microglial cell bodies normalized to controls after 7 days in the dark for 1 hour / day and flashing at 40 Hz, according to some embodiments. Figure 65C is a bar graph showing the average length of microglial primary processes normalized to controls after 7 days in the dark for 1 hour / day and flashing at 40 Hz, according to some embodiments. [Figure 66] 1 is a plot showing levels of soluble and insoluble Aβ peptide isoforms Aβ1-40 and Aβ1-42 in the visual cortex of a subject with and without visual gamma stimulation, according to some embodiments. [Figure 67] 67A-67B are plots showing whole brain Aβ peptide levels with and without transcranial gamma stimulation in a subject, according to some embodiments. [Figure 68] Figure 68A is a flow chart showing a study performed to determine whether gamma exposure and / or administration causes stress in subjects according to some embodiments, and Figure 68B is a bar graph showing corticosterone levels indicative of stress response in the subjects. [Figure 69]Figure 69A is a flow chart showing a study conducted to determine whether gamma exposure and / or administration reduces anxiety in subjects according to some embodiments. Figure 69B is an image showing an elevated plus maze apparatus. Figures 69C and 69D are images showing a representative trajectory of a subject during the elevated plus maze period. [Figure 70] 1 is a bar graph showing the average time subjects spent exploring the open and closed runways during the elevated plus maze session. [Figure 71] Figure 71A is a flow diagram illustrating a study conducted to determine whether gamma exposure and / or administration reduces stress and / or anxiety in subjects according to some embodiments. Figure 71B is an image showing an open field arena. Figures 71C and 71D are images showing representative trajectories of subjects during the open field test. [Figure 72] Figure 72A is a plot showing the average time spent by subjects in the center of the open field for each minute of the open field test. Figure 72B is a bar graph showing the average total time spent by subjects on the periphery of the open field during the open field test. [Figure 73] Figures 73A and 73B are schematic diagrams showing studies conducted to determine whether gamma exposure and / or administration according to some embodiments alters innate novelty-exploratory behavior in subjects. Figure 73C is a bar graph showing the average time subjects spent exploring a first novel object compared to a second novel object, according to the schematic diagram of Figure 73A. [Figure 74] FIG. 73C is a plot showing the average time per minute that subjects spent exploring the novel object according to the schematic diagram of FIG. 73B. [Figure 75] Figure 75A is a flow diagram showing testing performed using a fear conditioning paradigm to examine whether gamma exposure and / or administration affects subject learning and memory according to some embodiments. Figure 75B is a stimulus diagram showing tone testing with changing context as a function of time. [Figure 76]76A and 76B are bar graphs showing memory enhancement in subjects according to some embodiments. [Figure 77] Figure 77A is a flow diagram showing a study conducted to determine whether gamma exposure and / or administration improves memory in subjects, according to some embodiments. Figure 77B is a diagram showing a Morris water maze with a hidden platform in the goal quadrant. Figures 77C and 77D are images showing representative trajectories of subjects during a Morris water maze probe test. [Figure 78] Figure 78A is a plot showing the average time subjects spent finding the hidden platform during each day of the Morris water maze test. Figure 78B is a plot showing the average time subjects spent searching for the removed platform in the goal quadrant during each 30-second period. Figure 78C is a plot showing the average time subjects spent searching for the removed platform in the opposite quadrant during each 30-second period. [Figure 79] Figure 79A shows the Morris water maze test with the platform hidden in the first quadrant. Figure 79B shows the Morris water maze test with the platform hidden in the second quadrant, i.e., opposite the first quadrant, for reversal learning. Figure 79C is a plot showing the average time subjects spent finding the hidden platform in the Morris water maze reversal learning test for each day. [Figure 80] Figure 80A is a flow chart showing a study conducted to examine whether chronic gamma exposure and / or administration affects spatial learning and memory in subjects, according to some embodiments. Figure 80B is a plot showing the average time subjects spent finding the hidden platform in the Morris water maze test on each day. Figure 80C is a bar graph showing the average time subjects spent searching for the removed platform in the goal quadrant during a 30-second test. [Figure 81] Figure 81A is a flow diagram showing the test of Figure 80A extended to include reversal learning, and Figure 81B is a plot showing the mean time subjects spent finding the hidden platform in the Morris water maze reversal learning test on each day. [Figure 82]Figure 82A is a bar graph showing the average time subjects spent searching for the removed platform in the target quadrant during a 30-second test, and Figure 82B is a bar graph showing the average time subjects spent searching for the removed platform in the opposite quadrant. [Figure 83] FIG. 1 is a diagram of a timeline of studies conducted to examine the effects of gamma exposure and / or administration, according to some embodiments, on deoxyribonucleic acid (DNA) damage and neuronal loss in the visual cortex of subjects. [Figure 84] FIG. 1 shows a group of subjects in a study conducted to examine the effects of gamma exposure and / or administration, according to some embodiments. [Figure 85] 85 is a bar graph comparing the change in brain weight across the groups of subjects in FIG. 84 according to some embodiments. [Figure 86] 85 is a bar graph comparing lateral ventricle expression ratios across groups of subjects in FIG. 84 according to some embodiments. [Figure 87] 87A-87E are images showing representative lateral ventricles of the group of subjects in FIG. 84, according to some embodiments. [Figure 88] 88A-88C are brain anatomical diagrams showing brain regions of interest according to some embodiments. [Figure 89] 85 is a bar graph showing the mean V1 cortical thickness across the groups of subjects in FIG. 84 according to some embodiments. [Figure 90] 85 is a bar graph showing the mean thickness of the V1-NeuN positive cell layer across the groups of subjects in FIG. 84 according to some embodiments. [Figure 91] 91A-91E are images showing representative Hoechst- and / or NeuN-labeled cells from the group of subjects in FIG. 84, according to some embodiments. [Figure 92] 85 is a bar graph showing the mean thickness of the SS1 cortical layer across the groups of subjects in FIG. 84 according to some embodiments. [Figure 93]85 is a bar graph showing the mean thickness of SS1-NeuN positive cells across the groups of subjects in FIG. 84 according to some embodiments. [Figure 94] 94A-94E are images showing cells with Hoechst and / or NeuN labels across the groups of subjects in FIG. 84, according to some embodiments. [Figure 95] 85 is a bar graph showing the mean cortical thickness of the insular cortex across the group of subjects in FIG. 84 according to some embodiments. [Figure 96] 85 is a bar graph showing the mean thickness of the NeuN-positive cell layer of the insular cortex across the groups of subjects in FIG. 84 according to some embodiments. [Figure 97] 97A-97E are images showing representative Hoechst- and / or NeuN-labeled cells from the group of subjects in FIG. 84, according to some embodiments. [Figure 98] 85 is a bar graph comparing the amount of NeuN-positive cells in the visual cortex across the groups of subjects in FIG. 84 according to some embodiments. [Figure 99] 85 is a bar graph comparing the amount of γH2AX positive cells in the visual cortex across the groups of subjects in FIG. 84 according to some embodiments. [Figure 100] 85 is a series of images showing representative visual cortex samples of the group of subjects in FIG. 84, according to some embodiments. [Figure 101] 85 is a bar graph comparing the amount of NeuN-positive cells in the somatosensory cortex across the groups of subjects in FIG. 84 according to some embodiments. [Figure 102] 85 is a bar graph comparing the amount of γH2AX positive cells in the somatosensory cortex across the groups of subjects in FIG. 84 according to some embodiments. [Figure 103] 85 is a series of images showing representative somatosensory cortex samples of the group of subjects in FIG. 84 according to some embodiments. [Figure 104] 85 is a bar graph comparing the amount of NeuN-positive cell layer in the insular cortex across the groups of subjects in FIG. 84 according to some embodiments. [Figure 105] 85 is a bar graph comparing the amount of γH2AX positive cell layer in the insular cortex across the groups of subjects in FIG. 84 according to some embodiments. [Figure 106] 85 is a series of images showing representative samples of the insular cortex of the group of subjects in FIG. 84, according to some embodiments. [Figure 107] 85 is a bar graph comparing the amount of NeuN-positive cells in the hippocampus across the groups of subjects in FIG. 84 according to some embodiments. [Figure 108] 85 is a bar graph comparing the amount of γH2AX positive cells in the hippocampus across the groups of subjects in FIG. 84 according to some embodiments. [Figure 109] 85 is a series of images showing representative hippocampal samples from the group of subjects in FIG. 84 according to some embodiments. [Figure 110] 85 is a bar graph comparing visual cortex dot density across the groups of subjects in FIG. 84 according to some embodiments. [Figure 111] 85 is a bar graph comparing somatosensory cortex point density across the groups of subjects in FIG. 84 according to some embodiments. [Figure 112] 85 is a bar graph comparing point density of the insular cortex across the groups of subjects in FIG. 84 according to some embodiments. [Figure 113] 113A-113D are images showing Hoechst staining, VGluT1 marker, and / or GAD65 marker in representative samples, according to some embodiments. Figures 113E and 113F are images showing a method for quantification of points, according to some embodiments. [Figure 114] FIG. 10 is a stimulus diagram illustrating a click-train stimulus, according to some embodiments. [Figure 115] 1 is a flow diagram illustrating a study performed to determine whether auditory gamma exposure and / or administration induces microglial activation in the auditory cortex of a subject, according to some embodiments. [Figure 116]116A and 116B are bar graphs showing the average microglia count and process length ratio in the auditory cortex of a subject, respectively, according to some embodiments. [Figure 117] 117A and 117B are representative images of microglia in the auditory cortex of a subject, according to some embodiments. [Figure 118] 118A and 118B are magnified images of the microglial process lengths from FIGS. 117A and 117B, according to some embodiments. [Figure 119] 119A and 119B are enlarged images of the cell size of microglia from FIGS. 117A and 117B, according to some embodiments. [Figure 120] 120A and 120B are bar graphs showing the average number of microglia per image field in a subject's auditory cortex, according to some embodiments. [Figure 121] 121A and 121B are representative images of microglia in the auditory cortex of a subject, according to some embodiments. [Figure 122] 122A-122D are bar graphs showing levels of soluble Aβ isoforms Aβ1-40 and Aβ1-42 in the auditory cortex and hippocampus of a subject, according to some embodiments. [Figure 123] 123A-123D are bar graphs showing levels of insoluble Aβ isoforms Aβ1-40 and Aβ1-42 in the auditory cortex and hippocampus of subjects, according to some embodiments. [Figure 124] 124A-124D are representative images of microglia in the auditory cortex of a subject, according to some embodiments. [Figure 125] Figure 125A is a flow diagram showing a novel object recognition test. Figure 125B is a bar graph showing memory improvement according to some embodiments. [Figure 126]Figure 126A is a flow diagram showing a novel location recognition test. Figure 126B is a bar graph showing improvement in memory and / or discrimination according to some embodiments. [Figure 127] Figure 127A is a plot showing the mean time subjects spent finding the hidden platform during each day of the Morris water maze test. Figure 127B is a bar graph showing the mean time subjects spent searching for the removed platform in the target quadrant during the probe test. [Figure 128] Figure 128A is a series of representative immunofluorescence images showing enlarged vasculature in the visual cortex, according to some embodiments. Figure 128B is a bar graph showing vascular diameter in the visual cortex and showing an increase in vascular diameter after gamma exposure, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description In one aspect, the present disclosure provides methods, devices, and systems for preventing, alleviating, and / or treating a brain disorder or cognitive impairment / deficiency in a subject. In some embodiments, the brain disorder is dementia.

[0041] Cognitive function is highly dependent on the precise timing of oscillations in neural network activity, particularly gamma-frequency oscillations, i.e., rhythms (e.g., about 20 Hz to about 100 Hz, about 20 Hz to about 80 Hz, or about 20 Hz to about 50 Hz) that are implicated in focus and working memory. Because these oscillations arise from synaptic activity, they provide a direct link between the molecular properties of neurons and high-level coherent brain activity. Importantly, gamma oscillatory activity is disrupted in neural circuits impaired by molecular neuropathology in AD and may represent a major determinant of memory impairment in the disease. The causal relationship between pathology and impaired brain oscillations is unclear. Whether this has any beneficial effect has yet to be determined. However, manipulating brain rhythms could serve as a multi-targeted therapy for the treatment of dementia such as AD, and this could be achieved through non-invasive treatment.

[0042] In one aspect, the present disclosure provides devices, methods, and systems for enhancing or inducing gamma oscillations. In some embodiments, the enhancement or induction of gamma oscillations is by optogenetic methods. In other embodiments, the enhancement or induction of gamma oscillations is by behavioral methods. The present disclosure provides that the enhancement and / or induction of gamma oscillations by optogenetic, behavioral, or other methods reduces AD pathology.

[0043] In one aspect, the present disclosure provides devices, systems, and methods for restoring or inducing gamma oscillatory rhythms in subjects with dementia, in some embodiments, the dementia being AD, vascular dementia, frontotemporal dementia (FTD), and / or dementia with Lewy bodies. Thus, in some embodiments, the present disclosure provides devices, systems, and methods for treating dementia.

[0044] As used herein, the terms "treatment" or "treating" refer to both therapeutic treatment and prophylactic or preventive measures. In some embodiments, subjects in need of treatment include those who already have the disease or condition, as well as those who may develop the disease or condition, and the goal is to prevent, delay, or reduce the disease or condition. For example, in some embodiments, the devices, methods, and systems disclosed herein can be used to prevent, delay, or alleviate a disease or condition to which the subject is genetically predisposed, such as AD. In some embodiments, the devices, methods, and systems disclosed herein can be used to treat, alleviate, reduce symptoms, and / or slow the progression of a disease or condition for which the subject has already been diagnosed, such as AD.

[0045] As used herein, the term "subject" means a mammal, for example, a rodent, cat, dog, or primate. Preferably, the subject of the present invention is a human.

[0046] As used herein, the term "about" refers to plus or minus 10 percent of what it modifies.

[0047] Dementia is a disease characterized by loss of intellectual ability and / or memory impairment. Dementia includes, for example, AD, vascular dementia, dementia with Lewy bodies, Pick's disease, frontotemporal dementia (FTD), AIDS-related dementia, age-related cognitive impairment, and age-related memory impairment. Dementia may also be associated with neurological and / or psychiatric conditions, such as brain tumors, brain disorders, epilepsy, multiple sclerosis, Down syndrome, Rett syndrome, progressive supranuclear palsy, frontal lobe syndrome, schizophrenia, and traumatic brain injury.

[0048] AD is the most common neurodegenerative disease in developed countries. AD can be characterized by the accumulation of amyloid plaques composed of Aβ peptides and NFTs composed of tau protein. Clinically, AD is associated with progressive cognitive impairment characterized by loss of memory, function, language ability, judgment, and executive function. AD often leads to severe behavioral symptoms in its later stages.

[0049] Vascular dementia, sometimes also called cerebrovascular dementia, refers to a cerebrovascular accident (e.g., a cerebral hemispheric infarction) that generally has a fluctuating course with periods of improvement and gradual worsening. Vascular dementia may include one or more symptoms of disorientation, memory loss, and / or impaired judgment. Vascular dementia may be caused by separate multiple infarcts or may occur in combination with other vascular It may also be caused by vascular factors such as autoimmune vasculitis, such as that found in systemic lupus erythematosus, infectious vasculitis, such as Lyme disease, recurrent intracerebral hemorrhage, and / or stroke.

[0050] Frontotemporal dementia (FTD) is a progressive neurodegenerative disorder. Subjects with FTD typically exhibit significant behavioral and personality changes, often accompanied by language impairment.

[0051] Dementia with Lewy bodies is characterized by one or more of the following symptoms: dementia with overlapping features with those of AD, onset of Parkinson's disease features, and / or early onset of hallucinations. Dementia with Lewy bodies is generally characterized by daily fluctuations in symptom severity.

[0052] In some aspects, the present disclosure provides methods for preventing, alleviating, and / or treating dementia in a subject, comprising inducing synchronized gamma oscillations in the brain of the subject. In some embodiments, inducing gamma oscillations in a subject with a neurological disease or disorder or age-related decline acts to restore gamma oscillation rhythms that have been disrupted in the subject as a result of or in association with the disease or disorder or age-related decline.

[0053] In some embodiments, the induction of gamma oscillations is mediated by the isoform Aβ. 1-40 and Aβ 1-42 In some embodiments, the induction of gamma oscillations reduces the production of Aβ (e.g., isoform Aβ) from the brain of the subject. 1-40 and Aβ 1-42 ) clearance. In some embodiments, inducing gamma oscillations prevents the accumulation of Aβ in the subject's brain. In some embodiments, the methods provided herein reduce the level of Aβ in the subject's brain by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or more, compared to the level of Aβ in the subject's brain before treatment. In some embodiments, the level of Aβ in the subject's brain is reduced by at least about 50%, compared to the level of Aβ in the subject's brain before treatment.

[0054] In some embodiments, the level of Aβ in the subject's brain is reduced by reducing the cleavage of APP in the subject's brain. In some embodiments, the methods provided herein reduce the cleavage of APP in the subject's brain by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or more, compared to the level of APP cleavage in the subject's brain before treatment. In some embodiments, the level of APP cleavage in the subject's brain is reduced by at least about 50%, compared to the level of APP cleavage in the subject's brain before treatment. In some embodiments, the level of APP cleavage is measured by the level of C-terminal fragment β (β-CTF) in the subject's brain. In some embodiments, the level of APP cleavage in the brain is reduced by inhibiting β- and / or γ-secretase, e.g., by increasing the level of inhibition of β- and / or γ-secretase activity. In some embodiments, the methods provided herein reduce the aggregation of Aβ plaques in the subject's brain.

[0055] In some embodiments, the method improves cognitive performance and / or memory in a subject.

[0056] In another aspect, the present disclosure provides a method for inducing a neuroprotective profile or environment in the brain of a subject, comprising inducing synchronized gamma oscillations in the brain of the subject. For example, in some embodiments, the neuroprotective profile is associated with a neuroprotective microglial cell profile. In further embodiments, the neuroprotective profile is induced by or associated with increased activity of the M-CSF pathway. In some embodiments, the neuroprotective environment is associated with an anti-inflammatory signaling pathway. For example, in some embodiments, the anti-inflammatory signaling pathway is the anti-inflammatory microglial signaling pathway.

[0057] In some embodiments, the neuroprotective profile is associated with reduced or absent inflammatory glial cell activity, which is associated with the M1 phenotype of microglia and includes production of reactive oxygen species (ROS), the neurosecretory protein chromogranin A, the secretory cofactor cystatin C, NADPH oxidase, nitric oxide synthase enzymes such as iNOS, NF-κB-dependent inflammatory response proteins, and proinflammatory cytokines and chemokines (e.g., TNF, IL-1β, IL-6, and IFNγ).

[0058] In contrast, the M2 phenotype of microglia is associated with downregulation of inflammation and repair of inflammation-induced damage. Anti-inflammatory cytokines and chemokines (IL-4, IL-3, IL-10, and / or TGFβ) and increased phagocytic activity are associated with the M2 phenotype. Thus, in some embodiments, the methods provided herein induce a neuroprotective M2 phenotype in microglia. In some embodiments, the methods provided herein increase phagocytic activity in the brain of a subject. For example, in some embodiments, the methods provided herein increase microglial phagocytic activity such that clearance of Aβ is increased.

[0059] Gamma oscillations can include frequencies between about 20 Hz and about 100 Hz. Accordingly, in some embodiments, the present disclosure provides a method for preventing, alleviating, or treating dementia in a subject, comprising inducing gamma oscillations in the brain of the subject at frequencies between about 20 Hz and about 100 Hz, or between about 20 Hz and about 80 Hz, or between about 20 Hz and about 50 Hz, or between about 30 Hz and about 60 Hz, or between about 35 Hz and about 45 Hz, or about 40 Hz. Preferably, the gamma oscillations are about 40 Hz.

[0060] The stimulus may include a detectable change in the subject's internal or external environment that directly or ultimately induces gamma oscillations in at least one brain region. For example, the stimulus may be designed to stimulate electromagnetic radiation receptors (e.g., photoreceptors, infrared receptors, and / or ultraviolet receptors), mechanoreceptors (e.g., mechanical stress and / or strain), nociceptors (e.g., pain), sound receptors, electroreceptors (e.g., electric fields), magnetoreceptors (e.g., magnetic fields), hydroreceptors, chemoreceptors, thermoreceptors, osmoreceptors, and / or proprioceptors (i.e., position sense). The absolute threshold or minimum amount of sensation required to elicit a response from a receptor may vary depending on the type of stimulus and the subject. In some embodiments, the stimulus is adapted based on individual sensitivity.

[0061] In some embodiments, gamma oscillations are induced in a brain region-specific manner. For example, in some embodiments, gamma oscillations are induced in the hippocampus, visual cortex, barrel cortex, auditory cortex, or any combination thereof. By way of example, in some embodiments, gamma oscillations are induced in the visual cortex using phosphenes, and in other embodiments, gamma oscillations are induced in the auditory cortex using auditory stimuli of specific frequencies. In some embodiments, gamma oscillations are induced in multiple brain regions simultaneously using a combination of visual, auditory, and / or other stimuli. In some embodiments, gamma oscillations are induced in a virtual reality system.

[0062] In some embodiments, the subject receives stimuli through an environment configured to induce gamma oscillations, e.g., a chamber that passively or actively blocks out irrelevant stimuli (e.g., a light-tight or noise-canceling chamber). Alternatively, or in addition, the subject may receive stimuli through a system that includes, e.g., light-tight or noise-canceling features. In some embodiments, the subject receives visual stimuli through a stimulus-emitting device, e.g., eyewear designed to deliver the stimuli. The device may block other light. In some embodiments, the subject receives auditory stimuli through a stimulus-emitting device, e.g., headphones designed to deliver the stimuli. The device may filter out other noises.

[0063] In addition to at least one interface for emitting stimuli, some embodiments may include at least one processor (e.g., generating stimuli, controlling the emission of the stimuli, monitoring the emission / results of the stimuli, and / or processing feedback regarding the stimuli / results), at least one memory (e.g., storing processor-executable instructions, at least one stimuli, stimulus generation guidelines, feedback, and / or results), at least one communication interface (e.g., communicating with the subject, a healthcare provider, a caregiver, a clinical research investigator, a database, a monitoring application, etc.), and / or a detection device (e.g., detecting the stimuli and / or the subject and providing feedback regarding the stimuli and / or the subject, including whether gamma oscillations are being induced, the subject's susceptibility, cognitive function, physical or chemical changes, stress, safety, etc.).

[0064] In some embodiments, gamma oscillations are induced by visual stimuli, such as flashes of light at about 20 Hz to about 100 Hz. In certain embodiments, gamma oscillations are induced by flashes of light at about 20 Hz to about 50 Hz. In further embodiments, gamma oscillations are induced by flashes of light at about 35 Hz to about 45 Hz. In yet other embodiments, gamma oscillations are induced by flashes of light at about 40 Hz. In some embodiments, a subject is exposed to flashes of light at about 20 Hz to about 100 Hz, or about 20 Hz to about 50 Hz, or about 35 Hz to about 45 Hz, or about 40 Hz (e.g., by being placed in a chamber containing a light-emitting device or wearing a light-emitting device).

[0065] In some embodiments, gamma oscillations are induced by auditory stimuli, such as sound waves with a frequency of about 20 Hz to about 100 Hz, or about 20 Hz to about 80 Hz, or about 20 Hz to about 50 Hz, or about 35 Hz to about 45 Hz, or about 40 Hz. In some embodiments, the subject is exposed to auditory stimuli of about 20 Hz to about 100 Hz, about 20 Hz to about 80 Hz, about 20 Hz to about 50 Hz, about 35 Hz to about 45 Hz, or about 40 Hz (e.g., by being placed in a chamber equipped with an emitting noise-canceling device or by wearing an emitting noise-canceling device).

[0066] In some embodiments, the subject is exposed to the visual and / or auditory stimuli (e.g., placed in a chamber with an emitting light-blocking device or wearing an emitting light-blocking device) for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or more. In some embodiments, the subject is exposed to the stimuli (e.g., placed in a chamber with an emitting light-blocking device or wearing an emitting light-blocking device) for less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour. In some embodiments, the subject is exposed to the stimuli (e.g., placed in a chamber with an emitting light-blocking device or wearing an emitting light-blocking device) for less than 1 hour.

[0067] In some embodiments, a subject receives the methods provided herein. In other embodiments, a subject is treated with the methods provided herein on multiple separate occasions. A subject can be treated on a regular schedule or as symptoms arise or worsen. In some embodiments, long-term treatment can be effective in reducing soluble Aβ peptide and / or insoluble Aβ peptide (i.e., plaques).

[0068] In some embodiments, gamma oscillations are induced in a cell-type-specific manner. In some embodiments, gamma oscillations are induced in FS-PV interneurons. The term "fast-spiking" (FS), when used to describe a class of neurons, refers to the ability of neurons to discharge at high rates for extended periods with little adaptation or decay in spike frequency at spike height. Thus, these neurons can be sustained without significant modulation. FS neurons are capable of sustained high-frequency (e.g., equal to or higher than about 100 Hz or about 150 Hz) discharges. This property of FS neurons is due in large part to their expression of fast delayed rectifier channels, i.e., channels that activate and inactivate very rapidly.

[0069] In one aspect, the stimulus can be non-invasive. As used herein, the term "non-invasive" refers to devices, methods, and systems that do not require surgical intervention or treatment of the body, such as injection or implantation of a composition or device. For example, the stimulus can be visual (e.g., flashing light), auditory (e.g., sound vibration), and / or tactile (mechanical stimulus involving force, vibration, or movement).

[0070] In another embodiment, the stimulation can be invasive or at least partially invasive, for example, visual, auditory, and / or tactile stimulation can be combined with the injection or implantation of compositions (e.g., photosensitive proteins) or devices (e.g., integrated fiber optics and solid-state light sources).

[0071] Experimental data Gamma oscillations are reduced during hippocampal SWRs early in the disease in 5XFAD mice. Gamma impairments have been observed in multiple brain regions in several neurological and psychiatric disorders, including reduced spontaneous gamma synchronization in human patients with AD. Interestingly, reduced spontaneous gamma has also been demonstrated in vivo in two mouse models of AD (human amyloid precursor protein (hAPP) Tg mice and apolipoprotein E4 allele (APOE4) knock-in mice) and in another mouse model (Tg) in in vitro slice studies. However, it is unclear whether gamma oscillations are altered in other mouse models of AD, whether this occurs early in the disease progression, and whether gamma disruption influences disease progression.

[0072] To address these questions, we recorded neural activity from awake, behaving 5XFAD mice, an established AD model harboring five familial AD mutations. Specifically, 5XFAD mice express five distinct alleles of familial AD, including APP KM670 / 671NL (Swedish), APP I716V (Florida), APP V717I (London), PSEN1 M146L (A>C), and PSEN1 L286V. Therefore, 5XFAD mice were used as a model of AD amyloid pathology. In some embodiments, the neural activity was recorded from mice approximately 3 months old, at a time when their Aβ levels are elevated but before the onset of major plaque accumulation and the onset of learning and memory impairments. Figure 1 is a schematic diagram showing a mouse running a virtual linear maze on a spherical treadmill, according to some embodiments. Food-restricted mice can earn rewards by navigating the virtual linear maze on a spherical treadmill.

[0073] Neural activity can be recorded from the hippocampal subregion CA1. Figures 2A and 2B are electrical traces showing theta oscillations and sharp wave ripples (SWRs) recorded from the hippocampal CA1, according to some embodiments. In some embodiments, gamma oscillations in CA1 can be present during periods of overt activity, such as running, where theta oscillations (4-12 Hz) are observed, as shown in Figure 2A, as well as during resting and exploratory behaviors, where SWRs occur, as shown in Figure 2B.

[0074] Examination of power spectral densities during theta oscillations revealed no significant differences in slow gamma power (20 Hz to 50 Hz range) between 5XFAD mice and WT littermates. Figures 3A and 3B are plots showing the mean and standard deviation of normalized power spectra and normalized power spectral densities during theta cycles in 3-month-old Tg 5XFAD and WT mice, according to some embodiments. Figure 3A shows the mean and standard deviation of normalized power spectral densities during theta cycles in 3-month-old 5XFAD (n = 6 mice) and WT (n = 6 mice) mice. Figure 3B shows the mean and standard deviation of the power spectra obtained. In some embodiments, the power spectral density of each animal may be normalized to its peak (during theta). Figure 3B shows the normalized power spectral densities during theta periods for 3-month-old 5XFAD (n=6 mice) and WT (n=6 mice) mice.

[0075] As a next step, in some embodiments, we investigated gamma oscillations during SWRs, i.e., high-frequency oscillations between 150 and 250 Hz lasting approximately 50 to 100 milliseconds. SWRs are associated with bursts of collective activity in which patterns of spiking activity are replayed across the hippocampus. Previous studies have shown that slow gamma is elevated during SWRs and synchronized between CA3 and CA1. As a result, neurons across these hippocampal subregions tend to fire together during SWRs, as neurons are more likely to fire in phase with gamma. We identified SWRs (defined as the period when power in the ripple band between approximately 150 and 250 Hz exceeds the upper four standard deviations of the mean) and plotted spectrograms to examine power across a range of frequencies during these SWRs. In the spectrograms, we observed an increase in power above 100 Hz, indicative of high-frequency oscillations characteristic of SWRs, as well as an increase in power below approximately 50 Hz, indicative of a simultaneous increase in gamma power.

[0076] 4A and 4B are spectrograms showing SWRs for WT and 5XFAD mice, according to some embodiments. Figure 4A shows that the average SWR-triggered spectrogram for one WT mouse shows an increase in the gamma band 402 during SWR 404 at frequencies below 80 Hz, as shown in the right plot. Figure 4B shows that the average SWR-triggered spectrogram for one 5XFAD mouse shows an increase in the gamma band during SWR, but this increase is lower than in the WT mouse shown in Figure 4A.

[0077] In some embodiments, this study found that the instantaneous frequencies of these slow oscillations (ranging from 10 to 50 Hz, as further described herein) had a unimodal distribution centered around 40 Hz. Figures 5A-5C are plots showing the distribution of instantaneous gamma frequencies during SWRs, according to some embodiments. Figure 5A shows the distribution of instantaneous gamma frequencies during SWRs of the same mouse shown in Figure 4A peaking around 40 Hz (n=370 SWRs). Figure 5B shows that the distribution of instantaneous gamma frequencies during SWRs in 5XFAD and WT mice shows a distribution around 40 Hz for each recording period, and Figure 5C shows the mean and standard error of the mean (SEM) between animals (n = 820, 800, 679, 38, 1875, 57 gamma cycles per period for six 5XFAD animals, and 181, 1075, 919, 1622, 51, 1860, 1903 gamma cycles per period for six WT animals).

[0078] In some embodiments, these gamma oscillations during SWR in WT mice were then compared to those in 5XFAD littermates, revealing impaired gamma during SWR. Although gamma power increased from baseline during SWR in 5XFAD mice, as further described herein, gamma power during SWR was significantly lower in 5XFAD than in WT mice.

[0079] 6A is a series of graphs showing z-scored gamma power as a function of time from peak SWR in 5XFAD and WT mice, respectively, according to some embodiments. Figure 6A shows the mean and SEM, illustrating the increase in gamma power during SWR relative to baseline.

[0080] FIG. 6B is a plot showing the cumulative distribution of gamma power during SWR in 5XFAD and WT mice, according to some embodiments. The cumulative distribution of gamma power during SWR shows a significantly smaller increase in 5XFAD than in WT mice (rank sum test, p<1.0). 0 -5, n = 2166 SWR in six 5XFAD mice and 3085 SWR in six WT mice, median z-score for 5XFAD mice was 1.02 (0.39-1.87, 1st quartile-3rd quartile) and median z-score for WT mice was 1.18 (0.53-2.15, 1st quartile-3rd quartile)).

[0081] Figures 6C and 6D are plots showing the cumulative distribution of z-scored gamma power during 100 ms centered around the peak SWR for WT mouse 606 and 5XFAD mouse 608, as well as the mean and SEM (shaded) between animals, according to some embodiments (SWR per period n=514, 358, 430, 22, 805, 37 for 6 5XFAD animals and SWR per period 82, 311, 370, 776, 18, 710, 818 for 6 WT animals).

[0082] Figure 6E is a plot showing the cumulative distribution of z-scored gamma power during 100 ms around the peak of a large SWR in WT mouse 614 and 5XFAD mouse 616 (detection threshold above 6 standard deviations above the mean), according to some embodiments. As further described herein, data that was not normally distributed was subjected to a rank sum test. Figure 6E shows a significantly smaller increase in WT mouse 614 and 5XFAD mouse 616 (rank sum test, p<10 -5 , n = 1000 SWR in six 5XFAD mice and 1467 SWR in six WT mice).

[0083] In some instances, spiking was phase-modulated by these gamma oscillations in both groups, but the modulation of spiking by gamma phase was weaker in 5XFAD than in WT animals. This study showed that the depth of modulation may be significantly less in 5XFAD than in WT animals.

[0084] According to some embodiments, FIG. 7A is a plot showing the proportion of spikes as a function of gamma oscillation phase, and FIG. 7B is a plot showing the depth of modulation of spiking during SWR as a function of gamma phase (rank sum test, bootstrap method, p<10). -5 This is important when adjusting for multiple comparisons. For 5XFAD spike-gamma phase distributions (n ​​= 2500) and WT distributions (n ​​= 3000), the median modulation depth for 5XFAD mice was 0.35 (0.21-0.44, 1st-3rd quartile) and the median modulation depth for WT mice was 0.38 (0.29-0.47, 1st-3rd quartile). Error bars indicate the mean + / - SEM. Plot 704 shows a histogram of spiking modulation depth.

[0085] Figures 7C and 7D are plots showing the percentage of spikes in hippocampal CA1 during SWR as a function of the phase of gamma oscillations for each animal in 5XFAD and WT animals, as well as the mean and SEM across animals (spikes per period during SWR for six 5XFAD animals n=2475, 1060, 3092, 25, 6521, 123 and for six WT animals n=360, 4741, 1564, 2961, 88, 3058, 4270).

[0086] According to some embodiments, during large SWRs (detection thresholds above 6 standard deviations above the mean, as further described herein) in 3-month-old 5XFAD (n=6 mice) and WT (n=6 mice) mice, FIG. 7E is a plot showing the proportion of spikes as a function of the phase of gamma oscillations, and FIG. 7F is a plot showing the depth of modulation of spiking (rank sum test, bootstrap method; single asterisk indicates p<10). -10 Shown are 5XFAD spike-gamma phase distributions (n=2500 and WT distributions 3000). Error bars indicate mean + / - SEM.

[0087] This study also found that 5XFAD mice may have fewer SWRs per hour compared to WT during non-theta periods (rank sum test, p<10 -5 The non-theta period in six 5XFAD mice (n = 634) and six WT mice (n = 750) was median 0.07 Hz (0-0.17, 1st-3rd quartile) in 5XFAD mice and 0.12 Hz (0-0.24, 1st-3rd quartile) in WT mice; as noted above, the period further decreased when gamma power increased.

[0088] 8A and 8B are plots showing the percentage of SWRs per non-theta period for each animal (FIG. 8A) and all animals combined (FIG. 8B) for 5XFAD mice 802 and WT mice 804, according to some embodiments (rank sum test, p<10 -10 In six 5XFAD animals, non-theta cycles per period (n = 117, 210, 151, 55, 100, 1), and in six WT animals, non-theta cycles per period (n = 80, 68, 115, 95, 15, 159, 218). These results reveal impaired modulation of gamma oscillations and hippocampal CA1 spiking in a mouse model of AD prior to the development of primary amyloid plaque deposits and evidence of cognitive impairment.

[0089] Optogenetic stimulation of FS-PV interneurons at gamma frequencies drove gamma oscillations in the CA1 region of the hippocampus. The finding of gamma impairment during SWRs early in the progression of the disease in this mouse model of AD raises the question of whether gamma oscillations could influence the molecular and cellular pathophysiology of AD. To test this, we optogenetically drove gamma oscillations in FS-PV interneurons in the hippocampal CA1 of 2.5-month-old 5XFAD / PV-Cre double transgenic mice by expressing ChR2 in a Cre-dependent manner using a double-floxed inverted open reading frame (DIO) ChR2-EYFP adeno-associated virus (AAV). We performed studies to determine whether genetic induction of hippocampal gamma oscillations in mice impacts the molecular pathology of this mouse model of AD. Hippocampal gamma oscillations were genetically induced in awake, behaving WT and 5XFAD mice.

[0090] We generated an adeno-associated virus (i.e., AAV5 virus) containing a double-floxed, inverted open reading frame (DIO) of ChR2 linked to enhanced yellow fluorescent protein (EYFP) driven by the EF1α promoter. Figure 9 is a schematic diagram showing a viral vector (i.e., AAV5-DIO-ChR2-EYFP) for modulating the activation of specific cell types in a subject's brain, according to some embodiments. Viral expression was targeted to the CA1 region of the hippocampus in a cell-type-specific manner. In the presence of Cre recombinase, one of the two incompatible loxP variants is inverted to allow expression of ChR2.

[0091] The CA1 region of the hippocampus of 5XFAD mice was infected with either AAV-DIO-ChR2-EYFP or EYFp alone using a stereotactic viral injection method, which allows for precise, localized targeting of viral infection. In one embodiment, a ferrule containing a fiber optic cable (white bar) was placed approximately 0.3 mm above the targeted brain region during injection. After two weeks, allowing time for the mice to recover and for the virus to express in PV cells, CA1 interneurons were optogenetically manipulated.

[0092] 10A and 10B are schematic diagrams illustrating signal delivery to the CA1 region of a subject's hippocampus, according to some embodiments. In FIG. 10A, a mouse is shown running on a ball through a maze while receiving optogenetic gamma stimulation in the hippocampus, according to some embodiments. The arrows 1000 in FIGS. 10A and 10B indicate blue light flashing at approximately 40 Hz to activate the brain region.

[0093] In this example, a 200 mW 493 nm DPSS laser was connected to a patch cord with fiber channel / body contact connectors on each end. Approximately 1 mW of light stimulation was delivered for approximately 1 hour during the experiment. More specifically, blue light (e.g., 473 nm) was delivered at various frequencies, including theta (e.g., approximately 8 Hz), gamma (e.g., approximately 40 Hz), and randomly at approximately 40 Hz, through an optical fiber positioned directly over the CA1 region of the hippocampus. In some embodiments, no stimulation conditions were tested. According to some embodiments, the theta condition served as a frequency control, and the random condition controlled for periodic specificity.

[0094] After completion of the 1-hour stimulation, brain tissue was sectioned and frozen at -80°C for staining and enzyme-linked immunosorbent assay (ELISA) analysis. Figure 11 is an immunofluorescence image showing immunostaining of neural tissue in a subject with ChR2 and DAPI, according to some embodiments. In this example, Figure 11 shows DAPI (nuclei) and ChR2 staining in the hippocampus.

[0095] Figure 12A is an immunofluorescence image showing ChR2-EYFP expressed in PV+ interneurons, according to some embodiments. Figure 12A shows that ChR2-EYFP was strongly expressed in PV+ interneurons in the CA1 of 3-month-old 5XFAD / PV-Cre mice (scale bar = 100 μm). Figure 12B is a series of immunofluorescence images showing immunohistochemistry with anti-EYFP and anti-PV antibodies in the CA1 of 3-month-old 5XFAD / PV-Cre mice expressing AAV-DIO ChR2-EYFP, demonstrating EYFP expression only in PV+ cells (scale bar = 50 μm). To compare 5XFAD and WT mice, ChR2 was expressed in FS-PV interneurons in 5XFAD-negative littermates. To control for nonspecific effects of light stimulation, 5XFAD / PV-Cre double transgenic mice expressing AAV-DIO containing EYFP alone were used. In these mice, with the same genetic background and light delivery conditions, light delivery does not result in optogenetic stimulation. In some embodiments, FS-PV interneurons were selected at 40 Hz for two reasons. First, previous studies have shown that driving FS-PV interneurons at 40 Hz evoked maximal LFP responses. Second, in some embodiments, gamma disturbances were observed during SWRs, and the instantaneous gamma frequencies during SWRs formed a distribution centered around 40 Hz, as shown in Figures 5A-5C. In some embodiments, for electrophysiological recordings, 40 Hz stimulation periods were interleaved with periods of no stimulation or periods of stimulation delivered at randomized intervals selected from a Poisson distribution centered around 40 Hz, as described further herein.

[0096] 13A and 13B include schematic diagrams of FS-PV interneuron testing, electrical traces of local field potentials, and power spectral densities, according to some embodiments. Referring to FIG. 13A, 1302 shows electrical traces of local field potentials in CA1 before and during 40 Hz optogenetic activation of FS-PV interneurons. Plot 1304 shows the mean and standard deviation of power spectral densities during 40 Hz stimulation, random stimulation (stimulation at randomized intervals selected from a Poisson distribution centered around 40 Hz), or no stimulation of FS-PV interneurons in CA1 (n=4 5XFAD mice and 3 WT mice). Figure 13B shows the power spectral density of FS-PV interneurons in CA1 during 40 Hz stimulation 1306, random stimulation 1308, or no stimulation 1310 for each mouse (5XFAD mice n=4, 169, 130, 240, 73 40 Hz, 143, 129, 150, 72 random, and 278, 380, 52, and 215 no-stimulus cycles per animal; WT mice n=3, 65, 93, 91 40 Hz, 64, 93, 90 random, and 187, 276, 270 no-stimulus cycles per animal). Delivery of 1 ms 473 nm light pulses at 40 Hz resulted in an increase in power at 40 Hz in the LFP, as shown in plot 1306 of Figures 13A and 13B, while random stimulation did not result in an increase in power at 40 Hz, as shown in plot 1308 of Figures 13A and 13B.

[0097] Furthermore, in some embodiments, the light pulse effectively drove spikes 2-3 ms after light onset, with spikes per pulse being similar in both the random and 40 Hz conditions. Figures 14A and 14B include raw electrical traces after the onset of a 1 ms laser pulse, traces filtered for spikes after optogenetic stimulation, and plots of spike probability, according to some embodiments. Figure 14A shows an exemplary raw trace 1402 and a filtered trace for spikes (300-6000 Hz) 1404 after optogenetic stimulation 1406. Plot 1408 shows a histogram of spikes per pulse after the onset of a 1 ms laser pulse during 40 Hz stimulation, random stimulation, or no stimulation (n=345,762 40 Hz stimulations, 301,559 random pulse stimulations, and 32,350 no stimulations in four 5XFAD and three WT mice, separated by at least 500 ms from the 552 40 Hz stimulations, 543 random stimulations, and 1,681 no stimulation periods). Figure 14B shows the probability of spiking after the onset of a 1 ms laser pulse in response to 40 Hz stimulation 1412, random stimulation 1414, or no stimulation 1410, with an increase in spiking approximately 2-3 ms after laser pulse onset (5XFAD n=4, 87, 130, 8, 73 40 Hz stimulation, 85, 129, 5, 72 random stimulation, and 251, 379, 15, 215 no-stimulus periods per animal; and WT n=3, 65, 93, 91 40 Hz stimulation, 64, 93, 90 random stimulation, and 187, 277, 270 no-stimulus periods per animal). Error bars indicate the mean + / - SEM.

[0098] Thus, 40 Hz oscillations in CA1 were effectively driven by optogenetic stimulation of FS-PV interneurons. Previous studies have shown that Aβ peptide levels increase after increased neural activity and decrease after quiescence. In some embodiments, the random stimulation condition was used to control for overall changes in spiking activity induced by stimulation. In some embodiments, multi-unit firing rates were compared during interleaved periods of 40 Hz and random stimulation, and no significant differences in firing rates were observed between these conditions.

[0099] 15A is a histogram showing the difference in firing rate between 40 Hz and random stimulation periods, according to some embodiments. Figure 15A shows that both types of stimulation elicit similar amounts of spiking activity (Wilcoxon signed-rank test for median zero, p>0.6, n=538 stimulation periods from four 5XFAD and three WT mice, "ns" indicates not significant). Wilcoxon signed-rank test for median zero p>0.6 for the distribution of the difference in firing rate during 40 Hz and random stimulation for all mice combined: median -1.75×10 -5 Hz (-1.28-1.18Hz, 1st to 3rd quartile) stimulation period n = 538.

[0100] Figure 15B is a bar graph showing the multiunit firing rate per 40 Hz stimulation 1512, random stimulation 1514, and no stimulation 1510 cycle for each animal (rank sum test for each animal for three WT and four 5XFAD mice, p>0.09; medians and quartiles are shown in the figure; n=87, 130, 8, 65, 93, 91, 73 40 Hz stimulation cycles and 85, 129, 5, 64, 93, 90, 72 random stimulation cycles per mouse). Box plots show the median (white line within the box) and quartiles (top and bottom of the box). There was no significant difference in firing rate between 40 Hz and random stimulation in all animals, indicating that this random stimulation condition served as a control for spiking activity (rank sum test for each animal for three WT and four 5XFAD mice, p>0.09; medians and quartiles are shown in the figure; n=87, 130, 8, 65, 93, 91, 73 for 40 Hz stimulation periods and 85, 129, 5, 64, 93, 90, 72 for random stimulation periods per animal). We also investigated whether 40 Hz stimulation caused neuronal hyperactivity compared to no stimulation. In most animals, 40 Hz or random stimulation periods were significantly different. There was no significant difference in firing rate between random stimulation and no stimulation (rank sum test for two WT and two 5XFAD animals, p>0.25, 40 Hz stimulation cycles per animal n=8, 93, 91, 73 and baseline cycles 15, 277, 270, 215), or firing rate during 40 Hz or random stimulation was lower than during no stimulation (rank sum test for one WT and one 5XFAD animal, p<10 -5 This was significant when corrected for multiple comparisons (n ​​= 130, 65 for 40 Hz stimulation and 379, 187 for baseline periods per animal), indicating that 40 Hz stimulation did not cause neuronal hyperactivity. In one animal, activity was significantly higher during 40 Hz or random stimulation than during baseline (rank sum test for one 5XFAD mouse, p < 10 -5, 40 Hz stimulation period per animal, n = 87 and baseline period 251). Thus, in 6 of 7 animals, there is no evidence that 40 Hz optogenetic stimulation of FS-PV interneurons causes hyperactivity. Thus, in some embodiments, as shown in Figure 15A, this random condition did not induce gamma oscillations but did result in a similar amount of multi-unit spiking activity.

[0101] 16A shows electrical traces recorded from a subject's hippocampus during frequency-specific increases in stimulation of specific cell types in the CA1 region of the hippocampus, according to some embodiments. More specifically, FIG. 16A shows electrical traces recorded from a subject's hippocampus during frequency-specific increases in stimulation of FS-PV+ (i.e., gamma condition), according to some embodiments.

[0102] 16B is a power spectral density plot showing frequency-specific increases in local field potential power upon stimulation of specific cell types in the CA1 region of a subject's hippocampus, according to some embodiments. Notably, the power spectral density graph in FIG. 16B verifies the specificity of the stimulation. Local field potential (LFP) power was elevated only in the 40 Hz band 1600 during the gamma stimulation condition when FS-PV+ were activated with 40 Hz blue light pulses (n=4 mice per group). Neither the baseline nor the random stimulation condition showed enhancement at this frequency 1600.

[0103] Gamma stimulation reduced Aβ production in the CA1 region of the hippocampus. Accumulation of Aβ can lead to multiple neurotoxic events typical of AD pathology. Therefore, in some embodiments, we examined the effect of gamma stimulation on general Aβ peptide levels in 5XFAD mice. Three-month-old mice were used because they lack plaques in the hippocampus at this stage, allowing us to examine the dynamics of soluble Aβ independent of plaque burden. In some embodiments, 1 hour of stimulation of FS-PV interneurons significantly increased Aβ levels in the 40 Hz group compared to the EYFP control group in the CA1 region of the hippocampus, as measured by Aβ ELISA analysis. 1-4053.22% and Aβ 1-42 It was found that the β-amyloid ...

[0104] 17A and 17B show relative Aβ in 5XFAD / PV-Cre CA1 by one-way ANOVA across all mice combined, according to some embodiments. 1-40 and Aβ 1-42 A bar graph showing the levels of Aβ 1-40 For EYFP mice n = 8 and 40Hz mice n = 7, Aβ 1-42 (n=4 mice per group for each stimulation condition). The bar graph in Figure 17A shows the relative Aβ levels in 5XFAD / PV-Cre CA1 neurons under each stimulation condition. 1-40 The circles 1702 overlaid on the bars of the bar graph indicate individual data points for each group (5XFAD / PV-Cre mice per group: EYFP n=8, 40Hz n=7, 8Hz n=4, random n=6). In this figure, the note "ns" 1704 indicates not significant, an asterisk 1706 indicates p<0.05, and a double asterisk 1708 indicates p<0.01 by one-way ANOVA for all bar graphs. Figure 17B shows the relative Aβ levels in 5XFAD / PV-Cre CA1 for each stimulation condition. 1-42 The levels of EYFP (n=4, 4 5XFAD / PV-Cre mice per group) are shown. 0 Hz n=4, 8 Hz n=3, random n=3). Figures 17A and 17B show the mean and SEM.

[0105] Table 1 (below) shows the raw concentration (pg / ml) values, where significant differences were determined by Student's t-test (p<0.05) when comparing mice from the same litter receiving different conditions. Table 1 also shows the raw Aβ concentrations at ELISA dilutions for each experimental group. 1-40 and Aβ 1-42 Indicates the level of. [Table 1] JPEG2023101026000003.jpg224170 JPEG2023101026000004.jpg200170

[0106] In some embodiments, a comprehensive series of control experiments was performed to determine whether the effect was specific to frequency, cell type, and / or periodicity. To determine frequency specificity, FS-PV interneurons in 5XFAD / PV-Cre double transgenic mice were stimulated at 8 Hz, and no changes in Aβ levels were observed. FS-PV interneurons were then stimulated randomly, and the effect was specific to periodic stimulation. Indeed, amyloid levels did not decrease after random stimulation; instead, Aβ levels increased. 1-40 increased by 230.1%, and Aβ 1-42 increased by 133.8% (see e.g., Figures 17A and 17B, p<0.01 by one-way ANOVA for all mice combined, Aβ 1-40 For EYFP mice (n=8) and random mice (n=4), Aβ 1-42 n=3 mice per group. Comparison of mice from the same litter receiving different conditions showed significant differences by Student's t-test, p<0.01.

[0107] Finally, we examined the cell-type specificity of the effects of 8 Hz and 40 Hz stimulation on CamKII+ excitatory neurons in the hippocampal CA1 region using 5XFAD / αCamKII-Cre double transgenic mice. Figures 18A and 18B show the relative Aβ expression in 5XFAD / αCamKII-Cre CA1 neurons by one-way ANOVA, according to some embodiments. 1-40 and Aβ 1-42 Figure 18A is a bar graph showing the relative Aβ levels in 5XFAD / αCamKII-Cre CA1 under each stimulation condition. 1-40 Circles 1802 overlaid on the bars of the bar graph represent individual data points for each group (5XFAD / αCamKII-Cre mice per group, 40 Hz n=6, 8 Hz n=3, random n=3, by one-way ANOVA; the note "ns" 1804 indicates not significant, and an asterisk 1806 indicates p<0.001).

[0108] Figure 18B shows the relative Aβ levels in 5XFAD / αCamKII-Cre CA1 cells under each stimulation condition. 1-42 The levels shown are those of αCamKII-Cre mice per group (n=3). In some embodiments, driving CamKII+ excitatory neurons at 8 Hz or 40 Hz significantly reduced Aβ levels. 1-40 and Aβ 1-42 No significant differences were found in the levels of Aβ (see, e.g., Figures 18A and 18B, right, p>0.05 by one-way ANOVA, n=6 in 40 Hz mice and 3 in 8 Hz mice). 1-40 ), n = 3 mice per group (Aβ 1-42 ). When comparing mice from the same litter that underwent different conditions, there were no significant differences by Student's t-test (p>0.05). Similar to 5XFAD / PV-Cre mice, random stimulation also drove the generation of CamKII+ neurons, suggesting that Aβ 1-40 and a 257.6% increase in Aβ 1-42 (See e.g., Figures 18A and 18B, right, p<0.001 by one-way ANOVA, Aβ 1-40 For 40Hz mice n=5 and random mice 3, Aβ 1-42 n=3 mice per group. When comparing mice from the same litter that underwent different conditions, Aβ 1-40 The difference was significant by Student's t-test (p<0.001), and Aβ 1-42 p=0.13 by Student's t-test).

[0109] Thus, the decrease in Aβ peptide levels after 40 Hz stimulation may be specific to driving FS-PV interneurons. In some embodiments, to confirm these ELISA findings by immunohistochemistry, we performed Aβ labeling using a C-terminal specific antibody for β-amyloid that does not cross-react with APP in CA1.

[0110] Figure 19A is a series of images showing immunohistochemistry with anti-Aβ and anti-EEA1 antibodies in the CA1 region of the hippocampus in 5XFAD / PV-Cre mice under EYFP, 40 Hz, and random stimulation conditions (scale bar = 50 μm). Figure 19B is a series of bar graphs showing relative Aβ immunoreactivity normalized to EYFP in accordance with some embodiments (n = 4 mice per group, 1908 indicates p < 0.05, 1920 indicates p < 0.01 by one-way ANOVA).

[0111] FIG. 20A is a series of immunofluorescence images showing immunohistochemistry with an anti-Aβ antibody in the hippocampal CA1 region of 5XFAD / PV-Cre mice, according to some embodiments. Specifically, FIG. 20A is a series of immunofluorescence images showing immunohistochemistry with an anti-Aβ2002 (12F4) antibody in the hippocampal CA1 region of 5XFAD / PV-Cre mice under EYFP, 40 Hz, and random stimulation conditions (scale bar = 50 μm). FIG. 20B is a bar graph showing relative Aβ immunoreactivity normalized to EYFP, according to some embodiments. Specifically, FIG. 20B shows relative Aβ immunoreactivity normalized to EYFP (1 (n=4 mice per group, p<0.05 for 2004 and p<0.001 for 2006 by one-way ANOVA.) The intensity of Aβ labeling was significantly reduced by 39.5% after 40 Hz stimulation of FS-PV interneurons in 3-month-old 5XFAD / PV-Cre double transgenic mice and significantly increased by 187.0% after random stimulation when compared to the EYFP group (see, e.g., Figures 19A, 19B, 20A, and 20B, p<0.05 and p<0.01 by one-way ANOVA, n=4 mice per group).

[0112] Brain amyloid concentrations may depend on the rates of Aβ production and clearance. In some embodiments, Aβ peptides are produced by sequential proteolytic cleavage of APP by beta- and gamma-secretases. When BACE1 cleaves the APP holoprotein, CTF and NTF of APP may be produced. In some embodiments, to clarify how 40 Hz stimulation reduced Aβ levels, gamma-mediated APP cleavage was examined by measuring the levels of APP cleavage intermediates, CTF and NTF, after stimulation of FS-PV interneurons. After 40 Hz stimulation, a significant decrease in CTF was observed after 40 Hz stimulation compared to the EYFP group (p<0.05 and p<0.01 by one-way ANOVA, n=6 mice per group).

[0113] Figure 21A is a representative Western blot showing levels of APP (CT695), APP NTF (A8967), APP CTF (CT695), and β-actin (A5316) (loading control) in CA1 under EYFP, random, and 40 Hz stimulation conditions, with one mouse per lane and two biological replicates for each condition, according to some embodiments. Figure 21B is a bar graph showing relative immunoreactivity of APP CTF, according to some embodiments. Specifically, Figure 21B shows the relative (normalized to actin) immunoreactivity of APP CTF under 40 Hz versus EYFP and random conditions (n=6 mice per group; one asterisk 2102 indicates p<0.05, and two asterisks 2104 indicates p<0.01 by one-way ANOVA). Figure 21C is a series of Western blots showing levels of full-length APP2106 (CT695), APP CTF2108 (CT695), and β-actin 2112 (A5316, loading control) in CA1 according to some embodiments. Specifically, Figure 21C shows levels of full-length APP2106 (CT695), APP CTF2108 (CT695), and β-actin 2112 (A5316, loading control) in CA1 under EYFP, random, and 40 Hz stimulation conditions, with one mouse per lane and two biological replicates for each condition.

[0114] Figure 22A is a bar graph showing the relative (normalized to actin) immunoreactivity of APP NTF at 40 Hz for EYFP and random conditions (n=6 mice per group, one-way ANOVA, note "ns" 2204 indicates not significant, 2202 indicates p<0.05). Figure 22B is a bar graph showing the relative (normalized to actin) immunoreactivity of full-length APP in EYFP, random, and 40 Hz conditions (n=6 mice per group, one-way ANOVA, note "ns" 2204 indicates not significant, 2202 indicates p<0.05).

[0115] In some embodiments, after 40 Hz stimulation, a significant reduction in APP NTF levels was observed, 28.5% compared to the EYFP group and 28.2% compared to the random group (see, e.g., Figures 21A, 22A, and 21C; p<0.05 by one-way ANOVA; n=6 mice per group). Furthermore, full-length APP levels appeared similar among the various groups, indicating that the reduction in Aβ was not due to changes in precursor levels (see, e.g., Figures 21A, 22B, 21C; n=6 mice per group for APP experiments). In some embodiments, changes in full-length APP may be difficult to detect in this mouse model due to the relatively high abundance of APP compared to its cleavage products.

[0116] In some embodiments, APP processing occurs within the vesicular transport pathway, and previous studies have shown that after activity stimulation, APP is transported to recycling endosomes. Furthermore, enlarged early endosomes have been observed in brain tissue from AD patients and in human neurons from AD patients. In some embodiments, to test whether gamma stimulation affects endosome abundance in experimental animals, early endosomes in CA1 after 40 Hz and random stimulation were characterized using two markers: EEA1 (early endosomal antigen 1) and Rab5 (a Ras-related protein encoded by the RAB5A gene). Figure 23 is a series of immunofluorescence images showing immunohistochemistry with anti-Rab5 (ADI-KAp-GP006-E) antibody in 3-month-old 5XFAD / PV-Cre mice under EYFP, 40 Hz, and random stimulation conditions (scale bar = 50 μm).

[0117] Figure 24A is a bar graph showing relative EEA1 immunoreactivity normalized to EYFP (n=4 mice per group, one asterisk 2402 indicates p<0.05, two asterisks 2402 indicates p<0.01 by one-way ANOVA), according to some embodiments. Figure 24B is a bar graph showing relative Rab5 intensity levels in CA1 from 5XFAD / PV-Cre under EYFP, 40 Hz, and random stimulation conditions (n=3 mice per group, three asterisks 2408 indicates p<0.001 by one-way ANOVA), according to some embodiments. In some embodiments, EEA1 staining produced a punctate cytoplasmic and juxtamembrane pattern typical of early endosomes in neuronal cell bodies (see, e.g., Figure 19A). In some embodiments, Rab5 labeling was largely confined to the cell body and plasma membrane, represented by small, sparse puncta concentrated within endosomes and membrane compartments (see, e.g., Figure 23). Overall, early endosomal labeling of CA1 neurons showed a significant decrease in both EEA1 (39.7%) and Rab5 (40.1%) staining intensity after 40 Hz stimulation compared to EYFP controls (see, e.g., Figures 19A, 23, 24A; p<0.05 and p<0.001 by one-way ANOVA, respectively; n=2 sections from 3 mice per group). In contrast, random stimulation of FS-PV interneurons increased EEA1 staining intensity by 122% compared to EYFP controls (see, e.g., Figures 19A and 24A; p<0.01 by one-way ANOVA, n=2 sections from 3 mice per group). In some embodiments, treatment-dependent changes in EEA1 staining intensity were comparable to those of Aβ in CA1 (see, e.g., Figures 19A-B, 20A-B, 23, and 24A-B; p<0.05 by one-way ANOVA, n=2 sections from 3 mice per group). These results suggest that 40 Hz stimulation alters EEA1 and Rab5 in addition to the changes observed in CTF, indicating differences in general endosomal processing.

[0118] FIG. 25A shows the Aβ peptide isoform Aβ after different types of stimulation of the CA1 region of the hippocampus of a subject, according to some embodiments. 1-40This experiment shows that 1 hour of optogenetic stimulation of FS-PV+ at approximately 40 Hz reduces Aβ levels in the hippocampal CA1. 1-40 Excitatory cone stimulation at 8 Hz 506 and excitatory cone stimulation at 40 Hz 508 reduced Aβ levels. 1-40 Random 40 Hz stimulation 504, and especially random excitatory cone stimulation 510, did not significantly affect the levels of Aβ. 1-40 The levels of α-glucan-1, α-glucan-1, and α-glucan-1 were significantly increased (n = 4-9 animals per group).

[0119] FIG. 25B illustrates the Aβ peptide isoform Aβ following stimulation of specific cell types in the CA1 region of the hippocampus of a subject with gamma oscillations, according to some embodiments. 1-42 This experiment shows that 1 hour of optogenetic stimulation of FS-PV+ at approximately 40 Hz reduces Aβ in the hippocampal CA1. 1-42 The levels were decreased (n = 2-4 animals per group). Stimulation at 8 Hz 520, excitatory cone stimulation at 40 Hz 522, and excitatory cone stimulation at 8 Hz. Cone stimulation 524 is Aβ 1-42 Random 40 Hz stimulation518, and especially random excitatory cone stimulation526, increased the levels of Aβ. 1-42 significantly increased the levels of

[0120] Figure 25C is a series of images showing increased levels of full-length APP 528, 534 (normalized to actin 532) and decreased levels of CTFs (e.g., β-CTF) 530, 536 (normalized to actin 532) following stimulation of specific cell types in the CA1 region of a subject's hippocampus with gamma oscillations, according to some embodiments. Compared to a random 40 Hz control condition, FS-PV+ stimulation at 40 Hz reduced β-CTF levels of APP and increased full-length APP levels (n = 4-6 animals per group). Because β-CTF is an APP derivative generated during the amyloidogenic cleavage of APP by BACE1, high levels of β-CTF represent increased Aβ production.

[0121] Figures 26A-26B are immunofluorescence images showing endosomal levels (based on EEA1 levels) after different types of stimulation in the CA1 region of the hippocampus of a subject, according to some embodiments. Specifically, comparison of Figure 26B to Figure 26A shows that induction of gamma oscillations via 40 Hz stimulation of FS-PV+ reduces EEA1 levels (a marker of endosomal levels) compared to random FS-PV+ stimulation (n=3 mice per group, p=0.007), as measured by immunofluorescence. The reduced endosomal levels in these cells indicate a reduced interaction between APP and beta-secretase, which leads to reduced APP cleavage and Aβ production. Thus, this study demonstrated that gamma oscillations reduce AP production in an AD mouse model, as increased endosomal levels indicate increased APP processing and, therefore, Aβ production.

[0122] FIG. 27 is a bar graph showing the mean intensity values ​​(normalized to FAD) for the immunofluorescence images of FIGS. 26A-26B after different types of stimulation of the CA1 region of the hippocampus of a subject, according to some embodiments.

[0123] Gamma stimulation induced morphological transformation of microglia. In some embodiments, to further unbiasedly examine the cellular and molecular effects of 40 Hz stimulation, genome-wide RNA-seq was performed on hippocampal CA1 tissue from 5XFAD / PV-Cre double transgenic mice 1 hour after 40 Hz stimulation of FS-PV interneurons or no stimulation (EYFP). RNA-seq experiments yielded an average of 26,518,345 sequencing reads from three stimulated and three unstimulated mice. QC analysis of the data revealed a mean exon / intron ratio of 183, a mean exon / intergenic ratio of 272, and a mean percentage of ribosomal RNA reads of 3.6%. This analysis identified 523 differentially expressed genes (DEGs), of which 130 were upregulated and 393 were downregulated in response to 40 Hz stimulation.

[0124] Figure 28 is a heatmap showing differentially expressed genes determined by whole-transcriptome RNA-seq of the mouse hippocampal CA1 region with and without 40 Hz stimulation. Normalized z-score values ​​were calculated for each differentially expressed gene (columns). Colors represent relatively low and high levels of gene expression. Table 2 (below) shows 130 genes upregulated by 40 Hz stimulation of FS-PV interneurons (p<0.05 using Cufflinks 2.2 software (available from the Trapnell Lab, University of Washington, Seattle, WA) for assembling transcripts in RNA-seq samples, estimating their abundance, and testing for differential expression and regulation). [Table 2] JPEG2023101026000006.jpg178170

[0125] Table 3 (below) shows the 393 genes that were downregulated by 40 Hz stimulation of FS-PV interneurons (p<0.05 using Cufflinks 2.2 software (available from the Trapnell Lab, University of Washington, Seattle, WA)). [Table 3] JPEG2023101026000008.jpg249170 JPEG2023101026000009.jpg249170 JPEG2023101026000010.jpg89170

[0126] In some embodiments, upregulated genes generally had higher expression values ​​than downregulated genes. Figure 29 is a boxplot showing the FPKM values ​​of upregulated and downregulated genes in the EYFP and 40 Hz conditions according to some embodiments. The box indicates the median (black line within the box) and quartiles (top and bottom of the box), the whiskers represent the minimum and maximum values, and the circles represent outliers. The upregulated genes may be highly enriched in microglia. Specifically, approximately 35% of all upregulated genes had their highest expression in microglia (approximately 19% in neurons, 17% in endothelial cells, 14% in astrocytes, 9% in myelinating oligodendrocytes, 5% in oligodendrocyte progenitor cells, and 1% in newly formed oligodendrocytes).

[0127] Figure 30 is a pie chart showing the cell-type-specific expression patterns of identified upregulated genes after 40 Hz stimulation, according to some embodiments. Gene FPKM values ​​were calculated from published RNA-seq data from various brain cell types, including astrocytes, endothelial cells, microglia, myelinating oligodendrocytes (MOs), neurons, newly formed oligodendrocytes (NFo), and oligodendrocyte precursor cells (OPCs). Thus, the RNA-seq analysis strongly suggests that 1 hour of 40 Hz stimulation of FS-PV interneurons caused changes in the cellular state of microglia, which is important given the accumulating evidence that these cells play a role in AD pathology.

[0128] In some embodiments, to further explore the potential impact of 40 Hz stimulation on microglia, a series of publicly available RNA-seq datasets from microglia, peripheral macrophages, and neurons under different chemical and genetic perturbations were compared to the gene list from the characterization described in some embodiments herein using gene set enrichment analysis. Table 4 (below) shows the statistical significance based on GSEA of the correlation between genes up- or down-regulated by 40 Hz stimulation and published neuron-, microglia-, and macrophage-specific RNA-seq data under different chemical and genetic perturbations. [Table 4]

[0129] Interestingly, transcriptome changes following 40 Hz stimulation resembled those induced by increased neuronal activity (by NMDA and bicuculline) and less resembled those induced by resting activity (by tetrodotoxin). These findings further support the observation that 40 Hz stimulation of FS-PV interneurons does not reduce neuronal activity. Furthermore, the immediate-early genes Nr4a1, Arc, and Npas4, known to be upregulated by neuronal activity, were elevated after 1 hour of 40 Hz stimulation, as shown by both RNA-seq and RT-qPCR. Figure 31 is a bar graph showing RT-qPCR validation of specific gene targets in an RNA-seq dataset, according to some embodiments. The bar graph shows relative RNA levels (expression ratios) from EYFP3102 and 40 Hz stimulation 3104 conditions (Student's t-test: one asterisk indicates p<0.05, two asterisks indicate p<0.01, and three asterisks indicate p<0.001; n=3 mice per group). The most down-regulated genes were Grin4 and Camk2d (see, e.g., Figure 31, p<0.05, n=3 mice per group).

[0130] Additionally, the transcriptome results suggest a more phagocytic state of microglia. In some embodiments, upregulated genes were positively correlated with genomic changes induced by macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF), both of which are known to promote microglial uptake of Aβ. Figures 32A and 32B are plots showing the power spectral density of local field potentials recoded over the brain during 40 Hz light flashing, according to some embodiments. Figures 32A and 32B show no increase in power at 40 Hz, and therefore this effect is not due to photoelectric effects or electrical noise on the recording equipment (n = 4, 2, 1, 1, 17, 42, 36, 55, 53 for 40 Hz flashing periods from four recording sessions in three 5XFAD animals undergoing visual cortex recordings and five recording sessions in two 5XFAD and three WT mice undergoing hippocampal recordings). The mean (solid line) and standard deviation (shaded area) across recordings are shown on the left (Figure 32A) and per animal on the right (Figure 32B). Recordings with fewer than 3 blink periods 3202 had a noisier power spectral density than recordings with more data 3204, but none showed evidence of a 40 Hz peak. In some embodiments, RT-qPCR was performed to validate upregulated genes involved in known microglial functions. Cd68, B2m, Bst2, and I, which are associated with microglial phagocytosis, were also upregulated. Genes such as cam1 and Lyz2 were confirmed to be upregulated in the hippocampal CA1 region after 40 Hz stimulation.

[0131] Figure 33 is a bar graph showing RT-qPCR validation of specific gene targets in an RNA-seq dataset, according to some embodiments. Figure 33 shows relative RNA levels (expression ratios) in the EYFP3302 and 40 Hz stimulation 3304 conditions (one asterisk indicates p<0.05, two asterisks indicate p<0.01 by Student's t-test, n=6 mice per group). Other notable upregulated genes include the microglia-enriched transcriptional regulator Irf7, the cell adhesion and migration regulator Spp1, and the microglia proliferation markers Csf1r and Csf2ra (see, e.g., Figure 33, p<0.05 and p<0.01 by Student's t-test, n=6 mice per group). RT-qPCR also showed that expression levels of the pro-inflammatory genes Il6, Il1b (Il1-β), Itgam (CD11-b), and the anti-inflammatory gene Igf1 were unchanged (see, e.g., Figure 33, p>0.05 by Student's t-test, n=6 mice per group). Thus, the transcriptomic results described herein suggest that 40 Hz neuronal stimulation induced microglia into a state that promotes engulfment.

[0132] Given the observation that 40 Hz stimulation upregulated both phagocytosis-related genes and migration / cell adhesion-related genes, we examined the morphological characteristics of microglial activation. In some embodiments, we used an antibody recognizing the microglial marker Iba1 to label microglia in hippocampal CA1 sections from 5XFAD / PV-Cre mice after 1 hour of 40 Hz, random, or no stimulation (EYFP mice). Figure 34 is a series of immunofluorescence images showing immunohistochemistry with anti-Iba1 3402 (019-19741) and anti-Aβ 3404 (12F4) antibodies in the hippocampal CA1 region of 5XFAD / PV-Cre mice under EYFP, 40 Hz, and random stimulation conditions. Images were taken with a 40x objective (scale bar = 50 μm). Arrows indicate +Iba1 / +Aβ signals in cell bodies.

[0133] Figure 35A is a bar graph showing the number of microglia in the EYFP and 40 Hz conditions (n=2 sections from 4 mice per group), according to some embodiments. Figure 35B is a bar graph showing the diameter of microglial cell bodies normalized to EYFP in the EYFP, 40 Hz, and random stimulation conditions (n=2 sections from 4 mice per group), according to some embodiments. Figure 35C is a bar graph showing the average length of microglial primary processes or protrusions normalized to EYFP in the EYFP, 40 Hz, and random stimulation conditions. Figure 35D is a bar graph showing the percentage of Iba1-positive (microglial) cell bodies that are also Aβ-positive in the EYFP and 40 Hz stimulation conditions (n=2 sections from 4 mice per group), according to some embodiments. One-way ANOVA results show that the note "ns" 3502 indicates not significant, two asterisks 3504 indicate p<0.01, three asterisks 3506 indicate p<0.001, and four asterisks 3508 indicate p<0.0001.

[0134] First, we counted the number of Iba1+ microglia in six animals per condition and found approximately twice as many microglial cells in the 40 Hz group (15 microglial cells per 212.55 μm × 212.55 μm region of interest (ROI)) compared to the unstimulated EYFP condition (average of 8 microglial cells per ROI) (see e.g., Figures 34 and 35A, p<0.01 by one-way ANOVA, n=2 sections from 4 mice per group) and the random condition (average of 10 microglial cells per ROI) (see e.g., Figures 34 and 35A, p<0.05 by one-way ANOVA, n=2 sections from 4 mice per group). Previous studies have shown that the two main characteristics of phagocytic microglia are an increased cell body size and a decreased process length; therefore, these characteristics may be important for understanding the mechanism underlying the phagocytic microglia. We investigated how 40 Hz stimulation affected the microglial cell body diameter. In some embodiments, the diameter of each clearly labeled Iba1+ cell body within the field of view was measured. After 40 Hz stimulation, the diameter of microglial cell bodies was found to increase by 135.3% compared to no stimulation and 138.7% compared to random stimulation (see, e.g., Figures 34 and 35B; p<0.0001 by one-way ANOVA; n=2 sections from 4 mice per group). We also measured the length of primary microglial processes in each condition. We observed a 54.0% decrease in the length of primary microglial processes in the 40 Hz stimulation condition compared to the EYFP control and a 38.5% decrease compared to random stimulation (see, e.g., Figures 34 and 35C; p<0.0001 by one-way ANOVA; n=2 sections from 4 mice per group). These findings were not affected by Iba1 levels, as no differences in Iba1 expression were observed between conditions in the gene expression analysis described herein (see, e.g., Tables 2 and 3). Therefore, the increased cell body size and decreased process length observed after 40 Hz stimulation are morphological changes consistent with a shift of these microglia to a phagocytic state. Potential colocalization of Aβ within microglia was assessed by co-immunostaining with an Aβ antibody (12F4, which does not cross-react with APP) as a way to assess microglial Aβ uptake. In the CA1 neuropil, where Iba1+ cells are predominantly located, the ratio of the number of microglia with Aβ / Iba1 colocalization in cell bodies (ImageJ, Fuji colocalization plugin) to the total number of microglia increased by 54.9% compared to EYFP and 50.3% compared to the random condition after 40 Hz stimulation (see, e.g., Figures 34 and 35C; p<0.01 by one-way ANOVA, n=2 sections from 4 mice per group). Overlap of Iba1 / Aβ signals in microglial processes was excluded to avoid including potentially random non-phagocytic colocalization.

[0135] In some embodiments, 3D renderings of microglia from this tissue and movies from these renderings were generated to provide better resolution of the presence of Aβ signals within the microglia. Figure 36 shows a series of 3D renderings generated by merging the immunofluorescence images from Figure 34 rotated 0 degrees 3602, -25 degrees around the Y-axis 3604, and 30 degrees around the X-axis 3606, according to some embodiments. Images were captured with a 40x objective (scale bar = 50 μm). Overall, gene expression and morphological analysis suggest that 40 Hz stimulation affects microglial activity by increasing the recruitment of microglial cells to the stimulation site and enhancing their phagocytic activity, resulting in increased Aβ binding. Importantly, in some embodiments, no evidence of neuronal loss was observed by measuring the thickness of the CA1 cell layer using Hoechst nuclear staining. Mean CA1 volume was not significantly different between the EYFP and 40 Hz stimulation groups.

[0136] Figure 37A is a series of immunofluorescence images showing Hoechst immunohistochemistry in the hippocampal CA1 region of 5XFAD / PV-Cre with EYFP and 40 Hz stimulation conditions, according to some embodiments. Figure 37B is a bar graph showing estimated CA1 thickness in 5XFAD / PV-Cre with EYFP and 40 Hz stimulation conditions (n=4 mice per group, "ns" indicates not significant by Student's t-test), according to some embodiments.

[0137] Next, differential gene expression in 5XFAD mice infected with AAV-DIO-ChR2-EYFP and stimulated with 40 Hz FS-PV+ stimulation (treated) or control stimulation (CTRL) was assessed by genome-wide RNA-seq of the hippocampal CA1 after 1 hour of stimulation according to some embodiments. Figure 38A is a heatmap showing 523 differentially expressed genes (DEGs) determined by genome-wide RNA-seq of the hippocampal CA1 upon treatment or CTRL, according to some embodiments. Each row in Figure 38A represents a DEG, and the columns in Figure 38A represent the transcriptomic profiles of three individual control animals and three individual treated (40 Hz FS=PV+ stimulation) animals.

[0138] Figure 38B shows the overlap between DEGs upregulated in the treatment conditions of Figure 38A, according to some embodiments. In Figure 38B, induction of gamma oscillations via 40 Hz stimulation of FS-PV+ reduces Iba1 levels compared to random FS-PV+ stimulation, as measured by immunofluorescence (n=3 mice per group, p=0.006). Figure 38B shows that genes upregulated in the treatment conditions significantly and specifically overlap with microglial genes (i.e., MCSF genes) upregulated by anti-inflammatory microglial activation. Genes were more upregulated in microglial cells than in astrocytes, endothelial cells, myelinating oligodendrocytes (MOs), neurons, newly formed oligodendrocytes (NFOS), and oligodendrocyte progenitor cells (OPCs). Table 5 (below) shows the microglia / macrophage pathway of upregulated genes. [Table 5]

[0139] According to some embodiments, RT-qPCR was performed to validate specific gene targets from the RNA-seq dataset. Figure 39 is a bar graph showing RT-qPCR validation of specific gene targets in the RNA-seq dataset of Figure 38A, according to some embodiments. Specifically, Figure 39 shows the expression ratios (normalized to GAPDH) of specific gene targets in control and treatment conditions, such as the genes CSF1, CSF1R, Il-6, Il1-Beta, CD11-b, CYBA, Hmox1, H2-K1, Lgals3, and Icam1.

[0140] Figure 40 is a plot showing biological processes associated with the upregulated genes in Figure 38A, according to some embodiments. Importantly, the upregulated genes in Figure 40 are specifically associated with immune-related processes. The upregulated genes belonged to immune-related biological processes, including lymphocyte-mediated, adaptive immunity, and immunoglobulin-mediated processes. Figure 41 is a plot showing biological processes associated with the downregulated genes in Figure 38A, according to some embodiments. As shown in Figure 41, the downregulated genes belonged to biological processes including cell motility, intercellular signaling, synaptic transmission, locomotor behavior, and neuronal processes.

[0141] Figure 42A is a series of immunofluorescence images showing Iba1 levels after different types of stimulation of the CA1 region of a subject's hippocampus, according to some embodiments. Figure 42B is a bar graph showing mean intensity values ​​for the immunofluorescence images of Figure 42A, according to some embodiments. Figure 42A shows that endosomal levels are reduced by optogenetic enhancement of gamma rhythms. Induction of gamma oscillations via 40 Hz stimulation of FS-PV+ reduced the levels of EEA1 (an endosomal marker) as measured by immunofluorescence (n = 3 mice per group, p = 0.08). This result indicated that gamma oscillations reduce Aβ production in a mouse model of AD, as increased endosomal levels indicate increased APP processing and, therefore, Aβ production.

[0142] Taken together, the results of this study suggest that gamma rhythm restoration or showed that induction reverses the molecular pathology. Cell-type-specific and temporally precise reintroduction of gamma oscillations by optogenetics reverses the molecular pathology of Aβ isoforms, peptides that aggregate and initiate many of the degenerative cascades involved in AD neuropathology. 1-40 and Aβ 1-42 Furthermore, this treatment induced anti-inflammatory microglial signaling pathways that counteract immune mechanisms associated with neurodegeneration.

[0143] According to some embodiments, cell-type-specific and temporally controlled gamma oscillations can be induced in the hippocampus, visual cortex, barrel cortex, and / or auditory cortex without optogenetics.

[0144] Visual stimuli at gamma frequencies noninvasively drove gamma oscillations in the visual cortex. The robust reduction in Aβ levels by optogenetic stimulation at 40 Hz led us to explore other methods of inducing 40 Hz oscillations in the brain to ensure that this effect was not somehow specific to optogenetic manipulation or invasive methods. To investigate whether flashing lights could be used as a non-invasive method to induce 40 Hz oscillations in the visual cortex, in some embodiments, animals were exposed to cycles of 40 Hz or random flashing and continuous light interleaved with a dark cycle.

[0145] Figure 43A is a schematic diagram showing a mouse exposed to flashing light stimulation, according to some embodiments. To determine whether this flashing light alters Aβ, animals were exposed to 40 Hz flashing light for 1 hour, which coincides with the period of optogenetic stimulation that reduced Aβ described herein. The flashing light covered the animal's entire field of vision. As controls for molecular and cellular assays, 3-month-old 5XFAD mice were kept in constant darkness for 3 days or treated with 1 hour of constant light, 20 Hz flashing light, or 80 Hz flashing light (see, e.g., Figure 43A).

[0146] 43B includes plots of local field potential traces and power spectral densities in the visual cortex before and during 40 Hz light flashes. The mean (solid line) and standard deviation (shaded area) of power spectral densities are shown in the visual cortex during periods of 40 Hz light flashes 4302, random light flashes 4304, or darkness 4306 (n=4 5FXFAD mice from 5 recording periods), according to some embodiments. Figures 43C-43F are plots showing the power spectral density of local field potentials in the visual cortex for each mouse during 40 Hz light flashing, random light flashing, constant darkness, and constant light, respectively, according to some embodiments (n=5 recordings from four 5XFAD mice during 40 Hz light flashing, 47, 51, 61, 49, 16 random light flashing, 279, 302, 382, ​​294, 93 dark, and 47, 50, 64, 49, 15 light cycles). It was found that in the visual cortex, light flashing at 40 Hz increased the power of LFPs at 40 Hz (see, e.g., Figures 43B and 43C), whereas randomly spaced light flashing and darkness did not (see, e.g., Figures 43B, 43D, and 43E).

[0147] Figure 44A is a series of histograms showing the rate of spikes in the visual cortex as a function of time for four cycles of 40 Hz light flashing and as a function of equivalent durations for random light flashing, according to some embodiments. Figure 44A shows histograms of the rate of spikes in the visual cortex as a function of time 4402 for four cycles of 40 Hz light flashing or as a function of equivalent durations 4404 of random light flashing (n=4 5XFAD mice from 5 recording periods; bars indicate mean, error bars indicate SEM between animals). The upper bar indicates when the light was on 4406 or off 4408. In some embodiments, spiking increased and decreased as the light flashed on and off, resulting in spiking phases synchronized to the 40 Hz frequency during 40 Hz stimulation (histogram 4402 in Figure 44A), but no clear frequency emerged during random stimulation (histogram 4404 in Figure 44A).

[0148] Figure 44B is a series of electrical traces of local field potentials recorded over the brain during light flashes, according to some embodiments. In some embodiments, when recorded from saline directly over the brain, there was no increase in 40 Hz power during 40 Hz flashes, indicating that this effect was not due to photoelectric effects or electrical noise (see, e.g., Figures 32 and 44B). Similar to optogenetic stimulation, random flashes provided a control for global changes in activity due to light flashes.

[0149] Figure 45A is a histogram showing the difference in firing rate between 40 Hz light flashing and random light flashing (n=226 stimulation cycles from 5 recording periods in 4 5XFAD mice), according to some embodiments. Figure 45B is a plot showing multi-unit firing rates in the visual cortex during 40 Hz light flashing, random light flashing, dark, and light cycles, according to some embodiments. Figure 45B shows multi-unit firing rates in the visual cortex. Box plots show the median (white line within the box) and quartiles (top and bottom of the box). In all animals, firing rates were not significantly different between the 40 Hz flashing and random flashing conditions, indicating that this random stimulation condition served as a control for spiking activity (rank sum test for each of the five recording periods from four 5XFAD mice, p>0.06; medians and quartiles are shown in the figure; n=47, 51, 64, 49, 16 for 40 Hz flashing periods and 47, 50, 64, 50, 16 for random flashing periods per recording). There was no significant difference in firing rate between the 40 Hz flashing and light conditions, indicating that 40 Hz light flashing generally did not cause neuronal hyperexcitability (rank sum test for each of the five recording periods from four 5XFAD mice; p>0.2 for four recording periods, p<0.01 for one recording period, which was not significant when corrected for multiple comparisons. Median and quartiles are shown in the figure. 40 Hz periods per recording, n=47, 51, 64, 49, 16, and light periods 47, 50, 64, 49, 16). In one period, there was more activity with 40 Hz stimulation than in the dark condition. The difference in multiunit firing rate between 40 Hz and random flashing periods tended to be close to zero (see, e.g., 45A). Furthermore, when these cycles were compared within animals, no significant differences were observed (see, e.g., Figure 45B; rank sum test for each of the five recording periods from four 5XFAD mice, p>0.06; medians and quartiles are shown in the figure; n=47, 51, 64, 49, 16 gamma blink cycles per recording and 47, 50, 64, 50, 16 random blink cycles per recording).

[0150] Visual stimulation at gamma frequencies reduces Aβ levels in the visual cortex. Taking into account the effectiveness of optogenetic methods, a translational, non-invasive amyloid-reducing treatment was designed. Figure 46A is a schematic diagram illustrating an experimental paradigm according to some embodiments. As shown in Figure 46A, a first subset of AD model mice was placed in a first chamber 4600 equipped with a 40 Hz flashlight, and a second subset of AD model mice was placed in a second chamber 4602 that was kept dark. The animals in the first chamber 4600 were exposed to the 40 Hz flashlight for approximately 1 hour.

[0151] 46B and 46C show the Aβ peptide isoform Aβ, respectively, following the experimental paradigm of FIG. 46A, according to some embodiments. 1-40 and Aβ 1-42 Figure 46B is a plot further showing the change in baseline levels of Aβ in the visual cortex V1 of 5XFAD mice. 1-40 and Aβ 1-42 The results show that the level of Aβ was reduced. 1-40 and Aβ 1-42 Levels are expressed as pg / mL (n=6 animals per group).

[0152] Given that 40 Hz flashing light drives 40 Hz oscillations in the primary visual cortex and that optogenetic induction of 40 Hz oscillations reduces Aβ levels in the hippocampus, the goal was to determine whether 40 Hz flashing light could reduce Aβ levels in the visual cortex. For these experiments, in some embodiments, presymptomatic 3-month-old 5XFAD mice were used. The mice were placed in a dark box and exposed to either 40 Hz flashing light, constant light on (light), or constant light off (dark) for 1 hour.

[0153] 47A and 47B show Aβ levels in the visual cortex of 5XFAD mice in dark, light, 40 Hz blinking, 20 Hz blinking, 80 Hz blinking, 40 Hz blinking and picrotoxin (PTX), and random blinking conditions, respectively, according to some embodiments. 1-40 and Aβ 1-4247A and 47B show bar graphs depicting changes in baseline levels of Aβ (n=12 mice per group for dark; n=6 mice per group for light, 40 Hz blink, 20 Hz blink, 80 Hz blink, and PTX; n=4 mice per group for random blink, by one-way ANOVA; "ns" indicates not significant, one asterisk indicates p<0.05, two asterisks indicate p<0.01). Figures 47A and 47B show mean and SEM. Circles overlaid on the bar graph bars represent individual data points for each group. 1 hour after light exposure, Aβ levels in the visual cortex compared to the dark condition increased. 1-40 The level was reduced by 57.96% and Aβ 1-42 A 57.97% reduction in amyloid levels was observed (as measured by Aβ ELISA; see e.g., Figures 47A and 47B; p<0.05 by one-way ANOVA; n=6 mice per group). Compared to light controls, a 62.47% (Aβ ) reduction was observed after 1 hour of 40 Hz flashing. 1-40 ) and 68.55% (Aβ 1-42 ) was reduced (as measured by Aβ ELISA; see e.g., Figure 47; p<0.05 by one-way ANOVA; n=6 mice per group). Furthermore, this effect was specific to 40 Hz flashing, as neither 20 Hz, 80 Hz, nor random flashing significantly reduced Aβ levels compared to dark and light controls (see e.g., Figure 47; "ns" indicates not significant; n=6 mice per group).

[0154] In some embodiments, to test regional specificity, Aβ levels were examined in the somatosensory barrel cortex (BC) and no significant differences were found. Figure 48A shows relative Aβ levels in the barrel cortex of 5XFAD under dark and 40 Hz flashing conditions, according to some embodiments. 1-40 and Aβ 1-42Figure 47 is a bar graph showing the levels of Aβ (n=3 mice per group; "ns" indicates not significant by Student's t-test). Pretreatment of 5XFAD mice with a low dose of a GABA-A antagonist (picrotoxin, 0.18 mg / kg, which does not induce epileptic activity) completely abolished the effect of 40 Hz flickering on Aβ levels, indicating that GABAergic signaling from FS-PV interneurons is likely required for this effect (see, e.g., Figure 47; "ns" indicates not significant; n=6 mice per group).

[0155] To demonstrate that this effect is not specific to 5XFAD mice, we replicated this result in a different AD model, the APP / PS1 mouse, a well-validated model harboring two familial AD mutations (APP Swedish and PSEN1 delta E9). Figure 48B shows, according to some embodiments, the expression of Aβ in the visual cortex of APP / PS1 mice under dark and 40 Hz flashing conditions. 1-40 and Aβ 1-42 (n=5 mice per group for dark condition and n=4 mice per group for 40 Hz flashing condition. "ns" indicates not significant and one asterisk indicates p<0.05 by Student's t-test).

[0156] FIG. 48C shows Aβ expression in the visual cortex of WT under dark and 40 Hz flashing conditions, according to some embodiments. 1-40 and Aβ 1-42 1 is a bar graph showing the change in baseline levels of Aβ (n=11 mice per group for dark condition and n=9 mice per group for 40 Hz flashing condition. Single asterisk indicates p<0.05 by Student's t-test). In some embodiments, Aβ was significantly reduced by 20.80% in APP / PS1 mice after 40 Hz flashing treatment. 1-40 and 37.68% Aβ, which showed a decreasing trend 1-42 was found, but the latter was not significantly different from the dark condition (see, e.g., Figure 48B Aβ was detected by Student's t-test.1-40 p<0.05, Aβ 1-42 p<0.09, i.e., not significant. (n=5 mice per group for dark conditions, n=4 mice per group for 40 Hz flashing). In addition, in aged WT mice, endogenous mouse Aβ was significantly increased 1 hour after 40 Hz flashing. 1-40 A 58.2% reduction in Aβ was observed (see, e.g., Figure 48C; p<0.05 by Student's t-test; n=11 dark mice and n=9 40 Hz flashing mice). 1-42 was below detectable levels in both the blinking and control groups of these animals. 1-40 The decrease in β reveals that these results may not be limited to Tg APP expression or mutant APP; rather, they may extend to Aβ produced from APP with expression driven by its endogenous promoter. Figures 48A-48C show the mean and SEM.

[0157] Next, in some embodiments, we investigated whether 40 Hz flashing alters microglial activity in the visual cortex, using the same method as that used to demonstrate that 40 Hz optogenetic stimulation of FS-PV interneurons alters microglia in the hippocampal CA1. Figure 49 shows a series of immunofluorescence images showing immunohistochemistry with anti-Iba1 (019-19741) and anti-Aβ4904 (12F4) antibodies in the visual cortex of 5XFAD mice under dark and 40 Hz flashing conditions, according to some embodiments. Images were taken with a 40x objective (scale bar = 50 μm). Right: 120x zoom, arrows indicate +Iba1 / +Aβ signals in cell bodies.

[0158] Figure 50A is a bar graph showing microglia counts in dark and 40 Hz flashing conditions (n=2 sections from 4 mice per group; "ns" indicates not significant by Student's t-test), according to some embodiments. Figure 50B is a bar graph showing microglial soma diameters normalized to controls in dark and 40 Hz flashing conditions (n=2 sections from 4 mice per group; two asterisks indicate p<0.01 by Student's t-test), according to some embodiments. Figure 50C is a bar graph showing average microglial primary process lengths normalized to controls in dark and 40 Hz flashing conditions (n=2 sections from 4 mice per group; four asterisks indicate p<0.0001 by Student's t-test), according to some embodiments. Figure 50D is a bar graph showing the percentage of Iba1-positive (microglial) cell bodies that are also Aβ-positive under dark and 40 Hz flashing conditions (n=2 sections from 4 mice per group; double asterisks indicate p<0.01 by Student's t-test), according to some embodiments. Figures 50A-50D show the mean and SEM.

[0159] In some embodiments, Iba1 was used to label microglia in visual cortex sections of 5XFAD mice 1 hour after 40 Hz flashing or dark conditions (see, e.g., Figure 49). Microglia numbers were not different between dark and 40 Hz flashing conditions (see, e.g., Figures 49 and 50A; "ns" indicates not significant; n = 2 sections from 4 mice per group), but the diameter of microglial somata increased by 65.8% in the visual cortex after 40 Hz flashing compared to dark controls (see, e.g., Figures 49 and 50B; p < 0.01 by Student's t-test; n = 2 sections from 4 mice per group). The length of microglial primary processes decreased by 37.7% in the 40 Hz flashing condition compared to dark controls (see, e.g., Figures 49 and 50C; p < 0.0001 by Student's t-test; n = 2 sections from 4 mice per group). Because microglia in the visual cortex have a morphology indicative of enhanced phagocytic activity, in some embodiments, the number of microglia bearing Aβ was examined. For this experiment, visual cortex sections were co-labeled with Iba1 and Aβ (12F4) antibodies. Co-localization of Aβ / Iba1 within the cell body increased by 33.5% under the 40 Hz flashing condition, indicating that 40 Hz flashing resulted in more Aβ-bearing microglia than under the dark control condition (see, e.g., Figures 49 and 50D; p<0.01 by Student's t-test; n=2 sections from 4 mice per group).

[0160] In some embodiments, to provide better resolution of morphological changes in microglia, CLARITY was used to generate 3D renderings of microglia from 100 μm sections of the visual cortex, and movies were generated from these renderings. Figure 51 shows a series of 3D renderings (from immunofluorescence images) of Iba+ microglia under dark and 40 Hz flashing conditions from a CLARITY-processed 100 μm tissue section rotated 0° 5102, 45° around the X-axis 5104, and 45° around the Y-axis 5106. Images were taken with a 63x objective (scale bar = 15 μm). Finally, to demonstrate that microglia indeed phagocytose Aβ in 5XFAD mice, microglia from 5XFAD and WT animals were purified using fluorescence-activated cell sorting (FACS), and Aβ levels were analyzed by ELISA.

[0161] Figure 52A is a flow chart showing a method for isolating microglia from the visual cortex using fluorescence-activated cell sorting (FACS), according to some embodiments. The visual cortex was sectioned, and then single cells were suspended and labeled with CD11b and CD45 antibodies. The cells were then sorted by fluorescence-activated cell sorting (FACS) and lysed. Aβ 1-40 Figure 52B shows Aβ levels in microglia isolated from the visual cortex of 3-month-old 5XFAD and WT control animals using the method of Figure 52A, according to some embodiments. 1-40 (n=8 mice per group for 5XFAD and n=4 mice per group for WT mice. Single asterisk indicates p<0.05 by Student's t-test). Circles overlaid on the bars of the bar graph represent individual data points for each group.

[0162] Figure 53A is a series of immunofluorescence images showing immunohistochemistry with SVP38 antibody to detect synaptophysin in the visual cortex of 3-month-old 5XFAD mice under dark and 40 Hz flashing conditions, according to some embodiments. Images were taken with a 40x objective (scale bar = 50 μm). Right: Dark and 40 Hz flashing conditions at 100x. Figure 53B is a bar graph showing relative SVP38 intensity levels in the visual cortex of 5XFAD mice after dark and 40 Hz flashing conditions, according to some embodiments (n = 4 mice per group; "ns" indicates not significant by Student's t-test).

[0163] Microglia-specific levels of Aβ were significantly higher in 5XFAD animals compared to WT controls, with a mean of 27.2 pg / 10 in 5XFAD mice. 4 microglial levels, and 9.78 pg / 10 in WT control mice. 4 It was found to be at the level of microglia (see, e.g., Figures 52A and 52B; p<0.05 by Student's t-test; n=8 for 5XFAD and n=4 for WT mice). 1-42 was below detectable levels in both the blink and control groups in these animals. Overall, 40 Hz stimulation-induced microglial transformation in the visual cortex appeared similar to that occurring in the hippocampal CA1. Furthermore, synaptophysin levels did not change between dark and 40 Hz blink conditions, indicating that microglial activation did not significantly increase synaptic phagocytosis (see, e.g., Figures 53A and 53B; "ns" indicates not significant; n = 2 slices from 4 mice per group). Taken together, the data disclosed herein demonstrate that 40 Hz oscillations induced noninvasively via sensory stimulation can effectively reduce Aβ abundance and promote microglia / Aβ interactions in AD mouse models. Furthermore, 40 Hz stimulation could reduce Aβ in two distinct brain circuits, suggesting a common mechanism by which gamma oscillations reduce amyloid abundance and enhance microglial phagocytosis in various brain regions.

[0164] In further experiments, after 1 hour of exposure to darkness (no light), 20 Hz light flashes, 40 Hz light flashes, or 80 Hz light flashes, Aβ 1-42 The levels were evaluated here. Hz is a harmonic of 40 Hz. However, only 40 Hz flashes of light are Aβ 1-42 Figure 54A shows the level of Aβ peptide isoform Aβ after stimulation of the visual cortex of a subject with gamma oscillations according to some embodiments. 1-42 1 is a bar graph showing a decrease in

[0165] Another study was conducted to determine the Aβ 1-42 The timing of the decline in Aβ levels was assessed. Mice were exposed to either no light or a 40 Hz flash of light for 1 hour. 1-42 Levels of the Aβ peptide isoform Aβ were measured after 1 hour of treatment and again 24 hours after treatment ended. Figure 54B shows levels of the Aβ peptide isoform Aβ after stimulation of a subject's visual cortex with gamma oscillations according to some embodiments, and again 24 hours after stimulation. 1-42 Aβ levels were still reduced 24 hours after treatment, but this reduction was smaller than immediately after treatment.

[0166] Gamma frequency visual stimulation did not affect Aβ levels in the hippocampus. To determine whether visual stimulation by flashing lights affects brain circuits involved in AD, some embodiments examined the effects of flashing lights on the hippocampus, one of the brain regions affected early in the course of AD in humans. Figure 55A includes electrical traces and plots of power spectral density of local field potentials in the hippocampus before and during 40 Hz light flashes 5502, according to some embodiments. Mean (solid line) and standard deviation (shaded area) of power spectral density in CA1 during darkness 5504, 40 Hz light flashes 5506, and random light flashes 5508 (n=2 5XFAD mice and 3 WT mice).

[0167] Figure 55B is a series of histograms of spike rate in the hippocampus as a function of time 5510 for four cycles of 40 Hz light flashing and as a function of equivalent duration 5512 for random light flashing, respectively (n=2 5XFAD mice and 3 WT mice; bars indicate mean, error bars indicate SEM between animals), according to some embodiments. The upper bar indicates when the light was on (white) or off (black). For random stimulation, spiking was timed with the onset of light on, and additional light cycles occurred at random intervals, indicated by gray. Using the same method for examining the effects of light flashing in CA1 in the visual cortex disclosed herein, it was found that light flashing at 40 Hz increased power in LFPs recorded at 40 Hz (see, e.g., graph 5510 in Figures 55A and 55B), whereas randomly spaced light flashing (random flashing) and darkness did not (see, e.g., graph 4310 in Figures 50D and 43C). Spiking was also modulated by the 40 Hz flicker frequency during 40 Hz stimulation; however, this modulation appeared to be smaller than in the visual cortex (see, e.g., Figure 55B, hippocampus; Figure 44A, visual cortex).

[0168] Figure 56A is a histogram showing the difference in firing rate between 40 Hz light flashes and random light flashes (minimum n=168 stimulation cycles from five recording periods in two 5XFAD and three WT mice), according to some embodiments. Figure 56B is a plot showing multi-unit firing rate in CA1 during 40 Hz light flashes 5604, random flashes 5605, dark 5602, or light 5608 cycles, according to some embodiments. Box plots show the median (white line within the box) and quartiles (top and bottom of the box). There was no significant difference in firing rate between the 40 Hz flash and random flash conditions in all animals, indicating that this random stimulation condition served as a control for spiking activity (rank sum test for each of the five recording periods from two 5XFAD and three WT animals, p>0.2; median and quartiles are shown in the figure. 40 Hz flash cycles per recording, n=22, 54, 42, 71, 55; random flash cycles, n=12, 34, 32, 54, 36). There was no significant difference in firing rate between the 40 Hz flash and light conditions, indicating that 40 Hz light flashing did not generally cause neuronal hyperexcitability (rank sum test for each of the five recording periods from two 5XFAD and three WT animals, p>0.3; median and quartiles are shown in the figure. Recording per 40 Hz period n = 22, 54, 42, 71, 55 and light period 12, 34, 32, 54, 35).

[0169] As in the visual cortex, the difference in multiunit firing rates between 40 Hz and random flicker cycles tended to be close to zero (see, e.g., Figure 56A). Furthermore, when these cycles were compared within animals, no significant differences were found (see, e.g., Figure 56B; rank sum test, p>0.06, for each of the five recording periods from four 5XFAD mice; medians and quartiles are shown in the figure; n=22, 54, 42, 71, 55 for 40 Hz flicker cycles per recording, and 12, 34, 32, 54, 36 for random flicker cycles per recording).

[0170] In some embodiments, the effects of visual light flashes on Aβ levels in the hippocampus were examined using the same methods used in the visual cortex. According to some embodiments, Figure 57A shows the relative Aβ levels in the visual cortex of 5XFAD mice. 1-40 FIG. 57B is a bar graph showing the relative Aβ levels in the visual cortex of 5XFAD. 1-42 (n=4 mice per group; "ns" indicates not significant). In contrast to what was observed in the visual cortex, in CA1, Aβ levels were significantly higher 1 h after 40 Hz flashing or random stimulation. 1-40 and Aβ 1-42 No significant difference was observed in the Aβ levels after 40 Hz flashing or random flashing compared to dark conditions. 1-40 The levels of Aβ were 108.4% and 96.82% of those under dark conditions after 40 Hz and random flickering, respectively. 1-42 The levels of Aβ after 40 Hz and random flickering were 118.8% and 92.15% of those under dark conditions, respectively (see, e.g., Figures 57A and 57B; "ns" indicates not significant; n = 4 mice per group). Thus, 1 hour of 40 Hz light flickering did not significantly reduce Aβ levels in the hippocampus.

[0171] Chronic visual stimulation at gamma frequencies reduced plaque load in the visual cortex. We have previously described the impact of 40 Hz oscillations on amyloid burden in preplaque 5XFAD mice, either optogenetically or via visual stimulation via flashing light. The next step was to determine whether this treatment would be effective in animals already exhibiting plaque burden. To this end, in some embodiments, 6-month-old 5XFAD mice were used, as they develop extensive amyloid plaque pathology in many brain regions, including the visual cortex. Studies were conducted to determine what happens to advanced Aβ-related pathology after noninvasive gamma stimulation. To examine the duration of Aβ reduction in response to 1 hour of 40 Hz flashing, in some embodiments, Aβ levels were measured in the visual cortex 4, 12, and 24 hours after 1 hour of 40 Hz flashing or darkness.

[0172] 58A and 58B show relative Aβ levels in the visual cortex of 5XFAD at 1, 4, 12, and 24 hours after 1 hour of darkness or 40 Hz flashing treatment, respectively, according to some embodiments. 1-40 and Aβ 1-42 (n=4 mice per group for 4 and 12 hour wait, n=6 for 1 and 24 hour wait, n=12 for dark treatment. "ns" indicates not significant, one asterisk indicates p<0.05, two asterisks indicates p<0.01 by one-way ANOVA). The results show that after 4 hours, compared to dark control, Aβ levels were significantly elevated. 1-40 levels were reduced by 63.4%, and Aβ 1-42 By 12 hours, Aβ levels were reduced by 63.2% (see, e.g., Figure 58; p<0.01; n=4 mice per group). 1-40 levels decreased by 50.9%, while Aβ 1-42 The levels of soluble Aβ were not significantly different from the dark control (see, e.g., Figure 58; "ns" indicates not significant, p<0.01; n=4 mice per group). Finally, 24 hours after 1 hour of 40 Hz flashing treatment, soluble Aβ 1-40 and Aβ 1-42The levels of α-glucan were not significantly different in the 40 Hz flashing compared to the dark control condition (see, e.g., Figure 58; "ns" indicates not significant; n=6 mice per group for 24 h and n=4 mice per group for dark conditions). This indicates that the effect of the Hz blinking process is temporary.

[0173] Therefore, to destroy advanced plaque pathology, in some embodiments, mice were treated with 40 Hz flashing for 1 hour daily for 7 days, or dark conditions for controls. Figure 59A is a schematic diagram showing a 6-month-old mouse exposed to 1 hour of flashing per day for 7 days, according to some embodiments. Figure 59B shows the relative Aβ levels in the visual cortex of a 6-month-old 5XFAD mouse after 7 days under 1 hour / day dark or 40 Hz flashing conditions, according to some embodiments. 1-42 Figure 59C is a bar graph showing the levels of Aβ in the visual cortex of 6-month-old 5XFAD mice after 7 days in 1 hour / day darkness or 40 Hz flashing, according to some embodiments. 1-40 59B and 59C show the mean and SEM. Circles overlaid on the bar graphs represent individual data points for each group.

[0174] At the end of the 7-day period, the visual cortex was analyzed by ELISA and immunostaining. In some embodiments, the tissue was dissolved in phosphate-buffered saline (PBS) to extract the PBS-soluble Aβ fraction. A 1-hour 40 Hz flashing light for 7 days increased soluble Aβ levels in 6-month-old 5XFAD mice, as measured by ELISA. 1-40 and Aβ 1-42The levels of Aβ were found to be reduced by 60.5% and 51.7%, respectively (see, e.g., Figures 59B and 59C; p<0.05 and p<0.01 by Student's t-test; n=13 mice per group). The tissues were further treated with guanidine hydrochloride (HCl) to remove insoluble Aβ, which constitutes aggregated amyloid plaques. 1-40 and Aβ 1-42 The fractions were extracted. Insoluble Aβ 1-40 and Aβ 1-42 The levels of 40 The results showed that 500 Hz flashes disrupted pre-formed insoluble Aβ aggregates in 6-month-old mice (see, e.g., Figures 59B and 59C; p<0.01 and p<0.001 by Student's t-test; n=13 mice per group).

[0175] To determine how plaque burden was specifically affected, in some embodiments, immunohistochemical characterization was performed using an Aβ antibody (Cell Signaling Technology, D54D2). Figure 60A is a series of immunofluorescence images showing immunohistochemistry with an anti-Aβ (D5452) antibody in the visual cortex of 6-month-old 5XFAD mice after 7 days in the dark (top) or 40 Hz flashing (bottom) conditions, according to some embodiments (scale bar = 50 μm). Aβ signals observed intracellularly were excluded. Figure 60B is a bar graph showing the number of Aβ-positive plaque deposits in the visual cortex of 6-month-old 5XFAD mice after 7 days in the dark (top) or 40 Hz flashing conditions, according to some embodiments (n = 8 mice per group; three asterisks indicate p < 0.001 by Student's t-test). Figure 60C is a bar graph showing the area of ​​Aβ-positive plaques in the visual cortex of 6-month-old 5XFAD mice after 7 days in the dark or 40 Hz flashing conditions (n=8 mice per group; two asterisks indicate p<0.01 by Mann-Whitney test), according to some embodiments. Figures 60B and 60C show the mean and SEM.

[0176] Plaque abundance was quantified by counting the number of Aβ+ deposits ≥10 μm in diameter. 40 Hz flashing reduced the number of plaques to 11.0 compared to 33.5 in dark controls (see e.g., Figures 60A and 60B; p<0.01 by Student's t-test; n=8 mice per group). Furthermore, after 1 week of 40 Hz flashing treatment, plaque size (measured as the area of ​​the dense plaque area) was reduced by approximately 63.7% compared to dark controls (see e.g., Figures 60A and 60C; p<0.01 by Mann-Whitney test; n=8 mice per group). Taken together, these experiments identify a completely non-invasive treatment that has a profound effect on amyloid plaque pathology.

[0177] To determine whether 40 Hz flashing improves another important AD-related pathology, tau phosphorylation was examined using the TauP301S tauopathy mouse model. Four-month-old TauP301S Tg mice, which exhibit soma-localized phosphorylated tau at this age, were treated with either 40 Hz flashing or dark control conditions for 1 h daily for 7 days. To examine how 40 Hz flashing altered tau phosphorylation, immunohistochemical characterization of the visual cortex was performed using pTau antibodies against three distinct epitopes of pTau (S202, S396, and S400 / T403 / S404; 11834S, 9632S, 11837S) and the dendritic marker MAP2 as a control.

[0178] Figure 61A is a series of immunofluorescence images showing immunohistochemistry with anti-pTau6102 (S202) and anti-MAP2 6104 antibodies in 4-month-old P301S mice after 7 days in the dark (1 hr / day) or 40 Hz flashing conditions, according to some embodiments. Images were taken with a 40x objective (scale bar = 50 μm. The inset includes a 100x rendering of a representative cell body under the dark and 40 Hz flashing conditions). Figure 61B is a bar graph showing relative pTau (S202) intensity levels in the visual cortex of P301S mice after 7 days in the dark (1 hr / day) and 40 Hz flashing conditions, according to some embodiments (n = 8 mice per group. A single asterisk indicates p < 0.05 by Student's t-test). Figure 61C is a bar graph showing relative MAP2 intensity levels in the visual cortex of P301S after 7 days under 1 hour / day darkness and 40 Hz flashing conditions (n=8 mice per group; "ns" indicates not significant by Student's t-test), according to some embodiments. Figures 61B and 61C show the mean and SEM.

[0179] Figure 62A is a series of immunofluorescence images showing immunohistochemistry with anti-pTau6202 (S404) antibody in 4-month-old P301S mice after 7 days under 1 hour / day darkness and 40 Hz flashing conditions (scale bar = 50 μm), according to some embodiments. Figure 62B is a bar graph showing relative anti-pTau (S400 / T403 / S404) fluorescence intensity levels in the visual cortex of P301S mice after 7 days under 1 hour / day darkness and 40 Hz flashing conditions (n ​​= 8 mice per group. Double asterisks indicate p < 0.01 by Student's t-test), according to some embodiments. Figure 62B shows the mean and SEM.

[0180] Figure 63A is a series of immunofluorescence images showing immunohistochemistry with anti-pTau6302 (S396) antibody in 4-month-old P301S mice after 7 days under 1 hour / day dark and 40 Hz flashing conditions (scale bar = 50 μm), according to some embodiments. Figure 63B is a bar graph showing relative pTau (S396) fluorescence intensity levels in the visual cortex of P301S mice after 7 days under 1 hour / day dark and 40 Hz flashing conditions (n ​​= 8 mice per group; four asterisks indicate p < 0.0001 by Student's t-test), according to some embodiments.

[0181] As a result, under the 40 Hz flashing condition, the signal intensity of pTau(S202) was reduced by 41.2% and pTau(S400 / T403 / S404) by 42.3% compared to the dark control (see e.g., Figures 61A-B, 62A-B. p<0.01 by Student's t-test. n=2 sections from 8 mice per group), whereas MAP2 levels were unchanged (see e.g., Figures 61A and 61C. "ns" indicates not significant. n=2 sections from 4 mice per group). Staining with an antibody against pTau(S396) showed a trend in the same direction: 40 Hz flashing reduced pTau(S396) levels by 14.4% compared to the dark control (see e.g., Figures 63A-B. "ns" indicates significant). (n = 2 sections from 8 mice per group). Furthermore, there was less punctate cell body localization of pTau signal in response to 40 Hz flashing compared to dark controls. Although significant changes in tau phosphorylation were observed, no discernible differences in insoluble tau levels were observed between the 40 Hz flashing and dark control groups.

[0182] The effects of 40 Hz flashing on microglia in the TauP301S mouse model were evaluated. Figure 64 is a series of immunofluorescence images showing immunohistochemistry with anti-Iba1 (019-19741) antibody in 4-month-old P301S mice after 7 days under 1 hour / day dark and 40 Hz flashing conditions, according to some embodiments. Images were taken with a 40x objective (scale bar = 50 μm; inset includes 100x rendering of representative microglia under EYFP and 40 Hz stimulation conditions).

[0183] Figure 65A is a bar graph showing the number of microglia after 7 days in the dark for 1 hour / day and 40 Hz flashing light conditions (n=8 mice per group; "ns" indicates not significant by Student's t-test), according to some embodiments. Figure 65B is a bar graph showing the diameter of microglial cell bodies normalized to control after 7 days in the dark for 1 hour / day and 40 Hz flashing light conditions (n=8 mice per group; four asterisks indicate p<0.0001 by Student's t-test), according to some embodiments. Figure 65C is a bar graph showing the average length of microglial primary processes normalized to control after 7 days in the dark for 1 hour / day and 40 Hz flashing light conditions (n=8 mice per group; four asterisks indicate p<0.0001 by Student's t-test), according to some embodiments.

[0184] In some embodiments, microglia were labeled with anti-Iba1 antibodies in visual cortex slices from TauP301S mice after 7 days of 1 hour of 40 Hz flashing or dark exposure (see, e.g., Figure 64). In some embodiments, a trend toward a 29.50% increase in microglia numbers was observed in the 40 Hz flashing condition compared to dark controls, consistent with what was observed in the 5XFAD model (see, e.g., Figures 64 and 65A; "ns" indicates not significant; n=3 mice per group). Furthermore, the diameter of microglial somata increased by 49.00% in the visual cortex after 40 Hz flashing compared to dark controls (see, e.g., Figures 64 and 65B; p<0.0001 by Student's t-test; n=3 mice per group). The length of microglial primary processes was reduced by 39.08% in the 40 Hz flash group compared to dark controls (see, e.g., Figures 64 and 65C; p<0.0001 by Student's t-test; n=3 mice per group).

[0185] Taken together, these data from multiple models of AD pathology and in WT animals indicate that 40 Hz oscillations can attenuate amyloid pathology, as measured by reduced Aβ levels, and reduce tau phosphorylation. Furthermore, 40 Hz visual flickering can drive distinct morphological transformations of microglia in both amyloidosis and tauopathy models of AD pathology.

[0186] In another experiment, a subset of aged mice (i.e., 6 months old) was exposed to visual gamma stimulation for 7 days. The remaining mice were kept in the dark. Figure 66 is a plot showing the levels of both soluble and insoluble Aβ peptides (i.e., plaques) in the visual cortex of mice. As shown in Figure 66, the soluble isoform Aβ 1-40 6600, soluble isoform Aβ 1-42 6602, insoluble isoform Aβ 1-40 6604 and insoluble isoform Aβ 1-42 The levels of each of 6606 were significantly reduced in mice exposed to visual gamma stimulation.

[0187] 67A-67B show a graph of a subject's brain activity with and without transcranial gamma stimulation, according to some embodiments. 67A and 67B are plots showing Aβ peptide levels in 5xFAD mice. In Figure 67A, whole brain Aβ peptide levels remained stable without stimulation 6700 but decreased after 1 hour of transcranial gamma stimulation 6702 (n=1 animal per group). In Figure 67B, according to some embodiments, whole brain Aβ peptide levels were decreased in the hippocampus 6704 and cortex 6706 of 5xFAD mice after 40 Hz transcranial stimulation.

[0188] Gamma oscillations have long been thought to be associated with higher cognitive functions and sensory responses. In some embodiments, driving FS-PV interneurons using optogenetic methods enhanced LFPs at 40 Hz in mice. As disclosed herein, some embodiments demonstrate that driving 40 Hz oscillations and phase-locked spiking using optogenetics or noninvasive flashing light treatments in the 5XFAD mouse model resulted in a significant reduction in Aβ peptides in at least two distinct brain regions. This reduction was not due to a decrease in spiking activity, as Aβ peptide levels were significantly lower in response to 40 Hz stimulation than in random stimulation conditions, which produced a similar amount of multi-unit spiking activity without enhancing 40 Hz oscillations. While pyramidal cell firing rates may differ between these conditions, FS-PV interneurons or other cell types masked this change. In some embodiments, random optogenetic stimulation of FS-PV interneurons, combined with the same amount of direct stimulation of FS-PV interneurons, still did not reduce amyloid. On the contrary, optogenetic stochastic stimulation increased amyloid levels by more than threefold, while stochastic visual flashing did not produce significant changes, potentially indicating that some aspects of random stimulation have neurotoxic effects. In some embodiments, random stimulation did not result in an increase in gamma power, while a trend toward a slight increase in power was observed across a wide range of frequencies, from approximately 20 Hz to over 60 Hz. In some embodiments, a trend toward increased amyloid levels was observed with light flashing from 20 Hz to 80 Hz. Taken together, these results may suggest that driving activity at some frequencies below or above 40 Hz may increase amyloid levels. These results point to the need to understand how patterns of spiking activity influence molecular pathways and disease pathology.

[0189] The robust reduction in total amyloid levels is likely mediated by both reduced amyloid formation, associated with a decrease in EEA1 / Rab5-positive early endosomes, and increased amyloid endocytosis by microglia. Importantly, the Gene Set Enrichment Analysis (GSEA) statistical analysis (The Broad Institute, Cambridge, MA) disclosed herein demonstrated that the inflammatory M1 or anti-inflammatory M2 cellular state of classical macrophages did not correlate with either up- or down-regulated gene expression profiles after stimulation of neurons with 40 Hz oscillations. Indeed, the expression levels of the inflammatory genes Il6, Il1b, and Itgam, and the anti-inflammatory gene Igf1, remained unchanged after stimulation. Instead, multiple microglial pro-phagocytic genes and the cell adhesion / migration regulator Spp1 were activated by 40 Hz stimulation. Thus, driving 40 Hz gamma oscillations appears to induce a comprehensive neuroprotective response by recruiting both neurons and microglia. The fact that GABA-A antagonist treatment completely abolished the effect of 40 Hz stimulation on reducing Aβ levels strongly suggests that GABAergic signaling, most likely associated with FS-PV interneurons, is essential for these effects. Furthermore, in some embodiments, 40 Hz flickering stimulation reduced Aβ in multiple mouse models, including APP / PS1 and WT mice in addition to 5XFAD mice. This replication in multiple mouse models indicates that these findings may not be specific to one animal model and, importantly, may extend to situations where APP is expressed by its physiological promoter and Aβ arises from endogenous APP, as in WT animals. Furthermore, in some embodiments, 40 Hz oscillations reduced pTau in a mouse model of tauopathy, TauP301S. These results demonstrate that the protective effects of gamma stimulation generalize to other mouse models as well as to other pathogenic proteins. In summary, the findings disclosed herein reveal previously unknown cellular and molecular processes mediated by gamma oscillations and establish a functional connection between brain gamma rhythms, microglial function, and AD-related pathology. In some embodiments, the findings of impaired gamma oscillations converge with evidence of impaired gamma in different mouse models of AD (hAPP and apoE4) and report that gamma is altered in humans with AD. Searching for converging evidence from multiple mouse models of AD, including transgene and knock-in models, may demonstrate that these results are not simply due to transgene overexpression or other side effects in one model in particular. Together, these results from mice and humans indicate that multiple molecular pathways contributing to Aβ pathology converge to alter gamma oscillations in AD. The findings disclosed herein hold promise for novel therapeutic interventions for AD.

[0190] One theory of AD pathogenesis points to microglial dysfunction, specifically, the failure of microglia to remove pathological molecules, as a key mechanism for disease progression. Therefore, interventions that return microglia to an endocytic state, as 40 Hz stimulation does, have strong therapeutic potential. In experiments further described herein, driving gamma oscillations optogenetically or with flashing light did not cause neuronal hyperactivity. This approach is fundamentally different from previous AD therapies, and thus driving such patterned neural activity to induce endogenous repair may provide a novel treatment for AD.

[0191] Visual stimuli at gamma frequencies had a positive effect on the subjects' behavior. A study was conducted to determine whether gamma exposure and / or administration according to some embodiments causes any stress in subjects. Figure 68A is a flow chart illustrating this study. As shown in 6800 of Figure 68A, according to some embodiments, WT mice were exposed to either normal room light (N=8) or 40 Hz flashing light (N=8) for 1 hour per day for 7 consecutive days, i.e., days 1-7. On day 8, shown in 6802, blood was collected from the mice, and plasma was separated to determine corticosterone levels. In mice, corticosterone is the primary glucocorticoid involved in the stress response.

[0192] Figure 68B is a bar graph showing corticosterone (CORT) levels (pg / ml) in plasma collected from eight mice exposed to normal room light (NRL) and eight mice exposed to 40 Hz light flickering (40 Hz). No increase in corticosterone was observed in mice exposed to 40 Hz light flickering. Instead, the group of mice exposed to 40 Hz light flickering had lower corticosterone levels compared to the control group. For N=8 independent measurements per group, the T-distribution and p-value for corticosterone levels are: T(14)=0.827, p=0.422 (1) It was calculated that:

[0193] Another study was conducted to determine whether gamma exposure and / or administration according to some embodiments reduces anxiety in subjects. Figure 69A is a flow chart illustrating this study. As shown in 6900 of Figure 69A, according to some embodiments, WT mice were exposed to either normal room light (N=10) or 40 Hz flashing light (N=10) for 1 hour per day for 7 consecutive days, i.e., days 1-7. On day 8, shown in 6902, an elevated plus maze test was performed for a 10-minute period.

[0194] The elevated plus maze is a test used to measure anxiety in laboratory animals. This behavioral model is based on rodents' general aversion to open spaces, which leads to thigmotaxis, a preference for staying in closed spaces or near the edges of enclosed spaces. 69B is an image showing the elevated plus maze apparatus. This apparatus is a cross-shaped apparatus with two open (vertical) and two closed (horizontal) rungs. Anxiety is expressed by the animal spending more time in the closed rungs.

[0195] Figures 69C and 69D are images showing representative trajectories of subjects during the elevated plus maze. According to some embodiments, in Figure 69C, mice exposed to normal room light tended to stay in the walled runway and exhibited greater anxiety, while in Figure 69D, mice exposed to 40 Hz flashing light explored both the open and walled runways and exhibited relatively less anxiety.

[0196] 70 is a bar graph showing the total time spent exploring the unwalled and walled runways for 10 mice exposed to normal room light (NRL) and 10 mice exposed to 40 Hz light flashing (40 Hz), according to some embodiments. Mice exposed to 40 Hz light flashing, according to some embodiments, spent less total time in the walled runway and more total time in the unwalled runway, indicating less anxiety compared to the control group. For N=10 independent measurements per group, the T-distribution and p-value for the total time spent exploring the walled runway are: T(18)=-1.652, p=0.11 (2) It was calculated that:

[0197] For N=10 independent measurements per group, the T-distribution and p-value for the total time spent exploring the wall-free lane are: T(18)=-2.136, p=0.047 (3) It was calculated that:

[0198] Another study was conducted to determine whether gamma exposure and / or administration according to some embodiments reduces stress and / or anxiety in subjects. Figure 71A is a flow diagram illustrating this study. As shown in 7100 of Figure 71A, according to some embodiments, WT mice were exposed to either normal room light (N=8) or 40 Hz flashing light (N=8) for 1 hour per day for 7 consecutive days, i.e., days 1-7. On day 8, shown in 7102, a 5-minute open field test was performed.

[0199] The open field test is an experiment used to assess general locomotor activity and anxiety in laboratory animals. The behavioral model is based on anxiety generated by the paradoxical behavior of rodents, which avoid brightly lit areas but explore perceived threatening stimuli. Figure 71B shows an image of an open field arena. This open field arena may have walls to prevent escape, be marked with a grid, and be monitored using an infrared beam or video camera integrated with a software system. According to some embodiments, increased anxiety leads to decreased locomotor activity and a preference for the edge of the field, while decreased anxiety leads to increased exploratory behavior.

[0200] 71C and 71D are images showing representative trajectories of subjects during the open field test. According to some embodiments, in FIG. 71C, mice exposed to normal room light tended to prefer the edges of the arena and exhibited greater stress and / or anxiety, while in FIG. 71D, mice exposed to 40 Hz flashing light explored the center of the arena and exhibited relatively less stress and / or anxiety.

[0201] 72A and 72B are graphs showing the total time spent exploring the center and periphery of an open field arena for eight mice exposed to normal room light (NRL) and eight mice exposed to 40 Hz flashing light (40 Hz), according to some embodiments. Figure 72A is a plot of the average number of seconds spent in the center of the arena for each 5-minute period. Figure 72B is a bar graph of the total time spent on the periphery of the arena for all 5 minutes, averaged by minute.

[0202] According to some embodiments, compared to the control group, on average, mice exposed to 40 Hz light flashing spent significantly more time in the center of the arena during minutes 2, 4, and 5, thus exhibiting less stress and / or anxiety, which is also consistent with the elevated plus maze results. A repeated measures analysis of variance (RM ANOVA) was performed. For N=8 independent measurements per group, the F distribution and p-value for the mean time spent exploring the open field arena were: F(1,14)=4.860, p=0.045 (4) It was calculated that:

[0203] Another study was conducted to examine whether gamma exposure and / or administration according to some embodiments alters subjects' innate novelty-seeking behavior. Figures 73A and 73B are schematic diagrams illustrating this study using a novelty recognition task. In Figure 73A, two novel objects are presented in a familiar arena. In Figure 73B, one familiar object and one novel object are presented in a familiar arena. According to some embodiments, wild-type mice were exposed to either normal room light (N=8) or 40 Hz flashing light (N=8) for 1 hour per day for 7 consecutive days, i.e., days 1-7.

[0204] On day 8, the mice were exposed to the scenario of Figure 73A, i.e., two novel objects in a familiar arena, for 5 minutes. Figure 73C is a bar graph showing the percentage of time spent exploring novel object A versus the percentage of time spent exploring novel object B for eight mice exposed to normal room light (NRL) and eight mice exposed to 40 Hz flashing light (40 Hz), according to some embodiments. As shown in Figure 73C, each group showed equal preference for each object; that is, there were no differences in object exploration between the groups.

[0205] The mice were then exposed to the scenario of Figure 73B, i.e., one familiar object and one novel object in a familiar arena, for 5 minutes. Figure 74 is a plot showing the average number of seconds spent exploring the novel object every 5 minutes. According to some embodiments, compared to the control group, mice exposed to 40 Hz light flashing, on average, spent significantly more time exploring the novel object, particularly between minutes 1 and 3 and 5, thus demonstrating increased novelty-exploratory behavior. Friedman's nonparametric RM ANOVA was performed. The test statistic, χ , for the average time spent exploring the novel object for N=8 independent measurements per group was 2 and the p-value is: χ 2 (4,n=16)=16.088, p=0.003 (5) It was calculated that:

[0206] A Mann-Whitney U test was performed on the mean time spent exploring the novel object over the 3-minute period. For N=8 independent measurements per group, the U, Z, and p values ​​are: U=58.00, Z=2.731, p=0.005 (6) It was calculated that:

[0207] Another study was conducted to determine whether gamma exposure and / or administration according to some embodiments affects learning and memory in subjects. Figure 75A is a flow diagram illustrating a study using a fear conditioning paradigm. As shown at 7500 in Figure 75A, according to some embodiments, WT mice were exposed to normal gamma radiation for 1 hour per day for 7 consecutive days, i.e., days 1-7. Mice were exposed to either room light or a 40 Hz flashing light. On day 8, designated 7502, they were subjected to a mild two-tone shock pairing. Specifically, mice were placed in a novel arena pairing the first tone with a foot shock. These mice were conditioned to associate the context (i.e., tone) with the aversive experience (i.e., foot shock). For this first context, the t-distribution and p-value for the total time spent freezing were: T(24)=0.577, p=0.569 (7) It was calculated that:

[0208] On day 9, indicated by 7504, a tone test was conducted in the altered context. Figure 75B is a stimulus diagram showing tone tests as a function of time, including a first tone context 7506, a post-first tone context 7508, a second tone context 7510, and a post-second tone context 7512. For this test, mice were returned to the arena where the first tone was paired with the foot shock. When first tone context 7506 was applied, mice exposed to 40 Hz light flashes spent more time freezing, likely in anticipation of the foot shock and thus indicating a measure of memory. Mice exposed to 40 Hz light flashes also spent more time freezing than controls during the second tone context 7510, but freezing less during either post-tone context.

[0209] 76A and 76B are bar graphs illustrating memory enhancement, according to some embodiments. According to some embodiments, as shown in FIG. 76A, the percentage of time spent freezing during the first tone context 7506 and the second tone context 7510 was higher in mice exposed to 40 Hz light flashes compared to the control group, indicating enhanced memory association. Furthermore, according to some embodiments, mice exposed to 40 Hz light flashes showed stronger extinction after tone presentation. As shown in FIG. 76B, according to some embodiments, the percentage of time spent freezing during the first tone context 7506 and during the second tone context 7510 was higher in the control group compared to the mice exposed to 40 Hz light flashes, indicating enhanced memory specificity.

[0210] For the pre-tone context, an RM ANOVA was conducted between groups, and the F distribution and p-value for the mean time spent freezing was: F(1,24)=3.106, p=0.091 (8) It was calculated that:

[0211] For the first tone context, the t-distribution and p-value for total time spent freezing is: T(24)=-2.155, p=0.041 (9) It was calculated that:

[0212] For the second tone context, the t-distribution and p-value for total time spent freezing is: T(24)=-1.433, p=0.164 (10) It was calculated that:

[0213] For tone context, an RM ANOVA was conducted between groups, and the F distribution and p-value for the mean time spent freezing were: F(1,24)=4.559, p=0.043 (11) It was calculated that:

[0214] T-distribution and p-value for total time spent freezing after the first tone context teeth: T(24)=1.874, p=0.073 (12) It was calculated that:

[0215] For the second tone post-context, the t-distribution and p-value for total time spent freezing is: T(24)=2.223, p=0.036 (13) It was calculated that:

[0216] For post-tone context, an RM ANOVA was performed between groups, and the F distribution and p-value for the mean time spent freezing was: F(1,24)=6.646, p=0.017 (14) It was calculated that:

[0217] Another study was conducted to determine whether gamma exposure and / or administration according to some embodiments improves memory in subjects. Figure 77A is a flow diagram illustrating this study. As shown at 7700 in Figure 77A, according to some embodiments, WT mice were exposed to either normal room light or 40 Hz flashing light for 1 hour per day for 7 consecutive days, i.e., days 1-7. On day 8, shown at 7702, a Morris water maze test was performed.

[0218] The Morris water navigation task, or maze, is a test used to study spatial memory and learning in laboratory animals. The behavioral procedure involves placing a subject in a large circular pool with an invisible or visible platform that allows the subject to escape using habitual (recalling the movements required to reach the platform), geotactic (using visual cues to find the platform), or spatial (using distal cues as a reference point) strategies. Figure 77B shows the Morris water maze. The maze contains a circular pool of water divided into directional quadrants and a hidden platform 7704 in the southwest (SW) quadrant.

[0219] For weak training, the Morris water maze test was repeated twice daily for four consecutive days, i.e., days 8 through 11. Figure 78A is a plot showing the latency to find the platform for mice exposed to normal room light (NRL) and mice exposed to 40 Hz light flashing (40 Hz), according to some embodiments.

[0220] On day 12, a probe trial was conducted by removing the hidden platform from the Morris water maze. Figures 77C and 77D are images showing representative trajectories of subjects during the probe trial. According to some embodiments, in Figure 77C, mice exposed to normal room light appear to search for the platform throughout the pool, while in Figure 77D, mice exposed to 40 Hz light flashes appear to search more systematically, primarily in the SW quadrant. Figure 78B is a plot showing the total time (seconds per 30-second period) spent searching for the platform in the goal quadrant (i.e., SW quadrant), and Figure 78C is a plot showing the total time (seconds per 30-second period) spent searching for the platform in the opposite quadrant (i.e., NE quadrant). According to some embodiments, mice exposed to 40 Hz light flashes spent more time searching for the goal quadrant and less time searching for the opposite quadrant than controls, demonstrating enhanced spatial memory.

[0221] Reversal learning was performed using the same groups of mice from the Morris water maze test and the probe test. Figure 79A shows the Morris water maze with a platform 7900 hidden in the SW quadrant for this test. Figure 79B shows the Morris water maze with a platform 7902 hidden in the opposite NE quadrant for reversal learning.

[0222] For weak training, reversal learning was repeated twice daily for four consecutive days, i.e., days 14-17. Figure 79C is a plot showing the latency to find the platform for mice exposed to normal room light (NRL) and mice exposed to 40 Hz flashing light (40 Hz), according to some embodiments. Mice exposed to 40 Hz flashing light showed increased behavioral flexibility, despite not receiving further 40 Hz exposure after day 7.

[0223] According to some embodiments, another study was conducted to examine whether chronic gamma exposure and / or administration affects spatial learning and memory in subjects. Figure 80A is a flow diagram illustrating this study. As shown at 8000 in Figure 80A, according to some embodiments, C57BL / 6 mice were exposed to either normal room light (N=7) or 40 Hz flashing light (N=7) for 1 hour per day for 2 weeks. During the third week, shown at 8002, the mice continued to be exposed to either normal room light or 40 Hz flashing light for 1 hour each morning, and then were similarly subjected to the Morris water maze test each afternoon.

[0224] Figure 80B is a plot showing the latency to find the platform for mice exposed to normal room light (NRL) and mice exposed to 40 Hz light flashing (40 Hz) on days 1-4 of week 3. After week 3, the hidden platform was removed and a probe trial was conducted. Figure 80C is a bar graph showing the total time (in seconds per 30-second trial) spent searching for the platform within the target quadrant during the probe trial. According to some embodiments, similar to 1-week treatment, chronic 3-week treatment enhanced spatial learning.

[0225] Reversal learning was performed using the same groups of mice from Figures 80A-80C. Figure 81A is a flow chart showing an expanded study. As shown in 8100 of Figure 81A, according to some embodiments, C57BL / 6 mice were exposed to either normal room light or 40 Hz flashing light for one hour per day for two weeks. During the third week, shown in 8102, the mice continued to be exposed to either normal room light or 40 Hz flashing light for one hour each morning, and then similarly underwent the Morris water maze test each afternoon. During the fourth week, shown in 8104, the mice continued to be exposed to either normal room light or 40 Hz flashing light for one hour each morning, and then similarly underwent the Morris water maze retest each afternoon. Figure 81B is a plot showing the latency to find the platform for mice exposed to normal room light (NRL) and mice exposed to 40 Hz flashing light (40 Hz) on days 1-4 of week 4, according to some embodiments.

[0226] After the fourth week, the hidden platform was removed and a probe trial was conducted. Figure 82A is a bar graph showing the total time (in seconds per 30-second trial) spent searching for the platform in the target quadrant during the probe trial. Figure 82B is a bar graph showing the time spent in the opposite quadrant during the probe trial. Mice exposed to 40 Hz light flashes showed robust cognitive flexibility.

[0227] Visual stimulation at gamma frequencies provided anatomical, morphological, cellular, and molecular benefits. According to some embodiments, a study was conducted to examine the effects of gamma radiation exposure and / or administration on DNA damage and neuronal loss in the visual cortex of a subject. For this study, an inducible mouse model of p25 accumulation (i.e., creatine kinase carboxyl-terminal fragment p25Tg mice (CK-p25Tg mice)) was used. The CK-p25Tg mouse model exhibits key pathological features of AD, including significant neuronal loss in the forebrain, increased Aβ peptide production, tau pathology, DNA damage, and severe cognitive impairment. In this model, elevated Aβ peptide levels were observed prior to neuronal loss. Furthermore, reduced Aβ peptide production alleviated memory impairment in the CK-p25Tg mouse model, indicating that this event acts synergistically with the carboxyl-terminal fragment p25, leading to the development of neurodegeneration and memory impairment.

[0228] Figure 83 is a timeline 8300 showing changes in CK-p25Tg mice. After two weeks 8302, the mice exhibit DNA damage (e.g., biomarker γH2AX), increased Aβ peptides, and microglial activation. After six weeks 8304, the mice exhibit synaptic loss, neuronal loss, tau hyperphosphorylation, impaired long-term potentiation, and memory impairment.

[0229] Tests were conducted to compare groups of mice under different treatment regimens. Figure 84 shows a diagram of groups including CK control mice 8400, untreated CK-p25Tg mice 8402, CK-p25Tg mice 8404 treated with memantine (10 mg / kg daily), CK-p25Tg mice 8406 exposed to 40 Hz light flashing (1 hour daily for 6 weeks) according to some embodiments, and CK-p25Tg mice 8408 treated with memantine and further exposed to 40 Hz light flashing. Memantine is a drug used with some success to treat severe AD by blocking NMDA receptors, thereby affecting the glutamate system.

[0230] Gamma exposure and / or administration according to some embodiments has been shown to preserve and / or reduce changes in brain anatomy. For example, gamma exposure reduced and / or prevented CKp-25-induced brain weight loss. Figure 85 is a bar graph comparing brain weight changes in CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. Brain weight loss was significant in untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. However, according to some embodiments, CK-p25Tg mice exposed to 40 Hz light flashes maintained greater brain weight.

[0231] Gamma exposure and / or administration according to some embodiments has been shown to preserve brain morphology and / or reduce changes thereto. For example, gamma exposure reduced and / or prevented CKp-25-induced abnormal lateral ventricle enlargement in subjects. Figure 86 is a bar graph comparing the ratio of lateral ventricle enlargement in CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes with the enlargement in CK control mice as baseline. Lateral ventricle enlargement was significant in untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. According to some embodiments, the lateral ventricles of CK-p25Tg mice exposed to 40 Hz flashing light were much less dilated than the lateral ventricles of other CK-p25Tg mice.

[0232] Figures 87A-87E are images showing representative lateral ventricles from subjects in each group. The lateral ventricles were largest in untreated CK-p25Tg mice (Figure 87A), CK-p25Tg mice treated with memantine (Figure 87B), and CK-p25Tg mice treated with both memantine and 40 Hz flashing light (Figure 87C). As shown in Figure 87D, according to some embodiments, the lateral ventricles of CK-p25Tg mice exposed to 40 Hz flashing light were much less dilated. Figure 87E is an example of baseline lateral ventricle size in a CK control mouse.

[0233] 88A-88C are brain anatomical diagrams showing brain regions of interest for molecular characterization, according to some embodiments. FIG. 88A shows the visual cortex (V1) 8800, the somatosensory cortex (SS) 8810, and the cerebral cortex (C). 1) 8802, hippocampus 8804, and insular cortex 8806.

[0234] Gamma exposure and / or administration according to some embodiments has been shown to preserve and / or reduce changes to the cortical and neuronal layers of the visual cortex, for example, gamma exposure reduced and / or prevented CKp-25-induced loss of cortical and neuronal layers in a subject's visual cortex.

[0235] Cortical layer loss was measured using nuclear staining with Hoechst labeling (i.e., a blue fluorescent dye used to stain DNA). Neuronal layer loss was measured using NeuN, a neuronal nuclear antigen commonly used as a neuronal biomarker. Figure 89 is a bar graph showing the average thickness of the V1 cortical layer in each group, and Figure 90 is a bar graph showing the average thickness of the V1-NeuN-positive cell layer in each group.

[0236] Figures 91A-91E are images showing representative Hoechst- and / or NeuN-labeled cells from subjects in each group. Figure 91A shows an example of baseline V1 cortical layer (e.g., 837±9 μm) and V1 neuronal layer (e.g., 725±7 μm) thickness in CK control mice.

[0237] According to some embodiments, the V1 cortex gradually thinned in CK-p25Tg mice exposed to 40 Hz flashing light (Figure 91D, e.g., 855±9 μM), CK-p25Tg mice treated with both memantine and 40 Hz flashing light (Figure 91E, e.g., 821±22 μM), untreated CK-p25Tg mice (Figure 91B, e.g., 792±13 μM), and CK-p25Tg mice treated with memantine (Figure 91C, e.g., 788±9 μM).

[0238] According to some embodiments, the V1 neuronal layer in CK-p25Tg mice exposed to 40 Hz flashing light was indeed thicker than in CK control mice (Figure 91D, e.g., 743±9 μM), while it became progressively thinner than in CK control mice in CK-p25Tg mice treated with both memantine and 40 Hz flashing light (Figure 91E, e.g., 691±20 μM), untreated CK-p25Tg mice (Figure 91B, e.g., 666±14 μM), and CK-p25Tg mice treated with memantine (Figure 91C, e.g., 660±7 μM).

[0239] Gamma exposure and / or administration according to some embodiments has been shown to preserve and / or reduce changes to the cortical and neuronal layers in the somatosensory cortex, for example, gamma exposure reduced and / or prevented CKp-25-induced loss of cortical and neuronal layers in the somatosensory cortex of a subject.

[0240] FIG. 92 is a bar graph showing the average thickness of the SS1 cortical layer in each group, and FIG. 93 is a bar graph showing the average thickness of the SS1-NeuN positive cell layer in each group.

[0241] Figures 94A-94E are images showing representative Hoechst- and / or NeuN-labeled cells from subjects in each group. Figure 94A shows an example of baseline SS1 cortical layer (e.g., 846±10 μM) and SS1 neuronal layer (e.g., 707±8 μM) thickness in CK control mice.

[0242] The SS1 cortex was measured in CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments (Figure 94D, e.g., 834 ± 94 μM), CK-p25Tg mice treated with both memantine and 40 Hz light flashes (Figure 94E, e.g., 778 ± 13 μM), untreated CK-p25Tg mice (Figure 94B, e.g., 762 ± 17 μM), and The retinal septum gradually thinned in CK-p25Tg mice treated with memantine (Fig. 94C, e.g., 756 ± 11 μM).

[0243] The SS1 neuron layer in CK-p25Tg mice exposed to 40 Hz flashing light according to some embodiments was approximately the same thickness as that in CK control mice (Figure 94D, e.g., 705 ± 15 μM). However, the SS1 neuron layer became progressively thinner in CK-p25Tg mice treated with both memantine and 40 Hz flashing light (Figure 94E, e.g., 650 ± 11 μM), untreated CK-p25Tg mice (Figure 94B, e.g., 630 ± 13 μM), and memantine-treated CK-p25Tg mice (Figure 94C, e.g., 629 ± 9 μM).

[0244] Gamma exposure and / or administration according to some embodiments has been shown to preserve and / or reduce changes to the cortical and neuronal layers in the insular cortex, for example, gamma exposure reduced and / or prevented CKp-25-induced loss of cortical and neuronal layers in the insular cortex of a subject.

[0245] Figure 95 is a bar graph showing the average thickness of the cortical layer of the insular cortex in each group, and Figure 96 is a bar graph showing the average thickness of the NeuN-positive cell layer of the insular cortex in each group.

[0246] Figures 97A-97E are images showing representative Hoechst- and / or NeuN-labeled cells from subjects in each group. Figure 97A shows an example of baseline cortical (e.g., 1134±10 μM) and neuronal (e.g., 1010±11 μM) thicknesses of the insular cortex in CK control mice.

[0247] The cortical layer became progressively thinner in the insular cortex of CK-p25Tg mice exposed to 40 Hz flashing light according to some embodiments (Figure 97D, e.g., 1079±20 μM), CK-p25Tg mice treated with memantine (Figure 97C, e.g., 983±12 μM), CK-p25Tg mice treated with both memantine and 40 Hz flashing light (Figure 97E, e.g., 965±16 μM), and untreated CK-p25Tg mice (Figure 97B, e.g., 764±27 μM).

[0248] The neuronal layer became progressively thinner in the insular cortex of CK-p25Tg mice exposed to 40 Hz flashing light according to some embodiments (Figure 97D, e.g., 953±17 μM), untreated CK-p25Tg mice (Figure 97B, e.g., 861±30 μM), CK-p25Tg mice treated with memantine (Figure 97C, e.g., 850±18 μM), and CK-p25Tg mice treated with both memantine and 40 Hz flashing light (Figure 97E, e.g., 848±15 μM).

[0249] Gamma exposure and / or administration according to some embodiments has been shown to preserve and / or reduce changes in neuronal number and / or DNA damage, for example, gamma exposure reduced CKp-25-induced neuronal loss and DNA damage in the visual cortex of subjects.

[0250] Figure 98 is a bar graph comparing the amount of NeuN-positive cells as a percentage of NeuN-positive cells in CK control mice for CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. Thus, the percentage of NeuN-positive cells in CK control mice is 100% in CK control mice, but only about 80% in untreated CK-p25Tg mice, demonstrating the presence of NeuN in the CK-p25Tg mouse model. This confirms the loss of neurons. Treatment with memantine prevented some neuronal loss in CK-p25Tg mice compared to untreated controls. Exposure to 40 Hz flashing light, according to some embodiments, prevented most neuronal loss in CK-p25Tg mice. Thus, Figure 98 shows how 40 Hz visual flicker treatment, according to some embodiments, can preserve neurons in the visual cortex. However, the combination of memantine and exposure to 40 Hz flashing light did not equally prevent neuronal loss.

[0251] DNA double-strand breaks (DSBs) are an example of DNA damage in eukaryotic cells that causes genomic instability, leading to tumorigenesis and possibly accelerated aging. Phosphorylated histone H2AX (γH2AX) was used as a biomarker of cellular responses to DSBs. Figure 99 is a bar graph comparing the amount of γH2AX-positive cells in CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. γH2AX-positive cells were almost absent in CK control mice, but were highly abundant in untreated CK-p25Tg mice, indicating a large amount of DSBs and other DNA damage. Treatment with memantine reduced the amount of γH2AX-positive cells in CK-p25Tg mice compared to the untreated group. Exposure to 40 Hz flashing light, according to some embodiments, resulted in an even greater reduction in γH2AX-positive cells in CK-p25Tg mice. Thus, Figure 99 shows how 40 Hz flashing light treatment, according to some embodiments, can reduce DNA damage in the visual cortex. However, the combination of memantine and exposure to 40 Hz flashing light significantly increased the number of γH2AX-positive cells in CK-p25Tg mice.

[0252] Figure 100 is a series of images showing representative visual cortex samples from subjects in each group labeled with Hoechst staining (indicating cortical cells), green fluorescent protein or GFP (indicating CK-p25), γH2AX (indicating DSBs), or NeuN (indicating neurons).

[0253] Gamma exposure also reduced CKp-25-induced neuronal loss and DNA damage in the somatosensory cortex of subjects. Figure 101 is a bar graph comparing the amount of NeuN-positive cells, as a percentage of NeuN-positive cells, in CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. Thus, the percentage of NeuN-positive cells in CK control mice is 100%, but is closer to 80% in untreated CK-p25Tg mice, confirming neuronal loss in the CK-p25Tg mouse model. Except for the combination of exposure to 40 Hz flashing light, which prevented most neuronal loss in CK-p25Tg mice, treatment with memantine failed to prevent neuronal loss in CK-p25Tg mice compared to the untreated group. Thus, Figure 101 shows how 40 Hz visual flashing treatment according to some embodiments can preserve neurons in the somatosensory cortex.

[0254] Figure 102 is a bar graph comparing the amount of γH2AX positive cells in CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. Cells positive for γH2AX were absent in CK control mice, but were present in untreated CK-p25Tg mice. CK-p25Tg mice showed significantly more γH2AX-positive cells than CK-p25Tg mice, demonstrating a large number of DSBs and other DNA damage. Treatment with memantine reduced the amount of γH2AX-positive cells in CK-p25Tg mice compared to untreated mice. Exposure to 40 Hz flashing light, according to some embodiments, resulted in an even greater reduction in γH2AX-positive cells in CK-p25Tg mice. Thus, Figure 102 shows how 40 Hz visual flashing treatment, according to some embodiments, can reduce DNA damage in the somatosensory cortex. However, the combination of memantine and exposure to 40 Hz flashing light significantly increased the number of γH2AX-positive cells in CK-p25Tg mice.

[0255] Figure 103 is a series of images showing representative somatosensory cortex samples from subjects in each group labeled with NeuN (indicating neurons), γH2AX (indicating DSBs), GFP (indicating CK-p25), and / or Hoechst staining (indicating cortical cells).

[0256] Gamma exposure also reduced CKp-25-induced neuronal loss and DNA damage in the insular cortex of subjects. Figure 104 is a bar graph comparing the amount of NeuN-positive cells, as a percentage of NeuN-positive cells, in CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. Thus, the percentage of NeuN-positive cells in CK control mice is 100%, but is closer to 80% in untreated CK-p25Tg mice, confirming neuronal loss in the CK-p25Tg mouse model. Except for exposure to 40 Hz flashing light, which prevented minimal neuronal loss in CK-p25Tg mice, treatment with memantine prevented some neuronal loss in CK-p25Tg mice compared to the untreated group. Figure 104 thus illustrates how 40 Hz visual flashing treatment according to some embodiments can preserve neurons in the insular cortex.

[0257] Figure 105 is a bar graph comparing the amount of γH2AX-positive cells in CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz flashing light according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz flashing light. Cells positive for γH2AX were absent in CK control mice but were highly abundant in untreated CK-p25Tg mice, indicating abundant DSBs and other DNA damage. Treatment with memantine reduced the amount of γH2AX-positive cells in CK-p25Tg mice compared to the untreated group. Exposure to 40 Hz flashing light according to some embodiments resulted in a similar reduction in γH2AX-positive cells in CK-p25Tg mice. Thus, Figure 105 illustrates how 40 Hz visual flashing treatment according to some embodiments can reduce DNA damage in the insular cortex. However, the combination of memantine and exposure to 40 Hz flickering light significantly increased the number of γH2AX-positive cells in CK-p25Tg mice.

[0258] Figure 106 is a series of images showing representative samples of the insular cortex from subjects in each group labeled with NeuN (indicating neurons), γH2AX (indicating DSBs), GFP (indicating CK-p25), or Hoechst staining (indicating cortical cells).

[0259] Gamma exposure also reduced CKp-25-induced neuronal loss and DNA damage in the hippocampus of subjects. Figure 107 shows the effects of CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz flashing light according to some embodiments, and CK-p25Tg mice treated with memantine and 40 Hz flashing light according to some embodiments. Figure 107 is a bar graph comparing the amount of NeuN-positive cells, as a percentage of NeuN-positive cells in CK control mice, for CK-p25Tg mice treated with both 40 Hz light flashing and memantine. Thus, the percentage of NeuN-positive cells in CK control mice is 100%, while it is closer to 80% in untreated CK-p25Tg mice, confirming neuronal loss in the CK-p25Tg mouse model. Treatment with memantine, with or without exposure to 40 Hz light flashing, prevented some neuronal loss in CK-p25Tg mice compared to the untreated group, which showed minimal prevention of neuronal loss in CK-p25Tg mice. Thus, Figure 107 illustrates how 40 Hz visual flashing treatment, according to some embodiments, can preserve hippocampal neurons.

[0260] Figure 108 is a bar graph comparing the amount of γH2AX-positive cells in CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz flashing light according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz flashing light. Cells positive for γH2AX were absent in CK control mice but were highly abundant in untreated CK-p25Tg mice, indicating abundant DSBs and other DNA damage. Treatment with memantine reduced the amount of γH2AX-positive cells in CK-p25Tg mice compared to the untreated group. Exposure to 40 Hz flashing light according to some embodiments resulted in a greater reduction of γH2AX-positive cells in CK-p25Tg mice. Thus, Figure 108 illustrates how 40 Hz visual flashing treatment according to some embodiments can reduce DNA damage in the hippocampus. However, the combination of memantine and exposure to 40 Hz flickering light significantly increased the number of γH2AX-positive cells in CK-p25Tg mice.

[0261] Figure 109 is a series of images showing representative hippocampal samples from subjects in each group labeled with Hoechst staining (indicating cortical cells), GFP (indicating CK-p25), γH2AX (indicating DSBs), or NeuN (indicating neurons).

[0262] Gamma exposure and / or administration according to some embodiments has been shown to preserve synapses and / or reduce synaptic loss. Changes in synaptic connectivity can be quantified using markers specific for glutamatergic synapses (e.g., VGluT1, VGluT2, PSD95, and GluR2) and GABAergic synapses (e.g., GAD and VGAT).

[0263] For example, gamma exposure reduced CKp-25-induced synaptic loss in the visual cortex of subjects. Figure 110 is a bar graph comparing the puncta density of glutamatergic synapses (using VGluT1) and GABAergic synapses (using GAD65) as a percentage of the synaptic puncta density of baseline CK control mice for CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes.

[0264] Gamma exposure also reduced CKp-25-induced synaptic loss in the somatosensory cortex of subjects, and even increased synaptic puncta density. Figure 111 shows graphs of synaptic puncta density as a percentage of baseline CK control mice for CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes. 1 is a bar graph comparing point densities of glutamatergic synapses (using VGluT1) and GABAergic synapses (using GAD65).

[0265] Gamma exposure reduced CKp-25-induced synaptic loss in the insular cortex of subjects. Figure 112 is a bar graph comparing glutamatergic synaptic (using VGluT1) and GABAergic synaptic (using GAD65) puncta densities as a percentage of baseline synaptic puncta densities in CK control mice for CK control mice, untreated CK-p25Tg mice, CK-p25Tg mice treated with memantine, CK-p25Tg mice exposed to 40 Hz light flashes according to some embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashes.

[0266] Figure 113A is an image showing a representative sample with Hoechst staining (indicating cortical cells). Figure 113B is an image showing VGluT1 (indicating glutamatergic synapses) in this representative sample. Figure 113C is an image showing GAD65 (indicating GABAergic synapses) in this representative sample. Figure 113D is a merged image showing Hoechst staining, VGluT1, and GAD65 in this representative sample. Figures 113E and 113F show how to quantify points using GAD65. Figure 113E is a binary image of GAD65 converted from Figure 113C. As shown in Figure 113F, the binary image was quantified using ImageJ software (available from the National Institutes of Health, Bethesda, MD).

[0267] A study was conducted to determine whether gamma exposure and / or administration according to some embodiments affects the cerebral vasculature. Mice were placed in a dark box and exposed to either 40 Hz light-emitting diode (LED) flashing or constant off light (dark) for 1 hour. After stimulation, the mice were sacrificed and perfused. Brain sections were stained with fluorophore-conjugated lectins to fluorescently label blood vessels. Confocal imaging was used to measure changes in the size (i.e., vessel diameter) of the vasculature. One hour after 40 Hz LED flashing, vasodilation was observed.

[0268] Figure 128A is a series of representative immunofluorescence images showing enlarged vasculature in the visual cortex, according to some embodiments. Figure 128B is a bar graph showing vascular diameter in the visual cortex, illustrating an increase in vascular diameter after gamma exposure, according to some embodiments.

[0269] Thus, gamma exposure and / or administration has been shown to confer anatomical (e.g., prevention and / or reduction of brain weight loss and enlargement of the vasculature), morphological (e.g., prevention and / or reduction of abnormal ventricular enlargement and loss of cortical thickness), cellular (e.g., prevention and / or reduction of neuronal loss), and molecular (e.g., prevention and / or reduction of DNA damage and synaptic loss) benefits.

[0270] Furthermore, gamma exposure and / or administration has been shown to be neuroprotective: after gamma treatment, the CK-p25Tg mouse model, a model that otherwise exhibits elevated Aβ peptide levels, significant neuronal loss, DNA damage, synaptic loss, tau hyperphosphorylation, impaired long-term potentiation, and severe cognitive / memory impairment, showed relative preservation of neuronal structure and / or function (e.g., maintenance / prevention of disease severity and / or mitigation / attenuation of disease progression), and in some cases, suggested improvement of neuronal structure and / or function.

[0271] Gamma frequency auditory stimulation noninvasively induced microglial changes in subjects. In some embodiments, the gamma exposure and / or administration includes auditory stimulation. The auditory stimulation may include sound pulses or clicks. The sound stimulation may include a click train of about 35 sound pulses or clicks per second (clicks / sec) to about 45 clicks / sec. Figure 114 is a stimulus diagram illustrating a click train stimulus, according to some embodiments. The stimulus in Figure 114 has a click frequency of 40 clicks / second with 25 milliseconds between each click, and each click has a duration of 1 millisecond.

[0272] In some embodiments, the sound stimuli have a frequency between about 10 Hz and about 100 kHz, between about 12 Hz and about 28 kHz, between about 20 Hz and about 20 kHz, and / or between about 2 kHz and about 5 kHz. For example, each sound pulse or click in a click train may have a frequency of about 10 kHz.

[0273] In some embodiments, the sound stimuli have a sound pressure level between about 0 dB and about 85 dB, between about 30 dB and about 70 dB, and / or between about 60 dB and about 65 dB. For example, each sound pulse or click in a click train may have a sound pressure level of about 65 dB.

[0274] Auditory gamma stimulation, according to some embodiments, has been shown to induce changes in the cellular state of microglia in a subject. According to some embodiments, a study was conducted to determine whether auditory gamma exposure and / or administration induces microglial activation in the auditory cortex of a subject. A 40 Hz click-train stimulus similar to that shown in Figure 114 was used. This stimulus had a click frequency of approximately 40 clicks / second, with each click having a tone of approximately 10 kHz and a duration of approximately 1 ms at approximately 60-65 dB. This click-train stimulus was hypothesized to entrain PV+ interneurons in the auditory cortex, thereby exogenously modulating gamma oscillations in the auditory cortex.

[0275] Figure 115 is a flow diagram illustrating this study. In Figure 115, WT mice were housed in their home cage 11500. For one hour per day, for seven consecutive days (days 1-7), the mice were transferred to a behavior box (i.e., a soundproof room) 11502. Within the behavior box 11502, a first group of mice was exposed to silence, and a second group of mice was exposed to click train stimuli according to some embodiments. After each hour in the behavior box 11502, the mice were returned to their home cage 11500. On day 8, the mice were sacrificed for tissue collection and staining 11504.

[0276] The tissues were examined for microglial activation, as indicated by microglial cell levels, morphological changes in microglial cells, and cell size. Figure 116A is a bar graph showing the average number of microglia in mice exposed to silence compared to mice exposed to click train stimulation. According to some embodiments, more microglial cells were observed in mice exposed to click train stimulation. Figure 116B is a bar graph showing the average ratio of microglial process length in mice exposed to silence compared to mice exposed to click train stimulation. The average ratio of microglial process length was significantly lower in mice exposed to click train stimulation, according to some embodiments. Figure 116C is a bar graph showing the average ratio of microglial cell size in mice exposed to silence compared to mice exposed to click train stimulation. The mean microglial cell size ratio was significantly higher in mice exposed to click-train stimulation, indicating higher microglial activation according to some embodiments.

[0277] FIG. 117A is a representative image of a microglial cell in a mouse exposed to silence. FIG. 117B is a representative image of a microglial cell in a mouse exposed to click train stimulation, according to some embodiments. The microglial processes and cells are clearly different between FIG. 117A and 117B, according to some embodiments. FIG. 118A is a magnified image from FIG. 117B of a microglial cell from a mouse exposed to click train stimulation, according to some embodiments. One process 118 of a microglial cell 118B is a magnified image from FIG. 117A of a microglial cell from a mouse exposed to silence. One process 11802 of the microglial cell is highlighted, showing its length relative to the relatively short process 11800 of a microglial cell from a mouse exposed to click train stimulation according to some embodiments.

[0278] Figure 119A is a magnified image from Figure 117B of a microglial cell from a mouse exposed to click train stimulation, according to some embodiments. The location of this microglial cell, cell 11900, is highlighted. Meanwhile, Figure 119B is a magnified image from Figure 117A of a microglial cell from a mouse exposed to silence, with the location of this microglial cell, cell 11902, highlighted to illustrate its size relative to the relatively larger cell 11900 of the microglial cell from the mouse exposed to click train stimulation, thus indicating higher microglial activation, according to some embodiments.

[0279] Auditory gamma stimulation, according to some embodiments, has been shown to induce a microglial activation-like phenotype in subjects. The study in FIG. 115 was repeated in 5XFAD Tg mice, according to some embodiments. The tissues were examined for microglial cell levels, morphological changes in microglial cells (e.g., process length), and microglial activation (e.g., as indicated by cell size). FIG. 120A is a bar graph showing the average number of microglia per image field in mice exposed to silence (no stimulation) compared to mice exposed to click train stimulation (stimulation). Significantly more microglial cells were observed in mice exposed to click train stimulation according to some embodiments. FIG. 120B is a bar graph showing the average cell size ratio of microglia in mice exposed to silence (no stimulation) compared to mice exposed to click train stimulation (stimulation). This average cell size ratio was significantly higher in mice exposed to click train stimulation, indicating greater microglial activity according to some embodiments. 120C is a bar graph showing the average ratio of microglial process lengths in mice exposed to silence (no stimulation) compared to mice exposed to click train stimulation (stimulation). This average ratio of process lengths was significantly lower in mice exposed to click train stimulation according to some embodiments.

[0280] Figure 121A is a representative image of microglial cells in a mouse exposed to silence. Figure 121B is a representative image of microglial cells in a mouse exposed to click train stimulation according to some embodiments. The microglial processes and cells are clearly different between Figures 121A and 121B, with relatively shorter process lengths and larger cell sizes in microglia from mice exposed to click train stimulation according to some embodiments.

[0281] Gamma frequency auditory stimulation non-invasively reduces Aβ in the auditory cortex and hippocampus of subjects. Auditory gamma stimulation, according to some embodiments, has been shown to reduce Aβ levels in subjects. The study in FIG. 115 was performed on 6-month-old 5XFAD mice, according to some embodiments. This was repeated in Tg mice. On day 8, the auditory cortex and hippocampus were dissected. ELISA was used to measure the Aβ isoform. 1-40 Peptides and isoforms of Aβ 1-42 The levels of soluble and insoluble Aβ isoforms containing the peptide were measured. Insoluble Aβ was solubilized by treatment with 5 M guanidine HCl for 3 hours to solubilize the plaques.

[0282] Auditory gamma stimulation, according to some embodiments, has been shown to reduce levels of soluble Aβ in a subject. Figure 122A shows the levels of soluble Aβ isoforms in the auditory cortex of a mouse exposed to silence (no stimulation), according to some embodiments. 1-42 The levels of the peptide significantly increased in the auditory cortex of mice exposed to click-train stimulation (Stim). Small levels of soluble Aβ isoforms 1-42 1 is a bar graph showing peptides.

[0283] FIG. 122B shows soluble isoform Aβ in the auditory cortex of mice exposed to silence (no stimulation), according to some embodiments. 1-40 Relative to peptide levels, small levels of soluble Aβ isoforms in the auditory cortex of mice exposed to click-train stimulation (stimulation)1-40 1 is a bar graph showing peptides.

[0284] FIG. 122C shows soluble isoform Aβ in the hippocampus of mice exposed to silence (no stimulation), according to some embodiments. 1-42 Relative to peptide levels, much smaller levels of soluble Aβ isoforms in the hippocampus of mice exposed to click-train stimulation (stimulation) 1-42 1 is a bar graph showing peptides.

[0285] FIG. 122D shows soluble isoform Aβ in the hippocampus of mice exposed to silence (no stimulation), according to some embodiments. 1-40 Relative to peptide levels, small levels of soluble Aβ isoforms in the hippocampus of mice exposed to click-train stimulation (stimulation) 1-40 1 is a bar graph showing peptides.

[0286] Auditory gamma stimulation, according to some embodiments, has been shown to reduce levels of insoluble Aβ in a subject. Figure 123A shows the levels of insoluble Aβ isoform in the auditory cortex of a mouse exposed to silence (no stimulation), according to some embodiments. 1-42 Relative to the level of peptides, much smaller levels of the insoluble isoform Aβ in the auditory cortex of mice exposed to click-train stimulation (stimulation) 1-42 1 is a bar graph showing peptides.

[0287] FIG. 123B shows insoluble isoform Aβ in the auditory cortex of mice exposed to silence (no stimulation), according to some embodiments. 1-40 Relative to the level of peptides, small levels of insoluble isoform Aβ in the auditory cortex of mice exposed to click train stimulation (stimulation) 1-40 1 is a bar graph showing peptides.

[0288] FIG. 123C shows the insoluble isoform Aβ in the hippocampus of mice exposed to silence (no stimulation), according to some embodiments. 1-42Relative to the level of peptides, much smaller levels of the insoluble isoform Aβ in the hippocampus of mice exposed to click-train stimulation (stimulation) 1-42 1 is a bar graph showing peptides.

[0289] FIG. 123D shows insoluble isoform Aβ in the hippocampus of mice exposed to silence (no stimulation), according to some embodiments. 1-40 Relative to peptide levels, small levels of insoluble Aβ isoforms in the hippocampus of mice exposed to click-train stimulation (stimulation) 1-40 1 is a bar graph showing peptides.

[0290] Figure 124A is a representative image of microglial cells in a 5XFAD mouse exposed to click train stimulation according to some embodiments. Figure 124B is a representative image of microglial cells in a 5XFAD mouse exposed to silence. The microglial processes and cells are clearly different between Figures 124A and 124B, with the microglia from the 5XFAD mouse exposed to click train stimulation according to some embodiments having relatively shorter process lengths and larger cell sizes.

[0291] Figure 124C is a representative image of microglial cells in a WT mouse exposed to silence. Figure 124D is a representative image of microglial cells in a WT mouse exposed to click train stimulation according to some embodiments. The microglial processes and cells are clearly different between Figure 124C and Figure 124D, with relatively more protruding cells in microglia from a WT mouse exposed to click train stimulation according to some embodiments. The length of the stem is short and the cell size is large.

[0292] Thus, according to some embodiments, non-invasive auditory stimulation at gamma frequencies promoted gamma oscillations in the auditory cortex and hippocampus and a significant reduction in AD-related pathology.

[0293] Gamma frequency auditory stimuli had a positive effect on the subjects' behavior. Auditory gamma stimulation, according to some embodiments, has been shown to improve subject recognition. Figure 125A is a flow diagram illustrating a novel object recognition test conducted with 5XFAD mice exposed to click train stimulation and 5XFAD mice exposed to silence, according to some embodiments. This test assesses a subject's ability to recognize a novel object from a familiar object (i.e., recognition memory) based on the rodent's tendency to spend more time exploring the novel object than the familiar object. Subjects were compared using a recognition index, RI: JPEG2023101026000013.jpg17152

[0294] In FIG. 125A, 5XFAD mice were acclimated to environment 12500. At time T1, two novel objects 12502 were introduced. Then, at time T2, after a 1-hour rest, the mice were exposed to one familiar object and one novel object 12504, 12506 for 1 hour. FIG. 125B is a bar graph showing the results of a novel object recognition test in which mice exposed to click train stimuli had a higher RI, indicating that mice exposed to click train stimuli spent more time with the novel objects than the familiar objects, resulting in better recognition memory, according to some embodiments.

[0295] Auditory gamma stimulation, according to some embodiments, has been shown to improve object discrimination. Figure 126A is a flow diagram illustrating a novel location recognition test conducted with 5XFAD mice exposed to click train stimulation and silence according to some embodiments. This test assesses spatial memory and / or discrimination based on the tendency of rodents to spend more time exploring novelly located objects. Subjects were compared using a recognition index, RI: JPEG2023101026000014.jpg16165

[0296] In FIG. 126A, 5XFAD mice were acclimated to environment 12600. At time T1, two objects were introduced at a first location 12602. Then, at time T2, after a 1-hour rest, the mice were exposed to one of the objects in its original location and the other object at a novel second location 12604, 12606 for 1 hour. FIG. 126B is a bar graph showing the results of a novel location recognition test in which mice exposed to click train stimuli had a higher RI, indicating that, according to some embodiments, mice exposed to click train stimuli spent more time with the moved object than with the object that remained in the same location due to better spatial memory and / or discrimination.

[0297] Auditory gamma stimulation, according to some embodiments, has been shown to improve spatial memory in subjects. Morris water maze testing was performed using 5XFAD mice exposed to click-train stimulation and silence, according to some embodiments. As described above, this test assesses spatial and / or reference memory based on the distal cues used by subjects to navigate from a starting position around the perimeter of an open swimming arena and locate a submerged platform. This test was assessed over repeated trials, and spatial and / or reference memory was determined by a preference for the platform location when the platform was not present.

[0298] Figure 127A is a plot showing the average latency to find the platform for mice exposed to silence (no stimulation) and mice exposed to click train stimulation (stimulation) for each day, according to some embodiments. Figure 127B is a bar graph showing the results of a probe trial in which the platform was removed. Mice exposed to click train stimulation spent more time searching for the missing platform in the goal quadrant than mice exposed to silence, thus indicating that mice exposed to click train stimulation have better spatial and / or reference memory, according to some embodiments.

[0299] Thus, according to some embodiments, non-invasive auditory stimulation at gamma frequencies induced microglial activation, reduced AD-related (e.g., Aβ) pathology, and significantly alleviated cognitive impairment (e.g., in recognition, discrimination, and spatial memory). Because of simple and accessible administration options (including self-administration), auditory gamma stimulation has broad commercial potential, including but not limited to home or portable (e.g., using noise-canceling headphones) applications. In addition to the possibility of self-administration, clinicians and / or researchers may administer stimulation paradigms to subjects ranging from animal models to human patients according to some embodiments. Clinicians and / or researchers may find it useful to combine auditory gamma stimulation with various types of monitoring. For example, a treatment session may include placing the subject in a soundproof room or providing the subject with noise-canceling headphones or another device to limit interference. The subject may be monitored for beneficial brain state changes during stimulation, for example, using functional magnetic resonance imaging (fMRI).

[0300] Experimental Method animal All animal experiments were approved by the Committee for Animal Care of the Division of Comparative Medicine (Massachusetts Institute of Technology, Cambridge, MA). Adult (3-month-old) male double Tg 5XFAD Cre mice were generated by crossing 5XFAD Tg mice with Tg PV or CW2 promoter-driven Cre lines. Adult (5-month-old) male and female APP / PS1 mice were donated by Tonegawa Laboratory (Massachusetts Institute of Technology, Cambridge, MA). Adult (4-month-old) male TauP301S mice were obtained from the Jackson Laboratory. Aged WT mice (8 months old, C57Bl / 6) were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in groups of 3–5 under a standard 12-hour light / 12-hour dark cycle, and all experiments were performed during the light cycle. Food and water were available ad libitum unless otherwise noted. Litters were randomly assigned to each condition by the experimenter, who was blinded to the genotype of the animals during tissue processing and electrophysiological recording and analysis. No animals were excluded from the analysis.

[0301] AVV Vector Serotype 5 adeno-associated virus particles were obtained from the Vector Core Facility ( The AAV5 virus was obtained from the University of North Carolina at Chapel Hill, NC. This AAV5 virus contained ChR2 in a double-floxed, inverted open reading frame (DIO) fused to enhanced yellow fluorescent protein (EYFP) driven by the EF1α promoter (see Figure 9 for an example). The AAV DIO EYFP construct was used as a control.

[0302] Surgical procedure Three-month-old 5XFAD / PV-Cre or CW2 mice were treated with ketamine (1.1 mg kg -1 ) and xylazine (0.16 mg kg -1The rats were anesthetized with an intraperitoneal injection of a mixture of AAV DIO. A small craniotomy was performed on the left side, 2.0 mm from the posterior lobe to the fontanel point and 1.8 mm from the lateral lobe to the midline. Virus was delivered through a small incision in the dura with a glass micropipette attached to a Quintessential Stereotaxic Injector™ (available from Stoelting Co., Wood Dale, IL). The glass micropipette was lowered 1.2 mm below the brain surface. Virus (AAV DIO) was delivered to the brain via a small incision in the dura. A 1 μl bolus of ChR2-EYFP or AAV DIO-EYFP (2x10 viral molecules per ml) was administered in 0.075 μl increments into the CA1 region of the hippocampus. -1The injection was performed at 1000 rpm. The pipette was left in place for 5 minutes after injection and then retracted from the brain. A unilateral fiber optic implant (300 μm core diameter, available from Thorlabs Inc., Newton, NJ) was lowered approximately 0.9 mm below the brain surface at the injection site. Two machine screws, fixed at the anterior and posterior edges of the surgical site, were secured with dental adhesive to hold the implant in place. For electrophysiological recordings, adult (3-month-old) male 5XFAD / PV-Cre double transgenic mice and their 5XFAD-negative littermates (for CA1 recordings) or 5XFAD and their WT littermates (for visual cortex recordings) were anesthetized with isoflurane and placed in a stereotaxic frame. The scalp was shaved, eye ointment (e.g., Puralube® Vet Ointment (Dechra Pharmaceuticals PLC, Northwich, UK)) was applied to the eyes, and the surgical field was disinfected with Betadine® disinfectant (available from Purdue Products LP, Stamford, CT) and 70% ethanol. For CA1 recordings, a craniotomy (in mm from the fontanel: -2 A / P, 1.8 M / L) was performed and 1 μL of virus was delivered to the CA1 (as described above). The target craniotomy site for LFP recording was marked on the skull (in mm from the fontanel: -3.23 A / P, 0.98 M / L for CA1 and 2.8 A / P, 2.5 M / L for visual cortex), and three self-tapping screws (e.g., F000CE094, available from Morris Precision Screws and Parts, Southbridge, MA) were attached to the skull. A custom stainless steel head plate was attached using dental cement (e.g., C&B Metabond®, available from Parkell Inc., Edgewood, NY). On the day of the first recording session, an LFP craniotomy (e.g., 300–400 μm diameter) was created by first thinning the skull to approximately 100 μm using a dental drill and then creating a small opening using a 30-gauge needle.The craniotomy was then sealed with sterile silicone elastomer (eg, Kwik-Sil™ adhesive, available from World Precision Instruments, Inc., Sarasota, Fla.) until and for the duration of the recording that day.

[0303] Optogenetic stimulation protocol Hippocampal CA1 neurons were optogenetically manipulated 2-4 weeks after viral injection and implant placement, allowing time for the mice to recover, undergo behavioral training for electrophysiological animals, and for the virus to express in neurons. A 200mW 4793nm DPSS laser was connected to a patch cord with fiber channel / physical contact connectors at each end. Light stimulation of 1mW (measured from the end of the fiber) was delivered for 1 hour during the experiment. For molecular and biochemical analyses, each animal received one of three stimulation protocols: 8Hz, 10 ... For electrophysiological recordings, each animal received one of the following stimulation conditions: 40 Hz, or random stimulation (light pulses were delivered at a mean frequency of 40 Hz and at random intervals determined by a Poisson process), or, for electrophysiological recordings, each animal received all stimulation conditions interleaved during recording.

[0304] Visual stimulation protocol 5XFAD mice were treated with saline (control) or picrotoxin (0.18 mg / kg) 15 minutes before the experiment. For molecular and biochemical analyses, mice were then placed in a darkened room illuminated by an LED bulb and exposed to one of five stimulation conditions: dark, light, 20 Hz flashing, 40 Hz flashing, or 80 Hz flashing (12.5 ms light on, 12.5 ms light off) for 1 hour (see, e.g., Figure 43A). For electrophysiological recordings, each animal received dark, light, 40 Hz flashing, or random (light pulses delivered at random intervals determined by a Poisson process, with an average interval of 40 Hz) stimulation conditions interleaved in 10-second blocks during recording.

[0305] Behavioral training and virtual reality environments (VR) for electrophysiology For CA1 recordings, head-fixed animals were run on an 8-inch spherical treadmill supported by an air cushion in a virtual reality environment, as described by Harvey et al. Movement on the spherical treadmill was measured with an optical mouse and transmitted to virtual reality software running in the MATLAB® computing environment (software version 2013b, available from MathWorks, Natick, MA). The virtual environment consisted of a straight track and two small enclosures at the ends into which the animals could turn. Animals were rewarded with sweetened condensed milk (diluted 1:2 with water) at each end of the track for alternate visits to each end. Animals learned to run on this virtual straight track over approximately one week. The animals were allowed one week to recover from surgery and acclimatized to handling for one to two days before behavioral training began. During the first two days of training, animals were placed on the spherical treadmill with the virtual reality system turned off and were rewarded with undiluted sweetened condensed milk to allow them to learn to navigate the treadmill and become familiar with the testing environment. On the second day of training on the spherical treadmill, animals were food-restricted to motivate them to run. Animals were restricted to no more than 85% of their baseline body weight and typically exceeded 88% of their baseline body weight. From the third day until the end of training (usually 5–7 days), animals were placed on the treadmill for increasing periods (30 min–2 h) and run along a VR linear track. After traversing the length of the track, animals were rewarded with diluted (1:2) sweetened condensed milk at the end of the linear track. Between recording periods, animals were given a retraining period to maintain behavioral performance. For visual cortex recordings, animals were run on the spherical treadmill while exposed to dark, light, or flashing light conditions (described below for data collection). Prior to recording, animals were placed on the spherical treadmill (with the virtual reality system turned off) and taught to navigate the treadmill and become familiar with the testing environment by receiving a reward of undiluted sweetened condensed milk.

[0306] Electrophysiology data acquisition For optogenetic stimulation of CA1 during recording, a 300 μm-core optical fiber was advanced 900 μm into the brain through the craniotomy used to deliver the virus to CA1. Light pulses of 1 ms and 1 mW (measured from the end of the fiber) were delivered via a 473 nm DPSS (diode-pumped solid-state) laser (as described above). To avoid photoelectric artifacts, neural activity was recorded with a glass electrode. LFP electrodes were prepared from borosilicate glass pipettes (e.g., available from Warner Instruments, Hamden, CT) pulled to a fine tip using a filament-based micropipette puller (e.g., P-97 Flaming / Brown™ Micropipette Puller, available from Sutter Instrument Co., Novato, CA). These were then manually broken back to a diameter of approximately 10–20 μm and subsequently filled with sterile saline. For recording of CA1, the LFP electrode was inserted into the frontal plane through the LFP recording craniotomy. The electrode was advanced at a 60-degree angle backward and 45 degrees downward relative to the horizontal plane until clear electrophysiological signatures were observed in the pyramidal layer of the hippocampus (theta waves of approximately 600-1000 μV while the animal was running, clearly distinguishable SWRs during immobility, and multiple spikes exceeding 150 μV; see Figures 2A-2B). For recordings from the visual cortex, the LFP electrode was advanced vertically through the craniotomy to a depth of 600-900 μm until multiple spikes exceeding 150 μV were observed. Data were collected at a sampling rate of 20 kHz and band-pass filtered at 1 Hz-1 kHz. Animals were either allowed to run on a spherical treadmill for extended periods or allowed to rest. For optogenetic stimulation periods, data were recorded for 30 min before the onset of any stimulation. Stimulation was then delivered at gamma (40 Hz), random (as described in the optogenetic stimulation protocol), or theta (8 Hz) frequencies, with 10-second periods interleaved with 10-second baseline periods (no stimulation). In two animals, each type of stimulation or baseline was delivered in 5-minute periods instead of 10-second periods. Each 30-minute stimulation recording was followed by 5–30 minutes of no-stimulation recording. For the visual light flashing stimulation periods, LED strip lights around the animal were flashed at gamma (40 Hz), random (as described in the visual stimulation protocol), theta (8 Hz), or 20 Hz frequencies for 10-second periods, interleaved with 10-second light-off periods, or 10-second continuous-on periods. Between the light flashes, several recordings were made above the brain surface to ensure that these lights did not generate electrical or photoelectric noise during recording. The recording period ended after approximately 3–5 hours. Animals were 3–4 months old at the time of recording. Analysis of electrophysiological recordings

[0307] Spike Detection Spikes were detected by thresholding the bandpass signal from 300 to 6000 Hz. The threshold was the median of the filtered signal plus five times a robust estimate of the standard deviation of the filtered signal (median / 0.675) to avoid contamination of standard deviation measurements by spikes (see, e.g., Rossant et al., “Spike Sorting for Large, Dense Electrode Arrays,” bioRxiv doi:dx_doi_org_10.1101_015198 (Feb. 16, 2015)).

[0308] Local field potential (LFP) Recorded traces were down-resolution processed to 2 kHz and band-pass filtered between 1 and 300 Hz.

[0309] Theta and SWR detection When an animal runs or remains stationary, activity throughout the hippocampal network changes significantly; these changes are often referred to as distinct network states. These network states can be clearly distinguished by the presence or absence of LFP oscillations in different frequency bands. When the animal runs, large theta (4–12 Hz) oscillations were observed in CA1, as previously shown (see, e.g., Figure 2A). When the animal remains stationary, theta oscillations are no longer observed, and SWRs, i.e., high-frequency oscillations of 150–250 Hz lasting approximately 50–100 ms and associated with bursts of collective activity, were recorded (see, e.g., Figure 2B). SWRs were detected when the envelope amplitude of the filtered trace exceeded the upper four standard deviations of the mean for at least 15 ms (see, e.g., Figures 4A, 4B, 5A, 5B, 6A, 6B, 7B, and 8). The envelope amplitude was calculated by taking the absolute value of the Hilbert transform of the filtered LFP. The results disclosed herein have been confirmed to be valid when using a higher threshold for SWR detection, i.e., 6 standard deviations above the mean, which detects larger SWRs (see, e.g., Figures 6C and 7C). To detect theta (see, e.g., Figures 3A and 3C), the LFP was bandpass filtered for theta (4-12 Hz), delta (1-4 Hz), and beta (12-30 Hz) using FIR equiripple filters. The ratio of theta to delta and beta ("theta ratio") was calculated using the following equation: Theta envelope amplitude was calculated as theta envelope amplitude divided by the sum of beta envelope amplitudes. Theta periods were classified as such if the theta ratio exceeded one standard deviation above the mean for at least 2 seconds and this ratio reached a peak at least two standard deviations above the mean. Non-theta periods were classified as such if the theta ratio was less than 1 for at least 2 seconds. SWRs, theta periods, and non-theta periods were visually inspected to confirm that these criteria accurately detected SWRs, theta periods, and non-theta periods, respectively.

[0310] Power Spectrum Spectral analysis was performed using a multitaper method (e.g., Chronux open-source software available from Mitra Lab in Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, time-bandwidth product = 3, number of tapers = 5). To examine power spectra without stimulation (e.g., see Figures 3A and 3C), only theta periods were included. Theta periods longer than 5 seconds were divided into 5-second trials, and the average power spectral density was calculated for each animal across these trials. To examine power spectra during optogenetic stimulation (e.g., see Figures 13A and 6C) and visual stimulation (e.g., see Figures 43B and 43C), data were divided into 10-second trials of each stimulation condition or baseline period, and the average power spectral density was calculated for each animal across these trials.

[0311] Gamma in SWR Spectrograms were calculated using the multitaper method (e.g., Mitra Lab (Chronux open-source software available from Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Spectrograms were calculated for each SWR, including a 400-ms period before and after the SWR peak. Z-scored spectrograms were then calculated for each frequency band using the mean and standard deviation of the spectrograms calculated over the entire recording period, generating a normalized measure of power in units of standard deviation (see, e.g., Figures 4A, 4B, 5A, and 5B). The instantaneous frequency of gamma oscillations during SWRs was calculated by bandpass filtering the LFP for 10–50 Hz, taking the Hilbert transform, and then taking the inverse of the peak difference of the transformed signal (see, e.g., Figures 4A, 5A, and 6B). Gamma power before, during, and after SWRs was calculated by filtering the LFP for low gamma (20–50 Hz) and taking the envelope amplitude of the Hilbert transform to obtain the average gamma power in 100-ms bins centered on the SWR peak. This was normalized by the mean and standard deviation of the envelope amplitude for the entire recording period to obtain z-scored gamma power for each bin surrounding each SWR (see, e.g., Figures 6A and 7B). Phase modulation by gamma during SWRs was calculated by bandpass filtering the LFP for gamma (20-50 Hz), applying the Hilbert transform, and determining the phase of the resulting signal for each spike occurring during the SWR (see, e.g., Figure 7E). To measure differences in phase modulation between 5XFAD and WT animals, we used resampling with restoration. That is, a subset of 100 spikes was randomly selected from each recording to create a phase modulation distribution, which was repeated 500 times for each recording (see, e.g., Figures 6C and 7A). The depth of modulation was then measured by calculating the difference between the peak and trough for each distribution, divided by the sum of the peak and trough. Differences in firing during stimulation: To plot histograms of stimulus-evoked multiunit firing, spikes were binned into 2.5 ms intervals for 100 ms after the onset of each light in the pulse, and the proportion of spikes in each bin was calculated. The mean and SEM were then calculated across all lights in the cycle.To calculate the difference in multiunit firing rate between conditions, firing rate was calculated for each 10-second stimulus or baseline period (total spike count divided by the period duration). The difference in firing rate was taken between adjacent periods of the relevant type of stimulus (for optogenetic stimulation, gamma stimulation period minus baseline or random period firing rate; for light flashing stimulation, gamma). (Firing rate of the stimulus period minus baseline, continuous-on, or random period). Differences from all animals were plotted in histograms (see, e.g., Figures 14A and 44A), and median and quartiles of the differences per animal were plotted in boxplots (see, e.g., Figures 13B and 44A).

[0312] immunohistochemistry Mice were perfused with 4% paraformaldehyde under deep anesthesia, and brains were post-fixed overnight in 4% paraformaldehyde. Brains were sectioned at 40 μm using a vibratome (e.g., Leica VT100S, available from Leica Biosystems, Buffalo Grove, IL). Sections were permeabilized and blocked with 0.2% Triton X-100 and 10% normal donkey serum in PBS for 1 hour at room temperature. Sections were then incubated overnight in primary antibody PBS with 0.2% Triton X-100 and 10% normal donkey serum at 4°C. Primary antibodies were anti-EEA1 (BD Transduction Laboratories™ EEA1 (641057), available from BD Biosciences, San Jose, CA), anti-β amyloid (e.g., β Amyloid (D54D2) XP®, available from Cell Signaling Technology, Danvers, MA), anti-Iba1 (e.g., 019-19741, available from Wako Chemicals, Richmond, VA), anti-parvalbumin (e.g., ab32895, available from Abcam, Cambridge, MA), and anti-Rab5 (ADI-KAp-GP006-E, available from Enzo Life Sciences Inc., Farmingdale, NY). To confirm the ELISA experiments, the anti-Aβ antibody D54D2 was used because it allows colabeling with EEA1, and the anti-Aβ antibody 12F4 was used because it does not react with APP, allowing determination of whether this labeling is specific to Aβ. For co-labeling experiments, anti-Aβ antibody 12F4 (805501, available from BioLegend, San Diego, CA) was used. Primary antibodies were Alexa-Fluor 488 and Alex-Fluor 647 secondary antibodies (Molecular Probes), and neuronal nuclei were visualized with Hoechst 33342 (94403, available from Sigma-Aldrich, St. Louis, MO). Images were acquired using a confocal microscope (LSM 710, Zeiss™) with identical settings for all conditions. Images were quantified using ImageJ 1.42q by an experimenter blinded to the treatment groups.For each experimental condition, at least two coronal sections from at least three animals were used for quantification. For hippocampal CA1 imaging, analysis was limited to the pyramidal cell layer, except for Iba1+ cell analysis, which required the entire field of view to image a sufficient number of cells. ImageJ was used to measure the diameter of Iba1+ cell bodies and trace their course for length measurements. Additionally, the Coloc2 plugin was used to measure the colocalization of Iba1 and Aβ. Imaris x64 8.1.2 (available from Bitplane, Belfast, UK) was used for 3-D rendering. For the "plaque count," deposits ≥10 μm in size were included.

[0313] CLARITY Fixed brains were sliced ​​into 100 μm coronal sections in 1X PBS using a vibratome (e.g., Leica VT100S, available from Leica Biosystems, Buffalo Grove, IL). Sections containing the visual cortex were selected using the Allen Mouse Brain Atlas and incubated for 2 hours with shaking at 55°C in clearing buffer (pH 8.5-9.0, 200 mM sodium dodecyl sulfate, 20 mM lithium hydroxide monohydrate, 4 mM boric acid in ddH2O). Cleared sections were washed three times for 10 minutes in 1X PBST (0.1% Triton X-100 in 1X PBS) and placed in blocking solution (2% bovine serum albumin in 1X PBST) overnight with shaking at room temperature. Next, sections were washed three times in 1X PBST for 1 hour with shaking at room temperature. The sections were then incubated with anti-β-amyloid (805501, BioLegend) diluted 1:100 in 1X PBST. Sections were incubated with anti-Iba1 (Wako Chemicals, San Diego, CA) and anti-Iba1 (Wako Chemicals, Richmond, VA, 019-19741) primary antibodies for 2 hours at 4°C with shaking. After three additional 1-hour washes in 1X PBST, sections were incubated with a secondary antibody mixture diluted 1:100 in 1X PBS for 9 hours at room temperature with shaking. Primary antibody labeling was visualized using fragmented donkey anti-rabbit Alexa Fluor® 488 (ab175694) and anti-mouse 568 (ab150101) secondary antibodies (both available from Abcam, Cambridge, MA). Partway through this incubation period, Hoechst 33258 (Sigma-Aldrich, 94403) was spiked into each sample at a final dilution of 1:250. Sections were then washed overnight in 1X PBS with shaking at room temperature. Before mounting for imaging, slices were incubated in RIMS (refractive index matching solution: 75 g Histodenz, 20 mL 0.1 M phosphate buffer, 60 mL ddH2O) for 1 h with shaking at room temperature. Fluoromount G Mounting Medium (Electron Microscopy Sciences, Hatfield, PA, USA) was used to mount the slices on a coverslip (e.g., VistaVision™, VWR). Tissue sections were mounted onto microscope slides with Fluorescence Imaging (FIM) software (available from Fluorescence Imaging International, LLC, Radnor, PA). Images were acquired with a Zeiss™ LSM 880 microscope with Zen Black 2.1 software (Carl Zeiss Microscopy, Jena, Germany). Cross-sectional overview and cellular images used for 3-D reconstruction were captured using a Plan-Apochromat 63x / 1.4 Oil DIC objective. Imarisx64 8.1.2 (Bitplane™, Zurich, Switzerland) was used for 3-D rendering and analysis.

[0314] Western blot Hippocampal CA1 whole-cell lysates were prepared using tissue from 3-month-old male 5XFAD / PV-Cre mice. The tissue was homogenized in 1 ml of RIPA buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% Np-40, 0.5% sodium deoxycholate, 0.1% SDS) using a manual homogenizer (Sigma-Aldrich, St. Louis, MO). The homogenized tissue was incubated on ice for 15 minutes and then rotated at 4°C for 30 minutes. Cell debris was isolated and discarded by centrifugation at 14,000 rpm for 10 minutes. The lysates were quantified using a Nanodrop™ gel, and 25 μg of protein was loaded onto a 10% acrylamide gel. Proteins were transferred from the acrylamide gel to a PVDF membrane (e.g., Invitrogen™, Thermo Fisher Scientific) at 100 V for 120 minutes. The membranes were transferred to a 500-well plate (available from Thermo Fisher Scientific, Waltham, MA). The membranes were blocked with bovine serum albumin (5% w / v) diluted in TBS:Tween. The membranes were incubated in primary antibodies overnight at 4°C and in secondary antibodies for 90 minutes at room temperature. The primary antibodies were anti-APP (Invitrogen™ PAD CT695, available from Thermo Fisher Scientific, Waltham, MA), anti-APP (A8967, available from Sigma-Aldrich, St. Louis, MO), and anti-β-actin (ab9485, available from Abcam, Cambridge, MA). The secondary antibodies were horseradish peroxidase-conjugated (e.g., available from GE Healthcare, Marlborough, MA). Signal intensity was quantified using ImageJ 1.46a and normalized to the β-actin value. Tau protein solubility was examined using sequential protein extraction. The detergent-insoluble tau fraction was probed with an antibody against Tau5 (eg, AHB0042, available from Thermo Fisher Scientific, Waltham, MA).

[0315] ELISA Hippocampal CA1 or VC was isolated from male mice and treated with PBS or 5 M guanidine HCl. Dissolved mouse / human Aβ 1-40 or Aβ 1-42 Aβ measurements were performed using an ELISA kit (e.g., Invitrogen™, available from Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Tissues were lysed in phosphate-buffered saline (PBS) to extract the PBS-soluble Aβ fraction. This soluble Aβ fraction likely contained monomeric and oligomeric Aβ. The tissues were further treated with guanidine hydrochloride (HCl) to extract the insoluble Aβ fraction.

[0316] Genome-wide RNA sequencing Total RNA was extracted from hippocampal CA1 isolates using the RNeasy® kit (available from Qiagen, Hilden, Germany). Purified mRNA was used for RNA-seq library preparation using the BIOO NEXTflex™ kit (BIOO No. 5138-08) according to the manufacturer's instructions. Briefly, 1 μg of total mRNA was subjected to a sequential workflow of polyA purification, fragmentation, first- and second-strand synthesis, DNA end adenylation, and adapter ligation. These libraries were enriched by 15 cycles of PCR and cleaned with Agencourt® AMPure XP magnetic beads (available from Beckman Coulter Genomics, Danvers, MA). Library quality was assessed using an Advanced Analytical-Fragment Analyzer. Barcoded libraries were mixed equally for sequencing in a single lane on an Illumina HiSeq 2000 platform at the MIT BioMicro Center (Massachusetts Institute of Technology, Cambridge, MA). Raw fastq data from 50-base-pair single-end sequencing reads were aligned to the mouse mm9 reference genome using TopHat2.0 software (available from the Center for Computational Biology at Johns Hopkins University, Baltimore, MD; RNA-seq reads were aligned to a mammalian-sized genome using the ultra-high-throughput short-read aligner Bowtie, and then the mapping results were analyzed to identify inter-exon splice sites). Mapped reads were processed with the UCSC mm9 reference gene annotation using Cufflinks2.2 software (available from the Trapnell Lab at the University of Washington, Seattle, WA) to estimate transcript abundance and examine differential expression. Relative transcript abundance was measured in fragments per kilobase of exon per million fragments mapped (FPKM). Differential expression testing of genes between treated and untreated groups was performed using the Cuffdiff module (to identify significant changes in transcript expression, splicing, and promoter usage).Analysis was performed using Cufflinks 2.2 software (included as part of the Trapnell Lab, University of Washington, Seattle, WA) with an adjusted p-value for statistical significance of <0.05 (GEO accession: GSE77471).

[0317] To understand cellular and molecular mechanisms from RNA-seq data, 14 publicly available RNA-seq datasets were processed for cell-type-specific analysis. Additionally, 60 publicly available neuron-, microglia-, and macrophage-specific RNA-seq datasets under different chemical and genetic perturbations were downloaded and processed for GSEA statistical analysis using TopHat Cufflinks 2.2 software (available from the Trapnell Lab, University of Washington, Seattle, WA). Gene set enrichment analysis (GSEA) was used to determine whether defined gene sets from the RNA-seq data were significantly enriched in either direction in the ranked gene list from a specific perturbation study. Genes detected in the publicly available RNA-seq datasets were ranked from positive to negative by the log2 expression ratio (cases vs. controls). A defined gene set (in this case, up- or down-regulated genes in gamma treatment) was considered to be significantly correlated with perturbation-induced transcriptomic changes (either up- or down-regulated) if both the nominal p-value and the FDR q-value were less than 0.05. The sign of the calculated normalized enrichment score (NES) indicates whether the gene set is enriched at the top or bottom of the ranked list. Heat maps of differentially expressed genes were generated using a custom R script, and z-score values ​​across all libraries for each gene were calculated based on gene FPKM values. Boxplots for cell type specificity analysis were also generated by an R program based on gene FPKM values.

[0318] Quantitative RT-PCR CA1 was isolated from the hippocampus of 3-month-old male 5XFAD / PV-Cre mice. Tissue was rapidly frozen using liquid nitrogen and stored at -80°C. RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA (3 μg) was treated with DNase I (4 U, Worthington Biochemical Corporation, Lakewood, NJ) and purified using the RNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA) according to the manufacturer's instructions and eluted in 14 μl of DEPC-treated water. For each sample, 1 μg of RNA was reverse transcribed for 1 hour at 50°C in a 20 μl reaction volume containing a random hexamer mix and Superscript III reverse transcriptase (50 U, Invitrogen™, available from Thermo Fisher Scientific, Waltham, MA). First-strand cDNA was diluted 1:10, and 1 μl was used for RT-qPCR amplification in a 20 μl reaction (SsoFast™ EvaGreen® Supermix, Bio-Rad) containing primers (0.2 μM). Relative changes in gene expression were calculated using 2 -ΔΔCt The method was used to evaluate.

[0319] Isolation of microglia from the visual cortex. The V1 region was rapidly dissected and placed in ice-cold Hank's Balanced Salt Solution (HBSS) (Gibco™ 14175-095, available from Life Technologies). The tissue was then enzymatically digested using a Neural Tissue Dissociation Kit (P) (130-092-628, Miltenyi Biotec, Cambridge, MA) according to the manufacturer's protocol with minor modifications. Specifically, the tissue was enzymatically digested for 15 minutes at 37°C instead of 35 minutes, and the resulting cell suspension was passed through a MACS® SmartStrainer (352340, Falcon Cell Strainers, Sterile, Corning, NY), a 40 μm cell strainer instead of a 70 μm cell strainer. The resulting cell suspension was then stained with allophycocyanin (APC)-conjugated CD11b mouse clone M1 / 70.15.11.5 (130-098-088, Miltenyi Biotec, Cambridge, MA) and phycoerythrin (PE)-conjugated CD45 antibody (e.g., BD Pharmingen™, 553081). Fluorescence-activated cell sorting (FACS) was then used to purify CD11b- and CD45-positive microglial cells. These cells were sorted directly into 1X PBS (see, e.g., Figure 52A).

[0320] statistics For electrophysiological data that were not normally distributed, results were presented as medians and quartiles unless otherwise stated. A two-tailed Wilcoxon rank-sum test for equal medians was performed to determine whether the distributions were significantly different, or a Wilcoxon signed-rank test was performed to determine whether the distributions were significantly different from zero when the data were not assumed to be normally distributed. Variability was similar between statistically compared groups. Multiple comparisons were corrected using the Bonferroni method. Molecular and biochemical results are presented as means and SEM. Percentages reported in this disclosure are group means. All statistical analyses were performed using Prism GraphPad software (GraphPad software Inc., La Jolla, CA). Normality was confirmed according to D'Agostino & Normality was determined using the Pearson omnibus test. Variation was similar between statistically compared groups. Comparisons of normally distributed data from two groups were analyzed using a two-tailed unpaired t-test. Comparisons of normally distributed data from three or more groups were analyzed using one-way analysis of variance followed by Tukey's multiple comparison test. Comparisons of non-normally distributed data were performed using the Mann-Whitney test. Statistical tests, exact P values, and sample sizes (n) for each experiment are specified in the figure legends. Molecular and biochemical analyses were performed using a minimum of three biological replicates per condition.

[0321] Auditory gamma stimulus generation The following script, written in the MATLAB® programming language (available from MathWorks, Natick, Massachusetts), illustrates one method of generating auditory click train stimuli according to some embodiments: JPEG2023101026000015.jpg211156

[0322] conclusion While various inventive embodiments have been described and illustrated herein, various other methods and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein will readily occur to those skilled in the art, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the present disclosure is used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that, within the scope of the appended claims and their equivalents, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure relate to individual features, systems, articles, materials, kits, and / or methods described herein. Furthermore, combinations of two or more such features, systems, articles, materials, kits, and / or methods are included within the inventive scope of the present disclosure, unless such features, systems, articles, materials, kits, and / or methods are mutually inconsistent.

[0323] The above embodiments can be implemented in any of numerous ways. For example, the embodiments described herein may be implemented using hardware, software, or a combination thereof. When implemented in software, the software code may be executed on any suitable processor or group of processors, whether located on a single computer or distributed across multiple computers.

[0324] Further, it should be understood that a computer may be embodied in any of a number of forms, for example, a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. In addition, a computer may be embedded in a device not generally considered a computer but having suitable processing capabilities, including a personal digital assistant (PDA), a smartphone, or any other suitable portable or fixed electronic device.

[0325] A computer may also have one or more input and output devices. These devices may be used, among other things, to provide a user interface. Examples of output devices that may be used to provide a user interface include a printer or display screen for visual presentation of output and a speaker or other sound generating device for audible presentation of output. Examples of input devices that may be used for a user interface include a keyboard, a pointing device such as a mouse, a touchpad, and a discretization tablet. As another example, a computer may receive input information via voice recognition or in other audible formats.

[0326] Such computers may be interconnected by one or more networks of any suitable form, including local area networks or wide area networks, e.g., enterprise networks, and intelligent networks (IN) or the Internet. Such networks may be based on any suitable technology and operate according to any suitable protocol, and may include wireless networks, wired networks, or fiber optic networks.

[0327] The various methods or processes outlined herein may be coded as software executable on one or more processors using any one of a variety of operating systems or platforms. Further, such software may be written using any of a number of suitable programming languages ​​and / or programming or scripting tools, and may be compiled as executable machine code or intermediate code to run on a framework or virtual machine.

[0328] Also, various inventive concepts may be embodied in one or more methods, examples of which are provided. The acts performed as part of the method may be ordered in any suitable manner. Thus, embodiments may be constructed in which acts are performed in a different order than that described, which may include performing some acts simultaneously even though they are shown as sequential acts in the exemplary embodiment.

[0329] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0330] All definitions defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0331] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly indicated otherwise, should be understood to mean "at least one."

[0332] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, related or unrelated to those elements specifically identified, may be optionally included other than the elements expressly identified by the "and / or" clause. Thus, as a non-limiting example, when used in conjunction with open-ended terminology such as "comprising," a reference to "A and / or B" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0333] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as inclusive, i.e., the inclusion of at least one of a plurality of elements or a list of elements, but also of a plurality, and optionally, additional items not listed. Limiting terms expressly indicated otherwise, such as "only one" or "exactly one," or, when used in the claims, "consisting of," shall be deemed, for example, to indicate the inclusion of exactly one element of a plurality of elements or a list of elements. In general, the term "or" as used herein shall only be construed to indicate exclusive alternatives (i.e., "one or the other, but not both") when preceded by exclusive terms, such as "either," "one," "only one," or "exactly one." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0334] As used herein and in the claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically set forth in the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows for the optional inclusion of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") is used. "At least one" can refer, in one embodiment, to at least one, optionally including a plurality, of A and no B (optionally including elements other than B); in another embodiment, to at least one, optionally including a plurality, of B and no A (optionally including elements other than A); in yet another embodiment, to at least one, optionally including a plurality, of A and at least one, optionally including a plurality, of B (optionally including other elements);

[0335] In the claims, as well as in the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," etc., are to be understood to be open-ended, i.e., to mean including, but not limited to. As set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases.

Claims

1. A stimulus delivery device configured to provide non-invasive stimulation to a target brain region to induce in vivo synchronous gamma oscillations, the stimulus delivery device comprising at least: a light emitting device configured to emit a light stimulus to an eye of the subject; and a sound emitting device configured to emit sound stimuli to the ears of the subject; a stimulation emission device, at least one memory for storing stimulation parameters and processor-executable instructions; at least one processor communicatively coupled to the stimulus emitting device and the at least one memory, wherein, when the processor-executable instructions are executed, the at least one processor controls the stimulus emitting device to emit the stimuli according to the stimulation parameters, the parameters including a frequency between 35 Hz and 45 Hz that synchronously activates the brain region at that frequency; A system comprising:

2. A stimulus delivery device configured to provide non-invasive stimulation to a target brain region to induce in vivo synchronous gamma oscillations, the stimulus delivery device comprising: a light emitting device configured to emit a light stimulus to the subject's eyes, the light emitting device including a pair of glasses; or a sound emitting device including headphones and configured to emit sound stimuli to the ears of the subject; a stimulated emission device comprising: at least one memory for storing stimulation parameters and processor-executable instructions; at least one processor communicatively coupled to the stimulus emitting device and the at least one memory, wherein, when the processor-executable instructions are executed, the at least one processor controls the stimulus emitting device to emit the stimuli according to the stimulation parameters, the parameters including a frequency between 35 Hz and 45 Hz that synchronously activates the brain region at that frequency; A system comprising:

3. The system described in claim 1 or 2, configured to perform at least one of prevention, alleviation, and treatment of at least one of Aβ peptides, neuroinflammation, and cognitive function in the subject.

4. A system as described in claim 1 or 2, wherein the frequency is approximately 40 Hz.

5. The system described in claim 1 or 2, wherein the live in vivo synchronized gamma oscillations occur in specific cell types and are regulated by enzymes.

6. The system described in claim 5, wherein the specific cell type is an immunoreactive fast-spiking parvalbumin (FS-PV) interneuron.

7. The system described in claim 5 or 6, wherein the enzyme is at least one of an optogenetic activator, a microbial opsin, channelrhodopsin 2 (ChR2), and the vector AAV-DIO-ChR2-EYFP.

8. The system described in claim 1 or 2, comprising the light emitting device and further comprising an optical occlusion device for reducing ambient light to at least one eye of the subject, the optical occlusion device including the light emitting device for emitting the optical stimulus to the at least one eye for in vivo synchronous activation of at least one of the subject's visual cortex and hippocampus.

9. The system described in claim 1 or 2, comprising the sound emission device and further comprising a noise cancellation device for reducing ambient noise to at least one ear of the subject, the noise cancellation device including a speaker unit for emitting the sound stimulus to the at least one ear for in vivo synchronous activation of at least one of the subject's auditory cortex and hippocampus.

10. A system as described in claim 1 or 2, wherein the system includes a sound emitting device configured to generate a click train having a frequency of 12 Hz to 28 kHz or a sound pressure level of 30 dB to 70 dB.

11. The system described in claim 1 or 2, further comprising a detection device configured to detect the stimulus and provide feedback regarding the stimulus.

12. A system as described in claim 1 or 2, further comprising a neuroimaging device.

13. The system of claim 1, further comprising at least one communication interface for communicating with at least one of the subject, a healthcare provider, a caregiver, a clinical research investigator, a database, and a monitoring application.

14. A system as described in claim 1 or 2, wherein the stimulus emission device is configured to provide the stimulus to the subject for at least one hour per day over the exposure period.

15. The system described in claim 14, wherein the exposure period is at least one of 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and 1 month.

16. The system described in claim 15, wherein the exposure period is at least one week.

17. The system described in claim 1, wherein the frequency is a pulse frequency of approximately 35 pulses / second to approximately 45 pulses / second.

18. The system described in claim 17, wherein the stimulation parameters further include a repetition frequency in the range of approximately 35 Hz to approximately 45 Hz.

19. Further comprising stimulus generation guidelines stored in said memory; 20. The system of claim 18, wherein the at least one processor is configured to control the at least one light-emitting device to emit light pulses at the repetition frequency for at least one hour per day for at least seven days based at least in part on the stimulation parameters and the stimulation generation guidelines stored in the memory.

20. The processor is configured to: Induction of at least one change in the first microglial cells; Activation of pro-phagocytic genes in microglia; activation of the cell adhesion / migration regulator Spp1; reduction in soluble Aβ 1-40 and soluble Aβ 1-42 peptide levels; a reduction in insoluble Aβ 1-40 and insoluble Aβ 1-42 peptide levels; Decreased tau phosphorylation associated with pTau(S202) and pTau(S400 / T403 / S404); Recruitment of neurons and the first microglial cells to provide a neuroprotective response; and Increased blood vessel diameter 20. The system of claim 19, configured to induce at least one of:

21. Further comprising stimulus generation guidelines stored in the memory; The at least one processor controls the at least one sound-emitting device to emit sound pulses at a repetition frequency for at least one hour per day for at least seven days based at least in part on the stimulation parameters and the stimulation generation strategy stored in the memory, and to achieve the following therapeutic effect: inducing at least one change in second microglial cells in the auditory cortex of the subject; a reduction in soluble Aβ 1-40 and soluble Aβ 1-42 peptide levels in the auditory cortex of said subject; a reduction in the levels of insoluble Aβ 1-40 and insoluble Aβ 1-42 peptides in the auditory cortex of said subject; and Reduction of the levels of soluble Aβ 1-40 and soluble Aβ 1-42 peptides in the hippocampus of said subject 20. The system of claim 18, wherein the system is configured to direct at least one of:

22. The system of claim 2, further comprising at least one communication interface for communicating with at least one of the subject, a healthcare provider, a caregiver, and a clinical research investigator.

23. The system described in claim 2, wherein the frequency is a pulse frequency of approximately 35 pulses / second to approximately 45 pulses / second.

24. The system described in claim 23, wherein the stimulation parameters further include a repetition frequency in the range of approximately 35 Hz to approximately 45 Hz.

25. Further comprising stimulus generation guidelines stored in said memory; The at least one processor, based on the stimulation parameters and the stimulation generation guidelines stored in the memory, configured to control the at least one light emitting device to emit light pulses at the repetition rate for at least one hour per day for at least seven days; or 25. The system of claim 24, configured to control the at least one sound emitting device to emit sound pulses at the repetition frequency for at least one hour per day for at least seven days.

26. The processor is configured to: Induction of at least one change in the first microglial cells; Activation of pro-phagocytic genes in microglia; activation of the cell adhesion / migration regulator Spp1; reduction in soluble Aβ 1-40 and soluble Aβ 1-42 peptide levels; a reduction in insoluble Aβ 1-40 and insoluble Aβ 1-42 peptide levels; Decreased tau phosphorylation associated with pTau(S202) and pTau(S400 / T403 / S404); Recruitment of neurons and the first microglial cells to provide a neuroprotective response; Increased vessel diameter; inducing at least one change in second microglial cells in the auditory cortex of the subject; a reduction in soluble Aβ 1-40 and soluble Aβ 1-42 peptide levels in the auditory cortex of said subject; a reduction in the levels of insoluble Aβ 1-40 and insoluble Aβ 1-42 peptides in the auditory cortex of said subject; and Reduction of the levels of soluble Aβ 1-40 and soluble Aβ 1-42 peptides in the hippocampus of said subject 26. The system of claim 25, configured to induce at least one of:

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