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

Abstract

This disclosure provides systems and methods for preventing, reducing, and treating at least one of the following in the subject's brain: stimulation (e.g., light, sound, and / or touch) at a frequency (e.g., about 40 Hz) by inducing synchronized gamma oscillations in the subject's brain, for example, by using a stimulation-emitting device that synchronously activates specific cell types (e.g., flash-valley protein (FS-PV) immunoreactive interneurons) and / or brain regions (e.g., sensory cortex and / or hippocampus).

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610365088.X (22) Application Date 2016.11.23 (30) Priority Data 62 / 259,187 2015.11.24 US (62) Divisional Application Data 201680075447.2 2016.11.23 (71) Applicant Massachusetts Institute of Technology Address Massachusetts, USA (72) Inventors A.J. Mattorell C. Adie Aiken LH. Cai E. Brown H. Iacarino (74) Patent Agency Beijing Kunrui Law Firm 11494 Patent Attorney Feng Xinqin (51) Int.Cl. A61M 21 / 00 (2006.01) A61N 5 / 06 (2006.01) (54) Invention Title: Systems and Methods for Preventing, Alleviating and / or Treating Dementia (57) Abstract: This disclosure provides systems and methods for preventing, reducing and treating at least one of the following in the subject's brain: 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) by inducing synchronous γ oscillations at a frequency (e.g., about 40 Hz) of a specific cell type (e.g., flash-valley protein (FS-PV) immunoreactive interneurons) and / or brain regions (e.g., sensory cortex and / or hippocampus) using a stimulation-emitting device that, for example, synchronously activates a subject in vivo, emits stimuli (e.g., light, sound and / or touch) to prevent, reduce and treat at least one of the following in the subject: 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). Claims 1 page, Description 78 pages, Drawings 136 pages, CN 122399187 A 2026.07.17 CN 1 22 39 91 87 A 1. A method for at least one of increasing the number of microglia in at least one brain region of a subject, inducing morphological changes of said microglia consistent with a neuroprotective state, and promoting the activity of said microglia, comprising inducing synchronous gamma oscillations in said at least one brain region of the subject. 2. The method of claim 1, wherein said synchronous gamma oscillations upregulate at least one differentially expressed gene involved in the activity of said microglia in said at least one brain region of the subject. 3. The method of claim 2, wherein said at least one differentially expressed gene includes Nr4a1, Arc,At least one of Npas4, Cd68, B2m, Bsr2, Icam1, Lyz2, Irf7, Spp1, Csf1r, and Csf2ra. 4. The method of claim 1, wherein the morphological changes of the microglia consistent with the neuroprotective state include at least one of an increase in cell body size and a decrease in process length. 5. A method for reducing the amount of amyloid-β (Aβ) peptide in the hippocampus of a subject, the method comprising optogenetically stimulating fast flash-albumin (FS-PV) interneurons in the hippocampus using a plurality of light pulses, the FS-PV interneurons expressing optogenetic actuators thereby inducing in vivo synchronous gamma oscillations in the FS-PV interneurons that reduce the amount of Aβ peptide in the hippocampus. 6. The method of claim 5, wherein the plurality of light pulses have a pulse frequency of about 40 pulses / s. 7. The method of claim 6, wherein each of the plurality of light pulses has a duration of about 1 ms. 8. The method of any one of claims 5-7, wherein each of the plurality of light pulses has a wavelength of about 473 nm. 9. The method of any one of claims 5-8, wherein the optogenetic actuator is at least one of channel rhodopsin, halophilic rhodopsin, and paleorhodopsin. 10. The method of claim 9, wherein the optogenetic actuator is channel rhodopsin-2 (ChR2). Claims 1 / 1 page 2 CN 122399187 A Systems and methods for preventing, alleviating, and / or treating dementia

[0001] This application is a divisional application of patent application filed on November 23, 2016, with application number 202210905503.8, entitled "Systems and methods for preventing, alleviating, and / or treating dementia". Technical Field

[0002] This disclosure generally relates to systems and methods for preventing, alleviating, and / or treating dementia in a subject. More specifically, this disclosure relates to systems and methods for introducing synchronous gamma oscillations into at least one brain region of a subject. Background Art

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by a decline in memory, orientation, and reasoning. It is the most common form of dementia in the world, affecting approximately one in eight people over the age of 65, and is the sixth leading cause of death in the United States. The prevalence of this progressive neurodegenerative disease is estimated to increase by 40% over the next decade.

[0004] Histopathologically, AD can be characterized by the accumulation of amyloid plaques containing amyloid-β (Aβ) peptides and neurofibrillary tangles made of tau protein. Aβ peptides are 36-43 amino acid proteins whose normal physiological functions are not recognized. Aβ peptides are involved in β-secretase 1Aβ precursor protein (APP) is formed by the sequential proteolytic cleavage of APP via BACE1 and γ-secretase. C-terminal fragment β (β-CTF) is an APP derivative produced during the amyloidogenic cleavage of APP via 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 subjects with AD, Aβ peptides appear to aggregate into higher-order substances, forming soluble oligomers and insoluble plaques in a concentration-dependent manner. This aggregation can trigger a number of neurotoxic events, including disordered brain metabolism, neuroinflammation, reduced functional connectivity, synaptic and neuronal loss, and / or NFT formation.

[0005] A fundamental relationship between Aβ concentration and neuronal activity has been demonstrated. First, treatment of organoid hippocampal toe sections prepared from transgenic (Tg) mice overexpressing APP with tetrodotoxin reduced neuronal activity and subsequently reduced Aβ levels. Then, the opposite effect—increased neuronal activity—was observed after treatment with Stephania tetrandra toxin. Neuronal activity was also used to demonstrate the dynamic regulation of Aβ peptide concentration and final plaque deposition in vivo. In human AD patients, neuroimaging showed that the most severe plaque deposition likely corresponds to the most continuously active brain regions, termed the “default pattern network.”

[0006] Currently, there is no cure for AD, and treatment options do not suppress the pathological progression of AD, are primarily palliative, and / or may have a variety of concerning side effects. For example, prophylactic and / or therapeutic strategies targeting Aβ peptides and / or their precursors (e.g., Aβ immunotherapy and inhibition of β-secretase and γ-secretase) have been toxic and / or ineffective in reducing AD lesions in clinical trials. Clinical trials involving amyloid-β vaccines (e.g., bapineuzumab) have failed due to a lack of cognitive benefit. γ-secretase inhibitors (e.g., semagacestat) have caused clinical trials to fail because they exacerbated cognitive deficits in subjects. Even existing drugs like acetylcholinesterase inhibitors (e.g., donepezil and levamisole) and N-methyl-D-aspartate (NMDA) receptor antagonists (e.g., memantine) have shown only minor cognitive benefits.

[0007] The key microscopic pathological markers of AD are the presence of amyloid plaques, non-fibrillary tachycardia (NFT), and widespread neuronal loss. This accumulation of neuronal damage occurs over a time period and induces macroscopic circuit dysfunction in the brain, specifically, in the gamma power deficits during memory and attention tasks. These gamma oscillations (e.g., about 20 Hz to about 100 Hz, about 20 Hz to about 80 Hz, or about 20 Hz to about 50 Hz) are...The Hz primarily triggers and modulates flash-valley protein (FS-PV) interneurons.

[0008] In one aspect, this disclosure provides apparatus, 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 associated with AD, vascular dementia, frontotemporal dementia, Lewy body dementia, and / or age-related cognitive decline. The subject may be human or animal.

[0009] In one embodiment, the synchronized gamma oscillations have a frequency of about 20 Hz to about 50 Hz, such as about 40 Hz. The synchronized gamma oscillations can be induced in a cell type-specific manner. For example, the oscillations may correspond to synchronized activation of FS-PV interneurons. The synchronized gamma oscillations can be induced in a brain region-specific manner. For example, the oscillations may correspond to synchronized activation in at least one of the hippocampal region and the sensory cortex region.

[0010] In one embodiment, a method for preventing, alleviating, and / or treating dementia in a subject includes the steps of: controlling a stimulation emitting device to emit a stimulus and exposing the subject to the stimulus and / or applying the stimulus to the subject, thereby inducing in vivo synchronous gamma oscillations in at least one brain region of the subject. The stimulus may have a frequency of about 35 Hz to about 45 Hz, such as about 40 Hz. The stimulation 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 an optical fiber device. The duration of exposing the subject to the stimulus and / or applying the stimulus to the subject may be about one hour. Exposing the subject to the stimulus and / or applying the stimulus to the subject may be repeated within a time period. For example, exposing the subject to the stimulus and / or applying the stimulus to the subject may be repeated at least once per day within the time period. The time period may include, but is not limited to, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, two weeks, three weeks, and / or one month (or longer, such as once daily for the remaining lifespan of the subject).

[0011] In one aspect, a method for reducing the level (e.g., amount or ratio) of Aβ peptides in at least one brain region of a subject includes inducing synchronized gamma oscillations in said at least one brain region of the subject. The Aβ peptides may include one or more isoforms of Aβ peptides (e.g., isoform Aβ1-40, isoform Aβ1-42, and / or isoform Aβ1-43), soluble Aβ peptides, and / or insoluble Aβ peptides.

[0012] In some embodiments, the synchronized gamma oscillations are, for example, achieved by reducing the level (e.g., amount or ratio) of said at least one large brain region of the subject.The synchronous gamma oscillation reduces the production of Aβ peptide in at least one brain region of the subject by decreasing the levels (e.g., amount or ratio) of the C-terminal fragment (CTF) and / or N-terminal fragment (NTF) of APP in the brain region. The synchronous gamma oscillation reduces the cleavage of APP into CTF and NTF by BACE1 and / or gamma-secretase in at least one brain region of the subject. The synchronous gamma oscillation reduces the level (e.g., amount or ratio) of endosomes in at least one brain region of the subject. For example, the endosomes may be positive for early endosome antigen 1 (EEA1) and / or Ras-related proteins encoded by the RAB5A gene (Rab5). In some embodiments, the synchronous gamma oscillation promotes the clearance of Aβ peptide in at least one brain region of the subject. The synchronous gamma oscillation increases the uptake of Aβ peptide by microglia in at least one region of the subject.

[0013] In one aspect, a method for increasing the level (e.g., number or ratio) of microglia, morphological changes of said microglia consistent with a neuroprotective state, and / or the activity of said microglia in at least one brain region of a subject includes inducing synchronous gamma oscillations in said at least one brain region of the subject. The synchronous gamma oscillations may upregulate at least one differentially expressed gene in said at least one brain region of the subject involved in the activity of said microglia, such as Nr4a1, Arc, Npas4, Cd68, B2m, Bsr2, Icam1, Lyz2, Irf7, Spp1, Csf1r, and / or Csf2ra. The morphological changes of said microglia consistent with said neuroprotective state may include an increase in cell body size and / or a decrease in process length.

[0014] In one aspect, a method for reducing the level (e.g., amount or ratio) of Aβ peptide in the hippocampus of a subject includes optogenetically stimulating FS-PV-interneurons in the hippocampus using multiple light pulses, the FS-PV-interneurons expressing optogenetic actuators to modulate the period of in vivo synchronized gamma oscillations, measured by local field potential, in the excited neurons (e.g., FS-PV-interneurons) to reduce the level of Aβ peptide in the hippocampus. The light pulses may have a pulse frequency of about 40 pulses / s. Each light pulse may have a duration of about 1 ms. At least one light pulse may have a wavelength of about 473 nm. The optogenetic actuator may include channel rhodopsin, halophilic rhodopsin, and / or paleorhodopsin. For example, the optogenetic actuator may be channel rhodopsin-2 (ChR2).

[0015] In one aspect, a method for reducing the level (e.g., amount or ratio) of soluble and / or insoluble Aβ peptides in the visual cortex of a subject includes stimulating the subject with multiple light pulses at a pulse frequency of about 40 pulses / s, thereby inducing in vivo synchronous gamma oscillations that reduce the level of said soluble and / or insoluble Aβ peptides in the visual cortex.

[0016] In another aspect, a method for reducing the level (e.g., amount or ratio) of tau phosphorylation in the visual cortex of a subject includes stimulating the subject with multiple light pulses at a pulse frequency of about 40 pulses / s, thereby inducing in vivo synchronous gamma oscillations that reduce tau phosphorylation in the visual cortex.

[0017] In one aspect, a method for reducing the level (e.g., amount or ratio) 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 / s, thereby inducing in vivo synchronous gamma oscillations in at least one of the hippocampus and the auditory cortex to reduce the level of Aβ peptide in at least one of the hippocampus and the auditory cortex.

[0018] In one aspect, a system for preventing, reducing, and / or treating changes in the level (e.g., amount or ratio) of Aβ peptide, neuroinflammation, and / or cognitive function in a subject includes a stimulation firing device for in vivo synchronous activation of brain regions of the subject, at least one memory for storing stimulation parameters and processor-executable instructions, and at least one processor communicatively connected to the stimulation firing device and the at least one memory. After executing the processor-executable instructions, the at least one processor controls the stimulation firing device to fire the stimulus according to the stimulation parameters, the parameters including a frequency at which the brain region is synchronously activated, thereby preventing, reducing and / or treating the subject's Aβ peptide, neuroinflammation and / or dementia. The frequency may be from about 35 Hz to about 45 Hz, such as about 40 Hz. The in vivo synchronous activation may be enzyme-regulated and / or occur in specific cell types, such as in immune-responsive FS-PV-interneurons. The enzyme may include optogenetic activators, microbial opsins, ChR2 and / or the carrier AAV-DIO-ChR2-EYFP.

[0019] In one aspect, a system for preventing, reducing and / or treating changes in the level (e.g., amount or ratio) of Aβ peptide, neuroinflammation and / or cognitive function in a subject includes a light-blocking 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 light-blocking device may include a means for emitting light stimulation to the at least one eye for use by the subject's visual cortex.The light occlusion device may include a light emitting unit that is synchronously activated in vivo in at least one of the auditory cortex and hippocampus. The noise cancellation device may include a speaker unit for emitting sound stimuli to the at least one ear for synchronous activation in vivo in at least one of the auditory cortex and hippocampus of the subject. The system also includes at least one memory for storing processor-executable instructions and at least one processor communicatively connected to the light occlusion device and / or the noise cancellation device and the at least one memory. After executing the processor-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 at least one of the auditory cortex and hippocampus at the frequency. Alternatively or additionally, the at least one processor may control the noise cancellation device such that the speaker unit actuates the sound stimulus that synchronously activates at least one of the auditory cortex and hippocampus at the frequency.

[0020] In one aspect, a method for improving the cognitive function of a subject includes controlling at least one electroacoustic transducer to convert an electro-audio signal into a corresponding sound stimulus. In some embodiments, the auditory stimulus comprises a series of ticks having a tick frequency of approximately 35 ticks / s to approximately 45 ticks / s. The method further includes exposing the subject to the auditory stimulus and / or applying the stimulus to the subject to induce synchronized gamma oscillations in at least one brain region of the subject, the synchronized gamma oscillations causing the improvement in the subject's cognitive function. The cognitive function may include recognition, discrimination, and / or spatial memory.

[0021] In one aspect, a method for preventing, reducing, and / or treating changes in the level (e.g., amount or ratio) of Aβ peptides, neuroinflammation, and / or cognitive function in a subject includes controlling at least one electroacoustic transducer to convert an electroacoustic signal into a corresponding sound stimulus, said sound stimulus comprising a tick series having a tick frequency of about 35 ticks / s to about 45 ticks / s, and exposing said subject to said sound stimulus and / or administering said stimulus to said subject to induce synchronous gamma oscillations in at least one brain region of said subject, said synchronous gamma oscillations causing said prevention, reduction, and / or treatment of said Aβ peptide levels, neuroinflammation, and / or dementia in said subject.

[0022] The Aβ peptide may include one or more isoforms of Aβ peptides (e.g., isoform Aβ1-40, isoform Aβ1-42, and / or isoform Aβ1-43), soluble Aβ peptides, and / or insoluble Aβ peptides. The synchronized gamma oscillations can increase the number of microglia in at least one brain region of the subject and / or enhance the permeability of at least one brain region.The uptake of Aβ peptides via the microglia is used to prevent, reduce, and / or treat the Aβ peptide levels, neuroinflammation, and / or dementia in the subject. The at least one brain region may include the auditory cortex and / or the hippocampus.

[0023] The tick frequency may be approximately 40 ticks / s. Each tick in the tick series may have a duration of approximately 1 ms. Each tick in the tick series may have a frequency of approximately 10 Hz to approximately 100 kHz, approximately 12 Hz to approximately 28 kHz, approximately 20 Hz to approximately 20 kHz, and / or approximately 2 kHz to approximately 5 kHz. Each tick in the tick series may have a sound pressure level of approximately 0 dB to approximately 85 dB, approximately 30 dB to approximately 70 dB, and approximately 60 dB to approximately 65 dB.

[0024] The at least one electroacoustic transducer may include at least one earphone, and in this case, the method may include applying the at least one earphone around, above, and / or in the subject's at least one ear to direct the sound stimulus into the subject's at least one ear. The method may also include using passive noise isolation and / or active noise cancellation to reduce ambient noise.

[0025] In one aspect, a system for preventing, reducing, and / or treating changes in the level (e.g., amount or ratio) of Aβ peptide, neuroinflammation, and / or cognitive function of a subject includes: at least one electroacoustic transducer for converting an electroacoustic signal into a corresponding sound stimulus, the sound stimulus comprising a tick series having a tick frequency of about 35 ticks / s to about 45 ticks / s; at least one memory device for storing the electroacoustic signal and processor-executable instructions; and at least one processor communicatively connected to the at least one electroacoustic transducer and the at least one memory device. After executing the processor-executable instructions, the at least one processor controls the electroacoustic transducer to output the sound stimulus to at least one ear of the subject to induce synchronized gamma oscillations in at least one brain region of the subject, the synchronized gamma oscillations causing the subject's Aβ peptide levels, the prevention, reduction and / or treatment of neuroinflammation and / or dementia. Specification 4 / 78 pages 6 CN 122399187 A

[0026] The system may be fixed or portable. If the at least one electroacoustic transducer includes at least one earphone for the subject to wear around, above and / or in, the at least one ear to direct the sound stimulus to the subject's at least one ear and reduce ambient noise, the system may also include an earphone jack for transmitting the electro-audio signal to the at least one earphone. Alternatively or additionally, the system may include the sound...A neuroimaging scanner that monitors function in at least one brain region of the subject before, during, and / or after the output of the sound stimulation.

[0027] In one aspect, a method for preventing, alleviating, and / or treating dementia in a subject includes providing means for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0028] In one aspect, a method for maintaining and / or reducing blood levels (e.g., amounts) of glucocorticoids involved in a subject's stress response includes providing means for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0029] In one aspect, a method for preventing and / or reducing anxiety in a subject includes providing means for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0030] In one aspect, a method for maintaining and / or enhancing memory associations includes providing means for inducing synchronized gamma oscillations in at least one brain region of the subject. The memory associations may be based on spatial memory.

[0031] In one aspect, a method for maintaining and / or enhancing cognitive flexibility includes providing means for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0032] In one aspect, a method for maintaining and / or reducing anatomical and / or morphological changes in at least one brain region of a subject includes providing means for inducing synchronized gamma oscillations in said at least one brain region of the subject. The anatomy and / or morphology may include brain weight, lateral ventricle size, cortical thickness, neuronal layer thickness, and / or vascular diameter. The at least one brain region may include the subject's visual cortex, somatosensory cortex, and / or insular cortex.

[0033] In one aspect, a method for maintaining and / or reducing changes in the number of neurons, the quality of DNA in said neurons, and / or synaptic puncta density in at least one brain region of a subject includes providing means for inducing synchronized gamma oscillations in said at least one brain region of the subject. The at least one brain region may include the subject's visual cortex, somatosensory cortex, insular cortex, and / or hippocampus.

[0034] In one aspect, a device for inducing synchronized gamma oscillations in at least one brain region of a subject can prevent, alleviate, and / or treat dementia and / or anxiety in the subject, maintain and / or enhance the subject's memory associations and / or cognitive flexibility, and / or maintain and / or reduce anatomical, morphological, cellular, and molecular changes in the at least one brain region of the subject.

[0035] It should be recognized that all combinations of the foregoing concepts and other concepts discussed in more detail below (provided that such combinations are applicable)(The concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein. It should be recognized that terms explicitly adopted herein that may also appear in any disclosure incorporated by reference should be given the meaning most consistent with the specific concepts disclosed herein.

[0036] Other systems, methods, and features will become apparent to those skilled in the art upon review of the following figures and detailed description. All such additional systems, methods, and features are intended to be included within this specification, within the scope of the invention, and will be protected by the appended claims.

[0037] The invention comprises: Specification 5 / 78 pages 7 CN 122399187 A

[0038] 1. A method for preventing, alleviating, and treating at least one of dementia in a subject, comprising inducing synchronous gamma oscillations in at least one brain region of the subject.

[0039] 2. The method of claim 1, wherein the synchronous gamma oscillations have a frequency of about 20 Hz to about 50 Hz.

[0040] 3. The method of claim 2, wherein the synchronous gamma oscillation has a frequency of about 40 Hz.

[0041] 4. The method of any one of claims 1-3, wherein the synchronous gamma oscillation is induced in a cell type-specific manner.

[0042] 5. The method of claim 4, wherein the synchronous gamma oscillation corresponds to synchronous activation of flash-valley protein (FS-PV)-interneurons.

[0043] 6. The method of any one of claims 1-5, wherein the synchronous gamma oscillation is induced in a brain region-specific manner.

[0044] 7. The method of claim 6, wherein the synchronous gamma oscillation corresponds to synchronous activation in at least one of the hippocampus and the sensory cortex.

[0045] 8. The method of any one of claims 1-7, wherein the dementia is associated with at least one of Alzheimer's disease, vascular dementia, frontotemporal dementia, Lewy body dementia, and age-related cognitive decline.

[0046] 9. The method of any one of claims 1-8, wherein the subject is human.

[0047] 10. A method for preventing, alleviating, and treating at least one of dementia in a subject, comprising providing a stimulation emission device configured to emit a stimulus having at least one predetermined frequency for inducing in vivo synchronous gamma oscillations in at least one brain region of the subject.

[0048] 11. The method of claim 10, wherein the at least one predetermined frequency of the stimulus is about 35 Hz to about 45 Hz.

[0049] 12. The method of claim 11, wherein the at least one predetermined frequency of the stimulus is about 40 Hz.

[0050] 13. The method of any one of claims 10-12, wherein the stimulation emitting device is at least one of a tactile device, a light emitting device, and a sound emitting device.

[0051] 14. The method of claim 13, wherein the light emitting device is an optical fiber device.

[0052] 15. The method of any one of claims 10-14, wherein the duration of applying the stimulation to the subject is about one hour.

[0053] 16. The method of any one of claims 10-15, wherein applying the stimulation to the subject is repeated over a period of time.

[0054] 17. The method of claim 16, wherein applying the stimulation to the subject is repeated at least once per day over the period of time, the period of time being at least one of 2 days, 3 days, 4 days, 5 days, 6 days, one week, two weeks, three weeks, and one month.

[0055] 18. A method for maintaining and reducing at least one of the amounts of amyloid-β (Aβ) peptide in at least one brain region of a subject, comprising inducing synchronous gamma oscillations in the at least one brain region of the subject.

[0056] 19. The method of claim 18, wherein the Aβ peptide comprises at least one of isotype Aβ1-40 peptide and isotype Aβ1-42 peptide.

[0057] 20. The method of any one of claims 18 and 19, wherein the Aβ peptide comprises at least one of soluble Aβ peptide and insoluble Aβ peptide.

[0058] 21. The method of any one of claims 18-20, wherein the synchronous gamma oscillation reduces the production of Aβ peptide in at least one brain region of the subject.

[0059] 22. The method of claim 21, wherein the synchronous gamma oscillation reduces the amount of at least one of the C-terminal fragment (CTF) and N-terminal fragment (NTF) of amyloid precursor protein (APP) in at least one brain region of the subject.

[0060] 23. The method of claim 22, wherein the synchronous gamma oscillation reduces APP cleavage into CTF and NTF by at least one of β-secretase (BACE1) and gamma-secretase in at least one brain region of the subject.

[0061] 24. The method of claim 21, wherein the synchronous gamma oscillation reduces the number of endosomes in at least one brain region of the subject.

[0062] 25. The method of claim 24, wherein the endosomes are positive for at least one of early endosome antigen 1 (EEA1) and Ras-associated protein encoded by the RAB5A gene (Rab5).

[0063] 26. The method of any one of claims 18-25, wherein the synchronous gamma oscillation reduces the number of endosomes in at least one brain region of the subject.Oscillations promote the clearance of Aβ peptides in at least one brain region of the subject.

[0064] 27. The method of claim 26, wherein the synchronous gamma oscillations increase the uptake of Aβ peptides by microglia in at least one brain region of the subject.

[0065] 28. A method for at least one of increasing the number of microglia in at least one brain region of a subject, inducing morphological changes of the microglia consistent with a neuroprotective state, and promoting the activity of the microglia, comprising inducing synchronous gamma oscillations in at least one brain region of the subject.

[0066] 29. The method of claim 28, wherein the synchronous gamma oscillations upregulate at least one differentially expressed gene involved in the activity of microglia in at least one brain region of the subject.

[0067] 30. The method of claim 29, wherein the at least one differentially expressed gene includes at least one of Nr4a1, Arc, Npas4, Cd68, B2m, Bsr2, Icam1, Lyz2, Irf7, Spp1, Csf1r, and Csf2ra.

[0068] 31. The method of claim 28, wherein the morphological changes of the microglia consistent with the neuroprotective state include at least one of an increase in cell body size and a decrease in process length.

[0069] 32. A method for reducing the amount of amyloid-β (Aβ) peptide in the hippocampus of a subject, the method comprising optogenetically stimulating fast flash-valley protein (FS-PV) interneurons in the hippocampus using multiple light pulses, the FS-PV interneurons expressing optogenetic actuators thereby inducing in vivo synchronous γ oscillations in the FS-PV interneurons that reduce the amount of Aβ peptide in the hippocampus.

[0070] 33. The method of claim 32, wherein the plurality of light pulses have a pulse frequency of about 40 pulses / s.

[0071] 34. The method of claim 33, wherein each of the plurality of light pulses has a duration of about 1 ms.

[0072] 35. The method of any one of claims 32-34, wherein each of the plurality of light pulses has a wavelength of about 473 nm.

[0073] 36. The method of any one of claims 32-35, wherein the optogenetic actuator is at least one of channel rhodopsin, halophilic rhodopsin, and paleoporosis.

[0074] 37. The method of claim 36, wherein the optogenetic actuator is channel rhodopsin-2 (ChR2).

[0075] 38. A method for reducing soluble and insoluble amyloid-β (Aβ) peptides in the visual cortex of a subject.A method for reducing tau phosphorylation in the visual cortex of a subject, the method comprising stimulating the subject with a plurality of light pulses at a pulse frequency of about 40 pulses / s, thereby inducing in vivo synchronous gamma oscillations in the visual cortex that reduce the amount of at least one of the soluble Aβ peptides and insoluble Aβ peptides in the visual cortex.

[0076] 39. A method for reducing tau phosphorylation in the visual cortex of a subject, the method comprising stimulating the subject with a plurality of light pulses at a pulse frequency of about 40 pulses / s, thereby inducing in vivo synchronous gamma oscillations that reduce tau phosphorylation in the visual cortex.

[0077] 40. A method for reducing the amount of amyloid-β (Aβ) peptide in at least one of the hippocampus and auditory cortex of a subject, the method comprising stimulating the subject with a plurality of sound pulses at a pulse frequency of about 40 pulses / s, thereby inducing in vivo synchronous gamma oscillations in the at least one of the hippocampus and auditory cortex to reduce the amount of Aβ peptide in the at least one of the hippocampus and auditory cortex.

[0078] 41. A system for preventing, reducing, and treating at least one of changes in amyloid-β (Aβ) peptide, neuroinflammation, and cognitive function in a subject, the system comprising:

[0079] a stimulation firing device for in vivo synchronous activation of a brain region of the subject;

[0080] at least one memory for storing stimulation parameters and processor-executable instructions; and

[0081] at least one processor communicatively connected to the stimulation firing device and the at least one memory, wherein, after executing the processor-executable instructions, the at least one processor controls the stimulation firing device to fire the stimulation according to the stimulation parameters, the parameters including a frequency at which the brain region is synchronously activated,

[0082] thereby preventing, reducing, and treating at least one of the changes in Aβ peptide, neuroinflammation, and cognitive function in the subject.

[0083] 42. The system of claim 41, wherein the frequency is from about 35 Hz to about 45 Hz.

[0084] 43. The system of claim 42, wherein the frequency is about 40 Hz.

[0085] 44. The system of any one of claims 41-43, wherein the in vivo synchronous activation occurs in a specific cell type and is regulated by an enzyme.

[0086] 45. The system of claim 44, wherein the specific cell type is a flash-small albumin (FS-PV) immunoreactive interneuron.

[0087] 46.The system of any one of items 44 and 45, wherein the enzyme is at least one of optogenetic activator, microbial opsin, channel rhodopsin-2 (ChR2), and carrier AAV-DIO-ChR2-EYFP.

[0088] 47. A system for preventing, reducing, and treating changes in at least one of amyloid-β (Aβ) peptide, neuroinflammation, and cognitive function in a subject, the system comprising:

[0089] at least one of the following:

[0090] a light-blocking device for reducing ambient light to at least one eye of the subject, the light-blocking device comprising 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; and

[0091] a noise cancellation device for reducing ambient noise to at least one ear of the subject, the noise cancellation device comprising a loudspeaker 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;

[0092] at least one memory for storing processor-executable instructions; and

[0093] At least one processor communicatively connected to the light occlusion device, the noise cancellation device, and the at least one memory, wherein, upon execution of processor-executable instructions, the at least one processor controls at least one of the following:

[0094] The light occlusion device, causing the light emitting unit to emit light stimulation at a frequency that synchronously activates at least one of the visual cortex and the hippocampus at the same frequency; and

[0095] The noise cancellation device, causing the speaker unit to actuate sound stimulation at the frequency that synchronously activates at least one of the auditory cortex and the hippocampus at the same frequency,

[0096] thereby preventing, reducing, and treating at least one of the changes in at least one of the Aβ peptide, the neuroinflammation, and the cognitive function of the subject.

[0097] 48. A method for improving the cognitive function of a subject, the method comprising:

[0098] controlling at least one electroacoustic transducer to convert an electro-audio signal into a corresponding sound stimulus, the sound stimulus comprising a tick series having a tick frequency of about 35 ticks / s to about 45 ticks / s; and

[0099] applying the sound stimulus to the subject to induce synchronized gamma oscillations in at least one brain region of the subject, the synchronized gamma oscillations causing the improvement in the cognitive function of the subject.

[0100] 49. The method of claim 48, wherein the cognitive function comprises at least one of recognition, discrimination, and spatial memory.

[0101] 50. A method for preventing, reducing, and treating at least one of changes in amyloid-β (Aβ) peptide, neuroinflammation, and cognitive function in a subject, the method comprising:

[0102] controlling at least one electroacoustic transducer to convert an electroacoustic signal into a corresponding sound stimulus, the sound stimulus comprising a tick series having a tick frequency of about 35 ticks / s to about 45 ticks / s; and

[0103] administering the sound stimulus to the subject to induce synchronous gamma oscillations in at least one brain region of the subject, the synchronous gamma oscillations causing at least one of the prevention, reduction, and treatment of the changes in at least one of the Aβ peptide, the neuroinflammation, and the cognitive function in the subject.

[0104] 51. The method of claim 50, wherein the Aβ peptide comprises at least one of isotype Aβ1-40 peptide and isotype Aβ1-42 peptide.

[0105] 52. The method of any one of claims 50 and 51, wherein the Aβ peptide comprises at least one of soluble Aβ peptide and insoluble Aβ peptide.

[0106] 53. The method of claim 50, wherein the synchronous gamma oscillation prevents, reduces, and treats at least one of the changes in Aβ peptide, neuroinflammation, and cognitive function of the subject by increasing the number of microglia in the at least one brain region of the subject and enhancing the uptake of Aβ peptide by the microglia in the at least one brain region.

[0107] 54. The method of any one of claims 50-53, wherein the at least one brain region comprises at least one of the auditory cortex and the hippocampus.

[0108] 55. The method of any one of claims 50-54, wherein the tick frequency is about 40 ticks / s.

[0109] 56. The method of claim 55, wherein each tick in the tick series has a duration of about 1 ms.

[0110] 57. The method of any one of claims 50-56, wherein each tick in the ticking series has a frequency of about 10 Hz to about 100 kHz.

[0111] 58. The method of claim 57, wherein each tick in the ticking series has a frequency of about 12 Hz to about 28 kHz.

[0112] 59. The method of claim 58, wherein each tick in the ticking series has a frequency of about 20 Hz to about 20 kHz. Specification 9 / 78 pages 11 CN 122399187 A

[0113] 60. The method of claim 59, wherein each tick in the ticking series has a frequency of about 2 kHz to about 5 kHz.

[0114] 61. The method of any one of claims 50-60, wherein each tick in the tick series has a sound pressure level of about 0 dB to about 85 dB.

[0115] 62. The method of claim 61, wherein each tick in the tick series has a sound pressure level of about 30 dB to about 70 dB.

[0116] 63. The method of claim 62, wherein each tick in the tick series has a sound pressure level of about 60 dB to about 65 dB.

[0117] 64. The method of any one of claims 50-63, wherein the at least one electroacoustic transducer includes at least one earphone, and the method further includes applying the at least one earphone to at least one of the at least one ear of the subject around, above, and in, to direct the sound stimulus to the at least one ear of the subject.

[0118] 65. The method of any one of claims 50-64, further comprising using at least one of passive noise isolation and active noise cancellation to reduce ambient noise.

[0119] 66. A system for preventing, reducing, and treating at least one of changes in amyloid-β (Aβ) peptide, neuroinflammation, and cognitive function in a subject, the system comprising:

[0120] at least one electroacoustic transducer for converting an electroacoustic signal into a corresponding sound stimulus, the sound stimulus comprising a tick series having a tick frequency of about 35 ticks / s to about 45 ticks / s;

[0121] at least one memory device for storing the electroacoustic signal and processor-executable instructions; and

[0122] at least one processor communicatively connected to the at least one electroacoustic transducer and the at least one memory device, wherein, after executing the processor-executable instructions, the at least one processor controls the electroacoustic transducer to output the sound stimulus to at least one ear of the subject to induce synchronous gamma oscillations in at least one brain region of the subject, the synchronous gamma oscillations causing at least one of the prevention, reduction, and treatment of the changes in at least one of the Aβ peptide, the neuroinflammation, and the cognitive function in the subject.

[0123] 67. The system of claim 66, wherein:

[0124] the system is portable;

[0125] the at least one electroacoustic transducer includes at least one earphone for the subject to wear around, above, and in, at least one earphone to direct the sound stimulus to the subject's at least one ear and reduce ambient noise; and

[0126] the system further includes an earphone jack for transmitting the electro-audio signal to the at least one earphone.

[0127] 68. The system of claim 66, further comprising, before, during, and after, the output of the sound stimulus.

[0128] 69. A method for preventing, alleviating and treating at least one of dementia in a subject, comprising providing means for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0129] 70. A method for maintaining and reducing blood levels of glucocorticoids involved in a subject's stress response, comprising providing means for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0130] 71. A method for preventing and reducing at least one of anxiety in a subject, comprising providing means for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0131] 72. A method for maintaining and enhancing memory associations, comprising providing means for inducing synchronized gamma oscillations in at least one brain region of the subject. Specification 10 / 78 pages 12 CN 122399187 A

[0132] 73. The method of claim 72, wherein the memory association is based on spatial memory.

[0133] 74. A method for maintaining and enhancing at least one of the following: cognitive flexibility, comprising providing means for inducing synchronized gamma oscillations in at least one brain region of the subject.

[0134] 75. A method for maintaining and reducing at least one of the following: anatomical and morphological changes in at least one of the following: a subject's brain region, comprising providing means for inducing synchronized gamma oscillations in said at least one brain region of the subject.

[0135] 76. The method of claim 75, wherein said at least one of the following: anatomical and morphological changes, comprises at least one of brain weight, lateral ventricle size, cortical thickness, neuronal layer thickness, and vascular diameter.

[0136] 77. The method of claim 75, wherein said at least one brain region comprises at least one of the subject's visual cortex, somatosensory cortex, and insular cortex.

[0137] 78. A method for maintaining and reducing at least one of the following: changes in the number of neurons, the quality of deoxyribonucleic acid (DNA) in said neurons, and the density of synaptic plaques in at least one brain region of a subject, comprising providing means for inducing synchronized gamma oscillations in said at least one brain region of the subject.

[0138] 79. The method of claim 78, wherein said at least one brain region comprises at least one of the visual cortex, somatosensory cortex, insular cortex, and hippocampus of the subject.

[0139] 80. An apparatus for inducing synchronized gamma oscillations in at least one brain region of a subject to result in at least one of the following:

[0140] Preventing, alleviating, and / or treating dementia in the subject;

[0141] Preventing and / or reducing anxiety in the subject;

[0142] Maintaining and / or reducing blood levels of glucocorticoids involved in the subject's stress response;

[0143] Maintaining and / or enhancing memory associations in the subject;

[0144] Maintaining and / or enhancing cognitive flexibility in the subject;

[0145] Maintaining and / or reducing at least one of the anatomical and morphological changes in at least one of the at least one brain region of the subject; and

[0146] Maintaining and / or reducing at least one of the changes in the number of neurons, the quality of deoxyribonucleic acid (DNA) in the neurons, and the density of synaptic plaques in the at least one brain region of the subject.

[0147] 81. An apparatus comprising:

[0148] a stimulation transmitter configured to generate, according to a stimulation strategy, a stimulus inducing synchronized gamma oscillations in at least one brain region of a subject, so as to result in at least one of the following:

[0149] preventing, alleviating, and / or treating dementia in the subject;

[0150] preventing and / or reducing anxiety in the subject;

[0151] maintaining and / or reducing blood levels of glucocorticoids involved in the stress response of the subject;

[0152] maintaining and / or enhancing memory associations in the subject;

[0153] maintaining and / or enhancing cognitive flexibility in the subject;

[0154] maintaining and / or reducing at least one of anatomical and morphological changes in the at least one brain region of the subject; and

[0155] maintaining and / or reducing at least one of the number of neurons, the quality of deoxyribonucleic acid (DNA) in the neurons, and the density of synaptic plaques in the at least one brain region of the subject. Specification 11 / 78 pages 13 CN 122399187 A Description of Drawings

[0156] Those skilled in the art will understand 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 drawn to scale; in some cases, various aspects of the inventive subject matter disclosed herein may be exaggerated or enlarged in the drawings to facilitate understanding of different features. In the drawings, similar reference numerals generally refer to, for example, similar features (e.g., functionally similar and / or structurally similar elements).

[0157] FIG1 is a schematic diagram showing a mouse running through a virtual linear maze on a spherical treadmill according to some embodiments.

[0158] FIG2A and FIG2B are traces recorded from the CA1 of the hippocampus and showing the theta oscillations and sharp wave ripples (SWR) according to some embodiments.

[0159] FIG3A and FIG3B are diagrams showing the traces of three-month-old Tg 5XFAD mice and wild-type (WT) mice according to some embodiments.The graphs showing the mean and standard deviation of the normalized power spectrum and normalized power spectral density in mice during the θ time period.

[0160] Figures 4A and 4B are spectrograms showing the SWR of WT mice and 5XFAD mice according to some embodiments.

[0161] Figures 5A-5C are graphs depicting the distribution of instantaneous γ frequencies during the SWR process according to some embodiments.

[0162] Figure 6A is a series of graphs depicting the Z-score γ power as a function of time since the peak of SWR in 5XFAD mice and WT mice according to some embodiments. Figure 6B is a graph depicting the cumulative distribution of γ power during the SWR process in 5XFAD mice and WT mice according to some embodiments. Figures 6C and 6D are graphs depicting the cumulative distribution of Z-score γ power over a 100 ms period around the peak of SWR in WT mice and 5XFAD mice according to some embodiments. Figure 6E is a graph depicting the cumulative distribution of γ power during a large SWR process in 5XFAD mice and WT mice according to some embodiments.

[0163] Figure 7A is a graph depicting the peak fraction as a function of the phase of γ oscillation, and Figure 7B is a graph depicting the depth of peak modulation during the SWR process according to some embodiments. Figures 7C and 7D are graphs showing the peak fraction as a function of the phase of γ oscillation in the hippocampal CA1 during the SWR process according to some embodiments. Figure 7E is a graph depicting the peak fraction as a function of the phase of γ oscillation, and Figure 7F is a graph depicting the depth of peak modulation during a large SWR process according to some embodiments.

[0164] Figures 8A and 8B are graphs depicting the SWR rate / non-θ time period for each animal and all animals in combinations in 5XFAD and WT animals according to some embodiments.

[0165] Figure 9 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.

[0166] Figures 10A and 10B are schematic diagrams illustrating signal delivery to the CA1 region of the hippocampus of a subject according to some embodiments.

[0167] Figure 11 is an immunofluorescence image illustrating immunostaining of neural tissue of a subject using ChR2 and DAPI according to some embodiments.

[0168] Figure 12A is an immunofluorescence image illustrating ChR2-EYFP expressed in PV+ interneurons according to some embodiments. Figure 12B is a series of immunofluorescence images illustrating immunohistochemistry using anti-EYFP and anti-PV antibodies according to some embodiments.

[0169] Figures 13A and 13B include schematic diagrams of studies according to some embodiments, traces of local field potentials, and power spectral density of FS-PV-interneurons.

[0170] Figures 14A and 14B include the original traces according to some embodiments, the traces filtered for spikes after optogenetic stimulation, and the probability of spikes after a 1 ms laser pulse initiation. Specification 12 / 78 pages 14 CN 122399187 A

[0171] Figure 15A is a bar chart showing the difference in firing rate between 40-Hz stimulation periods and random stimulation periods according to some embodiments. Figure 15B is a bar chart showing the multi-unit firing rate for each animal for each 40-Hz stimulation period, random stimulation period, and no stimulation period according to some embodiments.

[0172] Figure 16A is a trace recorded from the hippocampus of a subject during a frequency-specific increase in stimulation of a specific cell type in the CA1 region of the hippocampus according to some embodiments. Figure 16B is a graph showing the power spectral density of the frequency-specific increase in local field potential power during stimulation of a specific cell type in the CA1 region of the hippocampus of a subject according to some embodiments.

[0173] Figures 17A and 17B are bar graphs depicting the relative Aβ1-40 and Aβ1-42 levels of 5XFAD / PV-Cre CA1 obtained by one-way ANOVA according to some embodiments.

[0174] Figures 18A and 18B are bar graphs depicting the relative Aβ1-40 and Aβ1-42 levels of 5XFAD / αCamKII-Cre CA1 obtained by one-way ANOVA according to some embodiments.

[0175] Figure 19A is a series of images showing the immunohistochemistry of anti-Aβ antibody and anti-EEA1 antibody in the hippocampal CA1 region according to some embodiments. Figure 19B is a series of bar graphs depicting the relative immunoreactivity of Aβ normalized to EYFP according to some embodiments.

[0176] Figure 20A is a series of immunofluorescence images showing the immunohistochemistry of anti-Aβ antibody in the hippocampal CA1 region of 5XFAD / PV-Cre according to some embodiments. Figure 20B is a bar chart depicting the relative immunoreactivity of Aβ normalized to EYFP according to some embodiments.

[0177] Figure 21A is a representative protein blot depicting the levels of APP (CT695), APP NTF (A8967), APP CTF (CT695), and β-actin (A5316) (load control) in CA1 according to some embodiments. Figure 21B is a bar chart depicting the 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 bar chart depicting the full-length APP 2106 (CT695), APP CTF 2108 (CT695), and β-actin 2112 in CA1 according to some embodiments.A series of Western blots at the level of (A5316, load control).

[0178] Figure 22A is a bar chart depicting the relative (normalized to actin) immunoreactivity of APP NTF at 40 Hz relative to EYFP and random conditions according to some embodiments. Figure 22B is a bar chart depicting the relative (normalized to actin) immunoreactivity of full-length APP at EYFP, random, and 40 Hz conditions according to some embodiments.

[0179] Figure 23 is a series of immunofluorescence images showing the immunohistochemistry of anti-Rab5 (ADI-KAp-GP006-E) antibody according to some embodiments.

[0180] Figure 24A is a bar chart representing the relative immunoreactivity of EEA1 normalized to EYFP, and Figure 24B is a bar chart depicting the relative Rab5 intensity level of CA1 from 5XFAD / PV-Cre under EYFP, 40 Hz, and random stimulation conditions according to some embodiments.

[0181] Figure 25A is a bar graph depicting the levels of Aβ peptide isoform Aβ1-40 after different types of stimulation of the CA1 region of the hippocampus of a subject according to some embodiments. Figure 25B is a bar graph depicting the decrease of Aβ peptide isoform Aβ1-42 after stimulation of a specific cell type in the CA1 region of the hippocampus of a subject using γ-oscillations according to some embodiments. Figure 25C is a series of images showing the decrease in CTF (e.g., β-CTF) levels and the increase in full-length APP (normalized to actin) levels after stimulation of a specific cell type in the CA1 region of the hippocampus of a subject using γ-oscillations according to some embodiments.

[0182] Figures 26A-26B are immunofluorescence images showing the endosome levels (based on EEA1 levels) after different types of stimulation of the CA1 region of the hippocampus of a subject according to some embodiments.

[0183] Figure 27 is a bar chart depicting the average intensity values ​​(normalized to FAD) of the immunofluorescence images of Figures 6A-6B after different types of stimulation of the CA1 region of the hippocampus of a subject according to some embodiments (page 13 / 78 of the specification, CN 122399187 A).

[0184] Figure 28 is a heatmap showing differentially expressed genes identified by whole transcriptome ribonucleic acid sequencing (RNA-seq) of the CA1 region of the mouse hippocampus with and without 40-Hz stimulation according to some embodiments.

[0185] Figure 29 is a box plot showing the FPKM values ​​of upregulated and downregulated genes in EYFP and 40-Hz conditions according to some embodiments.

[0186] Figure 30 is a pie chart showing the cell type-specific expression patterns of upregulated genes identified after 40-Hz stimulation according to some embodiments.

[0187] Figure 31 is a bar chart showing RT-qPCR validation of a specific gene target in an RNA-seq dataset according to some embodiments.

[0188] Figures 32A and 32B are graphs showing the power spectral density of the local field potential recorded above the brain during a 40-Hz light flickering performance according to some embodiments.

[0189] Figure 33 is a bar chart depicting RT-qPCR validation of a specific gene target in an RNA-seq dataset according to some embodiments.

[0190] Figure 34 is a series of immunofluorescence images showing immunohistochemistry of the CA1 region of the hippocampus in 5XFAD / PV-Cre mice using anti-Iba1 (019-19741) antibody and anti-Aβ (12F4) antibody under EYFP, 40-Hz and random stimulation conditions according to some embodiments.

[0191] Figure 35A is a bar chart depicting the number of microglia under EYFP and 40-Hz conditions according to some embodiments. Figure 35B is a bar chart depicting the diameter of microglial cell bodies normalized to EYFP under EYFP, 40-Hz, and random stimulation conditions according to some embodiments. Figure 35C is a bar chart depicting the average length of microglial primary processes normalized to EYFP under EYFP, 40-Hz, and random stimulation conditions according to some embodiments. Figure 35D is a bar chart depicting the percentage of Iba1-positive (microglial) cell bodies that are also Aβ-positive under EYFP and 40-Hz stimulation conditions according to some embodiments.

[0192] Figure 36 is a series of 3D renderings formed by combining immunofluorescence images from Figure 34 according to some embodiments.

[0193] Figure 37A is a series of immunofluorescence images showing the immunohistochemistry of the CA1 region of the hippocampus in 5XFAD / PV-Cre using Hoechst according to some embodiments. Figure 37B is a bar chart depicting the estimated CA1 thickness of 5XFAD / PV-Cre under EYFP and 40-Hz stimulation conditions according to some embodiments.

[0194] Figure 38A is a heatmap showing differentially expressed genes (DEGs) identified by genome-wide RNA-seq of hippocampal CA1 after 40-Hz FS-PV+ stimulation or control stimulation according to some embodiments. Figure 38B is a graph showing the overlap between upregulated DEGs under TREAT conditions in Figure 38A according to some embodiments.

[0195] Figure 39 is a bar chart depicting RT-qPCR validation of specific gene targets in the RNA-seq dataset of Figure 38A according to some embodiments.

[0196] Figure 40 is a graph showing the upregulated genes of Figure 38A and their associated biological processes according to some embodiments.

[0197] Figure 41 is a graph showing the downregulated genes of Figure 38A and their associated biological processes according to some embodiments.

[0198] Figure 42A is a series of immunofluorescence images showing the level of Iba1 in the CA1 region of the hippocampus of a subject after different types of stimulation according to some embodiments. Figure 42B is a bar chart depicting the average intensity values ​​of the immunofluorescence images of Figure 42A according to some embodiments.

[0199] Figure 43A is a schematic diagram showing mice exposed to light flickering stimulation according to some embodiments. Figure 43B includes curves of local field potential traces and power spectral density in the visual cortex before and during 40-Hz light flickering according to some embodiments. Figures 43C-43F are curves depicting the power spectral density of local field potential in the visual cortex according to some embodiments.

[0200] Figure 44A is a series of histograms depicting the fraction of spikes in the visual cortex as a function of time for four cycles and equivalent time periods of random light flickering for 40-Hz light flickering according to some embodiments. Figure 44B is a series of traces of local field potential recorded above the brain during light flickering according to some embodiments.

[0201] Figure 45A is a bar chart showing the difference in firing rate between 40-Hz light flickering and random light flickering according to some embodiments. Figure 45B is a graph showing the multi-unit firing rate in the visual cortex according to some embodiments.

[0202] Figure 46A is a schematic diagram showing an experimental example according to some embodiments. Figures 46B-46C are graphs further showing the changes in baseline levels of Aβ peptide isoforms Aβ1-40 and Aβ1-42 after the experimental example of Figure 46A according to some embodiments.

[0203] Figures 47A and 47B are bar charts depicting the changes in baseline levels of Aβ1-40 and Aβ1-42 in the 5XFAD visual cortex according to some embodiments.

[0204] Figure 48A is a bar graph depicting the changes in baseline levels of Aβ1-40 and Aβ1-42 in the 5XFAD barrel cortex under dark and 40-Hz scintillation conditions according to some embodiments. Figure 48B is a bar graph depicting the changes in baseline levels of Aβ1-40 and Aβ1-42 in the APP / PS1 visual cortex under dark and 40-Hz scintillation conditions according to some embodiments. Figure 48C is a bar graph depicting the changes in baseline levels of Aβ1-40 and Aβ1-42 in the WT visual cortex under dark and 40-Hz scintillation conditions according to some embodiments.

[0205] Figure 49 is a series of immunofluorescence images showing the immunohistochemistry of anti-Iba1 (019-19741) antibody and anti-Aβ (12F4) antibody in the 5XFAD visual cortex under dark and 40-Hz scintillation conditions according to some embodiments.

[0206] Figure 50A is a bar chart depicting the number of Iba1-positive cells (microglia) according to some embodiments. Figure 50B is a bar chart depicting the diameter of microglia bodies normalized to the control under dark and 40-Hz scintillation conditions according to some embodiments. Figure 50C is a bar chart depicting the average length of primary processes of microglia normalized to the control under dark and 40-Hz scintillation conditions according to some embodiments. Figure 50D is a bar chart depicting the percentage of microglia that are also Aβ-positive under dark and 40-Hz scintillation conditions according to some embodiments.

[0207] Figure 51 is a series of 3D renderings (from immunofluorescence images) of Iba+ microglia from 100 μm tissue sections treated with CLARITY according to some embodiments under dark and 40-Hz scintillation conditions. CLARITY is a method of making brain tissue transparent using, for example, an acrylamide-based hydrogel constructed from within the tissue and attached to the tissue.

[0208] Figure 52A is a flowchart illustrating 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 depicting the Aβ1-40 levels in microglia isolated from the visual cortex of three-month-old 5XFAD animals and WT control animals using the method of Figure 52A according to some embodiments.

[0209] Figure 53A is a series of immunofluorescence images showing immunohistochemistry of an antibody detecting SVP38, a synaptic vesicle protein, in the visual cortex of three-month-old 5XFAD animals under dark and 40-Hz flicker conditions according to some embodiments. Figure 53B is a bar graph depicting the relative SVP38 intensity levels in the 5XFAD visual cortex after dark and 40-Hz light flicker conditions according to some embodiments.

[0210] Figure 54A is a bar graph showing the decrease in Aβ peptide isotype Aβ1-42 after stimulating the visual cortex of subjects with γ-oscillations according to some embodiments. Figure 54B is a bar graph showing the levels of Aβ peptide isoform Aβ1-42 after γ-oscillation stimulation of the visual cortex of a subject according to some embodiments and again 24 hours after stimulation.

[0211] Figure 55A includes traces of local field potential and power spectral density in the hippocampus before and during 40-Hz light flicker according to some embodiments. Figure 55B is a series of histograms showing the fraction of spikes in the hippocampus as a function of time for four cycles and equivalent time periods of random light flicker according to some embodiments.

[0212] Figure 56A shows the firing rate between 40-Hz light flicker and random light flicker according to some embodiments.A bar chart showing the differences. Figure 56B is a graph showing the multi-unit firing rate in CA1 during 40-Hz light flicker according to some embodiments.

[0213] Figure 57A is a bar chart depicting the relative Aβ1-40 levels in the 5XFAD visual cortex according to some embodiments. Figure 57B is a bar chart depicting the relative Aβ1-42 levels in the 5XFAD visual cortex according to some embodiments.

[0214] Figure 58A is a bar chart depicting the relative Aβ1-40 levels in the 5XFAD visual cortex with recovery after 40-Hz light flicker conditions according to some embodiments. Figure 58B is a bar chart depicting the relative Aβ1-42 levels in the 5XFAD visual cortex with recovery after 40-Hz light flicker according to some embodiments.

[0215] Figure 59A is a schematic diagram showing a study according to some embodiments. Figure 59B is a bar graph depicting the relative Aβ1-42 levels in the visual cortex of six-month-old 5XFAD mice after seven days of exposure to darkness or 40-Hz flicker conditions for one hour / day according to some embodiments. Figure 59C is a bar graph showing the relative Aβ1-40 levels in the visual cortex of six-month-old 5XFAD mice after seven days of exposure to darkness or 40-Hz flicker conditions for one hour / day according to some embodiments.

[0216] Figure 60A is a series of immunofluorescence images of immunohistochemistry using Aβ antibodies in the visual cortex of six-month-old 5XFAD mice after seven days of exposure to darkness or 40-Hz flicker conditions for one hour / day according to some embodiments. Figure 60B is a bar graph depicting the number of Aβ-positive plaque deposits in the visual cortex of six-month-old 5XFAD mice after seven days of exposure to darkness or 40-Hz flicker conditions for one hour / day according to some embodiments. Figure 60C is a bar graph depicting the area of ​​Aβ-positive patches in the visual cortex of six-month-old 5XFAD mice after seven days of darkness or 40-Hz flicker conditions for one hour / day according to some embodiments.

[0217] Figure 61A is a series of immunofluorescence images showing immunohistochemistry of antiphosphorylated Tau (S202) antibody and antiMAP2 antibody in four-month-old P301S mice after seven days of darkness or 40-Hz flicker conditions for one hour / day according to some embodiments. Figure 61B is a bar graph depicting the relative phosphorylated Tau (pTau) (S202) intensity level in the P301S visual cortex after seven days of darkness and 40-Hz flicker conditions for one hour / day according to some embodiments. Figure 61C is a bar graph depicting the relative MAP2 intensity level in the P301S visual cortex after seven days of darkness and 40-Hz light flicker conditions for one hour / day according to some embodiments.

[0218] Figure 62A shows seven days after one hour / day of darkness and 40-Hz flicker conditions according to some embodiments.A series of immunofluorescence images of immunohistochemistry using anti-pTau 6202 (S404) antibody in 4-month-old P301S mice. Figure 62B is a bar graph depicting the relative pTau (S400 / T403 / S404) fluorescence intensity levels of the P301S visual cortex after seven days under darkness and 40-Hz flicker conditions for one hour / day according to some embodiments.

[0219] Figure 63A is a series of immunofluorescence images of immunohistochemistry using anti-pTau 6302 (S396) antibody in 4-month-old P301S mice after seven days under darkness and 40-Hz flicker conditions for one hour / day according to some embodiments. Figure 63B is a bar graph depicting the relative pTau (S396) fluorescence intensity levels of the P301S visual cortex after seven days under darkness and 40-Hz flicker conditions for one hour / day according to some embodiments.

[0220] Figure 64 is a series of immunofluorescence images showing the immunohistochemistry of anti-Iba1 antibody in four-month-old P301S mice after seven days of using the anti-Iba1 antibody under darkness and 40-Hz flicker conditions for one hour / day according to some embodiments.

[0221] Figure 65A is a bar graph depicting the number of microglia after seven days of using the anti-Iba1 antibody under darkness and 40-Hz flicker conditions for one hour / day according to some embodiments. Figure 65B is a bar graph depicting the diameter of microglia bodies normalized to the control after seven days of using the anti-Iba1 antibody under darkness and 40-Hz flicker conditions for one hour / day according to some embodiments. Figure 65C is a bar graph depicting the average length of the primary process of microglia normalized to the control after seven days of using the anti-Iba1 antibody under darkness and 40-Hz flicker conditions for one hour / day according to some embodiments.

[0222] Figure 66 is a graph showing the levels of soluble and insoluble Aβ peptide isoforms Aβ1-40 and Aβ1-42 in the visual cortex of subjects with and without visual gamma stimulation according to some embodiments.

[0223] Figures 67A-67B are graphs showing the levels of whole-brain Aβ peptides in subjects with and without transcranial gamma stimulation according to some embodiments.

[0224] Figure 68A is a flowchart showing a study implemented to examine whether gamma exposure and / or administration according to some embodiments results in stress in subjects. Figure 68B is a bar graph depicting the levels of corticosterone, indicating the stress response of subjects.

[0225] Figure 69A is a flowchart showing a study implemented to examine whether gamma exposure and / or administration according to some embodiments reduces anxiety in subjects. Figure 69B is an image showing an elevated cross maze apparatus. Figures 69C and 69D are images showing representative trajectories of subjects during an elevated cross maze session.

[0226] Figure 70 is a bar graph depicting the average time spent by subjects exploring the open and closed arms during an elevated cross maze session.

[0227] Figure 71A is a flowchart illustrating an implementation to examine whether gamma exposure and / or application according to some implementation schemes reduces stress and / or anxiety in subjects. Figure 71B is an image showing the open field site. Figures 71C and 71D are images showing representative trajectories of subjects during an open field test.

[0228] Figure 72A is a graph depicting the average amount of time spent by subjects at the center of the open field per minute during an open field test. Figure 72B is a bar graph depicting the average total time spent by subjects at the edges of the open field during an open field test.

[0229] Figures 73A and 73B are schematic diagrams illustrating an implementation to examine whether gamma exposure and / or application according to some implementation schemes alters subjects' inherent novelty-seeking behavior. Figure 73C is a bar graph depicting the average amount of time spent by subjects exploring the first novel item compared to the second novel item, according to the schematic diagram of Figure 73A.

[0230] Figure 74 is a graph depicting the average amount of time a subject spends exploring a novel object per minute during the process, according to the schematic diagram of Figure 73B.

[0231] Figure 75A is a flowchart illustrating a study using a fear conditioning paradigm to examine whether gamma exposure and / or administration according to some implementation schemes affects the subject's learning and memory. Figure 75B is a stimulus graph illustrating the tone test under changing environmental conditions as a function of time.

[0232] Figures 76A and 76B are bar graphs demonstrating enhanced memory in subjects according to some implementation schemes.

[0233] Figure 77A is a flowchart illustrating a study using an implementation to examine whether gamma exposure and / or administration according to some implementation schemes improves the subject's memory. Figure 77B is a graph illustrating the Morris water maze with a platform hidden in the target quadrant. Figures 77C and 77D are images illustrating representative trajectories of subjects during the Morris water maze detection test.

[0234] Figure 78A is a graph depicting the average amount of time spent by subjects each day in finding hidden platforms in the Morris water maze test. Figure 78B is a graph depicting the average amount of time spent by subjects in searching for removed platforms in the target quadrant every half minute. Figure 78C is a graph depicting the average amount of time spent by subjects in searching for removed platforms in the opposite quadrant every half minute.

[0235] Figure 79A is a graph showing the Morris water maze test with platforms hidden in the first quadrant. Figure 79B is a graph showing the Morris water maze test with platforms hidden in the second quadrant (opposite to the first quadrant) for back-learning. Figure 79C is from page 17 / 78 of the specification, CN 122399187 A.Figure 80A is a graph depicting the average amount of time subjects spend each day finding hidden platforms in the Morris water maze reverse learning test.

[0236] Figure 80A is a flowchart illustrating a study conducted to examine whether chronic gamma exposure and / or administration according to some implementation schemes affects subjects' spatial learning and memory. Figure 80B is a graph depicting the average amount of time subjects spend each day finding hidden platforms in the Morris water maze test. Figure 80C is a bar graph depicting the average amount of time subjects spend searching for removed platforms in the target quadrant during a 30-second trial.

[0237] Figure 81A is a flowchart illustrating a study of Figure 80A extended to include reverse learning. Figure 81B is a graph depicting the average amount of time subjects spend each day finding hidden platforms in the Morris water maze reverse learning test.

[0238] Figure 82A is a bar graph depicting the average amount of time subjects spend searching for removed platforms in the target quadrant during a 30-second trial. Figure 82B is a bar graph depicting the average amount of time subjects spend searching for removed platforms in the opposite quadrant.

[0239] Figure 83 is a timeline of studies conducted to examine the effects of gamma exposure and / or administration according to some embodiments on DNA damage and neuronal loss in the visual cortex of subjects.

[0240] Figure 84 is a graph showing groups of subjects for studies conducted to examine the effects of gamma exposure and / or administration according to some embodiments.

[0241] Figure 85 is a bar graph comparing changes in brain weight across groups of subjects in Figure 84 according to some embodiments.

[0242] Figure 86 is a bar graph comparing changes in folds of lateral ventricle expansion across groups of subjects in Figure 84 according to some embodiments.

[0243] Figures 87A-87E are images showing lateral ventricles representing groups of subjects in Figure 84 according to some embodiments.

[0244] Figures 88A-88C are brain anatomy diagrams showing brain regions of interest according to some embodiments.

[0245] Figure 89 is a bar graph depicting the mean thickness of the V1-cortex across groups of subjects in Figure 84 according to some embodiments.

[0246] Figure 90 is a bar graph depicting the average thickness of the V1-NeuN-positive cell layer in a group of subjects across Figure 84 according to some embodiments.

[0247] Figures 91A-91E are images showing cells with Hoechst and / or NeuN labels representing a group of subjects across Figure 84 according to some embodiments.

[0248] Figure 92 is a bar graph depicting the average thickness of the SS1-cortical layer in a group of subjects across Figure 84 according to some embodiments.

[0249] Figure 93 is a bar graph depicting the average thickness of the SS1-NeuN-positive cell layer in a group of subjects across Figure 84 according to some embodiments.

[0250] Figures 94A-94E are images showing Hoechst-labeled and / or NeuN-labeled cells in a group of subjects across Figure 84 according to some embodiments.

[0251] Figure 95 is a bar graph depicting the average thickness of the cortical layer of the insular cortex in a group of subjects across Figure 84 according to some embodiments.

[0252] Figure 96 is a bar graph depicting the average thickness of the NeuN-positive cell layer of the insular cortex in a group of subjects across Figure 84 according to some embodiments.

[0253] Figures 97A-97E are images showing cells representing a group of subjects across Figure 84 according to some embodiments, showing Hoechst-labeled and / or NeuN-labeled cells. Specification 18 / 78 pages 20 CN 122399187 A

[0254] Figure 98 is a bar graph comparing the amount of NeuN-positive cells in the visual cortex in a group of subjects across Figure 84 according to some embodiments.

[0255] Figure 99 is a bar graph comparing the amount of γH2AX-positive cells in the visual cortex across groups of subjects in Figure 84 according to some embodiments.

[0256] Figure 100 is a series of images showing visual cortex samples representing groups of subjects in Figure 84 according to some embodiments.

[0257] Figure 101 is a bar graph comparing the amount of NeuN-positive cells in the somatosensory cortex across groups of subjects in Figure 84 according to some embodiments.

[0258] Figure 102 is a bar graph comparing the amount of γH2AX-positive cells in the somatosensory cortex across groups of subjects in Figure 84 according to some embodiments.

[0259] Figure 103 is a series of images showing somatosensory cortex samples representing groups of subjects in Figure 84 according to some embodiments.

[0260] Figure 104 is a bar graph comparing the amount of NeuN-positive cells in the insular cortex across groups of subjects in Figure 84 according to some embodiments.

[0261] Figure 105 is a bar graph comparing the amount of γ H2AX-positive cells in the insular cortex across the subject group of Figure 84 according to some embodiments.

[0262] Figure 106 is a series of images showing insular cortex samples representing the subject group of Figure 84 according to some embodiments.

[0263] Figure 107 is a bar graph comparing the amount of NeuN-positive cells in the hippocampus across the subject group of Figure 84 according to some embodiments.

[0264] Figure 108 is a bar graph comparing the amount of γ H2AX-positive cells in the hippocampus across the subject group of Figure 84 according to some embodiments.

[0265] Figure 109 is a series of images showing hippocampal samples representing the subject group of Figure 84 according to some embodiments.

[0266] Figure 110 is a bar graph comparing the density of pigmented spots in the visual cortex across the subject group of Figure 84 according to some embodiments.

[0267] Figure 111 is a bar graph comparing the density of somatosensory cortex chromophores across groups of subjects in Figure 84 according to some embodiments.

[0268] Figure 112 is a bar graph comparing the density of insular cortex chromophores across groups of subjects in Figure 84 according to some embodiments.

[0269] Figures 113A-113D are images showing Hoechst staining, VGluT1 markers, and / or GAD65 markers in representative samples according to some embodiments. Figures 113E and 113F are images showing methods for chromophore quantification according to some embodiments.

[0270] Figure 114 is a stimulation graph showing a ticking series of stimuli according to some embodiments.

[0271] Figure 115 is a flowchart showing a study conducted to examine whether auditory gamma exposure and / or administration according to some embodiments induces microglial cell activation in the auditory cortex of subjects.

[0272] Figure 116A is a bar graph depicting the average number of microglial cells in the auditory cortex of subjects according to some embodiments. Figure 116B is a bar graph depicting the fold change in the length of microglia in the auditory cortex of a subject according to some embodiments. Specification 19 / 78 pages 21 CN 122399187 A

[0273] Figures 117A and 117B are representative images of microglia in the auditory cortex of a subject according to some embodiments.

[0274] Figures 118A and 118B are magnified images of the length of microglia from Figures 117A and 117B according to some embodiments.

[0275] Figures 119A and 119B are magnified images of the cell body size of microglia from Figures 117A and 117B according to some embodiments.

[0276] Figure 120A is a bar graph depicting the average number / image field of microglia in the auditory cortex of a subject according to some embodiments. Figure 120B is a bar chart depicting the mean fold change in cell body size of microglia in the auditory cortex of a subject according to some embodiments.

[0277] Figures 121A and 121B are representative images of microglia in the auditory cortex of a subject according to some embodiments.

[0278] Figures 122A-122D are bar charts depicting the levels of soluble Aβ isoforms Aβ1-40 and Aβ1-42 in the auditory cortex and hippocampus of a subject according to some embodiments.

[0279] Figures 123A-123D are bar charts depicting the levels of insoluble Aβ isoforms Aβ1-40 and Aβ1-42 in the auditory cortex and hippocampus of a subject according to some embodiments.

[0280] Figures 124A-124D are representative images of microglia in the auditory cortex of a subject according to some embodiments.

[0281] Figure 125A is a flowchart illustrating a novel object recognition test. Figure 125B is a bar graph demonstrating memory improvement according to some embodiments.

[0282] Figure 126A is a flowchart illustrating a novel object localization test. Figure 126B is a bar graph demonstrating memory and / or discrimination improvement according to some embodiments.

[0283] Figure 127A is a graph depicting the average amount of time a subject spends each day finding a hidden platform in a Morris water maze test. Figure 127B is a bar graph depicting the average amount of time a subject spends searching for a removed platform in the target quadrant during a detection test.

[0284] Figure 128A is a series of representative immunofluorescence images showing an enlarged vascular system in the visual cortex according to some embodiments. Figure 128B is a bar graph depicting the diameter of blood vessels in the visual cortex and showing the increase in blood vessel diameter after γ exposure according to some embodiments. Detailed Description

[0285] In one aspect, this disclosure provides methods, apparatus, and systems for preventing, alleviating, and / or treating brain disorders or cognitive impairments / deficiencies in subjects. In some embodiments, the brain condition is dementia.

[0286] Cognitive function depends primarily on neural network activity associated with attention and working memory, specifically the precise timing of oscillations of gamma frequencies (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). Because these oscillations arise from synaptic activity, they provide a direct link between the molecular properties of neurons and higher levels of coherent brain activity. Importantly, gamma oscillation activity is disrupted in neural circuits damaged by neuropathy in AD and can represent a key determinant of memory impairment in the disease. A causal relationship between the lesion and the impairment of brain oscillations has not yet been determined. However, driving brain rhythms can serve as a multi-target therapy for treating dementia such as AD and can be achieved through non-invasive therapies.

[0287] In one aspect, this disclosure provides apparatus, methods, and systems for enhancing or inducing gamma oscillations. In some embodiments, as described on pages 20 / 78 of CN 122399187 A, enhancement or induction of gamma oscillations is performed by optogenetic methods. In other embodiments, enhancement or induction of gamma oscillations is performed by behavioral methods. This disclosure provides for reducing AD lesions by enhancing and / or inducing gamma oscillations through optogenetic, behavioral, or other methods.

[0288] In one aspect, this disclosure provides apparatus, systems, and methods for restoring or inducing gamma oscillation rhythms in subjects suffering from dementia. In some embodiments, the dementia is AD, vascular dementia, frontotemporal dementia (FTD), and / or Lewy body dementia. Therefore, in some embodiments, this disclosure provides apparatus, systems, and methods for treating dementia.

[0289] As used herein, the term "treatment" refers to both therapeutic treatment and preventative or preventative measures. In some embodiments, subjects requiring treatment include those who already have a disease or condition and those who are likely to develop a disease or condition and whose aim is to prevent, delay, or reduce the disease or condition. For example, in some embodiments, the apparatus, methods, and systems disclosed herein may be used to prevent, delay, or reduce a disease or condition, such as AD, in which a subject is genetically susceptible. In some embodiments, the apparatus, methods, and systems disclosed herein may be used to treat, alleviate, reduce symptoms of a disease or condition, such as AD, that a subject has been diagnosed with and / or delay the progression of said disease or condition.

[0290] As used herein, the term "subject" means a mammal, such as a rodent, feline, canine, or primate. Preferably, the subject according to the invention is a human.

[0291] As used herein, the term "about" means ten percent of the object modified by "about".

[0292] Dementia is a condition characterized by loss of intellectual abilities and / or memory impairment. Dementia includes, for example, Alzheimer's disease (AD), vascular dementia, Lewy body dementia, Pick's disease, frontotemporal dementia (FTD), AIDS-related dementia, age-related cognitive impairment, and age-related memory impairment. Dementia can also be associated with neurological and / or psychiatric conditions such as brain tumors, brain lesions, epilepsy, multiple sclerosis, Down syndrome, Rett syndrome, progressive supranuclear palsy, frontal lobe syndrome, schizophrenia, and traumatic brain injury.

[0293] Alzheimer's disease (AD) is the most common neurodegenerative disease in developed countries. Histopathologically, AD is characterized by the accumulation of amyloid plaques containing Aβ peptides and non-fibrillary fibrosis (NFT) 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.

[0294] Vascular dementia, also known as cerebrovascular dementia, refers to cerebrovascular diseases (e.g., infarction in the cerebral hemispheres) that typically have a fluctuating course of improvement and gradual deterioration. Vascular dementia may include one or more symptoms such as disorientation, impaired memory, and / or impaired judgment. Vascular dementia can be caused by discrete multiple infarctions or other vascular etiologies, including, for example, autoimmune vasculitis, such as that found in systemic lupus erythematosus; infectious vasculitis, such as Lyme disease; recurrent intracerebral hemorrhage; and / or stroke.

[0295] Frontotemporal dementia (FTD) is a progressive neurodegenerative condition. Subjects with FTD typically exhibit marked behavioral and personality changes, often accompanied by language impairments.

[0296] Lewy body dementia is characterized by: the development of one or more symptoms of dementia with features overlapping with those of AD; the development of features of Parkinson's disease; and / or early development of hallucinations. Lewy body dementia is typically characterized by daily fluctuations in the severity of symptoms.

[0297] In some aspects, this disclosure provides methods for preventing, alleviating, and / or treating dementia in subjects, comprising inducing synchronized gamma oscillations in the subject's brain. In some embodiments, gamma oscillations are induced in subjects with neurological diseases or conditions or age-related decline to restore gamma oscillation rhythms that have been disrupted in the subject due to said disease or condition or age-related decline or associated with it.

[0298] In some embodiments, induced gamma oscillations reduce the generation of isotypes Aβ1-40 and Aβ1-42. In some embodiments, induced gamma oscillations enhance the clearance of Aβ (e.g., isotypes Aβ1-40 and Aβ1-42) from the subject's brain. In some embodiments, induced gamma oscillations prevent 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, relative 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%, relative to the level of Aβ in the subject's brain before treatment.

[0299] In some embodiments, the level of Aβ in the subject's brain is reduced by reducing the clearance of APP in the subject's brain. In some embodiments, the methods provided herein reduce APP clearance in the subject's brain by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or more, relative to the level of APP clearance in the subject's brain prior to treatment. In some embodiments, the level of APP clearance in the subject's brain is reduced by at least about 50%, relative to the level of APP clearance in the subject's brain prior to treatment. In some embodiments, the level of APP clearance is measured by the level of C-terminal fragment β (β-CTF) in the subject's brain. In some embodiments, the level of APP clearance in the brain is reduced via inhibition of β-secretase and / or γ-secretase (e.g., by increasing the level of inhibition of β-secretase and / or γ-secretase activity). In some embodiments, the methods provided herein reduce Aβ in the subject's brain.Aggregation of plaques.

[0300] In some embodiments, the method improves the cognitive abilities and / or memory of the subject.

[0301] In another aspect, this disclosure provides a method for inducing a neuroprotective feature or neuroprotective environment in the brain of a subject, comprising inducing synchronous gamma oscillations in the brain of the subject. For example, in some embodiments, the neuroprotective feature is associated with a neuroprotective microglial feature. In other embodiments, the neuroprotective feature is induced or associated with an 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 an anti-inflammatory microglial signaling pathway.

[0302] In some embodiments, the neuroprotective feature is associated with a decrease or absence of pro-inflammatory glial cell activity. Pro-inflammatory glial cell activity is associated with the M1 phenotype of microglia and includes the production of reactive oxygen species (ROS), the neurosecretory protein chromogranin A, the secretory cofactor cysteine ​​protease inhibitor C, NADPH oxidase, nitric oxide synthases such as iNOS, NF-κB-dependent inflammatory response proteins, and pro-inflammatory cytokines and chemokines (e.g., TNF, IL-1β, IL-6, and IFNγ).

[0303] In contrast, the M2 phenotype of microglia is associated with downregulation of inflammation and repair of inflammation-induced damage. Increased anti-inflammatory cytokines and chemokines (IL-4, IL-13, IL-10, and / or TGFβ) and phagocytic activity are associated with the M2 phenotype. Therefore, 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 the phagocytic activity of microglia, thereby increasing the clearance of Aβ.

[0304] Gamma oscillations may include about 20 Hz to about 100 Hz. Therefore, in some embodiments, this disclosure provides a method for preventing, alleviating, or treating dementia in a subject, comprising inducing gamma oscillations in the subject's brain at 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 30 Hz to about 60 Hz, or about 35 Hz to about 45 Hz, or about 40 Hz. Preferably, the gamma oscillations are about 40 Hz.

[0305] Stimulation may include any 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, stimulation may be designed to stimulate electromagnetic radiation receptors (e.g., photoreceptors, infrared receptors, etc.). (See page 22 / 78 of the specification, 24 CN 122399187 A)Receptors, including (and / or ultraviolet) receptors, mechanoreceptors (e.g., mechanical stress and / or strain), pain receptors (i.e., pain), sound receptors, electroreceptors (e.g., electric fields), magnetoreceptors (e.g., magnetic fields), hydrogen receptors, chemoreceptors, thermoreceptors, olfactory receptors, and / or proprioceptors (i.e., sense of position). The absolute threshold or minimum amount of sensation required to elicit a response from a receptor may be based on the type of stimulus and subject variation. In some embodiments, the stimulus is tuned based on individual sensitivity.

[0306] 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 flashes of light; and in other embodiments, gamma oscillations are induced in the auditory cortex using auditory stimuli at a specific frequency. In some embodiments, gamma oscillations are simultaneously induced in multiple brain regions using a combination of visual, auditory, and / or other stimuli. In some embodiments, gamma oscillations are induced in the virtual reality system.

[0307] In some embodiments, the subject receives stimulation through an environment configured to induce gamma oscillations (such as a room that passively or actively blocks irrelevant stimuli (e.g., light blocking or noise cancellation)). Alternatively or additionally, the subject may receive stimulation through a system that includes, for example, a light-blocking or noise-cancelling appearance. In some embodiments, the subject receives visual stimulation through a stimulation-emitting device (such as an eye-wearing device designed to deliver stimulation). The device may block other light. In some embodiments, the subject receives auditory stimulation through a stimulation-emitting device (such as headphones designed to deliver stimulation). The device may cancel other noise.

[0308] In addition to at least one interface for emitting stimuli, some embodiments may include at least one processor (for example, generating stimuli, controlling the emission of stimuli, monitoring the emission / outcome of stimuli, and / or processing feedback about stimuli / outcomes), at least one memory (for storing, for example, processor-executable instructions, at least one stimuli, stimulus generation strategy, feedback, and / or results), at least one communication interface (for communicating with, for example, subjects, healthcare providers, caregivers, clinical research investigators, databases, monitoring applications, etc.), and / or detection devices (for detecting and providing feedback about, for example, whether γ oscillations are induced and / or subjects, including whether γ oscillations are induced, subject sensitivity, cognitive function, physical or chemical changes, stress, safety, etc.).

[0309] In some embodiments, γ oscillations are induced by visual stimuli (such as flashes of light at about 20 Hz to about 100 Hz). In a particular embodiment, γ oscillations are induced by visual stimuli at about 20 Hz to about 50 Hz.In some embodiments, gamma oscillations are induced by flashes at frequencies of about 35 Hz to about 45 Hz. In yet another embodiment, gamma oscillations are induced by flashes at frequencies of about 40 Hz. In some embodiments, the subject receives (e.g., placed in a room with a light-blocking device or wearing a light-blocking device that emits) flashes at frequencies of 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.

[0310] In some embodiments, gamma oscillations are induced by auditory stimulation, such as sound at frequencies 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 receives (e.g., placed in a room with a noise cancellation device or wearing a noise cancellation device that emits) auditory stimulation at 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.

[0311] In some embodiments, the subject receives (e.g., placed in a room with a light blocking device or wearing a light blocking device that emits) visual and / or auditory stimulation for about one hour, about two hours, about three hours, about four hours, about five hours, or more. In some embodiments, the subject receives (e.g., placed in a room with a light blocking device or wearing a light blocking device that emits) stimulation for no more than about six hours, no more than about five hours, no more than about four hours, no more than about three hours, no more than about two hours, or no more than one hour. In some embodiments, the subject receives (e.g., placed in a room with a light-blocking device or wears a light-blocking device that emits light) stimulation for less than one hour.

[0312] In some embodiments, the subject experiences the methods provided herein. In other embodiments, the subject experiences treatment using the methods provided herein for multiple individual situations. The subject may receive treatment according to a regular schedule or when symptoms appear or worsen. In some embodiments, chronic treatment may be effective in reducing soluble Aβ peptides and / or insoluble Aβ peptides (i.e., plaques).

[0313] In some embodiments, γ oscillations are induced in a cell type-specific manner. In some embodiments, γ oscillations are induced in FS-PV-interneurons. When used to describe a class of neurons, the term "flash" (FS) refers to the ability of a neuron to fire at a high rate with very few spike frequencies or spikes that decay significantly over a long period of time. Thus, theseNeurons are capable of firing at sustained high frequencies (e.g., equal to or greater than about 100 Hz or about 150 Hz) without significant modulation. This characteristic of FS neurons is largely attributable to the expression of their fast-delay rectifier channels (in other words, channels that are activated and deactivated very quickly).

[0314] In one aspect, the stimulation can be non-invasive. As used herein, the term “non-invasive” refers to devices, methods, and systems that do not require surgical intervention or manipulation of the body (such as injection or implantation of compositions or devices). For example, the stimulation can be visual (e.g., flashing light), auditory (e.g., sound vibration), and / or tactile (mechanical stimulation using force, vibration, or movement).

[0315] In another aspect, the stimulation can be invasive or at least partially invasive. For example, visual, auditory, and / or tactile stimulation can be combined with injected or implanted compositions (e.g., photosensitive proteins) or devices (e.g., integrated optical fibers and solid-state light sources).

[0316] Experimental Data

[0317] Gamma oscillations were reduced in hippocampal toe SWR in 5XFAD mice in the early stages of the disease.

[0318] Gamma defects have been observed in multiple brain regions in several neurological and psychiatric disorders, including a reduction in spontaneous gamma synchronization in human patients with AD. Interestingly, reduced spontaneous gamma has also been found in two mouse models of AD in vivo (human amyloid precursor (hAPP) Tg mice and apolipoprotein E4 allele (APOE4) knock-in mice) and in in vitro slice studies of another mouse model (Tg CRND8 mice). However, it is unclear whether gamma oscillations are altered in other mouse models of AD, whether they occur early in disease progression, and whether gamma disruption affects disease progression.

[0319] To address these questions, neural activity from conscious behavioral 5XFAD mice (a well-established model of AD carrying five familial AD mutations) was recorded. Specifically, 5XFAD mice express five different familial AD alleles, including APP KM670 / 671NL (Sweden), APP I716V (Florida), APP V717I (London), PSEN1 M146L (A>C), and PSEN1 L286V. Therefore, 5XFAD mice are used as a model of AD amyloidosis. In some embodiments, neural activity is recorded from approximately 3 months of age (at which point the mice have elevated levels of Aβ), but prior to the onset of major plaque accumulation and the manifestation of learning and memory deficits. Figure 1 is a schematic diagram illustrating mice running through a virtual linear maze on a spherical treadmill according to some embodiments. Food-restricted mice are rewarded for running back and forth on the spherical treadmill through the virtual linear maze.

[0320] Neural activity from the CA1 subregion of the hippocampus can be recorded. Figures 2A and 2B are traces of theta oscillations and sharp wave ripples (SWR) recorded from the CA1 of the hippocampus according to some embodiments. In some embodiments, the gamma oscillations in the CA1 may be present during different time periods of activity, such as during running, when theta oscillations (4-12 Hz) are observed, as shown in Figure 2A, and during static and exploratory behaviors, when SWR occurs, as shown in Figure 2B.

[0321] The power spectral density during the theta oscillation process was examined, and no significant difference was found in the slow gamma activity (range of 20 Hz to 50 Hz) in 5XFAD mice and WT littermates. Figures 3A and 3B are graphs showing the mean and standard deviation of the normalized power spectrum and normalized power spectral density during the theta time period in three-month-old Tg 5XFAD mice and WT mice according to some embodiments. Figure 3A shows the mean and standard deviation of the normalized power spectrum during the θ time period in three-month-old 5XFAD (n = 6 mice) and WT (n = 6 mice) mice. In some embodiments, the power spectral density of each animal can be normalized to its peak (in θ). Figure 3B shows the normalized power spectral density during the θ time period in three-month-old 5XFAD (n = 6 mice) and WT (n = 6 mice) mice.

[0322] In some embodiments, as a next step, the γ oscillations during the SWR process are examined, which are high-frequency oscillations of 150-250 Hz lasting approximately 50-100 ms. SWR is associated with bursts of group activity (during which the pattern of peak activity re-evolves across the hippocampus). Previous work has shown that slow γ rises during the SWR process and is synchronized across CA3 and CA1. Therefore, neurons across these hippocampal subregions are more likely to fire together during the SWR process because neurons are more likely to fire a phase locked to γ. A study was conducted in which SWR (defined as a period of time in which power in a ripple band from about 150 Hz to about 250 Hz is above the average by more than four standard deviations) was identified and the spectrum was plotted to examine the power across a certain frequency range during these SWR processes. In the spectrum plot, increased power above 100 Hz indicating high-frequency oscillatory characteristics of SWR and concurrently increased power below about 50 Hz indicating gamma power were observed.

[0323] Figures 4A and 4B are spectrum plots showing SWR in WT mice and 5XFAD mice according to some embodiments. Figure 4A shows the spectrum plot of average SWR triggering in a WT mouse showing gamma power at a frequency below 80 Hz during SWR 404.The increase in band 402 is magnified in the right figure. Figure 4B shows the average SWR trigger spectrum of a 5XFAD mouse, illustrating the increase in the γ band during SWR, although this increase is lower than that in the WT mouse shown in Figure 4A.

[0324] In some embodiments, the study found that the instantaneous frequencies of these slower oscillations (10-50 Hz range, as further described herein) are a single-peak distribution centered around 40 Hz. Figures 5A-5C are graphs depicting the distribution of instantaneous γ frequencies during SWR according to some embodiments. Figure 5A shows the distribution of instantaneous γ frequencies during SWR of the same mouse with a peak around 40 Hz as shown in Figure 4A (n = 370 SWRs). Figure 5B shows the distribution of instantaneous γ frequencies during the SWR process in 5XFAD and WT mice, illustrating the distribution around 40 Hz for each recording session, and Figure 5C shows the mean and standard error of the mean (SEM) across animals (n = 820, 800, 679, 38, 1875, 57 γ cycles / session in the six 5XFAD animals, and n = 181, 1075, 919, 1622, 51, 1860, 1903 γ cycles / session in the six WT animals).

[0325] In some embodiments, these γ oscillations during the SWR process in WT mice are then compared with those in 5XFAD littermates, and a deficiency in γ during the SWR process is found: while γ power does increase from baseline in 5XFAD mice during the SWR process, the γ power during the SWR process is significantly smaller in 5XFAD mice than in WT mice, as further described herein.

[0326] Figure 6A is a series of plots depicting the z-score γ power as a function of time since the peak of SWR in 5XFAD mice and WT mice, respectively, according to some embodiments. Figure 6A shows the mean and SEM, and demonstrates the increase in γ power relative to baseline during the SWR process.

[0327] Figure 6B is a graph depicting the cumulative distribution of γ power during the SWR process in 5XFAD mice and WT mice, according to some embodiments. The cumulative distribution of γ power during the SWR process showed a significantly smaller increase in 5XFAD mice than in WT mice (rank-sum test, p < 10⁻⁵; n = 2166 SWRs in six 5XFAD mice and n = 3085 SWRs in six WT mice; median z-score of 1.02 (0.39–1.87, 25–75 percentile) in 5XFAD mice and 1.18 (0.53–2.15, 25–75 percentile) in WT mice).

[0328] Figures 6C and 6D are plots depicting the cumulative distribution of z-score γ power over a 100 ms period around the peak of SWR in WT mice 606 and 5XFAD mice 608 according to some embodiments, as well as the mean and SEM (shaded) curves across animals (n = 514, 358, 430, 22, 805, 37 SWR / session in six 5XFAD animals, and n = 82, 311, 370, 776, 18, 710, 818 SWR / session in six WT animals).

[0329] Figure 6E is a plot depicting the cumulative distribution of z-score γ power over a 100 ms period around the peak of large SWR (detection threshold greater than 6 standard deviations above the mean) in WT mice 614 and 5XFAD mice 616 according to some embodiments. All non-normally distributed data were subjected to a rank-sum test, as further described herein. Figure 6E shows a significantly smaller increase in WT mice 614 and 5XFAD mice 616 (rank-sum test, p < 10⁻⁵, n = 1000 SWRs in six 5XFAD mice and n = 1467 SWRs in six WT mice).

[0330] In some embodiments, the spikes are phase-modulated by these γ oscillations in both groups; however, the modulation of the spikes by the γ phase is weaker in 5XFAD animals than in WT animals. The study found that the depth of modulation can be significantly smaller in 5XFAD animals than in WT animals.

[0331] Figure 7A is a graph depicting the peak fraction as a function of the phase of the γ oscillation, and Figure 7B is a graph depicting the depth of peak modulation as a function of the γ phase during the SWR process in three-month-old 5XFAD (n = 6 mice) and WT (n = 6 mice) mice according to some embodiments (rank-sum test, bootstrap resampling, p < 10⁻⁵, which is important for controls for multiple comparisons, n = 2500 5XFAD peak-γ phase distributions and 3000 WT distributions, with a median modulation depth of 0.35 (0.21–0.44, 25–75 percent) in 5XFAD mice and 0.38 (0.29–0.47, 25–75 percent) in WT mice). Error bars indicate the mean + / - SEM. Figure 704 shows the frequency curve of the depth of the spike modulation

[0332] . Figures 7C and 7D are curves showing the spike fraction in the hippocampal CA1 during the SWR process as a function of the phase of the γ oscillation for each animal in 5XFAD animals and WT animals, as well as the mean and SEM curves across animals, according to some embodiments.Line graphs (n = 2475, 1060, 3092, 25, 6521, 123 spikes / sessions during the SWR process in six 5XFAD animals, and n = 360, 4741, 1564, 2961, 88, 3058, 4270 spikes / sessions during the SWR process in six WT animals).

[0333] Figure 7E is a graph depicting the spike fraction as a function of the phase of the γ oscillation, and Figure 7F is a graph depicting the depth of spike modulation during the large SWR process (detection threshold greater than 6 standard deviations above the mean, as further described herein) in three-month-old 5XFAD (n = 6 mice) and WT (n = 6 mice) mice (rank sum test, bootstrap resampling, an asterisk indicating p < 10-10, n = 2500 5XFAD spike-γ phase distribution and 3000 WT distribution). Error bars indicate the mean + / - SEM.

[0334] The study also found that, compared with WT, there were likely fewer SWRs / times in non-θ time periods in 5XFAD mice (rank-sum test, p < 10⁻⁵, n = 634 non-θ time periods in six 5XFAD mice and n = 750 non-θ time periods in six WT mice, with a median of 0.07 Hz (0–0.17, 25–75 percent) in 5XFAD mice and a median of 0.12 Hz (0–0.24, 25–75 percent) in WT mice), thereby further reducing the time periods of γ power elevation as disclosed above.

[0335] Figures 8A and 8B are plots depicting the SWR rate / non-θ time intervals for each animal (Figure 8A) and all animals in combinations according to some embodiments in 5XFAD mice 802 and WT mice 804 (rank-sum test, p < 10-10, n = 117, 210, 151, 55, 100, 1 non-θ time interval / session in six 5XFAD animals, and n = 80, 68, 115, 95, 15, 159, 218 non-θ time intervals / session in six WT animals). These results reveal evidence of defects in the modulation of γ oscillations and hippocampal CA1 spikes, as well as cognitive deficits, in mouse models of AD prior to the development of major amyloid plaque accumulation.

[0336] Optogenetic stimulation of FS-PV-interneurons at γ frequencies drives γ oscillations in the CA1 region of the hippocampus. Instruction manual, pages 26 / 78, CN 122399187 A

[0337] Early disease progression in this mouse model of AD during the SWR process revealed a γ deficiency, raising the question of whether γ oscillations can affect the molecular and cellular pathophysiology of AD. To test this question, 2.5-month-old 5X FAD / In FS-PV-interneurons of the hippocampal toe CA1 in PV-Cre double transgenic mice, a double-floxed inverted open reading frame (DIO) ChR2-EYFP adenovirus-associated virus (AAV) expresses ChR2 in a Cre-dependent manner to optogenetically drive γ oscillations. Studies were conducted to determine whether the genetic induction of hippocampal toe γ oscillations in mice affects molecular pathogenesis in mouse models of AD. Hippocampal toe γ oscillations were genetically induced in awake behavioral WT mice and 5XFAD mice.

[0338] An adenovirus-associated virus (i.e., AAV5 virus) with a double-floxed inverted open reading frame (DIO) ChR2 linked to enhanced yellow fluorescent protein (EYFP) driven by the EF1α promoter was generated. Figure 9 is a schematic diagram illustrating a viral vector (i.e., AAV5-DIO-ChR2-EYFP) used to modulate the activation of specific cell types in the brain of a subject 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 two incompatible loxP variants is flipped to achieve ChR2 expression.

[0339] The CA1 region of the hippocampus of 5XFAD mice is infected with AAV-DIO-ChR2-EYFP or EYFp-only constructs using a stereotactic viral injection method that allows precise regional targeting of viral infection. In one embodiment, at the time of injection, a cannula (white bar) containing a fiber optic cable is placed approximately 0.3 mm above the targeted brain region. After two weeks (which provides time for mouse recovery and viral expression in PV cells), CA1 interneurons are optogenetically manipulated.

[0340] Figures 10A and 10B are schematic diagrams illustrating the delivery of signals to the CA1 region of the hippocampus of a subject according to some embodiments. In Figure 10A, according to some embodiments, a mouse is shown running through a maze on a ball while being stimulated via optogenetic experience in the hippocampus. As shown in Figures 10A and 10B, arrow 1000 indicates blue light flashing at approximately 40 Hz to activate the brain region.

[0341] In the embodiment described, a 200-mW 493-nm DPSS laser is connected to a connector at each end using a fiber optic channel / physical contact connector. During the experiment, approximately 1 mW of optical stimulation is delivered for approximately one hour. More specifically, blue light (e.g., 473 nm) is delivered randomly at various frequencies (including θ (e.g., approximately 8 Hz), γ (e.g., approximately 40 Hz)) and also at approximately 40 Hz through an optical fiber positioned directly above the CA1 region of the hippocampus. In some embodiments, the stimulation condition is not tested. According to some embodiments, the θ condition serves as a frequency control, and the random condition is targeted at the rhythm.Sex-specificity was controlled.

[0342] After one hour of stimulation, the brain tissue was cut and frozen at -80°C for staining and enzyme-linked immunosorbent assay (ELISA) analysis. Figure 11 is an immunofluorescence image showing the immunostaining of the subject's neural tissue using ChR2 and DAPI according to some embodiments. In the embodiment, Figure 11 shows DAPI (nucleus) and ChR2 staining in the hippocampus.

[0343] Figure 12A is an immunofluorescence image showing ChR2-EYFP expressed in PV+ interneurons according to some embodiments. Figure 12A shows strong expression of ChR2-EYFP in PV+ interneurons in CA1 of three-month-old 5XFAD / PV-Cre mice (scale bar = 100 µm). Figure 12B shows a series of immunofluorescence images of immunohistochemistry in three-month-old 5XFAD / PV-Cre CA1 mice expressing AAV DIO ChR2-EYFP (which only shows EYFP expression in PV+ cells) under anti-EYFP and anti-PV antibody conditions. To compare 5XFAD and WT mice, ChR2 was expressed in FS-PV-interneurons in 5XFAD-negative littermates. As a control for the non-specific effects of light stimulation, 5XFAD / PV-Cre double transgenic mice expressing only EYFP-containing AAV-DIO were used. In these mice, under the same genetic background and light delivery conditions, light delivery did not induce 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 produces the largest LFP response. (Instructions for Use 27 / 78, page 29, CN 122399187 A) Second, in some embodiments, a γ deficiency is found in the SWR process, and the instantaneous γ frequency formation during the SWR process is distributed around 40 Hz, as shown in Figures 5A-5C. In some embodiments, for electrophysiological recording, the time periods of 40-Hz stimulation are interleaved with time periods of no stimulation or with time periods of stimulation delivered at random intervals (selected from a Poisson distribution centered at 40 Hz), as further described herein.

[0344] Figures 13A and 13B include schematic diagrams of the traces of local field potentials and the power spectral density of FS-PV-interneurons according to some embodiments. Referring to Figure 13A, 1302 is the trace of the local field potential in CA1 before and during the 40 Hz optogenetic drive of FS-PV-interneurons. Graph 1304 shows the CA1 in the process of 40-Hz stimulation, random stimulation (stimulation with random intervals selected from a Poisson distribution centered at 40 Hz), or no stimulation.Mean and standard deviation of power spectral density of FS-PV-interneurons (n ​​= four 5XFAD mice and three WT mice). Figure 13B shows the power spectral density of FS-PV-interneurons in CA1 of each mouse during 40-Hz stimulation 1306, random stimulation 1308, or no stimulation 1310 (n = four 5XFAD mice for 169, 130, 240, 73 40 Hz stimuli, 143, 129, 150, 72 random stimuli, and 278, 380, 52, 215 no stimulation time periods / animals; and n = three WT mice for 65, 93, 91 40 Hz stimuli, 64, 93, 90 random stimuli, and 187, 276, 270 no stimulation time periods). Delivering a 1 ms 473-nm light pulse at 40 Hz in an LFP causes an increase in power at 40 Hz, as shown in plot 1306 of Figures 13A and 13B, while random stimulation does not cause an increase in power at 40 Hz, as shown in plot 1308 of Figures 13A and 13B.

[0345] Furthermore, in some embodiments, the light pulse effectively drives the spike 2-3 ms after photoinitiation, and the spike / pulse ratio is similar in both random and 40 Hz conditions. Figures 14A and 14B include the original trace according to some embodiments, the trace filtered for the spike after photogenetic stimulation, and a graph of the spike probability after 1 ms laser pulse initiation. Figure 14A shows an exemplary original trace 1402 and a trace filtered for the spike (300-6000 Hz) 1404 after photogenetic stimulation 1406. Figure 1408 shows the peak / pulse frequency curves after a 1 ms laser pulse initiation during 40-Hz stimulation, random stimulation, or no stimulation (in four 5XFAD mice and three WT mice, n = 345762 40-Hz stimuli, 301559 random pulse stimuli, and 32350 no-stimulation periods, with a distance of at least 500 ms between 552 40-Hz stimuli, 543 random stimuli, and 1681 no-stimulation periods). Figure 14B illustrates the spike probabilities in response to 40-Hz stimulus 1412, random stimulus 1414, or no stimulus 1410 after a 1 ms laser pulse initiation, with spikes increasing approximately 2–3 ms after laser pulse initiation (n = four 5XFADs with 87, 130, 8, 73 40-Hz stimuli, 85, 129, 5, 72 random stimuli, and 251, 379, 15, 215 no-stimuli per animal; and n = three WTs with 65, 93, 91 40-Hz stimulus periods per animal, 64, 93, 90 random stimulus periods per animal, and 187, 277, 270...).

[0346] Thus, the 40-Hz oscillation in CA1 is 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 silencing neural activity. In some embodiments, random stimulation conditions are used as a control for overall changes in spike activity induced by stimulation. In some embodiments, multi-unit firing rates are compared during alternating periods of 40 Hz stimulation and random stimulation, and no significant differences in firing rates were found between these conditions.

[0347] Figure 15A is a bar chart showing the differences in firing rates between 40-Hz stimulation periods and random stimulation periods according to some embodiments. Figure 15A shows that the two types of stimulation elicit similar amounts of spike activity (for a Wilcoxon signed-rank test with zero median, p > 0.6, n = 538 stimulation periods from four 5XFAD mice and three WT mice, “ns” indicates non-significant). Wilcoxon signed-rank test for zero-median distribution of the difference in firing rate between 40 Hz stimulus and random stimulus in all mice, p > 0.6: median -1.75 × 10⁻⁵ Hz (-1.28 - 1.18 Hz, 25th - 75th percentile) n = 538 stimulus time intervals.

[0348] Figure 15B is a bar chart showing the multi-unit firing rate / 40-Hz stimulus 1512, random stimulus 1514, and no stimulus 1510 time periods for each animal (rank sum test for each animal (three WT mice and four 5XFAD mice), p > 0.09, median and quartiles are shown in the figure, n = 87, 130, 8, 65, 93, 91, 73 40-Hz stimulus time periods and 85, 129, 5, 64, 93, 90, 72 random stimulus time periods / mouse). The box plot shows the median (white line in the box) and quartiles (top and bottom of the box). In all animals, the firing rate was not significantly different between 40 Hz stimulation and random stimulation, thus demonstrating that random stimulation conditions act as a control for peaked activity (rank-sum test for each animal (three WT mice and four 5XFAD mice), p > 0.09, median and quartiles are shown in the figure, n = 87, 130, 8, 65, 93, 91, 73 40-Hz stimulation periods and 85, 129, 5, 64, 93, 90, 72 random stimulation periods / mouse). The presence or absence of 40-Hz stimulation relative to no stimulation was also examined. In most animals, 40 Hz stimulation or random stimulation was significantly different from no stimulation.The firing rates between stimuli were not significantly different (rank-sum test for each animal (2 WT and 2 5XFAD), p > 0.25, n = 8, 93, 91, 73 40-Hz stimulation periods and 15, 277, 270, 215 baseline periods / animal), or the firing rates during 40 Hz stimulation or random stimulation were lower than those during no-stimulation periods (rank-sum test for each animal (1 WT and 1 5XFAD), p < 10-5, which was significant when corrected for multiple comparisons, n = 130, 65 40-Hz stimulation periods and 379, 187 baseline periods / animal), thus indicating that 40-Hz stimulation did not lead to excessive neuronal activity. In one animal, there was significantly more activity under 40 Hz stimulation or random stimulation than at baseline (rank-sum test for 1 5XFAD mouse, p < 10⁻⁵, n = 87 40-Hz stimulation time periods and 251 baseline time periods / animal). Therefore, in six of seven animals, there was no evidence that 40 Hz optogenetic stimulation of FS-PV-interneurons resulted in excessive activity. Thus, in some embodiments, while random conditions do not induce γ oscillations, they do induce similar amounts of multi-unit spike activity, as shown in Figure 15A.

[0349] Figure 16A is a trace recorded from the hippocampus of a subject during a frequency-specific increase in stimulation of a specific cell type in the CA1 region of the hippocampus according to some embodiments. More specifically, Figure 16A is a record from the hippocampus of a subject during a frequency-specific increase in stimulation of FS-PV+ (i.e., γ condition) according to some embodiments.

[0350] Figure 16B is a power spectral density plot showing the frequency-specific increase in local field potential power during stimulation of specific cell types in the CA1 region of the hippocampus of a subject according to some embodiments. Specifically, the power spectral density plot in Figure 16B verifies the specificity of the stimulation. When FS-PV+ was activated by a 40-Hz blue light pulse, the local field potential (LFP) power was enhanced only at 40 Hz band 1600 during the γ stimulation condition (n = 4 mice / group). No enhancement was shown at this frequency 1600 under baseline or random stimulation conditions.

[0351] γ stimulation reduces Aβ production in the CA1 region of the hippocampus.

[0352] The accumulation of Aβ can trigger a variety of neurotoxic events typical of AD lesions. Therefore, in some embodiments, γ stimulation affects the overall Aβ peptide levels in examined 5XFAD mice. Three-month-old mice were used because plaques are not present in the hippocampus at this stage in these mice, thus allowing investigation of soluble Aβ dynamics independent of plaque load. In some implementations, it was found that in the CA1 region of the hippocampus, compared with the EYFP control group, at 40In the Hz group, one-hour stimulation of FS-PV-interneurons reduced Aβ1-40 by 53.22% and Aβ1-42 by 44.62%, as measured by Aβ ELISA analysis.

[0353] Figures 17A and 17B are bar graphs depicting the relative Aβ1-40 and Aβ1-42 levels of 5XFAD / PV-Cre CA1 obtained by one-way ANOVA when all mice were grouped together according to some embodiments (n = 8 EYFP mice and 740 Hz mice for Aβ1-40, n = 4 mice / group for Aβ1-42). The bar graph in Figure 17A represents the relative Aβ1-40 level of 5XFAD / PV-Cre CA1 in each stimulation condition. In the bar chart, circles 1702 superimposed on the bars indicate individual data points in each group (n = 8 EYFP5XFAD / PV-Cre mice, n = 7 40-Hz 5XFAD / PV-Cre mice, n = 4 8-Hz 5XFAD / PV-Cre mice, n = 6 randomized 5XFAD / PV-Cre mice / group). The symbol "ns" 1704 indicates non-significant, an asterisk 1706 indicates p < 0.05, and a double asterisk 1708 indicates p < 0.01. One-way ANOVA was performed for all bars in this figure. Figure 17B represents the relative Aβ1-42 levels of 5XFAD / PV-Cre CA1 in each stimulation condition (n = 4 EYFP mice, n = 4 40-Hz mice, n = 3 8 Hz mice, n = 3 randomized 5XFAD / PV-Cre mice / group). Figures 17A and 17B show the mean and SEM.

[0354] Table 1 (below) depicts the significantly different raw concentrations (pg / ml) when comparing mice from the same littermate mice receiving different conditions, p < 0.05, performed by Student's t-test. Table 1 shows the raw Aβ1-40 and Aβ1-42 levels for each experimental group in the case of ELISA dilution.

[0355] Table 1

[0356] Specification 30 / 78 pages 32 CN 122399187 A

[0357] Specification 31 / 78 pages 33 CN 122399187 A

[0358]

[0359] In some embodiments, a comprehensive set of control experiments is performed to determine whether the effect is specific for frequency, cell type, and / or rhythmicity. To determine frequency specificity, 5XFAD / PV-Cre dual-specificity was driven at 8 Hz.FS-PV interneurons from heterosex mice were stimulated, and no change in Aβ levels was observed. FS-PV interneurons were then driven randomly, and the effect was specific to rhythmic stimulation. In fact, amyloid levels did not decrease after random stimulation, and in fact, Aβ1-40 increased by 230.1% and Aβ1-42 by 133.8% (see, e.g., Figures 17A and 17B, p < 0.01, with all mice grouped together by one-way ANOVA: for Aβ1-40, n = 8 EYFP mice and n = 4 randomized mice; for Aβ1-42, n = 3 mice / group. Comparisons were made between mice from the same littermate receiving different conditions, and significant differences were observed, p < 0.01, by Student's t-test).

[0360] Finally, the cell type specificity of the effect was tested using 5XFAD / αCamKII-Cre bispecific mice by stimulating CamKII+ excitatory neurons in the CA1 of the hippocampus at 8 Hz and 40 Hz. Figures 18A and 18B are bar graphs depicting the relative Aβ1-40 and Aβ1-42 levels of 5XFAD / αCamKII-Cre CA1 obtained by one-way ANOVA according to some embodiments. Figure 18A represents the relative Aβ1-40 level of 5XFAD / αCamKII-Cre CA1 in each stimulation condition. The circles 1802 superimposed on the bars in the bar chart indicate individual data points in each group (n = 6 40-Hz 5XFAD / αCamKII-Cre mice, n = 3 8-Hz 5XFAD / αCamKII-Cre mice, n = 3 random 5XFAD / αCamKII-Cre mice / group, the symbol “ns” 1804 indicates non-significant, and the asterisk 1806 indicates p < 0.001, performed by one-way ANOVA).

[0361] Figure 18B represents the relative Aβ1-42 level of 5XFAD / αCamKII-Cre CA1 in each stimulus condition (n = 3 αCamKII-Cre mice / group). In some implementations, driving CamKII+ excitatory neurons at 8 Hz or 40 Hz did not produce significant differences in Aβ1-40 and Aβ1-42 levels (see, e.g., Figures 18A and 18B, right, p > 0.05, performed by one-way ANOVA, n = 6 mice at 40 Hz and 3 mice at 8 Hz (Aβ1-40), n = 3 mice / group (Aβ1-42). When comparing mice from the same littermate born to different conditions, they were not significantly different, p > 0.05, by Student's...(t-test performed). Similar to 5XFAD / PV-Cre mice, driving CamKII+ neurons with random stimulation also resulted in a 257.6% increase in Aβ1-40 and a 133.3% increase in Aβ1-42 (see, for example, Figures 18A and 18B, right, p < 0.001, manual 32 / 78 pages 34 CN 122399187 A). Performed by one-way ANOVA, for Aβ1-40, n = 5 40 Hz mice and 3 randomized mice, for Aβ1-42, n = 3 mice / group. Aβ1-40 was significantly different when compared to mice from the same littermate receiving different conditions, p < 0.001, performed by Student's t-test, and Aβ1-42 was significantly different, p = 0.13, performed by Student's t-test.

[0362] Therefore, the decrease in Aβ peptide levels after 40-Hz stimulation can be specific for driving FS-PV-interneurons. In some embodiments, to confirm these ELISA findings using immunohistochemistry, Aβ-labeling is performed using a β-amyloid C-terminus-specific antibody that does not cross-react with APP in CA1.

[0363] Figure 19A is a series of images showing the immunohistochemistry of anti-Aβ and anti-EEA1 antibodies in the hippocampal CA1 region according to some embodiments. Specifically, Figure 19A is a series of immunofluorescence images (scale bar = 50 µm) showing the immunohistochemistry of anti-Aβ 1902 (D54D2) and anti-EEA1 1904 (610457) antibodies in the hippocampal CA1 region of 5XFAD / PV-Cre under EYFP, 40-Hz, and random stimulation conditions. Figure 19B is a series of bar graphs depicting the relative immunoreactivity of Aβ normalized to EYFP according to some embodiments. Specifically, Figure 19B shows the relative immunoreactivity of Aβ normalized to EYFP (n = 4 mice / group, 1908 indicates p < 0.05 and 1920 indicates p < 0.01, performed by one-way ANOVA).

[0364] Figure 20A is a series of immunofluorescence images showing the immunohistochemistry of the CA1 region of the hippocampus in 5XFAD / PV-Cre using anti-Aβ antibodies according to some embodiments. Specifically, Figure 20A is a series of immunofluorescence images showing the immunohistochemistry of the CA1 region of the hippocampus in 5XFAD / PV-Cre using anti-Aβ 2002 (12F4) antibodies in EYFP, 40-Hz and random stimulation conditions (scale bar = 50 µm). Figure 20B depicts Aβ normalized to EYFP according to some embodiments.Bar graphs of relative immunoreactivity. Specifically, Figure 20B shows the relative immunoreactivity of Aβ normalized to EYFP (n = 4 mice / group, 2004 indicates p < 0.05 and 2006 indicates p < 0.001, performed by one-way ANOVA). When compared with the EYFP group, the intensity of Aβ-labeling was reduced by 39.5% after 40-Hz stimulation of FS-PV-interneurons in three-month-old 5XFAD / PV-Cre double transgenic mice, and significantly increased by 187.0% after random stimulation (see, for example, Figures 19A, 19B, 20A and 20B, p < 0.05 and p < 0.01, performed by one-way ANOVA, n = 4 mice / group).

[0365] Brain amyloid concentration can depend on the rate of Aβ production and clearance. In some embodiments, the Aβ peptide is generated by the sequential proteolytic cleavage of APP by β-secretase and γ-secretase. When BACE1 cleaves the entire APP protein, the CTF and NTF of APP are generated. In some embodiments, to elucidate how 40-Hz stimulation reduces Aβ levels, the effect of γ on APP cleavage was examined by measuring the levels of the cleavage intermediates CTF and NTF after stimulation of the FS-PV interneurons. Following 40-Hz stimulation, a significant 18.6% reduction in CTF was observed compared to the EYFP group and a significant 19.7% reduction compared to the randomized group (p < 0.05 and p < 0.01, performed by one-way ANOVA, n = 6 mice / group).

[0366] Figure 21A is a representative Western blot depicting the levels of APP (CT695), APP NTF (A8967), APP CTF (CT695), and β-actin (A5316) (load control) in CA1 under EYFP, random, and 40-Hz stimulation conditions according to some embodiments, one mouse / lane, with two biological replicates for each condition. Figure 21B is a bar graph depicting the relative immunoreactivity of APP CTF according to some embodiments. Specifically, Figure 21B shows the relative (normalized to actin) immunoreactivity of APP CTF at 40-Hz relative to EYFP and random conditions (n ​​= 6 mice / group, one asterisk 2102 indicates p < 0.05, and two asterisks 2104 indicate p < 0.01, performed by one-way ANOVA). Figure 21C is a series of Western blots depicting the levels of full-length APP 2106 (CT695), APP CTF 2108 (CT695), and β-actin 2112 (A5316, loading control) in CA1 according to some embodiments. Specifically, Figure 21C shows the levels in EYFP, randomized, and...Instructions for Use, Page 33 / 78, CN 122399187 A: Levels of full-length APP 2106 (CT695), APP CTF 2108 (CT695), and β-actin 2112 (A5316, load control) in CA1 under 40-Hz stimulation conditions, one mouse / lane, with two biological replicates for each condition.

[0367] Figure 22A is a bar chart depicting the relative (normalized to actin) immunoreactivity of APP NTF at 40-Hz relative to EYFP and random conditions (n ​​= 6 mice / group, symbol “ns” 2204 indicates non-significant, and 2202 indicates p < 0.05, performed by one-way ANOVA). Figure 22B is a bar chart depicting the relative (normalized to actin) immunoreactivity of full-length APP at EYFP, random, and 40-Hz conditions (n ​​= 6 mice / group, performed by one-way ANOVA).

[0368] In some embodiments, following 40-Hz stimulation, APP NTF levels were found to be significantly reduced by 28.5% compared to the EYFP group and by 28.2% compared to the randomized group (see, e.g., Figures 21A, 22A, and 21C, p < 0.05, performed by one-way ANOVA, n = 6 mice / group). Furthermore, the levels of full-length APP appeared to be similar across the groups, thus indicating that the reduction in Aβ was not due to changes in precursor levels (see, e.g., Figures 21A, 22B, and 21C, n = 6 mice / group in the APP experiment). In some embodiments, changes in full-length APP may be difficult to detect due to the relatively high abundance of APP compared to its cleavage products in this mouse model.

[0369] In some embodiments, APP processing occurs within the vesicular transport pathway, and previous work has shown that APP is transported to circulating endosomes after active stimulation. Furthermore, enlarged early endosomes have been observed in brain tissue from AD patients and in human neurons derived from AD patients. In some embodiments, to test whether gamma stimulation affects endosome abundance in experimental animals, two markers, EEA1 (early endosome antigen 1) and Rab5 (Ras-associated protein encoded by the RAB5A gene), were used to characterize early endosomes in CA1 after 40 Hz stimulation and random stimulation. Figure 23 is a series of immunofluorescence images (scale bar = 50 µm) showing the immunohistochemistry of three-month-old 5XFAD / PV-Cre mice using an anti-Rab5 (ADI-KAp-GP006-E) antibody under EYFP, 40-Hz, and random stimulation conditions.

[0370] Figure 24A is a bar graph representing the relative immunoreactivity of EEA1 normalized to EYFP according to some embodiments (n =Four mice per group, one asterisk 2402 indicates p < 0.05, and two asterisks 2402 indicate p < 0.01, by one-way ANOVA. Figure 24B is a bar chart depicting the relative Rab5 intensity levels of CA1 from 5XFAD / PV-Cre under EYFP, 40-Hz, and random stimulation conditions according to some embodiments (n = 3 mice per group, three asterisks 2408 indicate p < 0.001, by one-way ANOVA). In some embodiments, EEA1 staining produces dotted cytoplasmic and juxtamembranous patterns in the neuronal cell body, which is typical for early endosomes (see, e.g., Figure 19A). In some embodiments, Rab5 labeling is primarily confined to the cell body and plasma membrane, represented by small, thin patches concentrated within the endosome and membrane compartments (see, e.g., Figure 23). In summary, early endosome labeling of CA1 neurons demonstrated significantly reduced EEA1 (39.7%) and Rab5 (40.1%) staining intensities after 40-Hz stimulation compared to EYFP controls (see, e.g., Fig. 19A, Fig. 23, Fig. 24A, p < 0.05 and p < 0.001, performed by one-way ANOVA, n = 2 slices from 3 mice / group). In contrast, random stimulation of FS-PV-interneurons increased EEA1 staining intensity by 122% compared to EYFP controls (see, e.g., Fig. 19A and Fig. 24A, p < 0.01, performed by one-way ANOVA, n = 2 slices from 3 mice / group). In some embodiments, treatment-dependent changes in EEA1 staining intensity were similar to changes in Aβ in CA1 (see, e.g., Figures 19A-19B, 20A-20B, and 24A-24B, p < 0.05, performed by one-way ANOVA, n = 2 slices from 3 mice / groups). These results indicate that, in addition to the observed changes in CTF, 40-Hz stimulation alters EEA1 and Rab5, thus indicating differences in general endosomal treatment.

[0371] Figure 25A is a bar graph depicting the levels of the Aβ peptide isoform Aβ1-40 after different types of stimulation in the CA1 region of the hippocampus of subjects according to some embodiments. In the experiments described, one hour of optogenetic stimulation 502 of FS-PV+ at approximately 40 Hz reduced the level of Aβ1-40 in the hippocampal CA1. Excitatory pyramidal stimulation 506 at 8 Hz and excitatory pyramidal stimulation 508 at 40 Hz did not significantly affect the level of Aβ1-40. Random 40-Hz stimulation 504 and specifically random excitatory pyramidal stimulation (page 34 / 78, CN 122399187 A) significantly increased Aβ1-40 levels (n = 4-9 animals / group).

[0372] Figure 25B is a bar graph depicting the reduction of Aβ peptide isotype Aβ1-42 after stimulating specific cell types in the CA1 region of the hippocampus of subjects with gamma oscillations according to some embodiments. In the experiments, one hour of optogenetic stimulation 516 at approximately 40 Hz FS-PV+ reduced Aβ1-42 levels in the CA1 region of the hippocampus (n = 2-4 animals / group). Stimulation 520 at 8 Hz, excitatory pyramidal stimulation 522 at 40 Hz, and excitatory pyramidal stimulation 524 at 8 Hz increased Aβ1-42 levels. Random 40-Hz stimulation 518 and specifically random excitatory pyramidal stimulation 526 significantly increased Aβ1-42 levels.

[0373] Figure 25C is a series of images showing an increase in the levels of full-length APP 528, 534 (normalized to actin 532) and a decrease in the levels of CTF (e.g., β-CTF) 530, 536 (normalized to actin 532) after stimulating specific cell types in the CA1 region of the hippocampus of subjects according to some embodiments. FS-PV+ stimulation at 40 Hz decreased APP β-CTF levels and increased full-length APP levels compared to randomized 40-Hz control conditions (n ​​= 4-6 animals / group). Since β-CTF is an APP derivative generated during the amyloid cleavage of APP via BACE1, higher β-CTF levels represent increased Aβ production.

[0374] Figures 26A-26B are immunofluorescence images showing the endosome levels (based on EEA1 levels) after different types of stimulation in the CA1 region of the hippocampus of subjects according to some embodiments. Specifically, a comparison of Figure 26B with Figure 26A shows that FS-PV+ 40-Hz stimulation induced by γ oscillations reduced EEA1 levels (a marker of endosome levels) compared to random FS-PV+ stimulation at 900 Hz, as measured by immunofluorescence (n = 3 mice / group, p = 0.007). Reduced endosome levels in cells indicate a decreased interaction between APP and β-secretase, which leads to reduced APP cleavage and Aβ production. Therefore, the study shows that γ oscillations reduce AP production in an AD mouse model because increased endosome levels indicate increased APP treatment and thus increased Aβ production.

[0375] Figure 27 is a bar graph depicting the mean intensity values ​​(normalized to FAD) of immunofluorescence images for Figures 26A–26B after different types of stimulation of the CA1 region of the hippocampus in subjects according to some embodiments.

[0376] γ stimulation induces morphological transformation of microglia.

[0377] In some embodiments, in order to further explore the cellular and molecular effects of 40-Hz stimulation in an unbiased manner,Whole-genome RNA-seq of the CA1 region of the hippocampus was performed in 5XFAD / PV-Cre double transgenic mice after one hour of 40-Hz FS-PV-interneuron stimulation or no stimulation (EYFP). In the RNA-seq experiment, an average of 26,518,345 sequencing reads was obtained from three stimulated mice and three unstimulated mice. Data QC analysis revealed an average of 183 exon / intron ratios, an average of 272 exon / intergenic ratios, and an average of 3.6% of the percentage of ribosomal RNA reads. The analysis identified 523 differentially expressed genes (DEGs), of which 130 were upregulated and 393 were downregulated in response to 40-Hz stimulation.

[0378] Figure 28 is a heatmap showing the differentially expressed genes identified by whole-transcriptome RNA-seq of the CA1 region of the mouse hippocampus with and without 40-Hz stimulation. Normalized z-scores were calculated for each differentially expressed gene (row). Colors represent relatively low and high levels of gene expression. Table 2 (below) presents 130 genes upregulated by 40-Hz FS-PV-interneuron stimulation (p < 0.05, using Cufflinks 2.2 software (available from the Trapnell lab at the University of Washington, Seattle, Washington, for assembling transcripts, estimating their abundance, and testing differential expression and regulation in RNA-seq samples)).

[0379] Table 2 Specification 35 / 78 pages 37 CN 122399187 A

[0380] Specification 36 / 78 pages 38 CN 122399187 A

[0381]

[0382] Table 3 (below) presents 393 genes downregulated by 40-Hz FS-PV-interneuron stimulation (p < 0.05, using Cufflinks 2.2 software (available from the Trapnell lab at the University of Washington, Seattle, Washington)).

[0383] Table 3

[0384] Specification 37 / 78 pages 39 CN 122399187 A

[0385] Specification 38 / 78 pages 40 CN 122399187 A

[0386] Specification 39 / 78 pages 41 CN 122399187 A

[0387]

[0388] In some embodiments, the upregulated gene typically has a higher expression value than the downregulated gene. Figure 29 is a box plot showing the FPKM values ​​of upregulated and downregulated genes under EYFP and 40-Hz conditions according to some embodiments. The boxes show the median (black line in the box) and quartiles (top and bottom of the box), which represent the minimum and maximum values, and circles representOutliers. Upregulated genes are highly enriched in microglia. Specifically, about 35% of all upregulated genes have their highest expression in microglia (of which about 19% are in neurons, about 17% in endothelial cells, about 14% in astrocytes, about 9% in myelinated oligodendrocytes, about 5% in oligodendrocyte precursor cells, and about 1% in newly formed oligodendrocytes).

[0389] Figure 30 is a pie chart showing the cell type-specific expression patterns of upregulated genes identified after 40-Hz stimulation according to some embodiments. Gene FPKM values ​​were calculated based on publicly available RNA-seq data from different brain cell types, including astrocytes, endothelial cells, microglia, myelinated oligodendrocytes (MO), neurons, newly formed oligodendrocytes (NFo), and oligodendrocyte precursor cells (OPC). Therefore, RNA-seq analysis strongly suggests that one hour of 40-Hz stimulation of FS-PV-interneurons leads to changes in the cellular state of microglia, which is important given the growing evidence that these cells play a role in AD lesions.

[0390] In some embodiments, to further explore the potential role of 40-Hz stimulation for microglia, gene set enrichment analysis was used to compare a range of publicly available RNA-seq datasets from microglia, peripheral macrophages, and neurons under different chemical and genetic perturbations with a list of genes characterized from some embodiments described herein. Table 4 (below) shows the GSEA-based statistical significance of the correlations between genes upregulated and downregulated by 40-Hz stimulation and publicly available neuron, microglia, and macrophage-specific RNA-seq data under different chemical and genetic perturbations.

[0391] Table 4

[0392]

[0393] Interestingly, the transcriptomic changes following 40-Hz stimulation were more similar to those caused by increased neural activity (via NMDA and pasminoides) and less similar to those caused by silencing activity (via tetrodotoxin). These findings also support the finding that 40-Hz stimulation of FS-PV interneurons does not decrease neuronal activity. Furthermore, the immediately early genes Nr4a1, Arc, and Npas4, known to be upregulated by neuronal activity, are elevated after one hour of 40-Hz stimulation, as shown by both RNA-seq and RT-qPCR. Figure 31 is a bar chart showing RT-qPCR validation of specific gene targets in an RNA-seq dataset according to some embodiments. The bar chart shows the relative RNA levels (fold change) from EYFP 3102 and 40-Hz stimulation 3104 conditions.(One asterisk indicates p < 0.05, two asterisks indicate p < 0.01, and three asterisks indicate p < 0.001, performed by Student's t-test, n = 3 mice / group). Top downregulated genes were Grin4 and Camk2d (see, e.g., Figure 31, p < 0.05, n = 3 mice / group).

[0394] Additionally, transcriptomic results indicated 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 known to promote microglia Aβ uptake). Figures 32A and 32B are graphs showing the power spectral density of local field potentials recorded above the brain during 40 Hz light scintillation according to some embodiments. Figures 32A and 32B show no increase in power at 40 Hz, therefore the effect is not due to photoelectric effects or electrical noise on the recording equipment (n = 4, 2, 1, 1, 17, 42, 36, 55, 53 40-Hz flicker time intervals, from four recording sessions of three 5XFAD animals undergoing visual cortex recording and five recording sessions of two 5XFAD mice and three WT mice undergoing hippocampal toe recording). The mean (solid line) and standard deviation (shaded area) across recordings are shown in the left panel (Figure 32A), and the mean (solid line) and standard deviation (shaded area) for each animal are shown in the right panel (Figure 32B). Recording 3202, with fewer than 3 flicker time intervals, caused more noise in the power spectral density than recording 3204, which had more data, but neither showed evidence of a peak at 40 Hz. In some embodiments, RT-qPCR was performed to validate upregulated genes involved in known microglial cell function (page 41 / 78, CN 122399187 A). Genes associated with microglial cell phagocytosis (including Cd68, B2m, Bst2, Icam1, and Lyz2) were confirmed to be upregulated in the CA1 region of the hippocampus following 40-Hz stimulation.

[0395] Figure 33 is a bar chart depicting RT-qPCR validation of specific gene targets in an RNA-seq dataset according to some embodiments. Figure 33 shows the relative RNA levels (fold change) in EYFP 3302 and 40-Hz stimulation 3304 conditions (one asterisk indicates p < 0.05 and two asterisks indicate p < 0.01, performed by Student's t-test, n = 6 mice / 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, for example, Figure 33, p < 0.05 and p < 0.01, performed by Student's t-test, n = 6 mice / group). RT-qPCR also showed no change in the expression levels of pro-inflammatory genes Il6, Il1b (Il1-β), Itgam (CD11-b) and anti-inflammatory gene Igf1 (see, for example, Figure 33, p > 0.05, performed by Student's t-test, n = 6 mice / group). Therefore, the transcriptomic results described herein indicate that 40 Hz neuronal stimulation induces microglia into a state that promotes uptake.

[0396] Given that 40-Hz stimulation upregulates both phagocytosis-related genes and migration / cell adhesion-related genes, the morphological features of microglia activation were examined. In some embodiments, the microglia marker Iba1 is used to label microglia in CA1 sections of the hippocampus of 5XFAD / PV-Cre mice after one hour of 40 Hz, random or no stimulation (EYFP mice). Figure 34 is a series of immunofluorescence images showing the immunohistochemistry of the CA1 region of the hippocampus of 5XFAD / PV-Cre mice under EYFP, 40-Hz and random stimulation conditions using anti-Iba1 3402 (019-19741) antibody and anti-Aβ 3404 (12F4) antibody. Images were taken using a 40x objective (scale bar = 50 µm). Arrows indicate +Iba1 / +Aβ signals in the cell bodies.

[0397] Figure 35A is a bar graph depicting the number of microglia in EYFP and 40-Hz conditions according to some embodiments (n = 2 sections from 4 mice / groups). Figure 35B is a bar chart depicting the diameter of microglial cell bodies normalized to EYFP under EYFP, 40-Hz, and random stimulation conditions according to some embodiments (n = 2 slices from 4 mice / group). Figure 35C is a bar chart depicting the mean length of microglial primary processes or processes normalized to EYFP under EYFP, 40-Hz, and random stimulation conditions according to some embodiments. Figure 35D is a bar chart depicting the percentage of Iba1-positive (microglial) cell bodies that are also Aβ-positive under EYFP and 40-Hz stimulation conditions according to some embodiments (n = 2 slices from 4 mice / group). The symbol “ns” 3502 indicates non-significant, two asterisks 3504 indicate p < 0.01, three asterisks 3506 indicate p < 0.001, and four asterisks 3508 indicate p < 0.0001, performed by one-way ANOVA.

[0398] First, the number of Iba1+ microglia in 6 animals / conditions was counted and compared with the unstimulated EYFP condition (mean of 8 microglia / ROI) (see, e.g., Figures 34 and 35A, p < 0.01, performed by one-way ANOVA, n = 2 slices from 4 mice / group) and with the random condition (mean of 10 microglia / ROI) (see, e.g., Figures 34 and 35A, p < 0.05, performed by one-way ANOVA, n = 2 slices from 4 mice / group). Almost twice as many microglia were observed in the 40 Hz group (15 microglia / 212.55 μm x 212.55 μm region of interest (ROI)). Previous studies have shown that two key characteristics of phagocytic microglia are increased cell body size and decreased process length; therefore, how these characteristics are affected by 40-Hz stimulation was examined. In some implementations, the diameter of each clearly labeled Iba1+ cell body in the field of view was measured. It was found that after 40-Hz stimulation, the microglial cell body diameter increased by 135.3% compared to no stimulation and by 138.7% compared to the random condition (see, e.g., Figures 34 and 35B, p < 0.0001, performed by one-way ANOVA, n = 2 slices from 4 mice / group). The length of the primary microglial process in each condition was measured, and a 54.0% reduction in the length of the primary microglial process was observed in the 40-Hz stimulation condition compared to the EYFP control and a 38.5% reduction compared to the random stimulation (see, e.g., Figures 34 and 35C, p < 0.0001, performed by one-way ANOVA, n = 2 slices from 4 mice / group). These findings were not affected by Iba1 levels, as no differences in Iba1 expression were observed between conditions in the gene expression analyses described herein (see, for example, Tables 2 and 3). Therefore, the increase in cell body size and decrease in process length observed after 40-Hz stimulation are morphological changes consistent with the shift of these microglia toward a phagocytic state. Potential co-localization of Aβ within microglia was assessed as a means of evaluating microglia Aβ uptake following co-immunostaining with an Aβ antibody (12F4, which does not cross-react with APP). In CA1 neural fiber networks where Iba1+ cells are predominantly localized, the ratio of microglia with Aβ / Iba1 co-localization (ImageJ, Fuji co-localization plugin) in the cell body to the total number of microglia increased by 54.9% compared to the EYFP control and by 50% compared to the random condition after 40-Hz stimulation.0.3% (see, for example, Figures 34 and 35D, p < 0.01, performed by one-way ANOVA, n = 2 slices from 4 mice / group). Iba1 / Aβ signal overlap during microglia processing was excluded to avoid including potentially random non-phagocytic colocalization.

[0399] In some embodiments, to provide better resolution of the presence of Aβ signal within microglia, 3D renderings of microglia from this tissue and videos from these renderings are created. Figure 36 is a series of 3D renderings formed according to some embodiments by incorporating immunofluorescence images from Figure 34, 3602 rotated 0 degrees, 3604 rotated -25 degrees about the Y-axis, and 3606 rotated 30 degrees about the X-axis. Images were taken using a 40x objective (scale bar = 50 µm). In summary, gene expression and morphological analyses suggest that 40-Hz stimulation affects microglia activity by increasing recruitment of microglia to the stimulation site and enhancing their phagocytic activity, thereby leading to increased association with Aβ. Importantly, in some embodiments, no evidence of neuronal loss was found when measuring the thickness of the CA1 cell layer using nuclear staining with Hoechst. The mean CA1 volume was not significantly different between the EYFP and 40-Hz stimulation groups.

[0400] Figure 37A is a series of immunofluorescence images showing the immunohistochemistry of the CA1 region of the hippocampus in 5XFAD / PV-Cre under EYFP and 40-Hz stimulation conditions according to some embodiments. Figure 37B is a bar graph depicting the estimated CA1 thickness of 5XFAD / PV-Cre under EYFP and 40-Hz stimulation conditions according to some embodiments (n = 4 mice / group, “ns” indicates non-significant, performed by Student's t-test).

[0401] Next, according to some embodiments, differential gene expression in 5XFAD mice infected with AAV-DIO-ChR2-EYFP and stimulated with 40-Hz FS-PV+ stimulation (TREAT) or control stimulation (CTRL) was assessed by whole-genome RNA-seq of hippocampal CA1 after one hour of stimulation. Figure 38A is a heatmap showing 523 differentially expressed genes (DEGs) identified by whole-genome RNA-seq of hippocampal CA1 after TREAT or CTRL according to some embodiments. Each row in Figure 38A represents a DEG, and the columns in Figure 38A represent transcriptome maps of three individual control animals and three individual treated (40-Hz FS=PV+ stimulation) animals.

[0402] Figure 38B is a graph showing the overlap between DEGs upregulated in the TREAT condition in Figure 38A according to some embodiments. In Figure 38B, compared with random FS-PV+ stimulation, the expression of DEGs upregulated by FS-PV+...40-Hz stimulation induced γ oscillations that reduced Iba1 levels, as measured by immunofluorescence (n = 3 mice / group, p = 0.006). Figure 38B shows a significant and specific overlap between genes upregulated in TREAT conditions and microglia genes upregulated by anti-inflammatory microglia activation (i.e., MCSF genes). The genes were upregulated in microglia to a greater extent than in astrocytes, endothelial cells, myelinated oligodendrocytes (MOs), neurons, newly formed oligodendrocytes (NFOs), and oligodendrocyte precursor cells (OPCs). Table 5 (below) presents the microglia / macrophage pathways used for gene upregulation.

[0403] Table 5 Specification 43 / 78 pages 45 CN 122399187 A

[0404]

[0405] According to some embodiments, RT-qPCR was performed to validate specific gene targets from RNA-seq datasets. Figure 39 is a bar chart depicting RT-qPCR validation of specific gene targets in the RNA-seq dataset of Figure 38A according to some embodiments. Specifically, Figure 39 shows the fold change (normalized to GAPDH) of specific gene targets (including genes CSF1, CSF1R, ll-6, ll1-β, CD11-b, CYBA, Hmox1, H2-K1, Lgals3, and Icam1) under control and treatment conditions.

[0406] Figure 40 is a diagram illustrating the upregulated genes of Figure 38A and their associated biological processes according to some embodiments. Importantly, the upregulated genes in Figure 40 are specifically associated with immune-related processes. The upregulated genes belong to immune-related biological processes, including lymphocyte-mediated processes, adaptive immune processes, and immunoglobulin-mediated processes. Figure 41 is a diagram illustrating the downregulated genes of Figure 38A and their associated biological processes according to some embodiments. Downregulation of genes is a biological process, including cell movement, cell-cell signaling, synaptic transmission, motor behavior, and neuronal processes, as shown in Figure 41.

[0407] Figure 42A is a series of immunofluorescence images showing the level of Iba1 in the CA1 region of the hippocampus of subjects after different types of stimulation according to some embodiments. Figure 42B is a bar graph depicting the average intensity values ​​of the immunofluorescence images of Figure 42A according to some embodiments. Figure 42A shows that endosomal levels are reduced by optogenetic enhancement of γ rhythms. The level of EEA1 (a marker of endosomal) is reduced by γ oscillation induced by FS-PV+ 40-Hz stimulation, as measured by immunofluorescence (n = 3 mice / group, p = 0.08). The results show that γ oscillations reduce Aβ production in the AD mouse model because increased endosomal levels indicate increased APP treatment and therefore increased Aβ production.

[0408] Taken together, the results show that γThe restoration or induction of rhythms restores molecular lesions in mouse models of AD. Cell-type-specific and temporarily precise reintroduction of gamma oscillations via optogenetics reduces the production and enhances the clearance of isotypes Aβ1-40 and Aβ1-42, which aggregate to trigger numerous degenerative cascades involved in AD neuropathies. Furthermore, this treatment induces anti-inflammatory microglial signaling pathways, thereby counteracting immune mechanisms associated with neurodegeneration.

[0409] According to some embodiments, cell-type-specific and temporarily controlled gamma oscillations can be induced in the hippocampus, visual cortex, barrel cortex, and / or auditory cortex without optogenetics.

[0410] Visual stimulation at gamma frequencies noninvasively drives gamma oscillations in the visual cortex.

[0411] The significant reduction in Aβ levels achieved through optogenetic stimulation at 40 Hz led to the exploration of other ways to induce 40-Hz oscillations in the brain to ensure that the effect is not specific to optogenetic manipulation or invasive procedures in any way. To examine whether photo-scintillation can be used as a non-invasive method to induce 40-Hz oscillations in the visual cortex, in some embodiments, animals are exposed to continuous light alternating with periods of darkness for 40 Hz or random scintillation.

[0412] Figure 43A is a schematic diagram showing mice exposed to photo-scintillation stimulation according to some embodiments. To determine whether this photo-scintillation alters Aβ, animals are exposed to 40-Hz photo-scintillation for one hour, consistent with the duration of optogenetic stimulation that reduces Aβ as described herein. The photo-scintillation covers the entire visual field of the animals. As controls for molecular and cellular assays, three-month-old 5XFAD mice are kept in constant darkness for three days or treated with constant light or 20-Hz or 80-Hz scintillation for one hour (see, for example, Figure 43A).

[0413] Figure 43B includes graphs of local field potential traces and power spectral densities in the visual cortex before and during 40-Hz photo-scintillation according to some embodiments. The mean (solid line) and standard deviation (shaded area) of the power spectral density in the visual cortex during 40-Hz light flicker 4302, random light flicker 4304, or darkness 4306 processes are shown (n = 4 5FXFAD mice from 5 recording sessions). Figures 43C-43F are plots depicting the power spectral density of the local field potential in the visual cortex for each recording session per mouse during 40-Hz light flicker, random light flicker, constant darkness, and constant light processes according to some embodiments (n = 5 recordings from four 5XFAD mice, using 47, 51, 61, 49, 16...).40-Hz flicker, 47, 50, 64, 50, 16 random flickers, 279, 302, 382, ​​294, 93 dark periods, and 47, 50, 64, 49, 15 light periods. In the visual cortex, it was found that light flicker at 40 Hz increases the power in the LFP at 40 Hz (see, for example, Figures 43B and 43C), while random-interval light flicker and darkness do not increase said power (see, for example, Figures 43B, 43D, and 43E).

[0414] Figure 44A is a series of histograms depicting the peak fraction in the visual cortex as a function of time for four periods and equivalent time periods of random light flicker for 40-Hz light flicker according to some embodiments. Figure 44A shows a bar chart of the fraction of spikes in the visual cortex as a function of time for four cycles 4402 or equivalent time periods of random light flicker 4404 for 40-Hz light flicker (n = four 5XFAD mice from five recording sessions, bars indicate the mean and error bars indicate SEM across animals). The upper bars indicate the time when the light is on 4406 or off 4408. In some embodiments, the spikes increase and decrease as the light flicker is on and off, thereby causing a spike phase locked to the 40-Hz frequency during the 40-Hz stimulation process (bar chart 4402 in Figure 44A), but no distinct frequency is observed during the random stimulation process (bar chart 4404 in Figure 44A).

[0415] Figure 44B is a series of traces of local field potentials recorded above the brain during light flicker according to some embodiments. In some embodiments, when recorded from saline directly above the brain, no increase in 40 Hz power was observed during 40 Hz flickering, thus indicating that this effect is not due to photoelectric effects or electrical noise (see, for example, Figures 32 and 44B). As in the case of optogenetic stimulation, random flickering provides a control for the overall change in activity resulting from light flickering.

[0416] Figure 45A is a bar chart showing the difference in firing rates between 40 Hz light flickering and random light flickering according to some embodiments (n = 226 stimulation time periods from five recording sessions in four 5XFAD mice). Figure 45B is a graph showing the multi-unit firing rates in the visual cortex during 40 Hz light flickering, random light flickering, darkness, and light time periods according to some embodiments. Figure 45B shows the multi-unit firing rates in the visual cortex. The box plot shows the median (white line in the box) and quartiles (top and bottom of the box). In all animals, the firing rate was not significantly different between 40-Hz flicker and random flicker conditions, thus demonstrating that random stimulus conditions act as a control for spiked activity (rank-sum test against each of the five recorded sessions from four 5XFAD mice, p > 0.06, median and quartiles are shown in the figure, n =47, 51, 64, 49, and 16 40-Hz flickering time periods and 47, 50, 64, 50, and 16 random flickering time periods / records). There was no significant difference in firing rate between 40-Hz flickering and light conditions, indicating that 40-Hz light flickering generally does not lead to excessive neuronal excitability (rank-sum test for each of the five recording sessions from four 5XFAD mice, p > 0.2 for four recording sessions and p > 0.01 for one recording session; this was not significant when corrected for multiple comparisons, medians and quartiles are shown in the figure, n = 47, 51, 64, 49, and 16 40 Hz time periods and 47, 50, 64, 49, and 16 light time periods / records). Within a single session, more activity was observed in the 40-Hz stimulus than in the dark condition. The difference in multi-unit firing rate between 40 Hz and random flickering time periods tends to be close to zero (see, e.g., 45A); and no significant differences were found when comparing these time periods in animals (see, e.g., Figure 45B, rank-sum test for each of five recorded sessions from four 5XFAD mice, p > 0.06, median and quartiles shown in the figure, n = 47, 51, 64, 49, 16 γ flickering time periods and 47, 50, 64, 50, 16 random flickering time periods / recording). Specification 45 / 78 pages 47 CN 122399187 A

[0417] Visual stimulation at γ frequencies reduces Aβ levels in the visual cortex.

[0418] Given the efficacy of optogenetic methods, a translational, non-invasive amyloid reduction treatment was designed. Figure 46A is a schematic diagram illustrating an experimental paradigm according to some embodiments. As shown in Figure 46A, a first subgroup of AD model mice was placed in a first chamber 4600 with a 40-Hz flash, and a second subgroup of AD model mice was placed in a second chamber 4602 kept dark. The animals in the first chamber 4600 were exposed to the 40-Hz flash for approximately one hour.

[0419] Figures 46B and 46C are graphs that further illustrate the changes in baseline levels of Aβ peptide isoforms Aβ1-40 and Aβ1-42 after the experimental paradigm of Figure 46A, according to some embodiments. Figure 46B shows that 40-Hz light exposure in 5XFAD mice significantly reduced the levels of Aβ1-40 and Aβ1-42 in the visual cortex V1. The levels of Aβ1-40 and Aβ1-42 were presented as pg / mL (n = 6 animals / group).

[0420] Given that 40-Hz light flicker drives 40-Hz oscillations in the primary visual cortex and that 40-Hz oscillations optogenetically induce a decrease in Aβ levels in the hippocampus, the objective is to determine whether 40-Hz light flicker can reduce Aβ levels in the visual cortex.In some implementations, pre-symptom 5XFAD mice were used. The mice were placed in a dark chamber and exposed to 40-Hz light flickering, constant light on (light), or constant light off (dark) for one hour.

[0421] Figures 47A and 47B are bar graphs depicting the changes in baseline levels of Aβ1-40 and Aβ1-42 in the 5XFAD visual cortex under various embodiments under dark, light, 40-Hz flicker, 20-Hz flicker, 80-Hz flicker, 40-Hz flicker with PTX, and random flicker conditions (n ​​= 12 mice / group for dark; n = 6 mice / group for light, 40-Hz flicker, 20-Hz flicker, 80-Hz flicker, and PTX; n = 4 mice / group for random flicker; “ns” indicates non-significant, one asterisk indicates p < 0.05, and two asterisks indicate p < 0.01, performed by one-way ANOVA). Figures 47A and 47B show the mean and SEM. Circles superimposed on the bars in the bar graphs indicate individual data points in each group. One hour after light exposure, a 57.96% reduction in Aβ1-40 levels and a 57.97% reduction in Aβ1-42 levels were observed in the visual cortex compared to the dark conditions (as measured by Aβ ELISA, see, e.g., Figures 47A and 47B, p < 0.05, performed by one-way ANOVA, n = 6 mice / group). Compared to the light control, after one hour of 40-Hz flicker, amyloid levels were reduced by 62.47% (Aβ1-40) and 68.55% (Aβ1-42) (as measured by Aβ ELISA, see, e.g., Figure 47, p < 0.05, performed by one-way ANOVA, n = 6 mice / group). Furthermore, the effect is specific to 40-Hz flicker, as 20-Hz, 80-Hz, or random flicker did not significantly reduce Aβ levels compared to dark and light controls (see, for example, Figure 47, “ns” indicates non-significant, n = 6 mice / group).

[0422] In some embodiments, to test region specificity, Aβ levels in the somatosensory barrel cortex (BC) were examined and no significant differences were found. Figure 48A is a bar graph depicting relative Aβ1-40 and Aβ1-42 levels in the 5XFAD barrel cortex under dark and 40-Hz flicker conditions according to some embodiments (n = 3 mice / group; “ns” indicates non-significant, performed by Student's t-test). When 5XFAD mice were pretreated with a low dose of a GABA-A antagonist (tetracycline toxin, 0.18 mg / kg, which does not induce epileptic activity), the effect of 40-Hz flicker on Aβ levels was completely eliminated, thus indicatingγ-aminobutyric acid (GABAergic) signaling (most likely from FS-PV-interneurons) is necessary for this effect (see, for example, Figure 47, “ns” indicates non-significant, n = 6 mice / group).

[0423] To demonstrate that the effect is not specific to 5XFAD mice, this result was replicated in different AD models, APP / PS1 mice (a well-validated model with two familial AD mutations (APP Swedish and PSEN1 deltaE9)). Figure 48B is a bar graph depicting the changes in baseline levels of Aβ1-40 and Aβ1-42 in the APP / PS1 visual cortex under dark and 40-Hz flicker conditions according to some embodiments (n = 5 mice / group for dark conditions and n = 4 mice / group for 40-Hz flicker conditions; “ns.” indicates non-significant and an asterisk indicates p < 0.05, performed by Student's t-test).

[0424] Figure 48C is a bar graph depicting the changes in baseline levels of Aβ1-40 and Aβ1-42 in the WT visual cortex under dark and 40-Hz flicker conditions according to some embodiments (n = 11 mice / group for dark conditions and n = 9 mice / group for 40-Hz flicker conditions; an asterisk indicates p < 0.05, performed by Student's t-test). In some implementations, a trend was observed in APP / PS1 mice after 40-Hz flickering treatment that Aβ1-40 was significantly reduced by 20.80% and Aβ1-42 by 37.68%, although the latter was not significantly different from the dark condition (see, e.g., Figure 48B, Aβ1-40 p < 0.05, Aβ1-42 p < 0.09 – non-significant, performed by Student's t-test, n = 5 mice / group for dark, n = 4 mice / group for 40-Hz flickering). Furthermore, in aged WT mice, a 58.2% reduction in endogenous mouse Aβ1-40 was found after one hour of 40-Hz flickering (see, e.g., Figure 48C, p < 0.05, performed by Student's t-test, n = 11 dark mice and n = 9 40-Hz flickering mice). In these animals, Aβ1-42 was below detectable levels in both the scintillation group and the control group. The reduction in endogenous mouse Aβ1-40 in WT animals reveals that these results are not limited to Tg APP expression or mutant APP; rather, they extend to Aβ generated from APP, whose expression is driven by its endogenous promoter. Figures 48A-48C show the mean and SEM.

[0425] Next, in some embodiments, an investigation is conducted to determine whether 40-Hz flicker alters microglial cell activity in the visual cortex in the same way as 40-Hz optogenetic FS-PV-interneuron stimulation alters CA1 microglial cells in the hippocampus. Figure 49 is a series of immunofluorescence images showing immunohistochemistry of anti-Iba1 (019-19741) antibody and anti-Aβ 4904 (12F4) antibody in the 5X FAD visual cortex under dark and 40-Hz flicker conditions according to some embodiments. Images were taken using a 40x objective (scale bar = 50 µm). Right panel: 120X magnification; arrows indicate +Iba1 / +Aβ signals in cell bodies.

[0426] Figure 50A is a bar graph depicting the number of microglial cells under dark and 40-Hz flicker conditions according to some embodiments (n = 2 slices from 4 mice / group; “ns” indicates non-significant, performed by Student's t-test). Figure 50B is a bar chart depicting the diameter of microglial cell bodies normalized to the control under dark and 40-Hz flicker conditions according to some embodiments (n = 2 slices from 4 mice / group; two asterisks indicate p < 0.01, performed by Student's t-test). Figure 50C is a bar chart depicting the mean length of primary processes in microglial cells normalized to the control under dark and 40-Hz flicker conditions according to some embodiments (n = 2 slices from 4 mice / group; four asterisks indicate p < 0.0001, performed by Student's t-test). Figure 50D is a bar chart depicting the percentage of Iba1-positive (microglial) cell bodies that are also Aβ-positive under dark and 40-Hz flicker conditions according to some embodiments (n = 2 slices from 4 mice / group; two asterisks indicate p < 0.01, performed by Student's t-test). Figures 50A-50D show the mean and SEM.

[0427] In some embodiments, Iba1 was used to label microglia in visual cortical sections of 5XFAD mice after one hour of 40-Hz flicker or dark conditions (see, e.g., Fig. 49). While the number of microglia was not different between dark and 40-Hz flicker conditions (see, e.g., Fig. 49 and Fig. 50A, “ns” indicates non-significant, n = 2 sections from 4 mice / group), the microglia body diameter increased by 65.8% in the visual cortex after 40-Hz flicker compared to the dark control (see, e.g., Fig. 49 and Fig. 50B, p < 0.01, performed by Student's t-test, n =Two slices from 4 mice / group. The length of microglial primary processes was reduced by 37.7% under 40-Hz scintillation conditions compared to the dark control (see, e.g., Figures 49 and 50C, p < 0.0001, performed by Student's t-test, n = 2 slices from 4 mice / group). In some embodiments, the number of Aβ-carrying microglia is examined because microglia in the visual cortex have morphologies that indicate enhanced phagocytic activity. For this experiment, visual cortex slices were co-labeled with Iba1 and Aβ (12F4) antibodies. Aβ / Iba1 colocalization in cell bodies increased by 33.5% under 40-Hz flicker conditions, indicating that 40-Hz flicker induced more microglia carrying Aβ than the dark control (see, for example, Figures 49 and 50D, p < 0.01, performed by Student's t-test, n = 2 slices from 4 mice / groups, pages 47 / 78 of the specification, CN 122399187 A).

[0428] In some embodiments, to provide better resolution of morphological changes in microglia, CLARITY was used to create 3D renderings of microglia from 100 μm slices of the visual cortex, and videos were created from these renderings. Figure 51 is a series of 3D renderings (from immunofluorescence images) of Iba+ microglia from 100 μm tissue sections treated with CLARITY under dark and 40-Hz flicker conditions, 5102 rotated 0°, 5104 rotated 45° around the X-axis, and 5106 rotated 45° around the Y-axis. Images were taken using a 63x objective (scale bar = 15 µm). Finally, to demonstrate that microglia do indeed engulf 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.

[0429] Figure 52A is a flowchart illustrating a method for isolating microglia from the visual cortex using fluorescence-activated cell sorting (FACS) according to some embodiments. The visual cortex was cut, and single cells were then suspended and labeled with CD11b and CD45 antibodies. Subsequently, cells were sorted by fluorescence-activated cell sorting (FACS) and lysed. Aβ1-40 levels were analyzed by ELISA. Figure 52B is a bar graph depicting Aβ1-40 levels in microglia isolated from the visual cortex of three-month-old 5XFAD animals and WT control animals using the method of Figure 52A according to some implementation schemes (n = 8 mice / group for 5XFAD and n = 4 mice / group for WT mice; an asterisk indicates p < 0.05, according to Student's...).(t-test performed). Circles superimposed on the bars in the bar chart indicate individual data points in each group.

[0430] Figure 53A is a series of immunofluorescence images showing the immunohistochemistry of an SVP38 antibody for detecting synaptic vesicle proteins in the 5-month-old 5XFAD visual cortex under dark and 40-Hz flicker conditions according to some embodiments. Images were taken using a 40x objective (scale bar = 50 µm). Right panel: 100X dark and 40-Hz flicker conditions. Figure 53B is a bar chart depicting the relative SVP38 intensity levels in the 5XFAD visual cortex after dark and 40 Hz flicker conditions according to some embodiments (n = 4 mice / group; “ns” indicates non-significant, performed by Student's t-test).

[0431] Microglial-specific levels of Aβ were found to be significantly higher in 5XFAD animals than in WT controls, specifically at 27.2 pg / 10⁴ microglial cells in 5XFAD mice and at 9.78 pg / 10⁴ microglial cells in WT control mice (see, e.g., Figures 52A and 52B, p < 0.05, performed by Student's t-test, n = 8 for 5XFAD and n = 4 for WT mice). In these animals, Aβ1-42 was below detectable levels in both the scintillation and control groups. Overall, the microglial transformation induced by 40-Hz stimulation in the visual cortex appeared to be similar to the transformation occurring in the CA1 of the hippocampus. Furthermore, synaptic vesicle protein levels did not change between darkness and 40-Hz flash conditions, indicating that microglia activation did not significantly increase synaptic phagocytosis (see, for example, Figures 53A and 53B, “n.s.” indicates non-significant, n = 2 slices from 4 mice / groups). Taken together, the data disclosed herein demonstrate that 40-Hz oscillations induced noninvasively by sensory stimulation can effectively reduce Aβ abundance and promote microglia / Aβ interactions in an AD mouse model. In addition, 40-Hz stimulation reduced Aβ in two different brain circuits, thus demonstrating the general mechanism by which γ oscillations reduce amyloid abundance and enhance microglia phagocytosis in various brain regions.

[0432] In another experiment, Aβ1-42 levels were assessed one hour after exposure to darkness (no light), 20-Hz flash, 40-Hz flash, or 80-Hz flash, where 20 Hz and 80 Hz are harmonics of 40 Hz. However, only 40 Hz flash flicker significantly reduced Aβ1-42 levels. Figure 54A is a bar graph showing the reduction of Aβ peptide isoform Aβ1-42 after stimulating the visual cortex of subjects with gamma oscillations according to some embodiments.

[0433] Another study was conducted to assess the duration of the decrease in Aβ1-42 levels. Mice were exposed to darkness or 40-Hz flash for one hour. Aβ1-42 levels were determined after the one-hour treatment and again 24 hours after the treatment was completed. Figure 54B is a bar graph showing the levels of Aβ peptide isoform Aβ1-42 after stimulating the visual cortex of subjects with gamma oscillations according to some embodiments and again 24 hours after stimulation. Although Aβ levels remained decreased 24 hours after treatment, the decrease was smaller than that immediately following treatment.

[0434] Visual stimulation at gamma frequencies does not affect Aβ levels in the hippocampus.

[0435] In some embodiments, to determine whether visual stimulation by light flickering can affect brain circuits involved in AD, the effect of light flickering on the hippocampus, one of the brain regions in humans affected early in the AD process, is examined. Figure 55A includes traces of the local field potential and power spectral density in the hippocampus before and during 40-Hz light flashing 5502 according to some embodiments. The mean (solid line) and standard deviation (shaded area) of the power spectral density in CA1 during the processes of darkness 5504, 40-Hz light flashing 5506, and random light flashing 5508 (n = two 5XFAD mice and three WT mice).

[0436] Figure 55B is a series of bar graphs of the fraction of spikes in the hippocampus as a function of time for four cycles 5510 of 40-Hz light flashing or equivalent time periods of random light flashing 5512 according to some embodiments (n = two 5XFAD mice and three WT mice, bars indicate the mean and error bars indicate SEM across animals). The upper bars indicate the time when the light is on (white) or off (black). For random stimulation, the spikes are aligned with the start of the light being on, with additional time periods of light occurring at random intervals indicated by gray. Using the same method to examine the effect of light flicker in the visual cortex, such as in CA1 disclosed herein, it was found that light flicker at 40 Hz increased the power recorded in the LFP at 40 Hz (see, for example, Figure 5510 in Figures 55A and 55B), while random-interval light flicker (random flicker) and darkness did not increase said power (see, for example, Figure 4310 in Figures 50D and 43C). The spike was also modulated by the 40-Hz flicker frequency during the 40 H stimulation process; however, said modulation appeared to be smaller in the visual cortex (see, for example, Figure 55B, hippocampus, Figure 44A, visual cortex).

[0437] Figure 56A is a histogram showing the difference in firing rate between 40-Hz light flicker and random light flicker according to some embodiments (bottom n =168 stimulation time periods from 5 recorded sessions in two 5XFAD mice and three WT mice. Figure 56B is a plot showing the multi-unit firing rate in CA1 during 40-Hz light flicker 5604, random light flicker 5605, darkness 5602, or light 5608 time periods according to some implementation schemes. The box plot shows the median (white line in the box) and quartiles (top and bottom of the box). In all animals, the firing rate between 40-Hz flicker and random flicker conditions was not significantly different, thus showing that the random stimulation condition served as a control for peak activity (rank sum test for each of the 5 records from two 5XFAD animals and three WT animals, p > 0.2, median and quartiles are shown in the plot, n = 22, 54, 42, 71, 55 40-Hz flicker time periods and 12, 34, 32, 54, 36 random flicker time periods / records). There was no significant difference in firing rate between 40-Hz flicker and light conditions, indicating that 40-Hz light flicker generally does not lead to excessive neuronal excitability (rank-sum test for each of the five records from two 5XFAD animals and three WT animals, p > 0.3, median and quartiles are shown in the figure, n = 22, 54, 42, 71, 55 40 Hz time intervals and 12, 34, 33, 54, 35 light time intervals / records).

[0438] As in the visual cortex, the difference in multi-unit firing rate between 40 Hz and random flickering time periods tends to be close to zero (see, for example, Figure 56A), and no significant differences were found when comparing these time periods within animals (see, for example, Figure 56B, rank-sum test for each of five recorded sessions from four 5XFAD mice, p > 0.06, median and quartiles shown in the figure, n = 22, 54, 42, 71, 55 40-Hz flickering time periods and 12, 34, 32, 54, 36 random flickering time periods / recording).

[0439] In some embodiments, the effect of visual light flickering on Aβ levels in the hippocampus is examined using the same method used in the visual cortex. Figure 57A is a bar chart depicting relative Aβ1-40 levels in the 5XFAD visual cortex, and Figure 57B is a bar chart depicting relative Aβ1-42 levels in the 5XFAD visual cortex according to some implementation schemes (n = 4 mice / group; "ns" indicates non-significant). In CA1, no significant difference was found between Aβ1-40 and Aβ1-42 levels one hour after 40-Hz flicker or random stimulation (page 49 / 78, CN 122399187 A, manual). Aβ levels after 40-Hz flicker or random flicker were not significantly different from those under dark conditions: 40The Aβ1-40 levels after 40-Hz and random flickering were 108.4% and 96.82% of those under dark conditions, respectively, and the Aβ1-42 levels after 40-Hz and random flickering were 118.8% and 92.15% of those under dark conditions, respectively (see, for example, Figures 57A and 57B, “ns” indicates non-significant, n = 4 mice / group). Therefore, one hour of 40-Hz light flickering did not significantly reduce Aβ levels in the hippocampus.

[0440] Chronic visual stimulation at γ frequencies reduces plaque load in the visual cortex.

[0441] The affected amyloid abundance in pre-plaque 5XFAD mice when driven genetically or by visual stimulation via light flickering at 40-Hz oscillatory light has been examined and disclosed herein. The next objective is to determine whether this treatment is effective in animals that have shown plaque load. For this purpose, in some embodiments, six-month-old 5XFAD mice are used because they develop extensive amyloid plaque lesions in many brain regions, including the visual cortex. Tests were performed to determine what would happen in late Aβ-related lesions following noninvasive gamma stimulation. In some embodiments, to investigate the duration of Aβ reduction in response to one hour of 40-Hz flicker, Aβ levels were measured in the visual cortex at 4, 12, and 24 hours after one hour of 40-Hz flicker or dark conditions.

[0442] Figures 58A and 58B are bar graphs depicting the relative Aβ1-40 and Aβ1-42 levels in the 5XFAD visual cortex at 1, 4, 12, and 24 hours after one hour of dark or 40-Hz flicker treatment, respectively, according to some embodiments (n = 4 mice / group for 4 and 12 hours, n = 6 for 1 and 24 hours, and n = 12 for dark; “n.s.” indicates non-significant, one asterisk indicates p < 0.05, and two asterisks indicate p < 0.01, performed by one-way ANOVA). The results showed that, compared with the dark control, after 4 hours, Aβ1-40 levels decreased by 63.4% and Aβ1-42 levels decreased by 63.2% (see, e.g., Figure 58, p < 0.01, n = 4 mice / group). By 12 hours, Aβ1-40 levels had decreased by 50.9%, while Aβ1-42 levels were not significantly different from the dark control (see, e.g., Figure 58, “ns” indicates non-significant and p < 0.01, n = 4 mice / group). Finally, 24 hours after one hour of 40-Hz scintillation treatment, soluble Aβ1-40 and Aβ1-42 levels were not significantly different from those in the dark control condition (see, e.g., Figure 58, “ns” indicates non-significant, for 24 hours n = 6 mice / group and for dark n =4 mice / group). These results indicate that the effect of the 40-Hz flicker treatment is transient.

[0443] Thus, in some embodiments, mice were treated with 40-Hz flicker for one hour daily for seven days to disrupt late macular lesions, or as a control, they were treated with dark conditions. Figure 59A is a schematic diagram depicting six-month-old mice exposed to one hour of flicker / day for seven days according to some embodiments. Figure 59B is a bar graph showing the relative Aβ1-42 levels in the visual cortex of six-month-old 5XFAD mice after seven days of exposure to one hour / day of flicker in darkness or 40-Hz flicker conditions according to some embodiments (n = 13 mice / group, two asterisks indicate p < 0.01 and three asterisks indicate p < 0.001, Student's t-test). Figure 59C is a bar graph showing the relative Aβ1-40 levels in the visual cortex of six-month-old 5XFAD mice after seven days under one hour / day of darkness or 40-Hz flicker conditions according to some embodiments (n = 13 mice / group, one asterisk indicates p < 0.01 and two asterisks indicate p < 0.01, performed by Student's t-test). Figures 59B and 59C show the mean and SEM. Circles superimposed on the bars in the bar graphs indicate individual data points in each group.

[0444] At the end of the seven-day period, the visual cortex was analyzed by ELISA and immunostaining. In some embodiments, tissues were lysed in phosphate-buffered saline (PBS) to extract PBS-soluble Aβ fractions, and it was found that in six-month-old 5XFAD mice, seven days of one-hour 40-Hz scintillation reduced soluble Aβ1-40 and Aβ1-42 levels by 60.5% and 51.7%, respectively, as measured by ELISA (see, e.g., Figures 59B and 59C, p < 0.05 and p < 0.01, performed by Student's t-test, n = 13 mice / group). Tissues were further treated with guanidinochloride (HCl) to extract insoluble Aβ1-40 and Aβ1-42 fractions, which constitute aggregated amyloid plaques. The levels of insoluble Aβ1-40 and Aβ1-42 decreased by 43.7% and 57.9%, respectively, thereby indicating that 40-Hz scintillation disrupted insoluble Aβ aggregates that had formed in six-month-old mice (see, for example, Figures 59B and 59C, p < 0.01 and p < 0.001, performed by Student's t-test, n = 13 mice / group).

[0445] In some embodiments, to determine how plaque load was specifically affected, an immunization was performed using Aβ antibodies.Histochemical characterization (cell signaling techniques; D54D2). Figure 60A is a series of immunofluorescence images (scale bar = 50 µm) of the immunohistochemistry of the visual cortex of six-month-old 5XFAD mice after seven days using the Aβ (D5452) antibody under dark (top) or 40-Hz flicker (bottom) conditions for one hour / day according to some embodiments. Aβ signaling appearing in cells was excluded. Figure 60B is a bar graph depicting the number of Aβ-positive plaque deposits in the visual cortex of six-month-old 5XFAD mice after seven days under dark or 40-Hz flicker conditions for one hour / day according to some embodiments (n = 8 mice / group, three asterisks indicate p < 0.001, performed by Student's t-test). Figure 60C is a bar graph depicting the area of ​​Aβ-positive spots in the visual cortex of six-month-old 5XFAD mice after seven days under dark or 40-Hz flicker conditions for one hour / day according to some embodiments (n = 8 mice / group; two asterisks indicate p < 0.01, performed by the Mann-Whitney test). Figures 60B and 60C show the mean and SEM.

[0446] Spot abundance was quantified by counting the number of Aβ+ deposits with a diameter greater than or equal to about 10 μm. The 40-Hz flicker reduced the number of spots to 11.0 compared to a reduction to 33.5 in the dark control (see, for example, Figures 60A and 60B, p < 0.01, performed by Student's t-test, n = 8 mice / group). Furthermore, compared to the dark control, after one week of 40-Hz scintillation treatment, the plaque size (measured as the area of ​​the dense plaque region) was reduced by approximately 63.7% (see, e.g., Figures 60A and 60C, p < 0.01, performed by the Mann-Whitney test, n = 8 mice / group). Taken together, these experiments identify a completely non-invasive treatment with a significant effect on amyloid plaque lesions.

[0447] To determine whether 40-Hz scintillation improves another key AD-related lesion, the TauP301S tau proteinopathy mouse model was used to investigate tau phosphorylation. Four-month-old TauP301S Tg mice (which at this age show phosphorylated tau localized to the cell body) were treated daily for one hour under either 40-Hz scintillation or dark control conditions for seven days. To examine how 40-Hz scintillation alters tau phosphorylation, immunohistochemical characterization of the visual cortex was performed using pTau antibodies against three different epitopes of pTau (S202, S396, and S400 / T403 / S404; 11834S, 9632S, 11837S) and the dendritic marker MAP2 as a control.

[0448] Figure 61A is a series of immunofluorescence images showing the immunohistochemistry of anti-pTau 6102 (S202) antibody and anti-MAP2 6104 antibody in four-month-old P301S mice after seven days of immunohistochemistry under one hour / day of darkness or 40-Hz flicker conditions according to some embodiments. Images were taken using a 40x objective (scale bar = 50 µm; insets include 100x renderings of representative cell bodies under darkness and 40-Hz flicker conditions). Figure 61B is a bar graph depicting the relative pTau (S202) intensity levels of the P301S visual cortex after seven days of darkness and 40-Hz flicker conditions according to some embodiments (n = 8 mice / group; an asterisk indicates p < 0.05, performed by Student's t-test). Figure 61C is a bar graph depicting the relative MAP2 intensity levels of the P301S visual cortex after seven days under darkness and 40-Hz light flicker conditions for one hour / day according to some embodiments (n = 8 mice / group; “ns” indicates non-significant, performed by Student's t-test). Figures 61B and 61C show the mean and SEM.

[0449] Figure 62A is a series of immunofluorescence images (scale bar = 50 µm) showing the immunohistochemistry of 4-month-old P301S mice after seven days under darkness and 40-Hz flicker conditions for one hour / day according to some embodiments using anti-pTau 6202 (S404) antibody. Figure 62B is a bar graph depicting the relative pTau (S400 / T403 / S404) fluorescence intensity levels of the P301S visual cortex after seven days under dark and 40-Hz flicker conditions for one hour / day according to some embodiments (n = 8 mice / group; two asterisks indicate p < 0.01, performed by Student's t-test). Figure 62B shows the mean and specification page 51 / 78 53 CN 122399187 A SEM.

[0450] Figure 63A is a series of immunofluorescence images (scale bar = 50 µm) showing the immunohistochemistry of four-month-old P301S mice after seven days under dark and 40-Hz flicker conditions for one hour / day according to some embodiments using anti-pTau 6302 (S396) antibody. Figure 63B is a bar graph depicting the relative pTau (S396) fluorescence intensity levels of the P301S visual cortex after seven days under dark and 40-Hz flicker conditions for one hour / day according to some embodiments (n = 8 mice / group; four asterisks indicate p < 0.0001, performed by Student's t-test).

[0451] The results showed that the signal intensity of pTau (S202) was reduced by 41% under 40-Hz flicker conditions compared to the dark control.The signal intensity of pTau (S400 / T403 / S404) decreased by 42.3% (see, e.g., Figures 61A-61B, 62A-62B, p < 0.01, performed by Student's t-test, n = 2 slices from 8 mice / group), while MAP2 levels remained unchanged (see, e.g., Figures 61A and 61C, “ns” indicates non-significant, n = 2 slices from 4 mice / group). Staining with an antibody against pTau (S396) showed the same trend: 40-Hz flicker reduced pTau (S396) levels by 14.4% compared to the dark control (see, e.g., Figures 63A-63B, “ns” indicates non-significant, n = 2 slices from 8 mice / group). Furthermore, fewer spots and less cell-body localization of the pTau signal were observed in response to 40-Hz flicker compared to the dark control. Although significant changes in tau phosphorylation were observed, no discernible difference in the level of insoluble tau was observed between the 40-Hz scintillation treatment group and the dark control group.

[0452] The effect of 40-Hz scintillation on microglia in a TauP301S mouse model was assessed. Figure 64 is a series of immunofluorescence images showing the immunohistochemistry of four-month-old P301S mice after seven days of dark and 40-Hz scintillation conditions for one hour / day according to some embodiments, using an anti-Iba1 (019-19741) antibody. Images were taken using a 40x objective (scale bar = 50 µm; inset includes a 100x rendering of representative microglia in EYFP and 40-Hz scintillation conditions).

[0453] Figure 65A is a bar graph depicting the number of microglia after seven days of dark and 40-Hz scintillation conditions for one hour / day according to some embodiments (n = 8 mice / group; “ns” indicates non-significant, performed by Student's t-test). Figure 65B is a bar graph depicting the diameter of microglial cell bodies normalized to the control after seven days of one hour / day under darkness and 40-Hz flicker conditions according to some embodiments (n = 8 mice / group; four asterisks indicate p < 0.0001, performed by Student's t-test). Figure 65C is a bar graph depicting the mean length of the primary process of microglial cells normalized to the control after seven days of one hour / day under darkness and 40-Hz flicker conditions according to some embodiments (n = 8 mice / group; four asterisks indicate p < 0.0001, performed by Student's t-test).

[0454] In some embodiments, anti-Iba1 is used after seven days of one hour / day of 40-Hz flicker or darkness conditions.Antibody labeling of microglia in the visual cortex of TauP301S mice (see, e.g., Fig. 64). In some embodiments, a trend toward a 29.50% increase in the number of microglia was observed under 40-Hz flicker conditions compared to the dark control (see, e.g., Fig. 64 and Fig. 65A, “ns” indicates non-significant, n = 3 mice / group), consistent with observations made in the 5XFAD model (see, e.g., Fig. 50A). Furthermore, the microglia body diameter increased by 49.00% after 40-Hz flicker compared to the dark control (see, e.g., Fig. 64 and Fig. 65B, p < 0.0001, performed by Student's t-test, n = 3 mice / group). Compared to the dark control, the length of microglial primary processes was reduced by 39.08% in the 40-Hz flicker group (see, e.g., Figures 64 and 65C, p < 0.0001, performed by Student's t-test, n = 3 mice / group).

[0455] Taken together, these data from multiple models of AD lesions and in WT animals demonstrate that 40-Hz oscillations alleviate amyloidosis (as measured by a decrease in Aβ levels) and reduce tau phosphorylation. Furthermore, 40 Hz visual flicker drives different morphological transformations of microglia in both amyloidosis and tau disease models of AD lesions.

[0456] In another experiment, a subgroup of aged mice (i.e., six months old) were exposed to visual gamma stimulation for seven days. The remaining mice were kept in darkness. Figure 66 is a graph showing the levels of both soluble and insoluble Aβ peptides (i.e., spots) in the visual cortex of mice. As shown in Figure 66, the levels of each of the soluble isoform Aβ1-40 6600, soluble isoform Aβ1-42 6602, insoluble isoform Aβ1-40 6604, and insoluble isoform Aβ1-42 6606 were significantly reduced in mice exposed to visual gamma stimulation.

[0457] Figures 67A-67B are graphs showing the Aβ peptide levels in subjects with and without transcranial gamma stimulation according to some embodiments. In Figure 67A, the whole-brain Aβ peptide level remained unchanged without stimulation 6700, but decreased after one hour of transcranial gamma stimulation 6702 (n = 1 animal / group). In Figure 67B, according to some embodiments, whole-brain Aβ peptide levels decreased at hippocampus 6704 and cortical 6706 in 5xFAD mice after 40 oz transcranial stimulation.

[0458] Gamma oscillations have long been considered associated with higher cognitive function and sensory responses. In some embodiments,Optogenetic methods were used to drive FS-PV-interneurons to enhance LFP at 40 Hz in mice. As disclosed herein, it has been demonstrated in some embodiments that, in a 5XFAD mouse model, driving 40-Hz oscillations and phase-locked spikes using optogenetic or non-invasive photoscintillation treatment resulted in a significant reduction in Aβ peptide in at least two distinct brain regions. This reduction was not due to reduced spike activity, as Aβ peptide levels in response to 40-Hz stimulation were significantly lower than those under random stimulation conditions that produced a similar amount of multi-unit spike activity without enhancing 40-Hz oscillations. Cone cell firing rates may differ between these conditions, but firing of FS-PV-interneurons or other cell types masks this variation. In some embodiments, random optogenetic stimulation of FS-PV-interneurons provides the same amount of direct stimulation to FS-PV-interneurons but does not reduce amyloid. In fact, optogenetic random stimulation increased amyloid levels by more than three-fold, while random visual scintillation did not produce a significant change, which may indicate that some aspects of random stimulation have neurotoxic effects. While in some embodiments, random stimulation did not elicit an increased gamma power, a slight trend toward increased power was observed across a wide frequency range (approximately 20 Hz to greater than 60 Hz). In some embodiments, a trend toward increased amyloid levels was observed with light flicker at 20 Hz and 80 Hz. Taken together, these results suggest that driving activity at frequencies below or above 40 Hz can increase amyloid levels. These results highlight the need to understand how patterns of peak activity influence molecular pathways and disease pathogenesis.

[0459] The robust reduction in total amyloid levels may be mediated by reduced amyloid production, involving a decrease in early EEA1 / Rab5-positive endosomes and an increase in amyloid endocytosis via microglia. Importantly, the gene set enrichment analysis (GSEA) statistical analysis disclosed herein (The Broad Institute, Cambridge, Massachusetts) showed that the classic macrophage pro-inflammatory M1 or anti-inflammatory M2 cell state was not correlated with the upregulated or downregulated gene expression profile following neuronal stimulation via 40 Hz oscillations. In fact, the expression levels of pro-inflammatory genes Il6, Il1b, Itgam, and the anti-inflammatory gene Igf1 remained unchanged after stimulation. Conversely, the levels of microglia prophagocytic genes and the cell adhesion / migration regulator Spp1 were activated after 40-Hz stimulation. Therefore, it appears that driving the 40 Hz γ oscillation induces an overall neuroprotective response by recruiting both neurons and microglia. The fact that GABA-A antagonist treatment completely eliminated the effect of 40-Hz stimulation on reducing Aβ levels strongly suggests that γ-aminobutyric acid (GABA) signaling (most likely involved in...)And FS-PV-interneurons) are key to these effects. Furthermore, in some embodiments, 40-Hz scintillation stimulation reduces Aβ in multiple mouse models, including APP / PS1 and WT mice in addition to 5XFAD mice. This replication in multiple mouse models shows that these findings may not be specific to a single animal model, and importantly, can be extended to cases where APP is expressed via its physiological promoter and Aβ is generated from endogenous APP, as in WT animals. Furthermore, in some embodiments, 40-Hz oscillations have been found to reduce pTau in a mouse model of tau proteinopathy TauP301S, thus demonstrating that the protective effect of γ stimulation extends not only to other mouse models but also to other pathogenic proteins. In summary, the findings disclosed herein reveal previously unknown cellular and molecular processes mediated by γ oscillations and establish functional relationships between brain γ rhythms, microglia function, and AD-related lesions. In some implementations, the discovery of γ oscillation defects is pooled with evidence of γ defects in different mouse models of AD (hAPP and apoE4) and reports that γ is altered in humans with AD. By looking at pooled evidence from multiple mouse models of AD (including Tg and knock-in models), these results can be demonstrated to be due not only to transgene expression or other model-specific side effects. These results from mice and humans together show that multiple molecular pathways contributing to Aβ lesions collectively alter γ oscillations in AD. The findings disclosed herein hold promise for novel therapeutic interventions for AD.

[0460] One theory of AD etiology points to microglia dysfunction (specifically, the inability of microglia to clear pathological molecules) as a key mechanism for disease progression. Therefore, interventions that restore microglia to an endocytic state (as done by 40-Hz stimulation) have strong therapeutic potential. In experiments further described herein, optogenetically or by light flickering to drive γ oscillations did not result in excessive neuronal activity. Because this approach is fundamentally different from previous AD therapies, driving neural activity in this mode to trigger endogenous repair would provide novel therapeutic approaches for AD.

[0461] Visual stimulation at gamma frequencies has a positive effect on subject behavior.

[0462] Studies were conducted to examine whether gamma exposure and / or administration according to some embodiments resulted in any stress on the subjects. Figure 68A is a flowchart illustrating the study. As shown at 6800 in Figure 68A, WT mice were exposed to normal indoor light (N = 8) or 40-Hz light flicker (N = 8) for one hour / day for seven consecutive days, according to some embodiments.Day 7. On day 8, blood was collected from mice and plasma was separated to examine corticosterone levels, as shown at 6802. Corticosterone is the major glucocorticoid involved in stress response in mice.

[0463] Figure 68B is a bar graph depicting the levels (pg / ml) of corticosterone (CORT) in plasma collected from eight mice exposed to normal indoor light (NRL) and eight mice exposed to 40-Hz light flicker (40-Hz). No increase in corticosterone was observed in mice exposed to 40-Hz light flicker. Instead, the group of mice exposed to 40-Hz light flicker had lower levels of corticosterone compared to the control group. The T-distribution and p-value of corticosterone levels were calculated for N = 8 independent measurements / group as follows:

[0464] T(14) = 0.827; p = 0.422 (1)

[0465] Another study was conducted to examine whether gamma exposure and / or administration according to some implementation schemes reduced anxiety in subjects. Figure 68A is a flowchart illustrating the study. As shown at 6900 in Figure 69A, WT mice were exposed to normal indoor light (N = 10) or 40-Hz light flicker (N = 10) for one hour / day for seven consecutive days, from day 1 to day 7, according to some implementation schemes. On day 8, as shown at 6902, a ten-minute session of the elevated cross maze was performed.

[0466] The elevated cross maze is a test used to measure anxiety in laboratory animals. The behavioral pattern is based on the rodent’s general aversion to open spaces, which leads to tactile tendency, a preference for staying in enclosed spaces or near the edges of defined spaces. Figure 69B is an image illustrating the elevated cross maze apparatus. The apparatus is a cross shape with two open arms (vertical) and two closed arms (horizontal). Anxiety is expressed by the animal spending more time in the closed arms.

[0467] Figures 69C and 69D are images illustrating representative trajectories of subjects during the elevated cross maze session. According to some embodiments, in Figure 69C, mice exposed to normal indoor light tended to stay in the closed arm, indicating more anxiety, while in Figure 69D, mice exposed to 40-Hz light flicker explored both the open and closed arms, indicating relatively less anxiety.

[0468] Figure 70 is a bar graph depicting the total time spent exploring the open and closed arms by ten mice exposed to normal indoor light (NRL) and ten mice exposed to 40-Hz light flicker (40-Hz) according to some embodiments. According to some embodiments of a specification 54 / 78 pages 56 CN 122399187 A, mice exposed to 40-Hz light flicker spent less total time in the closed arm and more total time in the open arm compared to the control group, indicating less anxiety. For N = 10 independent measurements / group, during explorationThe T-distribution and p-value for the total time spent on the closed arm were calculated as follows:

[0469] T(18) = -1.652; p = 0.11 (2)

[0470] For N = 10 independent measurements / groups, the T-distribution and p-value for the total time spent on the open arm were calculated as follows:

[0471] T(18) = -2.136; p = 0.047 (3)

[0472] Another study was conducted to examine whether γ exposure and / or administration according to some implementation schemes reduced stress and / or anxiety in the subjects. Figure 71A is a flowchart illustrating the study. At 7100 in Figure 71A, WT mice were exposed to normal indoor light (N = 8) or 40-Hz light flicker (N = 8) for one hour / day for seven consecutive days, from day 1 to day 7, according to some implementation schemes. On day 8, as shown at 7102, a five-minute open field test was performed.

[0473] Open field testing was used to determine generalized motor activity levels and anxiety in laboratory mice. Behavioral patterns were based on anxiety resulting from the rodent's conflicting desire to avoid brightly lit areas and explore perceived dangerous stimuli. Figure 71B is an image showing an open field setting. The open field setting had walls to prevent escape and could be monitored using grid markings or infrared beams or cameras integrated with a software system. According to some embodiments, increased anxiety would result in less motor activity and a preference for the edges of the setting, while decreased anxiety would result in increased exploratory behavior.

[0474] Figures 71C and 71D are images showing representative trajectories of subjects during open field testing. According to some embodiments, in Figure 71C, mice exposed to normal indoor lighting tended to prefer the edges of the setting, indicating more stress and / or anxiety, while in Figure 71D, mice exposed to 40-Hz light flicker explored more of the center of the setting, indicating relatively less stress and / or anxiety.

[0475] Figures 72A and 72B are graphs depicting the total time spent exploring the center and edges of an open field by eight mice exposed to normal indoor light (NRL) and eight mice exposed to 40-Hz light flicker (40-Hz) according to some embodiments. Figure 72A is a graph of the average amount of seconds spent in the center of the field per minute over five minutes. Figure 72B is a bar graph of the total time spent at the edges of the field over the entire five-minute duration (average per minute).

[0476] On average, mice exposed to 40-Hz light flicker spent more time in the center of the field, significantly so during the 2nd, 4th, and 5th minutes, thus indicating less stress and / or anxiety compared to the control group, which is also consistent with the results of the elevated cross maze according to some embodiments. Repeated measures ANOVA was performed. For N =The F-distribution and p-value for the average time spent exploring the open field area for 8 independent measurements / groups were calculated as follows:

[0477] F(1,14) = 4.860; p = 0.045 (4)

[0478] Another study was conducted to examine whether gamma exposure and / or administration according to some implementation schemes altered the subjects’ inherent novelty-seeking behavior. Figures 73A and 73B are schematic diagrams illustrating studies using a novelty recognition task. In Figure 73A, two novel items were provided in a familiar field. In Figure 73B, a familiar item and a novel item were provided in a familiar field. Wild-type mice were exposed to normal indoor light (N = 8) or 40-Hz light flicker (N = 8) for one hour / day for seven consecutive days, from day 1 to day 7, according to some implementation schemes.

[0479] On day 8, the mice were exposed to the scenario in Figure 73A, with two novel items in a familiar field, for five minutes. Figure 73C is a bar graph depicting the percentage of time spent exploring novel item A versus the percentage of time spent exploring novel item B for eight mice exposed to normal indoor light (NRL) and eight mice exposed to 40-Hz light flicker (40-Hz) according to some embodiments. As shown in Figure 73C, each group shows equal preference for each item. That is, no difference in item exploration was observed between the groups. Specification 55 / 78 pages 57 CN 122399187 A

[0480] The mice were then exposed to the scenario in Figure 73B, a familiar item and a novel item in a familiar setting, for five minutes. Figure 74 is a graph depicting the average number of seconds spent per minute exploring the novel item over the five minutes. On average, according to some embodiments, mice exposed to 40-Hz light flicker spent significantly more time exploring the novel item, especially during the first 1–3 minutes and the 5th minute, thus indicating increased novelty-seeking behavior compared to the control group. Friedman nonparametric RM ANOVA was performed. For N = 8 independent measurements / groups, the test statistics χ² and p-value for the average time spent exploring novel items were calculated as follows:

[0481] χ²(4, n = 16) = 16.088; p = 0.003 (5)

[0482] The Mann-Whitney U test was performed for the average time spent exploring novel items during the 3-minute period. For N = 8 independent measurements / groups, the U, Z, and p-values ​​were calculated as follows:

[0483] U = 58.00; Z = 2.731; p = 0.005 (6)

[0484] Another study was conducted to examine whether γ exposure and / or administration according to some implementation schemes affected the subjects'Learning and memory. Figure 75A is a flowchart illustrating a study using the fear conditioning paradigm. As shown at 7500 in Figure 75A, WT mice were exposed to normal indoor light or 40-Hz light flashing for one hour / day for seven consecutive days, from day 1 to day 7, according to some implementation schemes. On day 8, as shown at 7502, the mice were subjected to a moderate two-tone-electric shock pairing. Specifically, the mice were introduced into a new setting where the first tone was paired with a foot shock. The mice conditioned to associate the environment (i.e., the tone) with the aversive experience (i.e., the foot shock). The T-distribution and p-value for the total time spent in freezing for this initial setting were calculated as follows:

[0485] T(24) = 0.577; p = 0.569 (7)

[0486] On day 9, as shown at 7504, the tone test was performed in a modified setting. Figure 75B is a stimulus plot showing the pitch test as a function of time, including a first pitch environment 7506, a first pitch post-environment 7508, a second pitch environment 7510, and a second pitch post-environment 7512. For the test, mice were returned to the area where the first pitch was paired with a leg shock. When the first pitch environment 7506 was applied, mice exposed to 40-Hz light flicker spent more time in rigidity, possibly anticipating the leg shock, thus indicating memory behavior. Compared to the control group, mice exposed to 40-Hz light flicker also spent more time in rigidity during the second pitch environment 7510, but less time in rigidity during the post-pitch environment.

[0487] Figures 76A and 76B are bar graphs demonstrating enhanced memory according to some embodiments. As shown in Figure 76A, according to some embodiments, mice exposed to 40-Hz light flicker, compared to the control group, spent a greater percentage of time in rigidity during the first tone environment 7506 and the second tone environment 7510, thus indicating enhanced memory association. Furthermore, according to some embodiments, mice exposed to 40-Hz light flicker exhibited stronger presentation after the fear extinction tone. As shown in Figure 76B, according to some embodiments, compared to mice exposed to 40-Hz light flicker, the control group, for the same reason, spent a greater percentage of time in rigidity during the first tone environment 7506 and the second tone environment 7510, thus indicating enhanced memory specificity.

[0488] For the pre-tone environment, RM ANOVA was performed across groups, and the F-distribution and p-value of the average time spent on stiffness were calculated as follows:

[0489] F(1,24) = 3.106; p = 0.091 (8)

[0490] For the first tone environment, the T-distribution and p-value of the total time spent on stiffness were calculated as follows:

[0491] T(24) = -2.155; p = 0.041 (9)

[0492] For the second tone environment, the T-distribution and p-value of the total time spent on stiffness are calculated as follows:

[0493] T(24) = -1.433; p = 0.164 (10) Specification 56 / 78 pages 58 CN 122399187 A

[0494] For the tone environment, RM ANOVA is performed between groups, and the F-distribution and p-value of the average time spent on stiffness are calculated as follows:

[0495] F(1,24) = 4.559; p = 0.043 (11)

[0496] For the first tone post-environment, the T-distribution and p-value of the total time spent on stiffness are calculated as follows:

[0497] T(24) = 1.874; p = 0.073 (12)

[0498] For the second tone post-environment, the T-distribution and p-value of the total time spent on stiffness are calculated as follows:

[0499] T(24) = 2.223; p = 0.036 (13)

[0500] For the post-tone environment, RM ANOVA was performed between groups, and the F-distribution and p-value of the average time spent on rigidity were calculated as follows:

[0501] F(1,24) = 6.646; p = 0.017 (14)

[0502] Another study was conducted to examine whether γ exposure and / or administration according to some implementation schemes improved the memory of the subjects. Figure 77A is a flowchart illustrating the study. As shown at 7700 in Figure 77A, WT mice were exposed to normal indoor light or 40-Hz light flashing for one hour / day for seven consecutive days, from day 1 to day 7, according to some implementation schemes. On day 8, as shown at 7702, the Morris water maze test was performed.

[0503] The Morris water navigation task or maze is a test used to study spatial memory and learning in laboratory mice. The behavioral procedure involved placing subjects in a large prototypical pool with invisible or visible platforms that allowed subjects to escape the water using praxic strategies (remembering the movement required to reach the platform), taxic strategies (using visual cues to locate the platform), or spatial strategies (using distance cues as reference points). Figure 77B is a diagram illustrating the Morris water maze. The maze comprises a circular pool with water divided into directional quadrants and a platform 7704 hidden in the southwest (SW) quadrant.

[0504] For weak training, the Morris water maze test was repeated twice daily for four consecutive days, from day 8 to day 11. Figure 78A is a graph depicting the delay in finding the platform by mice exposed to normal indoor light (NRL) and mice exposed to 40-Hz light flicker (40-Hz) according to some embodiments.

[0505] On day 12, a detection test was performed by removing the hidden platform from the Morris water maze. Figures 77C and 78A are also shown.Figure 77D is an image showing representative trajectories of subjects during the detection test. In Figure 77C, mice exposed to normal indoor lighting appear to search for the platform throughout the pool, while in Figure 77D, according to some embodiments, mice exposed to 40-Hz light flicker appear to search more methodically and primarily in the SW quadrant. Figure 78B is a graph depicting the total time (seconds per half-minute) spent searching for the platform in the target quadrant (i.e., the SW quadrant), while Figure 78C is a graph depicting the total time (seconds per half-minute) spent searching for the platform in the opposite quadrant (i.e., the NE quadrant). According to some embodiments, mice exposed to 40-Hz light flicker spent more time searching in the target quadrant and less time searching in the opposite quadrant than the control group, indicating enhanced spatial memory.

[0506] Reverse learning was performed using mice from the same group from the Morris water maze test and the detection test. Figure 79A is a diagram showing the Morris water maze in which platform 7900 is hidden in the SW quadrant as described in the experiment. Figure 79B is a diagram showing the Morris water maze in which platform 7902 is hidden in the opposite NE quadrant for reverse learning.

[0507] For weak training, reverse learning was repeated twice daily for four consecutive days, from day 14 to day 17. Figure 79C is a graph depicting the delay in finding the platform by mice exposed to normal indoor light (NRL) and mice exposed to 40-Hz light flicker (40-Hz) according to some embodiments. Although no further 40-Hz exposure was received after day 7, mice exposed to 40-Hz light flicker still showed increased behavioral flexibility.

[0508] Another study was conducted to examine whether chronic gamma exposure and / or administration according to some embodiments affected spatial learning and memory in the subjects. Figure 80A is a flowchart illustrating the study. As shown at 8000 in Figure 80A, according to some embodiments of a specification 57 / 78, page 59, CN 122399187 A, C57BL / 6 mice were exposed to normal indoor light (N = 7) or 40-Hz light flicker (N = 7) for one hour / day for two weeks. During the third week, as shown at 8002, the mice continued to be exposed to normal indoor light or 40-Hz light flicker for one hour each morning, and then subjected to the Morris water maze test each afternoon.

[0509] Figure 80B is a graph depicting the delay in finding the platform by mice exposed to normal indoor light (NRL) and mice exposed to 40-Hz light flicker (40-Hz) on days 1-4 of the third week. After the third week, a detection test was performed by removing the hidden platform. Figure 80C is a graph depicting the total time spent finding the platform in the target quadrant during the detection test.A bar graph of intervals (number of seconds in a 30-second trial). According to some embodiments, a chronic three-week treatment enhances spatial learning, similar to the one-week treatment.

[0510] Reverse learning was performed using mice from the same group as in Figures 80A-80C. Figure 81A is a flowchart illustrating the extended study. As shown at 8100 in Figure 81A, C57BL / 6 mice were exposed to normal indoor light or 40-Hz light flashing for one hour / day for two weeks, according to some embodiments. During the third week, as shown at 8102, the mice continued to be exposed to normal indoor light or 40-Hz light flashing for one hour each morning, and then also underwent the Morris water maze test each afternoon. During the fourth week, as shown at 8104, the mice continued to be exposed to normal indoor light or 40-Hz light flashing for one hour each morning, and then also underwent the Morris water maze reverse test each afternoon. Figure 81B is a graph depicting the delay in finding the platform by mice exposed to normal indoor light (NRL) and mice exposed to 40-Hz light flicker (40-Hz) on days 1-4 of week 4 according to some embodiments.

[0511] After week 4, a detection test was performed by removing the hidden platform. Figure 82A is a bar graph depicting the total time spent (seconds per 30-second trial) in finding the platform in the target quadrant during the detection test. Figure 82B is a bar graph depicting the time spent in the opposite quadrant during the detection test. Mice exposed to 40-Hz light flicker showed greater cognitive flexibility.

[0512] Visual stimulation at γ frequencies provides anatomical, morphological, cellular, and molecular benefits.

[0513] Studies were conducted to examine the effects of γ exposure and / or administration according to some embodiments on DNA damage and neuronal loss in the visual cortex of subjects. For the study, an inducible mouse model of p25 accumulation was used (i.e., creatine kinase-C-terminal p25 Tg mice (CK-p25 Tg mice)). The CK-p25 Tg mouse model exhibits key pathological markers of AD, including significant neuronal loss in the forebrain, increased Aβ peptide production, tau lesions, DNA damage, and severe cognitive impairment. In this model, increased Aβ peptide production was observed prior to neuronal loss; furthermore, reduced Aβ peptide production alleviated memory deficits in the CK-p25 Tg mouse model, indicating that this event synergizes with C-terminal p25, leading to the manifestation of neurodegeneration and memory impairment.

[0514] Figure 83 is a timeline diagram 8300 showing the changes in CK-p25 Tg mice. At two weeks later 8302, the mice exhibited DNA damage (e.g., biomarker γH2AX), increased Aβ peptide, and microglial cell activation. At six weeks later 8304,Mice exhibited synaptic loss, neuronal loss, tau hyperphosphorylation, long-term enhancement deficits, and memory impairment.

[0515] Studies were conducted to compare groups of mice under different treatment regimens. Figure 84 is a graph of the groups, including CK control mice 8400, untreated CK-p25 Tg mice 8402, CK-p25 Tg mice treated with memantine (10 mg / kg daily) 8404, CK-p25 Tg mice exposed to 40-Hz light flicker (one hour daily for 6 weeks) according to some embodiments 8406, and CK-p25 Tg mice treated with memantine and also exposed to 40-Hz light flicker 8408. Memantine is a drug with limited success for treating severe AD by blocking NMDA receptors, thereby acting on the glutamatergic system.

[0516] Gamma exposure and / or administration according to some embodiments are shown to protect brain anatomy and / or reduce changes in brain anatomy. For example, γ exposure reduces and / or prevents CKp-25-induced brain weight loss. Figure 85 is a bar graph comparing changes in brain weight in mice as described on pages 58 / 78 of the specification, 60 CN 122399187 A: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light flicker according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light flicker. Brain weight loss was evident in untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, and CK-p25 Tg mice treated with both memantine and 40-Hz light flicker. However, CK-p25 Tg mice exposed to 40-Hz light flicker according to some embodiments retained more brain weight.

[0517] γ exposure and / or application according to some embodiments shows protection against and / or reduction of changes in brain morphology. For example, γ exposure reduces and / or prevents CKp-25-induced aberrant lateral ventricular dilation in subjects. Figure 86 is a bar graph comparing the fold changes in lateral ventricular dilation in the following mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light flicker according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light flicker, with dilation in CK control mice as baseline. Lateral ventricular dilation in untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, and CK-p25 Tg mice treated with both memantine and 40-Hz light flicker.This is evident in Tg mice. CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments showed less lateral ventricle dilation than other CK-p25 Tg mice.

[0518] Figures 87A-87E are images showing the lateral ventricles of subjects representing each group. The lateral ventricles were largest in untreated CK-p25 Tg mice (Figure 87A), CK-p25 Tg mice treated with memantine (Figure 87B), and CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation (Figure 87C). As shown in Figure 87D, CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments showed significantly less lateral ventricle dilation. Figure 87E is an example of baseline lateral ventricle size in CK control mice.

[0519] Figures 88A-88C are brain anatomy diagrams showing brain regions of interest for molecular characterization according to some embodiments. Figure 88A includes the visual cortex (V1) 8800, the somatosensory cortex (SS1) 8802, the hippocampus 8804, and the insular cortex 8806.

[0520] γ exposure and / or application according to some embodiments are shown to protect the cortical and neuronal layers in the visual cortex and / or reduce changes in said cortical and neuronal layers. For example, γ exposure reduces and / or prevents CKp-25-induced loss of cortical and neuronal layers in the visual cortex of subjects.

[0521] Cortical layer loss was measured using nuclear staining with Hoechst markers (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 biomarker for neurons). Figure 89 is a bar graph depicting the average thickness of the V1-cortical layer in each group, and Figure 90 is a bar graph depicting the average thickness of the V1-NeuN-positive cell layer in each group.

[0522] Figures 91A-91E are images showing cells labeled with Hoechst and / or NeuN, representing subjects in each group. Figure 91A is an example of the baseline V1-cortical layer (e.g., 837 ± 9 µM) and V1-neuronal layer (e.g., 725 ± 7 µM) thickness in CK control mice.

[0523] The V1-cortical layer was progressively thinned in the following mice: CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments (Figure 91D, e.g., 855 ± 9 µM); CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation (Figure 91E, e.g., 821 ± 22 µM); untreated CK-p25 Tg mice (Figure 91B, e.g., 792 ± 13 µM); and CK-p25 Tg mice treated with memantine (Figure 91C, e.g., 788 ± 9 µM).

[0524] The V1 neuronal layer was actually thicker in CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments than in CK control mice (Fig. 91D, e.g., 743 ± 9 µM), but then progressively thinner than in CK control mice in the following mice: CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation (Fig. 91E, e.g., 691, specification 59 / 78 pages 61 CN 122399187 A ± 20 µM); untreated CK-p25 Tg mice (Fig. 91B, e.g., 666 ± 14 µM); and CK-p25 Tg mice treated with memantine (Fig. 91C, e.g., 660 ± 7 µM).

[0525] Showing γ exposure and / or application to protect the cortical and neuronal layers in the somatosensory cortex and / or reduce changes in said cortical and neuronal layers according to some embodiments. For example, γ exposure reduces and / or prevents CKp-25-induced cortical and neuronal layer loss in the somatosensory cortex of subjects.

[0526] Figure 92 is a bar chart depicting the mean thickness of the SS1-cortical layer in each group, and Figure 93 is a bar chart depicting the mean thickness of the SS1-NeuN-positive cell layer in each group.

[0527] Figures 94A-94E are images showing cells with Hoechst and / or NeuN labels representing subjects in each group. Figure 94A is an example of the baseline thickness of the SS1-cortical layer (e.g., 846 ± 10 µM) and the SS1-neuronal layer (e.g., 707 ± 8 µM) in CK control mice.

[0528] The SS1-cortical layer gradually thinned in the following mice: CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments (Fig. 94D, e.g., 834 ± 9 µM); CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation (Fig. 94E, e.g., 778 ± 13 µM); untreated CK-p25 Tg mice (Fig. 94B, e.g., 762 ± 17 µM); and CK-p25 Tg mice treated with memantine (Fig. 94C, e.g., 756 ± 11 µM).

[0529] The SS1-neuronal layer of CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments was almost the same thickness as that of the SS1-neuronal layer of CK control mice (Fig. 94D, e.g., 705 ± 15 µM). However, the SS1-neuron layer gradually thinned in the following mice: CK-p25 Tg mice treated with both methimazole and 40-Hz light scintillation (Fig. 94E, e.g., 650 ± 11 µM); and untreated CK-p25 Tg mice (Fig. 94B, e.g., 630 ± 13 µM).(µM); and CK-p25 Tg mice treated with memantine (Fig. 94C, e.g., 629 ± 9 µM).

[0530] γ exposure and / or administration according to some embodiments are shown to protect the cortical and neuronal layers in the insular cortex and / or reduce changes in said cortical and neuronal layers. For example, γ exposure reduces and / or prevents CKp-25-induced loss of the cortical and neuronal layers in the insular cortex of subjects.

[0531] Fig. 95 is a bar graph depicting the average thickness of the cortical layer in the insular cortex in each group, and Fig. 96 is a bar graph depicting the average thickness of the NeuN-positive cell layer in the insular cortex in each group.

[0532] Figs. 97A-97E are images showing cells with Hoechst and / or NeuN labels representing subjects in each group. Figure 97A is an example of the thickness of the baseline cortical layer (e.g., 1134 ± 10 µM) and neuronal layer (e.g., 1010 ± 11 µM) of the insular cortex in CK control mice.

[0533] The cortical layer gradually thins in the insular cortex of the following mice: CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments (Figure 97D, e.g., 1079 ± 20 µM); CK-p25 Tg mice treated with memantine (Figure 97C, e.g., 983 ± 12 µM); CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation (Figure 97E, e.g., 965 ± 16 µM); and untreated CK-p25 Tg mice (Figure 97B, e.g., 764 ± 27 µM).

[0534] The neuronal layer gradually thinned in the insular cortex of the following mice: CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments (Fig. 97D, e.g., 953 ± 17 µM); untreated CK-p25 Tg mice (Fig. 97B, e.g., 861 ± 30 µM); CK-p25 Tg mice treated with memantine (Fig. 97C, e.g., 850 ± 18 µM); and CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation (Fig. 97E, e.g., 848 ± 15 µM).

[0535] Changes in the number of neurons and / or damage to DNA protected and / or reduced neurons and / or damage to DNA are shown according to some embodiments. For example, γ exposure reduced CKp-25-induced neuronal loss and DNA damage in the visual cortex of subjects.

[0536] Figure 98 is a comparison of the percentage of NeuN-positive cells in the following mice as CK control mice. (NeuN-positive cells are shown on page 62 of the instruction manual, page 60 / 78, CN 122399187 A)Bar graph of sex cell counts: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light flicker according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light flicker. Therefore, the percentage of NeuN-positive cells in CK control mice was 100%, but only about 80% in untreated CK-p25 Tg mice, thus confirming neuronal loss in the CK-p25 Tg mouse model. Treatment with memantine prevented some neuronal loss in CK-p25 Tg mice compared to the untreated group. Exposure to 40-Hz light flicker according to some embodiments prevented the most neuronal loss in CK-p25 Tg mice. Therefore, Figure 98 illustrates how 40-Hz visual flicker treatment according to some embodiments can protect neurons in the visual cortex. However, the combination of memantine and exposure to 40-Hz light flicker did not prevent the same amount of neuronal loss.

[0537] DNA double-strand breaks (DSBs) are an example of DNA damage in eukaryotic cells that lead to genomic instability, thereby triggering tumorigenesis and potentially accelerated aging. Phosphorylated histone H2AX (γH2AX) serves as a biomarker for cellular responses to DSBs. Figure 99 is a bar graph comparing the amount of γH2AX-positive cells in the following mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with methimazole, CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments, and CK-p25 Tg mice treated with both methimazole and 40-Hz light scintillation. γH2AX-positive cells were almost absent in CK control mice but were very high in untreated CK-p25 Tg mice, indicating significant DSBs and other DNA damage. Treatment with methimazole reduced the number of γH2AX-positive cells in CK-p25 Tg mice compared to the untreated group. According to some embodiments, exposure to 40-Hz light flicker caused an even greater reduction in γH2AX-positive cells in CK-p25 Tg mice. Thus, Figure 99 illustrates how 40-Hz visual flicker treatment according to some embodiments can reduce DNA damage in the visual cortex. However, the combination of methimazole and exposure to 40-Hz light flicker significantly increased the number of γH2AX-positive cells in CK-p25 Tg mice.

[0538] Figure 100 is an illustration of the use of Hoechst staining agent (indicating cortical cells), green fluorescent protein or GFP (indicating CK-p25), γH2AX (indicating DSB), or NeuN for subjects representing each group.A series of images of visual cortical samples labeled with (indicator neurons).

[0539] γ exposure also reduced CKp-25-induced neuronal loss and DNA damage in the somatosensory cortex of the subjects. Figure 101 is a bar graph comparing the percentage of NeuN-positive cells in the following mice as CK control mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light flicker according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light flicker. Thus, the percentage of NeuN-positive cells in CK control mice was 100% in CK control mice, but closer to 80% in untreated CK-p25 Tg mice, thus confirming neuronal loss in the CK-p25 Tg mouse model. Compared to the untreated group, treatment with memantine did not prevent any neuronal loss in CK-p25 Tg mice, except in combination with exposure to 40-Hz light flicker, which prevented the most neuronal loss in CK-p25 Tg mice. Therefore, Figure 101 illustrates how 40-Hz visual flicker treatment according to some embodiments can protect neurons in the somatosensory cortex.

[0540] Figure 102 is a bar graph comparing the amount of γH2AX-positive cells in the following mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light flicker according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light flicker. γH2AX-positive cells were absent in CK control mice but were very high in untreated CK-p25 Tg mice, indicating significant DSB and other DNA damage. Compared to the untreated group, treatment with memantine reduced the number of γH2AX-positive cells in CK-p25 Tg mice. Exposure to 40-Hz light flicker according to some embodiments caused an even greater reduction in γH2AX-positive cells in CK-p25 Tg mice. Thus, Figure 102 illustrates how 40-Hz visual flicker treatment according to some embodiments (pages 61 / 78, CN 122399187 A) can reduce DNA damage in the somatosensory cortex. However, the combination of memantine and exposure to 40-Hz light flicker significantly increased the number of γH2AX-positive cells in CK-p25 Tg mice.

[0541] Figure 103 is an illustration of the use of NeuN (indicator neurons), γH2AX (indicator DSB), and GFP in subjects representing each group.A series of images of somatosensory cortical samples labeled with (indicating CK-p25) and / or Hoechst staining agent (indicating cortical cells).

[0542] γ exposure also reduced CKp-25-induced neuronal loss and DNA damage in the insular cortex of the subjects. Figure 104 is a bar graph comparing the amount of NeuN-positive cells as the percentage of NeuN-positive cells in the following mice as CK control mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation. Thus, the percentage of NeuN-positive cells in CK control mice was 100% in CK control mice, but closer to 80% in untreated CK-p25 Tg mice, thus confirming neuronal loss in the CK-p25 Tg mouse model. Compared to the untreated group, treatment with memantine prevented some neuronal loss in CK-p25 Tg mice, except in combination with exposure to 40-Hz light flicker, which prevented minimal neuronal loss in CK-p25 Tg mice. Therefore, Figure 104 illustrates how 40-Hz visual flicker treatment according to some embodiments can protect neurons in the insular cortex.

[0543] Figure 105 is a bar graph comparing the amount of γ H2AX-positive cells in the following mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light flicker according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light flicker. γ H2AX-positive cells were absent in CK control mice but were very high in untreated CK-p25 Tg mice, indicating significant DSB and other DNA damage. Compared to the untreated group, treatment with memantine reduced the number of γ H2AX-positive cells in CK-p25 Tg mice. Exposure to 40-Hz light flickering according to some embodiments induced a similar reduction in γ H2AX-positive cells in CK-p25 Tg mice. Thus, Figure 105 illustrates how 40-Hz visual flickering treatment according to some embodiments can reduce DNA damage in the insular cortex. However, the combination of memantine and exposure to 40-Hz light flickering significantly increased the number of γ H2AX-positive cells in CK-p25 Tg mice.

[0544] Figure 106 is an illustration of the use of NeuN (indicator neurons), γ H2AX (indicator DSB), and GFP in subjects representing each group.A series of images of insular cortical samples labeled with either CK-p25 (indicating CK-p25) or Hoechst stain (indicating cortical cells).

[0545] γ exposure also reduced CKp-25-induced neuronal loss and DNA damage in the hippocampus of the subjects. Figure 107 is a bar graph comparing the percentage of NeuN-positive cells in the following mice as CK control mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation. Thus, the percentage of NeuN-positive cells in CK control mice was 100% in CK control mice, but closer to 80% in untreated CK-p25 Tg mice, thus confirming neuronal loss in the CK-p25 Tg mouse model. Compared to the untreated group, treatment with or without exposure to 40-Hz light scintillation prevented some neuronal loss in CK-p25 Tg mice, with the untreated group showing the least neuronal loss prevention in CK-p25 Tg mice. Thus, Figure 107 illustrates how 40-Hz visual scintillation treatment according to some embodiments can protect neurons in the hippocampus.

[0546] Figure 108 is a bar graph comparing the amount of γ H2AX-positive cells in the following mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation. γ H2AX-positive cells were absent in CK control mice but were very high in untreated CK-p25 Tg mice, indicating significant DSB and other DNA damage. Treatment with memantine reduced the number of γ H2AX-positive cells in CK-p25 Tg mice compared to the untreated group. Exposure to 40-Hz light scintillation according to some embodiments resulted in a better reduction of γ H2AX-positive cells in CK-p25 Tg mice. Thus, Figure 108 illustrates how 40-Hz visual scintillation treatment according to some embodiments can reduce DNA damage in the hippocampus. However, the combination of memantine and exposure to 40-Hz light scintillation significantly increased the number of γ H2AX-positive cells in CK-p25 Tg mice.

[0547] Figure 109 shows the use of Hoechst staining agent (indicating cortical cells), GFP (indicating CK-p25), and γ H2AX-positive cells on subjects representing each group.A series of images of hippocampal samples labeled with H2AX (indicating DSB) or NeuN (indicating neurons).

[0548] γ exposure and / or application of protective synapses and / or reduced synaptic loss according to some embodiments are shown. Changes in synaptic connectivity can be quantified using specific markers for glutamatergic synapses (e.g., VGluT1, VGluT2, PSD95, and GluR2) and specific markers for γ-aminobutyric acid (GABAergic synapses) (e.g., GAD and VGAT).

[0549] For example, γ exposure reduces CKp-25-induced synaptic loss in the visual cortex of subjects. Figure 110 is a bar graph comparing the percentage of baseline synaptic plaque density in glutamatergic synapses (using VGluT1) and γ-aminobutyric acid (GABA) synapses in the following mice as CK control mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation.

[0550] γ exposure also reduced CKp-25-induced synaptic loss in the somatosensory cortex of the subjects and even increased synaptic plaque density. Figure 111 is a bar graph comparing the percentage of baseline synaptic plaque density in glutamatergic synapses (using VGluT1) and γ-aminobutyric acid (GABA) synapses (using GAD65) in the following mice as CK control mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation.

[0551] γ exposure also reduced CKp-25-induced synaptic loss in the insular cortex of the subjects. Figure 112 is a bar graph comparing the percentage of baseline synaptic chromatin density in glutamatergic synapses (using VGluT1) and γ-aminobutyric acid (GABA) synapses (using GAD65) in the following mice as CK control mice: CK control mice, untreated CK-p25 Tg mice, CK-p25 Tg mice treated with memantine, CK-p25 Tg mice exposed to 40-Hz light scintillation according to some embodiments, and CK-p25 Tg mice treated with both memantine and 40-Hz light scintillation.

[0552] 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 a representative sample. Figure 113C is an image showing VGluT1 (indicating glutamatergic synapses) in a representative sample.Images of GAD65 (indicating γ-aminobutyric acid synapses). Figure 113D is a combined image showing Hoechst staining agent, VGluT1, and GAD65 in a representative sample. Figures 113E and 113F illustrate the method of quantification of GAD65 spots using GAD65. Figure 113E is a binary image of GAD65 converted from Figure 113C. ImageJ software (available from the National Institutes of Health, Bethesda, Maryland) was used to quantify the binary image, as shown in Figure 113F.

[0553] Studies were conducted to examine whether γ exposure and / or administration according to some embodiments affected the cerebral vascular system. Mice were placed in a dark chamber and exposed to 40-Hz light-emitting diodes (LEDs) or constant light off (dark) for one hour. After stimulation, the mice were sacrificed and perfused. Brain slices were stained with lectins attached to fluorophores to fluorescently mark blood vessels. Changes in vascular system size (i.e., vessel diameter) were measured using confocal imaging. Vasodilation was observed after one hour of 40-Hz LED flashing.

[0554] Figure 128A is a series of representative immunofluorescence images illustrating the enlarged vascular system in the visual cortex according to some embodiments (63 / 78 pages, 65 CN 122399187 A). Figure 128B is a bar graph depicting the diameter of blood vessels in the visual cortex and showing the increase in blood vessel diameter after γ exposure according to some embodiments.

[0555] Thus, γ exposure and / or application are shown to provide anatomical benefits (e.g., prevention and / or reduction of brain weight loss and vascular system enlargement), morphological benefits (e.g., prevention and / or reduction of abnormal ventricular dilation and cortical thickness loss), cellular benefits (e.g., prevention and / or reduction of neuronal loss), and molecular benefits (e.g., prevention and / or reduction of DNA damage and synaptic loss).

[0556] Furthermore, γ exposure and / or application are shown to be neuroprotective. Following γ treatment, the CK-p25 Tg mouse model—which otherwise exhibited increased Aβ peptide levels, significant neuronal loss, DNA damage, synaptic loss, tau hyperphosphorylation, long-term enhancement deficits, and severe cognitive / memory impairment—showed relative protection of neuronal structure and / or function (e.g., maintaining and / or preventing disease progression and / or reducing / slowing disease progression), and in some cases indicated improvements in neuronal structure and / or function.

[0557] Auditory stimulation at γ frequencies noninvasively induces changes in microglia in subjects.

[0558] In some embodiments, γ exposure and / or administration includes auditory stimulation. Auditory stimulation may include sound pulses or ticks. Sound stimulation may include approximately 35 sound pulses or ticks / second (ticks / s) to approximately 45 ticks / s.A series of ticking sounds. Figure 114 is a stimulus diagram illustrating a series of ticking sounds according to some embodiments. The stimulus in Figure 114 has a ticking frequency of 40 ticks / s, with 25 ms between each tick and each tick having a duration of 1 ms.

[0559] In some embodiments, the sound stimulus has frequencies 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. For example, each sound pulse or tick in the ticking series has a frequency of about 10 kHz.

[0560] In some embodiments, the sound stimulus has a sound pressure level of about 0 dB to about 85 dB, about 30 dB to about 70 dB, and / or about 60 dB to about 65 dB. For example, each sound pulse or tick in the ticking series has a sound pressure level of about 65 dB.

[0561] According to some embodiments, auditory gamma stimulation is shown to induce changes in the microglial cell state of the subject. Studies were conducted to examine whether auditory gamma exposure and / or administration according to some embodiments induced microglial cell activation in the auditory cortex of subjects. A series of 40-Hz tick stimuli similar to those in Figure 114 were used, the stimuli having a tick frequency of approximately 40 ticks / s, wherein each tick had a duration of approximately 1 ms at a pitch of approximately 10 kHz and approximately 60-65 dB. It was hypothesized that the series of tick stimuli modulated PV+ neurons in the auditory cortex, thereby exogenously modulating gamma oscillations in the auditory cortex.

[0562] Figure 115 is a flowchart illustrating the study. In Figure 115, WT mice were housed in their housing cages 11500. The mice were moved to behavior boxes (i.e., soundproof chambers) 11502 one hour / day for seven consecutive days (day 1-day 7). While in behavior boxes 11502, according to some embodiments, a first group of mice was exposed to silence, and a second group of mice was exposed to a series of tick stimuli. After each hour in behavior box 11502, the mice were returned to their living cage 11500. On day 8, the mice were sacrificed for tissue collection and staining 11504.

[0563] The tissues were examined for microglial cell levels, microglial cell morphological changes, and microglial cell activation (e.g., indicated by cell body size). Figure 116A is a bar graph depicting the average number of microglial cells in mice exposed to silence (no stimulation) compared to mice exposed to a series of ticking stimuli (stimulation). More microglial cells were observed in mice exposed to a series of ticking stimuli according to some embodiments. Figure 116B is a bar graph depicting the number of microglial cells in mice exposed to silence (no stimulation) compared to mice exposed to a series of ticking stimuli (stimulation). (Instruction manual 64 / 78 pages 66)CN 122399187 A Bar graph showing the mean fold change in the length of microglia processes. The mean fold change in the length of microglia processes was significantly smaller in mice exposed to a series of tick stimuli according to some embodiments. Figure 116C is a bar graph depicting the mean fold change in the cell body size of microglia in mice exposed to silence (no stimulation) compared to mice exposed to a series of tick stimuli (stimulation). The mean fold change in the cell body size of microglia was significantly larger in mice exposed to a series of tick stimuli according to some embodiments, thus indicating greater microglia activation.

[0564] Figure 117A is a representative image of microglia in mice exposed to silence. Figure 117B is a representative image of microglia in mice exposed to a series of tick stimuli according to some embodiments. The processes and cell bodies of microglia are visibly different between Figure 117A and Figure 117B according to some embodiments. Figure 118A is a magnified image from Figure 117B of microglia from mice exposed to a series of ticking stimuli according to some embodiments. A protrusion 11800 of the microglia is highlighted. Meanwhile, Figure 118B is a magnified image from Figure 117A of microglia from mice exposed to a silent environment. A protrusion 11802 of the microglia is highlighted to show its length relative to the shorter protrusion 11800 from microglia from mice exposed to a series of ticking stimuli according to some embodiments.

[0565] Figure 119A is a magnified image from Figure 117B of microglia from mice exposed to a series of ticking stimuli according to some embodiments. The region of the cell body 11900 of the microglia is highlighted. Meanwhile, Figure 119B is a magnified image from Figure 117A of microglia from mice exposed to a silent environment. The region of cell body 11902 of microglia is highlighted to show its size relative to the larger cell body 11900 (thus indicating greater microglia activation) of microglia from mice exposed to a series of ticking stimuli according to some embodiments.

[0566] According to some embodiments, an auditory gamma stimulation-induced phenotype similar to microglia activation in subjects is shown. The study of Figure 115 was repeated using 5XFAD Tg mice according to some embodiments. The tissue was examined for microglia levels, morphological changes in microglia (e.g., process length), and microglia activation (e.g., indicated by cell body size). Figure 120A is a bar graph depicting the average number / image field of microglia in mice exposed to silence (no stimulation) compared to mice exposed to a series of ticking stimuli (stimulation).Significantly more microglia were observed in mice exposed to a series of ticking stimuli according to some embodiments. Figure 120B is a bar graph depicting the mean fold change in cell body size of microglia in mice exposed to silence (no stimulation) compared to mice exposed to a series of ticking stimuli (stimulation). The mean fold change in cell body size was significantly greater in mice exposed to a series of ticking stimuli according to some embodiments, thus indicating greater microglia activation. Figure 120C is a bar graph depicting the mean fold change in process length of microglia in mice exposed to silence (no stimulation) compared to mice exposed to a series of ticking stimuli (stimulation). The mean fold change in process length was significantly smaller in mice exposed to a series of ticking stimuli according to some embodiments.

[0567] Figure 121A is a representative image of microglia in mice exposed to silence. Figure 121B is a representative image of microglia in mice exposed to a series of ticking stimuli according to some embodiments. The processes and cell bodies of microglia are visibly different between Figure 121A and Figure 121B, where the processes of microglia from mice exposed to a series of ticking stimuli according to some embodiments are shorter and the cell bodies are larger.

[0568] Auditory stimulation at γ frequencies noninvasively reduces Aβ in the auditory cortex and hippocampus of subjects.

[0569] According to some embodiments, auditory γ stimulation has been shown to reduce the level of Aβ in subjects. The study in Figure 115 was repeated using six-month-old 5XFAD Tg mice according to some embodiments, page 67 of the specification 65 / 78, CN 122399187 A. On day 8, the auditory cortex and hippocampus were dissected. ELISA was used to measure the levels of soluble and insoluble Aβ isoforms (including isoform Aβ1-40 peptide and isoform Aβ1-42 peptide). Insoluble Aβ was treated with 5M guanidino-HCl for three hours to dissolve the plaques.

[0570] According to some embodiments, auditory gamma stimulation has been shown to reduce the level of soluble Aβ in subjects. Figure 122A is a bar graph depicting, according to some embodiments, the levels of soluble isotype Aβ1-42 peptide in the auditory cortex of mice exposed to silent (no stimulation) and the levels of soluble isotype Aβ1-42 peptide in the auditory cortex of mice exposed to a series of ticking stimuli (stimuli) at much smaller levels.

[0571] Figure 122B is a bar graph depicting, according to some embodiments, the levels of soluble isotype Aβ1-40 peptide in the auditory cortex of mice exposed to silent (no stimulation) and the levels of soluble isotype Aβ1-40 peptide in the auditory cortex of mice exposed to a series of ticking stimuli (stimuli) at much smaller levels.

[0572] Figure 122C is a bar graph depicting, according to some embodiments, the levels of soluble isotype Aβ1-40 peptide in the hippocampus of mice exposed to silent (no stimulation) and the levels of soluble isotype Aβ1-40 peptide in the auditory cortex of mice exposed to a series of ticking stimuli (stimuli) at much smaller levels.The levels of soluble isotype Aβ1-42 peptide in the hippocampus of mice exposed to a series of ticking stimuli (stimulation) are shown in a bar graph.

[0573] Figure 122D is a bar graph depicting, according to some embodiments, the levels of soluble isotype Aβ1-40 peptide in the hippocampus of mice exposed to a series of ticking stimuli (stimulation) are shown in a bar graph.

[0574] According to some embodiments, auditory gamma stimulation shows a reduction in the level of insoluble Aβ in subjects. Figure 123A is a bar graph depicting, according to some embodiments, the levels of insoluble isotype Aβ1-42 peptide in the auditory cortex of mice exposed to a series of ticking stimuli (stimulation) are shown in a bar graph.

[0575] Figure 123B is a bar graph depicting, according to some embodiments, the levels of insoluble isotype Aβ1-40 peptide in the auditory cortex of mice exposed to a series of ticking stimuli (stimuli) and the levels of insoluble isotype Aβ1-40 peptide in the auditory cortex of mice exposed to a series of ticking stimuli (stimuli) and the levels of insoluble isotype Aβ1-42 peptide in the hippocampus ...

[0577] Figure 123D is a bar chart depicting the levels of insoluble isotype Aβ1-40 peptide in the hippocampus of mice exposed to a series of ticking stimuli (stimulations) relative to the levels of insoluble isotype Aβ1-40 peptide in the hippocampus of mice exposed to a series of ticking stimuli (stimulations) according to some embodiments.

[0578] Figure 124A is a representative image of microglia in 5XFAD mice exposed to a series of ticking stimuli according to some embodiments. Figure 124B is a representative image of microglia in 5XFAD mice exposed to a series of ticking stimuli. The processes and cell bodies of microglia are visibly different between Figure 124A and Figure 124B, wherein the processes of microglia from 5XFAD mice exposed to a series of ticking stimuli according to some embodiments are shorter and the cell bodies are larger.

[0579] Figure 124C is a representative image of microglia in WT mice exposed to a series of ticking stimuli. Figure 124D is a representative image of microglia in WT mice exposed to a series of ticking stimuli according to some embodiments. The processes and cell bodies of the microglia are visibly different between Figure 124C and Figure 124D, where the microglia originate from...The microglia of WT mice exposed to a series of ticking stimuli showed shorter process lengths and larger cell bodies. (Instructions for Use, pages 66 / 78, CN 122399187 A)

[0580] Therefore, according to some embodiments, non-invasive auditory stimulation at γ frequencies promotes a significant reduction in γ oscillations and AD-related lesions in the auditory cortex and hippocampus.

[0581] Auditory stimulation at γ frequencies has a positive effect on subject behavior.

[0582] According to some embodiments, auditory γ stimulation has been shown to improve subject recognition ability. Figure 125A is a flowchart illustrating a novel object recognition test performed using 5XFAD mice exposed to a series of ticking stimuli according to some embodiments and 5XFAD mice exposed to silence. The test assesses a subject's ability to recognize novel objects from familiar objects (i.e., recognition memory) based on the tendency of rodents to spend more time exploring novel objects than exploring familiar objects. The recognition index (RI) was used to compare subjects:

[0583]

[0584] In Figure 125A, 5XFAD mice were acclimatized to environment 12500. At time T1, two novel items 12502 were introduced. Then at time T2, after a one-hour rest, the mice were exposed to a familiar item 12504 and a novel item 12506 for one hour. Figure 125B is a bar graph depicting the results of a novel item recognition test according to some embodiments, wherein mice exposed to a series of ticking stimuli have a higher RI, indicating that mice exposed to a series of ticking stimuli spend significantly more time on the new item than on the familiar item due to better recognition memory.

[0585] According to some embodiments, auditory gamma stimulation has been shown to improve the discrimination ability of subjects. Figure 126A is a flowchart showing a novel item localization test performed using 5XFAD mice exposed to a series of ticking stimuli according to some embodiments and 5XFAD mice exposed to silence. The test assesses spatial memory and / or discrimination based on the tendency of rodents to spend more time exploring newly located objects. The Recognition Index (RI) is used for comparison among subjects:

[0586]

[0587] In Figure 126A, 5XFAD mice are acclimatized to environment 12600. At time T1, two objects are introduced to a first location 12602. Then at time T2, after a one-hour rest, the mice are exposed to one of the objects 12604 at their first location and another object 12606 located in a new second location for one hour. Figure 126B is a bar graph depicting the results of a novel object localization test according to some embodiments, where mice exposed to a series of ticking stimuli have a higher RI, indicating that mice exposed to a series of ticking stimuli have better spatial memory and / or discrimination.Much more time is spent on moving objects than on objects held in the same position.

[0588] According to some embodiments, auditory gamma stimulation has been shown to improve spatial memory in subjects. The Morris water maze test was performed using 5XFAD mice exposed to a series of ticking stimuli according to some embodiments and 5XFAD mice exposed to silence. As described above, the test assesses spatial and / or reference memory based on the subjects' use of distance cues to navigate from a starting position around the perimeter of an open swimming area to locate a submerged escape platform. The test was assessed through repeated trials, and spatial and / or reference memory was determined by preference for the platform area when the platform was not present.

[0589] Figure 127A is a graph depicting the average delay in finding the platform by mice exposed to silence (no stimulation) and mice exposed to a series of ticking stimuli (stimuli) according to some embodiments. Figure 127B is a bar graph depicting the results of the detection test for platform removal. According to some embodiments, mice exposed to a series of ticking stimuli spent more time searching for the vanishing platform in the target quadrant than mice exposed to silence, thus indicating that mice exposed to a series of ticking stimuli had better spatial and / or reference memory.

[0590] Therefore, according to some embodiments, non-invasive auditory stimulation at γ frequencies induces microglial cell activation, reduces AD-related (e.g., Aβ) lesions, and significantly alleviates cognitive deficits (in terms of, for example, recognition, discrimination, and spatial memory). Auditory γ stimulation has the potential for a wide range of commercial applications, including but not limited to applications for home or mobile use (e.g., using noise-canceling headphones), in cases of simple and usable administration options (including self-administration). In addition to the potential for self-administration, clinicians and / or researchers can administer the stimulation to subjects ranging from animal models to human patients according to some embodiments. Clinicians and / or researchers may find it useful to combine auditory γ stimulation with various forms of monitoring. For example, a treatment session may include positioning the subject in a soundproof room or providing the subject with noise-canceling headphones or another device to limit interference. Subjects may be monitored during stimulation using, for example, functional magnetic resonance imaging (fMRI) for any beneficial changes in brain state.

[0591] Experimental Methods

[0592] Animals

[0593] All animal work was approved by the Committee for Animal Care of the Division of Comparative Medicine (MIT, Cambridge, Massachusetts). 5XFAD Tg mice were used in conjunction with Tg...Adult (3-month-old) male dual Tg 5XFAD Cre mice were generated by hybridization using the PV or CW2 promoter-driven Cre strain. Adult (5-month-old) male and female APP / PS1 mice were donated by the Tonegawa Laboratory (MIT, Cambridge, Massachusetts). 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, Maine). Mice were housed in groups of 3–5 according to a standard 12-hour light / 12-hour dark cycle, and all experiments were performed during the light cycle. Food and water were provided on an ad hoc basis unless otherwise specified. Litterctic mice were randomly assigned to each condition. The experimenters were genotypically unaware of the animals during tissue handling and electrophysiological recording and analysis. No animals were excluded from the analysis.

[0594] AAV Vector

[0595] Adenovirus-associated virus particles with serotype 5 were obtained from Vector Core Facility (University of North Carolina, Chapel Hill, North Carolina). The AAV5 virus contained ChR2 fused to an enhanced yellow fluorescent protein (EYFP) fused to a bilaterally defined inverted open reading frame (DIO) driven by the EF1α promoter (see, e.g., Figure 9). The AAV DIO EYFP construct was used as a control.

[0596] Surgical Procedure

[0597] Three-month-old 5XFAD / PV-Cre or CW2 mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (1.1 mg kg⁻¹) and acetaminophen (0.16 mg kg⁻¹). A small craniotomy was performed 2.0 mm posterior to the anterior skull and 1.8 mm superior to the left of the midline. Virus delivery was achieved via a glass micropipette attached to a Quintessential Stereotaxic Injector™ (available from Stoelting, Wood Dale, Illinois) through a small dura mater incision. The micropipette was lowered 1.2 mm below the brain surface. A 1 μl pellet of virus (AAV DIO ChR2 – EYFP or AAV DIO EYFP; 2 x 10¹² viral molecules / ml) was injected into the CA1 region of the hippocampus at 0.075 μl min⁻¹. The pipette was held in place for 5 min after injection and then retracted from the brain. A single fiber optic implant (300 μm core diameter, available from Thorlabs, Newton, New Jersey) was lowered 0.9 mm below the brain surface around the injection site.mm. Two small screws anchored at the anterior and posterior edges of the surgical site are joined using gutta-percha to secure the implant in place. For electrophysiological recording, adult (three-month-old) male 5XFAD / PV-Cr double transgenic mice, as well as 5XFAD-negative littermates (for CA1 recording) or 5XFAD and its WT littermates (for visual cortex recording), are anesthetized with isoflurane and placed in a stereotactic frame. The scalp is shaved, an ophthalmic ointment (e.g., Puralube® Vet ointment (Dechra Pharmaceuticals, Northwich, UK)) is applied to the eyes, and the surgical area is sterilized with Betadine® antibacterial agent (available from Purdue Products LP, Stamford, Connecticut) and 70% ethanol (pages 68 / 78, CN 122399187 A). For CA1 recording, a craniotomy was performed (in mm, from the anterior skull: -2 A / P, 1.8 M / L) to deliver 1 μL of virus to CA1 (as described above). Target craniotomy sites for LFP recording were marked on the skull (in mm, from the anterior skull: for CA1, -3.23 A / P, 0.98 M / L and for the visual cortex, 2.8 A / P, 2.5 M / L), and three self-tapping screws (e.g., F000CE094, available from Morris Precision Screws and Parts, Southbridge, Massachusetts) were attached to the skull, and a custom-made stainless steel top plate was attached using dental cement (e.g., C&B Metabond®, available from Parkell, Edgewood, New York). On the day of the first recording session, an LFP craniotomy (e.g., 300-400 μm diameter) was performed using a dental drill, which was done by first thinning the skull to approximately 100 μm thickness and then creating a small hole using a 30-gauge needle. The craniotomy skull was then sealed with a sterile silicone elastomer (e.g., Kwik-Sil™ adhesive, available from World Precision Instruments, Sarasota, Florida) until the day of the recording session and between recording sessions.

[0598] Optogenetic Stimulation Protocol

[0599] Two to four weeks after viral injection and implant placement (this allows the mice time to recover and undergo behavioral training for electrophysiology and time for viral expression in neurons), hippocampal CA1 neurons were optogenetically manipulated. 200 mW 4793 nm was applied to each end using a fiber optic channel / physical contact connector.The DPSS laser was connected to a plug-in cable. During the experiment, 1 mW (measured from the end of the fiber) of optical stimulation was delivered for one hour. For molecular and biochemical analyses, each animal received one of three stimulation protocols: 8 Hz, 40 Hz, or random stimulation (delivering light pulses at random intervals determined by a Poisson process with an average frequency of 40 Hz), or for electrophysiological recording, each animal received all stimulation conditions interleaved during the recording process.

[0600] Visual Stimulation Protocol

[0601] Fifteen minutes prior to the experiment, 5XFAD mice were treated with saline (control) or Stephania tetrandra toxin (0.18 mg / kg). For molecular and biochemical analyses, the mice were then placed in a dark room illuminated by LED lights and exposed to one of five stimulation conditions for one hour: darkness, light, 20-Hz flicker, 40-Hz flicker, or 80-Hz flicker (12.5 ms light on, 12.5 ms light off) (see, for example, Figure 43A). For electrophysiological recording, each animal was subjected to darkness, light, 40-Hz flicker, or random (with light pulses delivered at random intervals determined by a Poisson process with an average interval of 40 Hz) stimulation conditions interleaved during the recording process at 10 s intervals.

[0602] Behavioral training and virtual reality (VR) for electrophysiology

[0603] For CA1 recordings, head-fixed animals ran on an 8” spherical treadmill supported by an air cushion through a virtual reality environment, as described by Harvey et al. The motion of the spherical treadmill was measured by an optical mouse and fed into the virtual reality software, which was run in the MATLAB® computing environment (software version 2013b, available from MathWorks, Natick, Massachusetts). The virtual environment consisted of a linear track with two small fences at each end, where the animal could turn. The animal was rewarded with sweetened condensed milk (diluted 1:2 in water) at each end of the track due to alternating arrival at each end of the track. The animal learned to run on the virtual linear track over approximately one week. The animal was then allowed to rest for one week to recover from surgery. And acclimatize the animal to the operation for one to two days, then begin behavioral training. To learn to move on the treadmill and feel comfortable in the testing environment, for the first two days of training, the animal is placed on a spherical treadmill without wearing the virtual reality system and is rewarded with undiluted sweetened condensed milk. On the second day of training on the spherical treadmill, the animal's food is restricted to encourage running. The animal is limited to no more than 85% of its baseline weight and is typically weighed above 88% of its baseline weight. From the third day until the end of training (usually 5-7 days), the animal is placed on the treadmill to increase the amount of time spent running on the VR linear track (30 minutes).(min to 2 hours). After traversing the length of the track, the animal was rewarded with diluted (1:2) sweetened condensed milk at the end of the linear track. Between recording sessions, the animal was given a review training session to maintain behavioral performance. For visual instruction manual 69 / 78 pages 71 CN 122399187 A cortical recording, the animal ran on a spherical treadmill while being exposed to dark, light, or light flickering conditions (described in the data acquisition section below). Prior to recording, the animal learned to move on the treadmill and feel comfortable in the test environment by being placed on the spherical treadmill (without wearing a virtual reality system) and receiving a reward of undiluted sweetened condensed milk.

[0604] Electrophysiological Data Acquisition

[0605] For optogenetic stimulation of CA1, during recording, a 300 μm core fiber was advanced through the skull of an open craniotomy for delivering the virus to CA1 to a depth of 900 μm in the brain. A 1 ms and 1 mW (measured from the end of the fiber) light pulse was delivered via a 473 nm DPSS (diode-pumped solid-state) laser (as described above). To avoid photoelectric artifacts, a glass electrode was used to record neural activity. The LFP electrode was pulled from a borosilicate glass pipette (e.g., available from Warner Instruments, Hamden, Connecticut) to a fine tip via a filament-based micropipette puller (e.g., the P-97 Flaming / Brown™ micropipette puller, available from Sutter Instruments, Novato, California) to the fine tip, which was then manually broken down to a diameter of approximately 10–20 μm and subsequently filled with sterile saline. For CA1 recording, the LFP electrode was advanced through the craniotomy skull at an angle of 60 degrees posterior coronally and 45 degrees inferior horizontally until a clear electrophysiological signal was observed in the hippocampal pyramidal layer (approximately 600–1000 μV theta wave during animal running, clearly resolvable SWR during static processes, and multiple spikes greater than 150 μV; see, for example, Figures 2A–2B). For visual cortex recording, the LFP electrode was advanced vertically through the craniotomy skull to a depth of 600–900 μm, and multiple spikes greater than 150 μV were observed. Data were acquired at a sampling rate of 20 kHz and a bandpass filter of 1 Hz–1 kHz. The duration of animal running or resting on a spherical treadmill was extended. For optogenetic stimulation sessions, data were recorded 30 minutes before the start of any stimulation. The stimulus was then delivered at a frequency of γ (40 Hz), random (as described in optogenetic stimulation), or θ (8 Hz) for a duration of 10 s, interleaved with a 10 s baseline period (no stimulus).Incorrect. In both animals, each type of stimulus or baseline stimulus was delivered for 5 min intervals instead of 10 s intervals. Every 30 minutes of stimulus recording was followed by 5–30 minutes of no-stimulation recording. For visual light flickering stimulation sessions, LED strips around the animal's light were flickered for 10 s intervals at frequencies of γ (40 Hz), random (as described above in the visual stimulation protocol), θ (8 Hz), or 20 Hz, or for a continuous 10 s interval, alternating with 10 s intervals when the light was off. Some recording was made above the brain surface during the light flickering to ensure that the light did not generate electrical or photoelectric noise during recording. The recording session was terminated after approximately 3–5 hours. The animals were 3–4 months old at the time of recording. Analysis of electrophysiological recordings

[0606] Spike detection

[0607] Spikes were detected by thresholding the 300–6000 Hz bandpass signal. The threshold was the median of a robust estimate of the standard deviation of the filtered signal plus five times the standard deviation of the filtered signal (median / 0.675) to avoid the standard deviation measurement being contaminated by spikes (see, for example, Rossant et al., “Spike Sorting for Large, Dense Electrode Arrays”, bioRxiv doi: dx_doi_org_10.1101_015198 (February 16, 2015)).

[0608] Local Field Potential (LFP)

[0609] The recorded traces were downsampled to 2 kHz and then bandpass filtered between 1 Hz and 300 Hz.

[0610] θ and SWR Detection

[0611] The activity of the transhippocampal network changes significantly when the animal runs or stands quietly, and these changes are often referred to as different network states. These network states can be clearly distinguished by the presence or absence of LFP oscillations in different frequency bands. When the animal is running, large theta (4–12 Hz) oscillations are observed in CA1, as shown elsewhere (see, for example, Figure 2A). When the animal is standing still, the theta oscillations are no longer visible, and SWR is recorded, which is a high-frequency oscillation of 150–250 Hz lasting approximately 50–100 ms and associated with bursts of group activity (see, for example, Figure 2B). SWR is detected when the envelope amplitude of the filtered trace is greater than four standard deviations above the mean by at least 15 ms (see, for example, Figure 4A, Figure 4B, Figure 5A, Figure 5B, Figure 6A, Figure 6B, Figure 7B, and Figure 8). The envelope amplitude is calculated by taking the absolute value of the Hilbert transform of the filtered LFP. It has been confirmed that when using a higher threshold for SWR detection, i.e., above the mean...The results disclosed herein still hold when the detection SWR is 6 standard deviations (which detects larger SWRs) (see, for example, Figures 6C and 7C). To detect θ (see, for example, Figures 3A and 3C), the LFP is bandpass filtered for θ (4–12 Hz), δ (1–4 Hz), and β (12–30 Hz) using ripple filters such as FIR. The ratio of θ to δ and β ('θ ratio') is calculated as the θ envelope amplitude divided by the sum of the δ and β envelope amplitudes. θ time periods are categorized as times where the θ ratio is greater than one standard deviation above the mean for at least two seconds, and the ratio reaches a peak at least two standard deviations above the mean. Non-θ time periods are categorized as times where the θ ratio is less than one standard deviation for at least two seconds. The SWR, θ time periods, and non-θ time periods are visually inspected to ensure that these criteria accurately detect the SWR, θ time periods, and non-θ time periods, respectively.

[0612] Power Spectrum

[0613] Spectral analysis was performed using multitaper methods (e.g., Chronux open-source software, available from Mitra Laboratory in Cold Spring Harbor, New York, time-bandwidth product = 3, number of cones = 5). To examine the power spectrum in the absence of stimulation (see, e.g., Figures 3A and 3C), only θ time intervals were included: θ time intervals longer than 5 seconds were divided into 5-second trials, and the mean power spectral density was calculated for each animal with respect to these trials. To examine the power spectrum during optogenetic stimulation (see, e.g., Figures 13A and 6C) and visual stimulation (see, e.g., Figures 43B and 43C), the data were divided into 10-second trials for each stimulation condition or baseline time interval, and the mean power spectral density was calculated for each animal with respect to these trials.

[0614] γ during the SWR process

[0615] The spectrum was calculated using the multi-cone method (e.g., the Chronux open-source software, available from Mitra Labs at Cold Spring Harbor, Cold Spring Harbor, New York). The spectrum was calculated for each SWR (including a 400 ms window before and after the peak of the SWR). The z-score spectrum was then calculated in each frequency band using the average and standard deviation of the spectrum calculated across the entire recording session to create a normalized measurement of power in the unit of standard deviation (see, for example, Figures 4A, 4B, 5A, and 5B). The instantaneous frequency of the γ oscillation during the SWR process was calculated by performing a Hilbert transform for a 10–50 Hz bandpass filtered LFP and then taking the reciprocal of the difference between the peaks of the transformed signal (see, for example, Figures 4A, 5A, and 5B). The γ frequencies before, during, and after the SWR were calculated by performing a z-score spectrum for a low γ oscillation.The LFP (20–50 Hz) was filtered and the envelope amplitude of the Hilbert transform was taken to obtain the average γ power in the 100 ms bin centered at the SWR peak. This power was normalized by the mean and standard deviation of the envelope amplitude over the entire recording session to obtain the z-score γ power for each bin around each SWR (see, for example, Figures 6A and 7B). The phase modulation of γ during the SWR process was calculated by performing a Hilbert transform on the (20–50 Hz) filtered LFP for γ and determining the phase of the resulting signal for each spike in the SWR process (see, for example, Figure 7E). To measure the difference in phase modulation between 5XFAD and WT animals, resampling was used for replacement: a subgroup of 100 spikes from each recording was randomly selected to create the phase modulation distribution, and this was repeated 500 times for each recording (see, for example, Figures 6C and 7A). The modulation depth for the spike-γ phase distribution is then measured by calculating the difference between peaks and troughs divided by the sum of peaks and troughs for each distribution (see, for example, Figures 6C and 7A). Differences in firing rates during stimulation: To plot a multi-unit firing histogram of stimulus-evoked firing rates, spikes are binned into 2.5 ms bins for 100 ms after the start of each light on the pulse, and the spike fraction in each bin is calculated. The average and SEM are then calculated for all light across the time intervals. To calculate the difference in multi-unit firing rates between conditions, the firing rate is calculated for each 10-second time interval of the stimulus or baseline (total number of spikes divided by the duration of the time interval). The difference in firing rates between adjacent time intervals of the relevant type of stimulus is taken (firing rate in the γ-stimulation time interval minus the firing rate in the baseline or random time interval of the optogenetic stimulus; firing rate in the γ-stimulation time interval minus the firing rate in the baseline, continuous, or random time interval of the optogenetic stimulus). Differences from all animals were plotted in bar charts (see, for example, Figures 14A and 44A), and median and quartiles of differences for each animal were plotted in box plots (see, for example, Figures 13B and 44A).

[0616] Immunohistochemistry

[0617] Mice were perfused with 4% paraformaldehyde under deep anesthesia, and the brains were fixed overnight in 4% paraformaldehyde. The brains were sectioned at 40 μm using a vibratory microtome (e.g., a Leica VT100S, available from Leica Biosystems, Buffalo Grove, Illinois). The sections were permeabilized and blocked for one hour at room temperature in PBS containing 0.2% Triton X-100 and 10% normal donkey serum. The sections were then vibrated at 4°C in PBS containing 0.2% Triton X-100 andIncubate overnight in PBS containing primary antibodies in 10% normal donkey serum. The primary antibodies are anti-EEA1 (BD Transduction Laboratories™ EEA1 (641057), available from BD Biosciences, San Jose, California), 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, Virginia), anti-albumin (e.g., ab32895, available from Abcam, Cambridge, Massachusetts), and anti-Rab5 (ADI-KAp-GP006-E, available from Enzo Life Sciences, Farmingdale, New York). To confirm the ELISA assay, anti-Aβ antibody D54D2 was used because it allows co-labeling with EEA1, and anti-Aβ antibody 12F4 was used because it does not react with APP, thus allowing determination of whether the labeling is specific for Aβ. For the co-labeling assay, anti-Aβ antibody 12F4 (805501, available from BioLegend, San Diego, California) was used. Primary antibodies were visualized using Alexa-Fluor 488 and Alexa-Fluor 647 secondary antibodies (molecular probes), and neuronal nuclei were visualized using Hoechst 33342 (94403, available from Sigma-Aldrich, St. Louis, Missouri). Images were acquired under the same settings for all conditions using a confocal microscope (LSM 710; Zeiss™). Images were quantified using ImageJ 1.42q by an experimenter unaware of the treatment group. For each experimental condition, at least two coronal sections from at least three animals were used for quantification. For imaging of CA1 in the hippocampus, the analysis was limited to the cone cell layer, unlike the Iba1+ cell analysis, in which the entire field of view was required to image a sufficient number of cells. ImageJ was used to measure the diameter of the Iba1+ cell bodies and track the length measurement process. 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. The "number of spots" was counted, including deposits greater than or equal to 10 μm.

[0618] CLARITY

[0619] Fixed brain tissue was sectioned into 100 μM coronal sections using a vibratory microtome (e.g., Leica VT100S, available from Leica Biosystems, Buffalo Grove, Illinois) in 1XPBS. Sections containing the visual cortex were selected according to the Allen Mouse Brain Atlas and incubated for 2 hours in cleaning buffer (pH 8.5–9.0, ddH2O containing 200 mM sodium dodecyl sulfate, 20 mM lithium hydroxide monohydrate, and 4 mM boric acid) with shaking at 55°C. The cleaned sections were washed 3 x 10 min in 1XPBST (0.1% Triton-X100 / 1XPBS) and incubated overnight in blocking solution (2% fetal bovine serum / 1XPBST) with shaking at RT. Subsequently, three 1-hour washes were performed in 1XPBST with shaking at RT. The sections were then incubated for 2 days at 4°C with primary antibodies against β-amyloid (805501, available from BioLegend, San Diego, California) and anti-Iba1 (Wako Chemicals, Richmond, Virginia; 019-19741) diluted 1:100 in 1X PBST. Another set of 3 x 1 h washes in 1X PBST was performed, followed by incubation at RT with a 1:100 mixture of secondary antibodies diluted 1X PBS. Fragmented donkey anti-rabbit Alexa Fluor® 488 (ab175694) and anti-mouse 568 (ab150101) secondary antibodies (both available from Abcam, Cambridge, Massachusetts) were used to visualize the primary antibody markers. When this incubation period is halfway through (pages 72 / 78, CN 122399187 A, page 74), Hoechst 33258 (Sigma-Aldrich; 94403) is incorporated into each sample at a final dilution of 1:250. The sections are then washed overnight in 1xPBS at RT with shaking. Before mounting for imaging, the sections are incubated for one hour at RT in RIMS (refractive index matched solution: 75 g Histodenz, 20 mL 0.1 M phosphate buffer, 60 mL ddH2O) with shaking. The tissue sections are mounted onto microscope slides with coverslips (e.g., VistaVision™, available from VWR) using Fluoromount G mounting agent (Electron Microscopy Sciences, Hatfield, PA, USA).(Originally obtained from International, LLC, Radnor, PA). Images were acquired on a Zeiss™ LSM 880 microscope (Carl Zeiss Microscopy, Jena, Germany) with the accompanying Zen Black 2.1 software. Slice overview and cellular level images for 3-D reconstruction were taken 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.

[0620] Western Blotting

[0621] Whole-cell lysates of CA1 cells in the hippocampus were prepared using tissue from three-month-old male 5XFAD / PV-Cre mice. The tissue was homogenized in 1 ml RIPA (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, Missouri)), incubated on ice for 15 min, and rotated at 4°C for 30 min. Cell debris was separated and discarded by centrifugation at 14,000 rpm for 10 min. The lysate was quantified using nanodroplets and 25 µg of protein was loaded onto a 10% acrylamide gel. The protein was blotting at 100 μL. Transfer from acrylamide gel to a PVDF membrane (e.g., Invitrogen™, available from Thermo Fisher Scientific, Waltham, Massachusetts) for 120 min at room temperature. Block the membrane using fetal bovine serum albumin (5% w / v) diluted in TBS:Tween. Incubate the membrane overnight at 4°C with primary antibodies and then at room temperature for 90 min with secondary antibodies. Primary antibodies were anti-APP (Invitrogen™ PAD CT695, available from Thermo Fisher Scientific, Waltham, Massachusetts), anti-APP (A8967, available from Sigma-Aldrich, St. Louis, Missouri), and anti-β-actin (ab9485, available from Abcam, Cambridge, Massachusetts). Secondary antibodies were horseradish peroxidase-linked (e.g., available from GE Healthcare, Marlborough, Massachusetts). ImageJ was used. 1.46a Quantitative signal intensity and normalized to β-actin values.Tau protein solubility was examined using sequential protein extraction. Detergent-insoluble tau fractions were detected using an antibody against Tau5 (e.g., AHB0042, available from Thermo Fisher Scientific, Waltham, Massachusetts).

[0622] ELISA

[0623] Hippocampal toe CA1 or VC was isolated from male mice, lysed using PBS or 5 M guanidinium HCl, and subjected to Aβ measurements using a mouse / human Aβ1-40 or Aβ1-42 ELISA kit (e.g., Invitrogen™, available from Thermo Fisher Scientific, Waltham, Massachusetts) according to the manufacturer's instructions. Tissue was lysed in phosphate-buffered saline (PBS) to extract PBS-soluble Aβ fractions. Soluble Aβ fractions may contain monomeric Aβ and oligomeric Aβ. Tissue was also treated with guanidinium hydrochloride (HCl) to extract insoluble Aβ fractions.

[0624] Whole-genome RNA sequencing

[0625] Total RNA was extracted from the CA1 isolate in the hippocampus using the RNeasy® kit (available from Qiagen, Hilden, Germany). The purified mRNA was used for RNA-seq library preparation using the BIOO NEXTflex™ kit (BIOO# 5138-08) according to the manufacturer's instructions. Briefly, 1 μg of total mRNA was subjected to the following sequential workflow: poly-A purification, fragmentation, first flexible strand and second strand synthesis, DNA end-adenylation, and adaptor ligation. The library was enriched by 15 cycles of PCR and cleaned using Agencourt® AMPure XP magnetic beads (available from Beckman Coulter Genomics, Danvers, Massachusetts). The quality of the library was assessed using an advanced fragment analyzer. Barcode libraries were equally mixed for sequencing on a single lane on an Illumina HiSeq 2000 platform at the MIT BioMicro Center (MIT, Cambridge, Massachusetts), pages 73 / 78 (CN 122399187). The raw fastq data from 50-bp single-end sequencing reads were compared with a mouse mm9 reference matrix using TopHat 2.0 software (available from the Center for Computational Biology at Johns Hopkins University, Baltimore, Maryland) to align RNA-seq reads with a mammalian-sized genome using the ultra-high-throughput short-read aligner Bowtie, and then analyze the mapping results to identify splice sites between exons.Group comparisons were performed. Transcript abundance was estimated and tests were performed using UCSC mm9 reference gene cluster processing map reads with Cufflinks 2.2 software (available from the Trapnell Lab at the University of Washington, Seattle, Washington). Relative transcript abundance was measured by exon fragment / kilobase / million-mapped fragment (FPKM). Gene differential expression tests between treated and untreated groups were performed using the Cuffdiff module (used to find significant changes between transcript expression, splicing, and promoter usage, including as part of Cufflinks 2.2 software (available from the Trapnell Lab at the University of Washington, Seattle, Washington)) where the adjusted p-value for statistical significance was <0.05 (GEO accession: GSE77471).

[0626] To understand the cellular and molecular mechanisms of 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 using TopHat Cufflinks 2.2 software (available from the Trapnell lab at the University of Washington, Seattle, Washington) for GSEA statistical analysis. Gene set enrichment analysis (GSEA) was used to determine whether a defined gene set from the RNA-seq data was significantly enriched in the direction of a 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 value of the fold change (case relative to control). A defined gene set (in this case, genes upregulated or downregulated after γ treatment) was considered significantly associated with perturbation-induced transcriptome changes (upregulation or downregulation) when the nominal p-value and FDR q-value were less than 0.05. The sign of the calculated normalized enrichment score (NES) indicated whether the gene set was enriched at the top or bottom of the ranked list. Heatmaps of differentially expressed genes were generated using a custom R script, and z-scores were calculated for all libraries across each gene based on gene FPKM values. Box plots for cell type-specific analysis were also generated using an R program based on gene FPKM values.

[0627] Quantitative RT-PCR

[0628] CA1 was isolated from the hippocampus of three-month-old male 5XFAD / PV-Cre mice. The tissues were rapidly frozen using liquid nitrogen and stored at -80°C, and RNA was extracted using the RNeasy kit according to the manufacturer’s protocol (Qiagen (Hilden, Germany)). The RNA (3 μg) was treated with DNase I (4 U, Worthington Biochemicals).(Lakewood, New Jersey)) RNA was purified using the RNA Clean and Concentrator-5 Kit (Zymo Research (Irvine, California)) according to the manufacturer's instructions and eluted with 14 μl of DEPC-treated water. For each sample, 1 μg of RNA was reverse transcribed for one hour at 50°C in a 20 μl reaction volume containing a random hexamer mixture and Superscript III reverse transcriptase (50 U, Invitrogen™, available from Thermo Fisher Scientific, Waltham, Massachusetts). The 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). The relative changes in gene expression were assessed using the 2-ΔΔCt method.

[0629] Microglia were isolated from the visual cortex. The V1 region was quickly cut and placed in ice-cold Hanks balanced salt solution (HBSS) (Gibco™ 14175-095, available from Life Technologies). The tissue was then enzymatically lysed using the Neural Tissue Dissociation Kit (P) (130-092-628, Miltenyi Biotec, Cambridge, Massachusetts) with minor modifications to the manufacturer’s protocol. Specifically, the tissue was enzymatically lysed at 37°C for 15 minutes instead of 35 minutes, and the resulting cell suspension was passed through a 40 µm cell filter (352340, Falcon Cell Filter Instruction Manual 74 / 78, 76 CN 122399187 A, Sterile, Corning, New York) instead of the 70 µm MACS® Smart Filter. The resulting cell suspension was stained with allophycocyanin (APC)-conjugated CD11b mouse clone M1 / 70.15.11.5 (130-098-088, Miltenyi Biotec, Cambridge, Massachusetts) and phycoerythrin (PE)-conjugated CD45 antibody (e.g., BD Pharmingen™, 553081). CD11b and CD45-positive microglia were then purified using fluorescence-activated cell sorting (FACS). Cells were directly sorted into 1X PBS (see, e.g., Figure 52A).

[0630] Statistical

[0631] For non-normally distributed electrophysiological data, results are presented as medians and quartiles, unless otherwise indicated. When these medians and quartiles do not indicate that the data are normally distributed, a two-sided Wilcoxon rank-sum test for equal medians is performed to determine whether the distributions are significantly different, or a Wilcoxon signed-rank test is performed to determine whether the distributions are significantly different from zero. Fluctuations are statistically similar between groups being compared. The Bonferroni method is used to correct for multiple comparisons. Molecular and biochemical results are presented as means and SEM. Percentages stated in this disclosure are group means. All statistical analyses are performed using Prism GraphPad software (GraphPad Software, La Jolla, California). Normality is determined using the D'Agostino & Pearson universal normality test. Fluctuations are statistically similar between groups being compared. Comparisons of normally distributed data consisting of two groups are analyzed by a two-sided unpaired t-test. Comparisons of normally distributed data consisting of three or more groups are analyzed by one-way ANOVA, followed by the Tukey multiple comparison test. Comparative data for non-normally distributed data were performed using the Mann-Whitney test. The statistical test, exact p-value, and sample size (n) for each experiment were specified in the legend. Molecular and biochemical analyses were performed using the minimum of three biological replicates / conditions.

[0632] Auditory Gamma Stimulus Generation

[0633] The following script (available from MathWorks, Natick, Massachusetts), edited in the MATLAB® programming language, demonstrates one method for generating a series of auditory tick stimuli according to some implementation schemes:

[0634] Tick_Frequency = Input('Specify number of ticks / second:'); % Get the desired number of ticks / second from the keyboard

[0635] Tick_Duration = Input('Specify tick duration in milliseconds:'); % Get the desired tick duration from the keyboard

[0636] Sound_Frequency = Input('Specify sound frequency in Hertz:'); % Get the desired sound frequency in Hert...

Claims

1. A method for at least one of increasing the number of microglia in at least one brain region of a subject, inducing morphological changes of said microglia consistent with a neuroprotective state, and promoting the activity of said microglia, comprising inducing synchronous gamma oscillations in said at least one brain region of the subject.

2. The method of claim 1, wherein the synchronous gamma oscillation upregulates at least one differentially expressed gene involved in the activity of microglia in at least one brain region of the subject.

3. The method of claim 2, wherein the at least one differentially expressed gene includes at least one of Nr4a1, Arc, Npas4, Cd68, B2m, Bsr2, Icam1, Lyz2, Irf7, Spp1, Csf1r, and Csf2ra.

4. The method of claim 1, wherein the morphological changes of the microglia consistent with the neuroprotective state include at least one of an increase in cell body size and a decrease in process length.

5. A method for reducing the amount of amyloid-β (Aβ) peptide in the hippocampus of a subject, the method comprising optogenetically stimulating fast flash-albumin (FS-PV) interneurons in the hippocampus using multiple light pulses, the FS-PV interneurons expressing optogenetic actuators to induce in vivo synchronous gamma oscillations in the FS-PV interneurons that reduce the amount of Aβ peptide in the hippocampus.

6. The method of claim 5, wherein the plurality of optical pulses have a pulse frequency of about 40 pulses / s.

7. The method of claim 6, wherein each of the plurality of optical pulses has a duration of approximately 1 ms.

8. The method of any one of claims 5-7, wherein each of the plurality of optical pulses has a wavelength of about 473 nm.

9. The method of any one of claims 5-8, wherein the optogenetic actuator is at least one of channel rhodopsin, halophilic rhodopsin, and paleorhodopsin.

10. The method of claim 9, wherein the optogenetic actuator is channel rhodopsin-2 (ChR2).

Images