Systems and methods for the prevention, mitigation, and / or treatment of dementia

Abstract

Provided are systems and methods for at least one of preventing, reducing, and treating levels or changes in at least one of amyloid beta (Aβ) peptide, C-terminal fragment beta (β-CTF), beta-secretase (BACE1), gamma-secretase, neuroinflammation, and / or dementia (e.g., Alzheimer's disease or age-related decline). [Solution] The device is configured to emit stimuli that induce synchronous gamma oscillations in the brain at a frequency (e.g., approximately 40 Hz) that synchronously activates specific target cell types (e.g., fast-spiking parvalbumin (FS-PV) immunoreactive interneurons) and / or brain regions (e.g., sensory cortex and / or hippocampus) in vivo.

Description

[Technical Field]

[0001] Statement regarding government support This invention was made with government support under authorization number RF1 AG047661 granted by the National Institutes of Health. The government has the rights to this invention.

[0002] Cross-reference of related applications This application claims priority to U.S. Patent Application No. 62 / 259,187, entitled “System and Methods for Preventing, Mitigating, and / or Treating Dementia,” filed on 24 November 2015, pursuant to Section 119(e) of the U.S. Patent Act. The disclosure of said application is incorporated herein by reference in its entirety.

[0003] This disclosure relates, in general, to systems and methods for preventing, mitigating, and / or treating dementia in subjects. More specifically, this disclosure relates to systems and methods for inducing synchronous gamma oscillations in at least one brain region of a subject. [Background technology]

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

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

[0006] The fundamental relationship between Aβ concentration and neuronal activity has been demonstrated. Firstly, treatment of organoid hippocampal slices prepared from transgenic (Tg) mice overexpressing APP with tetrodotoxin reduced neuronal activity, followed by a decrease in Aβ levels. Subsequently, the opposite effect, namely an increase in neuronal activity, was observed with treatment with picrotoxin. Dynamic in vivo regulation of Aβ peptide concentration and final macula deposition has also been demonstrated using neuronal activity. In human AD patients, imaging of the nervous system shows that the most severe macula deposition may coincide with the most consistently active brain regions known as the “default mode network.”

[0007] To date, there is no cure for Alzheimer's disease (AD), and treatment options are largely palliative, do not suppress the pathological progression of AD, and / or may have multiple troublesome 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 found in clinical trials to be detrimental and / or ineffective in reducing AD lesions. Clinical trials using amyloid beta vaccines (e.g., bapineozumab) have failed due to insufficient effect on cognitive function. Gamma-secretase inhibitors (e.g., semagacestat) have failed in clinical trials due to worsening of cognitive impairment in subjects. Even existing drugs such as acetylcholinesterase inhibitors (e.g., donepezil and rivastigmine) and N-methyl-D-aspartate (NMDA) receptor antagonists (e.g., memantine) show only mild effects on cognitive function. [Overview of the project]

[0008] Key microscopic pathological features of Alzheimer's disease (AD) include amyloid plaques, neurofibromatosis (NFTs), and widespread neuronal loss. This accumulation of neuronal damage occurs over long periods, leading to macroscopic circuitry dysfunction in the brain, particularly gamma power deficiency during memory and concentration tasks. These gamma oscillations (e.g., approximately 20Hz–100Hz, 20Hz–80Hz, or 20Hz–50Hz) are primarily regulated by fast-spiking parvalbumin (FS-PV) interneurons.

[0009] In one embodiment, the Disclosure provides an apparatus, method, and system for preventing, mitigating, and / or treating dementia in a subject, comprising inducing synchronous gamma oscillations in at least one brain region of the subject. In some embodiments, the dementia is AD, vascular dementia, frontotemporal dementia, Lewy body dementia, and / or age-related cognitive decline. This relates to the subject, which may be a human or an animal.

[0010] In some embodiments, the synchronous gamma oscillation has a frequency of about 20 Hz to about 50 Hz, for example, about 40 Hz. The synchronous gamma oscillation can be induced in a cell type-specific manner. For example, the oscillation can be harmonized with the synchronous activation of FS-PV interneurons. The synchronous gamma oscillation can be induced in a brain region-specific manner. For example, the oscillation can be harmonized with the synchronous activation in at least one of the hippocampal and sensory cortical regions.

[0011] In one embodiment, a method for preventing, mitigating, and / or treating dementia in a subject includes the steps of controlling a stimulus-emitting device to emit a stimulus, exposing the subject to the stimulus, and / or administering the stimulus to the subject, thereby inducing synchronized gamma oscillations in vivo in at least one brain region of the subject. The stimulus may have a frequency of about 35 Hz to about 45 Hz, for example, about 40 Hz. The stimulus-emitting device may be a tactile device, a light-emitting device, and / or a sound-emitting device. For example, the light-emitting device may be a fiber optic device. The duration of the subject's exposure to the stimulus and / or the duration of the subject's administration of the stimulus may be about one hour. The exposure to the stimulus and / or the administration of the stimulus to the subject may be repeated over a period of time. For example, the exposure to the stimulus and / or the administration of the stimulus to the subject may be repeated at least once a day over that period. The period may be, but is not limited to, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, and / or one month (or a longer period, for example, once a day over the lifetime of the subject).

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

[0013] In some embodiments, the synchronous gamma oscillation reduces Aβ peptide production in the at least one brain region of the subject, for example, by reducing the level (e.g., amount or proportion) of the C-terminal fragment (CTF) and / or N-terminal fragment (NTF) of APP in the at least one brain region of the subject. The synchronous gamma oscillation may reduce the cleavage of APP to CTF and NTF by BACE1 and / or γ-secretase in the at least one brain region of the subject. The synchronous gamma oscillation may reduce the level (e.g., number or proportion) of endosomes in the at least one brain region of the subject. For example, the endosomes may be positive for early endosomal antigen 1 (EEA1) and / or Ras-related proteins encoded by the RAB5A gene (Rab5). In some embodiments, the synchronous gamma oscillation promotes Aβ peptide clearance in the at least one brain region of the subject. The synchronous gamma oscillation may increase the uptake of Aβ peptide by microglia in the at least one brain region of the subject.

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

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

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

[0017] In one embodiment, a method for reducing the level (e.g., amount or percentage) of taurinization in the visual cortex of a subject comprises stimulating the subject with multiple light pulses at a pulse frequency of approximately 40 pulses / second, thereby inducing synchronized gamma oscillations in vivo in the visual cortex, which reduces taurinization in the visual cortex.

[0018] In one embodiment, a method for reducing the level (e.g., amount or percentage) of Aβ peptide in a target hippocampus and / or auditory cortex includes stimulating the target with multiple sound wave pulses at a pulse frequency of approximately 40 pulses / second, thereby inducing synchronous gamma oscillations in vivo in at least one of the hippocampus and auditory cortex, which in turn reduce the level (e.g., amount or proportion) of Aβ peptide in a target hippocampus and / or auditory cortex. On the other hand, it reduces the level of Aβ peptide.

[0019] In one embodiment, a system for preventing, reducing, and / or treating the level (e.g., amount or percentage) or change in Aβ peptide, neuroinflammation, and / or cognitive function in a subject comprises a stimulus emitter for in vivo synchronous activation of a brain region of the subject, at least one memory for storing stimulus parameters and processor execution instructions, and at least one processor transmissibly connected to the stimulus emitter and the at least one memory. When the processor execution instructions are executed, the at least one processor controls the stimulus emitter to emit a stimulus according to the stimulus parameters, the parameters including a frequency that synchronously activates the brain region at that frequency, thereby preventing, reducing, and / or treating Aβ peptide, neuroinflammation, and / or dementia in the subject. The frequency may be about 35 Hz to about 45 Hz, for example, about 40 Hz. The in vivo synchronous activation may be regulated by enzymes and / or may occur in specific cell types, for example, immunoreactive FS-PV interneurons. Examples of such enzymes include optogenetic activators, microbial opsins, ChR2, and / or the vector AAV-DIO-ChR2-EYFP.

[0020] In one embodiment, a system for preventing, reducing, and / or treating the level (e.g., amount or percentage) or change of Aβ peptide, neuroinflammation, and / or cognitive function in a subject comprises a light-occluding device for reducing ambient light to at least one eye of the subject and / or a noise-canceling device for reducing ambient noise to at least one ear of the subject. The light-occluding device may comprise a light-emitting unit for emitting a light stimulus to at least one eye for synchronous in vivo activation of at least one of the visual cortex and hippocampus of the subject. The noise-canceling device may comprise a speaker unit for emitting an auditory stimulus to at least one ear for synchronous in vivo activation of at least one of the auditory cortex and hippocampus of the subject. The system also comprises at least one memory for storing processor execution instructions, and at least one processor transmissibly connected to the light-occluding device and / or the noise-canceling device and the at least one memory. When executing an execution instruction of the processor, the at least one processor may control the light-emitting unit to emit the light stimulus at a frequency that synchronously activates at least one of the visual cortex and the hippocampus at that frequency. Alternatively, or further, the at least one processor may control the noise cancellation unit to activate the sound stimulus at a frequency that synchronously activates at least one of the auditory cortex and the hippocampus at that frequency.

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

[0022] In one embodiment, a method for preventing, reducing, and / or treating the level (e.g., amount or percentage) or change in Aβ peptide, neuroinflammation, and / or cognitive function in a subject comprises controlling at least one electroacoustic transducer to convert an electrical audio signal into a corresponding sound stimulus, i.e., a sound stimulus such as a click train with a click frequency of approximately 35 clicks / second to approximately 45 clicks / second, and exposing the subject to the sound stimulus and / or administering the stimulus to the subject to induce synchronous gamma oscillations in at least one brain region of the subject. This includes the synchronous gamma oscillation resulting in the prevention, reduction, and / or treatment of levels of Aβ peptide, neuroinflammation, and / or dementia in the subject.

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

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

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

[0026] In one embodiment, a system for preventing, reducing, and / or treating the level (e.g., amount or percentage) or change in Aβ peptide, neuroinflammation, and / or cognitive function in a subject comprises at least one electroacoustic transducer for converting an electrical audio signal into a corresponding sound stimulus, i.e., a sound stimulus such as a click train with a click frequency of approximately 35 clicks / second to approximately 45 clicks / second; at least one memory device for storing the electrical audio signal and execution instructions for the processor; and at least one processor transmissibly connected to the at least one electroacoustic transducer and the at least one memory device. When the execution instructions for the processor are executed, the at least one processor controls the electroacoustic transducer to output a sound stimulus to at least one ear of the subject, inducing synchronous gamma oscillations in at least one brain region of the subject, the synchronous gamma oscillations resulting in the prevention, reduction, and / or treatment of the level of Aβ peptide, neuroinflammation, and / or dementia in the subject.

[0027] The system may be fixed or portable. If the at least one electroacoustic transducer comprises at least one headphone for wearing around, over, and / or inside at least one ear of the subject, directing an auditory stimulus to that at least one ear of the subject and reducing ambient noise, the system may further comprise a headphone interface for transmitting the electrical audio signal to the at least one headphone. Alternatively, or further, the system may comprise a neuroimaging device for monitoring the function of the at least one brain region of the subject before, during, and / or after the output of the auditory stimulus.

[0028] In one embodiment, a method for preventing, mitigating, and / or treating dementia in a subject includes providing a device for inducing synchronous gamma oscillations in at least one brain region of the subject.

[0029] In one embodiment, blood levels of glucocorticoids involved in the stress response in the subject. A method for maintaining and / or reducing (for example, a quantity) includes providing a device for inducing synchronous gamma oscillations in at least one brain region of the subject.

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

[0031] In one embodiment, a method for maintaining and / or enhancing memory associations includes providing a device for inducing synchronous gamma oscillations in at least one brain region of the subject. The memory associations may be based on spatial memory.

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

[0033] In one embodiment, a method for maintaining and / or reducing changes in biostructure and / or morphology in at least one brain region of a subject includes providing a device for inducing synchronous gamma oscillations in at least one brain region of the subject. The biostructure and / or morphology may include brain weight, lateral ventricular size, cutaneous thickness, neuronal thickness, and / or vascular diameter. The at least one brain region may include the visual cortex, somatosensory cortex, and / or insular cortex of the subject.

[0034] In one embodiment, a method for maintaining and / or reducing changes in the number of neurons, the quality of the DNA of the neurons, and / or synaptic point density in at least one brain region of a subject includes providing a device for inducing synchronous gamma oscillations in at least one brain region of the subject. The at least one brain region may be the visual cortex, somatosensory cortex, insula, and / or hippocampus of the subject.

[0035] In one embodiment, a device for inducing synchronous gamma oscillations in at least one brain region of a subject can prevent, reduce, and / or treat dementia and / or anxiety in the subject, maintain and / or enhance the flexibility of memory association and / or cognition in the subject, and / or maintain and / or reduce changes relating to biological structure, morphology, cells, and molecules in at least one brain region of the subject.

[0036] It should be understood that all combinations of the aforementioned concepts and further concepts discussed in more detail below (provided that such concepts are not contradictory) are intended to be part of the subject matter of the invention disclosed herein. In particular, all combinations of subject matter described in the last claims of this disclosure are intended to be part of the subject matter of the invention disclosed herein. It should also be understood that terms expressly used herein, which may also appear in any disclosure incorporated by reference, should be given meanings that are most consistent with the specific concepts of the disclosure herein.

[0037] Other systems, processes, and features will become apparent to those skilled in the art by examining the following drawings and detailed description. All such additional systems, processes, and features are included in this description, are within the scope of the present invention, and are intended to be protected by the appended claims.

[0038] Those skilled in the art will understand that the drawings are primarily for illustrative purposes and do not limit the scope of the subject matter of the invention as described herein. The drawings are not necessarily to scale, and in some instances, various aspects of the subject matter of the invention disclosed herein may be shown in exaggerated or enlarged in the drawings to facilitate understanding of different features. In drawings, similar reference numerals generally refer to similar features (e.g., functionally and / or structurally similar elements). [Brief explanation of the drawing]

[0039] [Figure 1] This is a schematic diagram showing a mouse running through a virtual linear maze on a spherical treadmill, according to several embodiments. [Figure 2] Figures 2A and 2B are electrical traces showing theta oscillations and sharp wave ripples (SWR) recorded from the hippocampus CA1 according to several embodiments. [Figure 3] Figures 3A and 3B are plots showing the normalized power spectra and the mean and standard deviation of the normalized power spectral density during the theta cycle of 3-month-old Tg 5XFAD and wild-type (WT) mice, according to several embodiments. [Figure 4] Figures 4A and 4B are spectrograms showing the SWR of WT mice and 5XFAD mice according to several embodiments. [Figure 5] Figures 5A to 5C are plots showing the distribution of instantaneous gamma frequencies in SWR according to several embodiments. [Figure 6]Figure 6A is a series of graphs showing the gamma power of the Z score as a function of time from the SWR peak in 5XFAD and WT mice, according to several embodiments. Figure 6B is a plot showing the cumulative distribution of gamma power in SWR in 5XFAD and WT mice, according to several embodiments. Figures 6C and 6D are plots showing the cumulative distribution of gamma power of the Z score over 100 milliseconds centered on the SWR peak in WT and 5XFAD mice, according to several embodiments. Figure 6E is a plot showing the cumulative distribution of gamma power in large SWR in 5XFAD and WT mice, according to several embodiments. [Figure 7] In some embodiments of SWR, Figure 7A is a plot showing the percentage of spikes as a function of gamma oscillation phase, and Figure 7B is a plot showing the modulation depth of the spiking. Figures 7C and 7D are plots showing the percentage of spikes in the hippocampus CA1 in SWR as a function of gamma oscillation phase, according to some embodiments. In some embodiments of large SWR, Figure 7E is a plot showing the percentage of spikes as a function of gamma oscillation phase, and Figure 7F is a plot showing the modulation depth of the spiking. [Figure 8] Figures 8A and 8B are plots showing the SWR rate per non-theta period for each animal and all animals combined, according to several embodiments, in 5XFAD and WT animals. [Figure 9] This is a schematic diagram showing viral vectors for regulating the activation of specific cell types in the target brain, according to several embodiments. [Figure 10] Figures 10A and 10B are schematic diagrams showing signal transmission to the CA1 region of the hippocampus in question, according to several embodiments. [Figure 11] These are immunofluorescence images showing immunostaining of nerve tissue in subjects having ChR2 and DAPI, according to several embodiments. [Figure 12]Figure 12A is an immunofluorescence image showing ChR2-EYFP expressed in PV+ interneurons according to several embodiments. Figure 12B is a series of immunofluorescence images showing immunohistochemistry with anti-EYFP and anti-PV antibodies according to several embodiments. [Figure 13] Figures 13A and 13B include schematic diagrams of FS-PV interneuron testing, electrical traces of local electric field potentials, and power spectral density according to several embodiments. [Figure 14] Figures 14A and 14B include, according to several embodiments, a raw electrical trace after the start of a 1-millisecond laser pulse, a trace after filtering for spikes following optogenetic stimulation, and a plot of spike probabilities. [Figure 15] Figure 15A is a histogram showing the difference in firing rates between 40 Hz stimulation cycles and random stimulation cycles, according to several embodiments. Figure 15B is a bar graph showing the multi-unit firing rates per 40 Hz stimulation, random stimulation, and no-stimulation cycle for each animal, according to several embodiments. [Figure 16] Figure 16A is an electrical trace recorded from the hippocampus of a subject during the frequency-specific increase in stimulation of a specific cell type in the CA1 region of the hippocampus of the subject, according to several embodiments. Figure 16B is a power spectral density plot showing the frequency-specific increase in local electric field potential power in stimulation of a specific cell type in the CA1 region of the hippocampus of the subject, according to several embodiments. [Figure 17] Figures 17A and 17B are bar graphs showing the relative Aβ1-40 and Aβ1-42 levels of 5XFAD / PV-Cre CA1 by one-way ANOVA, according to several embodiments. [Figure 18] Figures 18A and 18B are bar graphs showing the relative Aβ1-40 and Aβ1-42 levels of 5XFAD / αCamKII-Cre CA1 by one-way ANOVA, according to several embodiments. [Figure 19]Figure 19A is a series of images showing immunohistochemistry with anti-Aβ and anti-EEA1 antibodies in the hippocampal CA1 region according to several embodiments. Figure 19B is a series of bar graphs showing the relative immunoreactivity of Aβ normalized to EYFP according to several embodiments. [Figure 20] Figure 20A is a series of immunofluorescence images showing immunohistochemistry with an anti-Aβ antibody in the hippocampal CA1 region of 5XFAD / PV-Cre, according to several embodiments. Figure 20B is a bar graph showing the relative immunoreactivity of Aβ normalized to EYFP, according to several embodiments. [Figure 21] Figure 21A is a representative Western blot showing the levels of APP (CT695), APP NTF (A8967), APP CTF (CT695), and β-actin (A5316) (loading control) in CA1, according to several embodiments. Figure 21B is a bar graph showing the relative (normalized to actin) immunoreactivity of APP CTF at 40 Hz to EYFP and random conditions, according to several embodiments. Figure 21C is a series of Western blots showing the levels of full-length APP 2106 (CT695), APP CTF 2108 (CT695), and β-actin 2112 (A5316, loading control) in CA1, according to several embodiments. [Figure 22] Figure 22A is a bar graph showing the relative (actin-normalized) immunoreactivity of APP NTF at 40 Hz to EYFP and random conditions according to several embodiments. Figure 22B is a bar graph showing the relative (actin-normalized) immunoreactivity of full-length APP at EYFP, random, and 40 Hz conditions according to several embodiments. [Figure 23] A series of immunofluorescence images showing immunohistochemistry with anti-Rab5 (ADI-KAp-GP006-E) according to several embodiments. [Figure 24]Under EYFP, 40 Hz, and random stimulation conditions according to several embodiments, Figure 24A is a bar graph representing the relative immunoreactivity of EEA1 normalized to EYFP, and Figure 24B is a bar graph showing the relative Rab5 intensity levels of CA1 from 5XFAD / PV-Cre. [Figure 25] Figure 25A is a bar graph showing the levels of Aβ peptide isoforms Aβ1-40 after stimulation of different types in the CA1 region of the target hippocampus, according to several embodiments. Figure 25B is a bar graph showing the decrease in Aβ peptide isoforms Aβ1-42 after stimulation of a specific cell type in the CA1 region of the target hippocampus, according to several 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 relative to actin) levels after stimulation of a specific cell type in the CA1 region of the target hippocampus, according to several embodiments. [Figure 26] Figures 26A–26B are immunofluorescence images showing different types of endosomal levels (based on EEA1 levels) after stimulation of the CA1 region of the hippocampus in question, according to several embodiments. [Figure 27] According to several embodiments, this is a bar graph showing the average intensity values ​​(normalized with respect to FAD) for immunofluorescence images of the target CA1 region of the hippocampus after different types of stimulation, as shown in Figures 6A-6B. [Figure 28] This heatmap shows differentially expressed genes in the mouse hippocampal CA1 region, determined by whole transcriptome ribonucleic acid sequencing (RNA-seq) with and without 40 Hz stimulation, according to several embodiments. [Figure 29] Box plots showing the upregulated and downregulated FPKM values ​​of genes under EYFP and 40Hz conditions, according to several embodiments. [Figure 30] This is a pie chart showing the cell type-specific expression patterns of identified upregulated genes after 40 Hz stimulation, according to several embodiments. [Figure 31]This bar graph shows RT-qPCR validation of specific gene targets in RNA-seq datasets according to several embodiments. [Figure 32] Figures 32A and 32B are plots showing the power spectral density of local electric field potentials recoded over the brain while showing flashing lights at 40 Hz, according to several embodiments. [Figure 33] This bar graph shows RT-qPCR validation of specific gene targets in RNA-seq datasets according to several embodiments. [Figure 34] A series of immunofluorescence images showing immunohistochemistry of the hippocampal CA1 region of 5XFAD / PV-Cre mice with anti-Iba1(019-19741) and anti-Aβ(12F4) antibodies in EYFP, 40 Hz, and random stimulation conditions, according to several embodiments. [Figure 35] Figure 35A is a bar graph showing the number of microglia under EYFP and 40 Hz conditions, according to several embodiments. Figure 35B is a bar graph showing the diameter of microglial cells normalized to EYFP under EYFP, 40 Hz, and random stimulation conditions, according to several embodiments. Figure 35C is a bar graph showing the average length of the primary process of microglia normalized to EYFP under EYFP, 40 Hz, and random stimulation conditions, according to several embodiments. Figure 35D is a bar graph showing the percentage of Iba1-positive (microglia) cells that are also Aβ-positive under EYFP and 40 Hz stimulation conditions, according to several embodiments. [Figure 36] Figure 34 is a series of 3D renderings formed by integrating immunofluorescence images according to several embodiments. [Figure 37] Figure 37A is a series of immunofluorescence images showing Hoechst's immunohistochemistry of the hippocampal CA1 region with 5XFAD / PV-Cre, according to several embodiments. Figure 37B is a bar graph showing the estimated CA1 thickness of 5XFAD / PV-Cre under EYFP and 40 Hz stimulation conditions, according to several embodiments. [Figure 38]Figure 38A is a heatmap showing differentially expressed genes (DEGs) determined by genome-wide RNA-seq of hippocampal CA1 under 40 Hz FS-PV+ stimulation or control stimulation, according to several embodiments. Figure 38B shows the overlap between DEGs upregulated under the treatment conditions of Figure 38A, according to several embodiments. [Figure 39] According to several embodiments, Figure 38A is a bar graph showing RT-qPCR validation of specific gene targets in the RNA-seq dataset. [Figure 40] Figure 38A is a plot illustrating the biological processes associated with upregulated genes, according to several embodiments. [Figure 41] According to several embodiments, Figure 38A is a plot illustrating the biological processes associated with downregulated genes. [Figure 42] Figure 42A is a series of immunofluorescence images showing the levels of Iba1 after different types of stimulation of the CA1 region of the hippocampus in question, according to several embodiments. Figure 42B is a bar graph showing the average intensity values ​​relative to the immunofluorescence images in Figure 42A, according to several embodiments. [Figure 43] Figure 43A is a schematic diagram showing mice exposed to flashing light stimuli according to several embodiments. Figure 43B includes plots of local electric field potential traces and power spectral density in the visual cortex before and during flashing light at 40 Hz, according to several embodiments. Figures 43C–43F are plots showing the power spectral density of local electric field potentials in the visual cortex, according to several embodiments. [Figure 44] Figure 44A is a series of histograms showing the percentage of spikes in the visual cortex as a function of time for 4 cycles of 40 Hz light flashing and as a function of equivalent duration for random light flashing, according to several embodiments. Figure 44B is a series of electrical traces of local electric field potentials recorded on the brain while showing light flashing, according to several embodiments. [Figure 45]Figure 45A is a histogram showing the difference in firing rates between 40 Hz light flashing and random light flashing, according to several embodiments. Figure 45B is a plot showing multi-unit firing rates in the visual cortex, according to several embodiments. [Figure 46] Figure 46A is a schematic diagram showing an experimental paradigm according to several embodiments. Figures 46B-46C are plots showing further changes in baseline levels of Aβ peptide isoforms Aβ1-40 and Aβ1-42, respectively, after the experimental paradigm of Figure 46A, according to several embodiments. [Figure 47] Figures 47A and 47B are bar graphs showing changes in baseline levels of Aβ1-40 and Aβ1-42, respectively, in the visual cortex of the 5XFAD, according to several embodiments. [Figure 48] Figure 48A is a bar graph showing the change in baseline levels of Aβ1-40 and Aβ1-42 in the barrel cortex of 5XFAD under dark and 40 Hz flashing conditions, according to several embodiments. Figure 48B is a bar graph showing the change in baseline levels of Aβ1-40 and Aβ1-42 in the visual cortex of APP / PS1 under dark and 40 Hz flashing conditions, according to several embodiments. Figure 48C is a bar graph showing the change in baseline levels of Aβ1-40 and Aβ1-42 in the visual cortex of WT under dark and 40 Hz flashing conditions, according to several embodiments. [Figure 49] A series of immunofluorescence images showing immunohistochemistry of the visual cortex of 5XFAD with anti-Iba1(019-19741) and anti-Aβ(12F4) antibodies under dark and 40 Hz flashing conditions, according to several embodiments. [Figure 50]Figure 50A is a bar graph showing the number of Iba1-positive cells (microglia) according to several embodiments. Figure 50B is a bar graph showing the diameter of microglial cell bodies normalized to a control under dark and 40 Hz flashing conditions, according to several embodiments. Figure 50C is a bar graph showing the average length of the primary process of microglia normalized to a control under dark and 40 Hz flashing conditions, according to several embodiments. Figure 50D is a bar graph showing the percentage of microglia that are also Aβ-positive under dark and 40 Hz flashing conditions, according to several embodiments. [Figure 51] The following are a series of 3D renderings (from immunofluorescence images) of Iba+ microglia from CLARITY-treated 100 μm tissue pieces under dark and 40 Hz flashing conditions, according to several embodiments. CLARITY is a method of making brain tissue transparent, for example, using an acrylamide hydrogel composed within and bound to the tissue. [Figure 52] Figure 52A is a flowchart illustrating a method for isolating microglia from the visual cortex using fluorescence-activated cell classification (FACS) according to several embodiments. Figure 52B is a bar graph showing the levels of Aβ1-40 in microglia isolated from the visual cortex of 3-month-old 5XFAD and WT control animals using the method of Figure 52A, according to several embodiments. [Figure 53] Figure 53A is a series of immunofluorescence images showing immunohistochemistry with SVP38 antibody to detect synaptophysins in the visual cortex of 3-month-old 5XFADs under dark and 40 Hz flashing conditions, according to several embodiments. Figure 53B is a bar graph showing the relative SVP38 intensity levels in the visual cortex of 5XFADs after dark and 40 Hz flashing conditions, according to several embodiments. [Figure 54] Figure 54A is a bar graph showing the decrease in Aβ peptide isoforms Aβ1-42 after stimulation of the subject's visual cortex with gamma oscillation, according to several embodiments. Figure 54B is a bar graph showing the levels of Aβ peptide isoforms Aβ1-42 after stimulation of the subject's visual cortex with gamma oscillation, and further 24 hours after stimulation, according to several embodiments. [Figure 55] Figure 55A includes plots of the electrical trace and power spectral density of the local electric field potential in the hippocampus before and during flashing of 40 Hz light, according to several embodiments. Figure 55B is a series of histograms of the rate of spikes in the hippocampus as a function of time for 4 cycles of flashing 40 Hz light and as a function of an equivalent period for random flashing light, according to several embodiments. [Figure 56] Figure 56A is a histogram showing the difference in firing rates between 40Hz light flashing and random light flashing, according to several embodiments. Figure 56B is a plot showing the multi-unit firing rate in CA1 during 40Hz light flashing, according to several embodiments. [Figure 57] Figure 57A is a bar graph showing the relative levels of Aβ1-40 in the visual cortex of 5XFAD according to several embodiments. Figure 57B is a bar graph showing the relative levels of Aβ1-42 in the visual cortex of 5XFAD according to several embodiments. [Figure 58] Figure 58A is a bar graph showing the relative Aβ1-40 levels in the visual cortex of a 5XFAD with recovery after flashing 40 Hz light, according to several embodiments. Figure 58B is a bar graph showing the relative Aβ1-42 levels in the visual cortex of a 5XFAD with recovery after flashing 40 Hz light, according to several embodiments. [Figure 59] Figure 59A is a schematic diagram showing tests according to several embodiments. Figure 59B is a bar graph showing the relative levels of Aβ1-42 in the visual cortex of 6-month-old 5XFAD mice after 7 days under conditions of 1 hour / day of darkness or 40 Hz flashing, according to several embodiments. Figure 59C is a bar graph showing the relative levels of Aβ1-40 in the visual cortex of 6-month-old 5XFAD mice after 7 days under conditions of 1 hour / day of darkness or 40 Hz flashing, according to several embodiments. [Figure 60]Figure 60A is a series of immunofluorescence images showing immunohistochemistry with anti-Aβ antibody in the visual cortex of 6-month-old 5XFAD mice after 7 days under conditions of 1 hour / day of darkness or 40 Hz flashing, according to several embodiments. Figure 60B is a bar graph showing the number of Aβ-positive plaques deposited in the visual cortex of 6-month-old 5XFAD mice after 7 days under conditions of 1 hour / day of darkness or 40 Hz flashing, according to several embodiments. Figure 60C is a bar graph showing the area of ​​Aβ-positive plaques in the visual cortex of 6-month-old 5XFAD mice after 7 days under conditions of 1 hour / day of darkness or 40 Hz flashing, according to several embodiments. [Figure 61] Figure 61A is a series of immunofluorescence images showing immunohistochemistry with anti-phosphoTau(S202) and anti-MAP2 antibodies in 4-month-old P301S mice after 7 days under conditions of 1 hour / day darkness or 40 Hz flashing, according to several embodiments. Figure 61B is a bar graph showing the relative phosphoTau(pTau)(S202) intensity levels in the visual cortex of P301S mice after 7 days under conditions of 1 hour / day darkness and 40 Hz flashing, according to several embodiments. Figure 61C is a bar graph showing the relative MAP2 intensity levels in the visual cortex of P301S mice after 7 days under conditions of 1 hour / day darkness and 40 Hz flashing, according to several embodiments. [Figure 62] Figure 62A is a series of immunofluorescence images showing immunohistochemistry with anti-pTau6202(S404) antibody in 4-month-old P301S mice at 7 days after exposure to 1 hour / day of darkness and 40 Hz flashing, according to several embodiments. Figure 62B is a bar graph showing the relative anti-pTau(S400 / T403 / S404) fluorescence intensity levels in the visual cortex of P301S mice at 7 days after exposure to 1 hour / day of darkness and 40 Hz flashing, according to several embodiments. [Figure 63]Figure 63A is a series of immunofluorescence images showing immunohistochemistry with anti-pTau6302(S396) antibody in 4-month-old P301S mice at 7 days after exposure to 1 hour / day of darkness and 40 Hz flashing, according to several embodiments. Figure 63B is a bar graph showing the relative pTau(S396) fluorescence intensity levels in the visual cortex of P301S mice at 7 days after exposure to 1 hour / day of darkness and 40 Hz flashing, according to several embodiments. [Figure 64] A series of immunofluorescence images showing immunohistochemistry with anti-Iba1 antibody in 4-month-old P301S mice after 7 days under conditions of 1 hour / day of darkness and 40 Hz flashing, according to several embodiments. [Figure 65] Figure 65A is a bar graph showing the number of microglia after 7 days under 1 hour / day darkness and 40 Hz flashing conditions, according to several embodiments. Figure 65B is a bar graph showing the diameter of microglial cells normalized to a control after 7 days under 1 hour / day darkness and 40 Hz flashing conditions, according to several embodiments. Figure 65C is a bar graph showing the average length of primary processes of microglia normalized to a control after 7 days under 1 hour / day darkness and 40 Hz flashing conditions, according to several embodiments. [Figure 66] This plot shows the levels of soluble and insoluble Aβ peptide isoforms Aβ1-40 and Aβ1-42 in the target visual cortex with and without visual gamma stimulation, according to several embodiments. [Figure 67] Figures 67A–67B are plots showing whole-brain Aβ peptide levels with and without transcranial gamma stimulation of the subject, according to several embodiments. [Figure 68] Figure 68A is a flowchart showing a study conducted to investigate whether gamma exposure and / or administration according to several embodiments causes stress in subjects. Figure 68B is a bar graph showing corticosterone levels indicating the stress response in the subjects. [Figure 69]Figure 69A is a flowchart showing a study conducted to investigate whether gamma exposure and / or administration according to several embodiments reduces anxiety in subjects. Figure 69B is an image showing an elevated cross maze device. Figures 69C and 69D are images showing typical trajectories of the subjects during the elevated cross maze period. [Figure 70] This bar graph shows the average time spent by participants exploring the elevated cross maze during the period, both on paths without walls and paths with walls. [Figure 71] Figure 71A is a flowchart showing studies conducted to investigate whether gamma exposure and / or administration according to several embodiments reduces stress and / or anxiety in subjects. Figure 71B is an image showing an open-field arena. Figures 71C and 71D are images showing typical trajectories of subjects during open-field studies. [Figure 72] Figure 72A is a plot showing the average time spent by subjects at the center of the open field per minute during the open field trial. Figure 72B is a bar graph showing the average total time spent by subjects around the open field during the open field trial. [Figure 73] Figures 73A and 73B are schematic diagrams illustrating studies conducted to investigate whether gamma exposure and / or administration according to several embodiments alter the innate novelty-seeking behavior of subjects. Figure 73C is a bar graph showing the average time subjects spent exploring a first novelty object compared to a second novelty object, following the schematic diagram in Figure 73A. [Figure 74] Figure 73B is a schematic diagram showing the average time per minute that the subject spent searching for novel objects. [Figure 75] Figure 75A is a flowchart showing a test conducted using the fear conditioning paradigm to investigate whether gamma exposure and / or administration, according to several embodiments, affects the learning and memory of subjects. Figure 75B is a stimulus diagram showing a tone test with contextual changes as a function of time. [Figure 76]Figures 76A and 76B are bar graphs illustrating the enhancement of target memory according to several embodiments. [Figure 77] Figure 77A is a flowchart showing tests conducted to investigate whether gamma exposure and / or administration improves a subject's memory according to several embodiments. Figure 77B shows a Morris water maze with a platform hidden in the target quadrant. Figures 77C and 77D are images showing typical trajectories of a subject during a Morris water maze probe test. [Figure 78] Figure 78A is a plot showing the average time the subject spent finding the hidden platform in the Morris water maze test each day. Figure 78B is a plot showing the average time the subject spent searching for the removed platform in the target quadrant in each 30-second period. Figure 78C is a plot showing the average time the subject spent searching for the removed platform in the opposite quadrant in each 30-second period. [Figure 79] Figure 79A shows a Morris water maze test with a platform hidden in the first quadrant. Figure 79B shows a Morris water maze test with a platform hidden in the second quadrant, i.e., on the opposite side of the first quadrant, for reverse learning. Figure 79C is a plot showing the average time the subject spent finding the hidden platform in the Morris water maze reverse learning test each day. [Figure 80] Figure 80A is a flowchart showing tests conducted according to several embodiments to investigate whether long-term gamma exposure and / or administration affects subjects' spatial learning and memory. Figure 80B is a plot showing the average time subjects spent finding the hidden platform in the Morris water maze test each day. Figure 80C is a bar graph showing the average time subjects spent searching for the removed platform within the target quadrant during a 30-second test. [Figure 81] Figure 81A is a flowchart showing the test from Figure 80A extended to include reverse learning. Figure 81B is a plot showing the average time spent by the subjects to find the hidden platform in the Morris Water Maze Reverse Learning Test each day. [Figure 82]Figure 82A is a bar graph showing the average time the subject spent searching for the removed platform in the target quadrant during a 30-second test. Figure 82B is a bar graph showing the average time the subject spent searching for the removed platform in the opposite quadrant. [Figure 83] This is a schedule diagram of studies conducted to investigate the effects of gamma exposure and / or administration, according to several embodiments, on deoxyribonucleic acid (DNA) damage and neuronal loss in the target visual cortex. [Figure 84] This figure shows a group of subjects in studies conducted to investigate the effects of gamma exposure and / or administration according to several embodiments. [Figure 85] Figure 84 is a bar graph comparing the changes in brain weight across the groups of subjects, according to several embodiments. [Figure 86] Figure 84 is a bar graph comparing the expression ratio of the lateral ventricle across the groups of subjects, according to several embodiments. [Figure 87] Figures 87A to 87E are images showing representative lateral ventricles of the group of subjects in Figure 84, according to several embodiments. [Figure 88] Figures 88A to 88C are anatomical diagrams of the brain showing the target brain region according to several embodiments. [Figure 89] According to several embodiments, Figure 84 is a bar graph showing the average thickness of the V1 layer across the target group. [Figure 90] According to several embodiments, Figure 84 is a bar graph showing the average thickness of the V1-NeuN-positive cell layer across the group of subjects. [Figure 91] Figures 91A to 91E are images showing representative Hoechst-labeled and / or NeuN-labeled cells of the group of subjects in Figure 84, according to several embodiments. [Figure 92] According to several embodiments, Figure 84 is a bar graph showing the average thickness of the SS1 cortex across the target group. [Figure 93]According to several embodiments, Figure 84 is a bar graph showing the average thickness of SS1-NeuN-positive cells across the group of subjects. [Figure 94] Figures 94A–94E are images showing cells with Hoechst labeling and / or NeuN labeling across the group of subjects in Figure 84, according to several embodiments. [Figure 95] According to several embodiments, Figure 84 is a bar graph showing the average thickness of the cortex of the insular cortex across the group of subjects. [Figure 96] According to several embodiments, Figure 84 is a bar graph showing the average thickness of the NeuN-positive cell layer in the insular cortex across the group of subjects. [Figure 97] Figures 97A–97E are images showing representative Hoechst-labeled and / or NeuN-labeled cells of the group of subjects in Figure 84, according to several embodiments. [Figure 98] Figure 84 is a bar graph comparing the amount of NeuN-positive cells in the visual cortex across the groups of subjects, according to several embodiments. [Figure 99] According to several embodiments, Figure 84 is a bar graph comparing the amount of γH2AX-positive cells in the visual cortex across the groups of subjects. [Figure 100] Figure 84 is a series of images showing representative visual cortex samples from the group of subjects, according to several embodiments. [Figure 101] According to several embodiments, Figure 84 is a bar graph comparing the amount of NeuN-positive cells in the somatosensory cortex across the groups of subjects. [Figure 102] According to several embodiments, Figure 84 is a bar graph comparing the amount of γH2AX-positive cells in the somatosensory cortex across the groups of subjects. [Figure 103] Figure 84 is a series of images showing representative somatosensory cortical samples from the group of subjects, according to several embodiments. [Figure 104] According to several embodiments, Figure 84 is a bar graph comparing the amount of NeuN-positive cells in the insular cortex across the groups of subjects. [Figure 105] Figure 84 is a bar graph comparing the amount of γH2AX-positive cells in the insular cortex across the groups of subjects, according to several embodiments. [Figure 106] Figure 84 is a series of images showing representative insular cortical samples from the group of subjects, according to several embodiments. [Figure 107] According to several embodiments, Figure 84 is a bar graph comparing the amount of NeuN-positive cells in the hippocampus across the groups of subjects. [Figure 108] Figure 84 is a bar graph comparing the amount of γH2AX-positive cells in the hippocampus across the groups of subjects, according to several embodiments. [Figure 109] Figure 84 is a series of images showing representative hippocampal samples from the group of subjects, according to several embodiments. [Figure 110] According to several embodiments, Figure 84 is a bar graph comparing the point density of the visual cortex across the target group. [Figure 111] According to several embodiments, Figure 84 is a bar graph comparing the point density of the somatosensory cortex across the groups of subjects. [Figure 112] According to several embodiments, Figure 84 is a bar graph comparing the point density of the insular cortex across the target groups. [Figure 113] Figures 113A–113D are images showing Hoechst staining, VGluT1 marker, and / or GAD65 marker in representative samples according to several embodiments. Figures 113E and 113F are images showing point quantification methods according to several embodiments. [Figure 114] This is a stimulus diagram illustrating click-train stimuli according to several embodiments. [Figure 115] This flowchart illustrates studies conducted to investigate whether auditory gamma exposure and / or administration, according to several embodiments, induces microglial activation in the target auditory cortex. [Figure 116]Figure 116A is a bar graph showing the average number of microglia in the auditory cortex of a subject, according to several embodiments. Figure 116B is a bar graph showing the ratio of the lengths of microglial processes in the auditory cortex of a subject, according to several embodiments. [Figure 117] Figures 117A and 117B are representative images of microglia in the auditory cortex of a subject, according to several embodiments. [Figure 118] Figures 118A and 118B are magnified images of the length of microglial projections from Figures 117A and 117B, according to several embodiments. [Figure 119] Figures 119A and 119B are enlarged images of microglia cell sizes from Figures 117A and 117B, according to several embodiments. [Figure 120] Figure 120A is a bar graph showing the average number of microglia per image field in the auditory cortex of a subject, according to several embodiments. Figure 120B is a bar graph showing the average ratio of microglial cell sizes in the auditory cortex of a subject, according to several embodiments. [Figure 121] Figures 121A and 121B are representative images of microglia in the auditory cortex of a subject, according to several embodiments. [Figure 122] Figures 122A to 122D are bar graphs showing the levels of soluble Aβ isoforms Aβ1-40 and Aβ1-42 in the auditory cortex and hippocampus of a subject, according to several embodiments. [Figure 123] Figures 123A to 123D are bar graphs showing the levels of insoluble Aβ isoforms Aβ1-40 and Aβ1-42 in the auditory cortex and hippocampus of a subject, according to several embodiments. [Figure 124] Figures 124A to 124D are representative images of microglia in the auditory cortex of a subject, according to several embodiments. [Figure 125] Figure 125A is a flowchart illustrating a novel object recognition test. Figure 125B is a bar graph showing memory improvement according to several embodiments. [Figure 126]Figure 126A is a flowchart illustrating a novel location recognition test. Figure 126B is a bar graph showing improvements in memory and / or recognition according to several embodiments. [Figure 127] Figure 127A is a plot showing the average time the subject spent finding the hidden platform during each day's Morris water maze test. Figure 127B is a bar graph showing the average time the subject spent searching for the removed platform within the target quadrant during the probe test. [Figure 128] Figure 128A is a series of representative immunofluorescence images showing enlarged vascular structures in the visual cortex, according to several embodiments. Figure 128B is a bar graph showing vascular diameters in the visual cortex and the increase in vascular diameter after gamma exposure, according to several embodiments. [Modes for carrying out the invention]

[0040] Detailed explanation In one embodiment, the Disclosure provides methods, apparatus, and systems for preventing, mitigating, and / or treating brain damage or cognitive impairment / dementia in a subject. In some embodiments, the brain damage is dementia.

[0041] Cognitive function is highly dependent on the precise timing of gamma frequency oscillations in neural network activity, i.e., rhythms related to concentration and working memory (e.g., approximately 20Hz–100Hz, 20Hz–80Hz, or 20Hz–50Hz). Since these oscillations arise from synaptic activity, they provide a direct link between the molecular properties of neurons and high levels of consistent brain activity. Importantly, gamma oscillation activity is disrupted in neural circuits impaired by molecular neuropathology in AD, and may symbolize a major determinant of memory impairment in the disease. A link between pathology and impaired brain oscillations is likely. It is not yet confirmed whether there is a correlation. However, manipulating brain rhythms can act as a multi-target therapy for the treatment of dementia such as Alzheimer's disease (AD), and this can be achieved through non-invasive treatment.

[0042] In one embodiment, the Disclosure provides apparatus, methods, and systems for enhancing or inducing gamma oscillations. In some embodiments, the enhancement or induction of gamma oscillations is by optogenetic methods. In other embodiments, the enhancement or induction of gamma oscillations is by behavioral methods. The Disclosure states that the enhancement and / or induction of gamma oscillations by optogenetic, behavioral, or other methods reduces the lesions of Alzheimer's disease (AD).

[0043] In one embodiment, the present disclosure provides apparatus, systems, and methods for restoring or inducing gamma oscillatory rhythms in subjects with 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 devices, systems, and methods for treating dementia.

[0044] As used herein, the terms “treatment” or “treating” refer to both therapeutic treatment and prophylactic or preventive measures. In some embodiments, subjects requiring treatment include not only subjects who already have the disease or condition, but also subjects who may develop the disease or condition, and whose objective is to prevent, delay, or reduce the disease or condition. For example, in some embodiments, the devices, methods, and systems disclosed herein may be used to prevent, delay, or mitigate a disease or condition to which the subject is genetically predisposed, such as Alzheimer's disease (AD). In some embodiments, the devices, methods, and systems disclosed herein may be used to treat, mitigate, reduce, and / or delay the progression of a disease or condition to which the subject has already been diagnosed, such as the symptoms of AD.

[0045] As used herein, the term “subject” means mammals, such as rodents, cats, dogs, or primates. Preferably, the subject of the present invention is humans.

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

[0047] Dementia is a disorder characterized by loss of cognitive abilities and / or memory impairment. Examples of dementia include Alzheimer's disease (AD), vascular dementia, Lewy body dementia, Pick's disease, frontotemporal dementia (FTD), dementia due to AIDS, age-related cognitive impairment, and age-related memory impairment. Dementia may also be associated with neurological and / or psychiatric conditions, such as brain tumors, brain injuries, epilepsy, multiple sclerosis, Down syndrome, Rett syndrome, progressive supranuclear palsy, frontal lobe syndromes, schizophrenia, and traumatic brain injury.

[0048] Alzheimer's disease (AD) is the most common neurodegenerative disease in developed countries. AD may be characterized by the accumulation of amyloid plaques, which consist of Aβ peptides, and neurofibromatosis (NFTs), which consist of tau proteins. Clinically, AD is associated with progressive cognitive impairment characterized by loss of memory, function, language ability, judgment, and executive function. AD often leads to serious behavioral symptoms in its later stages.

[0049] Vascular dementia, sometimes called cerebrovascular dementia, generally refers to cerebrovascular disorders (e.g., infarctions of the cerebral hemispheres) that have a fluctuating course with periods of improvement and gradual deterioration. Vascular dementia may include one or more symptoms of disorientation, memory impairment, and / or impaired judgment. Vascular dementia can be caused by multiple separate infarctions, or by other blood vessels. It may also be caused by vascular factors, such as autoimmune vasculitis, like that seen in systemic lupus erythematosus; infectious vasculitis, such as Lyme disease; recurrent intracerebral hemorrhage; and / or stroke.

[0050] Frontotemporal dementia (FTD) is a progressive neurodegenerative disorder. Subjects with FTD generally exhibit prominent behavioral and personality changes and often have language dysfunction.

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

[0052] In some aspects, the present disclosure provides a method for preventing, reducing, and / or treating dementia in a subject, including inducing synchronous gamma oscillations in the subject's brain. In some embodiments, inducing gamma oscillations in a subject with a neurological disease or disorder or age-associated decline acts to restore a gamma oscillation rhythm disrupted in the subject as a result of, or in relation to, the disease or disorder or age-associated decline.

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

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

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

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

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

[0058] In contrast, the M2 phenotype of microglia is associated with downregulation of inflammation and repair of inflammation-induced damage. Increased anti-inflammatory cytokines and chemokines (IL-4, IL-3, 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 the subject. For example, in some embodiments, the methods provided herein increase the phagocytic activity of microglia so as to increase Aβ clearance.

[0059] Gamma oscillations may include a range of about 20 Hz to about 100 Hz. Accordingly, in some embodiments, the present disclosure provides methods for preventing, mitigating, or treating dementia in a subject, which include inducing gamma oscillations in the brain of the subject 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 30 to about 60 Hz, or about 35 Hz to about 45 Hz, or about 40 Hz. Preferably, the gamma oscillation is about 40 Hz.

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

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

[0062] In some embodiments, the subject is stimulated through an environment configured to induce gamma oscillations, such as a chamber that passively or actively blocks irrelevant stimuli (e.g., light shielding or noise cancellation). Alternatively, or further, the subject may be stimulated through a system that includes, for example, light shielding or noise cancellation features. In some embodiments, the subject receives visual stimuli through a stimulus emitter, such as eyeglasses designed to deliver stimuli. The device may block other light. In some embodiments, the subject receives auditory stimuli through a stimulus emitter, such as headphones designed to deliver stimuli. This can remove other noises.

[0063] In addition to at least one interface for emitting stimuli, some embodiments may include at least one processor (e.g., for generating stimuli, controlling the emission of said stimuli, monitoring the emission / outcomes of said stimuli, and / or processing feedback relating to said stimuli / outcomes), at least one memory (e.g., for storing instructions that the processor can execute, at least one stimuli, stimuli generation guidelines, feedback, and / or results), at least one communication interface (e.g., for communicating with said subject, healthcare providers, caregivers, clinical research investigators, databases, monitoring applications, etc.), and / or detection devices (e.g., for detecting said stimuli and / or said subject and providing feedback relating to said stimuli and / or said subject, including whether gamma oscillations are being induced, the subject's sensitivity, cognitive function, physical or chemical changes, stress, safety, etc.).

[0064] In some embodiments, gamma oscillations are induced by visual stimuli such as flashes of light in the range of approximately 20 Hz to 100 Hz. In certain embodiments, gamma oscillations are induced by flashes of light in the range of approximately 20 Hz to 50 Hz. In further embodiments, gamma oscillations are induced by flashes of light in the range of approximately 35 Hz to 45 Hz. In yet another embodiment, gamma oscillations are induced by flashes of light in the range of approximately 40 Hz. In some embodiments, the subject is exposed to flashes of light in the range of approximately 20 Hz to 100 Hz, or flashes of light in the range of approximately 20 Hz to 50 Hz, or flashes of light in the range of approximately 35 Hz to 45 Hz, or flashes of light in the range of approximately 40 Hz (for example, by being placed in a chamber equipped with a light-emitting shielding device, or by wearing a light-emitting shielding device).

[0065] In some embodiments, gamma oscillations are induced by auditory stimuli such as sound waves with frequencies of approximately 20 Hz to 100 Hz, or approximately 20 Hz to 80 Hz, or approximately 20 Hz to 50 Hz, or approximately 35 Hz to 45 Hz, or approximately 40 Hz. In some embodiments, the subject receives auditory stimuli of approximately 20 Hz to 100 Hz, approximately 20 Hz to 80 Hz, approximately 20 Hz to 50 Hz, approximately 35 Hz to 45 Hz, or approximately 40 Hz (for example, by being placed in a chamber equipped with a noise-canceling device, or by wearing a noise-canceling device).

[0066] In some embodiments, the subject receives visual and / or auditory stimuli (e.g., by being placed in a chamber equipped with a light-emitting device, or by wearing a light-emitting device) for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or longer. In some embodiments, the subject receives stimuli (e.g., by being placed in a chamber equipped with a light-emitting device, or by wearing a light-emitting device) for less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour. In some embodiments, the subject receives stimuli for less than 1 hour (e.g., by being placed in a chamber equipped with a light-emitting device, or by wearing a light-emitting device).

[0067] In some embodiments, the subject receives the method provided herein. In other embodiments, the subject is treated with the method provided herein on several separate occasions. The subject may be treated on a regular schedule or at the time symptoms appear or worsen. In some embodiments, long-term treatment may be effective in reducing soluble Aβ peptides and / or insoluble Aβ peptides (i.e., plaques).

[0068] In some embodiments, gamma oscillations are induced in a cell type-specific manner. In some embodiments, gamma oscillations are induced in FS-PV interneurons. The term "fast spiking" (FS), when used to describe a class of neurons, refers to the ability of a neuron to discharge at a high rate for a long period of time with little adaptation or attenuation of spike frequency at spike height. Thus, these neurons can sustain without significant regulation. Subsequent high-frequency discharges (e.g., equal to or higher than approximately 100 Hz or 150 Hz) are possible. This characteristic of FS neurons is largely due to the expression of fast, delayed-rectifying channels, i.e., channels that are activated and deactivated very rapidly.

[0069] In one embodiment, the stimulus may be non-invasive. As used herein, the term “non-invasive” means a device, method, and system that does not require any surgical intervention or procedure on the body, such as the injection or implantation of a composition or device. For example, the stimulus may be visual (e.g., flashing light), auditory (e.g., sound vibrations), and / or tactile (mechanical stimulation involving force, vibration, or movement).

[0070] In another embodiment, the stimulus may be invasive or at least partially invasive. For example, visual, auditory, and / or tactile stimuli may be combined with the injection or implantation of a composition (e.g., a photosensitive protein) or a device (e.g., integrated optical fibers and a solid-state light source).

[0071] Experimental data Gamma oscillations decrease during hippocampal SWR in the early stages of disease in 5XFAD mice. Gamma dysfunction has been observed in multiple brain regions in several neurological and psychiatric disorders, including decreased spontaneous gamma synchronization in human patients with AD. Interestingly, decreased spontaneous gamma has also been observed in vivo in two mouse models of AD (human amyloid precursor protein (hAPP) transgenic mice and apolipoprotein E4 allele (APOE4) knock-in mice), as well as in in vitro slice studies in another mouse model (transgenic mice). This has been observed in CRND8 mice. However, it is unclear whether gamma oscillations are altered in other mouse models of AD, whether this occurs early in disease progression, and whether gamma disturbances affect disease progression.

[0072] To address these issues, we recorded neural activity from arousal behavior 5XFAD mice, an established AD model possessing five familial AD mutations. Specifically, 5XFAD mice express five distinct alleles of familial AD, including APP KM670 / 671NL (Swedish type), APP I716V (Florida type), APP V717I (London type), PSEN1 M146L (A>C), and PSEN1 L286V. Therefore, 5XFAD mice were used as a model for amyloid pathology in AD. In some embodiments, the neural activity was recorded from mice approximately 3 months of age, at which point Aβ levels are elevated but before the onset of major plaque accumulation and the manifestation of learning and memory impairments. Figure 1 is a schematic diagram showing mice running through a virtual linear maze on a spherical treadmill according to some embodiments. Feeding restriction mice can receive rewards by moving back and forth through the virtual linear maze on the spherical treadmill.

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

[0074] When the power spectral density during theta oscillation was examined, no significant difference was observed in low-speed gamma power (in the range of 20 Hz to 50 Hz) between 5XFAD mice and WT littermates. Figures 3A and 3B are plots showing the normalized power spectrum and the mean and standard deviation of the normalized power spectral density during theta period of 3-month-old Tg 5XFAD and WT mice, according to several embodiments. Figure 3A shows the normalized power spectrum during the theta period of 3-month-old 5XFAD (n=6 mice) and WT (n=6 mice) mice. The mean and standard deviation of the power spectra are shown. In some embodiments, the power spectral density of each animal may be normalized to its peak (during theta). Figure 3B shows the normalized power spectral densities during theta period of 3-month-old 5XFAD (n=6 mice) and WT (n=6 mice) mice.

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

[0076] Figures 4A and 4B are spectrograms showing the SWR of WT mice and 5XFAD mice according to several embodiments. Figure 4A shows that the average SWR triggered spectrogram of one WT mouse shows an increase in the gamma band 402 within the SWR 404 at frequencies below 80 Hz, which is magnified in the plot on the right. Figure 4B shows that the average SWR triggered spectrogram of one 5XFAD mouse also shows an increase in the gamma band within the SWR, but this increase is lower than that in the WT mouse shown in Figure 4A.

[0077] In some embodiments, the study found that the instantaneous frequencies of these low-velocity vibrations (in the range of 10–50 Hz, as further described herein) exhibit a unimodal distribution centered at 40 Hz. Figures 5A–5C are plots showing the distribution of instantaneous gamma frequencies in the SWR according to some embodiments. Figure 5A shows the distribution of instantaneous gamma frequencies in the SWR of the same mouse shown with the peak around 40 Hz in Figure 4A (n=370SWR). Figure 5B shows that the distribution of instantaneous gamma frequencies during SWR in 5XFAD and WT mice is around 40 Hz for each recording period, and Figure 5C shows the mean and inter-animal mean standard errors (SEM) (for 6 5XFAD animals, gamma cycles per period n = 820, 800, 679, 38, 1875, 57, and for 6 WT animals, gamma cycles per period 181, 1075, 919, 1622, 51, 1860, 1903).

[0078] In some embodiments, when these gamma oscillations in the SWR of WT mice were subsequently compared with those in 5XFAD littermates, abnormalities were found in the gamma during the SWR. While gamma power increased from baseline during the SWR in 5XFAD mice, as will be further described herein, the gamma power during the SWR was significantly lower in 5XFAD mice than in WT mice.

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

[0080] Figure 6B is a plot showing the cumulative distribution of gamma power in SWR in 5XFAD and WT mice according to several embodiments. The cumulative distribution of gamma power in SWR shows a significantly smaller increase in 5XFAD mice than in WT mice (rank-sum test, p<1). 0 -56 5XFAD mice produced n=2166 SWRs and 6 WT mice produced 3085 SWRs. The median z-score for 5XFAD mice was 1.02 (0.39 to 1.87, first quartile to third quartile), and the median z-score for WT mice was 1.18 (0.53 to 2.15, first quartile to third quartile).

[0081] Figures 6C and 6D are plots showing the cumulative distribution of gamma power of z-scores over 100 milliseconds, centered on the SWR peaks of WT mice 606 and 5XFAD mice 608, according to several embodiments, as well as mean and inter-animal SEM (shaded) (for 6 5XFAD animals, SWR n=514, 358, 430, 22, 805, 37 per period, and for 6 WT animals, SWR n=82, 311, 370, 776, 18, 710, 818 per period).

[0082] Figure 6E is a plot showing the cumulative distribution of gamma power z-scores over 100 milliseconds, centered around a large SWR peak in WT mouse 614 and 5XFAD mouse 616, according to several embodiments (detection threshold is above the upper 6 standard deviations of the mean). As further described herein, rank-sum tests were performed on non-normally distributed data. Figure 6E shows a significantly smaller increase in WT mouse 614 and 5XFAD mouse 616 (rank-sum test, p<10). -5 (6 5XFAD mice showed n=1000 SWR, and 6 WT mice showed 1467 SWR).

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

[0084] According to several embodiments, in the SWR of 3-month-old 5XFAD (n=6 mice) and WT (n=6 mice), Figure 7A is a plot showing the proportion of spikes as a function of gamma oscillation phase, and Figure 7B is a plot showing the modulation depth of spiking in the SWR as a function of gamma phase (rank sum test, bootstrap method, p<10). -5 This is important when adjusting for multiple comparisons. 5XFAD spike-gamma phase distribution n=2500 and WT distribution 3000, median modulation depth 0.35 (0.21~0.44, first quartile~third quartile) for 5XFAD mice and median modulation depth 0.38 (0.29~0.47, first quartile~third quartile) for WT mice. Error bars indicate mean + / - SEM. Plot 704 shows a histogram of spiking modulation depth.

[0085] Figures 7C and 7D are plots showing the percentage of spikes in the hippocampus CA1 during SWR as a function of the phase of gamma oscillations for each animal in 5XFAD and WT animals, according to several embodiments, as well as the mean and inter-animal SEM (for 6 5XFAD animals, n=2475, 1060, 3092, 25, 6521, 123 spikes per period during SWR, and for 6 WT animals, 360, 4741, 1564, 2961, 88, 3058, 4270 spikes per period during SWR).

[0086] According to several embodiments, in 3-month-old 5XFAD (n=6 mice) and WT (n=6 mice) mice with a large SWR (detection threshold above the mean, 6 standard deviations above the mean; further described herein), Figure 7E is a plot showing the rate of spikes as a function of the phase of the gamma oscillation, and Figure 7F is a plot showing the depth of the spiking modulation (rank-sum test, bootstrap method, one asterisk indicates p<10). -10 The graph shows the 5XFAD spike-gamma phase distribution (n=2500) and the WT distribution (3000). Error bars indicate the mean + / - SEM.

[0087] This study also found that, in non-theta periods, 5XFAD mice may have a lower SWR per unit time compared to WT mice (rank-sum test, p<10). -5 In the 6 5XFAD mice, the non-theta period was n=634, and in the 6 WT mice, the non-theta period was 750, with a median of 0.07 Hz (0 to 0.17, first quartile to third quartile) for the 5XFAD mice and a median of 0.12 Hz (0 to 0.24, first quartile to third quartile) for the WT mice. As mentioned above, this period decreases further when gamma power increases.

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

[0089] Optogenetic stimulation of FS-PV interneurons at gamma frequencies drove gamma oscillations in the CA1 region of the hippocampus. The findings of gamma dysfunction during SWR in the early stages of disease progression in this mouse model of Alzheimer's disease (AD) raise the question of whether gamma oscillations can influence the molecular and cellular pathophysiology of AD. To investigate this, gamma oscillations were optogenetically driven in FS-PV interneurons of hippocampal CA1 in 2.5-month-old 5XFAD / PV-Cre dual transgenic mice by expressing ChR2 in a Cre-dependent manner using a dual-floxed reverse open reading frame (DIO) ChR2-EYFP adeno-associated virus (AAV). The study was conducted to confirm whether the gene induction of hippocampal gamma oscillations in mice affects the molecular pathology of the mouse model of AD. Hippocampal gamma oscillations were genetically induced in aroused behavioral WT and 5XFAD mice.

[0090] Adeno-associated viruses (i.e., AAV5 viruses) were constructed with a double-floxed, reverse-direction open reading frame (DIO)ChR2 conjugated to a highly sensitive yellow fluorescent protein (EYFP) driven by the EF1α promoter. Figure 9 is a schematic diagram showing viral vectors (i.e., AAV5-DIO-ChR2-EYFP) for regulating the activation of specific cell types in the brain of interest according to several embodiments. Viral expression targeted the CA1 region of the hippocampus in a cell type-specific manner. In the presence of Cre recombinase, one of two incompatible loxP variants inverts to enable ChR2 expression.

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

[0092] Figures 10A and 10B are schematic diagrams showing signal delivery to the CA1 region of the hippocampus in question, according to several embodiments. Figure 10A shows a mouse running on a ball and navigating a maze while receiving optogenetic gamma stimulation in the hippocampus, according to several embodiments. The arrow 1000 shown in Figures 10A and 10B indicates blue light that flashes at approximately 40 Hz to activate brain regions.

[0093] In this example, a 200mW 493nm DPSS laser was connected to a patch cord with fiber channel / physical contact connectors at each end. During the experiment, a light stimulus of approximately 1mW was delivered for approximately 1 hour. More specifically, blue light (e.g., 473nm) was delivered via an optical fiber positioned directly above the CA1 region of the hippocampus at various frequencies, including theta (e.g., approximately 8Hz), gamma (e.g., approximately 40Hz), and randomly at approximately 40Hz. In some embodiments, the stimulation conditions were not tested. According to some embodiments, the theta condition served as a frequency control, and the random condition adjusted for periodicity specificity.

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

[0095] Figure 12A shows immunofluorescence images of ChR2-EYFP expressed in PV+ interneurons according to several embodiments. Figure 12A shows that ChR2-EYFP was strongly expressed in PV+ interneurons of CA1 in 3-month-old 5XFAD / PV-Cre mice (scale bar = 100 μm). Figure 12B shows a series of immunofluorescence images (scale bar = 50 μm) of immunohistochemistry with anti-EYFP and anti-PV antibodies in 3-month-old 5XFAD / PV-Cre CA1 expressing AAV DIO ChR2-EYFP, which shows EYFP expression only in PV+ cells. To compare 5XFAD and WT mice, ChR2 was expressed in FS-PV interneurons of 5XFAD-negative littermates. For control against the nonspecific effects of photostimulation, 5XFAD / PV-Cre double transgenic mice expressing AAV-DIO containing only EYFP were used. In these mice, under identical genetic background and light delivery conditions, light delivery did not result in optogenetic stimulation. In some embodiments, FS-PV interneurons were selected at 40 Hz for two reasons. First, previous studies have shown that driving FS-PV interneurons at 40 Hz elicited the maximum LFP response. Second, in some embodiments, gamma impairment in SWR was found, and as shown in Figures 5A–5C, the instantaneous gamma frequencies in SWR formed a distribution centered around 40 Hz. In some embodiments, for electrophysiological recording, 40 Hz stimulation periods were alternated with no-stimulation periods or stimulation periods delivered at randomized intervals selected from a 40 Hz-centered Poisson distribution, as further described herein.

[0096] Figures 13A and 13B include schematic diagrams of the testing of FS-PV interneurons, electrical traces of local electric field potentials, and power spectral densities according to several embodiments. Referring to Figure 13A, 1302 is the electrical trace of the local electric field potential at CA1 before and during 40 Hz optogenetic drive of FS-PV interneurons. Plot 1304 shows the mean and standard deviation of power spectral density at CA1 during 40 Hz stimulation, random stimulation (stimulation at randomized intervals selected from a Poisson distribution centered at 40 Hz), or no stimulation of FS-PV interneurons (5XFAD mice n=4 and WT mice 3). Figure 13B shows the power spectral density of FS-PV interneurons in CA1 during 40Hz stimulation1306, random stimulation1308, or no stimulation1310 for each mouse (5XFAD mice n=4, 169, 130, 240, 73 per animal for 40Hz, 143, 129, 150, 72 for random, and 278, 380, 52, and 215 for no stimulation cycles; and WT mice n=3, 65, 93, 91 per animal for 40Hz, 64, 93, 90 for random, and 187, 276, 270 for no stimulation cycles). Delivery of a 1-millisecond 473nm optical pulse at 40Hz resulted in an increase in power at 40Hz in the LFP, as shown in plot 1306 of Figures 13A and 13B, whereas random stimulation did not result in an increase in power at 40Hz, as shown in plot 1308 of Figures 13A and 13B.

[0097] Furthermore, in some embodiments, the optical pulse effectively drove spikes 2–3 milliseconds after the onset of light, and the number of spikes per pulse was similar under both random and 40 Hz conditions. Figures 14A and 14B include plots of the raw electrical trace after the onset of a 1 ms laser pulse, the trace after filtering for spikes after optogenetic stimulation, and the probability of spikes, according to some embodiments. Figure 14A shows an exemplary raw trace 1402 and a filtered trace 1404 for spikes (300–6000 Hz) after optogenetic stimulation 1406. Plot 1408 shows a histogram of spikes per pulse after the start of a 1-millisecond laser pulse during 40Hz stimulation, random stimulation, or no stimulation (in 4 5XFAD and 3 WT mice, n=345762 times for 40Hz stimulation, 301559 times for random pulse stimulation, and 32350 times for no stimulation, with 552 40Hz stimulations, 543 random stimulations, and 1681 no-stimulation periods separated by at least 500 milliseconds). Figure 14B shows the probability of spikes after the start of a 1-millisecond laser pulse in response to a 40Hz stimulus 1412, a random stimulus 1414, or no stimulus 1410, with increased spiking approximately 2–3 milliseconds after the start of the laser pulse (5XFAD n=4, 87, 130, 8, 73 40Hz stimuli, 85, 129, 5, 72 random stimuli, and 251, 379, 15, 215 no-stimulation periods per animal, and WT n=3, 65, 93, 91 40Hz stimulus periods per animal, 64, 93, 90 random stimulus periods per animal, and 187, 277, 270 no-stimulation periods per animal). Error bars indicate mean + / - SEM.

[0098] Therefore, the 40 Hz oscillation in CA1 was effectively driven by optogenetic stimulation of FS-PV interneurons. Previous studies have shown that Aβ peptide levels increase after increased neuronal activity and decrease after quiescence. In some embodiments, the random stimulation condition was used to modulate the overall change in spiking activity induced by the stimulation. In some embodiments, multi-unit firing rates were compared in alternating periods of 40 Hz and random stimulation, and no significant difference in firing rates was observed under these conditions.

[0099] Figure 15A is a histogram showing the difference in firing rates between 40 Hz stimulation cycles and random stimulation cycles according to several embodiments. Figure 15A shows that both types of stimulation induce similar amounts of spiking activity (Wilcoxon signed-rank test against median zero, p>0.6, stimulation cycles n=538 from 4 5XFAD and 3 WT mice, "ns" indicates not significant). Distribution of the difference in firing rates during 40 Hz and random stimulation for all mice combined. Wilcoxon signed-rank test against zero p>0.6: median -1.75 × 10 -5 Hz (-1.28-1.18Hz, 1st to 3rd quartile) stimulation period n = 538.

[0100] Figure 15B is a bar graph showing the multi-unit firing rate per 1512 cycles of 40Hz stimulation, 1514 cycles of random stimulation, and 1510 cycles of no stimulation for each animal (rank-sum test for each animal of 3 WT and 4 5XFAD mice, p>0.09, median and quartiles shown in the figure, 40Hz stimulation cycles n=87, 130, 8, 65, 93, 91, 73 and random stimulation cycles 85, 129, 5, 64, 93, 90, 72 per mouse). The box plot shows the median (white line inside the box) and quartiles (top and bottom of the box). In all animals, there was no significant difference in firing rates between 40Hz and random stimulation, indicating that the random stimulation condition serves as a control for spiking activity (rank-sum test for each animal in 3 WT and 4 5XFAD mice, p>0.09, median and quartiles shown in figure, 40Hz stimulation periods per animal n=87, 130, 8, 65, 93, 91, 73 and random stimulation periods 85, 129, 5, 64, 93, 90, 72). We also investigated whether 40Hz stimulation caused neuronal hyperactivity compared to no stimulation. In most animals, 40Hz or There was no significant difference in firing rates between random stimulation and no stimulation (rank-sum test for each of the two WT and two 5XFAD animals, p>0.25, 40Hz stimulation periods per animal n=8, 93, 91, 73 and baseline periods 15, 277, 270, 215), or the firing rate during 40Hz or random stimulation was lower than during no stimulation (rank-sum test for each of the one WT and one 5XFAD animal, p<10). -5 This was significant when adjusted for multiple comparisons (40Hz stimulation period n=130, 65 and baseline period 379, 187 per animal), indicating that 40Hz stimulation did not cause neuronal hyperactivity. In one animal, activity was significantly higher with 40Hz or random stimulation than at baseline (rank-sum test for one 5XFAD mouse, p<10). -5(40 Hz stimulation period n=87 and baseline period 251) per animal. Therefore, in 6 out of 7 animals, there is no evidence that 40 Hz optogenetic stimulation of FS-PV interneurons causes hyperactivity. Thus, in some embodiments, as shown in Figure 15A, this random condition did not induce gamma oscillations but resulted in a similar amount of multi-unit spiking activity.

[0101] Figure 16A shows electrical traces recorded from the subject hippocampus during the frequency-specific increase of stimulation of a particular cell type in the CA1 region of the subject hippocampus, according to several embodiments. More specifically, Figure 16A shows traces recorded from the subject hippocampus during the frequency-specific increase of stimulation of FS-PV+ (i.e., gamma condition), according to several embodiments.

[0102] Figure 16B is a power spectral density plot showing frequency-specific increases in local electric field potential power in the CA1 region of the hippocampus in question, according to several embodiments. In particular, the power spectral density graph in Figure 16B verifies the specificity of the stimulation. Local electric field potential (LFP) power increased only in the 40 Hz band 1600 during gamma stimulation conditions when FS-PV+ was activated with a 40 Hz blue light pulse (n=4 mice per group). Neither baseline nor random stimulation conditions showed enhancement at this frequency 1600.

[0103] Gamma stimulation reduced Aβ production in the CA1 region of the hippocampus. Aβ accumulation can cause several neurotoxic events typical of AD lesions. Therefore, in some embodiments, the effects of gamma stimulation on overall Aβ peptide levels in 5XFAD mice were investigated. Three-month-old mice were used because plaques are absent in the hippocampus at this stage, allowing for the study of soluble Aβ dynamics independent of plaque loading. In some embodiments, 1 hour of stimulation of FS-PV interneurons resulted in Aβ levels in the 40Hz group compared to the EYFP control group, as measured by Aβ ELISA analysis in the CA1 region of the hippocampus. 1-4053.22% and Aβ 1-42 It was found that this reduced the amount by 44.62%.

[0104] Figures 17A and 17B show the relative Aβ of 5XFAD / PV-Cre CA1 by one-way ANOVA with all mice combined, according to several embodiments. 1-40 and Aβ 1-42 This is a bar graph showing the level (Aβ 1-40 Regarding this, EYFP mice n=8 and 40Hz mice 7, Aβ 1-42 For this, the number of mice per group was n=4. The bar graph in Figure 17A shows the relative Aβ of 5XFAD / PV-Cre CA1 under each stimulus condition. 1-40 This represents the level of Aβ. The circles 1702 superimposed on the bars of the bar graph indicate individual data points for each group (5XFAD / PV-Cre mice per group, EYFP n=8, 40Hz n=7, 8Hz n=4, random n=6). In this figure, one-way ANOVA was performed for all bar graphs, with note "ns" 1704 indicating non-significant, asterisk 1706 indicating p<0.05, and double asterisk 1708 indicating p<0.01. Figure 17B shows the relative Aβ of 5XFAD / PV-Cre CA1 under each stimulus condition. 1-42 This represents the level (5XFAD / PV-Cre mice per group, EYFP n=4, 4 (0Hz n=4, 8Hz n=3, random n=3). Figures 17A and 17B show the mean and SEM results.

[0105] Table 1 (below) shows the values ​​of raw Aβ (p<0.05) with a statistically significant difference, as determined by Student's t-test, when comparing mice from littermates subjected to different conditions. Table 1 shows the raw Aβ concentration (pg / ml) in ELISA dilution for each experimental group. 1-40 and Aβ 1-42 This indicates the level. [Table 1] JPEG0007837033000002.jpg224170 JPEG0007837033000003.jpg200170

[0106] In some embodiments, a comprehensive series of control experiments were conducted to determine whether the effect was specific to frequency, cell type, and / or periodicity. To determine frequency specificity, FS-PV interneurons in 5XFAD / PV-Cre dual transgenic mice were driven at 8 Hz, and no change in Aβ levels was observed. Next, when FS-PV interneurons were driven randomly, the effect was specific to periodic stimulation. In fact, amyloid levels did not decrease after random stimulation; on the contrary, Aβ levels increased instead. 1-40 It increased by 230.1%, Aβ 1-42 Aβ increased by 133.8% (see, for example, Figures 17A and 17B, p<0.01 by one-way ANOVA combining all mice, Aβ). 1-40 For comparison, EYFP mice (n=8) and random mice (n=4), Aβ 1-42 Each group consisted of n=3 mice. Mice from littermates under different conditions were compared. However, a statistically significant difference (p<0.01) was observed using Student's t-test.

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

[0108] Figure 18B shows the relative Aβ of 5XFAD / αCamKII-Cre CA1 under each stimulation condition. 1-42 Represents the level (n=3 αCamKII-Cre mice per group). In some embodiments, driving CamKII+ excitatory neurons at 8Hz or 40Hz results in Aβ 1-40 and Aβ 1-42 No significant difference was found in the levels (see, for example, Figures 18A and 18B, right, one-way ANOVA p>0.05, 40Hz mice n=6 and 8Hz mice 3(Aβ) 1-40 ), n=3 mice per group (Aβ 1-42 ). When comparing mice from littermates under different conditions, they showed no significant difference by Student's t-test (p>0.05). Similar to 5XFAD / PV-Cre mice, the driving of CamKII+ neurons in response to random stimulation also showed Aβ 1-40 A 257.6% increase and Aβ 1-42 This resulted in a 133.3% increase (see, for example, Figures 18A and 18B, right, p<0.001, Aβ by one-way ANOVA). 1-40 For comparison, 40Hz mice (n=5) and random mice (3), Aβ 1-42 For this group, n=3 mice were used. When comparing mice from littermates under different conditions, Aβ 1-40 The difference is statistically significant (p<0.001) according to the Student's t-test, and Aβ 1-42 (The result is p=0.13 by Student's t-test).

[0109] Therefore, the decrease in Aβ peptide levels after 40 Hz stimulation may be specific to the activation of FS-PV interneurons. In some embodiments, to confirm these ELISA findings by immunohistochemistry, Aβ was labeled using a C-terminal specific antibody of β-amyloid that does not cross-react with APP at CA1.

[0110] Figure 19A is a series of images showing immunohistochemistry of the hippocampal CA1 region with anti-Aβ and anti-EEA1 antibodies according to several embodiments. Specifically, Figure 19A is a series of immunofluorescence images showing immunohistochemistry of the hippocampal CA1 region with anti-Aβ1902(D54D2) and anti-EEA111904(610457) antibodies in 5XFAD / PV-Cre under EYFP, 40 Hz, and random stimulation conditions (scale bar = 50 μm). Figure 19B is a series of bar graphs showing the relative immunoreactivity of Aβ normalized to EYFP according to several embodiments. Specifically, Figure 19B shows the relative immunoreactivity of Aβ normalized to EYFP (n=4 mice per group, one-way ANOVA shows p<0.05 for 1908 and p<0.01 for 1920).

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

[0112] Brain amyloid concentration may depend on the production and clearance rate of Aβ. In some embodiments, Aβ peptides are produced by a series of proteolytic cleavages of APP by β and γ secretases. When BACE1 cleaves the APP holoprotein, CTF and NTF of APP may be produced. In some embodiments, to clarify how 40 Hz stimulation reduced Aβ levels, gamma-influenced APP cleavage was investigated by measuring the levels of CTF and NTF, which are cleavage intermediates of APP, after stimulation of FS-PV interneurons. A significant decrease in CTF after 40 Hz stimulation was observed in 18.6% of the EYFP group and 19.7% of the random group (p<0.05 and p<0.01 by one-way ANOVA, n=6 mice per group).

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

[0114] Figure 22A is a bar graph showing the relative (actin-normalized) immunoreactivity of APP NTF at 40 Hz for EYFP and random conditions (n=6 mice per group, one-way ANOVA; note "ns" 2204 indicates non-significant, 2202 indicates p<0.05). Figure 22B is a bar graph showing the relative (actin-normalized) immunoreactivity of full-length APP in EYFP, random, and 40 Hz conditions (one-way ANOVA, n=6 mice per group).

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

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

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

[0118] Figure 25A shows the Aβ peptide isoform Aβ after stimulation of different types of the CA1 region of the target hippocampus, according to several embodiments. 1-40This is a bar graph showing the level. In this experiment, 1 hour of optogenetic stimulation with FS-PV+ at approximately 40 Hz 502 resulted in Aβ in the hippocampal CA1. 1-40 The level was reduced. Excitatory pyramidal stimulation at 8 Hz (506) and 40 Hz (508) were Aβ 1-40 It did not have a significant effect on the level. Random 40Hz stimuli 504, and in particular random excitatory pyramidal stimuli 510, did not significantly affect the Aβ level. 1-40 This significantly increased the level of (n=4-9 animals per group).

[0119] Figure 25B shows the Aβ peptide isoform Aβ in the CA1 region of the target hippocampus after stimulation of a specific cell type in gamma oscillations according to several embodiments. 1-42 This is a bar graph showing the decrease in Aβ in the hippocampal CA1. In this experiment, 1 hour of optogenetic stimulation with FS-PV+ at approximately 40 Hz resulted in a decrease in Aβ in the hippocampal CA1. 1-42 The level was reduced (the number of animals per group n=2-4) ). Stimulation at 8Hz 520, excitatory pyramidal stimulation at 40Hz 522, and excitatory pyramidal stimulation at 8Hz 524 are Aβ 1-42 The level was increased. Random 40Hz stimuli 518, and in particular random excitatory pyramidal stimuli 526, Aβ 1-42 (This significantly increased the level of [the substance / condition].)

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

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

[0122] Figure 27 is a bar graph showing the average intensity values ​​(normalized to FAD) for the immunofluorescence images in Figures 26A-26B after different types of stimulation of the CA1 region of the hippocampus in question, according to several embodiments.

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

[0124] Figure 28 is a heatmap showing differentially expressed genes determined by whole transcriptome RNA-seq of the mouse hippocampal CA1 region with and without 40 Hz stimulation. Normalized z-scores were calculated for each differentially expressed gene (columns). Colors represent the relative low and high levels of gene expression. Table 2 (below) shows 130 genes upregulated by 40 Hz stimulation of FS-PV interneurons (p<0.05 using Cufflinks 2.2 software (available from Trapnell Lab, University of Washington, Seattle, Washington, for assembly of transcripts in RNA-seq samples, estimation of their abundances, and testing for differential expression and regulation)). [Table 2] JPEG0007837033000005.jpg178170

[0125] Table 3 (below) shows 393 genes downregulated by 40 Hz stimulation of FS-PV interneurons (p<0.05, obtained using Cufflinks 2.2 software, available from the Trapnell Lab, University of Washington, Seattle, Washington). [Table 3] JPEG0007837033000007.jpg249170 JPEG0007837033000008.jpg249170 JPEG0007837033000009.jpg89170

[0126] In some embodiments, upregulated genes generally had higher expression levels than downregulated genes. 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 inside the box) and quartiles (top and bottom of the box), the whiskers represent the minimum and maximum values, and the circles represent outliers. Upregulated genes may have been highly enriched in microglia. Specifically, about 35% of all upregulated genes had their highest expression in microglia (about 19% in neurons, about 17% in endothelial cells, about 14% in astrocytes, about 9% in myelinated oligodendrocytes, about 5% in oligodendrocyte progenitor cells, and about 1% in newly formed oligodendrocytes).

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

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

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

[0130] In addition, these transcriptome results suggest a greater phagocytic state of microglia. In some embodiments, the upregulated genes were positively correlated with genomic changes induced by macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF), both of which are known to promote Aβ uptake by microglia. Figures 32A and 32B are plots showing the power spectral density of local electric field potentials recoded over the brain while showing 40 Hz flashing lights, according to some embodiments. Figures 32A and 32B do not show an increase in power at 40 Hz, and therefore this effect is not due to photoelectric effects or electrical noise on the recording equipment (40 Hz flashing periods n=4, 2, 1, 1, 17, 42, 36, 55, 53 from 4 recording periods in 3 5XFAD animals receiving visual cortex recordings and 5 recording periods in 2 5XFAD and 3 WT mice receiving hippocampal recordings). The mean (solid line) and standard deviation (shaded area) between records are shown on the left (Figure 32A), and those for each animal are shown on the right (Figure 32B). Record 3202, with fewer than 3 flashing periods, had a noisier power spectral density than record 3204, which had more data, but none showed evidence of a 40 Hz peak. In some embodiments, RT-qPCR was performed to identify upregulated microglial cells involved in known microglial function. The identified genes were examined. It was confirmed that genes such as Cd68, B2m, Bst2, Icam1, and Lyz2, which are related to microglial phagocytosis, are upregulated in the hippocampal CA1 region after 40 Hz stimulation.

[0131] Figure 33 is a bar graph showing RT-qPCR validation of specific gene targets in RNA-seq datasets according to several embodiments. Figure 33 shows the relative RNA levels (expression ratios) under EYFP3302 and 40 Hz stimulation conditions (Student's t-test shows one asterisk = p<0.05, two asterisks = p<0.01, n=6 mice per group). Other notable upregulated genes include the microglia-rich transcription factor Irf7, the cell adhesion and migration factor Spp1, and the microglia proliferation markers Csf1r and Csf2ra (see, e.g., Figure 33, Student's t-test shows p<0.05 and p<0.01, n=6 mice per group). RT-qPCR also showed no change in the expression levels of the inflammatory genes Il6, Il1b (Il1-β), Itgam (CD11-b), and the anti-inflammatory gene Igf1 (see, e.g., Figure 33, p>0.05 by Student's t-test, n=6 mice per group). Therefore, the transcriptome results described herein suggest that 40 Hz neuronal stimulation induced microglia into a state that promotes uptake.

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

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

[0134] Firstly, when we counted the number of Iba1+ microglia in 6 animals per condition, we found that the 40Hz group had approximately twice as many microglia compared to the unstimulated EYFP condition (average 8 microglia cells per ROI) (see, e.g., Figures 34 and 35A, one-way ANOVA p<0.01, n=2 sections from 4 mice per group) and the random condition (average 10 microglia cells per ROI) (see, e.g., Figures 34 and 35A, one-way ANOVA p<0.05, n=2 sections from 4 mice per group) (15 microglia cells per 212.55 μm × 212.55 μm ROI). Previous studies have identified two main characteristics of phagocytic microglia as the size of the cell body. An increase in microglial primary process length and a decrease in process length have been shown in some cases, and therefore, we investigated how these features were affected by 40 Hz stimulation. In some embodiments, the diameter of each clearly labeled Iba1+ cell body in the field of view was measured. It was found that the diameter of microglial cell bodies increased by 135.3% after 40 Hz stimulation compared to no stimulation and by 138.7% compared to the random condition (see, e.g., Figures 34 and 35B, p<0.0001 by one-way ANOVA, n=2 sections from 4 mice per group). When the length of the primary process of microglia was measured under each condition, a decrease of 54.0% in the length of the primary microglial process was observed in the 40 Hz stimulation condition compared to the EYFP control and by 38.5% compared to the random stimulation (see, e.g., Figures 34 and 35C, p<0.0001 by one-way ANOVA, n=2 sections from 4 mice per group). These findings suggest that Iba1 levels did not affect the outcome, as no differences in Iba1 expression were observed between conditions in the gene expression analysis described herein (see, e.g., Tables 2 and 3). Therefore, the 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 to a phagocytic state. Potential co-localization of Aβ within microglia was evaluated as a method to assess microglial Aβ uptake during co-immunostaining with Aβ antibody (12F4, which does not cross-react with APP). In the CA1 neural network, where Iba1+ cells are predominantly located, the ratio of the number of microglia with Aβ / Iba1 co-localization of the cell body (ImageJ, Fuji co-localization plugin) to the total number of microglia increased by 54.9% compared to EYFP and by 50.3% compared to the random condition after 40 Hz stimulation (see, e.g., Figures 34 and 35C, p<0.01 by one-way ANOVA, n=2 sections from 4 mice per group). Overlap of Iba1 / Aβ signaling during microglial processes was excluded to avoid potentially random non-phagocytic co-localization.

[0135] In some embodiments, 3D renderings of microglia from this tissue and images from these renderings were formed to provide a better solution for the presence of Aβ signaling within microglia. Figure 36 is a series of 3D renderings formed by integrating immunofluorescence images from Figure 34 rotated 0°3602, -25°3604 around the Y axis, and 30°3606 around the X axis, according to some embodiments. Images were taken with a 40x objective lens (scale bar = 50 μm). Overall, gene expression and morphological analysis suggest that 40 Hz stimulation affects microglial activity by increasing the recruitment of microglial cells to the stimulation site and enhancing their phagocytic activity, resulting in increased binding to Aβ. Importantly, in some embodiments, no evidence of neuronal loss was observed by measuring the thickness of the CA1 cell layer using nuclear staining by Hoechst. There was no significant difference in mean CA1 volume between the EYFP and 40 Hz stimulation groups.

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

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

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

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

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

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

[0142] Overall, the results of this study showed that restoration or induction of gamma rhythm restored the molecular pathology in a mouse model of AD. Optogenetic cell-type specific and temporally precise reintroduction of gamma oscillations initiated multiple degenerative cascades involving the aggregation of the peptide isoform Aβ that contributes to AD neuropathology 1-40 and Aβ 1-42 production and enhanced their clearance. Furthermore, this treatment induced an anti-inflammatory microglial signaling pathway that counteracts immune mechanisms associated with neurodegeneration.

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

[0144] Visual stimulation at gamma frequency non-invasively drove gamma oscillations in the visual cortex. The strong reduction of Aβ levels by optogenetic stimulation at 40 Hz led to exploring other methods of inducing 40 Hz oscillations in the brain to confirm that this effect is not specific to optogenetic manipulation or invasive methods in any way. To investigate whether light flickering can be used as a non-invasive method to induce 40 Hz oscillations in the visual cortex, in some embodiments, animals were exposed to 40 Hz or random flickering and continuous light cycles alternating with dark cycles.

[0145] Figure 43A is a schematic diagram showing mice exposed to light flickering stimulation according to some embodiments. To determine whether this light flickering modifies Aβ, animals were exposed to light flickering at 40 Hz for 1 hour, which corresponded to the period of optogenetic stimulation that decreased Aβ described herein. The light flickering covered the entire visual field of the animal. As controls for molecular and cellular assays, 3-month-old 5XFAD mice were maintained in constant darkness for 3 days or treated with constant light or flickering light at 20 Hz or 80 Hz for 1 hour (see, for example, Figure 43A).

[0146] Figure 43B includes plots of local electric field potential traces and power spectral density in the visual cortex before and during flashing of 40 Hz light, according to several embodiments. The mean (solid line) and standard deviation (shaded area) of the power spectral density are shown in the visual cortex during periods of flashing 40 Hz light 4302, random flashing 4304, or darkness 4306 (5FXFAD mice n=4 from 5 recording periods). Figures 43C–43F are plots showing the power spectral density of local electric field potentials in the visual cortex for each recording period for each mouse, according to several embodiments, during the processes of 40 Hz light flashing, random light flashing, constant darkness, and constant light (40 Hz flashing at 47, 51, 61, 49, 16; random flashing at 47, 50, 64, 50, 16; darkness at 279, 302, 382, ​​294, 93; and light periods at 47, 50, 64, 49, 15, recordings from 4 5XFAD mice, n=5). In the visual cortex, it was found that 40 Hz light flashing increased the power of the LFP at 40 Hz (see, e.g., Figures 43B and 43C), while random interval light flashing and darkness did not increase it (see, e.g., Figures 43B, 43D, and 43E).

[0147] Figure 44A is a series of histograms showing the rate of spikes in the visual cortex as a function of time for 4 cycles of 40 Hz light flashing and as a function of equivalent duration for random light flashing, according to several embodiments. Figure 44A shows a histogram of the rate of spikes in the visual cortex as a function of time for 4 cycles of 40 Hz light flashing 4402 or as a function of equivalent duration for random light flashing 4404 (5XFAD mice n=4 from 5 recording periods, bars show mean and error bars show SEM between animals). The upper bars show when the light is on 4406 or off 4408. In some embodiments, spiking increased and decreased as the light flashed on and off, resulting in a spiking phase synchronized to the 40 Hz frequency during the 40 Hz stimulus (histogram in Figure 44A). Lamb 4402) did not show any clear frequencies during random stimulation (histogram 4404 in Figure 44A).

[0148] Figure 44B shows a series of electrical traces of local electric field potentials recorded over the brain during flashing lights, according to several embodiments. In some embodiments, when recorded from saline directly above the brain, no increase in 40 Hz power was observed during 40 Hz flashing, indicating that this effect was not due to the photoelectric effect or electrical noise (see, e.g., Figures 32 and 44B). Similar to optogenetic stimulation, random flashing provided a control for the overall change in activity due to flashing lights.

[0149] Figure 45A is a histogram showing the difference in firing rates between 40 Hz light flashing and random light flashing according to several embodiments (stimulus cycles n=226 from 5 recording periods in 4 5XFAD mice). Figure 45B is a plot showing multiunit firing rates in the visual cortex during 40 Hz light flashing, random light flashing, dark, and light cycles according to several embodiments. Figure 45B shows multiunit 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, there was no significant difference in firing rates between the 40 Hz flashing and random flashing conditions, indicating that the random stimulation condition served as a control for spiking activity (rank-sum test for each of the five recording periods from four 5XFAD mice, p>0.06, median and quartiles shown in the figure; 40 Hz flashing periods per recording n=47, 51, 64, 49, 16 and random flashing periods 47, 50, 64, 50, 16). There was no significant difference in firing rates between 40Hz flashing and light conditions, indicating that 40Hz flashing light generally did not induce neuronal hyperexcitability (rank-sum test for each of the five recording periods from four 5XFAD mice; p>0.2 for four recording periods, p<0.01 for one recording period, which is not significant when adjusted for the performance of multiple comparisons. Median and quartiles are shown in the figure. 40Hz periods per recording n=47, 51, 64, 49, 16 and light periods 47, 50, 64, 49, 16). In one period, activity was greater with 40Hz stimulation than in the dark condition. The difference in multi-unit firing rates between 40Hz and random flashing periods tended to be close to zero (see, e.g., 45A). Furthermore, no significant differences were observed when comparing these periods within the animals (see, for example, Figure 45B. Rank-sum test for each of the five recording periods from four 5XFAD mice, p>0.06, median and quartiles shown in the figure. Gamma flashing periods per recording n=47, 51, 64, 49, 16 and random flashing periods 47, 50, 64, 50, 16).

[0150] Visual stimuli at gamma frequencies reduce Aβ levels in the visual cortex. Considering the effectiveness of optogenetic methods, a translational and non-invasive amyloid reduction treatment was designed. Figure 46A is a schematic diagram showing an experimental paradigm according to several embodiments. As shown in Figure 46A, a first subset of AD model mice was placed in a first chamber 4600 equipped with a 40 Hz flash lamp, and a second subset of AD model mice was placed in a darkened second chamber 4602. The animals in the first chamber 4600 were exposed to the 40 Hz flash lamp for approximately 1 hour.

[0151] Figures 46B and 46C show, according to several embodiments, the Aβ peptide isoform Aβ, respectively, after the experimental paradigm of Figure 46A. 1-40 and Aβ 1-42 This plot further shows the change in baseline levels. Figure 46B shows that exposure to 40 Hz light in the visual cortex V1 of 5XFAD mice significantly increased Aβ 1-40 and Aβ 1-42 This indicates a decrease in level. Aβ 1-40 and Aβ 1-42 The levels are expressed as pg / mL (n=6 animals per group).

[0152] 40Hz flashing lights drive 40Hz vibrations in the primary visual cortex, and optic genes are generated from these 40Hz vibrations. Given that scientific induction reduced Aβ levels in the hippocampus, the objective became to determine whether flashing 40 Hz light could reduce Aβ levels in the visual cortex. For these experiments, in some embodiments, 3-month-old 5XFAD mice were used before the onset of the disease. These mice were placed in a dark box and exposed for one hour to either flashing 40 Hz light, constant light on (bright), or constant light off (dark).

[0153] Figures 47A and 47B show the Aβ in the visual cortex of the 5XFAD under several embodiments of dark, light, 40Hz flashing, 20Hz flashing, 80Hz flashing, 40Hz flashing, picrotoxin (PTX), and random flashing conditions, respectively. 1-40 and Aβ 1-42This is a bar graph showing the change in baseline levels (n=12 mice per group for dark, n=6 mice per group for light, 40Hz flashing, 20Hz flashing, 80Hz flashing, and PTX, and n=4 mice per group for random flashing; one-way ANOVA, where "ns" indicates non-significant, one asterisk indicates p<0.05, and two asterisks indicate p<0.01). Figures 47 and 47B show the mean and SEM. Circles superimposed on the bars in the bar graphs indicate individual data points for each group. Aβ in the visual cortex one hour after exposure compared to dark conditions. 1-40 The level decreased by 57.96%, Aβ 1-42 A 57.97% decrease in Aβ levels was observed (measured by ELISA; see, e.g., Figures 47A and 47B; p<0.05 by one-way ANOVA; n=6 mice per group). Compared to the light control, after 1 hour of 40 Hz flashing, amyloid levels were 62.47% (Aβ). 1-40 ) and 68.55% (Aβ 1-42 Aβ levels decreased (measured by ELISA for Aβ; see, e.g., Figure 47; p<0.05 by one-way ANOVA; n=6 mice per group). Furthermore, this effect was specific to 40Hz flashing, as none of 20Hz, 80Hz, or random flashing significantly reduced Aβ levels compared to the dark and light controls (see, e.g., Figure 47; "ns" indicates non-significant; n=6 mice per group).

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

[0155] To demonstrate that this effect is not specific to 5XFAD mice, the results were reproduced in a different AD model, APP / PS1 mice, a well-validated model with two familial AD mutations (APP Swedish type and PSEN1 delta E9). Figure 48B shows the Aβ in the visual cortex of APP / PS1 mice under dark and 40 Hz flashing conditions, according to several embodiments. 1-40 and Aβ 1-42 This is a bar graph showing the change in baseline levels (n=5 mice per group for the dark condition and n=4 mice per group for the 40Hz flashing condition. A Student's t-test indicates that "ns" is not statistically significant, and one asterisk indicates p<0.05).

[0156] Figure 48C shows the Aβ in the visual cortex of WT under dark and 40Hz flashing conditions, according to several embodiments. 1-40 and Aβ 1-42 This is a bar graph showing the change in baseline levels (for the dark condition, n=11 mice per group; for the 40Hz flashing condition, n=9 mice per group. A single asterisk indicates p<0 by Student's t-test). (showing 0.05). In some embodiments, Aβ was significantly reduced by 20.80% in APP / PS1 mice after 40Hz flashing treatment. 1-40 And Aβ showed a decreasing trend at 37.68%. 1-42 Aβ was found, but the latter did not show a significant difference from the dark condition (see, for example, Figure 48B. Aβ was found by Student's t-test).1-40 p<0.05, Aβ 1-42 p<0.09, i.e., not statistically significant. (For the dark condition, n=5 mice per group; for the 40Hz flashing condition, n=4 mice per group). In addition, in aged WT mice, endogenous mouse Aβ was observed 1 hour after 40Hz flashing. 1-40 A 58.2% decrease was observed (see, for example, Figure 48C; p<0.05 by Student's t-test; dark mice n=11 and 40Hz flashing mice n=9). Aβ 1-42 The levels were below detectable in both the flashing and control groups of these animals. Endogenous mouse Aβ in WT animals 1-40 The decrease suggests that these results may not be limited to Tg APP expression or mutant APP, but rather may extend to Aβ produced from APP with endogenous promoter-driven expression. Figures 48A-48C show mean and SEM results.

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

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

[0159] In some embodiments, microglia were labeled in visual cortex sections of 5XFAD mice after 1 hour of 40 Hz flashing or dark conditions using Iba1 (see, e.g., Figure 49). There was no difference in the number of microglia between the dark and 40 Hz flashing conditions (see, e.g., Figures 49 and 50A; "ns" indicates non-significant; n=2 sections from 4 mice per group), but the diameter of microglial cell bodies increased by 65.8% in the visual cortex after 40 Hz flashing compared to the dark control (see, e.g., Figures 49 and 50B; p<0.01 by Student's t-test; n=2 sections from 4 mice per group). The length of the primary process of microglia decreased by 37.7% under 40 Hz flashing conditions compared to the dark control (see, e.g., Figures 49 and 50C; p<0.0001 by Student's t-test; n=2 sections from 4 mice per group). Since microglia in the visual cortex have a morphology that shows enhanced phagocytic activity, in some embodiments, the number of microglia containing Aβ was investigated. For this experiment, sections of the visual cortex were co-labeled with Iba1 and Aβ(12F4) antibodies. Intracellular colocalization of Aβ / Iba1 increased by 33.5% under 40 Hz flashing conditions, which is equivalent to 40H We showed that flashing z resulted in microglia with more Aβ than the dark control condition (see, e.g., Figures 49 and 50D; p<0.01 by Student's t-test; n=2 sections from 4 mice per group).

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

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

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

[0163] The microglia-specific level of Aβ was significantly higher in 5XFAD animals compared to WT controls, at 27.2 pg / 10 4 microglia levels, while in WT control mice it was 9.78 pg / 10 4 microglia levels (see, e.g., FIGS. 52A and 52B. p < 0.05 by Student's t-test. n = 8 for 5XFAD and n = 4 for WT mice). Aβ 1-42 was below detectable levels for both the flicker and control groups in these animals. Overall, the transformation of microglia in the visual cortex induced by 40 Hz stimulation appeared similar to that which occurred in hippocampal CA1. Furthermore, synaptophysin levels did not change between dark conditions and 40 Hz flicker conditions, indicating that microglial activation did not significantly increase synaptic phagocytosis (see, e.g., FIGS. 53A and 53B. "n.s." indicates not significant. n = 2 sections from 4 mice per group). Taken together, the data disclosed herein show that non-invasively induced 40 Hz vibrations via sensory stimulation can effectively reduce the abundance of Aβ and promote microglia / Aβ interaction in an AD mouse model. Furthermore, 40 Hz stimulation may reduce Aβ in two different brain circuits, suggesting a general mechanism by which gamma vibrations reduce amyloid abundance and enhance microglial phagocytosis across various brain regions.

[0164] In further experiments, after 1 hour of exposure to darkness (no light), a 20 Hz flash, a 40 Hz flash, or an 80 Hz flash, Aβ 1-42 levels were evaluated. Here, 20 Hz and 80 Hz are harmonics of 40 Hz. However, only the flickering of the 40 Hz flash significantly reduced the Aβ 1-42 levels. FIG. 54A is a bar graph showing the reduction of Aβ peptide isoform Aβ 1-42 after stimulation of the visual field of a subject with gamma oscillations according to some embodiments.

[0165] Another test was conducted to evaluate the timing of the decrease in Aβ 1-42 levels. Mice were exposed to either no light or a 40 Hz flash for 1 hour. Aβ 1-42 levels were measured 1 hour after treatment and again 24 hours after the end of the treatment. FIG. 54B is a bar graph showing the levels of Aβ peptide isoform Aβ 1-42 after stimulation of the visual field of a subject with gamma oscillations according to some embodiments and further 24 hours after the stimulation. The Aβ levels were still decreased 24 hours after treatment, but this decrease was smaller than immediately after treatment.

[0166] Visual stimulation at gamma frequencies did not affect the levels of Aβ in the hippocampus. To determine whether visual stimulation by light flickering affects the brain circuits involved in AD, in some embodiments, the effect of light flickering on the hippocampus, which is one of the brain regions affected early in the course of AD in humans, was examined. FIG. 55A includes electrical traces of local field potentials and plots of power spectral density in the hippocampus before and during a 40 Hz light flicker 5502 according to some embodiments. Mean (solid line) and standard deviation (shaded area) of power spectral density between darkness 5504, 40 Hz light flicker 5506, and random light flicker 5508 in CA1 (5XFAD mice n = 2 and WT mice 3).

[0167] Figure 55B is a series of histograms of the rate of spikes in the hippocampus as a function of time 5510 for 4 cycles of 40 Hz light flashing and an equivalent function of duration 5512 for random light flashing, according to several embodiments (5XFAD mice n=2 and WT mice 3; bars indicate the mean and error bars indicate SEM between animals). The upper bars indicate when the light is on (white) or off (black). For random stimuli, spiking was timed to coincide with the start of the light on, and further light cycles were performed at random intervals indicated by gray. Using the same method as the method disclosed herein for investigating the effect of light flashing in CA1 in the visual cortex, it was found that light flashing at 40 Hz increases the power in LFP recorded at 40 Hz (see, e.g., graph 5510 in Figures 55A and 55B), but light flashing at random intervals (random flashing) and darkness do not increase (see, e.g., graph 4310 in Figures 50D and 43C). Spiking is also modulated by a 40Hz flashing frequency between 40Hz stimuli, however this modulation appeared to be less pronounced than in the visual cortex (see, e.g., Figure 55B, hippocampus; Figure 44A, visual cortex).

[0168] Figure 56A is a histogram showing the difference in firing rates between 40 Hz light flashing and random light flashing according to several embodiments (minimum n=168 stimulus cycles from 5 recording periods in 2 5XFAD and 3 WT mice). Figure 56B is a plot showing the multi-unit firing rates in CA1 during 40 Hz light flashing 5604, random flashing 5605, dark 5602, or light 5608 cycles according to several embodiments. The box plot shows the median (white line in the box) and quartiles (top and bottom of the box). In all animals, there was no significant difference in firing rates between the 40Hz flashing and random flashing conditions, indicating that this random stimulus condition serves as a control for spiking activity (rank-sum test for each of the five recording periods from two 5XFAD and three WT animals, p>0.2, median and quartiles shown in figure. 40Hz flashing periods per record n=22, 54, 42, 71, 55 and random flashing periods 12, 34, 32, 54, 36). There was no significant difference in the firing rate between light and dark conditions, indicating that the 40 Hz light flickering generally did not cause neuronal hyperexcitability (rank sum test for each of five recording periods from two 5XFAD and three WT animals, p > 0.3. Median and quartiles are shown in the figure. 40 Hz cycle per recording: n = 22, 54, 42, 71, 55 and light cycle: 12, 34, 32, 54, 35).

[0169] Similar to the visual cortex, the difference in multi-unit firing rate between 40 Hz and random flicker periods tended to be close to zero (see, for example, FIG. 56A). Also, when comparing these periods within animals, no significant difference was found (see, for example, FIG. 56B. Rank sum test for each of five recording periods from four 5XFAD mice, p > 0.06, median and quartiles are shown in the figure. 40 Hz flicker period per recording: n = 22, 54, 42, 71, 55 and random flicker period: 12, 34, 32, 54, 36).

[0170] In some embodiments, the effect of visual light flickering on the level of Aβ in the hippocampus was examined using the same method as used in the visual cortex. According to some embodiments, FIG. 57A is a bar graph showing the relative Aβ 1-40 level in the visual cortex of 5XFAD, and FIG. 57B is a bar graph showing the relative Aβ 1-42 level in the visual cortex of 5XFAD (n = 4 mice per group. "n.s." indicates not significant). In contrast to what was observed in the visual cortex, in CA1, there was no significant difference in the levels of Aβ 1-40 and Aβ 1-42 one hour after 40 Hz flickering or random stimulation. There was no significant difference in the Aβ level after 40 Hz flickering or random flickering compared to the dark condition. The levels of Aβ 1-40 were 108.4% and 96.82% of that in the dark condition respectively after 40 Hz and random flickering, and Aβ 1-42The levels were 118.8% and 92.15% after 40 Hz and random flashing, respectively, under dark conditions (see, e.g., Figures 57A and 57B; "ns" indicates non-significant; n=4 mice per group). Therefore, 1 hour of 40 Hz flashing did not significantly reduce Aβ levels in the hippocampus.

[0171] Long-term visual stimulation at gamma frequencies reduced mottled loading in the visual cortex. We have previously disclosed the amount of amyloid present in anterior plaque 5XFAD mice when 40 Hz vibrations were driven either optogenetically or by visual stimulation via flashing lights. The objective, then, was to determine whether this treatment is effective in animals already exhibiting plaque loading. For this purpose, in some embodiments, 6-month-old 5XFAD mice were used because they develop extensive amyloid plaque lesions in many brain regions, including the visual cortex. Tests were conducted to investigate what happens to advanced Aβ-related lesions after non-invasive gamma stimulation. To investigate the duration of Aβ reduction in response to 1 hour of 40 Hz flashing, in some embodiments, Aβ levels were measured in the visual cortex at 4, 12, and 24 hours after 1 hour of 40 Hz flashing or dark conditions.

[0172] Figures 58A and 58B show the relative Aβ in the visual cortex of the 5XFAD at 1, 4, 12, and 24 hours after 1 hour of dark or 40 Hz flashing treatment, according to several embodiments. 1-40 and Aβ 1-42 This is a bar graph showing the level of Aβ (n=4 mice per group for 4 and 12-hour waiting periods, n=6 for 1 and 24-hour waiting periods, and n=12 for dark treatment. One-way ANOVA shows that "ns" is not significant, with one asterisk indicating p<0.05 and two asterisks indicating p<0.01). This result shows that after 4 hours, compared to the dark control, Aβ 1-40 The level of Aβ decreased by 63.4%. 1-42The levels of Aβ decreased by 63.2% (see, for example, Figure 58; p<0.01; n=4 mice per group). By 12 hours, Aβ 1-40 The level of [unclear] decreased by 50.9%, while Aβ 1-42 The levels were not significantly different from the dark control (see, for example, Figure 58; "ns" indicates non-significant, p<0.01; n=4 mice per group). Finally, soluble Aβ 24 hours after 1 hour of 40 Hz flashing treatment. 1-40 and A β 1-42 The levels were not significantly different with 40Hz flashing compared to the dark control condition (see, for example, Figure 58; "ns" indicates non-significant. n=6 mice per group for 24 hours and n=4 mice per group under the dark condition). These results indicate that the effect of the 40Hz flashing treatment is temporary.

[0173] Therefore, in order to destroy the advanced plaque lesions, in some embodiments, mice were treated with 40 Hz flashing for 1 hour daily for 7 days, or in darkness for a control. Figure 59A is a schematic diagram showing 6-month-old mice exposed to 1 hour of flashing per day for 7 days according to some embodiments. Figure 59B shows the relative Aβ in the visual cortex of 6-month-old 5XFAD mice after 7 days under 1 hour / day of darkness or 40 Hz flashing conditions according to some embodiments. 1-42 This is a bar graph showing the levels of (n=13 mice per group; two asterisks indicate p<0.01, three asterisks indicate p<0.001; Student's t-test). Figure 59C shows the relative Aβ levels in the visual cortex of 6-month-old 5XFAD mice after 7 days under 1 hour / day of dark conditions or 40 Hz flashing conditions, according to several embodiments. 1-40 This is a bar graph showing the levels (n=13 mice per group. Student's t-test indicates that one asterisk means p<0.01, and two asterisks mean p<0.01). Figures 59B and 59C show the mean and SEM. The circles superimposed on the bars in the bar graph represent the individual data points for each group.

[0174] At the end of the 7-day period, this visual cortex was analyzed by ELISA and immunostaining. In some embodiments, the tissue was dissolved in phosphate-buffered saline (PBS) and the PBS-soluble Aβ fraction was extracted. In 6-month-old 5XFAD mice, soluble Aβ was measured by ELISA after 7 days of 1-hour 40Hz flashing. 1-40 and Aβ 1-42 The levels were found to have decreased by 60.5% and 51.7%, respectively (see, e.g., Figures 59B and 59C; p<0.05 and p<0.01 by Student's t-test; n=13 mice per group). The tissue was further treated with guanidine hydrochloride (HCl) to remove the insoluble Aβ that constitutes the aggregated amyloid plaques. 1-40 and Aβ 1-42 The fraction was extracted. Insoluble Aβ 1-40 and Aβ 1-42 The levels decreased by 43.7% and 57.9%, respectively, and 40 The Hz flashing signal demonstrated that it disrupted insoluble Aβ aggregates already formed in 6-month-old mice (see, e.g., Figures 59B and 59C; Student's t-test showed p<0.01 and p<0.001; n=13 mice per group).

[0175] To determine how plaque loading was specifically affected, in some embodiments, immunohistochemical characterization was performed using an Aβ antibody (Cell Signaling Technology, D54D2). Figure 60A is a series of immunofluorescence images showing immunohistochemistry with anti-Aβ (D5452) antibody in the visual cortex of 6-month-old 5XFAD mice after 7 days under 1 hour / day dark (top) or 40 Hz flashing (bottom) conditions, according to some embodiments (scale bar = 50 μm). Intracellular Aβ signals were excluded. Figure 60B is a bar graph showing the number of Aβ-positive plaque depositions in the visual cortex of 6-month-old 5XFAD mice after 7 days under 1 hour / day dark or 40 Hz flashing conditions, according to some embodiments (n=8 mice per group; Student's t-test, three asterisks indicate p<0.001). Figure 60C is a bar graph showing the area of ​​Aβ-positive plaques in the visual cortex of 6-month-old 5XFAD mice after 7 days under conditions of 1 hour / day of darkness or 40 Hz flashing, according to several embodiments (n=8 mice per group; two asterisks indicate p<0.01 by Mann-Whitney test). Figures 60B and 60C show the mean and SEM.

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

[0177] To determine whether 40 Hz flashing improves another important AD-related lesion, tau phosphorylation was investigated using a TauP301S tauopathy mouse model. Four-month-old TauP301S transgyroscope mice, exhibiting phosphorylated tau localized to the cell body at this age, were treated with either 40 Hz flashing or a dark control for one hour daily for seven days. To investigate how 40 Hz flashing altered tau phosphorylation, immunohistochemical characterization of the visual cortex was performed using pTau antibodies against three different pTau epitopes (S202, S396, and S400 / T403 / S404; 11834S, 9632S, 11837S) and the dendritic marker MAP2 as controls.

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

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

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

[0181] As a result, under the 40Hz flashing condition, the signal intensity of pTau(S202) decreased by 41.2% and pTau(S400 / T403 / S404) decreased by 42.3% compared to the dark control (see, e.g., Figures 61A-B, 62A-B. Student's t-test, p<0.01. Sections n=2 from 8 mice per group), while MAP2 levels did not change (see, e.g., Figures 61A and 61C. "ns" indicates non-significant change. per group) Sections were taken from 4 mice (n=2). Staining with antibodies against pTau(S396) showed a consistent direction. Specifically, 40Hz flashing reduced pTau(S396) levels by 14.4% compared to the dark control (see, for example, Figures 63A-B; "ns" indicates non-significant. Sections were taken from 8 mice per group, n=2). Furthermore, compared to the dark control, there was less punctate localization of the pTau signal in response to 40Hz flashing. Significant changes were observed in tau phosphorylation, but no discernible difference in insoluble tau levels was found between the 40Hz flashing treatment and the dark control group.

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

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

[0184] In some embodiments, microglia in visual cortex sections of TauP301S mice were labeled with anti-Iba1 antibody after 7 days of 1 hour of 40 Hz flashing or dark conditions per day (see, e.g., Figure 64). In some embodiments, compared to dark controls, the 40 Hz flashing condition showed a tendency to increase microglia by 29.50%, consistent with what was observed in the 5XFAD model (see, e.g., Figure 50A) (see, e.g., Figures 64 and 65A; "ns" indicates non-significant; n=3 mice per group). Furthermore, the diameter of microglial cells increased by 49.00% in the visual cortex after 40 Hz flashing compared to dark controls (see, e.g., Figures 64 and 65B; p<0.0001 by Student's t-test; n=3 mice per group). The length of the primary process in microglia was reduced by 39.08% in the 40 Hz flashing group compared to the dark control (see, e.g., Figures 64 and 65C; p<0.0001 by Student's t-test; n=3 mice per group).

[0185] In summary, these data from multiple models of AD lesions and from wild-type animals indicate that 40 Hz vibration can reduce amyloid lesions and decrease taurinary phosphorylation, as measured by a decrease in Aβ levels. Furthermore, 40 Hz visual flashing can drive distinct morphological changes in microglia in both amyloidosis and tauopathy models of AD lesions.

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

[0187] Figures 67A–67B are plots showing Aβ peptide levels with and without transcranial gamma stimulation of subjects, according to several embodiments. In Figure 67A, whole-brain Aβ peptide levels remained unchanged in the unstimulated state (6700) but decreased after 1 hour of transcranial gamma stimulation (6702) (n=1 animal per group). In Figure 67B, according to several embodiments, whole-brain Aβ peptide levels decreased in the hippocampus (6704) and cortex (6706) of 5xFAD mice after 40 Hz transcranial stimulation.

[0188] Gamma oscillations have long been associated with higher cognitive functions and sensory responses. In some embodiments, driving FS-PV interneurons using optogenetic methods enhanced LFP at 40 Hz in mice. As disclosed herein, in some embodiments, driving 40 Hz oscillations and phase-gated spiking using optogenetic or non-invasive light flashing treatments in a 5XFAD mouse model resulted in a significant decrease in Aβ peptide in at least two different brain regions. This decrease was not due to a decrease in spiking activity, as Aβ peptide levels were significantly lower in response to 40 Hz stimulation than under random stimulation conditions that produced similar amounts of multi-unit spiking activity without enhancing 40 Hz oscillations. Pyramidal cell firing rates may differ between these conditions, but FS-PV interneurons or other cell types masked this change. In some embodiments, random optogenetic stimulation of FS-PV interneurons with the same amount of direct stimulation as direct stimulation of FS-PV interneurons still did not reduce amyloid. On the contrary, optogenetic stochastic stimulation more than tripled amyloid levels, while stochastic visual flashing did not produce a significant change, which may indicate that some aspects of random stimulation have neurotoxic effects. In some embodiments, random stimulation did not result in an increase in gamma power, although a slight increase in power was observed over a wide frequency range from approximately 20 Hz to over 60 Hz. In some embodiments, a trend of increasing amyloid levels was observed with light flashing from 20 Hz to 80 Hz. Taken together, these results may suggest that driving activity at certain frequencies below or above 40 Hz may increase amyloid levels. These results highlight the need to understand how patterns of spiking activity affect molecular pathways and disease lesions.

[0189] The significant decrease in total amyloid levels is likely mediated by both reduced amyloid formation associated with a decrease in EEA1 / Rab5-positive early endosomes and increased amyloid endocytosis by microglia. Importantly, gene set enrichment analysis (GSEA) statistical analysis disclosed herein (The Broad Institute, Cambridge, Massachusetts) showed that the inflammatory M1 or anti-inflammatory M2 cellular status of classical macrophages did not correlate with either upregulated or downregulated gene expression profiles after neuronal stimulation with 40 Hz oscillations. Indeed, the expression levels of the inflammatory genes Il6, Il1b, Itgam, and the anti-inflammatory gene Igf1 did not change after stimulation. Instead, several microglial prophagic genes and the cell adhesion / migration regulator Spp1 were activated by 40 Hz stimulation. Thus, driving 40 Hz gamma oscillations appears to induce a comprehensive neuroprotective response by recruiting both neurons and microglia. The fact that GABA-A antagonist treatment completely neutralized the effect of 40Hz stimulation on Aβ levels strongly suggests that GABAergic signaling, most likely associated with FS-PV interneurons, is essential for these effects. Furthermore, in several embodiments, 40Hz flashing stimulation reduced Aβ in multiple mouse models, including APP / PS1 and WT mice in addition to 5XFAD mice. This replication across multiple mouse models suggests that these findings may not be specific to a single animal model, and importantly, as in WT animals, This indicates that APP is expressed by its physiological promoter, potentially extending to situations where Aβ arises from endogenous APP. Furthermore, in several embodiments, 40 Hz oscillations were found to reduce pTau in the mouse model of tauopathy, TauP301S, demonstrating that the protective effect of gamma stimulation generalizes to other mouse models as well as other pathogenic proteins. In summary, the findings disclosed herein elucidate previously unknown cellular and molecular processes mediated by gamma oscillations and establish functional connections between brain gamma rhythms, microglial function, and AD-related lesions. In several embodiments, findings of gamma oscillation impairment converge on evidence of gamma impairment in different AD mouse models (hAPP and apoE4), reporting that gamma is modified in humans with AD. By seeking convergent evidence from multiple AD mouse models, including Tg and knock-in models, it may be shown that these results are not simply due to transgene overexpression or other side effects, particularly for one model. These results from mice and humans, taken together, demonstrate that multiple molecular pathways contributing to Aβ lesions converge to alter gamma oscillations in AD. The findings disclosed herein offer potential for novel therapeutic interventions for AD.

[0190] One theory of Alzheimer's disease (AD) pathogenesis points to microglial dysfunction, specifically the inability of microglia to remove pathological molecules, as a key mechanism of disease progression. Therefore, interventions that restore microglia to an endocytic state, such as 40 Hz stimulation, have strong therapeutic potential. Experiments further described herein have shown that optogenetic or light-induced gamma oscillations did not induce neuronal hyperactivity. Because this method is fundamentally different from previous AD therapies, driving such patterned neuronal activity to induce endogenous repair may offer a novel therapeutic approach to AD.

[0191] Visual stimuli at gamma frequencies had a positive effect on the subjects' behavior. Studies were conducted to investigate whether gamma exposure and / or administration, according to several embodiments, induce any stress in subjects. Figure 68A is a flowchart illustrating these studies. As shown at 6800 in Figure 68A, according to several embodiments, WT mice were exposed to either normal indoor light (N=8) or flashing 40 Hz light (N=8) for one hour per day for seven consecutive days, i.e., days 1-7. On day 8, as shown at 6802, blood was collected from the mice, and plasma was separated to examine corticosterone levels. In mice, corticosterone is the major glucocorticoid involved in the stress response.

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

[0193] Another study was conducted to investigate whether gamma exposure and / or administration according to several embodiments reduces anxiety in subjects. Figure 69A is a flowchart illustrating this study. As shown at 6900 in Figure 69A, according to several embodiments, WT mice were exposed to either normal indoor light (N=10) or flashing 40 Hz light (N=10) for one hour per day for seven consecutive days, i.e., days 1-7. On day 8, as shown at 6902, an elevated cross maze was performed for a period of 10 minutes.

[0194] The elevated cruciform maze is a test used to measure anxiety in laboratory animals. This behavioral model is based on the general aversion of rodents to open spaces, which leads to tacticism, i.e., a preference for staying in enclosed spaces or near the edges of enclosed spaces. Figure 69B is an image showing the elevated cruciform maze apparatus. The apparatus is a cruciform with two open paths (vertical) and two walled paths (horizontal). Anxiety is expressed by the animal spending more time in the walled paths.

[0195] Figures 69C and 69D are images showing typical trajectories of subjects during the elevated cross maze. According to several embodiments, in Figure 69C, mice exposed to normal indoor light tended to stay in walled paths and showed greater anxiety, while in Figure 69D, mice exposed to flashing 40Hz light explored both walled and unwalled paths and showed relatively less anxiety.

[0196] Figure 70 is a bar graph showing the total time spent exploring open-air and walled paths by 10 mice exposed to normal indoor light (NRL) and 10 mice exposed to flashing 40 Hz light (40 Hz), according to several embodiments. According to several embodiments, mice exposed to flashing 40 Hz light spent less total time exploring walled paths and longer total time exploring open-air paths, and showed less anxiety compared to the control group. For N=10 independent measurements per group, the T-distribution and p-values ​​for total time spent exploring walled paths are: T(18) = -1.652, p = 0.11 (2) This was the calculation.

[0197] For N=10 independent measurements per group, the T-distribution and p-values ​​for the total time spent searching for wallless paths are: T(18) = -2.136, p = 0.047 (3) This was the calculation.

[0198] Another study was conducted to investigate whether gamma exposure and / or administration according to several embodiments reduces stress and / or anxiety in subjects. Figure 71A is a flowchart illustrating this study. As shown at 7100 in Figure 71A, according to several embodiments, WT mice were exposed to either normal indoor light (N=8) or flashing 40 Hz light (N=8) for one hour per day for seven consecutive days, i.e., days 1-7. On day 8, as shown at 7102, a five-minute open-field study was performed.

[0199] The open-field test is an experiment used to assay typical levels of spontaneous movement and anxiety in laboratory animals. The behavioral model is based on anxiety resulting from the contradictory movements of rodents, who avoid brightly lit areas but explore perceived threat stimuli. Figure 71B is an image showing an open-field arena. This open-field arena has walls to prevent escape and may be marked with a grid or monitored using an infrared beam or video camera integrated with a software system. According to some embodiments, increased anxiety leads to decreased spontaneous movement and a preference for the edge of the field, while decreased anxiety leads to increased exploratory behavior.

[0200] Figures 71C and 71D are images showing typical trajectories of subjects during an open-field test. According to several embodiments, in Figure 71C, mice exposed to normal indoor light tended to prefer the edge of the arena and showed greater stress and / or anxiety, while in Figure 71D, mice exposed to flashing 40 Hz light explored the center of the arena and showed relatively less stress and / or anxiety.

[0201] Figures 72A and 72B are graphs showing the total time spent exploring the center and perimeter of an open-field arena by eight mice exposed to normal indoor light (NRL) and eight mice exposed to flashing lights at 40 Hz (40 Hz), according to several embodiments. Figure 72A is a plot of the average number of seconds spent in the center of the arena every five minutes. Figure 72B is a bar graph of the total time spent around the arena over the entire five minutes, averaged per minute.

[0202] According to several embodiments, compared to the control group, mice exposed to flashing 40 Hz light spent significantly longer periods in the center of the arena during 2, 4, and 5 minutes, and consequently showed less stress and / or anxiety, which is also consistent with the results of the elevated cross maze. Repeated measures analysis of variance (RM ANOVA) was performed. For N=8 independent measurements per group, the F-distribution and p-values ​​for the mean time spent exploring the open field arena were: F(1,14)=4.860, p=0.045 (4) This was the calculation.

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

[0204] On day 8, these mice were exposed for 5 minutes to two novel objects in the scenario shown in Figure 73A, i.e., in a familiar arena. Figure 73C is a bar graph showing the percentage of time spent searching for novel object A versus the percentage of time spent searching for novel object B for 8 mice exposed to normal indoor light (NRL) and 8 mice exposed to flashing lights at 40 Hz (40 Hz), according to several embodiments. As shown in Figure 73C, each group showed an equal preference tendency for each object; that is, no difference was observed in object search between the groups.

[0205] Next, these mice were exposed for 5 minutes to the scenario in Figure 73B, i.e., one familiar object and one novel object in a familiar arena. Figure 74 is a plot showing the average number of seconds spent exploring the novel object every 5 minutes. According to several embodiments, compared to the control group, mice exposed to flashing 40 Hz light spent significantly longer time exploring the novel object, particularly between 1–3 minutes and 5 minutes, and consequently showed increased novelty-seeking behavior. Friedman's nonparametric RM ANOVA was performed. For N=8 independent measurements per group, the test statistic χ² was calculated for the average time spent exploring the novel object. 2 And the p-value is: χ 2 (4, n=16) = 16.088, p = 0.003 (5) This was the calculation.

[0206] A Mann-Whitney U test was performed on the average time spent searching for novel objects within a 3-minute period. For N=8 independent measurements per group, the U-value, Z-value, and p-value were: U=58.00, Z=2.731, p=0.005 (6) This was the calculation.

[0207] Gamma exposure and / or administration according to several embodiments affects the learning and memory of the subject. Another test was conducted to determine whether or not to administer the substance. Figure 75A is a flowchart illustrating a test using the fear conditioning paradigm. According to several embodiments, as shown at 7500 in Figure 75A, WT mice were exposed to either normal room light or flashing 40 Hz light for one hour per day for seven consecutive days, i.e., days 1-7. On day 8, shown at 7502, these mice were subjected to mild two-tone shock pairing. Specifically, the mice were placed in a new arena combining a first tone and a foot shock. These mice were conditioned to associate the context (i.e., the tone) with the aversive experience (i.e., the foot shock). For this first context, the T-distribution and p-values ​​for the total time spent freezing are: T(24)=0.577, p=0.569 (7) This was the calculation.

[0208] On day 9, indicated by 7504, the tone test was conducted in a modified context. Figure 75B is a stimulus diagram showing the tone test as a function of time, including the first tone context 7506, the first tone context after 7508, the second tone context 7510, and the second tone context after 7512. For this test, the mice were returned to the arena where the first tone was combined with a foot shock. When the first tone context 7506 was applied, mice exposed to 40 Hz flashing lights spent longer periods of freezing, presumably anticipating the foot shock, and thus indicating a measure of memory. Mice exposed to 40 Hz flashing lights also spent longer periods of freezing than the control group during the second tone context 7510, but freezing times were shorter during the periods after either tone context.

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

[0210] Regarding the context of tone, when RM ANOVA was performed between groups, the F-distribution and p-value for the mean time spent freezing were: F(1,24)=3.106, p=0.091 (8) This was the calculation.

[0211] Regarding the context of the first tone, the T-distribution and p-value for the total time spent in freezing are: T(24) = -2.155, p = 0.041 (9) This was the calculation.

[0212] Regarding the context of the second tone, the T-distribution and p-value for the total time spent in freezing are: T(24) = -1.433, p = 0.164 (10) This was the calculation.

[0213] Regarding the context of tone, when RM ANOVA was performed between groups, the F-distribution and p-value for the mean time spent freezing were: F(1,24)=4.559, p=0.043 (11) This was the calculation.

[0214] Regarding the context after the first tone, the T-distribution and p-value for the total time spent in freezing are: T(24)=1.874, p=0.073 (12) This was the calculation.

[0215] Regarding the context after the second tone, the T-distribution and p-value for the total time spent in freezing are: T(24)=2.223, p=0.036 (13) This was the calculation.

[0216] Regarding the context of tone, when RM ANOVA was performed between groups, the F-distribution and p-value for the mean time spent freezing were: F(1,24)=6.646, p=0.017 (14) This was the calculation.

[0217] Another study was conducted to investigate whether gamma exposure and / or administration according to several embodiments improved the memory of subjects. Figure 77A is a flowchart illustrating this study. According to several embodiments, as shown at 7700 in Figure 77A, WT mice were exposed to either normal room light or flashing 40 Hz light for one hour per day for seven consecutive days, i.e., days 1-7. On day 8, as shown at 7702, the Morris water maze test was performed.

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

[0219] For weaker training, the Morris water maze test was repeated twice a day for four consecutive days, i.e., from day 8 to day 11. Figure 78A is a plot showing the time it took for mice exposed to normal indoor light (NRL) and mice exposed to flashing 40 Hz light (40 Hz) to find the platform, according to several embodiments.

[0220] On day 12, the hidden platform was removed from the Morris water maze and the probe test was conducted. Figures 77C and 77D are images showing typical trajectories of the subjects during the probe test. According to some embodiments, in Figure 77C, mice exposed to normal room light appeared to search for the platform throughout the pool, while in Figure 77D, mice exposed to 40 Hz flashing lights appeared to search more systematically, primarily in the SW quadrant. Figure 78B is a plot showing the total time (seconds per 30 seconds) spent searching for the platform in the target quadrant (i.e., the SW quadrant), and Figure 78C is a plot showing the total time (seconds per 30 seconds) spent searching for the platform in the opposite quadrant (i.e., the NE quadrant). According to some embodiments, mice exposed to 40 Hz flashing lights spent more time searching the target quadrant and less time searching the opposite quadrant than the control group, demonstrating enhanced spatial memory.

[0221] Reverse learning was performed using mice from the same group as in the Morris water maze test and probe test. Figure 79A shows the Morris water maze with platform 7900 hidden in the SW quadrant in this test. Figure 79B shows a Morris water maze with platform 7902 hidden in the reverse NE quadrant for reverse learning.

[0222] For weak training, reverse learning was repeated twice a day for four consecutive days, i.e., from day 14 to day 17. Figure 79C is a plot showing the time it took for mice exposed to normal indoor light (NRL) and mice exposed to flashing 40 Hz light (40 Hz) to find the platform, according to several embodiments. Despite not receiving further 40 Hz exposure after day 7, mice exposed to flashing 40 Hz light showed increased behavioral flexibility.

[0223] In some embodiments, further studies were conducted to investigate whether long-term gamma exposure and / or administration affects spatial learning and memory in subjects. Figure 80A is a flowchart illustrating these studies. In some embodiments, as shown at 8000 in Figure 80A, C57BL / 6 mice were exposed to either normal room light (N=7) or flashing 40Hz light (N=7) for one hour per day for two weeks. During the third week, shown at 8002, these mice continued to be exposed to either normal room light or flashing 40Hz light for one hour each morning, and then subjected to the Morris water maze test each afternoon.

[0224] Figure 80B is a plot showing the time it took mice exposed to normal indoor light (NRL) and mice exposed to flashing 40Hz light (40Hz) to find the platform during days 1-4 of week 3. After week 3, the hidden platform was removed and the probe test was conducted. Figure 80C is a bar graph showing the total time (seconds per 30-second test) spent searching for the platform within the target quadrant during the probe test. According to several embodiments, the long-term 3-week treatment, as well as the 1-week treatment, enhanced spatial learning.

[0225] Reverse learning was performed using the same group of mice as shown in Figures 80A-80C. Figure 81A is a flowchart showing the extended test. As shown at 8100 in Figure 81A, according to several embodiments, C57BL / 6 mice were exposed to either normal indoor light or flashing 40Hz light for one hour per day for two weeks. During the third week, shown at 8102, these mice continued to be exposed to either normal indoor light or flashing 40Hz light for one hour each morning, and then subjected to the Morris water maze test again each afternoon. During the fourth week, shown at 8104, these mice continued to be exposed to either normal indoor light or flashing 40Hz light for one hour each morning, and then subjected to the Morris water maze test again each afternoon. Figure 81B is a plot showing the time it took for mice exposed to normal indoor light (NRL) and mice exposed to flashing 40Hz light (40Hz) to find the platform on days 1-4 of the fourth week, according to several embodiments.

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

[0227] Visual stimulation at gamma frequencies provided anatomical, morphological, cellular, and molecular benefits. In several embodiments, studies were conducted to investigate the effects of gamma exposure and / or administration on DNA damage and neuronal loss in the target visual cortex. For these studies, an inducible mouse model of p25 accumulation (i.e., creatine kinase carboxyl terminal fragment p25Tg mouse (CK-p25Tg mouse)) was used. The CK-p25Tg mouse model exhibits key pathological features of AD, including significant neuronal loss in the forebrain, increased Aβ peptide production, tau pathology, DNA damage, and severe cognitive impairment. In this model, elevated Aβ peptide levels were observed prior to neuronal loss, and furthermore, Aβ peptide... Reducing tide production mitigates memory impairment in the CK-p25Tg mouse model, suggesting that this event synergistically interacts with the carboxyl terminal fragment p25, leading to the development of neurodegeneration and memory impairment.

[0228] Figure 83 is a schedule diagram 8300 showing changes in CK-p25Tg mice. After 2 weeks 8302, the mice show DNA damage (e.g., biomarker γH2AX), increased Aβ peptide, and microglial activation. After 6 weeks 8304, the mice show synaptic loss, neuronal loss, tau hyperphosphorylation, long-term potentiation failure, and memory impairment.

[0229] A study was conducted to compare groups of mice under different treatment plans. Figure 84 shows a group including CK control mouse 8400, untreated CK-p25Tg mouse 8402, CK-p25Tg mouse 8404 treated with memantine (10 mg / kg daily), CK-p25Tg mouse 8406 exposed to flashing 40 Hz light (1 hour daily for 6 weeks) according to several embodiments, and CK-p25Tg mouse 8408 treated with memantine and further exposed to flashing 40 Hz light. Memantine is a drug that has achieved some success in treating severe AD by blocking NMDA receptors and thereby acting on the glutamate system.

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

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

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

[0233] Figures 88A–88C are anatomical diagrams of the brain showing the target brain region for molecular characterization according to several embodiments. Figure 88A includes the visual cortex (V1)8800, the somatosensory cortex (SS1)8802, the hippocampus8804, and the insular cortex8806.

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

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

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

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

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

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

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

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

[0242] The SS1 skin layer was exposed to flashing 40Hz light according to several embodiments of CK-p The chromosomes gradually became thinner in 25Tg mice (Figure 94D, e.g., 834±94 μM), CK-p25Tg mice treated with both memantine and 40 Hz flashing light (Figure 94E, e.g., 778±13 μM), untreated CK-p25Tg mice (Figure 94B, e.g., 762±17 μM), and CK-p25Tg mice treated with memantine (Figure 94C, e.g., 756±11 μM).

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

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

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

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

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

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

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

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

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

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

[0253] Gamma exposure also reduced CKp-25-induced neuronal loss and DNA damage in the somatosensory cortex of subjects. Figure 101 is a bar graph comparing the amount of NeuN-positive cells as a percentage of NeuN-positive cells in CK control mice for CK control mice, untreated CK-p25Tg mice, memantine-treated CK-p25Tg mice, CK-p25Tg mice exposed to 40 Hz light flashing according to several embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz light flashing. Thus, the percentage of NeuN-positive cells in CK control mice is 100% in CK control mice, but closer to 80% in untreated CK-p25Tg mice, supporting neuronal loss in the CK-p25Tg mouse model. Except for the combination with exposure to 40 Hz flashing lights, which prevented most neuronal loss in CK-p25Tg mice, treatment with memantine did not prevent neuronal loss in CK-p25Tg mice compared to the untreated group. Therefore, Figure 101 shows how 40 Hz visual flashing treatment according to several embodiments can preserve neurons in the somatosensory cortex.

[0254] Figure 102 shows CK control mice, untreated CK-p25Tg mice, memantine-treated CK-p25Tg mice, and mice exposed to flashing lights at 40 Hz according to several embodiments. This bar graph compares the amount of γH2AX-positive cells in untreated CK-p25Tg mice and in CK-p25Tg mice treated with both memantine and 40Hz light flashing. γH2AX-positive cells were absent in CK control mice but were significantly more numerous in untreated CK-p25Tg mice, exhibiting a large amount of DSBs and other DNA damage. Treatment with memantine reduced the amount of γH2AX-positive cells in CK-p25Tg mice compared to the untreated group. Exposure to 40Hz light flashing, according to several embodiments, resulted in a further significant reduction in γH2AX-positive cells in CK-p25Tg mice. Thus, Figure 102 illustrates how 40Hz visual flashing treatment, according to several embodiments, can reduce DNA damage in the somatosensory cortex. However, the combination of memantine and 40Hz light flashing exposure significantly increased the number of γH2AX-positive cells in CK-p25Tg mice.

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

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

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

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

[0259] Gamma exposure also causes CKp-25-induced neuronal loss and DN in the hippocampus of the affected individuals. A damage was also reduced. Figure 107 is a bar graph comparing the amount of NeuN-positive cells as a percentage of NeuN-positive cells in CK control mice for CK control mice, untreated CK-p25Tg mice, memantine-treated CK-p25Tg mice, CK-p25Tg mice exposed to 40 Hz flashing lights according to several embodiments, and CK-p25Tg mice treated with both memantine and 40 Hz flashing lights. Thus, the percentage of NeuN-positive cells in CK control mice is 100% in CK control mice, but closer to 80% in untreated CK-p25Tg mice, supporting neuronal loss in the CK-p25Tg mouse model. Treatment with memantine prevented some neuronal loss in CK-p25Tg mice, compared to the untreated group, which showed minimal prevention of neuronal loss in CK-p25Tg mice, regardless of exposure to 40 Hz flashing lights. Therefore, Figure 107 shows how 40Hz visual flashing processing according to several embodiments can conserve hippocampal neurons.

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

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

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

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

[0264] Gamma exposure not only reduced CKp-25-induced synaptic loss in the somatosensory cortex of subjects, but even increased synaptic point density. Figure 111 shows CK control mice, untreated CK-p25Tg mice, and memantine-treated CK-p25Tg mice. This bar graph compares the point density of glutamatergic synapses (using VGluT1) and GABAergic synapses (using GAD65) as a percentage of the synaptic point density of baseline CK control mice for CK-p25Tg mice exposed to 40Hz flashing light according to several embodiments, as well as CK-p25Tg mice treated with both memantine and 40Hz flashing light.

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

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

[0267] Studies were conducted to investigate whether gamma exposure and / or administration, according to several embodiments, affect the cerebrovascular system. Mice were placed in a dark box and exposed to either flashing 40 Hz light-emitting diodes (LEDs) or constant light-off (darkness) for 1 hour. After stimulation, the mice were sacrificed and perfused. Brain sections were stained with fluorophores-bound lectins to fluorescently label blood vessels. Changes in vascular structure size (i.e., vessel diameter) were measured using confocal imaging. Vasodilation was observed 1 hour after flashing 40 Hz LEDs.

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

[0269] Therefore, gamma exposure and / or administration have been shown to provide anatomical (e.g., prevention and / or reduction of brain weight and vascular structure enlargement), morphological (e.g., prevention and / or reduction of abnormal ventricular dilation and loss of cortical thickness), cellular (e.g., prevention and / or reduction of neuronal loss), and molecular (e.g., prevention and / or reduction of DNA damage and synaptic loss) benefits.

[0270] Furthermore, gamma exposure and / or administration have been shown to have neuroprotective effects. Following gamma treatment, CK-p25Tg mouse models, i.e., models otherwise exhibiting elevated Aβ peptide levels, significant neuronal loss, DNA damage, synaptic loss, tau hyperphosphorylation, chronic potentiation failure, and severe cognitive / memory impairment, showed relative preservation of neuronal structure and / or function (e.g., maintenance / prevention of disease severity and / or mitigation / reduction of disease progression), and in some cases, suggested improvement of neuronal structure and / or function.

[0271] Auditory stimulation at gamma frequencies non-invasively induced changes in the microglia of the target organisms. In some embodiments, gamma exposure and / or administration includes auditory stimulation. Auditory stimulation may include sound wave pulses or clicks. Auditory stimulation may include a click train of about 35 sound wave pulses or clicks per second (clicks / second) to about 45 clicks / second. Figure 114 is a stimulus diagram showing a click train stimulus according to some embodiments. The stimulus in Figure 114 has a click frequency of 40 clicks / second with 25 milliseconds between each click and each click having a duration of 1 millisecond.

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

[0273] In some embodiments, the sound stimulus has sound pressure levels of approximately 0 dB to approximately 85 dB, approximately 30 dB to approximately 70 dB, and / or approximately 60 dB to approximately 65 dB. For example, each sound wave pulse or click in a click train may have a sound pressure level of approximately 65 dB.

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

[0275] Figure 115 is a flowchart illustrating the experiment. In Figure 115, wild-type mice were housed in their home cages 11500. For one hour per day for seven consecutive days (days 1-7), these mice were moved to a behavioral chamber (i.e., a soundproof room) 11502. Inside the behavioral chamber 11502, the first group of mice were exposed to silence, while the second group of mice were exposed to click-train stimuli according to several embodiments. After each hour in the behavioral chamber 11502, the mice were returned to their home cages 11500. On day 8, these mice were sacrificed for tissue collection and staining 11504.

[0276] This tissue was examined for microglial activation, indicated by the level of microglial cells, morphological changes in microglial cells, and cell size. Figure 116A is a bar graph showing the average number of microglia in mice exposed to rest (no stimulation) compared to mice exposed to click-train stimulation (stimulation). According to several embodiments, more microglial cells were observed in mice exposed to click-train stimulation. Figure 116B is a bar graph showing the average ratio of microglial process lengths in mice exposed to rest (no stimulation) compared to mice exposed to click-train stimulation (stimulation). According to several embodiments, the average ratio of microglial process lengths was significantly lower in mice exposed to click-train stimulation. Figure 116C is a bar graph showing the average ratio of microglial cell sizes in mice exposed to rest (no stimulation) compared to mice exposed to click-train stimulation (stimulation). The mean ratio of microglial cell sizes was significantly higher in mice exposed to click-train stimulation, indicating higher microglial activation according to several embodiments.

[0277] Figure 117A is a representative image of microglial cells in mice exposed to silence. Figure 117B is a representative image of microglial cells in mice exposed to click-train stimulation according to several embodiments. Microglial processes and cells are There is a clear difference between Figure 117A and Figure 117B according to several embodiments. Figure 118A is a magnified image from Figure 117B of microglia cells from mice exposed to click-train stimulation according to several embodiments. One protrusion 11800 of the microglia cell is highlighted. On the other hand, Figure 118B is a magnified image from Figure 117A of microglia cells from mice exposed to silence. One protrusion 11802 of the microglia cell is highlighted, showing its length relative to the relatively short protrusion 11800 of the microglia cell from mice exposed to click-train stimulation according to several embodiments.

[0278] Figure 119A is a magnified image from Figure 117B of microglia cells from mice exposed to click-train stimulation according to several embodiments. The location of cell 11900 in this microglia cell is highlighted. On the other hand, Figure 119B is a magnified image from Figure 117A of microglia cells from mice exposed to silence. The location of cell 11902 in this microglia cell is highlighted to show its size relative to the relatively larger cell 11900 of the microglia cells from mice exposed to click-train stimulation, and thus to show higher microglial activation according to several embodiments.

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

[0280] Figure 121A is a representative image of microglial cells in mice exposed to silence. Figure 121B is a representative image of microglial cells in mice exposed to click-train stimulation according to several embodiments. The microglial processes and cells clearly differ between Figure 121A and Figure 121B, with the microglia from mice exposed to click-train stimulation according to several embodiments having relatively shorter processes and larger cell sizes.

[0281] Auditory stimulation at gamma frequencies non-invasively reduces Aβ in the target auditory cortex and hippocampus. Auditory gamma stimulation has been shown to reduce the level of Aβ in a subject, according to several embodiments. Figure 115 shows a test conducted in 6-month-old 5XFADs according to several embodiments. The procedure was repeated in transgygist mice. On day 8, the auditory cortex and hippocampus were resected. Isoform Aβ was identified using ELISA. 1-40 Peptides and isoform Aβ 1-42 The levels of soluble and insoluble Aβ isoforms containing peptides were measured. Insoluble Aβ was solubilized by treating it with 5M guanidine HCl for 3 hours.

[0282] According to some embodiments, auditory gamma stimulation reduces the level of soluble Aβ in the subject. This was demonstrated. Figure 122A shows the soluble isoform Aβ in the auditory cortex of mice exposed to silence (no stimulation) according to several embodiments. 1-42 Much lower levels of soluble isoform Aβ were observed in the auditory cortex of mice exposed to click-train stimulation compared to the peptide level. 1-42 This is a bar graph showing peptides.

[0283] Figure 122B shows the soluble isoform Aβ in the auditory cortex of mice exposed to silence (no stimulation) according to several embodiments. 1-40 Low levels of soluble isoform Aβ in the auditory cortex of mice exposed to click-train stimulation (stimulation), relative to peptide levels.1-40 This is a bar graph showing peptides.

[0284] Figure 122C shows the soluble isoform Aβ in the hippocampus of mice exposed to silence (no stimulation) according to several embodiments. 1-42 Much lower levels of soluble isoform Aβ in the hippocampus of mice exposed to click-train stimulation compared to peptide levels. 1-42 This is a bar graph showing peptides.

[0285] Figure 122D shows the soluble isoform Aβ in the hippocampus of mice exposed to silence (no stimulation) according to several embodiments. 1-40 Low levels of soluble isoform Aβ in the hippocampus of mice exposed to click-train stimulation (stimulation) relative to peptide levels. 1-40 This is a bar graph showing peptides.

[0286] Auditory gamma stimulation has been shown to reduce the level of insoluble Aβ in a subject, according to several embodiments. Figure 123A shows insoluble isoform Aβ in the auditory cortex of mice exposed to silence (no stimulation), according to several embodiments. 1-42 Much lower levels of insoluble isoform Aβ were observed in the auditory cortex of mice exposed to click-train stimulation compared to the peptide level. 1-42 This is a bar graph showing peptides.

[0287] Figure 123B shows the insoluble isoform Aβ in the auditory cortex of mice exposed to silence (no stimulation) according to several embodiments. 1-40 In contrast to peptide levels, small levels of the insoluble isoform Aβ were observed in the auditory cortex of mice exposed to click-train stimulation. 1-40 This is a bar graph showing peptides.

[0288] Figure 123C shows the insoluble isoform Aβ in the hippocampus of mice exposed to silence (no stimulation) according to several embodiments. 1-42Insoluble isoform Aβ is present at much lower levels in the hippocampus of mice exposed to click-train stimulation compared to peptide levels. 1-42 This is a bar graph showing peptides.

[0289] Figure 123D shows the insoluble isoform Aβ in the hippocampus of mice exposed to silence (no stimulation) according to several embodiments. 1-40 Low levels of insoluble isoform Aβ in the hippocampus of mice exposed to click-train stimulation (stimulation) relative to peptide levels. 1-40 This is a bar graph showing peptides.

[0290] Figure 124A is a representative image of microglial cells in 5XFAD mice exposed to click-train stimulation according to several embodiments. Figure 124B is a representative image of microglial cells in 5XFAD mice exposed to silence. The microglial processes and cells clearly differ between Figure 124A and Figure 124B, with the microglia from 5XFAD mice exposed to click-train stimulation according to several embodiments having relatively shorter processes and larger cell sizes.

[0291] Figure 124C is a representative image of microglia cells in WT mice exposed to silence. Figure 124D shows microglia exposed to click-train stimulation according to several embodiments. These are representative images of microglial cells in WT mice. The protrusions and cells of these microglia clearly differ between Figure 124C and Figure 124D, with relatively shorter protrusions and larger cell size in microglia from WT mice exposed to click-train stimulation according to several embodiments.

[0292] Therefore, according to several embodiments, non-invasive auditory stimulation at gamma frequencies promoted a significant reduction in gamma oscillations in the auditory cortex and hippocampus, as well as in AD-related lesions.

[0293] Auditory stimulation at gamma frequencies had a favorable effect on the subjects' behavior. Auditory gamma stimulation has been shown to improve object recognition according to several embodiments. Figure 125A is a flowchart showing a novel object recognition test conducted using 5XFAD mice exposed to click-train stimulation and 5XFAD mice exposed to silence according to several embodiments. This test assesses the ability of objects 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 familiar objects. Objects were compared using the recognition index RI: JPEG0007837033000012.jpg17152

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

[0295] Auditory gamma stimulation has been shown to improve object recognition according to several embodiments. Figure 126A is a flowchart showing a novel spatial recognition test conducted using 5XFAD mice exposed to click-train stimulation and 5XFAD mice exposed to silence according to several embodiments. This test assesses spatial memory and / or recognition based on the tendency of rodents to spend more time exploring objects in a novel location. Objects were compared using the recognition index RI: JPEG0007837033000013.jpg16165

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

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

[0298] Figure 127A is a plot showing the average time it took mice exposed to silence (no stimulation) and mice exposed to click-train stimulation (stimulation) each day to find the platform, according to several embodiments. Figure 127B is a bar graph showing the results of a probe test with the platform removed. Mice exposed to click-train stimulation spent more time searching for the platform that was not found in the target quadrant than mice exposed to silence, and consequently, according to several embodiments, mice exposed to click-train stimulation had better spatial and / or reference memory.

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

[0300] Experimental method animal All animal experiments were approved by the Committee for Animal Care of the Division of Comparative Medicine (Massachusetts Institute of Technology, Cambridge, Massachusetts). Adult (3-month-old) male double Tg 5XFAD Cre mice were produced by crossing 5XFAD Tg mice with Tg PV or CW2 promoter-driven Cre strains. Adult (5-month-old) male and female APP / PS1 mice were donated by Tonegawa Laboratory (Massachusetts Institute of Technology, Cambridge, Massachusetts). Adult (4-month-old) male TauP301S mice were obtained from Jackson Laboratory. Aged WT mice (8 months old, C57Bl / 6) were obtained from Jackson Laboratory (Bar Harbor, Maine). Mice were housed in groups of 3-5 in a standard 12-hour light / 12-hour dark cycle, and all experiments were conducted during the light cycle. Food and water were provided continuously unless otherwise noted. Lactate cubs were randomly assigned to each condition by the experimenter. The experimenter did not know the genotype of the animals during tissue processing, electrophysiological recording, and analysis. No animals were excluded from the analysis.

[0301] AVV Vector Adeno-associated virus particles of serotype 5 were obtained from the Vector Core Facility (University of North Carolina, Chapel Hill, North Carolina). This AAV5 virus contained ChR2 in a double-floxed, reverse-direction open reading frame (DIO) fused to a highly sensitive yellow fluorescent protein (EYFP) driven by the EF1α promoter (see, e.g., Figure 9). The AAV DIO EYFP construct was used as a control.

[0302] Surgical procedure Three-month-old 5XFAD / PV-Cre or CW2 mice were given ketamine (1.1 mg / kg). -1 ) and xylazine (0.16 mg kg -1The patient was anesthetized by intraperitoneal injection of a mixture of (AAV) and (DIO). A small craniotomy was performed on the left side, 2.0 mm from the posterior lobe to the fontanelle and 1.8 mm from the lateral lobe to the midline. The virus was administered through a small incision in the dura mater using a glass micropipette attached to a Quintessential Stereotaxic Injector (trademark) (available from Stoelting Co., Wooddale, Illinois). This glass micropipette was lowered to 1.2 mm below the brain surface. A 1 μl bolus of ChR2-EYFP or AAV DIO EYFP (2 x 10¹² virus molecules per 1 ml) is administered to the CA1 region of the hippocampus, with a dose of 0.075 μl. -1The injection was administered. The pipette was left in place for 5 minutes after injection, and then retracted from the brain. A unilateral fiber optic implant (core diameter 300 μm, available from Thorlabs Inc., Newton, New Jersey) was lowered to approximately 0.9 mm below the brain surface at the injection site. Two small screws fixed to the anterior and posterior edges of the surgical site were secured with dental adhesive to fix the implant in place. For electrophysiological recording, adult (3-month-old) male 5XFAD / PV-Cre double transgenic mice and 5XFAD-negative littermates (for CA1 recording), or 5XFAD and its WT littermates (for visual cortex recording) mice were anesthetized with isoflurane and placed in a stereotactic frame. The scalp was shaved, eye ointment (e.g., Puralube® Vet ointment (Dechra Pharmaceuticals PLC, Northwich, UK)) was applied to the eyes, and the surgical area was disinfected with Betadine® disinfectant (Purdue Products LP, available from Stamford, Connecticut) and 70% ethanol. For CA1 recording, a craniotomy was performed (in mm units, from the fontanelle: -2 A / P, 1.8 M / L), and 1 μL of virus was supplied to CA1 (as described above). The target craniotomy site for LFP recording was marked on the skull (in mm, from the fontanelle: -3.23 A / P, 0.98 M / L for CA1 and 2.8 A / P, 2.5 M / L for the visual cortex), three self-tapping screws (e.g., F000CE094, Morris Precision Screws and Parts, available from Southbridge, Massachusetts) were attached to this skull, and a custom stainless steel headplate was attached using dental cement (e.g., C&B Metabond®, Parkell Inc., available from Edgewood, New York). On the first recording day, an LFP craniotomy (e.g., 300-400 μm in diameter) was performed by first thinning the skull to approximately 100 μm using a dental drill, and then creating a small opening using a 30-gauge needle.The craniotomy was then sealed with sterile silicone elastomer (e.g., Kwik-Sil® adhesive, available from World Precision Instruments, Inc., Sarasota, Florida) until the day's record and throughout the record-keeping period.

[0303] Optogenetic stimulation protocol Two to four weeks after viral injection and implantation, allowing mice time to recover, undergo animal behavioral training for electrophysiology, and for the virus to express in the neurons, hippocampal CA1 neurons were optogenetically manipulated. A 200 mW 4793 nm DPSS laser was connected to a patch cord with fiber channel / physical contact connectors at each end. During the experiment, a 1 mW (measured from the fiber end) optical stimulus was delivered for one hour. For molecular and biochemical analysis, each animal received one of three stimulation protocols: 8 Hz, 40 Hz, or random stimulation (light pulses were delivered at an average frequency of 40 Hz at random intervals determined by a Poisson process). Alternatively, for electrophysiological recording, each animal received all alternating stimulation conditions during recording.

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

[0305] Behavioral training and virtual reality (VR) environments for electrophysiology For CA1 recording, as described by Harvey et al., animals with their heads fixed were run in a virtual reality environment on an 8-inch spherical treadmill supported by an air cushion. The movement of the spherical treadmill was measured with an optical mouse and sent to virtual reality software running in a MATLAB® computer environment (software version 2013b, MathWorks, available from Natick, Massachusetts). This virtual environment consisted of a straight track and two small enclosures at the ends where the animals could turn. For alternating visits to each end of the track, the animals were rewarded with sweetened condensed milk (diluted with water in a 1:2 ratio) at each end of the track. The animals learned to run on this virtual straight track for at least one week. These animals were allowed one week to recover from surgery, acclimatized to handling for 1-2 days, and then behavioral training was initiated. For the first two days of training, animals were placed on a spherical treadmill with the virtual reality system turned off and rewarded with undiluted sweetened condensed milk, in order to learn how to operate the treadmill and become accustomed to the test environment. On the second day of training on the spherical treadmill, the animals' food was restricted to motivate them to run. The animals were limited to at most 85% of their reference body weight, and usually exceeded 88%. From the third day until the end of training (usually 5-7 days), the animals were placed on the treadmill for extended periods (30 minutes to 2 hours) and made to run on a VR straight track. After traversing the length of the track, the animals were rewarded with diluted (1:2) sweetened condensed milk at the end of the straight track. During the recording period, the animals were given retraining periods to maintain their behavioral abilities. For visual cortex recording, animals were made to run on the spherical treadmill while being exposed to dark, light, or flashing light conditions (described in the data collection below). Prior to recording, the animals were placed on a spherical treadmill (with the virtual reality system turned off) and learned to operate the treadmill and become accustomed to the test environment by receiving undiluted sweetened condensed milk as a reward.

[0306] Electrophysiological data collection For optogenetic stimulation of CA1 during recording, a 300 μm core optical fiber was advanced into the brain to a depth of 900 μm through the craniotomy used to supply the virus to CA1. 1 millisecond and 1 mW (measured from the fiber end) optical pulses were supplied via a 473 nm DPSS (diode-excited solid-state) laser (as described above). To avoid photoelectric artifacts, neural activity was recorded using glass electrodes. The LFP electrodes were prepared by extending a filament-type micropipette puller (e.g., P-97 Flaming / Brown® micropipette puller, Sutter Instrument Co., Nabat, California) from a borosilicate glass pipette (e.g., Warner Instruments, available from Hampden, Connecticut) to a fine tip, then manually breaking it back to a diameter of approximately 10-20 μm, and finally filling it with sterile saline. For CA1 recording, the LFP electrode was advanced through an LFP recording craniotomy at a 60-degree posterior angle to the frontal plane and a 45-degree downward angle to the horizontal plane until a clear electrophysiological sign of the pyramidal layer of the hippocampus (theta waves of approximately 600–1000 μV during animal movement, a clearly distinguishable SWR in the immobile state, and multiple spikes exceeding 150 μV, e.g., see Figures 2A–2B) was observed. For visual cortex recording, the LFP electrode was advanced vertically through an LFP recording craniotomy to a depth of 600–900 μm, and multiple spikes exceeding 150 μV were observed. Data were collected at a sampling rate of 20 kHz and a bandpass filter of 1 Hz–1 kHz. Animals were either running on a spherical treadmill for an extended period or resting. For the optogenetic stimulation period, data was recorded for 30 minutes prior to the start of either stimulation. Subsequently, stimuli were supplied at gamma (40 Hz), random (described in the visual stimulation protocol), or theta (8 Hz) frequencies in 10-second cycles alternating with 10-second baseline cycles (no stimulation). In two animals, each type of stimulus or baseline was supplied in 5-minute cycles instead of 10-second cycles. After each 30-minute stimulation recording, 5–30 minutes of no stimulation recording was performed. For the visual light flashing stimulus period, LED strip lights around the animals were flashed at gamma (40 Hz), random (described above in the visual stimulation protocol), theta (8 Hz), or 20 Hz frequencies in 10-second cycles, or in 10-second continuous on cycles, alternating with 10-second light-off cycles. Several recordings were made on the brain surface during the light flashing to ensure that these lights did not generate electrical or photoelectric noise during recording. The recording period was terminated after approximately 3–5 hours. The animals were 3–4 months old at the time of recording. Analysis of electrophysiological records

[0307] Spike detection Spikes were detected by thresholding the bandpass signal in the 300–6000 Hz range. To avoid contamination of the standard deviation measurement by spikes, the threshold was the median of the filtered signal plus five times the robust estimate of the standard deviation of the filtered signal (median / 0.675) (see, for example, Rossant et al., “Spike Sorting for Large, Dense Electrode Arrays,” bioRxiv doi:dx_doi_org_10.1101_015198 (Feb. 16, 2015)).

[0308] Local electric field potential (LFP) The recorded traces were processed at a low resolution of 2kHz and then subjected to a bandpass filter between 1 and 300Hz.

[0309] Theta and SWR detection When animals are running or stationary, activity through the hippocampal network changes significantly, 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 animals were running, as shown elsewhere, large theta (4–12 Hz) oscillations were observed in CA1 (see, e.g., Figure 2A). When the animals were stationary, theta oscillations were no longer observed, and SWR, i.e., high-frequency oscillations of 150–250 Hz lasting approximately 50–100 milliseconds and associated with bursts of collective activity (see, e.g., Figure 2B), were recorded. SWR was detected when the envelope amplitude of the filtered trace exceeded at least the upper four standard deviations of the mean over 15 milliseconds (see, e.g., Figures 4A, 4B, 5A, 5B, 6A, 6B, 7B, and 8). The envelope amplitude was calculated by taking the absolute value of the Hilbert transform of the filtered LFP. The results disclosed herein have been confirmed to be effective for SWR detection when using the upper 6 standard deviations of the mean, which detects a higher threshold, i.e., a larger SWR (see, e.g., Figures 6C and 7C). To detect theta (see, e.g., Figures 3A and 3C), a ripple filter such as an FIR is used to bandpass the LFP for theta (4–12 Hz), delta (1–4 Hz), and beta (12–30 Hz). The Luther method was used. The ratio of theta to delta and beta ("theta ratio") was calculated as the theta envelope amplitude, obtained by dividing the ratio by the sum of the delta and beta envelope amplitudes. A theta period was classified as such if the theta ratio exceeded the upper one standard deviation of the mean for at least two seconds, and this ratio reached a peak of at least two standard deviations of the mean. A non-theta period was classified as such if this theta ratio was less than 1 for at least two seconds. The SWR, theta period, and non-theta period were visually inspected to confirm that these criteria accurately detected the SWR, theta period, and non-theta period, respectively.

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

[0311] Gamma during SWR The spectrogram was calculated using the multi-taper method (e.g., Mitra Lab). (In Cold Spring Harbor Laboratory, open-source software from Chronux, available from Cold Spring Harbor, New York). Spectrograms were calculated for each SWR, including a 400-millisecond period before and after the SWR peak. Subsequently, z-score spectrograms were calculated for each frequency band using the mean and standard deviation of the spectrograms calculated over the entire recording period to generate a normalized measure of power in units of standard deviation (see, e.g., Figures 4A, 4B, 5A, and 5B). The instantaneous frequency of gamma oscillations in the SWR was calculated by bandpass filtering the LFP for 10–50Hz, performing a Hilbert transform, and then taking the reciprocal of the difference in peaks of the transformed signal (see, e.g., Figures 4A, 5A, and 6B). Gamma power before, during, and after the SWR was calculated by filtering the LFP for low gamma (20–50Hz) and taking the envelope amplitude of the Hilbert transform to obtain the average gamma power in 100-millisecond bins centered on the SWR peak. This was normalized by the mean and standard deviation of the envelope amplitude over the entire recording period to obtain the gamma power of the z score for each bin around each SWR (see, e.g., Figures 6A and 7B). Phase modulation by gamma in the SWR was calculated by bandpass filtering the LFP against gamma (20–50 Hz), performing a Hilbert transform, and determining the phase of the resulting signal for each spike that occurred in the SWR (see, e.g., Figure 7E). Resampling and resampling was used to measure the difference in phase modulation between 5XFAD and WT animals. That is, a subset of 100 spikes was randomly selected from each recording to create a phase modulation distribution, and this was repeated 500 times for each recording (see, e.g., Figures 6C and 7A). The depth of modulation was then measured by calculating the peak-to-trough difference by dividing by the sum of peaks and troughs for each distribution, thereby measuring the spike-gamma phase distribution (see, e.g., Figures 6C and 7A). Difference in firing during stimulation: To plot the histogram of multi-unit firing induced by the stimulus, spikes were binned at 2.5-millisecond intervals for 100 milliseconds after the start of each light in the pulse, and the percentage of spikes in each bin was calculated. The average and SEM were then calculated over all light in the subsequent period.To calculate the difference in multi-unit firing rates between conditions, firing rates were calculated for each 10-second stimulation or baseline period (total number of spikes divided by the period duration). The difference in firing rates was taken between adjacent periods of related stimulation types (gamma stimulation periods for optogenetic stimulation). The firing rate was calculated as the period minus baseline or random period (for flashing light stimuli, the firing rate was calculated as the gamma stimulation period minus baseline, continuous on, or random period). Differences from all animals were plotted in histograms (see, e.g., Figures 14A and 44A), and the median and quartiles of the differences per animal were plotted in box plots (see, e.g., Figures 13B and 44A).

[0312] immunohistochemistry Mice were perfused with 4% paraformaldehyde under deep anesthesia, and their brains were fixed overnight with 4% paraformaldehyde. The brains were sectioned to 40 μm using a vibratome (e.g., Leica VT100S, available from Leica Biosystems, Buffalo Grove, Illinois). The sections were permeabilized and blocked at room temperature for 1 hour with PBS containing 0.2% Triton X-100 and 10% normal donkey serum. The sections were incubated overnight at 4°C in a PBS solution of primary antibody with 0.2% Triton X-100 and 10% normal donkey serum. The primary antibodies used were 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, Massachusetts), anti-Iba1 (e.g., 019-19741, available from Wako Chemicals, Richmond, Virginia), anti-parvalbumin (e.g., ab32895, available from Abcam, Cambridge, Massachusetts), and anti-Rab5 (ADI-KAp-GP006-E, available from Enzo Life Sciences Inc., Farmindale, New York). To confirm the ELISA experiment, anti-Aβ antibody D54D2 was used to enable co-labeling with EEA1, and anti-Aβ antibody 12F4 was used because it did not react with APP, allowing for determination of whether this labeling was specific to Aβ. For the co-labeling experiment, the anti-Aβ antibody 12F4 (805501, BioLegend, available from San Diego, California) was used. The primary antibody was Alexa-Fluor 488 and Alex-Fluor 647, and the secondary antibody (Molecular Probes), Hoechst 33342 (94403, Sigma-Aldrich, available from St. Louis, Missouri), which was used to visualize neuronal nuclei. Images were acquired using a confocal microscope (LSM 710, Zeiss®) with the same settings for all conditions. Images were quantified using ImageJ 1.42q by an experimenter who did not know which group was being treated.For each experimental condition, at least two coronal sections from at least three animals were used for quantification. For hippocampal CA1 imaging, this analysis was limited to the pyramidal cell layer, except in the case of Iba1+ cell analysis where the entire field of view was required to image a sufficient number of cells. The diameter of Iba1+ cell bodies was measured using ImageJ, and the length measurement process was traced. Furthermore, the colocalization of Iba1 and Aβ was measured using the Coloc2 plugin. Imaris x64 8.1.2 (Bitplane, available from Belfast, UK) was used for 3-D rendering. For the counting of "plaque count," deposits larger than 10 μm were included.

[0313] CLARITY The fixed brain was sliced ​​into 100 μM coronal sections in 1XPBS using a vibratome (e.g., Leica VT100S, available from Leica Biosystems, Buffalo Grove, Illinois). Sections containing the visual cortex were selected using the Allen Mouse Brain Atlas as a reference, and incubated in clarification buffer (200 mM sodium dodecyl sulfate, 20 mM lithium hydroxide monohydrate, 4 mM borate in ddH2O solution, pH 8.5–9.0) with shaking at 55°C for 2 hours. The clarified sections were washed in 1XPBST (0.1% Triton X-100 / 1XPBS) for 3 × 10 minutes, then placed in blocking solution (2% bovine serum albumin / 1XPBST) and shaken overnight at room temperature. Next, the sections were washed three times in 1XPBST for 1 hour each at room temperature with shaking. The sections were then 1 The sections were incubated with anti-β-amyloid (805501, BioLegend, available from San Diego, California) and anti-Iba1 (Wako Chemicals, Richmond, Virginia, 019-19741) primary antibodies diluted 1:100 in 1XPBST for 2 hours with shaking at 4°C. After a 3×1 hour wash in 1XPBST, the sections were incubated with a secondary antibody mixture diluted 1:100 in 1XPBS for 9 hours with shaking at room temperature. Primary antibody labeling was visualized using fragmented donkey anti-rabbit Alexa Fluor® 488 (ab175694) and anti-mouse 568 (ab150101) secondary antibodies (both Abcam, available from Cambridge, Massachusetts). During this incubation period, Hoechst 33258 (Sigma-Aldrich, 94403) was spiked into each sample at a final dilution of 1:250. The sections were then washed overnight in 1xPBS with shaking at room temperature. Before mounting for imaging, the slices were incubated in RIMS (Refractive Index Matching Solution: Histodenz 75g, 0.1M Phosphate Buffer 20mL, ddH2O 60mL) for 1 hour with shaking at room temperature. Mounting was done using Fluoromount G Mounting Medium (Electron Microscopy Sciences, Hatfield, Pennsylvania, USA) and coverslips (e.g., VistaVision®, VWR). Tissue sections were mounted on microscope slides along with tissue samples (available from International, LLC, Radnor, Pennsylvania). Images were acquired using a Zeiss® LSM 880 microscope with Zen Black 2.1 software (Carl Zeiss Microscopy, Jena, Germany). Overview and cellular-level images of the cross-sections used for 3-D reconstruction were captured using a Plan-Apochromat 63x / 1.4 Oil DIC objective lens. Imarisx64 8.1.2 (Bitplane® (Zurich, Switzerland)) was used for 3-D rendering and analysis.

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

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

[0316] Genome-wide RNA sequencing Total RNA was extracted from hippocampal CA1 isolates using the RNeasy® kit (available from Qiagen, Hilden, Germany). Purified mRNA was used for RNA-seq library preparation using the BIOO NEXTflex® kit (BIOO number 5138-08) as per the manufacturer's instructions. Briefly, 1 μg of total mRNA was subjected to a sequential workflow of poly(A) purification, fragmentation, first and second flex strand synthesis, DNA terminal adenylation, and adapter ligation. These libraries were enriched by 15 cycles of PCR and washed with Agencourt® AMPure XP magnetic beads (available from Beckman Coulter Genomics, Danvers, Massachusetts). Library quality was evaluated using an Advanced Analytical-fragment Analyzer. Barcoded libraries were homogeneously mixed for sequencing on a single lane of the Illumina HiSeq 2000 platform at the MIT BioMicro Center (Massachusetts Institute of Technology, Cambridge, Massachusetts). Raw fastq data from 50-base pair single-end sequencing reads were aligned to the mouse mm9 reference genome using TopHat2.0 software (available from the Center for Computational Biology, Johns Hopkins University, Baltimore, Maryland, for aligning RNA-seq reads to a mammalian-sized genome using the ultra-high-throughput short-read aligner Bowtie, and then analyzing the mapping results to identify interexonal splice sites). The mapped reads were processed with UCSCmm9 reference gene annotation using Cufflinks2.2 software (available from the Trapnell Lab, University of Washington, Seattle, Washington) to estimate transcript abundance and examine differential expression. Relative transcript abundance was measured in fragments per kilobase (FPKM) of exons per 1 million mapped fragments. Differential gene expression tests between treated and untreated groups were performed using the Cuffdiff module (to find significant changes in transcript expression, splicing, and promoter use).The analysis was performed using Cufflinks 2.2 software (included as part of the Trapnell Lab software, available from Seattle, Washington), with an adjusted p-value < 0.05 for statistical significance (GEO registration: GSE77471).

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

[0318] Quantitative RT-PCR CA1 was isolated from the hippocampus of 3-month-old male 5XFAD / PV-Cre mice. The tissue was rapidly frozen using liquid nitrogen and stored at -80°C. RNA was extracted using the RNeasy kit according to the manufacturer's protocol (Qiagen, Hilden, Germany). RNA (3 μg) was treated with DNase I (4 U, Worthington Biochemical Corporation, Lakewood, New Jersey), 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 at 50°C for 1 hour in a 20 μl reaction volume containing a random hexamer mixture and Superscript III reverse transcriptase (50 U, Invitrogen®, Thermo Fisher Scientific, available from Waltham, Massachusetts). The first strand cDNA was diluted 1:10, and 1 μl was used for RT-qPCR amplification in 20 μl of reaction mixture (SsoFast® EvaGreen® Supermix, Bio-Rad) containing primers (0.2 μM). The relative changes in gene expression were measured. -ΔΔCt The evaluation was conducted using legal methods.

[0319] Isolation of microglia from the visual cortex. The V1 region was rapidly cleaved and placed in ice-cold Hanks equilibrium salt solution (HBSS) (Gibco® 14175-095, available from Life Technologies). This tissue was then enzymatically digested using a Neural Tissue Dissociation Kit (P) (130-092-628, Miltenyi Biotec, Cambridge, Massachusetts), following the manufacturer's protocol with minor modifications. Specifically, the tissue was enzymatically digested at 37°C for 15 minutes instead of 35 minutes, and the resulting cell suspension was passed through a MACS® SmartStrainer, a 40 μm cell filter instead of 70 μm (352340, Falcon Cell Strainers, Sterile, Corning, New York). The resulting cell suspension was then stained with allophycocyanin (APC)-conjugated CD11b mouse clone M1 / 70.15.11.5 (130-098-088, Miltenyi Biotec, Cambridge, Massachusetts) and phycoerythrin (PE)-conjugated CD45 antibody (e.g., BD Pharmingen®, 553081). CD11b and CD45-positive microglia cells were then purified using fluorescence-activated cell classification (FACS). These cells were directly isolated into 1XPBS (see, for example, Figure 52A).

[0320] statistics For electrophysiological data that did not follow a normal distribution, results are presented as medians and quartiles unless otherwise specified. A two-tailed Wilcoxon rank-sum test was performed on equal medians to determine if the distributions were significantly different, or a Wilcoxon signed-rank test was performed to determine if these differed significantly from zero when the data were not assumed to be normally distributed. Variability was similar between statistically compared groups. Multiple comparisons were corrected using Bonferroni's method. Molecular and biochemical results are presented as means and SEM. Percentages mentioned in this disclosure are group means. All statistical analyses were performed using Prism GraphPad software (GraphPad software Inc.). The study was conducted using samples from La Jolla, California. Normality was determined using the D'Agostino & Pearson omnibus normality test. Variability was similar between statistically compared groups. Comparative data for normally distributed data consisting of two groups were analyzed using a two-tailed independent t-test. Comparative data for normally distributed data consisting of three or more groups were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. Comparative data for non-normally distributed data were analyzed using the Mann-Whitney test. The statistical test, exact p-value, and sample size (n) for each experiment are indicated in the legend of the figures. Molecular and biochemical analyses were performed using a minimum of three biological replicates per condition.

[0321] Auditory gamma stimulus generation The following script, written in MATLAB® programming language (available from MathWorks, Natick, Massachusetts), demonstrates one method for generating auditory click-train stimuli according to several embodiments: JPEG0007837033000014.jpg211156

[0322] conclusion While various embodiments relating to the invention have been described and explained herein, those skilled in the art will readily conceive of various other methods and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages thereof, and each of such variations and / or modifications will be considered to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will be able to understand all the parameters, dimensions, materials, and described herein. It will be readily understood that the configurations are intended to be illustrative, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific use or combination of uses in which the disclosure of this invention is intended. Those skilled in the art will be able to recognize or confirm many equivalents of the embodiments relating to the specific invention described herein by means of conventional experimentation alone. Therefore, it should be understood that the embodiments described herein are presented merely as examples, and that embodiments relating to the invention may be carried out in ways other than those specifically described and claimed, within the scope of the appended claims and their equivalents. Embodiments relating to the invention of this disclosure relate to individual features, systems, articles, materials, kits, and / or methods described herein. Furthermore, two or more combinations of such features, systems, articles, materials, kits, and / or methods are included within the scope of the invention of this disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not contradictory to each other.

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

[0324] Furthermore, it should be understood that a computer can be embodied in any of several forms, such as a rack-mount computer, a desktop computer, a laptop computer, or a tablet computer. In addition, a computer may be embedded in a device that is not generally considered a computer but has sufficient processing power, including a personal digital assistive device (PDA), a smartphone, or any other suitable portable or fixed electronic device.

[0325] Furthermore, a computer may have one or more input and output devices. These devices can, in particular, be used to provide a user interface. Examples of output devices that can be used to provide a user interface include a printer or display screen for visual presentation of output, and a speaker or other sound-generating device for audible presentation of output. Examples of input devices that can be used for a user interface include a keyboard, a pointing device, such as a mouse, a touchpad, and a discretized tablet. In another example, a computer may receive input information via speech recognition or in other audible forms.

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

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

[0328] Furthermore, various inventive concepts may be embodied in one or more methods for which examples are provided. The actions performed as part of the method may be ordered in any suitable manner. Therefore, embodiments may be configured in which the actions are performed in a different order than those described, which may include performing several actions simultaneously, even if they are shown as sequential actions in exemplary embodiments.

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

[0330] All definitions defined and used herein should be understood to govern the dictionary definition, the definition in documents incorporated by reference, and / or the ordinary meaning of the defined term.

[0331] In this specification and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly indicated otherwise.

[0332] The expression “and / or” as used herein and in the claims should be understood to mean “either or both” of the elements thus combined, that is, elements that are sometimes conjunctive and sometimes disjunctive. Multiple elements listed with “and / or” should be similarly interpreted as “one or more” of the elements thus combined. In addition to the elements explicitly identified by the “and / or” clause, other elements may be optionally included, whether related to or unrelated to those explicitly identified elements. Thus, as a non-restrictive example, when used in conjunction with open-ended terms such as “include,” a reference to “A and / or B” may refer to A only (optionally including elements other than B) in one embodiment, B only (optionally including elements other than A) in another embodiment, and both A and B (optionally including other elements) in yet another embodiment, and so on.

[0333] As used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as inclusive, i.e., including at least one of multiple elements or lists of elements, but also multiple, and optionally further items not on the list. Restrictive terms clearly indicated to be otherwise, such as “only one” or “strictly one,” or, as used in the claims, “consisting of,” shall be considered, for example, to include strictly one element from multiple elements or lists of elements. In general, as used herein, the term “or” shall be interpreted only as indicating an exclusive choice (i.e., “one or the other, but not both”) when preceded by an exclusive term, such as “either,” “one,” “only one,” or “strictly one.” As used in the claims, “essentially consisting of” shall have its usual meaning as used in the field of patent law.

[0334] The expression “at least one” as used herein and in the claims, referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the inclusion of elements other than those explicitly identified in the list of elements referred to by the expression “at least one,” whether related to or unrelated to the explicitly identified elements. Thus, as a non-limiting example, “at least one of A and B” (or equivalently, “at least one of A or B” or equivalently, “at least one of A and / or B”) may, in one embodiment, include at least one, and optionally more. In another embodiment, A may not include B (and may optionally include elements other than B); in yet another embodiment, B may include at least one or more elements and not A (and may optionally include elements other than A); and in yet another embodiment, A and at least one or more elements and B (and may optionally include other elements), and so on.

[0335] In the claims and in the specification, all transitional clauses, such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and “composed of,” should be understood to be open-ended, meaning they include but are not limited to. As stated in Section 2111.03 of the U.S. Patent and Trademark Office Examination Manual, only the transitional clauses “consisting of” and “consisting essentially of” are considered closed or semi-closed transitional clauses.

Claims

1. A device for treating Alzheimer's disease in a subject, The aforementioned device is At least one stimulus-releasing device that emits at least one non-invasive stimulus to the aforementioned object; At least one processor coupled to the at least one stimulus emission device and controlling the at least one stimulus emission device; and A memory connected to at least one of the processors, Executable instructions for at least one of the processors; and Stimulation parameters including a repetition frequency in the range of 35 Hz to 45 Hz for at least one non-invasive stimulus; and the memory for storing these parameters; Includes, The device is configured to emit a plurality of light pulses with pulse frequencies of 35 pulses / second to 45 pulses / second according to the stimulation parameters, and The system is configured to treat Alzheimer's disease in the subject by stimulating the subject with the light pulse to induce synchronized gamma oscillations in the subject's visual cortex and reducing the amount of phosphorylated tau protein in the subject's visual cortex. The aforementioned device.

2. The apparatus according to claim 1, wherein multiple light pulses have a frequency of 40 Hz.

3. The apparatus according to claim 1, wherein the target is a human.

4. At least one memory for storing stimulation parameters including frequencies between 35 Hz and 45 Hz; At least one processor communicating with the at least one memory, the at least one processor configured to control and monitor the emission of non-invasive visual stimuli; and Eyeglasses configured to emit a non-invasive visual stimulus having a pulse frequency of 35 pulses / second to 45 pulses / second over a certain period of time according to the aforementioned stimulation parameters, to induce synchronous gamma oscillations in at least one brain region of a subject, and to reduce the amount of phosphorylated tau protein in the at least one brain region of the subject, thereby treating Alzheimer's disease in the subject, A stimulus-releasing device that includes a stimuli-emitting device.

5. The stimulus emission device according to claim 4, wherein the non-invasive visual stimulus has a pulse frequency of 40 pulses / second.

6. The stimulus-releasing device according to claim 4, wherein the target is a human.

7. The stimulus-emitting device according to claim 4, wherein the non-invasive visual stimulus includes a pulse of light.

8. The stimulus emission device according to claim 4, wherein at least one brain region includes the target visual cortex.

9. A system for use in the treatment of Alzheimer's disease, At least one memory for storing stimulus parameters with a frequency of 35 Hz to 45 Hz; A signal generator that generates a signal having a frequency of 35 Hz to 45 Hz according to the aforementioned stimulus parameters; An emitter coupled to the signal generator, wherein, in accordance with the signal generated by the signal generator, a non-invasive administration of visual and / or auditory stimuli having a pulse frequency of 35 pulses / second to 45 pulses / second to a subject requiring treatment for Alzheimer's disease for a certain period of time induces synchronous gamma oscillations in at least one brain region of the subject, reduces the amount of phosphorylated tau protein in the at least one brain region of the subject, and thereby can treat Alzheimer's disease in the subject, The system comprising the above.

10. The system according to claim 9, wherein the visual stimulus and / or auditory stimulus have a pulse frequency of 40 pulses / second.

11. The system according to claim 9, wherein the emitter is a light-emitting body.

12. The system according to claim 11, wherein the light-emitting element is selected from the group consisting of optical fiber emitters and solid-state light sources.

13. The system according to claim 12, wherein the light-emitting element is a solid-state light source and includes at least one light-emitting diode (LED).

14. The system according to claim 9, wherein the discharger includes a display screen.

15. The system according to claim 9, wherein the emitter is a light-emitting body and further comprises a light-blocking device for reducing ambient light to at least one eye of an object.

16. The system according to claim 9, further comprising an imaging device for monitoring the function of at least one brain region of a target before, during, or after administration, or a combination thereof.

17. The system according to claim 9, wherein at least one brain region is the visual cortex of the target.

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