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

Abstract

To provide devices, systems and methods for treating dementia or Alzheimer's disease.SOLUTION: In one example, combined auditory and visual stimulation having a frequency of about 20 Hz to about 60 Hz, and more specifically about 40 Hz, are non-invasively delivered to the subject to induce synchronized gamma oscillations in at least one brain region of the subject. In particular, according to various treatment protocols and exposure protocols, combined auditory and visual stimulation (as opposed to auditory or visual stimulation alone) promotes a microglia response in the medial prefrontal cortex (mPFC). In general, combined auditory and visual stimulation induces an extended microglia clustering response in the auditory cortex, the visual cortex, and the mPFC.SELECTED DRAWING: Figure 7-7

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to each of the following U.S. Provisional Patent Application Specifications: U.S. Provisional Patent Application No. 62 / 570,250, filed on October 10, 2017, with the title "NEUROPROTECTIVE EFFECTS OF COMBINED SENSORY STIMULATION" (Attorney Docket No. MITX - 9699 / 00US); and U.S. Provisional Patent Application No. 62 / 570,929, filed on October 11, 2017, with the title "GAMMA ENTRAINMENT BINDS HIGHER ORDER BRAIN REGIONS AND OFFERS NEUROPROTECTION" (Attorney Docket No. MITX - 0070 / 00US). Each of these provisional patent application specifications is incorporated herein by reference in its entirety.

[0002] Statement Regarding Government Support This invention was made with government support under Grant No. RF1 AG047661 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

Background Art

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by decline in memory, orientation, and logical thinking. Alzheimer's disease is the most common dementia in the world, and in the United States, approximately 1 in 8 people over 65 years old are affected, making it the 6th leading cause of death. The prevalence of this progressive neurodegenerative disorder is estimated to increase by 40% over the next 10 years.

[0004] Histopathologically, Alzheimer's disease (AD) is characterized by the accumulation of amyloid plaques, which contain neurofibrillary tangles (NFTs) produced from amyloid-β (Aβ) peptide and tau protein. Aβ peptide is a protein consisting of 36-43 amino acids, and its normal physiological function has not yet been identified. Aβ peptide is related to β-secretase 1 (BACE1). 1) Amyloid precursor protein (APP) by γ-secretase It is formed by the sequential protein cleavage of the precursor protein. The C-terminal fragment β (β-CTF) is an APP derivative produced during the amyloid-forming cleavage of APP by BACE1 and is another indicator of Aβ peptide production. Under normal conditions, soluble Aβ peptide is produced and secreted by neurons. It is then removed from the brain via the cerebrospinal fluid (CSF) pathway. However, in subjects with AD, Aβ peptide is thought to aggregate into higher-order forms, forming soluble oligomers and insoluble plaques in a concentration-dependent manner. This aggregation can lead to many neurotoxic phenomena, including disruption of brain metabolism, neuroinflammation, decreased functional connectivity, synapse and neuronal loss, and / or NFT formation.

[0005] A fundamental relationship between Aβ concentration and neuronal activity has been reported. Firstly, treatment of organ-type hippocampal sections prepared from transgenic (Tg) mice overexpressing APP with tetrodotoxin reduced neuronal activity, followed by a decrease in Aβ levels. Subsequently, treatment with picrotoxin resulted in the opposite effect—an increase in neuronal activity. Dynamic regulation of Aβ peptide concentration using neuronal activity and eventual plaque deposition in vivo have also been demonstrated. Neuronal imaging of human AD patients Imaging studies suggest that the most severe plaque deposits may be located along the most consistently active brain region known as the "default-mode network."

[0006] Currently, there is no cure for Alzheimer's disease (AD), and treatment options do not inhibit the progression of the disease, are primarily temporary relief, 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- and γ-secretases) have been toxic in clinical trials and / or ineffective in reducing AD lesions. Clinical trials involving amyloid beta vaccines (e.g., bapineuzumab) have failed with no cognitive benefit. Gamma-secretase inhibitors (e.g., semagacestat) have failed in clinical trials and worsened cognitive impairment in subjects. Even existing drugs such as acetylcholinesterase inhibitors (e.g., donepezil and rivastigmine) and N-methyl-D-aspartate (NMDA) receptor antagonists (e.g., memantine) have only demonstrated mild cognitive function benefits. [Overview of the Initiative]

[0007] "SYSTEMS AND METHODS FOR PREVENTING, MITIGATING, AND / OR TREATING" filed on November 23, 2016 As disclosed in U.S. Patent Application No. 15 / 360,637, titled "DEMENTIA" (incorporated herein by whole reference), reduced amyloid load and morphological changes were induced in certain brain regions by inducing synchronized gamma oscillations in the brain through visual or auditory stimuli. However, the inventors recognize and understand that there is still a need for systems and methods for treating dementia and Alzheimer's disease that address circuit-wide diseases affecting multiple brain centers responsible for learning, memory, and most other higher brain functions.

[0008] In the aforementioned regard, this disclosure relates, at least in part, to combinations of auditory and visual stimuli that induce gamma oscillations in the target brain, following various techniques generally referred to herein as “Gamma Entrainment Using Sensory Stimuli” (GENUS). As disclosed herein, combinations of auditory and visual stimuli (e.g., combinations of visual GENUS and auditory GENUS) unexpectedly produced positive physiological and behavioral changes not observed with visual GENUS alone or auditory GENUS alone. The positive effects on the brain resulting from the combination of auditory and visual GENUS were not limited to the auditory cortex (AC) and hippocampus (HPC), but extended particularly to the induction of microglial clustering responses in the medial prefrontal cortex (mPFC) and a reduction in the overall amyloid load of the neocortex. The effects of the combination of auditory and visual GENUS have also been observed beyond the short-term frame of treatment / exposure (weeks).

[0009] In one embodiment, the Disclosure provides a device, method, and system for treating dementia or Alzheimer's disease in a subject where such treatment is needed, the method comprising the steps of non-invasively delivering a combination of auditory and visual stimuli having frequencies of about 20 Hz to about 60 Hz to the subject to induce synchronized gamma oscillations in at least one brain region of the subject. In some embodiments, dementia is associated with AD, vascular dementia, frontotemporal dementia, Lewy body dementia, and / or age-related cognitive decline. The subject may be human or animal.

[0010] In some embodiments, the combination of auditory and visual stimuli has a frequency of approximately 35 Hz to approximately 45 Hz, or approximately 40 Hz.

[0011] In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli induces a periodic spike response in at least 5% of the recording area of ​​at least one cortical region selected from the auditory cortex (AC), visual cortex (VC), hippocampus (HPC), and medial prefrontal cortex (mPFC). In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli induces a local field potential (LFP) of approximately 40 Hz in the mPFC.

[0012] In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli increases the microglial response in at least one cortical region. The cortical region may include the neocortex, AC, VC, HPC, and mPFC. In some embodiments, the microglial response is induced in the mPFC. In some embodiments, the microglial response is induced in multiple cortical regions. In some embodiments, the microglial response is induced throughout the neocortex.

[0013] In some embodiments, an increase in microglial response includes at least one effect selected from: an increase in the number of microglia within 25 micrometers of the amyloid plaque, an increase in the diameter of the microglial cell bodies, a decrease in the length of the microglial protrusions, and an increase in the number of microglial cells. In some embodiments, an increase in microglial response includes an increase of at least 10%, 20%, 30%, 40%, or 50% in the diameter of the microglial cell bodies. In some embodiments, an increase in microglial response includes a decrease of at least 10%, 20%, 30%, 40%, or 50% in the length of the microglial protrusions. In some embodiments, an increase in microglial response includes an increase of at least 10%, 20%, 30%, 40%, or 50% in the number of microglial cells.

[0014] In some embodiments, the increase in microglial response occurs after non-invasive delivery of a combination of auditory and visual stimuli for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0015] In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli includes a reduction in amyloid plaques in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. The cortical region may include the neocortex, AC, VC, HPC, and mPFC. In some embodiments, the microglial response is induced in the mPFC. In some embodiments, the microglial response is induced in multiple cortical regions. In some embodiments, the microglial response is induced throughout the neocortex.

[0016] In one embodiment, a reduction in amyloid plaques includes a reduction of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% in plaque size. In another embodiment, a reduction in amyloid plaques includes a reduction of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% in the number of plaques.

[0017] In some embodiments, the increase in microglial response occurs after non-invasive delivery of a combination of auditory and visual stimuli for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0018] In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli includes a reduction in amyloid-β (Aβ) peptide levels in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. The cortical region may include the neocortex, AC, VC, HPC, and mPFC. In some embodiments, the microglial response is mPFC It is induced in the following locations. In some embodiments, the microglia response is induced in multiple cortical regions. In some embodiments, the microglia response is induced throughout the neocortex.

[0019] In some embodiments, the reduction in the amount of Aβ peptide includes a reduction of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60%. In some embodiments, the Aβ peptide comprises at least one of the Aβ1-40 peptide isoform and the Aβ1-42 peptide isoform. In some embodiments, the Aβ peptide comprises at least one of the soluble Aβ peptide and the insoluble Aβ peptide.

[0020] In some embodiments, the increase in microglial response occurs after non-invasive delivery of a combination of auditory and visual stimuli for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0021] In a second embodiment, the Disclosure provides a method for treating dementia or Alzheimer's disease in a subject in need, the method comprising: controlling at least one visual stimulator emitting visual stimuli with a frequency of about 35 Hz to about 45 Hz; controlling at least one electroacoustic transducer converting an electrosound signal to a corresponding auditory stimulus with a frequency of about 35 Hz to about 45 Hz; and non-invasively delivering a combined stimulus to the subject, the combined stimulus comprising a synchronously aligned visual stimulus and an auditory stimulus, the combined stimulus inducing synchronized gamma oscillations in at least one brain region of the subject, the synchronized gamma oscillations resulting in improved cognitive function in the subject.

[0022] In some embodiments, the visual stimulus includes a repetition of 12.5 milliseconds of illumination followed by 12.5 milliseconds of deactivation. In some embodiments, the optogenetic stimulator is a light-emitting diode with a power of 40–80 W. In some embodiments, the auditory stimulus includes a 10 kHz tone reproduced at 40 Hz in load cycles of approximately 4%–80%. In some embodiments, the visual stimulus includes light flashing at 40 Hz for 10 seconds in load cycles of approximately 10%–80%.

[0023] In some embodiments, the visual and auditory stimuli are synchronized. In some embodiments, the visual and auditory stimuli are out of phase by -180 to 0 degrees or 0 to 180 degrees.

[0024] Naturally, all combinations of the aforementioned concepts and any additional concepts discussed in more detail below (assuming such concepts are not contradictory) are considered to be part of the subject matter of the invention disclosed herein. In particular, all combinations of subject matter described in the claims, which appear at the end of this disclosure, are considered to be part of the subject matter of the invention disclosed herein. Naturally, any terms explicitly used in any disclosure incorporated by reference must be given meanings that are most consistent with the specific concepts disclosed herein.

[0025] Further details and diagrams will be apparent to those skilled in the art. All such additional systems, processes, and features are included in this description, within the scope of the present invention, and are intended to be protected by the appended claims. [Brief explanation of the drawing]

[0026] The patent or application file must include at least one drawing created in color.

[0027] Those skilled in the art will understand that the drawings are for illustrative purposes only and that the information described herein is provided herein. It will be understood that this is not intended to limit the scope of the subject matter of the present invention. The drawings are not necessarily to a constant proportion, and in some examples, various aspects of the subject matter of the present invention disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate the understanding of different features. In the drawings, similar reference letters generally mean, for example, similar features (e.g., functionally similar and / or structurally similar elements).

[0028] [Figure 1-1] Figures 1A–1L show that 40 Hz auditory stimulation modulates spike activity in AC, CA1, and mPFC. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 1-9] Same as above. [Figure 1-10] Same as above. [Figure 1-11] Same as above. [Figure 1-12] Same as above. [Figure 1-13] Same as above. [Figure 1-14]Same as above. [Figure 1-15] Same as above. [Figure 2-1] Figures 2A–2J show that auditory GENUS improves cognitive and spatial memory tasks in 5XFAD mice. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 3-1] Figures 3A-3I show that auditory GENUS reduces amyloid loading in AC and HPC in 5XFAD mice. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above. [Figure 4-1] Figures 4A-4K show that auditory GENUS induces glial responses in AC and CA1 of 5XFAD mice. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 4-6] Same as above. [Figure 5-1] Figures 5A-5F show that auditory GENUS increases the association between amyloid and vascular systems. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 6-1] Figures 6A-6I show that the combination of auditory and visual GENUS induces a cluster-forming phenotypic response by microglia. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 6-5] Same as above. [Figure 6-6] Same as above. [Figure 6-7] Same as above. [Figure 6-8] Same as above. [Figure 6-9] Same as above. [Figure 7-1] Figures 7A–7J show that the combination of auditory GENUS and visual GENUS reduces amyloid loading in the mPFC and neocortex. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above. [Figure 7-7] Same as above. [Figure 7-8] Same as above. [Figure 8-1] Figures 8A–8R show that auditory stimuli at 20 Hz and 80 Hz modulate activity in the AC, CA1, and mPFC. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] Same as above. [Figure 8-6] Same as above. [Figure 8-7] Same as above. [Figure 8-8] Same as above. [Figure 8-9] Same as above. [Figure 8-10] Same as above. [Figure 8-11] Same as above. [Figure 8-12] Same as above. [Figure 8-13] Same as above. [Figure 8-14] Same as above. [Figure 8-15] Same as above. [Figure 8-16] Same as above. [Figure 8-17] Same as above. [Figure 8-18] Same as above. [Figure 9-1] Figures 9A-9L show that auditory GENUS does not affect mouse behavior. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above. [Figure 10-1] Figures 10A-10H show that auditory GENUS improves plaque load in APP / PS1 mice. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 11-1] Figures 11A-11H show that auditory GENUS induces a microglial response in APP / PS1 mice. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 12-1] Figures 12A-12L show that auditory GENUS reduces phosphorylated tau in P301S mice. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above. [Figure 12-6] Same as above. [Figure 12-7] Same as above. [Figure 13-1] Figures 13A–13U show that a combination of 40Hz auditory and visual stimuli modulates spike activity in AC, CA1, and mPFC. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. [Figure 13-5] Same as above. [Figure 13-6] Same as above. [Figure 13-7] Same as above. [Figure 13-8] Same as above. [Figure 13-9] Same as above. [Figure 13-10] Same as above. [Figure 13-11] Same as above. [Figure 13-12] Same as above. [Figure 13-13] Same as above. [Figure 13-14] Same as above. [Figure 13-15] Same as above. [Figure 13-16] Same as above. [Figure 13-17] Same as above. [Figure 13-18] Same as above. [Figure 13-19] Same as above. [Figure 13-20] Same as above. [Figure 13-21] Same as above. [Figure 14-1] Figures 14A to 14V show that auditory GENUS or visual GENUS alone for one week does not affect the lesions of the mPFC. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. [Figure 14-5] Same as above. [Figure 14-6] Same as above. [Figure 14-7] Same as above. [Figure 14-8] Same as above. [Figure 14-9] Same as above. [Figure 14-10] Same as above. [Figure 14-11] Same as above. [Figure 14-12] Same as above. [Figure 14-13] Same as above. [Figure 14-14] Same as above. [Figure 14-15] Same as above. [Figure 14-16] Same as above. [Modes for carrying out the invention]

[0029] The combination of auditory and visual sensory stimuli in gamma entrainment (GENUS) unexpectedly produces positive physiological and behavioral changes not observed with visual GENUS or auditory GENUS alone. The positive effects on the brain were not limited to the auditory cortex (AC) and hippocampus (HPC), but rather extended to the induction of microglial clustering responses in the medial prefrontal cortex (mPFC) and a reduction in amyloid loading across the neocortex. The effects of the auditory and visual GENUS combination have been observed beyond even shorter timeframes (weekly). Approximately one week of auditory and visual GENUS combination improves Alzheimer's disease (AD) lesions in brain regions affecting large-scale circuit networks. In particular, the auditory and visual GENUS combination results in significant reductions in amyloid loading in the AC, visual cortex (VC), hippocampal subregion CA1, and mPFC. The combination of auditory and visual GENUS induces microglial clustering and a reduction in amyloid in the medial prefrontal cortex. The combination of auditory and visual GENUS also results in a widespread reduction of amyloid plaques throughout the neocortex.

[0030] In one embodiment, the Disclosure provides a device, method, and system for treating dementia or Alzheimer's disease in a subject where such treatment is needed, the method comprising the steps of non-invasively delivering a combination of auditory and visual stimuli having frequencies of about 20 Hz to about 60 Hz to the subject to induce synchronized gamma oscillations in at least one brain region of the subject. In some embodiments, dementia is associated with AD, vascular dementia, frontotemporal dementia, Lewy body dementia, and / or age-related cognitive decline. The subject may be human or animal.

[0031] In some embodiments, the combination of auditory and visual stimuli has a frequency of approximately 35 Hz to approximately 45 Hz, or approximately 40 Hz.

[0032] In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli induces a periodic spike response in at least 5% of the recording area of ​​at least one cortical region selected from the auditory cortex (AC), visual cortex (VC), hippocampus (HPC), and medial prefrontal cortex (mPFC). In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli induces a local field potential (LFP) of approximately 40 Hz in the mPFC.

[0033] In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli increases the microglial response in at least one cortical region. The cortical region may include the neocortex, AC, VC, HPC, and mPFC. In some embodiments, the microglial response is induced in the mPFC. In some embodiments, the microglial response is induced in multiple cortical regions. In some embodiments, the microglial response is induced throughout the neocortex.

[0034] In some embodiments, an increase in microglial response includes at least one effect selected from: an increase in the number of microglia within 25 micrometers of the amyloid plaque, an increase in the diameter of the microglial cell bodies, a decrease in the length of the microglial protrusions, and an increase in the number of microglial cells. In some embodiments, an increase in microglial response includes an increase of at least 10%, 20%, 30%, 40%, or 50% in the diameter of the microglial cell bodies. In some embodiments, an increase in microglial response includes a decrease of at least 10%, 20%, 30%, 40%, or 50% in the length of the microglial protrusions. In some embodiments, an increase in microglial response includes a decrease of at least 10%, 20%, This includes increases of 30%, 40%, or 50%.

[0035] In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of non-invasive delivery of a combination of auditory and visual stimuli. In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of non-invasive delivery of a combination of auditory and visual stimuli. In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months of non-invasive delivery of a combination of auditory and visual stimuli. In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of non-invasive delivery of a combination of auditory and visual stimuli.

[0036] In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli includes a reduction in amyloid plaques in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. The cortical region may include the neocortex, AC, VC, HPC, and mPFC. In some embodiments, the microglial response is induced in the mPFC. In some embodiments, the microglial response is induced in multiple cortical regions. In some embodiments, the microglial response is induced throughout the neocortex.

[0037] In one embodiment, a reduction in amyloid plaques includes a reduction of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% in plaque size. In another embodiment, a reduction in amyloid plaques includes a reduction of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% in the number of plaques.

[0038] In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of non-invasive delivery of a combination of auditory and visual stimuli. In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of non-invasive delivery of a combination of auditory and visual stimuli. In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months of non-invasive delivery of a combination of auditory and visual stimuli. In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of non-invasive delivery of a combination of auditory and visual stimuli.

[0039] In some embodiments, non-invasive delivery of a combination of auditory and visual stimuli involves a reduction in amyloid-β (Aβ) peptide levels in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. The cortical region may include the neocortex, AC, VC, HPC, and mPFC. In some embodiments, the microglial response is induced in the mPFC. In some embodiments, the microglial response is induced in multiple cortical regions. In some embodiments, the microglial response is induced throughout the neocortex.

[0040] In some embodiments, the reduction in the amount of Aβ peptide includes a reduction of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60%. In some embodiments, the Aβ peptide comprises at least one of the Aβ1-40 peptide isoform and the Aβ1-42 peptide isoform. In some embodiments, the Aβ peptide comprises at least one of the soluble Aβ peptide and the insoluble Aβ peptide.

[0041] In some embodiments, the increase in microglial response occurs after non-invasive delivery of a combination of auditory and visual stimuli for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. or occurs after 10 weeks of non-invasive delivery of a combination of auditory and visual stimuli. In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months of non-invasive delivery of a combination of auditory and visual stimuli. In some embodiments, the increase in microglial response occurs after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of non-invasive delivery of a combination of auditory and visual stimuli.

[0042] In a second embodiment, the Disclosure provides a method for treating dementia or Alzheimer's disease in a subject in need, the method comprising: controlling at least one visual stimulator emitting visual stimuli with a frequency of about 35 Hz to about 45 Hz; controlling at least one electroacoustic transducer converting an electrosound signal to a corresponding auditory stimulus with a frequency of about 35 Hz to about 45 Hz; and non-invasively delivering a combined stimulus to the subject, the combined stimulus comprising a synchronously aligned visual stimulus and an auditory stimulus, the combined stimulus inducing synchronized gamma oscillations in at least one brain region of the subject, the synchronized gamma oscillations resulting in improved cognitive function in the subject.

[0043] In some embodiments, the visual stimulus includes a repetition of 12.5 milliseconds of illumination followed by 12.5 milliseconds of deactivation. In some embodiments, the optogenetic stimulator is a light-emitting diode with a power of 40–80 W. In some embodiments, the auditory stimulus includes a 10 kHz tone reproduced at 40 Hz in load cycles of approximately 4%–80%. In some embodiments, the visual stimulus includes light flashing at 40 Hz for 10 seconds in load cycles of approximately 10%–80%.

[0044] In some embodiments, the visual and auditory stimuli are synchronized. In some embodiments, the visual and auditory stimuli are out of phase by -180 to 0 degrees or 0 to 180 degrees. As used herein, “phase” refers to the time difference between the auditory and visual stimuli, expressed in degrees, where 0 degrees means simultaneous auditory and visual stimulation, and -180 degrees or +180 degrees means alternating visual and auditory stimuli.

[0045] As used herein, the terms “treatment” or “to treat” refer to both therapeutic actions and preventive or protective measures. In some embodiments, subjects requiring treatment include subjects who already have a disease or condition, subjects who are likely to develop a disease or condition, and subjects whose objective is the prevention, delay, or mitigation of a disease or condition. For example, in some embodiments, the devices, methods, and systems disclosed herein may be employed to prevent, delay, or mitigate a disease or condition to which a subject is genetically predisposed, such as AD. In some embodiments, the devices, methods, and systems disclosed herein may be employed to treat, alleviate, reduce, and / or slow the progression of symptoms of a disease or condition to which a subject has already been diagnosed, such as AD.

[0046] As used herein, the term "subject" means mammals such as rodents, felines, canines, or primates. The subject of the present invention is preferably humans.

[0047] As used herein, the term “approximately” refers to ±10 percent of the subject modified by “approximately.”

[0048] Dementia is a disorder characterized by a decline in 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), AIDS dementia, age-related cognitive impairment, and age-related memory impairment. Dementia can also be caused by neurological and / or psychiatric conditions, such as brain tumors, brain lesions, and epilepsy. It may also be associated with multiple sclerosis, Down syndrome, Rett syndrome, progressive supranuclear palsy, frontal lobe syndrome, schizophrenia, and traumatic brain injury.

[0049] Alzheimer's disease (AD) is the most common neurodegenerative disease in developed countries. Histopathologically, AD is characterized by the accumulation of amyloid plaques, which are composed of Aβ peptides and NFTs produced from tau protein. Clinically, AD is associated with progressive cognitive impairment characterized by a decline in memory, function, language ability, judgment, and executive function. Severe behavioral symptoms often develop in the later stages of AD.

[0050] Vascular dementia, sometimes referred to as cerebrovascular dementia, can also refer to cerebrovascular diseases (e.g., infarctions of the cerebral hemispheres). These typically have a fluid course with periods of improvement and gradual deterioration. Vascular dementia may include one or more of the following: disorientation, memory impairment, and / or impaired judgment. Vascular dementia may be caused by separate, complex infarctions, or by other vascular causes, such as autoimmune vasculitis found in systemic lupus erythematosus, infectious vasculitis such as Lyme disease, recurrent cerebral hemorrhage, and / or stroke.

[0051] Frontotemporal dementia (FTD) is a progressive neurodegenerative disorder. Patients with FTD generally exhibit significant behavioral and personality changes, often accompanied by speech impairment.

[0052] Lewy body dementia is characterized by the onset of dementia with overlapping characteristics with Alzheimer's disease, the manifestation of Parkinson's disease features, and / or the early onset of hallucinations. Lewy body dementia is generally characterized by day-to-day fluctuations in the severity of symptoms.

[0053] In some embodiments, the disclosure provides a method for preventing, mitigating, and / or treating dementia in a subject, the method comprising the step of inducing synchronized gamma oscillations in the subject's brain. In some embodiments, the induction of gamma oscillations in a subject suffering from a neurological disorder or impairment, or age-related decline, acts to restore disrupted gamma oscillation rhythms in the subject that are a result of, or related to, such disorder or impairment, or age-related decline.

[0054] In some embodiments, the induction of gamma oscillations is Aβ 1-40 and Aβ 1-42 It reduces the generation of isoforms. In some embodiments, the induction of gamma oscillations reduces the generation of Aβ (e.g., Aβ) from the brain of the subject. 1-40 Isoforms and Aβ 1-42 The clearance of isoforms is enhanced. In some embodiments, the induction of gamma oscillations prevents the accumulation of Aβ in the brain of the subject. In some embodiments, the methods presented herein reduce the Aβ level in the subject's brain by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or more than about 70% compared to the Aβ level in the subject's brain before treatment. In some embodiments, the Aβ level in the subject's brain is reduced by at least about 50% compared to the Aβ level in the subject's brain before treatment.

[0055] In some embodiments, Aβ levels in the target brain are reduced through a decrease in APP cleavage in the target brain. In some embodiments, the methods presented herein reduce APP cleavage levels in the target brain by approximately 10%, 20%, 30%, 40%, 50%, 60%, or 70% or more compared to pre-treatment APP cleavage levels in the target brain. In some embodiments, APP cleavage levels in the target brain are reduced by at least approximately 50% compared to pre-treatment APP cleavage levels in the target brain. In some embodiments, APP cleavage levels are measured by the level of C-terminal fragment β (β-CTF) in the target brain. In some embodiments, APP cleavage levels in the brain are reduced through inhibition of β and / or γ-secretase, for example, by an increase in the inhibition level of β and / or γ-secretase activity. Morphologically, the method presented herein reduces the aggregation of Aβ plaques in the target brain.

[0056] In some embodiments, the method of the present invention improves the cognitive abilities and / or memory of the subject.

[0057] In other embodiments, the disclosure includes a step of inducing a neuroprotective profile or neuroprotective environment within the brain of a subject, the step of inducing synchronized gamma oscillations within the brain of the subject. For example, in some embodiments, the neuroprotective profile is associated with a neuroprotective microglial cell profile. In further embodiments, the neuroprotective profile is induced by or associated with increased activity of the M-CSF pathway. In some embodiments, the neuroprotective environment is associated with an anti-inflammatory signaling pathway. For example, in some embodiments, the anti-inflammatory signaling pathway is an anti-inflammatory microglial signaling pathway.

[0058] In some embodiments, the neuroprotective profile is associated with reduced or absent pro-inflammatory glial cell activity. Pro-inflammatory glial cell activity is associated with the M1 phenotype of microglia and includes the production of reactive species of oxygen (ROS), the neurosecretory protein chromogranin A, the secretory cofactor cystatin C, NAPDH oxidase, nitric oxide synthase enzymes such as iNOS, NF-κB-dependent inflammatory response proteins, and pro-inflammatory cytokines and chemokines (e.g., TNF, IL-1β, IL-6, and IFNγ).

[0059] In contrast, the M2 phenotype in microglia is associated with downregulation of inflammation and recovery from inflammation-induced injury. Anti-inflammatory cytokines and chemokines (IL-4, IL-13, IL-10, and / or TGFβ), as well as increased phagocytic activity, are associated with the M2 phenotype. Therefore, in some embodiments, the methods presented herein induce a neuroprotective M2 phenotype in microglia. In some embodiments, the methods presented herein increase phagocytic activity in the target brain. For example, in some embodiments, the methods presented herein increase microglial phagocytic activity and increase Aβ clearance.

[0060] The gamma oscillations may include a range of about 20 Hz to about 100 Hz. In some embodiments, the disclosure provides a method for preventing, mitigating, or treating dementia in a subject, the method comprising the step of inducing gamma oscillations in the subject's brain of a range 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 Hz to about 60 Hz, or about 35 Hz to about 45 Hz, or about 40 Hz. The gamma oscillations are preferably about 40 Hz.

[0061] The stimulus may include any 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 (i.e., pain), sound receptors, electroreceptors (e.g., electric fields), magnetoreceptors (e.g., magnetic fields), hydroreceptors, chemoreceptors, thermoreceptors, osmoreceptors, and / or autoreceptors (i.e., proprioceptors). 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 object. In some embodiments, the stimulus is adapted according to individual sensitivity.

[0062] In some embodiments, gamma oscillations are induced in a brain region-specific manner. For example, in some embodiments, gamma oscillations are induced in the hippocampus, visual cortex, barrel cortex, auditory cortex, or their respective regions. Gamma oscillations are induced in combination. For example, in some embodiments, gamma oscillations are induced in the visual cortex using flashing 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.

[0063] In some embodiments, the subject receives stimuli through an environment configured to induce gamma oscillations, such as a chamber (e.g., light shielding or noise cancellation) that passively or actively blocks irrelevant stimuli. Alternatively, the subject may receive stimuli through a system that includes, for example, light shielding or noise cancellation aspects. In some embodiments, the subject receives visual stimuli through a stimulus-emitting device, such as eyeglasses designed to deliver stimuli. The device may block other light. In some embodiments, the subject receives auditory stimuli through a stimulus-emitting device, such as headphones designed to deliver stimuli. The device may cancel out other noise.

[0064] In addition to at least one stimulus emission interface, some embodiments may include at least one processor (e.g., for generating stimuli, controlling stimulus emission, monitoring stimulus emission / outcomes, and / or processing feedback on stimuli / outcomes), at least one memory (e.g., for storing processor-executable commands, at least one stimulus, stimulus generation strategies, feedback, and / or results, etc.), at least one communication interface (e.g., for exchanging information with subjects, healthcare providers, caregivers, principal investigators, databases, monitoring applications, etc.), and / or detection devices (e.g., for detecting and providing feedback on stimuli and / or subjects, including whether gamma oscillations have been induced, subject sensitivity, cognitive function, physical or chemical changes, stress, safety, etc.).

[0065] In some embodiments, gamma oscillations are induced by visual stimuli, such as flashing light in the range of approximately 20 Hz to 100 Hz. In certain embodiments, gamma oscillations are induced by flashing light in the range of approximately 20 Hz to 50 Hz. In further embodiments, gamma oscillations are induced by flashing light in the range of approximately 35 Hz to 45 Hz. In even further embodiments, gamma oscillations are induced by flashing light in the range of approximately 40 Hz. In some embodiments, the subject receives (or is placed in a chamber equipped with a light-shielding device that emits such flashing light, or wears a light-shielding device that emits such flashing light) flashing light in the range of approximately 20 Hz to 100 Hz, or flashing light in the range of approximately 20 Hz to 50 Hz, or flashing light in the range of approximately 35 Hz to 45 Hz, or flashing light in the range of approximately 40 Hz.

[0066] In some embodiments, gamma oscillations are induced by auditory stimuli such as sounds 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 (or is placed in a chamber equipped with a noise-canceling device that emits such auditory stimuli, or wears a noise-canceling device that emits such auditory stimuli).

[0067] In some embodiments, the subject receives visual and / or auditory stimuli for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours or more (e.g., being placed in a chamber equipped with a light-shielding device that emits such stimuli, or wearing a light-shielding device that emits such stimuli). In some embodiments, the subject receives stimuli for a period of about 6 hours or less, about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, or about 1 hour or less (e.g., (The subject is placed in a chamber equipped with a light-shielding device that emits such stimuli, or wears such a light-shielding device that emits such stimuli). In some embodiments, the subject receives the stimuli for a period of less than one hour (for example, by being placed in a chamber equipped with a light-shielding device that emits such stimuli, or by wearing such a light-shielding device that emits such stimuli).

[0068] In some embodiments, the subject receives the method provided herein. In other embodiments, the subject receives treatment using the method provided herein at multiple separate times. The subject may receive treatment on a regular schedule, or when symptoms occur or when symptoms worsen. In some embodiments, chronic treatment may be effective in reducing soluble Aβ peptides and / or insoluble Aβ peptides (i.e., plaques).

[0069] In some embodiments, gamma oscillations are induced in a cell-type-specific manner. In some embodiments, gamma oscillations are induced in FS-PV interneurons. When used to describe certain types of neurons, the term "fast-spiking" (FS) refers to the ability of neurons to discharge at high frequencies for extended periods with little to no adaptation of spike frequencies or attenuation of spike heights. Thus, these neurons can discharge at sustained, high frequencies (e.g., about 100 Hz or above about 150 Hz) without significant adaptation. This ability of FS neurons is largely due to the expression of their own fast-delay rectification channels, or in other words, the expression of channels that are activated and inactivated very rapidly.

[0070] 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 surgical intervention or bodily manipulation, 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).

[0071] In another embodiment, the stimulation may be invasive, or at least partially invasive. For example, visual, auditory, and / or tactile stimulation may be combined with the injection or transplantation of a composition (e.g., a photosensitive protein) or a device (e.g., an integrated fiber optic light source or a solid-state light source).

[0072] Experimental data

[0073] The experimental data related to the essence of the present invention as described herein, along with various drawings, are presented below. An overview is given first, followed by a detailed description.

[0074] Figure 1A shows the firing frequency adjustment of two estimated single units between 40 Hz auditory stimuli (left panel of each pair) and random stimuli (right panel of each pair) in AC. The scale represents auditory pulses. The bars of light represent randomly distributed pulses. The raster plot shows the spike response of two examples of estimated single units to 40 Hz auditory or random stimuli over 10 seconds.

[0075] Figure 1B shows the interval distribution between firing frequency peaks for all single units (n=292 units in 9 recording sessions with 5 mice; interval ratios centered on the interval between stimuli: P=0 40Hz vs. no stimulus, P=0 40Hz vs. random; z-test for the two ratios) for the conditions of no stimulus (labeled no stimulus), random stimulus (labeled random), and 40Hz auditory stimulus (labeled 40Hz stimulus) in AC.

[0076] Figure 1C shows an exemplary polar coordinate plot of firing frequency regulation compared to stimulus occurrence during a 40 Hz auditory stimulus (left, 0 = stimulus occurrence), vector intensity distribution of single-unit firing frequency regulation between 40 Hz auditory stimulus, random stimulus, and no stimulus (center, ****P<0.0001, P=4x10). -54 40Hz vs. no stimulation, P=2x10 -1340 Hz vs. random stimulation; Kolmogorov–Smirnov test), and Rayleigh statistic distribution of single-unit firing rate regulation (right, ****P < 0.0001, P = 5 x 10 -68 40 Hz vs. no stimulation, P = 6 x 10 -72 40 Hz vs. random stimulation; Kolmogorov–Smirnov test; 26 units had RS values for 40 Hz stimulation exceeding 30; 1 unit had a random stimulation RS value exceeding 30).

[0077] Figure 1D shows the distribution of mean firing rate values between stimulation conditions in AC.

[0078] Figure 1E shows the same content as Figure 1A for CA1.

[0079] Figure 1F shows the same content as Figure 1B for CA1 (in 10 recording sessions in 5 mice, n = 338 units. P = 0 40 Hz vs. no stimulation, P = 0 40 Hz vs. random stimulation; z-test for two proportions).

[0080] Figure 1G shows the same content as Figure 1C for CA1. (Center, ****P < 0.0001, P = 4 x 10 -40 40 Hz vs. no stimulation, P = 9 x 10 -11 40 Hz vs. random stimulation; Kolmogorov–Smirnov test; right, ****P < 0.0001, P = 1 x 10 -74 40 Hz vs. no stimulation, P = 2 x 10 -73 40 Hz vs. random stimulation; Kolmogorov–Smirnov test).

[0081] Figure 1H shows the same content as Figure 1D for CA1.

[0082] Figure 1I shows the same content as Figure 1A for mPFC.

[0083] Figure 1J shows the same content as Figure 1B for mPFC (in 7 recording sessions in 4 mice, n = 115 units. P = 0 40 Hz vs. no stimulation, P = 0 40 Hz vs. random stimulation; z-test for two proportions).

[0084] Regarding Figure 1K mPFC, the same content as in Figure 1C is shown. (Center, ****P<0.0001, P=2x10) -27 40Hz vs. no stimulation, P=4x10 -5 40Hz versus random stimulus; Kolmogorov-Smirnov test; right-handed, ****P<0.0001, P=1x10 -28 40Hz vs. no stimulation, P=5x10 -30 40Hz versus random stimulation; Kolmogorov-Smirnov test).

[0085] Figure 1L shows the same information as Figure 1D regarding mPFC.

[0086] Figure 2A shows the timeline of behavioral experiments on 5XFAD auditory GENUS mice.

[0087] Figure 2B shows the cognitive index of a novel object recognition (NOR) test in 5XFAD auditory GENUS mice (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; circles represent "n"; bar graphs show mean sem, ****P<0.0001, ns=no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0088] Figure 2C shows the average speed (cm / sec) during a novel object recognition test (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, and in the random frequency group). n=9, bar graph shows mean SEM, ns=no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0089] Figure 2D shows the total distance (cm) moved during a novel object recognition test (n=20 mice in the no-stimulus group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean SEM in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0090] Figure 2E shows the cognitive index of a novel object location (NOL) test in 5XFAD auditory GENUS mice (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean sem in the bar graph, ****P<0.0001, ns=no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0091] Figure 2F shows the average velocity (cm / sec) during a novel object position test (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean SEM in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0092] Figure 2G shows the total distance (cm) moved during a novel object position test (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean SEM in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0093] Figure 2H shows escape latency in a Morris water maze for 5XFAD mice treated with no stimulation, random frequency stimulation, and 40 Hz auditory stimulation (n=25 mice in the no-stimulation group, n=28 mice in the 40 Hz group, n=9 mice in the random frequency group; bar graphs show mean sem, ****P<0.0001, ns=no significant difference, two-way ANOVA with Tukey's multiple comparison test).

[0094] Figure 2I shows the time spent swimming in the target quarter circle during the probe trial (n=25 mice in the unstimulated group, n=28 mice in the 40Hz group, n=9 mice in the random frequency group; mean sem in the bar graph, *P<0.05, Kruskal-Wallis test with Dunn's multiple comparison test).

[0095] Figure 2J shows the number of platforms crossed during the probe trial (n=25 mice in the unstimulated group, n=28 mice in the 40Hz group, n=9 mice in the random frequency group; mean sem in the bar graph, **P<0.01, Kruskal-Wallis test with Dunn's multiple comparison test).

[0096] Figure 3A Relatively soluble Aβ in the auditory cortex (AC) and hippocampus (HPC) of 6-month-old 5XFAD mice after receiving auditory stimulation at 40 Hz, 8 Hz, 80 Hz, or random frequencies for 1 hour per day for 7 days. 1-42 The levels are shown. Normalized relative to the non-stimulated control (n=19 mice in the non-stimulated group, n=19 mice in the 40Hz group, n=4 mice in the 8Hz group, n=7 mice in the 80Hz group, n=6 mice in the random frequency group; mean SEM in the bar graph, ****P<0.0001, ns=no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0097] Figure 3B Insoluble Aβ 1-42 The same content as in Figure 3A is shown.

[0098] Figure 3C shows the results of immunohistochemical staining using anti-Aβ (D54D2, green) antibody in the AC of 6-month-old 5XFAD mice after receiving auditory GENUS stimulation for 1 hour per day for 7 days, or without stimulation (n=7 mice per group, scale bar, 50 μm).

[0099] Regarding Figure 3D CA1, the same content as Figure 3C is shown.

[0100] Figure 3E shows the mean number of Aβ-positive plaques in AC and CA1 (n=7 mice per group, mean sem in bar graph, *P<0.05, ****P<0.0001, independent two-group Mann-Whitney test).

[0101] Figure 3F shows the mean area of ​​Aβ-positive plaques in AC and CA1 (n=7 mice per group, mean sem in bar graph, **P<0.01, ***P<0.001, independent two-group Mann-Whitney test).

[0102] Figure 3G shows the results of immunohistochemical staining using anti-Aβ (12F4, red) antibody in the AC of 6-month-old 5XFAD mice after receiving auditory GENUS stimulation for 1 hour per day for 7 days, or without stimulation (insert, 20x, scale bar, 50 μm).

[0103] Regarding Figure 3H CA1, the same content as in Figure 3G is shown.

[0104] Figure 3I shows the mean intensity values ​​of Aβ(12F4) (12F4 antibody) normalized compared to the unstimulated control (n=7 mice per group, mean sem in the bar graph, ****P<0.0001, independent two-group Mann-Whitney test).

[0105] Figure 4A shows the results of immunohistochemical staining using anti-Iba1 (019-19741, green) and anti-Aβ (12F4, red) antibodies in the AC of 5XFAD mice after receiving auditory GENUS stimulation for 1 hour per day for 7 days, or without stimulation (n=8 mice per group, scale bar, 50 μm).

[0106] Regarding CA1 in Figure 4B, the same content as in Figure 4A is shown.

[0107] Figure 4C shows the number of Iba1-positive microglia in AC and CA1 (n=8 mice per group, mean sem in bar graph, *P<0.05, independent two-group Mann-Whitney test).

[0108] Figure 4D shows the diameter of Iba1-positive microglial cells in AC and CA1, normalized to the unstimulated control group (n=8 mice per group, mean sem in bar graph, ****P<0.0001, independent two-group Mann-Whitney test).

[0109] Figure 4E shows the mean lengths of major processes of Iba1-positive microglia in AC and CA1, normalized to the unstimulated control group (n=8 mice per group, mean sem in bar graph, ****P<0.0001, independent two-group Mann-Whitney test).

[0110] Figure 4F shows the mean process branching of Iba1-positive microglia in AC and CA1, normalized to the unstimulated control group (n=8 mice per group, mean sem in bar graph, P<0.05, **P<0.01, independent two-group Mann-Whitney test).

[0111] Figure 4G shows the proportion of microglial cells in AC and CA1 that are both Iba1-positive and Aβ-positive (n=8 mice per group, mean sem in bar graph, **P<0.01, ***P<0.001, independent two-group Mann-Whitney test).

[0112] Figure 4H shows the results of immunohistochemical staining using anti-S100B (ab868, purple) and anti-GFAP (ab4674, gray) antibodies in the AC of 5XFAD mice after receiving auditory GENUS stimulation for 1 hour per day for 7 days, or without stimulation (n=8 mice per group, scale bar, 50 μm).

[0113] Regarding Figure 4I CA1, the same content as in Figure 4H is shown.

[0114] Figure 4J shows the number of S100B-positive astrocytes in AC and CA1 (n=8 mice per group, *P<0.05, independent two-group Mann-Whitney test).

[0115] Figure 4K shows the same information as Figure 4J regarding GFAP-positive astrocytes.

[0116] Figure 5A shows the results of immunohistochemical staining using lectin staining (DL-1174, green) in the AC of 6-month-old 5XFAD mice after receiving auditory GENUS stimulation for 1 hour per day for 7 days, or without stimulation (scale bar, 50 μm).

[0117] Regarding Figure 5B CA1, the same content as in Figure 5A is shown.

[0118] Figure 5C shows the percentage change in AC and CA1 vascular diameter in 6-month-old 5XFAD mice after receiving auditory GENUS stimulation for 1 hour per day for 7 days, or without stimulation. Normalized to the unstimulated control (n=7 mice per group, mean sem in bar graph, ****P<0.0001, independent two-group Mann-Whitney test).

[0119] Figure 5D shows the results of immunohistochemical staining using anti-LRP1 (28320, red), anti-Aβ (AB9234, green), and lectin staining (DL-1174, gray) in the AC of 6-month-old 5XFAD mice after receiving auditory GENUS stimulation for 1 hour per day for 7 days, or without stimulation (n=8 mice per group, scale bar, 50 μm).

[0120] Regarding Figure 5E CA1, the same content as in Figure 5D is shown.

[0121] Figure 5F shows the proportion of Aβ-LRP1 colocalization in AC and CA1 of 5XFAD mice after receiving auditory GENUS stimulation for 1 hour per day for 7 days, or without stimulation (n=8 mice per group, *P<0.05, independent two-group Mann-Whitney test).

[0122] Figure 6A shows the modulation of single-unit firing frequency between 40 Hz auditory-visual stimuli (left, bottom). The raster plot shows the spike response of two examples of estimated single units to 40 Hz auditory stimuli or random stimuli for 10 seconds (left, top). Vector intensity distributions for 40 Hz auditory-visual stimuli, random auditory-visual stimuli, and no-stimulus periods (right, ****P<0.0001, P=9x10).-59 40Hz vs. no stimulation, P=1x10 -13 40Hz vs. randomized; Kolmogorov-Smirnov test).

[0123] Regarding CA1 in Figure 6B, the same content as in Figure 6A is shown. (Right, ****P<0.0001, P=6x10) -41 40Hz vs. no stimulation, P=2x10 -11 40Hz vs. randomized, Kolmogorov-Smirnov test).

[0124] Figure 6C shows the same information as Figure 6A regarding mPFC. (Right, ****P<0.0001, P=2x10) -23 40Hz vs. no stimulation, P=9x10 -5 40Hz vs. randomized, Kolmogorov-Smirnov test).

[0125] Figure 6D shows the results of immunohistochemical staining and 3D reconstruction using IMARIS (method) with anti-Iba1 antibody (019-19741) and anti-Aβ antibody (12F4) in AC, VC, CA1, and mPFC of 6-month-old 5XFAD mice that received 1 hour of stimulation per day for 7 days without stimulation (n=6 mice per group; top inset: Example of IMARIS use to quantify the number of microglia around a 25 μm radius centered on an amyloid plaque. Plaques are shown as red dots, microglia as green dots, and white arrows point to clusters. bottom inset: Enlarged integrated image of AC. Scale bar, 20 μm).

[0126] Regarding the combination GENUS in Figure 6E, it is shown as in Figure 6D.

[0127] Figure 6F shows the mean microglial cell body diameters in AC, VC, CA1, and mPFC of 6-month-old 5XFAD mice after receiving combined GENUS (A+V stimulation) for 1 hour per day for 7 days, or without stimulation, normalized to an unstimulated control (n=6 mice in the unstimulated control group, n=7 mice in the combined GENUS group, bar graph, mean sem, ****P<0.0001, independent two-group Mann-Whitney test).

[0128] Figure 6G shows the mean microglial process lengths in AC, VC, CA1, and mPFC of 6-month-old 5XFAD mice after receiving combined GENUS for 1 hour per day for 7 days, or without stimulation, normalized to an unstimulated control (n=6 mice in the unstimulated control group, n=7 mice in the combined GENUS group, mean sem in the bar graph, **P<0.01, ****P<0.0001, independent two-group Mann-Whitney test).

[0129] Figure 6H shows the number of microglia per unit area in AC, VC, CA1, and mPFC of 6-month-old 5XFAD mice after receiving combined GENUS for 1 hour per day for 7 days, or without stimulation (n=6 mice in the unstimulated control group, n=7 mice in the combined GENUS group, bar graph, mean sem, *P<0.05, **P<0.01, independent two-group Mann-Whitney test).

[0130] Figure 6I shows the mean number of microglia around a 25 μm radius of plaques in AC, VC, CA1, and mPFC after or without combined GENUS treatment (n=6 mice per group, bar graph, mean sem, ns=not significant, *P<0.05, independent two-group Mann-Whitney test).

[0131] Figure 7A shows the results of immunohistochemical staining of anti-Aβ plaque antibody (D54D2, green) in AC, VC, CA1, and mPFC of 6-month-old 5XFAD mice that received 1 hour of stimulation per day for 7 days without stimulation (images taken with a 40x objective lens, scale bar, 50 μm).

[0132] Regarding the combination GENUS in Figure 7B, it is shown as in Figure 7A.

[0133] Figure 7C. Normalized comparison of 6-month-old 5XFAD mice after receiving 1 hour per day for 7 days, no stimulation, 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)80 Hz, and combined (A+V) random frequency stimulation compared to a no-stimulation control. This shows the mean plaque core area in AC, CA1, mPFC, and VC of the mice (n=12 mice per group, bar graph, mean sem, ns=not significant, *P<0.05, Kruskal-Wallis test with Dunn's multiple comparison test).

[0134] Figure 7D shows the mean number of plaques in AC, CA1, mPFC, and VC of 6-month-old 5XFAD mice after receiving no stimulation, 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)80 Hz, and combined (A+V) random frequency stimulation for 7 days at 1 hour per day, normalized compared to a no-stimulation control (n=12 mice per group, in bar graph, mean sem, ns=not significant, *P<0.05, Kruskal-Wallis test with Dunn's multiple comparison test).

[0135] Figure 7E Relatively soluble Aβ in the mPFC of 6-month-old 5XFAD mice after receiving 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)8 Hz, or combined (A+V) random frequency stimulation for 7 days at 1 hour per day, normalized compared to a no-stimulation control. 1-42 Levels are shown (n=4-5 mice per group, bar graph, mean sem, ns=not significant, *P<0.05, Kruskal-Wallis test with Dunn's multiple comparison test).

[0136] Figure 7F Soluble Aβ 1-42 This is shown in Figure 7E (ns = not significant, *P < 0.05).

[0137] Figure 7G shows the results of immunohistochemical staining of anti-Aβ plaque (D54D2, white) antibody in the whole brain (sagittal plane of 25 μm brain sections) of SHIELD-treated 6-month-old 5XFAD mice after 7 days of no stimulation for 1 hour per day (light sheet microscope, scale bar, 700 μm).

[0138] Regarding the combination (A+V)GENUS in Figure 7H, it is shown as in Figure 7G.

[0139] Figure 7I shows the mean number of cortical plaques after no stimulation or combined (A+V)GENUS (n=6 mice per group, bar graph, mean sem, *P<0.05, independent two-group Mann-Whitney test).

[0140] Figure 7J Mean cortical plaque volume (μm³) after combination (A+V) GENUS 3 This shows (n=6 mice per group, bar graph, mean sem, *P<0.05, independent two-group Mann-Whitney test).

[0141] Figure 8A shows the average LFP response to auditory mapping tones used to detect the auditory cortex (left). The blue area indicates when a 50-millisecond mapping tone was played. An example of clustered estimation single units is shown (right).

[0142] Figure 8B shows the power spectral density (PSD) response to periods of 40 Hz auditory flashing stimulation and no stimulation. The mean and standard deviation for the entire recording day are shown (left), and the power spectral LFP response in AC to auditory flashing for all recording days is shown (showing the recording site with the highest 40 Hz peak during 40 Hz auditory flashing per recording depth; see Methods) (right).

[0143] Figure 8C shows the average firing rate (FR) of a single unit in AC during periods of auditory stimulation and no stimulation at 40 Hz (left). The difference in average firing rates between multiple stimulation conditions for a single unit in AC is concentrated around 0 Hz (right, ****P<0.0001 40 Hz-no stimulation, all others ns; Wilcoxon signed test for zero median).

[0144] Figure 8D shows the estimated single-unit firing frequency modulation in response to a 20 Hz auditory flashing stimulus (left, bottom). The raster plot shows the spikes in the response to the 10-second stimulus (left, top). Distribution of intervals between peaks in the firing frequency response to the 20 Hz auditory stimulus (right, ratio of intervals centered on the interval between stimuli: P=0 20 Hz vs. no stimulus; z-test for the two ratios).

[0145] Figure 8E shows the firing frequency regulation of the same unit as indicated by D in response to an 80 Hz auditory flashing stimulus (left, bottom). The raster plot shows the spikes in the response to the 10-second stimulus (left, top). Distribution of intervals between peaks in the firing frequency response to the 80 Hz auditory stimulus (right, ratio of intervals centered on the interval between stimuli: P=0 80 Hz vs. no stimulus; z-test for the two ratios).

[0146] Figure 8F shows the vector intensity distributions for auditory stimuli at 20 Hz and 80 Hz, and for the no-stimulation condition (left, ****P<0.0001, P=3x10). -61 20Hz vs. no stimulation, P=3x10 -61 Rayleigh statistical distribution (80Hz vs. no stimulus; Kolmogorov-Smirnov test), with auditory stimuli of 20Hz and 80Hz, and no stimulus (right, ****P<0.0001, P=3x10) -73 20Hz vs. no stimulation, P=1x10 -68 80Hz versus no stimulation; Kolmogorov-Smirnov test; 54 units had 20Hz stimulation RS values ​​greater than 30; 28 units had 80Hz stimulation RS values ​​greater than 30.

[0147] Figure 8G shows an example of theta rhythm, a characteristic of the hippocampus, used to detect CA1.

[0148] Regarding Figure 8H CA1, the same content as in B is shown.

[0149] Regarding Figure 8I CA1, the same content as C is shown (right, ns; Wilcoxon signed test for the zero median).

[0150] Regarding Figure 8J CA1, the same content as D is shown (right, P=0 20Hz vs no stimulation; z-test for the two ratios).

[0151] Regarding Figure 8K CA1, the same content as E is shown (right, P=0 80Hz vs. no stimulation; z-test for the two ratios).

[0152] Regarding Figure 8L CA1, the same content as F is shown. (Left, ****P<0.0001, P=1x10) -40 20Hz vs. no stimulation, P=9x10 -45 80Hz vs. no stimulation; Kolmogorov-Smirnov test; right, ****P<0.0001, P=1x10 -71 20Hz vs. no stimulation, P=8x10 -73 80Hz versus no stimulation; Kolmogorov-Smirnov test).

[0153] Figure 8M shows a tissue biopsy image illustrating the probe trace and the recording location within the mPFC. The red arrows indicate the recording location.

[0154] Regarding Figure 8N mPFC, the same content as in B is shown.

[0155] Regarding mPFC in Figure 8O, the same content as in C is shown (right, ns; Wilcoxon signed test for the zero median).

[0156] Regarding mPFC in Figure 8P, the same content as in D is shown (right, P=0 20Hz vs. no stimulation; (Z-test for two proportions).

[0157] Regarding mPFC in Figure 8Q, the same content as in E is shown (right, P=0 80Hz vs. no stimulation; z-test for the two ratios).

[0158] Regarding mPFC in Figure 8R, the same content as F is shown. (Left, ****P<0.0001, P=1x10) -23 20Hz vs. no stimulation, P=6x10 -24 80Hz vs. no stimulation; Kolmogorov-Smirnov test; right, ****P<0.0001, P=2x10 -17 20Hz vs. no stimulation, P=4x10 -26 80Hz versus no stimulation; Kolmogorov-Smirnov test).

[0159] Figure 9A shows the time (seconds) spent by 5XFAD mice stimulated with no stimulation, 40Hz frequency, and random frequency to explore familiar and novel objects during the NOR test (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; bar graphs show mean sem, **P<0.01, ****p<0.0001, ns=no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0160] Figure 9B shows the time (minutes) required for mice to reach the 20-second total object search requirement during the NOR test (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean sem in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0161] Figure 9C shows the time (seconds) spent by 5XFAD mice, stimulated with no stimulation, 40Hz frequency, and random frequency, to explore objects in familiar and novel locations during the NOL test (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; bar graphs show mean sem, ***P<0.001, ****p<0.0001, ns=no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0162] Figure 9D shows the time (minutes) required for mice to reach the 20-second total object search requirement during the NOL test (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean sem in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0163] Figure 9E shows the average speed (cm / sec) during habituation (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean SEM in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0164] Figure 9F shows the total distance (cm) traveled during habituation (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean SEM in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0165] Figure 9G shows the time (seconds) spent at the center of the behavioral chamber during habituation (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean SEM in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0166] Figure 9H shows the time (seconds) spent around the behavioral chamber during habituation (n=20 mice in the no-stimulation group, n=20 mice in the 40Hz group, n=9 mice in the random frequency group; mean sem in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0167] Figure 9I shows the average swimming speed (cm / sec) in the Morris water maze (n=25 mice in the no-stimulation group, n=28 mice in the 40Hz group, n=9 mice in the random frequency group; mean SEM in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0168] Figure 9J shows the mean velocity (cm / sec) during 1-hour periods of no stimulation, auditory GENUS, or random frequency stimulation (n=6 mice in the no-stimulation group, n=6 mice in the 40Hz group, n=6 mice in the random frequency group; mean sem in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0169] Figure 9K shows the total distance (cm) traveled during 1 hour of no stimulation, auditory GENUS, or random frequency stimulation (n=6 mice in the no stimulation group, n=6 mice in the 40Hz group, n=6 mice in the random frequency group; mean sem in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0170] Figure 9L shows the time (seconds) spent at 2 cm / sec during 1 hour of no stimulation, auditory GENUS, or random frequency stimulation (n=6 mice in the no stimulation group, n=6 mice in the 40 Hz group, n=6 mice in the random frequency group; mean sem in the bar graph, ns = no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0171] Figure 10A Relatively soluble Aβ in the auditory cortex (AC) and hippocampus (HPC) of 6-month-old 5XFAD mice after receiving auditory stimulation at 40 Hz, 8 Hz, 80 Hz, or random frequencies for 1 hour per day for 7 days. 1-40 The levels are shown. Normalized relative to the non-stimulated control (Note: ELISA for 80Hz and random frequency HPC samples was unsuccessful and could not be reported. n=19 mice in the non-stimulated group, n=19 mice in the 40Hz group, n=4 mice in the 8Hz group, n=7 mice in the 80Hz group, and n=6 mice in the random frequency group. Mean SEM in the bar graph, **P<0.01, ns=no significant difference, Kruskal-Wallis test with Dunn's multiple comparison test).

[0172] Figure 10B. Relatively soluble Aβ in the auditory cortex (AC) and hippocampus (HPC) of 6-month-old APP / PS1 mice after receiving auditory GENUS for 1 hour per day for 7 days. 1-42 The levels are shown. Normalized relative to the non-stimulation control (n=4 mice in the non-stimulation group, n=4 mice in the 40Hz group, mean SEM in the bar graph, *P<0.05, independent two-group Mann-Whitney test).

[0173] Figure 10C AC and CA1 in 9-month-old APP / PS1 mice after receiving auditory GENUS for 1 hour per day for 7 days ("Target area", ROI: region of The mean plaque count is shown in the interest group. Normalized relative to the non-stimulated control (n=5 mice in the non-stimulated group, n=5 mice in the 40Hz group, mean SEM in the bar graph, *P<0.05, independent two-group Mann-Whitney test).

[0174] Figure 10D shows the mean plaque core area in AC and CA1 in 9-month-old APP / PS1 mice after receiving auditory GENUS for 1 hour per day for 7 days. Normalized to non-stimulation control (n=5 mice in the non-stimulation group, n=5 mice in the 40Hz group, mean sem in bar graph, *P<0.05, independent two-group Mann-Whitney test). .

[0175] Figure 10E shows the average intensity values of Aβ (12F4) in the AC and CA1 of 9-month-old APP / PS1 mice after receiving auditory GENUS for 7 days at 1 hour per day. Normalized to the unstimulated control (n = 5 mice in the unstimulated group, n = 5 mice in the 40 Hz group, mean s.e.m. in the bar graph, *P < 0.05, Mann-Whitney test for two independent groups).

[0176] Figure 10F shows the average number of plaques in the AC and CA1 of 6-month-old 5XFAD mice after receiving auditory GENUS for 7 days at 1 hour per day and then 7 days without stimulation. Normalized to the unstimulated control (n = 6 mice in the unstimulated group, n = 6 mice in the 40 Hz group, mean s.e.m. in the bar graph, n.s. = no significant difference, Mann-Whitney test for two independent groups).

[0177] Figure 10G shows the average plaque core area in the AC and CA1 of 6-month-old 5XFAD mice after receiving auditory GENUS for 7 days at 1 hour per day and then 7 days without stimulation. Normalized to the unstimulated control (n = 6 mice in the unstimulated group, n = 6 mice in the 40 Hz group, mean s.e.m. in the bar graph, n.s. = no significant difference, Mann-Whitney test for two independent groups).

[0178] Figure 10H shows the average intensity values of Aβ (12F4) in the AC and CA1 of 6-month-old 5XFAD mice after receiving auditory GENUS for 7 days at 1 hour per day and then 7 days without stimulation. Normalized to the unstimulated control (n = 6 mice in the unstimulated group, n = 6 mice in the 40 Hz group, mean s.e.m. in the bar graph, n.s. = no significant difference, Mann-Whitney test for two independent groups).

[0179] Figure 11A shows the diameter of Iba1-positive microglial cells in AC and CA1 in 9-month-old APP / PS1 mice after receiving auditory GENUS for 1 hour per day for 7 days. Normalized to unstimulated controls (n=5 mice per group, mean sem in bar graph, *P<0.05, independent two-group Mann-Whitney test).

[0180] Figure 11B shows the mean lengths of Iba1-positive major protrusions in AC and CA1 in 9-month-old APP / PS1 mice after receiving auditory GENUS for 1 hour per day for 7 days. Normalized to non-stimulated controls (n=5 mice per group, mean sem in bar graph, *P<0.05, independent two-group Mann-Whitney test).

[0181] Figure 11C shows the number of Iba1-positive microglia in AC and CA1 in 9-month-old APP / PS1 mice after receiving auditory GENUS for 1 hour per day for 7 days. Normalized to unstimulated controls (n=5 mice per group, mean sem in bar graph, *P<0.05, independent two-group Mann-Whitney test).

[0182] Figure 11D shows the diameter of Iba1-positive microglial cells in AC and CA1 in 6-month-old 5XFAD mice after 7 days of auditory GENUS stimulation for 1 hour per day, followed by 7 days of no stimulation. Normalized to the unstimulated control (n=6 mice per group, mean sem in bar graph, ns=no significant difference, independent two-group Mann-Whitney test).

[0183] Figure 11E shows the mean length of major processes of Iba1-positive microglia in AC and CA1 in 6-month-old 5XFAD mice after 7 days of auditory GENUS stimulation for 1 hour per day, followed by 7 days of no stimulation. Normalized to the unstimulated control (n= per group). Six mice, bar graph shows mean SEM, ns = no significant difference, independent two-group Mann-Whitney test).

[0184] Figure 11F shows the number of Iba1-positive microglia in AC and CA1 in 6-month-old 5XFAD mice after 7 days of auditory GENUS stimulation for 1 hour per day, followed by 7 days of no stimulation. Normalized to the unstimulated control (n=6 mice per group, mean sem in bar graph, *P<0.05, ns=no significant difference, independent two-group Mann-Whitney test).

[0185] Figure 11G shows the results of immunohistochemical staining of CLARITY-treated brain sections in CA1 of 6-month-old wild-type and 5XFAD mice using anti-GFAP antibody (ab4674, red) and lectin-staining antibody (DL-1174, green) (n=5 mice per group, scale bar, 50 μm).

[0186] Figure 11H shows the number of GFAP-positive cells (number per control image, using IMARIS) in CA1 of 6-month-old wild-type and 5XFAD mice (n=5 mice per group, mean sem in bar graph, **P<0.01, independent two-group Mann-Whitney test).

[0187] Figure 12A shows the results of immunohistochemical staining of AC in 6-month-old P301S mice using anti-p-tau antibody (T181, red) for 7 days with 1 hour of stimulation per day, either without stimulation or after auditory GENUS stimulation (images taken with a 40x objective lens, scale bar, 50 μm).

[0188] Regarding CA1 in Figure 12B, the same content as in Figure 12A is shown.

[0189] Figure 12C shows the relative intensity levels of p-tau (T181) in AC and CA1 of P301S mice after 7 days of no stimulation or auditory GENUS stimulation at 1 hour per day, normalized to an unstimulated control (n=10 mice per group, ***P<0.001, ****P<0.0001, independent two-group Mann-Whitney test).

[0190] Figure 12D shows the results of immunohistochemical staining of AC in 6-month-old P301S mice using anti-p-tau antibody (S396, green) for 7 days with or without auditory GENUS stimulation at 1 hour per day (scale bar, 50 μm).

[0191] Figure 12E CA1 shows the same content as Figure 12D.

[0192] Figure 12F shows the relative intensity levels of p-tau (S396) in AC and CA1 of P301S mice after 7 days of no stimulation or auditory GENUS stimulation at 1 hour per day, normalized to an unstimulated control (n=10 mice per group, ****P<0.0001, independent two-group Mann-Whitney test).

[0193] Figure 12G shows representative Western blotting results indicating the levels of p-tau (S396) and total tau in AC of 6-month-old P301S mice after 7 days of no stimulation or auditory GENUS stimulation at 1 hour per day.

[0194] Figure 12H shows the same information as Figure 12G regarding the hippocampus.

[0195] Figure 12I shows the levels of p-tau (T181) and total tau in the hippocampus of 6-month-old P301S mice after 1 hour of stimulation per day for 7 days, either without stimulation or after auditory GENUS stimulation. The results of a typical Western blotting test are shown.

[0196] Figure 12J shows the relative immunoreactivity of p-tau (S396) normalized to total tau (from Western blotting of G) in P301S mice after 7 days of no stimulation or auditory GENUS stimulation at 1 hour per day (n=3 mice per group, bar graph, mean sem, *P<0.05, independent two-group Mann-Whitney test).

[0197] Figure 12K shows the relative immunoreactivity of p-tau (S396) normalized to total tau (from Western blotting of H) in the HPC of P301S mice after 7 days of 1 hour per day, with no stimulation or auditory GENUS stimulation (n = 2 mice in the 40 Hz group, n = 3 mice in the non-stimulated group, mean ± s.e.m. in the bar graph, ***P < 0.001, Mann-Whitney test for two independent groups).

[0198] Figure 12L shows the relative immunoreactivity of p-tau (T181) normalized to total tau (from Western blotting of I) in the hippocampus of P301S mice after 7 days of 1 hour per day, with no stimulation or auditory GENUS stimulation (n = 2 mice in the 40 Hz group, n = 3 mice in the non-stimulated group, mean ± s.e.m. in the bar graph, **P < 0.01, Mann-Whitney test for two independent groups).

[0199] Figure 13A shows the power spectral density (PSD) responses to 40 Hz auditory-visual flickering stimulation periods and non-stimulated periods. Mean and standard deviation over the entire recording day (left), power spectral LFP responses in the AC to 4 Hz auditory-visual flickering stimulation over all recording days (showing the recording site with the highest 40 Hz peak between 40 Hz auditory-visual flickers per recording depth. See methods) (right).

[0200] Figure 13B shows the following firing rate modulations of the putative single units shown in Figure 13A in response to auditory-visual random stimulation. The upper raster plot shows the spikes of a single unit in response to 10 s of random stimulation (left). Distribution of intervals between peaks in the firing rate response to auditory-visual stimulation in the AC (middle, proportion of intervals centered on the interval between stimuli: P = 0 for 40 Hz vs. no stimulation, P = 0 for 40 Hz vs. random stimulation; z-test for the two proportions), Rayleigh statistical distribution of the single unit response to 40 Hz auditory-visual stimulation (right, ****P < 0.0001, P = 2x10 -79 40 Hz vs. no stimulation, P = 1x10 -7240Hz versus random stimulation; Kolmogorov-Smirnov test; 20 units had 40Hz stimulus RS values ​​greater than 30. 2 units had random stimulus RS values ​​greater than 30.

[0201] Figure 13C shows the average firing frequency of a single unit under all auditory-visual stimulus conditions.

[0202] Figure 13D shows the estimated single-unit firing frequency modulation in response to a 20 Hz auditory-visual flashing stimulus (left, bottom). The raster plot shows the spikes in the response to the 10-second stimulus (left, top). Distribution of intervals between peaks in the firing frequency response to the 20 Hz auditory-visual stimulus (right, ratio of intervals centered on the interval between stimuli: P=0 20 Hz vs. no stimulus; z-test for the two ratios).

[0203] Figure 13E shows the firing frequency regulation of the same unit as shown in D in response to an 80 Hz auditory-visual flashing stimulus (left, bottom). The raster plot shows the spikes in the response to the 10-second stimulus (left, top). Distribution of intervals between peaks in the firing frequency response to the 80 Hz auditory-visual stimulus (right, ratio of intervals centered on the interval between stimuli: P=0 80 Hz vs. no stimulus; z-test for the two ratios).

[0204] Figure 13F shows that the vector intensity distribution of auditory stimuli at 20 Hz and 80 Hz is higher than that of the no-stimulation condition (left, ****P<0.0001, 2x10). -63 20Hz vs. no stimulation, P=1x10 -56 (80Hz vs. no stimulation; Kolmogorov-Smirnov test), the Rayleigh statistical distribution of auditory stimuli at 20Hz and 80Hz is higher than that of no stimulation (right, ****P<0.0001, P=P = 1x10⁻¹⁰). -88 20Hz vs. no stimulation, P=5x10 -72 80Hz versus no stimulation; Kolmogorov-Smirnov test; 50 units had 20Hz stimulation RS values ​​greater than 30; 19 units had 80Hz stimulation RS values ​​greater than 30.

[0205] Figure 13G shows that the difference in the average firing frequency of a single unit in AC across multiple stimulus conditions is concentrated around 0 Hz (*P<0.05 20Hz-80Hz, *P<0.05 20Hz-40Hz, all others ns; Wilcoxon signed test for zero median).

[0206] Regarding Figure 13H CA1, the same content as A is shown.

[0207] Regarding Figure 13I CA1, the same content as B is shown (right, ****P<0.0001, P=1×10 -71 40Hz vs. no stimulation, P=1x10 -71 40Hz vs. random stimuli; Kolmogorov-Smirnov test. Center, the ratio of intervals between stimuli: P=0 40Hz vs. no stimulation, P=0; 40Hz vs. random stimulation; z-test for the two proportions).

[0208] Regarding Figure 13J CA1, the same content as C is shown.

[0209] Regarding Figure 13K CA1, the same content as in D is shown (right, P=0 20Hz vs no stimulation; z-test for the two ratios).

[0210] Regarding Figure 13L CA1, the same content as E is shown (right, P=0 80Hz vs. no stimulation; z-test for the two ratios).

[0211] Regarding Figure 13M CA1, the same content as F is shown. (Left, ****P<0.0001, P=8x10) -43 20Hz vs. no stimulation, P=8x10 -40 80Hz vs. no stimulation; Kolmogorov-Smirnov test; right, ****P<0.0001, P=2x10 -70 20Hz vs. no stimulation, P=1x10 -57 80Hz versus no stimulation; Kolmogorov-Smirnov test).

[0212] Regarding Figure 13N CA1, the same content as in G is shown (all ns; Wilcoxon signed test for the zero median).

[0213] Regarding mPFC in Figure 13O, the same content as in A is shown.

[0214] Regarding mPFC in Figure 13P, the same content as B is shown (right, ****P<0.0001, P=5x10). -23 40Hz vs. no stimulation, P=3x10 -21 40Hz vs. random stimulation; Kolmogorov-Smirnov test. Proportion of intervals centered on the interval between stimuli: P=0. 40Hz vs. no stimulation, P=0. 40Hz vs. random stimulation; z-test for the two proportions).

[0215] Regarding mPFC in Figure 13Q, the same content as C is shown.

[0216] Regarding mPFC in Figure 13R, the same content as in D is shown (right, P=0 20Hz vs. no stimulation). (Z-test for two proportions).

[0217] Regarding mPFC in Figure 13S, the same content as in E is shown (right, P=0 80Hz vs. no stimulation; z-test for the two ratios).

[0218] Regarding Figure 13T mPFC, the same content as F is shown. (Left, ****P<0.0001, P=1x10) -23 20Hz vs. no stimulation, P=2x10 -25 80Hz vs. no stimulation; Kolmogorov-Smirnov test; right, ****P<0.0001, P=1x10 -23 20Hz vs. no stimulation, P=8x10 -25 80Hz versus no stimulation; Kolmogorov-Smirnov test).

[0219] Regarding mPFC in Figure 13U, the same content as in G is shown (*P<0.05 40Hz-no stimulation, all others ns; Wilcoxon signed test for zero median).

[0220] Figure 14A shows the results of immunohistochemical staining with anti-Iba1 (019-19741, green) and anti-Aβ (12F4, red) antibodies in AC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of auditory GENUS stimulation for 1 hour per day (insert plot magnification, 100x; scale bar, 50 μm).

[0221] Figure 14B shows the results of immunohistochemical staining with anti-Iba1 (019-19741, green) and anti-Aβ (12F4, red) antibodies in VC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of 1 hour of visual GENUS stimulation per day (insert plot magnification, 100x; scale bar, 50 μm).

[0222] Figure 14C shows the mean microglial cell body diameters in AC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of auditory GENUS at 1 hour per day, normalized to an unstimulated control (n=6 mice per group, bar graph, mean sem, ns=no significant difference, **P<0.01, independent two-group Mann-Whitney test).

[0223] Figure 14D shows the mean microglial process lengths in AC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of auditory GENUS at 1 hour per day, normalized to an unstimulated control (n=6 mice per group, bar graph, mean sem, ns=no significant difference, **P<0.01, independent two-group Mann-Whitney test).

[0224] Figure 14E shows the number of microglia per unit area in AC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of auditory GENUS at 1 hour per day (n=6 mice per group, bar graph, mean sem, ns=no significant difference, **P<0.01, independent two-group Mann-Whitney test).

[0225] Figure 14F shows the mean number of microglia around a 25 μm radius of plaques in AC, CA1, and mPFC after or without auditory GENUS (n=6 mice per group, bar graph, mean sem, ns=not significant, independent two-group Mann-Whitney test).

[0226] Figure 14G shows the mean microglial cell body diameters in VC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of visual GENUS at 1 hour per day, normalized to an unstimulated control (n=6 mice per group, bar graph, mean sem, ns=no significant difference, *P<0.05, independent two-group Mann-Whitney test).

[0227] Figure 14H Visual GEN 1 hour per day for 7 days, normalized to no-stimulus control. This shows the mean microglial process lengths in VC, CA1, and mPFC of 6-month-old 5XFAD mice after US (n=6 mice per group, bar graph, mean sem, ns=no significant difference, *P<0.05, independent two-group Mann-Whitney test).

[0228] Figure 14I shows the number of microglia per unit area in VC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of visual GENUS at 1 hour per day (n=6 mice per group, bar graph, mean sem, ns=no significant difference, independent Mann-Whitney test of two groups).

[0229] Figure 14J shows the mean number of microglia around a 25 μm radius of plaques in VC, CA1, and mPFC after or without visual GENUS stimulation (n=6 mice per group, bar graph, mean sem, ns=not significant, independent two-group Mann-Whitney test).

[0230] Figure 14K shows the mean microglial cell body diameters in AC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)80 Hz, or combined (A+V) random frequency stimulation, normalized to a no-stimulation control (n=6 mice per group, bar graph, mean sem, ns=no significant difference, *P<0.05, **P<0.01, independent two-group Mann-Whitney test).

[0231] Figure 14L shows the mean microglial process lengths in AC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)80 Hz, or combined (A+V) random frequency stimulation at 1 hour per day, normalized compared to a no-stimulation control (n=6 mice per group, bar graph, mean sem, ns=no significant difference, *P<0.05, independent two-group Mann-Whitney test).

[0232] Figure 14M shows the number of microglia per unit area in AC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)80 Hz, or combined (A+V) random frequency stimulation, normalized compared to a no-stimulation control (n=6 mice per group, bar graph, mean sem, ns=no significant difference, *P<0.05, independent two-group Mann-Whitney test).

[0233] Figure 14N shows the results of immunohistochemical staining of anti-Aβ plaque (D54D2, green) antibodies in AC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of auditory GENUS at 1 hour per day (n=6 mice per group, scale bar, 50 μm).

[0234] Figure 140 shows the results of immunohistochemical staining of anti-Aβ plaque (D54D2, green) antibodies in VC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of visual GENUS at 1 hour per day (n=6 mice per group, scale bar, 50 μm).

[0235] Figure 14P shows the mean plaque core area per target region, normalized compared to the unstimulated control (n=6 animals per group, bar graph, mean sem, ns=no significant difference, *P<0.05, independent two-group Mann-Whitney test).

[0236] Figure 14Q shows the mean number of plaques in AC, CA1, and mPFC after auditory GENUS, normalized compared to the unstimulated control (n=6 animals per group, bar graph, mean sem, ns=no significant difference, *P<0.05, ***P<0.001, independent 2-group man- Whitney Houston quiz.

[0237] Figure 14R shows the mean plaque core area per target region, normalized compared to the unstimulated control (n=6 animals per group, bar graph, mean sem, ns=no significant difference, *P<0.05, independent two-group Mann-Whitney test).

[0238] Figure 14S shows the mean number of plaques in VC, CA1, and mPFC after visual GENUS, normalized to the unstimulated control (n=6 animals per group, bar graph, mean sem, ns=no significant difference, *P<0.05, independent two-group Mann-Whitney test).

[0239] Figure 14T Relative soluble Aβ in AC and HPC of 6-month-old 5XFAD mice after receiving 7 days of 1 hour of stimulation per day, normalized to a no-stimulation control, with no stimulation, 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)8 Hz, and combined (A+V) random frequency stimulation. 1-42Levels are shown (n=4-5 mice per group, bar graph, mean sem, ns=not significant, *P<0.05, Kruskal-Wallis test with Dunn's multiple comparison test).

[0240] Figure 14U Relative insoluble Aβ in AC and HPC of 6-month-old 5XFAD mice after receiving no stimulation, 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)8 Hz, and combined (A+V) random frequency stimulation for 7 days at 1 hour per day, normalized compared to a no-stimulation control. 1-42 Levels are shown (n=4-5 mice per group, bar graph, mean sem, ns=not significant, *P<0.05, Kruskal-Wallis test with Dunn's multiple comparison test).

[0241] Figure 14V shows the mean Aβ(12F4) intensity values ​​(12F4 antibody) in AC, CA1, and mPFC of 6-month-old 5XFAD mice after receiving no stimulation, 40 Hz auditory stimulation, combined (A+V)GENUS, combined (A+V)80 Hz, and combined (A+V) random frequency stimulation for 7 days at 1 hour per day, normalized compared to a no-stimulation control (n=4-5 mice per group, bar graph, mean sem, ns=not significant, *P<0.05, Kruskal-Wallis test with Dunn's multiple comparison test).

[0242] 40Hz auditory stimulation modulates spike activity in the AC, CA1, and mPFC.

[0243] The inventors first determined whether auditory tonal stimuli could induce GENUS in the auditory cortex (AC), hippocampal microregion CA1, and medial prefrontal cortex (mPFC). The inventors presented animals with tonal sequences of 20 Hz, 40 Hz, and 80 Hz, or randomly distributed sequences (10 kHz tones with a length of 1 millisecond played every 12.5 milliseconds, 25 milliseconds, and 50 milliseconds, or at random intervals of an average of 25 milliseconds between tones; hereinafter referred to as "auditory flashing stimuli"). During tonal presentation, the inventors performed electrophysiological recordings in the AC, CA1, and mPFC of 3-8 month old male wild-type (C57BL6J) mice running and resting on a spherical treadmill using a 32-channel silicon probe. To locate the AC, a series of 50-millisecond auditory mapping tones (hereinafter referred to as "mapping stimuli") were played back at various depths until a transient LFP response was detected around 20 milliseconds from the start of the tone (Figure 8A). CA1 was positioned based on electrophysiological features, and the mPFC recording location was confirmed from tissue structure after the last recording was made in each animal (Figures 8G and 8M).

[0244] After reaching the target area, the animals were alternately presented with periods of silence and auditory flashing stimuli while neural activity was recorded. These stimuli were at 20Hz, 40Hz, 80Hz, and random auditory flashing stimuli. The stimulus blocks cycled. The estimated firing frequency of a single unit periodically increased and decreased with each tone, tuning to the 40 Hz auditory flashing stimulus (Figures 1A, 1E, and 1I, blue). The unit was also regulated by random stimuli. When all random pulses were aligned, a change occurred in post-stimulus firing frequency regulation. This suggests that the single unit responded to random stimulus pulses. However, random auditory tone sequences did not induce periodic firing regulation (Figures 1A, 1E, and 1I, orange). Tuning varied between single units in both phase distribution and amplitude. During the flashing stimulus, neurons fired in response to the stimulus, but not in every cycle, often firing across a wide range of phases (Figures 1A, 1E, and 1I). The interval between peak firing frequency during the auditory flashing stimulus was approximately 25 milliseconds for most of the single units (equivalent to 40 Hz); 75% for AC, 79% for CA1, and 74% for mPFC. (Figures 1B, 1F, and 1J).

[0245] In contrast, during the baseline period without tone and during the period of random tone, the intervals between peaks had a broad distribution, with less than 11% of cells in AC, less than 12% of cells in CA1, and less than 16% of cells in mPFC having peak intervals of approximately 25 milliseconds (i.e., firing frequency was not modulated at 40 Hz; Figures 1B, 1F, and 1J). Intensity modification was quantified by examining the single-unit firing frequency in response to the stimulus phase and calculating its vector intensity (VS) (Figures 1C, 1G, and 1K, left). The vector intensity value ranges from 0 to 1, where 0 represents a uniform firing distribution that is not modulated by the stimulus (VS=0), and 1 represents a distribution where neurons fire only in response to a specific stimulus phase (VS=1). The vector intensity distribution of single-unit responses to 40 Hz auditory stimuli ranged from 0.002 to 1 for AC, 0.0005 to 1 for HPC, and 0.1 to 0.6 for PFC, all significantly higher than the no-stimulus and random-stimulus conditions (Figures 1C, 1G, and 1K, center). Since vector intensity measures stimulus-induced modulation, the random-stimulus vector intensity was also significantly higher than the no-stimulus condition. However, vector intensity does not quantify the periodicity of modulation. Random stimulation elicited single-unit responses but did not induce periodic firing modulation.

[0246] Similarly, the Rayleigh statistical distribution for single units during 40 Hz auditory stimulation was significantly higher than in the no-stimulation and random-stimulation controls (Figure 1C, 1G, and 1K, right). The mean firing frequency of single neurons was similar between 40 Hz auditory flashing stimulation and no-stimulation controls, random-stimulation, and 20 Hz and 80 Hz auditory flashing stimulation (Figure 1D, 1H, and 1L; Figure 8C, 1I, and 1O, right). Local electric field potentials in AC showed high power at 40 Hz during auditory flashing stimulation, but this effect varied between recording location, recording session, and response latency to mapping tones (Figure 8B, 1H, and 1N). These findings suggest that 40 Hz auditory flashing stimulation reliably induced GENUS in AC, CA1, and mPFC.

[0247] Auditory GENUS improves memory in 5XFAD mice.

[0248] We hypothesized that auditory GENUS could influence hippocampal neuronal activity and then evaluated its effects on hippocampus-dependent learning and memory in 6-month-old 5XFAD mice (Figure 2A). We used 6-month-old 5XFAD mice, which is when behavioral impairments first become apparent. For all of the following experiments, we performed a one-week auditory GENUS regimen: specifically, the mice were placed in a quiet chamber and exposed to a 10 kHz auditory tone sequence of 1 milliseconds in length at a frequency of 40 Hz (i.e., 40 (10 kHz) tones / second) for 1 hour / day for 7 days. We began by acclimatizing the mice to the behavioral chamber, and 24 hours later, we administered novel object recognition (NOR) and novel object position (NOL) memory tests. These assess the ability to remember the identity and position of objects under specific circumstances and are behaviors known to be affected in human AD subjects. These tests measure behavioral ability using cognitive indices. This index Each of these represents the ratio of the time spent searching for new objects or objects in new locations to the total time spent searching for them.

[0249] During habituation, none of the auditory GENUS group, random frequency group, or non-stimulation group showed significant changes in average speed, total distance, time spent in the center, or time spent in the periphery, suggesting that these three groups did not show differences in general activity or anxiety-like behavior (Figures 9E-9H). After auditory GENUS, 5XFAD mice showed significantly higher cognitive indices of 65.50±1.40% in the object task and 61.41±2.0% in the spatial memory task. On the other hand, the non-stimulation control group and the random frequency control group showed no preference for either novel or newly moved objects in the two tests (Figures 2B and 2E). There were no significant differences in distance traveled or average speed during the task period among these three groups. This suggests that these effects were not due to general differences in activity (Figures 2C, 2D, 2F, and 2G).

[0250] The amount of time spent exploring new and familiar objects during the No Object Search (NOR) was examined. Mice after auditory GENUS spent significantly more time exploring new objects, while the non-stimulation control group and the random frequency control group did not exhibit an exploration preference (Figure 9A). Similarly, mice after auditory GENUS spent significantly more time exploring objects in new locations (NOL), while the non-stimulation control group and the random frequency control group did not exhibit an exploration preference (Figure 9C). As an additional control measure to examine differences in exploration activity, the inventors measured the amount of time (minutes) that mice spent during the object task to reach the 20-second object exploration requirement. The inventors observed no significant difference in the time taken to reach the object exploration requirement among these three groups (Figures 9B and 9D).

[0251] To further analyze the effects of auditory GENUS on hippocampus-dependent behavior, the inventors conducted the Morris Water Maze Test. This test measures the ability to remember the location of a hidden platform in association with ambient cues. Mice gradually learned the location of the hidden platform through successive trials, and spatial memory of the platform location was measured by escape latency. Escape latency is the amount of time it took an individual mouse to find the hidden platform. During the training phase, all three groups successfully learned the location of the hidden platform. However, the escape latency of the auditory GENUS-treated groups was consistently and significantly shorter than that of both the non-stimulation control group and the random frequency control group (Figure 2H). There were no significant differences in swimming speed among the three groups (Figure 9I). During the probe trial, or when the hidden platform was removed from the tank, auditory GENUS-treated mice spent significantly longer searching the quadrant where the missing platform had been and exhibited a higher number of crossovers over the previous platform location compared to both the non-stimulation control group and the random frequency control group (Figures 2I and 2J).

[0252] As a final behavioral measurement, the inventors examined the activity of 5XFAD mice during 1 hour of auditory GENUS, in a non-stimulated state, or with random frequency stimulation, and observed no significant differences in average speed (cm / sec) or distance traveled (cm) (Figures 9J and 9K). To investigate whether there were differences in "sleep" states or rest periods, the inventors measured the amount of time mice spent at less than 2 cm / sec among the 1 hour stimulation groups. The inventors observed no significant differences among these three groups (Figure 9L). In summary, these results suggest that auditory GENUS may improve cognitive and spatial memory in 6-month-old 5XFAD mice.

[0253] Auditory GENUS reduces amyloid loading in the AC and hippocampus of 5XFAD mice.

[0254] Since auditory GENUS had beneficial effects on cognitive function, the inventors of this invention... We decided to investigate whether the underlying amyloid lesions in 5XFAD mice could be modified. Previously, we have shown the improvement effect of visual GENUS on amyloid loading in younger 3-month-old mice. This specification aims to study in detail the effect of auditory GENUS on 6-month-old mice, which are in a more advanced stage of AD and exhibit greater amyloid plaque loading. Mice were placed in a quiet chamber and exposed to a series of different auditory tonal frequencies, including 40 Hz, 8 Hz, 80 Hz, and random frequency stimulation, or were left unstimulated. 24 hours after the completion of 7 days of stimulation, we analyzed amyloid loading in the AC and whole hippocampus (HPC) by enzyme-linked immunosorbent assay (ELISA). After 40 Hz auditory stimulation, we compared soluble Aβ compared to unstimulated controls or additional frequency controls. 1-42 The levels decreased to 51.48±4.98% in AC and 46.89±3.89% in HPC, while the soluble Aβ1-40 levels in AC and HPC decreased to 20.65%, respectively. We confirmed a decrease to ±3.21% and 34.15±4.83% (Figures 3A and 10A). Similarly, insoluble Aβ 1-42 The levels decreased to 36.68±3.21% in AC and 43.84±2.42% in HPC (Figure 3B). Insoluble Aβ 1-40 It could not be detected by ELISA in either the auditory GENUS or the unstimulated control.

[0255] Our results indicate that the observed reduction in amyloid was specific to 40 Hz stimulation, and that Aβ levels did not change significantly with 8 Hz, 80 Hz, or random frequency stimulation compared to the unstimulated control. To determine whether these effects are applicable to other AD mouse models, and to determine whether our results are specific to the 5XFAD model, we examined Aβ levels in 6-month-old APP / PS1 transgenic mice after 7 days of auditory GENUS. This mouse is a proven AD model. We examined soluble Aβ 1-42 We found that the levels were significantly reduced compared to the unstimulated control group by 48.39±3.50% in AC and 35.54±4.27% in HPC (Figure 10B).

[0256] Next, the inventors examined plaque load in the 5XFAD mouse model using immunohistochemical analysis with a β-amyloid-specific antibody (Cell Signaling Technology; D54D2) (Figures 3C and 3D). Compared to the unstimulated control, the number of plaques was significantly reduced to 45.73±2.818% and 59.30±2.083% in AC and CA1 mice, respectively, after 7 days of auditory GENUS (Figure 3E). The size of the plaques was also significantly reduced to 54.37±5.603% and 40.70±5.321% in AC and CA1 mice, respectively (Figure 3F). 1-42 Specific immunohistochemical analysis revealed Aβ concentrations of 45.35±0.011% and 43.21±0.0285% for AC and CA1, respectively. 1-42A significant reduction in deposition was suggested (Figures 3G-3I). To investigate the dynamics of plaque load after 40Hz stimulation, the inventors used a β-amyloid-specific antibody (Cell Immunohistochemical staining using Signaling Technology (D54D2) was performed on 5XFAD mice. These mice were initially given auditory GENUS for one week, followed by seven days of no stimulation. A slight decrease in average plaque number, plaque area, and amyloid intensity was observed, but the difference was not statistically significant (see Figures 10F-10H). To investigate plaque load after auditory GENUS in another AD model, the inventors used 9-month-old APP / PS1 mice. 9-month-old mice were used because 6-month-old mice do not exhibit significant plaque expression.

[0257] A significant decrease in plaque number was observed, from 52.65±7.53% in AC and 62.90±15.5% in CA1. Plaque size also significantly decreased, from 67.90±6.18% in AC and 64.06±15.2% in CA1. 1-42 Aβ using antibody (BioLegend; 12F4) 1-42 Specific immunohistochemical analysis showed that Aβ was superior in AC and CA1, respectively, compared to the unstimulated control. 1-42 Deposition: 38.77±4.21% and 4 A significant reduction of 7.63 ± 6.08% was suggested (Figures 10C-10E). In summary, these results demonstrate that auditory GENUS can synchronize gamma activity in AC and CA1 and reduce amyloid loading in an AD mouse model.

[0258] Auditory GENUS induces glial and vascular responses in 5XFAD mice.

[0259] Evidence is accumulating that microglia are responsible for changes in neuronal activity and play some role in AD lesions (Allen and Barres, 2005; Mosher and Wyss-Coray, 2014; Walker and Lue, 2015). Given the ability of AC and HPC to reduce amyloid loading, we investigated whether auditory GENUS could stimulate changes in microglial responses in 6-month-old 5XFAD mice. Microglia have been shown to change their cellular morphology during activation, including phagocytosis (Davies et al., 2016), and indeed, our previous research demonstrated that 1 hour of visual GENUS was sufficient to induce microglial morphological changes consistent with VC activation and increased phagocytic activity (Iaccarino et al., 2016). Using antibodies against the microglial marker Iba1 (Figures 4A and 4B), approximately 60% more microglia were observed in both AC and CA1 in the auditory GENUS group compared to unstimulated controls (Figure 4C). Compared to the unstimulated control, after auditory GENUS, the microglial cell body area increased to 70.60±4.78% in AC and 117.17±10.4% in CA1 (Figure 4D). Furthermore, compared to the unstimulated control, the inventors found that the microglial process length decreased to 46.44±3.2% (AC) and 50.875±4.8% (CA1), and the process branching increased to 36.00±9.5% (AC) and 143.813±29.9% (CA1) (Figures 4E and 4F). To evaluate Aβ uptake by microglia, the inventors used Iba1 antibody and Aβ 1-42 The co-localization of Aβ within microglia was measured by co-immunostaining of tissue sections using an Aβ-specific antibody (12F4, see Methods). The percentage of microglia in which microglial cell bodies co-localized with Aβ increased to 58.75±1.25% in AC and 61.33±3.71% in CA1 after auditory GENUS compared to the unstimulated control (Figure 4G).

[0260] To investigate whether a microglial response occurs after auditory GENUS in other AD mouse models, we measured microglial morphology in 9-month-old APP / PS1 mice after 7 days of auditory GENUS. Similar to the results observed in 5XFAD microglia after 7 days of auditory GENUS (see Figures 4A-4G), we observed a significant increase in microglial cell body diameter and number, as well as a significant decrease in mean process length, in AC and CA1 compared to unstimulated controls (Figures 11A-11C).

[0261] To understand the long-term effects of the microglial response in 5XFAD mice after auditory GENUS, we examined the morphology of microglia after a 7-day unstimulated period following a 1-week auditory GENUS. A trend similar to that observed in amyloid (Figures 10F-10H) was observed. Specifically, there was a non-significant increase in microglial cell body diameter, a decrease in mean process length, and an increase in microglia number in the auditory cortex (see Figures 11D-11F). However, compared to the unstimulated control, a significant increase in the number of microglia in CA1 was observed (41.70 ± 6.75%).

[0262] Astrocytes are another major glial cell type in the central nervous system and are essential for maintaining homeostasis, synaptic pruning, waste clearance, and other important biological processes such as regulating cerebral blood flow (Chung et al., 2015, Kisler et al., 2017). Reactive-like astrocytes produce glial cell fibrillary acidic protein (GFAP). (Eng et al., 1971). To investigate whether there was a baseline change in the number of reactive astrocytes between 6-month-old 5XFAD mice and WT littermate control mice, the inventors performed CLARITY on 100 μm CA1 brain sections and stained them with GFAP antibody (Figure 11G). 5XFAD mice were observed to have significantly fewer GFAP-positive astrocytes compared to WT controls (Figure 11H). This result is consistent with reports that other AD transgenic mouse models exhibit similar glial cell dysfunction (Rodriguez et al., 2009). To determine if auditory GENUS can affect astrocyte reactivity, we administered either 1 hour / day auditory GENUS or a non-stimulation control to 6-month-old 5XFAD mice for 7 days, and then immunostained brain sections with antibodies against GFAP and S100 calcium-binding protein B (S100B), another protein shown to be expressed in reactive-like astrocytes (Figures 4H and 4I). GFAP-positive astrocytes increased to 27.66±0.954% and 18.14±0.799% in AC and CA1, respectively. S100B-positive astrocytes increased to 21.83±1.07% in AC and 15.57±0.869% in CA1 (Figures 4J and 4K). This change observed in astrocyte count after auditory GENUS indicates a potential increase in astrocyte viability.

[0263] Astrocytes are known to play a crucial role in regulating the brain's vascular network, and there is accumulating evidence suggesting that dysfunction of this network in Alzheimer's disease can worsen the disease. Amyloid clearance from the brain is multifaceted, and various processes mediated by the vascular system, such as through the glial-lymphatic system and through transport by endocytotic receptor lipoprotein receptor-related protein 1 (LRP1), have been proposed.

[0264] To investigate potential changes in the vascular system, we first stained 5XFAD brain sections after auditory GENUS using tomato lectin (Lycopersicon esculentum), an efficient marker of vascular endothelium (Figures 5A and 5B). Interestingly, we observed increases in vascular diameter of 49.70±7.80% (AC) and 104.70±10.96% (CA1) after auditory GENUS compared to an unstimulated control (Figure 5C). Furthermore, we explored whether amyloid-vascular interactions change after auditory GENUS. We investigated whether auditory GENUS could affect the co-localization of LRP1 and Aβ by staining LRP1 and Aβ in brain sections of 5XFAD mice exposed to auditory GENUS for 7 days at 1 hour / day, or unstimulated mice (Figures 5D and 5E). LRP1 has been shown to play a crucial role in the transport and systemic excretion of Aβ via the vascular system (Storck et al., 2016). In the unstimulated control group, co-localization of Aβ and LRP1 was observed at 8.17±2.70% (AC) and 6.97±1.73% (CA1). On the other hand, in the auditory GENUS group, the co-localization of Aβ and LRP1 was significantly increased to 17.71±2.78% and 16.50±3.90% in AC and CA1, respectively (Figure 5F). In summary, these results suggest that one possible explanation for the decrease in Aβ levels in AC and CA1 after auditory GENUS is increased Aβ clearance via microglia and changes in the vascular system.

[0265] Auditory GENUS reduces tau phosphorylation in the AC and hippocampus.

[0266] Another classic pathological feature of AD is the accumulation of phosphorylated tau aggregates. Phosphorylation of tau at specific amino acid residues associated with AD has been shown to alter its cytoskeletal support function and reduce its solubility, and this has been proposed to be a major cause of neuronal damage. To investigate whether auditory GENUS could influence the lesions of another AD-related mouse model, we used tau P301S mice. Tau P301S mice were 6 months old. As mice begin to exhibit spatial and contextual learning deficits with age, the inventors investigated whether auditory GENUS could lead to a decrease in phosphorylated tau in AC and HPC of 6-month-old Tau P301S mice. Immunohistochemical staining of brain sections from these mice (Figures 12A, 12B, 12D, and 12E) showed that auditory GENUS reduced tau phosphorylation to 36.20±2.828% (AC) and 38.70±2.737% (CA1) with threonine-181 (T181), and to 37.90±3.469% (AC) and 40.80±4.528% (CA1) with serine-396 (S396) (Figures 12C and 12F). Western blotting (WB) experiments confirmed immunohistochemical staining results for tau phosphorylation at S396, showing a decrease in phosphorylation of 33.83±0.20% and 43.20±1.50% in AC and whole hippocampal tissue compared to total tau, respectively (Figures 12G, 12H, 12J, and 12K). WB analysis showed a 34.50±1.61% decrease in phosphorylated T181 tau in the hippocampus, but the difference was not significant in AC (Figures 12I and 12L). In summary, these results suggest that auditory GENUS may reduce the level of AD-related hyperphosphorylated epitopes and that auditory GENUS may influence lesions in a tauopathy mouse model.

[0267] This study demonstrates that the combination of auditory and visual GENUS induces a cluster-forming phenotypic response by microglia.

[0268] To date, our research has shown that auditory GENUS can reduce amyloid levels and induce glial and vascular changes in the cortical sensory area and hippocampus. Therefore, we decided to investigate whether a combination of auditory and visual GENUS could elicit a greater cellular effect. We first determined whether a combination of 40 Hz auditory tone stimulation and 40 Hz flashing light could synchronize neuronal responses in the AC, CA1, and mPFC. We recorded neuronal activity in the AC, CA1, and mPFC of 3-8 month old male wild-type (C57BL6J) mice while they ran or rested on a spherical treadmill, using a 32-channel silicon probe while presenting 1-millisecond auditory tones and 12.5-millisecond light pulses at a frequency of 40 Hz. The spikes periodically increased and decreased during each tone and flashing period, synchronizing to 40 Hz during the auditory-visual stimulus combination (Figures 6A-6C, left). The vector intensity distribution was significantly higher during 40 Hz auditory-visual stimulation than during random stimulation or no stimulation conditions (Figures 6A-6C, right). Therefore, single neuronal spikes in AC, CA1, and mPFC were more significantly tuned to 40 Hz during auditory-visual stimulation than during the baseline period. Local electric field potentials in AC, HPC, and mPFC showed high power at 40 Hz during auditory-visual flashing stimulation, but the effect was very small in mPFC (Figures 13A, 13H, and 13O). Thus, 40 Hz tone and light stimulation induced GENUS in AC, CA1, and mPFC.

[0269] Small differences were observed in the mPFC between auditory stimuli and auditory-visual stimulus combinations in local electric field potential (LFP) responses and single-unit mean firing frequency. A slight increase in LFP power was observed at 40 Hz during combined stimuli, but not during auditory-only stimuli (Figures 8N and 6O). Furthermore, the distribution of mean firing frequency differences between combined stimuli and the baseline had a median significantly different from zero, but not significantly different from zero for auditory-only stimuli (Figures 8O and 6U).

[0270] After 7 days of combined GENUS at 1 hour / day, the inventors examined the morphological characteristics of microglia and their interaction with Aβ in AC, VC, and CA1 (Figures 6D and 6E). Since higher cognitive regions are known to process complex patterns of sensory stimuli, the inventors investigated whether combined GENUS could similarly induce a microglial effect in the medial prefrontal cortex (mPFC). Microglia were compared to unstimulated controls in terms of somatic cell area. A significant increase was observed in the soma area, but the process length was found to be significantly decreased (Figures 6F and 6G). Microglia also significantly increased in AC, VC, CA1, and mPFC after the combined GENUS (Figure 6H). Microglia in the auditory or visual stimulation single-target groups (Figures 14A-14D, 14G, and 14H) showed a decrease in process length and an increase in somatic cell area in AC, VC, and CA1, but not in mPFC.

[0271] In contrast to visual GENUS, auditory GENUS showed a significant increase in microglia count in CA1. However, neither auditory nor visual GENUS alone induced a significant change in microglia count in the mPFC (Figures 14E and 14I). These findings suggest that, after one week of GENUS, only the combination of auditory and visual stimulation, and not auditory or visual stimulation alone, promoted the microglial response in the mPFC.

[0272] Interestingly, microglia in the combined GENUS group appeared to exhibit altered activity by creating an inclusion effect surrounding amyloid deposits. To further elucidate the microglia-Aβ clustering phenotype, we generated three-dimensional (3D) renderings from AC, VC, CA1, and mPFC images taken from 5XFAD brain sections after combined GENUS and from an unstimulated control (shown in the "3D Reconstruction" column of Figures 6D and 6E). Using IMARIS imaging software (see Methods), we generated 3D surfaces of amyloid deposits (red spots) and microglial cells (green spots) and quantified the proximity and number of microglia within a 25 μm radius of the amyloid deposits (rightmost inset, Figures 6D and 6E; video examples showing the microglia-Aβ clustering phenotype after combined GENUS and from an unstimulated control are presented in Additional Videos 1 and 2). Compared to unstimulated controls, significant increases were observed in the number of microglia surrounding a 25 μm radius around amyloid plaques after combined GENUS: 48.88±0.651% in AC, 31.56±1.11% in VC, and 38.64±0.959% in mPFC (Figure 6I). A non-significant increase of 33.05±2.65% was also observed in CA1. To verify whether the microglia-Aβ clustering phenotype is specific to combined GENUS, the inventors analyzed the number of microglia within a 25 μm radius of amyloid deposition after auditory GENUS alone or visual GENUS alone. No significant difference in the number of microglia per plaque was observed between GENUS mice and unstimulated mice (Figures 14F and 14J).

[0273] Next, the inventors addressed the frequency specificity of microglial responses in 6-month-old 5XFAD mice after 7 days of 40 Hz auditory GENUS, combined GENUS, 80 Hz, or random frequency stimulation in the AC, CA1, and mPFC. Compared to additional frequencies and unstimulated controls, a significant increase in microglial cell body diameter and number, as well as a significant decrease in mean process length, was observed in the AC and CA1 after 40 Hz auditory stimulation and combined GENUS.

[0274] Compared to 40 Hz auditory stimulation, additional frequencies, and a no-stimulation control, only combined GENUS induced a microglial response in the mPFC (Figure 14K-14M). These results indicate that combined GENUS enhances the microglial response through changes in neuronal activity. Therefore, we conclude that combined GENUS induces an extended microglial cluster formation response in the AC, VC, and mPFC.

[0275] Rather than auditory or visual stimulation alone, the combined use of auditory and visual genus reduces amyloid loading in the mPFC.

[0276] Based on research results on microglial reactions in AC, VC, CA1, and mPFC, we investigated whether combined GENUS could also alter amyloid levels in these regions 7 days after 1 hour of stimulation. Immunohistochemical analysis using anti-Aβ antibody (D54D2) showed a reduction in plaque area (56.34±6.35% in AC, 71.50±6.51% in VC, and 69.73±6.48% in CA1) and number (50.02±3.74% in AC, 50.60±10.9% in VC, and 48.80±11.1% in CA1) after combined GENUS compared to the unstimulated control. Surprisingly, these results showed a 59.64±8.71% reduction in plaque size and a twofold reduction in plaque number in the mPFC in the combined GENUS group compared to the unstimulated control (Figures 7A-7D). Neither auditory GENUS alone nor visual GENUS alone could induce a reduction in amyloid plaque staining in mPFCs, suggesting that the response was specific to the combination GENUS (Figure 14N-14S). Visual GENUS did not show any change in plaque size or number in CA1. Both auditory GENUS alone and combination GENUS treatment reduced soluble Aβ as measured by Aβ-ELISA in AC and HPC. 1-42 and insoluble Aβ 1-42This showed a decrease in amyloid levels. On the other hand, combined random flashing, 8Hz, or 80Hz stimulation did not have a significant effect on amyloid levels in AC or HPC (Figures 14T and 14U).

[0277] Next, 6-month-old 5XFAD mice were treated with sensory stimuli of various frequencies to determine whether the reduction in Aβ in the mPFCs was type-specific (comparison of auditory stimulation alone and combinations) or frequency-specific. Using Aβ-ELISA, changes in amyloid levels in the mPFCs were measured, revealing differences in soluble or insoluble Aβ between combined 8Hz and 40Hz auditory stimuli, combined random frequency stimulation, and no stimulation. 1-42 No significant difference was observed. In contrast, the combined GENUS group showed higher levels of soluble Aβ in the mPFC compared to the unstimulated group. 1-42 A decrease of 59.58 ± 7.26%, and insoluble Aβ 1-42 This showed a decrease of 34.17 ± 8.20% (Figures 7E and 7F).

[0278] Furthermore, plaque loading was measured in 6-month-old 5XFAD mice after 7 days of 40 Hz auditory GENUS, combined GENUS, 80 Hz, or random frequency stimulation using immunohistochemical analysis with a β-amyloid-specific antibody (Cell Signaling Technology; D54D2). A significant reduction in mean plaque number was observed in AC and CA1 after 40 Hz auditory stimulation and combined GENUS. However, only combined GENUS resulted in a significant reduction in plaque number in mPFCs compared to the additional frequency control and the unstimulated control (Figures 7C and 7D). 1-42 Aβ using antibodies 1-42 Specific immunohistochemical staining analysis showed a significant decrease in Aβ in AC and CA1 after 40 Hz auditory stimulation and combined GENUS. However, only combined GENUS resulted in a significant decrease in immunohistochemical intensity in mPFCs (Figure 14V).

[0279] The reduction in amyloid loading in the mPFC suggests that combined GENUS affects a wider range of cortical regions. To determine the overall effect of combined GENUS on the total amyloid plaque abundance in the cortex, we performed whole-brain SHIELD treatment (method) on 6-month-old 5XFAD mice after one week of combined GENUS, and immunostained amyloid plaques (using D54D2 antibody) (Figures 7G and 7H). Using a light-sheet microscope, 3D analysis of the plaques revealed a 37% and 34% reduction in total plaque volume and number in the neocortex compared to the unstimulated control (Figures 7I and 7J; video examples showing 3D whole-brain SHIELD samples with immunostained plaques after combined GENUS and from the unstimulated control are presented in additional videos 3 and 4). In summary, these results suggest that combined GENUS significantly reduces the total amyloid plaque loading in the neocortex of the 5XFAD mouse model. This is shown to be the case.

[0280] method

[0281] animal

[0282] All animal studies were approved by the Animal Experimentation Committee of the Department of Comparative Medicine, Massachusetts Institute of Technology, and the Animal Experimentation Committee of Georgia Institute of Technology. Mice were housed in groups of five or fewer in a standard 12-hour light / 12-hour dark cycle. All experiments were conducted during the light cycle. Electrophysiological experiments were conducted at Georgia Institute of Technology, and male (1-3 months old) wild-type mice (C57Bl / 6) were obtained from the Jackson laboratory. Mice were housed in a reverse 12-hour light / 12-hour dark cycle, and all experiments were conducted during the dark cycle. Food and water were provided without restriction.

[0283] Surgical procedure

[0284] All surgeries were performed as described in Iaccarino and Singer et al., 2016. Briefly, adult mice (2-3 months old) were anesthetized with isoflurane prior to the headplate placement surgery. The custom stainless steel headplate was fixed using dental cement (C&B Metabond, Perkel), and the target craniotomy sites for LFP recording were marked on the skull (in mm, from the anterior apex (bregma): -2.0 anterior / posterior, ±1.8 medial / lateral for the CA1 target, -2.0 to -3.0 anterior / posterior, ±1.8 medial / lateral for the auditory cortex target, and +1.3 to +1.4 anterior / posterior, ±1.0 medial / lateral for the prefrontal cortex target). Craniotomy was subsequently performed on mice 3-8 months old. Before the first recording session, a craniotomy opening (200-500 μm in diameter) was created by thinning the skull using a dental drill and drilling holes with a 27-gauge needle. If not recorded, sterile silicone elastomer (Kwik-Sil) was used. The craniotomy site was sealed with WPI (Whole Body Injection).

[0285] Electrophysiological Records

[0286] During recording, animals with their heads fixed ran on an airborne 8-inch (approximately 20.3 cm) spherical treadmill. All animals had already learned to move on the treadmill until comfortable, with occasional doses of sweetened condensed milk (1:2 water dilution). Animals remained on the ball for up to 5 hours, with multiple periods of running and resting. A single-shank 32-channel probe (NeuroNexus) was advanced to the target location. The recording area extended to 250 μm. For auditory cortical recordings, the probe was advanced along the frontal plane at a 45-degree angle from the vertical line to a depth of 3–4.15 mm. A series of 50-millisecond tones at 5, 10, 15, and 20 kHz were presented, and auditory responses at the average LFP were detected. For CA1 recordings, the probe was advanced vertically through the craniotomy area to a depth of 1.14–2.05 mm until electrophysiological features of the pyramidal layer of the hippocampus (large theta waves and sharp ripples, 150+ μV spikes on multiple channels) were observed. For prefrontal cortex recordings, the probe was advanced at a 49-degree angle from the frontal plane and a 20-degree angle from the vertical line to a depth of 1.48–2.15 mm. When data was collected at multiple depths during the same recording session, the new depths were mapped to confirm that the recording site remained at the target location (for AC, n=9 depths were recorded in 9 sessions with 5 mice; for CA1, n=12 depths were recorded in 10 sessions with 5 mice; for mPFC, n=7 depths were recorded in 7 sessions with 4 mice). Data were analyzed using the Intan RHD2000 Evaluation System with ground pellets as reference values ​​for 20k The data was acquired at a sampling rate of Hz.

[0287] Auditory and visual stimuli for electrophysiological recording

[0288] Animals were presented with alternating 10-second stimulus blocks and 10-second baseline periods. Stimulus blocks consisted of auditory stimuli only (20 Hz, 40 Hz, 80 Hz, or randomized stimulation frequencies; pulses were delivered at 25-millisecond average intervals, with randomized pulse intervals determined from a uniform distribution) or a combination of auditory and visual stimuli. The stimulus blocks were alternated to ensure that observations were not due to temporal changes in neuronal responses. All auditory pulses were 1 millisecond in length and 10 kHz in pitch. All visual pulses were 50% load cycles of the stimulation frequency (25 milliseconds, 12.5 milliseconds, or 6.25 milliseconds in length). For combined stimuli, auditory and visual pulses were aligned to the start of each pulse.

[0289] Data acquisition

[0290] The data was acquired at a sampling rate of 20 kHz using the Intan RHD2000 Evaluation System.

[0291] Spike detection

[0292] The unprocessed trace was band-pass filtered between 300 and 6,000 Hz. Then, spikes were detected using a threshold (median / 0.675) obtained by adding 5 times the estimated standard deviation to the median of the filtered signal.

[0293] Spike sorting and single-unit stability

[0294] Spike detection and sorting were performed using MountainSort automated spike sorting, followed by manual curation based on visual inspection of waveforms and cross-correlograms. Prior to manual curation, a quality threshold was applied, and only units with a peak SNR of 1 or greater, less than 10% noise overlap, and more than 95% isolation from other units, forming cleanly isolated single units, were included. To account for unstable periods where single units disappear during recording, a stability criterion was applied, and only stable periods (no sudden disappearance of single unit firing frequency) were considered in the analysis. The firing frequency (FR) for each unit was computer-calculated during the recording session. The firing frequencies were clustered into two distributions, low FR and high FR, using k-means clustering. Units with FR below 10% of the high FR mean were further analyzed to identify stable recording periods defined as the longest period where the FR was 2 standard deviations higher than the low FR mean.

[0295] Local electric field potential

[0296] The LFP was acquired by downsampling the unprocessed trace to 2kHz and applying a bandpass filter from 1 to 300Hz.

[0297] Recording area for analysis

[0298] AC and CA1 data were analyzed across multiple channels. For AC, the lower 16 of the 32 channels were used over a 375 μm range. The lowest channel of the probe was used to determine the location of AC, while the higher 16 channels were not considered the primary target region. The determination was made. For CA1, all functional channels on the probe were analyzed up to 250 μm (27 / 32 or 31 / 32). For both AC and CA1, the highest channel on the probe was used as the probe reference for power spectral analysis. Similar results were obtained using ground as the reference.

[0299] Histological examination of the prefrontal cortex

[0300] During the final mPFC recording in each animal, the probe was coated with Dil and inserted to the target depth. Mice were anesthetized (isoflurane) and transcardiac perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS), and the brains were post-fixed overnight with 4% paraformaldehyde in 1x PBS. The brains were sectioned to a thickness of 100 μm using a Leica VT1000S vibratome (Leica). The sections were stained with 0.2% 1 mMol DAPI in 1x PBS and mounted on microscope slides using Vectashield specimen medium. Images were acquired on a Zeiss Axio Observer Z1 inverted epifluorescence microscope with Zen Blue 2 software.

[0301] Power Spectrum

[0302] Power spectral density analysis was performed using the multitaper method in the Chronux toolbox (time-band product = 3, taper number = 5). LFP traces were divided into 10-second trials for each stimulus condition. The average power spectral density was computer-calculated for each animal across these trials, relative to a ground pellet in saline on the skull (within the same recording day and recording depth). Initially, power spectral density analysis was calculated for all recording sites of AC, CA1, and mPFC. From each recording depth, traces with the largest 40Hz peak in response to a 40Hz flashing stimulus were included in the analysis. Traces per depth shown in the presented data are those with the largest 40Hz peak in response to the auditory flashing stimulus.

[0303] Ignition during flashing stimulation

[0304] For each stimulus frequency, the single-unit peri-stimulus time histogram (PSTH) contains four stimulus cycles.

number

number

[0305] Average ignition frequency

[0306] For each stimulus condition, the average firing frequency of each single unit was calculated by computer. For each unit, only the stable period contributed to the calculation of the average firing frequency (see spike sorting and single-unit stability above). The difference in average firing frequencies between stimulus conditions was calculated within each unit by taking the difference in average firing frequencies for each condition of that unit.

[0307] 40Hz visual flashing stimulus protocol

[0308] For biochemical and immunohistochemical analysis, 5XFAD mice were placed in a dark chamber, illuminated with a light-emitting diode (LED) bulb, and exposed to one of the following four stimulation conditions: darkness, 8 Hz, 40 Hz (12.5 ms on, 12.5 ms off, 60 W), or random (light pulses were given at random intervals determined by a uniform distribution with an average of 25 ms) for 1 hour for 7 days.

[0309] 40Hz auditory sound series stimulation protocol

[0310] For biochemical, immunohistochemical, or behavioral analysis, 5XFAD, APP / PS1, or P301S mice were placed in a dimly lit chamber in a quiet room, soundproofed with sound-insulating foam (McMaster-Carr, 5692T49). A speaker (AYL, AC-48073) was placed out of reach of the mice in the chamber. The mice were exposed to one of five stimulus conditions: no tone, 8 Hz tone, 40 Hz tone, 80 Hz tone, or randomly delivered tone (auditory tones were delivered at random intervals determined by a uniform distribution with an average of 25 milliseconds). The stimulus condition-like tone consisted of a 10 kHz tone delivered at 60 dB for a duration of 1 millisecond. For electrophysiological recording, after probe placement, the room lights were turned off, and the animals were presented with alternating 10-second auditory-only stimuli and visual-auditory stimuli, interleaved with 10-second periods of no light or sound. For the auditory-only stimuli, a 10kHz tone was played at 40Hz with a 4% load cycle. For the auditory-visual stimuli, the auditory stimulus was accompanied by ambient light flashing at 40Hz for 10 seconds with a 50% load cycle. The stimuli were presented in this manner for 20-minute sessions, with 1-10 minute pauses between sessions during which the animals' behavior was checked.

[0311] Protocol of parallel 40Hz auditory and visual stimuli

[0312] For biochemical, immunohistochemical, or behavioral analysis, 5XFAD mice were placed in a dark chamber illuminated by an LED light and simultaneously exposed to auditory tones. Mice were exposed to one of four stimuli: darkness / stillness, flashing light at 40 Hz, auditory tones at 40 Hz, flashing light and auditory tones at 40 Hz in parallel, or random flashing light / tone stimulation.

[0313] Immunohistochemistry

[0314] Mice were anesthetized (2:1 ketamine / xylazine) and transcardiac perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were post-fixed overnight in 4% paraformaldehyde in PBS. The brains were sectioned to a thickness of 40 μm using a Leica VT1000S vibratome (Leica). The sections were permeabilized and blocked at room temperature for 2 hours in PBS containing 0.3% Triton X-100 and 10% donkey serum. The sections were incubated overnight at 4°C in PBS containing 0.3% Triton X-100 and 10% donkey serum, along with the primary antibody. The primary antibodies were as follows: anti-β-amyloid (Cell Signaling Technology; D54D2), anti-Iba1 (Wako Chemicals; 019-19741), anti-glial cell fibrous acidic protein (GFAP) (Abcam; ab4674), anti-S100B (Abcam; ab868), anti-LRP1 (Abcam; 28320), DyLight 488-labeled Lycopersicon Esculentum (tomato) lectin (Vector Laboratories; DL-1174), anti-amyloid oligomer (Millipore Sigma; AB9234), anti-phosphotau (Ser396) (Cell Signaling Technology; 9632), anti-phosphotau (Thr181) (Cell Signaling Technology; 12885), Hoechst 33342 (Thermo Fisher). Scientific (H3570). The reason anti-Aβ antibody 12F4 was used is that it does not react with APP. This allows for determination of whether the label is Aβ-specific, and enables co-labeling with Iba1. The anti-amyloid oligomer antibody AB9234 was used for co-labeling with LRP1. The following day, brain sections were incubated with a fluorescently conjugated secondary antibody (Jackson ImmunoResearch) at room temperature for 2 hours, and the nuclei were stained with Hoechst 33342 (Invitrogen). Images were acquired using a confocal microscope (LSM 710; Zeiss) with the same settings and a 40x objective lens for all conditions.Images were quantified using ImageJ 1.42q by blinded experimenters for each treatment group. Two coronal sections from each animal were used for quantification for each experimental condition. Unless otherwise noted in the figure captions, scale bars represent 50 μm. ImageJ was used to measure the diameter of Iba1+ cell bodies, and processes were traced for length measurement. The Coloc2 plugin was used to measure the colocalization of Iba1 and Aβ. Microglia process branching was quantified using Imarisx64 8.1.2 (Bitplane, Zurich, Switzerland). The "Particle Analysis" function in ImageJ was used to count the number and area of ​​plaques. Depositions of at least 10 μm were included, and a set threshold was used for both the control and experimental groups.

[0315] Vasculature-Aβ colocalization analysis

[0316] The ImarisColoc module was used to quantify the signal co-localization between two separate source channels (i.e., lectin and AB, and lectin and LRP1) in 3D. Thresholds were set on these source channels to mask intensities arising from noise or background signals. ImarisColoc then created a new channel containing only voxels co-localized within the thresholded source channels, which was presented for relevant statistical analysis.

[0317] Microglia-Aβ cluster formation analysis

[0318] Using IMARIS, we analyzed the clustering pattern of microglia around amyloid plaques in 40 μM sections. Using the Surface module, we detected plaques (red) based on the 12F4 signal and rendered them in 3D. Next, Iba1-positive microglia were counted using the Spots module, and spheres were placed on the cell bodies of each cell (green). Finally, we ran Spots Close To Surface XTension. A subset of spots closer to the surface object than the specified 25 μM threshold was identified, and spots outside this range were excluded. The algorithm measures the distance from the center of the spot to the nearest point on the surface object in 3D space. This allows for the quantification of microglia aggregation near the plaque.

[0319] Clarity immunohistochemical staining in brain sections

[0320] Mice were perfused with ice-cold PBS (1X), followed by perfusion with ice-cold 1X PBS solution of 4% PFA and 1% glutaraldehyde. The brains were dissected in 4% PFA / 1% glutaraldehyde solution and post-fixed at 4°C for 72 hours. Fixation was terminated by incubating the brains in an inactivation solution (1X PBS solution of 4% acrylamide, 1M glycine, and 0.1% Triton-X100) at room temperature for 48 hours. After washing with 1X PBS, the brains were cut into 100 μM coronal sections on a vibratome (Leica VT100S) in 1X PBS. Sections containing the target regions (i.e., the auditory cortex and hippocampus) were selected with reference to the Allen Mouse Brain Atlas and incubated in clearing buffer (pH 8.5-9.0, 200 mM sodium dodecyl sulfate, 20 mM lithium hydroxide monohydrate, 4 mM boric acid in ddH2O solution) for 2-4 hours with shaking at 55°C. The cleaned sections were washed in 1xPBST (0.1% Triton-X100 / 1xPBS) for 3 x 15 minutes and left overnight in blocking solution (2% bovine serum albumin / 1xPBST) at RT. Subsequently, three 1-hour washes in 1xPBST were performed with shaking at RT. The sections were incubated in weakly bound buffer (pH 8.5-9.0, 37.75 mM Na2HPO4, 3.53 mM KH2PO4, 0.02% sodium azide PBST solution) for 1 hour at RT, and then transferred to primary antibody diluted 1:100 in 1x weakly bound buffer at 37°C for 12 hours. Reversal buffer (pH 7.4, 37.75 mM Na2HPO4, 3.53 mM KH2PO4, 0.02% sodium azide PBST solution) was then added in fractional amounts every hour for 6 hours until the tissue volume equaled the volume of primary antibody solution added. A separate set of 3 x 1 hour washes was performed in 1x PBST, and then the sections were incubated at RT for 12 hours using Hoechst. The sections were incubated in a 1x PBS mixture of 33258 (1:250) (Sigma-Aldrich, 94403) and a secondary antibody (1:100). The sections were then washed overnight in 1x PBS and incubated at RT for 1 hour in RIMS (Refractive Index Matching Solution: 75g Histodenz, 20mL of 0.1M phosphate buffer, 60mL of ddH2O) before mounting. Brain sections were mounted on microscope slides using coverslips while in RIMS (VWR VistaVision, VWR International, LLC, Radnor, Pennsylvania).

[0321] Images were acquired on a Zeiss LSM 880 microscope using the included Zen Black 2.1 software (Carl Zeiss Microscopy, Jena, Germany). Z-stack images were captured with a 0.4–0.5 μm step size, 4.1 ms pixel dwell, average 2, and a resolution of 1024x1024, suitable for 3D reconstruction. Imarisx64 8.3.1 (Bitplane, Zurich, Switzerland) was used for 3D rendering and analysis.

[0322] Processing and clearing of the entire mouse brain

[0323] Brains of 5XFAD mice were treated according to the SHIELD protocol. Briefly, 5XFAD mice were perfused transcardiacly with ice-cold PBS, followed by perfusion with 20 mL of 4% PFA-containing SHIELD-OFF solution. The brains were dissected and post-fixed in the same solution at 4°C for 24 hours. The brains were then left overnight in a PFA-free SHIELD-OFF solution. The brains were incubated in OFF solution at 4°C. Then, they were incubated in SHIELD-ON solution at 37°C for 24 hours. After fixation, the brains were incubated in a clearing aqueous solution containing 200 mM sodium dodecyl sulfate (SDS), 20 mM lithium hydroxide monohydrate, 40 mM boric acid, and pH 8.5–9.0. The brains were then cleared for several days using SmartClear Pro (LifeCanvas Technologies, Cambridge, Massachusetts) based on probabilistic electrotransport (Kim et al., PNAS, 2015) until clear.

[0324] Immunostaining of the cleared hemisphere

[0325] The cleared hemispheres were stained over two days using the modified probabilistic electrotransport method eTANGO (Kim et al., PNAS, 2015) with 15 µl of β-amyloid antibody (CST, #51374) conjugated with Alexa Fluor-488.

[0326] Light sheet microscope

[0327] Immunostained samples were incubated with hProtos (105 ml DI aqueous solution of 3 g diatrizoic acid, 5 g N-methyl-d-glutamine, and 125 g iohexol) and optically cleared, then mounted in acrylic holders using 2% low-melting-point agarose in hProtos. A custom-made light-sheet microscope equipped with a 10x clarity-optimized objective lens was used to image the entire hemisphere, using 488 channels for beta-amyloid visualization and 647 channels for autofluorescence.

[0328] Image processing of the cleared whole brain, plaque detection, and atlas alignment.

[0329] The acquired image data was light-corrected using CIDRE, open-source software implemented in Matlab. The resulting processed images were stitched together using Terastitcher in Imaris (trademark) (Bitplane (registered trademark)) and used for 3D visualization. 2D visualization of representative sections was performed using ImageJ (National Institutes of Health). Automated plaque detection was performed using a combination of open-source ClearMap software, a custom cell classification neuron network model, and Elastix. Candidate plaques were placed as "spots" using ClearMap's spot detection module. First, background subtraction was performed section by section using a grayscale morphological top-hat deformation with disk structure elements having outer and inner pixel sizes of (21,21). Next, local maximums of the data were detected by applying a 3D maximum filter with disk structure elements of size (7,7,4). These local maximums were filtered with an intensity threshold of 100. The pixel capacity corresponding to the center position of each spot is also calculated using 3D watershed deformation with the spot center as the seed point. Subsequently, all plaque candidates with a volume smaller than a sphere with a diameter of 10 microns were excluded. True plaques were identified from candidate plaques using a convolutional neural network (CNN) model as a classification plaque / non-plaque classifier implemented in Keras® with the Theano® backend. The CNN input is a 32x32 pixel bounding box centered on the candidate plaque center, and the output is a two-element one-hot vector representing the plaque and non-plaque categories. The architecture consists of 12 total convolution layers, each rectified line (ReLU) The process involves activation of the ar unit (ar unit) followed by batch normalization: three 64 2x2 kernels, three 128 2x2 kernels, followed by three 192 2x2 kernels, one 256 2x2 kernel, one 256 1x1 kernel, and one 2 1x1 kernel. 2x2 subsampling is performed after the third, sixth, and ninth superposition layers. Dropout with a ratio of 0.5 is applied after the last nine superposition / batch normalization layers for regularization. After the final superposition layer, global average pooling and subsequent softmax activation are applied to generate the final categorical vector. During training, cross-category entropy loss was used along with the Adam optimization tool with default parameters. The CNN was trained over 400 epochs, including 64 batch sizes, on approximately 10,000 manually annotated plaques augmented with random rotation, shear, and inversion using Keras® Image Data Generator. The resulting model was then used to classify plaques from detected spots for all samples. To perform atlas alignment, autofluorescent channel images were first downsampled to atlas resolution, and then affine and B-spline transformation parameters were calculated using Elastix to perform 3D image registration using the resampled autofluorescent images as fixed images and the atlas as moving images. The resulting alignment parameters were applied to the plaque locations (output from the CNN model) to transform the plaques into atlas space. A CSV file (segmented according to the Allen Brain Atlas) containing information on the number and quantity of plaques for each brain region was then generated.

[0330] Western blot

[0331] The hippocampus and auditory cortex were dissected from 6-month-old male 5XFAD mice, and lysates were prepared. The tissue was then mixed with 1 ml of RIPA (50 mM Tris HCl, pH 8.0, 150 mM). The lysate was homogenized using a hand homogenizer (Sigma) in NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS-) buffer, incubated on ice for 15 minutes, and rotated at 4°C for 30 minutes. Cellular residue was isolated and discarded by centrifugation at 14,000 rpm for 10 minutes. The lysate was quantified using Nanodrops, and 25 μg of protein was loaded onto a 10% acrylamide gel. The protein was transferred from the acrylamide gel to a PVDF membrane (Invitrogen) at 100 V for 120 minutes. The membrane was blocked with bovine serum albumin (5% w / v) diluted with TBS:Tween. The membrane was incubated overnight in primary antibody at 4°C and then incubated at room temperature for 90 minutes in secondary antibody. The primary antibodies were anti-phosphotau (Ser396) and anti-phosphotau (Thr181). The secondary antibody was LI-COR IRDye secondary antibody. Signal intensity was quantified using ImageJ 1.46a and normalized to the value of total tau Tau5 (Thermo Fisher Scientific; AHB0042).

[0332] ELISA

[0333] The major auditory cortex, medial prefrontal cortex, and hippocampus were isolated from 6-month-old 5XFAD male mice and then treated with Aβ. 42 or Aβ 40 Aβ levels were measured using an ELISA kit (Invitrogen) according to the manufacturer's instructions. Insoluble Aβ was treated with 5M guanidine / 50mM Tris HCl (pH 8.0) buffer before ELISA measurement.

[0334] Behavioral experiment

[0335] Recognition of new objects

[0336] The novel object recognition (NOR) task, as previously described (Leger et al., 2013), consisted of a habituation phase followed by training and examination the following day. 24 hours prior to training, mice were habituated for 5 minutes in an open examination arena (40cmL x 40cmW x 35cmH), during which the total distance (cm), center dwell time (seconds), and velocity (cm / second) were calculated (TSE Systems). During training, mice were placed in the same box containing two identical objects in opposite corners. Mice were given a total of 20 seconds of object interaction time (within a maximum 10-minute time frame) and immediately removed from the arena. Object memory was examined 1 hour later using the same procedure as during training, except that one object was replaced by a new object in its position. Object search was recorded when the snout touched either object, and the recognition index, RI=T 新規 / (T 新規 +T 慣れた ), calculated by the formula, T 新規 and T 慣れた These figures represent the time spent on new objects and familiar objects, respectively.

[0337] new object position

[0338] The novel position recognition (NOL) task was performed using the same procedure as the object recognition task, except that two identical objects were used for both training and testing, and one of the objects was moved to a new position during the testing period.

[0339] Morris Water Maze Test

[0340] The spatial reference memory test was conducted in a ring-shaped tank (1.2 m in diameter) filled with white, opaque water at approximately 22°C. Reference cues consisted of different colors and shapes and were placed along the walls surrounding the tank. A fixed platform (10 cm in diameter) was placed inside the tank in a target quadrant. During the test, the platform was submerged, and mice were placed inside the tank at one of seven randomly placed points facing the tank walls. Mice were given 60 seconds to search for the platform, and if they could not find it, they were gently guided to the platform. Mice were placed on the platform for 15 seconds. Two trials were conducted per day, with a one-hour interval between trials. During the trials, the mice's feet were gently dried and warmed on a heating pad. The mice's behavior was video recorded using TSE Systems. Escape latency, or the time it took for the mouse to reach the platform, was scored in each trial and averaged over each test day. On day 6, the platform was removed, and a memory test (probe test) was conducted. The time spent in each of the four quadrants and the number of times the area where the platform was located was crossed were recorded. Swimming speed was automatically recorded.

[0341] conclusion

[0342] The embodiments relating to the inventions of this disclosure cover the individual features, systems, articles, materials, kits, and / or methods described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the inventions of this disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not inconsistent with each other.

[0343] Furthermore, various inventive concepts may be embodied in one or more methods, and examples of this have been provided. Actions performed as part of a method can be ordered by any suitable means. Therefore, embodiments may be constructed in which the actions are performed in a different order than those illustrated, and Even when shown as an action following an exemplary embodiment, this may include performing some of the actions simultaneously.

[0344] All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference.

[0345] All definitions defined and used herein should be understood to govern dictionary definitions, definitions incorporated by reference in documents, and / or the ordinary meanings of the terms defined.

[0346] As used herein and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly stated otherwise.

[0347] As used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the combined elements, that is, elements that are sometimes conjunctive and sometimes disjunctive. The multiple elements listed in “and / or” should be interpreted as “one or more” of the elements that are coordinately connected. Other elements may be present, as they see fit, whether related to or unrelated to the elements specifically identified by the “and / or” clause. Thus, as a non-restrictive example, a reference to “A and / or B” when used in conjunction with unrestrictive language such as “equipped with,” may refer in one embodiment to A only (optionally including elements other than B), in another embodiment to B only (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), and so on.

[0348] 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, that is, including at least one of a number of elements or a list of elements, and optionally additional items not on the list, but also including two or more. Only terms that explicitly indicate the opposite, such as “only one of” or “exactly one of” or, as used in the claims, “consisting of,” shall be considered to include exactly one element of a number of elements or a list 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, not both”) when preceded by terms of exclusivity such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting of” as used in the claims shall have the usual meaning as used in the field of patent law.

[0349] As used herein and in the claims, the phrase “at least one” in relation 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 all elements specifically enumerated in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the possibility that elements other than those specifically identified in the list of elements, to which the phrase “at least one” refers, may exist, whether related to or unrelated to the specifically identified elements. Thus, as a non-restrictive 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, In one embodiment, B does not exist and optionally includes two or more A's, or at least one A (optionally including elements other than B); in another embodiment, A does not exist and optionally includes two or more B's, or at least one B (optionally including elements other than A); and in yet another embodiment, optionally includes two or more A's, or at least one A, and optionally includes two or more B's, or at least one B (optionally including other elements), and so on.

[0350] In the claims and the above specification, all transitional phrases, such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and similar, are understood to be unrestrictive, meaning they include but are not limited to them. Only the transitional phrases “consisting of” and “consisting essentially of” are considered closed or semi-closed transitional phrases, respectively, as described in Section 2111.03 of the U.S. Patent and Trademark Office's Patent Examination Procedure Manual.

[0351] The present invention may also be in the following embodiments. (1) A method for treating dementia or Alzheimer's disease in a subject in need thereof, comprising A) non-invasively delivering a combination of auditory and visual stimuli having frequencies of approximately 20 Hz to approximately 60 Hz to the subject, thereby inducing synchronized gamma oscillations in at least one brain region of the subject. (2) The method according to (1) above, wherein the combination of auditory and visual stimuli has a frequency of approximately 35 Hz to approximately 45 Hz. (3) The method according to (2) above, wherein the combination of the auditory stimulus and the visual stimulus has a frequency of approximately 40 Hz. (4) The method according to any one of (1) to (3) above, wherein A) induces a periodic spike response in recording areas of 5% or more of at least one cortical region selected from the auditory cortex (AC), visual cortex (VC), hippocampus (HPC), and medial prefrontal cortex (mPFC). (5) The method according to any one of (1) to (4) above, wherein A) induces a local electric field potential (LFP) of approximately 40 Hz in the mPFC. (6) The method according to any one of (1) to (5) above, wherein A) increases the microglial response in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. (7) The method according to (6) above, wherein A1) includes at least one of increasing the number of microglia within 25 micrometers of the amyloid plaque, increasing the diameter of the microglial cell bodies, decreasing the length of the microglial processes, and increasing the number of microglial cells. (8) The method according to (7) above, wherein A1) is performed by increasing the diameter of the microglial cell bodies by at least 10%, 20%, 30%, 40%, or 50%. (9) The method according to (7) above, wherein A1) reduces the length of the microglial protrusions by at least 10%, 20%, 30%, 40%, or 50%. (10) The method according to (7) above, wherein A1) increases the number of microglial cells by at least 10%, 20%, 30%, 40%, or 50%. (11) The method according to any one of (6) to (10) above, wherein at least one cortical region includes the mPFC. (12) The method according to any one of (6) to (11) above, wherein A1) occurs after non-invasive delivery of a combination of auditory and visual stimuli over several days. (13) The method according to (12) above, wherein A1) occurs after 7 days of non-invasive delivery of a combination of auditory and visual stimuli. (14) The method according to any one of (1) to (13) above, wherein A) reduces amyloid plaques in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. (15) The method according to (14) above, wherein A2) reduces the size of the plaque by at least about 50%. (16) The method according to (14) above, wherein A2) reduces the number of plaques by at least about 50%. (17) The method according to any one of (14) to (16) above, wherein at least one cortical region includes the mPFC. (18) The method according to any one of (14) to (17) above, wherein A2) occurs after non-invasive delivery of a combination of auditory and visual stimuli over several days. (19) The method according to (18) above, wherein A2) occurs after 7 days of non-invasive delivery of a combination of auditory and visual stimuli. (20) The method according to any one of (1) to (19) above, wherein A) reduces the amount of amyloid-β (Aβ) peptide in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. (21) The method according to (20) above, wherein A3) reduces the amount of the Aβ peptide by at least 50%. (22) In A3), the Aβ peptide is Aβ 1-40 Peptide isoforms and Aβ 1-42 The method according to either (20) or (21) above, comprising at least one of the peptide isoforms. (23) The method according to any one of (20) to (22) above, wherein the Aβ peptide comprises at least one of a soluble Aβ peptide and an insoluble Aβ peptide. (24) The method according to any one of (20) to (23) above, wherein at least one cortical region includes the mPFC. (25) The method according to any one of (20) to (24) above, wherein A3) occurs after non-invasive delivery of a combination of auditory and visual stimuli over several days. (26) The method according to (25) above, wherein A3) occurs after 7 days of non-invasive delivery of a combination of auditory and visual stimuli. (27) A method for treating dementia or Alzheimer's disease in a subject in need thereof, comprising controlling at least one visual stimulator that emits a visual stimulus at a frequency of about 35 Hz to about 45 Hz; controlling at least one electroacoustic transducer that converts an electrosound signal into a corresponding auditory stimulus at a frequency of about 35 Hz to about 45 Hz; and non-invasively delivering a combined stimulus to the subject, the combined stimulus comprising the visual and auditory stimuli arranged in synchronous order, the combined stimulus inducing synchronized gamma oscillations in at least one brain region of the subject, the synchronized gamma oscillations resulting in an improvement in the cognitive function of the subject. (28) The method according to (27), wherein the visual stimulus comprises repeated periods of 12.5 milliseconds of illumination followed by 12.5 milliseconds of deactivation. (29) The method according to either (27) or (28) above, wherein the visual stimulation device is a light-emitting diode having a power of 40 to 80 watts. (30) The method according to any one of (27) to (29) above, wherein the auditory stimulus includes a 10kHz tone reproduced at 40Hz in load cycles of approximately 4% to approximately 80%. (31) The method according to any one of (27) to (30) above, wherein the visual stimulus includes light that flashes at 40 Hz for 10 seconds at a load cycle of about 10% to about 80%. (32) The method according to any one of (27) to (31) above, wherein the visual stimulus and the auditory stimulus are synchronized. (33) The method according to any one of (27) to (31) above, wherein the visual stimulus and the auditory stimulus are out of phase by -180 to 0 degrees or 0 to 180 degrees. (34) The method according to any one of (1) to (33) above, wherein the dementia is associated with at least one of Alzheimer's disease, vascular dementia, frontotemporal dementia, Lewy body dementia, and age-related memory impairment. (35) A method for treating dementia or Alzheimer's disease in a subject in need thereof, comprising A) non-invasively delivering a combination of auditory and visual stimuli having a frequency of about 35 Hz to about 45 Hz to the subject to induce synchronized gamma oscillations in at least one brain region of the subject, wherein A) comprises A1) inducing a periodic spike response in the medial prefrontal cortex (mPFC) of the subject, A2) inducing a local electric field potential (LFP) of about 40 Hz in the mPFC, and A3) increasing the microglial response in the mPFC. (36) The method according to (35), wherein A) includes A3), and A3) includes at least one of increasing the number of microglia within 25 micrometers of an amyloid plaque, increasing the diameter of the microglial cell body, decreasing the length of the microglial protrusions, and increasing the number of microglial cells. (37) The method according to (36) above, wherein A3) increases the diameter of the microglial cell bodies by at least 10%, 20%, 30%, 40%, or 50%. (38) The method according to (36) above, wherein A3) reduces the length of the microglial protrusions by at least 10%, 20%, 30%, 40%, or 50%. (39) The method according to (36) above, wherein A3) increases the number of microglial cells by at least 10%, 20%, 30%, 40%, or 50%. (40) The method according to any one of (36) to (39) above, wherein A3) occurs after non-invasive delivery of a combination of auditory and visual stimuli over several days. (41) The method according to (40) above, wherein A3) occurs after 7 days of non-invasive delivery of a combination of auditory and visual stimuli.

Claims

1. A visual stimulation device configured to deliver a visual stimulus containing multiple light pulses to a target, At least one electroacoustic transducer configured to deliver an auditory stimulus including multiple auditory pulses, wherein the combined stimulus of the visual stimulus and the auditory stimulus is delivered to the target to induce synchronized gamma oscillations in at least one brain region of the target, At least one processor connected to communicate with the at least one visual stimulation device and the at least one electroacoustic transducer. A system equipped with, When executing a processor-executable instruction, the at least one processor controls the at least one visual stimulus device to emit a visual stimulus at a frequency of 20 Hz to 60 Hz, and controls the at least one electroacoustic transducer to convert an electroacoustic signal into an auditory stimulus at a frequency of 20 Hz to 60 Hz. Here, the visual stimulus and the auditory stimulus are synchronized and aligned, have a fixed phase relationship with a phase difference of zero degrees, and each of the plurality of light pulses has a longer duration than each of the plurality of auditory pulses. Therefore, a system configured to treat at least one of the aforementioned dementia or Alzheimer's disease.

2. The system according to claim 1, wherein the visual stimulus and the auditory stimulus have a frequency of approximately 40 Hz.

3. The system according to claim 1, wherein multiple auditory pulses include a 10 kHz tone reproduced at 40 Hz in load cycles ranging from approximately 4% to approximately 80%.

4. The system according to claim 3, wherein the load cycle is approximately 4%.

5. The system according to claim 2, wherein multiple light pulses include light that flashes at 40 Hz for 10 seconds at load cycles of approximately 10% to approximately 80%.

6. The system according to claim 5, wherein the load cycle is approximately 50%.

7. The system according to claim 1, further comprising a light-shielding device that reduces ambient light to the target eye.

8. The system according to claim 1, further comprising a noise-canceling device for reducing ambient noise around at least one ear of the subject.

9. The system according to claim 1, wherein the duration of each of the multiple light pulses is approximately 12.5 milliseconds.

10. The system according to claim 1, wherein the duration of each of the multiple sound pulses is approximately 1 millisecond.

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