阅读说明：本技术 用于预防、减轻和/或治疗痴呆的系统和方法 (Systems and methods for preventing, alleviating and/or treating dementia ) 是由 L-H·蔡 A·J·马托雷尔 H-J·苏 E·博伊登 于 2018-09-19 设计创作，主要内容包括：用于治疗有需要的受试者中的痴呆或阿尔茨海默氏病的装置、系统和方法。在一个实例中,向所述受试者非侵入性地递送具有约20Hz至约60Hz且更具体地约40Hz的频率的组合的听觉和视觉刺激,以在所述受试者的至少一个脑区中引起同步的γ振荡。特别地,根据各种治疗和暴露方案,组合的听觉和视觉刺激(与单独的听觉或视觉刺激相反)促进内侧前额叶皮层(mPFC)中的小神经胶质反应。更一般而言,组合的听觉和视觉刺激在听觉皮层、视觉皮层和所述mPFC中引起延长的小神经胶质簇聚反应。(Devices, systems, and methods for treating dementia or alzheimer's disease in a subject in need thereof. In one example, the combined auditory and visual stimuli having a frequency of about 20Hz to about 60Hz, and more specifically about 40Hz, are delivered non-invasively to the subject to induce synchronized gamma oscillations in at least one brain region of the subject. In particular, combined auditory and visual stimuli (as opposed to auditory or visual stimuli alone) promote microglial responses in the medial prefrontal cortex (mPFC) according to various treatment and exposure regimens. More generally, the combined auditory and visual stimuli elicit a prolonged microglial clustering response in the auditory cortex, visual cortex, and the mPFC.)

1. A method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of:

A) non-invasively delivering to the subject a combined auditory and visual stimulus having a frequency of about 20Hz to about 60Hz to induce synchronized gamma oscillations in at least one brain region of the subject.

2. The method of claim 1, wherein in a), the combined auditory and visual stimulus has a frequency of about 35Hz to about 45 Hz.

3. The method of claim 2, wherein in a), the combined auditory and visual stimulus has a frequency of about 40 Hz.

4. The method of any one of claims 1 to 3, wherein A) comprises causing periodic spike responses in 5% or more of the recording sites in at least one cortical area selected from the group consisting of the Auditory Cortex (AC), the Visual Cortex (VC), the Hippocampus (HPC), and the medial prefrontal cortex (mPFC).

5. The method of any of claims 1 to 4 wherein A) comprises inducing a Local Field Potential (LFP) in the mPF at about 40 Hz.

6. The method of any one of claims 1 to 5, wherein A) comprises:

A1) increasing microglial response in at least one cortical region selected from neocortex, said AC, said VC, said HPC, and said mPF.

7. The method of claim 6, wherein a1) comprises at least one of:

increasing the amount of microglia within 25 micron amyloid plaques;

increasing microglial body diameter;

reducing the microglial projection length; and

increase microglial cell count.

8. The method of claim 7, wherein a1) comprises increasing the microglia cell body diameter by at least 10%, 20%, 30%, 40%, or 50%.

9. The method of claim 7, wherein a1) comprises reducing the microglial projection length by at least 10%, 20%, 30%, 40%, or 50%.

10. The method of claim 7, wherein a1) comprises increasing microglia count by at least 10%, 20%, 30%, 40%, or 50%.

11. The method of any one of claims 6 to 10, wherein the at least one cortical region comprises the mPFC.

12. The method of any one of claims 6 to 11, wherein a1) is produced after a number of days of non-invasively delivering combined auditory and visual stimuli.

13. The method of claim 12, wherein a1) is generated 7 days after non-invasively delivering the combined auditory and visual stimulus.

14. The method of any one of claims 1 to 13, wherein a) comprises:

A2) reducing amyloid plaques in at least one cortical region selected from the neocortex, the AC, the VC, the HPC, and the mPF.

15. The method of claim 14, wherein a2) comprises reducing plaque size by at least about 50%.

16. The method of claim 14, wherein a2) comprises reducing the number of patches by at least about 50%.

17. The method of any one of claims 14 to 16, wherein the at least one cortical region comprises the mPFC.

18. The method of any one of claims 14 to 17, wherein a2) is produced after a number of days of non-invasively delivering combined auditory and visual stimuli.

19. The method of claim 18, wherein a2) is generated after 7 days of non-invasively delivering the combined auditory and visual stimulus.

20. The method of any one of claims 1 to 19, wherein a) comprises:

A3) reducing the amount of amyloid- β (A β) peptide in at least one cortical region selected from the neocortex, the AC, the VC, the HPC, and the mPF.

21. The method of claim 20, wherein a3) comprises reducing the amount of a β peptide by at least 50%.

22. The method of any one of claims 20 and 21, wherein in A3), the a β peptide comprises isoform a β_1-40Peptides and isoform a β_1-42At least one peptide.

23. The method of any one of claims 20 to 22, wherein in a3), the a β peptide comprises at least one of a soluble a β peptide and an insoluble a β peptide.

24. The method of any one of claims 20 to 23, wherein the at least one cortical region comprises the mPFC.

25. The method of any one of claims 20 to 24, wherein a3) is produced after a plurality of days of non-invasively delivering combined auditory and visual stimuli.

26. The method of claim 25, wherein a3) is generated after 7 days of non-invasively delivering the combined auditory and visual stimulus.

27. A method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of:

controlling at least one visual stimulator to emit visual stimuli at a frequency of about 35Hz to about 45 Hz;

controlling at least one electro-acoustic transducer to convert an electrical audio signal into a corresponding auditory stimulus having a frequency of about 35Hz to about 45 Hz; and

non-invasively delivering to the subject a combined stimulus comprising a visual stimulus and an audio stimulus that are in simultaneous alignment, the combined stimulus causing simultaneous gamma oscillation in at least one brain region of the subject that results in an improvement in cognitive function of the subject.

28. The method of claim 27, wherein the visual stimulus comprises repeatedly turning on the light for 12.5ms and then turning off the light for 12.5 ms.

29. The method of any one of claims 27 and 28, wherein the visual stimulator is a light emitting diode with a power of 40-80W.

30. The method of any of claims 27-29, wherein the auditory stimulus comprises a 10kHz tone played at 40Hz with a duty cycle of about 4% to about 80%.

31. The method of any of claims 27-30, wherein the visual stimulus comprises light flashing at 40Hz for a period of 10s at a duty cycle of about 10% to about 80%.

32. The method of any one of claims 27 to 31, wherein the visual stimulus and the auditory stimulus are synchronized.

33. The method of any of claims 27 to 31, wherein the visual stimulus and the auditory stimulus are different by a phase of from-180 degrees to 0 degrees or from 0 degrees to 180 degrees.

34. The method of any one of claims 1-33, wherein the dementia is associated with at least one of alzheimer's disease, vascular dementia, frontotemporal dementia, dementia with lewy bodies, and age-related cognitive decline.

35. A method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of:

A) non-invasively delivering to the subject a combined auditory and visual stimulus having a frequency of about 35Hz to about 45Hz to induce synchronized gamma oscillation in at least one brain region of the subject, wherein A) comprises at least one of:

A1) causing a periodic spike response in the medial prefrontal cortex (mPFC) of the subject;

A2) inducing a Local Field Potential (LFP) of about 40Hz in the mPF; and

A3) increasing microglial response in the mPFCs.

36. The method of claim 35, wherein a) comprises A3), and wherein A3) comprises at least one of:

increasing the amount of microglia within 25 micron amyloid plaques;

increasing microglial body diameter;

reducing the microglial projection length; and

increase microglial cell count.

37. The method of claim 36, wherein a3) comprises increasing the microglia cell body diameter by at least 10%, 20%, 30%, 40%, or 50%.

38. The method of claim 36, wherein a3) comprises reducing the microglial projection length by at least 10%, 20%, 30%, 40%, or 50%.

39. The method of claim 36, wherein a3) comprises increasing microglia count by at least 10%, 20%, 30%, 40%, or 50%.

40. The method of any one of claims 36 to 39, wherein A3) is produced after a plurality of days of non-invasively delivering combined auditory and visual stimuli.

41. The method of claim 40, wherein A3) was generated 7 days after non-invasively delivering the combined auditory and visual stimulus.

Background

Alzheimer's Disease (AD) is a progressive neurodegenerative disease characterized by a decline in memory, orientation, and reasoning abilities. It is the most common form of dementia in the world, affecting approximately one eighth of people over 65 years of age, and is the sixth leading cause of death in the united states. Over the next decade, the prevalence of this progressive neurodegenerative disease is estimated to increase by 40%.

Histopathologically, AD can be characterized by the accumulation of amyloid plaques, which contain the amyloid- β (a β) peptide and the Neuronal Fibrillar Tangles (NFTs) made from tau protein. A.beta.peptide is a 36-43 amino acid protein whose normal physiological function has not been determined. The A β peptide is formed by sequential proteolytic cleavage of Amyloid Precursor Protein (APP) by β -secretase 1(BACE1) and γ -secretase. The C-terminal fragment β (β -CTF) is an APP derivative produced during amyloidogenic cleavage of APP by BACE1 and thus is another indicator of A β peptide production. Under normal conditions, soluble a β peptides are produced and secreted by neurons and subsequently cleared from the brain via the cerebrospinal fluid (CSF) pathway. However, in subjects with AD, a β peptides appear to aggregate in a concentration-dependent manner into higher order species to form soluble oligomers and insoluble plaques. This aggregation may trigger a number of neurotoxic events including brain metabolic disorders, neuroinflammation, decreased functional connectivity, loss of synapses and neurons, and/or NFT formation.

A fundamental relationship between a β concentration and neuronal activity has been demonstrated. First, treatment of hippocampal organotypic brain slices prepared from transgenic (Tg) mice overexpressing APP with tetrodotoxin reduced neuronal activity, followed by a β levels. The opposite effect was then observed upon treatment with tetrandrine-an increase in neuronal activity. Neuronal activity has also been used to demonstrate dynamic modulation of a β peptide concentration and eventual plaque deposition in vivo. In human AD patients, neuroimaging shows that the most severe plaque deposits are likely aligned with the always most active brain region called the "default-mode network".

Currently AD is not curable and treatment options do not inhibit the pathological progression of AD, are mainly palliative, and/or may have a variety of disturbing side effects. For example, in clinical trials, prophylactic and/or therapeutic strategies directed to a β peptides and/or their precursors (e.g., a β immunotherapy and inhibition of β -and γ -secretases) are toxic and/or ineffective in reducing AD lesions. Clinical trials involving amyloid beta vaccines (e.g., baclizumab) failed due to a lack of cognitive benefit. Gamma-secretase inhibitors (e.g., semazet) have failed in clinical trials by exacerbating cognitive deficits in the subject. Even existing drugs like acetylcholinesterase inhibitors (e.g. donepezil and rivastigmine) and N-methyl-D-aspartate (NMDA) -receptor antagonists (e.g. memantine) show only slight cognitive benefits.

Disclosure of Invention

As disclosed in U.S. patent application serial No. 15/360,637 filed on 23/11/2016 (herein incorporated by reference in its entirety) and entitled "SYSTEMS AND METHODS FOR PREVENTING, MITIGATING, AND/OR TREATING DEMENTIA," the induction of synchronized gamma oscillations in the brain via visual OR auditory stimuli results in a reduction in amyloid burden and morphological changes in some brain regions. However, the inventors have recognized and appreciated that there remains a need for systems and methods for treating dementia and alzheimer's disease that address all-circuit disorders affecting multiple brain centers that are important responsible for learning and memory as well as other higher brain functions.

In light of the foregoing, the present disclosure is directed, at least in part, to combined auditory and visual stimulation to induce Gamma oscillations in the brain of a subject according to various techniques generally referred to herein as "Gamma induction Using sensory stimulation" or "Gamma ENtrainment". Combined auditory and visual stimuli (e.g., combined visual and auditory GENUS) as disclosed herein unexpectedly generate positive physiological and behavioral changes that are not observed for visual or auditory GENUS alone. The positive effects on the brain produced by the combined auditory and visual GENUS are not limited to the Auditory Cortex (AC) and Hippocampus (HPC), but rather, significantly, they extend to cause microglial clustering reactions in the medial prefrontal cortex (mPFC) and reduce amyloid burden throughout the neocortex. Furthermore, the effects of combined auditory and visual GENUS are observed over a shorter treatment/exposure time frame (on the order of weeks).

In one aspect, the present disclosure provides devices, methods, and systems for treating dementia or alzheimer's disease in a subject in need thereof, the methods comprising the steps of: non-invasively delivering to the subject a combined auditory and visual stimulus having a frequency of about 20Hz to about 60Hz to induce synchronized gamma oscillations in at least one brain region of the subject. In some embodiments, the dementia is associated with AD, vascular dementia, frontotemporal dementia, dementia with lewy bodies, and/or age-related cognitive decline. The subject may be a human or an animal.

In some embodiments, the combined auditory and visual stimulus has a frequency of about 35Hz to about 45Hz, or a frequency of about 40 Hz.

In some embodiments, non-invasively delivering the combined auditory and visual stimulus elicits a periodic spike response in 5% or more of the recording sites in at least one cortical region selected from the group consisting of the Auditory Cortex (AC), the Visual Cortex (VC), the Hippocampus (HPC), and the medial prefrontal cortex (mPFC). In some embodiments, non-invasively delivering the combined auditory and visual stimulus induces a Local Field Potential (LFP) in the mPFC at about 40 Hz.

In some embodiments, non-invasively delivering the combined auditory and visual stimulus increases the microglial response in at least one cortical region. Cortical regions may include neocortex, AC, VC, HPC, and mPF. In some embodiments, a microglial reaction is induced in the mPFC. In some embodiments, a microglial response is elicited in multiple cortical regions. In some embodiments, the microglial response is elicited across the entire neocortex.

In some embodiments, increasing the microglial response comprises a treatment selected from increasing the number of microglia within an amyloid plaque of 25 microns; increasing microglial body diameter; reducing the microglial projection length; and at least one effect of increasing microglial cell count. In embodiments, increasing the microglial response comprises increasing the microglial cell body diameter by at least 10%, 20%, 30%, 40%, or 50%. In embodiments, increasing the microglial response comprises reducing the microglial projection length by at least 10%, 20%, 30%, 40%, or 50%. In embodiments, increasing the microglial response comprises increasing the microglial cell count by at least 10%, 20%, 30%, 40%, or 50%.

In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 days of non-invasive delivery of the combined auditory and visual stimulus.

In some embodiments, non-invasively delivering the combined auditory and visual stimulus comprises reducing amyloid plaques in at least one cortical region selected from the group consisting of neocortex, AC, VC, HPC, and mPFC. Cortical regions may include neocortex, AC, VC, HPC, and mPF. In some embodiments, a microglial reaction is induced in the mPFC. In some embodiments, a microglial response is elicited in multiple cortical regions. In some embodiments, the microglial response is elicited across the entire neocortex.

In embodiments, reducing amyloid plaques comprises reducing plaque size by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. In embodiments, reducing amyloid plaques comprises reducing the number of plaques by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 days of non-invasive delivery of the combined auditory and visual stimulus.

In some embodiments, non-invasively delivering the combined auditory and visual stimulus comprises reducing the amount of amyloid- β (Α β) peptide in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. Cortical regions may include neocortex, AC, VC, HPC, and mPF. In some embodiments, a microglial reaction is induced in the mPFC. In some embodiments, a microglial response is elicited in multiple cortical regions. In some embodiments, the microglial response is elicited across the entire neocortex.

In embodiments, reducing the amount of a β peptide comprises reducing the amount by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. In some embodiments, the a β peptide comprises at least one of an isoform a β 140 peptide and an isoform a β 1-42 peptide. In some embodiments, the a β peptide comprises at least one of a soluble a β peptide and an insoluble a β peptide.

In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 days of non-invasive delivery of the combined auditory and visual stimulus.

In a second aspect, the present disclosure provides a method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of: controlling at least one visual stimulator to emit visual stimuli at a frequency of about 35Hz to about 45 Hz; controlling at least one electro-acoustic transducer to convert an electrical audio signal into a corresponding auditory stimulus having a frequency of about 35Hz to about 45 Hz; and non-invasively delivering to the subject a combined stimulus comprising a visual stimulus and an audio stimulus in simultaneous alignment, the combined stimulus causing simultaneous gamma oscillations in at least one brain region of the subject. The synchronized gamma oscillations result in an improvement in cognitive function of the subject.

In some embodiments, the visual stimulus comprises repeatedly turning the light on for 12.5ms and then off for 12.5 ms. In some embodiments, the optogenetic stimulator is a light emitting diode with a power of 40-80W. In some embodiments, the auditory stimulus comprises a 10kHz tone played at 40Hz with a duty cycle of about 4% to about 80%. In some embodiments, the visual stimulus comprises light flashing at 40Hz for a period of 10 seconds at a duty cycle of about 10% to about 80%.

In some embodiments, the visual stimulus and the auditory stimulus are synchronized. In some embodiments, the visual stimulus and the auditory stimulus are different by from-180 degrees to 0 degrees or from 0 degrees to 180 degrees.

It should be understood that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided that such concepts do not contradict each other) are considered a part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are considered part of the inventive subject matter disclosed herein. It should also be understood that terms explicitly employed herein that may also appear in any disclosure incorporated by reference should be given the most consistent meaning to the particular concepts disclosed herein.

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

Drawings

This patent or application document contains at least one drawing executed in color.

Those skilled in the art will appreciate that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The figures are not necessarily to scale; in some instances, aspects of the subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate an understanding of various features. In the drawings, like reference characters generally refer to the same features (e.g., functionally similar and/or structurally similar elements), for example.

Fig. 1A-1L show that 40Hz auditory stimulation modulates spike activity in AC, CA1, and mPFC.

Fig. 2A-2J show that auditory GENUS improves recognition and spatial memory tasks in 5XFAD mice.

Figures 3A-3I show that auditory GENUS reduces amyloid burden in AC and HPC of 5XFAD mice.

Fig. 4A-4K show that auditory GENUS elicited glial responses in AC and CA1 in 5XFAD mice.

Fig. 5A-5F show the association of auditory GENUS in increasing amyloid-vasculature.

Fig. 6A-6I show that combined auditory and visual GENUS elicits a clustering phenotype response through microglia.

Fig. 7A-7J show that combined auditory and visual GENUS reduces amyloid burden in mPFC and neocortex.

Fig. 8A to 8R show that 20Hz and 80Hz auditory stimuli modulate activity in AC, CA1 and mPFC.

Fig. 9A-9L show that auditory GENUS does not affect mouse behavior.

FIGS. 10A-10H show that auditory GENUS improves plaque load in APP/PS1 mice.

FIGS. 11A-11H show that auditory GENUS elicits a microglial response in APP/PS1 mice.

Fig. 12A-12L show that auditory GENUS reduces phosphorylated tau in P301S mice.

Fig. 13A-13U show that 40Hz combined auditory and visual stimuli modulated the spike activity in AC, CA1, and mPFC.

Fig. 14A-14V show that 1 week auditory or visual GENUS does not affect mPFC lesions only.

Detailed Description

The combined auditory and visual Gamma Entrainment (GENUS) using sensory stimuli unexpectedly generated positive physiological and behavioral changes not observed for visual or auditory GENUS alone. The positive effects on the brain are not limited to the Auditory Cortex (AC) and Hippocampus (HPC), but extend to causing microglial clustering reactions in the medial prefrontal cortex (mPFC) and reducing amyloid burden throughout the neocortex. Furthermore, the effects of combined auditory and visual GENUS are observed over a short time frame (on the order of weeks). The combined auditory and visual GENUS of about one week improves Alzheimer's Disease (AD) lesions in brain regions spanning large loop networks. In particular, combined auditory and visual GENUS significantly reduced amyloid burden in AC, Visual Cortex (VC), hippocampus CA1, and mPFC. The combined auditory and visual GENUS produces microglial clustering and reduced amyloid in the medial prefrontal cortex. Combined auditory and visual GENUS broadly reduces amyloid plaques throughout the neocortex.

In one aspect, the present disclosure provides devices, methods, and systems for treating dementia or alzheimer's disease in a subject in need thereof, the methods comprising the steps of: non-invasively delivering to the subject a combined auditory and visual stimulus having a frequency of about 20Hz to about 60Hz to induce synchronized gamma oscillations in at least one brain region of the subject. In some embodiments, the dementia is associated with AD, vascular dementia, frontotemporal dementia, dementia with lewy bodies, and/or age-related cognitive decline. The subject may be a human or an animal.

In some embodiments, the combined auditory and visual stimulus has a frequency of about 35Hz to about 45Hz, or a frequency of about 40 Hz.

In some embodiments, non-invasively delivering the combined auditory and visual stimulus elicits a periodic spike response in 5% or more of the recording sites in at least one cortical region selected from the group consisting of the Auditory Cortex (AC), the Visual Cortex (VC), the Hippocampus (HPC), and the medial prefrontal cortex (mPFC). In some embodiments, non-invasively delivering the combined auditory and visual stimulus induces a Local Field Potential (LFP) in the mPFC at about 40 Hz.

In some embodiments, non-invasively delivering the combined auditory and visual stimulus increases the microglial response in at least one cortical region. Cortical regions may include neocortex, AC, VC, HPC, and mPF. In some embodiments, a microglial reaction is induced in the mPFC. In some embodiments, a microglial response is elicited in multiple cortical regions. In some embodiments, the microglial response is elicited across the entire neocortex.

In some embodiments, increasing the microglial response comprises a treatment selected from increasing the number of microglia within an amyloid plaque of 25 microns; increasing microglial body diameter; reducing the microglial projection length; and at least one effect of increasing microglial cell count. In embodiments, increasing the microglial response comprises increasing the microglial cell body diameter by at least 10%, 20%, 30%, 40%, or 50%. In embodiments, increasing the microglial response comprises reducing the microglial projection length by at least 10%, 20%, 30%, 40%, or 50%. In embodiments, increasing the microglial response comprises increasing the microglial cell count by at least 10%, 20%, 30%, 40%, or 50%.

In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 days of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 months of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 years of non-invasive delivery of the combined auditory and visual stimuli.

In some embodiments, non-invasively delivering the combined auditory and visual stimulus comprises reducing amyloid plaques in at least one cortical region selected from the group consisting of neocortex, AC, VC, HPC, and mPFC. Cortical regions may include neocortex, AC, VC, HPC, and mPF. In some embodiments, a microglial reaction is induced in the mPFC. In some embodiments, a microglial response is elicited in multiple cortical regions. In some embodiments, the microglial response is elicited across the entire neocortex.

In embodiments, reducing amyloid plaques comprises reducing plaque size by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. In embodiments, reducing amyloid plaques comprises reducing the number of plaques by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 days of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 months of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 years of non-invasive delivery of the combined auditory and visual stimuli.

In some embodiments, non-invasively delivering the combined auditory and visual stimulus comprises reducing the amount of amyloid- β (Α β) peptide in at least one cortical region selected from the neocortex, AC, VC, HPC, and mPFC. Cortical regions may include neocortex, AC, VC, HPC, and mPF. In some embodiments, a microglial reaction is induced in the mPFC. In some embodiments, a microglial response is elicited in multiple cortical regions. In some embodiments, the microglial response is elicited across the entire neocortex.

In embodiments, reducing the amount of a β peptide comprises reducing the amount by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. In some embodiments, the a β peptide comprises at least one of an isoform a β 140 peptide and an isoform a β 1-42 peptide. In some embodiments, the a β peptide comprises at least one of a soluble a β peptide and an insoluble a β peptide.

In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 days of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 months of non-invasive delivery of the combined auditory and visual stimulus. In some embodiments, the enhanced microglial response is produced after 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 years of non-invasive delivery of the combined auditory and visual stimuli.

In a second aspect, the present disclosure provides a method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of: controlling at least one visual stimulator to emit visual stimuli at a frequency of about 35Hz to about 45 Hz; controlling at least one electro-acoustic transducer to convert an electrical audio signal into a corresponding auditory stimulus having a frequency of about 35Hz to about 45 Hz; and non-invasively delivering to the subject a combined stimulus comprising a visual stimulus and an audio stimulus in simultaneous alignment, the combined stimulus causing simultaneous gamma oscillations in at least one brain region of the subject. The synchronized gamma oscillations result in an improvement in cognitive function of the subject.

In some embodiments, the visual stimulus comprises repeatedly turning the light on for 12.5ms and then off for 12.5 ms. In some embodiments, the optogenetic stimulator is a light emitting diode with a power of 40-80W. In some embodiments, the auditory stimulus comprises a 10kHz tone played at 40Hz with a duty cycle of about 4% to about 80%. In some embodiments, the visual stimulus comprises light flashing at 40Hz for a period of 10 seconds at a duty cycle of about 10% to about 80%.

In some embodiments, the visual stimulus and the auditory stimulus are synchronized. In some embodiments, the visual stimulus and the auditory stimulus are different by from-180 degrees to 0 degrees or from 0 degrees to 180 degrees. As used herein, "phase" refers to the lag between auditory and visual stimuli expressed in degrees, where 0 degrees means simultaneous auditory and visual stimuli, and-180 or +180 refers to alternating visual and auditory stimuli.

As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. In some embodiments, subjects in need of treatment include those already with the disease or disorder as well as those who may develop the disease or disorder, and in the latter subjects, the aim is to prevent, delay or reduce the disease or disorder. For example, in some embodiments, the devices, methods, and systems disclosed herein can be employed to prevent, delay, or reduce a disease or disorder genetically susceptible to a subject, such as AD. In some embodiments, the devices, methods, and systems disclosed herein can be employed to treat, alleviate, reduce symptoms of, and/or delay progression of a disease or disorder (such as AD) that a subject has been diagnosed with.

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

The term "about" as used herein means plus or minus ten percent of the subject modified by "about".

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

AD is the most common neurodegenerative disease in developed countries. AD is histopathologically characterized by the accumulation of amyloid plaques, consisting of the a β peptide and NFT made from tau protein. Clinically, AD is associated with progressive cognitive impairment characterized by loss of memory, function, language ability, judgment, and executive function. AD often causes severe behavioral symptoms in its later stages.

Vascular dementia, which may also be referred to as cerebrovascular dementia, refers to cerebrovascular disorders (e.g., cerebral hemispheric infarction) which often have a fluctuating process with periods of improvement and gradual deterioration. Vascular dementia may include one or more symptoms of disorientation, memory impairment, and/or impaired judgment. Vascular dementia may be caused by discrete multiple infarcts or other vascular causes including, for example, autoimmune vasculitis, such as found in systemic lupus erythematosus; infectious vasculitis, such as lyme disease; recurrent cerebral hemorrhage; and/or a midwind.

Frontotemporal dementia (FTD) is a progressive neurodegenerative disease. Subjects with FTD often exhibit marked behavioral and character changes, often accompanied by language impairment.

Dementia with lewy bodies is characterized by one or more of the following symptoms: dementia progression with features overlapping those of AD; development of the parkinsonian features; and/or early development of hallucinations. Dementia with lewy bodies is generally characterized by daily fluctuations in symptom severity.

In some aspects, the present disclosure provides methods for preventing, alleviating, and/or treating dementia in a subject, the methods comprising causing synchronized gamma oscillations in the brain of the subject. In some embodiments, inducing gamma oscillation in a subject having a neurological disease or disorder or age-related decline is used to restore a gamma oscillation rhythm disrupted in the subject by or associated with the disease or disorder or age-related decline.

In some embodiments, the induction of gamma oscillation reduces isoform a β_1-40And A β_1-42In some embodiments, the induction of gamma oscillation enhances clearance of a β (e.g., isoform a β) from the brain of the subject_1-40And A β_1-42) In some embodiments, the methods provided herein reduce a β levels in the brain of a subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or more relative to a β levels in the brain of a subject before treatment.

In some embodiments, a β levels in the brain of the subject are reduced by reducing cleavage of APP in the brain of the subject. In some embodiments, the methods provided herein reduce APP cleavage in the brain of a subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or more relative to the level of APP cleavage in the brain of a subject prior to treatment. In some embodiments, the level of APP cleavage in the brain of the subject is reduced by at least about 50% relative to the level of APP cleavage in the brain of the subject prior to treatment. In some embodiments, the level of APP cleavage is measured by the level of C-terminal fragment β (β -CTF) in the brain of the subject. In some embodiments, the level of APP cleavage in the brain is reduced by inhibiting β -and/or γ -secretase, such as by increasing the level of inhibition of β -and/or γ -secretase activity. In some embodiments, the methods provided herein reduce aggregation of Α β plaques in the brain of the subject.

In some embodiments, the method improves cognitive ability and/or memory in the subject.

In another aspect, the present disclosure provides a method for inducing a neuroprotective posture or a neuroprotective environment in the brain of a subject, the method comprising inducing synchronized gamma oscillations in the brain of the subject. For example, in some embodiments, the neuroprotective posture is associated with a neuroprotective microglial cell contour. In additional embodiments, the neuroprotective posture is caused by or associated with an increase in 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 microglia signaling pathway.

In some embodiments, the neuroprotective posture is associated with a decrease or lack of pro-inflammatory glial activity. Proinflammatory glial cell activity is associated with the M1 phenotype in microglia and includes the production of Reactive Oxygen Species (ROS), the neurosecretory protein chromogranin A, the secreted cofactor cystatin C, NADPH oxidase, nitric oxide synthase enzymes such as iNOS, NF-. kappa.B-dependent inflammatory response proteins, and proinflammatory cytokines and chemokines (e.g., TNF, IL-1. beta., IL-6, and IFN. gamma.).

In contrast, the M2 phenotype of microglia is associated with the down-regulation of inflammation and the repair of inflammation-induced injury. Increases in anti-inflammatory cytokines and chemokines (IL-4, IL-13, IL-10 and/or TGF β) as well as phagocytic activity were associated with the M2 phenotype. Thus, in some embodiments, the methods provided herein cause the neuroprotective M2 phenotype in microglia. In some embodiments, the methods provided herein increase phagocytic activity in the brain of a subject. For example, in some embodiments, the methods provided herein increase phagocytic activity of microglia, such that the clearance of a β is increased.

The gamma oscillations may comprise about 20Hz to about 100 Hz. Thus, in some embodiments, the present disclosure provides methods for preventing, alleviating, or treating dementia in a subject, the method comprising causing gamma oscillations in the brain of the subject of about 20Hz to about 100Hz, or about 20Hz to about 80Hz, or about 20Hz to about 50Hz, or about 30Hz to about 60Hz, or about 35Hz to about 45Hz, or about 40 Hz. Preferably, the gamma oscillation is about 40 Hz.

The stimulus may include any detectable change in the internal or external environment of the subject that directly or ultimately causes 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), mechanical receptors (e.g., mechanical stress and/or strain), nociceptors (i.e., pain), acoustic receptors, electrical receptors (e.g., electric fields), magnetic receptors (e.g., magnetic fields), water receptors, chemoreceptors, heat receptors, osmoreceptors, and/or proprioceptors (i.e., position senses). The absolute threshold or minimum sensory amount required to elicit a response from a subject may vary based on the type of stimulus and the subject. In some embodiments, the stimulation is adjusted based on the individual's sensitivity.

In some embodiments, the gamma oscillations are induced in a brain region specific manner. For example, in some embodiments, the gamma oscillations are induced in the hippocampus, visual cortex, tubal cortex, auditory cortex, or any combination thereof. For example, in some embodiments, gamma oscillations are induced in the visual cortex using a flash of light; while in other embodiments, an auditory stimulus of a particular frequency is used to induce gamma oscillations in the auditory cortex. In some embodiments, a combination of visual, auditory, and/or other stimuli is used to simultaneously induce gamma oscillations in multiple brain regions. In some embodiments, gamma oscillations are induced in the virtual reality system.

In some embodiments, the subject receives stimulation via an environment configured to cause gamma oscillation, such as a chamber that passively or actively blocks extraneous stimulation (e.g., light blocking or noise cancellation). Alternatively or additionally, the subject may receive stimulation via a system that includes, for example, light blocking or noise cancellation aspects. In some embodiments, the subject receives the visual stimulus via a stimulus-emitting device (such as glasses designed to deliver the stimulus). The device may block other light. In some embodiments, the subject receives auditory stimulation via a stimulation emitting device (such as an earpiece designed to deliver the stimulation). The device may eliminate other noise.

In addition to at least one interface for emitting stimulation, some embodiments may also include at least one processor (e.g., to generate stimulation, control the emission of stimulation, monitor the emission of stimulation/results, and/or process feedback regarding stimulation/results), at least one memory (storing, for example, processor-executable instructions, at least one stimulation, stimulation generation strategies, feedback, and/or results), at least one communication interface (communicating with, e.g., a subject, a health care provider, a caregiver, a clinical research investigator, a database, a monitoring application, etc.) and/or a detection device (detecting and providing feedback regarding, e.g., stimuli and/or the subject, including whether gamma oscillations are induced, sensitivity of the subject, cognitive function, physical or chemical changes, stress, safety, etc.).

In some embodiments, the gamma oscillations are caused by a visual stimulus (such as a flash of light from about 20Hz to about 100 Hz). In a particular embodiment, the gamma oscillations are caused by a flash of light from about 20Hz to about 50 Hz. In further embodiments, the gamma oscillations are caused by a flash of light from about 35Hz to about 45 Hz. In other embodiments, the gamma oscillations are caused by a flash of light at about 40 Hz. In some embodiments, the subject receives (e.g., is placed in a room with or wears a light-blocking device that emits) a flash of light from about 20Hz to about 100Hz, or from about 20Hz to about 50Hz or from about 35Hz to about 45Hz, or about 40 Hz.

In some embodiments, the gamma oscillations are caused by an auditory stimulus such as a sound having a frequency of about 20Hz to about 100Hz, or about 20Hz to about 80Hz, or about 20Hz to about 50Hz, or about 35Hz to about 45Hz, or about 40 Hz. In some embodiments, the subject receives (e.g., is placed in a room with or wears a noise cancellation device that emits) auditory stimuli of about 20Hz to about 100Hz, about 20Hz to about 80Hz, about 20Hz to about 50Hz, about 35Hz to about 45Hz, or about 40 Hz.

In some embodiments, the subject receives (e.g., is placed in a room with or wears a light blocking device that emits) the visual and/or auditory stimuli for about one hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or more. In some embodiments, the subject receives (e.g., is placed in a room with or wears a light blocking device that emits) the stimulus for no more than about 6 hours, no more than about 5 hours, no more than about 4 hours, no more than about 3 hours, no more than about 2 hours, or no more than about one hour. In some embodiments, the subject receives (e.g., is placed in a room with or wears a light blocking device that emits) the stimulus for less than one hour.

In some embodiments, the subject is subjected to the methods provided herein. In other embodiments, the subject is treated with the methods provided herein on multiple separate occasions. The subject may be treated on a regular basis or as symptoms appear or worsen. In some embodiments, chronic treatment is effective to reduce soluble a β peptide and/or insoluble a β peptide (i.e., plaques).

In some embodiments, the gamma oscillations are induced in a cell type specific manner. In some embodiments, the gamma oscillations are induced in the FS-PV-interneuron. When used to describe a class of neurons, the term "fast firing" (FS) refers to the ability of a neuron to fire at a high rate for a long period of time with little peak frequency adaptation or attenuation of peak height. Thus, these neurons are capable of sustained high frequency (e.g., equal to or greater than about 100Hz or about 150Hz) discharge without significant adaptation. This characteristic of FS neurons can be largely attributed to the expression of their rapidly delayed rectifying channels (in other words, channels that are activated and deactivated very rapidly).

In one aspect, the stimulation may be non-invasive. The term "non-invasive" as used herein refers to devices, methods, and systems that do not require surgical intervention or manipulation of the body, such as injection or implantation of a composition or device. For example, the stimulus may be visual (e.g., flashing light), audio (e.g., sound vibration), and/or tactile (mechanical stimulus induced by force, vibration, or motion).

In another aspect, the stimulation may be invasive or at least partially invasive. For example, visual, audio, and/or tactile stimulation may be combined with injection or implantation of a composition (e.g., light-sensitive protein) or device (e.g., integrated fiber optic and solid state light source).

Experimental data

Experimental data relating to the inventive concepts described herein are set forth below in conjunction with various figures, which are summarized first and then described in detail.

Fig. 1A shows the firing rate adjustment of two putative single units during 40Hz auditory stimulation (left panel of each pair) and random stimulation (right panel of each pair) in AC. Punctuation indicates an auditory pulse; the light bars indicate randomly distributed pulses. The grid plot shows the spiking response of the single units inferred by both embodiments to a 10 second 40Hz auditory or random stimulus.

Figure 1B shows the distribution of the intervals between the peaks of the excitation rate in AC under no stimulation (marked as no stimulation), random (marked as random) and 40Hz auditory stimulation (marked as 40Hz stimulation) for all individual units (n-292 units over 9 recording sessions in 5 mice the interval ratio around the interval between excitations: P-040 Hz versus no stimulation, P-040 Hz versus random; z-test was performed for both ratios).

Fig. 1C shows an exemplary polar plot of firing rate modulation during 40Hz auditory stimulation relative to the start of stimulation (left, stimulation started at 0), vector intensity distribution (center, P) during 40Hz auditory stimulation, random, and no stimulation for a single unit of firing rate modulation<0.0001，P＝4x10^-5440Hz vs. no stimulation, P2 x10^-1340Hz vs. random; kolmogorov-smirnov test), and distribution of single unit firing rate adjusted rayleigh statistics (right, P<0.0001，P＝5x10^-6840Hz vs. no stimulation, P6 x10^-7240Hz vs. random, Kolmogorov-Similnov test; stimulation RS values of 26 units at 40Hz are greater than 30; random stimulation RS value of 1 unit greater than 30).

Fig. 1D shows the distribution of average firing rate values between stimulation conditions in AC.

Fig. 1E shows the same thing as fig. 1A for CA 1.

Fig. 1F shows the same as fig. 1B for CA1 (n 338 units in 10 recording sessions in 5 mice, P040 Hz versus no stimulation, P040 Hz versus random; z-test for both scales).

Fig. 1G shows the same contents (center, P) as fig. 1C for CA1<0.0001，P＝4x10^-4040Hz vs. no stimulation, P9 x10^-1140Hz vs. random; kolmogorov-smirnov test; right side, P<0.0001，P＝1x10^-7440Hz vs. no stimulation, P2 x10^-7340Hz vs. random; kolmogorov-smirnov test).

Fig. 1H shows the same for CA1 as fig. 1D.

FIG. 1I shows the same for mPF as in FIG. 1A.

Fig. 1J shows the same for mPFC as fig. 1B (n 115 units in 7 recording sessions in 4 mice, P040 Hz versus no stimulation, P040 Hz versus random; z-test for both scales).

Fig. 1K shows the same contents (center, P) as fig. 1C for mPFC<0.0001，P＝2x10^-2740Hz vs. no stimulation, P4 x10^-540Hz vs. random; kolmogorov-smirnov test; right side, P<0.0001，P＝1x10^-2840Hz vs. no stimulation, P5 x10^-3040Hz vs. random; kolmogorov-smirnov test).

FIG. 1L shows the same for mPF as in FIG. 1D.

Figure 2A shows a timeline of a behavioral experiment for a 5XFAD auditory GENUS mouse.

Figure 2B shows the recognition index of the New Object Recognition (NOR) test of 5XFAD auditory GENUS mice (n-20 mice in the non-stimulated group, n-20 mice in the 40Hz group, n-9 in the random frequency group, circles indicate 'n', mean s.e.m. in the bar graph, P <0.0001, n.s. not significant, kruscarl-voris test and dunen multiple comparison test).

Fig. 2C shows the average velocity (cm/s) during the new object identification test (n-20 mice in the non-stimulated group, n-20 mice in the 40Hz group, n-9 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

Fig. 2D shows recent total distances (cm) during the new object identification test (n 20 mice in the non-stimulated group, n 20 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

Figure 2E shows the recognition indices of the New Object Localization (NOL) test for 5XFAD auditory GENUS mice (n 20 mice in the non-stimulated group, n 20 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, P <0.0001, n.s. not significant, kruscarl-voris test and dunen multiple comparison test).

Fig. 2F shows the average velocity (cm/s) during the new object localization test (n 20 mice in the non-stimulated group, n 20 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

Fig. 2G shows the recent total distance (cm) during the new object localization test (n 20 mice in the non-stimulated group, n 20 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

Figure 2H shows the escape latency(s) in the Morris water maze for 5XFAD non-stimulated, random frequency and 40Hz auditory stimuli mice (n 25 mice in the non-stimulated group, n 28 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, P <0.0001, n.s. not significant, two-factor analysis of variance and graph-based multiple comparisons test).

Figure 2I shows the time(s) taken to swim in the target quadrant during the probation test (n 25 mice in the non-stimulated group, n 28 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, P <0.05, kruscarl-wallis test and dunne multiple comparison test).

Figure 2J shows the number of platform crossings during the probing trial (n 25 mice in the non-stimulated group, n 28 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, # P <0.01, kruscarl-wallis test and dunn multiple comparison test).

FIG. 3A shows the relative solubility A β in the Auditory Cortex (AC) and Hippocampus (HPC) of 6-month-old 5XFAD mice after auditory stimulation at 40Hz, 8Hz, 80Hz, or random frequency (1 hour per day for 7 days) normalized to non-stimulated control_1-42Levels (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 in the random frequency group, mean values in the bar graphs s.e.m.,. P<0.0001, n.s.: not significant, kruskal-wallis test and dunne multiple comparison test).

FIG. 3B shows the same thing as in FIG. 3A for insoluble A β 1-42.

Figure 3C shows immunohistochemistry with anti- Α β (D54D2, green) antibodies in 6 month old ACs in 5XFAD mice (n-7 mice per group, scale bar, 50 μm) after auditory GENUS or no stimulation (1 hour per day for 7 days).

Fig. 3D shows the same for CA1 as in fig. 3C.

Figure 3E shows the average number of Α β positive plaques in AC and CA1 (average s.e.m. P <0.05 in bar graph, n.7 mice per group, p.p < 0.0001; unpaired mann-whitney test).

Figure 3F shows the average area of Α β positive plaques in AC and CA1 (average s.e.m. P <0.01, P <0.001 in bar graph per group of n-7 mice; unpaired mann-whitney test).

Figure 3G shows immunohistochemistry with anti- Α β (12F4, red) antibody in 6 month old AC of 5XFAD mice (inset, 20-fold, scale bar, 50 μm) after auditory GENUS or no stimulation (1 hour per day for 7 days).

Fig. 3H shows the same for CA1 as in fig. 3G.

Figure 3I shows the average intensity values of Α β (12F4) (12F4 antibodies) normalized to the non-stimulated control (average s.e.m.; P <0.0001 in bar graph, unpaired mann-whitney test, for each group of 7 mice).

FIG. 4A shows immunohistochemistry with anti-Iba 1 (019-.

Fig. 4B shows the same for CA1 as in fig. 4A.

Figure 4C shows the number of Iba1 positive microglia in AC and CA1 (average s.e.m. in bar graph,. P < 0.05; unpaired mann-whitney test, 8 mice per group).

Figure 4D shows the diameter of Iba1 positive microglia bodies in AC and CA1 normalized to non-stimulated controls (average s.e.m. P <0.0001 in bar graph, n-8 mice per group; unpaired mann-whitney test).

Figure 4E shows the average length of Iba1 positive microglia protodentates in AC and CA1 normalized to non-stimulated controls (average s.e.m. P <0.0001 in bar graph, n-8 mice per group; unpaired mann-whitney test).

Figure 4F shows the average dentition branches of Iba1 positive microglia in AC and CA1 normalized to non-stimulated controls (n-8 mice per group, average s.e.m. in bar graph,. P <0.05,. P < 0.01; unpaired mann-whitney test).

Figure 4G shows the percentage of Iba1 positive microglia bodies that were also Α β positive in AC and CA1 (average s.e.m. in bar graphs, P <0.01, P <0.001 per group of n-8 mice; unpaired mann-whitney test).

Figure 4H shows immunohistochemistry with anti-S100B (ab868, purple) and anti-GFAP (ab4674, grey) antibodies in AC in 5XFAD mice (n-8 per group, scale bar, 50 μm) after 7 days of non-irritating or auditory GENUS at 1 hour per day.

Fig. 4I shows the same for CA1 as in fig. 4H.

Figure 4J shows the number of S100B positive astrocytes in AC and CA1 (n ═ 8 mice per group,. P < 0.05; unpaired mann-whitney test).

Fig. 4K shows the same contents as in fig. 4I for GFAP-positive astrocytes.

FIG. 5A shows immunohistochemistry (scale bar, 50 μm) generated with lectin stain (DL-1174, green) in AC of 6-month old 5XFAD mice after 7 days of non-irritating or auditory GENUS at 1 hour per day.

Fig. 5B shows the same for CA1 as in fig. 5A.

Figure 5C shows the percent fold change in vascular diameter in AC and CA1 of 6 month old 5XFAD mice after 7 days of 1 hour per day without stimulation or auditory GENUS normalized to a non-stimulated control (n-7 mice per group, mean s.e.m. in bar graph,. P < 0.0001; unpaired mann-whitney test).

Figure 5D shows immunohistochemistry generated with anti-LRP 1(28320, red), anti- Α β (AB9234, green) and lectin stain (DL-1174, grey) antibodies in the AC of 6 month old 5XFAD mice (n-8 mice per group, scale bar, 50 μm) after 7 days of non-irritating or auditory GENUS at 1 hour per day.

Fig. 5E shows the same for CA1 as in fig. 5D.

Figure 5F shows the percentage of Α β -LRP1 co-localisation in AC and CA1 of 5XFAD mice after 7 days of 1 hour per day of non-irritating or auditory GENUS (n ═ 8 mice per group,. P < 0.05; unpaired mann-whitney test).

Fig. 6A shows the firing rate modulation (left, bottom) of a single unit during 40Hz audio-visual stimulation. The grid plot shows the spike response (left, top) of the single unit inferred by both embodiments to a 10 second 40Hz auditory or random stimulus. Vector intensity distribution of 40Hz audio-visual stimuli, random audio-visual stimuli and periods of no stimulation (right, P)<0.0001，P＝9x10^-5940Hz vs. no stimulation, P1 x10^-1340Hz vs. random; kolmogorov-smihrThe nover test).

Fig. 6B shows the same as fig. 6A for CA1 (right side, P)<0.0001，P＝6x10^-4140Hz vs. no stimulation, P2 x10^-1140Hz vs. random; kolmogorov-smirnov test).

Fig. 6C shows the same contents as fig. 6A for mPFC (right side, P)<0.0001，P＝2x10^-2340Hz vs. no stimulation, P9 x10^-540Hz vs. random; kolmogorov-smirnov test).

FIG. 6D shows immunohistochemistry and 3D reconstruction using IMARIS (method) of anti-Iba 1(019-19741) and anti-Abeta (12F4) antibodies in AC, VC, CA1 and mPF C of 6 month old 5XFAD mice after 7 days of no stimulation at 1 hour per day (n ═ 6 mice per group, top inset: example using IMARIS to quantify the amount of microglia at a radius of 25 μm around the amyloid plaques.

Fig. 6E shows the same for combined GENUS as in fig. 6D.

Figure 6F shows the mean microglial body diameters in AC, VC, CA1 and mPFC of 6 month old 5XFAD mice 7 days after 1 hour stimulation or combined GENUS (a + V stimulation) normalized to the non-stimulated control (n ═ 6 mice in the no control group, n ═ 7 mice in the combined GENUS group, mean s.e.m.,. P <0.0001 in the bar graph; unpaired mann-whitney test).

Figure 6G shows the average microglial neurite length in AC, VC, CA1 and mPFC of 6 month old 5XFAD mice normalized to non-stimulated control, 7 days after 1 hour of stimulation or combination GENUS per day (n ═ 6 mice in the no control group, n ═ 7 mice in the combination GENUS group, mean s.e.m. in the bar graphs, × <0.01 × P < 0.0001; unpaired mann-whitney test).

Figure 6H shows the microglial counts per region of interest in AC, VC, CA1, and mPFC of 6-month-old 5XFAD mice after 7 days of no stimulation or combined GENUS per 1 hour day (n ═ 6 mice in the no control group, n ═ 7 mice in the combined GENUS group, mean s.e.m. in the bar graphs, # P <0.05, # P <0.01, unpaired mann-whitney test).

Figure 6I shows the average number of microglia around a radius of 25 μm for plaques in AC, VC, CA1 and mPFC after no stimulation or combination of GENUS (n ═ 6 mice per group, average s.e.m., n.s. in bar graph, not significant, P < 0.05; unpaired mann-whitney test).

Fig. 7A shows immunohistochemistry (images taken with a 40-fold objective lens, scale bar, 50 μm) of anti- Α β plaque (D54D2, green) antibodies in AC, VC, CA1 and mPFC in 5XFAD mice of 6 months of age after 7 days of no stimulation at 1 hour per day.

Fig. 7B shows the same contents as in fig. 7A for the combined GENUS.

Figure 7C shows the average plaque core area in AC, CA1, mPFC and VC for 6 month old 5 ad mice after stimulation at 1 hour per day, 40Hz auditory stimuli, combination (a + V) GENUS, combination (a + V)80Hz and combination (a + V) random frequency for 7 days normalized to non-stimulated controls (n ═ 12 mice per group, mean xfsem, n.s. insignificant in bar graphs, × P <0.05, kruscarl-wallis test and dunen multiple comparison test).

Figure 7D shows the average plaque numbers in AC, CA1, mPFC and VC for 6 month old 5XFAD mice after stimulation for 1 hour per day, 40Hz auditory stimulation, combination (a + V) GENUS, combination (a + V)80Hz, and combination (a + V) random frequency for 7 days normalized to non-stimulated controls (n-12 mice per group, mean s.e.m., n.s. not significant in bar graphs, P <0.05, kruscarl-willis test and dunen multiple comparison test).

FIG. 7E shows the relative solubility of A β in mPCCs of 6 month old 5XFAD mice stimulated at 40Hz auditory stimuli, combination (A + V) GENUS, combination (A + V)8Hz, or combination (A + V) random frequencies for 7 days at 1 hour per day normalized to non-stimulated control_1-42Levels (4-5 mice per group, mean s.e.m. in bar graph, n.s. not shownH, P<0.05, kruskal-voris test and dunne multiple comparison test).

Fig. 7F shows the same as in fig. 7E for insoluble a β 1-42 (n.s.: not significant, P < 0.05).

Figure 7G shows SHIELD-treated immunohistochemistry (light sheet illumination microscope, scale bar, 700 μm) of whole brains (sagittal plane of 25 μm sections of brain) against Α β plaques (D54D2, white) in 6-month-old 5XFAD mice after 7 days of no stimulation at 1 hour per day.

Fig. 7H shows the same contents as in fig. 7G for the combined (a + V) GENUS.

Figure 7I shows the average number of cortical plaques (average s.e.m. in bar graphs, P < 0.05; unpaired mann-whitney test) after non-stimulated or combined (a + V) GENUS.

FIG. 7J shows the mean cortical plaque volume (μm) after combined (A + V) GENUS³) (n-6 mice per group, average s.e.m. in bar graph, P<0.05; unpaired mann-whitney test).

Fig. 8A shows the average LFP response to auditory mapping tones used to detect the auditory cortex (left). The blue area indicates when the 50ms mapped tone is played. Example of clustering putative single units (right).

Fig. 8B shows the Power Spectral Density (PSD) response to 40Hz auditory flicker stimulation and non-stimulation periods with mean and standard deviation across recording days (left), the power spectral LFP response in AC to auditory flicker for all recording days (showing the recording site with the maximum 40Hz peak during 40Hz auditory flicker per recording depth, see method) (right).

Figure 8C shows the average Firing Rate (FR) of a single unit in AC during 40Hz auditory stimulation versus no stimulation period (left), the average firing rate difference of a single unit between multiple stimulation conditions in AC center around 0Hz (right, P < 0.000140 Hz-no stimulation, all other n.s.; wiegmann test on zero median).

Fig. 8D shows the excitation rate adjustment of the putative single unit in response to 20Hz audio flicker stimulation (left, bottom), and the grid plot shows the spike in response to 10s stimulation (left, top). The distribution of the intervals between peaks in the excitation rate response to 20Hz audio stimuli (on the right, the ratio of intervals around the inter-stimulus interval: P020 Hz versus no stimulus; z-test for both ratios).

Fig. 8E shows the same unit of firing rate adjustment (left, bottom) as shown at D in response to an 80Hz audio flicker stimulus, and the grid plot shows the spikes (left, top) in response to a 10s stimulus. The distribution of the intervals between peaks in the response to the excitation rate of an 80Hz audio stimulus (on the right, the ratio of intervals around the inter-stimulus interval: P080 Hz vs. no stimulus; z-test for both ratios).

Fig. 8F shows the vector intensity distribution (left, P) of 20Hz and 80Hz auditory stimuli relative to the non-irritating condition<0.0001，P＝3x10^-6120Hz vs. no stimulation, P3 x10^-6180Hz versus no stimulation; kolmogorov-smirnov test) and rayleigh statistical distribution of 20Hz and 80Hz auditory stimuli versus non-stimulation (right, P<0.0001，P＝3x10^-7320Hz vs. no stimulation, P1 x10^-6880Hz versus no stimulation; kolmogorov-smirnov test; a 20Hz stimulation RS value of 54 units is greater than 30; 80Hz stimulation RS values of 28 units are greater than 30).

Fig. 8G shows an example for detecting the theta rhythm (sign of hippocampus) of CA 1.

Fig. 8H shows the same as B for CA 1.

Fig. 8I shows the same as C for CA1 (right, n.s.; wilson signed rank test for zero median).

Fig. 8J shows the same as D for CA1 (on the right, P020 Hz versus no stimulation; z test for both scales).

Fig. 8K shows the same as E for CA1 (right, P080 Hz versus no stimulation; z test for both scales).

Fig. 8L shows the same as F for CA1 (left, P)<0.0001，P＝1x10^-4020Hz vs. no stimulation, P9 x10^-4580Hz versus no stimulation; kolmogorov-smirnov test; right side, P<0.0001，P＝1x10^-7120Hz vs. no stimulation, P8 x10^-7380Hz versus no stimulation; kolmogorov-smirnov test).

Fig. 8M shows a histological image showing the probe trajectory and recording position in the mPFC. The red arrow indicates the recording position.

Fig. 8N shows the same contents as B for mPFC.

Fig. 8O shows the same as C for mPFC (right, n.s.; wilson signed rank test for zero median).

Fig. 8P shows the same as D for mPFC (on the right, P020 Hz versus no stimulation; z test for both ratios).

Fig. 8Q shows the same as E for mPFC (right, P080 Hz versus no stimulation; z test for both scales).

Fig. 8R shows the same contents as F for mPFC (left, P)<0.0001，P＝1x10^-2320Hz vs. no stimulation, P6 x10^-2480Hz versus no stimulation; kolmogorov-smirnov test; right side, P<0.0001，P＝2x10^-1720Hz vs. no stimulation, P4 x10^-2680Hz versus no stimulation; kolmogorov-smirnov test).

Figure 9A shows the time (seconds) spent exploring familiar and new objects during NOR testing of 5XFAD non-stimulated, 40Hz and random frequency stimulated mice (n ═ 20 mice in the non-stimulated group, n ═ 20 mice in the 40Hz group, n ═ 9 in the random frequency group, mean values in the bar graph s.e.m., (0.0001), n.s.. insignificant, kruscarl-wallis test and dunen multiple comparison test).

Figure 9B shows the time (minutes) required for a mouse to reach 20s total object exploration requirement during the NOR test (n ═ 20 mice in the non-stimulated group, n ═ 20 mice in the 40Hz group, n ═ 9 in the random frequency group, mean s.e.m. in the bar graph, n.s.: not significant, kruscarl-wallis test and dunne multiple comparison test).

Figure 9C shows the time (seconds) spent exploring objects in familiar and new locations during the NOL test of 5XFAD non-stimulated, 40Hz and random frequency stimulated mice (n ═ 20 mice in the non-stimulated group, n ═ 20 mice in the 40Hz group, n ═ 9 in the random frequency group, mean values in the bar graph s.e.m.,. P <0.001,. P <0.0001, n.s. insignificant, kruscarl-wallis test and dunen multiple comparison test).

Figure 9D shows the time (minutes) required for the mice to reach 20s total object exploration requirement during the NOL test (n ═ 20 mice in the non-stimulated group, n ═ 20 mice in the 40Hz group, n ═ 9 in the random frequency group, mean s.e.m. in the bar graph, n.s.: not significant, kruskarl-wallis test and dunne multiple comparison test).

Fig. 9E shows the average velocity (cm/s) during habituation (n 20 mice in the non-stimulated group, n 20 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in bar graph, n.s. not significant, krustal-wallis test and dunne multiple comparison test).

Fig. 9F shows the total distance (cm) traveled during habituation (n 20 mice in the non-stimulated group, n 20 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test)

Fig. 9G shows the time (seconds) spent in the center of the behavioral chamber during habituation (n 20 mice in the non-stimulated group, n 20 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

Fig. 9H shows the time (seconds) spent in the periphery of the behavioral chamber during habituation (n-20 mice in the non-stimulated group, n-20 mice in the 40Hz group, n-9 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

Fig. 9I shows the average swimming speed (cm/s) during the Morris water maze (n 25 mice in the non-stimulated group, n 28 mice in the 40Hz group, n 9 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

Fig. 9J shows the average velocity (cm/s) during 1 hour of non-stimulation, auditory GENUS, or random frequency stimulation (n ═ 6 mice in the non-stimulated group, n ═ 6 mice in the 40Hz group, n ═ 6 in the random frequency group, average s.e.m. in the bar graph, n.s.: not significant, kruscarl-wallis test and dunne multiple comparison test).

Fig. 9K shows the total distance (cm) traveled during 1 hour of non-stimulation, auditory GENUS, or random frequency stimulation (n 6 mice in the non-stimulated group, n 6 mice in the 40Hz group, n 6 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

Fig. 9L shows the time (seconds) spent at 2cm/s during 1 hour of non-irritating, auditory GENUS or random frequency stimulation (n 6 mice in the non-irritating group, n 6 mice in the 40Hz group, n 6 in the random frequency group, mean s.e.m. in the bar graph, n.s. not significant, kruscarl-wallis test and dunne multiple comparison test).

FIG. 10A shows the relative solubility A β in the Auditory Cortex (AC) and Hippocampus (HPC) of 6-month-old 5XFAD mice after auditory stimulation at 40Hz, 8Hz, 80Hz, or random frequency (1 hour per day for 7 days) normalized to non-stimulated control_1-40Levels (note: ELISA for 80Hz and random frequency HPC samples was unsuccessful and not 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, n-6 in the random frequency group, mean s.e.m. in the bar graph, P<0.01, n.s. -not significant, kruskal-wallis test and dunne multiple comparison test).

FIG. 10B shows auditory sensation of 6-month-old APP/PS1 mice after auditory GENUS (1 hour per day for 7 days) normalized to non-stimulated controlRelative solubility A β in cortex (AC) and Hippocampus (HPC)_1-42Levels (n-4 mice in the non-stimulated group, n-4 mice in the 40Hz group, mean s.e.m. in bar graph, P<0.05, unpaired mann-whitney test).

Figure 10C shows the average plaque number in AC and CA1 ("region of interest", ROI) of 9 month old APP/PS1 mice (n 5 mice in the non-stimulated group, n 5 mice in the 40Hz group, mean s.e.m. in the bar graph, P <0.05, unpaired mann-whitney test) after auditory GENUS (1 hour per day for 7 days) normalized to non-stimulated control.

Figure 10D shows the average plaque core area in AC and CA1 of 9 month old APP/PS1 mice (n 5 mice in the non-stimulated group, n 5 mice in the 40Hz group, average s.e.m. in the bar graph, P <0.05, unpaired mann-whitney test) after auditory GENUS (1 hour per day for 7 days) normalized to non-stimulated control.

Figure 10E shows the average intensity values (12F4 antibody) of Α β (12F4) in AC and CA1 of 9 month old APP/PS1 mice (n-5 mice in the non-stimulated group, n-5 mice in the 40Hz group, average s.e.m. in the bar graph, P <0.05, unpaired mann-whitney test) after auditory GENUS (1 hour per day for 7 days) normalized to non-stimulated control.

Figure 10F shows the average plaque numbers in AC and CA1 for 6 month old 5XFAD mice after 7 days of no stimulation after auditory GENUS (1 hour per day for 7 days) normalized to a non-stimulated control (n 6 mice in the non-stimulated group, n 6 mice in the 40Hz group, average s.e.m., n.s. not significant in the bar graph, unpaired mann-whitney test).

Figure 10G shows the average plaque core area in AC and CA1 of 6 month old 5XFAD mice after 7 days of no stimulation after auditory GENUS (1 hour per day for 7 days) normalized to a non-stimulated control (n 6 mice in the non-stimulated group, n 6 mice in the 40Hz group, mean s.e.m., n.s. not significant in the bar graph, unpaired mann-whitney test).

Figure 10H shows the average intensity values (12F4 antibody) for Α β (12F4) in AC and CA1 of 6 month old 5XFAD mice after 7 days of no stimulation (1 hour per day for 7 days) normalized to non-stimulated control (n ═ 6 mice in the non-stimulated group, n ═ 6 mice in the 40Hz group, average s.e.m., n.s. in the bar graph, unpaired mann-whitney test).

Figure 11A shows the diameter of Iba1 positive microglia bodies in AC and CA1 of 9 month old APP/PS1 mice (n ═ 5 mice per group, mean s.e.m. in bar graphs,. P <0.05, unpaired mann-whitney test) normalized to non-stimulated controls after auditory GENUS (1 hour per day for 7 days).

Figure 11B shows the average length of Iba1 positive microglia in AC and CA1 of 9 month old APP/PS1 mice (n ═ 5 mice per group, average s.e.m. in bar graphs,. P <0.05, unpaired mann-whitney test) normalized to non-stimulated control after auditory GENUS (1 hour per day for 7 days).

Figure 11C shows the number of Iba1 positive microglia in AC and CA1 of 9 month old APP/PS1 mice (n ═ 5 mice per group, mean s.e.m. in bar graphs,. P <0.05, unpaired mann-whitney test) normalized to non-stimulated controls after auditory GENUS (1 hour per day for 7 days).

Figure 11D shows the diameter of Iba1 positive microglia in AC and CA1 of 6 month old 5XFAD mice after 7 days of no stimulation after auditory GENUS (1 hour per day for 7 days) normalized to non-stimulated control (n ═ 6 mice per group, mean s.e.m., n.s. insignificant in bar graph, unpaired mann-whitney test).

Figure 11E shows the average length of Iba1 positive microglial processes in AC and CA1 (average s.e.m., n.s.: insignificant, unpaired mann-whitney test in bar graphs, 6 month old 5XFAD mice after 7 days of no stimulation normalized to non-stimulated control (6 mice per group, average s.e.m., n.s.: insignificant in bar graphs).

Figure 11F shows the number of Iba1 positive microglia in AC and CA1 of 6 month old 5XFAD mice after 7 days of no stimulation after auditory GENUS (1 hour per day for 7 days) normalized to non-stimulated control (n ═ 6 mice per group, mean s.e.m. in bar graph, # P <0.05, n.s.: insignificant, unpaired mann-whitney test).

Figure 11G shows immunohistochemistry of category treated brain slices generated with anti-GFAP (ab4674, red) and lectin stain (DL-1174, green) antibodies in CA1 of 6 month old WT and 5XFAD mice (n ═ 5 mice per group, scale bar, 50 μm).

Figure 11H shows the number of GFAP positive cells in CA1 (per image of interest, using IMARIS) for 6-month old WT and 5XFAD mice (average s.e.m. P <0.01 in bar graph, unpaired mann-whitney test per group of n-5 mice).

Fig. 12A shows immunohistochemistry (images taken with a 40-fold objective lens, scale bar, 50 μm) generated with anti-pTau (T181, red) antibody in AC of 6-month old P301S mice after 7 days of no stimulation or auditory GENUS at 1 hour per day.

Fig. 12B shows the same for CA1 as in fig. 12A.

Figure 12C shows relative pTau (T181) intensity levels in AC and CA1 of P301S mice (n ═ 10 mice per group, P <0.001, × P < 0.0001; unpaired mann-whitney test) normalized to non-stimulated control, after 7 days of non-stimulated or auditory GENUS at 1 hour per day.

Fig. 12D shows immunohistochemistry (scale bar, 50 μm) generated with anti-pTau (S396, green) antibody in AC of 6 month old P301S mice after 7 days of non-irritating or auditory GENUS at 1 hour per day.

Fig. 12E shows the same for CA1 as in fig. 12D.

Fig. 12F shows the relative pTau (S396) intensity levels in AC and CA1 in P301S mice (n ═ 10 mice per group, P × < 0.0001; unpaired mann-whitney test) after 7 days of non-stimulated or auditory GENUS at 1 hour per day normalized to non-stimulated control.

Fig. 12G shows a representative western blot showing the levels of pTau (S396) and total tau in ACs of 6 months of age P301S mice 7 days after 1 hour per day of non-irritating or auditory GENUS.

Fig. 12H shows the same contents as in fig. 12G for the hippocampus.

Fig. 12I shows representative western blots showing levels of pTau (T181) and total tau in the hippocampus of 6-month old P301S mice after 7 days of non-irritating or auditory GENUS at 1 hour per day.

Fig. 12J shows the relative immunoreactivity of pTau (S396) normalized to total tau (from western blot in G) in ACs from P301S mice 7 days after 1 hour per day of no stimulation or auditory GENUS (average s.e.m. P <0.05 in bar graph per group of n-3 mice; unpaired mann-whitney test).

Figure 12K shows the relative immunoreactivity of pTau (S396) normalized to total tau (from western blot in H) in HPCs of P301S mice 7 days after 1 hour per day of non-stimulated or auditory GENUS (2 mice in the 40Hz group and 3n in the non-stimulated group, mean s.e.m. in the bar graph,. P < 0.001; unpaired mann-whitney test).

Figure 12L shows the relative immunoreactivity of pTau (T181) normalized to total tau (from western blot in I) in hippocampus of P301s mice after 7 days of 1 hour per day without stimulation or auditory GENUS (2 mice in the 40Hz group and 3n in the non-stimulated group, mean s.e.m. in bar graphs, < P < 0.01; unpaired mann-whitney test).

Fig. 13A shows the Power Spectral Density (PSD) response to 40Hz audio-visual flicker stimulation and the periods of no stimulation with mean and standard deviation across recording days (left), the power spectral LFP response in AC to audio-visual flicker stimulation for all recording days (showing the recording sites with the maximum 40Hz peak during 40Hz audio-visual flicker per recording depth, see method) (right).

FIG. 13B illustrates the firing rate adjustment (lower) of the putative single unit to audiovisual random stimulus shown in FIG. 13A; the grid plot (top) shows the spikes (left) of a single unit to 10s random stimulation. Distribution of intervals between excitation rate response peaks to an audiovisual stimulus in AC (center, thorn)Interval ratio around the laser interval: p040 Hz is non-irritating, P040 Hz is random; z test for two ratios), rayleigh statistical distribution of single units versus 40Hz audio-visual stimuli (right, P)<0.0001，P＝2x10^-7940Hz vs. no stimulation, P1 x10^-7240Hz vs. random; kolmogorov-smirnov test; a 40Hz stimulation RS value of 20 units is greater than 30; the 2 units of random stimulation RS value is greater than 30.

Fig. 13C shows the single unit average firing rate during all audiovisual stimulation conditions.

Fig. 13D shows the excitation rate adjustment of the putative single unit in response to a 20Hz audio-visual flicker stimulus (left, bottom), and the grid plot shows the spike in response to a 10s stimulus (left, top). The distribution of the intervals between peaks in the response to the firing rate of 20Hz audio-visual stimuli (on the right, the ratio of intervals around the inter-stimulus interval: P020 Hz versus no stimulus; z-test was performed for both ratios).

Fig. 13E shows the same unit of firing rate adjustment (left, bottom) as shown at D in response to an 80Hz audio visual flicker stimulus, and the grid plot shows the spikes (left, top) in response to a 10s stimulus. The distribution of the intervals between peaks in the response to the firing rate of an 80Hz audio-visual stimulus (on the right, the ratio of intervals around the inter-stimulus interval: P080 Hz vs. no stimulus; z-test for both ratios).

Figure 13F shows the vector intensity distribution of 20Hz and 80Hz auditory stimuli above the no stimulation condition (left, P)<0.0001，P＝2x10^-6320Hz vs. no stimulation, P1 x10^-5680Hz versus no stimulation; kolmogorov-smirnov test) and 20Hz and 80Hz auditory stimuli were higher than the non-stimulated rayleigh statistical distribution (right, P<0.0001，P＝1x10^-8820Hz vs. no stimulation, P5 x10^-7280Hz versus no stimulation; kolmogorov-smirnov test; a 20Hz stimulation RS value of 50 units is greater than 30; 19 units of 80Hz stimulation RS values greater than 30).

Figure 13G shows the average firing rate difference between the individual units in AC centers between multiple stimulation conditions around 0Hz ([ P < 0.0520 Hz-80 Hz, [ P < 0.0520 Hz-40 Hz, all other n.s.; wilson symbol rank test on zero median).

Fig. 13H shows the same as a for CA 1.

Fig. 13I shows the same as B for CA1 (right, P)<0.0001，P＝1x10^-7140Hz vs. no stimulation, P1 x10^-7140Hz vs. random; kolmogorov-smirnov test; center, interval ratio around the inter-stimulus interval: p040 Hz is non-irritating, P040 Hz is random; z-test for two ratios).

Fig. 13J shows the same as C for CA 1.

Fig. 13K shows the same as D for CA1 (on the right, P020 Hz versus no stimulation; z test for both scales).

Fig. 13L shows the same as E for CA1 (right, P080 Hz versus no stimulation; z test for both scales).

Fig. 13M shows the same as F for CA1 (left, P)<0.0001，P＝8x10^-4320Hz vs. no stimulation, P8 x10^-4080Hz versus no stimulation; kolmogorov-smirnov test; right side, P<0.0001，P＝2x10^-7020Hz vs. no stimulation, P1 x10^-5780Hz versus no stimulation; kolmogorov-smirnov test).

Fig. 13N shows the same as G for CA1 (all n.s.; wilson signed rank test for zero median).

Fig. 13O shows the same contents as a for mPFC.

Fig. 13P shows the same as B for mPFC (right, P)<0.0001，P＝5x10^-2340Hz vs. no stimulation, P3 x10^-2140Hz vs. random; kolmogorov-smirnov test; center, interval ratio around the inter-stimulus interval: p040 Hz is non-irritating, P040 Hz is random; z-test for two ratios).

FIG. 13Q shows the same as C for mPF.

Fig. 13R shows the same as D for mPFC (on the right, P020 Hz versus no stimulation; z test for both ratios).

Fig. 13S shows the same as E for mPFC (right, P080 Hz versus no stimulation; z test for both scales).

Fig. 13T shows the same contents as F for mPFC (left, P)<0.0001，P＝1x10^-2320Hz vs. no stimulation, P2 x10^-2580Hz versus no stimulation; kolmogorov-smirnov test; right side, P<0.0001，P＝1x10^-2320Hz vs. no stimulation, P8 x10^-2580Hz versus no stimulation; kolmogorov-smirnov test).

Fig. 13U shows the same as G for mPFC (./P < 0.0540 Hz — no stimulation, all other n.s.; weissen sign rank test for zero median).

FIG. 14A shows immunohistochemistry (inset, 100-fold; scale bar, 50 μm) for anti-IBa 1(019-19741, green) and anti-Abeta (12F4, red) antibodies in AC, CA1 and mPFC of 6-month old 5XFAD mice after 7 days of auditory GENUS per day 1 hour.

FIG. 14B shows immunohistochemistry (inset, 100-fold; scale bar, 50 μm) for anti-IBa 1(019-19741, green) and anti-Abeta (12F4, red) antibodies in VC, CA1 and mPFC of 6 month old 5XFAD mice after 7 days of visual GENUS1 hour per day.

Figure 14C shows mean microglia cell body diameters in AC, CA1 and mPFC of 6 month old 5XFAD mice 7 days after 1 hour auditory GENUS per day normalized to non-stimulated control (n ═ 6 mice per group, mean s.e.m. in bar graph, n.s.: insignificant,. P < 0.01; unpaired mann-whitney test).

Figure 14D shows the average microglial neurite length in AC, CA1, and mPFC of 6 month old 5XFAD mice after 7 days of 1 hour auditory GENUS per day normalized to non-stimulated control (n ═ 6 mice per group, average s.e.m. in bar graph, n.s.: not significant,. P < 0.01; unpaired mann-whitney test).

Figure 14E shows the microglial counts per region of interest in AC, CA1 and mPFC for 6 month old 5XFAD mice after 7 days of 1 hour auditory GENUS per day (n ═ 6 mice per group, mean s.e.m. in bar graph, n.s. ═ insignificant, × P < 0.01; unpaired mann-whitney test).

Figure 14F shows the average number of microglia around a 25 μm radius of plaques in AC, CA1 and mPFC after no stimulation or auditory GENUS (average s.e.m., n.s. not significant in bar graph, unpaired mann-whitney test per group of 6 mice).

Figure 14G shows mean microglia cell body diameters in VC, CA1, and mPFC in 6 month old 5XFAD mice after 7 days of 1 hour visual GENUS per day normalized to non-stimulated control (n ═ 6 mice per group, mean s.e.m. in bar graph, n.s.: not significant,. P < 0.05; unpaired mann-whitney test).

Figure 14H shows the average microglial neurite length in VC, CA1, and mPFC in 6 month old 5XFAD mice after 7 days of 1 hour visual GENUS per day normalized to no-stimulation control (n ═ 6 mice per group, average s.e.m. in bar graph, n.s.: insignificant,. P < 0.05; unpaired mann-whitney test).

Figure 14I shows the microglial counts per region of interest in VC, CA1, and mPFC for 6 month old 5XFAD mice after 7 days of 1 hour visual GENUS per day (n.6 mice per group, mean s.e.m. in bar graph, n.s. not significant, unpaired mann-whitney test).

Figure 14J shows the average number of microglia around a 25 μm radius of plaques in VC, CA1 and mPFC after no stimulation or visual GENUS (average s.e.m., n.s. not significant in bar graph, unpaired mann-whitney test per group of 6 mice).

Figure 14K shows the mean microglial body diameters in AC, CA1 and mPFC for 6 month old 5XFAD mice after 1 hour per day of 40Hz auditory stimulation, combination (a + V) GENUS, combination (a + V)80Hz or combination (a + V) random frequency stimulation for 7 days normalized to non-stimulated controls (n ═ 6 mice per group, mean s.e.m., n.s. not significant in bar graphs,. P <0.05,. P < 0.01; unpaired mann-whitney test).

Figure 14L shows the average microglial neurite length in AC, CA1, and mPFC of 6 month old 5XFAD mice after 1 hour per day of 40Hz auditory stimulation, combination (a + V) GENUS, combination (a + V)80Hz, or combination (a + V) random frequency stimulation for 7 days normalized to non-stimulated controls (n ═ 6 mice per group, mean s.e.m., n.s. not significant in the bar graph, P <0.05, unpaired mann-whitney test).

Figure 14M shows microglial counts per region of interest in AC, CA1, and mPFC for 6 month old 5XFAD mice after 7 days of 40Hz auditory stimulation, combined (a + V) GENUS, combined (a + V)80Hz, or combined (a + V) random frequency stimulation normalized to non-stimulated controls (n ═ 6 mice per group, mean s.e.m., n.s. not significant in bar graphs,. P <0.05, unpaired mann-whitney test).

Figure 14N shows immunohistochemistry for anti- Α β plaque (D54D2, green) antibodies in AC, CA1 and mPFC in 6 month old 5XFAD mice after 1 hour of auditory GENUS7 days per day (N ═ 6 mice per group, scale bar, 50 μm).

Figure 14O shows immunohistochemistry for anti- Α β plaque (D54D2, green) antibody in VC, CA1 and mPFC in 6 month old 5XFAD mice after 1 hour visual GENUS7 days per day (n ═ 6 mice per group, scale bar, 50 μm).

Figure 14P shows the average plaque core area per region of interest normalized to no-stimulation control (n 6 per group, mean s.e.m., n.s. not significant in bar graph, P <0.05, unpaired mann-whitney test).

Figure 14Q shows the average plaque number in AC, CA1 and mPFC after auditory GENUS normalized to non-irritating controls (n 6 per group, average s.e.m. in bar graph, n.s. not significant, P <0.05, P <0.001, unpaired mann-whitney test).

Figure 14R shows the average plaque core area per region of interest normalized to the non-stimulated control (n 6 per group, mean s.e.m., n.s. not significant in bar graph, P <0.05, unpaired mann-whitney test).

Figure 14S shows the average plaque number in VC, CA1 and mPFC after normalization to non-irritant control, visual GENUS (average s.e.m., n.s. not significant in bar graph,. P <0.05, unpaired mann-whitney test).

FIG. 14T shows the relative solubility A β in AC and HPC of 6 month old 5XFAD mice after stimulation at 1 hour per day of no stimulation, 40Hz auditory stimulation, combination (A + V) GENUS, combination (A + V)8Hz, and combination (A + V) random frequency for 7 days, normalized to a no-stimulation control_1-42Levels (n.e.m., mean s.e.m., n.s. not significant, p.p. in bar graphs, 4-5 per group, p.s. not significant<0.05, kruskal-voris test and dunne multiple comparison test).

FIG. 14U shows the relative insolubility of A β in AC and HPC of 6 month old 5XFAD mice after stimulation at 1 hour per day with no stimulation, 40Hz auditory stimulation, combination (A + V) GENUS, combination (A + V)8Hz, and combination (A + V) random frequency for 7 days, normalized to a non-stimulated control_1-42Levels (n.e.m., mean s.e.m., n.s. not significant, p.p. in bar graphs, 4-5 per group, p.s. not significant<0.05, kruskal-voris test and dunne multiple comparison test).

Figure 14V shows the a β (12F4) mean intensity values (12F4 antibody) in AC, CA1 and mPFC for 6 month old 5XFAD mice after 1 hour per day stimulation, 40Hz auditory stimulation, combination (a + V) GENUS, combination (a + V)80Hz, and combination (a + V) random frequency stimulation for 7 days normalized to non-stimulated controls (n-4-5 mice per group, mean s.e.m., n.s. -not significant in bar graphs, × P <0.05, kruscarl-wallis test and engenden multiple comparison test).

40Hz auditory stimulation modulates spike activity in AC, CA1 and mPFC

We first determined whether auditory tone stimulation can produce GENUS in the Auditory Cortex (AC), hippocampus subregion CA1, and medial prefrontal cortex (mPFC). We present the animal with a 20Hz, 40Hz, 80Hz tone sequence, or a randomly spaced tone sequence (1 ms long, 10kHz tones are played every 12.5ms, 25ms, 50ms, or randomized inter-tone intervals of 25ms on average, hereafter referred to as "auditory flicker stimuli"). During tone presentation, we performed electrophysiological recordings using 32-channel silicon probes in AC, CA1, and mPFC in 3-8 month old male wild type (C57BL6J) mice running or resting on a ball bench. To locate the AC, a series of 50ms auditory mapping tones (hereinafter "mapping stimuli") are played at different depths until a transient LFP response is detected about 20ms after the onset of the tone (fig. 8A). CA1 was located based on electrophysiological markers and the mPFC recording location was confirmed histologically after final recording in each animal (fig. 8G and 8M).

After we reached the target area, the animals were presented with staggered periods of quiet and auditory blinking stimulation while neural activity was recorded. The stimulation blocks alternate between 20Hz, 40Hz, 80Hz and random auditory flicker stimulation. The presumed single unit firing rate increases and decreases periodically with each tone, thereby entraining an auditory flicker stimulus at 40Hz (fig. 1A, 1E and 1I, blue). The units are also adjusted by random stimulation. When all random pulses are aligned, the firing rate adjustment changes after stimulation, indicating that the individual units respond to the random stimulation pulses. However, the random sequence of auditory tones does not cause periodic excitation modulation (fig. 1A, 1E and 1I, orange). Entrainment varies between single units in both phase distribution and amplitude. During the blinking stimulation, neurons fire according to the stimulation, but they do not fire every cycle and typically fire over a wide range of phases (fig. 1A, 1E, and 1I). In most individual units, the interval between excitation rate peaks during auditory flicker stimulation is about 25ms (equivalent to 40 Hz); 75% in AC, 79% in CA1, and 74% in mPF. (FIG. 1B, FIG. 1F and FIG. 1J).

In contrast, during the baseline period without tones and the period with random tones, the intervals between peaks have a wider distribution, with less than 11% of the cells in AC, 12% of the cells in CA1, and 16% of the cells in mPF having peak intervals of about 25ms (i.e., the firing rate was not adjusted at 40 Hz; FIG. 1B, FIG. 1F, and FIG. 1J). The modulation intensity is quantified by considering the single unit firing rate as a function of the stimulation phase and calculating its vector intensity (VS) (fig. 1C, 1G and 1K, left). The vector intensity values range from 0 to 1, where 0 represents a uniform excitation distribution that is not conditioned by the stimulation (VS ═ 0), and 1 represents a distribution that only excites neurons to a particular stimulation phase (VS ═ 1). The distribution of vector intensities of the single unit responses to 40Hz auditory stimuli ranges from 0.002 to 1 in AC, from 0.0005 to 1 in HPC, from 0.1 to 0.6 in PFC, and is significantly higher than the distribution of non-irritating and random stimuli (fig. 1C, 1G and 1K, center). The random stimulation vector intensity is also significantly higher than the non-stimulated condition because the vector intensity measures the modulation by the stimulation. However, the vector intensity does not quantify the periodicity of the modulation, and while a random stimulus does elicit a single unit response, it does not elicit a periodic excitation modulation.

Similarly, the rayleigh statistical distribution of individual units during 40Hz auditory stimulation was significantly higher than that of non-stimulated and randomly stimulated controls (fig. 1C, 1G and 1K, right). The average firing rates of individual neurons were similar between the 40Hz auditory scintillant stimulation and the controls without stimulation, random stimulation, 20Hz and 80Hz auditory scintillant stimulation (FIG. 1D, FIG. 1H and FIG. 1L; FIG. 8C, FIG. 1I and FIG. 1O, right). The local field potentials in the AC show increased power at 40Hz during auditory flicker stimulation, but the effect varies between recording position, recording phase and reaction latency to the mapped tone (fig. 8B, 1H and 1N). These findings indicate that 40Hz auditory scintillant stimulation is a strong cause of GENUS in AC, CA1 and mPFC.

Auditory GENUS improves memory performance of 5XFAD mice

Assuming auditory GENUS can affect hippocampal neural activity, we next evaluated its effect on hippocampus-related learning and memory in 6-month-old 5XFAD mice (fig. 2A). We used 5XFAD animals at 6 months of age, as this is when behavioral impairment first became apparent. For all subsequent experiments, we performed 1 week of auditory GENUS; specifically, mice were placed in a quiet room and exposed to a 1ms long sequence of 10kHz auditory tones, 1 hour per day for 7 days, at a frequency of 40Hz (thus, 40(10kHz) tones/second). We first habituated mice to the behavioral compartment 24 hours prior to testing for New Object Recognition (NOR) and New Object Localization (NOL) memory efficiencies that assessed the ability to remember the identity or placement of an object in a particular environment (i.e., behavior known to be affected in human AD subjects). These tests measure behavioral performance using an identification index, which is the percentage of time spent exploring a new object or a new location object, respectively, over the duration of the exploration.

During habituation, none of the auditory GENUS, random frequency, or unstimulated groups showed significant changes in mean velocity, total distance, time spent in the center, or time spent in the periphery, indicating that three groups did not show differences in general activity or anxiety-like behavior (fig. 9E-9H). After auditory GENUS, the 5XFAD mice exhibited significantly higher recognition index for object memory tasks (65.50 ± 1.40%) and significantly higher recognition index for position memory tasks (61.41 ± 2.0%), while the non-stimulated and random frequency controls showed no preference for new objects or new displaced objects in both tests (fig. 2B and 2E). During the task period between the three groups, there was no significant difference in distance traveled or average speed, indicating that these effects are not attributable to general differences in activity (fig. 2C, 2D, 2F, and 2G).

The amount of time spent exploring new and familiar objects during NOR was examined; we observed that mice after auditory GENUS spent a significantly higher amount of time on new objects, while the non-stimulated and random frequency control group showed no exploration preference (fig. 9A). Similarly, we observed that mice after auditory GENUS spent a significantly higher amount of time on objects in new locations (NOLs), while the non-stimulated and random frequency control group showed no exploratory preference (fig. 9C). As an additional control measure to check for differences in exploration activities, we measured the amount of time (minutes) it took for the mouse to reach the 20s target exploration requirement during the object task. We observed no significant difference in the time taken to reach the object exploration requirements between the three groups (fig. 9B and 9D).

To further characterize the effect of auditory GENUS on hippocampus-related behavior, we performed the Morris water maze test, which measures the ability to remember the location of hidden platforms relative to ambient cues. Mice learn the location of the hidden platform gradually during successive trials, and their spatial memory of the platform location is measured by the escape latency, which is the amount of time it takes for an individual mouse to find the hidden platform. During the training phase, all three groups were able to successfully learn the location of the hidden platform, however, the escape latency of the group receiving auditory GENUS was consistently and significantly shorter than both the non-stimulated and random frequency control groups (fig. 2H). There was no significant difference in swimming speed between the three groups (fig. 9I). During the probing trial, or when the hidden platform was removed from the bin, mice receiving auditory GENUS spent a significantly longer period of time exploring the quadrant containing the missing platform compared to both the non-stimulated and random frequency control groups, and showed more crossover on the previous platform positions (fig. 2I and 2J).

As a final behavioral measure, we examined the activity of 5XFAD mice during 1 hour auditory GENUS, no stimulation or random frequency stimulation, and observed no significant difference in average velocity (cm/s) and distance traveled (cm) (fig. 9J and 9K). To explore whether there were differences in the "sleep" state or resting phase, we measured the amount of time mice spent at 2cm/s during the 1 hour stimulation group. We observed no significant differences between the three groups (fig. 9L). Together, these results show that auditory GENUS can improve recognition and spatial memory in 6-month old 5XFAD mice.

Auditory GENUS reduces amyloid burden in AC and hippocampus of 5XFAD mice

The beneficial effects of auditory GENUS on cognitive function led us to investigate whether potential amyloidosis could be modified in a 5XFAD mouse model. Previously, we showed an improvement in amyloid burden in younger 3-month old mice by visual GENUS. Here, our aim was to further study the effect of auditory GENUS on 6-month-old mice, which are in a more advanced AD state and are shownMice were placed in a quiet chamber and exposed to a range of different auditory tone sequence frequencies (including 40Hz, 8Hz, 80Hz, random frequency stimulation) or no stimulation.7 days after stimulation was complete 24 hours, we analyzed amyloid loading in AC and whole Hippocampus (HPC) by a β enzyme-linked immunosorbent assay (ELISA). after 40Hz auditory stimulation, we observed soluble a β compared to no tone or additional frequency control_1-42Levels were reduced by 51.84. + -. 4.98% in AC and 46.89. + -. 3.89% in HPC, whereas soluble A β in AC and HPC_1-40The levels were reduced by 20.65 ± 3.21% and 34.15 ± 4.83%, respectively (fig. 3A and fig. 10A)_1-42Levels were reduced by 36.68 ± 3.21% in AC and 43.84 ± 2.42% in HPC (fig. 3B) — insoluble a β could not be detected by ELISA in both auditory GENUS or non-irritating controls_1-40。

Our results indicate that the observed amyloid reduction is specific to 40Hz stimulation compared to non-stimulated controls, as none of the 8Hz, 80Hz, and random frequency stimulations significantly changed A β levels to determine whether these effects are applicable to other mouse models of AD and whether our results are specific to our 5XFAD model, we examined A β levels in 6-month old APP/PS1 transgenic mice (i.e., a well-validated model of AD) after 7 days of auditory GENUS_1-4248.39 ± 3.50% was significantly reduced in AC and 35.54 ± 4.27% was significantly reduced in HPC (fig. 10B).

Next, we examined the plaque load in the 5XFAD mouse model using β -amyloid specific antibody (Cell Signaling Technology; D54D2) in immunohistochemical analysis (FIGS. 3C and 3D.) the number of plaques was significantly reduced by 45.73 + -2.818% and 59.30 + -2.083% (FIG. 3E) in AC and CA1, respectively, after 7 days of auditory GENUS compared to the non-irritating control, the plaque size was also significantly reduced by 54.37 + -5.603% and 40.70 + -5.321% (FIG. 3F) in AC and CA1, respectively, versus A β_1-42Analysis of specific immunostaining indicates A β_1-42Deposit is significantly reduced in AC and CA1, respectively45.35 ± 0.011% and 43.21 ± 0.0285% (fig. 3G to 3I). to examine the dynamics of plaque burden after 40Hz stimulation, we performed immunohistochemical analysis in 5XFAD mice using β -amyloid specific antibodies (Cell Signaling Technology; D54D2), which were first subjected to auditory GENUS for one week and then not stimulated for the next 7 days.

We observed a significant reduction in plaque number of 52.65 ± 7.53% in AC, 62.90 ± 15.5% in CA1, a significant reduction in plaque size of 67.90 ± 6.18% in AC, 64.06 ± 15.2% in CA1, using a β_1-42A β by antibody (BioLegend; 12F4)_1-42Analysis of specific immunostaining indicated that A β compared to the non-stimulated control_1-42The deposits were significantly reduced by 38.77 ± 4.21% and 47.63 ± 6.08% in AC and CA1, respectively (fig. 10C to 10E). Collectively, these results demonstrate that auditory GENUS can entrain gamma activity in AC and CA1 and reduce amyloid burden in AD mouse models.

Auditory GENUS elicited glial and vascular responses in 5XFAD mice

There is increasing evidence that microglia respond to changes in neuronal activity and play a role in AD pathology (Allen and Barres, 2005; Mosher and Wyss-Coray, 2014; Walker and Lue, 2015). Our ability to reduce amyloid burden in AC and HPC led us to examine whether auditory GENUS can stimulate changes in microglial responses in 6-month-old 5XFAD mice. Microglia have been shown to change their cellular morphology during an activation state involving phagocytosis (Davies et al, 2016), and indeed, our earlier studies demonstrated that 1 hour of visual GENUS was sufficient to cause morphological changes in microglia consistent with activation in VC and increased phagocytic activity (Iaccarino et al, 2016). Using a small antibodyAntibodies to glial marker Iba1 (fig. 4A and 4B), we observed approximately 60% more microglia in both AC and CA1 in the auditory GENUS group compared to the non-stimulated control (fig. 4C) following auditory GENUS the microglia somatic area increased 70.60 ± 4.78% in AC and 117.17 ± 10.4% in CA1 (fig. 4D) compared to the non-stimulated control we also found that microglia length decreased 46.44 ± 3.2% (AC) and 50.875 ± 4.8% (CA1) and odontic branching increased 36.00 ± 9.5% (AC) and 143.813 ± 29.9% (CA1) (fig. 4E and 4F) compared to the non-stimulated control_1-42We observed that the percentage of microglia with cell bodies co-localized with a β increased 58.75 ± 1.25% in AC and 61.33 ± 3.71% in CA1 after auditory GENUS compared to the non-stimulated control (fig. 4G).

To examine whether microglial reactions occurred after auditory GENUS in other AD mouse models, we measured the microglial morphology from 9-month-old APP/PS1 mice after 7 days of auditory GENUS. Similar to our results seen in 5XFAD microglia after 7 days of auditory GENUS (see fig. 4A-4G), we observed a significant increase in microglial body diameter and count and a significant decrease in average neurite length in AC and CA1 compared to the non-stimulated control (fig. 11A-11C).

To understand the longitudinal effects of microglial responses in 5XFAD mice after auditory GENUS, we examined microglial morphology 7 days after auditory GENUS without stimulation. We observed a trend similar to that from amyloid (fig. 10F to 10H), specifically no significant increase in microglia cell body diameter, a decrease in mean neurite length, and an increase in microglial count in the auditory cortex (see fig. 11D to 11F). However, we did see a significant increase in microglia count in CA1 (41.70 ± 6.75% increase) compared to the non-stimulated control.

Astrocytes are another primary glial cell in the central nervous system and are critical for homeostatic maintenance, synaptic pruning, waste clearance, and other important biological processes, such as regulating cerebral blood flow (Chung et al, 2015; Kisler et al, 2017). Reactive astrocytes express Glial 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 and WT littermate control mice, we performed classification on 100 μm CA1 brain slices stained for GFAP antibody (fig. 11G). We observed a significant reduction in the number of GFAP-positive astrocytes in 5XFAD mice compared to WT controls (fig. 11H). This observation is in line with reports showing other AD transgenic mouse models with similar glial atrophy (rodri i guez et al, 2009). To determine whether auditory GENUS might affect astrocytic reactivity, we subjected 6-month-old 5XFAD mice to 7 days of auditory GENUS or non-irritating controls for 1 hour per day, and then immunostained their brain slices with antibodies against GFAP and S100 calcium binding protein B (S100B), S100B being another protein that was demonstrated to be expressed in reactive astrocytes (fig. 4H and fig. 4I). Astrocytes positive for GFAP increased 27.66 ± 0.954% and 18.14 ± 0.799% in AC and CA1, respectively, and S100B positive astrocytes increased 21.83 ± 1.07% in AC and 15.57 ± 0.869% in CA1 (fig. 4J and 4K). This observed change in the number of astrocytes after auditory GENUS may indicate a potential increase in astrocyte survival.

Astrocytes are known to play an important role in regulating the vascular network of the brain, and there is increasing evidence that dysfunction of this network in AD may exacerbate the pathology. Clearance of amyloid from the brain is multifaceted, and various processes have been proposed that are performed through the vasculature, such as through the lymphatic system and via transport of the endocytic receptor lipoprotein receptor-related protein 1(LRP 1).

To investigate potential changes in the vasculature, we first used tomato lectin (tomato), a potent marker of vascular endothelium, to stain 5XFAD brain slices after auditory GENUS (fig. 5A and 5B). Interestingly, we observed an increase in vessel diameter of 49.70 ± 7.80% (AC) and 104.70 ± 10.96% (CA1) after auditory GENUS compared to the non-irritating control (fig. 5C). We further explored whether amyloid-vascular interactions were altered following auditory GENUS. We examined whether auditory GENUS might affect co-localization of Α β with LRP1, which has been shown to play an important role in Α β transport and systemic elimination by staining LRP1 and Α β via the vasculature in brain slices from 5XFAD mice exposed to 1 hour/day auditory GENUS or no stimulation for 7 days (Storck et al, 2016) (fig. 5D and 5E). In the non-stimulated control, we observed co-localization of Α β with LRP1 at 8.17 ± 2.70% (AC) and 6.97 ± 1.73% (CA1), whereas in the auditory GENUS group, the co-localization of Α β with LRP1 increased significantly to 17.71 ± 2.78% and 16.50 ± 3.90%, respectively, in AC and CA1 (fig. 5F). Together, these results suggest that one explanation for the decrease in Α β levels in AC and CA1 following auditory GENUS may be increased clearance of Α β by small glia and alterations in vasculature.

Auditory GENUS reduces tau phosphorylation in AC and hippocampus

Another classical pathological hallmark of AD is the accumulation of phosphorylated tau aggregates. Tau phosphorylation at specific amino acid residues associated with AD has been shown to alter its cytoskeletal support function and reduce its solubility, thus being considered a major damage to neurons. To investigate whether auditory GENUS might affect lesions in another mouse model associated with AD, we used Tau P301S mice. Since tau P301S mice began to exhibit spatial and contextual learning disabilities at 6 months of age, armchair we examined whether auditory GENUS could lead to a decrease in phosphorylated tau in AC and HPC in 6-month-old tau P301S mice. Immunohistochemical analysis of brain slices from these mice (fig. 12A, 12B, 12D, and 12E) indicated that auditory GENUS reduced tau phosphorylation at threonine-181 (T181) by 36.20 ± 2.828% (AC) and 38.70 ± 2.737% (CA1), and at serine-396 (S396) by 37.90 ± 3.469% (AC) and 40.80 ± 4.528% (CA1) (fig. 12C and 12F). Western Blot (WB) experiments confirmed immunohistochemical results of tau phosphorylation at S396, which showed decreased phosphorylation in AC and whole hippocampus tissues by 33.83 ± 0.20% and 43.20 ± 1.50%, respectively, compared to total tau (fig. 12G, 12H, 12J and 12K). WB analysis indicated that phosphorylated T181 tau was reduced by 34.50 ± 1.61% in hippocampus, but not significantly different in AC (fig. 12I and 12L). Together, our results show that auditory GENUS can reduce the levels of AD-associated hyperphosphorylated tau epitopes, and that auditory GENUS can affect lesions in mouse models of tauopathies.

Combined auditory and visual GENUS elicits a clustering phenoresponse in microglia

Thus, our findings demonstrate that auditory GENUS can reduce amyloid levels and cause alterations in the glial and vasculature of the cortical sensory area and hippocampus. This prompted us to investigate whether combining auditory and visual GENUS could cause more profound cellular effects. We first determined whether a combination of 40Hz auditory tone stimulation and 40Hz light flashing can entrain neural responses in AC, CA1 and mPFC. We presented a combination of 1ms long auditory tones and 12.5mm long light pulses to 3-8 month old male wild type (C57BL6J) mice at a 40Hz frequency while recording neural activity in AC, CA1, and mPFC using 32-channel silicon probes as the animals run or rest on a ball race table. The spikes increase and decrease periodically with respect to each tone and lighting period, thereby entraining to 40Hz (fig. 6A-6C, left) during the combined audio-visual stimulus. The vector intensity distribution was significantly higher during 40Hz auditory-visual stimulation than under random stimulation or no stimulation conditions (fig. 6A-6C, right). Thus, the spikes of individual neurons in AC, CA1, and mPFC were significantly more clipped to 40Hz during the auditory-visual stimulation period than during the baseline period. AC. Local field potentials in HPC and mPFC showed increased power at 40Hz during audiovisual flicker stimulation, however, the effect was small in mPFC (fig. 13A, 13H and 13O). Thus, 40Hz tone plus light stimulation induces GENUS in AC, CA1, and mPFC.

Although small, we observed differences in the Local Field Potential (LFP) response and the single unit mean firing rate of the auditory and combined auditory-visual stimuli of mPFC. There was a small increase in LFP power at 40Hz during combined stimulation, but not during auditory stimulation alone (fig. 8N and 6O). In addition, the distribution of the mean firing rate differences between the combined stimuli and baseline had a median value significantly different from zero, whereas the median value did not significantly differ from zero with respect to auditory stimuli only (fig. 8O and 6U).

After 1 hour per day for 7 days of combined GENUS, we examined the morphological features of microglia in AC, VC, and CA1 and their interaction with Α β (fig. 6D and 6E). Since higher order cognitive regions are known to handle multimodal sensory stimuli, we examined whether the combined GENUS can also cause microglial effects in the medial prefrontal cortex (mPFC). We found that microglia showed a significant increase in somatic cell area, while the projection length was significantly reduced, compared to the non-stimulated control (fig. 6F and 6G). Microglial numbers also increased significantly in AC, VC, CA1 and mPFC after combined GENUS (fig. 6H). Microglia in the individual auditory or visual stimulation groups (fig. 14A-14D, 14G, and 14H) showed reduced projection lengths and increased somatic cell areas in AC, VC, and CA1, but not in mPFC.

In contrast to visual GENUS, auditory GENUS showed a significant increase in microglial count in CA 1; neither auditory or visual GENUS alone caused significant changes in microglia count in mPFC (fig. 14E and 14I). These findings show that only combined auditory and visual stimuli (rather than auditory or visual stimuli alone) promoted the microglial response in mPFC after 1 week of GENUS.

Interestingly, the microglia in the combined GENUS group appeared to show activity changes by showing encapsulation around amyloid deposits. To better address the phenotype of clustered microglia Α β, we created a three-dimensional (3D) rendering from AC, VC, CA1 and mPFC images taken from a 5XFAD brain slice after combining GENUS and no-stimulus controls (shown in "3D reconstruction bar" in fig. 6D and 6E). Using IMARIS imaging software (see methods), we created 3D surfaces of amyloid deposits (red dots) and microglia (green dots) and quantified the proximity and number of microglia within a 25 μm radius of amyloid deposits (rightmost inset, fig. 6D and 6E, exemplary videos are provided in supplementary videos 1 and 2 showing clustered microglia Α β phenotype after combining GENUS and non-stimulated controls). We observed a significant increase in the number of microglia within a 25 μm radius around amyloid plaques 48.88 ± 0.651% in AC, 31.56 ± 1.11% in VC, and 38.64 ± 0.959% in mPFC after combination with GENUS compared to the non-stimulated control (figure 6I). We also observed a non-significant increase in CA1 of 33.05 ± 2.65%. To examine whether the clustered microglia Α β phenotype was a specific phenotype after combining GENUS, we analyzed the number of microglia within a 25 μm radius of the amyloid deposit after auditory or visual GENUS alone. We observed that the amount of microglia per plaque was not significantly different between GENUS and non-stimulated mice (fig. 14F and 14J).

Next, we solved the frequency specificity of microglial responses in 6-month-old 5XFAD mice 7 days after 40Hz auditory GENUS, combined GENUS, 80Hz, or random frequency stimulation in AC, CA1, and mPFC. We observed a significant increase in microglia body diameter and count and a significant decrease in mean neurite length in AC and CA1 after 40Hz auditory stimulation and combined GENUS compared to the otherwise frequency and non-stimulated controls.

In comparison to 40Hz auditory stimuli, additional frequency and non-stimulated controls, only GENUS in combination resulted in a microglial response in mPFC (fig. 14K-14M). These results indicate that the combined GENUS enhances microglial response through alterations in neuronal activity. Therefore, we conclude that the combined GENUS causes prolonged microglia clustering in AC, VC and mPFC.

Simultaneous auditory and visual GENUS, rather than auditory or visual alone, reduces amyloid burden in mPFCs

The microglial responses we observed in AC, VC, CA1 and mPF, prompted us to investigate whether the combined GENUS could also alter amyloid levels in these regions 7 days after 1 hour stimulation, using an anti-A β antibody (D54D2)Demonstrates, compared to non-stimulated controls, a reduction in plaque area (56.34 ± 6.35% in AC, 71.50 ± 6.51% in VC, 69.73 ± 6.48% in CA1) and a reduction in number (50.02 ± 3.74% in AC, 50.60 ± 10.9% in VC, 48.80 ± 11.1% in CA1) after combined GENUS surprisingly, our results demonstrate, compared to non-stimulated controls, a reduction of 59.64 ± 8.71% in plaque size in mPFC and a reduction of 2-fold in plaque number in combined GENUS group (fig. 7A to 7D), neither auditory nor visual GENUS alone causes a reduction in amyloid staining in mPFC, which suggests a specific response to combined GENUS1 for both plaque size or number change (fig. 14N to 14S), visual GENUS 36 β, both visual GENUS and auditory GENUS treatment show a soluble change in ELISA β and 3a soluble gea-ELISA_1-42And insoluble A β_1-42The levels were reduced, while the combined random scintillant, 8Hz or 80Hz stimulation had no significant effect on amyloid levels in AC or HPC (fig. 14T and 14U).

Next, we treated 6-month-old 5XFAD mice with various frequencies of sensory stimulation to determine whether the reduction of A β in mFC is specific to the type of stimulation (auditory versus combinatorial only) or frequency A β -ELISA was used to measure changes in amyloid levels in mFC, we observed soluble or insoluble A β_1-42In contrast, the combined GENUS group showed soluble A β in the mPF as compared to the non-stimulated group_1-42Reduced by 59.58 + -7.26%, and insoluble A β_1-42A reduction of 34.17 ± 8.20% (fig. 7E and 7F).

Furthermore, we measured plaque burden via immunohistochemical analysis using β -amyloid specific antibody (CellSignaling Technology; D54D2) in 5XFAD mice 6 months of age after 40Hz auditory GENUS, combined GENUS, 80Hz, or random frequency 7 days We observed that the mean plaque number in AC and CA1 was significantly reduced after 40Hz auditory stimulation and combined GENUS, however, compared to frequency-only and non-stimulated controls alone, GENUS resulted in a significant reduction in the number of plaques in mPFCs (FIGS. 7C and 7D). use of A β_1-42Antibody pair A β_1-42Analysis of specific immunostaining indicated that a β in AC and CA1 decreased significantly after 40Hz auditory stimulation and combination of GENUS, however, only combination of GENUS resulted in a significant decrease in immunostaining intensity in mPFC (fig. 14V).

The reduction in amyloid burden in mPFC indicates that the combined GENUS affects a broader cortical region. To determine the overall effect of combined GENUS on amyloid plaque abundance in the entire cortex, we performed whole brain SHIELD treatment (method) in 5XFAD mice 6 months of age after combined GENUS1 weeks and immunostained amyloid plaques (using D54D2 antibody) (fig. 7G and 7H). Using light sheet microscopy to analyze plaques 3D, we found that the total plaque volume and number in the neocortex was reduced by 37% and 34% respectively compared to non-stimulated controls (fig. 7I and 7J, exemplary videos are provided in supplementary videos 3 and 4 showing 3D whole brain SHIELD samples immunostained for plaques after combining GENUS and non-stimulated controls). Together, these results indicate that the combined GENUS significantly reduced amyloid plaque load across neocortex of the 5XFAD mouse model.

Method of producing a composite material

Animal(s) production

All animal work was approved by the Massachusetts institute of technology, comparative institutional animal Care Committee of medical department and the institutional animal Care and use Committee of Georgia institute of technology. Mice were placed in groups of no more than five in a standard 12 hour light/12 hour dark cycle; all experiments were performed during the light cycle. Electrophysiological experiments were performed at the Georgia institute of technology and male (1-3 month old) WT mice (C57Bl/6) were obtained from the Jackson laboratory. Mice were placed in opposing 12-hour light/12-hour dark cycles, during which all experiments were performed. Food and water were provided ad libitum.

Surgical operation

All surgical procedures were performed as described by Iaccarino and Singer et al (2016). Briefly, adult (2-3 month old) mice were anesthetized with isoflurane prior to headboard placement surgery. Custom-made stainless steel headboards were fixed using dental cement (C & B Metabond, Parkell) and the target craniotomy site for LFP recording was marked on the skull (in mm from the anterior halide: -2.0 anterior/posterior, +/-1.8 medial/lateral for targeting CA1, -2.0 to-3.0 anterior/posterior, +/-1.8 medial/lateral for targeting the auditory cortex, and +1.3 to +1.4 anterior/posterior, +/-1.0 medial/lateral for targeting the prefrontal cortex). Craniotomies were then performed in 3-8 month old mice. Prior to the first recording stage, craniotomies (200 and 500 μm diameter) were performed by thinning the skull with dental drill and then making a hole with a 27-pin. When not recorded, the craniotomy was sealed with sterile silicone elastomer (KWik-Sil WPI).

Electrophysiological recording

During recording, the head-mounted animal runs on an air bearing 8-inch spherical running platform. All animals had previously learned to maneuver on the race table until they were comfortable, while occasionally receiving sweetened condensed milk (1:2 water dilution). The animal is on the ball for up to 5 hours and has a number of periods of running and rest in the course of this. A single handle 32 channel probe (NeuroNexus) is advanced to the target location. The recording sites span 250 m. For auditory cortical recordings, the probe is advanced at a 45 angle from perpendicular parallel to the coronal plane to a depth of 3-4.15 mm. A series of 50ms tones at 5, 10, 15 and 20kHz are presented to detect an auditory response in the mean LFP. For CA1 recordings, the probe was advanced vertically through craniotomy to a depth of 1.14-2.05 mm until the hippocampus pyramidal layer electrophysiology characteristics (large theta and sharp wave ripples, 150+ μ V spikes on multiple channels) were observed. For prefrontal cortex recordings, the probe is advanced from the vertical at a 20 angle, from the coronal plane at a 49 angle, to a depth of 1.48-2.15 mm. If data is collected at multiple depths during the same recording phase; new depths are indicated in order to ensure that the position of the recording site remains in the target position (for AC, n-9 recording depths from 9 stages in 5 mice, 12 recording depths for CA1, 10 stages in 5 mice, for mPCF, n-7 recording depths from 7 stages in 4 mice). Data was acquired at a sampling rate of 20kHz using the inten RHD2000 evaluation system, which uses the milled pellets as a reference.

Auditory and visual stimulation of electrophysiological recording

The animals were presented with 10s stimulation blocks interleaved with the 10s baseline period. The stimulation blocks alternate between 20Hz, 40Hz, 80Hz auditory or auditory and visual only stimulation or random stimulation (pulses are delivered with randomized inter-pulse intervals, which are determined by a uniform distribution with an average interval of 25 ms). The stimulation blocks are staggered to ensure that the observed results are not due to neuronal response changes over time. All auditory pulses are 10kHz tones 1ms long. All visual impulses were at a stimulation frequency (length of 25ms, 12.5ms, or 6.25ms) of 50% duty cycle. For combined stimulation, the auditory and visual pulses are aligned with the beginning of each pulse.

Data acquisition

Data was acquired at a sampling rate of 20kHz using the inten RHD2000 evaluation system.

Peak detection

The original trace was band-pass filtered between 300-6,000 Hz. The spike is then detected by adding five times the estimated standard deviation (median/0.675) to the median threshold of the filtered signal.

Spike order and stability of single unit

Spike detection and ranking was done using mountain initial sort automation spike ranking, followed by manual management based on visual inspection of the waveform and cross-correlation plot. Prior to manual management, a quality threshold is applied to include only units with peak SNR greater than or equal to 1, less than 10% overlap with noise, and greater than 95% isolation for other units, which results in well-separated single units. To account for periods of instability during which a single unit of recording is lost, a stabilization criterion is applied such that only the period of stability (without sudden loss of the firing rate of a single unit) will be considered in the analysis. During the course of the recording session, the activation rate (FR) per unit is calculated. The firing rates were clustered into two distributions, i.e., low FR and high FR, using k-means clustering. Further analysis identified a stable recording period for units where FR decreased to below 10% of the high FR average, defined as the longest length of time that FR was 2 standard deviations above the low FR average.

Local field potential

The LFP is obtained by down-sampling the original trace to 2kHz and band-pass filtering between 1 and 300 Hz.

Recording sites for analysis

Data in AC and CA1 were analyzed on multiple channels. In AC, the next 16 channels of the 32 channels spanning 375m are utilized because the lowest channel on the probe is used to determine the location of the AC and the highest 16 channels are determined not to be in the main region of interest. For CA1, all active channels on the probe spanning 250m were analyzed (27/32 or 31/32). The highest channel on the probe in both AC and CA1 is used as the probe reference for power spectrum analysis. Similar results were obtained using the ground as a reference.

Prefrontal cortex histology

During the final mPFC recording for each animal, the probe was coated with DiI and inserted to the target depth. Mice were intracardiac perfused under anesthesia (isoflurane) with 4% paraformaldehyde in Phosphate Buffered Saline (PBS), and then brains were fixed in 4% paraformaldehyde in 1xPBS overnight. The brains were cut into 100 μm thick slices with a Leica VT1000S vibrating microtome (Leica). Sections were stained with 0.2% 1mMol DAPI in 1xPBS and mounted onto microscope slides using Vectashield mounting medium. Images were acquired on a Zeiss Axio Observer Z1 inverted epi-fluorescence microscope using the attendant Zen Blue 2 software.

Power spectrum

Power spectral density analysis was performed using the multi-cone method from the chrinux toolbox (time-bandwidth product 3, cone 5). The LFP trace was divided into 10s trials for each stimulation condition. Mean power spectral densities (on the same day and depth of recording) were calculated for each animal in these trials, with reference to a milling pellet in saline above the skull. Power spectral density analysis was initially calculated for all recording sites in AC, CA1 and mPFC. From each recording depth, the trace with the maximum 40Hz peak in response to the 40Hz flicker stimulus was included in the analysis. The trace displayed in the presented data had the largest 40Hz peak per depth in response to the auditory flicker stimulus.

Excitation during scintillation stimulation

A single unit ambient stimulation time histogram (PSTH) for each stimulation frequency contains four stimulation cycles10 bins per cycle to show the spikes of the entire stimulation sequence. PSTH is calculated for all individual units by binning the spikes of 2 stimulation cycles before and after the start of each ignition or audio-on pulse. As in random stimulation conditions, no randomly distributed pulse times are used to calculate the stimulation histogram. The firing rate is calculated in each bin by dividing the number of spikes per bin by the total number of pulses and the bin size. To quantify the excitation rate periodicity in relation to the stimulation frequency, the time interval between excitation rate peaks was calculated for all single unit histograms. The peak of each PSTH is the maximum firing rate within one stimulation interval. To quantify the firing rate adjustment of the stimulus and calculate the cycle statistics, the spike times around the stimulus were converted to radians: (peripheral-stimulation spike time) 2 pi (stimulation frequency) vector intensity was calculated using the method from CircStat toolbox; rayleigh statistics using the equation RS 2nVS²Calculations where n is the total spike count and VS is the vector intensity (Berens, 2009; Ma et al, 2013).

Average excitation rate

The firing rate was averaged for each individual unit for each stimulation condition. Only the stabilization period per unit contributes to the average FR calculation (see spike ordering and single unit stability, above). By taking the average FR difference per unit under each condition, the average motor rate difference between stimulation conditions was calculated within each unit.

40Hz visual flicker stimulation protocol

For biochemical and immunohistochemical analysis, 5XFAD mice were placed in a dark room illuminated by a Light Emitting Diode (LED) bulb and exposed to one of four stimulation conditions: dark, 8Hz, 40Hz (12.5 ms on, 12.5ms off, 60W) or random (delivering light pulses at random intervals determined by a uniform distribution with an average of 25ms) stimulation for 1 hour for seven days.

40Hz auditory tone stimulation protocol

For biochemical, immunohistochemical, or behavioral analysis, 5XFAD, APP/PS1, or P301S mice were placed in a dark room with acoustic foam (McMaster-Carr, 5692T 49). The speaker (AYL, AC-48073) was placed over the room in a position inaccessible to the mouse. Mice were exposed to one of five stimulation conditions: no tone, 8Hz tone, 40Hz tone, 80Hz tone, or randomly delivered tones (auditory tones delivered at random intervals determined by an even distribution of 25ms on average). The tone of the stimulation condition consists of a 10kHz tone of 1ms duration and delivered at 60 dB. For electrophysiological recording, after placement of the probe, the lights in the room were turned off and the animal was presented with alternating 10s audio-only and audiovisual stimulation periods interleaved with 10s no light or tone periods. For pure audio stimulation, a tone of 10kHz is played at 40Hz with a 4% duty cycle. For audiovisual stimulation, the audio stimulus is accompanied by ambient light that flashes at 40Hz for a period of 10s at a 50Hz duty cycle. The stimuli are presented in this manner for 20 minutes with 1-10 minutes pauses between sessions to check the behavior of the animals.

Simultaneous 40Hz auditory and visual stimulation protocol

For biochemical, immunohistochemical, or behavioral analysis, 5XFAD mice were placed in a dark room illuminated by an LED bulb and simultaneously exposed to auditory tone sequences. Mice were exposed to one of four stimuli: darkness/silence, 40Hz light flicker, a 40Hz auditory tone sequence, simultaneous 40Hz light flicker and auditory tone, or random light flicker/tone stimulation.

Immunohistochemistry

Mice were intracardiac perfused under anesthesia (2:1, ketamine/bupropion) with 4% paraformaldehyde in Phosphate Buffered Saline (PBS), and then brains were fixed in 4% paraformaldehyde in PBS overnight. The brains were cut into 40 μm thick slices with a Leica VT1000S vibrating microtome (Leica). Sections were permeabilized and blocked in PBS containing 0.3% Triton X-100 and 10% donkey serum for 2 hours at room temperature. Sections were incubated overnight at 4 ℃ in primary antibody in PBS containing 0.3% Triton X-100 and 10% donkey serum. The first antibody is: anti-beta-amyloid (Cell Signaling Technology; D54D2), anti-Iba 1(Wako Chemicals; 019-. The anti-a β antibody 12F4 was used because it did not react with APP, which allowed us to determine if our label was specific for a β, and allowed co-labeling with Iba 1. Anti-amyloid oligomer antibody AB9234 was used for co-labeling with LRP 1. The next day, brain slices were incubated with fluorescently conjugated secondary antibodies (Jackson ImmunoResearch) for 2 hours at room temperature and nuclei were stained with Hoechst33342 (Invitrogen). Images were acquired at the same settings under all conditions using a confocal microscope (LSM 710; Zeiss) with a 40-fold objective. Images were quantified using imagej1.42q by an experimenter blinded to the treatment group. For each experimental condition, two coronal sections from each animal were used for quantification. The scale bar is 50 μm unless otherwise indicated in the legend. ImageJ was used to measure the diameter of Iba1+ cell bodies and to follow the processes for length measurements. In addition, the Coloc2 insert was used to measure co-localization of Iba1 and A β. The microglial dentition branches were quantified using imarisx648.1.2(Bitplane, zurich, switzerland). The "analyze particles" function in ImageJ was used to count the number and area of spots, including at least 10 μm of sediment, and set thresholds were used for both control and experimental groups.

vasculature-Abeta co-localization assay

The imaris coloc module was used to quantify signal co-localization between two independent source channels (i.e., lectin and AB, lectin and LRP1) in 3D. These source channels are thresholded to mask any intensity from noise or background signals. The ImarisColoc then generates a new channel containing only voxels co-located within the threshold set of source channels and presents a statistical analysis of the correlation.

Microglial Abeta clustering analysis

IMARIS was used to analyze the pattern of microglial clustering around amyloid plaques in 40uM sections. The surface module is used to detect and 3D render the plaque (red) based on the 12F4 signal. Iba1 positive microglia were then counted using the dot module, which placed one sphere on each cell's somatic cell (green). Finally, the points close to the surface XTENsion are run to find a subset of points closer to the surface object than the defined 25 μ M threshold and to exclude points that fall outside this range. The algorithm measures the distance from the center of the point to the nearest point of the surface object in 3D space, which allows quantification of the microglial aggregation in the vicinity of the plaque.

Clarity immunostaining in brain slices

Mice were perfused with ice-cold PBS (1X) and then with 4% PFA, 1% glutaraldehyde in ice-cold 1 xPBS. The brains were dissected out and post-fixed for 72 hours at 4 ℃ in a 4% PFA/1% glutaraldehyde solution. Immobilization was terminated by incubating the brain in inactivation solution (4% acrylamide, 1M glycine, 0.1% Triton-X100 in 1X PBS) for 48 hours at RT. After washing with 1xPBS, the brains were cut into 100uM coronal sections on a vibrating microtome (Leica VT100S) in 1 xPBS. Referring to the Allen mouse brain map, sections containing the region of interest (i.e., auditory cortex and hippocampus) were selected and incubated in clear buffer (pH8.5-9.0, 200mM sodium dodecyl sulfate, 20mM lithium hydroxide monohydrate, 4mM boric acid in ddH2O) for 2-4 hours, shaken at 55 ℃, cleaned sections were washed in 1xPBST (0.1% Triton-X100/1xPBS) for 3X 15 minutes, and placed in blocking solution (2% bovine serum albumin/1 xPBST) overnight at RT. Subsequently, three 1 hour washes were performed in 1x PBST, i.e., shaking at RT. Sections were incubated in weak binding buffer (pH8.5-9.0, 37.75mM Na2HPO4, 3.53mM KH2PO4, 0.02% sodium azide in PBST) at RT for 1 hour, then transferred to primary antibody, diluted to 1:100 in 1X weak binding buffer at 37 ℃ over 12 hours, then reverse buffer (pH7.4, 37.75mM Na2HPO4, 3.53mM KH2PO4 in 0.02% sodium azide in PBST) was added in aliquots every hour over 6 hours to equal the volume of primary antibody solution plus the volume of tissue. Another set of 3X1 hour washes in 1xPBST was performed before RT incubation of the sections in a 1xPBS mixture of Hoechst33258(1:250) (Sigma-Aldrich, 94403) and secondary antibody (1:100) for 12 hours. Sections were then washed in 1xPBS overnight and incubated in RIMS (refractive index matching solution: 75g Histodenz, 20mL0.1M phosphate buffer, 60mL ddH2O) for 1 hour at RT before mounting. Brain sections were mounted on microscope slides using coverslips in RIMS (VWR VistaVision, VWR International, LLC, Radnor, PA).

Images were obtained on a Zeiss LSM880 microscope, which was accompanied by Zen black2.1 software (carl Zeiss microscope, jena, germany). The Z-stack images were acquired with a step size of 0.4-0.5 μm, a pixel dwell of 4.1ms, an average of 2, a resolution of 1024 x 1024 suitable for 3D reconstruction. Imarisx648.3.1(Bitplane, Zurich, Switzerland) was used for 3-D rendering and analysis.

Treatment and clearance of whole mouse brain

The brains of 5XFAD mice were treated according to the SHIELD protocol. Briefly, 5XFAD mice were perfused intracardially with ice-cold PBS followed by 20mL of SHIELD-OFF solution containing 4% PFA. Brains were dissected and postfixed in the same solution for 24 hours at 4C. The brains were then incubated overnight at 4C in SHIELD-OFF solution without PFA. The brains were then incubated in SHIELD-ON solution for 24 hours at 37C. After fixation, brains were incubated in clear aqueous solution containing 200mM Sodium Dodecyl Sulfate (SDS), 20mM lithium hydroxide monohydrate, 40mM boric acid, pH 8.5-9.0. The brain was then cleaned using SmartClear Pro (LifeCanvas Technologies, cambridge, massachusetts) based on random electrical transport (Kim et al, PNAS, 2015) for several days until clear.

Immunostaining of cleared whole hemisphere

Cleared hemispheres were stained with 15ul beta-amyloid antibody coupled to Alexa Fluor-488(CST, #51374) using eTANGO (i.e., a modified random electrotransfer method) over 2 days (Kim et al, PNAS, 2015).

Light sheet microscope

Immunostained samples were incubated with hProtos (3g diatrizoic acid, 5g N-methyl-d-glutamine, 125g iohexol in 105ml deionized water) for optical clarification and then fixed in hProtos using 2% cryomelt agarose on acrylic acid holders. The entire hemisphere was imaged using a custom light sheet microscope equipped with a 10-fold gradient optimized objective lens for using the 488 channel for β -amyloid visualization and the 647 channel for autofluorescence.

Clear whole brain image processing, patch detection and atlas alignment

Illumination correction of acquired image data using CIDRE (i.e., an open source software package implemented in Matlab), followed by Imaris^TM The resulting processed images were stitched together using Terastitcher for 3D visualization, while ImageJ (national institute of health) was used to create representative piece-by-piece 2D visualizations. Automated plaque detection was performed using a combination of open source ClearMap software, custom cell classification neural network model, and Elastix. The point detection module of ClearMap locates the candidate blob as a "blob". First, background subtraction is performed piece by using a gray-scale morphological top-hat transform with a disk structure element having a major and minor diameter pixel size of (21, 21). Next, local maxima of the data are detected by applying a 3D maximum filter with a disk structure element size of (7,7,4) and these local maxima are filtered using an intensity threshold 100. A pixel volume corresponding to each point center position is also computed using a 3D watershed transform seeded with the point center. All candidate plaques with a volume less than 10 microns in diameter were then filtered out. Identifying true plaques from candidate plaques using a Convolutional Neural Network (CNN) model as in Keras^TMAnd Theano^TMA classification blob/non-blob classifier implemented at the back-end. The CNN input is a 32x 32 pixel bounding box centered on the candidate blob center, and the output is a two-element-thermal vector representing the blob and non-blob classes. The architecture consists of 12 total convolutional layers, each convolutional layer having a rectifying linear unit (ReLU) activationAnd then batch normalization was performed: 3 with 64 cores of 2x2, 3 with 128 cores of 2x2, next 3 with 1922 x2, 1 with 256 cores of 2x2, 1 with 256 cores of 1x1, and 1 with 2 cores of 1x 1. Subsampling by 2x2 is performed after the third, sixth, and ninth convolution layers, and dryout with a rate of 0.5 is applied after the last nine convolution/batch normalization layers for regularization. After the last convolutional layer, the global average pool is applied, followed by softmax activation to generate the final classification vector. During training, the Adam optimizer uses the classification cross-entropy loss using default parameters. Keras for CNN use^TMThe image data generator trained 400 epochs on approximately 10,000 manual plaque annotations, 64 in batch size, enhanced with random rotations, cuts, and reflections. The resulting model was then used to classify plaques from the checkpoints of all samples. To perform atlas alignment, the autofluorescence channel images are first down-sampled to atlas resolution, and then affine and B-long strip transformation parameters are calculated using Elastix for 3D image registration, where the re-sampled autofluorescence images are fixed images and the atlas is a moving image. The resulting alignment parameters are applied to the plaque locations (output from the CNN model) to convert the plaque into atlas space, and then a CSV file is generated with plaque count and volume information for each brain region (subdivided according to the allen brain atlas).

Western blot

Hippocampus and auditory cortex were dissected and lysates prepared from 6-month-old males, 5 XFAD. Tissues were homogenized in 1ml RIPA (50mM Tris HCl pH 8.0, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) buffer with a hand homogenizer (Sigma), incubated on ice for 15 minutes, and spun at 4 ℃ for 30 minutes. Cell debris was separated and discarded by centrifugation at 14,000 r.p.m. for 10 minutes. Lysates were analyzed using nanodrop quantification and then 25 μ g of protein was loaded on a 10% acrylamide gel. Proteins were transferred from acrylamide gels to PVDF membrane (Invitrogen) at 100V for 120 min. Used in TBS: membrane was blocked with diluted bovine serum albumin (5% w/v) in Tween. The membrane was incubated overnight at 4 ℃ in the primary antibody and for 90 minutes at room temperature in the secondary antibody. The first antibody was anti-phosphorylated tau (Ser396) and anti-phosphorylated tau (Thr 181). The second antibody was LI-COR IRDye second antibody. Signal intensities were quantified using ImageJ 1.46a and normalized to the value of total Tau5 (Thermo Fisher Scientific; AHB 0042).

ELISA

Primary auditory cortex, medial prefrontal cortex and hippocampus were isolated from 6-month-old 5XFAD males and used A β according to the manufacturer's instructions₄₂Or A β₄₀ELISA kit (Invitrogen) A β measurements were performed before ELISA measurements, insoluble A β was treated with 5M guanidine/50 mM Tris HCl (pH 8.0) buffer.

Behavior experiment

New object recognition

As previously described (Leger et al, 2013), the New Object Recognition (NOR) task included a habituation phase, and then training and testing were performed the next day. 24 hours prior to training, mice were habituated to an open testing arena (40cm long x 40cm wide x 35cm high) for 5 minutes, during which the total distance (cm), time in the center(s) and speed (cm/s) were calculated (TSE system). During training, mice were placed in the same box with two identical objects placed in opposite corners. The mice were allowed a total of 20 seconds of object interaction time (maximum time range 10 minutes) and then immediately removed from the arena. After 1 hour, object memory was tested using the same procedure during training, except that one object was replaced by a new object in its place. When the mouth and nose part contacts any object, the object exploration is recorded, and the identification index RI is T_New/(T_New+T_{Familiarity with}) Calculation of where T_NewAnd T_{Familiarity with}Indicating the time spent with respect to a new object and a familiar object, respectively.

New object positioning

The new location identification (NOL) task is performed using the same process as the object identification task, except that two identical objects are used for both training and testing, and one object is moved to a new location during testing.

Morris Water maze test

The spatial reference memory test was performed in a circular box (diameter, 1.2m) filled with white opaque water at about 22 ℃. Reference threads consisting of different colors and shapes are placed along the walls around the tank. Inside the water tank is a fixed platform (diameter, 10cm) located in the target quadrant. During the test, the platform was flooded and the mouse was placed into the box at one of seven points randomly facing the walls of the box. The mouse was provided with 60 seconds to search for the platform and if no platform was found, the mouse was gently guided to the platform. The animals were kept on the platform for 15 s. The test was performed twice daily with 1 hour interval between tests. Between trials, mice were lightly ground dry and warmed on a heating pad. Video recordings of mouse behaviour were made using the TSE system. Each trial scored escape latency or time it took the mouse to reach the platform and averaged on each test day. On day 6, the platform was removed and a memory test (probe test) was performed. The time spent in each of the 4 quadrants and the number of passes of the area where the platform was used were recorded. Swimming speed was recorded automatically.

Conclusion

Inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods (if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent) is included within the inventive scope of the present disclosure.

Moreover, various inventive concepts may be embodied as one or more methods, examples of which have been provided. The actions performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed which perform acts in an order different than illustrated, which may include performing some acts concurrently, even though shown as sequential acts in the illustrative embodiments.

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

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

The indefinite articles "a" and "an" as used herein in the specification and in the claims are to be understood as meaning "at least one" unless explicitly indicated to the contrary.

The phrase "and/or" as used in the specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that are present together in some cases and not continuously present in other cases. Multiple elements listed with "and/or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language (e.g., "including"), reference to "a and/or B" may refer in one embodiment to a alone (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than a); in yet another embodiment, both a and B (optionally including other elements); and so on.

As used herein in the specification and claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items in a list are separated, "or" and/or "should be interpreted as being inclusive, i.e., including at least one (and also more than one) of the plurality or list of elements, and optionally other unlisted items. Merely explicitly stating the opposite terms, such as "only one" or "exactly one", or "consisting of …" when used in the claims will refer to comprising exactly one element of a plurality or list of elements, for example. In general, the term "or" as used herein should only be construed to indicate an exclusive alternative (i.e., "one or the other, but not both") before exclusive terms such as "either," one, "" only one, "or" exactly one. "consisting essentially of …" when used in the claims shall have its ordinary meaning as used in the patent law field.

As used herein in the specification and claims, the phrase "at least one," when referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that, in addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, other elements are optionally present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, in one embodiment, "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") can refer to at least one, optionally including more than one, a, with no B present (and optionally including elements other than B); in another embodiment, may refer to at least one, optionally including more than one, B, with no a present (and optionally including elements other than a); in yet another embodiment, may refer to at least one, optionally containing more than one a, and at least one, optionally containing more than one B (and optionally containing other elements); and so on.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," including, "" carrying, "" having, "" containing, "" involving, "" containing, "" consisting of … and the like are to be understood to be open-ended, i.e., to mean including but not limited to. As described in the united states patent office patent examination program manual, section 2111.03, only the transition phrases "consisting of …" and "consisting essentially of …" should be closed or semi-closed transition phrases, respectively.