Gamma‐patterned sensory stimulation reverses synaptic plasticity deficits in rat models of early Alzheimer's disease

Non‐invasive sensory stimulation in the range of the brain's gamma rhythm (30–100 Hz) is emerging as a new potential therapeutic strategy for the treatment of Alzheimer's disease (AD). Here, we investigated the effect of repeated combined exposure to 40 Hz synchronized sound and light stimuli on hippocampal long‐term potentiation (LTP) in vivo in three rat models of early AD. We employed a very complete model of AD amyloidosis, amyloid precursor protein (APP)‐overexpressing transgenic McGill‐R‐Thy1‐APP rats at an early pre‐plaque stage, systemic treatment of transgenic APP rats with corticosterone modelling certain environmental AD risk factors and, importantly, intracerebral injection of highly disease‐relevant AD patient‐derived synaptotoxic beta‐amyloid and tau in wild‐type animals. We found that daily treatment with 40 Hz sensory stimulation for 2 weeks fully abrogated the inhibition of LTP in all three models. Moreover, there was a negative correlation between the magnitude of LTP and the level of active caspase‐1 in the hippocampus of transgenic APP animals, which suggests that the beneficial effect of 40 Hz stimulation was dependent on modulation of pro‐inflammatory mechanisms. Our findings support ongoing clinical trials of gamma‐patterned sensory stimulation in early AD.

K E Y W O R D S 40 Hz sensory stimulation, Alzheimer's disease aqueous brain extract, amyloid precursor protein transgenic rats, beta-amyloid, long-term potentiation, tau

| INTRODUCTION
After many years of intensive preclinical and clinical research, there is growing hope that effective diseasemodifying therapies for Alzheimer's disease (AD) are now an achievable goal.Agents that directly pharmacologically target major neuropathological hallmarks of the disease including beta-amyloid (Aß) plaques, taucontaining neurofibrillary tangles and neuroinflammation are the main focus of attention (Cummings et al., 2022;van Dyck et al., 2023).In addition, the therapeutic potential of non-pharmacological approaches, including those targeting brain network dysfunction, is gaining considerable traction (Chen et al., 2022;Manippa et al., 2022).Abnormal brain network activity in patients with AD includes a marked reduction in gamma band electroencephalogram oscillations (γ, 30-100 Hz) (Casula et al., 2022), a feature that also has been observed in many animal models (Bazzigaluppi et al., 2018;Driver et al., 2007;Iaccarino et al., 2016).Considering the welldocumented role of gamma rhythm in cognition (Griffiths et al., 2019), much recent research has focussed on the development of potential AD treatments based on non-invasive electrical and sensory brain stimulation protocols that induce or restore gamma (Mably & Colgin, 2018;Traikapi & Konstantinou, 2021).Moreover, recent small clinical studies indicate reasonable safety and good tolerability of gamma-patterned sensory stimulation (Chan et al., 2022;He et al., 2021).
Preclinical studies in amyloid precursor protein (APP)-and tau-transgenic mouse models showed that gamma range sensory stimulation, in particular visual and auditory stimulation at 40 Hz, alone or combination, beneficially affected behavioural deficits in these animals (Adaikkan et al., 2019;Martorell et al., 2019).Somewhat surprisingly, the behavioural improvement was accompanied by marked reductions in Aß plaque and phosphorylated tau accumulation as well as neuroinflammatory markers in the brains of the transgenic mice.
Although the studies in transgenic mice indicate that 40 Hz sensory stimulation may have disease-modifying properties, little is known about the action of such stimulation in other animal AD models.Here, we investigated whether therapeutically relevant visual and auditory stimulation synchronized at 40 Hz could reverse the inhibition of long-term potentiation (LTP), an electrophysiological correlate of learning and memory, in APP-transgenic rats at an early, pre-plaque, stage of amyloidosis.Because Aß oligomer-mediated proinflammatory mechanisms are strongly implicated in causing the LTP deficit in these rats (Qi et al., 2014(Qi et al., , 2018;;Zhang et al., 2017), we also examined caspase-1 cleavage, a measure of NOD-, LRR-and pyrin domain-containing protein 3 (NLRP3) inflammasome activation, one of the key elements of the brain's innate immune response to Aß (Heneka et al., 2013;Rand & Cooper, 2021).Furthermore, we employed disease-relevant aqueous extract of AD brain to test the ability of 40 Hz sensory stimulation to reverse the persistent LTP deficit induced by intracerebral injection of patient-derived synaptotoxic Aß and tau.

| Animals
All experiments were carried out in accordance with the approval of the Health Products Regulatory Authority, Ireland.Animals had free access to food and water and a 12-h lights on/off cycle.Male transgenic rats (5-7 months old; Figures 1 and 2) expressing human APP751 with Swedish and Indiana mutations under the control of the murine Thy1.2 promoter (McGill-R-Thy1-APP) (Leon et al., 2010) and their wild-type Wistar Han littermates were genotyped commercially by Transnetyx (Cordova, TN, USA) using real-time PCR.Some McGill-R-Thy1-APP rats (Figure 2) received three doses of corticosterone (10 mg/kg/day, Sigma) in polyethylene glycol 400 (1 ml/kg, Sigma) as vehicle subcutaneously (s.c.) (Qi et al., 2021).Male Lister Hooded wild-type rats (3-5 months old) were used for experiments with AD human brain aqueous extract from the temporal cortex of an 87-year-old man (Figure 3), prepared and characterized as described previously (Ondrejcak et al., 2018), that contained both synaptotoxic Aß and tau (AD6 extract, Ondrejcak et al., 2023).Human brain tissue was used in accordance with the guidelines of Trinity College Dublin Faculty of Health Science Ethics Committee (under approval 16014).

| Surgery
The rats were anaesthetized with urethane (1.5 g/kg, intraperitoneal, i.p.), and core body temperature was maintained at 37.5 ± .5 C. As previously described (Klyubin et al., 2014), both recording (targeted at 3.8 mm posterior to bregma and 2.5 mm lateral to midline) and stimulating (targeted at 4.6 mm posterior to bregma and 3.8 mm lateral to midline) electrodes, constructed from Teflon coated tungsten twisted-pair wires (75 μm inner core diameter, 112 μm external diameter), were positioned in the stratum radiatum of area CA1.Screw electrodes located over the contralateral cortex were used as reference and earth.
To investigate the effect of AD extract in Lister Hooded wild-type rats, intracerebroventricular (i.c.v., coordinates: .5 mm posterior to bregma and 1.2 mm right of midline, depth 3.8 mm) injections were carried out under recovery anaesthesia using a mixture of ketamine (60 mg/kg) and medetomidine (.4 mg/kg) (both i.p.) (Klyubin et al., 2014).Electrophysiological recordings were carried out under urethane anaesthesia 21 days later.

| In vivo electrophysiology
Excitatory postsynaptic potentials (EPSPs) were recorded from the stratum radiatum in the CA1 area of the dorsal hippocampus in response to stimulation of the ipsilateral Schaffer collateral/commissural pathway (Klyubin  p20) in the dorsal hippocampus, normalized to beta-actin and then expressed relative to levels of p20 in wild-type animals.As a positive control, we used the spleen homogenate of a 6-month-old wild-type rat (Spleen).(d) Correlation analysis of LTP magnitude and caspase-1 activation as measured by p20 level in each rat across all three experimental groups.et al., 2014).Test stimuli were delivered to the Schaffercollateral/commissural pathway every 30 s to evoke field EPSPs that were 50% maximum amplitude.To induce LTP, we used either our standard high-frequency stimulation (HFS) protocol consisting of one set of 10 trains of 20 stimuli at test pulse intensity at 200 Hz with an intertrain interval of 2 s or, in the case of Figure 2, a strong (sHFS) protocol consisting of three sets of 10 trains of 20 stimuli at high intensity (75% maximum) at 400 Hz with an inter-train interval of 2 s and an inter-set interval of 5 min or, in the case of Figure 3f, a weak (wHFS) protocol consisting of one set of 10 trains of 10 stimuli at test pulse intensity at 100 Hz with an inter-train interval of 2 s.

| Forty hertz sensory stimulation
Rats housed in pairs in transparent cages were placed in a light-and sound-insulated modified rat cage holding cabinet (Scantainer) with background dimmed light during their 12-h light on phase for either 1-or 2-h daily session.The cabinet contained an Arduino-controlled system composed of light-emitting diodes and audio speakers similar to that previously described (Singer et al., 2018).The light (pulse power 260 μW) was synchronized with the sound (pulse intensity 80 dB) and applied at 40 Hz or, as a control, at a random frequency (10-80 Hz) continuously for 1 (Figures 2 and 3) or 2 h (Figure 1).In the case of corticosterone-treated animals (Figure 2), the 40 Hz sensory stimulation was applied during the 12-h light off phase, with no background light inside the modified Scantainer.LTP experiments were carried out 1 day after the last sensory stimulation session.
During the first 1-h 40 Hz sensory stimulation session for wild-type animal pairs, movement behaviour was automatically analysed from video recordings using EthoVision ® XT (Version 16, Noldus, Wageningen, Netherlands).Recordings were made with an over-head camera and using an MPEG-4 video file format.Off-line automated video-tracking was conducted continuously blindly.Movement was detected as changes at pixel level from one frame to the next.Parameters included sample rate (.33/s), background noise filter (1), compression artefacts filter (on) and immobility threshold (1%).

| Western blot
Western blotting was performed as previously described (Chen et al., 2020;Zhang et al., 2022).Briefly, rats were decapitated after electrophysiological recording, and the brains were taken out immediately.Hippocampi were separated on the ice-cold surface and frozen immediately.Proteins were extracted by homogenizing in CHAPS lysis buffer, and concentrations were determined by BCA protein assay (ThermoFisher, 23227).Proteins were separated in 4% to 12% midi protein gels (ThermoFisher, WG1402A) by electrophoresis and transferred to PVDF membrane (Merck Millipore, IPVH00010).Then, the membranes were blocked with 5% non-fat milk (Cell Signaling Technology, 9999S) for 1 h in room temperature and incubated with primary antibodies for overnight at 4 C.The primary antibody was anti-Caspase-1 (p20) (AdipoGen, AG-20B-0042, 1:1,000).After washing with TBST, the membranes were incubated with the corresponding HRP-conjugated secondary anti-mouse IgG (Dako, P0447, 1:1,000) antibody.Finally, the bands were detected with enhanced chemiluminescence reagents (Merck Millipore, WBKLS0500) and quantified using Image Lab software (Version 6.1.0build 7, Bio-Rad Laboratories, Inc.).Values shown are normalized to beta-actin and then expressed relative to levels of p20 in wild-type animals.

| Statistical analysis
The strength of synaptic transmission is expressed as a percentage of the EPSP amplitude recorded over a 30 min baseline recording period.The magnitude of LTP was measured at 3 h post-HFS and expressed as the mean ± SEM % baseline.No data were excluded.Control experiments were interleaved randomly throughout experimental sets.Sample sizes were chosen to ensure adequate statistical power comparable to previously published papers.Experimental data were normally distributed.For statistical analysis and graphical display, EPSP amplitude measurements were grouped into 10 min epochs.Oneway ANOVA followed by Bonferroni's multiple comparison tests was used to compare the magnitude of LTP between multiple groups.Paired and unpaired Student's t tests were used to compare within one group and between two groups, respectively.One-way ANOVA was also used to analyse the Western blots and a parametric Gaussian distribution was assumed for correlation analysis (with Pearson's r).Two-way ANOVA followed by Bonferroni's multiple comparison was used to analyse animal movement.A p value of <.05 was considered statistically significant.Statistical analyses were performed in Prism 10.0.0.

| RESULTS
To investigate any potential beneficial effects of light and sound stimulation synchronized at 40 Hz, we studied pre-plaque transgenic animals first (Figure 1a,b).Consistent with our previous reports (Qi et al., 2014(Qi et al., , 2019)), whereas 200 Hz HFS induced robust synaptic LTP in the hippocampus of wild-type rats (Untreated WT, p = .0059,compared with pre-HFS baseline), LTP was only transient in age-matched pre-plaque APP transgenic rats, being completely blocked at 3 h post-HFS (Untreated TG, p = .1775,compared with pre-HFS baseline, and p = .0167,compared with Untreated WT).A 2-week treatment consisting of 2-h daily sessions completely abrogated the LTP deficit in transgenic rats (40 Hz TG, p = .0071,compared with pre-HFS baseline; p > .9999,compared with Untreated WT; and p = .0009,compared with Untreated TG; Figure 1b).Reducing the 40 Hz sensory stimulation session to 1 h/day had similar beneficial effects in transgenic rats (148 ± 7%, p = .0029,compared with pre-HFS baseline, and p = .0006,compared with Untreated TG, n = 4; graphical representation of the data is not shown).
Because of our previous discovery of the involvement of pro-inflammatory mechanisms in LTP inhibition in McGill APP transgenic rats (Qi et al., 2018), we also measured caspase-1 cleavage in the same animals postmortem (Figure 1a,b).Using Western blotting for caspase-1 p20 subunit, we observed a modest, but not statistically significant, increase in the level of activated caspase-1 in Untreated TG with respect to Untreated WT and 40 Hz TG groups ( p = .0652,one-way ANOVA, Figure 1c).We wondered if the ability of HFS to trigger LTP in these animals was related to the extent of caspase-1 activation in individual rats across the three experimental groups.Interestingly, there was a highly significant negative correlation between the level of F I G U R E 2 Reversal of corticosterone-exacerbated LTP deficit in APP transgenic rats by 40 Hz sensory stimulation.(a) Strong high-frequency stimulation (400 Hz, sHFS) triggered similar magnitude of LTP in both wild-type (untreated WT) and transgenic (untreated TG) animals.(b) Transgenic and wild-type rats received 3 daily s.c.injections of corticosterone (CORT) followed by 2-week treatment with auditory and visual stimulation (1 h/day) synchronized at 40 Hz.Four weeks after CORT and $24 h after the last sensory stimulation session, sHFS was applied under urethane anaesthesia.(c) While sHFS triggered robust LTP in wild-type rats pre-treated with corticosterone (WT CORT), the same pretreatment strongly inhibited LTP in transgenic rats (TG CORT).Forty hertz sensory stimulation reversed the LTP deficit in transgenic animals (TG CORT 40 Hz).(d) The summary bar chart shows the magnitude of LTP during the last 10 min for data in (a) and (c).Insets show representative field EPSP traces at the times indicated.Calibration bars: vertical, 1 mV; horizontal, 10 ms.Values are mean ± SEM. # p < .05,## p < .01,### p < .001compared with pre-HFS, paired t test; ** p < .01,*** p < .001one-way ANOVA followed by Bonferroni's multiple-comparison tests.
active caspase-1 in the dorsal hippocampus and the magnitude of LTP (Figure 1d).
While AD can have a strong genetic component, in most cases, environmental factors, such as lifetime stress, are known to significantly exacerbate symptoms and likely accelerate disease progression (Klyubin et al., 2022).Recently, we reported that brief exposure of APP transgenic rats to elevated glucocorticoids triggers a persistent exacerbation of LTP deficits (Qi et al., 2021).We, therefore, examined the effects of 40 Hz sensory stimulation in APP transgenic rats that previously had been briefly exposed to elevated levels of corticosterone (Figure 2b-d).Consistent with our previous report (Qi et al., 2021), a strong 3 Â 400 Hz HFS protocol induced similar magnitude LTP in untreated wild-type and transgenic animals (Untreated TG, p = .0004,compared with pre-sHFS baseline, and p = .9846,compared with Untreated WT; Figure 2a).We also confirmed our previous findings (Qi et al., 2021) that three daily injections of corticosterone (3 Â 10 mg/kg daily) selectively and persistently inhibited sHFS-induced LTP in transgenic rats for at least 4 weeks (TG CORT, p = .3248,compared with pre-sHFS baseline, and p = .0014,compared with WT CORT, Figure 2c,d).Importantly, 1 h daily 40 Hz sensory stimulation for 2 weeks (Figure 2b) reversed the additional LTP deficit in corticosteroneinjected transgenic rats (TG CORT 40 Hz, p = .0023,compared with pre-sHFS baseline, and p = .7069,compared with Untreated WT; Figure 2c,d).
Finally, we employed a highly disease-relevant, nontransgenic model (Klyubin et al., 2014), in which wildtype rats received a single i.c.v.injection of aqueous extract of AD brain under recovery anaesthesia and LTP inducibility was tested under non-recovery anaesthesia 3 weeks later (Figure 3a).Previously, we found that this particular extract of AD brain caused an Aß-and taudependent persistent inhibition of LTP, being prevented by immunodepletion of either Aß or tau (AD6 extract, Ondrejcak et al., 2023).Remarkably, daily 1 h long sensory stimulation at 40 Hz reversed the LTP inhibition in this model as well (40 Hz, p = .0011,compared with pre-HFS baseline, and p > .9999,compared with Untreated; Figure 3b).In contrast, control random frequency (10-80 Hz) visual and auditory stimulation did not reverse inhibition of LTP in AD brain extract-injected rats (Random, p = .4676,compared with pre-HFS baseline; p = .0005,compared with Untreated; and p = .0002,compared with 40 Hz; Figure 3b).
Of note, the 40 Hz sensory stimulation neither affected LTP induced by our standard (200 Hz) HFS nor enhance a relatively small LTP induced by a weak HFS (100 Hz) conditioning protocol in wild-type animals (Untreated vs. 40 Hz, p = .2204and p = .8828,respectively; Figure 3c,e,f).Moreover, while brief 40 Hz light stimulation has been reported to disturb performance of a behavioural exploration task by wild-type mice (Soula et al., 2023), we did not observe a significant effect of combined audio and visual stimulation on spontaneous movement of wild-type rats under our experimental conditions ( p = .8205,Untreated versus 40 Hz, 12 rats in each group, Figure 3d).

| DISCUSSION
Our results provide strong evidence that combined auditory and visual stimulation synchronized at 40 Hz has the ability to reverse LTP deficits in three rat models of early AD, transgenic APP overexpression, gene x environmental corticosterone interaction and delayed effect of AD brain extract.Indeed, gamma rhythms that support synaptic plasticity under physiological conditions (Zarnadze et al., 2016) are impaired in AD patients (Guntekin et al., 2022), and normalizing any deficits likely will be important for restoring normal synaptic functioning.Apart from uncovering the beneficial effect of 40 Hz sensory stimulation in transgenic APP rats, a very complete model of AD amyloidosis (Leon et al., 2010), our findings in AD brain extract-injected wild-type animals help close the gap between preclinical studies and therapeutic implications because these aqueous extracts contain highly disease-relevant patientderived synaptotoxic Aß and tau (Hong et al., 2018;Ondrejcak et al., 2018).
How then might gamma-patterned sensory stimulation alleviate AD-associated hippocampal LTP deficits?One possibility is that repeated 40 Hz sensory stimulation promotes clearance of synaptotoxic Aß by microglia.In transgenic plaque-bearing mice prolonged presentation of either 40 Hz light or sound stimulation reduced both soluble and insoluble Aß load (Martorell et al., 2019), probably by microglial engulfment (Singer et al., 2018), and decreased the number of Aß-positive neurons (Park et al., 2020).Interestingly, combined visual and auditory 40 Hz stimulation, similar to what we used in this study, was more effective in clustering of microglia around plaques than either alone (Martorell et al., 2019).Additionally, in pre-plaque 3xTg-AD mice 40 Hz light flicker significantly reduced the number of Aß-positive neurons in the hippocampus as well (Park et al., 2022).
A possibly related mechanism, gamma-patterned brain stimulation may have triggered anti-inflammatory processes.Our data showing a negative correlation between LTP magnitude and caspase-1 activation (Figure 1d) are consistent with this hypothesis.Indeed, pre-plaque 3xTg-AD (Park et al., 2022) and Tau P301S (Adaikkan et al., 2019) mice had a reduced inflammatory profile after exposure to 40 Hz light flicker.Moreover, 40 Hz transauricular vagal nerve electrical stimulation reduced hippocampal expression of NLRP3, caspase-1, interleukin-1ß and interleukin-18, which was accompanied by microglia-associated reduction in both soluble and insoluble Aß deposition in APP/PS1 mice (Yu et al., 2023).
It is also possible that 40 Hz sensory stimulation interacted with tau in the case of the model using patientderived synaptotoxic Aß and tau (Figure 3a,b).In tau overexpressing neurofibrillary tangle-bearing mice 40 Hz light flicker improved synaptic integrity markers such as brainderived neurotrophic factor (BDNF) and synaptophysin (Adaikkan et al., 2019;Park et al., 2020).On the other hand, Jeong et al. (2021) reported that 40 Hz transcranial electrical stimulation reversed impaired hippocampal LTP in the absence of changes in BDNF expression in young 5xFAD mice without tau deposition.
The attempts to restore/influence gamma power in AD are still at an investigational stage, as no changes in AD biomarkers in humans after such treatment have been reported to date (Manippa et al., 2022).However, gamma-patterned AD treatment has a clear clinical potential because a steady state gamma induced in sensory cortex can propagate into deeper brain areas in rodents (Adaikkan et al., 2019;Li et al., 2021;Martorell et al., 2019;Soula et al., 2023) and humans (Jones et al., 2019).Given the importance of corticosteroids in mediating circadian rhythms (Oster et al., 2017), our data on beneficial effects of 40 Hz sensory stimulation in corticosterone-injected APP transgenic rats (Figure 2), which are in accord with findings that similar sensory stimulation improves the circadian clock in APP/PS1 mice (Yao et al., 2020) and sleep in AD patients (Cimenser et al., 2021), support this approach.In conclusion, results of this study may lead to development of effective non-invasive treatment as a new potential disease-modifying therapy for AD.Values are mean ± SEM. # p < .05,## p < .01,### p < .001#### p < .0001compared with pre-HFS, paired t test; *** p < .001,one-way ANOVA followed by Bonferroni's multiple-comparison tests.

F
I G U R E 1 Daily 40 Hz sensory stimulation abrogates the LTP deficit in pre-plaque APP transgenic rats.(a) Schematic diagram of the treatment regimen.Animals were treated daily (2 h/day) for 2 weeks with auditory and visual stimulation synchronized at 40 Hz.The following day, the animals were anaesthetized, and LTP was measured in vivo.Post-mortem samples of the hippocampus were collected for subsequent Western blotting.(b) Our standard high-frequency stimulation (200 Hz, HFS)-induced LTP was impaired in transgenic (untreated TG) rats compared with age-matched wild-type (untreated WT) littermates.Sensory stimulation at 40 Hz restored LTP in TG animals to a level indistinguishable from untreated WT rats.The summary bar chart of LTP magnitude during the last 10 min is in the righthand panel.Insets show representative field EPSP traces at the times indicated.Calibration bars: vertical, 1 mV; horizontal, 10 ms.Values are mean ± SEM. ## p < .01compared with pre-HFS, paired t test; *p < .05,*** p < .001,one-way ANOVA followed by Bonferroni's multiplecomparison tests.(c) Representative Western blotting and quantification analysis of the level of active caspase-1 (

F
I G U R E 3 Forty hertz sensory stimulation reverses persistent LTP inhibition by synaptotoxic AD brain-derived Aß and tau in wild-type rats without affecting control LTP or spontaneous animal behaviour.(a) Schematic diagram of treatment regimen.(b) Forty hertz, in contrast to random (10-80 Hz), sensory stimulation reversed the persistent inhibition of standard (200 Hz) HFSinduced LTP caused by a single i.c.v.injection of AD brain aqueous extract.Untreated animals served as an additional interleaved control group.In a different group of naïve rats, (c) 40 Hz sensory stimulation had no effect on the rats' spontaneous movement during the first one-hour session in the stimulation cabinet (d), or LTP induced by either our standard 200 Hz (HFS, e), or a weak 100 Hz (wHFS, f) protocol after a 2-week sensory treatment schedule.Left-hand panel shows the time course of LTP.Summary bar chart of LTP magnitude during the last 10 min is in the righthand panel.Insets show representative field EPSP traces at the times indicated.Calibration bars: vertical, 1 mV; horizontal, 10 ms.