Background: Alzheimer's disease (AD) is characterized by presence of extracellular amyloid plaques, intracellular tau tangles, and extensive neuronal cell death. In addition to neurons, astrocytes modulate neuronal network activity through tripartite synapses. Also, astrocytes are increasingly recognized for their involvement in AD pathology. Aberrant astrocytic calcium signaling has been implicated in AD pathological processes, including disrupted synaptic transmission, dysregulated glutamate homeostasis, and impaired vascular function via astrocytic endfeet. Previous investigations have assessed compartment-specific astrocytic calcium transients, yet most employed a restricted range of metrics. Thus, comprehensive analyses of calcium dynamics within individual astrocytic compartments in mouse models of amyloidosis are lacking. Objective: To analyze spontaneous calcium transients within distinct astrocytic compartments in APP/PS1 mice. Methods: Using in vivo multiphoton imaging of Yellow Cameleon 3.6, a genetically encoded calcium indicator targeted to astrocytes in APP/PS1 mice, we analyzed spontaneous calcium transients in cortical astrocytes at 4-6 months of age. We quantified event rate, activity duration, area under the curve (AUC), and peak amplitude across four compartments: soma, processes, microdomains, and endfeet. Correlation analyses were used to assess astrocyte synchrony and distance-dependent activity relationships. Results: In APP/PS1 mice, somas exhibited increased activity duration and peak amplitude, while processes and microdomains showed reduced duration, AUC, and amplitude despite higher event rates. Endfeet showed reductions in all parameters. Correlation analyses revealed enhanced astrocyte synchrony in APP/PS1 mice, with distance-dependent correlation decay observed only in nontransgenic controls. Conclusions: These findings highlight compartment-specific disruptions of astrocytic calcium activity caused by amyloidosis.

Sleep abnormalities and dysfunction of gamma band (30-80 Hz) activity generated by parvalbumin (PV) interneurons are early characteristics of Alzheimer's disease (AD) which correlate with the severity of amyloid-β deposition (Aβ) and cognitive impairment. However, the timing of these alterations in vivo with respect to disease progression is unclear. Here, in longitudinal recordings from APP/PS1/PV-cre (AD mice) from 3-6 months, we found reduced sleep slow-wave power (0.5-4 Hz) in hippocampus and medial prefrontal cortex in AD mice as young as 3 months old, compared to non-AD (PV-cre) mice, well before overt pathology. This finding was primarily due to reductions in the NREM delta range (1.5-4 Hz), a hallmark of restorative functions of sleep. In contrast, beta (15-30 Hz) power linked to insomnia was significantly higher across all sleep-wake states. Loss of deep NREM sleep was not compensated by an increase in NREM sleep time, instead NREM sleep during the dark (active) phase was slightly but significantly lower in AD mice. 40-Hz auditory steady-state responses and associated evoked calcium responses of hippocampal PV neurons recorded using fiber photometry were also impaired by 3 months old. However, Y-maze performance in 3- and 6-month-old AD mice was not significantly different from non-AD mice. These results reveal reduced deep sleep and PV-associated 40-Hz activity as very early changes amenable to early intervention occurring prior to cognitive deficits. Furthermore, they establish APP/PS1 mice as a good model to causally test the relationship between sleep, PV neuronal activity and amyloid-mediated pathology.

Red light is considered less phototoxic than blue light and is widely used in both research and photobiomodulation therapy. The difference in the response to brain exposure to light between young and old mammals is currently unknown. We found that brain exposure to blue light caused local damage in the cerebral cortex in both young and old mice. Brain exposure to red light did not have any noticeable effect on young mice. However, it caused a marked reduction in electroencephalogram power, damaged fiber bundles throughout the brain, and brought about a coma-like state in old mice. The effect of red light on electroencephalogram power was dose-dependent and particularly strong in the theta range. When delivered at a lower intensity but over a longer period, red light produced a similar reduction in electroencephalogram power and brain damage as those seen in the mice treated with higher irradiation over a shorter period. These results indicate that the impact of light on electroencephalogram and brain tissue strongly depends not only on the light wavelength, duration and intensity of the exposure, but also on the age of the animal and type of tissue exposed to light.

Since endothelial dysfunction is implicated in stroke pathogenesis and is featured with suppressed endothelial nitric oxide (NO) synthase (eNOS) and NO deficiency, restoring endothelial NO represents a promising approach to treating stroke injury. We have shown that near-infrared II (NIR-II) laser treatment enhances the phosphorylation of eNOS at serine 1176 (S1176) and augments NO production. Here, we further determine the beneficial effect of NIR-II laser on stroke injury. Wild-type (WT) C57BL/6J and unphosphorylatable eNOS mutant mice with S1176A mutation (S1176A) were treated by NIR-II laser (1064 nm) at an irradiance of 50 mW/cm2 (5 min daily for 4 days) prior to the middle cerebral artery occlusion (MCAO, 30 min) by a filament. The ischemic and reperfusion (30 min) values of cerebral blood flow (CBF) were confirmed by laser Doppler flowmetry. Neurological deficit and infarct volume was evaluated 48 h after reperfusion. One- and two-way ANOVAs and the Kruskal-Wallis followed by Dunn's tests were used for statistical evaluation. The NIR-II laser-pretreated WT mice demonstrated significantly increased CBF reperfusion and decreased infarct volume compared with untreated mice. The neurologic scoring showed less severe deficits in the laser-treated mice than in the non-treated mice (1.1 vs. 1.8). To determine the involvement of S1176-dependent eNOS phosphorylation in NIR-II effects, we further treated S1176A mice. MCAO followed by reperfusion resulted in decreased CBF and an increased infarct volume in S1176A mice compared to WT mice. The infarct volume was significantly larger in laser-treated S1176A compared to laser-treated WT mice. Neurological deficit in S1176A mice showed no improvement with the laser pretreatment (2.3 vs. 2.6) and was significantly more severe in the laser-treated S1176A compared to the laser-treated WT mice. In conclusion, pretreatment with a 1064 nm laser at 50 mW/cm2 improved stroke outcomes via S1176 eNOS phosphorylation. NIR-II photobiomodulation offers a non-invasive and low-risk treatment for stroke. This new modality using a physical parameter could lead to the development of innovative therapies to prevent and treat a wide array of cerebrovascular diseases.

BACKGROUND: The current management of patients with stroke with intravenous thrombolysis and endovascular thrombectomy is effective only when it is timely performed on an appropriately selected but minor fraction of patients. The development of novel adjunctive therapy is highly desired to reduce morbidity and mortality with stroke. Since endothelial dysfunction is implicated in the pathogenesis of stroke and is featured with suppressed endothelial nitric oxide synthase (eNOS) with concomitant nitric oxide deficiency, restoring endothelial nitric oxide represents a promising approach to treating stroke injury. METHODS: This is a preclinical proof-of-concept study to determine the therapeutic effect of transcranial treatment with a low-power near-infrared laser in a mouse model of ischemic stroke. The laser treatment was performed before the middle cerebral artery occlusion with a filament. To determine the involvement of eNOS phosphorylation, unphosphorylatable eNOS S1176A knock-in mice were used. Each measurement was analyzed by a 2-way ANOVA to assess the effect of the treatment on cerebral blood flow with laser Doppler flowmetry, eNOS phosphorylation by immunoblot analysis, and stroke outcomes by infarct volumes and neurological deficits. RESULTS: Pretreatment with a 1064-nm laser at an irradiance of 50 mW/cm 2 improved cerebral blood flow, eNOS phosphorylation, and stroke outcomes. CONCLUSIONS: Near-infrared II photobiomodulation could offer a noninvasive and low-risk adjunctive therapy for stroke injury. This new modality using a physical parameter merits further consideration to develop innovative therapies to prevent and treat a wide array of cardiovascular diseases.

Endothelial dysfunction featuring insufficient endothelial nitric oxide synthase (eNOS) and accompanying nitric oxide (NO) deficiency is implicated in the pathogenesis of cardiovascular diseases. Restoring endothelial NO represents a promising approach to treating cerebrovascular diseases, including stroke. Low-power near-infrared (NIR) light shows diverse beneficial effects, broadly defined as photobiomodulation (PBM). The literature reports that PBM increases bioavailable NO. These lines of evidence indicate that PBM could be used to treat cerebrovascular diseases. Recent investigations revealed that PBM improved stroke outcomes in animal models via augmenting NO signalling and other pathways. However, clinical trials of PBM using NIR light in the NIR-I window (630–900 nm) have yet to demonstrate the beneficial effect of PBM on ischaemic stroke. Since NIR light in the NIR-II window (1000–1700 nm) with the largest penetration depth into tissues compared to NIR I has also been reported to augment NO bioavailability and cerebral blood flow ameliorating stroke injury, PBM using NIR-II light may be suitable for therapeutic use. This new non-pharmacological modality using a physical parameter of NIR-II laser could provide a new avenue for therapeutic strategies for cerebrovascular diseases. Since impaired NO production has been associated with neurological abnormalities, this novel therapeutic approach could be broadly explored to treat various disease conditions such as traumatic brain injury, stroke, and Alzheimer’s disease. This review summarises recent findings on PBM in treating stroke and discusses its potential to treat other neurological diseases.

Alzheimer’s disease (AD) is a progressive neurodegenerative condition marked by memory impairments and distinct histopathological features such as amyloid-beta (Aβ) accumulations. Alzheimer’s patients experience sleep disturbances at early stages of the disease. APPswe/PS1dE9 (APP) mice exhibit sleep disruptions, including reductions in non-rapid eye movement (NREM) sleep, that contribute to their disease progression. In addition, astrocytic calcium transients associated with a sleep-dependent brain rhythm, slow oscillations prevalent during NREM sleep, are disrupted in APP mice. However, at present it is unclear whether restoration of circuit function by targeting astrocytic activity could improve sleep in APP mice. To that end, APP mice expressing channelrhodopsin-2 (ChR2) targeted to astrocytes underwent optogenetic stimulation at the slow oscillation frequency. Optogenetic stimulation of astrocytes significantly increased NREM sleep duration but not duration of rapid eye movement (REM) sleep. Optogenetic treatment increased delta power and reduced sleep fragmentation in APP mice. Thus, optogenetic activation of astrocytes increased sleep quantity and improved sleep quality in an AD mouse model. Astrocytic activity provides a novel therapeutic avenue to pursue for enhancing sleep and slowing AD progression.

BACKGROUND:Alzheimer's disease (AD) patients exhibit memory disruptions and profound sleep disturbances, including disruption of deep non-rapid eye movement (NREM) sleep. Slow-wave activity (SWA) is a major restorative feature of NREM sleep and is important for memory consolidation. METHODS:We generated a mouse model where GABAergic interneurons could be targeted in the presence of APPswe/PS1dE9 (APP) amyloidosis, APP-GAD-Cre mice. An electroencephalography (EEG) / electromyography (EMG) telemetry system was used to monitor sleep disruptions in these animals. Optogenetic stimulation of GABAergic interneurons in the anterior cortex targeted with channelrhodopsin-2 (ChR2) allowed us to examine the role GABAergic interneurons play in sleep deficits. We also examined the effect of optogenetic stimulation on amyloid plaques, neuronal calcium as well as sleep-dependent memory consolidation. In addition, microglial morphological features and functions were assessed using confocal microscopy and flow cytometry. Finally, we performed sleep deprivation during optogenetic stimulation to investigate whether sleep restoration was necessary to slow AD progression. RESULTS:APP-GAD-Cre mice exhibited impairments in sleep architecture including decreased time spent in NREM sleep, decreased delta power, and increased sleep fragmentation compared to nontransgenic (NTG) NTG-GAD-Cre mice. Optogenetic stimulation of cortical GABAergic interneurons increased SWA and rescued sleep impairments in APP-GAD-Cre animals. Furthermore, it slowed AD progression by reducing amyloid deposition, normalizing neuronal calcium homeostasis, and improving memory function. These changes were accompanied by increased numbers and a morphological transformation of microglia, elevated phagocytic marker expression, and enhanced amyloid β (Aβ) phagocytic activity of microglia. Sleep was necessary for amelioration of pathophysiological phenotypes in APP-GAD-Cre mice. CONCLUSIONS:In summary, our study shows that optogenetic targeting of GABAergic interneurons rescues sleep, which then ameliorates neuropathological as well as behavioral deficits by increasing clearance of Aβ by microglia in an AD mouse model.

Studying the biology of sleep requires accurate and efficient assessment of the sleep stages. However, analysis of sleep-wake cycles in mice and other laboratory animals remains a time-consuming and laborious process. In this study, we developed a Python script and a process for the streamlined analysis of sleep data that includes real-time processing of electroencephalogram (EEG) and electromyogram (EMG) signals that is compatible with commercial sleep-recording software that supports user datagram protocol (UDP) communication. The process consists of EEG/EMG data acquisition, automated threshold calculation for real-time determination of sleep stages, sleep staging and EEG power spectrum analysis. It also allows data storage in the format that facilitates further analysis of the sleep pattern in mice. The described method is aimed at increasing efficiency of sleep stage scoring and analysis in mice thus facilitating sleep research. • A process of EEG/EMG recording and streamline analysis of sleep-wake cycle in real time in mice. • The compatibility with commercial sleep-recording software that can generate a UDP stream. • The capability of further analysis of recorded data by an open-source software.

Alzheimer’s disease (AD) is characterized by progressive memory loss and cognitive decline. These impairments correlate with early alterations in neuronal network activity in AD patients. Disruptions in the activity of individual neurons have been reported in mouse models of amyloidosis. However, the impact of amyloid pathology on the spontaneous activity of distinct neuronal types remains unexplored in vivo. Here we use in vivo calcium imaging with multiphoton microscopy to monitor and compare the activity of excitatory and two types of inhibitory interneurons in the cortices of APP/PS1 and control mice under isoflurane anesthesia. We also determine the relationship between amyloid accumulation and the deficits in spontaneous activity in APP/PS1 mice. We show that somatostatin-expressing (SOM) interneurons are hyperactive, while parvalbumin-expressing interneurons are hypoactive in APP/PS1 mice. Only SOM interneuron hyperactivity correlated with proximity to amyloid plaque. These inhibitory deficits were accompanied by decreased excitatory neuron activity in APP/PS1 mice. Our study identifies cell-specific neuronal firing deficits in APP/PS1 mice driven by amyloid pathology. These findings highlight the importance of addressing the complexity of neuron-specific deficits to ameliorate circuit dysfunction in Alzheimer’s disease.

Regulation of receptor tyrosine kinase (RTK) activity is necessary for studying cell signaling pathways in health and disease. We developed a generalized approach for engineering RTKs optically controlled with far-red light. We targeted the bacterial phytochrome DrBphP to the cell surface and allowed its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. Systematic optimization of these constructs has resulted in optically regulated epidermal growth factor receptor, HER2, TrkA, TrkB, FGFR1, IR1, cKIT and cMet, named eDrRTKs. eDrRTKs induced downstream signaling in mammalian cells in tens of seconds. The ability to activate eDrRTKs with far-red light enabled spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays. We validated eDrTrkB performance in mice and found that minimally invasive stimulation in the neocortex with penetrating via skull far-red light-induced neural activity, early immediate gene expression and affected sleep patterns.

Non-invasive stimulation technologies are emerging as potential treatment options for a range of neurodegenerative disorders. Experimental evidence suggests that stimuli-evoked changes in slow brain rhythms may mitigate or even prevent neuropathological and behavioral impairments. Slow wave activity is prevalent during sleep and can be triggered non-invasively by sensory stimulation targeting the visual system or directly via activation of neurons locally using optogenetics. Here, we developed new tools for delivering visual stimulation using light-emitting diodes in freely moving mice while awake and during sleep. We compared these tools to traditional optogenetic approaches used for local stimulation of neurons in the cerebral cortex. We then used these tools to compare the effects of low-frequency visual versus optogenetic stimulations on the slow wave activity and sleep pattern in mice. Visual stimulation effectively enhanced slow wave activity without disrupting the sleep pattern. Optogenetic stimulation of cortical GABAergic neurons increased NREM sleep. These results suggest that visual stimulation can be effective at boosting slow wave activity without having adverse effects on sleep and thus holds great potential as a non-invasive stimulation treatment strategy.

Alzheimer’s disease (AD) is characterized by synaptic loss and neuronal network dysfunction. These network deficits are mediated by early alterations in neuronal firing rates that coincide with amyloid plaque accumulation. Mounting evidence supports that inhibitory networks are impaired in AD, but the mechanisms driving these inhibitory deficits are poorly understood. Here we use in vivo multiphoton calcium imaging to determine the relationship between amyloid accumulation and the spontaneous activity of excitatory neurons and inhibitory interneurons in an APP/PS1 mouse model of Alzheimer’s disease. We show that somatostatin-expressing (SOM) interneurons are hyperactive, while parvalbumin-expressing interneurons are hypoactive in APP/PS1 mice. Only SOM interneuron hyperactivity correlated with proximity to amyloid plaque. These inhibitory deficits were accompanied by decreased excitatory neurons activity and decreased pairwise activity correlations in APP/PS1 mice. Our study identifies cell-specific interneuronal firing deficits driven by amyloid pathology in APP/PS1 mice and provides new insights for targeting inhibitory circuits in Alzheimer’s disease.

Sleep abnormalities are widely reported in patients with Alzheimer's disease (AD) and are linked to cognitive impairments. Sleep abnormalities could be potential biomarkers to detect AD since they are often observed at the preclinical stage. Moreover, sleep could be a target for early intervention to prevent or slow AD progression. Thus, here we review changes in brain oscillations observed during sleep, their connection to AD pathophysiology and the role of specific brain circuits. Slow oscillations (0.1–1 Hz), sleep spindles (8–15 Hz) and their coupling during non-REM sleep are consistently reduced in studies of patients and in AD mouse models although the timing and magnitude of these alterations depends on the pathophysiological changes and the animal model studied. Changes in delta (1–4 Hz) activity are more variable. Animal studies suggest that hippocampal sharp-wave ripples (100–250 Hz) are also affected. Reductions in REM sleep amount and slower oscillations during REM are seen in patients but less consistently in animal models. Thus, changes in a variety of sleep oscillations could impact sleep-dependent memory consolidation or restorative functions of sleep. Recent mechanistic studies suggest that alterations in the activity of GABAergic neurons in the cortex, hippocampus and thalamic reticular nucleus mediate sleep oscillatory changes in AD and represent a potential target for intervention. Longitudinal studies of the timing of AD-related sleep abnormalities with respect to pathology and dysfunction of specific neural networks are needed to identify translationally relevant biomarkers and guide early intervention strategies to prevent or delay AD progression.

Alzheimer's disease (AD) is the major cause of dementia, characterized by the presence of amyloid-beta plaques and neurofibrillary tau tangles. Plaques and tangles are associated with sleep-wake cycle disruptions, including the disruptions in non-rapid eye movement (NREM) slow wave sleep (SWS). Alzheimer's patients spend less time in NREM sleep and exhibit decreased slow wave activity (SWA). Consistent with the critical role of SWS in memory consolidation, reduced SWA is associated with impaired memory consolidation in AD patients. The aberrant SWA can be modeled in transgenic mouse models of amyloidosis and tauopathy. Animal models exhibited slow wave impairments early in the disease progression, prior to the deposition of amyloid-beta plaques, however, in the presence of abundant oligomeric amyloid-beta. Optogenetic rescue of SWA successfully halted the amyloid accumulation and restored intraneuronal calcium levels in mice. On the other hand, optogenetic acceleration of slow wave frequency exacerbated amyloid deposition and disrupted neuronal calcium homeostasis. In this review, we summarize the evidence and the mechanisms underlying the existence of a positive feedback loop between amyloid/tau pathology and SWA disruptions that lead to further accumulations of amyloid and tau in AD. Moreover, since SWA disruptions occur prior to the plaque deposition, SWA disruptions may provide an early biomarker for AD. Finally, we propose that therapeutic targeting of SWA in AD might lead to an effective treatment for Alzheimer's patients.

Interleukin (IL)-18 is a pro-inflammatory cytokine that is a member of the IL-1 family. IL-18 is activated by inflammatory stimuli including the gram negative bacterial cell wall component lipopolysaccharide (LPS) and influenza—both of which alter sleep and slow-wave activity (SWA). IL-18 is also enhanced in individuals with pro-inflammatory conditions such as sepsis, type 2 diabetes, and cancer. We previously found that the nucleotide-binding domain leucine-rich family pyrin containing 3 (NLRP3) inflammasome is enhanced by sleep loss and LPS. NLRP3 inflammasome activation is a primary mechanism for the activation of both IL-1beta (IL-1β) and IL-18 by its corresponding pathogen associated molecular patterns including extracellular adenosine tri-phosphate and LPS. Thus, we examined sleep architecture responses to pro-inflammatory sleep promoting stimuli in mice lacking IL-18 [i.e., IL-18 knockout (KO) mice]. IL-18 KO and wild-type (WT) control mice were sleep deprived for 6 h prior to dark onset using the gentle handling method, allowed to sleep ad libitum, or intracerebroventricular infusions of LPS, IL-18 protein, or vehicle. Polysomnography was performed in the mice after the preceding treatment and sleep states and SWA were analyzed. Significance was set at p < 0.05. IL-18 KO and WT mice both had significantly enhanced non-rapid eye movement (NREM) sleep and SWA responses after sleep deprivation or LPS compared to ad libitum sleep conditions and vehicle administration, respectively. However, a significant interaction was observed between genotypes and the treatments where WT mice exhibited significant enhancements in NREM sleep and SWA after sleep deprivation and LPS compared to IL-18 KO mice. Infusion of IL-18 significantly enhanced NREM sleep and SWA in both IL-18 KO and WT mice rescuing the sleep and SWA response of IL-18 KO mice. These data indicate that IL-18 is, in part, involved in homeostatic sleep regulation. Moreover, these findings are consistent with the idea that IL-18 like IL-1β is, in part, activated by NLRP3 inflammasomes to promote sleep and SWA by its respective pathogen associated molecular patterns and pattern recognition receptors. Veteran Affairs 1I01RX00928 (MRZ), 1R21NS106406 (DG)

Inflammasomes are protein complexes that are activated by specific pathogen-associated molecular patterns (PAMPs) through corresponding pattern recognition receptors (PRRs) that stimulate caspase-1 to activate interleukin (IL)-beta (IL-1β) and IL-18 into their mature forms. Multiple inflammasomes exist that are activated by unique stimuli. The nucleotide-binding domain leucine-rich family pyrin containing 3 (NLRP3) inflammasome is activated by lipopolysaccharide (LPS) through toll-like receptor 4, NLRP1 is activated by muramyl dipeptide (MDP) through the nucleotide-binding oligomerization domain-containing protein-2 receptor, and retinoic acid-inducible gene-I (RIGI) is activated by double-stranded DNA including the synthetic deoxythymidylic acid sodium salt (poly dA:dT) through RIG-I-like receptors. NLRP3 inflammasomes are activated by sleep loss and LPS to enhance non-rapid eye movement (NREM) sleep and slow-wave activity (SWA). We determined if additional PAMPS and inflammasomes are involved in modulating sleep. Caspase-1 knockout (KO) mice and wild-type (WT) controls were given LPS, MDP, poly dA:dT or the vehicle intracerebroventricularly. Sleep architecture was analyzed by polysomnography. Inflammasome-related markers including NLRP3, NLRP1, RIG-I, caspase-1, IL-1β, and IL-18 were determined in somatosensory cortices by real-time polymerase chain reaction analysis. Significance was set at p < 0.05. LPS, MDP, and poly (dA:dT) significantly enhanced NREM sleep and SWA in WT mice. However, LPS, MDP, and poly (dA:dT) did not significantly alter sleep states or SWA in caspase-1 KO mice. In the somatosensory cortex, LPS enhanced the expression of NLRP3, caspase-1, IL-1β, and IL-18 but not NLRP1 or RIG-I in WT mice. Additionally, MDP enhanced the expression of NLRP1, caspase-1, IL-1β, and IL-18 but not NLRP3 or RIG-I in WT mice. Poly (dA:dT) enhanced the expression of RIG-I, caspase-1, IL-1β, and IL-18 but not NLRP3 or NLRP1 in WT mice. Caspase-1 KO mice showed similar enhancements in gene expression of upstream inflammasome components with the corresponding stimuli but not for IL-1β or IL-18. Our findings suggest that multiple types of inflammasomes modulate sleep by the activation of their specific corresponding PAMPS and PRRs. Veteran Affairs 1I01RX00928 (MRZ), 1R21NS106406 (DG)

Slow-wave activity (SWA) is an oscillatory neocortical activity occurring in the electroencephalogram delta (δ) frequency range (~0.5-4 Hz) during nonrapid eye movement sleep. SWA is a reliable indicator of sleep homeostasis after acute sleep loss and is involved in memory processes. Evidence suggests that cortical neuronal nitric oxide synthase (nNOS) expressing neurons that coexpress somatostatin (SST) play a key role in regulating SWA. However, previous studies lacked selectivity in targeting specific types of neurons that coexpress nNOS-cells which are activated in the cortex after sleep loss. We produced a mouse model that knocks out nNOS expression in neurons that coexpress SST throughout the cortex. Mice lacking nNOS expression in SST positive neurons exhibited significant impairments in both homeostatic low-δ frequency range SWA production and a recognition memory task that relies on cortical input. These results highlight that SST+/nNOS+ neurons are involved in the SWA homeostatic response and cortex-dependent recognition memory.

Slow-wave activity (SWA) in the electroencephalogram during slow-wave sleep (SWS) varies as a function of sleep-wake history. A putative sleep-active population of neuronal nitric oxide synthase (nNOS)-containing interneurons in the cerebral cortex, defined as such by the expression of Fos in animals euthanized after protracted deep sleep, may be a local regulator of SWA. We investigated whether electrophysiological responses to activation of these cells are consistent with their role of a local regulator of SWA. Using a Cre/loxP strategy, we targeted the population of nNOS interneurons to express the light-activated cation channel Channelrhodopsin2 and the histological marker tdTomato in mice. We then performed histochemical and optogenetic studies in these transgenic mice. Our studies provided histochemical evidence of transgene expression and electrophysiological evidence that the cerebral cortex was responsive to optogenetic manipulation of these cells in both anesthetized and behaving mice. Optogenetic stimulation of the cerebral cortex of animals expressing Channelrhodopsin2 in nNOS interneurons triggered an acute positive deflection of the local field potential that was followed by protracted oscillatory events only during quiet wake and slow wave sleep. The response during wake was maximal when the electroencephalogram (EEG) was in a negative polarization state and abolished when the EEG was in a positive polarization state. Since the polarization state of the EEG is a manifestation of slow-wave oscillations in the activity of underlying pyramidal neurons between the depolarized (LFP negative) and hyperpolarized (LFP positive) states, these data indicate that sleep-active cortical neurons expressing nNOS function in sleep slow-wave physiology.