Thomas S. Kilduff

Sleep and wakefulness are associated with distinct cortical patterns of rhythmic activity ¹ . During low-arousal states such as slow-wave sleep, synchronous low-frequency rhythms dominate activity across widespread cortical regions. Although inhibitory neurons are increasingly recognized as key regulators of cortical state ^2–4 , the circuit mechanisms that coordinate synchronized activity across local and distant neocortical networks in vivo remain poorly understood. Here we show in mice that cells co-expressing somatostatin (Sst) and chondrolectin (Chodl)—which constitute a sparse and genetically distinct class of neocortical GABAergic inhibitory neurons—are selectively active during low-arousal states and mostly silent during periods of high arousal. In contrast to most neocortical inhibitory neurons, Sst-Chodl cells, despite being extremely sparse, exert widespread influence across the neocortex, through long-range axons that target multiple regions simultaneously. Selective activation of Sst-Chodl cells is sufficient to promote the multi-region cortical synchronization that is characteristic of low-arousal states and to induce sleep. Together, these findings show that long-range Sst-Chodl inhibitory neurons not only track behavioural state, but can also actively promote synchronized cortical activity and sleep behaviour, highlighting that cortical circuits have a key role in sleep regulation, alongside established subcortical mechanisms.

Poor sleep quality and reduced sleep duration are associated with Alzheimer’s disease (AD)-related β-amyloid (Aβ) pathologies. We conducted two studies of sleep/wake, activity and body temperature in AppNL-G-F mice, a strain that exhibits three mutations in the human App gene associated with elevated risk for early onset AD. First, AppNL-G-F mice were compared to wildtype (WT) littermates at 14-18 and 18-22 months of age and, at both ages, were found to exhibit more Wake and less NREM and REM sleep than WT littermates. This long wake/short sleep phenotype was evident during the dark phase at 14-18 months but occurred in both the light and dark phases at 18-22 months. AppNL-G-F mice had fewer short (260 sec) Wake bouts and were hyperactive at 18-22 months, which undoubtedly contributed to the increased Wake/reduced sleep. Despite this reduced sleep phenotype, AppNL-G-F mice were no sleepier than WT mice and the sleep homeostat was functional in both strains. In the second study, sex differences in these parameters were assessed at 18-24 months. Reduced sleep was evident in both sexes of AppNL-G-F mice but was clearly more evident in females. Wake and REM sleep bout durations were longer in both sexes of AppNL-G-F mice than in WT littermates. EEG spectral power during NREM sleep was reduced in female AppNL-G-F mice between 4.88-10.50 Hz compared to WT mice whereas, during REM sleep, both male and female AppNL-G-F mice exhibited reduced spectral power in the theta range. These results suggest that Aβ deposition may impair state transition mechanism(s) in AppNL-G-F mice and demonstrate that, as in human AD patients, the long wake/short sleep phenotype was more evident in female AppNL-G-F mice, thus supporting the use of this strain as a model to investigate interventions that mitigate AD burden during early disease stages.

Poor sleep quality and reduced sleep duration are associated with Alzheimer’s disease (AD)-related β-amyloid (Aβ) pathologies. We conducted two studies of sleep/wake, activity and body temperature in App ^NL-G-F mice, a strain that exhibits three mutations in the human App gene associated with elevated risk for early onset AD. First, App ^NL-G-F mice were compared to wildtype (WT) littermates at 14-18 and 18-22 months of age and, at both ages, were found to exhibit more Wake and less NREM and REM sleep than WT littermates. This long wake/short sleep phenotype was evident during the dark phase at 14-18 months but occurred in both the light and dark phases at 18-22 months. App ^NL-G-F mice had fewer short (260 sec) Wake bouts and were hyperactive at 18-22 months, which undoubtedly contributed to the increased Wake/reduced sleep. Despite this reduced sleep phenotype, App ^NL-G-F mice were no sleepier than WT mice and the sleep homeostat was functional in both strains. In the second study, sex differences in these parameters were assessed at 18-24 months. Reduced sleep was evident in both sexes of App ^NL-G-F mice but was clearly more evident in females. Wake and REM sleep bout durations were longer in both sexes of App ^NL-G-F mice than in WT littermates. EEG spectral power during NREM sleep was reduced in female App ^NL-G-F mice between 4.88-10.50 Hz compared to WT mice whereas, during REM sleep, both male and female App ^NL-G-F mice exhibited reduced spectral power in the theta range. These results suggest that Aβ deposition may impair state transition mechanism(s) in App ^NL-G-F mice and demonstrate that, as in human AD patients, the long wake/short sleep phenotype was more evident in female App ^NL-G-F mice, thus supporting the use of this strain as a model to investigate interventions that mitigate AD burden during early disease stages.

The manual evaluation of mouse sleep studies is labor-intensive and time-consuming. Although several approaches for automatic sleep stage classification have been proposed, no automatic pipeline for detecting a specific mouse phenotype has yet been developed. Here, we present a fully automated pipeline for estimating the probability of Narcolepsy Type 1 (NT1) in the hypocretin-tTA;TetO-Diphteria toxin A (DTA) mouse model using unlabeled electroencephalographic (EEG) and electromyographic (EMG) data. The pipeline is divided into three modules: (1) automatic sleep stage classification, (2) feature extraction, and (3) phenotype classification. We trained two automatic sleep stage classifiers, UsleepEEG and UsleepEMG, using data from 83 wild-type (WT) mice. We next computed features such as EEG spectral power bands, EMG root mean square, and bout metrics from 11 WT and 19 DTA mice. The features were used to train an L1-penalized logistic regression classifier in a Leave-One-Subject-Out approach, achieving an accuracy of 97%. Finally, we validated the pipeline in a held-out dataset of EEG/EMG recordings at four different timepoints during disease development in seven DTA mice, finding that the pipeline captured disease progression in all mice. While our pipeline generalizes well to data from other laboratories, it is sensitive to artifacts, which should be considered in its application. With this study, we present a pipeline that facilitates a fast assessment of NT1 probability in the DTA model and thus can accelerate large-scale evaluations of NT1 treatments.

Introduction: This study aimed to investigate the role of TAAR2-9 in sleep/wake regulation, given TAAR1's known involvement in modulating neurotransmitter release and sleep patterns.Methods: Male TAAR2-9 knockout (KO) and wild-type (WT) mice were compared using baseline sleep/wake patterns, responses to sleep deprivation, effects of TAAR1 agonists, and dopaminergic markers. EEG recordings and tyrosine hydroxylase immunohistochemistry were used for analysis.Results: KO mice exhibited lower delta and theta power and higher gamma power, with fragmented sleep characterized by 16% more NREM sleep during the dark phase and 23% more REM sleep during the light phase compared to WT mice. High doses of the TAAR1 agonist RO5256390 increased wakefulness and reduced NREM sleep, while both RO5256390 and the partial agonist RO5263397 suppressed REM sleep in KO mice. Elevated tyrosine hydroxylase levels in the ventral tegmental area suggested dopaminergic involvement in these altered sleep patterns.Discussion: TAAR2-9 modulates sleep/wake states and interacts with TAAR1. These findings highlight the therapeutic potential of targeting TAARs 2-9 in sleep-related neuropsychiatric disorders. Further research is needed to elucidate their roles.

Trace amine-associated receptor 1 (TAAR1) is a negative regulator of dopamine (DA) release. The partial TAAR1 agonist RO5263397 promotes wakefulness and suppresses NREM and REM sleep in rodents and non-human primates. We tested the hypothesis that the TAAR1-mediated effects on sleep/wake regulation were due, in part, to DA release. Male C57BL6/J mice (n = 8) were intraperitoneally administered the D1R antagonist SCH23390, the D2R antagonist eticlopride, a combination of D1R + D2R antagonists, or saline at ZT5.5, followed 30 min later by RO5263397 or vehicle per os. EEG, EMG, subcutaneous temperature, and activity were recorded across the 8 treatments and sleep architecture was analyzed for 6 h post-dosing. As described previously, RO5263397 increased wakefulness and delayed NREM and REM sleep onset. D1, D2, and D1 + D2 pretreatment reduced RO5263397-induced wakefulness for 1–2 h after dosing but only the D1 antagonist significantly reduced the TAAR1-mediated increase in NREM latency. Neither the D1 nor the D2 antagonist affected the TAAR1-mediated suppression of REM sleep. These results suggest that, whereas the TAAR1 effects on wakefulness are mediated, in part, through the D2R, D1R activation plays a role in reversing the TAAR1-mediated increase in NREM sleep latency. In contrast, the TAAR1-mediated suppression of REM sleep appears not to involve D1R or D2R mechanisms.

Trace amine-associated receptor 1 (TAAR1) has gained attention for its roles in modulating neural systems, sleep/wake control, and as a therapeutic target for neuropsychiatric disorders. Although TAARs 2-9 were initially identified as non-canonical olfactory receptors, recent studies have identified extra-nasal receptor distribution of multiple TAARs. To evaluate whether TAARs 2-9 have a role in arousal state regulation, we investigated sleep/wake control in male TAAR2-9 knockout (KO) mice. After determination of baseline sleep/wake patterns, the homeostatic response to sleep deprivation and response to TAAR1 agonists were compared between KO and C57BL/6J mice. Although the EEG of TAAR2-9 KO mice had lower power in the delta and theta bands and higher power in the gamma range, sleep/wake states were readily identified. KO mice had more NREM sleep during the dark phase and more REM sleep during the light phase. Sleep/wake was fragmented in KO mice with shorter Wake and REM bouts during the dark phase and more REM bouts during the light phase. KO mice exhibited more REM sleep during a sleep latency test but the homeostatic response to sleep loss did not differ between the strains. A high dose of the TAAR1 agonist RO5256390 increased Wake and reduced NREM sleep in KO mice whereas RO5256390 and the partial TAAR1 agonist RO5263397 suppressed REM sleep. The number of tyrosine hydroxylase-immunoreactive neurons in the ventral tegmental area was significantly elevated in KO mice. These dopaminergic and sleep/wake alterations in TAAR2-9 KO mice highlight the need for further elucidation of the functions of TAAR2-9.

Trace amine-associated receptor 1 (TAAR1) is known to negatively regulate dopamine (DA) release. The partial TAAR1 agonist RO5263397 promotes wakefulness and suppresses NREM and REM sleep in mice, rats, and non-human primates. We tested the hypothesis that the TAAR1-mediated effects on sleep/wake were due, at least in part, to DA release. Male C57BL6/J mice (n=8) were intraperitoneally administered the D1R antagonist SCH23390, the D2R antagonist eticlopride, a combination of D1R+D2R antagonists or saline at ZT5.5, followed 30 min later by RO5263397 or vehicle (10% DMSO in DI water) at ZT6 per os . EEG, EMG, subcutaneous temperature, and activity were recorded in each mouse across the 8 treatment conditions and sleep architecture was analyzed for 6 hours post-dosing. Consistent with our previous reports, RO5263397 increased wakefulness as well as the latency to NREM and REM sleep. D1, D2, and D1+D2 pretreatment reduced RO5263397-induced wakefulness during the first 1-2 hours after dosing, but only the D1+D2 combination attenuated the wake-promoting effect of RO5263397 from ZT6-8, mostly by increasing NREM sleep. Although D1+D2 antagonism blocked the wake-promoting effect of RO5263397, only the D1 antagonist significantly reduced the TAAR1-mediated increase in NREM latency. Neither the D1 nor the D2 antagonist affected TAAR1-mediated suppression of REM sleep. These results suggest that, whereas TAAR1 effects on wakefulness are mediated in part through the D2R, D1R activation plays a role in reversing the TAAR1-mediated increase in NREM sleep latency. By contrast, TAAR1-mediated suppression of REM sleep appears not to involve D1R or D2R mechanisms.

Sleep and wakefulness are associated with distinct cortical patterns of rhythmic activity. During low-arousal states such as slow wave sleep, synchronous low-frequency rhythms dominate activity across widespread cortical regions. Although inhibitory neurons are increasingly recognized as key players of cortical state, the in vivo circuit mechanisms coordinating synchronized activity across local and distant neocortical networks remain poorly understood. Here, we show that somatostatin and chondrolectin co-expressing cells (Sst-Chodl), a sparse and genetically distinct class of neocortical GABAergic inhibitory neurons, are selectively active during low-arousal states and largely silent during periods of high arousal. In contrast to most neocortical inhibitory neurons, Sst-Chodl cells, despite being extremely sparse, exert widespread influence across the neocortex via long-range axons that simultaneously target multiple regions. Selective activation of Sst-Chodl cells is sufficient to promote multi-region cortical synchronization characteristic of low-arousal states and to induce sleep. Together, these findings show that long-range Sst-Chodl inhibitory neurons not only track behavioral state but can actively promote sleep-like cortical activity and sleep behavior, highlighting an important contribution of cortical circuits to sleep regulation alongside established subcortical mechanisms.

For nearly a century, evidence has accumulated indicating that the lateral hypothalamus (LH) contains neurons essential to sustain wakefulness. While lesion or inactivation of LH neurons produces a profound increase in sleep, stimulation of inhibitory LH neurons promotes wakefulness. To date, the primary wake-promoting cells that have been identified in the LH are the hypocretin/orexin (Hcrt) neurons, yet these neurons have little impact on total sleep or wake duration across the 24-h period. Recently, we and others have identified other LH populations that increase wakefulness. In the present study, we conducted microendoscopic calcium imaging in the LH concomitant with EEG and locomotor activity (LMA) recordings and found that a subset of LH neurons that express Ca ²⁺ /calmodulin-dependent protein kinase IIα (CaMKIIα) are preferentially active during wakefulness. Chemogenetic activation of these neurons induced sustained wakefulness and greatly increased LMA even in the absence of Hcrt signaling. Few LH CaMKIIα-expressing neurons are hypocretinergic or histaminergic while a small but significant proportion are GABAergic. Ablation of LH inhibitory neurons followed by activation of the remaining LH CaMKIIα neurons induced similar levels of wakefulness but blunted the LMA increase. Ablated animals showed no significant changes in sleep architecture but both spontaneous LMA and high theta (8 to 10 Hz) power during wakefulness were reduced. Together, these findings indicate the existence of two subpopulations of LH CaMKIIα neurons: an inhibitory population that promotes locomotion without affecting sleep architecture and an excitatory population that promotes prolonged wakefulness even in the absence of Hcrt signaling.