Activity of estrogen receptor ß expressing neurons in the medial amygdala regulates preference toward receptive females in male mice.

The processing of information regarding the sex and reproductive state of conspecific individuals is critical for successful reproduction and survival in males. Generally, male mice exhibit a preference toward the odor of sexually receptive (RF) over nonreceptive females (XF) or gonadally intact males (IM). Previous studies suggested the involvement of estrogen receptor beta (ERß) expressed in the medial amygdala (MeA) in male preference toward RF. To further delineate the role played by ERß in the MeA in the neuronal network regulating male preference, we developed a new ERß-iCre mouse line using the CRISPR-Cas9 system. Fiber photometry Ca2+ imaging revealed that ERß-expressing neurons in the postero-dorsal part of the MeA (MeApd-ERß+ neurons) were more active during social investigation toward RF compared to copresented XF or IM mice in a preference test. Chemogenetic inhibition of MeApd-ERß+ neuronal activity abolished a preference to RF in "RF vs. XF," but not "RF vs. IM," tests. Analysis with cre-dependent retrograde tracing viral vectors identified the principal part of the bed nucleus of stria terminalis (BNSTp) as a primary projection site of MeApd-ERß+ neurons. Fiber photometry recording in the BNSTp during a preference test revealed that chemogenetic inhibition of MeApd-ERß+ neurons abolished differential neuronal activity of BNSTp cells as well as a preference to RF against XF but not against IM mice. Collectively, these findings demonstrate for the first time that MeApd-ERß+ neuronal activity is required for expression of receptivity-based preference (i.e., RF vs. XF) but not sex-based preference (i.e., RF vs. IM) in male mice.

Association between idiopathic hypersomnia and a genetic variant in the PER3 gene.

We aim to identify genetic markers associated with idiopathic hypersomnia, a disabling orphan central nervous system disorder of hypersomnolence that is still poorly understood. In our study, DNA was extracted from 79 unrelated patients diagnosed with idiopathic hypersomnia with long sleep time at the National Reference Center for Narcolepsy-France according to very stringent diagnostic criteria. Whole exome sequencing on the first 30 patients with idiopathic hypersomnia (25 females and 5 males) allowed the single nucleotide variants to be compared with a control population of 574 healthy subjects from the French Exome project database. We focused on the identification of genetic variants among 182 genes related to the regulation of sleep and circadian rhythm. Candidate variants obtained by exome sequencing analysis were then validated in a second sample of 49 patients with idiopathic hypersomnia (37 females and 12 males). Our study characterised seven variants from six genes significantly associated with idiopathic hypersomnia compared with controls. A targeted sequencing analysis of these seven variants on 49 other patients with idiopathic hypersomnia confirmed the relative over-representation of the A➔C variant of rs2859390, located in a potential splicing-site of PER3 gene. Our findings support a genetic predisposition and identify pathways involved in the pathogeny of idiopathic hypersomnia. A variant of the PER3 gene may predispose to idiopathic hypersomnia with long sleep time.

Ventral pallidal GABAergic neurons control wakefulness associated with motivation through the ventral tegmental pathway.

The ventral pallidum (VP) regulates motivation, drug addiction, and several behaviors that rely on heightened arousal. However, the role and underlying neural circuits of the VP in the control of wakefulness remain poorly understood. In the present study, we sought to elucidate the specific role of VP GABAergic neurons in controlling sleep-wake behaviors in mice. Fiber photometry revealed that the population activity of VP GABAergic neurons was increased during physiological transitions from non-rapid eye movement (non-REM, NREM) sleep to either wakefulness or REM sleep. Moreover, chemogenetic and optogenetic manipulations were leveraged to investigate a potential causal role of VP GABAergic neurons in initiating and/or maintaining arousal. In vivo optogenetic stimulation of VP GABAergic neurons innervating the ventral tegmental area (VTA) strongly promoted arousal via disinhibition of VTA dopaminergic neurons. Functional in vitro mapping revealed that VP GABAergic neurons, in principle, inhibited VTA GABAergic neurons but also inhibited VTA dopaminergic neurons. In addition, optogenetic stimulation of terminals of VP GABAergic neurons revealed that they promoted arousal by innervating the lateral hypothalamus, but not the mediodorsal thalamus or lateral habenula. The increased wakefulness chemogenetically evoked by VP GABAergic neuronal activation was completely abolished by pretreatment with dopaminergic D1 and D2/D3 receptor antagonists. Furthermore, activation of VP GABAergic neurons increased exploration time in both the open-field and light-dark box tests but did not modulate depression-like behaviors or food intake. Finally, chemogenetic inhibition of VP GABAergic neurons decreased arousal. Taken together, our findings indicate that VP GABAergic neurons are essential for arousal related to motivation.

The rostromedial tegmental nucleus is essential for non-rapid eye movement sleep.

The rostromedial tegmental nucleus (RMTg), also called the GABAergic tail of the ventral tegmental area, projects to the midbrain dopaminergic system, dorsal raphe nucleus, locus coeruleus, and other regions. Whether the RMTg is involved in sleep-wake regulation is unknown. In the present study, pharmacogenetic activation of rat RMTg neurons promoted non-rapid eye movement (NREM) sleep with increased slow-wave activity (SWA). Conversely, rats after neurotoxic lesions of 8 or 16 days showed decreased NREM sleep with reduced SWA at lights on. The reduced SWA persisted at least 25 days after lesions. Similarly, pharmacological and pharmacogenetic inactivation of rat RMTg neurons decreased NREM sleep. Electrophysiological experiments combined with optogenetics showed a direct inhibitory connection between the terminals of RMTg neurons and midbrain dopaminergic neurons. The bidirectional effects of the RMTg on the sleep-wake cycle were mimicked by the modulation of ventral tegmental area (VTA)/substantia nigra compacta (SNc) dopaminergic neuronal activity using a pharmacogenetic approach. Furthermore, during the 2-hour recovery period following 6-hour sleep deprivation, the amount of NREM sleep in both the lesion and control rats was significantly increased compared with baseline levels; however, only the control rats showed a significant increase in SWA compared with baseline levels. Collectively, our findings reveal an essential role of the RMTg in the promotion of NREM sleep and homeostatic regulation.

Sleep and Wakefulness Are Controlled by Ventral Medial Midbrain/Pons GABAergic Neurons in Mice.

Sleep-wake behavior is controlled by a wide range of neuronal populations in the mammalian brain. Although the ventral midbrain/pons (VMP) area is suggested to participate in sleep-wake regulation, the neuronal mechanisms have remained unclear. Here, we found that nonspecific cell ablation or selective ablation of GABAergic neurons by expressing diphtheria toxin fragment A in the VMP in male mice induced a large increase in wakefulness that lasted at least 4 weeks. In contrast, selective ablation of dopaminergic neurons in the VMP had little effect on wakefulness. Chemogenetic inhibition of VMP GABAergic neurons also markedly increased wakefulness. The wake-promoting effect of the VMP GABAergic neuron ablation or inhibition was attenuated to varying degrees by the administration of dopamine D1 or D2/3 receptor antagonists and abolished by the administration of both antagonists together. In contrast, chemogenetic activation of VMP GABAergic neurons very strongly increased slow-wave sleep and reduced wakefulness. These findings suggest that VMP GABAergic neurons regulate dopaminergic actions in the sleep-wake behavior of mice.SIGNIFICANCE STATEMENT Current understanding of the neuronal mechanisms and populations that regulate sleep-wake behavior is incomplete. Here, we identified a GABAergic ventral midbrain/pons area that is necessary for controlling the daily amount of sleep and wakefulness in mice. We also found that these inhibitory neurons control wakefulness by suppressing dopaminergic systems. Surprisingly, activation of these neurons strongly induced slow-wave sleep while suppressing wakefulness. Our study reveals a new brain mechanism critical for sleep-wake regulation.

Miniaturized microscope with flexible light source input for neuronal imaging and manipulation in freely behaving animals.

Simultaneous imaging and manipulation of a genetically defined neuronal population can provide a causal link between its activity and function. Here, we designed a miniaturized microscope (or 'miniscope') that allows fluorescence imaging and optogenetic manipulation at the cellular level in freely behaving animals. This miniscope has an integrated optical connector that accepts any combination of external light sources, allowing flexibility in the choice of sensors and manipulators. Moreover, due to its simple structure and use of open source software, the miniscope is easy to build and modify. Using this miniscope, we demonstrate the optogenetic silencing of hippocampal CA1 neurons using two laser light sources-one stimulating a calcium sensor (i.e., jGCaAMP7c) and the other serving as an optogenetic silencer (i.e., Jaws). This new miniscope can contribute to efforts to determine causal relationships between neuronal network dynamics and animal behavior.

Concise Review: Regulatory Influence of Sleep and Epigenetics on Adult Hippocampal Neurogenesis and Cognitive and Emotional Function.

Neural stem and progenitor cells continue to generate new neurons in particular regions of the brain during adulthood. One of these neurogenic regions is the dentate gyrus (DG) of the hippocampus, which plays an important role in cognition and emotion. By exploiting this innate neuronal regeneration mechanism in the DG, new technologies have the potential to promote resistance to or recovery from brain dysfunction or degeneration. However, a deeper understanding of how adult DG neurogenesis is regulated by factors such as sleep and epigenetic modifications of gene expression could lead to further breakthroughs in the clinical application of neural stem and progenitor cells. In this review, we discuss the functions of adult-born DG neurons, describe the epigenetic regulation of adult DG neurogenesis, identify overlaps in how sleep and epigenetic modifications impact adult DG neurogenesis and memory consolidation, and suggest ways of using sleep or epigenetic interventions as therapies for neurodegenerative and psychiatric disorders. By knitting together separate strands of the literature, we hope to trigger new insights into how the functions of adult-generated neurons are directed by interactions between sleep-related neural processes and epigenetic mechanisms to facilitate novel approaches to preventing and treating brain disorders such as depression, post-traumatic stress disorder, and Alzheimer's disease. Stem Cells 2018;36:969-976.

Dietary Zinc Acts as a Sleep Modulator.

While zinc is known to be important for many biological processes in animals at a molecular and physiological level, new evidence indicates that it may also be involved in the regulation of sleep. Recent research has concluded that zinc serum concentration varies with the amount of sleep, while orally administered zinc increases the amount and the quality of sleep in mice and humans. In this review, we provide an exhaustive study of the literature connecting zinc and sleep, and try to evaluate which molecular mechanism is likely to be involved in this phenomenon. A better understanding should provide critical information not only about the way zinc is related to sleep but also about how sleep itself works and what its real function is.

A novel GABAergic population in the medial vestibular nucleus maintains wakefulness and gates rapid eye movement sleep.

Body rocking can either induce sleep or arousal. That is, the vestibular sense influences sleep-wake states. Neuronal interactions between sleep-wake systems and vestibular systems, however, remain unclear. In this study, we found that GABAergic neurons in the lateral part of the medial vestibular nucleus (LMVN), a primary vestibular afferent projection site, control sleep-wake states. Specific inhibition of LMVN GABAergic neurons revealed that the firing of LMVN GABAergic neurons underlies stable wakefulness and smooth transitions from non-rapid-eye-movement (NREM) sleep to rapid eye movement (REM) sleep and that LMVN GABAergic neurons do not affect body balance control in freely moving conditions. Selective axonal tracing of LMVN GABAergic neurons indicated that LMVN GABAergic neurons send axons not only to areas involved in vestibular and oculomotor functions but also to areas regulating sleep-wake states. Our findings suggest that LMVN GABAergic neurons stabilize wakefulness and gate the entry into REM sleep through the use of vestibular information.

Optochemical control of slow-wave sleep in the nucleus accumbens of male mice by a photoactivatable allosteric modulator of adenosine A2A receptors.

Optochemistry, an emerging pharmacologic approach in which light is used to selectively activate or deactivate molecules, has the potential to alleviate symptoms, cure diseases, and improve quality of life while preventing uncontrolled drug effects. The development of in-vivo applications for optochemistry to render brain cells photoresponsive without relying on genetic engineering has been progressing slowly. The nucleus accumbens (NAc) is a region for the regulation of slow-wave sleep (SWS) through the integration of motivational stimuli. Adenosine emerges as a promising candidate molecule for activating indirect pathway neurons of the NAc expressing adenosine A2A receptors (A2ARs) to induce SWS. Here, we developed a brain-permeable positive allosteric modulator of A2ARs (A2AR PAM) that can be rapidly photoactivated with visible light (λ > 400 nm) and used it optoallosterically to induce SWS in the NAc of freely behaving male mice by increasing the activity of extracellular adenosine derived from astrocytic and neuronal activity.

Positive allosteric adenosine A2A receptor modulation suppresses insomnia associated with mania- and schizophrenia-like behaviors in mice.

Background: Insomnia is associated with psychiatric illnesses such as bipolar disorder or schizophrenia. Treating insomnia improves psychotic symptoms severity, quality of life, and functional outcomes. Patients with psychiatric disorders are often dissatisfied with the available therapeutic options for their insomnia. In contrast, positive allosteric modulation of adenosine A2A receptors (A2ARs) leads to slow-wave sleep without cardiovascular side effects in contrast to A2AR agonists. Methods: We investigated the hypnotic effects of A2AR positive allosteric modulators (PAMs) in mice with mania-like behavior produced by ablating GABAergic neurons in the ventral medial midbrain/pons area and in a mouse model of schizophrenia by knocking out of microtubule-associated protein 6. We also compared the properties of sleep induced by A2AR PAMs in mice with mania-like behavior with those induced by DORA-22, a dual orexin receptor antagonist that improves sleep in pre-clinical models, and the benzodiazepine diazepam. Results: A2AR PAMs suppress insomnia associated with mania- or schizophrenia-like behaviors in mice. A2AR PAM-mediated suppression of insomnia in mice with mania-like behavior was similar to that mediated by DORA-22, and, unlike diazepam, did not result in abnormal sleep. Conclusion: A2AR allosteric modulation may represent a new therapeutic avenue for sleep disruption associated with bipolar disorder or psychosis.

Arousal effect of caffeine depends on adenosine A2A receptors in the shell of the nucleus accumbens.

Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A(2A) receptors (A(2A)Rs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A(2A)Rs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A(2A)Rs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A(2A)Rs were focally removed from the NAc core or other A(2A)R-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain.

Motivational and Valence-Related Modulation of Sleep/Wake Behavior are Mediated by Midbrain Dopamine and Uncoupled from the Homeostatic and Circadian Processes.

Motivation and its hedonic valence are powerful modulators of sleep/wake behavior, yet its underlying mechanism is still poorly understood. Given the well-established role of midbrain dopamine (mDA) neurons in encoding motivation and emotional valence, here, neuronal mechanisms mediating sleep/wake regulation are systematically investigated by DA neurotransmission. It is discovered that mDA mediates the strong modulation of sleep/wake states by motivational valence. Surprisingly, this modulation can be uncoupled from the classically employed measures of circadian and homeostatic processes of sleep regulation. These results establish the experimental foundation for an additional new factor of sleep regulation. Furthermore, an electroencephalographic marker during wakefulness at the theta range is identified that can be used to reliably track valence-related modulation of sleep. Taken together, this study identifies mDA signaling as an important neural substrate mediating sleep modulation by motivational valence.

Rapid eye movement sleep is initiated by basolateral amygdala dopamine signaling in mice.

The sleep cycle is characterized by alternating non-rapid eye movement (NREM) and rapid eye movement (REM) sleeps. The mechanisms by which this cycle is generated are incompletely understood. We found that a transient increase of dopamine (DA) in the basolateral amygdala (BLA) during NREM sleep terminates NREM sleep and initiates REM sleep. DA acts on dopamine receptor D2 (Drd2)-expressing neurons in the BLA to induce the NREM-to-REM transition. This mechanism also plays a role in cataplectic attacks-a pathological intrusion of REM sleep into wakefulness-in narcoleptics. These results show a critical role of DA signaling in the BLA in initiating REM sleep and provide a neuronal basis for sleep cycle generation.

Hypothalamic modulation of adult hippocampal neurogenesis in mice confers activity-dependent regulation of memory and anxiety-like behavior.

Adult hippocampal neurogenesis plays a critical role in memory and emotion processing, and this process is dynamically regulated by neural circuit activity. However, it remains unknown whether manipulation of neural circuit activity can achieve sufficient neurogenic effects to modulate behavior. Here we report that chronic patterned optogenetic stimulation of supramammillary nucleus (SuM) neurons in the mouse hypothalamus robustly promotes neurogenesis at multiple stages, leading to increased production of neural stem cells and behaviorally relevant adult-born neurons (ABNs) with enhanced maturity. Functionally, selective manipulation of the activity of these SuM-promoted ABNs modulates memory retrieval and anxiety-like behaviors. Furthermore, we show that SuM neurons are highly responsive to environmental novelty (EN) and are required for EN-induced enhancement of neurogenesis. Moreover, SuM is required for ABN activity-dependent behavioral modulation under a novel environment. Our study identifies a key hypothalamic circuit that couples novelty signals to the production and maturation of ABNs, and highlights the activity-dependent contribution of circuit-modified ABNs in behavioral regulation.

Lateral habenula glutamatergic neurons projecting to the dorsal raphe nucleus promote aggressive arousal in mice.

The dorsal raphe nucleus (DRN) is known to control aggressive behavior in mice. Here, we found that glutamatergic projections from the lateral habenula (LHb) to the DRN were activated in male mice that experienced pre-exposure to a rival male mouse ("social instigation") resulting in heightened intermale aggression. Both chemogenetic and optogenetic suppression of the LHb-DRN projection blocked heightened aggression after social instigation in male mice. In contrast, inhibition of this pathway did not affect basal levels of aggressive behavior, suggesting that the activity of the LHb-DRN projection is not necessary for the expression of species-typical aggressive behavior, but required for the increase of aggressive behavior resulting from social instigation. Anatomical analysis showed that LHb neurons synapse on non-serotonergic DRN neurons that project to the ventral tegmental area (VTA), and optogenetic activation of the DRN-VTA projection increased aggressive behaviors. Our results demonstrate that the LHb glutamatergic inputs to the DRN promote aggressive arousal induced by social instigation, which contributes to aggressive behavior by activating VTA-projecting non-serotonergic DRN neurons as one of its potential targets.

A cluster of mesopontine GABAergic neurons suppresses REM sleep and curbs cataplexy.

Physiological rapid eye movement (REM) sleep termination is vital for initiating non-REM (NREM) sleep or arousal, whereas the suppression of excessive REM sleep is promising in treating narcolepsy. However, the neuronal mechanisms controlling REM sleep termination and keeping sleep continuation remain largely unknown. Here, we reveal a key brainstem region of GABAergic neurons in the control of both physiological REM sleep and cataplexy. Using fiber photometry and optic tetrode recording, we characterized the dorsal part of the deep mesencephalic nucleus (dDpMe) GABAergic neurons as REM relatively inactive and two different firing patterns under spontaneous sleep-wake cycles. Next, we investigated the roles of dDpMe GABAergic neuronal circuits in brain state regulation using optogenetics, RNA interference technology, and celltype-specific lesion. Physiologically, dDpMe GABAergic neurons causally suppressed REM sleep and promoted NREM sleep through the sublaterodorsal nucleus and lateral hypothalamus. In-depth studies of neural circuits revealed that sublaterodorsal nucleus glutamatergic neurons were essential for REM sleep termination by dDpMe GABAergic neurons. In addition, dDpMe GABAergic neurons efficiently suppressed cataplexy in a rodent model. Our results demonstrated that dDpMe GABAergic neurons controlled REM sleep termination along with REM/NREM transitions and represented a novel potential target to treat narcolepsy.

Induction of narcolepsy-like symptoms by orexin receptor antagonists in mice.

Orexins/hypocretins are hypothalamic neuropeptides that promote and stabilize wakefulness by binding to the orexin receptor type-1 (OX1R) and type-2 (OX2R). Disruption of orexinergic signaling results in the sleep disorder narcolepsy in mice, rats, dogs, and humans. The orexin receptor antagonist suvorexant promotes sleep by blocking both OX1R and OX2R. Whereas suvorexant has been clinically approved for the treatment of insomnia because it is well tolerated in experimental animals as well as in human patients, a logical question remains as to why orexin receptor antagonists do not induce overt narcolepsy-like symptoms. Here we show that acute and chronic suvorexant promotes both rapid eye movement (REM) and non-rapid eye movement (NREM) sleep without inducing cataplexy in mice. Interestingly, chronic suvorexant increases OX2R mRNA and decreases orexin mRNA and peptide levels, which remain low long after termination of suvorexant administration. When mice are chronically treated with suvorexant and then re-challenged with the antagonist after a 1-week washout, however, cataplexy and sleep-onset REM (SOREM) are observed, which are exacerbated by chocolate administration. Heterozygous orexin knockout mice, with lower brain orexin levels, show cataplexy and SOREM after acute suvorexant administration. Furthermore, we find that acute suvorexant can induce cataplexy and SOREM in wild-type mice when co-administered with chocolate under stress-free (temporally anesthetized) conditions. Taken together, these results suggest that suvorexant can inhibit orexin synthesis resulting in susceptibility to narcolepsy-like symptoms in mice under certain conditions.

Open-Source Software for Real-time Calcium Imaging and Synchronized Neuron Firing Detection.

We developed Carignan, a real-time calcium imaging software that can automatically detect activity patterns of neurons. Carignan can activate an external device when synchronized neural activity is detected in calcium imaging obtained by a one-photon (1p) miniscope. Combined with optogenetics, our software enables closed-loop experiments for investigating functions of specific types of neurons in the brain. In addition to making existing pattern detection algorithms run in real-time seamlessly, we developed a new classification module that distinguishes neurons from false-positives using deep learning. We used a combination of convolutional and recurrent neural networks to incorporate both spatial and temporal features in activity patterns. Our method performed better than existing neuron detection methods for false-positive neuron detection in terms of the F1 score. Using Carignan, experimenters can activate or suppress a group of neurons when specific neural activity is observed. Because the system uses a 1p miniscope, it can be used on the brain of a freely-moving animal, making it applicable to a wide range of experimental paradigms.

Medial Parabrachial Nucleus Is Essential in Controlling Wakefulness in Rats.

Activation of the parabrachial nucleus (PB) in the brainstem induced wakefulness in rats, suggesting which is an important nucleus that controls arousal. However, the sub-regions of PB in regulating sleep-wake cycle is still unclear. Here, we employ chemogenetics and optogenetics strategies and find that activation of the medial part of PB (MPB), but not the lateral part, induces continuous wakefulness for 10 h without sleep rebound in neither sleep amount nor the power spectra. Optogenetic activation of glutamatergic MPB neurons in sleeping rats immediately wake rats mediated by the basal forebrain (BF) and lateral hypothalamus (LH), but not the ventral medial thalamus. Most importantly, chemogenetic inhibition of PB neurons decreases wakefulness for 10 h. Conclusively, these findings indicate that the glutamatergic MPB neurons are essential in controlling wakefulness, and that MPB-BF and MPB-LH pathways are the major neuronal circuits.