Nacc1 Mutation in Mice Models Rare Neurodevelopmental Disorder with Underlying Synaptic Dysfunction.

A missense mutation in the transcription repressor Nucleus accumbens-associated 1 (NACC1) gene at c.892C>T (p.Arg298Trp) on chromosome 19 causes severe neurodevelopmental delay ( Schoch et al., 2017). To model this disorder, we engineered the first mouse model with the homologous mutation (Nacc1+/R284W ) and examined mice from E17.5 to 8 months. Both genders had delayed weight gain, epileptiform discharges and altered power spectral distribution in cortical electroencephalogram, behavioral seizures, and marked hindlimb clasping; females displayed thigmotaxis in an open field. In the cortex, NACC1 long isoform, which harbors the mutation, increased from 3 to 6 months, whereas the short isoform, which is not present in humans and lacks aaR284 in mice, rose steadily from postnatal day (P) 7. Nuclear NACC1 immunoreactivity increased in cortical pyramidal neurons and parvalbumin containing interneurons but not in nuclei of astrocytes or oligodendroglia. Glial fibrillary acidic protein staining in astrocytic processes was diminished. RNA-seq of P14 mutant mice cortex revealed over 1,000 differentially expressed genes (DEGs). Glial transcripts were downregulated and synaptic genes upregulated. Top gene ontology terms from upregulated DEGs relate to postsynapse and ion channel function, while downregulated DEGs enriched for terms relating to metabolic function, mitochondria, and ribosomes. Levels of synaptic proteins were changed, but number and length of synaptic contacts were unaltered at 3 months. Homozygosity worsened some phenotypes including postnatal survival, weight gain delay, and increase in nuclear NACC1. This mouse model simulates a rare form of autism and will be indispensable for assessing pathophysiology and targets for therapeutic intervention.

Elevated TNF-α Leads to Neural Circuit Instability in the Absence of Interferon Regulatory Factor 8.

Interferon regulatory factor 8 (IRF8) is a transcription factor necessary for the maturation of microglia, as well as other peripheral immune cells. It also regulates the transition of microglia and other immune cells to a pro-inflammatory phenotype. Irf8 is also a known risk gene for multiple sclerosis and lupus, and it has recently been shown to be downregulated in schizophrenia. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. Here, we show by RNAseq from Irf8 -/- male and female mouse brains that several genes involved in regulation of neural activity are dysregulated. We then show that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to lethal seizures in male and female Irf8 -/- mice. Finally, we identify that TNF-α is elevated specifically in microglia in the CNS, and genetic or acute pharmacological blockade of TNF-α in the Irf8 -/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. Our data further suggest that neuronal function is impacted by loss of IRF8, a factor involved in neuropsychiatric and neurodegenerative diseases.SIGNIFICANCE STATEMENT Here, we identify a previously unknown and key role for interferon regulator factor 8 (IRF8) in regulating neural excitability and seizures. We further determine that these effects on neural circuits are through elevated TNF-α in the CNS. As IRF8 has most widely been studied in the context of regulating the development and inflammatory signaling in microglia and other immune cells, we have uncovered a novel function. Further, IRF8 is a risk gene for multiple sclerosis and lupus, IRF8 is dysregulated in schizophrenia, and elevated TNF-α has been identified in a multitude of neurologic conditions. Thus, elucidating these IRF8 and TNF-α-dependent effects on brain circuit function has profound implications for understanding underlying, therapeutically relevant mechanisms of disease.

Reassessing the Role of Histaminergic Tuberomammillary Neurons in Arousal Control.

The histaminergic neurons of the tuberomammillary nucleus (TMNHDC) of the posterior hypothalamus have long been implicated in promoting arousal. More recently, a role for GABAergic signaling by the TMNHDC neurons in arousal control has been proposed. Here, we investigated the effects of selective chronic disruption of GABA synthesis (via genetic deletion of the GABA synthesis enzyme, glutamic acid decarboxylase 67) or GABAergic transmission (via genetic deletion of the vesicular GABA transporter (VGAT)) in the TMNHDC neurons on sleep-wake in male mice. We also examined the effects of acute chemogenetic activation and optogenetic inhibition of TMNHDC neurons upon arousal in male mice. Unexpectedly, we found that neither disruption of GABA synthesis nor GABAergic transmission altered hourly sleep-wake quantities, perhaps because very few TMNHDC neurons coexpressed VGAT. Acute chemogenetic activation of TMNHDC neurons did not increase arousal levels above baseline but did enhance vigilance when the mice were exposed to a behavioral cage change challenge. Similarly, acute optogenetic inhibition had little effect upon baseline levels of arousal. In conclusion, we could not identify a role for GABA release by TMNHDC neurons in arousal control. Further, if TMNHDC neurons do release GABA, the mechanism by which they do so remains unclear. Our findings support the view that TMNHDC neurons may be important for enhancing arousal under certain conditions, such as exposure to a novel environment, but play only a minor role in behavioral and EEG arousal under baseline conditions.SIGNIFICANCE STATEMENT The histaminergic neurons of the tuberomammillary nucleus of the hypothalamus (TMNHDC) have long been thought to promote arousal. Additionally, TMNHDC neurons may counter-regulate the wake-promoting effects of histamine through co-release of the inhibitory neurotransmitter, GABA. Here, we show that impairing GABA signaling from TMNHDC neurons does not impact sleep-wake amounts and that few TMNHDC neurons contain the vesicular GABA transporter, which is presumably required to release GABA. We further show that acute activation or inhibition of TMNHDC neurons has limited effects upon baseline arousal levels and that activation enhances vigilance during a behavioral challenge. Counter to general belief, our findings support the view that TMNHDC neurons are neither necessary nor sufficient for the initiation and maintenance of arousal under baseline conditions.

Non-equilibrium critical dynamics of bursts in θ and δ rhythms as fundamental characteristic of sleep and wake micro-architecture.

Origin and functions of intermittent transitions among sleep stages, including short awakenings and arousals, constitute a challenge to the current homeostatic framework for sleep regulation, focusing on factors modulating sleep over large time scales. Here we propose that the complex micro-architecture characterizing the sleep-wake cycle results from an underlying non-equilibrium critical dynamics, bridging collective behaviors across spatio-temporal scales. We investigate θ and δ wave dynamics in control rats and in rats with lesions of sleep-promoting neurons in the parafacial zone. We demonstrate that intermittent bursts in θ and δ rhythms exhibit a complex temporal organization, with long-range power-law correlations and a robust duality of power law (θ-bursts, active phase) and exponential-like (δ-bursts, quiescent phase) duration distributions, typical features of non-equilibrium systems self-organizing at criticality. Crucially, such temporal organization relates to anti-correlated coupling between θ- and δ-bursts, and is independent of the dominant physiologic state and lesions, a solid indication of a basic principle in sleep dynamics.

Genetic Activation, Inactivation, and Deletion Reveal a Limited And Nuanced Role for Somatostatin-Containing Basal Forebrain Neurons in Behavioral State Control.

Recent studies have identified an especially important role for basal forebrain GABAergic (BFVGAT) neurons in the regulation of behavioral waking and fast cortical rhythms associated with cognition. However, BFVGAT neurons comprise several neurochemically and anatomically distinct subpopulations, including parvalbumin-containing BFVGAT neurons and somatostatin-containing BFVGAT neurons (BFSOM neurons), and it was recently reported that optogenetic activation of BFSOM neurons increases the probability of a wakefulness to non-rapid-eye movement (NREM) sleep transition when stimulated during the rest period of the animal. This finding was unexpected given that most BFSOM neurons are not NREM sleep active and that central administration of the synthetic somatostatin analog, octreotide, suppresses NREM sleep or increases REM sleep. Here we used a combination of genetically driven chemogenetic and optogenetic activation, chemogenetic inhibition, and ablation approaches to further explore the in vivo role of BFSOM neurons in arousal control. Our findings indicate that acute activation or inhibition of BFSOM neurons is neither wakefulness nor NREM sleep promoting and is without significant effect on the EEG, and that chronic loss of these neurons is without effect on total 24 h sleep amounts, although a small but significant increase in waking was observed in the lesioned mice during the early active period. Our in vitro cell recordings further reveal electrophysiological heterogeneity in BFSOM neurons, specifically suggesting at least two distinct subpopulations. Together, our data support the more nuanced view that BFSOM neurons are electrically heterogeneous and are not NREM sleep or wake promoting per se, but may exert, in particular during the early active period, a modest inhibitory influence on arousal circuitry.SIGNIFICANCE STATEMENT The cellular basal forebrain (BF) is a highly complex area of the brain that is implicated in a wide range of higher-level neurobiological processes, including regulating and maintaining normal levels of electrocortical and behavioral arousal. The respective in vivo roles of BF cell populations and their neurotransmitter systems in the regulation of electrocortical and behavioral arousal remains incompletely understood. Here we seek to define the neurobiological contribution of GABAergic somatostatin-containing BF neurons to arousal control. Understanding the respective contribution of BF cell populations to arousal control may provide critical insight into the pathogenesis of a host of neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, schizophrenia, and the cognitive impairments of normal aging.

Preventing acute neurotoxicity of CNS therapeutic oligonucleotides with the addition of Ca2+ and Mg2+ in the formulation.

Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography (EEG) and electromyography (EMG) in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca2+ and Mg2+ up to molar equivalents of the phosphodiester (PO)/phosphorothioate (PS) bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establishes the importance of adding Ca2+ and Mg2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides.

Identification and characterization of a sleep-active cell group in the rostral medullary brainstem.

Early transection and stimulation studies suggested the existence of sleep-promoting circuitry in the medullary brainstem, yet the location and identity of the neurons comprising this putative hypnogenic circuitry remains unresolved. In the present study, we sought to uncover the location and identity of medullary neurons that might contribute to the regulation of sleep. Here we show the following in rats: (1) a delimited node of medullary neurons located lateral and dorsal to the facial nerve-a region we termed the parafacial zone (PZ)-project to the wake-promoting medial parabrachial nucleus; (2) PZ neurons express c-Fos after sleep but not after wakefulness and hence are sleep active; and (3) cell-body-specific lesions of the PZ result in large and sustained increases (50%) in daily wakefulness at the expense of slow-wave sleep (SWS). Using transgenic reporter mice [vesicular GABA/glycine transporter (Vgat)-GFP], we then show that >50% of PZ sleep-active neurons are inhibitory (GABAergic/glycinergic, VGAT-positive) in nature. Finally, we used a Cre-expressing adeno-associated viral vector and conditional Vgat(lox/lox) mice to selectively and genetically disrupt GABA/glycinergic neurotransmission from PZ neurons. Disruption of PZ GABAergic/glycinergic neurotransmission resulted in sustained increases (40%) in daily wakefulness at the expense of both SWS and rapid eye movement sleep. These results together reveal the location and neurochemical identity of a delimited node of sleep-active neurons within the rostral medullary brainstem.

Beyond the symptom: the biology of fatigue.

A workshop titled "Beyond the Symptom: The Biology of Fatigue" was held virtually September 27-28, 2021. It was jointly organized by the Sleep Research Society and the Neurobiology of Fatigue Working Group of the NIH Blueprint Neuroscience Research Program. For access to the presentations and video recordings, see: https://neuroscienceblueprint.nih.gov/about/event/beyond-symptom-biology-fatigue. The goals of this workshop were to bring together clinicians and scientists who use a variety of research approaches to understand fatigue in multiple conditions and to identify key gaps in our understanding of the biology of fatigue. This workshop summary distills key issues discussed in this workshop and provides a list of promising directions for future research on this topic. We do not attempt to provide a comprehensive review of the state of our understanding of fatigue, nor to provide a comprehensive reprise of the many excellent presentations. Rather, our goal is to highlight key advances and to focus on questions and future approaches to answering them.

The waking brain: an update.

Wakefulness and consciousness depend on perturbation of the cortical soliloquy. Ascending activation of the cerebral cortex is characteristic for both waking and paradoxical (REM) sleep. These evolutionary conserved activating systems build a network in the brainstem, midbrain, and diencephalon that contains the neurotransmitters and neuromodulators glutamate, histamine, acetylcholine, the catecholamines, serotonin, and some neuropeptides orchestrating the different behavioral states. Inhibition of these waking systems by GABAergic neurons allows sleep. Over the past decades, a prominent role became evident for the histaminergic and the orexinergic neurons as a hypothalamic waking center.

Two novel mouse models of slow-wave-sleep enhancement in aging and Alzheimer's disease.

Aging and Alzheimer's disease (AD) are both associated with reduced quantity and quality of the deepest stage of sleep, called slow-wave-sleep (SWS). Slow-wave-sleep deficits have been shown to worsen AD symptoms and prevent healthy aging. However, the mechanism remains poorly understood due to the lack of animal models in which SWS can be specifically manipulated. Notably, a mouse model of SWS enhancement has been recently developed in adult mice. As a prelude to studies assessing the impact of SWS enhancement on aging and neurodegeneration, we first asked whether SWS can be enhanced in animal models of aging and AD. The chemogenetic receptor hM3Dq was conditionally expressed in GABAergic neurons of the parafacial zone of aged mice and AD (APP/PS1) mouse model. Sleep-wake phenotypes were analyzed in baseline condition and following clozapine-N-oxide (CNO) and vehicle injections. Both aged and AD mice display deficits in sleep quality, characterized by decreased slow wave activity. Both aged and AD mice show SWS enhancement following CNO injection, characterized by a shorter SWS latency, increased SWS amount and consolidation, and enhanced slow wave activity, compared with vehicle injection. Importantly, the SWS enhancement phenotypes in aged and APP/PS1 model mice are comparable to those seen in adult and littermate wild-type mice, respectively. These mouse models will allow investigation of the role of SWS in aging and AD, using, for the first time, gain-of SWS experiments.

Validation of DREADD agonists and administration route in a murine model of sleep enhancement.

BACKGROUND: Chemogenetics is a powerful tool to study the role of specific neuronal populations in physiology and diseases. Of particular interest, in mice, acute and specific activation of parafacial zone (PZ) GABAergic neurons expressing the Designer Receptors Activated by Designer Drugs (DREADD) hM3Dq (PZGABA-hM3Dq) enhances slow-wave-sleep (SWS), and this effect lasts for up to 6 h, allowing prolonged and detailed study of SWS. However, the most widely used DREADDs ligand, clozapine N-oxide (CNO), is metabolized into clozapine which has the potential of inducing non-specific effects. In addition, CNO is usually injected intraperitoneally (IP) in mice, limiting the number and frequency of repeated administration. NEW METHODS: The present study is designed to validate the use of alternative DREADDs ligands-deschloroclozapine (DCZ) and compound 21 (C21)-and a new administration route, the voluntary oral administration. RESULTS: We show that IP injections of DCZ and C21 dose-dependently enhance SWS in PZGABA-hM3Dq mice, similar to CNO. We also show that oral administration of CNO, DCZ and C21 induces the same sleep phenotype as compared with IP injection. COMPARISON WITH EXISTING METHODS AND CONCLUSION: Therefore, DCZ and C21 are powerful alternatives to the use of CNO. Moreover, the voluntary oral administration is suitable for repeated dosing of DREADDs ligands.

Orexin/hypocretin and histamine: distinct roles in the control of wakefulness demonstrated using knock-out mouse models.

To determine the respective role played by orexin/hypocretin and histamine (HA) neurons in maintaining wakefulness (W), we characterized the behavioral and sleep-wake phenotypes of orexin (Ox) knock-out (-/-) mice and compared them with those of histidine-decarboxylase (HDC, HA-synthesizing enzyme)-/- mice. While both mouse strains displayed sleep fragmentation and increased paradoxical sleep (PS), they presented a number of marked differences: (1) the PS increase in HDC(-/-) mice was seen during lightness, whereas that in Ox(-/-) mice occurred during darkness; (2) contrary to HDC(-/-), Ox(-/-) mice had no W deficiency around lights-off, nor an abnormal EEG and responded to a new environment with increased W; (3) only Ox(-/-), but not HDC(-/-) mice, displayed narcolepsy and deficient W when faced with motor challenge. Thus, when placed on a wheel, wild-type (WT), but not littermate Ox(-/-) mice, voluntarily spent their time in turning it and as a result, remained highly awake; this was accompanied by dense c-fos expression in many areas of their brains, including Ox neurons in the dorsolateral hypothalamus. The W and motor deficiency of Ox(-/-) mice was due to the absence of Ox because intraventricular dosing of orexin-A restored their W amount and motor performance whereas SB-334867 (Ox1-receptor antagonist, i.p.) impaired W and locomotion of WT mice during the test. These data indicate that Ox, but not HA, promotes W through enhanced locomotion and suggest that HA and Ox neurons exert a distinct, but complementary and synergistic control of W: the neuropeptide being more involved in its behavioral aspects, whereas the amine is mainly responsible for its qualitative cognitive aspects and cortical EEG activation.

The neuroanatomy and neurochemistry of sleep-wake control.

Sleep-wake control is dependent upon multiple brain areas widely distributed throughout the neural axis. Historically, the monoaminergic and cholinergic neurons of the ascending arousal system were the first to be discovered, and it was only relatively recently that GABAergic and glutamatergic wake- and sleep-promoting populations have been identified. Contemporary advances in molecular-genetic tools have revealed both the complexity and heterogeneity of GABAergic NREM sleep-promoting neurons as well as REM sleep-regulating populations in the brainstem such as glutamatergic neurons in the sublaterodorsal nucleus. The sleep-wake cycle progresses from periods of wakefulness to non-rapid eye movement (NREM) sleep and subsequently rapid eye movement (REM) sleep. Each vigilance stage is controlled by multiple neuronal populations, via a complex regulation that is still incompletely understood. In recent years the field has seen a proliferation in the identification and characterization of new neuronal populations involved in sleep-wake control thanks to newer, more powerful molecular genetic tools that are able to reveal neurophysiological functions via selective activation, inhibition and lesion of neuroanatomically defined sub-types of neurons that are widespread in the brain, such as GABAergic and glutamatergic neurons.1,2.

Suprachiasmatic VIP neurons are required for normal circadian rhythmicity and comprised of molecularly distinct subpopulations.

The hypothalamic suprachiasmatic (SCN) clock contains several neurochemically defined cell groups that contribute to the genesis of circadian rhythms. Using cell-specific and genetically targeted approaches we have confirmed an indispensable role for vasoactive intestinal polypeptide-expressing SCN (SCNVIP) neurons, including their molecular clock, in generating the mammalian locomotor activity (LMA) circadian rhythm. Optogenetic-assisted circuit mapping revealed functional, di-synaptic connectivity between SCNVIP neurons and dorsomedial hypothalamic neurons, providing a circuit substrate by which SCNVIP neurons may regulate LMA rhythms. In vivo photometry revealed that while SCNVIP neurons are acutely responsive to light, their activity is otherwise behavioral state invariant. Single-nuclei RNA-sequencing revealed that SCNVIP neurons comprise two transcriptionally distinct subtypes, including putative pacemaker and non-pacemaker populations. Altogether, our work establishes necessity of SCNVIP neurons for the LMA circadian rhythm, elucidates organization of circadian outflow from and modulatory input to SCNVIP cells, and demonstrates a subpopulation-level molecular heterogeneity that suggests distinct functions for specific SCNVIP subtypes.

Differential Role of Pontomedullary Glutamatergic Neuronal Populations in Sleep-Wake Control.

Parafacial zone (PZ) GABAergic neurons play a major role in slow-wave-sleep (SWS), also called non-rapid eye movement (NREM) sleep. The PZ also contains glutamatergic neurons expressing the vesicular transporter for glutamate, isoform 2 (Vglut2). We hypothesized that PZ Vglut2-expressing (PZVglut2) neurons are also involved in sleep control, playing a synergistic role with PZ GABAergic neurons. To test this hypothesis, we specifically activated PZVglut2 neurons using the excitatory chemogenetic receptor hM3Dq. Anatomical inspection of the injection sites revealed hM3Dq transfection in PZ, parabrachial nucleus (PB), sublaterodorsal nucleus (SLD) or various combinations of these three brain areas. Consistent with the known wake- and REM sleep-promoting role of PB and SLD, respectively, chemogenetic activation of PBVglut2 or SLDVglut2 resulted in wake or REM sleep enhancement. Chemogenetic activation of PZVglut2 neurons did not affect sleep-wake phenotype during the mouse active period but increased wakefulness and REM sleep, similar to PBVglut2 and SLDVglut2 activation, during the rest period. To definitively confirm the role of PZVglut2 neurons, we used a specific marker for PZVglut2 neurons, Phox2B. Chemogenetic activation of PZPhox2B neurons did not affect sleep-wake phenotype, indicating that PZ glutamatergic neurons are not sufficient to affect sleep-wake cycle. These results indicate that PZ glutamatergic neurons are not involved in sleep-wake control.

Newly identified sleep-wake and circadian circuits as potential therapeutic targets.

Optogenetics and chemogenetics are powerful tools, allowing the specific activation or inhibition of targeted neuronal subpopulations. Application of these techniques to sleep and circadian research has resulted in the unveiling of several neuronal populations that are involved in sleep-wake control, and allowed a comprehensive interrogation of the circuitry through which these nodes are coordinated to orchestrate the sleep-wake cycle. In this review, we discuss six recently described sleep-wake and circadian circuits that show promise as therapeutic targets for sleep medicine. The parafacial zone (PZ) and the ventral tegmental area (VTA) are potential druggable targets for the treatment of insomnia. The brainstem circuit underlying rapid eye movement sleep behavior disorder (RBD) offers new possibilities for treating RBD and neurodegenerative synucleinopathies, whereas the parabrachial nucleus, as a nexus linking arousal state control and breathing, is a promising target for developing treatments for sleep apnea. Therapies that act upon the hypothalamic circuitry underlying the circadian regulation of aggression or the photic regulation of arousal and mood pathway carry enormous potential for helping to reduce the socioeconomic burden of neuropsychiatric and neurodegenerative disorders on society. Intriguingly, the development of chemogenetics as a therapeutic strategy is now well underway and such an approach has the capacity to lead to more focused and less invasive therapies for treating sleep-wake disorders and related comorbidities.

An inverse agonist of the histamine H(3) receptor improves wakefulness in narcolepsy: studies in orexin-/- mice and patients.

Narcolepsy is characterized by excessive daytime sleepiness (EDS), cataplexy, direct onsets of rapid eye movement (REM) sleep from wakefulness (DREMs) and deficiency of orexins, neuropeptides that promote wakefulness largely via activation of histamine (HA) pathways. The hypothesis that the orexin defect can be circumvented by enhancing HA release was explored in narcoleptic mice and patients using tiprolisant, an inverse H(3)-receptor agonist. In narcoleptic orexin(-/-) mice, tiprolisant enhanced HA and noradrenaline neuronal activity, promoted wakefulness and decreased abnormal DREMs, all effects being amplified by co-administration of modafinil, a currently-prescribed wake-promoting drug. In a pilot single-blind trial on 22 patients receiving a placebo followed by tiprolisant, both for 1 week, the Epworth Sleepiness Scale (ESS) score was reduced from a baseline value of 17.6 by 1.0 with the placebo (p>0.05) and 5.9 with tiprolisant (p<0.001). Excessive daytime sleep, unaffected under placebo, was nearly suppressed on the last days of tiprolisant dosing. H(3)-receptor inverse agonists could constitute a novel effective treatment of EDS, particularly when associated with modafinil.

Activation of the GABAergic Parafacial Zone Maintains Sleep and Counteracts the Wake-Promoting Action of the Psychostimulants Armodafinil and Caffeine.

We previously reported that acute and selective activation of GABA-releasing parafacial zone (PZVgat) neurons in behaving mice produces slow-wave-sleep (SWS), even in the absence of sleep deficit, suggesting that these neurons may represent, at least in part, a key cellular substrate underlying sleep drive. It remains, however, to be determined if PZVgat neurons actively maintain, as oppose to simply gate, SWS. To begin to experimentally address this knowledge gap, we asked whether activation of PZVgat neurons could attenuate or block the wake-promoting effects of two widely used wake-promoting psychostimulants, armodafinil or caffeine. We found that activation of PZVgat neurons completely blocked the behavioral and electrocortical wake-promoting action of armodafinil. In some contrast, activation of PZVgat neurons inhibited the behavioral, but not electrocortical, arousal response to caffeine. These results suggest that: (1) PZVgat neurons actively maintain, as oppose to simply gate, SWS and cortical slow-wave-activity; (2) armodafinil cannot exert its wake-promoting effects when PZVgat neurons are activated, intimating a possible shared circuit/molecular basis for mechanism of action; (3) caffeine can continue to exert potent cortical desynchronizing, but not behavioral, effects when PZVgat neurons are activated, inferring a shared and divergent circuit/molecular basis for mechanism of action; and 4) PZVgat neurons represent a key cell population for SWS induction and maintenance.

Functionally Complete Excision of Conditional Alleles in the Mouse Suprachiasmatic Nucleus by Vgat-ires-Cre.

Mice with targeted gene disruption have provided important information about the molecular mechanisms of circadian clock function. A full understanding of the roles of circadian-relevant genes requires manipulation of their expression in a tissue-specific manner, ideally including manipulation with high efficiency within the suprachiasmatic nuclei (SCN). To date, conditional manipulation of genes within the SCN has been difficult. In a previously developed mouse line, Cre recombinase was inserted into the vesicular GABA transporter (Vgat) locus. Since virtually all SCN neurons are GABAergic, this Vgat-Cre line seemed likely to have high efficiency at disrupting conditional alleles in SCN. To test this premise, the efficacy of Vgat-Cre in excising conditional (fl, for flanked by LoxP) alleles in the SCN was examined. Vgat-Cre-mediated excision of conditional alleles of Clock or Bmal1 led to loss of immunostaining for products of the targeted genes in the SCN. Vgat-Cre+; Clockfl/fl; Npas2m/m mice and Vgat-Cre+; Bmal1fl/fl mice became arrhythmic immediately upon exposure to constant darkness, as expected based on the phenotype of mice in which these genes are disrupted throughout the body. The phenotype of mice with other combinations of Vgat-Cre+, conditional Clock, and mutant Npas2 alleles also resembled the corresponding whole-body knockout mice. These data indicate that the Vgat-Cre line is useful for Cre-mediated recombination within the SCN, making it useful for Cre-enabled technologies including gene disruption, gene replacement, and opto- and chemogenetic manipulation of the SCN circadian clock.