General Regulatory Factors Control the Fidelity of Transcription by Restricting Non-coding and Ectopic Initiation.

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.

Harnessing olfactory bulb oscillations to perform fully brain-based sleep-scoring and real-time monitoring of anaesthesia depth.

Real-time tracking of vigilance states related to both sleep or anaesthesia has been a goal for over a century. However, sleep scoring cannot currently be performed with brain signals alone, despite the deep neuromodulatory transformations that accompany sleep state changes. Therefore, at heart, the operational distinction between sleep and wake is that of immobility and movement, despite numerous situations in which this one-to-one mapping fails. Here we demonstrate, using local field potential (LFP) recordings in freely moving mice, that gamma (50-70 Hz) power in the olfactory bulb (OB) allows for clear classification of sleep and wake, thus providing a brain-based criterion to distinguish these two vigilance states without relying on motor activity. Coupled with hippocampal theta activity, it allows the elaboration of a sleep scoring algorithm that relies on brain activity alone. This method reaches over 90% homology with classical methods based on muscular activity (electromyography [EMG]) and video tracking. Moreover, contrary to EMG, OB gamma power allows correct discrimination between sleep and immobility in ambiguous situations such as fear-related freezing. We use the instantaneous power of hippocampal theta oscillation and OB gamma oscillation to construct a 2D phase space that is highly robust throughout time, across individual mice and mouse strains, and under classical drug treatment. Dynamic analysis of trajectories within this space yields a novel characterisation of sleep/wake transitions: whereas waking up is a fast and direct transition that can be modelled by a ballistic trajectory, falling asleep is best described as a stochastic and gradual state change. Finally, we demonstrate that OB oscillations also allow us to track other vigilance states. Non-REM (NREM) and rapid eye movement (REM) sleep can be distinguished with high accuracy based on beta (10-15 Hz) power. More importantly, we show that depth of anaesthesia can be tracked in real time using OB gamma power. Indeed, the gamma power predicts and anticipates the motor response to stimulation both in the steady state under constant anaesthetic and dynamically during the recovery period. Altogether, this methodology opens the avenue for multi-timescale characterisation of brain states and provides an unprecedented window onto levels of vigilance.

Morphine-Induced Dendritic Spine Remodeling in Rat Nucleus Accumbens Is Corticosterone Dependent.

BACKGROUND: Chronic morphine treatments produce important morphological changes in multiple brain areas including the nucleus accumbens. METHODS: In this study, we have investigated the effect of chronic morphine treatment at a relatively low dose on the morphology of medium spiny neurons in the core and shell of the nucleus accumbens in rats 1 day after the last injection of a chronic morphine treatment (5 mg/kg once per day for 14 days). Medium spiny neurons were labeled with 1,1' dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate crystal and analyzed by confocal laser-scanning microscope. RESULTS: Our results show an increase of thin spines and a decrease of stubby spines specifically in the shell of morphine-treated rats compared with control. Since morphine-treated rats also presented an elevation of corticosterone level in plasma, we explored whether spine alterations induced by morphine treatment in the nucleus accumbens could be affected by the depletion of the hormone. Thus, bilaterally adrenalectomized rats were treated with morphine in the same conditions. No more alteration in stubby spines in the shell was detected in morphine-treated rats with a depletion of corticosterone, while a significant increase was observed in mushroom spines in the shell and stubby spines in the core. Regarding the thin spines, the increase observed with morphine compared with saline was lower in adrenalectomized rats than in nonadrenalectomized animals. CONCLUSION: These results indicate that dendritic spine remodeling in nucleus accumbens following chronic morphine treatment at relatively low doses is dependent on corticosterone levels.

Glucose Induces Slow-Wave Sleep by Exciting the Sleep-Promoting Neurons in the Ventrolateral Preoptic Nucleus: A New Link between Sleep and Metabolism.

Sleep-active neurons located in the ventrolateral preoptic nucleus (VLPO) play a crucial role in the induction and maintenance of slow-wave sleep (SWS). However, the cellular and molecular mechanisms responsible for their activation at sleep onset remain poorly understood. Here, we test the hypothesis that a rise in extracellular glucose concentration in the VLPO can promote sleep by increasing the activity of sleep-promoting VLPO neurons. We find that infusion of a glucose concentration into the VLPO of mice promotes SWS and increases the density of c-Fos-labeled neurons selectively in the VLPO. Moreover, we show in patch-clamp recordings from brain slices that VLPO neurons exhibiting properties of sleep-promoting neurons are selectively excited by glucose within physiological range. This glucose-induced excitation implies the catabolism of glucose, leading to a closure of ATP-sensitive potassium (KATP) channels. The extracellular glucose concentration monitors the gating of KATP channels of sleep-promoting neurons, highlighting that these neurons can adapt their excitability according to the extracellular energy status. Together, these results provide evidence that glucose may participate in the mechanisms of SWS promotion and/or consolidation. SIGNIFICANCE STATEMENT: Although the brain circuitry underlying vigilance states is well described, the molecular mechanisms responsible for sleep onset remain largely unknown. Combining in vitro and in vivo experiments, we demonstrate that glucose likely contributes to sleep onset facilitation by increasing the excitability of sleep-promoting neurons in the ventrolateral preoptic nucleus (VLPO). We find here that these neurons integrate energetic signals such as ambient glucose directly to regulate vigilance states accordingly. Glucose-induced excitation of sleep-promoting VLPO neurons should therefore be involved in the drowsiness that one feels after a high-sugar meal. This novel mechanism regulating the activity of VLPO neurons reinforces the fundamental and intimate link between sleep and metabolism.

Temporal regulation of peripheral BDNF levels during cocaine and morphine withdrawal: comparison with a natural reward.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a neurotrophin that has long been studied in the field of addiction and its importance in regulating drug addiction-related behavior has been widely demonstrated. The aim of our study was to analyze the consequences of a repeated exposure to drugs of abuse or natural reward on plasma BDNF levels during withdrawal. METHODS: Rats were chronically injected with morphine (subcutaneously, 5mg/kg) or cocaine (intraperitoneally, 20mg/kg) or fed with a butter biscuit (per os, 4g) once per day for 14 days. Blood collection was performed on the 1st (withdrawal day 1 or WD1) or on (WD14), either at the same time point rats had been exposed to drugs or natural reward or at a different time point (used to quantify basal brain-derived neurotrophic factor levels). RESULTS: Cocaine treatment led to a rapid (WD1) and persistent (WD14) decrease of basal BDNF levels compared with saline-treated animals, whereas morphine induced an increase on WD14 without any alteration on WD1. On the contrary, the natural reward induced a significant increase of basal brain-derived neurotrophic factor levels only on WD1. The analysis of BDNF levels at the usual time point at which animals had been exposed showed that both drugs, but not the natural reward, increased BDNF levels compared with basal levels. CONCLUSION: Our data highlight that only drugs of abuse are able to persistently alter BDNF levels and to induce specific variations of this neurotrophic factor at the usual hour of injection.

Diversity of GABAergic interneurons in layer VIa and VIb of mouse barrel cortex.

Neocortical layer VI modulates the thalamocortical transfer of information and has a significant impact on sensory processing. This function implicates local γ-aminobutyric acidergic (GABAergic) interneurons that have only been partly described at the present time. Here, we characterized 85 layer VI GABAergic interneurons in acute slices of mouse somatosensory barrel cortex, using whole-cell current-clamp recordings, single-cell reverse transcription-polymerase chain reaction, and biocytin labeling followed by Neurolucida reconstructions. Unsupervised clustering based on electrophysiological molecular and morphological properties disclosed 4 types of interneurons. The 2 major classes were fast-spiking cells transcribing parvalbumin (PV) (51%) and adapting interneurons transcribing somatostatin (SOM) (26%). The third population (18%) transcribed neuropeptide Y (NPY) and appeared very similar to neurogliaform cells. The last class (5%) was constituted by well-segregated GABAergic interneurons transcribing vasoactive intestinal peptide (VIP). Using transgenic mice expressing GFP under the control of the glutamic acid decarboxylase 67k (GAD67) promoter, we investigated the densities of GABAergic cells immunolabeled against PV, SOM, VIP, and NPY through the depth of layer VI. This analysis revealed that PV and NPY translating interneurons concentrate in the upper and lower parts of layer VI, respectively. This study provides an extensive characterization of the properties of layer VI interneurons.

Kinetochore component function in C. elegans oocytes revealed by 4D tracking of holocentric chromosomes.

During cell division, chromosome congression to the spindle center, their orientation along the spindle long axis and alignment at the metaphase plate depend on interactions between spindle microtubules and kinetochores, and are pre-requisite for chromosome bi-orientation and accurate segregation. How these successive phases are controlled during oocyte meiosis remains elusive. Here we provide 4D live imaging during the first meiotic division in C. elegans oocytes with wild-type or disrupted kinetochore protein function. We show that, unlike in monocentric organisms, holocentric chromosome bi-orientation is not strictly required for accurate chromosome segregation. Instead, we propose a model in which initial kinetochore-localized BHC module (comprised of BUB-1Bub1, HCP-1/2CENP-F and CLS-2CLASP)-dependent pushing acts redundantly with Ndc80 complex-mediated pulling for accurate chromosome segregation in meiosis. In absence of both mechanisms, homologous chromosomes tend to co-segregate in anaphase, especially when initially mis-oriented. Our results highlight how different kinetochore components cooperate to promote accurate holocentric chromosome segregation in oocytes of C. elegans.

Synergistic stabilization of microtubules by BUB-1, HCP-1, and CLS-2 controls microtubule pausing and meiotic spindle assembly.

During cell division, chromosome segregation is orchestrated by a microtubule-based spindle. Interaction between spindle microtubules and kinetochores is central to the bi-orientation of chromosomes. Initially dynamic to allow spindle assembly and kinetochore attachments, which is essential for chromosome alignment, microtubules are eventually stabilized for efficient segregation of sister chromatids and homologous chromosomes during mitosis and meiosis I, respectively. Therefore, the precise control of microtubule dynamics is of utmost importance during mitosis and meiosis. Here, we study the assembly and role of a kinetochore module, comprised of the kinase BUB-1, the two redundant CENP-F orthologs HCP-1/2, and the CLASP family member CLS-2 (hereafter termed the BHC module), in the control of microtubule dynamics in Caenorhabditis elegans oocytes. Using a combination of in vivo structure-function analyses of BHC components and in vitro microtubule-based assays, we show that BHC components stabilize microtubules, which is essential for meiotic spindle formation and accurate chromosome segregation. Overall, our results show that BUB-1 and HCP-1/2 do not only act as targeting components for CLS-2 at kinetochores, but also synergistically control kinetochore-microtubule dynamics by promoting microtubule pause. Together, our results suggest that BUB-1 and HCP-1/2 actively participate in the control of kinetochore-microtubule dynamics in the context of an intact BHC module to promote spindle assembly and accurate chromosome segregation in meiosis.

VIP, CRF, and PACAP act at distinct receptors to elicit different cAMP/PKA dynamics in the neocortex.

The functional significance of diverse neuropeptide coexpression and convergence onto common second messenger pathways remains unclear. To address this question, we characterized responses to corticotropin-releasing factor (CRF), pituitary adenylate cyclase-activating peptide (PACAP), and vasoactive intestinal peptide (VIP) in rat neocortical slices using optical recordings of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) sensors, patch-clamp, and single-cell reverse transcription-polymerase chain reaction. Responses of pyramidal neurons to the 3 neuropeptides markedly differed in time-course and amplitude. Effects of these neuropeptides on the PKA-sensitive slow afterhyperpolarization current were consistent with those observed with cAMP/PKA sensors. CRF-1 receptors, primarily expressed in pyramidal cells, reportedly mediate the neocortical effects of CRF. PACAP and VIP activated distinct PAC1 and VPAC1 receptors, respectively. Indeed, a selective VPAC1 antagonist prevented VIP responses but had a minor effect on PACAP responses, which were mimicked by a specific PAC1 agonist. While PAC1 and VPAC1 were coexpressed in pyramidal cells, PAC1 expression was also frequently detected in interneurons, suggesting that PACAP has widespread effects on the neuronal network. Our results suggest that VIP and CRF, originating from interneurons, and PACAP, expressed mainly by pyramidal cells, finely tune the excitability and gene expression in the neocortical network via distinct cAMP/PKA-mediated effects.

Molecular and electrophysiological features of GABAergic neurons in the dentate gyrus reveal limited homology with cortical interneurons.

GABAergic interneurons tend to diversify into similar classes across telencephalic regions. However, it remains unclear whether the electrophysiological and molecular properties commonly used to define these classes are discriminant in the hilus of the dentate gyrus. Here, using patch-clamp combined with single cell RT-PCR, we compare the relevance of commonly used electrophysiological and molecular features for the clustering of GABAergic interneurons sampled from the mouse hilus and primary sensory cortex. While unsupervised clustering groups cortical interneurons into well-established classes, it fails to provide a convincing partition of hilar interneurons. Statistical analysis based on resampling indicates that hilar and cortical GABAergic interneurons share limited homology. While our results do not invalidate the use of classical molecular marker in the hilus, they indicate that classes of hilar interneurons defined by the expression of molecular markers do not exhibit strongly discriminating electrophysiological properties.

Serotonin 3A receptor subtype as an early and protracted marker of cortical interneuron subpopulations.

To identify neocortical neurons expressing the type 3 serotonergic receptor, here we used transgenic mice expressing the enhanced green fluorescent protein (GFP) under the control of the 5-HT(3A) promoter (5-HT(3A):GFP mice). By means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reversed-transcriptase polymerase chain reaction on acute brain slices of 5-HT(3A):GFP mice, we identified 2 populations of 5-HT(3A)-expressing interneurons within the somatosensory cortex. The first population was characterized by the frequent expression of the vasoactive intestinal peptide and a typical bipolar/bitufted morphology, whereas the second population expressed predominantly the neuropeptide Y and exhibited more complex dendritic arborizations. Most interneurons of this second group appeared very similar to neurogliaform cells according to their electrophysiological, molecular, and morphological properties. The combination of 5-bromo-2-deoxyuridine injections with 5-HT(3A) mRNA detection showed that cortical 5-HT(3A) interneurons are generated around embryonic day 14.5. Although at this stage the 5-HT(3A) receptor subunit is expressed in both the caudal ganglionic eminence and the entopeduncular area, homochronic in utero grafts experiments revealed that cortical 5-HT(3A) interneurons are mainly generated in the caudal ganglionic eminence. This protracted expression of the 5-HT(3A) subunit allowed us to study specific cortical interneuron populations from their birth to their final functional phenotype.

Breathing-driven prefrontal oscillations regulate maintenance of conditioned-fear evoked freezing independently of initiation.

Brain-body interactions are thought to be essential in emotions but their physiological basis remains poorly understood. In mice, regular 4 Hz breathing appears during freezing after cue-fear conditioning. Here we show that the olfactory bulb (OB) transmits this rhythm to the dorsomedial prefrontal cortex (dmPFC) where it organizes neural activity. Reduction of the respiratory-related 4 Hz oscillation, via bulbectomy or optogenetic perturbation of the OB, reduces freezing. Behavioural modelling shows that this is due to a specific reduction in freezing maintenance without impacting its initiation, thus dissociating these two phenomena. dmPFC LFP and firing patterns support the region's specific function in freezing maintenance. In particular, population analysis reveals that network activity tracks 4 Hz power dynamics during freezing and reaches a stable state at 4 Hz peak that lasts until freezing termination. These results provide a potential mechanism and a functional role for bodily feedback in emotions and therefore shed light on the historical James-Cannon debate.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity.

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Classification of NPY-expressing neocortical interneurons.

Neuropeptide Y (NPY) is an abundant neuropeptide of the neocortex involved in numerous physiological and pathological processes. Because of the large electrophysiological, molecular, and morphological diversity of NPY-expressing neurons their precise identity remains unclear. To define distinct populations of NPY neurons we characterized, in acute slices of rat barrel cortex, 200 cortical neurons of layers I-IV by means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reverse transcriptase-PCR designed to probe for the expression of well established molecular markers for cortical neurons. To classify reliably cortical NPY neurons, we used and compared different unsupervised clustering algorithms based on laminar location and electrophysiological and molecular properties. These classification schemes confirmed that NPY neurons are nearly exclusively GABAergic and consistently disclosed three main types of NPY-expressing interneurons. (1) Neurogliaform-like neurons exhibiting a dense axonal arbor, were the most frequent and superficial, and substantially expressed the neuronal isoform of nitric oxide synthase. (2) Martinotti-like cells characterized by an ascending axon ramifying in layer I coexpressed somatostatin and were the most excitable type. (3) Among fast-spiking and parvalbumin-positive basket cells, NPY expression was correlated with pronounced spike latency. By clarifying the diversity of cortical NPY neurons, this study establishes a basis for future investigations aiming at elucidating their physiological roles.

Degenerative abnormalities in transgenic neocortical neuropeptide Y interneurons expressing tau-green fluorescent protein.

The introduction of a reporter gene into bacterial artificial chromosome (BAC) constructs allows a rapid identification of the cell type expressing the gene of interest. Here we used BAC transgenic mice expressing a tau-sapphire green fluorescent protein (GFP) under the transcriptional control of the neuropeptide Y (NPY) genomic sequence to characterize morphological and electrophysiological properties of NPY-GFP interneurons of the mouse juvenile primary somatosensory cortex. Electrophysiological whole-cell recordings and biocytin injections were performed to allow the morphological reconstruction of the recorded neurons in three dimensions. Ninety-six recorded NPY-GFP interneurons were compared with 39 wild-type (WT) NPY interneurons, from which 23 and 19 were reconstructed, respectively. We observed that 91% of the reconstructed NPY-GFP interneurons had developed an atypical axonal swelling from which emerge numerous ramifications. These abnormalities were very heterogeneous in shape and size. They were immunoreactive for the microtubule-associated protein tau and the lysosomal-associated membrane protein 1 (LAMP1). Moreover, an electron microscopic analysis revealed the accumulation of numerous autophagic and lysosomal vacuoles in swollen axons. Morphological analyses of NPY-GFP interneurons also indicated that their somata were smaller, their entire dendritic tree was thickened and presented a restricted spatial distribution in comparison with WT NPY interneurons. Finally, the morphological defects observed in NPY-GFP interneurons appeared to be associated with alterations of their electrophysiological intrinsic properties. Altogether, these results demonstrate that NPY-GFP interneurons developed dystrophic axonal swellings and severe morphological and electrophysiological defects that could be due to the overexpression of tau-coupled reporter constructs.

Functional role of epsilon-tubulin in the assembly of the centriolar microtubule scaffold.

Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as delta-, epsilon-, or eta-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of epsilon-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules.

Glutamatergic Control of Microvascular Tone by Distinct GABA Neurons in the Cerebellum.

The tight coupling between increased neuronal activity and local cerebral blood flow, known as functional hyperemia, is essential for normal brain function. However, its cellular and molecular mechanisms remain poorly understood. In the cerebellum, functional hyperemia depends almost exclusively on nitric oxide (NO). Here, we investigated the role of different neuronal populations in the control of microvascular tone by in situ amperometric detection of NO and infrared videomicroscopy of microvessel movements in rat cerebellar slices. Bath application of an NO donor induced both NO flux and vasodilation. Surprisingly, endogenous release of NO elicited by glutamate was accompanied by vasoconstriction that was abolished by inhibition of Ca2+-phopholipase A2 and impaired by cyclooxygenase and thromboxane synthase inhibition and endothelin A receptor blockade, indicating a role for prostanoids and endothelin 1 in this response. Interestingly, direct stimulation of single endothelin 1-immunopositive Purkinje cells elicited constriction of neighboring microvessels. In contrast to glutamate, NMDA induced both NO flux and vasodilation that were abolished by treatment with a NO synthase inhibitor or with tetrodotoxin. These findings indicate that NO derived from neuronal origin is necessary for vasodilation induced by NMDA and, furthermore, that NO-producing interneurons mediate this vasomotor response. Correspondingly, electrophysiological stimulation of single nitrergic stellate cells by patch clamp was sufficient to release NO and dilate both intraparenchymal and upstream pial microvessels. These findings demonstrate that cerebellar stellate and Purkinje cells dilate and constrict, respectively, neighboring microvessels and highlight distinct roles for different neurons in neurovascular coupling.

Multiparametric characterization of neuronal subpopulations in the ventrolateral preoptic nucleus.

The characterization of neuronal properties is a necessary first step toward understanding how the ventrolateral preoptic nucleus (VLPO) neuronal network regulates slow-wave sleep (SWS). Indeed, the electrophysiological heterogeneity of VLPO neurons suggests the existence of subtypes that could differently contribute in SWS induction and maintenance. The aim of the present study was to define cell classes in the VLPO using an unsupervised clustering classification method. Electrophysiological features extracted from 289 neurons recorded in whole-cell patch-clamp allowed the identification of three main classes of VLPO neurons subdivided into five distinct subpopulations (cluster 1, 2a, 2b, 3a and 3b). The high occurrence of a low-threshold calcium spike (LTS) was one of the most distinctive features of cluster 1 and 3. Since sleep-promoting neurons are generally identified by their ability to generate an LTS and by their inhibitory response to noradrenaline (NA), 189 neurons from our dataset were also tested for this neurotransmitter. Neurons from cluster 3 were the most frequently inhibited by NA. Biocytin labeling and Neurolucida reconstructions of 112 neurons furthermore revealed a small dendritic arbor of cluster 3b neurons compared, in particular, to cluster 2b neurons. Altogether, we performed an exhaustive characterization of VLPO neuronal subtypes that is a crucial step toward a better understanding of the neuronal network within the VLPO and thereby sleep physiology.

Serotonin differentially modulates excitatory and inhibitory synaptic inputs to putative sleep-promoting neurons of the ventrolateral preoptic nucleus.

The role of serotonin (5-HT) in sleep-wake regulation has been a subject of intense debate and remains incompletely understood. In the ventrolateral preoptic nucleus (VLPO), the main structure that triggers non-rapid eye movement (NREM) sleep, putative sleep-promoting (PSP) neurons were shown ex vivo to be either inhibited (Type-1) or excited (Type-2) by 5-HT application. To determine the complex action of this neurotransmitter on PSP neurons, we recorded spontaneous and miniature excitatory and inhibitory postsynaptic currents (sEPSCs, sIPSCs, mEPSCs and mIPSCs) in response to bath application of 5-HT. We established in mouse acute VLPO slices that 5-HT reduces spontaneous and miniature EPSC and IPSC frequencies to Type-1 neurons, whereas 5-HT selectively increases sIPSC and mIPSC frequencies to Type-2 VLPO neurons. We further determined that Type-1 neurons display a lower action potential threshold and a smaller soma size than Type-2 neurons. Finally, single-cell RT-PCR designed to identify the 13 serotonergic receptor subtypes revealed the specific mRNA expression of the 5-HT1A,B,D,F receptors by Type-1 neurons. Furthermore, the 5-HT2A-C,4,7 receptors were found to be equivalently expressed by both neuronal types. Altogether, our results establish that the excitatory and inhibitory inputs to Type-1 and Type-2 VLPO PSP neurons are differentially regulated by 5-HT. Electrophysiological, morphological and molecular differences were also identified between these two neuronal types. Our results provide new insights regarding the orchestration of sleep regulation by 5-HT release, and strongly suggest that Type-2 neurons could play a permissive role, whereas Type-1 neurons could have an executive role in sleep induction and maintenance.

Astrocyte-derived adenosine is central to the hypnogenic effect of glucose.

Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity.