NMDA Receptors in the Lateral Preoptic Hypothalamus Are Essential for Sustaining NREM and REM Sleep.

The lateral preoptic (LPO) hypothalamus is a center for NREM and REM sleep induction and NREM sleep homeostasis. Although LPO is needed for NREM sleep, we found that calcium signals were, surprisingly, highest in REM sleep. Furthermore, and equally surprising, NMDA receptors in LPO were the main drivers of excitation. Deleting the NMDA receptor GluN1 subunit from LPO abolished calcium signals in all cells and produced insomnia. Mice of both sexes had highly fragmented NREM sleep-wake patterns and could not generate conventionally classified REM sleep. The sleep phenotype produced by deleting NMDA receptors depended on where in the hypothalamus the receptors were deleted. Deleting receptors from the anterior hypothalamic area (AHA) did not influence sleep-wake states. The sleep fragmentation originated from NMDA receptors on GABA neurons in LPO. Sleep fragmentation could be transiently overcome with sleeping medication (zolpidem) or sedatives (dexmedetomidine; Dex). By contrast, fragmentation persisted under high sleep pressure produced by sleep deprivation (SD), mice had a high propensity to sleep but woke up. By analyzing changes in δ power, sleep homeostasis (also referred to as "sleep drive") remained intact after NMDA receptor ablation. We suggest NMDA glutamate receptor activation stabilizes firing of sleep-on neurons and that mechanisms of sleep maintenance differ from that of the sleep drive itself.SIGNIFICANCE STATEMENT Insomnia is a common affliction. Most insomniacs feel that they do not get enough sleep, but in fact, often have good amounts of sleep. Their sleep, however, is fragmented, and sufferers wake up feeling unrefreshed. It is unknown how sleep is maintained once initiated. We find that in mice, NMDA-type glutamate receptors in the hypothalamus are the main drivers of excitation and are required for a range of sleep properties: they are, in fact, needed for both sustained NREM sleep periods, and REM sleep generation. When NMDA receptors are selectively reduced from inhibitory preoptic (PO) neurons, mice have normal total amounts of sleep but high sleep-wake fragmentation, providing a model for studying intractable insomnia.

Reproducing the dopamine pathophysiology of schizophrenia and approaches to ameliorate it: a translational imaging study with ketamine.

Patients with schizophrenia show increased striatal dopamine synthesis capacity in imaging studies. The mechanism underlying this is unclear but may be due to N-methyl-D-aspartate receptor (NMDAR) hypofunction and parvalbumin (PV) neuronal dysfunction leading to disinhibition of mesostriatal dopamine neurons. Here, we develop a translational mouse model of the dopamine pathophysiology seen in schizophrenia and test approaches to reverse the dopamine changes. Mice were treated with sub-chronic ketamine (30 mg/kg) or saline and then received in vivo positron emission tomography of striatal dopamine synthesis capacity, analogous to measures used in patients. Locomotor activity was measured using the open-field test. In vivo cell-type-specific chemogenetic approaches and pharmacological interventions were used to manipulate neuronal excitability. Immunohistochemistry and RNA sequencing were used to investigate molecular mechanisms. Sub-chronic ketamine increased striatal dopamine synthesis capacity (Cohen's d = 2.5) and locomotor activity. These effects were countered by inhibition of midbrain dopamine neurons, and by activation of PV interneurons in pre-limbic cortex and ventral subiculum of the hippocampus. Sub-chronic ketamine reduced PV expression in these cortical and hippocampal regions. Pharmacological intervention with SEP-363856, a novel psychotropic agent with agonism at trace amine receptor 1 (TAAR1) and 5-HT1A receptors but no appreciable action at dopamine D2 receptors, significantly reduced the ketamine-induced increase in dopamine synthesis capacity. These results show that sub-chronic ketamine treatment in mice mimics the dopaminergic alterations in patients with psychosis, that this requires activation of midbrain dopamine neurons, and can be ameliorated by activating PV interneurons and by a TAAR1/5-HT1A agonist. This identifies novel therapeutic approaches for targeting presynaptic dopamine dysfunction in patients with schizophrenia and effects of ketamine relevant to its therapeutic use for  treating major depression.

Tonic GABAergic inhibition, via GABAA receptors containing αßƐ subunits, regulates excitability of ventral tegmental area dopamine neurons.

The activity of midbrain dopamine neurons is strongly regulated by fast synaptic inhibitory γ-Aminobutyric acid (GABA)ergic inputs. There is growing evidence in other brain regions that low concentrations of ambient GABA can persistently activate certain subtypes of GABAA receptor to generate a tonic current. However, evidence for a tonic GABAergic current in midbrain dopamine neurons is limited. To address this, we conducted whole-cell recordings from ventral tegmental area (VTA) dopamine neurons in brain slices from mice. We found that application of GABAA receptor antagonists decreased the holding current, indicating the presence of a tonic GABAergic input. Global increases in GABA release, induced by either a nitric oxide donor or inhibition of GABA uptake, further increased this tonic current. Importantly, prolonged inhibition of the firing activity of local GABAergic neurons abolished the tonic current. A combination of pharmacology and immunohistochemistry experiments suggested that, unlike common examples of tonic inhibition, this current may be mediated by a relatively unusual combination of α4ßƐ subunits. Lastly, we found that the tonic current reduced excitability in dopamine neurons suggesting a subtractive effect on firing activity.

Mitogen-activated protein kinase phosphatase-2 deletion modifies ventral tegmental area function and connectivity and alters reward processing.

Mitogen-activated protein kinases (MAPKs) regulate normal brain functioning, and their dysfunction is implicated in a number of brain disorders. Thus, there is great interest in understanding the signalling systems that control MAPK functioning. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in foetal development, the immune system, cancer and synaptic plasticity and memory. In the present study, we performed an unbiased investigation using MKP-2-/- mice to assess whether MKP-2 plays a global role in modulating brain function. Local cerebral glucose utilization is significantly increased in the ventral tegmental area (VTA) of MKP-2-/- mice, with connectivity analysis revealing alterations in VTA functional connectivity, including a significant reduction in connectivity to the nucleus accumbens and hippocampus. In addition, spontaneous excitatory postsynaptic current frequency, but not amplitude, onto putative dopamine neurons in the VTA is increased in MKP-2-/- mice, which indicates that increased excitatory drive may account for the increased VTA glucose utilization. Consistent with modified VTA function and connectivity, in behavioural tests MKP-2-/- mice exhibited increased sucrose preference and impaired amphetamine-induced hyperlocomotion. Overall, these data reveal that MKP-2 plays a role in modulating VTA function and that its dysfunction may contribute to brain disorders in which altered reward processing is present.

Transcriptional profiling aligned with in situ expression image analysis reveals mosaically expressed molecular markers for GABA neuron sub-groups in the ventral tegmental area.

γ-Aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) provide local inhibitory control of dopamine neuron activity and send long-range projections to several target regions including the nucleus accumbens. They play diverse roles in reward and aversion, suggesting that they be comprised of several functionally distinct sub-groups, but our understanding of this diversity has been limited by a lack of molecular markers that might provide genetic entry points for cell type-specific investigations. To address this, we conducted transcriptional profiling of GABA neurons and dopamine neurons using immunoprecipitation of tagged polyribosomes (RiboTag) and RNAseq. First, we directly compared these two transcriptomes in order to obtain a list of genes enriched in GABA neurons compared with dopamine neurons. Next, we created a novel bioinformatic approach, that used the PANTHER (Protein ANalysis THrough Evolutionary Relationships) gene ontology database and VTA gene expression data from the Allen Mouse Brain Atlas, from which we obtained 6 candidate genes: Cbln4, Rxfp3, Rora, Gpr101, Trh and Nrp2. As a final step, we verified the selective expression of these candidate genes in sub-groups of GABA neurons in the VTA (and neighbouring substantia nigra pars compacta) using immunolabelling. Taken together, our study provides a valuable toolbox for the future investigation of GABA neuron sub-groups in the VTA.

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target.

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. ß-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling.

Notch signalling stabilises boundary formation at the midbrain-hindbrain organiser.

The midbrain-hindbrain interface gives rise to a boundary of particular importance in CNS development as it forms a local signalling centre, the proper functioning of which is essential for the formation of tectum and cerebellum. Positioning of the mid-hindbrain boundary (MHB) within the neuroepithelium is dependent on the interface of Otx2 and Gbx2 expression domains, yet in the absence of either or both of these genes, organiser genes are still expressed, suggesting that other, as yet unknown mechanisms are also involved in MHB establishment. Here, we present evidence for a role for Notch signalling in stabilising cell lineage restriction and regulating organiser gene expression at the MHB. Experimental interference with Notch signalling in the chick embryo disrupts MHB formation, including downregulation of the organiser signal Fgf8. Ectopic activation of Notch signalling in cells of the anterior hindbrain results in an exclusion of those cells from rhombomeres 1 and 2, and in a simultaneous clustering along the anterior and posterior boundaries of this area, suggesting that Notch signalling influences cell sorting. These cells ectopically express the boundary marker Fgf3. In agreement with a role for Notch signalling in cell sorting, anterior hindbrain cells with activated Notch signalling segregate from normal cells in an aggregation assay. Finally, misexpression of the Notch modulator Lfng or the Notch ligand Ser1 across the MHB leads to a shift in boundary position and loss of restriction of Fgf8 to the MHB. We propose that differential Notch signalling stabilises the MHB through regulating cell sorting and specifying boundary cell fate.

Somatostatin neurons in prefrontal cortex initiate sleep-preparatory behavior and sleep via the preoptic and lateral hypothalamus.

The prefrontal cortex (PFC) enables mammals to respond to situations, including internal states, with appropriate actions. One such internal state could be 'tiredness'. Here, using activity tagging in the mouse PFC, we identified particularly excitable, fast-spiking, somatostatin-expressing, γ-aminobutyric acid (GABA) (PFCSst-GABA) cells that responded to sleep deprivation. These cells projected to the lateral preoptic (LPO) hypothalamus and the lateral hypothalamus (LH). Stimulating PFCSst-GABA terminals in the LPO hypothalamus caused sleep-preparatory behavior (nesting, elevated theta power and elevated temperature), and stimulating PFCSst-GABA terminals in the LH mimicked recovery sleep (non-rapid eye-movement sleep with higher delta power and lower body temperature). PFCSst-GABA terminals had enhanced activity during nesting and sleep, inducing inhibitory postsynaptic currents on diverse cells in the LPO hypothalamus and the LH. The PFC also might feature in deciding sleep location in the absence of excessive fatigue. These findings suggest that the PFC instructs the hypothalamus to ensure that optimal sleep takes place in a suitable place.

Lrrn1 is required for formation of the midbrain-hindbrain boundary and organiser through regulation of affinity differences between midbrain and hindbrain cells in chick.

The midbrain-hindbrain boundary (MHB) acts as an organiser/signalling centre to pattern tectal and cerebellar compartments. Cells in adjacent compartments must be distinct from each other for boundary formation to occur at the interface. Here we have identified the leucine-rich repeat (LRR) neuronal 1 (Lrrn1) protein as a key regulator of this process in chick. The Lrrn family is orthologous to the Drosophila tartan/capricious (trn/caps) family. Differential expression of trn/caps promotes an affinity difference and boundary formation between adjacent compartments in a number of contexts; for example, in the wing, leg and eye imaginal discs. Here we show that Lrrn1 is expressed in midbrain cells but not in anterior hindbrain cells. Lrrn1 is down-regulated in the anterior hindbrain by the organiser signalling molecule FGF8, thereby creating a differential affinity between these two compartments. Lrrn1 is required for the formation of MHB--loss of function leads to a loss of the morphological constriction and loss of Fgf8. Cells overexpressing Lrrn1 violate the boundary and result in a loss of cell restriction between midbrain and hindbrain compartments. Lrrn1 also regulates the glycosyltransferase Lunatic Fringe, a modulator of Notch signalling, maintaining its expression in midbrain cells which is instrumental in MHB boundary formation. Thus, Lrrn1 provides a link between cell affinity/compartment segregation, and cell signalling to specify boundary cell fate.

Cohesin-dependence of neuronal gene expression relates to chromatin loop length.

Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here, we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in mouse primary cortical neurons. Immediate early genes (IEGs) remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the IEGs Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterized by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend on cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts.

Nitric oxide synthase regulates morphogenesis of zebrafish spinal cord motoneurons.

Nitric oxide (NO) is a signaling molecule that is synthesized in a range of tissues by the NO synthases (NOSs). In the immature nervous system, the neuronal isoform of NOS (NOS1) is often expressed during periods of axon outgrowth and elaboration. However, there is little direct molecular evidence to suggest that NOS1 influences these processes. Here we address the functional role of NOS1 during in vivo zebrafish locomotor circuit development. We show that NOS1 is expressed in a population of interneurons that lie close to nascent motoneurons of the spinal cord. To determine how this protein regulates spinal network assembly, we perturbed NOS1 expression in vivo with antisense morpholino oligonucleotides. This treatment dramatically increased the number of axon collaterals formed by motoneuron axons, an effect mimicked by pharmacological inhibition of the NO/cGMP signaling pathway. In contrast, exogenous elevation of NO/cGMP levels suppressed motor axon branching. These effects were not accompanied by a change in motoneuron number, suggesting that NOS1 does not regulate motoneuron differentiation. Finally we show that perturbation of NO signaling affects the ontogeny of locomotor performance. Our findings provide evidence that NOS1 is a key regulator of motor axon ontogeny in the developing vertebrate spinal cord.

GABA and glutamate neurons in the VTA regulate sleep and wakefulness.

We screened for novel circuits in the mouse brain that promote wakefulness. Chemogenetic activation experiments and electroencephalogram recordings pointed to glutamatergic/nitrergic (NOS1) and GABAergic neurons in the ventral tegmental area (VTA). Activating glutamatergic/NOS1 neurons, which were wake- and rapid eye movement (REM) sleep-active, produced wakefulness through projections to the nucleus accumbens and the lateral hypothalamus. Lesioning the glutamate cells impaired the consolidation of wakefulness. By contrast, activation of GABAergic VTA neurons elicited long-lasting non-rapid-eye-movement-like sleep resembling sedation. Lesioning these neurons produced an increase in wakefulness that persisted for at least 4 months. Surprisingly, these VTA GABAergic neurons were wake- and REM sleep-active. We suggest that GABAergic VTA neurons may limit wakefulness by inhibiting the arousal-promoting VTA glutamatergic and/or dopaminergic neurons and through projections to the lateral hypothalamus. Thus, in addition to its contribution to goal- and reward-directed behaviors, the VTA has a role in regulating sleep and wakefulness.

nNOS-Expressing Neurons in the Ventral Tegmental Area and Substantia Nigra Pars Compacta.

GABA neurons in the VTA and SNc play key roles in reward and aversion through their local inhibitory control of dopamine neuron activity and through long-range projections to several target regions including the nucleus accumbens. It is not clear whether some of these GABA neurons are dedicated local interneurons or if they all collateralize and send projections externally as well as making local synaptic connections. Testing between these possibilities has been challenging in the absence of interneuron-specific molecular markers. We hypothesized that one potential candidate might be neuronal nitric oxide synthase (nNOS), a common interneuronal marker in other brain regions. To test this, we used a combination of immunolabelling (including antibodies for nNOS that we validated in tissue from nNOS-deficient mice) and cell type-specific virus-based anterograde tracing in mice. We found that nNOS-expressing neurons, in the parabrachial pigmented (PBP) part of the VTA and the SNc were GABAergic and did not make detectable projections, suggesting they may be interneurons. In contrast, nNOS-expressing neurons in the rostral linear nucleus (RLi) were mostly glutamatergic and projected to a number of regions, including the lateral hypothalamus (LH), the ventral pallidum (VP), and the median raphe (MnR) nucleus. Taken together, these findings indicate that nNOS is expressed by neurochemically- and anatomically-distinct neuronal sub-groups in a sub-region-specific manner in the VTA and SNc.

Phasic Stimulation of Midbrain Dopamine Neuron Activity Reduces Salt Consumption.

Salt intake is an essential dietary requirement, but excessive consumption is implicated in hypertension and associated conditions. Little is known about the neural circuit mechanisms that control motivation to consume salt, although the midbrain dopamine system, which plays a key role in other reward-related behaviors, has been implicated. We, therefore, examined the effects on salt consumption of either optogenetic excitation or chemogenetic inhibition of ventral tegmental area (VTA) dopamine neurons in male mice. Strikingly, optogenetic excitation of dopamine neurons decreased salt intake in a rapid and reversible manner, despite a strong salt appetite. Importantly, optogenetic excitation was not aversive, did not induce hyperactivity, and did not alter salt concentration preferences in a need-free state. In addition, we found that chemogenetic inhibition of dopamine neurons had no effect on salt intake. Lastly, optogenetic excitation of dopamine neurons reduced consumption of sucrose following an overnight fast, suggesting a more general role of VTA dopamine neuron excitation in organizing motivated behaviors.

Excitatory Pathways from the Lateral Habenula Enable Propofol-Induced Sedation.

The lateral habenula has been widely studied for its contribution in generating reward-related behaviors [1, 2]. We have found that this nucleus plays an unexpected role in the sedative actions of the general anesthetic propofol. The lateral habenula is a glutamatergic, excitatory hub that projects to multiple targets throughout the brain, including GABAergic and aminergic nuclei that control arousal [3-5]. When glutamate release from the lateral habenula in mice was genetically blocked, the ability of propofol to induce sedation was greatly diminished. In addition to this reduced sensitivity to propofol, blocking output from the lateral habenula caused natural non-rapid eye movement (NREM) sleep to become highly fragmented, especially during the rest ("lights on") period. This fragmentation was largely reversed by the dual orexinergic antagonist almorexant. We conclude that the glutamatergic output from the lateral habenula is permissive for the sedative actions of propofol and is also necessary for the consolidation of natural sleep.