A brainstem-hypothalamus neuronal circuit reduces feeding upon heat exposure.

Empirical evidence suggests that heat exposure reduces food intake. However, the neurocircuit architecture and the signalling mechanisms that form an associative interface between sensory and metabolic modalities remain unknown, despite primary thermoceptive neurons in the pontine parabrachial nucleus becoming well characterized1. Tanycytes are a specialized cell type along the wall of the third ventricle2 that bidirectionally transport hormones and signalling molecules between the brain's parenchyma and ventricular system3-8. Here we show that tanycytes are activated upon acute thermal challenge and are necessary to reduce food intake afterwards. Virus-mediated gene manipulation and circuit mapping showed that thermosensing glutamatergic neurons of the parabrachial nucleus innervate tanycytes either directly or through second-order hypothalamic neurons. Heat-dependent Fos expression in tanycytes suggested their ability to produce signalling molecules, including vascular endothelial growth factor A (VEGFA). Instead of discharging VEGFA into the cerebrospinal fluid for a systemic effect, VEGFA was released along the parenchymal processes of tanycytes in the arcuate nucleus. VEGFA then increased the spike threshold of Flt1-expressing dopamine and agouti-related peptide (Agrp)-containing neurons, thus priming net anorexigenic output. Indeed, both acute heat and the chemogenetic activation of glutamatergic parabrachial neurons at thermoneutrality reduced food intake for hours, in a manner that is sensitive to both Vegfa loss-of-function and blockage of vesicle-associated membrane protein 2 (VAMP2)-dependent exocytosis from tanycytes. Overall, we define a multimodal neurocircuit in which tanycytes link parabrachial sensory relay to the long-term enforcement of a metabolic code.

A hypothalamic dopamine locus for psychostimulant-induced hyperlocomotion in mice.

The lateral septum (LS) has been implicated in the regulation of locomotion. Nevertheless, the neurons synchronizing LS activity with the brain's clock in the suprachiasmatic nucleus (SCN) remain unknown. By interrogating the molecular, anatomical and physiological heterogeneity of dopamine neurons of the periventricular nucleus (PeVN; A14 catecholaminergic group), we find that Th+/Dat1+ cells from its anterior subdivision innervate the LS in mice. These dopamine neurons receive dense neuropeptidergic innervation from the SCN. Reciprocal viral tracing in combination with optogenetic stimulation ex vivo identified somatostatin-containing neurons in the LS as preferred synaptic targets of extrahypothalamic A14 efferents. In vivo chemogenetic manipulation of anterior A14 neurons impacted locomotion. Moreover, chemogenetic inhibition of dopamine output from the anterior PeVN normalized amphetamine-induced hyperlocomotion, particularly during sedentary periods. Cumulatively, our findings identify a hypothalamic locus for the diurnal control of locomotion and pinpoint a midbrain-independent cellular target of psychostimulants.

Unified Classification of Molecular, Network, and Endocrine Features of Hypothalamic Neurons.

Peripheral endocrine output relies on either direct or feed-forward multi-order command from the hypothalamus. Efficient coding of endocrine responses is made possible by the many neuronal cell types that coexist in intercalated hypothalamic nuclei and communicate through extensive synaptic connectivity. Although general anatomical and neurochemical features of hypothalamic neurons were described during the past decades, they have yet to be reconciled with recently discovered molecular classifiers and neurogenetic function determination. By interrogating magnocellular as well as parvocellular dopamine, GABA, glutamate, and phenotypically mixed neurons, we integrate available information at the molecular, cellular, network, and endocrine output levels to propose a framework for the comprehensive classification of hypothalamic neurons. Simultaneously, we single out putative neuronal subclasses for which future research can fill in existing gaps of knowledge to rationalize cellular diversity through function-determinant molecular marks in the hypothalamus.

Hypothalamic cell diversity: non-neuronal codes for long-distance volume transmission by neuropeptides.

Volume transmission is a mode of intercellular communication using cerebral liquor to deliver signal molecules over long distances and allow their action for extended periods. For hypothalamic neuropeptides, nerve endings amongst ependymal cells are seen as a site of release into the cerebrospinal fluid. Recent single-cell RNA-seq data identify tanycytes and ventricular ependyma as alternative sources by being unexpectedly rich in neuroactive substances. This notion, coupled with circuit analysis showing regionalized innervation of periventricular ependyma by intrahypothalamic neurons, could allow for the integration of hypothalamic neuronal activity patterns with brain-wide activity changes upon metabolic challenges through phasic volume transmission primed by neuron-ependyma coupling. Here, we discuss emerging data for an ependymal interface and its breaches in neuropsychiatric disease.

Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon acute stress.

Stress-induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. Here, we show that hypothalamic corticotropin-releasing hormone-releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal-regulated kinase 1 and tyrosine hydroxylase with the Ca2+-sensor secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress-induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identify a multimodal pathway that is rate-limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long-lasting cortical excitability following acute stress.

Molecular diversity of corticotropin-releasing hormone mRNA-containing neurons in the hypothalamus.

Hormonal responses to acute stress rely on the rapid induction of corticotropin-releasing hormone (CRH) production in the mammalian hypothalamus, with subsequent instructive steps culminating in corticosterone release at the periphery. Hypothalamic CRH neurons in the paraventricular nucleus of the hypothalamus are therefore considered as 'stress neurons'. However, significant morphological and functional diversity among neurons that can transiently produce CRH in other hypothalamic nuclei has been proposed, particularly as histochemical and molecular biology evidence associates CRH to both GABA and glutamate neurotransmission. Here, we review recent advances through single-cell RNA sequencing and circuit mapping to suggest that CRH production reflects a state switch in hypothalamic neurons and thus confers functional competence rather than being an identity mark of phenotypically segregated neurons. We show that CRH mRNA transcripts can therefore be seen in GABAergic, glutamatergic and dopaminergic neuronal contingents in the hypothalamus. We then distinguish 'stress neurons' of the paraventricular nucleus that constitutively express secretagogin, a Ca2+ sensor critical for the stimulus-driven assembly of the molecular machinery underpinning the fast regulated exocytosis of CRH at the median eminence. Cumulatively, we infer that CRH neurons are functionally and molecularly more diverse than previously thought.

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes.

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

Lack of presynaptic interaction between glucocorticoid and CB1 cannabinoid receptors in GABA- and glutamatergic terminals in the frontal cortex of laboratory rodents.

Corticosteroid and endocannabinoid actions converge on prefrontocortical circuits associated with neuropsychiatric illnesses. Corticosteroids can also modulate forebrain synapses by using endocannabinoid effector systems. Here, we determined whether corticosteroids can modulate transmitter release directly in the frontal cortex and, in doing so, whether they affect presynaptic CB1 cannabinoid receptor- (CB1R) mediated neuromodulation. By Western blotting of purified subcellular fractions of the rat frontal cortex, we found glucocorticoid receptors (GcRs) and CB1Rs enriched in isolated frontocortical nerve terminals (synaptosomes). CB1Rs were predominantly presynaptically located while GcRs showed preference for the post-synaptic fraction. Additional confocal microscopy analysis of cortical and hippocampal regions revealed vesicular GABA transporter-positive and vesicular glutamate transporter 1-positive nerve terminals endowed with CB1R immunoreactivity, apposing GcR-positive post-synaptic compartments. In functional transmitter release assay, corticosteroids, corticosterone (0.1-10 microM) and dexamethasone (0.1-10 microM) did not significantly affect the evoked release of [(3)H]GABA and [(14)C]glutamate in superfused synaptosomes, isolated from both rats and mice. In contrast, the synthetic cannabinoid, WIN55212-2 (1 microM) diminished the release of both [(3)H]GABA and [(14)C]glutamate, evoked with various depolarization paradigms. This effect of WIN55212-2 was abolished by the CB1R neutral antagonist, O-2050 (1 microM), and was absent in the CB1R KO mice. CB2R-selective agonists did not affect the release of either neurotransmitter. The lack of robust presynaptic neuromodulation by corticosteroids was unchanged upon either CB1R activation or genetic inactivation. Altogether, corticosteroids are unlikely to exert direct non-genomic presynaptic neuromodulation in the frontal cortex, but they may do so indirectly, via the stimulation of trans-synaptic endocannabinoid signaling.

Distribution and classification of aggrecan-based extracellular matrix in the thalamus of the rat.

Extracellular matrix molecules take part in functional isolation and stabilization of neuronal compartments but form a vivid interface between neuronal elements at the same time. Previous studies have shown that the accumulation of extracellular matrix, especially its typical phenotypic form, termed perineuronal nets, correlates not only with the functional properties of the single neuron but also with the functional properties of the whole brain area. In contrast to recent advances in investigating neocortex, the present study mapped the occurrence and phenotypic appearance of aggrecan-based matrix accumulation throughout the rat thalamus. Results showed that divisions of thalamus that relay information to cortical fields known rather for their plastic properties exibit a poor matrix immunoreactivity, whereas matrix accumulation is more enhanced in nuclei connected to primary cortical regions. In addition to perineuronal nets, extracellular matrix condensed in another peculiar form, in 2-5-µm, large, round or oval structures, as described by Brückner et al. ([ 2008] Neuroscience 151:489-504) as axonal coats (ACs). Multiple labelling experiments showed that specific excitatory afferents were not ensheathed with these structures. At the same time, inhibitory endings were occasionally enwrapped in ACs. Electron microscopic analysis showed that aggrecan-immunoreactive profiles were present mostly around inhibitory terminals but also in all neuronal compartments. We suggest that aggrecan-based extracellular matrix is formed by both pre- and postsynaptic elements and is preferably associated with inhibitory terminals in the extracellular space.