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.

Ca-sensor proteins are generally implicated in insulin release through SNARE interactions. Here, secretagogin, whose expression in human pancreatic islets correlates with their insulin content and the incidence of type 2 diabetes, is shown to orchestrate an unexpectedly distinct mechanism. Single-cell RNA-seq reveals retained expression of the TRP family members in β-cells from diabetic donors. Amongst these, pharmacological probing identifies Ca-permeable transient receptor potential vanilloid type 1 channels (TRPV1) as potent inducers of secretagogin expression through recruitment of Sp1 transcription factors. Accordingly, agonist stimulation of TRPV1s fails to rescue insulin release from pancreatic islets of glucose intolerant secretagogin knock-out() mice. However, instead of merely impinging on the SNARE machinery, reduced insulin availability in secretagogin mice is due to β-cell loss, which is underpinned by the collapse of protein folding and deregulation of secretagogin-dependent USP9X deubiquitinase activity. Therefore, and considering the desensitization of TRPV1s in diabetic pancreata, a TRPV1-to-secretagogin regulatory axis seems critical to maintain the structural integrity and signal competence of β-cells.

Neurotransmitter release from the presynaptic terminal is under very precise spatial and temporal control. Following neurotransmitter release, synaptic vesicles are recycled by endocytosis and refilled with neurotransmitter. During the exocytosis event leading to release, SNARE proteins provide most of the mechanical force for membrane fusion. Here, we show one of these proteins, Syntaxin1A, is SUMOylated near its C-terminal transmembrane domain in an activity-dependent manner. Preventing SUMOylation of Syntaxin1A reduces its interaction with other SNARE proteins and disrupts the balance of synaptic vesicle endo/exocytosis, resulting in an increase in endocytosis. These results indicate that SUMOylation regulates the emerging role of Syntaxin1A in vesicle endocytosis, which in turn, modulates neurotransmitter release and synaptic function.

SummaryThe rapid, activity-dependent quantal presynaptic release of neurotransmitter is vital for brain function. The complex process of vesicle priming, fusion, and retrieval is very precisely controlled and requires the spatiotemporal coordination of multiple protein-protein interactions. Here, we show that posttranslational modification of the active zone protein Rab3-interacting molecule 1α (RIM1α) by the small ubiquitin-like modifier 1 (SUMO-1) functions as a molecular switch to direct these interactions and is essential for fast synaptic vesicle exocytosis. RIM1α SUMOylation at lysine residue K502 facilitates the clustering of CaV2.1 calcium channels and enhances the Ca2+ influx necessary for vesicular release, whereas non-SUMOylated RIM1α participates in the docking/priming of synaptic vesicles and maintenance of active zone structure. These results demonstrate that SUMOylation of RIM1α is a key determinant of rapid, synchronous neurotransmitter release, and the SUMO-mediated “switching” of RIM1α between binding proteins provides insight into the mechanisms underpinning synaptic function and dysfunction.

Although extensively studied postnatally, the functional differentiation of cholecystokinin (CCK)-containing interneurons en route towards the cerebral cortex during fetal development is incompletely understood. Here, we used CCKBAC/DsRed mice encoding a CCK promoter-driven red fluorescent protein to analyze the temporal dynamics of DsRed expression, neuronal identity, and positioning through high-resolution developmental neuroanatomy. Additionally, we developed a dual reporter mouse line (CCKBAC/DsRed::GAD67gfp/+) to differentiate CCK-containing interneurons from DsRed+ principal cells during prenatal development. We show that DsRed is upregulated in interneurons once they exit their proliferative niche in the ganglionic eminence and remains stably expressed throughout their long-distance migration towards the cerebrum, particularly in the hippocampus. DsRed+ interneurons, including a cohort coexpressing calretinin, accumulated at the palliosubpallial boundary by embryonic day 12.5. Pioneer DsRed+ interneurons already reached deep hippocampal layers by embryonic day 14.5 and were morphologically differentiated by birth. Furthermore, we probed migrating interneurons entering and traversing the cortical plate, as well as stationary cells in the hippocampus by patch-clamp electrophysiology to show the first signs of Na+ and K+ channel activity by embryonic day 12.5 and reliable adult-like excitability by embryonic day 18.5. Cumulatively, this study defines key positional, molecular, and biophysical properties of CCK+ interneurons in the prenatal brain.