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.

Endocannabinoids (eCBs) have recently been identified as axon guidance cues shaping the connectivity of local GABAergic interneurons in the developing cerebrum. However, eCB functions during pyramidal cell specification and establishment of longrange axonal connections are unknown. Here, we show that eCB signaling is operational in subcortical proliferative zones from embryonic day 12 in the mouse telencephalon and controls the proliferation of pyramidal cell progenitors and radial migration of immature pyramidal cells. When layer patterning is accomplished, developing pyramidal cells rely on eCB signaling to initiate the elongation and fasciculation of their long-range axons. Accordingly, CB 1 cannabinoid receptor (CB1R) null and pyramidal cellspecific conditional mutant (CB 1R f/f,NEX-Cre ) mice develop deficits in neuronal progenitor proliferation and axon fasciculation. Likewise, axonal pathfinding becomes impaired after in utero pharmacological blockade of CB 1Rs. Overall, eCBs are fundamental developmental cues controlling pyramidal cell development during corticogenesis.excitation ͉ glutamate ͉ layer patterning ͉ neocortex ͉ neurogenesis P yramidal cell specification follows a sequential scenario in the developing cerebrum: commitment of progenitor cells to the neuronal lineage occurs in the subcortical proliferative ventricular zone (VZ) and subventricular zone (SVZ). Immature pyramidal cells undergo radial migration to populate the cortical plate (CP) (1), where they acquire layer-specific neurochemical and morphological diversity (2). Pyramidal cell positioning and patterning of their corticofugal and intracortical axons is in part achieved via transcriptional control acting throughout cellular identification (2). However, epigenetic microenvironmental cues, provided by neural progenitors, radial glia, and immature neurons, are also fundamental in attaining cortical cell identity with particularly robust effects on pathfinding and directional growth of long-range axons (3).Endocannabinoids [eCBs; anandamide (AEA) and 2-arachidonoylglycerol] control various forms of synaptic plasticity at cortical glutamatergic synapses in the postnatal brain (4) through functional CB 1 cannabinoid receptors (CB 1 Rs) (5). During brain development, eCBs control neuronal fate decision (6), interneuron migration (7), and axonal specification (8). Developmental eCB actions are underpinned by a temporally defined assembly of functional eCB signaling networks with coincident expression of sn-1-diacylglycerol lipases (DAGL␣/␤) (9) and N-arachidonoyl-phosphatidyl ethanolamine (NAPE)-selective phospholipase D involved in eCB synthesis, fatty-acid amide hydrolase (FAAH) (an enzyme preferentially degrading AEA), and CB 1 Rs (8). The selective axonal targeting of CB 1 Rs and DAGLs in immature neurons suggests that eCBs may function in either cell-autonomous (6, 9) or target-derived (8) manner to control axonal elongation and postsynaptic target selection, respectively.Although recent findings in both mammals (8) and nonmammalian v...

Endocannabinoids, particularly 2-arachidonoyl glycerol (2-AG), impact the directional turning and motility of a developing axon by activating CB 1 cannabinoid receptors (CB 1 Rs) in its growth cone. Recent findings posit that sn-1-diacylglycerol lipases (DAGL␣/␤) synthesize 2-AG in the motile axon segment of developing pyramidal cells. Coincident axonal targeting of CB 1 Rs and DAGLs prompts the hypothesis that autocrine 2-AG signaling facilitates axonal outgrowth. However, DAGLs alone are insufficient to account for the spatial specificity and dynamics of 2-AG signaling. Therefore, we hypothesized that local 2-AG degradation by monoacylglycerol lipase (MGL) must play a role. We determined how subcellular recruitment of MGL is temporally and spatially restricted to establish the signaling competence of 2-AG during axonal growth. MGL is expressed in central and peripheral axons of the fetal nervous system by embryonic day 12.5. MGL coexists with DAGL␣ and CB 1 Rs in corticofugal axons of pyramidal cells. Here, MGL and DAGL␣ undergo differential axonal targeting with MGL being excluded from the motile neurite tip. Thus, spatially confined MGL activity generates a 2-AG-sensing microdomain and configures 2-AG signaling to promote axonal growth. Once synaptogenesis commences, MGL disperses in stationary growth cones. The axonal polarity of MGL is maintained by differential proteasomal degradation because inhibiting the ubiquitin proteasome system also induces axonal MGL redistribution. Because MGL inactivation drives a CB 1 R-dependent axonal growth response, we conclude that 2-AG may act as a focal protrusive signal for developing neurons and whose regulated metabolism is critical for attaining correct axonal complexity.

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly D 9-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptordriven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB 1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB 1 cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs 1 , 2 . Nevertheless, a developmental blueprint integrating molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development remains unresolved 3 . Here, we combine single-cell RNA-seq on 51,199 cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with GWAS-based disease phenotyping and genetic lineage reconstruction to show that 9 glial and 33 neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes arising from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. Differentiation of GABA and dopamine but not glutamate neurons relies on quasi-stable intermediate states with a pool of GABA progenitors giving rise to dopamine cells 4 . An unexpected abundance of chemotropic proliferation and guidance cues commonly implicated in dorsal (cortical) patterning 5 was found in the hypothalamus. Particularly, Slit / Robo loss-of-function impacted both the production and positioning of periventricular dopamine neurons. Overall, we uncover molecular principles shaping the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to endow a virtually infinite adaptive potential throughout life.