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The hypothalamus is a brain region rich in functionally segregated neurons. Here Romanov and colleagues use single-cell RNA sequencing to distinguish 62 neuronal subtypes and define their neuropeptide and neurotransmitter makeup. They then show that onecut-3-containing dopamine neurons populate the periventricular area and are wired into the circadian circuitry. 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.

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.

Psychostimulant use is an ever-increasing socioeconomic burden, including a dramatic rise during pregnancy. Nevertheless, brain-wide effects of psychostimulant exposure are incompletely understood. Here, we performed Fos-CreERT2–based activity mapping, correlated for pregnant mouse dams and their fetuses with amphetamine, nicotine, and caffeine applied acutely during midgestation. While light-sheet microscopy-assisted intact tissue imaging revealed drug- and age-specific neuronal activation, the indusium griseum (IG) appeared indiscriminately affected. By using GAD67gfp/+ mice we subdivided the IG into a dorsolateral domain populated by γ-aminobutyric acidergic interneurons and a ventromedial segment containing glutamatergic neurons, many showing drug-induced activation and sequentially expressing Pou3f3/Brn1 and secretagogin (Scgn) during differentiation. We then combined Patch-seq and circuit mapping to show that the ventromedial IG is a quasi-continuum of glutamatergic neurons (IG-Vglut1⁺) reminiscent of dentate granule cells in both rodents and humans, whose dendrites emanate perpendicularly toward while their axons course parallel with the superior longitudinal fissure. IG-Vglut1⁺ neurons receive VGLUT1⁺ and VGLUT2⁺ excitatory afferents that topologically segregate along their somatodendritic axis. In turn, their efferents terminate in the olfactory bulb, thus being integral to a multisynaptic circuit that could feed information antiparallel to the olfactory–cortical pathway. In IG-Vglut1⁺ neurons, prenatal psychostimulant exposure delayed the onset of Scgn expression. Genetic ablation of Scgn was then found to sensitize adult mice toward methamphetamine-induced epilepsy. Overall, our study identifies brain-wide targets of the most common psychostimulants, among which Scgn⁺/Vglut1⁺ neurons of the IG link limbic and olfactory circuits.

Pain signals are transmitted by multisynaptic glutamatergic pathways. Their first synapse between primary nociceptors and excitatory spinal interneurons gates the sensory load. In this pathway, glutamate release is orchestrated by Ca2+-sensor proteins, with N-terminal EF-hand Ca2+-binding protein 2 (NECAB2) being particular abundant. However, neither the importance of NECAB2+ neuronal contingents in dorsal root ganglia (DRGs) and spinal cord nor the function determination by NECAB2 has been defined. A combination of histochemical analyses and single-cell RNA-sequencing showed NECAB2 in small- and medium-sized C- and Aδ D-hair low-threshold mechanoreceptors in DRGs, as well as in protein kinase C γ excitatory spinal interneurons. NECAB2 was downregulated by peripheral nerve injury, leading to the hypothesis that NECAB2 loss of function could limit pain sensation. Indeed, Necab2-/- mice reached a pain-free state significantly faster after peripheral inflammation than did WT littermates. Genetic access to transiently activated neurons revealed that a mediodorsal cohort of NECAB2+ neurons mediates inflammatory pain in the mouse spinal dorsal horn. Here, besides dampening excitatory transmission in spinal interneurons, NECAB2 limited pronociceptive brain-derived neurotrophic factor (BDNF) release from sensory afferents. Hoxb8-dependent reinstatement of NECAB2 expression in Necab2-/- mice then demonstrated that spinal and DRG NECAB2 alone could control inflammation-induced sensory hypersensitivity. Overall, we identify NECAB2 as a critical component of pronociceptive pain signaling, whose inactivation offers substantial pain relief.

Timely and efficient information transfer at synapses is fundamental to brain function. Synapses are highly dynamic structures that exhibit long-lasting activity-dependent alterations to their structure and transmission efficiency, a phenomenon termed synaptic plasticity. These changes, which occur through alterations in presynaptic release or in the trafficking of postsynaptic receptor proteins, underpin the formation and stabilisation of neural circuits during brain development, and encode, process and store information essential for learning, memory and cognition. In recent years, it has emerged that the ubiquitin-like posttranslational modification SUMOylation is an important mediator of several aspects of neuronal and synaptic function. Through orchestrating synapse formation, presynaptic release and the trafficking of postsynaptic receptor proteins during forms of synaptic plasticity such as long-term potentiation, long-term depression and homeostatic scaling, SUMOylation is being increasingly appreciated to play a central role in neurotransmission. In this review, we outline key discoveries in this relatively new field, provide an update on recent progress regarding the targets and consequences of protein SUMOylation in synaptic function and plasticity, and highlight key outstanding questions regarding the roles of protein SUMOylation in the brain.

Pain signals are transmitted by multisynaptic glutamatergic pathways. Their first synapse between primary nociceptors and excitatory spinal interneurons gates the sensory load. In this pathway, glutamate release is orchestrated by Ca2+sensor proteins, with N-terminal EF-hand Ca2+-binding protein 2 (NECAB2) being particular abundant. However, neither the importance of NECAB2+ neuronal contingents in dorsal root ganglia (DRGs) and spinal cord nor the function determination by NECAB2 has been defined. A combination of histochemical analyses and single-cell RNA-sequencing showed NECAB2 in small- and medium-sized C- and Að D-hair low-threshold mechanoreceptors in DRGs, as well as in protein kinase C Y excitatory spinal interneurons. NECAB2 was downregulated by peripheral nerve injury, leading to the hypothesis that NECAB2 loss of function could limit pain sensation. Indeed, Necab2-/- mice reached a pain-free state significantly faster after peripheral inflammation than did WT littermates. Genetic access to transiently activated neurons revealed that a mediodorsal cohort of NECAB2· neurons mediates inflammatory pain in the mouse spinal dorsal horn. Here, besides dampening excitatory transmission in spinal interneurons, NECAB2 limited pronociceptive brain-derived neurotrophic factor (BDNF) release from sensory afferents. Hoxb8-dependent reinstatement of NECAB2 expression in Necab2-/- mice then demonstrated that spinal and DRG NECAB2 alone could control inflammation-induced sensory hypersensitivity. Overall, we identify NECAB2 as a critical component of pronociceptive pain signaling, whose inactivation offers substantial pain relief.

Pain signals are transmitted by multisynaptic glutamatergic pathways. Their first synapse between primary nociceptors and excitatory spinal interneurons gates the sensory load. In this pathway, glutamate release is orchestrated by [Ca.sup.2+]-sensor proteins, with N-terminal EF-hand [Ca.sup.2+]-binding protein 2 (NECAB2) being particular abundant. However, neither the importance of [NECAB2.sup.+] neuronal contingents in dorsal root ganglia (DRGs) and spinal cord nor the function determination by NECAB2 has been defined. A combination of histochemical analyses and single- cell RNA-sequencing showed NECAB2 in small- and medium-sized C- and A[delta] D-hair low-threshold mechanoreceptors in DRGs, as well as in protein kinase C [gamma] excitatory spinal interneurons. NECAB2 was downregulated by peripheral nerve injury, leading to the hypothesis that NECAB2 loss of function could limit pain sensation. Indeed, [Necab2.sup.-/-] mice reached a pain-free state significantly faster after peripheral inflammation than did WT littermates. Genetic access to transiently activated neurons revealed that a mediodorsal cohort of [NECAB2.sup.+] neurons mediates inflammatory pain in the mouse spinal dorsal horn. Here, besides dampening excitatory transmission in spinal interneurons, NECAB2 limited pronociceptive brain-derived neurotrophic factor (BDNF) release from sensory afferents. Hoxb8-dependent reinstatement of NECAB2 expression in [Necab2.sup.-/-] mice then demonstrated that spinal and DRG NECAB2 alone could control inflammation-induced sensory hypersensitivity. Overall, we identify NECAB2 as a critical component of pronociceptive pain signaling, whose inactivation offers substantial pain relief.

The hypothalamus contains the highest diversity of neurons in the brain, which use-dependently co-release neurotransmitters and neuropeptides. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here, we mapped neuronal identities in the hypothalamus by single-cell RNA-sequencing. We distinguish 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic codes for synaptic neurotransmission and harboring the ability of task-dependent neurotransmitter switching. We identify dopamine neurons that uniquely co-express Onecut3 and Nmur2 genes, and place 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 because their neuromedin S+ inputs originate from pacemaker 2/3 (Per2/3)-expressing neurons and their tyrosine hydroxylase phosphorylation is circadian regulated. Overall, our catalogue of neuronal subclasses presents new understanding of hypothalamic organization and function.

Abstract Psychostimulant use is an ever-increasing socioeconomic burden, including a dramatic rise during pregnancy. Nevertheless, brain-wide effects of psychostimulant exposure are incompletely understood. Here, we performed Fos-CreERT2-based activity mapping, correlated for pregnant mouse dams and their fetuses with amphetamine, nicotine and caffeine applied acutely during mid-gestation. While light-sheet microscopy-assisted intact tissue imaging revealed drug- and age-specific neuronal activation, the indusium griseum (IG) appeared indiscriminately affected. By using GAD67gfp/+ mice we subdivided the IG into a dorsolateral domain populated by GABA interneurons and a ventromedial segment containing glutamatergic neurons, many showing drug-induced activation and sequentially expressing Pou3f3/Brn1 and secretagogin (Scgn) during differentiation. We then combined Patch-seq and circuit mapping to show that the ventromedial IG is a quasi-continuum of glutamatergic neurons (IG-Vglut1+) reminiscent to dentate granule cells in both rodents and humans, whose dendrites emanate perpendicularly towards, while their axons course parallel with the superior longitudinal fissure. IG-Vglut1+ neurons receive Vglut1+ and Vglut2+ excitatory afferents that topologically segregate along their somatodendritic axis. In turn, their efferents terminate in the olfactory bulb, thus being integral to a multi-synaptic circuit that could feed information antiparallel to the olfactory-cortical pathway. In IG-Vglut1+ neurons, prenatal psychostimulant exposure delayed the onset of Scgn expression. Genetic ablation of Scgn was then found to sensitize adult mice towards methamphetamine-induced epilepsy, suggesting a role for this Ca2+-binding protein in scaling IG-Vglut1+ neuronal excitability. Overall, our study identifies brain-wide targets of the most common psychostimulants, among which Scgn+/Vglut1+ neurons of the IG link limbic and olfactory circuits. Significance statement Drug abuse during pregnancy is a significant socioeconomic problem. The use of psychostimulants is particularly common during pregnancy even though a risk to the developing fetus is significant. Here, we show that short-lived exposure to amphetamine, nicotine and caffeine during pregnancy induces neuronal activation in the fetal brain with the indusium griseum (IG), a brain area situated parallel to the central surface of the cortical hemispheres, becoming indiscriminately activated. By using mouse genetics, we find that psychostimulants preferentially target glutamatergic IG neurons, and delay their differentiation postnatally. Notably, the expressional onset of secretagogin, a Ca2+-sensor amenable for synaptic integration, is deregulated. This is significant because these neurons are integral to a multi-synaptic neuronal pathway that links limbic and olfactory circuits. As such, genetic deletion of secretagogin brings about heightened sensitivity to psychostimulants, manifesting as epileptiform discharges. Cumulatively, we describe a novel psychostimulant-sensitive neuronal subtype and its circuit arrangement whose developmental delay seems critical for behavioral abnormalities in offspring prenatally exposed to the most common psychostimulants.