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.

T helper (Th) cell activation is required for the adaptive immune response. Formation of the immunological synapse (IS) between Th cells and antigen-presenting cells is essential for Th cell activation. IS formation induces the polarization and redistribution of many signaling molecules; however, very little is known about organelle redistribution during IS formation in Th cells. We show that formation of the IS induced cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. Using total internal reflection microscopy, we found that upon stimulation, the distance between the IS and mitochondria was decreased to values <200 nm. Consequently, mitochondria close to the IS took up more Ca 2؉ than the ones farther away from the IS. The redistribution of mitochondria to the IS was necessary to maintain Ca 2؉ influx across the plasma membrane and Ca 2؉ -dependent Th cell activation. Our results suggest that mitochondria are part of the signaling complex at the IS and that their localization close to the IS is required for Th cell activation.calcium ͉ lymphocyte ͉ mitochondria

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Formation of an immunological synapse (IS) between APC and T cells activates calcium entry through ORAI channels, which is indispensable for T cell activation. Successful proliferation and maturation of naive T cells is possible only if premature inactivation of ORAI channels is prevented. Although it is undisputed that calcium entry through ORAI channels is required for T cell function, it is not known if calcium influx is uniformly distributed over the plasma membrane or if preferential local calcium entry sites (for instance, at the IS) exist. In this study, we show that mitochondrial positioning determines the magnitude of local calcium entry anywhere in the plasma membrane by reducing local calcium-dependent channel inactivation: if mitochondria are close to any given local calcium entry site, calcium influx is large; if they are not close, calcium influx is small. Following formation of the IS, mitochondria are preferentially translocated to the IS in a calcium influx-dependent manner but independent of the exact calcium influx site. Mitochondrial enrichment at the IS favors local calcium entry at the IS without the necessity to enrich ORAI channels at the IS. We conclude that local calcium entry rather than global calcium entry is the preferential mechanism of calcium entry at stable ISs in Th cells.

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.

Activation of the adaptive immune response requires the interaction between antigen-presenting cells and T-cells. This cell-cell interaction, called the immunological synapse (IS), facilitates the activation of several T-cell receptor (TCR)-mediated signalling cascades including a rise in the cytosolic Ca2+ concentration ([Ca2+]i) through the activation of CRAC/ORAI1 channels. These channels are opened after depletion of intracellular Ca2+ stores and inactivated by the inflowing Ca2+ itself. We show by epifluorescence, 2-photon and total internal reflection microscopy, that a large fraction of mitochondria was moved to the immediate vicinity of the IS, a process highly dependent on the actin cytoskeleton. Mitochondrial movement to the IS was required to sustain the CRAC/ORAI1-mediated Ca2+ influx. Disruption of the actin cytoskeleton prevented mitochondrial movement and subsequent [Ca2+]i rises. The increased [Ca2+]i signals following interaction of the IS with mitochondria correlated with an enhanced T-cell proliferation. Our results show that actin cytoskeleton-mediated movement of mitochondria into the vicinity of the IS is required to sustain Ca2+ influx and permit efficient T-cell activation. This project was funded by the Deutsche Forschungsgemeinschaft (SFB 530 and GRK 845) and a grant from the Saarland University (HOMFOR).

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The objective of the study was to analyze the role of T cell polarization and T cell shape changes for calcium (Ca) signals and Ca dependent T cell activation. We have used sophisticated live cell imaging technology to quantify cytoskeletal components, mitochondria and other organelle rearrangements during and after formation of the immunological synapse (IS) in parallel with Ca signals. We found that T cell polarization after formation of the IS was paralleled by drastic cell shape changes. These changes were dependent on the actin cytoskeleton and they induced a very intimate proximity between mitochondria and the plasma membrane at the IS. The mitochondrial translocation to the IS was Ca dependent but did not require local Ca influx at the IS. The shape change-dependent mitochondrial translocation to the plasma membrane prevented ORAI1 channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the cell shape to control Ca influx. Our results suggest that cell shape changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca dependent T cell activation. Organelle polarization during IS formation is an important mechanism to modulate Ca dependent T cell activation. This project was funded by the Deutsche Forschungsgemeinschaft (Graduate Research School 1326, SFB 530, project A3) and a HOMFOR grant from the Medical Faculty.