A hypothalamic circuit for circadian regulation of corticosterone secretion.

The secretion of cortisol in humans and corticosterone (Cort) in rodents follows a daily rhythm which is important in readying the individual for the daily active cycle and is impaired in chronic depression. This rhythm is orchestrated by the suprachiasmatic nucleus (SCN) which governs the activity of neurons in the paraventricular nucleus of the hypothalamus that produce the corticotropin-releasing hormone (PVHCRH neurons). The dorsomedial nucleus of the hypothalamus (DMH) serves as a crucial intermediary, being innervated by the SCN both directly and via relays in the subparaventricular zone, and projecting axons to the PVH, thereby exerting influence over the cortisol/corticosterone rhythm. However, the role and synaptic mechanisms by which DMH neurons regulate the daily rhythm of Cort secretion has not been explored. We found that either ablating or acutely inhibiting the DMH glutamatergic (DMHVglut2) neurons resulted in a 40-70% reduction in the daily peak of Cort. Deletion of the Vglut2 gene within the DMH produced a similar effect, highlighting the indispensable role of glutamatergic signaling. Chemogenetic stimulation of DMHVglut2 neurons led to an increase of Cort levels, and optogenetic activation of their terminals in the PVH in hypothalamic slices directly activated PVHCRH neurons through glutamate release (the DMHVglut2 → PVHCRH pathway). Similarly, ablating, inhibiting, or disrupting GABA transmission by DMH GABAergic (DMHVgat) neurons diminished the circadian peak of Cort, particularly under constant darkness conditions. Chemogenetic stimulation of DMHVgat neurons increased Cort, although with a lower magnitude compared to DMHVglut2 neuron stimulation, suggesting a role in disinhibiting PVHCRH neurons. Supporting this hypothesis, we found that rostral DMHVgat neurons project directly to GABAergic neurons in the caudal ventral part of the PVH and adjacent peri-PVH area (cvPVH), which directly inhibit PVHCRH neurons, and that activating the DMHVgat terminals in the cvPVH in brain slices reduced GABAergic afferent input onto the PVHCRH neurons. Finally, ablation of cvPVHVgat neurons resulted in increased Cort release at the onset of the active phase, affirming the pivotal role of the DMHVgat → cvPVHVgat → PVHCRH pathway in Cort secretion. In summary, our study delineates two parallel pathways transmitting temporal information to PVHCRH neurons, collectively orchestrating the daily surge in Cort in anticipation of the active phase. These findings are crucial to understand the neural circuits regulating Cort secretion, shedding light on the mechanisms governing this physiological process and the coordinated interplay between SCN, DMH, and PVH.

Afferent projections to the Calca /CGRP-expressing parabrachial neurons in mice.

The parabrachial nucleus (PB), located in the dorsolateral pons, contains primarily glutamatergic neurons which regulate responses to a variety of interoceptive and cutaneous sensory signals. The lateral PB subpopulation expressing the Calca gene which produces the neuropeptide calcitonin gene-related peptide (CGRP) relays signals related to threatening stimuli such as hypercarbia, pain, and nausea, yet the afferents to these neurons are only partially understood. We mapped the afferent projections to the lateral part of the PB in mice using conventional cholera toxin B subunit (CTb) retrograde tracing, and then used conditional rabies virus retrograde tracing to map monosynaptic inputs specifically targeting the PB Calca /CGRP neurons. Using vesicular GABA (vGAT) and glutamate (vGLUT2) transporter reporter mice, we found that lateral PB neurons receive GABAergic afferents from regions such as the lateral part of the central nucleus of the amygdala, lateral dorsal subnucleus of the bed nucleus of the stria terminalis, substantia innominata, and the ventrolateral periaqueductal gray. Additionally, they receive glutamatergic afferents from the infralimbic and insular cortex, paraventricular nucleus, parasubthalamic nucleus, trigeminal complex, medullary reticular nucleus, and nucleus of the solitary tract. Using anterograde tracing and confocal microscopy, we then identified close axonal appositions between these afferents and PB Calca /CGRP neurons. Finally, we used channelrhodopsin-assisted circuit mapping to test whether some of these inputs directly synapse upon the PB Calca /CGRP neurons. These findings provide a comprehensive neuroanatomical framework for understanding the afferent projections regulating the PB Calca /CGRP neurons.

A spatially-resolved transcriptional atlas of the murine dorsal pons at single-cell resolution.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.