Unveiling Hypothalamic Molecular Signatures via Retrograde Viral Tracing and Single-Cell Transcriptomics.

Despite the importance of hypothalamic neurocircuits in regulating homeostatic and survival-related behaviors, our understanding of the intrinsic molecular identities of neural components involved in these complex multi-synaptic interactions remains limited. In this study, we constructed a Cre recombinase-dependent pseudorabies virus (PRVs) capable of crossing synapses, coupled with transcriptome analysis of single upstream neurons post-infection. By utilizing this retrograde nuclear Connect-seq (nuConnect-seq) approach, we generated a single nuclei RNA-seq (snRNA-seq) dataset of 1,533 cells derived from the hypothalamus of CRH-IRES-Cre (CRH-Cre) mice. To ensure the technical validity of our nuConnect-seq dataset, we employed a label transfer technique against an integrated reference dataset of postnatal mouse hypothalamus comprising 152,524 QC-passed cells. The uniqueness of our approach lies in the integration of diverse datasets for validation, providing a more nuanced diversity of hypothalamic cell types. The presented validated dataset may deepen our understanding of hypothalamic neurocircuits and underscore the essential role of comprehensive integrated transcriptomic data for technical validity.

Olfactory modulation of stress-response neural circuits.

Stress responses, which are crucial for survival, are evolutionally conserved throughout the animal kingdom. The most common endocrine axis among stress responses is that triggered by corticotropin-releasing hormone neurons (CRHNs) in the hypothalamus. Signals of various stressors are detected by different sensory systems and relayed through individual neural circuits that converge on hypothalamic CRHNs to initiate common stress hormone responses. To investigate the neurocircuitry mechanisms underlying stress hormone responses induced by a variety of stressors, researchers have recently developed new approaches employing retrograde transsynaptic viral tracers, providing a wealth of information about various types of neural circuits that control the activity of CRHNs in response to stress stimuli. Here, we review earlier and more recent findings on the stress neurocircuits that converge on CRHNs, focusing particularly on olfactory systems that excite or suppress the activities of CRHNs and lead to the initiation of stress responses. Because smells are arguably the most important signals that enable animals to properly cope with environmental changes and survive, unveiling the regulatory mechanisms by which smells control stress responses would provide broad insight into how stress-related environmental cues are perceived in the animal brain.

Inhibition of high-fat diet-induced inflammatory responses in adipose tissue by SF1-expressing neurons of the ventromedial hypothalamus.

Inflammation and thermogenesis in white adipose tissue (WAT) at different sites influence the overall effects of obesity on metabolic health. In mice fed a high-fat diet (HFD), inflammatory responses are less pronounced in inguinal WAT (ingWAT) than in epididymal WAT (epiWAT). Here we show that ablation and activation of steroidogenic factor 1 (SF1)-expressing neurons in the ventromedial hypothalamus (VMH) oppositely affect the expression of inflammation-related genes and the formation of crown-like structures by infiltrating macrophages in ingWAT, but not in epiWAT, of HFD-fed mice, with these effects being mediated by sympathetic nerves innervating ingWAT. In contrast, SF1 neurons of the VMH preferentially regulated the expression of thermogenesis-related genes in interscapular brown adipose tissue (BAT) of HFD-fed mice. These results suggest that SF1 neurons of the VMH differentially regulate inflammatory responses and thermogenesis among various adipose tissue depots and restrain inflammation associated with diet-induced obesity specifically in ingWAT.

Melanin-concentrating hormone-producing neurons in the hypothalamus regulate brown adipose tissue and thus contribute to energy expenditure.

KEY POINTS: Melanin-concentrating hormone (MCH) neuron-ablated mice exhibit increased energy expenditure and reduced fat weight. Increased brown adipose tissue (BAT) activity and locomotor activity-independent energy expenditure contributed to body weight reduction in MCH neuron-ablated mice. MCH neurons send inhibitory input to the medullary raphe nucleus to modulate BAT activity. ABSTRACT: Hypothalamic melanin-concentrating hormone (MCH) peptide robustly affects energy homeostasis. However, it is unclear whether and how MCH-producing neurons, which contain and release a variety of neuropeptides/transmitters, regulate energy expenditure in the central nervous system and peripheral tissues. We thus examined the regulation of energy expenditure by MCH neurons, focusing on interscapular brown adipose tissue (BAT) activity. MCH neuron-ablated mice exhibited reduced body weight, increased oxygen consumption, and increased BAT activity, which improved locomotor activity-independent energy expenditure. Trans-neuronal retrograde tracing with the recombinant pseudorabies virus revealed that MCH neurons innervate BAT via the sympathetic premotor region in the medullary raphe nucleus (MRN). MRN neurons were activated by MCH neuron ablation. Therefore, endogenous MCH neuron activity negatively modulates energy expenditure via BAT inhibition. MRN neurons might receive inhibitory input from MCH neurons to suppress BAT activity.

[Use of Transsynaptic Viral Tracers for Observing Neural Circuit Control of Physiological Responses].

Central neural circuits in the brain receive and integrate environmental and internal information to enable the animals to execute appropriate behaviors and physiological responses. Communication between the brain and peripheral organs via peripheral neural circuits maintains energy homeostasis in the body. Therefore it is important to investigate the anatomical organization of central and peripheral neural circuits for elucidating the mechanisms of energy homeostasis. Transsynaptic viral tracers can travel through connected neurons via synaptic connections and have been used to delineate the anatomical organization of neural circuits with specific functions. Herein, I review our recent studies investigating neural circuits and their involvement in physiological changes using transsynaptic tracers.

Connect-seq to superimpose molecular on anatomical neural circuit maps.

The mouse brain contains about 75 million neurons interconnected in a vast array of neural circuits. The identities and functions of individual neuronal components of most circuits are undefined. Here we describe a method, termed "Connect-seq," which combines retrograde viral tracing and single-cell transcriptomics to uncover the molecular identities of upstream neurons in a specific circuit and the signaling molecules they use to communicate. Connect-seq can generate a molecular map that can be superimposed on a neuroanatomical map to permit molecular and genetic interrogation of how the neuronal components of a circuit control its function. Application of this method to hypothalamic neurons controlling physiological responses to fear and stress reveals subsets of upstream neurons that express diverse constellations of signaling molecules and can be distinguished by their anatomical locations.