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The mouse brain contains ~100 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 reveal subsets of upstream neurons that express diverse constellations of signaling molecules and can be distinguished by their anatomical locations.

Key points MCH neuron-ablated mice exhibit increased energy expenditure and reduced fat weight. Increased 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. This article is protected by copyright. All rights reserved.

A central objective in deciphering the nervous system in health and disease is to define the connections of neurons. The propensity of neurotropic viruses to spread among synaptically-linked neurons makes them ideal for mapping neural circuits. So far, several classes of viral neuronal tracers have become available and provide a powerful toolbox for delineating neural networks. In this paper, we review the recent developments of neurotropic viral tracers and highlight their unique properties in revealing patterns of neuronal connections.

Deregulated activation of RAS/extracellular signal-regulated kinase (ERK) signaling and defects in retinoic acid receptor (RAR) signaling are both implicated in many types of cancers. However, interrelationships between these alterations in regulating cancer cell fates have not been fully elucidated. Here, we show that RAS/ERK and RAR signaling pathways antagonistically interact with each other to regulate colorectal cancer (CRC) cell fates. We show that RAR signaling activation promotes spontaneous differentiation of CRC cells, while ERK activation suppresses it. Our microarray analyses identify genes whose expression levels are upregulated by RAR signaling. Notably, one of these genes, MKP4, encoding a member of dual-specificity phosphatases for mitogen-activated protein (MAP) kinases, mediates ERK inactivation upon RAR activation, thereby promoting the differentiation of CRC cells. Moreover, our results also show that RA induction of RAR target genes is suppressed by the ERK pathway activation. This suppression results from the inhibition of RAR transcriptional activity, which is shown to be mediated through an RIP140/histone deacetylase (HDAC)-mediated mechanism. These results identify antagonistic interactions between RAS/ERK and RAR signaling in the cell fate decision of CRC cells and define their underlying molecular mechanisms.

New studies show that POMC neurons linked to appetite suppression also play a key role in stress hormone responses., Mammals exhibit instinctive reactions to danger critical to survival, including surges in blood stress hormones. Hypothalamic corticotropin-releasing hormone neurons (CRHNs) control stress hormones but how diverse stressors converge on CRHNs is poorly understood. We used sRNA profiling to define CRHN receptors for neurotransmitters and neuromodulators and then viral tracing to localize subsets of upstream neurons expressing cognate receptor ligands. Unexpectedly, one subset comprised POMC (proopiomelanocortin)–expressing neurons in the arcuate nucleus, which are linked to appetite suppression. The POMC neurons were activated by one psychological stressor, physical restraint, but not another, a predator odor. Chemogenetic activation of POMC neurons induced a stress hormone response, mimicking a stressor. Moreover, their silencing markedly reduced the stress hormone response to physical restraint, but not predator odor. These findings indicate that POMC neurons involved in appetite suppression also play a major role in the stress hormone response to a specific type of psychological stressor.

Summary: The gustatory system plays an important role in sensing appetitive and aversive tastes for evaluating food quality. In mice, taste signals are relayed by multiple brain regions, including the parabrachial nucleus (PBN) of the pons, before reaching the gustatory cortex via the gustatory thalamus. Recent studies show that taste information at the periphery is encoded in a labeled-line manner, such that each taste modality has its own receptors and neuronal pathway. In contrast, the molecular identity of gustatory neurons in the CNS remains unknown. Here, we show that SatB2-expressing neurons in the PBN play a pivotal role in sweet taste transduction. With cell ablation, in vivo calcium imaging, and optogenetics, we reveal that SatB2PBN neurons encode positive valance and selectively transmit sweet taste signals to the gustatory thalamus. : While the molecular mechanism of peripheral taste system has been extensively investigated, the molecular identity of gustatory neurons in the CNS remains unknown. Fu et al. demonstrate that SatB2-expressing neurons in the mouse parabrachial nucleus encode positive valence and selectively transmit sweet taste signals to the gustatory thalamus. Keywords: taste, optogenetics, parabrachial nucleus, SatB2, positive valence

The mechanisms by which odors induce instinctive behaviors are largely unknown. Odor detection in the mouse nose is mediated by >1, 000 different odorant receptors (ORs) and trace amine-associated receptors (TAARs). Odor perceptions are encoded combinatorially by ORs and can be altered by slight changes in the combination of activated receptors. However, the stereotyped nature of instinctive odor responses suggests the involvement of specific receptors and genetically programmed neural circuits relatively immune to extraneous odor stimuli and receptor inputs. Here, we report that, contrary to expectation, innate odor-induced behaviors can be context-dependent. First, different ligands for a given TAAR can vary in behavioral effect. Second, when combined, some attractive and aversive odorants neutralize one another's behavioral effects. Both a TAAR ligand and a common odorant block aversion to a predator odor, indicating that this ability is not unique to TAARs and can extend to an aversive response of potential importance to survival. In vitro testing of single receptors with binary odorant mixtures indicates that behavioral blocking can occur without receptor antagonism in the nose. Moreover, genetic ablation of a single receptor prevents its cognate ligand from blocking predator odor aversion, indicating that the blocking requires sensory input from the receptor. Together, these findings indicate that innate odor-induced behaviors can depend on context, that signals from a single receptor can block innate odor aversion, and that instinctive behavioral responses to odors can be modulated by interactions in the brain among signals derived from different receptors.

Instinctive reactions to danger are critical to the perpetuation of species and are observed throughout the animal kingdom. The scent of predators induces an instinctive fear response in mice that includes behavioural changes, as well as a surge in blood stress hormones that mobilizes multiple body systems to escape impending danger. How the olfactory system routes predator signals detected in the nose to achieve these effects is unknown. Here we identify a specific area of the olfactory cortex in mice that induces stress hormone responses to volatile predator odours. Using monosynaptic and polysynaptic viral tracers, we found that multiple olfactory cortical areas transmit signals to hypothalamic corticotropin-releasing hormone (CRH) neurons, which control stress hormone levels. However, only one minor cortical area, the amygdalo-piriform transition area (AmPir), contained neurons upstream of CRH neurons that were activated by volatile predator odours. Chemogenetic stimulation of AmPir activated CRH neurons and induced an increase in blood stress hormones, mimicking an instinctive fear response. Moreover, chemogenetic silencing of AmPir markedly reduced the stress hormone response to predator odours without affecting a fear behaviour. These findings suggest that AmPir, a small area comprising