Research Article

Behavioral state coding by molecularly defined paraventricular hypothalamic cell type ensembles

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Science  16 Oct 2020:
Vol. 370, Issue 6514, eabb2494
DOI: 10.1126/science.abb2494

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How neuron types encode behavioral states

What is the contribution of molecularly defined cell types to neural coding of stimuli and states? Xu et al. aimed to evaluate neural representation of multiple behavioral states in the mouse paraventricular hypothalamus. To achieve this goal, they combined deep-brain two-photon imaging with post hoc validation of gene expression in the imaged cells. The behavioral states could be well predicted by the neural response of multiple neuronal clusters. Some clusters were broadly tuned and contributed strongly to the decoding of multiple behavioral states, whereas others were more specifically tuned to certain behaviors or specific time windows of a behavioral state.

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Structured Abstract


Brain function is often compared to an orchestral ensemble, where subgroups of neurons that have similar activity are analogous to different types of instruments playing a musical score. Brains are composed of specialized neuronal subtypes that can be efficiently classified by gene expression profiles measured by single-cell RNA sequencing (scRNA-seq). Are these molecularly defined cell types the “instruments” in the neural ensemble? To address this question, we examined the neural ensemble dynamics of the hypothalamic paraventricular nucleus (PVH), a small brain region that is important for behavior states such as hunger, thirst, and stress. Past work has emphasized specialized behavioral state–setting roles for different PVH cell types, but it is not clear whether the dynamics of the PVH ensemble support this view.


We considered three possibilities for how PVH neurons could be involved in encoding behavioral states: (i) PVH neurons of a molecularly defined cell type may respond similarly and be specialized for a behavioral state as a “labeled-line,” (ii) molecularly defined cell types may show unrelated activity patterns and be irrelevant to behavioral state coding, and (iii) molecularly defined neurons may respond similarly within a type, but behavioral state may be encoded by combinations of cell types. To evaluate the role of molecularly defined cell types in the neural ensemble, it is important to monitor activity in many individual neurons with subsecond temporal resolution along with quantitative gene expression information about each cell. For this, we developed the CaRMA (calcium and RNA multiplexed activity) imaging platform in which deep-brain two-photon calcium imaging of neuron activity is performed in mice during multiple behavioral tasks. This is followed by ex vivo multiplexed RNA fluorescent in situ hybridization to measure gene expression information in the in vivo–imaged neurons.


We simultaneously imaged calcium activity in hundreds of PVH neurons from 10 cell types across 11 behavioral states. Within a molecularly defined cell type, neurons often showed similar activity patterns such that we could predict functional responses of individual neurons solely from their quantitative gene expression information. Behavioral states could be decoded with high accuracy based on combinatorial assemblies of PVH cell types, which we called “grouped-ensemble coding.” Labeled-line coding was not observed. The neuromodulatory receptor gene neuropeptide receptor neuropeptide Y receptor type 1 (Npy1r) was usually the most predictive gene for neuron functional response and was expressed in multiple cell types, analogous to the “conductor” of the PVH neural ensemble.


Our results validated molecularly defined neurons as important information processing units in the PVH. We found correspondence between the gene expression hierarchies used for molecularly defined cell type classification and functional activity hierarchies involving coordination by neuromodulation. CaRMA imaging offers a solution to the problem of how to rapidly evaluate the function of the panoply of cell types being uncovered with scRNA-seq. CaRMA imaging bridges a gap between the abstract digital elements typically described in systems neuroscience with the “wetware” associated with traditional molecular neuroscience. Merging these two areas is essential to understanding the relationships of gene expression, brain function, behavior, and ultimately neurological diseases.

CaRMA imaging reveals combinatorial cell type coding of behavior states.

CaRMA imaging records calcium dynamics of PVH neurons across multiple behavioral states followed by gene expression profiling. Combinatorial assemblies of PVH cell types encoded behavioral states. The PVH neural activity ensemble was split by Npy1r expression into two main cell classes that were subdivided into cell types. Thus, neuromodulation coordinates cell types for grouped-ensemble coding to represent different survival behaviors such as eating, drinking, and stress.


Brains encode behaviors using neurons amenable to systematic classification by gene expression. The contribution of molecular identity to neural coding is not understood because of the challenges involved with measuring neural dynamics and molecular information from the same cells. We developed CaRMA (calcium and RNA multiplexed activity) imaging based on recording in vivo single-neuron calcium dynamics followed by gene expression analysis. We simultaneously monitored activity in hundreds of neurons in mouse paraventricular hypothalamus (PVH). Combinations of cell-type marker genes had predictive power for neuronal responses across 11 behavioral states. The PVH uses combinatorial assemblies of molecularly defined neuron populations for grouped-ensemble coding of survival behaviors. The neuropeptide receptor neuropeptide Y receptor type 1 (Npy1r) amalgamated multiple cell types with similar responses. Our results show that molecularly defined neurons are important processing units for brain function.

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