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Leveraging single-cell spatial transcriptomics and connectomics to resolve brain circuit function in psychiatry

Neuropsychopharmacology (2025)Cite this article

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Understanding how brain circuits contribute to neuropsychiatric disorders remains a major challenge, in part because functionally distinct neurons are frequently intermingled within densely interconnected networks, making it difficult to assign specific behavioral functions to individual cell types. Moreover, neurons that appear to share a common cellular phenotype, anatomical location, and projection pattern can often exert different or even opposite effects on behavior. For example, subpopulations of dopamine neurons in the ventral tegmental area (VTA) contribute to both reward and aversion. Single-nucleus RNA sequencing (snRNA-seq) has revealed at least 11 transcriptionally distinct neuronal subtypes in the rat VTA, including dopaminergic populations that co-express markers for glutamate or GABA co-release [1]. Complementary optogenetic studies demonstrated that dopamine neurons in lateral areas of the VTA project preferentially to the nucleus accumbens and mediate reward, while dopamine neurons in medial VTA project to prefrontal cortex and contribute to aversion [2]. These findings highlight the limitations of classifying neurons solely by transmitter type or brain structure and underscore the need for integrative approaches that incorporate molecular, spatial, and connectomic data to resolve functional heterogeneity.

The MAPseq and BARseq procedures, developed by the Zador laboratory, are high-throughput methods that generate connectomic data with single-neuron resolution [3, 4]. MAPseq uses a virus to label individual neurons with unique RNA barcodes, enabling identification of long-range projections by sequencing barcodes from the injection site and downstream targets [3]. BARseq builds on this approach by combining RNA barcoding with in situ sequencing, allowing projection patterns and gene expression to be mapped concurrently with single-neuron resolution while preserving spatial context [4]. Because BARseq currently profiles only a limited set of gene transcripts (~100), its integration with higher-density spatial transcriptomic (Spatial-seq) platforms enables more comprehensive molecular characterization of projection-defined neurons. Several widely used Spatial-seq platforms use various sequencing methodologies, including whole-transcriptome next-generation sequencing (Visium HD), in situ sequencing (Xenium), and in situ hybridization (MERFISH).

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Funding

This work was supported by grants from the National Institute on Drug Abuse (DA053629 and DA020686 to PJK).

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  1. Nash Family Department of Neuroscience and Friedman Brain Institute (FBI), Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY, USA

    Sarah M. Claypool & Paul J. Kenny

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  1. Sarah M. Claypool
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  2. Paul J. Kenny
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SC drafted the manuscript. SC and PJK reviewed and edited the manuscript. Both authors contributed to the conceptualization of the work and approved the final version of the manuscript.

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Correspondence to Paul J. Kenny.

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Competing interests

PJK is co-founder and a shareholder in Eolas Therapeutics, Inc. PJK serves as a consultant for EpiVario, Exavir, and Kallyope, and has research support from Eli Lilly. SC declares no competing interests.

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Claypool, S.M., Kenny, P.J. Leveraging single-cell spatial transcriptomics and connectomics to resolve brain circuit function in psychiatry. Neuropsychopharmacol. (2025). https://doi.org/10.1038/s41386-025-02178-0

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  • DOI: https://doi.org/10.1038/s41386-025-02178-0

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