Single-Cell Resolution of Individual Variation in Hypothalamic Neurons Allows Targeted Manipulation Affecting Social Motivation
2025-03-06
Pre-printed on bioRxiv as:
S. Sarafinovska, S.K. Koester, L.Z. Fang, J.W. Thorpe, S.M. Chaturvedi, J. Ji, E.F. Jones, D. Selmanovic, D.J. Kornbluth, M.R. Barrett, G.M. Rurak, S.E. Maloney, M.C. Creed, R.D. Mitra, J.D. Dougherty. Single-Cell Resolution of Individual Variation in Hypothalamic Neurons Allows Targeted Manipulation Affecting Social Motivation. bioRxiv. 2025.03.10.642464. (2025). doi: https://doi.org/10.1101/2025.03.10.642464
Sarafinovska, S., Koester, S.K., Fang, L.Z., Thorpe, J.W., Chaturvedi, S.M., Ji, J., Jones, E.F., Selmanovic, D., Kornbluth, D.J., Barrett, M.R., Rurak, G.M., Maloney, S.E, Creed, M.C., Mitra, R.D., Dougherty, J.D.
Despite decades of research, connecting molecular and cellular phenotypes to complex behavioral traits remains an elusive goal. Social motivation exhibits individual trait variation, which we hypothesize is mediated by molecular and cellular variability across hypothalamic neurons. To test this, we generated single-nucleus RNA-sequencing profiles of >120,000 neurons from tuberal hypothalamus and adjacent thalamus in 36 mice, balanced across sex and autism-associated mutation, with all mice assessed for social motivation. First, we show that molecular activation patterns predict behavior across individuals: specifically, activation of paraventricular Agtr1a+ (angiotensin receptor 1a) neurons predicted reduced social behavior. Subsequent inhibition of AGTR1A with telmisartan—an FDA-approved antihypertensive—improved social orienting. Second, we show natural variation in neuronal proportions—likely arising from stochastic developmental events—is sufficient to shape adult behavior even among genetically-identical individuals: we identified multiple neuronal populations whose relative abundance predicted social reward-seeking behavior. Chemogenetic inhibition of one such population, Nxph4+ neurons of the postero-lateral hypothalamus, suppressed multiple aspects of social motivation. This work establishes proof-of-principle for an approach where single-cell genomics precisely maps neural substrates governing behavior. This approach revealed that stochastic variations in neuronal architecture deterministically influence social motivation, and enabled identification of therapeutically-actionable targets with immediate translational potential for disorders with social deficits.
Following a social operant conditioning paradigm (Maloney et al., Cell Rep 2023), tuberal hypothalamus and neighboring thalamus were harvested from Myt1l mice (n=36, split by sex and genotype) 30 minutes after task completeion. Nuclei were isolated (per Sarafinovska et al., protocols.io 2025) and barcoded libraries prepared using ScaleBio snRNAseq (Scale Biosciences 2020008).
The raw and processed data used to generate these results are available on GEO at accession number GSE291295.
We thank members of the Dougherty lab for helpful discussions and feedback, as well as Dr. John Constantino and Benjamin Boros for critical reading and manuscript suggestions. Thanks to Anthony Fischer for assistance with transporting Cohort 1 mice, Dr. Lex Kravitz for providing 3D brain matrices for dissections, and Kyle Kniepkamp for 3D printing support. We appreciate the expertise and services of the DNA Sequencing and Innovation Lab (DSIL) and the Genome Technology Access Center at the McDonnell Genome Institute (GTAC@MGI). We thank Dominic Skinner and Fatjon Leti from Scale Bio for technical support with the methodological and computational pipeline. We are grateful to Justin Wang and the Kravitz lab for donating DREADDs viruses. Additionally, we thank Dr. Christelle Anaclet for sharing the detailed DCZ voluntary oral administration protocol and Dr. Zachary Knight and the Knight Lab for generously donating Nxph4-Cremut/wt breeders. This work was funded by grants from the National Institute of Mental Health (RF1MH126723 to J.D.D. and R.D.M.; R01MH124808 to JDD, SEM; R01DA058755 and R01DA056829 to M.C.C.) and Simons Foundation Autism Research Initiative (734069 to J.D.D). S.S. was supported in part by the Autism Science Foundation (22-007).
Maloney, S. E. et al. A comprehensive assay of social motivation reveals sex-specific roles of autism-associated genes and oxytocin. Cell Rep. Methods 3, 100504 (2023).
Sarafinovska, S., Yen, A., & Chaturvedi, S. Isolation of nuclei from fresh frozen brain sections. protocols.io (2025). doi: 10.17504/protocols.io.dm6gp9qjdvzp/v1