DECIPHERING ASTROCYTIC INVOLVEMENT IN VALENCE PROCESSING WITHIN THE DORSAL RAPHE NUCLEUS (DRN)

The dorsal raphe nucleus (DRN) is a heterogeneous structure comprising diverse neuronal subpopulations. Beyond being the principal source of serotonergic innervation across the brain, it also contains dopaminergic neurons (DRN_DA) and astrocytes (DRN_Astro), among other cells. The complexity of DRN microcircuit modulation is further highlighted by anatomical studies indicating that the DRN receives inputs from other neuromodulatory nuclei, such as the Locus Coeruleus (LC), the brain’s primary source of norepinephrine (NE) and a key structure involved in threat-related responses. While the involvement of DRN serotonergic (DRN_5-HT) and DRN_DA neurons in valence processing is emerging, the contribution of DRN_Astro and LC_NE remains elusive. In this study, we investigated how DRN_Astro, and regulation of their activity by local or afferent projections, shape DRN_5-HT activity and influence stimulus valence processing within the DRN. To this end, we combined intersectional viral strategies with in vivo fiber photometry, ex vivo two-photon Ca²⁺ imaging, and machine learning–based behavioral analysis to investigate Pavlovian learning using stimuli of diverse valence. Our results indicate that aversive stimuli enhance DRN_Astro Ca²⁺ activity and noradrenergic signaling within the DRN, with optogenetic stimulation of LC projections eliciting freezing-like behavior in mice. Furthermore, targeted viral manipulation of astrocytic activity influences DRN circuit dynamics in response to both rewarding and aversive stimuli, consistent with a role for astrocytes in shaping serotonergic system output. Together, these findings identify DRN_Astro as active participants in DRN valence encoding and reveal a previously underappreciated astrocyte–serotonergic neuron interaction that may be critical for adaptive emotional behavior.