NEUROCHEMICAL DIVERSITY OF PONTINE CORTICOTROPIN-RELEASING HORMONE NEURONS IN MICE

Corticotropin-releasing hormone (CRH)–expressing neurons near the pontine midline have been variably assigned to the median raphe, paramedian raphe, or reticulotegmental nucleus, and their neurotransmitter identities remain unresolved. To clarify their anatomical and neurochemical profiles, we examined CRH neurons in the pons of C57BL/6 and NMRI adult mice (N = 5 per strain) using RNAscope in situ hybridization combined with immunohistochemistry. Crh mRNA expression was assessed together with markers for serotonergic (Tph), GABAergic (Gad1), and glutamatergic (Vglut1, Vglut2, Vglut3) phenotypes, and anatomical localization was referenced to the Allen Mouse Brain Atlas and Paxinos and Franklin stereotaxic coordinates. We identified two distinct CRH neuronal populations: a dorsal group that co-expressed Gad1 without Vglut1/2/3, indicative of a GABAergic phenotype consistent with the median raphe, and a ventral group lacking Gad1 but robustly co-expressing Vglut2 (with a subset also expressing Vglut1), consistent with a glutamatergic identity, characteristic of the reticulotegmental nucleus. Neither population co-expressed Tph, and both showed minimal Vglut3 co-expression, excluding serotonergic identity. These findings demonstrate pronounced neurochemical heterogeneity among pontine CRH neurons and support re-assignment of a substantial subset to reticulotegmental rather than raphe nuclei. This distinction refines our understanding of brainstem CRH circuits, with implications for how stress- and arousal-related neural pathways are organized and function across neurotransmitter systems.