Multiscale single-cell analysis reveals unique phenotypes of raphe 5-HT neurons projecting to the forebrain

Serotonergic neurons of the raphe nuclei exhibit anatomical, neurochemical and elecrophysiological heterogeneity that likely underpins their specific role in multiple behaviors. However, the precise organization of serotonin (5-HT) neurons to orchestrate 5-HT release patterns throughout the brain is not well understood. We compared the electrophysiological and neurochemical properties of dorsal and median raphe 5-HT neurons projecting to the medial prefrontal cortex (mPFC), amygdala (BLA) and dorsal hippocampus (dHP), combining retrograde tract tracing with brain slice electrophysiology and single-cell RT-PCR in Pet1-EGFP mice. Our results show that 5-HT neurons projecting to the dHP and the mPFC and the BLA form largely non-overlapping populations and that BLA-projecting neurons have characteristic excitability and membrane properties. In addition, using an unbiased clustering method that correlates anatomical, molecular and electrophysiological phenotypes, we find that 5-HT neurons with projections to the mPFC and the dHP segregate from those projecting to the BLA. Single-cell gene profiling showed a restricted expression of the peptide galanin in the population of 5-HT neurons projecting to the mPFC. Finally, cluster analysis allowed identifying an atypical subtype of 5-HT neuron with low excitability, long firing delays and preferential expression of the vesicular glutamate transporter type 3. Overall, these findings allow to define correlated anatomical and physiological identities of serotonin raphe neurons that help understanding how discrete raphe cells subpopulations account for the heterogeneous activities of the midbrain serotonergic system.