Projection-defined median raphe Pet⁺ subpopulations are diversely implicated in seizure

The raphe nuclei, the primary resource of forebrain 5-HT, play an important but heterogeneous role in regulating subcortical excitabilities. Fundamental circuit organizations of different median raphe (MR) subsystems are far from completely understood. In the present study, using cell-specific viral tracing, Ca²⁺ fiber photometry and epilepsy model, we map out the forebrain efferent and afferent of different MR Pet⁺ subpopulations and their divergent roles in epilepsy. We found that Pet^MR neurons send both collateral and parallel innervations to different downstream regions through different subpopulations. Notably, CA3-projecting Pet^MR subpopulations are largely distinct from habenula (Hb)-projecting Pet^MR subpopulations in anatomical distribution and topological organization, while majority of the CA3-projecting Pet^MR subpopulations are overlapped with the medial septum (MS)-projecting Pet^MR subpopulations. Further, using Ca²⁺ fiber photometry, we monitor activities of Pet^MR neurons in hippocampal-kindling seizure, a classical epilepsy model with pathological mechanisms caused by excitation-inhibition imbalance. We found that soma activities of Pet^MR neurons are heterogeneous during different periods of generalized seizures. These divergent activities are contributed by different projection-defined Pet^MR subpopulations, manifesting as increased activities in CA3 but decreased activity in Hb resulting from their upstream differences. Together, our findings provide a novel framework of MR subsystems showing that projection-defined MR Pet⁺ subpopulations are topologically heterogenous with divergent circuit connections and are diversely implicated in seizures. This may help in the understanding of heterogeneous nature of MR 5-HTergic subsystems and the paradox roles of 5-HTergic systems in epilepsy.