Neurochemically and Hodologically Distinct Ascending VGLUT3 versus Serotonin Subsystems Comprise the r2-<i>Pet1</i>Median Raphe

Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions modulate affective, memory-related, and circadian behaviors. Some express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 ( Tph2 ) encoding rate-limiting enzyme [5-hydroxytryptamine (5-HT)] synthesis, but abundant vesicular glutamate transporter type 3 Vglut3 synaptic vesicle-associated transporter. Genetic fate maps show these nonclassical, putatively glutamatergic in MR arise embryonically from same progenitor cell compartment—hindbrain rhombomere (r2)—as TPH2 + neurons. Well established is distribution efferents en masse r2-derived, -neurons; unknown relationship between efferent targets specific constituent source-neuron subgroups identified as r2- -high versus . Using male female mice, we found axonal boutons segregated anatomically largely by VGLUT3 identity. The former present suprachiasmatic nucleus, paraventricular nucleus thalamus, olfactory bulb; latter are hippocampus, cortex, septum. Thus Tph2- high Vglut3- likely regulate distinct brain encased interneuron somata, forming specialized presynaptic “baskets” or /5-HT identity; this suggests that some may local networks, perhaps differential kinetics via signaling. Fibers other (non-r2-derived) were observed many baskets, suggesting multifaceted regulation. Collectively, findings inform organization new circuit nodes therapeutic considerations. SIGNIFICANCE STATEMENT Our match bouton neurochemical identity distant bodies brainstem raphe. results significant because they suggest disparate neuronal subsystems derive precursor cells embryonic compartment (r2). Of subsystems, one appears serotonergic, expected given expression PET1, projects bulb, nucleus. Another expresses VGLUT3, principally transmission, septum, cortex. boutons—those co-positive—comprise encasing interneurons, control networks Results consideration.