Neurochemically and hodologically distinct ascending VGLUT3 versus serotonin subsystems comprise the r2- Pet1 median raphe.

Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 (Tph2 ) encoding the rate-limiting enzyme for serotonin (5-hydroxytryptamine, 5-HT) synthesis, but abundant for vesicular glutamate transporter 3 ( Vglut3 ) encoding a synaptic-vesicle associated glutamate transporter. Genetic fate maps show these non-classical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment - hindbrain rhombomere 2 (r2) - as serotonergic TPH2+ MR Pet1 neurons. Well established is the distribution of efferents en masse from r2- derived, Pet1 -neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2- Pet1Tph2 -high versus r2- Pet1Vglut3 -high Using male and female mice, we found r2- Pet1 axonal boutons segregated anatomically largely by serotonin+ versus VGLUT3+ identity. The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum. Thus r2- Pet1Tph2- high and r2- Pet1Vglut3- high neurons likely regulate distinct brain regions and behaviors. Some r2- Pet1 boutons encased interneuron somata, forming specialized presynaptic "baskets" of VGLUT3+ or VGLUT3+ /5-HT+ identity; this suggests that some r2- Pet1Vglut3- high neurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling. Fibers from other Pet1 neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation. Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerations. Significance statement Our findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe. The results are significant because they suggest that disparate neuronal subsystems derive from Pet1+ precursor cells of the embryonic progenitor compartment rhombomere 2 (r2). Of these r2- Pet1 neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus. Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex. Some r2- Pet1 boutons-those that are VGLUT3+ or VGLUT3+ /5-HT+ co-positive-comprise "baskets" encasing interneurons, suggesting they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling. Results inform brain organization and circuit nodes for therapeutic consideration.