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- Pet1T...