Molecular and Functional Characterization of Diverse Pet1 Lineage-Defined Neuron Subtypes of the Brainstem Raphe System

Abstract

Serotonergic (5HT) neurons regulate numerous behaviors and physiological functions and are commensurately implicated in diverse human disorders. This breadth of functional roles is likely rooted in intrinsic molecular and functional differences among 5HT neurons, which if elucidated would inform 5HT system organization, etiologies of 5HT-associated disorders, and possible functional subtype-specific therapeutic leads. Work towards these ends is presented here, including a collaborative project with lab colleague Dr. Benjamin Okaty involving comprehensive molecular characterization of mouse 5HT neurons. Findings show that embryonically derived 5HT neuron sublineages impart upon the mature 5HT neuronal system an intrinsic and persistent molecular organization that distinguishes mature 5HT neuron subtypes. This work led to identification of a molecular 5HT neuron subtype enriched for transcripts encoded by genes implicated by human gene variant association studies in autism and schizophrenia. This adult 5HT neuron subtype mapped directly onto the embryonic 5HT neuron sublineage previously identified as r2Hoxa2/P neurons—a nomenclature reflecting birth in rhombomere 2 of the embryonic hindbrain and expression of both Hoxa2 and the 5HT neuron differentiation factor gene Pet1. We found that r2Hoxa2/P neurons can be further subdivided into two molecularly distinct groups: one appearing classically serotonergic and another more glutamatergic. To better understand how the unique transcriptomic profile of r2Hoxa2/P neurons relates to subtype-specific functional properties, we queried r2Hoxa2/P neurons across a range of cellular, circuit, and behavioral parameters. Subtype-specific neuron silencing and conditional gene knockout revealed that r2Hoxa2/P neurons contribute to modulation of features of sensorimotor gating and social behavior, respectively. Towards identifying potential downstream circuits and cells involved in behavior modulation, we mapped r2Hoxa2/P neuron axonal efferent boutons, revealing novel target areas, morphological specializations, and putative cell type-specific targets. We further queried how differential expression of serotonergic- versus glutamatergic-type genes in r2Hoxa2/P neuron subgroups relates to neurotransmitter immunophenotypes of axonal boutons. Findings show region-dependent separation of boutons containing 5HT versus Vglut3 (a vesicular transporter that packages the excitatory neurotransmitter glutamate), suggesting additional specializations. In summary, this work contributes to establishing genome-wide molecular definitions of 5HT neuron sublineages and sheds light on specialized 5HT neuron subtypes with relevance by extension to human disorders.