Neurochemically distinct median raphe subsystems: unique hodology and presynaptic specializations

Abstract

From their somata residing in the brainstem, neurons expressing the master serotonergic regulator gene Pet1 (aka Fev) extend axonal projections to forebrain regions underlying such diverse functions as sleep, memory, olfaction, and emotional processing. Recent research has shown that Pet1 neurons previously considered a serotonergic monolith are in fact highly diverse molecularly and hodologically, and further, that this diversity underlies subpopulation differences in function. Molecularly distinct subpopulations of Pet1 neurons are not randomly distributed within the raphe nuclei but arise in part through developmental patterning (and associated gene expression). The second rhombomere (r2) of the developing hindbrain gives rise to two groups of Pet1 cells: one classically serotonergic and one primarily glutamatergic (expressing high levels of vesicular glutamate transporter 3, VGLUT3, but with low expression of other serotonin neuron identity genes despite expression of Pet1). We mapped the projections arising from these two groups, finding r2-Pet1 boutons of distinct (serotonergic vs. glutamatergic) phenotypes largely segregated to separate target regions, excepting the septum, which exhibited a mixed phenotype. Serotonin+ r2-Pet1 boutons predominated in olfactory bulb, thalamus, and suprachiasmatic nucleus and VGLUT3+ boutons in the hippocampus, septum, and cortex. These results, combined with additional retrograde viral tracing and bouton phenotyping experiments, support a model of separable neuronal subsystems within the r2-Pet1 lineage. Furthermore, some r2-Pet1 neurons were observed to form pericellular baskets, an intricate presynaptic specialization involving axons decorating the soma and proximal dendrites of their postsynaptic partner. We found that r2-Pet1 pericellular baskets exhibit regional target specificity for certain subtypes of GABAergic neurons, often interneurons. We also revealed that pericellular baskets in the septum are commonly “composites” formed from axons of multiple Pet1 neurons, indeed from multiple Pet1 lineages, converging to form baskets on the same postsynaptic target cell. Further, these septal baskets were enriched for serotonin+/VGLUT3+ co-positive boutons, indicating they are potentially specialized sites for co-transmission of glutamate and serotonin. We discuss at length the potential function of these pericellular baskets, as well as their roles as centers for neurotransmitter convergence deployed by Pet1 neurons as well as across different classes of neurons expressing diverse neurotransmitter phenotypes. Through this research, we aim to inform brain organization and new circuit nodes for potential therapeutic considerations.