A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons.

Benjamin W Okaty,Yoonjeung Chang,Krissy A Lyon,Nikita Sturrock,Olga V Alekseyenko,Rebecca A Senft,Susan M Dymecki,Yasmin Escobedo Lozoya

doi:10.7554/elife.55523

Abstract

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons - the most molecularly distinct subtype - possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

Highlights

Brainstem neurons that synthesize the monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) (Baker et al, 1991a; Baker et al, 1991b; Baker et al, 1990; Dahlstroem and Fuxe, 1964; Ishimura et al, 1988; Steinbusch, 1981; Steinbusch et al, 1978) derive from embryonic precursors that express the transcription factor PET1 upon terminal cell division (Hendricks et al, 1999)
Fluorescent labeling of Pet1-lineage dorsal raphe nucleus (DR) neurons was achieved in mice of the following genotypes: (1) Tg(Fev-flpe)1Dym (Jensen et al, 2008); En1tm2(cre)wrst (Kimmel et al, 2000); GT(ROSA)26Sortm8(CAG-mCherry,EGFP)Dym (referred to as RC-FrePe, a dual Flpe- and Cre-dependent fluorescent reporter inserted into the ROSA26 (R26) locus; Brust et al, 2014; Dymecki et al, 2010; Okaty et al, 2015), in which Pet1-lineage neurons derived from the En1+ isthmus and rhombomere 1 (r1) embryonic progenitor domains are marked by EGFP expression or (2) Pet1-Flpe; GT(ROSA)26Sortm3.2(Cag-EGFP,CHRM3*/ mCherry/Htr2a)Pjen (Sciolino et al, 2016), in which all Pet1 neurons are EGFP-labeled (Cre was not utilized in these experiments, only EGFP, not hM3Dq, was expressed)
Gad1 and Gad2 are paralogous genes that encode two distinct forms of the gamma-aminobutyric acid (GABA) synthetic enzyme glutamate decarboxylase (Erlander et al, 1991), and we found a sizable group of Pet1 neurons (~50%) that express high levels of Tph2 and Gad2, and to a lesser extent Gad1 (Figure 1—figure supplement 2A,B, clusters one and two), which could be further subdivided into five sub-clusters at finer resolution (Figure 1B, C, clusters two-six)

Summary

Introduction

Brainstem neurons that synthesize the monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) (Baker et al, 1991a; Baker et al, 1991b; Baker et al, 1990; Dahlstroem and Fuxe, 1964; Ishimura et al, 1988; Steinbusch, 1981; Steinbusch et al, 1978) derive from embryonic precursors that express the transcription factor PET1 (alias FEV) upon terminal cell division (Hendricks et al, 1999). PET1 shapes the serotonergic identity of neurons by regulating expression of genes required for 5-HT biosynthesis, packaging in synaptic vesicles, reuptake, and metabolism (Hendricks et al, 2003; Krueger and Deneris, 2008; Liu et al, 2010; Wyler et al, 2015; Wyler et al, 2016), though some Pet1-lineage cells in the brain have ambiguous phenotypes with respect to their ability to synthesize and release 5-HT (Alonso et al, 2013; Barrett et al, 2016; Okaty et al, 2015; Pelosi et al, 2014; Sos et al, 2017). Assembling a taxonomy of Pet neuron subtypes based on molecular and cellular properties and linking identified Pet neuron subtypes to specific biological functions is

Methods

Results

Conclusion