Figure 1.

Isolation and sequencing of RNA from samples enriched for either GABA or dopamine neurons. (a) Representative images showing triple immunolabelling for HA, GABAARα1 and TH in the VTA from a VGATCre:RiboTag mouse (top panels) and double immunofluorescence for HA and TH in the VTA from a DATCre:RiboTag mouse (bottom panels). In each case HA immunoreactivity is selectively localised to the target neuronal population as illustrated by co‐localisation with GABAARα1 (in HA+/TH‐ cells (yellow arrows) but not in TH+ cells (blue arrows)) in the top panels and TH in the bottom panels. (b) Graph showing mean (+SEM) percentage of cells (n = 481) that were either HA+, TH+, or HA+ and TH+. (c) Graph showing mean (+SEM) percentage of HA+ (TH‐) cells (n = 278) that expressed GABAARα1. (d) Graph showing mean (+SEM) GABA neuron‐specific transcripts (red box; slc32a1, Gad1, Gad2, Gabra1, Pvalb) significantly enriched in samples enriched for GABA neurons compared to dopamine neurons (P‐Adj < 0.05). In contrast, dopamine neuron‐related transcripts (green box; Dat, Th, Aadc) were not enriched (P‐Adj > 0.05). (e) Heatmap depicting the 15,015 mRNA transcripts detected in GABA and dopamine neurons (each row represents a different transcript). Expression levels (reads per kilobase of transcript per million mapped reads; RPKM) were scaled and sorted by z‐score. The red‐green scale illustrates level of enrichment (red = enriched; green = de‐enriched). Each column represents a different RNA sample (biological repeat) for GABA (VGATCre:RiboTag) and dopamine (DA; DATCre:RiboTag). (f) Volcano plot illustrating genes that were differentially expressed (p‐Adj < 0.05) in GABA and dopamine neurons (each dot represents an individual gene). 7,530 genes were differentially expressed (green and red dots), 3,364 of which were significantly enriched in GABA neurons (red dots), including GABA neuron marker genes (Gad1, Gad2, Gabra1 and Slc32a1; labelled in black). [Colour figure can be viewed at http://www.wileyonlinelibrary.com]