Abstract
γ‐Aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) provide local inhibitory control of dopamine neuron activity and send long‐range projections to several target regions including the nucleus accumbens. They play diverse roles in reward and aversion, suggesting that they be comprised of several functionally distinct sub‐groups, but our understanding of this diversity has been limited by a lack of molecular markers that might provide genetic entry points for cell type‐specific investigations. To address this, we conducted transcriptional profiling of GABA neurons and dopamine neurons using immunoprecipitation of tagged polyribosomes (RiboTag) and RNAseq. First, we directly compared these two transcriptomes in order to obtain a list of genes enriched in GABA neurons compared with dopamine neurons. Next, we created a novel bioinformatic approach, that used the PANTHER (Protein ANalysis THrough Evolutionary Relationships) gene ontology database and VTA gene expression data from the Allen Mouse Brain Atlas, from which we obtained 6 candidate genes: Cbln4, Rxfp3, Rora, Gpr101, Trh and Nrp2. As a final step, we verified the selective expression of these candidate genes in sub‐groups of GABA neurons in the VTA (and neighbouring substantia nigra pars compacta) using immunolabelling. Taken together, our study provides a valuable toolbox for the future investigation of GABA neuron sub‐groups in the VTA.
Highlights
The tagged exon lies downstream of the wild‐ type (WT) exon 4, which is flanked by LoxP sites
We first used immunofluorescence to confirm the selective expression of Rpl22‐HA in γ‐aminobutyric acid (GABA) neurons and dopamine neurons in the ventral tegmental area (VTA) of RiboTag mice crossed with either VGATCre or DATCre mice, respectively
Our final list of seven candidate genes included neuronal nitric oxide synthase (NOS1), which provides a useful validation of our approach, because we have recently shown that it is expressed in a sub‐population of GABA neurons in the VTA (Paul et al, 2018)
Summary
Γ‐Aminobutyric acid (GABA) neurons make up around one‐third of neurons in the ventral tegmental area (VTA) (Nair‐Roberts et al, 2008; Olson & Nestler, 2007) These inhibitory neurons send long‐range axonal projections to several regions, including the nucleus accumbens (NAc) and ventral pallidum, and make local synaptic connections with dopamine neurons (Breton et al, 2018; Omelchenko & Sesack, 2009; Taylor et al, 2014). One key mechanism by which opiates have their rewarding effects in the VTA is through direct inhibition of a subset of GABA neurons, leading to disinhibition of dopamine neurons (Fields & Margolis, 2015) Given these apparently diverse roles of GABA neurons in the VTA, it seems likely that this population is comprised of several functionally and anatomically heterogeneous sub‐ groups. We tested a number of candidates using immunofluorescence, revealing several molecular markers for sub‐groups of GABA neurons within the VTA
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