Multiplexed dopamine neurons predominate in the ventral midbrain of young macaques

Dopamine (DA) is important in many fundamental behaviors, including positive and negative reinforcement, incentive salience, and decision-making. This behavioral diversity is now known to be due, in part, to neurotransmitter diversity, based on rodent models. To address DA neuron transmitter properties in higher species, we examined the ventral midbrain in 5 young macaques (3 male, 2 females, 3-6 years) using RNAscope in situ hybridization for tyrosine hydroxylase (TH), vesicular glutamate transporter 2 (VGluT2) and glutamic acid dehydroxylase 1 (GAD1) across the A10 (VTA; 'midline VTA' nuclei and parabrachial nucleus; PBP), the A9 (substantia nigra, pars compact, SNc) and the A8 (retrorubral field, RRF) subregions. We followed up with immunocytochemical studies in the same cohort to infer extent of mRNA and protein matches. There were 7 mRNA phenotypes, with TH-mRNA containing cells forming the largest proportions of all neurons, as expected. Surprisingly, multiplexed TH+ neurons were much more frequent than TH-single labeled neurons overall (TH-VGluT2, 22% and TH-VGluT2-GAD1, 23% compared to TH-single labeled neurons, 19%). GAD1 mRNA co-expression mainly occurred in 'triple labeled' cells, i.e. those with VGluT2- and TH mRNA expression. VGluT2 mRNA single-labeled neurons represented only 8%, and GAD1 mRNA single-labeled neurons comprised 20%, of the total population. Proportions of cellular phenotypes were similar across the A10-A9-A8 subregions. Most DA neurons in the young macaque contain multiple transmitters, indicating an important role for fast synaptic transmission alongside dopaminergic transmission in all subregions. We discuss the developmental and circuit implications of these findings in higher primates.