The ventral tegmental area (VTA) plays a role in motivation and reward. Recent studies demonstrated that in addition to neurons containing dopamine (DA) or ?-amino butyric acid (GABA), glutamatergic neurons are present in the VTA. The VTA glutamatergic neurons express transcripts encoding the vesicular glutamate transporter type 2 (VGluT2). To investigate whether VTA glutamatergic neurons establish local synapses, we marked axon terminals of VTA neurons by local injection of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHAL). Then, we performed double immunolabeling and electron microcopy analysis to determine if PHAL-positive terminals contain VGluT2. We observed that within the VTA, the PHAL- positive terminals established asymmetric or symmetric synapses predominantly on dendrites, 65% of all PHAL-positive terminals that made recognizable synapses contained VGluT2, and 35% lacked it. Double PHAL-positive/VGluT2-positive terminals established predominantly asymmetric synapses (83%), and the majority of PHAL-positive terminals lacking VGluT2 formed symmetric synapses (68%).? To determine whether DAergic neurons are a target of VTA glutamatergic neurons, we performed triple fluorescence immunolabeling using antibodies against PHAL, VGluT2 and tyrosine hydroxylase (TH, a marker for DAergic neurons). By confocal fluorescence microscopy we observed double labeled PHAL-positive/ VGluT2-positive varicosities that made contacts on TH-positive dendrites. By electron microscopy, we further determined that PHAL-positive/ VGluT2-positive axon terminals formed synaptic contacts on some dendrites that contained or lacked TH immunoreactivity. In vitro whole cell recording from midbrain slide preparations showed that the blocking of action potential activity decreases the frequency of excitatory events in the VTA.? In summary, we provide anatomical and electrophysiological evidence supporting the notion that in the VTA local glutamatergic neurons establish intrinsic synapses.
