Parkinson's disease is caused by the loss of dopamine (DA) producing neurons in the midbrain that send their axons to the dorsal striatum (dStr), whereas addiction occurs when DA release from DA axons in the nucleus accumbens (NAc) becomes dysregulated by drugs of abuse. Increasing the complexity of DA transmission, DA axons in the dStr and NAc co-release DA the inhibitory transmitter gamma-aminobutyric acid (GABA). Data from the Rice Lab shows that co-released GABA activates GABA_A receptors to inhibit DA release. Given previous work showing that mRNA for ɑ3-subunit containing GABA_A receptors (ɑ3-GABA_ARs) is found in 90% of midbrain DA neurons, we sought to establish whether ɑ3-GABA_ARs are localized to DA axons.

To identify the presence of GABA_A receptors on DA axons, we used immunohistochemistry with confocal fluorescence microscopy of coronal sections of fixed mouse striatal tissue. Sections were cut on a freezing microtome then incubated with primary antibodies against the DA-synthesizing enzyme tyrosine hydroxylase (TH) and against ɑ3-subunits. After rinsing these sections, fluorescent-tagged secondary antibodies were applied that recognize each primary antibody.

We first optimized procedures to visualize ɑ3-GABA_ARs and established the specificity of the primary antibody for ɑ3-subunits. High magnification of immuno-stained images in striatum showed evidence of co-localization of ɑ3-subunits on TH immunolabeled DA axons in both the dStr and NAc. These data were important to validate other findings in which immuno-electron microscopy (i-EM) was used to visualize the subcellular location of ɑ3-subunits using the same antibody and concentration. Quantification of i-EM data revealed that approximately 50% of DA axons express ɑ3-GABA_AR.

These anatomical data combined with functional DA-release data introduce a novel mechanism by which co-released GABA acts to autoinhibit DA signaling.