Ultrastructural analysis of synaptic inputs to dopamine neurons in the substantia nigra pars compacta and the ventral tegmental area




Copas, Charlotte

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Dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) form the foundation of the nigrostriatal pathway and are most notably discussed in the context of Parkinson’s disease (PD) pathology. The ventral tegmental area (VTA) which resides just medial to the SNc is a second influential DA output center and is the relay station for the mesocortical and mesolimbic pathways. Despite an ever-growing body of literature investigating the broad effects of their outputs due to their influential roles in movement execution and reinforcement learning, respectively, one question that remains unanswered is how these DA neurons are modulated by their afferent inputs. Previous research shows that glutamate (Glut), acetylcholine (ACh), and γ-aminobutyric acid (GABA) are a few of the major neurotransmitters that modulate the activity of midbrain DA neurons. Interestingly, these findings also suggest that there is a population of terminals that co-localize both ACh and GABA vesicles, implying that DA neurons are under extremely fine-tuned afferent control. The aims of this study were to describe the morphology of putative Glut, ACh, and GABA vesicles, determine if ACh and GABA are indeed packaged in the same terminals, determine the frequency of co-transmission, and finally investigate whether there is heterogeneity in the prevalence of these inputs between the medial and lateral adult mouse SNc and VTA. Immunohistochemical staining for tyrosine hydroxylase (TH), vesicular GABA transporter (VGAT), vesicular acetylcholine transporter (VAChT), and vesicular glutamate transporter 2 (VGLUT2) was employed to visualize synaptic terminals at the confocal level. Here, we show that there is in fact overlap between VGAT and VAChT in terminals on DA dendrites, however this occurs much less frequently than ACh or GABA only inputs. Using electron microscopy (EM), several distinct types of synapses are observed in these regions. Putative cholinergic synapses are filled with large uniform-sized round vesicles ~50-60 nm diameter, GABAergic type terminals with small oblong vesicles that vary greatly in size but average at ~50-80 nm in length and ~20-30 nm in width, and glutamate (Glut) terminals with small-sized round vesicles (~30-45 nm diameter). Some terminals showed a mixture of oblong vesicles and large round vesicles, and to our surprise, terminals mixed with oblong, and small round vesicles whose diameter was consistent with that of Glut vesicles were also present, prompting us at add a fifth category of synapse, putative Glut/GABA. In total 613 synapses were categorized, on average 200 per region, using the criteria above. At the EM level we found that across all regions pure GABA terminals rarely appeared and were often were co-localized with either large or small round vesicles. In addition, glutamate was the most common neurotransmitter found opposing DA dendrites in the VTA and was observed at a higher frequency compared to the medial SNc. Similarly, the lateral SNc has a significantly higher incidence of mixed ACh/GABA terminals compared to the VTA. The results of this study offer a glimpse into the regulation of these essential modulatory neurons, adding a small piece of the puzzle to the ongoing investigation concerning basic ultrastructure and normal functioning of the SNc and VTA.



substantia nigra, ventral tegmental area, dopamine, parkinson's disease, ultrastructure, electron microscopy, neurotransmitter, inputs