Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat

Abstract

Midbrain dopamine neurons in the ventral tegmental area, substantia nigra and retrorubral field play key roles in reward processing, learning and memory, and movement. Within these midbrain regions and admixed with the dopamine neurons, are also substantial populations of GABAergic neurons that regulate dopamine neuron activity and have projection targets similar to those of dopamine neurons. Additionally, there is a small group of putative glutamatergic neurons within the ventral tegmental area whose function remains unclear. Although dopamine neurons have been intensively studied and quantified, there is little quantitative information regarding the GABAergic and glutamatergic neurons. We therefore used unbiased stereological methods to estimate the number of dopaminergic, GABAergic and glutamatergic cells in these regions in the rat. Neurons were identified using a combination of immunohistochemistry (tyrosine hydroxylase) and in situ hybridization (glutamic acid decarboxylase mRNA and vesicular glutamate transporter 2 mRNA). In substantia nigra pars compacta 29% of cells were glutamic acid decarboxylase mRNA-positive, 58% in the retrorubral field and 35% in the ventral tegmental area. There were further differences in the relative sizes of the GABAergic populations in subnuclei of the ventral tegmental area. Thus, glutamic acid decarboxylase mRNA-positive neurons represented 12% of cells in the interfascicular nucleus, 30% in the parabrachial nucleus, and 45% in the parainterfascicular nucleus. Vesicular glutamate transporter 2 mRNA-positive neurons were present in the ventral tegmental area, but not substantia nigra or retrorubral field. They were mainly confined to the rostro-medial region of the ventral tegmental area, and represented approximately 2-3% of the total neurons counted ( approximately 1600 cells). These results demonstrate that GABAergic and glutamatergic neurons represent large proportions of the neurons in what are traditionally considered as dopamine nuclei and that there are considerable heterogeneities in the proportions of cell types in the different dopaminergic midbrain regions.

Fig. 1

In situ hybridization for GAD (blue reaction product) combined with immunohistochemistry for TH (brown reaction product) in sections of the ventral midbrain. (A) GAD positive neurons in the IP ventral to the VTA. (B) Low-power micrograph of the nigral complex. Large numbers of GAD-positive neurons are present in the SNpr but also among the densely distributed TH-positive neurons in the SNpc and pars lateralis (lSN). (C) Higher-power image of the SNpr showing the clear differentiation between GAD-positive neurons (black arrows) and TH-positive neurons (gray arrow). Note that the in situ labeling is confined to the perikaryon, whereas the TH immunolabeling also extends into dendritic processes. (D) High-power image of the RRF showing GAD-positive neurons (black arrows) among the brown TH-positive neurons. (E, F) High-power images of subdivisions of the VTA (E: paranigral VTA; F: parabrachial). Some of the GAD-positive neurons are indicated by black arrows. Scale bars=200 μm (A, B); 100 μm (D, E); 50 μm (C, F).

Fig. 2

In situ hybridization for VGluT2 (blue reaction product) combined with immunohistochemistry for TH (brown reaction product). (A) VGluT2-positive neurons in the red nucleus located dorsal–medial to the VTA, which is heavily immunostained for TH. (B) Higher-power image of VGluT2-positive neurons in the red nucleus (note the occasional TH-positive process coursing among the glutamatergic neurons). (C) A VGluT2-positive neuron (black arrow) in the RLi of VTA. (D) A VGluT2-positive neuron (black arrow) in the parabrachial region of the VTA (PBP). Scale bars=200 μm (A); 100 μm (C); 50 μm (B, D).

Fig. 3

Distribution of VGluT2-positive neurons in the VTA. (A) Blue dots indicate the located of single VGluT2-positive neurons in a single animal. Schematics are derived from the atlas of Paxinos and Watson (2005); the distance from bregma is indicated on the left of each plot. (B) Graph illustrating the decrease in the number of VGluT2-positive neurons counted per section going from rostral to caudal regions of the VTA (data obtained from four animals, error bars represent standard error of the mean). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.