Relative contributions and mapping of ventral tegmental area dopamine and GABA neurons by projection target in the rat

Abstract

The ventral tegmental area (VTA) is a heterogeneous midbrain structure that contains dopamine (DA), GABA, and glutamate neurons that project to many different brain regions. Here, we combined retrograde tracing with immunocytochemistry against tyrosine hydroxylase (TH) or glutamate decarboxylase (GAD) to systematically compare the proportion of dopaminergic and GABAergic VTA projections to 10 target nuclei: anterior cingulate, prelimbic, and infralimbic cortex; nucleus accumbens core, medial shell, and lateral shell; anterior and posterior basolateral amygdala; ventral pallidum; and periaqueductal gray. Overall, the non-dopaminergic component predominated VTA efferents, accounting for more than 50% of all projecting neurons to each region except the nucleus accumbens core. In addition, GABA neurons contributed no more than 20% to each projection, with the exception of the projection to the ventrolateral periaqueductal gray, where the GABAergic contribution approached 50%. Therefore, there is likely a significant glutamatergic component to many of the VTA's projections. We also found that VTA cell bodies retrogradely labeled from the various target brain regions had distinct distribution patterns within the VTA, including in the locations of DA and GABA neurons. Despite this patterned organization, VTA neurons comprising these different projections were intermingled and never limited to any one subregion. These anatomical results are consistent with the idea that VTA neurons participate in multiple distinct, parallel circuits that differentially contribute to motivation and reward. While attention has largely focused on VTA DA neurons, a better understanding of VTA subpopulations, especially the contribution of non-DA neurons to projections, will be critical for future work.

Keywords: GABA; RRID: AB_2278725; RRID: AB_390204; RRID: SCR_003070; dopamine; immunocytochemistry; retrograde; ventral tegmental area.

Figure 1.

Methods summary. (a) Representations of coronal sections of the target nuclei receiving retrograde tracer injections. Coordinates are anterior-posterior from bregma (Paxinos & Watson, 1998). For surgical coordinates, see Table 2. (b) Seven days after tracer injection (left), horizontal sections of the VTA were made for analysis of the distributions of retrogradely labeled neurons within the VTA (right) (−8.10 mm ventral from the skull surface) (Paxinos & Watson, 1998). (c) Example brightfield image of a horizontal slice containing the VTA, 50 μm thick. The sampling regions are indicated (−8.10 mm ventral) Scale bar 2 mm. (d) Fluorescent image of a horizontal slice containing the VTA, 50 μm thick, immunocytochemically labeled for TH (green) and depicting the sampling regions (−8.10 mm ventral). (e) Horizontal VTA slices immunocytochemically labeled for TH representing the dorsal-ventral (DV) extent of the VTA. Approximate coordinates are dorsal-ventral from the skull surface (Paxinos & Watson, 1998). Scale bar 500 μm.

Figure 2.

Overall quantification of retrogradely labeled VTA neurons for each projection: laterality, and dopaminergic and GABAergic contributions. (a) Ipsilateral (ipsi) and contralateral (contra) VTA projection densities for ACC, PrL, IL, VP, ABLA, PBLA, and vlPAG. (b) Ipsilateral and contralateral VTA projection densities for NAcC, NAcMs, and NAcLs. (c) The proportions of ipsilateral retrogradely labeled neurons that were co-labeled with either TH (TH-DiI) or GAD (GAD-DiI) for ACC, PrL, IL, VP, ABLA, PBLA, and vlPAG. (d) The proportions of ipsilateral retrogradely labeled VTA neurons that project to the NAc sub-regions and were co-labeled for TH (TH-DiI). (e) The proportions of contralateral retrogradely labeled neurons that were co-labeled with either TH (TH-DiI) or GAD (GAD-DiI) for ACC, PrL, IL, VP, ABLA, PBLA, and vlPAG. (f) The proportions of contralateral retrogradely labeled VTA neurons that project to the NAc sub-regions and were co-labeled for TH (TH-DiI). All results are presented as mean ± S.E.M.

Figure 3.

Projections from the VTA to the anterior cingulate cortex (ACC). (a) Example DiI injection site in the ACC (AP 1.6 mm). Scale bar 2 mm. (b) Summary of all DiI injection sites (red circles) in the ACC (AP 1.6 mm) (Paxinos & Watson, 1998). n=5 (c) Retrogradely labeled neurons were detected in all sampling regions throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Example images of DiI labeled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrows indicate TH-DiI co-labeled neurons. Arrowheads indicate DiI only neurons. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with TH. (f) Representative images of DiI filled cells and immunofluorescent labeling for GAD. Scale bar 50 μm. Arrows indicate GAD-DiI co-labeled neurons. Arrowheads represent DiI only neurons. (g) Within each sampling region, the color indicates the percentage of DiI neurons co-localized with GAD.

Figure 4.

Projections from the VTA to the prelimbic cortex (PrL). (a) Example DiI injection site in the PrL (AP 3.2 mm). Scale bar 2 mm. (b) Summary of all DiI injection sites (red circles) in the PrL (AP 3.2 mm) (Paxinos & Watson, 1998). n=12 (c) Retrogradely labeled neurons were detected in all but one sampling region throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI labeled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrows indicate TH-DiI co-labeled neurons. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with TH. (f) Representative images of DiI filled cells and immunofluorescent labeling for GAD. Scale bar 50 μm. Arrow indicates GAD-DiI co-labeled neuron. (g) Within each sampling region, the color indicates the percentage of DiI neurons co-localized with GAD.

Figure 5.

Projections from the VTA to the infralimbic cortex (IL). (a) Example DiI injection site in the IL (AP 2.7 mm). Scale bar 2 mm (b) Summary of all DiI injection sites (red circles) in the IL (AP 2.7 mm) (Paxinos & Watson, 1998). n=7 (c) Retrogradely labeled neurons were detected in all sampling regions ipsilateral to the injection site throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI filled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrows indicate TH-DiI co-labeled neurons. Arrowheads indicate DiI only neurons. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with TH across animals. (f) Representative images of DiI filled cells and immunofluorescent labeling for GAD. Scale bar 50 μm. Arrows indicate GAD-DiI co-labeled neurons. (g) Within each sampling region, the color indicates the percentage of DiI neurons co-localized with GAD.

Figure 6.

Projections from the VTA to the nucleus accumbens core (NAcC). (a) Example DiI injection site in the NAcC (AP 1.0 mm). Scale bar 2 mm. (b) Summary of all DiI injection sites (red circles) in the NAcC (AP 1.0 mm) (Paxinos & Watson, 1998). n=3 (c) Retrogradely labeled neurons were detected in all but two sampling regions throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI filled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrows indicate TH-DiI co-labeled neurons. Arrowheads indicate DiI only neurons. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with TH.

Figure 7.

Projections from the VTA to the nucleus accumbens medial shell (NAcMs). (a) Example DiI injection site in the NacMs (AP 1.6 mm). Scale bar 2 mm. (b) Summary of all DiI injection sites (red circles) in the NAcMs (AP 1.6 mm) (Paxinos & Watson, 1998). n=3 (c) Retrogradely labeled neurons were detected in all ipsilateral sampling regions throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI filled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrows indicate TH-DiI co-labeled neurons. Arrowheads indicate DiI only neurons. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-localized with TH.

Figure 8.

Projections from the VTA to the nucleus accumbens lateral shell (NAcLs). (a) Example DiI injection site in the NAcLs (AP 1.6 mm). Scale bar 2mm. (b) Summary of all DiI injection sites (red circles) in the NAcLs (AP 1.6 mm) (Paxinos & Watson, 1998). n=3. (c) Retrogradely labeled neurons were detected in all sampling regions throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depth from dorsal to ventral is approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI filled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrows indicate DiI, TH co-labeled neurons. Arrowheads indicate DiI(+), TH(−) neurons. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-localized with TH.

Figure 9.

Projections from the VTA to the ventral pallidum (VP). (a) Example DiI injection site in the VP (AP −0.2 mm). Scale bar 2 mm. (b) Summary of all DiI injection sites (red circles) in the VP (AP −0.2 mm) (Paxinos & Watson, 1998). n=7 (c) Retrogradely labeled neurons were detected in all sampling regions throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI filled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrows indicate TH-DiI co-labeled neurons. Arrowheads indicate DiI only neurons. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with TH. (f) Representative images of DiI filled cells and immunofluorescent labeling for GAD. Scale bar 50 μm. Arrows indicate GAD-DiI co-labeled neurons. Arrowheads represent DiI only neurons. (g) Within each sampling region, the color indicates the percentage of DiI neurons co-localized with GAD.

Figure 10.

Projections from the VTA to the anterior basolateral amygdala (ABLA). (a) Example DiI injection site in the ABLA (AP −2.56 mm). Scale bar 2 mm. (b) Summary of all DiI injection sites (red circles) in the ABLA (AP −2.56 mm) (Paxinos & Watson, 1998). n=3. (c) Retrogradely labeled neurons were detected in all sampling regions throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI filled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrow indicates TH-DiI co-labeled neuron. Arrowheads indicate DiI only neurons. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with TH. (f) Representative images of DiI filled cells and immunofluorescent labeling for GAD. Scale bar 50 μm. Arrows indicate GAD-DiI co-labeled neurons. Arrowhead represents DiI only neuron. (g) Within each sampling region, the color indicates the percentage of DiI neurons co-localized with GAD.

Figure 11.

Projections from the VTA to the posterior basolateral amygdala (PBLA). (a) Example DiI injection site in the PBLA (AP −3.3 mm) Scale bar 2 mm. (b) Summary of all DiI injection sites (red circles) in the PBLA (AP −3.3 mm) (Paxinos & Watson, 1998). n=7 (c) Retrogradely labeled neurons were detected in all sampling regions throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI filled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrow indicates TH-DiI co-labeled neuron. Arrowhead indicates DiI only neuron. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with TH. (f) Representative images of DiI filled cells and immunofluorescent labeling for GAD. Scale bar 50 μm. Arrow indicates GAD-DiI co-labeled neuron. Arrowhead represents DiI only neuron. (g) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with GAD.

Figure 12.

Projections from the VTA to the ventrolateral periaqueductal gray (vlPAG). (a) Example DiI injection site in the vlPAG (DV −5.8 mm). Scale bar 2 mm. (b) Summary of all DiI injection sites (red circles) in the vlPAG (DV −5.8 mm) (Paxinos & Watson, 1998). n=6 (c) Retrogradely labeled neurons were distributed relatively evenly across both ipsilateral and contralateral sampling regions throughout the DV extent of the VTA. Dot size indicates the mean percentage of retrogradely labeled neurons within that sampling window compared to the total projection. The slice depths from dorsal to ventral are approximately −7.8, −8.0, −8.2 and −8.4 mm. (d) Representative images of DiI filled cells (magenta) and immunofluorescent labeling for TH (green). Scale bar 50 μm. Arrow indicates TH-DiI co-labeled neuron. (e) Within each sampling region, the color indicates the percentage of DiI neurons co-labeled with TH. (f) Representative images of DiI filled cells and immunofluorescent labeling for GAD. Scale bar 50 μm. Arrow indicates GAD-DiI co-labeled neuron. (g) Within each sampling region, the color indicates the percentage of DiI neurons co-localized with GAD.