Identification of rat ventral tegmental area GABAergic neurons

Abstract

The canonical two neuron model of opioid reward posits that mu opioid receptor (MOR) activation produces reward by disinhibiting midbrain ventral tegmental area (VTA) dopamine neurons through inhibition of local GABAergic interneurons. Although indirect evidence supports the neural circuit postulated by this model, its validity has been called into question by growing evidence for VTA neuronal heterogeneity and the recent demonstration that MOR agonists inhibit GABAergic terminals in the VTA arising from extrinsic neurons. In addition, VTA MOR reward can be dopamine-independent. To directly test the assumption that MOR activation directly inhibits local GABAergic neurons, we investigated the properties of rat VTA GABA neurons directly identified with either immunocytochemistry for GABA or GAD65/67, or in situ hybridization for GAD65/67 mRNA. Utilizing co-labeling with an antibody for the neural marker NeuN and in situ hybridization against GAD65/67, we found that 23±3% of VTA neurons are GAD65/67(+). In contrast to the assumptions of the two neuron model, VTA GABAergic neurons are heterogeneous, both physiologically and pharmacologically. Importantly, only 7/13 confirmed VTA GABA neurons were inhibited by the MOR selective agonist DAMGO. Interestingly, all confirmed VTA GABA neurons were insensitive to the GABA(B) receptor agonist baclofen (0/6 inhibited), while all confirmed dopamine neurons were inhibited (19/19). The heterogeneity of opioid responses we found in VTA GABAergic neurons, and the fact that GABA terminals arising from neurons outside the VTA are inhibited by MOR agonists, make further studies essential to determine the local circuit mechanisms underlying VTA MOR reward.

Figure 1. Mean density of GABA neurons in VTA: approximately 20% are GABAergic.

In horizontal brain slices containing the VTA and the SN the general neural marker NeuN was cytochemically visualized with the secondary label DAB (brown); GABAergic neurons were labeled using in situ hybridization against GAD65/67 mRNA (black grains). (A, left) Low magnification example image of one of the slices used to estimate the percentage of neurons that are GABAergic. Scale bar: 400 µm. Schematic (right) illustrates slice orientation and anatomical landmarks. (IPF: interpeduncular fossa; IPN: interpeduncular nucleus; ML: medial lemniscus; MT: medial terminal nucleus of the accessory optic track; SNc: substantia nigra pars compacta; SNr: substantia nigra pars reticulata; R: rostral; C: caudal; M: medial; L: lateral). Higher magnification images (color coded rectangles show location in (A)) show GAD65/67(+) and GAD65/67(−) in the SNr (B - yellow border), the SNc (C – red border), and the VTA (D – blue border with higher magnification section below). (E), Mean percentage of GAD65/67(+) neurons in VTA, SNc, and SNr neurons and, for comparison, our prior estimates of % TH(+) neurons .

Figure 2. Identification of GABA neurons following whole cell recordings.

(A) Example where immunocytochemistry against GABA (green) was used to identify GABAergic VTA neurons filled with biocytin (red). (Top row) An example GABA(+) recorded cell, and (Lower row) an example GABA(−) recorded cell. (B) In other cases we used in situ hybridization against GAD65/67 mRNA to test whether recorded neurons were GABAergic. In these examples of positive colabeling (left) and a lack of colabeling (right), filled cells are labeled with DAB (brown), and in situ label is visualized with digoxin (purple).

Figure 3. Locations of recorded VTA GABA neurons.

Sample includes neurons throughout the VTA. Schematic diagrams of ventral, middle, and dorsal horizontal slices containing the VTA. (R: rostral; C: caudal; M: medial; L: lateral).

Figure 4. Plot of soma crossectional areas and input resistances for all confirmed GABAergic neurons.

There was a trend towards an inverse relationship between these two measures. P = 0.09.

Figure 5. Most VTA GABA neurons are I _h(+).

(A) Example GABAergic neuron that has a large I _h (far left, scale bars: 100 pA and 50 ms). The neuron was filled with biocytin (left; red; scale bar: 20 µm) and immunocytochemically confirmed GABA(+) (middle, green). (B) Example I _h(−) GABAergic neuron (far left, scale bars: 50 pA and 50 ms) that was filled with biocytin (left; scale bar: 20 µm) and confirmed GABA(+). (C) Histogram of I _h magnitudes measured in GABA/GAD(+) and GABA/GAD(−) neurons. (D) For comparison, histogram of I _h magnitudes from recorded VTA dopamine neurons identified with TH immunocytochemistry.

Figure 6. VTA neurons express HCN protein.

(A) Immunocytochemical colabeling in VTA tissue for GAD and HCN4, one of the channels expressed in the VTA that produces the I _h, yields examples of GAD(+) neurons that are HCN4(+) (top) or HCN4(−) (bottom), consistent with the physiology. Scale bars: 20 µm. (B) Immunocytochemical colabeling for HCN4 and TH in VTA slices shows that HCN4 is expressed in some TH(−) neurons. Scale bar: 20 µm. (C) The HCN4 antibody was not non-specifically labeling all neurons as HCN4 colabeling with the neural marker NeuN shows that some VTA neurons were cytochemically HCN4(−). Scale bar: 20 µm.

Figure 7. The distribution of action potential durations in VTA GABA neurons overlaps with that of cytochemically confirmed dopamine neurons.

Neurons recorded on the same day were identified post hoc as GABA/GAD(+) or GABA/GAD(−), and their AP duration distributions are overlapping (top). This is consistent with data from TH(+) and TH(−) neurons where the range AP durations of TH(−) neurons spans the range for TH(+) neurons (bottom).

Figure 8. The spontaneous firing rates of VTA GABA neurons are similar to those of dopamine neurons ex vivo.

(A) Example traces of AP activity from a confirmed VTA GABA neuron (left) and a confirmed dopamine neuron (right) firing at similar frequencies. (B) For each spontaneously firing neuron, the ISI CV is plotted against the firing rate of the neuron.

Figure 9. Some, but not all, VTA GABA neurons are inhibited by activation of the MOR.

Example current clamp traces from identified VTA GABA neurons tested with bath application of 1 µM DAMGO. (A) GABA neuron showing robust inhibition by DAMGO (B) GABA neuron unresponsive to DAMGO.

Figure 10. GABA_B receptor activation inhibits VTA dopamine neurons, but not VTA GABA neurons.

(A) Example current clamp traces from cytochemically identified GABA neuron lacking a response to the GABA_B receptor agonist baclofen (1 µM). (Bi) An unidentified neuron recorded on the same day as GABA(+) neurons that were insensitive to baclofen shows a significant hyperpolarization in response to baclofen (1 µM; bar). (Bii) Example of a cytochemically identified dopamine neuron hyperpolarized by baclofen (1 µM; bar). Scale bars: 5 mV and 5 min. (C) Average trace of identified GABA neurons (n = 6) tested with bath application of baclofen (1 µM) compared to the averages of all quiescent confirmed dopamine neurons (n = 8). (D) Average trace of 4 neurons significantly inhibited by baclofen (1 µM); the inhibition was reversed by the GABA_B receptor antagonist CGP35348 (10 µM). (E) Table of all observed baclofen responses across confirmed GABA, dopamine, and I _h(+), TH(−) neurons. TH-identified neurons include spontaneously firing, quiescent (from panel C), and voltage-clamp experiments.