Tonic GABAergic inhibition, via GABAA receptors containing αβƐ subunits, regulates excitability of ventral tegmental area dopamine neurons

Abstract

The activity of midbrain dopamine neurons is strongly regulated by fast synaptic inhibitory γ-Aminobutyric acid (GABA)ergic inputs. There is growing evidence in other brain regions that low concentrations of ambient GABA can persistently activate certain subtypes of GABA_A receptor to generate a tonic current. However, evidence for a tonic GABAergic current in midbrain dopamine neurons is limited. To address this, we conducted whole-cell recordings from ventral tegmental area (VTA) dopamine neurons in brain slices from mice. We found that application of GABA_A receptor antagonists decreased the holding current, indicating the presence of a tonic GABAergic input. Global increases in GABA release, induced by either a nitric oxide donor or inhibition of GABA uptake, further increased this tonic current. Importantly, prolonged inhibition of the firing activity of local GABAergic neurons abolished the tonic current. A combination of pharmacology and immunohistochemistry experiments suggested that, unlike common examples of tonic inhibition, this current may be mediated by a relatively unusual combination of α4βƐ subunits. Lastly, we found that the tonic current reduced excitability in dopamine neurons suggesting a subtractive effect on firing activity.

Keywords: addiction; extra-synaptic; midbrain; synaptic transmission.

FIGURE 1

VTA dopamine neurons exhibit a tonic GABAergic current. (a) An example raw trace of holding current change during picrotoxin (PTX, 100 µM) application. (b) An example of average holding current baseline values before and after PTX or bicuculline (BIC, 10 µM) showing a decrease in holding current. (c) Graph showing the mean (+SEM) change in holding current following application of PTX and BIC (PTX: p = 0.00632, BIC: p = 0.0409). Tonic currents were defined as the difference between the average holding current (I _hold) during baseline versus that obtained during drug application as ΔI _hold. *p < 0.05, **p < 0.01

FIGURE 2

Tonic GABAergic current is sensitive to exogenous GABA concentration and maintained by firing activity of VTA GABA neurons. (a) Example traces and (b) graph of mean (+SEM) ΔI _hold effect showing that bath application of SNAP (400 µM) and NO‐711 (20 µM) increases I _hold (SNAP: p = 0.0073, NO‐711: p = 0.013). (c) Graph showing the mean (+SEM) effect of SNAP and NO‐711 on sIPSC amplitude, decay time and frequency (SNAP; Amplitude: p = 0.8806, Decay time: p = 0.8610, Frequency: p = 0.1970, NO‐711; Amplitude: p = 0.0917, Decay time: p = 0.1630, Frequency: p = 0.0771). (d) Example traces and (e) graph of mean (+SEM) ΔI _hold effect showing that pro‐longed incubation with TTX (1 µM), to wash‐off exogenous GABA, eliminates the effect of GABA_A receptor antagonists, PTX (100 µM), BIC (10 µM) and GABAzine (100 µM) on ΔI _hold (PTX: p = 0.5928, BIC: p = 0.9660, GABAzine: p = 0.9589). *p < 0.05, **p < 0.01

FIGURE 3

Zinc blocks tonic GABAergic current with little effect on synaptic currents. (a) An example trace showing that bath application of low concentration of Zinc (20 µM) leads to a reduction in I _hold, consistent with the presence of a tonic current. (b) Graph of mean (+SEM) ΔI _hold effect of Zinc and PTX on the holding current (Zinc: p = 0.0366, Zinc + PTX: p = 0.0492). (c) An example trace and averages (grey trace is SEM) of mIPSCs before and after zinc application showing no effect on postsynaptic IPSCs. (d) Graph of mean (+SEM) effect of zinc on mIPSC amplitude, decay time and frequency (Amplitude: p = 0.2684, Decay time: p = 0.8043, Frequency: p = 0.8097). (e) An example trace showing that bath application of THIP (5 µM) has only a small effect on I _hold. (f) Graph of mean (+SEM) ΔI _hold effect of THIP on the holding current (p = 0.0398). *p < 0.05

FIGURE 4

Ɛ subunit containing (but not θ) GABA_A receptors are located extrasynaptically in VTA dopamine neurons. (a, i), Representative immunofluorescent images showing that the θ subunit is sparsely expressed in the VTA and rarely colocalized with TH. (ii) Orthogonal view of confocal image showing rare co‐localization of θ and gephyrin (Geph). (b, i) Representative immunofluorescent images showing that the Ɛ subunit is expressed in the VTA in TH+ neurons and shows little co‐localization with gephyrin. (ii) Orthogonal view shows appositive expression of ε and gephyrin. (c) The percentage of colocalized signals against total gephyrin + puncta in both TH+ and TH‐ structures (ε: 2.77% ± 0.73 (TH+), 2.39% ± 0.49 (TH‐), n = 13 [N = 4), θ: 1.58% ± 0.38 (TH+), 0.35% ± 0.12 (TH‐), n = 11 [N = 4]). (d) The percentage of subunit expression in the TH+ structure against total subunit expression (ε: 60.02% ± 3.16, n = 13 [N = 4), θ: 33.07% ± 2.70, n = 11 [N = 4]). White circles indicate co‐localization of subunits and gephyrin and yellow arrowhead indicate apposition of subunits and gephyrin. Scale bars: (i) 20 µm, inset 5 µm, (ii) 10 µm

FIGURE 5

Amongst α1‐4, the α4 subunit is rarely expresses colocalized with postsynaptic markers in VTA dopamine neurons. (a) α1 subunit expresses selectively to non‐TH+ structure. (ii) Co‐localization with gephyrin was found in non‐TH dendritic structures (TH+: 6.45% ± 1.67, TH‐: 67.36% ± 3.53, n = 10 [N = 4]). (b) α2 subunit expresses strongly on TH+ structures. (ii) Co‐localization with gephyrin was found in TH+ structures (TH+: 43.34% ± 5.79, TH‐: 1.91% ± 0.59, n = 10 [N = 4]). (c) α3 subunit expresses on both TH+ and TH‐ structures and colocalized with gephyrin (TH+: 9.71% ± 1.26, TH‐: 9.73% ± 1.63, n = 10 [N = 3]). (d) α4 subunit expresses predominantly to TH+ structures, while these signals rarely colocalized with gephyrin (TH+: 0.11% ± 0.08, TH‐: 0.51% ± 0.19, n = 11 [N = 5]). (e) The percentage of subunit expression in the TH+ structure against total subunit expression (α1: 6.44% ± 1.10, n = 10 [N = 4), α2: 70.09% ± 3.85, n = 10 [N = 4), α3: 46.26% ± 5.18, n = 10 [N = 3), α4: 54.59% ± 2.93, n = 11 [N = 5]). White circles indicate co‐localization of subunits and gephyrin and yellow arrowhead indicate apposition of subunits and gephyrin. Scale bars: (i) 20 µm, inset 5 µm, (ii) 10 µm

FIGURE 6

Tonic GABA current reduces excitability in VTA dopamine neurons. (a) Example traces of firing activity in response to current injections of increasing amplitude (starting holding potential of −60 mV) before and during the blockade of the tonic GABAergic current with PTX. (b) Graph of mean (+SEM) firing activity showing an increase in excitability following blockade of the tonic current (Group: p = 0.0116, Step: p = <0.0001, Interaction: p = 0.011). (c) Graph of mean (+SEM) input resistance (Rm) showing an increase after the blockade of the tonic GABAergic current with PTX (Before‐after interaction: p = 0.0093). *p < 0.05, **p < 0.01