Dopamine modulation of GABA tonic conductance in striatal output neurons

Abstract

We previously reported greater GABAA receptor-mediated tonic currents in D2+ striatopallidal than D1+ striatonigral medium spiny neurons (MSNs) are mediated by alpha5-subunit-containing receptors. Here, we used whole-cell recordings in slices from bacterial artificial chromosome transgenic mice to investigate the link between subunit composition, phosphorylation, and dopamine receptor activation. Whole-cell recordings in slices from delta-subunit knock-out mice demonstrate that while MSNs in wild-type mice do express delta-subunit-containing receptors, this receptor subtype is not responsible for tonic conductance observed in the acute slice preparation. We assessed the contribution of the beta1- and beta3-subunits expressed in MSNs by their sensitivity to etomidate, an agonist selective for beta2- or beta3-subunit-containing GABAA receptors. Although etomidate produced substantial tonic current in D2+ neurons, there was no effect in D1+ neurons. However, with internal PKA application or dopamine modulation, D1+ neurons expressed tonic conductance and responded to etomidate application. Our results suggest that distinct phosphorylation of beta3-subunits may cause larger tonic current in D2+ striatopallidal MSNs, and proper intracellular conditions can reveal tonic current in D1+ cells.

Figure 1.

The δ-subunit does not contribute to tonic current. A, Illustrative records from MSNs in a δ−/− mouse showing differential block in tonic current with BMR application. Right, all-points histogram and Gaussian fit from each segment. B, Summary results show that the tonic current expression in a δ−/− mouse resembled the pattern of tonic current expressed in D₂+ and D₁+ MSN from BAC D₂ EGFP mice, suggesting that the δ-subunit is not responsible for the differential tonic currents between D₂+ and D₁+ MSN. C, THIP application in the presence of TTX did not induce tonic current in δ−/− MSNs (n = 6), as it did in both D₂+ (n = 17) and D₁+ (n = 6) MSNs from BAC D₂ EGFP mice, confirming that the δ-subunit is not present.

Figure 2.

Etomidate's direct effects are selective for β3-containing receptors. A, Representative currents from HEK 293 cells transfected with α2β1γ2 or α2β3γ2 receptors with GABA (3 μm for α2β1γ2, 1 μm for α2β3γ2) and etomidate (3 μm) application, showing β3-subunit selectivity for direct and potentiating effects. Calibration: 200 pA, 10 s. B, Representative currents from HEK 293 cells transfected with α5β1γ2 or α5β3γ2 receptors with GABA (250 nm for α5β1γ2 and α5β3γ2) and etomidate (3 μm) application, showing β3-subunit selectivity for direct effects only. Calibration: 50 pA, 10 s. C, Summary of etomidate's direct effects on striatally relevant GABA_A receptors (α2β1γ2, n = 6; α2β3γ2, n = 10; α5β1γ2, n = 5; α5β3γ2, n = 7). D, Summary of etomidate's potentiating effects on GABA currents evoked by EC10 concentrations of GABA on each receptor type (α2β1γ2, n = 4; α2β3γ2, n = 5; α5β1γ2, n = 13; α5β3γ2, n = 6).

Figure 3.

Etomidate selectively activates tonic receptors in D₂+ MSNs. A, Representative current traces of a D₂+ and a D₁+ MSN showing etomidate-elicited currents in the D₂+, but not the D₁+, MSN. B, Representative current traces of two individual MSNs, showing the direct effect of etomidate (3 μm), response to 1 μm GABA, and etomidate's (3 μm) potentiating effects with 1 μm GABA, all in 0.5 μm TTX. C, Summary data of etomidate's direct (n = 10, D₂+; n = 8, D₁+ MSN) and potentiating (n = 7, D₂+; n = 5, D₁+ MSN) effects to 1 μm GABA on D₂+ and D₁+ tonic current.

Figure 4.

Etomidate does not affect striatal synaptic GABA_A receptors. A, Examples of mIPSCs in D₂+ and D₁+ neurons before (gray) and after (black) etomidate application. Averaged mIPSC traces are normalized and overlaid to demonstrate that etomidate had little effect on current decay. B, Summary of phasic data demonstrating that etomidate had little effect on the frequency (n = 8 and 8), amplitude (n = 8 and 8), weighted tau (n = 8 and 6), or rise time (n = 3 and 3) in both D₂+ and D₁+ MSNs.

Figure 5.

Internal PKA application modulates tonic current. A, Representative traces of two individual MSNs where PKA was supplemented in the internal solution, showing BMR-sensitive tonic current in both cells. B, Summary graph for tonic current in D₂+ and D₁+ MSN in control conditions (D₂+, n = 12; D₁+, n = 10) and in conditions with internal PKA application (D₂+, n = 10; D₁+, n = 15). C, Summary graph of the changes in mIPSC decay with internal PKA application (D₂+, n = 14; D₁+, n = 21) compared with control (D₂+, n = 31; D₁+, n = 26). D, Representative traces from simultaneous dual recording of D₁+–D₂+ MSNs, showing etomidate responses with internal PKA application. E, Summary graph for current induced by etomidate with internal PKA application. The D₁+ etomidate response increases significantly with internal PKA application, whereas D₂+ responses decrease slightly (n = 10 and 8, D₂+ and D₁+, respectively, in CsCl; n = 9 and 12 in PKA).

Figure 6.

Dopamine agonists alter MSN tonic conductances. A, Representative current traces from individual D₂+ and D₁+ MSN illustrating that the D₂ agonist, quinpirole (10 μm), reduces tonic current in the D₂+ MSN, whereas it does not affect tonic currents in the D₁+ MSN. B, Representative traces of a simultaneous dual recording between a D₂+ and D₁+ MSN illustrating that the D₁ agonist, SKF-81297 (10 μm), induces a tonic current in the D₁+ MSN but also reduces it in the D₂+ MSN. C, Summary graph showing effects on tonic current with quinpirole and SKF-81297 application on D₂+ (n = 5 and 3) and D₁+ (n = 6 and 4). D, Summary graph of phasic currents of both D₂+ and D₁+ in response to their respective agonists (n = 6 and 5 for D₂+, n = 8 and 5 for D₁+). E, Representative current trace from a D₁+ neuron where etomidate (3 μm) was given before and during SKF-81297 (10 μm) application. SKF-81297 was given for over 5 min before coapplication with etomidate to allow full drug action.

Figure 7.

Dopamine modulates MSN cell excitability. A, Representative current-clamp recording from a D₂+ MSN illustrating the responses to a series of depolarizing current injections (20 pA steps) from −70 mV, recorded with K-gluconate internal in the absence and presence of quinpirole (10 μm) and BMR (25 μm). B, Representative example of a D₁+ MSN in the same conditions as A, but with the D₁-like selective agonist, SKF-81297 (10 μm). C, Summary plot showing the averaged rheobase current in D₂+ (n = 5) and D₁+ (n = 7) MSNs with dopamine agonist and BMR application. D, Summary of action potential firing frequency in response to increasing depolarizing current injections recorded with K-gluconate internal solution in D₂+ MSNs (■) in the absence and presence of 10 μm quinpirole (□) and 25 μm BMR (●). Data derive from the same cells in C. E, Summary of action potential firing frequency in D₁+ MSNs (■) in the absence and presence of 10 μm SKF-81297 (□) and 25 μm BMR (●). *Significance to D₂+ control cells; ^#significance between D₂+ and D₁+ cells. Calibration: 20 μm, 500 ms.

Figure 8.

Tonic conductance is mediated through a phosphorylated β3-subunit. Under basal conditions (little to no dopamine), D₂ receptors do not activate the G_i/o protein to inhibit PKA phosphorylation, and the β1- and β3-subunits are basally phosphorylated. Because the phosphorylated β3-subunit yields increased currents and may be more plentiful than extrasynaptic β1-subunits, D₂+ MSN display tonic current. Without dopamine, D₁ receptors do not activate the G_s/G_olf protein to promote PKA phosphorylation, and the dephosphorylated β3-subunits do not mediate increased conductance, resulting in smaller tonic current in the D₁+ than the D₂+ MSN. More abundant β1-subunit expression in D₁+ MSNs results in increased current only during GABA application. When stimulated, the D₂ receptor activates the G_i/o protein to inhibit PKA activity, dephosphorylating the β1/β3-subunits. A dephosphorylated β3-subunit results in smaller tonic currents compared with basal conditions. During D₁ stimulation, the G_s/G_olf protein activates cAMP and PKA pathways to phosphorylate the β3-subunit and increase tonic currents.