Dopamine D(2)-like receptors selectively block N-type Ca(2+) channels to reduce GABA release onto rat striatal cholinergic interneurones

Abstract

1. The modulatory roles of dopamine (DA) in inhibitory transmission onto striatal large cholinergic interneurones were investigated in rat brain slices using patch-clamp recording. 2. Pharmacologically isolated GABA(A) receptor-mediated IPSCs were recorded by focal stimulation within the striatum. Bath application of DA reversibly suppressed the amplitude of evoked IPSCs in a concentration-dependent manner (IC(50), 10.0 microM). 3. A D(2)-like receptor agonist, quinpirole (3-30 microM), also suppressed the IPSCs, whereas a D(1)-like receptor agonist, SKF 81297, did not affect IPSCs. Sulpiride, a D(2)-like receptor antagonist, blocked the DA-induced suppression of IPSCs (apparent dissociation constant (K(B)), 0.36 microM), while a D(1)-like receptor antagonist, SCH 23390 (10 microM), had no effect. 4. DA (30 microM) reduced the frequency of spontaneous miniature IPSCs (mIPSCs) without changing their amplitude distribution, suggesting that GABA release was inhibited, whereas the sensitivity of postsynaptic GABA(A) receptors was not affected. The effect of DA on the frequency of mIPSCs was diminished when extracellular Ca(2+) was replaced by Mg(2+) (5 mM), indicating that DA affected the Ca(2+) entry into the presynaptic terminal. 5. An N-type Ca(2+) channel selective blocker, omega-conotoxin GVIA (omega-CgTX, 3 microM), suppressed IPSCs by 65.4 %, whereas a P/Q-type Ca(2+) channel selective blocker, omega-agatoxin IVA (omega-Aga-IVA, 200 nM), suppressed IPSCs by 78.4 %. Simultaneous application of both blockers suppressed IPSCs by 95.9 %. Assuming a 3rd power relationship between Ca(2+) concentration and transmitter release, the contribution of N-, P/Q- and other types of Ca(2+) channels to presynaptic Ca(2+) entry is estimated to be, respectively, 29.8, 40.0 and 34.5 % at this synapse. After the application of omega-CgTX, DA (30 microM) no longer affected IPSCs. In contrast, omega-Aga-IVA did not alter the level of suppression by DA, suggesting that the action of DA was selective for N-type Ca(2+) channels. 6. A G protein alkylating agent, N-ethylmaleimide (NEM), significantly reduced the DA-induced suppression of IPSCs. 7. These results suggest that presynaptic D(2)-like receptors are present on the terminals of GABAergic afferents to striatal cholinergic interneurones, and down-regulate GABA release by selectively blocking N-type Ca(2+) channels through NEM-sensitive G proteins.

Figure 1. Identification of striatal cholinergic neurones

A, a striatal cholinergic neurone filled with Lucifer Yellow (0.1 %) in the dorsolateral striatum in a coronal slice (17-day-old rat). The dorsal side is at the top and the lateral side is to the right. B, Nomarski view of another striatal cholinergic neurone at a higher magnification. C, the neurone in B filled with Lucifer Yellow. Scale bar applies to both B and C. D, membrane potentials recorded from another large striatal neurone in response to hyperpolarizing current pulses applied intracellularly through Cs⁺-filled electrodes. Voltage sags during hyperpolarizing current pulses, characteristic of striatal cholinergic neurones, were identified.

Figure 2. Characteristics of GABAergic inhibitory postsynaptic currents (IPSCs) recorded in striatal cholinergic neurones

A, current-voltage (I-V) relationship of IPSCs evoked at 0.2 Hz in the presence of CNQX (5 μm), d-AP5 (25 μm) and strychnine (0.5 μm). Average traces of 10 IPSCs at each potential are shown, and the peak current magnitude is plotted against V_h. IPSCs reversed at potential close to the Cl⁻ equilibrium potential E_Cl (+2.6 mV). The holding current was -22 pA at -60 mV. B, IPSCs were blocked by bath-applied bicuculline (Bic, 10 μm). The amplitude of IPSCs evoked at 0.2 Hz is plotted against time. Traces a-c are averages of 20 consecutive IPSCs during the indicated periods.

Figure 3. Inhibition of GABAergic IPSCs by dopamine (DA)

IPSCs were evoked at 0.2 Hz. V_h, -60 mV. A, time course of the inhibitory effect of DA (30 μm) on the amplitude of evoked IPSCs. Each point represents the mean amplitude of five consecutive responses. DA was bath applied during the indicated period. The traces a-c show averages of 20 consecutive IPSCs during the indicated periods. B, concentration-dependent inhibition of IPSCs by DA. Numbers of cells are shown in parentheses. Error bars indicate s.e.m. The data were fitted to a logistic function (superimposed curve; see Methods) to estimate the IC₅₀ value (10.0 μm), maximum inhibition (by 65.4 %) and Hill slope (1.1). C, effect of the endogenous DA uptake system upon DA-induced suppression of IPSCs. For the same cell (numbers of cells shown in parentheses), the effect of DA (3 and 30 μm) was examined in the absence and presence of the DA uptake inhibitor GBR 12909 (GBR, 10 μm). GBR significantly enhanced the action of DA at 3 μm, but not at 30 μm.

Figure 4. Effect of DA receptor agonists on evoked IPSCs

All IPSCs were evoked at 0.2 Hz. V_h, -60 mV. A, lack of effect of a D₁-like receptor agonist, SKF 81297 (30 μm). B, inhibitory effect of a D₂-like receptor agonist, quinpirole (30 μm). Each trace is the average of 20 IPSCs. C, histograms summarizing the mean inhibitory effect of SKF 81297 (30 μm), quinpirole (3 and 30 μm) and DA (30 μm). Error bars indicate s.e.m. Values for SKF 81297, 3 μm quinpirole, 30 μm quinpirole and DA were 8.8 ± 5.9 % (n = 9), 34.1 ± 6.6 % (n = 4), 55.8 ± 5.6 % (n = 9) and 48.6 ± 1.7 % (n = 46), respectively. The effects of 30 μm quinpirole and 30 μm DA were not significantly different (P > 0.05).

Figure 5. Effects of DA receptor antagonists on DA-induced inhibition of evoked IPSCs

Effects of DA on IPSCs (0.2 Hz; V_h, -60 mV) were compared in the absence and presence of a D₁-like receptor antagonist, SCH 23390 (10 μm; A), or a D₂-like receptor antagonist, sulpiride (5 μm; B). Traces are averages of 20 consecutive IPSCs. Antagonists were applied for 7 min before a second application of DA. The concentration of DA ranged from 0.3 μm to 1 mm and concentration-response curves in the absence (same as in Fig. 3B) and presence of each antagonist are superimposed in the graphs on the right. Error bars indicate s.e.m. and numbers of cells are given in parentheses. A, the concentration-response curve for IPSC inhibition remained unchanged by SCH 23390 (▪) compared with the curve in the absence of SCH 23390 (□). The estimated IC₅₀ value, maximum inhibition and Hill slope were 7.8 μm, 62.0 % and 1.1, respectively. B, the concentration-response curve was shifted to the right by sulpiride (•), with the estimated IC₅₀ value, maximum inhibition and Hill slope being 150 μm, 66.8 % and 1.2, respectively. □, curve in the absence of sulpiride.

Figure 6. Effect of DA on spontaneous miniature IPSCs (mIPSCs)

The mIPSCs were recorded in the presence of TTX (0.3 μm), CNQX (5 μm), d-AP5 (25 μm) and strychnine (0.5 μm). V_h, -60 mV. A, consecutive traces recorded in control, 3 min after application of bicuculline (10 μm), and 15 min after washout. B, consecutive traces recorded before and 3 min after application of 30 μm DA. C and D, cumulative probability distribution of inter-event intervals (C) and peak amplitudes (D) of mIPSCs from the same neurone shown in B, comparing distributions in the absence (open symbols) and presence (filled symbols) of DA. Control data contain 364 events per 10 min period, and DA data contain 181 events per 10 min period. The inter-event interval was increased, whereas the mIPSC amplitude was unaffected by DA. Insets: histograms of inter-event intervals and amplitudes. E, superimposed traces of averaged mIPSCs in control (364 events) and during application of DA (181 events). The mean amplitudes of mIPSCs in control and in the presence of DA were 25.0 and 25.7 pA, respectively.

Figure 7. Lack of DA-induced effect on mIPSCs in Ca²⁺-free solution

A, the mIPSCs were recorded in nominally Ca²⁺-free, high-Mg²⁺ (5 mm) aCSF containing TTX (0.3 μm), CNQX (5 μm), d-AP5 (25 μm) and strychnine (0.5 μm). V_h, -60 mV. Consecutive traces recorded before and 3 min after application of DA (30 μm). B and C, cumulative probability distribution of inter-event intervals (B) and amplitudes (C) of mIPSCs in Ca²⁺-free solution before (131 events per 10 min period) and during DA application (126 events per 10 min period). In Ca²⁺-free solution, DA no longer affected the distribution. Inset in C: superimposed traces of averaged mIPSCs in control (131 events) and during application of DA (126 events). The mean amplitudes of mIPSCs in control and during DA application were 28.2 and 28.4 pA, respectively. D, histograms summarizing the inhibitory effect of DA (30 μm) on the frequency of mIPSCs in Ca²⁺ concentrations of 2.4 and 0 mm. Values for 2.4 mm Ca²⁺ and Ca²⁺-free solutions were 47.2 ± 2.4 % (n = 8) and 5.6 ± 2.4 % (n = 7), respectively. The effect in the nominally Ca²⁺-free solution was not significant (P > 0.05).

Figure 8. Multiple Ca²⁺ channel subtypes are responsible for inhibitory synaptic transmission onto striatal cholinergic neurones

A-C, time course of the inhibitory effect of ω-CgTX (3 μm) and ω-Aga-IVA (200 nm) on the amplitude of evoked IPSCs (0.2 Hz; V_h, -60 mV). Each point represents the mean amplitude of five consecutive responses. Toxins were bath applied during the indicated periods. Superimposed traces on the right in A-C are averages of 20 consecutive IPSCs during the indicated periods. D, histograms summarizing the suppression of evoked IPSCs by Ca²⁺ channel blockers. Values for ω-CgTX, ω-Aga-IVA, and both toxins are 65.4 ± 3.3 % (n = 11), 78.4 ± 2.7 % (n = 7) and 95.9 ± 1.5 % (n = 6), respectively. E, histograms summarizing the estimated fractions of Ca²⁺ channel subtypes contributing to GABAergic synaptic transmission onto striatal cholinergic neurones. The proportions of N-, P/Q- and other types were 0.298, 0.400 and 0.345, respectively (sum = 1.04), assuming a third power relation between presynaptic Ca²⁺ concentration and postsynaptic response amplitude.

Figure 9. DA-induced inhibition of evoked IPSCs is selectively coupled to N-type Ca²⁺ channels

IPSCs were evoked at 0.2 Hz. Vh, -60 mV. Top panels in A and B, time course of effects of DA (30 μm) on IPSCs before and after application of ω-CgTX (3 μm, A) or ω-Aga-IVA (200 nm, B). Each point represents the mean amplitude of five consecutive IPSCs. DA, ω-CgTX and ω-Aga-IVA were applied in the bath during the indicated periods. Toxins were applied after IPSCs had recovered from the inhibition induced by an initial application of DA. After the toxin-induced suppression had reached steady state, DA was applied again. Bottom panels in A and B, averaged traces of 20 consecutive IPSCs during the indicated periods. C, histograms summarizing the effects of ω-CgTX (3 μm) and ω-Aga-IVA (200 nm) on the DA (30 μm)-induced inhibition. After the effect of ω-CgTX had reached steady state, DA no longer affected IPSCs (2.9 ± 1.4 % inhibition, n = 6). In contrast, DA still had an effect (55.7 ± 7.6 % inhibition, n = 5) after the effect of ω-Aga-IVA had reached steady state. The effects obtained by application of DA alone (44.2 ± 5.9 %, n = 5) and DA in the presence of ω-Aga-IVA (55.7 ± 7.6 %, n = 5) were not significantly different (P > 0.05).

Figure 10. Effect of N-ethylmaleimide (NEM) on the DA-induced inhibition of the evoked IPSCs

A, top, time course of the inhibition of IPSCs (0.2 Hz; V_h, -60 mV) by DA (30 μm), which was blocked by NEM (50 μm). Each point represents the mean amplitude of five consecutive IPSCs. DA and NEM were applied in the bath during the indicated periods. After IPSCs had recovered from the initial DA-induced suppression, NEM was applied for 5 min and DA was applied again in the presence of NEM. Bottom, averaged traces of 20 consecutive IPSCs during the indicated periods. B, histograms summarizing the effects of DA with and without NEM. DA-induced inhibitory effect in the presence of NEM (6.9 ± 5.2 %, n = 6) was significantly (P < 0.05) smaller than that of DA alone (44.3 ± 2.2 %, n = 6).