Differential tonic GABA conductances in striatal medium spiny neurons

Abstract

Medium spiny neurons (MSNs) provide the principal output for the dorsal striatum. Those that express dopamine D2 receptors (D2+) project to the globus pallidus external and are thought to inhibit movement, whereas those that express dopamine D1 receptors (D1+) project to the substantia nigra pars reticulata and are thought to facilitate movement. Whole-cell and outside-out patch recordings in slices from bacterial artificial chromosome transgenic mice examined the role of GABA(A) receptor-mediated currents in dopamine receptor D1+ striatonigral and D2+ striatopallidal MSNs. Although inhibitory synaptic currents were similar between the two neuronal populations, D2+ MSNs showed greater GABA(A) receptor-mediated tonic currents. TTX application abolished the tonic current to a similar extent as GABA(A) antagonists, suggesting a synaptic origin of the ambient GABA. Low GABA concentrations produced larger whole-cell responses and longer GABA channel openings in D2+ than in D1+ MSNs. Recordings from MSNs in alpha1-/- mice and pharmacological analysis of tonic currents suggested greater expression of alpha5-containing GABA(A) receptors in D2+ than in D1+ MSNs. As a number of disorders such as Parkinson's disease, Huntington's chorea, and tardive dyskinesia arise from an imbalance between these two pathways, the GABA(A) receptors responsible for tonic currents in D2+ MSNs may be a potential target for therapeutic intervention.

Figure 1.

Characteristics of D₁⁺ and D₂⁺ medium spiny neurons. A, Examples of differential interference contrast (left) and fluorescence (right) confocal micrographs of a corticostriatal slice prepared from a BAC D₂ EGFP mouse. EGFP expression was used to classify MSNs as D₁⁺ or D₂⁺. Scale bar, 20 μm. B, Examples of current-clamp recordings with K-gluconate internal, demonstrating responses to a series of hyperpolarizing and depolarizing current injections (20 pA steps) from RMPs of −65 mV in a D₁⁺ and D₂⁺ MSN. Calibration: 20 mV, 100 pA.

Figure 2.

D₂⁺ MSNs demonstrate larger GABA_A receptor-mediated tonic currents than D₁⁺ MSNs. A, Representative traces from a D₁⁺ MSN (left) and a D₂⁺ MSN (right) demonstrate that BIC (25 μm) blocked sIPSCs in both cell types but only revealed an endogenous GABA-mediated tonic current in the D₂⁺ MSN. The remaining sEPSCs in both cell types were blocked by NBQX (5 μm) with no effects on the tonic current. B, The mean of the baseline current during BIC application from the representative traces in A were adjusted to 0, and the amplitude distributions were drawn from segments immediately preceding BIC application. The non-skewed sides of the amplitude histograms were fit with a Gaussian, the peak of which was used to determine the absolute magnitude of tonic current blocked by BIC. C, Summary of tonic currents blocked by BIC (25 μm) with KCl internal (black bars; n = 23 and 44) and with CsCl internal (white bars; n = 7 and 9) in D₁⁺ and D₂⁺ MSNs, respectively. D, Representative traces from a D₁⁺ MSN (left) and a D₂⁺ MSN (right) demonstrate that blockade of voltage-gated Na⁺ channels with TTX (0.5 μm) reduces the frequency and amplitude of postsynaptic events in both cell types but only alters the amount of holding current required to maintain the holding voltage at −60 mV in the D₂⁺ MSN. E, Amplitude distributions representing the tonic current abolished by TTX, drawn from segments in D as described above for BIC (B). F, Summary of tonic current blocked by TTX (0.5 μm) from recordings with KCl internal (black bars; n = 11 and 32) and with CsCl internal (white bars; n = 6 and 7) in D₁⁺ and D₂⁺ MSNs, respectively.

Figure 3.

GABA_A receptor-mediated sIPSCs and mISPCs do not differ between D₁⁺ and D₂⁺ MSNs. A, Representative traces showing sIPSCs in a D₁⁺ MSN (top) and a D₂⁺ MSN (bottom). Calibration: 1 s, 25 pA. Summary of the mean amplitude (B), frequency of events (C), tau values (D), and rise time (E) for sIPSCs in D₁⁺ and D₂⁺ MSNs. sIPSC values were measured using KCl internal (black bars; n = 6 and 12) and with CsCl internal (white bars; n = 8 and 10) from D₁⁺ and D₂⁺ MSNs, respectively. G, Representative traces demonstrate mIPSCs in a D₁⁺ MSN (top) and a D₂⁺ MSN (bottom). Calibration: 1 s, 25 pA. Summary of the mean amplitude (H), frequency of events (I), tau values (J), and rise time (K) for mIPSCs in D₁⁺ and D₂⁺ MSNs. mIPSCs values were measured from the same cells represented in B–D using KCl internal (black bars) and CsCl internal (white bars) by subsequent application of TTX (500 nm), NBQX (5 μm), and strychnine (500 nm). Cumulative probability plots for the average mIPSC (F), amplitude, and interevent interval (L) from D₁⁺ MSNs (dashed black line; n = 9) and D₂⁺ MSNs (solid gray line; n = 10) recorded with CsCl internal.

Figure 4.

Greater sensitivity to exogenously applied GABA in D₂⁺ than in D₁⁺ MSNs. Representative traces (A) and summary dose–response data (B) of the whole-cell currents elicited by increasing doses of GABA applied in the presence of TTX (0.5 μm), NBQX (5 μm), and strychnine (0.5 μm) in D₁⁺ and D₂⁺ MSNs. The EC₅₀ for GABA was 107.3 ± 0.5 and 18.2 ± 1.9 μm and the Hill coefficient was 1.04 ± 0.05 and 1.75 ± 0.18 for D₁⁺ and D₂⁺ MSNs, respectively. All recordings were performed with KCl internal solution, and data were derived from at least sixteen cells in each group. Data on whole-cell currents induced by 0.5 μm and 5 μm GABA (C) for D₁⁺ MSNs (white bar) and D₂⁺ MSNs (black bar) are illustrated for clarity. D, A summary of whole-cell currents elicited by 0.5 μm and 5 μm THIP in D₁⁺ MSNs (white bars) and D₂⁺ MSNs (black bars). E, Dose–response data of whole-cell recordings of THIP elicited currents normalized to currents elicited by a saturating dose of GABA (2 mm; GABA_sat) in each cell. For both D and E, recordings were performed in the presence of TTX (0.5 μm), NBQX (5 μm), and strychnine (0.5 μm), and data were derived from at least six MSNs in each group.

Figure 5.

GABA channel currents in excised patches from D₁⁺ and D₂⁺ MSNs. A, Representative examples of currents elicited by three concentrations of GABA (100 nm, 500 nm, and 2 mm) in patches excised from a D₁⁺ and a D₂⁺ MSN and held at −80 mV. Dashed lines illustrate the main conductance level derived from channel current histograms shown in B. Histograms were fit with a Gaussian, and the peak of the curve defined the main conductance level. The main opening levels for the channel currents elicited by 500 nm GABA in the patches represented in A were 2.18 ± 0.09 pA for the D₁⁺ MSN and 2.19 ± 0.08 pA for the D₂⁺ MSN. C, Overlapping open time distributions with a multiple Gaussian fit on a logarithmic scale for the patches illustrated in A elicited by 500 nm GABA. Time constants of the distributions indicated by arrows were 0.41 ms (fast) and 1.9 ms (slow) (81% fast component) and 0.39 ms (fast) and 1.85 ms (slow) (59% fast component) for the D₁⁺ and the D₂⁺ MSN, respectively. D, Current to voltage relationship for the main open state of GABA-elicited currents in patches excised from D₁⁺ (n = 9) and D₂⁺ (n = 8) MSNs. The slope conductance from the linear fits illustrated was 27.9 ± 0.4 and 27.5 ± 0.4 pS for the D₁⁺ and the D₂⁺ MSNs, respectively. E, Summary of mean open times for the channel openings at the main conductance for three GABA concentrations. F, Summary of the open time distributions for the two main open time constants as in C at each concentration tested. G, Summary of the percentage contribution of the fast open time to the fit constant at the three concentrations of GABA.

Figure 6.

The role of the α1 subunit in MSN tonic and phasic inhibition. A, Representative traces demonstrate the occurrence of sIPSCs and the response to BIC in two examples of MSNs in striatal slices derived from a postnatal day 19 α1^−/− mouse. B, Overlaying representative traces of sIPSCs (top) and mIPSCs (bottom) from MSNs in α1^−/− (gray) and BAC D₂ EGFP (black) mice. C, Comparison of the distributions of BIC-sensitive tonic currents in 21 MSNs from α1^−/− mice and a sample of 15 D₁⁺ and 15 D₂⁺ MSNs from BAC D₂ EGFP mice illustrates a similar scatter. D, Summary of mean tau values for sIPSCs and mIPSCs in MSNs from 12 α1^−/− (white bars) and a pool of six D₁⁺ and six D₂⁺ MSNs from age-matched BAC D₂ EGFP mice (black bars).

Figure 7.

L655,703 antagonizes GABA_A receptor-mediated tonic currents in D₂⁺ MSNs. A, Representative trace showing the effects of L655,703 (10 μm) on sIPSCs and on BIC-sensitive tonic current in a D₂⁺ MSN. Calibration: 20 ms, 30 pA. B, Summary of tonic current blocked by L655,703 (50 nm, n = 4; 10 μm, n = 8) in D₂⁺ MSNs. C, Representative traces showing the effects of L655,703 (10 μm) on sIPSCs and BIC-sensitive tonic currents elicited by bath application of 1 μm GABA in a D₁⁺ MSN and a D₂⁺ MSN. Calibration: 20 ms, 30 pA. D, Summary of the percentage blockade by L655,708 of the BIC-sensitive tonic current elicited by bath application of 1 μm GABA in D₁⁺ MSNs (100 nm, n = 3; 10 μm, n = 5) and in D₂⁺ MSNs (50 nm, n = 2; 10 μm, n = 4). E, Representative traces showing overlapping sIPSCs recorded in a D₁⁺ MSN (top) and a D₂⁺ MSN (middle) in the presence (gray trace) and the absence (black trace) of 10 μm L655,703, as well as the summary of the changes in sIPSC amplitude (Amp) and decay time (Tau) produced by 10 μm L655,703 in six D₁⁺ and seven D₂⁺ MSNs (bottom) represented as percentage of baseline values. Calibration: 20 ms, 30 pA.

Figure 8.

Blocking GABA_A receptor currents reduces action potential firing frequency in D₂⁺ MSNs. A, Representative example of a current-clamp recording from a D₂⁺ MSN illustrating the responses to a series of hyperpolarizing and depolarizing current injections (20 pA steps) recorded with K-gluconate internal in the absence and the presence of 10 μm gabazine. Calibration: 15 mV, 80 pA. B, Summary of action potential firing frequency in response to increasing depolarizing current injections recorded with K-gluconate internal solution in D₁⁺ (■) and D₂⁺ (▲) MSNs the absence or the presence [D₁⁺ (□) and D₂⁺ (△)] of 10 μm gabazine. Data derive from six D₁⁺ and seven D₂⁺ MSNs. C, Representative example of a D₂⁺ MSN demonstrates action potential firing evoked by stimulation of glutamatergic afferents and assessed using extracellular recording in cell-attached configuration in the absence (left) or the presence (right) of 50 nm L655,703. Calibration: 3 ms, 40 pA.