Control of spontaneous firing patterns by the selective coupling of calcium currents to calcium-activated potassium currents in striatal cholinergic interneurons

Abstract

The spontaneous firing patterns of striatal cholinergic interneurons are sculpted by potassium currents that give rise to prominent afterhyperpolarizations (AHPs). Large-conductance calcium-activated potassium (BK) channel currents contribute to action potential (AP) repolarization; small-conductance calcium-activated potassium channel currents generate an apamin-sensitive medium AHP (mAHP) after each AP; and bursts of APs generate long-lasting slow AHPs (sAHPs) attributable to apamin-insensitive currents. Because all these currents are calcium dependent, we conducted voltage- and current-clamp whole-cell recordings while pharmacologically manipulating calcium channels of the plasma membrane and intracellular stores to determine what sources of calcium activate the currents underlying AP repolarization and the AHPs. The Cav2.2 (N-type) blocker omega-conotoxin GVIA (1 microM) was the only blocker that significantly reduced the mAHP, and it induced a transition to rhythmic bursting in one-third of the cells tested. Cav1 (L-type) blockers (10 microM dihydropyridines) were the only ones that significantly reduced the sAHP. When applied to cells induced to burst with apamin, dihydropyridines reduced the sAHPs and abolished bursting. Depletion of intracellular stores with 10 mM caffeine also significantly reduced the sAHP current and reversibly regularized firing. Application of 1 microM omega-conotoxin MVIIC (a Cav2.1/2.2 blocker) broadened APs but had a negligible effect on APs in cells in which BK channels were already blocked by submillimolar tetraethylammonium chloride, indicating that Cav2.1 (Q-type) channels provide the calcium to activate BK channels that repolarize the AP. Thus, calcium currents are selectively coupled to the calcium-dependent potassium currents underlying the AHPs, thereby creating mechanisms for control of the spontaneous firing patterns of these neurons.

Figure 1.

Firing properties of striatal cholinergic interneurons in vitro. a, A 20 s trace depicting a spontaneous firing pattern of a cholinergic interneurons in vitro, which includes transitions from tonic to burst discharge. The bursts are followed by prominent hyperpolarizations. b, Action-potential waveform of a cholinergic interneuron. c, During spontaneous discharge, each action potential is followed by a medium AHP. d, A somatic current injection elicits a slow AHP after its termination.

Figure 2.

A calcium-dependent apamin-sensitive outward current contributes to the medium AHPs. a, A brief 10 ms voltage step to -2 mV from a holding potential of -57 mV elicits an outward tail current (black trace), which is abolished by bath application of 25 nm apamin (red trace), an effect that is partially reversed after washout (Wash; blue trace). b, This current (black trace) is abolished in a calcium-free bathing solution (red trace) but then fully recovers after washing in normal ACSF (Wash; blue trace). Inset, In current clamp, the reduction of this current in a calcium-free bath corresponds to a reduction in the medium AHPs and leads to an increased firing rate (red trace) relative to control (black trace). This effect is reversible after the wash-in of normal ACSF (blue trace). Thus, the outward current generated by this short pulse will be called the mAHP current. The current traces are averages of five trials.

Figure 3.

Reduction of the mAHP current by Ca_v2.2 channel blockade can induce burst discharge. a, One micromolar GVIA, which blocks Ca_v2.2 channels, reduces the mAHP current (gray trace) relative to control (black trace). Inset, The mean ± SEM value across cells of the mAHP current before (black boxes) and after (gray boxes) application of various calcium-current blockers: 10 μm dihydropyridines (dihyd), which block Ca_v1 channels; 1 μm GVIA; 1 μm MVIIC, which blocks Ca_v2.1/2.2 channels; and 100 nm SNX-482 (SNX), which blocks Ca_v2.3 channels. The current was measured 25 ms after the end of the pulse and averaged over five traces taken in a 20 s interval. Only the GVIA treatment elicited a significant reduction in the value of the mAHP current (^*p < 0.001; two-tailed paired t test; values of n indicate the number of cells). b, In current clamp, application of 1 μm GVIA reduced the mAHP after a 60 pA, 1 s somatic current injection (gray trace) relative to control (black trace), without affecting the sAHP that followed the termination of the pulse. c, Mean ± SD (calculated from 5 trials conducted in a 20 s interval) of the mAHP current measured from a cell that was bathed sequentially in (1) a combination of 1 μm GVIA and 100 nm of the Ca_v2.1 blocker ω-agatoxin TK (CnTx+AgTx), (2) 10 μm nimodipine, and finally in (3) normal ACSF (Wash). d, Traces of the spontaneous discharge of a neuron in control and after bath application of 1 μm GVIA, which caused it to burst.

Figure 4.

The calcium-dependent slow AHP current is selectively sensitive to dihydropyridine treatment. a, An 800 ms step to -2 mV from a holding potential of -57 mV elicits a large and long-lasting outward current (black trace) that is abolished in the calcium-free bath (light gray trace) and that partially recovers after wash-in (Wash) of normal ACSF (dark gray trace). The current traces are averages of five single trials measure during a 70 s interval. This sAHP current was measured as the temporal mean of the averaged current trace calculated between 975 and 1025 ms past the end of the pulse (arrow) to avoid contamination by the mAHP current. b, The sAHP current (black trace) is reduced by bath application of 10 μm nimodipine (light gray trace). This block was reversible after washout (Wash) of the drug (dark gray trace). Inset, The mean ± SEM value across cells of the sAHP current before and after application of the various calcium-current blockers (the same format as in Fig. 3a). Only the dihydropyridine (dihyd) treatment caused a significant reduction in this current (^*p < 0.005; two-tailed paired t test; values of n indicate the number of cells). c, In current clamp, application of 10 μm nifedipine reduced the sAHP after a 100 pA, 1 s somatic current injection (gray trace) relative to control (black trace). d, Mean ± SD of the sAHP current measured from a cell that was bathed sequentially in (1) 1 μm TTX, (2) a combination of 2 μm GVIA and 2 μm MVIIC (CnTx), (3) 100 nm SNX, (4) 20 μm nifedipine (Nif), and finally in (5) normal ACSF (Wash). SNX, SNX-482.

Figure 5.

Spontaneous sAHPs during apamin-induced bursting are dihydropyridine sensitive. a, Preincubation of the slice in apamin caused stereotyped burst discharge with prominent regenerative hyperpolarization. Addition of barium (plus 100 nm apamin) blocked the regenerative hyperpolarization but did not suppress the bursting. Subsequent addition of 10 μm nimodipine to the existing mixture dramatically reduced the sAHPs, thereby leading to variable lengths of bursts, each with a variable number of action potentials. b, Application of 10 μm nimodipine alone to a bursting cell preincubated in apamin also reduced the hyperpolarizations and lead to irregular single-spiking discharge.

Figure 6.

The role of intracellular stores in generating the sAHP current and the spontaneous discharge of cholinergic interneurons. a, The sAHP current, but not the early phase of the tail current, recorded in control (black trace) is reduced by bath application of 10 mm caffeine (light gray trace) and subsequently partially recovers after washing in normal ACSF (Wash; dark gray trace). Each trace is an average of five trials measured during an interval of 70 s. Inset, Time course of the mean ± SD of the sAHP current during caffeine treatment and the subsequent washing in of normal ACSF. b, The mean ± SEM value across cells of the sAHP current before (black boxes) and after (gray boxes) depleting intracellular stores with 10 mm caffeine (caff); 15 μm ryanodine (ryan), which selectively affects ryanodine-sensitive stores; or 1 μm XeC, which selectively blocks IP₃-mediated calcium release from stores. These drugs significantly reduced the sAHP current by 66, 36, and 35%, respectively (^*p < 0.05; two-tailed paired t test; values of n indicate the number of cells). c, Caffeine treatment causes a cholinergic interneuron displaying an irregular spontaneous discharge pattern that includes prominent hyperpolarizations to transition to rapid single-spiking discharge. This effect is reversed after washing out (Wash) the drug. Same neuron as that depicted in a.

Figure 7.

BK channels activated by calcium influx through Ca_v2.1 channels contribute to action potential repolarization. a, Action-potential waveform before (black trace) and after (gray trace) bath application of 1 μm MVIIC. Inset, The mean ± SEM across cells of the spike width before and after application of the various calcium-current blockers. Same format as in Figure 3a, with the additional treatment with 10 mm caffeine (caff). Only MVIIC generated a significant increase in the width of the action potential (^*p < 0.05; two-tailed Wilcoxon signed-ranks test; values of n indicate the number of cells). dihyd, Dihydropyridine; SNX, SNX-482. b, The effect of MVIIC was occluded by 1 mm TEA, indicating that the sole effect of blocking Ca_v2.1 channels is mediated by their contribution to the activation of BK channels. The control action-potential waveform was shifted in the hyperpolarizing direction to correct for a 6.5 mV offset.