Chronic sulpiride treatment mimics the haloperidol effect on DA neuron intrinsic pacemaker activity. Whole-cell current clamp recording from cultured DA neurons treated with vehicle (A) and sulpiride (B, chronic Sul) for 6 days. Spontaneous firing activity was recorded without current injection. Broken lines indicate -40 mV. C, D Interspike interval (ISI) histograms from 60 s of data from the cells shown in A and B, respectively. Mean pacemaker ISI (E) and coefficient of variation of interspike interval (CV_ISI, F) from cultured DA neurons (vehicle, n = 9; chronic Sul, n = 10). ^*p < 0.05.

Chronic blockade of D autoreceptors upregulates A-type K⁺ currents through cAMP-PKA signaling pathway in cultured DA neurons. A, 8-Br-cAMP (1 mM) prevents further upregulation of A-type K⁺ current density by sulpiride. B, The PKA inhibitor H-89 (1 μM) blocks the effect of sulpiride on A-type K⁺ current density. Transient currents density was obtained by the protocol described in Figure. 3B. ^*p < 0.05; NS, not significant.

Reversible recapitulation of the effect of chronic autoreceptor blockade on DA pacemaker activity by virtual A-type K⁺ channels. A, Pacemaker frequency is inversely proportional to A-type K⁺ current density. Spontaneous firing activity and K⁺ currents were recorded from the same DA neurons treated chronically with vehicle (○, n = 8) or sulpiride (•, n = 8). Frequency from each group was plotted against A-type current density from its corresponding group. B, Adding and then removing 150 nS of A-type K⁺ channel conductance with the dynamic clamp alters spontaneous firing activity of a cultured DA neuron (left panels). Spontaneous firing activity was recorded without current injection. Broken lines indicate -40 mV. Histograms of interspike interval (right panels) from 40 s of data for each condition. The mean interspike interval (C) and the coefficient of variation of interspike interval (D) from independent cultured DA neurons (n = 6). The augmentation of A-type K⁺ channel conductance with the dynamic clamp increased mean value of ISI and CV_ISI, respectively. ^*p < 0.05.

Chronic haloperidol treatment alters DA neuron intrinsic pacemaker activity in vitro. Whole-cell current clamp recordings from cultured DA neurons treated with vehicle (A) or haloperidol (B, chronic Hal) for 6 days. Spontaneous firing activity was recorded without current injection. Broken lines indicate -40 mV. C, D Interspike interval (ISI) histograms from 60 s of data from the cells shown in A and B, respectively. Mean ISI (E) and coefficient of variation of interspike interval (CV_ISI, F) from cultured DA neurons (vehicle, n = 4; chronic Hal, n = 4). ^*p < 0.05, ^**p < 0.01.

Chronic sulpiride treatment increases A-type K⁺ current in DA neurons. A, Left, Whole-cell current clamp recordings from cultured DA neurons treated with vehicle or sulpiride (chronic Sul) for 6 days. Spontaneous activity was recorded when current clamp was released from a holding potential of -80 mV. Right, Mean first spike latency (vehicle = 7; chronic Sul = 7) and interspike interval between first and second spikes (vehicle = 8; chronic Sul = 6). ^*p < 0.05. B, Left, Whole-cell voltage clamp recordings from DA neurons treated chronically with vehicle or sulpiride (chronic Sul). Transient currents were obtained by subtracting noninactivating currents (inset, right traces) from total outward currents (inset, left traces). Currents were evoked by voltage steps in 20 mV increments to -20 to +60 mV from a holding potential of -90 mV for total currents (insets: Horizontal bars equal 100 ms. Vertical bars equal 5 nA). Noninactivating currents were evoked with the same protocol following a 500 ms prepulse to -40 mV. Right, Mean current densities of transient K⁺ current at 60 mV in vehicle (n=26) and sulpiride treated DA neurons (n = 29). C, Mean current density of noninactivating K⁺ currents (I_D) at 60 mV is not altered by sulpiride (vehicle, n = 9; chronic Sul, n = 9). D, Mean transient K⁺ current density from cultured DA neurons treated chronically with vehicle (n = 9) or haloperidol (chronic Hal, n = 10). E, The D2 agonist quinpirole blocks the increase in A-type channel activity induced by the antagonist sulpiride. The right pair of bars shows that the effect of sulpiride seen in the left pair of bars is abolished in the presence of quinpirole. ^*p < 0.05; NS, not significant.

Upregulation of A-type K⁺ channel activity occurs without changing gating. A, Example steady state inactivation current traces. Prepulses were delivered in 20 mV steps from -140 mV and current was measured at +60 mV. The holding potential was -90 mV. B, Voltage dependence of activation and inactivation of A-type current in DA neurons treated chronically with vehicle or sulpiride. For activation curves, the peak amplitudes were measured from transient K⁺ currents generated by the same protocols described in Figure 3B. Steady state inactivation curves were generated by prepulsing to the indicated membrane potential for 0.5 s prior to the test pulse. Membrane conductances (g) at different voltage levels were obtained by dividing the peak transient K⁺ currents by the current driving force and normalized to conductance at +60 mV (g_max). The mean data from both groups were fitted with Boltzmann functions, which yielded half-activation potentials of -26.9 ± 1.6 mV for vehicle (○, n = 6) and -29.2 ± 1.0 mV for sulpiride (•, n = 7). Half-inactivation potentials were -91.1 ± 1.3 for vehicle (◇, n = 4) and -87.3 ± 1.8 mV for sulpiride (◆, n = 4). C, Recovery from inactivation with a double pulse protocol. Voltage was stepped to +60 mV with varying interpulse intervals from a holding potential of -90 mV. D, Mean data from vehicle (○, n = 3) and sulpiride (•, n = 5) treated DA neurons. E, Time to peak for vehicle (open bar, n = 15) and sulpiride (filled bar, n = 15) treated DA neurons at +60 mV. F, Inactivation time constant at 60 mV for vehicle (open bar, n = 15) and sulpiride (filled bar, n = 15) treated DA neurons.

A-type channels control the rate and regularity of pacemaker activity in perforated patch clamped dopamine neurons. A, Action potential activity before (left) and after (right) adding 200 nS of virtual A-type channel conductance. Change in mean ISI (B) and CV_ISI (C) induced by adding virtual A-type channels (n = 6). D, Action potential activity before (left) and after (right) subtracting 200 nS of virtual A-type channel conductance. Change in mean ISI (D) and CV_ISI (E) induced by subtracting virtual A-type channels (n = 6). Dashed lines indicate -40 mV. ^*p < 0.05. Wilcoxon signed rank test was used in B because data was not normally distributed.