The Cav3-Kv4 complex acts as a calcium sensor to maintain inhibitory charge transfer during extracellular calcium fluctuations

Abstract

Synaptic transmission and neuronal excitability depend on the concentration of extracellular calcium ([Ca](o)), yet repetitive synaptic input is known to decrease [Ca](o) in numerous brain regions. In the cerebellar molecular layer, synaptic input reduces [Ca](o) by up to 0.4 mm in the vicinity of stellate cell interneurons and Purkinje cell dendrites. The mechanisms used to maintain network excitability and Purkinje cell output in the face of this rapid change in calcium gradient have remained an enigma. Here we use single and dual patch recordings in an in vitro slice preparation of Sprague Dawley rats to investigate the effects of physiological decreases in [Ca](o) on the excitability of cerebellar stellate cells and their inhibitory regulation of Purkinje cells. We find that a Ca(v)3-K(v)4 ion channel complex expressed in stellate cells acts as a calcium sensor that responds to a decrease in [Ca]o by dynamically adjusting stellate cell output to maintain inhibitory charge transfer to Purkinje cells. The Ca(v)3-K(v)4 complex thus enables an adaptive regulation of inhibitory input to Purkinje cells during fluctuations in [Ca](o), providing a homeostatic control mechanism to regulate Purkinje cell excitability during repetitive afferent activity.

Figure 1.

A physiologically relevant decrease of [Ca]_o shifts the V_h of I_A. A, Inactivation plots of stellate cell I_A recorded in 1.5 or 0.1 mm [Ca]_o from a holding potential of −110 mV, followed by a series of preconditioning voltages of 10 mV steps to 0 mV, and current amplitude tested with a test pulse to −30 mV (see inset). B, The dose–response curve of the relationship between [Ca]_o and mean I_A V_h in stellate cells. Voltage commands are as in A. C, Dose–response curve between [Ca]_o and K_v4.2 V_h expressed in tsA-201 cells for a Ca_v3–K_v4 complex in the presence or absence of expressed KChIP3 protein. Voltage commands are as in A. D, The effects of varying [Ca]_o on the density of I_A and I_T in two different stellate cells. I_A was first evoked by a step from −80 to −45 mV in 1.5 or 1.1 mm [Ca]_o, and I_T was then isolated in the same cells by perfusion of 20 mm 4-AP. On the right are plots of the mean densities of I_A and I_T as a function of [Ca]_o. Current densities are averages of 10 traces and normalized to membrane capacitance.

Figure 2.

Repetitive 10 Hz climbing fiber stimulation evokes a decrease in stellate cell I_T. A, Dual recordings of I_T in a stellate cell (SC) and spike discharge in a Purkinje cell (PC) soma recorded in the cell layer directly below the SC. Synaptic responses are pharmacologically blocked in the SC to allow activation of complex spikes in the Purkinje cell by climbing fiber (CF; arrows) stimulation. I_T is evoked by a step from −110 to −45 mV (5 Hz), and CF stimuli are interleaved with the step commands using a 10 pulse 10 Hz CF stimulus. Recordings are shown for control (left), every second CF stimulus during the 10 pulse train (middle), and 1.0 s after the end of the train (Recovery; right). The dashed line below recordings marks the control I_T amplitude for comparison. B, Mean values for I_T amplitude normalized and color coded with respect to the mean of control pulses preceding CF stimulation (n = 6). The period of 10 Hz CF stimulation is shown bounded by a box. C, Expanded and superimposed representative records of I_T in control, immediately post CF stimulation, and recovery 1.0 s after the stimulus train, as for experiments in A and B. D, Bar plots of the mean change in I_T amplitude immediately following CF stimulation (Post CF) and 1.0 s following the end of the stimulus train. For display purposes, stimulus and capacitative artifacts are digitally reduced in A and C and a slight shift in baseline inward current during CF stimulation is removed from I_T recordings in A. Sample values are shown in brackets within the bar plot. *p < 0.05.

Figure 3.

Repetitive 10 Hz climbing fiber stimulation evokes a decrease in stellate cell I_A. A, Dual recordings of I_A in a stellate cell (SC) and spike discharge in a Purkinje cell (PC) soma recorded in the cell layer directly below. Synaptic responses are pharmacologically blocked in the SC to allow selective activation of complex spikes in the Purkinje cell through climbing fiber (CF; arrows) stimulation. I_A is evoked by a step from −85 to −30 mV (5 Hz), and CF stimuli are interleaved with the step commands using a 10 pulse, 10 Hz CF stimulus. Recordings are shown for control (left), every second CF stimulus during the 10 pulse train (middle), and after 2.4 s recovery (right). The dashed line above recordings marks the control I_A amplitude for comparison. B, Mean values for I_A amplitude normalized and color coded with respect to the mean of six control pulses preceding CF stimulation when recorded in 1.5 mm [Ca]_o (n = 10) or 2.2 mm [Ca]_o (n = 4). The period of 10 Hz CF stimulation is shown bounded by a box. C, Expanded and superimposed representative records of I_A in 1.5 or 2.2 mm [Ca]_o in control, during CF stimulation, and recovery 2.4 s after the stimulus train, as for experiments in A and B. D, Bar plots of the mean change in I_A amplitude immediately following the CF stimulus train in 1.5 or 2.2 mm [Ca]_o. Note the rapid decrease in peak I_A during CF stimulation in 1.5 mm [Ca]_o and the lack of effect in 2.2 mm [Ca]_o to offset stimulus-induced changes in [Ca]_o. For display purposes, recordings are truncated in A. Stimulus and capacitative artifacts were digitally reduced in A and C. Sample values are shown in brackets within bar plots. ***p < 0.001.

Figure 4.

Low [Ca]_o increases the gain and lowers the threshold of stellate cell firing. A, B, Lowering [Ca]_o from 1.5 to 1.1 mm significantly increases the frequency of spontaneous stellate cell spike discharge in on-cell recordings and gain of intracellular spike discharge evoked by square wave current pulses to calculate current–frequency plots in B. C, Bar plots of the effect of 1.1 mm [Ca]_o on gain of firing and its occlusion by pretreatment with mibefradil (Mibef; 500 nm) or infusion of an antibody against KChIP3 (1:100) through the patch electrode, but not by an antibody against KChIP1. D, Recordings and scatter plot reveal a significant decrease in the absolute voltage threshold for spike discharge (dashed lines) in 1.1 mm [Ca]_o. Sample values are shown in brackets. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5.

Simulating a selective shift in I_A voltage for inactivation through dynamic clamp. A, The stellate cell I_A V_h [Ca]_o dose–response curve (top) and shift in I_A h_∞ for 1.5 or 1.1 mm [Ca]_o (bottom). The dashed lines (top) represent the calculated V_h for I_A in the presence of 1.5 mm [Ca]_o (blue) or 1.1 mm [Ca]_o (green; Fig. 1B). Fits of the mean inactivation curves of stellate cell I_A (bottom) for 1.5 and 1.1 mm [Ca]_o are used to iteratively calculate the h_∞ values and difference currents for I_A (I_diff). Dashed lines indicate the h_∞ values calculated for either condition at −70 mV. I_diff is then added or subtracted to simulate conditions of 1.5 or 1.1 mm [Ca]_o in real time during the spike (25 μs sampling interval). B, C, Recordings of single spike responses (top row) performed in the presence of 1.5 mm [Ca]_o or 1.1 mm [Ca]_o, but modulated through dynamic clamp according to the predicted availability of I_A at the alternate [Ca]_o. The second row represents the I_A predicted for the [Ca]_o indicated, the third row the I_A predicted to be available at the alternate [Ca]_o, and the fourth row the difference current calculated to dynamically add or subtract to simulate the alternate [Ca²⁺]_o condition. The I_diff was inward in B and had a depolarizing effect on membrane potential, and outward in C with a hyperpolarizing effect on membrane potential.

Figure 6.

Dynamic subtraction or addition of I_A modulates the gain and threshold of stellate cell firing. A, B, A comparison of the effects of recording spike output in a stellate cell maintained in 1.5 mm [Ca]_o and then subtracting I_A to simulate a shift to 1.1 mm [Ca]_o (A), and the same cell now perfused with 1.1 mm [Ca]_o before (black) and after dynamic clamp injection of I_A (red) according to the predicted availability of I_A in 1.5 mm [Ca]_o (B). Representative traces of current-evoked spike output and the associated current–frequency plots are shown. C, Plots of the average effect of either dynamically subtracting I_A in cells perfused with 1.5 mm [Ca]_o (left column) or dynamically adding I_A to cells perfused in 1.1 mm [Ca]_o (right column) on the gain of firing frequency and spike voltage threshold. Lines on current–frequency plots represent best-fit calculations to the data, and sample values are shown at the base of bar plots. *p < 0.05; **p < 0.01.

Figure 7.

Inhibitory charge transfer of sIPSCs to Purkinje cells is maintained in low [Ca]_o. A, mIPSCs recorded from a Purkinje cell in the presence of 200 nm TTX to block presynaptic spike discharge in 1.5 and 1.1 mm [Ca]_o. B, Bar plots of average mIPSC frequency reveal a significant decrease in mIPSC frequency in low [Ca]_o. C, sIPSCs recorded from a Purkinje cell in 1.5 mm [Ca]_o and 1.1 mm [Ca]_o. The internal electrolyte was a CsCl-based solution, and DNQX and dl-AP5 were bath applied to block excitatory synaptic transmission. Lowering [Ca]_o to 1.1 mm increases the amplitude of sIPSCs, while subsequent perfusion of the GABA_A blocker picrotoxin (PTX; 50 μm) abolishes all sIPSCs. D, A cumulative distribution function of sIPSC amplitudes indicating an increase in the proportion of large-amplitude sIPSCs with application of 1.1 mm [Ca]_o compared to 1.5 mm [Ca]_o. E, Mean bar plot of sIPSC frequency measured in 1.5 and 1.1 mm [Ca]_o and after perfusion of picrotoxin indicate that sIPSC frequency is maintained in low [Ca]_o and significantly decreases in the presence of picrotoxin. F, Bar plots of the total charge transfer in Purkinje cell recordings in the presence of 1.5 or 1.1 mm [Ca]_o and after perfusion of picrotoxin. Sample values are shown within bar plots. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8.

Inhibitory charge transfer of evoked IPSCs to Purkinje cells (PCs) is maintained in low [Ca]_o. Dual recordings of stellate (SC) and Purkinje cells were used to record presynaptic spike discharge and postsynaptic IPSCs in 1.5 or 1.1 mm [Ca]_o in the presence of synaptic blockers. Stellate cells were held below threshold and presynaptic spike discharge evoked using 20 1 s current pulses while holding Purkinje cells at −65 mV to record inward IPSCs. A, Recordings from a synaptically connected SCs and PCs. Dashed lines indicate stellate cell spikes giving rise to eIPSCs. B, Superimposed spike-triggered averages of IPSCs and bar plots of eIPSC amplitudes in 1.5 or 1.1 mm [Ca]_o. Top traces include only eIPSCs directly associated with presynaptic spikes, while lower traces are all responses including transmission failures. SEM is indicated by shaded regions. C–E, Bar plots of spike and eIPSC properties from identified stellate–Purkinje cell pairs in 1.5 and 1.1 mm [Ca]_o. Reducing [Ca]_o from 1.5 to 1.1 mm significantly increases eIPSC failure rate in C and increases current-evoked stellate cell spike count in D. E, Mean values for spike train charge transfer from stellate to Purkinje cells in 1.5 and 1.1 mm [Ca]_o, calculated as the product of spike count in D and fits (inset) to the mean eIPSCs shown in lower records of B. Charge transfer in 1.1 mm [Ca]_o was calculated for both the actual spike count and for a hypothetical situation (1.1 hypoth) where spike count did not change from that recorded in 1.5 mm [Ca]_o. F, Bar plots of Purkinje cell spontaneous firing rates during on-cell recordings. The rate of Purkinje cell firing is not significantly different during perfusion of 1.5 or 1.1 mm [Ca]_o. Addition of picrotoxin (50 μm) reveals that the level of Purkinje cell excitability would be significantly higher in 1.1 mm [Ca]_o without the associated increase in GABAergic inhibition in the network. Sample values are shown in brackets within bar plots. *p < 0.05; **p < 0.01.

Figure 9.

Diagrammatic representations of the sequence of events proposed for climbing fiber (CF)-induced reductions in [Ca]_o, stellate cell ionic currents, and adaptive inhibitory control of Purkinje cell (PC) excitability. A, A text-based sequence of events related to CF-induced shifts in [Ca]_o and I_T and I_A in stellate cells. B, A schematic diagram and flow chart of the entire sequence of events underlying adaptive inhibition by stellate cells to maintain a homeostatic balance of inhibitory charge transfer (tone) on Purkinje cells during repetitive CF inputs. Text in green indicates CF-induced changes in [Ca]_o and I_T, and text in red indicates events that lead to shifts in I_A availability in stellate cells. The center circle indicates the change in amplitude and pattern of GABAergic IPSPs in Purkinje cells. Under control conditions, I_A availability is high and stellate cell excitability is low (low gain). Repetitive CF input reduces [Ca]_o and I_T, leading to a decrease in I_A availability through the Ca_v3–K_v4 complex (V_h shifts left). An associated increase in stellate cell firing (high gain) compensates for a reduction in release probability (P_r) when [Ca]_o decreases. When CF input stops, [Ca]_o and I_A V_h return to normal, and stellate cell excitability is reduced when I_A availability increases by way of the Ca_v3–K_v4 complex.