Two Distinct Sets of Ca2+ and K+ Channels Are Activated at Different Membrane Potentials by the Climbing Fiber Synaptic Potential in Purkinje Neuron Dendrites

Abstract

In cerebellar Purkinje neuron dendrites, the transient depolarization associated with a climbing fiber (CF) EPSP activates voltage-gated Ca²⁺ channels (VGCCs), voltage-gated K⁺ channels (VGKCs), and Ca²⁺-activated SK and BK K⁺ channels. The resulting membrane potential (V_m) and Ca²⁺ transients play a fundamental role in dendritic integration and synaptic plasticity of parallel fiber inputs. Here we report a detailed investigation of the kinetics of dendritic Ca²⁺ and K⁺ channels activated by CF-EPSPs, based on optical measurements of V_m and Ca²⁺ transients and on a single-compartment NEURON model reproducing experimental data. We first measured V_m and Ca²⁺ transients associated with CF-EPSPs at different initial V_m, and we analyzed the changes in the Ca²⁺ transients produced by the block of each individual VGCCs, of A-type VGKCs and of SK and BK channels. Then, we constructed a model that includes six active ion channels to accurately match experimental signals and extract the physiological kinetics of each channel. We found that two different sets of channels are selectively activated. When the dendrite is hyperpolarized, CF-EPSPs mainly activate T-type VGCCs, SK channels, and A-type VGKCs that limit the transient V_m ∼ <0 mV. In contrast, when the dendrite is depolarized, T-type VGCCs and A-type VGKCs are inactivated and CF-EPSPs activate P/Q-type VGCCs, high-voltage activated VGKCs, and BK channels, leading to Ca²⁺ spikes. Thus, the potentially activity-dependent regulation of A-type VGKCs, controlling the activation of this second set of channels, is likely to play a crucial role in signal integration and plasticity in Purkinje neuron dendrites.SIGNIFICANCE STATEMENT The climbing fiber synaptic input transiently depolarizes the dendrite of cerebellar Purkinje neurons generating a signal that plays a fundamental role in dendritic integration. This signal is mediated by two types of Ca²⁺ channels and four types of K⁺ channels. Thus, understanding the kinetics of all of these channels is crucial for understanding PN function. To obtain this information, we used an innovative strategy that merges ultrafast optical membrane potential and Ca²⁺ measurements, pharmacological analysis, and computational modeling. We found that, according to the initial membrane potential, the climbing fiber depolarizing transient activates two distinct sets of channels. Moreover, A-type K⁺ channels limit the activation of P/Q-type Ca²⁺ channels and associated K⁺ channels, thus preventing the generation of Ca²⁺ spikes.

Keywords: calcium channels; cerebellar Purkinje neuron; climbing fiber; neuron modeling; neuronal dendrites; potassium channels.

Figure 1.

V_m calibration protocol and illustration of NEURON model. A, Left, Fluorescence reconstruction of a PN with three ROIs (R1, R2, and R3). From the resting V_m (−67 mV), negative or positive current pulses of 1 s duration were delivered from the recording electrode. Right, Somatic V_m and the VSD-ΔF/F₀ signals in R1-R3 associated with the current pulses. The VSD-ΔF/F₀ signal in each region is converted into millivolts to quantify the V_m transient associated with the CF-EPSP, assuming that the resting V_m is uniform (int state) and that the hyperpolarizing pulse spreads into the dendrites without attenuation (hyp state). The protocol also allows determining the dendritic V_m associated with the strongest depolarizing pulse (dep state). B, A dendritic region of ∼17 × 17 μm² is approximated with a cylinder of 4 μm diameter and 20 μm length in the NEURON model. The model contains P/Q-type and T-type Ca²⁺ channels; SK, BK, A-type K⁺ channels; and a generic HVAK. It includes four buffers: a fast immobile buffer, the Ca²⁺ indicator (either Fura-FF or OG5N), Parvalbumin, and Calbindin D-28k. It also includes Ca²⁺ extrusion and a LEAK channel.

Figure 2.

Combined V_m and Ca²⁺ transients associated with the CF-EPSP. A, Bottom, Fluorescence reconstruction of a representative PN with two ROIs indicated (R1 and R2). Top, Somatic V_m associated with a CF-EPSP at three different initial V_m: hyperpolarized (hyp blue trace); intermediate (int green trace); and depolarized (dep red trace). B, Top, Dendritic V_m in R1 and R2 calibrated as illustrated in Figure 1A corresponding to the somatic CF-EPSPs in A. Bottom, Corresponding FuraFF ΔF/F₀ signals. C, Analysis of the V_m and Ca²⁺ maxima (max) associated with signals in R1 reported in B; the first max of the V_m and Ca²⁺ transients is calculated within the first 4 ms after the CF stimulation; the second max of the V_m and Ca²⁺ transients is calculated between 4 and 14 ms after the CF stimulation. Blue traces represent the hyp state. Green traces represent the int state. Red traces represent the dep state. D, Mean ± SD for 19 regions in 12 cells analyzed as illustrated in C. The hyp states (blue columns) were with initial V_m between −87 mV and −74 mV. The int states (green columns) were with initial V_m between −68 mV and −61 mV. The dep states (red columns) were with initial V_m between −54 and −46 mV. *Significant increase in the max (p < 0.005, paired t test).

Figure 3.

Dendritic Ca²⁺ channels activated by the CF-EPSP. A, Left, Fluorescence reconstruction of a PN with two ROIs indicated (R1 and R2): R1 is next to a pipette delivering 1 μm of the P/Q-type VGCC inhibitor AgaIVA; R2 is >50 μm from the application pipette. Right, Top, Somatic V_m associated with CF-EPSPs in control conditions and after local application of AgaIVA at three different initial V_m: hyp (blue trace); int (green trace); and dep (red trace). Right, Bottom, The corresponding OG5N ΔF/F₀ signals. B, In another PN, same as A, but with the pipette delivering the T-type VGCC inhibitors ML (5 μm) and NNC (30 μm). C, In another PN, same as A, but with the pipette delivering both the P/Q VGCC inhibitor AgaIVA (1μm) and the T-type VGCC inhibitors ML (5 μm) and NNC (30 μm). D, In three other PNs, from a region next to a pipette delivering a VGCC blocker, the OG5N ΔF/F₀ signals associated with CF-EPSPs in control conditions and after local application of 20 μm of the L-type VGCC inhibitor Isr, of 5 μm of the N-type VGCC inhibitor PD or 1 μm of the R-type VGCC inhibitor SNX at the three different initial V_m.

Figure 4.

Quantitative analysis of dendritic Ca²⁺ channels activated by the CF-EPSP. Left, From two representative cells, OG5N ΔF/F₀ signals associated with CF-EPSPs in control conditions at three different initial V_m: hyp (blue trace); int (green trace); and dep (red trace); superimposed (gray traces) are the OG5N ΔF/F₀ signals after local application of either 1 μm of the P/Q-type VGCC inhibitor AgaIVA or of the T-type VGCC blockers ML (5 μm) and NNC (30 μm); the first max of the Ca²⁺ transient is calculated within the first 4 ms after the CF stimulation; the second max of the Ca²⁺ transient is calculated between 4 and 14 ms after the CF stimulation; the percentages from control ΔF/F₀ maxima after application of the VGCC blockers are reported above or below the arrows. Right, Mean ± SD of the percentages from control ΔF/F₀ maxima after application of the VGCC inhibitors AgaIVA (N = 6 cells), ML+NNC (N = 6 cells), AgaIVA+ML+NNC (N = 6 cells), Isr (N = 4 cells), PD (N = 4 cells), or SNX (N = 4 cells). Gray columns represent the statistics of the first max. White columns represent the statistics of the second max. *Significant change in the max (p < 0.005, paired t test).

Figure 5.

Dendritic A-type VGKCs activated by the CF-EPSP. A, Left, Fluorescence reconstruction of a PN with an ROI indicated next to a pipette delivering 1 μm of the A-type VGKC inhibitor AmmTx3. Right, Top, Somatic V_m associated with CF-EPSPs in control conditions and after local application of AmmTx3 at three different initial V_m: hyp (blue trace); int (green trace); and dep (red trace). Right, Bottom, The corresponding OG5N ΔF/F₀ signals. B, Left, From the cell in A, OG5N ΔF/F₀ signals associated with CF-EPSPs in control conditions at the three different initial V_m; superimposed (gray traces) are the OG5N ΔF/F₀ signals after local application of either 1 μm AmmTx3; the first max of the Ca²⁺ transient is calculated within the first 4 ms after the CF stimulation; the percentages from control ΔF/F₀ maxima after application of the VGCC blockers are reported above the arrows. Right, Mean ± SD of the percentages from control ΔF/F₀ maxima after application of AmmTx3 (N = 6 cells). Gray columns represent the statistics of the first max. White columns represent the statistics of the second max. *Significant change in the max (p < 0.005, paired t test).

Figure 6.

Dendritic Ca²⁺-activated K⁺ channels activated by the CF-EPSP. A, Left, Fluorescence reconstruction of a PN with an ROI indicated next to a pipette delivering 1 μm of the BK channel inhibitor iberiotoxin. Right, Top, Somatic V_m associated with CF-EPSPs in control conditions and after local application of iberiotoxin at three different initial V_m: hyp (blue trace); int (green trace); and dep (red trace). Right, Bottom, The corresponding OG5N ΔF/F₀ signals. B, In another PN, same as A, but with the pipette delivering 1 μm of the SK channel inhibitor apamin. C, Mean ± SD of the percentages from control ΔF/F₀ maxima after application of iberiotoxin (N = 5 cells) or apamin (N = 6 cells). Gray columns represent the statistics of the first max. White columns represent the statistics of the second max. D, Left, From the cell in A, OG5N ΔF/F₀ signal associated with the CF-EPSP at dep state in control condition and after addition of iberiotoxin (gray trace). Right, From the cell in B, OG5N ΔF/F₀ signal associated with the CF-EPSP at hyp state in control condition and after addition of apamin (gray trace).

Figure 7.

NEURON model of 4 PN dendritic compartments reproducing V_m and Ca²⁺ transients associated with the CF-EPSP. Left, Experimental dendritic V_m and Ca²⁺ transients associated with CF-EPSPs from 4 selected cells at three different initial V_m: hyperpolarized (hyp, blue trace); intermediate (int, green trace); depolarized (dep, red trace). Right, Simulations of dendritic V_m and Ca²⁺ transients associated with CF-EPSPs reproducing experimental data (gray traces).

Figure 8.

Simulations of block of P/Q-type VGCCs, T-type VGCCs, A-type VGKCs, BK and SK Ca²⁺-activated K⁺ channels from a NEURON model. Simulated Ca²⁺ transients (OG5N) associated with CF-EPSPs at three different initial V_m in control condition and after reduction of 90% of one individual channels from Cell 1 model variant reported in Figure 7. For each case of 90% channel reduction, traces under control conditions are reported in gray.

Figure 9.

Individual currents extracted from the NEURON model. Ca²⁺ currents of P/Q and T channels and K⁺ currents of A, BK, SK, and HVAK channels from the simulations of Cell 1 model variant reported in Figure 7. Simulations were at hyp (blue traces), int (green traces), and dep (red traces) states in control conditions, and at hyp (purple traces) after blocking 90% of A-type VGKCs, superimposed to currents in control conditions (gray traces).

Figure 10.

Channel activation following CF-EPSPs at hyperpolarized and depolarized states. A, In control conditions, at hyp state, the CF-EPSP activates T-type channels, which activate SK channels, and A channels, which limit activation P/Q and HVAK channels; at dep state, the CF-EPSP activates P/Q-type channels, which activate BK channels, and HVAK channels, while T channels and A channels are inactivated. B, When A channels are blocked or inactivated, at hyp state, the CF-EPSP also activates P/Q-type channels, which activate BK channels, and HVAK channels.