Distinct Actions of Voltage-Activated Ca2+ Channel Block on Spontaneous Release at Excitatory and Inhibitory Central Synapses

Abstract

At chemical synapses, voltage-activated calcium channels (VACCs) mediate Ca²⁺ influx to trigger action potential-evoked neurotransmitter release. However, the mechanisms by which Ca²⁺ regulates spontaneous transmission have not been fully determined. We have shown that VACCs are a major trigger of spontaneous release at neocortical inhibitory synapses but not at excitatory synapses, suggesting fundamental differences in spontaneous neurotransmission at GABAergic and glutamatergic synapses. Recently, VACC blockers were reported to reduce spontaneous release of glutamate and it was proposed that there was conservation of underlying mechanisms of neurotransmission at excitatory and inhibitory synapses. Furthermore, it was hypothesized that the different effects on excitatory and inhibitory synapses may have resulted from off-target actions of Cd²⁺, a nonselective VACC blocker, or other variations in experimental conditions. Here we report that in mouse neocortical neurons, selective and nonselective VACC blockers inhibit spontaneous release at inhibitory but not at excitatory terminals, and that this pattern is observed in culture and slice preparations as well as in synapses from acute slices of the auditory brainstem. The voltage dependence of Cd²⁺ block of VACCs accounts for the apparent lower potency of Cd²⁺ on spontaneous release of GABA than on VACC current amplitudes. Our findings indicate fundamental differences in the regulation of spontaneous release at inhibitory and excitatory synapses by stochastic VACC activity that extend beyond the cortex to the brainstem.SIGNIFICANCE STATEMENT Presynaptic Ca²⁺ entry via voltage-activated calcium channels (VACCs) is the major trigger of action potential-evoked synaptic release. However, the role of VACCs in the regulation of spontaneous neurotransmitter release (in the absence of a synchronizing action potential) remains controversial. We show that spontaneous release is affected differently by VACCs at excitatory and inhibitory synapses. At inhibitory synapses, stochastic openings of VACCs trigger the majority of spontaneous release, whereas they do not affect spontaneous release at excitatory synapses. We find this pattern to be wide ranging, holding for large and small synapses in the neocortex and brainstem. These findings indicate fundamental differences of the Ca²⁺ dependence of spontaneous release at excitatory and inhibitory synapses and heterogeneity of the mechanisms of release across the CNS.

Keywords: VGCC; calcium channel; mEPSC; mIPSC; minis; spontaneous.

Figure 1.

Cd²⁺ reduces spontaneous release of GABA but not glutamate in acute neocortical slices. A, Exemplary current traces showing mEPSCs (red) before (top trace) and during (middle trace) application of 100 μm Cd²⁺. Calibration: 20 pA, 100 ms. The bottom trace shows superimposed average mEPSCs after normalization for amplitude. Calibration: 5 ms. B, Histogram of average mEPSC amplitude in control conditions and in the last 2 min of Cd²⁺ application. Open circles linked with lines represent average mEPSC amplitudes from individual experiments (n = 6). C, Exemplary current traces showing mIPSCs (blue) before (top trace) and during (middle trace) application of 100 μm Cd²⁺. Calibration: 60 pA, 100 ms. The bottom trace shows superimposed average mIPSCs after normalization for amplitude. Calibration: 5 ms. D, Histogram of average mIPSC amplitude in control conditions and in the last 120 s of Cd²⁺ application (n = 6). E, F, Exemplary (open circles) and normalized average (closed circles) diary plots showing the effect of 100 μm Cd²⁺ (bar and dotted lines) on mEPSC (red) and mIPSC (blue) frequency (mean ± SEM) versus time. Average effects measured over the last 5 min of drug application relative to basal frequency (averaged over the 5 min before application) are shown in this figure and diary plots in Figures 4 and 5.

Figure 2.

Cd²⁺ apparently blocks VACC currents more potently than it inhibits spontaneous release of GABA. A, VACC currents activated by steps from −70 to −10 mV were substantially reduced by 1 μm (red) or 100 μm Cd²⁺ (blue) B, Average normalized diary plot of VACC current amplitude versus time following application of 0, 1, 3, 10, 30, 100, and 300 μm Cd²⁺ (n = 12). The bath contained 1.1 mm Mg²⁺ and 1.1 mm Ca²⁺, and micromolar [Cd²⁺] is indicated by the logarithmic scale above the plot. Currents were activated every 10 s, and each cell was normalized according to the average current recorded over the first 60 s. C, Concentration–effect relationship for normalized VACC currents recorded at steady-state [Cd²⁺] as indicated in B. D, Current traces showing mIPSCs before and during 30 μm Cd²⁺ (red). E, Average normalized diary plot of mIPSC frequency versus time following application of 0, 1, 3, 10, 30, 100, and 300 μm Cd²⁺ (n = 10). The bath contained 1.1 mm Mg²⁺ and 1.1 mm Ca²⁺, and micromolar [Cd²⁺] is indicated by the logarithmic scale above the plot. Each cell was normalized according to the average mIPSC frequency recorded over the first 200 s. F, Concentration–effect relationship for normalized mIPSC frequency recorded at steady-state [Cd²⁺] as indicated by the logarithmic scale above the plot in E.

Figure 3.

Voltage dependence of Cd²⁺ block of VACC currents. A, Exemplary traces of 100 μm Cd²⁺-subtracted currents recorded at −40, −20, and −10 mV voltage-step protocols under control conditions (black) and in 1 μm Cd²⁺ (red). Notice the decrease in block at more hyperpolarized steps. B, I–V curve for VACC currents measured over the last 2 ms of the 10 ms voltage step (I_8–10) from the same experiment in A. C, Plot of the peak amplitude of the tail currents from the same experiment versus the voltage step from the same experiment in A. Tail currents recorded in control conditions are indicated in black, and those recorded in the presence of 1 μm Cd²⁺ are indicated in blue. Tail current amplitude is measured as the minimum occurring within 200 μs after the voltage step. D, Plot of VACC current amplitude (red, I_8–10) and tail current amplitude (blue, I_Tail) in 1 μm Cd²⁺ divided by that measured in control conditions and plotted against membrane potential (n = 4). Note the reduced block by Cd²⁺ at more hyperpolarized potentials and the increase in variability of the steady-state current at positive potentials. E, Exemplary traces of currents recorded in control conditions (black) and in 1 μm Cd²⁺ (red) with prepulse and postpulse currents (S1 and S2, respectively) recorded at −10 mV, where the current amplitude was maximal and the middle pulse was 130 mV. The middle pulse was increased in duration from 0 to 15 ms. Note the presence of the Cd²⁺-sensitive outward current resulting from the middle pulse. F, Plot of the ratio of the peak amplitude of the current measured (S_x) to the peak amplitude of the current measured for S1 under control conditions versus the duration of the middle pulse (n = 3). S_x described S2 under control conditions (black) or in the presence of 1 μm Cd²⁺ at either S1 (blue) or S2 (red). Error bars indicate SEM.

Figure 4.

MVIIC reduces spontaneous release of GABA but not glutamate in acute neocortical slices. A, Exemplary current traces showing mEPSCs (red) before (top trace) and during (middle trace) application of 5 μm MVIIC. Calibration: 100 ms. The bottom trace shows superimposed average mEPSCs after normalization for amplitude. Calibration: 5 ms. B, Histogram of average mEPSC amplitudes in control conditions and in the last 120 s of MVIIC application. Open circles linked with lines represent average mEPSC amplitudes from individual experiments (n = 5). C, Exemplary current traces showing mIPSCs (blue) before (top trace) and during (middle trace) application of 5 μm MVIIC. Calibration: 100 ms. The bottom trace shows superimposed average mIPSCs after normalization for amplitude. Calibration: 5 ms. D, Histogram of average mIPSC amplitudes in control conditions and in the last 120 s of MVIIC application (n = 6). E, F, Exemplary (open circles) and normalized average (closed circles) diary plots showing the effect of 5 μm MVIIC (bar and dotted lines) on mEPSC (red) and mIPSC (blue) frequency (mean ± SEM) versus time.

Figure 5.

Cd²⁺ reduces spontaneous release of GABA and glycine but not glutamate in acute auditory brainstem slices. A, Exemplary current traces showing mEPSCs (red) before (top trace) and during (middle trace) application of 50 μm Cd²⁺. Asterisks denote individual release events. The bottom trace shows superimposed average mEPSCs after normalization for amplitude. B, Exemplary current traces showing mIPSCs (blue) before (top trace) and during (middle trace) application of 50 μm Cd²⁺. Note simultaneously recorded mEPSCs denoted in red. The bottom trace shows superimposed average mIPSCs after normalization for amplitude. C, D, Exemplary (open circles) and normalized average (closed circles) diary plots showing the effect of 50 μm Cd²⁺ (bar and dotted lines) on mEPSC (red) and mIPSC (blue) frequency (mean ± SEM) versus time. Calibrations: A, B, Top, middle traces, 20 pA, 100 ms; bottom traces, 5 ms.

Figure 6.

An alternative external solution does not affect resistance of mEPSC frequency to Cd²⁺. A, Exemplary current traces show mEPSCs recorded before and after the application of 100 μm Cd²⁺ in control conditions and in an alternative extracellular solution that contained lower Na⁺ concentration and higher glucose and HEPES concentrations. B, Histogram showing the average effect of Cd²⁺ in control and alternative solutions (n = 6). Average mEPSC frequency was measured over the 200 s before Cd²⁺ application and used to normalize each recording. The average mEPSC frequencies represent steady-state data collected over a 100 s epoch once the test solutions had been applied for 100 s.