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 Ca influx to trigger action potential-evoked neurotransmitter release. However, the mechanisms by which Ca 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 Cd, 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 Cd block of VACCs accounts for the apparent lower potency of Cd 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. Presynaptic Ca 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 Ca dependence of spontaneous release at excitatory and inhibitory synapses and heterogeneity of the mechanisms of release across the CNS.

Calcium dependence of spontaneous neurotransmitter release

“…Additionally, laboratories that detected VACC regulation of mEPSC frequency at small central neurons used external solutions with higher K + , Ca 2+ , glucose, and buffer concentrations (Ermolyuk et al, ; Xu et al, ). However, switching to solutions with higher K + , Ca 2+ , glucose, and buffer concentrations did not change the sensitivity of mEPSCs to VACC blockers, indicating that any relative depolarization must have arisen for other reasons (Tsintsadze et al, ). It is possible that the preparations were developmentally distinct.…”

“…In cultured hippocampal neurons from neonatal rats, Cd 2+ , a nonselective VACC blocker, did not affect mEPSC frequency (Abenavoli et al, ). In cultured mouse neurons from the neocortex, VACC block with Cd 2+ or MVIIC did not reduce mEPSC frequency (Tsintsadze, Williams, Weingarten, von Gersdorff, & Smith, ; Vyleta & Smith, ). In hippocampal, neocortical, cerebellar, and brainstem slices from mice, spontaneous release of glutamate was also independent of VACC activity (Eggermann, Bucurenciu, Goswami, & Jonas, ; Tsintsadze et al, ; Yamasaki, Hashimoto, & Kano, ).…”

“…Consistent with this idea, Cd 2+ does permeate VACCs (Chow, ) and directly increases Fluo‐4 fluorescence (Hinkle, Shanshala, & Nelson, ; Johnson & Spence, ; Lopin, Thevenod, Page, & Jones, ). However, since spontaneous excitatory transmission remained intact in the presence of Cd 2+ (Tsintsadze et al, ; Vyleta & Smith, ), this argues against the vesicle depletion hypothesis. Moreover, Cd 2+ was effective on spontaneous release from inhibitory synapses but not excitatory synapses recorded simultaneously, making it unlikely that Cd 2+ was poisoning synaptic transmission via a VACC‐independent pathway (Tsintsadze et al, ).…”

“…However, since spontaneous excitatory transmission remained intact in the presence of Cd 2+ (Tsintsadze et al, ; Vyleta & Smith, ), this argues against the vesicle depletion hypothesis. Moreover, Cd 2+ was effective on spontaneous release from inhibitory synapses but not excitatory synapses recorded simultaneously, making it unlikely that Cd 2+ was poisoning synaptic transmission via a VACC‐independent pathway (Tsintsadze et al, ). Furthermore, in studies in which Cd 2+ was ineffective at reducing mEPSCs, specific VACC blockers, like MVIIC, were also ineffective (Tsintsadze et al, ; Vyleta & Smith, ).…”

“…Interestingly, in similar experiments using Cd 2+ as the VACC blocker, the mIPSC frequency–VACC current relationship was reversed (Figure d). This increase in the effectiveness of Cd 2+ on VACC currents recorded at −10 mV compared with spontaneous release at resting membrane potential is mainly attributable to the increased block by Cd 2+ at depolarized membrane potentials (Chow, ; Tsintsadze et al, ). However, since spontaneous release of GABA relies on coincident VACCs opening (Williams et al, ), the relative loss of affinity of Cd 2+ on mIPSC frequency may also arise from its flickery block of VACCs (Lansman, Hess, & Tsien, ), which, unlike irreversible blockers, does not preclude multiple VACC openings (Figure ).…”