Calcium dependence of spontaneous neurotransmitter release

Abstract

Spontaneous release of neurotransmitters is regulated by extracellular [Ca²⁺ ] and intracellular [Ca²⁺ ]. Curiously, some of the mechanisms of Ca²⁺ signaling at central synapses are different at excitatory and inhibitory synapses. While the stochastic activity of voltage-activated Ca²⁺ channels triggers a majority of spontaneous release at inhibitory synapses, this is not the case at excitatory nerve terminals. Ca²⁺ release from intracellular stores regulates spontaneous release at excitatory and inhibitory terminals, as do agonists of the Ca²⁺ -sensing receptor. Molecular machinery triggering spontaneous vesicle fusion may differ from that underlying evoked release and may be one of the sources of heterogeneity in release mechanisms.

Keywords: VGCC (voltage-gated calcium channels); calcium; minis; spontaneous release.

Figure 1

Random variability in the rate of spontaneous release. a Fluorescent micrograph labeled for synaptophysin (green) and DAPI (blue) illustrates that alarge number of nerve terminals may synapse onto a singleneocortical neuron. Spontaneous release will increase with the number of synaptic contacts, along with other factors including temperature. b Exemplary trace of miniature inhibitory post-synaptic currents (mIPSCs) from cortical cultured neurons recorded at −70 mV under previously described conditions (Williams et al. 2012). Horizontal bars above the trace indicate the traces used in c and d. The stochastic nature of spontaneous release results in a variable frequency and amplitude of events as shown. c Expanded time course from the trace in b to indicate basal mIPSC frequency. d Expanded timecourse from the trace in b to show a portion of the recording where a high frequency “burst” of mIPSCs was taking place.

Figure 2

Non-linear actions of VACC blockers on VACC currents and mIPSCs. a Cartoon of VACC current through two single channels indicating the effects of different types of channel blockers. The center illustration shows block by a selective “irreversible” blocker for N-type VACCs (GVIa, 1μM). The illustration on the right shows the result of blocking channels with Cd²⁺, which is nonselective and results in “flickery” block of the current (Lansman et al, 1986). b Plot of normalized mIPSC frequency versus normalized VGCC current amplitude in the presence and absence of a saturating dose of Aga Iva (300 nM), a selective P/Q-type channel antagonist. VACC currents were elicited by steps from −70 to −10 mV and mIPSC frequency was recorded at −70 mV, both in cultured neocortical neurons as previously described (Tsintsadze et al. 2017). Data are normalized average frequency (n=4) and average current (n=5) and represent the fraction the mIPSCs and VACC currents that are sensitive to a saturating dose of Cd²⁺ (300 μM). c Plot of normalized mIPSC frequency versus normalized VGCC current amplitude in the presence and absence of a saturating dose of GVIa (1μM), a selective N-type channel antagonist. Data are average frequency (n=9) and VACC current amplitude (n=5) recorded and normalized as for b. d Plot of normalized mIPSC frequency (n= 9) versus normalized VGCC current amplitude (n=13) in the presence of 0, 1, 3, 10, 30, 100, and 300 μM Cd²⁺. Recordings made as previously described(Tsintsadze et al. 2017). Note the differences in the shapes of the curves in b and c from the curve in d. If more than one channel contributes to spontaneous release, the relationship between release probability and the size of the Ca²⁺ current will be supralinear.

Figure 3

Single channel currents and Ca²⁺ domains. (Upper panel) Illustration of VACC current through two single channels in 1.1 and 6 mM [Ca²⁺]_o. Note that the single channel current in 6 mM [Ca²⁺]_o is approximately 3-fold larger than physiological [Ca²⁺]_o (Weber et al. 2010). (Lower panel) Illustration showing the side view of the Ca²⁺ domain with 2 synaptic vesicles in 1.1 and 6 mM [Ca²⁺]_o. With a higher [Ca²⁺]_o, the Ca²⁺ domain may also increase 3-fold.

Figure 4

Spontaneous release is increased by external calcium in cultured neocortical neurons without MVR. a and g Exemplary mIPSC and mEPSCs, respectively, in 1.1 (top, black) and 6 (bottom, blue/red) mM [Ca²⁺]_o. Recordings were made as reported previously (Vyleta and Smith 2011; Williams et al. 2012). b and h Histogram of inter-event interval (IEI) for mIPSCs or mEPSCs in 1.1 (black) and 6 (blue/red) mM [Ca²⁺]_o from the same experiments in a and g. The signal-to-noise is reduced by plotting the square root of the ordinate and using logarithmic binning. Histograms are fit with double exponentials. c and i Histogram of amplitude of mIPSCs or mEPSCs in 1.1 (black) and 6 (blue/red) mM [Ca²⁺]_o from the same experiments in a and g. d – k Cumulative probability plots for IEI and amplitude of mIPSCs or mEPSCs in 1.1 (black) and 6 (blue/red) mM [Ca²⁺]_o from the same experiments in a and g. f and l Average time constants (1st and 2nd Taus) from the double exponential fit of the IEI histograms for mIPSCs (n=9) and mEPSCs (n=8). Black bars indicate time constants in 1.1 [Ca²⁺]_o and 6 blue/red bars represent time constants in 6 mM [Ca²⁺]_o.