Impact of vesicular glutamate leakage on synaptic transmission at the calyx of Held

Abstract

Glutamate leaks out of synaptic vesicles when the transvesicular proton gradient is dissipated in isolated vesicle preparations. In the nerve terminal, however, it is controversial whether glutamate can leak out of vesicles. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate in whole-cell dialysis or by blocking vacuolar ATPase using bafilomycin A1 (Baf) at the calyx of Held in mouse brainstem slices. Presynaptic glutamate washout or Baf application reduced the mean amplitude and frequency of spontaneous miniature (m)EPSCs and the mean amplitude of EPSCs evoked every 10 min. The percentage reduction of mEPSC amplitude was much less than that of EPSC amplitude or mEPSC frequency, and tended to reach a plateau. The mean amplitude of mEPSCs after glutamate washout or Baf application remained high above the detection limit, deduced from the reduction of mEPSC amplitude by the AMPA receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione. Membrane capacitance measurements from presynaptic terminals indicated no effect of glutamate washout on exocytosis or endocytosis of synaptic vesicles. We conclude that glutamate can leak out of vesicles unless it is continuously taken up from presynaptic cytosol. However, the magnitude of glutamate leakage was small and had only a minor effect on synaptic responses. In contrast, prominent rundowns of EPSC amplitude and mEPSC frequency observed after glutamate washout or Baf application are likely to be caused by accumulation of unfilled vesicles in presynaptic terminals retrieved after spontaneous and evoked glutamate release.

Activity and cytosolic Na+regulates synaptic vesicle endocytosis

“…5). These results are consistent with previous experiments in the calyx of Held (Hori and Takahashi, 2012; Takami et al, 2017). Another possible mechanism for Na + effects on endocytosis is through effects on [Ca 2+ ].…”

Vesicular GABA Uptake Can Be Rate Limiting for Recovery of IPSCs from Synaptic Depression

“…After bath application of Baf (5 mM), IPSCs ran down with a time constant of 11.4 ± 2.3 min (n = 5; Figure S1A) like that after GABA washout ( Figure 1B; p = 0.57, Student's t test). The IPSC rundown was faster during higher frequency stimulation (Figure S1A), suggesting an additional contribution of empty vesicles accumulated by recycling (Takami et al, 2017). The blocking effect of Baf was irreversible ( Figure S1B), confirming that vesicular transmitter refilling is indispensable for recovery from depression.…”

“…Presynaptic membrane capacitance changes were then evoked by depolarizing pulses in the presence of GABA (5 mM) or in its absence (Figures 2C and 2D). No significant difference was found between them ( Figure 2D; p = 0.99, repeated-measures ANOVA), indicating that presynaptic cytosolic transmitter concentration has no effect on SV availability at inhibitory synapses as at excitatory synapses (Hori and Takahashi, 2012;Takami et al, 2017). from the mean IPSC amplitude 5-10 min after GABA loading.…”

Age-related defects in short-term plasticity are reversed by acetyl-L-carnitine at the mouse calyx of Held

“…Median event amplitudes were reported to avoid potential skewing due to rare but large events. Previous studies at this synapse in juvenile mice report quantal events are ~39 pA using similar threshold detection methods (Takami et al, 2017).…”