Imaging of Ca2+ responses mediated by presynaptic L-type channels on GABAergic boutons of cultured hippocampal neurons

We have previously demonstrated that L-type Ca2+ channels are involved in post-tetanic potentiation (PTP) of GABAergic IPSCs in cultured hippocampal neurons. Here we have used intracellular Fluo-3 to detect [Ca2+]i in single GABAergic boutons in response to stimulation that evokes PTP. During control stimulation of the presynaptic GABAergic neuron at 40 Hz for 1-2 s, ΔF/F0 increased rapidly to a peak value and started to decline shortly after the train ended, returning to baseline within 10-20 s. The L-type channel blocker, isradipine (5 μM), had no significant effect on the amplitude or kinetics of the Ca2+ signal. Following blockade of N- and P/Q-type Ca2+-channels, the amplitude was reduced by 52.9 ± 3%. Isradipine caused a reduction of the remaining response (by 26.6 ± 5%, P < 0.01), that was fully reversible on washing. The L-type channel “agonist”, BayK 8644 (8 μM), caused a significant enhancement of the peak (by 18.7% ± 7%, P < 0.05). The rising phase of the Ca2+ signal, which is related to the rate of entry of Ca2+ into the bouton, was decreased by isradipine (by 25.5 ± 6%, P < 0.05) and enhanced by BayK 8644 (by 45.2% ± 16%, P < 0.05). These Ca2+ imaging experiments support the putative role of L-type channels in PTP of GABAergic synapses on cultured hippocampal neurons. We expect L-channels to be few in number, although they may couple strongly to intracellular signalling cascades that could amplify a signal that regulates synaptic vesicle turnover in the GABAergic boutons.