Imaging of Ca2+ responses mediated by presynaptic L-type channels on GABAergic boutons of cultured hippocampal neurons.
We have previously demonstrated that L-type Ca(2+) channels are involved in post-tetanic potentiation (PTP) of GABAergic IPSCs in cultured hippocampal neurons. Here we have used intracellular Fluo-3 to detect [Ca(2+)](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, DeltaF/F(0) 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 microM), had no significant effect on the amplitude or kinetics of the Ca(2+) signal. Following blockade of N- and P/Q-type Ca(2+)-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 microM), caused a significant enhancement of the peak (by 18.7%+/-7%, P<0.05). The rising phase of the Ca(2+) signal, which is related to the rate of entry of Ca(2+) 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 Ca(2+) 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.
Holmgaard
Kim
K
Institute of Physiology and Biophysics, Building 1160, University of Aarhus, DK-8000 Aarhus C, Denmark.
Jensen
Kimmo
K
Lambert
John D C
JD
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000378:metabolism; D002478:Cells, Cultured; D006624:Hippocampus / Q000166:cytology / Q000502:physiology*; D017275:Isradipine / Q000494:pharmacology; D008564:Membrane Potentials; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D017729:Presynaptic Terminals / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D017661:Receptors, Presynaptic / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000502:physiology*
D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D017661:Receptors, Presynaptic; D005680:gamma-Aminobutyric Acid; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D002118:Calcium; D017275:Isradipine