Developmental increase in asynchronous GABA release in cultured hippocampal neurons

Developmental changes in GABAergic synaptic transmission were examined in c ultured hippocampal neurons using patch-clamp recordings and Ca2+ imaging. In paired recordings, tetanization of the presynaptic GABAergic neuron with 80 pulses at either 40 or 80 Hz was accompanied by tetanic depression of i nhibitory postsynaptic responses. In neurons that had been cultured for mor e than two weeks, asynchronous inhibitory postsynaptic currents often appea red during the tetanus and continued for several seconds following stimulat ion. There was little asynchronous activity in neurons that had been cultur ed for shorter times. However, no age-related changes were observed in the amplitude of single synchronous inhibitory postsynaptic currents, paired-pu lse depression or post-tetanic potentiation of inhibitory postsynaptic curr ents. Following equimolar replacement of extracellular Ca2+ with strontium ions (Sr2+), Single autaptic inhibitory postsynaptic currents were depresse d in amplitude and asynchronous inhibitory postsynaptic currents were prese nt on the decaying phase. Sr2+-induced asynchronous inhibitory postsynaptic currents showed no dependence on age in culture. Imaging of Ca2+ in single GABAergic boutons was performed by including Fluo-3 in the patch pipette. During action potential firing induced by stimulating at 80 Hz for 1 s, int racellular calcium [Ca2+](i) increased rapidly in individual boutons. Follo wing the stimulus, [Ca2+](i) decayed back to baseline within 10-15 s. The h alf-time of decay increased from 1.7 +/- 0.2 s at 15 days in vitro to 4.0 /- 0.2 s at 30 days in vitro (P < 0.05), with a developmental profile that closely matched the increase in asynchronous inhibitory postsynaptic curren ts. We propose that the increase in tetanus-induced asynchronous GABA-relea se during the first month of synapse maturation in vitro is caused by a slo wing of the Ca2+-clearing mechanisms in the GABAergic boutons. This results in larger and more prolonged elevations of [Ca2+](i) during tetanic stimul ation, which leads to enhanced asynchronous transmitter release. We propose that the results of this study demonstrate a potentially importa nt aspect of synapse maturation during development, and also imply that GAB A release is up-regulated in conditions of decreased Ca2+ buffering and cle aring. (C) 2000 IBRO. Published by Elsevier Science Ltd. All rights reserve d.