Developmental increase in asynchronous GABA release in cultured hippocampal neurons

Abstract

Developmental changes in GABAergic synaptic transmission were examined in cultured hippocampal neurons using patch-clamp recordings and Ca 2+ 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 inhibitory postsynaptic responses. In neurons that had been cultured for more than two weeks, asynchronous inhibitory postsynaptic currents often appeared during the tetanus and continued for several seconds following stimulation. There was little asynchronous activity in neurons that had been cultured for shorter times. However, no age-related changes were observed in the amplitude of single synchronous inhibitory postsynaptic currents, paired-pulse depression or post-tetanic potentiation of inhibitory postsynaptic currents. Following equimolar replacement of extracellular Ca 2+ with strontium ions (Sr 2+), single autaptic inhibitory postsynaptic currents were depressed in amplitude and asynchronous inhibitory postsynaptic currents were present on the decaying phase. Sr 2+-induced asynchronous inhibitory postsynaptic currents showed no dependence on age in culture. Imaging of Ca 2+ 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, intracellular calcium [Ca 2+] i increased rapidly in individual boutons. Following the stimulus, [Ca 2+] i decayed back to baseline within 10–15 s. The half-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 currents. We propose that the increase in tetanus-induced asynchronous GABA-release during the first month of synapse maturation in vitro is caused by a slowing of the Ca 2+-clearing mechanisms in the GABAergic boutons. This results in larger and more prolonged elevations of [Ca 2+] i during tetanic stimulation, which leads to enhanced asynchronous transmitter release. We propose that the results of this study demonstrate a potentially important aspect of synapse maturation during development, and also imply that GABA release is up-regulated in conditions of decreased Ca 2+ buffering and clearing.