Real-Time Measurements of Synaptic Autoinhibition Produced by Serotonin Release in Cultured Leech Neurons

Abstract

We studied autoinhibition produced immediately after synaptic serotonin (5-HT) release in identified leech Retzius neurons, cultured singly or forming synapses onto pressure-sensitive neurons. Cultured Retzius neurons are isopotential, thus allowing accurate recordings of synaptic events using intracellular microelectrodes. The effects of autoinhibition on distant neuropilar presynaptic endings were predicted from model simulations. Following action potentials (APs), cultured neurons produced a slow hyperpolarization with a rise time of 85.4 ± 5.2 ms and a half-decay time of 252 ± 17.4 ms. These inhibitory postpotentials were reproduced by the iontophoretic application of 5-HT and became depolarizing after inverting the transmembranal chloride gradient by using microelectrodes filled with potassium chloride. The inhibitory postpotentials were reversibly abolished in the absence of extracellular calcium and absent in reserpine-treated neurons, suggesting an autoinhibition due to 5-HT acting on autoreceptors coupled to chloride channels. The autoinhibitory responses increased the membrane conductance and decreased subsequent excitability. Increasing 5-HT release by stimulating with trains of ten pulses at 10 or 30 Hz produced 23 ± 6 and 47 ± 2% of AP failures, respectively. These failures were reversibly abolished by the serotonergic antagonist methysergide (140 μM). Moreover, reserpine-treated neurons had only 5 ± 4% of failures during trains at 10 Hz. This percentage was increased to 35 ± 4% by iontophoretic application of 5-HT. Increases in AP failures correlated with smaller postsynaptic currents. Model simulations predicted that the autoinhibitory chloride conductance reduces the amplitude of APs arriving at neuropilar presynaptic endings. Altogether, our results suggest that 5-HT autoinhibits its subsequent release by decreasing the excitability of presynaptic endings within the same neuron.