Membrane Capacitance Measurements of Isolated Bipolar Cell Synaptic Terminals in Goldfish Retinal Slices

Abstract

Abstract: : Purpose: The synaptic terminals of goldfish retinal bipolar cells have been used to study neurotransmitter release due to their large size and simple morphology, which makes them highly suitable for patch-clamp and membrane capacitance measurements. Previous recordings have used acutely dissociated bipolar cell terminals but it is unknown whether the dissociation proceedure affects terminal function. Methods: Whole cell voltage-clamp recordings were made from visually identified bipolar cell terminals in the goldfish retinal slice preparation. Terminals which were isolated from the rest of the cell due to severing of the axon during the slicing proceedure were readily identified by the capacitative current response to a 10 mV voltage step, and could be confirmed as isolated by filling with the fluorescent dye Lucifer Yellow. Results: The Ca₂₊ current and capacitance increase evoked by a 200 ms voltage step from -60 to -10 mV were measured in terminals in slices and compared with dissociated terminals. It was found that the peak Ca₂₊ current was significantly greater in terminals in slices (328.4 ± 25.5 pA, n = 16, versus 182.6 ± 28.5 pA, n = 9, p < 0.01). However the evoked capacitance increase was not significantly different between the two preparations (slice 133.7 ± 14.2 fF, n = 16, versus dissociated 153.2 ± 27.2 fF, n = 9). Bipolar cells exhibit pronounced paired pulse depression of release, which is not correlated with a depression of Ca₂₊ influx. In dissociated terminals the depression induced by a 2 ms depolarising step recovered with a double exponential time-course with time constants of 1.03 s and 11.8 s. Increasing the stimulus duration to 25 ms evoked a larger Ca₂₊ influx and capacitance jump but did not change the rate of recovery of release. It was found that the rates of recovery for both 2 ms and 25 ms stimulus durations were not significantly different in terminals in slices compared with dissociated cells. Conclusion: Dissociated bipolar cell terminals appear to behave in a manner very similar to terminals in situ with regard to the kinetics of Ca₂₊-induced exocytosis.

Keywords: 330 bipolar cells • 594 synapse • 402 excitatory neurotransmitters