The interference reflection microscopy (IRM) technique allows the direct visualization of close contacts between surface membrane and a glass coverslip, and has been used to study cell motility and membrane adhesion (Izzard & Lochner, 1976). Here we demonstrate that this technique can also be used to monitor changes in membrane area associated with exocytosis and endocytosis at a presynaptic terminal.

Depolarizing bipolar cells were isolated from the retina of humanely killed goldfish and whole-cell recordings made using an electrode placed on the soma. The area of close contact between the synaptic terminal and glass coverslip was visualized by IRM as a homogenous black region of destructive interference. This ‘footprint’ had an average area of ~40 mm² (10Ð15 % of the area of the terminal). Exocytosis was triggered by a step depolarization from -70 to -10 mV and images of the footprint recorded at 25 Hz.

Depolarizations of 20 ms or longer caused the area of the footprint (A_f) to expand, indicating an increase in the amount of membrane in contact with the glass. A 500 ms depolarisation increased A_f significantly by 4.6 ± 0.8 % (mean ± S.E.M., n = 16, Student’s paired t test, P < 0.01). Immediately following repolarization, A_f recovered fully with a time constant of about 1 s. Changes in A_f were directly proportional to changes in total membrane surface area measured simultaneously using the capacitance technique. A 3 % increase in global capacitance caused a 1 % increase in A_f (n = 3). We conclude that changes in the area of the footprint measured by IRM are an accurate reflection of changes in membrane surface area associated with exocytosis and endocytosis.

A 5 s depolarization increased the A_f in three kinetically distinct phases. The first and most rapid phase was complete in < 80 ms and represented an increase in A_f of 0.5Ð1 %. The second phase was slower; A_f increased by 2 % in 2 s. During the third phase, A_f increased continuously at a rate of ~0.5 % s^-1. The recovery in A_f following a longer stimulus was relatively complex; often there was a brief further increase (representing exocytosis driven by residual calcium) followed by a fall with fast (t ~1 s) and slow (t > 10 s) components. The kinetics of exocytosis and endocytosis measured by IRM were very similar to those measured in bipolar cell terminals using the capacitance technique or the membrane dye FM 1-43 (Neves & Lagnado, 1999).

The use of IRM provides some advantages over the use of the capacitance technique (Gillis, 1995). IRM allows a continuous monitor of increases in membrane surface area and can be applied to cells with complex morphologies.

All procedures accord with current UK legislation.