Multiple components of membrane retrieval in synaptic terminals revealed by changes in hydrostatic pressure

Membrane retrieval following exocytosis in synaptic terminals is fast and compensatory, however, little is known about the factors that regulate or contribute to this special form of endocytosis. We used whole-terminal capacitance measurements to examine the effect of hydrostatic pressure on compensatory endocytosis in single synaptic terminals of retinal bipolar neurons. We report that a small increase in hydrostatic pressure reversibly inhibits compensatory endocytosis. Elevation in hydrostatic pressure does not block all membrane retrieval, however. A small, fast component of endocytosis persists, while a slower component is inhibited. When the hydrostatic pressure is then stepped back to a near-neutral setting, an even slower form of endocytosis is observed that restores the resting membrane capacitance to baseline. Thus even when endocytosis is temporally uncoupled from calcium entry and exocytosis, it can still be compensatory, indicating that presynaptic surface area is highly regulated. Our results suggest that at least two distinct mechanisms of membrane retrieval contribute to compensatory endocytosis. Given its dramatic inhibitory effect on membrane retrieval, we suggest that hydrostatic pressure be carefully controlled when studying endocytosis in the whole cell recording configuration.