Abstract
Purpose::
Bipolar cells play a vital role in the transfer of visual information from photoreceptors to ganglion cells. To better understand synaptic signaling in the retina and set the stage for the application of genetic and molecular tools, we characterized the kinetics of refilling of the releasable pool of synaptic vesicles and membrane retrieval in mouse rod bipolar cells (RBCs).
Methods::
RBCs were acutely isolated from adult mouse retina. Exocytosis and endocytosis were tracked using membrane capacitance measurements, and intraterminal calcium was monitored using a calcium-sensitive fluorescent indicator dye (Zhou et al., 2006). The number of active zones was quantified by confocal microscopy in cells double-labeled for PKC and ribeye.
Results::
A pulse-duration protocol established that a 1s depolarization (-70 mV to 0 mV) was sufficient to deplete the entire releasable pool (29±2 fF; n=43), corresponding to about 34 vesicles/ribbon-style active zone, without evoking a second component of release. The time course of pool refilling was measured with a double-pulse protocol. The first 1s pulse depleted the pool and the second, given at a variable time later, probed the state of refilling. Simultaneous measurement of intraterminal calcium ruled out contributions from changes in calcium entry. The time course of refilling was best described by a double expontential function with time constants of 400ms and 8s (n=86). Endocytosis typically followed exocytosis. The time constant of endocytosis, described by a single exponential function, ranged from 2s to 26s (500ms pulse, n=11). Longer time constants were associated with a larger, preceding calcium transient, whereas time constants on the order of a few seconds were only observed when the calcium transient was < 500nM (1s pulse; n=31). In addition, a brief, calcium-dependent delay often preceded the start of onset of endocytosis (500ms pulse; n=7/11).
Conclusions::
Both exocytosis and endocytosis are calcium-regulated processes in mouse RBCs. Given the time course of endocytosis, it is likely that the fast component of pool refilling draws from preformed vesicles.
Keywords: bipolar cells • synapse • calcium