Characterization of Calcium Channels at Individual Presynaptic Terminals of Lamprey Reticulospinal Axons

Stimulus evoked calcium (Ca2+) influx through voltage-gated Ca2+ channels, localized in the presynaptic release face membrane, gates neurotransmitter release. Differing Ca2+ requirements for release have been proposed in different presynaptic terminals, with release being either gated by Ca2+ influx through a large number of open channels or through the opening of very few channels. Enumeration of the number of Ca2+ channels at a terminal and more importantly the number that open in response to a stimulus is critical to determining the Ca2+ requirement for release. Recording Ca2+ currents at the release face membrane is thus crucial and recording access to the release face membrane at individual presynaptic terminals an absolute necessity. This has not been possible in most presynaptic terminals including the lamprey reticulospinal synapse. Such recordings have only successfully been achieved in two calyceal presynaptic terminals. The acutely dissociated reticulospinal axon preparation presents a significant breakthrough, permitting direct recording from the release face membrane of individual terminals. Ca2+ channels were characterized at individual terminals by immunohistochemistry and low noise single channel electrophysiological recordings. Multiple subtypes of Ca2+ channels, N-, P/Q-, R- and L-type, were found to be present in these terminals. Number of channels at a terminal was determined for each of the subtypes; N-type (4-10; mean 6), P/Q-type (3-9; mean 6), R-type (4-32; mean 12) and L-type (3-17; mean 10). A small number of Ca2+ channels (68, mean 33) were found to be present at a terminal. Furthermore, a very small number (3-6, mean 4) of Ca2+ channels opened with a stimulus, suggesting that Ca2+ influx through few open channels may gate release in these terminals. This result, which is the first determination of Ca2+ channel activity at single active zone central synapses, has profound implications for the reliability and function of evoked neurotransmitter release in the central nervous system.