Presynaptic Direct Structures-Function Analysis

The accepted model for chemical transmission at the neuromuscular junction is that when an action potential reaches the nerve terminal, voltage-sensitive calcium channels open which leads to a cascade of events resulting in vesicles fusing with the presynaptic membrane to release their contents into the synaptic cleft. The number of active zones within synapses, and not necessarily the size or numbers of synapses per se, is correlated to the degree of synaptic efficacy. Additional factors besides structural elements have a role in synaptic efficacy, but the purpose of this study is to focus on structural correlations. Investigating the function of individual synapses is essential to understanding the mechanisms that influence the efficacy of chemical synaptic transmission. The known simplicity of the synaptic structure at the crayfish opener neuromuscular junction and its quantal nature of release allow an assessment of discrete synapses within the motor nerve terminals. By analyzing areas of a quantal current trace using modern statistical techniques, it is noted that the >gold standard= of directly counting quantal events is unstable in determining the number of release sites (n) and the probability of release at these sites (p) when the probability of release is low. This classical approach estimates one site even when there are clearly multiple sites operating. Modern methods used in this investigation estimate the probability of release at each site (p) to determine whether new sites are recruited as the firing frequency is increased. The proposed investigations directly correlate quantal parameters by various statistical methods to synaptic structure. An established method of identifying and recording from a synaptic site allows for its subsequent serial reconstruction using electron microscopic techniques. Thus, the same region can be serial sectioned and reconstructed from electron micrographs to directly correlate function to synaptic ultra structure. This effort will provide a clearer understanding of the mechanisms employed by structural elements that influence synaptic efficacy and mechanisms underlying frequency facilitation of transmitter release in crustaceans.