VITA for Physiological and Anatomical Assessment of Synapses at the Crayfish Neuromuscular Junction-phd of Johnstone

The crayfish, Procambarus clarkii, has a multitude of ideal sites in which synaptic transmission may be studied. Its opener muscle, being innervated by a single excitatory neuron is a good model for studying the structure/function of… (More) The crayfish, Procambarus clarkii, has a multitude of ideal sites in which synaptic transmission may be studied. Its opener muscle, being innervated by a single excitatory neuron is a good model for studying the structure/function of neuromuscular junctions since the preparation is identifiable from animal to animal and the nerve terminals are visible using a vital dye. This allows ease in finding a suitable site to record from in each preparation and offers the ability to relocate it anatomically. Marking a recorded site and rebuilding it through electron microscopy gives good detail of synaptic struture for assesment. In the first of these studies, low output sites known as stems (which lie between varicosities) were used to reduce n (number of release sites) in order to minimize synaptic complexity so individual quantal events could be analyzed by their unique parameters (area, peak, tau, rise time and latency). This was in attempt to uncover specific quantal signatures that could be traced back to the structure of the area recorded. It was found that even at stem regions synaptic structure is still complex having multiple synapses each of which could harbor a number of AZs. This gives insight as to how quantal analysis should be treated. Even low output synapses n must be treated at the AZ level. Synaptic depression was studied at the crayfish extensor muscle. By depressing the phasic neuron and recording from the muscle it appears that depression is a presynaptic phenomenon. The use of 5-HT gave insight to vesicular dynamics within the nerve terminal, by delaying depression and increasing maximum EPSP amplitude. TEM of phasic nerve terminals reveals no change in numbers of dock or RRP vesicles. Short term facilitation and vesicular dynamics were studied with the use of 5-HT and a neurotoxin TBOA, which blocks the glutamate transporter. In this study I showed differential mechanisms that control RRP and RP vesicles. By blocking glutamate reuptake, the RRP is depleted as shown by reduced EPSPs, but recovered with 5-HT application. The understanding of vesicle dynamics in any system has relevance for all chemical synapses. (Less)

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