Early studies on transmitter release showed it to be intermittent and probabilistic in nature. Furthermore, the probability of transmitter release from adjacent release sites is highly non-uniform. This probabilistic nature of release has been suggested to form the basis of learning and memory. However, while numerous manipulations of terminals have been shown to affect the probability of release, the mechanisms by which the probability of release is regulated in vivo are still unknown. The primary aims of this thesis were: firstly, to examine some factors that have been suggested to affect transmitter release probability in sympathetic varicosities; secondly, to examine the morphology of terminals in which the probability of release has been pharmacologically enhanced; and thirdly, to examine the role of NSF in transmitter release.
To address the first aim, transmitter release was measured using a focal extracellular electrode placed over pairs of visualised varicosities. The vesicle density, release machinery staining intensity and co-localisation and the calcium influx following single impulse nerve stimulation were measured. Transmitter release probability varied by more than 55-fold between sympathetic varicosities. The distributions of both the synaptic vesicle densities and the release machinery protein co-localisation were both non-uniform but significantly different from the distribution of transmitter release probabilities. Given the non-linear relationship between calcium and transmitter release, the variation in stimulus induced calcium influx was shown to closely match the distribution of transmitter release probabilities. These results suggest that the variation in transmitter release probability is strongly influenced by small variations in calcium influx but is also affected by variation in the calcium sensitivity of a terminal.
To address the second aim, the release machinery protein staining intensity and co-localisation were examined in vasa deferentia from chronically morphine treated mice. Chronic exposure to morphine has been shown to produce counter-adaptive changes in the terminal that culminate in a significant increase in the probability of release, which is unmasked during acute withdrawal. The results of this study showed an increase in the staining intensity and an increase in the co-localisation of SV2 and synaptotagmin 1. These results confirmed the suggestion made in the first chapter that transmitter release probability might be affected by variation in the levels of individual release machinery proteins.
To test whether other pre-junctionally acting drugs could induce these counter-adaptive increases in transmitter release probability, vasa deferentia were acutely and chronically exposed to the á2-adrenoceptor agonist clonidine. In control preparations, clonidine (0.5 µM) completely abolished evoked transmitter release. The inhibitory effect of clonidine was reduced by increasing [Ca2+]o from 2 to 4 mM and the stimulation frequency from 0.1 to 1 Hz. The nerve terminal impulse was not affected by concentrations of clonidine that completely abolished evoked transmitter release. Sympathetic varicosities developed a tolerance to clonidine (0.5 µM) following 7 to 9 days of chronic exposure to clonidine. Acute withdrawal of preparations following chronic clonidine treatment resulted in a significant (P < 0.005) enhancement of neurotransmitter release above control levels. These findings suggested that chronic pre-junctional inhibition of transmitter release results in counter adaptive changes leading to an increase in the probability of release.
Finally, to address the third aim, intact nerve-muscle preparations from cane toads (Bufo marinus) were treated with the N-ethylmaleimide (NEM) sensitive fusion protein (NSF) inhibitor, NEM. Treatment with NEM (10 µM) caused significant increases in both the amplitude of evoked release and the frequency of spontaneous release without affecting the amplitude of spontaneous release. NEM treatment also resulted in reduced levels of facilitation, increased depression and a decrease in the synaptic delay. The effects of NEM on transmitter release were not due to inhibition of pertussis toxin sensitive adenosine receptors. A model of NSF action is proposed that suggests NSF has a dual role in vesicle priming. The first involves ATP dependent dissociation of cis-SNARE complexes and is insensitive to NEM. The second role for NSF is a NEM-sensitive, negative regulation of trans- SNARE formation. In the absence of NEM, this negative regulation prevents trans-SNARE formation until after the arrival of the depolarisation-induced calcium signal.
In summary, the results of this thesis suggest that the probability of transmitter release is susceptible to regulation by several factors including vesicle density, the levels of individual release machinery proteins and the calcium influx. These results provide valuable insight in to the in vivo mechanisms of transmitter release regulation.