D. Knight

Functional analysis of neurotransmission at β2‐laminin deficient terminals

β2‐Laminin is important for the formation of neuromuscular junctions in vertebrates. Previously, we have inactivated the gene that encodes for β2‐laminin in mice and observed predominantly prejunctional structural defects. In this study, we have used both intra‐ and extracellular recording methods to investigate evoked neurotransmission in β2‐laminin‐deficient mice, from postnatal day 8 (P8) through to day 18 (P18). Our results confirmed that there was a decrease in the frequency of spontaneous release, but no change in the postjunctional response to such release. Analysis of evoked neurotransmission showed an increase in the frequency of stimuli that failed to elicit an evoked postjunctional response in the mutants compared to litter mate controls, resulting in a 50 % reduction in mean quantal content at mutant terminals. Compared to littermate controls, β2‐laminin‐deficient terminals showed greater synaptic depression when subjected to high frequency stimulation. Furthermore, the paired pulse ratio of the first two stimuli was significantly lower in β2‐laminin mutant terminals. Statistical analysis of the binomial parameters of release showed that the decrease in quantal content was due to a decrease in the number of release sites without any significant change in the average probability of release. This suggestion was supported by the observation of fewer synaptic vesicle protein 2 (SV2)‐positive varicosities in β2‐laminin‐deficient terminals and by ultrastructural observations showing smaller terminal profiles and increased Schwann cell invasion in β2‐laminin mutants; the differences between β2‐laminin mutants and wild‐type mice were the same at both P8 and P18. From these results we conclude that β2‐laminin plays a role in the early structural development of the neuromuscular junction. We also suggest that transmitter release activity may act as a deterrent to Schwann cell invasion in the absence of β2‐laminin.

Effect of chronic clonidine treatment on transmitter release from sympathetic varicosities of the guinea‐pig vas deferens

Previous studies have demonstrated that chronic pre‐synaptic inhibition of transmitter release by morphine evokes a counter‐adaptive response in the sympathetic nerve terminals that manifests itself as an increase in transmitter release during acute withdrawal. In the present study we examined the possibility that other pre‐synaptically acting drugs such as clonidine also evoke a counter‐adaptive response in the sympathetic nerve terminals. In chronically saline treated (CST) preparations, clonidine (0.5 μM) completely abolished evoked transmitter release from sympathetic varicosities bathed in an extracellular calcium concentration ([Ca2+]o) of 2 mM. 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 (NTI) 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 – 9 days of chronic exposure to clonidine. Acute withdrawal of preparations following chronic clonidine treatment (CCT) resulted in a significant (P<0.005) enhancement of neurotransmitter release (3.75 times) above control levels observed in CST preparations. The present findings demonstrate an enhancement of neurotransmitter release from sympathetic varicosities following acute withdrawal from chronic clonidine treatment.