Second, continued exposure to MbCD elicited a dramatic decrease in EJP amplitude and failure of impulse propagation to synaptic terminals in the cold-acclimatized group, but neither of these effects occurred in the warm-acclimatized group. Third, extended treatment with MbCD decreased MLN4924 Metabolic Enzyme/Protease inhibitor quantal release in the cold-acclimatized group but increased release in the warm-acclimatized group, despite a comparable decrease in the amplitude of evoked single quantal events in both groups ; both effects in the cold-acclimatized group were irreversible. Fourth, application of MbCD LEE011 yielded a rapid, transient increase in the amplitude of muscle depolarization evoked by exogenous L-glutamate, but this effect was only significant in the cold-acclimatized group. Finally, although exposure to MbCD subsequently decreased responses of muscle fibres to L-glutamate in both experimental groups, the effect was more pronounced and irreversible in preparations from cold-acclimatized animals. Results from direct, quantitative lipid analyses enabled assessment of whether some or all of these differences related to cholesterol levels. The ability of MbCD to reduce transmitter release in the cold-acclimatized but not in the warm-acclimatized group was most likely related to its effect on impulse propagation. Impulses failed to propagate to the synaptic terminals in preparations from cold-acclimatized crayfish after 6- 10 min of exposure to MbCD, when cholesterol levels were significantly reduced. In the warm-acclimatized group, however, impulse propagation was not disrupted, even though cholesterol levels were significantly reduced by the end of the exposure to MbCD. Despite little difference in the ratio of cholesterol to total lipids in neuromuscular preparations from cold and warm-acclimatized crayfish, analyzing cholesterol levels separately in nerve and muscle revealed substantially more cholesterol in nerves, and comparison between the warm- and cold-acclimatized groups revealed significantly less neuronal cholesterol in the warmacclimatized group. As cholesterol is important in synapse formation and is a major component of secretory vesicles, the difference in cholesterol content between nerve and muscle tissues is perhaps not surprising. Similarly, it is known that cold-acclimatization can result in increased levels of membrane cholesterol in some ectothermic animals. Notably, in preparations from cold-acclimatized crayfish, treatment with MbCD caused little change in cholesterol levels from muscle, but a substantial change in cholesterol levels from nervous tissue. In preparations from warm-acclimatized crayfish, this effect reversed; only in muscle from warm-acclimatized animals did 3 min of exposure to MbCD significantly decrease cholesterol levels.