Such cellular processes include excitability, ion channel activity, exocytosis, endocytosis / vesicle recycling, neurotransmitter uptake and storage and postCT99021 synaptic receptor localization. Such studies make a case that cholesterol may be a critical membrane component for all the key steps in chemical synaptic transmission. The sole use of MbCD to examine and identify cholesteroldependent functions, however, poses at least two main limitations. First, it can be challenging to demonstrate definitively that a physiological effect is caused by a change in cholesterol level rather than some non-specific effect of MbCD, such as interaction with and removal of phospholipids. Some studies employing MbCD do not even report cholesterol levels, although many do provide corroborating evidence showing the same physiological effect with inhibitors of cholesterol synthesis, including an assessment of gross changes in the levels of cholesterol. Thus, the second limitation is that such qualitative assessments of global membrane cholesterol concentration may not correlate with the local concentrations at the functional sites of interest. The use of multiple pharmacological tools to confirm the direct role of cholesterol in any given mechanism thus remains the most secure approach. Why do these issues exist? As noted above, there is considerable evidence that cyclodextrins exert pleiotropic effects on membranes, removing other lipidic components, as well as depleting cholesterol from both fluid and cholesterol-enriched microdomains. Thus, effects of MbCD on physiological functions should be interpreted with caution and always confirmed with alternative approaches. One such alternative approach to investigating cholesteroldependent physiological effects is suggested by observations that thermal acclimatization for periods of weeks can alter the content of cholesterol and other lipids in ectothermic animals. This raised the question of whether or not animals acclimatized to different temperatures, whose own metabolism would generate differences in levels of cholesterol and other membrane lipids, would respond similarly to cholesterol-depletion with MbCD. Thus, here we used acclimatization temperature as a tool to address both the acute effects of MbCD and the postulated roles of cholesterol in the physiology of chemical neurotransmission. We chose to address this question using crayfish, since these animals are known to adapt reasonably rapidly to decreases in temperature, assuming a more ��quiescent�� state. In addition, MbCD is recognized to elicit several presynaptic effects on crayfish neuromuscular preparations, including: failure of impulses to propagate through axonal branches, reduction in the amplitude of excitatory junctional potentials, modest enhancement of evoked transmitter release from directly stimulated synaptic terminals and enhancement of Evofosfamide spontaneous transmitter release.