Temperature-Dependent Phase Behavior of the Synaptosomal Membranes from Mammalian and Marine Invertebrate Synaptosomes

Given reports of raft domains in rodent brain synaptosomes, we probed for their conservation in a poikilotherm, the Woods Hole squid Loligo. Because rafts are described as liquid-ordered phase-separated domains, we expected phase transition temperatures above body temperature if raft function is important to neuronal activity. We tested this hypothesis by comparing synaptosomes, intact nerve endings, from animals that live at two very different temperatures: mouse (body temperature-37°C) and squid (body temperature-20°C). We measured the temperature-dependence of the lipid phase of intact synaptosomes in the absence of exogenous probes by using line-width and spinning sideband intensities of lipid hydrocarbon chain resonances, using proton magic angle spinning NMR spectra as a function of temperature between 0 and 40°C. We also determined the phase behavior of total synaptosomal lipids by repeating the NMR protocol on extracted lipids. To our surprise, there was no detectable signal for liquid-ordered lipid phases at the body temperature of either species. However, there was a difference in the temperature of the onset of order as synaptosomes were cooled below body temperature: the phase state of mouse synaptosomal membranes changes drastically below 24°C, whereas this change occurs below 8°C for the squid synaptosomal membranes. We then measured the composition of synaptosomes in terms of total lipid heads and tails, and the main difference arises from high concentrations of omega-3 poly-unsaturated fatty acids and cholesterol in the squid. Fluorescence microscopy images of squid synaptosomes stained with lipid dyes confirmed the formation of domains below, but not above the phase transition temperatures obtained from NMR measurements. Thus although the membranes of synaptosomes contain lipids that can phase-separate, these lipids remain in the liquid-disordered state at the usual physiological temperatures for squid and mouse.