High and low temperatures affect rat hippocampal synaptosome’s viability and functions

Introduction: Synaptosomes are sealed particles that contain mitochondria, cytoskeleton and vesicles which are necessary to synaptic events like neurotransmitter release and uptake in the nervous system. However, the effect of high and low temperatures on synaptosome membrane integrity and function during a time course after its extraction is less known. The purpose of this study was to assess synaptosome viability and function at 37, 4°C and room temperature (RT) during 6 hours after its extraction. Methods: Hippocampi of 40 male Wistar rats were used for synaptosome preparation. To ensure synaptosome membrane integrity and function, lactate dehydrogenase activity (LDH) and GABA uptake were assessed during 6 successive hours after their extraction at 37, 4°C and RT. Results: Our results showed that at 37°C, synaptosome membrane integrity was reduced 3 hours but at 4°C and RT, it occurred 5 hours following their extraction. The results of synaptosome function analysis coincide with LDH enzyme assay data, meaning that GABA uptake faced a 50% reduction from the initial value at 37°C after 3 hours and at RT after 5 hours. We also found that GABA uptake was reduced at 4°C in the first hour after extraction because the low temperature inhibits GABA transporters. Conclusion: Synaptosomes preserved their viability and function at RT, 37 and 4°C at least for 3 hours after extraction and reduced over time. For long term application of synaptosomes, it is better to keep them at 4°C. iD Physiol Pharmacol 22 (2018) 73-81 D ow nl oa de d fr om p pj . p hy ph a. ir at 1 0: 19 + 03 30 o n M on da y N ov em be r 4t h 20 19 Synaptosome viability and function Physiol Pharmacol 22 (2018) 73-81 | 74 by Whittaker in the late 1950s and detected as detached synapses by electron microscopy (Whittaker, 1993; Whittaker and Gray, 1962). Synaptosomes are acquired by homogenization of fresh brain tissue in an appropriate isotonic solution which causes the nerve terminals to be separated from their axon stalks. Then, the membrane of presynaptic terminals is resealed, which contains cytoplasm, cytoskeleton, mitochondria and synaptic vesicles with the presynaptic and occasionally postsynaptic membranes (Dunkley et al., 2008; Whittaker et al., 1964). Synaptosomes have widely been applied as an in vitro model to investigate the molecular mechanisms of brain synapses specially storage, release, and uptake of neurotransmitters. As a result, our knowledge about the function of synapse ending at a physiological, cellular and molecular level was enhanced (Breukel et al., 1997; Dunkley et al., 1988; Dunkley et al., 2008; Whittaker, 1993). Different procedures have been developed to isolate synaptosomes including ultrafiltration, electrophoresis, sucrose and percoll gradients; however, a median-speed centrifuge technique seems to be more appropriate (Dunkley et al., 1988; Dunkley et al., 2008; Enriquez et al.,1990; Kamat et al., 2014; Stadler and Tashiro, 1979). Synaptosomes can be obtained from any part of the brain tissues. In addition, the nerves of the non-neurological tissue are also involved in the synaptosomes preparation (Jonakait et al.,1979). If a chemical active ingredient is found abundantly in synaptosomes, it can be assumed as a neurotransmitter or coneurotransmitter. Moreover, mixed fraction synaptosomes can be applied to assess the mechanisms of neurotransmitters release (Breukel et al., 1997). Exposure of adult albino rats to the high ambient temperature at 35 °C for 2-12 hours or at 45 °C for 12 hours increased the acetylcholinesterase activity of their brain synaptosomes (Mukhopadhyay and Poddar, 1990). Several studies have investigated the effects of organophosphate compound on the GABA uptake of synaptosomes in cerebral cortex, cerebellum and hippocampus, suggesting that the GABA uptake was optimal one hour after synaptosome extraction (Ghasemi et al., 2007; Pourabdolhossein et al., 2009; Shahroukhi, et al., 2007). Furthermore, Hosseini et al. reported 35 minute is the optimal time for GABA release by rat cerebral synaptosomes (Hosseini et al., 2004). Incubation of synaptosomes at room temperature (22-25 °C) gradually increases the percentage of microtubule containing synaptosomes (41-47%) while its stabilization at low temperature leads to a significant reduction in the number of synaptosomes (Hajos et al., 1979). Despite the significance of synaptosomes in pharmaceutical, structural and functional studies, there is no empirical data on synaptosomes viability and function at different temperatures and time points after their extraction. Thus, the main ambition behind this study was to examine the synaptosomes survival and function at different temperatures and time points by measuring lactate dehydrogenase enzyme (LDH) and GABA uptake. We used hippocampal synaptosome due to its high synaptic contacts ratio compared to the other brain regions (Cragg, 1975). Materials and methods