Sahand Asharfpour

Bio: Sahand Asharfpour is an academic researcher from Babol University of Medical Sciences. The author has contributed to research in topics: Hippocampal formation & Synaptosome. The author has an hindex of 1, co-authored 1 publications receiving 2 citations.

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

Abstract: 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

Non-cholinergic effects of paraoxon on [3h]-GABA release from rat cerebellar giant synaptosomes

Abstract: Diethyl p-nitrophenyl phosphate (paraoxon) is the active toxic metabolite of parathion. Some evidences indicate that OPs affect the GABA system via noncholinergic mechanisms. The purpose of this study was to investigate the effects of paraoxon on K+-evoked [3H]-GABA release from cerebellar synaptosomes. Adult male rats (200 ± 30 g; 3-4 months old) were sacrificed by decapitation and the cerebellum was removed immediately and homogenized. Homogenate was centrifuged twice at 1000 × g for 5 min (all in 0-4 ?C). Synaptosomes were incubated with [3H]-GABA (S.A 99 Ci/mmol, 0.1 µm). Then, aliquots of the synaptosomal suspension were layered on microporous filters at the bottom of superfusion chambers (14900 Superfusion System, Raiteri,s Method, UGO BASILE, Italy). Following 34 minutes of superfusion (time required to equilibrate the system, t = 0), fractions were collected every minute and the radioactivity in the different samples was quantified by liquid-scintillation counting. At t = 8 (s1) and t = 28 (s2), synaptosomes were depolarized with KCl (30 mM). Paraoxon was added to the superfusion medium concomitantly with the second stimulus (s2) and the ratio of s2 /s1 release was compared between the control and test groups. Present data indicate that paraoxon increases spontaneous and K+-evoked [3H]-GABA release from rat cerebellar giant synaptosomes, possibly via noncholinergic mechanisms.