| Objective:Central nervous system toxicities and generalized convulsions arefrequently happened in local anesthetics. It has been shown that intracellular Ca2+ in hippocampal CAi neurons is elevated during convulsion. This Ca2+ overload has been suggested to be involved in local anesthetics-induced convulsion brain damage. However, the influx pathways mediating the raised intracellular Ca2+ during convulsion are still unclear. It is becoming increasingly recognizd that voltaged-gated calcium channels (VGCCs), particularly the L-type, can be a route for toxic levels of Ca'+ influx after a number of insults. Furthermore, VGCCs represent a major Ca2+ entry pathway even during the activation of ligand-gated channels such as NMDA receptors. In addition, L-type Ca2+ channels are prevalent in hippocampal pyramidal neurons, contributing 30%~50% of total calcium current. So, we speculate L-type calcium channels likely play a crucial role in hippocampal CA1 pyramidal neuronal damage during local anesthetics-induced convulsions. The aim of our present study was to examine whether L-type calcium currents in CA1 pyramidal neurons of adult rat hippocampal were changed after giving several concentrations of Lidocaine using cell-attached configuration of patch clamp techniques.Methods:Pyramidal cells in hippocampal CAi region were dissociated acutely from SD adult rats weighting 200-250g using procedures as described previously. Briefly, SD rats were anesthetized with chloral hydrate (i.p., 40mg/100g weight) and then decapitated. Brains were quickly removed, iced, and blocked for slicing. The blocked tissue was cut into 400um- thick slices with a Vibroslice whilst bathed in a low Ca2+, HEPES-buffered salt solution. Slices were incubated for 1-6 h at room temperature (20-22癈) in a NaHCOj-buffered saline bubbled with 95% O2/5% CO2. Slices were then removed into the low Ca~+ buffer, and CAi region of hippocampus was dissected out with two pinheads and placed into an oxygenated chamber containing pronase (Sigma protease Type XIV, 1-1.5 g/L) in HEPES-buffered HBSS (Sigma) at 33癈. After 30-45 min of enzyme digestion, tissue was rinsed three times in the low Ca +, HEPES-buffered saline and dissociated mechanically with a graded series of fire-polished Pasteur pipettes. The cell suspension was then placed into a 35 mm Lux petri dish mounted on the stage of a microscope (Olympus IX-70) containing HEPES-buffered HBSS saline. After allowing the cell to settle, the solution bathing the cells was changed to our recording solution. The concentrations of Lidocaine were 0, 2, 4, 8, 16, 32 ug/ml. Lidocaine was respectively infused recording solution in turn in our experimentation.Cell-attached recording pipettes were pulled from glass capillary tubes using a micropipette puller (model PC-10). The pipette resistance was 6-9 MQ. and the seal resistance was in excess of 5 GQ. Recordings were obtained according to standard patch-clamp methods using an Patch/Whole Cell Clamp Amplifier (CEZ-2400), with the current filtered at 1 kHz and sampled at 5 kHz. Voltage pulses were delivered at 5sec intervals. Linear leak and capacitive currents were subtracted digitally. Electrophysiological recordings were performed at room temperature (22-24癈). Voltage commands were generated, and current responses were recorded and analyzed using a computerized acquisition and storage system (pCLAMP; version 8.0, Axon Instruments).Results:1. The acute isolated adult rat hippocampal CAi pyramidal neurons preserved fine activity. Every plump intact neurons had a clean smooth surface, exhibited a well-kept long axon and some proximal dendritic structures. The L-type VGCCs were distinguished, and the electro-physiological and pharmacological properties of L-type Ca2+ channel in hypothalamic neurons were confirmed. The characteristics of L-type Ca2+ channel in cell-attached patches in hippocampal neurons were consistent with those of reported previously: high activation threshold, no obvious inactivation, high selectivity to Ca2+,...
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