Effects Of Nicotine On The Activity Of Paraventricular Nucleus Magnocellular Neurons In Vitro In Rats

[Object i ve]Using whole-cell patch-clamp recording accompanied with biocytin staining technique and neuropharmacology methods to study the effective mechanisms of nicotine on the activity of magnocellular paraventricular nucleus (PVN) neurons in vitro in rats.[Methods]Male Wistar rats (2-3week old) were anesthetized by a small amount of isofluran, the brain was isolated and immerged into ice-cold artificial cerebral spinal fluid (ACSF). Coronal slices were 250μm in thickness, including PVN, and were prepared from P10-21-day rats using a vibratome. After incubated one hour in ACSF which was bubbled with 95% O2 and 5% CO2 at room temperature. The ACSF contained (in mM):118 NaCl,3 KCl,1 MgCl2.6H2O,1 NaH2PO4.2H2O,25 NaHCO3,10 D-Glucose,2 CaCl2; PH:7.3-7.4, with osmolarity adjusted to 295-305 mOsM. The recording electrodes were filled with 10 μl internal solution, with the resistance of 4-6 MΩ. The effects of nicotine on spontaneous firing rate, membrane potential and postsynaptic potential of PVN magnocellular neurons were examined by whole cell patch-clamp recording accompanied with biocytin staining technique and neuropharmacology methods. Whole-cell patch clamp was performed by an Axopatch-700B amplifier and acquired using Clampex 10.4 software and a Digidata 1440A analog-to-digital interface. At the end of experiments, the slices were fixed in 4% paraformaldehyde in 0.1 PBS (pH 8) at room temperature for 24 hours. Tissue was reacted overnight in avidin-biotin complex (ABC Elite kit, Vector Laboratories, Burlingame, CA) at room temperature. Biocytin was detected using 3, 3’-diaminobenzidine tetrahydrochloride histochemistry. The histological characterization was determined using a microscopy (Leica 500B). Electrophysiological data were analyzed using Clampfit 10.4 software. Differences between the mean values recorded under control and test conditions were evaluated by Student’s paired t-test using SPSS software. Differences were considered significant at P< 0.05.[Results](1) Under current-clamp recording conditions, application of nicotine (1μM) induced a decrease in spontaneous firing rate accompanied with a hyperpolarization of membrane potential in 38.7% PVN magnocellular neurons.(2) The nicotine induced inhibition of spontaneous activity of PVN manocellular neurons was dose-dependent. The half inhibition concentration (IC50) is 17 μM.(3) The nicotine induced-inhibition of spontaneous activity of PVN magnocellular neurons was sensitive to tetradotoxin (TTX,0.5 μM). Application of TTX blocked spontaneous spike firing and nicotine-induced hyperpolarization.(4) Application of nicotine induced an increase in frequency of inhibitory postsynaptic potentials (IPSPs), without changing the amplitude of IPSPs. [Conelusion] Our present results indicated that nicotine significantly decreased the spontaneous firing rate of PVN magnocellular neurons via enhancing presynaptic inhibitiory inputs, suggesting that nicotine inhibited the neuronal spontaneous activity and modulated secretion of neuronal bioactive substance in PVN magnocellular neurons through presynaptic mechanisms.