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

[Aim]The hypothalamus paraventricular nucleus (PVN) parvocellular neurons control the activity of autonomic nerves, which play an important role in regulation of cardiovascular function, neuronal endocrine activity and energy metabolism. In vivo experimental studies indicate that nicotine affects food intake, energy metabolism and neuronal endocrine activates via activation of presynaptic nicotinic receptors. However, the mechanisms of nicotine affects PVN parvocellular neurons in vitro are currently unclear. Therefore, we here using whole cell patch-clamp recording accompanied with biocytin staining technique and neuropharmacology methods to study the effects of nicotine on the spontaneous spike firing activity and excitatory postsynaptic currents (EPSCs) of rat hypothalamic PVN parvocellular neurons, and to examine the dose-dependency property of the nicotinic effect and concentration for 50% of maximal effect (EC50). We also explored the histological property of the nicotine sensitive PVN parvocellular neurons. Our study will understand the effective mechanisms of nicotine on the activity of PVN parvocellular neurons, which is contribute to interpret the mechanisms of nicotine regulates the functional activity of cardiovascularand hypothalamic neuronal endocrine.[Material and Methods]Postnatal 14-21-day old male SD rats were deeply anesthetized with isofluran, and killed by decapitation. The brain was rapidly isolated and immerged into ice-cold artificial cerebral spinal fluid (ACSF). The PVN included coronal slices were 250μm in thickness, which were prepared using an automatic vibratome (Leica 1200S). The slices were incubated in ACSF which was bubbled with 95%O2 and 5%CO2 at room temperature for over than 1 hour. The ACSF contained (in mM):118 NaCl,3 KCl,1 MgCl2·6H2O,1 NaH2PO4·2H20,25 NaHCO3,10 D-Glucose,2 CaCl2; PH:7.3-7.4, with osmolarity adjusted to 295-305 mOsM. The recording electrodes were filled with 10μl internal solution, with the resistance of 4-6 MΩ. The effects of nicotine on spontaneous firing rate, membrane potential and postsynaptic potential of PVN parvocellular 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) The somas of PVN parvocellular neurons were 12.6±1.1 μm in diameter. The number of dendrites was 2-6. The mean frequency of spontaneous spike firing was 2.21±0.24 Hz, and the mean membrane potential was -54.7±1.3 mV.(2) Under current-clamp recording conditions, application of nicotine (1μM) induced an increase in spontaneous firing rate accompanied with a depolarization of membrane potential in 46.6% PVN parvocellular neurons.(3) The nicotine induced-increase in spontaneous activity in PVN parvocellular neurons was dose-dependent. The concentration for 50% of maximal effect (EC50) is 13μM.(4) Application of nicotine induced an increase in frequency of excitatory postsynaptic currents (EPSCs), but without changing the amplitude of EPSCs, suggesting that nicotine induced-excitation of PVN parvocellular neurons may be related to the presynaptic action.[Conclusions] Nicotine excites nicotinergic afferents of PVN induces depolarization of membrane potential in a part of PVN parvocellular neurons, resulting in increases in spontaneous spike firing and sEPSCs.