Effects Of Urocortin Ⅱ On The Activity Of Paraventricular Nucleus Parvocellular Endocrine Neurons In Vitro In Rats

[Objective]Using whole-cell patch-clamp recording technique, neuropharmacology and imunohistochemistry methods to explore the mechanisms of UCN Ⅱ affect the spontaneous spike firing activity and membrane potential of paraventricular nucleus (PVN) parvocellular endocrine neurons in vitro in rats.[Methods]Wistar rats (15-26-day old) were used in this study. After the rats were anesthetized by isofluran, the whole brain was isolated and put into ice-cold artificial cerebral spinal fluid (ACSF). PVN including coronal slices were prepared using a vibratome. The thickness of the slices was 250 μm. The slices were incubated in 95% O2 and 5%CO2 bubbled ACSF at 24-25 ℃ for 1 hour. The compositions of ACSF were as following:118 mM NaCl,3 mM KCl,1 mM MgCl2.6H2O,1 mM NaH2PO4.2H2O,25 mM NaHCO3,10 mM D-Glucose,2 mM CaCl2; PH:7.25-7.35, with osmolarity adjusted to 295-300 mOsM. The recording electrodes were filled with 7 μl internal solution, which consisting of 120 mM potassium gluconate,10 mM HEPES,1 mM EGTA,5 mM KC1,3.5 mM MgCl2,4 mM NaCl,8 mM biocytin,4 mM Na2ATP and 0.2 mM Na2GTP. The pH was adjusted to 7.3 with KOH. Patch pipette resistances were 5-7 MΩ in the bath, with series resistances in the range of 10-20 M The whole cell recordings from PVN parvocellular neurons were performed by whole cell path clamp recording system. The acquired data were saved in the hard disc. After the electrophysiological recording experiments ended, the slices were fixed in 4% paraformaldehyde in 0.1 PBS (PH 8) at room temperature over 24 hours, and then the slices was stained in avidin-biotin complex(DAB staining). The location and histological characterization of the recording neurons in the issue were determined using a microscopy and the staining results were acquired through taking pictures. Electrophysiological data were analysis 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 mode, PVN parvocellular endocrine neurons were sensitive to injected currents, but they expressed less inward rectification, transient outward rectification, low threshold spikes (LTS) and hyperpolarization activated inward currents (Ih).2. Under current-clamp conditions, UCN II induced a significant decrease in spike firing rate of 34.7% PVN parvocellular endocrine neurons, which were recovery after washout. The UCN Ⅱ-induced inhibition in spontaneous spike firing rate was enhanced by increasing UCN II concentration.3. UCN II induced inhibition of spontaneous spike firing accompanied with a hyperpolarization of membrane potential. UCN Ⅱ-induced hyperpolarization of membrane potential was insensitive to tetrodotoxin (TTX).4. In the presence of TTX, UCN Ⅱ-induced hyperpolarization of membrane potential was dose-dependent. The half-inhibition concentration (IC50) is about 37 nM.5. UCN II significantly inhibited the number of injected current-evoked action potentials, and significantly decreased input resistance.6. Current-voltage relationship curves indicated that the reversal potential of UCN II-sensitive currents was close to potassium equilibrium potential.[Conclusion]Our present study indicated that UCN II activated potassium channels resulting in a hyperpolarization and a decrease in spontaneous firing rate of a subpopulation PVN parvocellular endocrine neuron. Our results suggested that UCN II directly acted on a part of PVN parvocellular endocrine neurons and involved in regulation of these neuronal endocrine function.