Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Animals and reagents

Wistar rats, ¡á, 250*20 g, were purchased from Animal Center of Beijing Medical University. CORT, urethane and a-chloralose were purchased from Sigma Chemicals Co. Ltd.

Recording of LTP in hippocampal dentate gyrus in anesthetized rat

The experiment was conducted according to the method employed by Zhang et al. Briefly, Wistar rats were anesthetized by injection of a mixture of urethane and a-chloralose (ip, 1 g/kg and 25 mg/kg respectively) and fixed in a conventional stereotaxic frame (Narishige Co. Ltd, Japan). A bipolar stainless-steel electrode connected with a electronic stimulator (SEN-3201, Nihon Kohden Co. Ltd, Japan) was inserted into the left entorhinal cortex (8.1 mm posterior to bregma, 4.4 mm lateral to midline and approximately 3.0 mm below the dura) to stimulate the perforant pathway. A monopolar recording electrode connected with a microelectrode amplifier (MEZ-8201, Nihon Kohden Co. Ltd, Japan) was placed in the granule cell layer of the ipsilateral dentate gyrus (3.5 mm posterior to bregma, 2.0 mm lateral to midline and about 3.5 mm below the dura). The depths of both stimulating and recording electrodes were adjusted to obtain a desired response displayed on an oscilliscope (VC-10, Nihon Kohden Co. Ltd, Japan). A single test stimulus (0.08 ms duration) was applied at a constant interval of 30 seconds and the evoked field potential was recorded extracellularly and printed out using a pen-printer. The stimulating intensity was set at a level of 50 % of the population spike (PS) of the maximum amplitude. A brief tetanic stimulation (60 Hz, 30 pulses) was applied at the same stimulus intensity to generate LTP after 60~70 minutes calibration until the amplitude of PS became stable. The evoked potentials were recorded for 60 minutes after the tetanus. The effects of COTR on both the time-course curves of potentiation and the area under the curve (AUC) from 5~60 minutes after application of tetanus were assessed. LTP was considered to occur when the tetanus-potentiated spike amplitude was maintained at a level of 30 % higher above the baseline and lasted at least for 30 minutes.

Primary hippocampal and cerebral cortex culture

The whole brains were taken from day 18 embryo Wistar rats (Animal Center of Beijing Medical University) and the hippocampi and cerebral cortex were dissected. Primary neuronal cultures of the hippocampus and cerebral cortex were prepared as Banker and Cowan. The hippocampi and cerebral cortex were pooled and enzymatically dispersed with 0.125% trypsin and 0.01% DNase I (Sigma) at 37 ¡æ for 20 min. the cells were washed and counted in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) containing 2 mM glutamine, 5.0 mM glucose , 10 % fetal calf serum (FCS) and 10 % horse serum, and then plated into 96-well plates which had been coated with 0.1 ml of 10 ug/mL of poly-D-lysine (Sigma) at a density of 5.0*104 cells/well in 100 uL. The cultures were incubated at 37 ¡æ in 5 % CO2-95 % air. The medium was changed twice a week until use.

Ca2+ currents recording

Hippocampal neurons in 7~10 days were used and recordings of evoked Ca2+ currents were carried out at 20~25 ¡æ using the whole-cell patch-clamp technique as previously described by Sakmann. The patch-pipette was filled with solution containing CsCl 140, HEPES 10, egtazic acid 10 and ATP 2 mM with the electric resistance of 1~5 MW. The extarcellular solution contained NaCl 140, KCl 5, Ca2Cl 3, MgCl2 1, HEPES 10, glucose 10, tetraethylammonium chloride 140, tetrodotoxin 0.001 mM. Following seal formation and prior to entering the whole-cell mode, electrode capacitance was neutralized by using the capacitance compensation circuitry of Axopatch 1D (Axon Instruments, Foster City, CA). In the whole-cell mode, the Axopatch was further adjusted to correct for 80~85 % of the series resistance. Command potential sequences were delivered to the patch-clamp amplifier and the data were simultaneously collected with a computer. Evoked currents were filtered at 10 kHz (-3 dB, 8-pole low-pass Bessel filter, Frequency Devices, Haverhill, MA), digitally sampled at 500 usec per point and stored on magnetic media in digital form for later analysis. Capacitative and leakage currents digitally subtracted from all records, which was carried out on line by using pCLAMP 5.5.1 (Axon Instruments).

Applications of drugs

CORT was applied by a puff pipette connected to a pressure injector (BH-2, Medical System Co. Ltd). The puff pipette was consisted of 3 microtubes with a diameter of 5~10 um. The distance between the pipette and the recorded neuron was 20~30 um, and 50~60 kPa of N2 pressure was delivered.

Statistics

The results from LTP experiment were expressed with means*standard errors (x*se). The amplitudes of CORT-induced Ca2+ currents (ICa) were measured with the program of pCLAMP 5.5.1. Mean values were given with means*standard deviation (x*s). The program of Statistics Analysis System (SAS) was used for ANOVA analysis followed by Duncan test.

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