The Modulation Of Potassium Channels In Neonatal Rat Cortical Neurons By Gadolinium

ObjectiveNeurons from rat cerebral cortex were cultured and their growth process was observed.The effect of extracellular Gd3+ on the IA of cortical neurons was investigated by using the whole-cell patch-clamp technique in order to analyze how the different concentration of Gd3+ modulates the IA channels and explore the clues for explaining the structure-function relationship of IA.MethodsThe cortical neurons from newborn Wistar rat were cultured,and the whole-cell patch-clamp technique was used to investigate the changes in activation and inactivation currents of IA in the cultured cortical neurons after extracellular application of Gd3+ in concentrations of 1,10,100,1000μmol/L1.Cell culture and observation:The neonatal rat brain was taken and the cortex was made into suspension with aids of enzyme-digestion and mechanical dissociation,and then the cells were seeded onto the culture plates,and the morphology and growth of neurons were observed on days 1,3,5,7 and 9 under the inverted phase contrast microscope.2.Whole-cell patch-clamp recording:The bright neurons with smooth appearance and uniform reflecting light were chosen on days 7 through 10. Electrodes with tip resistant of 4-6MQ were moved onto neuron to establish the whole-cell mode and carry out whole-cell patch clamp recording. The IA steady-state activation curves and inactivation curves were recorded and fitted with a Boltzmann equationResults1.The morphological observation of the cultured cells:one hour after planted,some cells started to adhere to the containter,later,most cells had adhered and showed their shapes in ellipse,fusiform or irregularity,and some of them with small protrusions already.As time went,the branches became more and longer.The cultured cells growed the fastest on days 5 and the protrusions formed reticular formation on days 7 through 10.2.Effect of extracellular Gd3+ on the IA of the cultured cells:The different concentration of Gd3+ reduced IA peak current,and with the increase of the concentration,Gd3+ suppressed the amplitude of IA further,1,10,100,1000μmol/LGd3+ on the IA of the primary cultured neurons reduced IA peak currents by (10.139±5.549)%,(10.139±5.549)%,(21.24±7.011)%,(32.527±8.485)% and (45.199±10.104)% respectively.3.Effect of extracellular Gd3+ on the IA steady-state activation curves: Contract to the control,the Gd3+ application in all concentrations had no effect on Vh of IA,and had no effect on k of IA in its conentrations of 1,10,100 u mol/L,but increased k of IA statistically in concentrations of 1000μmol/L4.Effect of extracellular Gd3+ on the IA steady-state inactivation curve:Contract to the control,the Gd3+ application in all concentrations had no effect on Vh of IA,and had no effect on k of IA in its conentrations of 1,10,100μmol/L, but increased k of IA statistically in concentrations of 1000μmol/LConclusions1.The primary cultured neurons from neonatal rat cerebral cortex growed the fastest on days 3 through 5 and their protrusions formed reticular formation on days 7 through 10.2.The different concentration of Gd3+ reduced IA peak current in a concent-ration-dependent manner.3.The different concentration of Gd3+ failed to affect steady-state activation curve of IA4.The different concentration of Gd3+failed to affect steady-state inactivat-ion curve of IA...