HCN Currents Protect Hypoxic-ischemic Damage Of Hippocampal CA1 Pyramidal Neuron In Neonatal Rats

Objective:By using the immunohistochemistry and whole-cell patch-clamp techniques,investigate the distribution of hyperpolarization-activated cyclic nucleotide-gated cation channel(HCN)channels in the hippocampal CA1 region,research the change of Ih before and after HI treatment,observe the effect of HCN channel antagonist on cell viability after hypoxic-ischemic treatment,in order to find new treatment target for HIE.Methods:1.ImmumohistochemistryImmunohistochemistry techniques for HCN1-4 channel subtype labeling were from neonatal Sprague-Dawley rats hippocampal slices and the distribution of HCN channel protein in the CA1 area and pyramidal neurons were observed by using the laser scanning confocal microscope.Quantification of neuronal death under control,hypoxic-ischemic treatment with or without Cs Cl(HCN channel antagonist)in the hippocampal CA1 region were determined by To-pro-3 staining.2.Electrophysiologic recordingBy using the whole-cell patch-clamp technique,Ih was recorded in hippocampal CA1 pyramidal neurons from acutely dissociated brain slices.The amplitude of Ih,activation kinetics,and spontaneous discharge before and after hypoxic-ischemic treatment were observed.Results:1.HCN1-4 channels were expressed in hippocampal CA1 region heterogeneously.2.Membrane hyperpolarization could generate a voltage-dependent,fast activated and slowly deactivated Ih,which was mediated by HCN channel.3.The amplitude of Ih in pyramidal neurons was increased after hypoxicischemic treatment.On the contrary,the frequency of spontaneous discharge was decreased.4.Hippocampal CA1 pyramidal neurons gradually died after the hypoxic-ischemic treatment with time.HCN channel antagonist Cs Cl further increased the neuronal damage.Conclusion:HCN channels are expressed in hippocampal CA1 of neonatal rats.Amplitude of Ih currents are increased after hypoxic-ischemic treatment in CA1 pyramidal neurons,whereas the frequency of spontaneous discharge was decreased.Moreover,the antagonist of HCN channel could aggravate the neuronal death.Our results indicatethat the increase of Ih amplitude may protect the neuron after hypoxic-ischemic injury.