The Roles And Mechanisms Of HCN Channels And GABAA Receptor In Thalamocortical Circuit Involving In The General Anesthesia Of Propofol

Background:The mechanisms of general anesthetics is always an important issue to be solved.In recent studies,combined with γ-aminobutyric acid A(GABAA)receptor,a hyperpolarization activated potassium ion channel,hyperpolarization-activated,cyclic nucleotide gated(HCN)ion channel,which is widely expressed in central nervous system,especillay in cortex and thalamus,has been demonstrated to be involved in the hypnotic actions of general anesthetics.However,the effects of HCN ion channels and GABAA receptors on the hypnotic actions of general anesthetics,and the interaction between the HCN ion channels and GABAA receptors are still unclear.Objective:The effects of the HCN channels blocker,ZD-7288,and the GABAA receptors agonist,GABA,on the propofol induced loss-of-righting reflex(LORR)in mice was studied by pharmacological methods,and explore the effect of HCN ion channels and GABAA receptors on the hypnotic actions of general anesthetics along with their interaction.Methods:We firstly detected the expression of HCN channels and GABAA receptor in cortex and thalamus by RT-PCR.C57/BL6 male mice,weighing 22 ± 2 g,were used to induce the LORR by tail vein injection of propofol,and the median effective concentration(LORR EC50)of LORR for propofol was caculated.We then examined the effects of intracerebroventricular injection of ZD-7288 and GABA on the LORR EC50 of propofol and LORR,and evaluated the interaction between HCN ion channels and GABAA receptors.Co-immunoprecipitation was used to identify the interaction between HCN channels and GABAA receptor in cortex and thalamus.Results:HCN1 and HCN2 channels were highly expressed in both cortex and thalamus while the expression of HCN3 and HCN4 channels were relative low.Intracerebroventricular injection of different concentration of ZD-7288(0～50 μg)significantly decreased the LORR EC50 of propofol;the similar results were observed after intracerebroventricular injection of GABA(0～150 μg),the LORR EC50 of propofolwas dramatically reduced after the after intracerebroventricular injection of GABA in a dose-dependent manner.Interestingly,the intracerebroventricular injection of ZD-7288(50 μg)and GABA(150 μg)produced LORR in mice which indicated that ZD-7288 or GABA could simulate the hypnotic action of propofol.Moreover,a synergistic action between ZD-7288 and GABA was observed by pharmacological measure.Co-immunoprecipitation technique indicated that both HCN1 and HCN2 channels co-localized with GABAA receptor in cortex and thalamus.Conclusion:The hypnotic actions of propofol were closely correlated with HCN ion channels and GABAA receptors,and there was a synergistic action existed in the hypnotic action of propofol between HCN ion channels and GABAA receptors.Background:With some insight into the principle of general anesthesia,it is found that there are many inconsistencies in the role of general anesthetics between the molecular level and their macro effects.Recent studies indicated that the changes in electroencephalogram(EEG),especially the effect of general anesthetics on low frequency EEG,during general anesthesia were related to the effects of general anesthetics.HCN ion channels and GABAA receptor have been considered to be the important mediators involved in the generation of low frequency EEG.However,the exact mechanisms are still unclear.Objective:To investigate the effect of propofol,HCN ion channel antagonist and GABAA receptor agonist on the low frequency EEG,especially the slow wave oscillation,we analyze the role of HCN ion channel and/or GABAA receptor in propofol-induced slow wave oscillation(<1 Hz)of EEG.Methods:Firstly,in order to clarify the effect of intravenous propofol on EEG in mice,adult male C57/BL6 mice,weighing 22 ± 2 g,metal electrodes were used to embedded in the scalp.The fontal lobe was implanted in one metal electrode while the occipital lobe was implanted in two metal electrodes and one of the electrodes was used as the reference electrode.The EEG recording system was quickly connected to observe the changes of EEG after the tail vein injection of propofol.Subsequently,we observed the effect of intracerebroventricular injection of HCN channel blocker ZD-7288 and GABAA receptor agonist GABA on propofol-induced EEG.Finally,we investigated the effects of the single use of ZD-7288 or GABA and the combining use of ZD-7288 and GABA on EEG.Results:Tail vain injection of propofol significantly increased the slow wave oscillation of mice in a dose-dependent manner.Intracerebroventricular injection of ZD-7288 and GABA promote the propofol-induced slow wave oscillation.Intracerebroventricular injection of propofol,ZD-7288 or GABA could also increased the slow wave oscillation in dose-dependent manner in mice.The results indicated that the HCN ion channels and GABAA receptor were involved in propofol-indcued slow wave oscillation in mice by analyzing the effects of intracerebroventricular injection of LORR EC50 of ZD-7288 and GABA on the slow wave oscillation.Conclusion:Intravenous injection of propofol could induce the slow wave oscillation and the intracerebroventicular injection of ZD-7288 and GABA significantly increased these effects.We concluded that HCN channels and GABAA receptor were involved in propofol-induced slow wave oscillation.Background:The cerebral electrical activity has been implicated to be involved in the general anesthetics status induced by general anesthesia.Specially,the slow wave oscillation resulting from the thalamocortical circuit is closely correlated with the general anesthesia.HCN channels and GABAA receptor are widely expressed in various neurons in thalamocortical circuit,which are considered to play an important role in signal transduction of thalamocortical circuit.However,the exact mechanisms are still unclear.Objective:We first identify the cellular localization of HCN channels and GAB AA receptor in thalamocortical Then we investigate the effect of propofol,ZD-7288 and GABA on resting membrane potential,membrane input resistance,excitatory post-synaptic current and inhibitory post-synaptic current in reticular thalamic neuron and cortical pyramidal neuron.Finally,the effect of propofol,ZD-7288 and GABA on the signal transduction between reticular thalamic neuron and cortical pyramidal neuron is illustrated.By these expreiments,we will analyze the role of HCN channels and GABAA receptor in these effects.Methods:Immunoflourescent staining was used to identify the cellular localization of HCN channels and GABAA receptor in thalamocortical circuit,and whole cell patch clamp was used to record the resting membrane potential,membrane input resistance,excitatory post-synaptic current and inhibitory post-synaptic current.The effect of propofol,ZD-7288 and GABA on these index was then studied.We also used the electrical stimulation technique to investigate the signal transduction between the reticular thalamic neuron and cortical pyramidal neuron and determine the effect of propofol,ZD-7288 and GABA on the signal transduction.Results:HCN1 and HCN2 channel were widely co-localized with the GABAA receptor in cortical layer V pyramidal neuron and reticular thalamic neuron.Propofol,ZD-7288 and GABA significantly increased the resting membrane potential,membrane input resistance,and up-regulated the frequency and ampitude of inhibitory post-synaptic current in cortical pyramidal neuron and reticular thalamic neuron.However,there was hardly no effect on excitatory post-synaptic current.The evoked inhibitory post-synaptic current was recorded after stimulating the reticular thalamic neuron.Furthermore,propofol,ZD-7288 and GABA significantly increased the density of the evoked inhibitory post-synaptic current.Conclusion:Thalamocortical circuit plays an important role in propofol-induced general anesthesia.HCN channels and GABAA receptor are involved in the signal transduction in thalamocortical circuit.