The Role Of Hippocampal TASK-3 Ion Channels In Memory Inhibition Induced By Sevoflurane And Its Mechanism

Part I: The effects of sevoflurane anesthesia on hippocampus-dependent fear memory in miceObjective: The inhibition of memory by general anesthetics is an important connotation of general anesthesia.Sevoflurane is a commonly used inhaled anesthetic in clinic,and its inhibitory effect on memory has been widely confirmed.Currently,the hippocampus is considered to be a core brain region that regulates memory function.However,the mechanisms by which sevoflurane inhibits the hippocampus-dependent memory remain unclear.The contextual fear conditioning experiment and inhibitory avoidance experiment are currently well-recognized behavioral methods for detecting the hippocampus-dependent fear memory,which are widely used to evaluate the effects of anesthetics on memory function.Therefore,this part of the experiment first investigated the effects of different concentrations of sevoflurane anesthesia on hippocampus-dependent fear memory in mice.Methods: The C57BL/6 mice(male,6-8 weeks)were used as experimental subjects.The median effective concentration(EC50)of loss of righting reflex(LORR)in mice induced by sevoflurane inhalation was determined by righting reflex test.The effects of different concentrations of sevoflurane(0.1×,0.2×,0.3× LORR EC50)on hippocampus-dependent fear memory in mice were tested by contextual fear conditioning test and inhibitory avoidance test.In the contextual fear conditioning test,the percentage of freezing time and number of freeing bouts of mice were used as evaluation indexes.In the inhibitory avoidance experiment,the latency of avoiding darkness and the number of times entering the darkroom of mice were used as evaluation indexes.Results: 1)The LORR EC50 of mice induced by sevoflurane inhalation was 1.35%(95% CI,1.3to 1.4%).The effect of sevoflurane on hippocampus-dependent fear memory of mice was respectively detected at 0.1×,0.2×,0.3× LORR EC50,and the corresponding concentration of sevoflurane was 0.14%,0.27%,0.41%.2)In the contextual fear conditioning experiment,compared with the control group,sevoflurane at the concentration of 0.14% significantly reduced the percentage of freezing time(30.2 ± 1.0% vs.9.4 ± 0.6%,P < 0.001)and number of freezing bouts(18.3 ± 0.7 vs.11.6 ± 0.6,P < 0.001)in mice.With the increase of sevoflurane concentration,the percentage of freezing time and number of freezing bouts in sevoflurane-anesthetized mice were further decreased,and the differences were significant compared with the previous concentration.3)In the inhibitory avoidance experiment,compared with the control group,sevoflurane at the concentration of 0.14% significantly reduced the latency of avoiding darkness(577.8 ± 9.6 s vs.167.3 ± 10.8 s,P < 0.001)and increased the number of times entering the dark room(1.8 ± 0.5 vs.6.1 ± 0.7,P < 0.001)in mice.With the increase of sevoflurane concentration,the latency of avoiding darkness of sevoflurane-anesthetized mice was further shortened,and the number of times entering the darkroom was further increased,and the differences of effect were significant compared with the previous concentration.Conclusion: Sevoflurane can effectively suppress the hippocampus-dependent fear memory in mice at 0.14%(0.1× LORR EC50),and the inhibitory effect of sevoflurane on memory was enhanced in a dose-dependent manner.Part ?: The effects of sevoflurane anesthesia on local field potentials and theta rhythm in hippocampal CA3 region of miceObjective: The hippocampus is a core brain area for learning and memory.The activity of neurons in the hippocampal circuit is characterized by rhythmic fluctuations of local field potentials(LFPs).Theta rhythm is one of the characteristic rhythms of LFPs,which can be produced in hippocampal CA3 region and has been widely demonstrated to be involved in memory function.A large number of studies have shown that the regulation of theta rhythm in the brain can affect the memory function.Meanwhile,successful memory formation is accompanied by a decrease in the power of theta rhythm.This suggests that theta rhythm is involved in regulating the formation of memory and is an important electrophysiological indicator of memory function.It is not clear whether theta rhythm is involved in the inhibitory effect of sevoflurane on memory.Therefore,this part of the experiment explored the effects of sevoflurane anesthesia,which can inhibit memory,on LFPs and theta rhythm in hippocampal CA3 region of mice.Methods: The C57BL/6 mice(male,6-8 weeks)were used as experimental subjects.A metal electrode was respectively implanted in bilateral hippocampal CA3 area of mice(-1.7 mm to Bregma;2.1 mm from midline;2.1 mm under the skull),and a reference electrode was implanted above the cerebellum(-2.0 mm from Lambda,0.0 mm from midline).After electrodes implantation and recovery for one-week for experimental animals,the metal electrodes were connected to the Power Lab EEG recording and analysis system to record the changes of LFPs(1-50 Hz)in hippocampal CA3 region of mice before and during sevoflurane anesthesia(0.27%,0.2× LORR EC50),and the behavior of mice was recorded with the camera system.After LFPs recording,the location of the electrodes was confirmed by brain dissection.Results:1)After sevoflurane anesthesia,the amplitude,power density and power spectrum amplitude of low frequency rhythm(1-30 Hz)in LFPs(1-50 Hz)of hippocampal CA3 region in mice and the total power of each band in this frequency range were higher than those before anesthesia(P < 0.05),while the amplitude,power density,power spectrum amplitude and total power of 30-50 Hz band were not significantly different from those before anesthesia.2)Compared with those before sevoflurane anesthesia,the amplitude,power density and power spectral density of theta rhythm(4-12 Hz)in LFPs were significantly increased after sevoflurane anesthesia.After sevoflurane anesthesia,the total power of theta rhythm in LFPs was also significantly higher than that before anesthesia(27.6 ± 3.7 d B vs.51.8 ± 6.2 d B,P < 0.05).Conclusion: Sevoflurane enhanced the amplitude,power density,power spectral density and total power of the theta rhythm in hippocampus CA3 region while it inhibited memory.It is inferred that the inhibitory effect of sevoflurane on memory is related to the enhancement of theta rhythm in hippocampal CA3 region.Part ?: TASK-3 ion channels mediate the regulation of sevoflurane on hippocampal theta rhythm and memory functionObjective: According to the above studies,sevoflurane can enhance the theta rhythm of hippocampal CA3 region in mice while inhibiting memory,but the mechanism is unclear.TASK-3 ion channels are one of the targets of sevoflurane,which are abundantly expressed in the hippocampus,and can mediate the enhancement of theta rhythm induced by halothane.We speculated that the TASK-3 ion channels in hippocampal CA3 region may be a potential target for sevoflurane to regulate hippocampal theta rhythm.Therefore,this part of the experiment explored the role of TASK-3 ion channels in the regulation of hippocampal theta rhythm and memory function by sevoflurane.Methods: Firstly,whole-cell patch clamp technique was used to detect the effect of sevoflurane(0.5 m M)on the function of TASK-3 ion channels transfected into HEK293 cells.Then,the adeno-associated viruses expressing NT(none-target)sh RNA(control group)or TASK-3 sh RNA(experimental group)were injected into the CA3 region of the bilateral hippocampus of the mice,and the knockdown efficiency was detected by immunofluorescence technique.Subsequently,the in vivo LFPs recording technique was used to detect the effect of knockdown of TASK-3 ion channels on the regulation of the hippocampal theta rhythm by sevoflurane.Finally,the contextual fear conditioning test and inhibitory avoidance test were used to detect the effect of knockdown of TASK-3 ion channels on the inhibitory effect of sevoflurane on memory function.Results: 1)The whole-cell patch clamp experiment showed that sevoflurane(0.5 m M)could activate the TASK-3 ion channels and increase the outward current of potassium ions.2)The immunofluorescence results showed that compared with the mice in NT sh RNA injection group,the expression of TASK-3 ion channels in hippocampal CA3 region ofmice in TASK-3 sh RNA injection group was significantly decreased,and the number of positive cells labeled by TASK-3 antibody was significantly reduced(P < 0.001).3)The results of LFPs recordings in vivo showed that after sevoflurane anesthesia,the amplitude,power density and total power of hippocampal theta rhythm in mice of NT sh RNA injection group were increased compared with those before anesthesia,while,the amplitude,power density and total power of hippocampal theta rhythm in mice of TASK-3 sh RNA injection group were not significantly different from those before anesthesia,suggesting that the sevoflurane-induced enhancement of hippocampal theta rhythm was inhibited after knocking down the expression of TASK-3 ion channels.4)The results of the contextual fear conditioning test showed that,compared with the mice in NT sh RNA injection group,knockdown of TASK-3 ion channels reduced the movement trajectory and average movement speed of sevoflurane-anesthetized mice(32.0 ± 0.4 mm/s vs.22.0 ± 0.4 mm/s,P < 0.001),and increased the percentage of freezing time(6.8 ± 0.3% vs.17.0 ± 0.2%,P < 0.001)and number of freezing bouts(7.8 ± 0.3 vs.16.9 ± 0.2,P < 0.001)of sevoflurane-anesthetized mice,indicating that the inhibitory effect of sevoflurane on hippocampus-dependent fear memory in mice was weakened after the expression of TASK-3 ion channels in hippocampal CA3 region was down-regulated by sh RNA.5)The results of inhibitory avoidance test showed that compared with the mice in NT sh RNA injection group,knockdown of TASK-3 ion channels prolonged the latency of dark avoidance of sevoflurane-anesthetized mice(131.4 ± 9.9 s vs.305.3 ± 13.5 s,P < 0.001),and decreased the number of times entering the darkroom of sevoflurane-anesthetized mice(12.6 ± 0.8 vs.5.3 ± 0.5,P < 0.001),indicating that the inhibitory effect of sevoflurane on hippocampus-dependent fear memory in mice was weakened after the expression of TASK-3 ion channels in hippocampal CA3 region was down-regulated by sh RNA.Conclusion: Knockdown of TASK-3 ion channels expression in hippocampal CA3 region of mice attenuated the enhancing effect of sevoflurane on hippocampal theta rhythm and decreased the inhibitory effect of sevoflurane on memory.It is inferred that the enhancement of hippocampal theta rhythm mediated by TASK-3 ion channels is involved in the inhibitory effect of sevoflurane on hippocampus-dependent fear memory in mice.Part ?: The role of TASK-3 ion channels in the inhibitory effect of propofol on memoryObjective: The above studies found that sevoflurane can inhibit memory by enhancing the TASK-3 ion channels-mediated hippocampal theta rhythm.In clinical anesthesia,both intravenous anesthetics and inhaled anesthetics have been widely used,and both of them can inhibit memory.However,the role of TASK-3 ion channels in inhibitory effect of intravenous anesthetics-propofol on memory is not clear.Therefore,this part of the experiment explored the role of TASK-3 ion channels in the regulation of hippocampal theta rhythm and memory function by propofol.Methods: The C57BL/6 mice(male,6-8 weeks)were used as experimental subjects.The median effective concentration(EC50)of loss of righting reflex(LORR)in mice induced by intraperitoneal injection of propofol was determined by righting reflex test.The effects of different concentrations of propofol(0.1×,0.2×,0.3× LORR EC50)on hippocampus-dependent fear memory in mice were tested by contextual fear conditioning test and inhibitory avoidance test.The in vivo local field potentials technique was used to record the changes of local field potentials of hippocampal CA3 region of mice before and during propofol anesthesia.The expression of TASK-3 ion channels in CA3 region of bilateral hippocampus of mice was knocked down by adeno-associated virus exogenously using stereotaxic injection technique to detect the effect of TASK-3 knockdown on the regulation of hippocampal theta rhythm and memory function by propofol.Results: 1)The LORR EC50 of mice induced by intraperitoneal injection of propofol was 70.8 mg/kg(95% CI,65.1 to 76.4 mg/kg).The effect of propofol on hippocampus-dependent fear memory of mice was respectively detected at 0.1×,0.2×,0.3× LORR EC50,and the corresponding concentration of propofol was 7.1 mg/kg?14.2 mg/kg?21.2 mg/kg.2)In the contextual fear conditioning experiment,there was no statistical difference in the percentage of freezing time and number of freezing bouts between propofol-anesthetized mice and control mice when propofol was used at 7.1 mg/kg and 14.2 mg/kg.When propofol was used at 21.2 mg/kg,the percentage of freezing time and number of freezing bouts in propofol-anesthetized mice were significantly lower than those in the control group(34.8 ± 1.0% vs.22.8 ± 1.6%,21.1 ± 0.6 vs.14.1 ± 0.6,P < 0.001).The results showed that propofol could inhibit the hippocampus-dependent fear memory in mice at 21.2 mg/kg.3)In the inhibitory avoidance experiment,there was no statistical difference in the latency of avoiding darkness and number of times entering the darkroom between propofol-anesthetized mice and control mice when propofol was used at 7.1 mg/kg and 14.2 mg/kg.When propofol was used at 21.2 mg/kg,the latency of avoiding darkness of propofol-anaesthetized mice was significantly shorter than that of control mice(565.7 ± 11.4 s vs.362.9 ± 18.5 s,P < 0.001),and the number of times entering dark room was significantly higher than that of control mice(0.9 ± 0.3 vs.8.4 ± 0.9,P < 0.001).It is suggested that propofol can inhibit the hippocampus-dependent fear memory in mice at 21.2 mg/kg.4)Compared with that before propofol anesthesia,there was no significant change in theta rhythm(amplitude,power density,power spectral density,total power)in hippocampal CA3 region of mice after propofol anesthesia at non-memory inhibition concentration(14.2 mg/kg).After propofol anesthesia at memory inhibition concentration(21.2 mg/kg),the amplitude,power density,power spectral density and total power of theta rhythm in hippocampal CA3 region of mice were significantly increased,and this enhancement remained unchanged after knocking down the expression of TASK-3 ion channels.The results indicated that TASK-3 ion channels could not mediate the effect of propofol on hippocampal theta rhythm.5)Compared with the mice in NT sh RNA injection group,the percentage of freezing time and number of freezing bouts of propofol-anesthetized mice were not significantly changed after knockdown of TASK-3 ion channels,and the latency of avoiding darkness and number of times entering the dark chamber of propofol-anesthetized mice were also not significantly changed after knockdown of TASK-3 ion channels.These results indicated that TASK-3 ion channels could not mediate the inhibitory effect of propofol on memory.Conclusion: Propofol can enhance the hippocampal theta rhythm when it inhibits memory.TASK-3 ion channels do not play a role in the regulation of hippocampal theta rhythm and memory function by propofol.