The Role And Mechanisms Of Hippocampal CA1 Ripple Rhythmic Oscillations Involved In Amnesia Induced By Isoflurane

BackgroundGeneral anesthetics have been widely used throughout clinical practice and scientific research,but the mechanisms of their various pharmacological effects(amnesia,unconsciousness,immobility,analgesia,etc.)are still unsolved mysteries in the field of biomedicine.The amnesia of intraoperative events in patients receiving general anesthesia is one of the important connotations of general anesthesia.The amnestic effect of general anesthetics can prevent the generation of intraoperative memory effectively,and prevent the painful memory caused by unintended intraoperative recovery of consciousness.Studies have found that volatile anesthetics can exert amnestic effects at subanesthetic concentrations(lower than those required to induce unconsciousness and immobility effects),resulting in anterograde amnesia.However,the neurobiological mechanism underlying its effects has not been fully elucidated.Understanding the amnestic mechanisms of general anesthetics will help develop safer and more effective general anesthetics and promote a greater understanding of the mechanisms of general anesthesia and memory.Therefore,it is of great clinical merit and scientific implications to study amnestic mechanisms of general anesthetics.Clinical concentrations of volatile anesthetics can act on multiple molecular targets such as ion channels and neurotransmitter receptors to produce anesthetic effects.Studies reported that ion channels and/or neurotransmitter receptors involved in modulating neuronal excitability may contribute to the amnestic effects of volatile anesthetics,including hyperpolarization-activated cyclic nucleotide-gated channels 1,α4-γ-aminobutyric acid receptors,and TWIK-related acid-sensitive K~+channels 3.However,increasing evidence suggests that the contribution of independent molecular targets to the amnestic effect of volatile anesthetics is limited.There remain certain limitations.The amnestic mechanism of volatile anesthetics is still difficult to explain.Studying the amnestic mechanisms of volatile anesthetics at the system network level may be a breakthrough for current studies on the mechanisms of general anesthesia.Hippocampus plays an important role in stimulus perception,memory encoding,consolidation,and retrieval.Target receptors associated with the effects of general anesthesia are widely expressed in the hippocampus.Therefore,the hippocampus is a key brain region to study the mechanisms of amnesia induced by general anesthetics.Complex memory processes require the joint participation of a functional network composed of neuronal populations.Local field potentials(LFPs)in the hippocampus reflect the synchronized firing activity of neuronal populations and play an important role in memory formation and function.Studies have shown that volatile anesthetics at clinical concentrations can affect hippocampal neuronal excitability and LFPs.However,how subanesthetic concentrations of volatile anesthetics affect hippocampal neuronal excitability and LFPs at the system network level,and their relationship with amnestic effects,remains to be explored.Ripple rhythmic oscillations are LFPs in the hippocampal cornu ammonis 1(CA1)evoked by the excitatory inputs from the CA3 region,which are closely related to the synchronous firing activity of pyramidal neurons and interneurons in CA1 region.Studies have confirmed that Ripple rhythmic oscillations in the hippocampal CA1 region are involved in information transmission between brain regions,and play a key role in memory consolidation,retrieval,and multiple neurocognitive functions.Prolonging the duration of Ripple rhythmic oscillations in the hippocampal CA1 region by optogenetic stimulation in vivo can significantly improve the performance in hippocampus-dependent memory tasks in rats.In addition,it has been reported that some clinical concentrations of general anesthetics may suppress the amplitude and duration of hippocampal CA1 Ripple rhythmic oscillations.However,whether subanesthetic concentrations of volatile anesthetics could affect and modulate hippocampal CA1 Ripple rhythmic oscillations,through which mechanisms,and its relationship with amnestic effects,remains unclear.In summary,the amnestic mechanisms of general anesthetics are urgent major scientific problems at present.Combining neuronal firing activity and LFPs,and exploring the amnestic mechanisms of volatile anesthetics at the system network level may be a breakthrough for current studies on the mechanisms of general anesthesia.Therefore,the following scientific hypothesis is proposed based on the above background:subanesthetic concentrations of volatile anesthetics may mediate the effects on Ripple rhythmic oscillations by regulating the excitability and firing patterns of pyramidal neurons and interneurons in the hippocampal CA1 region,and contribute to its mechanisms of amnestic effect.ObjectivesTo study the role and mechanisms of hippocampal CA1 Ripple rhythmic oscillations involved in amnesia induced by volatile anesthetics isoflurane in mice.Part I:To study how isoflurane affects hippocampus-dependent memory,exerts amnestic effect,and its relationship with unconsciousness and immobility in mice.Part II:To study how subanesthetic concentrations of isoflurane affect LFPs in the hippocampal CA1 region,focusing on the effects on Ripple rhythmic oscillations in mice.Part III:To study how subanesthetic concentrations of isoflurane affect the excitability and firing patterns of pyramidal neurons and interneurons in the hippocampal CA1 region in mice.Part IV:To simulate and verify whether subanesthetic concentrations of isoflurane mediate the effects on Ripple rhythmic oscillations by regulating the excitability and firing patterns of pyramidal neurons and interneurons in the hippocampal CA1 region using neural simulation technology.MethodsPart I:Adult C57BL/6 mice(male/female,7-8 weeks)were used for this study.Fear-conditioning and inhibitory avoidance animal behavioral experiments were used to detect the effects of isoflurane on hippocampus-dependent memory.Righting reflex and tail-clamping reflex animal behavioral experiments were used to detect the potencies of unconsciousness and immobility induced by isoflurane.Experiment 1:Fear-conditioning and inhibitory avoidance animal behavioral experiments were used to detect the effects of a series of concentrations of isoflurane on hippocampus-dependent memory in mice,and calculated its median effective concentration(EC50).Experiment 2:Righting reflex and tail-clamping reflex animal behavioral experiments were used to detect the effects of a series of concentrations of isoflurane on the righting reflex and tail-clamping reflex in mice,and calculated the EC50 for loss of righting reflex(LORR)(unconsciousness)and loss of response to tail-clamping(LRTC)(immobility).Part II:Adult C57BL/6 mice(male/female,7-8 weeks)were used for this study.LFPs recording in vivo experiments were used to study the effects of the subanesthetic concentration of isoflurane on LFPs,especially hippocampal CA1 Ripple rhythmic oscillations.The open-field tests were used to examine the effects of the subanesthetic concentration of isoflurane on the activity state of mice,and observed the relationship between the activity states of mice and Ripple rhythmic oscillations.Experiment 1:Electrodes were implanted into the hippocampal CA1 region to establish an LFPs recording model in vivo.The effects of the subanesthetic concentration of isoflurane on LFPs in the hippocampal CA1 region were recorded,including the overall LFPs(0.1-500 Hz),Delta(0.1-4 Hz),Theta(4-12 Hz),and Ripple rhythmic oscillations.Raw waves,peak frequency,power spectral density,and spectrogram of these rhythmic oscillations were recorded and analyzed.The effects of the subanesthetic concentration of isoflurane on hippocampal CA1 Ripple rhythmic oscillations were further analyzed,including amplitude,frequency,duration,interarrival time,and rate.Experiment 2:The effects of the subanesthetic concentration of isoflurane on the activity of mice without head-fixed cable were examined under the same experimental protocol as the LFPs recording using the open-field test.The movement trajectories,travel distance,mean speed,and percentage of resting time were measured before and after isoflurane exposure.Part III:Neonatal(male/female,postnatal day 7 to 10)and adult C57BL/6 mice(male/female,7-8 weeks)were used for this study.Preparation of acute hippocampal slices from neonatal and adult mice.Patch-clamp electrophysiological experiments were used to explore the effects of the subanesthetic concentration of isoflurane on the excitability and firing patterns of pyramidal neurons and interneurons in the hippocampal CA1 region.The action potential frequency,resting membrane potential,current-voltage curve,and input resistance were measured before and after isoflurane exposure.Part IV:Based on the electrophysiological experimental data from Part III,the hippocampal CA1 Ripple rhythmic oscillations were simulated before and after the subanesthetic concentration of isoflurane exposure using neural simulation technology.The simulated results were compared with the real results recorded in mice to verify whether the subanesthetic concentration of isoflurane mediates the effects on hippocampal CA1 Ripple rhythmic oscillations by regulating the excitability and firing patterns of pyramidal neurons and interneurons.ResultsPart I:1.The results of fear-conditioning experiments showed that isoflurane suppressed the percentage of freezing time in a concentration-dependent manner.The EC50 of isoflurane for suppressing context memory was 0.23%(95%CI,0.21%-0.25%),and no significant sex difference existed(P=0.147).The EC50 of isoflurane for suppressing cue-tone memory was 0.39%(95%CI,0.36%-0.42%),and no significant sex difference existed(P=0.561).2.The results of inhibitory avoidance experiments showed that isoflurane suppressed the latency to enter the dark compartment in a concentration-dependent manner.The EC50of isoflurane for suppressing inhibitory avoidance memory was 0.20%(95%CI,0.16%-0.23%),and no significant sex difference existed(P=0.985).3.The results of the righting reflex experiments showed that the EC50 of isoflurane for inducing LORR was 0.82%(95%CI,0.80%-0.83%),and no significant sex difference existed(P=0.086).4.The results of the tail-clamping reflex experiments showed that the EC50 of isoflurane for inducing LRTC was 1.32%(95%CI,1.25%-1.39%),and no significant sex difference existed(P=0.666).Part II:1.The results of LFPs recording in vivo experiments showed that the subanesthetic concentration of isoflurane(0.5%)significantly enhanced the peak frequency of hippocampal CA1 Ripple rhythmic oscillations(P=0.002),while suppressed its power spectral density(P=0.008)compared with the baseline.However,there were no significant effects on the peak frequency(P=0.904)and power spectral density(P=0.127)of Delta rhythmic oscillations.There were also no significant effects on the peak frequency(P=0.554)and power spectral density(P=0.091)of Theta rhythmic oscillations.2.The results of Ripple rhythmic oscillations showed that the subanesthetic concentration of isoflurane(0.5%)significantly reduced the amplitude(P=0.003),duration(P<0.001)and rate(P=0.002),and significantly increased its interarrival time(P<0.001)and frequency(P<0.001)compared with the baseline.3.The results of the open-field test showed that the subanesthetic concentration of isoflurane(0.5%)caused the hyperactivity at the initial anesthesia induction phase(the first4 minutes).The movement distance(P<0.001)and mean speed(P<0.001)were significantly increased,while the percentage of resting time was significantly decreased(P<0.001)compared with the control.Subsequently,mild sedation was induced with decreased mobility,which returned rapidly to baseline after isoflurane washout.In addition,there were no significant sex differences in the effects of the subanesthetic concentration of isoflurane(0.5%)on the distance,mean speed,and the percentage of resting time.4.The results of the open-field test and LFPs recording in vivo experiments showed that although mice showed hyperactivity and decreased activity successively under the subanesthetic concentration of isoflurane(0.5%),the power of hippocampal CA1 Ripple rhythmic oscillations was continuously suppressed.And there was no obvious relationship with the changes in the movement state of the mice.Part III:1.The results of patch-clamp electrophysiological experiments on adult hippocampal slices showed that the subanesthetic concentration of isoflurane(0.12-0.15 mmol/L,equivalent to～0.5%in vivo)increased action potential frequency of pyramidal neurons at current injections of 60 p A(P=0.034),90 p A(P=0.024),120 p A(P=0.022)and 150 p A(P=0.013),while decreased action potential frequency of interneurons at current injections of 120 p A(P=0.012)and 150 p A(P=0.004).Isoflurane depolarized the resting membrane potential in pyramidal neurons(P=0.007),but had no effects on its current-voltage curve(P=0.164)and input resistance(P=0.370).Different from pyramidal neurons,isoflurane had no effects on its resting membrane potential(P=0.260),current-voltage curve(P=0.289)and input resistance(P=0.128).2.Consistent with the results of adult mice,the subanesthetic concentration of isoflurane(0.12-0.15 mmol/L)increased action potential frequency of pyramidal neurons at current injections of 30 p A(P<0.001),while decreased action potential frequency of interneurons at current injections of 60 p A(P=0.031),90 p A(P=0.048),120 p A(P=0.014)and 150 p A(P=0.001).Isoflurane depolarized the resting membrane potential in pyramidal neurons(P=0.027),but had no effects on its input resistance(P=0.409).Meanwhile,isoflurane had no effects on its resting membrane potential(P=0.133)and input resistance(P=0.983).Part IV:1.The results of neural simulation showed that the characteristics of hippocampal CA1 Ripple rhythmic oscillations simulated by this model were consistent with the real results recorded in vivo.Thus,this model could be used to simulate changes in the hippocampal CA1 Ripple rhythmic oscillations before and after the subanesthetic concentration of isoflurane exposure.2.The simulation results based on the electrophysiological data of adult or neonatal mice showed that the subanesthetic concentration of isoflurane increased the action potential frequency of pyramidal neurons by～36.08%or～22.78%,and decreased the action potential frequency of interneurons by～29.11%or～18.57%compared with the control.These trends were consistent with electrophysiological results.3.The simulation results of hippocampal CA1 Ripple rhythmic oscillations of adult or neonatal mice showed that the subanesthetic concentration of isoflurane decreased the amplitude(P<0.001;P<0.001)and duration(P<0.001;P<0.001),and increased its frequency(P=0.002;P=0.002)and interarrival time(P<0.001;P<0.001)compared with the control.Normalized effects of the subanesthetic concentration of isoflurane on Ripple rhythmic oscillations in model were similar to the effects seen on LFPs recordings in vivo,including amplitude,duration,frequency,and interarrival time.Conclusions1.Subanesthetic concentrations of isoflurane(lower than those required to induce unconsciousness and immobility effects)can significantly suppress hippocampus-dependent memory and exert its amnestic effect.2.The subanesthetic concentration of isoflurane(0.5%)can significantly affect the hippocampal CA1 Ripple rhythmic oscillations.3.The subanesthetic concentration of isoflurane(0.12-0.15 mmol/L,equivalent to～0.5%in vivo)can differentially modulate the excitability and firing patterns of pyramidal neurons and interneurons in the hippocampal CA1 region.4.The subanesthetic concentration of isoflurane mediates the effects on Ripple rhythmic oscillations by differentially regulating the excitability and firing patterns of pyramidal neurons and interneurons in the hippocampal CA1 region,which may be one of the important mechanisms responsible for the amnestic effect of isoflurane.