| Objective:The development of general anesthetics and related technologies of general anesthesia has played a key role in promoting the progress of modern medicine.General anesthesia is the most important progress in clinic,especially in the surgery.How general anesthetics produce anesthesia is an important basic scientific problem.It is related to the advancement of medicine,the safety of general anesthesia as well as the research of new general anesthetics.Inhaled anesthetic is a kind of general anesthetics widely used in clinic,which produces general anaesthesia through inhalation administration of respiratory system.Compared with the clear mechanism of intravenous general anesthetics,mechanism of inhaled anesthetics is not clear.Taking isoflurane as an example,the effect of isoflurane can be amnesia,that is,inhibition of learning and memory,sedation and hypnosis and analgesia.At the same time,with the increase of drug concentration,hyperactivity and inhibition of cardiovascular system and respiratory system and other side effects could happen.Therefore,different concentrations of isoflurane can regulate the central nervous system bi-directionally and produce excitatory and inhibitory effects.Sodium channels are the most basic ion channels in excitable tissues and cells,which directly mediate the generation and synaptic transmission of excitatory action potentials in the nervous system.Voltage-dependent sodium channel(Nav)directly mediates the ascending branch of action potential,and another voltage-independent sodium leak channel(NALCN)is an important background cation leak current channel for maintaining neuronal resting membrane potential,which can regulate the rhythm of central nervous system and neuronal excitability.In this study,combined with whole animal behavior test,electrophysiological patch clamp,RNA interference,immunofluorescence staining and computer model simulation,we confirmed that:(1)Isoflurane at the clinical concentration inhibits the excitability of hippocampal pyramidal neurons through Nav;(2)Isoflurane at MAC and sub-MAC concentrations contributes to its bidirectional modulation of excitabilities of hippocampal pyramidal neurons;(3)Isoflurane differentially regulates hippocampal pyramidal neurons and fast-spiking interneurons to affect hippocampal ripples and regulate memory.Part 1Materials and Methods:The sodium currents and excitability of pyramidal neurons in CA1 of acute mice hippocampal slices were recorded by whole-cell patch clamp.To analyze the effects of clinical concentration of isoflurane on the gating characteristics of voltage-dependent sodium channels Nav,and to observe the effects of isoflurane on three kinds of sodium currents,namely transient,persistent and resurgent sodium currents.In addition,to investigate the effect of isoflurane on action potential and other excitatory indexes of pyramidal neurons under clinical anesthetic concentration.Results:(1)The 50% inhibitory concentration of isoflurane on transient sodium current was 1.0 ± 0.3 m V(~3.7 MAC [Minimum alveolar concentration])when physiological holding membrane potential was-70 m V.While as the holding potential was-120 m V,isoflurane had a weak inhibitory effect on sodium current(50% inhibitory concentration: 3.6 ± 0.7 m M,~13.3 MAC).(2)Isoflurane at anesthetic concentration(0.55 m M,~2 MAC)shifted the steady-state inactivation curve of voltage-dependent sodium channels Nav by-6.5 ± 1.0 m V(n = 11,P < 0.0001),which means isoflurane accelerated the inactivation of Nav,but did not affect the activation of Nav at this concentration.At the same time,isoflurane increased the recovery time constant of Nav from 7.5 ± 0.6 ms to 12.7 ± 1.3 ms(n = 13,P < 0.001).(3)Isoflurane also reduced persistent sodium current density(50% inhibitory concentration: 0.4 ± 0.1 m M,~1.5 MAC)and resurgent sodium current.(4)Isoflurane(0.55 m M,~2 MAC)reduced the number of action potentials of pyramidal neurons,decreased the amplitudes of individual action potentials,and hyperpolarized the resting membrane potentials(RMPs)of pyramidal neurons from-54.6 ± 2.3 m V to-58.7 ± 2.1 m V(n = 16,P = 0.001),thus reducing the excitability of pyramidal neurons.Part 2Materials and Methods:Immunofluorescence staining was used to determine the distribution of sodium leak channel(NALCN)in the central nervous system.The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices,including NALCN currents,and Nav currents.Isoflurane-induced hyperactivities of C57BL/6J mice were evaluated four weeks after intracerebral injection of adeno-associated virus.The NALCN expression plasmid was constructed in vitro and transfected into HEK 293 T cells.The direct effects of isoflurane at sub-MAC and MAC concentrations on NALCN were investigated by cell electrophysiological patch clamp.Results:(1)NALCN was widely expressed throughout the cortex and hippocampus of mice.(2)Isoflurane at sub-anesthetic concentrations(0.5 MAC)increased the spontaneous firing rates of CA3 pyramidal neurons from 0.9 ± 0.2 Hz to 1.4 ± 0.2 Hz(n = 6,P = 0.008),while anesthetic concentrations of isoflurane(1.5 MAC)decreased the firing rates from 0.7 ± 0.2 Hz to 0.5 ± 0.2 Hz(n = 7,P = 0.001).Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance,but minimally inhibited Nav currents.Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes.Isoflurane at sub-MAC concentrations depolarized resting membrane potentials(RMPs)of neurons while hyperpolarized the RMPs at anesthetic concentrations.(3)Isoflurane at low concentrations induced hyperactivity in vivo,which was diminished in NALCN knockdown mice.(4)Isoflurane did not activate NALCN conductance per se in transfected HEK 293 T cells.Part 3Materials and Methods:Potency of isoflurane were measured in mice in vivo(both sexes)for memory impairment,loss of righting reflex and loss response to tail-clamping.Hippocampal ripples were measured by placing brain recording electrodes in the CA1 subfield.Whole-cell patch clamp recording was used to explore the effects of isoflurane on the excitabilities of hippocampal pyramidal neurons and fast-spiking interneurons.A simulation model of ripples based on the firing frequency of hippocampal CA1 neurons was used to validate the effects of isoflurane on neuronal excitability in vitro and ripples in vivo.Results:(1)Isoflurane at 0.5% impaired hippocampus-dependent fear memory by 97.4%,but only produce 0% loss of righting reflex.(2)Isoflurane at 0.5% reduced the amplitude(41.8 ± 13.2 μV vs.38.2 ± 12.9 μV,n = 9,P = 0.003),rate(538 ± 81 beats/min vs.462 ± 66 beats/min,n = 9,P = 0.002)and duration(48.0 ± 8.8 ms vs.36.3 ± 4.8 ms,n = 9,P < 0.001)of ripples and increased the inter-arrival time(68.5 ± 6.1 ms vs.78.1 ± 7.0 ms,n = 9,P < 0.001)and frequency(145 ± 3 Hz vs.148 ± 3 Hz,n = 9,P = 0.001).(3)In acute hippocampal slices,isoflurane at same concentration(0.4-0.5 MAC)depressed the frequency of action potentials in fast-spiking interneurons(n = 10,F(1,108)= 9.03,P = 0.003)while slightly enhancing the frequency of action potentials in hippocampal CA1 pyramidal neurons(n = 11,F(1,120)= 4.743,P = 0.031).(4)The simulated effects of isoflurane on hippocampal CA1 ripples were comparable to the recordings of local field potentials in vivo.Conclusion:Different concentrations of isoflurane modulate neuronal excitabilities through different effects on Nav and NALCN sodium channels;low concentrations of isoflurane differently regulate hippocampal pyramidal neurons and fast-spiking interneurons to affect hippocampal ripples and mediate anterograde amnesia. |
PDF Full Text Request