The Role Of GABAergic System In Basal Forebrain During Sevoflurane Anesthesia

Since the general anesthetics were first used in surgical clinic in 1846,modern surgery has developed faster than ever.In 2015,the Lancet reports that there are about 318 million operations around the world every year,and in most of them general anesthesia has been applied.How do general anesthetics work and obtain the loss/recover of consciousness effect? We still have no idea about this question at present.To clarify the mechanism of general anesthetics is helpful not only in safer application of anesthetics in clinics as well as the invention of new anesthetics,but also for understanding other neurological mechanisms,such as formation of consciousness.As Nature Review Neuroscience reported,understanding the molecular mechanism of general anesthetics is one of the important scientific issues to be solved in the field of neuroscience.Since anesthetics appeared,people often compare the anesthesia as sleep,both of which show a lower level of awareness and inhibition of responses to external environmental stimulation.A growing number of studies have found that general anesthetics can partly act via the sleep-arousal system in the brain.The basal forebrain is located in front of the hypothalamus,where contains mainly GABAergic,glutamic and cholinergic neurons.As the last part of ascending reticular activating system,the basal forebrain plays an important role in promoting the awareness in the cortex,transformation of sleep-waking phase and maintaining sleep homeostasis regulation,however its role in anesthesia are poorly understood.Sevoflurane is widely used in clinics.Previous studies have found that inhaled anesthetics isoflurane and halothane can increase neurotransmitter release at the synaptic level,so it means that GABAergic neuron is likely to be one of the targets for inhalation anesthetics.However,there are few studies focusing on the effects of general anesthetics on inhibitory interneurons.In present study,we observed the electrical activities of GABAergic neurons and postsynaptic pyramidal neurons in the basal forebrain area by patch clamp technique under the ACSF infusion with sevoflurane,following with anesthesia behavioral experiments,to reveal the role of different types of neurons in the anesthesia effect and explore whether the GABAergic neurons in basal forebrain area participate in and adjust the mechanism of anesthesia-awakening process.Experiment 1:Effect of sevoflurane on the electrical activity of GABAergic interneurons and postsynaptic pyramidal neurons in the basal forebrain areaObjective: To explore the effect of sevoflurane on the membrane potential,spikes frequency from GABAergic interneurons and the inhibitory postsynaptic current from postsynaptic pyramidal neurons in the basal forebrain area.Methods: We used 4-6 weeks male C57BL/6 mice brain slices.The slices were constantly perfused under room temperature.We used whole-cell current clamp mode to record spontaneous action potential,resting membrane potential and action potential discharge by current injection from the GABAergic interneurons before and after given sevoflurane infusion.And then used whole-cell voltage clamp mode to record spontaneous inhibitory postsynaptic current(s IPSC)and small inhibitory postsynaptic current(m IPSC)of pyramidal neurons in basal forebrain before and after given sevoflurane infusion.Results: We used current-clamp,K+ internal solution to examine the effects of sevofluraneon spontaneous action potential,resting membrane potential and action potential discharge by current injection recorded from GABAergic interneurons.Application of sevoflurane increased the frequency of action potential(P<0.001),and depolarized the resting membrane potential,and added the number of action potential discharge by current injection(P<0.05).Then we used voltage-clamp,Cs+ internal solution to examine application of sevoflurane on s IPSC and m IPSC of pyramidal neurons in basal forebrain.Application of sevoflurane could significantly increased the frequency(control: 2.0±0.2 Hz,sevoflurane: 2.8±0.4 Hz,P<0.05)and amplitude(control: 28.1±2.7 p A,sevoflurane: 47.6±4.1 p A,P<0.05)of s IPSC;Application of sevoflurane had little influence on frequency and amplitude of m IPSC in pyramidal neurons.Experiment 2 Effect of microinjection of GABAergic/glutamic receptor agonist/antagonist into basal forebrain on sevoflurane anesthesiaObjective : Microinjection of GABA/ glutamic receptor agonist/antagonist into BF to explore GABAergic and glutamic system in the regulation of sevoflurane anesthesia.Methods: According to the random grouping method,the 120 C57BL/6 male mice(8-12 weeks)were divided into 24 groups.Firstly,the mice were anesthetized with 0.3ml/100 g of 10% chloral hydrate by intraperitoneal injection,and the microinjection cannula was placed into the BF area of mice.After 5 days of rest,the mice were subjected to the experiment of sevoflurane anesthesia.The mice were put in an anesthesia box that was fulfilled with 1.0 MAC(2.4%)sevoflurane and we recorded the time of loss of righting reflex(LORR).Fifteen minutes after LORR,the same group of mice were given a dose of 0.3? l vehicle and two different doses of GABA/glutamic receptor agonist/antagonist into the BF area in 2-3 min,respectively.At the time of 30 minutes after LORR,the introduction of sevoflurane was stopped and we recorded the time of the recovery of righting reflex,which is regarded as the emergence time.Results: Compared with the control group,microinjection muscimol(GABA receptor agonist)to BF area,in both 50 pmol and 100 pmol groups,prolonged emergence timeafter sevoflurane anesthesia(P< 0.05);Microinjection 100 pmol bicuculline(GABA receptor antagonist)also prolonged emergence time following sevoflurane anesthesia,in comparison to the control group(P< 0.05).Compared with the control group,microinjection NMDA receptor agonist glutamate/NMDA into BF area,in both 1 nmol and 2 nmol groups,decreased the emergence time in sevoflurane anesthsia(P< 0.05);While microinjection NMDA receptor antagonist DL-AP5/MK-801 into BF area prolonged the emergence time in comparison to the control group(P<0.05).Microinjection of 1 nmol AMPA receptor agonist into BF area decreased emergence time following sevoflurane anesthesia(P<0.05),and microinjection AMPA receptor antagonist into BF area prolonged emergence time(P<0.05).Conclusion: In this study we proved: sevoflurane can directly excited GABAergic interneurons and increase the inhibitory postsynaptic current of pyramidal neurons in basal forebrain area;after the local microinjection of GABA agonists and antagonists,both of them prolonged emergence time after sevoflurane anesthesia,and microinjection NMDA/ AMPA agonist decreased the emergence time.NMDA/AMPA antagonist prolonged emergence time.In conclusion,the experiment proved that regulating excitatory and inhibitory neurotransmitter can significantly influence on emergence following anesthesia,and the effect of sevoflurane on the activation of GABAergic interneurons in basal forebrain area plays an important role in regulating anesthesia.