Orexinergic Neurons In Pef-BF Neural Circuit Involved In General Anesthesia-awakening Awareness Transformation

BackgroundGeneral anesthetics have been widely used in clinical surgery because of their unique role in mediating the disappearance and recovery of reversible consciousness.However,the mechanism of general anesthetics has not been elucidated and has become one of the important scientific problems to be solved urgently in the field of anesthesiology.The analysis of the characteristics of general anesthesia and the elucidation of its core mechanism not only have a profound impact on the application of general anesthesia,but also greatly promote the understanding of complex neural activities such as cognition and consciousness in the field of basic science.Previous studies have shown that the Orexin neurotransmitter system in the Perifornical area(Pef)of the hypothalamus is the trigger to regulate the conversion of general anesthesia to arousal consciousness.At the same time,traditional neuropharmacological studies further confirmed that exogenous administration of Orexin A and Orexin B neurotransmitters in the basal forebrain(BF)accelerated the recovery of consciousness after general anesthesia.It is suggested that BF is the key brain region in which Orexin neurotransmitter system regulates the conversion of general anesthesia to arousal consciousness.We speculate that BF receives direct innervation from the Orexinergic neuron in the Pef of the hypothalamus,and this neural projection plays an important role in the conversion of general anesthesia to arousal consciousness.However,previous studies have been limited to exogenous pharmacological regulation,and it is difficult to directly certify the role of endogenous Orexin neurotransmitter system in the loss and recovery of consciousness induced by general anesthetics.Therefore,by constructing transgenic Hcrt-cre rats(specifically labeled Orexinergic neurons),combined with photogenetics and other cutting-edge neurobiological techniques,To explore the Involvement of Orexinergic neurons in Pef-BF Neural Circuit in General Anesthesia-Awakening Awareness Transformation.Objectives(1)To construct Hcrt-cre transgenic rats and verify the efficiency and specificity of gene knockout.(2)The Orexinergic neurons in Pef were specifically activated by photogenetics,and then observe the impact on the the state of consciousness and EEG characteristics under isoflurane anesthesia.(3)The Orexinergic neurons projection in BF were specifically activated by photogenetics.then observe the impact on the state of consciousness and EEG characteristics under isoflurane anesthesia.Method(1)Hcrt-cre rat was established by CRISPR/Cas9 technique.The structure of Hcrt gene was analyzed and Cre-WPRE-pA element was inserted between 5’UTR and coding region.SgRNA is designed on Exon1.The EGE system developed by Company(Biocytogen Inc,Beijing,China).The knockin gene was identified by PCR and Southern blot.(2)We crossed rat with Cre-dependent tdTomato reporter knock-in rat(B-Tdtomato cKI rats),provided by Biocytogen Inc for verification of Cre expression.Perfusing their offsprings,the brain slices containing Pef were continuously cut with a thickness of 35 μ M.the whole process paid attention to avoiding light to prevent the fluorescence quenching.Then immunofluorescence staining was carried out: according to the order of rinsing,blocking,incubating the first antibody,incubating the second antibody,DAPI staining and sealing.The fluorescence co-expressed rate(n=9)was calculated.(3)Hcrt-cre rats,eight weeks old,were unilaterally injected AAV-DIO-ChR2-mCherry virus(n=3)in Pef.After the expression of the virus,the brain slices containing Pef were continuously cut,then immunofluorescence staining.The fluorescence double labeling coincidence rate(n=9)was calculated after confocal photography.(4)Hcrt-cre rats,3 weeks old,were bilaterally injected AAV-DIO-ChR2-mCherry virus(n=4)in Pef.After virus successful expression,stimulation in PefLH with blue light(473 nm)on brain slices in vitro effectively activated the light-sensitive channel-expressed neurons.(5)Male Hcrt-cre rats aged 8-10 weeks were randomly divided into two groups(n=6),the experimental group and the control group were injected AAV-DIO-ChR2-mCherry and AAV-DIO-mCherry virus into the Pef,respectively.The ceramic fiber for light stimulation was embedded in the 0.1mm upward at the virus injection site,and the EEG electrode was embedded in the cranial surface for EEG recording.After four weeks expression,light was given to measure the loss and recovery of righting reflex time.(6)Male Hcrt-cre rats aged 8-10 weeks were randomly divided into two groups(n=6),Grouping and methods are same as(5),and 1.4% isoflurane and pure oxygen 1.5L/min were inhaled in both groups.10 mins light stimulation(20 Hz,10 mW,10 ms)was given after 40 min anesthesia.PowerLab EEG monitoring system was used to record the whole duration of EEG,and Matlab software was used to analyze the changes of burst suppression rate of EEG before and after stimulation.(7)Male Hcrt-cre rats aged 8-10 weeks were randomly divided into two groups(n=6),Virus injection same as(5),The ceramic fiber for light stimulation was embedded in BF,and the EEG electrode was embedded in the cranial surface for EEG recording.After four weeks expression,light was given to measure the loss and recovery of righting reflex time.(8)Male Hcrt-cre rats aged 8-10 weeks were randomly divided into two groups(n=6),Grouping and methods are same as(7),1.4% isoflurane and pure oxygen 1.5L/min were inhaled in both groups.10 mins light stimulation(20 Hz,10 mW,10 ms)was given after 40 min anesthesia.PowerLab EEG monitoring system was used to record the whole duration of EEG,and Matlab software was used to analyze the changes of burst suppression rate of EEG before and after stimulation.(9)Male Hcrt-cre rats aged 8-10 weeks were randomly divided into two groups(n=6),Grouping and methods are same as(7).After four weeks expression,light was given to measure the loss and recovery of righting reflex time.One week after recovery,0.8% isoflurane and pure oxygen 1.5L/min were inhaled in both groups.10 mins light stimulation(20 Hz,10 mW,10 ms)was given after 30 min anesthesia.PowerLab EEG monitoring system was used to record the whole duration of EEG,and Matlab software was used to analyze the time-frequency changes of EEG before and after stimulation.The awakening behavior of rats was recorded by camera and video recording software,and the score was recorded.Results(1)Construction of Hcrt-cre transgenic rats and verification of the specificity of Orexinergic neuron labeling.1)The identification of F0 generation and F1 generation showed that Hcrt-cre rats were successfully constructed by CRISPR/Cas9.F0 rats with expected genotype confirmed by tail genomic DNA PCR and sequencing were mated with SD rats to establish germline-transmitted F1 founders.F1 founders were genotyped by tail genomic PCR/DNA sequencing and Southern blot examination was performed to further confirm the genotype and correctly recombined without random insertion.2)After immunofluorescence staining in heterozygous offspring,tdTomato and Orexin-A were co-labeled in the lateral hypothalamic area,and 98.80 ±0.16% of Orexin-A positive neurons expressed tdTomato,.91.92 ±0.80% of tdTomato positive neurons were Orexin-A positive.(2)To verify the transfection efficiency and function of photosensitive channel protein ChR2 on Orexinergic neurons in Pef in vitro.1)After injection of ChR2-mCherry virus,immunofluorescence staining also showed that 95.07 ±1.00% of Orexin-A positive neurons expressed mCherry,and 91.92 ±0.80% of mCherry positive neurons showed Orexin-A positive in lateral hypothalamic.2)After clamping Orexin neurons,Orexinergic neurons showed robust depolarization and spiking following 500 ms illumination in current-clamp mode and inward current in voltage-clamp mode.Optical stimulation at 1-30 Hz evoked different frequency spiking in current-clamp mode.The fidelity response of Orexinergic neurons to light pulses at frequencies was up to 20 Hz(3)Orexinergic neurons in Pef were activated by blue light in vivo.The effects of Orexinergic neurons in Pef on the loss and recovery time of righting reflex(LoRR & RoRR)under 1.4% isoflurane anesthesia and the characteristics of EEG spectrum change were observed.During the optical stimulation,there was a significant change in EEG traces and power spectrum in the ChR2-mCherry group.Most obviously,the burst suppression of EEG during 1.4% isoflurane maintenance was largely inhibited.Statistical analysis of a total of 20 minutes of EEG optical stimulation for 10 minutes and the other two 5 minutes before and after light stimulations)demonstrated the significant decline of burst suppression ratio(BSR)during the optical stimulation.Optical stimulation of the Orexinergic neurons in PefLH also had a significant effect on shortening arousal time(923.3 ± 72.42 s vs.493.3 ± 30.62 s,P=0.0003),but changes of the induction process was not detectable.(4)Orexinergic neurons in Pef were activated by blue light in vivo.The effects of Orexinergic neurons in Pef on the loss and recovery time of righting reflex(LoRR & RoRR)under 1.4% isoflurane anesthesia and the characteristics of EEG spectrum change were observed.1)Light activation of Orexinergic terminals in BF caused a similar change of EEG as what was shown after orxinergic cell bodies were stimulated.Specifically,there was a significant decline of BSR during the 10-minute period of light stimulation.Light stimulation of Orexinergic terminals in BF also had a significant effect in promoting arousal,shown as a shortened time to RORR(936.7 ± 68.15 s,vs.674.2 ± 48.42 s,P=0.0105),but no effect on the induction process of anaesthesia.2)During the optical stimulation,EEG of ChR2-mCherry group was changed from the anaesthesia state to an awake-like state,and recovered back to normal anaesthesia state after the stimulation.In particular,optical stimulation produced a significant decrease of delta power(63.15 ± 1.23% vs.42.14 ± 3.47%,P=0.0002),while the power in theta(16.54 ± 0.80% vs.22.34 ± 1.87%,P= 0.0172)and beta(4.33 ± 0.35% vs.10.98 ± 1.79%,P=0.0045)bands markedly increased in ChR2-mCherry rats.There was no statistical difference of EEG power spectrum in the mCherry animals.Upon optic stimulation,movement of the rats was evidently improved.The total score of ChR2-mCherry rats was significantly higher than that of mCherry rats.(P=0.0022)ConclusionsHcrt-cre knock-in rats were generated by using CRISPR/Cas9 technology.The selective expression of Cre was verified by crossing the Hcrtcre rat with a cre-dependent tdTomato reporter knock-in rat and co-staining the Orexinergic neurons with tdTomato.By using this genetic animal,we found that the activation of specific Orexinergic neuronal cell bodies or their terminals in the BF with optic technique shortened the time to RORR,and induced a decrease of burst suppression ratio under 1.4% isoflurane anaesthesia.During the anaesthesia of 0.8% isoflurane,optical stimulation of Orexinergic terminals at BF reduced the power percentage of delta wave in EEG,and even promoted the movement recovery in the anaesthetized rats.In conclusion,through the successful construction and application of Hcrt-cre rats,we finally confirmed that BF can directly mediate the pro-emergence role of Orexinergic neurons during isoflurane anesthesia.Hcrt-cre rats can be used as an effective method for the study of Orexin neurons.It provides a new tool for further study of the multifold physiological function of Orexinergic neurons in rats.