| BackgroundSince the first public demonstration of ether anesthesia in 1846,the mechanism of general anesthesia becomes a huge mystery,which has been listed as one of the 125 most important scientific questions we do not know by the journal of Science in 2005.To clarify the mechanism of general anesthesia,especially its underlying neural circuit,will be benefit to not only the safety of clinical practice,but also the development of new anesthetics.As part of the ventral ascending reticular activating system(ARAS),the basal forebrain(BF)participates in the regulation of sleep,feeding,drinking,fluid homeostasis,learning,rewarding,attention and other behaviors.Previous studies have found that the BF may be one of the downstream targets of orexinergic neurons,the application of orexin-A or orexin-B intra the BF could induce wakefulness during the anesthesia of isoflurane,sevoflurane and propofol,indicating that the BF plays an important role in the anesthesia-wake transitions.In the BF,there are mainly three types of neurons,including GABAergic,glutamatergic and cholinergic neurons.Previously studies found that both of the glutamatergic and cholinergic neurons in the BF could promote wakefulness,while the GABAergic neurons have the opposite effect to promote anesthesia.However,other researchers have found that the GABAergic neurons in the BF exhibit much more powerful pro-arousal effect in sleep regulation than glutamatergic and cholinergic neurons.Considering the BF has extensive long projections throughout the brain,it’s of significant importance to clarify the exact projections and their effects on the regulation of anesthesia.In this study,channelrhodopsin-assisted circuit mapping(CRACM)was used to explore the downstream projections of BF GABAergic,glutamatergic and cholinergic neurons.And then,we further explored the function of the BF GABAergic neurons in anesthesia-wake transitions by combining electrophysiologic and optogenetic techniques in cre driver mice lines.This present study is aimed to enhance our understating of the effects of the BF on the regulation of wake and anesthesia,and provides evidence and insight on the general anesthesia associated nuclei and their projections.Objectives(1)To clarify the long projections of the GABAergic,glutamatergic and cholinergic neurons in the BF by channelrhodopsin-assisted circuit mapping(CRACM).(2)To verify the physiological function of the GABAergic projections from the BF to the lateral hypothalamus(LH),lateral habenula(LHb),central medial thalamic(CMT)and ventral tegmental area(VTA)by in vitro electrophysiological tests.(3)To observe the effects of activating the BF GABAergic long projections to LH,LHb,CMT and VTA on the induction time and emergence time and electroencephalogram(EEG)changes under isoflurane anesthesia using optogenetic technique.Method(1)Vgat-Cre,Vglut2-Cre and Ch AT-Cre mice,aged 6-8 weeks old,were unilaterally injected AAV-DIO-Ch R2-m Cherry virus(n=3)into BF.After the expression of virus for 3 weeks,the brains were post-fixed,dehydrated and then sliced continuously(40μm thickness).The slices containing BF was further stained to verify the specificity of virus transfection by GABAergic,glutamatergic and cholinergic neuron markers,respectively.One from every three slices were mounted and cover-slipped.At last,the long projections of different types of BF neurons were observed using whole slide imaging.(2)Three weeks old Vgat-Cre mice were unilaterally injected AAV-DIO-Ch R2-m Cherry virus(n=3)in the BF.After viral expression for 3 weeks,slices for electrophysiological examines are prepared,and the neurons in the LH,LHb,CMT and VTA were patched.And then,optical stimulation(blue light 473 nm)was applied in above regions to activate BF GABAergic transfected terminals,and the changes of postsynaptic current were recorded.Furthermore,the antagonists of GABA receptors were used to identify the interacted type of receptors.(3)Male Vgat-Cre mice,aged 6-8 weeks old,were randomly divided into two groups (n=6),the mice in the experimental group were injected with AAV-DIO-Ch R2-m Cherry,while those in the control group were microinjected with AAV-DIO-m Cherry virus into the BF,respectively.At the same time,the optical fiber was embedded in LH,LHb,CMT or VTA,respectively.After 3 weeks recovery,opto-stimulation(20 Hz,30 ms,10 m W from tips)was applied to activate the above neural projections,and we investigated the time of loss righting reflex(LORR)and recovery of righting reflex(RORR)under 1.4%isoflurane anesthesia,successively.(4)Male Vgat-Cre,aged 6-8 weeks,were randomly divided into two groups(n=5),the procedure of virus microinjection and optical fiber implantation were the same as described in part(3),and three screws were implanted in the skull as EEG electrodes.Three weeks later,1.0%isoflurane and pure oxygen 1.0 L/min were delivered in both groups.The EEG was continuously monitored by Power Lab software during the whole experiments.After 30 min isoflurane anesthesia,1 min optical stimulation(473 nm,20 Hz,30 ms,10 m W from tips)was delivered for activation,and the changes of burst-suppression ratio(BSR)before and during optical stimulation was analyzed by Matlab software.(5)Taking the same procedure as in(4),after 0.8%isoflurane anesthesia for 30 min,we compared the changes of power spectrum density for 1 min before and during optical stimulation by the software of Matlab.Results(1)Tracing the efferent projections from the BF GABAergic,glutamatergic and cholinergic neurons in the whole brain.1)The BF GABAergic neurons mainly project to the medial septal nucleus(MS),CMT,LHb,medial habenula nucleus(MHb),LH,VTA and mesencephalic reticular formation(MRF).2)The BF glutamatergic neurons mainly project to the MS,lateral septal nucleus(LS)and LHb.3)The BF cholinergic neurons mainly project to the MS,basolateral amygdaloid nucleus(BLA),hippocampus(HIP)and magnocellular part of red nuclei(m RN).Only a few projection fibers from the BF cholinergic neurons could be found in the LH.(2)The effects of the BF GABAergic projections on the regulation of anesthesia and wake.1)After optical activation of the BF GABAergic terminals in vitro brain slices, some of the neurons in the LH,LHb,CMT and VTA showed postsynaptic current.Among them,the postsynaptic current in the LH,LHb and CMT could be blocked by GABA_A receptor antagonist,while the effect in VTA could only be blocked by GABA_B receptor antagonist.2)Compared with the control group,activation of the projections from the BFGABAergic neurons to the LH significantly shortened the time of RORR(500.8±36.16 s vs 317.8±15.26 s,P=0.0009)but did not influence the LORR time(316.7±15.12 s vs 338.0±18.64 s,P=0.3949).Under 1.0%isoflurane anesthesia,activation of this projections could reduce the BSR from 62.65± 2.51%to 21.89±4.15%(P<0.0001).There was a significant change in EEG power spectrum by optical stimulation,which produced a significant decrease of δpower(38.95±0.99%vs 24.51±2.78%,P=0.0022),while the power inγbands (17.17±2.17%vs 30.09±4.46%,P=0.0288)markedly increased under 0.8% isoflurane anesthesia.3)Compared with the control group,activation of the projections from the BF GABAergic neurons to the LHb markedly shortened the time of RORR (440.7±15.88 s vs 324.5±10.06 s,P=0.0001),but the time of LORR(319.8±8.15s vs 335.0±13.50 s,P=0.3587)remained unchanged.Optical stimulation of this projections reduced the BSR(59.00±1.81%vs 32.70±4.95%,P=0.0117)under 1.0%isoflurane anesthesia.When the mice were anesthetized by 0.8%isoflurane,a significant decrease ofδpower(40.63±2.59%vs 25.36±3.00%,P=0.0309)was observed,while the power ofβbands(13.92±1.20%vs 22.64±2.61%,P=0.0350) andγbands(15.06±0.83%vs 25.37±3.00%,P=0.0364)increased.4)Compared with the control group,activation of the projections from the BF GABAergic neurons to the CMT shortened the time of RORR(517.2±8.35 s vs 452.3±25.60 s,P=0.0368)without changing the LORR time(357.3±12.98 s vs 369.5±10.17 s,P=0.4776).Optical stimulation of this projections decreased the BSR(67.55±2.63%vs 33.76±6.84%,P=0.0082)under 1.0%isoflurane anesthesia.We also observed a significant decrease ofδpower(42.19±1.69%vs29.14±2.01%,P=0.0010),while the power ofβbands(12.13±0.99%vs17.89±0.70%,P=0.0006)andγbands(14.29±0.86%vs 23.92±2.40%,P=0.0296) increased under 0.8%isoflurane anesthesia.5)Compared with the control group,activation of the projections from the BF GABAergic neurons to the VTA reduced the time of RORR(470.2±28.06 s vs 315.0±18.81 s,P=0.0002)with no effect on the LORR time(289.5±14.79 s vs 310.0±12.82 s,P=0.2733).Activation of this projections decreased the BSR(69.78±1.26%vs 38.23±2.35%,P=0.0003)under 1.0%isoflurane anesthesia.Meanwhile,theδpower decreased(36.43±1.90%vs 26.40±3.53%,P=0.0183),whereas the power ofβbands(13.98±0.45%vs 21.07±2.27%,P=0.0347)andγbands(13.96±0.56%vs 21.74±1.95%,P=0.0064)increased under 0.8% isoflurane anesthesia.ConclusionsIn this study,the exact efferent projections of the BF GABAergic,glutamatergic and cholinergic neurons in the whole brain was observed by channelrhodopsin-assisted circuit mapping(CRACM)in cre driver mice lines.On this basis,we explored the effect of the BF GABAergic terminals activation on postsynaptic current in the LH,LHb,CMT and VTA neurons by in vitro electrophysiologic tests,and confirmed that all of the above efferent projections from the BF are functional projections.Among them,the projections from the BF GABAergic neurons to the LH,LHb,CMT could be mediated by GABA_Areceptor while the projections to the VTA are mainly through GABA_Breceptor.We further explored the function of the above BF GABAergic projections in wake-anesthesia transitions.Optogenetic activation of the BF GABAergic projections to the LH,LHb, CMT and VTA could accelerate the emergence from anesthesia without changing the induction time.During the maintenance of isoflurane anesthesia,the electroencephalogram(EEG)showed that activation of these BF GABAergic projections could reduce the depth of anesthesia by decreasing the BSR and altering the relative pectrum density.All these results indicate the pro-arousal effect of the above BF GABAergic projections on the wake-anesthesia regulation.Combined with previous study,it is possible that BF GABAergic neurons could participate in the regulation of wake-anesthesia transitions through multiple projections. |
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