| Objective: The midbrain dopamine system plays a very important role in physiological sleep and anesthesia.Dorsal raphe nucleus(DRN)is a heterogeneous nucleus located on the ventral side of the midbrain aqueduct,which is involved in regulating social interactions,rewards,and sleep-wake.Studies have found that dopamine(DA)neurons in DRN play an important role in regulating sleep-wake processes and can effectively promote wakefulness of physiological sleep.But it is not clear whether DRN DA neurons effect the loss and recovery of consciousness in the general anesthesia,so this experiment ues: 1.fiber optic recording technology of living brain nerve calcium signal changes to observe the activity of DRN DA neurons before and after anesthesia with intravenous general anesthesia propofol and inhaled general anesthetic isoflurane;2.chemical genetics / optogenetics technology to activate / inhibit DRN DA neurons specifically and record the changes of LORR 、RORR and corresponding cortical electroencephalography(EEG),and analyze their effects on loss and recovery of consciousness/behavior during propofol / isoflurane anesthesia;3.spontaneous inhibitory postsynaptic currents(s IPSC)as recording indicators to analyze propofol / isoflurane inhibiting synaptic transmission of DRN DA neurons.That exploring the mechanism of DRN DA neurons in propofol / isoflurane general anesthesia provide new ideas for further study the mechanism of general anesthesia.Methods: 1.Calcium signal: 6 DAT-cre mice were selected to establish a calcium signal model(r AAV EFLa-DIO-GCa MP6s-WPRE-PA n = 6).After three weeks of modeling,experiments were carried out to monitor the fluorescence intensity of DA neurons of DRN in propofol / isoflurane general anesthesia in real time and analyze the effect of propofol / isoflurane on the activity of DA neurons of DRN 2.Chemical genetics: 24 DAT-cre mice were selected to establish the chemical genetic experiment group model(activation group: r AAV-EFLa-DIO-h M3D(Gq)-EGFP-WPRES,n=8,inhibition group: r AAV-EFLa-DIO-h M4D(Gi)-EGFP-WP RES,n = 8)and control group model(r AAV-EFLa-DIO-m Cherry-WPRE-PA,n = 8).After three weeks,the mice were divided into normal saline(NS)group(I ntraperitoneal injection of NS:1mg/kg)and clozapine-N-oxide(CNO)group(Int raperitoneal injection of CNO:1mg/kg).The changes of behavioral and EEG dur ing propofol / isoflurane general anesthesia were recorded,and the effects of D A neurons activity of DRN on propofol / isoflurane general anesthesia were an alyzed 3.Photogenetics: 24 DAT-cre mice were selected to establish the experimental group model of photogenetics(activation group: r AAV-EFLa-DIO-h Ch R2-m Cher ry-WPRE-PA n = 8,inhibition group: r AAv-EFLa-DIO-e Np HR-m Cherry-WPREPA,n = 8)and control group model(r AAv-EFLa-DIO-m Cherry-WPRE-PA,n = 8),After three weeks of modeling,experiments were carried out to record th e changes of behavior and EEG in mice during propofol / isoflurane general a nesthesia with or without light stimulation,and analyze the effect of the activit y DRN DA neurons on propofol / isoflurane general anesthesia 4.In vitro electrophysiology: Selecting 14-21 days healthy SD rats,and establishing the whole cell recording mode successfully.Taking s IPSC as the recording index to record the changes of s IPSC’s frequency and amplitude of DRN DA neurons before and after perfusion of propofol / isoflurane.Analyzing the effect of propofol / isoflurane on DRN DA neurons,and using immunofluorescence technology to identify DRN DA neuronsResults: 1.The results of calcium signal show that:(1)Propofol: After a single intravenous injection of propofol(20mg / kg),the righting reflex disappeared immediately,and the activity of DA neurons in DRN decreased significantly(P<0.05)and remained stable,while it increased significantly(P<0.05)when the righting reflex recovered and lower than pre-aesthesia level.(2)Isoflurane: After the mice were placed in the induction box containing 1.4% isoflurane,the activity of DA neurons in DRN began to decrease gradually(P<0.001)and decline slowly during the loss of consciousness,while it increased gradually(P<0.001)when the righting reflex of mice recovered but lower than pre-aesthesia level.2.The results of chemogenetics / photogenetics showed that: Chemogenetics activation group(M3 group):(1)Propofol: Compared to the NS group of the M3 group,the CNO group in the same group reduce the RORR time significantly(P<0.001).This group reduce the radio of δ wave(P<0.0001)and increase the radio of β wave(P<0.05),while the ratio of other frequency bands did not significantly change.Compared to the CNO group of m Cherry group,the CNO group in the M3 group reduce the RORR time significantly(P<0.05).This group reduce the radio of δ wave(P<0.001),while the ratio of other frequency bands did not significantly change.(2)Isoflurane: Compared to the NS group of the M3 group,the CNO group in the same group reduce the RORR time significantly(P<0.01),but the LORR time did not change.This group reduce the radio of δ wave(P<0.01),while the ratio of other frequency bands did no significantly change.Compared to the CNO group of m Cherry group,the CNO group in the M3 group reduce the RORR time significantly(P<0.01),but the LORR time did not change.This group reduce the radio of δ wave(P<0.05),while the ratio of other frequency bands did not significantly change.Chemogenetics inhibition group(M4 group):(1)Propofol: Compared to the NS group of the M4 group,the CNO group in the same group increase the RORR time significantly(P<0.01).This group increase the radio of δ wave(P<0.001)and reduce the radio of γ wave(P<0.05),while the ratio of other frequency bands did not significantly change.Compared to the CNO group of m Cherry group,the CNO group in the M4 group increase the RORR time significantly(P<0.01).This group increase the radio of δ wave(P<0.001),while the ratio of other frequency bands did not significantly change.(2)Isoflurane: Compared to the NS group of the M4 group,the CNO group in the same group increase the RORR time significantly(P<0.01),but the LORR time did not change.This group increase the radio of δ wave(P<0.0001),while the ratio of other frequency bands did not significantly change.Compared to the CNO group of m Cherry group,the CNO group in the M4 group increase the RORR time significantly(P<0.001),but the LORR time did not change.This group increase the radio of δ wave(P<0.0001),while the ratio of other frequency bands did not significantly changed.Optogenetics activation group(Ch R2 group):(1)Propofol: Compared to the Light-off group of the Ch R2 group,the Light-on group in the same group reduce the RORR time significantly(P<0.001).This group reduce the radio of δ wave(P<0.0001),but the ratio of other frequency bands did not significantly change.Compared to the Light-on group of m Cherry group,the Light-on group in the Ch R2 group reduce the RORR time significantly(P<0.01).This group reduce the radio of δ wave(P<0.05),while the ratio of other frequency bands did not significantly change.(2)Isoflurane: Compared to the Light-off group of the Ch R2 group,the Light-on group in the same group reduce the RORR time significantly(P<0.01),but the LORR time did not change.This group reduce the radio of δ wave(P<0.001),while the ratio of other frequency bands did no significantly change.Compared to the Light-on group of m Cherry group,the Light-on group in the Ch R2 group reduce the RORR time significantly(P<0.001),but the LORR time did not change.This group reduce the radio of δ wave(P<0.05)and increase the radio of γ wave(P<0.01),while the ratio of other frequency bands did not significantly change.Optogenetics inhibition group(Np HR group):(1)Propofol: Compared to the Light-off group of the Np HR group,the Light-on group in the same group did not change the RORR、LORR time.This group increase the radio of δ wave(P<0.01),while the ratio of other frequency bands did not significantly change.Compared to the Light-on group of m Cherry group,the Light-on group in the Np HR group did not change the RORR、LORR time and the radio of the all frequency bands.(2)Isoflurane: Compared to the Light-off group of the Np HR group,the Light-on group in the same group did not change the RORR、LORR time.This group increase the radio of δ wave(P<0.0001),while the ratio of other frequency bands did no significantly change.Compared to the Light-on group of m Cherry group,the Light-on group in the Np HR group did not change the RORR、LORR time.This group increase the radio of δ wave(P<0.01),while the ratio of other frequency bands did not significantly changed.In vitro electrophysiological results showed that:(1)Propofol: After perfusion of 10mm propofol,the frequency of IPSC produced by DRN DA neurons increased,but the amplitude did not change;after perfusion of gabazine(GABAA receptor blocker),the current disappeared.(2)Isoflurane:After perfusion of 0.55 m M isoflurane,the frequency and amplitude of IPSC produced by DRN DA neurons increased;after perfusion of gabazine,the current disappeared.Conclusion: 1.DRN DA neurons may be involved in the induction and recovery of propofol / isoflurane anesthesia 2.The activity of DRN DA neurons has a significant effect on the anesthetic effect of propofol / isoflurane 3.Propofol may indirectly affects DA neurons through DRN GABA neurons 4.The inhibitory effect of isoflurane on DRN DA neurons may involve GABAA receptors... |
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