The States of Different 5-HT Receptors Located in the Dorsal Raphe Nucleus Are Crucial for Regulating the Awakening During General Anesthesia

General anesthesia is widely used in various clinical practices due to its ability to cause loss of consciousness. However, the exact mechanism of anesthesia-induced unconsciousness remains unclear. It is generally thought that arousal-related brain nuclei are involved. 5-Hydroxytryptamine (5-HT) is closely associated with sleep arousal. Here, we explore the role of the 5-HT system in anesthetic awakening through pharmacological interventions and optogenetic techniques. Our data showed that exogenous administration of 5-hydroxytryptophan (5-HTP) and optogenetic activation of 5-HT neurons in the dorsal raphe nucleus (DR) could significantly shorten the emergence time of sevoflurane anesthesia in mice, suggesting that regulation of the 5-HT system using both endogenous and exogenous approaches could mediate delayed emergence. In addition, we first discovered that the different 5-HT receptors located in the DR, known as 5-HT autoreceptors, are essential for the regulation of general anesthetic awakening, with 5-HT1A and 5-HT2A/C receptors playing a regulatory role. These results can provide a reliable theoretical basis as well as potential targets for clinical intervention to prevent delayed emergence and some postoperative risks.


Introduction
General anesthesia, which can cause temporary depression of the central nervous system (CNS), is a drug-induced, reversible coma.It is widely used in surgery or a variety of diagnostic medicine, clinically manifested by unconsciousness, analgesia, amnesia, and akinesia [1,2].Previous studies have shown that under general anesthesia, the brain responds to somatosensory, auditory, and visual stimuli rather than being silent or unresponsive to external stimuli [3][4][5].Delayed emergence, as a common postoperative complication, is closely associated with general anesthesia, which will result in an increased incidence of cognitive decline and some serious cardiovascular events [6][7][8], thus increasing the risk and difficulty of anesthesia management and severely affecting the quality of the awakening period.Therefore, it is important to study the specific mechanism of delayed emergence from general anesthesia.However, due to the complexity of the mechanisms underlying delayed emergence, the current understanding of it remains elusive.
HaiXiang Ma, LeYuan Gu, YuLing Wang, QingXu, Yuanli Zhang, and WeiHui Shao contributed equally to this work.Some studies have reported that the state of the brain during general anesthesia is similar to that during nonrapid eye movement (NREM) sleep [9].As a result, the neurotransmitters associated with sleep-wake behavior may play an important role in regulating anesthetic awakening.5-Hydroxytryptamine (5-HT) is involved in many functions of the central nervous system, including sleep-wake behavior, cognition, emotion, etc. [10,11].Related studies have shown that central 5-HT level fluctuates during anesthesia, suggesting that the 5-HT system plays a regulatory role in general anesthesia [12].The dorsal raphe nucleus (DR) is a main source of 5-HT in the CNS and projects to the cerebral cortex, limbic system (hippocampus, amygdala), basal ganglia, and hypothalamus [13][14][15][16].Although a previous study has revealed that 5-HT neurons in the DR were implicated in arousal from isoflurane anesthesia, whether increasing 5-HT synthesis by exogenous administration affects anesthetic awakening has also not been verified [17].In addition, sevoflurane is currently the most commonly used general anesthetic because inhalation of sevoflurane can result in a shorter emergence time compared with isoflurane [18].However, there are fewer studies on the involvement of 5-HT neurons and 5-HT receptors in the regulation of sevoflurane anesthesia awakening, and the specific mechanism of action is not clear.Based on this, we chose the 5-HT system as the target of our study to investigate its role in the regulation of consciousness and its neuronal mechanism in sevoflurane anesthesia.Although studies have illustrated the regulatory role of different 5-HT receptors in the past, they have only focused on postsynaptic 5-HT heteroreceptors located in downstream targets.Based on studies showing the importance of the 5-HT autoreceptors in the DR in regulating 5-HT neuronal activity, further studies on the role of 5-HT autoreceptors in the DR in general anesthetic awakening are urgently needed.
In the present study, we explored whether exogenous 5-HT supplementation or endogenous manipulation of the 5-HT neurons in the DR could affect the emergence time of sevoflurane anesthesia.Using pharmacology, optogenetics, Fos expression, and cortical electroencephalogram (EEG), we found that 5-HT neurons in the DR and different 5-HT receptors are involved in modulating awakening from sevoflurane anesthesia.More importantly, by intranuclear microinjection, we found that the states of different 5-HT receptors located in the DR played a key role.Notably, we took the lead in combining endogenous and exogenous administrations to conduct the experiments and test our hypothesis.Our study may provide an important potential intervention target for exploring the mechanism of anesthetic awakening and clinical regulation of the postanesthesia recovery phase.

Animals
This study was approved by the Animal Advisory Committee of Zhejiang University, and all experimental procedures were in line with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.Wildtype C57BL/6 J mice were purchased from the Animal Experiment Center of Zhejiang University.All mice were housed and bred in the SPF-Class Housing of Laboratory of Animal Center of Zhejiang University School of Medicine and raised in a standard condition (indoor temperature 25 ℃, ambient humidity 65%, 12-h light/dark cycle) with rodent food and water ad libitum.In order to avoid interference from gender and female estrous cycle, only male mice were used, and all the experiments were completed between 9:00 and 15:00.The TPH2-ChETA (E123T mutation in Channelrhodopsin 2 [ChR2])-expressing mice were generated by viral delivery of pAAV-TPH2 PRO-ChETA-EYFP-WPRES-PAS under the control of the promoter of TPH2 into the DR, and post 3 weeks, TPH2-ChETA expression in the DR was determined by immunohistochemistry upon completion of optogenetics experiments.The promoter for TPH2 that we used in the present study has been used previously to be effectively infected 5-HT neurons in the DR by our group to mediate the balance between reward and aversion [19] (Table 1).

Stereotactic Surgery
C57BL/6 J mice were anesthetized with 3.5% chloral hydrate and head-fixed in a stereotaxic apparatus (68,030, RWD Life Science Inc., Shenzhen, China), as previously described.Throughout the entire surgical process, a heating pad was used to keep the body temperature of anesthetized mice around 37 °C.According to the 4th edition of the mouse brain atlas, the target brain coordinates were located on the skull surface (DRN: AP − 4.60 mm, ML − 0.05 mm, DV − 3.10 mm, 10° right; parococele: AP − 0.45 mm, ML − 1.00 mm, DV − 2.50 mm).After the coordinates were confirmed, the skull was exposed and cleaned with 75% alcohol.Then, a miniature handheld cranial drill penetrated the skull without causing any damage to the dura mater or cerebral parenchyma.Bone fragments and blood scabs were removed with the tip of a 1-ml syringe.Virus of pAAV-TPH2 PRO-ChETA-EYFP-WPRES-PAS was used for optogenetic experiments, which were provided by Sheng BO, Co., Ltd.(Shanghai, China), and the sequences of vectors were designed by Kazuki Nagayasu as the gift to the study (Department of Molecular Pharmacology Graduate School of Pharmaceutical Sciences, Kyoto University).Viruses were microinjected at a rate of 40 nl/min into confirmed coordinates via a gauge needle by an Ultra Micro Pump (160,494 F10E, WPI) of approximately 100 nl; the syringe was not removed until 15 min after the end of infusion to allow diffusion of the viruses.Wet cotton balls with normal saline are placed on the surface of the skull to keep the skull and scalp moist, helping to ease skin tension and facilitate suturing.The incision was smeared with erythromycin ointment to avoid infection.Then the mice were resuscitated on the thermostatic electric blanket, and put back into the feeding cage after confirming reactivation.Wait until the virus has been infected for 3-4 weeks before proceeding to the next step.The position of virus expression and cannula was confirmed after the experiment, that is, whether the fluorescence expression of the virus and the cannula were buried in the target brain region.

Behavioral Tests
Behavioral tests were adopted to examine the induction and emergence times.Mice were freely placed in an inhalation anesthesia box for 30 min to adapt to the environment.The gas flow into the anesthesia box is 2 L/min, with 80% oxygen and 20% nitrogen.The bottom of the anesthesia box was preheated with an electric heating pad for 15 min prior to the experiment to keep the temperature in the anesthesia box appropriate during the experiment.Soda lime was spread on the bottom of the box to absorb the CO2.Anesthesia was induced by 8% sevoflurane with 100% O2 at a flow rate of 2 L/min.Sevoflurane for anesthesia was purchased from RWD Life Science Inc (Shenzhen, China).The box was rotated 90° every 15 s until the mice exhibited a behaved loss of righting reflex (LORR) and could not turn themselves prone onto all four limbs.The induction time was defined as the interval from the start of sevoflurane inhalation to the LORR.Then the concentration of sevoflurane changed to 2% for anesthesia maintenance.At the 30th min of anesthesia, the sevoflurane ceased, and rapid oxygen administration at a rate of 2 L/min was used to swiftly eliminate the remaining sevoflurane from the anesthesia box and tube.The anesthesia box was rotated as described above to estimate the moment at which mice could turn independently from supine with at least three paws reaching the bottom of the box.Emergence time was defined as the moment between the termination of anesthesia to the recovery of the righting reflex (RORR).

Effect of IP Administration of 5-HTP on Induction Time, Emergence Time, and Activity of 5-TH Neurons from Sevoflurane Anesthesia
5-HTP was dissolved in saline for intraperitoneal (IP) administration.IP injections of 5-HTP (50 mg/kg, 100 mg/ kg), a precursor to 5-HT synthesis, were administered to C57BL/6 J mice, followed by another IP injection 24 h later.One hour later, the mice were placed in an anesthesia box for induction and maintenance, and the induction time was recorded.The sevoflurane volatilization tank was closed at the 30th minute of anesthesia to stop inhalation anesthesia and the emergence time was recorded.According to the program of sevoflurane anesthesia, the analysis of co-expression between c-fos and TPH2 in the DR from the groups with and without sevoflurane anesthesia was performed post the emergence time.From the same program of sevoflurane anesthesia, the analysis of co-expression between c-fos and TPH2 in the DR from the groups with and without treatment with 5-THP under sevoflurane anesthesia was performed post the emergence time.

The Effects of ActivatingTPH2-ChETA Neurons by Optogenetics in the DR on the Induction Time, Emergence Time, and Activity of the DR Under Sevoflurane Anesthesia
For the optogenetic experiment, 3 weeks after pAAV-TPH2 PRO-ChETA-EYFP-WPRES-PAS was injected into the DR of C57BL/6 J mice, optical fiber (diameter 200 μm, numerical aperture 0.22) was implanted into the DR.After the fiber was embedded for 1 week, the optogenetics experiments were performed.The laser was connected to the core of the mice's heads by the optical fiber bouncing lining.The 5-HT neurons (expressing ChETA) in the DR were activated by blue light (465 nm, 20 Hz, 20-ms pulse width, 15 mW, and 20 min).The output parameters of the laser were adjusted according to the laser intensity detected by the optical power meter at the end of the core, and the laser range within 15 mW was controlled.The induction time and emergence time of sevoflurane anesthesia were recorded and analyzed statistically.After finishing the experiments, the co-expression between TPH2 and ChETA and the placement of fiber were confirmed by immunohistochemistry and histology.
According to the program of sevoflurane anesthesia, the analysis of the co-expression between c-fos and TPH2 was performed in the groups treatment with and without photostimulation under sevoflurane anesthesia post the emergence time.

Effect of ICV Administration of Different 5-HT Receptor Agonists and Antagonists on the Induction Time and Emergence Time of Sevoflurane Anesthesia
The same batch of 8-week-old wild-type C57BL/6 J mice was used, with an intracerebroventricular (ICV) guide cannula implanted for 1 week.The mice were administered agonists/antagonists of different 5-HT receptors through the lateral ventricle and placed in an anesthesia box for anesthesia induction and maintenance, and the anesthesia induction time was recorded.At 15 min of anesthesia, 5-HT1A receptor agonist 8-OH-DPAT (3 mg/ml, 6 mg/ml) or 5-HT1A receptor antagonist WAY 100635 (5 mg/ml, 10 mg/ml) or 5-HT2A/C receptor agonist DOI (1 mg/ml, 2 mg/ml) or 5-HT2A receptor antagonist Ketanserin (KET) (1 mg/ml, 2 mg/ml) 2000 nL were administered via a lateral ventricular catheter.The anesthesia was terminated at the 30th min, and the time of emergence was recorded.

Effect of Intranuclear Administration in the DR of Different 5-HT Receptors Agonists and Antagonists on Induction Time and Emergence Time of Sevoflurane Anesthesia
The same batch of 8-week-old wild-type C57BL/6 J mice with DR guide cannula implantation for 1 week was used, and agonists/antagonists of different 5-HT receptors were microinjected into the nucleus through cannulas.C57BL/6 J mice were placed in an anesthesia box for anesthesia induction and maintenance, and the time spent on induction was recorded.After the 15 min of anesthesia, 8-OH-DPAT (1.5 mg/ml, 3 mg/ml, 6 mg/ml) or WAY 100635 (5 mg/ml, 10 mg/ml, 15 mg/ml) or DOI (1 mg/ml, 2 mg/ml) or KET (1 mg/ml, 2 mg/ml) 200 nL were administered through the cannulas.At the 30th min following anesthesia, the anesthesia was terminated, and the time of awakening was recorded as emergence time.

Lateral Ventricle Catheterization and EEG Electrode Implantation
After leveling the skull and drilling the target region, a shallow concave is drilled in the left anterior bregma as well as the left and right anterior of the posterior fontanelle respectively (without drilling through the skull).The screw in a shallow concave was fixed, with exposed 1/3 of the skull surface and firmly connected as the standard.A wire is attached to the screws in the left anterior bregma and the right anterior of the posterior fontanelle to implant EEG electrodes.The screws are then tightened until the top of the screw meets the brain tissue, after which the cannula and screws are secured.Two wires were then welded to the electrode.After the cannula, screws, electrodes, and mouse cranium were securely fastened and inserted into the inner core of the cannula, the mice weights were once more measured and recorded.After the cannula, screw, electrode, and skull were bonded together, mice were weighed once more each night, and the data were gathered.The cannula is placed directly inside the desired region of the brain if electrode implantation is not necessary.After firmly bonding the cannula, screw, electrode, and cranium and inserting them into the cannula's inner core, the mice were weighed once more, and the results were recorded.
The surgical site was covered with erythromycin ointment, and the mice were fed alone for a week while they recovered.

Immunohistochemistry and Histology
Histology was used to verify the placement position of the optical fiber cannula tip for the DR in each mouse.
After fixation, placed in a 4% PFA refrigerator for fixation for about 16 h and saturation in 30% sucrose for about 24 h, each mouse brain was sectioned at 30-μm-thick coronal slices using a freezing microtome (CM30503, Leica Biosystems, Buffalo Grove, IL, USA).The brain sections were first washed in PBS for 5 min each and incubated in a blocking solution for 2.5 h at room temperature.Then for c-fos or TPH2 staining in the DR, sections were incubated at 4 °C overnight in a solution of mouse anti-c-fos primary antibody (1:1000 dilution, Abcam, UK) or mouse anti-TPH2 primary antibody (1:100 dilution, T0678, Sigma-Aldrich, UK), followed by incubation of the secondary antibodies, containing anti-mouse Alexa 488 (1:1000; A32766, Thermo Fisher Scientific, Waltham, MA, USA), anti-rabbit Alexa 546 (1:1000; A10036, Thermo Fisher Scientific), or anti-rabbit Alexa 488 (1:1000; A10523, Thermo Fisher Scientific) for 1 h at room temperature.
After washing three times with PBS for 15 min each, the sections were mounted onto glass slides and incubated in DAPI (1:4000; Cat#C1002; Beyotime Biotechnology; Shanghai, China) for 7 min at room temperature.The glass slides were then sealed with 60% glycerol.The Nikon A1 laser-scanning confocal microscope (Nikon, Tokyo, Japan) was used to capture all of the images, and ImageJ (NIH, Bethesda, MD, USA) was used to count and analyze the number of immunopositive cells.Stereological cell counting of co-expression cells were captured using a Nikon A1 confocal microscope through a 20 × objective (numerical aperture 0.75).All images were taken at a resolution of 1024 × 1024 pixels at room temperature.Co-expression of c-fos and TPH2 cells was counted in three sections (n = 3) from each mouse, and coexpression of c-fos, EYFP, and TPH2 cells were counted in six sections (n = 6) from each mouse.Notably, mice with implantations outside of the targeted brain structure were not used in our studies.

Statistical Analysis
All data are expressed as the mean ± standard error of the mean (SEM).Statistical analyses were performed with GraphPad Prism TM 8.0 and SPSS version 22.0 (SPSS Software Inc., Chicago, IL, USA).Comparison between the two groups: If the data conformed to the normal distribution, Student's T-test was used, including independent sample T-test and paired sample T-test.If the data did not meet the normal distribution, Mann-Whitney U or Wilcoxon signed-rank test was used.Levene test was used to test the homoscedasticity of each other.After the data is in line with normal distribution and homoscedasticity, one-way ANOVA was used to compare three groups and above, and two-way ANOVA followed by Bonferroni's test was used to compare double-factor.P values less than 0.05 were considered statistically significant (Table 2).

The Emergence Time of Sevoflurane Anesthesia Was Significantly Reduced by IP Injection of 5-HTP for Exogenous Supplementation of 5-HT
To explore the role of 5-HT in the induction time and emergence time of sevoflurane anesthesia, we selected wild-type C57BL/6 J mice at 8 weeks after birth for the experiment.C57BL/6 J mice were intraperitoneally injected with 5-HTP at different doses (50, 100 mg/kg), the precursor of 5-HT, and then intraperitoneally injected with 5-HTP again 24 h later.Compared with the control group, IP injection of 5-HTP (50 mg/kg) had no significant effect on the induction time (P > 0.05), whereas IP injection of 5-HTP (100 mg/kg) prolonged the induction time (P < 0.05, Fig. 1B).However, compared with the control group and 5-HTP (50 mg/kg) group, the emergence time was significantly reduced in the 5-HTP group (100 mg/kg) (P < 0.05, Fig. 1C).These results showed that IP injection of 5-HTP could dose-dependently prolong the induction time and shorten the emergence time, indicating that 5-HT played a key role in modulating the emergence from sevoflurane anesthesia, and exogenous supplementation of 5-HT might have the effect of promoting awakening.Next, we would like to investigate which structure was the target of 5-HTP.Since 5-HTP can pass through the blood-brain barrier before being converted to 5-HT in the brain, we speculated that the DR, which is the biggest nucleus in the brain that synthesizes and releases 5-HT, would be its possible target.We observed the changes in the co-expression of c-fos, which is commonly accepted as a proxy of neuronal activity, and tryptophan hydroxylase (TPH2), a key enzyme for 5-HT synthesis, in the DR by immunohistochemistry.Under sevoflurane anesthesia, compared with the control group, the co-expression of c-fos and TPH2 in the DR was significantly increased in the 5-HTP treatment group (P < 0.05, Fig. 1D-F).This result indicated that IP injection of 5-HTP, in addition to exogenously increasing the level of 5-HT, may also promote emergence from sevoflurane anesthesia by activating 5-HT neurons in the DR.

The Activity of 5-HT Neurons in the DR Was Significantly Reduced Under Sevoflurane Anesthesia
To further investigate whether IP injection of 5-HTP mediated the awaking from sevoflurane anesthesia by targeting the DR, we again observed the changes in the co-expression of the c-fos and TPH2 in the DR by immunohistochemistry. Compared with the control group without the sevoflurane anesthesia, the co-expression of c-fos and TPH2 was significantly reduced in the group with the sevoflurane anesthesia, indicating that the activity of 5-HT neurons in the DR was suppressed under sevoflurane anesthesia (P < 0.05, Fig. 2).This result suggested that 5-HT neurons in the DR could be a major contributor to delayed emergence.did not perform photostimulation (No PS) to observe the interrelation between 5-HT neurons and emergence time (Fig. 3A).Although activation of DR 5-HT neurons showed little influence on the induction time (P > 0.05, Fig. 3B), the emergence time was significantly shortened in 15-min and 20-min blue light irradiation at DR compared with the control group (P < 0.01, Fig. 3C).Next, we examined the neuronal expression of c-fos in the DR of C57BL/6 J mice with and without photostimulation to investigate whether photostimulation increased the excitability of DR 5-HT neurons.Under sevoflurane anesthesia, c-fos expression in DR 5-HT neurons was significantly increased with photostimulation (20-ms pulse duration, 20 Hz, at 15 mW for 20 min (P < 0.05, Fig. 4) in comparison with the control group without photostimulation.This result indicated that the reduction in emergence time induced by photostimulation occurred via the activation of 5-HT neurons.These data indicated that specific endogenous activation of 5-HT neurons in the DR can significantly reduce the emergence time while having no significant effect on induction time.

Different 5-HT Receptors Agonists/Antagonists Injected into the Lateral Ventricle Only Affected the Emergence Time of Sevoflurane Anesthesia
The above results have shown that 5-HT signaling could facilitate emergence from general anesthesia.Next, we began to explore the effects of another important component of the 5-HT system, the different 5-HT receptors, on anesthesia induction and emergence time.We selected wild-type 8-week-old C57BL/6 J mice for lateral ventricle catheterization and then administered agonists/ antagonists of different 5-HT receptors by ICV.5-HT1A receptor agonist 8-OH-DPAT, 5-HT1A receptor antagonist WAY100635, 5-HT2A/2C receptor agonist DOI, or 5-HT2A receptor antagonist KET 2000 nL were administered through the lateral ventricle cannula.These four drugs had no effect on the induction time (Fig. 5B-E).However, for emergence time, compared with the control group, administration of 8-OH-DPAT (6 mg/ml), DOI (2 mg/ml), or KET (2 mg/ml) reduced emergence time, with 8-OH-DPAT and KET having a greater effect on shortening emergence time (P < 0.01 or 0.01 < P < 0.05, Fig. 5F, H, I).In contrast, administration of WAY100635 (10 mg/ml) prolonged the emergence time (P < 0.01, Fig. 5G).These results indicated that activation of 5-HT1A receptor could significantly shorten the emergence time.However, for 5-HT2A/2C receptors, both antagonism of 5-HT2A receptors and simultaneous activation of 5-HT2A and 2C receptors could shorten the emergence time.The 5-HT1A and 2C receptors may promote awakening, while 5-HT2A receptors inhibit awakening.Taken together, the interaction between different 5-HT receptors can play an important role in modulating awakening from general anesthesia.

Different 5-HT Receptors Agonists/Antagonists Microinjected into the DR Only Affected the Emergence Time of Sevoflurane Anesthesia
To further clarify the effects of different 5-HT receptors in the DR on induction time and emergence time of sevoflurane anesthesia, the drugs mentioned above were microinjected into the DR via guide cannulas.8-OH-DPAT or WAY 100635 or DOI or KET 200 nL were administered through the cannulas (Fig. 6A).Intra-DR microinjection of these four drugs had no significant effect on sevoflurane induction time (P > 0.05, Fig. 6B-E).However, DR intranuclear injection of 8-OH-DPAT, KET, or DOI could shorten the emergence time in a dose-dependent manner, among which high concentrations of 8-OH-DPAT and DOI were more significant(P < 0.01 or 0.01 < P < 0.05, Fig. 6F, H, I).In contrast, microinjection of WAY 100635 into the DR resulted in prolonged emergence time (P < 0.01, Fig. 6G).It can be found that activation of 5-HT1A receptor can shorten the emergence time, and activation of 5-HT2A/C receptor and inhibition of 5-HT2A receptor both led to shorter emergence times.These results were consistent with those of the pharmacological intervention in the lateral ventricle.Overall, intranuclear injection selectively targets 5-HT receptors in the DR and avoids adverse pharmacological effects, and the results of these two parts highlight the involvement of different 5-HT receptors in the regulation of emergence from sevoflurane anesthesia.

Activation of 5-HT1A Receptor Alleviated General Anesthesia-Induced Depression of EEG Activity Without Affecting the Depth of Anesthesia
In order to investigate the effects of sevoflurane anesthesia and 5-HT1A receptor agonist 8-OH-DPAT on the brain electrical activity, cortical EEG was recorded and analyzed under different states of anesthesia.We selected the same batch of wild-type C57BL/6 J mice at 8 weeks after birth for lateral ventricle catheterization and cortical electrode implantation; 5-HT1A receptor agonist 8-OH-DPAT was administered through the lateral ventricle, and EEG was recorded (Fig. 7A).There was no significant difference in the induction time of sevoflurane anesthesia in the 8-OH-DPAT (6 mg/ml) group compared with the vehicle group (P > 0.05, Fig. 7B, n = 8), whereas the emergence time of sevoflurane anesthesia was significantly reduced in the 8-OH-DPAT (6 mg/ml) group compared with the vehicle group (P < 0.001, Fig. 7C, n = 8).
Cortical EEG of mice was recorded as "before sevoflurane on," "during sevoflurane anesthesia," and "after sevoflurane off" (Fig. 7A).We found that the cortical EEG voltage amplitude peak (power) of both groups showed a substantial decrease after LORR, and the cortical EEG voltage was at a low level during the maintenance of anesthesia.When sevoflurane inhalation was stopped and RORR occurred in mice, i.e., after the mice woke up, the cortical EEG voltage was restored, but their EEG activity was still lower than before anesthesia (Fig. 7D-H).
For Delta and Theta waves, the average proportions reduced considerably in the two groups following anesthesia compared to the condition of being conscious, while the decline in the 8-OH-DPAT group (0.01 < P < 0.05) was smaller than that in the vehicle group (P < 0.01, Fig. 7I-J).For Alpha waves, the average proportion of Alpha waves in the vehicle group was significantly lower during the maintenance and emergence phases of anesthesia compared to the conscious state (P < 0.01).However, the average proportion of Alpha waves in the 8-OH-DPAT group decreased during the maintenance phase of anesthesia, but did not show a statistical difference, and further decreased when the mice were in the emergence phase.Compared with the state of being conscious, there were significant differences (P < 0.05, Fig. 7K).For Beta waves, the vehicle group decreased significantly during the maintenance and emergence phases of anesthesia compared to the conscious state (P < 0.01 or 0.01 < P < 0.05).However, in the 8-OH-DPAT group, there was no statistical difference in the proportion of Beta wave during the maintenance and emergence phases of anesthesia compared to the conscious state (P > 0.05, Fig. 7L).For Gamma wave, its proportion in the vehicle group decreased after anesthesia but showed no statistical difference compared with the state of being conscious (P > 0.05), but significantly decreased during the emergence state (P < 0.01).For the 8-OH-DPAT group, the Gamma wave proportion decreased significantly under anesthesia and during the emergence state (P < 0.05, Fig. 7M).
We found that there was no statistical difference in the proportion of each wave between the two groups in different states, suggesting that the ICV injection of 8-OH-DPAT had no significant effect on the depth of anesthesia.However, it is worth noting that during the maintenance and emergence phases of anesthesia, the amplitude of each wave in the 8-OH-DPAT group decreased less than that in the vehicle group, showing that activation of 5-HT1A receptor could alleviate anesthesia-induced depression of EEG activity, which was beneficial for promoting postanesthesia awakening and preventing the occurrence of delayed emergence (Fig. 7).

Discussion
The mechanisms of emergence from general anesthesia and reversible loss of consciousness under general anesthesia are the focus of research in the field of anesthesia.Delayed emergence is a common and serious complication associated with general anesthesia and requires high concern.Although initial progress has been made to identify the causes of delayed emergence, the exact pathogenesis of it remains still unexplored.Herein, our results provide new insights into the role of the 5-HT system to elucidate the cause of delayed emergence.Our study showed that both IP injection of 5-HTP and activation of 5-HT neurons in the DR could significantly shorten the emergence time of sevoflurane anesthesia.In addition, intracerebroventricular injection and intranuclear microinjection of different 5-HT receptor agonists or antagonists indicated that the interaction between 5-HT1A and 5-HT2A/C receptors was involved in the regulation of emergence, with the 5-HT1A receptor located in the DR playing a key role.Finally, we also observed the effect of anesthesia on EEG in mice by cortical EEG recording, indicating that activating the 5-HT1A receptor had no significant effect on the depth of anesthesia, but can alleviate the inhibition of EEG activity caused by anesthesia and shorten the emergence time.
Considering the distribution of 5-HT both in the peripheral and central nervous system, based on our observation already made by peripheral administration of 5-HTP via IP injection, we further explored whether ICV administration of 5-HT receptor agonists or antagonists would affect via the central pathway.Similar effects were observed with ICV administrations of 5-HT receptor agonists, consistent with the effects of IP injections of 5-HTP.Furthermore, we performed DR intranuclear injection to achieve precise localization and selective targeting of the DR, thus avoiding adverse pharmacological effects [20].
Our study has the following innovation points: Firstly, we improved the efficiency of 5-HT synthesis and transmission in the brain via IP injection of 5-HTP, suggesting that exogenous 5-HT supplementation could regulate emergence from sevoflurane anesthesia.Secondly, pharmacological methods in our study represent a progressive experimental process from the periphery to the lateral ventricles to the DR, namely from the periphery to the center, and revealed the different roles of different 5-HT receptors in anesthetic awakening through the activation/inhibition What's more, the effect of sevoflurane anesthesia on the brain electrical activity was explored for the first time by recording cortical EEG.These results provide potential targets for intervention to prevent delayed awakening in our clinical practice.
The arousal of the brain depends on many regions that can simultaneously receive different arousal signals.The DR is closely linked to various brain regions, suggesting to us that specific intervention of the activity of serotonergic neurons in the DR may alter the neural activity of its downstream targets and thus regulate the anesthetic awakening through a more complex mechanism.Meanwhile, the noradrenergic, histaminergic, dopaminergic, glutamatergic, cholinergic, and GABAergic systems in different brain regions are also involved in sleep and arousal [20][21][22][23][24][25].The ascending reticular activating system plays a very vital role in maintaining the awakening state of the organism.As an important part of the ascending activation system, 5-HT plays an important role in anesthesia-arousal regulation.Some studies have shown that the concentration of 5-HT in the different brain regions, including the prefrontal cortex and hippocampus, would be reduced during general anesthesia, indicating the 5-HT system may play a role during general anesthesia [12,26].And these alterations in serotonergic neurotransmission may be associated with changes in arousal and sleep.The level of 5-HT was significantly reduced in slow wave sleep (SWS), whereas the activity of 5-HT neurons in the DR was markedly increased when mice transitioned from sleep to arousal [27,28], which is consistent with our findings.However, recent studies have shown that the role of 5-HT in sleep and wakefulness cannot be simply generalized as sleep-promoting or wakefulness-promoting.The specific role of 5-HT on ).F Compared with the vehicle group, the emergence time of 6 mg/mL 8-OH-DPAT group was significantly reduced (P < 0.01).G Compared with the vehicle group, the emergence time of 10 mg/mL WAY 100635 group was significantly increased (P < 0.01).H Compared with the vehicle group, the emergence time of the 2 mg/ ml DOI group was significantly reduced (0.01 < P < 0.05).I Compared with the vehicle group, the emergence time of the 2 mg/ml KET group was significantly reduced (P < 0.01).J Staining results of the position of the lateral ventricular cannula.Data are mean ± SD sleep or wakefulness may vary and change from time to time as the timing and degree of activation of the central 5-HT system vary [29,30].These studies, combined with our findings, imply a bidirectional regulatory role for DR 5-HT neurons and highlight the importance of further studies to measure the activation of these neurons during wakefulness, sleep, or anesthesia.Meanwhile, 5-HTP can promote anesthetic awakening and alter the activity of DR There was no statistical difference in induction time among all groups (P > 0.05, n = 8).F-I Emergence time of sevoflurane anesthesia in groups with different concentrations of 8-OH-DPAT, WAY 100635, DOI, and KET (n = 8).F Compared with the vehicle group, the emergence time of 3 mg/mL and 6 mg/mL 8-OH-DPAT groups was significantly reduced (P < 0.01).G Compared with vehicle, the emergence time of 10 mg/mL and 15 mg/mL WAY 100635 group was significantly increased (P < 0.05).H Compared with the vehicle group, the emergence time of the 2 mg/ ml DOI group was significantly reduced (P < 0.01).I Compared with the vehicle group, the emergence time of the 2 mg/ ml KET group was significantly reduced (P < 0.05).J Staining results of the position of the DR cannula, Data are mean ± SD 5-HT neurons.On the one hand, this effect may be due to the passage of 5-HTP through the blood-brain barrier and its subsequent conversion to 5-HT in the brain, acting on 5-HT autoreceptors in the DR, thus causing altered neuronal activity.On the other hand, decarboxylation of 5-HTP produces serotonin, which is then converted to melatonin (N-acetyl-5-methoxytryptamine), and melatonin can regulate circadian rhythms [31].The level of melatonin in the mouse brain may increase after IP injection of 5-HTP, and its effect on delayed emergence from anesthesia needs to be further investigated [32].
5-HT can bind to a variety of receptors with different characteristics and participate in different signal transduction pathways, further adding to the postsynaptic complexity of the 5-HT system, giving the serotonergic system the ability to regulate various behavioral functions [33,34].5-HT1A receptor is a metabolic receptor that can hyperpolarize membrane potential by activating G protein-coupled receptor potassium channel [35].Relevant studies have shown that the systematic application of 5-HT1A receptor agonist flesinoxan or 8-OH-DPAT can promote arousal [36].In this study, we found that activating the 5-HT1A receptor could significantly shorten the emergence time.This result supports the important role of the 5-HT1A receptor in recovery from general anesthesia.For the 5-HT2A/C receptor, we found that injection of 5-HT2A/C receptor agonist DOI into the lateral ventricle and DR can significantly shorten the recovery time, while injection of 5-HT2A receptor antagonist KET can also shorten the recovery time, suggesting the 5-HT2A receptor has an inhibitory effect in general anesthesia awakening.This may be because KET also has mild α1 adrenergic receptor and histamine H1 receptor-blocking effects and needs to be further studied.Notably, by intranuclear injection in the DR, we found the states of different 5-HT autoreceptors (5-HT1ARauto) in the DR play a very important regulatory role.In a recent study, it was discovered that under physiological conditions, the 5-HT1ARauto in the DR has an entirely different effect from the postsynaptic 5-HT1A receptor in other parts of the brain.The activation of somatodendritic 5-HT1ARauto in the DR suppresses the neural firing of serotonergic neurons, which decreases 5-HT release in the cortex, hippocampus, and other parts of the brain [37].These results suggest that 5-HT autoreceptors in the DR and 5-HT heteroreceptors at downstream targets may play distinct roles, and it is therefore crucial to investigate the role of 5-HT autoreceptors in general anesthetic awakening.
Our study differs from a previous study in the following ways [17]: Firstly, IP injection of 5-HTP for exogenous 5-HT supplementation may regulate emergence from sevoflurane anesthesia by activating the DR 5-HT neurons.As 5-HTP is a food supplement that can also be a major component of some drugs, future clinical trials, such as cohort studies, could be conducted to further define the specific role of this component in general anesthetic awakening, which would be of great significance in ameliorating the serious post-anesthetic clinical issue of delayed awakening.Secondly, by DR intranuclear microinjection, we found an important role for the 5-HT autoreceptors in the DR in regulating general anesthetic awakening, which has not been previously studied.The concentration of intercellular 5-HT in the DR is critically regulated by 5-HT autoreceptors, which has great implications for the mechanism of general anesthetic awakening.Our results, combined with those of others, could open a promising window into the mechanisms of anesthetic awakening.
Some studies have reported that activation of postsynaptic 5-HT1A receptors expressed by GABAergic cells located in the DR would be expected to indirectly facilitate the activity of 5-HT neurons [38].For the 5-HT2A/C receptor, they have striking amino acid homology.They are primarily coupled to Gq protein, and their actions are mediated by the activation of phospholipase C, with a resulting depolarization of the host cell.The 5-HT2A and 5-HT2C receptor-containing neurons are predominantly GABAergic interneurons and projection neurons [39][40][41].Thus, activation of 5-HT2A and 5-HT2C receptors expressed in GABAergic neurons located in the DR would result in a decrease in the firing rate of the 5-HT neurons.Therefore, different 5-HT receptors on GABAergic neurons in the DR may act differently on 5-HT neurons and thus cause different effects, which can be considered as the existence of complementary mechanisms controlling the functional activity of 5-HT neurons.Further investigations are needed to obtain more solid evidence.

Limitations
There are some limitations in our study.First of all, although we have experimentally verified the role of 5-HT1A, 2A, and 2C receptors in general anesthesia emergence, there are many other 5-HT receptors, such as 5-HT3 and 5-HT6 receptors, similarly have a role in promoting awakening, and the exact role of these receptors in general anesthesia awakening remains to be further investigated.Secondly, the cholinergic ascending arousal system, GABAergic sleep-promoting system, dopaminergic system, orexinergic system, and noradrenergic system are also implicated in the mechanisms of sleep-wake cycle and general anesthesia.Even while we have shown that the 5-HT system plays a significant part in general anesthesia awakening, its involvement through interactions with other neural networks cannot be ruled out.What's more, DR projects widely to different regions of the brain, whereas we did not investigate possible projection pathways and focused only on nuclei.In summary, there are currently no well-established theoretical studies on the specific mechanisms of awakening from anesthesia, and deeper investigations are still needed in the future.

Fig. 1
Fig. 1 IP injection of 5-HTP reduced the emergence time of sevoflurane anesthesia and increased the co-expression of c-fos and TPH2 in the DR.A Experimental process of IP injection of 5-HTP.B Compared with the control group (n = 6) and 50 mg/kg 5-HTP group (n = 8), intraperitoneal administration of 100 mg/kg 5-HTP group had a longer induction time (n = 6, P < 0.05).C Compared with the control group (n = 6) and 50 mg/kg 5-HTP group (n = 8), intraperitoneal administration of 100 mg/kg 5-HTP group had a shorter emer-

Fig. 2
Fig.2The co-expression of c-fos and TPH2 in the DR was significantly reduced under sevoflurane anesthesia.A Immunofluorescence images of co-expression of TPH2 and c-fos in the control group (without sevoflurane anesthesia).B Immunofluorescence images of co-expression of TPH2 and c-fos in the sevoflurane anesthesia group.C Compared with the control group without sevoflurane anesthesia, the co-expression of c-fos and TPH2 in the DR was significantly reduced in the sevoflurane anesthesia group (n = 6/group, P < 0.05)

Fig. 3
Fig. 3 Optogenetic activation of DR 5-HT neurons reduced the emergence time of sevoflurane anesthesia.A Experimental process of activation of 5-HT neurons in the DR by optogenetics.B Compared with the control group (no PS, n = 9), there was no significant difference in the induction time of mice in each experimental group (PS 15 mW 5 min: n = 10, PS 15 mW 15 min: n = 8, PS 15 mW 20 min: n = 8, P > 0.05).C Compared with the control group (no PS, n = 9), the emergence time of the PS (15 mW 15 min) group and the PS (15 mW 20 min) group was significantly decreased (PS 15 mW 15 min: n = 8, PS 15 mW, 20 min: n = 8, P < 0.01).D Schematic diagram of ChETA virus injection in DR and optical fiber location and colocalization with TPH2.E Immunohistochemistry of C57BL/6 J mice identified co-expressed neurons of ChETA and TPH2 and pinpointed the position of the fiber tip.Thermal damage induced by light stimulation was not observed in the region around the fiber tip.F1-H2 Localized expression of ChETA on the surface of DR 5-HT neurons of C57BL/6 J mice.F1, F2 Neuronal immunostaining of EYFP, a surrogate marker for ChETA, in 5-HT neurons in the DR of a coronal brain slice.G1, G2 Immunostaining of TPH2.H1, H2 Merged images showing the co-expression of TPH2 and EYFP in 5-HT neurons.Confocal image magnifications: f1-h1, 10 × ; f2-h2, 20 × ; f3-h3, 40 × .Data are mean ± SD.No PS, no photostimulation; PS, photostimulation ◂

Fig. 4 c
Fig. 4 c-fos expression was significantly increased in the DR by the photostimulation of TPH2-ChETA neurons in the DR.A Neuronal immunostaining showing co-localization of c-fos, TPH2, and EYFP in a C57BL/6 J mouse that underwent the implantation of a fiber optic cannula without PS.B Immunostaining showing the co-localization of c-fos, TPH2, and EYFP in a C57BL/6 J mouse exposed to PS at 15 mW for 20 min.C Quantification of c-fos( +)/EYFP( +)/TPH2( +) cells in C57BL/6 J mice with and without PS.Significantly more c-fos( +)/EYFP( +)/ TPH2( +) cells were observed in the PS group than in the No PS group (P < 0.05).No PS, no photostimulation; PS, photostimulation

Fig. 5
Fig. 5 Effects of different 5-HT agonists/antagonists injected into the lateral ventricle (ICV) on induction time and emergence time of sevoflurane anesthesia.A Specific experimental protocol for lateral ventricle administration.B-E Induction time of sevoflurane anesthesia in different concentrations of 8-OH-DPAT, WAY 100635, DOI, and KET groups.There was no statistical difference in induction time among all groups (P > 0.05, n = 8).F-I Emergence time of sevoflurane anesthesia in groups with different concentrations of 8-OH-DPAT, WAY 100635, DOI, and KET (n = 8).F Compared with the vehicle group, the emergence time of 6 mg/mL 8-OH-DPAT group was significantly reduced (P < 0.01).G Compared with the vehicle group, the emergence time of 10 mg/mL WAY 100635 group was significantly increased (P < 0.01).H Compared with the vehicle group, the emergence time of the 2 mg/ ml DOI group was significantly reduced (0.01 < P < 0.05).I Compared with the vehicle group, the emergence time of the 2 mg/ml KET group was significantly reduced (P < 0.01).J Staining results of the position of the lateral ventricular cannula.Data are mean ± SD

Fig. 6
Fig.6 Effects of 5-HT agonist/ antagonist microinjected into the DR on induction time and emergence time of sevoflurane anesthesia.Specific experimental protocol for intranuclear microinjection of DR.B-E Induction time of sevoflurane anesthesia in different concentrations of 8-OH-DPAT, WAY 100635, DOI, and KET groups.There was no statistical difference in induction time among all groups (P > 0.05, n = 8).F-I Emergence time of sevoflurane anesthesia in groups with different concentrations of 8-OH-DPAT, WAY 100635, DOI, and KET (n = 8).F Compared with the vehicle group, the emergence time of 3 mg/mL and 6 mg/mL 8-OH-DPAT groups was significantly reduced (P < 0.01).G Compared with vehicle, the emergence time of 10 mg/mL and 15 mg/mL WAY 100635 group was significantly increased (P < 0.05).H Compared with the vehicle group, the emergence time of the 2 mg/ ml DOI group was significantly reduced (P < 0.01).I Compared with the vehicle group, the emergence time of the 2 mg/ ml KET group was significantly reduced (P < 0.05).J Staining results of the position of the DR cannula, Data are mean ± SD

Fig. 7
Fig.7 Effects of vehicle/administration of 8-OH-DPAT in the lateral ventricle on EEG activity.A Specific experimental protocols for EEG recording.B There was no significant difference in the induction time of sevoflurane anesthesia in the 8-OH-DPAT (6 mg/ml) group compared with the vehicle group (P > 0.05, n = 8).C Compared with the vehicle group, the emergence time of sevoflurane anesthesia in the 8-OH-DPAT (6 mg/ml) group was significantly reduced.D-E EEG in the vehicle and 8-OH-DPAT groups.F-H EEG power spectrum of mice at the state of being conscious, anesthetized, and awakened (n = 8).I-M The proportions of Delta, Theta, Alpha, Beta, and Gamma waves in the two groups under different states.N Schematic diagram of cortical EEG recording and drug delivery device in the lateral ventricle.Data are mean ± SD ◂

Table 1
Summary of experimental groups of C57BL/6 J mice

Table 2
Statistical analysis