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Generation of the unconsciousness associated with arousal during the initial stage of anesthesia by midazolam is critical for general anesthesia, however, the exact mechanism remains unknown. Here, firstly, we found that the destruction of noradrenergic neurons in the locus coeruleus (LCNE) could prolong the emergence time of midazolam-induced anesthesia. Secondly, the same results were found by activation of the noradrenergic pathway between the LC and the ventrolateral preoptic nucleus (VLPO) using optogenetics and chemogenetics approaches, respectively. Thirdly, this effect was mediated by alpha 1 and beta adrenergic receptors rather than alpha2 adrenergic receptors in the VLPO. Moreover, the noradrenergic pathway to modulate the arousal between the LC and VLPO was controlled by GABAA receptors in the LC and VLPO in our models. Our data demonstrate that activation of the NEergic pathway between the LC and VLPO can promote arousal to prevent delayed recovery from midazolam-induced 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.

In order to explore the mechanism of general anesthesia emergence, based on the common clinical phenomenon-delayed emergence, we explore the role of 5-hydroxytryptamine (5-HT) neurons in the dorsal raphe nucleus in promoting awakening from sevoflurane anesthesia in mice model. In this study, C57BL/6J male mice were selected to specifically activate or inhibit 5-HT neurons in the dorsal raphe nucleus (DRN) and different 5-HT receptors by intraperitoneal, lateral ventricle, intranuclear or DRN injection of agonists/antagonists and optogenetics during the sevoflurane anesthesia to record and observe the anesthesia induction and emergence time of mice. Through intraventricular infusion and intranuclear microinjection of 5-HT and the agonists or antagonists of different 5-HT receptors, our data showed that 5-HT and 5-HT1A and 2A/C receptors, especially 5-HT1A receptor, are involved in the regulation of delayed awakening mediated by DRN 5-HT neurons. This can provide a reliable theoretical basis as well as potential targets for clinical intervention to prevent delayed emergence and some postoperative risks.Graphical Abstract

Sudden unexpected death of epilepsy (SUDEP) is the leading cause of of death in patients with epilepsy. Due to the complicated pathogenesis of SUDEP, however, the exact mechanism of SUDEP remains elusive. Currently, although it is recognized that the seizure-induced respiratory arrest (S-IRA) may be a main cause for SUDEP, other factors resulting in SUDEP can not be excluded e.g arrhythmias. Our previous findings indicated that the incidence of S-IRA and SUDEP evoked by acoustic stimulation or pentetrazol (PTZ) injection was significantly reduced by atomoxetine, a norepinephrine reuptake inhibitor (NRI), suggesting that noradrenergic neurotransmission modulates S-IRA and SUDEP. Given that norepinephrine acts on the target to modulate respiratory and circulation function by targeting adrenergic receptor α and beta (a-AR and β-AR) and the arrhythmias can be contributed to SUDEP. Meanwhile, to further test whether cardiac factors are implicated in S-IRA and SUDEP, we choose esmolol hydrochloride, a selective antagonist of β1-AR to test it in our models. Our findings demonstrated that the lower incidence of S-IRA and SUDEP evoked by acoustic stimulation or PTZ injection in DBA/1 mice by administration with atomoxetine was significantly reversed by intraperitoneal (IP) of esmolol hydrochloride. Importantly, the data of electrocardiogram (ECG) showed that the cardiac arrhythmia including the ventricular tachycardia, ventricular premature beat and atrioventricular block can be evoked by acoustic stimulation or PTZ injection in our model. Administration of atomoxetine significantly reduced these arrhythmias and the incidence of S-IRA and SUDEP in our models. However, administration of esmolol hydrochloride with the dose without affecting ECG and mortality changing of DBA/1 significantly blocking the protective effects of atomoxetine on S-IRA and SUDEP in our models. Thus, the dysfunction of respiratory and circulation may be implicated in the pathogenesis of S-IRA and SUDEP. Enhancing the central norepinephrinergic neurotransmission in the brain contributes to inhibition of seizure-induced respiratory arrest by targeting β1-AR locating in the cardiomyocytes. Furthermore, the suppression effects of S-IRA by atomoxetine was significantly reversed by the norepinephrine neuronal degradation in the LC in our models. Furthermore, PTZ-induced Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine, activity but not TH content from the serum of left ventricle and the whole heart tissue was reduced following the S-IRA. Our findings will show a new light on decoding the pathogenesis of SUDEP concerning the pathway between the LC and heart.

Sudden unexpected death in epilepsy (SUDEP) is the fatal cause leading to the death of epilepsy patients, especially those with anti-epileptic drug resistance. However, the underlying mechanism of SUDEP remains elusive. Our previous study demonstrated that enhancement of serotonin (5-HT) synthesis by intraperitoneal (IP) injection of 5-hydroxytryptophan (5-HTP) significantly reduced the incidence of seizure-induced respiratory arrest (S-IRA) in DBA/1 mice SUDEP models. Given that 5-HT2A receptor (5-HT2AR) plays an important role in mediating the respiration system in brain, we hypothesized that 5-HT2AR is of great significance to modulate S-IRA and SUDEP. To test this hypothesis, we examined whether the decreased incidence of S-IRA evoked by either acoustic stimulation or PTZ injection by administration with 5-HTP will be blocked by administration with ketanserin (KET), a selective antagonist of 5HT2AR, in DBA/1 mice SUDEP models to examine the role of 5-HT2AR in modulating S-IRA. Our results suggested that the decreased incidence of S-IRA by 5-Hydroxytryptophan (5-HTP), a precursor for central nervous system (CNS) serotonin (5-HT) synthesis, was significantly reversed by IP or intracerebroventricularly (ICV) injection of ketanserin in our models. Furthermore, given that 5-HT2AR locating in the pre-Botzinger complex (PBC) which plays a key role in regulating the respiratory rhythm, we wonder whether the lower incidence of S-IRA by IP with 5-HTP was significantly reversed by KET which inhibits 5-HT2AR in the above models and acts on the 5-HT2AR in pre-Botzinger complex .To test our hypothesis, we activated the neural circuit between dorsal raphe nucleus and the pre-Botzinger complex by optogenetics technology. Our results indicated that the lower incidence of S-IRA evoked by PTZ by stimulating the TPH2-ChETA, a variant of channelrhodopsin-2 (ChR2) neurons in the dorsal raphe nucleus (DR) in DBA/1 mice and the suppressant effects was significantly reversed by administration of KET in bilateral pre-Botzinger complex without changing electroencephalogram (EEG) activity. The neural circuit between DR and the pre-Botzinger complex was confirmed by injection of CTB555, a nerve tracer, in DR or the pre-Botzinger complex, respectively. Taken together, our findings suggested that targeting the neural circuit between the DR and the pre-Botzinger complex in which 5-HT2AR plays a critical role in regulating this process would be promising to prevent SUDEP. Keywords: sudden unexpected death in epilepsy; 5-Hydroxytryptophan (5-HTP); ketanserin (KET); pre-Botzinger complex; dorsal raphe nucleus; 5HT2A receptor; respiration rhythm.