Use of corticotropin-releasing hormone receptor antagonists for the preparation of inhalational anaesthesia antagonists

Abstract

The present application relates to the technical field of inhalation anesthetic antagonism, and particularly relates to the application of a corticotropin-releasing hormone receptor antagonist in the preparation of an inhalation anesthetic antagonist. The present application first discovers that the corticotropin-releasing hormone receptor of the dorsal raphe nucleus is a key target of the action of an inhalation anesthetic, and that the oculomotor nerve parvocellular nucleus-dorsal raphe nucleus neural loop mediates the effect of an inhalation anesthetic, and that a corticotropin-releasing hormone receptor antagonist can block the excitatory effect of urocortin on the neurons of the dorsal raphe nucleus, thereby weakening the effect of an inhalation anesthetic. The present application first applies a corticotropin-releasing hormone receptor antagonist in the preparation of an inhalation anesthetic antagonist, fills the technical gap in this field, is conducive to the rapid postoperative recovery of patients under general anesthesia, and has a broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of anesthetic antagonist technology, specifically to the application of adrenocorticotropic hormone-releasing hormone receptor antagonists in the preparation of inhaled anesthetic antagonists. Background Technology

[0002] Globally, hundreds of millions of patients undergo general anesthesia each year, but the mechanism of action of general anesthetics remains a mystery worldwide. General anesthetics are broadly classified into intravenous and inhaled anesthetics. Currently, combined intravenous and inhaled anesthesia is commonly used in clinical practice, employing both intravenous and inhaled anesthetics for induction and maintenance. After surgery, antagonists are often used to accelerate patient recovery during the recovery process. However, to date, the only commonly used antagonists in clinical practice are those targeting intravenous general anesthetics (such as flumazenil). Flumazenil's mechanism of action is to competitively antagonize the binding site of benzodiazepines on GABA-A receptors. While inhaled anesthetics (such as sevoflurane, isoflurane, and desflurane) also primarily act on GABA-A receptors, their binding sites differ from those of benzodiazepines, and flumazenil cannot block these sites.

[0003] In the prior art, some studies have attempted to identify molecular targets for inhaled anesthetics. For example, Chinese patent application CN202210611703.2 discloses the application of growth hormone secretagogue receptor (GHSR) antagonists in the preparation of inhaled anesthetic resuscitation agents, finding that sevoflurane specifically activates GHSR-positive neurons in the oculomotor nerve accessory nucleus (EW) region. However, this approach targets the GHSR, and the affected brain region is the oculomotor nerve accessory nucleus itself. This nucleus is mainly responsible for pupillary regulation and oculomotor reflexes, and has a weaker connection with the core brain regions for general anesthesia consciousness recovery (such as the cortex, thalamus, and brainstem arousal system). Summary of the Invention

[0004] The present invention aims to provide the application of adrenocorticotropic hormone-releasing hormone receptor antagonists in the preparation of inhaled anesthetic antagonists, so as to solve the technical problem that there are no antagonists for inhaled anesthetics in the prior art.

[0005] The technical solution of this invention: the application of adrenocorticotropic hormone-releasing hormone receptor antagonists in the preparation of inhaled anesthetic antagonists.

[0006] Compared with the prior art, the present invention has the following beneficial effects: (1) It provides an antagonist for inhaled anesthetics for the first time. In the prior art, there are only antagonists for intravenous general anesthetics (such as flumazenil, sugammadextrose sodium, etc.) in clinical practice, and there is a lack of antagonists for inhaled anesthetics. The present invention discovers that the corticotropin-releasing hormone receptor of the dorsal raphe nucleus is the key target of the action of inhaled anesthetics, and for the first time uses corticotropin-releasing hormone receptor antagonists to prepare inhaled anesthetic antagonists, filling the technical gap in this field; (2) It can effectively shorten the recovery time after inhaled anesthesia: corticotropin-releasing hormone receptor antagonists can block the excitatory effect of urocortin on neurons of the dorsal raphe nucleus, thereby weakening the anesthetic effect of inhaled anesthetics, significantly shortening the recovery time of anesthesia, and facilitating the rapid recovery of patients after general anesthesia; (3) It can effectively delay the onset time of inhaled anesthesia: corticotropin-releasing hormone receptor antagonists can delay the onset time of inhaled anesthetics, providing a more flexible operating window for clinical anesthesia; (4) The mechanism of action is clear and the target is clear. This invention reveals for the first time the key role of the oculomotor nerve accessory nucleus → dorsal raphe nucleus neural circuit in inhalation anesthesia, and clarifies that the adrenocorticotropic hormone-releasing hormone receptor of the dorsal raphe nucleus is the target of inhalation anesthetics, providing a clear theoretical basis for the development of inhalation anesthetic antagonists; (5) Wide applicability: Adrenocorticotropic hormone-releasing hormone receptor antagonists can antagonize a variety of inhalation anesthetics (including but not limited to sevoflurane, isoflurane, desflurane, etc.), and can be administered through various routes such as intravenous administration, intramuscular injection, subcutaneous injection, and inhalation administration, and has good conversion potential.

[0007] Preferably, the inhaled anesthetic antagonist is a drug that inhibits the activity of neurons in the dorsal raphe nucleus, which receive monosynaptic projections from neurons in the accessory oculomotor nucleus. This invention demonstrates for the first time that the target of inhaled anesthesia is primarily the neurons in the dorsal raphe nucleus that receive projections from the accessory oculomotor nucleus. This region exhibits high expression of corticotropin-releasing hormone receptors (CRHs). Furthermore, by directly applying a CRH-releasing hormone receptor antagonist to the dorsal raphe nucleus region, the anesthetic effect of inhaled anesthetics can be effectively reduced, especially accelerating recovery.

[0008] Preferably, the corticotropin-releasing hormone receptor antagonist is a corticotropin-releasing hormone receptor 1 antagonist and / or a corticotropin-releasing hormone receptor 2 antagonist.

[0009] Preferably, the antagonist is the adrenocorticotropic hormone-releasing hormone receptor 1 antagonist NBI27914.

[0010] Preferably, the antagonist is administered via intravenous, intramuscular, subcutaneous or intradermal injection, inhalation, or intracerebral injection. The appropriate administration method can be selected based on the patient's specific condition to achieve accelerated recovery after inhalation anesthesia.

[0011] Preferably, the site of intracerebral injection is the dorsal raphe nucleus region of the brainstem. Neurons in this region receive abundant projections from oculomotor nerve accessory nucleus neurons activated by inhaled anesthetics and exhibit high expression of corticotropin-releasing hormone receptors. Therefore, using the dorsal raphe nucleus as the injection site effectively promotes recovery after inhalation anesthesia. Furthermore, when studying the mechanisms of inhalation anesthesia and recovery, a rapid recovery model of inhalation anesthesia can be established by injecting an antagonist into the dorsal raphe nucleus region.

[0012] Preferably, in mice, the dose of the corticotropin-releasing hormone receptor 1 antagonist NBI27914 administered into the dorsal raphe nucleus region of the brainstem is 0.5 μL, at a concentration of 10 nmol / μL. Using the above concentration and dose, the awakening of mice after inhalation anesthesia can be effectively promoted.

[0013] Preferably, the concentration for inhalation anesthesia is 0.1–1.5 MAC. These concentrations are standard for inhalation anesthesia administration and can be adjusted within this range as needed. Attached Figure Description

[0014] Figure 1 This is an example of Fos-TRAP combined with anterograde transsynaptic circuit tracing technology used in Example 1 to track brain images of downstream projection regions of the oculomotor nerve accessory nucleus activated by inhaled anesthetics. Figure 1 a is a schematic diagram of the experimental design process. Figure 1 The cell bodies of DRN neurons downstream of the oculomotor nerve accessory nucleus activated by inhaled anesthetic were tracked by bFos-TRAP combined with anterograde transsynaptic circuit tracing technology (green fluorescence). Figure 2 This is a diagram illustrating the effect of the adrenocorticotropic hormone-releasing hormone receptor 1 antagonist NBI27914 on isolated brain slices in Example 2. Figure 2 a is a representative record of the inward current. Figure 2 b is the effect statistics (Mean ± SEM, n=10); Figure 3 This is an in vivo experimental diagram of the adrenocorticotropic hormone-releasing hormone receptor 1 antagonist NBI27914 from Example 3, wherein: Figure 3 a is a schematic diagram of the anesthesia experimental procedure and the placement of the drug delivery catheter at the midline dorsal nucleus. Figure 3 b shows representative raw EEG-EMG recordings from the anesthesia experiment in the control group and the NBI27914 group. Figure 3 c. Figure 3 d represents the statistical graphs of the time to enter anesthesia and the time to wake up (Mean ± SEM, n=8). Detailed Implementation

[0015] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the materials, reagents, etc. used are all commercially available. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods.

[0016] Example 1: Tracking experiment of brain regions downstream of the accessory oculomotor nerve nucleus activated by inhaled anesthetic. This embodiment utilizes Fos-TRAP transgenic technology and anterograde transsynaptic circuit tracing technology to screen and identify downstream projection brain regions of Edinger-Westphal nucleus (EW) neurons activated by inhaled anesthetics throughout the whole brain, in order to discover key neural circuits of inhaled anesthetic action.

[0017] 1. Experimental materials and equipment (1) Experimental animals: Foss animals aged 8-12 weeks were selected. 2A-iCreERT2 Transgenic mice; (2) Drugs and reagents: sevoflurane (inhalation anesthetic, concentration 1.5-2.0 MAC), tamoxifen (150 mg / kg), AAV-hSyn-DIO-EGFP-T2a-Her2CT9-pA and AAV-UL26.5p-DIO-cmgD-WPRE-pA (anterograde transsynaptic helper virus), and transsynaptic tracer virus based on HSV-H129 (Hs06); (3) Main equipment: stereotaxic instrument, animal anesthesia machine, vibrating slicer, fluorescence microscope.

[0018] 2. Experimental Procedure Step 1: Transsynaptic assisted viral injection Select 8-12 week old Fos 2A-iCreERT2Transgenic mice. First, the mice were fixed on a stereotaxic apparatus to expose the skull. Using the anterior fontanelle as the origin, the accessory nucleus of the oculomotor nerve was located (coordinates: bregma AP -3.55 mm, ML 0.0 mm, DV -3.0 mm). Helper virus AAV-hSyn-DIO-EGFP-T2a-Her2CT9-pA and AAV-UL26.5p-DIO-cmgD-WPRE-pA were mixed at a volume ratio of 1:1. The mixture was injected into the accessory nucleus of the oculomotor nerve using a microsyringe at a total injection volume of 50 nL and an injection rate of 20 nL / min. The needle was left in place for 3 minutes after injection and then slowly withdrawn.

[0019] Step 2: Tamoxifen-induced Fos-TRAP One week later, mice were intraperitoneally injected with tamoxifen (150 mg / kg) once daily for 5 consecutive days. After the fifth tamoxifen injection, the mice were placed in an anesthesia induction box and anesthetized for 2 hours with oxygen (1.5 L / min) and sevoflurane (1.5 MAC) to label neurons activated by inhalation anesthesia. The timing of tamoxifen injection and anesthesia activation should ensure that activated neurons have sufficient time to express Cre recombinase.

[0020] Step 3: Transsynaptic viral injection One week later, the transsynaptic tracer virus Hs06 based on HSV-H129 was injected in situ into the oculomotor nerve accessory nucleus of mice. The injection volume was 50-100 nL, and the injection rate was 20 nL / min. The virus can be recognized by the outer membrane protein expressed by the helper virus and spread anterogradely along the synapse to the downstream first-order neurons.

[0021] Step 4: Tissue processing and fluorescence observation Five to seven days after viral injection, mice were perfused with 0.1M PBS and 4% paraformaldehyde. Brain tissue was then removed and fixed in 4% paraformaldehyde at 4°C for 2 hours, followed by dehydration in 30% sucrose solution at 4°C. The brain tissue containing the dorsal raphe nucleus was cut into 40 μm coronal sections using a cryostat, mounted, and observed under a fluorescence microscope.

[0022] 3. Experimental Results like Figure 1 Fluorescence microscopy revealed that the cell bodies of neurons in the dorsal raphe nucleus region of the brainstem significantly expressed abundant green fluorescence (see...). Figure 1 (as indicated by arrow b), while no green fluorescent marker was observed in the dorsal raphe nucleus region of the control group (unanesthetized mice).

[0023] The results of this experiment show that the dorsal raphe nucleus neurons receive monosynaptic high-abundance projections from oculomotor accessory nucleus neurons activated by inhaled anesthetics, confirming the existence of the oculomotor accessory nucleus → dorsal raphe nucleus neural circuit, suggesting that the dorsal raphe nucleus is a key downstream target for the anesthetic effect of inhaled anesthetics.

[0024] Example 2: In vitro electrophysiological experiment of NBI27914, an adrenocorticotropic hormone-releasing hormone receptor 1 antagonist. Example 1 revealed that the dorsal raphe nucleus is an important downstream nucleus of the oculomotor accessory nucleus. Example 2 verified the effect of the neurotransmitter urocortin (urocortin, an endogenous adrenocorticotropic hormone-releasing hormone receptor 1 agonist) released by the oculomotor accessory nucleus on the firing activity of neurons in the dorsal raphe nucleus at the level of isolated brain slices, and whether the adrenocorticotropic hormone-releasing hormone receptor 1 specific antagonist NBI27914 could block this effect.

[0025] 1. Experimental materials and equipment (1) Experimental animals: C57BL / 6J mice, 6-8 weeks old; (2) Drugs: Urocortin (100 nM), NBI27914 (200 nM); (3) Main equipment: vibratory slicer, patch clamp recording system.

[0026] 2. Experimental Procedure Step 1: Preparation of ex vivo brain slices containing the dorsal raphe nucleus Prepare artificial cerebrospinal fluid (composition: 124 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose), and freeze at -20℃ for later use. Anesthetize mice, decapitate them quickly, and rapidly excise the brain tissue into ice-cold artificial cerebrospinal fluid in a culture dish, trimming the tissue into blocks. Use a vibratory microtome to cut brain slices containing the dorsal raphe nucleus, with a thickness of 250-400 μm. Transfer the target brain slices to an incubation solution at 32℃, continuously purging with a mixed gas (95% O2 + 5% CO2), and incubate for approximately 15 minutes for subsequent patch-clamp recording experiments.

[0027] Step 2: Whole-cell patch-clamp recording The brain slice was transferred to the patch-clamp recording slot and continuously perfused with artificial cerebrospinal fluid containing a gas mixture at a flow rate of 2–3 mL / min. The target region (dorsolateral raphe nucleus) was located under low magnification, and the microarray was switched to high magnification to observe cell state. The microelectrode was slowly moved above the recording neuron using a microscopic three-dimensional manipulator. The relative position and sealing status of the electrode and recording neuron were detected using test pulses (5 mV, 5 ms). A brief period of negative pressure was applied to seal the electrode until a Giga (>10) was formed. 9Ω) sealing. The neuron's membrane potential is clamped at -60 mV to rupture the membrane, forming a whole-cell recording mode.

[0028] The membrane potential was clamped at -60 mV in voltage clamp mode, and the baseline level was recorded for 5 minutes. Then, artificial cerebrospinal fluid containing urocortin (100 nM) was perfused, and the changes in cell membrane current were observed for 5 minutes. Then, artificial cerebrospinal fluid containing NBI27914 (200 nM) was perfused again, and the changes in cell membrane current were observed for 5 minutes.

[0029] 3. Experimental Results Experimental results are as follows Figure 2 As shown: Urocortin treatment: After perfusion with urocortin, neurons in the dorsal raphe nucleus region produced significant inward currents (excitation effect), with an average current amplitude of -79.6 ± 19.6 pA, which was statistically significant compared with the baseline (p<0.01).

[0030] NBI27914 Blockage: After perfusion of NBI27914, the inward current effect induced by Urocortin was significantly blocked, and the average current amplitude was reduced to -50.3 ± 18.5 pA, which was statistically significant compared with the Urocortin group (p<0.01).

[0031] Experiments have shown that Urocortin exerts its excitatory effect by activating corticotropin-releasing hormone receptor 1 (CRHRP1) on neurons in the dorsal raphe nucleus, and that the CRHRP1 antagonist NBI27914 can effectively block this effect, providing direct pharmacological evidence for intervention in this pathway at the whole animal level.

[0032] Example 3: In vivo pharmacodynamic study of NBI27914, an adrenocorticotropic hormone-releasing hormone receptor 1 antagonist. Example 3 verifies, at the whole animal level, whether intracerebral microinjection of the adrenocorticotropic hormone-releasing hormone receptor 1 antagonist NBI27914 targeting the dorsal raphe nucleus region can affect the effect of inhalation anesthesia (onset time and recovery time) in mice, thus confirming that adrenocorticotropic hormone-releasing hormone receptor 1 is a functional target of inhalation anesthetics.

[0033] 1. Experimental materials and equipment (1) Experimental animals: C57BL / 6J mice, 8-12 weeks old, weighing 20-25g; (2) Drug: NBI27914 (dosage 0.5 μL, concentration 10 nmol / μL); (3) Main equipment: stereotaxic instrument, EEG-EMG recording system, animal anesthesia machine.

[0034] 2. Experimental Procedure Step 1: Implantation of the drug delivery catheter and EEG-EMG electrodes in the dorsal raphe nucleus. Mice were anesthetized with isoflurane and fixed to a stereotaxic instrument. The hair on the head was then shaved, the skin was cut, and the skull plane was adjusted to be horizontal. Four holes were drilled: one hole on the left side anterior to the fontanelle (coordinates: AP 1.50 mm, ML ±1.50 mm, for mounting an EEG screw); one hole on each side of the parietal lobe (coordinates: AP -3.50 mm, ML ±3.00 mm, for mounting an EEG screw); one hole in the mid-cerebellum (for mounting a reference screw); and a hole was drilled in the dorsal raphe nucleus of the target brain region (coordinates: AP -4.40 mm, ML ±1.10 mm, DV -2.37 mm, 20° angle).

[0035] During the surgery, EEG screws and reference screws were first installed, and a drug delivery catheter was placed in the target dorsal suture nucleus region. Subsequently, EEG screws in the prefrontal and unilateral parietal lobes were welded to the reference screws, and electromyographic electrodes were installed. Finally, all electrodes (EEG and electromyographic) and the drug delivery catheter were fixed to the skull surface with dental cement. The mice were allowed to recover for 7 days post-surgery.

[0036] Step 2: Anesthesia Experiment (Administration + Anesthesia + Recording) The animal anesthesia machine was placed in a room with an ambient temperature of 22°C and oxygen was pre-circulated (1.5 L / min). Adult mice were placed in an anesthesia induction box, and electroencephalography (EEG) and electromyography (EMG) were recorded simultaneously.

[0037] The experiment was divided into two groups (n=8): a control group (injected solvent) and an NBI27914 group (injected antagonist). Twenty minutes before anesthesia, NBI27914 (0.5 μL, 10 nmol / μL) or an equal volume of solvent was injected via a micro-infusion pump through a drug delivery catheter.

[0038] Five minutes after baseline recording, the inhaled anesthetic vaporizer was opened and adjusted to 1.5 MAC for 20 minutes of anesthesia. The vaporizer was then closed and recording continued until the mouse was fully awake. Anesthesia was performed on the same mouse every three days, and only mice with accurate catheter placement were included in the data analysis.

[0039] Recorded data: (1) Time of entering anesthesia: the time from the time the vaporizer was turned off until the EEG showed high amplitude low frequency slow waves and the electromyography activity was significantly reduced; (2) Time of awakening: the time from the time the vaporizer was turned off until the EEG resumed continuous rhythmic activity and the electromyography activity was restored.

[0040] 3. Experimental Results like Figure 3 The experimental results shown indicate that: (1) Anesthesia onset time: 179.3 ± 18.8 seconds in the control group and 296.6 ± 15.8 seconds in the NBI27914 group, which was about 94% delayed compared with the control group. The difference was statistically significant (p<0.001).

[0041] (2) Awakening time: 336.7 ± 27.1 seconds in the control group and 258.3 ± 26.3 seconds in the NBI27914 group, which was about 50% shorter than that in the control group. The difference was statistically significant (p<0.001).

[0042] Experimental results show that administration of the corticotropin-releasing hormone receptor 1 antagonist NBI27914 to the dorsal raphe nucleus region significantly antagonizes the anesthetic effect of sevoflurane, manifested as delayed onset and accelerated recovery. This fully demonstrates that corticotropin-releasing hormone receptor 1 in the dorsal raphe nucleus region is a key target for the anesthetic effect of inhaled anesthetics, and its antagonists can serve as dedicated recovery-promoting drugs for inhaled anesthesia.

[0043] Example 4: Effects of different routes of administration of NBI27914 on recovery from inhalation anesthesia Example 4 verifies the promoting effect of NBI27914 on the recovery from inhalation anesthesia through different administration methods (intravenous injection and intraperitoneal injection) to expand the operability of its application.

[0044] 1. Experimental Procedure Thirty C57BL / 6J mice were randomly divided into three groups (n=10 / group): In the dorsal raphe nucleus drug administration group, NBI27914 (0.5 μL, 10 nmol / μL) was micro-injected into the dorsal raphe nucleus 20 minutes before anesthesia. Intravenous administration group: NBI27914 (1 mg / kg, dissolved in 100 μL normal saline) was injected via the tail vein 20 minutes before anesthesia. Intraperitoneal administration group: NBI27914 (5 mg / kg, dissolved in 200 μL normal saline) was injected intraperitoneally 20 minutes before anesthesia. The anesthesia experimental protocol was the same as in Example 3, and the recovery time was recorded.

[0045] 2. Experimental Results (1) The awakening time of the dorsal raphe nucleus drug administration group was 6.0 ± 0.9 minutes; (2) Time to awakening in the intravenous administration group: 7.8 ± 1.0 minutes; (3) The awakening time of the intraperitoneal drug administration group was 8.5 ± 1.2 minutes.

[0046] All three groups showed significantly shorter recovery times compared to the control group (solvent control, recovery time 12.5 ± 1.1 minutes) in Example 3 (p<0.01). Direct administration to the dorsal raphe nucleus (DRN) was most effective, and systemic administration (intravenous and intraperitoneal) was also effective. This demonstrates that NBI27914, administered systemically (intravenously and intraperitoneally), can significantly shorten the recovery time from inhalation anesthesia, showing good potential for clinical translation. The optimal effect of direct administration to the DRN suggests that the DRN is a key target brain region for corticotropin-releasing hormone receptor 1 antagonists to antagonize the effects of inhalation anesthesia.

[0047] The above embodiments use the CRFR1 antagonist NBI27914 as an example for verification, but the scope of protection of the present invention is not limited thereto. The corticotropin-releasing hormone receptor (CRHR) includes two subtypes, CRHR1 and CRHR2, both expressed in the dorsal raphe nucleus region and both activated by urocortin. Therefore, both CRHR1 and CRHR2 antagonists can be used to block the excitatory effect of urocortin on dorsal raphe nucleus neurons, thereby achieving antagonism in inhalation anesthesia. Those skilled in the art will understand that, in addition to NBI27914, other CRHR1 antagonists (such as Antalarmin) and CRHR2 antagonists (such as Astressin2-B and Antisauvagine-30) can also be used to achieve the technical effects of the present invention.

[0048] The English-Chinese translations of the professional terms appearing in the above embodiments are shown in the table below:

Claims

1. Application of adrenocorticotropic hormone-releasing hormone receptor antagonists in the preparation of inhaled anesthetic antagonists.

2. The application according to claim 1, characterized in that, The inhaled anesthetic antagonist is a drug that inhibits the activity of dorsal raphe nucleus neurons, which receive monosynaptic projections from accessory neurons of the autooptic nerve.

3. The application according to claim 2, characterized in that, The adrenocorticotropic hormone-releasing hormone receptor antagonist is an adrenocorticotropic hormone-releasing hormone receptor 1 antagonist and / or an adrenocorticotropic hormone-releasing hormone receptor 2 antagonist.

4. The application according to claim 3, characterized in that, The antagonist is the adrenocorticotropic hormone-releasing hormone receptor 1 antagonist NBI27914.

5. The application according to claim 4, characterized in that, The antagonist can be administered via intravenous, intramuscular, subcutaneous or intradermal injection, inhalation, or intracerebral injection.

6. The application according to claim 5, characterized in that, The site of administration for brain injection is the dorsal raphe nucleus region of the brain.

7. The application according to claim 6, characterized in that, In mice, the dose of the adrenocorticotropic hormone-releasing hormone receptor 1 antagonist NBI27914 was 0.5 μL in the dorsal raphe nucleus region of the brain, at a concentration of 10 nmol / μL.

8. The application according to any one of claims 1-7, characterized in that, The concentration of the inhaled anesthetic is 0.1–1.5 MAC.

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