Propofol Regulates Arousal by Enhancing Inhibitory Synaptic Transmission of Noradrenergic Neurons in the Locus Coeruleus of Adult Male Mice

Propofol Regulates Arousal by Enhancing Inhibitory Synaptic Transmission of Noradrenergic Neurons in the Locus Coeruleus of Adult Male Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Propofol Regulates Arousal by Enhancing Inhibitory Synaptic Transmission of Noradrenergic Neurons in the Locus Coeruleus of Adult Male Mice Tatsuya Abe, Miyuki Kurabe, Yuka Nakamura, Mika Sasaki, Yutaka Seino, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6585147/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Locus coeruleus-noradrenergic (LC-NA) neurons have been implicated to be involved in the effects of general anesthetics. However, the contribution of LC-NA neurons during propofol anesthesia remains unknown. We aimed to elucidate the mechanism of action of propofol in the LC-NA neurons. LC-NA neurons from adult male mice were identified by targeted expression of fluorescent proteins. Whole-cell patch-clamp recordings were performed to analyze the effects of propofol on action potentials and synaptic transmission. The results showed that propofol concentration-dependently decreased action potential frequencies. Propofol also increased the frequency of spontaneous inhibitory postsynaptic currents and prolonged their decay time. The presence of GABA A receptor antagonist bicuculline prevented these effects. Inhibitory tonic currents were evoked only at high concentration of propofol. In behavioral experiments, bicuculline injection into the LC significantly shortened the return of righting reflex time following propofol anesthesia. We demonstrated that clinical doses of propofol induce a facilitatory effect on phasic GABAergic neural currents and direct action on GABAA receptors in LC-NA neurons. The enhancement of inhibitory effects mediated by GABA A receptors in LC-NA neurons is considered one of the mechanisms underlying the anesthetic effects of propofol. Biological sciences/Neuroscience Biological sciences/Physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction General anesthesia and natural sleep share the feature of reversible loss of consciousness, implicating a common neural circuit.[ 1 ] Natural sleep is regulated by the interaction of sleep and wake control neurons.[ 2 ] The locus coeruleus (LC), a wake control center in the brainstem, contains noradrenergic neurons that release noradrenaline (NA) widely into brain regions that promote arousal or regulate various behaviors, including sleep-wake cycles.[ 3 ] Some reports indicate that animals lacking adrenergic neurotransmission have delayed arousal from anesthesia.[ 4 , 5 ] Selective chemogenetic or optogenetic[ 6 – 9 ] activation of LC-NA neurons results in rapid arousal from anesthesia, suggesting the LC is a critical site of action in general anesthesia. Propofol is a short-acting intravenous anesthetic that targets γ-aminobutyric acid type A (GABA A ) receptors. Although its overall mechanism of action remains unclear, it is thought to affect various receptors and neuronal nuclei,[ 10 , 11 ] including the LC. One of its action in the LC is to inhibit action potential firing via GABA A receptors in rats.[ 12 ] Similarly, in zebrafish LC-NA neurons, propofol suppresses action potential firing by inhibiting excitatory transmission.[ 5 ] The induction of inhibitory tonic currents has been implicated in propofol’s action as a general anesthetic.[ 13 , 14 ] In hippocampal neurons, this tonic extrasynaptic GABA A component is more sensitive to anesthetics than the effect on phasic currents, and it is thought to be crucial in suppressing neuronal excitability.[ 15 ] However, few studies have comprehensively analyzed the effects on inhibitory and excitatory synaptic transmissions in pre- and postsynaptic components of LC-NA neurons in mammals. We hypothesized that propofol suppresses the excitability of LC-NA neurons by enhancing GABA A receptor-mediated inhibitory synaptic transmission in LC-NA neurons. Herein, we observed the activity of fluorescently-identified LC-NA neurons to distinguish them from LC-GABA neurons in murine brain slices. Using whole-cell patch clamp techniques, we examined the effects of propofol on action potential firing and excitatory or inhibitory synaptic transmission, revealing enhancement of inhibitory synaptic transmission in LC-NA neurons. Additionally, behavioral experiments were conducted to determine whether the identified inhibitory mechanisms to LC were involved in the anesthetic effects of propofol. Results Characterization of the LC-NA Neurons AAV-PRSx8-ChR2-mCherry was injected into the LC to confirm mCherry expression specific to LC-NA neurons (Fig. 1 a). Immunostaining revealed co-expression of tyrosine hydroxylase (TH, a marker of NAergic neurons) and mCherry (Fig. 1 b). Whole-cell patch-clamp recordings from mCherry-labeled LC-NA neurons showed spontaneous action potentials (4.16 ± 2.75 Hz) and a membrane potential of − 44.99 ± 3.88 mV (n = 16). NA perfusion suppressed action potential firing via negative feedback[ 16 ] (Fig. 1 d). In the presence of TTX, miniature IPSCs were blocked by bicuculline, a GABA A receptor antagonist, and fully abolished with additional strychnine (n = 6; Fig. 1 e). Miniature EPSCs were reduced to approximately 30% after the perfusion of CNQX, a non-NMDA receptor antagonist (n = 4; Fig. 1 f). Most currents disappeared after additional treatment with AP5, an NMDA receptor antagonist. Propofol Inhibits Action Potentials in a Concentration-Dependent Manner To analyze the effects of propofol on LC-NA neurons, we examined the action potential frequency and membrane potential changes in current-clamp recordings. Propofol at 3 µM did not affect the firing frequency; however, the dose of 30 µM significantly suppressed action potential frequencies (3 µM; 3.20 ± 1.82 Hz to 3.40 ± 2.45 Hz; P = 0.579, n = 5: 30 µM; 3.96 ± 2.99 Hz to 3.03 ± 2.56 Hz; P = 0.045, n = 10: Fig. 2 a, b, d). The dose at 300 µM further abolished the firing in most neurons (3.74 ± 2.90 Hz to 0.36 ± 0.70 Hz; P = 0.043, n = 6; Fig. 2 c). Propofol inhibited the action potential firing in a concentration-dependent manner (suppression ratio: 3 µM, 4.65 ± 30.97%, n = 5; 10 µM, − 12.38 ± 27.42%, n = 5; 30 µM, 27.81 ± 33.48%, n = 10; 100 µM, 32.83 ± 24.68%, n = 6; 300 µM, 83.42 ± 24.98%, n = 6; Fig. 2 e). The membrane potential was not changed at 3 and 30 µM, while it was changed at 300 µM (-3.61 ± 3.56 mV). Propofol Increases the Frequency and Decay Time of Spontaneous and Miniature IPSCs Voltage-clamp recordings showed that 30 µM propofol increased the frequency of spontaneous IPSCs (2.26 ± 1.14 Hz to 2.91 ± 1.16 Hz, P = 0.025, n = 8) and prolonged the decay time (10.57 ± 4.48 ms to 14.57 ± 6.66 ms, P = 0.007, n = 8) without changes in amplitude (14.21 ± 4.40 pA to 14.25 ± 2.58 pA; P = 0.970, n = 8; Fig. 3 a, b). Propofol at 3 µM did not alter the frequency (2.34 ± 0.93 Hz to 2.28 ± 0.83 Hz, P = 0.624, n = 5), amplitude (13.69 ± 2.31 pA to 12.95 ± 1.65 pA; P = 0.207, n = 5), and decay time (8.76 ± 1.93 ms to 8.94 ± 1.03 ms; P = 0.706, n = 5) of spontaneous IPSCs (Fig. 3 c, d). Propofol at 300 µM increased the frequency of spontaneous IPSCs (1.47 ± 0.84 Hz to 4.12 ± 1.69 Hz; P = 0.026, n = 4) without changes in amplitude (7.22 ± 2.37 pA to 7.50 ± 2.25 pA; P = 0.718, n = 4). The decay time was significantly prolonged (20.19 ± 4.80 ms to 44.95 ± 10.81 ms; P = 0.010, n = 4; Fig. 3 e, f). One of the spontaneous IPSC data did not show a clear spontaneous IPSC waveform, so that data was used only for the analysis of the tonic current. Propofol (300 µM) induced the tonic current (54.14 ± 45.26 pA, n = 5; Fig. 3 f), but propofol at 3 and 30 µM did not. Next, we examined the effects of propofol on miniature IPSCs in the presence of TTX (0.5 µM). Propofol (30 µM) increased the frequency of miniature IPSCs (1.73 ± 0.69 Hz to 2.65 ± 1.39 Hz; P = 0.014, n = 11) and prolonged the decay time (9.85 ± 4.86 ms to 15.98 ± 6.62 ms; P < 0.001, n = 11; Fig. 4 a, b). Amplitudes showed an increasing trend from 14.42 ± 3.49 pA to 15.68 ± 4.73 pA ( P = 0.098, n = 11). Propofol Does Not Affect Spontaneous and Miniature EPSCs Next, we examined the effects of propofol on EPSCs. Propofol (30 µM) did not alter the frequency, amplitude, and decay time of spontaneous and miniature EPSCs (Fig. 5 ). No significant changes were observed in the frequency (10.38 ± 8.86 Hz to 10.58 ± 9.58 Hz, P = 0.772, n = 6), amplitude 12.44 ± 4.44 pA to 13.31 ± 5.04 pA; P = 0.128, n = 6), and decay time (3.68 ± 1.81 ms to 3.69 ± 1.82 ms; P = 0.950, n = 6) of spontaneous EPSCs (Fig. 5 a, b). The frequency (5.04 ± 2.31 Hz to 5.11 ± 2.69 Hz; P = 0.787, n = 9), amplitude (16.12 ± 2.09 pA to 16.92 ± 3.23 pA; P = 0.325, n = 9), and decay time (3.20 ± 0.69 ms to 3.82 ± 0.83 ms; P = 0.134, n = 9) of miniature EPSCs also did not change significantly (Fig. 5 c, d). GABA A Receptor Antagonists Prevent Propofol Effects We next investigated the receptors involved in the effects of propofol on inhibitory transmission and action potentials. Because propofol is a GABA A receptor agonist,[ 17 , 18 ] we performed experiments in the presence of bicuculline. Bicuculline (20 µM) prevented the propofol-induced increase in frequency and prolongation of decay time (frequency; 0.30 ± 0.17 to 0.28 ± 0.18 Hz, P = 0.246, n = 5: amplitude; 9.67 ± 3.55 to 7.70 ± 2.32 pA, P = 0.146, n = 5: decay time; 6.43 ± 4.08 ms to 4.48 ± 1.63 ms, P = 0.181, n = 5: Fig. 6 a, b). Likewise, the propofol-induced reduction in action potential disappeared in the presence of bicuculline (Fig. 6 c). Under the bicuculline application, the frequency remained unaltered (4.74 ± 3.65 Hz to 4.34 ± 3.66 Hz, P = 0.319, n = 6; Fig. 6 d). Microinjection of GABA A Receptor Antagonists into the LC Attenuates Anesthetic Propofol Effects In vitro experiments showed that propofol suppresses the action potential of LC-NA neurons by enhancing inhibitory synaptic transmission via GABA A receptors. To determine whether this GABA A receptor-mediated mechanism affects propofol-induced anesthesia in vivo, we further examined the effects of bicuculline to the LC on the arousal in anesthetized mice. Vehicle or bicuculline was microinjected into the LC, and behavioral tests were conducted under the propofol anesthesia (naïve group, n = 8; vehicle group, n = 6; bicuculline group, n = 6; Fig. 7 a). Only animals in which the cannula tip position was at the LC were included in the analysis (Fig. 7 b), in which the data from two animals in the bicuculline group and five animals in the vehicle group were excluded. No significant difference in the LORR time after intraperitoneal propofol administration (100 mg/kg) was observed among the three groups (naïve, 206.9 ± 40.50 s; vehicle, 191.7 ± 52.88 s; bicuculline, 233.0 ± 26.30 s; P = 0.241, one-way ANOVA: Fig. 7 c). However, the return of RORR time was significantly shorter in the bicuculline group than that in the vehicle group (naïve,1536 ± 537.5 s; vehicle, 2058 ± 630.8 s; bicuculline, 872.7 ± 184.4 s; P = 0.002, one-way ANOVA followed by Tukey’s post hoc comparisons: Fig. 7 c). As bicuculline affected only the RORR time, the GABA A receptor-mediated inhibitory effect of propofol on LC-NA neurons is suggested to be involved in suppression of the arousal from anesthesia in vivo. Discussion We show here that propofol suppresses arousal by enhancing inhibitory synaptic transmission via GABA A receptors in LC. We directly measured synaptic responses in fluorescently identified LC-NA neurons, allowing precise analysis of inhibitory modulation in these neurons. Electrophysiological analysis revealed that propofol inhibits LC-NA neuron activity through two mechanisms. First, propofol suppresses the excitability of LC-NA neurons by promoting phasic inhibitory currents via increased GABA release from presynaptic terminals. Second, it prolongs the decay time of GABA A receptors in the postsynaptic LC-NA neurons. Contrarily, tonic currents were observed only at high concentrations. This indicates that phasic, rather than tonic, inhibition is involved in the anesthetic effect of propofol in LC-NA neurons. In behavioral experiments, the administration of a GABA A receptor antagonist to the LC did not affect the induction time of propofol anesthesia, whereas the arousal time was significantly shortened. These results suggest that propofol prevents the arousal phase by enhancing inhibitory synaptic transmission in LC-NA neurons. Propofol has been considered to induce tonic inhibition by acting on extrasynaptic GABA A receptors.[ 5 ] Tonic inhibition is typically more sensitive to anesthetics than phasic inhibition.[ 15 ] However, we noted tonic inhibition only with higher propofol concentrations. In some brain regions, phasic inhibition is more sensitive to propofol than tonic inhibition.[ 19 , 20 ] General anesthetics enhance inhibitory synaptic transmission via GABA A receptor, with regional differences in sensitivity. The human genome comprises 18 different GABA A receptor subunits (α1–6, β1–4, γ1–3, δ, ε, and ρ1–3, 5) which form a pentameric structure containing a central Cl - channel.[ 21 ] Subtypes composed of α1–3βγ2 subunits are involved in phasic currents, whereas extrasynaptic subtypes composed of α4–6βδ subunits are involved in tonic currents. The α4 subunit is also involved in propofol-induced tonic currents.[ 22 ] Although GABA A receptor subunits expressed in the LC remain unclear, local differences in GABA A receptor subunit composition and propofol sensitivity in mouse LC-NA neurons may explain the differential sensitivity to tonic and phasic inhibition. In this study, only high propofol concentrations (300 µM) induced tonic inhibition. In rats, 10 mg/kg of propofol produces anesthesia, and the blood concentration 4 minutes after 5 mg/kg of propofol administration is 14.2 µM.[ 23 ] However, it is important to note that in vitro concentrations in brain slices differ from in vivo blood concentrations. Taking into account the rate of protein binding and other factors, a free concentration of 0.63 µM may be sufficient to achieve clinical brain concentrations.[ 24 ] On the other hand, brain slices have extremely low diffusion coefficients, and the depth to the recording cells must be considered, so higher concentrations than clinical blood concentrations may be required in in vitro. For these reasons, a concentration of tens of µM is considered clinically relevant. Thus, phasic rather than tonic inhibition is thought to be involved in the effect of propofol on LC-NA neurons at clinical concentrations. Our results demonstrate that propofol increases GABA release from presynaptic terminals onto LC-NA neurons. Presynaptic mechanisms regulating LC activity have been proposed to be influenced by distally-located neurons. Electrical stimulation of the pontine inhibitory area, including the middle portion of the pontine reticular nucleus or the gigantocellular reticular nucleus, significantly reduces the firing rate of LC neurons.[ 25 ] Whether this inhibition results from direct inhibitory projections from these nuclei or from disynaptic pathways, e.g., activation of local GABAergic neurons, remains unclear. Our data suggest that local increases in synaptic GABA regulate LC-NA neuron activity, which is consistent with propofol inducing an inward, depolarizing current in GABAergic terminals.[ 19 ] Although the presynaptic propofol action might involve voltage-dependent mechanisms, a propofol-induced increase in IPSC frequency was also observed under voltage-dependent NA channel blockade by TTX. Furthermore, no effects of propofol on glutamatergic terminals were observed, suggesting that the presynaptic site of action is specific to GABAergic nerve terminals. Another example of LC neuron regulation is presynaptic inhibition of afferent glutamate- and corticotropin-releasing factor-positive axons by κ-opiate receptors in the LC.[ 26 ] Since activation of these receptors reduces LC neuron responses, their relationship to propofol requires further examination. A subpopulation of GABA neurons in the LC regulates LC-NA neurons.[ 27 ] Specific manipulation and physiological /molecular examination of LC-GABAergic neurons will clarify the mechanism of local propofol action via GABAergic receptors. Propofol prolonged the decay times of spontaneous and miniature IPSCs, which is consistent with the previous observations in other central nervous system neurons. Propofol increases the duration of spontaneous and endogenous inhibitory synaptic responses by reducing desensitization of postsynaptic GABA A receptors in cultured hippocampal neurons[ 28 ] and dissociated spinal dorsal horn neurons.[ 29 ] This decay time prolongation could be due to a reduction in postsynaptic GABA A receptor desensitization. Low propofol concentrations prolong the decay time of spontaneous IPSCs in isolated neurons from the nucleus tractus solitarius.[ 19 ] Since IPSC decay is dominated by the ion channel closure following ligand removal,[ 21 ] an increased decay time is indicative of a prolonged time to inactivation of the Cl − channel of the GABAergic receptor. Thus, the propofol-induced prolongation of the decay time may reflect the inhibition of LC-NA neuron excitability. In behavioral experiments, GABA A receptor inhibition in the LC did not affect the loss of righting reflex time but shortened the return of righting reflex time after propofol administration. LC GABA A receptor inhibition might facilitate NA release by blocking the inhibitory effects of propofol via GABA A receptors in LC-NA neurons, resulting in a shorter time to arousal. Chemogenetic activation of LC neurons and optogenetic activation of orexinergic terminals of the LC only shortened the return of righting reflex time during isoflurane anesthesia.[ 7 , 30 ] Our results also suggest that the LC does not contribute to loss of consciousness during propofol administration but regulates arousal. Propofol may inhibit arousal triggering in the LC via GABA receptors. This study has some limitations. We used only male mice, although sex differences in anesthetic effects exist in humans[ 31 ] and rodents.[ 32 , 33 ] Dexmedetomidine’s sedative effects and propofol’s anesthetic effects in humans are influenced by the menstrual cycle.[ 34 ] In rodents, the estrous cycle affects arousal from anesthesia with dexmedetomidine but not from isoflurane, sevoflurane, or propofol.[ 35 ] To assess the influence of the menstrual cycle, future studies should include male and female mice. In conclusion, propofol enhances inhibitory effect by promoting presynaptic GABA release and directly interacting with postsynaptic GABA A receptors in LC-NA neurons. In LC-NA neurons, propofol produces phasic inhibition at lower concentrations than tonic inhibition. These inhibitory effects are considered to prevent arousal from propofol anesthesia. Methods Animals Male C57BL/6 mice (Jackson Laboratory Japan, Yokohama, Japan) were used in all experiments. Mice were housed in plastic cages at 22 ± 2°C under a 12-h light-dark cycle with water and food ad libitum. All animal experiments were approved by the Niigata University Animal Experiment Ethics Committee (approval numbers: SA00873, SD01480) and conducted in accordance with the Regulations for Animal Experiments at Niigata University and the guidelines of the Science Council of Japan. The study was conducted and the data were reported per the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.[ 36 ] All efforts were made to minimize animal suffering and minimize the number of mice studied in experiments. Production of AAV Vectors Adenoassociated virus (AAV) vectors expressing ChR2-mCherry under the control of PRSx8, an NA-specific synthetic promoter (AAV1-PRSx8-ChR2-mCherry, 4.3 × 10 12 gc/ml), were generated as previously described.[ 37 ] ChR2-mCherry fragment derived from pAAV-CAG-ChR2-mCherry (AV-1-20938M, Penn Vector Core) was subcloned into BamHI/EcoRI site of pAAV-PRSx8-hM3Dq (provided by Michihiro Mieda, Kanazawa University, Japan)[ 38 ] in a substitution of hM3Dq. AAVpro 293T cells (632273, Takara Bio Inc., Shiga, Japan) were transfected with helper (pHelper; Takara Bio Inc.), rep/cap (pAAV2/1; Penn Vector Core), and pAAV-PRSx8-ChR2-mCherry plasmids. AAV particles in the cells and supernatants were collected and purified by an iodixanol gradient. The AAV was then concentrated in phosphate-buffered saline containing 0.001% Pluronic F68. The titer of AAV was determined by using AAVpro titration kit (Takara Bio Inc.) and Thermal Cycler Dice Real Time System III (Takara Bio Inc.). Virus Injection Mice (4–5 weeks old) were anesthetized with medetomidine (0.75 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg). Hair was removed above the injection site, and mice were fixed in a stereotaxic frame (ST-7R-HT and SR-10AR, Narishige, Tokyo, Japan). After exposing the skull, a hole was drilled above the injection site (anteroposterior, − 5.4 mm; mediolateral, 0.9 mm; dorsoventral, 3.0–3.6 mm) to place an injection needle (33 gauge, NF33BL, World Precision Instruments, Sarasota, FL). AAV1-PRSx8-ChR2-mCherry (400 nl) was slowly injected into each side using an injection pump (IMS-20, Narishige) and a 10-µl injection syringe (Nanofil syringe, World Precision Instruments). After the needle was slowly removed, skull defects were filled with bone wax (Tokyo M.I., Tokyo, Japan), and the skin was sutured. A medetomidine antagonist (atipamezole 0.75 mg/kg) was then administered intraperitoneally. Electrophysiological Recordings Experiments were performed in 7-week-old mice, 3–4 weeks after virus injection. Animals were decapitated after urethane anesthesia (1.5 g/kg, intraperitoneal), and brains were rapidly extracted and immediately placed in ice-cold Krebs solution (in mM: NaCl 117, KCl 3.6, CaCl 2 2.5, MgCl 2 1.2, NaH 2 PO 4 1.2, NaHCO 3 25, and D-glucose 11.5, saturated with 95% O 2 and 5% CO 2 ). LC-containing coronal slices (300 µm) were prepared with a microslicer (NLS-MT, Dosaka EM, Kyoto, Japan). The slices were then placed in Krebs solution at 23–25°C for 1 h. For recordings, the slice was placed in the recording chamber and perfused with Krebs solution at 31–32°C (4 ml/min). Electrodes were fabricated from thin-walled, borosilicate, glass-capillary (TW150F-4, World Precision Instruments) using a puller (P-97, Sutter Instrument, Novato, CA). For voltage-clamp recordings, the electrodes were filled with a cesium-based intracellular solution containing (in mM): Cs 2 SO 4 110, CaCl 2 0.5, MgCl 2 2, EGTA 5, HEPES 5, TEA 5, and ATP-Mg 5. For current-clamp recordings, the electrodes were filled with a potassium gluconate-based intracellular solution containing (in mM): K-gluconate 135, KCl 5, CaCl 2 0.5, MgCl 2 2, EGTA 5, HEPES 5, TEA 5, and ATP-Mg 5. The resistance of the electrodes was 4–6 MΩ. The slices were visualized with a fluorescence microscope (E600FN, Nikon, Tokyo, Japan) equipped with a water immersion lens (40×/0.8 W) and a CMOS camera (C11578-36U, Hamamatsu Photonics, Hamamatsu, Japan). LC-NA neurons were identified based on mCherry expression, cell size, and location relative to the fourth ventricle. After establishing whole-cell configuration, neurons were voltage-clamped at either − 70 mV to record excitatory postsynaptic currents (EPSCs) or 0 mV to record inhibitory postsynaptic currents (IPSCs). Action potentials were recorded in the current clamp mode (I = 0). The signals were recorded using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA), filtered at 2 kHz, and digitized at 5 kHz. Data were collected and analyzed using pCLAMP (10.6) Software Suite (Molecular Devices) and Mini Analysis 6.0 software (Synaptosoft, Decatur, GA). Drug Application For electrophysiological experiments, propofol (2,6-diisopropylphenol), tetrodotoxin (TTX; 0.5 µM), bicuculline (20 µM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM), and D(-)-2-amino-5-phosphonovaleric acid (AP5; 50 µM) were purchased from Wako (Osaka, Japan). Strychnine (2 µM) was purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions, prepared by dissolving the drugs in distilled water or dimethyl sulfoxide at 1000 times the final concentration, were diluted in Krebs solution to the final concentration immediately before use. Behavioral Experiments Behavioral experiments were compared among three groups: naïve (no microinjection), bicuculline-treated (microinjection of bicuculline), and vehicle-treated group (microinjection of saline). For LC microinjections, 4-week-old mice of the bicuculline and vehicle groups were implanted with stainless-steel cannulas (26 gauge; P1 Technologies, Roanoke, VA) using the same method as for virus injection. The guide cannula with the dummy cannula (P1 Technologies) was unilaterally positioned adjacent to the right LC (anteroposterior, − 5.4 mm; mediolateral, right 0.9 mm; dorsoventral, 3.4 mm). The guide cannula was fixed to the skull using dental cement. Mice were allowed to recover for approximately 5 days before the experiments. To avoid the bias of circadian rhythms, behavioral experiments were conducted between 9:00 and 16:00. In the bicuculline and vehicle groups, microinjection into the LC was performed before propofol administration. The bicuculline stock solution was dissolved in 0.9% saline by a second researcher so that the experimenter was blinded to which drug was used. After removing the dummy cannula, a PE10 tube (Becton Dickinson, Franklin Lakes, NJ) was connected to the internal cannula (33 gauge; P1 Technologies), and a unilateral microinjection of 0.2 µl bicuculline (50 µg) or vehicle was given over 2 min using a 10 µl Hamilton syringe (Hamilton Company, Reno, NV). All groups were evaluated for the time of sedation after intraperitoneal administration of 100 mg/kg propofol (1% Diprivan, Sandoz, Tokyo, Japan). Loss of righting reflex (LORR) time was defined as the time from the start of propofol administration until the animal could not completely right itself. Return of righting reflex (RORR) time was defined as the time from the start of administration to the animal managed to right itself.[ 39 ] Intraperitoneal administration was selected over intravenous injection to allow measurement of LORR, as the slower induction enabled more stable observation. Microinjection sites were identified by the location of the cannula track in frozen sections and immunohistochemical staining. In this experiment, n refers to the number of animals, with each used only once. Outcome assessors were blinded to group assignments during behavioral evaluation. Immunohistochemistry Mice were euthanized in accordance with the Regulations for Animal Experiments at Niigata University, ensuring ethical considerations and adherence to animal welfare standards. Specifically, isoflurane was administered at a concentration of 4% with an oxygen flow rate of 2 L/min to facilitate rapid and humane euthanasia while minimizing distress. This procedure was approved by the relevant ethics committee and conducted in accordance with standard protocols to maintain research integrity. Immediately, mice were transcardially perfused with 20 ml of saline, followed by 20 ml of 4% paraformaldehyde (Mildform 10 N, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). The brain was removed and post-fixed in 4% paraformaldehyde overnight at 4°C. Then, the samples were cryoprotected using 20% sucrose in 0.1 M phosphate buffer overnight at 4°C. The sample was cut to size, embedded entirely with FSC22 frozen section medium (Leica Biosystems, Wetzlar, Germany), and stored at − 70°C until sectioning. LC-containing sections (10 µm) were serially made using a frozen microtome (CM1520, Leica Biosystems), mounted on APS-coated glass slides (Matsunami Glass Ind Ltd., Osaka, Japan), and stored at − 70°C until staining. The sections were washed twice with TNT buffer (0.1 M Tris-HCl [pH 7.5], 0.15 M NaCl, 0.03% Tween 20) and blocked using Blocking One Histo blocking buffer (Nacalai Tesque, Kyoto, Japan) at 23–25°C for 1 h. Following the removal of the blocking buffer, the sections were incubated in a humidified chamber at 4°C for 48 h with the following primary antibodies: rabbit anti-mCherry (1:800; #26765-1-AP, Proteintech, Rosemont, IL, USA) and mouse anti-tyrosine hydroxylase antibody (1:2000; #22941, Immunostar, Hudson, WI). After washing with TNT buffer, the sections were incubated with Cy3-conjugated goat anti-rabbit IgG (1:500; #111-167-003, Jackson Immuno Research Laboratories Inc., West Grove, PA) or FITC-conjugated donkey anti-mouse IgG (1:500; #AP192F, Merck KGaA, Darmstadt, Germany) at 4°C overnight in a humidified chamber. All antibodies were diluted with 0.1% Tween 20 in TNB buffer (0.1 M Tris-HCl-buffered saline [pH 7.5] containing 1% blocking reagent). The sections were washed twice with TNT buffer, embedded in VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA), and visualized using a fluorescence microscope (BX 53, Olympus, Tokyo, Japan) equipped with a digital camera system (DP73, Olympus). Statistical Analysis Sample sizes were determined based on previous publications analyzing the effects of general anesthetics in mice.[ 22 , 40 ] All numerical data are shown as mean ± standard deviation. Statistical significance was set as P < 0.05. Parametric tests were used if the data set passed the assumptions required for parametric analysis including level of measurement, normality, and homogeneity of variances. We used paired t-test and one-way analysis of variance [ANOVA] followed by Tukey’s post hoc comparisons for statistical analyses. GraphPad Prism 9 (GraphPad Software, Boston, MA) was used for statistical analysis. Declarations Competing Interests Statement The authors declare no competing interests. Author Contribution T.A., M.K., Y.S., K.F., and H.B. devised the study design. T.A. and M.K. led the experiment, acquired, analyzed, and interpreted the data, and prepared the manuscript. T.A. and M.K. contributed equally to this work. Y.N., M.S., and M.U. acquired, analyzed, and interpreted the data. M.K. and Y.S. obtained funding and revised the manuscript. K.F., M.U., and H.B. supervised the project and revised the manuscript. All the authors read and approved the final manuscript. Acknowledgement We would like to thank Editage (http://www.editage.com) for editing and reviewing this manuscript for English language. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Moody, O. A. et al. The neural circuits underlying general anesthesia and sleep. Anesth. 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B. & Pearce, R. A. Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro. Br. J. Anaesth. 93 , 810–817 (2004). Mileykovskiy, B. Y., Kiyashchenko, L. I., Kodama, T., Lai, Y. Y. & Siegel, J. M. Activation of pontine and medullary motor inhibitory regions reduces discharge in neurons located in the locus coeruleus and the anatomical equivalent of the midbrain locomotor region. J. Neurosci. 20 , 8551–8558 (2000). Kreibich, A. et al. Presynaptic inhibition of diverse afferents to the locus ceruleus by κ-opiate receptors: a novel mechanism for regulating the central norepinephrine system. J. Neurosci. 28 , 6516–6525 (2008). Breton-Provencher, V. & Sur, M. Active control of arousal by a locus coeruleus GABAergic circuit. Nat. Neurosci. 22 , 218–228 (2019). Bai, D., Pennefather, P. S., MacDonald, J. F. & Orser, B. A. The general anesthetic propofol slows deactivation and desensitization of GABA(A) receptors. J. 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Anaesth. 127 , 435–446 (2021). Fu, F. et al. Propofol EC50 for inducing loss of consciousness is lower in the luteal phase of the menstrual cycle. Br. J. Anaesth. 112 , 506–513 (2014). Vincent, K. F. et al. Oestrous cycle affects emergence from anaesthesia with dexmedetomidine, but not propofol, isoflurane, or sevoflurane, in female rats. Br. J. Anaesth. 131 , 67–78 (2023). Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8 , e1000412 (2010). Nakamura, Y. et al. Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord. eLife . 12 (2023). Hasegawa, E., Yanagisawa, M., Sakurai, T. & Mieda, M. Orexin neurons suppress narcolepsy via 2 distinct efferent pathways. J. Clin. Invest. 124 , 604–616 (2014). Luo, D., Chen, S. Y. & Zhang, Y. Effects of different injection methods of propofol anesthesia on the behavior and electroencephalography recording in mice. Ibrain 8 , 109–116 (2022). Wang, D. et al. GABAergic Neurons in the Dorsal-Intermediate Lateral Septum Regulate Sleep-Wakefulness and Anesthesia in Mice. Anesthesiology 135 , 463–481 (2021). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6585147","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":468143240,"identity":"61107b31-967d-49aa-ae4d-ee697172ee89","order_by":0,"name":"Tatsuya Abe","email":"","orcid":"","institution":"Niigata University Medical and Dental Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tatsuya","middleName":"","lastName":"Abe","suffix":""},{"id":468143241,"identity":"e0e3d8ea-9b67-4830-8e46-7cdb512611ee","order_by":1,"name":"Miyuki Kurabe","email":"data:image/png;base64,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","orcid":"","institution":"Niigata University Medical and Dental Hospital","correspondingAuthor":true,"prefix":"","firstName":"Miyuki","middleName":"","lastName":"Kurabe","suffix":""},{"id":468143242,"identity":"1872475b-56c2-4837-b5a1-533aecf62ea6","order_by":2,"name":"Yuka Nakamura","email":"","orcid":"","institution":"Niigata University","correspondingAuthor":false,"prefix":"","firstName":"Yuka","middleName":"","lastName":"Nakamura","suffix":""},{"id":468143243,"identity":"531e7b22-83d9-4c83-bcc8-538d4e17afb7","order_by":3,"name":"Mika Sasaki","email":"","orcid":"","institution":"Gifu University","correspondingAuthor":false,"prefix":"","firstName":"Mika","middleName":"","lastName":"Sasaki","suffix":""},{"id":468143244,"identity":"c4edd5d8-dcb0-4919-951b-d89ee852a1fd","order_by":4,"name":"Yutaka Seino","email":"","orcid":"","institution":"Niigata University Medical and Dental Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yutaka","middleName":"","lastName":"Seino","suffix":""},{"id":468143245,"identity":"6bfb996e-86bd-42ec-ad3e-069448f9b435","order_by":5,"name":"Kenta Furutani","email":"","orcid":"","institution":"Niigata University Medical and Dental Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kenta","middleName":"","lastName":"Furutani","suffix":""},{"id":468143246,"identity":"06346f79-46a3-42b6-a258-b759c1b7db8b","order_by":6,"name":"Masaki Ueno","email":"","orcid":"","institution":"Niigata University","correspondingAuthor":false,"prefix":"","firstName":"Masaki","middleName":"","lastName":"Ueno","suffix":""},{"id":468143247,"identity":"ea70c88d-9fc1-4834-830e-85f8d3caee74","order_by":7,"name":"Hiroshi Baba","email":"","orcid":"","institution":"Niigata University Medical and Dental Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Baba","suffix":""}],"badges":[],"createdAt":"2025-05-03 16:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6585147/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6585147/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-22005-2","type":"published","date":"2025-10-28T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84227925,"identity":"b7ef9236-24fe-4ac5-bbb1-d55b53c729dd","added_by":"auto","created_at":"2025-06-09 13:18:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83625,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of LC-NA neurons by fluorescent labeling with AAV and electrophysiological characterization of identified LC-NA neurons. (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e) Schematic of AAV1-PRSx8-ChR2-mCherry injection into the LC of C57BL/6 mice. (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e) Expression of mCherry in the LC and co-expression with TH. The area enclosed by the white dashed rectangle is magnified. Scale bars: 20 µm (upper) and 40 µm (lower). (\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e) Differential interference microscopy (left) and epifluorescence microscopy (right) images of the mCherry-labeled LC neuron. Scale bar: 25 µm. (\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e) NA (40 µM) perfusion suppresses action potential firing in LC-NA neurons. (\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e) Representative traces of mIPSCs at a holding potential of 0 mV in LC-NA neurons (left) and changes in the frequency of mIPSCs (right). The occurrence of mIPSCs is largely reduced by bicuculline, a GABA\u003csub\u003eA\u003c/sub\u003e receptor antagonist, and the remaining currents are almost completely suppressed by strychnine, a glycine receptor antagonist. (\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e) Representative traces of mEPSCs at a holding potential of −70 mV (left) in LC-NA neurons and changes in the frequency of mEPSCs (right). Miniature EPSCs are almost completely inhibited by the non-NMDA antagonist CNQX and the NMDA antagonist AP5. AAV, adeno-associated virus; AP5, D(-)-2-amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; GABA\u003csub\u003eA\u003c/sub\u003e, γ-aminobutyric acid type A; LC, locus coeruleus; mEPSCs, miniature excitatory postsynaptic currents; mIPSCs, miniature inhibitory postsynaptic currents; NA, noradrenaline; NMDA, \u003cem\u003eN\u003c/em\u003e-methyl-D-aspartate; TH, tyrosine hydroxylase; TTX, tetrodotoxin.\u003c/p\u003e","description":"","filename":"Online1.png","url":"https://assets-eu.researchsquare.com/files/rs-6585147/v1/2ab36f8c548a39b712b6bcd0.png"},{"id":84229189,"identity":"f1d7a232-1860-425f-9d49-1a7772708175","added_by":"auto","created_at":"2025-06-09 13:34:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87244,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol suppresses action potentials in LC-NA neurons. (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e) Representative traces of propofol-induced inhibition of action potentials (propofol 3 µM, \u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e; propofol 30 µM, \u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e; propofol 300 µM, \u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e) recorded from LC-NA neurons in the current-clamp mode. Expanded traces are shown in the lower panel. (\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e) The action potential frequency is significantly decreased by 30 µM propofol (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, paired t-test, n = 10). (\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e) Concentration dependency of the percentage of propofol-induced frequency suppression from baseline (3 µM, n = 5; 10 µM, n = 5; 30 µM, n = 10; 100 µM, n = 6; 300 µM, n = 6). Data are shown as mean ± standard deviation. LC, locus coeruleus; NA, noradrenaline.\u003c/p\u003e","description":"","filename":"Online2.png","url":"https://assets-eu.researchsquare.com/files/rs-6585147/v1/5d9d60dd8fb7d82e54a60431.png"},{"id":84227932,"identity":"535eb906-8425-46cf-a37a-c781470fcc87","added_by":"auto","created_at":"2025-06-09 13:18:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":186479,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol increases frequency and decay time of spontaneous IPSCs in LC-NA neurons. (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e) Representative traces of sIPSCs in the voltage-clamp mode (holding potential 0 mV) with propofol (30 μM). Expanded traces are shown in the lower panel. (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e) Analysis of the sIPSC data. Propofol significantly increases the frequency and prolongs the decay time of sIPSCs (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, paired t-test, n = 8). (\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e) Representative traces of sIPSCs with propofol (3 μM). Expanded traces are shown in the lower panel. (\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e) Analysis of the sIPSC data. Propofol does not alter the frequency, amplitude, or decay time of the sIPSCs (paired t-test, n = 5). (\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e) Representative traces of sIPSCs with propofol (300 μM). Expanded traces are shown in the lower panel. (\u003cem\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/em\u003e) Analysis of the sIPSC and the outward current data. Propofol significantly increases the frequency and prolongs the decay time of sIPSCs (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, paired t-test, n = 4). Propofol induced the tonic current (n = 5). LC, locus coeruleus; NA, noradrenaline; sIPSCs, spontaneous inhibitory postsynaptic currents.\u003c/p\u003e","description":"","filename":"Online3.png","url":"https://assets-eu.researchsquare.com/files/rs-6585147/v1/ece31a55983673304f5bcfac.png"},{"id":84228814,"identity":"96e8a765-0fec-4642-97de-53459cb0d9ff","added_by":"auto","created_at":"2025-06-09 13:26:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133311,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol increases frequency and decay time of miniature IPSCs in LC-NA neurons. (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e) Representative traces of mIPSCs in the voltage-clamp mode (holding potential 0 mV) in the presence of TTX with propofol (30 μM). Expanded traces are shown in the lower panel. (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e) Analysis of the mIPSCs data. Propofol significantly increases the frequency (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, paired t-test, n = 11) and prolongs the decay time (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, paired t-test, n = 11) of mIPSCs. The amplitude shows an increasing trend, but no significant difference is observed (\u003cem\u003eP\u003c/em\u003e= 0.098, paired t-test, n = 11). LC, locus coeruleus; NA, noradrenaline; mIPSCs, miniature inhibitory postsynaptic currents; TTX, tetrodotoxin.\u003c/p\u003e","description":"","filename":"Online4.png","url":"https://assets-eu.researchsquare.com/files/rs-6585147/v1/dcdfdcb0572645a15078651d.png"},{"id":84228819,"identity":"16ad961b-8600-4a56-87f1-c40593e7b95d","added_by":"auto","created_at":"2025-06-09 13:26:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":137120,"visible":true,"origin":"","legend":"\u003cp\u003ePropofol does not alter the activities of EPSCs in LC-NA neurons. (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e) Representative traces of sEPSCs in the voltage-clamp mode (holding potential −70 mV) with propofol (30 μM). Expanded traces are shown in the lower panel. (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e) Analysis of the sEPSCs data. Propofol does not alter the frequency, amplitude, or decay time of sEPSCs (paired t-test, n = 6). (\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e) Representative traces of mEPSCs in the voltage-clamp mode (holding potential −70 mV) in the presence of TTX with propofol (30 μM). Expanded traces are shown in the lower panel. (\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e) Analysis of the mEPSCs data. Propofol does not alter the frequency, amplitude, or decay time of mEPSCs (paired t-test, n = 11). LC, locus coeruleus; mEPSCs, miniature excitatory postsynaptic current; NA, noradrenaline; sEPSCs, spontaneous excitatory postsynaptic current; TTX, tetrodotoxin.\u003c/p\u003e","description":"","filename":"Online5.png","url":"https://assets-eu.researchsquare.com/files/rs-6585147/v1/6d051f6fca9db4c9f3cd31b2.png"},{"id":84230351,"identity":"bd492ff5-43dc-4ef2-b8af-e2f4e82f311e","added_by":"auto","created_at":"2025-06-09 13:42:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169635,"visible":true,"origin":"","legend":"\u003cp\u003eGABA\u003csub\u003eA \u003c/sub\u003ereceptor antagonists suppress the effects of propofol on mIPSCs and action potentials. (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e) Representative traces showing the effect of propofol (30 μM) on mIPSCs in the presence of bicuculline in the voltage-clamp mode (holding potential −70 mV). Expanded traces are shown in the lower panel. (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e) Analysis of mIPSCs in the presence of bicuculline before and after propofol. Propofol does not alter the frequency, amplitude, or decay time of mIPSCs in the presence of bicuculline (paired t-test, n = 5). (\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e) Representative traces showing the effect of propofol (30 μM) on action potentials in the presence of bicuculline in the current-clamp mode. (\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e) Analysis of action potentials in the presence of bicuculline before and after propofol. Propofol does not alter the action potential frequency in the presence of bicuculline (paired t-test, n = 6). GABA\u003csub\u003eA\u003c/sub\u003e, γ-aminobutyric acid type A; mIPSCs, miniature inhibitory postsynaptic currents; TTX, tetrodotoxin.\u003c/p\u003e","description":"","filename":"Online6.png","url":"https://assets-eu.researchsquare.com/files/rs-6585147/v1/05ccbe3e60429addc59333d0.png"},{"id":84227946,"identity":"86cc0036-374d-4348-83f4-634b4b0bb025","added_by":"auto","created_at":"2025-06-09 13:18:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":96569,"visible":true,"origin":"","legend":"\u003cp\u003eGABA\u003csub\u003eA\u003c/sub\u003e antagonist bicuculline shortens the arousal time in propofol anesthesia. (\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e) The time courses of the behavioral experiment in naïve, bicuculline, and vehicle groups are shown. A microinjection cannula was implanted in the LC 5 days prior to the injection and behavioral analysis. (\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e) Schematic representation of the microinjection sites. Locations in yellow represent the LC. Circles represent the bicuculline group (n = 6), and triangles represent the vehicle group (n = 6). Scale bar: 500 µm. (\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e) LORR times are not significantly different between the groups (one-way ANOVA, left). The RORR time is significantly shorter in the bicuculline group than in the vehicle group (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, one-way ANOVA followed by Tukey’s post hoc comparisons, right). Naïve, n = 8; vehicle, n = 6; bicuculline, n = 6. Data are given as means ± standard deviations. GABA\u003csub\u003eA\u003c/sub\u003e, γ-aminobutyric acid type A; i.p., intraperitoneal; LC, locus coeruleus; LORR, loss of righting reflex; RORR, return of righting reflex.\u003c/p\u003e","description":"","filename":"Online7.png","url":"https://assets-eu.researchsquare.com/files/rs-6585147/v1/f9392e2fee0dfd2a3123ec3c.png"},{"id":95040461,"identity":"16ef1179-a0a6-4de7-9eb0-e2eaf7748497","added_by":"auto","created_at":"2025-11-03 16:09:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1954343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6585147/v1/c072425c-3444-4671-8098-83c3ba737dfa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Propofol Regulates Arousal by Enhancing Inhibitory Synaptic Transmission of Noradrenergic Neurons in the Locus Coeruleus of Adult Male Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGeneral anesthesia and natural sleep share the feature of reversible loss of consciousness, implicating a common neural circuit.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Natural sleep is regulated by the interaction of sleep and wake control neurons.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] The locus coeruleus (LC), a wake control center in the brainstem, contains noradrenergic neurons that release noradrenaline (NA) widely into brain regions that promote arousal or regulate various behaviors, including sleep-wake cycles.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Some reports indicate that animals lacking adrenergic neurotransmission have delayed arousal from anesthesia.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Selective chemogenetic or optogenetic[\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] activation of LC-NA neurons results in rapid arousal from anesthesia, suggesting the LC is a critical site of action in general anesthesia.\u003c/p\u003e \u003cp\u003ePropofol is a short-acting intravenous anesthetic that targets γ-aminobutyric acid type A (GABA\u003csub\u003eA\u003c/sub\u003e) receptors. Although its overall mechanism of action remains unclear, it is thought to affect various receptors and neuronal nuclei,[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] including the LC. One of its action in the LC is to inhibit action potential firing via GABA\u003csub\u003eA\u003c/sub\u003e receptors in rats.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Similarly, in zebrafish LC-NA neurons, propofol suppresses action potential firing by inhibiting excitatory transmission.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] The induction of inhibitory tonic currents has been implicated in propofol\u0026rsquo;s action as a general anesthetic.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] In hippocampal neurons, this tonic extrasynaptic GABA\u003csub\u003eA\u003c/sub\u003e component is more sensitive to anesthetics than the effect on phasic currents, and it is thought to be crucial in suppressing neuronal excitability.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] However, few studies have comprehensively analyzed the effects on inhibitory and excitatory synaptic transmissions in pre- and postsynaptic components of LC-NA neurons in mammals. We hypothesized that propofol suppresses the excitability of LC-NA neurons by enhancing GABA\u003csub\u003eA\u003c/sub\u003e receptor-mediated inhibitory synaptic transmission in LC-NA neurons.\u003c/p\u003e \u003cp\u003eHerein, we observed the activity of fluorescently-identified LC-NA neurons to distinguish them from LC-GABA neurons in murine brain slices. Using whole-cell patch clamp techniques, we examined the effects of propofol on action potential firing and excitatory or inhibitory synaptic transmission, revealing enhancement of inhibitory synaptic transmission in LC-NA neurons. Additionally, behavioral experiments were conducted to determine whether the identified inhibitory mechanisms to LC were involved in the anesthetic effects of propofol.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCharacterization of the LC-NA Neurons\u003c/p\u003e \u003cp\u003eAAV-PRSx8-ChR2-mCherry was injected into the LC to confirm mCherry expression specific to LC-NA neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Immunostaining revealed co-expression of tyrosine hydroxylase (TH, a marker of NAergic neurons) and mCherry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Whole-cell patch-clamp recordings from mCherry-labeled LC-NA neurons showed spontaneous action potentials (4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.75 Hz) and a membrane potential of \u0026minus;\u0026thinsp;44.99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88 mV (n\u0026thinsp;=\u0026thinsp;16). NA perfusion suppressed action potential firing via negative feedback[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In the presence of TTX, miniature IPSCs were blocked by bicuculline, a GABA\u003csub\u003eA\u003c/sub\u003e receptor antagonist, and fully abolished with additional strychnine (n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Miniature EPSCs were reduced to approximately 30% after the perfusion of CNQX, a non-NMDA receptor antagonist (n\u0026thinsp;=\u0026thinsp;4; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Most currents disappeared after additional treatment with AP5, an NMDA receptor antagonist.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePropofol Inhibits Action Potentials in a Concentration-Dependent Manner\u003c/p\u003e \u003cp\u003eTo analyze the effects of propofol on LC-NA neurons, we examined the action potential frequency and membrane potential changes in current-clamp recordings. Propofol at 3 \u0026micro;M did not affect the firing frequency; however, the dose of 30 \u0026micro;M significantly suppressed action potential frequencies (3 \u0026micro;M; 3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82 Hz to 3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45 Hz; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.579, n\u0026thinsp;=\u0026thinsp;5: 30 \u0026micro;M; 3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99 Hz to 3.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56 Hz; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045, n\u0026thinsp;=\u0026thinsp;10: Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, d). The dose at 300 \u0026micro;M further abolished the firing in most neurons (3.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.90 Hz to 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70 Hz; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Propofol inhibited the action potential firing in a concentration-dependent manner (suppression ratio: 3 \u0026micro;M, 4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;30.97%, n\u0026thinsp;=\u0026thinsp;5; 10 \u0026micro;M, \u0026minus;\u0026thinsp;12.38\u0026thinsp;\u0026plusmn;\u0026thinsp;27.42%, n\u0026thinsp;=\u0026thinsp;5; 30 \u0026micro;M, 27.81\u0026thinsp;\u0026plusmn;\u0026thinsp;33.48%, n\u0026thinsp;=\u0026thinsp;10; 100 \u0026micro;M, 32.83\u0026thinsp;\u0026plusmn;\u0026thinsp;24.68%, n\u0026thinsp;=\u0026thinsp;6; 300 \u0026micro;M, 83.42\u0026thinsp;\u0026plusmn;\u0026thinsp;24.98%, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The membrane potential was not changed at 3 and 30 \u0026micro;M, while it was changed at 300 \u0026micro;M (-3.61\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56 mV).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePropofol Increases the Frequency and Decay Time of Spontaneous and Miniature IPSCs\u003c/p\u003e \u003cp\u003eVoltage-clamp recordings showed that 30 \u0026micro;M propofol increased the frequency of spontaneous IPSCs (2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 Hz to 2.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 Hz, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025, n\u0026thinsp;=\u0026thinsp;8) and prolonged the decay time (10.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.48 ms to 14.57\u0026thinsp;\u0026plusmn;\u0026thinsp;6.66 ms, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007, n\u0026thinsp;=\u0026thinsp;8) without changes in amplitude (14.21\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40 pA to 14.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58 pA; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.970, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Propofol at 3 \u0026micro;M did not alter the frequency (2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93 Hz to 2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 Hz, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.624, n\u0026thinsp;=\u0026thinsp;5), amplitude (13.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31 pA to 12.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65 pA; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.207, n\u0026thinsp;=\u0026thinsp;5), and decay time (8.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93 ms to 8.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 ms; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.706, n\u0026thinsp;=\u0026thinsp;5) of spontaneous IPSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). Propofol at 300 \u0026micro;M increased the frequency of spontaneous IPSCs (1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 Hz to 4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69 Hz; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026, n\u0026thinsp;=\u0026thinsp;4) without changes in amplitude (7.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37 pA to 7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 pA; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.718, n\u0026thinsp;=\u0026thinsp;4). The decay time was significantly prolonged (20.19\u0026thinsp;\u0026plusmn;\u0026thinsp;4.80 ms to 44.95\u0026thinsp;\u0026plusmn;\u0026thinsp;10.81 ms; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010, n\u0026thinsp;=\u0026thinsp;4; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f). One of the spontaneous IPSC data did not show a clear spontaneous IPSC waveform, so that data was used only for the analysis of the tonic current. Propofol (300 \u0026micro;M) induced the tonic current (54.14\u0026thinsp;\u0026plusmn;\u0026thinsp;45.26 pA, n\u0026thinsp;=\u0026thinsp;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), but propofol at 3 and 30 \u0026micro;M did not. Next, we examined the effects of propofol on miniature IPSCs in the presence of TTX (0.5 \u0026micro;M). Propofol (30 \u0026micro;M) increased the frequency of miniature IPSCs (1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69 Hz to 2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39 Hz; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014, n\u0026thinsp;=\u0026thinsp;11) and prolonged the decay time (9.85\u0026thinsp;\u0026plusmn;\u0026thinsp;4.86 ms to 15.98\u0026thinsp;\u0026plusmn;\u0026thinsp;6.62 ms; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;11; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Amplitudes showed an increasing trend from 14.42\u0026thinsp;\u0026plusmn;\u0026thinsp;3.49 pA to 15.68\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73 pA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.098, n\u0026thinsp;=\u0026thinsp;11).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePropofol Does Not Affect Spontaneous and Miniature EPSCs\u003c/p\u003e \u003cp\u003eNext, we examined the effects of propofol on EPSCs. Propofol (30 \u0026micro;M) did not alter the frequency, amplitude, and decay time of spontaneous and miniature EPSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). No significant changes were observed in the frequency (10.38\u0026thinsp;\u0026plusmn;\u0026thinsp;8.86 Hz to 10.58\u0026thinsp;\u0026plusmn;\u0026thinsp;9.58 Hz, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.772, n\u0026thinsp;=\u0026thinsp;6), amplitude 12.44\u0026thinsp;\u0026plusmn;\u0026thinsp;4.44 pA to 13.31\u0026thinsp;\u0026plusmn;\u0026thinsp;5.04 pA; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.128, n\u0026thinsp;=\u0026thinsp;6), and decay time (3.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81 ms to 3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82 ms; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.950, n\u0026thinsp;=\u0026thinsp;6) of spontaneous EPSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). The frequency (5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31 Hz to 5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69 Hz; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.787, n\u0026thinsp;=\u0026thinsp;9), amplitude (16.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09 pA to 16.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23 pA; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.325, n\u0026thinsp;=\u0026thinsp;9), and decay time (3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69 ms to 3.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 ms; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.134, n\u0026thinsp;=\u0026thinsp;9) of miniature EPSCs also did not change significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGABA\u003csub\u003eA\u003c/sub\u003e Receptor Antagonists Prevent Propofol Effects\u003c/p\u003e \u003cp\u003eWe next investigated the receptors involved in the effects of propofol on inhibitory transmission and action potentials. Because propofol is a GABA\u003csub\u003eA\u003c/sub\u003e receptor agonist,[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] we performed experiments in the presence of bicuculline. Bicuculline (20 \u0026micro;M) prevented the propofol-induced increase in frequency and prolongation of decay time (frequency; 0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 to 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 Hz, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.246, n\u0026thinsp;=\u0026thinsp;5: amplitude; 9.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.55 to 7.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32 pA, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.146, n\u0026thinsp;=\u0026thinsp;5: decay time; 6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;4.08 ms to 4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63 ms, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.181, n\u0026thinsp;=\u0026thinsp;5: Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Likewise, the propofol-induced reduction in action potential disappeared in the presence of bicuculline (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Under the bicuculline application, the frequency remained unaltered (4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;3.65 Hz to 4.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.66 Hz, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.319, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMicroinjection of GABA\u003csub\u003eA\u003c/sub\u003e Receptor Antagonists into the LC Attenuates Anesthetic Propofol Effects\u003c/p\u003e \u003cp\u003eIn vitro experiments showed that propofol suppresses the action potential of LC-NA neurons by enhancing inhibitory synaptic transmission via GABA\u003csub\u003eA\u003c/sub\u003e receptors. To determine whether this GABA\u003csub\u003eA\u003c/sub\u003e receptor-mediated mechanism affects propofol-induced anesthesia in vivo, we further examined the effects of bicuculline to the LC on the arousal in anesthetized mice. Vehicle or bicuculline was microinjected into the LC, and behavioral tests were conducted under the propofol anesthesia (na\u0026iuml;ve group, n\u0026thinsp;=\u0026thinsp;8; vehicle group, n\u0026thinsp;=\u0026thinsp;6; bicuculline group, n\u0026thinsp;=\u0026thinsp;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Only animals in which the cannula tip position was at the LC were included in the analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), in which the data from two animals in the bicuculline group and five animals in the vehicle group were excluded. No significant difference in the LORR time after intraperitoneal propofol administration (100 mg/kg) was observed among the three groups (na\u0026iuml;ve, 206.9\u0026thinsp;\u0026plusmn;\u0026thinsp;40.50 s; vehicle, 191.7\u0026thinsp;\u0026plusmn;\u0026thinsp;52.88 s; bicuculline, 233.0\u0026thinsp;\u0026plusmn;\u0026thinsp;26.30 s; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.241, one-way ANOVA: Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). However, the return of RORR time was significantly shorter in the bicuculline group than that in the vehicle group (na\u0026iuml;ve,1536\u0026thinsp;\u0026plusmn;\u0026thinsp;537.5 s; vehicle, 2058\u0026thinsp;\u0026plusmn;\u0026thinsp;630.8 s; bicuculline, 872.7\u0026thinsp;\u0026plusmn;\u0026thinsp;184.4 s; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, one-way ANOVA followed by Tukey\u0026rsquo;s post hoc comparisons: Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). As bicuculline affected only the RORR time, the GABA\u003csub\u003eA\u003c/sub\u003e receptor-mediated inhibitory effect of propofol on LC-NA neurons is suggested to be involved in suppression of the arousal from anesthesia in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe show here that propofol suppresses arousal by enhancing inhibitory synaptic transmission via GABA\u003csub\u003eA\u003c/sub\u003e receptors in LC. We directly measured synaptic responses in fluorescently identified LC-NA neurons, allowing precise analysis of inhibitory modulation in these neurons. Electrophysiological analysis revealed that propofol inhibits LC-NA neuron activity through two mechanisms. First, propofol suppresses the excitability of LC-NA neurons by promoting phasic inhibitory currents via increased GABA release from presynaptic terminals. Second, it prolongs the decay time of GABA\u003csub\u003eA\u003c/sub\u003e receptors in the postsynaptic LC-NA neurons. Contrarily, tonic currents were observed only at high concentrations. This indicates that phasic, rather than tonic, inhibition is involved in the anesthetic effect of propofol in LC-NA neurons. In behavioral experiments, the administration of a GABA\u003csub\u003eA\u003c/sub\u003e receptor antagonist to the LC did not affect the induction time of propofol anesthesia, whereas the arousal time was significantly shortened. These results suggest that propofol prevents the arousal phase by enhancing inhibitory synaptic transmission in LC-NA neurons.\u003c/p\u003e \u003cp\u003ePropofol has been considered to induce tonic inhibition by acting on extrasynaptic GABA\u003csub\u003eA\u003c/sub\u003e receptors.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Tonic inhibition is typically more sensitive to anesthetics than phasic inhibition.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] However, we noted tonic inhibition only with higher propofol concentrations. In some brain regions, phasic inhibition is more sensitive to propofol than tonic inhibition.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] General anesthetics enhance inhibitory synaptic transmission via GABA\u003csub\u003eA\u003c/sub\u003e receptor, with regional differences in sensitivity. The human genome comprises 18 different GABA\u003csub\u003eA\u003c/sub\u003e receptor subunits (α1–6, β1–4, γ1–3, δ, ε, and ρ1–3, 5) which form a pentameric structure containing a central Cl\u003csup\u003e-\u003c/sup\u003e channel.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Subtypes composed of α1–3βγ2 subunits are involved in phasic currents, whereas extrasynaptic subtypes composed of α4–6βδ subunits are involved in tonic currents. The α4 subunit is also involved in propofol-induced tonic currents.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Although GABA\u003csub\u003eA\u003c/sub\u003e receptor subunits expressed in the LC remain unclear, local differences in GABA\u003csub\u003eA\u003c/sub\u003e receptor subunit composition and propofol sensitivity in mouse LC-NA neurons may explain the differential sensitivity to tonic and phasic inhibition.\u003c/p\u003e \u003cp\u003eIn this study, only high propofol concentrations (300 µM) induced tonic inhibition. In rats, 10 mg/kg of propofol produces anesthesia, and the blood concentration 4 minutes after 5 mg/kg of propofol administration is 14.2 µM.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] However, it is important to note that in vitro concentrations in brain slices differ from in vivo blood concentrations. Taking into account the rate of protein binding and other factors, a free concentration of 0.63 µM may be sufficient to achieve clinical brain concentrations.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] On the other hand, brain slices have extremely low diffusion coefficients, and the depth to the recording cells must be considered, so higher concentrations than clinical blood concentrations may be required in in vitro. For these reasons, a concentration of tens of µM is considered clinically relevant. Thus, phasic rather than tonic inhibition is thought to be involved in the effect of propofol on LC-NA neurons at clinical concentrations.\u003c/p\u003e \u003cp\u003eOur results demonstrate that propofol increases GABA release from presynaptic terminals onto LC-NA neurons. Presynaptic mechanisms regulating LC activity have been proposed to be influenced by distally-located neurons. Electrical stimulation of the pontine inhibitory area, including the middle portion of the pontine reticular nucleus or the gigantocellular reticular nucleus, significantly reduces the firing rate of LC neurons.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Whether this inhibition results from direct inhibitory projections from these nuclei or from disynaptic pathways, e.g., activation of local GABAergic neurons, remains unclear. Our data suggest that local increases in synaptic GABA regulate LC-NA neuron activity, which is consistent with propofol inducing an inward, depolarizing current in GABAergic terminals.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Although the presynaptic propofol action might involve voltage-dependent mechanisms, a propofol-induced increase in IPSC frequency was also observed under voltage-dependent NA channel blockade by TTX. Furthermore, no effects of propofol on glutamatergic terminals were observed, suggesting that the presynaptic site of action is specific to GABAergic nerve terminals. Another example of LC neuron regulation is presynaptic inhibition of afferent glutamate- and corticotropin-releasing factor-positive axons by κ-opiate receptors in the LC.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] Since activation of these receptors reduces LC neuron responses, their relationship to propofol requires further examination. A subpopulation of GABA neurons in the LC regulates LC-NA neurons.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Specific manipulation and physiological /molecular examination of LC-GABAergic neurons will clarify the mechanism of local propofol action via GABAergic receptors.\u003c/p\u003e \u003cp\u003ePropofol prolonged the decay times of spontaneous and miniature IPSCs, which is consistent with the previous observations in other central nervous system neurons. Propofol increases the duration of spontaneous and endogenous inhibitory synaptic responses by reducing desensitization of postsynaptic GABA\u003csub\u003eA\u003c/sub\u003e receptors in cultured hippocampal neurons[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and dissociated spinal dorsal horn neurons.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] This decay time prolongation could be due to a reduction in postsynaptic GABA\u003csub\u003eA\u003c/sub\u003e receptor desensitization. Low propofol concentrations prolong the decay time of spontaneous IPSCs in isolated neurons from the nucleus tractus solitarius.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Since IPSC decay is dominated by the ion channel closure following ligand removal,[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] an increased decay time is indicative of a prolonged time to inactivation of the Cl\u003csup\u003e−\u003c/sup\u003e channel of the GABAergic receptor. Thus, the propofol-induced prolongation of the decay time may reflect the inhibition of LC-NA neuron excitability.\u003c/p\u003e \u003cp\u003eIn behavioral experiments, GABA\u003csub\u003eA\u003c/sub\u003e receptor inhibition in the LC did not affect the loss of righting reflex time but shortened the return of righting reflex time after propofol administration. LC GABA\u003csub\u003eA\u003c/sub\u003e receptor inhibition might facilitate NA release by blocking the inhibitory effects of propofol via GABA\u003csub\u003eA\u003c/sub\u003e receptors in LC-NA neurons, resulting in a shorter time to arousal. Chemogenetic activation of LC neurons and optogenetic activation of orexinergic terminals of the LC only shortened the return of righting reflex time during isoflurane anesthesia.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] Our results also suggest that the LC does not contribute to loss of consciousness during propofol administration but regulates arousal. Propofol may inhibit arousal triggering in the LC via GABA receptors.\u003c/p\u003e \u003cp\u003eThis study has some limitations. We used only male mice, although sex differences in anesthetic effects exist in humans[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and rodents.[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] Dexmedetomidine’s sedative effects and propofol’s anesthetic effects in humans are influenced by the menstrual cycle.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] In rodents, the estrous cycle affects arousal from anesthesia with dexmedetomidine but not from isoflurane, sevoflurane, or propofol.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] To assess the influence of the menstrual cycle, future studies should include male and female mice.\u003c/p\u003e \u003cp\u003eIn conclusion, propofol enhances inhibitory effect by promoting presynaptic GABA release and directly interacting with postsynaptic GABA\u003csub\u003eA\u003c/sub\u003e receptors in LC-NA neurons. In LC-NA neurons, propofol produces phasic inhibition at lower concentrations than tonic inhibition. These inhibitory effects are considered to prevent arousal from propofol anesthesia.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eAnimals\u003c/p\u003e\u003cp\u003eMale C57BL/6 mice (Jackson Laboratory Japan, Yokohama, Japan) were used in all experiments. Mice were housed in plastic cages at 22 ± 2°C under a 12-h light-dark cycle with water and food \u003cem\u003ead libitum.\u003c/em\u003e All animal experiments were approved by the Niigata University Animal Experiment Ethics Committee (approval numbers: SA00873, SD01480) and conducted in accordance with the Regulations for Animal Experiments at Niigata University and the guidelines of the Science Council of Japan. The study was conducted and the data were reported per the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] All efforts were made to minimize animal suffering and minimize the number of mice studied in experiments.\u003c/p\u003e\u003cp\u003eProduction of AAV Vectors\u003c/p\u003e\u003cp\u003eAdenoassociated virus (AAV) vectors expressing ChR2-mCherry under the control of PRSx8, an NA-specific synthetic promoter (AAV1-PRSx8-ChR2-mCherry, 4.3 × 10\u003csup\u003e12\u003c/sup\u003e gc/ml), were generated as previously described.[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] ChR2-mCherry fragment derived from pAAV-CAG-ChR2-mCherry (AV-1-20938M, Penn Vector Core) was subcloned into BamHI/EcoRI site of pAAV-PRSx8-hM3Dq (provided by Michihiro Mieda, Kanazawa University, Japan)[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] in a substitution of hM3Dq. AAVpro 293T cells (632273, Takara Bio Inc., Shiga, Japan) were transfected with helper (pHelper; Takara Bio Inc.), rep/cap (pAAV2/1; Penn Vector Core), and pAAV-PRSx8-ChR2-mCherry plasmids. AAV particles in the cells and supernatants were collected and purified by an iodixanol gradient. The AAV was then concentrated in phosphate-buffered saline containing 0.001% Pluronic F68. The titer of AAV was determined by using AAVpro titration kit (Takara Bio Inc.) and Thermal Cycler Dice Real Time System III (Takara Bio Inc.).\u003c/p\u003e\u003cp\u003eVirus Injection\u003c/p\u003e\u003cp\u003eMice (4–5 weeks old) were anesthetized with medetomidine (0.75 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg). Hair was removed above the injection site, and mice were fixed in a stereotaxic frame (ST-7R-HT and SR-10AR, Narishige, Tokyo, Japan). After exposing the skull, a hole was drilled above the injection site (anteroposterior, − 5.4 mm; mediolateral, 0.9 mm; dorsoventral, 3.0–3.6 mm) to place an injection needle (33 gauge, NF33BL, World Precision Instruments, Sarasota, FL). AAV1-PRSx8-ChR2-mCherry (400 nl) was slowly injected into each side using an injection pump (IMS-20, Narishige) and a 10-µl injection syringe (Nanofil syringe, World Precision Instruments). After the needle was slowly removed, skull defects were filled with bone wax (Tokyo M.I., Tokyo, Japan), and the skin was sutured. A medetomidine antagonist (atipamezole 0.75 mg/kg) was then administered intraperitoneally.\u003c/p\u003e\u003cp\u003eElectrophysiological Recordings\u003c/p\u003e\u003cp\u003eExperiments were performed in 7-week-old mice, 3–4 weeks after virus injection. Animals were decapitated after urethane anesthesia (1.5 g/kg, intraperitoneal), and brains were rapidly extracted and immediately placed in ice-cold Krebs solution (in mM: NaCl 117, KCl 3.6, CaCl\u003csub\u003e2\u003c/sub\u003e 2.5, MgCl\u003csub\u003e2\u003c/sub\u003e 1.2, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.2, NaHCO\u003csub\u003e3\u003c/sub\u003e 25, and D-glucose 11.5, saturated with 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e). LC-containing coronal slices (300 µm) were prepared with a microslicer (NLS-MT, Dosaka EM, Kyoto, Japan). The slices were then placed in Krebs solution at 23–25°C for 1 h.\u003c/p\u003e\u003cp\u003eFor recordings, the slice was placed in the recording chamber and perfused with Krebs solution at 31–32°C (4 ml/min). Electrodes were fabricated from thin-walled, borosilicate, glass-capillary (TW150F-4, World Precision Instruments) using a puller (P-97, Sutter Instrument, Novato, CA). For voltage-clamp recordings, the electrodes were filled with a cesium-based intracellular solution containing (in mM): Cs\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 110, CaCl\u003csub\u003e2\u003c/sub\u003e 0.5, MgCl\u003csub\u003e2\u003c/sub\u003e 2, EGTA 5, HEPES 5, TEA 5, and ATP-Mg 5. For current-clamp recordings, the electrodes were filled with a potassium gluconate-based intracellular solution containing (in mM): K-gluconate 135, KCl 5, CaCl\u003csub\u003e2\u003c/sub\u003e 0.5, MgCl\u003csub\u003e2\u003c/sub\u003e 2, EGTA 5, HEPES 5, TEA 5, and ATP-Mg 5. The resistance of the electrodes was 4–6 MΩ.\u003c/p\u003e\u003cp\u003eThe slices were visualized with a fluorescence microscope (E600FN, Nikon, Tokyo, Japan) equipped with a water immersion lens (40×/0.8 W) and a CMOS camera (C11578-36U, Hamamatsu Photonics, Hamamatsu, Japan). LC-NA neurons were identified based on mCherry expression, cell size, and location relative to the fourth ventricle. After establishing whole-cell configuration, neurons were voltage-clamped at either − 70 mV to record excitatory postsynaptic currents (EPSCs) or 0 mV to record inhibitory postsynaptic currents (IPSCs). Action potentials were recorded in the current clamp mode (I = 0). The signals were recorded using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA), filtered at 2 kHz, and digitized at 5 kHz. Data were collected and analyzed using pCLAMP (10.6) Software Suite (Molecular Devices) and Mini Analysis 6.0 software (Synaptosoft, Decatur, GA).\u003c/p\u003e\u003cp\u003eDrug Application\u003c/p\u003e\u003cp\u003eFor electrophysiological experiments, propofol (2,6-diisopropylphenol), tetrodotoxin (TTX; 0.5 µM), bicuculline (20 µM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM), and D(-)-2-amino-5-phosphonovaleric acid (AP5; 50 µM) were purchased from Wako (Osaka, Japan). Strychnine (2 µM) was purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions, prepared by dissolving the drugs in distilled water or dimethyl sulfoxide at 1000 times the final concentration, were diluted in Krebs solution to the final concentration immediately before use.\u003c/p\u003e\u003cp\u003eBehavioral Experiments\u003c/p\u003e\u003cp\u003eBehavioral experiments were compared among three groups: naïve (no microinjection), bicuculline-treated (microinjection of bicuculline), and vehicle-treated group (microinjection of saline). For LC microinjections, 4-week-old mice of the bicuculline and vehicle groups were implanted with stainless-steel cannulas (26 gauge; P1 Technologies, Roanoke, VA) using the same method as for virus injection. The guide cannula with the dummy cannula (P1 Technologies) was unilaterally positioned adjacent to the right LC (anteroposterior, − 5.4 mm; mediolateral, right 0.9 mm; dorsoventral, 3.4 mm). The guide cannula was fixed to the skull using dental cement. Mice were allowed to recover for approximately 5 days before the experiments.\u003c/p\u003e\u003cp\u003eTo avoid the bias of circadian rhythms, behavioral experiments were conducted between 9:00 and 16:00. In the bicuculline and vehicle groups, microinjection into the LC was performed before propofol administration. The bicuculline stock solution was dissolved in 0.9% saline by a second researcher so that the experimenter was blinded to which drug was used. After removing the dummy cannula, a PE10 tube (Becton Dickinson, Franklin Lakes, NJ) was connected to the internal cannula (33 gauge; P1 Technologies), and a unilateral microinjection of 0.2 µl bicuculline (50 µg) or vehicle was given over 2 min using a 10 µl Hamilton syringe (Hamilton Company, Reno, NV). All groups were evaluated for the time of sedation after intraperitoneal administration of 100 mg/kg propofol (1% Diprivan, Sandoz, Tokyo, Japan). Loss of righting reflex (LORR) time was defined as the time from the start of propofol administration until the animal could not completely right itself. Return of righting reflex (RORR) time was defined as the time from the start of administration to the animal managed to right itself.[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] Intraperitoneal administration was selected over intravenous injection to allow measurement of LORR, as the slower induction enabled more stable observation. Microinjection sites were identified by the location of the cannula track in frozen sections and immunohistochemical staining. In this experiment, n refers to the number of animals, with each used only once. Outcome assessors were blinded to group assignments during behavioral evaluation.\u003c/p\u003e\u003cp\u003eImmunohistochemistry\u003c/p\u003e\u003cp\u003e Mice were euthanized in accordance with the Regulations for Animal Experiments at Niigata University, ensuring ethical considerations and adherence to animal welfare standards. Specifically, isoflurane was administered at a concentration of 4% with an oxygen flow rate of 2 L/min to facilitate rapid and humane euthanasia while minimizing distress. This procedure was approved by the relevant ethics committee and conducted in accordance with standard protocols to maintain research integrity. Immediately, mice were transcardially perfused with 20 ml of saline, followed by 20 ml of 4% paraformaldehyde (Mildform 10 N, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). The brain was removed and post-fixed in 4% paraformaldehyde overnight at 4°C. Then, the samples were cryoprotected using 20% sucrose in 0.1 M phosphate buffer overnight at 4°C. The sample was cut to size, embedded entirely with FSC22 frozen section medium (Leica Biosystems, Wetzlar, Germany), and stored at − 70°C until sectioning. LC-containing sections (10 µm) were serially made using a frozen microtome (CM1520, Leica Biosystems), mounted on APS-coated glass slides (Matsunami Glass Ind Ltd., Osaka, Japan), and stored at − 70°C until staining. The sections were washed twice with TNT buffer (0.1 M Tris-HCl [pH 7.5], 0.15 M NaCl, 0.03% Tween 20) and blocked using Blocking One Histo blocking buffer (Nacalai Tesque, Kyoto, Japan) at 23–25°C for 1 h. Following the removal of the blocking buffer, the sections were incubated in a humidified chamber at 4°C for 48 h with the following primary antibodies: rabbit anti-mCherry (1:800; #26765-1-AP, Proteintech, Rosemont, IL, USA) and mouse anti-tyrosine hydroxylase antibody (1:2000; #22941, Immunostar, Hudson, WI). After washing with TNT buffer, the sections were incubated with Cy3-conjugated goat anti-rabbit IgG (1:500; #111-167-003, Jackson Immuno Research Laboratories Inc., West Grove, PA) or FITC-conjugated donkey anti-mouse IgG (1:500; #AP192F, Merck KGaA, Darmstadt, Germany) at 4°C overnight in a humidified chamber. All antibodies were diluted with 0.1% Tween 20 in TNB buffer (0.1 M Tris-HCl-buffered saline [pH 7.5] containing 1% blocking reagent). The sections were washed twice with TNT buffer, embedded in VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA), and visualized using a fluorescence microscope (BX 53, Olympus, Tokyo, Japan) equipped with a digital camera system (DP73, Olympus).\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eSample sizes were determined based on previous publications analyzing the effects of general anesthetics in mice.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] All numerical data are shown as mean ± standard deviation. Statistical significance was set as \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Parametric tests were used if the data set passed the assumptions required for parametric analysis including level of measurement, normality, and homogeneity of variances. We used paired t-test and one-way analysis of variance [ANOVA] followed by Tukey’s post hoc comparisons for statistical analyses. GraphPad Prism 9 (GraphPad Software, Boston, MA) was used for statistical analysis.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eCompeting Interests Statement\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.A., M.K., Y.S., K.F., and H.B. devised the study design. T.A. and M.K. led the experiment, acquired, analyzed, and interpreted the data, and prepared the manuscript. T.A. and M.K. contributed equally to this work. Y.N., M.S., and M.U. acquired, analyzed, and interpreted the data. M.K. and Y.S. obtained funding and revised the manuscript. K.F., M.U., and H.B. supervised the project and revised the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Editage (http://www.editage.com) for editing and reviewing this manuscript for English language.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMoody, O. A. et al. The neural circuits underlying general anesthesia and sleep. \u003cem\u003eAnesth. Analg\u003c/em\u003e. \u003cb\u003e132\u003c/b\u003e, 1254\u0026ndash;1264 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown, R. E., Basheer, R., McKenna, J. T., Strecker, R. E. \u0026amp; McCarley, R. W. Control of sleep and wakefulness. \u003cem\u003ePhysiol. 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GABAergic Neurons in the Dorsal-Intermediate Lateral Septum Regulate Sleep-Wakefulness and Anesthesia in Mice. \u003cem\u003eAnesthesiology\u003c/em\u003e \u003cb\u003e135\u003c/b\u003e, 463\u0026ndash;481 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6585147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6585147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLocus coeruleus-noradrenergic (LC-NA) neurons have been implicated to be involved in the effects of general anesthetics. However, the contribution of LC-NA neurons during propofol anesthesia remains unknown. We aimed to elucidate the mechanism of action of propofol in the LC-NA neurons. LC-NA neurons from adult male mice were identified by targeted expression of fluorescent proteins. Whole-cell patch-clamp recordings were performed to analyze the effects of propofol on action potentials and synaptic transmission. The results showed that propofol concentration-dependently decreased action potential frequencies. Propofol also increased the frequency of spontaneous inhibitory postsynaptic currents and prolonged their decay time. The presence of GABA\u003csub\u003eA\u003c/sub\u003e receptor antagonist bicuculline prevented these effects. Inhibitory tonic currents were evoked only at high concentration of propofol. In behavioral experiments, bicuculline injection into the LC significantly shortened the return of righting reflex time following propofol anesthesia. We demonstrated that clinical doses of propofol induce a facilitatory effect on phasic GABAergic neural currents and direct action on GABAA receptors in LC-NA neurons. The enhancement of inhibitory effects mediated by GABA\u003csub\u003eA\u003c/sub\u003e receptors in LC-NA neurons is considered one of the mechanisms underlying the anesthetic effects of propofol.\u003c/p\u003e","manuscriptTitle":"Propofol Regulates Arousal by Enhancing Inhibitory Synaptic Transmission of Noradrenergic Neurons in the Locus Coeruleus of Adult Male Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 13:18:15","doi":"10.21203/rs.3.rs-6585147/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-07T02:35:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-02T23:13:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232607402236855003904336291984044631729","date":"2025-06-23T17:34:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-17T13:22:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64918635251769189252911599770962026749","date":"2025-06-08T12:44:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-05T13:14:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-31T11:19:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-30T16:06:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-09T03:53:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-09T03:52:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6c72c28-5a26-4acb-9033-c05cf61aba6b","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49702577,"name":"Biological sciences/Neuroscience"},{"id":49702578,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-11-03T16:04:45+00:00","versionOfRecord":{"articleIdentity":"rs-6585147","link":"https://doi.org/10.1038/s41598-025-22005-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-28 15:57:09","publishedOnDateReadable":"October 28th, 2025"},"versionCreatedAt":"2025-06-09 13:18:15","video":"","vorDoi":"10.1038/s41598-025-22005-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-22005-2","workflowStages":[]},"version":"v1","identity":"rs-6585147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6585147","identity":"rs-6585147","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}