Male Sprague-Dawley rats weighing 260–300 g (n=72) were housed in an isolated chamber at 20–22°C under a 12 h light/dark cycle (lights on at 8:30 AM). Food and water were available ad libitum. Twenty-four rats were used for righting reflex behavioral assays, and the remaining rats were used for in vivo electrophysiological recordings. Forty-eight neurons were recorded from forty-eight rats. All animals were supplied by the laboratory animal center of the Third Military Medical University (Chongqing, China). Animal experiments were approved by the Zunyi Medical College Animal Care and Use Committee (approval number: 2016126). All efforts were made to minimize the number of rats and their suffering.

For behavioral experiments, rats were anesthetized with sodium pentobarbital [50 mg/kg, intraperitoneally (i.p.)] and atropine sulfate (0.2 mg, i.p.). Anesthetized animals were mounted onto a stereotaxic device (68505; RWD Life Science, China) in a flat-skull position. The core body temperature was maintained at 37–38°C using a heat-controlled pad equipped with a rectal probe (Stoelting Co., Wood Dale, IL, USA). Sterilized guide cannulas were implanted as described previously (17,18). A pair of sterilized guide cannulas made of 23G stainless steel tubes and plugged internally with 30G stylets were stereotaxically implanted 2.0 mm above the VLPO (anteroposterior, −0.35 mm; left-right, ± 1.2 mm; dorsoventral, −7.0 mm from bregma). Stereotaxic positioning was defined according to the brain atlas of Paxinos and Watson (2007). Guide cannulas were fixed to the skull using dental cement.

To induce lesions in the VLPO, rats were anesthetized and kept in place as mentioned above. The skull of each rat was exposed, and a glass micropipette (10–12-µm tip diameter) was lowered into the VLPO region stereotaxically. Fifteen nanoliters of ibotenic acid solution (10 nmol; Sigma, St. Louis, MO, USA) (n=24) or saline (n=24) were injected into the VLPO bilaterally using a microinjection injector (Nanoliter 2000, World Precision Instruments, Sarasota, FL, USA) as described previously (16). The glass micropipette was slowly withdrawn after 5 min. The two holes above VLPO were filled with bone wax. The wound was stitched with sutures and closed with wound clips. A prophylactic dose of penicillin (50 ku/kg, intramuscular injection) was injected into each rat following surgery. After seven days of recovery, in vivo electrophysiology experiments were conducted.

In rodents, loss of consciousness can be measured by the LoRR and resumption of consciousness can be analyzed by the RoRR (19,20). Fig. 1A illustrates the experimental design used to quantify the LoRR and RoRR after the VLPO was microinjected with artificial cerebrospinal fluid (ACSF) (126 mM Na⁺, 3 mM K⁺, 2 mM Ca²⁺, 1.2 mM Mg²⁺, 150 mM Cl⁻, pH=7.4) and a bolus injection of propofol (11 mg/kg, i.v.). After seven days of recovery from cannulas implantation surgery, rats (n=24) were put into a Plexiglas inhalation anesthesia induction chamber for 10-min acclimation (Fig. 1A; t=−40 min). Then, oxygen containing 2% isoflurane was introduced into the chamber at 2 l/min (Fig. 1A; t=−30 min). The 30G stylet from the guide cannula was removed from the isoflurane-anesthetized rats, and a 31G injection cannula was inserted. An injection cannula was attached to a dual-channel microinjection syringe with PE tubing under the control of an injection pump (302; RWD Life Science), and then a 24G intravenous catheter was implanted and fixed to the rat's tail vein. Isoflurane administration was subsequently discontinued. The ACSF (t=0 min; 0.2 µl/side; n=24) (Fig. 1A) was microinjected into the bilateral VLPO at an infusion speed of 0.2 µl/min for 1 min (t=1 to t=2) (Fig. 1A). Five min later (t=7 min) (Fig. 1A), propofol (11 mg/kg, n=24) was bolus injected into the tail vein. The induction time of propofol was quantified as the time (sec) to LoRR; the resumption time was quantified as the time to RoRR (sec). Three days later, the twenty-four rats were subdivided into GABAA agonist group (n=12) and GABAA antagonist group (n=12). Rats in the agonist group were microinjected with the GABAA agonist 4,5,6,7-tetrahydroisoxazolo (5,4-c) pyridin-3-ol (THIP), also called Gaboxadol (t=0; n=12) (Fig. 1B). Rats in the GABAA antagonist group received the same dose of gabazine (t=0; n=12) (Fig. 1C), and the timeline and procedures were the same as in the ACSF group.

After a recovery period of seven days, the extracellular single-unit recording was performed from noradrenergic neurons of the LC (LC-NA) with VLPO lesions (n=24) and sham lesions (n=24) rats. Each group was subdivided into a consciousness group (n=6, without propofol administration), a low propofol concentration group (n=6; 20 mg/kg/h, i.v.), medium concentration group (n=6; 40 mg/kg/h, i.v.) and a high concentration propofol group (n=6; 60 mg/kg/h, i.v.).

The protocol of extracellular single-unit recordings in non-anesthetized rats was refereed to previously report (21). Briefly, the rats were progressively habituated to the head-restrained position (7–14 days) by placing them inside a plastic chamber, painlessly restraining the head with a head holder and preventing large body movements with a cotton-coated plastic cover. The eyes were covered to reduce visual stimulation. After 7–14 days of habituation, the rats could be kept in a sphinx position for 3–6 consecutive h without showing any signs of discomfort. If any signs of discomfort were seen, the rats were freed from the restrained position.

Then A 24G intravenous catheter was implanted and fixed as described above. Rats were removed from the induction chamber and mounted onto a stereotaxic device with blunt ear bars. Propofol was continuously delivered via a syringe pump (WZS-50F6; Smith Medical, Plymouth, MN, US) connected to the intravenous catheter in propofol administration groups. LC-NA extracellular recordings were obtained as previously described (21). A single barrel glass micropipette was filled with 0.5% sodium acetate and lowered into the LC (3.5 mm caudal to lambda, 0.85–1.0 mm lateral to the midline, 5–6.5-mm below the cortical surface) by using a micromanipulator (Mini 25; Luigs & Neumann, Ratingen, Germany) according to coordinates defined in the brain atlas of Paxinos and Watson (2007). The impedance of the micropipette was 10–20 MΩ, measured in physiological saline. Recording was performed during a period of at least 5 min, with only one cell measured from each rat. The recorded extracellular signals were amplified using a patch clamp amplifier (EPC10; HEKA Elektronik, Lambrecht, Germany), bandpass filtered (100–10,000 Hz) and saved to a computer installed with PATCHMASTER (Heka) and Mini Analysis System (Synaptosoft, Inc., Fort Lee, NJ, USA) for offline analysis. The body temperature of rats was maintained at 37–38°C during the recording period.

The discharge patterns of NA-LC neurons were identified using standard criteria (22,23). They display a positive-negative action potential shape with a notch on the ascending limb (Fig. 2A) and a typical, biphasic excitation-inhibition response to noxious stimulation of the contralateral hind paw (Fig. 2B). We pinched the skin of the rat's hind paw using toothed forceps for cutaneous nociceptive stimulation to induce this characteristic firing response. The noxious stimulation procedure was only operated in anesthetized rats.

After behavioral and electrophysiology experiments, rats were deeply anesthetized with sodium pentobarbital. Cold 100 ml physiological saline was perfused through the ascending aorta, followed by 200 ml 4% paraformaldehyde. The brains were then removed, post-fixed for 24 h at 4°C in paraformaldehyde, and transferred to glass containers filled with 30% sucrose for 48 h at 4°C. 20-µm coronal sections containing microinjection or lesion sites were cut and stained with hematoxylin-eosin for histological verification (Fig. 3). Stereotaxic coordinates were identified according to the brain atlas of Paxinos and Watson (2007).

Statistical analyses were performed using SPSS 17.0 for Windows. All data were expressed as means ± standard deviation. A P-value of <0.05 was considered statistically significant using paired-samples t-test in behavioral experiments. N refers to the number of rats studied. The firing frequencies of noradrenergic neurons in different groups were compared using independent-samples t-test. The firing frequencies of noradrenergic neurons in the same group were compared using one-way ANOVA, and n refers to the number of neurons analyzed.

To determine whether GABAergic neurons are involved in unconsciousness induced by propofol. We microinjected GABAA agonist (THIP) or GABAA antagonist (gabazine) into VLPO and observed the LoRR and RoRR. Fig. 4A and B illustrates how ACSF and THIP microinjection into the VLPO of the same rats (n=12) affected propofol-mediated anesthesia and subsequent recovery. Microinjection of THIP into the VLPO decreased LoRR (23.67±8.24 sec to 16.83±6.41 sec, P<0.05) (Fig. 4A) and increased RoRR (694.00±126.73 sec to 762.83±126.28 sec, P<0.05) (Fig. 4B) before propofol administration. (Fig. 4C and D) illustrates the effects of ACSF and the GABAA antagonist gabazine on propofol-mediated anesthesia and recovery in the same rats (n=12). Microinjection of gabazine into the VLPO increased LoRR (20.42±4.14 sec to 26.83±6.28 sec, P<0.05) (Fig. 4C) and decreased RoRR (605.67±81.79 sec to 562.58±78.05 sec, P<0.05, Fig. 4D) before propofol administration.

To explore the effect of propofol on LC firing activity, we anesthetized the VLPO sham-lesion and VLPO lesion rats with different concentrations of propofol. Fig. 5 shows that propofol inhibited the spontaneous firing activity of LC neurons in a dose-dependent manner. In the VLPO sham-lesion group, the spontaneous firing activity of LC neurons in medium propofol concentration (40 mg/kg/h) was reduced to 2.98±0.6 Hz compared with 6.54±1.16 Hz of the low propofol concentration (20 mg/kg/h) group (P<0.05). The spontaneous firing activity of LC neurons in high propofol concentration (60 mg/kg/h) was reduced to 1.36±0.9 Hz compared with 2.98±0.6 Hz of the medium propofol concentration (40 mg/kg/h) group (P<0.05). The firing activity of LC neurons in medium propofol concentration was reduced to 2.98±0.6 Hz compared with 7.32±1.33 Hz of the consciousness group (P<0.05). And the firing activity of LC neurons in high propofol concentration was reduced to 1.36±0.9 Hz compared with 7.32±1.33 Hz of the consciousness group (P<0.05). However, there's no statistical significant differences in the firing activity between consciousness and low propofol concentration group, (Fig. 5A).

The similar inhibitory effect was observed in the VLPO lesion group. In this group, the spontaneous firing activity of LC neurons in medium propofol concentration rats was 2.80±0.46 Hz compared with 5.89±0.93 Hz in the low propofol concentration group (P<0.05). The spontaneous firing activity of LC neurons in high propofol concentration rats was 1.57±0.85 Hz compared with 2.80±0.46 Hz in the medium propofol concentration group (P<0.05). The firing activity of LC neurons in medium propofol concentration was reduced to 2.80±0.46 Hz compared with 6.99±2.03 Hz of the consciousness group (P<0.05). And the firing activity of LC neurons in high propofol concentration was reduced to 1.57±0.85 Hz compared with 6.99±2.03 Hz of the consciousness group (P<0.05). In addition, there's no statistical significant differences in the firing activity between consciousness and low propofol concentration group, (Fig. 5B). Nevertheless, our experimental results showed that firing activities were not significantly different between sham-lesion and VLPO lesion rats after administration of the same concentration of propofol (P>0.05), (Fig. 5C).