Female animals are known to exhibit less anxiety than male animals in behavioral assessments performed in an open space.30 We first performed open field test and compared time spent in the center of the open field test (see below) between C57BL/6 male and female mice. In male mice, the time spent in the center of the 2nd trial was decreased to less than half of it (48 ± 12.5 % of the first trail, n = 6). That of female mice was not largely changed (74.6% of the first trial, see Results), suggesting that anxiety in male mice is not stable even without anesthesia and surgery. To detect behavioral changes including anxiety following anesthesia and surgery, a recent study developed a battery of behavioral analyses using female animals.9 In this study, we therefore used female animals (C57BL/6 mice (SLC, Hamamatsu, Japan)) to detect behavioral and neuronal changes following anesthesia and surgery. The animals were housed in cages with food and water available ad libitum. The room was maintained at 20°C with a 12-h light/dark cycle. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Hyogo College of Medicine, and were performed in accordance with the institutional guidelines for animal experiments and carried out in accordance with the ARRIVE guidelines 2.0. Every effort was made to reduce the number of animals used.
Behavioral tests and electrophysiological recordings from mice after anesthesia and surgery
Mice were randomly divided into three groups: control, Anesthesia/surgery, and anesthesia/surgery (dexmedetomidine). Mice in the anesthesia/surgery and anesthesia/surgery (dexmedetomidine) groups received a laparotomy under general anesthesia (see below). In the two groups, a battery of behavior tests was conducted 6, 9, and 24 h after surgery. The same battery of behavioral tests was also performed 24 h before the surgery to obtain baseline behavioral data. In the control group, mice were placed in their home cages, and the battery of behavioral tests was administered at the same four time points as in other two groups. We performed in vitro whole-cell patch-clamp recordings for PFC neurons in acute brain slice preparations from the mice of all three groups within 14 days after anesthesia and surgery.
Anesthesia and surgery
Laparotomy was performed in 8–10-week-old mice under isoflurane anesthesia, similar to previous studies.9,31 Anesthesia was induced and maintained using 1.5% isoflurane in 100% O2 in a transparent acrylic box for 15 min, after which anesthesia was maintained via a cone-shaped mask over the nose. A longitudinal incision was made from the xiphoid to pubic symphysis (0.5 cm) on the skin, abdominal muscles, and peritoneum, after which the incision was sutured with 5-0 vicryl thread. The procedure required approximately 10 min. The mouse was then transferred back into the box, and was under anesthesia for 2 h in total. EMLA cream® (2.5% lidocaine and 2.5% propitocaine, Astra Pharmaceuticals) was applied onto the incision at the end of the procedure and at 8-h intervals for one day. Dexmedetomidine hydrochloride at a dose of 5 µg/kg (intraperitoneal) and a mixture of MK-801, a noncompetitive N-methyl-d-aspartate receptor antagonist at a dose of 0.5 mg/kg (intraperitoneal), and CP 465022, a noncompetitive α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist at a dose of 10 mg/kg (subcutaneously) were administered 20 min before the surgery.
Buried food test
The buried food test was performed as described in previous studies with modifications.32,33 We used ten 45-mg pieces of a purified pellet of F-0021J (BrainScience idea Co., Ltd.). The mice were habituated to the testing room for 1 h. The test cages (30 × 5 × 15 cm) were prepared with clean bedding (height, 3 cm) and a round-shaped case (diameter, 2 cm) containing the pellets. The case was buried in the bedding (0.5 cm below to the surface), and the location of the case in the test cage was changed at random. The mouse was placed in the center of the test cage, and the time to start eating was measured. If the mouse did not find the pellet in 5 min, we ended the trial and considered 300 s as the latency period. We used a new test cage, bedding, and gloves for each mouse.
Open field test
Open field test was performed as described in previous studies with modifications.9,34 The open field apparatus was a white plexiglass square box (40 × 40 × 40 cm). The mouse was gently placed at the center of the box under dim lighting and was allowed to move freely for 5 min. The mouse in the box was monitored by a video camera, and the behavioral trajectory was analyzed with Any-maze behavior tracking system software (Stoelting Co., Wood Dale, IL). The total distance traveled in the box, the time spent in the center of the open field, the freezing time, and the latency to reach the center of the open field at the first attempt were analyzed. The floor of the open field was cleaned with 70% ethanol solution after every test.
Y maze test
Y maze test was performed as described in previous studies, with modifications.9,35,36 The Y maze apparatus was made of black-colored plexiglass. The apparatus consisted of three arms (30 × 5 × 15 cm) with an angle of 120° between each arm. The three arms were the start arm, in which the mouse starts to explore (always open); the novel arm, which is blocked at the first trial, but opened at the second trial; and the other arm (always open). The start arm and the other arm were randomly designed to avoid the spatial memory error. The Y maze test consisted of 2 trials separated by an inter-trial interval (ITI). The first trial (training) was performed for 10 min and allowed the mouse to explore 2 arms (start arm and other arm) of the maze, with the novel arm being blocked. After an ITI of 1 h, the second trial (retention) was performed. For the second trial, the mouse was placed back in the maze in the same starting arm, with free access to all three arms for 5 min. The maze was placed in a quiet and illustrated room. The behavior was recorded by a video camera mounted above the maze, and the number of entries and the time spent in each arm were assessed with the Any-maze behavior tracking system software. The time spent in and entries into the novel arm were used as an indication of spatial recognition memory. Each of the arms of the Y maze was cleaned with 70% ethanol solution before testing.
In vitro whole-cell patch-clamp recording
Mice that underwent the behavioral tests were rapidly decapitated after cervical dislocation, and the brain was quickly removed and submerged in a cold artificial cerebrospinal fluid (ACSF) solution containing (in mM) 124 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 25 NaHCO3, 1 NaH2PO4, and 10 glucose equilibrated with 95% O2-5% CO2. Coronal brain slices (300 µm) at the level of the PFC were prepared using a vibratome.37-39 The brain slices were transferred to a submerged recovery chamber with the ACSF solution at room temperature for at least 1 h. Individual slices were then put into a recording chamber in which the ACSF solution was continuously perfused at 32-33 ℃. Whole-cell patch-clamp recordings were made from single layer Ⅱ/Ⅲ pyramidal neurons visualized with infrared differential interference contrast optics (BX50WI, Olympus, Tokyo, Japan)39. The patch pipettes (3-5 MΩ) were filled with a potassium-based intracellular solution containing (in mM): K-glucose 135, CaCl2 0.5, MgCl2 2, EGTA 5, ATP-Mg 5, and HEPES 5; pH: 7.2 for recordings of EPSCs and membrane potentials, or a cesium-based intracellular solution containing (in mM): Cs2SO4 110, TEA-Cl 5, CaCl2 0.5, MgCl2 2, EGTA 5, ATP-Mg 5 and HEPES-CsOH 5; pH: 7.2 for recordings of IPSCs. EPSCs and IPSCs were recorded under voltage-clamp conditions at a holding potential of -70 mV and 0 mV, respectively. Membrane potentials and action potentials (APs) were recorded under current-clamp conditions. APs were evoked by 500-ms depolarizing pulses from 0 to 150 pA with 30-pA steps through the recording electrode from a membrane potential of -70 mV. The access resistance (15-30 MΩ) was monitored throughout the experiment. Data were discarded if the resistance changed by more than 20% during the recording. The recording signals were amplified with a patch-clamp amplifier (MultiClamp 700A; Molecular Devices, Sunnyvale, CA, USA), low-pass filtered at 5 kHz, digitized with an analog-to-digital converter (Digidata 1321A; Molecular Devices), and stored on a personal computer using a data acquisition program (pCLAMP version 12.3; Molecular Devices) with a sampling frequency of 10-20 kHz. Synaptic events were analyzed using a software (Minianalysis version 6.0.7; Synaptosoft, Fort Lee, NJ, USA). Only cells exhibiting a stable resting membrane potential (less than –50 mV) were used in this study. The following criteria were used to measure action potential threshold and amplitude. The first action potential evoked by the minimum injection current was used for all measurements. Action potential threshold is the voltage where a sharp upward rise of the depolarizing phase of the action potential occurs, while action potential amplitude is the difference in voltage between the threshold and its peak amplitude
Dexmedetomidine was obtained from Maruishi Pharmaceutical Co., LTD. (Osaka, Japan) and was dissolved in saline (1 µg/mL). MK-801 and CP 465022 were obtained from Tocris Bioscience (Bristol, England) and dissolved in saline at 25 µg/mL and 2 mg/mL, respectively.
All numerical data are shown as mean ± SD. Statistical analyses were performed with EZR40 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to perform statistical functions frequently used in biostatistics. Student’s unpaired t tests was used to compare the firing properties, the parameters of EPSCs and IPSCs between control and anesthesia/surgery group. Student’s paired t tests was used to compare the firing frequency between before and after stimuli. Two-way repeated analysis of variance (ANOVA) followed by post hoc Bonferroni test was used to compare the frequency-current relationship of action potentials and the cumulative distributions of inter-event interval of spontaneous EPSCs. The Kruskal-Wallis test followed by post hoc Steel-Dwass was used to analyze the behavioral test results of the three groups (control, anesthesia/surgery, anesthesia/surgery (dexmedetomidine)) and the frequency of spontaneous EPSCs for the three groups (anesthesia/surgery, anesthesia/surgery (MK-801 and CP465022), anesthesia/surgery (dexmedetomidine)). In all cases, p < 0.05 was considered to be statically significant. “n” refers to the number of animals or neurons.