Acepromazine

Mice were anesthetized using a mixture of ketamine (100 mg/kg IP) and acepromazine (5 mg/kg IP). A rostrocaudal incision was made in the scalp, and a small neodymium magnet (4.57 mm × 4.57 mm × 2.03 mm, 375 mg) was attached to the center of the dorsal surface of the cranium using cyanoacrylate or dental resin; the magnet was mounted with the axis of the N-S poles parallel to the dorsoventral plane of the head. The mice were allowed to recover for 1–2 weeks prior to testing. After magnet implantation, mice were tested multiple times over 4–6 weeks, with a minimum of 1–2 weeks between tests; importantly, it was previously reported that administration of a 5-HT_2A agonist at weekly intervals does not alter HTR sensitivity (Gewirtz and Marek, 2000 (link)). To record head movements, the mice were placed in a 12.5 cm diameter glass beaker surrounded by ~150 turns of #30 enameled magnet wire. The output of the coil was recorded using a Powerlab/8SP with LabChart v.7.3.2 (ADInstruments, Colorado Springs, CO, USA). Coil voltage was amplified, low-pass filtered (10 kHz cut-off frequency) to remove radiofrequency interference, and sampled at 40 kHz. Data were imported into the Matlab programming environment (MathWorks, Natick, MA, USA) for Fourier analysis. Voltage and frequency peaks were detected using the LabChart cyclic measurements analysis module. For HTR analysis, the LabChart data were digitally band-pass filtered (40–200 Hz), and responses were identified by manually searching for sinusoidal wavelets with the following characteristics: (1) waveform and spectrum consistent with 40–160 Hz activity; (2) contain more than 2 bipolar peaks; (3) amplitude exceeds the background noise level; and (3) duration <120 ms.
In most experiments, the magnetometer-based assessment of head twitch was combined with simultaneous video recordings. The behavior of the mice within the magnetometer coil was captured at 30-Hz using a CCD video camera located above the glass beaker, digitized, and stored on a PC as an AVI file. Subsequently, head twitches were counted by an observer blind to the treatment and the magnetometer data (Halberstadt et al., 2011 (link)). Potential responses were analyzed frame-by-frame using VirtualDub v.1.9.11 and were counted as head twitches if there was evidence of torsional head movement over consecutive frames. For the experiment with SKF38393, the duration of each grooming bout (including licking the paws, legs, fur, body, tail, or genitals, and washing the head, face, and ears) was assessed by an observer blind to the treatment.

Muscle strength and fatigability were analyzed in EDL muscles using an ASI muscle contraction system (Aurora Scientific), equipped with a 300C-LR dual mode force transducer and a 701C stimulator. Mice were anaesthetized by intra-peritoneal injection of 100 mg/kg ketamine, 10 mg/kg xylazine, and 2 mg/kg acepromazine. EDL muscles were exposed after removing TA muscles. The proximal and distal tendons of EDL muscle were then tied using 4-0 surgical suture and EDL muscles were carefully excised and mounted between two platinum electrode plates while continuously perfused with oxygenated Ringer solution in the experimental chamber.⁴⁶ Muscle optimal length (L_o) was determined using a series of 1 Hz stimulation and set at the length that generates the maximal force. Stimulus output was set at 120% of the voltage that elicits maximal force. Muscles were first equilibrated using three 500 ms, 150 Hz tetani at 1 min intervals and then subjected to a force frequency, a single sustained high frequency tetanus (150 Hz, 2 sec) and/or a repetitive tetanic fatiguing (60 successive 50Hz, 500ms tetani) protocol. Muscle force was recorded using a Dynamic Muscle Control software and analysed using Clampfit 10.0 software. Muscle cross-sectional area and specific force were calculated as described previously⁴⁶ .