In vivo electrophysiology for ACC recording

In vivo preparations were made as described previously [38]. Under urethane anesthesia (1.2–1.5 g/kg, i.p.), rats were mechanically ventilated after tracheostomy and bilateral thoracotomy was performed. After the head was fixed in a stereotaxic apparatus (Model SR-6R, Narishige, Tokyo, Japan), a craniotomy is performed using a dental drill to open a hole above the ACC according to the stereotaxic coordinates. A tungsten electrode (impedance, 10 MΩ, A-M systems, Sequim, WA) was placed into the ACC, and conventional extracellular recordings were obtained as shown previously [39] with an AC differential amplifier (DAM 80, World Precision Instruments, Sarasota, FL). Data were filtered (300 to 5 kHz) and digitized (10 kHz). Unit firings were sorted with Offline Sorter software (version 3, Plexon, Dallas, TX). Putative pyramidal neurons in the ACC were identified based on their waveforms as shown previously [40]. In the case of drug microinjection into the ACC, a cannula was inserted through the same hole, and the tip was placed in the vicinity (approximately 1 mm) of the recording electrode. Drugs (noradrenaline, 50 μg/0.5 μL; phenylephrine, 5 μg/0.5 μL; Isoproterenol, 37 μg/0.5μL) diluted in normal saline were microinjected over a 5 min period. For the LC electrical stimulation, a hole was further opened on the skull above the cerebellum ipsilateral to the recording site, and a combined bipolar stimulating electrode-tungsten extracellular recording electrode (1 MΩ, A-M systems, Sequim, WA) was stereotaxically inserted into the floor of the fourth ventricle above the LC. The electrode was then lowered and placed into the LC if neuronal activity is considered to be obtained from LC neurons. LC neurons were identified based on their characteristic spontaneous firing and responses to contralateral cutaneous noxious stimulation as described previously [41, 42]. Trains of electrical pulses (duration 200 μs, 50 times, 4 ms interval) were then applied with an interval of 2 s for 1–2 min.