Cyberamp

Electrophysiological experiments were carried out as described previously^{20 (link),70 (link)}. For recordings of the monosynaptic reflex, the intact ex vivo spinal cord preparation was perfused continuously with oxygenated (95% O₂/5% CO₂) aCSF at ∼10 mL/min. The dorsal root and ventral root of the L1 segment were placed into suction electrodes for stimulation and recording, respectively. The extracellular recorded potentials were acquired (DC, 3 kHz, Cyberamp, Molecular Devices) in response to a brief (0.2 ms) stimulation (A365, current stimulus isolator, World Precision Instruments) of the L1 dorsal root. The maximum responses were acquired following supramaximal stimulation intensity (ranging between 2 and 10 times the Threshold of stimulation). The stimulus Threshold was defined as the current at which a minimal evoked response was recorded in three out of five trials. Recordings were fed to an A/D interface (Digidata 1440A, Molecular Devices) and acquired with Clampex (version 10.2, Molecular Devices) at a sampling rate of 10 kHz. Data were analyzed offline using Clampfit (version 10.2, Molecular Devices). Measurements were taken from averaged traces of five trials elicited at 0.1 Hz. The temperature of the physiological solution ranged between 21 and 25 °C.

Recordings were performed in awake, head-fixed animals for no longer than 4 h while their body temperature was supported using a homeothermic pad (FHC, Bowdoin, ME, USA). Custom-made, borosilicate glass capillaries (OD 1.5 mm, ID 0.86 mm; resistance 8–12 MΩ; taper length ~8 mm; tip diameter ~1 μm) (Harvard Apparatus, Holliston, MA, USA) filled with 2 M NaCl were used for electrophysiological Purkinje cell recordings. Electrodes were positioned stereotactically using an electronic pipette holder (SM7; Luigs & Neumann, Ratingen, Germany). Neurons were recorded extracellularly in both medial (vermis) and lateral (paravermis and hemisphere) areas of the cerebellar cortex, mainly from lobules VI–X. Purkinje cells were identified by the characteristic occurrence of both complex spikes and simple spikes and a minimal pause in simple spike firing following each complex spike of 10 ms. ECoGs were filtered online using a 1–100 Hz band pass filter and a 50 Hz notch filter. Single unit extracellular recordings and ECoGs were simultaneously sampled at 20 kHz (Digidata 1322A, Molecular Devices LLC, Axon Instruments, Sunnyvale, CA, USA), amplified, and stored for off-line analysis (CyberAmp and Multiclamp 700A, Molecular Devices LLC, Axon Instruments, Sunnyvale, CA, USA). ECoG traces were down sampled to 300 Hz.

Aortic rings were placed in a 10 mL tissue bath filled with Krebs–Henseleit solution, bubbled with 95% O₂ and 5% CO₂ and maintained at 37 °C in a circulating water bath. The rings were placed between two stainless steel wires; one wire was fixed to a manipulator, and the other was connected to an isometric force transducer (Radnoti Glass Technology Inc., Monrovia, CA, USA). The force transducer was connected to a CyberAmp (Axon Instruments, Foster City, CA, USA) and the signal acquired with a Digidata 1200 (Axon Instruments, Foster City, CA, USA) in the Axoscope subroutine of pClamp (version 9; Axon Instruments, Foster City, CA, USA). Aortic rings were precontracted with 1 µM phenylephrine and, when contraction was stable (this was set at 100%), we added 30 µM ACPA to the bath. We performed analyses using the Clampfit subroutine. The experiments were performed in duplicate, and the data averaged. Results are expressed as the mean ± standard error of the measurements from at least 8 aorta rings from at least four different rats.