Dp 304

DC-coupled recordings were obtained from both VRs and DRs using monopolar suction electrodes realized by pulling tight-fitting glass pipettes (1.5 mm outer diameter, 0.225 mm wall thickness; Hilgenberg, Germany). The tip of each pipette was filled with Krebs physiological solution and, using micromanipulators, was positioned on targeted VRs and DRs. Then, a gentle negative pressure was applied to draw the root extremity inside the pipette. Afterwards, the pipette was moved close to the origin of the targeted VR and, through further pressure, the entire root was suctioned into the glass pipette. Electrodes were connected to a differential amplifier (DP-304, Warner Instruments, Hamden, CT, USA; high-pass filter = 0.1 Hz, low-pass filter = 10 kHz, gain X 1000). Analog signals were filtered through a noise eliminator (D400, Digitimer Ltd, UK), then digitized (Digidata 1440, Molecular Devices Corporation, Downingtown, PA, USA; digital Bessel low-pass filter at 10 Hz; sampling rate = 5 kHz) and visualized real-time with the software Clampex 10.7 (Molecular Devices Corporation, Downingtown, PA, USA).

In each group, a 1 min recording of LSG activity was obtained. The tungsten-coated microelectrode was inserted into the fascia of the LSG, and the chest wall was connected to a group lead. The LSG-generated electrical signals were recorded using a Power Lab data acquisition system (8/35, AD Instruments, New South Wales, Australia) and an amplifier (DP-304, Warner Instruments, Hamden, CT). Prior to recording, bandpass filters (300–1 kHz) were established and amplification was set to 30–50 times. Neural activity was recorded based on frequency and amplitude, with deflections having a signal-to-noise ratio >3:1 defined as neural activity. The amplitude and frequency were manually determined, as previously described in our studies.^{8 (link)}