Model 1700

For each experiment, a single neonatal spinal cord was extracted as described above. The isolated spinal cord was submerged in physiological saline (composition in mM: NaCl, 129; KCl, 4; CaCl₂, 2.5; MgCl₂, 1.14; NaH₂PO₄, 0.5; NaHCO₃, 25; glucose, 10, adjusted to pH = 7.4 with HCI) that had been superfused with oxygen (95% O₂/5% CO₂) before recording. The bath temperature was kept constant at 24 °C. Locomotor-like activity was induced by bath applying glutamate (10 mM stock, frozen, and stored at −20 °C). Neurons in the ventral horns were recorded with inspect-pin electrodes connected to a differential A/C amplifier (Model1700, A-M Systems, Sequim, WA) and a data acquisition system (PowerLab 8/30, ADInstruments, Dunedin, New Zealand). Records were digitized and recorded in LabChart 7.3 (ADInstruments) at a sampling rate of 20 kHz.

Electrophysiological recordings were performed separately outside the MRI bore. The animals, first anesthetized with 1.5% isoflurane, were placed in a stereotaxic frame (David Kopf instrument, CA). Body temperature was maintained at 37 °C using a heating pad (DC temperature controller; FHC Inc., Bowdoin, ME, USA). The skull was exposed and multiple holes (1 mm diameter) were made through the skull with a dental drill for insertion of micro-electrodes. The multiple tungsten microelectrodes (<1.0 MΩ, with 1 μm tip and 0.127-mm shaft diameter, Alpha Omega Engineering, Nazareth, Israel) was positioned at left visual cortex (AP −2.9 mm, ML −2.5 mm, DV −0.8 mm from the bregma), left retrosplenial cortex (AP −2.3 mm, ML −0.3 mm, DV −0.8 mm from the bregma) and left ventromedial hypothalamus (AP −1.6 mm, ML −0.3 mm, DV −5.5 mm from the bregma). After surgery, isoflurane concentration was changed to 0.8–1.0%, which is the same concentration as the fMRI experiment. Electrodes were connected to a differential AC amplifier Model 1700 (AM systems, Sequim, WA, USA), via a Model 1700 head stage (AM systems, Sequim, WA, USA). LFPs were continuously recorded for 6 min. LFP signals in three regions were simultaneously acquired at 10 kHz sampling rate using dedicated data acquisition software (Power Lab, AD Instruments, Dunedin, New Zealand). The reference electrode was positioned on the scalp.

Teflon-coated, silver wire electrodes (75 μm diameter) with exposed ball tips (50–100 μm diameter) were used to record the vocal motor volley, hereafter referred to as a fictive vocalization (VOC), from the vocal nerves that innervate the vocal muscles attached to the swim bladder. Signals were amplified 1000-fold and band-pass filtered (300–5000 Hz) with a differential AC amplifier (Model 1700, A-M Systems). VOCs were evoked by current pulses delivered to vocal midbrain areas via insulated tungsten electrodes (5 MΩ impedance; A-M Systems). For display purposes, electrical artifacts were truncated in the illustrations (marked by black arrowheads in Figures 1, 2, 5, and 6). Current pulses were delivered via a constant current source (model 305-B, World Precision Instruments). A stimulus generator (A310 Accupulser, World Precision Instruments) was used to generate TTL pulses with a standard stimulus of five pulses at 200 Hz. Each pulse train equaled one stimulus delivery with inter-stimulus intervals of 1 s. During recordings, inter-pulse intervals (100–300 Hz) and total pulse number (2–10) varied. Occasionally, VOCs also occurred spontaneously.

The signals were collected in a large Faraday cage and first amplified by a gain of 100 (Model 1700, A-M Systems, WA) with filters setting of 10 Hz–10 kHz, and then sampled at 25kHz through a National Instruments data acquisition board (PCI 6071) controlled by custom-designed MATLAB codes. Stimulus-triggered averaging (STA) was used (N = 10) to suppress the background neural activity and other sources of random noise. The averaged signal was further band-pass filtered in MATLAB (10 Hz– 1 kHz) before analyzing. Voltage transitions within 2ms window around the rising edge of the square pulses were taken as the induced voltage at the corresponding depth. The first derivative of the voltage data with respect to depth was computed as the electric field (E-field). All field measurements were made exclusively in the vertical direction (VE-field) in this study. Horizontal E-field measurements would require derivation of the voltage measurements made through different brain penetrations and lead to large calculation errors due to even slight changes in the absolute value of the voltages measured, which could easily occur from repositioning of the recording reference electrode between penetrations.