Grass model p511

Glass micropipettes were pulled from borosilicate glass (OD 1.5 mm, ID 1.12 mm) on a horizontal puller (Sutter Instrument Co., Model P-97). The tip of the micropipette was coated with a fluorescence dye (Vybrant® Dil Cell-labeling solution, Life Technologies, reference V22885, MW: 933.88), filled with artificial cerebrospinal fluid (ACSF; 124 mM NaCl, 3 mM KCl, 1.5 mM CaCl₂, 1 mM MgSO₄, 25 mM NaHCO₃, 0.5 mM NaH₂PO₄, and 30mM D-glucose), and combined with an optic fiber (200 μm, Doric Lenses or ThorLabs), which was placed 500–1000 μm above the tip of the electrode. Transfected mice were placed in the stereotaxic frame as described above. A window was drilled through the skull of sufficient size to allow a free range of movement to the combined electrode/fiber. The recording electrode was connected to a headstage (Siskiyou) and the signal amplified (Grass Model P511, Grass Instruments) and sampled at 20 kHz (PowerLab 16SP, ADInstruments). Breathing-modulated units were found 4.6 to 4.9 mm below the cerebellar surface and 1.1 to 1.3 mm lateral to the midline. Post-mortem, the placement of the combined electrode/fiber was confirmed before immunostaining to preserve the dye using a fluorescence microscope (Axioplan2 Imaging, Carl Zeiss).

To record neuronal activity, transfected mice were placed in the stereotaxic frame as described above. An optical cannula was implanted at a 27° angle from vertical, tilted across the midline from right to left, down to preBötC and glued to the skull with Metabond (Parkell). A window was drilled through the skull of sufficient size to allow the recording electrode free range of movement to record on the left side of the brain. Respiratory-modulated units were found 4.6 to 4.9 mm below the cerebellar surface and 1.1 to 1.3 mm lateral to the midline. A glass electrode (~3 µm inner tip diameter; filled with aCSF containing (in mM) 124 NaCl, 3 KCl, 1.5 CaCl₂, 1 MgSO₄, 25 NaHCO₃, 0.5 NaH₂PO₄, and 30 d-glucose) was connected to a headstage (Siskiyou) and the signal amplified (Grass Model P511; Grass Instruments) and sampled at 20 kHz (PowerLab 16SP; ADInstruments). Due to a laser artifact apparent at the start and end of laser pulses, we used a single 1 s pulse for photostimulation to reduce noise. Most neural recordings were stable for 5 – 10 min.