Electrophysiological recording
Electrophysiological recordings were conducted with etched tungsten microelectrodes (18–20 MΩ, 1 V at 1 kHz; FHC, Inc., Bowdoinham, ME, USA). For NTS recordings, the electrode was lowered into the caudal medulla above the rostral NTS located 2.7 mm anterior and 1.8 mm lateral the obex and ~1.0 mm below the dorsal surface of the brainstem. For PbN recordings, the electrode was lowered through the cerebellum above the pons located 5.4 mm anterior and 1.8 mm lateral to the obex and 5–6 mm below the cerebellar surface. Electrophysiological activity was digitized with an analog-to-digital interface (Model 1401, Cambridge Electronic Designs, Cambridge, UK) and was processed with Spike2 software (Cambridge Electronic Designs, Cambridge, UK). The signal was amplified (Model P511, Grass Technologies, West Warwick, RI, USA) and monitored online with a speaker, oscilloscope and Spike2 software. Single cells were identified by periodically delivering a 0.1-M NaCl solution followed by a water rinse as the electrode was slowly lowered through the brain. Cell isolation was based on the consistency of the waveform shape using template matching and principal component analysis. A signal-to-noise ratio of 3:1 was required for cell isolation. Isolated cells were tested with the exemplars of the four basic taste qualities yielding the “response profile” of the cell, defined as the relative response rates across tastants. Water was presented before tastant delivery and 5 s after tastant delivery. This allowed the assessment of tastant-mediated alteration of the water response. The cell was tested for as long as it remained isolated allowing for multiple presentations of the same stimulus. The precise timing of each spike (1 ms precision) was calculated with respect to the onset of each stimulus delivery, including water.