Ex vivo electrophysiological recording of the carotid sinus nerve (CSN) activity

Rats were deeply anesthetized with ketamine/xylazine (0.1 ml/100 g, i.p.), both carotid bifurcations were removed “en bloc.” The excised tissue was placed in a petri dish containing ice-cold Tyrode solution (in mM: 125 NaCl, 5 KCl, 2 MgSO₄, 1.2 NaH₂PO₄, 25 NaHCO₃, 1.8 CaCl₂, 5 sucrose, and 10 glucose, pH 7.4) bubbled with carbogen (95% O₂ + 5% CO₂). The carotid sinus nerve (CSN) was then carefully dissected and cleaned from surrounding connective tissue and its activity was recorded in vitro using standard methods used in our group (Joseph et al., 2012). Briefly, the preparation was placed in the recording chamber (volume = 5.4 ml) and a catheter placed on the inlet of the perfusion solution entering the chamber was passed through the common carotid artery to improve perfusion. The recording chamber was maintained at 36°C (TC2Bip temperature controller; Cell Micro-Controls; Norfolk, VA, USA) and perfused at a flow rate of 6 mL/min with Tyrode solution bubbled with 5% CO₂ balance in O₂; pH 7.4. The CSN was drawn up into the tip of a glass suction electrode (Model # 573000, A-M Systems Inc., Carlsborg, WA) for activity recording. Sufficient suction was applied to seal the electrode tip against connective tissue encircling the junction of the carotid body and CSN. A grounding electrode was placed in the recording chamber. The neural signal was fed to a differential input head-stage pre-amplifier, filtered (30–1500 Hz), and amplified (Neurolog modules NL100AK, NL104A, NL126, NL106). The signal was then processed by an A/D converter (Micro 1401-2 Cambridge Electronic Design (CED), Cambridge, UK) for display of raw activity and frequency histograms on a computer running the Spike 2 software (CED). Chemoreceptor discharges are discriminated in Spike 2 as action potentials with amplitude of 25% above baseline noise and which responded to a decrease in perfusate PO₂ with a reversible increase in discharge frequency.