In situ study on anaesthetized snakes

A total of nine snakes were anaesthetized by an intramuscular injection of pentobarbital (30 mg kg⁻¹, Sygehusapotekerne, Denmark) and mechanically ventilated with a Harvard Apparatus mechanical ventilator at 5–10 breaths min⁻¹ and a tidal volume of approximately 60 ml kg⁻¹. The heart was exposed by a 7 cm right ventrolateral incision. To measure blood pressures, occlusive PE50 catheters filled with heparinized saline (50 IU ml⁻¹) were inserted in the vertebral artery (P_sys) and in the sinus venosus via the right jugular vein (P_SV). A flared catheter was inserted through a small incision in the right atrial lumen (P_RA), the incision was closed with a tight suture. Electrocardiograms were recorded from 3 of the 9 snakes. All catheters were connected to disposable pressure transducers (Baxter Edward, model PX600, Irvine, CA) and the signals were amplified using an in-house built amplifier. The pressure transducers were calibrated against a static water column prior to each experiment and maintained at heart level. All pressure measures were recorded with a MP100 data acquisition system (Biopac Systems, Goleta, CA) at 200 Hz. No less than 30 min after catheterization injections of autonomic agonists were given in the vertebral artery in a volume of 1 ml kg⁻¹ followed by a flush of approximately 0.2 ml kg⁻¹ heparinized saline. After each agonist, the maximal response was compared to the pre-injection baseline. Agonists were given in the following order; adrenalin (5 µg kg⁻¹), acetylcholine (2 µg kg⁻¹, 22 mmol kg⁻¹), isoproterenol (0.1 µmol kg⁻¹), phenylephrine (10 µg kg⁻¹). Hereafter we delivered blockade of muscarinic and β-adrenergic receptors with atropine (4 mg kg⁻¹) and propranolol (4 mg kg⁻¹) respectively. The efficacy of the blockade was verified with acetylcholine and adrenalin.