2003) including NK-1R-positive excitatory neurons (Gray em et al /em

2003) including NK-1R-positive excitatory neurons (Gray em et al /em . powerful and help to conquer opiate-induced respiratory major depression. Hence, 5-HT1AR activation stabilizes the rhythmicity of deep breathing during opiate medication of pain. cat experiments Experiments were performed on 10 adult pet cats of either sex weighing 2.8C3.8 kg using methods that have been explained in detail in other published studies from this laboratory (Lalley rat experiments Sprague Dawley rats of either sex (250C350 g) were anaesthetized by intraperitoneal injection of pentobarbital (60 mg kg?1). The depth of anaesthesia was tested having a forepaw pinch. In case GKA50 of reflex responses, P19 an additional dose of pentobarbital was applied (approx. one-tenth of the initial dose). The trachea and right femoral vein were canulated with polyethylene tubing to record respiratory air flow and for drug and fluid injections, respectively. The inspiratory/expiratory airflow was recorded using a MacLab-linked pressure transducer connected to the tracheal tubing. Nociceptive responses were assessed from the tail-flick (TF) response. High-intensity light adequate to stimulate nociceptors was applied to marked places (1 cm from the tip of the tail, four places at an interval of 1 1 cm), and the TF was quantified by measurement of the latency from warmth on to the evoked withdrawal response. The average TF response latency ideals of three consecutive tests before drug application were used as baseline. To avoid cells damage, the heating was halted when the TF latency exceeded 300 per cent of control, concluding TF response GKA50 abolishment. To prevent central hypoxia, the animals were insufflated with oxygen during the whole experiment and ventilated artificially with space air flow at low rate of recurrence (10C15 breath min?1) after opioid-induced apnoea. Artificial air flow was immediately halted after detection of the 1st indications of spontaneous breathing motions evoked by drug application. At GKA50 the end of the experiments, all animals were sacrificed using an overdose of pentobarbital that produced irreversible cardiac arrest. (c) Perfused brainstemCspinal wire preparation of rat or mouse The experiments within the brainstemCspinal wire preparation were performed on Sprague-Dawley rats (P22CP32) or C57BL mice (P20CP25) as explained originally (Paton 1996). For isolating the brainstemCspinal wire from higher mind areas, animals were deeply anaesthetized with halothane until apnoea occurred and they were unresponsive to a forepaw pinch. Animals were then decerebrated in the pre-collicular level and cerebellectomized, bisected below the diaphragm and the skin was eliminated. The upper body was placed in a recording chamber and perfused retrogradely via the thoracic aorta with artificial cerebrospinal fluid (ACSF, comprising in mM: 1.25 MgSO4; 1.25 KH2PO4; 5 KCl; 125 NaCl; 2.5 CaCl2; 25 NaHCO3; 10 glucose, 70 Ficoll (0.1785 mM)), and gassed with carbogen (5% CO2/95% O2; pH 7.35). The GKA50 perfusate was warmed to 30C as measured in the skull foundation, filtered twice and re-circulated. Norcuronium-bromide at 0.5 mg 200 ml?1 was added for immobilization. The perfusion pressure was arranged to 45C65 mm Hg. Using a glass suction electrode, phrenic nerve discharges were recorded as an index of central respiratory rhythm. Drugs were added to the perfusate for specific pharmacological manipulation of 5-HTR and glycine receptors. (d) Rhythmic brainstem slice preparation of rat Inspiratory neurons in the pre-B?tC were recorded in whole-cell mode before harvesting the cytosol for single-cell RTCPCR analysis. Transverse slices (600 m) were cut from your caudal medulla at the level of the pre-B?tC having a vibroslicer (Campden Tools, Loughborough, UK) and stored in ACSF at room temp (20C23C) for at least 30 min before experiments were started. They were then transferred to a recording chamber and kept submerged by nylon fibres for mechanical stabilization. The chamber was mounted on an upright microscope (Axioscope FS; Zeiss, Oberkochen, Germany) and perfused continually with ACSF (20C23C) at a circulation rate of 5C10 ml min?1. Whole-cell voltage-clamp recordings were obtained having a Multiclamp 700A amplifier (Axon Tools). Patch electrodes were drawn from borosilicate glass capillaries (Biomedical Tools, Zlpich, Germany) on a horizontal pipette puller (Zeitz-Instrumente, Munich, Germany) and filled with patch solution comprising (in mM): GKA50 125 KCl, 1 CaCl2, 2 MgCl2, 4 Na2ATP, 10 EGTA, 10.