All gas conditions were administered by a flow

metre gas-mixing pump (Cameron Instruments GF-3/MP). O2 (Raytech quadralyser 224A) and CO2 (Beckman LB2) gas analysers were used to monitor gas composition inside screening assay the animal chamber for all experimental protocols. Each animal was used once and received only one injection of PPADS or vehicle. All recording experiments were carried out at ambient temperature (24.5 ± 0.5 °C). Upon completion of the experiments, animals were anesthetized with 2,2,2-tribromoethanol and perfused intracardially with saline followed by 4% paraformaldehyde. The brain was removed and stored in 4% paraformaldehyde for 4 h. Following fixation, paraformaldehyde solution was replaced for 20% saccharose (48 h, at 4 °C) to cryoprotect the tissue prior to processing. Tissue was frozen, sectioned on a cryostat at −20 °C (40 μm-thick coronal sections) and stained by the

Nissl method for light microscopy. The see more location of injection was determined by the distance between the centre of injection and the caudal pole of facial nucleus (Paxinos and Watson, 1998). Only rats where the site of microinjection was located in the rostral and caudal aspect of the MR were considered for data analysis. Values are reported as means ± SEM. V˙E, VT and fR measurements were taken before CO2 exposure, at 5, 10, 20 and 30 min during hypercapnia and after CO2 exposure. Statistical analyses of the data were performed using a two-way ANOVA and Duncan’s

test for post hoc comparisons (Sigma Stat, Systat Selleck Afatinib Software Inc., Point Richmond, CA, USA). Data was considered statistically significant when p < 0.05. Representative photomicrographs of typical sites of microinjections into the rostral MR and caudal MR are shown in Fig. 1A and B, respectively. In addition, diagrams of transverse sections of the brainstem showing the rostro-caudal distribution of microinjections sites are shown in Fig. 1C. These rostro-caudal sites are representative for all animals that received PPADS microinjections and underwent hypercapnic exposure protocol. Note in Fig. 1C that the rostral microinjections were located in the RMg nucleus (n = 7) and the caudal microinjections in the ROb nucleus (n = 5). For rostral MR (RMg) microinjection centre ranged from 10.5 to 11.58 mm caudal to bregma, while for caudal MR (ROb) microinjection ranged from 12.1 to 13.1 mm caudal to bregma. Fig. 2 summarizes data indicating that neither antagonism of P2X receptors (PPADS: 0.02 M; n   = 8) nor microinjection of 50 nL of the vehicle (saline, 0.9% NaCl; n   = 7) in the rostral or caudal MR changed baseline V  T, fR and V˙E (p > 0.05) ( Fig. 2, panels A–C). Data for rostral and caudal MR are plotted together in Fig. 2. Microinjection of PPADS into both rostral and caudal MR did not change body temperature compared with the vehicle group (37.4 ± 0.03 vs. 37.5 ± 0.04 (p > 0.