Cgp52432

Rabbit polyclonal IgG anti-bovine serum albumin was purchased from MP Biochemical. Alexa fluor 488 conjugated bovine serum albumin was obtained from Invitrogen. Anti-β-actin, anti-pERK and anti-p38 MAPK antibodies were purchased from SantaCruz Biotechnology. Anti-GABA_BR2, anti-pCREB, and anti-active caspase-3 antibodies were obtained from Cell Signaling Incorporated. CGP52432 was obtained from Abcam (Cambridge, MA, USA). Anti-GABA_BR2 antibody was obtained from Thermo Scientific (Rockford, IL). Hematoxylin and eosin was obtained from Fischer Scientific (Pittsburgh, PA, USA). Anti-GAPDH and anti-TNF-α, and the ApopTag (TUNEL kit) were obtained from Millipore. All other reagents utilized in this study were obtained from Sigma Aldrich (St Louis, MO, USA) unless otherwise stated. Naphthol AS-D chloroacetate esterase (NACE) kit was obtained from Sigma Aldridge.

To synaptically isolate respiratory networks, a cocktail was used consisting of AMPA receptor blocker 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX; 10 μM), NMDA receptor blocker D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 50 μM), GABA_(A) receptor blocker bicuculline methobromide (10 μM), group 2 MGluR blocker LY 341,495 (500 nM), GABA_(B) receptor blocker CGP 52,432 (10 μM) (all from Abcam, Waltham MA), and glycine receptor blocker strychnine (2 μM; Sigma-Aldrich, St Louis MO). To block gap junctions, the selective, reversible CX-43 and CX-36 channel blocker meclofenamic acid sodium (MFA; 60–100 μM; Sigma-Aldrich, St Louis MO) was applied alone or in conjunction with the blocker cocktail. To confirm results obtained using MFA, the broad-spectrum gap junction blocker carbenoxolone (CBX; 100 μM; Sigma-Aldrich, St Louis MO) was applied following synaptic blockade. NA⁺_(V) conductances were blocked with tetrodotoxin (TTX; 1 μM; Sigma-Aldrich, St Louis MO). All solutions were prepared the day of the experiment and recirculated to enable longer recordings with low solution volumes.

Drugs applied to the slice perfusion solution were: SCH50911 (20–50 μM), CYN 154806 (20 μM), and somatostatin (SST, 1–2 μM) from Tocris (UK), Tetrodotoxin (1 μM), CGP52432 (20 μM), HC030031 (80 μM), NBQX (10 μM), APV (50 μM), MPEP (50 μM), PPADS (100 μM) from Abcam (UK).