Anisomycin

3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), sGC inhibitor ODQ, non-competitive selective PDE inhibitor IBMX, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PKA inhibitor H89 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). The sGC activator BAY 41-2272 and NF-κB inhibitor Ro 106-9920 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The extracellular regulated MAP kinase (ERK) inhibitor U0126, p38 MAP kinases inhibitor SB202190, c-Jun N-terminal kinases (JNK) inhibitor SP600125, HIF-1α inhibitor CAY10585, p38 activator anisomycin, and phosphatidylinositol-3-kinases (PI3K) inhibitor wortmannin were purchased from Cayman Chemical (Ann Arbor, MI, USA).

For the cannula placement procedure, mice were anesthetized with isoflurane through an anesthetic vaporizer, secured to the stereotaxic instrument, and the cannula made from a 25-gauge needle was inserted bilaterally into LA and basolateral amygdala (relative to bregma: –1.2 mm anteroposterior; ±3.5 mm mediolateral; –4.3 mm dorsoventral) (16 (link), 36 (link)). Dental cement secured the cannula and bone anchor screw in place. Mice recovered for 4–5 days before any subsequent testing was carried out. A 10 μL Hamilton syringe connected to a 30-gauge injector was inserted 1 mm past the cannula tip to inject the selective ASIC1a inhibitor, PcTX-1 (100 nM, Allomone Labs) or anisomycin (62.5 μg/μL, Cayman Chemical). The chemicals were diluted in 1 μL artificial cerebrospinal fluid (ACSF), pH 7.3, and injected over 5 minutes each side. The injection sites were mapped postmortem by sectioning the brain (10 μm coronal) and examining Cresyl violet staining using a Nissl Stain Kit (FD Neuro Technologies). Only animals that had a correctly placed cannula in the amygdala were included in the statistical analysis.