Model 1004

HF was induced by continuous infusion of isoproterenol with an osmotic pump, following the conventional procedure in this model [9 (link)]. Briefly, mice were anesthetized with a mixture of inhaled isoflurane and oxygen-enriched air (1.25% during induction and 1% during maintenance) and a small incision was made on the back of each animal between the shoulder blades after removing the hair from the area using depilatory cream. The skin was carefully separated from underlying connective tissues using blunt-ended scissors and an osmotic mini-pump (Alzet, model 1004) containing isoproterenol (Sigma Aldrich) at 30 mg/kg per day dissolved in sterile 0.9% NaCl solution or only 0.9% NaCl solution (for healthy controls) was implanted subcutaneously for delivery pharmacological agent or placebo for 30 days and the incision was sutured with surgical staples (Autoclip, Fine Science Tools). The procedure was performed under aseptic conditions. The surgery platform was continuously warmed to maintain body temperature until the end of anesthesia. Buprenorphine (0.1 mg/kg) was subcutaneously administered 10 minutes before surgery and after 24 hours. Suture staples were removed 7 days after surgery.

ASMko and wt mice were anesthetised with isoflurane, and i.c.v. delivery cannulas (brain alzet kit III) were implanted with a stereotaxic frame (KOPF Instruments) at the following coordinates according to the bregma: AP, −0.5 mm; ML, 1 mm; and DV, −2.2 mm. Osmotic minipumps (Alzet; model #1004) were filled with Ca074Me (Sigma‐Aldrich) or vehicle (0.9% NaCl, 1.5% DMSO), equilibrated in 0.9% NaCl at 37°C for 48 h, attached to the i.c.v. cannula tubing and subcutaneously implanted at the back. Ca074Me was administered at 1 mg/ml at a rate of 0.25 μl/h as previously described (Hook et al, 2008). Animals were housed individually. One month after implantation, behavioural testing was performed and then mice were sacrificed for histological analyses.