Apamin

In 18 P8 rats, pups were prepared for electrophysiological recording as described above and transferred to the recording rig. Once a pup started cycling between sleep and wake, a 0.5 µl microsyringe was lowered stereotaxically into the IO and 100 nl of apamin (Abcam, Cambridge, MA; 1 µM, dissolved in 0.9% saline, n = 8) or saline (n = 10) was injected over 1 min. During preparation of the drug or vehicle, fluorogold (4%, Fluorochrome, Denver, CO) was added to the solutions for subsequent assessment of the extent of drug diffusion. After a 15-min period to allow for diffusion, the microsyringe was withdrawn and a recording electrode was lowered in its place into the IO and activity was recorded for 30 min. At the end of the experiment, the pup was sacrificed and its brain was prepared for histology as described above.

Tetrodotoxin, phentolamine, iberiotoxin, NS-1619, Y-27632, apamin, NS-309, and ryanodine were purchased from Abcam (Cambridge, MA, USA). Atropine, propranolol, suramin, nisoldipine, and SKF-96365 were purchased from Sigma-Aldrich (St. Louis, MO USA). Bisindolylmaleimide 1 and H₂O₂ were obtained from Cayman Chemical (Ann Arbor, MI, USA) and Fisher Scientific (Pittsburg, PA, USA), respectively.
NS-1619, nisoldipine, NS 309, and SKF-96365 were dissolved in dimethylsulphoxide (DMSO). ryanodine was dissolved in 100% ethanol. All other drugs were prepared with water. Final ethanol and DMSO concentrations in the bath solution did not exceed 0.1% and were shown not to affect spontaneous contractions or bladder function in previous reports [35 (link)–37 (link)].