M3262

Mice were anesthetized with an intraperitoneal injection of Avertin, 125 mg per kg of body weight of 2,2,2-tribromoethanol diluted in 2-methyl-2-butanol (#T48402; and #240486; respectively, MilliporeSigma, Burlington, MA, USA), followed by proparacaine eye drops (0.5% Proparacaine Hydrochloride Ophthalmic Solution; Sandoz Inc., Princeton, NY, USA) for topical anesthesia. Pupils were dilated by instillation of 5% phenylephrine and 0.5% tropicamide eye drops (Massachusetts Eye and Ear Infirmary Pharmacy, Boston, MA, USA). Consequently, a conjunctival incision was made temporally and a sclerotomy was created approximately 3–4 mm from the limbus. A 33- gauge needle (Hamilton Custom Needles: Length: 10.00mm/Point Style: 4/Angle: 20, #7803–05; Hamilton Company, Reno, NV, USA) connected to a 10-µl syringe (Hamilton, 701RN SYR, #7635–01; Hamilton Company, Reno, NV, USA) was then inserted into the intravitreal cavity and 2 µl of NMDA (100 nmoles, #M3262; MilliporeSigma, Burlington, MA, USA) or vehicle (phosphate buffer saline-PBS) were injected slowly into the intravitreal space. Special care was given to avoid hitting the lens. At the end of the procedure antibiotic ophthalmic ointment (Bacitracin Zinc Ointment; Fougera Pharmaceuticals Inc., Melville, NY, USA) was applied to prevent potential infection. Any eyes with cataract and/or hemorrhage were excluded from the analysis.

To permit direct activation of spinal nociceptive circuitry, animals received intrathecal delivery of the glutamate receptor agonist N-methyl-D-aspartate (NMDA) (M3262, Sigma Aldrich, St. Louis, MO) or the NK1 receptor agonist substance P (sP) (S6883, Sigma Aldrich, St. Louis, MO). Two days after BoNT-B or vehicle pre-treatment, anesthesia was induced and maintained with 2.5% isoflurane. Maintenance anesthesia was administered via a nosecone. Mice were then intrathecally (i.t.) injected between the L5 and L6 vertebrae with 5 μL of NMDA (0.25nM), sP (3.71 pM) or saline. A flick of the tail upon insertion of the needle was taken to indicate appropriate needle placement. Mice remained anesthetized and the spinal cords were harvested 5 min post-injection for western blot analysis of pAkt and pGluA1.