Pilocarpine hydrochloride

To induce epilepsy, rats were injected with pilocarpine hydrochloride (380 mg/kg, i.p.; Sigma-Aldrich, UK), a muscarinic cholinergic agonist. In order to prevent the peripheral cholinergic effects of pilocarpine, rats were pre-treated with scopolamine methyl nitrate, as a cholinergic antagonist (1 mg/kg, i.p.; Sigma-Aldrich, UK) 30 minutes before the injection of a single dose of pilocarpine hydrochloride. Then the treated rats were monitored for a period of 3 to 4 hours after injection of pilocarpine, and the seizure severity was graded according to Racine’s criteria[33 (link)]. Only rats were included in the present work if they exhibited stage 4 or 5 seizure score. In order to terminate the sustained seizure that lasted for 3 hours, diazepam (7 mg/kg, i.p.) was injected, and an additional dose of diazepam (3 mg/kg, i.p.) was administered every 2 hours, if needed, and then the animals were cared and fed with Hartmann’s solution till recovery[34 (link)]. Two weeks after pilocarpine injection, the occurrence of spontaneous seizures was recorded during chronic phase using daily video monitoring 8 hours/day for two weeks.

Five days after behavioral testing, animals from both groups (Anx and Non-Anx) were injected with pilocarpine for SE induction, as described elsewhere (Mello and Covolan, 1996 (link)). Briefly, scopolamine methyl bromide (1 mg/kg, i.p., Sigma) was administered, 30 min prior to pilocarpine hydrochloride (320 mg/kg, i.p., Merck), to reduce peripheral effects. Some animals presented a generalized convulsive (stage 4 or 5) seizure that turned into continuous seizures in the form of limbic motor seizures with intense salivation, rearing, upper extremity clonus and falling, lasting up to 90–150 min, which characterized SE (Mello and Covolan, 1996 (link)). Thionembutal (25 mg/kg, i.p., Cristalia, Brazil) was administered 90 min after SE onset to suppress or attenuate SE. On the following 2–3 days after SE, animals were fed with a mixture of sweetened milk and Gatorade at least three times/day. During this 2–3 day period, fruit slices (bananas and oranges) were placed on the floor of the cage. Animals that received pilocarpine but did not progress into continuous limbic seizure activity (NoSE), also received thionembutal. The control group was injected with an equivalent amount of saline followed by injection of sodium thionembutal 90 min later.

We purchased pilocarpine hydrochloride, scopolamine hydrobromide, and scopolamine
methylbromide from Merck (Darmstadt, Germany). We dissolved all drugs in a
sodium chloride solution (0.9%). We applied all drugs intraperitoneally at the
following concentrations: pilocarpine (1 mg/kg), scopolamine (1 mg/kg) and
methylscopolamine (1 mg/kg). Cholinergic antagonists were applied 25 min before
the fMRI experiment started. To effectively reduce peripheral effects of
pilocarpine on hemodynamic parameters, such as mean arterial blood pressure,^{21 (link)}
this drug was always applied in combination with a cholinergic antagonist
(methylscopolamine or scopolamine); i.e., in these experiments methylscopolamine
was applied 25 min before the start of the fMRI measurement and pilocarpine
5 min later. Methylscopolamine (1 mg/kg) alone increased heart rate but had as
described previously no effect on blood pressure, core temperature, and motor activity.^{22 (link)}