Pentylenetetrazol Seizure Threshold Test (mouse, rat)
The pentylenetetrazol (PTZ) or Metrazol test detects the ability of a test compound to raise the chemoconvulsant-induced seizure threshold of an animal and thus protect it from exhibiting a clonic, forebrain seizure. In the subcutaneous (SC) threshold test, following the administration of a test compound PTZ at a dose of 85 mg/kg for CF-1 mice, 45 mg/kg for C57BL/6 mice, and 68 mg/kg for Sprague Dawley CD rats is injected into a loose fold of skin in the midline of the neck at a predetermined timepoint based on the time of peak effect (TPE) of the test drug. The animals are placed in isolation cages to minimize stress and observed for the next 30 min for the presence or absence of a seizure. An episode of approximately 3 to 5 seconds of clonic spasms of the fore and/or hind limbs, jaws, or vibrissae is taken as the endpoint. Animals not displaying fore and/or hind limb clonus, jaw chomping, or vibrissae twitching are considered protected. Initial qualitative screen for anticonvulsant activity in the SC PTZ test is performed with N = 4 male mice or rats/dose/time point. Three doses and two timepoints based on supporting data are selected to examine the efficacy of the test compound. Quantification of the effective dose that confers protection in 50% of animals treated with the compound (ED50) is conducted at the TPE and is described separately.
The intravenous (IV) PTZ seizure threshold test is not routinely performed but is useful for evaluating whether a compound increases or decreases the threshold at which a seizure can be induced by PTZ. The timed IV infusion of PTZ to mice is used as a chemoconvulsant test to differentiate those compounds that lower seizure threshold and, as such, may be proconvulsant, from those compounds that elevate seizure threshold, and are thus anticonvulsant. Male CF1 mice (n=10 per dose level) are injected 2 minutes apart with either the vehicle or one of two doses (ED50 and TD50) of the test compound previously determined to be effective in screening seizure tests and motor impairment assessment. At the previously determined TPE, 0.5% PTZ solution is infused in a cannulated lateral tail vein of a mouse at a constant rate of 0.34 ml/min. The time in seconds from the start of the infusion to the appearance of the "first twitch" and the onset of sustained clonus, or 90 seconds if the time has passed with no seizure is recorded. An increase in mg/kg of PTZ to first twitch or to clonus indicates the test compound increases seizure threshold and is potentially an anticonvulsant, whereas a decrease indicates that the test substance decreases seizure threshold and may have proconvulsant activity.