Behavioral Characterization of Pentylenetetrazole-induced Seizures: Moving Beyond the Racine Scale

Summary

The protocol introduces an innovative method for analyzing and visualizing behaviors associated with pentylenetetrazole-induced seizures in mice. This approach extends beyond traditional Racine's scores, providing a more comprehensive evaluation of the diversity and temporal dynamics of ictal behaviors.

Abstract

Detailed behavioral characterization of seizures is essential in animal models of epilepsy. Although various protocols exist for inducing seizures, the Racine scale remains the most widely adopted method for its quantification. Initially developed to score the progressive recruitment of behaviors during electrical kindling of limbic structures, the scale has since become the standard for classifying seizures across models with varying etiologies.

However, careful examination of behavior following pentylenetetrazole (PTZ) administration, a commonly used seizure model, reveals that the Racine scale cannot capture the full spectrum of behavioral alterations induced by this GABAergic antagonist. After a single subcutaneous high-dose PTZ injection, mice first exhibit non-convulsive behaviors such as hypotonia, reduced ambulation, behavioral arrest, and tremors. These are succeeded by ictal behaviors, including spasms, myoclonic jerks, and limbic seizures, though only the latter is quantified by the Racine scale.

To overcome these limitations, a refined approach is employed that combines video tagging with animal tracking to distinguish spasms and myoclonus as distinct behavioral expressions from limbic seizures. These early behaviors may facilitate the recruitment of limbic structures, eventually triggering limbic seizures. The method is straightforward to implement and enables a more comprehensive assessment of PTZ-induced acute seizures. Its utility is further evident in the PTZ kindling protocol, in which repeated low doses of PTZ result in temporal changes in spasms, myoclonus, and limbic seizure severity. This approach holds potential for improving reproducibility in seizure and epilepsy research.

Introduction

Behavioral characterization of seizures is the most employed readout in studies of the pathophysiology of epilepsy and seizure activity, and it is widely used for the screening of new antiseizure medication^1,2. Most of the work published in the last 50 years used the progressive score system developed by Ronald J. Racine to describe behavioral seizure severity during amygdala and hippocampal kindling³. The Racine scale showed a strong correlation between behavior and electrophysiological paroxysmal activity in rats³ and cats⁴. It classifie....

Protocol

All experimental procedures were approved by the local ethical committee of the Federal University of Rio Grande do Norte (Protocol number 043/2022, License number 307.043/2022). Male C57BL/6 mice ranging from 3 to 4 months old were used in this protocol, with a recommended sample size of 6-10 animals according to established PTZ induction protocols¹⁶.

1. Preparation of the observational arena

1. Use a square or circular arena with at least 10 x 10 cm surface area and 30 cm of wall height. Ensure that the arena is well-illuminated. If necessary, use infrared lighting to guarantee saturated i....

Results

To evaluate the coding strategy and its ability to detail ictal behaviors during PTZ-induced seizures, three doses of PTZ were first tested and their effects on C57BL/6 mice observed (40, 60, and 80 mg/kg, N = 7, 5, and 5, respectively). Behavioral outcomes observed across three doses of PTZ revealed clear dose-dependent differences after a single injection. Animals treated with 40 mg/kg PTZ displayed spasms but no limbic seizures or myoclonus. In contrast, animals treated with 60 and 80 mg/kg consistently expressed spas.......

Discussion

This study employed a categorization of behaviors based on motor expressions to provide a more comprehensive framework for analyzing seizure dynamics. This approach enables quantification of key parameters, including latency, duration, and frequency of spasms, myoclonus, and limbic seizures. Results from weekly PTZ injections demonstrated that repeated exposure significantly alters the timing and frequency of limbic seizures, with a notable reduction in latency to the first spasms and limbic seizures, alongside an increa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Solution	Fresenius Kabi Brasil Ltda	74MK3168	Closed system for intravenous use; injectable solution, 250 mL.
Analytical Scale	Shimadzu	ATY224	Maximum capacity: 220 g, Minimum capacity: 10 mg, Readability: 0.1 mg. Serial No: 1311070096
Camera	Jortan	Jortan-8816AHD	IR CCD camera, NTSC system, 2.0 MP resolution, 3.6 mm lens, DC 12 V power supply.
Digital Lux Meter	Minipa	MLM-1011	Measures light intensity in lux units.
Hypodermic Needle	Descarpack	353101	Size 26.5 G.
Mice	-	-	Male C57BL/6 wild type mice. Two-three months old.
Pentylenetetrazole	Sigma-Aldrich (Merck)	P6500-25G	Powder, CNS stimulant. Purchased: 25 g. Storage: -25 °C.
Software: MouseLabTracker	Giuliano Vilela	-	Open-source software, reference paper: DOI: 10.1016/j.jneumeth.2006.04.005. Site: https://neuro.ufrn.br/softwares/mouselabtracker
Software: Solomon Coder	András Péter	-	Open-source software, v.19.08.02. Site:  https://solomon.andraspeter.com/
Thermo-Hygrometer	Hikari	HK-T240	Measures temperature and humidity.