Elsevier

Experimental Neurology

Volume 219, Issue 1, September 2009, Pages 284-297
Experimental Neurology

Behavioral and cognitive alterations, spontaneous seizures, and neuropathology developing after a pilocarpine-induced status epilepticus in C57BL/6 mice

https://doi.org/10.1016/j.expneurol.2009.05.035Get rights and content

Abstract

Many patients with epilepsy suffer from psychiatric comorbidities including depression, anxiety, psychotic disorders, cognitive, and personality changes, but the mechanisms underlying the association between epilepsy and psychopathology are only incompletely understood. Animal models of epilepsy, such as the pilocarpine model of acquired temporal lobe epilepsy (TLE), are useful to study the relationship between epilepsy and behavioral dysfunctions. In the present study, we examined behavioral and cognitive alterations, spontaneous seizures, and neuropathology developing after a pilocarpine-induced status epilepticus in the C57BL/6 (B6) inbred strain of mice, which is commonly used as background strain for genetically modified mice. For this study, we used the same pilocarpine ramping-up dosing protocol and behavioral test battery than in a previous study in NMRI mice, thus allowing direct comparison between these two mouse strains. All B6 mice that survived SE developed epilepsy with spontaneous recurrent seizures. Epileptic B6 mice exhibited significant increases of anxiety-related behavior in the open field and light–dark box, increased locomotor activity in the open field, elevated plus maze, hole board, and novel object exploration tests, and decreased immobility in the forced swimming and tail suspension tests. Furthermore, spatial learning and memory were severely impaired in the Morris water maze, although hippocampal damage was much less severe than previously determined in NMRI mice. B6 mice in which pilocarpine did not induce SE but only single seizures did not exhibit any detectable neurodegeneration, but differed behaviorally from sham controls in several tests of the test battery used. Our data indicate that the pilocarpine model of TLE in B6 mice is ideally suited to study the neurobiological mechanisms underlying the association between seizures, brain damage and psychopathology.

Introduction

In recent years, the pilocarpine model of temporal lobe epilepsy (TLE) has become the most popular and widely used rodent model of this common and difficult-to-treat type of epilepsy (Cavalheiro et al., 2006, Curia et al., 2008). In this model, the cholinomimetic convulsant pilocarpine is used to induce a status epilepticus (SE), which is followed by hippocampal damage and development of spontaneous recurrent seizures (SRS). The model has initially been described in rats, but is increasingly used in mice (Cavalheiro et al., 2006, Curia et al., 2008). Surprisingly, although the acute convulsive and neurodegenerative effects of pilocarpine in mice were first reported in 1984 (Turski et al., 1984) and SRS following a pilocarpine-induced SE in mice in 1996 (Cavalheiro et al., 1996), to our knowledge the long-term behavioral and cognitive alterations occurring in this model have not been fully characterized in this species since then. In patients, TLE is often associated with behavioral alterations, such as depression, anxiety and psychosis, and impaired cognitive performance (Boro and Haut, 2003, Swinkels et al., 2005, Cornaggia et al., 2006, Hoppe et al., 2007, Marcangelo and Ovsiew, 2007). Memory impairment in patients with TLE is thought to be a consequence of the hippocampal alterations that are associated with TLE, whereas the neurobiological mechanisms of the relationship between TLE and psychiatric disorders are poorly understood (Devinsky, 2003, Motamedi and Meador, 2003, Swinkels et al., 2005, Hoppe et al., 2007). Animal models of epilepsy may help to enhance our understanding of causal mechanisms underlying the association between epilepsy and behavioral abnormalities (Post, 2004, Majak and Pitkanen, 2004, Heinrichs and Seyfried, 2006).

In the NMRI outbred strain of mice, we have recently reported that pilocarpine-induced epilepsy is associated with significant increases of anxiety-related behavior and impairment of learning and memory, thus indicating that pilocarpine-treated mice reflect several of the behavioral and cognitive disturbances that are associated with TLE in humans (Gröticke et al., 2007). In the present study, we examined development of SRS, neurodegeneration and behavioral and cognitive alterations after a pilocarpine-induced SE in the C57BL/6 (B6) inbred strain of mice, using the same pilocarpine dosing protocol and behavioral test battery than in our previous study in NMRI mice (Gröticke et al., 2007). Pilocarpine has previously been used to induce SE and subsequent SRS in B6 mice (Shibley and Smith, 2002, Borges et al., 2003, Peng et al., 2004, Houser et al., 2008), but, to our knowledge, behavioral alterations developing after SE have not been described in this strain, except for one study in which pilocarpine-treated B6 mice were evaluated in an open field and water maze shortly after SE (Mohajeri et al., 2004). Our goal was to directly compare the various long-term consequences of pilocarpine-induced SE in B6 mice with those previously determined by us in NMRI mice, thus allowing to determine the impact of genetic background in this model of TLE.

Section snippets

Animals

Mice (C57BL/6NCrl) were obtained from Charles River (Sulzfeld, Germany) at an age of about 5–6 weeks. Female mice were used to allow comparison with previous studies of our group on pilocarpine in mice (Gröticke et al., 2007, Müller et al., in press) and to ease housing in groups for the long period needed to perform experiments in models of epilepsy. After arriving at our Department, animals were housed in groups under controlled conditions (temperature: 21 ± 0.5 °C; humidity: 55 ± 5%), under a

Induction of SE by pilocarpine and development of spontaneous seizures after SE

By the ramping-up protocol used in a total of 33 B6 mice, 14 animals developed and survived SE, one did not exhibit an SE, and 18 animals died. In order to allow group comparisons between pilocarpine-treated mice with and without SE, we added four B6 mice that did not develop SE after pilocarpine, but were injected and tested at a different time than the total of 33 mice. As in humans, SE was defined by continuous or intermittent seizures without full recovery between seizures (Lowenstein, 1999

Discussion

The B6 mouse is one of the most widely used inbred mouse strains in biomedical research, especially in behavioral studies. Furthermore, B6 mice are commonly used as background strain for genetically modified mice. In studies on induction of seizures by various convulsive agents, including pilocarpine, B6 mice were generally reported to exhibit a lower sensitivity than various other inbred or outbred mouse strains (Engstrom and Woodbury, 1988, Kosobud and Crabbe, 1990, Schauwecker and Steward,

Acknowledgments

We thank Dr. Larry C. Schmued (Division of Neurotoxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA) for advice on the interpretation of FJC-labeled cells, Dr. Katrin Hoffmann for some preliminary experiments with pilocarpine in B6 mice, and Tobias Kalair for skillful technical assistance. The study was supported by a PhD-scholarship (to C.J.M.) from the Konrad-Adenauer-Stiftung (Sankt Augustin, Germany). Ina Gröticke acknowledges the

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