Background

The mouse pilocarpine model of TLE is an experimental paradigm that has been widely used as an animal model of human mTLE. This model has been used for testing long-term efficacy of novel therapies for suppressing SRS and improving cognitive and neuropathological changes associated with severe mTLE. This rodent model of TLE recapitulates many of the neuropathological hallmarks in mTLE, including severe seizures, loss of GABAergic interneurons in the dentate gyrus, Ammon’s Horn sclerosis, and cognitive impairments (Swartz et al., 2006). Although some patients with mTLE can be treated successfully anti-convulsant medications, about one-third experience intractable pharmaco-resistant seizures and surgical removal of the seizure foci are required. If the foci are located in the mesial temporal lobes, surgical removal may not be feasible due to the role of the hippocampus in memory formation and consolidation. For these patient populations, novel treatments are needed. The cost-effective pilocarpine model of TLE in rodents provides an experimental system for testing novel therapies because a high percentage of rodents develop SRS that can be monitored by video electroencephalography (v-EEG) for weeks or months. Novel treatments that suppress seizures can also be studied for their effects on the neuropathological changes observed in this model, including mossy fiber sprouting (Henderson et al., 2014), dispersion of the granule cell layer (Scharfman et al., 2000), and aberrant neurogenesis (Parent et al., 1997).

Pilocarpine hydrochloride (referred to as pilocarpine) is a drug that directly stimulates muscarinic cholinergic receptors in the central nervous system and in the parasympathetic branch of the peripheral nervous system. As a parasympathomimetic drug, pilocarpine induces exocrine gland secretion and stimulates smooth muscle contractions in the gut and secretory glands. It is also a chemoconvulsant that is used to elicit limbic seizures in rodents. Pilocarpine is typically administered by subcutaneous (Walter et al., 2007) or intraperitoneal (i.p.) injections in rodents to induce a condition of continuous generalized seizures, called Status Epilepticus (SE). After the mice develop SE in this model, the experimenter terminates the seizures by injecting a sedative dose of benzodiazepine, a class of drugs that acts on gamma-aminobutyric acid-A (GABA-A) receptors in the brain. Typically, rodents that have experienced one or more hours of SE, will develop TLE with high-frequency interictal spike, wave activity, and SRS (Mello et al., 1993; Wozny et al., 2003; Knopp et al., 2005; Henderson et al., 2014).

In a comprehensive study of the multiple factors contributing to the development of SRS in the rodent pilocarpine model, the strongest correlations were found between dentate gyrus GABAergic neuron loss and seizure frequency (i.e., disease severity) (Buckmaster et al., 2017). Many human TLE patients and rodents subjected to pilocarpine-induce SE show loss of somatostatin-expressing GABAergic interneurons in the hippocampus (Robbins et al., 1991; Borges et al., 2003; Hoffman et al., 2016). In the mouse pilocarpine model, GABAergic interneurons in the hilus of the dentate gyrus and CA1 that co-express somatostatin and Striatal Enriched Tyrosine Phosphatase (STEP) were found to be highly vulnerable to SE (Buckmaster and Jongen-Relo, 1999; Choi et al., 2007; Zhang et al., 2009). Associated with the loss of GABAergic interneurons, studies reported reduced synaptic inhibition (Kobayashi and Buckmaster, 2003), despite the fact that many of the GABAergic interneurons in the dentate gyrus and CA1 that survive after SE sprout additional inhibitory projections (Wittner et al., 2002; Bausch, 2005; Zhang et al., 2009; Thind et al., 2010). Loss of both somatic and distal dendritic GABAergic inhibition to granule cells occurs shortly after SE, but precedes the development of SRS, suggesting that loss of GABAergic inhibition onto granule cells in the dentate gyrus is an important predictor of whether TLE will develop (Kobayashi and Buckmaster, 2003).

Because of the strong link between loss of GABAergic inhibition and development of TLE, we and other groups have tested whether transplantation of GABAergic progenitors, from either the embryonic mouse forebrain or generated in vitro from human pluripotent stem cells, suppresses pilocarpine-induced TLE (Maisano et al., 2012; Henderson et al., 2014; Anderson et al., 2018). Additionally, recent work shows that this model can be used in combination with molecular and electrophysiological approaches, such as retroviral labeling and optogenetics, to evaluate cellular mechanisms responsible for seizure suppression following transplantation (Gupta et al., 2019). The methods detailed in this protocol for induction of SE in both male and female rodents can be adapted to different inbred strains of mice with low mortality; a high incidence of the mice that experience SE will subsequently develop SRS and the typical histopathological hallmarks of TLE (Walter et al., 2007; Henderson et al., 2014; Anderson et al., 2018; Gupta et al., 2019).

In the following protocol, we provide the detailed methods for the successful induction of SE with multiple subthreshold doses of pilocarpine in inbred strains of mice. This method builds upon number of prior studies. In a comparison of 8 inbred strains of mice, Schauwecker and colleagues found significant differences in the susceptibility to pilocarpine, mortality and neuropathological changes after SE (Schauwecker, 2012). Doses ranging between 300 and 400 mg/kg were reported to induce SE successfully. Higher single doses of pilocarpine were correlated with more successful induction of SE but also increased mortality (Turski et al., 1984; Cavalheiro et al., 1991; Curia et al., 2008; Buckmaster and Haney, 2012; Kelly and Coulter, 2017). In contrast, multiple subthreshold doses of pilocarpine were effective for inducing SE and SRS, with much lower mortality rates (Glien et al., 2001; Groticke et al., 2007).