Statistical parametric mapping reveals regional alterations in cannabinoid CB1 receptor distribution and G-protein activation in the 3D reconstructed epileptic rat brain.

A large percentage of patients with epilepsy are refractory to conventional anticonvulsant treatment; therefore, epilepsy is a neurologic condition that requires novel insights for treatment and seizure prevention (Hauser, 1990; Annegers et al., 1996; Herman, 2002). Cannabinoids are known to have CB1-receptor–dependent anticonvulsant effects in both in vitro and in vivo seizure models (Wallace et al., 2001, 2002; Marsicano et al., 2003; Blair et al., 2006; Gholizadeh et al., 2007; Armstrong et al., 2009), and the endogenous cannabinoid system has been shown to play a role in modulating epileptic seizure frequency and duration (Wallace et al., 2003). Previously our laboratory demonstrated that following pilocarpine-induced status epilepticus (SE), animals with acquired epilepsy (AE) exhibit a long-term redistribution of CB1 receptors in the hippocampus, including layer-specific alterations in CB1-receptor immunoreactivity (IR) and binding sites accompanied by changes in CB1-receptor–mediated G-protein activity (Falenski et al., 2007). However, the regional whole-brain extent of CB1-receptor adaptations in this model has not been reported. The CB1 receptor is among the most abundant G-protein–coupled receptors in brain, with a distribution consistent with behavioral effects of cannabinoid administration (Herkenham et al., 1991; Jansen et al., 1992; Tsou et al., 1998). Autoradiographic techniques such as [3H] ligand binding and agonist-stimulated [35S]GTPγS autoradiography have been used to characterize regional changes in CB1-receptor binding and G-protein activation in a number of paradigms (Sim et al., 1996; Berrendero et al., 1998; Dean et al., 2001; Sim-Selley & Martin, 2002). Conventionally, these data are analyzed using a region of interest (ROI) approach. However, ROI analyses are normally hypothesis driven, and effects of interest outside boundaries of predefined ROIs could be missed. To address this limitation, established whole-brain based approaches, such as statistical parametric mapping (SPM), have allowed for localization of significant effects of interest in an unbiased manner (Friston et al., 1990, 1995b). Previously, SPM has been utilized in autoradiographic data sets mapping cerebral blood flow (Nguyen et al., 2004; Dubois et al., 2008) and was recently adapted to study CB1-receptor–mediated G-protein activity in three-dimensional (3D) reconstructed mouse brain (Nguyen et al., 2010, 2012). In this study, the pilocarpine model was used to prepare rats with AE that were evaluated 1 year post-SE. Control and epileptic brains were evaluated using [3H]WIN55,212 (WIN) and WIN-stimulated [35S]GTPγS autoradiography to determine changes in receptor binding and G-protein activation, respectively. SPM was used to identify significant changes in CB1-receptor binding and G-protein activation in reconstructed brain images, and subsequently confirmed by ROI analysis. CB1 receptor expression was evaluated using immunohistochemistry (IHC). Examination of the plasticity of the CB1 receptor at the whole-brain level in this model is critical for understanding the overall role that CB1 receptors and the endogenous cannabinoid system plays in AE.