Inverse relationship of cannabimimetic (R+)WIN 55, 212 on behavior and seizure threshold during the juvenile period

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Abstract

Cannabinoids have anti-convulsant effects in both in vivo and in vitro models of status epilepticus. Since the development of spontaneous seizures and neuronal vulnerability are age-dependent, we hypothesized that the anti-convulsant effects of cannabimimetics are also age-dependent. We administered a single injection of varied doses of (R+)WIN 55,212 (0.5, 1, 5 mg/kg) to postnatal (P) day 20 rats 90 min prior to induction of kainate (KA)-induced status epilepticus. The highest dose of (R+)WIN 55,212 (5 mg/kg) resulted in rapid onset of behavioral stupor, loss of balance, stiffening and immobility while standing on hind legs or laying flat in prone position; lower doses had minimal or no behavioral effect. After KA administration, seizure scores and electroencephalography (EEG) recordings were inversely related to (R+)WIN 55,212 dosage whereby higher doses were associated with high seizures scores and synchronous epileptiform activity and low doses with low seizure scores and diminished spiking in the EEG. Immunohistochemistry revealed a dose-dependent reduction in CB1 receptor expression with increasing concentrations of (R+)WIN 55,212 in presence or absence of KA seizures. Nissl and NeuN staining showed hippocampal injury was attenuated only when seizures were mild following low doses of WIN 55,212 (0.5, 1 mg/kg), consistent with the level of CB1 expression. Since low doses abolished seizures without psychotropic side-effects further study may facilitate a groundbreaking cannabamimetic therapeutic strategy to treat early-life seizures. Higher doses had adverse effects on behavior and failed to prevent seizures and protect CA1 neurons possibly due to inactivation or loss of CB1 receptors.

Highlights

► Low doses of WIN 55,212 prevent kainate seizures in juvenile rats. ► Moderate doses of WIN 55,212 induce catatonic behaviors and pro-convulsant effects. ► Spatiotemporal changes in CB1R expression may regulate the seizure threshold.

Introduction

Marijuana usage is highest during adolescence when the brain is still developing which may subsequently lead to beneficial and non-beneficial effects. For instance, the active psychotropic constituent of marijuana, ∆9-tetrahydrocannabinol, was reported to impair short-term memory and learning, cause neuronal cell death of the hippocampus after chronic exposures, and increase the risk of developing psychotic disorders with maturity (Scallet, 1991, Lichtman and Martin, 1996, Chan et al., 1998, Pattij et al., 2008, Ranganathan and D'Souza, 2006, Bossong and Niesink, 2010). In addition, retrospective cohort studies of women smoking one or more marijuana cigarettes a day during the third trimester of pregnancy documented prolonged startle responses, disturbances in sleep cycling, and increased motility in the offspring (Fried, 1982, Scher et al., 1988). Experimentally, 15 days of daily prenatal treatment of the synthetic cannabimimetic, [(R+)WIN 55,212 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-napthylenylcarbonyl)-6H-pyrrolol[3,2,1-1]quinolin-6-one] to pregnant rats (0.5 mg/kg) caused in the offspring transient impairment of a number of cognitive functions as well as altered glutamatergic transmission (Ferraro et al., 2009). On the other hand, cannabinoids also have a long history of medicinal effects. They have been used in various forms to treat a variety of disorders such as cancer, multiple sclerosis, spinal cord injuries, glaucoma, ischemia, and epilepsy due to their analgesic, appetite stimulant, antiemetic, antioxidant, anti-inflammatory and anticonvulsant properties (reviews: Consroe, 1998, Hampson et al., 2003, Amar, 2006). Interestingly, cannabinoids have also been proposed to improve memory and cognition that may be useful to treat Alzheimer's disease (Grotenhermen, 2005).

In experimental epilepsy models in adult rats or in primary hippocampal cultures, synthetic and endogenous cannabinoids were shown to attenuate neuronal excitation and epileptic activity (Shen et al., 1996, Wallace et al., 2003, Blair et al., 2006, Deshpande et al., 2006, Deshpande et al., 2007). Accordingly, stimulation of CB1 receptors with endocannabinoids, anandamide or 2-arachidonylglycerol(2-AG), attenuates epileptic seizures and the associated neuronal cell loss (Deshpande et al., 2006, Deshpande et al., 2007). Anticonvulsant effects in a number of structures were attributed to reducing presynaptic release of glutamate via activation of presynaptic G protein-coupled cannabinoid type-1 (CB1) receptors, a negative feedback mechanism (Shen and Thayer, 1999, Ameri and Simmet, 2000, Wallace et al., 2001, Wallace et al., 2002, Freund et al., 2003, Marsicano et al., 2003, Blair et al., 2006, Deshpande et al., 2007, Haj-Dahmane and Shen, 2009, Zarrindast et al., 2009).

In adult rats, the expression of the CB1 receptor is ubiquitously expressed throughout the hippocampus, frontal cortex, basal ganglia and cerebellum, regions responsible for seizure susceptibility, higher cognitive function, and motor coordination (Mailleux and Vanderhaeghen, 1992, Köfalvi et al., 2005, Mackie, 2005). In early life, robust expression of CB1 receptors also occurs and these receptors were described to play critical roles in normal brain development and maturation (Harkany et al., 2007). However, there is currently a paucity of research on the anticonvulsant effect of cannabinoids in development, particularly during the adolescent period when the brain first becomes vulnerable to seizure-induced brain damage (Albala et al., 1984, Nitecka et al., 1984: Holmes and Thompson, 1988, Sankar et al., 1999, Liu et al., 2006). One study showed WIN 55,212 had anticonvulsant activity in young mice (20–28 days old) treated with pilocarpine (Wallace et al., 2001, Wallace et al., 2002). In immature rat pups (P10), another study showed seizures produce long-lasting delayed increases in CB1 receptor expression within the hippocampus as determined by Western blotting (Schuchmann et al., 2006). In contrast, increases and decreases in expression have been observed in adults depending on the time and area examined (Falenski et al., 2007, Falenski et al., 2009). We hypothesized that the cannabimimetic (+ R)WIN 55,212 would have anticonvulsant activity and protect the juvenile brain from kainate (KA)-induced seizures as observed in adults but that these effects may be age and dose-dependent which may be critical to potential clinical outcomes. Since synthetically derived cannabinoids or endogenous up-regulation of endocannabinoids in humans with epilepsy have attractive therapeutic potential, we tested various doses of (+ R)WIN 55,212 on the seizure threshold, injury, and CB1 receptor expression distribution of the hippocampus at the end of the 3rd postnatal week of development in a juvenile rat model of status epilepticus.

Section snippets

Drug administration of CB1 receptor agonist R(+)WIN 55,212

Male Sprague–Dawley rats from postnatal (P) day P20 (40–50 g) were used in accordance with NIH guidelines. Animals were housed in single cages with their lactating mother until sacrifice (P23), given food and water ad libitum, and kept on a 12-h light/dark cycle at room temperature (55% humidity) in our own accredited animal facility. Animals were divided into control and experimental groups. A single injection of (R+)WIN 55,212 (0.5, 1 or 5 mg/kg, i.p., n = 10, n = 7, n = 15, respectively) (Tocris,

Dose dependent behavior induced by cannabinoid

In order to investigate whether a selective CB1 agonist is neuroprotective and an effective anticonvulsant against KA seizures in the juvenile period, (R+)WIN 55,212 was administered to P20 rats at three doses (0.5, 1 and 5 m g/kg) 90 min before induction of seizures with KA. Based on animals studies conducted in adult rats or juvenile mice (Wallace et al., 2002, Wallace et al., 2003) no significant behavioral changes were expected, however, pronounced behavioral manifestations were observed in

Discussion

Previously, we have illustrated age-dependent differences in gene and protein expression of glutamatergic systems as a result of status epilepticus supporting that seizures have different effects at different ages (Friedman, 2006). The present study tested the effects of the exogenous cannabinoid agonist, (R+)WIN 55,212, on behavior, seizure threshold, and CB1 distribution during a critical stage in development, an age when the brain first becomes sensitive to seizure-induced hippocampal

Acknowledgements

We would like to thank the Neuroscience Department and Academic Fellowship for providing funds to conduct the experiments. We also would like to thank two OMSIII scholars, Jared S Honigman and Kristin M DiGregorio for their technical assistance with the Western.

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