Administered and Endogenously Released Kappa Opioids Decrease Pilocarpine-Induced Seizures and Seizure-Induced Histopathology

The effects of kappa opioids on seizures and seizure-induced histopathology were investigated with the pilocarpine model of temporal lobe epilepsy. Rats treated with the kappa opioid receptor agonist U50488h before pilocarpine showed: 1) increased seizure latency; 2) decreased seizure duration; 3) decreased mossy fiber sprouting; and 4) increased hilar neuron survival when compared with rats pretreated with saline. Behavioral effects of U50488h were blocked by the kappa opioid receptor antagonist norbinaltorphimine (nBNI), whereas the changes caused by U50488h in the histological response to pilocarpine were not blocked by nBNI. Rats treated with nBNI before pilocarpine exhibited: 1) increased incidence of seizures; 2) increased mossy fiber sprouting; and 3) increased hilar neuron loss when compared with rats treated with pilocarpine alone. These changes suggest a protective role of endogenously released kappa opioids in this seizure model. The location of functional kappa opioid receptors in the rat dentate gyrus was documented electrophysiologically to enable correlation with kappa opioid effects on histopathology. The kappa selective agonist, U69593, reversibly decreased the amplitude of excitatory postsynaptic potentials in the middle molecular layer of the dentate gyrus from the ventral but not the more dorsal portion of the hippocampal formation. Thus, kappa opioids decreased the severity and incidence of behavioral seizures and secondarily decreased seizure-induced histopathology via the decreased incidence of seizures. Complex partial epilepsy of temporal lobe origin is one of the most common forms of epilepsy (Hauser and Kurland, 1975) and is often associated with histopathology termed Ammon’s horn sclerosis (Margerison and Corsellis, 1966). Ammon’s horn sclerosis is characterized by neuronal loss in the hilar, CA3 and CA1, but not CA2 regions of the hippocampal formation (Babb and Brown, 1987) as well as sprouting of the granule cell axons (mossy fibers) into the inner molecular layer of the dentate gyrus (Nadler et al., 1980; Sutula et al., 1989; Houser et al., 1990). Pharmacotherapy for TLE is generally unsatisfactory because approximately 40% of the patients with this disease are not responsive to currently available anticonvulsant medications (Elwes et al., 1984; Mattsen et al., 1985). Kappa opioids may represent one alternative treatment. Kappa opioids decrease excitatory neurotransmission in numerous brain regions (McFadzean et al., 1987; Moore et al., 1988; Wagner et al., 1992) and, in the guinea pig hippocampus, decrease excitatory transmission by modulating glutamate release from presynaptic terminals (Gannon and Terrian, 1991; Simmons et al., 1994). Kappa opioids have been reported to act as anticonvulsants in a variety of epilepsy models (see Tortella, 1988; Simmons and Chavkin, 1996) and specifically the kappa opioid receptor agonists, PD117302 and U69593, inhibit acute pilocarpine-induced seizures and neurotoxicity in mice (Przewlocka et al., 1994). Kappa opioids also are neuroprotective both in vivo (Hall and Pazara, 1988; Hayward et al., 1992; Mackay et al., 1993; Widmayer et al., 1994) and in vitro (DeCoster et al., 1994). In the present study, the pilocarpine model of TLE was used to investigate the anticonvulsant and neuroprotective effects of both administered and endogenously released kappa opioids. The pilocarpine model is a well established model of TLE in which a single high dose (300–400 mg/kg; Turski et al., 1989) of the cholinergic agonist, pilocarpine, produces behavioral and electroencephalographic seizures (Turski et al., 1983). This acute phase of seizure activity is followed by a chronic phase in which animals exhibit recurrent spontaneous seizures. Systemic administration of many anticonvulsant drugs used to treat human forms of epilepsy prevent acute pilocarpine-induced seizures in rats. The effectiveness of these drugs against the chronic recurrent sponReceived for publication May 19, 1997. 1 This work was supported by USPHS grant NS33898. 2 Present address: Dept. of Medicine (Neurology), Box 3676, Duke University Medical Center, Durham, NC 27710. ABBREVIATIONS: ANOVA, analysis of variance; fEPSPs, field excitatory postsynaptic potentials; LSD, least significant difference; nBNI, norbinaltorphimine; SE, status epilepticus; TLE, temporal lobe epilepsy. 0022-3565/98/2843-1147$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 3 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 284:1147–1155, 1998 1147 at A PE T Jornals on Jne 4, 2017 jpet.asjournals.org D ow nladed from taneous seizures has not been reported (Turski et al., 1989). Pilocarpine-induced seizures also lead to hippocampal pathology which mirrors human Ammon’s horn sclerosis, including cell loss and mossy fiber sprouting (see Mello et al., 1992). The aims of this study were 1) to replicate previous results in mice using an alternative kappa opioid receptor agonist, U50488h (Lahti et al., 1982; VonVoigtlander et al., 1983) and 2) to investigate the hypothesis that endogenous kappa opioids act as anticonvulsants and neuroprotectants against pilocarpine-induced seizures. Blockade of kappa opioid receptors with the selective antagonist, nBNI (Takemori et al., 1988a) was used to investigate the effects of endogenous kappa opioids on acute pilocarpine-induced seizures and histopathology. Furthermore, the location of functional kappa opioid receptors and the physiological effects of kappa opioid receptor activation in the rat dentate gyrus were documented electrophysiologically by the kappa selective agonist U69593 (Lahti et al., 1985). The location of functional kappa opioid receptors within the dentate gyrus was then correlated with histopathological changes. Materials and Methods Pilocarpine and opioid injections. Male Sprague-Dawley rats (110–150 g; Bantin and Kingman, Bellevue, WA) were injected with pilocarpine to induce chronic epilepsy (Turski et al., 1983) as described previously (Bausch and Chavkin, 1997). Rats were injected with methyl-scopolamine nitrate (1 mg/kg in saline i.p.; Sigma, St. Louis, MO) 30 min before pilocarpine hydrochloride injection (275– 375 mg/kg in saline i.p.; Sigma) to minimize the peripheral effects of pilocarpine (Baez et al., 1976, Turski et al., 1983). Control animals also received methyl-scopolamine, but were injected with saline instead of pilocarpine. Animals were observed for 2.5 hr and monitored for 6 to 8 hr after injection with pilocarpine. To reduce the mortality rate, all rats were administered diazepam (4 mg/kg i.p.) after 1 hr of SE, and every 2 hr as necessary thereafter to control seizures (Mello et al., 1993). All animals that received pilocarpine were given rat chow soaked in Gatorade and sucrose for 2 days after injection. In experiments investigating the effects of kappa opioids, U50488h (Research Biochemicals International, Natick, MA) was injected (10–20 mg/kg in saline s.c.) 45 min before pilocarpine injection. Although U69593 generally is used for selective activation of the kappa-1 type opioid receptor, the related benzeneacetamide analog, U50488h, was used for systemic treatment because it penetrates the blood brain barrier more readily (Bianchi, 1989). nBNI (Research Biochemicals International, Natick, MA) was injected (10 mg/kg in saline s.c.) 0.5, 2 or 18 hr before the pilocarpine injection. In some experiments in which nBNI was given 18 hr before pilocarpine, a second smaller dose of nBNI (0.4 mg/kg in saline s.c.) was administered 2 hr before pilocarpine injection. Kappa opioid injection protocols were derived from previous studies investigating the effects of U50488h and nBNI on physiological measures (Takemori et al., 1988b; Milanes et al., 1991; Leyton and Stewart, 1992; Veeranna and Bhargava, 1993). Pilocarpine was given at a dose of 325–375 mg/kg (Turski et al., 1983) in U50488h experiments, and it was given at a lower dose (275 mg/kg) in studies of endogenous kappa opioid action to avoid a ceiling effect. Rats in the ‘no pilocarpine’ control group were pretreated with saline (n 5 6), U50488h (n 5 6) or nBNI (n 5 6) and were then injected with methyl-scopolamine nitrate. Finally, all rats in this control group were injected with saline instead of pilocarpine. Data from all animals receiving ‘no pilocarpine’ were averaged since there were no significant differences in histological or behavioral responses. Histology. Rats were sacrificed by CO2 narcosis 4 weeks after treatment. The brains were removed, blocked and immersion fixed for 1 hr in 0.1% sodium sulfide followed by 2 to 3 days in 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Brains were cut into 40-mm transverse sections using a freezing sliding microtome, sections placed into 0.1 M phosphate buffer and mounted onto subbed glass slides. Mounted sections were then stained with cresyl violet or with neo-Timm stain (Holm and Geneser, 1991). For the neo-Timm stain, sections were postfixed through 95%, 70% and 50% ethanol, rehydrated in distilled water for 20 to 30 min, then dipped in 0.5% gelatin and allowed to dry overnight. Slides were developed in a solution of 0.11% silver lactate, 0.85% hydroquinone, 30% gum arabic colloid (all w/v) in 0.2 M citrate buffer for 1 to 1.5 hr and then rinsed, counterstained with Neutral Red, dehydrated, cleared and coverslipped. Images were collected with a Leitz Dialux 20 microscope for figures 2 and 3 or with a Nikon Diaphot microscope with Image 1 software for analysis of cresyl violet-stained sections. Histological data analysis. Sections for analysis were taken from horizontal stereotaxic coordinates B 25.60, IA 4.40 to B 25.32, IA 4.68 and B 27.34, IA 2.66 to B 27.10, IA 2.90 according to the atlas of Paxinos and Watson (1986). These regions correspond to the middle and ventral portions of the hippocampus, respectively. One section