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Summary
Purpose: Several results support the conclusion that the cannabinoid system has a role in generation and cessation of epileptic seizures. The aim of this study was to evaluate the effects of intracerebroventricular AM-251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1-receptor antagonist, and ACEA (arachidonyl-2-chloroethylamide), a CB1-receptor agonist, on penicillin-induced epileptiform activity in rats.
Methods: In the first set of experiments, 30 min after penicillin injection, AM-251, at doses of 0.125, 0.25, 0.5, and 1 μg, was administered intracerebroventricularly (i.c.v.). In the second set of experiments, 30 min after penicillin injection, ACEA, at doses of 2.5, 5, 7.5, and 15 μg (i.c.v.), was administered. In the third set of experiments, AM-251, at doses of 0.125 and 0.25 μg (i.c.v.), was administered 10 min before ACEA (7.5 μg, i.c.v.) injection.
Results: ACEA, at a dose of 7.5 μg, significantly decreased the frequency of penicillin-induced epileptiform activity without changing the amplitude. ACEA, at doses of 2.5, 5, and 15 μg, had no impact on either frequency or amplitude of epileptiform activity. AM-251, at doses of 0.25 and 0.50 μg, significantly increased the frequency of epileptiform activity. AM-251, at a dose of 0.25 μg (i.c.v.), was the most effective in changing the frequency of penicillin-induced epileptiform activity, and it also caused status epilepticus—like activity. AM-251, at doses of 0.125 and 0.25 μg, 10 min before ACEA (7.5 μg), reversed the anticonvulsant action of ACEA.
Discussion: The results of the present study provide electrophysiologic evidence for the role of CB1 receptors in regulating the frequency of epileptiform activity in the model of penicillin-induced epilepsy. To elucidate the precise mechanism of cannabinoid action in the brain during seizure, more advanced electrophysiologic and neurochemical studies are required.
All types of epilepsy are recurrent, unprovoked seizures caused by an uncontrolled electrical discharge from nerve cells in selective regions of the brain (Dichter, 1994). Epileptogenic processes have been associated with imbalance between excitatory and inhibitory control systems in the brain (Brailowsky & Garcia, 1999).
There has been increasing interest in the use of the cannabinoids in many disorders, including epilepsy (Gordon & Devinsky, 2001). An abundant series of the cannabinoid analogs has been tested in experimental models of seizure activity (in vivo and in vitro) (Wallace et al., 2001, 2002; Blair et al., 2006; Deshpande et al., 2007a). It has been demonstrated that the endocannabinoid system plays an important role in regulating seizure activity in the brain (Wallace et al., 2003; Luszczki et al., 2006; Deshpande et al., 2007a). Wallace et al. (2001) demonstrated that the cannabinoid (Δ9-tetrahydrocannabinol) and cannabimimetic (WIN 55,212-2) compounds are anticonvulsant in the maximal electroshock model of epilepsy. They also suggested that this cannabinoid anticonvulsant effect was cannabinoid CB1-receptor—dependent (Wallace et al., 2001). WIN 55,212-2 was also shown to totally suppress the spontaneous recurrent epileptiform discharges via CB1-receptor activation in the models of pilocarpine, neuronal culture models of acquired epilepsy, and status epilepticus (Wallace et al., 2003; Blair et al., 2006). In addition, activation of CB1 receptors by WIN 55212-2 significantly suppressed both the frequency and amplitude of spontaneous inhibitory synaptic currents to about 50% of control (Nakatsuka et al., 2003). The suppressive effects were completely abolished in the presence of the CB1-receptor antagonist, AM-251 (Nakatsuka et al., 2003). Deshpande et al. (2007a) reported that the CB1-receptor antagonist AM-251 disrupts the endocannabinoid tone by blocking CB1-receptor activation, which caused development of status epilepticus in populations of epileptic neurons in the hippocampal neuronal culture model. They also noted that the induction of status epilepticus—like activity by CB1-receptor antagonist (AM-251) was reversible and could be overcome by higher concentrations of CB1-receptor agonists (WIN 55,212-2) (Deshpande et al., 2007a). Conversely, it has been shown recently that AM-251 decreased the spike firing, suggesting an anticonvulsant role in the piriform cortical model of epilepsy in rat (Dennis et al., 2008; Ma et al., 2008). Luszczki et al. (2006) used CB1-receptor agonist arachidonyl-2-chloroethylamide (ACEA), which exhibits greater than 1,400-fold preferential affinity for CB1 over CB2 (Hillard et al., 1999), in the mouse maximal electroshock-induced seizure model. They reported that the systemic administration of ACEA, at doses of 1.25 and 2.5 mg/kg, increased the threshold by 4% and 9%, whereas ACEA, at doses of 5 and 7.5 mg/kg, produced a 63% and 123% increase in the electroconvulsive threshold in mice (Luszczki et al., 2006). In addition, the systemic administration of ACEA, at a dose of 5 mg/kg, antagonized cocaine-induced convulsive seizures (Hayase et al., 2001). However, Moreira et al. (2007) reported that the systemic administration of CB1-receptor antagonist has yielded contradictory data, with the anxiogenic (Rodgers et al., 2005; Patel & Hillard, 2006) and anxiolytic activity (Griebel et al., 2005). Moreira et al. (2007) suggested that the local injections of CB1-receptor agonist and antagonist may help to clarify this controversy. Therefore, we decided for the first time, to investigate the effects of intracerebroventricular (i.c.v.) administration of ACEA, a CB1-receptor agonist, and AM-251, a CB1-receptor antagonist, on penicillin-induced epileptiform activity in rats. We used penicillin to induce epileptiform activity in rats in the present study, which is a widely used method for inducing epileptiform activity by applying penicillin to the cerebral cortex (Holmes et al., 1987). The application of penicillin to the neocortex results in synchronous discharge of neurons which, bears an electrophysiologic resemblance to human focal interictal epileptic discharges (Purpura et al., 1972).
Materials and Methods
Animals
Adult male rats weighing 220—260 g (Ondokuz Mayis University of Turkey) were used throughout this study after at least 1 week of acclimatization. All described procedures were approved by the local ethics committee. Animals were housed in groups of 3—4 and were allowed free access to food and water, except for the short time that the animals were removed from their cages for the experiments. All animals were kept in a temperature controlled (22 ± 1°C) environment on a 12-h light/dark cycle. Rats were assigned to the following experiments and groups: intracortical (i.c.) delivery of (1) 2.5 μl artificial cerebrospinal fluid [aCSF containing (mm): NaCl, 124; KCl, 5; KH2PO4, 1.2; CaCl2, 2.4; MgSO4, 1.3; NaHCO3, 26; glucose, 10; HEPES, 10; pH 7.4 when saturated with 95% O2 and 5% CO2] (i.c.); (2) 500 units penicillin (2.5 μl, i.c.); (3) 500 units penicillin (2.5 μl, i.c.) + 2.5 μg ACEA (i.c.v.); (4) 500 units penicillin (2.5 μl, i.c.) + 5 μg ACEA (i.c.v.); (5) 500 units penicillin (2.5 μl, i.c.) + 7.5 μg ACEA (i.c.v.); (6) 500 units penicillin (2.5 μl, i.c.) + 15 μg ACEA (i.c.v.); (7) 500 units penicillin (2.5 μl, i.c.) + 0.125 μg AM-251 (i.c.v.); (8) 500 units penicillin (2.5 μl, i.c.) + 0.25 μg AM-251 (i.c.v.); (9) 500 units penicillin (2.5 μl, i.c.) + 0.5 μg AM-251 (i.c.v.); (10) 500 units penicillin (2.5 μl, i.c.) + 1 μg AM-251 (i.c.v.); (11) 500 units penicillin (2.5 μl, i.c.) + 0.125 μg AM-251 (i.c.v.) + 7.5 μg ACEA (i.c.v.); (12) 500 units penicillin (2.5 μl, i.c.) + 0.25 μg AM-251 (i.c.v.) + 7.5 μg ACEA (i.c.v.); (13) 7.5 μg ACEA (i.c.v.); (14) 0.25 μg AM-251 (i.c.v.); (15) 1 μl dimethylsulfoxide (DMSO)/saline (3:7 volume/volume, i.c.v.). Each animal group was composed of seven rats.
Induction of epileptiform activity
The animals were anesthetized with urethane (1.25 g kg−1, i.p.) and placed in a stereotaxic frame. Rectal temperature was maintained between 36.0 and 37.5°C using a feedback-controlled heating system. A polyethylene cannula was introduced into the right femoral artery to monitor blood pressure, which was kept above 110 mm Hg during the experiments (mean 118 ± 7 mm Hg). All contact and incision points were infiltrated with procaine hydrochloride to minimize possible sources of pain.
The left cerebral cortex was exposed by craniotomy (5 mm posterior to bregma and 3 mm lateral to sagittal sutures). The epileptic focus was produced by 500 units of penicillin G potassium injection (1 mm beneath the brain surface by a Hamilton microsyringe type 701N; infusion rate 0.5 μl/min) (Ayyildiz et al., 2007).
Drug and drug administration
AM-251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and ACEA (arachidonyl-2-chloroethylamide) (Sigma Chemical Co., St. Louis, MO, U.S.A.) were used in the experiments. AM-251 and ACEA were dissolved in dimethylsulfoxide (DMSO) to which was added sterile physiologic saline (final solution DMSO/saline 3:7 volume/volume, respectively), and the requisite doses were administered intracerebroventricularly in a volume of 1 μl. Intracerebroventricular injections were administered into the left lateral ventricle of each rat through a stereotaxic apparatus, with the coordinates of 0.8 mm posterior to the bregma, 2.0 mm lateral to the midline, and 4.2 mm ventral to the surface of the skull based on the atlas of the rat brain (Paxinos & Watson, 1986). Penicillin was prepared in the sterile distilled water and administered intracortically in a volume of 2.5 μl. In the first set of experiments, CB1-receptor agonist ACEA, at doses of 2.5, 5, 7.5, and 15 μg (i.c.v.), was administered 30 min after penicillin (i.c.) application. In the second set of experiments, CB1-receptor antagonist AM-251, at doses of 0.125, 0.25, 0.5, and 1 μg, was administered 30 min after penicillin (i.c.) application. In the third set of experiments, the animals received AM-251, at doses of 0.125 and 0.25 μg (i.c.v.), 10 min before an effective dose of ACEA (7.5 μg, i.c.v.). The effective doses of AM-251 (0.25 μg, i.c.v.) and ACEA (7.5 μg, i.c.v.) were administered alone in a volume of 1 μl.
Electrocorticography recordings
Two Ag—AgCl ball electrodes were placed over the left somatomotor cortex (electrode coordinates: first electrode, 2 mm lateral to sagittal suture and 1 mm anterior to bregma; second electrode, 2 mm lateral to sagittal suture 5 mm posterior to bregma). The common reference electrode was fixed on the pinna. The electrocorticography (ECoG) activity was continuously monitored on a four-channel recorder (PowerLab, 4/SP, AD Instruments, Castle Hill, Australia). All recordings were made under anesthesia and stored on a computer. The frequency and amplitude of epileptiform ECoG activity was analyzed off-line.
Data analysis
The results are given as the means ± standard error of the mean (SEM). Statistical comparisons were made using SigmaStat software (SPSS Science, Chicago, IL, U.S.A.). Data analysis was performed using one-way analysis of variance (ANOVA) and Bonferroni tests for comparisons. For all statistical tests, p < 0.05 was considered statistically significant.
Results
We used the penicillin model of epilepsy, which was previously used in our laboratory (Ayyildiz et al., 2007; Bosnak et al., 2007). To induce epileptiform activity, 500 units penicillin was administered intracortically. Epileptiform activity began within 2—4 min. It reached a constant level as to the frequency and amplitude in the 30 min and lasted for 3—5 h. As we reported before, the means of spike frequency and amplitude were 29 ± 2 spike/min, 1,007 ± 193 μV, respectively (Fig. 1A) (Ayyildiz et al., 2007).
Figure 2 shows the effect of single administration (i.c.v.) of different doses (2.5, 5, 7.5, and 15 μg) of ACEA on the penicillin-induced epileptiform activity. ACEA, at a dose of 7.5 μg (i.c.v.), significantly decreased the mean of frequency of epileptiform activity in the 60 min after ACEA injection without changing the amplitude. ACEA, at doses of 2.5, 5, and 15 μg, did not significantly change either the mean of frequency or amplitude of epileptiform activity during the experiments (Fig. 2). The mean spike frequency of epileptiform activity was 20.4 ± 3.2, 23.2 ± 4.9, 13.5 ± 3.7, and 21.6 ± 4.6 spike/min, and the mean amplitude was 860 ± 66, 720 ± 70, 640 ± 80, and 940 ± 142 μV after 80 min from ACEA injection in the 2.5, 5, 7.5 and 15 μg ACEA-administered groups, respectively. (Fig. 1B—E).
Figure 3 shows the effect of single administration (i.c.v.) of different doses (0.125, 0.25, 0.50, and 1.0 μg, i.c.v.) of CB1-receptor antagonist AM-251 on penicillin-induced epileptiform activity. Bonferroni test revealed a significant proconvulsant effect for AM-251, at doses of 0.25 and 0.50 μg, with a maximal effect at the dose of 0.25 μg. AM-251, at a dose of 0.25 μg, also caused the development of status epilepticus—like activity (Fig. 1G). AM-251, at doses of 0.25 and 0.5 μg (i.c.v.), increased the mean spike frequency of epileptiform activity in the 30 and 40 min after AM-251 injection, without changing the amplitude, respectively (Fig. 3). The other two doses of AM-251 (0.125 and 1 μg, i.c.v.) did not significantly change either the mean of frequency or amplitude of epileptiform activity. The mean spike frequency of epileptiform activity was 31.6 ± 2.2, 60.6 ± 7.9, 59.4 ± 8.7, and 24.2 ± 2.1 spike/min, and the mean amplitude was 1,002 ± 123, 940 ± 112, 768 ± 138, and 940 ± 164 μV after 40 min from AM-251 injection in the 0.125, 0.25, 0.5 and 1 μg AM-251-administered groups, respectively (Fig. 1F—I). Moreover, to evaluate the role of CB1-receptor activation, we used the most effective dose and noneffective dose of CB1-receptor antagonist, AM-251 (0.25 and 0.125 μg) 10 min before CB1-receptor agonist, ACEA (7.5 μg, i.c.v.). AM-251, at doses of 0.125 and 0.25 μg, fully inhibited the anticonvulsant effects of ACEA. The spike frequency of epileptiform activity in the presence of AM-251 (0.125 and 0.25 μg, i.c.v.) and ACEA (7.5 μg, i.c.v.) was similar to the frequency recorded in the presence of AM-251 alone (Fig. 1J, K). The intracerebroventricular injection of AM-251 (0.25 μg), ACEA (7.5 μg), DMSO/saline (1 μl), and the intracortical injection of aCSF (2.5 μl), did not cause any change in the frequency or amplitude of ECoG activity with respect to the control base line in the nonpenicillin-injected animals (Fig. 1L).
Discussion
Several studies have shown that the cannabinoids have anticonvulsant properties, which are mediated through activation of the cannabinoid CB1 receptors in the model of the maximal electroshock of grand-mal seizure (Wallace et al., 2001, 2002), the rat pilocarpine model of acquired epilepsy (Wallace et al., 2003; Falenski et al., 2007), the in vitro hippocampal neuronal culture models of acquired epilepsy and status epilepticus (Blair et al., 2006; Deshpande et al., 2007a), and the pentylenetetrazole (PTZ) model of myoclonic seizures in mice (Shafaroodi et al., 2004; Gholizadeh et al., 2007). In the present study, we demonstrated that the intracerebroventricular administration of CB1-receptor antagonist AM-251, at doses of 0.25 and 0.50 μg, caused marked increase in the frequency of penicillin-induced epileptiform activity in rat, whereas the intracerebroventricular administration of CB1-receptor agonist ACEA, at a dose of 7.5 μg, decreased the frequency of epileptiform activity.
We used ACEA, at doses of 2.5, 5, 7.5, and 15 μg (i.c.v.), in the present study. ACEA, at a dose of 7.5 μg (i.c.v.), showed anticonvulsant activity. The other doses of ACEA used in this study did not change either the frequency or amplitude of epileptiform activity. This finding is in-line with the previous reports on anticonvulsant effects of CB1-receptor agonist, ACEA (Luszczki et al., 2006; Gholizadeh et al., 2007; Bahremand et al., 2008). The intraperitoneal administration of CB1-receptor agonist ACEA, at doses of 2—8 mg/kg, induced an anticonvulsant effect against the PTZ model of myoclonic seizures (Bahremand et al., 2008). Gholizadeh et al. (2007) reported that ACEA, at doses of 0.1, 0.5, and 1 mg/kg, i.p., did not alter the seizure threshold, whereas ACEA, at a dose of 2 mg/kg, i.p., significantly increased the threshold. In addition, it was reported that ACEA, at doses of 5 and 7.5 mg/kg, i.p., increased the electroconvulsive threshold, whereas ACEA, at a dose of 15 mg/kg, i.p., did not alter significantly the threshold in mice (Luszczki et al., 2006).
In the current study, CB1-receptor antagonist AM-251, at doses of 0.25 and 0.5 μg (i.c.v.), increased the frequency of epileptiform activity without changing the amplitude. AM-251, at a dose of 0.25 μg (i.c.v.), was the most effective dose in changing the frequency of penicillin-induced epileptiform activity. AM-251, at a dose of 0.25 μg (i.c.v.), also caused the development of status epilepticus—like activity in this group. The other doses of AM-251 (0.125 and 1 μg) did not affect either the frequency or amplitude of epileptiform activity. Our data are also consistent with recent electrophysiologic studies, which confirm that the administration of CB1-receptor antagonist, AM-251, causes an increase in epileptiform activity (Wallace et al., 2002, 2003; Shafaroodi et al., 2004; Deshpande et al., 2007a). CB1-receptor antagonist AM-251, at different doses (0.75—3 mg/kg, i.p.), showed proconvulsant effect, with a maximal effect at 1 mg/kg, PTZ-induced seizure model in mice (Shafaroodi et al., 2004). In addition, AM-251 (1 μm) produced continuous epileptiform discharges in the epileptic neurons, causing the development of status epilepticus—like activity and a break down in the ability of neurons to terminate the seizure activity in the hippocampal neuronal culture model of acquired epilepsy (Deshpande et al., 2007a). It is important to note that AM-251 (1 μm) alone did not produce any hyperexcitability in the control neurons (Deshpande et al., 2007b). Furthermore, the blockade of CB1-receptor function with the selective antagonist SR141716A (10 mg/kg, i.p.), which is structurally similar to AM-251, significantly reduced the maximal seizure threshold and significantly increased the epileptic seizure frequency and duration in the pilocarpine model of temporal lobe epilepsy (Wallace et al., 2002, 2003). In contrast, Naderi et al. (2008) reported that the systemic administration of AM-251, at doses of 0.25, 0.5, 1, 2, and 4 mg/kg, did not produce significant effect in the electroshock-induced seizure in mice. Moreover, AM-251 decreased the spike firing, suggesting an anticonvulsant role in the piriform cortical model of epilepsy in rat, and AM-251 increased inhibitory neurotransmission in the mouse cerebellum (Dennis et al., 2008; Ma et al., 2008). In the presence of CB1-receptor antagonist AM-251, at the most effective (0.25 μg) and noneffective doses (0.125 μg), the anticonvulsant effect of ACEA was absent in this study, confirming that the anticonvulsant property of ACEA is mediated via activation of the cannabinoid CB1 receptors. This confirms the results of previous studies, which reported that the effective dose of CB1-receptor antagonist AM-251 (1 μm) reversed the anticonvulsant effect of CB1-receptor agonist WIN 55,212-2 on status epilepticus activity in the model of hippocampal neuronal culture (Deshpande et al., 2007b) and in bicuculline-induced convulsion in newborn rats (Koda et al., 2005). In addition, the effective dose of CB1-receptor antagonist SR141716A, which is structurally similar to AM-251, completely abolished the anticonvulsant effect of CB1-receptor agonist WIN 55,212-2 in the maximal electroshock model in mice (Wallace et al., 2001).
The intracerebroventricular injections of both compounds, AM-251 and ACEA, presented bell-shaped dose—effect curves in this study. Contradictory results exist concerning dose—response relationships of CB1-receptor agonist and antagonist compounds. Several studies indicate that the administration of CB1-receptor agonist and antagonist compounds produced a dose-dependent effect (Wallace et al., 2001; Blair et al., 2006; Deshpande et al., 2007c; Naderi et al., 2008). Conversely, CB1-receptor antagonist AM-251 showed proconvulsant effect between 0.75—3 mg/kg (i.p.) at the PTZ-induced seizure model in mice, but the maximal effect was observed at the dose of 1 mg/kg (Shafaroodi et al., 2004). De Oliveira Alvares et al. (2005) noted that only the intermediate dose (5.5 ng) of AM-251 was effective in the inhibitory avoidance task and that the absence of effect with a larger dose (55 ng) may be the consequence of nonspecific binding in the hippocampus. ACEA, administered alone at a dose of 15 mg/kg (i.p.), underwent a rapid metabolic degradation by the fatty-acid amide hydrolase in mice to inactive metabolites, and thus this cannabinoid CB1-receptor agonist had no impact on the threshold for electroconvulsions, whereas low doses of ACEA had anticonvulsant activity (Luszczki et al., 2006). In addition, Moreira et al., (2007) found a bell-shaped dose—response for anandamide, which is an analog of ACEA, suggesting a possible explanation that anandamide may inhibit the release of either glutamate or γ-aminobutyric acid (GABA), depending on the administered dose. Unfortunately, the results of the present study do not clarify the certain mechanism related to the bell-shaped dose—response relationship of CB1-receptor agonist and antagonist compounds.
According to the γ-aminobutyric acid hypothesis of epilepsy, an insufficient GABAergic inhibition was suggested as one of the reasons for central hyperexcitability (Corda et al., 1991; Meldrum, 1995). In addition, it was reported that penicillin blocks synaptic transmission by the inhibitory neurotransmitter (Macdonald & Barker, 1977). On the other hand, recent findings have suggested that CB1 receptors have been localized presynaptically on GABAergic interneurons (Hoffman & Lupica, 2000; Katona et al., 2001; Freund et al., 2003) and glutamatergic terminals, albeit at much lower levels than on GABAergic terminals (Freund et al., 2003; Katona et al., 2006; Kawamura et al., 2006). Howlett et al., (2004) reported that CB1 receptors are among the most abundant G protein—coupled receptors in the brain, their densities being similar to the levels of GABA and glutamate-gated ion channels. Vaughan and Christie (2005) also noted that the action of these cannabinoid agents is different from that of conventional anticonvulsant agents, since the cannabinoids activate presynaptic CB1 receptors, causing decreased neurotransmitter release with the resultant dampening of neuronal excitability. In light of these studies, a plausible mechanism for the anticonvulsant actions of cannabinoids in the penicillin-induced model may cause a compensatory shift to occur in the balance between CB1-receptor—mediated inhibition of glutamate and GABA release. Further studies are, however, required to demonstrate the exact mechanism of action.
In conclusion, the application of CB1-receptor agonist, ACEA, partially suppressed penicillin-induced epileptiform activity. CB1-receptor antagonist, AM-251, caused an increase in the frequency of epileptiform activity. It also caused continuous epileptiform discharges that meet the criteria for status epilepticus—like activity. Moreover, CB1-receptor antagonism can reverse the anticonvulsant effect of ACEA. The results of the present study show for the first time that the intracerebroventricular injection of cannabinoid agents could also reveal a role of the cannabinoid system in penicillin-induced epileptic activity.
Source, Graphs and Figures: The effects of intracerebroventricular AM-251, a CB1-receptor antagonist, and ACEA, a CB1-receptor agonist, on penicillin-induced epileptiform activity in rats - Kozan - 2009 - Epilepsia - Wiley Online Library
Purpose: Several results support the conclusion that the cannabinoid system has a role in generation and cessation of epileptic seizures. The aim of this study was to evaluate the effects of intracerebroventricular AM-251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1-receptor antagonist, and ACEA (arachidonyl-2-chloroethylamide), a CB1-receptor agonist, on penicillin-induced epileptiform activity in rats.
Methods: In the first set of experiments, 30 min after penicillin injection, AM-251, at doses of 0.125, 0.25, 0.5, and 1 μg, was administered intracerebroventricularly (i.c.v.). In the second set of experiments, 30 min after penicillin injection, ACEA, at doses of 2.5, 5, 7.5, and 15 μg (i.c.v.), was administered. In the third set of experiments, AM-251, at doses of 0.125 and 0.25 μg (i.c.v.), was administered 10 min before ACEA (7.5 μg, i.c.v.) injection.
Results: ACEA, at a dose of 7.5 μg, significantly decreased the frequency of penicillin-induced epileptiform activity without changing the amplitude. ACEA, at doses of 2.5, 5, and 15 μg, had no impact on either frequency or amplitude of epileptiform activity. AM-251, at doses of 0.25 and 0.50 μg, significantly increased the frequency of epileptiform activity. AM-251, at a dose of 0.25 μg (i.c.v.), was the most effective in changing the frequency of penicillin-induced epileptiform activity, and it also caused status epilepticus—like activity. AM-251, at doses of 0.125 and 0.25 μg, 10 min before ACEA (7.5 μg), reversed the anticonvulsant action of ACEA.
Discussion: The results of the present study provide electrophysiologic evidence for the role of CB1 receptors in regulating the frequency of epileptiform activity in the model of penicillin-induced epilepsy. To elucidate the precise mechanism of cannabinoid action in the brain during seizure, more advanced electrophysiologic and neurochemical studies are required.
All types of epilepsy are recurrent, unprovoked seizures caused by an uncontrolled electrical discharge from nerve cells in selective regions of the brain (Dichter, 1994). Epileptogenic processes have been associated with imbalance between excitatory and inhibitory control systems in the brain (Brailowsky & Garcia, 1999).
There has been increasing interest in the use of the cannabinoids in many disorders, including epilepsy (Gordon & Devinsky, 2001). An abundant series of the cannabinoid analogs has been tested in experimental models of seizure activity (in vivo and in vitro) (Wallace et al., 2001, 2002; Blair et al., 2006; Deshpande et al., 2007a). It has been demonstrated that the endocannabinoid system plays an important role in regulating seizure activity in the brain (Wallace et al., 2003; Luszczki et al., 2006; Deshpande et al., 2007a). Wallace et al. (2001) demonstrated that the cannabinoid (Δ9-tetrahydrocannabinol) and cannabimimetic (WIN 55,212-2) compounds are anticonvulsant in the maximal electroshock model of epilepsy. They also suggested that this cannabinoid anticonvulsant effect was cannabinoid CB1-receptor—dependent (Wallace et al., 2001). WIN 55,212-2 was also shown to totally suppress the spontaneous recurrent epileptiform discharges via CB1-receptor activation in the models of pilocarpine, neuronal culture models of acquired epilepsy, and status epilepticus (Wallace et al., 2003; Blair et al., 2006). In addition, activation of CB1 receptors by WIN 55212-2 significantly suppressed both the frequency and amplitude of spontaneous inhibitory synaptic currents to about 50% of control (Nakatsuka et al., 2003). The suppressive effects were completely abolished in the presence of the CB1-receptor antagonist, AM-251 (Nakatsuka et al., 2003). Deshpande et al. (2007a) reported that the CB1-receptor antagonist AM-251 disrupts the endocannabinoid tone by blocking CB1-receptor activation, which caused development of status epilepticus in populations of epileptic neurons in the hippocampal neuronal culture model. They also noted that the induction of status epilepticus—like activity by CB1-receptor antagonist (AM-251) was reversible and could be overcome by higher concentrations of CB1-receptor agonists (WIN 55,212-2) (Deshpande et al., 2007a). Conversely, it has been shown recently that AM-251 decreased the spike firing, suggesting an anticonvulsant role in the piriform cortical model of epilepsy in rat (Dennis et al., 2008; Ma et al., 2008). Luszczki et al. (2006) used CB1-receptor agonist arachidonyl-2-chloroethylamide (ACEA), which exhibits greater than 1,400-fold preferential affinity for CB1 over CB2 (Hillard et al., 1999), in the mouse maximal electroshock-induced seizure model. They reported that the systemic administration of ACEA, at doses of 1.25 and 2.5 mg/kg, increased the threshold by 4% and 9%, whereas ACEA, at doses of 5 and 7.5 mg/kg, produced a 63% and 123% increase in the electroconvulsive threshold in mice (Luszczki et al., 2006). In addition, the systemic administration of ACEA, at a dose of 5 mg/kg, antagonized cocaine-induced convulsive seizures (Hayase et al., 2001). However, Moreira et al. (2007) reported that the systemic administration of CB1-receptor antagonist has yielded contradictory data, with the anxiogenic (Rodgers et al., 2005; Patel & Hillard, 2006) and anxiolytic activity (Griebel et al., 2005). Moreira et al. (2007) suggested that the local injections of CB1-receptor agonist and antagonist may help to clarify this controversy. Therefore, we decided for the first time, to investigate the effects of intracerebroventricular (i.c.v.) administration of ACEA, a CB1-receptor agonist, and AM-251, a CB1-receptor antagonist, on penicillin-induced epileptiform activity in rats. We used penicillin to induce epileptiform activity in rats in the present study, which is a widely used method for inducing epileptiform activity by applying penicillin to the cerebral cortex (Holmes et al., 1987). The application of penicillin to the neocortex results in synchronous discharge of neurons which, bears an electrophysiologic resemblance to human focal interictal epileptic discharges (Purpura et al., 1972).
Materials and Methods
Animals
Adult male rats weighing 220—260 g (Ondokuz Mayis University of Turkey) were used throughout this study after at least 1 week of acclimatization. All described procedures were approved by the local ethics committee. Animals were housed in groups of 3—4 and were allowed free access to food and water, except for the short time that the animals were removed from their cages for the experiments. All animals were kept in a temperature controlled (22 ± 1°C) environment on a 12-h light/dark cycle. Rats were assigned to the following experiments and groups: intracortical (i.c.) delivery of (1) 2.5 μl artificial cerebrospinal fluid [aCSF containing (mm): NaCl, 124; KCl, 5; KH2PO4, 1.2; CaCl2, 2.4; MgSO4, 1.3; NaHCO3, 26; glucose, 10; HEPES, 10; pH 7.4 when saturated with 95% O2 and 5% CO2] (i.c.); (2) 500 units penicillin (2.5 μl, i.c.); (3) 500 units penicillin (2.5 μl, i.c.) + 2.5 μg ACEA (i.c.v.); (4) 500 units penicillin (2.5 μl, i.c.) + 5 μg ACEA (i.c.v.); (5) 500 units penicillin (2.5 μl, i.c.) + 7.5 μg ACEA (i.c.v.); (6) 500 units penicillin (2.5 μl, i.c.) + 15 μg ACEA (i.c.v.); (7) 500 units penicillin (2.5 μl, i.c.) + 0.125 μg AM-251 (i.c.v.); (8) 500 units penicillin (2.5 μl, i.c.) + 0.25 μg AM-251 (i.c.v.); (9) 500 units penicillin (2.5 μl, i.c.) + 0.5 μg AM-251 (i.c.v.); (10) 500 units penicillin (2.5 μl, i.c.) + 1 μg AM-251 (i.c.v.); (11) 500 units penicillin (2.5 μl, i.c.) + 0.125 μg AM-251 (i.c.v.) + 7.5 μg ACEA (i.c.v.); (12) 500 units penicillin (2.5 μl, i.c.) + 0.25 μg AM-251 (i.c.v.) + 7.5 μg ACEA (i.c.v.); (13) 7.5 μg ACEA (i.c.v.); (14) 0.25 μg AM-251 (i.c.v.); (15) 1 μl dimethylsulfoxide (DMSO)/saline (3:7 volume/volume, i.c.v.). Each animal group was composed of seven rats.
Induction of epileptiform activity
The animals were anesthetized with urethane (1.25 g kg−1, i.p.) and placed in a stereotaxic frame. Rectal temperature was maintained between 36.0 and 37.5°C using a feedback-controlled heating system. A polyethylene cannula was introduced into the right femoral artery to monitor blood pressure, which was kept above 110 mm Hg during the experiments (mean 118 ± 7 mm Hg). All contact and incision points were infiltrated with procaine hydrochloride to minimize possible sources of pain.
The left cerebral cortex was exposed by craniotomy (5 mm posterior to bregma and 3 mm lateral to sagittal sutures). The epileptic focus was produced by 500 units of penicillin G potassium injection (1 mm beneath the brain surface by a Hamilton microsyringe type 701N; infusion rate 0.5 μl/min) (Ayyildiz et al., 2007).
Drug and drug administration
AM-251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and ACEA (arachidonyl-2-chloroethylamide) (Sigma Chemical Co., St. Louis, MO, U.S.A.) were used in the experiments. AM-251 and ACEA were dissolved in dimethylsulfoxide (DMSO) to which was added sterile physiologic saline (final solution DMSO/saline 3:7 volume/volume, respectively), and the requisite doses were administered intracerebroventricularly in a volume of 1 μl. Intracerebroventricular injections were administered into the left lateral ventricle of each rat through a stereotaxic apparatus, with the coordinates of 0.8 mm posterior to the bregma, 2.0 mm lateral to the midline, and 4.2 mm ventral to the surface of the skull based on the atlas of the rat brain (Paxinos & Watson, 1986). Penicillin was prepared in the sterile distilled water and administered intracortically in a volume of 2.5 μl. In the first set of experiments, CB1-receptor agonist ACEA, at doses of 2.5, 5, 7.5, and 15 μg (i.c.v.), was administered 30 min after penicillin (i.c.) application. In the second set of experiments, CB1-receptor antagonist AM-251, at doses of 0.125, 0.25, 0.5, and 1 μg, was administered 30 min after penicillin (i.c.) application. In the third set of experiments, the animals received AM-251, at doses of 0.125 and 0.25 μg (i.c.v.), 10 min before an effective dose of ACEA (7.5 μg, i.c.v.). The effective doses of AM-251 (0.25 μg, i.c.v.) and ACEA (7.5 μg, i.c.v.) were administered alone in a volume of 1 μl.
Electrocorticography recordings
Two Ag—AgCl ball electrodes were placed over the left somatomotor cortex (electrode coordinates: first electrode, 2 mm lateral to sagittal suture and 1 mm anterior to bregma; second electrode, 2 mm lateral to sagittal suture 5 mm posterior to bregma). The common reference electrode was fixed on the pinna. The electrocorticography (ECoG) activity was continuously monitored on a four-channel recorder (PowerLab, 4/SP, AD Instruments, Castle Hill, Australia). All recordings were made under anesthesia and stored on a computer. The frequency and amplitude of epileptiform ECoG activity was analyzed off-line.
Data analysis
The results are given as the means ± standard error of the mean (SEM). Statistical comparisons were made using SigmaStat software (SPSS Science, Chicago, IL, U.S.A.). Data analysis was performed using one-way analysis of variance (ANOVA) and Bonferroni tests for comparisons. For all statistical tests, p < 0.05 was considered statistically significant.
Results
We used the penicillin model of epilepsy, which was previously used in our laboratory (Ayyildiz et al., 2007; Bosnak et al., 2007). To induce epileptiform activity, 500 units penicillin was administered intracortically. Epileptiform activity began within 2—4 min. It reached a constant level as to the frequency and amplitude in the 30 min and lasted for 3—5 h. As we reported before, the means of spike frequency and amplitude were 29 ± 2 spike/min, 1,007 ± 193 μV, respectively (Fig. 1A) (Ayyildiz et al., 2007).
Figure 2 shows the effect of single administration (i.c.v.) of different doses (2.5, 5, 7.5, and 15 μg) of ACEA on the penicillin-induced epileptiform activity. ACEA, at a dose of 7.5 μg (i.c.v.), significantly decreased the mean of frequency of epileptiform activity in the 60 min after ACEA injection without changing the amplitude. ACEA, at doses of 2.5, 5, and 15 μg, did not significantly change either the mean of frequency or amplitude of epileptiform activity during the experiments (Fig. 2). The mean spike frequency of epileptiform activity was 20.4 ± 3.2, 23.2 ± 4.9, 13.5 ± 3.7, and 21.6 ± 4.6 spike/min, and the mean amplitude was 860 ± 66, 720 ± 70, 640 ± 80, and 940 ± 142 μV after 80 min from ACEA injection in the 2.5, 5, 7.5 and 15 μg ACEA-administered groups, respectively. (Fig. 1B—E).
Figure 3 shows the effect of single administration (i.c.v.) of different doses (0.125, 0.25, 0.50, and 1.0 μg, i.c.v.) of CB1-receptor antagonist AM-251 on penicillin-induced epileptiform activity. Bonferroni test revealed a significant proconvulsant effect for AM-251, at doses of 0.25 and 0.50 μg, with a maximal effect at the dose of 0.25 μg. AM-251, at a dose of 0.25 μg, also caused the development of status epilepticus—like activity (Fig. 1G). AM-251, at doses of 0.25 and 0.5 μg (i.c.v.), increased the mean spike frequency of epileptiform activity in the 30 and 40 min after AM-251 injection, without changing the amplitude, respectively (Fig. 3). The other two doses of AM-251 (0.125 and 1 μg, i.c.v.) did not significantly change either the mean of frequency or amplitude of epileptiform activity. The mean spike frequency of epileptiform activity was 31.6 ± 2.2, 60.6 ± 7.9, 59.4 ± 8.7, and 24.2 ± 2.1 spike/min, and the mean amplitude was 1,002 ± 123, 940 ± 112, 768 ± 138, and 940 ± 164 μV after 40 min from AM-251 injection in the 0.125, 0.25, 0.5 and 1 μg AM-251-administered groups, respectively (Fig. 1F—I). Moreover, to evaluate the role of CB1-receptor activation, we used the most effective dose and noneffective dose of CB1-receptor antagonist, AM-251 (0.25 and 0.125 μg) 10 min before CB1-receptor agonist, ACEA (7.5 μg, i.c.v.). AM-251, at doses of 0.125 and 0.25 μg, fully inhibited the anticonvulsant effects of ACEA. The spike frequency of epileptiform activity in the presence of AM-251 (0.125 and 0.25 μg, i.c.v.) and ACEA (7.5 μg, i.c.v.) was similar to the frequency recorded in the presence of AM-251 alone (Fig. 1J, K). The intracerebroventricular injection of AM-251 (0.25 μg), ACEA (7.5 μg), DMSO/saline (1 μl), and the intracortical injection of aCSF (2.5 μl), did not cause any change in the frequency or amplitude of ECoG activity with respect to the control base line in the nonpenicillin-injected animals (Fig. 1L).
Discussion
Several studies have shown that the cannabinoids have anticonvulsant properties, which are mediated through activation of the cannabinoid CB1 receptors in the model of the maximal electroshock of grand-mal seizure (Wallace et al., 2001, 2002), the rat pilocarpine model of acquired epilepsy (Wallace et al., 2003; Falenski et al., 2007), the in vitro hippocampal neuronal culture models of acquired epilepsy and status epilepticus (Blair et al., 2006; Deshpande et al., 2007a), and the pentylenetetrazole (PTZ) model of myoclonic seizures in mice (Shafaroodi et al., 2004; Gholizadeh et al., 2007). In the present study, we demonstrated that the intracerebroventricular administration of CB1-receptor antagonist AM-251, at doses of 0.25 and 0.50 μg, caused marked increase in the frequency of penicillin-induced epileptiform activity in rat, whereas the intracerebroventricular administration of CB1-receptor agonist ACEA, at a dose of 7.5 μg, decreased the frequency of epileptiform activity.
We used ACEA, at doses of 2.5, 5, 7.5, and 15 μg (i.c.v.), in the present study. ACEA, at a dose of 7.5 μg (i.c.v.), showed anticonvulsant activity. The other doses of ACEA used in this study did not change either the frequency or amplitude of epileptiform activity. This finding is in-line with the previous reports on anticonvulsant effects of CB1-receptor agonist, ACEA (Luszczki et al., 2006; Gholizadeh et al., 2007; Bahremand et al., 2008). The intraperitoneal administration of CB1-receptor agonist ACEA, at doses of 2—8 mg/kg, induced an anticonvulsant effect against the PTZ model of myoclonic seizures (Bahremand et al., 2008). Gholizadeh et al. (2007) reported that ACEA, at doses of 0.1, 0.5, and 1 mg/kg, i.p., did not alter the seizure threshold, whereas ACEA, at a dose of 2 mg/kg, i.p., significantly increased the threshold. In addition, it was reported that ACEA, at doses of 5 and 7.5 mg/kg, i.p., increased the electroconvulsive threshold, whereas ACEA, at a dose of 15 mg/kg, i.p., did not alter significantly the threshold in mice (Luszczki et al., 2006).
In the current study, CB1-receptor antagonist AM-251, at doses of 0.25 and 0.5 μg (i.c.v.), increased the frequency of epileptiform activity without changing the amplitude. AM-251, at a dose of 0.25 μg (i.c.v.), was the most effective dose in changing the frequency of penicillin-induced epileptiform activity. AM-251, at a dose of 0.25 μg (i.c.v.), also caused the development of status epilepticus—like activity in this group. The other doses of AM-251 (0.125 and 1 μg) did not affect either the frequency or amplitude of epileptiform activity. Our data are also consistent with recent electrophysiologic studies, which confirm that the administration of CB1-receptor antagonist, AM-251, causes an increase in epileptiform activity (Wallace et al., 2002, 2003; Shafaroodi et al., 2004; Deshpande et al., 2007a). CB1-receptor antagonist AM-251, at different doses (0.75—3 mg/kg, i.p.), showed proconvulsant effect, with a maximal effect at 1 mg/kg, PTZ-induced seizure model in mice (Shafaroodi et al., 2004). In addition, AM-251 (1 μm) produced continuous epileptiform discharges in the epileptic neurons, causing the development of status epilepticus—like activity and a break down in the ability of neurons to terminate the seizure activity in the hippocampal neuronal culture model of acquired epilepsy (Deshpande et al., 2007a). It is important to note that AM-251 (1 μm) alone did not produce any hyperexcitability in the control neurons (Deshpande et al., 2007b). Furthermore, the blockade of CB1-receptor function with the selective antagonist SR141716A (10 mg/kg, i.p.), which is structurally similar to AM-251, significantly reduced the maximal seizure threshold and significantly increased the epileptic seizure frequency and duration in the pilocarpine model of temporal lobe epilepsy (Wallace et al., 2002, 2003). In contrast, Naderi et al. (2008) reported that the systemic administration of AM-251, at doses of 0.25, 0.5, 1, 2, and 4 mg/kg, did not produce significant effect in the electroshock-induced seizure in mice. Moreover, AM-251 decreased the spike firing, suggesting an anticonvulsant role in the piriform cortical model of epilepsy in rat, and AM-251 increased inhibitory neurotransmission in the mouse cerebellum (Dennis et al., 2008; Ma et al., 2008). In the presence of CB1-receptor antagonist AM-251, at the most effective (0.25 μg) and noneffective doses (0.125 μg), the anticonvulsant effect of ACEA was absent in this study, confirming that the anticonvulsant property of ACEA is mediated via activation of the cannabinoid CB1 receptors. This confirms the results of previous studies, which reported that the effective dose of CB1-receptor antagonist AM-251 (1 μm) reversed the anticonvulsant effect of CB1-receptor agonist WIN 55,212-2 on status epilepticus activity in the model of hippocampal neuronal culture (Deshpande et al., 2007b) and in bicuculline-induced convulsion in newborn rats (Koda et al., 2005). In addition, the effective dose of CB1-receptor antagonist SR141716A, which is structurally similar to AM-251, completely abolished the anticonvulsant effect of CB1-receptor agonist WIN 55,212-2 in the maximal electroshock model in mice (Wallace et al., 2001).
The intracerebroventricular injections of both compounds, AM-251 and ACEA, presented bell-shaped dose—effect curves in this study. Contradictory results exist concerning dose—response relationships of CB1-receptor agonist and antagonist compounds. Several studies indicate that the administration of CB1-receptor agonist and antagonist compounds produced a dose-dependent effect (Wallace et al., 2001; Blair et al., 2006; Deshpande et al., 2007c; Naderi et al., 2008). Conversely, CB1-receptor antagonist AM-251 showed proconvulsant effect between 0.75—3 mg/kg (i.p.) at the PTZ-induced seizure model in mice, but the maximal effect was observed at the dose of 1 mg/kg (Shafaroodi et al., 2004). De Oliveira Alvares et al. (2005) noted that only the intermediate dose (5.5 ng) of AM-251 was effective in the inhibitory avoidance task and that the absence of effect with a larger dose (55 ng) may be the consequence of nonspecific binding in the hippocampus. ACEA, administered alone at a dose of 15 mg/kg (i.p.), underwent a rapid metabolic degradation by the fatty-acid amide hydrolase in mice to inactive metabolites, and thus this cannabinoid CB1-receptor agonist had no impact on the threshold for electroconvulsions, whereas low doses of ACEA had anticonvulsant activity (Luszczki et al., 2006). In addition, Moreira et al., (2007) found a bell-shaped dose—response for anandamide, which is an analog of ACEA, suggesting a possible explanation that anandamide may inhibit the release of either glutamate or γ-aminobutyric acid (GABA), depending on the administered dose. Unfortunately, the results of the present study do not clarify the certain mechanism related to the bell-shaped dose—response relationship of CB1-receptor agonist and antagonist compounds.
According to the γ-aminobutyric acid hypothesis of epilepsy, an insufficient GABAergic inhibition was suggested as one of the reasons for central hyperexcitability (Corda et al., 1991; Meldrum, 1995). In addition, it was reported that penicillin blocks synaptic transmission by the inhibitory neurotransmitter (Macdonald & Barker, 1977). On the other hand, recent findings have suggested that CB1 receptors have been localized presynaptically on GABAergic interneurons (Hoffman & Lupica, 2000; Katona et al., 2001; Freund et al., 2003) and glutamatergic terminals, albeit at much lower levels than on GABAergic terminals (Freund et al., 2003; Katona et al., 2006; Kawamura et al., 2006). Howlett et al., (2004) reported that CB1 receptors are among the most abundant G protein—coupled receptors in the brain, their densities being similar to the levels of GABA and glutamate-gated ion channels. Vaughan and Christie (2005) also noted that the action of these cannabinoid agents is different from that of conventional anticonvulsant agents, since the cannabinoids activate presynaptic CB1 receptors, causing decreased neurotransmitter release with the resultant dampening of neuronal excitability. In light of these studies, a plausible mechanism for the anticonvulsant actions of cannabinoids in the penicillin-induced model may cause a compensatory shift to occur in the balance between CB1-receptor—mediated inhibition of glutamate and GABA release. Further studies are, however, required to demonstrate the exact mechanism of action.
In conclusion, the application of CB1-receptor agonist, ACEA, partially suppressed penicillin-induced epileptiform activity. CB1-receptor antagonist, AM-251, caused an increase in the frequency of epileptiform activity. It also caused continuous epileptiform discharges that meet the criteria for status epilepticus—like activity. Moreover, CB1-receptor antagonism can reverse the anticonvulsant effect of ACEA. The results of the present study show for the first time that the intracerebroventricular injection of cannabinoid agents could also reveal a role of the cannabinoid system in penicillin-induced epileptic activity.
Source, Graphs and Figures: The effects of intracerebroventricular AM-251, a CB1-receptor antagonist, and ACEA, a CB1-receptor agonist, on penicillin-induced epileptiform activity in rats - Kozan - 2009 - Epilepsia - Wiley Online Library
