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.

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.

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.

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; KH 2 PO 4 , 1.2; CaCl 2 , 2.4; MgSO 4 , 1.3; NaHCO 3 , 26; glucose, 10; HEPES, 10; pH 7.4 when saturated with 95% O 2 and 5% CO 2 ] (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.

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 1 Open in figure viewer PowerPoint (A) The intracortical injection of penicillin (500 IU) induced epileptiform activity on ECoG. (B) The intracerebroventricular (i.c.v.) administration of CB1‐receptor agonist ACEA, at a dose of 2.5 μg, did not influence either the mean frequency or amplitude of penicillin‐induced epileptiform activity. (C) The administration of ACEA (i.c.v.), at a dose of 5 μg, did not influence either the mean frequency or amplitude of penicillin‐induced epileptiform activity. (D) The administration of ACEA (i.c.v.), at a dose of 7.5 μg, significantly decreased the mean frequency of epileptiform activity in the 60 min after ACEA injection without changing the amplitude. (E) The administration of ACEA (i.c.v.), at a dose of 15 μg, did not influence either the mean frequency or amplitude of penicillin‐induced epileptiform activity. (F) The intracerebroventricular administration of CB1‐receptor antagonist AM‐251, at a dose of 0.125 μg, did not influence either the mean frequency or amplitude of penicillin‐induced epileptiform activity. (G) The administration of AM‐251 (i.c.v.), at a dose of 0.25 μg, caused marked increase in the frequency of penicillin‐induced epileptiform activity in the 30 min after AM‐251 injection without changing the amplitude. AM‐251 (0.25 μg) also caused the development of status epilepticus‐like activity. (H) The administration of AM‐251 (i.c.v.), at a dose of 0.5 μg, caused increase in the frequency of penicillin‐induced epileptiform activity in the 40 min after AM‐251 injection without changing the amplitude. (I) The administration of AM‐251 (i.c.v.), at a dose of 1 μg, did not influence either the mean frequency or amplitude of penicillin‐induced epileptiform activity. (J) The administration of the noneffective dose of CB1‐receptor antagonist, AM‐251 (0.125 μg, i.c.v.) 10 min before CB1‐receptor agonist, ACEA (7.5 μg, i.c.v.) fully inhibited the anticonvulsant effects of ACEA. (K) The administration of the most effective dose of CB1‐receptor antagonist, AM‐251 (0.25 μg, i.c.v.) 10 min before CB1‐receptor agonist, ACEA (7.5 μg, i.c.v.) fully inhibited the anticonvulsant effects of ACEA. (L) Presents baseline ECoG activity before penicillin or the injection of other substances.

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 2 Open in figure viewer PowerPoint The effects of intracerebroventricular administration of CB1‐receptor antagonist, AM‐251 on the mean spike frequency of penicillin‐induced epileptiform activity. 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, respectively. The best and earlier effects appeared in the 0.25 μg (i.c.v.) administered group. AM‐251, at doses of 0.125 and 1 μg (i.c.v.), did not influence the mean frequency of epileptiform activity. *p < 0.05, **p < 0.01, ***p < 0.001.

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).