Reduction of pentylenetetrazol-induced convulsions by the octadecaneuropeptide ODN☆

Peptides 20 (1999) 1431–1436

Reduction of pentylenetetrazol-induced convulsions by the octadecaneuropeptide ODN夞 Juana Garcia de Mateos–Vercherea, Jerome Leprinceb, Marie-Christine Tononb, Hubert Vaudryb,*, Jean Costentina a

Institut Fe´de´ratif de Recherches Multidisciplinaires sur les Peptides (IFRMP 23), Unite´ de Neuropsychopharmacologie, CNRS, UPRES-A 6036, Faculte´ de Me´decine et Pharmacie, Universite´ de Rouen, 76800 Saint–Etienne du Rouvray, France b Laboratoire de Neuroendocrinologie Cellulaire et Mole´culaire, INSERM U413, UA CNRS, Universite´ de Rouen, 76821 Mont–Saint–Aignan, France Received 18 March 1999; accepted 27 July 1999

Abstract Intracerebroventricular injection of the octadecaneuropeptide ODN in mouse, at doses of 12.5–1000 ng, reduced the percentage of convulsing animals and increased the latency of convulsions elicited by pentylenetetrazol (50 mg/kg, intraperitoneal [IP]). ODN also reduced the percentage of mortality induced by pentylenetetrazol (100 mg/kg, IP). The COOH-terminal octapeptide fragment of ODN was approximately equally effective but acted more rapidly than ODN to reverse the convulsant effect of pentylenetetrazol. ODN (100 ng, intracerebroventricular [ICV]) increased the convulsion latency and reduced the percentage of animals that convulsed after the administration of the inverse agonist of benzodiazepine receptors DMCM (13 mg/kg, IP), whereas the benzodiazepine receptor antagonist flumazenil (1 mg/kg, subcutaneously) abrogated the protective effect of ODN (100 ng, ICV) on pentylenetetrazol-induced convulsions. ODN (100 ng, ICV) also reduced the percentage of DBA/2J mice displaying audiogenic convulsions. In contrast, ODN did not reduce the percentage of mice displaying tonic or clonic convulsions when electrical interauricular stimulations were applied. It is concluded that ODN, or more likely a proteolytic fragment derived from ODN, reduces pentylenetetrazol-induced convulsions through activation of central-type benzodiazepine receptors. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Octadecaneuropeptide (ODN); Endozepines; Seizures; Pentylenetetrazol; DMCM; Flumazenil; Mouse

1. Introduction The octadecaneuropeptide ODN (Gln-Ala-Thr-Val-Gly Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys) is a proteolytic fragment of an 86-amino acid residue polypeptide called diazepam-binding inhibitor (DBI) that has been originally identified on the basis of its ability to displace diazepam from its binding sites in brain homogenates [8]. DBI and its processing products, including ODN, are generally designated by the generic term endozepines [18]. Intracerebroventricular injection (ICV) of endozepines

夞 This work was supported by CNRS (UPRESA 6036), INSERM (U 413) and the Conseil Re´gional de Haute–Normandie. J.L. was the recipient of a scholarship from ORIL-SERVIER laboratories and the Conseil Re´gional de Haute–Normandie. * Corresponding author. Tel.: ⫹33-235-660821; fax: ⫹33-235664413. E-mail address: [email protected] (H. Vaudry)

induces proconflict behavior and reverses the anticonflict action of diazepam [5,6,8]. In particular, ODN has been reported to enhance aggressive interaction [10] and to increase anxiety in rodents [1,7]. The mechanism of action of endozepines is not fully understood. It has been initially proposed that these peptides act as inverse agonists of central-type benzodiazepine receptors (CBR) [6] and thereby inhibit the activity of ␥-aminobutyric acid type A (GABAA) receptors [3,12,17]. Such inverse agonists, including ␤-carbolines, generally induce convulsions [13] although several ␤-carboline derivatives have been reported to display anticonvulsant effects [13,19]. It has also been reported that very high doses of DBI (⬵100 ␮g) provoke convulsions in rat [6]. Paradoxically, ODN had no effect whereas the C terminal fragments of ODN mimicked the convulsant action of DBI [6]. These latter observations prompted us to examine the effect of much lower doses (12.5–1000 ng) of ODN and its C terminal octapeptide moiety. In the present study we have investigated the

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effects of ODN on convulsions elicited in mice by pentylenetetrazol (PTZ), a compound that induces clonic seizure activity by blocking the GABAA receptor-associated chloride channel [21], and has thus been widely used to evaluate the effects of anticonvulsant drugs acting on the GABAAbenzodiazepine receptor complex.

2. Method 2.1. Animals Male Swiss albino mice CD1 (weighing 22–25 g) and DBA/2J (weighing 20 –21 g) (Charles River, Saint–Aubin le`s Elbeuf, France) were housed 20 per cage (Makrolon, 40 ⫻ 25 ⫻ 18 cm) under fixed environmental conditions (22 ⫾ 1°C; 12:12 h light/dark cycle; lights on at 7:00 a.m.) with free access to water and food (U.A.R., France). Experiments were carried out between 9:00 a.m. and 5:00 p.m. The animals were subjected to only one test and were killed immediately after. Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators. 2.2. Electrically- and chemically-induced seizures in mice 2.2.1. Electroconvulsions Clonic or tonic convulsions were provoked by electrical stimulation (50 mA, 52 Hz, 0.2 s) applied through auricular electrodes, by using an Ugo–Basile apparatus (Varese, Italy). The percentage of lethality and the percentage of convulsing animals were determined. The data were collected from 30 mice per group. 2.2.2. Chemical convulsions Chemical convulsions were induced by either PTZ (50 or 100 mg/kg, IP) or methyl 6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate (DMCM; 13 mg/kg, IP). The animals were subsequently placed individually into Perspex cages (L ⫽ 20, W ⫽ 11, H ⫽ 13 cm) and observed during 5 min. The latency for convulsion, the percentage of convulsing animals and the percentage of lethality were determined. The anticonvulsant potency of 100 ng ODN was evaluated through the modification of the ED50 of PTZ-induced convulsions, in groups of 20 –50 mice. 2.3. Time course of the effects of ODN and its COOHterminal octapeptide fragment The effects of ICV injection of saline or 100 ng ODN were compared 15, 45, 90, and 180 min after the injection, using 10 saline- and 10 ODN-treated mice for each considered time. Two animals (one saline- and one ODN-treated) were tested simultaneously in two different cages, by the same observer. The effects of ICV injection of saline or the

octapeptide fragment of ODN (100 ng) were compared 15, 45, and 90 min after the injection, by using 10 saline- and 10 octapeptide-treated mice for each considered time. 2.4. Audiogenic convulsions Male DBA/2J mice were exposed to sound stimulation with a tone of 90 –110 dB and 14 kHz for 300 s, 90 min after ICV injection of saline or 100 ng ODN. The animals were placed individually in a sound insulated box with a Plexiglas box inside (L ⫽ 24, W ⫽ 15, H ⫽ 14 cm) and the sound stimulation began immediately. The latency for convulsions and the percentage of mice displaying clonic convulsions were determined. 2.5. Drugs Rat ODN (Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-AspArg-Pro-Gly-Leu-Leu-Asp-Leu-Lys) and the octapeptide (Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys) were synthetized by the solid phase methodology, as previously described [11]. The peptides were dissolved in saline, just before ICV injection. PTZ (Sigma Chemical, St. Louis, MO, USA) was dissolved in saline. DMCM (a gift from Schering, Berlin, Germany) was dissolved in 0.01 N HCl. Flumazenil (Anexate™, Roche, France, solution for injection) was diluted in saline. 2.6. ICV injections ICV injections of ODN or its octapeptide fragment (10 ␮l) were made in the left ventricle, in about 3 s, with a microsyringe (Hamilton, 50 ␮l) connected to a needle (diameter 0.5 mm) of which the median part of the bevel protruded only 3.5 mm from a guard limiting its penetration into the brain of manually immobilized mice, according to the procedure of Haley and McCormick [9]. 2.7. Statistics The data are expressed as means ⫾ SEM. Differences between groups were assessed by Student’s t-test, by ␹2 test and by two-way ANOVA.

3. Results Intracerebroventricular injections of ODN, at doses ranging from 12.5 to 1000 ng (6.5–523 pmol), did not elicit any convulsions (data not shown). Administration of PTZ (50 mg/kg, IP) induced clonic seizures in 80% of mice. Injection of 25-ng ODN provoked a significant increase in the clonic convulsion latency. The effect of ODN peaked at the 100-ng dose and vanished at the 1000-ng dose (Fig. 1A). In addition, ICV injection of 100-ng ODN significantly decreased the percentage of convulsing mice (Fig. 1A): only

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Fig. 1. Effects of ODN or its COOH-terminal octapeptide fragment on the convulsions elicited by pentylenetetrazol. Mice were injected ICV with saline or ODN (A) or octapeptide (B). The animals received an IP injection of pentylenetetrazol (50 mg/kg) 90 min later. Mean ⫾ SEM of 10 mice per group. Student’s t-test: *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001. ␹2-test: a P ⬍ 0.05; bP ⬍ 0.01.

30% of the animals injected with 100-ng ODN displayed clonic seizures compared to 79% in the control group (pooled data of 90 mice per group; ␹2-test, P ⬍ 0.001). Administration of graded doses of PTZ (40, 50, 60, 80 mg/kg, IP) induced a dose-dependent increase in the per-

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centage of convulsing animals (ED50 ⫽ 44 ⫾ 0.2 mg/kg). ICV injection of 100-ng ODN significantly increased the ED50 to 52 ⫾ 0.5 mg/kg (Student’s t-test, P ⬍ 0.001; data not shown). Injection of the COOH-terminal octapeptide fragment of ODN (6.25–1000 ng, i.e. 7.1–1097 pmol, ICV) significantly increased the clonic convulsion latency (Fig. 1B). At a dose of 25 ng, the octapeptide significantly reduced the percentage of convulsing animals (Fig. 1 B). The time-courses of the effects of ODN and its COOHterminal octapeptide fragment (100 ng each) are compared in Table 1. The increase in clonic convulsion latency and the decrease in the percentage of convulsing animals culminated 90 min after ICV injection of ODN. The octapeptide fragment had a more rapid onset of effect (15 min) on clonic convulsion latency than ODN. Administration of 100 mg/kg PTZ killed 90% of the animals. ICV injection of 100-ng ODN 90 min before IP administration of 100 mg/kg PTZ significantly reduced the mortality (Table 2). Administration of DMCM (13 mg/kg, IP) induced clonic seizures in 70% of the mice. Injection of ODN (100 ng, ICV) 90 min before DMCM administration significantly reduced the percentage of convulsing animals (Table 2). To determine the mode of action of ODN, mice were treated with the benzodiazepine receptor antagonist flumazenil (1 mg/kg, SC) 5 min before administration of PTZ (50 mg/kg, IP). Flumazenil significantly attenuated the effect of an ICV injection of ODN (100 ng, 90 min before PTZ administration) on the clonic convulsion latency and the percentage of convulsing animals (Table 3). In DBA/2J mice, audiogenic stimulations (90 –110 dB, 14 000 Hz, 300 s) induced clonic convulsions in 75% of control animals (10 ␮l saline, ICV). Injection of ODN (100 ng, ICV) 90 min before exposure to the audiogenic stimulation significantly reduced the proportion of convulsing mice to 30% (␹2-test, P ⬍ 0.01). Concurrently, ODN sig-

Table 1 Time course of the effects of ODN and its COOH-terminal octapeptide fragment on the convulsions elicited by pentylenetetrazol ICV and IP injection time before testing

Clonic convulsion latency (s) Saline 10 ␮l

ODN 100 ng/10 ␮l

Saline 10 ␮l

ODN 100 ng/10 ␮l

15 min 45 min 90 min 180 min

115 ⫾ 16 118 ⫾ 24 108 ⫾ 7 129 ⫾ 11

157 ⫾ 28 216 ⫾ 30* 222 ⫾ 13*** 181 ⫾ 23ns

70% 70% 77% 80%

70%ns 50%ns 23%a 70%ns

15 min 45 min 90 min

Percentage of convulsing animals

ns

Saline 10 ␮l

Octapeptide 100 ng/10 ␮l

Saline 10 ␮l

Octapeptide 100 ng/10 ␮l

107 ⫾ 12 90 ⫾ 10 128 ⫾ 7

198 ⫾ 16*** 146 ⫾ 7** 149 ⫾ 7*

80% 80% 80%

40%ns 50%ns 60%ns

Mice were injected ICV with 10 ␮l saline or 100 ng ODN (upper part) or 100 ng of its octapeptide fragment (lower part), at various times before IP administration of pentylenetetrazol (50 mg/kg). Means ⫾ SEM of 10 mice per group except the 100-ng ODN group at 90 min and its control (n ⫽ 30). * P ⬍ 0.05; *** P ⬍ 0.001; ns not significantly different, as compared to saline controls (Student’s t-test). a P ⬍ 0.05; ns not significantly different, as compared to saline controls (␹2-test).

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Table 2 Effects of ODN on the convulsions and lethality caused by pentylenetetrazol or by DMCM Treatments

Convulsion latency(s)

50 mg/kg pentylenetetrazol, IP Saline 10 ␮l, ICV ODN 100 ng/10 ␮l, ICV

109 ⫾ 6 200 ⫾ 14***

100 mg/kg pentylenetetrazol, IP Saline 10 ␮l, ICV ODN 100 ng/10 ␮l, ICV 13 mg/kg DMCM, IP Saline 10 ␮l, ICV ODN 100 ng/10 ␮l, ICV

Percentage of convulsing animals

Percentage of lethality

78% 28%b

0% 0%

75 ⫾ 5 86 ⫾ 7ns

100% 95%ns

90% 60%a

161 ⫾ 5 205 ⫾ 10**

70% 40%a

0% 0%

Mice were injected ICV with 10 ␮l saline or 100 ng ODN 90 min before IP administration of pentylenetetrazol (50 or 100 mg/kg) or DMCM (13 mg/kg). Means ⫾ SEM of 50 mice in experiments with 50 mg/kg pentylenetetrazol. Means ⫾ SEM of 20 mice in the experiments with 100 mg/kg pentylenetetrazol or DMCM. *** P ⬍ 0.001; ns not significantly different as compared to the saline controls (Student’s t-test). a P ⬍ 0.05; b P ⬍ 0.01; ns not significantly different as compare to saline controls (␹2-test).

nificantly increased the convulsion latency from 112 ⫾ 12 s to 175 ⫾ 12 s (mean ⫾ SEM of 20 mice per group; Student’s t-test, P ⬍ 0.001). In CD1 mice, electrical interauricular stimulation (50 mA, 52 Hz, 0.2 s) induced either clonic or tonic convulsions. ICV injection of 100-ng ODN did not significantly modify the percentage of animals displaying tonic convulsions (63.3% vs. 53.3% in control mice) or clonic convulsions (36% vs. 46.7% in control mice). Although electrical interauricular stimulation did not cause mortality in the control group, 20% of the animals died in the ODN-treated group (30 mice per group; ␹2 -test, P ⬍ 0.01). 4. Discussion It has been previously reported that ICV injection of high doses of DBI (10 nmol ⬵100 ␮g) induces tonic and/or Table 3 Effects of ODN in the absence or presence of flumazenil on the convulsions elicited by pentylenetetrazol Treatment

Convulsion latency (s)

Percentage of convulsing animals

Saline 10 ␮l ODN 100 ng/10 ␮l Flumazenil 1 mg/kg Flumazenil 1 mg/kg ⫹ ODN 100 ng/10 ␮l

137 ⫾ 3 189 ⫾ 6 130 ⫾ 3 133 ⫾ 6a

80% 33%b 80% 60%c

Flumazenil (1 mg/kg) was administered SC 85 min after ICV injection of either saline (10 ␮l) or ODN (100 ng). Five minutes after the latter injection, the animals were injected with pentylenetetrazol (50 mg/kg) and they were placed into individual cages for testing. Data are means ⫾ SEM from 40 mice per group. a Significantly different as compared to the ODN-treated group (ANOVA: F(35.6;46.7) ⫽ 28.4; P ⬍ 0.001). ␹2-test. b P ⬍ 0.001 as compared to saline ICV injected mice. c P ⬍ 0.05 as compared to ODN ICV injected mice.

clonic convulsions in rat [6]. In contrast, ODN, even at the very high dose of 100 nmol (⬵190 ␮g) had no effect whereas the shorter COOH-terminal octapeptide, heptapeptide, hexapeptide moieties provoked convulsions. In the present study, we have investigated the effect of 200- to 20 000-fold lower doses of ODN and its octapeptide fragment on PTZ-induced clonic convulsions in mice. For doses ranging from 25 to 100 ng (13–52 pmol), ODN increased the latency of the convulsions elicited by PTZ and, at a dose of 100 ng, ODN reduced the percentage of convulsing mice. The maximum effect of ODN was observed after a long delay (90 min) whereas the effect of the C terminal octapeptide fragment on the convulsion latency occurred much faster (15 min). Similar observations have been reported regarding the anxiogenic effects of ODN and its COOH-terminal fragment [7]. These data strongly suggest that the anticonvulsant effect of ODN requires the formation of a biologically active proteolytic fragment, as already proposed for the anxiogenic effect of the peptide [7]. We also found that the protective effect of ODN on PTZ-induced clonic convulsions was maximum at the dose of 100 ng (52 pmol) whereas a 1000-ng (523- pmol) dose had no effect. Such a biphasic response has already been observed for the anxiogenic effect of ODN [7]. These data indicate that ODN may act as a prodrug displaying a certain affinity for the same receptor as its cleavage fragment, but exhibiting distinct intrinsic activity. On this receptor, ODN would behave as an antagonist whereas the active fragment would operate as an agonist or a partial agonist. Thus, shortly after the administration of a high dose of ODN the antagonistic activity would prevail over the agonistic activity of its processing product(s). It is commonly believed that an antagonism of PTZinduced clonic seizures is predictive of an efficacy against absence seizures and of an anxiolytic effect. Drugs that are active in this seizure model generally interact with

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GABAergic neurotransmission. On the other hand, prevention of electrically-induced tonic kindlimb extension in rodents is predictive of an efficacy against primary or secondarily generalized seizures. Drugs that are active in this latter seizure model may either affect voltage-dependent Na⫹ channels or block NMDA-type excitatory amino acid receptors (for review see Ref. [14]). The fact that ODN prevented PTZ-evoked convulsions but did not protect against electroconvulsions (and even provoked lethal effects) indicates that endozepines could have beneficial effects in absence seizures but not in generalized tonic-clonic seizures. Previous studies have shown that endozepines can interact either with CBR [6,16] or with peripheral-type benzodiazepine receptors (PBR) [2]. In rat, unilateral intrahippocampal injection of very high doses of the octapeptide (150 nmol ⬵136 ␮g) or the nonapeptide (100 nmol ⬵104 ␮g) elicit convulsions that can be prevented by the PBR antagonist PK 11195 but not the CBR antagonist flumazenil [20]. In contrast, several lines of evidence indicate that, in our model, the action of ODN is mediated through CBR:1) PTZ interacts with the chloride channel, which is an integral part of the GABAA-CBR complex; 2) ODN reversed the clonic seizures induced by DMCM that acts as an inverse agonist of CBR; 3) the protective effect of ODN on PTZinduced convulsions was significantly reduced by the CBR antagonist flumazenil. It thus seems that the protective effect of ODN on PTZ-induced convulsions can be accounted by its agonistic activity on the GABAA-benzodiazepine receptor complex. The present study has shown that ODN has no effect on convulsions elicited by electrical interauricular stimulation in CD1 mice. It is conceivable that electrical stimulation may induce the release of massive amounts of endogenous endozepines that would mask the effects of moderate doses of ODN injected ICV. In contrast, ODN attenuated the frequency and augmented the latency of audiogenic convulsions in DBA/2J mice, a strain of mice that is genetically prone to epileptic seizure [15]. The fact that audiogenic convulsions can be prevented by CBR agonists [4] indicates that, in this model as in PTZ-induced convulsions, ODN acts as a direct agonist of CBR. It should be noticed however that, on both models, ODN had only a modest effect on the percentage of convulsing animals and on the duration of convulsion latency. On the other hand, it has been previously shown that ODN, acting through benzodiazepine receptors, induces anxiogenic effects in rodents [7], indicating that the peptide can also act as an inverse agonist of CBR. Taken together, these data suggest that, in rodents, ODN, or more likely a shorter biologically active peptide generated by proteolytic cleavage of ODN can act on CBR either as a partial agonist (anticonvulsant effect) or as a partial inverse agonist (anxiogenic effect) depending on the subunit composition of the GABAA-benzodiazepine receptor complex.

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