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Effects of 2-chloroadenosine on cortical epileptic afterdischarges in immature rats
Pharmacological Reports
Copyright © 2010 by Institute of Pharmacology Polish Academy of Sciences
2010, 62, 6267 ISSN 1734-1140
Effects of 2-chloroadenosine on cortical epileptic afterdischarges in immature rats Marie Pometlová1, Hana Kubová2, Pavel Mareš2 Charles University, Third Medical School, Department of Normal, Pathological and Clinical Physiology, Ke Karlovu 4, CZ-12000 Prague 2, Czech Republic
Institute of Physiology, Academy of Sciences of the Czech Republic, Department of Developmental Epileptology, Vídenská 1083, CZ-14220 Prague 4, Czech Republic Correspondence: Pavel Mare, e-mail:
[email protected]
Abstract: Adenosine may represent an endogenous anticonvulsant in the brain. This study focused on the possible anticonvulsant action of an adenosine agonist, 2-chloroadenosine, against cortical epileptic afterdischarges (ADs) in immature rats. Three age groups of rat pups with implanted electrodes were studied: 12-, 18- and 25-days-old. The compound, 2-chloroadenosine, was injected after the first successful stimulation at doses of 1, 4 or 10 mg/kg intraperitoneally, and stimulation at the same intensity was repeated three more times. Movements directly elicited by stimulation, as well as clonic seizures accompanying electroencephalography (EEG) ADs, were markedly suppressed in only the 18-day-old animals. The effects in the 12- and especially the 25-day-old rats were moderate. The duration of the ADs decreased in all three age groups with 2-chloroadenosine treatment, and the shortest AD duration was seen in the treated, 12-day-old rats. The AD suppression also lasted longer in this age group than it did in the older animals. After a brief suppression of the second AD, the treated, 25-day-old group exhibited a significant AD rebound during the third and fourth stimulations. Taken together, our data show that 2-chloroadenosine exhibits an anticonvulsant effect that is dose- and age-dependent. Key words: cerebral cortex, electrical stimulation, afterdischarges, 2-chloroadenosine, ontogeny, rat
Abbreviations: AD – cortical afterdischarges, GABA – g-aminobutyric acid
Introduction The adenosinergic system is an important inhibitory modulator in the brain [5, 14]. A possible role of adenosine in epilepsies has been repeatedly reviewed [11, 13, 30]. A role for adenosine in the regulation of epileptic activity has been demonstrated in animal
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Pharmacological Reports, 2010, 62, 6267
models [7, 12] as well as in human tissue [15]. In agreement with these findings are data for the proconvulsant and/or convulsant action of the adenosine antagonists, caffeine and others [9, 16, 28], as well as their interaction with antiepileptic drugs [8] in adult animals. In the first developmental study, Trommer et al. [33] demonstrated a proconvulsant effect of aminophylline in a rat kindling model. Related to that study, we demonstrated a higher sensitivity of immature two- and three-week-old rats to the convulsant action of aminophylline [24]. Similarly, subconvulsant doses of aminophylline markedly potentiated the cortical epileptic afterdischarges (ADs) and a spread
Anticonvulsant action of 2-chloroadenosine
Marie Pometlová et al.
of this activity into the limbic system [4]. On the other hand, developmental data on the anticonvulsant action of adenosine and its agonists are missing. The question of whether adenosine agonists could serve as anticonvulsant drugs cannot be answered without data from immature experimental animals. Nearly one half of all epilepsies start during infancy and childhood [20]. Some age-dependent forms of epilepsy represent a serious therapeutic problem [29]. To begin studies on the anticonvulsant action of the adenosinergic system in developing rats, cortical epileptic ADs were used as a model. In this model, cortical epileptic ADs induced by the rhythmic electrical stimulation of the sensorimotor cortical region allows for the study of at least three phenomena: movements elicited by the stimuli, EEG ADs and clonic seizures accompanying the ADs. We have data on the anticonvulsant effects of drugs influencing various neurotransmitter systems, including g-aminobutyric acid A (GABA-A) [26, 31], GABA-B [27], and excitatory amino acids [23]). We have also described a proconvulsant effect of the adenosine antagonist theophylline (an active compound of aminophylline) in this model [4]. 2-Chloroadenosine was chosen as an agonist of the adenosine receptors because of the availability of data on its action in adult animals [1, 6, 7, 12, 18, 19].
Methods Experiments were performed using three different age groups of immature Wistar rats; the rats were 12-, 18and 25-days-old. The level of brain development in the 12-day-old rats corresponds to the early postnatal stage of the human brain. In rats, postnatal day 18 is the earliest age at which a spike-and-wave EEG rhythm can be recorded, while postnatal day 25 is close to the start of sexual maturation. This project was approved by Animal Care and Use Committee to be in agreement with the Animal Protection Law of the Czech Republic and is fully compatible with the European Community Council directives 86/609/EEC. Flat silver cortical electrodes were implanted into animals epidurally under ether anesthesia. Stimulation electrodes were placed on the right sensorimotor (frontal) cortex, while recording electrodes were placed on the left sensorimotor, parietal and occipital cortex, as well as on the right occipital cortex. A reference electrode was placed on the nasal bone,
a grounding electrode in the occipital bone. The whole surgical preparation lasted less than 15 min and the animals were allowed to recover for 1 h. Righting, placing and suckling reflexes were examined and only then was the stimulation initiated. Initial experiments were performed with classical paper EEG recording, later the system was replaced with a paperless recording (Kaminskij Biomedical Systems, Prague) with a digitalization rate of 500 Hz. The stimulator with a constant current output generated 1 ms biphasic pulses in 15 s series with 8 Hz frequency. Threshold intensity for elicitation of an epileptic AD lasting more than 3 s was established and this intensity was applied during the whole experiment (i.e. four times at 10 min intervals). The first AD always served as the control AD. Five minutes after the end of the first AD, 2-chloroadenosine (Sigma, St. Louis, MO, USA) was administered by intraperitoneal injection. The following three doses of 2-chloroadenosine were used in these experiments: 1, 4, and 10 mg/kg. Pattern and duration of ADs were evaluated. The intensity of the movements elicited by stimulation, as well as clonic seizures accompanying the spike-andwave type of ADs were quantified by means of a modified Racine’s 5-point scale [22, 32]. Movements induced by individual stimuli reflect the excitability of the motor cortex and the motor system up to the muscles of the contralateral forelimb. Clonic seizures indicate the spread of epileptic activity generated in the thalamocortical circuits into the motor system. Statistical comparison between the control and the three dosage groups was performed by one-way analysis of variance (ANOVA). Comparison of the four ADs in each age and dose group were analyzed by one-way repeated measure (RM) ANOVA. The Holm-Sidak test was used for subsequent pairwise comparison (SigmaStat® SYSTAT). The level of statistical significance was set at 5%.
Results Control animals
The first stimulation elicited an AD in all of the animals. The duration of the first AD in the 12-day-old rats was significantly longer than in the two older groups. Control rats (injected with saline after the first AD) continued to generate ADs after all subsequent Pharmacological Reports, 2010, 62, 6267
63
Fig. 1. Electrocorticographic recording of a cortical epileptic afterdischarge (AD) in an 18-day-old rat. Individual leads from top to bottom: left frontal (LF), left occipital (LO) and right occipital (RO) regions, always in a reference connection. At the beginning, the last second of stimulatio n is recorded and then the AD is registered. The numbers over the AD denote motor phenomena according to the modified Racines scale (see Methods). Time mark is 2 s and amplitude calibration is 0.5 mV
stimulations. The intensity of the movements directly bound to the stimulation remained stable with repeated stimulations in the 12- and 18-day-old rats, while the intensity of the movements moderately decreased in the 25-day-old rats. The duration of ADs tended to increase during the experiments in the two younger groups, but did not change in the 25-day-old rats (Fig. 1). The intensity of the clonic seizures was not significantly changed in the youngest group, while the intensity of the clonic seizures significantly decreased in the fourth AD in the 18-day-old animals and in the second and fourth ADs in 25-day-old rats. During stimulation, as well as during clonic seizures, the 12-day-old rats reared only exceptionally (always with support) and the maximal intensity was usually grade 3.
Movements induced by stimulation (Fig. 2)
The injection of 2-chloroadenosine did not significantly influence the intensity of movements directly bound to stimulation in the 12-day-old rats compared to the appropriate stimulations in the control group. On the other hand, the 18-day-old animals exhibited a significantly lower intensity of movements in the second and third stimulation after all three doses of 2-chloroadenosine compared with the appropriate stimulations in the control animals. The decrease in movement intensity during the fourth stimulation was significant only at the highest dose of the agonist. Significant effects with respect to movement intensity were seen only exceptionally in the 25-day-old rats. 64
Pharmacological Reports, 2010, 62, 6267
Fig. 2.
Intensity of movements during stimulation (mean ± SEM) in
12-, 18- and 25-day-old rats (from top to bottom, see numbers on the right). Abscissae are the first to the fourth stimulation and ordinates indicate the 5-point scale (see Methods). For individual doses see in set. Asterisks indicate a significant difference in comparison with the appropriate first stimulation in each group
Anticonvulsant action of 2-chloroadenosine
Marie Pometlová et al.
Fig. 3.
Relative duration of afterdischarges (ADs). The first AD for
each dosage group was taken as 100%. Details are the same as in
Fig. 4. Intensity of clonic convulsions accompanying afterdischarges (ADs). Details are found in Figure 2
Figure 2, only the ordinates indicate the duration of the ADs. Upper panel (12-day-old rats): n means complete suppression of AD
in the animals given 4 and 10 mg/kg of 2-chloroadenosine compared to the control group. Duration of ADs (Fig. 3) Intensity of clonic seizures (Fig. 4)
The two highest doses of 2-chloroadenosine markedly shortened the ADs after the second and third stimulation in the 12-day-old rats. The ADs were completely suppressed in the second stimulation series after injection of 2-chloroadenosine at the 10 mg/kg dose. No effect of 2-chloroadenosine was observed on the fourth AD. The effect of 2-chloroadenosine was even shorter in the 18-day-old rats. A significant shortening of the ADs was observed only in the second stimulation series after injection of the 4 and 10 mg/kg dosages of 2-chloroadenosine. In contrast, the 4 and 10 mg/kg doses tended to prolong the fourth ADs, but due to the high variability of the data significance was not reached. Similar effects were recorded in the 25-day-old animals: the second ADs were shortened, but the third and fourth ADs were significantly longer
The results for the intensity of clonic seizures were similar to the results of the movements elicited by stimulation. A significant decrease in the intensity of seizures was found in the 12-day-old rats, but only in the rats treated with the 4 mg/kg dose of 2-chloroadenosine in the second AD. Injection of 10 mg/kg of 2-chloroadenosine resulted in no ADs at all after the second stimulation. The 18-day-old rats exhibited a significant decrease in the intensity of the seizures after all three doses of 2-chloroadenosine in the second and third ADs. No significant effect on seizure intensity was found at any dose for the fourth AD. The oldest group exhibited only one significant effect with respect to seizure intensity; the 10-mg/kg dose resulted in less intense seizures in the second AD. Pharmacological Reports, 2010, 62, 6267
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Discussion The developmental changes seen in the control animals correspond with our previous results [22]. Changes in ADs were observed with age. The EEG pattern was formed by rhythmic sharp waves, while the spike-and-wave rhythm was recorded in the two older animal groups. These data are in agreement with the development of other rhythmic phenomena of thalamocortical origin [25]. The duration of ADs was longer in the youngest group of animals and is probably due to immature mechanisms of arresting seizures and may be responsible for the postictal refractoriness (Mareš et al., unpublished results). The development of motor abilities is reflected in the failure of the 12day-old rats to rear during stimulation and/or during ADs. The ability to rear fully develops in the third postnatal week [3]. We treated the animals with 2-chloroadenosine to determine whether it possesses anticonvulsant activity. 2-Chloroadenosine exhibited an anticonvulsant action in all three age groups; however, there were differences in the motor and EEG phenomena. Movements during stimulation and clonic seizures were markedly suppressed in the 18-day-old rats, while the effects in the other two age groups were negligible. These data indicate that the spread of activity from the cerebral cortex and the thalamocortical system to lower levels of the motor system is especially sensitive to the inhibitory action of the adenosinergic system at postnatal day 18. In contrast, the generation of epileptic activity in the 18-day-old rats was affected less than in the youngest group. These data indicate that the site of action is not at the cortical level. EEG ADs were markedly influenced in the 12-day-old rat pups. The shortening of the ADs in the 18- and especially the 25-day-old animals was only short-lasting. In addition, there was a clear rebound phenomenon, as demonstrated by a significant prolongation of the fourth ADs in the oldest group. The marked shortening of ADs in the 12-day-old rats is in agreement with our study on the action of aminophylline in the same model. In our previous study, we observed that the prolongation was the largest in the same age group [4]. The convulsant action of high doses of aminophylline was also stronger in the younger age groups than in the 25-day-old rats [24]. Differences in the effects of aminophylline on amygdala kindling between 15-day-old and adult rats also proved a higher effi-
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cacy of manipulation of the adenosinergic system in immature animals [33]. The higher sensitivity of the immature brain to the adenosine antagonist aminophylline [4, 24, 33], as well as to an agonist 2-chloroadenosine demonstrated in the present experiments, does not correspond with the ontogeny of adenosine receptors. Binding of [3H]cyclohexyladenosine is present in all of the studied brain structures at birth and the binding maximum (Bmax) increases, especially during the first three postnatal weeks. In contrast, the dissociation constant (Kd) remains stable [17, 21]. Adenosine receptors in the cerebral cortex exhibit a steep (nearly ten-fold) increase between postnatal days 1 and 21 [17]. This contradiction cannot be explained presently and remains to be studied. Our results also contradict the data of Descombes et al. [10]. However, their in vivo experiments were performed in the hippocampal CA3 field and the development of the cerebral cortex and the hippocampus may differ. However, the presence of a significant increase in GTP [g-35S]-binding following N6-cyclopentyladenosine stimulation of A1 receptors in the cerebral cortex of 14-day-old rats support our findings [2]. Dragunow et al. [12] classified adenosine as an endogenous anticonvulsant. Adenosinergic system also possesses anticonvulsant action in immature rodents, but the rebound effect found in our experiments needs to be analyzed and the peripheral (cardiovascular) effects of adenosine agonists must be taken into account. Additionally, it is necessary to differentiate the effect of 2-chloroadenosine on A1 and A2 receptors using specific agonists or antagonists. Furthermore, the possible negative side effects of adenosine agonists should be studied.
Acknowledgments:
This study was supported by a grant (No. NR9184/3) from the Grant Agency of the Ministry of Health of the Czech Republic and by a Research Project AV0Z 50110509.
References: 1. Abdul-Ghani AS, Attwell PJ, Bradford HF: The protective effect of 2-chloroadenosine against the development of amygdala kindling and on amygdala-kindled seizures. Eur J Pharmacol, 1997, 325, 7–14.
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2. Adén U, Leverin A-L, Hagberg H, Fredholm BB: Adenosine A receptor agonism in the immature rat brain and heart. Eur J Pharmacol, 2001, 426, 185–192. 3. Altman J, Sudarshan K: Postnatal development of locomotion in the laboratory rat. Anim Behav, 1975, 23, 896–920. 4. Bernášková K, Mareš P: Proconvulsant effect of aminophylline on cortical epileptic afterdischarges varies during ontogeny. Epilepsy Res, 2000, 39, 183–190. 5. Boison D: Adenosine as a modulator of brain activity. Drug News Perspect 2007, 20, 607–611. 6. Borowicz KK, £uszczki J, Czuczwar SJ: 2-Chloroadenosine, a preferential agonist of adenosine A1 receptors, enhances the anticonvulsant activity of carbamazepine and clonazepam in mice. Eur Neuropsychopharmacol, 2002, 12, 173–179. 7. Cavalheiro EA, Calderazzo Filho LS, Bortolotto ZA, Mello L, Turski L: Anticonvulsant role of adenosine. Pol J Pharmacol, 1987, 39, 537–543. 8. Chroœciñska-Krawczyk M, Ratnaraj N, Patsalos PN, Czuczwar SJ: Effect of caffeine on the anticonvulsant effects of oxcarbazepine, lamotrigine and tiagabine in a mouse model of generalized tonic-clonic seizures. Pharmacol Rep, 2009, 61, 819–826. 9. Chu N-S: Caffeine- and aminophylline-induced seizures. Epilepsia, 1981, 22, 85–94. 10. Descombes S, Avoli M, Psarropoulou C: A comparison of the adenosine-mediated synaptic inhibition in the CA3 area of immature and adult rat hippocampus. Develop Brain Res, 1998, 110, 51–59. 11. Dragunow M: Purinergic mechanisms in epilepsy. Progr Neurobiol, 1988, 31, 85–108. 12. Dragunow M, Goddard GV, Laverty R: Is adenosine an endogenous anticonvulsant? Epilepsia, 1985, 26, 480–487. 13. Dunwiddie TV: Adenosine and suppression of seizures. In: Jasper’s Basic Mechanisms of the Epilepsies. Eds. Delgado-Escueta A, Wilson WA, Olsen RW, Porter RJ, Lippincott Williams and Wilkins, Philadelphia, 1999, 1001–1010. 14. Dunwiddie TV, Masino SA: The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci, 2001, 24, 31–55. 15. During MJ, Spencer DD: Adenosine: a potential mediator of seizure arrest and postictal refractoriness. Ann Neurol, 1992, 32, 618–624. 16. Francis A, Fochtmann L: Caffeine augmentation of electroconvulsive seizures. Psychopharmacology, 1994, 115, 320–324. 17. Geiger JD, LaBella FS, Nagy JI: Ontogeny of adenosine receptors in the central nervous system of the rat. Develop Brain Res, 1984, 13, 97–104. 18. George B, Kulkarni SK: Modulation of lithium-pilocarpineinduced status epilepticus by adenosinergic agents. Meth Find Exp Clin Pharmacol, 1997, 19, 329–333.
19. Handforth A, Treiman DM: Effect of an adenosine antagonist and an adenosine agonist on status entry and severity in a model of limbic status epilepticus. Epilepsy Res, 1994, 18, 29–42. 20. Hauser WA, Annegers JF, Kurland LT: Incidence of epilepsy and unprovoked seizures in Rochester, Minnesota: 1935–1984. Epilepsia, 1993, 34, 543–568. 21. Marangos PJ, Patel J, Stivers J: Ontogeny of adenosine binding sites in rat forebrain and cerebellum. J Neurochem, 1982, 39, 267–270. 22. Mareš P, Haugvicová R, Kubová H: Unequal development of thresholds for various phenomena induced by cortical stimulation in rats. Epilepsy Res 2002, 49, 35–43. 23. Mareš P, Kubová H: What is the role of neurotransmitter systems in cortical seizures? Physiol Res, 2008, 57, Suppl 3, S111–S120. 24. Mareš P, Kubová H, Czuczwar SJ: Aminophylline exhibits convulsant action in rats during ontogenesis. Brain Dev, 1994, 16, 296–300. 25. Mareš P, Marešová D, Trojan S, Fischer J: Ontogenetic development of rhythmic thalamo-cortical phenomena in the rat. Brain Res Bull, 1982, 8, 765–769. 26. Mareš P, Šlamberová R: Efficacy of bretazenil against cortical epileptic aferdischarges increases during early ontogeny in rats. Pharmacol Rep, 2006, 58, 519–525. 27. Mareš P, Tabashidze N: Contradictory effects of GABA-B receptor agonists on cortical epileptic afterdischarges in immature rats. Brain Res Bull, 2008, 75, 173–178. 28. Morgan PF, Deckert J, Jacobson KA, Marangos PJ, Daly JW: Potent convulsant actions of the adenosine receptor antagonist, xanthine amine congener (XAC). Life Sci, 1989, 45, 719–728. 29. Nabbout R, Dulac O: Epileptic syndromes in infancy and childhood. Curr Opin Neurol, 2008, 21, 161–166. 30. Pagonopoulou O, Efthimiadou A, Asimakopoulos B, Nikolettos NK: Modulatory role of adenosine and its receptors in epilepsy: Possible therapeutic approaches. Neurosci Res, 2006, 56, 14–20. 31. Polášek R, Kubová H, Šlamberová R, Mareš P, Vorlíèek J: Suppression of cortical epileptic afterdischarges in developing rats by anticonvulsants increasing GABAergic inhibition. Epilepsy Res, 1996, 25, 177–184. 32. Racine RJ: Modification of seizure activity by electrical stimulation: II. Motor seizures. Electroenceph Clin Neurophysiol, 1972, 32, 281–294. 33. Trommer BL, Pasternak JF, Suyeoka GM: Proconvulsant effects of aminophylline during amygdala kinding in developing rats. Dev Brain Res, 1989, 46, 169–172.
Received:
February 11, 2009; in revised form: July 17, 2009.
Pharmacological Reports, 2010, 62, 6267
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Copyright © 2010 by Institute of Pharmacology Polish Academy of Sciences
2010, 62, 6267 ISSN 1734-1140
Effects of 2-chloroadenosine on cortical epileptic afterdischarges in immature rats Marie Pometlová1, Hana Kubová2, Pavel Mareš2 Charles University, Third Medical School, Department of Normal, Pathological and Clinical Physiology, Ke Karlovu 4, CZ-12000 Prague 2, Czech Republic
Institute of Physiology, Academy of Sciences of the Czech Republic, Department of Developmental Epileptology, Vídenská 1083, CZ-14220 Prague 4, Czech Republic Correspondence: Pavel Mare, e-mail:
[email protected]
Abstract: Adenosine may represent an endogenous anticonvulsant in the brain. This study focused on the possible anticonvulsant action of an adenosine agonist, 2-chloroadenosine, against cortical epileptic afterdischarges (ADs) in immature rats. Three age groups of rat pups with implanted electrodes were studied: 12-, 18- and 25-days-old. The compound, 2-chloroadenosine, was injected after the first successful stimulation at doses of 1, 4 or 10 mg/kg intraperitoneally, and stimulation at the same intensity was repeated three more times. Movements directly elicited by stimulation, as well as clonic seizures accompanying electroencephalography (EEG) ADs, were markedly suppressed in only the 18-day-old animals. The effects in the 12- and especially the 25-day-old rats were moderate. The duration of the ADs decreased in all three age groups with 2-chloroadenosine treatment, and the shortest AD duration was seen in the treated, 12-day-old rats. The AD suppression also lasted longer in this age group than it did in the older animals. After a brief suppression of the second AD, the treated, 25-day-old group exhibited a significant AD rebound during the third and fourth stimulations. Taken together, our data show that 2-chloroadenosine exhibits an anticonvulsant effect that is dose- and age-dependent. Key words: cerebral cortex, electrical stimulation, afterdischarges, 2-chloroadenosine, ontogeny, rat
Abbreviations: AD – cortical afterdischarges, GABA – g-aminobutyric acid
Introduction The adenosinergic system is an important inhibitory modulator in the brain [5, 14]. A possible role of adenosine in epilepsies has been repeatedly reviewed [11, 13, 30]. A role for adenosine in the regulation of epileptic activity has been demonstrated in animal
62
Pharmacological Reports, 2010, 62, 6267
models [7, 12] as well as in human tissue [15]. In agreement with these findings are data for the proconvulsant and/or convulsant action of the adenosine antagonists, caffeine and others [9, 16, 28], as well as their interaction with antiepileptic drugs [8] in adult animals. In the first developmental study, Trommer et al. [33] demonstrated a proconvulsant effect of aminophylline in a rat kindling model. Related to that study, we demonstrated a higher sensitivity of immature two- and three-week-old rats to the convulsant action of aminophylline [24]. Similarly, subconvulsant doses of aminophylline markedly potentiated the cortical epileptic afterdischarges (ADs) and a spread
Anticonvulsant action of 2-chloroadenosine
Marie Pometlová et al.
of this activity into the limbic system [4]. On the other hand, developmental data on the anticonvulsant action of adenosine and its agonists are missing. The question of whether adenosine agonists could serve as anticonvulsant drugs cannot be answered without data from immature experimental animals. Nearly one half of all epilepsies start during infancy and childhood [20]. Some age-dependent forms of epilepsy represent a serious therapeutic problem [29]. To begin studies on the anticonvulsant action of the adenosinergic system in developing rats, cortical epileptic ADs were used as a model. In this model, cortical epileptic ADs induced by the rhythmic electrical stimulation of the sensorimotor cortical region allows for the study of at least three phenomena: movements elicited by the stimuli, EEG ADs and clonic seizures accompanying the ADs. We have data on the anticonvulsant effects of drugs influencing various neurotransmitter systems, including g-aminobutyric acid A (GABA-A) [26, 31], GABA-B [27], and excitatory amino acids [23]). We have also described a proconvulsant effect of the adenosine antagonist theophylline (an active compound of aminophylline) in this model [4]. 2-Chloroadenosine was chosen as an agonist of the adenosine receptors because of the availability of data on its action in adult animals [1, 6, 7, 12, 18, 19].
Methods Experiments were performed using three different age groups of immature Wistar rats; the rats were 12-, 18and 25-days-old. The level of brain development in the 12-day-old rats corresponds to the early postnatal stage of the human brain. In rats, postnatal day 18 is the earliest age at which a spike-and-wave EEG rhythm can be recorded, while postnatal day 25 is close to the start of sexual maturation. This project was approved by Animal Care and Use Committee to be in agreement with the Animal Protection Law of the Czech Republic and is fully compatible with the European Community Council directives 86/609/EEC. Flat silver cortical electrodes were implanted into animals epidurally under ether anesthesia. Stimulation electrodes were placed on the right sensorimotor (frontal) cortex, while recording electrodes were placed on the left sensorimotor, parietal and occipital cortex, as well as on the right occipital cortex. A reference electrode was placed on the nasal bone,
a grounding electrode in the occipital bone. The whole surgical preparation lasted less than 15 min and the animals were allowed to recover for 1 h. Righting, placing and suckling reflexes were examined and only then was the stimulation initiated. Initial experiments were performed with classical paper EEG recording, later the system was replaced with a paperless recording (Kaminskij Biomedical Systems, Prague) with a digitalization rate of 500 Hz. The stimulator with a constant current output generated 1 ms biphasic pulses in 15 s series with 8 Hz frequency. Threshold intensity for elicitation of an epileptic AD lasting more than 3 s was established and this intensity was applied during the whole experiment (i.e. four times at 10 min intervals). The first AD always served as the control AD. Five minutes after the end of the first AD, 2-chloroadenosine (Sigma, St. Louis, MO, USA) was administered by intraperitoneal injection. The following three doses of 2-chloroadenosine were used in these experiments: 1, 4, and 10 mg/kg. Pattern and duration of ADs were evaluated. The intensity of the movements elicited by stimulation, as well as clonic seizures accompanying the spike-andwave type of ADs were quantified by means of a modified Racine’s 5-point scale [22, 32]. Movements induced by individual stimuli reflect the excitability of the motor cortex and the motor system up to the muscles of the contralateral forelimb. Clonic seizures indicate the spread of epileptic activity generated in the thalamocortical circuits into the motor system. Statistical comparison between the control and the three dosage groups was performed by one-way analysis of variance (ANOVA). Comparison of the four ADs in each age and dose group were analyzed by one-way repeated measure (RM) ANOVA. The Holm-Sidak test was used for subsequent pairwise comparison (SigmaStat® SYSTAT). The level of statistical significance was set at 5%.
Results Control animals
The first stimulation elicited an AD in all of the animals. The duration of the first AD in the 12-day-old rats was significantly longer than in the two older groups. Control rats (injected with saline after the first AD) continued to generate ADs after all subsequent Pharmacological Reports, 2010, 62, 6267
63
Fig. 1. Electrocorticographic recording of a cortical epileptic afterdischarge (AD) in an 18-day-old rat. Individual leads from top to bottom: left frontal (LF), left occipital (LO) and right occipital (RO) regions, always in a reference connection. At the beginning, the last second of stimulatio n is recorded and then the AD is registered. The numbers over the AD denote motor phenomena according to the modified Racines scale (see Methods). Time mark is 2 s and amplitude calibration is 0.5 mV
stimulations. The intensity of the movements directly bound to the stimulation remained stable with repeated stimulations in the 12- and 18-day-old rats, while the intensity of the movements moderately decreased in the 25-day-old rats. The duration of ADs tended to increase during the experiments in the two younger groups, but did not change in the 25-day-old rats (Fig. 1). The intensity of the clonic seizures was not significantly changed in the youngest group, while the intensity of the clonic seizures significantly decreased in the fourth AD in the 18-day-old animals and in the second and fourth ADs in 25-day-old rats. During stimulation, as well as during clonic seizures, the 12-day-old rats reared only exceptionally (always with support) and the maximal intensity was usually grade 3.
Movements induced by stimulation (Fig. 2)
The injection of 2-chloroadenosine did not significantly influence the intensity of movements directly bound to stimulation in the 12-day-old rats compared to the appropriate stimulations in the control group. On the other hand, the 18-day-old animals exhibited a significantly lower intensity of movements in the second and third stimulation after all three doses of 2-chloroadenosine compared with the appropriate stimulations in the control animals. The decrease in movement intensity during the fourth stimulation was significant only at the highest dose of the agonist. Significant effects with respect to movement intensity were seen only exceptionally in the 25-day-old rats. 64
Pharmacological Reports, 2010, 62, 6267
Fig. 2.
Intensity of movements during stimulation (mean ± SEM) in
12-, 18- and 25-day-old rats (from top to bottom, see numbers on the right). Abscissae are the first to the fourth stimulation and ordinates indicate the 5-point scale (see Methods). For individual doses see in set. Asterisks indicate a significant difference in comparison with the appropriate first stimulation in each group
Anticonvulsant action of 2-chloroadenosine
Marie Pometlová et al.
Fig. 3.
Relative duration of afterdischarges (ADs). The first AD for
each dosage group was taken as 100%. Details are the same as in
Fig. 4. Intensity of clonic convulsions accompanying afterdischarges (ADs). Details are found in Figure 2
Figure 2, only the ordinates indicate the duration of the ADs. Upper panel (12-day-old rats): n means complete suppression of AD
in the animals given 4 and 10 mg/kg of 2-chloroadenosine compared to the control group. Duration of ADs (Fig. 3) Intensity of clonic seizures (Fig. 4)
The two highest doses of 2-chloroadenosine markedly shortened the ADs after the second and third stimulation in the 12-day-old rats. The ADs were completely suppressed in the second stimulation series after injection of 2-chloroadenosine at the 10 mg/kg dose. No effect of 2-chloroadenosine was observed on the fourth AD. The effect of 2-chloroadenosine was even shorter in the 18-day-old rats. A significant shortening of the ADs was observed only in the second stimulation series after injection of the 4 and 10 mg/kg dosages of 2-chloroadenosine. In contrast, the 4 and 10 mg/kg doses tended to prolong the fourth ADs, but due to the high variability of the data significance was not reached. Similar effects were recorded in the 25-day-old animals: the second ADs were shortened, but the third and fourth ADs were significantly longer
The results for the intensity of clonic seizures were similar to the results of the movements elicited by stimulation. A significant decrease in the intensity of seizures was found in the 12-day-old rats, but only in the rats treated with the 4 mg/kg dose of 2-chloroadenosine in the second AD. Injection of 10 mg/kg of 2-chloroadenosine resulted in no ADs at all after the second stimulation. The 18-day-old rats exhibited a significant decrease in the intensity of the seizures after all three doses of 2-chloroadenosine in the second and third ADs. No significant effect on seizure intensity was found at any dose for the fourth AD. The oldest group exhibited only one significant effect with respect to seizure intensity; the 10-mg/kg dose resulted in less intense seizures in the second AD. Pharmacological Reports, 2010, 62, 6267
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Discussion The developmental changes seen in the control animals correspond with our previous results [22]. Changes in ADs were observed with age. The EEG pattern was formed by rhythmic sharp waves, while the spike-and-wave rhythm was recorded in the two older animal groups. These data are in agreement with the development of other rhythmic phenomena of thalamocortical origin [25]. The duration of ADs was longer in the youngest group of animals and is probably due to immature mechanisms of arresting seizures and may be responsible for the postictal refractoriness (Mareš et al., unpublished results). The development of motor abilities is reflected in the failure of the 12day-old rats to rear during stimulation and/or during ADs. The ability to rear fully develops in the third postnatal week [3]. We treated the animals with 2-chloroadenosine to determine whether it possesses anticonvulsant activity. 2-Chloroadenosine exhibited an anticonvulsant action in all three age groups; however, there were differences in the motor and EEG phenomena. Movements during stimulation and clonic seizures were markedly suppressed in the 18-day-old rats, while the effects in the other two age groups were negligible. These data indicate that the spread of activity from the cerebral cortex and the thalamocortical system to lower levels of the motor system is especially sensitive to the inhibitory action of the adenosinergic system at postnatal day 18. In contrast, the generation of epileptic activity in the 18-day-old rats was affected less than in the youngest group. These data indicate that the site of action is not at the cortical level. EEG ADs were markedly influenced in the 12-day-old rat pups. The shortening of the ADs in the 18- and especially the 25-day-old animals was only short-lasting. In addition, there was a clear rebound phenomenon, as demonstrated by a significant prolongation of the fourth ADs in the oldest group. The marked shortening of ADs in the 12-day-old rats is in agreement with our study on the action of aminophylline in the same model. In our previous study, we observed that the prolongation was the largest in the same age group [4]. The convulsant action of high doses of aminophylline was also stronger in the younger age groups than in the 25-day-old rats [24]. Differences in the effects of aminophylline on amygdala kindling between 15-day-old and adult rats also proved a higher effi-
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cacy of manipulation of the adenosinergic system in immature animals [33]. The higher sensitivity of the immature brain to the adenosine antagonist aminophylline [4, 24, 33], as well as to an agonist 2-chloroadenosine demonstrated in the present experiments, does not correspond with the ontogeny of adenosine receptors. Binding of [3H]cyclohexyladenosine is present in all of the studied brain structures at birth and the binding maximum (Bmax) increases, especially during the first three postnatal weeks. In contrast, the dissociation constant (Kd) remains stable [17, 21]. Adenosine receptors in the cerebral cortex exhibit a steep (nearly ten-fold) increase between postnatal days 1 and 21 [17]. This contradiction cannot be explained presently and remains to be studied. Our results also contradict the data of Descombes et al. [10]. However, their in vivo experiments were performed in the hippocampal CA3 field and the development of the cerebral cortex and the hippocampus may differ. However, the presence of a significant increase in GTP [g-35S]-binding following N6-cyclopentyladenosine stimulation of A1 receptors in the cerebral cortex of 14-day-old rats support our findings [2]. Dragunow et al. [12] classified adenosine as an endogenous anticonvulsant. Adenosinergic system also possesses anticonvulsant action in immature rodents, but the rebound effect found in our experiments needs to be analyzed and the peripheral (cardiovascular) effects of adenosine agonists must be taken into account. Additionally, it is necessary to differentiate the effect of 2-chloroadenosine on A1 and A2 receptors using specific agonists or antagonists. Furthermore, the possible negative side effects of adenosine agonists should be studied.
Acknowledgments:
This study was supported by a grant (No. NR9184/3) from the Grant Agency of the Ministry of Health of the Czech Republic and by a Research Project AV0Z 50110509.
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Received:
February 11, 2009; in revised form: July 17, 2009.
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