J Neural Transm DOI 10.1007/s00702-014-1234-y
TRANSLATIONAL NEUROSCIENCES - ORIGINAL ARTICLE
A1 not A2A adenosine receptors play a role in cortical epileptic afterdischarges in immature rats Pavel Maresˇ
Received: 6 January 2014 / Accepted: 28 April 2014 Ó Springer-Verlag Wien 2014
Abstract Endo- as well as exogenous adenosine exhibits anticonvulsant action. Participation of individual types of adenosine receptors was studied in present experiments in immature rats. Cortical epileptic afterdischarges were used as a model in rat pups 12, 18 and 25 days old. CCPA, an agonist of A1 adenosine receptors, decreased markedly duration of afterdischarges whereas DPCPX, an antagonist of A1 receptors, exhibited strong proconvulsant action. Action of either drug was best expressed in 12-day-old rats and it decreased with age. Drugs influencing A2A adenosine receptors (agonist CGS21680 and antagonist ZM241385) did not exhibit systematic effects in our model. Motor phenomena accompanying cortical stimulation or epileptic afterdischarge were never influenced by any of the four drugs studied. A1 adenosine receptors are important in the model of cortical seizures, especially in the youngest group studied. Keywords Adenosine receptors Epileptic afterdischarges Cerebral cortex Ontogeny Rat
Introduction Adenosine was proposed as an endogenous anticonvulsant (Dragunow 1988). Modulatory adenosinergic system, which decreases the excitability of wide parts of the brain was described (Fredholm et al. 2005) and influencing this system by systemic or local application may be a new way
P. Maresˇ (&) Department of Developmental Epileptology, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic e-mail: [email protected]
how to suppress seizures (Boison 2005, 2013a; Li et al. 2008; Pagonopoulou et al. 2006; Szybala et al. 2009). Caffeine, a nonspecific antagonist of adenosine receptors, exhibits proconvulsant (Francis and Fochtmann 1994; Kulkarni et al. 1997) or (in very high doses) convulsant action (Chu 1981; Morgan and Durcan 1990). High doses also inhibit phosphodiesterase (Nehlig et al. 1992); analogous action was demonstrated for aminophylline composed of adenosine receptor antagonist theophylline and ethylenediamine (Albertson et al. 1983; Handforth and Treiman 1994; Kulkarni et al. 1997). 2-Chloroadenosine, a nonspecific adenosine receptor agonist, possesses anticonvulsant activity (Abdul-Ghani et al. 1997; Dunwiddie and Worth 1982; Handforth and Treiman 1994; Khan et al. 2000; Pourgholami et al. 1997; Turski et al. 1985). Our previous experiments demonstrated anticonvulsant action of 2-chloroadenosine also in immature rats: it was found to suppress cortical epileptic afterdischarges (Pometlova´ et al. 2010) as well as motor seizures elicited by pentetrazol, i.e., convulsant drug acting on GABAA receptors (Maresˇ 2010). Opposite, proconvulsant or convulsant effect was observed in immature animals with caffeine (Tchekalarova et al. 2010) and with another nonspecific adenosine receptor antagonist aminophylline (Berna´sˇkova´ and Maresˇ 2000; Hung et al. 2002; Maresˇ et al. 1994; Trommer et al. 1989). Developmental studies of the role of adenosine in epileptic seizures are important because at least half of human epilepsies start in infancy or early childhood (Hauser and Hesdorffer 1990). Adenosine exerts its action through four types of G-protein coupled receptors: A1, A2A, A2B and A3. The A1 and A2A represent major types of adenosine receptors in the brain. The A1 adenosine receptors are widely distributed especially in cortical structures (cerebral cortex, hippocampus), A2A receptors are nearly exclusively
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localized in basal ganglia, their representation in other brain structures is low (Jacobson 2009; Ribeiro et al. 2002). Anticonvulsant action is usually ascribed to activation of A1 adenosine receptors, but there are some results indicating also a role of A2A receptors (for review Dunwiddie and Masino 2001; Pagonopoulou et al. 2006). Recently, we demonstrated an important role of A1 but not A2A adenosine receptors in convulsions elicited by pentetrazol in immature rats (Maresˇ 2010), but the participation of these two types of adenosine receptors may differ in models of various types of seizures—for e.g., activation of adenosine A2 receptors exhibits proconvulsant effect in hyperthermic seizures in immature rats (Fukuda et al. 2010). Brain structures representing generators of different types of seizures may also play a role. Two types of convulsive seizures elicited by pentetrazol have generators in the brainstem (generalized tonic–clonic seizures) or in basal forebrain (minimal clonic seizures—Browning and Nelson 1985). Epileptic afterdischarges (ADs) elicited by cortical stimulation (a model of different type of epileptic– myoclonic seizures routinely used in our laboratory, e.g., Maresˇ et al. 2002; Maresˇ and Kubova´ 2006; Lojkova´Janecˇkova´ et al. 2009) were chosen in the present study because of the site of origin in cortico-thalamo-cortical circuit and a spread of epileptic activity into a motor system (Maresˇ et al. 2002). This model of reactive seizures allows to evaluate not only EEG epileptic activity, but also motor phenomena accompanying stimulation and EEG seizures (Maresˇ et al. 2013). Adenosine was found to be important for the arrest of seizures in slices from resected human epileptic hippocampi (During and Spencer 1992) as well as in animal models of seizures (Boison 2013b), but no detailed discrimination of the role of individual types of adenosine receptors was presented. In addition, different brain structures may use various neurotransmitter mechanisms to arrest seizures (Velı´sˇek and Maresˇ 1992). We hypothesized that A1 adenosine receptors play a leading role in cortical afterdischarges.
Methods The experiments were approved by Animal Care and Use Committee of the Institute of Physiology, Academy of Sciences of the Czech Republic to be in agreement with Animal Protection Law of the Czech Republic and European Community Council directives 86/609/EEC.
2002). Cortical flat silver electrodes were implanted epidurally under ether anesthesia. Stimulation electrodes were placed over right sensorimotor area (AP -1 and ?1; L 2.5 mm), recording electrodes over left sensorimotor (AP 0; L 2.5), parietal and occipital areas and over right occipital region. Coordinates for parietal and occipital electrodes were recalculated from adult coordinates (AP 3, L 3 and AP 6, L 4 mm, respectively) on the basis of bregma–lambda distance. Reference electrode was inserted into nasal bone, grounding electrode was over cerebellum. The whole assembly was fixed to the scull with a fastcuring dental acrylic. Surgical preparation lasted 10–12 min, then the animals were allowed to recover for 1 h (righting, placing and suckling reflexes as well as spontaneous locomotion were normal at that time) and only then the experiment started. Body temperature of 12- and 18-day-old rats was maintained by means of a pad heated electrically on 34 °C (i.e., the temperature in the nest). Drugs Drugs specific for subtypes of adenosine receptors were tested. A specific A1 receptor agonist CCPA (2-chloro-N6cyclopentyladenosine) was administered in doses of 0.5 and 1 mg/kg i.p., an A1 receptor antagonist DPCPX (8cyclopentyl-1,3-dipropylxantine) in doses 1 and 2 mg/kg i.p., an A2A receptor agonist CGS 21680 (4-[2-[[6-amino9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride) in doses 0.5 and 5 mg/kg i.p. and A2A receptor antagonist ZM 241385 (4-(2-(7-amino-2-(2-furyl)[1,2,4]triazolol[2,3a][1,3,5]thiazin-5-ylamino)ethyl)phenol) in doses 1 and 5 mg/kg i.p. The doses were chosen on the basis of our previous experiments with pentetrazol-induced seizures to be directly comparable (Maresˇ 2010). All drugs were purchased from Tocris Biosciences (Bristol, UK). CCPA (concentration 1 mg/ml) was put into suspension with a drop of Tween 80, CGS 21680, DPCPX and ZM 241385 were dissolved in dimethylsulfoxide (1, 2 and 1 mg in 1 ml, respectively), then diluted with distilled water in a ratio 1:1. All drugs were prepared freshly just before the start of the experiment. Control group for CCPA was formed by rats injected with saline, control animals for the other three drugs received 50 % dimethylsulfoxide (DMSO) in a volume corresponding to the 5-mg/kg dose of CGS21680 and ZM241385 injected (i.e., 2.5 ml/kg) a common group for all three drugs was formed. Individual age and dose groups were formed by 7–10 animals.
Animals Stimulation and recording A total of 249 Wistar albino male rat pups 12, 18 and 25 days old were used. Cortical epileptic afterdischarges cannot be reliably elicited in younger animals (Maresˇ et al.
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Stimulation series lasting 15 s consisted from biphasic pulses with 0.5-ms duration applied at 8-Hz frequency.
Role of adenosine receptors in cortical epileptic afterdischarges Fig. 1 EEG recordings of afterdischarges in 12- (left graphs) and 25-day-old rats (right graphs). Upper row represents control afterdischarges, lower row third afterdischarges after administration of DPCPX in a dose of 1 mg/kg i.p. Individual leads in each graph (from top to bottom): LF left frontal, LP left parietal, LO left occipital, RO right occipital cortical region. Time mark 2 s, amplitude calibration 1 mV
Stimulator with constant current output was driven by a computer. At first, intensity generating afterdischarges with at least 3-s duration (Fig. 1) was found and then used throughout the experiment. As in our previous studies (Maresˇ et al. 2002; Maresˇ 2013), the intensity had to be higher in 12-day-old rats (starting with a 5-mA intensity, sometimes it was necessary to go up to 10.5 mA) than in the two older groups (usually 3, in a few cases 4.5 or 5 mA). Stimulation series were applied six times, interval between these series was 10 min. Five min after the first successful stimulation the drugs were administered. Electrocorticographic activity was recorded 20 s before stimulation, during stimulation and AD and at least 20 s after the end of the AD. Activity was amplified, collected and digitalized at a 2-kHz frequency and saved using CED Power1401 system and Spike 2 software. Motor phenomena were coded directly into the recording. Duration of ADs was measured and intensity of motor phenomena was classified by means of a modified Racine‘s 5-point scale (Maresˇ et al. 2002). The highest grade of motor phenomena was used for statistical evaluation. Statistics Duration of the six ADs (actual values in seconds) in each age and dose group was compared with repeated-measures ANOVA. Corresponding ADs in control and two dose groups were evaluated with one-way ANOVA. Subsequent pairwise comparison was always performed with HolmSidak test. All statistical evaluation was done with SigmaStatÒ software (SYSTAT Inc.), original data were used for these calculations but columns in figures represent
relative duration of ADs—the first one in each age and dose group was taken as 100 %. The level of statistical significance was set at 5 %.
Results Duration of afterdischarges CCPA (Fig. 2). Control animals exhibited statistically significant prolongation of cortical afterdischarges with repeated stimulations in all three studied age groups. This effect was best expressed in 12-day-old animals. Both doses of specific agonist of adenosine A1 receptors CCPA not only blocked this prolongation, but markedly decreased the duration of the third to sixth ADs in 12- and 18-day-old rats if compared to the first predrug ADs as well as to the corresponding ADs in the control groups. In contrast, such effect was absent in 25-day-old animals. The fifth and sixth ADs after both 0.5- and 1-mg/kg doses were significantly longer than the first, control AD. The only difference was in the fourth AD where the level of significance was reached only in the control group. DPCPX (Figs.1, 3). Control animals injected with DMSO exhibited a progressive prolongation of ADs in all age groups. Again, this effect was best expressed in 12-day-old rats. An antagonist of A1 adenosine receptors DPCPX led to a dose-dependent excessive prolongation of ADs in 12-day-old rats; the 2-mg/kg dose resulted in 17-fold increase in duration. Similar but not so strong effect was present also in 18-day-old rats. Significant prolongation of ADs was found in the second AD, i.e.,
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Fig. 2 Effects of adenosine A1 receptor agonist CCPA on relative duration of afterdischarges (mean ? SEM) in three age groups—from top to bottom 12-, 18- and 25-day-old rats. Duration of the first, predrug AD was always taken as 100 %, and duration of subsequent ADs was related to this value. Abscissae: the first to sixth AD; ordinates: duration of ADs in percents of the first one. White columns—control rats injected after the first AD with saline, obliquely stripped columns—animals given 0.5 and/or 1 mg of CCPA (see inset). Asterisks denote significant difference in comparison with the first AD, circles denote significant difference from corresponding control ADs
5 min after the injection of DPCPX. Third and fourth ADs in treated animals were significantly longer than corresponding control ADs. In contrast, the only significant difference between control- and DPCPX-treated 25-dayold animals was found in the second and third ADs, especially after the 2-mg/kg dose. CGS 21680 (Fig. 4). Either dose of adenosine A2A receptor agonist CGS 21680 resulted in a tendency to attenuation of progressive prolongation of ADs in 12-dayold rats. No systematic changes of ADs duration were observed in 18- and 25-day-old animals. ZM 241385 (Fig. 5). Antagonist of A2A receptors ZM 241385 did not exhibit any marked action in 12-day-old
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Fig. 3 Effects of adenosine A1 receptor antagonist DPCPX on relative duration of afterdischarges (mean ? SEM) in three age groups—from top to bottom 12-, 18- and 25-day-old rats. Details as in Fig. 2, only stripped columns demonstrate groups with 1- and 2-mg/ kg doses of DPCPX
rats. The only difference was in the second AD—both doses of ZM 241385 led to a significant prolongation, whereas a change in control animals did not reach the level of significance. Eighteen-day-old rats exhibited a slight tendency to suppression of progressive prolongation of ADs; there was only a tendency to an increase of ADs duration after the 5-mg/kg dose in contrast to significant changes in control and 1-mg/kg groups. An unexpected result was suppression of prolongation of ADs after the 1but not 5-mg/kg dose of ZM241385 in 25-day-old animals, but the differences between treated and control groups did not reach the level of significance. Motor phenomena Intensity of movements directly elicited by stimulation and/or clonic seizures accompanying ADs was classified as stage 3 (clonic movements of forelimbs) in nearly all
Role of adenosine receptors in cortical epileptic afterdischarges
Fig. 4 Effects of adenosine A2A receptor agonist CGS 21680 on relative duration of afterdischarges (mean ? SEM) in three age groups—from top to bottom 12-, 18- and 25-day-old rats. Details as in Fig. 2, only stripped columns demonstrate groups with 0.5- and 5-mg/ kg doses of CGS 21680
stimulations in all age and dose groups, stage 4 (i.e., rearing) was seen exceptionally in 18- and 25-day-old rats. There was no significant difference under the influence of the four drugs studied, i.e., motor phenomena were not affected by any drug used (data not shown).
Discussion Both drugs influencing A1 type of adenosine receptors resulted in marked systematic changes of epileptic afterdischarges, whereas effects of agonist of A2A receptors CGS 21680 were negligible and the A2A receptor antagonist exhibited marked effect only in a low dose in 25-dayold rats. Cortical epileptic afterdischarges represent a model of different type of human seizures—myoclonic ones. Their ontogeny was described in detail in our laboratory:
Fig. 5 Effects of adenosine A2A receptor antagonist ZM 241385 on relative duration of afterdischarges (mean ? SEM) in three age groups—from top to bottom 12-, 18- and 25-day-old rats. Details as in Fig. 2, only stripped columns demonstrate groups with 1- and 5-mg/ kg doses of ZM 241385
threshold intensities of stimulation current are lowest in the third and fourth week of postnatal life in rats (Maresˇ et al. 2002). Higher threshold in 12-day-old rats is due to immaturity of connections between cortical and thalamic neurons (Scheibel et al. 1976) making synchronization of their activity difficult. Cortical ADs might be suppressed by various drugs in adult (Kubova´ et al. 1996) as well as in developing rats (Kubova´ et al. 1993; Pola´sˇek et al. 1996; Krsˇek et al. 1998; Pometlova´ et al. 2010). Pharmacological sensitivity of EEG and motor phenomena markedly differs—both movements during stimulation and clonic seizures accompanying ADs are resistant to antiepileptic drugs. The only class of drugs markedly suppressing motor phenomena are benzodiazepines (Kubova´ et al. 1993). Our results are in agreement with data from adult animals—not only adenosine (Kulkarni et al. 1997) and its nonspecific agonist 2-chloroadenosine (Abdul-Ghani et al. 1997; Dunwiddie and Worth 1982; Handforth and Treiman 1994; Khan et al. 2000; Pourgholami et al. 1997; Turski
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et al. 1985), but also agonists of A1 adenosine receptors CPA and CCPA (Adami et al. 1995; de Sarro et al. 1999; Malhotra and Gupta 1997) were found to have anticonvulsant action. Adenosine A1 receptor antagonist DPCPX was found to block anticonvulsant action of adenosine and CPA (Malhotra and Gupta 1997) and in high doses it induced seizures (de Sarro et al. 1999). Failure of drugs influencing A2A adenosine receptors in our experiment is in contradiction with some literary data, demonstrating anticonvulsant action of 2HE-NECA and CGS21680 against audiogenic seizures (de Sarro et al. 1999) and 2HE-NECA against pentetrazol-induced seizures (Adami et al. 1995) in adult rodents. There are two points which could explain this difference: (1) different models used, and (2) mature vs. developing brain. The most probable possibility that both factors participate in the difference should be tested in future. Effects of A1 receptor agonist CCPA in the present experiments were similar to those of 2-chloroadenosine (Pometlova´ et al. 2010) not only in the effect on duration of ADs, but also in an ontogenetic profile—a decrease of activity in 12- to 25-day-old rats. Corresponding developmental profile was found also for proconvulsant effect of A1 receptor antagonist DPCPX—prolongation of ADs was most expressed in 12-day-old rat pups. This developmental profile is in contradiction with data on the ontogeny of adenosine receptors in rodents. Both A1 and A2 adenosine receptors were found to be present in many brain structures at birth, and their Bmax increases during the first three postnatal weeks (Geiger et al. 1984; Marangos et al. 1982), an increase was described also for adenosine uptake sites (Morgan et al. 1987). Data for cerebral cortex demonstrated that postnatal increase of binding in A1 receptors is much higher than in A2A receptors (Johansson et al. 1997). An increase of Bmax measured with [3H]cyclohexyladenosine was found between P1 and P21 (Geiger et al. 1984), or between prenatal values and P25 (Morgan et al. 1987). An explanation of the contradictory development of anticonvulsant activity and binding might be explained by developmental changes in coupling of adenosine receptors to G proteins or to another part of metabolic cascade. Such changes were demonstrated in another metabotropic receptor—GABAB—during postnatal development (Kagan et al. 2012). Explanation might be also in an interaction with other major neurotransmitter systems—e.g., GABAergic inhibitory system finishes its maturation in the third postnatal week in rats overtaking a role of the main inhibitory system (Sanchez and Jensen 2001). Importance of adenosinergic inhibitory modulation might thus decrease. Failure of all four drugs to affect motor phenomena connected with stimulation or afterdischarges was surprising, especially in A2A drugs known to induce
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kataplexia (Wardas et al. 2003; Wu et al. 2009). Our previous experiments with pentetrazol-induced seizures demonstrated that rat pups pretreated with high doses of CGS21680 or ZM241385 did not exhibit spontaneous activity, but they did react physically to injection— administration of pentetrazol (Maresˇ 2010). It is highly improbable that the effects of DPCPX are mediated by an interaction with phosphodiesterase. There are data for DPCPX demonstrating that IC50 for inhibition of phosphodiesterase activity is about 10,000-fold higher than the inhibition constant for antagonism at A1 adenosine receptors (Ukena et al. 1993).
Conclusions Adenosine A1 receptors, but not A2A receptors, were demonstrated to play a role in cortical epileptic afterdischarges in immature rats. Action of drugs affecting A1 adenosine receptors is strongest in 12-day-old rat pups and decreases with age. Our future studies will be focused on the role of adenosine in arrest of seizures and immediate postictal period. References Abdul-Ghani AS, Attwell PJ, Bradford HF (1997) The protective effect of 2-chloroadenosine against the development of amygdala kindling and on amygdala-kindled seizures. Eur J Pharmacol 326:7–14 Adami M, Bertorelli R, Ferri N, Foddi MC, Ongini E (1995) Effect of repeated administration of selective adenosine A1 and A2A receptor agonists on pentylenetetrazole-induced convulsions in the rat. Eur J Pharmacol 294:383–389 Albertson TE, Stark LG, Joy RM, Bowyer JF (1983) Aminophylline and kindled seizures. Exp Neurol 81:703–713 Berna´sˇkova´ K, Maresˇ P (2000) Proconvulsant effect of aminophylline on cortical epileptic afterdischarges varies during ontogeny. Epilepsy Res 39:183–190 Boison D (2005) Adenosine and epilepsy: from therapeutic rationale to new therapeutic strategies. Neuroscientist 11:25–36 Boison D (2013a) Role of adenosine in status epilepticus: a potential new target? Epilepsia 54(Suppl 6):20–22. doi:10.1111/epi.12268 Boison D (2013b) Adenosine and seizure termination: endogenous mechanisms. Epilepsy Curr 13(1):35–37. doi:10.5698/15357511-13.1.35 Browning RA, Nelson BK (1985) Variation in threshold and pattern of electroshock-induced seizures in rats depending on site of stimulation. Life Sci 37:2205–2211 Chu N-S (1981) Caffeine- and aminophylline-induced seizures. Epilepsia 22:85–94 De Sarro G, De Sarro A, Di Paola ED, Bertorelli R (1999) Effects of adenosine receptor agonists and antagonists on audiogenic seizure-sensible DBA/2 mice. Eur J Pharmacol 371:137–145 Dragunow M (1988) Purinergic mechanisms in epilepsy. Progr Neurobiol 31:85–108 Dunwiddie TV, Masino SA (2001) The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci 24:31–55
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TRANSLATIONAL NEUROSCIENCES - ORIGINAL ARTICLE
A1 not A2A adenosine receptors play a role in cortical epileptic afterdischarges in immature rats Pavel Maresˇ
Received: 6 January 2014 / Accepted: 28 April 2014 Ó Springer-Verlag Wien 2014
Abstract Endo- as well as exogenous adenosine exhibits anticonvulsant action. Participation of individual types of adenosine receptors was studied in present experiments in immature rats. Cortical epileptic afterdischarges were used as a model in rat pups 12, 18 and 25 days old. CCPA, an agonist of A1 adenosine receptors, decreased markedly duration of afterdischarges whereas DPCPX, an antagonist of A1 receptors, exhibited strong proconvulsant action. Action of either drug was best expressed in 12-day-old rats and it decreased with age. Drugs influencing A2A adenosine receptors (agonist CGS21680 and antagonist ZM241385) did not exhibit systematic effects in our model. Motor phenomena accompanying cortical stimulation or epileptic afterdischarge were never influenced by any of the four drugs studied. A1 adenosine receptors are important in the model of cortical seizures, especially in the youngest group studied. Keywords Adenosine receptors Epileptic afterdischarges Cerebral cortex Ontogeny Rat
Introduction Adenosine was proposed as an endogenous anticonvulsant (Dragunow 1988). Modulatory adenosinergic system, which decreases the excitability of wide parts of the brain was described (Fredholm et al. 2005) and influencing this system by systemic or local application may be a new way
P. Maresˇ (&) Department of Developmental Epileptology, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic e-mail: [email protected]
how to suppress seizures (Boison 2005, 2013a; Li et al. 2008; Pagonopoulou et al. 2006; Szybala et al. 2009). Caffeine, a nonspecific antagonist of adenosine receptors, exhibits proconvulsant (Francis and Fochtmann 1994; Kulkarni et al. 1997) or (in very high doses) convulsant action (Chu 1981; Morgan and Durcan 1990). High doses also inhibit phosphodiesterase (Nehlig et al. 1992); analogous action was demonstrated for aminophylline composed of adenosine receptor antagonist theophylline and ethylenediamine (Albertson et al. 1983; Handforth and Treiman 1994; Kulkarni et al. 1997). 2-Chloroadenosine, a nonspecific adenosine receptor agonist, possesses anticonvulsant activity (Abdul-Ghani et al. 1997; Dunwiddie and Worth 1982; Handforth and Treiman 1994; Khan et al. 2000; Pourgholami et al. 1997; Turski et al. 1985). Our previous experiments demonstrated anticonvulsant action of 2-chloroadenosine also in immature rats: it was found to suppress cortical epileptic afterdischarges (Pometlova´ et al. 2010) as well as motor seizures elicited by pentetrazol, i.e., convulsant drug acting on GABAA receptors (Maresˇ 2010). Opposite, proconvulsant or convulsant effect was observed in immature animals with caffeine (Tchekalarova et al. 2010) and with another nonspecific adenosine receptor antagonist aminophylline (Berna´sˇkova´ and Maresˇ 2000; Hung et al. 2002; Maresˇ et al. 1994; Trommer et al. 1989). Developmental studies of the role of adenosine in epileptic seizures are important because at least half of human epilepsies start in infancy or early childhood (Hauser and Hesdorffer 1990). Adenosine exerts its action through four types of G-protein coupled receptors: A1, A2A, A2B and A3. The A1 and A2A represent major types of adenosine receptors in the brain. The A1 adenosine receptors are widely distributed especially in cortical structures (cerebral cortex, hippocampus), A2A receptors are nearly exclusively
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localized in basal ganglia, their representation in other brain structures is low (Jacobson 2009; Ribeiro et al. 2002). Anticonvulsant action is usually ascribed to activation of A1 adenosine receptors, but there are some results indicating also a role of A2A receptors (for review Dunwiddie and Masino 2001; Pagonopoulou et al. 2006). Recently, we demonstrated an important role of A1 but not A2A adenosine receptors in convulsions elicited by pentetrazol in immature rats (Maresˇ 2010), but the participation of these two types of adenosine receptors may differ in models of various types of seizures—for e.g., activation of adenosine A2 receptors exhibits proconvulsant effect in hyperthermic seizures in immature rats (Fukuda et al. 2010). Brain structures representing generators of different types of seizures may also play a role. Two types of convulsive seizures elicited by pentetrazol have generators in the brainstem (generalized tonic–clonic seizures) or in basal forebrain (minimal clonic seizures—Browning and Nelson 1985). Epileptic afterdischarges (ADs) elicited by cortical stimulation (a model of different type of epileptic– myoclonic seizures routinely used in our laboratory, e.g., Maresˇ et al. 2002; Maresˇ and Kubova´ 2006; Lojkova´Janecˇkova´ et al. 2009) were chosen in the present study because of the site of origin in cortico-thalamo-cortical circuit and a spread of epileptic activity into a motor system (Maresˇ et al. 2002). This model of reactive seizures allows to evaluate not only EEG epileptic activity, but also motor phenomena accompanying stimulation and EEG seizures (Maresˇ et al. 2013). Adenosine was found to be important for the arrest of seizures in slices from resected human epileptic hippocampi (During and Spencer 1992) as well as in animal models of seizures (Boison 2013b), but no detailed discrimination of the role of individual types of adenosine receptors was presented. In addition, different brain structures may use various neurotransmitter mechanisms to arrest seizures (Velı´sˇek and Maresˇ 1992). We hypothesized that A1 adenosine receptors play a leading role in cortical afterdischarges.
Methods The experiments were approved by Animal Care and Use Committee of the Institute of Physiology, Academy of Sciences of the Czech Republic to be in agreement with Animal Protection Law of the Czech Republic and European Community Council directives 86/609/EEC.
2002). Cortical flat silver electrodes were implanted epidurally under ether anesthesia. Stimulation electrodes were placed over right sensorimotor area (AP -1 and ?1; L 2.5 mm), recording electrodes over left sensorimotor (AP 0; L 2.5), parietal and occipital areas and over right occipital region. Coordinates for parietal and occipital electrodes were recalculated from adult coordinates (AP 3, L 3 and AP 6, L 4 mm, respectively) on the basis of bregma–lambda distance. Reference electrode was inserted into nasal bone, grounding electrode was over cerebellum. The whole assembly was fixed to the scull with a fastcuring dental acrylic. Surgical preparation lasted 10–12 min, then the animals were allowed to recover for 1 h (righting, placing and suckling reflexes as well as spontaneous locomotion were normal at that time) and only then the experiment started. Body temperature of 12- and 18-day-old rats was maintained by means of a pad heated electrically on 34 °C (i.e., the temperature in the nest). Drugs Drugs specific for subtypes of adenosine receptors were tested. A specific A1 receptor agonist CCPA (2-chloro-N6cyclopentyladenosine) was administered in doses of 0.5 and 1 mg/kg i.p., an A1 receptor antagonist DPCPX (8cyclopentyl-1,3-dipropylxantine) in doses 1 and 2 mg/kg i.p., an A2A receptor agonist CGS 21680 (4-[2-[[6-amino9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride) in doses 0.5 and 5 mg/kg i.p. and A2A receptor antagonist ZM 241385 (4-(2-(7-amino-2-(2-furyl)[1,2,4]triazolol[2,3a][1,3,5]thiazin-5-ylamino)ethyl)phenol) in doses 1 and 5 mg/kg i.p. The doses were chosen on the basis of our previous experiments with pentetrazol-induced seizures to be directly comparable (Maresˇ 2010). All drugs were purchased from Tocris Biosciences (Bristol, UK). CCPA (concentration 1 mg/ml) was put into suspension with a drop of Tween 80, CGS 21680, DPCPX and ZM 241385 were dissolved in dimethylsulfoxide (1, 2 and 1 mg in 1 ml, respectively), then diluted with distilled water in a ratio 1:1. All drugs were prepared freshly just before the start of the experiment. Control group for CCPA was formed by rats injected with saline, control animals for the other three drugs received 50 % dimethylsulfoxide (DMSO) in a volume corresponding to the 5-mg/kg dose of CGS21680 and ZM241385 injected (i.e., 2.5 ml/kg) a common group for all three drugs was formed. Individual age and dose groups were formed by 7–10 animals.
Animals Stimulation and recording A total of 249 Wistar albino male rat pups 12, 18 and 25 days old were used. Cortical epileptic afterdischarges cannot be reliably elicited in younger animals (Maresˇ et al.
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Stimulation series lasting 15 s consisted from biphasic pulses with 0.5-ms duration applied at 8-Hz frequency.
Role of adenosine receptors in cortical epileptic afterdischarges Fig. 1 EEG recordings of afterdischarges in 12- (left graphs) and 25-day-old rats (right graphs). Upper row represents control afterdischarges, lower row third afterdischarges after administration of DPCPX in a dose of 1 mg/kg i.p. Individual leads in each graph (from top to bottom): LF left frontal, LP left parietal, LO left occipital, RO right occipital cortical region. Time mark 2 s, amplitude calibration 1 mV
Stimulator with constant current output was driven by a computer. At first, intensity generating afterdischarges with at least 3-s duration (Fig. 1) was found and then used throughout the experiment. As in our previous studies (Maresˇ et al. 2002; Maresˇ 2013), the intensity had to be higher in 12-day-old rats (starting with a 5-mA intensity, sometimes it was necessary to go up to 10.5 mA) than in the two older groups (usually 3, in a few cases 4.5 or 5 mA). Stimulation series were applied six times, interval between these series was 10 min. Five min after the first successful stimulation the drugs were administered. Electrocorticographic activity was recorded 20 s before stimulation, during stimulation and AD and at least 20 s after the end of the AD. Activity was amplified, collected and digitalized at a 2-kHz frequency and saved using CED Power1401 system and Spike 2 software. Motor phenomena were coded directly into the recording. Duration of ADs was measured and intensity of motor phenomena was classified by means of a modified Racine‘s 5-point scale (Maresˇ et al. 2002). The highest grade of motor phenomena was used for statistical evaluation. Statistics Duration of the six ADs (actual values in seconds) in each age and dose group was compared with repeated-measures ANOVA. Corresponding ADs in control and two dose groups were evaluated with one-way ANOVA. Subsequent pairwise comparison was always performed with HolmSidak test. All statistical evaluation was done with SigmaStatÒ software (SYSTAT Inc.), original data were used for these calculations but columns in figures represent
relative duration of ADs—the first one in each age and dose group was taken as 100 %. The level of statistical significance was set at 5 %.
Results Duration of afterdischarges CCPA (Fig. 2). Control animals exhibited statistically significant prolongation of cortical afterdischarges with repeated stimulations in all three studied age groups. This effect was best expressed in 12-day-old animals. Both doses of specific agonist of adenosine A1 receptors CCPA not only blocked this prolongation, but markedly decreased the duration of the third to sixth ADs in 12- and 18-day-old rats if compared to the first predrug ADs as well as to the corresponding ADs in the control groups. In contrast, such effect was absent in 25-day-old animals. The fifth and sixth ADs after both 0.5- and 1-mg/kg doses were significantly longer than the first, control AD. The only difference was in the fourth AD where the level of significance was reached only in the control group. DPCPX (Figs.1, 3). Control animals injected with DMSO exhibited a progressive prolongation of ADs in all age groups. Again, this effect was best expressed in 12-day-old rats. An antagonist of A1 adenosine receptors DPCPX led to a dose-dependent excessive prolongation of ADs in 12-day-old rats; the 2-mg/kg dose resulted in 17-fold increase in duration. Similar but not so strong effect was present also in 18-day-old rats. Significant prolongation of ADs was found in the second AD, i.e.,
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Fig. 2 Effects of adenosine A1 receptor agonist CCPA on relative duration of afterdischarges (mean ? SEM) in three age groups—from top to bottom 12-, 18- and 25-day-old rats. Duration of the first, predrug AD was always taken as 100 %, and duration of subsequent ADs was related to this value. Abscissae: the first to sixth AD; ordinates: duration of ADs in percents of the first one. White columns—control rats injected after the first AD with saline, obliquely stripped columns—animals given 0.5 and/or 1 mg of CCPA (see inset). Asterisks denote significant difference in comparison with the first AD, circles denote significant difference from corresponding control ADs
5 min after the injection of DPCPX. Third and fourth ADs in treated animals were significantly longer than corresponding control ADs. In contrast, the only significant difference between control- and DPCPX-treated 25-dayold animals was found in the second and third ADs, especially after the 2-mg/kg dose. CGS 21680 (Fig. 4). Either dose of adenosine A2A receptor agonist CGS 21680 resulted in a tendency to attenuation of progressive prolongation of ADs in 12-dayold rats. No systematic changes of ADs duration were observed in 18- and 25-day-old animals. ZM 241385 (Fig. 5). Antagonist of A2A receptors ZM 241385 did not exhibit any marked action in 12-day-old
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Fig. 3 Effects of adenosine A1 receptor antagonist DPCPX on relative duration of afterdischarges (mean ? SEM) in three age groups—from top to bottom 12-, 18- and 25-day-old rats. Details as in Fig. 2, only stripped columns demonstrate groups with 1- and 2-mg/ kg doses of DPCPX
rats. The only difference was in the second AD—both doses of ZM 241385 led to a significant prolongation, whereas a change in control animals did not reach the level of significance. Eighteen-day-old rats exhibited a slight tendency to suppression of progressive prolongation of ADs; there was only a tendency to an increase of ADs duration after the 5-mg/kg dose in contrast to significant changes in control and 1-mg/kg groups. An unexpected result was suppression of prolongation of ADs after the 1but not 5-mg/kg dose of ZM241385 in 25-day-old animals, but the differences between treated and control groups did not reach the level of significance. Motor phenomena Intensity of movements directly elicited by stimulation and/or clonic seizures accompanying ADs was classified as stage 3 (clonic movements of forelimbs) in nearly all
Role of adenosine receptors in cortical epileptic afterdischarges
Fig. 4 Effects of adenosine A2A receptor agonist CGS 21680 on relative duration of afterdischarges (mean ? SEM) in three age groups—from top to bottom 12-, 18- and 25-day-old rats. Details as in Fig. 2, only stripped columns demonstrate groups with 0.5- and 5-mg/ kg doses of CGS 21680
stimulations in all age and dose groups, stage 4 (i.e., rearing) was seen exceptionally in 18- and 25-day-old rats. There was no significant difference under the influence of the four drugs studied, i.e., motor phenomena were not affected by any drug used (data not shown).
Discussion Both drugs influencing A1 type of adenosine receptors resulted in marked systematic changes of epileptic afterdischarges, whereas effects of agonist of A2A receptors CGS 21680 were negligible and the A2A receptor antagonist exhibited marked effect only in a low dose in 25-dayold rats. Cortical epileptic afterdischarges represent a model of different type of human seizures—myoclonic ones. Their ontogeny was described in detail in our laboratory:
Fig. 5 Effects of adenosine A2A receptor antagonist ZM 241385 on relative duration of afterdischarges (mean ? SEM) in three age groups—from top to bottom 12-, 18- and 25-day-old rats. Details as in Fig. 2, only stripped columns demonstrate groups with 1- and 5-mg/ kg doses of ZM 241385
threshold intensities of stimulation current are lowest in the third and fourth week of postnatal life in rats (Maresˇ et al. 2002). Higher threshold in 12-day-old rats is due to immaturity of connections between cortical and thalamic neurons (Scheibel et al. 1976) making synchronization of their activity difficult. Cortical ADs might be suppressed by various drugs in adult (Kubova´ et al. 1996) as well as in developing rats (Kubova´ et al. 1993; Pola´sˇek et al. 1996; Krsˇek et al. 1998; Pometlova´ et al. 2010). Pharmacological sensitivity of EEG and motor phenomena markedly differs—both movements during stimulation and clonic seizures accompanying ADs are resistant to antiepileptic drugs. The only class of drugs markedly suppressing motor phenomena are benzodiazepines (Kubova´ et al. 1993). Our results are in agreement with data from adult animals—not only adenosine (Kulkarni et al. 1997) and its nonspecific agonist 2-chloroadenosine (Abdul-Ghani et al. 1997; Dunwiddie and Worth 1982; Handforth and Treiman 1994; Khan et al. 2000; Pourgholami et al. 1997; Turski
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et al. 1985), but also agonists of A1 adenosine receptors CPA and CCPA (Adami et al. 1995; de Sarro et al. 1999; Malhotra and Gupta 1997) were found to have anticonvulsant action. Adenosine A1 receptor antagonist DPCPX was found to block anticonvulsant action of adenosine and CPA (Malhotra and Gupta 1997) and in high doses it induced seizures (de Sarro et al. 1999). Failure of drugs influencing A2A adenosine receptors in our experiment is in contradiction with some literary data, demonstrating anticonvulsant action of 2HE-NECA and CGS21680 against audiogenic seizures (de Sarro et al. 1999) and 2HE-NECA against pentetrazol-induced seizures (Adami et al. 1995) in adult rodents. There are two points which could explain this difference: (1) different models used, and (2) mature vs. developing brain. The most probable possibility that both factors participate in the difference should be tested in future. Effects of A1 receptor agonist CCPA in the present experiments were similar to those of 2-chloroadenosine (Pometlova´ et al. 2010) not only in the effect on duration of ADs, but also in an ontogenetic profile—a decrease of activity in 12- to 25-day-old rats. Corresponding developmental profile was found also for proconvulsant effect of A1 receptor antagonist DPCPX—prolongation of ADs was most expressed in 12-day-old rat pups. This developmental profile is in contradiction with data on the ontogeny of adenosine receptors in rodents. Both A1 and A2 adenosine receptors were found to be present in many brain structures at birth, and their Bmax increases during the first three postnatal weeks (Geiger et al. 1984; Marangos et al. 1982), an increase was described also for adenosine uptake sites (Morgan et al. 1987). Data for cerebral cortex demonstrated that postnatal increase of binding in A1 receptors is much higher than in A2A receptors (Johansson et al. 1997). An increase of Bmax measured with [3H]cyclohexyladenosine was found between P1 and P21 (Geiger et al. 1984), or between prenatal values and P25 (Morgan et al. 1987). An explanation of the contradictory development of anticonvulsant activity and binding might be explained by developmental changes in coupling of adenosine receptors to G proteins or to another part of metabolic cascade. Such changes were demonstrated in another metabotropic receptor—GABAB—during postnatal development (Kagan et al. 2012). Explanation might be also in an interaction with other major neurotransmitter systems—e.g., GABAergic inhibitory system finishes its maturation in the third postnatal week in rats overtaking a role of the main inhibitory system (Sanchez and Jensen 2001). Importance of adenosinergic inhibitory modulation might thus decrease. Failure of all four drugs to affect motor phenomena connected with stimulation or afterdischarges was surprising, especially in A2A drugs known to induce
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kataplexia (Wardas et al. 2003; Wu et al. 2009). Our previous experiments with pentetrazol-induced seizures demonstrated that rat pups pretreated with high doses of CGS21680 or ZM241385 did not exhibit spontaneous activity, but they did react physically to injection— administration of pentetrazol (Maresˇ 2010). It is highly improbable that the effects of DPCPX are mediated by an interaction with phosphodiesterase. There are data for DPCPX demonstrating that IC50 for inhibition of phosphodiesterase activity is about 10,000-fold higher than the inhibition constant for antagonism at A1 adenosine receptors (Ukena et al. 1993).
Conclusions Adenosine A1 receptors, but not A2A receptors, were demonstrated to play a role in cortical epileptic afterdischarges in immature rats. Action of drugs affecting A1 adenosine receptors is strongest in 12-day-old rat pups and decreases with age. Our future studies will be focused on the role of adenosine in arrest of seizures and immediate postictal period. References Abdul-Ghani AS, Attwell PJ, Bradford HF (1997) The protective effect of 2-chloroadenosine against the development of amygdala kindling and on amygdala-kindled seizures. Eur J Pharmacol 326:7–14 Adami M, Bertorelli R, Ferri N, Foddi MC, Ongini E (1995) Effect of repeated administration of selective adenosine A1 and A2A receptor agonists on pentylenetetrazole-induced convulsions in the rat. Eur J Pharmacol 294:383–389 Albertson TE, Stark LG, Joy RM, Bowyer JF (1983) Aminophylline and kindled seizures. Exp Neurol 81:703–713 Berna´sˇkova´ K, Maresˇ P (2000) Proconvulsant effect of aminophylline on cortical epileptic afterdischarges varies during ontogeny. Epilepsy Res 39:183–190 Boison D (2005) Adenosine and epilepsy: from therapeutic rationale to new therapeutic strategies. Neuroscientist 11:25–36 Boison D (2013a) Role of adenosine in status epilepticus: a potential new target? Epilepsia 54(Suppl 6):20–22. doi:10.1111/epi.12268 Boison D (2013b) Adenosine and seizure termination: endogenous mechanisms. Epilepsy Curr 13(1):35–37. doi:10.5698/15357511-13.1.35 Browning RA, Nelson BK (1985) Variation in threshold and pattern of electroshock-induced seizures in rats depending on site of stimulation. Life Sci 37:2205–2211 Chu N-S (1981) Caffeine- and aminophylline-induced seizures. Epilepsia 22:85–94 De Sarro G, De Sarro A, Di Paola ED, Bertorelli R (1999) Effects of adenosine receptor agonists and antagonists on audiogenic seizure-sensible DBA/2 mice. Eur J Pharmacol 371:137–145 Dragunow M (1988) Purinergic mechanisms in epilepsy. Progr Neurobiol 31:85–108 Dunwiddie TV, Masino SA (2001) The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci 24:31–55
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