Pharmacological Reports Copyright © 2006 2006, 58, 519–525 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences

Efficacy of bretazenil against cortical epileptic afterdischarges increases during early ontogeny in rats

Pavel Mareš1, Romana Šlamberová1,2

Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeòská 1083, CZ-142 20 Prague 4, Czech Republic Charles University, 3rd Medical School, Department of Normal, Pathological and Clinical Physiology, Ke Karlovu 4, CZ-120 00 Prague 2, Czech Republic

Correspondence: Pavel Mareš, e-mail: [email protected]

Abstract: The effect of a benzodiazepine partial agonist bretazenil on cortical epileptic afterdischarges was studied in 12-, 18- or 25-day-old rat pups with implanted electrodes. Afterdischarges were induced by low-frequency stimulation of sensorimotor area four times in each animal and bretazenil (0.1; 1; 5; 10; or 25 mg/kg) and/or solvent were administered intraperitoneally between the first and second stimulation. Bretazenil was able to decrease the duration of afterdischarges as well as intensity of clonic seizures accompanying these afterdischarges in all age groups in a dose-dependent manner. The efficacy of bretazenil was age-dependent: the most marked effect on duration of afterdischarges was observed in 25-day-old rats. It is in sharp contrast with our earlier data for full agonists clonazepam and midazolam demonstrating the strongest action in the youngest group.

Key words: cerebral cortex, epileptic afterdischarges, benzodiazepine partial agonist, ontogeny, rat

Introduction experimental seizures [36]. Their excellent anticon- vulsant effect is compromised by rapid development Among main types of epileptic seizures all but ab- of tolerance in experimental animals [23] as well as in sence seizures are due to a predominance of excitation patients [5]. This phenomenon is clearly expressed for over inhibition in the central nervous system [12]. full agonists of benzodiazepine receptors like diaze- Therefore, the common way how to suppress epileptic pam and clonazepam [29]. One of the possibilities seizures is potentiation of inhibitory systems namely how to avoid this unwanted effect is a replacement of the main, GABAergic one. Augmentation of GABAergic full agonists by partial agonists [10]. There are results inhibition was described as the main mechanism of indicating that tolerance either does not develop at all action for many classical (phenobarbital, valproate, or, at least, it is not marked [11, 31]. Absence of toler- benzodiazepines) as well as new (vigabatrin, tiaga- ance does not mean that there are no signs of with- bine) antiepileptic drugs [9]. Comparing the preclini- drawal [30]. One of the first partial agonists of benzo- cal efficacy of various anticonvulsants the most po- diazepine binding sites tested in adult animals was tent drugs described up to now are benzodiazepines. bretazenil (Ro 16-6028 – [27]). Among other drugs Their doses in the range of hundreds of micrograms synthesized later Ro 19-8022 deserves attention. Both per kilogram were found to abolish different types of drugs exhibit excellent anticonvulsant action in vari-

Pharmacological Reports, 2006, 58, 519–525 519 ous seizure models in adult rodents [15, 27]. Our Materials and Methods laboratory demonstrated marked action of bretazenil against episodes of spike-and-wave rhythm elicited by low doses of pentetrazole in adult rats [4]. In addi- tion, the anticonvulsant action of bretazenil and Ro Animal procedures 19-8022 in immature rats was demonstrated using motor seizures elicited by pentetrazole as a model The experiments were approved by Animal Care and [21, 22]. To determine the spectrum of anticonvulsant Use Committee of the Institute of Physiology to be in action of bretazenil during postnatal development, we agreement with Animal Protection Law of the Czech decided to test its action in another model of epileptic Republic (fully compatible with European Commu- seizures in immature rats, cortical epileptic afterdis- nity Council directives 86/609/EEC). Albino Wistar charges (ADs). Benzodiazepines clonazepam, mida- rats from the breeding station of the Institute of Physi- zolam and clobazam were found to be effective in this ology were used. They were maintained under stan- model in adult as well as developing rats [17, 20, 33]. dard conditions (light/dark cycle 12/12 h, humidity Other dugs augmenting the action of GABAergic in- 60%, temperature 23 ± 1oC). Experiments were per- hibitory system (inhibitor of GABA transporter formed in three age groups: 12, 18, and 25 days old GAT-1 NNC 711 [1-(2-([(diphenylmethylene) amino] (the day of birth was taken as day 0). oxy) ethyl)-1,2,4,6-tetrahydro-3-pyridinecarboxylic acid Flat epidural silver electrodes were placed under light hydrochloride] [3], neurosteroids [24]) as well as an- ether anesthesia: two stimulation electrodes over the tagonists of excitatory amino acid receptors [25, 32]) right sensorimotor region (AP = + 1 and –1; L = 2 mm), exhibited effects against cortical ADs. In addition, recording electrodes over the left sensorimotor region this model offers a possibility to evaluate four differ- (AP = 0; L = 2 mm) and over both occipital regions. ent phenomena: The coordinates for occipital electrodes were calcu- – movements directly bound to stimulation, i.e. a di- lated from those for adult rats (AP = 6; L = 4 mm) on rect activation of motor system; the basis of the bregma – lambda distance (8 mm was – epileptic AD formed by spike-and-wave (S + W) taken as an average adult value). An indifferent elec- rhythm which is with high probability generated in trode was inserted into the nasal bone. The whole as- the thalamocortical system (like spike-and-wave epi- sembly was fixed to the skull by means of fast curing sodes appearing spontaneously in genetically absence dental acrylic. The animals were allowed to recover epilepsy rats from Strasbourg [1]); for at least one hour after the interruption of ether an- – clonic movements accompanying S + W ADs, i.e. esthesia; they were examined neurologically (righting involvement of the motor system; and placing reflexes), fed with a 5% solution of su- – transition into another type of ADs characterized by crose and only then recording started. EEG activity huge delta waves with or without superimposed fast was recorded for 20 s before stimulation, during spikes accompanied by behavioral arrest and (towards stimulation and at least for 1 min after the end of the end of AD) by automatisms. This last phenome- stimulation. Behavior of animals was marked directly non is elicited only by high intensities of stimulation in the EEG recording. current and is difficult to be evoked in immature rats Series of biphasic rectangular pulses (a total of 120 [26]. It is due to a spread of epileptic activity into the pulses of 1 ms duration and 8 Hz frequency) were limbic system; its EEG and behavioral patterns are generated by a constant current stimulator. Intensity similar to those induced by electrical stimulation of of the current eliciting an afterdischarge (AD) of at limbic structures e.g. dorsal hippocampus [8]. The first phenomenon represents a measure of ba- least 1 s duration was found for each rat (it varied sic excitability of the motor system tested at the corti- from 2.5 to 5 mA). Animals in which the 5-mA cur- cal level. The other three phenomena are clearly epi- rent did not elicit an AD of at least 1-s duration were leptic demonstrating different faces of epileptogenesis not taken for the experiment. These stimulation series – generation and spread of epileptic seizures. Our model were repeated four times with a 10-min interval be- is similar to Voskuyl’s model but they pay attention tween the end of the AD and the beginning of the sub- mostly to the stimulation-bound phenomena [7, 38]. sequent stimulation.

520 Pharmacological Reports, 2006, 58, 519–525 Bretazenil and cortical afterdischarges in immature rats Pavel Mareš et al.

Drug treatment

Bretazenil (a generous gift of Hoffman La Roche Co., Basel, Switzerland) was dissolved in a three- component solvent composed of propylene glycol, ethanol and water at a ratio 5:2:3 by volume. Concen- tration of the solution was different to inject always the same volume (1 ml/kg). Freshly prepared solution was administered ip 5 min after the end of the first AD in doses of 0.1; 1; 5; 10 or 25 mg/kg. Control ani- mals received the solvent again in a volume of 1 ml/kg ip. Each age and dose group consisted from 8 to 10 rat pups. Additional groups of 25-day-old rats Fig. 1. An electrocorticographic recording of an afterdischarge of the were formed to avoid ethanol as a solvent: bretazenil spike-and-wave type in 12- (above) and 25-day-old rat. The end of stimu- (1 mg) was suspended in 1 ml of isotonic saline using lation is marked by arrows. Individual leads from top to bottom: LF = left frontal, LO = left occipital, RO = right occipital cortical region in reference one drop of Tween 80 and administered ip at a dose of connection. Time mark 2 s, amplitude calibration 0.5 mV and/or 1 mV 0.1 (tenfold diluted with saline) and/or 1 mg/kg. Cor- responding control group was injected with saline with a drop of Tween 80. To have more information about duration of the effect, six stimulations were ap- Results plied instead of four in the basic experiment. Three basic parameters were evaluated in our model: intensity of movements accompanying stimu- All control as well as experimental rats exhibited in- lation, duration of ADs and intensity of clonic sei- tense movements of head and forelimbs synchronous zures accompanying ADs. Intensities of current used with individual pulses during the first stimulation. in the present experimental series were not sufficient The end of stimulation was followed by an AD (Fig. 1) to elicit a transition into the limbic type of ADs. formed by spike-and-wave rhythm (rhythmic sharp A modified five-point scale of Racine [16, 26, 28] delta waves in 12-day-old rat pups – at this age was used to quantify the intensity of motor phenom- a spike-and-wave rhythm cannot be generated [3, 26]) ena accompanying stimulation as well as ADs. The and accompanied by clonic seizures of forelimb and duration of ADs was measured from EEG recordings. head muscles. Features of ADs as well as of motor phenomena were not changed by bretazenil. Transi- tion into the second type of ADs was never seen in Statistics control or experimental animals in the present study.

Duration of ADs from four stimulations for each age Movements elicited by stimulation (Fig. 2) and dose group was statistically evaluated by means of one-way repeated measures ANOVA, correspond- Intensity of these movements did not change with re- peated stimulations in control 12-day-old rat pups ing stimulations in individual dose groups were evalu- whereas a significant decrease in intensity of move- ated using one-way ANOVA. Data derived from Raci- ments was observed during the second stimulation in ne’s scale were evaluated by means of one-way Fried- 18-day-old rats and during the second, third and man repeated measures ANOVA on ranks, comparison fourth stimulations in 25-day-old animals. Bretazenil of individual dose groups was performed with at doses of 1, 10, and 25 mg/kg significantly de- Kruskal-Wallis ANOVA on ranks. Subsequent com- creased the intensity of movements during all parison with the control values (the first, predrug AD postdrug stimulations in 12-day-old rats. The same ef- or the corresponding AD in the control group) was al- fect was seen after all doses of bretazenil in 18-day- ways made with Dunnett’s test (SigmaStat® SPSS). old animals. A decrease in the intensity in the 25- The level of statistical significance was set at 5%. day-old group observed after all doses of bretazenil

Pharmacological Reports, 2006, 58, 519–525 521 No of Stim.

Fig. 2. Intensity of movements (mean + SEM) induced directly by cor- tical stimulation. From top to bottom: rats 12, 18, and 25 days old. Ab- Fig. 3. Intensity of stimulation-bound movements, relative duration of scissa: four stimulations; ordinate: score according to a 5-point scale afterdischarges and intensity of clonic seizures accompanying after- (see Materials and Methods). Black columns denote control groups discharges (from top to bottom) under the influence of bretazenil ad- with the injection of solvens, other columns denote groups with differ- ministered as a suspension in 25-day-old rats. Control animals (white ent doses of bretazenil injected always 5 min after the first stimulation columns) were injected with a saline with one drop of Tween 80 for (see inset). Asterisks mark statistically significant differences in com- 1 ml. Details as in Figure 2, only ordinate in the middle graph – rela- parison with the appropriate first, predrug stimulation tive duration of ADs (the mean value of the first one is taken as 100%). Circles denote significant differences in comparison with corre- sponding AD in the control group dissolved in DMSO cannot be ascribed to bretazenil because similar change was observed in control ani- 12-day-old rats the lowest dose of bretazenil (0.1 mg/kg) mals. The action of bretazenil independent of DMSO blocked the progressive increase in duration of ADs, was demonstrated in this age group by means of drug a tendency to shortening of post-drug ADs appeared administered as suspension (Fig. 3). The changes after higher doses. A statistically significant shorten- when compared with the first, predrug stimulation ing was observed after the 5- and 25-mg/kg doses. were dose-dependent: the lower dose (0.1 mg/kg) did Eighteen-day-old animals exhibited a suppression of not change the score whereas the 1-mg/kg dose sig- prolongation with the two lowest doses and the doses nificantly decreased the intensity of movements dur- of 5 mg/kg and higher led to a shortening of ADs. The ing the third, fourth and fifth stimulation. If the inten- most marked action of bretazenil was observed in 25- sity of movements was compared with that in the con- day-old rats: all doses (even the 0.1-mg/kg) resulted in trol group both doses resulted in a significant decrease. a significant shortening of ADs. The same was observed with the 0.1- and 1-mg/kg doses of bretazenil adminis- Duration of afterdischarges (Fig. 4) tered as a suspension (Fig. 3).

Control animals exhibited a progressive prolongation of ADs with repeated stimulations. The level of statis- Intensity of clonic seizures (Fig. 5) tical significance (in comparison with the first AD) was reached by the fourth AD in 12- and 18-day-old An average intensity of seizures never surpassed the rat pups and by the third AD in 25-day-old animals. In intensity of movements directly bound to stimulation.

522 Pharmacological Reports, 2006, 58, 519–525 Bretazenil and cortical afterdischarges in immature rats Pavel Mareš et al.

Repeated elicitation of ADs did not significantly change seizure severity in any control group. Effects of bretazenil again differed according to the age. Twelve-day-old rat pups exhibited a significant de- crease in seizure severity only in the fourth AD after the 1- and 5-mg/kg doses, in the third and fourth AD after the 10-mg/kg dose and in all ADs after the high- est dose. In contrast, 18-day-old rats exhibited a de- crease in intensity of seizures after the doses of 1 mg/kg and higher, and all doses of bretazenil were efficient in all ADs in 25-day-old rats. A possible role of DMSO was excluded in the additional groups of 25-day-old rats with administration of bretazenil in suspension (Fig. 3). The higher dose of bretazenil (1 mg/kg) signifi- cantly decreased the intensity of clonic seizures in the second, third, fourth and fifth AD in comparison with the first AD. When compared to corresponding clonic seizures in the control group, only the third and fourth seizures exhibited lower intensity.

Fig. 4. Duration of cortical afterdischarges (mean ± SEM). Details as in Figure 2, only ordinates – relative duration of the afterdischarges (for the direct comparison the mean of the first AD is always taken as 100%). The absolute values for the 12-, 18-, and 25-day-old groups were 15.3 ± 1.2, 10.8 ± 0.8, and 8.8 ± 0.9 s (M ± SEM), respectively. Discussion The duration of ADs in 12-day-old rat pups was significantly longer that in the two older groups

Bretazenil was found to be highly efficient in our Intensity of seizures model in all age groups. Among the three evaluated Bretazenil phenomena, the intensity of movements elicited by 5 control 12 0.1 mg/kg stimulation and/or accompanying ADs exhibited 4 1mg/kg * 5mg/kg higher sensitivity to bretazenil action than AD dura- 3 * *** 10 mg/kg * * 25 mg/kg tion. This is in agreement with repeatedly described Score 2 effects of benzodiazepines – motor seizures are much 1 easier to be suppressed than epileptic EEG phenom- 0 ena [18, 39]. This effect was better expressed in 12- 5 18 and 18-day-old rats than in 25-day-old ones; the old- 4 est group studied exhibited the most marked action of * * * * * 3 * * * * * * bretazenil against ADs. It may be concluded that bre- * Score 2 tazenil similarly as benzodiazepines clonazepam, mi-

1 dazolam [17, 20] and clobazam [33] influenced

0 spread of activity (from sensorimotor cortex during 5 stimulation and from thalamocortical generator of 25 4 spike-and-wave rhythm during ADs) to the generator

3 of the clonic (“minimal”) pattern localized in the ba- * * * * * * * * * 2 * * sal forebrain [6] much more than the proper generator Score * * * * 1 of EEG spike-and-wave ADs (a thalamocortical mechanism [1, 35]). A marked effect of benzodi- 0 1234No of ADs azepines on stimulation-bound movements was also described by Hoogerkamp et al. [13]. The effects of bretazenil on transition into the second type of ADs Fig. 5. Intensity of clonic seizures accompanying afterdischarges (mean + SEM). Details as in Figure 2 could not be analyzed in the present series of ex-

Pharmacological Reports, 2006, 58, 519–525 523 periments because the intensity of stimulation (2.5 to seizures in 12-day-old rats but this sensitivity de- 5 mA) was much lower than threshold intensities neces- creases with age [22]. In contrast, the action of bre- sary for elicitation of the second type of ADs (from 9 to tazenil against EEG cortical ADs was less marked in 12 mA in the three age groups studied [26]). The role of 12- than in 25-day-old rats. This difference may be solvent (especially of ethanol in the solvent) is not a cru- due to different generators of these epileptic phenom- cial one because data for drugs dissolved in the tricompo- ena: in contrast to cortical ADs with a thalamocortical nent solvent (bretazenil – present study, clonazepam [20], mechanism [1], generalized tonic-clonic seizures are clobazam [33]) are the same as for a water-soluble benzo- generated in the brainstem [6] and even spinal cord is diazepine midazolam [20] and the two control groups able to produce this type of seizures [37]. It has to be (one with the tricomponent solvent, the other with iso- due to a caudo-cranial gradient of maturation of the tonic saline) tested in the study with clonazepam and mi- central nervous system [14]. dazolam [20] did not differ from each other. Direct proof The action of bretazenil in the used model differs was brought by the additional groups treated with a sus- from that of benzodiazepine full agonists clonazepam pension of bretazenil in which there was a marked action and midazolam. This might be ascribed either to its of the drug whereas solvent did not significantly influ- partial agonistic properties or to a subunit composition ence the ADs. of the GABAA/benzodiazepine receptor/chloride iono- There are two differences between the effects of bre- phore supramolecular complex which changes during tazenil in the present study and in a model of motor sei- development and varies in different brain structures. zures induced by systemic administration of pentetrazole The marked anticonvulsant action of bretazenil as well [22]: as its less serious effects on motor performance in com- 1. Minimal metrazol (pentetrazole) seizures (clonic parison with diazepam and clonazepam [34] make further seizures with a pattern identical to that seen in cortical research on this drug as potential antiepileptic reasonable stimulation model) are suppressed by bretazenil only in spite of the fact that clinical trials with bretazenil as an in age groups exhibiting this type of seizures under anxiolytic did not bring the expected results [2]. control conditions, i.e. in 18-day-old and older ani- mals. In contrast, younger rats (7- and 12-day-old ones) exhibited an increased incidence of these mini- Acknowledgments: This study was supported by a grant No. 305/05/2581 from the mal seizures under the influence of bretazenil like un- Grant Agency of the Czech Republic and the Research Project der the influence of clonazepam and/or midazolam AV0Z 50110509. [18, 19]. 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