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Anticonvulsant and behavioral effects of GABAB receptor positive modulator CGP7930 in immature rats
Epilepsy & Behavior 28 (2013) 113–120
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Anticonvulsant and behavioral effects of GABAB receptor positive modulator CGP7930 in immature rats Pavel Mareš ⁎, Kateřina Tichá, Anna Mikulecká Department of Developmental Epileptology, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
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Article history: Received 23 December 2012 Revised 9 April 2013 Accepted 15 April 2013 Available online 22 May 2013 Keywords: Cortical afterdischarges Motor performance Behavior GABAB receptors Development Rat
a b s t r a c t Possible anticonvulsant action of GABAB receptor positive allosteric modulator CGP7930 was studied in cortical epileptic afterdischarges (ADs) in rat pups 12, 18, and 25 days old. Afterdischarges were induced by six series of stimulation of sensorimotor cortex, and CGP7930 (20 or 40 mg/kg i.p.) was administered after the first AD. In addition, the effects of CGP7930 on sensorimotor performance and behavior in open field and elevated plus maze were assessed. CGP7930 decreased duration of ADs in 12-day-old but not in older rats. Motor phenomena (movements accompanying stimulation and clonic seizures) were not changed. CGP7930 only moderately affected sensorimotor performance, altered slightly spontaneous behavior in the open field, and did not influence behavior in the elevated plus maze in terms of an adaptive form of learning or anxiety-like behavior. Marked anticonvulsant action with subtle deficits in sensorimotor performance in 12-day-old rats suggests a possible use of CGP7930 as an age-specific anticonvulsant. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Gamma-aminobutyric acid (GABA), the main inhibitory neurotransmiter in the central nervous system, binds to at least two types of receptors, ionotropic GABAA and metabotropic glutamate GABAB. Many antiepileptic drugs have GABAA receptors as the target [1]. In contrast, there are contradictory results concerning a role of GABAB receptor system in models of epileptic seizures. It is due to pre- as well as postsynaptic localization of these receptors. Presynaptic receptors may be autoreceptors decreasing release of GABA from presynaptic terminals and also heterosynaptic receptors on glutamatergic endings suppressing release of this excitatory transmitter [2]. Antagonists of GABAB receptors suppress genetical as well as pharmacological models of absence seizures but potentiate models of convulsive audiogenic seizures and they can induce convulsions in cortical and limbic structures in adult laboratory animals [3,4]. Because, in addition to GABAA receptors, (generally accepted as inhibitory since the end of the first week of postnatal life) the GABAB system represents an important inhibitory system at an early stage of brain development [5], we started to study the action of GABAB receptor agonists and antagonists in developing rats. Our previous study showed that an antagonist of GABAB receptors, CGP 35348, increases duration of cortical epileptic afterdischarges (ADs) in immature rats [6]. Low-frequency rhythmic stimulation of ⁎ Corresponding author at: Institute of Physiology, Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic. Fax: +420 24106 2488. E-mail address: [email protected] (P. Mareš). 1525-5050/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yebeh.2013.04.011
sensorimotor cortical region elicits at least three different phenomena: (1) movements directly bound to individual stimuli, (2) epileptic afterdischarges characterized by spike-and-wave rhythm in the EEG (if intensity is high enough), and (3) clonic seizures of head and forelimb muscles [7,8]. Repeated stimulations with constant intensity resulted in progressive prolongation of ADs, especially marked in 12-day-old rats. This probably reflects an immaturity of mechanisms arresting seizures. The two agonists studied, baclofen and SKF97541, exhibit a mixture of anticonvulsant and proconvulsant effects in the same age groups in this model [9]. The finding that the action of these two agonists is not identical – SKF97541 possesses more anticonvulsant and less proconvulsant activities than baclofen – led us to study the effect of GABAB receptor positive allosteric modulator CGP7930 [10]. The highest doses of CGP7930 (20 and 40 mg/kg) used in a study of PTZ-induced seizures exhibited anticonvulsant action against generalized seizures in rats 7, 12, 18, and 25 days old and in adults [11]. Positive allosteric modulators potentiate only active GABAB receptors, and therefore, their actions should be more specific and with milder unwanted effects [12,13] as was demonstrated in mice [14]. The allosteric modulator CGP7930 also has beneficial effects [13]. It is active against drug abuse — a decrease in craving was described in experimental animals (cocaine [15,16], nicotine [17]), and its moderate anxiolytic action as well as antidepressant profile were also found [18,14]. Moreover, we examined an effect of this drug on sensorimotor performance using a series of age-appropriate tests, spontaneous locomotor, and exploratory behavior in the open field (OF) and behavior in the elevated plus maze (EPM) similarly as in a previous study [19].
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2. Methods 2.1. Animals Male rats of Wistar strain 12, 18, and 25 days old were used. To study the effects of CGP7930 on epileptic afterdischarges, each age and dose group consisted of 7–8 animals. The animals for behavioral studies were brought to the experimental room 1 h before testing. To assess the behavior in the OF, one control and three groups treated with different doses of CGP7930 were formed in each age group. The same animals were tested for motor performance. Different groups of 18- and 25-day-old animals were used to assess behavior in the EPM and sensorimotor performance on the rotarod. To prevent litter effects on statistical analysis, the animals were selected from different litters. Each age and dose group in the behavioral part of our study consisted of 10 animals. The project was approved by the Animal Care and Use Committee of the Institute of Physiology to be in agreement with the Animal Protection Law of the Czech Republic and with European Community Council directive 86/609/EEC. 2.2. Drugs CGP7930 (3,5-bis(1,1-dimethylethyl)-4-hydroxy-b,b-dimethylbenzenepropanol, Tocris Bioscience, UK) was dissolved in dimethylsulfoxide (DMSO) in a concentration of 5 mg/1 ml. Doses of 5-, 10-, 20-, and 40-mg/kg dose were used to test anticonvulsant effects. The same doses were used for behavioral testing, except for the dose of 40 mg/kg, because in a pilot experiment, we found that this dose suppressed locomotion. Control animals were injected with DMSO (8 ml/kg in electrophysiological experiments and 4 ml/kg in behavioral experiments, i.e., the amount corresponding to the highest dose of CGP7930, 40 and 20 mg/kg, respectively). 2.3. Elicitation of cortical epileptic afterdischarges Cortical flat epidural stimulation and recording electrodes were implanted under ether anesthesia and fixed to the skull by means of fast curing dental acrylic. Surgery lasted less than 15 min, and then the animals were left to recover for 1 h. Electrical stimulation (15-s series of 1-ms biphasic pulses with 8-Hz frequency) started at an intensity of 3 mA. This intensity was sufficient to elicit ADs in two older groups, and higher intensities (average value was 4.2 ± 0.44 mA) had to be used in 12-day-old rats. Our previous experiments demonstrated threshold intensities for spike-and-wave type of ADs — 1.54 ± 0.18 mA in 12-, 0.93 ± 0.08 mA in 18-, and 0.98 ± 0.06 mA in 25-day-old rats, respectively [9]. Suprathreshold stimulation (approximately three times threshold) was repeated six times with 20-min intervals, and CGP7930 (5, 10, 20, or 40 mg/kg i.p.) or DMSO in a volume of 8 ml/kg was injected intraperitoneally 10 min after the end of the first AD. Electroencephalogram (amplified and digitalized at a rate of 500 Hz, Kaminskij Biomedical Systems, Prague) was recorded for 20 s before stimulation and at least 1 min after the end of AD. Motor phenomena were marked directly into EEG recording. Incidence, pattern, and duration of ADs and motor phenomena accompanying stimulation and ADs were evaluated. For quantification of motor phenomena, a modified Racine's scale was used [8,20]. During the experiment, the body temperature of the two younger age groups was maintained by means of a pad electrically heated to 34 °C (i.e., temperature in the nest). 2.4. Behavioral experiments 2.4.1. Open-field (OF) test The animals were placed individually in the center of an arena (48 × 48 × 30 cm). The test was performed three times for 5 min at 20 min (session 1), 60 min (session 2), and 24 h (session 3) after
drug/vehicle administration. The following behavioral variables were evaluated: locomotor activity expressed as the distance moved, exploratory behavior as the number of rearings (both with and without support together), and duration of self-grooming. Each animal was tested for motor performance after exposure to OF. 2.4.2. Sensorimotor tests Four tests appropriate for the individual age groups were employed considering the time of appearance and maturation of some sensorimotor reflexes: negative geotaxis for 12- and 18-day-old rats, wire mesh ascending and rotarod for 18- and 25-day-old rats, and bar holding for all age groups. All the tests were performed three times, at 30, 60 min, and 24 h after CGP7930 or DMSO administration. 2.4.2.1. Negative geotaxis. The rats were individually placed on an inclined surface (30°), with the head facing downwards. The ability of pups to turn to 180° was measured. The maximal duration of this test was 60 s. 2.4.2.2. Wire mesh ascending. The rats were put at the lower end of the wire mesh (45 × 15 cm inclined at a 70° angle) placed at an edge of the desk. The time to reach a platform connected to the upper end of the mesh was measured with a limit of 120 s. 2.4.2.3. Bar holding. An animal was held by the nape, and its forepaws were allowed to touch a wooden bar (25 cm long, 1 cm in diameter suspended 25 cm above a soft surface). The time of fore- and hindlimb grasping was measured with a limit of 120 s. 2.4.2.4. Rotarod test. The animals were individually placed on a drum (diameter of 60 mm, width of each drum 85 mm) with a rough surface rotating at a speed of 5 rpm. Their heads were directed against the rotation, and the time to stay on the drum was measured with a limit of 120 s. The test was repeated three times in a close succession, and the average of the three values was taken as a result. 2.4.3. Elevated plus maze (EPM) test Two open and two closed arms (30 × 10 cm, walls of the closed arms 30 cm high) connected by a central space (10 × 10 cm) were 50 cm above the floor. An animal was placed at the end of one open arm with the head directed to the periphery, and the transfer latency (the time it takes for the animal to move from the open arm to either one of the enclosed arms) was measured. A number of studies validated the utility of the EPM for the assessment of an adaptive form of spatial memory. Because during the initial exposure to the EPM an animal acquires phobic avoidance of the open arms and retains strong memory for this threat for a certain time, the transfer latency was significantly shortened when the repeated exposure to the EPM took place. After measurement of transfer latency, the rat was allowed to move freely in the maze for 5 min for the assessment of anxiety-like behavior. The percent of the time spent on open arms [(open arm time / total time) × 100] as an index of anxiety-like behavior and the number of entries into closed arms as an index of locomotion were calculated. The test was performed three times at 20 min (session 1), 60 min (session 2), and 24 h (session 3) after the drug administration. After completion of each session in the EMP, the animals were submitted to the rotarod test. The behavior in the OF and the EPM was recorded by a video camera and evaluated off-line using the programs EthoVision and Observer (Noldus Information Technology). After each animal exposure, the OF and the EPM were wiped clean. 2.4.4. Statistics Duration of ADs and intensity of motor phenomena in each age and dose group were evaluated with repeated measure ANOVA, and a
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comparison of corresponding ADs among control and two drug groups was done with one-way ANOVA both with a subsequent Holm–Sidak test. The data from the OF and EPM tests were analyzed with a twoway repeated measure ANOVA with between-groups factor (treatment) and time of testing as repeated measure. Because the data from sensorimotor performance did not always meet the criteria for normal distribution, the nonparametric tests were used: the Kruskal–Wallis test for comparison of the individual age groups and the Friedman test for comparison of the individual sessions. Student–Newman–Keul's test was used for subsequent comparisons. The level of significance was always set at P b 0.05. SigmaStat® software (SPSS) was used for all calculations.
3. Results 3.1. Epileptic afterdischarges (ADs) Afterdischarges were elicited in all animals included in this study. A tendency to a shorter duration of the first, control AD with increasing age did not reach the level of significance. There was a difference
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in frequency of sharp EEG elements in the ADs, and the 12-day-old rats exhibited slower activity than the 25-day-old ones (Fig. 1).
3.1.1. Control animals The duration of the first AD elicited before the drug or DMSO administration did not differ among the three age groups; it was 7.77 ± 0.79, 7.37 ± 0.59, and 8.21 ± 0.75 s in 12-, 18-, and 25-dayold rats (N = 36, 39, and 39), respectively. Repeated elicitation of seizures resulted in an increase of duration of ADs in the control animals (Fig. 1). Significant prolongation (in comparison with the first AD recorded before the administration of DMSO) was found for the 3rd, 4th, 5th, and 6th ADs in the 12-day-old and for the 4th to 6th ADs in the 18-day-old DMSO-treated rats. Changes in the 25-day-old animals did not reach the level of significance (Fig. 2); the outlined prolongation of the 3rd to 6th ADs was due to a transition into the limbic type of ADs in a few animals. Afterdischarges of the limbic type characterized by delta waves usually with superimposed fast beta activity accompanied by behavioral automatisms are usually very long; therefore, this transition in a subset of animals results not only in a longer average duration but also in a huge increase in variability. Motor phenomena were not significantly affected by DMSO in any age group.
Fig. 1. Original EEG recordings of afterdischarges in a 12- (top) and a 25-day-old (bottom) rat. Individual leads in either rat from top to bottom: left frontal, left parietal, left occipital and right occipital region. Time mark 1 s, amplitude calibration at each trace on the left (±0.5 mV).
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Fig. 2. Effects of CGP7930 on relative duration of cortical epileptic afterdischarges in 12-, 18-, and 25-day-old rats (from top to bottom). Injections were made 5 min after the end of the first AD. The first AD was always taken as 100%, and subsequent ADs were related to this first one. Abscissa — number of ADs, ordinates — duration in percents of the first AD in each age and dose group. White columns — DMSO controls, cross-hatched columns — 20-mg/kg dose, dark columns — 40-mg/kg dose. Asterisks denote significant difference in comparison with the appropriate first AD, circles — with corresponding AD in the control group.
3.1.2. CGP7930-treated animals The doses 10, 20, and 40 mg/kg of CGP7930 abolished the progressive prolongation of ADs in the 12-day-old rats. Comparison of corresponding ADs demonstrated that ADs in these experimental groups were shorter than those in the DMSO group. Nearly all these differences were statistically significant (Fig. 2). There were only a few changes after the administration of any dose of CGP7930 in the 18as well as in the 25-day-old animals. Outlined differences in the 25-day-old rats were not statistically significant, and the level of significance was not reached even when the limbic parts of ADs were not taken into account. Motor phenomena directly connected with stimulation of sensorimotor cortex as well as clonic seizures accompanying ADs were not affected by CGP7930 — both motor phenomena were usually classified as Racine's stage 3, only exceptionally did rearing, i.e., stage 4, appear. Average varied between 3.0 and 3.2 without any significant difference (data not shown). 3.2. Open-field test In the 12-day-old rats, CGP7930 did not significantly affect distance moved in the open-field test; locomotion was decreased in the control animals and increased after the dose of 10 mg/kg in the second session compared with the corresponding first session. In the third session, the controls and the animals treated with a dose of 10 mg/kg walked for a longer distance compared with the second session (Fig. 3). In the 18-day-old rats, the 5-mg/kg dose decreased the distance moved only in the first session. While in the control animals the
Fig. 3. Effect of CGP7930 on locomotor activity in the open field in 12-, 18- and 25-day-old rats. Abscissa: the 1st, 2nd, and 3rd sessions (i.e., at 30 min, 60 min, and 24 h after drug administration); ordinate: mean + S.E.M. for distance moved. P b 0.05: * Compared with control group. # Compared with the 1st session. + Compared with the 2nd session. 0 Compared with the dose of 5 mg/kg.
distance moved decreased with repeated session, in the animals treated with 5-mg/kg dose, the distance moved decreased in the second session and increased in the third session compared with the first and second ones (Fig. 3). For the rearing number, we did not find a significant effect of treatment with CGP7930. As for duration of grooming, all three doses of CGP7930 decreased grooming behavior in the third session (Fig. 4). In the 25-day-old rats, a significant increase in the distance moved was found in the animals treated with the 5-mg/kg dose compared with the controls 20 min after the drug administration. Further, both higher doses decreased the distance moved compared with the 5-mg/kg dose. Finally, there was a significant decrease in the second session in the animals treated with the 5- and 20-mg/kg doses of CGP7930 as well as in the third session in all the experimental animals (Fig. 3). For the rearing number in 25-day-old rats, a significant increase was found in the animals treated with the 5-mg/kg dose compared with the controls in the first session. The similar effect was found in the third session with the doses of 5 and 10 mg/kg. In contrast, all doses of CGP7930 significantly decreased the number of rearings in
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Fig. 4. Effect of CGP7930 on rearing and grooming behavior in the open-field test in 18-day-old rats (left side) and in 25-day-old rats (right side). Details are as in Fig. 2.
the second session. The doses of 5 and 10 mg/kg of CGP7930 decreased grooming duration only in the third session. 3.3. Sensorimotor performance In the 12-day-old rats, the 10-mg/kg dose of CGP7930 increased the latency to negative geotaxis 20 min after administration, whereas the dose of 20 mg/kg increased this latency 20 min and 60 min after the drug administration compared with the controls. Repeated measure showed that both higher doses decreased the latency of the negative geotaxis response 24 h after the drug administration. As for the bar-holding test, only the dose of 5 mg/kg of CGP7930 decreased significantly the time to hold the bar 20 and 60 min after the drug injection. In the 18-day-old rats, the negative geotaxis response was not significantly affected by CGP7930 administration. The dose of 5 mg/kg shortened the latency to ascending the wire mesh 20 min after the drug administration. In contrast, the dose of 20 mg/kg significantly increased the time for wire mesh ascending in comparison with the control and 5-mg/kg dose groups. Surprisingly, the sensorimotor performance was not significantly affected in the most demanding tests — bar-holding and rotarod tests. Repeated measure showed an increased time spent on the rotated drum 24 h after the drug administration. In the 25-day-old rats, any dose of CGP7930 tested did not affect significantly the performance in ascending the wire mesh. The dose of 20 mg/kg decreased the time to hold the bar 20 min after the drug administration but improved the performance when repeated 24 h later. Finally, the sensorimotor performance was not significantly altered by drug administration in the rotarod test except for the dose of 20 mg/kg in the first session. Repeated measure showed an improvement in rotarod performance in the control — as well as CGP7930-treated animals (Table 1). 3.4. Elevated plus maze test The CGP7930 did not affect the transfer latency with the exception of shorter latency in the 25-day-old animals pretreated with the
5-mg/kg dose in the first session. With repeated exposure to the EPM, both the control and experimental animals acquired a phobic avoidance of the open arms and retained this form of adaptive learning for at least 24 h. This phenomenon was more expressed in 25than in 18-day-old animals (Fig. 5). There were no significant changes in the percent of time spent in the open arms (an index of anxietylike behavior) as well as in the closed arm entries (an index of locomotion) (Fig. 5). 4. Discussion Anticonvulsant action of GABAB receptor positive allosteric modulator CGP7930 against cortical epileptic afterdischarges was found only in 12-day-old rats in contrast to our recent data with PTZinduced convulsions when the 20- and 40-mg/kg doses affected generalized seizures in all age groups [11]. Different actions in the two models can be due to generators in different brain structures. Generator of generalized tonic–clonic seizures was localized to the brainstem [21], and cortical ADs characterized by spike-and-wave rhythm in the EEG are generated by corticothalamic mechanisms [22,23] with a spread of activity into the motor system. Receptors of GABAB type are widely distributed in the rodent brain [24,25], and they can be demonstrated at early stages of development [26]. As structures responsible for generation of cortical afterdischarges are concerned, GABAB receptors were demonstrated early postnatally in both the cerebral cortex and the thalamus, and they peak at postnatal day 14 [27,28]. Laminar distribution of GABA R1 protein in the cerebral cortex with the highest presence in layers V and VIb is mature at postnatal day 12 [29]. Inactivity of CGP7930 after the second postnatal week indicated in our study could be explained by unequal development of postsynaptic and presynaptic GABAB receptors. Presynaptic GABAB receptors in somatosensory cortex are functional much earlier than postsynaptic ones, and they play a role in regulating not only GABA release [30] but also glutamate release. Maturation of postsynaptic GABAB receptors in the somatosensory cortex takes place in the second and the
24.5 ± 7.1 110.3 ± 9.7a 120 ± 0.0a 20.9 ± 6.5 94.4 ± 12.9 85.8 ± 11.7a 22.9 ± 5.7 82.1 ± 14.2⁎ 56.6 ±16.1⁎ 33.4 ± 5.7 120 ± 0.0 119.0 ± 1.0 26.6 ± 5.6 112.5 ± 5.3 93.1 ± 11.2 23.9 ± 6.4 109 ± 11.0 70.9 ± 15.6 30.7 ± 5.6 120 ± 0.0 120 ± 0.0a 15.7 ± 2.3 120 ± 0.0 113.3 ± 6.7a Values are mean ± S.E.M. ⁎ P b 0.05 compared with control rats. a Compared with session 1 (30 min after drug administration). b Compared with the dose of 5 mg/kg.
14.7 ± 3.5 117.4 ± 2.6 80.3 ± 15.9 15.1 ± 2.9 120 ± 0.0 109.4 ± 10.6a 21.9 ± 6.6 116.6 ± 2.5 69.9 ± 13.2 24 ± 5.7 108.7 ± 7.6 70.3 ± 14.9
5.5 4.2⁎,b 13.7 14.8a ± ± ± ± 17.1 18.9 79 73 2.7 7.8⁎,b 14.6 11.7 ± ± ± ± 10.5 34.4 48.9 85.8 4.6 6.1⁎,b 13.6 10.9 ± ± ± ± 12.6 33.9 39.7 44.6 3.1 3.7 16.0 16.9 ± ± ± ± 8.6 16.9 82.7 64.1 5.4 10.0 7.9 11.2 ± ± ± ± 12.4 30.4 33.2 95.1 3.1 10.0 8.5 15.1 ± ± ± ± 9.3 37.5 41.4 58.8 5.7 2.6 13.9 13.6 ± ± ± ± 8.6 10.4 71.8 63.1 0.6 2.3 10.1 14.3 ± ± ± ± 4.9 13.2 48.3 79.9 1.0 1.5⁎ 12.3 12.4 ± ± ± ± 7 13.7 48.3 59.8 2.6 1.2a 14.3 14.4 ± ± ± ± 9.4 8.4 68.7 65.8 1.1 3.1a 11.3 13.2 ± ± ± ± 5.5 14.4 46 65.9 0.8 2.8 11.8 10.9 ± ± ± ± 6.2 23.9 46 47.8
15.6 ± 3.7⁎ 13.6 ± 0.9
Session 2 Session 1
20.3 ± 6.8⁎ 14.4 ± 1.8 5.2 ± 0.2a 20.7 ± 7.5 11.2 ± 5.6 11.7 ± 1.5 14.4 ± 5.2⁎ 12.3 ± 1.7 4.2 ± 0.6 10.1 ± 1.3 3.7 ± 0.6 7.2 ± 0.7⁎ 5.6 ± 1.3 8.1 ± 0.7 6.2 ± 1.2 9.8 ± 1.2 7.8 ± 1.2 12.1 ± 1.0 5.5 ± 0.9 14 ± 1.7
12 days Negative geotaxis Bar holding 18 days Negative geotaxis Wire mesh Bar holding Rotarod 25 days Wire mesh Bar holding Rotarod
20-mg/kg CGP7930
Session 3 Session 2 Session 1
10-mg/kg CGP7930
Session 3 Session 2 Session 1
5-mg/kg CGP7930
Session 3 Session 1 Testing
Session 2 Controls Group
Table 1 Effect of CGP7930 on sensorimotor performance in 12-, 18-, and 25-day-old rats.
6.5 ± 1.3a 14.2 ± 3.2
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Session 3
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third week of postnatal life [31]. Our data from cortical ADs suggest that GABAB heteroreceptors at glutamatergic terminals might be responsible for the demonstrated anticonvulsant effect. Later maturation of presynaptic homoreceptors and postsynaptic receptors might mask the anticonvulsant effect. The explanation concerning the longer duration of afterdischarges in 12-day-old rats found in some our previous studies (e.g. [32]) but not in the present series of experiments seems improbable. First, there was no significant difference in the present study; the same is true for threshold current intensities — they only tended to be higher in the 12-day-old animals than in the two older groups [8]. Second, prolongation of cortical ADs found after the administration of a GABAB receptor antagonist CGP46381 was marked in the 12-, present in 18-, and absent in 25-day-old rats (Mareš — submitted). Third, some drugs affecting other neurotransmitter systems exhibit the strongest action in the 25-day-old rats with minor effects in the 12-day-old ones (benzodiazepine agonist bretazenil [33]; NMDA receptor antagonists dizocilpine and 2-amino-7-phosphonoheptanoic acid [34]). In addition, effects of CGP7930 against pentylenetetrazol-induced seizures are more marked in the 12- than in 25-day-old rats [11] in spite of the fact that sensitivity of these two age groups to convulsant action of pentylenetetrazol is the same [35]. Comparison of anticonvulsant action of CGP7930 with that of full GABAB receptor agonists, baclofen and SKF97541, argues for the positive allosteric modulator. Neither agonist decreased the duration of afterdischarges in the 12-day-old rats; on the contrary, they prolonged afterdischarges especially in the 18- and 25-day-old animals [9]. There is no “mirror” action of an antagonist of GABAB receptors to agonists or to CGP7930. An antagonist CGP35348 markedly increased duration of cortical afterdischarges in all three age groups [6]; the same is true for another antagonist CGP46381 (experiments in progress). It is in agreement with an increase of persistent network activity under the influence of GABAB receptor antagonist CGP35348 demonstrated in vitro [36]. It may be concluded that the three classes of drugs influence at least partially different subsets of GABAB receptors. Twelve-day-old rats exhibit the highest sensitivity to anticonvulsant action of CGP7930 against cortical ADs as well as against convulsions elicited by pentylenetetrazol [11]. Our results in this age group are in agreement with presentation of GABAB receptors as the important inhibitory system at early stages of development [5] and indicate that it might be possible to search for an anticonvulsant drug specific for developing brain among positive allosteric modulators of GABAB receptors. Our results from the sensorimotor performance tests indicate that administration of CGP7930 in doses up to 20 mg/kg affect subtle sensorimotor performance in immature rats. All animals irrespective of the age were able to perform the given task and improved their performance with the repeated exposure. In the bar holding and rotarod, the most demanding tests of sensorimotor motor performance [37,38], the 25-day-old animals treated with higher dose had a worse performance only in the first exposure. Locomotion (expressed as distance moved in the open field) was not affected by either CGP7930 doses in the 12-day-old rats. The suppression of locomotion and rearing caused by the lower dose of CGP7930 in the first session and the converse increase in both of these behaviors in the 25-day-old rats seem to indicate a different sensitivity to the drug. Generally, rearing behavior is taken as an index of rodent emotionality (more rears being associated with exploratory behavior and/or alert state [39]). The grooming behavior distinguishable at about 20 days of age and reflecting the maturation of motor capabilities [40] was suppressed 24 h after the drug administration in both ages, except for the highest dose administered in the 25-day-old rats. The grooming behavior may serve many functions depending on the context in which this behavior occurs [41]. In a novel environment, grooming behavior is a response to stress and serves to lower the anxiety produced by a stressful experience; therefore, grooming restores the homeostasis of the animal [42,43].
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Fig. 5. Effect of CGP7930 on behavior in the elevated plus maze. Left graphs: 18-day-old rats, right graphs: 25-day-old rats. From top to bottom: transfer latency(s), time spent (%) in open arm, and number of closed arm entries. Abscissa: the 1st, 2nd, and 3rd sessions (i.e., at 30 min, 60 min, and 24 h after drug administration); ordinate: mean + S.E.M. P b 0.05: # Compared with the 1st session.
Regarding behavior in EPM, the CGP7930 did not affect transfer latency, an index of learning and memory, except for the lower dose in the 25-day-old rats in the first exposure that could be explained by a short-time stimulatory effect. Moreover, the drug did not induce an anxiolytic-like response and did not affect locomotion. With repeated exposure to the EPM or OF, a decreased behavioral response occurred. Generally, prior experience with the behavioral procedures alters subsequent responsiveness; animals simply experience environmental novelty, which subsequently influences their behavioral response. Thus, the waning of a response elicited by repeated exposure to a novel stimulus is a phenomenon commonly termed as habituation (for review see [44]). Marked anticonvulsant action with only minor deficits in sensorimotor performance in 12-day-old rats suggests a possible use of positive allosteric modulators of GABAB receptors as age-specific anticonvulsants. Acknowledgments This study was supported by a grant no. P304/10/1274 of the Grant Agency of the Czech Republic, a grant no. 200117 of the Grant Agency of Charles University, and projects LC-554 of the Ministry of Education and AV0Z 50110509 of the Academy of Sciences of the
Czech Republic. The authors would like to thank Ms. Irina Necheva for her excellent technical assistance. References [1] Rogawski MA, Löscher W. The neurobiology of antiepileptic drugs. Nat Rev Neurosci 2004;5:553–64. [2] Pinard A, Seddik R, Bettler B. GABAB receptors. Physiological functions and mechanisms of diversity. Adv Pharmacol 2010;58:231–55. [3] Leung LS, Canning KJ, Shen B. Hippocampal afterdischarges after GABA(B)-receptor blockade in the freely moving rat. Epilepsia 2005;46:203–16. [4] Vergnes M, Boehrer A, Simler S, Bernasconi R, Marescaux C. Opposite effects of GABAB receptor antagonists on absences and convulsive seizures. Eur J Pharmacol 1997;332:245–55. [5] Gaiarsa JL, McLean H, Congar P, Leinekugel X, Khazipov R, Tseeb V, et al. Postnatal maturation of gamma-aminobutyric acid A and B-mediated inhibition in the CA3 hippocampal region of the rat. J Neurobiol 1995;26:339–49. [6] Mareš P. GABA-B receptor antagonist CGP 35348 interferes with an arrest of cortical epileptic afterdischarges in developing rats. Epilepsy Res 2010;92:125–33. [7] Mareš P, Kubová H. Electrical stimulation-induced models of seizures. In: Pitkanen A, Schwartzkroin PA, Moshé SL, editors. Models of seizures and epilepsy. Amsterdam: Elsevier; 2006. p. 153–9. [8] 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. [9] Mareš P, Tabashidze N. Contradictory effects of GABA-B receptor agonists on cortical epileptic afterdischarges in immature rats. Brain Res Bull 2008;75:173–8.
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Anticonvulsant and behavioral effects of GABAB receptor positive modulator CGP7930 in immature rats Pavel Mareš ⁎, Kateřina Tichá, Anna Mikulecká Department of Developmental Epileptology, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
a r t i c l e
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Article history: Received 23 December 2012 Revised 9 April 2013 Accepted 15 April 2013 Available online 22 May 2013 Keywords: Cortical afterdischarges Motor performance Behavior GABAB receptors Development Rat
a b s t r a c t Possible anticonvulsant action of GABAB receptor positive allosteric modulator CGP7930 was studied in cortical epileptic afterdischarges (ADs) in rat pups 12, 18, and 25 days old. Afterdischarges were induced by six series of stimulation of sensorimotor cortex, and CGP7930 (20 or 40 mg/kg i.p.) was administered after the first AD. In addition, the effects of CGP7930 on sensorimotor performance and behavior in open field and elevated plus maze were assessed. CGP7930 decreased duration of ADs in 12-day-old but not in older rats. Motor phenomena (movements accompanying stimulation and clonic seizures) were not changed. CGP7930 only moderately affected sensorimotor performance, altered slightly spontaneous behavior in the open field, and did not influence behavior in the elevated plus maze in terms of an adaptive form of learning or anxiety-like behavior. Marked anticonvulsant action with subtle deficits in sensorimotor performance in 12-day-old rats suggests a possible use of CGP7930 as an age-specific anticonvulsant. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Gamma-aminobutyric acid (GABA), the main inhibitory neurotransmiter in the central nervous system, binds to at least two types of receptors, ionotropic GABAA and metabotropic glutamate GABAB. Many antiepileptic drugs have GABAA receptors as the target [1]. In contrast, there are contradictory results concerning a role of GABAB receptor system in models of epileptic seizures. It is due to pre- as well as postsynaptic localization of these receptors. Presynaptic receptors may be autoreceptors decreasing release of GABA from presynaptic terminals and also heterosynaptic receptors on glutamatergic endings suppressing release of this excitatory transmitter [2]. Antagonists of GABAB receptors suppress genetical as well as pharmacological models of absence seizures but potentiate models of convulsive audiogenic seizures and they can induce convulsions in cortical and limbic structures in adult laboratory animals [3,4]. Because, in addition to GABAA receptors, (generally accepted as inhibitory since the end of the first week of postnatal life) the GABAB system represents an important inhibitory system at an early stage of brain development [5], we started to study the action of GABAB receptor agonists and antagonists in developing rats. Our previous study showed that an antagonist of GABAB receptors, CGP 35348, increases duration of cortical epileptic afterdischarges (ADs) in immature rats [6]. Low-frequency rhythmic stimulation of ⁎ Corresponding author at: Institute of Physiology, Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic. Fax: +420 24106 2488. E-mail address: [email protected] (P. Mareš). 1525-5050/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yebeh.2013.04.011
sensorimotor cortical region elicits at least three different phenomena: (1) movements directly bound to individual stimuli, (2) epileptic afterdischarges characterized by spike-and-wave rhythm in the EEG (if intensity is high enough), and (3) clonic seizures of head and forelimb muscles [7,8]. Repeated stimulations with constant intensity resulted in progressive prolongation of ADs, especially marked in 12-day-old rats. This probably reflects an immaturity of mechanisms arresting seizures. The two agonists studied, baclofen and SKF97541, exhibit a mixture of anticonvulsant and proconvulsant effects in the same age groups in this model [9]. The finding that the action of these two agonists is not identical – SKF97541 possesses more anticonvulsant and less proconvulsant activities than baclofen – led us to study the effect of GABAB receptor positive allosteric modulator CGP7930 [10]. The highest doses of CGP7930 (20 and 40 mg/kg) used in a study of PTZ-induced seizures exhibited anticonvulsant action against generalized seizures in rats 7, 12, 18, and 25 days old and in adults [11]. Positive allosteric modulators potentiate only active GABAB receptors, and therefore, their actions should be more specific and with milder unwanted effects [12,13] as was demonstrated in mice [14]. The allosteric modulator CGP7930 also has beneficial effects [13]. It is active against drug abuse — a decrease in craving was described in experimental animals (cocaine [15,16], nicotine [17]), and its moderate anxiolytic action as well as antidepressant profile were also found [18,14]. Moreover, we examined an effect of this drug on sensorimotor performance using a series of age-appropriate tests, spontaneous locomotor, and exploratory behavior in the open field (OF) and behavior in the elevated plus maze (EPM) similarly as in a previous study [19].
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2. Methods 2.1. Animals Male rats of Wistar strain 12, 18, and 25 days old were used. To study the effects of CGP7930 on epileptic afterdischarges, each age and dose group consisted of 7–8 animals. The animals for behavioral studies were brought to the experimental room 1 h before testing. To assess the behavior in the OF, one control and three groups treated with different doses of CGP7930 were formed in each age group. The same animals were tested for motor performance. Different groups of 18- and 25-day-old animals were used to assess behavior in the EPM and sensorimotor performance on the rotarod. To prevent litter effects on statistical analysis, the animals were selected from different litters. Each age and dose group in the behavioral part of our study consisted of 10 animals. The project was approved by the Animal Care and Use Committee of the Institute of Physiology to be in agreement with the Animal Protection Law of the Czech Republic and with European Community Council directive 86/609/EEC. 2.2. Drugs CGP7930 (3,5-bis(1,1-dimethylethyl)-4-hydroxy-b,b-dimethylbenzenepropanol, Tocris Bioscience, UK) was dissolved in dimethylsulfoxide (DMSO) in a concentration of 5 mg/1 ml. Doses of 5-, 10-, 20-, and 40-mg/kg dose were used to test anticonvulsant effects. The same doses were used for behavioral testing, except for the dose of 40 mg/kg, because in a pilot experiment, we found that this dose suppressed locomotion. Control animals were injected with DMSO (8 ml/kg in electrophysiological experiments and 4 ml/kg in behavioral experiments, i.e., the amount corresponding to the highest dose of CGP7930, 40 and 20 mg/kg, respectively). 2.3. Elicitation of cortical epileptic afterdischarges Cortical flat epidural stimulation and recording electrodes were implanted under ether anesthesia and fixed to the skull by means of fast curing dental acrylic. Surgery lasted less than 15 min, and then the animals were left to recover for 1 h. Electrical stimulation (15-s series of 1-ms biphasic pulses with 8-Hz frequency) started at an intensity of 3 mA. This intensity was sufficient to elicit ADs in two older groups, and higher intensities (average value was 4.2 ± 0.44 mA) had to be used in 12-day-old rats. Our previous experiments demonstrated threshold intensities for spike-and-wave type of ADs — 1.54 ± 0.18 mA in 12-, 0.93 ± 0.08 mA in 18-, and 0.98 ± 0.06 mA in 25-day-old rats, respectively [9]. Suprathreshold stimulation (approximately three times threshold) was repeated six times with 20-min intervals, and CGP7930 (5, 10, 20, or 40 mg/kg i.p.) or DMSO in a volume of 8 ml/kg was injected intraperitoneally 10 min after the end of the first AD. Electroencephalogram (amplified and digitalized at a rate of 500 Hz, Kaminskij Biomedical Systems, Prague) was recorded for 20 s before stimulation and at least 1 min after the end of AD. Motor phenomena were marked directly into EEG recording. Incidence, pattern, and duration of ADs and motor phenomena accompanying stimulation and ADs were evaluated. For quantification of motor phenomena, a modified Racine's scale was used [8,20]. During the experiment, the body temperature of the two younger age groups was maintained by means of a pad electrically heated to 34 °C (i.e., temperature in the nest). 2.4. Behavioral experiments 2.4.1. Open-field (OF) test The animals were placed individually in the center of an arena (48 × 48 × 30 cm). The test was performed three times for 5 min at 20 min (session 1), 60 min (session 2), and 24 h (session 3) after
drug/vehicle administration. The following behavioral variables were evaluated: locomotor activity expressed as the distance moved, exploratory behavior as the number of rearings (both with and without support together), and duration of self-grooming. Each animal was tested for motor performance after exposure to OF. 2.4.2. Sensorimotor tests Four tests appropriate for the individual age groups were employed considering the time of appearance and maturation of some sensorimotor reflexes: negative geotaxis for 12- and 18-day-old rats, wire mesh ascending and rotarod for 18- and 25-day-old rats, and bar holding for all age groups. All the tests were performed three times, at 30, 60 min, and 24 h after CGP7930 or DMSO administration. 2.4.2.1. Negative geotaxis. The rats were individually placed on an inclined surface (30°), with the head facing downwards. The ability of pups to turn to 180° was measured. The maximal duration of this test was 60 s. 2.4.2.2. Wire mesh ascending. The rats were put at the lower end of the wire mesh (45 × 15 cm inclined at a 70° angle) placed at an edge of the desk. The time to reach a platform connected to the upper end of the mesh was measured with a limit of 120 s. 2.4.2.3. Bar holding. An animal was held by the nape, and its forepaws were allowed to touch a wooden bar (25 cm long, 1 cm in diameter suspended 25 cm above a soft surface). The time of fore- and hindlimb grasping was measured with a limit of 120 s. 2.4.2.4. Rotarod test. The animals were individually placed on a drum (diameter of 60 mm, width of each drum 85 mm) with a rough surface rotating at a speed of 5 rpm. Their heads were directed against the rotation, and the time to stay on the drum was measured with a limit of 120 s. The test was repeated three times in a close succession, and the average of the three values was taken as a result. 2.4.3. Elevated plus maze (EPM) test Two open and two closed arms (30 × 10 cm, walls of the closed arms 30 cm high) connected by a central space (10 × 10 cm) were 50 cm above the floor. An animal was placed at the end of one open arm with the head directed to the periphery, and the transfer latency (the time it takes for the animal to move from the open arm to either one of the enclosed arms) was measured. A number of studies validated the utility of the EPM for the assessment of an adaptive form of spatial memory. Because during the initial exposure to the EPM an animal acquires phobic avoidance of the open arms and retains strong memory for this threat for a certain time, the transfer latency was significantly shortened when the repeated exposure to the EPM took place. After measurement of transfer latency, the rat was allowed to move freely in the maze for 5 min for the assessment of anxiety-like behavior. The percent of the time spent on open arms [(open arm time / total time) × 100] as an index of anxiety-like behavior and the number of entries into closed arms as an index of locomotion were calculated. The test was performed three times at 20 min (session 1), 60 min (session 2), and 24 h (session 3) after the drug administration. After completion of each session in the EMP, the animals were submitted to the rotarod test. The behavior in the OF and the EPM was recorded by a video camera and evaluated off-line using the programs EthoVision and Observer (Noldus Information Technology). After each animal exposure, the OF and the EPM were wiped clean. 2.4.4. Statistics Duration of ADs and intensity of motor phenomena in each age and dose group were evaluated with repeated measure ANOVA, and a
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comparison of corresponding ADs among control and two drug groups was done with one-way ANOVA both with a subsequent Holm–Sidak test. The data from the OF and EPM tests were analyzed with a twoway repeated measure ANOVA with between-groups factor (treatment) and time of testing as repeated measure. Because the data from sensorimotor performance did not always meet the criteria for normal distribution, the nonparametric tests were used: the Kruskal–Wallis test for comparison of the individual age groups and the Friedman test for comparison of the individual sessions. Student–Newman–Keul's test was used for subsequent comparisons. The level of significance was always set at P b 0.05. SigmaStat® software (SPSS) was used for all calculations.
3. Results 3.1. Epileptic afterdischarges (ADs) Afterdischarges were elicited in all animals included in this study. A tendency to a shorter duration of the first, control AD with increasing age did not reach the level of significance. There was a difference
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in frequency of sharp EEG elements in the ADs, and the 12-day-old rats exhibited slower activity than the 25-day-old ones (Fig. 1).
3.1.1. Control animals The duration of the first AD elicited before the drug or DMSO administration did not differ among the three age groups; it was 7.77 ± 0.79, 7.37 ± 0.59, and 8.21 ± 0.75 s in 12-, 18-, and 25-dayold rats (N = 36, 39, and 39), respectively. Repeated elicitation of seizures resulted in an increase of duration of ADs in the control animals (Fig. 1). Significant prolongation (in comparison with the first AD recorded before the administration of DMSO) was found for the 3rd, 4th, 5th, and 6th ADs in the 12-day-old and for the 4th to 6th ADs in the 18-day-old DMSO-treated rats. Changes in the 25-day-old animals did not reach the level of significance (Fig. 2); the outlined prolongation of the 3rd to 6th ADs was due to a transition into the limbic type of ADs in a few animals. Afterdischarges of the limbic type characterized by delta waves usually with superimposed fast beta activity accompanied by behavioral automatisms are usually very long; therefore, this transition in a subset of animals results not only in a longer average duration but also in a huge increase in variability. Motor phenomena were not significantly affected by DMSO in any age group.
Fig. 1. Original EEG recordings of afterdischarges in a 12- (top) and a 25-day-old (bottom) rat. Individual leads in either rat from top to bottom: left frontal, left parietal, left occipital and right occipital region. Time mark 1 s, amplitude calibration at each trace on the left (±0.5 mV).
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Fig. 2. Effects of CGP7930 on relative duration of cortical epileptic afterdischarges in 12-, 18-, and 25-day-old rats (from top to bottom). Injections were made 5 min after the end of the first AD. The first AD was always taken as 100%, and subsequent ADs were related to this first one. Abscissa — number of ADs, ordinates — duration in percents of the first AD in each age and dose group. White columns — DMSO controls, cross-hatched columns — 20-mg/kg dose, dark columns — 40-mg/kg dose. Asterisks denote significant difference in comparison with the appropriate first AD, circles — with corresponding AD in the control group.
3.1.2. CGP7930-treated animals The doses 10, 20, and 40 mg/kg of CGP7930 abolished the progressive prolongation of ADs in the 12-day-old rats. Comparison of corresponding ADs demonstrated that ADs in these experimental groups were shorter than those in the DMSO group. Nearly all these differences were statistically significant (Fig. 2). There were only a few changes after the administration of any dose of CGP7930 in the 18as well as in the 25-day-old animals. Outlined differences in the 25-day-old rats were not statistically significant, and the level of significance was not reached even when the limbic parts of ADs were not taken into account. Motor phenomena directly connected with stimulation of sensorimotor cortex as well as clonic seizures accompanying ADs were not affected by CGP7930 — both motor phenomena were usually classified as Racine's stage 3, only exceptionally did rearing, i.e., stage 4, appear. Average varied between 3.0 and 3.2 without any significant difference (data not shown). 3.2. Open-field test In the 12-day-old rats, CGP7930 did not significantly affect distance moved in the open-field test; locomotion was decreased in the control animals and increased after the dose of 10 mg/kg in the second session compared with the corresponding first session. In the third session, the controls and the animals treated with a dose of 10 mg/kg walked for a longer distance compared with the second session (Fig. 3). In the 18-day-old rats, the 5-mg/kg dose decreased the distance moved only in the first session. While in the control animals the
Fig. 3. Effect of CGP7930 on locomotor activity in the open field in 12-, 18- and 25-day-old rats. Abscissa: the 1st, 2nd, and 3rd sessions (i.e., at 30 min, 60 min, and 24 h after drug administration); ordinate: mean + S.E.M. for distance moved. P b 0.05: * Compared with control group. # Compared with the 1st session. + Compared with the 2nd session. 0 Compared with the dose of 5 mg/kg.
distance moved decreased with repeated session, in the animals treated with 5-mg/kg dose, the distance moved decreased in the second session and increased in the third session compared with the first and second ones (Fig. 3). For the rearing number, we did not find a significant effect of treatment with CGP7930. As for duration of grooming, all three doses of CGP7930 decreased grooming behavior in the third session (Fig. 4). In the 25-day-old rats, a significant increase in the distance moved was found in the animals treated with the 5-mg/kg dose compared with the controls 20 min after the drug administration. Further, both higher doses decreased the distance moved compared with the 5-mg/kg dose. Finally, there was a significant decrease in the second session in the animals treated with the 5- and 20-mg/kg doses of CGP7930 as well as in the third session in all the experimental animals (Fig. 3). For the rearing number in 25-day-old rats, a significant increase was found in the animals treated with the 5-mg/kg dose compared with the controls in the first session. The similar effect was found in the third session with the doses of 5 and 10 mg/kg. In contrast, all doses of CGP7930 significantly decreased the number of rearings in
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Fig. 4. Effect of CGP7930 on rearing and grooming behavior in the open-field test in 18-day-old rats (left side) and in 25-day-old rats (right side). Details are as in Fig. 2.
the second session. The doses of 5 and 10 mg/kg of CGP7930 decreased grooming duration only in the third session. 3.3. Sensorimotor performance In the 12-day-old rats, the 10-mg/kg dose of CGP7930 increased the latency to negative geotaxis 20 min after administration, whereas the dose of 20 mg/kg increased this latency 20 min and 60 min after the drug administration compared with the controls. Repeated measure showed that both higher doses decreased the latency of the negative geotaxis response 24 h after the drug administration. As for the bar-holding test, only the dose of 5 mg/kg of CGP7930 decreased significantly the time to hold the bar 20 and 60 min after the drug injection. In the 18-day-old rats, the negative geotaxis response was not significantly affected by CGP7930 administration. The dose of 5 mg/kg shortened the latency to ascending the wire mesh 20 min after the drug administration. In contrast, the dose of 20 mg/kg significantly increased the time for wire mesh ascending in comparison with the control and 5-mg/kg dose groups. Surprisingly, the sensorimotor performance was not significantly affected in the most demanding tests — bar-holding and rotarod tests. Repeated measure showed an increased time spent on the rotated drum 24 h after the drug administration. In the 25-day-old rats, any dose of CGP7930 tested did not affect significantly the performance in ascending the wire mesh. The dose of 20 mg/kg decreased the time to hold the bar 20 min after the drug administration but improved the performance when repeated 24 h later. Finally, the sensorimotor performance was not significantly altered by drug administration in the rotarod test except for the dose of 20 mg/kg in the first session. Repeated measure showed an improvement in rotarod performance in the control — as well as CGP7930-treated animals (Table 1). 3.4. Elevated plus maze test The CGP7930 did not affect the transfer latency with the exception of shorter latency in the 25-day-old animals pretreated with the
5-mg/kg dose in the first session. With repeated exposure to the EPM, both the control and experimental animals acquired a phobic avoidance of the open arms and retained this form of adaptive learning for at least 24 h. This phenomenon was more expressed in 25than in 18-day-old animals (Fig. 5). There were no significant changes in the percent of time spent in the open arms (an index of anxietylike behavior) as well as in the closed arm entries (an index of locomotion) (Fig. 5). 4. Discussion Anticonvulsant action of GABAB receptor positive allosteric modulator CGP7930 against cortical epileptic afterdischarges was found only in 12-day-old rats in contrast to our recent data with PTZinduced convulsions when the 20- and 40-mg/kg doses affected generalized seizures in all age groups [11]. Different actions in the two models can be due to generators in different brain structures. Generator of generalized tonic–clonic seizures was localized to the brainstem [21], and cortical ADs characterized by spike-and-wave rhythm in the EEG are generated by corticothalamic mechanisms [22,23] with a spread of activity into the motor system. Receptors of GABAB type are widely distributed in the rodent brain [24,25], and they can be demonstrated at early stages of development [26]. As structures responsible for generation of cortical afterdischarges are concerned, GABAB receptors were demonstrated early postnatally in both the cerebral cortex and the thalamus, and they peak at postnatal day 14 [27,28]. Laminar distribution of GABA R1 protein in the cerebral cortex with the highest presence in layers V and VIb is mature at postnatal day 12 [29]. Inactivity of CGP7930 after the second postnatal week indicated in our study could be explained by unequal development of postsynaptic and presynaptic GABAB receptors. Presynaptic GABAB receptors in somatosensory cortex are functional much earlier than postsynaptic ones, and they play a role in regulating not only GABA release [30] but also glutamate release. Maturation of postsynaptic GABAB receptors in the somatosensory cortex takes place in the second and the
24.5 ± 7.1 110.3 ± 9.7a 120 ± 0.0a 20.9 ± 6.5 94.4 ± 12.9 85.8 ± 11.7a 22.9 ± 5.7 82.1 ± 14.2⁎ 56.6 ±16.1⁎ 33.4 ± 5.7 120 ± 0.0 119.0 ± 1.0 26.6 ± 5.6 112.5 ± 5.3 93.1 ± 11.2 23.9 ± 6.4 109 ± 11.0 70.9 ± 15.6 30.7 ± 5.6 120 ± 0.0 120 ± 0.0a 15.7 ± 2.3 120 ± 0.0 113.3 ± 6.7a Values are mean ± S.E.M. ⁎ P b 0.05 compared with control rats. a Compared with session 1 (30 min after drug administration). b Compared with the dose of 5 mg/kg.
14.7 ± 3.5 117.4 ± 2.6 80.3 ± 15.9 15.1 ± 2.9 120 ± 0.0 109.4 ± 10.6a 21.9 ± 6.6 116.6 ± 2.5 69.9 ± 13.2 24 ± 5.7 108.7 ± 7.6 70.3 ± 14.9
5.5 4.2⁎,b 13.7 14.8a ± ± ± ± 17.1 18.9 79 73 2.7 7.8⁎,b 14.6 11.7 ± ± ± ± 10.5 34.4 48.9 85.8 4.6 6.1⁎,b 13.6 10.9 ± ± ± ± 12.6 33.9 39.7 44.6 3.1 3.7 16.0 16.9 ± ± ± ± 8.6 16.9 82.7 64.1 5.4 10.0 7.9 11.2 ± ± ± ± 12.4 30.4 33.2 95.1 3.1 10.0 8.5 15.1 ± ± ± ± 9.3 37.5 41.4 58.8 5.7 2.6 13.9 13.6 ± ± ± ± 8.6 10.4 71.8 63.1 0.6 2.3 10.1 14.3 ± ± ± ± 4.9 13.2 48.3 79.9 1.0 1.5⁎ 12.3 12.4 ± ± ± ± 7 13.7 48.3 59.8 2.6 1.2a 14.3 14.4 ± ± ± ± 9.4 8.4 68.7 65.8 1.1 3.1a 11.3 13.2 ± ± ± ± 5.5 14.4 46 65.9 0.8 2.8 11.8 10.9 ± ± ± ± 6.2 23.9 46 47.8
15.6 ± 3.7⁎ 13.6 ± 0.9
Session 2 Session 1
20.3 ± 6.8⁎ 14.4 ± 1.8 5.2 ± 0.2a 20.7 ± 7.5 11.2 ± 5.6 11.7 ± 1.5 14.4 ± 5.2⁎ 12.3 ± 1.7 4.2 ± 0.6 10.1 ± 1.3 3.7 ± 0.6 7.2 ± 0.7⁎ 5.6 ± 1.3 8.1 ± 0.7 6.2 ± 1.2 9.8 ± 1.2 7.8 ± 1.2 12.1 ± 1.0 5.5 ± 0.9 14 ± 1.7
12 days Negative geotaxis Bar holding 18 days Negative geotaxis Wire mesh Bar holding Rotarod 25 days Wire mesh Bar holding Rotarod
20-mg/kg CGP7930
Session 3 Session 2 Session 1
10-mg/kg CGP7930
Session 3 Session 2 Session 1
5-mg/kg CGP7930
Session 3 Session 1 Testing
Session 2 Controls Group
Table 1 Effect of CGP7930 on sensorimotor performance in 12-, 18-, and 25-day-old rats.
6.5 ± 1.3a 14.2 ± 3.2
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third week of postnatal life [31]. Our data from cortical ADs suggest that GABAB heteroreceptors at glutamatergic terminals might be responsible for the demonstrated anticonvulsant effect. Later maturation of presynaptic homoreceptors and postsynaptic receptors might mask the anticonvulsant effect. The explanation concerning the longer duration of afterdischarges in 12-day-old rats found in some our previous studies (e.g. [32]) but not in the present series of experiments seems improbable. First, there was no significant difference in the present study; the same is true for threshold current intensities — they only tended to be higher in the 12-day-old animals than in the two older groups [8]. Second, prolongation of cortical ADs found after the administration of a GABAB receptor antagonist CGP46381 was marked in the 12-, present in 18-, and absent in 25-day-old rats (Mareš — submitted). Third, some drugs affecting other neurotransmitter systems exhibit the strongest action in the 25-day-old rats with minor effects in the 12-day-old ones (benzodiazepine agonist bretazenil [33]; NMDA receptor antagonists dizocilpine and 2-amino-7-phosphonoheptanoic acid [34]). In addition, effects of CGP7930 against pentylenetetrazol-induced seizures are more marked in the 12- than in 25-day-old rats [11] in spite of the fact that sensitivity of these two age groups to convulsant action of pentylenetetrazol is the same [35]. Comparison of anticonvulsant action of CGP7930 with that of full GABAB receptor agonists, baclofen and SKF97541, argues for the positive allosteric modulator. Neither agonist decreased the duration of afterdischarges in the 12-day-old rats; on the contrary, they prolonged afterdischarges especially in the 18- and 25-day-old animals [9]. There is no “mirror” action of an antagonist of GABAB receptors to agonists or to CGP7930. An antagonist CGP35348 markedly increased duration of cortical afterdischarges in all three age groups [6]; the same is true for another antagonist CGP46381 (experiments in progress). It is in agreement with an increase of persistent network activity under the influence of GABAB receptor antagonist CGP35348 demonstrated in vitro [36]. It may be concluded that the three classes of drugs influence at least partially different subsets of GABAB receptors. Twelve-day-old rats exhibit the highest sensitivity to anticonvulsant action of CGP7930 against cortical ADs as well as against convulsions elicited by pentylenetetrazol [11]. Our results in this age group are in agreement with presentation of GABAB receptors as the important inhibitory system at early stages of development [5] and indicate that it might be possible to search for an anticonvulsant drug specific for developing brain among positive allosteric modulators of GABAB receptors. Our results from the sensorimotor performance tests indicate that administration of CGP7930 in doses up to 20 mg/kg affect subtle sensorimotor performance in immature rats. All animals irrespective of the age were able to perform the given task and improved their performance with the repeated exposure. In the bar holding and rotarod, the most demanding tests of sensorimotor motor performance [37,38], the 25-day-old animals treated with higher dose had a worse performance only in the first exposure. Locomotion (expressed as distance moved in the open field) was not affected by either CGP7930 doses in the 12-day-old rats. The suppression of locomotion and rearing caused by the lower dose of CGP7930 in the first session and the converse increase in both of these behaviors in the 25-day-old rats seem to indicate a different sensitivity to the drug. Generally, rearing behavior is taken as an index of rodent emotionality (more rears being associated with exploratory behavior and/or alert state [39]). The grooming behavior distinguishable at about 20 days of age and reflecting the maturation of motor capabilities [40] was suppressed 24 h after the drug administration in both ages, except for the highest dose administered in the 25-day-old rats. The grooming behavior may serve many functions depending on the context in which this behavior occurs [41]. In a novel environment, grooming behavior is a response to stress and serves to lower the anxiety produced by a stressful experience; therefore, grooming restores the homeostasis of the animal [42,43].
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Fig. 5. Effect of CGP7930 on behavior in the elevated plus maze. Left graphs: 18-day-old rats, right graphs: 25-day-old rats. From top to bottom: transfer latency(s), time spent (%) in open arm, and number of closed arm entries. Abscissa: the 1st, 2nd, and 3rd sessions (i.e., at 30 min, 60 min, and 24 h after drug administration); ordinate: mean + S.E.M. P b 0.05: # Compared with the 1st session.
Regarding behavior in EPM, the CGP7930 did not affect transfer latency, an index of learning and memory, except for the lower dose in the 25-day-old rats in the first exposure that could be explained by a short-time stimulatory effect. Moreover, the drug did not induce an anxiolytic-like response and did not affect locomotion. With repeated exposure to the EPM or OF, a decreased behavioral response occurred. Generally, prior experience with the behavioral procedures alters subsequent responsiveness; animals simply experience environmental novelty, which subsequently influences their behavioral response. Thus, the waning of a response elicited by repeated exposure to a novel stimulus is a phenomenon commonly termed as habituation (for review see [44]). Marked anticonvulsant action with only minor deficits in sensorimotor performance in 12-day-old rats suggests a possible use of positive allosteric modulators of GABAB receptors as age-specific anticonvulsants. Acknowledgments This study was supported by a grant no. P304/10/1274 of the Grant Agency of the Czech Republic, a grant no. 200117 of the Grant Agency of Charles University, and projects LC-554 of the Ministry of Education and AV0Z 50110509 of the Academy of Sciences of the
Czech Republic. The authors would like to thank Ms. Irina Necheva for her excellent technical assistance. References [1] Rogawski MA, Löscher W. The neurobiology of antiepileptic drugs. Nat Rev Neurosci 2004;5:553–64. [2] Pinard A, Seddik R, Bettler B. GABAB receptors. Physiological functions and mechanisms of diversity. Adv Pharmacol 2010;58:231–55. [3] Leung LS, Canning KJ, Shen B. Hippocampal afterdischarges after GABA(B)-receptor blockade in the freely moving rat. Epilepsia 2005;46:203–16. [4] Vergnes M, Boehrer A, Simler S, Bernasconi R, Marescaux C. Opposite effects of GABAB receptor antagonists on absences and convulsive seizures. Eur J Pharmacol 1997;332:245–55. [5] Gaiarsa JL, McLean H, Congar P, Leinekugel X, Khazipov R, Tseeb V, et al. Postnatal maturation of gamma-aminobutyric acid A and B-mediated inhibition in the CA3 hippocampal region of the rat. J Neurobiol 1995;26:339–49. [6] Mareš P. GABA-B receptor antagonist CGP 35348 interferes with an arrest of cortical epileptic afterdischarges in developing rats. Epilepsy Res 2010;92:125–33. [7] Mareš P, Kubová H. Electrical stimulation-induced models of seizures. In: Pitkanen A, Schwartzkroin PA, Moshé SL, editors. Models of seizures and epilepsy. Amsterdam: Elsevier; 2006. p. 153–9. [8] 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. [9] Mareš P, Tabashidze N. Contradictory effects of GABA-B receptor agonists on cortical epileptic afterdischarges in immature rats. Brain Res Bull 2008;75:173–8.
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