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Anticonvulsant action of three neurosteroids against cortical epileptic afterdischarges in immature rats
Brain Research Bulletin 68 (2005) 179–184
Anticonvulsant action of three neurosteroids against cortical epileptic afterdischarges in immature rats Pavel Mareˇs ∗ Institute of Physiology, Academy of Sciences of the Czech Republic, Department of Developmental Epileptology, V´ıdeˇnsk´a 1083, CZ 142 20 Prague 4, Czech Republic Received 13 June 2005; received in revised form 9 August 2005; accepted 22 August 2005 Available online 12 September 2005
Abstract Neurosteroids exhibit anticonvulsant action probably by positive modulatory influence on GABA-A receptors. The action of three neurosteroids was tested against cortical epileptic afterdischarges in immature rats with implanted electrodes. Afterdischarges (ADs) were elicited by rhythmic electrical stimulation (biphasic pulses at 8 Hz frequency for 15 s) of sensorimotor cortical region with a slightly suprathreshold current intensity. Drugs were administered intraperitoneally after the first afterdischarge and stimulation was repeated five more times with the same intensity. Allopregnanolone in doses of 20 and 30 mg/kg i.p. was found to be active in 12-day-old rats; there was no effect in 18-day-old rats and only a tendency in 25-day-old ones. Therefore, the effects of pregnanolone and a new derivative THDOC-conjugate (20 and 40 mg/kg) were compared with those of allopregnanolone (40 mg/kg) only in 12- and 25-day-old rats in the second part of study. All three neurosteroids blocked progressive prolongation of repeated ADs seen in control 12-day-old rats. In addition, pregnanolone was able to shorten the ADs. In contrast, duration of ADs in 25-day-old animals was significantly shorter than the duration of the first, predrug AD only after administration of the 40 mg/kg dose of pregnanolone; if corresponding ADs in the control and drug groups were compared, pregnanolone and THDOC-conjugate led to significantly shorter ADs, changes after allopregnanolone administration were statistically significant only in the fourth AD. None of the studied neurosteroids was able to suppress movements directly bound to stimulation as well as clonic seizures accompanying afterdischarges. Among the three drugs studied, pregnanolone was found to be the most potent one. As developmental changes are concerned, the youngest animals exhibited the highest sensitivity to anticonvulsant action of neurosteroids. © 2005 Elsevier Inc. All rights reserved. Keywords: Neurosteroids; Sensorimotor cortex; Electrical stimulation; Epileptic afterdischarges; Ontogeny; Rat
1. Introduction One of the main targets of antiepileptic drugs is GABA-A receptor [26]. Many classical as well as new antiepileptics potentiate GABAergic inhibition; this potentiation can be realized in different ways—by agonists at the benzodiazepine binding site or at the binding site in the chloride channel, by an increase of synthesis or release of GABA, by an inhibition of reuptake of GABA or by an inhibition of GABA catabolism (for review [15]). Another possibility is positive allosteric modulation of GABA-A receptors exhibited by neurosteroids [13]. Anticonvulsant action of progesterone was discovered by Selye [27] and since that time the same action was described for
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progesterone metabolites (e.g. pregnanolone, allopregnanolone) as well as for synthetic steroids [7,9]. There are numerous data about anticonvulsant action of neurosteroids from experiments in adult laboratory animals demonstrating efficacy against various chemically- [1,5,7,9,10,29], as well as electrically-induced seizures [8]. On the other hand there are only a few results from immature rats: ganaxolone exhibited anticonvulsant action in flurothyl model [14], allopregnanolone was found to be active against pentetrazol-induced motor seizures (Mareˇs et al., submitted for publication). Our study demonstrated not only quantitative (animals in the first 2 weeks of postnatal life are more sensitive than older ones) but also qualitative changes of anticonvulsant action of allopregnanolone during ontogeny. To have more data on development of the anticonvulsant action of allopregnanolone we decided to study its effects in another model routinely used in our laboratory—cortical epileptic afterdischarges. This model allows to measure effects of drugs
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on excitability of motor system or at least of the motor cortex (movements directly elicited by rhythmic electrical stimulation of sensorimotor cortical area), on epileptic afterdischarges characterized by spike-and-wave rhythm in the EEG generated in the thalamocortical system and on clonic seizures accompanying these afterdischarges due to a spread of epileptic activity to the motor system. This type of epileptic seizures can be reliably elicited since postnatal day 12 [17,18]. Among new epalons synthesized in the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences in Prague and tested in our laboratory triethylammonium 3␣-hydroxy-20-oxo-5␣-pregnan-21-yl hydrogensuccinate (THDOC-conjugate) was found to have anticonvulsant activity similar to that of allopregnanolone (Mareˇs et al. [19]). Therefore, we added the second series of experiments with this drug and another naturally occurring neurosteroid–pregnanolone (3␣-hydroxy-5-pregnan-20-one), stereoisomer of allopregnanolone, to compare efficacy of these three drugs. The first series of experiments (with allopregnanolone) was performed in 12-, 18- and 25-day-old rats. The anticonvulsant effect was clearly seen in 12- and 25-day-old animals. Therefore the second series with all three neurosteroids was made only in these two age groups. 2. Materials and methods Three age groups of immature rats from the breeding colony of the Institute of Physiology were used in the present experiments. Animals were kept with their mothers up to the experiment under controlled conditions (temperature 22 ± 1 ◦ C, humidity 50–60%, 12/12 h light/dark cycle, lights-on at 6:00 p.m.). An average weight of 12-, 18- and 25-day-old rat pups was 27.0 ± 0.3, 38.9 ± 0.9, and 63.7 ± 0.9 g (mean ± S.E.M.), respectively. Experiments were performed according to the Animal Protection Law of the Czech Republic (fully compatible with European Community Council directives 86/609/EEC) and approved by Animal Care and Use Committee of the Institute of Physiology. Surgical preparation was made under ether anesthesia. Cortical flat silver electrodes were implanted epidurally: two stimulation electrodes over right sensorimotor area (AP +1 and −1 mm; L 2.5 mm in relation to bregma), and four recording electrodes: over left sensorimotor area (AP 0 mm, L 2.5 mm), left parietal and left and right occipital areas. Coordinates for the parietal and occipital electrodes were calculated from adult values AP 3 mm, L 3 mm and AP 6 mm, L 4 mm, respectively. This recalculation was based on the ratio between the actual bregma- distance in individual animals and an adult value (8 mm). Reference electrode was inserted into the nasal bone, ground electrode into the occipital bone. The whole assembly was fixed to the skull with a fast curing dental acrylic, then the anesthesia was interrupted and the animals were allowed to rest for at least 1 h. Stimulation series lasting 15 s (1 ms biphasic pulses at a frequency of 8 Hz) were generated by a constant current stimulator. The threshold intensity for elicitation of an epileptic afterdischarge was established. This intensity varied from 3 to 5 mA. Rats not responding to the 5 mA current were not included into the study. Stimulation series with the threshold intensity was applied six times. Interval between stimulation series was different in the two experiments. Experiment 1 was performed on three age groups of rats (12-, 18- and 25-days-old), interval between stimulation series was 20 min and only allopregnanolone was studied. Because of short duration of anticonvulsant action of the three neurosteroids found in the parallel experiments with pentetrazol-induced seizures (Mareˇs et al. [19]) interstimulation interval was shortened to 10 min in Experiment 2. In this experiment, all three drugs were tested in two age groups—12and 25-day-old animals. Neurosteroids – allopregnanolone (3␣-hydroxy-5␣-pregnan-20-one), pregnanolone (3␣-hydroxy-5-pregnan-20-one) and triethylammonium 3␣hydroxy-20-oxo-5␣-pregnan-21-yl hydrogensuccinate (THDOC-conjugate)
synthesized in the Institute of Organic Chemistry and Biochemistry – were freshly put into suspension using a drop of Tween 80. Concentration of all three drugs was 10 mg/ml. Experiment 1: allopregnanolone in doses of 20 or 30 mg/kg was injected intraperitoneally 10 min after the end of the first afterdischarge. Experiment 2: allopregnanolone in a dose of 40 mg/kg and the other two drugs in doses of 20 or 40 mg/kg were administered intraperitoneally 5 min after the end of the first afterdischarge. Doses of neurosteroids were chosen on the basis of our results on the action of these drugs against pentetrazol-induced seizures; the doses used in the present study exhibited an anticonvulsant action against generalized tonic–clonic seizures (Mareˇs et al. [19]). Control animals (siblings) received an injection of saline with a drop of Tween in a volume corresponding to the higher volume of allopregnonanolone, i.e. 3 ml/kg in Experiment 1 and 4 ml/kg in Experiment 2. Each age and dose group in either experiment was formed by 8–12 rat pups. EEG was recorded in reference connections; activity was digitized at a rate of 500 Hz and saved at the harddisc. A system fabricated by Kaminskij-Biomedical Research Systems, Prague, Czech Republic, was used. Registration started 20 s before stimulation, continued during stimulation and afterdischarge and at least for 1 min after the end of the afterdischarge. Behavioral phenomena were coded directly into the recording. Intensity of seizures was qualified according to a Racine’s scale modified in point 1 [21,24]: 1, activities not synchronous with stimuli and/or sharp graphoelements in the EEG; 2, clonic seizures of head; 3, clonic seizures of forepaws; 4, clonic seizures of forepaws + rearing; 5, clonic seizures of forepaws + rearing + falling. Intensity of movements during stimulation as well as of clonic seizures accompanying ADs was classified by the highest grade observed. Duration of ADs was measured and averages and standard errors of the mean were calculated. Statistical comparison (SigmaStat® SPSS) was performed by repeated measures ANOVA (comparison of individual stimulation series in the same age and dose group) or by ANOVA (comparison of corresponding afterdischarges among the dose groups), the pairwise comparison was made with Tukey’s test. In the case of the 40 mg/kg dose of allopregnanolone (duration of ADs was compared between two groups—control and allopregnanolone) t-test was used. The level of statistical significance was set at 5%.
3. Results 3.1. Experiment 1 Rhythmic electrical stimulation of the sensorimotor cortical area was in all animals accompanied by jerks of the head and contralateral forelimbs. These jerks spread to other limbs, 18- and 25-day-old rats sometimes exhibited rearing with clonic jerks of both forelimbs (stage 4)—a phenomenon not observed in 12-day-old rat pups. The first stimulation always induced an afterdischarge characterized by spike-and-wave rhythm in 18and 25-day-old rats and by rhythmic sharp waves in 12-dayold animals (Fig. 1). All these sharp epileptic graphoelements were accompanied by clonic seizures (individual jerks were synchronous with spikes and/or sharp waves). Repetition of stimulation in control groups resulted in a progressive prolongation of afterdischarges—significant changes were found in 12and 18-day-old rats (F = 8.028 and 5.436; p < 0.001 and =0.001, respectively). ADs after the fifth and sixth stimulation were longer than the first, predrug one. The outlined difference in 25day-old animals did not reach the level of statistical significance (p = 0.067). Severity of movements accompanying stimulation and/or afterdischarges expressed as an average score did not change with repeated stimulations (data not shown). Duration of afterdischarges was influenced by allopregnanolone in 12-day-old rats (Fig. 2). A tendency to shortening of afterdischarges was observed after the second stimulation and
P. Mareˇs / Brain Research Bulletin 68 (2005) 179–184
Fig. 1. EEG recordings of afterdischarges in 12- (top) and 25-day-old (bottom) rats. In both parts, C means control animal, P rat receiving pregnanolone in a dose of 40 mg/kg i.p. Six lines in recordings from each animal represent from top to bottom six successive afterdischarges, an arrow denotes moment of pregnalone or solvent injection. Left part of each recording represents last 9 s before stimulation and the first second of stimulation; then 13 s are omitted and the right part represents the last second of stimulation and 39 s after the end of stimulation. Time mark, 2 s; amplitude calibration, 1 mV.
then a slow progressive increase of ADs duration took place after the higher (30 mg/kg) dose. The 20 mg/kg dose only delayed the significant prolongation to the sixth afterdischarge. There was no effect of allopregnanolone in 18-day-old rats and only a shortlasting tendency to a shortening of afterdischarges in 25-day-old animals (Fig. 2). Differences among the corresponding afterdischarges in the three groups never reached the level of statistical significance in spite of the very long first afterdischarge in the group designated for the higher dose of allopregnanolone. Motor phenomena during stimulation as well as severity of clonic seizures accompanying afterdischarges were not influenced by either dose of allopregnanolone in any age group. 3.2. Experiment 2 Based on the results of Experiment 1, the 18-day-old group was omitted. Control 12-day-old group with the 10 min intervals exhibited a clear-cut prolongation of afterdischarges (F = 2.740, p = 0.023). The second to sixth afterdischarges were significantly
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Fig. 2. Average duration (+S.E.M.) of afterdischarges in 12-, 18- and 25-day-old rats (from top to bottom). Allopregnanolone or solvent was injected 10 min after the first afterdischarge. Abscissa from left to right: first to sixth afterdischarge (interval between stimulations was 20 min); ordinate: duration in seconds. Individual column, see inset. Asterisks, significant difference in comparison with the appropriate first afterdischarge; double cross, significant difference in comparison with the corresponding afterdischarge in the control group.
longer than the first one. Allopregnanolone (40 mg/kg) abolished the progressive prolongation of afterdischarges in this age group and the third to sixth afterdischarges were significantly shorter than corresponding ones in the control group (Fig. 3). The effect of allopregnanolone was not so marked in 25-day-old rats. Control animals exhibited an increase in duration of ADs (F = 2.724, p = 0.038); the fourth and fifth afterdischarges were significantly longer than the first, preinjection one. Allopregnanolone shifted the increase in duration to the fifth and sixth afterdischarges (Fig. 3). The difference between corresponding afterdischarges in the control and allopregnanolone groups was significant only in the fourth afterdischarge. Pregnanolone reliably abolished the prolongation of afterdischarges in 12-day-old rats (Fig. 1) so that there was no significant difference among the six ADs in either dose group. All postdrug afterdischarges (with the exception of the second afterdischarge after the 20 mg/kg dose) were significantly shorter than corresponding ones in the control group (Fig. 4). In 25-day-old animals, the 40 mg/kg dose of pregnanolone resulted in a significant difference in duration of ADs (F = 8.432, P < 0.001). It not only blocked the progressive increase of afterdischarge duration observed in the control group (Fig. 1), but it significantly shortened the second and third afterdischarges (Fig. 4). In contrast, a moderate but significant prolongation was found since the fourth
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Fig. 3. Average duration (+S.E.M.) of afterdischarges in 12- and 25-day-old rats. Allopregnanolone was injected 5 min after the first afterdischarge. Details as in Fig. 2 only the interval between stimulations was 10 min.
Fig. 4. Average duration (+S.E.M.) of afterdischarges in 12- and 25-day-old rats. Pregnanolone was injected 5 min after the first afterdischarge. Details as in Fig. 2 only the interval between stimulations was 10 min.
afterdischarge in the 20 mg/kg group (F = 3.607, p = 0.008 for this group; Fig. 4). Differences between drug groups and the control one exhibited a dependence on the dose—they lasted longer after the higher dose (the second, third, fourth and fifth afterdischarges were shorter than corresponding ones in the control group) than after the lower dose of pregnanolone (Fig. 4). In addition, the higher dose of pregnanolone markedly decreased the amplitude of spikes and waves in this age group (Fig. 1). THDOC-conjugate also blocked the progressive prolongation of afterdischarges with repeated stimulations in 12-day-old rat pups (Fig. 5). If corresponding afterdischarges were compared among the three groups, the higher dose of THDOCconjugate resulted in all postinjection afterdischarges significantly shorter than the control ones; such a difference was found only in the third afterdischarge in the group with the 20 mg/kg dose. The prolongation of afterdischarges was not abolished in 25-day-old rats (F = 4.521 and 4.017, p = 0.01 and 0.023 for the 20 and 40 mg/kg group, respectively) but the difference between the duration of afterdischarges in the control and either THDOCconjugate group was significant in most cases (Fig. 5). Direct comparison of the effects of all three neurosteroids could be performed only for the 40 mg/kg dose. Significant differences in duration (ADs after pregnanolone administration were shorter than after the other two drugs) were found in the second, third and fourth ADs in 12-day-old rats (F = 6.037, 7.697 and 6.115, p = 0.007, 0.002 and 0.006, respectively). ADs in 25-day-old rats were also shorter after pregnanolone than after
the other two neurosteroids. Statistical significance was reached for the fourth and fifth ADs (F = 6.858 and 3.628, p = 0.004 and 0.039, respectively). The difference among the third ADs stayed below the level of significance (p = 0.061). Motor phenomena (movements directly elicited by stimulation and clonic seizures) were not influenced by the three neurosteroids studied (data not shown). 4. Discussion Our results confirmed developmental difference between the 12-day-old animals and the two older groups in the electroencephalographic pattern of afterdischarges and motor phenomena [17]. The absence of spike-and-wave rhythm in 12-day-old rats is in agreement with maturation of rhythmic thalamocortical phenomena [16] and with an immaturity of the cerebral cortex expressed as a low incidence of bursting neurons [6] as well as a poor synchronization of activities of individual neurons during epileptic phenomena [23]. Failure of rearing in this age group corresponds to their inability to rear during spontaneous behavior [3]. All three neurosteroids exhibit an anticonvulsant action in the model of cortical epileptic afterdischarges in 12- as well as 25day-old rat pups. Failure of allopregnanolone in 18-day-old rats cannot be explained at the moment. We found a similar irregularity in the action of ketamine in the same model [11]. The reasons for this exception have to be analyzed in future. Comparing the
P. Mareˇs / Brain Research Bulletin 68 (2005) 179–184
Fig. 5. Average duration (+S.E.M.) of afterdischarges in 12- and 25-day-old rats. THDOC-conjugate was injected 5 min after the first afterdischarge. Details as in Fig. 2 only the interval between stimulations was 10 min.
results of Experiment 2 pregnanolone appears to be more efficient than the other two neurosteroids. This difference was better seen in 25- than in 12-day-old rats. The three neurosteroids only blocked the prolongation of afterdischarges with repetitive stimulations in 12-day-old rats. In contrast, pregnanolone was able to shorten the second and third afterdischarge in 25-day-old rats. Abolition of prolongation and shortening of afterdischarges can be taken as two different phenomena. Prolongation of afterdischarges is an expression of changes following a previous afterdischarge similar to partial kindling as described by Racine et al. [25]. Decrease of afterdischarge duration might be taken as an effect on generation of seizures or – more probably – as a potentiation of mechanisms arresting seizures. Comparison of the present results with our previous data on the action of other drugs augmenting GABAergic inhibition reveals a similarity of action of neurosteroids with that of benzodiazepines and phenobarbital which are equally effective in both 12- and 25-day-old rats [12,22]. In contrast, progabide (both GABA-A and GABA-B agonist) – [20] and NNC-711 (GABA uptake inhibitor) – [28] and valproate exhibit markedly higher efficacy in 25- than in 12-day-old rats [2,22]. It might be hypothesized that targets for drugs influencing directly the opening of chloride channels – benzodiazepines and phenobarbital – are mature at the age of 12 days. Developmental data on benzodiazepine receptors are in agreement with this possible explanation [4]. Drugs acting at more general level – inhibitor of GABA-transporter NNC-711, mixed GABA-A and GABA-
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B agonist progabide and a drug with many possible but not exactly proved actions valproate – are more efficient in 25- than in 12-day-old rats. The developmental profile of the action of neurosteroids studied in the our experiment is close to the drugs opening chloride channel. Unfortunately, there are no comparable data on the ontogeny of allopregnanolone or pregnanolone binding to the supramolecular complex of GABA-A receptor to support this possibility. Absence of effects of all three neurosteroids on motor phenomena was surprising because we found a short-lasting derangement of motor abilities of rat pups after allopregnanolone administration expressed especially in 12-day-old rats (Mareˇs et al., submitted for publication). Therefore, we expected at least a change in the ability to rear during stimulation and clonic seizures in 18- and 25-day-old rats. Electrical stimulation and epileptic activity are probably so strong activators of the motor system that a moderate disability could be overcome. An additional factor – possible differences in the action of various neurosteroids on motor performance – remains to be studied in future. Among drugs potentiating GABAergic inhibition clonazepam is an exception because it very potently suppress clonic seizures preserving shortened afterdischarges without any motor correlate. On the other hand, it does not affect movements directly elicited by electrical stimulation of sensorimotor cortex [12]. The effects of other mentioned drugs is not so marked: NNC-711, progabide and valproate decrease the intensity of both stimulation-bound movements and clonic seizures in 25- and 18-day-old rats; this effect is only moderate in 12day-old animals [2,22]. Phenobarbital exhibits an effect on both motor phenomena only in the highest dose used (40 mg/kg i.p.). The only developmental change is higher efficacy of phenobarbital against clonic seizures in 12-, if compared with 18- and 25-day-old rats [22]. It can be concluded that the mechanism of potentiation of GABAergic inhibition is very important for the anticonvulsant action of drugs modifying this main inhibitory system. All three neurosteroids studied exhbited an anticonvulsant action against epileptic afterdischarges induced by electrical stimulation of sensorimotor cortical region in immature rats. Unfortunately, the new derivative was less potent than pregnanolone, a natural metabolite of progesterone. Acknowledgements This study was supported by research projects no. S 5011007 and AV0Z 50110509 of the Academy of Sciences of the Czech Republic. The author would like to express thanks to A. Kasal, D. Sc from the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences in Prague for a supply of neurosteroids and to Ms. Irina Necheva for excellent technical assistance. References [1] D. Belelli, M.B. Bolger, K.W. Gee, Anticonvulsant profile of the progesteron metabolite 5␣-pregnan-3␣-ol-20-one, Eur. J. Pharmacol. 166 (1989) 325–329.
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Anticonvulsant action of three neurosteroids against cortical epileptic afterdischarges in immature rats Pavel Mareˇs ∗ Institute of Physiology, Academy of Sciences of the Czech Republic, Department of Developmental Epileptology, V´ıdeˇnsk´a 1083, CZ 142 20 Prague 4, Czech Republic Received 13 June 2005; received in revised form 9 August 2005; accepted 22 August 2005 Available online 12 September 2005
Abstract Neurosteroids exhibit anticonvulsant action probably by positive modulatory influence on GABA-A receptors. The action of three neurosteroids was tested against cortical epileptic afterdischarges in immature rats with implanted electrodes. Afterdischarges (ADs) were elicited by rhythmic electrical stimulation (biphasic pulses at 8 Hz frequency for 15 s) of sensorimotor cortical region with a slightly suprathreshold current intensity. Drugs were administered intraperitoneally after the first afterdischarge and stimulation was repeated five more times with the same intensity. Allopregnanolone in doses of 20 and 30 mg/kg i.p. was found to be active in 12-day-old rats; there was no effect in 18-day-old rats and only a tendency in 25-day-old ones. Therefore, the effects of pregnanolone and a new derivative THDOC-conjugate (20 and 40 mg/kg) were compared with those of allopregnanolone (40 mg/kg) only in 12- and 25-day-old rats in the second part of study. All three neurosteroids blocked progressive prolongation of repeated ADs seen in control 12-day-old rats. In addition, pregnanolone was able to shorten the ADs. In contrast, duration of ADs in 25-day-old animals was significantly shorter than the duration of the first, predrug AD only after administration of the 40 mg/kg dose of pregnanolone; if corresponding ADs in the control and drug groups were compared, pregnanolone and THDOC-conjugate led to significantly shorter ADs, changes after allopregnanolone administration were statistically significant only in the fourth AD. None of the studied neurosteroids was able to suppress movements directly bound to stimulation as well as clonic seizures accompanying afterdischarges. Among the three drugs studied, pregnanolone was found to be the most potent one. As developmental changes are concerned, the youngest animals exhibited the highest sensitivity to anticonvulsant action of neurosteroids. © 2005 Elsevier Inc. All rights reserved. Keywords: Neurosteroids; Sensorimotor cortex; Electrical stimulation; Epileptic afterdischarges; Ontogeny; Rat
1. Introduction One of the main targets of antiepileptic drugs is GABA-A receptor [26]. Many classical as well as new antiepileptics potentiate GABAergic inhibition; this potentiation can be realized in different ways—by agonists at the benzodiazepine binding site or at the binding site in the chloride channel, by an increase of synthesis or release of GABA, by an inhibition of reuptake of GABA or by an inhibition of GABA catabolism (for review [15]). Another possibility is positive allosteric modulation of GABA-A receptors exhibited by neurosteroids [13]. Anticonvulsant action of progesterone was discovered by Selye [27] and since that time the same action was described for
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progesterone metabolites (e.g. pregnanolone, allopregnanolone) as well as for synthetic steroids [7,9]. There are numerous data about anticonvulsant action of neurosteroids from experiments in adult laboratory animals demonstrating efficacy against various chemically- [1,5,7,9,10,29], as well as electrically-induced seizures [8]. On the other hand there are only a few results from immature rats: ganaxolone exhibited anticonvulsant action in flurothyl model [14], allopregnanolone was found to be active against pentetrazol-induced motor seizures (Mareˇs et al., submitted for publication). Our study demonstrated not only quantitative (animals in the first 2 weeks of postnatal life are more sensitive than older ones) but also qualitative changes of anticonvulsant action of allopregnanolone during ontogeny. To have more data on development of the anticonvulsant action of allopregnanolone we decided to study its effects in another model routinely used in our laboratory—cortical epileptic afterdischarges. This model allows to measure effects of drugs
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on excitability of motor system or at least of the motor cortex (movements directly elicited by rhythmic electrical stimulation of sensorimotor cortical area), on epileptic afterdischarges characterized by spike-and-wave rhythm in the EEG generated in the thalamocortical system and on clonic seizures accompanying these afterdischarges due to a spread of epileptic activity to the motor system. This type of epileptic seizures can be reliably elicited since postnatal day 12 [17,18]. Among new epalons synthesized in the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences in Prague and tested in our laboratory triethylammonium 3␣-hydroxy-20-oxo-5␣-pregnan-21-yl hydrogensuccinate (THDOC-conjugate) was found to have anticonvulsant activity similar to that of allopregnanolone (Mareˇs et al. [19]). Therefore, we added the second series of experiments with this drug and another naturally occurring neurosteroid–pregnanolone (3␣-hydroxy-5-pregnan-20-one), stereoisomer of allopregnanolone, to compare efficacy of these three drugs. The first series of experiments (with allopregnanolone) was performed in 12-, 18- and 25-day-old rats. The anticonvulsant effect was clearly seen in 12- and 25-day-old animals. Therefore the second series with all three neurosteroids was made only in these two age groups. 2. Materials and methods Three age groups of immature rats from the breeding colony of the Institute of Physiology were used in the present experiments. Animals were kept with their mothers up to the experiment under controlled conditions (temperature 22 ± 1 ◦ C, humidity 50–60%, 12/12 h light/dark cycle, lights-on at 6:00 p.m.). An average weight of 12-, 18- and 25-day-old rat pups was 27.0 ± 0.3, 38.9 ± 0.9, and 63.7 ± 0.9 g (mean ± S.E.M.), respectively. Experiments were performed according to the Animal Protection Law of the Czech Republic (fully compatible with European Community Council directives 86/609/EEC) and approved by Animal Care and Use Committee of the Institute of Physiology. Surgical preparation was made under ether anesthesia. Cortical flat silver electrodes were implanted epidurally: two stimulation electrodes over right sensorimotor area (AP +1 and −1 mm; L 2.5 mm in relation to bregma), and four recording electrodes: over left sensorimotor area (AP 0 mm, L 2.5 mm), left parietal and left and right occipital areas. Coordinates for the parietal and occipital electrodes were calculated from adult values AP 3 mm, L 3 mm and AP 6 mm, L 4 mm, respectively. This recalculation was based on the ratio between the actual bregma- distance in individual animals and an adult value (8 mm). Reference electrode was inserted into the nasal bone, ground electrode into the occipital bone. The whole assembly was fixed to the skull with a fast curing dental acrylic, then the anesthesia was interrupted and the animals were allowed to rest for at least 1 h. Stimulation series lasting 15 s (1 ms biphasic pulses at a frequency of 8 Hz) were generated by a constant current stimulator. The threshold intensity for elicitation of an epileptic afterdischarge was established. This intensity varied from 3 to 5 mA. Rats not responding to the 5 mA current were not included into the study. Stimulation series with the threshold intensity was applied six times. Interval between stimulation series was different in the two experiments. Experiment 1 was performed on three age groups of rats (12-, 18- and 25-days-old), interval between stimulation series was 20 min and only allopregnanolone was studied. Because of short duration of anticonvulsant action of the three neurosteroids found in the parallel experiments with pentetrazol-induced seizures (Mareˇs et al. [19]) interstimulation interval was shortened to 10 min in Experiment 2. In this experiment, all three drugs were tested in two age groups—12and 25-day-old animals. Neurosteroids – allopregnanolone (3␣-hydroxy-5␣-pregnan-20-one), pregnanolone (3␣-hydroxy-5-pregnan-20-one) and triethylammonium 3␣hydroxy-20-oxo-5␣-pregnan-21-yl hydrogensuccinate (THDOC-conjugate)
synthesized in the Institute of Organic Chemistry and Biochemistry – were freshly put into suspension using a drop of Tween 80. Concentration of all three drugs was 10 mg/ml. Experiment 1: allopregnanolone in doses of 20 or 30 mg/kg was injected intraperitoneally 10 min after the end of the first afterdischarge. Experiment 2: allopregnanolone in a dose of 40 mg/kg and the other two drugs in doses of 20 or 40 mg/kg were administered intraperitoneally 5 min after the end of the first afterdischarge. Doses of neurosteroids were chosen on the basis of our results on the action of these drugs against pentetrazol-induced seizures; the doses used in the present study exhibited an anticonvulsant action against generalized tonic–clonic seizures (Mareˇs et al. [19]). Control animals (siblings) received an injection of saline with a drop of Tween in a volume corresponding to the higher volume of allopregnonanolone, i.e. 3 ml/kg in Experiment 1 and 4 ml/kg in Experiment 2. Each age and dose group in either experiment was formed by 8–12 rat pups. EEG was recorded in reference connections; activity was digitized at a rate of 500 Hz and saved at the harddisc. A system fabricated by Kaminskij-Biomedical Research Systems, Prague, Czech Republic, was used. Registration started 20 s before stimulation, continued during stimulation and afterdischarge and at least for 1 min after the end of the afterdischarge. Behavioral phenomena were coded directly into the recording. Intensity of seizures was qualified according to a Racine’s scale modified in point 1 [21,24]: 1, activities not synchronous with stimuli and/or sharp graphoelements in the EEG; 2, clonic seizures of head; 3, clonic seizures of forepaws; 4, clonic seizures of forepaws + rearing; 5, clonic seizures of forepaws + rearing + falling. Intensity of movements during stimulation as well as of clonic seizures accompanying ADs was classified by the highest grade observed. Duration of ADs was measured and averages and standard errors of the mean were calculated. Statistical comparison (SigmaStat® SPSS) was performed by repeated measures ANOVA (comparison of individual stimulation series in the same age and dose group) or by ANOVA (comparison of corresponding afterdischarges among the dose groups), the pairwise comparison was made with Tukey’s test. In the case of the 40 mg/kg dose of allopregnanolone (duration of ADs was compared between two groups—control and allopregnanolone) t-test was used. The level of statistical significance was set at 5%.
3. Results 3.1. Experiment 1 Rhythmic electrical stimulation of the sensorimotor cortical area was in all animals accompanied by jerks of the head and contralateral forelimbs. These jerks spread to other limbs, 18- and 25-day-old rats sometimes exhibited rearing with clonic jerks of both forelimbs (stage 4)—a phenomenon not observed in 12-day-old rat pups. The first stimulation always induced an afterdischarge characterized by spike-and-wave rhythm in 18and 25-day-old rats and by rhythmic sharp waves in 12-dayold animals (Fig. 1). All these sharp epileptic graphoelements were accompanied by clonic seizures (individual jerks were synchronous with spikes and/or sharp waves). Repetition of stimulation in control groups resulted in a progressive prolongation of afterdischarges—significant changes were found in 12and 18-day-old rats (F = 8.028 and 5.436; p < 0.001 and =0.001, respectively). ADs after the fifth and sixth stimulation were longer than the first, predrug one. The outlined difference in 25day-old animals did not reach the level of statistical significance (p = 0.067). Severity of movements accompanying stimulation and/or afterdischarges expressed as an average score did not change with repeated stimulations (data not shown). Duration of afterdischarges was influenced by allopregnanolone in 12-day-old rats (Fig. 2). A tendency to shortening of afterdischarges was observed after the second stimulation and
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Fig. 1. EEG recordings of afterdischarges in 12- (top) and 25-day-old (bottom) rats. In both parts, C means control animal, P rat receiving pregnanolone in a dose of 40 mg/kg i.p. Six lines in recordings from each animal represent from top to bottom six successive afterdischarges, an arrow denotes moment of pregnalone or solvent injection. Left part of each recording represents last 9 s before stimulation and the first second of stimulation; then 13 s are omitted and the right part represents the last second of stimulation and 39 s after the end of stimulation. Time mark, 2 s; amplitude calibration, 1 mV.
then a slow progressive increase of ADs duration took place after the higher (30 mg/kg) dose. The 20 mg/kg dose only delayed the significant prolongation to the sixth afterdischarge. There was no effect of allopregnanolone in 18-day-old rats and only a shortlasting tendency to a shortening of afterdischarges in 25-day-old animals (Fig. 2). Differences among the corresponding afterdischarges in the three groups never reached the level of statistical significance in spite of the very long first afterdischarge in the group designated for the higher dose of allopregnanolone. Motor phenomena during stimulation as well as severity of clonic seizures accompanying afterdischarges were not influenced by either dose of allopregnanolone in any age group. 3.2. Experiment 2 Based on the results of Experiment 1, the 18-day-old group was omitted. Control 12-day-old group with the 10 min intervals exhibited a clear-cut prolongation of afterdischarges (F = 2.740, p = 0.023). The second to sixth afterdischarges were significantly
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Fig. 2. Average duration (+S.E.M.) of afterdischarges in 12-, 18- and 25-day-old rats (from top to bottom). Allopregnanolone or solvent was injected 10 min after the first afterdischarge. Abscissa from left to right: first to sixth afterdischarge (interval between stimulations was 20 min); ordinate: duration in seconds. Individual column, see inset. Asterisks, significant difference in comparison with the appropriate first afterdischarge; double cross, significant difference in comparison with the corresponding afterdischarge in the control group.
longer than the first one. Allopregnanolone (40 mg/kg) abolished the progressive prolongation of afterdischarges in this age group and the third to sixth afterdischarges were significantly shorter than corresponding ones in the control group (Fig. 3). The effect of allopregnanolone was not so marked in 25-day-old rats. Control animals exhibited an increase in duration of ADs (F = 2.724, p = 0.038); the fourth and fifth afterdischarges were significantly longer than the first, preinjection one. Allopregnanolone shifted the increase in duration to the fifth and sixth afterdischarges (Fig. 3). The difference between corresponding afterdischarges in the control and allopregnanolone groups was significant only in the fourth afterdischarge. Pregnanolone reliably abolished the prolongation of afterdischarges in 12-day-old rats (Fig. 1) so that there was no significant difference among the six ADs in either dose group. All postdrug afterdischarges (with the exception of the second afterdischarge after the 20 mg/kg dose) were significantly shorter than corresponding ones in the control group (Fig. 4). In 25-day-old animals, the 40 mg/kg dose of pregnanolone resulted in a significant difference in duration of ADs (F = 8.432, P < 0.001). It not only blocked the progressive increase of afterdischarge duration observed in the control group (Fig. 1), but it significantly shortened the second and third afterdischarges (Fig. 4). In contrast, a moderate but significant prolongation was found since the fourth
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Fig. 3. Average duration (+S.E.M.) of afterdischarges in 12- and 25-day-old rats. Allopregnanolone was injected 5 min after the first afterdischarge. Details as in Fig. 2 only the interval between stimulations was 10 min.
Fig. 4. Average duration (+S.E.M.) of afterdischarges in 12- and 25-day-old rats. Pregnanolone was injected 5 min after the first afterdischarge. Details as in Fig. 2 only the interval between stimulations was 10 min.
afterdischarge in the 20 mg/kg group (F = 3.607, p = 0.008 for this group; Fig. 4). Differences between drug groups and the control one exhibited a dependence on the dose—they lasted longer after the higher dose (the second, third, fourth and fifth afterdischarges were shorter than corresponding ones in the control group) than after the lower dose of pregnanolone (Fig. 4). In addition, the higher dose of pregnanolone markedly decreased the amplitude of spikes and waves in this age group (Fig. 1). THDOC-conjugate also blocked the progressive prolongation of afterdischarges with repeated stimulations in 12-day-old rat pups (Fig. 5). If corresponding afterdischarges were compared among the three groups, the higher dose of THDOCconjugate resulted in all postinjection afterdischarges significantly shorter than the control ones; such a difference was found only in the third afterdischarge in the group with the 20 mg/kg dose. The prolongation of afterdischarges was not abolished in 25-day-old rats (F = 4.521 and 4.017, p = 0.01 and 0.023 for the 20 and 40 mg/kg group, respectively) but the difference between the duration of afterdischarges in the control and either THDOCconjugate group was significant in most cases (Fig. 5). Direct comparison of the effects of all three neurosteroids could be performed only for the 40 mg/kg dose. Significant differences in duration (ADs after pregnanolone administration were shorter than after the other two drugs) were found in the second, third and fourth ADs in 12-day-old rats (F = 6.037, 7.697 and 6.115, p = 0.007, 0.002 and 0.006, respectively). ADs in 25-day-old rats were also shorter after pregnanolone than after
the other two neurosteroids. Statistical significance was reached for the fourth and fifth ADs (F = 6.858 and 3.628, p = 0.004 and 0.039, respectively). The difference among the third ADs stayed below the level of significance (p = 0.061). Motor phenomena (movements directly elicited by stimulation and clonic seizures) were not influenced by the three neurosteroids studied (data not shown). 4. Discussion Our results confirmed developmental difference between the 12-day-old animals and the two older groups in the electroencephalographic pattern of afterdischarges and motor phenomena [17]. The absence of spike-and-wave rhythm in 12-day-old rats is in agreement with maturation of rhythmic thalamocortical phenomena [16] and with an immaturity of the cerebral cortex expressed as a low incidence of bursting neurons [6] as well as a poor synchronization of activities of individual neurons during epileptic phenomena [23]. Failure of rearing in this age group corresponds to their inability to rear during spontaneous behavior [3]. All three neurosteroids exhibit an anticonvulsant action in the model of cortical epileptic afterdischarges in 12- as well as 25day-old rat pups. Failure of allopregnanolone in 18-day-old rats cannot be explained at the moment. We found a similar irregularity in the action of ketamine in the same model [11]. The reasons for this exception have to be analyzed in future. Comparing the
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Fig. 5. Average duration (+S.E.M.) of afterdischarges in 12- and 25-day-old rats. THDOC-conjugate was injected 5 min after the first afterdischarge. Details as in Fig. 2 only the interval between stimulations was 10 min.
results of Experiment 2 pregnanolone appears to be more efficient than the other two neurosteroids. This difference was better seen in 25- than in 12-day-old rats. The three neurosteroids only blocked the prolongation of afterdischarges with repetitive stimulations in 12-day-old rats. In contrast, pregnanolone was able to shorten the second and third afterdischarge in 25-day-old rats. Abolition of prolongation and shortening of afterdischarges can be taken as two different phenomena. Prolongation of afterdischarges is an expression of changes following a previous afterdischarge similar to partial kindling as described by Racine et al. [25]. Decrease of afterdischarge duration might be taken as an effect on generation of seizures or – more probably – as a potentiation of mechanisms arresting seizures. Comparison of the present results with our previous data on the action of other drugs augmenting GABAergic inhibition reveals a similarity of action of neurosteroids with that of benzodiazepines and phenobarbital which are equally effective in both 12- and 25-day-old rats [12,22]. In contrast, progabide (both GABA-A and GABA-B agonist) – [20] and NNC-711 (GABA uptake inhibitor) – [28] and valproate exhibit markedly higher efficacy in 25- than in 12-day-old rats [2,22]. It might be hypothesized that targets for drugs influencing directly the opening of chloride channels – benzodiazepines and phenobarbital – are mature at the age of 12 days. Developmental data on benzodiazepine receptors are in agreement with this possible explanation [4]. Drugs acting at more general level – inhibitor of GABA-transporter NNC-711, mixed GABA-A and GABA-
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B agonist progabide and a drug with many possible but not exactly proved actions valproate – are more efficient in 25- than in 12-day-old rats. The developmental profile of the action of neurosteroids studied in the our experiment is close to the drugs opening chloride channel. Unfortunately, there are no comparable data on the ontogeny of allopregnanolone or pregnanolone binding to the supramolecular complex of GABA-A receptor to support this possibility. Absence of effects of all three neurosteroids on motor phenomena was surprising because we found a short-lasting derangement of motor abilities of rat pups after allopregnanolone administration expressed especially in 12-day-old rats (Mareˇs et al., submitted for publication). Therefore, we expected at least a change in the ability to rear during stimulation and clonic seizures in 18- and 25-day-old rats. Electrical stimulation and epileptic activity are probably so strong activators of the motor system that a moderate disability could be overcome. An additional factor – possible differences in the action of various neurosteroids on motor performance – remains to be studied in future. Among drugs potentiating GABAergic inhibition clonazepam is an exception because it very potently suppress clonic seizures preserving shortened afterdischarges without any motor correlate. On the other hand, it does not affect movements directly elicited by electrical stimulation of sensorimotor cortex [12]. The effects of other mentioned drugs is not so marked: NNC-711, progabide and valproate decrease the intensity of both stimulation-bound movements and clonic seizures in 25- and 18-day-old rats; this effect is only moderate in 12day-old animals [2,22]. Phenobarbital exhibits an effect on both motor phenomena only in the highest dose used (40 mg/kg i.p.). The only developmental change is higher efficacy of phenobarbital against clonic seizures in 12-, if compared with 18- and 25-day-old rats [22]. It can be concluded that the mechanism of potentiation of GABAergic inhibition is very important for the anticonvulsant action of drugs modifying this main inhibitory system. All three neurosteroids studied exhbited an anticonvulsant action against epileptic afterdischarges induced by electrical stimulation of sensorimotor cortical region in immature rats. Unfortunately, the new derivative was less potent than pregnanolone, a natural metabolite of progesterone. Acknowledgements This study was supported by research projects no. S 5011007 and AV0Z 50110509 of the Academy of Sciences of the Czech Republic. The author would like to express thanks to A. Kasal, D. Sc from the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences in Prague for a supply of neurosteroids and to Ms. Irina Necheva for excellent technical assistance. References [1] D. Belelli, M.B. Bolger, K.W. Gee, Anticonvulsant profile of the progesteron metabolite 5␣-pregnan-3␣-ol-20-one, Eur. J. Pharmacol. 166 (1989) 325–329.
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