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The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats Deborah Lonsdale⁎, Kirk Nylen, W. McIntyre Burnham The University of Toronto Epilepsy Research Program and the University of Toronto, Department of Pharmacology, Canada
A R T I C LE I N FO
AB S T R A C T
Article history:
Progesterone is a neurosteroid that modulates neuronal excitability. The anticonvulsant
Accepted 2 May 2006
effects of progesterone are largely mediated by the actions of its metabolites. The purpose of
Available online 19 June 2006
this study was to measure the anticonvulsant effects of progesterone, 5alphadihydroprogesterone, and allopregnanolone against amygdala-kindled seizures in male
Keywords:
rats. The amygdala kindling model is a model of human complex partial seizures with
Progesterone
secondary generalization. A bipolar electrode was chronically implanted in the right
5alpha-dihydroprogesterone
amygdala of male Wistar rats. All subjects were kindled to 30 stage 5 seizures and stability
Allopregnanolone
tested. Multiple doses of progesterone, 5alpha-dihydroprogesterone, or allopregnanolone
Kindling
were administered in separate dose–response studies. The antiseizure effects of each
Complex partial seizure
compound were determined. A progesterone time–response study was also conducted. At 30 min after injection, progesterone had an ED50 of 65.3 mg/kg against the secondarily generalized seizure and an ED50 of 114 mg/kg against the focal seizure. 5alphadihydroprogesterone had a low ED50 of 6.2 mg/kg against both the generalized component of the amygdala-kindled seizure and the focal seizure. Allopregnanolone had an ED50 of 15.2 mg/kg against the secondarily generalized seizure and was not effective against the focal seizure. Progesterone is an effective anticonvulsant against the secondarily generalized component of amygdala-kindled seizures in male rats. Progesterone is only effective against the focal seizure at high ataxic doses. 5alpha-dihydroprogesterone is a potent anticonvulsant against both the kindled amygdala focal discharge and the secondarily generalized seizure. Allopregnanolone is an effective anticonvulsant against the secondarily generalized component of the seizure, but not against the amygdala focal discharge. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Epilepsy is a chronic neurological disorder in which patients experience spontaneous, recurrent seizures. Although the most commonly recommended therapy is drug treatment, up to 40% of patients do not achieve adequate control of their seizures (Shorvon, 1996) on existing drugs. New medications
with novel mechanisms of action are needed to help those patients whose seizures are resistant to the drugs that are currently available. Progesterone is a steroid hormone produced by the ovaries, placenta, testes, adrenal glands, glia and neurons. For many years, progesterone was considered to be primarily a reproductive hormone. In the early 1940s, however, Selye
⁎ Corresponding author. University of Toronto, Department of Pharmacology, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8. Fax: 416-971-2433. E-mail address: [email protected] (D. Lonsdale). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.05.005
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demonstrated that progesterone has sedative, anesthetic, and anticonvulsant properties in animals (Selye, 1942). During the 1980s, renewed interest in progesterone's CNS effects led to studies of mechanism and the classification of progesterone as a neurosteroid (Baulieu and Robel, 1990). A neurosteroid is any steroid compound that is synthesized de novo in the brain. Progesterone is a neurosteroid that modulates neuronal excitability (Baulieu and Schumacher, 2000). Progesterone is metabolized to 5alpha-dihydroprogesterone (5alpha-DHP) by the enzyme 5alpha-reductase. 5alpha-DHP is then reduced via 3alpha-hydroxysteroid dehydrogenase to tetrahydroprogesterone—usually called allopregnanolone (see Fig. 1). Progesterone and progesterone metabolites found in the brain may be synthesized locally or derived from peripheral sources. In animal seizure models, there is evidence that the anticonvulsant effects of progesterone are largely mediated by its reduced metabolites 5alpha-dihydroprogesterone and allopregnanolone (Frye et al., 2002; Lonsdale and Burnham, 2003). Some of this evidence has come from experiments involving finasteride. Finasteride is an inhibitor of both 5alpha-reductase isoenzymes in rodents. When finasteride is used to block the reduction of progesterone to 5alpha-DHP in mice, the anticonvulsant properties of progesterone are reduced in a dose-dependent manner (Kokate et al., 1999). Reddy and Rogawski (2004) also demonstrated that the anticonvulsant potency of progesterone is not reduced in progesterone receptor knockout mice. There is clinical evidence that the administration of progesterone may decrease the frequency of seizures in women with epilepsy (Backstrom et al., 1984; Herzog, 1999) and that conversion to metabolites is important for its anticonvulsant effects (Herzog and Frye, 2003). The anticonvulsant effects of progesterone, therefore, are believed to be due to the action of 5alpha-DHP and allopregnanolone. These effects are seen within 15 min after s.c. or i.p. administration (Lonsdale and Burnham, 2003). The rapid onset of these effects suggests that the compounds are acting via cell surface receptors. Allopregnanolone is known to be a positive modulator of the GABAA receptor, increasing chloride ion flux and reducing neuronal excitability (Lambert et al., 1995). The mechanism of action of 5alpha-DHP has yet to be determined. In previous experiments, we determined that progesterone and its 5alpha-reduced metabolites are anticonvulsant when tested against amygdala-kindled seizures in adult female rats (Lonsdale and Burnham, 2003). In our kindling studies, rats were kindled to 30 stage 5 seizures to ensure that the seizure threshold at the focus is
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stable (Racine, 1972). Mohammad et al. (1998) also investigated the anticonvulsant effects of progesterone (but not its metabolites) against amygdala-kindled seizures in rats. In the Mohammad et al. study, however, the rats were kindled to only 5 stage 5 seizures, and seizure thresholds may not have been as stable. Stable thresholds are necessary in drug testing to ensure that any effects seen are due to the drug and not due to an unstable seizure threshold. The rat amygdala kindling preparation is a pharmacologically validated model of human complex partial seizures secondarily generalized (Albright and Burnham, 1980). Complex partial seizures are common in the adult population and are frequently drug-resistant (Blume, 2002). The purpose of the present study was to determine whether progesterone and its metabolites are anticonvulsant against amygdala-kindled seizures in male rats. If the metabolite 5alpha-dihydroprogesterone is as effective an anticonvulsant in amygdala-kindled males as it was in amygdala-kindled females, it (or an analog) would represent an exciting, novel candidate for the treatment of complex partial seizures clinically. Given the problem of drugresistant complex partial seizures, such compounds are greatly needed.
2.
Results
The number of stimulations required to kindle rats to 30 stage 5 seizures ranged from 36 to 41. Prior to each drug study, all subjects had reproducible stage 5 seizures (Racine, 1972). Fig. 2 presents a time – response curve for progesterone following an injection of 160 mg/kg. Progesterone's anticonvulsant effects against the secondarily generalized component of the seizures began to be seen at 20 min after injection and were maximal (100% suppression) 40 min following injection. Progesterone's anticonvulsant effects against the focal electrographic seizures began to be seen at 20 min and were maximal (37.5%) at 40 min after injection. Fig. 3 presents a dose–response curve for progesterone. At 30 min post-injection, the median effective dose (ED50) of progesterone against secondarily generalized seizures was 65.3 mg/kg. Progesterone's ED50 against the focal electrographic seizures was 114 mg/kg—a dose that approached the TD50 of 141.2 mg/kg. Administration of the vehicle, betacyclodextrin, to each subject had no effect on the seizures and did not cause ataxia.
Fig. 1 – Enzymatic reduction of progesterone to 5alpha-dihydroprogesterone by 5alpha-reductase followed by reduction of 5alpha-dihydroprogesterone to THP (allopregnanolone) by 3alpha-hydroxysteroid dehydrogenase.
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Fig. 2 – A graph of the time–response curve for the suppression of amygdala-kindled seizures by progesterone (N = 8). A single, high dose of progesterone was administered (160 mg/kg), and the kindling stimulus was applied at varying post-injection intervals.
Fig. 4 – The percentage of subjects showing scores of ≥2 on the Loscher ataxia scale at varying doses of progesterone. These are the same subjects whose seizure-suppression scores are presented in Fig. 3 (N = 9).
exhibit ataxia at doses above 10 mg/kg. The TD50 was 26.6 mg/kg. Fig. 4 is a graph of the percentage of subjects showing scores of ≥ 2 on the Loscher ataxia scale at various doses of progesterone. Subjects began to show ataxia at doses above 40 mg/kg. The TD50 was 141.2 mg/kg. Fig. 5 presents a dose–response curve for 5alpha-DHP. At 15 min after injection, 5alpha-DHP had a low ED50 of 6.2 mg/kg against both the secondarily generalized seizures and the focal seizures. Subjects did not exhibit ataxia at any dose of 5alpha-DHP. Fig. 6 presents a dose–response curve for allopregnanolone. At 15 min after injection, allopregnanolone had an ED50 of 15.2 mg/kg against the generalized component of the amygdala-kindled seizures. Although allopregnanolone suppressed 100% of the secondarily generalized seizures at a dose of 30 mg/kg, it did not suppress the focal electrographic seizures at any dose. Fig. 7 is a graph of the percentage of subjects showing ataxia scores of ≥ 2 on the Loscher ataxia scale at various doses of allopregnanolone. Subjects began to
Fig. 3 – A graph of the dose–response curve for the suppression of amygdala-kindled seizures by progesterone (N = 9). Progesterone (varying doses) was injected, and the kindling stimulus was administered 30 min later. Only one dose was administered to a subject on any given test day.
3.
Discussion
This study was performed to determine the anticonvulsant effects of progesterone and its metabolites 5alpha-DHP and allopregnanolone in the amygdala kindling model in male rats. Our protocol for testing the anticonvulsant effects of steroids followed the procedures described by Albright and Burnham (1980). All subjects were kindled to 30 stage 5 seizures and stability tested in order to ensure stable seizure thresholds. In our time–response study, progesterone's anticonvulsant effects were not evident at 10 min post-injection. Mohammad et al. (1998) reported that, in their study, progesterone was anticonvulsant at a 10 min injection–test interval. This may reflect the difference in the vehicles used in the two studies.
Fig. 5 – A graph of the dose–response curve for the suppression of amygdala-kindled seizures by 5alpha-dihydroprogesterone (N = 7). 5alpha-DHP was injected, and the kindling stimulus was administered 15 min later. Only one dose was administered to a subject on any given test day.
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Fig. 6 – A graph of the dose–response curve for the suppression of amygdala-kindled seizures by allopregnanolone (N = 8). Allopregnanolone was injected, and the kindling stimulus was administered 15 min later. Only one dose was administered to a subject on any given test day.
Mohammad et al. (1998) used propylene glycol to dissolve progesterone, while our laboratory used beta-cyclodextrin. In our study, the maximal effect (100% suppression of secondarily generalized seizures) was seen from 40 to 80 min postinjection. This is consistent with our previous study in female rats, in which the anticonvulsant effects of progesterone were seen over an even more extended time period. It is perplexing that Mohammad et al. (1998) did not observe anticonvulsant effects at 30 min after injection of progesterone. Progesterone is converted to 5alpha-DHP, which is then reduced to allopregnanolone. Both 5alpha-DHP and allopregnanolone have been shown to have anticonvulsant effects (Lonsdale and Burnham, 2003; Reddy and Rogawski, 2001). It is possible that insufficient concentrations of metabolites are produced when a dose of 75 mg/kg is given to male rats. In our progesterone dose–response study, however, an 80 mg/kg progesterone dose produced anticonvulsant effects at 30 min. The progesterone dose–response study was conducted at 30 min post-injection. Progesterone had an ED50 of 65.3 mg/kg against the generalized component of the amygdala-kindled seizure and a much higher ED50 of 114 mg/kg against the focal component of the seizure. These results are consistent with the results seen in our previous work (Lonsdale and Burnham, 2003) in female rats, which demonstrated that progesterone was anticonvulsant only at high doses. Mohammad et al. (1998) also reported that progesterone was anticonvulsant against amygdala-kindled seizures in male rats only at high doses > 75 mg/kg at an injection–test interval of 10 min. Using the Loscher ataxia scale, we determined that the TD50 for progesterone at the time of seizure testing was 141.2 mg/kg. Progesterone generally has a low therapeutic index when used as an anticonvulsant—it tends to suppress amygdala-kindled seizures, particularly the focal component, only at doses that approach the toxic dose (Lonsdale and Burnham, 2003; Mohammad et al., 1998). In the dose–response study, 5alpha-DHP was anticonvulsant against both the secondarily generalized seizures and the focal electrographic seizures, with a low ED50 of 6.2 mg/kg
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against both components of the seizure. These results are similar to the results we reported in amygdala-kindled female rats (Lonsdale and Burnham, 2003). In the female study, however, 5alpha-DHP suppressed 100% of the secondarily generalized seizures and had a lower ED50 (2.9 mg/kg) against secondarily generalized seizures. 5alpha-DHP had an ED50 of 4.3 mg/kg against focal amygdala seizures and totally suppressed 60% of these seizures. In both male and female rats, 5alpha-DHP was a very effective anticonvulsant with no observable toxicity at the effective dose. Allopregnanolone was anticonvulsant against the secondarily generalized component of the amygdala-kindled seizure but was not effective against the focal component of the seizure at any dose. In male rats, allopregnanolone had an ED50 of 15.2 mg/kg against the secondarily generalized seizure. The TD50 for allopregnanolone in male rats was 26.6 mg/kg. The therapeutic index was much lower in males (1.8) than in female rats (Lonsdale and Burnham, submitted for publication). 5alpha-DHP was the most effective anticonvulsant against the amygdala focal seizures that model complex partial seizures in humans. Its low ED50 against both the focal and secondarily generalized components of the amygdala-kindled seizure, combined with the absence of toxicity at anticonvulsant doses, suggests that 5alpha-DHP (or an analog) may be a candidate for human use against complex partial seizures. Although 5alpha-DHP may bind weakly to and activate the cytosolic progesterone receptor (Rupprecht et al., 1993), its anticonvulsant effects are rapid, and this suggests action at a cell surface receptor. Weiler and Wiebe (2000) have characterized a cell surface receptor for 5alpha-DHP. In their study, binding of 5alpha-DHP occurred only in the plasma membrane fraction, not in the nuclear and cytosolic fractions, of the tissue. Studies designed to detect a binding site for 5alphaDHP in rat brain tissue are currently under way in our laboratory. The amygdala focal seizure produced by kindling is highly drug-resistant (Albright and Burnham, 1980). This is an excellent model in which to test compounds that could be used in the treatment of human complex partial seizures. The model is under-utilized. Drug testing in this model is often more expensive and requires a much greater time
Fig. 7 – The percentage of subjects showing scores of ≥2 on the Loscher ataxia scale at varying doses of allopregnanolone. These are the same subjects whose seizure-suppression scores are presented in Fig. 6 (N = 8).
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commitment than testing in other animal seizure models. It is, however, important to test the anticonvulsant effects of compounds in this model. New medications for the treatment of patients with complex partial seizures are urgently needed. Progesterone is present in the brains of both male and female humans (Hammond et al., 1983) and rodents (Corpechot et al., 1993). Following combined gonadectomy and adrenalectomy, progesterone and 5alpha-dihydroprogesterone persist in the brain tissue, but not the plasma, of both male and female rats (Corpechot et al., 1993). The results of this study in male rats suggest that the progesterone metabolite 5alpha-DHP, or its analog, may be an effective anticonvulsant in male as well as in female epilepsy patients.
was from the skull surface. The incisor bar was set at +5.0. Subjects weighed 300 – 400 g at the time of surgery.
4.4.
Two weeks (minimum) after surgery, kindling was begun. The kindling stimulus was a 1-s train of 60-Hz biphasic squarewave pulses at an intensity of 400 microamps (peak-to-peak). The stimulation was produced by a Grass model S-88 stimulator in series with two PSIU 6 stimulus isolation units (Grass Instruments, Quincy, MA, USA). Electrographic activity (EEG) was recorded on a model 6 electroencephalogram (Grass Instruments). Frequencies <5 Hz or >60 Hz were filtered out. Subjects were kindled to a criterion of 30 stage 5 (Racine, 1972) seizures.
4.5.
4.
Experimental procedure
4.1.
Subjects
Adult male Wistar rats (60 days old; Charles River, Quebec, Canada) were housed individually in 24 × 24 × 45-cm transparent, plastic cages. The animals were provided with food (Purina rat chow) and water ad libitum. The vivarium was kept at a constant temperature (21°C) and maintained on a 12h light/dark cycle (lights on at 07:00 h). The research protocol was approved by the Animal Care Committee (Faculty of Medicine) of the University of Toronto.
4.2.
Procedure for threshold measurement/stability testing
After the thirtieth stage 5 seizure, the afterdischarge threshold of each subject was determined by using the ascending-series technique (Pinel et al., 1976). This technique involves a stepwise increase in stimulation until an EEG afterdischarge is seen and a behavioral seizure occurs. The current that first produced an afterdischarge with a generalized seizure was considered the afterdischarge threshold. Subjects were then tested for stability at an intensity of 120% of their afterdischarge threshold. Stability testing involved triggering a seizure at 120% of threshold every second day for a period of 10 days. The average weight of subjects prior to drug testing was 703 g.
Drugs 4.6.
Progesterone and 5alpha-dihydroprogesterone were obtained from Sigma Chemical Co. Allopregnanolone was obtained from Steraloids Inc. The vehicle beta-cyclodextrin also was obtained from Sigma. Drugs were dissolved in a 45% solution of beta-cyclodextrin in physiologic saline. Fresh solutions were made on each day of drug testing. Injections for the progesterone and allopregnanolone studies were administered intraperitoneally. Injections for the 5alpha-dihydroprogesterone study were given s.c. into the loose skin on the back of the subjects' necks. Drug concentration was held constant (28 mg/ml for progesterone; 2.5 mg/ml for 5alpha-dihydroprogesterone; and 5 mg/ml for allopregnanolone). The volume of injection varied for different doses. A control dose of vehicle, matched in volume to the volume of the largest drug dose, was administered as part of every dose–response curve.
4.3.
Procedure for kindling
Procedure for surgery
Two weeks after arrival in the vivarium, during which animals were handled daily, subjects were anesthetized with Ketamine (50 mg/kg)/Xylazine (10 mg/kg) and implanted with a bipolar electrode (MS303/1; Plastics One, Roanoke, VA, USA) aimed at the right basolateral amygdala. The following coordinates were used: anterior–posterior, −1.0; lateral, +4.8; and ventral, −8.5. Anterior–posterior and lateral measurements were from bregma, whereas the ventral measurement
Procedure for progesterone time–response curve
A progesterone time–response study was then initiated, according to a modification of the test paradigm of Albright and Burnham (1980). Eight male Wistar rats that had reached criterion were injected i.p. on every second day with 160 mg/ kg of progesterone and then seizure-tested at one of the following times after injection: 10, 20, 40, 80 or 160 min. Each animal received a seizure stimulus equal to 120% of its afterdischarge threshold. The procedure was repeated every second day until all rats had been tested at all post-injection intervals. The presence or absence of afterdischarge and behavioral seizures was noted for each rat. The behavioral seizure was considered to be suppressed if a rearing or a rearing and falling convulsion was absent (stages 4 and 5 by Racine's 1972 classification). The focal seizure was considered to be suppressed if the stimulus was followed by <4 s of afterdischarge in the EEG. A 4-s minimum was chosen because afterdischarges <4 s are hard to score because of the switch artifact. A 48-h intertest interval was chosen to assure that all drugs from a previous test had been eliminated before the following test. All tests were performed between 7:00 and 10:00 a.m.
4.7.
Procedure for progesterone dose–response curve
A progesterone dose–response study was initiated 2 weeks after the progesterone time–response study. Nine male Wistar rats that had reached criterion were injected i.p. on
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every second day with one of the following doses of progesterone: 0, 40, 80, 120, or 160 mg/kg. Each rat received all five doses (in randomized order) on different test days. Each subject served as its own control and received a dose of the vehicle beta-cyclodextrin. Thirty minutes after the injection, each rat received a seizure stimulus equal to 120% of its afterdischarge threshold. Seizures were scored according to the criteria described above.
4.8.
Procedure for toxicity assessment
Immediately before each seizure test, animals were assessed for toxicity by using Loscher's ataxia scale (Loscher et al., 1987). Loscher's ataxia test involves observation of movement impairment. The five stages of impairment include: stage 1, slight ataxia in hind legs (tottering of the hind quarters), no decrease in abdominal muscle tone; stage 2, more pronounced ataxia, slight decrease in abdominal muscle tone, with flat body position; stage 3, further increase in ataxia and more marked decrease in abdominal muscle tone, with the flat body posture more pronounced, splayed hind legs, crawling during forward locomotion; stage 4, marked ataxia, animals lose balance during forward locomotion, total loss of abdominal muscle tonus, with flat body posture, splayed hind legs, and dragging during forward locomotion; stage 5, very marked ataxia with frequent loss of balance during forward locomotion, loss of abdominal muscle tone. Toxicity and seizure testing were repeated until all rats had been tested at all doses and with vehicle.
4.9. Procedure for 5alpha-dihydroprogesterone dose–response curve A different group of rats was used for the 5alpha-DHP dose– response study. Seven male Wistar rats were injected s.c. on every second day with vehicle (45% beta-cyclodextrin in physiologic saline) or one of the following doses of 5alphadihydroprogesterone: 0, 2.5, 5, or 7.5 mg/kg. Doses > 7.5 mg/kg could not be tested because of solubility problems. A 15 min injection–test interval was used in previous metabolite studies. Therefore, 15 min after the injection, each animal received a seizure stimulus equal to 120% of its afterdischarge threshold. Each rat received all four doses (in randomized order) on different test days. Each subject served as its own control and received a dose of vehicle (cyclodextrin). Seizures were scored according to the procedure described for the progesterone time–response study. Subjects were assessed for toxicity just prior to seizure testing.
4.10.
Procedure for allopregnanolone dose–response curve
A separate group of rats was used for the allopregnanolone dose–response study. Eight male Wistar rats were injected i.p. on every second day with the beta-cyclodextrin vehicle or one of the following doses of allopregnanolone: 0, 1.25, 5, 10, 20, or 30 mg/kg. A 15 min injection–test interval was used in previous metabolite studies. Therefore, 15 min following the injection, each animal received a seizure stimulus equal to 120% of its afterdischarge threshold. Each rat received all six
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doses (in randomized order) on different test days. Each subject served as its own control and received a dose of vehicle (cyclodextrin). Seizures were scored according to the protocol described for the progesterone time–response study. Subjects were assessed for ataxia immediately before seizure testing.
Acknowledgments This research was supported by a grant from the Canadian Institute of Health Research and a studentship from the Savoy Foundation.
REFERENCES
Albright, P.S., Burnham, W.M., 1980. Development of a new pharmacological seizure model: effects of anticonvulsants on cortical- and amygdala-kindled seizures in the rat. Epilepsia 21, 681–689. Backstrom, T., Zetterlund, B., Blom, S., Romano, M., 1984. Effects of intravenous progesterone infusions on the epileptic discharge frequency in women with partial epilepsy. Acta Neurol. Scand. 69, 240–248. Baulieu, E.E., Robel, P., 1990. Neurosteroids: a new brain function? J. Steroid Biochem. Mol. Biol. 37, 395–403. Baulieu, E., Schumacher, M., 2000. Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination. Steroids 65, 605–612. Blume, W.T., 2002. Complex partial seizures: clinical description and diagnosis. In: Burnham, W.M., Carlen, P.L., Hwang, P.A. (Eds.), Intractable Seizures: Diagnosis, Treatment and Prevention. Kluwer Academic/Plenum Publishers, New York, pp. 9–18. Corpechot, C., Young, J., Calvel, M., Wehrey, C., Veltz, J.N., Touyer, G., Mouren, M., Prasad, V.V.K., Banner, C., Sjovall, J., Baulieu, E.E., Robel, P., 1993. Neurosteroids: 3α-hydroxy-5αpregnan-20-one and its precursors in the brain, plasma, and steroidogenic glands of male and female rats. Endocrinology 133, 1003–1009. Frye, C.A., Rhodes, M.E., Walf, A., Harney, J., 2002. Progesterone reduces pentylenetetrazol-induced ictal activity of wild-type mice but not those deficient in type 1 5alpha-reductase. Epilepsia 43 (Suppl. 5), 14–17. Hammond, G.L., Hirvonen, J., Vihko, R., 1983. Progesterone, androstenedione, testosterone, 5α-dihydrotestosterone and androsterone concentrations in specific regions of the human brain. J. Steroid Biochem. 18, 185–189. Herzog, A.G., 1999. Progesterone therapy in women with epilepsy: a 3-year follow-up. Neurology 52, 1917–1918. Herzog, A.G., Frye, C.A., 2003. Seizure exacerbation associated with inhibition of progesterone metabolism. Ann. Neurol. 53, 390–391. Kokate, T.G., Banks, M.K., Magee, T., Yamaguchi, S., Rogawski, M.A., 1999. Finasteride, a 5α-reductase inhibitor, blocks he anticonvulsant activity of progesterone in mice. J. Pharmacol. Exp. Ther. 288, 679–684. Lambert, J.J., Belelli, O., Hill-Venning, C., Peters, J.A., 1995. Neurosteroids and GABAA receptor function. Trends Pharmacol. Sci. 16, 295–303. Lonsdale, D., Burnham, W.M., 2003. The anticonvulsant effects of progesterone and 5alpha-dihydroprogesterone on amygdala-kindled seizures in rats. Epilepsia 44, 1494–1499. Loscher, W., Honack, D., Hashem, A., 1987. Anticonvulsant efficacy of clonazepam and the β-carboline ZK 93423 during chronic
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treatment in amygdala-kindled rats. Eur. J. Pharmacol. 143, 403–414. Mohammad, S., Abolhassan, A., Pourgholami, M.H., 1998. Evaluation of the anticonvulsant profile of progesterone in male amygdala-kindled rats. Epilepsy Res. 30, 195–202. Pinel, J.P., Skelton, R., Mucha, R.F., 1976. Kindling-related changes in afterdischarge “thresholds”. Epilepsia 17, 197–206. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation, II: motor seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281–294. Reddy, D.S., Rogawski, M.A., 2001. Enhanced anticonvulsant activity of neuroactive steroids in a rat model of catamenial epilepsy. Epilepsia 42, 337–344. Reddy, D.S., Castaneda, D.C., O'Malley, B.W., Rogawski, M.A., 2004. Anticonvulsant activity of progesterone and neurosteroids in
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats Deborah Lonsdale⁎, Kirk Nylen, W. McIntyre Burnham The University of Toronto Epilepsy Research Program and the University of Toronto, Department of Pharmacology, Canada
A R T I C LE I N FO
AB S T R A C T
Article history:
Progesterone is a neurosteroid that modulates neuronal excitability. The anticonvulsant
Accepted 2 May 2006
effects of progesterone are largely mediated by the actions of its metabolites. The purpose of
Available online 19 June 2006
this study was to measure the anticonvulsant effects of progesterone, 5alphadihydroprogesterone, and allopregnanolone against amygdala-kindled seizures in male
Keywords:
rats. The amygdala kindling model is a model of human complex partial seizures with
Progesterone
secondary generalization. A bipolar electrode was chronically implanted in the right
5alpha-dihydroprogesterone
amygdala of male Wistar rats. All subjects were kindled to 30 stage 5 seizures and stability
Allopregnanolone
tested. Multiple doses of progesterone, 5alpha-dihydroprogesterone, or allopregnanolone
Kindling
were administered in separate dose–response studies. The antiseizure effects of each
Complex partial seizure
compound were determined. A progesterone time–response study was also conducted. At 30 min after injection, progesterone had an ED50 of 65.3 mg/kg against the secondarily generalized seizure and an ED50 of 114 mg/kg against the focal seizure. 5alphadihydroprogesterone had a low ED50 of 6.2 mg/kg against both the generalized component of the amygdala-kindled seizure and the focal seizure. Allopregnanolone had an ED50 of 15.2 mg/kg against the secondarily generalized seizure and was not effective against the focal seizure. Progesterone is an effective anticonvulsant against the secondarily generalized component of amygdala-kindled seizures in male rats. Progesterone is only effective against the focal seizure at high ataxic doses. 5alpha-dihydroprogesterone is a potent anticonvulsant against both the kindled amygdala focal discharge and the secondarily generalized seizure. Allopregnanolone is an effective anticonvulsant against the secondarily generalized component of the seizure, but not against the amygdala focal discharge. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Epilepsy is a chronic neurological disorder in which patients experience spontaneous, recurrent seizures. Although the most commonly recommended therapy is drug treatment, up to 40% of patients do not achieve adequate control of their seizures (Shorvon, 1996) on existing drugs. New medications
with novel mechanisms of action are needed to help those patients whose seizures are resistant to the drugs that are currently available. Progesterone is a steroid hormone produced by the ovaries, placenta, testes, adrenal glands, glia and neurons. For many years, progesterone was considered to be primarily a reproductive hormone. In the early 1940s, however, Selye
⁎ Corresponding author. University of Toronto, Department of Pharmacology, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8. Fax: 416-971-2433. E-mail address: [email protected] (D. Lonsdale). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.05.005
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demonstrated that progesterone has sedative, anesthetic, and anticonvulsant properties in animals (Selye, 1942). During the 1980s, renewed interest in progesterone's CNS effects led to studies of mechanism and the classification of progesterone as a neurosteroid (Baulieu and Robel, 1990). A neurosteroid is any steroid compound that is synthesized de novo in the brain. Progesterone is a neurosteroid that modulates neuronal excitability (Baulieu and Schumacher, 2000). Progesterone is metabolized to 5alpha-dihydroprogesterone (5alpha-DHP) by the enzyme 5alpha-reductase. 5alpha-DHP is then reduced via 3alpha-hydroxysteroid dehydrogenase to tetrahydroprogesterone—usually called allopregnanolone (see Fig. 1). Progesterone and progesterone metabolites found in the brain may be synthesized locally or derived from peripheral sources. In animal seizure models, there is evidence that the anticonvulsant effects of progesterone are largely mediated by its reduced metabolites 5alpha-dihydroprogesterone and allopregnanolone (Frye et al., 2002; Lonsdale and Burnham, 2003). Some of this evidence has come from experiments involving finasteride. Finasteride is an inhibitor of both 5alpha-reductase isoenzymes in rodents. When finasteride is used to block the reduction of progesterone to 5alpha-DHP in mice, the anticonvulsant properties of progesterone are reduced in a dose-dependent manner (Kokate et al., 1999). Reddy and Rogawski (2004) also demonstrated that the anticonvulsant potency of progesterone is not reduced in progesterone receptor knockout mice. There is clinical evidence that the administration of progesterone may decrease the frequency of seizures in women with epilepsy (Backstrom et al., 1984; Herzog, 1999) and that conversion to metabolites is important for its anticonvulsant effects (Herzog and Frye, 2003). The anticonvulsant effects of progesterone, therefore, are believed to be due to the action of 5alpha-DHP and allopregnanolone. These effects are seen within 15 min after s.c. or i.p. administration (Lonsdale and Burnham, 2003). The rapid onset of these effects suggests that the compounds are acting via cell surface receptors. Allopregnanolone is known to be a positive modulator of the GABAA receptor, increasing chloride ion flux and reducing neuronal excitability (Lambert et al., 1995). The mechanism of action of 5alpha-DHP has yet to be determined. In previous experiments, we determined that progesterone and its 5alpha-reduced metabolites are anticonvulsant when tested against amygdala-kindled seizures in adult female rats (Lonsdale and Burnham, 2003). In our kindling studies, rats were kindled to 30 stage 5 seizures to ensure that the seizure threshold at the focus is
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stable (Racine, 1972). Mohammad et al. (1998) also investigated the anticonvulsant effects of progesterone (but not its metabolites) against amygdala-kindled seizures in rats. In the Mohammad et al. study, however, the rats were kindled to only 5 stage 5 seizures, and seizure thresholds may not have been as stable. Stable thresholds are necessary in drug testing to ensure that any effects seen are due to the drug and not due to an unstable seizure threshold. The rat amygdala kindling preparation is a pharmacologically validated model of human complex partial seizures secondarily generalized (Albright and Burnham, 1980). Complex partial seizures are common in the adult population and are frequently drug-resistant (Blume, 2002). The purpose of the present study was to determine whether progesterone and its metabolites are anticonvulsant against amygdala-kindled seizures in male rats. If the metabolite 5alpha-dihydroprogesterone is as effective an anticonvulsant in amygdala-kindled males as it was in amygdala-kindled females, it (or an analog) would represent an exciting, novel candidate for the treatment of complex partial seizures clinically. Given the problem of drugresistant complex partial seizures, such compounds are greatly needed.
2.
Results
The number of stimulations required to kindle rats to 30 stage 5 seizures ranged from 36 to 41. Prior to each drug study, all subjects had reproducible stage 5 seizures (Racine, 1972). Fig. 2 presents a time – response curve for progesterone following an injection of 160 mg/kg. Progesterone's anticonvulsant effects against the secondarily generalized component of the seizures began to be seen at 20 min after injection and were maximal (100% suppression) 40 min following injection. Progesterone's anticonvulsant effects against the focal electrographic seizures began to be seen at 20 min and were maximal (37.5%) at 40 min after injection. Fig. 3 presents a dose–response curve for progesterone. At 30 min post-injection, the median effective dose (ED50) of progesterone against secondarily generalized seizures was 65.3 mg/kg. Progesterone's ED50 against the focal electrographic seizures was 114 mg/kg—a dose that approached the TD50 of 141.2 mg/kg. Administration of the vehicle, betacyclodextrin, to each subject had no effect on the seizures and did not cause ataxia.
Fig. 1 – Enzymatic reduction of progesterone to 5alpha-dihydroprogesterone by 5alpha-reductase followed by reduction of 5alpha-dihydroprogesterone to THP (allopregnanolone) by 3alpha-hydroxysteroid dehydrogenase.
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Fig. 2 – A graph of the time–response curve for the suppression of amygdala-kindled seizures by progesterone (N = 8). A single, high dose of progesterone was administered (160 mg/kg), and the kindling stimulus was applied at varying post-injection intervals.
Fig. 4 – The percentage of subjects showing scores of ≥2 on the Loscher ataxia scale at varying doses of progesterone. These are the same subjects whose seizure-suppression scores are presented in Fig. 3 (N = 9).
exhibit ataxia at doses above 10 mg/kg. The TD50 was 26.6 mg/kg. Fig. 4 is a graph of the percentage of subjects showing scores of ≥ 2 on the Loscher ataxia scale at various doses of progesterone. Subjects began to show ataxia at doses above 40 mg/kg. The TD50 was 141.2 mg/kg. Fig. 5 presents a dose–response curve for 5alpha-DHP. At 15 min after injection, 5alpha-DHP had a low ED50 of 6.2 mg/kg against both the secondarily generalized seizures and the focal seizures. Subjects did not exhibit ataxia at any dose of 5alpha-DHP. Fig. 6 presents a dose–response curve for allopregnanolone. At 15 min after injection, allopregnanolone had an ED50 of 15.2 mg/kg against the generalized component of the amygdala-kindled seizures. Although allopregnanolone suppressed 100% of the secondarily generalized seizures at a dose of 30 mg/kg, it did not suppress the focal electrographic seizures at any dose. Fig. 7 is a graph of the percentage of subjects showing ataxia scores of ≥ 2 on the Loscher ataxia scale at various doses of allopregnanolone. Subjects began to
Fig. 3 – A graph of the dose–response curve for the suppression of amygdala-kindled seizures by progesterone (N = 9). Progesterone (varying doses) was injected, and the kindling stimulus was administered 30 min later. Only one dose was administered to a subject on any given test day.
3.
Discussion
This study was performed to determine the anticonvulsant effects of progesterone and its metabolites 5alpha-DHP and allopregnanolone in the amygdala kindling model in male rats. Our protocol for testing the anticonvulsant effects of steroids followed the procedures described by Albright and Burnham (1980). All subjects were kindled to 30 stage 5 seizures and stability tested in order to ensure stable seizure thresholds. In our time–response study, progesterone's anticonvulsant effects were not evident at 10 min post-injection. Mohammad et al. (1998) reported that, in their study, progesterone was anticonvulsant at a 10 min injection–test interval. This may reflect the difference in the vehicles used in the two studies.
Fig. 5 – A graph of the dose–response curve for the suppression of amygdala-kindled seizures by 5alpha-dihydroprogesterone (N = 7). 5alpha-DHP was injected, and the kindling stimulus was administered 15 min later. Only one dose was administered to a subject on any given test day.
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Fig. 6 – A graph of the dose–response curve for the suppression of amygdala-kindled seizures by allopregnanolone (N = 8). Allopregnanolone was injected, and the kindling stimulus was administered 15 min later. Only one dose was administered to a subject on any given test day.
Mohammad et al. (1998) used propylene glycol to dissolve progesterone, while our laboratory used beta-cyclodextrin. In our study, the maximal effect (100% suppression of secondarily generalized seizures) was seen from 40 to 80 min postinjection. This is consistent with our previous study in female rats, in which the anticonvulsant effects of progesterone were seen over an even more extended time period. It is perplexing that Mohammad et al. (1998) did not observe anticonvulsant effects at 30 min after injection of progesterone. Progesterone is converted to 5alpha-DHP, which is then reduced to allopregnanolone. Both 5alpha-DHP and allopregnanolone have been shown to have anticonvulsant effects (Lonsdale and Burnham, 2003; Reddy and Rogawski, 2001). It is possible that insufficient concentrations of metabolites are produced when a dose of 75 mg/kg is given to male rats. In our progesterone dose–response study, however, an 80 mg/kg progesterone dose produced anticonvulsant effects at 30 min. The progesterone dose–response study was conducted at 30 min post-injection. Progesterone had an ED50 of 65.3 mg/kg against the generalized component of the amygdala-kindled seizure and a much higher ED50 of 114 mg/kg against the focal component of the seizure. These results are consistent with the results seen in our previous work (Lonsdale and Burnham, 2003) in female rats, which demonstrated that progesterone was anticonvulsant only at high doses. Mohammad et al. (1998) also reported that progesterone was anticonvulsant against amygdala-kindled seizures in male rats only at high doses > 75 mg/kg at an injection–test interval of 10 min. Using the Loscher ataxia scale, we determined that the TD50 for progesterone at the time of seizure testing was 141.2 mg/kg. Progesterone generally has a low therapeutic index when used as an anticonvulsant—it tends to suppress amygdala-kindled seizures, particularly the focal component, only at doses that approach the toxic dose (Lonsdale and Burnham, 2003; Mohammad et al., 1998). In the dose–response study, 5alpha-DHP was anticonvulsant against both the secondarily generalized seizures and the focal electrographic seizures, with a low ED50 of 6.2 mg/kg
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against both components of the seizure. These results are similar to the results we reported in amygdala-kindled female rats (Lonsdale and Burnham, 2003). In the female study, however, 5alpha-DHP suppressed 100% of the secondarily generalized seizures and had a lower ED50 (2.9 mg/kg) against secondarily generalized seizures. 5alpha-DHP had an ED50 of 4.3 mg/kg against focal amygdala seizures and totally suppressed 60% of these seizures. In both male and female rats, 5alpha-DHP was a very effective anticonvulsant with no observable toxicity at the effective dose. Allopregnanolone was anticonvulsant against the secondarily generalized component of the amygdala-kindled seizure but was not effective against the focal component of the seizure at any dose. In male rats, allopregnanolone had an ED50 of 15.2 mg/kg against the secondarily generalized seizure. The TD50 for allopregnanolone in male rats was 26.6 mg/kg. The therapeutic index was much lower in males (1.8) than in female rats (Lonsdale and Burnham, submitted for publication). 5alpha-DHP was the most effective anticonvulsant against the amygdala focal seizures that model complex partial seizures in humans. Its low ED50 against both the focal and secondarily generalized components of the amygdala-kindled seizure, combined with the absence of toxicity at anticonvulsant doses, suggests that 5alpha-DHP (or an analog) may be a candidate for human use against complex partial seizures. Although 5alpha-DHP may bind weakly to and activate the cytosolic progesterone receptor (Rupprecht et al., 1993), its anticonvulsant effects are rapid, and this suggests action at a cell surface receptor. Weiler and Wiebe (2000) have characterized a cell surface receptor for 5alpha-DHP. In their study, binding of 5alpha-DHP occurred only in the plasma membrane fraction, not in the nuclear and cytosolic fractions, of the tissue. Studies designed to detect a binding site for 5alphaDHP in rat brain tissue are currently under way in our laboratory. The amygdala focal seizure produced by kindling is highly drug-resistant (Albright and Burnham, 1980). This is an excellent model in which to test compounds that could be used in the treatment of human complex partial seizures. The model is under-utilized. Drug testing in this model is often more expensive and requires a much greater time
Fig. 7 – The percentage of subjects showing scores of ≥2 on the Loscher ataxia scale at varying doses of allopregnanolone. These are the same subjects whose seizure-suppression scores are presented in Fig. 6 (N = 8).
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commitment than testing in other animal seizure models. It is, however, important to test the anticonvulsant effects of compounds in this model. New medications for the treatment of patients with complex partial seizures are urgently needed. Progesterone is present in the brains of both male and female humans (Hammond et al., 1983) and rodents (Corpechot et al., 1993). Following combined gonadectomy and adrenalectomy, progesterone and 5alpha-dihydroprogesterone persist in the brain tissue, but not the plasma, of both male and female rats (Corpechot et al., 1993). The results of this study in male rats suggest that the progesterone metabolite 5alpha-DHP, or its analog, may be an effective anticonvulsant in male as well as in female epilepsy patients.
was from the skull surface. The incisor bar was set at +5.0. Subjects weighed 300 – 400 g at the time of surgery.
4.4.
Two weeks (minimum) after surgery, kindling was begun. The kindling stimulus was a 1-s train of 60-Hz biphasic squarewave pulses at an intensity of 400 microamps (peak-to-peak). The stimulation was produced by a Grass model S-88 stimulator in series with two PSIU 6 stimulus isolation units (Grass Instruments, Quincy, MA, USA). Electrographic activity (EEG) was recorded on a model 6 electroencephalogram (Grass Instruments). Frequencies <5 Hz or >60 Hz were filtered out. Subjects were kindled to a criterion of 30 stage 5 (Racine, 1972) seizures.
4.5.
4.
Experimental procedure
4.1.
Subjects
Adult male Wistar rats (60 days old; Charles River, Quebec, Canada) were housed individually in 24 × 24 × 45-cm transparent, plastic cages. The animals were provided with food (Purina rat chow) and water ad libitum. The vivarium was kept at a constant temperature (21°C) and maintained on a 12h light/dark cycle (lights on at 07:00 h). The research protocol was approved by the Animal Care Committee (Faculty of Medicine) of the University of Toronto.
4.2.
Procedure for threshold measurement/stability testing
After the thirtieth stage 5 seizure, the afterdischarge threshold of each subject was determined by using the ascending-series technique (Pinel et al., 1976). This technique involves a stepwise increase in stimulation until an EEG afterdischarge is seen and a behavioral seizure occurs. The current that first produced an afterdischarge with a generalized seizure was considered the afterdischarge threshold. Subjects were then tested for stability at an intensity of 120% of their afterdischarge threshold. Stability testing involved triggering a seizure at 120% of threshold every second day for a period of 10 days. The average weight of subjects prior to drug testing was 703 g.
Drugs 4.6.
Progesterone and 5alpha-dihydroprogesterone were obtained from Sigma Chemical Co. Allopregnanolone was obtained from Steraloids Inc. The vehicle beta-cyclodextrin also was obtained from Sigma. Drugs were dissolved in a 45% solution of beta-cyclodextrin in physiologic saline. Fresh solutions were made on each day of drug testing. Injections for the progesterone and allopregnanolone studies were administered intraperitoneally. Injections for the 5alpha-dihydroprogesterone study were given s.c. into the loose skin on the back of the subjects' necks. Drug concentration was held constant (28 mg/ml for progesterone; 2.5 mg/ml for 5alpha-dihydroprogesterone; and 5 mg/ml for allopregnanolone). The volume of injection varied for different doses. A control dose of vehicle, matched in volume to the volume of the largest drug dose, was administered as part of every dose–response curve.
4.3.
Procedure for kindling
Procedure for surgery
Two weeks after arrival in the vivarium, during which animals were handled daily, subjects were anesthetized with Ketamine (50 mg/kg)/Xylazine (10 mg/kg) and implanted with a bipolar electrode (MS303/1; Plastics One, Roanoke, VA, USA) aimed at the right basolateral amygdala. The following coordinates were used: anterior–posterior, −1.0; lateral, +4.8; and ventral, −8.5. Anterior–posterior and lateral measurements were from bregma, whereas the ventral measurement
Procedure for progesterone time–response curve
A progesterone time–response study was then initiated, according to a modification of the test paradigm of Albright and Burnham (1980). Eight male Wistar rats that had reached criterion were injected i.p. on every second day with 160 mg/ kg of progesterone and then seizure-tested at one of the following times after injection: 10, 20, 40, 80 or 160 min. Each animal received a seizure stimulus equal to 120% of its afterdischarge threshold. The procedure was repeated every second day until all rats had been tested at all post-injection intervals. The presence or absence of afterdischarge and behavioral seizures was noted for each rat. The behavioral seizure was considered to be suppressed if a rearing or a rearing and falling convulsion was absent (stages 4 and 5 by Racine's 1972 classification). The focal seizure was considered to be suppressed if the stimulus was followed by <4 s of afterdischarge in the EEG. A 4-s minimum was chosen because afterdischarges <4 s are hard to score because of the switch artifact. A 48-h intertest interval was chosen to assure that all drugs from a previous test had been eliminated before the following test. All tests were performed between 7:00 and 10:00 a.m.
4.7.
Procedure for progesterone dose–response curve
A progesterone dose–response study was initiated 2 weeks after the progesterone time–response study. Nine male Wistar rats that had reached criterion were injected i.p. on
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every second day with one of the following doses of progesterone: 0, 40, 80, 120, or 160 mg/kg. Each rat received all five doses (in randomized order) on different test days. Each subject served as its own control and received a dose of the vehicle beta-cyclodextrin. Thirty minutes after the injection, each rat received a seizure stimulus equal to 120% of its afterdischarge threshold. Seizures were scored according to the criteria described above.
4.8.
Procedure for toxicity assessment
Immediately before each seizure test, animals were assessed for toxicity by using Loscher's ataxia scale (Loscher et al., 1987). Loscher's ataxia test involves observation of movement impairment. The five stages of impairment include: stage 1, slight ataxia in hind legs (tottering of the hind quarters), no decrease in abdominal muscle tone; stage 2, more pronounced ataxia, slight decrease in abdominal muscle tone, with flat body position; stage 3, further increase in ataxia and more marked decrease in abdominal muscle tone, with the flat body posture more pronounced, splayed hind legs, crawling during forward locomotion; stage 4, marked ataxia, animals lose balance during forward locomotion, total loss of abdominal muscle tonus, with flat body posture, splayed hind legs, and dragging during forward locomotion; stage 5, very marked ataxia with frequent loss of balance during forward locomotion, loss of abdominal muscle tone. Toxicity and seizure testing were repeated until all rats had been tested at all doses and with vehicle.
4.9. Procedure for 5alpha-dihydroprogesterone dose–response curve A different group of rats was used for the 5alpha-DHP dose– response study. Seven male Wistar rats were injected s.c. on every second day with vehicle (45% beta-cyclodextrin in physiologic saline) or one of the following doses of 5alphadihydroprogesterone: 0, 2.5, 5, or 7.5 mg/kg. Doses > 7.5 mg/kg could not be tested because of solubility problems. A 15 min injection–test interval was used in previous metabolite studies. Therefore, 15 min after the injection, each animal received a seizure stimulus equal to 120% of its afterdischarge threshold. Each rat received all four doses (in randomized order) on different test days. Each subject served as its own control and received a dose of vehicle (cyclodextrin). Seizures were scored according to the procedure described for the progesterone time–response study. Subjects were assessed for toxicity just prior to seizure testing.
4.10.
Procedure for allopregnanolone dose–response curve
A separate group of rats was used for the allopregnanolone dose–response study. Eight male Wistar rats were injected i.p. on every second day with the beta-cyclodextrin vehicle or one of the following doses of allopregnanolone: 0, 1.25, 5, 10, 20, or 30 mg/kg. A 15 min injection–test interval was used in previous metabolite studies. Therefore, 15 min following the injection, each animal received a seizure stimulus equal to 120% of its afterdischarge threshold. Each rat received all six
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doses (in randomized order) on different test days. Each subject served as its own control and received a dose of vehicle (cyclodextrin). Seizures were scored according to the protocol described for the progesterone time–response study. Subjects were assessed for ataxia immediately before seizure testing.
Acknowledgments This research was supported by a grant from the Canadian Institute of Health Research and a studentship from the Savoy Foundation.
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