0361-9230/92 35.00 + .OO Copyright 0 1992 Pergamon Press Ltd.
Brain Research Bulletin, Vol. 29, pp. 51 l-514, 1992 Printed in the USA. All rights reserved.
Epileptogenic Activity in the Amygdala is not Affected by the Amidine Steroid, R 5 135 0.
M. S. MYSLOBODSKY
Psychobiology Research Unit, Department of Psychology, Tel-Aviv University, Ramat Aviv 69978, Israel Received
KOFMAN, 0.. R. TARRASCH, M. MINTZ AND M. S. MYSLOBODSKY. Epileptogenic activity in the amygdulu is not affected by the amidine steroid, R 5135. BRAIN RES BULL 29(3/4) 51 I-514, 1992.-The synthetic steroid amidine 3-a-hydroxy-l6-
imino-5-&17aza androstan- I l-one (R 5 135) is known to elicit long-lasting spiking in the cortex in the presence of neocortical damage. R 5 I35 administered to amygdaloid-kindled and naive rats resulted in regular, high-amplitude spiking in the cortex but only occasionally elicited small-amplitude spikes in the amygdala (AMY) and hippocampus (HPC). Interictal spikes from the AMY of kindled rats were not synchronized with cortical spikes induced by the steroid. Given that R 5135 is known to be a GABA, receptor antagonist, these findings suggest that GABAA receptors in AMY and HPC may have lower affinity for 3~ hydroxysteroids. Amygdala
MORE than four decades ago, Beach (3) speculated that sex steroids alter thresholds of synaptic events in the CNS. This hunch was repeatedly supported years later when it appeared that steroids tune the neuronal activity to display a wide range of effects from proconvulsant to anticonvulsant phenomena (14,20,30,32-34). More recently, interest in these effects was rekindled by findings that some steroids and their metabolites are endogenous brain compounds (“neurosteroids”) that act at the level of the cell membrane of steroid-sensitive neurons ( 16,I7). Neurosteroids have been associated with agonistic and antagonistic actions at the type-A GABAA receptor (2,19). A synthetic steroid amidine, 3-a-hydroxy- 16-imino-5-&17aza androstan- 1l-one (R 5 I35), described a decade ago ( 1S), is an interesting example of a uniquely potent GABAA receptor antagonist. Its affinity for the GABAA receptor is about 500 times greater than the reference GABA receptor antagonist bicuculline (15). Administered systemically in small doses, it alters brain excitability and ultimately establishes a long-lasting period of epileptic cortical spiking in rats (21) and rabbits (22). These spikes were confined to the electrodes associated with neocortical damage and were accompanied by meager motor manifestations (21,22). Also, R 5 135-induced spikes were highly resistant to anticonvulsants (21) as has been reported for interictal spikes in other forms of experimental epilepsy involving tissue damage (7).
Because a steady period of interictal spikes follows seizures kindled by amygdaloid stimulation (8,12,3 1) and because steroids are known to play a role in the modulation of seizure thresholds in the amygdala (AMY) (29) we expected to find considerable interaction between amygdaloid (kindled) ictal and interictal discharges and steroid-induced epileptogenicity. METHOD
Six male, albino rats were anesthetized with Equithesin (3 mg/kg, IP) and implanted with silver ball epidural electrodes over the visual cortex (4 mm lateral to the lambda) and a reference electrode in the nasal bone. A bipolar electrode (two twisted 0.2-mm Telfon-insulated stainless steel wires) was implanted in the AMY at coordinates: I mm posterior to bregma, 4.55 mm lateral to midline, and 9 mm below cortical surface. The electrodes, attached to microminiature pins, were fixed to the skull with acrylic dental cement reinforced by jeweller’s screws. Electrocortigram (ECoG) recording and kindling were conducted in an electrostatically shielded cage 27 X 26 X 46 cm, fitted on the sides with mirror panels. Animals were attached to cable assembly that plugged into a mercury swivel mounted on the top of the cage. Electrical activity was monitored with a Beckman type R Dynograph (bandwidth 0.53-30 Hz; Beckman Instruments, Fullerton, CA)
’ Requests for reprints should be addressed to 0. Kofman at his current address: Department of Behavioral Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva, Israel.
FIG. I, Cortical (a) and amygdala AMY (b) tracing of electrical activity after IP injection of the steroid derivative R 5 135. Note that epileptiform activity has not spread to the ipsilateral AMY. Arrowheads denote application of the kindling stimulus. Numbers below arrowheads denote seizure stage as described in the text. The small arrows denote the appearance of spontaneous seizures.
Seven days after surgery, each rat was placed in the shielded chamber and AMY kindling was initiated according to the procedure described in Myslobodsky and Valenstein (23). A 2-s train of square-wave pulses (100 KS; 60 Hz) was applied repeatedly to define the individual threshold intensity for eliciting subcortical afterdischarge. This train was then delivered once daily and the kindling stage was classified as follows: stage 0, no motor response: stage I, mouth and facial movements; stage 2, head nodding; stage 3, forelimb clonus; stage 4, rearing; stage 5, rearing and falling with loss of postural control (25.27); with the addition of stage 6 to designate multiple episodes of rearing and falling. Several days after the rat attained a stage 6 seizure, it was placed in the experimental chamber and electrical activity was recorded continuously from cortical and AMY electrodes. Following baseline recording, the rat was injected IP with R 5 135 (2 mg/kg) until regular spiking developed in the ECoG, usually within 15 min (2 I). Thereafter, the AMY was stimulated by a train of pulses at threshold intensity pseudorandomly to produce fully developed and aborted seizures. At the end of the experiment, animals were anesthetized with Nembutal and perfused via the heart with 10% formol-saline. Brains were removed and 25-pm frozen sections were cut through the electrode sites to monitor electrode locations.
Consistent with our previous observations. administration of R 5 I35 elicited lasting quasiperiodic spiking in the ECoG at a rate of approximately I/s. In contrast, in no case were spikes obtained in the recordings from AMY. Following repeated application of the kindling stimuli to the AMY, fully kindled epilepsy (stages 5-6) appeared, accompanied by high-frequency spiking at both AMY and cortical sites. Typical interictal spiking was recorded from the AMY electrodes in no apparent synchrony with steroid-induced spikes in the cortex. The situation did not change when electrical stimuli were repeatedly applied to a fully kindled animal at intervals from 2- 15 min. resulting in seizures of varying intensity (from stages 2-6) (Fig. 1). To determine if the absence of spiking was specific to the AMY. eight naive rats were implanted with recording electrodes in the cortex, AMY, and dorsal hippocampus (HPC). Seven days after surgery, R 5 135 was injected in two to three repeated doses of 1 mg/kg IP and electroencephalogram (EEG) was recorded. In all rats. highamplitude regular spiking was obtained from the cortical electrode, whereas in the HPC and AMY low-amplitude spikes were noticed. The appearance of spiking at subcortical sites was significantly delayed in comparison to cortical spikes. In one rat. spikes were obtained in both the HPC and AMY when a cumulative dose of 3 mg/kg was attained. This dose accelerated the frequency of spikes in the cortex and only then did high-
amplitude spikes in the form of cyclic driving appear in the subcortical leads. Thus, the AMY, known to be particularly susceptible to epileptogenic activity (4,l I), appeared to be immune to the neurotoxic effects of R 5 135. Spikes induced in the cortex with R 5 I35 failed to codevelop in the AMY or propagate from the cortical foci at a later stage when AMY was sensitized by kindling (Fig. 1). Although AMY seizures were associated with a shortlasting reduction in the frequency of cortical spikes, which lasted up to several seconds, spikes in the cortex showed limited dependence upon electrically evoked or spontaneous after-discharges originating from the depth electrode, in keeping with stereo EEG recordings in epileptic patients (1). It was shown elsewhere that R 5 135 is neurotoxic in the presence of neocortical damage, such as that inflicted by electrode implants (22). The coincidental appearance of R 5 135 neurotoxicity and brain injury suggests an excess extracellular accumulation of glutamate that is thought to lead to an influx of Ca2+ and thereby to destruction of postsynaptic neuronal cells (10). For example, glucocorticoid pretreatment prior to kainic acid (KA) administration potentiated damage to hippocampal neurons (28). Purdy and Paul (26) refer to their unpublished findings showing that pregnenolone sulphate augments NMDA receptor-mediated influx of Cazc into nerve cells. The AMY and HPC, like the cortex, must have been traumatized by the electrode implant; even a 50-pm needle or a glass capillary inserted into the cortex causes increased expansion of Evans blue-labeled albumin (24). Hence, it remains unclear why excitotoxic con-
sequences of trauma were obtained in the cortex but not in the AMY and HPC. The AMY has long been recognized to be responsive to steroids (29), contains glutamate (9) and responds with seizure discharges to local injections of KA (5), suggesting that, like cortex, it should be susceptible to steroid potentiation of the toxic effects of excessive glutamate. The susceptibility of AMY and HPC to toxic effects of KA was further demonstrated by the inability of noncompetitive antagonists of the NMDA receptor, ketamine, phencyclidine, and MK 80 1, to antagonize the spread of seizure activity to AMY and CA1 of HPC, even though cortical seizures were attenuated by these drugs (6). Therefore, it seems unlikely that diminished sensitivity to steroids or to the neurotoxic effects of excitatory amino acids could account for the conspicuous resistance of the AMY and HPC to R 5135. An alternate explanation is that R 5 135 is less effective in AMY due to differential distribution of GABA* receptors. Binding of the specific GABAA antagonist SR 95531 revealed a significantly greater density of GABA* receptors in cortex than in AMY (13). Furthermore, the GABAA receptor is known for its diversity. Its numerous subunits are currently being recognized (18,35). Thus, the resistance of the AMY to R 5135 may be attributed to some isoforms of GABAA receptor present in the AMY that have exceptionally low affinity for 3-a-hydroxysteroids compared to the neocortical isoforms. ACKNOWLEDGEMENTS
R 5 I35 was kindly supplied by Dr. P. Hunt, Roussel-UCLAF. We thank Z. Elazar for use of his facilities.
REFERENCES 1. Angelery, F.; Ferro-Milone, F.; Par&i, S. Electrical activity and reactivity of the rhinencephalic pararhinencephalic and thalamic structures: Prolonged implantation of electrodes in man. Electroenceph. Clin. Neurophysiol. 16: IOO-129; 1964. Baulieu, E. E.; Robe], P. Neurosteroids-a new brain function. J. Steroid Biochem. Mol. Biol. 37:395-403; 1990. Beach, F. A. Hormones and behavior. New York: P. B. Hoeber; 1948. Ben-Ari, Y. The amygdaloid complex: INSERM Symposium 20. Amsterdam: Elsevier; 198 1. Ben-A& Y. The role of seizures in kainic acid induced brain damage. In: Fuxe, K.; Roberts, P.; Schwartz, R., eds. Excitotoxins. New York: Plenum Press; 1982:184. 6. Clifford, D. B.; Olney, J. W.; Benz, A. M.; Fuller, T. A.; Zorumski, C. F. Ketamine, phencyclidine and MK-80 1 protect against kainic acid-induced seizure-related brain damage. Epilepsia 3 1:382-390; 1990. 7. Elazar, Z.; Blum, B. Effects of drugs on interictal spikes and afterdischarges in experimental epilepsy. Arch. Int. Pharmacodyn. Ther. 189:310-318; 1971. 8. Engel, J.; Ackermann, R. F. lnterictal EEG spikes correlate with decreased, rather than increased, epileptogenicity in amygdaloid kindled rats. Brain Res. 190:543-548; 1980. 9. Fallon, J. H.; Ben-Ari, Y. Chairman’s comments. In: Ben-Ari, Y., ed. The amygdaloid complex. INSERM Symposium No. 20. Elsevier: Amsterdam; 1981:151-162. 10. Farooqui, A. A.: Horrocks, L. A. Excitatory amino acid receptors, neural membrane phospholipid metabolism and neurological disorders. Brain Res. Rev. 16:171-191; 1991. 11. Goddard, G. V. Development of epileptic seizures through brain stimulation of low intensity. Nature 214: 1020; 1965. 12. Gotman, J. Relationship between triggered seizures, spontaneous seizures and interictal spiking in the kindling model of epilepsy. Exp. Neurol. 84:269-273; 1984. 13. Heaulme. M.; Chambon, J. P.; Leyris, R.; Wermuth, C. G.; Biziero,
K. Characterization of the binding of [3H]SR 9553 1, a GABA* antagonist, to rat brain membranes. J. Neurochem. 48:1677-1686; 1987. Heuser, G.; Ling, G. M.; Buchwald, N. A. Sedation and seizures as dose-dependent effects of steroids. Arch. Neurol. 13: 195-203; 1965. Hunt, P.; Clements-Jewery, S. A steroid derivative, R 5 135, antagonizes the GABA/benzodiazepine receptor interaction. Neuropharmacology 20:357-36 I; 1981. Jo, D. H.; Abdallah, M. A.; Young, J.; Baulieu, E. E.; Robel, P. Pregnenolone, dehydroepiandrosterone and their sulfate and fatty acid esters in the rat brain. Steroids 54:287-297; 1989. Le Goascogne, C.; Robel, P.; Gouezou, M.; Sananes, N.; Baulieu, E. E.; Waterman, M. Neurosteroids: Cytochrome P-450, in rat brain. Science 237:1212-1215: 1987. Luddens, H.; Wisden, W. Function and pharmacology of multiple GABA, receptor subunits. Trends Pharmacol. Sci. 12:49-5 1; 199 1. Majewska, M. D.; Demirgoren, S.; Spivak, C. E.; London, E. D. The neurosteroid dehydroepiandrosterone sulfate is an allosteric antagonist of the GABA, receptor. Brain Res. 526: 143-146; 1990. Michael, R. P. Oestrogens in the central nervous system. Br. Med. Bull. 2:87-90; 1965. Myslobodsky, M. S.; Kofman, 0. Regular and lasting neocortical spiking produced by systemic administration of a steroid derivative in the rat. Neuropharmacology 22: 157- 164; 1983. Myslobodsky, M. S.; Mintz, M.; Tarrasch, R.; Bar-Ziv, J. A steroid derivative (RU 5135) exhibits epileptogenicity in the presence of deficient blood-brain barrier. Pharmacol. Biochem. Behav. 38:327331; 1991. Myslobodsky, M. S.; Valenstein, E. Amygdaloid kindling and the GABA system. Epilepsia 21: 163-175; 1980. Persson, L.; Rosengren, L. Increased blood-brain barrier permeability around cerebral stab wounds, aggravated by acute ethanol intoxication. Acta Neurol. Stand. 56:7-16; 1977.
25. Pinel, J. P.; Rovner. L. 1. Experimental epileptogenesis: Kindlinginduced epilepsy in rats. Exp. Neurol. 58:335-346; 1978. 26. Purdy, R. H.; Paul, S. Neuroactive steroids. FASEB (in press). 27. Racine, R. J. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalog. Clin. Neurophysiol. 32:269279; 1972. 28. Sapolsky. R. Glucocorticoid toxicity in the hippocampus: Temporal aspects of synergy with kainic acid. Neuroendocrinology 43:440444; 1986. 29. Terasawa. E.; Timiras, P. S. Electrical activity during estrous cycle of the rat: Cyclic changes in the limbic structures. Endocrinology 83:207-216: 1968. 30. Timiras, P. S. Role of hormones in development of seizures. In: Jasper. H. H.; Ward, A. A.: Pope, A.. eds. Basic mechanism of epilepsies. Boston: Little, Brown & Co.; 1969:727-736.
3 I. Wada. J. A.: Sato, M.: Corcoran, M. E. Persistent seizure susceptibility and recurrent spontaneous seizures in kindled cats. Epilepsia 15: 465-478; 1974. 32. Woodbury, D. M. Effect of adrenocortical steroids and adrenocorticotropic hormone on electroshock seizure threshold. J. Pharmacol. Exp. Ther. 105:27-36: 1952. 33. Woolley. D. E.; Timiras, P. S. The gonad-brain relationship: Effects of female sex hormones on electroshock convulsions in the rat. Endocrinology 70: 196-209; 1962. 34. Woolley. D. E.; Timiras, P. S. Estrous and circadian periodicity and electroshock convulsions in rats. Am. J. Physiol. 202:379-382; 1962. 35. Zhang, J. H.; Sato. M.; Tohyama, M. Region specific expression of the mRNAs encoding beta subunits (beta I. beta 2 and beta 3) of GABAA receptor in the rat brain. J. Comp. Neurol. 303:637-657: 1991.