~euro~hur~ueoIogyVol. 23, No. 9, pp. 1001-1007,I984 Printed in Great Britain. All rights reserved
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LIMITATIONS OF A SIMPLE MODEL FOR THE STUDY OF ANTICONVULSANTS G. T. GOLDEN and R. G. FARIEI_LO* Department of Neurology and Pha~acology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107 and Research and Neurology Services, Veterans Administration Hospital, Coatesville, PA 19320, U.S.A. (Accepted 17 August 1983)
S~mary-Direct GABA agonists that suppress spikes induced by penicillin in cats failed to do so in rats. Phenytoin and Iarge doses of THIP increased the rate of spiking activity of the peniciilin focus. Only progabide caused marked, initial, short-lasting suppression and a modest reduction of frequency of spikes for 1hr. Homotaurine (3APS) reduced the amplitude and changed the morphology of the contralateral “mirror” spike. Antagonism of penicillin-induced spikes in rats is considered to be an unsuitable parameter for the screening of anticonvulsant agents. Key words: direct GABA agonists, phenytoin, penicillin spikes, rats.
The screening of potential anticonvulsant drugs is still based on their efficacy in preventing electricallyand chemically-induced convulsions in rodents (Swinyard, 1969). The clinical thera~utic spectrum of the anticonvulsants in the various forms of human epilepsy does not always match the expectation derived from the experimental data. Thus, there is a recognized need for experimental models of epilepsy that can be used as a reliable predictor of anticonvulsant effects of a given drug. It has been suggested that the acute penicillin focus in cats may represent a suitable model for testing potential anticonvulsants to be used in the treatment of partial seizures (Worms, Depoortere, Durand, Morselli, Lloyd and Bartholini, 1982). Since impairment of GABA-mediated inhibition is thought to be at least partly responsible for the generation of spikes induced by penicillin (Anderson and Rutledge, 1979; Cutler and Young, 1979; Dingledine and Gjeerstad, 197S), potential anticonvulsants with direct GABAmimetic action should be particularly effective in suppressing these experimental models of epilepsy. The rat offers a great economical and practical advantage over the cat in large-scale screening studies. Homotaurine (3-aminopropanesulfonic acid, 3APS) is a powerful, specific GABA agonist with anticonvulsant properties against various models of epilepsy (Adembri, Bartolini, Bartolini, Giotti and Zilletti, 1974; Farielfo, 1979; Fariello and Golden, 1980; Fariello, Golden and Pisa, 1982). Progabide
and [4,5,6,7-tetrahydroxyisoxasolo(4,5,-c) pyridine-301, THIP] are presently undergoing evaluation as clinical anticonvulsant agents (Dam, Gram, Philbert, Hansen, Lyon, Christen~n and Angelo, 1983). The present authors have, therefore, investigated the effects of various direct GABA agonists on the acute penicillin focus in rats and compared them to the action of diphenylhydantoin, a widely used drug for the treatment of partial seizures.
METHODS Animal surgery
Male Sprague-Dawley Albino rats, weighing between 250 and 300 g were used for this study. Under urethane anesthesia (1 g/kg, i.p.) the calvarium was exposed and burr holes were hand-drilled over the prefrontal cortex, 2 mm rostra1 and 1.8 mm lateral to bregma. Under microscopic guidance (40 x magnification), the dura was cut with dural scissors. A glass microelectrode (20-30 p tip), containing an aqueous solution of sodium G ~nicillin, was stereotaxically lowered 1.2 mm into the cortex underlying the dura opening. By means of an air pressure injection, 5 ~1 of penicillin was injected over a 5 min period. A femoral vein catheter was also inserted. The EEG recordings were obtained with screw electrodes positioned as outlined in the Figures, using a Grass Polygraph Model 6 with low and high band pass filters set at 1 and 35. Isolated spikes were seen __ emanating from the area of application 3-14 min *Address correspondence and reprint requests to Dr after injection of penicillin. No ictal discharge of Ruggero G. Fariello, Thomas Jefferson University, behavioral correlates of the spikes were ever seen in Room 9606, Eleventh and Chestnut Streets, Philadelthese experimental conditions. phia, PA 19107, U.S.A.
T. GOLDENand R. G. FARIELLO
Preparation of drugs
homotaurine at 40mg/kg. Similarly, the injection of an equal volume of dimethylsufoxide did not change The drugs, THIP (Lundbeck, Copenhagen) and any of the observed variables. The effects on frehomotaurine (3APS) (Aldrich Company) were disquency of spike after THIP (lOmg/kg) progabide solved in distilled water and the pH of the solution (25 mg/kg) and homotaurine (80mg/kg) are illusadjusted to a value of 6.8 to 7.6. Dilantin was used trated in Figs I and 2; here they are compared to the in the commercially available injectable form. In spiking rates in epileptic naive (nontreated) animals, preliminary experiments, progabide was dissolved in animals treated with dimethylsufoxide and animals tween 80, propyleneglycol or ethanol. All solutions treated with diphenylhydantoin. In general, no statisstill contained a largely suspended form of the drug tically significant reduction of frequency of spike, was and caused severe respiratory and electrographic noted except for progabide at 1 and 1.5 hr (P I 0.1) suppression upon intravenous administration. Only and THIP at 2 hr (P < .0.2). A statistically significant dimethylsufoxide (DMSO) completely dissolved progabide in small volumes. However, the solution is increase in spike activity was observed with phenytoin at 1.5 hr (P I 0.2). A closer analysis of the highly hyperosmolar. To avoid severe hemolysis, immediate effect of the injection of drugs demonsmall volumes (0.5 ml) were injected over a long strated that progabide markedly depressed the ampliperiod that varied from 5 to 7 min. In control experitude and less so the frequency of spikes in the first ments, injections of equal volume and pH of vehicle l-6min (Fig. 3). The effect was associated with a solvents, including dimethylsufoxide were administered. Some compounds were administered at doses marked suppression of background EEG activity. The suppression of the background, however, was effective in other epileptic models: homotaurine (Fariello and Golden, 1980), progabide (Worms et of much shorter duration than the suppression of spiking as shown in Fig. 3. al., 1982) and THIP (Christensen and KrogsgaardIn general, the amplitude and duration of spikes Larsen, 1983). were not affected. However, homotaurine (80 mg/kg) Thirty minutes after the appearance of spikes, induced in the first 4-8 min a 36% reduction in various solutions of the drugs were injected. Their effect on the frequency and amplitude of the spikes amplitude of the spike transmitted to the contralateral homologous area (Fig. 4). Also after homowas observed, both on the area of primary applicataurine, a change in the morphology of spikes was tion of penicillin and on the contralateral homolonoted because of the appearance of a small, but clear, gous (mirror) area. Spike histograms were obtained fast pre-positive component prior to the major negaby averaging the frequency of spike for 2 min periods tive deflection on the projected spike. The slow, at 30 min intervals. Injections of THIP were given in negative wave component observed on the primary doses of 5, 10 and 20 mg/kg and of progabide, in focal spike was also not clearly detectable (Fig. 4). doses of 15, 25 and 50 mg/kg. To avoid massive At 20mg/kg, THIP, in three animals, induced a hemolysis, the injection rate for progabide was very slow. Dilantin was injected at a dose of 40 mg/kg and steady, remarkable enhancement of spike activity with the following sequence of events. Within 15 min, homotaurine at doses of 40 and 80 mg/kg. the spikes turned into duplets with an increase of the rate of spiking up to 180% from control levels. Within RESULTS 3&4Omin, triplets were observed without a further No changes were seen after injections of THIP at increase in the rate of occurrence. From 60 min on, the dose of 5 mg/kg, progabide at 15 mg/kg and bursts of polyspikes were alternating with suppressed
Control n =6
1. The graph compares the frequency of spikes of the neocortical penicillin focus of naive (saline treated) rats and rats treated with progabide (PROG) (25 mg/kg) or THIP (10 mg/kg).
GABA agonists on penicillin spikes
DMSO n- 4
3 APS n=4
Fig. 2. Comparison of the frequency of spikes in rats treated with homotaurine (3APS) (40mg/kg), phenytoin (DPH) (20 mg/kg), dimethylsufoxide (DMSO) (75% 1 ml) and control animals.
background activity (Fig. 5). The total duration of the spiking activity as computed in control animals was not changed by any of the pharmacological agents tested. DISCUSSION
In general, GABA agonists failed to suppress, in a sustained way, spikes induced by penicilhn in rats. The most powerful agent in that regard was progabide which immediately and transiently reduced amplitude and frequency of spikes together with the suppression of the background EEG. Also, a significant, although modest effect, was noted at 1 and 1.5 hr after injection of 25 mg/kg of progabide. The acute cortical focus induced by penicillin in cats behaves in a remarkably different way in response to the administration of direct GABA agonists. The transient, complete suppression of spikes as observed in cats with homotaurine (Fariello, 1979) and progabide (Worms et al., 1982) was not seen in rats. Whereas GABA is thought to play a major role in cortical inhibition in cats (Dreifuss, Kelly and Krjevie, 1969), it has been suggested that in the rat cortex, glycine may be the mediator of such a function (Bernardi, Cherubini, Marciani and Stanzione, 1979; Marciani, Stanzione, Cherubini and Bernardi, 1980). It is not known if penicillin possesses any anti-glycine action which may be responsible for the appearance of spikes in rats. Nevertheless, even in the absence of such an antagonistic effect of glycine, it may be postulated that the intracortical segmental inhibition required to counteract paroxysmal, epileptiform activity in rats is mediated by non-GABAergic, possibly glycinergic mechanisms (Marciani et al., 1980), thus explaining the relative ineffectiveness of GABA agonists. Each drug demonstrated a different action, stressing its individuality and the limitation of its categorization under the common denominator of direct
GABA agonists. Properties other than their capability of binding and activating the GABA receptor complexes might have influenced their action. Particularly, the degree of permeability to the blood-brain barrier (BBB), their half-life and the production of metabolites that have as yet, unknown effects could have been partly responsible for the differential effect. A different permeability through the blood-brain barrier which is excellent for progabide, good for THIP and probably minimal for homotaurine should have resulted only in a different potency, but not in different types of effects. The results of the present studies conflict with the observation of a modest anticonvulsant effect of muscimol on spikes induced by penicillin in the rat (Collins, 1980). A reduction of spike frequency after 0.9 and 1.5 mg/kg (i.p.) of muscimol was noted, but no data was provided on the number of animals or the quantification and statistical significance of the reduction in spikes. Occasional pro-convulsant effects were also noted as ictal discharges in the studies of Collins and appeared less frequently after muscimol, but lasted longer and presented a more severe behavioral component. In the absence of quantified data, a comparison of the present results with those experiments is impossible. However, an important variable that separates the two experimental conditions and might account for the discrepancy is that the animals in this study were under urethane anesthesia, whereas the study with muscimol was performed in alert rats that had recovered from halothane. Furthermore, it has been demonstrated that after systemic administration of muscimol, over 90% of what is found in the brain consists of metabolites of muscimol of unknown pharmacological activity (Moroni, Forchetti, Krogsgaard-Larsen and Guidotti, 1982), the effects of which may have been responsible for the observed diminution of spikes. Another consideration concerns the change of
G. T. GOLDEN and R. G. FARIELLO
GABA agonists on penicillin spikes
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G.T. GOLDEN and R. G. FARIELLO
GABA agonists on penicillin spikes
morphology and amplitude of spikes in the mirror area after large doses of homotaurine. The decrease in amplitude and the appearance of a very sharp positive pre-potential may be achieved by a reduction of the area receiving transcallosal input from the primary focus, together with a sharpening of the sink function of radially-oriented neuronal dendrites. In other studies, it has been demonstrated that taurine, a non-GABAmimetic inhibitory amino acid suppressed mirror spikes in cats (Fariello, Lloyd and Hornykiewicz, 1975). Phenytoin also increases prepositivity of spikes induced by penicillin in the primary focal area (Bustamante, Lueders, Pippinger and Goldensohn, 1980). Therefore, the effect of homotaurine may be related to a nonspecific taurine-like effect or to the activation of a subpopulation of GABA receptors (Meldrum, 1980) which results in reduction of transcallosal transmission of spikes either by enhancement of excitation in deep cortical layers (disinhibition) or by potentiation of superficial, previously masked inhibitory postsynaptic potentials. In conclusion, direct GABA agonists had minimal or no suppressant effects on spikes induced by penicillin in rats. Since they are very effective in suppressing penicillin-induced spikes in cats, a different neurochemical or physiological mechanism is likely to underlie this epileptic phenomenon in the two species. Among the drugs tested, progabide demonstrated the strongest antagonistic effect, confirming the broad spectrum of anticonvulsant activity of this new compound (Worms, et al., 1982). The failure of spikes induced by penicillin in rats to respond to the administration of established (phenytoin) and novel anticonvulsant agents casts serious doubts on the suitability of this model for screening potential antiepileptic drugs.
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