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Relative convulsant potencies of structural analogues of penicillin
Brain Research, 114 (1976) 139-143 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
139
Relative convulsant potencies of structural analogues of penicillin
MICHAEL J. GUTNICK*, HANS VAN DUIJN and NATHAN CITRI Department of Neurology, Hadassah University Hospital, Jerusalem, Israel, (M.J.G.), Department of Neurology, Valeriuskliniek, Amsterdam, The Netherlands (H.v.D.) and Institute of Microbiology, Hebrew University ( N.C.), Jerusalem (Israel)
(Accepted May 25th, 1976)
Penicillin has long been recognized as a potent convulsant agent. Topical application of this antibiotic to the cortical surface has become one of the most commonly employed techniques for investigation of the cellular mechanisms of focal epileptogenesis 1,8,1z,14,17-z°. Despite widespread interest in the convulsant nature of penicillin, relatively little is known about the relationship between the structure of the compound and its epileptogenic properties. Penicillins are all derivatives of 6-aminopenicillanic acid (6-APA) which consists of a thiazolidine ring and a fl-lactam ring. It has been shown that the epileptogenic properties of penicillin, like its antimicrobial and antigenic properties, are dependent on the integrity of the fl-lactam bond, and convulsant activity is lost when that bond is hydrolysed by flglactamase (penicillinase) enzymes 11. N-acyl substituents of 6-APA enhance its otherwise low antimicrobial potency. The nature of the side chain determines, in large part, the antimicrobial activity of the antibiotic and its reactivity with fl-lactamases 6. In virtually all studies of the neural effects of penicillin, only one of the many fl-lactam antibiotics, benzylpenicillin (penicillin G) has been used. However, it is known that other penicillin analogues are also epileptogenic 5,1°,zz,2z. In order to assess the effects of structure of the side chains on the convulsive properties of the penicillins, we determined the relative epileptogenic potencies of 5 derivatives of 6-APA and one of the closely related cephalosporins. As a measure of epileptogenic potency, we determined for each antibiotic the minimum concentration necessary to induce epileptiform discharge when applied topically to the cortical surface. Experiments were performed in pentobarbital-anesthetized cats and in urethane-anesthetized rats. Animals were mounted in a stereotaxic frame and the bone overlying the cortex was removed bilaterally. EEG activities were led from chlorided silver ball electrodes to RC preamplifiers, and from there to an inkwriter and an oscilloscope. At the time of each experiment, the sodium salt of the antibiotic under study was dissolved in normal saline, and a dilution series was pre-
* Present address: Department of Comparative Medicine, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel.
140
BENZYLPENICILLIN
[]CATS
PHENOXYMETHYLPENICILLIN
] OXACILLIN
~J~i~i~i~i~il;::::~i;]
RATS
,
METHICILLIN
AMPICILLIN
CEPHALOTHIN
L
[i~i~i~i~:ii~i~:~ii~!iii~i~J!~ii~i!~::iii~ji!i!!!~i~i~:ii~i:~iiiiii~i~i~ii!!!~!~!~!ii~i~ii~i~i~i:i:~ i
I
O
i
0050
0100
0150
0200
1 0.250
moles / liter
Fig. 1. Threshold concentrations of 6 fl-lactam antibiotics in the cat and rat. The length of each column represents the mean value for 4 experiments. Horizontal lines indicate standard deviation for drugs whose threshold concentrations varied from trial to trial. pared. A gelfoam pledgette (1 cu.mm) was saturated with 2-5 #1 of the most dilute solution, and placed directly onto the pial surface through a small hole in the dura. If no spontaneous interictal discharges appeared in the E E G within ten minutes, the next highest concentration was applied. Thus, the threshold concentration was the lowest concentration of antibiotic required to induce an epileptogenic focus within 10 min. In order to avoid the possible effects of the spread of antibiotic over the cortical surface, each rat was used for only one drug trial. In cats, two different compounds were tested in each animal, one on the pericruciate gyrus and the other on the contralateral supersylvian gyrus. In no case was convulsive potency related to the area of the cortex used. Threshold concentrations were determined for benzylpenicillin, oxacillin, ampicillin, phenoxymethylpencillin, methicillin, and cephalothin (a penicillin-like cephalosporin). Results are shown in Fig. 1. Although all of the compounds were epileptogenic, their convulsive potencies varied. With each of the penicillin analogues, significantly higher concentrations were required in rats than in cats to induce epileptiform discharge. The most effective of the compounds were benzylpencillin and phenoxymethylpenicillin, which were equipotent. Required concentrations were 0.012-0.014 M (68008500 IU/ml) in cats, and 0.017-0.022 M (10,200-13,000 IU/ml) in rats. A ten-fold increase in concentration was necessary to induce epileptogenesis with methicillin and with ampicillin. In cats, the minimum effective concentration of oxacillin varied considerably from experiment to experiment. However, in rats this compound was clearly more potent than ampicillin or methicillin. As compared with the penicillin
141 derivatives, cephalothin was an extremely weak convulsant agent, requiring concentrations well over 0.2 M to produce a focus. With the exception of the differences in threshold concentration, there were no other features that clearly distinguished the convulsant potencies of these compounds. Although amplitudes and waveforms of interictal discharges varied somewhat from focus to focus, this variability was not related to the antibiotic used, and all of the compounds induced epileptiform waves which were essentially similar. The amount of time required for foci to develop and interictal discharge rates are summarized in Table I for experiments in cats. Although these data seemed to suggest a tendency for foci induced by more potent agents to develop sooner and to be more active, an analysis of variance showed that the apparent differences between the antibiotics were not statistically significant. The structure common to all penicillin analogues, 6-APA, was relatively ineffective as a convulsant agent. Three attempts were made in rats to induce epileptogenesis with this compound at concentrations as high as 0.5 M. In only one experiment did a transient focus develop. In this case, epileptiform discharges appeared rarely, were of extremely low amplitude, and disappeared entirely from the E E G within 15 min. These data demonstrate that, as with other biological actions of the penicillins, convulsive properties are greatly enhanced by the addition of side chains to the fllactam ring of the 6-APA nucleus. Moreover, modification of the side chain is associated with modification of epileptogenic potency. Cephalothin, one of the structurallyrelated cephalosporins, had weaker convulsive properties than any of the 6-APA derivatives. However, since this was the only cephalosporin studied, we cannot say whether low epileptogenic activity is a general feature of this entire other family of fl-lactam antibiotics. The mechanisms underlying these structure-function relationships are as yet unclear. It is unlikely that the differences we observed in epileptogenic potency were due to differences in the ability of analogues to penetrate through the pia and into the cortex. If this were the case, threshold concentrations would be expected to depend on TABLE I Time required for a focus to develop and interictal discharge rates following application of threshold concentrations of 6 fl-lactam antibiotics in the cat
Each value is the mean ± standard deviation for 4 experiments. Interictal discharge rate was measured over a period of 15 min.
Benzylpenicillin Phenoxymethylpenicillin Oxacillin Methicillin Ampicillin Cephalothin
Time to first interictal discharge (min)
Interietal discharges/min
3.2 -tz 1.6 2.8 i 1.1 4.8 ~= 2.6 5.2 d= 1.8 5.8 ~: 1.8 6.5 d= 2.3
20 ± 6 27 =L 15 15 ± 9 15 -t_ 9 14 -t- 10 8:5 2
142 the lipophilic properties of the compounds 16. The lipid solubilities of all of these antibiotics have been measured 4 and do not correlate with the relative convulsive potencies reported here. In this regard, it should also be noted that foci produced by less potent antibiotics did not require a significantly longer time to develop. It thus seems probable that the differences in threshold concentration actually reflect differences in the abilities of these compounds to produce their effect on the neurons of the cortical focus. In view of the profound effects of anesthetic drugs on cortical epileptogenesis 2°, it is likely that they can influence the minimum concentration of a convulsant agent required to produce a focus. Thus, our use of barbiturate anesthesia might explain why the threshold concentration of benzylpenicillin which we found in cats was considerably higher than the minimum dose of 1,000-5,000 IU/ml suggested by Prince 2°. Anesthetic effects might also account for the finding that with each of the penicillin analogues, higher concentrations were required to produce foci in urethane-anesthetized rats than in pentobarbital-anesthetized cats. It is significant that although the threshold concentration of a compound differed when determined under different experimental conditions, the relative potencies of the antibiotics did not vary. In both cats and rats, the order of convulsant potencies was benzylpenicillin = phenoxymethylpenicillin ~> oxacillin ~ methicillin ampicillin > cephalothin. This order is different from that reported by Gerald et al. 10. These authors injected several penicillin analogues intracerebrally in mice and ranked the convulsant potencies on the basis of subsequent behavioral seizure manifestations. However, the discrepancy is resolved by considering the minimum concentrations they required to produce any behavioral effect. When determined in this way, the relative potencies in their experiments are consistent with those reported here. Knowledge of the relative convulsant potencies of various penicillin analogues may be of use in the study of the basic mechanisms of penicillin epileptogenesis. In an effort to elucidate these mechanisms several investigators have examined the effects of the antibiotic in a variety of vertebrate and invertebrate preparations which lack the organizational complexity of the mammalian cortex and where synaptic and membrane properties are more readily studied2,3,7,9,15,21, 24. Although several different actions of penicillin have been described, it is difficult to assess the relationship between these various effects and the convulsant nature of the antibiotic. Only benzylpenicillin has been used for these studies. It is possible that determination of dose-response relationships for several penicillin analogues might be helpful in evaluating the relevance of an effect to epileptogenesis. We would expect that, if a neural action of benzylpenicillin is related to the drug's convulsant properties, the relative potencies of various other penicillin derivatives for producing that action will be the same as the relative potencies for inducing cortical epileptiform discharge.
1 Ayala, G. F., Dichter, M., Gumnit, R. J., Matsumoto, H. and Spencer, W. A., Genesis of epileptic interictal spikes, Brain Research, 52 (1973) 1-17. 2 Ayala, G. F., Lin, S. and Vasconetto, C., Penicillinas an epileptogenicagent: its effecton an isolated neuron, Science, 167 (1970) 1257-1260.
143 3 Ayala, G. F., Spencer, W. A. and Gumnit, R. J., Penicillin as an epileptogenic agent: its effect on an isolated synapse, Science, 171 (1971) 915-917. 4 Biagi, G. L., Guerra, M. C., Barbaro, A. M. and Gamba, M. F., Influence of lipophilic character on the antibacterial activity of cephalosporins and penicillins, J. reed. Chem., 13 (1970) 511-516. 5 Blum, P. and Matsen, J. M., Obvious seizures from carbenicillin, Minn. Med., 54 (1971) 697-699. 6 Citri, N., Penicillinase and other fl-lactamases. In P. D. Boyer (Ed.), The Enzymes, Vol. 4, Academic Press, New York, 1971, pp. 23-46. 7 Davidoff, R., Penicillin and presynaptic inhibition in the amphibian spinal cord, Brain Research, 36 (1972) 218-222. 8 Dichter, M. and Spencer, W. A., Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographical features, J. NeurophysioL, 32 (1969) 649-662. 9 Futamachi, K. J. and Prince, D. A., Effect of penicillin at an excitatory synapse, Brain Research, 1O0 (1975) 589-597. 10 Gerald, M. C., Massey, J. and Sparado, D. C., Comparative convulsant activity of various penicillins after intracerebral injection in mice, J. Pharm. Pharmacol., 25 (1973) 104-108. 11 Gutnick, M. J., and Prince, D. A., Penicillinase and the convulsant action of penicillin, Neurology, 21 (1971) 759-764. 12 Hochner, B., Spira, M. E. and Werman, R., Penicillin decreases chloride conductance in crustacean muscle: a model for the epileptic neuron, Brain Research, 107 (1976) 85-103. 13 Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: lnterictal manifestations, Exp. NeuroL, 9 (1964) 286-304. 14 Matsumoto, H., Ayala, G. F. and Gumnit, R. J., Neuronal behavior and triggering mechanisms in the cortical epileptic focus, J. Neurophysiol., 32 (1969) 688-703. 15 Meyer, H. and Prince, D. A., Convulsant actions of penicillin: effects on inhibitory mechanisms, Brain Research, 53 (1973) 477-482. 16 Oldendrof, W. H., Lipid solubility and drug penetration of the blood brain barrier, Proc. Soc. exp. Biol. (N. Y.), 147 (1974) 813-816. 17 Prince, D. A., Inhibition in "epileptic" neurons, Exp. NeuroL, 21 (1968) 307-321. 18 Prince, D. A., The depolarization shift in "epileptic" neurons, Exp. Neurol., 21 (1968) 467-485. 19 Prince, D. A., Microelectrode studies of penicillin foci. In H. H. Jasper, A. A. Ward, Jr. and A. Pope (Eds.), Basic Mechanisms of the Epilepsies, Little Brown, Boston, 1969, pp. 320-238. 20 Prince, D. A., Topical convulsants and metabolic antagonists. In D. P. Purpura, J. K. Penry, D. Tower, D. M. Woodbury and R. Walter (Eds.), Experimental Models of Epilepsy, Raven Press, New York, 1972, pp. 51-83. 21 Spira, M. E. and Bennett, M. V. L., Penicillin induced seizure activity in the hatchet fish, Brain Research, 43 (1972) 235-241. 22 Van Hartesveldt, C., Petit, T. L. and Isaacson, R. L., Epileptogenic effects of several penicillins and penicillin-related compounds in rat cortex, Epilepsia, 16 (1975) 449-455. 23 Weihrauch, T. R., Homer, D. and Krieglestein, J., The neurotoxic effect of benzylpenicillin and semisynthetic penicillins in the rabbit, Proc. Europ. Soc. Study Drug Toxicity, 15 (1973) 52-56. 24 Wilson, W. A. and Escueta, A. V., Common effects of pentylenetetrazol and penicillin, Brain Research, 72 (1974) 168-171.
139
Relative convulsant potencies of structural analogues of penicillin
MICHAEL J. GUTNICK*, HANS VAN DUIJN and NATHAN CITRI Department of Neurology, Hadassah University Hospital, Jerusalem, Israel, (M.J.G.), Department of Neurology, Valeriuskliniek, Amsterdam, The Netherlands (H.v.D.) and Institute of Microbiology, Hebrew University ( N.C.), Jerusalem (Israel)
(Accepted May 25th, 1976)
Penicillin has long been recognized as a potent convulsant agent. Topical application of this antibiotic to the cortical surface has become one of the most commonly employed techniques for investigation of the cellular mechanisms of focal epileptogenesis 1,8,1z,14,17-z°. Despite widespread interest in the convulsant nature of penicillin, relatively little is known about the relationship between the structure of the compound and its epileptogenic properties. Penicillins are all derivatives of 6-aminopenicillanic acid (6-APA) which consists of a thiazolidine ring and a fl-lactam ring. It has been shown that the epileptogenic properties of penicillin, like its antimicrobial and antigenic properties, are dependent on the integrity of the fl-lactam bond, and convulsant activity is lost when that bond is hydrolysed by flglactamase (penicillinase) enzymes 11. N-acyl substituents of 6-APA enhance its otherwise low antimicrobial potency. The nature of the side chain determines, in large part, the antimicrobial activity of the antibiotic and its reactivity with fl-lactamases 6. In virtually all studies of the neural effects of penicillin, only one of the many fl-lactam antibiotics, benzylpenicillin (penicillin G) has been used. However, it is known that other penicillin analogues are also epileptogenic 5,1°,zz,2z. In order to assess the effects of structure of the side chains on the convulsive properties of the penicillins, we determined the relative epileptogenic potencies of 5 derivatives of 6-APA and one of the closely related cephalosporins. As a measure of epileptogenic potency, we determined for each antibiotic the minimum concentration necessary to induce epileptiform discharge when applied topically to the cortical surface. Experiments were performed in pentobarbital-anesthetized cats and in urethane-anesthetized rats. Animals were mounted in a stereotaxic frame and the bone overlying the cortex was removed bilaterally. EEG activities were led from chlorided silver ball electrodes to RC preamplifiers, and from there to an inkwriter and an oscilloscope. At the time of each experiment, the sodium salt of the antibiotic under study was dissolved in normal saline, and a dilution series was pre-
* Present address: Department of Comparative Medicine, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel.
140
BENZYLPENICILLIN
[]CATS
PHENOXYMETHYLPENICILLIN
] OXACILLIN
~J~i~i~i~i~il;::::~i;]
RATS
,
METHICILLIN
AMPICILLIN
CEPHALOTHIN
L
[i~i~i~i~:ii~i~:~ii~!iii~i~J!~ii~i!~::iii~ji!i!!!~i~i~:ii~i:~iiiiii~i~i~ii!!!~!~!~!ii~i~ii~i~i~i:i:~ i
I
O
i
0050
0100
0150
0200
1 0.250
moles / liter
Fig. 1. Threshold concentrations of 6 fl-lactam antibiotics in the cat and rat. The length of each column represents the mean value for 4 experiments. Horizontal lines indicate standard deviation for drugs whose threshold concentrations varied from trial to trial. pared. A gelfoam pledgette (1 cu.mm) was saturated with 2-5 #1 of the most dilute solution, and placed directly onto the pial surface through a small hole in the dura. If no spontaneous interictal discharges appeared in the E E G within ten minutes, the next highest concentration was applied. Thus, the threshold concentration was the lowest concentration of antibiotic required to induce an epileptogenic focus within 10 min. In order to avoid the possible effects of the spread of antibiotic over the cortical surface, each rat was used for only one drug trial. In cats, two different compounds were tested in each animal, one on the pericruciate gyrus and the other on the contralateral supersylvian gyrus. In no case was convulsive potency related to the area of the cortex used. Threshold concentrations were determined for benzylpenicillin, oxacillin, ampicillin, phenoxymethylpencillin, methicillin, and cephalothin (a penicillin-like cephalosporin). Results are shown in Fig. 1. Although all of the compounds were epileptogenic, their convulsive potencies varied. With each of the penicillin analogues, significantly higher concentrations were required in rats than in cats to induce epileptiform discharge. The most effective of the compounds were benzylpencillin and phenoxymethylpenicillin, which were equipotent. Required concentrations were 0.012-0.014 M (68008500 IU/ml) in cats, and 0.017-0.022 M (10,200-13,000 IU/ml) in rats. A ten-fold increase in concentration was necessary to induce epileptogenesis with methicillin and with ampicillin. In cats, the minimum effective concentration of oxacillin varied considerably from experiment to experiment. However, in rats this compound was clearly more potent than ampicillin or methicillin. As compared with the penicillin
141 derivatives, cephalothin was an extremely weak convulsant agent, requiring concentrations well over 0.2 M to produce a focus. With the exception of the differences in threshold concentration, there were no other features that clearly distinguished the convulsant potencies of these compounds. Although amplitudes and waveforms of interictal discharges varied somewhat from focus to focus, this variability was not related to the antibiotic used, and all of the compounds induced epileptiform waves which were essentially similar. The amount of time required for foci to develop and interictal discharge rates are summarized in Table I for experiments in cats. Although these data seemed to suggest a tendency for foci induced by more potent agents to develop sooner and to be more active, an analysis of variance showed that the apparent differences between the antibiotics were not statistically significant. The structure common to all penicillin analogues, 6-APA, was relatively ineffective as a convulsant agent. Three attempts were made in rats to induce epileptogenesis with this compound at concentrations as high as 0.5 M. In only one experiment did a transient focus develop. In this case, epileptiform discharges appeared rarely, were of extremely low amplitude, and disappeared entirely from the E E G within 15 min. These data demonstrate that, as with other biological actions of the penicillins, convulsive properties are greatly enhanced by the addition of side chains to the fllactam ring of the 6-APA nucleus. Moreover, modification of the side chain is associated with modification of epileptogenic potency. Cephalothin, one of the structurallyrelated cephalosporins, had weaker convulsive properties than any of the 6-APA derivatives. However, since this was the only cephalosporin studied, we cannot say whether low epileptogenic activity is a general feature of this entire other family of fl-lactam antibiotics. The mechanisms underlying these structure-function relationships are as yet unclear. It is unlikely that the differences we observed in epileptogenic potency were due to differences in the ability of analogues to penetrate through the pia and into the cortex. If this were the case, threshold concentrations would be expected to depend on TABLE I Time required for a focus to develop and interictal discharge rates following application of threshold concentrations of 6 fl-lactam antibiotics in the cat
Each value is the mean ± standard deviation for 4 experiments. Interictal discharge rate was measured over a period of 15 min.
Benzylpenicillin Phenoxymethylpenicillin Oxacillin Methicillin Ampicillin Cephalothin
Time to first interictal discharge (min)
Interietal discharges/min
3.2 -tz 1.6 2.8 i 1.1 4.8 ~= 2.6 5.2 d= 1.8 5.8 ~: 1.8 6.5 d= 2.3
20 ± 6 27 =L 15 15 ± 9 15 -t_ 9 14 -t- 10 8:5 2
142 the lipophilic properties of the compounds 16. The lipid solubilities of all of these antibiotics have been measured 4 and do not correlate with the relative convulsive potencies reported here. In this regard, it should also be noted that foci produced by less potent antibiotics did not require a significantly longer time to develop. It thus seems probable that the differences in threshold concentration actually reflect differences in the abilities of these compounds to produce their effect on the neurons of the cortical focus. In view of the profound effects of anesthetic drugs on cortical epileptogenesis 2°, it is likely that they can influence the minimum concentration of a convulsant agent required to produce a focus. Thus, our use of barbiturate anesthesia might explain why the threshold concentration of benzylpenicillin which we found in cats was considerably higher than the minimum dose of 1,000-5,000 IU/ml suggested by Prince 2°. Anesthetic effects might also account for the finding that with each of the penicillin analogues, higher concentrations were required to produce foci in urethane-anesthetized rats than in pentobarbital-anesthetized cats. It is significant that although the threshold concentration of a compound differed when determined under different experimental conditions, the relative potencies of the antibiotics did not vary. In both cats and rats, the order of convulsant potencies was benzylpenicillin = phenoxymethylpenicillin ~> oxacillin ~ methicillin ampicillin > cephalothin. This order is different from that reported by Gerald et al. 10. These authors injected several penicillin analogues intracerebrally in mice and ranked the convulsant potencies on the basis of subsequent behavioral seizure manifestations. However, the discrepancy is resolved by considering the minimum concentrations they required to produce any behavioral effect. When determined in this way, the relative potencies in their experiments are consistent with those reported here. Knowledge of the relative convulsant potencies of various penicillin analogues may be of use in the study of the basic mechanisms of penicillin epileptogenesis. In an effort to elucidate these mechanisms several investigators have examined the effects of the antibiotic in a variety of vertebrate and invertebrate preparations which lack the organizational complexity of the mammalian cortex and where synaptic and membrane properties are more readily studied2,3,7,9,15,21, 24. Although several different actions of penicillin have been described, it is difficult to assess the relationship between these various effects and the convulsant nature of the antibiotic. Only benzylpenicillin has been used for these studies. It is possible that determination of dose-response relationships for several penicillin analogues might be helpful in evaluating the relevance of an effect to epileptogenesis. We would expect that, if a neural action of benzylpenicillin is related to the drug's convulsant properties, the relative potencies of various other penicillin derivatives for producing that action will be the same as the relative potencies for inducing cortical epileptiform discharge.
1 Ayala, G. F., Dichter, M., Gumnit, R. J., Matsumoto, H. and Spencer, W. A., Genesis of epileptic interictal spikes, Brain Research, 52 (1973) 1-17. 2 Ayala, G. F., Lin, S. and Vasconetto, C., Penicillinas an epileptogenicagent: its effecton an isolated neuron, Science, 167 (1970) 1257-1260.
143 3 Ayala, G. F., Spencer, W. A. and Gumnit, R. J., Penicillin as an epileptogenic agent: its effect on an isolated synapse, Science, 171 (1971) 915-917. 4 Biagi, G. L., Guerra, M. C., Barbaro, A. M. and Gamba, M. F., Influence of lipophilic character on the antibacterial activity of cephalosporins and penicillins, J. reed. Chem., 13 (1970) 511-516. 5 Blum, P. and Matsen, J. M., Obvious seizures from carbenicillin, Minn. Med., 54 (1971) 697-699. 6 Citri, N., Penicillinase and other fl-lactamases. In P. D. Boyer (Ed.), The Enzymes, Vol. 4, Academic Press, New York, 1971, pp. 23-46. 7 Davidoff, R., Penicillin and presynaptic inhibition in the amphibian spinal cord, Brain Research, 36 (1972) 218-222. 8 Dichter, M. and Spencer, W. A., Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographical features, J. NeurophysioL, 32 (1969) 649-662. 9 Futamachi, K. J. and Prince, D. A., Effect of penicillin at an excitatory synapse, Brain Research, 1O0 (1975) 589-597. 10 Gerald, M. C., Massey, J. and Sparado, D. C., Comparative convulsant activity of various penicillins after intracerebral injection in mice, J. Pharm. Pharmacol., 25 (1973) 104-108. 11 Gutnick, M. J., and Prince, D. A., Penicillinase and the convulsant action of penicillin, Neurology, 21 (1971) 759-764. 12 Hochner, B., Spira, M. E. and Werman, R., Penicillin decreases chloride conductance in crustacean muscle: a model for the epileptic neuron, Brain Research, 107 (1976) 85-103. 13 Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: lnterictal manifestations, Exp. NeuroL, 9 (1964) 286-304. 14 Matsumoto, H., Ayala, G. F. and Gumnit, R. J., Neuronal behavior and triggering mechanisms in the cortical epileptic focus, J. Neurophysiol., 32 (1969) 688-703. 15 Meyer, H. and Prince, D. A., Convulsant actions of penicillin: effects on inhibitory mechanisms, Brain Research, 53 (1973) 477-482. 16 Oldendrof, W. H., Lipid solubility and drug penetration of the blood brain barrier, Proc. Soc. exp. Biol. (N. Y.), 147 (1974) 813-816. 17 Prince, D. A., Inhibition in "epileptic" neurons, Exp. NeuroL, 21 (1968) 307-321. 18 Prince, D. A., The depolarization shift in "epileptic" neurons, Exp. Neurol., 21 (1968) 467-485. 19 Prince, D. A., Microelectrode studies of penicillin foci. In H. H. Jasper, A. A. Ward, Jr. and A. Pope (Eds.), Basic Mechanisms of the Epilepsies, Little Brown, Boston, 1969, pp. 320-238. 20 Prince, D. A., Topical convulsants and metabolic antagonists. In D. P. Purpura, J. K. Penry, D. Tower, D. M. Woodbury and R. Walter (Eds.), Experimental Models of Epilepsy, Raven Press, New York, 1972, pp. 51-83. 21 Spira, M. E. and Bennett, M. V. L., Penicillin induced seizure activity in the hatchet fish, Brain Research, 43 (1972) 235-241. 22 Van Hartesveldt, C., Petit, T. L. and Isaacson, R. L., Epileptogenic effects of several penicillins and penicillin-related compounds in rat cortex, Epilepsia, 16 (1975) 449-455. 23 Weihrauch, T. R., Homer, D. and Krieglestein, J., The neurotoxic effect of benzylpenicillin and semisynthetic penicillins in the rabbit, Proc. Europ. Soc. Study Drug Toxicity, 15 (1973) 52-56. 24 Wilson, W. A. and Escueta, A. V., Common effects of pentylenetetrazol and penicillin, Brain Research, 72 (1974) 168-171.