Epilepsia, 17: 129-135, 1976.
Raven Press, New York
The Effect of Experimentally Induced Focal Epilepsy on Operant Responding in the Rat Harvey L. Edmonds, Jr College of Pharmacy, Washington State University, Pullman, Washington 991 63 (Received in revised form January 6,1976) INTRODUCTION The effect of generalized epileptic seizures on learning and memory is well-known. However, the effects of interictal discharges on the various aspects of behavior have not been studied extensively. In a recent review, Woodruff (1974) concluded that electrographic spike discharges per se may not be responsible for impairments in learning and performance. He further stated that the presence of behavioral seizures does not necessarily disrupt operant performance. On the other hand, Aquino-Cias (1972) showed that the retention efficiency of rats in a behavioral situation was impaired by “interictal subclinical epileptic activity.” Rennick et al. (1969) observed a diminution in the ability of epileptic patients to perform a complex series of psychomotor tasks during the appearance of. EEG spike volleys. The apparent conflict in these reports emphasizes the need for further study. Since interictal spike discharges commonly occur in cases of focal epilepsy (Woodruff, 1974), focal seizures were experimentally induced in rats in order to quantitate the influence of focal abnormalities on operant behavior. The intracortical injection of epileptogens such as penicillin and conjugated estrogens (PremarinB) is well-suited to this purpose. Small amounts of either substance will produce transient interictal spike discharges with occasional brief afterdischarges (AD) (Marcus et al., 1966, Woodruff, 1974) and minimal cytoarchitectural disruption (Okada, 1971). Thus focal K e y W o r d s : Epilepsy
-
Operant Behavior
-Rat
seizures of short duration and mild intensity can be repeatedly induced at will in the same animals with little permanent neurological impairment. In addition, bicuculline was used as a topical epileptogen. Curtis et al. (1972) proposed that penicillin and bicuculline have the same mechanism of action. If this is so, they should produce similar epileptic responses.
METHODS AND MATERIALS The subjects were adult male SpragueDawley albino rats obtained from the campus breeding facility. The animals were kept in a room with regulated 1 2 hr light-dark cycle and housed in individual suspended cages. The animals were given free access to water and sufficient lab chow to maintain themselves at 85% of their full-fed weight. Each animal was weighed daily. Dose-response studies on the EEG and behavioral effects of intracortical injection of sodium penicillin G (Squibb), bicuculline (Sigma), and Premarin@ Intravenous (Ayerst) were performed to determine doses suitable for subsequent behavioral tests. After each rat was anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and placed in a stereotactic instrument, its scalp and underlying muscles were reflected bilaterally. Stainless steel recording screws were fixed in the skull overlying the frontal sinus and bilaterally 4 mm posterior to bregma, 3 mm lateral to midline. A device for accurate intracortical injection (Edmonds and Stark, 1974) was implanted unilaterally 2 mm posterior to bregma, 3 mm lateral to midline
129
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over the sensorimotor cortex. The recording screws were attached to a connector made from a portion of Cambion integrated circuit socket (#703-3897-01-0316). The device and connector were held in place with dental acrylic cement. Following recovery from surgery, 60 animals were divided into groups of 5 and, after control EEG recordings, were injected intracortically to an approximate depth of 0.8 mm with one of the following solutions: buffered sodium penicillin G 50, 100, or 200 international units (IU) (pH 6.2 in saline); bicuculline 0.002, 0.004, 0.025, or 0.05% at pH 5.0 in citrate buffer; PremarinB Intravenous 0.5, 1.0, or 2.0% at pH 7.2 in commercial diluent; isotonic saline adjusted to pH 5.0 with citrate buffer; Premarin diluent. The volume of each injection was 5 pl. The rats receiving the saline or Premarin diluent solutions were injected daily for 1 0 consecutive days to determine the effect of repeated intracortical injections on EEG activity. Operant studies were carried out in Plexiglas Skinner boxes. Depression of a lever in the box initially resulted in the presentation of a 40-mg food pellet (Noyes). After shaping, two groups of 6 rats each were trained to make 20 lever depressions to obtain one food pellet (FR 20 schedule). This particular schedule was used in order to generate high stable response rates that are sensitive to drug-induced behavioral alteration (Edmonds et al., 1975). The operant session was 25 min in length. After each animal achieved an average weekly response rate of greater than 30 responses per minute, it was implanted in the manner previously discussed. One animal died during surgery so that the penicillin group consisted of 6 subjects, whereas the Premarin group contained 5. These 11 rats were used in all subsequent behavioral studies. After recovery from surgery, the subjects were again required to perform in the operant paradigm. FR 20 response rates were recorded daily for 5 consecutive days and served to establish control response rates to which later rates could be compared. For each of the next 1 0 days, after a 5-min control EEG recording, the rats were injected intracortically with a 5-pl solution containing either 100 IU of penicillin or a 1% solution of Premarin. The control recordings served to insure the absence of EEG abnormalities prior to injection. The animals were then
tested in the 25-min operant session. Operant responses were recorded on Lehigh Valley electromechanical counters. The EEG was monitored on a Tektronix 5103N dual beam oscilloscope and recorded on Model 7 Grass electronencephalograph. The topical epileptogens often produced discrete largeamplitude mono- or biphasic events of 20-50 msec duration as exemplified by the bicuculline trace in Fig. 1. However, these epileptic spikes occasionally became polyphasic and widely varied in duration. At times it was difficult to distinguish between many individual spikes occurring in rapid succession and a single polyspike (e.g., Premarin trace, Fig. 1).Previous experience in this laboratory has shown that this distinction is even more difficult in higher mammals such as the cat or dog. To unify data analysis in all species and to facilitate automated procedures, the term “spike” was defined as: (1) a mono- or polyphasic electrical event with a threshold amplitude between 3 and 50 times base-line EEG; (2) a duration of less than 2 sec; (3) followed by at least 2 sec of shbthreshold amplitude. Except in the unusual case of bicuculline, interictal episodes of less than 2 sec were not typically associated with obvious clinical signs of epileptic activity. The spiking frequency was determined by counting five random 1-min segments throughout each record. An afterdischarge (AD) was defined as a polyphasic event of threshold amplitude, longer than 2 sec in duration and followed by at least 2 sec of relative electrical. quiescence. The number of AD was determined for each session. The total amount of AD activity (TAA) was calculated by multiplying the duration of each AD (to the nearest 1 0 sec) by the number of AD. Clinical seizure severity was quantified by means of an arbitrary 0-4 scale: 0 = no sign of seizure activity; 1 = localized unilateral twitching or clonus; 2 = unilateral twitching or clonus; 3 = generalized twitching or clonus without the loss of the righting reflex; 4 = generalized twitching, clonus, or tonic extension with the loss of the righting reflex. The effect of each of the indices of seizure activity on operant responding was determined by comparing response rates following intracortical injection with preinjection response rates. These rates were normalized by expressing each subject’s daily rate as a percent of the
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PENICILLIN 100 units
I
/
I
\\
\
BlCUCULLlNE .05 %
I
500pr
Rot 3 13/24/75)
5 sec
FIG. 1. Effect of epileptogenesis on the EEG. Electroencephalographic abnormalities induced following intracortical injection of sodium penicillin G (100 IU), Premarin (l%), and bicuculline HCI (0.05%). average preinjection control value for that animal. Statistical significance was determined by use of the unpaired Student’s f-test. RESULTS Figure 1 demonstrates the effects of intracortical injection of these epileptogens on the EEG. Each of the agents produced focal abnormalities characterized by a spiking focus with brief paroxysmal AD. Bicuculline induced the most intense epileptiform activity. The amplitude of both spikes and AD was substantially larger than that of penicillin or Premarin. This intense activity was also manifested by severe clonic movements associated with each spike. The action of bicuculline was brief and the animal’s behavior so disrupted that it was unsuitable for subsequent operant studies. Doses of penicillin less than 100 IU or Premarin less than 1%infrequently produced focal epileptic activity. Larger doses produced extensive AD activity and marked behavioral impairment. Thus 100 IU of penicillin and 1%Premarin solutions were employed in the operant studies. Neither repeated injections of saline nor the Premarin diluent produced any signs of EEG or behavioral abnormality.
Penicillin and Premarin in these doses resulted in EEG spike activity within 75 sec, and the first AD generally appeared in less than 5 min. Paroxysmal AD activity continued for 60-90 min. Epileptic patterns always subsided within 2 hr after intracortical injection. In contrast, the onset of spiking following bicuculline (0.025 and 0.05%) was less than 10 sec, and the first AD typically appeared within 40 sec. AD activity usually stopped within 20 min; in no case did epileptic patterns persist for longer than 40 min. The frequency of spiking was approximately 1 Hz in both the Premarin and penicillin groups. Figure 1 shows the slow and irregular pattern commonly seen after bilateral application of estrogenic substances (Marcus et al., 1966). This was seen in only one rat unilaterally injected with Premarin and in none of the penicillin- or bicuculline-treated rats. The EEG tracings of Fig. 1 were typical of most recordings throughout the experiment. Except in the case of bicuculline, clinical seizures were seldom severe enough to prevent quantification of spike and AD activity. Movement artifacts associated with operant performance were uncommon and of small amplitude relative t o that of the epileptic spikes and AD. In no way did the presence of movement
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5
LCC
FIG. 2. Effect of 1%Premarin on operant responding. Relationship between operant response patterns and marked epileptic activity. Each downward deflection of the pen on the bottom tracing represents a bar press. The animal is quite capable of high rates of responding during periods of spike discharge. Only after the development of an afterdischarge is the responding disrupted. artifacts obscure EEG tracings and subsequent analysis during operant sessions (Fig. 2). The effect of repeated intracortical injections of the convulsants is shown in Fig. 3. The variability of response in the penicillin group was t o o great t o observe any consistent trends. The number of subjects exhibiting seizure activity after daily Premarin injections decreased over time. The TAA increased in those subjects with electrographic seizures. In operant behavioral studies, daily injection of Premarin produced a spiking focus with AD in 65% of the cases. Only once did a spiking focus with no AD develop. Penicillin caused spiking with AD 32% of the time, whereas in 30% of the injections a spiking focus without AD was seen. Comparison of the cases of spiking with AD (Fig. 3) showed penicillin to produce 40% fewer AD than Premarin. Thirty
percent of the Premarin and 38% of the penicillin injections resulted in no EEG or behavioral abnormalities. Figure 4 indicates that the act of intracortical injection per se (No Spike Group) did not disrupt the operant responding. The response rate was significantly depressed in penicillintreated rats that developed spiking with no AD (Fig. 4). This epileptic pattern occurred in only 1 Premarin-treated rat. The existence of AD in either drug group disrupted bar-pressing behavior and significantly decreased response rates (Fig. 4). The average severity of clinical seizures for the 2 groups was almost the same (penicillin = 2.7 versus Premarin = 2.3) (Fig. 5). Seizures of even mild clinical severity were generally associated with a marked decrease in overall response rate. Despite EEG and clinical signs of focal
TOTAL AFTERDISCHARGE ACTIVITY
INCIDENCE OF AFTERDISCHARGE
300 1
0 LI
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LOO
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FIG. 3. Effect of chronic intracortical injection of convulsants. Left graph shows the number of animals exhibiting electrographic signs of seizure activity following repeated injections of penicillin or Premarin. The incidence of seizures decreased following repeated Premarin administration, but the penicillin results were too variable to note a trend. Right graph depicts the average amount of AD activity recorded in rats that demonstrated electrographic seizures. The amount of AD activity tended to increase following repeated Premarin administration and decreased after chronic penicillin injections.
FOCAL EPILEPSY AND RAT OPERANT BEHAVIOR P E N I C I L L I 1UO N
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PREMARI N 1%
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7 CONTKOI.
NO
SPIKk.
*
SPLKL 0NI.Y
CONTKOI.
AD
NO SPIKL
SPIKt: ONLY
AD
p 4 11. I J O j
FIG. 4. Influence of penicillin and Premarin-induced discharges and afterdischarges on the operant responding of rats. The N o Spike Group represents subjects in which intracortical injection of an epileptogen failed to elicit spike discharges. In the Spike Only Group, epileptogens induced spike discharges, but no AD. The ordinate is expressed as percent of preinjection control response rates. The penicillin group consisted of 6 animals whereas the Premarin group had 5. N refers to the number of intracortical penicillin or Premarin injections during the 10-day period that resulted in a particular EEG pattern. epileptic activity, several of the animals performed at, or near, their preinjection rates. In these animals spike discharges had no demonstrable effect on responding. Only the advent of an AD disrupted bar-press behavior. An example of such a rat’s performance is shown in
Fig. 2. Note the cessation of responding only during and shortly after an AD. It should be emphasized that this pattern was the exception, not the rule. Spike discharges, with or without AD, tended to decrease responding in most subjects.
Prernariir 1%
P c n l c l l l l l I 100 I . U .
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g2y-f2, “0
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=
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seizure sign
spike
*
= pC.01
8/1/75
FIG. 5. Effect of the severity of clinical seizures on operant response rates. Seizure severity was classified as: No Spike - intracortical injection resulting in no EEG or clinical seizure signs; 1 localized unilateral clonus; 2 - unilateral clonus; 3 - generalized clonus without loss of righting reflex; 4 - generalized clonus or tonic extension with loss of righting reflex.
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DISCUSSION Intracortical injection of either penicillin or Premarin resulted in epileptic activity, but there were differences in the quality of the response. Premarin produced an all-or-none response; 22% of the injections had no effect, whereas 78% demonstrated spiking foci with recurring AD. Penicillin caused a graded epileptic response: 38% of the injections had no effect, 30% produced only spiking, and 32% resulted in spiking and AD. The decreased number of subjects exhibiting seizure activity following repeated Premarin injections was probably due t o fibrotic occlusion of the lumen of the implanted device. Such occlusion was observed at postmortem examination. In the absence of occlusion, the epileptogenic action of Premarin was enhanced over time. This appeared to be due t o a specific action of Premarin since it was not observed in the penicillin or control groups. A possible explanation for the increased sensitivity to Premarin is enhanced enzymatic inhibition. Baxter (1970) noted that estrogens inhibit the activity of glutamic acid decarboxylase,the enzyme responsible for the synthesis of the putative inhibitory neurotransmitter, gamma aminobutyric acid (GABA). The present study confirms the finding of Aquino-Cias et al. (1972) and Rennick et al. (1969) that a regular spiking focus in the absence of AD activity may produce decrements in operant responding. However, it may be that the magnitude of the disruptive influence of the focus on operant performance is related to the particular operant task. Had the subjects in the present study been required to perform on a low-response rate schedule like a Fixed Interval 1 min, the same amount of focal discharge might not have produced the same performance decrement as that seen on the FR 20 schedule (Kelleher and Morse, 1968). The marked difference in the response of individual rats t o the effects of the topical epileptogens is of special interest. The operant performance of some animals was surprisingly resistant to the disruptive influences of the convulsant agents. These animals continued to respond at a normal rate except during the actual occurrence of an AD. Yet other animals’ response rates were greatly decreased even though AD activity comprised less than 3% of
the duration of the operant session. There is no clear explanation for the marked variability at this time. Since idiopathic seizures are not uncommon in rodents, it may be that some of the subjects had a genetically determined increase in seizure susceptibility. Previous studies (Curtis et al., 1972) suggested that penicillin and bicuculline exert their epileptogenic action by the same mechanism, i.e., a competive inhibition of GABA. Recently, several authors (Miller and McLennan, 1974; Shank et al., 1974) have questioned the specificity of bicuculline for GABA receptors. Further, Florio and Longo (1972) observed differential effects of bicuculline upon the EEG following intravenous and topical administration in the rabbit. Intravenous injection of 0.2 mg/kg induced paroxysmal 3-4 Hz biphasic spike-and-wave complexes similar to those seen after parenteral administration of pentylenetetrazol. Topical application of a 0.02% solution to the sensorimotor cortex produced highvoltage monophasic spiking which occasionally terminated in a generalized seizure. If, as Curtis suggests, bicuculline and penicillin act in like fashion, one would predict that intracortical injections would produce similar EEG alterations. The results of the present study are not consistent with this prediction. The differences in onset, intensity, and duration of epileptic activity suggest that the two agents do not work by the same mechanism. In addition, diazepam effectively blocks bicuculline seizures in low doses (Edmonds, 1975) but is not effective in altering penicillin seizures in the rat (Edmonds et al., 1974). Conversely, a new anticonvulsant agent code, SC-13504, blocks penicillin seizures (Edmonds et al., 1974), but not those caused by bicuculline (Edmonds, 1975). The present study suggests that the ability of bilateral cortical applications of Premarin to produce the characteristics 3/sec spike-andwave pattern of petit mal epilepsy may be related more t o the method and site of application than to the intrinsic epileptic qualities of the Premarin solution. Unilateral injections of both Premarin and penicillin typically produce l/sec sharp spikes. The similar time course, intensity, and EEG patterns of epileptiform activity induced by these two agents suggest that they may possess similar mechanisms of action.
FOCAL EPILEPSY AND R A T OPERANT BEHAVIOR ACKNOWLEDGMENTS This investigation was supported in part by funds provided for biological and medical research by the State of Washington Initiative Measure No. 171. The author wishes to thank Karen Phillips and Kurt Schanzenbach for their technical assistance.
REFERENCES Aquino-Cias J, Aneiros-Riba R, Fernandez-Yero F, and Hernandez-Mesa N. Effects of epileptic foci in the visual cortex of the rat on passive avoidance learning. Physiol Behav 8:957-961, 1972. Baxter CF. The nature of gamma amino butyric acid. In: Abel Lajtha (Ed), Handbook of Neurochemistry, Vol 111. Plenum Press, New York, 1970, pp 401-435. Curtis DR, Game CJA, Johnston GAR, McCulloch RM, and Maclachlan RM. The convulsive action of penicillin. Brain Res 43~242-245.1972. Edmonds HL. The effect of anticonvulsants on seizures induced by penicillin, picrotoxin and bicuculline. Proc Soc Neurosci 5:723, 1975. Edmonds HL and Stark LG. Application of substances t o the brain of small animals. Res C o m m u n C h e m Pathol Pharmacol 7 ~ 6 4 1 - 6 4 41974. , Edmonds HL, Stark LG, and Albertson TE. The effect of anticonvulsants on F R performance in the rat. Proc West Pharmacol Soc 8:246-250, 1975. Florio V and Longo VG. Electroencephalographic effects of bicuculline. Physiol Behav 9 ~ 2 8 3 - 2 8 51972. , Kelleher RT and Morse WH. Determinants of the specificity of behavioral effects of drugs. In: Reviews o f Physiology, Biochemistry and Experimental Pharmacology. SpringerVerlag, Berlin, 1968, pp 1-56. Marcus EM, Watson CW, and Goldman PL. Effects of steroids on cerebral electrical activity. Arch Neurol 15:521-532, 1966. Miller J T and McLennan H. The action of
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b i c u c u l line upon acetylcholine-induced excitations of central neurons. Neuropharmacology 13:785-787, 1974. Okada K. Ultrastructure of penicillin-induced epileptic lesion of the cerebral cortex in cats. J Neurophysiol Exp Neurol 30:120, 1971. Rennick M, Perez-Borja C, and Rodin A. Transient mental deficits associated with recurrent prolonged epileptic clouded states. Epilepsia 10:397-405, 1969. Shank RP, Pong SF, Freeman AF, and Graham CT Jr. Bicuculline and picrotoxin as antagonists of gamma aminobutyrate and neuromuscular inhibition in the lobster. Brain Res 72:71-78, 1974. Woodruff ML. Subconvulsive epileptiform discharge and behavioral impairment. Behav Biol 11:431-458, 1974.
SUMMARY Dose-response studies were performed on freely moving rats bearing chronically implanted electrodes. The effects of intracortical injections of penicillin, bicuculline, and conjugated estrogens (PremarinB) were determined on EEG activity and mator performance. Bicuculline in doses of 0.025 and 0.05% regularly produced spike and afterdischarge (AD) patterns of rapid onset (
Raven Press, New York
The Effect of Experimentally Induced Focal Epilepsy on Operant Responding in the Rat Harvey L. Edmonds, Jr College of Pharmacy, Washington State University, Pullman, Washington 991 63 (Received in revised form January 6,1976) INTRODUCTION The effect of generalized epileptic seizures on learning and memory is well-known. However, the effects of interictal discharges on the various aspects of behavior have not been studied extensively. In a recent review, Woodruff (1974) concluded that electrographic spike discharges per se may not be responsible for impairments in learning and performance. He further stated that the presence of behavioral seizures does not necessarily disrupt operant performance. On the other hand, Aquino-Cias (1972) showed that the retention efficiency of rats in a behavioral situation was impaired by “interictal subclinical epileptic activity.” Rennick et al. (1969) observed a diminution in the ability of epileptic patients to perform a complex series of psychomotor tasks during the appearance of. EEG spike volleys. The apparent conflict in these reports emphasizes the need for further study. Since interictal spike discharges commonly occur in cases of focal epilepsy (Woodruff, 1974), focal seizures were experimentally induced in rats in order to quantitate the influence of focal abnormalities on operant behavior. The intracortical injection of epileptogens such as penicillin and conjugated estrogens (PremarinB) is well-suited to this purpose. Small amounts of either substance will produce transient interictal spike discharges with occasional brief afterdischarges (AD) (Marcus et al., 1966, Woodruff, 1974) and minimal cytoarchitectural disruption (Okada, 1971). Thus focal K e y W o r d s : Epilepsy
-
Operant Behavior
-Rat
seizures of short duration and mild intensity can be repeatedly induced at will in the same animals with little permanent neurological impairment. In addition, bicuculline was used as a topical epileptogen. Curtis et al. (1972) proposed that penicillin and bicuculline have the same mechanism of action. If this is so, they should produce similar epileptic responses.
METHODS AND MATERIALS The subjects were adult male SpragueDawley albino rats obtained from the campus breeding facility. The animals were kept in a room with regulated 1 2 hr light-dark cycle and housed in individual suspended cages. The animals were given free access to water and sufficient lab chow to maintain themselves at 85% of their full-fed weight. Each animal was weighed daily. Dose-response studies on the EEG and behavioral effects of intracortical injection of sodium penicillin G (Squibb), bicuculline (Sigma), and Premarin@ Intravenous (Ayerst) were performed to determine doses suitable for subsequent behavioral tests. After each rat was anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and placed in a stereotactic instrument, its scalp and underlying muscles were reflected bilaterally. Stainless steel recording screws were fixed in the skull overlying the frontal sinus and bilaterally 4 mm posterior to bregma, 3 mm lateral to midline. A device for accurate intracortical injection (Edmonds and Stark, 1974) was implanted unilaterally 2 mm posterior to bregma, 3 mm lateral to midline
129
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over the sensorimotor cortex. The recording screws were attached to a connector made from a portion of Cambion integrated circuit socket (#703-3897-01-0316). The device and connector were held in place with dental acrylic cement. Following recovery from surgery, 60 animals were divided into groups of 5 and, after control EEG recordings, were injected intracortically to an approximate depth of 0.8 mm with one of the following solutions: buffered sodium penicillin G 50, 100, or 200 international units (IU) (pH 6.2 in saline); bicuculline 0.002, 0.004, 0.025, or 0.05% at pH 5.0 in citrate buffer; PremarinB Intravenous 0.5, 1.0, or 2.0% at pH 7.2 in commercial diluent; isotonic saline adjusted to pH 5.0 with citrate buffer; Premarin diluent. The volume of each injection was 5 pl. The rats receiving the saline or Premarin diluent solutions were injected daily for 1 0 consecutive days to determine the effect of repeated intracortical injections on EEG activity. Operant studies were carried out in Plexiglas Skinner boxes. Depression of a lever in the box initially resulted in the presentation of a 40-mg food pellet (Noyes). After shaping, two groups of 6 rats each were trained to make 20 lever depressions to obtain one food pellet (FR 20 schedule). This particular schedule was used in order to generate high stable response rates that are sensitive to drug-induced behavioral alteration (Edmonds et al., 1975). The operant session was 25 min in length. After each animal achieved an average weekly response rate of greater than 30 responses per minute, it was implanted in the manner previously discussed. One animal died during surgery so that the penicillin group consisted of 6 subjects, whereas the Premarin group contained 5. These 11 rats were used in all subsequent behavioral studies. After recovery from surgery, the subjects were again required to perform in the operant paradigm. FR 20 response rates were recorded daily for 5 consecutive days and served to establish control response rates to which later rates could be compared. For each of the next 1 0 days, after a 5-min control EEG recording, the rats were injected intracortically with a 5-pl solution containing either 100 IU of penicillin or a 1% solution of Premarin. The control recordings served to insure the absence of EEG abnormalities prior to injection. The animals were then
tested in the 25-min operant session. Operant responses were recorded on Lehigh Valley electromechanical counters. The EEG was monitored on a Tektronix 5103N dual beam oscilloscope and recorded on Model 7 Grass electronencephalograph. The topical epileptogens often produced discrete largeamplitude mono- or biphasic events of 20-50 msec duration as exemplified by the bicuculline trace in Fig. 1. However, these epileptic spikes occasionally became polyphasic and widely varied in duration. At times it was difficult to distinguish between many individual spikes occurring in rapid succession and a single polyspike (e.g., Premarin trace, Fig. 1).Previous experience in this laboratory has shown that this distinction is even more difficult in higher mammals such as the cat or dog. To unify data analysis in all species and to facilitate automated procedures, the term “spike” was defined as: (1) a mono- or polyphasic electrical event with a threshold amplitude between 3 and 50 times base-line EEG; (2) a duration of less than 2 sec; (3) followed by at least 2 sec of shbthreshold amplitude. Except in the unusual case of bicuculline, interictal episodes of less than 2 sec were not typically associated with obvious clinical signs of epileptic activity. The spiking frequency was determined by counting five random 1-min segments throughout each record. An afterdischarge (AD) was defined as a polyphasic event of threshold amplitude, longer than 2 sec in duration and followed by at least 2 sec of relative electrical. quiescence. The number of AD was determined for each session. The total amount of AD activity (TAA) was calculated by multiplying the duration of each AD (to the nearest 1 0 sec) by the number of AD. Clinical seizure severity was quantified by means of an arbitrary 0-4 scale: 0 = no sign of seizure activity; 1 = localized unilateral twitching or clonus; 2 = unilateral twitching or clonus; 3 = generalized twitching or clonus without the loss of the righting reflex; 4 = generalized twitching, clonus, or tonic extension with the loss of the righting reflex. The effect of each of the indices of seizure activity on operant responding was determined by comparing response rates following intracortical injection with preinjection response rates. These rates were normalized by expressing each subject’s daily rate as a percent of the
FOCAL EPILEPSY AND RAT OPERANT BEHAVIOR
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PENICILLIN 100 units
I
/
I
\\
\
BlCUCULLlNE .05 %
I
500pr
Rot 3 13/24/75)
5 sec
FIG. 1. Effect of epileptogenesis on the EEG. Electroencephalographic abnormalities induced following intracortical injection of sodium penicillin G (100 IU), Premarin (l%), and bicuculline HCI (0.05%). average preinjection control value for that animal. Statistical significance was determined by use of the unpaired Student’s f-test. RESULTS Figure 1 demonstrates the effects of intracortical injection of these epileptogens on the EEG. Each of the agents produced focal abnormalities characterized by a spiking focus with brief paroxysmal AD. Bicuculline induced the most intense epileptiform activity. The amplitude of both spikes and AD was substantially larger than that of penicillin or Premarin. This intense activity was also manifested by severe clonic movements associated with each spike. The action of bicuculline was brief and the animal’s behavior so disrupted that it was unsuitable for subsequent operant studies. Doses of penicillin less than 100 IU or Premarin less than 1%infrequently produced focal epileptic activity. Larger doses produced extensive AD activity and marked behavioral impairment. Thus 100 IU of penicillin and 1%Premarin solutions were employed in the operant studies. Neither repeated injections of saline nor the Premarin diluent produced any signs of EEG or behavioral abnormality.
Penicillin and Premarin in these doses resulted in EEG spike activity within 75 sec, and the first AD generally appeared in less than 5 min. Paroxysmal AD activity continued for 60-90 min. Epileptic patterns always subsided within 2 hr after intracortical injection. In contrast, the onset of spiking following bicuculline (0.025 and 0.05%) was less than 10 sec, and the first AD typically appeared within 40 sec. AD activity usually stopped within 20 min; in no case did epileptic patterns persist for longer than 40 min. The frequency of spiking was approximately 1 Hz in both the Premarin and penicillin groups. Figure 1 shows the slow and irregular pattern commonly seen after bilateral application of estrogenic substances (Marcus et al., 1966). This was seen in only one rat unilaterally injected with Premarin and in none of the penicillin- or bicuculline-treated rats. The EEG tracings of Fig. 1 were typical of most recordings throughout the experiment. Except in the case of bicuculline, clinical seizures were seldom severe enough to prevent quantification of spike and AD activity. Movement artifacts associated with operant performance were uncommon and of small amplitude relative t o that of the epileptic spikes and AD. In no way did the presence of movement
H. L. EDMONDS, JR,
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5
LCC
FIG. 2. Effect of 1%Premarin on operant responding. Relationship between operant response patterns and marked epileptic activity. Each downward deflection of the pen on the bottom tracing represents a bar press. The animal is quite capable of high rates of responding during periods of spike discharge. Only after the development of an afterdischarge is the responding disrupted. artifacts obscure EEG tracings and subsequent analysis during operant sessions (Fig. 2). The effect of repeated intracortical injections of the convulsants is shown in Fig. 3. The variability of response in the penicillin group was t o o great t o observe any consistent trends. The number of subjects exhibiting seizure activity after daily Premarin injections decreased over time. The TAA increased in those subjects with electrographic seizures. In operant behavioral studies, daily injection of Premarin produced a spiking focus with AD in 65% of the cases. Only once did a spiking focus with no AD develop. Penicillin caused spiking with AD 32% of the time, whereas in 30% of the injections a spiking focus without AD was seen. Comparison of the cases of spiking with AD (Fig. 3) showed penicillin to produce 40% fewer AD than Premarin. Thirty
percent of the Premarin and 38% of the penicillin injections resulted in no EEG or behavioral abnormalities. Figure 4 indicates that the act of intracortical injection per se (No Spike Group) did not disrupt the operant responding. The response rate was significantly depressed in penicillintreated rats that developed spiking with no AD (Fig. 4). This epileptic pattern occurred in only 1 Premarin-treated rat. The existence of AD in either drug group disrupted bar-pressing behavior and significantly decreased response rates (Fig. 4). The average severity of clinical seizures for the 2 groups was almost the same (penicillin = 2.7 versus Premarin = 2.3) (Fig. 5). Seizures of even mild clinical severity were generally associated with a marked decrease in overall response rate. Despite EEG and clinical signs of focal
TOTAL AFTERDISCHARGE ACTIVITY
INCIDENCE OF AFTERDISCHARGE
300 1
0 LI
50
2 00
25
LOO
0.
I
5
10 d a y s
FIG. 3. Effect of chronic intracortical injection of convulsants. Left graph shows the number of animals exhibiting electrographic signs of seizure activity following repeated injections of penicillin or Premarin. The incidence of seizures decreased following repeated Premarin administration, but the penicillin results were too variable to note a trend. Right graph depicts the average amount of AD activity recorded in rats that demonstrated electrographic seizures. The amount of AD activity tended to increase following repeated Premarin administration and decreased after chronic penicillin injections.
FOCAL EPILEPSY AND RAT OPERANT BEHAVIOR P E N I C I L L I 1UO N
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FIG. 4. Influence of penicillin and Premarin-induced discharges and afterdischarges on the operant responding of rats. The N o Spike Group represents subjects in which intracortical injection of an epileptogen failed to elicit spike discharges. In the Spike Only Group, epileptogens induced spike discharges, but no AD. The ordinate is expressed as percent of preinjection control response rates. The penicillin group consisted of 6 animals whereas the Premarin group had 5. N refers to the number of intracortical penicillin or Premarin injections during the 10-day period that resulted in a particular EEG pattern. epileptic activity, several of the animals performed at, or near, their preinjection rates. In these animals spike discharges had no demonstrable effect on responding. Only the advent of an AD disrupted bar-press behavior. An example of such a rat’s performance is shown in
Fig. 2. Note the cessation of responding only during and shortly after an AD. It should be emphasized that this pattern was the exception, not the rule. Spike discharges, with or without AD, tended to decrease responding in most subjects.
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FIG. 5. Effect of the severity of clinical seizures on operant response rates. Seizure severity was classified as: No Spike - intracortical injection resulting in no EEG or clinical seizure signs; 1 localized unilateral clonus; 2 - unilateral clonus; 3 - generalized clonus without loss of righting reflex; 4 - generalized clonus or tonic extension with loss of righting reflex.
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H. L. EDMONDS, JR.
DISCUSSION Intracortical injection of either penicillin or Premarin resulted in epileptic activity, but there were differences in the quality of the response. Premarin produced an all-or-none response; 22% of the injections had no effect, whereas 78% demonstrated spiking foci with recurring AD. Penicillin caused a graded epileptic response: 38% of the injections had no effect, 30% produced only spiking, and 32% resulted in spiking and AD. The decreased number of subjects exhibiting seizure activity following repeated Premarin injections was probably due t o fibrotic occlusion of the lumen of the implanted device. Such occlusion was observed at postmortem examination. In the absence of occlusion, the epileptogenic action of Premarin was enhanced over time. This appeared to be due t o a specific action of Premarin since it was not observed in the penicillin or control groups. A possible explanation for the increased sensitivity to Premarin is enhanced enzymatic inhibition. Baxter (1970) noted that estrogens inhibit the activity of glutamic acid decarboxylase,the enzyme responsible for the synthesis of the putative inhibitory neurotransmitter, gamma aminobutyric acid (GABA). The present study confirms the finding of Aquino-Cias et al. (1972) and Rennick et al. (1969) that a regular spiking focus in the absence of AD activity may produce decrements in operant responding. However, it may be that the magnitude of the disruptive influence of the focus on operant performance is related to the particular operant task. Had the subjects in the present study been required to perform on a low-response rate schedule like a Fixed Interval 1 min, the same amount of focal discharge might not have produced the same performance decrement as that seen on the FR 20 schedule (Kelleher and Morse, 1968). The marked difference in the response of individual rats t o the effects of the topical epileptogens is of special interest. The operant performance of some animals was surprisingly resistant to the disruptive influences of the convulsant agents. These animals continued to respond at a normal rate except during the actual occurrence of an AD. Yet other animals’ response rates were greatly decreased even though AD activity comprised less than 3% of
the duration of the operant session. There is no clear explanation for the marked variability at this time. Since idiopathic seizures are not uncommon in rodents, it may be that some of the subjects had a genetically determined increase in seizure susceptibility. Previous studies (Curtis et al., 1972) suggested that penicillin and bicuculline exert their epileptogenic action by the same mechanism, i.e., a competive inhibition of GABA. Recently, several authors (Miller and McLennan, 1974; Shank et al., 1974) have questioned the specificity of bicuculline for GABA receptors. Further, Florio and Longo (1972) observed differential effects of bicuculline upon the EEG following intravenous and topical administration in the rabbit. Intravenous injection of 0.2 mg/kg induced paroxysmal 3-4 Hz biphasic spike-and-wave complexes similar to those seen after parenteral administration of pentylenetetrazol. Topical application of a 0.02% solution to the sensorimotor cortex produced highvoltage monophasic spiking which occasionally terminated in a generalized seizure. If, as Curtis suggests, bicuculline and penicillin act in like fashion, one would predict that intracortical injections would produce similar EEG alterations. The results of the present study are not consistent with this prediction. The differences in onset, intensity, and duration of epileptic activity suggest that the two agents do not work by the same mechanism. In addition, diazepam effectively blocks bicuculline seizures in low doses (Edmonds, 1975) but is not effective in altering penicillin seizures in the rat (Edmonds et al., 1974). Conversely, a new anticonvulsant agent code, SC-13504, blocks penicillin seizures (Edmonds et al., 1974), but not those caused by bicuculline (Edmonds, 1975). The present study suggests that the ability of bilateral cortical applications of Premarin to produce the characteristics 3/sec spike-andwave pattern of petit mal epilepsy may be related more t o the method and site of application than to the intrinsic epileptic qualities of the Premarin solution. Unilateral injections of both Premarin and penicillin typically produce l/sec sharp spikes. The similar time course, intensity, and EEG patterns of epileptiform activity induced by these two agents suggest that they may possess similar mechanisms of action.
FOCAL EPILEPSY AND R A T OPERANT BEHAVIOR ACKNOWLEDGMENTS This investigation was supported in part by funds provided for biological and medical research by the State of Washington Initiative Measure No. 171. The author wishes to thank Karen Phillips and Kurt Schanzenbach for their technical assistance.
REFERENCES Aquino-Cias J, Aneiros-Riba R, Fernandez-Yero F, and Hernandez-Mesa N. Effects of epileptic foci in the visual cortex of the rat on passive avoidance learning. Physiol Behav 8:957-961, 1972. Baxter CF. The nature of gamma amino butyric acid. In: Abel Lajtha (Ed), Handbook of Neurochemistry, Vol 111. Plenum Press, New York, 1970, pp 401-435. Curtis DR, Game CJA, Johnston GAR, McCulloch RM, and Maclachlan RM. The convulsive action of penicillin. Brain Res 43~242-245.1972. Edmonds HL. The effect of anticonvulsants on seizures induced by penicillin, picrotoxin and bicuculline. Proc Soc Neurosci 5:723, 1975. Edmonds HL and Stark LG. Application of substances t o the brain of small animals. Res C o m m u n C h e m Pathol Pharmacol 7 ~ 6 4 1 - 6 4 41974. , Edmonds HL, Stark LG, and Albertson TE. The effect of anticonvulsants on F R performance in the rat. Proc West Pharmacol Soc 8:246-250, 1975. Florio V and Longo VG. Electroencephalographic effects of bicuculline. Physiol Behav 9 ~ 2 8 3 - 2 8 51972. , Kelleher RT and Morse WH. Determinants of the specificity of behavioral effects of drugs. In: Reviews o f Physiology, Biochemistry and Experimental Pharmacology. SpringerVerlag, Berlin, 1968, pp 1-56. Marcus EM, Watson CW, and Goldman PL. Effects of steroids on cerebral electrical activity. Arch Neurol 15:521-532, 1966. Miller J T and McLennan H. The action of
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b i c u c u l line upon acetylcholine-induced excitations of central neurons. Neuropharmacology 13:785-787, 1974. Okada K. Ultrastructure of penicillin-induced epileptic lesion of the cerebral cortex in cats. J Neurophysiol Exp Neurol 30:120, 1971. Rennick M, Perez-Borja C, and Rodin A. Transient mental deficits associated with recurrent prolonged epileptic clouded states. Epilepsia 10:397-405, 1969. Shank RP, Pong SF, Freeman AF, and Graham CT Jr. Bicuculline and picrotoxin as antagonists of gamma aminobutyrate and neuromuscular inhibition in the lobster. Brain Res 72:71-78, 1974. Woodruff ML. Subconvulsive epileptiform discharge and behavioral impairment. Behav Biol 11:431-458, 1974.
SUMMARY Dose-response studies were performed on freely moving rats bearing chronically implanted electrodes. The effects of intracortical injections of penicillin, bicuculline, and conjugated estrogens (PremarinB) were determined on EEG activity and mator performance. Bicuculline in doses of 0.025 and 0.05% regularly produced spike and afterdischarge (AD) patterns of rapid onset (
