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Pentylenetetrazol: Inhibitory avoidance behavior, brain seizure activity, and Z3H]lysine incorporation into brain proteins of different mouse strains
BEHAVIORAL BIOLOGY 21, 236--250 (1977)
Pentylenetetrazol: Inhibitory Avoidance Behavior, Brain Seizure Activity, and [3H]Lysine Incorporation into Brain Proteins of Different Mouse Strains I ]3. M I C H A E L IUVONE, 2 CARL
A. BOAST, 3 HARRY E. GRAY,
AND ADRIAN J. D U N N 4
Department of Neuroscience, University of Florida College of Medicine, Gainesville, Florida 32610 The effects of pentylenetetrazol (PTZ,Metrazol) on step-through inhibitory avoidance behavior, brain seizure activity, and [ZH]lysine incorporation into brain proteins were examined in Swiss/ICR and C57B1/6J mice. Immediate posttraining administration of PTZ (60 mg/kg) resulted in significant retention deficits in both strains of mice when tested 24 hr later. In Swiss/ICR mice, subconvulsive doses of PTZ (50 mg/kg) were sufficient to produce amnesia, and there was no correlation between the presence or absence of convulsions and the induction of amnesia. Electrographic recordings from the cerebral cortex and hippocampus indicated that behavorial convulsions always accompanied sustained high-voltage spiking activity in the hippocampus and cortex. Both convulsive and nonconvulsive doses of PTZ inhibited the incorporation of [3H]lysine into brain proteins during the first 10 rain after training and PTZ administration. There was no correlation between the presence or absence of convulsions and the degree of inhibition of [SH]lysine incorporation in the brains of Swiss/ICR mice receiving 50 mg/kg of PTZ. PTZ also inhibited [3H]lysine incorporation into liver proteins. In C57B 1/6J mice administered a convulsive dose of PTZ, the inhibition of [3H]lysine incorporation showed the following regional distribution: cortex > diencephalon + striatum > hippocampus = brainstem. Thus, PTZ both produced amnesia and inhibited the incorporation of [3H]lysine into brain proteins in the absence of gross electrographic abnormalities, but neither of these effects was related to the presence or absence of convulsions. i We are indebted to Dr. S. F. Zornetzer for useful discussion and to Dr. H. D. Rees and B. Bergert for technical assistance. This research was supported by a Grant from the U.S. National Institute of Mental Health (MH25486), PMI and HEG were supported by an NIMH Training Grant to the Center for Neurobiological Sciences of the University of Florida (MHI0320). CAB was supported by an NIAAA Grant (AA00200). AJD is an Alfred P. Sloan Foundation Neurobiology Fellow. 2 Present address: Laboratory for Preclinical Pharmacology, NIMH, St. Elizabeths Hospital, Washington, D.C. 20032. 3 Present address: Department of Psychology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. 4 To whom correspondence should be addressed. 236 Copyright© 1977by AcademicPress, Inc. All rightsof reproductionm any formreserved.
ISSN 0091-6773
PTZ: BEHAVIOR AND PROTEIN SYNTHESIS
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The chemical convulsant pentylenetetrazol (PTZ;Metrazol) has been reported to cause retrograde amnesia (RA) in experimental animals (Pearlman et al., 1961; Weissman, 1967; Palfai and Chillag, 1971). Vinnitsky and Abuladze (1971) reported that, although ether prevented the PTZ-induced convulsions, EEG seizures persisted and animals were amnesic, leading them to suggest that brain seizure activity was necessary for the induction of amnesia. However, Van Buskirk and McGaugh (1974) showed that doses of PTZ that do not cause cortical seizures can induce a retention deficit in a step-through inhibitory avoidance task in Ha/ICR mice. Thus, neither behavioral convulsions nor cortical seizure activity appears to be necessary for the amnestic effect of PTZ. Recent evidence suggests that PTZ is not amnestic in all strains of mice; while subconvulsive doses of PTZ produced a performance deficit in the inhibitory avoidance behavior of Ha/ICR mice, neither subconvulsive nor zonvulsive doses of PTZ caused amnesia in C57B 1/6J mice (Van Buskirk and McGaugh, 1974). Antibiotic inhibitors of protein synthesis can be ~mnestic (Barondes, 1970), and electroconvulsive shock (ECS), which is ~mnestic (McGaugh, 1966), has been shown to inhibit brain protein syn:hesis (Cotman et al., 1971 ; Dunn, 1971). The examination of the biochemcal and electroencephalographic effects of PTZ in susceptible and non~usceptible strains of mice would provide data indicating which chemical md electrical changes were related to the production of amnesia and ~vhich changes were nonspecific effects of the drug. Therefore, we examned the effects of PTZ on inhibitory avoidance behavior, the incorporaion of [~H]lysine into brain protein, and the electroencephalographic tctivity of C57BI/6J and Swiss/ICR strains of mice. MATERIALS AND METHODS
Male Swiss/ICR mice (Flow Laboratories, Dublin, Va.), 20-34 g, male 257B 1/6J mice (Jackson Laboratories, Bar Harbor, Maine), 18-23 g, and nale CD-1 Mice (Caesarean-derived HaM/ICR, Charles River's ~aboratories, Wilmington, Mass.), 35-46 g, were housed seven to a cage n a room with lights on from 7 AM to 7 PM. All experiments were performed between 11 AM and 2 PM. Behavioral procedures. Mice were trained in the single-trial stephrough inhibitory avoidance task of Jarvik and Kopp (1967) as previously [escribed (Boast et al., 1975). The footshock intensity was 300/~A. The ime elapsed between placing the mouse in the outer chamber and its ntry into the inner chamber (initial step-through latency) was recorded. ~ollowing footshock, each mouse was immediately removed from the pparatus and was injected intraperitoneally with either 0.9% saline (3 d/g) or PTZ (Sigma Chemical Co.) in 0.9% saline. The animals were ,bserved for 10 min following the injection, and the occurrence of convulions was noted.
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Twenty-four hours after the training sessions each mouse was replaced in the step-through apparatus. A retention test step-through latency (STL) was recorded for each mouse. Subtraction of the individual initial STL from the retention STL resulted in an STL difference score (ASTL) used for statistical analysis. Animals not stepping into the inner chamber within 300 sec were removed from the apparatus and were assigned a ASTL of 300 sec. Electrophysiological procedures. The effects of PTZ on brain electrical activity were assessed in Swiss/ICR and C57B1/6J mice. Bipolar electrodes, constructed by twisting together two strands of 62.5-/xm nichrome wire (Johnson Matthey Metals, Ltd., London, England), were bilaterally implanted in seven Swiss/ICR mice and four C57B1/6J mice. Standard stereotaxic techniques were used to implant the electrodes. During the surgical procedure the mice were anesthetized with Nembutal (50 mg/kg), and atropine sulfate (0.02 mg/mouse) was administered to help prevent respiratory difficulties. One electrode in each mouse was aimed at the hippocampal formation, 2.5 mm anterior to lambda, 1.6 mm lateral to midline, and 1.8 mm ventral to brain surface, while the other was intended to lie in the cortex dorsal to the hippocampus on the contralateral side (i.e., same coordinates except DV = 1.2 mm ventral to brain surface). Amphenol pins soldered to the electrodes were then inserted into a miniature connecting strip which was held in place with dental cement. A small jeweler's screw inserted in the skull over the nasal sinus served to anchor the head plug assembly and also was used to ground the mouse electrically. Six to ten days after surgery each mouse was connected to a lightweight recording cable and was placed in a Plexiglass recording chamber. The electrical activity was recorded by a Grass Model 7B polygraph. Lowand high-frequency filters were set at 1 and 35 Hz, respectively, and a 60-Hz notch filter was used. Sensitivity settings ranged from 200 to 1500 /xV/cm, depending on the animals. The recordings were generally artifact free. Following a brief recording of normal activity some of the mice in each strain (four Swiss/ICR, two C57B 1/6J) were injected with saline. Electrical activity was then recorded for 10 min. Then PTZ (50 mg/kg) was injected, and recording continued for 10 rain. Additional mice in each strain (three Swiss/ICR, two C57B1/6J) received only PTZ injections. During the recording session the mice were observed, and abnormalities (convulsions, rapid hind-limb extension) were recorded. Following the recording session the mice were anesthetized with ether and were intracardially perfused with 0.9% saline followed by 10% formalin. The brains were removed and placed in formalin; 30-/xm frozen sections through the electrode tracts were stained with cresyl violet to determine electrode placements.
PTZ: BEHAVIOR AND PROTEIN SYNTHESIS
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Biochemical procedures. The mice were injected subcutaneously with ither 30 or 75 /zCi of [4,5-3H]lysine (Amersham-Searle, Arlington teights, Ill.: Batch No. 15, specific activity 13 Ci/mmole). [4,5-3H]lysine vas used in these experiments because, during the short pulse employed, here is virtually no metabolism of this precursor except to protein and ritiated water (Dunn and Bergert, 1976). The subjects were sacrificed by ervical dislocation 10 rain after this lysine injection. Radioactivity in ,rotein and in the free amino acid pool was determined as previously escribed (Rees et al., 1974). The results are expressed as a relative adioactivity (RR) to correct for [3H]lysine uptake (RR = TCA-insoluble isintegrations per minute/TCA-soluble disintegrations per minute). RESULTS
'ehavioral Experiments Pilot data had indicated that a 50-mg/kg dose of PTZ produced convulions in less than half of the mice, whereas 60 mg/kg produced convulions in most animals. Thus, we studied retention in a one-trial inhibitory voidance task in Swiss/ICR mice, uninjected or injected with saline or 50 r 60 mg/kg of PTZ. Initial step-through latencies were not statistically ifferent among any of the groups tested. Kruskal-Wallis nonparametric nalysis of variance indicated a difference in the ASTL scores of the four wiss/ICR groups (H(3) = 23.68, P < 0.001). Subsequent analysis with ~e Mann-Whitney U test indicated that both the 60- (P < 0.02) and the 3-mg/kg dose (P < 0.05) resulted in a significant retention deficit relative ) the saline-injected or uninjected mice. No significant difference was bserved between the ASTL scores of the 50- and the 60-mg/kg PTZ roups. The median ASTL scores for these groups are presented in Fig. 1. ~s indicated, 11 of the 12 subjects in the 60-mg/kg group had behavioral 3nvulsions, while only 2 of 13 subjects were observed to have convulons after 50 mg/kg of PTZ. There was no correlation between the resence or absence of a convulsion and the ASTL in either drug injected -oup. Thus, we conclude that behavioral convulsions are not required for le manifestation of a PTZ-induced retention deficit in inhibitory ¢oidance in Swiss/ICR mice. Two groups of C57B l/6J mice were used: a saline-injected group and a "oup injected with 60 mg/kg of PTZ. Only the convulsive dose of PTZ as used since it had been reported previously that this dose did not apair performance in this strain (Van Buskirk and McGaugh, 1974). ince the first experiment gave equivocal results, the experiment was .,peated, and the data from both experiments are shown in Fig. 2. In the "st experiment, a Mann-Whitney U test did not reveal a significant fference between PTZ- and saline-injected groups (P > 0.05). However, wen of the PTZ-injected animals had ASTLs of 300 sec. Correction for
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IUVONE ET AL.
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FIG. 1. Median step-through latency difference scores (ASTL) for Swiss/ICR mice injected immediately after training with: SAL, saline; 50PTZ, 50 mg/kg of PTZ; 60PTZ, 60 mg/kg of PTZ; or N, uninjected. ASTLs of individual mice are indicated: nonconvulsing mice by circles and convulsing mice by squares. Both PTZ-injected groups were significantly impaired relative to saline and uninjected groups.
these ties using a Wilcoxon midrank test suggested that PTZ did cause significant amnesia (P < 0.03). In the second experiment, the PTZ was significantly amnestic compared to saline (P < 0.01). Thus, we conclude that PTZ (60 mg/kg) did cause significant amnesia in C57B 1/6J mice. Two animals in each experiment did not convulse. There was no correlation between the presence or absence of convulsions and the ASTL.
Electrophysiological Experiments In all animals the hippocampal electrode was in the hippocampus. The cortical electrode, however, was also in the hippocampus in many animals. In those animals with electrodes in both hippocampus and cortex no differences in the type or degree of electrical abnormalities were observed between these regions. Neither were differences noted among the various hippocampal subfields sampled (CA 1, CA3, and dentate gyrus). Thus, the recorded abnormalities will not be discussed with respect to electrode placement. Saline injections had no effect on the electrical activity recorded, did not result in any behavioral abnormalities, and did not detectably alter the response to PTZ in either strain studied. Four of the seven Swiss/ICR mice tested had convulsions during the 10-min observation period following PTZ. Prior to these convulsions some isolated high-voltage spikes
PTZ: BEHAVIOR AND PROTEIN SYNTHESIS
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PTZ
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/ere observed in three of these four mice. These isolated spikes were "equently associated with rapid hind-limb extension and, occasionally, 1ith loss of the righting reflex. Brain seizures were observed during each ehavioral convulsion. In the three nonconvulsing mice only one brief pisode of spiking was observed in one mouse. This consisted of four or ve spikes in 10-15 sec of recorded activity. Three of the four C57B 1/6J mice tested had convulsions. Prior to these onvulsions, epileptiform spikes were noted in each of the three animals. ;rain seizures were observed during the behavioral convulsions. Spike bnormalities were also observed in the mouse which did not exhibit a ~nvulsion. Figure 3 shows examples of the electrographic activity of oth mouse strains.
iochemicaI Experiments To determine the effect of PTZ on the incorporation of lysine into brain rotein during the post-training period, mice were trained in the steplrough avoidance task and were injected with saline or PTZ as in the ehavioral experiments. The subjects were injected with [4,5-3H]lysine at arious times after training and were killed 10 min later. In the first 10 min ~ter training and drug injection both doses of PTZ significantly inhibited H]lysine incorporation into brain protein of Swiss/ICR mice as mea-
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~red by the relative radioactivity (Table 1). The high dose of PTZ (60 g/kg), which produced convulsions in 12 of 15 mice, decreased the RR 20% (P < 0.001), while the lower dose of PTZ (50 mg/kg), which ;sulted in convulsions in only 7 of 18 subjects, decreased the RR by 17% ' < 0.01). The high dose of PTZ significantly reduced (by 19%) the ~corporation of radioactivity in the TCA-insoluble fraction (P < 0.05). he 50-mg/kg dose of PTZ also reduced the radioactivity of this fraction 5%), but this effect was not statistically significant. Fifteen to twentyve and fifty to sixty minutes after training and drug injection, 60 mg/kg of -FZ reduced the RR and acid-insoluble radioactivity (Table 1), but these :fects were not statistically significant. At no time after injection did ther dose of PTZ have any significant effect on the dried acid-soluble tdioactivity (free lysine). To determine whether the inhibition of lysine incorporation was caused y convulsions or brain seizures, we divided the animals which received 3 mg/kg of PTZ into two groups: those that convulsed and those that did ot. Although the brains of subjects which convulsed had slightly more tdioactivity in the dried acid-soluble (free lysine) and the acid-insoluble )rotein) fractions than did the subjects which had not convulsed, neither f these changes was significant (Table 2). There was no difference beveen the RRs of the convulsing and nonconvulsing mice. This indicates lat the inhibition of lysine incorporation was not dependent on the ccurrence of behavioral convulsions or brain seizures. In support of this onclusion is the observation that PTZ (75 mg/kg) inhibited (60%) the incororation of lysine into protein in the livers of CD-1 mice. This liver ffect was in fact larger than the brain effect in the same animals (Table 3). We also examined the effects of PTZ on lysine incorporation into brain roteins of C57B1/6J mice. To determine the sites of action of PTZ, four rain regions were examined. In the first 10 min after training and drug dministration, PTZ (60 mg/kg) significantly inhibited the incorporation of ~dioactivity into the acid-insoluble fraction and the RR of all four brain ~gions (Table 4). The cerebral cortex showed the largest decrease in RR 11%), followed by the diencephalon-striatum (3 I%), with smaller reducons in hippocampus (26%) and brain stem (24%). There was no indicaon of a bimodal distribution in the inhibition of [3H]lysine incorporation. DISCUSSION
ehavioral Experiments The amnestic effects of PTZ observed in this study are in agreement ,ith those previously described (Pearlman et al., 1961; Weissman, 1967; alfai and Chillag, 1971). Palfai and Kurtz (1973) have previously shown lat the PTZ-induced amnesia is time dependent and, thus, consistent ,ith retrograde amnesia rather than a performance deficit. The induction
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f amnesia by 50 mg/kg of PTZ, which produced convulsions in only 2 of 3 mice, confirms the finding of Van Buskirk and McGaugh (1974) that abconvulsive doses of PTZ can produce a retention deficit in a steplrough inhibitory avoidance task in mice. It also agrees with the results f Essman (1968) who found that blocking the PTZ-induced convulsions lith lidocaine did not attenuate the amnesia. Our results indicate that PTZ induces RA in both Swiss/ICR and '.57B1/6J mice, while Van Buskirk and McGaugh (1974) were unable to emonstrate any effect of PTZ on the performance of C57B1/6J mice in ae same task. Careful inspection of our data indicates a bimodal distribu:on in the response of subjects to PTZ. When tested 24 hr after training, 10 '.57B 1/6J mice which received PTZ had perfect retention scores (~STL = 00), while 18 identically treated mice had poor retention scores (ASTL < 63). The physiological or genetic bases of this bimodal distribution are not nown, but the existence of this distribution suggests that the conflict etween the results of our study and those of Van Buskirk and McGaugh 1974) may be the result of sampling a nonhomogeneous population.
'lectrophysiological Experiments In all convulsing subjects the behavioral convulsions were always acompanied by hippocampal and cortical seizures. The onset and offset of ~ese seizures corresponded well with the beginning and end of the ehavioral convulsion (Fig. 3). Similar seizure activity in response to PTZ as been reported previously (Van Buskirk and McGaugh, 1974; Palfai nd Kurtz, 1976). In the Swiss/ICR mice, the occurrence of sustained high-voltage spiking 1 the hippocampus and cortex was always accompanied by behavorial onvulsions. We conclude that cortical and hippocampal seizures do not ccur in PTZ-treated Swiss/ICR mice that do not exhibit behavioral onvulsions. However, this does not exclude the possibility of electrical bnormalities in other brain regions of the nonconvulsing mice. Spiking ctivity occurred in one C57B 1/6J mouse that did not have a convulsion. urther studies with a larger sample are needed to determine whether eizure activity without convulsions is common in C57BI/6J mice.
iochemical Experiments Geiger et al. (1960) reported that PTZ at a convulsive dose caused an cute inhibition of the incorporation of [14C]glucose into protein of perlsed cat brain, suggestive of a decreased protein synthesis. We have )und that PTZ, in the first 10 min following administration, significantly lhibited [3H]lysine incorporation into brain protein of Swiss/ICR, '57B1/6J and CD-1 mice. Of the brain regions sampled, the degree of lhibition of lysine incorporation was greatest in the cortex (cortex > iencephalon + striatum > hippocampus -~ brainstem). This inhibition of
248
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protein synthesis could be related to the small decrease in cerebral RNA content observed following PTZ (Essman, 1968). Since the protein synthesis that appears to be necessary for the formation of long-term memory has been reported to occur within a few minutes after training (Barondes and Cohen, 1968), the rapid onset of protein synthesis inhibition may be important if this is the mechanism by which amnesia is induced by PTZ. An important question is whether the inhibition of lysine incorporation into protein was related to the occurrence of convulsions. No difference was observed in the degree of inhibition of lysine incorporation into brain protein between PTZ-injected Swiss/ICR mice that convulsed and those that did not, suggesting that the inhibition was not dependent upon the convulsions. Since, in Swiss/ICR mice, electrocortical seizure activity was not observed in the nonconvulsing mice, inhibition of incorporation is probably unrelated to the occurrence of generalized electrical abnormalities. This conclusion is further supported by the observation that PTZ resulted in an inhibition of lysine incorporation into protein of the liver, which was even greater than that occurring in the brain. Thus, the effects of PTZ are not confined to electrically excitable tissue, suggesting that the inhibition of lysine incorporation may be related to chemical changes induced by the drug. Gross and Woodbury (1972) noted that PTZ produced a dose-dependent increase in the short-circuit transmembrane current of the toad bladder, which was associated with an increase in the K + flux. If this change in membrane permeability also occurs in mouse tissues, alterations in the intracellular and extracellular concentrations of Na ÷ and K ÷ would be expected to occur. Ribosomal protein is sensitive to changes in the Na ÷ and K ÷ concentrations (Zomzely et al., 1964; Dunn, 1968), which could account for the PTZ-induced decreases in protein synthetic activity. A similar mechanism has been proposed to explain the alterations in protein synthesis caused by ECS (Dunn, 1971). The relationship of ionic imbalances and protein synthesis to apparent memory has been explored by Mark and Watts (1971). They observed that treatment of chicks with the Na ÷, K+-ATPase inhibitor, ouabain, resulted in a retention deficit 24 hr after training. Cycloheximide and ouabain produced no larger impairment of apparent memory than ouabain alone, but the combination of drugs was more effective than cycloheximide alone (Watts and Mark, 1971). These findings suggest the possibility that both ionic and metabolic disturbances may be involved in the apparent memory loss induced by PTZ. Barondes and Cohen (1968) reported that more than 80% inhibition of protein synthesis is required for antibioticinduced amnesia. This is much greater than the inhibition caused by PTZ. However, since Na ÷, K÷-pump inhibitors potentiate the amnestic effects of cycloheximide, the combined ionic and metabolic effects of PTZ may be responsible for the observed retention deficits.
PTZ: BEHAVIOR AND PROTEIN SYNTHESIS
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We intended to test whether PTZ inhibited the incorporation of H]lysine into brain proteins when administered under conditions that •oduce RA, and whether the inhibition of lysine incorporation occurred C57B1/6J mice, a strain of mice reported not to be susceptible to FZ-induced amnesia (Van Buskirk and McGaugh, 1974). Since our data dicate that PTZ can produce amnesia in C57B1/6J mice in the steprough inhibitory avoidance task, it was not possible to compare the ochemical effects of PTZ in strains that were susceptible and resistant to e amnestic effect of PTZ. There was no evidence of a bimodal distribum of the inhibition of cerebral protein synthesis by PTZ in either strain m i c e to parallel the amnestic effects. Our data show that PTZ both hibits the incorporation of [SH]lysine into brain protein and produces anesia in the absence of gross electrographic abnormalities. While it was ~t possible to establish a casual relationship between the inhibition of rebral protein synthesis and RA, the present data provide further evi'nce for such a relationship. REFERENCES rondes, S. H. (1970). Cerebral protein synthesis inhibitors block long term memory. Int. Rev. Neurobiol. 12, 177-205. rondes, S. H., and Cohen. H. D. (1968). Memory impairment after subcutaneous injection of acetoxycycloheximide. Science 160, 556-557. ast, C. A., Zornetzer, S. F.. and Hamrick, M. R. (1975). Electrolytic lesions of various hippocampal subfields in the mouse: Differential effects on short- and long-term memory. Behav. Biol. 14, 85-94. tman, C. W., Banker, G., Zornetzer, S. F., and McGaugh, J. L. (1971). Electroshock effects on brain protein synthesis: Relation to brain seizures and retrograde amnesia. Science 173, 454-456. nn, A. J. (1968). "Protein Synthesis in Rat Brain." Ph.D. Thesis, University of Cambridge. nn, A. (1971). Brain protein synthesis after electroshock. Brain Res. 35, 254-259. nn, A. J., and Bergert, B. J. (I 976). Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions. J. Neurochem. 26, 369-375. sman, W. B. (1968), Retrograde amnesia in seizure-protected mice: Behavioral and biochemical effects of pentylenetetrazol. Physiol. Behav. 3, 549-552. iger, A., Horvath, N., and Kawakita, Y. (1960). The incorporation of 14C derived from glucose into the proteins of the brain cortex, at rest and during activity. J. Neurochem. 5, 311-322. )ss, G. J., and Woodbury, D. M. (1972). Effects of pentylenetetrazol on ion transport in the isolated toad bladder. J. Pharmacol. Exp. Ther. 181, 257-272. vik, M. E., and Kopp, R. (1967). An improved 1-trial passive avoidance learning situation. Psychol. Rep. 21, 221-224. rk, R. F., and Watts, M. E. (1971). Drug inhibition of memory formation in chickens. I. Long-term memory. Proc. Roy. Soc. B 178, 439-454. Gaugh, J. L. (1966). Time-dependent processes in memory storage. Science 153, 13511358. fai, T., and Chillag. D. (1971). Time dependent memory deficits produced by pen-
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tylenetetrazol (Metrazol): The effects of reinforcement magnitude. Physiol. Behav. 7, 439-442. Palfai, T., and Kurtz, P. (1973). Time-dependent effects of Metrazol on memory. Pharmacol. Biochem. Behav. 1, 55-59. Palfai, T., and Kurtz, P. (1976). Dose-related effects of Metrazol on retention and EEG. Pharmacol. Biochem. Behav. 4, 123-127. Pearlman, C. A., Sharpless, S. K., and Jarvik, M. E. (1961). Retrograde amnesia produced by anesthetic and convulsive agents. J. Comp. Physiol. Psychol. 54, 109-112. Rees, H. D., Brogan, L. L., Entingh, D. J., Dunn, A. J., Shinkman, P. G., DamstraEntingh, T., Wilson, J. E., and Glassman, E. (1974). Effect of sensory stimulation on the uptake and incorporation of radioactive lysine into protein of mouse brain and liver. Brain Res. 68, 143-156. Van Buskirk, R., and McGaugh, J. L. (1974). Pentylenetetrazol-induced retrograde amnesia and brain seizures in mice. Psychopharmacologia 40, 77-90. Vinnitsky, I. M., and Abuladze, G. V. (1971). Retrogradnaya amneziya, vyzyvaemaya korazolom, na fone deistviya narkozo. Zh. Vyssh. Nerv. Deyatel. im. I. P. Pavlova 21, 572-575. Watts, R. F., and Mark, M. E. (1971). Drug inhibition of memory formation in chickens. II. Short-term memory. Proc. Roy. Soc. B 178, 455-464. Weissman, A. (1967). Drugs and retrograde amnesia. Int. Rev. Neurobiol. 10, 167-198. Zomzely, C. E., Roberts, S., and Rapaport, D. (1964). Characteristics of amino acid incorporation into proteins of microsomal and ribosomal preparations of rat cerebral cortex. J. Neurochem. 11, 567-582.
Pentylenetetrazol: Inhibitory Avoidance Behavior, Brain Seizure Activity, and [3H]Lysine Incorporation into Brain Proteins of Different Mouse Strains I ]3. M I C H A E L IUVONE, 2 CARL
A. BOAST, 3 HARRY E. GRAY,
AND ADRIAN J. D U N N 4
Department of Neuroscience, University of Florida College of Medicine, Gainesville, Florida 32610 The effects of pentylenetetrazol (PTZ,Metrazol) on step-through inhibitory avoidance behavior, brain seizure activity, and [ZH]lysine incorporation into brain proteins were examined in Swiss/ICR and C57B1/6J mice. Immediate posttraining administration of PTZ (60 mg/kg) resulted in significant retention deficits in both strains of mice when tested 24 hr later. In Swiss/ICR mice, subconvulsive doses of PTZ (50 mg/kg) were sufficient to produce amnesia, and there was no correlation between the presence or absence of convulsions and the induction of amnesia. Electrographic recordings from the cerebral cortex and hippocampus indicated that behavorial convulsions always accompanied sustained high-voltage spiking activity in the hippocampus and cortex. Both convulsive and nonconvulsive doses of PTZ inhibited the incorporation of [3H]lysine into brain proteins during the first 10 rain after training and PTZ administration. There was no correlation between the presence or absence of convulsions and the degree of inhibition of [SH]lysine incorporation in the brains of Swiss/ICR mice receiving 50 mg/kg of PTZ. PTZ also inhibited [3H]lysine incorporation into liver proteins. In C57B 1/6J mice administered a convulsive dose of PTZ, the inhibition of [3H]lysine incorporation showed the following regional distribution: cortex > diencephalon + striatum > hippocampus = brainstem. Thus, PTZ both produced amnesia and inhibited the incorporation of [3H]lysine into brain proteins in the absence of gross electrographic abnormalities, but neither of these effects was related to the presence or absence of convulsions. i We are indebted to Dr. S. F. Zornetzer for useful discussion and to Dr. H. D. Rees and B. Bergert for technical assistance. This research was supported by a Grant from the U.S. National Institute of Mental Health (MH25486), PMI and HEG were supported by an NIMH Training Grant to the Center for Neurobiological Sciences of the University of Florida (MHI0320). CAB was supported by an NIAAA Grant (AA00200). AJD is an Alfred P. Sloan Foundation Neurobiology Fellow. 2 Present address: Laboratory for Preclinical Pharmacology, NIMH, St. Elizabeths Hospital, Washington, D.C. 20032. 3 Present address: Department of Psychology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. 4 To whom correspondence should be addressed. 236 Copyright© 1977by AcademicPress, Inc. All rightsof reproductionm any formreserved.
ISSN 0091-6773
PTZ: BEHAVIOR AND PROTEIN SYNTHESIS
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The chemical convulsant pentylenetetrazol (PTZ;Metrazol) has been reported to cause retrograde amnesia (RA) in experimental animals (Pearlman et al., 1961; Weissman, 1967; Palfai and Chillag, 1971). Vinnitsky and Abuladze (1971) reported that, although ether prevented the PTZ-induced convulsions, EEG seizures persisted and animals were amnesic, leading them to suggest that brain seizure activity was necessary for the induction of amnesia. However, Van Buskirk and McGaugh (1974) showed that doses of PTZ that do not cause cortical seizures can induce a retention deficit in a step-through inhibitory avoidance task in Ha/ICR mice. Thus, neither behavioral convulsions nor cortical seizure activity appears to be necessary for the amnestic effect of PTZ. Recent evidence suggests that PTZ is not amnestic in all strains of mice; while subconvulsive doses of PTZ produced a performance deficit in the inhibitory avoidance behavior of Ha/ICR mice, neither subconvulsive nor zonvulsive doses of PTZ caused amnesia in C57B 1/6J mice (Van Buskirk and McGaugh, 1974). Antibiotic inhibitors of protein synthesis can be ~mnestic (Barondes, 1970), and electroconvulsive shock (ECS), which is ~mnestic (McGaugh, 1966), has been shown to inhibit brain protein syn:hesis (Cotman et al., 1971 ; Dunn, 1971). The examination of the biochemcal and electroencephalographic effects of PTZ in susceptible and non~usceptible strains of mice would provide data indicating which chemical md electrical changes were related to the production of amnesia and ~vhich changes were nonspecific effects of the drug. Therefore, we examned the effects of PTZ on inhibitory avoidance behavior, the incorporaion of [~H]lysine into brain protein, and the electroencephalographic tctivity of C57BI/6J and Swiss/ICR strains of mice. MATERIALS AND METHODS
Male Swiss/ICR mice (Flow Laboratories, Dublin, Va.), 20-34 g, male 257B 1/6J mice (Jackson Laboratories, Bar Harbor, Maine), 18-23 g, and nale CD-1 Mice (Caesarean-derived HaM/ICR, Charles River's ~aboratories, Wilmington, Mass.), 35-46 g, were housed seven to a cage n a room with lights on from 7 AM to 7 PM. All experiments were performed between 11 AM and 2 PM. Behavioral procedures. Mice were trained in the single-trial stephrough inhibitory avoidance task of Jarvik and Kopp (1967) as previously [escribed (Boast et al., 1975). The footshock intensity was 300/~A. The ime elapsed between placing the mouse in the outer chamber and its ntry into the inner chamber (initial step-through latency) was recorded. ~ollowing footshock, each mouse was immediately removed from the pparatus and was injected intraperitoneally with either 0.9% saline (3 d/g) or PTZ (Sigma Chemical Co.) in 0.9% saline. The animals were ,bserved for 10 min following the injection, and the occurrence of convulions was noted.
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Twenty-four hours after the training sessions each mouse was replaced in the step-through apparatus. A retention test step-through latency (STL) was recorded for each mouse. Subtraction of the individual initial STL from the retention STL resulted in an STL difference score (ASTL) used for statistical analysis. Animals not stepping into the inner chamber within 300 sec were removed from the apparatus and were assigned a ASTL of 300 sec. Electrophysiological procedures. The effects of PTZ on brain electrical activity were assessed in Swiss/ICR and C57B1/6J mice. Bipolar electrodes, constructed by twisting together two strands of 62.5-/xm nichrome wire (Johnson Matthey Metals, Ltd., London, England), were bilaterally implanted in seven Swiss/ICR mice and four C57B1/6J mice. Standard stereotaxic techniques were used to implant the electrodes. During the surgical procedure the mice were anesthetized with Nembutal (50 mg/kg), and atropine sulfate (0.02 mg/mouse) was administered to help prevent respiratory difficulties. One electrode in each mouse was aimed at the hippocampal formation, 2.5 mm anterior to lambda, 1.6 mm lateral to midline, and 1.8 mm ventral to brain surface, while the other was intended to lie in the cortex dorsal to the hippocampus on the contralateral side (i.e., same coordinates except DV = 1.2 mm ventral to brain surface). Amphenol pins soldered to the electrodes were then inserted into a miniature connecting strip which was held in place with dental cement. A small jeweler's screw inserted in the skull over the nasal sinus served to anchor the head plug assembly and also was used to ground the mouse electrically. Six to ten days after surgery each mouse was connected to a lightweight recording cable and was placed in a Plexiglass recording chamber. The electrical activity was recorded by a Grass Model 7B polygraph. Lowand high-frequency filters were set at 1 and 35 Hz, respectively, and a 60-Hz notch filter was used. Sensitivity settings ranged from 200 to 1500 /xV/cm, depending on the animals. The recordings were generally artifact free. Following a brief recording of normal activity some of the mice in each strain (four Swiss/ICR, two C57B 1/6J) were injected with saline. Electrical activity was then recorded for 10 min. Then PTZ (50 mg/kg) was injected, and recording continued for 10 rain. Additional mice in each strain (three Swiss/ICR, two C57B1/6J) received only PTZ injections. During the recording session the mice were observed, and abnormalities (convulsions, rapid hind-limb extension) were recorded. Following the recording session the mice were anesthetized with ether and were intracardially perfused with 0.9% saline followed by 10% formalin. The brains were removed and placed in formalin; 30-/xm frozen sections through the electrode tracts were stained with cresyl violet to determine electrode placements.
PTZ: BEHAVIOR AND PROTEIN SYNTHESIS
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Biochemical procedures. The mice were injected subcutaneously with ither 30 or 75 /zCi of [4,5-3H]lysine (Amersham-Searle, Arlington teights, Ill.: Batch No. 15, specific activity 13 Ci/mmole). [4,5-3H]lysine vas used in these experiments because, during the short pulse employed, here is virtually no metabolism of this precursor except to protein and ritiated water (Dunn and Bergert, 1976). The subjects were sacrificed by ervical dislocation 10 rain after this lysine injection. Radioactivity in ,rotein and in the free amino acid pool was determined as previously escribed (Rees et al., 1974). The results are expressed as a relative adioactivity (RR) to correct for [3H]lysine uptake (RR = TCA-insoluble isintegrations per minute/TCA-soluble disintegrations per minute). RESULTS
'ehavioral Experiments Pilot data had indicated that a 50-mg/kg dose of PTZ produced convulions in less than half of the mice, whereas 60 mg/kg produced convulions in most animals. Thus, we studied retention in a one-trial inhibitory voidance task in Swiss/ICR mice, uninjected or injected with saline or 50 r 60 mg/kg of PTZ. Initial step-through latencies were not statistically ifferent among any of the groups tested. Kruskal-Wallis nonparametric nalysis of variance indicated a difference in the ASTL scores of the four wiss/ICR groups (H(3) = 23.68, P < 0.001). Subsequent analysis with ~e Mann-Whitney U test indicated that both the 60- (P < 0.02) and the 3-mg/kg dose (P < 0.05) resulted in a significant retention deficit relative ) the saline-injected or uninjected mice. No significant difference was bserved between the ASTL scores of the 50- and the 60-mg/kg PTZ roups. The median ASTL scores for these groups are presented in Fig. 1. ~s indicated, 11 of the 12 subjects in the 60-mg/kg group had behavioral 3nvulsions, while only 2 of 13 subjects were observed to have convulons after 50 mg/kg of PTZ. There was no correlation between the resence or absence of a convulsion and the ASTL in either drug injected -oup. Thus, we conclude that behavioral convulsions are not required for le manifestation of a PTZ-induced retention deficit in inhibitory ¢oidance in Swiss/ICR mice. Two groups of C57B l/6J mice were used: a saline-injected group and a "oup injected with 60 mg/kg of PTZ. Only the convulsive dose of PTZ as used since it had been reported previously that this dose did not apair performance in this strain (Van Buskirk and McGaugh, 1974). ince the first experiment gave equivocal results, the experiment was .,peated, and the data from both experiments are shown in Fig. 2. In the "st experiment, a Mann-Whitney U test did not reveal a significant fference between PTZ- and saline-injected groups (P > 0.05). However, wen of the PTZ-injected animals had ASTLs of 300 sec. Correction for
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IUVONE ET AL.
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FIG. 1. Median step-through latency difference scores (ASTL) for Swiss/ICR mice injected immediately after training with: SAL, saline; 50PTZ, 50 mg/kg of PTZ; 60PTZ, 60 mg/kg of PTZ; or N, uninjected. ASTLs of individual mice are indicated: nonconvulsing mice by circles and convulsing mice by squares. Both PTZ-injected groups were significantly impaired relative to saline and uninjected groups.
these ties using a Wilcoxon midrank test suggested that PTZ did cause significant amnesia (P < 0.03). In the second experiment, the PTZ was significantly amnestic compared to saline (P < 0.01). Thus, we conclude that PTZ (60 mg/kg) did cause significant amnesia in C57B 1/6J mice. Two animals in each experiment did not convulse. There was no correlation between the presence or absence of convulsions and the ASTL.
Electrophysiological Experiments In all animals the hippocampal electrode was in the hippocampus. The cortical electrode, however, was also in the hippocampus in many animals. In those animals with electrodes in both hippocampus and cortex no differences in the type or degree of electrical abnormalities were observed between these regions. Neither were differences noted among the various hippocampal subfields sampled (CA 1, CA3, and dentate gyrus). Thus, the recorded abnormalities will not be discussed with respect to electrode placement. Saline injections had no effect on the electrical activity recorded, did not result in any behavioral abnormalities, and did not detectably alter the response to PTZ in either strain studied. Four of the seven Swiss/ICR mice tested had convulsions during the 10-min observation period following PTZ. Prior to these convulsions some isolated high-voltage spikes
PTZ: BEHAVIOR AND PROTEIN SYNTHESIS
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PTZ
SAL
PTZ
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/ere observed in three of these four mice. These isolated spikes were "equently associated with rapid hind-limb extension and, occasionally, 1ith loss of the righting reflex. Brain seizures were observed during each ehavioral convulsion. In the three nonconvulsing mice only one brief pisode of spiking was observed in one mouse. This consisted of four or ve spikes in 10-15 sec of recorded activity. Three of the four C57B 1/6J mice tested had convulsions. Prior to these onvulsions, epileptiform spikes were noted in each of the three animals. ;rain seizures were observed during the behavioral convulsions. Spike bnormalities were also observed in the mouse which did not exhibit a ~nvulsion. Figure 3 shows examples of the electrographic activity of oth mouse strains.
iochemicaI Experiments To determine the effect of PTZ on the incorporation of lysine into brain rotein during the post-training period, mice were trained in the steplrough avoidance task and were injected with saline or PTZ as in the ehavioral experiments. The subjects were injected with [4,5-3H]lysine at arious times after training and were killed 10 min later. In the first 10 min ~ter training and drug injection both doses of PTZ significantly inhibited H]lysine incorporation into brain protein of Swiss/ICR mice as mea-
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~red by the relative radioactivity (Table 1). The high dose of PTZ (60 g/kg), which produced convulsions in 12 of 15 mice, decreased the RR 20% (P < 0.001), while the lower dose of PTZ (50 mg/kg), which ;sulted in convulsions in only 7 of 18 subjects, decreased the RR by 17% ' < 0.01). The high dose of PTZ significantly reduced (by 19%) the ~corporation of radioactivity in the TCA-insoluble fraction (P < 0.05). he 50-mg/kg dose of PTZ also reduced the radioactivity of this fraction 5%), but this effect was not statistically significant. Fifteen to twentyve and fifty to sixty minutes after training and drug injection, 60 mg/kg of -FZ reduced the RR and acid-insoluble radioactivity (Table 1), but these :fects were not statistically significant. At no time after injection did ther dose of PTZ have any significant effect on the dried acid-soluble tdioactivity (free lysine). To determine whether the inhibition of lysine incorporation was caused y convulsions or brain seizures, we divided the animals which received 3 mg/kg of PTZ into two groups: those that convulsed and those that did ot. Although the brains of subjects which convulsed had slightly more tdioactivity in the dried acid-soluble (free lysine) and the acid-insoluble )rotein) fractions than did the subjects which had not convulsed, neither f these changes was significant (Table 2). There was no difference beveen the RRs of the convulsing and nonconvulsing mice. This indicates lat the inhibition of lysine incorporation was not dependent on the ccurrence of behavioral convulsions or brain seizures. In support of this onclusion is the observation that PTZ (75 mg/kg) inhibited (60%) the incororation of lysine into protein in the livers of CD-1 mice. This liver ffect was in fact larger than the brain effect in the same animals (Table 3). We also examined the effects of PTZ on lysine incorporation into brain roteins of C57B1/6J mice. To determine the sites of action of PTZ, four rain regions were examined. In the first 10 min after training and drug dministration, PTZ (60 mg/kg) significantly inhibited the incorporation of ~dioactivity into the acid-insoluble fraction and the RR of all four brain ~gions (Table 4). The cerebral cortex showed the largest decrease in RR 11%), followed by the diencephalon-striatum (3 I%), with smaller reducons in hippocampus (26%) and brain stem (24%). There was no indicaon of a bimodal distribution in the inhibition of [3H]lysine incorporation. DISCUSSION
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f amnesia by 50 mg/kg of PTZ, which produced convulsions in only 2 of 3 mice, confirms the finding of Van Buskirk and McGaugh (1974) that abconvulsive doses of PTZ can produce a retention deficit in a steplrough inhibitory avoidance task in mice. It also agrees with the results f Essman (1968) who found that blocking the PTZ-induced convulsions lith lidocaine did not attenuate the amnesia. Our results indicate that PTZ induces RA in both Swiss/ICR and '.57B1/6J mice, while Van Buskirk and McGaugh (1974) were unable to emonstrate any effect of PTZ on the performance of C57B1/6J mice in ae same task. Careful inspection of our data indicates a bimodal distribu:on in the response of subjects to PTZ. When tested 24 hr after training, 10 '.57B 1/6J mice which received PTZ had perfect retention scores (~STL = 00), while 18 identically treated mice had poor retention scores (ASTL < 63). The physiological or genetic bases of this bimodal distribution are not nown, but the existence of this distribution suggests that the conflict etween the results of our study and those of Van Buskirk and McGaugh 1974) may be the result of sampling a nonhomogeneous population.
'lectrophysiological Experiments In all convulsing subjects the behavioral convulsions were always acompanied by hippocampal and cortical seizures. The onset and offset of ~ese seizures corresponded well with the beginning and end of the ehavioral convulsion (Fig. 3). Similar seizure activity in response to PTZ as been reported previously (Van Buskirk and McGaugh, 1974; Palfai nd Kurtz, 1976). In the Swiss/ICR mice, the occurrence of sustained high-voltage spiking 1 the hippocampus and cortex was always accompanied by behavorial onvulsions. We conclude that cortical and hippocampal seizures do not ccur in PTZ-treated Swiss/ICR mice that do not exhibit behavioral onvulsions. However, this does not exclude the possibility of electrical bnormalities in other brain regions of the nonconvulsing mice. Spiking ctivity occurred in one C57B 1/6J mouse that did not have a convulsion. urther studies with a larger sample are needed to determine whether eizure activity without convulsions is common in C57BI/6J mice.
iochemical Experiments Geiger et al. (1960) reported that PTZ at a convulsive dose caused an cute inhibition of the incorporation of [14C]glucose into protein of perlsed cat brain, suggestive of a decreased protein synthesis. We have )und that PTZ, in the first 10 min following administration, significantly lhibited [3H]lysine incorporation into brain protein of Swiss/ICR, '57B1/6J and CD-1 mice. Of the brain regions sampled, the degree of lhibition of lysine incorporation was greatest in the cortex (cortex > iencephalon + striatum > hippocampus -~ brainstem). This inhibition of
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protein synthesis could be related to the small decrease in cerebral RNA content observed following PTZ (Essman, 1968). Since the protein synthesis that appears to be necessary for the formation of long-term memory has been reported to occur within a few minutes after training (Barondes and Cohen, 1968), the rapid onset of protein synthesis inhibition may be important if this is the mechanism by which amnesia is induced by PTZ. An important question is whether the inhibition of lysine incorporation into protein was related to the occurrence of convulsions. No difference was observed in the degree of inhibition of lysine incorporation into brain protein between PTZ-injected Swiss/ICR mice that convulsed and those that did not, suggesting that the inhibition was not dependent upon the convulsions. Since, in Swiss/ICR mice, electrocortical seizure activity was not observed in the nonconvulsing mice, inhibition of incorporation is probably unrelated to the occurrence of generalized electrical abnormalities. This conclusion is further supported by the observation that PTZ resulted in an inhibition of lysine incorporation into protein of the liver, which was even greater than that occurring in the brain. Thus, the effects of PTZ are not confined to electrically excitable tissue, suggesting that the inhibition of lysine incorporation may be related to chemical changes induced by the drug. Gross and Woodbury (1972) noted that PTZ produced a dose-dependent increase in the short-circuit transmembrane current of the toad bladder, which was associated with an increase in the K + flux. If this change in membrane permeability also occurs in mouse tissues, alterations in the intracellular and extracellular concentrations of Na ÷ and K ÷ would be expected to occur. Ribosomal protein is sensitive to changes in the Na ÷ and K ÷ concentrations (Zomzely et al., 1964; Dunn, 1968), which could account for the PTZ-induced decreases in protein synthetic activity. A similar mechanism has been proposed to explain the alterations in protein synthesis caused by ECS (Dunn, 1971). The relationship of ionic imbalances and protein synthesis to apparent memory has been explored by Mark and Watts (1971). They observed that treatment of chicks with the Na ÷, K+-ATPase inhibitor, ouabain, resulted in a retention deficit 24 hr after training. Cycloheximide and ouabain produced no larger impairment of apparent memory than ouabain alone, but the combination of drugs was more effective than cycloheximide alone (Watts and Mark, 1971). These findings suggest the possibility that both ionic and metabolic disturbances may be involved in the apparent memory loss induced by PTZ. Barondes and Cohen (1968) reported that more than 80% inhibition of protein synthesis is required for antibioticinduced amnesia. This is much greater than the inhibition caused by PTZ. However, since Na ÷, K÷-pump inhibitors potentiate the amnestic effects of cycloheximide, the combined ionic and metabolic effects of PTZ may be responsible for the observed retention deficits.
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We intended to test whether PTZ inhibited the incorporation of H]lysine into brain proteins when administered under conditions that •oduce RA, and whether the inhibition of lysine incorporation occurred C57B1/6J mice, a strain of mice reported not to be susceptible to FZ-induced amnesia (Van Buskirk and McGaugh, 1974). Since our data dicate that PTZ can produce amnesia in C57B1/6J mice in the steprough inhibitory avoidance task, it was not possible to compare the ochemical effects of PTZ in strains that were susceptible and resistant to e amnestic effect of PTZ. There was no evidence of a bimodal distribum of the inhibition of cerebral protein synthesis by PTZ in either strain m i c e to parallel the amnestic effects. Our data show that PTZ both hibits the incorporation of [SH]lysine into brain protein and produces anesia in the absence of gross electrographic abnormalities. While it was ~t possible to establish a casual relationship between the inhibition of rebral protein synthesis and RA, the present data provide further evi'nce for such a relationship. REFERENCES rondes, S. H. (1970). Cerebral protein synthesis inhibitors block long term memory. Int. Rev. Neurobiol. 12, 177-205. rondes, S. H., and Cohen. H. D. (1968). Memory impairment after subcutaneous injection of acetoxycycloheximide. Science 160, 556-557. ast, C. A., Zornetzer, S. F.. and Hamrick, M. R. (1975). Electrolytic lesions of various hippocampal subfields in the mouse: Differential effects on short- and long-term memory. Behav. Biol. 14, 85-94. tman, C. W., Banker, G., Zornetzer, S. F., and McGaugh, J. L. (1971). Electroshock effects on brain protein synthesis: Relation to brain seizures and retrograde amnesia. Science 173, 454-456. nn, A. J. (1968). "Protein Synthesis in Rat Brain." Ph.D. Thesis, University of Cambridge. nn, A. (1971). Brain protein synthesis after electroshock. Brain Res. 35, 254-259. nn, A. J., and Bergert, B. J. (I 976). Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions. J. Neurochem. 26, 369-375. sman, W. B. (1968), Retrograde amnesia in seizure-protected mice: Behavioral and biochemical effects of pentylenetetrazol. Physiol. Behav. 3, 549-552. iger, A., Horvath, N., and Kawakita, Y. (1960). The incorporation of 14C derived from glucose into the proteins of the brain cortex, at rest and during activity. J. Neurochem. 5, 311-322. )ss, G. J., and Woodbury, D. M. (1972). Effects of pentylenetetrazol on ion transport in the isolated toad bladder. J. Pharmacol. Exp. Ther. 181, 257-272. vik, M. E., and Kopp, R. (1967). An improved 1-trial passive avoidance learning situation. Psychol. Rep. 21, 221-224. rk, R. F., and Watts, M. E. (1971). Drug inhibition of memory formation in chickens. I. Long-term memory. Proc. Roy. Soc. B 178, 439-454. Gaugh, J. L. (1966). Time-dependent processes in memory storage. Science 153, 13511358. fai, T., and Chillag. D. (1971). Time dependent memory deficits produced by pen-
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tylenetetrazol (Metrazol): The effects of reinforcement magnitude. Physiol. Behav. 7, 439-442. Palfai, T., and Kurtz, P. (1973). Time-dependent effects of Metrazol on memory. Pharmacol. Biochem. Behav. 1, 55-59. Palfai, T., and Kurtz, P. (1976). Dose-related effects of Metrazol on retention and EEG. Pharmacol. Biochem. Behav. 4, 123-127. Pearlman, C. A., Sharpless, S. K., and Jarvik, M. E. (1961). Retrograde amnesia produced by anesthetic and convulsive agents. J. Comp. Physiol. Psychol. 54, 109-112. Rees, H. D., Brogan, L. L., Entingh, D. J., Dunn, A. J., Shinkman, P. G., DamstraEntingh, T., Wilson, J. E., and Glassman, E. (1974). Effect of sensory stimulation on the uptake and incorporation of radioactive lysine into protein of mouse brain and liver. Brain Res. 68, 143-156. Van Buskirk, R., and McGaugh, J. L. (1974). Pentylenetetrazol-induced retrograde amnesia and brain seizures in mice. Psychopharmacologia 40, 77-90. Vinnitsky, I. M., and Abuladze, G. V. (1971). Retrogradnaya amneziya, vyzyvaemaya korazolom, na fone deistviya narkozo. Zh. Vyssh. Nerv. Deyatel. im. I. P. Pavlova 21, 572-575. Watts, R. F., and Mark, M. E. (1971). Drug inhibition of memory formation in chickens. II. Short-term memory. Proc. Roy. Soc. B 178, 455-464. Weissman, A. (1967). Drugs and retrograde amnesia. Int. Rev. Neurobiol. 10, 167-198. Zomzely, C. E., Roberts, S., and Rapaport, D. (1964). Characteristics of amino acid incorporation into proteins of microsomal and ribosomal preparations of rat cerebral cortex. J. Neurochem. 11, 567-582.