TYROSINE HYDROXYLASE INHIBITION BY CYCLOHEXIMIDE AND ANISOMYCIN IS NOT RESPONSIBLE FOR THEIR AMNESIC EFFECT

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1 Brain Research, 82 (1974) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands TYROSINE HYDROXYLASE INHIBITION BY CYCLOHEXIMIDE AND ANISOMYCIN IS NOT RESPONSIBLE FOR THEIR AMNESIC EFFECT LARRY R. SQUIRE, RONALD KUCZENSKI AND SAMUEL H. BARONDES Department of Psychiatry, University of California, San Diego School of Medicine, La Jolla, Calif and Veterans Administration Hospital, San Diego, Calif (U.S.A.) (Accepted August 24th, 1974) SUMMARY It has been reported that the administration of cycloheximide, a protein synthesis inhibitor, depresses brain tyrosine hydroxylase activity as measured in vitro. This finding raised the possibility that the amnesic effect of this drug could be due to reduction of norepinephrine synthesis rather than to inhibition of protein synthesis required for long-term memory. We have found that (1) amnesic doses of cycloheximide and anisomycin, two protein synthesis inhibitors, produced measurable depression of tyrosine hydroxylase activity although much of the depression may be due to isotope dilution rather than true inhibition, and (2) a-methyl-p-tyrosine a competitive inhibitor of tyrosine hydroxylase, in doses which depressed tyrosine hydroxylase activity as much as or more than either cycloheximide or anisomycin, did not affect memory. Therefore the effect of protein synthesis inhibitors on brain tyrosine hydroxylase activity is not sufficient to explain their amnesic effect. INTRODUCTION Three classes of protein synthesis inhibitors represented by puromycin, cycloheximide and anisomycin impair long-term memory of discrimination learning in mice if administered before training 3,4,19,21. These findings suggest that cerebral protein synthesis is required for the formation of long-term memory. The possibility that side effects of these drugs could be responsible for their amnesic effects has been repeatedly considered. For example puromycin produces occult seizures in mice 6 but cycloheximide produces amnesia without exerting this effect on brain electrical activity. Also both cycloheximide and anisomycin affect locomotor activity 16,17,2~,22, but the effects on activity can be dissociated from the effects on memory 17,21. Recently, Flexner et al. reported that brain tyrosine hydroxylase activity was reduced by about

2 242 L.R. SQUIRE et al. 25 ~ 90 rain after injection of cycloheximide or acetoxycycloheximide s. On the basis of this finding they suggested that the amnesic effects of these drugs might be mediated by norepinephrine depletion due to tyrosine hydroxylase inhibition, rather than to the inhibition of synthesis of proteins required for long-term memory storage. To evaluate this possibility we compared the effects of cycloheximide, anisomycin, and a-methyl-p-tyrosine (ampt) on brain tyrosine hydroxylase and memory. We have found that injection of cycloheximide or anisomycin depressed brain tyrosine hydroxylase activity, as determined by two assays. The same doses of these drugs inhibited brain protein synthesis by about 95 ~ and impaired long-term memory. However, repeated injection of ampt, a competitive inhibitor of tyrosine hydroxylase, did not affect long-term memory in spite of the fact that this drug depressed brain tyrosine hydroxylase as much as or more than the protein synthesis inhibitors. These results indicate that the effect of protein synthesis inhibitors on brain tyrosine hydroxylase is not responsible for their amnesic effects. MATERIALS AND METHODS Tyrosine hydroxylase assays L-[3,5-3H]Tyrosine (30 Ci/mmole) and L-[1-14C]tyrosine (50 mci/mmole) were obtained from New England Nuclear Corp. e-tyrosine [side chain: -2,3-ZH] (12 Ci/ mmole) was obtained from Amersham-Searle Corp. The synthetic cofactor, 2-amino- 4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine (DMPH D was obtained from Calbiochem. To replicate the tissue concentrations in the assay described by Flexner et al. 8, mouse forebrains, anterior to the midbrain, were homogenized in ice cold water (1/3, w/v) containing 1 ~ Triton X-100, and centrifuged at g for 30 min. Aliquots of the supernatant were assayed for tyrosine hydroxylase activity at 37 C according to the procedure of Nagatsu et al. 13 with modifications as previously described 11, at 0.3 mm L-[3,5-ZH]tyrosine and a final ph in the assay of 5.8 (assay I). Alternatively, tyrosine hydroxylase activity was measured by the method of Kuczenski and SegaP z (assay II). Tissue was homogenized in 0.32 M sucrose (1/2, w/v) and aliquots of the whole homogenate were incubated with L-[1-14C]tyrosine. The evolved ~4CO2 has been shown to be a measure of tyrosine hydroxylase activity confined in synaptosomes and varies directly with L-DOPA formedll The accumulation of [3H]tyrosine into a crude mitochondrial fraction enriched in synaptosomes was determined using a Millipore filtration technique as previously described ~2. None of the drugs used altered the rate of accumulation of [ZH]tyrosine into this fraction. Drugs Cycloheximide (Sigma Chemical Co.) and anisomycin (the generous gift of Nathan Belcher, Pfizer Inc.) were dissolved in 0.15 M NaC1 and injected subcutaneously in doses of 120 mg/kg and 150 mg/kg, respectively, ampt (D,L-a-methyl-ptyrosine methyl ester HC1, Sigma Chemical Co.) was dissolved in 0.15 M NaC1 and injected intraperitoneally (80 mg/kg or 160 mg/kg), one or more times.

3 TYROS1NE HYDROXYLASE AND MEMORY 243 Subjects and training procedure Hybrid offspring of C3H male and Balb/c female mice were obtained from Simonson Laboratories (Gilroy, Calif.) at 8 weeks of age and introduced to the experimental situation at 12 weeks of age. The mice were trained to perform an object discrimination in the automated Deutsch Carousel by a modification of the method described previously 19. Each mouse was restrained by the tail in the center of a circular platform 15 in. in diameter. A black cylinder 9 in. high and in. in diameter resting on the platform enclosed the apparatus and effectively limited the animal's vision to the apparatus itself. The mouse was then rotated from trial to trial to one of three stations located around the perimeter of the platform. At each station the mouse touched the correct one of two metal objects to escape footshock. The correct object was a vertically oriented screw (0.25 in. in diameter, 2 in. long) and the incorrect object, a horizontal stainless steel block 1.87 in. across, 0.50 in. high, and 0.75 in. deep. A correct trial was scored when the mouse touched the screw before touching the block. An incorrect trial was scored when the mouse touched the block before touching the screw. The intertrial interval was 15 sec. The entire operation was controlled by a PDP-8/L computer. In the learning experiments all mice were given 20 trials during training. Retention was tested by giving an additional 20 trials at a later time. Retention was evidenced by an increase in the number of correct trials obtained in the second test session. RESULTS Biochemical effects Subcutaneous injection of cycloheximide (120 mg/kg) or anisomycin (150 mg/kg) inhibited rat brain tyrosine hydroxylase activity to a small but significant degree under the conditions of assay I (Table I). Cycloheximide and anisomycin both significantly inhibited tyrosine hydroxylase activity as measured by assay II (Fig. 1A). With either drug the enzyme activity was maximally decreased by about 30 % at 3 h. With cycloheximide, significant inhibition was still detectable 9 h after injection; with anisomycin no significant inhibition remained 6 h after injection. TABLE I TYROSINE HYDROXYLASE ACTIVITY 90 M1N AFTER SUBCUTANEOUS INJECTION OF THE INDICATED DRUGS AS DETERMINED BY ASSAY I Drug N Tyrosine hydroxylase activity % Change (nmoles/g/h) ± S.E. Saline =k 3.2 Cycloheximide (120 mg/kg) " Anisomycin (150 mg/kg) " * Significantly different from saline group, P < 0.01.

4 244 L.R. SQUIRE et al. I ANISOM CIN 150 mg/kg 120 mg/kg I-,- 40 = ioo ~ I I I,I I J~(61 a METHYL-p-TYROSlNE l(m t t t t mg/kg mg/kg mg/k 9 mg/k9 B i i i i i ~.t i HOURS AFTER INJECTION Fig. 1. Effects of cycloheximide, anisomycin and a-methyl-p-tyrosine on brain tyrosine hydroxylase. A: mice were injected with anisomycin (150 mg/kg) or cycloheximide (120 mg/kg), and brain tyrosine hydroxylase activity was determined by assay II (see text) at the indicated times after injection. The numbers in parentheses indicate the number of mice at each point. Asterisks denote values significantly lower than control (P < 0.01). B: mice were injected with a-methyl-p-tyrosine (80 mg/kg) every 1.5 h, for a total of 4 injections, and brain tyrosine hydroxylase activity was determined by assay II (see text) at the indicated time after the first, second and fourth injections. A As measured by assay II, a single injection of ampt (80 mg/kg) reduced tyrosine hydroxylase activity by 30 ~ in one-half hour, but the effect did not last as long as 3 h (Fig. 2). A single dose of ampt at 160 mg/kg decreased the enzyme activity about 60 ~ in 1 h, and at the higher dose of ampt the inhibition was still significant 3 h after injection but not 6 h afterward. To produce profound and sustained inhibition of tyrosine hydroxylase we administered four injections of ampt (80 mg/kg) 1.5 h apart and achieved about 30~ inhibition of the enzyme for about 6 h (Fig. 1B). Behavioral effects Either cycloheximide (120 mg/kg) or anisomycin (150 mg/kg) injected 30 min before training produced significant impairment of memory when mice were tested one day later (Table II), which confirms our previous results 19,21. In the present task, a smaller dose of anisomycin (20 mg/kg) produced significant impairment of memory tested one day later, although the degree of amnesia was not as great as that produced by 150 mg/kg.

5 ]?YROSINE HYDROXYLASE AND MEMORY I I I I I I J,,..z I00 Z--, "160 mg/kg ~ I I 1 I I I I 0 I HOURS AFTER INJECTION Fig. 2. Effect of single doses of a-methyl-p-tyrosine on brain tyrosine hydroxylase. Mice were injected with a-methyl-p-tyrosine (80 mg/kg or 160 mg/kg), and brain tyrosine hydroxylase activity was determined by assay I1 (see text) at the indicated times after injection. Six mice contributed to each point. Asterisks denote values significantly different from control (P < 0.01). Single injections of 80 or 160 mg/kg of ampt 30 min before training had no effect on memory measured one day later (Table II). Likewise, repeated injections of 80 mg/kg ampt had no effect on memory tested either one day or 4 days after training (Table II). Finally, the effect of the small dose of anisomycin (20 mg/kg) combined with a single dose of 80 mg/kg of ampt did not differ significantly from that of the small dose of anisomycin alone (Table II). TABLE II EFFECTS OF ANISOMYCIN, CYCLOHEXIMIDE AND Ct-METHYL-p-TYROSINE ON PERFORMANCE AFTER TRAINING Mice were given the indicated drugs 30 min before training for 20 trials. Retention was tested by giving an additional 20 trials 1 day later. Group 8 was tested 4 days after training. Drug Dose (mg/kg) N Trials correct training Trials correct retention 1 Saline Anisomycin '* 3 Anisomycin ' 4 Cycloheximide "* 5 ampt ampt ampt (4 injections)*** ampt (4 injections)*** Anisomycin ~ 20~ ampt t 801 * Significantly lower than saline group, P < ** Significantly lower than saline group, P < *** Administered 30 min before training and every 1.5 h thereafter for a total of 4 injections, each 80 mg/kg.

6 246 L.R. SQUIRE et al. DISCUSSION The present studies confirm the report 8 that cycloheximide results in a measurable inhibition of rat brain tyrosine hydroxylase assayed by the method of Nagatsu et al. 13 (assay I). In our hands the effects were somewhat less than those reported by Flexner et al. 8. In addition anisomycin, another inhibitor of protein synthesis in brain that causes amnesia, resulted in measurable inhibition of brain tyrosine hydroxylase under the conditions of assay I. Cycloheximide is known to elevate brain tyrosine levels 8, presumably by blocking utilization of tyrosine for protein synthesis. From the measurements made by Flexner et al. 8 of tyrosine levels in the mouse brain before and after injection of cycloheximide, we calculated that in assay I endogenous tyrosine represented about 3 ~ of the total tyrosine in the controls and 7 ~ of the total tyrosine in cycloheximide-treated animals. Therefore, a portion of the inhibition oftyrosine hydroxylase activity by cycloheximide in assay I may have been an artifact of dilution of the radioactive substrate tyrosine. It is unlikely that 2-fold to 3-fold increases in brain tyrosine produce a true depression of tyrosine hydroxylase since 3-fold increases in brain tyrosine had no detectable effects on the rate of catecholamine synthesis measured in vivo 5. Because of the similar effects of cycloheximide and anisomycin and their strikingly different molecular structures it seems unlikely that the inhibition of tyrosine hydroxylase after cycloheximide is caused by a metabolite of that drug, as suggested by Flexner et al. 8. It seems likely that anisomycin, like cycloheximide, would elevate brain tyrosine levels by blocking utilization of tyrosine for protein synthesis. Therefore a portion of the inhibition of tyrosine hydroxylase by anisomycin may also be attributable to dilution of radioactive tyrosine. The cause of the remaining small effect in assay I is unclear, but might be due to natural degradation of the enzyme and failure to replace it because of inhibition of protein synthesis. In contrast with assay I, tyrosine hydroxylase activity measured by assay II was strikingly inhibited by both anisomycin and cycloheximide. Assay II was designed to determine the effects of drugs in undisrupted synaptosomes where the enzyme is concentrated in vivo and where the vast majority of normal catecholamine synthesis occurs. We chose to use a small amount of exogenous tyrosine of high specific activity in this assay so that it would not interfere with competition between endogenous tyrosine and the ampt associated with the tissue. In the case of anisomycin and cycloheximide, which produce increases in endogenous tyrosine levels 8, isotope ailution could cause a striking overestimation of in vivo inhibition of tyrosine hydroxylase measured by assay II. This may account for the striking inhibition observed in assay II with anisomycin and cycloheximide and the slight inhibition observed in assay I. Assay II is, however, required for evaluating effects of ampt after in vivo injection. This drug is a competitive inhibitor of tyrosine hydroxylase with respect to the substrate tyrosine. Inhibition of this enzyme by ampt is dependent on the concentration to which the enzyme is exposed. Using assay II, we assume that the ampt effect on tyrosine hydroxylase is due to the drug being present in synaptosomes along with the tyrosine hydroxylase. However, any leakage of the drug in the diluted assay mixture

7 TYROSINE HYDROXYLASE AND MEMORY 247 would serve to underestimate the actual in vivo inhibition of tyrosine hydroxylase by ampt. Despite this, in vivo administration of ampt produced marked inhibition of tyrosine hydroxylase as measured by assay II. It is noteworthy that in mouse brain, ampt (100 mg/kg, a dose comparable to that used in this study) decreases catecholamine levels in whole brain by 20 ~ within 1 h and by 50 ~ within 4 h 2a. Based on these more direct studies, our results with assay II probably underestimate the degree of inhibition of catecholamine synthesis by ampt. Despite the fact that the measured inhibition of the enzyme by ampt was at least as great as that produced by anisomycin, no amnesic effect was observed with ampt. Therefore, the reduction in tyrosine hydroxylase activity produced by anisomycin is not sufficient to explain its amnesic effect. When one considers that the inhibition of tyrosine hydroxylase activity (assay II) by the protein synthesis inhibitors may be largely due to isotope dilution, as suggested by the very small effects seen in assay I, this conclusion is difficult to dispute. Because of evidence implicating the adrenergic system in long-term memory storage 4,7,9,1,14,15, we also investigated the possibility that a dose of anisomycin that was insufficient to produce marked amnesia by itself might be more amnesic if combined with ampt. We found that ampt (80 mg/kg) in combination with anisomycin (20 mg/kg) produced no greater effect on retention than did anisomycin alone. This finding, however, does not preclude the participation of the biogenic amines in arousal or in other processes required for long-term memory storage. The disruption of the adrenergic system might have to be more severe than that produced by ampt in the doses we used. Whatever the role of biogenic amines in memory formation, the present results indicate that the inhibition of tyrosine hydroxylase produced by cycloheximide or anisomycin is not sufficient to explain the amnesia they produce. All available evidence is therefore consistent with the suggestion that these drugs block memory by preventing the synthesis of brain proteins required for long-term memory storage 1,2,1s,20. ACKNOWLEDGEMENTS The authors thank Professor J. A. Deutsch for his continuing interest and Hasker P. Davis and Patrick Russo for assistance in the experiments. This research was supported by Grants MH24600 and MH18282 from the National Institutes of Health and by a grant from the Alfred P. Sloan Foundation. L. S. was supported by a Clinical Investigatorship from the Veterans Administration. REFERENCES 1 BARONDES, S.H., Cerebral protein synthesis inhibitors block long-term memory, Int. Rev. Neurobiol., 13 (1970) BARONDES, S. H., Protein synthesis-dependent and protein synthesis-independent memory storage processes. In D. D~uTscIq AND J. A. DEUTSCH (Eds.), Short Term Memory, Academic Press, New York, in press.

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