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The dependence of brain ATP content on cerebral electroshock current
442
Brain Research, 61 (1973) 442-445 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The dependence of brain ATP content on cerebral electroshock current
ADRIAN DUNN* Division of Chemical Neurobiology, Department of Biochemistry, School of Medicine, University of North Carolina, Chapel Hill, N. C. 27514 (U.S.A.)
(Accepted July 2nd, 1973)
A question of continuing interest is the nature of the biochemical lesion responsible for the amnesia caused by cerebral electroshock. Very many studies have been concerned with this problem, but apparently the effect of electroshock is so gross that almost all brain chemicals that have been studied are affected (see ref. 6). It is likely that many of the changes result from the convulsions induced by the electroshock. Since the occurrence of bodily convulsions is not necessary for the amnesia3,7,9,11,12, examination of the biochemical effects of subconvulsive electroshock may simplify the problem. The convulsions associated with electroshock may be prevented either by the use of anesthetics or by decreasing the electroshock current. The effect of subconvulsive electroshock on the incorporation of radioactive amino acids into brain protein has been examined using both these techniques. Cotman et aL ~ showed that diethyl ether, which inhibited the convulsions but not the brain seizure, did not prevent the inhibition of amino acid incorporation caused by the electroshock. Significant inhibitions of the incorporation were only observed when brain seizures occurred. Dunn 4 showed that the inhibition of amino acid incorporation was linearly correlated with the current used in the electroshock; the presence or absence of convulsions did not especially disturb this correlation. A similar relationship between electroshock current and retrograde amnesia has also been observed 8,7, 9,12. Thus a convenient way of studying the interrelationships of the effects of electroshock is to relate the behavioral, electrophysiological and biochemical data to the electroshock current. The depression of amino acid incorporation into brain protein may be caused by a depression of cerebral ATP levels or the cation imbalance caused by the electroshock4, s. Severe disturbances of brain ATP are induced by the electroconvulsive shock s, but, since these may be prevented by administration of oxygen 1, the disturbances may be provoked by the anoxia associated with the convulsions, rather than the electroshock itself. Nevertheless, anesthetization of mice with secobarbitone prevented the convulsions but not the depression of ATP associated with electroconvul* Present address: Dept. of Neuroscience, University of Florida College of Medicine, Gainesville, Fla. 32601, U.S.A.
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443
sive shock (ECS) 8. To resolve this problem, the A T P content of brain was measured in relation to the electroshock current. Male Swiss-Webster mice (30 g) were administered an electroshock of 0.2 sec duration from transcorneal electrodes as previously described 4. The electroshocking current was controlled and varied as desired. At the required time following electroshock the mice were plunged head first into liquid nitrogen and held there until the brains were completely frozen (2 min). Pieces of the frozen forebrain were then chiselled out, powdered frozen in a mortar with a pestle, and immediately homogenized in 5 ml of 5 ~ trichloroacetic acid. The macromolecular precipitate was centrifuged out at 10,000 × g for 10 min at 0 °C. The trichloroacetic acid was then extracted from the supernatant with diethyl ether and the p H of the supernatant adjusted to 6.8 with 0.5 N and 0.1 N N a O H still at 0 °C. A T P in this neutralized supernatant was then assayed by the hexokinase-glucose-6-phosphate dehydrogenase system using an ATP Stat-Pack (Calbiochem., La Jolla, Calif.) as described by the manufacturers. Protein in the precipitate was determined after dissolution in 0.3 N N a O H by the method of Lowry et al. lo. It was preferred to relate the A T P to the protein of the sample rather than the brain weight to obviate weighing errors due to the presence of ice, and so that only tissue properly homogenized would be determined in both assays. First the effect of the electroshock current from 0 to 25 m A on the A T P content 15 sec after the shock was determined (Fig. 1). There is a clear correlation (P < 0.0005) between the two variables. Animals that did not convulse were very similarly correlated (P < 0.0005). Animals that did convulse had A T P levels that were slightly more depressed than those that did not, and the correlation with electroshock current was also significant (P < 0.05). The lesser slope of this correlation suggests that maximal depression levels may be approached in these conditions. Essentially iden-
25 z
20 0
0 0
0 ~. ~ 0
15 E
"6 I0 E E
0 ELECTROSHOCK CURRENT (rnA)
Fig. 1. Mice were electroshocked transcorneally with the current designated for 0.2 sec. Fifteen seconds later they were dropped into liquid nitrogen. The ATP content was determined as described in the text. Each circle is the mean of the results from one mouse. Filled circles are used for animals that exhibited behavioral convulsions. The lines are regression lines obtained by least squares analysis. Solid line: all mice (r = 0.90, P < 0.0005); dashes: mice that did not convulse (r = 0.82, P < 0.0005) ; dots and dashes: mice that convulsed (r = 0.51, P <~ 0.05)
444
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T I M E (SECONDS)
Fig. 2. Mice were electroshocked with a current of 15 mA. At the time designated they were dropped into liquid nitrogen and the ATP content of the frozen brain determined as described in the text. Mice in this experiment and that of Fig. 1 were analyzed together. The results are presented as mean 4- S.E.M. The open circle at zero time is the mean of the data for sham-shocked mice. * Significantly different from control P < 0.01 (2-tailed t-test). tical results were obtained in an independent experiment. Fig. 2 shows the time course of the effect of 15 mA ECS on brain ATP. There was a significant depression when the mice were frozen 2, 15 or 30 sec after the ECS (P < 0.01, 2-tailed t-test) being maximal at 30 sec (57 ~o inhibition). The small depression at 60 sec (13 ~ ) was not quite significant (P < 0.1) and the A T P was completely recovered by 2 min. The effect of electroshock current on brain A T P is very similar to that previously observed on amino acid incorporation into protein 4,5. The regression lines for the two biochemical measures correlate remarkably: 50 ~ depression of ATP was observed at about 15 m A ; 50 ~ depression of amino acid incorporation occurred at about 14 mA. Thus it seems very unlikely that the two effects are unrelated, and very likely that the inhibition of amino acid incorporation is caused by the depression of ATP. These results are in agreement with those of King et al. 8, who found that secobarbitone anesthesia did not prevent the depression of brain ATP caused by electroshock in mice. The time course of the effect of ECS on ATP in the latter study is also very similar to that observed in the present work. We may conclude that the major effects of electroshock on brain energy metabolites are central, and result from an increased energy demand. Presumably this energy is used by the ion pumps to restore the balance of cations disturbed by the brain seizures. We cannot in these studies adequately distinguish between the effect of the electroshock itself and that of the secondary brain seizures. However, the threshold for brain seizures under the conditions of the present study was probably about 2-3 m A (McGaugh and Zornetzer, personal communication)and no significant ATP depression was observed at this current, so the seizures may well have been a major factor. Since ATP is of such central importance in cerebral metabolism, it seems likely that most brain chemicals will at least be temporarily affected by electroshock. The problem of locating the specific lesion responsible for the retrograde amnesia will therefore be much more difficult.
SHORT COMMUNICATIONS
445
The technical assistance o f L o f t o n H a r r i s a n d the advice o f Dr. A. T. M i l l e r a n d his colleagues is gratefully a c k n o w l e d g e d . This research was s u p p o r t e d by a g r a n t f r o m the University o f N o r t h C a r o l i n a at C h a p e l Hill (VC337).
1 COLLINS, R. C., POSNER, J. B., AND PLUM, F., Cerebral energy metabolism during electroshock seizures in mice, Amer. J. Physiol., 218 (1970) 943-950. 2 COTMAN,C. W., BANKER,G., ZORNETZER,S. F., AND McGAuGI-I,J. L., Electroshock effects on brain protein synthesis: relation to brain seizures and retrograde amnesia, Science, 173 (1971) 454--456. 3 DORFMANN,L. J., AND JARWK, M. E., A parametric study of electroshock-induced retrograde amnesia in mice, Neuropsychologia, 6 (1968) 373-380. 4 DUNN, A., Brain protein synthesis after electroshock, Brain Research, 35 (1971) 254-259. 5 DUNN, A., GIUDIrTA, A., WILSON,J. E., AND GL~SSMAN,E., The effect of electroshock on brain RNA and protein synthesis and its possible relationship to behavioral effects. In M. FINK, J. McGAuGH AND S. I~TY (Eds.), Psychobiology of ECT, Winston and Son, New York, 1973, In press. 6 ESSMAN,W. B., Neurochemical changes in ECS and ECT, Seminars in Psychiatry, 4 (1972) 67-77. 7 J~tvm, M. E., AND KOpP, R., Transcorneal electroconvulsive shock and retrograde amnesia in mice, J. comp. physioL Psychol., 64 (1967) 431-433. 8 KING, L.J., LOWRY, L.H., PASSONNEAU,J.V., AND VENSON, V., Effects of convulsants on energy reserves in the cerebral cortex, J. Neurochem., 14 (1967) 599-611. 9 LEE-TENG, E., Retrograde amnesia in relation to subconvulsive and convulsive currents in the chick, 3". comp. physioL Psychol., 67 (1969) 135-139. 10 LOWRY, O.H., ROSEBROUGH, N.J., FARR, A; L., AND RANDALL, R.J., Protein measurement with the folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 11 McGAuOH, J. L., AND ALPERN, H. P., Effects of electroshock on memory: Amnesia without convulsions, Science, 152 (1966) 665-666. 12 RAY, O. S., AND BARRETT, R. J., Disruptive effects of electroconvulsive shock as a function of current levels and mode of delivery, J. cutup, physiol. Psychol., 67 (1969) 110-116.
Brain Research, 61 (1973) 442-445 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The dependence of brain ATP content on cerebral electroshock current
ADRIAN DUNN* Division of Chemical Neurobiology, Department of Biochemistry, School of Medicine, University of North Carolina, Chapel Hill, N. C. 27514 (U.S.A.)
(Accepted July 2nd, 1973)
A question of continuing interest is the nature of the biochemical lesion responsible for the amnesia caused by cerebral electroshock. Very many studies have been concerned with this problem, but apparently the effect of electroshock is so gross that almost all brain chemicals that have been studied are affected (see ref. 6). It is likely that many of the changes result from the convulsions induced by the electroshock. Since the occurrence of bodily convulsions is not necessary for the amnesia3,7,9,11,12, examination of the biochemical effects of subconvulsive electroshock may simplify the problem. The convulsions associated with electroshock may be prevented either by the use of anesthetics or by decreasing the electroshock current. The effect of subconvulsive electroshock on the incorporation of radioactive amino acids into brain protein has been examined using both these techniques. Cotman et aL ~ showed that diethyl ether, which inhibited the convulsions but not the brain seizure, did not prevent the inhibition of amino acid incorporation caused by the electroshock. Significant inhibitions of the incorporation were only observed when brain seizures occurred. Dunn 4 showed that the inhibition of amino acid incorporation was linearly correlated with the current used in the electroshock; the presence or absence of convulsions did not especially disturb this correlation. A similar relationship between electroshock current and retrograde amnesia has also been observed 8,7, 9,12. Thus a convenient way of studying the interrelationships of the effects of electroshock is to relate the behavioral, electrophysiological and biochemical data to the electroshock current. The depression of amino acid incorporation into brain protein may be caused by a depression of cerebral ATP levels or the cation imbalance caused by the electroshock4, s. Severe disturbances of brain ATP are induced by the electroconvulsive shock s, but, since these may be prevented by administration of oxygen 1, the disturbances may be provoked by the anoxia associated with the convulsions, rather than the electroshock itself. Nevertheless, anesthetization of mice with secobarbitone prevented the convulsions but not the depression of ATP associated with electroconvul* Present address: Dept. of Neuroscience, University of Florida College of Medicine, Gainesville, Fla. 32601, U.S.A.
SHORT COMMUNICATIONS
443
sive shock (ECS) 8. To resolve this problem, the A T P content of brain was measured in relation to the electroshock current. Male Swiss-Webster mice (30 g) were administered an electroshock of 0.2 sec duration from transcorneal electrodes as previously described 4. The electroshocking current was controlled and varied as desired. At the required time following electroshock the mice were plunged head first into liquid nitrogen and held there until the brains were completely frozen (2 min). Pieces of the frozen forebrain were then chiselled out, powdered frozen in a mortar with a pestle, and immediately homogenized in 5 ml of 5 ~ trichloroacetic acid. The macromolecular precipitate was centrifuged out at 10,000 × g for 10 min at 0 °C. The trichloroacetic acid was then extracted from the supernatant with diethyl ether and the p H of the supernatant adjusted to 6.8 with 0.5 N and 0.1 N N a O H still at 0 °C. A T P in this neutralized supernatant was then assayed by the hexokinase-glucose-6-phosphate dehydrogenase system using an ATP Stat-Pack (Calbiochem., La Jolla, Calif.) as described by the manufacturers. Protein in the precipitate was determined after dissolution in 0.3 N N a O H by the method of Lowry et al. lo. It was preferred to relate the A T P to the protein of the sample rather than the brain weight to obviate weighing errors due to the presence of ice, and so that only tissue properly homogenized would be determined in both assays. First the effect of the electroshock current from 0 to 25 m A on the A T P content 15 sec after the shock was determined (Fig. 1). There is a clear correlation (P < 0.0005) between the two variables. Animals that did not convulse were very similarly correlated (P < 0.0005). Animals that did convulse had A T P levels that were slightly more depressed than those that did not, and the correlation with electroshock current was also significant (P < 0.05). The lesser slope of this correlation suggests that maximal depression levels may be approached in these conditions. Essentially iden-
25 z
20 0
0 0
0 ~. ~ 0
15 E
"6 I0 E E
0 ELECTROSHOCK CURRENT (rnA)
Fig. 1. Mice were electroshocked transcorneally with the current designated for 0.2 sec. Fifteen seconds later they were dropped into liquid nitrogen. The ATP content was determined as described in the text. Each circle is the mean of the results from one mouse. Filled circles are used for animals that exhibited behavioral convulsions. The lines are regression lines obtained by least squares analysis. Solid line: all mice (r = 0.90, P < 0.0005); dashes: mice that did not convulse (r = 0.82, P < 0.0005) ; dots and dashes: mice that convulsed (r = 0.51, P <~ 0.05)
444
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25 z bJ I'o z0
~,
E F'~
5
i ~ i 0 I 30
I 60
I 120
//
i 500
T I M E (SECONDS)
Fig. 2. Mice were electroshocked with a current of 15 mA. At the time designated they were dropped into liquid nitrogen and the ATP content of the frozen brain determined as described in the text. Mice in this experiment and that of Fig. 1 were analyzed together. The results are presented as mean 4- S.E.M. The open circle at zero time is the mean of the data for sham-shocked mice. * Significantly different from control P < 0.01 (2-tailed t-test). tical results were obtained in an independent experiment. Fig. 2 shows the time course of the effect of 15 mA ECS on brain ATP. There was a significant depression when the mice were frozen 2, 15 or 30 sec after the ECS (P < 0.01, 2-tailed t-test) being maximal at 30 sec (57 ~o inhibition). The small depression at 60 sec (13 ~ ) was not quite significant (P < 0.1) and the A T P was completely recovered by 2 min. The effect of electroshock current on brain A T P is very similar to that previously observed on amino acid incorporation into protein 4,5. The regression lines for the two biochemical measures correlate remarkably: 50 ~ depression of ATP was observed at about 15 m A ; 50 ~ depression of amino acid incorporation occurred at about 14 mA. Thus it seems very unlikely that the two effects are unrelated, and very likely that the inhibition of amino acid incorporation is caused by the depression of ATP. These results are in agreement with those of King et al. 8, who found that secobarbitone anesthesia did not prevent the depression of brain ATP caused by electroshock in mice. The time course of the effect of ECS on ATP in the latter study is also very similar to that observed in the present work. We may conclude that the major effects of electroshock on brain energy metabolites are central, and result from an increased energy demand. Presumably this energy is used by the ion pumps to restore the balance of cations disturbed by the brain seizures. We cannot in these studies adequately distinguish between the effect of the electroshock itself and that of the secondary brain seizures. However, the threshold for brain seizures under the conditions of the present study was probably about 2-3 m A (McGaugh and Zornetzer, personal communication)and no significant ATP depression was observed at this current, so the seizures may well have been a major factor. Since ATP is of such central importance in cerebral metabolism, it seems likely that most brain chemicals will at least be temporarily affected by electroshock. The problem of locating the specific lesion responsible for the retrograde amnesia will therefore be much more difficult.
SHORT COMMUNICATIONS
445
The technical assistance o f L o f t o n H a r r i s a n d the advice o f Dr. A. T. M i l l e r a n d his colleagues is gratefully a c k n o w l e d g e d . This research was s u p p o r t e d by a g r a n t f r o m the University o f N o r t h C a r o l i n a at C h a p e l Hill (VC337).
1 COLLINS, R. C., POSNER, J. B., AND PLUM, F., Cerebral energy metabolism during electroshock seizures in mice, Amer. J. Physiol., 218 (1970) 943-950. 2 COTMAN,C. W., BANKER,G., ZORNETZER,S. F., AND McGAuGI-I,J. L., Electroshock effects on brain protein synthesis: relation to brain seizures and retrograde amnesia, Science, 173 (1971) 454--456. 3 DORFMANN,L. J., AND JARWK, M. E., A parametric study of electroshock-induced retrograde amnesia in mice, Neuropsychologia, 6 (1968) 373-380. 4 DUNN, A., Brain protein synthesis after electroshock, Brain Research, 35 (1971) 254-259. 5 DUNN, A., GIUDIrTA, A., WILSON,J. E., AND GL~SSMAN,E., The effect of electroshock on brain RNA and protein synthesis and its possible relationship to behavioral effects. In M. FINK, J. McGAuGH AND S. I~TY (Eds.), Psychobiology of ECT, Winston and Son, New York, 1973, In press. 6 ESSMAN,W. B., Neurochemical changes in ECS and ECT, Seminars in Psychiatry, 4 (1972) 67-77. 7 J~tvm, M. E., AND KOpP, R., Transcorneal electroconvulsive shock and retrograde amnesia in mice, J. comp. physioL Psychol., 64 (1967) 431-433. 8 KING, L.J., LOWRY, L.H., PASSONNEAU,J.V., AND VENSON, V., Effects of convulsants on energy reserves in the cerebral cortex, J. Neurochem., 14 (1967) 599-611. 9 LEE-TENG, E., Retrograde amnesia in relation to subconvulsive and convulsive currents in the chick, 3". comp. physioL Psychol., 67 (1969) 135-139. 10 LOWRY, O.H., ROSEBROUGH, N.J., FARR, A; L., AND RANDALL, R.J., Protein measurement with the folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 11 McGAuOH, J. L., AND ALPERN, H. P., Effects of electroshock on memory: Amnesia without convulsions, Science, 152 (1966) 665-666. 12 RAY, O. S., AND BARRETT, R. J., Disruptive effects of electroconvulsive shock as a function of current levels and mode of delivery, J. cutup, physiol. Psychol., 67 (1969) 110-116.