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Inhibitory effect of intraspinal injection of 6-hydroxydopamine on the clonic convulsion in maximal electroshock seizure
Brain Research, 169 (1979) 189-193 © Elsevier/North-Holland biomedical Press
189
Inhibitory effect of intraspinal injection of 6-hydroxydopamine on the clonic convulsion in maximal electroshock seizure
RYOZO OISHI, NORIKO SUENAGA, TADASHI HIDAKA and TAKEO FUKUDA* Department of Pharmacology, Faculty of Medicine, Kyushu University 60, Fukuoka 812 (Japan)
(Accepted February 15th, 1979)
Reduction of brain monoamines by reserpine or other amine-depleting agents inhibits the postdecapitation convulsionT, 10. In previous studies we have reported that intraventricular injection of 6-hydroxydopamine (6-OHDA) inhibits both the clonic convulsion in maximal electroshock seizure and the decapitation convulsion in mice s, and there are close correlations between norepinephrine (NE) content of the spinal cord and the latency or the duration of decapitation convulsion in 6-OHDA-treated rats la. However, there is still no evidence that spinal NE is essential for the development of the clonic convulsion induced by electroshock. The present study was carried out to clarify the correlation between the clonic convulsion in maximal electroshock seizure and N E content of the spinal cord. Male CF-1 strain mice, supplied by Kyushu University Institute of Laboratory Animals, weighing 25-35 g were used in the experiment. 6-OHDA at a dose of 4, 8 or 12/~g dissolved in 2 #l of 0.9 % saline-0.1 ~ ascorbic acid solution was injected into the spinal cord (at the level of T7) through a 0.3 mm diameter needle connected to a motor-driven microsyringe at a rate of 1 pl/min under ether anesthesia. Maximal electroshock seizure was induced by a constant current of 50 mA, 0.2 see through corneal electrodes 4 days after the treatment with 6-OHDA. Each mouse was killed by decapitation 7 days after the injection of 6-OHDA and the decapitation convulsion was recorded. The brain was removed and NE contents of the spinal cord (caudal to T7) and whole brain were determined fluorometrically as described by Shellenberger and Gordon 12. Both maximal electroshock seizure and the decapitation convulsion were recorded according to our previous method a. Immediately following electroshock or decapitation, the mouse was placed on the round plastic plate (1 mm thickness; 23 cm diameter), to which 3 accelerometers were attached, and which was placed on a soft sponge rubber. Convulsive movement detected by 3 accelerometers connected in parallel was amplified and recorded by a polygraph (SAN-EI 141-6). At the same time, the output from the amplifier was integrated and recorded on a separate * Present address: Department of Pharmacology, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan.
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Fig. 1. Recording of maximal electroshock seizure (A) and decapitation convulsion (B). Trace (a) represents the input from 3 accelerometers, and trace (b) represents the integral of trace (a). Abbreviations: ES, electroshock; DC, decapitation.
TABLE 1 Effect o f intraspinal injection o f 6-OHDA on maximal electroshock seizure ( M E S ) , decapitation convulsion ( DC) and N E content o f the brain Maximal electroshock and decapitation were performed 4 and 7 days after intraspinal injection of 6-OHDA respectively. Each value is the mean 4- S.E.M. Abbreviations: TE, tonic extension; CL, clonic convulsion. 6-OHDA
Number of mice TE in MES Duration (sec) CL in MES Duration (sec) Intensity(%)§ DC Latency (sec) Duration (sec) Intensity (%)§ N E content (~g/g) Spinal cord Whole brain
Vehicle
4 [tg
8 #g
12 #g
9
7
8
7
9.3 ± 0.4
11.4 ± 0.5**
10.7 ± 0.6
10.3 ± 1.1
4.4 ± 0.4 100.0 4- 11.0
4.2 ± 0.9 49.5 i 8.3**
1.9 ± 0.7*** 34.1 :k 19.0'*
6.7 4- 0.4 t2.7 4- 1.0 100.0 4- 5.7
8.1 4- 1.2 13.2 4- 1.7 84.6 4- 18.8
14.6 4- 2.5"**(7)~ 7.6 4- 2.1" 37.9 4- 13.7"**
13.7 4- 1.1"** (3)§§ 4.0 4- 1.9"** 7.9 4- 4.9***
0.28 ± 0.05 0.46 4- 0.02
0.13 4- 0.05*** 0.45 4- 0.02
0.07 ± 0.03*** 0.45 ± 0.01
0.35 4- 0.01 0.46 4- 0.01
0.2 ± 0.1"** 2.6 4- 1.7'**
§ Each value is expressed as % of the mean value of vehicle group. §§ Number of mice which appeared in convulsion. * Significantly different from the vehicle group, P < 0.05; ** P <0.01; *** P < 0.001 (two-tailed Student's t-test).
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Fig. 2. Correlation between the intensity of the clonic convulsion in maximal electroshock seizure and spinal norepinephrine content (A) or the intensity of the decapitation convulsion (B) in 6-OHDAtreated mice. Ordinate: spinal norepinephrine content (A) and the intensity of the decapitation convulsion (B). Abscissa: the intensity of the clonic convulsion in maximal electroshock seizure. Each point represents the value of individual mouse, r = 0.773 (P < 0.01) in A, and r = 0.771 (P < 0.01) in B.
channel (Fig. 1). The area of integral was determined as the value of the intensity of convulsion. Table I shows the effects of intraspinal injection of 6-OHDA on maximal electroshock seizure, decapitation convulsion and NE contents in the brain. The duration of tonic extension in maximal electroshock seizure was significantly prolonged only by treatment with 4 #g of 6-OHDA, whereas both the duration and the intensity of the clonic convulsion were reduced in a dose-dependent manner by treatment with 6-OHDA. The clonic convulsion did not appear in 2 or 5 mice of the group treated with 8 or 12/zg of 6-OHDA, respectively. The latency of decapitation convulsion was prolonged by treatment with 6-OHDA, and the convulsion did not appear in 1 or 4 mice of the group treated with 8 or 12 #g of 6-OHDA, respectively. In addition, both the duration and the intensity of decapitation convulsion were reduced. Spinal NE content was also decreased in a dose-dependent manner by treatment with 6-OHDA, but NE content of the whole brain was not affected. There were significantly positive correlations between the intensity of the clonic convulsion in maximal electroshock seizure and either spinal NE content (Fig. 2A) or the intensity of decapitation convulsion (Fig. 2B) in 6-OHDA-treated mice. Correlation coefficient (r) was 0.773 in the former, and 0.771 in the latter. There were also significant correlations between spinal NE content and the intensity of decapitation convulsion, the duration of decapitation convulsion or the duration of the clonic
192 convulsion in maximal electroshock seizure (r ---- 0.809, 0.586 and 0.745, respectively; P < 0.01). In our previous report s, we found that intraventricular injection of 6-OHDA inhibited the clonic convulsion in maximal electroshock seizure. The data obtained in the present study revealed that intraspinal injection of 6-OHDA produced the inhibition of the clonic convulsion and decrease in spinal NE content only. Furthermore, there was a significant correlation between the intensity of the clonic convulsion and spinal NE content. In addition, we found that intraspinal injection of 6 - O H D A did not affect the spinal 5-hydroxytryptamine content (unpublished data). These results suggest that spinal NE is an essential substance for the development of clonic convulsion in maximal electroshock seizure. Prolonged effect of intraspinal injection of 6 - O H D A on the tonic extension in maximal electroshock seizure is similar to that of intracisternal or intraventricular injection of 6 - O H D A 6,8. However, the role of spinal NE on the tonic extension does not seem to be important, since these effects are not dependent on the dose of 6OHDA. Decapitation convulsion was inhibited in a dose-dependent manner by intraspinal injection of 6 - O H D A and its intensity showed a high correlation with spinal NE content. These results further support the hypothesis 5,11,13 that spinal NE neurons play an important role on the appearance of the decapitation convulsion. And6n and his co-workers 1-4 have reported that N E receptors which increase the spinal flexor reflex are present in the spinal cord. Although there may be some differences between flexor reflex and these clonic convulsions, it is clear that bulbospinal NE neurons have an important role to play in motor integration.
1 And6n, N. E., Corrodi, H., Fuxe, K., HOkfelt, B., H6kfelt, T., Rydin, C. and Svensson, T., Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci., 9 (1970) 513-523. 2 And6n, N. E. and Fuxe, K., A new dopamine-fl-hydroxylaseinhibitor: effects on the noradrenaline concentration and on the action of L-DOPA in the spinal cord, Brit. J. Pharmacol., 43 (1971) 747-756. 3 And6n, N. E., Jukes, M. G. M., Lundberg, A. and Vyklicky, L., A new spinal flexor reflex, Nature (Lond.), 202 (1964) 1344-1345. 4 Austin, J. H., Nygren, L. G. and Fuxe, K., A system for measuring the noradrenaline receptor contribution to the flexor reflex, Med. Biol., 54 (1976) 352-363. 5 Bourn, W. M., Geiger, P. F. and Jobe, P. C., Influence of norepinephrine and 5-hydroxytryptamine in post-decapitation convulsions in rats, Res. Commun. Psychol. Psychiat. Behav., 2 (1977) 9-20. 6 Browning, F. E. and Maynert, E. W., Effect of intracisternal 6-hydroxydopamine on seizure susceptibility in rats, Europ. J. Pharmaeol., 50 (1978) 97-101. 7 Eichbaum, F. W. and Yasaka, W. J., Inhibition of post-decapitation convulsions by reserpine, Experientia (Basel), 29 (1973) 816-817. 8 Fukuda, T., Araki, Y. and Suenaga, N., Inhibitory effects of 6-hydroxydopamine on the clonic convulsions induced by electroshock and decapitation, Neuropharmacology, 14 (1975) 579-583. 9 Fukuda, T., Oishi, R., Suenaga, N., Hidaka, T. and Ohba, T., Effects of monoamine related drugs on the clonic convulsions induced by electroshock and decapitation, Jap. J. Neurosci., 4 (1978) 24-25 (in Japanese). 10 Kamat, U. G. and Sheth, U. K., The role of central monoamines in decapitation convulsions of mice, Neuropharmacology, 10 (1971) 571-579.
193 11 Richardson, J. S. and Jacobowitz, D. M., Depletion of brain norepinephrine by intraventricular injection of 6-hydroxydopa: a biochemical, histochemical and behavioral study in rats, Brain Research, 58 (1973) 117-133. 12 Shellenberger, M. K. and Gordon, J. H., A rapid simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas, Analyt. Biochem., 39 (1971) 356-372. 13 Suenaga, N., Yamada, K. and Fukuda, T., Correlation between central catecholamine level and postdecapitation convulsion in rats treated with 6-hydroxydopamine, Brain Research, 122 (1977) 165-169.
189
Inhibitory effect of intraspinal injection of 6-hydroxydopamine on the clonic convulsion in maximal electroshock seizure
RYOZO OISHI, NORIKO SUENAGA, TADASHI HIDAKA and TAKEO FUKUDA* Department of Pharmacology, Faculty of Medicine, Kyushu University 60, Fukuoka 812 (Japan)
(Accepted February 15th, 1979)
Reduction of brain monoamines by reserpine or other amine-depleting agents inhibits the postdecapitation convulsionT, 10. In previous studies we have reported that intraventricular injection of 6-hydroxydopamine (6-OHDA) inhibits both the clonic convulsion in maximal electroshock seizure and the decapitation convulsion in mice s, and there are close correlations between norepinephrine (NE) content of the spinal cord and the latency or the duration of decapitation convulsion in 6-OHDA-treated rats la. However, there is still no evidence that spinal NE is essential for the development of the clonic convulsion induced by electroshock. The present study was carried out to clarify the correlation between the clonic convulsion in maximal electroshock seizure and N E content of the spinal cord. Male CF-1 strain mice, supplied by Kyushu University Institute of Laboratory Animals, weighing 25-35 g were used in the experiment. 6-OHDA at a dose of 4, 8 or 12/~g dissolved in 2 #l of 0.9 % saline-0.1 ~ ascorbic acid solution was injected into the spinal cord (at the level of T7) through a 0.3 mm diameter needle connected to a motor-driven microsyringe at a rate of 1 pl/min under ether anesthesia. Maximal electroshock seizure was induced by a constant current of 50 mA, 0.2 see through corneal electrodes 4 days after the treatment with 6-OHDA. Each mouse was killed by decapitation 7 days after the injection of 6-OHDA and the decapitation convulsion was recorded. The brain was removed and NE contents of the spinal cord (caudal to T7) and whole brain were determined fluorometrically as described by Shellenberger and Gordon 12. Both maximal electroshock seizure and the decapitation convulsion were recorded according to our previous method a. Immediately following electroshock or decapitation, the mouse was placed on the round plastic plate (1 mm thickness; 23 cm diameter), to which 3 accelerometers were attached, and which was placed on a soft sponge rubber. Convulsive movement detected by 3 accelerometers connected in parallel was amplified and recorded by a polygraph (SAN-EI 141-6). At the same time, the output from the amplifier was integrated and recorded on a separate * Present address: Department of Pharmacology, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan.
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Fig. 1. Recording of maximal electroshock seizure (A) and decapitation convulsion (B). Trace (a) represents the input from 3 accelerometers, and trace (b) represents the integral of trace (a). Abbreviations: ES, electroshock; DC, decapitation.
TABLE 1 Effect o f intraspinal injection o f 6-OHDA on maximal electroshock seizure ( M E S ) , decapitation convulsion ( DC) and N E content o f the brain Maximal electroshock and decapitation were performed 4 and 7 days after intraspinal injection of 6-OHDA respectively. Each value is the mean 4- S.E.M. Abbreviations: TE, tonic extension; CL, clonic convulsion. 6-OHDA
Number of mice TE in MES Duration (sec) CL in MES Duration (sec) Intensity(%)§ DC Latency (sec) Duration (sec) Intensity (%)§ N E content (~g/g) Spinal cord Whole brain
Vehicle
4 [tg
8 #g
12 #g
9
7
8
7
9.3 ± 0.4
11.4 ± 0.5**
10.7 ± 0.6
10.3 ± 1.1
4.4 ± 0.4 100.0 4- 11.0
4.2 ± 0.9 49.5 i 8.3**
1.9 ± 0.7*** 34.1 :k 19.0'*
6.7 4- 0.4 t2.7 4- 1.0 100.0 4- 5.7
8.1 4- 1.2 13.2 4- 1.7 84.6 4- 18.8
14.6 4- 2.5"**(7)~ 7.6 4- 2.1" 37.9 4- 13.7"**
13.7 4- 1.1"** (3)§§ 4.0 4- 1.9"** 7.9 4- 4.9***
0.28 ± 0.05 0.46 4- 0.02
0.13 4- 0.05*** 0.45 4- 0.02
0.07 ± 0.03*** 0.45 ± 0.01
0.35 4- 0.01 0.46 4- 0.01
0.2 ± 0.1"** 2.6 4- 1.7'**
§ Each value is expressed as % of the mean value of vehicle group. §§ Number of mice which appeared in convulsion. * Significantly different from the vehicle group, P < 0.05; ** P <0.01; *** P < 0.001 (two-tailed Student's t-test).
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Fig. 2. Correlation between the intensity of the clonic convulsion in maximal electroshock seizure and spinal norepinephrine content (A) or the intensity of the decapitation convulsion (B) in 6-OHDAtreated mice. Ordinate: spinal norepinephrine content (A) and the intensity of the decapitation convulsion (B). Abscissa: the intensity of the clonic convulsion in maximal electroshock seizure. Each point represents the value of individual mouse, r = 0.773 (P < 0.01) in A, and r = 0.771 (P < 0.01) in B.
channel (Fig. 1). The area of integral was determined as the value of the intensity of convulsion. Table I shows the effects of intraspinal injection of 6-OHDA on maximal electroshock seizure, decapitation convulsion and NE contents in the brain. The duration of tonic extension in maximal electroshock seizure was significantly prolonged only by treatment with 4 #g of 6-OHDA, whereas both the duration and the intensity of the clonic convulsion were reduced in a dose-dependent manner by treatment with 6-OHDA. The clonic convulsion did not appear in 2 or 5 mice of the group treated with 8 or 12/zg of 6-OHDA, respectively. The latency of decapitation convulsion was prolonged by treatment with 6-OHDA, and the convulsion did not appear in 1 or 4 mice of the group treated with 8 or 12 #g of 6-OHDA, respectively. In addition, both the duration and the intensity of decapitation convulsion were reduced. Spinal NE content was also decreased in a dose-dependent manner by treatment with 6-OHDA, but NE content of the whole brain was not affected. There were significantly positive correlations between the intensity of the clonic convulsion in maximal electroshock seizure and either spinal NE content (Fig. 2A) or the intensity of decapitation convulsion (Fig. 2B) in 6-OHDA-treated mice. Correlation coefficient (r) was 0.773 in the former, and 0.771 in the latter. There were also significant correlations between spinal NE content and the intensity of decapitation convulsion, the duration of decapitation convulsion or the duration of the clonic
192 convulsion in maximal electroshock seizure (r ---- 0.809, 0.586 and 0.745, respectively; P < 0.01). In our previous report s, we found that intraventricular injection of 6-OHDA inhibited the clonic convulsion in maximal electroshock seizure. The data obtained in the present study revealed that intraspinal injection of 6-OHDA produced the inhibition of the clonic convulsion and decrease in spinal NE content only. Furthermore, there was a significant correlation between the intensity of the clonic convulsion and spinal NE content. In addition, we found that intraspinal injection of 6 - O H D A did not affect the spinal 5-hydroxytryptamine content (unpublished data). These results suggest that spinal NE is an essential substance for the development of clonic convulsion in maximal electroshock seizure. Prolonged effect of intraspinal injection of 6 - O H D A on the tonic extension in maximal electroshock seizure is similar to that of intracisternal or intraventricular injection of 6 - O H D A 6,8. However, the role of spinal NE on the tonic extension does not seem to be important, since these effects are not dependent on the dose of 6OHDA. Decapitation convulsion was inhibited in a dose-dependent manner by intraspinal injection of 6 - O H D A and its intensity showed a high correlation with spinal NE content. These results further support the hypothesis 5,11,13 that spinal NE neurons play an important role on the appearance of the decapitation convulsion. And6n and his co-workers 1-4 have reported that N E receptors which increase the spinal flexor reflex are present in the spinal cord. Although there may be some differences between flexor reflex and these clonic convulsions, it is clear that bulbospinal NE neurons have an important role to play in motor integration.
1 And6n, N. E., Corrodi, H., Fuxe, K., HOkfelt, B., H6kfelt, T., Rydin, C. and Svensson, T., Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci., 9 (1970) 513-523. 2 And6n, N. E. and Fuxe, K., A new dopamine-fl-hydroxylaseinhibitor: effects on the noradrenaline concentration and on the action of L-DOPA in the spinal cord, Brit. J. Pharmacol., 43 (1971) 747-756. 3 And6n, N. E., Jukes, M. G. M., Lundberg, A. and Vyklicky, L., A new spinal flexor reflex, Nature (Lond.), 202 (1964) 1344-1345. 4 Austin, J. H., Nygren, L. G. and Fuxe, K., A system for measuring the noradrenaline receptor contribution to the flexor reflex, Med. Biol., 54 (1976) 352-363. 5 Bourn, W. M., Geiger, P. F. and Jobe, P. C., Influence of norepinephrine and 5-hydroxytryptamine in post-decapitation convulsions in rats, Res. Commun. Psychol. Psychiat. Behav., 2 (1977) 9-20. 6 Browning, F. E. and Maynert, E. W., Effect of intracisternal 6-hydroxydopamine on seizure susceptibility in rats, Europ. J. Pharmaeol., 50 (1978) 97-101. 7 Eichbaum, F. W. and Yasaka, W. J., Inhibition of post-decapitation convulsions by reserpine, Experientia (Basel), 29 (1973) 816-817. 8 Fukuda, T., Araki, Y. and Suenaga, N., Inhibitory effects of 6-hydroxydopamine on the clonic convulsions induced by electroshock and decapitation, Neuropharmacology, 14 (1975) 579-583. 9 Fukuda, T., Oishi, R., Suenaga, N., Hidaka, T. and Ohba, T., Effects of monoamine related drugs on the clonic convulsions induced by electroshock and decapitation, Jap. J. Neurosci., 4 (1978) 24-25 (in Japanese). 10 Kamat, U. G. and Sheth, U. K., The role of central monoamines in decapitation convulsions of mice, Neuropharmacology, 10 (1971) 571-579.
193 11 Richardson, J. S. and Jacobowitz, D. M., Depletion of brain norepinephrine by intraventricular injection of 6-hydroxydopa: a biochemical, histochemical and behavioral study in rats, Brain Research, 58 (1973) 117-133. 12 Shellenberger, M. K. and Gordon, J. H., A rapid simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas, Analyt. Biochem., 39 (1971) 356-372. 13 Suenaga, N., Yamada, K. and Fukuda, T., Correlation between central catecholamine level and postdecapitation convulsion in rats treated with 6-hydroxydopamine, Brain Research, 122 (1977) 165-169.