Brain Research, 169 (1979) 393-397 © Elsevier/North-Holland Biomedical Press
393
Progressive increase of motor activity induced by 5-HTP in the rat below a complete section of the spinal cord
PAUL BEDARD, HUGUES BARBEAU, Gt~RALD BARBEAU and MICHEL F1LION Depts. of Anatomy and Physiology, Faculty of Medicine, Laval University, Quebec (Canada)
(Accepted February 15th, 1979)
The greater part if not the whole amount of serotonin or 5-hydroxytryptamine (5-HT) present in the spinal cord I is contained within neurons whose cell bodies are located in the medulla 5. The immediate precursor of 5-HT, 5-hydroxytryptophan (5HTP), increases the excitability of spinal motoneurons located below a complete cord transection3, 9. It was shown that this occurs in the cat 9 and dog 7 not only immediately but also 3 weeks after spinalization, when all bulbospinal serotoninergic neurons are likely to be degenerated. On the other hand, the latency of the effect of 5-HTP was shown to be shorter in chronic than in acute spinal cats ~,9. This suggests that spinal neurons become more sensitive to 5-HT after spinal cord transection. We reinvestigated the effects of 5-HTP on motoneuronal activity in spinal rats. We used a simple method to quantify and determine the time course of change of motoneuronal activity induced by injections of 5-HTP at different times after spinalization. In female Wistar rats weighing 200 g, a complete spinal transection at the T-5 level was made under sodium thiamylal anesthesia. This was done by removing a portion of the cord by microdissection of vessels, and of nervous tissue under visual control through a microscope. The completeness of the section was further verified postmortem by visual and histological examination. For the next 10 days the rats were housed in individual cages and their bladders emptied manually twice a day. At intervals varying from one to 25 days after the section, silver wires were twisted around the quadriceps femoris and a reference electrode was fixed to the hip bone. These electrodes were linked by insulated wires coursing under the skin to a connector fixed on the back of the animal. Electromyographic (EMG) signals were amplified and displayed by a pen recorder (Nihon-Kohden RM45). The signals were also rectified, integrated and simultaneously displayed by the pen recorder. The integration was automatically reset at a fixed level. This produced deflections of the pen at a frequency proportional to the amount of E M G activity. The number of deflections per period of 5 min was counted to produce graphs. During the recording sessions, the animal was * Address correspondence to P.B. at: Laboratoires de neurobiologie, Pavilion Notre-Dame, Hbpital de I'Enfant-J6sus, 2075 ave de Vitr6, Quebec, Qu6. GIJ 5B3 Canada.
394 E.I.
/ J
E .L;~
r --
.......
T
,+..... .,,m,++++_ + +
.rr+:r~,V++.~mr-~:.+~. ++ . . . . . . . .
+.:;: ;;;~
,f
+"+P +O0~°/KG
l
-o'PRo 1OmG/'Ko + ~,+uTE
Fig. 1. E M G tracing o f the right quadriceps femoris muscle o f a chronic spinal rat. The upFer tracing is the integrated E M G . N o t e the rapid onset o f the effect of 5-HTP and the rapid return to the baseline level o f activity after cyproheptadine, t0 mg/kg. The n u m b e r of peaks per 5 rain periods o n the integrated E M G tracings is used for quantification.
% OF CONTROL
~AL
RAT
30 Zl 19
100(
p
~Tr
5001
107 6
I00
=3 ANt) 2 O/*VS =
I
. . . . . . .
-2o
I
6 t5
I
=
so
=
ill
70
+Es
HTP I00 HG/KG
Fig. 2. Graph showing the progressive increase of the amplitude of the effect of 5-FITP on the spontaneous E M G activity o f the quadriceps muscle at increasing intervals after spinal transection: See text.
395 placed on a small padded platform with the paralyzed hindlimbs hanging through holes. After a period of acclimatization of 20 min, the EMG activity was recorded during the following 20 rain and considered as baseline activity. All drugs were injected intraperitoneally except apomorphine which was injected subcutaneously. They were dissolved in saline containing a minimal quantity of HCI. DL-5-HTP (Sigma, 100 mg/kg, i.p.) barely modified the spontaneous EMG activity in the paralyzed hindlimbs up to 4 days after transection. At this time, however, an increase of the spontaneous EMG activity following 5-HTP became more and more apparent. Fig. 1 shows raw and integrated EMG recordings of the right quadriceps femoris made 15 days after transection. Less than one minute after the injection of 5-HTP, at the arrow, the increase of EMG activity is obvious. At the same time atethoid movements, torsion of the trunk and walking movements were observed. Also there was penile erection, ejaculation and defecation. The effect lasted 3-4 h but could be blocked completely within 2 min (or prevented) by cyproheptadine (10 mg/kg). Fig. 2 shows that the amplitude of the EMG response to a standard dose of oL-5HTP ( 100 mg/kg) progressively increases with time. A set of 12 curves are displayed on the same coordinates. Each curve plots the amplitude of the EMG response of the quadriceps femoris obtained in different animals between the first and the 30th day. The amplitude of the responses was normalized with respect to the level of EMG activity maintained before the injection of 5-HTP and taken as 100 ~o. Note that the 5HTP-induced muscle activity increases during the first 10 rain and then persists as a plateau for at least one hour. Responses of peak amplitude were obtained twenty days after transection. The effect of 5-HTP in the chronic rat (more than 15 days after spinal transection) was completely suppressed in 2 rats by RO-4-4602 (800 mg/kg i.p.) N-DL-SerylN'- (2,2,4, trihydroxybenzyl) hydrazine (Hoffman LaRoche) injected 30 rain beforehand. At such doses, this drug is a potent inhibitor of the L-aromatic decarboxylase in the peripheral but also in the central nervous system4. The following drugs were tried in chronic spinal rats (more than 15 days after transection) and were without effect: 5-hydroxytryptamine(Calbiochem) 10 mg/kg i.p. itself and its main metabolite 5-hydroxyindole acetic acid (5-HIAA) (Calbiochem) at a dose of l0 mg/kg i.p. Other drugs were also without effect in the same conditions: imipramine 15 mg/kg (Ciba-Geigy), and fluoxetine, 50 mg/kg (Lilly), two inhibitors of the reuptake of 5-HT; L-dihydroxyphenylalanine 0--DOPA), 100 mg/kg, (Hoffman LaRoche), dissolved in the same manner and at the same pH as 5-HTP; apomorphine hydrochloride, 0.5 mg/kg (Merck, Sharp and Dohme); tetrabenazine, 20 mg/kg (Hoffman LaRoche). On the other hand, phenoxybenzamine (Hoffman LaRoche), at a dose of 4 mg/kg, did not block the stimulating action of DL-5-HTP, 100 mg/kg. Our results confirm the previous reports of a stimulating effect of 5-HTP on the spontaneous EMG activity of the hindlimbs in spinalized animals3, 9. They strongly suggest that the active substance is the 5-HT that is formed inside the central nervous system by the decarboxylation of the injected 5-HTP. This is demonstrated by the lack
396 of effect of 5-HT itself, injected systemically, of its main metabolite (5-HIAA) and of substances known to increase the effects of endogenous 5-HT (tetrabenazine, imipramine and fluoxetine). The possibility that 5-HT acted non specifically on non serotoninergic receptors (dopamine, noradrenaline, ACh, GABA, etc.) is not entirely excluded but is unlikely as L-DOPA and apomorphine were without effect and also in view of the powerful blocking action of cyproheptadine hydrochloride (10 mg/kg) but not of phenoxybenzamine. It is interesting to note the lack of action of L-DOPA on spontaneous EMG activity as opposed to reflexes2. The most striking element in our results is the progressive increase in the amplitude of the motor response to the same dose of 5-HTP during the 30 days following spinal transection. One possible interpretation of this fact is that the effect of 5-HT on its receptors on spinal neurons does not change after transection but that the excitability of motor neurons is increased for other reasons. There is in fact a 4-fold increase in the baseline level of E M G activity during the 15 days that follow transection. An alternative explanation is that 5-HT produces a greater effect because of denervation supersensitivity of 5-HT receptors. Our own results do not allow us at present to choose between these two explanations but the latter hypothesis is supported by the results of Nygren and Olson 8 who destroyed 5-HT pathways chemically but observed an increase in extensor reflexes studied semi-quantitatively one day after spinal transection. The use of integrated spontaneous EMG activity has more quantitative value than subjective estimation of the amplitude of reflexes. The dramatic increase of the motor response to 5-HTP, whatever is its exact mechanism, makes the chronic spinal preparation a very sensitive model for studying serotoninergic mechanisms in the central nervous system without having to use potentiating drugs. Moreover, the complete disappearance of the 5-HT axons will allow us to distinguish pre- from postsynaptic effects of pharmacological agents. This research was supported by the Medical Research Council of Canada. P.B. was a Scholar of the Canadian Life Insurance Association.
1 Anden, N. E,, Distribution of monoamines and dihydroxyphenylalanine decarboxylase activity in the spinal cord, Acta physiol, scand., 64 (1965) 197-203. 2 Anden, N. E., Jukes, M. G. M. and Lundberg, A., Spinal reflexes and monoamine liberation, Nature (Lond.), 202 (1964) 1222-1223. 3 Anderson, E. G. and Shibuya, T., The effects of 5-hydroxytryptophan and L-tryptophan on spinal synaptic activity, J. Pharmacol. exp. Ther., 153 (1966) 352-360. 4 Bartholini, G., Bates, H. M., Buckard, W. P., and Pletseher, A., Increase of cerebral catecholamines caused by 3-4-dihydroxyphenylalanine after inhibition of peripheral decarboxylase, Nature (Lond.), 215 (1967) 852-853. 5 Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N., Cellular localization of monoamines in the spinal cord, Acta physiol, scand., 69 (1964) 112-119.
397 6 Clineschmidt, B. V., Pierce, J. E. and Sjoerdsma, A., Interactions of tricyclic antidepressants and 5-hydroxyindolealkylamine precursors on spinal monosynaptic reflex transmission, J. PharmacoL exp. Ther., 179 (1971) 312-323. 7 Martin, W. and Eades, C. B., The action of tryptamine on the dog spinal cord and its relationship to the agonistic actions of LSD-like psychotogens Psyehopharmaeologia (BerL), 17 (1970) 242 -257. 8 Nygren, L., Fuxe, K., Jonsson, G. and Olson, L., Functional regeneration of 5-hydroxytryptamine nerve terminals in the rat spinal cord following 5,6-dihydroxytryptamine induced degeneration, Brain Research, 78 (1974) 377-394. 9 Shibuya, T. and Anderson, E. G., The influence of chronic cord transection on the effects of 5-hydroxytryptophan, L-tryptophan and pargyline on spinal neuronal activity, J. Pharmaeol. Exp. Ther., 164 (1968) 185-190.
393
Progressive increase of motor activity induced by 5-HTP in the rat below a complete section of the spinal cord
PAUL BEDARD, HUGUES BARBEAU, Gt~RALD BARBEAU and MICHEL F1LION Depts. of Anatomy and Physiology, Faculty of Medicine, Laval University, Quebec (Canada)
(Accepted February 15th, 1979)
The greater part if not the whole amount of serotonin or 5-hydroxytryptamine (5-HT) present in the spinal cord I is contained within neurons whose cell bodies are located in the medulla 5. The immediate precursor of 5-HT, 5-hydroxytryptophan (5HTP), increases the excitability of spinal motoneurons located below a complete cord transection3, 9. It was shown that this occurs in the cat 9 and dog 7 not only immediately but also 3 weeks after spinalization, when all bulbospinal serotoninergic neurons are likely to be degenerated. On the other hand, the latency of the effect of 5-HTP was shown to be shorter in chronic than in acute spinal cats ~,9. This suggests that spinal neurons become more sensitive to 5-HT after spinal cord transection. We reinvestigated the effects of 5-HTP on motoneuronal activity in spinal rats. We used a simple method to quantify and determine the time course of change of motoneuronal activity induced by injections of 5-HTP at different times after spinalization. In female Wistar rats weighing 200 g, a complete spinal transection at the T-5 level was made under sodium thiamylal anesthesia. This was done by removing a portion of the cord by microdissection of vessels, and of nervous tissue under visual control through a microscope. The completeness of the section was further verified postmortem by visual and histological examination. For the next 10 days the rats were housed in individual cages and their bladders emptied manually twice a day. At intervals varying from one to 25 days after the section, silver wires were twisted around the quadriceps femoris and a reference electrode was fixed to the hip bone. These electrodes were linked by insulated wires coursing under the skin to a connector fixed on the back of the animal. Electromyographic (EMG) signals were amplified and displayed by a pen recorder (Nihon-Kohden RM45). The signals were also rectified, integrated and simultaneously displayed by the pen recorder. The integration was automatically reset at a fixed level. This produced deflections of the pen at a frequency proportional to the amount of E M G activity. The number of deflections per period of 5 min was counted to produce graphs. During the recording sessions, the animal was * Address correspondence to P.B. at: Laboratoires de neurobiologie, Pavilion Notre-Dame, Hbpital de I'Enfant-J6sus, 2075 ave de Vitr6, Quebec, Qu6. GIJ 5B3 Canada.
394 E.I.
/ J
E .L;~
r --
.......
T
,+..... .,,m,++++_ + +
.rr+:r~,V++.~mr-~:.+~. ++ . . . . . . . .
+.:;: ;;;~
,f
+"+P +O0~°/KG
l
-o'PRo 1OmG/'Ko + ~,+uTE
Fig. 1. E M G tracing o f the right quadriceps femoris muscle o f a chronic spinal rat. The upFer tracing is the integrated E M G . N o t e the rapid onset o f the effect of 5-HTP and the rapid return to the baseline level o f activity after cyproheptadine, t0 mg/kg. The n u m b e r of peaks per 5 rain periods o n the integrated E M G tracings is used for quantification.
% OF CONTROL
~AL
RAT
30 Zl 19
100(
p
~Tr
5001
107 6
I00
=3 ANt) 2 O/*VS =
I
. . . . . . .
-2o
I
6 t5
I
=
so
=
ill
70
+Es
HTP I00 HG/KG
Fig. 2. Graph showing the progressive increase of the amplitude of the effect of 5-FITP on the spontaneous E M G activity o f the quadriceps muscle at increasing intervals after spinal transection: See text.
395 placed on a small padded platform with the paralyzed hindlimbs hanging through holes. After a period of acclimatization of 20 min, the EMG activity was recorded during the following 20 rain and considered as baseline activity. All drugs were injected intraperitoneally except apomorphine which was injected subcutaneously. They were dissolved in saline containing a minimal quantity of HCI. DL-5-HTP (Sigma, 100 mg/kg, i.p.) barely modified the spontaneous EMG activity in the paralyzed hindlimbs up to 4 days after transection. At this time, however, an increase of the spontaneous EMG activity following 5-HTP became more and more apparent. Fig. 1 shows raw and integrated EMG recordings of the right quadriceps femoris made 15 days after transection. Less than one minute after the injection of 5-HTP, at the arrow, the increase of EMG activity is obvious. At the same time atethoid movements, torsion of the trunk and walking movements were observed. Also there was penile erection, ejaculation and defecation. The effect lasted 3-4 h but could be blocked completely within 2 min (or prevented) by cyproheptadine (10 mg/kg). Fig. 2 shows that the amplitude of the EMG response to a standard dose of oL-5HTP ( 100 mg/kg) progressively increases with time. A set of 12 curves are displayed on the same coordinates. Each curve plots the amplitude of the EMG response of the quadriceps femoris obtained in different animals between the first and the 30th day. The amplitude of the responses was normalized with respect to the level of EMG activity maintained before the injection of 5-HTP and taken as 100 ~o. Note that the 5HTP-induced muscle activity increases during the first 10 rain and then persists as a plateau for at least one hour. Responses of peak amplitude were obtained twenty days after transection. The effect of 5-HTP in the chronic rat (more than 15 days after spinal transection) was completely suppressed in 2 rats by RO-4-4602 (800 mg/kg i.p.) N-DL-SerylN'- (2,2,4, trihydroxybenzyl) hydrazine (Hoffman LaRoche) injected 30 rain beforehand. At such doses, this drug is a potent inhibitor of the L-aromatic decarboxylase in the peripheral but also in the central nervous system4. The following drugs were tried in chronic spinal rats (more than 15 days after transection) and were without effect: 5-hydroxytryptamine(Calbiochem) 10 mg/kg i.p. itself and its main metabolite 5-hydroxyindole acetic acid (5-HIAA) (Calbiochem) at a dose of l0 mg/kg i.p. Other drugs were also without effect in the same conditions: imipramine 15 mg/kg (Ciba-Geigy), and fluoxetine, 50 mg/kg (Lilly), two inhibitors of the reuptake of 5-HT; L-dihydroxyphenylalanine 0--DOPA), 100 mg/kg, (Hoffman LaRoche), dissolved in the same manner and at the same pH as 5-HTP; apomorphine hydrochloride, 0.5 mg/kg (Merck, Sharp and Dohme); tetrabenazine, 20 mg/kg (Hoffman LaRoche). On the other hand, phenoxybenzamine (Hoffman LaRoche), at a dose of 4 mg/kg, did not block the stimulating action of DL-5-HTP, 100 mg/kg. Our results confirm the previous reports of a stimulating effect of 5-HTP on the spontaneous EMG activity of the hindlimbs in spinalized animals3, 9. They strongly suggest that the active substance is the 5-HT that is formed inside the central nervous system by the decarboxylation of the injected 5-HTP. This is demonstrated by the lack
396 of effect of 5-HT itself, injected systemically, of its main metabolite (5-HIAA) and of substances known to increase the effects of endogenous 5-HT (tetrabenazine, imipramine and fluoxetine). The possibility that 5-HT acted non specifically on non serotoninergic receptors (dopamine, noradrenaline, ACh, GABA, etc.) is not entirely excluded but is unlikely as L-DOPA and apomorphine were without effect and also in view of the powerful blocking action of cyproheptadine hydrochloride (10 mg/kg) but not of phenoxybenzamine. It is interesting to note the lack of action of L-DOPA on spontaneous EMG activity as opposed to reflexes2. The most striking element in our results is the progressive increase in the amplitude of the motor response to the same dose of 5-HTP during the 30 days following spinal transection. One possible interpretation of this fact is that the effect of 5-HT on its receptors on spinal neurons does not change after transection but that the excitability of motor neurons is increased for other reasons. There is in fact a 4-fold increase in the baseline level of E M G activity during the 15 days that follow transection. An alternative explanation is that 5-HT produces a greater effect because of denervation supersensitivity of 5-HT receptors. Our own results do not allow us at present to choose between these two explanations but the latter hypothesis is supported by the results of Nygren and Olson 8 who destroyed 5-HT pathways chemically but observed an increase in extensor reflexes studied semi-quantitatively one day after spinal transection. The use of integrated spontaneous EMG activity has more quantitative value than subjective estimation of the amplitude of reflexes. The dramatic increase of the motor response to 5-HTP, whatever is its exact mechanism, makes the chronic spinal preparation a very sensitive model for studying serotoninergic mechanisms in the central nervous system without having to use potentiating drugs. Moreover, the complete disappearance of the 5-HT axons will allow us to distinguish pre- from postsynaptic effects of pharmacological agents. This research was supported by the Medical Research Council of Canada. P.B. was a Scholar of the Canadian Life Insurance Association.
1 Anden, N. E,, Distribution of monoamines and dihydroxyphenylalanine decarboxylase activity in the spinal cord, Acta physiol, scand., 64 (1965) 197-203. 2 Anden, N. E., Jukes, M. G. M. and Lundberg, A., Spinal reflexes and monoamine liberation, Nature (Lond.), 202 (1964) 1222-1223. 3 Anderson, E. G. and Shibuya, T., The effects of 5-hydroxytryptophan and L-tryptophan on spinal synaptic activity, J. Pharmacol. exp. Ther., 153 (1966) 352-360. 4 Bartholini, G., Bates, H. M., Buckard, W. P., and Pletseher, A., Increase of cerebral catecholamines caused by 3-4-dihydroxyphenylalanine after inhibition of peripheral decarboxylase, Nature (Lond.), 215 (1967) 852-853. 5 Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N., Cellular localization of monoamines in the spinal cord, Acta physiol, scand., 69 (1964) 112-119.
397 6 Clineschmidt, B. V., Pierce, J. E. and Sjoerdsma, A., Interactions of tricyclic antidepressants and 5-hydroxyindolealkylamine precursors on spinal monosynaptic reflex transmission, J. PharmacoL exp. Ther., 179 (1971) 312-323. 7 Martin, W. and Eades, C. B., The action of tryptamine on the dog spinal cord and its relationship to the agonistic actions of LSD-like psychotogens Psyehopharmaeologia (BerL), 17 (1970) 242 -257. 8 Nygren, L., Fuxe, K., Jonsson, G. and Olson, L., Functional regeneration of 5-hydroxytryptamine nerve terminals in the rat spinal cord following 5,6-dihydroxytryptamine induced degeneration, Brain Research, 78 (1974) 377-394. 9 Shibuya, T. and Anderson, E. G., The influence of chronic cord transection on the effects of 5-hydroxytryptophan, L-tryptophan and pargyline on spinal neuronal activity, J. Pharmaeol. Exp. Ther., 164 (1968) 185-190.
