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Effects of agonists and antagonists of serotonin on spontaneous hindlimb EMG activity in chronic spinal rats
Neuropharmircobgy Vol. 20, pp. 99 to 107 Pergamon Press Ltd 1981. Printed in Great Britain
EFFECTS OF AGONISTS AND ANTAGONISTS OF SEROTONIN ON SPONTANEOUS HrNDLIMB EMG ACTIVITY IN CHRONIC SPINAL RATS* H. BARBEAU,M. FILION and P. BEDARD~ Departments of Physiology and Anatomy, FacultC de Mtdecine, Universit6 Lavai, Centre de recherche en neurobiologie, H&pita1 de I’Enfant-J&s, 1401, 1Se rue Q&bee, Que. GlJ 124, Canada (Accepted 5 July 1980)
Summary-After spinal transection at elicited discharges in both Rexor and ively after the transection until there increase may correspond in part to
the T-5 level in tats, 5-hydroxytryptophane (S-HTP) 100 mgjkg (i.p.) extensor mu&es of the hindlimbs. This effect increased progresswas a lo-fold increase of the response at the twentieth day. This denervation supersensitivity. The effect of 5-HTP could be suppressed by cyproheptadine, pizotiline, SQ 10 631 (a cinanserin derivative) chlorpromazine and also by methysergide. It could be mimicked by LSD, 5 MEDOMT, BOL 148, quipazine, and to some extent by tryptamine and methysergide. Drugs having a presynaptic action and drugs acting on noradrenaline, dopamine, acetylcholine receptors were without effect. The present model of integrated electromyography (EMG) in chronic spinal rats is a simple and sensitive method to study agonists and antagonists of 5-hydroxytry~tamine (S-HT) in the lumbar segment of the spinal cord. The absence of S-HT terminals makes it possible to distinguish drugs acting on presynaptic mechanisms.
5-Hydroxytryptamine (S-HT) was first detected in the spinal cord with biochemical techniques by Amin, Crawford and Gaddum (1954). This observation was confirmed and extended by several groups (Anderson and Holgerson, 1966; Carlsson, Magnusson and Rosengren, 1963). With the histofluorescence technique, it was demonstrated that 5-HT is located in neuronal cell bodies of the medulla oblongata, in axons coursing downward and ending around anterior horn cells, and also in the posterior and lateral horns (Carlsson, Falck, Fuxe and Hillarp, 1964; Chouchkov, 1974). This same pattern of distribution of serotoninergic terminals was confirmed recently by Segu and Calas (1978) using quantitative radioautography. Following transection of the spinal cord, the level of 5-HT below the lesion initially increases, then drops in five days (And& Haggendal, Magnusson and Rosengren, 1964; Magnusson, 1973) while there is a rapid decrease of the 5-hydroxyindoiea~tic acid (S-HIAA) level. In the spinal rat, S-hydroxytryptophan (S-HTP) the immediate precursor of S-HT increases reflexes in extensor muscles and, in large doses, induces athetoid movements of the hindlimbs (Anden, Carlsson and Haggendal, 1969). In spinalized animals (cats, frogs
and rats), S-HTP, has been shown to increase monosynaptic reflexes (MSR) but to decrease polysynaptic reflexes (Anderson and Shibuya, 1966; Saade, Chanelet and Lonchampt, 1971). Dorsal root potentials are decreased by 5-HTP in the rat (Anden, Jukes, Lundberg and Vyklicky, 1966). In a previous study using the integrated EMG, it was shown that 5-HTP enhanced the spontaneous motor activity of the paralysed hindlimbs of chronic spinal rats (Bedard, Barbeau, Barbeau and Filion, 1979). Several compounds are known to mimic or block the effect of 5-HT in both the peripheral and central nervous system presumabIy by acting on serotonergic receptors. Most of these drugs, however, whether they are recognized mostly as agonists or antagonists, may have the opposite effect, depending on several factors, including dose, time species and organ under study. It appeared that the present model, in view of its’ relative simplicity and great sensitivity, would be useful to study qualitatively and quantitatively drugs which mimic (agonists) and drugs which block (antagonists) the effect of 5-HT on neurons of the lumbar segment of the spinal cord. The absence of 5-HT terminals below the lesion should make it possible to ~stinguish drugs that act on presynaptic from those acting on postsynaptic mechanisms.
*Supported by the Medical Research Council of Canada. t Scholar of the Canadian Life Insurance Association. Key words: serotonin (agonists-antagonists), rat spinal cord.
_ __-__^-MOIHUUY
NP. 20;2 -A
In a group of 30 female Wistar rats weighing between 200 and 3OOg, a complete spinal transection 99
loo
H. BARBEAUet al.
Fig. 1. Schematic drawing of the restraining device used to record EMG activity in chronic spinal rats. The animal is maintained on a padded platform by means of wide elastic bands, its hindlimbs hanging through holes in the platform; EMG wires are run subcutaneously to a connector fixed to the back of the animal.
was performed at the T-5 level, under sodium thiamylal anesthesia. This was done by microdissection of vessels and of nervous tissue under visual control through a dissection microscope. The completeness of the section was further verified post mortem by visual and histological examination. All animals received 200,ooO I.U. of penicillin-G intramuscularly immediately after the intervention. For the next 10 days the rats were housed in individual cages and their bladder emptied manually twice a day. At intervals varying from 1 to 25 days after the section, the skin was opened around the right thigh and silver wires twisted around the right quadriceps and biceps femoris. A reference electrode was fixed to the hip bone. These electrodes were then linked by insulated wires coursing under the skin to a connector fixed on the back of the animal (Fig. 1). 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 EMG activity (Fig. 3). The number of deflections per period of 5 min was counted to produce the graphs in Figures 4 and 5. During the recording sessions, the animal was placed on a small padded platform (Fig. 1) with access to food and water and with the paralysed hindlimbs hanging through holes. After a period of acclimatization of 20 min, the activity was recorded during the following 20min and considered as baseline activity. The effect of the drugs under evaluation was estimated by comparing the number of peaks/5 min observed after the injection to values obtained during the 20 min preceding the injection. Except for apomorphine, which was injected subcutaneously, all drugs were injected intraperitoneally unless stated otherwise. They were given to animals which had been spinalized at least 15 days previously. They were dissolved in saline containing a minimal quantity of HCl: L-tryptophan: 100 mg/kg (Calbiothem); or_-5-hydroxytryptophan (DL-5-HTP): 3 or 100 mg/kg (Sigma); serotonin creatine sulfate (5-HT): 10 mg/kg (Calbiochem); 5-hydroxyindoleacetic acid
(5-HIAA): 10 mg/kg (Calbiochem); L-dihydroxyphenylalanine (L-DOPA): 100 mg/kg (Hoffman LaRache); N-DL-seryl-N’-(2,-2,-4, trihydroxybenzyl) hydrazine (RO-4-4602): 800 mg/kg (HoffmanLaRoche); thiamylal sodium (Surital): 3.0 mg/kg 2.5’\& (ParkeDavis); apormorphine hydrochloride 0.5 mg/kg (Merck, Sharp & Dohme); tetrabenazine: 20 mg/kg (Hoffman-LaRoche); phenoxybenamine: 4 mg/kg (Hoffman-LaRoche); clonidine: 1.0 mg/kg (Boehringer-Ingelheim); fenfluramine : 15 mg/kg (Servier); Arecoline hydrobromide: 10 mg/kg (Sigma); o-lysergic acid diethylamide (LSD): 0.01, 0.05, 0.1, 0.5 and l.Omg/kg (Sandoz Ltd, Basle); (5-methoxy N-Ndimethyltryptamine (5 MeO-DOMT): 5 mg/kg (Sigma); tryptamine: 10 mg/kg (Sigma); methysergide maleate: l-3-5 and 10 mg/kg (Sandoz Ltd, Basle), mescaline: 3 mg/kg (Aldrich); fluoxetine HCl: 5 mg/kg (Lilly), imipramine: 15 mg/kg (Ciba-Geigy); amitryptiline: 10 mg/kg (Merck Frosst Laboratories); chlorimipramine: 15 mg/kg (Ciba-Geigy, Basle); quipazine: 5-50 mg/kg (Miles laboratories); harmaline hydrochloride: l-200 mg/kg (Fluka Basle); cyproheptadine: lOmg/kg (Merck, Sharp & Dohme); (S 10-631: 2-chloro-2’-(3 dimethylaminopropylthio) cinnamanilide hydrochloride 4mg/kg (E. R. Squibb & Sons Ltd): pizotilin 10 mg/kg (Sandoz Ltd; chlorpromazine: 50 mg/kg (Poulenc Ltd); brom-LSD (Bol-14% HTA): 1.5 mg/kg (Sandoz S.A. Basle). At various intervals after spinal transection, some of the animals were sacrificed by decapitation; segments of the spinal cord above and below the section were dissected out for determination of 5-HT. according to the method of Maickel, Cox, Saillant and Miller (1968). RESULTS Histological In the rats sacrificed 30 days after spinal cord transection there was a marked degeneration of all descending tracts below the lesion. Only the posterior funiculus, made up of ascending fibers. appeared spared. The large motoneurons in the ventral and lateral horns were intact. Above the transection all tracts, and especially the posterior funiculus, appeared degenerated. Biochemical There was a marked decrease in the level of 5-HT 4 days after the transection (Fig. 2). As reported previously by Magnusson (1973) there was a concomitant increase of the 5-HT level above the lesion.
Pharmacological
As reported elsewhere (Bedard et a/., 1979) 5-HTP, lOOmg/kg (i.p.) had a rapid effect on the EMG activity (Fig. 3) that increased progressively in the twenty days following the lesion (Fig. 4).
5-HT and the rat spinal cord
101
5-HYDROXYTRYPTAMINE
1
N
3
5
7
9
DAYS
11
Fig. 2. The effect of spinal transection on the levels of 5-HT above (solid line) and below (dotted line) the lesion. Note that within 4-S days, the level of 5-HT in the lumbar segment fell below the limit of
sensitivity of the method of Maickel, while there was a relative increase in the level of S-HT above the lesion. Each point is the mean of three to 6 determinations + SEM. Drugs which block the effect of .5-HTP Figure 3 shows that the effect of 5-HTP (100 mg/kg) in chronic spinal rats was completely antagonized by cyproheptadine (10 mg/kg). This suppression lasted more than one hour. Pizotilin (BC-105) (10 mg/kg), a derivative of cyproheptadine, also antagonized completely the effect of 5-HTP (Table 1). Another derivative of cyproheptadine, SQ-10631
#
w-n’
100
(4mg/kg) (Fig. 5), had a similar but much briefer effect on 5-HTP-induced motor activity. Note that the effect of SQ-10631 lasted only 12-l 5 min and subsequently the action of 5-HTP rapidly returned to maximal level. Chlorpromazine (50 mg/kg) also antagonized the effect of 5-HTP. The same drugs were given 30min prior to 5-HTP and completely prevented its effect. However, SQ-10631 was probably ineffective because of its short action (see Fig. 5).
t@*mG +
mf’RG
WIG/KG
Fig. 3. Electromyograph tracings of the flexor (F) and extensor (E) muscles of the right hindlimbs of a chronic spinal rat. The two middle tracings are the Integrated EMGs of respectively, the extensor (EI) and flexor (FI) muscles. Note the rapid effect of S-HTP, which is of comparable amplitude in both the extensor and flexor muscles. Note also the rapid return to the baseline level of activity after cyproheptadine lOmg/kg. The number of peaks/5 min periods on the integrated EMG tracings was used for quantification in the next figures.
H.
102
BARBEAU
PROGRESSIVE %
OF
INCREASE
MOTOR RESPONSE A SPINAL CORD
CONTROL
et
al.
IN
TO SHTP TRANSSECTION
THE FOLLOWING
1500 1
0
l-4
S-IO
13-15
19-30
DAYS AFTER TRANSSECTION
Fig. 4. Integrated EMG activity in peaks/S
min (see Fig. 3) expressed in percent of the baseline level after a standard dose (100 mg/kg, i.p.) of DL-5-HTP. Note the progressive increase in the response after spinal transection with a maximum reached at about the 2lX-30 days. Each bar represents the mean of at least 5 experiments k SEM.
Drugs which mimic the effect oj5-HTP acid diethylamide at doses of D-Lysergic 0.01-l mg/kg, quipazine, 10 mg/kg, 5 MeO-DMT, 5 mg/kg and fenfluramine, 15 mg/kg, all had a marked 5-HTP-like action ranging between 500 and 900% of the baseline level (Figs 5 and 6). It was found however that when methysergide was given at doses of 5 and 10 mg/kg after 5-HTP (100 mg/kg), it markedly decreased the motor response to the latter drug (Fig. 5F). The response to increasing doses of LSD
Table 1. The effects of different drugs on the activity hindlimbs,
Treatment DL-5-HTP 5-ME DOMT Tryptamine Methysergide LSD Quipazine BOL Harmaline Mescaline Fluoxetine Imipramine Amitryptiline Fenfluramine Cyproheptadine Pizotiline SQ 10,631* Chlorpromazine Phenoxybenzamine
in chronic
iv 5 3 3 3 4 3 3 4 4 3 3 3 3 4 4 3 3 3
spinal rats (see text) Dose (mg/kg)
On effect activity of hindlimbs
100.0 5.0 100.0 5.0 0.5 10.0 1.5 20.0 5.0 5.0 15.0
s S S S+B S S S
10.0 15.0 10.0 10.0 4.0 50.0 4.0
S B B B B
N: number of experiments. S: stimulates motor activity in hindlimbs. B: blocks or decreases markedly the effect of 5-HTP. (-): No effect.
of
(0.01-l mg/kg/pg) grew exponentially producing a dose-response curve R = 0.94 (Fig. 6). Tryptamine, at lOOmg/kg, had a weak effect and its action lasted only 30min. The EMG activity was increased by BOL-148 by 200% for more than 1 hr. Methysergide had a dual effect. Given alone, at doses ranging between 1 and lOmg/kg (i.p.), it had a 5-HT-like action on motor activity, although it was somewhat weaker (between 100 and 300% of the baseline level Fig. 6B). However, as has been mentioned in the preceding paragraph, methysergide also had S-HT blocking properties when given before or after 5-HTP. Drugs believed to act via presynaptic mechanisms Mescaline at 5 mg/kg, imipramine (15 mg/kg), fluoxetine (5 mg/kg) and amitryptiline (10 mg/kg), all inhibitors of the reuptake mechanism, had no effect in this preparation. Harmaline, a reversible monoamine oxidase inhibitor (10 mg/kg) and tetrabenazine, a short acting reserpine-like compound (20 mg/kg) were also without effect. Drugs aficting
mainly other neurotransmitters
Such drugs were given to investigate whether the effects observed after 5-HTP and related agonists were specific or could be mimicked by stimulation of receptors or pathways using other neurotransmitters. Precursors or agonists of the following were administered: Noradrenaline: L-DOPA, 100 mg/kg, Clonidine (100 mg/kg); Dopamine: L-DOPA, 100 mg/kg, apomorphine hydrochloride 0.5 mg/kg; Acetylcholine: arecoline 10 mg/kg; GABA: Baclofen 10 mg/kg. None produced any stimulation of spontaneous motor activity of the hindlimbs. Phenoxybenzamine (4 mg/kg) and atropine, 5 mg/kg, were without effect
5-HT and the rat spinal cord %
OF
1500
SPINAL
CONTROL
103
RAT
1
E
1000-
1
1
1
500-
100 I -20
I -10
t 0
I
IO
SMEDOMT %
OF
CONTROL
%
OF
CONTROL
I
I
50
1,
70
MINUTES
5MG/KG SPINAL
LSD
I 30
RA7
0 SMG/KG SPINAL
RAl
(C) 1500
1000
500
-20
-10
0
IO METHY SMG/KG
70 CYPRO
IOMG/KG
MINUTES
%
OF
SPINAL
WROL
RAT
0
4 I
i
-20
8
WTP 46 OF
8
0 IOOMG/KG
% OF CONTROL
,
7o#@MnEs
t&/KG
50
SCJ ~.6JI
SPINAL
1
RA7
30
SHTP
I
so
CYPGQ 0
iCWG/KG SPINAL
-lo
8
50
CONTROL
-20
4
i
1
lo
0
-0
70
MWUTES
4MG/KG
RAT
(F)
-20
-10
0
5HTP
30
IO
IOOMG/KG
50
70 MINUTES
METHY 5MG/KG
Fig. 5. The effect of different drugs which mimic or block the effect of 5-HTP on the EMG activity of the right quadriceps femoris in chronic spinal rats. In Figure 5(A), (B) and (C) are shown three examples of tracings obtained with drugs which mimic the effect of 5-HTP. (A = SMeO-DOMT. B = LSD, C = ~ethysergide.) In (D). (E) and (F) are shown the effect of three drugs (D = cyproheptadine, E = SQ10631. F = methysergide) which can inhibit the effect of 5-HTP IO0 mg/kg). 104
105
5-HT and the rat spinal cord %
OF
SPINAL
CONTROL
RAT
*
R=O
94
.
LSD
%
OF
SPINAL
CONTROL
RAl
(B) *
*
R=O 95
METHYSERGIDE
:
100,
10, I
05
,
I 06
I
I
07
I
1
OE
I
I 09
I
1
LOG
IO
DOSE
Fig. 6. Dose-response curve describing the effect of: (A) LSD 25; (B) Methysergide. The asterisk represents the EMG activity in peaks/5 min expressed as percentage of the baseline level for one experiment, plotted against the log 10 of the dose. The larger dots represent the calculated mean for each dose. Note that the curves fit the straight line that would be expected of dose-response curves. The linear regression coefficient (R) is given for each curve.
on the motor 5-HTP.
activity
elicited
by lOOmg/kg
of DL
DISCUSSION
As reported previously (Bedard et al., 1979), S-HT and S-HIAA injected systemically do not reproduce the effect of 5-HTP, and inhibition of the L-aromatic decarboxylase prevents the effect of 5-HTP. This sug-
gests that 5-HTP is decarboxylated inside the central nervous system and that the active compound is 5-HT itself. A striking element in the present 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 (Fig. 4). A possible explanation of this phenomenon is that 5-HT produces a greater effect because of denervation supersensitivity
106
H. BARBEAU et al.
of 5-HT receptors. This suggestion is supported by the findings of Nygren, Fuxe, Jonsson and Olson (1974) who studied semi-quantitatively the amplitude of extensor reflexes 1 day after spinal transection in tats. In their work, previous administration (15 days or more) of 5-6-dihydroxytryptamine (5-6DHT) produced a marked increase in the amplitude of the response which however, returned to baseline level as the 5-HT levels increased again 3 months after the administration of 5-6DHT. In the rat spinal cord, quipazine, tryptamine, fenfluramine, LSD, 5-MeO-DOMT, BOL 148 and methysergide mimicked the effect of 5-HTP. Harmaline, mescaline, fluoxetine, imipramine and amitryptiline given in a single dose did not have any effect. This suggests that in order to be active the latter drugs required the presence of 5-HT axons, which were all degenerated in the present preparation. Mescaline was thought by Haigler and Aghajanian (1973) to be a direct agonist at the postsynaptic 5-HT receptor. The present authors have to agree with Maj, Palider and Rawlow (1977) that the action of mescaline on the 5-HT system, at least in the spinal cord, is mostly presynaptic. Blockers of the cyproheptadine family (SQ 10631, pizotiline) appear to be the most potent in this model. Methysergide and LSD, are both considered as potent blockers of 5-HT receptors in the periphery. In this preparation, LSD had agonistic properties at all doses and even the largest dose (1 mg/kg) was ineffective in blocking the effect of 5-HTP. As LSD is very lipid soluble and readily crosses the blood brain barrier, the difference observed between the peripheral and central effects is probably not due to the fact that a smaller dose is reaching the 5-HT receptors in the central nervous system. The fact that LSD blocks the 5-HTP-induced increase in MSR in the cat (Banna and Anderson, 1968) and that LSD is not as pctent as 5-HTP in inducing the Wet Dog Shakes (Bedard and Pycock, 1977) may be due to species or dose differences and/or concomitant stimulation of other receptors (e.g. dopaminergic) by LSD. Tryptamine was found by Martin and Eades (1970) in chronic spinal dogs and by Nozaki and Bell (1976) in rats, to stimulate, like 5-HTP, flexor reflexes. Both reports stated that the effect of tryptamine but not that of 5-HTP was antagonized by cyproheptadine. The presence of two different receptors was therefore suggested. The present results with a slightly larger dose of cyproheptadine do not permit such a distinction and suggest a common site of action. Methysergide on the other hand had some 5-HT blocking properties as it markedly decreased the effect of 5-HTP at doses of 5 and 10 mg/kg. However, at doses ranging between 1 and 10 mg/kg, methysergide also had some 5-HT-like properties, which increased in a dose-dependent manner (R = 0.95) (Fig. 6B). Methysergide therefore appears to have, in the same dose range, both agonistic and antagonistic properties on the 5-HT receptors of the lumbar neurons. The
agonistic effect confirms clinical results obtained by Bedard and Bouchard (1974). Fanciullaci, Granchi and Sicuteri (1976) also found methysergide to be a partial agonist of 5-HT receptors in vascular smooth muscles in man. This effect of 5-HTP is not reproduced by drugs acting on other systems (e.g. catecholamines, acetylcholine, GABA) and is antagonized by cyproheptadine and related drugs. It is therefore concluded that in the rat spinal cord, LSD, tryptamine, methysergide, fenfluramine, 5-Me DOMT and quipazine act as 5-HT agonists and that their effects are antagonized by several 5-HT antagonists, such as cyproheptadine, pizotitine, cinanserin and chlorpromazine. Methysergide has a mixed action. The present model of the chronic spinal rat is a relatively simple and sensitive method to study qualitatively and quantitatively agonists and antagonists of the effect of 5-HT on neurons of the lumbar segment of the spinal cord. It is in fact possible that the same drug may behave as an agonist or an antagonist depending not only on the species under study but also on the region of the CNS being investigated. The absence of 5-HT terminals below the lesion allows drugs that act on presynaptic to be distinguished from those acting on postsynaptic mechanisms. Ackrlowledyenlents-We wish to thank Mrs L. Bertrand, G. GagnC, Mr G. Guano and J. Simard for technical assistance, and M. Baron for secretarial assistance.
REFERENCES Amin, A. H., Crawford, T. B. and Gaddum, J. H. (1954). The distribution of substance P and 5-HT in the central nervous system of the dog. J. Physiol., Land. 126:59&618. Anden, N. E., Carlsson, N. A. and Haggendal, L. J. (1969). Adrenergic mechanisms. A. Rev. Pharmnc. 9: 119-134. Anden, N. E., Haggendal, J., Magnusson, T. and Rosengren, E. (1964). The time course of disappearance of noradrenaline and Shydroxytryptamine in the spinal cord after transection. Acta physiol. stand. 62: I 15-I 18. Anden, N. E. Jukes, M., Lundberg, A. and Vyklicky, L. (1966). The effect of DOPA on the spinal cord. I. Influence on transmission from primary afferents. Acta physiol. stand. 67: 3733386. Anderson, E. G. and Holgerson, L. 0. (1966). The distribution of Shydroxytryptamine and norepinephrine in cat spinal cord. J. Neurochem. 13: 479485. Anderson, E. G. and Shibuya, T. (1966). The effects of Shydroxytryptophan and I-tryptophan on spinal synaptic activity. J. Pharmac. exp. Ther. 153: 352-360. Banna, N. R. and Anderson, E. G. (1968). The effects 01 5-hydroxytryptamine antagonist on spinal neuronal activity. J. Pharmac. exp. Ther. 162: 319-325. BCdard, P., Barbeau, H., Barbeau, G. and Filion, M. (1979). Progressive increase in the motor response to S-HT in the rat after chronic section of the spinal cord. Bruin Res. 169: 393-397. Bedard, P. and Bouchard, R. (1974). Methysergide and myoclonus. Lnncet 7860 1: 788. Bedard, P. and Pycock, C. J. (1977). Wet-dog shake behaviour in the rat: a possible quantitative model of central 5-hydroxytryptamine. Neuropharmacology 16: 663-670.
S-HT and the rat spinal cord Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N. (1964). Cellular localization of monoamines in the spinal cord. Acta pkysiol. stand. 69, 112-l 19. Carisson, A., Magnusson, T. and Rosengren, E. (1963). 5-ydroxytryptamine of the spinal cord normally and after transection. Experientia 17: 359. Chouchkov, Ch. N. (1974). Zur lokalisation von Biogenen amin im riivckenmark der Ratte. Histochemistry 41: 167-173. Fanciullacci, M., Granchi, G. and Sicuteri, F. (1976). Ergotamine and methysergide as serotonin partial agonists in migraine. Headacke 16: 226-23 1. Haigler, H. J. and Aghajanian, G. K. (1973). Mescaline and LSD: direct and indirect effects on serotonin-containing neurons in brain. Eur. J. Pharmac. 21: 53-60. Magnusson, T. (1973). Effect of chronic transection on Dopamine, Noradrenaline and %hydroxytryptamine in the rat spinal cord. Naunyn-Sckmiedebergs Arch. Pkarmat. 278: 13-22.
Maickel, R. P., Cox, R. H. Jr, Saillant, J. and Miller, F. P. (1968). A method for the determination of serotonin and norepinephrine in discrete areas of rat brain. Int. .I. Neuropharmac. 7: 275-281.
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Maj, J., Palider, W. and Rawlow, A. (1977). The influence of mescaline on the flexor reflex of the limb of the spinal rat. J. Pkarm. Pkarmac. 26: 177-178. Martin, W. R. and Eades, C. G. (1970). The action of tryptamine on the dog spinal cord and its relationship to the agonist actions of LSD-like psychotogens. Psyckopkarmacologia 17: 242-257.
Nozaki, M. and Bell, J. A. (1976). Responses of flexor reflex to LSD, tryptamine, 5-HTP, methoxamine and Damphetamine in acute and chronic spinal rats. Fedn Proc. Fedn Am. Sees exp. Biol. 35: 270.
Nygren, L., Fuxe, K., Jonsson, G. and Olson, L. (1974). Functional regeneration of 5-hydroxytryptamine nerve terminals in the rat spinal cord following S-6-dihvdroxvtryptamine induced- degeneration. grain R&. 7& 377-394.
Saade, N., Chanelet, J. and Lonchampt, P. (1971). Application de la technique de l’injection micrortgionale zI l’etude de l’action de la strotonine sur i’activitti rbflexe de la grenouille spinale. Sot. Biol. 165 (4): 784-792. Segi, L. and Calas, A. (1978). The topographical distribution of serotoninergic terminals in the spinal cord of the cat: quantitative radioautographic studies. Brain Res. 153 : 449-464.
EFFECTS OF AGONISTS AND ANTAGONISTS OF SEROTONIN ON SPONTANEOUS HrNDLIMB EMG ACTIVITY IN CHRONIC SPINAL RATS* H. BARBEAU,M. FILION and P. BEDARD~ Departments of Physiology and Anatomy, FacultC de Mtdecine, Universit6 Lavai, Centre de recherche en neurobiologie, H&pita1 de I’Enfant-J&s, 1401, 1Se rue Q&bee, Que. GlJ 124, Canada (Accepted 5 July 1980)
Summary-After spinal transection at elicited discharges in both Rexor and ively after the transection until there increase may correspond in part to
the T-5 level in tats, 5-hydroxytryptophane (S-HTP) 100 mgjkg (i.p.) extensor mu&es of the hindlimbs. This effect increased progresswas a lo-fold increase of the response at the twentieth day. This denervation supersensitivity. The effect of 5-HTP could be suppressed by cyproheptadine, pizotiline, SQ 10 631 (a cinanserin derivative) chlorpromazine and also by methysergide. It could be mimicked by LSD, 5 MEDOMT, BOL 148, quipazine, and to some extent by tryptamine and methysergide. Drugs having a presynaptic action and drugs acting on noradrenaline, dopamine, acetylcholine receptors were without effect. The present model of integrated electromyography (EMG) in chronic spinal rats is a simple and sensitive method to study agonists and antagonists of 5-hydroxytry~tamine (S-HT) in the lumbar segment of the spinal cord. The absence of S-HT terminals makes it possible to distinguish drugs acting on presynaptic mechanisms.
5-Hydroxytryptamine (S-HT) was first detected in the spinal cord with biochemical techniques by Amin, Crawford and Gaddum (1954). This observation was confirmed and extended by several groups (Anderson and Holgerson, 1966; Carlsson, Magnusson and Rosengren, 1963). With the histofluorescence technique, it was demonstrated that 5-HT is located in neuronal cell bodies of the medulla oblongata, in axons coursing downward and ending around anterior horn cells, and also in the posterior and lateral horns (Carlsson, Falck, Fuxe and Hillarp, 1964; Chouchkov, 1974). This same pattern of distribution of serotoninergic terminals was confirmed recently by Segu and Calas (1978) using quantitative radioautography. Following transection of the spinal cord, the level of 5-HT below the lesion initially increases, then drops in five days (And& Haggendal, Magnusson and Rosengren, 1964; Magnusson, 1973) while there is a rapid decrease of the 5-hydroxyindoiea~tic acid (S-HIAA) level. In the spinal rat, S-hydroxytryptophan (S-HTP) the immediate precursor of S-HT increases reflexes in extensor muscles and, in large doses, induces athetoid movements of the hindlimbs (Anden, Carlsson and Haggendal, 1969). In spinalized animals (cats, frogs
and rats), S-HTP, has been shown to increase monosynaptic reflexes (MSR) but to decrease polysynaptic reflexes (Anderson and Shibuya, 1966; Saade, Chanelet and Lonchampt, 1971). Dorsal root potentials are decreased by 5-HTP in the rat (Anden, Jukes, Lundberg and Vyklicky, 1966). In a previous study using the integrated EMG, it was shown that 5-HTP enhanced the spontaneous motor activity of the paralysed hindlimbs of chronic spinal rats (Bedard, Barbeau, Barbeau and Filion, 1979). Several compounds are known to mimic or block the effect of 5-HT in both the peripheral and central nervous system presumabIy by acting on serotonergic receptors. Most of these drugs, however, whether they are recognized mostly as agonists or antagonists, may have the opposite effect, depending on several factors, including dose, time species and organ under study. It appeared that the present model, in view of its’ relative simplicity and great sensitivity, would be useful to study qualitatively and quantitatively drugs which mimic (agonists) and drugs which block (antagonists) the effect of 5-HT on neurons of the lumbar segment of the spinal cord. The absence of 5-HT terminals below the lesion should make it possible to ~stinguish drugs that act on presynaptic from those acting on postsynaptic mechanisms.
*Supported by the Medical Research Council of Canada. t Scholar of the Canadian Life Insurance Association. Key words: serotonin (agonists-antagonists), rat spinal cord.
_ __-__^-MOIHUUY
NP. 20;2 -A
In a group of 30 female Wistar rats weighing between 200 and 3OOg, a complete spinal transection 99
loo
H. BARBEAUet al.
Fig. 1. Schematic drawing of the restraining device used to record EMG activity in chronic spinal rats. The animal is maintained on a padded platform by means of wide elastic bands, its hindlimbs hanging through holes in the platform; EMG wires are run subcutaneously to a connector fixed to the back of the animal.
was performed at the T-5 level, under sodium thiamylal anesthesia. This was done by microdissection of vessels and of nervous tissue under visual control through a dissection microscope. The completeness of the section was further verified post mortem by visual and histological examination. All animals received 200,ooO I.U. of penicillin-G intramuscularly immediately after the intervention. For the next 10 days the rats were housed in individual cages and their bladder emptied manually twice a day. At intervals varying from 1 to 25 days after the section, the skin was opened around the right thigh and silver wires twisted around the right quadriceps and biceps femoris. A reference electrode was fixed to the hip bone. These electrodes were then linked by insulated wires coursing under the skin to a connector fixed on the back of the animal (Fig. 1). 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 EMG activity (Fig. 3). The number of deflections per period of 5 min was counted to produce the graphs in Figures 4 and 5. During the recording sessions, the animal was placed on a small padded platform (Fig. 1) with access to food and water and with the paralysed hindlimbs hanging through holes. After a period of acclimatization of 20 min, the activity was recorded during the following 20min and considered as baseline activity. The effect of the drugs under evaluation was estimated by comparing the number of peaks/5 min observed after the injection to values obtained during the 20 min preceding the injection. Except for apomorphine, which was injected subcutaneously, all drugs were injected intraperitoneally unless stated otherwise. They were given to animals which had been spinalized at least 15 days previously. They were dissolved in saline containing a minimal quantity of HCl: L-tryptophan: 100 mg/kg (Calbiothem); or_-5-hydroxytryptophan (DL-5-HTP): 3 or 100 mg/kg (Sigma); serotonin creatine sulfate (5-HT): 10 mg/kg (Calbiochem); 5-hydroxyindoleacetic acid
(5-HIAA): 10 mg/kg (Calbiochem); L-dihydroxyphenylalanine (L-DOPA): 100 mg/kg (Hoffman LaRache); N-DL-seryl-N’-(2,-2,-4, trihydroxybenzyl) hydrazine (RO-4-4602): 800 mg/kg (HoffmanLaRoche); thiamylal sodium (Surital): 3.0 mg/kg 2.5’\& (ParkeDavis); apormorphine hydrochloride 0.5 mg/kg (Merck, Sharp & Dohme); tetrabenazine: 20 mg/kg (Hoffman-LaRoche); phenoxybenamine: 4 mg/kg (Hoffman-LaRoche); clonidine: 1.0 mg/kg (Boehringer-Ingelheim); fenfluramine : 15 mg/kg (Servier); Arecoline hydrobromide: 10 mg/kg (Sigma); o-lysergic acid diethylamide (LSD): 0.01, 0.05, 0.1, 0.5 and l.Omg/kg (Sandoz Ltd, Basle); (5-methoxy N-Ndimethyltryptamine (5 MeO-DOMT): 5 mg/kg (Sigma); tryptamine: 10 mg/kg (Sigma); methysergide maleate: l-3-5 and 10 mg/kg (Sandoz Ltd, Basle), mescaline: 3 mg/kg (Aldrich); fluoxetine HCl: 5 mg/kg (Lilly), imipramine: 15 mg/kg (Ciba-Geigy); amitryptiline: 10 mg/kg (Merck Frosst Laboratories); chlorimipramine: 15 mg/kg (Ciba-Geigy, Basle); quipazine: 5-50 mg/kg (Miles laboratories); harmaline hydrochloride: l-200 mg/kg (Fluka Basle); cyproheptadine: lOmg/kg (Merck, Sharp & Dohme); (S 10-631: 2-chloro-2’-(3 dimethylaminopropylthio) cinnamanilide hydrochloride 4mg/kg (E. R. Squibb & Sons Ltd): pizotilin 10 mg/kg (Sandoz Ltd; chlorpromazine: 50 mg/kg (Poulenc Ltd); brom-LSD (Bol-14% HTA): 1.5 mg/kg (Sandoz S.A. Basle). At various intervals after spinal transection, some of the animals were sacrificed by decapitation; segments of the spinal cord above and below the section were dissected out for determination of 5-HT. according to the method of Maickel, Cox, Saillant and Miller (1968). RESULTS Histological In the rats sacrificed 30 days after spinal cord transection there was a marked degeneration of all descending tracts below the lesion. Only the posterior funiculus, made up of ascending fibers. appeared spared. The large motoneurons in the ventral and lateral horns were intact. Above the transection all tracts, and especially the posterior funiculus, appeared degenerated. Biochemical There was a marked decrease in the level of 5-HT 4 days after the transection (Fig. 2). As reported previously by Magnusson (1973) there was a concomitant increase of the 5-HT level above the lesion.
Pharmacological
As reported elsewhere (Bedard et a/., 1979) 5-HTP, lOOmg/kg (i.p.) had a rapid effect on the EMG activity (Fig. 3) that increased progressively in the twenty days following the lesion (Fig. 4).
5-HT and the rat spinal cord
101
5-HYDROXYTRYPTAMINE
1
N
3
5
7
9
DAYS
11
Fig. 2. The effect of spinal transection on the levels of 5-HT above (solid line) and below (dotted line) the lesion. Note that within 4-S days, the level of 5-HT in the lumbar segment fell below the limit of
sensitivity of the method of Maickel, while there was a relative increase in the level of S-HT above the lesion. Each point is the mean of three to 6 determinations + SEM. Drugs which block the effect of .5-HTP Figure 3 shows that the effect of 5-HTP (100 mg/kg) in chronic spinal rats was completely antagonized by cyproheptadine (10 mg/kg). This suppression lasted more than one hour. Pizotilin (BC-105) (10 mg/kg), a derivative of cyproheptadine, also antagonized completely the effect of 5-HTP (Table 1). Another derivative of cyproheptadine, SQ-10631
#
w-n’
100
(4mg/kg) (Fig. 5), had a similar but much briefer effect on 5-HTP-induced motor activity. Note that the effect of SQ-10631 lasted only 12-l 5 min and subsequently the action of 5-HTP rapidly returned to maximal level. Chlorpromazine (50 mg/kg) also antagonized the effect of 5-HTP. The same drugs were given 30min prior to 5-HTP and completely prevented its effect. However, SQ-10631 was probably ineffective because of its short action (see Fig. 5).
t@*mG +
mf’RG
WIG/KG
Fig. 3. Electromyograph tracings of the flexor (F) and extensor (E) muscles of the right hindlimbs of a chronic spinal rat. The two middle tracings are the Integrated EMGs of respectively, the extensor (EI) and flexor (FI) muscles. Note the rapid effect of S-HTP, which is of comparable amplitude in both the extensor and flexor muscles. Note also the rapid return to the baseline level of activity after cyproheptadine lOmg/kg. The number of peaks/5 min periods on the integrated EMG tracings was used for quantification in the next figures.
H.
102
BARBEAU
PROGRESSIVE %
OF
INCREASE
MOTOR RESPONSE A SPINAL CORD
CONTROL
et
al.
IN
TO SHTP TRANSSECTION
THE FOLLOWING
1500 1
0
l-4
S-IO
13-15
19-30
DAYS AFTER TRANSSECTION
Fig. 4. Integrated EMG activity in peaks/S
min (see Fig. 3) expressed in percent of the baseline level after a standard dose (100 mg/kg, i.p.) of DL-5-HTP. Note the progressive increase in the response after spinal transection with a maximum reached at about the 2lX-30 days. Each bar represents the mean of at least 5 experiments k SEM.
Drugs which mimic the effect oj5-HTP acid diethylamide at doses of D-Lysergic 0.01-l mg/kg, quipazine, 10 mg/kg, 5 MeO-DMT, 5 mg/kg and fenfluramine, 15 mg/kg, all had a marked 5-HTP-like action ranging between 500 and 900% of the baseline level (Figs 5 and 6). It was found however that when methysergide was given at doses of 5 and 10 mg/kg after 5-HTP (100 mg/kg), it markedly decreased the motor response to the latter drug (Fig. 5F). The response to increasing doses of LSD
Table 1. The effects of different drugs on the activity hindlimbs,
Treatment DL-5-HTP 5-ME DOMT Tryptamine Methysergide LSD Quipazine BOL Harmaline Mescaline Fluoxetine Imipramine Amitryptiline Fenfluramine Cyproheptadine Pizotiline SQ 10,631* Chlorpromazine Phenoxybenzamine
in chronic
iv 5 3 3 3 4 3 3 4 4 3 3 3 3 4 4 3 3 3
spinal rats (see text) Dose (mg/kg)
On effect activity of hindlimbs
100.0 5.0 100.0 5.0 0.5 10.0 1.5 20.0 5.0 5.0 15.0
s S S S+B S S S
10.0 15.0 10.0 10.0 4.0 50.0 4.0
S B B B B
N: number of experiments. S: stimulates motor activity in hindlimbs. B: blocks or decreases markedly the effect of 5-HTP. (-): No effect.
of
(0.01-l mg/kg/pg) grew exponentially producing a dose-response curve R = 0.94 (Fig. 6). Tryptamine, at lOOmg/kg, had a weak effect and its action lasted only 30min. The EMG activity was increased by BOL-148 by 200% for more than 1 hr. Methysergide had a dual effect. Given alone, at doses ranging between 1 and lOmg/kg (i.p.), it had a 5-HT-like action on motor activity, although it was somewhat weaker (between 100 and 300% of the baseline level Fig. 6B). However, as has been mentioned in the preceding paragraph, methysergide also had S-HT blocking properties when given before or after 5-HTP. Drugs believed to act via presynaptic mechanisms Mescaline at 5 mg/kg, imipramine (15 mg/kg), fluoxetine (5 mg/kg) and amitryptiline (10 mg/kg), all inhibitors of the reuptake mechanism, had no effect in this preparation. Harmaline, a reversible monoamine oxidase inhibitor (10 mg/kg) and tetrabenazine, a short acting reserpine-like compound (20 mg/kg) were also without effect. Drugs aficting
mainly other neurotransmitters
Such drugs were given to investigate whether the effects observed after 5-HTP and related agonists were specific or could be mimicked by stimulation of receptors or pathways using other neurotransmitters. Precursors or agonists of the following were administered: Noradrenaline: L-DOPA, 100 mg/kg, Clonidine (100 mg/kg); Dopamine: L-DOPA, 100 mg/kg, apomorphine hydrochloride 0.5 mg/kg; Acetylcholine: arecoline 10 mg/kg; GABA: Baclofen 10 mg/kg. None produced any stimulation of spontaneous motor activity of the hindlimbs. Phenoxybenzamine (4 mg/kg) and atropine, 5 mg/kg, were without effect
5-HT and the rat spinal cord %
OF
1500
SPINAL
CONTROL
103
RAT
1
E
1000-
1
1
1
500-
100 I -20
I -10
t 0
I
IO
SMEDOMT %
OF
CONTROL
%
OF
CONTROL
I
I
50
1,
70
MINUTES
5MG/KG SPINAL
LSD
I 30
RA7
0 SMG/KG SPINAL
RAl
(C) 1500
1000
500
-20
-10
0
IO METHY SMG/KG
70 CYPRO
IOMG/KG
MINUTES
%
OF
SPINAL
WROL
RAT
0
4 I
i
-20
8
WTP 46 OF
8
0 IOOMG/KG
% OF CONTROL
,
7o#@MnEs
t&/KG
50
SCJ ~.6JI
SPINAL
1
RA7
30
SHTP
I
so
CYPGQ 0
iCWG/KG SPINAL
-lo
8
50
CONTROL
-20
4
i
1
lo
0
-0
70
MWUTES
4MG/KG
RAT
(F)
-20
-10
0
5HTP
30
IO
IOOMG/KG
50
70 MINUTES
METHY 5MG/KG
Fig. 5. The effect of different drugs which mimic or block the effect of 5-HTP on the EMG activity of the right quadriceps femoris in chronic spinal rats. In Figure 5(A), (B) and (C) are shown three examples of tracings obtained with drugs which mimic the effect of 5-HTP. (A = SMeO-DOMT. B = LSD, C = ~ethysergide.) In (D). (E) and (F) are shown the effect of three drugs (D = cyproheptadine, E = SQ10631. F = methysergide) which can inhibit the effect of 5-HTP IO0 mg/kg). 104
105
5-HT and the rat spinal cord %
OF
SPINAL
CONTROL
RAT
*
R=O
94
.
LSD
%
OF
SPINAL
CONTROL
RAl
(B) *
*
R=O 95
METHYSERGIDE
:
100,
10, I
05
,
I 06
I
I
07
I
1
OE
I
I 09
I
1
LOG
IO
DOSE
Fig. 6. Dose-response curve describing the effect of: (A) LSD 25; (B) Methysergide. The asterisk represents the EMG activity in peaks/5 min expressed as percentage of the baseline level for one experiment, plotted against the log 10 of the dose. The larger dots represent the calculated mean for each dose. Note that the curves fit the straight line that would be expected of dose-response curves. The linear regression coefficient (R) is given for each curve.
on the motor 5-HTP.
activity
elicited
by lOOmg/kg
of DL
DISCUSSION
As reported previously (Bedard et al., 1979), S-HT and S-HIAA injected systemically do not reproduce the effect of 5-HTP, and inhibition of the L-aromatic decarboxylase prevents the effect of 5-HTP. This sug-
gests that 5-HTP is decarboxylated inside the central nervous system and that the active compound is 5-HT itself. A striking element in the present 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 (Fig. 4). A possible explanation of this phenomenon is that 5-HT produces a greater effect because of denervation supersensitivity
106
H. BARBEAU et al.
of 5-HT receptors. This suggestion is supported by the findings of Nygren, Fuxe, Jonsson and Olson (1974) who studied semi-quantitatively the amplitude of extensor reflexes 1 day after spinal transection in tats. In their work, previous administration (15 days or more) of 5-6-dihydroxytryptamine (5-6DHT) produced a marked increase in the amplitude of the response which however, returned to baseline level as the 5-HT levels increased again 3 months after the administration of 5-6DHT. In the rat spinal cord, quipazine, tryptamine, fenfluramine, LSD, 5-MeO-DOMT, BOL 148 and methysergide mimicked the effect of 5-HTP. Harmaline, mescaline, fluoxetine, imipramine and amitryptiline given in a single dose did not have any effect. This suggests that in order to be active the latter drugs required the presence of 5-HT axons, which were all degenerated in the present preparation. Mescaline was thought by Haigler and Aghajanian (1973) to be a direct agonist at the postsynaptic 5-HT receptor. The present authors have to agree with Maj, Palider and Rawlow (1977) that the action of mescaline on the 5-HT system, at least in the spinal cord, is mostly presynaptic. Blockers of the cyproheptadine family (SQ 10631, pizotiline) appear to be the most potent in this model. Methysergide and LSD, are both considered as potent blockers of 5-HT receptors in the periphery. In this preparation, LSD had agonistic properties at all doses and even the largest dose (1 mg/kg) was ineffective in blocking the effect of 5-HTP. As LSD is very lipid soluble and readily crosses the blood brain barrier, the difference observed between the peripheral and central effects is probably not due to the fact that a smaller dose is reaching the 5-HT receptors in the central nervous system. The fact that LSD blocks the 5-HTP-induced increase in MSR in the cat (Banna and Anderson, 1968) and that LSD is not as pctent as 5-HTP in inducing the Wet Dog Shakes (Bedard and Pycock, 1977) may be due to species or dose differences and/or concomitant stimulation of other receptors (e.g. dopaminergic) by LSD. Tryptamine was found by Martin and Eades (1970) in chronic spinal dogs and by Nozaki and Bell (1976) in rats, to stimulate, like 5-HTP, flexor reflexes. Both reports stated that the effect of tryptamine but not that of 5-HTP was antagonized by cyproheptadine. The presence of two different receptors was therefore suggested. The present results with a slightly larger dose of cyproheptadine do not permit such a distinction and suggest a common site of action. Methysergide on the other hand had some 5-HT blocking properties as it markedly decreased the effect of 5-HTP at doses of 5 and 10 mg/kg. However, at doses ranging between 1 and 10 mg/kg, methysergide also had some 5-HT-like properties, which increased in a dose-dependent manner (R = 0.95) (Fig. 6B). Methysergide therefore appears to have, in the same dose range, both agonistic and antagonistic properties on the 5-HT receptors of the lumbar neurons. The
agonistic effect confirms clinical results obtained by Bedard and Bouchard (1974). Fanciullaci, Granchi and Sicuteri (1976) also found methysergide to be a partial agonist of 5-HT receptors in vascular smooth muscles in man. This effect of 5-HTP is not reproduced by drugs acting on other systems (e.g. catecholamines, acetylcholine, GABA) and is antagonized by cyproheptadine and related drugs. It is therefore concluded that in the rat spinal cord, LSD, tryptamine, methysergide, fenfluramine, 5-Me DOMT and quipazine act as 5-HT agonists and that their effects are antagonized by several 5-HT antagonists, such as cyproheptadine, pizotitine, cinanserin and chlorpromazine. Methysergide has a mixed action. The present model of the chronic spinal rat is a relatively simple and sensitive method to study qualitatively and quantitatively agonists and antagonists of the effect of 5-HT on neurons of the lumbar segment of the spinal cord. It is in fact possible that the same drug may behave as an agonist or an antagonist depending not only on the species under study but also on the region of the CNS being investigated. The absence of 5-HT terminals below the lesion allows drugs that act on presynaptic to be distinguished from those acting on postsynaptic mechanisms. Ackrlowledyenlents-We wish to thank Mrs L. Bertrand, G. GagnC, Mr G. Guano and J. Simard for technical assistance, and M. Baron for secretarial assistance.
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S-HT and the rat spinal cord Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N. (1964). Cellular localization of monoamines in the spinal cord. Acta pkysiol. stand. 69, 112-l 19. Carisson, A., Magnusson, T. and Rosengren, E. (1963). 5-ydroxytryptamine of the spinal cord normally and after transection. Experientia 17: 359. Chouchkov, Ch. N. (1974). Zur lokalisation von Biogenen amin im riivckenmark der Ratte. Histochemistry 41: 167-173. Fanciullacci, M., Granchi, G. and Sicuteri, F. (1976). Ergotamine and methysergide as serotonin partial agonists in migraine. Headacke 16: 226-23 1. Haigler, H. J. and Aghajanian, G. K. (1973). Mescaline and LSD: direct and indirect effects on serotonin-containing neurons in brain. Eur. J. Pharmac. 21: 53-60. Magnusson, T. (1973). Effect of chronic transection on Dopamine, Noradrenaline and %hydroxytryptamine in the rat spinal cord. Naunyn-Sckmiedebergs Arch. Pkarmat. 278: 13-22.
Maickel, R. P., Cox, R. H. Jr, Saillant, J. and Miller, F. P. (1968). A method for the determination of serotonin and norepinephrine in discrete areas of rat brain. Int. .I. Neuropharmac. 7: 275-281.
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Nozaki, M. and Bell, J. A. (1976). Responses of flexor reflex to LSD, tryptamine, 5-HTP, methoxamine and Damphetamine in acute and chronic spinal rats. Fedn Proc. Fedn Am. Sees exp. Biol. 35: 270.
Nygren, L., Fuxe, K., Jonsson, G. and Olson, L. (1974). Functional regeneration of 5-hydroxytryptamine nerve terminals in the rat spinal cord following S-6-dihvdroxvtryptamine induced- degeneration. grain R&. 7& 377-394.
Saade, N., Chanelet, J. and Lonchampt, P. (1971). Application de la technique de l’injection micrortgionale zI l’etude de l’action de la strotonine sur i’activitti rbflexe de la grenouille spinale. Sot. Biol. 165 (4): 784-792. Segi, L. and Calas, A. (1978). The topographical distribution of serotoninergic terminals in the spinal cord of the cat: quantitative radioautographic studies. Brain Res. 153 : 449-464.