Non-serotonergic depression of spinal monosynaptic reflex transmission by 5-hydroxytryptophan

Neuropharmocology Vol. 20. pp. 109 lo 114 Pergamon Press Ltd 1981.Printed I” Great

0028.?9OS/El/O201-0loSS02.00/0 Britain

NON-SEROTONERGIC DEPRESSION OF SPINAL MONOSYNAPTIC REFLEX TRANSMISSION BY 5-HYDROXYTRYPTOPHAN Program

in Pharmacology,

E. D. HALL Northeastern Ohio Universities Rootstown, OH 44272, U.S.A. 21 July

(Accepred

College

of Medicine,

1980)

Summary-Administration of a 50mg/kg (i.v.) dose of either L-, or the racemic o,L-mixture of 5-hydroxytryptophan (5HTP) in acute unanesthetized spinal (C-1) cats produced a depression of the lumbar monosynaptic response (MSR) amplitude when the reflex was evoked by supramaximal triceps surae (TS) nerve stimulation. Neither pretreatment with the serotonin receptor antagonists cincanserin or cyproheptadine, nor the L-aromatic amino acid decarboxylase inhibitor methyldopa antagonized the depressant effect of S-HTP on the MSR in TS-stimulated animals. Administration of amitriptyline, a selective serotonin reuptake inhibitor, reversed the depression in the MSR amplitude produced by S-HTP and produced an increase in the MSR above the original pre-5-HTP control level. These data suggest that 5-HTP has a depressant action upon spinal monosynaptic reflex transmission that is unrelated to an increase in spinal serotonergic activity. The site of the depressant effect of 5-HTP within the MSR pathway is postulated to be the Ia afferent terminal. This non-serotonergic 5-HTP action should be considered when interpreting the results of neuropharmacologic studies employing 5-HTP as a serotonin precursor

5-Hydroxytryptophan (5-HTP) is the immediate precursor for the production of 5-hydroxytryptamine (i.e. serotonin) through enzymatic conversion by L-aromatic amino acid decarboxylase in peripheral tissues and the central nervous system. Considering this fact and the ability of 5-HTP to cross readily the blooddbrain barrier in contrast to serotonin which does not, 5-HTP has been employed extensively for the study of CNS serotonergic mechanisms. For instance, numerous studies have used 5-HTP to investigate the modulatory influence of a bulbospinal serotonergic system on segmental spinal reflexes (Anden, Jukes and Lundberg, 1964; Anderson and Shibuya, 1966; Banna and Anderson, 1968; Bedard, Barbeau, Barbeau and Filion, 1979; Clineschmidt, Pierce and Sjoerdsma, 1971; Myslinski and Anderson, 1978; Sastry and Sinclair, 1976a, b; 1977; Shibuya and Anderson, 1968). Anderson and Shibuya (1966) have found that intravenous administration of 5-HTP produces a dose-related increase in cat lumbar spinal monosynaptic reflex (MSR) transmission when the distally-sectioned dorsal root is the site of afferent stimulation. Agents, such as methysergide, cinanserin and cyproheptadine, which are recognized as selective serotonin receptor blockers, have been demonstrated to antagonize the MSR increase by 5-HTP (Banna and Anderson, 1968). Furthermore, pretreatment with inhibitors of L-aromatic amino acid decarboxylase, has been shown to prevent the facilitaKey words: 5-hydroxytryptophan serotonin, cyproheptadine, cinanserin, methyldopa, spinal cord, monosynaptic transmission. 109

tory MSR actions of 5-HTP (Shibuya and Anderson. 1968). Thus, it has been postulated that the excitatory actions of 5-HTP on spinal MSR transmission are mediated through uptake of 5-HTP into the terminals of bulbospinal serotonergic neurons and increased production and spontaneous release of serotonin. The present study, in contrast to those employing predominantly whole dorsal root afferent activation, has examined the spinal MSR actions of 5-HTP in acute spinal cats in which a peripheral motor nerve (i.e. triceps surae) is the site of afferent stimulation. Thus, the experiments reported in this paper involving stimulation of an homologous group of Ia afferent fibers have revealed an additional spinal MSR depressant action of 5-HTP that does not appear related to serotonin production.

METHODS

Thirty-four adult cats of either sex (2-3 kg) were employed in this study. In all cats, a tracheotomy was performed under halothane anesthesia and the spinal cord was transected at the C-l level. Anesthesia was then terminated and positive pressure ventilation with room air begun. One carotid artery was cannulated for blood pressure recording. Both carotids were ligated and the vertebral arteries were mechanically compressed to produce brain ischemia. A dorsal laminectomy exposed the spinal cord from L4 to S2. The popliteal fossa of one hindlimb was dissected to expose the triceps surae (TS) nerves (i.e. soleus, medial and lateral gastrocnemius) for stimulation with bipolar platinum hook electrodes. The re-

110

E. D. HALL

flex response was recorded at the distally transected ipsilateral L7 or Sl ventral roots (VR). Stimuli were square pulses of 0.2 msec duration applied once every 5 set via an isolation unit. The stimulus strength was adjusted to 1.2 times that necessary to evoke the maximum monosynaptic and short latency polysynaptic responses. In 5 experiments, the distally transected L7 or Sl dorsal root (DR) was used for stimulation (OS msec stimulus duration) instead of the TS nerves. The reflex discharges were displayed on a cathode ray oscilloscope. Prior to and at varying times after drug administrations, 10 successive responses were averaged on a Tracer-Northern 1505 signal averager, the display of which was photographed and measured. Complete neuromuscular paralysis was produced using 3 mg/kg gallamine triethiodide intravenously and maintained with subsequent doses as required. Either L- or D,r_-5-hydroxytryptophan (5-HTP) (Sigma) was dissolved in physiological saline with gentle warming (25 mg/ml) and administered in a single dose per animal of 50 mg/‘kg (i.v.) over 10 min via an infusion pump (Anderson and Shibuya, 1966: Clineschmidt et ul., 197 I). Amitriptyline chloride (Merck, Sharp & Dohme) was given in saline in a dose of 5 mg/kg (5 mg/ml, i.v.) over 5 min beginning 60 min after 5-HTP (Clineschmidt et ul.. 1971). In 5 experiments, 200 mg/kg (i.v.) of methyldopa (Sigma) was injected over 10 min beginning 30 min prior to 5-HTP administration. Methyldopa (20 mgiml) was dissolved in saline by warming. In another 5 animals.

140 120

0

DR-VR

n=5

0

TS-VR

n=8

cyproheptadine chloride (Merck, Sharp & Dohme) 2 mg/kg (iv.) was given over 5 min beginning at 15 min before the 5-HTP test dose. In an additional 6 experiments, cinanserin hydrochloride (Squibb) 4 mg/kg (iv.) was given over 5 min at 15 min before 5-HTP. Statistical analyses of the effects of various drug treatments on the MSR responses to 5-HTP, the effects of 5-HTP in the DR-VR and TS-VR and the actions of L-. vs I~,I.-5-HTP were carried out with a repeated measures analysis of variance. In addition. the Student’s t-test (one-tailed) was used to assess differences at individual time points, RESULTS 6jfect.s

of

D,L-5-HTP

in DR-VR

L’S TS-VR

Figure 1 shows the striking difference in the action of U,L-5-HTP in DR-VR vs TS-VR animals. Administration of the 50 mg/kg (i.v.) test dose in the DR-VR preparations produced a progressive increase in the amplitude of the MSR as shown in the top curve (Fig. 1). The average peak increase from 5 animals was 77.0”;, and occurred at 60 min after drug injection. The peak actually approached a maximum plateau at 30min. In preparations in which the TS nerves were the site of supramaximal stimulation, the MSR effects of o.~-5-HTP were consistently depressant rather than facilitatory. In the TS-VR experiments, a progressive

t

;

‘p

50

mg/kg

;o

D.L -

,‘o

4b

;

$0

MINUTES

5-HTP

Fig. 1. Effect of I>,L-5-HTP on the lumbar spinal monosynaptic reflex amplitude in preparations where L7 or SI dorsal root was supramaximally stimulated vs those in which the triceps surae nerves were activated. (Bars represent SEM. II = number of animals.)

the

prep-

arations

111

after D, L- 5-HTP 50 mglkg i. v.

Before

I mV

L

5 msec

Fig. 2. Typical examples of the effects of D,L-S-HTP on the lumbar spinal monosynaptic root-ventral root vs triceps surae nerve-ventral root preparations. Traces are summated successive ventral root responses.

.--

:reflex in dorsal averages

of 10

.-

fi 50 @kg D,L - 5 - HTP

20

1,”

40

50

i30_

70

80

90

5KUk9 Amitriptyline

Fig. 3. Effect of D,L-5-HTP followed by amitriptyline on the lumbar spinal monosynaptic reflex amplitude in triceps surae-ventral root preparations. (Bars represent SEM, II = number of animals.)

E. D.

112

100. 4

2 z E % z :: E

0

After

0

Control

Methyldopo

I

60.

I

40

-

I

20

-

I

200

n=5

n=8

00.

IO

HALL

20

(f

1;

20

30

40

50

6b

50 mg/kg

mg/kg

Methyldopa

70

00

Min

5 mg/kg

D.L-5-HTP

Amitrlptyline

Fig. 4. Effect of IXL-5-HTP on the lumbar spinal monosynaptic reflex amplitude in triceps surae-ventral root animals pretreated with methyldopa (2OOmg/kg infused i.v. over IOmin). (Bars represent SEM. II = number of animals.) decline in the MSR amplitude was seen with a maximum of 41.1% below control at 20 min after administration. After the point of peak decline in the TS-VR cats (30 min), the MSR began to increase slowly up to a level of 23.6% below the initial amplitude by 60 min. The difference in response between the DR-VR and TS-VR preparations was significant by Student’s t-test, single tailed (P < 0.05) at 10, 20, 30 and 40 min after D,L-5-HTP. Figure 2 shows typical examples of the effect of D,L-5-HTP in a DR-VR preparation as compared to a TS-VR one. Eficts

of amitriptyline

after

D,L-5-HTP

in

TS-VR

preparations

Figure 3 illustrates the effects of amitriptyline following D,L-5-HTP in TS-VR preparations. Amitriptyline. which selectively blocks the presynaptic neuronal reuptake of released serotonin (Maas, 1975) produced a rapid reversal of the depressant actions of KI,L-5-HTP as the MSR increased within the 5 min infusion time to a peak level of 43.5% above the pre-D,L-5-HTP level. This facilitatory action of amitriptyline was rather short-lived; however, as the effect waned over the ensuing 20min with the MSR falling toward the original control amplitude. Figure 3 also displays the minimal effects of 5-HTP and amitriptyline on mean arterial pressure. Eflects of c_vproheptadine or cinanserin the D. L-~-H TP monosynaptic

pretreatment

on

depression

Five animals were pretreated with cyproheptadine and another 6 with cinanserin, both selective serotonin receptor antagonists, prior to D, L-5-HTP administration to determine whether the depressant

action of 5-HTP was serotonin nor cinanserin cyproheptadine 5-HTP induced MSR decrease. actually potentiated the maximum the MSR by over 40%, although statistically significant by repeated of variance. Cinanserin pretreatment cause a slight augmentation of the 5-HTP.

mediated. Neither antagonized the Cyproheptadine S-HTP decrease in this effect was not measures analysis also appeared to depressant effect of

Effect

on the D.L-5-HTP

of methyldopa

monosynaptic

pretreatment

depression

In order to test further whether the depressant effects of D,L-5-HTP on the TS-VR MSR transmission were mediated through an increase in spinal serotonergic activity, methyldopa was administered to 5 cats 30min prior to the I,,L-5-HTP challenge. Methyldopa, which is known to block L-aromatic amino acid decarboxylase and thus inhibit the production of serotonin from 5-HTP (Nickerson and Ruedy, 1975). enhanced rather than attenuated the MSR depressant action of subsequent D,L-5-HTP (Fig. 4). The mean depression was greater in the methyldopa-treated animals by 12-50% comparing the means at the various time points. Also in Figure 4. it should be noted that methyldopa produced a slight depression of the MSR but that this apparent direct effect began to wane before the D,L-5-HTP was administered. At 60 min after 5-HTP, amitriptyline was given to provide a test of the completeness of the methyldopa blockade of serotonin production from the exogenous 5-HTP. Figure 4 shows that amitriptyline injection produced only about a 202, increase in the MSR above the maximum level of the prior D,L-5-HTP de-

S-HTP spinal reflexes crease in methyldopa-pretreated preparations as compared to approximately a 73% rise in the non-methyldopa-treated experiments (P < 0.05 by t-test at 5 min). Assuming that the MSR facilitation by amitriptyline is serotonergic (Ciineschmidt et al, 1971; Maas, 1975), this would suggest that serotonin synthesis from the pharmacologically administered D,L-5-HTP was indeed attenuated by methyldopa. DISCUSSION

These results demonstrate a depressant action of S-HTP on monosynaptic transmission in the cat spinal cord that has been neither consistently observed nor adequately investigated in other studies in which 5-HTP has been employed at a similar dose to study the influence of bulbospinal serotonergic pathway(s) on spinal reflexes (Anden et nl., 1964; Anderson and Shibuya, 1966; Banna and Anderson, 1968; Shibuya and Anderson, 1968; Clineschmidt et al., 1971; Sastry and Sinclair, 1976a,b; 1977). Furthermore, the present data strongly suggest that this action of 5-HTP is not mediated by serotonin. The non-serotonergic nature of the MSR depression by 5-HTP in triceps surae-ventral root preparations is demonstrated by the results of three separate experiments. (1) Administration of amitriptyline, which selectively inhibits the presynaptic reuptake of serotonin (Maas, 1975) and is known to facilitate the cat MSR by a serotonergic mechanism (Clineschmidt et al., 1971), rapidly reversed the 5-HTP-induced MSR depression and produced a facilitation of the MSR (Fig. 3). (2) Pretreatment with cyproheptadine or cinanserin, well-known selective serotonin receptor antagonists, did not block the depression by subsequent 5-HTP. (3) Prior administration of the centrally-acting decarboxylase inhibitor methyldopa, which blocks the enzymatic conversion of 5-HTP to serotonin, also failed to prevent the MSR attenuation by 5-HTP (Fig. 4). The assumption that serotonin production was indeed blocked by methlydopa is supported by the fact that the facilitatory action of amitriptyline was greatly reduced after methyldopa pretreatment. Others have in fact noted a spinal depressant action of 5-HTP in cats and provided evidence that the effect may not be mediated by serotonin. First of all, Anden et al. (1964) have shown that 5-HTP suppressed transmission from flexor, but not extensor reflex afferents to motor neurons. In the former instance the effect was not antagonized by decarboxylase inhibition and thus may be a direct action of 5-HTP. Secondly, Anderson and Shibuya (1966) have observed that 5-HTP decreased both lumbar polysynaptic transmission and the dorsal root reflex. Furthermore, the effects were not fully preventable by the prior administration of selective serotonin receptor blocking drugs (Banns and Anderson, 1968). On the other hand, Anderson et al. (1966) and Shibuya and Anderson (1968) have not reported a

113

5-HTP-induced decrease in the cat spinal MSR as was shown here. This discrepancy is in part explained by the fact that these previous investigators employed both dorsal root-ventral root and peripheral nerveventral root preparations with no indication of how many of each type were used. As shown in Figures 1 and 2 in this report, the action of 5-HTP in dorsal root-ventral root preparations was indeed facilitatory while, in triceps surae nerve-ventral root animals, the effect of the same 5-HTP dose was consistently depressant. In addition, Sastry and Sinclair (1976a) have noted that “5-HTP produced somewhat variable results on the unconditioned MSR when administered alone”. The disparity between the MSR facilitatory action of 5-HTP in dorsal ventral-root preparations and the depressant effect observed here in animals with triceps surae stimulation is perhaps understandable if one considers the relative balance between the previously described serotonergic spinal facilitatory effects of 5-HTP on the MSR vs the non-serotonergic depressant action. In addition to the serotonergic action of 5-HTP to enhance motor neuron excitability (Anden et al., 1964; Anderson and Shibuya, 1966; Myslinski and Anderson, 1978), pharmacologically administered 5-HTP acting via a serotonergic mechanism has also been reported to decrease both presynaptic (Sastry and Sinclair, 1977) and recurrent (Sastry and Sinclair, 1976b) inhibition of the MSR illicited by quadriceps nerve activation, but not that evoked by posterior biceps Ia stimulation (Sastry and Sinclair, 1977). Thus, the increased motor neuron excitability and the decreased presynaptic and recurrent inhibition in the case of some Ia-motor neuron paths (e.g. quadriceps) would tend to facilitate the MSR while a direct depressant action of 5-HTP at some point in the reflex pathway, perhaps on the Ia afferent terminal, would act to decrease the reflex. Therefore, in preparations employing whole lumbar 7 dorsal root stimulation the net result of the two facilitatory effects of 5-HTP vs the one depressant effect would be a reflex enhancement. In the TS-VR preparation, the overall effect of 5-HTP is one of MSR depression perhaps because of a possible lack of antagonism of segmental inhibitory influences as is the case with posterior biceps-ventral root animals (Sastry and Sinclair, 1976b, 1977). In other words, regarding the triceps surae MSR, there may be only two relevant actions of 5-HTP, namely an increase in motor neuron responsiveness plus a depression of the Ia afferent terminal with the latter predominating. However, subsequent administration of amitriptyline may quite conceivably enhance the serotonergic action of 5-HTP to increase further motor neuron excitability with the balance then being shifted toward MSR facilitation (Fig. 3). The significance of the present demonstration of a non-serotonergic action of 5-HTP on spinal monosynaptic reflex transmission is 2-fold. First, while it certainly does not challenge the existence of a seroto-

ii4

E. D. HALL

nergic spinal system which may serve to modulate motor output, it does suggest that use of 5-HTP to study CNS seratonergic mechanisms sfiould be thorough enough in terms of technicme and dose so as to exclude direct effects of S-HTP. Secondly, the present results may have at least some relevance to an understanding of the antidepressant efficacy, or lack of efficacy, of 5-HTP in certain cases of psychiatric depression (Zarcone, Berger, Brodie, Sach and Barchas, f977f assuming that 5-HTP may have direct depressant effect on neuronal excitabihty and synaptic transmission at higher CNS centers. Acknowfedgem~nts-The author gratefully acknowledges the dedicated technical assistance of Mrs Brigitte Hirst, the graphic design of MS Jan Weiner and the generous gifts of a~tr~ptyline chloride and ~~oheptad~n~ chioride from Merck, Sharp & Dohme and cinanserin hydrochloride from Squibb. This research was supported by a grant from the Amyotropbic Lateral Sclerosis Society of America, by NIMH (Small Grant) 31887-01 and by NIMH 34111.01.

Progressive increase of motor activity induced by 5sHTP in the rat below a complete section of the spinal cord. Bruin Res. 169: 393-397. C~ines~hm~dt, B. V., Fierce, J. E. and Sjoerdsma, A. (19X). Interactions of tricyefic antidepressants and %hydroxy~ndo~ealkyI~m;ne precursors on spinal monosynapti~ reflex transmission.-J. Pharmnr. exp.. Ilher. 179: Si2-323. Maas, J. W. (1975). Bionenic amines and deoression. Biochemical and pharmacological separation of two types of depression. Arch. gen. Psychiar. 32: 1357-l 36 I. Myslinski, N. R. and Anderson, E. G. (1978). The effect of serotonin precursors on x- and ~-motoneuron activity. J. P~urm~c. exp. Ther. 204: 19-26. Nickerson, M. and Ruedy, J. (1975). Anfihyperte~sive agents and tbe drug therapy of hypertension. In: The Pharmocalogical Basis of Therapeutics (Goodman, L. S. and Gilman, A., Eds), pp. 705-726. Macmillan, New York. Sastry, B. S. R. and Sinclair, J. G. (1976a). Serotonln involvement in the blockade of bulbospina~ inhibition of the spinat monosvnautic reflex. Brain Res. 115: 423-436. Sastry,B. S. R. and~Si&air, J. G. (1976b). Tonic inhibitory influence 0r a supraspinal monaaminergic system on recurrent inhibition of an extensor monosynaptic reflex. Bruin

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Sastry, B. S. R. and Sinclair, J. G. (1977). Tonic inhibitory influence of a supraspinal mon~arn~nerg~&system on presynaptic inb~bition of an extensor monosynaptic reflex. Brain Res. 124: 105-I 20. Shibuya, T. and Anderson, E. G. (1968). The infiuence of chronic cord transection on the effects of 5-hydroxytryp tophan, r.-tryptophan and pargyline on spinal synaptic activity. J. Phurmac. exp. Ther. 164: 185-190. Zarcone, Y. P., Berger, P. A., Brodie, H. K. H., Sack, R. and Barchas, J. LXff977). The indoleamine hypothesis of depression: an overview and pilot study. Dis. Nrn;. 5~s. August 646653.