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Electrical stimulation of the nucleus raphe magnus in the rat. Effects on 5-HT metabolism in the spinal cord
Brain Research, 194 (1980) 377-389 © Elsevier/North-Holland Biomedical Press
377
ELECTRICAL STIMULATION OF THE NUCLEUS RAPHE MAGNUS IN THE RAT. EFFECTS ON 5-HT METABOLISM IN THE SPINAL CORD
S. BOURGOIN, J. L. OLIVERAS, J. BRUXELLE, M. H A M O N and J. M. BESSON
Groupe N B - - I N S E R M U. 114, Colldge de France, l l,place Marcelin Berthelot, 75231 Paris Cedex 05 and ( J.L.O., J. B. and J.M.B.) Unitd de Recherches de Neurophysiologie Pharmacologique, I N S E R M U. 161, 2, rue d'Aldsia, 75014 Paris (France) (Accepted January 24th, 1980)
Key words: serotonin - - electrical stimulation - - raphe magnus - - spinal cord
SUMMARY
The direct electrical stimulation (with biphasic pulses of 1 msec, 10 pulses/sec, 200/,A, for 30 min) of the nucleus raphe magnus in chloral hydrate anaesthesized rats produced a significant acceleration ( + 5 0 ~ ) of 5-HT synthesis in the spinal cord as revealed by the increased rate of 5-HTP accumulation occurring at this level after the blockade of central 5-HTP decarboxylase with benserazid. In contrast, no change was detected in 5-HT metabolism in the forebrain of stimulated rats. The acceleration of 5-HT synthesis was likely not due to an increased availability of tryptophan for the rate-limiting enzyme, tryptophan hydroxylase, since the concentration of this amino acid was changed neither in the spinal cord, nor in the forebrain of stimulated rats. The measurement of tryptophan hydroxylase activity in soluble extracts from the spinal cord of control and stimulated rats revealed that the acceleration in 5-HT synthesis produced by the electrical stimulation of the nucleus raphe magnus was not associated with a persisting activation of this enzyme. Although one cannot completely exclude that a short-lasting activation of tryptophan hydroxylase, no longer detectable in soluble extracts, has occurred in the spinal cord of stimulated rats, the present findings rather suggest that the rate of 5-HT synthesis can be controlled by factors other than only the concentration of tryptophan and the intrinsic activity of tryptophan hydroxylase in serotoninergic neurons. The demonstration of an acceleration of 5-HT synthesis in bulbospinal serotoninergic neurons under stimulating conditions close to those producing analgesia in rats further supports the role of these neuronal systems in the physiological mechanisms of pain control.
INTRODUCTION
Among the various and complex .mechanisms involved in the regulation of
378 monoamines synthesis in the CNS, those triggered by the nerve impulse flow in monoaminergic neurons have raised considerable interest. Attempts to select appropriate experimental models for further analyzing the relationship between the firing and the neurotransmitter synthesis rates in monoaminergic neurons generally converge to those consisting of direct electrical stimulation of nerve fibres or perikaryons. Using this approach, Roth and coworkers ~4,29 reached the conclusion that the accelerated synthesis of noradrenaline (NA) and dopamine (DA) following the electrical stimulation of the locus coeruleus and of the substantia nigra, respectively, were due to the activation of tyrosine hydroxylase, the first enzyme involved in the biosynthesis of catecholamines (CA) in the CNS. Furthermore, they were able to propose a working hypothesis on the molecular mechanisms triggered by the depolarization of nerve terminals and ending at the activation of this key enzyme24,'~9 In the case of serotoninergic neurons, several authors1,13,19,3t have also observed that the direct electrical stimulation of cell bodies or fibres may result in an enhancement of the rate of 5-HT synthesis. Using the method developed by Carlsson et al. 1° to measure the rate of tryptophan hydroxylation in vivo, Herr et a1.19 reached the conclusion that the nerve impulse flow-induced change in 5-HT synthesis involves that very enzymic step. Although they could eliminate possible changes in tryptophan levels as being responsible for the increased synthesis observed ~9, several other mechanisms may produce an acceleration of tryptophan hydroxylation. Indeed, in the CNS, not only the concentration of tryptophan but also those of oxygen and of tetrahydrobiopterin (the putative endogenous pterin cofactor of aromatic amino acid hydroxylase) are lower than the respective Kms of tryptophan hydroxylase (EC 1.14.16.4)2L Therefore, an increase in the concentration of oxygen or/and of that of tetrahydrobiopterin in tissues may well trigger an acceleration of tryptophan hydroxylation. Furthermore, alterations of enzyme effectors or of tryptophan hydroxylase itself (activation) may also be involved in the acceleration of 5-HT synthesis induced by the electrical stimulation of the raphe nuclei. In an attempt to further explore the mechanisms triggering the acceleration of 5HT synthesis in stimulated neurons, we first selected experimental conditions leading to a reproducible increase in 5-HT synthesis as the result of electrical current passing through the raphe. Then, we extracted tryptophan hydroxylase from stimulated tissues in order to detect any possible alteration consecutive to in vivo electrical stimulation of serotoninergic neurons. The growing interest for the bulbospinal serotoninergic pathway with respect to the central physiology, notably the control of pain transmission2,4, led us to study the effects of electrical stimulation of cell bodies in the nucleus raphe magnus on the synthesis of 5-HT in nerve terminals in the spinal cord. This raphe nucleus has been shown by several groups to contain the majority of cell bodies of serotoninergic neurons descending within the spinal cord tl,25. MATERIALSAND METHODS Chemicals. Adenosine-triphosphate (ATP, Boerhinger-Mannheim), DL-6-methyl-
379 5,6,7,8-tetrahydropterin (6-MPH4, Calbiochem), chloral hydrate (Prolabo), Benserazid (Ro 4.4602, Hoffmann-LaRoche). Other compounds (salts, indoles... ) were from usual sources (Merck, Prolabo). Animals. Adult male Sprague-Dawley (Charles River strain) rats weighing 350--400 g were used. Before the experiments, they were kept for at least 10 days in a controlled environment (24 °C, 60~ relative humidity, alternate cycles of 12 h light and 12 h darkness, food and water ad libitum). Chloralhydrate (400 mg/kg, 1 ml/rat) and benserazid (800 mg/kg, 2.5 ml/rat) were dissolved in saline and administered i.p.
Electrical stimulation of the nucleus raphe magnus Five to 10 min following an i.p. injection of chloral hydrate (400 mg/kg), rats were positioned in a stereotaxic apparatus (La Pr6cision Cin6matographique, Asni6res, France). The skull was drilled and a bipolar concentric stimulating electrode was lowered into the nucleus raphe magnus (A = --3; H = ---0.5; L = 0, with the zero at the middle of the interauricular axis). On occasion, the electrode was placed laterally to the nucleus raphe magnus (L = 1). The structures were identified according to the stereotaxic atlas of Fitkov~, and Marsala 14. Concentric electrodes were made of stainless steel. The external diameter of the outer core was equal to 0.25 mm. The inner core was sharpened to a diameter of 30/~m at the tip which protruded by 0.20 mm from the outer core. Electrical stimulation consisted of biphasic rectangular pulses (1 msec, 10/sec, 100 or 200 #A) and was maintained for 30 min. Histological control of the electrode location revealed that such stimulating conditions did not produce any detectable lesion even at the tip. Sham animals were prepared in the same way except that no current was passed through the electrode during the 30 min period they were maintained in the stereotaxic frame. Biochemical analyses At the end of the stimulation period or 30 min later (see Results), rats were decapitated and their brain and spinal cord were rapidly removed. The brain stem and the cerebellum were isolated by a coronal section in front of anterior colliculi. This piece of tissue was immediately immersed in a saline solution containing 3 ~ formaldehyde. Histological control of the electrode location proceeded as described previously28. The remaining forebrain and the srinal cord were homogenized in 6 ml and 4 ml, respectively, of an ice-cold ethanol-water solution (74:16, v]v) containing 0.05 ~ of EDTA and 0.05 ~o of ascorbic acid. After a storage at --30 °C for 12 h, homogenates were centrifuged and 5-HT, tryptophan, 5-hydroxytryptophan (5-HTP) and 5-hydroxyindole acetic acid (5-HIAA) in the clear supernatant were selectively extracted by ion-exchange and adsorption chromatography. They were finally measured using appropriate spectrofluorimetric methods. The complete procedure is described in details elsewhere a. When the activity of tryptophan hydroxylase was measured, tissues were homogenized in two steps. A first homogenate was obtained by disruption in 2 (for the
380 forebrain) or 3 (for the spinal cord) vols. (v/w) of 0.05 M Tris.acetate, pH 7.6, containing 2 mM/3-mercaptoethanol, using a Polytron PT l 0 0 D apparatus. Homogenates were then centrifuged at 35,000 g for 30 min at 4 cC and the resulting supernatants were used as the final sources of tryptophan hydroxylase. The enzymic activity was measured on 0.2 ml aliquots of the supernatants with 0.15 mM tryptophan and 0.16 mM 6-MPH i as the pterin cofactor. The reaction proceeded for 15 min at 37 °C under air atmosphere and the final product, 5-HTP, was measured spectrofluorimetrically (see ref. 16 for details). Since these assay conditions were subsaturating, they were appropriate to detect any change (inhibition, activation or induction) in tryptophan hydroxylase intrinsic activity a~i. The second homogenate was obtained by mixing the remaining tissue supernatants with the corresponding pellets using the Polytron PT 10 OD disrupter. This was achieved after the addition of ice-cold ethanol (4:1 (v/v) of the first homogenate) and EDTA (0.05% final concentration). The resulting mixture was then treated as above for the biochemical determinations of indolic compounds. No significant difference in the respective fluorescence blanks and recoveries (75-95 °/o) was noted for tryptophan, 5-HTP, 5-HT and 5-HIAA when homogenization was carried out by the usual method (with ethanol-water 74:16, v/v) or by the two step procedure described above (data not shown). Statistical calculations were performed as proposed by Snedecor and Cochran ~2. When the P-value was higher than 0.05 (Student's t-test), a difference was consisted to be non-significant. RESULTS
Effects of chloral hydrate anaesthesia on 5-HT metabolism in the spinal cord and the forebrain of adult rats The rate of 5-HT synthesis was estimated by measuring the accumulation of 5HTP in tissues after the blockade of 5-HTP decarboxylase 1° by benserazid. For this series of experiments, 4 groups of rats were used; A, control rats; B, animals treated with chloral hydrate (400 mg/kg) 40 min before death; C, animals treated with benserazid (800 mg/kg i.p.) 30 rain before death; and D, those receiving both treatments. As shown in Table 1, anaesthesia for 40 min in rats treated with chloral hydrate alone (group B) did not significantly alter the endogenous levels of tryptophan, 5-HT and 5-HIAA in the spinal cord. In contrast, slight but significant elevations of 5-HT ( ÷ 1 7 ~ ) and 5-HIAA ( + 2 6 ~ ) levels were detected in their forebrain (Table II). Comparison of data obtained in groups C and D indicates that chloral hydrate anaesthesia failed to modify the rate of 5-HTP accumulation in tissues after the blockade of 5-HTP decarboxylase. However, anaesthesia did partly antagonize the benserazid-induced reduction in 5-HT levels. Thus, both in the spinal cord (Table I) and in the forebrain (Table I1), the 5-HT levels in anaesthetized animals treated with benserazid (group D) were not significantly different from those found in controls (group A).
381 TABLE I
Effects o f anaesthesia with chloral hydrate on 5 - H T metabolism in the spinal cord Four groups of 8 rats were used. Two groups were anaesthesized with chloral hydrate (400 mg/kg i.p.). Ten to 20 min after the onset of anaesthesia, 8 rats together with 8 control animals were treated with benserazid (800 mg/kg i.p.). Animals were killed 30 min after benserazid or 40 min after chloral hydrate anesthesia. Eight untreated rats served as 'controls'. Each value (mean 4- S.E.M. of 8 individual determinations) is expressed as/~g/g of fresh tissue.
A B C D
Controls Chloral hydrate Benserazid Chloral hydrate 4benserazid
TRP
5-HTP
5-HT
5-H1A A
5.51 -4- 0.31 4.93 -- 0.31 6.24 4- 0.22
--0.144 4- 0.008
0.524 4- 0.023 0.538 4- 0.025 0.427 zk 0.018"
0.429 -4- 0.036 0.460 -4- 0.015 0.375 -4- 0.030
6.05 ± 0.25
0.129 -4- 0.005
0.463 -4- 0.024
0.403 ± 0.024
* P < 0.05 when compared to the control group. TABLE II
Effects o f anaesthesia with chloral hydrate on 5 - H T metabolism in the forebrain The protocol is described in the legend to Table I. Each value (mean 4- S.E.M. of 8 individual det,rm±nations) is expressed as/~g/g of fresh tissue.
TRP A B C D
Controls Chloral hydrate Benserazid Chloral hydrate 4benserazid
5-HTP
5-HT
5-HIA A
4.79 4- 0.17 4.59 4- 0.24 5.54 4- 0.22
--0.170 4- 0.009
0.445 i 0.017 0.522 -4- 0.018" 0.353 4- 0.017"
0.418 -4- 0.020 0.525 4- 0.017" 0.382 4- 0.030
5.37 -4- 0.12
0.154 4- 0.006
0.435 -4- 0.032
0.451 4- 0.025
* P < 0.05 when compared to respective control values. TABLE IIl
Effects o f electrical stimulation o f the nucleus raphe magnus on 5-HTmetabolism in the spinal cord A: rats were anaesthesized with chloral hydrate (400 mg/kg i.p.) and positioned in a stereotaxic apparatus. The stimulating electrode was placed as described in 'Materials and Methods' but current was passed through only the rats in the 'stimulated' group. All rats received an i.p. injection of benserazid (800 mg/kg) as soon as the electrode was correctly postioned. They were killed 30 min later (i.e. at the end of the stimulation period for the 'stimulated' group). B: the same protocol as in A was used except that benserazid was administered 30 rain after the animals were positioned in the stereotaxic apparatus for electrode implantation ('sham') or continuous stimulation ('stimulated'). All rats were maintained in the stereotaxic frame for 30 min following the drug injection but with no stimulation. They were killed by decapitation and biochemical analyses were performed as described in 'Materials and Methods'. Each value (in .ug/g of fresh tissue) is the mean -4- S.E.M. of 8-14 individual determinations.
I RP A Sham Stimulated B Sham Stimulated
6.13 6.29 6.03 6.39
-4- 0.27 -4- 0.20 4- 0.36 4- 0.42
5-HTP
5-HT
0.134 -4- 0.015 0 . 2 0 1 i 0.012" 0.132 4- 0.008 0.185 -4- 0.008*
0.483 0.485 0.471 0.482
* P < 0.05 when compared to respective 'sham' values.
5-HIAA -4- 0.025 4- 0.018 -4- 0.017 -4- 0.021
0.399 -4- 0.021 0.443 4- 0.017 0.361 4- 0.019 0.411 4- 0.017
382 In conclusion, although chloral hydrate anaesthesia did alter 5-HT metabolism in the CNS (increases in 5-HT and 5-HIAA levels in the forebrain likely involving a reduced utilization of 5-HT and a partial blockade of 5-H1AA efflux respectively), the observed modifications were less pronounced than after halothane, nitrous oxide8 or ether 7 inhalation. This led us to choose chloral hydrate as a more appropriate anaesthetic in subsequent experiments.
Effects of electrical stimulation of the nucleus raphe magnus on 5-HT metabolism in the C NS of chloral hydrate-anaesthesized rats (a) 5-HT metabolism during the stimulation period. Immediately after the administration of benserazid, electrical stimulation of the nucleus raphe magnus was turned on and maintained for 30 min, i.e. till death. When the intensity current was equal to 200/tA, this stimulation resulted in a significant increase ( + 50 ~, Table III) in the rate of 5-HTP accumulation in the spinal cord when compared to that found in control rats. Decreasing the intensity by half markedly reduced this effect since only a small increment in the rate of 5-HTP accumulation was observed in the spinal cord (-~- 12 °/o,P > 0.05) of rats electrically stimulated for 30 min with biphasic pulses of 100 /zA (data not shown). The electrical stimulation of the nucleus raphe magnus with a current of 100 #A (data not shown) or 200/~A (Table IV) did not affect the rate of 5-HTP accumulation in the forebrain. As shown in Tables III and IV, neither tryptophan, 5-HT nor 5-HIAA levels were modified by the electrical stimulation of the nucleus raphe magnus. However, it should be noted that a slight non-significant increase in the concentration of 5-HIAA was regularly observed (in 3 independent experiments) particularly in the spinal cord of stimulated rats (Table III). Detailed analysis of the electrode sites producing significant increases in the rate of 5-HTP accumulation indicated that those precisely located in the nucleus raphe magnus were more efficient (m ---- +56 ± 11 ~) than those outside (+32 ~ 6 ~o) (Fig. 1). However, even when the tip of the electrode was situated 0.2 mm laterally to the nucleus, electrical stimulation was still efficient in producing a significant acceleration of 5-HTP formation (Fig. 1). (b) 5-HT metabolism after the stimulation period. Whereas benserazid treatment was applied for the whole stimulation period in the preceding experimental situation (a), in this second series of experiments, the 5-HTP decarboxylase inhibitor was injected at the end of the stimulation period. Animals were then killed 30 min later. Under these conditions, only the 200/~A current was used. Like with the previous experimental design (a), the rate of 5-HTP accumulation remained unaltered in the forebrain (Table IV) whereas it increased significantly in the spinal cord (Table III) of stimulated animals. However, the increment in the rate of 5HTP formation (+40.2 ~) occurring during the post-stimulation period (conditions b) was slightly less pronounced than that ( + 5 0 ~ ) observed during the stimulation period (conditions a).
383
ao' O o 4-4602
©
Fig. 1. Cross section of the brain stem at the level of the nucleus raphe magnus showing the sites where passing current triggered an acceleration of 5-HTP synthesis in the spinal cord. Benserazid (Ro 4-4602, 800 mg/kg i.p.) was administered to chloral hydrate anaesthesized rats at the beginning of the electrical stimulation. Animals were killed 30 min later, at the end of the stimulation period. Each figure corresponds to the per cent increase in 5-HTP accumulation resulting from the stimulation at the indicated site. Rm, nucleus raphe magnus. The control value (100 %) is the mean of 5-HTP accumulated in the spinal cord of 14 rats placed under the same conditions except that no current was passedthrough the electrode.
As already noted under the previous experimental conditions (a), tryptophan, 5HT and 5-HIAA levels were not significantly affected by the stimulation. However, as before, 5-HIAA levels in the spinal cord were slightly increased in stimulated animals ( + 1 4 Z , P > 0.05). Histological control of the electrode locations (Fig. 2) revealed that the more efficient sites were situated in the nucleus raphe magnus or just in its vicinity. When the electrode tip reached zones situated at least 0.25 mm beyond the nucleus sides, the rate of 5-HTP accumulation was hardly affected by the electrical stimulation ( + 7 and + 14 ~o, see Fig. 2, and data not shown for sites located farther than those indicated in this Fig.). TABLE IV
Effects of electrical stimulation of the nucleus raphe magnus on 5-HTmetabolism in the forebrain The protocol is described in the legend to Table IlL Each value (in/~g/g of fresh tissue) is the mean 4- S.E.M. of 8-14 individual determinations.
TRP A Sham Stimulated B Sham Stimulated
5.25 5.38 5.42 6.05
+ 0.47 4- 0.24 -4- 0.32 -4- 0.31
5-HTP
5-HT
0.159 0.171 0.162 0.177
0.453 0.453 0.450 0.438
4- 0.018 4- 0.016 4- 0.012 4- 0.011
5-HIAA 44+ i
0.029 0.024 0.019 0.019
0.455 0.491 0.515 0.520
44± +
0.023 0.022 0.056 0.019
384
Fig. 2. Cross section of the brain stem at the level of the nucleus raphe magnus showing the sites where electrical stimulation produced a persisting acceleration of 5-HTP synthesis in the spinal cord. Chloral hydrate anaesthesized rats were maintained for 1 h in a stereotaxic frame. For the first 30 min, current was applied to the indicated sites under the conditions described in 'Materials and Methods'. Benserazid (800 mg/kg i.p.) was then administered and rats were killed 30 min later. Each figure corresponds to the per cent increase in 5-HTP accumulation following electrical stimulation at the indicated site. Rm, nucleus raphe magnus; VII, nucleus originis nervi facialis; P, tractus corticospinalis.
Current spread in the vicinity oJ,"the nucleus raphe magnus in the course of its electrical stimulation with 200 #A biphasic pulses Since the electrical stimulation of sites located outside the nucleus raphe magnus also produced marked elevations in the rate of 5-HTP formation, the current should most likely spread from the electrode tip to reach serotoninergic cell bodies in this nucleus. Observations of the animal during electrical stimulation was, in this respect, very informative. Thus, in a few cases, rhythmic contractions of face muscles and vibrissae oscillations occurred in phase with the current. As shown in Fig. 3, this was seen when the electrode tip was between the nucleus raphe magnus and the nucleus originis nervi facialis (VII) which contains the cell bodies of neurons innervating face muscles. Accordingly, under the present experimental conditions, the current was probably spreading enough to produce efficient excitation of neurons inside a sphere of about 0.20 m m radius a r o u n d the electrode tip.
Effects of electrical stimulation of the nucleus raphe magnus on the activity of tryptophan hydroxylase in soluble extracts from the spinal cord Since the rate of 5-HTP accumulation was increased not only during but alsoJor the 30 minfollowing the electrical stimulation, this implied that the full effect o f raphe stimulation on 5-HT synthesis would be occurring just at the end of the stimulation period. This time was chosen to look for possible changes in t r y p t o p h a n hydroxylase activity in tissues from stimulated rats.
385
•
face
movements(lO/S)
no effect
Fig. 3. Sites of electrical stimulation producing face movements. When the electrode was positioned at the black dots, passing current produced face movements indicating that stimulation reached the VII nucleus. Therefore, even when the electrode was not in the right position but only adjacent to the nucleus raphe magnus, the current diffusion was large enough to produce excitation of serotoninergic neurons Thus, with the electrode located at the two black dots on the left, we still observed a slight increase ( ± 7; ± 14 ~ ; see Fig. 2) in the rate of 5-HTP formation in the spinal cord. Rm, nucleus raphe magnus; VII, nucleus originis nervi facialis.
TABLE V Effects o f electrical stimulation o f the nucleus raphe magnus on tryptophan hydroxylase activity in vivo and in vitro in the spinal cord
Four groups of 6 rats were used. Two groups were anaesthesized with chloralhydrate (400 mg/kg i.p.) and positioned in a stereotaxic apparatus. These and 6 other animals received benserazid (800 mg/kg i.p.) 30 min before death. Electrical stimulation of the nucleus raphe magnus was set on just after the i.p. injection of the 5-HTP decarboxylase inhibitor as described in 'Materials and Methods'. The in vivo accumulation of 5-HTP is expressed as nmol/g tissue/l 5 min. Tryptophan hydroxylase activity (in vitro) was assayed in the 35,0D0 g sup~rnatant of spinal cord homogenates with or without 0.5 m M ATP, 5 m M Mg ~+ and 50/~M Ca 2÷ (phgsphorytating mixture 16). It is expressed as nmol 5-HTP formed/g of fresh tissue and per 15 min. Each value is the mean 4- S.E.M. of 6 individual determinations. 5-HTP in vivo
in vitro ÷ ATP ÷ Mg 2+ + Ca 2+
Control Benserazid Benserazid -- chloral hydrate Benserazid + chloral hydrate ± electrical stimulation
-0.326 i' 0.018 0.312 ± 0.018
10.81 ± 0.57 9.44 ± 0.85 9.33 ± 0.59
14.53 ± 0.67** 13.31 i 0.98** 12.74 ± 0.71 **
0.449 ± 0.041"
9.64 4- 0.47
14.00 ± 1.18"*
* P -< 0.05 when compared to corresponding values in the two other groups. P < 0.05 when compared to tryptophan hydroxylase activity in the same group but in the absence of ATP, Mg ~+, Ca 2+.
* *
386 Although the electrical stimulation induced a significant increase in the rate of 5HTP formation in vivo, no change in the activity of tryptophan hydroxylase was detected in soluble extracts from the spinal cord of stimulated rats (Table V). In addition, the activation of this enzyme by the ATP, Mg 2+, Ca `)'- mixture (see ref. 16) was similarly pronounced whether tryptophan hydroxylase was extracted from control or from stimulated animals (Table V). The comparison of the rate of tryptophan hydroxylation in the spinal cord in vivo with the in vitro activity of soluble tryptophan hydroxylase from the same tissues revealed that the latter was 20-30 times higher than the former value (Table V). This was true for control and stimulated animals (Table V). DISCUSSION The present data demonstrated that the synthesis of 5-HT increased in the terminals of serotoninergic bulbospinal neurons when their cell bodies were electrically stimulated. Therefore, they extend to descending serotoninergic neurons the findings already reported for those located in the anterior raphe nuclei and projecting rostrally into the forebrain la,19,31. The comparison of experimental conditions for stimulating the anterior raphe nuclei19, 3~ with those presently used for the stimulation of the posterior nucleus raphe magnus indicates that an intensity current of 200/~A was required to produce a significant acceleration of 5-HT synthesis in terminal areas. However, this does not mean that such an intensity was necessary at the very level of serotoninergic neurons since histological control suggested that current spread from sites located outside the nucleus raphe magnus was sufficient to produce excitation of serotoninergic neurons. In this respect, further comparison with available data in the literature is impossible since the histological control of the exact locations of the stimulated sites was never reported. Although most serotoninergic cell bodies are located in raphe nuclei, a nonnegligible proportion is situated outside 2~,a3 and therefore, the effective stimulation sites lateral to the nucleus raphe magnus might well involve extra raphe neurons also projecting into the spinal cord. However, experiments in the cat consisting of selective lesion of the nucleus raphe magnus within the limits described by Berman 3 have shown that at least 70 ~ of serotoninergic terminals in the spinal cord belong to neurons situated in this nucleus 25. It is therefore very unlikely that the electrical stimulation of less than 30 ~ of these extra raphe neurons would be as efficient as that at the very level of the nucleus raphe magnus to produce an acceleration of 5-HT synthesis in the spinal cord ~see Figs. 1 and 2). Current spread from the electrode tip to the nucleus raphe magnus was more likely the main factor responsible for the activation of spinal 5-HT synthesis occurring when extra raphe sites were electrically stimulated. Under normal conditions, tryptophan hydroxylase, the rate limiting enzyme for the synthesis of 5-HT, is not saturated by tryptophan ~8. Therefore, fluctuations in the concentration of this amino acid in brain tissues generally trigger parallel modifications in the rate of 5-HT synthesis 18. In the present case, since tryptophan levels were not altered in tissues from stimulated animals, it can be argued that the acceleration of
387 5-HTP formation was not the consequence of a primary increase in the concentration of the precursor amino acid in the spinal cord (see also refs. 19 and 31). However, one cannot completely rule out that the tryptophan concentration actually increased in serotoninergic neurons in a very discrete pool directly involved in the enzymic hydroxylation process (see ref. 18). Unfortunately, the measurement of tryptophan concentration in the immediate vicinity of tryptophan hydroxylase is not yet possible. Apparently, the acceleration of 5-HT synthesis occurring in electrically-stimulated serotoninergic neurons was not the consequence of an activation of tryptophan hydroxylase since no significant alteration in the intrinsic activity of this enzyme was detected in soluble extracts from the spinal cord after electrical stimulation of the nucleus raphe magnus. This observation is in contrast with recent in vitro findings demonstrating that K+-induced depolarization of 5-HT neurons did produce a marked activation of the soluble enzymeSA5. The difference between in vitro depolarization and in vivo electrical stimulation was further substantiated when considering the effects of in vitro activating conditions on tryptophan hydroxylase: whereas Ca z÷dependent phosphorylation (with the ATP, Mg 2+, Ca 2+ mixture) increased the activity of tryptophan hydroxylase extracted from tissues of stimulated (this paper) or nonstimulated animals 16, it was no longer effective to further enhance the activity of the enzyme extracted from tissues depolarized in vitro 15. Since the assay conditions for the measurement of tryptophan hydroxylase activity in vitro are very different from those in 5-HT neurons, it can be argued that they are not suitable for detecting an activation of this enzyme which would be induced in vivo. As already discussed by Meek and Lofstrandh2a, artificial in vitro conditions resulted in tryptophan hydroxylase activity markedly higher than that measured in vivo, possibly meaning that numerous regulatory properties were lost under in vitro conditions. However, it has to be emphasized that these in vitro assay conditions have been shown quite appropriate for detecting the activation of tryptophan hydroxylase resulting from in vivo treatment v~ithmethiothepin17 or from depolarization of brain slices15. Another criticism to the experimental approach used in the present study may concern the time elapsing between the end of the stimulation and the extraction of tryptophan hydroxylase from tissues. If the putative in vivo activation of the enzyme was rapidly reversible, this time would have been long enough to allow tryptophan hydroxylase to return to a non-activated state. However, the effect of electrical stimulation of the nucleus raphe magnus on the rate of 5-HT synthesis in the spinal cord was still observed during the 30 minfollowing the stimulation period indicating that the return to a non-activated state might be a slow-acting process. Furthermore, previous examples of enzymic activation either by in vivo electrical stimulation (tyrosine hydroxylase24, 29) or by in vitro depolarization (tyrosine and tryptophan hydroxylasesg,l~, a°) have shown that the enzyme alteration persisted under drastic conditions such as freezing and thawing of tissues and homogenates. Therefore, it seems reasonable to conclude that the electrical stimulation of the nucleus raphe magnus induced an acceleration of 5-HT synthesis which was not associated with an activation of tryptophan hydroxylase. As recently discussed for catecholaminergic
388 neurons 12,2°, the present findings indicated that the rate o f the neurotransmitte~ synthesis m a y increase in the absence o f any significant change in the activity o f the rate-limiting enzyme. Reciprocally, an activation o f the rate-limiting enzyme m a y well occur w i t h o u t p r o d u c i n g an acceleration o f the n e u r o t r a n s m i t t e r synthesis6, I.~. Since the conditions used in this study were close to those which have been shown to induce analgesia in rats a n d cats 26 '~8, it can be concluded that increased activity o f descending serotoninergic p a t h w a y s does occur during electrically i n d u c e d analgesia in animals. In this respect, the present findings further confirm the role of the bulbospinal serotoninergic system in the physiological mechanisms o f pain control 2,4. ACKNOWLEDGEMENTS This research was s u p p o r t e d by grants from I N S E R M ( A T P 76.71), D G R S T , D R E T (79.077) a n d R h 6 n e Poulenc S.A. The excellent technical assistance o f Miss F. A r t a u d is gratefully acknowledged. W e are indebted to Dr. W. Haefely ( H o f f m a n n - L a Roche, Basel) for generous gifts o f benserazid.
REFERENCES 1 And6n, N. E., Carlsson, A., Hillarp, N. A. and Magnusson, T., 5-Hydroxytryptamine release by nerve stimulation of the spinal cord, Life Sci., 3 (1964) 473-478. 2 Basbaum, A. I. and Fields, H. L., Endogenous pain control mechanisms: review and hypothesis, Ann. Neurol., 4 (1978) 451-462. 3 Berman, A. L., The Brain Stem of the Cat: a Cytoarchitectonic Atlas with Stereotaxic Coordinates, University of Wisconsin Press, Madison, Wisc., 1968. 4 Besson, J. M., Dickenson, A. H., Le Bars, D. and Oliveras, J. L., Opiate analgesia: the physiology and pharmacology of spinal pain systems. In C. Dumont (Ed.), Advances in Pharmacology and Therapeutics - - Neuropsychopharmacology, Vol. 5, Pergamon Press, 1979, pp. 61 81. 5 Boadle-Biber, M. C., Activation of tryptophan hydroxylase from central neurons by calcium and depolarization, Biochem. Pharmacol., 27 (1978) 1069-1079. 6 Boarder, M. R. and Fillenz, M., Absence of increased tyrosine hydroxylation after induction of brain tyrosine hydroxylase following reserpine administration, Biochem. PharmacoL, 28 (1979) 1675-1677. 7 Bourgoin, S., Morot-Gaudry, Y., Glowinski, J. and Hamon, M., Stimulating effect of short term ether anaesthesia on central 5-HT synthesis and utilization in the mouse brain, Europ. J. PharmacoL, 22 (1973) 209-211. 8 Bourgoin, S., Ternaux, J. P., Boireau, A., H6ry, F. and Hamon, M., Effects of halothane and nitrous oxide anaesthesia on 5-HT turnover in the rat brain, Naunyn Schmiedeberg's Arch. exp. Path. Pharmak., 288 (1975) 109-121. 9 Bustos, G., Roth, R. H. and Morgenroth, V. H. III, Activation of tyrosine hydroxylase in rat striatal slices by K+-depolarization. Effect of ethanol, Biochem. Pharmaeol., 25 (1976) 2493-2497. I 0 Carlsson, A., Davis, J. N., Kehr, W., Lindqvist, M. and Atack, C. V., Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in vivo in brain, using an inhibitor of the aromatic amino acid decarboxylase, Naunyn Schmiedeberg 's Arch. exp. Path. Pharmak., 275 (1972) 153-168. 11 Dahlstrbm, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system, II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Actaphysiol. scand., Suppl. 247 (1965) 1-36. 12 Di Chiara, G., Onali, P. L., Tissari, A. H., Porceddu, M. L., Morelli, M. and Gessa, G. L., Destruction ofpost-synaptic dopamine receptors prevents neuroleptic-induced activation of striatal tyrosine hydroxylase but not dopamine synthesis stimulation, Life Sci., 23 (1978) 691-696. 13 Eccleston, D., Ritchie, I. M. and Roberts, M. H. T., Long term effects of midbrain stimulation on
389 5-hydroxyindole synthesis in rat brain, Nature (Lond.), 226 (1970) 84-85. 14 Fifkov~, E. and Marsala, J., Stereotaxic atlases of the cat, rabbit and rat. In J. Bures, M. Petran and J. Zachar (Eds.), Electrophysiological Methods in Biological Research, Academic Press, New York, N.Y., 1967, pp. 636-691. 15 Hamon, M., Bourgoin, S., Artaud, F. and Glowinski, J., The role of intra neuronal 5-HT and of tryptophan hydroxylase activation in the control of 5-HT synthesis in rat brain slices incubated in K+-enriched medium, J. Neurochem., 33 (1979) 1031-1042. 16 Hamon, M., Bourgoin, S., H6ry, F. and Simonnet, G., Activation of tryptophan hydroxylase by adenosine triphosphate, magnesium and calcium, Molec. Pharmacol., 14 (1978) 99-110. 17 Hamon, M., Bourgoin, S., H6ry, F., Ternaux, J. P. and Glowinski, J., In vivo and in vitro activation of rat brain stem soluble tryptophan bydroxylase, Nature (Lond.), 260 (1976) 61-63. 18 Hamon, M. and Glowinski, J., Regulation of serotonin synthesis, Life Sci., 15 (1974) 1533-1548. 19 Herr, B. E., Gallager, D. W. and Roth, R. H., Tryptophan hydroxylase activation in vivo following stimulation of central serotonergic neurons, Biochem. PharmacoL, 24 (1975) 2019-2033. 20 Kapatos, G. and Zigmond, M. J., Effect of haloperidol on dopamine synthesis and tyrosine hydroxylase in striatal synaptosomes, J. Pharmacol. exp. Ther., 208 (1979) 468~,75. 21 Kaufman, S., Properties of the pterin dependent aromatic amino acid hydroxylases. In G. E. W. Wolstenholme and D. W. Fitzsimons (Eds.), Aromatic Amino Acids in the Brain, Elsevier/Excerpta Medica-North-Holland, 1974, pp. 85-108. 22 L6ger, L., Wiklund, L., Descarries, L. and Persson, M., Description of an indolaminergic cell component in the cat locus coeruleus: a fluorescence histochemical and radioautographic study, Brain Research, 168 (1979) 43-56. 23 Meek, J. L. and Lofstrandh, S., Tryptophan hydroxylase in discrete brain nuclei: comparison of activity in vitro and in vivo, Europ. J. Pharmacol., 37 (1976) 377-380. 24 Murrin, L. C., Morgenroth, V. H. III and Roth, R. H., Dopaminergic neurons: effects of electrical stimulation on tyrosine hydroxylase, Molec. Pharmacol., 12 (1976) 1070-1081. 25 Oliveras, J. L., Bourgoin, S., H6ry, F., Besson, J. M. and Hamon, M., The topographical distribution of serotoninergic terminals in the spinal cord of the cat; biochemical mapping by the combined use of microdissection and microassay procedures, Brain Research, 138 (1977) 393-406. 26 Oliveras, J. L., Guilbaud, G. and Besson, J. M., A map of serotoninergic structures involved in stimulation producing analgesia in unrestrained freely moving cats, Brain Research, 164 (1979) 317-322. 27 Oliveras, J. L., Hosobuchi, Y., Bruxelle, J., Passot, C. and Besson, J. M., Analgesic effects induced by electrical stimulation of the nucleus raphe magnus in the rat; interaction with morphine analgesia, Proc. 7th Int. Congress Pharmacol. (Paris), 1 (1978)280. 28 Oliveras, J. L., Redjemi, F., Guilbaud, G. and Besson, J. M., Analgesia induced by electrical stimulation of the inferior centralis nucleus of the raphe in the cat, Pain, 1 (1975) 139-145. 29 Roth, R. H., Morgenroth, V. H. III and Salzman, P. M., Tyrosine hydroxylase: allosteric activation induced by stimulation of central noradrenergic neurons. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 289 (1975) 327-343. 30 Roth, R. H. and Salzman, P. M., Role of calcium in the depolarization induced activation of tyrosine hydroxylase. In E. Usdin, N. Weiner and M. B. H. Youdim (Eds.), Structure and Function of Monoamine Enzymes. Marcel Dekker, New York, N.Y., 1977, pp. 149-168. 31 Shields, P. J. and Eccleston, D., Effect of electrical stimulation of rat midbrain on 5-hydroxytryptamine synthesis as determined by a sensitive radioisotope method, J. Neurochem., 19 (1972) 265272. 32 Snedecor, G. W. and Cochran, W. G., Statistical Methods, 6th edn., Iowa State College Press, Ames, 1967. 33 Steinbusch, H. W. H., Distribution of serotonin-like immunoreactivity in the central nervous system and pituitary of the rat. In B. Haber (Ed.), Serotonin-Current Aspects of Neurochemistry and Function, Plenum, New York, N.Y., in press.
377
ELECTRICAL STIMULATION OF THE NUCLEUS RAPHE MAGNUS IN THE RAT. EFFECTS ON 5-HT METABOLISM IN THE SPINAL CORD
S. BOURGOIN, J. L. OLIVERAS, J. BRUXELLE, M. H A M O N and J. M. BESSON
Groupe N B - - I N S E R M U. 114, Colldge de France, l l,place Marcelin Berthelot, 75231 Paris Cedex 05 and ( J.L.O., J. B. and J.M.B.) Unitd de Recherches de Neurophysiologie Pharmacologique, I N S E R M U. 161, 2, rue d'Aldsia, 75014 Paris (France) (Accepted January 24th, 1980)
Key words: serotonin - - electrical stimulation - - raphe magnus - - spinal cord
SUMMARY
The direct electrical stimulation (with biphasic pulses of 1 msec, 10 pulses/sec, 200/,A, for 30 min) of the nucleus raphe magnus in chloral hydrate anaesthesized rats produced a significant acceleration ( + 5 0 ~ ) of 5-HT synthesis in the spinal cord as revealed by the increased rate of 5-HTP accumulation occurring at this level after the blockade of central 5-HTP decarboxylase with benserazid. In contrast, no change was detected in 5-HT metabolism in the forebrain of stimulated rats. The acceleration of 5-HT synthesis was likely not due to an increased availability of tryptophan for the rate-limiting enzyme, tryptophan hydroxylase, since the concentration of this amino acid was changed neither in the spinal cord, nor in the forebrain of stimulated rats. The measurement of tryptophan hydroxylase activity in soluble extracts from the spinal cord of control and stimulated rats revealed that the acceleration in 5-HT synthesis produced by the electrical stimulation of the nucleus raphe magnus was not associated with a persisting activation of this enzyme. Although one cannot completely exclude that a short-lasting activation of tryptophan hydroxylase, no longer detectable in soluble extracts, has occurred in the spinal cord of stimulated rats, the present findings rather suggest that the rate of 5-HT synthesis can be controlled by factors other than only the concentration of tryptophan and the intrinsic activity of tryptophan hydroxylase in serotoninergic neurons. The demonstration of an acceleration of 5-HT synthesis in bulbospinal serotoninergic neurons under stimulating conditions close to those producing analgesia in rats further supports the role of these neuronal systems in the physiological mechanisms of pain control.
INTRODUCTION
Among the various and complex .mechanisms involved in the regulation of
378 monoamines synthesis in the CNS, those triggered by the nerve impulse flow in monoaminergic neurons have raised considerable interest. Attempts to select appropriate experimental models for further analyzing the relationship between the firing and the neurotransmitter synthesis rates in monoaminergic neurons generally converge to those consisting of direct electrical stimulation of nerve fibres or perikaryons. Using this approach, Roth and coworkers ~4,29 reached the conclusion that the accelerated synthesis of noradrenaline (NA) and dopamine (DA) following the electrical stimulation of the locus coeruleus and of the substantia nigra, respectively, were due to the activation of tyrosine hydroxylase, the first enzyme involved in the biosynthesis of catecholamines (CA) in the CNS. Furthermore, they were able to propose a working hypothesis on the molecular mechanisms triggered by the depolarization of nerve terminals and ending at the activation of this key enzyme24,'~9 In the case of serotoninergic neurons, several authors1,13,19,3t have also observed that the direct electrical stimulation of cell bodies or fibres may result in an enhancement of the rate of 5-HT synthesis. Using the method developed by Carlsson et al. 1° to measure the rate of tryptophan hydroxylation in vivo, Herr et a1.19 reached the conclusion that the nerve impulse flow-induced change in 5-HT synthesis involves that very enzymic step. Although they could eliminate possible changes in tryptophan levels as being responsible for the increased synthesis observed ~9, several other mechanisms may produce an acceleration of tryptophan hydroxylation. Indeed, in the CNS, not only the concentration of tryptophan but also those of oxygen and of tetrahydrobiopterin (the putative endogenous pterin cofactor of aromatic amino acid hydroxylase) are lower than the respective Kms of tryptophan hydroxylase (EC 1.14.16.4)2L Therefore, an increase in the concentration of oxygen or/and of that of tetrahydrobiopterin in tissues may well trigger an acceleration of tryptophan hydroxylation. Furthermore, alterations of enzyme effectors or of tryptophan hydroxylase itself (activation) may also be involved in the acceleration of 5-HT synthesis induced by the electrical stimulation of the raphe nuclei. In an attempt to further explore the mechanisms triggering the acceleration of 5HT synthesis in stimulated neurons, we first selected experimental conditions leading to a reproducible increase in 5-HT synthesis as the result of electrical current passing through the raphe. Then, we extracted tryptophan hydroxylase from stimulated tissues in order to detect any possible alteration consecutive to in vivo electrical stimulation of serotoninergic neurons. The growing interest for the bulbospinal serotoninergic pathway with respect to the central physiology, notably the control of pain transmission2,4, led us to study the effects of electrical stimulation of cell bodies in the nucleus raphe magnus on the synthesis of 5-HT in nerve terminals in the spinal cord. This raphe nucleus has been shown by several groups to contain the majority of cell bodies of serotoninergic neurons descending within the spinal cord tl,25. MATERIALSAND METHODS Chemicals. Adenosine-triphosphate (ATP, Boerhinger-Mannheim), DL-6-methyl-
379 5,6,7,8-tetrahydropterin (6-MPH4, Calbiochem), chloral hydrate (Prolabo), Benserazid (Ro 4.4602, Hoffmann-LaRoche). Other compounds (salts, indoles... ) were from usual sources (Merck, Prolabo). Animals. Adult male Sprague-Dawley (Charles River strain) rats weighing 350--400 g were used. Before the experiments, they were kept for at least 10 days in a controlled environment (24 °C, 60~ relative humidity, alternate cycles of 12 h light and 12 h darkness, food and water ad libitum). Chloralhydrate (400 mg/kg, 1 ml/rat) and benserazid (800 mg/kg, 2.5 ml/rat) were dissolved in saline and administered i.p.
Electrical stimulation of the nucleus raphe magnus Five to 10 min following an i.p. injection of chloral hydrate (400 mg/kg), rats were positioned in a stereotaxic apparatus (La Pr6cision Cin6matographique, Asni6res, France). The skull was drilled and a bipolar concentric stimulating electrode was lowered into the nucleus raphe magnus (A = --3; H = ---0.5; L = 0, with the zero at the middle of the interauricular axis). On occasion, the electrode was placed laterally to the nucleus raphe magnus (L = 1). The structures were identified according to the stereotaxic atlas of Fitkov~, and Marsala 14. Concentric electrodes were made of stainless steel. The external diameter of the outer core was equal to 0.25 mm. The inner core was sharpened to a diameter of 30/~m at the tip which protruded by 0.20 mm from the outer core. Electrical stimulation consisted of biphasic rectangular pulses (1 msec, 10/sec, 100 or 200 #A) and was maintained for 30 min. Histological control of the electrode location revealed that such stimulating conditions did not produce any detectable lesion even at the tip. Sham animals were prepared in the same way except that no current was passed through the electrode during the 30 min period they were maintained in the stereotaxic frame. Biochemical analyses At the end of the stimulation period or 30 min later (see Results), rats were decapitated and their brain and spinal cord were rapidly removed. The brain stem and the cerebellum were isolated by a coronal section in front of anterior colliculi. This piece of tissue was immediately immersed in a saline solution containing 3 ~ formaldehyde. Histological control of the electrode location proceeded as described previously28. The remaining forebrain and the srinal cord were homogenized in 6 ml and 4 ml, respectively, of an ice-cold ethanol-water solution (74:16, v]v) containing 0.05 ~ of EDTA and 0.05 ~o of ascorbic acid. After a storage at --30 °C for 12 h, homogenates were centrifuged and 5-HT, tryptophan, 5-hydroxytryptophan (5-HTP) and 5-hydroxyindole acetic acid (5-HIAA) in the clear supernatant were selectively extracted by ion-exchange and adsorption chromatography. They were finally measured using appropriate spectrofluorimetric methods. The complete procedure is described in details elsewhere a. When the activity of tryptophan hydroxylase was measured, tissues were homogenized in two steps. A first homogenate was obtained by disruption in 2 (for the
380 forebrain) or 3 (for the spinal cord) vols. (v/w) of 0.05 M Tris.acetate, pH 7.6, containing 2 mM/3-mercaptoethanol, using a Polytron PT l 0 0 D apparatus. Homogenates were then centrifuged at 35,000 g for 30 min at 4 cC and the resulting supernatants were used as the final sources of tryptophan hydroxylase. The enzymic activity was measured on 0.2 ml aliquots of the supernatants with 0.15 mM tryptophan and 0.16 mM 6-MPH i as the pterin cofactor. The reaction proceeded for 15 min at 37 °C under air atmosphere and the final product, 5-HTP, was measured spectrofluorimetrically (see ref. 16 for details). Since these assay conditions were subsaturating, they were appropriate to detect any change (inhibition, activation or induction) in tryptophan hydroxylase intrinsic activity a~i. The second homogenate was obtained by mixing the remaining tissue supernatants with the corresponding pellets using the Polytron PT 10 OD disrupter. This was achieved after the addition of ice-cold ethanol (4:1 (v/v) of the first homogenate) and EDTA (0.05% final concentration). The resulting mixture was then treated as above for the biochemical determinations of indolic compounds. No significant difference in the respective fluorescence blanks and recoveries (75-95 °/o) was noted for tryptophan, 5-HTP, 5-HT and 5-HIAA when homogenization was carried out by the usual method (with ethanol-water 74:16, v/v) or by the two step procedure described above (data not shown). Statistical calculations were performed as proposed by Snedecor and Cochran ~2. When the P-value was higher than 0.05 (Student's t-test), a difference was consisted to be non-significant. RESULTS
Effects of chloral hydrate anaesthesia on 5-HT metabolism in the spinal cord and the forebrain of adult rats The rate of 5-HT synthesis was estimated by measuring the accumulation of 5HTP in tissues after the blockade of 5-HTP decarboxylase 1° by benserazid. For this series of experiments, 4 groups of rats were used; A, control rats; B, animals treated with chloral hydrate (400 mg/kg) 40 min before death; C, animals treated with benserazid (800 mg/kg i.p.) 30 rain before death; and D, those receiving both treatments. As shown in Table 1, anaesthesia for 40 min in rats treated with chloral hydrate alone (group B) did not significantly alter the endogenous levels of tryptophan, 5-HT and 5-HIAA in the spinal cord. In contrast, slight but significant elevations of 5-HT ( ÷ 1 7 ~ ) and 5-HIAA ( + 2 6 ~ ) levels were detected in their forebrain (Table II). Comparison of data obtained in groups C and D indicates that chloral hydrate anaesthesia failed to modify the rate of 5-HTP accumulation in tissues after the blockade of 5-HTP decarboxylase. However, anaesthesia did partly antagonize the benserazid-induced reduction in 5-HT levels. Thus, both in the spinal cord (Table I) and in the forebrain (Table I1), the 5-HT levels in anaesthetized animals treated with benserazid (group D) were not significantly different from those found in controls (group A).
381 TABLE I
Effects o f anaesthesia with chloral hydrate on 5 - H T metabolism in the spinal cord Four groups of 8 rats were used. Two groups were anaesthesized with chloral hydrate (400 mg/kg i.p.). Ten to 20 min after the onset of anaesthesia, 8 rats together with 8 control animals were treated with benserazid (800 mg/kg i.p.). Animals were killed 30 min after benserazid or 40 min after chloral hydrate anesthesia. Eight untreated rats served as 'controls'. Each value (mean 4- S.E.M. of 8 individual determinations) is expressed as/~g/g of fresh tissue.
A B C D
Controls Chloral hydrate Benserazid Chloral hydrate 4benserazid
TRP
5-HTP
5-HT
5-H1A A
5.51 -4- 0.31 4.93 -- 0.31 6.24 4- 0.22
--0.144 4- 0.008
0.524 4- 0.023 0.538 4- 0.025 0.427 zk 0.018"
0.429 -4- 0.036 0.460 -4- 0.015 0.375 -4- 0.030
6.05 ± 0.25
0.129 -4- 0.005
0.463 -4- 0.024
0.403 ± 0.024
* P < 0.05 when compared to the control group. TABLE II
Effects o f anaesthesia with chloral hydrate on 5 - H T metabolism in the forebrain The protocol is described in the legend to Table I. Each value (mean 4- S.E.M. of 8 individual det,rm±nations) is expressed as/~g/g of fresh tissue.
TRP A B C D
Controls Chloral hydrate Benserazid Chloral hydrate 4benserazid
5-HTP
5-HT
5-HIA A
4.79 4- 0.17 4.59 4- 0.24 5.54 4- 0.22
--0.170 4- 0.009
0.445 i 0.017 0.522 -4- 0.018" 0.353 4- 0.017"
0.418 -4- 0.020 0.525 4- 0.017" 0.382 4- 0.030
5.37 -4- 0.12
0.154 4- 0.006
0.435 -4- 0.032
0.451 4- 0.025
* P < 0.05 when compared to respective control values. TABLE IIl
Effects o f electrical stimulation o f the nucleus raphe magnus on 5-HTmetabolism in the spinal cord A: rats were anaesthesized with chloral hydrate (400 mg/kg i.p.) and positioned in a stereotaxic apparatus. The stimulating electrode was placed as described in 'Materials and Methods' but current was passed through only the rats in the 'stimulated' group. All rats received an i.p. injection of benserazid (800 mg/kg) as soon as the electrode was correctly postioned. They were killed 30 min later (i.e. at the end of the stimulation period for the 'stimulated' group). B: the same protocol as in A was used except that benserazid was administered 30 rain after the animals were positioned in the stereotaxic apparatus for electrode implantation ('sham') or continuous stimulation ('stimulated'). All rats were maintained in the stereotaxic frame for 30 min following the drug injection but with no stimulation. They were killed by decapitation and biochemical analyses were performed as described in 'Materials and Methods'. Each value (in .ug/g of fresh tissue) is the mean -4- S.E.M. of 8-14 individual determinations.
I RP A Sham Stimulated B Sham Stimulated
6.13 6.29 6.03 6.39
-4- 0.27 -4- 0.20 4- 0.36 4- 0.42
5-HTP
5-HT
0.134 -4- 0.015 0 . 2 0 1 i 0.012" 0.132 4- 0.008 0.185 -4- 0.008*
0.483 0.485 0.471 0.482
* P < 0.05 when compared to respective 'sham' values.
5-HIAA -4- 0.025 4- 0.018 -4- 0.017 -4- 0.021
0.399 -4- 0.021 0.443 4- 0.017 0.361 4- 0.019 0.411 4- 0.017
382 In conclusion, although chloral hydrate anaesthesia did alter 5-HT metabolism in the CNS (increases in 5-HT and 5-HIAA levels in the forebrain likely involving a reduced utilization of 5-HT and a partial blockade of 5-H1AA efflux respectively), the observed modifications were less pronounced than after halothane, nitrous oxide8 or ether 7 inhalation. This led us to choose chloral hydrate as a more appropriate anaesthetic in subsequent experiments.
Effects of electrical stimulation of the nucleus raphe magnus on 5-HT metabolism in the C NS of chloral hydrate-anaesthesized rats (a) 5-HT metabolism during the stimulation period. Immediately after the administration of benserazid, electrical stimulation of the nucleus raphe magnus was turned on and maintained for 30 min, i.e. till death. When the intensity current was equal to 200/tA, this stimulation resulted in a significant increase ( + 50 ~, Table III) in the rate of 5-HTP accumulation in the spinal cord when compared to that found in control rats. Decreasing the intensity by half markedly reduced this effect since only a small increment in the rate of 5-HTP accumulation was observed in the spinal cord (-~- 12 °/o,P > 0.05) of rats electrically stimulated for 30 min with biphasic pulses of 100 /zA (data not shown). The electrical stimulation of the nucleus raphe magnus with a current of 100 #A (data not shown) or 200/~A (Table IV) did not affect the rate of 5-HTP accumulation in the forebrain. As shown in Tables III and IV, neither tryptophan, 5-HT nor 5-HIAA levels were modified by the electrical stimulation of the nucleus raphe magnus. However, it should be noted that a slight non-significant increase in the concentration of 5-HIAA was regularly observed (in 3 independent experiments) particularly in the spinal cord of stimulated rats (Table III). Detailed analysis of the electrode sites producing significant increases in the rate of 5-HTP accumulation indicated that those precisely located in the nucleus raphe magnus were more efficient (m ---- +56 ± 11 ~) than those outside (+32 ~ 6 ~o) (Fig. 1). However, even when the tip of the electrode was situated 0.2 mm laterally to the nucleus, electrical stimulation was still efficient in producing a significant acceleration of 5-HTP formation (Fig. 1). (b) 5-HT metabolism after the stimulation period. Whereas benserazid treatment was applied for the whole stimulation period in the preceding experimental situation (a), in this second series of experiments, the 5-HTP decarboxylase inhibitor was injected at the end of the stimulation period. Animals were then killed 30 min later. Under these conditions, only the 200/~A current was used. Like with the previous experimental design (a), the rate of 5-HTP accumulation remained unaltered in the forebrain (Table IV) whereas it increased significantly in the spinal cord (Table III) of stimulated animals. However, the increment in the rate of 5HTP formation (+40.2 ~) occurring during the post-stimulation period (conditions b) was slightly less pronounced than that ( + 5 0 ~ ) observed during the stimulation period (conditions a).
383
ao' O o 4-4602
©
Fig. 1. Cross section of the brain stem at the level of the nucleus raphe magnus showing the sites where passing current triggered an acceleration of 5-HTP synthesis in the spinal cord. Benserazid (Ro 4-4602, 800 mg/kg i.p.) was administered to chloral hydrate anaesthesized rats at the beginning of the electrical stimulation. Animals were killed 30 min later, at the end of the stimulation period. Each figure corresponds to the per cent increase in 5-HTP accumulation resulting from the stimulation at the indicated site. Rm, nucleus raphe magnus. The control value (100 %) is the mean of 5-HTP accumulated in the spinal cord of 14 rats placed under the same conditions except that no current was passedthrough the electrode.
As already noted under the previous experimental conditions (a), tryptophan, 5HT and 5-HIAA levels were not significantly affected by the stimulation. However, as before, 5-HIAA levels in the spinal cord were slightly increased in stimulated animals ( + 1 4 Z , P > 0.05). Histological control of the electrode locations (Fig. 2) revealed that the more efficient sites were situated in the nucleus raphe magnus or just in its vicinity. When the electrode tip reached zones situated at least 0.25 mm beyond the nucleus sides, the rate of 5-HTP accumulation was hardly affected by the electrical stimulation ( + 7 and + 14 ~o, see Fig. 2, and data not shown for sites located farther than those indicated in this Fig.). TABLE IV
Effects of electrical stimulation of the nucleus raphe magnus on 5-HTmetabolism in the forebrain The protocol is described in the legend to Table IlL Each value (in/~g/g of fresh tissue) is the mean 4- S.E.M. of 8-14 individual determinations.
TRP A Sham Stimulated B Sham Stimulated
5.25 5.38 5.42 6.05
+ 0.47 4- 0.24 -4- 0.32 -4- 0.31
5-HTP
5-HT
0.159 0.171 0.162 0.177
0.453 0.453 0.450 0.438
4- 0.018 4- 0.016 4- 0.012 4- 0.011
5-HIAA 44+ i
0.029 0.024 0.019 0.019
0.455 0.491 0.515 0.520
44± +
0.023 0.022 0.056 0.019
384
Fig. 2. Cross section of the brain stem at the level of the nucleus raphe magnus showing the sites where electrical stimulation produced a persisting acceleration of 5-HTP synthesis in the spinal cord. Chloral hydrate anaesthesized rats were maintained for 1 h in a stereotaxic frame. For the first 30 min, current was applied to the indicated sites under the conditions described in 'Materials and Methods'. Benserazid (800 mg/kg i.p.) was then administered and rats were killed 30 min later. Each figure corresponds to the per cent increase in 5-HTP accumulation following electrical stimulation at the indicated site. Rm, nucleus raphe magnus; VII, nucleus originis nervi facialis; P, tractus corticospinalis.
Current spread in the vicinity oJ,"the nucleus raphe magnus in the course of its electrical stimulation with 200 #A biphasic pulses Since the electrical stimulation of sites located outside the nucleus raphe magnus also produced marked elevations in the rate of 5-HTP formation, the current should most likely spread from the electrode tip to reach serotoninergic cell bodies in this nucleus. Observations of the animal during electrical stimulation was, in this respect, very informative. Thus, in a few cases, rhythmic contractions of face muscles and vibrissae oscillations occurred in phase with the current. As shown in Fig. 3, this was seen when the electrode tip was between the nucleus raphe magnus and the nucleus originis nervi facialis (VII) which contains the cell bodies of neurons innervating face muscles. Accordingly, under the present experimental conditions, the current was probably spreading enough to produce efficient excitation of neurons inside a sphere of about 0.20 m m radius a r o u n d the electrode tip.
Effects of electrical stimulation of the nucleus raphe magnus on the activity of tryptophan hydroxylase in soluble extracts from the spinal cord Since the rate of 5-HTP accumulation was increased not only during but alsoJor the 30 minfollowing the electrical stimulation, this implied that the full effect o f raphe stimulation on 5-HT synthesis would be occurring just at the end of the stimulation period. This time was chosen to look for possible changes in t r y p t o p h a n hydroxylase activity in tissues from stimulated rats.
385
•
face
movements(lO/S)
no effect
Fig. 3. Sites of electrical stimulation producing face movements. When the electrode was positioned at the black dots, passing current produced face movements indicating that stimulation reached the VII nucleus. Therefore, even when the electrode was not in the right position but only adjacent to the nucleus raphe magnus, the current diffusion was large enough to produce excitation of serotoninergic neurons Thus, with the electrode located at the two black dots on the left, we still observed a slight increase ( ± 7; ± 14 ~ ; see Fig. 2) in the rate of 5-HTP formation in the spinal cord. Rm, nucleus raphe magnus; VII, nucleus originis nervi facialis.
TABLE V Effects o f electrical stimulation o f the nucleus raphe magnus on tryptophan hydroxylase activity in vivo and in vitro in the spinal cord
Four groups of 6 rats were used. Two groups were anaesthesized with chloralhydrate (400 mg/kg i.p.) and positioned in a stereotaxic apparatus. These and 6 other animals received benserazid (800 mg/kg i.p.) 30 min before death. Electrical stimulation of the nucleus raphe magnus was set on just after the i.p. injection of the 5-HTP decarboxylase inhibitor as described in 'Materials and Methods'. The in vivo accumulation of 5-HTP is expressed as nmol/g tissue/l 5 min. Tryptophan hydroxylase activity (in vitro) was assayed in the 35,0D0 g sup~rnatant of spinal cord homogenates with or without 0.5 m M ATP, 5 m M Mg ~+ and 50/~M Ca 2÷ (phgsphorytating mixture 16). It is expressed as nmol 5-HTP formed/g of fresh tissue and per 15 min. Each value is the mean 4- S.E.M. of 6 individual determinations. 5-HTP in vivo
in vitro ÷ ATP ÷ Mg 2+ + Ca 2+
Control Benserazid Benserazid -- chloral hydrate Benserazid + chloral hydrate ± electrical stimulation
-0.326 i' 0.018 0.312 ± 0.018
10.81 ± 0.57 9.44 ± 0.85 9.33 ± 0.59
14.53 ± 0.67** 13.31 i 0.98** 12.74 ± 0.71 **
0.449 ± 0.041"
9.64 4- 0.47
14.00 ± 1.18"*
* P -< 0.05 when compared to corresponding values in the two other groups. P < 0.05 when compared to tryptophan hydroxylase activity in the same group but in the absence of ATP, Mg ~+, Ca 2+.
* *
386 Although the electrical stimulation induced a significant increase in the rate of 5HTP formation in vivo, no change in the activity of tryptophan hydroxylase was detected in soluble extracts from the spinal cord of stimulated rats (Table V). In addition, the activation of this enzyme by the ATP, Mg 2+, Ca `)'- mixture (see ref. 16) was similarly pronounced whether tryptophan hydroxylase was extracted from control or from stimulated animals (Table V). The comparison of the rate of tryptophan hydroxylation in the spinal cord in vivo with the in vitro activity of soluble tryptophan hydroxylase from the same tissues revealed that the latter was 20-30 times higher than the former value (Table V). This was true for control and stimulated animals (Table V). DISCUSSION The present data demonstrated that the synthesis of 5-HT increased in the terminals of serotoninergic bulbospinal neurons when their cell bodies were electrically stimulated. Therefore, they extend to descending serotoninergic neurons the findings already reported for those located in the anterior raphe nuclei and projecting rostrally into the forebrain la,19,31. The comparison of experimental conditions for stimulating the anterior raphe nuclei19, 3~ with those presently used for the stimulation of the posterior nucleus raphe magnus indicates that an intensity current of 200/~A was required to produce a significant acceleration of 5-HT synthesis in terminal areas. However, this does not mean that such an intensity was necessary at the very level of serotoninergic neurons since histological control suggested that current spread from sites located outside the nucleus raphe magnus was sufficient to produce excitation of serotoninergic neurons. In this respect, further comparison with available data in the literature is impossible since the histological control of the exact locations of the stimulated sites was never reported. Although most serotoninergic cell bodies are located in raphe nuclei, a nonnegligible proportion is situated outside 2~,a3 and therefore, the effective stimulation sites lateral to the nucleus raphe magnus might well involve extra raphe neurons also projecting into the spinal cord. However, experiments in the cat consisting of selective lesion of the nucleus raphe magnus within the limits described by Berman 3 have shown that at least 70 ~ of serotoninergic terminals in the spinal cord belong to neurons situated in this nucleus 25. It is therefore very unlikely that the electrical stimulation of less than 30 ~ of these extra raphe neurons would be as efficient as that at the very level of the nucleus raphe magnus to produce an acceleration of 5-HT synthesis in the spinal cord ~see Figs. 1 and 2). Current spread from the electrode tip to the nucleus raphe magnus was more likely the main factor responsible for the activation of spinal 5-HT synthesis occurring when extra raphe sites were electrically stimulated. Under normal conditions, tryptophan hydroxylase, the rate limiting enzyme for the synthesis of 5-HT, is not saturated by tryptophan ~8. Therefore, fluctuations in the concentration of this amino acid in brain tissues generally trigger parallel modifications in the rate of 5-HT synthesis 18. In the present case, since tryptophan levels were not altered in tissues from stimulated animals, it can be argued that the acceleration of
387 5-HTP formation was not the consequence of a primary increase in the concentration of the precursor amino acid in the spinal cord (see also refs. 19 and 31). However, one cannot completely rule out that the tryptophan concentration actually increased in serotoninergic neurons in a very discrete pool directly involved in the enzymic hydroxylation process (see ref. 18). Unfortunately, the measurement of tryptophan concentration in the immediate vicinity of tryptophan hydroxylase is not yet possible. Apparently, the acceleration of 5-HT synthesis occurring in electrically-stimulated serotoninergic neurons was not the consequence of an activation of tryptophan hydroxylase since no significant alteration in the intrinsic activity of this enzyme was detected in soluble extracts from the spinal cord after electrical stimulation of the nucleus raphe magnus. This observation is in contrast with recent in vitro findings demonstrating that K+-induced depolarization of 5-HT neurons did produce a marked activation of the soluble enzymeSA5. The difference between in vitro depolarization and in vivo electrical stimulation was further substantiated when considering the effects of in vitro activating conditions on tryptophan hydroxylase: whereas Ca z÷dependent phosphorylation (with the ATP, Mg 2+, Ca 2+ mixture) increased the activity of tryptophan hydroxylase extracted from tissues of stimulated (this paper) or nonstimulated animals 16, it was no longer effective to further enhance the activity of the enzyme extracted from tissues depolarized in vitro 15. Since the assay conditions for the measurement of tryptophan hydroxylase activity in vitro are very different from those in 5-HT neurons, it can be argued that they are not suitable for detecting an activation of this enzyme which would be induced in vivo. As already discussed by Meek and Lofstrandh2a, artificial in vitro conditions resulted in tryptophan hydroxylase activity markedly higher than that measured in vivo, possibly meaning that numerous regulatory properties were lost under in vitro conditions. However, it has to be emphasized that these in vitro assay conditions have been shown quite appropriate for detecting the activation of tryptophan hydroxylase resulting from in vivo treatment v~ithmethiothepin17 or from depolarization of brain slices15. Another criticism to the experimental approach used in the present study may concern the time elapsing between the end of the stimulation and the extraction of tryptophan hydroxylase from tissues. If the putative in vivo activation of the enzyme was rapidly reversible, this time would have been long enough to allow tryptophan hydroxylase to return to a non-activated state. However, the effect of electrical stimulation of the nucleus raphe magnus on the rate of 5-HT synthesis in the spinal cord was still observed during the 30 minfollowing the stimulation period indicating that the return to a non-activated state might be a slow-acting process. Furthermore, previous examples of enzymic activation either by in vivo electrical stimulation (tyrosine hydroxylase24, 29) or by in vitro depolarization (tyrosine and tryptophan hydroxylasesg,l~, a°) have shown that the enzyme alteration persisted under drastic conditions such as freezing and thawing of tissues and homogenates. Therefore, it seems reasonable to conclude that the electrical stimulation of the nucleus raphe magnus induced an acceleration of 5-HT synthesis which was not associated with an activation of tryptophan hydroxylase. As recently discussed for catecholaminergic
388 neurons 12,2°, the present findings indicated that the rate o f the neurotransmitte~ synthesis m a y increase in the absence o f any significant change in the activity o f the rate-limiting enzyme. Reciprocally, an activation o f the rate-limiting enzyme m a y well occur w i t h o u t p r o d u c i n g an acceleration o f the n e u r o t r a n s m i t t e r synthesis6, I.~. Since the conditions used in this study were close to those which have been shown to induce analgesia in rats a n d cats 26 '~8, it can be concluded that increased activity o f descending serotoninergic p a t h w a y s does occur during electrically i n d u c e d analgesia in animals. In this respect, the present findings further confirm the role of the bulbospinal serotoninergic system in the physiological mechanisms o f pain control 2,4. ACKNOWLEDGEMENTS This research was s u p p o r t e d by grants from I N S E R M ( A T P 76.71), D G R S T , D R E T (79.077) a n d R h 6 n e Poulenc S.A. The excellent technical assistance o f Miss F. A r t a u d is gratefully acknowledged. W e are indebted to Dr. W. Haefely ( H o f f m a n n - L a Roche, Basel) for generous gifts o f benserazid.
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