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Role of the raphe nuclei in the regulation of adrenal tyrosine hydroxylase
Brain Research, 122 (1977) 183-190
183
© Elsevier/North-Holland BiomedicalPress, Amsterdam - Printed in The Netherlands
Role of the raphe nuclei in the regulation of adrenal tyrosine hydroxylase
MARYKA QUIK, THEODORE L. SOURKES, BERNARDO O. DUBROVSKY and SERGE GAUTHIER Department of Psychiatry, Allan Memorial Institute, McGill University, Montreal, Quebec 1t3.4 1.4 l (Canada)
(Accepted November llth, 1976)
A variety of conditions, such as immobilizations tress zl, cold exposure 32, swim stress 2a, psychosocial situations 6 and the administration of certain drugs~, 33, which produce a sustained increase in peripheral sympathetic activity lead to an increase of tyrosine hydroxylase (TH, tyrosine-3-monooxygenase, EC 1.14.16.2) in the adrenal medulla. This enzyme catalyzes the rate-limiting step in catecholamine biosynthesis 23. Evidence has accumulated that transsynaptic influences play a vital role in the regulation of TH activity12,21,33; that is, the augmented enzyme levels14,17 occur through centrally mediated mechanisms. A role for a serotoninergic system in the control of adrenal TH has been suggested since destruction of serotonin (5-HT) nerve terminals with 5,7-dihydroxytryptamine, blockade of 5-HT receptors with methiothepin and depletion of brain 5-HT with p-chlorophenylalanine (PCPA) increase adrenal TH activitys,30. FurthErmore, these treatments potentiate the induction of adrenal TH produced by electrical stimulation of a mesencephalic site 25 or by the administration of drugs that stimulate central dopamine receptors8, ~0. This suggests that a central serotoninergic system has a net inhibitory role in the regulation of adrenal TH. To determine whether control of adrenal TH activity by the serotoninergic system involved in this regulatory process could be localized to a specific brain region the effect of lesions of the raphe nuclei, areas rich in 5-HT cell bodies4, 9, on adrenal TH activity was investigated. For the lesions, Sprague-Dawley rats, weighing 150 g, were anesthetized with sodium pentobarbital and placed in a stereotaxic instrument. A burr hole was made in the midline over the midbrain raphe nuclei. Anodal electrolytic lesions were produced by passing a current of 2 mA for 10 sec through a stainless steel monopolar electrode of contact diameter and exposed length of 0.25 mm and 0.5 mm, respectively. A large electrode clipped to the wound margin was connected to the cathode. Stereotaxic coordinates (according to K6nig and Klippe119) were anterior ( + ) 350/~m and vertical (--) 2.6 mm for the medial raphe nucleus, and anterior ( + ) 350/zm and vertical (--) 0.6 mm for the dorsal raphe nucleus. Sham-operated control animals were treated in the same manner except that the electrode was not lowered intracranially; for shamlesioned controls the electrode was placed as for the lesion but no current was passed.
184 TABLE 1 Effect o f medial and dorsal raphe nuclei lesions on adrenal TH activity and brain monoanline cont'entrations
The lesions were produced as described in Materials and Methods. In exp. A, results were pooled from rats killed 6 and 11 days after the lesions. In exps. B and C, the rats were killed 10 days after the lesion. The adrenal TH activity and forebrain monoamine concentrations were then determined. Means t S.E.M. are shown. Sham-operated controls did not have the electrode placed intracranially. Experiment Group
Number Tyrosine hydrox- Brain amine (#g/g) o f rats ylase (nmoles ................ DOPA/h per pair 5-HT DA NA adrenals)
A
11
52.83 ± 1.75
0.32 i 0.02
6 20 10 11 19
90.09 41.26 44.10 54.65 75.99
0.12 0.38 0.19 0.35 0.28
B C
Sham operated Dorsal plus medial lesion Sham-operated Dorsal lesion Sham-operated Medial lesion
:k 8.19'* ± 1.34 ± 2.04 i 2.88 = 2.49**
1.50 zk 1.07 0.25 i 0.01
± 0.02"'1.76 ± ± 0.01 1.49 ± 4- 0.02* 1.43 ± ± 0.01 1.26 ± :k 0.02* 1.23 ±
0.13 0.05 0.06 0.10 0.05
0.23 0.29 0.26 0.28 0.29
± 0.02 :k 0.01 ± 0.01" ± 0.01 _%0.01
Significance of difference from controls : ** P < 0.001 ; * P < 0.05.
A n i m a l s were killed by decapitation. The brains were r e m o v e d a n d the f o r e b r a i n (minus h i p p o c a m p u s a n d h y p o t h a l a m u s ) was weighed a n d h o m o g e n i z e d in 10 ml 0.4 N p e r c h l o r i c acid for a m i n e determinations. F o r histological verification o f electrode placement, the b r a i n stems were i m m e r s e d in 10 ~ f o r m a l i n for 2-3 weeks; frozen sections were then cut at 45 # m a n d stained with cresyl violet. The a d r e n a l s were also rem o v e d a n d placed in ice-cold 0.3 M sucrose. The fat a n d connective tissue were cut away, a n d each p a i r o f a d r e n a l s was weighed a n d h o m o g e n i z e d in 0.9 ml o f 0.3 M sucrose with a Teflon homogenizer. A n aliquot o f the h o m o g e n a t e (0.2 ml) was used for assay o f T H activity. T H was assayed a c c o r d i n g to the m e t h o d o f N a g a t s u et al. 27 with certain modifications as previously described 29. F o r d e t e r m i n a t i o n o f b r a i n amines, f o r e b r a i n s were h o m o g e n i z e d in 10 ml 0.4 N perchloric acid; the m a t e r i a l was centrifuged a n d the s u p e r n a t a n t b r o u g h t to p H 7.5-8.0 by a d d i t i o n o f 4 N N a O H c o n t a i n i n g 250 m M N a ~ E D T A . The n o r a d r e n a l i n e ( N A ) a n d d o p a m i n e ( D A ) were then a d s o r b e d on an a l u m i n a c o l u m n ~ (0.5 c m x 2.0 cm), previously equilibrated with a solution 10 m M with respect to b o t h N a 2 E D T A a n d Na2S2Oa, a n d o f p H 8.0; 5-HT was a b s o r b e d on an A m b e r l i t e c o l u m n 3 (0.5 c m x 2.0 cm) equilibrated with 0.2 M NaH~PO4 o f p H 6.7. This c o l u m n was placed below the a l u m i n a c o l u m n to receive the m a t e r i a l flowing f r o m it. The c a t e c h o l a m i n e s were eluted f r o m the a l u m i n a with 4 ml 0.1 N perchloric acid a n d the 5 - H T was released f r o m the A m b e r l i t e c o l u m n s with 10 m g ~ ascorbic acid in 2 N NC1. T h e fluorescence o f 5-HT solutions was r e a d i m m e d i a t e l y ; the N A a n d D A were assayed later by the t r i h y d r o x y i n d o l e technique o f Laverty a n d Taylor2L T o determine whether alterations in a d r e n a l T H activity after t r e a t m e n t with 5 , 7 - d i h y d r o x y t r y p t a m i n e , m e t h i o t h e p i n o r P C P A s,3° are due to generalized inter-
185 ference with central serotonin systems or to dysfunction of specific nuclei, a lesion was made either in the dorsal raphe nucleus (B7) or in the medial raphe nucleus (B8). Some rats were lesioned in both sites. These two nuclei contain most of the 5-HT cell bodies whose axons project to the telencephalon 4,9 and their destruction markedly depletes forebrain 5-HT 16,2°,24. After the combined lesion the adrenal T H activity was significantly (P < 0.001) increased (Table I, exp. A); the 5-HT concentration in the forebrain decreased to 38 ~ of control. When the lesion was restricted to the dorsal raphe nucleus (Table I, exp. B), adrenal T H activity remained at control level, despite the 42 ~ decrease in forebrain 5-HT. After a lesion of the medial raphe nucleus (exp. C), a highly significant increase in adrenal T H activity was observed; the decrease in forebrain 5-HT was only 20 ~ . No significant changes were observed in the D A and N A concentrations in exps. A and C (Table I); however, in exp. B, there was a very small but statistically significant decrease in brain NA. This indicates that the lesions were relatively specific for 5-HT neurons, a fact which is confirmed by the histological sections (Fig. 1). In Table I the biochemical data from the lesioned rats have been compared to those from controls which had been sham-operated but which had not had the electrode lowered intracranially. To ensure that the results obtained are due to the lesion, and not to non-specific damage, sham-operated controls were also compared with sham-lesioned controls. Table II shows that in the case of the dorsal raphe lesion the two types of control have similar adrenal TH activity and brain amine concentrations. In the case of sham lesion of the medial raphe nuclei, only the forebrain
TABLE II The effect of electrode placement in the medial and/or dorsal raphe nuclei on adrenal TH activity and brain monoamine concentrations
Sham-operated controls and sham-lesioned controls were treated in the same way as lesioned rats except that (a) for sham-operated controls the electrode was not placed intracranially and (b) for the sham-lesioned controls the electrode was in place but no current was passed. The rats were killed 10 days after treatment, at which time the adrenal TH activity and brain amine concentrations were determined as described in the text. Means 4- S.E.M. are shown. Group
Sham-operated for dorsal lesion Sham-lesioned for dorsal lesion Sham-operated for medial or combined lesion Sham-lesioned for medial or combined lesion
Number Tyrosine hydroxylof rats ase (nmolesDOPA/h perpair adrenals)
Brain amines (Itg/g) 5-HT
DA
NA
20
41.26 4- 1.34
0.38 4- 0.01
1.49 4-4-0.05
0.29 4-4-0.01
10
40.07 4- 1.51
0.41 4- 0.03
1.58 4-4-0.12
0.30 4-4-0.01
22
53.74 4- 1.65
0.33 4- 0.01
1.37 4- 0.07
0.27 4- 0.01
21
55.13 4- 2.31
0.25 4- 0.02*
1.35 4- 0.05
0.26 4- 0.01
* Significance of difference from sham-operated group: P < 0.01.
A
B
Fig. 1.
C
Fig. 1. Frontal plane section (45/~m) through caudal midbrain showing a representative lesion of (A) the medial and dorsal raphe nuclei, (B) the medial raphe nucleus, (C) the dorsal raphe nucleus and (D) the reticular formation.
188 TABLE 111 Effect o['lesion ht reticular formation and in medial raphe nucleus on adrenal TH activity
The lesions were produced as described in the text. Means ± S.E.M. are shown. Significance of difference between the two means: P < 0.025. Group
Number of rats
Tyros±hehydroxylase (nmoles DOPA/h per pair adrenals)
Medial raphe lesion Unilateral lesion of reticular formation
5 4
72.49 ± 4.83 55.48 ± 1.25
5-HT is decreased. This is probably due to damage to the dorsal raphe nucleus in the passage of the electrode to the medial nucleus. Because some of the lesions which elicited an increase in adrenal T H activity involved portions of the reticular formation (e.g., Fig. 1A), a further set of controls was obtained in which the reticular formation was deliberately damaged while sparing the medial raphe nucleus (Fig. 1D). The coordinates used were anterior ( + ) 350 pro, vertical (--) 2.6 ram, lateral ( + ) 1.2 ram. The results (Table III) show that rats with such lesions have mean adrenal T H activity within the range of sham-operated or sham-lesioned animals (Table II) whereas those with the lesion restricted to the medial raphe nucleus had elevated values. It has been shown that in most conditions raphe neurons have a slow, regular firing pattern1, 2,13 and appear to exert a tonic inhibitory influence on neurons which they innervate 7,1~. This suggests a possible homeostatic function and there is evidence that serotonin has such a role in the regulation of states of sleep 18, aggression 11, locomotor activity 1°,1~ and sexuality 31. It is frequently presumed that whole brain 5-HT is involved in the physiological processes in which serotonin is implicated and no attempt is made to determine whether there is a separate contribution of specific raphe nuclei. The results of the lesion experiments show that an increase is obtained in adrenal T H activity when the medial raphe nucleus is lesioned (Table I, exps. A and C; Table III) but not when the dorsal raphe nucleus is damaged (Table ]). Moreover, lesions of the medial raphe nucleus are accompanied by only a modest decline in 5-HT concentrations of the forebrain as compared to dorsal raphe nucleus lesions. This indicates that the midbrain raphe system, specifically the medial raphe nucleus, exerts a tonic inhibitory influence on the transsynaptic induction of adrenal tyros±he hydroxylase. As the enzyme is rate controlling in the biosynthesis of NA and adrenaline, the two transmitters secreted from the adrenal medulla to meet the demands of the body in response to stress, this may imply that the medial raphe nucleus plays an important regulatory role during certain stress situations. This research was supported by grants of the Medical Research Council (Canada). M. Quik held a Studentship of the M.R.C.S. Gauthier is a Fellow of the M.R.C.
189 1 Aghajanian, G. K., Foote, W. E. and Sheard, M. H., Lysergic acid diethylamide: Sensitive neuronal
units in the midbrain raphe, Science, 161 (1968) 706-708. 2 Aghajanian, G. K., Foote, W. E. and Sheard, M. H., Action of psychotogenic drugs on single midbrain raphe neurons, J. Pharmacol. exp. Ther., 171 (1970) 178-187. 3 Ahtee, L., Sharman, D. F. and Vogt, M., Acid metabolites of monoamines in avian brain; effects of probenecid and reserpine, Brit. J. Pharmaeol., 38 (1970) 72-85. 4 And~n, N. E., Dahlstr6m, A., Fuxe, K., Larsson, K., Olson, L. and Ungerstedt, U., Ascending monoamineneurons to the telencephalon and diencephalon, Actaphysiol. scand., 67 (1966) 313-326. 5 Anton, A. H. and Sayre, D. F., The distribution of dopamine and DOPA in various animals and a method for their determination in diverse biological material, J. PharmacoL exp. Ther., 138 (1962) 360--375. 6 Axelrod, J., Mueller, R. A., Henry, J. P. and Stephens, P. M., Changes in enzymes involved in the biosynthesis and metabolism of noradrenaline and adrenaline after psychosocial stimulation, Nature (Lond.), 225 (1970) 1059-1060. 7 Bloom, F. E., Hoffer, B. J., Siggins, G. R., Barker, J. L. and Nicoll, R. A., Effects of serotonin on central neurons: microiontophoretic administration, Fed. Proc., 31 (1972) 97-106. 8 Breese, G. R., Cooper, B. R. and Mueller, R.A., Evidence for involvementof 5-hydroxytryptamine in the actions of amphetamine, Brit. J. PharmacoL, 52 (1974) 307-314. 9 Dahlstr~Sm,A. and Fuxe, K., Evidence for the existence of monoamine-containingneurons in the central nervous system, Acta physioL scand., Suppl. 232 (1964) 1-55. 10 Fibiger, H. C. and Campbell, B. A., The effect of parachlorophenylalamine on spontaneous locomotor activity in the rat, Neuropharmacology, 10 (1971) 25-32. 11 Grant, L. D., Coscina, D. V., Grossman, S. P. and Freedman, D. X., Muricide after serotonin depleting lesions of midbrain raphe nuclei, Pharmacol. Biochem. Behav., 1 (1973) 77-80. 12 Guidotti, A., Zivkovic, B., Pfeiffer, R. and Costa, E., Involvement of 3',5'-cyclic adenosine monophosphate in the increase of tyrosine hydroxylase activity elicited by cold exposure, NaunynSchmiedeberg's Arch. exp. Path. Pharmak., 278 (1973) 195-206. 13 Haigler, H. J. and Aghajanian, G. K., Lysergic acid diethylamide and serotonin: a comparison of effects on serotonergic neurons and neurons receiving a serotonergic input, J. Pharmacol. exp. Ther., 188 (1974) 688-699. 14 Hoeldtke, R., Lloyd, T. and Kaufman, S., An immunochemical study of the induction of tyrosine hydroxylase in rat adrenal glands, Biochem. biophys. Res. Commun., 57 (1974) 1045-1053. 15 Jacobs, B. L., Eubank, E. E. and Wise, W. D., Effect of indolealkylaminemanipulations on locomotor activity in rats, Neuropharmacology, 13 (1974) 575-583. 16 Jacobs, B. L., Wise, W. D. and Taylor, K. M., Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats, Brain Research, 79 (1974) 353-361. 17 Joh, T. H., Geghman, C. and Reis, D., Immunochemicaldemonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2767-2771. 18 Jouvet, M., Neurophysiology of the states of sleep, Physiol. Rev., 47 (1967) 117-177. 19 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963, 162 pp. 20 Kostowski, W., Giacalone, E., Garattini, S. and Valzelli, L., Studies on behavioral and biochemical changes in rats after lesions of midbrain raphe, Europ. J. PharmacoL, 4 (1968) 371-376. 21 Kvetnansky, R., Weise, V. K. and Kopin, I. J., Elevation of adrenal tyrosine hydroxylase and phenylethanolamine-N-methyltransferase by repeated immobilization of rats, Endocrinology, 87 (1970) 1323-1329. 22 Laverty, R. and Taylor, K. M., The fluorometric assay of catecholamines and related compounds: improvements and extensions to the trihydroxyindole technique, Analyt. Biochem., 22 (1968) 269-279. 23 Levitt, M., Spector, S. Sjoerdsma, A. and Udenfriend, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart., J. Pharmacol. exp. Ther., 148 (1965) 1-8. 24 Lorens, S. A. and Guldberg, H. C., Regional 5-hydroxytryptamine following selective midbrain raphe lesions in the rat, Brain Research, 78 (1974) 45-56. 25 Mueller, R. A., Smith, R. D., Breese, G. R. and Cooper, B. R., Modulation of adrenal tyrosine hydroxylase induction by serotonin nerve function. In E. Usdin and S. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, New York, 1973, pp. 187-189.
190 26 Mueller, R. A., Thoenen, H. and Axelrod, J., Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. exp. Ther., 169 (1969) 74 79. 27 Nagatsu, T., Levitt, M. and Udenfriend, S., A rapid and single radioassay for tyrosine hydroxylase activity, Analyt. Biochem., 9 (1964) 122-126. 28 Otten, U., Paravicini, U., Oesch, F. and Thoenen, H., Time requirements for the single steps of trans-synaptic induction of tyrosine hydroxylase in the peripheral nervous system, NaunynSchmiedeberg's Arch. exp. Peth. Pharmak., 280 (1973) 117 127. 29 Quik, M. and Sourkes, T. L., Regulation of adrenal tyrosine hydroxylase: neuronal versus local control studied with apomorphine, Biochem. Pharmacol., 25 (1976) 1157-1166. 30 Quik, M. and Sourkes, T. L., Central dopaminergic and serotoninergic systems in the regulation of adrenal tyrosine hydroxylase, J. Neurochem., in press. 31 Tagliamonte, A., Tagliamonte, P., Gessa, G. L. and Brodie, B. B., Compulsive sexual activity induced by p-chlorophenylalanine in normal and pinealectomized male rats, Science, 166 (1969) 1433-1435. 32 Thoenen, H., Induction of tyrosine hydroxylase in peripheral and central adrenergic neurons by cold-exposure of rats, Nature (Lond.), 228 (1970) 861-862. 33 Thoenen, H., Mueller, R. A. and Axelrod, J., Trans-synapticinduction ofadrenal tyrosine hydroxylase, J. Pharmacol. exp. Ther., 169 (1969) 249-254.
183
© Elsevier/North-Holland BiomedicalPress, Amsterdam - Printed in The Netherlands
Role of the raphe nuclei in the regulation of adrenal tyrosine hydroxylase
MARYKA QUIK, THEODORE L. SOURKES, BERNARDO O. DUBROVSKY and SERGE GAUTHIER Department of Psychiatry, Allan Memorial Institute, McGill University, Montreal, Quebec 1t3.4 1.4 l (Canada)
(Accepted November llth, 1976)
A variety of conditions, such as immobilizations tress zl, cold exposure 32, swim stress 2a, psychosocial situations 6 and the administration of certain drugs~, 33, which produce a sustained increase in peripheral sympathetic activity lead to an increase of tyrosine hydroxylase (TH, tyrosine-3-monooxygenase, EC 1.14.16.2) in the adrenal medulla. This enzyme catalyzes the rate-limiting step in catecholamine biosynthesis 23. Evidence has accumulated that transsynaptic influences play a vital role in the regulation of TH activity12,21,33; that is, the augmented enzyme levels14,17 occur through centrally mediated mechanisms. A role for a serotoninergic system in the control of adrenal TH has been suggested since destruction of serotonin (5-HT) nerve terminals with 5,7-dihydroxytryptamine, blockade of 5-HT receptors with methiothepin and depletion of brain 5-HT with p-chlorophenylalanine (PCPA) increase adrenal TH activitys,30. FurthErmore, these treatments potentiate the induction of adrenal TH produced by electrical stimulation of a mesencephalic site 25 or by the administration of drugs that stimulate central dopamine receptors8, ~0. This suggests that a central serotoninergic system has a net inhibitory role in the regulation of adrenal TH. To determine whether control of adrenal TH activity by the serotoninergic system involved in this regulatory process could be localized to a specific brain region the effect of lesions of the raphe nuclei, areas rich in 5-HT cell bodies4, 9, on adrenal TH activity was investigated. For the lesions, Sprague-Dawley rats, weighing 150 g, were anesthetized with sodium pentobarbital and placed in a stereotaxic instrument. A burr hole was made in the midline over the midbrain raphe nuclei. Anodal electrolytic lesions were produced by passing a current of 2 mA for 10 sec through a stainless steel monopolar electrode of contact diameter and exposed length of 0.25 mm and 0.5 mm, respectively. A large electrode clipped to the wound margin was connected to the cathode. Stereotaxic coordinates (according to K6nig and Klippe119) were anterior ( + ) 350/~m and vertical (--) 2.6 mm for the medial raphe nucleus, and anterior ( + ) 350/zm and vertical (--) 0.6 mm for the dorsal raphe nucleus. Sham-operated control animals were treated in the same manner except that the electrode was not lowered intracranially; for shamlesioned controls the electrode was placed as for the lesion but no current was passed.
184 TABLE 1 Effect o f medial and dorsal raphe nuclei lesions on adrenal TH activity and brain monoanline cont'entrations
The lesions were produced as described in Materials and Methods. In exp. A, results were pooled from rats killed 6 and 11 days after the lesions. In exps. B and C, the rats were killed 10 days after the lesion. The adrenal TH activity and forebrain monoamine concentrations were then determined. Means t S.E.M. are shown. Sham-operated controls did not have the electrode placed intracranially. Experiment Group
Number Tyrosine hydrox- Brain amine (#g/g) o f rats ylase (nmoles ................ DOPA/h per pair 5-HT DA NA adrenals)
A
11
52.83 ± 1.75
0.32 i 0.02
6 20 10 11 19
90.09 41.26 44.10 54.65 75.99
0.12 0.38 0.19 0.35 0.28
B C
Sham operated Dorsal plus medial lesion Sham-operated Dorsal lesion Sham-operated Medial lesion
:k 8.19'* ± 1.34 ± 2.04 i 2.88 = 2.49**
1.50 zk 1.07 0.25 i 0.01
± 0.02"'1.76 ± ± 0.01 1.49 ± 4- 0.02* 1.43 ± ± 0.01 1.26 ± :k 0.02* 1.23 ±
0.13 0.05 0.06 0.10 0.05
0.23 0.29 0.26 0.28 0.29
± 0.02 :k 0.01 ± 0.01" ± 0.01 _%0.01
Significance of difference from controls : ** P < 0.001 ; * P < 0.05.
A n i m a l s were killed by decapitation. The brains were r e m o v e d a n d the f o r e b r a i n (minus h i p p o c a m p u s a n d h y p o t h a l a m u s ) was weighed a n d h o m o g e n i z e d in 10 ml 0.4 N p e r c h l o r i c acid for a m i n e determinations. F o r histological verification o f electrode placement, the b r a i n stems were i m m e r s e d in 10 ~ f o r m a l i n for 2-3 weeks; frozen sections were then cut at 45 # m a n d stained with cresyl violet. The a d r e n a l s were also rem o v e d a n d placed in ice-cold 0.3 M sucrose. The fat a n d connective tissue were cut away, a n d each p a i r o f a d r e n a l s was weighed a n d h o m o g e n i z e d in 0.9 ml o f 0.3 M sucrose with a Teflon homogenizer. A n aliquot o f the h o m o g e n a t e (0.2 ml) was used for assay o f T H activity. T H was assayed a c c o r d i n g to the m e t h o d o f N a g a t s u et al. 27 with certain modifications as previously described 29. F o r d e t e r m i n a t i o n o f b r a i n amines, f o r e b r a i n s were h o m o g e n i z e d in 10 ml 0.4 N perchloric acid; the m a t e r i a l was centrifuged a n d the s u p e r n a t a n t b r o u g h t to p H 7.5-8.0 by a d d i t i o n o f 4 N N a O H c o n t a i n i n g 250 m M N a ~ E D T A . The n o r a d r e n a l i n e ( N A ) a n d d o p a m i n e ( D A ) were then a d s o r b e d on an a l u m i n a c o l u m n ~ (0.5 c m x 2.0 cm), previously equilibrated with a solution 10 m M with respect to b o t h N a 2 E D T A a n d Na2S2Oa, a n d o f p H 8.0; 5-HT was a b s o r b e d on an A m b e r l i t e c o l u m n 3 (0.5 c m x 2.0 cm) equilibrated with 0.2 M NaH~PO4 o f p H 6.7. This c o l u m n was placed below the a l u m i n a c o l u m n to receive the m a t e r i a l flowing f r o m it. The c a t e c h o l a m i n e s were eluted f r o m the a l u m i n a with 4 ml 0.1 N perchloric acid a n d the 5 - H T was released f r o m the A m b e r l i t e c o l u m n s with 10 m g ~ ascorbic acid in 2 N NC1. T h e fluorescence o f 5-HT solutions was r e a d i m m e d i a t e l y ; the N A a n d D A were assayed later by the t r i h y d r o x y i n d o l e technique o f Laverty a n d Taylor2L T o determine whether alterations in a d r e n a l T H activity after t r e a t m e n t with 5 , 7 - d i h y d r o x y t r y p t a m i n e , m e t h i o t h e p i n o r P C P A s,3° are due to generalized inter-
185 ference with central serotonin systems or to dysfunction of specific nuclei, a lesion was made either in the dorsal raphe nucleus (B7) or in the medial raphe nucleus (B8). Some rats were lesioned in both sites. These two nuclei contain most of the 5-HT cell bodies whose axons project to the telencephalon 4,9 and their destruction markedly depletes forebrain 5-HT 16,2°,24. After the combined lesion the adrenal T H activity was significantly (P < 0.001) increased (Table I, exp. A); the 5-HT concentration in the forebrain decreased to 38 ~ of control. When the lesion was restricted to the dorsal raphe nucleus (Table I, exp. B), adrenal T H activity remained at control level, despite the 42 ~ decrease in forebrain 5-HT. After a lesion of the medial raphe nucleus (exp. C), a highly significant increase in adrenal T H activity was observed; the decrease in forebrain 5-HT was only 20 ~ . No significant changes were observed in the D A and N A concentrations in exps. A and C (Table I); however, in exp. B, there was a very small but statistically significant decrease in brain NA. This indicates that the lesions were relatively specific for 5-HT neurons, a fact which is confirmed by the histological sections (Fig. 1). In Table I the biochemical data from the lesioned rats have been compared to those from controls which had been sham-operated but which had not had the electrode lowered intracranially. To ensure that the results obtained are due to the lesion, and not to non-specific damage, sham-operated controls were also compared with sham-lesioned controls. Table II shows that in the case of the dorsal raphe lesion the two types of control have similar adrenal TH activity and brain amine concentrations. In the case of sham lesion of the medial raphe nuclei, only the forebrain
TABLE II The effect of electrode placement in the medial and/or dorsal raphe nuclei on adrenal TH activity and brain monoamine concentrations
Sham-operated controls and sham-lesioned controls were treated in the same way as lesioned rats except that (a) for sham-operated controls the electrode was not placed intracranially and (b) for the sham-lesioned controls the electrode was in place but no current was passed. The rats were killed 10 days after treatment, at which time the adrenal TH activity and brain amine concentrations were determined as described in the text. Means 4- S.E.M. are shown. Group
Sham-operated for dorsal lesion Sham-lesioned for dorsal lesion Sham-operated for medial or combined lesion Sham-lesioned for medial or combined lesion
Number Tyrosine hydroxylof rats ase (nmolesDOPA/h perpair adrenals)
Brain amines (Itg/g) 5-HT
DA
NA
20
41.26 4- 1.34
0.38 4- 0.01
1.49 4-4-0.05
0.29 4-4-0.01
10
40.07 4- 1.51
0.41 4- 0.03
1.58 4-4-0.12
0.30 4-4-0.01
22
53.74 4- 1.65
0.33 4- 0.01
1.37 4- 0.07
0.27 4- 0.01
21
55.13 4- 2.31
0.25 4- 0.02*
1.35 4- 0.05
0.26 4- 0.01
* Significance of difference from sham-operated group: P < 0.01.
A
B
Fig. 1.
C
Fig. 1. Frontal plane section (45/~m) through caudal midbrain showing a representative lesion of (A) the medial and dorsal raphe nuclei, (B) the medial raphe nucleus, (C) the dorsal raphe nucleus and (D) the reticular formation.
188 TABLE 111 Effect o['lesion ht reticular formation and in medial raphe nucleus on adrenal TH activity
The lesions were produced as described in the text. Means ± S.E.M. are shown. Significance of difference between the two means: P < 0.025. Group
Number of rats
Tyros±hehydroxylase (nmoles DOPA/h per pair adrenals)
Medial raphe lesion Unilateral lesion of reticular formation
5 4
72.49 ± 4.83 55.48 ± 1.25
5-HT is decreased. This is probably due to damage to the dorsal raphe nucleus in the passage of the electrode to the medial nucleus. Because some of the lesions which elicited an increase in adrenal T H activity involved portions of the reticular formation (e.g., Fig. 1A), a further set of controls was obtained in which the reticular formation was deliberately damaged while sparing the medial raphe nucleus (Fig. 1D). The coordinates used were anterior ( + ) 350 pro, vertical (--) 2.6 ram, lateral ( + ) 1.2 ram. The results (Table III) show that rats with such lesions have mean adrenal T H activity within the range of sham-operated or sham-lesioned animals (Table II) whereas those with the lesion restricted to the medial raphe nucleus had elevated values. It has been shown that in most conditions raphe neurons have a slow, regular firing pattern1, 2,13 and appear to exert a tonic inhibitory influence on neurons which they innervate 7,1~. This suggests a possible homeostatic function and there is evidence that serotonin has such a role in the regulation of states of sleep 18, aggression 11, locomotor activity 1°,1~ and sexuality 31. It is frequently presumed that whole brain 5-HT is involved in the physiological processes in which serotonin is implicated and no attempt is made to determine whether there is a separate contribution of specific raphe nuclei. The results of the lesion experiments show that an increase is obtained in adrenal T H activity when the medial raphe nucleus is lesioned (Table I, exps. A and C; Table III) but not when the dorsal raphe nucleus is damaged (Table ]). Moreover, lesions of the medial raphe nucleus are accompanied by only a modest decline in 5-HT concentrations of the forebrain as compared to dorsal raphe nucleus lesions. This indicates that the midbrain raphe system, specifically the medial raphe nucleus, exerts a tonic inhibitory influence on the transsynaptic induction of adrenal tyros±he hydroxylase. As the enzyme is rate controlling in the biosynthesis of NA and adrenaline, the two transmitters secreted from the adrenal medulla to meet the demands of the body in response to stress, this may imply that the medial raphe nucleus plays an important regulatory role during certain stress situations. This research was supported by grants of the Medical Research Council (Canada). M. Quik held a Studentship of the M.R.C.S. Gauthier is a Fellow of the M.R.C.
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