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Increased tyrosine hydroxylase activity in the locus coeruleus of rat brain stem after reserpine treatment and cold stress
Brain Research, 70 (1974) 547-552 ~"~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
547
Increased tyrosirm hydroxylase activity in the locus coeruleus of rat brain stem after reserpine treatment and cold stress
R. E. ZIGMOND, F. SCHON AND L. L. IVERSEN
MRC Neurochemical Pharmacology Unit, Department of Pharmacology, Medical School, Cambridge CB2 2QD (Great Britain) (Accepted January 18th, 1974)
Increases in the activity of tyrosine hydroxylase, the rate-limiting enzyme in noradrenaline biosynthesis, have been reported in adrenergic neurones in both the peripheral and central nervous system after administration of reserpine and after cold stress t4,2°,~1. The mechanism of this increase in enzyme activity has been studied most thoroughly in the rat superior cervical ganglion and in the adrenal gland after reserpine treatment. In these tissues, reserpine seems to act through a trans-synaptic mechanism; an increased synthesis of new tyrosine hydroxylase molecules occurs in response to increased firing of preganglionic neurones induced by autonomic reflexes after the drug treatment7,s,15, 26. The increased enzyme activity appears first in adrenergic cell bodies and several days later in sympathetic nerve terminals "3. Reserpine also leads to increases in tyrosine hydroxylase in the brain stem. A 55 % increase in enzyme activity was reported in the rabbit following two injections of the drug (2.5 mg/kg/day) 14 and a 26}/o increase in the rat after 8 daily injections (0.5 mg/kg/day) 1. Segal et al. found a 40 °J(, increase in tyrosine hydroxylase activity in the rat midbrain after 8 daily injections (0.5 mg/kg/day), but 11o significant change was found if the treatment lasted only 7 days t7. These effects of reserpine in the central nervous system are in general smaller and require more prolonged drug treatment than in the peripheral nervous system. Since tyrosine hydroxylase in the brain stem is found both in cell bodies and in nerve terminals and since there are several groups of catecholaminergic neurones in this area of the brain which may not all respond to reserpine in the same way, we have examined the effects of reserpine on tyrosine hydroxylase in a specific portion of the rat brain stem enriched in catecholaminergic cell bodies. The area chosen for this purpose was the region of the locus coeruleus, a nucleus which includes a large number of densely packed cells that fluoresce using the FalckHillarp technique for visualizing catecholaminergic neurones 3. (This area was designated A6 in the original fluorescent study of Dahlstr6m and FuxeS.) Pharmacological, immunochemical, and biochemical evidence suggests that the fluorescent cell bodies in the locus coeruleus are noradrenergic",4,a,9-aL Ventral to this cell group are
54~
StIORl (~)MMt,NI~ ~.IIONS
Fig. 1. Dissection procedure for removal ol the locus coeruleus, a : dorsal view of the {~l~m~lcm ,.ho\~ing the two halves of the cerebellum (() displaced laterally and the 3 indentations at the level of the cerebellar pedtlncle (indicated by arrowheadsL The two dashed lines indicate cuts made ~o istilale the coronal section containing the locus coeruteus (L). The caudal portion of the pons medtt!la is referred to as region 3 in the text. b: a coronal section through the pons at the level of the Itxus coeruleus stained for acetylcholinesterase according to lhe method of Shuie and Lewis ~, showing promineni enzyme activity in the locus coeruleus (L) and the trigeminal motor nucleus {TI. Cuts made in the locus coeruleus disseciion are represented by the dashed lines. The remaining areas 1 and 2 were taken as control regions (see text). This micrograph was kindly supplied by Dr. P. R. t.ewis, Department oi" Physiology, University of Cambridge, The calibration bar I mm in each picture
a n u m b e r o f larger cell bodies, p r o b a b l y also noradrenergic, which are part o f the ' s u b c o e r u l e u s a r e a 'a,16. The dissection o f the locus coeruteus was d e v e l o p e d with the aid o f the F a l c k Hillarp histochemical technique. By removing areas o f the dorsal pons i m d looking for the characteristic cluster o f fluorescent cell bodies on histochemical s e c t i o n s the following dissection p r o c e d u r e was arrived at based on the use o f easily visible landmarks. This technique allows the r e p r o d u c i b l e dissection o f the locus coeruteus in a piece of tissue weighing 2.5-3.0 rag. The b r a i n is t a k e n from the skull and placed on ice. The c e r e b e l l u m is transected in the midline and displaced laterally oil either side, exposing the dorsal surface o f the brain stem (Fig. ta). The cerebellum is then removed by cutting the cerebellar pedttncles at the lateral b o r d e r of the pons. A line can be defined visually in a c o r o n a l plane at the level o f the cerebeltar peduncles by 3 deep i n d e n t a t i o n s on the dorsal surface o f the pons (Fig. la). These are f o r m e d by the sulcus m e d i a n u s dorsalis a l o n g the midline and the two sulci limitantes on either side 'ca. T w o c o r o n a l cuts are m a d e as shown in Fig. ta, one t h r o u g h the i n d e n t a t i o n s and the other 1-1.5 mm rostralty. The resulting c o r o n a l section is placed with its caudal surface up. The 3 sulci can be seen clearly from this perspective (Fig. lb); A h o r i z o n t a l cut is m a d e t h r o u g h the section at a d e p t h c o r r e s p o n d i n g to the ventral tip o f the sulcus m e d i a n u s dorsalis ( a p p r o x i m a t e l y 1 m m from the d o r s a l surface)l The medial p o r t i o n between the two sulci limitantes is then removed and the re-
549
SHORT COMMUNICATIONS
Fig. 2. Portion of a methylene blue-stained section from the locus coeruleus dissection showing the clustered cell bodies of the locus coeruleus. The insert taken from an adjacent section shows these cell bodies are intensely fluorescent after treatment by the Falck-Hillarp technique a. Calibration bar 20 Mm.
m a i n i n g piece o f tissue is t a k e n as the ' l o c u s c o e r u l e u s s a m p l e ' (Fig. I b). M i c r o g r a p h s d e m o n s t r a t i n g the presence o f locus c o e r u l e u s cell b o d i e s in the s a m p l e are s h o w n in Fig. 2. T h e t y r o s i n e h y d r o x y l a s e a c t i v i t y o f the locus c o e r u l e u s s a m p l e was c o m p a r e d with 3 o t h e r parts o f the b r a i n s t e m : (1) the a r e a b e t w e e n the two sulci limitantes (Fig. lb), (2) the rest o f the c o r o n a l section described a b o v e (Fig. lb), and (3) the r e m a i n d e r o f the p o n s m e d u l l a c a u d a l to this c o r o n a l section (Fig. la). Tissues were
TABLE I D I S T R I B U T I O N ()F T Y R O S I N E H Y D R O X Y L A S E A C T I V I T Y IN D I F F E R E N T P A R I S OF T H E M E D U L L A PONS
Tyrosine hydroxylase (T-OH) activity was measured by a modification of the method of Hendry and Iversen". Tetra-hydrobiopterin was used as cofactor and the final concentration of k-[aH]tyrosine was 51 /~M. Samples were incubated at 37 C for 15 rain. A description of the method of dissection of the 4 brain samples is given in Fig. la and b and in the text. The data are the means : S.E.M. from 6 animals. Sample
T-OH aclivity (nmo/es ' a H / D O P A ./brnled/g/h)
o Tola[ T-OH activit.v
% Total wet weight
Locus coeruleus
37.3 ~ 4.2 6.4 ~k 1.2 3.6 0.2 3.8 5_ 1.9
23 9 14 60
2.9 1.3 18.4 77.3
1
2 3
550
SIi()RI (()MMI '
weighed, homogenized in 4 vol. of 5 mM Tris buffer (pH 6) containing tl. i ';, l riton X-100 and assayed for tyrosine hydroxylase activity (T-OH) by the method described by Hendry and iversen 6. The tyrosine hydroxylase activity per mg wet weight in the tocu.~ coeruleus sample was considerably higher than in the other 3 areas (Table 1). Although the locus coeruleus sample represented only about 3 °//o of the weight of the entire pons and medulla it contained 23 o(, of the tyrosine hydroxylase activity present m that part of the brain. The activity of the enzyme was also high compared to other areas of the central nervous system. For instance, the locus coeruteus sample contained about twice as much tyrosine hydroxylase activity per mg wet weight as the hypothalamus and more than 20 times as much as the hippocampus. Rats treated with two doses of reserpine (5 mg/kg subcutaneously) at 24 h intervals showed a 380 o~, increase m tyrosine hydroxylase activity in the locus coeruleus sample when assayed 48 h after the first injection (Fig. 3). The enzyme activity was also increased by cold stress. After 66 h at 4 ~"C. there was an 84°~, increase in tyrosine hydroxylase activity in this area (Fig. 3). When the ability of reserpine to increase tyrosine hydroxylase activity was compared in the locus coeruleus and in the rest of the pons plus medulla, the former area showed a much larger response. In these experiments the enzyme was assayed 72 h after a single injection of reserpine (5 mg/kg). The drug caused a 138 '~,, increase in the locus coeruleus sample and only a 307',i increase in the rest of the pons plus medulla (Table ii). The results show that both reserpine treatment and cold stress can stimulate tyrosine hydroxylase activity in the area of the locus coeruteus. Although there is a large concentration of noradrenergic cell bodies in this area of the brain there are
g~
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O u
400 t/) ~. 30C
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RESERPINE TREATMENT
COLD STRESS
Fig. 3. Effect of reserpine treatment and cold stress on tyrosine hydroxylase (T-OH) activity in the locus coeruleus. Reserpine treated animals were given two injections of reserpine (5 mg/kg) subcutaneously in 2 0 ~ ascorbic acid at 0 a n d 24 h a n d were killed at 48 h. Control animals were injected with ascorbic acid alone. Cold stressed rats were housed individually in metal cages m a cold r o o m (4 "C~ for 66 h. Control rats were housed similarly but kept at r o o m temperature (23 °C). The data expressed as tyrosine hydroxylase activity as a percentage of the appropriate control group were calculated on the basis of the a m o u n t of D O P A formed per locus coeruleus sample. The means _+~S.E.M. are shown. The number of animals per group is shown in each bar. In these experiments 6,7-dimethyl-tetrahydropteridine was used as cofactor and the final concentration of L-[ZH]tyrosine was 5.3 ¢tM.
551
SHORT COMMUNICATIONS
TABLE I1 COMPARISON OF THE EFFECTOF RESERPINEON TYROSINE HYDROXYLASEACTIVITYIN THE LOCUSCOERUEEUS A N D T H E REST OF T H L P O N S P L U S M E D U L L A
Animals were pretreated with a single injection of reserpine (5 mg/kg, s.c.) 72 h before sacrifice. Details of the dissection procedure are given in Fig. I, and the assay conditions are given in the legend to Table I. The data are the means :[ S.E.M. from 6 animals.
T-OH activity as % control
Vehicle controls Reserpine treated
Locus eoeru[etls
Rest oi meaTl//.;-l?OtlS
100 z I1 238 ! 37
100 : 7 130 9
also some noradrenergic terminals in the locus coeruleus and in the adjacent mesencephalic nucleus of the trigeminal nerve "4. Thus we cannot rule out the possibility that some of the increased tyrosine hydroxylase activity in the area may be located in nerve ternrinals. However the finding that large increases in tyrosine hydroxylase activity are found at short intervals after reserpine treatment (e.g. 2 days) strongly suggests that much of the increased activity is present in noradrenergic cell bodies. The increase in tyrosine hydroxylase activity is higher in the locus coeruleus sample than in the rest of the pons and medulla. It is not yet clear whether this is due to a selective effect of reserpine (either direct or trans-synaptic) on the ceils of the locus coeruleus or whether because of the large number of noradrenergic cell bodies in this region the effect is easier to detect there at short time intervals after drug treatment. The cell bodies of the locus coeruleus and the subcoeruleus area are known to project to lnany areas of the brain (e.g. hippocampus, cerebral cortex, hypothalamus) le.uS.l¢i,ts,ei. If the stimulation of tyrosine hydroxylase activity with reserpine in the central nervous system is analogous to that seen in the periphery one should be able to detect a delayed increase in enzyme activity in such areas of termination. In fact the appearance of increased tyrosine hydroxylase activity reported in the lnidbrain after 8 daily injections of reserpine may reflect the arrival of newly synthesized enzyme in terminals of noradrenergic neurones whose cell bodies are in the pons and medulla rather than a stimulation of enzyme activity in dopaminergic cell bodies in the midbraii317. Experiments in progress in our laboratory show such a delayed increase in tyrosine hydroxylase activity in the hypothalamus and hippocampus. This work was done during the tenure of a British-American Research Fellowship of the American Heart Association and the British Heart Foundation and an Alfred P. Sloan Foundation Research Fellowship to R . E . Z . F . S . is a Medical Research Council Scholar. l BESSON, M. J., CHERAMY, A., GAUCHY, C., AND MUSACCHIO, J. M., Effects of some psychotropic drugs on lyrosine hydroxylase activity in different structures of the rat brain, Europ. J. Pharmacol., 22 (1973) 181-186.
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SHORT COMMUNICATIONS
2 CORRODI, H., Fbx~, K., HAMBEI','.,,k.R,B., AND LJUN(IDAHL, A., Studies on central al~d peripheral noradrenaline neurones using a new dopamine-/;-hydroxylase inhibitor, Europ, J PharmucoL, 12 (1970) 145-155. 3 DAHLSTR(}M,A., AND FUXlL K., Evidence for the existence of monoamine-containing neurons m the central nervous system. 1. Demonstration of monoamines in the cell bodies ol" brain stem neurons, Acta physiol, stand., 62, Suppl. 232 (1964) 1 -55. 4 GOLDSTEIN, M., FUXE, K., AND HOKFt-I r, I . , Characterization and tissue Iocalisation t,,f catecholamine synthesizing enzymes, PharmaeoL Rev.. 24 (1972) 283 309. 5 HARTMAN, B. K., Zme, D., An1) UDVNFRIeXD,S., The use of dopamine-/J-hydroxyla.~c as a marker for the central noradrenergic nervous system in rat brain, Proc. nat. Acad. Sci. r Wash ). 69 (1972) 2722-2726. 6 HENDRY, 1. A., AND IVERSEN, L. l_., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical sympathetic ganglion, Brain Research, 29 (1971) 159 162. 7 I~;Go, A., A>aDVOCiT, M., Preganglionic sympathetic activity in normal and in reserpine-treated cats, a. Physiol. (Lond.), 150 (1960) 114 133. 8 JoH, T. H., GE(IHmAN, C., ANt) REIS, D., lmmunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine, Proc. nat. Aead. Sci. (Wash.), 70 (1973) 2767 2771. 9 KORF, J., ACmAJANIAN,G. K., AND ROTH, R. H., Stimulation and destruction of the locus coeruleus; opposite effects on 3-methoxy-4--hydroxyphenylglycolsulfate levels in the rat cerebral cortex, Eurap. a. Pharmaeol., 21 (1973) 305 310. 10 KoRr, J., ROVrl, R. H., AND AGHAJANIAN,G. K., Alterations in turnover and endogenous levels of norepinephrine in cerebral cortex following electrical stimulation and acute axotomy of cerebral noradrenergic pathways, Europ. J. PharnutcoL, 23 (1973) 276-282. 11 KUHAR, M. J., ROXH, R. H., AYO AGHMANJAN, G. K., Synthesis of catecholamines m the locus coeruleus from aH-tyrosine ht vivo, Biachem. Pharmacol., 21 (1972) 2280-2282. 12 LolZOU, L. A., Projections of the nucleus locus coeruleus in the albino rat, Brain Research, 15 (1969) 563-566. 13 MAEDA, T., ET SHIMIZU, N.. Projections ascendantes du locus coeruleus et d'autres neurones anainergiques pontiques au niveau du prosene6phale du rat. Brain Research. 36 ~1972~ 19-35. 14 MU~LLEm R. A., TrtoENvY. H.. ANt) AXrLaOD. J.. Increase in tyrosine hydroxylase activity alter reserpine administration, J. PharmaeoL exp. Ther.. 169 (1969) 74-79. 15 MUELt,ZR, R, A., THOENEN, H.. AND AXELROD. J., Inhibition of trans-synaptically increased tyrosine hydroxylase activity by cycloheximide and actinomycin D. Molec. PharmacoL. 5 11969) 463 469. 16 OLSON, L., AXD FUXE, K., Further mapping out of central noradrenaline neuron system: projections of the 'subcoeruleus' area. Brain Research. 43 H972) 289-295. 17 SEGAL, D. S., SULLIVAN, J. L.. KUr:ZENSKI. R. T.. ANt) MANDELL, A. J.. Effects of long-term reserpine treatment on brain tyrosme hydroxylase and behavioral activity. Science, 173 (1971) 847 -849. 18 SEGAL, M., PICKEL, V., AND BLOOM.l-:., The projections of the nucleus locus coeruleus: an autoradiographic study, Life Sci., 13 11973t 817-821, 19 SHUTE, C. C. D., AND LEWIS. P. R,. The ascending cholinergic reticular system: Neocortical. olfactory and subcortical projections, Brain, 90 (1967) 497-520. 20 THOENEN, H., Induction of tyrosine hydroxylase in peripheral and central adrenergic neurones by cold exposure of rats, Nature ( L o n d . . 228 t'1970) 86t-862. 21 THOENEN, H., MUELLER, t . a.. AND AXELROD. J., Increased tyrosine hydroxylase activity after drug induced alteration of sympathetic transmission. Nature Lond. ;. 221 (1969) 1264. 22 THOtNEN, H,, MUELLER, R. A.. AND AXELROD. J.. Trans-synaptic induction of adrenal tyrosine hydroxylase, J. Pharmacol. exp. Ther.. 169 (1969) 249-254. 23 THOENEN, H,, MUELLER, R. A.. AND AXELROD. J.. Phase differences in the induction of tyrosine hydroxylase in cell body and nerve terminals of sympathetic neurons. Proc. nat Acad. Sci. (Wash.), 65 (1970) 58-62. 24 UNGERSTEOT, U., Stereotaxic mapping of the monoamine pathways in the rat brain. Acra physiol. seand., Suppl. 367 (1971) 1-48. 25 ZEMAN,W., AND INNES, J. R. M., Craigie's Nemoanatomy o/the Rat, Academic Press. New York, 1963, pp. 22--27. 26 ZlGMOND, R. E., AND MACKAY, A. V. P., Dissociation of stimulatory and synthetic phases in the trans-synaptic induction of tyrosine hydroxylase, Nature (Lond.), 247 (t974) 112 1t3.
547
Increased tyrosirm hydroxylase activity in the locus coeruleus of rat brain stem after reserpine treatment and cold stress
R. E. ZIGMOND, F. SCHON AND L. L. IVERSEN
MRC Neurochemical Pharmacology Unit, Department of Pharmacology, Medical School, Cambridge CB2 2QD (Great Britain) (Accepted January 18th, 1974)
Increases in the activity of tyrosine hydroxylase, the rate-limiting enzyme in noradrenaline biosynthesis, have been reported in adrenergic neurones in both the peripheral and central nervous system after administration of reserpine and after cold stress t4,2°,~1. The mechanism of this increase in enzyme activity has been studied most thoroughly in the rat superior cervical ganglion and in the adrenal gland after reserpine treatment. In these tissues, reserpine seems to act through a trans-synaptic mechanism; an increased synthesis of new tyrosine hydroxylase molecules occurs in response to increased firing of preganglionic neurones induced by autonomic reflexes after the drug treatment7,s,15, 26. The increased enzyme activity appears first in adrenergic cell bodies and several days later in sympathetic nerve terminals "3. Reserpine also leads to increases in tyrosine hydroxylase in the brain stem. A 55 % increase in enzyme activity was reported in the rabbit following two injections of the drug (2.5 mg/kg/day) 14 and a 26}/o increase in the rat after 8 daily injections (0.5 mg/kg/day) 1. Segal et al. found a 40 °J(, increase in tyrosine hydroxylase activity in the rat midbrain after 8 daily injections (0.5 mg/kg/day), but 11o significant change was found if the treatment lasted only 7 days t7. These effects of reserpine in the central nervous system are in general smaller and require more prolonged drug treatment than in the peripheral nervous system. Since tyrosine hydroxylase in the brain stem is found both in cell bodies and in nerve terminals and since there are several groups of catecholaminergic neurones in this area of the brain which may not all respond to reserpine in the same way, we have examined the effects of reserpine on tyrosine hydroxylase in a specific portion of the rat brain stem enriched in catecholaminergic cell bodies. The area chosen for this purpose was the region of the locus coeruleus, a nucleus which includes a large number of densely packed cells that fluoresce using the FalckHillarp technique for visualizing catecholaminergic neurones 3. (This area was designated A6 in the original fluorescent study of Dahlstr6m and FuxeS.) Pharmacological, immunochemical, and biochemical evidence suggests that the fluorescent cell bodies in the locus coeruleus are noradrenergic",4,a,9-aL Ventral to this cell group are
54~
StIORl (~)MMt,NI~ ~.IIONS
Fig. 1. Dissection procedure for removal ol the locus coeruleus, a : dorsal view of the {~l~m~lcm ,.ho\~ing the two halves of the cerebellum (() displaced laterally and the 3 indentations at the level of the cerebellar pedtlncle (indicated by arrowheadsL The two dashed lines indicate cuts made ~o istilale the coronal section containing the locus coeruteus (L). The caudal portion of the pons medtt!la is referred to as region 3 in the text. b: a coronal section through the pons at the level of the Itxus coeruleus stained for acetylcholinesterase according to lhe method of Shuie and Lewis ~, showing promineni enzyme activity in the locus coeruleus (L) and the trigeminal motor nucleus {TI. Cuts made in the locus coeruleus disseciion are represented by the dashed lines. The remaining areas 1 and 2 were taken as control regions (see text). This micrograph was kindly supplied by Dr. P. R. t.ewis, Department oi" Physiology, University of Cambridge, The calibration bar I mm in each picture
a n u m b e r o f larger cell bodies, p r o b a b l y also noradrenergic, which are part o f the ' s u b c o e r u l e u s a r e a 'a,16. The dissection o f the locus coeruteus was d e v e l o p e d with the aid o f the F a l c k Hillarp histochemical technique. By removing areas o f the dorsal pons i m d looking for the characteristic cluster o f fluorescent cell bodies on histochemical s e c t i o n s the following dissection p r o c e d u r e was arrived at based on the use o f easily visible landmarks. This technique allows the r e p r o d u c i b l e dissection o f the locus coeruteus in a piece of tissue weighing 2.5-3.0 rag. The b r a i n is t a k e n from the skull and placed on ice. The c e r e b e l l u m is transected in the midline and displaced laterally oil either side, exposing the dorsal surface o f the brain stem (Fig. ta). The cerebellum is then removed by cutting the cerebellar pedttncles at the lateral b o r d e r of the pons. A line can be defined visually in a c o r o n a l plane at the level o f the cerebeltar peduncles by 3 deep i n d e n t a t i o n s on the dorsal surface o f the pons (Fig. la). These are f o r m e d by the sulcus m e d i a n u s dorsalis a l o n g the midline and the two sulci limitantes on either side 'ca. T w o c o r o n a l cuts are m a d e as shown in Fig. ta, one t h r o u g h the i n d e n t a t i o n s and the other 1-1.5 mm rostralty. The resulting c o r o n a l section is placed with its caudal surface up. The 3 sulci can be seen clearly from this perspective (Fig. lb); A h o r i z o n t a l cut is m a d e t h r o u g h the section at a d e p t h c o r r e s p o n d i n g to the ventral tip o f the sulcus m e d i a n u s dorsalis ( a p p r o x i m a t e l y 1 m m from the d o r s a l surface)l The medial p o r t i o n between the two sulci limitantes is then removed and the re-
549
SHORT COMMUNICATIONS
Fig. 2. Portion of a methylene blue-stained section from the locus coeruleus dissection showing the clustered cell bodies of the locus coeruleus. The insert taken from an adjacent section shows these cell bodies are intensely fluorescent after treatment by the Falck-Hillarp technique a. Calibration bar 20 Mm.
m a i n i n g piece o f tissue is t a k e n as the ' l o c u s c o e r u l e u s s a m p l e ' (Fig. I b). M i c r o g r a p h s d e m o n s t r a t i n g the presence o f locus c o e r u l e u s cell b o d i e s in the s a m p l e are s h o w n in Fig. 2. T h e t y r o s i n e h y d r o x y l a s e a c t i v i t y o f the locus c o e r u l e u s s a m p l e was c o m p a r e d with 3 o t h e r parts o f the b r a i n s t e m : (1) the a r e a b e t w e e n the two sulci limitantes (Fig. lb), (2) the rest o f the c o r o n a l section described a b o v e (Fig. lb), and (3) the r e m a i n d e r o f the p o n s m e d u l l a c a u d a l to this c o r o n a l section (Fig. la). Tissues were
TABLE I D I S T R I B U T I O N ()F T Y R O S I N E H Y D R O X Y L A S E A C T I V I T Y IN D I F F E R E N T P A R I S OF T H E M E D U L L A PONS
Tyrosine hydroxylase (T-OH) activity was measured by a modification of the method of Hendry and Iversen". Tetra-hydrobiopterin was used as cofactor and the final concentration of k-[aH]tyrosine was 51 /~M. Samples were incubated at 37 C for 15 rain. A description of the method of dissection of the 4 brain samples is given in Fig. la and b and in the text. The data are the means : S.E.M. from 6 animals. Sample
T-OH aclivity (nmo/es ' a H / D O P A ./brnled/g/h)
o Tola[ T-OH activit.v
% Total wet weight
Locus coeruleus
37.3 ~ 4.2 6.4 ~k 1.2 3.6 0.2 3.8 5_ 1.9
23 9 14 60
2.9 1.3 18.4 77.3
1
2 3
550
SIi()RI (()MMI '
weighed, homogenized in 4 vol. of 5 mM Tris buffer (pH 6) containing tl. i ';, l riton X-100 and assayed for tyrosine hydroxylase activity (T-OH) by the method described by Hendry and iversen 6. The tyrosine hydroxylase activity per mg wet weight in the tocu.~ coeruleus sample was considerably higher than in the other 3 areas (Table 1). Although the locus coeruleus sample represented only about 3 °//o of the weight of the entire pons and medulla it contained 23 o(, of the tyrosine hydroxylase activity present m that part of the brain. The activity of the enzyme was also high compared to other areas of the central nervous system. For instance, the locus coeruteus sample contained about twice as much tyrosine hydroxylase activity per mg wet weight as the hypothalamus and more than 20 times as much as the hippocampus. Rats treated with two doses of reserpine (5 mg/kg subcutaneously) at 24 h intervals showed a 380 o~, increase m tyrosine hydroxylase activity in the locus coeruleus sample when assayed 48 h after the first injection (Fig. 3). The enzyme activity was also increased by cold stress. After 66 h at 4 ~"C. there was an 84°~, increase in tyrosine hydroxylase activity in this area (Fig. 3). When the ability of reserpine to increase tyrosine hydroxylase activity was compared in the locus coeruleus and in the rest of the pons plus medulla, the former area showed a much larger response. In these experiments the enzyme was assayed 72 h after a single injection of reserpine (5 mg/kg). The drug caused a 138 '~,, increase in the locus coeruleus sample and only a 307',i increase in the rest of the pons plus medulla (Table ii). The results show that both reserpine treatment and cold stress can stimulate tyrosine hydroxylase activity in the area of the locus coeruteus. Although there is a large concentration of noradrenergic cell bodies in this area of the brain there are
g~
z
500
[]CONTROL
O u
400 t/) ~. 30C
[7t EXPERIMENTAL 11 /.
~'~
<
~
~-o'rI0C ~
~'~'~
RESERPINE TREATMENT
COLD STRESS
Fig. 3. Effect of reserpine treatment and cold stress on tyrosine hydroxylase (T-OH) activity in the locus coeruleus. Reserpine treated animals were given two injections of reserpine (5 mg/kg) subcutaneously in 2 0 ~ ascorbic acid at 0 a n d 24 h a n d were killed at 48 h. Control animals were injected with ascorbic acid alone. Cold stressed rats were housed individually in metal cages m a cold r o o m (4 "C~ for 66 h. Control rats were housed similarly but kept at r o o m temperature (23 °C). The data expressed as tyrosine hydroxylase activity as a percentage of the appropriate control group were calculated on the basis of the a m o u n t of D O P A formed per locus coeruleus sample. The means _+~S.E.M. are shown. The number of animals per group is shown in each bar. In these experiments 6,7-dimethyl-tetrahydropteridine was used as cofactor and the final concentration of L-[ZH]tyrosine was 5.3 ¢tM.
551
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TABLE I1 COMPARISON OF THE EFFECTOF RESERPINEON TYROSINE HYDROXYLASEACTIVITYIN THE LOCUSCOERUEEUS A N D T H E REST OF T H L P O N S P L U S M E D U L L A
Animals were pretreated with a single injection of reserpine (5 mg/kg, s.c.) 72 h before sacrifice. Details of the dissection procedure are given in Fig. I, and the assay conditions are given in the legend to Table I. The data are the means :[ S.E.M. from 6 animals.
T-OH activity as % control
Vehicle controls Reserpine treated
Locus eoeru[etls
Rest oi meaTl//.;-l?OtlS
100 z I1 238 ! 37
100 : 7 130 9
also some noradrenergic terminals in the locus coeruleus and in the adjacent mesencephalic nucleus of the trigeminal nerve "4. Thus we cannot rule out the possibility that some of the increased tyrosine hydroxylase activity in the area may be located in nerve ternrinals. However the finding that large increases in tyrosine hydroxylase activity are found at short intervals after reserpine treatment (e.g. 2 days) strongly suggests that much of the increased activity is present in noradrenergic cell bodies. The increase in tyrosine hydroxylase activity is higher in the locus coeruleus sample than in the rest of the pons and medulla. It is not yet clear whether this is due to a selective effect of reserpine (either direct or trans-synaptic) on the ceils of the locus coeruleus or whether because of the large number of noradrenergic cell bodies in this region the effect is easier to detect there at short time intervals after drug treatment. The cell bodies of the locus coeruleus and the subcoeruleus area are known to project to lnany areas of the brain (e.g. hippocampus, cerebral cortex, hypothalamus) le.uS.l¢i,ts,ei. If the stimulation of tyrosine hydroxylase activity with reserpine in the central nervous system is analogous to that seen in the periphery one should be able to detect a delayed increase in enzyme activity in such areas of termination. In fact the appearance of increased tyrosine hydroxylase activity reported in the lnidbrain after 8 daily injections of reserpine may reflect the arrival of newly synthesized enzyme in terminals of noradrenergic neurones whose cell bodies are in the pons and medulla rather than a stimulation of enzyme activity in dopaminergic cell bodies in the midbraii317. Experiments in progress in our laboratory show such a delayed increase in tyrosine hydroxylase activity in the hypothalamus and hippocampus. This work was done during the tenure of a British-American Research Fellowship of the American Heart Association and the British Heart Foundation and an Alfred P. Sloan Foundation Research Fellowship to R . E . Z . F . S . is a Medical Research Council Scholar. l BESSON, M. J., CHERAMY, A., GAUCHY, C., AND MUSACCHIO, J. M., Effects of some psychotropic drugs on lyrosine hydroxylase activity in different structures of the rat brain, Europ. J. Pharmacol., 22 (1973) 181-186.
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