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On the projections from the locus coeruleus noradrenaline neurons: The cerebellar innervation
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Short Communications On the projections from the locus coieruleus noradrenaline neurons: The cerebellar innervation In 1964 locus coeruleus was found to be built up entirely~of catecholamine (CA) containing cell bodies TM. From studies on anterograde and retrograde degeneration of the noradrenergic neurons after lesions in the mesencephalon ~ indirect evidence was obtained that the locus coeruleus cell bodies contained noradrenaline (NA). In recent work using dopamine-fl-hydroxylase inhibitors 6 and immunohistochemical techniques for the cellular demonstration of dopamine-fl-hydroxylasO2 definite evidence has been obtained that the CA nerve cells of locus coeruleus contain NA. It is known that the locus coeruleus N A neurons give rise to ascending monosynaptic pathways to the tel- and diencephalon 1, and recently Ungerstedt 27 has provided evidence that they preferentially innervate the cortex cerebri where fine networks of NA nerve terminals are found in all layers of the cortex 4,13. The present investigation was mainly concerned with the study of the origin of the sparse plexus of fine NA nerve terminals found in all parts of the cortex cerebelli 17. Previous findings had suggested that part of the cerebellar N A innervation originated from N A nerve cells in the lateral reticular nucleus and in the locus coeruleus area of the pons a. The present findings demonstrate that the cerebellar N A innervation, in the same way as the cerebral cortical N A nerve terminals, originates mainly from the locus coeruleus, and suggest that a single locus coeruleus N A nerve TABLE 1 TYPES OF EXPERIMENTS PERFORMED
Number of rats within parentheses. Experiments
7-day-old rats 1-month-old rats Cerebellectomy (7-day-old rats) Cerebellectomy (1-month-old rats) Unilateral cut in front of locus coeruleus (7-day-old rats) Unilateral cut in front of locus coeruleus (l-month-old-rats) Unilateral cuts in front of locus coeruleus which were too dorsally or ventrally located
Day of killing after the operation
(10) (8) 1 (2)
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Fig. 1. Locus coeruleus of a 7-day-old rat. Sagittal section. Moderately fluorescent bundles of fibers with varicose-like enlargements are seen to leave the locus coeruleus NA nerve cells, The tibe~-s traverse the nuc. tr. n. mesencephalici in a rostral direction. : 190.
cell is capable of innervating all cortices of the brain. Male Sprague-Dawley rats were used. About half of the rats were 7 days of age: the rest were one month of age. For number of rats see Table i. Young rats were chosen, since they more easily exhibit retrograde cell body changes. Operations were performed under fluothane-N20-Oz anesthesia. The following experiments were performed to elucidate the origin of the N A innervation of the cortex cerebelli (.see also Table 1). (1) With the help of a line glass cannula as much as possible of the cerebellum was removed by suction. The operation was most successful in the 7-day-old rats in which practically all of the cerebellum could be removed. The rats were killed 1--7 days after the operation. The brains were removed and subjected to histochemical fluorescence analysis of CA 7,~,16. Usually, serial sagittal sections of whole brains were made but also several serial transversal and horizontal sections were prepared. (2) With the help of a fine knife (I.5 m m × 0.5 mm) similar to that used by Halasz 15, cuts were made in front of the locus coeruleus to damage the NA fibers localized dorsally in the tegmentum of mesencephalon and which probably originate mainly from the locus coeruleus. Cuts were made in both 1 week- and l-month-old rats. The rats were killed 1-7 days following the operation. The whole brains were dissected out and subjected to histochemicat fluorescence analysis of CA. In 7-day-old rats the locus coeruleus N A cell bodies exhibited a moderate to strong green fluorescence intensity, and moderately fluorescent axons with varicoselike enlargements could be seen to leave most of the cell bodies in a rostral direction (Fig. 1) so as to form a distinct bundle in the dorsal tegmentum just ventrolateral to the fasciculus longitudinalis medialis. The individual fibers lie aggregated in bundles. This rostral projection originated mainly from the cranial part, particularly the dorsoBrain Research, 28 (1971) 165-171
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Fig. 2. Cerebellectomy 1 day after operation. 8-day-old rat. Frontal section. The cut cerebellar NA fibers are deformed and exhibit a strong fluorescence. They are seen to leave the locus coeruleus NA nerve cells, which exhibit signs of retrograde changes (swollen cell bodies, displaced nuclei and increased fluorescence intensities). × 120. lateral portion. In mature rats these fibers are not easily seen unless the intraneuronal amine levels are increased by pharmacological treatment or interruption of the axons 10. The detailed course of the rostral pathway has been described by Ungerstedt 27. In addition, a medial fiber projection to the raphe was observed in horizontal sections (see also ref. 9) which probably partly represents crossing fibers (Fig. 4). This fiber projection partly originated from the most ventromedial part of the locus coeruleus which contains the largest cell bodies and exhibits the strongest fluorescence intensity. The axons had varicose-like enlargements which exhibited a moderate fluorescence intensity. No bundle formation was observed. In l-month-old rats the fibers of the rostral projection were only weakly fluorescent, whereas the fibers of the medial projection still showed a moderate intensity. The fluorescence intensity in the NA nerve cell bodies had also decreased somewhat so that only a weak to moderate intensity was observed as in mature rats. Cerebellectorn),. The best results were obtained in the youngest rats. Strongly green fluorescent fibers could be seen in the remaining parts of the white matter. The NA fibers were swollen and deformed as described in previous papers 10 and reached the white matter of the cerebellum by passing dorsally medial to the pedunculus cerebellaris medius close to the fourth ventricle in the grey matter surrounding the lateral part of the ventricle. The N A fibers could be traced all the way to the dorsolateral locus coeruleus NA cell bodies, which appeared swollen and showed increased fluorescence intensities and displaced nuclei (Fig. 2). This was true also for the N A cell bodies in the roof of the fourth ventricle (group A4, see ref. 9) which represents the caudal extension of the dorsolateral part of the locus coeruleus. The retrograde cell body changes were not evident in the 1-month-old rats. The other CA cell groups within the medulla
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Fig. 3. Cerebellar cortex 3,days following mesencephalic transection of the dorsal ascending NA bundle which originates from the locus coeruleus (cf. Fig, 1). Operation performed in a 7-day-old ral. The sparse plexus of cerebellar NA nerve terminals exhibit strong fluorescence. In an untreated animal no or only a very weak fluorescence can be observed. The present results are probably best explained as the result of an increased transport of amine granules into the cerebellar NA fibers duc Io damage of the ascending cerebral NA fibers originating from the same cell bodies. 120 Fig. 4. Pontic midline area at the level of the locus coeruleus. I-month-old-rat. Horizontal section: Anterior up; posterior down. The medial fiber projections from the locus coeru/eus are seen to meet in the midline. No bundle formation is observed. The fibers have varicose-like enlargements and exhibit a moderate green fluorescence, i 80. o b l o n g a t a , pons a n d m e s e n c e p h a l o n did n o t show any clearcut changes in a n y of the rats studied. The N A nerve terminals in the brain, e.g., m the cortex cerebri, did n o t show any certain changes in fluorescence intensity. Cut in.front of the locus coeruleus. A g a i n the best results were o b t a i n e d in the youngest rats. The extent of the lesion was easily evaluated in sagittal sections. The cuts which missed the dorsal N A b u n d l e from the locus coeruleus or were too ventrally located, so t h a t they also damaged other ascending N A fibers (see ref. I), were regarded as controls. The cuts were located 0.5-1 m m in front of the locus coeruleus. I n the experimental rats that showed only damage to the dorsal N A fibers a piling up o f C A fluorescence was found on the caudal side o f the lesion in the NA tibers which could be seen to originate mainly from the locus coeruleus. Retrograde cell body changes were present in most of the locus coeruleus N A cell bodies especially in those of the dorsolateral part including g r o u p A4 in the r o o f o f the fourth ventricle (see ref. 9). Seven days after the operation, the N A nerve terminals of the cortex cerebelli showed marked increases in fluorescence intensity t Fig. 3). They exhibited a m o d e r a t e to strong green fluorescence. N o r m a l l y they are barely visible in the fluorescence microscope. I n contrast, several N A nerve terminal systems m the rostral parts of the b r a i n h a d disappeared. It was thus possible to confirm the disappearance o f the N A nerve terminals in the cortex cerebri on the lesioned side as d e m o n s t r a t e d by Ungerstedt2L F u r t h e r m o r e . a considerable disappearance of fluorescent N A nerve
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terminals was observed in the colliculus, the geniculate bodies and in the thalamus on the lesioned side. When the cuts were too dorsally located no change in the central NA neurons was observed. If the cuts extended too ventrally retrograde cell body changes were observed in practically all of the locus coeruleus NA cells and also in NA cell bodies other than in the locus coeruleus area. Furthermore, disappearance of fluorescence was also observed in practically all parts of the prosencephalon on the lesioned side (see ref. 1). The present experiments give good evidence that the major part of the cerebellar N A innervation originates from the dorsolateral part of the locus coeruleus NA nerve cells (groups A6 and A4; ref. 9). Thus, retrograde cell body changes occurred in these NA cell bodies followirtg cerebellectomy and the cerebellar NA fibers could be traced to these NA cell bodies. The results are in agreement with recent electrical stimulation experiments on the locus coeruleus showing an increase of NA release in the cerebellar cortex of the stimulated side (Arbuthnott et al., unpublished data). Additional support for this view has also been obtained in this laboratory by making specific lesions of the locus coeruleus 27 resulting in the disappearance of NA nerve terminals in the cortex cerebelli. Since retrograde cell body changes were found in the dorsolateral parts of the locus coeruleus cells both following cerebellectomy and following cutting of the ascending NA bundle to the cortex cerebri, it is likely that the individual N A cell bodies in the dorsolateral locus coeruleus can innervate both the cortex cerebri and the cortex cerebelli. This is further supported by the present observation that following lesion of the dorsal NA pathway to the cortex cerebri there was a marked increase in the intraneuronal amine contents of the entire cerebellar NA network on the lesioned side. These increases are best explained as the result of an increased transport of amine granules from the cell bodies into the remaining intact fibers of the cells, since the amine granules no longer can be transported along the ascending axons (see ref. 2). The reason why it was so easy to observe increases in the fluorescence intensity of the cerebellar N A terminals is due to the fact that these terminals normally have a low NA content and therefore a low fluorescence intensity in contrast to e.g. those of the hypothalamus. No increase in the intraneuronal amine content was seen in the cortex cerebri following cerebellectomy. This is explained by the fact that the cerebellar NA innervation represents only a small part of the innervation area of the locus coeruleus neurons, thus causing a proportionally much smaller increase in axonal flow towards the cerebral cortex. Thus, the present data in combination with the previous findings of Ungerstedt ')7 concerning the N A innervation of the cortex cerebri favor the view that one single NA neuron in the dorsolateral locus coeruleus area can monosynapticaIly innervate both the cortex cerebri and cerebeIli. This enables these neurons to immediately and simultaneously influence the neural activity in practically all cortical areas of the brain. Furthermore, it is likely that the ascending NA pathway on its course from the locus coeruleus to the cortex cerebri gives collaterals to the colliculi, the geniculate bodies and parts of the thalamus. Brain Research, 28 (1971) 165-171
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In view of the results of Jouvet's grouplS,20, 9t. it seems likely that this system is important for the induction of paradoxical sleep, since there is an increase in NA turnover during the rebound phase of paradoxical sleep '~. DOPA induces a normal paradoxical sleep in reserpine-pretreated rats, and the N A receptor blocking agent phenoxybenzamine diminishes paradoxical sleep "4 as does destruction of the locus coeruleus area. The cortical locus coeruleus system innervates also the geniculate bodies, as shown in the present study. Therefore this system may be involved also in the regulation of ponto-geniculo-occipital discharges found in paradoxical sleep; these discharges are particularly frequent after reserpine treatment or destruction of the locus coeruleus area 20. N o t only paradoxical sleep, but also the maintenance of cortical activation appears to be dependent on the locus coeruleus NA neurons. Thus. it has bcen found that D O P A causes an increase m the waking state, and destruction of the NA neurons increases cortical synchronization ag. The importance of the locus coeruleus NA system for cortical arousal is al so shown by the marked inhibition of the activity of this system occurring after treatment with minor tranquillizers and barbiturates s,z'~ and by the high sensitivity of this system to the releasing action of amphetamine r'. It seems a likely possibility that the activating effects of the locus coeruleus NA neurons on all cortices form an important component of the reticular activating s3 stem. The activation of the NA neurons may be important for many behavioral performances such as conditioned avoidance behavior a4 and formation of memory traces e~. which probably requires an alert cerebral cortex. The medial fiber projection from the locus coeruleus to the raphe may constitute a partially crossed fiber system, responsible for part of the cortical innervation and at least part of the innervation of the vagal area. raphe area and the hypothalamus in view of Loizou's findings '~a of a disappearance of NA nerve terminals m the latter areas following bilateral locus coeruleus lesions. This possibility is now being investigated. In view of the atonia occurring in the neck musculature m paradoxical sleep it is of interest that this projection probably heavily innervates the nuc. n. accessorii, the nucleus responsible for the motor innervation of these muscles. This work has been supported by Grants B71-14X-715-06C and B71-14X3185-01 from the Swedish Medical Research Council and by a Grant from Magn. Bergvalls Stiftelse. The skillful technical assistance of Mrs. Karin Andreasson. Mrs. Agneta Eliasson and Mrs. Barbro Norstedt is gratefully acknowledged. Department of Histology, Karolinska Institute S-104 Ol Stockholm 60 (Sweden)
LARS OLSON K JELL FUXE
1 ANDEN, N.-E., DAHLSTROM,A., FUXE, K., LARSSON, K., OLSON, L., AND UNGERSTEDT~U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta physioL scand., 67 (1966) 313-326. 2 ANDI~N, N.-E., FUXE, K., AND LARSSON, K., Effect of large mesencephalic-diencephalic lesions Brain Research, 28 (1971) 165-171
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on the NA, DA and 5-HT neurons of the central nervous system, Experientia (Basel), 22 (1966) 842-847. ANDEN, N.-E., FtrxE, K., AND UNGERSTEDT, U., Monoamine pathways to the cerebellar and cerebral cortex, Experientia (Basel), 23 (1967) 838-839. BLACKSTAD, T., FUXE, K., AND HOKFELT, T., Noradrenaline nerve terminals in the hippocampa[ region of the rat and the guinea pig, Z. Zellforsch., 78 (1967) 463-473. CARLSSON, A., FUXE, K., HAMBERGER, B., AND LINDQVIST, M., Biochemical and histochernical studies on the effects of imipramine-like drugs and (+)-amphetamine on central and peripheral catecholamine neurons, Acta physiol, scand., 67 (1966) 481-497. CORRODI, H., FUXE, K., HAMBERGER, B., AND LJONGDAHL,/~., Studies on central and peripheral noradrenaline neurons using a new dopamine-fl-hydroxylase inhibitor, Europ. J. Pharmacol., 12 (1970) 145 155. CORRODI, H., AND JONSSON, G., The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology, J. Histochem. Cytochem., 15 (1967) 65-78. CORRODI, H., Ft~XE, K., LIDBRINK, P., AND OLSON, L., Minor tranquillizers, stress and central catecholamine neurons, Brain Research, 29 0971) in press. DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. DAHLSTR(~M,A., AND FUXE, K., A method for the demonstration of monoamine containing nerve fibres in the central nervous system, Acta physiol, stand., 60 (1964) 293-294. FALCK, B., HILLARP, N.-A., THIEME, G., AND TORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. FUXE, K., GOLDSTEIN, M., H()KFELT, Y., AND HYUB, J. T., lmmunohistochemical localization of dopamine-fl-hydroxylase in the peripheral and central nervous system, Res. Commun. Chem. Pathol. Pharmacol., 1 (1970) 627-636. FUXE, K., HAMBERGER, B., AND HOKFELT, T., Distribution of noradrenaline nerve terminals in cortical areas of the rat, Brain Research, 8 (1968) 125-131. FuxE, K., AND HANSSON, L., Central catecholamine neurons and conditioned avoidance behaviour, Psychopharmacologia (Berl.), 11 (1967) 439-447. HALASZ, B., The endocrine effects of isolation of the hypothalamus from the rest of the brain. In W. F. GANONG AND L. MARTINI (Eds.), Frontiers in Neuroendocrinology, Oxford Univ. Press, London, 1969, pp. 307-342. HILLARP, N.-/~., FUXE, K., AND DAHLSTR6M, A., Central monoamine neurons. In U.S. VON EULER, S. ROSELL AND B. UVNXS (Eds.), Mechanisms on Release of Biagenie Amines, Pergamon Press, London, 1966, pp. 31 37. HOKVEL'V,T., AND FUXE, K., Cerebellar monoamine nerve terminals, a new type of afferent fibres to the cortex cerebelli, Exp. Brain Res., 9 (1969) 63-72. JONES, B. E., Cateeholamine Containing Neurons in the Brain Stem of the Cat and their Role in Waking, Thesis, Tixier et Fils, Lyon, 1969, pp. 1-87. JONES, B. E., BOBILLIER, P., ET JOUVET, M., Effets de la destruction des neurones contenant des catdcholamines du m6senc6phale sur le cycle veille sommeils du chat, C.R. Soc. Biol. (Paris), 163 (1969) 176-180. JOUVET, M., Neurophysiology of the states of sleep, Physiol. Rev., 47 (1967) 117 177. JOUVET, M., Biogenic amines and the state of sleep, Se&nce, 163 (1969) 32-41. LIDBRINK, P., CORRODI, H., FUXE, K., AND OLSON, L., Decreases in brain catecholamine turnover after barbiturates or meprobamate treatment of stressed and unstressed rats, To be published. Lolzou, L. A., Projections of the nucleus locus coeruleus in the albino rat, Brain Research, 15 (1969) 563-566. MATSUMOTO, I., AND WATANABE, S., Paradoxical sleep: Effects of adrenergic blocking agents, Proc. Jap. Acad., 43 (1967) 680-683. PUJOL, J. F., MOURET, J., JOUVET, M., AND GLOWINSKI, J., Increased turnover of cerebral norepinephrine during rebound of paradoxical sleep in the rat, Science, 159 (1968) 112-114. ROBERTS, R. B., FLEXNER, J. B., AND FLEXNER, L. B., Some evidence for the involvement of adrenergic sites in the memory trace, Proc. nat. Acad. Sci. (Wash.), 66 (1970) 31~313. UNGERSTEDT, U., Noradrenaline pathways in the rat brain: Principal architecture, Science, in press.
(Accepted January 21st, 1971 )
Brain Research, 28 (1971) 165-171
BRAIN RESEARCH
Short Communications On the projections from the locus coieruleus noradrenaline neurons: The cerebellar innervation In 1964 locus coeruleus was found to be built up entirely~of catecholamine (CA) containing cell bodies TM. From studies on anterograde and retrograde degeneration of the noradrenergic neurons after lesions in the mesencephalon ~ indirect evidence was obtained that the locus coeruleus cell bodies contained noradrenaline (NA). In recent work using dopamine-fl-hydroxylase inhibitors 6 and immunohistochemical techniques for the cellular demonstration of dopamine-fl-hydroxylasO2 definite evidence has been obtained that the CA nerve cells of locus coeruleus contain NA. It is known that the locus coeruleus N A neurons give rise to ascending monosynaptic pathways to the tel- and diencephalon 1, and recently Ungerstedt 27 has provided evidence that they preferentially innervate the cortex cerebri where fine networks of NA nerve terminals are found in all layers of the cortex 4,13. The present investigation was mainly concerned with the study of the origin of the sparse plexus of fine NA nerve terminals found in all parts of the cortex cerebelli 17. Previous findings had suggested that part of the cerebellar N A innervation originated from N A nerve cells in the lateral reticular nucleus and in the locus coeruleus area of the pons a. The present findings demonstrate that the cerebellar N A innervation, in the same way as the cerebral cortical N A nerve terminals, originates mainly from the locus coeruleus, and suggest that a single locus coeruleus N A nerve TABLE 1 TYPES OF EXPERIMENTS PERFORMED
Number of rats within parentheses. Experiments
7-day-old rats 1-month-old rats Cerebellectomy (7-day-old rats) Cerebellectomy (1-month-old rats) Unilateral cut in front of locus coeruleus (7-day-old rats) Unilateral cut in front of locus coeruleus (l-month-old-rats) Unilateral cuts in front of locus coeruleus which were too dorsally or ventrally located
Day of killing after the operation
(10) (8) 1 (2)
3 (3)
7 (4)
1 (2)
3 (1)
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3 (2)
7 (4)
7 (4) 3 (3)
7 (3) Brain Research, 28 (1971) 165-171
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Fig. 1. Locus coeruleus of a 7-day-old rat. Sagittal section. Moderately fluorescent bundles of fibers with varicose-like enlargements are seen to leave the locus coeruleus NA nerve cells, The tibe~-s traverse the nuc. tr. n. mesencephalici in a rostral direction. : 190.
cell is capable of innervating all cortices of the brain. Male Sprague-Dawley rats were used. About half of the rats were 7 days of age: the rest were one month of age. For number of rats see Table i. Young rats were chosen, since they more easily exhibit retrograde cell body changes. Operations were performed under fluothane-N20-Oz anesthesia. The following experiments were performed to elucidate the origin of the N A innervation of the cortex cerebelli (.see also Table 1). (1) With the help of a line glass cannula as much as possible of the cerebellum was removed by suction. The operation was most successful in the 7-day-old rats in which practically all of the cerebellum could be removed. The rats were killed 1--7 days after the operation. The brains were removed and subjected to histochemical fluorescence analysis of CA 7,~,16. Usually, serial sagittal sections of whole brains were made but also several serial transversal and horizontal sections were prepared. (2) With the help of a fine knife (I.5 m m × 0.5 mm) similar to that used by Halasz 15, cuts were made in front of the locus coeruleus to damage the NA fibers localized dorsally in the tegmentum of mesencephalon and which probably originate mainly from the locus coeruleus. Cuts were made in both 1 week- and l-month-old rats. The rats were killed 1-7 days following the operation. The whole brains were dissected out and subjected to histochemicat fluorescence analysis of CA. In 7-day-old rats the locus coeruleus N A cell bodies exhibited a moderate to strong green fluorescence intensity, and moderately fluorescent axons with varicoselike enlargements could be seen to leave most of the cell bodies in a rostral direction (Fig. 1) so as to form a distinct bundle in the dorsal tegmentum just ventrolateral to the fasciculus longitudinalis medialis. The individual fibers lie aggregated in bundles. This rostral projection originated mainly from the cranial part, particularly the dorsoBrain Research, 28 (1971) 165-171
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Fig. 2. Cerebellectomy 1 day after operation. 8-day-old rat. Frontal section. The cut cerebellar NA fibers are deformed and exhibit a strong fluorescence. They are seen to leave the locus coeruleus NA nerve cells, which exhibit signs of retrograde changes (swollen cell bodies, displaced nuclei and increased fluorescence intensities). × 120. lateral portion. In mature rats these fibers are not easily seen unless the intraneuronal amine levels are increased by pharmacological treatment or interruption of the axons 10. The detailed course of the rostral pathway has been described by Ungerstedt 27. In addition, a medial fiber projection to the raphe was observed in horizontal sections (see also ref. 9) which probably partly represents crossing fibers (Fig. 4). This fiber projection partly originated from the most ventromedial part of the locus coeruleus which contains the largest cell bodies and exhibits the strongest fluorescence intensity. The axons had varicose-like enlargements which exhibited a moderate fluorescence intensity. No bundle formation was observed. In l-month-old rats the fibers of the rostral projection were only weakly fluorescent, whereas the fibers of the medial projection still showed a moderate intensity. The fluorescence intensity in the NA nerve cell bodies had also decreased somewhat so that only a weak to moderate intensity was observed as in mature rats. Cerebellectorn),. The best results were obtained in the youngest rats. Strongly green fluorescent fibers could be seen in the remaining parts of the white matter. The NA fibers were swollen and deformed as described in previous papers 10 and reached the white matter of the cerebellum by passing dorsally medial to the pedunculus cerebellaris medius close to the fourth ventricle in the grey matter surrounding the lateral part of the ventricle. The N A fibers could be traced all the way to the dorsolateral locus coeruleus NA cell bodies, which appeared swollen and showed increased fluorescence intensities and displaced nuclei (Fig. 2). This was true also for the N A cell bodies in the roof of the fourth ventricle (group A4, see ref. 9) which represents the caudal extension of the dorsolateral part of the locus coeruleus. The retrograde cell body changes were not evident in the 1-month-old rats. The other CA cell groups within the medulla
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Fig. 3. Cerebellar cortex 3,days following mesencephalic transection of the dorsal ascending NA bundle which originates from the locus coeruleus (cf. Fig, 1). Operation performed in a 7-day-old ral. The sparse plexus of cerebellar NA nerve terminals exhibit strong fluorescence. In an untreated animal no or only a very weak fluorescence can be observed. The present results are probably best explained as the result of an increased transport of amine granules into the cerebellar NA fibers duc Io damage of the ascending cerebral NA fibers originating from the same cell bodies. 120 Fig. 4. Pontic midline area at the level of the locus coeruleus. I-month-old-rat. Horizontal section: Anterior up; posterior down. The medial fiber projections from the locus coeru/eus are seen to meet in the midline. No bundle formation is observed. The fibers have varicose-like enlargements and exhibit a moderate green fluorescence, i 80. o b l o n g a t a , pons a n d m e s e n c e p h a l o n did n o t show any clearcut changes in a n y of the rats studied. The N A nerve terminals in the brain, e.g., m the cortex cerebri, did n o t show any certain changes in fluorescence intensity. Cut in.front of the locus coeruleus. A g a i n the best results were o b t a i n e d in the youngest rats. The extent of the lesion was easily evaluated in sagittal sections. The cuts which missed the dorsal N A b u n d l e from the locus coeruleus or were too ventrally located, so t h a t they also damaged other ascending N A fibers (see ref. I), were regarded as controls. The cuts were located 0.5-1 m m in front of the locus coeruleus. I n the experimental rats that showed only damage to the dorsal N A fibers a piling up o f C A fluorescence was found on the caudal side o f the lesion in the NA tibers which could be seen to originate mainly from the locus coeruleus. Retrograde cell body changes were present in most of the locus coeruleus N A cell bodies especially in those of the dorsolateral part including g r o u p A4 in the r o o f o f the fourth ventricle (see ref. 9). Seven days after the operation, the N A nerve terminals of the cortex cerebelli showed marked increases in fluorescence intensity t Fig. 3). They exhibited a m o d e r a t e to strong green fluorescence. N o r m a l l y they are barely visible in the fluorescence microscope. I n contrast, several N A nerve terminal systems m the rostral parts of the b r a i n h a d disappeared. It was thus possible to confirm the disappearance o f the N A nerve terminals in the cortex cerebri on the lesioned side as d e m o n s t r a t e d by Ungerstedt2L F u r t h e r m o r e . a considerable disappearance of fluorescent N A nerve
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terminals was observed in the colliculus, the geniculate bodies and in the thalamus on the lesioned side. When the cuts were too dorsally located no change in the central NA neurons was observed. If the cuts extended too ventrally retrograde cell body changes were observed in practically all of the locus coeruleus NA cells and also in NA cell bodies other than in the locus coeruleus area. Furthermore, disappearance of fluorescence was also observed in practically all parts of the prosencephalon on the lesioned side (see ref. 1). The present experiments give good evidence that the major part of the cerebellar N A innervation originates from the dorsolateral part of the locus coeruleus NA nerve cells (groups A6 and A4; ref. 9). Thus, retrograde cell body changes occurred in these NA cell bodies followirtg cerebellectomy and the cerebellar NA fibers could be traced to these NA cell bodies. The results are in agreement with recent electrical stimulation experiments on the locus coeruleus showing an increase of NA release in the cerebellar cortex of the stimulated side (Arbuthnott et al., unpublished data). Additional support for this view has also been obtained in this laboratory by making specific lesions of the locus coeruleus 27 resulting in the disappearance of NA nerve terminals in the cortex cerebelli. Since retrograde cell body changes were found in the dorsolateral parts of the locus coeruleus cells both following cerebellectomy and following cutting of the ascending NA bundle to the cortex cerebri, it is likely that the individual N A cell bodies in the dorsolateral locus coeruleus can innervate both the cortex cerebri and the cortex cerebelli. This is further supported by the present observation that following lesion of the dorsal NA pathway to the cortex cerebri there was a marked increase in the intraneuronal amine contents of the entire cerebellar NA network on the lesioned side. These increases are best explained as the result of an increased transport of amine granules from the cell bodies into the remaining intact fibers of the cells, since the amine granules no longer can be transported along the ascending axons (see ref. 2). The reason why it was so easy to observe increases in the fluorescence intensity of the cerebellar N A terminals is due to the fact that these terminals normally have a low NA content and therefore a low fluorescence intensity in contrast to e.g. those of the hypothalamus. No increase in the intraneuronal amine content was seen in the cortex cerebri following cerebellectomy. This is explained by the fact that the cerebellar NA innervation represents only a small part of the innervation area of the locus coeruleus neurons, thus causing a proportionally much smaller increase in axonal flow towards the cerebral cortex. Thus, the present data in combination with the previous findings of Ungerstedt ')7 concerning the N A innervation of the cortex cerebri favor the view that one single NA neuron in the dorsolateral locus coeruleus area can monosynapticaIly innervate both the cortex cerebri and cerebeIli. This enables these neurons to immediately and simultaneously influence the neural activity in practically all cortical areas of the brain. Furthermore, it is likely that the ascending NA pathway on its course from the locus coeruleus to the cortex cerebri gives collaterals to the colliculi, the geniculate bodies and parts of the thalamus. Brain Research, 28 (1971) 165-171
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In view of the results of Jouvet's grouplS,20, 9t. it seems likely that this system is important for the induction of paradoxical sleep, since there is an increase in NA turnover during the rebound phase of paradoxical sleep '~. DOPA induces a normal paradoxical sleep in reserpine-pretreated rats, and the N A receptor blocking agent phenoxybenzamine diminishes paradoxical sleep "4 as does destruction of the locus coeruleus area. The cortical locus coeruleus system innervates also the geniculate bodies, as shown in the present study. Therefore this system may be involved also in the regulation of ponto-geniculo-occipital discharges found in paradoxical sleep; these discharges are particularly frequent after reserpine treatment or destruction of the locus coeruleus area 20. N o t only paradoxical sleep, but also the maintenance of cortical activation appears to be dependent on the locus coeruleus NA neurons. Thus. it has bcen found that D O P A causes an increase m the waking state, and destruction of the NA neurons increases cortical synchronization ag. The importance of the locus coeruleus NA system for cortical arousal is al so shown by the marked inhibition of the activity of this system occurring after treatment with minor tranquillizers and barbiturates s,z'~ and by the high sensitivity of this system to the releasing action of amphetamine r'. It seems a likely possibility that the activating effects of the locus coeruleus NA neurons on all cortices form an important component of the reticular activating s3 stem. The activation of the NA neurons may be important for many behavioral performances such as conditioned avoidance behavior a4 and formation of memory traces e~. which probably requires an alert cerebral cortex. The medial fiber projection from the locus coeruleus to the raphe may constitute a partially crossed fiber system, responsible for part of the cortical innervation and at least part of the innervation of the vagal area. raphe area and the hypothalamus in view of Loizou's findings '~a of a disappearance of NA nerve terminals m the latter areas following bilateral locus coeruleus lesions. This possibility is now being investigated. In view of the atonia occurring in the neck musculature m paradoxical sleep it is of interest that this projection probably heavily innervates the nuc. n. accessorii, the nucleus responsible for the motor innervation of these muscles. This work has been supported by Grants B71-14X-715-06C and B71-14X3185-01 from the Swedish Medical Research Council and by a Grant from Magn. Bergvalls Stiftelse. The skillful technical assistance of Mrs. Karin Andreasson. Mrs. Agneta Eliasson and Mrs. Barbro Norstedt is gratefully acknowledged. Department of Histology, Karolinska Institute S-104 Ol Stockholm 60 (Sweden)
LARS OLSON K JELL FUXE
1 ANDEN, N.-E., DAHLSTROM,A., FUXE, K., LARSSON, K., OLSON, L., AND UNGERSTEDT~U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta physioL scand., 67 (1966) 313-326. 2 ANDI~N, N.-E., FUXE, K., AND LARSSON, K., Effect of large mesencephalic-diencephalic lesions Brain Research, 28 (1971) 165-171
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on the NA, DA and 5-HT neurons of the central nervous system, Experientia (Basel), 22 (1966) 842-847. ANDEN, N.-E., FtrxE, K., AND UNGERSTEDT, U., Monoamine pathways to the cerebellar and cerebral cortex, Experientia (Basel), 23 (1967) 838-839. BLACKSTAD, T., FUXE, K., AND HOKFELT, T., Noradrenaline nerve terminals in the hippocampa[ region of the rat and the guinea pig, Z. Zellforsch., 78 (1967) 463-473. CARLSSON, A., FUXE, K., HAMBERGER, B., AND LINDQVIST, M., Biochemical and histochernical studies on the effects of imipramine-like drugs and (+)-amphetamine on central and peripheral catecholamine neurons, Acta physiol, scand., 67 (1966) 481-497. CORRODI, H., FUXE, K., HAMBERGER, B., AND LJONGDAHL,/~., Studies on central and peripheral noradrenaline neurons using a new dopamine-fl-hydroxylase inhibitor, Europ. J. Pharmacol., 12 (1970) 145 155. CORRODI, H., AND JONSSON, G., The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology, J. Histochem. Cytochem., 15 (1967) 65-78. CORRODI, H., Ft~XE, K., LIDBRINK, P., AND OLSON, L., Minor tranquillizers, stress and central catecholamine neurons, Brain Research, 29 0971) in press. DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. DAHLSTR(~M,A., AND FUXE, K., A method for the demonstration of monoamine containing nerve fibres in the central nervous system, Acta physiol, stand., 60 (1964) 293-294. FALCK, B., HILLARP, N.-A., THIEME, G., AND TORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. FUXE, K., GOLDSTEIN, M., H()KFELT, Y., AND HYUB, J. T., lmmunohistochemical localization of dopamine-fl-hydroxylase in the peripheral and central nervous system, Res. Commun. Chem. Pathol. Pharmacol., 1 (1970) 627-636. FUXE, K., HAMBERGER, B., AND HOKFELT, T., Distribution of noradrenaline nerve terminals in cortical areas of the rat, Brain Research, 8 (1968) 125-131. FuxE, K., AND HANSSON, L., Central catecholamine neurons and conditioned avoidance behaviour, Psychopharmacologia (Berl.), 11 (1967) 439-447. HALASZ, B., The endocrine effects of isolation of the hypothalamus from the rest of the brain. In W. F. GANONG AND L. MARTINI (Eds.), Frontiers in Neuroendocrinology, Oxford Univ. Press, London, 1969, pp. 307-342. HILLARP, N.-/~., FUXE, K., AND DAHLSTR6M, A., Central monoamine neurons. In U.S. VON EULER, S. ROSELL AND B. UVNXS (Eds.), Mechanisms on Release of Biagenie Amines, Pergamon Press, London, 1966, pp. 31 37. HOKVEL'V,T., AND FUXE, K., Cerebellar monoamine nerve terminals, a new type of afferent fibres to the cortex cerebelli, Exp. Brain Res., 9 (1969) 63-72. JONES, B. E., Cateeholamine Containing Neurons in the Brain Stem of the Cat and their Role in Waking, Thesis, Tixier et Fils, Lyon, 1969, pp. 1-87. JONES, B. E., BOBILLIER, P., ET JOUVET, M., Effets de la destruction des neurones contenant des catdcholamines du m6senc6phale sur le cycle veille sommeils du chat, C.R. Soc. Biol. (Paris), 163 (1969) 176-180. JOUVET, M., Neurophysiology of the states of sleep, Physiol. Rev., 47 (1967) 117 177. JOUVET, M., Biogenic amines and the state of sleep, Se&nce, 163 (1969) 32-41. LIDBRINK, P., CORRODI, H., FUXE, K., AND OLSON, L., Decreases in brain catecholamine turnover after barbiturates or meprobamate treatment of stressed and unstressed rats, To be published. Lolzou, L. A., Projections of the nucleus locus coeruleus in the albino rat, Brain Research, 15 (1969) 563-566. MATSUMOTO, I., AND WATANABE, S., Paradoxical sleep: Effects of adrenergic blocking agents, Proc. Jap. Acad., 43 (1967) 680-683. PUJOL, J. F., MOURET, J., JOUVET, M., AND GLOWINSKI, J., Increased turnover of cerebral norepinephrine during rebound of paradoxical sleep in the rat, Science, 159 (1968) 112-114. ROBERTS, R. B., FLEXNER, J. B., AND FLEXNER, L. B., Some evidence for the involvement of adrenergic sites in the memory trace, Proc. nat. Acad. Sci. (Wash.), 66 (1970) 31~313. UNGERSTEDT, U., Noradrenaline pathways in the rat brain: Principal architecture, Science, in press.
(Accepted January 21st, 1971 )
Brain Research, 28 (1971) 165-171