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Catecholaminergic terminals in the developing and adult rat cerebellum
Braht Research, 132 (1977) 355-36l
355
Elsevier/North-Holland Biomedical Press
Catecholaminergic terminals in the developing and adult rat cerebellum
TETSURO YAMAMOTO, MASATSUNE ISHIKAWA and CHIKAKO TANAKA* Department of Physiolog.v, htstitute Jbr Brahz Research, { M.I.) Department o[" Neurosurgery, Faculty qf Medichle, Kyoto Ulliversity and (C.T.) Department o[" Pharmacology, Kobe University School of Medicine, Kobe (Japa~t)
(Accepted May I lth, 1977)
With the formaldehyde histofluorescence method of Falck and Hillarp (FA method) a, catecholaminergic (CA), most probably noradrenergic (NA), terminals have been demonstrated in the cerebellar cortex 1,4,~, and they were found to be derived from the NA neurons in the locus coeruleus (LC) la and other pontine NA neurons e. It has also been reported that NA terminals in the cerebellar cortex made synaptic contacts with Purkinje cell dendrites and dendritic spines 1 and depressed the Purkinje cell discharge ~. The CA terminals in adult rat cerebellum are apparently quite low in density'L Seiger and Olson reported CA terminals in the cerebellar cortex in newly born rats after monoamine oxidase inhibition 1'~. The recently developed glyoxylic acid histofluorescence method combined with Vibratome (GA method) was found to be more sensitive than the FA method for demonstrating catecholamines s. Thus, the rich innervation of CA terminals in the rat diencephalon, where a low density of CA terminals has been reported, can be readily made visible 9. Using the GA method, we carried out studies on the density and innervation pattern of CA terminals in the rat cerebellum with special attention given to the aspect of postnatal development. Twenty male and female Wistar rats were used in this study, including four aged 0 and 1 day, eight aged 4, 5, 6 and 8 days, four aged 4 and 5 weeks, and four adult rats over 6 months. Under pentobarbital sodium anesthesia (30 mg/kg, i.p.), the rats were perfused via ascending aorta or the left ventricle with ice-cold 2'j'J~glyoxylic acid solution in a Krebs-Ringer bicarbonate buffer adjusted to pH 7.0 with N a O H . One hundred milliliter was perfused during about 3 rain for adult and 4-5-week-old rats. For the rats younger than I week, 10 20 ml was perfused. Thereafter, the brain was quickly removed and stored in the ice-cold glyoxylic acid solution. The cerebellum was mounted on a metal bar and sectioned in parallel to the course of the folium in a thickness of 20-40 #m in a Vibratome. The sections were immersed for 5 rain in the ice-cold GA perfusion medium, and then spread out on the glass slides. After drying with a hairdryer, the sections were put on a hot plate at 100 °C for 10 rain. All preparations were examined using a fluorescence microscope equipped with Schott BG 12 as primary * To whom reprint requests showed be addressed.
357
Fig. 2. a: 4-day-old rat cerebellum. Innervation pattern of CA terminals is not so different from that of neonatal rat. In the Purkinje cell (P) and molecular layer (M), note that the density increases. E, external granular layer; X, artifact, x 64. b: 4-week-old rat cerebellum. In the Purkinje cell (P) and molecular layers (M), a moderately dense innervation of CA terminals is evident. The terminal arborization is less conspicuous than in the adult rat. G, granular layer; X, artifact, x 64. c: adult rat cerebellum. CA terminals are evident in all cortical layers. Most fibers run perpendicular to the cortical surface and branch in a T-division. Note dense innervation of fine varicose terminals in the molecular layer (M). In the upper part of the granular layer (G), fine varicose fibers are also visible. P, Purkinje cell layer. ,: 64.
filter and Zeiss 50 as secondary filter. F o r this study, the hemispheric portion of the cerebellum was m a i n l y used. In the rats aged 0 a n d I day, CA terminals were quite evident in the cerebellar cortex. CA varicosities at this stage were as fine as those in the molecular layer of the adult rat as a d o m i n a n t type. A moderately dense network of CA fibers with fine varicosities was observed in the undifferentiated internal g r a n u l a r layer which contained i m m a t u r e Purkinje cells a n d g r a n u l a r cells. A small n u m b e r of CA terminals was observed a r o u n d the Purkinje cells. There were no CA terminals observed in the external g r a n u l a r layer (Fig. la and b). In rats aged 4-5 days and in those a r o u n d the end of the first postnatal week, the i n n e r v a t i o n pattern of CA terminals was quite similar to that of neonatal rats. However, the density was more a b u n d a n t . As the Purkinje cells and the molecular layer developed, the CA terminals increased in density in both layers (Fig. 2a).
Fig. 1.0-day-old rat cerebellum, a: a moderately dense innervation of CA terminals is observed in the undifferentiated internal granular layer (Ul). A few varicose terminals are visible around the immature Purkinje cells. E, external granular layer, x 64. b: the same preparation as in (a) stained with toluidine blue. Note that the internal granular layer and Purkinje cell layer have not yet differentiated. Lightly stained cells with large somata are immature Purkinje cells and densely stained small cells which are intermingled are immature granule cells. ~ 64,
358
Fig. 3. Adult rat cerebellum, a: Purkinje cell and molecular layer. In the molecular layer, there are many CA terminals with fine varicosities, especially in the lower part. Here rather smooth fibers with elongated finer varicosities (~') can be clearly observed. P, Purkinje cell layer. :~ 160. b : granular layer. In the lower part of the granular layer, rather smooth fibers with elongated finer varicosities (~) are visible under high magnification. These fibers appear to continue distally to the fine varicosities, ~< 160.
359 In rats aged 4-5 weeks, a different pattern of innervation was observed and such was the same as is seen in adult animals. In the granular layer, there were dense CA terminals with finer varicosities and the fluorescence was weaker here than in the molecular layer. In the Purkinje cell and molecular layers, a moderately dense innervation of CA terminals was observed. These were the fibers with fine varicosities which were larger and more intense than those in the granular layer. These CA varicose fibers ran perpendicular to the cortical surface and branched in a T-division parallel to the surface. They appeared to be less arborized than in the adult (Fig. 2b), In adult rats, CA terminals were observed in all cortical layers (Fig. 2c), while such were hardly visible in the white matter. In the Purkinje cell and molecular layers (Figs. 2c and 3a), there were many CA terminals with fine varicosities, particularly in the lower part of molecular layer. The varicosities herein were of two different types: one type showed fibers with fine varicosities as seen in the cerebral cortex and the other revealed rather smooth fibers with elongated finer varicosities. The former were predominant, and most of them ran perpendicular to the cortical surface and then branched in a T-division running parallel to the course of the folium. In the lower part of this layer, a small number of rather smooth fibers was observed (Fig. 3a). In the granular layer (Fig. 2c), fibers with fine varicosities were seen mainly in the upper part of this layer, and some were closely apposed to the neuronal perikarya. In the lower part (Fig. 3b), rather smooth fibers with elongated finer varicosities were mainly observed. In this work~ a rich innervation of CA terminals was demonstrated in the cerebellum both in developing and adult rats. Using the GA method, these CA terminals can be visualized with a high degree of sensitivity. It should be noted that two different types of CA terminals are present in the adult rat cerebellum; one is composed of fibers with fine varicosities as in the cerebral cortex, another with rather smooth fibers with elongated finer varicosities. The former is probably identical to NA terminals as previously noted with the FA method1,6,13. The latter may be regarded as preterminal axons since they appeared to be in continuity with CA terminals of fine varicosities. Except for the above finding, the innervation pattern in the adult rat cerebellum is much the same as that reported in the chicken 12. Some CA fibers in the molecular layer ran in a pattern similar to that seen in parallel fibers. In the density of CA terminals, the rat cerebellum had less dense innervation than was seen in chicken and such may be due to the species difference as indicated by Mugnaini and Dahl v~. Nevertheless~ with the GA method, more numerous CA terminals were observed in the rat cerebellar cortex than when the FA method was applied. On the ontogeny of NA neurons, the first appearance in embryos showed a crown rump length of 1l ram, corresponding to day 14 of gestation 14. Using [3H]thymidine autoradiography, it was revealed that NA neurons in the LC began to differentiate on days 10-13 of gestation, while differentiation of cerebellar Purkinje cells commenced on days 14-15 v. Our present observations in the rat cerebellar cortex indicate that CA terminals innervation is present at birth and such findings are in agreement with data reported by Seiger and Olson ~5. Recently, Lor6n et al. 1° have, with a modified GA method, shown CA terminals in the cerebellar cortex of neonatal rats, without pharmacological pretreatment. During the first postnatal week CA
360 terminals were o b s e r v e d mostly in the internal g r a n u l a r layer. A s Purkinje cells and the m o l e c u l a r layer develop, these terminals increase in these two layers a n d at the f o u r t h p o s t n a t a l week the i n n e r v a t i o n p a t t e r n is identical to t h a t seen in the a d u l t rats. W o o d w a r d et al. 17 suggested that in rats the chemosensitivity of" Purkinje cell to p u t a t i v e n e u r o t r a n s m i t t e r s such as N A , adenosine cyclic m o n o p h o s p h a t e , g a m m a a m i n o b u t y r i c acid etc., precedes the onset o f synaptogenesis which occurred on the third p o s t n a t a l day. The present findings provide m o r p h o l o g i c a l evidence for the existence o f c a t e c h o l a m i n e r g i c terminals at birth. These terminals m a y be a l r e a d y in a functional state at the time when synapses have n o t yet developed. Recently, West and Del C e r r o t6 d e m o n s t r a t e d t h a t the synapses are recognizable even on the 19th d a y o f e m b r y o n i c stage in the m o l e c u l a r layer o f the vermis. They considered most o f these early synapses to be derived from i m m a t u r e parallel fibers and climbing fibers and did n o t refer to the C A terminals. The possibility that some o f these early synapses m a y be C A t e r m i n a l s has to be considered. T h e r e is an interesting a s s u m p t i o n t h a t C A terminals influence the d e v e l o p m e n t o f other neuronal elements in the early p o s t n a t a l period. M a e d a et al. H reported that destruction o f the LC, the origin o f N A terminals in the cerebral a n d cerebellar cortex in n e w b o r n rats, results in an i m m a t u r e d e v e l o p m e n t o f n e u r o n a l elements o f the cerebral cortex. The observation t h a t the a p p e a r a n c e o f C A terminals antedates the m a t u r a t i o n o f Purkinje cells suggests the role o f C A terminals in m a t u r a t i o n o f neuronal elements in the cerebellar cortex as well as in the cerebral cortex. This study was s u p p o r t e d by G r a n t 157059 from the M i n i s t r y o f E d u c a t i o n , Science a n d Culture, J a p a n . T h a n k s are due to M. O h a r a for assistance with the manuscript.
1 Bloom, F. E., Hoffer, B. J. and Siggins, G. R., Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. !. Localization of the fibers and their synapses, Brain Research, 25 (1971) 501-521. 2 Chu, N, S. and Bloom, F. E., The catecholamine containing neurons in the cat dorsolateral pontine tegmentum: distribution of the cell bodies and some axonal projections, Brain Research, 66 (1974) 1-21. 3 Falck, B., Hillarp, N.-A., Thieme, G. and Torp, A., Fluorescence of catechotamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 4 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervOus system. IV. Distribution of monoamine nerve terminals in the central nervous system, Acta physiol, scand., 64, Suppl. 247 (1965) 39-85. 5 Hoffer, B. J., Siggins, G. R., Oliver, A. P. and Bloom, F. E,, Activation of the pathway from locus coeruleus to rat cerebellar Purkinje neurons: pharmacological evidence for noradrenergic central inhibition, J. Pharmacol. exp. Ther., 184 (1973) 553-569. 6 H6kfelt, F. and Fuxe, K., Cerebellar monoamine nerve terminals, a new type of afferent fibers to the cortex cerebelli, Exp. Brain Res., 9 (1969) 63-72. 7 Lauder, J. M. and Bloom, F. E., Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat, I. Cell differentiation, J. comp. Neurol., 155 (1974) 469-482. 8 Lindvall, O. and Bj6rklund, A., The glyoxylic acid fluorescence histochemistry method: a detailed account of the methodology for the visualization of central catecholamine neurons, Histochemistry, 39 (1974) 97-127. 9 Lindvall, O., Bj6rklund, A., Nobin, A. and Steveni, U., The adrenergic innervation of the rat
361
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I1 12 13 14 15 16 17
thalamus as revealed by the glyoxylic acid fluorescence method, J. comp. Neurol., 154 (1974) 317 348. Lor6n, 1., Bj6rklund, A. and Lindvall, O., The catecholamine systems in the developing rat brain : improved visualization by a modified glyoxylic acid formaldehyde method, Brain Research, 117 (1976) 313-318. Maeda, T., Tohyama, M. and Shimizu, N., Modification of postnatal development of neocortex in rat brain with experimental deprivation of locus coeruleus, Brain Research, 70 (1974) 515-520. Mugnaini, E. and DaN, A. L., Mode of distribution of aminergic fibers in the cerebellar cortex of the chicken, J. comp. Neurol., 162 (1975) 417--432. Olson, L. and Fuxe, K., On the projection from the locus coeruleus noradrenaline neurons: the cerebellar innervation, Brain Research, 28 (1971) 165-171. Olson, L. and Seiger /~., Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Geseh., 137 (1972) 301 316. Seiger, /~. and Olson, L., Late prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Gesch., 140 (1973) 281 318. West, M. J. and Del Cerro, M., Early formation of synapses in the molecular layer of the fetal rat cerebellum, J. comp. Neurol., 165 (1976) 137-160. Woodward, D. J., Hoffer, B. J., Siggins, G. R. and Bloom, F. E., The ontogenetic development of synaptic junctions, synaptic activation and responsiveness to neurotransmitter substances in rat cerebellar Purkinje cells, Brain Research, 34 (1971) 73-97.
355
Elsevier/North-Holland Biomedical Press
Catecholaminergic terminals in the developing and adult rat cerebellum
TETSURO YAMAMOTO, MASATSUNE ISHIKAWA and CHIKAKO TANAKA* Department of Physiolog.v, htstitute Jbr Brahz Research, { M.I.) Department o[" Neurosurgery, Faculty qf Medichle, Kyoto Ulliversity and (C.T.) Department o[" Pharmacology, Kobe University School of Medicine, Kobe (Japa~t)
(Accepted May I lth, 1977)
With the formaldehyde histofluorescence method of Falck and Hillarp (FA method) a, catecholaminergic (CA), most probably noradrenergic (NA), terminals have been demonstrated in the cerebellar cortex 1,4,~, and they were found to be derived from the NA neurons in the locus coeruleus (LC) la and other pontine NA neurons e. It has also been reported that NA terminals in the cerebellar cortex made synaptic contacts with Purkinje cell dendrites and dendritic spines 1 and depressed the Purkinje cell discharge ~. The CA terminals in adult rat cerebellum are apparently quite low in density'L Seiger and Olson reported CA terminals in the cerebellar cortex in newly born rats after monoamine oxidase inhibition 1'~. The recently developed glyoxylic acid histofluorescence method combined with Vibratome (GA method) was found to be more sensitive than the FA method for demonstrating catecholamines s. Thus, the rich innervation of CA terminals in the rat diencephalon, where a low density of CA terminals has been reported, can be readily made visible 9. Using the GA method, we carried out studies on the density and innervation pattern of CA terminals in the rat cerebellum with special attention given to the aspect of postnatal development. Twenty male and female Wistar rats were used in this study, including four aged 0 and 1 day, eight aged 4, 5, 6 and 8 days, four aged 4 and 5 weeks, and four adult rats over 6 months. Under pentobarbital sodium anesthesia (30 mg/kg, i.p.), the rats were perfused via ascending aorta or the left ventricle with ice-cold 2'j'J~glyoxylic acid solution in a Krebs-Ringer bicarbonate buffer adjusted to pH 7.0 with N a O H . One hundred milliliter was perfused during about 3 rain for adult and 4-5-week-old rats. For the rats younger than I week, 10 20 ml was perfused. Thereafter, the brain was quickly removed and stored in the ice-cold glyoxylic acid solution. The cerebellum was mounted on a metal bar and sectioned in parallel to the course of the folium in a thickness of 20-40 #m in a Vibratome. The sections were immersed for 5 rain in the ice-cold GA perfusion medium, and then spread out on the glass slides. After drying with a hairdryer, the sections were put on a hot plate at 100 °C for 10 rain. All preparations were examined using a fluorescence microscope equipped with Schott BG 12 as primary * To whom reprint requests showed be addressed.
357
Fig. 2. a: 4-day-old rat cerebellum. Innervation pattern of CA terminals is not so different from that of neonatal rat. In the Purkinje cell (P) and molecular layer (M), note that the density increases. E, external granular layer; X, artifact, x 64. b: 4-week-old rat cerebellum. In the Purkinje cell (P) and molecular layers (M), a moderately dense innervation of CA terminals is evident. The terminal arborization is less conspicuous than in the adult rat. G, granular layer; X, artifact, x 64. c: adult rat cerebellum. CA terminals are evident in all cortical layers. Most fibers run perpendicular to the cortical surface and branch in a T-division. Note dense innervation of fine varicose terminals in the molecular layer (M). In the upper part of the granular layer (G), fine varicose fibers are also visible. P, Purkinje cell layer. ,: 64.
filter and Zeiss 50 as secondary filter. F o r this study, the hemispheric portion of the cerebellum was m a i n l y used. In the rats aged 0 a n d I day, CA terminals were quite evident in the cerebellar cortex. CA varicosities at this stage were as fine as those in the molecular layer of the adult rat as a d o m i n a n t type. A moderately dense network of CA fibers with fine varicosities was observed in the undifferentiated internal g r a n u l a r layer which contained i m m a t u r e Purkinje cells a n d g r a n u l a r cells. A small n u m b e r of CA terminals was observed a r o u n d the Purkinje cells. There were no CA terminals observed in the external g r a n u l a r layer (Fig. la and b). In rats aged 4-5 days and in those a r o u n d the end of the first postnatal week, the i n n e r v a t i o n pattern of CA terminals was quite similar to that of neonatal rats. However, the density was more a b u n d a n t . As the Purkinje cells and the molecular layer developed, the CA terminals increased in density in both layers (Fig. 2a).
Fig. 1.0-day-old rat cerebellum, a: a moderately dense innervation of CA terminals is observed in the undifferentiated internal granular layer (Ul). A few varicose terminals are visible around the immature Purkinje cells. E, external granular layer, x 64. b: the same preparation as in (a) stained with toluidine blue. Note that the internal granular layer and Purkinje cell layer have not yet differentiated. Lightly stained cells with large somata are immature Purkinje cells and densely stained small cells which are intermingled are immature granule cells. ~ 64,
358
Fig. 3. Adult rat cerebellum, a: Purkinje cell and molecular layer. In the molecular layer, there are many CA terminals with fine varicosities, especially in the lower part. Here rather smooth fibers with elongated finer varicosities (~') can be clearly observed. P, Purkinje cell layer. :~ 160. b : granular layer. In the lower part of the granular layer, rather smooth fibers with elongated finer varicosities (~) are visible under high magnification. These fibers appear to continue distally to the fine varicosities, ~< 160.
359 In rats aged 4-5 weeks, a different pattern of innervation was observed and such was the same as is seen in adult animals. In the granular layer, there were dense CA terminals with finer varicosities and the fluorescence was weaker here than in the molecular layer. In the Purkinje cell and molecular layers, a moderately dense innervation of CA terminals was observed. These were the fibers with fine varicosities which were larger and more intense than those in the granular layer. These CA varicose fibers ran perpendicular to the cortical surface and branched in a T-division parallel to the surface. They appeared to be less arborized than in the adult (Fig. 2b), In adult rats, CA terminals were observed in all cortical layers (Fig. 2c), while such were hardly visible in the white matter. In the Purkinje cell and molecular layers (Figs. 2c and 3a), there were many CA terminals with fine varicosities, particularly in the lower part of molecular layer. The varicosities herein were of two different types: one type showed fibers with fine varicosities as seen in the cerebral cortex and the other revealed rather smooth fibers with elongated finer varicosities. The former were predominant, and most of them ran perpendicular to the cortical surface and then branched in a T-division running parallel to the course of the folium. In the lower part of this layer, a small number of rather smooth fibers was observed (Fig. 3a). In the granular layer (Fig. 2c), fibers with fine varicosities were seen mainly in the upper part of this layer, and some were closely apposed to the neuronal perikarya. In the lower part (Fig. 3b), rather smooth fibers with elongated finer varicosities were mainly observed. In this work~ a rich innervation of CA terminals was demonstrated in the cerebellum both in developing and adult rats. Using the GA method, these CA terminals can be visualized with a high degree of sensitivity. It should be noted that two different types of CA terminals are present in the adult rat cerebellum; one is composed of fibers with fine varicosities as in the cerebral cortex, another with rather smooth fibers with elongated finer varicosities. The former is probably identical to NA terminals as previously noted with the FA method1,6,13. The latter may be regarded as preterminal axons since they appeared to be in continuity with CA terminals of fine varicosities. Except for the above finding, the innervation pattern in the adult rat cerebellum is much the same as that reported in the chicken 12. Some CA fibers in the molecular layer ran in a pattern similar to that seen in parallel fibers. In the density of CA terminals, the rat cerebellum had less dense innervation than was seen in chicken and such may be due to the species difference as indicated by Mugnaini and Dahl v~. Nevertheless~ with the GA method, more numerous CA terminals were observed in the rat cerebellar cortex than when the FA method was applied. On the ontogeny of NA neurons, the first appearance in embryos showed a crown rump length of 1l ram, corresponding to day 14 of gestation 14. Using [3H]thymidine autoradiography, it was revealed that NA neurons in the LC began to differentiate on days 10-13 of gestation, while differentiation of cerebellar Purkinje cells commenced on days 14-15 v. Our present observations in the rat cerebellar cortex indicate that CA terminals innervation is present at birth and such findings are in agreement with data reported by Seiger and Olson ~5. Recently, Lor6n et al. 1° have, with a modified GA method, shown CA terminals in the cerebellar cortex of neonatal rats, without pharmacological pretreatment. During the first postnatal week CA
360 terminals were o b s e r v e d mostly in the internal g r a n u l a r layer. A s Purkinje cells and the m o l e c u l a r layer develop, these terminals increase in these two layers a n d at the f o u r t h p o s t n a t a l week the i n n e r v a t i o n p a t t e r n is identical to t h a t seen in the a d u l t rats. W o o d w a r d et al. 17 suggested that in rats the chemosensitivity of" Purkinje cell to p u t a t i v e n e u r o t r a n s m i t t e r s such as N A , adenosine cyclic m o n o p h o s p h a t e , g a m m a a m i n o b u t y r i c acid etc., precedes the onset o f synaptogenesis which occurred on the third p o s t n a t a l day. The present findings provide m o r p h o l o g i c a l evidence for the existence o f c a t e c h o l a m i n e r g i c terminals at birth. These terminals m a y be a l r e a d y in a functional state at the time when synapses have n o t yet developed. Recently, West and Del C e r r o t6 d e m o n s t r a t e d t h a t the synapses are recognizable even on the 19th d a y o f e m b r y o n i c stage in the m o l e c u l a r layer o f the vermis. They considered most o f these early synapses to be derived from i m m a t u r e parallel fibers and climbing fibers and did n o t refer to the C A terminals. The possibility that some o f these early synapses m a y be C A t e r m i n a l s has to be considered. T h e r e is an interesting a s s u m p t i o n t h a t C A terminals influence the d e v e l o p m e n t o f other neuronal elements in the early p o s t n a t a l period. M a e d a et al. H reported that destruction o f the LC, the origin o f N A terminals in the cerebral a n d cerebellar cortex in n e w b o r n rats, results in an i m m a t u r e d e v e l o p m e n t o f n e u r o n a l elements o f the cerebral cortex. The observation t h a t the a p p e a r a n c e o f C A terminals antedates the m a t u r a t i o n o f Purkinje cells suggests the role o f C A terminals in m a t u r a t i o n o f neuronal elements in the cerebellar cortex as well as in the cerebral cortex. This study was s u p p o r t e d by G r a n t 157059 from the M i n i s t r y o f E d u c a t i o n , Science a n d Culture, J a p a n . T h a n k s are due to M. O h a r a for assistance with the manuscript.
1 Bloom, F. E., Hoffer, B. J. and Siggins, G. R., Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. !. Localization of the fibers and their synapses, Brain Research, 25 (1971) 501-521. 2 Chu, N, S. and Bloom, F. E., The catecholamine containing neurons in the cat dorsolateral pontine tegmentum: distribution of the cell bodies and some axonal projections, Brain Research, 66 (1974) 1-21. 3 Falck, B., Hillarp, N.-A., Thieme, G. and Torp, A., Fluorescence of catechotamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 4 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervOus system. IV. Distribution of monoamine nerve terminals in the central nervous system, Acta physiol, scand., 64, Suppl. 247 (1965) 39-85. 5 Hoffer, B. J., Siggins, G. R., Oliver, A. P. and Bloom, F. E,, Activation of the pathway from locus coeruleus to rat cerebellar Purkinje neurons: pharmacological evidence for noradrenergic central inhibition, J. Pharmacol. exp. Ther., 184 (1973) 553-569. 6 H6kfelt, F. and Fuxe, K., Cerebellar monoamine nerve terminals, a new type of afferent fibers to the cortex cerebelli, Exp. Brain Res., 9 (1969) 63-72. 7 Lauder, J. M. and Bloom, F. E., Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat, I. Cell differentiation, J. comp. Neurol., 155 (1974) 469-482. 8 Lindvall, O. and Bj6rklund, A., The glyoxylic acid fluorescence histochemistry method: a detailed account of the methodology for the visualization of central catecholamine neurons, Histochemistry, 39 (1974) 97-127. 9 Lindvall, O., Bj6rklund, A., Nobin, A. and Steveni, U., The adrenergic innervation of the rat
361
10
I1 12 13 14 15 16 17
thalamus as revealed by the glyoxylic acid fluorescence method, J. comp. Neurol., 154 (1974) 317 348. Lor6n, 1., Bj6rklund, A. and Lindvall, O., The catecholamine systems in the developing rat brain : improved visualization by a modified glyoxylic acid formaldehyde method, Brain Research, 117 (1976) 313-318. Maeda, T., Tohyama, M. and Shimizu, N., Modification of postnatal development of neocortex in rat brain with experimental deprivation of locus coeruleus, Brain Research, 70 (1974) 515-520. Mugnaini, E. and DaN, A. L., Mode of distribution of aminergic fibers in the cerebellar cortex of the chicken, J. comp. Neurol., 162 (1975) 417--432. Olson, L. and Fuxe, K., On the projection from the locus coeruleus noradrenaline neurons: the cerebellar innervation, Brain Research, 28 (1971) 165-171. Olson, L. and Seiger /~., Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Geseh., 137 (1972) 301 316. Seiger, /~. and Olson, L., Late prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Gesch., 140 (1973) 281 318. West, M. J. and Del Cerro, M., Early formation of synapses in the molecular layer of the fetal rat cerebellum, J. comp. Neurol., 165 (1976) 137-160. Woodward, D. J., Hoffer, B. J., Siggins, G. R. and Bloom, F. E., The ontogenetic development of synaptic junctions, synaptic activation and responsiveness to neurotransmitter substances in rat cerebellar Purkinje cells, Brain Research, 34 (1971) 73-97.