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Out-of-phase development of dendritic spines in locus coeruleus and nucleus raphe dorsalis in rats of three age groups
Developmental Brain Research, 4 (1982) 487-490 Elsevier Biomedical Press
487
Short Communications
Out-of-phase development of dendritic spines in locus coeruleus and nucleus raphe dorsalis in rats of three age groups P. J. MORGANE, T. KEMPER, L. CINTRA* and S. DIAZ-CINTRA* Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545 (U.S.A.) (Accepted March 31st, 1982) Key words: locus coeruleus - - nucleus raphe dorsalis - - dendritic spines - - dendritic plasticity - Golgi studies - - morphometric studies - - amine nuclei - - age changes in dendritic spines
We have previously carried out morphometric Golgi studies of the 3 cell types, ovoid, multipolar and fusiform, which we have identified in the nucleus raphe dorsalis and nucleus locus co~ruleus of rats. In the present study we have compared the dendritic spine densities of cells of the nucleus raphe dorsalis and nucleus locus cozruleus during development from 30 to 90 and 90 to 220 days of age in rats. Between 30 and 90 and 90 and 220 days of age dendritic spines on both primary and secondary dendrites of all 3 cell types in each nucleus showed exact out-of-phase development between the two nuclei though all 3 cell types within each nucleus were in phase with each other. Thus, all 3 cell types in the nucleus raphe dorsalis between 30 and 90 days showed a significant increase in spines on primary and secondary dendrites while all 3 cell types in the locus coeruleus during this period showed a significant decrease in primary and secondary dendritic spines. Between 90 and 220 days all cell types in the locus coeruleus showed a significant increase in dendritic spines while in the nucleus raphe dorsalis there was a significant decrease in dendritic spines on 2 of the 3 cell types, the exception being a non-significant decrease in primary dendritic spines in the ovoid cells. This out-of-phase development of dendritic spines between locus coeruleus and dorsal raphe may have important functional implications since the dorsal raphe and locus coeru leus are mutually interactive and are involved, via their widespread projection systems, in many biochemical, physiological and bzhavioral regulations. T h e nucleus r a p h e dorsalis a n d locus coeruleus c o m p r i s e the m a j o r s e r o t o n i n a n d n o r e p i n e p h r i n e cell g r o u p s in the b r a i n a n d have been shown to be i m p l i c a t e d in a wide variety o f b e h a v i o r s a n d physiological activities. Histofluorescence m a p p i n g , i m m u n o h i s t o c h e m i c a l analysis a n d a x o n t r a n s p o r t studies have revealed w i d e s p r e a d p r o j e c t i o n s f r o m b o t h o f these nuclei to the spinal cord, lower b r a i n stem, d i e n c e p h a l o n a n d telencephalon, including a l m o s t all areas o f the c e r e b r a l cortex6,7A1,20, zT. The nucleus r a p h e dorsalis has been r e p o r t e d to consist largely, b u t n o t exclusively, o f s e r o t o n i n - c o n t a i n i n g n e u r o n s while the locus coeruleus has been shown to consist p r i m a r i l y o f n o r e p i n e p h r i n e c o n t a i n i n g cells (refs. 1, 2, 4, 17, 24, 30, 37). C o m b i n e d histofluorescence a n d G o l g i studies have revealed 3 cell types in b o t h o f these nuclei. T h e studies o f Pfister a n d Danner2~,26 indicate t h a t m o n o a m i n e fluorescence is confined to the m u l t i p o l a r elements. H o w e v e r ,
Steinbusch et al. a4, using i m m u n o c y t o c h e m i c a l m e t h o d s , f o u n d t h a t at least 2 cell types in the nucleus r a p h e dorsalis were s e r o t o n i n - c o n t a i n i n g , p a r ticularly the m e d i u m sized fusiform a n d large multip o l a r neurons. A host o f fluorescence, a x o n transport, i m m u n o c y t o c h e m i c a l , a n d physiological studies have revealed interconnections between locus coeruleus a n d r a p h e nuclei3,11-13,17-19,22,28,30, 37. These reciprocal relations have been widely interp r e t e d as key factors in regulating m e t a b o l i s m o f the m o n o a m i n e s s e r o t o n i n a n d n o r e p i n e p h r i n e in these nuclei a n d in their p r o j e c t i o n fields t h e r e b y playing a role in sleep--waking oscillations a n d v a r i o u s other behaviors14-16,23,29,32,33. Since the r a p h e a n d locus coeruleus have been i m p l i c a t e d in m a n y electrophysiological a n d behavioral activities a n d r e c i p r o c a l l y interact, it is o f special interest to s t u d y t h e m d e v e l o p m e n t a l l y in t e m p o r a l relation to each other. A c c o r d i n g l y , we
* On leave from Instituto de Investigaciones Biom6dicas, Universidad Nacional Aut6noma de M6xico, Apartado Postal 70228, Mexico 20, Mexico. 0165-3806/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
488 have used multiple quantitative parameters to examine all 3 cell types, i.e. the ovoid, fusiform and multipolar, which we have identified in each nuclear formation 5,s,9. In rats of 30, 90 and 220 days of age we have previously described the normal morphometric development of these cell types in the nucleus raphe dorsalis 8 and recently have carried out similar cytomorphometric analyses in the locus coeruleus:L In the present studies we are comparing dendritic spine development in the nucleus raphe dorsalis and locus coeruleus across 3 age groups of animals. In these experiments we have used 5 Charles River C.D. Sprague-Dawley rats of each of 3 age groups (30, 90, 220 days of age) bred and raised in our laboratories. In each of the 15 animals the nucleus locus coeruleus and nucleus raphe dorsalis were studied from sections taken from the same rat. The rapid Golgi technique was applied in each instance as described in our previous studiesS, 8,9. As in our previous studies we classified the cells in both nucleus raphe dorsalis and locus coeruleus into 3 types, ovoid, fusiform and multipolar. A total of one hundred cells were selected randomly from all parts of each nucleus in a total of 5 animals at each age as follows: at each age 35 fusiform, 40 multipolar and 35 ovoid cells were selected representing the first consecutively encountered well-impregnated neurons in each sampling area. The boundaries of the nucleus raphe dorsalis were defined similar to those described by Steinbusch et al. 34 while the boundaries of the locus coeruleus were established similar to those described by Swanson as. For each cell the following measures were taken: major and minor axes, number, diameter and linear extent of primary and secondary dendrites and number of synaptic spines on the primary and secondary dendrites and on the cell bodies. Age-related changes other than for synaptic spines were infrequent. O f 96 of these comparisons across these various parameters only 7 achieved statistical significance. In the nucleus raphe dorsalis all 3 cell types, ovoid, fusiform and multipolar, showed an increase in synaptic spines on both primary and secondary dendrites between 30 and 90 days followed by a decrease between 90 and 220 days of age (Fig. 1). In the locus coeruleus diametrically opposite age-related changes were noted with. all 3 cell types showing a decrease in synaptic spines on both
5PINE DENSITY PRIMARY DENDRITE
50~90 90L220
~GE ,~doys)
SECONDARY DENDRITE 30-90 90-220
Ft./S/FORM ~ L z
+ 75 +50~
+ 50-
+32% +25-
I
RAPHE DORSALtS
*18%
• 16%
~LOCUS COERULEUS 0
-25-
-50
MULTI,°OL~I? C,ELL +50-
4-254
o-
1-24% H
~* +18%
-19%
-25-
-2B% -50
OVOID CELL
+75.
+50
+86%
+67%
-
+34% +25-
15%
O--9% - 25-39%
-50
-45%
Fig. 1. Graph showing percent change in dendritic spines across two age periods in primary and secondary dendrites of nucleus raphe dorsalis and nucleus locus coeruleus. Significance levels: * P 0.05; ** P " 0.01 ; *** P -J 0.001. primary and secondary dendrites between 30 and 90 days followed by an increase between 90 and 220 days. As shown in Fig. 1, of these age-related changes in both nuclei, all but one achieved statistical significance. In the case of perisomatic spines there were significant changes in 8 out of 12 measures (3 cell types, 2 age groups x 2 nuclei) and these were exactly in register with the changes in dendritic spines. These studies show that within the nucleus raphe dorsalis and locus coeruleus all 3 cell types showed synchronous age-related increases and decreases in synaptic spines on primary and secondary dendrites and on the cell body which, when the two nuclei
489 were compared, were in diametrically opposite directions. In our studies o n the dendritic spine develo p m e n t in the nucleus raphe dorsalis we find an agerelated increase followed by a decrease in dendritic spines which is similar to patterns described by L u n d 21 and Felten and C u m m i n g s 10. N o previous studies have assessed such dendritic development in the locus coeruleus, t h o u g h Sievers 31 described early increases in locus coeruleus dendritic spines followed by a decrease t h r o u g h 30 days o f age. The significance o f the synchrony of neuronal development within each of these nuclei can only be speculated on as the timing and location o f the afferent projections and local circuits within these nuclei are still largely undetermined. According to the c o m b i n e d Nissl, histochemical and Golgi studies of Pfister and D a n n e r ~5,26 on the rat nucleus raphe dorsalis and locus coeruleus, the multipolar cell is monoaminergic, the fusiform cell is a n o n - m o n o aminergic projection cell and the ovoid cell a local circuit cell. Thus, 3 apparently different types of cells in each nucleus are showing simultaneous development o f synaptic spines at 3 different locations, the primary and secondary dendrites and on the cell body. However, Steinbusch et al. 34, 1 Aghajanian, G. K., Wang, R. Y. and Baraban, J., Serotonergic and non-serotonergic neurons of the dorsal raphe: reciprocal changes in firing induced by peripheral nerve stimulation, Brain Research, 153 (1978) 169-175. 2 Albanese, A. and Butcher, L. L., Acetylcholinesterase and catecholamine distribution in the locus coeruleus of the rat, Brain Res. Bull., 5 (1980) 127-134. 3 Baraban, J. and Aghajanian, G. K., Noradrenergic innervation of serotonergic neurons in the dorsal raphe: demonstration by electron microscopic autoradiography, Brain Research, 204 (1981) 1-11. 4 Belin, M. F., Aguera, M., Tappaz, M., Jouvet, M. and Pujol, J. F., Identification des neurones accumulant le GABA dans le noyau dorsal du raphe, C.R. Acad. Sci. (Paris), 287 (1978) 865-869. 5 Cintra, L., Diaz-Cintra, S., Kemper, T. and Morgane, P. J., Nucleus locus coeruleus: a morphometric Golgi study in rats of three age groups, Brain Research, (in press). 6 Dahlstr6m, 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. 7 Dahlstr6m, A. and Fuxe, K., Evidence for the existence. of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta physioL scand., 64 Suppl., 247 (1965) 1-36. 8 Diaz-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and
using cytoarchitectonic and immunohistochemical methods, noted that serotonin immunoreactive neurons closely resemble both the medium-sized fusif o r m and large multipolar neurons observed in normal material. Whether the synchrony o f dendritic spine develo p m e n t on all cell types in each nucleus represents a specific or non-specific effect o f afferent projections to all these synaptic locations in all cell types in these nuclei or is a response to the development o f local circuits within each nucleus remains to be determined. The inverse relationship between spine develo p m e n t in the nucleus raphe dorsalis and locus coeruleus is a finding one would not anticipate from their apparent close anatomical and neurophysiological interactions. It will be interesting in physiological experiments to determine the significance o f the synaptic spine increase in the nucleus raphe dorsalis which antedates that shown by the locus coeruleus. Supported by N I H G r a n t HD-06364, N I C H H D , N S F G r a n t B N S 79-22507, F o g a r t y International Fellowship G r a n t 5 F05 T w o 2693-02, and Fellowship G r a n t 27234 C O N A C Y T (Mexico).
9
10 11
12 13
14 15
Morgane, P. J., Nucleus raphe dorsalis: a morphometric Golgi study in rats of three age groups, Brain Research, 207 (1981) 1-16. Diaz-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and Morgane, P. J., The effects of protein deprivation on the nucleus raphe dorsalis: a morphometric Golgi study in rats of three age groups, Brain Research, 221 (1981) 243-255. Felten, D. L. and Cummings, J. P., Ontogeny of medullary raphe nuclei in the rabbit brain stem: a Golgi study, Brain Res. Bull., 6 (1981) 413--425. 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) 37-85. Grzanna, R. and Molliver, M. E., The locus coeruleus in the rat: an immunohistochemical delineation, Neuroscience, 5 (1980) 21-40. Jones, G., Segal, M., Foote, S. L. and Bloom, F. E., Locus coeruleus neurons in freely moving rats exhibit pronounced alterations of discharge rate during sensory stimulation and stages of sleep-wake cycles. In E. Usdin, I. J. Kopin and J. Barchas (Eds.), Catecholamines Basic and Clinical Frontiers, Pergamon Press, New York, 1979, pp. 643-645. Jouvet, M., The role of monoamines and acetylcholinecontaining neurons in the regulation of the sleep-waking cycle, Rev. Physiol., 64 (1972) 166-307. Kostowski, W., Two noradrenergic systems in the brain
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18
19
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21 22
23
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and their interactions with other monoaminergic neurons, Pol. J. Pharmaeol. Pharm., 31 (1979) 425 436. Kostowski, W., Samanin, R., Bareggi, S. R., Marc, V., Garattini, S. and Valzelli, L., Biochemical aspects of the interaction between midbrain raphe and locus coeruIeus in the rat, Brain Research, 82 (1974) 178-182. Levitt, P. and Moore, R. Y., Developmental organization of raphe serotonin neurons groups in the rat, Anat. Embryol., 154 (1978) 241 251. Levitt, P. and Moore, R. Y., Origin and organization of brainstem catecholamine innervation in the rat, J. comp. NeuroL, 186 (1979) 505-528. Lidbrink, P. and Jonsson, G., Specificity of 6-hydroxydopamine induced lesions of the ascending locus coeruleus noradrenaline neurons after intracranial injections. In G. Jonsson, T. Malmfors and C. Sachs (Eds.), Chemical Tools in Catecholamine Research, Vol. 1, North-Holland, Amsterdam, 1975, pp. 267 271. Lidov, H. G. W., Grzanna, R. and Molliver, M. E., The serotonin innervation of the cerebral cortex in the rat - an immunohistochemical analysis, Neuroscience, 5 (1980) 207--227. Lund, R. D., Development and Plastkqty o f the Brain, Oxford University Press, New York, 1978, pp. 166-180. Morgane, P. J. and Jacobs, M. S., Raphe projections to the locus coeruleus in the rat, Brain Res. Bull., 4 (1979) 519 534. Morgane, P. J. and Stern, W. C., Chemical anatomy of brain circuits in relation to sleep and wakefulness. In E. Weitzman (Ed.), Advances in Sleep Research, VoL I, Spectrum Publications, New York, 1974, pp. 1-131. Ochi, J. and Shimizu, K., Occurrence of dopamine-containing neurons in the midbrain rapfie nuclei of the rat, Neurosci. Lett., 8 (1978) 317-320. Pfister, V. C. and Danner, H., Fluorescence histochemical and neurohistological investigations on the locus coeruleus of the rat, Acts Histochem., 66 (1980) 101-113. Pfister, V. C. and Danner, H., Fluorescence histochemical and neurohistological investigations on the nucleus raphe dorsalis of the rat, Aeta Histoehem., 66 (1980) 253-261. Pickel, V. M., Segal, M. and Bloom, F. E., A radioautographic study of the efferent pathways of the nucleus locus coeruleus, J. comp. Neurol., 155 (1974) 15~41.
28 Pickel, V. M., Joh, T. H. and Reis. I)..1+, Imlnunocylochemical evidence for serolooergic innervalkm of uoradrenergic neurons in nucleus locus coeruteu~ I~, S. Garattini, J. F. Pujol and R. Samanin (Eds.), /merac/l~m, Betweett Putative i~eln'olr~lnsnliller.s l'11 /he l]l'(lilh Rztvcli Press, New York, 1978, pp. 369+382. 29 Pujol, d. F., Keane, P., McRae, A., I.cwis, t',+ 19. and Renaud, B., Biochemical evidence for serotonergic control of the locus coeruleus. In S. Garattini, J. 12. Ptuol and R. Saminin (Eds.), lnleraclitms Between Ptllali~., ~\em'otraHsmitters in the Brain, Raven Press, Ne~ York, i97~, pp. 401 410. 30 Saavedra, J. M., Grobecker, H. and Zivin, J.+ (atccholamines in the raphe nuclei of the rat, Brain Research, 114 (1976) 339 345. 31 Sievers, J., Lolova, I., Jenner, S., Klemm, H. P. and Sievers, H., Morphological and biochemical studies on the ontogenesis of the nucleus locus coeruleus, Bihliotheca Anatomies, 19 (1981) 52-130. 32 Smith, B. and Sweet, W., Monoaminergic regulation of central nervous system function: It. Noradrenergic sys+ tems, Neurosurger.v, 3 (1978) 109- 119. 33 Smith, B. and Sweet, W., Monoaminergic regulation of central nervous system function: 11. Serotonergic systems, Neurosurgeo,, 3 (1978)257 272. 34 Steinbusch, H. W. M., Nieuwenhuys, R., Verhofstad. A. A. J. and Van Der Kooy, D., The nucleus raphe dorsalis of the rat and its projection upon the caudato-putamen. A combined cytoarchitectonic, immunohistochemical and retrograde transport study, .I. Physiol. (Paris'), 77 (198 I 157 174+ 35 Swanson, L W., 1he locus coeruleus: a cytoarchitectonic, Golgi and immunohistochemical study in the albino rat+ Brain Research, I10 (1976) 39 56. 36 Swanson, L. W. and Hartman, B. K., ][he central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine fi-hydroxylase as a marker, J. eomp. Neural., 163 (1975) 467 506. 37 Versteeg, D. H. G., Van der Gugten, J., de Jong, W. and Palkovits, M., Regional concentrations of noradrenaline and dopamine in rat brain, Brain Research, ll3 (19761 563 574.
487
Short Communications
Out-of-phase development of dendritic spines in locus coeruleus and nucleus raphe dorsalis in rats of three age groups P. J. MORGANE, T. KEMPER, L. CINTRA* and S. DIAZ-CINTRA* Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545 (U.S.A.) (Accepted March 31st, 1982) Key words: locus coeruleus - - nucleus raphe dorsalis - - dendritic spines - - dendritic plasticity - Golgi studies - - morphometric studies - - amine nuclei - - age changes in dendritic spines
We have previously carried out morphometric Golgi studies of the 3 cell types, ovoid, multipolar and fusiform, which we have identified in the nucleus raphe dorsalis and nucleus locus co~ruleus of rats. In the present study we have compared the dendritic spine densities of cells of the nucleus raphe dorsalis and nucleus locus cozruleus during development from 30 to 90 and 90 to 220 days of age in rats. Between 30 and 90 and 90 and 220 days of age dendritic spines on both primary and secondary dendrites of all 3 cell types in each nucleus showed exact out-of-phase development between the two nuclei though all 3 cell types within each nucleus were in phase with each other. Thus, all 3 cell types in the nucleus raphe dorsalis between 30 and 90 days showed a significant increase in spines on primary and secondary dendrites while all 3 cell types in the locus coeruleus during this period showed a significant decrease in primary and secondary dendritic spines. Between 90 and 220 days all cell types in the locus coeruleus showed a significant increase in dendritic spines while in the nucleus raphe dorsalis there was a significant decrease in dendritic spines on 2 of the 3 cell types, the exception being a non-significant decrease in primary dendritic spines in the ovoid cells. This out-of-phase development of dendritic spines between locus coeruleus and dorsal raphe may have important functional implications since the dorsal raphe and locus coeru leus are mutually interactive and are involved, via their widespread projection systems, in many biochemical, physiological and bzhavioral regulations. T h e nucleus r a p h e dorsalis a n d locus coeruleus c o m p r i s e the m a j o r s e r o t o n i n a n d n o r e p i n e p h r i n e cell g r o u p s in the b r a i n a n d have been shown to be i m p l i c a t e d in a wide variety o f b e h a v i o r s a n d physiological activities. Histofluorescence m a p p i n g , i m m u n o h i s t o c h e m i c a l analysis a n d a x o n t r a n s p o r t studies have revealed w i d e s p r e a d p r o j e c t i o n s f r o m b o t h o f these nuclei to the spinal cord, lower b r a i n stem, d i e n c e p h a l o n a n d telencephalon, including a l m o s t all areas o f the c e r e b r a l cortex6,7A1,20, zT. The nucleus r a p h e dorsalis has been r e p o r t e d to consist largely, b u t n o t exclusively, o f s e r o t o n i n - c o n t a i n i n g n e u r o n s while the locus coeruleus has been shown to consist p r i m a r i l y o f n o r e p i n e p h r i n e c o n t a i n i n g cells (refs. 1, 2, 4, 17, 24, 30, 37). C o m b i n e d histofluorescence a n d G o l g i studies have revealed 3 cell types in b o t h o f these nuclei. T h e studies o f Pfister a n d Danner2~,26 indicate t h a t m o n o a m i n e fluorescence is confined to the m u l t i p o l a r elements. H o w e v e r ,
Steinbusch et al. a4, using i m m u n o c y t o c h e m i c a l m e t h o d s , f o u n d t h a t at least 2 cell types in the nucleus r a p h e dorsalis were s e r o t o n i n - c o n t a i n i n g , p a r ticularly the m e d i u m sized fusiform a n d large multip o l a r neurons. A host o f fluorescence, a x o n transport, i m m u n o c y t o c h e m i c a l , a n d physiological studies have revealed interconnections between locus coeruleus a n d r a p h e nuclei3,11-13,17-19,22,28,30, 37. These reciprocal relations have been widely interp r e t e d as key factors in regulating m e t a b o l i s m o f the m o n o a m i n e s s e r o t o n i n a n d n o r e p i n e p h r i n e in these nuclei a n d in their p r o j e c t i o n fields t h e r e b y playing a role in sleep--waking oscillations a n d v a r i o u s other behaviors14-16,23,29,32,33. Since the r a p h e a n d locus coeruleus have been i m p l i c a t e d in m a n y electrophysiological a n d behavioral activities a n d r e c i p r o c a l l y interact, it is o f special interest to s t u d y t h e m d e v e l o p m e n t a l l y in t e m p o r a l relation to each other. A c c o r d i n g l y , we
* On leave from Instituto de Investigaciones Biom6dicas, Universidad Nacional Aut6noma de M6xico, Apartado Postal 70228, Mexico 20, Mexico. 0165-3806/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
488 have used multiple quantitative parameters to examine all 3 cell types, i.e. the ovoid, fusiform and multipolar, which we have identified in each nuclear formation 5,s,9. In rats of 30, 90 and 220 days of age we have previously described the normal morphometric development of these cell types in the nucleus raphe dorsalis 8 and recently have carried out similar cytomorphometric analyses in the locus coeruleus:L In the present studies we are comparing dendritic spine development in the nucleus raphe dorsalis and locus coeruleus across 3 age groups of animals. In these experiments we have used 5 Charles River C.D. Sprague-Dawley rats of each of 3 age groups (30, 90, 220 days of age) bred and raised in our laboratories. In each of the 15 animals the nucleus locus coeruleus and nucleus raphe dorsalis were studied from sections taken from the same rat. The rapid Golgi technique was applied in each instance as described in our previous studiesS, 8,9. As in our previous studies we classified the cells in both nucleus raphe dorsalis and locus coeruleus into 3 types, ovoid, fusiform and multipolar. A total of one hundred cells were selected randomly from all parts of each nucleus in a total of 5 animals at each age as follows: at each age 35 fusiform, 40 multipolar and 35 ovoid cells were selected representing the first consecutively encountered well-impregnated neurons in each sampling area. The boundaries of the nucleus raphe dorsalis were defined similar to those described by Steinbusch et al. 34 while the boundaries of the locus coeruleus were established similar to those described by Swanson as. For each cell the following measures were taken: major and minor axes, number, diameter and linear extent of primary and secondary dendrites and number of synaptic spines on the primary and secondary dendrites and on the cell bodies. Age-related changes other than for synaptic spines were infrequent. O f 96 of these comparisons across these various parameters only 7 achieved statistical significance. In the nucleus raphe dorsalis all 3 cell types, ovoid, fusiform and multipolar, showed an increase in synaptic spines on both primary and secondary dendrites between 30 and 90 days followed by a decrease between 90 and 220 days of age (Fig. 1). In the locus coeruleus diametrically opposite age-related changes were noted with. all 3 cell types showing a decrease in synaptic spines on both
5PINE DENSITY PRIMARY DENDRITE
50~90 90L220
~GE ,~doys)
SECONDARY DENDRITE 30-90 90-220
Ft./S/FORM ~ L z
+ 75 +50~
+ 50-
+32% +25-
I
RAPHE DORSALtS
*18%
• 16%
~LOCUS COERULEUS 0
-25-
-50
MULTI,°OL~I? C,ELL +50-
4-254
o-
1-24% H
~* +18%
-19%
-25-
-2B% -50
OVOID CELL
+75.
+50
+86%
+67%
-
+34% +25-
15%
O--9% - 25-39%
-50
-45%
Fig. 1. Graph showing percent change in dendritic spines across two age periods in primary and secondary dendrites of nucleus raphe dorsalis and nucleus locus coeruleus. Significance levels: * P 0.05; ** P " 0.01 ; *** P -J 0.001. primary and secondary dendrites between 30 and 90 days followed by an increase between 90 and 220 days. As shown in Fig. 1, of these age-related changes in both nuclei, all but one achieved statistical significance. In the case of perisomatic spines there were significant changes in 8 out of 12 measures (3 cell types, 2 age groups x 2 nuclei) and these were exactly in register with the changes in dendritic spines. These studies show that within the nucleus raphe dorsalis and locus coeruleus all 3 cell types showed synchronous age-related increases and decreases in synaptic spines on primary and secondary dendrites and on the cell body which, when the two nuclei
489 were compared, were in diametrically opposite directions. In our studies o n the dendritic spine develo p m e n t in the nucleus raphe dorsalis we find an agerelated increase followed by a decrease in dendritic spines which is similar to patterns described by L u n d 21 and Felten and C u m m i n g s 10. N o previous studies have assessed such dendritic development in the locus coeruleus, t h o u g h Sievers 31 described early increases in locus coeruleus dendritic spines followed by a decrease t h r o u g h 30 days o f age. The significance o f the synchrony of neuronal development within each of these nuclei can only be speculated on as the timing and location o f the afferent projections and local circuits within these nuclei are still largely undetermined. According to the c o m b i n e d Nissl, histochemical and Golgi studies of Pfister and D a n n e r ~5,26 on the rat nucleus raphe dorsalis and locus coeruleus, the multipolar cell is monoaminergic, the fusiform cell is a n o n - m o n o aminergic projection cell and the ovoid cell a local circuit cell. Thus, 3 apparently different types of cells in each nucleus are showing simultaneous development o f synaptic spines at 3 different locations, the primary and secondary dendrites and on the cell body. However, Steinbusch et al. 34, 1 Aghajanian, G. K., Wang, R. Y. and Baraban, J., Serotonergic and non-serotonergic neurons of the dorsal raphe: reciprocal changes in firing induced by peripheral nerve stimulation, Brain Research, 153 (1978) 169-175. 2 Albanese, A. and Butcher, L. L., Acetylcholinesterase and catecholamine distribution in the locus coeruleus of the rat, Brain Res. Bull., 5 (1980) 127-134. 3 Baraban, J. and Aghajanian, G. K., Noradrenergic innervation of serotonergic neurons in the dorsal raphe: demonstration by electron microscopic autoradiography, Brain Research, 204 (1981) 1-11. 4 Belin, M. F., Aguera, M., Tappaz, M., Jouvet, M. and Pujol, J. F., Identification des neurones accumulant le GABA dans le noyau dorsal du raphe, C.R. Acad. Sci. (Paris), 287 (1978) 865-869. 5 Cintra, L., Diaz-Cintra, S., Kemper, T. and Morgane, P. J., Nucleus locus coeruleus: a morphometric Golgi study in rats of three age groups, Brain Research, (in press). 6 Dahlstr6m, 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. 7 Dahlstr6m, A. and Fuxe, K., Evidence for the existence. of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta physioL scand., 64 Suppl., 247 (1965) 1-36. 8 Diaz-Cintra, S., Cintra, L., Kemper, T., Resnick, O. and
using cytoarchitectonic and immunohistochemical methods, noted that serotonin immunoreactive neurons closely resemble both the medium-sized fusif o r m and large multipolar neurons observed in normal material. Whether the synchrony o f dendritic spine develo p m e n t on all cell types in each nucleus represents a specific or non-specific effect o f afferent projections to all these synaptic locations in all cell types in these nuclei or is a response to the development o f local circuits within each nucleus remains to be determined. The inverse relationship between spine develo p m e n t in the nucleus raphe dorsalis and locus coeruleus is a finding one would not anticipate from their apparent close anatomical and neurophysiological interactions. It will be interesting in physiological experiments to determine the significance o f the synaptic spine increase in the nucleus raphe dorsalis which antedates that shown by the locus coeruleus. Supported by N I H G r a n t HD-06364, N I C H H D , N S F G r a n t B N S 79-22507, F o g a r t y International Fellowship G r a n t 5 F05 T w o 2693-02, and Fellowship G r a n t 27234 C O N A C Y T (Mexico).
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