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Differential projections of the nucleus raphe dorsalis and nucleus raphe centralis as revealed by autoradiography
Brain Research, 85 (1975) 205-210 ~;) Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands
205
DIFFERENTIAL PROJECTIONS OF THE NUCLEUS RAPHE DORSALIS AND NUCLEUS RAPHE CENTRALIS AS REVEALED BY AUTORADIOGRAPHY
P. BOBILLIER, F. PETITJEAN, D. SALVERT, M. LIGIER AND S. SEGUIN
Ddpartement de M~decine Exp~rimentale, Universit~ Claude-Bernard, 8, avenue Rockefeller, 69373 Lyon C~dex 2 (France)
Classic neuroanatomical techniques2,a, 10 have not permitted a selective delineation of the projections of the rostral raphe nuclei. More recently histofluorescent techniques, combined with specific chemical lesions (i.e. neurotoxic dihydroxytryptamines) 1,5, have demonstrated several pathways originating from the pontine indolamine cell groups, but the exact contribution of the respective projections of both nuclei raphe dorsalis and raphe centralis superior has not been demonstrated. Therefore we used the recently developed autoradiographic fibre-tracing technique4,7 to visualise the nerve projection system of these two nuclei. This note is a preliminary step to understand better the relative involvement of the raphe nuclei in the mechanism of sleep, and to show a functional relationship with neuroanatomical biochemistry. Two groups of two normal adult cats were anaesthetised (Nembutal 30 mg/kg) and stereotaxically injected (Horsley-Clarke Instrument) with 2/~Ci of L-[14C]leucine (The Radiochemical Centre, spec. act. 348 mCi/mmole in physiological saline) by using a Hamilton syringe. The injection (in a volume of 0.5/zl) was aimed either at the nucleus raphe dorsalis or nucleus raphe centralis superior according to their described topography 10. Two days after the injection, the animals were killed by KC1 i.v. injection. Frontal frozen sections (48 #m thick) of the whole brain were cut following the Ullberg autoradiographic technique 11 by which each section is attached to adhesive tape (No. 810, 3M company). The tape-mounted sections were pressed against Kodirex X-ray films (Kodak). Tissue sections were stained simultaneously with cresyl violet for macroscopic localisation of the labelled material on a standard stereotaxic atlas 6. The site of injection of the nucleus raphe dorsalis (Fig. 2, AP0 plane) extends ventrally in the periaqueductal grey matter for about 2 mm in each plane, between the dorsal tegmenti nuclei of Gudden and the oculomotor nuclei. Some projections descend vertically above the nucleus interpeduncularis and border laterally the nucleus raphe centralis superior. More rostrally another portion extends vertically between the red nuclei. At the level of mesencephalon the main part of the fibres deviate from the midline and proceed in the median forebrain bundle dorsolaterally to the nucleus interpeduncularis (Fig. 2, A4 plane). More dorsally, the autoradio-
206
A
E
B
• II | EE~lr
F
Fig. 1. Autoradiograms of cat frontal brain sections showing the distribution of radioactivity after a local injection of [14C]leucineinto: (I) the nucleus raphe centralis superior. A: position of the labelled ascending pathway dorsolateral to the nucleus interpeduncularis. B: position of the main pathway into the median forebrain bundle region with projections to the nucleus habenula lateralis via the tractus habenulo-peduncularis. C: labelled terminals in the hippocampus and cortex entorhinalis. (2) The nucleus raphe dorsalis. D: labelling of the corpus geniculatum laterale with its innervating peduncle above the substantia nigra. Note the absence of labelling of the mammillary peduncles and the small density of radioactivity in the habenular complex. E: projections into the lateral hypothalamus, the corpus striatum and the nucleus caudatus bordering the capsula interna. F: labelling of the nucleus caudatus and the preoptic area. graphic traces suggest the existence of short axons spreading out diffusely to the mesencephalic reticular f o r m a t i o n , the periaqueductal grey matter (mainly the ventrolateral part) a n d the nucleus raphe centralis. I n the A7 plane, a p o r t i o n o f the fibres follow the external a n d internal limits o f the tractus h a b e n u l o - p e d u n c u l a r i s a n d terminate
207
A4
)
A17 Fig. 2. Nucleus raphe dorsalis: schematic representation of the injection site and distribution of orthogradely labelled bundles outlined in a stereotaxic atlas of the cat brain, by reconstruction from serial autoradiographic sections. The horizontal lines signify the plane HCO. Dots mark the density of the radioactivity. in the nucleus parafascicularis and the nucleus habenula lateralis. In this plane, the corpus geniculatum laterale (mainly its ventral part) is also innervated by a small group of fibres, which leaves the main bundle in a lateral direction above the substantia nigra and ascends along the internal side of the tractus opticus (Fig. 1D). Further rostrally the main pathway continues to follow the region of the median
208 forebrain bundle as far as the level of the zona incerla and extends ventrally and dorsally in the Forel fields with heavy labelling of the hypothalamic region (Fig. 2, Ala plane). Some fibres appear to cross the mammillary bodies dorsally. A few fibres ascend along the lamina medulla externa. Important projections are observed in the corpus striatum and particularly in the nucleus caudatus whose main innervating peduncle runs along the internal side of the capsula interna (Fig. 1E). The thalamic innervation seems to be more particularly focussed on the nucleus periventricularis anterior. A slight labelling is also present in the stria terminalis. In front of the hypothalamus, the rest of the fibres spread diffusely through the median and lateral preoptic region (Fig. 1F), the area amygdalea, the olfactive tubercle, the septum area, the diagonal band of Broca and the nucleus accumbens (Fig. 2, A17 plane). The olfactory bulb is also labelled through the tractus olfactorius. A slight labelling is noted in the frontal neocortex. We have not observed autoradiographic traces in the cingulate area nor in the hippocampus. The site of injection of the nucleus raphe centralis superior (Fig. 3, AP0 plane) covers approximately the group of neurons described by Taber. Some dorsal projections are observed along the midline between the two medial longitudinal fasciculi as far as the nucleus raphe dorsalis and the periaqueducta[ grey matter. The pontine reticular formation receives diffuse projections, with heavy labelling localised in the nucleus reticularis pontis oralis. Two important groups of parallel, thin fibres, spread out dorsovertically, occupy vertically the parasagittal region of the isthmic tegmentum on both sides of the nucleus linearis intermedius and deviate ventrally at the level of the nucleus interpeduncularis (Fig. IA). In the mesencephalon the main bundle runs along the fibre bundles of the nucleus raphe dorsalis, though being slightly more dorsomedial. There is a diffuse labelling of the mesencephalic reticular formation (Fig. 3, A4 plane). The nucleus interpeduncularis, the tractus habenulo-peduncularis and the nucleus habenula lateralis are highly labelled (Fig. 1B). The mammillary peduncles and the mammillary bodies also receive projections (Fig. 3, A8 plane). The innervation of the thalamus is more intense than by the nucleus raphe dorsalis though we could not find a distinct labelling of the lateral geniculate body. This innervation is mainly into the nucleus parafascicularis and the nucleus medialis dorsalis. There also seem to be some projections toward the anterior complex of the thalamus. A small amount of radioactivity is observed in the corpus striatum while contrary to the nucleus raphe dorsalis, the caudal part of the nucleus caudatus does not show labelling. Further rostrally the ventral terminal projections are similar to that of the nucleus raphe dorsalis, however, with a much more intense labelling of the diagonal band of Broca (Fig. 3, A17 plane). A large proportion of the ascending fibres enters in the cingulum and spreads out caudally to the cingulate area and the major part of the neocortical formations. Large projections were found in the cortex entorhinalis and in the ventral, dorsal and posterior formation hippocampalis: macroautoradiographs show high density in the granular layer of the gyrus dentatus (Fig. lC). The main innervating peduncle of the hippocampus seems to go through the fornix, the stria terminalis and the fimbria hippocampi. The efferent connections of both main anterior groups of the raphe nuclei
209
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A17 Fig. 3. Nucleus raphe centralis superior: schematic representation of the injection site and distribution of orthogradely labelled bundles outlined in a stereotaxic atlas of the cat brain, by reconstruction from serial autoradiographic sections. The horizontal lines signify the plane HCO. Dots mark the density of the radioactivity. follow a pathway almost identical until the hypothalamic region, but the anatomical localisation of their terminal projections presents clearly some specificity, which probably parallels some specific function in the central nervous system. For example, the cortex entorhinalis, the hippocampus, the neocortex and the mammillary bodies are exclusively or mainly innervated by the nucleus raphe centralis superior while the
210 nucleus r a p h e dorsalis m a i n l y innervates the lateral geniculate b o d y a n d the c o r p u s striatum. The l a t t e r p a t h w a y is in a g r e e m e n t with the d e m o n s t r a t i o n o f the afferents to the rat c a u d o p u t a m e n by the h o r s e r a d i s h p e r o x i d a s e technique s. The present findings confirm a n d extend the o r g a n i s a t i o n o f the a s c e n d i n g i n d o l a m i n e n e u r o n system, p r o v i d e d by the fluorescence histochemical a p p r o a c h 1,~. To a large extent they also s u p p o r t s o m e previous o b s e r v a t i o n s describing the p r o j e c t i o n s o f the r a p h e c o m p l e x into the m i d b r a i n l i m b i c circuit a. This w o r k was s u p p o r t e d by C . N . R . S . (L.A. 162), 1.N.S.E.R.M. (U. 52) a n d D . R . M . E . ( G r a n t 73-198).
l BJ()RKLUND, A., NOB1N, A., AND STENEVI, U., The use of neurotoxic dihydroxytryptamines as tools for morphological studies and localized lesioning of central indolamine neurons, Z. Zellforsch., 145 (1973) 479-501. 2 BRODAL, A., TABER, E., AND WALBERG,F,, The raphe nuclei of the brain stem in the cat. II. Efferent connections, J. comp. Neurol., 114 (1960) 239-260. 3 BRODAL,A., WALBERG,F., AND TABER, E., The raphe nuclei of the brain stem in the cat. llI. Afferent connections, J. comp. Neurol., 114 (1960) 261-281. 4 COWAN, W. M., GOTTLIEa, D. I., HENDRICKSON,A. E., PRICE, J. L., AND WOOLSEY,T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-41. 5 FUXE, K., AND JONSSON,G., Further mapping of central 5-hydroxytryptamine neurons: studies
6
7 8
9 10
I1
with the neurotoxic dihydroxytryptamines. In E. COSTA, G. L. GESSAAND M. SANDLER(Eds.), Serotonin: New Vistas, Advances in Biochem. Psychopharmacol., Vol. 10, Raven Press, New York, N.Y., 1974, pp. 13-33. JASPER,H., ANDAJMONEMARSAN,C., A Stereotaxic Atlas of the Diencephalon of the Cat, National Research Council of Canada, Ottawa, 1953. LASEK,R. J., JOSEPH, B. S., AND WHITLOCK,D. G., Evaluation of a radioautographic neuroanatomical tracing method, Brain Research, 8 (1968) 319-336. NAUTA, H. J. W., PRITZ, M.B., AND LASEK, R. J., Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. NAUTA,W. J. H., Hippocampal projections and related neural pathways to the mid-brain in the cat, Brain, 81 0958) 319-340. TABER, E., BRODAL, A., AND WALBERO, F., The raphe nuclei of the brain stem in the cat. I. Normal topography and cytoarchitecture and general discussion, J. comp. Neurol., 114 (1960) 161-188. ULLBERG, S., Studies on the distribution and fate of SaS-labelled benzylpenicillin in the body. Acta radiol. (Stockh.), Suppl. 118 0954) l-110.
205
DIFFERENTIAL PROJECTIONS OF THE NUCLEUS RAPHE DORSALIS AND NUCLEUS RAPHE CENTRALIS AS REVEALED BY AUTORADIOGRAPHY
P. BOBILLIER, F. PETITJEAN, D. SALVERT, M. LIGIER AND S. SEGUIN
Ddpartement de M~decine Exp~rimentale, Universit~ Claude-Bernard, 8, avenue Rockefeller, 69373 Lyon C~dex 2 (France)
Classic neuroanatomical techniques2,a, 10 have not permitted a selective delineation of the projections of the rostral raphe nuclei. More recently histofluorescent techniques, combined with specific chemical lesions (i.e. neurotoxic dihydroxytryptamines) 1,5, have demonstrated several pathways originating from the pontine indolamine cell groups, but the exact contribution of the respective projections of both nuclei raphe dorsalis and raphe centralis superior has not been demonstrated. Therefore we used the recently developed autoradiographic fibre-tracing technique4,7 to visualise the nerve projection system of these two nuclei. This note is a preliminary step to understand better the relative involvement of the raphe nuclei in the mechanism of sleep, and to show a functional relationship with neuroanatomical biochemistry. Two groups of two normal adult cats were anaesthetised (Nembutal 30 mg/kg) and stereotaxically injected (Horsley-Clarke Instrument) with 2/~Ci of L-[14C]leucine (The Radiochemical Centre, spec. act. 348 mCi/mmole in physiological saline) by using a Hamilton syringe. The injection (in a volume of 0.5/zl) was aimed either at the nucleus raphe dorsalis or nucleus raphe centralis superior according to their described topography 10. Two days after the injection, the animals were killed by KC1 i.v. injection. Frontal frozen sections (48 #m thick) of the whole brain were cut following the Ullberg autoradiographic technique 11 by which each section is attached to adhesive tape (No. 810, 3M company). The tape-mounted sections were pressed against Kodirex X-ray films (Kodak). Tissue sections were stained simultaneously with cresyl violet for macroscopic localisation of the labelled material on a standard stereotaxic atlas 6. The site of injection of the nucleus raphe dorsalis (Fig. 2, AP0 plane) extends ventrally in the periaqueductal grey matter for about 2 mm in each plane, between the dorsal tegmenti nuclei of Gudden and the oculomotor nuclei. Some projections descend vertically above the nucleus interpeduncularis and border laterally the nucleus raphe centralis superior. More rostrally another portion extends vertically between the red nuclei. At the level of mesencephalon the main part of the fibres deviate from the midline and proceed in the median forebrain bundle dorsolaterally to the nucleus interpeduncularis (Fig. 2, A4 plane). More dorsally, the autoradio-
206
A
E
B
• II | EE~lr
F
Fig. 1. Autoradiograms of cat frontal brain sections showing the distribution of radioactivity after a local injection of [14C]leucineinto: (I) the nucleus raphe centralis superior. A: position of the labelled ascending pathway dorsolateral to the nucleus interpeduncularis. B: position of the main pathway into the median forebrain bundle region with projections to the nucleus habenula lateralis via the tractus habenulo-peduncularis. C: labelled terminals in the hippocampus and cortex entorhinalis. (2) The nucleus raphe dorsalis. D: labelling of the corpus geniculatum laterale with its innervating peduncle above the substantia nigra. Note the absence of labelling of the mammillary peduncles and the small density of radioactivity in the habenular complex. E: projections into the lateral hypothalamus, the corpus striatum and the nucleus caudatus bordering the capsula interna. F: labelling of the nucleus caudatus and the preoptic area. graphic traces suggest the existence of short axons spreading out diffusely to the mesencephalic reticular f o r m a t i o n , the periaqueductal grey matter (mainly the ventrolateral part) a n d the nucleus raphe centralis. I n the A7 plane, a p o r t i o n o f the fibres follow the external a n d internal limits o f the tractus h a b e n u l o - p e d u n c u l a r i s a n d terminate
207
A4
)
A17 Fig. 2. Nucleus raphe dorsalis: schematic representation of the injection site and distribution of orthogradely labelled bundles outlined in a stereotaxic atlas of the cat brain, by reconstruction from serial autoradiographic sections. The horizontal lines signify the plane HCO. Dots mark the density of the radioactivity. in the nucleus parafascicularis and the nucleus habenula lateralis. In this plane, the corpus geniculatum laterale (mainly its ventral part) is also innervated by a small group of fibres, which leaves the main bundle in a lateral direction above the substantia nigra and ascends along the internal side of the tractus opticus (Fig. 1D). Further rostrally the main pathway continues to follow the region of the median
208 forebrain bundle as far as the level of the zona incerla and extends ventrally and dorsally in the Forel fields with heavy labelling of the hypothalamic region (Fig. 2, Ala plane). Some fibres appear to cross the mammillary bodies dorsally. A few fibres ascend along the lamina medulla externa. Important projections are observed in the corpus striatum and particularly in the nucleus caudatus whose main innervating peduncle runs along the internal side of the capsula interna (Fig. 1E). The thalamic innervation seems to be more particularly focussed on the nucleus periventricularis anterior. A slight labelling is also present in the stria terminalis. In front of the hypothalamus, the rest of the fibres spread diffusely through the median and lateral preoptic region (Fig. 1F), the area amygdalea, the olfactive tubercle, the septum area, the diagonal band of Broca and the nucleus accumbens (Fig. 2, A17 plane). The olfactory bulb is also labelled through the tractus olfactorius. A slight labelling is noted in the frontal neocortex. We have not observed autoradiographic traces in the cingulate area nor in the hippocampus. The site of injection of the nucleus raphe centralis superior (Fig. 3, AP0 plane) covers approximately the group of neurons described by Taber. Some dorsal projections are observed along the midline between the two medial longitudinal fasciculi as far as the nucleus raphe dorsalis and the periaqueducta[ grey matter. The pontine reticular formation receives diffuse projections, with heavy labelling localised in the nucleus reticularis pontis oralis. Two important groups of parallel, thin fibres, spread out dorsovertically, occupy vertically the parasagittal region of the isthmic tegmentum on both sides of the nucleus linearis intermedius and deviate ventrally at the level of the nucleus interpeduncularis (Fig. IA). In the mesencephalon the main bundle runs along the fibre bundles of the nucleus raphe dorsalis, though being slightly more dorsomedial. There is a diffuse labelling of the mesencephalic reticular formation (Fig. 3, A4 plane). The nucleus interpeduncularis, the tractus habenulo-peduncularis and the nucleus habenula lateralis are highly labelled (Fig. 1B). The mammillary peduncles and the mammillary bodies also receive projections (Fig. 3, A8 plane). The innervation of the thalamus is more intense than by the nucleus raphe dorsalis though we could not find a distinct labelling of the lateral geniculate body. This innervation is mainly into the nucleus parafascicularis and the nucleus medialis dorsalis. There also seem to be some projections toward the anterior complex of the thalamus. A small amount of radioactivity is observed in the corpus striatum while contrary to the nucleus raphe dorsalis, the caudal part of the nucleus caudatus does not show labelling. Further rostrally the ventral terminal projections are similar to that of the nucleus raphe dorsalis, however, with a much more intense labelling of the diagonal band of Broca (Fig. 3, A17 plane). A large proportion of the ascending fibres enters in the cingulum and spreads out caudally to the cingulate area and the major part of the neocortical formations. Large projections were found in the cortex entorhinalis and in the ventral, dorsal and posterior formation hippocampalis: macroautoradiographs show high density in the granular layer of the gyrus dentatus (Fig. lC). The main innervating peduncle of the hippocampus seems to go through the fornix, the stria terminalis and the fimbria hippocampi. The efferent connections of both main anterior groups of the raphe nuclei
209
APo ~
•
A4
I
i(~
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•
~
"
A17 Fig. 3. Nucleus raphe centralis superior: schematic representation of the injection site and distribution of orthogradely labelled bundles outlined in a stereotaxic atlas of the cat brain, by reconstruction from serial autoradiographic sections. The horizontal lines signify the plane HCO. Dots mark the density of the radioactivity. follow a pathway almost identical until the hypothalamic region, but the anatomical localisation of their terminal projections presents clearly some specificity, which probably parallels some specific function in the central nervous system. For example, the cortex entorhinalis, the hippocampus, the neocortex and the mammillary bodies are exclusively or mainly innervated by the nucleus raphe centralis superior while the
210 nucleus r a p h e dorsalis m a i n l y innervates the lateral geniculate b o d y a n d the c o r p u s striatum. The l a t t e r p a t h w a y is in a g r e e m e n t with the d e m o n s t r a t i o n o f the afferents to the rat c a u d o p u t a m e n by the h o r s e r a d i s h p e r o x i d a s e technique s. The present findings confirm a n d extend the o r g a n i s a t i o n o f the a s c e n d i n g i n d o l a m i n e n e u r o n system, p r o v i d e d by the fluorescence histochemical a p p r o a c h 1,~. To a large extent they also s u p p o r t s o m e previous o b s e r v a t i o n s describing the p r o j e c t i o n s o f the r a p h e c o m p l e x into the m i d b r a i n l i m b i c circuit a. This w o r k was s u p p o r t e d by C . N . R . S . (L.A. 162), 1.N.S.E.R.M. (U. 52) a n d D . R . M . E . ( G r a n t 73-198).
l BJ()RKLUND, A., NOB1N, A., AND STENEVI, U., The use of neurotoxic dihydroxytryptamines as tools for morphological studies and localized lesioning of central indolamine neurons, Z. Zellforsch., 145 (1973) 479-501. 2 BRODAL, A., TABER, E., AND WALBERG,F,, The raphe nuclei of the brain stem in the cat. II. Efferent connections, J. comp. Neurol., 114 (1960) 239-260. 3 BRODAL,A., WALBERG,F., AND TABER, E., The raphe nuclei of the brain stem in the cat. llI. Afferent connections, J. comp. Neurol., 114 (1960) 261-281. 4 COWAN, W. M., GOTTLIEa, D. I., HENDRICKSON,A. E., PRICE, J. L., AND WOOLSEY,T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-41. 5 FUXE, K., AND JONSSON,G., Further mapping of central 5-hydroxytryptamine neurons: studies
6
7 8
9 10
I1
with the neurotoxic dihydroxytryptamines. In E. COSTA, G. L. GESSAAND M. SANDLER(Eds.), Serotonin: New Vistas, Advances in Biochem. Psychopharmacol., Vol. 10, Raven Press, New York, N.Y., 1974, pp. 13-33. JASPER,H., ANDAJMONEMARSAN,C., A Stereotaxic Atlas of the Diencephalon of the Cat, National Research Council of Canada, Ottawa, 1953. LASEK,R. J., JOSEPH, B. S., AND WHITLOCK,D. G., Evaluation of a radioautographic neuroanatomical tracing method, Brain Research, 8 (1968) 319-336. NAUTA, H. J. W., PRITZ, M.B., AND LASEK, R. J., Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. NAUTA,W. J. H., Hippocampal projections and related neural pathways to the mid-brain in the cat, Brain, 81 0958) 319-340. TABER, E., BRODAL, A., AND WALBERO, F., The raphe nuclei of the brain stem in the cat. I. Normal topography and cytoarchitecture and general discussion, J. comp. Neurol., 114 (1960) 161-188. ULLBERG, S., Studies on the distribution and fate of SaS-labelled benzylpenicillin in the body. Acta radiol. (Stockh.), Suppl. 118 0954) l-110.