The raphe nuclei of the cat brain stem: A topographical atlas of their efferent projections as revealed by autoradiography

449

Bruin Research,

0

113 (1976) 449486 Elsevier Scientific Publishing Company, Amsterdam

- Printed in The Netherlands

Research Reports

THE

RAPHE

ATLAS

OF

NUCLEI

OF THE

CAT

THEIR

EFFERENT

BRAIN

STEM:

PROJECTIONS

A TOPOGRAPHICAL AS

REVEALED

BY

AUTORADIOGRAPHY

PIERRE BOBILLIER, MONIQUE TOURET

SOLANGE SEGUIN, FRANCOISE and MICHEL JOUVET

Department of Experimental

Medicine,

PETITJEAN,

DENISE

SALVERT,

University Claude Bernard, 8, avenue Rockfeller, Lyon (France)

(Accepted February 4th, 1976)

SUMMARY

Stereotaxic rior, rapht

injections

dorsalis,

of [14C]leucine

raphe magnus

were made in nuclei rapht

and raphe pontis

centralis

supe-

of the cat. The organization

of

the regional connections was outlined in a stereotaxic atlas using the autoradiographic tracing method: the majority of the ascending pathways from the rostra1 raphe nuclei are directed

mainly

through

a ventrolateral

Tsai, with some lateral extensions

bundle

to the substantia

via the ventral

tegmental

nigra, and then through

area of the fields

of Fore1 and the zona incerta. More rostrally the fibers are joined to the medial forebrain bundle through the hypothalamic region up to the preoptic area or the diagonal band of Broca. Multiple divisions leave this tract towards the epithalamic or the intralaminar thalamic ansa lenticularis.

nuclei, the stria terminalis, the septum, the capsula interna and the The bulk of the rostra1 projections terminates in the frontal lobe,

while some labeling projections

of nucleus

n. interpeduncularis,

is scarcely

distributed

(n.) raphi! centralis the mammillary

throughout superior

bodies

the rest of the neocortex.

are specifically

and the hippocampal

associated formation

The

with the while

the n. raphe dorsalis innervates selectively the lateral geniculate bodies, striatum, piriform lobes, olfactory bulb and amygdala. The rest of the ascending fibers form the centrolateral or the dorsal ascending tracts radiating either in the reticular mesencephalic formation or in the periventricular gray matter. On the contrary there are heavy descending projections from n. rapht centralis superior which distribute to the main nuclei of the brain stem, the central gray matter and the cerebellum. The ascending projections from the caudal raphe nuclei are much less dense. They disseminate mainly in the colliculus superior, the pretectum, the nucleus of the posterior commissure, the preoculomotor complex and the intralaminar nuclei of the thalamus. From n. raphe pontis, a dense labeling is selectively localized at then. paraventricularis

450 hypothalami with some rostral extensions to limbic areas. Diffuse caudal anti rostrai projections from both nuclei are observed in the mesencephalic, pontobulbar reticular formation and the cerebellum. The main differences come from the specific localization of their descending bulbospinal tracts inside the lateroventral funiculus o[ the spinal cervical cord.

INTRODUCTION Some cytologic46, anatomic 7,ee,24-26,zz,3s-4°, ontogenetic41,4r), electrophysiologic36,4~ and behavioraP 7,18,21,43 as well as biochemicaP s,')7,43 arguments militate strongly for an individual system of projections specific to each of the raph6 nuclei. In regards to the possible functional role attributed to some of these nuclei 9, for example, in the regulation of the states of sleep 20, the knowledge of the respective nervous connections of the nuclear complex of the raph6 has a fundamental importance in every study of physiological and biochemical neuroanatomy. Although the topography of the cat 44,46 and rat 49 raph6 nuclei is well known, their efferent and afferent connectionsV,8,3a,49 are not precisely characterized because of the limitations of the degeneration technique used. Thus, only a few anatomical works had been devoted to their study until the discovery of the histofluorescent techniques which have permitted the localization of fluorescing neurons containing serotonin (5-HT) in almost all of these nuclei 4Az,35. However, the somewhat impaired sensitivity of the method has not permitted a clear delineation of the pathways of the 5-HT-containing raph6 groups B1-B9 in the rat 5,15,48. On the other hand, the percentage of raph6 cells which contain 5-HT is not yet known. In order to get some information concerning the eventual function of the raphe system, the present study was undertaken to provide a comparative stereotaxic atlas of each regional projections of the following raph6 nuclei of the cat brain stem raph6 complex: nuclei raph6 dorsalis (RD), raph6 centralis superior (NCS), raph6 pontis (RPo) and raph6 magnus (RM). Experiments were carried out using the fibertracing technique which is based on the orthograde axoplasmic transport of macromolecular material previously labeled by the neuronal incorporation of radioactive precursors, and subsequently visualized by autoradiographya1,13,23. These isotope injections, of course, label not only 5-HT neurons, and hence this study cannot be interpreted as a pure demonstration of 5-HT pathways. Some preliminary results of the differential proiections of RD and NCS have been reported elsewhere 6. METHODS Because of the number and the complexity of the projections of the raph6 nuclei, we prefered to neglect the subcollular anatomical details. Thus, autoradiographic preparations for macroscopic viewing appeared to be the most suitable and rapid method. In fact, this technique is easy, reproducible, allows a rapid scanning of a great number of possible radioactive sites, yields plates of good contrast and, most

451 importantly, has a higher efficiency than that of the classical techniques of dipping or stripping film 37. The radioisotope chosen was L-[U-14C]leucine (The Radiochemical Centre, spec. act. 348 mCi/mmole). On the day of the experiment, the commercially available solution was evaporated under nitrogen and redissolved in double-distilled water to obtain a final radioactivity of 4 #Ci//zl. All the injections were made with a 5-/A Hamilton syringe and a 26 S gauge hypodermic needle. In order to achieve a more restricted labeling area, we used a glass micropipette with a tip diameter of 50/~m in two cats. The volume (0.5/zl) was delivered over a 10-rain interval. The needle was left in place another 10 rain before being withdrawn. A total of 13 adult cats were studied. The animals were anesthetized with Nembutal and placed in a Horsley-Clarke stereotaxic apparatus. Injections were made at the following coordinates: APo, HC-1.5 (RD); P1, HC-5 (NCS); P3, HC-7.5 (RPo); P6, HC-8.5 (RM). Two days after the injection, the animals were sacrificed by i.v. injection of a saturated KCI solution. Then the animals were placed in the stereotaxic apparatus and stereotaxic frontal sections of the whole brain (15-20 m m thick) were rapidly removed and freeze-dried. Frontal frozen sections (48-/~m thick) of each piece of tissue were cut and fixed to adhesive tape according to the Ullberg 4v autoradiographic technique. The tape-mounted sections were applied against Kodirex X-ray films (Kodak) and exposed at 4 °C for a few hours or days at the site of injection to a maximum of one month for the most distant structures. The films were developed and fixed with K o d a k - L X 24 and AL 4. Alternating serial tissue sections of the same thickness were stained simultaneously with cresyl violet. With this procedure and using a microprojector ( × 10), the macroscopic localization of the blackening areas of the film due to the presence of labeled material in the tissue was charted in the coordinates of the stereotaxic atlases of Jasper and Ajmone-Marsan 19 and of Berman 3. Reproducible autoradiographic sections were obtained with at least two cats for each nucleus. The international nomenclature for cerebral structures was used whenever possible (Nomina anatomica33). Uniform blackening or denser and limited blackening aggregates were interpreted as being either areas of terminal fields or fiber bundles. In order to determine the background due to the general diffusion of the leucine which had not been incorporated by the neurons at the site of injection, one additional cat was injected in the saphenous vein with the same quantity of radioactive leucine as in the experimental animals. After a survival time of 48 h, the brain was treated as above. Following one month of exposure, no significant background could be observed in any region of the brain.

Combination of autoradiographic and lesion studies In a further experiment utilizing two groups of two adult cats, electrolytic lesions were made in either the R D or RM. The labeling of these nuclei was realized by injection of [14C]leucine either after or before the lesion. In the last case the lesion was made 30 rain after the injection of the radioisotope in order to obtain an optimum proteic labeling of the neurons z. This was done to eliminate the possible comparti-

Z

.......

~

ITI

J~

453 mentalization impairing the free diffusion of the labeled product into the tissue surrounding t h e lesion w h e n it p r e c e d e s the injection. F i n a l l y , a c o n t r o l e x p e r i m e n t was d o n e o n a c a t a f t e r a l e s i o n o f the N C S . S u b s e q u e n t l y an i n j e c t i o n o f [14C]leucine was m a d e l a t e r a l l y to t h a t n u c l e u s ( L : 1.5 ram) in the s a m e f r o n t a l plane, in the p a r a m e d i a n r e t i c u l a r f o r m a t i o n o f the i s t h m i c t e g m e n t a l field. RESULTS

The analysis of the injection site shows that the radioactivity remains localized almost entirely in the cellular architecture of the raph6 nuclei as described by Taber et al. 46. The area of efficient diffusion was estimated to remain in a sphere of 2 mm diameter. When the injection was made in the rostral pole (RD) (Fig. 7, plane PO. 2), we observed no extension of the passive diffusion to the nuclei tegmenti dorsalis and ventralis of Gudden. A more selective labeling was obtained for the RPo (Fig. 15, plane PYl) with a glass micropipette. The nuclei NCS (Fig. 7, plane PO.2) and RM (Fig. 14, plane P7.1 ; Fig. 2E) were selectively injected due to their relatively large spatial disposition. However we cannot exclude a possible extension of the labeling area of the R M t o t h e n u c l e u s r a p h 6 pallidus.

(I) Projections of the nucleus raphd dorsalis ( A ) Ascending projections The ascending projections from the RD (Figs. 7-13) can be schematically g r o u p e d into t h r e e o r g a n i z e d s y s t e m s o f fibers: d o r s a l , m e d i a l a n d v e n t r o l a t e r a l .

Fig. 1. Differential distribution of radioactivity into some brain regions following the injection of [taC]leucine (2/tCi in 0.5 pl) respectively into the nucleus raph6 centralis superior (left half of the figure) and the nucleus raphe dorsalis (right half of the figure). The survival time of cats was two days. A: emergence of the ventrolateral ascending bundle as a bilateral paramedian lamina with fibers crossing through the decussation of the brachium conjunctivum. Profuse dispersion of label is seen into the mesencephalic reticular formation while dense label is centered over the nucleus interpeduncularis. Some pontocerebellar fibers spread out ventrolaterally. Note the absence of labeling of the trochlear nucleus. B: localization of the ventrolateral ascending bundle dorsolaterally to the nucleus interpeduncularis. C: labeled ventrolateral ascending bundle extending through the ventral tegmental area of Tsai, the Ford's fields and the zona incerta. Terminal fields of projection are seen successively into the mammillary bodies, the nucleus parafascicularis, the periaqueductal gray matter and the nucleus habenulae lateralis. D: distribution of labeling into the hippocampus and the cortex entorhinalis. E: localization of the ventrolateral ascending bundle curving around the medial border of the red nucleus to join the ventral tegmental area. Mediodorsally a fine tract of dense labeling constitutes the dorsal periaqueductal ascending bundle with some distribution of radioactivity over the lateral gray matter. F: ventrolateral ascending bundle with its two components respectively localized in the ventral tegmental area and the Ford's field Hl. Note the absence of labeling of the mammillary bodies and the smaller density of radioactivity in the habenular complex as compared to C. In addition there is an extension of the labeling to the lateral geniculate bodies and to the reticular division of the substantia nigra. G: the ventrolateral ascending bundle extends largely beyond the medial forebrain bundle through the rostral lateral hypothalamic region where fibers are moving in several directions; dorsally along the internal border of the capsula interna towards the nucleus caudatus and the stria terminalis, laterally towards the putamen and the globus pallidus and ventrolaterally towards the amygdala. Note the absence of labeling of the nucleus ventromedialis hypothalami. H: heavily labeled projections into the preoptic area, the striatum and the capsula interna.

"¢!

TI

.¢1

455 (1) The d o r s a l system is f o r m e d by fibers which emerge r o s t r o - d o r s a l l y from the R D a n d follow the l a t e r a l a n d ventral p a r t s o f the a q u e d u c t (Fig. 1E). This d o r s a l p e r i a q u e d u c t a l a n d p e r i v e n t r i c u l a r ascending system c o n t r i b u t e s to the heavy innervation o f the s u b s t a n t i a grisea centralis o f the m e s e n c e p h a l o n a n d diencephalon. (2) The m e d i a l system can be c h a r a c t e r i z e d either i m m e d i a t e l y in front o f the R D o r d e p a r t i n g progressively f r o m the m a i n v e n t r o l a t e r a l bundle (see below). A n i m p o r t a n t n u m b e r o f fibers crosses the midline t h r o u g h the decussation o f the b r a c h i u m c o n j u n c t i v u m . I n the same plane, o t h e r fibers deviate l a t e r o v e n t r a l l y to ramify, either in the mesencephalic r e t i c u l a r f o r m a t i o n o r in the medial lemniscus a n d along the s u p e r i o r b o r d e r o f the s u b s t a n t i a nigra. (3) The v e n t r o l a t e r a l system is f o r m e d by the m a i n ascending bundle which proceeds in the v e n t r o l a t e r a l region o f the m e s o d i e n c e p h a l o n . First, this bundle ascends t h r o u g h the fasciculus l o n g i t u d i n a l i s medialis a n d the o c u l o m o t o r complex then, m o s t o f its fibers d i p d o w n vertically a l o n g each side o f the midline t h r o u g h the b r a c h i u m c o n j u n c t i v u m a n d further on, a l o n g the nucleus linearis intermedius. The greatest p a r t o f the fibers continue to descend vertically a l o n g the d o r s o l a t e r a l b o r d e r o f the i n t e r p e d u n c u l a r nucleus to j o i n the ventral t e g m e n t a l a r e a o f Tsai (Fig. 1E a n d F) with a lateral extension t e r m i n a t i n g into the s u b s t a n t i a n i g r a (mainly the p a r s c o m p a c t a a n d pars reticularis). At the level of the mesencephalic junction, the m a i n bundle progresses always ventrally a n d laterally to the m a m m i l l a r y bodies which r e m a i n unlabeled. Then the fibers sweep d o r s o l a t e r a l l y in the z o n a incerta to dissociate r o u g h l y in two c o m p o nents o f high density localized respectively in the F o r e l ' s fields H 1 a n d H2 (Fig. 1F). These two m a i n c o m p o n e n t s , m o r e or less intermingled, o c c u p y rostrally the lateral

Fig. 2. Differential distribution of the radioactivity into some representative brain regions following injection of [14C]leucine respectively into the nucleus raph6 pontis (left half of the figure) and the nucleus raph6 magnus (right half of the figure). A: position of the ventrolateral ascending bundle which surrounds the dorsal and the ventral parts of the red nucleus. The main labeled fibers of the dorsal ascending bundle are running through the medial longitudinal bundle. Note the absence of labeling of the oculomotor nuclei. Heavy projections distribute into the nuclei of the posterior commissure, the mesencephalic reticular formation, the pretectal area and the periaqueductal gray matter. B: position of the remaining fibers of the ventrolateral ascending bundle into the lateral hypothalamic region. Some labeled fibers are clearly seen crossing ventrally through the supraoptic decussation while others extend laterally towards the nucleus amygdaloideus centralis via the ansa lenticularis. Some label is observed successively into the nucleus supraopticus, the nucleus paraventricularis hypothalami and the nucleus reticularis thalami. C: fading out of the more rostral fibers into the diagonal band of Broca. E and D show respectively the injection of [14C]leucine into the nucleus raph6 magnus and the corresponding electrolytic lesion which suppresses both the ascending and descending projections of this nucleus (cresyl violet). F: heavily labeled fibers leave the paramedian ascending bundle in a dorsolateral direction to diffuse into the dorsal pontine tegmentum. The dorsal ascending fibers travel within the medial longitudinal bundle. Note the absence of label in the nucleus tegmenti ventralis of Gudden while some appear in the cerebellar cortex. G : distribution of labeling through the optic tectum, the periaqueductal gray matter and the mesencepbalic reticular formation. The ventrolateral and the dorsal ascending labeled fibers course respectively through the ventral tegmentum outside the nucleus interpeduncularis and the medial longitudinal bundle. Note the absence of labeling of the trochlear nucleus (5 7<).

456

Fig. 3. A: heavily labeled descending projections through the lateral and the paramedian bulbar reticular formation following injection of [HC]leucine into the nucleus raph6 magnus. Some label is seen in the inferior olivary complex and in the nucleus intercalatus. B and C: differential bulbospinal descending bundles coming respectively from the nucleus raph6 magnus (B) and the nucleus raph6 pontis (C). These tracts are localized in either the ventral or the lateral funiculus and near the central canal of the spinal cervical cord. Accumulation of label is centered over the dorsal horn (5 ":).

457 TABLE

I

Differential labeling of the nuclei raphd centralis superior ( C), raphd dorsalis ( D), raphd magnus ( M) and raphd pontis (P) in various parts of the brain The variations of density were estimated subjectively according to the following symbols: ÷ ÷ ~ , high density; -t + , moderate density; ÷ , low density; -f - - , very low or indistinct fibres or terminal areas of labeling.

Nuclei o[" the CNS

C

D

M

P

(1) Medulla cervicalis Cornu ventrale (2) Myelencephalon nucleus cochlearis dorsalis nucleus cochlearis ventralis nucleus cuneatus nucleus dorsalis nervi vagi nucleus intercalatus nucleus nervi facialis nucleus nervi hypoglossi nucleus olivaris inferior nucleus olivaris inferior dorsalis nucleus olivaris inferior medialis nucleus prepositus hypoglossi nucleus raph6 magnus nucleus raph6 obscurus nucleus raph6 pallidus nucleus reticularis gigantocellularis nucleus reticularis lateralis nucleus reticularis medullae oblongatae nucleus reticularis paramedianus nucleus tractus solitarii nucleus vestibularis inferior nucleus vestibularis lateralis nucleus vestibularis medialis nucleus vestibularis superior substantia grisea centralis (3) Metencephalon cerebellum colliculus inferior nucleus corporis trapezoidei nucleus locus coeruleus nucleus motorius nervi trigemini nucleus nervi abducentis nucleus olivaris superior nucleus olivaris superior lateralis nucleus olivaris superior medialis nucleus parabrachialis dorsalis nucleus parabrachialis ventralis nucleus raph6 centralis superior nucleus raph6 dorsalis nucleus raph~ pontis nucleus reticularis parvocellularis nucleus reticularis pontis caudalis nucleus reticularis pontis oralis

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4. +4

458 ( Table I contimted) Nuclei o[tDe CNS

D

nucleus reticularis tegmenti pontis nucleus sensorius superio~ nervi trigemini nucleus tractus spinalis nervi trigemini nucleus tegmenti dorsalis Gudden nucleus tegmenti ventralis Gudden substantia grisea centralis (4) Mesencephalon area pretectalis area tegmentalis ventralis Tsai formatio reticularis nucleus commissurae posterior nucleus Darkschewitsch nucleus Edinger-Westphal nucleus interpeduncularis nucleus interstitialis Cajal nucleus linearis intermedius nucleus linearis rostralis nucleus tuber stratum griseum colliculi superioris substantia grisea centralis substantia nigra pars lateralis substantia nigra zona compacta substantia nigra zona reticulata (5) Diencephalon area hypothalamica dorsalis area hypothalamica lateralis area hypotha[amica posterior corpus mammillare nucleus anterior dorsalis nucleus centralis, pars lateralis nucleus centralis, pars medialis nucleus dorsalis corporis geniculati lateralis nucleus centrimediani nucleus dorsomedialis hypothalami nucleus entopeduncularis nucleus habenulae lateralis nucleus habenulae medialis nucleus medialis dorsalis nucleus parafascicularis nucleus parataenialis nucleus paraventricularis hypothalami nucleus paraventricularis anterior nucleus praethalamicus nucleus reticularis nucleus reuniens nucleus rhomboideus nucleus subparafascicularis nucleus subthalamicus nucleus suprachiasmaticus

i

i

P

M

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459 (Continued Table 1) Nuclei o f the C N S

nucleus supra opticus nucleus ventralis corporis geniculati lateralis nucleus ventralis, pars posterolateralis nucleus ventralis, pars posteromedialis nucleus ventromedialis regio preoptica (6) Telencephalon area amygdaloidea anterior area pyriformis bulbus olfactorius corpus amygdaloideum cortex entorhinalis diagonal band of Broca frontal neocortex globus pallidus gyms cinguli gyrus dentatus gyrus parahippocampalis indusium griseum nucleus accumbens nucleus amygdaloideus centralis nucleus caudatus putamen rest of neocortex septum stria medullaris stria terminalis subiculum tuberculum olfactorium

C

D

M

P

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-t + + + + +

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+ -t-

+

+ ~ 4 .~

h y p o t h a l a m i c region following the medial forebrain b u n d l e while extending largely b e y o n d it. Multiple collaterals ramify m a i n l y in the h y p o t h a l a m i c region (Table I). Some fibers radiate ventrally either towards the nucleus supraopticus a n d nucleus suprachiasmaticus or controlaterally to the lateral h y p o t h a l a m u s via the supraoptic commissure. Several dorsal a n d lateral divisions leave the m a i n bundle along its ventrodiencephalic course: on one side, the dorsal division is mostly a p p a r e n t at two levels, either densely along the fasciculus retroflexus (Fig. IF) or with a mere heterogenous diffusion along the d o r s o m e d i a n b o r d e r of the Forel's field H I . These fibers c o n t r i b u t e to the i n n e r v a t i o n of the nuclei parafascicularis, subparafascicularis, h a b e n u l a lateralis a n d that of the midline thalamic nuclei (Table 1). O n the other side, the lateral division divides into two c o m p o n e n t s : the first one is c o m p o s e d of fibers proceeding in the mere external region of the z o n a incerta, m o v i n g dorsally in an oblique direction to distribute in the lateral geniculate nucleus (Fig. IF). The second lateral peduncle

460

Fig. 4. A: electrolytic lesion of the nucleus iaph6 centralis superior associated wilh injection of i ~~( i[eucine laterally in the tegmental reticular formation. B: Dorsally some bilateral projection~ arc seen in the periaqueductal gray matter. C: the corresponding labeled ascending projections arc ~uJ~ning through the mesencephalic reticular formation. A heavily labeled tract is seen running ros~rally inside the peduncular tract (5 ~.

runs through the ansa lenticularis to ramify densely in the direction of the main amygdala nuclei as well as in the area amygdaloidea anterior and the cortex pyriformis. Part of this peduncle moves dorsally through the nucleus entopeduncularis to diffuse in the globus pallidus and the putamen (Fig. I G). In front of the hypothalamus, the ventrolateral system sweeps diffusely towards the preoptic region (Fig. I H) and the remaining fibers diverge in several directions: some fibers ascend along the internal border of the internal capsula to radiate in the nucleus praethalamicus, the fundus caudati and the whole caudate nucleus (with denser labeling in the caudal part). A dorsal peduncle moves up in a dorsal and medial direction along the fornix to terminate in the septum nuclei and the nucleus accumbens. The more rostral fibers join the gyrus cinguli, the rostral part, via the cingulum. Finally a fourth group of fibers moves medially and ventrally to invade the diagonal band of Broca and to distribute to the olfactory tubercle, the area amygdaloidea anterior, the cortex praepyriformis and the olfactory bulb. All along its diencephalic course the main ascending bundle distributes laterally an important number of fibers which follow the internal capsule and more rostrally the external capsule to radiate into the neocortex (mainly the frontal neocortex). No projections can be distinguished in the hippocampal formation.

(B) Descendingprojections The descending projections (Figs. 5 and 6) of the R D are p o o r and no significant labeling was observed in the pontine reticular formation. These fibers emerge from the caudal part of the R D in the periaqueductal gray matter with a lateral extension above the brachium conjunctivum. This system distributes to the dorsal tegmental nucleus, the locus coeruleus and the marginal nuclei of the brachium conjunctivum.

461

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Figs. 5-13. Schematic representation of the efferent connections from the nucleus raph6 centralis superior (C, left half of the section) and the nucleus raph6 dorsalis (D, right half of the section) as outlined in a stereotaxic atlas of the cat brain (according to Berman 3 for planes P 12.7-A 7.5 or to Jasper and Ajmone-MarsanTM for planes A 8.5-A 18.5), by reconstruction from serial autoradiographic and Nissl stained sections. The coordinates are rostral (A) and caudal (P) to the interauricular line. The site of injection and the labeled bundles are represented by heavier dots than the terminal projection areas. The horizontal line represents the plane HCO except for the planes P 7.1, P 10, P 11.6 and P 12.7 (HC-5).

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47O Finally a small contingent ~)llows the fasciculus longitudinalis medialis to finish ~t~ course principally in the nucleus olivarius inferior, pars medialis. No innerwltioll of the cerebellum could be seen.

(lI) Projections of the nucleus raphe centralis superior (A) Ascending projections The ascending systems of the NCS (Figs. 7-10) are similar to the ascending systems originating from the RD. (1) The periaqueductal ascending dorsal system is rather poor. The fibers emerging from the dorsal board of the NCS ascend vertically along the midline and distribute into the substantia grisea centralis of the mesencephalon. Only the RD and the ventral tegmental nucleus of Gudden present a rather important bulk of labeling. (2) The medial bundle moves up laterally and rostrally from the lateral board of the NCS. It is responsible for the dense labeling of the medial two-thirds of the mesencephalic reticular formation (Fig. I A and B). (3) The main ventrolateral bundle occupies vertically the parasagittal region of the isthmic tegmentum (Fig. I A) where numerous fibers ascend through the decussation of the brachium conjunctivum. Then the greatest part of the fibers deviate laterally to reach the ventral area of Tsai (Fig. l B). The nucleus linearis intermedius is only slightly labeled while the interpeduncular nucleus and the substantia nigra, pars compacta, exhibit a dense labeling. There is an important lateral extension of the fibers at the level of the medial lemniscus and through the region of the red nucleus. The mesodiencephalic course has a similar topography to that of the projections of the RD, but there are important differences concerning some secondary branchings: the mammillary body is richly innervated (Fig. 1C). The thalamus, except the metathalamus, also receives numerous diffuse projections localized ventrally to the nucleus subthalamicus, nucleus entopeduncularis, and dorsally to the epithalamus (mainly the nucleus habenularis lateralis) and to the group of the median and midline nuclei (Table I). A dense part of the fibers of this system follows the fasciculus retroflexus on each side of this bundle (Fig. 1C). In a more rostral plane a small contingent ascends with the fasciculus mammillothalamicus through the lamina medullaris medialis to contribute partly to the innervation of the gyrus cinguli. Further on, some collaterals innervate principally the area hypothalamica lateralis, posterior and dorsalis. Among the more rostral projection areas some are similar to that of the RD (Table l) while the olfactory bulb, the area amygdalae, the cortex pyrilbrmis and the striatum are not significantly labeled; on the contrary, the paleocortical formations (gyrus dentatus, hippocampus and cortex entorhinalis) are highly innervated (Fig. t D) by two systems of dorsal fibers. The first one ascends along the fornix and then incurves caudally to join the fimbria hippocampi. While the second one ascends through the cingulum and then curves back spraying out caudally all along the indusium griseum and distributing some collaterals to the gyrus cinguli.

471

(B) Descending projections (Figs. 6 and 7) The very dense descending projections of the NCS (Figs. 5 and 6) can be differentiated into three descending medial cerebellar, dorsal and paramedian bundles. The medial cerebellar bundle emerges from the ventral border of the NCS. It incurves immediately laterally (Fig. IA) and then ascends along the external board of the medial lemniscus. The fibers then move in a rostro-caudal direction to join the brachium pontis and distribute into the deep nuclei of the cerebellum and the cortex cerebelli. (2) The fibers of the dorsal descending bundle are joined in a sagittal lamina emerging progressively from the dorsal board of the nucleus, as they ascend inside the fasciculus longitudinalis medialis and distribute into the subtantia grisea centralis. This system sends numerous collaterals along its way. They are aimed successively at the nucleus incertus, the dorsal tegmental nucleus of Gudden and the locus coeruleus. A lateral branching reaches the dorsolateral and ventromedial marginal nuclei of the brachium conjunctivum. More caudally these fibers follow the dorsal region of the fasciculus longitudinalis medialis, then the dorsolateral border of the genu nervi facialis to finally fade out progressively through the nucleus intercalatus. (3) The paramedian bundle is composed by an important contingent of fibers emerging from the caudal part of the NCS. These fibers sweep vertically along the raph6 overlapping it largely on the reticular formation. They successively invade the nucleus reticularis tegmenti pontis, the RPo and the RM. A part of these fibers move in a dorsal direction along the fasciculus longitudinalis medialis, contributing to the constitution of the descending dorsal bundle. More caudally this contingent subdivides into two distinct peduncles: the first one forms a narrow paramedian lamina, which descends at the level of the nuclei raph6 pallidus and obscurus. The second peduncle moves aside progressively from the midline to densely occupy the region of the nucleus reticularis paramedianus. In the pons and medulla (Table 1) the descending dorsal bundle sends many collaterals towards some cranial nerve nuclei. The paramedian bundle shares a large part in the innervation of the main nuclei of the reticular formation of the pons and medulla, and the remaining cranial nerve nuclei. The cochlear complex, mainly the pars dorsalis, the superior and inferior olivary complex also receive some projections.

(llI) Projections of the nucleus raph~ pontis ( A ) Ascending projections The ascending projections of the RPo (Figs. 15-17) are organized into three well-separated systems: a dorsal ascending bundle, a cerebellar bundle and a ventrolateral bundle. The dorsal bundle is constituted by fibers ascending from the dorsal pole of the RPo along the raph6 and the fasciculus longitudinalis medialis up to the substantia grisea centralis. An important lateral density is localized in the region of the locus

472

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Figs. 14-17. Schematic representation of the efferent connections from the nucleus raph6 magnus (M, left half of the section) and the nucleus raph6 pontis (P, right half of the section) as outlined in a stereotaxic atlas of the cat brain. The rostral projections have been described every 3 mm.

473

Fig. 15.

474

.x,

Fig. 16.

4~ ~h

476 coeruleus and the marginal nuclei of the brachium conjunctivum. More rostr,llly a small diffuse peduncle progresses in the ventral part of the mesodiencephalic substantia grisea and appears to fade out in the nucleus periventricularis anterior. A second contigent accompanies the dorsal region of the fasciculus Iongitudinalis medialis during its mesencephalic course and then diffuses in the preoculomotor complex. (2) The medial cerebellar bundle is represented by an important group of ventral fibers moving rostrally from the ventrolateral bundle (see below) in a more lateral direction through the fibrae pontis transversae. Then they turn back dorsocaudally along the medial and lateral lemniscus. This peduncle progresses further along in the brachium pontis to distribute itself rather densely both in the cerebellar cortex and the cerebellar nuclei. (13) The ventro-lateral bundle, which is more important, comes from the rostral pole of the RPo. The fibers run through the nucleus reticularis tegmenti pontis and progressively sweep ventrolateralty to form a dense laminar mass localized between the ventral border of both NCS and interpeduncular nucleus and the mediolateral region of the isthmic tegmentum. Dorsolaterally there is a diffuse dispersion of short axons through the pontine reticular formation. In the caudal part of the mesencephalon, the ventrolateral bundle leaves an important peduncle which moves aside the laterodorsal region of the laminar mass precedently described. These fibers ascend vertically through the mesencephalic reticular formation (Fig. 2A) outside of the ventral tegmental nucleus of Gudden and on both sides of the nucleus cuneiformis. Then they ramify densely in the substantia grisea centralis of the mesencephalon. While some collaterals invade the lateral division of the RD, others terminate in the superior colliculus. The remaining fibers of the main bundle form two distinct branchings on both sides of the red nucleus (Fig. 2A). The dorsal fibers innervate the nuclei of the posterior commissure and the pretectal region while the ventral fibers subdivide at the level of the Forel's field in two components which innervate the thalamic nuclei (Table I). At the level of the hypothalamus, a high degree of labeling is localized in the nucleus paraventricularis hypothalami while an area of small density spreads out through the area hypothalamica lateralis, posterior and dorsalis. Some fibers move in a ventral direction towards the controlateral lateral hypothalamus via the supra-optic commissure (Fig. 2B), while some others ascend along the superior board of the optic tract in the direction of the nucleus supraopticus and nucleus suprachiasmaticus. The more rostral fibers fade out successively in the preoptic region, the septum and the diagonal band of Broca (Fig. 2C). Few extend through the internal capsule and appear to project very slightly to the neocortex. Some run through the ansa lenticularis and are responsible for a clear labeling limited to the nucleus amygdalae centralis medialis. There are no projections either in the striatum or in the hippocampus.

477

t

1 M

P

Fig. 18. Schematic representation of the descending projections from the nucleus raph6 magnus (M, left half of the section) and the nucleus raph6 pontis (P, right half of the section) as outlined in a transverse section of the 7th cervical segment of the cat's spinal cord (according to Papez~4).

( B) Descendingprojections The RPo distributes abundant projections in all the reticular formation (Figs. 14, 15 and 18) through three different bundles. (1) The dorsal fibers ascend vertically to the periventricular gray matter along the fasciculus longitudinalis medialis. Then they turn back caudally and run mainly in the dorsolateral region of the fasciculus longitudinalis medialis and continue through the nucleus intercalatus. Some collaterals are also disseminated towards several brain stem nuclei (Table 1). (2) The distribution of the lateral fibers through the reticular formation is very heterogeneous. Some spread out laterally in the plane of the RPo, while others leave the descending course of the paramedian bundle to terminate in most of the nuclei of the pontine and bulbar reticular formation. (3) The paramedian fibers form a compact group, overlapping the reticularis tegmenti ponti and largely laying out caudally, upwards along the raph6 and laterally in the reticular formation. This bundle descends successively through the caudal raph6 nuclei and then the nucleus reticularis paramedianus. Some of its fibers cross through the ventral commissure of the cord while descending more caudally in the cervical medulla where they subdivide further on in the medioventral region of the ventral horn (funiculus ventralis) along the tractus cerebrospinalis ventralis and the tractus tectospinalis. The fibers which follow the tractus spinocerebellaris dorsalis are localized in the funiculus lateralis (Fig. 3C). A few projections are observed in the substantia grisea.

(IV) Projections of the nucleus raphe magnus (A) Ascending projections The ascending projections of the RM present the same general organization as their homologues of the RPo (Figs. 15-17).

478 (1) The dorsal bundle distributes in the substantia grisea centvalis and laterally, sending several ramifications towards the locus coeruleus and the dorsolateral and ventromedian areas of the brachium conjunctivum. Further on, the projections fade out rapidly and no labeling of the nucleus periventricularis anterior is observed. The dispersion of the fibers ascending along the fasciculus longitudinalis medialis is similar to that of the RPo however with a denser labeling localized in the nucleus of Darkschewitsch. (2) The main ascending bundle originates from the rostral pole of the RM. It forms a paramedian triangular mass whose base is lying on the dorsal surface of the trapezoid bodies. During their frontal progression through the RPo and the nucleus reticularis tegmenti pontis a part of the fibers deviate gradually from the midlinc to ascend successively around the ventrolateral region of the NCS (with some slight projections in its medial part) and the interpeduncular nucleus. Then, they extend more laterally between the dorsal surface of the median division of the nucleus pontis to the central area of the reticular formation (Fig. 2F). A great number of fibers spread throughout the pontine and mesencephalic reticular formation. Some dorsolateral extensions are observed towards some cranial nerve nuclei. At the level of the isthmic tegmentum the main ascending bundle gives rise to two secondary branchings: the caudal fibers innervate the superior colliculus and the substantia grisea centralis (Fig. 2G). More rostrally the remaining fibers are responsible for the labeling of the pretectal area, the nuclei of the posterior commissure and the thalamic region (Table l). Finally, the hypothalamic region and the diagonal band of Broca appear to be the terminal site of the more rostral fibers.

(B) Descendingprojections The important descending efferent systems of the RM (Figs. 14 and 18) also have some similarities with those of the RPo. The topography of the bulbospinal pathways is however somewhat different. (1) A diffuse system of lateral fibers spreads throughout the pontine reticular formation with some ventral ramifications to the inferior olivary complex and the nucleus facialis. (2) A dorsal descending bundle leaves the dorsolateral border of the RM to ascend along the fasciculus longitudinalis medialis. Then the fibers run back proceeding caudally in the mediodorsal part of this bundle, being localized laterally inside the genu nervi facialis and the nucleus hypoglossi. Some fibers cross along the dorsal commissure of the spinal cord and descend over the central canal, intermingling with the dorsal fasciculus proprius (Fig. 3B). (3) The majority of the fibers leave the caudal pole of the RM to form the paramedian group. This bundle runs through the raph6 nuclei and gradually spreads ventrolaterally along the nucleus reticularis paramedianus, the nucleus reticularis lateralis and the tractus spinocerebetlaris ventralis to reach the spinal cord (Fig. 3A). Then these fibers are localized ventrolaterally at the ventral horn. The more lateral fibers descend with the tractus spinocerebellaris ventralis while the mediodorsal ones

479 cross first at the level of the ventral commissure of the spinal cord (dessatio pyramidum) before moving ventrally along the ventral fasciculus proprius towards the tractus vestibulospinalis. Finally some terminal areas of projections occur mainly in the ventral horn (Fig. 3B). (4) From the dorsal border of the RM ascends the inferior cerebellar bundle via the restiform body. A slight labeling is observed in the cerebellar cortex (Fig. 2F).

(V) Control injections after raphd lesions In the first experiment, [14C]leucine was injected in the RD or in the RM either before or after the lesion (Fig. 2D and E) to determine whether intact neurons or those localized at the periphery of the lesion site and in the passive diffusion path of the radioisotope were actively labeled in their axonal projections. In the two cases we observed the disappearance of the projections from these two nuclei. One must note however that the lesion of the RM was somewhat more important and extend over the nucleus reticularis gigantocellularis. In the second experiment, the injection of [14C]leucine was placed laterally in the isthmic reticular formation following the lesion of the NCS (Fig. 4A and B). In this case the ascending or descending labeling is quite different from the typical one observed after injection in the NCS. It occurs ipsilaterally in the central tegmental field (Fig. 4C), dorsally to the red nucleus and reaches the Forel's field H l and the lateral part of the zona incerta. Some bilateral descending labeling is observed in the dorsal tegmental nucleus of Gudden, the substantia grisea centralis and the nucleus locus coeruleus. However no labeling is observed in the marginal nuclei of the brachium conjunctivum, the pontine reticular formation, the cranial nerve nuclei or the cerebellum. DISCUSSION

(1) Validity of the method As shown by numerous studies 11 the injected labeled amino acids are specifically taken up by the cell bodies. This eliminates the putative cause of errors which may be encountered when using lesion and degeneration technique (i.e. associated lesion of'fibres de passage'). However, the limitation of this method should be emphasized: the distinction between 'fibres de passage' and the areas of terminal projections is not easy when densely labeled bundles are running through some nuclei, i.e. the raph6 nuclei, the dorsolateral region of the pontine tegmentum and the oculomotor nuclei. However, in the majority of the cases these difficulties have been overcome by varying the times of exposure. Furthermore, compared with the corresponding Nissl stained sections, the macroautoradiographic technique appears to be sufficiently precise since we could easily distinguish a differential labeling at the level of structures as small as the habenular nuclei. Finally the diminution of the times of exposure, the reproduction of the results and the rapidity of reading the slides has convinced us that, concerning the global and regional outline of the anatomical projections of the raph6 system, this technique may present some advantages to the dipping one. Of course

480 another approach would be to use this latter technique in a more detailed ~tud~. Another, more important, drawback concerning the autohistoradiographic method consists of the uncertainty of the exact extension of the cellular labeling area responsible for the orthograde transport of labeled molecules. From the work of others ~4 and from our studies (different time exposures at the level of the injection site), we have to admit the existence of a gradient of labeling whose intensity decreases rapidly from the site of injection of the radioisotope and its area of gliosis. The results of our control studies show that if a lesion is made either 30 min before or' after the labeling of a raph6 nucleus, there is no labeling of the projection of this nucleus. Thus the passive transport of labeled elements outside the area of injection cannot be responsible for any further transport. On the other hand, we have shown that an injection of [~4C]leucine immediately parasagittal to a lesion of the NCS gives rise to labeled projections whose topography is ipsilateral and different from those ol the NCS. Of course, and this criticism is very hard to refute, it could still be objected that some labeled fibers extending forward from the injection site originate lateral to the raph6 region but immediately cross over to the opposite side. In such a case, these fibers would thus be interrupted at their decussation by lesions in the midlinc.

(II) General organization of the projections These projections can be summarized as follows. Most of the fibers emerging from both the anterior and posterior nuclear groups of the raph6 (according to the terminology of Taber et al. 46) can be grouped into 4 common and organized bipolar systems, ascending or descending. Some of these systems follow classical anatomical pathways: an ascending ventrolateral system via the ventral tegmental region of Tsai, then the enlarged medial forebrain bundle; a dorsal ascending and descending periventricular system which closely parallel the dorsal longitudinal bundle of Schfiltz; a paramedian descending system partly along the median longitudinal bundle; a medial and inferior cerebellar system. Finally a fifth group, less well individualized, is represented by the centrolateral diffuse system which distributes mainly to the reticular formation. Our results also show the organization and the wealth of the monosynaptic projections of the raph6 nuclei which are related to most cerebral structures. Globally the main areas of projections are represented rostrally by the mesencephalic region, the non-specific thalamic nuclei, the hypothalamic junction, the enlarged prosencephalolimbic system, the striatum and the neocortex; caudally by the pontine and bulbar reticular formation, the cranial nerve nuclei, the spinal cervical cord and the cerebellum. There is much evidence that the projections of the 4 raph6 nuclei are also topically organized (see Table 1). Thus some structures are innervated more or less densely together by 2 or 3 nuclei or even simultaneously by the 4 raph6 nuclei. However some terminal fields are specifically related to one nucleus. Some important descending pathways from the caudal raph6 have also different localizations through the spinal cord (Fig. 18). Finally we should again emphasize the rather peculiar projections of the RPo which is connected rostrally to the limbic system, dorsally to the cerebellum and caudally to the medulla.

481 Our data confirm and extend largely the results obtained in the cat with other techniques (lesion, degeneration). Thus Nauta and Kuypers 31 and Brodal et al. v observed long ascending fibers to the mesodiencephalon, and probably beyond, originating from the rostral group while descending fibers were exhibited from RM. Some projections were also discovered from the RPo to the deep cerebellar nuclei. In a more recent study TM following lesion of the substantia grisea centralis and the rostral part of RD, some degenerating fibers could be seen rostrally in the field of Forel, the ventral tegmental area and caudally in the nucleus olivaris inferior. Our data can also be compared with those recently obtained with the horseradish peroxidase technique (HRP). Thus Llamas et al. '~6 have shown that the projections towards the gyrus proreus come from both the NCS and RD. But they failed to find any efferents from these nuclei to the occipital or parietal cortex. However following injection of HRP in the occipital cortex, Leger et al. 25 could visualize the reaction product in a few neurons localized in the RD. This finding is in accordance with our results since we have found that the projection to the frontal cortex was much more important than other cortical areas. These authors have also shown that there are projections from the RD to the caudate nucleus (personal communication) and the lateral geniculate body '~4. Each of the RD, RPo and RM nuclei send projections to the locus coeruleus 38. Furthermore Kuypers and Maisky '~2 have demonstrated that many neurons in the area of the bulbar raph6 distribute fibers to the spinal cord. In the rat, the connections of both NCS and RD have been studied by autoradiographic and degeneration techniques 10. The general picture agrees with the pattern found in the cat although the respective projections of the two nuclei could not be differentiated due to the overlapping areas of each nucleus. However some important differences can be noted between these two species. First, the nucleus suprachiasmaticus and the nucleus supraopticus are not labeled in the rat. In other words one must emphasize in the rat, as well as in the cat, the absence of labeling of the nucleus hypothalami ventromedialis. The following other structures, which are labeled in the cat and do not seem to receive projections in the rat, are respectively the lateral geniculate nuclei, the cortex pyriformis and the neocortex. Only a few projections were found in the ventral part of the caudate nucleus in the rat. But the more important differences concern the absence of descending projections to the cranial nerve nuclei of the brain stem and caudally below the nucleus originis n. facialis and the absence of cerebellar innervation. Several explanations could be advanced in respect to these discrepancies. First as noted by the authors, there may be a more selective uptake of [3H]proline by different populations of neurons as compared to [14C]leucine. The limits of the sensitivity of the method c(.uld also fail to detect label in terminal areas at a long distance from the cell bodies or in structures poorly innervated such as the lateral geniculate body and the nucleus suprachiasmaticus. Most likely the main differences could be assigned to some degree of variability among species.

(11I) The problem of 5-HT-containing neurons Many histochemical and biochemical studies combined with experimental

482 lesions z,:',L:',~ have demonstrated the serotoninergic nature of the long asceiicl!ng

and descending efferent prctiections arising from the raph6 nuclei complex ~ho~c topography is in accordance with our data in the cat. There is less e~idence thai ~lssumes a similar correspondance in the cat since the non-specific labeling of the raph~5 neurons by the [14C]leucine does #tot allow differentiation of the serotoninergic neurons from those whose nature and organization remain unknown. In fact while the dist,ibution of 5-HT-containing neurons in the raphe nuclei of the cat have been described :~:~, the histofluorescence mapping of the indoleaminergic pathways is still lacking. Conclusion

In conclusion, the main ascending connections of the brain stem raph6 nuclei allow us to extend the concept of the midbrain limbic circuit '~9. Indeed we can include the raph6 system in the ascending pathways of the limbic system : ascending component of Schiiltz dorsal longitudinal fasciculus originating throughout the substantia grisea centralis and the system of the mammillary peduncle arising from both nuclei of Gudden. Furthermore as suggested by Nauta ~° the frontal lobe, which receives the most important neocortical projections from the raph6 anterior group, can be considered as the neocortical representation of this limbic system. Secondary branchings of this system lead to the midbrain tegmentum and to the intralaminar nuclei of the thalamus. It appears also that the raph6 nuclei by their projections to the reticular formation have close relations with the reticular ascending activating system of Muruzzi and Magoun 2s. Finally by their multiple connections with the limbic system, the reticular formation and the catecholaminergic system, the raphe nuclei appe~/r to play a strategic role in the regulation of many functions. ABBREVIATIONS A, AA, ACM, AD, AL, ATV, C, CA, CAI, CC,

CCA, CCI, CCS,

CD, CE, CG, CI, CL, CLA, CM, CO, COD, COV,

nucleusaccumbens; area amygdaloidea anterior; nucleusamygdalae centralis medlalis: nucleusanterior dorsalis: ansa lenticularis; area tegmentalis venlralis Tsai; nucleus caudatus; commissuraanterior: capsulainterna; cortexcerebelli; corpuscallosum; commissuracolliculi inferioris; commissuracolliculi superioris; cornu dorsale: capsulaexterna : corpus amygdaloideum; colliculusinferior: cornu laterale : claustrum; nucleuscentri mediani; chaismaopticurn; nucleuscochlearis dorsalis nucleuscochlearis ventralis;

CP, CSO,

commissuraposterior commissurasupraopticae; CT, corpus trapezoideum; CU, nucleuscuneatus; CV, cornu ventrale; DBB, diagonalband of Broca; DCGL, nucleus dorsalis corporis geniculati lateralis; DP, decussatiopyrarnidum; DPCS, decussatio pedunculorum cerebellarium superiorum; DR, radix dorsalis; DT, decussationestegmenti; EN, nucleusentopeduncularis; ENT, cortexentorhinalis ; F, fornix; FCD, funduscaudati; FD, funiculusdorsalis ; FL, funiculuslateralis; FLM, fasciculuslongitudinalis medians: FR, fasciculusretroflexus; FRM, formatio reticularis mesencephali; FRP, formatio reticularis ponti;

483 FV, G, GC, GM,

funiculus ventralis; genu nervi facialis; gyrus cinguli ; nucleus marginalis corporis geniculati medialis ; GP, globus pallidus ; HD, area hypothalamica dorsalis; HDM, nucleus hypothalami dorso medialis; HI, hippocampus ; HIA, area hypothalamica anterior; HL, area hypothalamica lateralis; HP, area hypothalamica posterior; HI, forel H 1 ; H2, forel H2; 1, indusium griseum ; IC, nucleus interstitialis cajal; IO, nucleus olivaris inferior; 1OD, nucleus olivaris inferior dorsalis; IOM, nucleus olivaris inferior medialis; IP, nucleus inter peduncularis; LC, nucleus locus coeruleus; LI, nucleus linearis intermedius; LL, lemniscus lateralis; LLV, nucleus lemnisci lateralis ventralis; LM, lemniscus medialis; LR, nucleus linearis rostralis; MFB, fasciculus medialis prosencephali; Mm, corpus mammillare; NCP, nucleus commissurae posterioris; NCS, nucleus raph6 centralis superior; NCT, nucleus corporis trapezoidei; NCU, nucleus cuneiformis ; ND, nucleus Darkschewitsch; NE, nucleus Edinger Westphal; NHM, nucleus habenulae medialis; NHL, nucleus habenulae lateralis; NHVM, nucleus hypothalami ventromedialis; NIC, nucleus intercalatus ; NMD, nucleus medialis dorsalis; NPD, nucleus parabrachialis dorsalis; NPR, nucleus praethalamicus; NPV, nucleus parabrachialis ventralis; NRD, nucleus reticularis medullae oblongatae pars dorsalis; NRP, nucleus reticularis paramedianus; NRV, nucleus reticularis medullae oblongatae pars ventralis; NSC, nucleus suprachiasmaticus ; NSO, nucleus supraopticus; NTD, nucleus tegmenti dorsalis Gudden; NTS, nucleus tractus solitarii; NTV, nucleus tegmenti ventralis Gudden; NTV, nucleus tractus spinalis nervi trigemini ; NVM, nucleus ventromedialis; NVPL, nucleus ventralis posterolateralis; NVPM, nucleus ventralis posteromedialis; N Ill nucleus originis nervi oculomotorii;

N IV, NVM, NVS, N VI, V VII, NX, N XII, P, PC, PC1, PCM, PCS, PF, PH, PI, PO,

POR, PRT, PT, PU, PVA, PV H,

R, RD, RE, RET, RGI, RH, RL, RM,

RO, RP, RPC, RPOC,

RPO, RPOO, RTP, SA, SB, SC, SGC, SM, SNC, SNL, SNR, SO, SOL, SOM, SPF, SPT, STH, ST, SUM, TM, TML,

nucleus originis nervi trochlearis; nucleus motorius nervi trigemini; nucleus sensorius superior nervi trigemini ; nucleus originis nervi abducentis; nucleus originis nervi facialis; nucleus originis dorsalis vagi; nucleus originis nervi hypoglossi; tractus corticospinalis ; pedunculus cerebri; pedunculus cerebellaris inferior; pedunculus cerebellaris medialis; pedunculus cerebellaris superior; nucleus parafascicularis ; nucleus prepositus hypoglossi; cortex pyriformis ; nuclei pontis ; regio preoptica ; area pretectalis; nucleus parataenialis; putamen ; nucleus periventricularis anterior; nucleus paraventricularis hypothalami; nucleus ruber; nucleus raph6 dorsalis; nucleus reuniens; nucleus reticularis ; nucleus reticularis gigantocellularis; nucleus rhomboideus ; nucleus reticularis lateralis; nucleus raph6 magnus; nucleus raph6 obscurus; nucleus raph6 pallidus; nucleus reticularis parvocellularis; nucleus reticularis pontis caudalis; nucleus raph6 pontis; nucleus reticularis pontis oralis; nucleus reticularis tegmenti pontis; striae acusticae; subiculum; nucleus subcoeruleus ; substantia grisea centralis; stria medullaris ; substantia nigra zona compacta ; substantia nigra pars lateralis; substantia nigra zona reticularis; stratum opticum colliculi superioris; nucleus olivaris superior lateralis; nucleus olivaris superior medialis; nucleus subparafascicularis ; septum; nucleus subthalamicus; stria terminalis ; decussatio supramammillaris; tractus mesencephalicus nervi trigemini; nucleus centra[is, pars lateralis ;

484 TMM, TMT,

nucleus centralis, pars medialis; tractus mammillothalamicus; TO, tractus opticus; TOL, tractus olfactorius lateralis: TS, tractus solitarius: TS V, tractus spinalis nervi trigemini; TU, tuberculum olfactoriunl ; VGCL, nucleus ventralis corporis geniculati lateralis :

Vl, VL, VM, VR. VS. V, Z I,

nucleus vestibularls inferior; nucleus vestibularis laleralis; nucleus vestibularis medialis; radix ventralis: nucleus veslibularis superior nervus trigeminalis zona incerta ;

NOTE ADDED IN PROOF After this paper was submitted for publication, two new reports on the projections o f the raph6 nuclei appeared (Shinnar, S., Maciewicz, R. J. a n d Shofer, R. J., A raph6 projection to cat cerebellar cortex, Brain Research, 97 (1975) 139-143 a n d T a b e r Pierce, E., Foote, W. E. a n d Hobsen, J. A., The efferent c o n n e c t i o n of the nucleus raph6 dorlaris, Brain Research, 107 (1976) 137-144). ACKNOWLEDGEMENTS This study was s u p p o r t e d by C N R S (LA 162), D R M E (74-232) a n d I N S E R M ( U 52). The a u t h o r s wish to t h a n k Miss A. M c R a e for her careful correction of the manuscript.

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