Brain Research, 107 (1976) !37-144 © ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands

137

The efferent connection of the nucleus raphe dorsalis

ELIZABETH TABER PIERCE, W A R R E N E. FOOTE AND J. ALLEN HOBSON Department of Anatomy, Harvard Medical School, Boston, Mass. 02115, (W.E.F.) Department of Psychiatry, Massachusetts General Hospital, Boston, Mass. 02114 and (J.A.H.) Laboratory of Neurophysiology, Massachusetts Mental Health Center, Boston, Mass. 02115 (U.S.A.)

(Accepted January 28th, 1976)

The probability that the raphe and reticular nuclei might be interconnected was noted by Ram6n y Cajal iv. Renewed interest in this possibility had followed the discovery that the raphe contains serotonergic neurons2,10 with serotonin implicated as the neurotransmitterS,a in a variety of physiological states ranging from psychosis to sleep. The reciprocal firing of raphe 16 and reticular neurons 11 in the sleep cycle has suggested that one functional consequence might be sleep cycle regulation by reciprocal interaction of raphe and reticular cell groups 15. A detailed anatomical study concerning the projections and terminal sites of the individual raphe nuclei is basic to a full understanding of these suggested reciprocal interactions. To this end the following results are presented based upon observations using the autoradiographic technique. In this report the distribution of efferents from the caudal portion of the dorsal raphe nucleus (DRN), the largest raphe nucleus, is documented and confilms many of the rostral connections suggested by Bobillier et al. in the cat 3 and by Conrad et al. in the rat v. This study also shows that many caudal brain stem structures, including the pontine and medullary reticular nuclei, receive significant projections. These data thus indicate the presence, within the DRN, of subpopulations with selective projections to rostral and caudal target sites. The isotope (L-[2-aH]proline, 5 Ci/mmole, New England Nuclear) was concentrated by evaporation under vacuum the day before injection, and diluted with saline (0.9 N) to a concentration of 50 pCi//zl just prior to surgery. Six cats were anesthetized with sodium pentobarbital (Nembutal, 40 mg/kg) and placed in a stereotaxic apparatus. A midline incision was made in the scalp and the underlying fascia and muscle retracted. A burr hole was made in the bone overlying the cerebellum and the dura incised. The anterior midline portion of the cerebellum was removed by gentle suction until the floor of the fourth ventricle was exposed. A glass micropipette, 30 #m in external diameter, connected by polyethylene tubing to a 10 pl Hamilton syringe was stereotaxically positioned and 0.5 #l injected mechanically over a 30 min period. The pipette was withdrawn and the injection site washed with saline. A plastic prosthesis was used to close the defect in the skull and the muscles and the skin sutured. The

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Fig. 1. Efferent pathways 0arge dots) and terminal sites (small dots) emanating from the caudal portion of nucleus raphe dorsalis (J) illustrated in selected drawings of transverse sections of cat's brain. Abbreviations for this and subsequent figures: A, nuclei amygdaloidalis; C, nucleus cunoatus; CB, cingulum; CS, nucleus centralis superior; DT, nucleus dorsalis tegmenti (yon Gudden); HB, nuclei habenulae; I, inter; IO, nuclei olivaris inferior; IP, nucleus interpeduncularis; LGB, nuclei corpus lateralis geniculati; MB, nuclei mammillaris; MFB, pedunculus medialis prosenccphalon (medial forebrain bundle); MGB, nuclei corporis medialis goniculati; MLF, fasciculus longitudinalis mexiialis; RN, nucleus rutmr; ST, stria terrninalis; V, nucleus nervi motorius trigemini; VII, nucleus nervi facialis; VIII, nuclei vestibularis; XII, nucleus nervi hypoglossi.

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Fig. 2. A: site of isotope injection in caudal portion of nuclcusfaphe dorsalis, x 250, toluidinc blue. B: labeled axons in nucleus cuneiformis, x 400, toluidine blue. C-E: density variations in labeling classified heavy (C), moderate (D), and light (E). x400, toluidine blue.

141 TABLE I DENSITY OF TERMINAL LABELING AT VARIOUS TARGET SITES FOLLOWING INJECTION OF [3H]PROLINE IN THE NUCLEUS RAPHE DORSALIS

Site

Terminal density Heavy

Medium

Light

Periaqueductal gray (ventral and lateral area) N. interpeduncularis N. substantia nigra (zona compacta)

Periaqueductal gray (dorsal area) N. ruber N. III (lateral)

Colliculus superior (stratum profundis) Area pretectalis N. III

N. cuneiformis N. subcuneiformis

N. Edinger Westphal

Ascending Mesencephalon

Diencephalon

Corpus mammillaris N. medialis N. lateralis

N. premammillaris N. hypothalamicus dorsalis N. hypothalamicus anterior N. rhomboidalis N. centromedian N. intermediodorsalis

N. anterior N. supramammillaris Area hypothalamicus posterior Area hypothalamieus N. periventralis lateralis N. habenularis lateralis N. mediodorsalis N. parataenialis N. habenularis medialis Telencephalon

N. N. N. N.

septi lateralis septi accumbens accumbens amygdaloideus medialis Hippocampus

N. globus pallidus Gyrus cingulus

Descending Rhombencephaion

N. pontis centralis oralis N. pontis centralis caudalis N. gigantocellularis N. medullae oblongatae centralis, ventralis N. raphe magnus N. N. raphe obscurus N. praepositus hypoglossi N. parvocellularis N. N. tractus solitari N.

All sensory and motor cranial nerve nuclei

paragigantocellularis lateralis

paragigantocellularis dorsalis reticularis paramedianus No cuneatus lateralis N. olivaris inferior (specific areas)

Injection level

N. locus coeruleus N. parabrachialis dorsalis N. parabrachialis medialis

142 animals were maintained for 5 days post-operatively in order for slow transport of the amino acid to occur. The animals were then anesthetized with sodium pentobarbital and perfused through the heart with saline followed by 10~ formol-saline. The brain was removed 4 h later, post-fixed for one week, dehydrated, cleared, embedded in paraffin and sectioned serially in either the sagittal or transverse plane. Every tenth section was mounted. The slides were deparaffinized, coated in the dark with Kodak NTB2 liquid nuclear emulsion and exposed at 4 °C in light-tight boxes with Drierite for selected periods of time, ranging from two weeks to three months (in order to determine optimum exposure times), developed with Kodak D-1 9 at 16 °C for 3 min, fixed in hypo for 5 min, stained with toluidine blue, coverslipped with Permount and examined with the light microscope for the presence of silver grains.

(A) Site of injection of label In the three cases with an injection in the caudal pole of the nucleus raphe dorsalis, the location and spread of the isotope were similar. The injection area (Fig. 2A) also included the dorsal tegmental nuclei of von Gudden and the dorsal tip of the nucleus centralis superior. Neurons in these areas were heavily labeled. There was no spread of label to other nuclei.

(B) Pathways (Fig. 1) (1) Ascending. From the ventral border of the dorsal raphe nucleus fibers coursed ventrally along the lateral edge of the nucleus centralis superior; upon reaching the lateral border of the nucleus interpeduncularis the fibers turned rostrally to ascend within the medial forebrain bundle. At rostral levels, numerous labeled fibers were found within the diagonal band of Broca, stria medullaris, stria terminalis, cingulate bundle, fornix and the fasciculus retroflexus. In addition numerous dispersed ascending fibers (Fig. 2B) were located within the periaqueductal gray, the medial longitudinal fasciculus and the tegmentum. (2) Descending. From the injection site many labeled fibers descended within a broad area ventral to the medial longitudinal fasciculus and lateral to the raphe. More dispersed descending fibers were located within the periaqueductal gray and tegmentum. Descending fibers in decreasing numbers were traced to caudal medullary levels.

(C) Terminals Areas containing a density of silver grains above background count were considered terminal sites of neurons labeled with the isotope. The density of the silver grains was characterized as heavy (Fig. 2C), medium (Fig. 2D) and light (Fig. 2E). The terminal sites are shown in Fig. 1 and Table I. Many of the projections are paramedian with respect to the brain stem core. Other significant observations are: the ventral nucleus of the lateral geniculate body was the only geniculate area to receive any (very light) labeling, and the supraoptic nucleus and putamen received none. By contrast supraependymal areas in all ventricles were labeled with light to heavy intensity. The present results reveal notable similarities and some differences from previous studies of DRN efferents; most of the rostral terminal sites found in earlier autoradiographic studiesz,7 have been confirmed except that no projection to the

143 caudate nucleus was found and only a very light projection to the ventral lateral geniculate body was seen. The differences are probably due to the more restricted and/or more caudal placement of the injection sites in this study, a fact which may also account for the extensive descending projections seen which were not previously evident in the autoradiographic data. Although moderately labeled, the pontine targets found here are extensive, ranging throughout the brain stem and including other raphe nuclei as well as cranial nerve nuclei. Some of the pontine terminals were found to be coextensive with degenerating terminals observed after lesions of the dorsal and deep tegmental nuclei4, however, the raphe efferents observed went beyond the most caudal level found for these lesions. In addition, the terminal sites observed in this study along the ventricular side of the ependyma is consistent with the findings of previous studies1, is demonstrating the presence of serotonin nerve terminals in the ependyma of the ventricles and the cerebral aqueduct. These data and those of Bobillier e t al. 3 also suggest that the DRN and the nucleus centralis superior contain subpopulations of neurons which project differently to selective terminal sites. Leger e t al. ~4 report afferents to the dorsal nucleus of the lateral geniculate body from the anterior portion of the dorsal raphe nucleus; results which further substantiate this hypothesis. The cytoarchitecture of the two nuclei has been described for the cat z0 and, in each nucleus, more than one cell type is recognized. Further, the configuration of each nucleus is unique: the DRN contains a central portion flanked by bilateral dorsal and ventral wings, while the nucleus centralis superior contains a central portion of medium sized cells surrounded by a peripheral zone of smaller cells. It is conceivable that the central and peripheral regions of these nuclei contain varying proportions of 5-HT neurons and that neurons in different regions of the nuclei have distinctive terminal destinations. Use of a retrograde tracing method, such as horseradish peroxidase, would help to substantiate this hypothesis. Experiments designed to show selective uptake of 5-HT in cell soma would also be helpful in determining the position and size of serotonin-containing neurons. The more interesting question of the function of this perviously unidentified descending serotonergic pathway remains a matter for speculation. The descending projection may be related to the raphe-reticular connections noted by Ram6n y Caja117 in which case one would want to investigate the possibility that this pathway forms one part of the substrate for reciprocal raphe-reticular interaction. For their help in this project appreciation is due Louise Edwards, Nora Friel, Peter Ley and Tom McKenna. This work was funded by research grants Milton Fund (E.T.P.), MH 13923 (A.H.), and NS 12050 (W.F.).

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The efferent connection of the nucleus raphe dorsalis.

Brain Research, 107 (1976) !37-144 © ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands 137 The efferent connection of the nu...
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