Nuclei of the Human Raphe

Okajimas Folia Anat. Jpn., 89(1): 15 – 22, May, 2012

Nuclei of the human raphe

By

Masakazu SHIBATA1), Noboru GOTO2), Jun GOTO2) and Naoko NONAKA3)

1)Kanagawa University of Human Services, Faculty of Health and Social Services, 10-1 Heiseicho 1, Yokosuka 238-8522, Japan 2)Showa University School of Medicine, First Department of Anatomy, Tokyo, Japan 3)Showa University School of Dentistry, Department of Oral Anatomy & Developmental Biology, Tokyo, Japan

– Received for Publication, May 28, 2012 –

Key Words: Raphe, Raphe nucleus, Serotonin, Brainstem, K-B stain

Summary: Along the raphe of the brain stem, a series of small neuronal groups can be observed in the medulla oblongata, the pons and the mesencephalon. The neurons located in and adjacent to the raphe are considered to produce mainly serotonin (5-HT). The groups of nuclei containing 5-HT were first reported in experimental animals in the early 1960s1). The presence of such nuclei, however, has not yet been brought to light in the human brainstem except the few atlases2,3), although in several neuroanatomy textbooks4), extrapolated data are shown in the form of drawings as if they were the data from the human brain. The aim of this study is to present microscopic photos of such raphe nuclei made from serial sections of the human brainstem, and to clarify the differences between findings in human and textbook drawings from animal data.

Researches on the raphe nuclei has been reported in complete serial transverse sections. Then we stained every experimental animals since the early 1900s5). However, in 5th section alternatively with Kultschitzky and K-B meth- the human brainstem, such group nuclei have not yet been ods. Details of the staining methods have been given in studied precisely. We made serial sections of the human previous reports6). brainstem and cerebellum to present precise findings of the raphe group nuclei.

Results Material and Methods According to our careful observations of the serial We used the brain stem and cerebellum from a 63-year- brainstem sections, the following groups of raphe nuclei old Japanese female cadaver. The cause of death had no can be recognized: (1) nucleus linealis oralis, (2) nucleus direct or indirect connection with the central or peripher- linealis intermedius, (3) nucleus raphe medialis, (4) nu­ al nervous system, and no gross pathological alterations cleus supratrochlealis (or nucleus raphe dorsalis), (5) nu- were observed. We injected 12% formaldehyde solution cleus raphe pontis, (6) nucleus centralis superior medialis, into the femoral artery prior to removing the whole brain (7) nucleus centralis superior dorsalis, (8) nucleus raphe the next day. After further fixation in 3.7% formaldehyde magnus, (9) nucleus raphe pallidus, (10) nucleus raphe ob- solution for one week, we cut the brainstem and cerebel- scurus intraraphalis, and (11) nucleus raphe obscurus ex- lum as one block, and immersed the block into secondary traraphalis. chromic acid fixative6) for two weeks at room temperature. Then we renewed the fixative to continue the immersion another week at 37°C in an incubator. The block was washed using a pipette washer with siphon mechanism, Locations of the Nuclei and Neuronal Findings and was dehydrated in a series of graded alcohol and fol- lowed by embedding in celloidine (nitrocellulose). The Precise nuclear locations and their neuronal findings celloidine block was sliced at 40 μm thickness to make are described below.

Correspondence: Masakazu Shibata, Kanagawa University of Human Services, Faculty of Health and Social Services, 1-10-1 Heiseicho, Yokosuka 238-8522, Japan, TEL: +81-46-828-2778, FAX: +81-46-828-2779, E-mail: [email protected] 16 M. Shibata et al.

(1) nucleus linealis oralis (Plate I) (11) nucleus raphe obscurus extraraphalis (Plate IV) This nucleus is located between both sides of the nu­ This nucleus is attached to the raphe, forming a small cleus ruber at the upper level of the mesencephalon. The mass, but its location is also uncertain. The neuronal soma included neurons are quite small in number, and the neu- sizes are the smallest in the raphe groups and measure ronal soma sizes are around 20 μm in diameter. 10 –20 μm in diameter. (2) nucleus linealis intermedius (Plate I) This nucleus is located caudally to the nucleus linealis oralis at the caudal level of the mesencephalon. The neu- rons are packed together and are in larger numbers com- Discussion pared to the nucleus linealis oralis. The soma sizes of included neurons are around 20 –30 μm. (3) nucleus raphe medialis (Plate II) The idea of the grouping of raphe nuclei first came This nucleus is located among the nerve fibers of the from the ordinary staining method used in rabbit brains5). decussatio pedunclorum cerebellarium superiorum. The However, it took more than half a century to discover that somata of the neurons are 30 μm in diameter and are scat- neurons containing serotonin were found not only in the tered among the fibers. raphe nuclei, but also in the surrounding area and the sub- (4) nucleus supratrochlealis (equivalent to nucleus raphe stantia grisea centralis1). Some authors considered the dorsalis in animals, Plate II) ­raphe group nuclei to be phylogenetically old7). The raphe This nucleus occupies the ventral part of substantia gri- nuclear group in humans has been described by several sea centralis which begins at the same level of the nucleus authors2,3,8). Although the midsagittal projection of the nervi trochlealis toward the oral direction. The included ­raphe nuclear group in the human brainstem is fairly small neurons are aggregated together, and are the largest in in size and of elongated shape, except for the nucleus su- number among the raphe group. The somata of neurons pratrochlearis (or nucleus raphe dorsalis in animals, fig. measure 30 – 40 μm in diameter. 1), midsagittal drawings of raphe nuclear groups shown in (5) nucleus raphe pontis (Plate II) textbooks are represented as being larger and not elongat- This nucleus can be observed between the decussatio ed (fig. 2, cat4)). These differences might be derived from pedunclorum cerebellarium superiorum and the fasciculus differences between humans and animals. In animals, ra- longitudinalis medialis. The included neurons are packed phe group nuclei can be divided into two groups: oral and together, forming a small mass. The soma sizes of neurons caudal. In humans, however, this classification remains are 20 – 40 μm in diameter. undecided4). Generally speaking, in animals the caudal (6) nucleus centralis superior medialis (Plate III) groups include the nucleus raphe obscurus, the nucleus ra- The location of this nucleus is evident, even under the phe pallidus and the nucleus raphe magnus, while the oral myelin sheath stain, along the raphe at the upper level of groups include the nucleus raphe pontis, the nucleus raphe the pons. The included neuronal somata are about 30 μm centralis superior, the nucleus raphe dorsalis and the nu- in diameter. cleus raphe linealis4). The caudal groups are considered to (7) nucleus centralis superior dorsalis (Plate III) project to spinal levels, while the oral groups are consid- This nucleus is located near the fourth ventricle at ered to have extensive projections to various nuclei in the the same level as the nucleus centralis superior me­ brainstem. Projections to diencephalic structures are most dialis. The somata of neurons measure 20 – 40 μm in di­ numerous from the nuclei raphe medialis et dorsalis and ameter. from the nucleus raphe pontis. The nucleus raphe medialis (8) nucleus raphe magnus (Plate III) and the nucleus raphe dorsalis also project widely to telen- This nucleus can be observed at the middle level of cephalic structures4). Even though in human raphe group the pons (the same level as the abducent nucleus). The nuclei, the nucleus supratrochlealis (which corresponds to included neuronal somata measure 30 – 60 μm in di­ nucleus raphe dorsalis in animals) consists of a very large ameter. This nucleus contains large size neurons which number of neurons compared to the other nuclei, such ap- are scattered in the nucleus, as the name “magnus” indi- pearance is not contradictory to the developmental en- cates. largement of the human telencephalon. To elicit proof (9) nucleus raphe pallidus (Plate IV) of neuronal connections in humans, it is indispensable to This nucleus is located between both sides of lemnis- precisely examine postmortem brains with various types cus medialis at the lowest level of the pons. The soma of lesions, for example cerebrovascular diseases, cerebral sizes of neurons vary from 20 – 60 μm in diameter. This neoplasms, degenerative diseases, or simple aging, etc. in nucleus contains various sizes of neuronal somata. order to examine the precise sites of lesions, and also to (10) nucleus raphe obscurus intraraphalis (Plate IV) examine secondary degenerative changes of raphe group The location of this nucleus in the raphe is uncertain. nuclei with serial brainstem sections. However, these pro- The neurons are very small in number, however, and their jections have not yet been confirmed in human raphe nu- soma sizes are 40 –50 μm in diameter. clei at present. Nuclei of the human raphe 17

Fig. 1. Localization of human raphe nuclei.

Fig. 2. Midsagittal drawing of the brainstem indicating the positions of the raphe nuclei (cat, Parent, 1996) ● project to the diencephalon and telencephalon ● project to the spinal cord 18 M. Shibata et al.

References 5) Kohnstamm O: Ueber die gekreutz-aufsteigenden Spinalbahnen und ihre Beziehung zum Gowers’schen Strang. Neurol Centralbl 1900; 1) Dahlström A, Fuxe K: Evidence for the existence of monoamine- 19:242–249. containing neurons in the central nervous system. I. Demonstra- 6) Goto N: Discriminative staining methods for the nervous system: tion of monoamines in the cell bodies of brain stem neurons. Acta Luxol fast blue – periodic acid – Schiff – hematoxylin triple stain Physiol Scand, 1964; 62, Suppl 232:1–55. and subsidiary staining methods. Stain Technol 1987; 62:305– 2) Olszewski J, Baxter D: Cytoarchtecture of the human brain stem. 315. 2nd ed, S. Karger, Basel, 1982. 7) Kappers CU Ariëns, Huber GC, Crosby EC: The comparative anat- 3) Goto N: Atlas of the human brainstem and cerebellum. Medical Tri- omy of the nervous system of vertebrates, including man. Hafner bune, Tokyo, 1989. Bul Co, New York, 1960. 4) Parent A: Carpenter’s human neuroanatomy. 9th ed, Williams & 8) Braak H: Über die Kerngebiete des menschlichen Hirnstammes. II. Wilkins, Baltimore, A Waverly Co, 1996; 455. Die Raphekerne. Z Zellforsch 1970; 107:123–141.

ABBREVIATIONS for Plates

4VE: fourth ventricle; ABN: abducent nucleus; BA: basilar artery; BICOL: brachium of inferior colliculus; BSCOL: brachium of superior collicu- lus; CAQ: cerebral aqueduct; CBH: cerebellar hemisphere; CBV: cerebellar vermis; CGS: central gray substance; CMCB: corpus medullaris cer- ebelli; CRC: crus cerebri; CTT: central tegmental tract; DCON: dorsal cochlear nucleus; DNCB: dentate nucleus of cerebellum; DTN: decussation of trochlear nerve; ENCB: emboliform nucleus of cerebellum; E-W: Edinger-Westphal; FBP: fibrae pyramidales; FCE: foramen cecum; FLO: floc- culus; FN: facial nucleus; FN(7): facial nerve; FNCB: fastigeal nucleus of cerebellum; GFN: genu of facial nerve; GNCB: globosus nucleus of cerebellum; GPN(9): glossopharyngeal nerve; ICBP: inferior cerebellar peduncle; ICOLN: inferior collicular nucleus; IPF: interpeduncular fossa; IVN: inferior vestibular nucleus; LGB: lateral geniculate body; LF: longitudinal fibers; LL: lateral lemniscus; LVN: lateral vestibular nucleus; MCBP: middle cerebellar peduncle; MGB: medial geniculate body; ML: medial lemniscus; MLF: medial longitudinal fascicle; MNTN: mesence- phalic nucleus of trigeminal nerve; MVN: medial vestibular nucleus; Ncsd: nucleus centralis superior dorsalis; Ncsm: nucleus centralis superior medialis; NLC: nucleus loci celulei; Nlim: nucleus linealis intermedius; Nlo: nucleus linealis oralis; NO: nodulus; NR: nucleus ruber; Nroe: nucle- us raphe obscurus extraraphalis; Nroi: nucleus raphe obscurus intraraphalis; Nrm: nucleus raphe magnus; Nrp: nucleus raphe pallidus; Nst: nucleus supratrochlealis; OLN: olivary nucleus; OMN: oculomotor nucleus; OMN(3): oculomotor nerve; PCA: posterior cerebral artery; PINB: pineal body; PN: pontine nuclei; PTHN: posterior thalamic nucleus; RABN: radix of abducent nerve; RF: reticular formation; RFN: radix of facial nerve; SCN: superior central nucleus; SCOL: superior colliculus; SN: substantia nigra; STNTN: spinal tract nucleus of trigeminal nerve; STTN: spinal tract of trigeminal nerve; SVN: superior vestibular nucleus; TPF: transverse pontine fibers; TRN: trochlear nucleus; VA: vertebral artery; VC(8): vestibulocochlear nerve; VCON: ventral cochlear nucleus; Staining methods: Kultschitzky’s myelin sheath stain (scale bar = 10 mm) Klüber-Barrera’s method Nuclei of the human raphe 19

Explanation of Figures

Plate I

1. Nucleus linealis oralis 2. Nucleus linealis intermedius 20 M. Shibata et al.

Plate II

3. Nucleus raphe medialis 4. Nucleus supratrochlealis (= Nucleus raphe dorsalis) 5. Nucleus raphe pontis Nuclei of the human raphe 21

Plate III

6. Nucleus centralis superior medialis 7. Nucleus centralis superior dorsalis 8. Nucleus raphe magnus 22 M. Shibata et al.

Plate IV

9. Nucleus raphe pallidus 10. Nucleus raphe obscurus intraraphalis 11. Nucleus raphe obscurus extraraphalis