Brain Research, 101 (1976) 385-410 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

385

Research Reports

MORPHOLOGY AND FINE STRUCTURE OF THE FELINE NEONATAL MEDULLARY RAPHE NUCLEI

G E O F F R E Y Q. FOX*, G E O R G E D. PAPPAS AND D O M I N I C K P. P U R P U R A

Department of Neuroscience, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461 ( U.S A.) (Accepted July 16th, 1975)

SUMMARY

A light and electron microscopic study of the caudal medullary raphe nuclei of neonatal kittens reveals that these nuclei are composed of three size classes of neurons with several possible subclasses. Internuclearly projecting dendritic arborizations in the transverse plane and intranuclear projections in the sagittal plane are common features of large and medium size class neurons of raphe nuclei magnus and obscurus though not for the cells of nucleus raphe pallidus. A positive correlation exists between neuron size and density of axosomatic and axodendritic synapses, which suggests that the large class neurons are the first to receive input in synaptogenesis, which is occurring at this time. A wide variety of synaptic forms and integration is also a characteristic feature within these nuclei, though it is not clear whether this morphological variance represents a phylogenic and/or ontogenic trend or just an expression of the multifunctional nature of this region.

INTRODUCTION

Several lines of inquiry during the past decade have suggested an important role of midline nuclei of the brain stem in a variety of complex functional activities. Histofluorescence studies indicate that neurons of the raphe nuclei are the major source of 5-hydroxytryptamine in the central nervous system1~. The raphe nuclei have been * Present address: Max-Planck Institut fiir Biophysicalische Chemic, 3400 Grttingen-Nikolausberg am Fassberg, Postfach 968, G.F.R.

386 implicated m the regulation of sleep-wakefulness activities TM, possibly through mechamsms of interaction with noradrenerglc systems of the locus coeruleus2~ and ponto-mesencephalic tegmentum23. Additionally, evidence has accumulated to the effect that serotonergic neurons of the brain stem raphe system influence synaptic operations in spinal afferent projection pathways subserving nociception45. Several morphological slmllarltles exist between raphe and more laterally situated reticular nuclei. Afferent and efferent connectmns of raphe neurons are overtly similar to those of reticular neurons 8,9.41. Preliminary electron microscope studies suggest further fine structural simflarmes between raphe and reticular neurons m adult ammals 24. The present study characterizes the morphological features of some raphe nucle~ in the postnatal kitten through the use of both light and electron mxcroscopic approaches. This report details observations on the fine structure of medullary raphe nuclei m the immature feline brain, whereas the following report consxders developmental aspects that serve to identify features of growth and synaptogenesis in this neuronal organization of the lower brain stem. MATERIALS AND METHODS

Kittens, 2, 4, 10, 14 and 17 days of age, were used m this study. The animals were first anesthetized with sodium pentobarbital (0.1 ml/100 g body weight) and perfused, via the left ventricle, with a variety of mixed aldehyde solutions in either a phosphate or cacodylate buffer. The mixed aldehyde solutions consisted of formaldehyde and glutaraldehyde in varying concentrations (1-5 %) of total perfusate. These solutions were introduced either seriatim or mixed together as one perfusion solution. No particular combination of either concentration or application of the two aldehydes was found to be superior. The phosphate-buffered aldehydes were judged slightly better than the cacodylate-buffered solutions in the preservatmn of the immature brain stem. Per animal 600-800 ml of perfusate were used. This was delivered by a drip bottle suspended approximately 5-6 ft. above the animal. Perfuslon time lasted about 20-25 ram. Following perfuslon, the brain was carefully removed from the skull and successive transverse sections of approximately 1 mm thickness were cut with a clean razor blade through the length of the medulla. These sections were then individually postfixed in 1% buffered OsO4, dehydrated in a graded series of ethanols (50-100 %), and finally flat embedded in Epon 812. Upon polymerization, the flat embedded tissue was trimmed, oriented and mounted on Epon blanks made from '00' gelatin capsules. The light microscopy portion of this study was conducted by examination of (1) Golgi-Cox material with Nissl counterstain, and (2) 1 #m sections cut from the above tissue blocks prepared for electron microscopy. These sections were mounted on glass slides and stained with a 1 7ooaqueous solution of toluidine blue. All thin sections (sliver) for electron microscopy were cut on a Porter-Blum

387 MT-2 ultramicrotome and mounted on 150 mesh grids. They were doubly stained with 5 0 ~ ethanolic uranyl acetate and lead citrate a6 and then examined with a Philips 200 electron microscope. RESULTS

The brain stem of kittens ranging in age from 2 to 17 days was examined by light and electron microscopy to characterize the topographic, cytoarchitectonic and ultrastructural features of the medullary raphe nuclei. The 3 caudal raphe nuclei lie along the midsagittal plane of the medulla extending rostrally from the anterior edge of the pyramidal decussation to the caudal edge of the pons. The ventral nucleus raphe pallidus (n.RP) and the dorsal nucleus raphe obscurus (n.RO) occupy the caudal half of the medulla merging anteriorally with the nucleus raphe magnus (n.RM) which occupies the remaining rostral half of the medulla. The following results are grouped into 3 broad categories: (A) The neuron populations. (B) Topology and cytoarchitecture. (C) Synapses.

(A) Neuron populations The 3 nuclei of the medullary raphe consist of heterogeneous populations of neurons that can be divided into 3 classes based on cell body size: large, medium and small. These 3 classes are represented in varying proportions in the different nuclei and evidence is presented here for the existence of numerous subclasses.

Large class The large neuron class is represented by a multipolar cell whose perikaryon ranges in size from 25 to 50 #m (Figs. 1B, 1D, 2D, 3B and 4). The typical neuron in this class contains a large centrally spherical nucleus 16-18 /~m in diameter with diffuse chromatin and frequent and sometimes extensive indentations of the nuclear membrane (Fig. 4). A large prominent nucleolus with a conspicuous nucleolema and pars amorpha component is often present (Fig. 4). The soma is bounded by a perikaryal membrane, which may be studded by a moderately dense population of somatic spines. The presence or absence of somatic spines may serve as a useful criteria for further characterization of this neuronal class (Fig. 8A and B). The soma contains an extensive and evenly distributed population of cytoplasmic organelles consisting of pleomorphic mitochondria, numerous Golgi complexes, vesicles, lysosomes, smooth endoplasmic reticulum, tubules and filaments, all of which gives the general appearance of a 'metabolically active' cell (Fig. 4). The rough, granular endoplasmic reticulum (RER) appears as two types. The first, referred to as Nissl body-rough endoplasmic reticulum (NB-RER), is characterized by clumps of short, non-oriented, studded endoplasmic membranes (Fig. 4), while the second consists of ordered, laminar stacks of granular membranes (Fig. 8B).

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389 Both types are associated with conspicuous numbers of free ribosomes and ribosomal rosettes. An additional point of interest with regard to these two types of RER is that there appears to be some correspondence between firstly, laminar RER and cells with smooth perikaryal membranes (Fig. 8B) and secondly, between NB-RER and neurons with somatic spines (Fig. 8A). The primary dendritic projections of large neurons are of large caliber (up to 11 #m) and number from about 4 to 6 per plane of section (Figs. 1D, 2D, 3B). Dendrites are elaborated in several planes and divide approximately 30 # m from the soma, this giving rise to finer caliber secondary dendrites, which in turn may further subdivide into tertiary and then quaternary branches (Figs. 2D, 3B). This neuron class is most represented in the n.RM, where it makes up close to 30 ~o of the neuron population. Medium class

The medium size class of neurons ranges in cell body size from 15 to 30 ffm and represents the largest proportion of neurons in all 3 nuclei. Two distinct varieties of medium size neurons have been identified in this study, both of which are represented in all 3 nuclei. The first, which is stellate in appearance, is the most numerous and is characterized by a spherical or tear-drop cell body with medium caliber dendrites projecting in all planes (Figs. 1A, 1C, 2A, 3A, 3B, 5). The second variety consists of neurons whose cell bodies are fusiform in shape and which give rise to large primary dendrites that appear more often to be oriented in the transverse plane (Figs. 1A, 1B, 2B, 2C, 2D, 3B, 6). Stellate neuron. The stellate neuron cell body contains an eccentric nucleus, spherical in shape, but characteristically flattened and indented towards the cell center (Fig. 5). The cytoplasmic component of the cell body resembles that of the large neuron class with respect to the extent and variety of cytoplasmic organelles. Extensive Golgi networks, pleomorphic mitochondria, lysosomes, tubules, vesicles and ribosomes give a similar impression of high metabolic activity (Fig. 5). The RER within this neuron is exclusively in the NB-RER configuration (Fig. 5). Elaboration of the dendritic tree varies between transverse and sagittal orientations of the stellate neuron. From 4 to 5 medium caliber primary dendrites project

Fig. 1. Light micrographs of transverse sections through the 3 medullary raphe nuclei showing the arrangement, density and diversity of the neuronal populations. A: nucleus raphe obscurus, consisting of predominantly medium size class neurons which form two vecticallaminae whose lateral boundries (arrowhead) are the medial longitudinal fasciculus and medial lemniscus. B: region common to the 3 medullary raphe nuclei showing large multipolar neurons (L), a medium fusiform neuron (F) and a small class neuron (S). C: nucleus raphe paUidus, composed of medium class fusiform (F) stellate, with eccentric nucleus (E) and small class (S) neurons. D: nucleus raphe magnus, two large class neurons (L) and a medium class neuron (M) are present in this least densely packed nucleus. 1 ffm toluidine blue stained sections, x 375.

390

391 radially from the cell body in the transverse plane (Fig. 2A), while a somewhat greater number (up to 7) of both medium and fine caliber dendrites project in the sagittal plane (Fig. 3A, 3B). In either case, the medium caliber dendrites frequently divide some 20-30 # m from the soma. The fine caliber processes do not undergo further subdivision, nor is there tertiary branching to any significant degree (Fig. 3A, 3B). Fusiform neuron. The subclass of fusiform neurons is seen in Figs. 2B, 2C, 2D, 3B and 6. The cell bodies fall within the range of 15 #m × 30/~m with the long axis oriented in the transverse plane either laterally or dorsoventrally (Fig. 2B, 2C, 2D). The cell body contains a spherical and frequently elaborately indented nucleus (Fig. 6). The general composition of the cytoplasm is similar to that described above for the large class and medium class stellate neurons though several differences can be noted. The RER is similar to the large class neurons with both NB-RER and laminar RER (not shown) present (Fig. 6). The perikaryon, however, like the stellate neuron, is devoid of somatic spines (Fig. 6). Finally, the variety of cytoplasmic organelles, though essentially the same, is less extensive (e.g., Golgi complexes) giving this neuron a more 'quiescent' appearance. In transverse section 4-6 large caliber dendrites project from the cell body generally in the direction of the long axis. They soon branch giving rise to medium caliber secondary dendrites (Fig. 2B, 2C, 2D). These secondary dendrites sometimes possess distinct spines (Fig. 2B) and frequently further divide into fine tertiary processes (Fig. 2B, 2C, 2D).

Small class This neuron has a spherical cell body 10-15 # m in diameter with 2-3 fine caliber non-branching dendrites projecting from it (Figs. 1B, 1C, 2C, 3B). A spherical, partially flattened and indented nucleus occupies a major portion of the cell body (Fig. 7). The cytoplasm, generally 'washed out' in appearance due to the difficulty of preserving this cell, is uncomplicated, relative to the large and medium class neurons, with short RER membranes distributed throughout, some Golgi networks, and a modest number of mitochondria, vacuoles and ribosomes (Fig. 7). Often one finds a satellite glial cell associated with this neuron (Fig. 7).

(B) Topology and cytoarchitecture The 3 caudal raphe nuclei seem to have but two points in common between them (1) their anatomlc location, the midsagittal plane of the medulla, and (2) their Fxg. 2. Light mlcrographs of transverse Golgi-Cox stained medullary rapbe nuclei sections illustrating some typical neurons and their dendritic arborizatlons. A: medium class stellate neuron with spherical cell body and long, straight, relatively non-branching primary dendrites that project internuclearly. B: medmm class fusiform neuron with large caliber primary dendrites some of which immediately branch. Dendrlttc spines are conspicuous. C: medium class fusiform neuron with similar dendritic pattern as (B) above. A fine caliber axon is projecting off the cell body (arrowhead). In close proximity is a small class neuron (S) with spherical cell body and 3 straight non-branching dendrites. D: large class multlpolar neuron (L) and medium fusiform neuron (F). The large class neuron has multiple large caliber dendrites projecting radially off a spherical cell body. Golgi-Cox sections with Nissl counterstain. × 250.

Fig. 3 Light m l c r o g r a p h s o f G o l g l - C o x stained mldsaglttal sections of nucleus raphe obscurus (A) a n d nucleus r a p h e m a g n u s (B). A" the dendritic projections m this plane of section are m o r e n u m e r o u s per cell body t h a n in the transverse plane T h e y remain lntranuclearly d~str~buted and characteristically bend at the dorsal a n d ventral borders o f this nucleus (arrowhead) B. large multipolar ([,), m e d i u m stellate (E), m e d i u m fuslform (F) and small class n e u r o n s (S) are present here The large n e u r o n s exhibit a dendritic pattern smldar to that seen in transverse sections (Fig 2D) T h e fustform n e u r o n is dorsoventrally oriented a n d the small n e u r o n h a s typically fe~ dendrites GolgJ Cox ~agittal sections wzth Nlssl c o u n t e r s t a m 120.

393

Fig. 4. Electron micrograph of large multipolar neuron from nucleus raphe magnus. The centrally placed spherical nucleus contains a distinct nucleolus (Nuc) with prominent pars amorpha and fibrillar component. Chromatin material is evenly dispersed and the nuclear membrane invaginations are common. The cytoplasm contains the Nlssl body configuration of rough endoplasmic reticulum (ER). Stacked Golgi membrane complexes and many mitochondria are scattered throughout. Numerous axomatic synapses and somatic spines can be seen on careful examination of the cell surface. Three primary dendritic trunks (D) are prolecting from the cell body. × 3500.

n e u r o n a l m o r p h o l o g y o f large, m e d i u m a n d small class n e u r o n s described above. A d d i t i o n a l m o r p h o l o g i c a l criteria such as neural p a c k i n g density, the relative p r o p o r t i o n s a n d d i s t r i b u t i o n o f n e u r o n a l classes, spatial configuration o f each nucleus, dendritic p a t t e r n s a n d synaptic o r g a n i z a t i o n are distinctively different for each nucleus.

394

h g 5 A m e d i u m class stellate neuron from nucleus raphe palhdus with an eccentr~,, nucleus flattened along Its cytoplasmLc pole A d~stmct nucleolus ( N u c ) a n d nuclear indentations are characteristic Golgi complexes (g) are scattered t h r o u g h o u t the cell body and extend into the basal portions of a primary dendrite (D ~, R o u g h endoplasmlc retlculum ts m the Nlssl body configuration 4~00

395

Fig. 6. Two medium class fusiform neurons. These cells are characterized by their elongate cell body, the long axis of which is frequently oriented in either a dox soventral or lateral direction. The centrally placed spherical nucleus shows wide variation in degree of nuclear indentations. The cytoplasm contains mitochondria, Golgi complexes, vacuoles and rough endoplasmtc reticulum either in the Nissl body configuration (shown here) or as stacked lamellae. × 3700.

Fig 7 Small class n e u r o n from nucleus r a p h e m a g n u s Typically spherical m profile, this neuron cell body Is d o m i n a t e d by a spherical nucleus which m a y be centrally o~ eccentricall) positmned. T h e perikaryon typically a p p e a i s "washed out" a n d sparse m organelles, probably as a result o f suscept~blIJty to the preparatory process 9000

397

Nucleus raphe obscurus (n.RO ) The n.RO consists of two parasagittal laminae of moderately packed neurons separated from each other by a midsagittal lamina of primarily non-myelinated fibers. This nucleus is bounded by the medial longitudinal fasciculus (MLF) and the medial tectospinal tracts (MTS) (Fig. 1A). The medium sized class of neurons comprises the greatest proportion of neurons in this nucleus with the large and small class making up not more than 25 % of the total. The medium size neurons are frequently found situated within the interstices of the longitudinal myelinated fiber bundles of the MLF and the MTS where they give off medium and large caliber dendrites that course laterally between these tracts and enter the reticular formation (Fig. 1A). Thus, in transverse section, the majority of neurons project their dendrites laterally in an internuclear fashion. Dorsoventral projections are also quite commonly seen (Fig. 1A) but when followed for sufficient distance are found to also exit laterally. In sagittal section, the dendritic pattern appears to be primarily intranuclearly distributed with regards to the stellate neurons. Their dendrites sharply bend on approaching the dorsal and ventral boundaries of the nucleus (Fig. 3A). This particular subclass of neurons, therefore, has both and inter- and intra-nuclear dendritic coverage with the internuclear dendrites invading primarily reticular space rather than adjacent raphe nuclei.

Nucleus raphe pallidus (n.RP) This nucleus is characterized by a single mass of relatively densely packed neurons which in transverse section is vaguely pyramidal in outline (Fig. 1A) and in sagittal section is spherical to oval in outline. The medium size class neurons again make up the major portion of the neuron population with stellates of the predominant type. The small class makes up about 15 ~o and the large class somewhat higher up to perhaps 25 ~. The frequency of large class neurons increases anteriorally in this nucleus. The dendritic distribution of this nucleus is somewhat different from that of the n.RO, particularly with respect to the stellate neurons m transverse section. The dendritic tree is projected in an internuclear circumferential pattern passing into the n.RO as well as the lateral reticular areas. Branches pass in close proximity to the inferior olive and the pyramidal tracts, though no data are available as to whether they enter these areas. In sagittal section, the dendritic projections also extend beyond the limits of the nucleus and do not display the abrupt bending typical of this neuron type in the n.RO.

Nucleus raphe magnus (n.RM) The nucleus magnus occupies the largest volume of the 3 caudal raphe muclei. Its neuronal population is the least densely packed and most evenly represented between the 3 morphologic classes of neurons - - each of which makes up approximately one-third of the neuronal population. The dendritic fields generated by both the large and medium class neurons

398 appear to be similar to that of the n.RO. The large class neuron elaborates a spherical dendritic field in which the transverse plane projects internuclearly and the sagittal plane remains intranuclear (Fig. 3B). The same general pattern seems to hold for the medmm class neurons, though the extent of their dendritic fields ~s more difficult to determine because of the indistinct boundaries of th~s nucleus. It does appear that both the stellate and fusfform neurons project mternuclearly in the transverse plane, whereas the dendrmc field, as seen in sagittal section, is less far reaching and probably ntranuclear for the stellate neurons and absent for the fuslform cells (Fig 3B).

(C) Synapses S) naptogenesis Morphological diversity appears to increase m magnitude at the synaptic level of orgamzatlon in the caudal raphe nuclei. This statement is based on the relative complexity of synaptic terminal morphology and the large variety of synaptic contact combinations encountered m these nuclei. This morphological and organizational complexity is evident in the neonatal period. Further synaptogenesis and secondary dendritic growth are, however, occurring at these times 15, but this seems to be more of a quantitative contribution than a qualitative one. A positive correlation exists between neuron size and synaptic density with the order of coverage being first axodendritic then axosomatic. Large neurons are almost completely invested with axodendritlc and axosomatic synapses by 2 days of age, whereas the medium class neurons are less well covered and the small class are almost devoid of synapses. By 17 days of age, more axosomatic synapses are found on the medium class of neurons, whereas no such increase is noted on the small class neurons. Apparently, the large neurons are the first to receive their afferent input followed by increased numbers of axosomatic synapses in the medium class neurons by 17 days of age.

S ynaptic morphology Presynaptic specializations. The presynaptic axonal boutons occur in a wide variety of sizes and shapes and contain a number of structures. Glycogen, mitochondria, clear irregular vesicles and smooth tubular elements may be present, but the common feature for all is the synaptic vesicle. At least 2 distinct morphological types of vesicles can be identified: (1) a clear variety, 40-60 nm m diameter, and (2) a dense-core variety, 70-100 nm in diameter (Figs. 8B, 10). The clear vesicle is most frequently round (Figs. 8B, 11), though a flattened form is also present (Fig. 9B). Axosomatic and axodendritic synapses may contain either spherical (Fig. 8B) or flattened (FJg. 9B) vesicle populations and no correlations were found relating vesicle geometry to synaptic loci. Dense-core vesicles are found in axonal terminals in distinctively fewer numbers than clear vesicles. They may be found in both axosomatic and axodendritic synapses (Figs. 8B, 10A) but appear to be more readily associated with spherical vesicle populations (Figs. 8B, 10A, 11A), though this latter point has not been verified statistically.

Fig. 8. Examples of two types of perlkaryal membrane surfaces (P) seen on large class neurons. In A, the surface is studded with numerous somatic spines (S), while in B the surface is smooth. In both examples, axosomatic synapses abound. A, × 28,000; B, x 32,000.

F~g 9 Electron m l c r o g r a p h s s h o w i n g a x o n s (Ax) s y n a p s m g s+multaneously u p o n two adjacent celJ bodies (Pa a n d P2). I n A, the a x o n contains spherical vesicles a n d s o m e dense-core vesicles, while m B the a x o n c o n t a i n s a pleomorphlc flattened vesicle population with s o m e glycogen particles (g) A spherical vesicle a x o s o m a t l c synapse can be seen adjacent to this latter synapse (arrow) A, , 21,000, B, 32,000

Fig. 10. A: an axosomatic synapse from nucleus raphe magnus containing dense-core vesicles and clear spherical vesicles. The longitudinal profile shows an axon making numerous contacts (arrowheads) upon the cell body. x 45,000. B: an axon (Ax) forming an axosomatic (S) and an axoden0riti¢ (d) synapse, x 38,000.

Fig. 11 Two examples of embedded axosomatlc synapses from nucleus raphe magnus In A, the axon terminal (Ax) contains spherical vesicles and in B it contains pleomorphlc vesicles The subsynaptlc c~stern (arrowheads), formed from endoplasmlc retlculum, is always present m conjunctmn with th~s synaptic form and may be studded (A) or devotd (B) of ribosomes A, 50,000, B, 43,000

403 Both clear and dense-core vesicles can occasionally be found in the cytoplasm of the soma and dendrites of all large and medium class neurons, and it is not unusual to find dense-core vesicles in the vicinity of the Golgi complexes. A variable amount of dense material closely associated with the presynaptic membrane is frequently found at the 'contact zone' of both axosomatic and axodendritic synapses (Figs. 8, 11A). The synaptic cleft area is often filled with an amorphous dense material, the synaptic gap substance, and the pre- and postsynaptic membranes are typically more separated from one another (approximately 20 nm) than in adjacent areas of membrane apposition (Fig. 8B). Postsynaptic specializations. Spinous projections are perhaps the most distinctive postsynaptic structure found. They occur on both dendrites and somas though the somatic spines are restricted to the large class neurons (Fig. 8A). Somatic spines are, in addition, somewhat more elongate and irregular than dendritic spines (Fig. 8A), though both types contain a variety of organelles such as mitochondria, coated vesicles, multivesicular bodies, irregular vesicles and subsynaptic dense bodies. The postsynaptic membrane, similar to the presynaptic membrane, has variable amounts of dense material associated with it, particularly in the region of the 'contact zone' (Fig. 10B). Subsynaptic cisterns formed from either rough or smooth endoplasmic reticular membranes occur in conjunction with axosomatic and axodendritic synapses (Fig. 11). Synaptic organization. The variety of form and contact seen in the axosomatic and axodendritic synapses of the caudal raphe nuclei suggests a complex organizational framework. Axosomatic synapses occur most frequently in the following order: (1) large class neurons, (2) fusiform medium class neurons, (3) stellate medium class neurons, (4) small class neurons. The synaptic contact may have a single or multiple 'contact zone' (Fig. 10A), spherical or pleomorphic vesicles (Fig. 11), varying amounts of dense pre- and postsynaptic membrane accumulations (Fig. 8B) and varying synaptic cleft widths and contents. Large class neurons have axosomatic synapses which terminate on flattened perikaryal surfaces (Fig. 8B) or dense spinous surfaces (Fig. 8A). They, along with fusiform medium class neurons, also possess the embedded synaptic form most often found in n.RP and n.RM (Fig. 11). This synapse contains either spherical or pleomorphic vesicles (Fig. l lA, B) and is always seen in conjunction with a subsynaptic cistern (Fig. 11). The embedded synapse is more rarely found as an axodendritic contact but here also is typically in association with a subsynaptic cistern. A still rarer finding is a somatodendritic synapse arising from a stellate neuron in n.RO (Fig. 12). The larger, elaborate synaptic bouton is connected to the soma by a slender stalk and contains a pleomorphic population of vesicles with some glycogen particles. It contacts a dendritic spine at what is interpreted to be multiple 'contact zones'. No other types of somatodendritic, -axonal, or -somatic synapses were found in these nuclei. Axodendritic synapses display a similar degree of variation in their mode of contact, though less extreme than axosomatic synapses. Axonal terminals are found

Fig 12. A rare s o m a t o d e n d r m c synapse from nucleus raphe palhdus seen contacting a spree of ,~ dendrite (dl The umqueness of this synaptlc from is seen m the large engulfing bouton c o n t a m m g pleomorphlc vesicles (v) and attached to the cell body by a narrow stalk (arrowl The nucleus Inl of the neuron Js seen above 18,500

405 forming 'contact zones' on dendritic trunks and spines. Most of these terminals contain spherical vesicles, though pleomorphic and dense-core vesicles are also present. Axonal terminals are frequently found contacting more than one postsynaptic element, as shown in Figs. 9A, 9B and 10B. No axo-axonic or dendrodendritic synapses were found in these nuclei. DISCUSSION

Identification of raphe nuclei of the brain stem as functionally distinct systems with discrete ascending and descending projections is based largely on studies involving the histochemical localization of 5-HT in these elementslz. The hypothesis that such serotonergic systems arising in brain stem raphe nuclei play a major role in the phenomenological aspects of slow-wave or quiet sleep19,20 has given further impetus to the view of functional discreteness of the raphe system. However, more recent physiological and biochemical studies have tended to emphasize the complex interactions between raphe and reticular neurons including elements of the locus coeruleus21,aa. The present observations are consonant with these latter suggestions in respect to similarities in fine structural features and morphological relations between raphe and neighboring neurons of reticular nuclei. Anatomically, the medullary raphe nuclei (MRN) are closely related to the surrounding brain stem reticular formation (BSRF). Dendritic arborizations noted in this study and by others2Z,35,44 extend well into the medial medullary reticular nuclei and axonal projections from MRN clearly pass through these same regionsS, 9. Recent findings indicate that the morphological diversity of BSRF neurons represents only a variation of a basic reticular neuronal type22, or isodendritic neuron aS. Leontovich and Zhukova 22 suggest that the reticular neuron is a distinctive cell apart from specific motor or sensory neurons found at similar levels in the BSRF. Their argument is based primarily on the character of the dendritic tree, i.e., reticular neurons have 'few long, straight, poorly ramified dendrites with long thorns and are characterized by the small size of the cell body in comparison to the length and thickness of the dendrites'2L Ram6n-Moliner and Nauta a5 also consider dendritic complexity as the basis for their classification scheme. The lsodendritic neuron is one which posesses a simple dendritic arborization pattern and represents a phylogenetically old cell type of pluripotential character. The MRN neurons of this study meet the criteria of this isodendritic neuron having, in the majority of cases, few long, straight dendrites, with proximal branches that are considerably shorter than the distal segments. In this respect, medullary raphe nuclei might be included as morphological components of the reticular formation as proposed earlier by Leontovich and Zhukova22. Diversity of cell body size and shape within the MRN corresponds well with the neuron populations of the adjacent medullary reticular formation, as seen in this study and noted by others22,41,44. Little is known, however, concerning the significance of this diversity. Studies of the BSRF by Brodal and Rossi 7 and of the raphe system by Brodal et al.8, a did not show preferential retrogade degeneration related to cell

406 size, though a later study by Petras a0 showed that the ~gmnt' neurons of the pons and medulla are closely assocmted with retlcutospinal and spinoretlcular fibers. Walberg 4'', m an earher study, suggested that cell body size may only be of ~mportance at the intraclass level of organization. The present study has revealed some ultrastructural differences which appear referable to cell size and form in the MRN. Large class neurons are distinguishable from medium class neurons by their obvious relative size difference, their high frequency of laminar RER, somatic sprees (when present), and a high density of axosomatic synapses. The two varieties of medium sized neurons (fusiform and stellate) differ from one another with respect to a centrally (fusiform) or eccentrically (stellate) positioned nucleus, presence or absence of elaborate laminar RER (always seen m profiles judged to be fuslform), and density of axosomatic synapses. A more extenswe analysis of these two forms, however, Js necessary before a d~stmctive subclassification can be made. Small class neurons, besides their relative size difference, lack all of the differentiating criteria mentioned above Perhaps the foremost d~stinguishing feature is evident in axosomatic synaptology. Four different categories which relate axosomatxc synapses to cell body sJze and form can be recognized from this study and are summarized as follows:

Cell body size and form

Axosomatic synapses

(1) Large multipolar neuron (a) Smooth surfaced (b) Spinous surface

Well covered

(2) Medium fusiform neuron Smooth surfaced

Sparse to well covered

(3) Medium stellate neuron Smooth surfaced

Sparsely covered

(4) Small spherical neuron Smooth surfaced

None to sparsely covered

Bowsher and Westman 5,6, examining several BSRF nuclei, have postulated the existence of two distinct neuron populations based on quantitative differences of their somadendritic synaptology and on some specific somal and dendritic differences. Their polydendritic neuron is the largest of the two, multipolar m form with a centrally placed nucleus, somatic and dendritic spines and much of its surface covered by synapses. The oligodendritic neuron is smaller with few dendrites, no somatic or dendritic spines and sparse synaptic coverage. In this study, the large multipolar neurons correspond well to the polydendritic form, while the majority of medium and possibly all of the small class neurons correspond to the oligodendritic form. The axosomatic coverage of M R N neurons, as outlined above, is very similar to that reported by Bowsher and WestmanS, 6. However, this may be considered, at least in part, a reflection of synaptogenesis occurring during the neonatal period 15.

407 The somas of adult M R N neurons are well covered with synapses z4 but in the latter study no mention was made concerning possible relative differences that might exist between neuronal groups. In the present ontogenetic study, there is a suggestion of a differential sequential development of axosomatic synapses which may be referable to cell body size. Thus, the largest neurons are the first to complete their afferent somatic imput, the smallest neurons are the last. Alternatively, it may be argued that axosomatic synaptogenesis occurs at equal rates for all cell sizes and that relative differences persist into adulthood. There is no indication from the present study as to when synaptogenesis began or is completed for any of the 3 neuronal size classes. It must be remembered that the large class neuron is well covered with synapses, but not completely covered, even in adult animals z4. Axonal boutons containing either spherical or flattened (pleomorphic) vesicles as well as mixtures of both geometries 10 can be found along the entire perikaryal surface (soma and dendrite) of M R N neurons. Spherical vesicle synapses are seen most abundantly, though a possible shift towards higher densities of pleomorphic vesicles may be taking place 11. Controversy exists concerning the functional significance of these vesicle geometries4,26,z7,29,40, 42,43. Two unusual synapt~c forms were encountered in the MRN. The first of these, the embedded synapse, is found most frequently on the somas of large class and fus~form medium class neurons though occasionally it is seen on their dendrites. This embedded synapse is always found in association with a subsynaptic cistern, formed as an extension of either granular or agranular endoplasmic reticulum. In this regard, the cistern must be considered as an integral part of this synaptic complex 11,27. Another unusual synaptic form encountered was a somatodendritic synapse. Somatodendritic synapses have been reported in frog optic tectum zS, frog paraventricular organ 31, mouse olfactory bulb 17, rat spinal cord 16, and monkey lateral geniculate nucleus 47. In all these cases, the somatodendritic synapse displays a remarkable degree of morphological similarity. The presynaptic component consists of only a few clear spherical or pleomorphic vesicles in close proximity to the perikaryon. The 'contact zone' displays variable pre- and postsynaptic membrane densities, while the postsynaptic dendritic element is generally devoid of structural elements. Somatodendritic synapses closely resemble dendrodendritic synapses as seen, for example, in thalamic nuclei88, 84 and olfactory bulb 82. The somatodendritic synapse found in this study is notably different in this regard for it presents a definitive structure quite unlike anything described previously. Nothing definitive is known concerning the functional properties of these somatodendritic synapses. They have been found on small, possibly sensory, neurons which lack axons 31,47. They are suggested to be inhibitory because of their somal origin 88 and their pleomorphic vesicle population 47. Somatodendritic synapses have not been previously identified in either BSRF or M R N and the example presented in this report was found on a medium size class neuron assumed to be a Golgi type II. This was a rare observation and is presently considered as 'non-typical' of the synaptology of the MRN. A final synaptic combination of interest is that found frequently in the more

408 densely packed neurons of n.RP and n.RO. This consisted o1" an axonal boutoll simultaneously contacting two adjacent somal membranes Similar s o m a - a x o n - s o m a synaptlc combinations have been shown in frog oculomotor nucleus ~'v and in cat spinal cord 11. Peters e t al. 'z9 have shown a somewhat similar dendrite-axon-dendrlte combination in rat spinal cord. It has been established that raphe neurons are uniquely revolved in the productlon of brain serotonm (5-HT). Early histofluorescent studies by Dahlstr6m and Fuxe 12 showed that 5-HT ~s localized m medium size spherical neurons of n.RO and in the medium size spherical, spmdle and multipolar neurons of n.RM. A majority of the neurons in M R N appeared to fluoresce with the amount of fluorescence distributed rather evenly throughout the neuron cell body. Studies on prenatal ammals~, a7 have shown that the 5-HT system develops very early m embryogenesls Bzogemc amines m the CNS are believed to be correlated with the presence ol dense-core vesicles within the size range of about 50-110 nm 1-a,18. A 50 nm vesicle species has been localized m regions which characteristically contain monoamlne neuronsla, 18. Larger dense-core veslcles (60-110 nm range) have been found in neurons from both monoamlnergic and non-monoammerglc reglons TM. Similar s~ze dense-core vesicles (1.e., 75-90 nm) have also been found in regenerating proximal nerve stumps 28 adding support to the view that dense-core vesicles contain a variety of products. This study found the medium size dense-core vesicle (about 60-110 nm) ubiquitous to all 3 raphe nuclei. They are most noticeable m axosomatlc and axodendritic boutons though they have been frequently seen m dendrites ~'5. They are commonly found, singularly, throughout the cytoplasm of large and medium size neurons and often in assocmtlon w~th the Golgl complex, but never with rough or smooth endoplasmic reticulum. Though many dense-core vesicles are m ewdence m the M R N , correspondence with fluorescence studies is tacking. The extensive fluorescence seen m the cell bodies of M R N neurons from the studies of Dahlstr6m and Fuxe 12 and Seiger and Olson 37 cannot be accounted for by the few dense-core vesicles randomly scattered throughout M R N neurons as seen m th~s study Similarly the above two reports make no special mention concerning the fluorescence emanating from axonal boutons w~thin the M R N where the maximal concentration of dense-core vesicles has been found in this study. The direct relatlonsh~p between 5-HT and dense-core vesicles remains unclear and will require further mqulry. ACKNOWLEDGEMENTS

This study was supported in part by the following grants: N I H I F I O NS2417-01 NSRB; N I M H N I H 5 T I M H 6 4 1 8 ; N I H NSO7512; N I H NS11431. A preliminary account of these results has been presented 14.

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