Anat. Embryol. 149, 323--346 (1976) 9 by Springer-Verlag 1976
The Uhrastructure of the Nucleus of the Oculomotor Nerve (Somatic Efferent Portion) of the Cat G i o v a n n i Tredici, Giuliano Pizzini, a n d Sergio Milanesi Istituto di Anatomia Umana Normale, Universit~ di Milano, Italy Received March 2, 1976
Summary. The ultrastructure of the somatic efferent portion of the nucleus of the oculomotor nerve was studied in four adult cats. The neuronal population is composed of neurons of variable size. A continuous pattern of morphological aspects is evident between the large neurons, which show abundant cytoplasm with well developed organelles, and the small neurons which have a reduced amount of cytoplasm. The dendrites are generally smooth, with few short spines. Axo-somatic and axo-dendritic synapses are numerous. Synaptic boutons arc also present on the axon hillock. The neuropil is characterized by the occurrence of small groups of dendrites which may be in direct touch with their membranes. Direct membrane appositions may also occur between neighbouring neurons and between the cell somata and tangentially running dendrites. Generally beneath the site of apposition there is accumulation of mitochondria, multivesicular bodies, coated vesicles and moderately dense amorphous material. The morphological features suggest the possibility of cellular interchanges at the sites of direct membrane apposition. Five types of synaptic boutons were recognized on the basis of their vesicular content, the presence of abundant filaments in the pre-synaptic bag~ the occm'rence of post-synaptic specializations. The different synaptic types and their distribution are similar to those reported in the spinal motor nuclei. Many of the synapses make synaptic contacts with two or more post-synaptic elements. Axo-axonic synapses were also observed. Key words: Oculomotor nucleus - - Ultrastructure - - Synapses - - Membrane apposition - Motoneurons. Ocular m o v e m e n t s are performed i n a very fine a n d precise way to p e r m i t fixation, vergence, s c a n n i n g a n d persuit m o v e m e n t s (Robinson, 1968). The a c t i v i t y of the extraocular muscles is u n d e r the control of the n e u r o n s of the oeulomotor nuclei (Warwick, 1964). These nuclei take p a r t i n m a n y complicated reflex arches at the b r a i n stem level (Bender a n d Shanzer, 1964; Tarlov, 1972; Schwindt et M., 1974; H i g h s t e i n et al., 1974) a n d are u n d e r the influence of m a n y s u p r a t e n t o r i a l eentres (Mettler, 1964; Sehlag a n d Sehlag-Rey, 1970; P r e e h t et al., 1974; Graybiel a n d t t a r t w i e g , 1974). S y n c h r o n i z a t i o n of the a c t i v i t y of the n e u r o n s controlling the different extraocular muscles a n d m u t u a l influence b e t w e e n groups of n e u r o n s m u s t be postulated to e x p l a i n some of the peculiar functions of the oculomotor system. Such f u n c t i o n a l properties are i n d e e d p a r t l y achieved t h r o u g h short-axon n e u r o n s c o n n e c t i n g the oeulomotor nuclei (Baker a n d IIighstein, 1975; Maciewicz et al., 1975) b u t p r o b a b l y also direct i n t e r n e u r o n a l influences p l a y a n i m p o r t a n t role i n r e g u l a t i n g the a c t i v i t y of the different n e u r o n s w i t h i n the same nucleus. Generally, electrical coupling of n e u r o n s a n d c o n s e q u e n t s y n c h r o n i z a t i o n of their 5 A n a l Embryol.
324
G. Tredici et al.
a c t i v i t y is a c h i e v e d t h r o u g h electrotonic j u n c t i o n s (Bennett, 1972), which have t h e i r morphological c o u n t e r p a r t in t h e sites of close m e m b r a n e a p p o s i t i o n (gap j u n c t i o n s : B r i g h t m a n a n d l~eese, 1969; P a p p a s a n d W a x m a n , 1972). N e i t h e r morphological nor electrophysiological studies have r e p o r t e d electrotonic coupling in t h e o c u l o m o t o r nuclei ol m a m m a l s , whereas electrotonic coupeling has been r e p o r t e d (Kriebel et al., 1969) a n d electrotonic synapses morphologically d e m o n s t r a t e d ( W a x m a n a n d P a p p a s , 1971; Schuster, 1972)in t h e nucleus of t h e oculomotor nerve of some i n f r a m a m m a l i a n species. U l t r a s t r u c t u r a l studies ( W a x m a n a n d P a p p a s , 1970; B a k a n d Choi, 1974) on t h e oculomotor nuclei of m a m m a l s are few a n d t h e fine s t r u c t u r e of t h e nucleus of t h e o c u l o m o t o r nerve in p a r t i c u l a r has n e v e r been e x t e n s i v e l y r e p o r t e d previously. W e therefore investg a t e d t h e u l t r a s t r u e t n r e of this nucleus in t h e cat with p a r t i c u l a r i n t e r e s t in t h e s y n a p t i e o r g a n i z a t i o n a n d cellular relationships.
Material and Methods
This study was carried out on four adult cats, weighing from 2 to 3.5 kg. The animals were anesthetized with Nembutal, rinsed through the addominal aorta with a phosphate buffer 0.24 M solution and perfused with 19% glutaraldehyde for a short time then followed by 3 % glutaraldehyde, both in a phosphate buffer 0.24 M. The brain was kept in the last fixative solution overnight and next morning serial laminae, 1 mm thick, of the brain stem were cut. The laminae were post-fixed in 2% OsO4 in a phosphate buffer 0.18 M for 3 4 h, dehydrated in ethanol and embedded in Epon 812. Ultrathin sections of the nuclei nervi oeulomotorii principalis and nucleus Perlia (Tabor 1958) of the oculomotor complex were stained with 3% aleoholic uranyl acetate, contrasted with lead citrate, and observed with an Elmiskop I eleetronmicroseope. Observations
a) Neurons The n e u r o n a l p o p u l a t i o n of the nucleus of t h e oculomotor nerve is composed of neurons of v a r i a b l e size, r a n g i n g from 10 to 40 ~m, with an u n i m o d a l distribut i o n a n d a m e a n largest d i a m e t e r of 25 ~m. The shape m a y be either poligonal or s t a r - s h a p e d in t h e larger neurons, b u t e l o n g a t e d a n d fusiform in t h e s m a l l e r ones. The neurons (Figs. 1, 2) d e p i c t f a i r l y u n i f o r m u l t r a s t r u c t u r a l features, although differences in d i s t r i b u t i o n a n d o r g a n i z a t i o n of t h e c y t o p l a s m i c organelles are e v i d e n t b e t w e e n t h e large a n d the small cellular profiles. The nucleus is g e n e r a l l y large, r o u n d in shape a n d shows a p r o m i n e n t nucleolus. I d e n t a t i o n s of t h e n u c l e a r m e m b r a n e are rare. N u c l e a r bodies (Figs. 1, 2) ( W e b e r a n d F r o m rues, 1963) a n d occasionally fibrillar a n d r o d l e t s (Siegesmnnd e t a l . , 1964) occur in t h e nucleoplasm. The c y t o p l a s m (Fig. 3) is rich in r o u g h e n d o p l a s m i c r e t i c u l u m which often forms large or i n t e r m e d i a t e sized masses of Nissl bodies. These masses are e v e n l y
Fig. 1. A large neuron of the nucleus of the oculomotor nerve. Nuclear bodies (arrows) are evident in the round central nucleus. The cytoplasm shows large and medium-sized Nissl bodies (Nb). The Golgi complexes (G) are mainly perinuelear. The neuron is covered by several synaptic boutons. The large block arrows point to the emergence of two dendrites. • 4,000 Fig. 2. A small neuron showing reduced cytoplasm and less developed Nissl bodies. Few synaptic boutons cover the neuron. The nucleus contains nuclear bodies (arrows). In the sur-
rounding neuropil a longitudinally cut dendrite (block arrow) and the nearby small transversally cut dendrites form a small cluster of dendrites, some of which (asterisks) show direct apposition of their plasma membrane. •
326
G. Tredici e~ al.
Ultrastructure of the Oculomotor Nucleus of the Cat
327
distributed in a well-ordered pattern in the somatic cytoplasm and extend in the large main dendrites which taper off from the cell body. Often the ribosomes lie free, clustered in small rosettes or in short chains, between the eisternae. The smaller neuron profiles have less developed Nissl bodies which m a y be reduced to small arrays of few cisternae in the smallest neurons. Large Golgi complexes occur frequently expeeially in a perinuelear position, but are also widely distributed throughout the soma and in the main branches of the dendritic tree. The agranular retieulum is also well developed. Some cisternae m a y lie just beneath (50-60 nm) the plasmamembrane (hypolemmal cisternae, Palay and Chan-Palay, 1974) and m a y be connected with thin elongated eisternae (sub-surface cisternae, Rosenbluth, 1962) which are often in a sub-synaptie position and are closely apposed to the plasmamembrane (10-20 nm). Connections of the subsurface cisternae with the cisternae of the rough endoplasmie retieulum as well as continuity between the agranular and rough reticulmn were also observed. Mitochondria, free ribosomes, lysosome-like particles, lipofuseine-like pigments, microtubuli, and neurofilaments are numerous and widely distributed, oecurring frequently in the spaces free of the Nissl bodies. The profiles of the small neurons (Fig. 2) still show a large nucleus, but the surrounding cytoplasm is reduced and its organelles less developed. However, between the large and small neurons there is a continous pattern of aspects, making the a t t e m p t to classify the neuronal profiles, in single sections, in either of the two groups, a difficult task. The cellular membrane is generally smooth; spiny extrusions occur rarely. Several synaptie boutons contact the cellular soma; tile sites devoid of terminals are covered b y glial laminar processes. The extension of synaptic contacts is higher in the large neurons whereas in the small neurons the cellular profiles are mainly enveloped by the glial processes. I-Iowever exceptions to this model do occur. From the large cells 2-4 large main dendrites (Fig. 1) gradually taper off without any changes in the characteristics of the cytoplasm. In the small neurons the emergence of the dendrites is more abrupt and the dendrites are thinner and contain mainly neurofilaments and mierotubuli similar to the dendritic profiles of the neuropil. Nissl bodies, Golgi complexes, ribosomial rosettes however, m a y be observed in large and medium sized dendrites of the neuropil. The profiles of the dendrites are generally smooth. Spiny extrusions were observed at the basis of the large dendrites or in the fine branches of the neuropil, but were much less frequent and not as developed as those described in other sites of the central nervous system (Gray, 1959; Pappas and Purpura, 1961; I-Ierdon, 1963; Peters and Palay, 1966; Peters and Kaiserman-Abramof, 1970; Tredici etal., 1973; Palay and Chan-Palay, 1973).
Fig. 3. The cytoplasm of a large neuron. Cisternae of the I~ER are organized in Nissl bodies (Nb). }Iany of the ribosomes lie free between the cisternae forming rosettes or short chains. Golgi complexes (G) are numerous. A subsurface cisterna (small arrows) partially underlie an axo-somatic synaptic bouton which also shows a "puncture adhaerens" (block arrow).
• 12,000
328
G. Tredici etal.
The dendrites are covered by numerous synaptie contacts which prevail at the basis of the emerging dendrites, but we did not observe any typical elustering and disposition as observed in the proximal part of the dendrites of the neurons of the red nucleus (Trediei et al., 1973). The axon hillock (Fig. 4) shows the typical aspects described in other neurons (Patsy et al., 1968; Peters et al., 1968; Conradi, 1969c). Longitudinal arrays of microtubuli start in the axon hillock and extend in the initial segment. The plasma membrane is coated underneath by dense material. Synaptie contacts m a y be observed on the axon hillock but rarely do they extend far in the initial segment.
b) The Neuro~)il Besides the numerous synaptie boutons which contact small dendrites and the myelinated and unmyelinated pro-terminal fibres, the most prominent feature of the neuropil in the nucleus of the oeulomotor nerve is the occurrence of m a n y dendritic profiles of intermediate size which can be followed for some distance in the plane of the section (Figs. 2, 5). Often 2-3 of such dendrites run parallel and m a y come in touch with other dendrites running in an orthogonal plane. Sm0oI1 clusters of 3-5 dendrites are therefore formed and these groups, although they are not so extensive nor so well organized in large bundles as in other sites of the central nervous system (Seheibel and Seheibel, 1970; Matthews etal., 1971 ; Marsh et al., 1972; Peters and Walsh, 1972; Fleischauer etal., 1972; Kerns and Peters, 197~), are nevertheless well apparent in low power electron mierographs and even with the light microscope. The dendrites which run parallel or eriss-eross to each other are generally separated by the interposition of small synaptie fields (Fig. 5) or by thin, single or arrayed, glial laminae (Fig. 6), but sometimes direct apposition of dendrite membranes is observed (vide infra). The pro-terminal myelinated fibres generally lose their myelin sheath far from the terminal bouton, but occasionally it is lost at the terminal bag. Many Ranvier nodes have been observed but no nodal synapses similar to those reported in some inframammalian species (Waxmann and Pappas, 1971), were ever observed at this level. However, branching of the fibres at the Ranvier node is not uncommon. Similarly fibres devoid of myelin m a y divide just prior to the formation of the terminal bontons. Astroeyte processes containing fibrillar structures or small round glial processes are intermingled with the fibres and the synaptic boutons contributing further to the complexity of the neuropil. The glial processes are arranged in such a fashion as to form loose and discontinuous meshes. Therefore the glial architecture does nor separate nor define different synaptic fields as observed in the red nucleus (Tredici et al., 1973). Glia] laminae are less common and occur mainly interposed between parallel running dendrites or neighl3ouring neurons. Glial cells do not deserve much mention as they are similar, in cytological features, position, and number to those observed in m a n y other sites of the CNS (see for example: Peters etal., 1970; Ling et al., 1973).
c) The Synapses Five different types of synaptic boutons were recognized mainly on the basis of, the features of the pre-synaptie vesicles, the presence of abundant fibrillar
Fig. 4a and b. Contigous images of the axon hillock and initial segment of a neuron. The arrows point to several synaptic boutons on the axon hillock. The synaptic complex is evident only in some boutons. Bundles of microtubuli (small arrows) are evident in the initial segment of the axon. • 16,000
Fig. 5. A small group of dendrites in the neuropil. Small laminar glial septa (arrows) or interposed synaptie boutons separate the dendrites. • 7,500 Fig. 6. A higher magnification of a dendritic cluster of the neuropil. Two parallel running dendrites (D) are separated by ~ thin glial lamina. • 37,500
Ultrastructure of the Oculomotor Nucleus of the Cat
331
structures in the pre-synaptic bag, and the occurrence of specialized post-synaptic structures. Type I boutons (Figs. 7, 8) are terminal knobs which contain clear, round vesicles, 40-50 mn in size. They are the most prominent group of synaptic terminals in the nucleus of the oculomotor nerve, making up 60% of the total number of the synapses. Few type I boutons were shown to degenerate after cortical ablation and were considered the terminals of monosynaptic cortico-oculomotor fibres (Trediei et al., 1976). The size of type I boutons is variable; small boutons (0.5-1.5 t~m) are very nnmerous and evenly distributed in all parts of the neuron. Boutons of intermediate size (1.5-3 ~m) and larger are less common and occur expecially on the cellular soma or large dendrites. Generally type I boutons show a symmetrical thickening of the pre- and post-synaptic membrane, but asymmetrical thickening or intermediate type of synaptic junctions are not infrequent. It is therefore difficult to assign this group of boutons to either of the two classes proposed by Gray (1959). Moreover the extension of the synaptic complex is variable and independent of the thickening or not of the post-synaptic membrane. The cleft at the synaptic complex is generally 15-20 nm and is often much narrower than in the other parts of the synaptic apposition. Conversely in some boutons the cleft appears to be wider at the synaptic complex (25-30 nm) while it is reduced in the sites devoid of membrane specialization. Moderately dense material is generally apparent in the synaptic cleft. The small boutons of type I generally show only one synaptic complex, rarely associated with a puncture adhaerens (Fig. 9). Intermediate and large sized boutons have often two or more synaptic complexes and several puncta adhaerentia. These boutons may establish synaptie contacts with two or more different postsynaptie elements (Fig. 10). Besides the differences in size and pattern of contacts, type I boutons may also differ in the electron density of the matrix of the pre-synaptie bag and in the number, distribution and size of the vesicles. Sometimes the vesicles are unusually large, sometimes they arc few and sparse or conversely densely packed and arranged in a para-erystalline fashion. At present it is impossible to decide whether these morphological differences are expressions of a different source of the axons (Chan-Palay, 1973) or of different functional states of the terminals. ~re have therefore not taken these differences as further criteria for subdi~Tiding the synaptie bontons of this group. In the pre-synaptic bag of type I boutons glycogen granules and dense core vesicles m a y be frequently observed. Dense core vesicles are generally located peripherally in the boutons and their number ranges from 1 to 10. Boutons with a preponderance of dense core vesicles, similar to those reported in the troehlear nucleus of the cat (Bak and Choi, 1974), were not observed. Type I I boutons (Fig. 3) are similar in size and vesicular content to the intermediate or large boutons of type I but differ by the oeeurrenee of a sub-surface eisterna (Rosenbluth, 1962). The eisterna m a y underlie the entire extension of the synaptie contact but more frequently only a part of the site of apposition is subtended by the cisterna. I n this ease the bouton m a y show a synaptie complex or a puncture adhaerens. When the cisterna underlies the entire bouton no membrane speeializations are evident. Sometimes the sub-surface cisterna may be related to a stack of 4-5 cisternae of the granular endoplasmic retieulum.
332
G. Tredici et al.
Fig. 7. Synaptic boutons of the neuropil. Some boutons (R) have round clear vesicles while bouton P has round as well as flattened vesicles. Glial processes and laminae (asterisks) separate the synaptic boutons. A site of direct apposition of two neighbouring boutons is also shown (arrows). • 37,500
Type II boutons establish synaptic contacts almost exclusively on the large dendrites and on the cellular soma. Oeeasionally boutons of this type come in touch with other dendritic profiles, but membrane specializations or accumulations of vesicles which could indicate the occurrence of a synaptic junction were never observed. This group of b o u t o n s makes u p 5% of the bo~tons observed in the oculomotor nucleus. Type I I I b o u t o n s (Figs. 10, 11) are very few (less t h a n 2%) a n d are characterized b y the occurrence of a single row of dense bodies i n s u b s y n a p t i c position (Taxi, 1961; Milehaud a n d Pappas, 1966). T h e y resemble in m a n y aspects, the
Fig. 8. Axo-deadritic synapses containing different vesicles. A synaptic bouton (R) contains clear round vesicles; another bouton (P) has flattened vesicles. }
The Uhrastructure of the Nucleus of the Oculomotor Nerve (Somatic Efferent Portion) of the Cat G i o v a n n i Tredici, Giuliano Pizzini, a n d Sergio Milanesi Istituto di Anatomia Umana Normale, Universit~ di Milano, Italy Received March 2, 1976
Summary. The ultrastructure of the somatic efferent portion of the nucleus of the oculomotor nerve was studied in four adult cats. The neuronal population is composed of neurons of variable size. A continuous pattern of morphological aspects is evident between the large neurons, which show abundant cytoplasm with well developed organelles, and the small neurons which have a reduced amount of cytoplasm. The dendrites are generally smooth, with few short spines. Axo-somatic and axo-dendritic synapses are numerous. Synaptic boutons arc also present on the axon hillock. The neuropil is characterized by the occurrence of small groups of dendrites which may be in direct touch with their membranes. Direct membrane appositions may also occur between neighbouring neurons and between the cell somata and tangentially running dendrites. Generally beneath the site of apposition there is accumulation of mitochondria, multivesicular bodies, coated vesicles and moderately dense amorphous material. The morphological features suggest the possibility of cellular interchanges at the sites of direct membrane apposition. Five types of synaptic boutons were recognized on the basis of their vesicular content, the presence of abundant filaments in the pre-synaptic bag~ the occm'rence of post-synaptic specializations. The different synaptic types and their distribution are similar to those reported in the spinal motor nuclei. Many of the synapses make synaptic contacts with two or more post-synaptic elements. Axo-axonic synapses were also observed. Key words: Oculomotor nucleus - - Ultrastructure - - Synapses - - Membrane apposition - Motoneurons. Ocular m o v e m e n t s are performed i n a very fine a n d precise way to p e r m i t fixation, vergence, s c a n n i n g a n d persuit m o v e m e n t s (Robinson, 1968). The a c t i v i t y of the extraocular muscles is u n d e r the control of the n e u r o n s of the oeulomotor nuclei (Warwick, 1964). These nuclei take p a r t i n m a n y complicated reflex arches at the b r a i n stem level (Bender a n d Shanzer, 1964; Tarlov, 1972; Schwindt et M., 1974; H i g h s t e i n et al., 1974) a n d are u n d e r the influence of m a n y s u p r a t e n t o r i a l eentres (Mettler, 1964; Sehlag a n d Sehlag-Rey, 1970; P r e e h t et al., 1974; Graybiel a n d t t a r t w i e g , 1974). S y n c h r o n i z a t i o n of the a c t i v i t y of the n e u r o n s controlling the different extraocular muscles a n d m u t u a l influence b e t w e e n groups of n e u r o n s m u s t be postulated to e x p l a i n some of the peculiar functions of the oculomotor system. Such f u n c t i o n a l properties are i n d e e d p a r t l y achieved t h r o u g h short-axon n e u r o n s c o n n e c t i n g the oeulomotor nuclei (Baker a n d IIighstein, 1975; Maciewicz et al., 1975) b u t p r o b a b l y also direct i n t e r n e u r o n a l influences p l a y a n i m p o r t a n t role i n r e g u l a t i n g the a c t i v i t y of the different n e u r o n s w i t h i n the same nucleus. Generally, electrical coupling of n e u r o n s a n d c o n s e q u e n t s y n c h r o n i z a t i o n of their 5 A n a l Embryol.
324
G. Tredici et al.
a c t i v i t y is a c h i e v e d t h r o u g h electrotonic j u n c t i o n s (Bennett, 1972), which have t h e i r morphological c o u n t e r p a r t in t h e sites of close m e m b r a n e a p p o s i t i o n (gap j u n c t i o n s : B r i g h t m a n a n d l~eese, 1969; P a p p a s a n d W a x m a n , 1972). N e i t h e r morphological nor electrophysiological studies have r e p o r t e d electrotonic coupling in t h e o c u l o m o t o r nuclei ol m a m m a l s , whereas electrotonic coupeling has been r e p o r t e d (Kriebel et al., 1969) a n d electrotonic synapses morphologically d e m o n s t r a t e d ( W a x m a n a n d P a p p a s , 1971; Schuster, 1972)in t h e nucleus of t h e oculomotor nerve of some i n f r a m a m m a l i a n species. U l t r a s t r u c t u r a l studies ( W a x m a n a n d P a p p a s , 1970; B a k a n d Choi, 1974) on t h e oculomotor nuclei of m a m m a l s are few a n d t h e fine s t r u c t u r e of t h e nucleus of t h e o c u l o m o t o r nerve in p a r t i c u l a r has n e v e r been e x t e n s i v e l y r e p o r t e d previously. W e therefore investg a t e d t h e u l t r a s t r u e t n r e of this nucleus in t h e cat with p a r t i c u l a r i n t e r e s t in t h e s y n a p t i e o r g a n i z a t i o n a n d cellular relationships.
Material and Methods
This study was carried out on four adult cats, weighing from 2 to 3.5 kg. The animals were anesthetized with Nembutal, rinsed through the addominal aorta with a phosphate buffer 0.24 M solution and perfused with 19% glutaraldehyde for a short time then followed by 3 % glutaraldehyde, both in a phosphate buffer 0.24 M. The brain was kept in the last fixative solution overnight and next morning serial laminae, 1 mm thick, of the brain stem were cut. The laminae were post-fixed in 2% OsO4 in a phosphate buffer 0.18 M for 3 4 h, dehydrated in ethanol and embedded in Epon 812. Ultrathin sections of the nuclei nervi oeulomotorii principalis and nucleus Perlia (Tabor 1958) of the oculomotor complex were stained with 3% aleoholic uranyl acetate, contrasted with lead citrate, and observed with an Elmiskop I eleetronmicroseope. Observations
a) Neurons The n e u r o n a l p o p u l a t i o n of the nucleus of t h e oculomotor nerve is composed of neurons of v a r i a b l e size, r a n g i n g from 10 to 40 ~m, with an u n i m o d a l distribut i o n a n d a m e a n largest d i a m e t e r of 25 ~m. The shape m a y be either poligonal or s t a r - s h a p e d in t h e larger neurons, b u t e l o n g a t e d a n d fusiform in t h e s m a l l e r ones. The neurons (Figs. 1, 2) d e p i c t f a i r l y u n i f o r m u l t r a s t r u c t u r a l features, although differences in d i s t r i b u t i o n a n d o r g a n i z a t i o n of t h e c y t o p l a s m i c organelles are e v i d e n t b e t w e e n t h e large a n d the small cellular profiles. The nucleus is g e n e r a l l y large, r o u n d in shape a n d shows a p r o m i n e n t nucleolus. I d e n t a t i o n s of t h e n u c l e a r m e m b r a n e are rare. N u c l e a r bodies (Figs. 1, 2) ( W e b e r a n d F r o m rues, 1963) a n d occasionally fibrillar a n d r o d l e t s (Siegesmnnd e t a l . , 1964) occur in t h e nucleoplasm. The c y t o p l a s m (Fig. 3) is rich in r o u g h e n d o p l a s m i c r e t i c u l u m which often forms large or i n t e r m e d i a t e sized masses of Nissl bodies. These masses are e v e n l y
Fig. 1. A large neuron of the nucleus of the oculomotor nerve. Nuclear bodies (arrows) are evident in the round central nucleus. The cytoplasm shows large and medium-sized Nissl bodies (Nb). The Golgi complexes (G) are mainly perinuelear. The neuron is covered by several synaptic boutons. The large block arrows point to the emergence of two dendrites. • 4,000 Fig. 2. A small neuron showing reduced cytoplasm and less developed Nissl bodies. Few synaptic boutons cover the neuron. The nucleus contains nuclear bodies (arrows). In the sur-
rounding neuropil a longitudinally cut dendrite (block arrow) and the nearby small transversally cut dendrites form a small cluster of dendrites, some of which (asterisks) show direct apposition of their plasma membrane. •
326
G. Tredici e~ al.
Ultrastructure of the Oculomotor Nucleus of the Cat
327
distributed in a well-ordered pattern in the somatic cytoplasm and extend in the large main dendrites which taper off from the cell body. Often the ribosomes lie free, clustered in small rosettes or in short chains, between the eisternae. The smaller neuron profiles have less developed Nissl bodies which m a y be reduced to small arrays of few cisternae in the smallest neurons. Large Golgi complexes occur frequently expeeially in a perinuelear position, but are also widely distributed throughout the soma and in the main branches of the dendritic tree. The agranular retieulum is also well developed. Some cisternae m a y lie just beneath (50-60 nm) the plasmamembrane (hypolemmal cisternae, Palay and Chan-Palay, 1974) and m a y be connected with thin elongated eisternae (sub-surface cisternae, Rosenbluth, 1962) which are often in a sub-synaptie position and are closely apposed to the plasmamembrane (10-20 nm). Connections of the subsurface cisternae with the cisternae of the rough endoplasmie retieulum as well as continuity between the agranular and rough reticulmn were also observed. Mitochondria, free ribosomes, lysosome-like particles, lipofuseine-like pigments, microtubuli, and neurofilaments are numerous and widely distributed, oecurring frequently in the spaces free of the Nissl bodies. The profiles of the small neurons (Fig. 2) still show a large nucleus, but the surrounding cytoplasm is reduced and its organelles less developed. However, between the large and small neurons there is a continous pattern of aspects, making the a t t e m p t to classify the neuronal profiles, in single sections, in either of the two groups, a difficult task. The cellular membrane is generally smooth; spiny extrusions occur rarely. Several synaptie boutons contact the cellular soma; tile sites devoid of terminals are covered b y glial laminar processes. The extension of synaptic contacts is higher in the large neurons whereas in the small neurons the cellular profiles are mainly enveloped by the glial processes. I-Iowever exceptions to this model do occur. From the large cells 2-4 large main dendrites (Fig. 1) gradually taper off without any changes in the characteristics of the cytoplasm. In the small neurons the emergence of the dendrites is more abrupt and the dendrites are thinner and contain mainly neurofilaments and mierotubuli similar to the dendritic profiles of the neuropil. Nissl bodies, Golgi complexes, ribosomial rosettes however, m a y be observed in large and medium sized dendrites of the neuropil. The profiles of the dendrites are generally smooth. Spiny extrusions were observed at the basis of the large dendrites or in the fine branches of the neuropil, but were much less frequent and not as developed as those described in other sites of the central nervous system (Gray, 1959; Pappas and Purpura, 1961; I-Ierdon, 1963; Peters and Palay, 1966; Peters and Kaiserman-Abramof, 1970; Tredici etal., 1973; Palay and Chan-Palay, 1973).
Fig. 3. The cytoplasm of a large neuron. Cisternae of the I~ER are organized in Nissl bodies (Nb). }Iany of the ribosomes lie free between the cisternae forming rosettes or short chains. Golgi complexes (G) are numerous. A subsurface cisterna (small arrows) partially underlie an axo-somatic synaptic bouton which also shows a "puncture adhaerens" (block arrow).
• 12,000
328
G. Tredici etal.
The dendrites are covered by numerous synaptie contacts which prevail at the basis of the emerging dendrites, but we did not observe any typical elustering and disposition as observed in the proximal part of the dendrites of the neurons of the red nucleus (Trediei et al., 1973). The axon hillock (Fig. 4) shows the typical aspects described in other neurons (Patsy et al., 1968; Peters et al., 1968; Conradi, 1969c). Longitudinal arrays of microtubuli start in the axon hillock and extend in the initial segment. The plasma membrane is coated underneath by dense material. Synaptie contacts m a y be observed on the axon hillock but rarely do they extend far in the initial segment.
b) The Neuro~)il Besides the numerous synaptie boutons which contact small dendrites and the myelinated and unmyelinated pro-terminal fibres, the most prominent feature of the neuropil in the nucleus of the oeulomotor nerve is the occurrence of m a n y dendritic profiles of intermediate size which can be followed for some distance in the plane of the section (Figs. 2, 5). Often 2-3 of such dendrites run parallel and m a y come in touch with other dendrites running in an orthogonal plane. Sm0oI1 clusters of 3-5 dendrites are therefore formed and these groups, although they are not so extensive nor so well organized in large bundles as in other sites of the central nervous system (Seheibel and Seheibel, 1970; Matthews etal., 1971 ; Marsh et al., 1972; Peters and Walsh, 1972; Fleischauer etal., 1972; Kerns and Peters, 197~), are nevertheless well apparent in low power electron mierographs and even with the light microscope. The dendrites which run parallel or eriss-eross to each other are generally separated by the interposition of small synaptie fields (Fig. 5) or by thin, single or arrayed, glial laminae (Fig. 6), but sometimes direct apposition of dendrite membranes is observed (vide infra). The pro-terminal myelinated fibres generally lose their myelin sheath far from the terminal bouton, but occasionally it is lost at the terminal bag. Many Ranvier nodes have been observed but no nodal synapses similar to those reported in some inframammalian species (Waxmann and Pappas, 1971), were ever observed at this level. However, branching of the fibres at the Ranvier node is not uncommon. Similarly fibres devoid of myelin m a y divide just prior to the formation of the terminal bontons. Astroeyte processes containing fibrillar structures or small round glial processes are intermingled with the fibres and the synaptic boutons contributing further to the complexity of the neuropil. The glial processes are arranged in such a fashion as to form loose and discontinuous meshes. Therefore the glial architecture does nor separate nor define different synaptic fields as observed in the red nucleus (Tredici et al., 1973). Glia] laminae are less common and occur mainly interposed between parallel running dendrites or neighl3ouring neurons. Glial cells do not deserve much mention as they are similar, in cytological features, position, and number to those observed in m a n y other sites of the CNS (see for example: Peters etal., 1970; Ling et al., 1973).
c) The Synapses Five different types of synaptic boutons were recognized mainly on the basis of, the features of the pre-synaptie vesicles, the presence of abundant fibrillar
Fig. 4a and b. Contigous images of the axon hillock and initial segment of a neuron. The arrows point to several synaptic boutons on the axon hillock. The synaptic complex is evident only in some boutons. Bundles of microtubuli (small arrows) are evident in the initial segment of the axon. • 16,000
Fig. 5. A small group of dendrites in the neuropil. Small laminar glial septa (arrows) or interposed synaptie boutons separate the dendrites. • 7,500 Fig. 6. A higher magnification of a dendritic cluster of the neuropil. Two parallel running dendrites (D) are separated by ~ thin glial lamina. • 37,500
Ultrastructure of the Oculomotor Nucleus of the Cat
331
structures in the pre-synaptic bag, and the occurrence of specialized post-synaptic structures. Type I boutons (Figs. 7, 8) are terminal knobs which contain clear, round vesicles, 40-50 mn in size. They are the most prominent group of synaptic terminals in the nucleus of the oculomotor nerve, making up 60% of the total number of the synapses. Few type I boutons were shown to degenerate after cortical ablation and were considered the terminals of monosynaptic cortico-oculomotor fibres (Trediei et al., 1976). The size of type I boutons is variable; small boutons (0.5-1.5 t~m) are very nnmerous and evenly distributed in all parts of the neuron. Boutons of intermediate size (1.5-3 ~m) and larger are less common and occur expecially on the cellular soma or large dendrites. Generally type I boutons show a symmetrical thickening of the pre- and post-synaptic membrane, but asymmetrical thickening or intermediate type of synaptic junctions are not infrequent. It is therefore difficult to assign this group of boutons to either of the two classes proposed by Gray (1959). Moreover the extension of the synaptic complex is variable and independent of the thickening or not of the post-synaptic membrane. The cleft at the synaptic complex is generally 15-20 nm and is often much narrower than in the other parts of the synaptic apposition. Conversely in some boutons the cleft appears to be wider at the synaptic complex (25-30 nm) while it is reduced in the sites devoid of membrane specialization. Moderately dense material is generally apparent in the synaptic cleft. The small boutons of type I generally show only one synaptic complex, rarely associated with a puncture adhaerens (Fig. 9). Intermediate and large sized boutons have often two or more synaptic complexes and several puncta adhaerentia. These boutons may establish synaptie contacts with two or more different postsynaptie elements (Fig. 10). Besides the differences in size and pattern of contacts, type I boutons may also differ in the electron density of the matrix of the pre-synaptie bag and in the number, distribution and size of the vesicles. Sometimes the vesicles are unusually large, sometimes they arc few and sparse or conversely densely packed and arranged in a para-erystalline fashion. At present it is impossible to decide whether these morphological differences are expressions of a different source of the axons (Chan-Palay, 1973) or of different functional states of the terminals. ~re have therefore not taken these differences as further criteria for subdi~Tiding the synaptie bontons of this group. In the pre-synaptic bag of type I boutons glycogen granules and dense core vesicles m a y be frequently observed. Dense core vesicles are generally located peripherally in the boutons and their number ranges from 1 to 10. Boutons with a preponderance of dense core vesicles, similar to those reported in the troehlear nucleus of the cat (Bak and Choi, 1974), were not observed. Type I I boutons (Fig. 3) are similar in size and vesicular content to the intermediate or large boutons of type I but differ by the oeeurrenee of a sub-surface eisterna (Rosenbluth, 1962). The eisterna m a y underlie the entire extension of the synaptie contact but more frequently only a part of the site of apposition is subtended by the cisterna. I n this ease the bouton m a y show a synaptie complex or a puncture adhaerens. When the cisterna underlies the entire bouton no membrane speeializations are evident. Sometimes the sub-surface cisterna may be related to a stack of 4-5 cisternae of the granular endoplasmic retieulum.
332
G. Tredici et al.
Fig. 7. Synaptic boutons of the neuropil. Some boutons (R) have round clear vesicles while bouton P has round as well as flattened vesicles. Glial processes and laminae (asterisks) separate the synaptic boutons. A site of direct apposition of two neighbouring boutons is also shown (arrows). • 37,500
Type II boutons establish synaptic contacts almost exclusively on the large dendrites and on the cellular soma. Oeeasionally boutons of this type come in touch with other dendritic profiles, but membrane specializations or accumulations of vesicles which could indicate the occurrence of a synaptic junction were never observed. This group of b o u t o n s makes u p 5% of the bo~tons observed in the oculomotor nucleus. Type I I I b o u t o n s (Figs. 10, 11) are very few (less t h a n 2%) a n d are characterized b y the occurrence of a single row of dense bodies i n s u b s y n a p t i c position (Taxi, 1961; Milehaud a n d Pappas, 1966). T h e y resemble in m a n y aspects, the
Fig. 8. Axo-deadritic synapses containing different vesicles. A synaptic bouton (R) contains clear round vesicles; another bouton (P) has flattened vesicles. }
