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The fine structure of neurons and synapses of the habenula of the cat with special reference to sub-junctional bodies
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BRAIN RESEARCH
T H E F I N E S T R U C T U R E OF N E U R O N S A N D SYNAPSES OF T H E H A B E N U L A OF T H E CAT W I T H SPECIAL R E F E R E N C E TO SUBJ U N C T I O N A L BODIES
MONIQUE MILHAUD* AND GEORGE D. PAPPAS** Department of Anatomy, Columbia University, College of Physicians and Surgeons, New York, N. Y. (u.s.A.).
(Received May 16th, 1966)
INTRODUCTION The two habenulas are deep nuclei of the epithalamus, symmetrically placed on each side of the third ventricle. The major connections of each habenula are: (1) the stria medullaris which relate the habenula to the olfactory system, (2) the habenulotectal and tecto-habenular tracts which are interconnections between the habenula and the superior colliculi, (3) the habenulo-tegmental tracts which discharge impulses from the habenula to the dorsal tegmental nuclei, and (4) the largest and best known efferent path from each habenula, the habenulo-peduncular tract or fasciculus retro-flexus of Meynert which arises from the habenula and ends in the interpeduncular nucleus. Each habenula may be divided into medial and lateral nuclei, the lateral one continuing in the fiber bundles of the stria medullaris. The purpose of this study is to relate the fine structure of the neurons and the synaptic organization at the light and electron microscopical levels. MATERIAL AND METHODS The brains of 6 adult cats were perfused under pressure with 2.5~o glutaraldehyde phosphate buffered at p H 7.4. The habenula nuclei were removed and placed into fresh cold fixative for a total of 2 h. After many changes of cold phosphate buffer, the tissue was left in it overnight. Then the tissue was post-fixed with cold 1 ~o osmium tetroxide phosphate buffered at p H 7.4 for 1 h, dehydrated, and embedded in Epon 812. Both thick and thin sections were cut on a Porter Blum microtome M.T. 2. Thick sections of 1/z were stained with toluidine blue in 0.5 ~ aqueous solution and examined * Attach6 de Recherche ~ l'Institut National de la Sant6 et Recherches M6dicales (INSERM) (France). Supported in part by a NATO fellowship. Permanent address: Laboratoire de Microscopic Electronique, Division Risler, La Sall~tri/~re, Paris 13 (France). ** Supported by a Public Health Service research career award (I-K3-NB-21,947-01) from the National Institute of Neurological Diseases and Blindness. Brain Research, 3 (1966) 158-173
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with the light microscope. Thin sections were stained with warm aqueous uranyl acetate solution for 30 rain and examined with an R C A - E M U 3F electron microscope. RESULTS
Two types of neurons can be discriminated with the light microscope in the lateral as well as in the medial nucleus (Figs. 1 and 2). The larger neurons are about
Fig. 1. Light micrograph of a I-/~ thick section of the medial nucleus of the habenula stained with toluidine blue. Neurons can be differentiated into two sizes, N1 being larger than Nz. Throughout the medial nucleus the neuronal cell bodies are more-or-less evenly dispersed. Often neuronal somata can be found in close apposition to each other (at arrows). Many blood vessels (BV) are present throughout the neuropil. × 2500. 15 by 20 # in diameter and the smaller ones about 10 # in diameter. Both types have one or more processes and contain many dark granulations in their cytoplasm. The neurons are more densely placed in the medial nucleus than in the lateral, where they are broken up by myelinated fiber bundles. The cells are often arranged side by side regardless of their size (Fig. 2). Many blood vessels are present throughout the neuropil. Oligodendroglial cells are readily identified when they are close to the neuronal cell bodies (Fig. 2). Electron microscopic examination shows that there is no significant distinction between the two sizes of neurons except the amount of cytoplasm. The usual cytoplasmic organelles, such as Golgi apparatus, mitochondria, granular endoplasmic reticulum which is not arranged in Nissl bodies but distributed throughout the perikaryon, and occasional dense core bodies, are present. The neuronal cytoplasm is characterized by many dense granular lysozome-like bodies someBrain Research, 3 (1966) 158-173
Fig. 2. Light micrograph of a 1-# thick section of the lateral nucleus of the habenula stained with toluidine blue. The neurons are fewer in number and more dispersed than in the medial nucleus. Both large and small neurons are present in the lateral nucleus as in the medial. A large neuron (N1) is shown in close apposition with a smaller one (Na). Oligodendroglia (G) are closely applied to the neuronal somata. The neuropil of the lateral nucleus contains more myelinated fibers than the medial. Bv ~ blood vessels; My ~ myelinated fibers, x 2500.
Fig. 3. Electron micrograph of a portion of the cytoplasm of a neuron in the medial nucleus. Many dark granular bodies (DB) of varying densities characterize these neurons. At times the dense body is found in close contact to mitochondria (at arrows). N = nucleus; E R = granular endoplasmic reticulum; G = Golgi complex. × 13,800.
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Fig. 4. Electron micrograph of two closely applied neurons with no intervening processes. An axon (A) forms a synaptic junction with one of them. M = mitochondria; DB = dense bodies; ER = endoplasmi¢ reticulum, x 12,000. times in close association with mitochondria (Fig. 3). Multivesicular bodies are abundant in the perikaryon regions as well as in the processes. They are frequent in dendrites and are often found in proximity to synapses 17. Occasionally 2 or 3 neurons, especially in the medial nucleus, are side by side without any glial interposition (Figs. 4, 5 and 6). The apposing neuronal membranes are separated by the extracellular space of about 200 A found throughout the neuropil of the CNS 11. Occasionally a few intervening processes do occur in restricted areas (Fig. 5). The area of somasoma contiguity may be as much as 12 if, which represents the entire length of neuronal apposition (Fig. 4). Sometimes, desmosome-like specializations 'joining' these cells are found (Fig. 6). Two types of glial cells are readily identified in the neuropil, the fibrous astrocytes and oligodendrocytes close to the neuronal cell bodies. Dendritic trunks, some of which are very large, are characterized by their high content of microtubules and long mitochondria (Fig. 7). Many typical synapses are present on dendritic trunks (Fig. 7). The presynaptic process is characterized by the presence of clusters of clear vesicles, mitochondria, and some larger vesicles about 800-1200 A in diameter having a dense core. Both the pre- and postsynaptic membranes show Brain Research, 3 (1966) 158-173
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Fig. 5. Higher magnification of a p o r t i o n of a region of c o n t a c t b e t w e e n two neurons. T h e extracellular space o f a b o u t 180-200 A is m a i n t a i n e d between t h e m . Occasionally a few intervening processes (P) m a y be found. N = n u c l e u s ; M ~ m i t o c h o n d r i a ; D B ~ dense bodies. ×, 44,800,
Fig. 6. A n o t h e r p o r t i o n of the contact between two s o m a t a . At arrows, a desmosome-like specialization is apparent. T h e extracellular space is m a i n t a i n e d t h r o u g h o u t the n e u r o n a l m e m b r a n e apposition. D B ~ dense bodies; M = m i t o c h o n d r i a ; N = nucleus. × 44,800.
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Fig. 7. A cross section of a dendrite. Circular profiles of microtubules (MT) are present throughout. Two axons (A) form synaptic junctions on the surface of the dendrite. The postsynaptic membrane has more dense material accumulated than the presynaptic one. In addition about 500 A from the postsynaptic membrane some round dense discrete bodies may be found (at arrows), x 49,400.
Fig. 8. An axo-dendritic junction in the neuropil of the lateral nucleus. The axon (A) contains a large accumulation of clear vesicles. In the dendrite about 500 A from the postsynaptic membrane a single row of dense bodies is found. Often a multivesicular body is present near the dense bodies (MV). × 60,000.
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Fig. 9. T h e axon (A) of the axo-dendritic synapse contains fewer vesicles t h a n one s h o w n in Fig. 8. T h e microtubules (MT) of the dendrite are seen in longitudinal section. A row o f postsynaptic bodies (at arrows) is present. F := fibrils of a fibrous astrocytic process. ~ 50,000.
Fig. 10. Axo-dendritic s y n a p s e in the medial nucleus, T h e a x o n h a s a large a c c u m u l a t i o n of vesicles (V). A row of discrete p o s t s y n a p t i c bodies is present in the dendrite. M = mitochondria, x 43,000.
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Fig. 1I. In this axo-dendritic synapse, in addition to clear synaptic vesicles (V) a few larger densecored vesicles (DC) are present. Postsynaptic dense bodies are seen at arrow. MT = longitudinal array of microtubules. × 41,000. accumulation of dense material though this is usually in greater amount in the latter. There is material of intermediate density filling the synaptic gap and usually appearing condensed into stria perpendicular to the membranes. Occasionally some clear vesicles are open into the cleft from the presynaptic side (Fig. 9). In addition to these usual synaptic features, a single row of dense bodies is present in the postsynaptic process in about one-third of the junctions (Figs. 7, 8, 9, 10 and 11). These bodies are round, usually discrete, and about 200-250 A in diameter. They are regularly arranged about 200 A apart from center to center. Usually 2-12 are found in thin sections. They are located at about 500 A from the postsynaptic membrane and are distinct from it. They are composed of a fine dense granular material and are not membrane-bounded. They are seen as circular profiles in both longitudinal (Figs. 9, 10 and 11) and cross sections (Figs. 7 and 8), and always appear in a single row in the micrographs. Because of their regular spacing and their invariable appearance in a single row, it is assumed that they are spherical and are arranged in an hexagonal array. This hexagonal arrangement must have 200 A sides and be located at a distance of 500 A from the postsynaptic membrane. This hypothesis seems to be confirmed by the appearance of axo-dendritic synapses in tangential sections where the synaptic region loses its distinct morphological features because of the plane of section, but where hexagonal array of postsynaptic bodies is clearly seen adjacent to the synapse (Figs. 12a and b). In a three dimensional reconstruction drawing made from electron micrographs, a plaque made up of postsynaptic spherical bodies in an hexagonal array 500 A from the postsynaptic membrane is illustrated in Fig. 12c. Dendritic spines are present with some frequency in the habenula. They are always found in sections with two axons surrounding the neck and forming synapses Brain Research, 3 (1966) 158-173
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Fig. 12a, b and c. Tangential sections through axo-somatic synapses (a and b). Because of the plane of section, pre- and postsynaptic dense material appears continuous. In such a tangential section the postsynaptic bodies are seen in an hexagonal array (at arrows). × 41,000. In Fig. 12c a three-dimensional drawing shows the relationship of the hexagonally-packed subjunctional bodies. The plaque of dense bodies is about 500 A from the postsynaptic dendritic membrane. The bodies are about 250 .~ in diameter arranged hexagonally, having 200/~ sides.
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9
Fig. 13a, b and c. Electron micrographs of sections through three dendritic spines in the habenula. The central core of the spine contains a row of postsynaptic dense bodies which do not appear as discrete as in other synapses (at arrows). Two axonal processes form synapses onto the sides of the spine process. The axons contain large accumulations of clear vesicles (V) and a few large dense-cored vesicles (DC). The row of postsynaptic bodies which is present in all the spines observed is about 500 A from the postsynaptic membrane. A multivesicular body (MV) is sometimes found at the base of the spine. The synaptic gap substance can be seen condensed as striae perpendicular to the preand postsynaptic membrane in Fig. 13c. MT = microtubules. Figs. 13a and b, 49,400; Fig. 13c, 43,000.
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on both sides but not at the bulbous tip 13. The occurrence o f p o s t s y n a p t i c bodies in spines is very consistent. They always appear as a single row of regularly arranged dense bodies forming the central axis or core of the neck o f the spine. This row is about 500 ~ from the postsynaptic m e m b r a n e (Figs. 13a, b and c). Axo-somatic synapses are c o m m o n and may occur more frequently on the larger neurons than on the smaller ones. In about one-third of the axo-somatic junctions, postsynaptic bodies are seen with the same features as in the axo-dendritic junctions (Fig. 14).
Fig. 14. Axo-somatic synapses on a large neuron. Of the three synaptic junctions shown in this micrograph only the middle one shows a row of postsynaptic bodies (at arrows). V = clear synaptic vesicles; DC -= dense-cored vesicles; M = mitochondria; ER = granular endoplasmic reticulum. × 52,400.
Most frequently, the axons in the habenula have a few o f the dense-cored bodies beside the clear vesicles, not only in the synaptic region but sometimes t h r o u g h o u t the length o f the axon. Axo-axonic synapses are infrequently found. They are identified only if one of the axons of the pair is presynaptic to an axo-somatic or axo-dendritic synapse (Fig. 15). The fact o f the synaptic contact between two axons is assumed by the presence o f synaptic vesicles in both processes and of the postsynaptic thickenings which m a y indicate the polarity of the transmission. In any case, postsynaptic bodies associated with an axo-axonic contact have not been found. Brain Research, 3 (1966) 158-173
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In addition to these synaptic specializations and to the desmosome-like thickenings between adjacent neuronal cell bodies, dendro-dendritic junctions can be occasionally identified by having an equal amount of dense material in the cytoplasm in close apposition to each membrane. The gap between the apposing membranes at these regions is somewhat enlarged (Fig. 16a and b). In Fig. 16a, axon A forms a
Fig. 15. Profile of three axons (A1, As and As) containing synaptic vesicles. Axon A1forms a synapse with dendrite D. Axon A1 may also be forming a synapse with A2. In this axo-axonic junction axon As is probably postsynaptic to A1 because of the greater accumulation of dense material onto its apposing membrane. × 35,000.
synapse with dendrite D1. In Fig. 16b, an axon forms a synapse with dendrite D2. In both micrographs dendrite D2 forms a desmosome-like junction with dendrite D1 and has, in addition, two structures associated with postsynaptic processes of the habenula: sub-junctional dense bodies (at arrows) and a multivesicular body (MV). COMMENTS The two types of neurons in the habenula are present in both nuclei. In the medial nucleus there are more cells per unit area than in the lateral one where they are broken up by myelinated fiber bundles. The neuronal soma-soma apposition observed in the habenula has already been described in other regions. Green and Maxwell 10 have observed that in the adult Brain Research, 3 (1966) 158-173
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Fig. 16a a n d b. T w o adjacent dendrites f o r m a d e s m o s o m e - l i k e j u n c t i o n where there is a symmetrical a n d equal a c c u m u l a t i o n of dense material on b o t h sides as well as an enlarged extracellular space at this site. A row o f dense bodies (at arrows) is f o u n d in o n e of t h e dendrites. In addition, at a short distance f r o m these sub-junctional dense bodies a multivesicular b o d y (MV) is present. In Fig. 16a, a x o n (A) f o r m s a synapse with dendrite D1 a n d in Fig. 16b axon (A) f o r m s a synapse with dendrite D2. In b o t h micrographs, dendrites Dz have rows o f dense bodies as well as a multivesicular body. However, dendrite D1 c o n t a i n s n o n e of t h e m o r p h o l o g i c a l features o f a presynaptic process. M T cross section of microtubules. × 35,700.
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hippocampus the pyramidal cell bodies appear to be in contiguity without any enlargement of the extracellular space at these sites. As a result, in this area, the extraneuronal space around the pyramidal bodies is very limited. Pappas and Purpura is have noticed the same cell body to cell body appositions in the hippocampus of neonatal kittens, but without any desmosome-like junctions. However, desmosomelike specializations at soma-somatic 'contacts' have been observed between granule cells of the cerebellum by Gray 7 in the adult and by Shofer et al. 2° in the immature cerebellum. They have been described as equilateral densities associated with the apposing membranes and they resemble synaptic contacts except that they show no asymmetry or polarity and their functional interpretation, if not of adherence, is quite obscureL Axons forming synaptic junctions in the habenula are evenly dispersed throughout the neuropil. Most of them occur on dendritic trunks regardless of their size, fewer are seen on neuronal somata and on dendritic spines, while only very rarely are any seen on other axons. The morphological characteristics of synapses in the habenula are the same as in other regions. In the presynaptic process clusters of clear vesicles as well as the granular dense-cored vesicles are found. These larger vesicles have been discussed recently in their relation to catecholamine metabolism in the midbrainlL The densities associated with the pre- and the postsynaptic membranes, in axo-dendritic as in axosomatic synapses are variable and do not seem to be limited to two discrete types as described by Gray 5 in the cortex, since many intermediate forms may be present. The synaptic cleft substance sometimes appears to be organized into stria perpendicular to the apposing synaptic membranes, or condensed as an intermediate line parallel to the membrane 15. In the habenula, the gap substance in most instances appears to be in a striated form. In the postsynaptic process the dense bodies in the habenula are similar to some structures noticed in other regions of the central nervous systemL They have been observed also in the locus coeruleus and in the nucleus interpeduncularislL Recently, Mugnaini 14 has found similar postsynaptic bodies in the nucleus vestibularis lateralis and in the granular layer of the cerebellar cortex of the cat. They resemble the postsynaptic bars described by Taxi 22 in the autonomic ganglia of the frog in that they are distinct from the postsynaptic densities closely associated to the membrane, but they differ in that they are hexagonally arranged. More striking is the fact that these postsynaptic bodies are present in all dendritic spines observed in the habenula as well as the interpeduncular nucleus. Such a specialized structure in spines has not been described in other regions. The spine apparatus, particularly prominent in the rat cortex 6, has not been found in the feline habenula. At about the same distance where the spine apparatus has been described, a multivesicular body is sometimes present (Fig. 13). The significance of the postsynaptic bodies is unknown. The question arises whether there is any relationship between the presence of postsynaptic bodies and the high concentration of monoamine-oxydase in the habenulal, 2a. For the present there are no data to support such an inference. Brain Research, 3 (1966) 158-173
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Axo-axonic synapses have been identified by Gray 8 in the spinal cord, by Colonnier and Guillery2 in the lateral geniculate nucleus and by Pappas et al. 16 throughout various other regions of the thalamus. Axo-axonic synapses have been postulated by Eccles 3 to be the morphological basis of presynaptic inhibition. They may also form the basis for presynaptic inhibition of inhibition16. Unfortunately no electrophysiological study of the habenula has been reported. Therefore no correlation between morphological data and functional activities can be made with respect to this structure. This statement is equally applicable to studies reporting a major participation of the habenula in reflex controlling blood volume4. The most puzzling finding is that dense bodies similar to those described above and in a previous communication13 as 'postsynaptic' are found in dendro-dendritic junctions. As in all desmosome-like junctions the densities closely associated with the apposing membranes are symmetrical and the extracellular space between them, at this site, is somewhat enlarged2, 9. The dendro-dendritic junctions shown in Fig. 16a and b have all the morphological features of a synapse except for the complete lack of vesicles. Recently dendro-dendritic synapses have been described in the olfactory bulb 19. Their appearance differs from the dendro-dendritic junctions observed in the habenula by two important features: (1) the presence of clusters of vesicles at the presynaptic site of contact and (2) the asymmetrical localization of the dense material associated with apposing membranes. In addition, Rall et al. 19 present physiological data to support the evidence of dendro-dendritic transmission in the olfactory bulb. In the habenula, the significance of a specialized dendro-dendritic junction where one dendrite has the morphological features of a postsynaptic process is, to say the least, unknown. For present purposes, these dense bodies described here may be designated as sub-junctional dense bodies, without further implications as to their functional significance. SUMMARY
Two types of neurons are present through the lateral and medial habenula. Occasionally these form soma-soma appositions without any intervening processes. About one-third of the axo-dendritic and axo-somatic synapses have discrete dense bodies in an hexagonal array in the postsynaptic process. All the dendritic spines observed have a single row of these dense bodies. Axo-axonic synapses are present but are rare. Desmosome-like structures in dendro-dendritic junctions are occasionally found containing dense bodies, similar to those found in the postsynaptic process in some synaptic junctions. The significance of the presence of sub-junctional dense bodies in the habenula is unknown. ACKNOWLEDGEMENTS
This work was supported in part by Public Health Service grants from the National Institute of Neurological Diseases and Blindness (NB-02314-07 and NB03448-05) and the United Cerebral Palsy and Educational Foundation. Brain Research, 3 (1966) 158-173
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REFERENCES 1 BOUCHAUD, CL., COUTEAUX, R., ET GAUTRON, J., L'inhibition des monoamine-oxydases du cerveau du rat par la fl-phenylisopropylhydrazine et l'harmaline. Etude histochimique de l'action de ces inhibiteurs et de leur antagonisme, C.R. Acad. Sci. (Paris), 260 (1965) 348-351. 2 COLONr,aER, M., AND GUILLERY, R. W., Synaptic organization in the lateral geniculate nucleus of the monkey, Z. Zellforsch., 62 (1964) 333-355. 3 ECCLES,J., Physiology of Synapses, Academic Press, New York, 1963. 4 FAUR~, J., LE NOV~NE, J., VINCENT,D. ET BENSCH,EL., R6actions bio61ectriques de l'habenula et du tronc c6r6bral It l'hypovol6mie exp6rimentale, Rev. NeuroL, 112 (1962) 258-266. 5 GRAY, E. G., Axo-somatic and axo-dendritic synapses of the cerebral cortex - - an electron microscope study, J. Anat. (Lond.), 93 (1959a) 420-433. 6 GRAY, E. G., Electron microscopy of synaptic contacts on dendrite spines of the cerebral cortex, Nature, 183 (1959b) 1592-1593. 7 GRAY, E. G., The granule cells, mossy synapses and Purkinje spine synapses of the cerebellum: Light and electron microscope observations, J. Anat. (Lond.), 95 (1961) 345. 8 GRAY, E. G., A morphological basis for presynaptic inhibition, Nature, 193 (1962) 82-83. 9 GRAY, E. G,, AND GUILLERY,R. W., Synaptic morphology in the normal and degenerating nervous system. In BOURNE AND DANIELLI (Eds.), International ReviewofCytology, Vol. 19, New York, Academic Press, 1966. 10 GREEN, J. D., AND MAXWELL, D. S., Hippocampal electrical activity. Morphological aspects, Electroenceph. clin. Nearophysiol., 13 (1961) 837-846. 11 HORSTMANN, E., AND MEVES, H., Die Feinstruktur des molekularen Rindengraues und ihre physiologische Bedeutung, Z. Zellforsch., 49 (1959) 569-604. 12 LENN, N. J., Electron microscopic observations on monoamine containing brain stem neurons in normal and drug treated rats, Anat. Rec., 153 0965) 399--406. 13 MILHAUD, M., AND PAPPAS, G. D., Postsynaptic bodies in the habenula and interpeduncular nuclei of the cat, J. Cell Biol., (in press). 14 MUGNA~NI,E., Personal communication, 1966. 15 PAPPAS, G. O., Electron microscopy of neuronal junctions involved in transmission in the central nervous system. In K. RODAHL (Ed.), Nerve as a Tissue, Harper and Row, New York, 1966. 16 PAPPAS, G. D., COHEN, E. B., AND PURPURA, D. P., Fine structure of synaptic and nonsynaptic neuronal relations in the thalamus of the cat. In D. P. PURPtrRA AND M. YAHR (Eds.), The Thalamus, Columbia University Press, 1966. 17 PAPPAS, G. D., AND PURPtrRA, D. P., Fine structure of dendrites in cat superficial neocortex neuropil, Exp. Neurol., 4 (1961) 507-630. 18 PAPPAS, G. D., AND PURPURA, D. P., Electron microscopic studies of immature feline hippocampus, Anat. Rec., 148 (1964) 319. 19 RALL, W., SHEPHERD, G. M., REESE, T. S., AND BRIGHTMAN,M. W., Dendro-dendritic synaptic pathway for inhibition in the olfactory bulb, Exp. Neurol., 14 (1966) 44-56. 20 SHOFER,R. J., PAPPAS, G. D., AND PLrRPURA,D. P., Radiation induced changes in morphological and physiological properties of immature cerebellar cortex. In T. J. HALEY AND R. S. SNIDER (Eds.), Second Symposium on Response of the Nervous System to Ionizing Radiations, Little Brown, Boston, 1964, pp. 476-508. 21 SMITH, B., MAO in pineal, neurohypophysis and brain of the albino rat, J. Anat. (Lond.), 97 (1963) 81-86. 22 TAXI, J., Etude de l'ultrastructure des zones synaptiques dans les ganglions sympathiques de la grenouille, C.R. Acad. Sci. (Paris), 252 (1961) 174-176.
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BRAIN RESEARCH
T H E F I N E S T R U C T U R E OF N E U R O N S A N D SYNAPSES OF T H E H A B E N U L A OF T H E CAT W I T H SPECIAL R E F E R E N C E TO SUBJ U N C T I O N A L BODIES
MONIQUE MILHAUD* AND GEORGE D. PAPPAS** Department of Anatomy, Columbia University, College of Physicians and Surgeons, New York, N. Y. (u.s.A.).
(Received May 16th, 1966)
INTRODUCTION The two habenulas are deep nuclei of the epithalamus, symmetrically placed on each side of the third ventricle. The major connections of each habenula are: (1) the stria medullaris which relate the habenula to the olfactory system, (2) the habenulotectal and tecto-habenular tracts which are interconnections between the habenula and the superior colliculi, (3) the habenulo-tegmental tracts which discharge impulses from the habenula to the dorsal tegmental nuclei, and (4) the largest and best known efferent path from each habenula, the habenulo-peduncular tract or fasciculus retro-flexus of Meynert which arises from the habenula and ends in the interpeduncular nucleus. Each habenula may be divided into medial and lateral nuclei, the lateral one continuing in the fiber bundles of the stria medullaris. The purpose of this study is to relate the fine structure of the neurons and the synaptic organization at the light and electron microscopical levels. MATERIAL AND METHODS The brains of 6 adult cats were perfused under pressure with 2.5~o glutaraldehyde phosphate buffered at p H 7.4. The habenula nuclei were removed and placed into fresh cold fixative for a total of 2 h. After many changes of cold phosphate buffer, the tissue was left in it overnight. Then the tissue was post-fixed with cold 1 ~o osmium tetroxide phosphate buffered at p H 7.4 for 1 h, dehydrated, and embedded in Epon 812. Both thick and thin sections were cut on a Porter Blum microtome M.T. 2. Thick sections of 1/z were stained with toluidine blue in 0.5 ~ aqueous solution and examined * Attach6 de Recherche ~ l'Institut National de la Sant6 et Recherches M6dicales (INSERM) (France). Supported in part by a NATO fellowship. Permanent address: Laboratoire de Microscopic Electronique, Division Risler, La Sall~tri/~re, Paris 13 (France). ** Supported by a Public Health Service research career award (I-K3-NB-21,947-01) from the National Institute of Neurological Diseases and Blindness. Brain Research, 3 (1966) 158-173
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with the light microscope. Thin sections were stained with warm aqueous uranyl acetate solution for 30 rain and examined with an R C A - E M U 3F electron microscope. RESULTS
Two types of neurons can be discriminated with the light microscope in the lateral as well as in the medial nucleus (Figs. 1 and 2). The larger neurons are about
Fig. 1. Light micrograph of a I-/~ thick section of the medial nucleus of the habenula stained with toluidine blue. Neurons can be differentiated into two sizes, N1 being larger than Nz. Throughout the medial nucleus the neuronal cell bodies are more-or-less evenly dispersed. Often neuronal somata can be found in close apposition to each other (at arrows). Many blood vessels (BV) are present throughout the neuropil. × 2500. 15 by 20 # in diameter and the smaller ones about 10 # in diameter. Both types have one or more processes and contain many dark granulations in their cytoplasm. The neurons are more densely placed in the medial nucleus than in the lateral, where they are broken up by myelinated fiber bundles. The cells are often arranged side by side regardless of their size (Fig. 2). Many blood vessels are present throughout the neuropil. Oligodendroglial cells are readily identified when they are close to the neuronal cell bodies (Fig. 2). Electron microscopic examination shows that there is no significant distinction between the two sizes of neurons except the amount of cytoplasm. The usual cytoplasmic organelles, such as Golgi apparatus, mitochondria, granular endoplasmic reticulum which is not arranged in Nissl bodies but distributed throughout the perikaryon, and occasional dense core bodies, are present. The neuronal cytoplasm is characterized by many dense granular lysozome-like bodies someBrain Research, 3 (1966) 158-173
Fig. 2. Light micrograph of a 1-# thick section of the lateral nucleus of the habenula stained with toluidine blue. The neurons are fewer in number and more dispersed than in the medial nucleus. Both large and small neurons are present in the lateral nucleus as in the medial. A large neuron (N1) is shown in close apposition with a smaller one (Na). Oligodendroglia (G) are closely applied to the neuronal somata. The neuropil of the lateral nucleus contains more myelinated fibers than the medial. Bv ~ blood vessels; My ~ myelinated fibers, x 2500.
Fig. 3. Electron micrograph of a portion of the cytoplasm of a neuron in the medial nucleus. Many dark granular bodies (DB) of varying densities characterize these neurons. At times the dense body is found in close contact to mitochondria (at arrows). N = nucleus; E R = granular endoplasmic reticulum; G = Golgi complex. × 13,800.
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Fig. 4. Electron micrograph of two closely applied neurons with no intervening processes. An axon (A) forms a synaptic junction with one of them. M = mitochondria; DB = dense bodies; ER = endoplasmi¢ reticulum, x 12,000. times in close association with mitochondria (Fig. 3). Multivesicular bodies are abundant in the perikaryon regions as well as in the processes. They are frequent in dendrites and are often found in proximity to synapses 17. Occasionally 2 or 3 neurons, especially in the medial nucleus, are side by side without any glial interposition (Figs. 4, 5 and 6). The apposing neuronal membranes are separated by the extracellular space of about 200 A found throughout the neuropil of the CNS 11. Occasionally a few intervening processes do occur in restricted areas (Fig. 5). The area of somasoma contiguity may be as much as 12 if, which represents the entire length of neuronal apposition (Fig. 4). Sometimes, desmosome-like specializations 'joining' these cells are found (Fig. 6). Two types of glial cells are readily identified in the neuropil, the fibrous astrocytes and oligodendrocytes close to the neuronal cell bodies. Dendritic trunks, some of which are very large, are characterized by their high content of microtubules and long mitochondria (Fig. 7). Many typical synapses are present on dendritic trunks (Fig. 7). The presynaptic process is characterized by the presence of clusters of clear vesicles, mitochondria, and some larger vesicles about 800-1200 A in diameter having a dense core. Both the pre- and postsynaptic membranes show Brain Research, 3 (1966) 158-173
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Fig. 5. Higher magnification of a p o r t i o n of a region of c o n t a c t b e t w e e n two neurons. T h e extracellular space o f a b o u t 180-200 A is m a i n t a i n e d between t h e m . Occasionally a few intervening processes (P) m a y be found. N = n u c l e u s ; M ~ m i t o c h o n d r i a ; D B ~ dense bodies. ×, 44,800,
Fig. 6. A n o t h e r p o r t i o n of the contact between two s o m a t a . At arrows, a desmosome-like specialization is apparent. T h e extracellular space is m a i n t a i n e d t h r o u g h o u t the n e u r o n a l m e m b r a n e apposition. D B ~ dense bodies; M = m i t o c h o n d r i a ; N = nucleus. × 44,800.
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Fig. 7. A cross section of a dendrite. Circular profiles of microtubules (MT) are present throughout. Two axons (A) form synaptic junctions on the surface of the dendrite. The postsynaptic membrane has more dense material accumulated than the presynaptic one. In addition about 500 A from the postsynaptic membrane some round dense discrete bodies may be found (at arrows), x 49,400.
Fig. 8. An axo-dendritic junction in the neuropil of the lateral nucleus. The axon (A) contains a large accumulation of clear vesicles. In the dendrite about 500 A from the postsynaptic membrane a single row of dense bodies is found. Often a multivesicular body is present near the dense bodies (MV). × 60,000.
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Fig. 9. T h e axon (A) of the axo-dendritic synapse contains fewer vesicles t h a n one s h o w n in Fig. 8. T h e microtubules (MT) of the dendrite are seen in longitudinal section. A row o f postsynaptic bodies (at arrows) is present. F := fibrils of a fibrous astrocytic process. ~ 50,000.
Fig. 10. Axo-dendritic s y n a p s e in the medial nucleus, T h e a x o n h a s a large a c c u m u l a t i o n of vesicles (V). A row of discrete p o s t s y n a p t i c bodies is present in the dendrite. M = mitochondria, x 43,000.
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Fig. 1I. In this axo-dendritic synapse, in addition to clear synaptic vesicles (V) a few larger densecored vesicles (DC) are present. Postsynaptic dense bodies are seen at arrow. MT = longitudinal array of microtubules. × 41,000. accumulation of dense material though this is usually in greater amount in the latter. There is material of intermediate density filling the synaptic gap and usually appearing condensed into stria perpendicular to the membranes. Occasionally some clear vesicles are open into the cleft from the presynaptic side (Fig. 9). In addition to these usual synaptic features, a single row of dense bodies is present in the postsynaptic process in about one-third of the junctions (Figs. 7, 8, 9, 10 and 11). These bodies are round, usually discrete, and about 200-250 A in diameter. They are regularly arranged about 200 A apart from center to center. Usually 2-12 are found in thin sections. They are located at about 500 A from the postsynaptic membrane and are distinct from it. They are composed of a fine dense granular material and are not membrane-bounded. They are seen as circular profiles in both longitudinal (Figs. 9, 10 and 11) and cross sections (Figs. 7 and 8), and always appear in a single row in the micrographs. Because of their regular spacing and their invariable appearance in a single row, it is assumed that they are spherical and are arranged in an hexagonal array. This hexagonal arrangement must have 200 A sides and be located at a distance of 500 A from the postsynaptic membrane. This hypothesis seems to be confirmed by the appearance of axo-dendritic synapses in tangential sections where the synaptic region loses its distinct morphological features because of the plane of section, but where hexagonal array of postsynaptic bodies is clearly seen adjacent to the synapse (Figs. 12a and b). In a three dimensional reconstruction drawing made from electron micrographs, a plaque made up of postsynaptic spherical bodies in an hexagonal array 500 A from the postsynaptic membrane is illustrated in Fig. 12c. Dendritic spines are present with some frequency in the habenula. They are always found in sections with two axons surrounding the neck and forming synapses Brain Research, 3 (1966) 158-173
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Fig. 12a, b and c. Tangential sections through axo-somatic synapses (a and b). Because of the plane of section, pre- and postsynaptic dense material appears continuous. In such a tangential section the postsynaptic bodies are seen in an hexagonal array (at arrows). × 41,000. In Fig. 12c a three-dimensional drawing shows the relationship of the hexagonally-packed subjunctional bodies. The plaque of dense bodies is about 500 A from the postsynaptic dendritic membrane. The bodies are about 250 .~ in diameter arranged hexagonally, having 200/~ sides.
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9
Fig. 13a, b and c. Electron micrographs of sections through three dendritic spines in the habenula. The central core of the spine contains a row of postsynaptic dense bodies which do not appear as discrete as in other synapses (at arrows). Two axonal processes form synapses onto the sides of the spine process. The axons contain large accumulations of clear vesicles (V) and a few large dense-cored vesicles (DC). The row of postsynaptic bodies which is present in all the spines observed is about 500 A from the postsynaptic membrane. A multivesicular body (MV) is sometimes found at the base of the spine. The synaptic gap substance can be seen condensed as striae perpendicular to the preand postsynaptic membrane in Fig. 13c. MT = microtubules. Figs. 13a and b, 49,400; Fig. 13c, 43,000.
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on both sides but not at the bulbous tip 13. The occurrence o f p o s t s y n a p t i c bodies in spines is very consistent. They always appear as a single row of regularly arranged dense bodies forming the central axis or core of the neck o f the spine. This row is about 500 ~ from the postsynaptic m e m b r a n e (Figs. 13a, b and c). Axo-somatic synapses are c o m m o n and may occur more frequently on the larger neurons than on the smaller ones. In about one-third of the axo-somatic junctions, postsynaptic bodies are seen with the same features as in the axo-dendritic junctions (Fig. 14).
Fig. 14. Axo-somatic synapses on a large neuron. Of the three synaptic junctions shown in this micrograph only the middle one shows a row of postsynaptic bodies (at arrows). V = clear synaptic vesicles; DC -= dense-cored vesicles; M = mitochondria; ER = granular endoplasmic reticulum. × 52,400.
Most frequently, the axons in the habenula have a few o f the dense-cored bodies beside the clear vesicles, not only in the synaptic region but sometimes t h r o u g h o u t the length o f the axon. Axo-axonic synapses are infrequently found. They are identified only if one of the axons of the pair is presynaptic to an axo-somatic or axo-dendritic synapse (Fig. 15). The fact o f the synaptic contact between two axons is assumed by the presence o f synaptic vesicles in both processes and of the postsynaptic thickenings which m a y indicate the polarity of the transmission. In any case, postsynaptic bodies associated with an axo-axonic contact have not been found. Brain Research, 3 (1966) 158-173
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In addition to these synaptic specializations and to the desmosome-like thickenings between adjacent neuronal cell bodies, dendro-dendritic junctions can be occasionally identified by having an equal amount of dense material in the cytoplasm in close apposition to each membrane. The gap between the apposing membranes at these regions is somewhat enlarged (Fig. 16a and b). In Fig. 16a, axon A forms a
Fig. 15. Profile of three axons (A1, As and As) containing synaptic vesicles. Axon A1forms a synapse with dendrite D. Axon A1 may also be forming a synapse with A2. In this axo-axonic junction axon As is probably postsynaptic to A1 because of the greater accumulation of dense material onto its apposing membrane. × 35,000.
synapse with dendrite D1. In Fig. 16b, an axon forms a synapse with dendrite D2. In both micrographs dendrite D2 forms a desmosome-like junction with dendrite D1 and has, in addition, two structures associated with postsynaptic processes of the habenula: sub-junctional dense bodies (at arrows) and a multivesicular body (MV). COMMENTS The two types of neurons in the habenula are present in both nuclei. In the medial nucleus there are more cells per unit area than in the lateral one where they are broken up by myelinated fiber bundles. The neuronal soma-soma apposition observed in the habenula has already been described in other regions. Green and Maxwell 10 have observed that in the adult Brain Research, 3 (1966) 158-173
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Fig. 16a a n d b. T w o adjacent dendrites f o r m a d e s m o s o m e - l i k e j u n c t i o n where there is a symmetrical a n d equal a c c u m u l a t i o n of dense material on b o t h sides as well as an enlarged extracellular space at this site. A row o f dense bodies (at arrows) is f o u n d in o n e of t h e dendrites. In addition, at a short distance f r o m these sub-junctional dense bodies a multivesicular b o d y (MV) is present. In Fig. 16a, a x o n (A) f o r m s a synapse with dendrite D1 a n d in Fig. 16b axon (A) f o r m s a synapse with dendrite D2. In b o t h micrographs, dendrites Dz have rows o f dense bodies as well as a multivesicular body. However, dendrite D1 c o n t a i n s n o n e of t h e m o r p h o l o g i c a l features o f a presynaptic process. M T cross section of microtubules. × 35,700.
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hippocampus the pyramidal cell bodies appear to be in contiguity without any enlargement of the extracellular space at these sites. As a result, in this area, the extraneuronal space around the pyramidal bodies is very limited. Pappas and Purpura is have noticed the same cell body to cell body appositions in the hippocampus of neonatal kittens, but without any desmosome-like junctions. However, desmosomelike specializations at soma-somatic 'contacts' have been observed between granule cells of the cerebellum by Gray 7 in the adult and by Shofer et al. 2° in the immature cerebellum. They have been described as equilateral densities associated with the apposing membranes and they resemble synaptic contacts except that they show no asymmetry or polarity and their functional interpretation, if not of adherence, is quite obscureL Axons forming synaptic junctions in the habenula are evenly dispersed throughout the neuropil. Most of them occur on dendritic trunks regardless of their size, fewer are seen on neuronal somata and on dendritic spines, while only very rarely are any seen on other axons. The morphological characteristics of synapses in the habenula are the same as in other regions. In the presynaptic process clusters of clear vesicles as well as the granular dense-cored vesicles are found. These larger vesicles have been discussed recently in their relation to catecholamine metabolism in the midbrainlL The densities associated with the pre- and the postsynaptic membranes, in axo-dendritic as in axosomatic synapses are variable and do not seem to be limited to two discrete types as described by Gray 5 in the cortex, since many intermediate forms may be present. The synaptic cleft substance sometimes appears to be organized into stria perpendicular to the apposing synaptic membranes, or condensed as an intermediate line parallel to the membrane 15. In the habenula, the gap substance in most instances appears to be in a striated form. In the postsynaptic process the dense bodies in the habenula are similar to some structures noticed in other regions of the central nervous systemL They have been observed also in the locus coeruleus and in the nucleus interpeduncularislL Recently, Mugnaini 14 has found similar postsynaptic bodies in the nucleus vestibularis lateralis and in the granular layer of the cerebellar cortex of the cat. They resemble the postsynaptic bars described by Taxi 22 in the autonomic ganglia of the frog in that they are distinct from the postsynaptic densities closely associated to the membrane, but they differ in that they are hexagonally arranged. More striking is the fact that these postsynaptic bodies are present in all dendritic spines observed in the habenula as well as the interpeduncular nucleus. Such a specialized structure in spines has not been described in other regions. The spine apparatus, particularly prominent in the rat cortex 6, has not been found in the feline habenula. At about the same distance where the spine apparatus has been described, a multivesicular body is sometimes present (Fig. 13). The significance of the postsynaptic bodies is unknown. The question arises whether there is any relationship between the presence of postsynaptic bodies and the high concentration of monoamine-oxydase in the habenulal, 2a. For the present there are no data to support such an inference. Brain Research, 3 (1966) 158-173
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Axo-axonic synapses have been identified by Gray 8 in the spinal cord, by Colonnier and Guillery2 in the lateral geniculate nucleus and by Pappas et al. 16 throughout various other regions of the thalamus. Axo-axonic synapses have been postulated by Eccles 3 to be the morphological basis of presynaptic inhibition. They may also form the basis for presynaptic inhibition of inhibition16. Unfortunately no electrophysiological study of the habenula has been reported. Therefore no correlation between morphological data and functional activities can be made with respect to this structure. This statement is equally applicable to studies reporting a major participation of the habenula in reflex controlling blood volume4. The most puzzling finding is that dense bodies similar to those described above and in a previous communication13 as 'postsynaptic' are found in dendro-dendritic junctions. As in all desmosome-like junctions the densities closely associated with the apposing membranes are symmetrical and the extracellular space between them, at this site, is somewhat enlarged2, 9. The dendro-dendritic junctions shown in Fig. 16a and b have all the morphological features of a synapse except for the complete lack of vesicles. Recently dendro-dendritic synapses have been described in the olfactory bulb 19. Their appearance differs from the dendro-dendritic junctions observed in the habenula by two important features: (1) the presence of clusters of vesicles at the presynaptic site of contact and (2) the asymmetrical localization of the dense material associated with apposing membranes. In addition, Rall et al. 19 present physiological data to support the evidence of dendro-dendritic transmission in the olfactory bulb. In the habenula, the significance of a specialized dendro-dendritic junction where one dendrite has the morphological features of a postsynaptic process is, to say the least, unknown. For present purposes, these dense bodies described here may be designated as sub-junctional dense bodies, without further implications as to their functional significance. SUMMARY
Two types of neurons are present through the lateral and medial habenula. Occasionally these form soma-soma appositions without any intervening processes. About one-third of the axo-dendritic and axo-somatic synapses have discrete dense bodies in an hexagonal array in the postsynaptic process. All the dendritic spines observed have a single row of these dense bodies. Axo-axonic synapses are present but are rare. Desmosome-like structures in dendro-dendritic junctions are occasionally found containing dense bodies, similar to those found in the postsynaptic process in some synaptic junctions. The significance of the presence of sub-junctional dense bodies in the habenula is unknown. ACKNOWLEDGEMENTS
This work was supported in part by Public Health Service grants from the National Institute of Neurological Diseases and Blindness (NB-02314-07 and NB03448-05) and the United Cerebral Palsy and Educational Foundation. Brain Research, 3 (1966) 158-173
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REFERENCES 1 BOUCHAUD, CL., COUTEAUX, R., ET GAUTRON, J., L'inhibition des monoamine-oxydases du cerveau du rat par la fl-phenylisopropylhydrazine et l'harmaline. Etude histochimique de l'action de ces inhibiteurs et de leur antagonisme, C.R. Acad. Sci. (Paris), 260 (1965) 348-351. 2 COLONr,aER, M., AND GUILLERY, R. W., Synaptic organization in the lateral geniculate nucleus of the monkey, Z. Zellforsch., 62 (1964) 333-355. 3 ECCLES,J., Physiology of Synapses, Academic Press, New York, 1963. 4 FAUR~, J., LE NOV~NE, J., VINCENT,D. ET BENSCH,EL., R6actions bio61ectriques de l'habenula et du tronc c6r6bral It l'hypovol6mie exp6rimentale, Rev. NeuroL, 112 (1962) 258-266. 5 GRAY, E. G., Axo-somatic and axo-dendritic synapses of the cerebral cortex - - an electron microscope study, J. Anat. (Lond.), 93 (1959a) 420-433. 6 GRAY, E. G., Electron microscopy of synaptic contacts on dendrite spines of the cerebral cortex, Nature, 183 (1959b) 1592-1593. 7 GRAY, E. G., The granule cells, mossy synapses and Purkinje spine synapses of the cerebellum: Light and electron microscope observations, J. Anat. (Lond.), 95 (1961) 345. 8 GRAY, E. G., A morphological basis for presynaptic inhibition, Nature, 193 (1962) 82-83. 9 GRAY, E. G,, AND GUILLERY,R. W., Synaptic morphology in the normal and degenerating nervous system. In BOURNE AND DANIELLI (Eds.), International ReviewofCytology, Vol. 19, New York, Academic Press, 1966. 10 GREEN, J. D., AND MAXWELL, D. S., Hippocampal electrical activity. Morphological aspects, Electroenceph. clin. Nearophysiol., 13 (1961) 837-846. 11 HORSTMANN, E., AND MEVES, H., Die Feinstruktur des molekularen Rindengraues und ihre physiologische Bedeutung, Z. Zellforsch., 49 (1959) 569-604. 12 LENN, N. J., Electron microscopic observations on monoamine containing brain stem neurons in normal and drug treated rats, Anat. Rec., 153 0965) 399--406. 13 MILHAUD, M., AND PAPPAS, G. D., Postsynaptic bodies in the habenula and interpeduncular nuclei of the cat, J. Cell Biol., (in press). 14 MUGNA~NI,E., Personal communication, 1966. 15 PAPPAS, G. O., Electron microscopy of neuronal junctions involved in transmission in the central nervous system. In K. RODAHL (Ed.), Nerve as a Tissue, Harper and Row, New York, 1966. 16 PAPPAS, G. D., COHEN, E. B., AND PURPURA, D. P., Fine structure of synaptic and nonsynaptic neuronal relations in the thalamus of the cat. In D. P. PURPtrRA AND M. YAHR (Eds.), The Thalamus, Columbia University Press, 1966. 17 PAPPAS, G. D., AND PURPtrRA, D. P., Fine structure of dendrites in cat superficial neocortex neuropil, Exp. Neurol., 4 (1961) 507-630. 18 PAPPAS, G. D., AND PURPURA, D. P., Electron microscopic studies of immature feline hippocampus, Anat. Rec., 148 (1964) 319. 19 RALL, W., SHEPHERD, G. M., REESE, T. S., AND BRIGHTMAN,M. W., Dendro-dendritic synaptic pathway for inhibition in the olfactory bulb, Exp. Neurol., 14 (1966) 44-56. 20 SHOFER,R. J., PAPPAS, G. D., AND PLrRPURA,D. P., Radiation induced changes in morphological and physiological properties of immature cerebellar cortex. In T. J. HALEY AND R. S. SNIDER (Eds.), Second Symposium on Response of the Nervous System to Ionizing Radiations, Little Brown, Boston, 1964, pp. 476-508. 21 SMITH, B., MAO in pineal, neurohypophysis and brain of the albino rat, J. Anat. (Lond.), 97 (1963) 81-86. 22 TAXI, J., Etude de l'ultrastructure des zones synaptiques dans les ganglions sympathiques de la grenouille, C.R. Acad. Sci. (Paris), 252 (1961) 174-176.
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