Ultrastructural changes in the gracile nucleus of the spontaneously diabetic BB rat.

The present study describes the structural changes in the gracile nucleus of the spontaneously diabetic BB rat. At 3-7 days post-diabetes, axons, axon terminals and dendrites showed electron-dense degeneration. Degenerating axons were characterized by swollen mitochondria, vacuolation, accumulation of glycogen granules, tubulovesicular elements, neurofilaments and dense lamellar bodies. Degenerating axon terminals consisted of an electron-dense cytoplasm containing swollen mitochondria, vacuoles and clustering of synaptic vesicles. These axon terminals made synaptic contacts with cell somata, dendrites and other axon terminals. Degenerating dendrites were postsynaptic to normal as well as degenerating axon terminals. At 1-3 months post-diabetes, degenerating electron-dense axons, axon terminals and dendrites were widely scattered in the neuropil. Macrophages containing degenerating electron-dense debris were also present. At 6 months post-diabetes, the freshly degenerating neuronal elements encountered were similar to those observed at 3-7 days. However, there were more degenerating profiles at 6 months post-diabetes compared to the earlier time intervals. Terminally degenerating axons were vacuolated and their axoplasm appeared amorphous. It is concluded that degenerative changes occur in the gracile nucleus of the spontaneously diabetic BB rat.     

Mapping of experimental axon degeneration by electron microscopy of golgi preparations

Summary For the mapping of the terminal area of transected axons within the central nervous system, electron microscopy has recently been adopted. A greater accuracy is thereby obtained than with silver impregnation and light microscopy, since it becomes possible to determine the kinds of structure (soma, dendrites, spines) with which the degenerating boutons establish synaptic contact. In the present study this technique was extended by Golgi impregnation of such material with the aim of making possible classification of the postsynaptic neuron. A few days after transection of a pathway (commissural fibres to the hippocampus being used as a model in this study) the brain was fixed by perfusion with phosphate buffered formalin with sucrose. This was followed by immersion in an osmium tetroxide-potassium dichromate mixture (Dalton's fixative without sodium chloride) later replaced by a solution of silver nitrate. Satisfactory Golgi impregnation of nerve cells and processes was obtained. By careful trimming, and reembedding of selected areas, blocks for ultramicrotomy could be obtained which contained only one type of impregnated cell, e.g., hippocampal pyramidal cells.The relation of basal dendrites of such cells to degenerating boutons of commissural fibres was studied. Numerous examples of contact between degenerating boutons and spines belonging to the basal dendrites were seen. Although the Golgi precipitate obscured the postsynaptic substance in the spines, a number of features at the sites of contact were considered strong indication that many of the contacts were synapses and not merely the result of random juxtaposition. This combined procedure is supposed to be applicable to other problems and to other parts of the nervous system as well.This study was supported in part by Grant NB 02215 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. This ais is gratefully acknowledged. The author is indebted to Mrs. J. L. Vaaland and Mr. B. V. Johansen for valuable technical assistance.     

Cell types and neuronal connections in cultures of mammalian central nervous tissue

Summary Various neuronal cell types surviving in cultures of cerebellum, brain stem and cerebral cortex of new-born rats and kittens were described. The regenerative power of neurons in these explants was expressed by the growth of new processes that reached a length of several millimeters. However, nerve fiber regeneration and growth followed an entirely erratic pattern as evidenced by sometimes extensive fiber convolutions.Feed-back collaterals of axonic processes that returned fibers to their own cell bodies or their dendrites were a common feature in many neurons. Short chains of interconnected neurons were detected in a number of cultures. The connections between these neurons were established by apposition of fine axon terminals to presumable postsynaptic cell bodies or dendrites but not by true synaptic boutons. In numerous instances, synapses of the bouton type that appeared morphologically normal could be shown to represent remnants of severed connections since the presynaptic fibers proved to be isolated fiber fragments. Under the sterile conditions of the culture environment these structures apparently persisted without considerable morphological alteration. Evidently, most neurons in our cultures were reduced to the status of isolated cells due to extensive de-afferentiation at the time of explantation. Moreover, there was no evidence for the establishment of new synaptic connections between regenerating neurons.This work was supported by Research Grant NB 03114-05 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service.Dedicated to my esteemed teacher, Prof. Dr. W. Bargmann, on the occasion of his 60th birthday.Sincere appreciation is expressed to Mrs. Eleanor Morris for her assistance in the management of the cultures, and to Mr. E. E. Pitsinger, Jr. for his photographic assistance.     

Ultrastructural changes in the gracile nucleus of alloxan-induced diabetic rats

The present study reports ultrastructural changes in the gracile nucleus of male Wistar rats after alloxan-induced diabetes. During the acute phase (3-7 days) degenerating electron-dense dendrites and axon terminals were dispersed in the neuropil. Degenerating dendrites were characterized by an electron-dense cytoplasm, swollen mitochondria, dilated endoplasmic reticulum and randomized ribosomes. Degenerating axon terminals were characterized by an electron-dense cytoplasm and clustering of small spherical agranular vesicles. Degenerating axon terminals may form the central element or part of a synaptic glomerulus. Macrophages were present in the neuropil and in the process of engulfing neuronal elements. During the medium phase (1-6 months), most of the degenerating dendrites and axon terminals had been engulfed or removed by macrophages. During the late phase (9-12 months), a second wave of degeneration occurred in the gracile nucleus, similar to the acute phase.     

Ultrastructural changes in the superficial layers of the superior colliculus in Galago crassicaudatus (primates) after eye enucleation

Summary Several types of terminals were found in the three superficial collicular layers of Galago. At least two axon terminals with round vesicles (R1 and R2) could be distinguished on the basis of vesicle packing and electron density of the cytoplasmic and mitochondrial matrices. R1 axon terminals were characterized by aggregations of vesicles in an electron lucent cytoplasm and mitochondria with a relatively dark matrix, while in R2 axon terminals the vesicles were more evenly distributed in an electron dense cytoplasm and the mitochondrial matrix was pale. R2 endings occurred in clusters in the stratum griseum superficiale; they were absent in the stratum zonale. R1 endings were found in all three superficial collicular layers. Both types of R terminals made asymmetrical contacts with small dendrites, dendritic spines and F profiles. Profiles containing flattened vesicles and establishing symmetrical contacts were numerous, and many could be identified as dendrites by accepting as criteria for dendrites evenly spaced microtubules, clusters of ribosomes and the fact that these F profiles were postsynaptic to other terminals. F terminals were presynaptic to other F profiles, dendrites and somata; they were postsynaptic to R terminals and took part in serial synapses. Dendrodendritic contacts were frequent, somatodendritic contacts rare. After eye enucleation most R2 axon terminals underwent the electron dense degenerative reaction. The degeneration process was a lengthy one; many degenerating boutons were found 30 days after axotomy and some persisted up to 180 days postoperatively. There was strong indication that the superior colliculus received more crossed than uncrossed retinofugal fibers. The crossed and uncrossed retinocollicular axons terminated in two different substrata of the stratum griseum superficiale.This study was supported by N.I.H. Grant RR-00165 to Yerkes Regional Primate Research Center and N.I.H Grant EY 00638-03 to J. Tigges. — The opportunity to use the electron microscopic facilities of the Fernbank Science Center for the initial stage of this work is gratefully acknowledged.     

Projections to the median eminence and the arcuate nucleus with special reference to monoamine systems: Effects of lesions

The external layer of the median eminence (ELME) and the arcuate nucleus of male rats were studied with the Falck-Hillarp technique and electron microscopy of aldehyde-OsO4 or KMnO4 fixed material after various types of hypothalamic deafferentation experiments with the Haláz knife. Special reference was paid to the monoamine systems and the results can be summarized as follows. 1. The main monoaminergic input to the ELME comes from the arcuate nucleus-periventricular area via a dorsal approach. A horizontal transection through the arcuate nucleus decreases the percentage of monoamine boutons i.e. boutons with small granular vesicles, from 31.6% in the controls to 4.4% in the lesion group, whereas only a small effect is seen after anterior (or complete) deafferentations. 2. A major input to the ELME enters the basal hypothalamus at the anterior-lateral aspects (see Réthelyi and Halsáz, 1970). The fibers cut after anterior deafferentations in all probability mainly come from cell bodies localized in the anterior hypothalamus or even further rostrally but some may represent Na axons ascending from the lower brain stem. 3. The degeneration course of nerve endings in the ELME both after anterior deafferentations as well as after lesions in the arcuate nucleus is rapid (within 2-3 days) and morphologically characterized by an initial aggregation of large dence cored vesicles seemingly to electron dense bodies within the boutons and probably also to a closer spacing of the small electron lucent synaptic vesicles (see Raisman, 1972). This type of degeneration seems to take place both in monoamine and non-monoamine neurons. 4. Degenerating boutons are found in the arcuate nucleus after anterior and complete deafferentations. Thus, the anterior hypothalamus may exert an "indirect" control of the pituitary gland via synapses on arcuate neurons although quantitavely the direct influence through the projection to the ELME is of more importance. 5. After anterior deafferentations enlarged axons containing large amounts of large dense cored vesicles and other organelles are found caudally of the cut indicating the existence of rostral projections from the medial hypothalamus.     

The reticular substance of the medulla oblongata of the albino rat

Summary The region of the reticular giganto-cellular nucleus, perfused with formalin and postfixed in osmium tetroxide, was studied with histochemical and electron microscopic techniques. The perikarya of the neurons have two zones. The peripheral cytoplasm contains Nissl bodies, mitochondria, and free RNP particles. The juxtanuclear cytoplasm contains the Golgi complex, mitochondria, RNP particles and dense bodies. The nucleus is indented and has a prominent nucleolus and a paranucleolar body. Dense bodies are found along the axon and dendrites as well. Three different types of synapses are described and two types of synaptic vesicles (spherical and ellipsoidal) are shown.The capillary endothelium shows microvilli and marginal flaps. The endothelial cytoplasm contains vacuoles, micropinocytotic vesicles, and a few dense bodies. Processes of pericapillary cells, surrounded by a basement membrane, also contain dense bodies. The dense bodies found in the neurons and endothelial cells show acid phosphatase activity. On the basis of their morphology and their enzymatic activity these bodies are identified as lysosomes.Partially supported by a school grant No RF 62051 from the Rockefeller Foundation, New York, USA, and Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.Fellows of the Consejo National de Investigaciones Científicas y Técnicas, Argentina. The authors wish to thank Dr. Mario H. Burgos for his constant interest and criticism, and to Dr. Eduardo Rodriguez Echandia and Dr. Fabio L. Sacerdote for their valuable assistance.     

Neuronal and synaptic organization of the lateral geniculate nucleus of the tree shrew,Tupaia glis

Summary The ultrastructural study of the lateral geniculate nucleus (LGN) of the tree shrew (Tupaia glis) revealed two types of neurons: (1) a large thalamocortical relay cell (TCR), which may bear cilia, and (2) a small Golgi type-II interneuron (IN) with an invaginated nucleus. The narrow rim of pale cytoplasm of the IN contains fewer lysosomes and fewer Nissl bodies than the cytoplasm of the TCR. The IN perikarya, which in some cases establish somatosomatic contacts, frequently contain flattened or pleomorphic synaptic vesicles. The ratio of TCR to IN is 31.Three types of axon terminals were observed in the LGN. Two of them contain round synaptic vesicles but differ in size. The large RL boutons undergo dark degeneration after enucleation; they are the terminals of retino-geniculate fibers. The smaller RS boutons show dark degeneration after ablation of the visual cortex; they are the terminals of the cortico-geniculate fibers. The third type of bouton (F₁ does not degenerate after either intervention. The boutons of this type are filled with flattened vesicles and are believed to be intrageniculate terminals. F₂-profiles were interpreted as presynaptic dendrites of the IN. The characteristic synaptic glomeruli found in the LGN contain in their center an optic terminal. These optic terminals establish synaptic contacts with dendrites or spine-like dendritic protrusions of TCRs as well as with presynaptic dendrites. Synaptic triads were also seen. The distribution of the individual types of synaptic contacts in layers 3 and 4 was determined. Layer 4 contains only one third of the retino-geniculate synapses and of the synaptic contacts of F₁-terminals.     

Degenerated boutons in the fundus striati (nucleus accumbens septi) after lesion of the parafascicular nucleus in the cat

Summary An attempt has been made to reveal which of the nine different types of synapses in the fundus striati, discriminated in a previous study, degenerate following experimental lesions in the parafasciculo-center median complex of the cat. Two types of synaptic contacts were found to be degenerated two days after the lesion was performed: (1) the axo-spinous type IV, characterized by densely-packed, small, round vesicles and a strong asymmetric thickening, and, (2) the axo-dendritic or axo-somatic type VII, again characterized by small, round vesicles in a dense accumulation and an asymmetric thickening. After two days of survival the original characteristics of the boutons could still be recognized in both types of synapses.A positive correlation exists between the location and extent of the coagulation foci in the parafascicular nucleus and the appearance of degenerated boutons in the fundus striati. Therefore, the conclusion that the parafasciculofundus neurons terminate as type IV or type VII boutons is entirely justified. Additionally, the role of the special types of boutons in the synaptic organization of the fundus striati has beeen discussed.Dr. J. W. Chung on leave of absence from the Department of Anatomy, Catholic Medical College, Seoul, KoreaHerrn Professor Dr. Drs. h. c. Wolfgang Bargmann zum 70. Geburtstag gewidmet     

Septal afferents to the area dentata terminate on vasoactive intestinal polypeptide (VIP)-like immunoreactive,non-pyramidal neurons

Summary Thermic lesions in the medial septum of adult rats result in dark degeneration of terminal boutons in the stratum moleculare and hilus of the area dentata. While most of the degenerating terminals are in synaptic contact with non-reactive cells, part of them end on dendrites of VIP-like immunoreactive neurons.     

Acetylcholinesterase activity at the synapses of presynaptic boutons with presumed alpha-motoneurons in chicken ventral horn. Light- and electron-microscopic studies

Acetylcholinesterase (AChE) activity at the synapses of presynaptic boutons on presumed alpha-motoneurons in the chicken ventral horn was studied histochemically at the light- and electron-microscope levels. At the light-microscope level, many dot-like AChE-active sites were observed on the soma and dendrites of presumed alpha-motoneurons. On electron microscopy, reaction products for AChE activity were observed mainly in the synaptic clefts of the four kinds of presynaptic boutons: (1) S type boutons, (2) boutons containing small, spherical, dense cored vesicles (diameter range, 60-105 nm) and spherical, clear vesicles, (3) boutons containing medium-sized, spherical, dense cored vesicles (65-115 nm) and spherical, clear vesicles, and (4) boutons containing large, spherical, dense cored vesicles (80-130 nm) and spherical, clear vesicles. In the light of previous physiological and biochemical studies, the present results suggest the possibility that each of these presynaptic boutons which are AChE-active in their synaptic clefts may contain acetylcholine, substance P, or enkephalins which acts as a neurotransmitter or modulator.     

Types of degenerating geniculocortical axon terminals and their contribution to layer IV of area 17 in the squirrel monkey (Saimiri)

Summary Radiofrequency lesions were made in the lateral geniculate nuclei of six squirrel monkeys. The resulting degenerating terminals and their postsynaptic structures in layer IV of area 17 were quantitatively categorized on photomontages covering large areas of neuropil. Two to five days after the lesion, numerous axon terminals were affected by a variety of degenerative changes, i.e., enlargement and distortion of synaptic vesicles, neurofilamentous hyperplasia, electron-lucent and electron-dense reactions. Based on the aggregation of electron-dense material beneath the postsynaptic membrane, the degenerating terminals were considered to be of the asymmetric type. Among the degenerating boutons were the largest axon endings that occur in layer IV. Three days postoperatively, degenerating boutons contributed an average of 16.2% to the total synapse population; five days postoperatively, the average had increased to 19.3 %. The percentage of degenerating boutons on individual montages, however, amounted to as much as 29%. This amount probably reflects more closely the actual contribution of the geniculocortical fiber system to layer IV of striate cortex. The postsynaptic structure most frequently contacted by degenerating axon endings was the dendritic spine, followed by dendrites of small diameter. To account for the diversity of degenerative changes in the same fiber system, we offer the tentative suggestion that heterogeneously degenerating axon terminals arise from a heterogeneous population of neurons in the lateral geniculate nucleus, i.e., from magnocellular versus parvocellular laminae.     

Long-term effects of alloxan-induced diabetes on the nucleus ventralis posterolateralis in the thalamus of the rat.

The present paper describes the long-term ultrastructural changes in the nucleus ventralis posterolateralis of the thalamus of male Wistar rats after alloxan-induced diabetes. Degenerating dendrites were characterized by an electron-dense cytoplasm with scattered endoplasmic reticulum and ribosomes. Degenerating axon terminals were characterized by an electron-dense cytoplasm and clustering of small spherical agranular vesicles. Degenerating axon terminals formed axosomatic synapses with seemingly normal cell bodies and axodendritic synapses with normal as well as degenerating dendrites. Degenerating axons (both myelinated and unmyelinated) were readily encountered in the neuropil. Activated microglial and astrocytic cells in the neuropil were in the process of phagocytosis or had residua in their cytoplasm.     

The Fine Structure of Degenerating Nerve Fibers, their Sheaths, and their Terminations in the Central Nerve Cord of the Cockroach (Periplaneta americana)

The connectives above and below the second thoracic ganglion and nerves to and from the mesothoracic leg were severed in Periplaneta americana. Isolated ganglia and severed nerve cord were examined in the electron microscope. In the connectives, sheaths of degenerating fibers remain continuous but become thicker and more dense. There is increase in number and more haphazard disposition of the neuroglial processes which ensheath the axons. The cytoplasm contains vacuoles. Dense droplets normally intercalated between the layers of neuroglial processes ensheathing the axons are strikingly increased in number. The axoplasm with its organelles forms dense clumps. Mitochondria in axons are enlarged, the intramitochondrial matrix is more dense, and the internal folds are disorganized. In ganglia, mitochondrial changes in terminal parts of the axons appear similar to those described in the parent axons in the connective. The synaptic portions of nerve fibers appear very dense. Alterations of the sheath are minimal. Synaptic particles in the degenerating axoplasmic coagulum undergo only slight morphological changes and are still present up to 6 days after severance of their nerve fibers. It is difficult to assess whether there are any alterations in the total number of synaptic particles during degeneration.     

Retinohypothalamic pathway in the duck (Anas platyrhynchos)

Summary Retinohypothalamic connections were studied in the duck after unilateral optic nerve transection using both light and electron microscopic techniques. Degenerated endings of optic fibers were found only in a circumscribed part of the anterior hypothalamic area, i.e. the ventral region of the contralateral suprachiasmatic nucleus. Images of degenerating boutons were observed in frozen sections (method according to Johnstone-Bowsher), and their presence confirmed by electron microscopic examination. These degenerating boutons make synaptic contacts with dendrites or dendritic spines of neurons of the suprachiasmatic nucleus.In the same material, the decussation of the optic chiasma was studied with the light microscope. Uncrossed retinal fibers were found in the marginal optic tract, the basal optic root and occasionally also in the isthmo-optic tract.Dedicated to Professor Dr. W. Bargmann on the occasion of his 70th birthdaySupported by the DGRST and European Training Program Brain and Behaviour ResearchI wish to express my gratitude to Professor Andreas Oksche, who repeatedly offered me the scientific facilities at the Department of Anatomy of the University of Giessen, and who provided me with valuable neuroanatomical suggestions throughout the progress of these studies.     

Synapses in the cerebral ganglion of adult Ciona intestinalis

Summary Electron microscopy of the synaptic morphology of synapses in the cerebral ganglion of the adult ascidian (sea squirt) Ciona intestinalis reveals that the synapses are restricted to the central neuropil of the ganglion. Many of the synapses show a polarity of structure such that pre and post synaptic parts can be identified. The vesicles in the presynaptic bag are of two main diameters 80 and 30 nm respectively. The large vesicles have electron dense contents that vary both in their capacity and dimensions.The pre and postsynaptic membranes are more electron dense than the surrounding membranes, but they are only slightly thicker. Both the pre and post synaptic membranes have electron dense dots some 10 nm in diameter associated with their cytoplasmic surfaces. Sometimes the presynaptic membrane has larger peg-like projections between the vesicles. Associated with the post synaptic membrane are tubules some 10 nm in diameter. These tubules may be the dots cut obliquely.The synaptic cleft material is more electron dense than the surrounding intercellular material, and in it there is a dense line made up of granules about 3–5 nm in diameter. This dense line is usually mid way between the pre and post synaptic membranes, but may be nearer the postsynaptic membrane.No tight junctions between adjacent nerve process profiles have been observed.I wish to thank Professors J. Z. Young, F. R. S. and E. G. Gray for much advice and encouragement, also Dr. R. Bellairs for the use of electron microscope facilities and Mr. R. Moss and Mrs. J. Hamilton for skillful technical assistance.     

Distribution of reticulospinal fibers and theri endings in the lumbar segments of the rat spinal cord]

After stereotaxis lexions in the nucleus reticularis gigantocellularis of the modulla oblongata and nucleus reticularis pontis caudalis, the distribution of degenerating nerve fibers in the lumbar segments of the spinal cord has been studied by silver impregnation methods of Nauta and Fink-Heimer. Degenerating reticulo-spinal fibers and fragments of axonal terminations were found in the area of n. motorius ventro-medialis and n. motorius ventro-lateralis, as well as partly in n. motorius dorso-lateralis close to motoneurons and their dendrites. Mainly they pass into layers VII and VIII. This fact indicates the existence of direct-reticulo-motoneuronal synaptic connections in rats, which coincides with electrophysiological data.     

Synaptic connections of substance P-immunoreactive nerve terminals in the substantia nigra of the rat

Summary A monoclonal antibody that recognises the C-terminal part of substance P was used to study immunoreactive structures in the substantia nigra by the unlabeled antibody, peroxidase-antiperoxidase procedure. Immunoreactivity was present in nerve fibres in all parts of the substantia nigra, particularly in the pars reticulata and pars lateralis. Electron microscopically two types of bouton immunoreactive for substance P were found: Type 1 contained large electron-lucent vesicles, occasional large granulated vesicles and formed symmetrical synapses with dendrites. Type 2 boutons contained smaller, round electron-lucent vesicles, many large granular vesicles and formed asymmetrical synapses (having prominent postjunctional dense bodies) with dendrites and perikarya.Immunoreactive fibres with varicosities that had been identified light microscopically were studied in serial sections in the electron microscope. Each identified varicosity contained synaptic vesicles and formed a single synapse. An individual fibre formed boutons of only one kind (type 1 or type 2) and could form multiple synapses with the same neuron. Thus, an identified fibre in the pars compacta had eight varicosities, each of which was in synaptic contacts (type 2) with the dendrites or soma of the same neuron.The results are consistent with the concept that substance P is a synaptic transmitter in the substantia nigra and indicate that neurons in this region may receive a significant input from substance P-containing afferents, and that there are at least two types of such afferent fibres.     

Terminal degeneration in the mediodorsal cerebral cortex of the lizard Agama agama: Light and electron microscopy

The degeneration of axon terminals in the small-celled part of the mediodorsal cortex (sMDC) of the lizard Agama agama has been studied after lesions in the dorsal cortex at various survival periods. The Fink-Heimer stain was used to map and demonstrate terminal degeneration with the light and electron microscope. Electron microscopy was used to identify and describe degenerating boutons ultrastructurally. One sham-operated and three unoperated animals served as controls. Between 6 and 21 days postsurgically, degenerating terminals can be seen through 80% of the superficial plexiform layer, the zone adjacent to the cellular layer remaining free of degeneration. Swelling of dendrites in the outer part of the superficial plexiform layer and increased numbers of vacuolar invaginations, both present at short (24 hr–6 days; peak at 48–54 hr) survival periods, can be regarded as reaction to the surgical trauma. Degeneration of axon terminals takes three forms, all of the electron-dense type: gray boutons, degenerating bouton-dendritic spine complexes surrounded or engulfed by glia, and degeneration debris inside glial processes. Several forms of terminal degeneration occur concomitantly at any short (3–12 days) survival time. At longer survival times (15–21 days) only debris is present. From 6 days on, considerable numbers of degenerating structures are present, but the majority of degenerating boutons and debris are associated with reactive glia rather than with dendrites. From these observations it is concluded that in this lizard application of the combined degeneration-Golgi-EM technique would probably lead to little success. Electron microscopy of Fink-Heimer-stained sections suggests that degenerating bouton-dendritic spine complexes and degeneration debris accumulate silver particles, whereas gray boutons do not.