Routes of excretion of neuronal lysosomal dense bodies after ventricular infusion of leupeptin in the rat: a study using ubiquitin and PGP 9.5 immunocytochemistry

Summary To determine the rate and routes of removal of lysosomal, lipofuscin-like dense bodies from neurons, the protease inhibitor, leupeptin, was infused into the lateral ventricle of rats for up to nine days. After seven days a number of animals were then allowed to recover. The formation and later disappearance of dense bodies was followed by morphology and immunocytochemistry. After 48 h of infusion lysosomal dense bodies in large numbers appeared in cortical, hippocampal and cerebellar neurons, which also showed increased ubiquitin immunoreactivity, as well as in other cell types. By 3–4 days ubiquitin-immunoreactive dense bodies were equally distributed between neurons and astroglia. After seven to nine days of infusion ubiquitin immunoreactive dense bodies filled neuronal perikarya, dendrites and expanded initial segments of many axons and were abundant in glial processes. All dense bodies studied by electron microscopy were ubiquitin immunoreactive. After four days of recovery dense bodies were markedly fewer in neuronal perikarya, and virtually all were now within glial processes. From 7 to 28 days of recovery, when most neurons appeared normal, lipofuscin bodies remained in axon initial segments and in reduced numbers in glial processes, particularly around blood vessels and beneath the pia of hippocampus and of cerebellar cortex. Thus, neurons probably have a steady passage of short lived proteins through the lysosomal excretory pathway. The observed temporal sequence of events on recovery suggests that secondary lysosomes probably pass rapidly from neuronal perikarya and dendrites to astrocytes and thus to the vascular bed or pia-arachnoid. The mechanism of cell-to-cell transfer is not clear from this study.     

The fine structure of human digital arterio-venous anastomoses (Hoyer-Grosser's organs)

Summary Hoyer-Grosser's organs were studied in human digital biopsies. The fine structure of both the supplying arteries and collecting veins was found to be inconspicuous.Endothelial cells in the AV canals form a continuous layer. They are characterized by their rich content of specific organelles (Weibel-Palade bodies), especially in the venous segments. The epitheloid zone is composed of a variety of ramified smooth muscle cells (RSM). These appear either dense, when well provided with bundles of myofilaments, or clear, when including only a few myofilaments. The nuclei of dense RSM show condensed chromatin, while those of clear RSM are larger with loose chromatin texture. In addition, all transitional forms occur. Cell organelles are rarely seen within all types of RSM. The cytoplasmic processes reach each other as well as endothelial cells. The preservation of our material did not allow observation of specialized membrane contacts in these zones. All RSM are invested with a regular basal lamina and well provided with surface vesicles. Profiles of free basal lamina material and faint collagen (argyrophil) fibers are seen in the wide intercellular spaces.RSM poor in myofilaments are interpreted to represent epitheloid cells of light microscopy. Their number constantly decreases from the arterial segment of the AV canal to the venous segment. Here the cytoplasmic processes of RSM become less pronounced and the wall of the anastomotic segment continuously changes to that of the collecting vein. Dense RSM rich in myofilaments are compared with pacemaker cells found in the ureter. Both internal and external elastic membranes are absent in AV canals.A dense network of unmyelinated nerve fibers is found in the adventitial layer of the AV canal, especially in its arterial segment. The axons branch out from small dermal nerves which also contain two or three myelinated axons. The myelin sheaths terminate where the axons reach the adventitia of the AV canals. Axon varicosities filled with mitochondria are thought to be terminals of myelinated axons and are interpreted as receptory. Axon varicosities with synaptic-type vesicles are assumed to be terminals of sympathetic and parasympathetic fibers. All axon profiles are confined to the adventitial layer of the anastomotic segment.     

Gastrointestinal autonomic nerve (GAN) tumor. Ultrastructural evidence for a newly recognized entity

We describe three cases of spindle cell tumors arising in the gastrointestinal tract, each with ultrastructural features that recapitulate the ultrastructural morphology of the enteric autonomic nervous system. These features include the presence of small, dense core granules in the synthetic-secretory Golgi structures, sparsely distributed within the ample tumor-cell cytoplasm, near plasma-membrane surfaces and within axons. Tumor cells had elongated processes and axons contained small granular vesicles, clear vesicles, or large, dense vesicles. Specific features diagnostic for smooth-muscle cell, Schwann cell, or fibroblast cellular origin were absent.     

Investigations on the ultrastructural cytology of branchiomeric paragangliomas (author's transl)

The paragangliomas of the jugular and carotid bodies are growth processes which show varying histological and cytological patterns. The real nature of these lesions is still a matter of discussion. In this study 6 jugular and 3 carotid body tumours were investigated using the electron microscope. The main components were irregular cells with short and long cytoplasmic processes closely packed and mostly arranged in complexes. The ultrastructural cytology of the jugular and carotid bodies showed no striking differences. The great majority of the cells exhibits a clear similarity to the cells of normal paraganglia, especially to chief cells of carotid bodies (type I cells). But there were some modifications in the cellular structure including the abnormal structure of mitochondria and a greater variation in the morphology of the dense cored granules. The cytoplasm was dominated by differing populations of mitochondria comprising such of the crista type and such showing tubulus-like structures with a dense matrix and very often intramitochondrial bodies. The characteristic feature was the occurrence of randomly distributed dense cored granules measuring 700-800 A in diameter. These granules display a homogeneous electron dense or moderately dense somewhat granular core which is separated from limiting membrane by a clear electron lucent halo. A further population of granules was of a greater dimension (3,000-4,000 A) revealing an oval or cigar-like shape. In contrast to the former type the homogeneous dense cores of these granules showed a smooth contour and the lucent halo was very narrow. In general, singular exocytotic phenomena could be supposed. More frequently, however, signs of granulolysis were encountered. Free ribosomes, arranged often as polysomes, sparse, disorderly distributed microfilaments of 60-80 A diameter, few 240 A-microtubules and some lysosomal structures are also observed. Usually the cell clusters abutted directly upon the vascular connective tissue. In other cases cytoplasmic processes are intercalated resembling sustentacular cells. Fenestrated capillaries known to be typical for the paraganglionic tissues were only seldom detected. In the interstitial tissue typical collagen fibres are found showing a periodicity of 600-700 A, but occasionally fibres with a periodicity of about 1,200 A could be seen resembling fibrous-long-spacing collagen. Synaptic junctions and nerves or axons were always lacking.     

The structure of Schwann cells in unmyelinated fibres. A qualitative and quantitative electron microscope study

The structure, size and distribution of many cytoplasmic components of Schwann cells associated with unmyelinated axons in lizard thoracic spinal roots were analysed under the electron microscope. The percentages of Schwann cell cytoplasmic area occupied by the following cytoplasmic components were determined: mitochondria, Golgi apparatus, granular endoplasmic reticulum, multivesicular bodies, smooth endoplasmic reticulum, lipofuscin granules, peroxisome-like bodies, autophagic vacuoles, dense bodies and lipid droplets. A linear correlation was found between the sectional areas of the mitochondria and granular endoplasmic reticulum of the Schwann cell and both length of Schwann cell plasma membrane profile and size of the related axoplasm. The structure of Schwann cells associated with unmyelinated axons and that of Schwann cells associated with myelinated axons were compared in the same species and in the same region of the peripheral nervous system using the same fixative and the same preparation technique. Some differences were detected in the organization of the granular endoplasmic reticulum, in the presence of cilia and in the percentages of cytoplasm occupied by various components. The hypothesis that Schwann cell mitochondria and granular endoplasmic reticulum are involved in the production and storage of proteins for the plasma membrane of this cell as well as the hypothesis that these organelles are involved in the production and storage of protein metabolites which are subsequently transferred to the related axons seem applicable not only to Schwann cells associated with myelinated axons (Pannese et al., in press), but also to those associated with unmyelinated ones.     

Different kinds of axon terminals forming symmetric synapses with the cell bodies and initial axon segments of layer II/III pyramidal cells. III. Origins and frequency of occurrence of the terminals

Summary The cell bodies of the layer II/III pyramidal cells in rat visual cortex receive three morphologically distinct types of axon terminals. These axon terminals all form symmetric synapses and have been termed large, medium-sized, and dense axon terminals. The present study shows that each of these different kinds of axon terminals contains gamma-aminobutyric acid (GABA) which suggests that they are inhibitory. From an analysis of the profiles of 50 cell bodies it is calculated that the average layer II/III pyramidal cell has 65 axosomatic synapses, of which 43 are formed by medium-sized terminals, 10 by large terminals/and 12 by dense terminals. Comparison of these different kinds of axon terminals with labelled axon terminals of known origin suggests that the medium-sized terminals are derived from smooth multipolar cells with unmyelinated axons, and that at least some of the dense terminals originate from bipolar cells that contain vasoactive intestinal polypeptides. The source of the large axon terminals is not known, but it is suggested that they originate from multipolar non-pyramidal cells with myelinated axons.Since the initial axon segments of these same neurons receive GABAergic axon terminals from chandelier cells, at least four different types of neurons provide inhibition to the cell bodies and axons of layer II/III pyramidal cells. This serves as an illustration; of the complexity of the neuronal circuits in which pyramidal cells are involved.     

An ultrastructural study of two cases of adrenal ganglioneuroma

Tissue samples of adrenal ganglioneuromas were obtained from two patients: 35- and 47-year-old males. Light microscopic studies showed that these tumors contained Schwann cells and ganglion cells. Electron microscopic examinations revealed numerous unmyelinated and myelinated axons surrounded by Schwann cells. The ganglion cells in the tumors had abundant organelles, such as well-developed cisternae of the rough endoplasmic reticulum, many profiles of the Golgi apparatus, mitochondria, lysosomes, microtubules and neurofilaments. Electron dense cored granules resembling catecholamine granules were present in the ganglion cell bodies and neural processes. These features resmebled those of normal sympathetic ganglion cells. It is concluded that adrenal ganglioneuroma originates from sympathetic ganglion tissue. This study was presented in part at the 26th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Kochi, October 5–7, 1994.     

The distribution of intrinsic cortical axons in area 3b of cat primary somatosensory cortex

Summary The morphology of single neurons in area 3b of cat primary somatosensory (SI) cortex was examined after horseradish peroxidase (HRP) injections. Neurons were labeled either by intracellular injection of HRP following intracellular recording or by small extracellular iontophoretic HRP injections. Both pyramidal and nonpyramidal neurons were labeled and reconstructed from serial sections. Their axons had local, interlaminar and interareal patterns of termination. Most neurons formed local axonal fields around their cell bodies and dendrites. Pyramidal neurons in cortical layer IV sent axons up into layers II and III, neurons in layers II and III sent axons down to layer V, and layer V neurons sent axons to layer VI as well as back to the upper layers. Layer VI neurons sent axons back to the upper cortical layers in a unique bowl-shaped pattern. The horizontal distribution of axons of pyramidal cells in layer III was extremely widespread. Axons of layer III neurons in area 3b terminated within 3b and area 1, but not in other areas of SI. Layer III neurons in area 1 distributed axon collaterals to all fields of SI as well as projecting a main axon to motor cortex. In general, the axon collaterals of area 3b pyramidal cells outside layer III remained confined to area 3b. Most of the nonpyramidal neurons labeled were basket cells in layers III and VI. These neurons formed dense axonal fields around their cell bodies, and none of their axons could be followed into the underlying white matter. The results of the present study demonstrate that area 3b somatosensory cortical neurons and their axons are vertically organized in a manner similar to that reported for other sensory cortical areas. They also show that widespread horizontal connections are formed by pyramidal neurons of layer III, and that these horizontal axons can travel for great distances in the cortical grey matter.     

Degeneration and regeneration of autonomic nerve endings in the anterior part of rhesus monkey ciliary muscle

Summary The autonomic nerve plexus of the ciliary muscle was examined with the electron microscope in normal rhesus monkeys of different ages. In the anterior region of the muscle, at the boundary with the poorly innervated scierai spur and trabecular meshwork, 3.8–7.1% of the axons exhibit either degenerative or regenerative features. The cytoplasm of degenerating axons contains lamellated, dense and multivesicular bodies, vesicles, whorls of filaments, and membranous debris. The plasma membrane is often discontinuous and, on occasion, axonal debris and degenerative organelles are freely dispersed in the connective tissue spaces of the muscle. Degenerating axons contain a granular reaction product when stained for acid phosphatase activity. Regenerating axons are characterized by tightly packed mitochondria, glycogen particles, and aggregates of synaptic vesicles; they synapse with muscle cells and are negative to the acid phosphatase reaction.A quantitative analysis showed that in the anterior region of the ciliary muscle degenerating and regenerating axons increase in number with age, although the total number of axonal profiles remains constant. In the age groups examined, degenerating axons occurred with the same frequency as regenerating axons, thus, the age-dependent increase in axonal degeneration is accompanied by a parallel increase in axonal regeneration. We conclude that autonomie nerve endings in the anterior part of the ciliary muscle undergo a continuous process of renewal that is more prominent in old age.     

Ensheathment of the olfactory nerves in the adult rat

The ensheathment of the olfactory nerve fibres is achieved by cooperation of two cell types. The olfactory ensheathing cells have a rounded outer surface enclosed in a continuous single basal lamina, and enclose an inner compartment from which overlapping processes of the same and adjacent cells enwrap interweaving territories of tightly apposed aligned axons. The olfactory nerve fibroblasts are highly flattened, dense cells generating multiple layers of very thin processes encircling individual or groups of olfactory ensheathing cells. This paper illustrates the unique ultrastructural features of this ensheathment.     

The action of capsaicin on primary afferent central terminals in the superficial dorsal horn of newborn mice

Capsaicin was injected subcutaneously (50 mg/kg) into 10 mice on days 2 or 3 after birth, and 12 h, 3 and 5 days later the distribution and structure of degenerated primary afferent central axons or terminals (C-terminals) in the lumbar spinal dorsal horn were examined by electron microscopy. Degenerated terminal axons with dense or lamellar bodies or higher electron density were conspicuous 12 h after treatment with capsaicin. Severely degenerated unmyelinated axons, including dense or lamellar bodies engulfed by microglial cells, were numerous in the most superficial (marginal) layer, but rarely seen in the substantia gelatinosa. Two types of primary afferent central terminals in the substantia gelatinosa showed various extents of degeneration: small dark C-terminals (CI-terminals) with densely packed agranular synaptic vesicles, and large light ones (CII-terminals) with less dense agranular synaptic vesicles and a few granular synaptic vesicles. Thus, many central axon terminals of dorsal root ganglion (DRG) neurons that are sensitive to capsaicin enter the marginal layer and substantia gelatinosa. Degenerated primary afferent central axons or terminals markedly decreased in the superficial dorsal horn 3 and 5 days after capsaicin treatment, still, there were many degenerating DRG neurons at this time as shown by our previous study. Previously we also reported that fewer slightly degenerating unmyelinated dorsal root axons and small DRG neurons appear at 12 h and larger DRG neurons degenerate later than smaller ones after treatment with capsaicin. As a result, the discovery of many severely degenerated terminal axons in the superficial dorsal horn soon after treatment supports the idea that capsaicin first acts on the central terminals and that this is followed by damage to larger DRG neurons.     

Ultrastructural study of lamellar and nucleolus-like bodies in the harderian gland during postnatal development of the hamster (Mesocricetus auratus)

Membranous structures identified as lamellar bodies (LBs) and dense intracytoplasmic bodies referred to as nucleolus-like bodies (NLBs) have been found in the hamster Harderian gland during neonatal stages. Both structures appear between 8 and 12 postnatal days, coinciding with the beginning of secretory activity. LBs in males and NLBs in both sexes gradually decrease in number with further differentiation of the glandular cells. The morphological features of these cytoplasmic structures are described, and their origin as well as their possible functional significance are discussed.     

Ultrastructural heterogeneity of gangliogliomas

Gangliogliomas are rare brain tumors, composed of neuronal and glial cells mixed in a different proportion. The basic histopathological pattern of gangliogliomas is well recognized but the variable microscopic appearance still can pose a challenge to the neuropathologist. The authors reanalyzed their series of gangliogliomas in the files of two departments of neuropathology. All analyzed tumors fulfilled the WHO histological criteria of ganglioglioma. Seven tumors were examined by electron microscopy. The following ultrastructural features were graded: presence of dense-cored vesicles, synaptic vesicles, synapses and intermediate filaments, abundant basal membranes, dystrophic neurites, autophagic vacuoles, and multivesicular bodies. Most of the neoplastic neurons were large, polyglonal or oval with well-developed subcellular organelles, round nuclei, and prominent nucleoli. In most cases there were abundant dense core vesicles, observed in both the tumor cell bodies as well as in their processes. Synapses were typically observed. Intermediate filaments were abundant in all tumors. The most intriguing ultrastructural finding was abundant presence of autophagic vacuoles. In 4 cases, multivesicular bodies were observed. All of the tumors with multivesicular bodies also contained abundant autophagic vacuoles.     

Fine structural study of the spinal cord and spinal ganglia in mice afflicted with a hereditary sensory neuropathy, dystonia musculorum

Dystonia musculorum in mice is a hereditary autosomal recessive disorder, characterized by a progressive neuromuscular incoordination. This paper describes the ultrastructural changes in the spinal cord and compares and correlates the results with changes in the spinal ganglia in dystonic mice. Ganglion cells exhibited various stages of degeneration and pyknosis. The dorsal roots of the spinal nerves showed severe degeneration and loss of myelinated fibres accompanied by fibrosis, whilst the ventral roots appeared normal. Nerve cells within the dorsal and intermediate grey matter (laminae I to VII) of the spinal cord showed chromatolysis, atrophy, and necrosis. Boutons exhibited glycogen accumulation or an increase in their electron density. Axonal changes consisted of focal swellings, marked accumulation of neurofilaments, membranous and dense bodies, and disintegration of axoplasm. Myelin sheath degeneration of Wallerian type and degenerating axons were prominent in the dorsal, lateral and ventral white columns of the spinal cord. Glial reactions in the spinal cord were limited to mild hypertrophy and hyperplasia of astrocytic processes. The process of phagocytic activity was not intense in spite of the presence of an abundance of degenerating myelin and cell debris. This study showed that the ultrastructural changes in the spinal cord are more severe than those seen with routine light microscopy. The detection of definite neuronal degeneration of the dorsal root ganglia and spinal cord suggests that the defect apparently operates at the level of cell bodies, as well as axons, of the primary and second order sensory neurons.     

Ultrastructural identification of specialized endocytic compartments in macrophages of the thymic cortico-medullary zone and germinal centers of peripheral lymphatic organs of the rat.

In this study transmission electron microscopy was used to investigate the ultrastructural features of macrophages which are strategically positioned in the thymic cortico-medullary zone and the dark zone of germinal centers of peripheral lymphatic organs in adult Wistar rats. We show that this, morphologically distinct, type of macrophage displays the entire range of cytoplasmic inclusions, which structurally closely correspond to those of endosomal/MHC-II-enriched compartments of antigen presenting cells. The macrophages of the cortico-medullary zone and germinal centers contain numerous multivesicular bodies, as well as various kinds of cytoplasmic inclusions ranging from single to aggregated multivesicular/multilamellar bodies to large vacuoles. These multilamellar inclusions are composed of elongated, irregularly shaped cisternae, with abundance of internal membrane profiles and dense bodies. Often, cortico-medullary zone and germinal center macrophages contain the typical multilamellar bodies. Polysaccharides are detected by the thiocarbohydrazide-silver proteinate method within the dense bodies of these macrophages. The functional significance of cortico-medullary zone and germinal center macrophages is briefly discussed.     

针刺“足三里”穴下丘脑血管加压素(Vp)样细胞的形态学变化——免疫组织化学法

采用免疫组化PAP技术,在电针大鼠“足三里”穴位后,观察丘脑下部Vp样神经元的变化。发现视上核与室旁核Vp样大神经元数目增多、胞体胀大,并伸出细长突起。室旁核少数串珠状神经纤维可插入第三脑室壁的细胞之间。在视交叉上核腹内侧Vp小神经元密集,而背外侧反应阳性神经元较少。视上核与室旁核间的神经元岛的细胞体积增大、轴索变长伸向视上区。该区的串珠状纤维集合成束达正中隆起的外层。以上实验结果提示,电针“足三里”可促进下丘脑有关核团Vp样物质的合成与分泌。     

The lack of a structured blood-brain barrier in the onychophoran Peripatus acacioi

Summary Onychophorans are ‘living fossils’ frequently purported to have evolved from the same ancestor as the arthropods and annelids. In the CNS ofPeripatus acacioi, beneath an outer acellular neural lamella, glial cells ensheath the cerebral ganglion and the nerve cords. These glial cells are, however, attenuated and rather few in number and, although they interdigitate with one another, they seem to lack intercellular junctions. Exogenous tracers penetrate between them and into the underlying neuropile, suggesting that there is no structural blood-brain barrier. Throughout the nervous tissue, extracellular spaces occur which contain banded collagen fibrils embedded in a matrix material. Thin glial cell processes, characterized by dense filaments, surround these regions and frequently form hemi-desmosomes with the extracellular matrix. The peripheral nerve cell bodies have a range of diameters; some have the characteristics of neurosecretory neurons. Granules in such neurons are produced by the Golgi saccules and associated fenestrated membranes which also possess many coated vesicles. Comparable granules are also found in axonal tracts, but no distinct peripheral neurohaemal areas have been found. Lysosomes are common in the nerve cell bodies and are frequently in the form of multivesicular bodies or large phagocytic vacuoles. Beneath the outer nerve cells lie many tracheae, arranged as a ring around the central neuropile which consists of glial processes, extracellular matrix, axons and nerve terminals. These nerve terminals occur throughout the central neuropile and are characterized by dense pyramidal presynaptic specializations and postsynaptic subsurface cisternae. The nervous system ofPeripatus is relatively simple in its organization, in the lack of glial intercellular junctions and in the ready accessibility of substances from the external milieu.     

Bunina bodies in dendrites of patients with amyotrophic lateral sclerosis

We studied the brains of two cases of amyotrophic lateral sclerosis with dementia. Bunina bodies were found in the motor neurons of cranial nerve nuclei (trigeminal, facial and hypoglossal nerves) as well as in the spinal motoneurons. They appeared mostly in the cytoplasm and occasionally in the neuronal processes. However, the present electron microscopic study disclosed clearly that Bunina bodies were present not only in the cell body but also in the dendrites. No Bunina bodies were observed in the axons. It is inferred that the Bunina bodies were degenerative products formed as a result of a protein metabolism disorder.     

Age-related changes in the optic nerve of Sprague-Dawley rats: An ultrastructural and immunohistochemical study

The optic nerve is a unique part of the central nervous system. It lacks neuronal cell bodies and consists of axons of the retinal ganglion cells together with the supporting neuroglial cells. In the present study, aging of the optic nerve was studied in female Sprague-Dawley rats aged 3, 12, 24 and 30 months old, ultrastructurally, immunohistochemically and morphometrically trying to answer the question why aging is a common risk factor for many ocular diseases especially glaucoma. Additionally, studying the optic nerve aging offered a good opportunity to gain further insight into the effects of aging on white matter. Both nerve fibers and neuroglial cells demonstrated several age related changes which were more profound in 30 months old rats. Optic nerve axons displayed watery degeneration and dark degeneration. Myelin disturbances including widening, whorls, splitting and vacuolations of the myelin lamellae were also observed. Neuroglial cells appeared to be more frequent than in younger rats especially microglia cells and developed dense cytoplasmic inclusions. GFAP-positive astrocytes delineated age-related progressive increase in number, size as well as length and thickness of their processes. CD68 immunohistochemical staining revealed age-related changes in the morphology, location and number of CD68 positive microglia cells.