THE FINE STRUCTURE OF NEURONS

1. Thin sections of representative neurons from intramural, sympathetic and dorsal root ganglia, medulla oblongata, and cerebellar cortex were studied with the aid of the electron microscope. 2. The Nissl substance of these neurons consists of masses of endoplasmic reticulum showing various degrees of orientation; upon and between the cisternae, tubules, and vesicles of the reticulum lie clusters of punctate granules, 10 to 30 mµ in diameter. 3. A second system of membranes can be distinguished from the endoplasmic reticulum of the Nissl bodies by shallower and more tightly packed cisternae and by absence of granules. Intermediate forms between the two membranous systems have been found. 4. The cytoplasm between Nissl bodies contains numerous mitochondria, rounded lipid inclusions, and fine filaments.     

The chicken trigeminal ganglion. I. An anatomical analysis of the neuron types in the adult

An anatomical analysis of the chicken trigeminal ganglion was made using light microscopy on specimens prepared by usual chemical fixation or freeze-drying methods and by electron microscopy. Two types of neurons were consistently seen, dark and light cells. Dark cells contained a dense cytoplasm with Nissl substance distributed evenly throughout, whereas light cells had a less dense cytoplasm containing clumps of Nissl substance. The Nissl bodies in light cells contained only a few small cisternae of granular endoplasmic reticulum as compared with many stacked cisternae in Nissl bodies of dark cells. The ratio of dark to light cells was approximately 62:38 in all regions of the ganglion. Dark cells were consistently smaller than light cells. In the seven-day old chick, the mean diameters of the dark and light neurons were 21.4 μ and 29.5 μ respectively; in the adult the values were 29.9 μ and 39.7 μ respectively. It is concluded that the dark and light cells belong to two distinct neuronal cell populations.     

Regional differences in the ultrastructure of Purkinje cells of the rat

Summary In the albino rat, perikaryal diameter, volume density of the granular endoplasmic reticulum and Golgi apparatus, and lumenal diameter of cisternae of the granular endoplasmic reticulum are larger in Purkinje cells of lobule Via (neocerebellum) than in those of lobule X (archicerebellum). In contrast, only the surface density of cisternae of the granular endoplasmic reticulum is larger in Purkinje cells of lobule X. The cisternae of granular endoplasmic reticulum are arranged into conspicuous Nissl bodies parallel to the nuclear membrane, but the content of ribosomes and polysemes is markedly less in lobule-X cells than in cells from lobule VI a. These results indicate qualitative and quantitative differences between the metabolically important organelles in Purkinje cells of the neo- and archicerebellum (cf. Larsell 1952).Supported by a grant from the Deutsche Forschungsgemeinschaft (La 184/7)     

Unusual intracytoplasmic lamellar bodies in a malignant gonadal stromal tumor

Characteristic intracytoplasmic lamellar bodies were found in a malignant gonadal stromal tumor. These bodies consisted of the stacks of up to 200 tubular cisternae arranged in parallel. Each cisterna had a circular section in tangential view and a diameter of about 85 nm. The cisternae on the outermost side of these lamellar bodies tended to be dilated and adorned with ribosomes. The ends of cisternae were often contiguous with rough-surfaced endoplasmic reticulum. The latter feature is also seen in annulate lamellae, but periodically spaced annuli or discontinuities characteristic of annulate lamellae were never observed. Furthermore, fine ribosomal granules resembling a rosary were recognizable along the whole circumference of the outer surface of each cisterna. The unique structure we describe is a cytoplasmic organelle which, like annulate lamellae, is closely associated with the endoplasmic reticulum and is presumed to be related to the genesis of rough-surfaced endoplasmic reticulum in tumor cells.     

Unusual cytoplasmic bodies in chromaffin cells of the adrenal medulla of the viscacha (Lagostomus maximus maximus)

Complex structures can be seen associated with large areas of endoplasmic reticulum of the chromaffin cells in the viscacha adrenal medulla. These structures are cylindrical in shape and are formed by chains of globular subunits. A central lumen is observed within which there are bodies resembling chromaffin granules. Parallel cisternae of the endoplasmic reticulum are arranged surrounding these bodies. The functional significance of these structures in unknown.     

Ultrastructural investigation of ACTH immunoreactivity in arcuate and supraoptic nuclei of the rat

Summary Fine structural localization of an ACTH-like substance was obtained in neurons of the rat arcuate nucleus using immuno-electron microscopy, whereas it could not be confirmed that ACTH-containing cell bodies are present in the supraoptic nucleus. The immunoreactive cells of the arcuate nucleus appeared to be more numerous than the unreactive neurons. Immunostaining was carried out before embedding in resin. Empty vesicles of irregular shape were found in dendrites of immunoreactive arcuate neurons, but their significance and nature remain enigmatic. The reaction product was distributed uniformly throughout the cytoplasm of the ACTH-positive cells, except that the mitochondria, rough endoplasmic reticulum and Golgi vesicles and cisternae were devoid of PAP molecules. This distribution differed from the localization reported in ACTH-secreting cells of the rat anterior pituitary, where the reaction product was found in the rough endoplasmic reticulum and Golgi complex as well as in secretory granules.     

Immunocytochemical localization of parathyroid hormone in hamster parathyroid gland

The immunocytochemical localization of parathyroid hormone was examined in the hamster parathyroid gland by using the protein A-gold technique. Protein A-gold particles were concentrated over secretory granules, large secretory granules thought to be storage granules and Golgi vacuoles. No protein A-gold particles were detected over large vacuolar bodies and cisternae of the granular endoplasmic reticulum.     

Specialized formations of endoplasmic reticulum in parenchyma cells ofMimosa pudica L.

Summary Parenchyma cells ofMimosa pudica display close associations between two or more cisternae of the endoplasmic reticulum. These associations form simplified types of lamellar bodies in which inner paired lamellae have lost their ribonucleoprotein granules and are separated by a dense layer.     

The ultrastructure of oogenesis and yolk formation in Labidocera aestiva (Copepoda: Calanoida)

Yolk formation in the oocytes of the free-living, marine copepod, Labidocera aestiva (order Calanoida) involves both autosynthetic and heterosynthetic processes. Three morphologically distinct forms of endogenous yolk are produced in the early vitellogenic stages. Type 1 yolk spheres are formed by the accumulation and fusion of dense granules within vesicular and lamellar cisternae of endoplasmic reticulum. A granular form of type 1 yolk, in which the dense granules within the cisternae of endoplasmic reticulum do not fuse, appears to be synthesized by the combined activity of endoplasmic reticulum and Golgi complexes. Type 2 yolk bodies subsequently appear in the ooplasm but their formation could not be attributed to any particular oocytic organelle. In the advanced stages of vitellogenesis, a single narrow layer of follicle cells becomes more developed and forms extensive interdigitations with the oocytes. Extra-oocytic yolk precursors appear to pass from the hemolymph into the follicle cells and subsequently into the oocytes via micropinocytosis. Pinocytotic vesicles fuse in the cortical ooplasm to form heterosynthetically derived type 3 yolk bodies.     

Reticular and areticular Nissl bodies in sympathetic neurons of a lizard

Sympathetic ganglia of the horned lizard, Phrynosoma cornutum, were fixed in OsO(4) and imbedded in methacrylate. Thin sections were cut for electron microscopy. Some adjacent thick sections were cut for light microscopy and were stained in acidified, dilute thionine both before and after digestion by RNase. In the light microscope two types of Nissl bodies are found, both removable by RNase: (1) a deep, diffuse, indistinctly bounded, metachromatic variety, and (2) a superficial, dense, sharply delimited, orthochromatic sort. Electron microscopically, the former ("reticular" Nissl bodies) corresponds to the granulated endoplasmic reticular structure of Nissl material previously described by others, whereas the latter ("areticular" Nissl bodies) comprises compact masses of particles of varying internal density and devoid of elements of endoplasmic reticulum. The constituent particles of the areticular Nissl material are 4 to 8 x the diameter of single ribonucleoprotein granules of the reticular Nissl substance and seem, near zones of junction with the reticular type, to arise by clustering of such granules with subsequent partial dispersion of the substance of the granules into an added, less dense material. It is suggested that the observed orthochromasia of the areticular Nissl substance is due to accumulation of a large amount of protein bound to RNA and, further, that these Nissl bodies may represent storage depots of RNA and protein.     

"Reticular" and "Areticular" Nissl Bodies in Sympathetic Neurons of a Lizard

Sympathetic ganglia of the horned lizard, Phrynosoma cornutum, were fixed in OsO₄ and imbedded in methacrylate. Thin sections were cut for electron microscopy. Some adjacent thick sections were cut for light microscopy and were stained in acidified, dilute thionine both before and after digestion by RNase. In the light microscope two types of Nissl bodies are found, both removable by RNase: (1) a deep, diffuse, indistinctly bounded, metachromatic variety, and (2) a superficial, dense, sharply delimited, orthochromatic sort. Electron microscopically, the former ("reticular" Nissl bodies) corresponds to the granulated endoplasmic reticular structure of Nissl material previously described by others, whereas the latter ("areticular" Nissl bodies) comprises compact masses of particles of varying internal density and devoid of elements of endoplasmic reticulum. The constituent particles of the areticular Nissl material are 4 to 8 x the diameter of single ribonucleoprotein granules of the reticular Nissl substance and seem, near zones of junction with the reticular type, to arise by clustering of such granules with subsequent partial dispersion of the substance of the granules into an added, less dense material. It is suggested that the observed orthochromasia of the areticular Nissl substance is due to accumulation of a large amount of protein bound to RNA and, further, that these Nissl bodies may represent storage depots of RNA and protein.     

Annulate lamellae,lamellar bodies and subsurface cisternae in neurons of the avian hyperstriatum accessorium

Summary Structures identified as annulate lamellae, lamellar bodies and subsurface cisternae were found in neurons of the hyperstriatum accessorium of the avian forebrain. Annulate lamellar arrays with up to six lamellae were present in the larger somata. The lamellae were made up of fused smooth-surfaced cisternae forming pores or annuli and were surrounded by a dense filamentous to granular material. Stacks of nonfenestrated, parallel, regularly spaced cisternae, designated as lamellar bodies, also appeared in the cytoplasm. When flattened they were reminiscent of the electron dense subsurface cisternae. Continuity could be demonstrated between peripherally located subsurface cisternae and lamellar bodies. The dense filamentous to finely granular substance was also located between these structures. Annulate lamellae, lamellar bodies and subsurface cisternae were always observed in conjunction with the rough endoplasmic reticulum. The functional significance of these structural associations is considered.     

Structural changes in spinal ganglion neurons of lizards after cold-exposure

We studied structural changes in spinal ganglion neurons that occur in lizards exposed to the cold, both at the light and electron microscope levels. Two types of perikaryal changes were found in the cold-exposed animals: (a) In 25% of all ganglion neurons, the central region of the perikaryon was devoid of Nissl bodies and a narrow peripheral zone stained deeply basophilic. Electron microscopic examination of these cells showed that mitochondria, Golgi complexes and other organelles were assembled in the central region of the perikaryon, while most cisternae of granular endoplasmic reticulum and free polysomes were confined to the periphery of the perikaryon. These changes seem to take place mainly in dark neurons. (b) In 8.6% of all ganglion neurons, Nissl bodies were present throughout the perikaryon, but separated by large, clear spaces. Under the electron microscope, these clear spaces were filled with large numbers of densely packed filaments. It seems that mainly light neurons undergo this type of structural change. The degree of nuclear eccentricity was significantly greater in the neurons of cold-exposed animals than in controls. The nucleolar volume was significantly increased and both the percentages of nuclei with two nucleoli and of nuclei with 'vacuolated' nucleoli were significantly greater in neurons displaying structural changes than in the other neurons. The structural modifications observed in spinal ganglion neurons of cold-exposed lizards closely resemble those seen in the same lizard neurons following axonal section. They could be due to a) metabolic changes induced by low temperature and fasting, b) alterations in the flow of nerve impulses from the periphery, or c) impaired retrograde transport of trophic substances from the periphery to the cell body.     

内质网及其标志酶在离体培养脊髓神经元中的发育变化

In an attempt to elucidate the relationship between synapse formation and cell development, the morphology and cytochemistry of the endoplasmic reticulum and its enzymic marker, glucose-6-phosphatase (G-6-Pase), in cultured mouse spinal neurons were investigated ultrastructurally. It was found that in the early period of the development, neurons were characterized by scarceness of organelles; only a few of granular or agranular endoplasmic reticulum and mitochondria were seen. The endoplasmic reticulum and nuclear envelope were packed specifically with G-6-Pase resection product but the product was weak. After a period of culture, most of the neurons had well-developed endoplasmic reticulum, Golgi apparatus, mitochondria and microtubules, etc. The Golgi apparatus was relatively large, having some cisternae associated with vesicles. Either concave of convex face of the saccules was labeled by thiamine pyrophosphatase (TPPase) specifically. GERL, labeled by cytidine monophosphatase (CMPase), was also seen close to the inner or outer face of some Golgi apparatus. The endoplasmic reticulum at this stage was distributed throughout the cytoplasm, including that in dendrites; its enzyme marker (G-6-Pase) localized consistently within the lumen of all endoplasmic reticulum, nuclear space and subsurface cisternae, and frequently in the concave saccules of the Golgi apparatus. After a long-term culture, some neurons became "aged". The endoplasmic reticulum cisternae enlarged and G-6-Pase reaction reduced. Along with the neuronal development, especially maturation of the endoplasmic reticulum and its enzymic marker, synapse formation was begun at the neuropile area. The axo-dendritic synapses always occurred between the axonal terminals and dendrites where the endoplasmic reticulum had showed positive G-6-Pase reactions. Considering the fact, it suggests that the appearance and change of these specific enzymes may be related to the maturation of the neurons in vitro, and also related to the synapse formation between neurons.     

Substructure of cisternal organelles of neuronal perikarya in immature rat brains revealed by quick-freeze and deep-etch techniques

Summary Membrane-bounded organelles possessing cisternae, i.e., rough endoplasmic reticulum and Golgi apparatus, in immature rat central neurons were examined by quick-freeze and deep-etch techniques to see how their intracisternal structures are organized and how ribosomes are associated with the membrane of the endoplasmic reticulum. Cisternae of endoplasmic reticulum, 60–100 nm wide, were bridged with randomly-distributed strands (trabecular strands, 12.5 nm in mean diameter). Luminal surfaces of cisternae of the endoplasmic reticulum were decorated with various-sized globular particles, some as small as intramembrane particles, and others as large as granules formed by soluble proteins seen in the cytoplasm. A closer examination revealed much thinner strands (3.3. nm in mean diameter). Such thin strands were short, usually winding toward the luminal surface, and sometimes touching the luminal surface with one end. Ribosomes appeared to be embedded into the entire thickness of cross-fractured membranes of endoplasmic reticulum, that is, their internal portions appeared to be situated at almost the same level as the cisternal luminal surface. From the internal portion of ribosomes, single thin strands occasionally protruded into the lumen, suggesting that these thin strands were newly synthesized polypeptides. A horizontal separation within ribosomes appeared to occur at the same level as the hydrophobic middle of the membrane of the endoplasmic reticulum. Interiors of the Golgi apparatus cisternae, which were much narrower than cisternae of endoplasmic reticulum, were similarly bridged with trabecular strands, but the Golgi trabecular strands were thinner and more frequent. Their cisternal lumina were also dotted with globular particles. No identifiable profiles corresponding to the thin strands in the endoplasmic reticulum were observed. Golgi cisternae showed a heterogeneous distribution of membrane granularity; the membrane in narrow cisternal space was granule-rich, while that in expanded space was granule-poor, suggesting a functional compartmentalization of the Golgi cisternae.     

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Summary Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance.Nucleolus In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as nucleolar shadow, overlaid by a few small irregular bodies of higher electron density than other nuclear contents.Nucleus The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas.Nissl Substance In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed.The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.This investigation was supported by USPHS Grants NB 03114-04, NB 00690-11 and 5 T 1 GM 495 from the National Institutes of Health, Bethesda, Maryland.Acknowledgement. Mrs. Eleanor W. Morris and Mr. Edwin E. Pitsinger, Jr. gave indispensible aid with the management of the cultures and with photographic procedures.     

ELECTRON MICROSCOPE STUDIES ON DEVELOPING CRAYFISH OOCYTES WITH SPECIAL REFERENCE TO THE ORIGIN OF YOLK

The endoplasmic reticulum is composed, in places, of stacks of parallel cisternae which are limited by membranes having great numbers of ribosomes attached to their outer surface. These are connected with other cisternae of similar structure but with fewer ribosomes and without preferred orientation. The latter extend in all directions from the stacked cisternae, branching and anastomosing freely so that the entire system of membrane-limited cisternae appears interconnected; a morphological condition suitable to serve as the basis for an active transport system. Within the stacked cisternae appear granules about 40 to 60 mµ in diameter. These are thought to represent the precursors of proteinaceous yolk, and the hypothesis is advanced that most of the intracisternal granules are synthesized here, possibly under the influence of the ribosomes. They then "flow" into and along the unoriented cisternae to regions where they collect, expand the cisternae, and undergo transformation into finely granular, relatively large proteinaceous yolk bodies. The mitochondria are somewhat pleomorphic, often show atypical cristae, and frequently contain a few dense granules. Lipid is abundant. Other cytoplasmic components are illustrated.     

内质网及其标志酶在离体培养脊髓神经元中的发育变化

本文采用形态学与细胞化学相结合的方法,在超微结构水平观察了与突触酶、受体和结构蛋白的合成有关的内质网和高尔基复合体、GERL以及它们的标志酶的发育变化。结果表明神经元本身有一发育过程,发育早期的细胞器较少,成熟时逐渐增多,以内质网和高尔基复合体最为明显。用G一6一Pase、TPPase和CMPase可分别标记内质网及同源结构、高尔基复合体的成熟而膜囊和GERL。这些酶的出现及阳性水平与神经元的发育呈同步关系。可作为判断细胞分化程度和功能状态的指标。G-6-Pase还分布在突触后树突的内质网中,突触形成大都从含G-6-Pase阳性内质网的树突开始。本文对内质网及G-6-Pase在神经元中的发育变化及功能进行了讨论。     

Mechanisms of protein yolk synthesis and deposition in crustacean oocytes

Summary Electron microscope studies on the oocytes of several crustacean species demonstrate that the protein yolk arises within vesicular and lamellar forms of the rough-surfaced endoplasmic reticulum. The vesicular form of the endoplasmic reticulum may have its origin from a blebbing process of the outer layer of the nuclear envelope. Disc-shaped granules, representing precursor elements of the yolk granules, appear within the vesicular and lamellar profiles of endoplasmic reticulum. Autoradiographic results suggest that the ribosomes attached to the endoplasmic reticulum take part in the biosynthesis of yolk proteins. Numerous disc-shaped granules accumulate within the cisternae of the endoplasmic reticulum, but eventually they undergo a transformation into a finely granular yolk granule. Thus, both the origin and growth of protein yolk granules occur within membranes constituting the endoplasmic reticulum. The results provide evidence that intra-ooplasmic synthesis of yolk protein occurs in these oocytes.This investigation was supported by research grants (HD-00699; GM-09229) and a Career Development Award (GM-11,524) from the National Institutes of Health, U.S. Public Health Service.     

A comparative ultrastructural study of the trigeminal ganglion in the rat and chicken

The neurons of the trigeminal ganglia of the rat and chicken were characterized by means of light microscopic, electron microscopic, and histochemical methods. Light microscopy disclosed four types of neurons, based on the characteristics of Nissl granules: (1) large neurons with diffusely distributed and very fine granules, (2) neurons containing coarse and sparsely distributed Nissl granules, (3) neurons containing dense Nissl granules of varying size, and (4) small neurons with granules concentrated peripherally. Electron microscopy allowed further definition of these four types of neurons by the length and arrangement of flattened cisterns of granular endoplasmic reticulum (gER) and the number of neurofilaments. Type 1 cells were largest, with a mean nuclear area of 139.8 ± 28.3 μm². Type 4 cells were smallest, with a mean nuclear area of 74.6 ± 20.9 μm². The mean nuclear areas of type 2 and 3 cells were intermediate to those of the type 1 and 4 cells. Type 3 and 4 neurons lacked neurofilaments. Four forms of Golgi apparatus were found: (1) large bent grains forming a network throughout the soma, (2) dispersed fine granular deposits, (3) fine or small granules, and (4) coarse bent deposits arranged confluently in the perinuclear zone. In some rat neurons, the concentration of acid phosphatase reaction products suggested a high enzymatic activity, whereas the chicken ganglion cells showed no such concentration. These findings are discussed and compared with the classifications of previous studies.