The nervous system in planarians: Peripheral and gastrodermal plexuses,pharynx innervation,and the relationship between central nervous system structure and the acoelomate organization

The Champy-Maillet osmium tetroxide-zinc iodide technique and a new method using azur B-sodium thioglycolate were used to study the general nervous tissue structure in planarians. A subepidermal and a submuscular nerve plexus, partially reported by earlier authors, are described, and a gastrodermal plexus is reported for the first time in triclads. The possible functions for each one of these plexuses are discussed. By the Champy-Maillet method, the innervation within the parenchyma appears as an array of numerous single nerve fibers that course between the parenchyma cells making apparent synaptic contacts. The pharynx has outer and inner nerve nets similar in structure to the submuscular nerve plexus. Both nerve nets are connected to each other by radial nerves. The central nervous system has a sponge-like structure with many lacunae filled with cell bodies, dorso-ventral muscle fibers, parenchymal cell processes and excretory ducts. The existence of this sponge-like nervous tissue structure is discussed in relation to the still incomplete centralization of the nervous tissue in these organisms, to the lack of a true vascular system and to the acoelomate level of organization. A comparison with the nervous tissue structure of more advanced groups like polyclads and nemertines is suggested.     

Elektronenmikroskopische Untersuchungen an menschlichen Leukozyten nach Osmium-Zink-Imprägnation

Zusammenfassung Leukozytenkonzentrate des peripheren Blutes von gesunden Versuchs-personen wurden in 2,5%iger Glutaraldehyd-Lösung (0,1 M Na-Cacodylat-Puffer, pH 7,3) vorfixiert und nach der Methode von Maillet (1959) mit OsO₄ + ZnJ₂ inkubiert. Dickschnitte wurden mit dem Mikroanalysator (Siemens) auf ihren Gehalt an Osmium, Zink und Jod analysiert und die Dünnschnitte mit dem Elektronenmikroskop untersucht. Bei der Mikroanalyse ließen sich Osmium und Zink, nicht aber Jod nachweisen. Die elektronenmikroskopischen Untersuchungen ergaben folgende Resultate: Beträgt der pH-Wert der Inkubationslösung 5,6, so findet sich eine starke Os/Zn-Imprägnation des Golgi-Apparates, des Kernspaltes und des Endoplasmatischen Retikulums bei sämtlichen Arten der weißen Blutzellen. Die Matrix der Mitochondrien dieser Zellen ist im allgemeinen weniger stark imprägniert. Auch in den Lysosomen der Monozyten und Plasmazellen sind geringfügige Os/Zn-Präzipitate nachweisbar. Es findet keine Reaktion in den Leukozytengranula und in den Zentriolen statt. Dagegen zeigen die Lipidtropfen der eosinophilen Granulozyten eine sehr starke, homogene Os/Zn-Imprägnation. Diese ist bereits nach 40 min Inkubation voll ausgeprägt. Bei einer Schnittkontrastierung mit Uranylacetat werden die Os/Zn-Präzipitate wieder herausgelöst, wenn die Kontrastierung länger als 2 min dauert. Eine Bleikontrastierung hat keinen Einfluß auf die Os/Zn-Präzipitate.Bei Erhöhung des pH-Wertes der Inkubationslösung auf 6,2 kommt es zur starken Imprägnation des Externums der eosinophilen Granula, während Kernspalt, Golgi-Apparat und Mitochondrienmatrix keine Reaktion zeigen. Die Imprägnationsdichte der Lipidtropfen der eosinophilen Granulozyten ist pH-unabhängig.

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Electron microscopic studies on human leukocytes after osmium-zinc-impregnation

Summary Buffy coats of normal human peripheral blood were fixed in 2.5% glutaraldehyde (buffered with 0.1 M Na-cacodylate, pH 7.3) and incubated with a mixture of osmium tetroxide and zinc iodide (pH 5.2) after the method of Maillet (1959). Utilizing an electron probe microanalyser (Siemens), the authors demonstrated the presence of osmium and zinc in the specimen, whereas the presence of iodide could not be proved with certainty. The ratio of osmium and zinc in the precipitates ranges from 3/2 to 4/1. Electron microscopic studies of the leukocytes have lead to the following results: the Golgi apparatus, the perinuclear cleft and the endoplasmic reticulum (ER) of all types of leukocytes are strongly impregnated with osmium/zinc, whereas the matrices of the mitochondria are less impregnated. Osmium/zinc precipitates have also been detected in the lysosomes of monocytes and plasma cells. No reaction was evident in the leukocytic granules or centrioles. The presence of lipid droplets in eosinophils has been demonstrated. These droplets show a dense homogeneous osmium/zinc impregnation which reaches its maximum after 40 minutes of incubation, compared with much longer incubation times (5–6 hours) for other cellular components. Setting the pH of the osmium tetroxide-zinc iodide solution to 6.2 resulted in a dense impregnation of the externum of the eosinophilic granules. At this same pH, the perinuclear cleft, Golgi apparatus and mitochondrial matrix did not show a definite reaction. The extent of impregnation of the lipid droplets of the eosinophils showed no dependency on pH. Care must be taken when a stain containing uranyl acetate is used since this substance dissolves the Os/Zn precipitates. Exposure of the precipitates to the uranyl acetate for longer than 2 minutes results in gradual dissolution within complete dissolution in 10 minutes.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

THE FINE LOCALIZATION OF NUCLEOSIDE TRIPHOSPHATASE ACTIVITY IN THE RETINA OF THE FROG

Nucleoside triphosphatase (NTPase) activity was demonstrated at the submicroscopic level in the frog retina by the Wachstein-Meisel method utilizing various purine and pyrimidine nucleosides. Under the electron microscope magnesium-activated NTPase was localized in the outer and inner segments, and in the plexiform layers. NTPase active sites in the cones were localized diffusely in the 70 to 80 A interspaces between the double membranes of the stacked lamellae and in the investing cytoplasm. In the rods, on the other hand, sites of activity were observed at the periphery of the stacked lamellae as discrete electron opaque deposits measuring 1000 to 1500 A which interdigitated between the lamellae for short distances. Deposits of reaction product appeared more numerous in rods of dark-adapted frogs stimulated with monochromatic light with a wave length of 510 mµ. Enzyme activity was also observed in mitochondria of the rod and cone ellipsoids. In the outer and inner plexiform layers NTPase active sites were present on and between the membranes of axons and the plasma membranes of some of the neurons.     

Presynaptic tubular structures in photoreceptor cells

Summary After the application of fixatives including phosphotungstic acid or a mixture of osmium tetroxide and zinc iodide, complex tubular structures are evident in the presynaptic side of the synapses between photoreceptor and bipolar cells of the rat's retina. In the first case only the limiting membranes are visualized, while in the second only the content of the tubules is stained. These tubules seem to be related, on a morphological ground, with the formation of synaptic vesicles. These tubular structures are not observed when fixation is done with osmium tetroxide or glutaraldehyde-osmium tetroxide.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, and from National Institutes of Health, U.S.A., (5 RO1 NS 06953-05 NEUA).We want to express our gratitude to Mrs. Haydée Agoff de Zimman and Mr. Alberto Saénz for their skillful technical assistance.     

‘Disk shedding’ in the cone outer segments of the teleost,Poecilia reticulata P.

Summary Electron microscopical observations show that the cones in the retina of the diurnal Poecilia reticulata shed their membranous outer segment disks. This occurs at the side of the disk which is open to the extracellular space. Shedding is observed in single and twin cones and occurs at any level of the outer segment. The disks are not discarded in packages or as single disks, but are shed in small vesicular portions. This mode of disk shedding may explain why in cone outer segments radioactively labelled replacement protein is diffusely distributed.The authors wish to thank Dr. C. Wise of this Department for helpful discussions     

Morphological changes induced by -SH reagents in rod photoreceptor outer segments

Summary Rat retinas were treated in vitro with -SH reagents and stained with zinc iodide-osmium tetroxide (ZIO). Dithioerythritol (DTE), an -S-S-reducing agent, increased the electron opaque deposits observed after ZIO staining in the intraand extradiskal spaces of the rods. N-ethyl-maleimide (NEM), an -SH blocking agent, applied directly or after DTE, blocks the ZIO reaction. Furthermore, after treatment with NEM, distorted tubular and vesicular structures are substituted for the stacks of disks. These results strongly suggest that ZIO reacts with -SH groups in rod outer segments. They also indicate that SH-groups play an important role in the structural organization of rod outer segments.Supported by Grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina and Fight for Sight, Inc. N.Y. United StatesI am grateful to Miss Margarita López for her skilful technical assistance and to Mr. Alberto Saenz for the electron micrographs     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

The zinc iodide-osmium tetroxide staining-fixative of Maillet. Nature of the precipitate studied by x-ray microanalysis and detection of Ca2+-affinity subcellular sites in a tonic smooth muscle.

A mechanism of osmium reduction during zinc iodide-osmium tetroxide (ZIO) fixation is proposed. X-ray powder microanalyses of ZIO precipitates formed both in the presence or absence of tissues are identical with those of CuOsO4 and CuRuO4. Therefore, and based on indexation methods, ZnOsO4 was found to be the formula of the ZIO mixture reduction; this zinc osmate has an orthorhombic crystalline lattice. In smooth muscle preparations, ZIO electron dense deposits are localized in both cisternae of the sarcoplasmic reticulum and in mitochondria after a short fixation time. According to the microanalysis results, the zinc osmate has been associated to Ca2+ high affinity sites since Zn2+ is either replacing Ca2+ and/or displacing it by having a higher affinity for Ca2+ binding sites. Consequently, the ZIO mixture might be useful in revealing some Ca2+ storage sites in cells. This hypothesis was tested in ABRM preparations by selectively depleting sites which are known to bind Ca2+. In this case, the sarcoplasmic reticulum only retains the staining deposits after a short ZIO fixation. It is likely that OsO4 alone, used as fixative in cytology might be due to the formation of metallic osmates (e.g., divalent osmates like CaOsO4). In addition, of course, reduction of osmium during tissue fixation is accompanied by oxidation of double bonds of lipoproteic complexes or unsaturated lipids, and oxidation of sulfhydryl groups and amino groups.     

Regeneration of the goldfish retina after exposure to different doses of ouabain

Summary After ouabain-induced degeneration, the retina of the goldfish shows a remarkable regeneration capacity. The extent of the damage depends on the dose of ouabain used in the experiment. After intraocular injection of 7l 10^–5 M ouabain, the ganglion cells and the cells of the inner nuclear layer (INL) become necrotic except for most of the outer horizontal cells, some bipolar cells, and Müller cells. The outer nuclear layer (ONL) and the marginal growth zone at the ora serrata remain intact; the plexiform layers become spongy. The degenerated material is removed by the proliferated reactive macroglial cells and invading macrophages. The degenerated cellular elements of the retina are replaced by mitosis of neuroblasts in the marginal growth zone and of cells in the ONL.After intraocular injection of a 5-fold higher dose of ouabain (7 l 5·10^–5M), the degeneration of the retina proceeds more rapidly and completely. In this experiment, the ONL is destroyed and the receptor outer segments are phagocytosed by cells of the pigment epithelium. In contrast to the regeneration of the amphibian retina, in the goldfish cells of the pigment epithelium do not participate by metaplastic transformation in the regeneration of the retina. The only source of cellular regeneration of the retina after complete destruction of its differentiated neural elements is the marginal growth zone, which is highly resistant to ouabain. The rate of mitoses in this region is strongly increased. The derivatives of these cells spread out tangentially over the entire fundus of the eye in a concentric manner. In this regenerate, mitotic processes continue in a radial direction, resulting in thickening and layering of the new retinal formation.     

The intracytoplasmic channel in pigment epithelial cells of the chick retina

Summary The fine structure of pigment epithelial cells in the chick retina was studied by electron microscopy with a special attention to the intracytoplasmic channel which is considered to be an important passage of metabolites from the choroidal side to the vitreal side. The chick retina was fixed either by perfusion with glutaraldehyde followed by osmium tetroxide or by immersion in situ with osmium tetroxide before removal of the eyeball. The infoldings appearing in the basal zone of the retinal pigment epithelial cell were provided with the gear-like projection which was encountered as their bottom in many cases, suggesting selective absorption of proteins. It was noticed that certain interspaces of the infoldings were continuous to tubular elements of the agranular endoplasmic reticulum. Moreover, the tubular elements were found in association with such other cellular components as nuclear envelope, mitochondria, fuscin granules and plasma membrane surrounding the outer segment of photoreceptor. The pigment epithelial cell appeared to be continuous with the photoreceptor through the pores of their plasma membranes. The presence of a certain intracytoplasmic channel from the choroid to the photoreceptor is considered to facilitate the transport of metabolites in the pigment epithelial cell.Part of these observations was presented at the Sixth International Congress for Electron Microscopy, Kyoto in 1966.I wish to thank Prof. Gonpachiro Yasuzumi for his valuable advices and discussions through this study.     

Production of the raw-starch digesting amylase of Aspergillus sp. K-27

Summary Aspergillus sp. K-27, isolated from soil, produced extracellular glucoamylase and -amylase using wheat starch as a carbon source, and its productivity was doubled by the addition of -methyl-d-glucoside to the medium. The crude enzyme preparation, which was found to be a mixture of 70% glucoamylase and 30% -amylase, well degraded not only cereal starches but also tuber and root starches, and the initial velocity for potato starch was 72% of that for corn starch.     

Structure and postnatal development of photoreceptors and their synapses in the retina of the tree shrew (Tupaia belangen)

Summary The all cone retina of the tree shrew (Tupaia belangeri) was examined in the adult and early postnatal stages by light and electron microscopy. Rods are not as rare as previously thought, but make up about 4% of the photoreceptors. They are relatively short and narrow cells, which stain (toluidine blue) more intensively and lie more proximal than cones. Among the cones three morphological varieties could be distinguished. Most cones stain lightly but have a light or a dark giant mitochondrion in their inner segment; a third type stains darker but occurs only rarely. All cones possess extensive radial processes (lateral fins) around the basal part of their inner segments. Such fins are well known from reptiles and birds, but have only once been described in a mammal (gray squirrel).The maturation of the retina in Tupaia belangeri proceeds centrifugally, i.e., from the vitreal to the scleral side, as in most mammals. A few synapses are already present at birth in the outer and inner plexiform layers, but seem to be more advanced in the latter. Such early synapses are small and have only few synaptic vesicles; they appear almost mature by day 14. The light-sensitive outer segments develop last. The first disks are seen by day 10, but regular membrane stacks are only present by day 18. Thus, it seems that the retina is functional when the young first open their eyes, which occurs around day 18.     

Electron microscopic observations on isolated mitochondrial membranes,prepared by various histological methods

Summary The pictures of isolated mitochondrial membranes, as seen on the electron-microscope, depend very much on the method of specimen preparation. Subunits of linear dimensions of about 25 m, (electron transport particles) are observed in carbon-replicas of the membranes and in specimens treated with trypsin or pepsin (0.02% for 30 mins) and shadowed with platinum. A three-layered structure of the unit membrane is seen in sections of specimens fixed with osmium tetroxide or formalin followed by post-fixation with osmium tetroxide. But fixation with potassium permanganate or with formalin, followed by post-fixation with potassium permanganate reveals an electron-dense globular structural element in the unit membrane. An electron-transparent ultrastructural element of the unit membrane is observed after treatment with trypsin (0.2% for 5 mins) and fixation with osmium tetroxide. Unsectioned specimens treated with 0.02% trypsin for 30 mins show a honeycomb-like structure of the membrane. Thus, part of the results appear to support the concept of a mosaic-like structure of the unit membrane, whereas other results are in agreement with the classical concept of a three-layered structure.The authors wish to express their gratitude to Dr. Sina Rosenthal, Department of Physiological Chemistry, Humboldt University, Berlin, who prepared the isolated membranes, to Mr. E. Fischer, Head Technician of the Department of Electron Microscopy, Greifswald University, who took most of the electron micrographs, to Mr. G. Bartsch, Department of Electron Microscopy, Greifswald University, and especially to Prof. W. Bargmann and to Doz. E. Lindner, Department of Anatomy, Kiel University, for many valuable suggestions.     

Localization of GABAA receptors in the rabbit retina

The distribution of gamma-aminobutyric acid_A (GABA_A) receptors in the rabbit retina is investigated and compared with the distribution of GABAergic neurons using immunocytochemical methods. Antibodies against the 1, 2/3, and 2 subunits of the GABA_A receptor label subpopulations of bipolar, amacrine and ganglion cells. Double labeling experiments show that the 2 subunit is colocalized with the 1 and the 2/3 subunits in bipolar, amacrine and ganglion cells. Electron microscopy reveals that in the outer plexiform layer, GABA_A receptor immunoreactivity is present on dendrites of cone bipolar cells adjacent to the cone pedicles. Bipolar cell dendrites are also receptor-positive at synapses from interplexiform cells. Some receptor immunoreactivity is found intracellularly in processes of horizontal cells. In the inner plexiform layer, GABA_A receptor immunoreactivity is present on both rod bipolar and cone bipolar axon terminals at putative GABAergic input sites. Amacrine and ganglion cell processes in sublamina a and b are also labeled.     

Whorl-like outer segments in the retina of the mole (Scalopus aquaticus)

The retina of the common mole, Scalopus aquaticus, has been studied with the transmission electron microscope. Structures examined include: pigment epithelium, outer and inner segments of the sensory cells, and synaptic ribbons of the outer plexiform layer. Rods and cones described in species by previous light microscopic studies are not seen with electron microscopic techniques. Instead, the mole retina contains peculiar outer segments consisting of whorls of membranes. These whorls have some similarities to dystrophic retinas of several vertebrates. The possibility of their being caused by extraordinary light exposure is discussed, also. The appearance of the sensory cells suggests that they are functional receptors.     

The zinc iodide-osmium tetroxide staining-fixative of Maillet. Nature of the precipitate studied by X-ray microanalysis and detection of Ca2+-affinity subcellular sites in a tonic smooth muscle

Summary A mechanism of osmium reduction during zinc iodide-osmium tetroxide (ZIO) fixation is proposed.X-ray powder microanalyses of ZIO precipitates formed both in the presence or absence of tissues are identical with those of CuOsO₄ and CuRuO₄. Therefore, and based on indexation methods, ZnOsO₄ was found to be the formula of the ZIO mixture reduction; this zinc osmate has an orthorhombic crystalline lattice.In smooth muscle preparations, ZIO electron dense deposits are localized in both cisternae of the sarcoplasmic reticulum and in mitochondria after a short fixation time.According to the microanalysis results, the zinc osmate has been associated to Ca²⁺ high affinity sites since Zn²⁺ is either replacing Ca²⁺ and/or displacing it by having a higher affinity for Ca²⁺ binding sites. Consequently, the ZIO mixture might be useful in revealing some Ca²⁺ storage sites in cells. This hypothesis was tested in ABRM preparations by selectively depleting sites which are known to bind Ca²⁺. In this case, the sarcoplasmic reticulum only retains the staining deposits after a short ZIO fixation.It is likely that OsO₄ alone, used as fixative in cytology might be due to the formation of metallic osmates (e.g., divalent osmates like CaOsO₄). In addition, of course, reduction of osmium during tissue fixation is accompanied by oxidation of double bonds of lipoproteic complexes or unsaturated lipids, and oxidation of sulfhydryl groups and amino groups.     

The verification of cytochemical tests for ATP-ase activity in plant cells using X-ray microanalysis

Summary Electron microprobe analysis (EMMA 4) was carried out on two types of electron opaque deposit found in thin sections of barley root tips as a result of cytochemical tests for ATP-ase activity.The granular type of deposit, which mainly occurs in radial and tangential cell walls of epidermal and sub-epidermal cells, was shown to contain lead, whereas lead was absent from the opaque globular deposits, which are much more generally distributed and always associated with membrane structures. Thus the latter deposits, which contain osmium and have often been interpreted as ATP-ase reaction products, should be regarded as artifacts of the fixation and rinsing procedures. It is suggested that calcium may play a role in the formation of the osmiophilic deposits.     

Changes of lysosomal enzymes during hereditary degeneration and histogenesis of retina in mice

Summary During the post-natal development of the retina in mice, macrophages which are selectively stained for N-Acetyl--glucosaminidase enter the retina through the vascular route. Most of these cells finally occupy the outer and the inner levels of the inner nuclear layer adjoining the plexiform layers and are transformed into very small cells which persist in the adult retina without further change.In mice with hereditary retinal degeneration (rd rd) these -glucosaminidase positive macrophages enter the outer nuclear layer of the retina, soon after the onset of degeneration undergo extensive hypertrophy and rapidly phagocytize the degenerating photoreceptor cells. After the digestion of the ingested materials the enzyme activity is very much reduced and the cells become smaller in size. They eventually acquire the morphological features seen in the normal retina.     

Histochemical studies on catecholaminergic cells in the carp retina

Histochemical studies on catecholaminergic cells were conducted with the carp (Cyprinus carpio) retina. Catecholamine (CA)-containing cell bodies appear sparsely distributed among amacrine cells in the innermost cellular row of the inner nuclear layer (INL) and occasionally in the outer half part of the inner plexiform layer (IPL); only exceptionally are they found among ganglion cells. The fluorescent cells interspersed with the amacrine cells and in the IPL send their fiber processes toward both the outer plexiform layer (OPL) and the IPL; the fine fibers form dense networks in the INL and IPL. Pretreatment of the fish with intramuscular injection of reserpine (20 hr prior to enucleation) completely depleted CA from the retina. The fluorescence of catecholaminergic cells was enhanced, and the number of fluorescent cells visible was increased, by intravitreous injection ofl-DOPA, DA, and NA (3 hr prior to enucleation). A combination of pretreatment with intramuscular reserpine and intravitreous NA was particularly effective. These results indicate that catecholamines may play an important role in the modulation of the membrane potential of horizontal cells.     

l-[3H]lysine binding to rat retinal membrane: II. effect of kainic acid,d,l-α-aminoadipic acid,iodoacetic acid,and modification by dark-exposure

The rat retina and the different brain regions contain membranes sites that bindl-lysine in the nanomolar range. These binding sites undergo changes in different experimental conditions, thus: I) intraocular injection of kainic acid induces a reduction of the density ofl-lysine binding sites, II)d,l--aminoadipic acid injected into the eye enhances both kinetic parameters (B _max andK _d) ofl-[³H]lysine binding sites, III) the intraperitoneal injection of iodoacetic acid decreases the sensitivity for its ligand binding sites, and IV) the exposure to darkness of the rats reducesl-[³H]lysine binding in the retina, thalamus, hypothalamus and superior colliculus, but not in the occipital cortex; such a decrease appears to be characterized, at least in the retina, by a lower sensitivity of the binding sites forl-lysine after the exposure to darkness. The results show thatl-lysine binding sites are located on kainic acid-sensitive cells and can be involved in the physiological mechanism of vision.