Structural changes of drosopterinosomes (red pigment granules) during the erythrophore differentiation of the frog,Rana japonica,with reference to other pigment-containing organelles

Summary Structural changes in drosopterinosomes (red pigment granules) of Rana japonica in the process of erythrophore differentiation were studied by light and electron microscopy. On the basis of the degree of pterinosome differentiation, three types can be recognized: Typ-I drosopterinosomes appear first during metamorphosis and have clear limiting membranes and amorphous materials within. Those of type-II are found in abundance shortly after metamorphosis and have inner structures, consisting of fibrillae and/or small lamellae in dense concentric arrangement. Type-III is found abundantly in adults and acquires an almost homogeneously electron-dense mature morphology, probably from the deposition of electron-dense materials. On the basis of counts of pterinosomes, a successive transformation from type I to III is suggested. The differences among red drosopterinosomes, yellow sepiapterinosomes in xanthophore and melanosomes are not always distinguishable electron microscopically. Discrimination is possible by careful examination of lamellar patterns characteristic of the respective granules and by a simultaneous application of light and electron microscopy. From this viewpoint, a re-evaluation of the identification of granules previously reported was effected.This work was supported by a grant in aid to T. H. from the Ministry of Education (No. 92112, 1971).     

Surface fine structure of the subfornical organ in the Japanese quail,Coturnix coturnix japonica

The surface ultrastructure of the subfornical organ (SFO) was investigated in the Japanese quail. The SFO consists of a body and a stalk. The body of the SFO can be divided into rostral and caudal parts. On the rostral part, each ependymal cell possesses a short central solitary cilium; clustered cilia are also occasionally seen. Microvilli are abundant. On the caudal part, cells with a solitary cilium are fewer in number, and clustered cilia are rarely found. Microvilli are not as abundant as on the rostral part. In addition, large bulbous protrusions, tufts of small protrusions, deep funnel-shaped hollows, small pinocytotic invaginations and possible cerebrospinal fluid-contacting axons are sporadically observed on the surface of various regions of the body. Each ependymal cell of the stalk has a wide apical surface. A central solitary cilium, microvilli and other structures are observed more rarely on the stalk than on the body, while clustered cilia are not seen on the stalk. These structures are compared with those of the mammalian SFO and further discussed in relation to the possible dipsogenic receptor function for angiotensin II.     

Ultrastructural immunocytochemical evidence for peptidergic neurotransmission in the pond snail Lymnaea stagnalis

Summary Three neuronal systems of the pond snail Lymnaea stagnalis were immunocytochemically investigated at the ultrastructural level with the unlabeled peroxidase-antiperoxidase technique. Preliminary electrophysiological and cell-filling investigations have shown that a cluster of neurons which reacts positively with an antiserum against the molluscan cardio-active peptide FMRFamide, sends axons to the penis retractor muscle. In this muscle anti-FMRF-amide (aFM) positive axons form neuro-muscular synapses with (smooth) muscle fibers. The morphological observations suggest the aFM immunoreactive system to be involved in peptidergic neurotransmission. In the right parietal ganglion a large neuron (LYAC) is penetrated by aFM positive axons which form synapse-like structures (SLS) with the LYAC. The assumption that the SLS represent the morphological basis for peptidergic transmission is sustained by the observation that iontophoretical application of synthetic FMRFamide depolarizes the LYAC. The axons of a group of pedal anti-vasopressin (aVP) positive cells run in close vicinity to the cerebral ovulation (neuro-)-hormone producing cell system (CDC system) Synapses or SLS between the two systems were not observed. The fact that (bath) application of arg-vasopressin induces bursting in the CDC, may indicate that the vasopressin-like substance of the aVP cells is released non-synaptically.     

New types of islet cells in a cyclostome,Petromyzon marinus L.

Four types of acidophilic granular cells, in addition to B-cells, are identified in the islet organ of anadromous specimens of two subspecies of Petromyzon marinus by light and electron microscopy. Three of these acidophils (PI, PII and PIV-cells) occur in both the cranial and hepatic islets while a fourth type (PIII-cell) has only been found in the hepatic islet of some animals. The granules of the PI-cells stain with ponceau de xylidine, give a distinct tryptophan reaction and in ultrastructural examination show large, dense granules. The PII-cells contain unusual crystals and appear to be a non-secretory stage of the PI. The PIII-cells stain deep-red and acid fuchsin. They contain very large, dense granules and some lysosomes. PIV-cells stain selectively with phosphotungstic acid-hematoxylin and ultrastructurally, contain small, more or less dense granules. It appears that PI- and PIV-cells develop directly from B-cells, while the PIII-cells derive from PI-cells. despite their direct or indirect origin from B-cells, the PI-, PIII- and PIV-cells show characteristic features of functionally independent endocrine cells. Petromyzon marinus may be an ideal model for the understanding of phylogenetic and pathological interrelationships between islet and gastrointestinal hormones. It is clear that the interpretation of the islet organ of the cyclostomes, which has been generally considered a source of insulin only, requires a revaluation.     

Scanning and transmission electron microscopy of the subfornical organ of the grass frog (Rana pipiens)

Summary The ventricular surface of the subfornical organ of the frog is made up of ependymal cells with numerous apical microvilli, occasional cytoplasmic protrusions and many vacuoles projecting into the lumen of the third ventricle. Between these cells dendrites of cerebrospinal fluid-contacting neurons reach the ventricle to terminate in bulbous enlargements. In addition, flask-shaped encephalo-chromaffin cells, containing granulated vesicles and aggregates of filaments in their cytoplasm, project into the cerebrospinal fluid. Surrounding the centrally located capillaries are enlarged dendrites and axons of heterogeneous morphology, some of which appear to originate within the subfornical organ, intermingled with dendrites and axons of normal structure. The glial cells in this region, especially the microglial cells, often contain large lipofuscin inclusions, suggestive of degeneration and subsequent phagocytosis of some of the enlarged dendrites and axons. The normally scarce neurosecretory peptidergic axons become more evident and form typical Herring bodies in stalk-transected animals. Neuronal perikarya of varying morphology are predominantly located peripheral to the region of enlarged dendrites and axons. Supraependymal macrophages are particularly numerous on the subfornical organ.Abbreviations used CSF cerebrospinal fluid - SEM scanning electron microscope, scanning electron microscopy - SFO subfornical organ - TEM transmission electron microscope, transmission electron microscopy Supported, in part, by NIH grant NB 07492The skillful technical assistance of J.G. Linner and the secretarial assistance of Ann Gerdom are gratefully acknowledged. The SEM studies were made possible through a grant from the Graduate College of Iowa State University and the use of the SEM facility in the Department of Botany     

Surface morphology of the subfornical organ in the rabbit's brain

Summary The ventricular surface of the subfornical organ of the rabbit's brain was studied with scanning and transmission electron microscopic techniques. The ependymal covering was found to consist of hexagonal cells with convex apical surfaces. From the center of each cellular surface a single kinocilium up to 6 m in length protrudes into the liquor. It is usually covered with secretory material having the shape of pearlstrings. The surface aspect of the subfornical organ suggests secretion into the liquor by emptying of giant vacuoles which originate below the ependyma in nerve cells, move towards the surface, develop pressure while flattening their ependymal cover and finally erupt, leaving collapsed ependyma- and/or nerve cells bag on the surface of the organ. A second mechanism of more granular secretion by ependymal cells appears possible.We are indebted to Fräulein E. Östermann, Frau L. Schulze and Frau H. Zuther-Witzsch for excellent technical assistance.     

Structure and ultrastructure of the frog motor endplate

Summary The frog motor endplate in its simplest form consists of an elongated, slender nerve ending embedded in a gutter-like depression of the sarcolemma. This nerve terminal contains the usual synaptic organelles. It is covered by a thin coating of Schwann cell cytoplasm which embraces the terminal with thin finger-like processes from both sides, thereby sub-dividing it into 300–1000 regularly spaced compartments. The individual synaptic compartments correspond to the strings of varicosities or grape-like configurations of motor nerve terminals in endplates of other species and in the cerebral neuropil of vertebrates.Each compartment contains one or more bar-like densities of the presynaptic membrane, active zones, which are associated with the attachment sites between synaptic vesicles and plasmalemma. Active zones have a regular transverse arrangement and occur at specific loci opposite the junctional folds. The attachment sites for synaptic vesicles are at the edges of the bars which are bilaterally delineated by a double row of 10 nm particles attached to the A-face. The structural appearance of vesicle attachment sites in freeze-etch replicas corresponds to that of micropinocytosis. The active zones are often fragmented and the frequency of their association with vesicle attachment sites is highly variable.The junctional folds are characterized by specific sites in which intramembranous particle aggregations occur at relatively high packing density (7500/m²). These sites are located opposite the active zones at the juxtaneural lips, a location where one would expect ACh-sensitive receptors on the postsynaptic membrane.This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 38, Projekt N), The Swiss National Foundation for Scientific Research (Grants Nr. 3 82372 and 3 77472) and the Dr. Eric Slack-Gyr Foundation Zürich.     

Ultrastructure of epidermis of Salamandra salamandra followed throughout ontogenesis

Summary Ventral epidermal ultrastructure of the amphibian urodele Salamandra salamandra is described and followed throughout its life cycle.Tadpoles were divided into five categories on the basis of the organization of their epidermis and the ultrastructure of its cells. In newly hatched tadpoles the epidermis is arranged in two layers and four types of cells were recognized. The number of epidermal layers increases in the metamorphosing tadpole. At this stage the layers become organized in four strata. Metamorphosis involves the disappearance of some cell types and the appearance of others, typical of the adult epidermis.The significance of these ontogenetic changes in epidermal ultrastructure is discussed in respect to aquatic and terrestrial life habits.     

Parenchymal fine structure of the subfornical organ in the Japanese quail,Coturnix coturnix japonica

Summary The parenchyma of the subfornical organ (SFO) of the Japanese quail was studied by light and electron microscopy. The SFO consists of ependymal, intermediate, and basal (perimeningeal) layers. In the intermediate layer, neurons, glial cells, and their processes are found. Axons containing dense core granules approximately 80 nm in diameter are numerous, some of which make synaptic contact with the neuronal perikarya or dendrites. Synaptic vesicles in some axons contain a dense dot in the interior after treatment with 5-hydroxydopamine. The activity of the SFO, which is probably concerned with elicitation of drinking by angiotensin II, may be regulated at least partly by afferent monoaminergic axons. Capillaries with a non-fenestrated endothelium are occasionally found in the parenchyma. The basal layer is occupied by glial processes abutting on the digitating layer of perivascular connective tissue of meningeal vessels. The endothelium of these vessels is occasionally fenestrated. Trypan blue injected systemically accumulated in the SFO, but not in the deeper areas of the brain. The absence of a blood-brain barrier is suggested in the SFO.     

Scanning electron microscopy of the wall of the third ventricle in the brain of Rana temporaria. Part IV.

Summary The surface specializations of the wall of the third cerebral ventricle of Rana temporaria were investigated with the scanning electron microscope. These specializations can be divided into three types: cilia, large bulbous protrusions, and microvillus-like protrusions.Most parts of the ventricular surface are densely ciliated. In contrast, other regions are either scantily ciliated or devoid of cilia. Four areas of the ventricular surface are studded with numerous large bulbous protrusions. These large protrusions can be divided into two types: One type consists of intraventricular end bulbs of dendrites of secretory neurons. The other type is represented by large cytoplasmic extensions of ependymal cells.In the third ventricle of Rana, microvillus-like surface specializations of ependymal cells are ubiquitous structures. Generally, filiform protrusions of varying length are the predominant type. The microvillus-like specializations are transient structures, the number of which varies according to different physiological states of the ependymal cells.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Histological and ultrastructural studies of the basal disk of Hydra

Summary The glandulomuscular cells of Hydra are located exclusively in the basal disk. They are derived from epithelio-muscular cells which have been forced proximally. Light and electron microscopical studies show that prior to their destruction and elimination, the transformed epithelio-muscular cells (i.e. the glandulomuscular cells) undergo certain striking morphological and physiological changes. Golgi complexes and elements of rough E. R. increase remarkably in activity, and individually or jointly produce at least six types of morphologically different droplets. One additional type of droplet is thought to originate from neighboring digestive cells. Although the chemical nature of the individual droplets is uncertain, it is known that some are Alcian blue and PAS positive and contain hyaluronic acid. These evidences suggest the presence of an acid mucopolysaccharide material, the adhesive agent which attaches the animal to a substrate. The myonemes contain thick (200 Å in diameter) and thin (60 Å in diameter) filaments as in epithelio-muscular cells. There are also filaments of intermediate sizes and large fibers (770 Å in diameter). The myonemes are oriented radially with respect to the aboral pore and therefore in addition to contributing to the contraction and relaxation of the body column, they apparently regulate the opening and closing of the aboral pore. Although there is no evidence to substantiate the mechanism for transformation of epithelio-muscular cells to glandulomuscular cells as well as cell death of the latter cell types, these problems are discussed briefly.This investigation was supported by The National Science Foundation, Grant Number GB-27395.With the technical assistance of Linda M. Bookman.     

Distribution of pectin and arabinogalactan protein epitopes during organogenesis from androgenic callus of wheat

The distribution of several arabinogalactan protein and pectic epitopes were studied during organogenesis in androgenic callus of wheat. In cell wall of mature and degenerating parenchyma cells, the arabinogalactan epitopes JIM4, JIM14, JIM16 or LM2 were expressed differently according to the cells location. LM2 was observed also in meristematic cells of regenerated shoot buds and leaves. Anti-pectin JIM7 labelled the wall of meristematic cells but fluorescence was strongest in outer walls of surface cells of callus and shoot buds coated by extracellular matrix surface network (ECMSN). During leaves growth the ECMSN disappeared, and JIM7 fluorescence decreased. JIM5 epitope was abundant in the cell walls lining the intercellular spaces of callus parenchyma and in tricellular junctions within regenerated buds and leaves.     

The hypothalamo-hypophysial system of the frog Rana temporaria

Summary The median eminence (ME) of the adult frog, Rana temporaria, was studied by means of electron microscopy including quantitative electron-microscopic autoradiography. In frogs captured in May and June numerous peptidergic neurosecretory fibres extending via the internal zone to the pars nervosa display large swellings containing few granules, mitochondria, neurotubules and cisternae of the smooth endoplasmic reticulum. In addition, few secretory globules up to 1.5 m in diameter occur in these varicosities. In animals collected during the autumn period many of these neurosecretory swellings filled with neurosecretory granules and polymorphic inclusions resemble Herring bodies. Three types of granule-containing neurosecretory fibres were observed in the external zone (EZ) of the ME of adult R. temporaria. Peptidergic A₁- and A₂-type fibres are characterized by granules 150–220 nm and 100–160 nm in diameter, respectively. Monoaminergic fibres of type B with granules approximately 100 nm in diameter represent 50% of all neurosecretory elements in the EZ of the frog ME; 12% of the total number of granule-bearing axons in the EZ actively taking up radiolabelled 5-hydroxytryptophan are thought to be serotoninergic terminals. Neurosecretory terminals of all types and glial vascular endfeet establish direct contacts with the perivascular space of the primary portal capillaries. Some neurosecretory terminals are separated from the lumen of the third ventricle by a thin cytoplasmic lamella of tanycytes. The possible physiological significance of this structural pattern is discussed.     

Continuity between the ventricular and subarachnoid cerebrospinal fluid in an amphibian,Rana pipiens

Summary Continuity between the ventricular and subarachnoid cerebrospinal fluid has been investigated in Rana pipiens. The structure of the posterior tela, a deficient membrane situated at the extreme caudal end of the roof of the fourth ventricle, has been studied using whole membrane mounts and by light microscopy of resin embedded tissue. The ependymal component consists of columnar and rounded cells which form a regular syncytium enclosing round and oval fenestrations. Small fenestrations are covered on the subarachnoid side by elongated pial cells and thus do not give total continuity between the fourth ventricle and the subarachnoid space. Large fenestrations, on the other hand, are accompanied by equivalent pial fenestrations giving direct access between the fluid compartments. Towards the caudal end the fenestrations break up and the numbers of ependymal and pial cells decrease, the caudal end itself being characterised by a small remaining clump of ependyma and pia or of pia alone.Flow through the tela has been studied using fluorescein-labelled dextran placed in the intraventricular space. Infusion into the lateral ventricle and subsequent localisation by fluorescence microscopy shows the marker to be in the fourth ventricle, in the fenestrations of the posterior tela and in the subarachnoid space overlying the tela. Infusion of the marker followed by freezing and examination of the cut heads on a freezing microtome, shows fluorescence throughout the ventricular system, in the subarachnoid space adjacent to the posterior tela and also along the dorsal subarachnoid space of the spinal cord.     

The optic tracts of Rana temporaria and a possible retino-preoptic pathway

Summary The optic tracts and centres of optic terminals of Rana temporaria have been investigated with silver impregnation techniques after unilaterally cutting the optic nerve and autoradiographically after injection of a mixture of tritiated amino acids into the vitreous body of the left eye.The observations on the course of the optic tracts and on the optic terminals in the thalamus and optic tectum are to a great extent in agreement with those of other authors. The probability of a retino-preoptic pathway is supported by the detection of fibres running in dorsal direction in front of the place where the optic nerve penetrates the brain. In horizontal sections these fibres can be seen deviating from the optic tract, covering a short distance in frontal direction and then turning upward under an angle of about 90 degrees. They disappear between the ventral aldehyde-fuchsin positive cells of the preoptic nucleus. The ventral and median parts of the ipsilateral preoptic nucleus contain fragments of degenerated fibres.Autoradiographic data are also in favour of the presence of an ipsilateral retino-preoptic tract. After carefully counting the number of grains over left and right preoptic nucleus, it appeared that over the ventral and median parts of the left preoptic nucleus more grains occur than over the identical areas of the right one.It is a pleasure to acknowledge Prof. Dr. J. C. van de Kamer and Dr. F. C. G. van de Veerdonk for their helpful encouragement and constructive suggestions, Dr. L. Boomgaart for checking and amending the English writing. Thanks are also due to Miss Ans de Groot and Miss Thera Verstappen for their technical assistance and to the staff of the photographic department for making the illustrations.