On the development of the cerebellum of the trout, Salmo gairdneri. II. Early development

Summary The early histogenesis of the cerebellum of Salmo gairdneri RICHARDSON, 1836 has been studied in fish ranging in length from about 5 to 14 mm, both with light microscopical and electron microscopical techniques. Structurally, the matrix cells correspond to those of other vertebrates. Mitoses occur predominantly at the ventricular surface, but peripheral mitoses are found as well, particularly in the period of highest mitotic activity. Mantle cell somata can be distinguished from the elongated matrix cells on the basis of their rounded shape. The neurogenetic and gliogenetic periods overlap considerably. Presumably the first mantle cells are all neuroblasts: as soon as the mantle layer starts to form, axonal profiles are found. In a slightly later stage glial differentiation is manifest in the radial processes contacting the meningeal surface. In young stages a distinction between neuroblasts and glioblasts can only be made on the basis of the structure of their processes. Processes of glioblasts can be distinguished form axons and dendrites by their paucity of microtubules. Dendrites, appearing in late-embryonic stages, contain the same organelles as axons, but in larger amounts. The first differentiation of mantle cell somata is an increase of rough endoplasmic reticulum, and that to a lesser degree in glioblasts than in neuroblasts. Neuronal nuclei are rounded and more electronlucent than those of mantle cells. Apart from zonulae adhaerentes between the internal processes of matrix cells, puncta adhaerentia occur frequently in the cerebellar anlage. However, they rarely occur on young neurons. The possible significance of these junctions is discussed. The present study indicates that growth cones and filopodia are characteristic of most and probably of all types of cells in the early developing cerebellum. Growth cones contain much vesicular and tubular endoplasmic reticulum and in filopodia a fine filamentous network is present. In the somata of mantle cells growth areas were found, i.e. areas under the cell membrane with a similar content as growth cones. It is suggested that these areas anticipate the outgrowth of a new process.     

On the development of the cerebellum of the trout, Salmo gairdneri. I. Patterns of cell migration

Patterns of cell migration in the cerebellum of Salmo gairneri RICHARDSON, 1836 were studied in fish ranging in length from 4.5 to 230 mm. Sagittal and transverse series were stained with haematoxylin-eosin or according to Nissl or Golgi. The cerebellum of the trout comprises three main parts, i.e. the massive corpus cerebelli, the folded valvula cerebelli and the transversely oriented lobus vestibulolateralis. The early cerebellar anlage is a simple plate, which is delimited from the tectum mesencephali by the fissura rhombo-mesencephalica. The histogenesis may be divided into three phases. During the first phase the matrix layer produces the mantle layer. During the second phase the three typical cerebellar layers are formed. The third phase is characterized by growth. As regards the first phase, the mantle layer develops throughout almost the entire extent of the cerebellar anlage. Only in a narrow paramedian strip (matrix zone M) this layer does fail to appear. In regions where the mantle layer is formed, the matrix no longer occupies the whole width of the wall and is termed the ventricular matrix. The largest part of the ventricular matrix is gradually exhausted. However, in some places this matrix persists as a layer of proliferating cells. This holds for the matrices of the caudal border of the cerebellum; matrix zone L, surrounding the lateral recesses of the fourth ventricle, and matrix zone P, connecting the matrix zones L. The mantle layer produced in the first phase of histogensis mainly develops into the ganglionic layer. The second phase of histogenesis is characterized by the formation of a secondary matrix. Newly produced cells of the matrix zones M, L and P migrate away from their sites of origin towards the regions where a mantle layer prevsiously has been formed. The majority of these cells develops into granule cells. Migration of the cells produced in the first phase of histogenesis occurs in the radial direction. Because of the curvature of the cerebellum this direction changes with respect to the main longitudinal axis of the brain from region to region. The migration paths of granule cells show variable directions, namely (a) tangential followed by radial, for granule cells in the corpus cerebelli and in the medial parts of the valvula cerebelli and the lobus vestibulolaterialis, (b) tangential, for granule cells in the lateral parts of the valvula and (c) radial, for granule cells in the lateral parts of the lobus vestibulolateralis. The analysis of these migration patterns elucidates both the histogenesis and the morphogenesis of the cerebellum of the trout.     

On the development of the cerebellum of the trout, Salmo gairdneri. III. Development of neuronal elements

Summary The differentiation of the cerebellar neurons and of their afferent fibres has been studied in young specimens ofSalmo gairdneri Richardson, 1836. Both light microscopic preparations, stained with haematoxylin-eosin or according to Bodian, Nissl, Klüver-Barrera or Golgi, and electron microscopic preparations were used. The ventricular matrix layer gives rise to the large neurons of the cerebellum,i.e. Purkinje, eurydendroid and Golgi cells; the secondary matrix produces the smaller neurons,i.e. the granule and stellate cells. The afferent fibres of the cerebellum are the mossy and the climbing fibres. The identification of the cell types, originating from either the ventricular matrix or from the secondary matrix, can be made earlier on the basis of the structure of their processes than on the basis of the structure of their somata. The development of the cerebellar neurons in the trout corresponds in many respects to that in higher vertebrates. In general, differentiation is characterized by a decrease in the number of free ribosomes and an increase of the other organelles, particularly of rough endoplasmic reticulum. The ganglionic layer contains, in addition to the Purkinje cells, the eurydendroid cells. The axons of these elements were in some cases observed to leave the cerebellum, whereas the axons of Purkinje cells are mainly confined to the ganglionic layer. In the trout the development of the granule cells shows a varied pattern. The mature shape of the axons of these elements depends on the migration paths followed by their precursors. T-shaped processes occur in all parts of the cerebellum and unbranched processes only in the valvula. The opinion held for mammals that the more superficial a parallel fibre is situated in the molecular layer the later it has been formed, is not valid for the trout. A number of secondary matrix cells performs tangential migration, not in the submeningeal region but deeper in the molecular layer,viz. under bundles of parallel fibres. The granule cells originating from these deeper matrix cells extend their axons at a lower level than the parallel fibres which have been formed previously. Throughout development dark cells are found in osmiumstained material. Their dark aspect is due to the presence of a fine filamentous network and of many free ribosomes in the cytoplasm. These immature cells are considered to be migratory.     

On the development of the cerebellum of the trout, Salmo gairdneri. IV. Development of the pattern of connectivity

Summary Synaptogenesis has been studied in the corpus cerebelli of the troutSalmo gairdneri, Richardson, 1836. The first synapses are observed in hatchlings and occur between parallel fibres and the shafts of Purkinje dendrites. Subsequently the axosomatic synapses of Purkinje axon collaterals on the neurons of the ganglionic layer appear, and finally the synapses made by climbing fibres and mossy fibres, and by stellate cell axons develop. Young synapses in the cerebellum of the trout resemble the mature structures so closely that the criteria for the identification of the latter can also be applied to the former. The number of parallel fibre synapses and of Purkinje axon collateral synapses increases considerably during development. Eurydendroid cells, the axons of which leave the cerebellum, receive an abundance of Purkinje axon collaterals on their somata and main dendritic trunks. Mossy fibre synapses are numerous in the granular layer. Climbing fibre contacts and synapses of stellate cell axons, both with Purkinje cells, are found occasionally. the following pattern of connectivity is proposed. The main input-output system is formed by the mossy fibres, the granule cells, the Purkinje cells and the eurydendroid cells. Additional pathways are formed by (1) the mossy fibres, granule cells and eurydendroid cells, and (2) the climbing fibres, Purkinje cells and eurydendroid cells. The afferent-efferent systems, mentioned above, are influenced by a number of internuncial elements: (1) The Golgi cells receive their input from the parallel fibres and contact with their axon collaterals the dendrites of granule cells. (2) Axon collaterals of Purkinje cells are in synaptic relation with Golgi cells. (3) Axon collaterals of Purkinje cells impinge upon the somata and main dendrites of other Purkinje cells. (4) Stellate cells, which derive their input from the parallel fibres, synapse with the dendrites and somata of Purkinje cells. The possible functional roles of all of these neuronal elements are discussed.     

Oocyte structure and early vitellogenesis in the trout,Salmo gairdneri

Light and electron microscopic studies have been made on young oocytes of the trout; i.e., during previtelline and early stages of vitellogenesis. Balbiani's basophilic body has been followed from its origin in the youngest oocyte to its disappearance at the beginning of vitellogenesis. It assumes the form of a duplex body composed of a non-basophilic component (idiosome) and a highly basophilic portion, the pallial substance. Electron micrographs reveal the Balbiani body to be a dense, finely granular substance apparently unrelated to the Golgi apparatus or mitochondria. Its role in oogenesis is not clear, but probably constitutes an essential precursor substance (perhaps RNA) necessary for oocyte growth and vitellogenesis. At given periods in early oocyte growth, an extensively developed system of endoplasmic reticulum is formed and the cisternae contain numerous granules. These intracisternal granules probably represent precursor elements for a specialized type of yolk substance. The granules collect in dilated regions of the smooth-surfaced cisternae where the latter undergo anastomosis. Here the granules undergo transformation into a finely granular particle. Older and more vitellogenetic oocytes have not been included in this study.     

Acousticolateral and visual processing and their interaction in the torus semicircularis of the trout, Salmo gairdneri

The hypothesis that synaptic transmission between the auditory nerve and the cochlear nucleus is mediated by an excitatory amino acid acting through N-methyl-D-aspartate (NMDA) receptors was examined in an in vitro preparation of the chicken brainstem. The ability of various bath-applied excitatory amino acid receptor antagonists to inhibit synaptically-evoked responses was assessed by recording field potentials from nucleus magnocellularis (NM) following electrical stimulation of the cochlear nerve. Antagonists that selectively block responses mediated by NMDA receptors, such as D-alpha-aminoadipate and 2-amino-5-phosphonovalerate, were without effect on evoked transmission in NM. In contrast, antagonists that additionally act on non-NMDA receptors, such as cis-2,3-piperidine dicarboxylate and gamma-D-glutamylglycine, reversibly suppressed transmission. The results indicate that (1) transmission in the chicken auditory system is mediated by non-NMDA receptors, and (2) a substance(s) chemically akin to aspartate and glutamate may be the transmitter used by the auditory nerve in NM.     

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: An ultrastructural study

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdneri, kept at 14°C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epthelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cell types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.     

Accessory cells in the gill epithelium of the freshwater rainbow trout Salmo gairdneri

Two types of mitochondria-rich cells were identified in the gill epithelium of the freshwater-adapted rainbow trout, Salmo gairdneri, after selective impregnation of their tubular system with reduced osmium. A first type consisted of large cells with a poorly developed and loosely anastomosed tubular system; thus, that resembled the chloride cells commonly encountered in the gill epithelium of freshwater-adapted euryhaline fishes. A second type comprised smaller cells with an extensively developed and tightly anastomosed tubular system. These never reached the basal lamina of the gill epithelium and were adjacent to chloride cells, to which they were linked by shallow apical junctions (100-200 nm); thus, they resembled accessory cells, which are currently found in the gill epithelium of sea-water-adapted fishes but are usually lacking in freshwater living fishes. Transfer of the freshwater-adapted trout into seawater induced the proliferation of the tubular system in the chloride cells and the formation of lateral plasma membrane interdigitations between accessory cells and the apical portion of the chloride cells. The length of the apical junction sealing off this ex tended intercellular space was reduced to 20-50 nm. The tubular system of the accessory cells was not modified. The extension of the tubular system in the chloride cells of the seawater-adapted fishes indicated that, as in most euryhaline fishes, these cells have a role in the adaptation of the rainbow trout to seawater. In contrast, the function of the presumptive accessory cells in fresh water trout remains to be established.     

Enzyme- and immuno-histochemical study of the thymic stroma in the rainbow trout, Salmo gairdneri, Richardson.

Owing to the lack of data about thymic non-lymphoid cells in fish we decided to perform a histochemical characterization of these cells in order to ascertain their relationships to other thymic components. In the present study we analyze the enzyme-histochemical patterns for acid phosphatase, alkaline phosphatase, non-specific sigma-naphthyl acetate esterase and 5' nucleotidase activities, as well as the presence of keratin demonstrated by immunoperoxidase staining, in the non-lymphoid cell populations of the thymus of the rainbow trout, Salmo gairdneri. According to their location in the organ, morphology and histochemical reactivities, we were able to define seven different subpopulations of keratin-positive epithelial cells: 1) Epithelial cells limiting with the capsular and septal connective tissues; 2) Subcapsular epithelial cells; 3) Stellate epithelial cells of the inner thymic zone; 4) Large, ovoid epithelial cells of the inner thymic zone; 5) Acidophilic epithelial cells of the outer thymic zone; 6) Cystic cells; and 7) Goblet cells. The significance of the heterogeneity of the epithelial cell (EC) population, its specific distribution in the organ, which apparently conforms distinct cell microenvironments, as well as the possible phylogenetical relationships between these microenvironments and the classical cortex and medulla of the mammalian thymus, are discussed.     

Single nephron filtration rates (SNGFR) in the trout,Salmo gairdneri

1. Trout kidney slices incubated in the presence of¹⁴C-sodium ferrocyanide accumulated radioactivity. Uptake by individual nephroi increased with time, stabilizing after 30 min. The greater the concentration of radioactivity the greater the uptake per mm of tubule, while reduced specific activity inhibited¹⁴C uptake.
2. The renal excretory pattern of¹⁴C-sodium ferrocyanide was compared with that of³H-inulin in fresh water adapted trout. The renal clearance rates of the substances were similar and linearly related over a range or urine flows, providing plasma ferrocyanide concentrations were between 1.5 and 2.5 mM.¹⁴C-ferrocyanide is thus a reliable marker for glomerular activity in this species.
3. A protocol for the determination of single nephron glomerular filtration rates (SNGFR) in trout is described. The method exploits the ability to visualize ferrocyanide as Prussian Blue within nephroi after a single pulse injection superimposed upon a constant infusion of¹⁴C-ferrocyanide. The effects of surgical procedure and extraluminal contamination are assessed.
4. The SNGFR of freshwater trout, 1.31±0.2 nl/min is approximately 65% less than that observed in sea water adapted fish (3.74±1.12 nl/min). The overall, total kidney glomerular filtration rate, (GFR) is 142.6±17.4 μl/min/kg in fresh water animals and 20.1±0.9 μl/min/kg in sea water fish.
5. It is concluded that whilst SNGFRs of sea water animals are higher than those of fresh water animals, filtration is distributed to nephron populations selected to meet the homeostatic demands of the organism.

     

The production of a macrophage-activating factor from rainbow trout Salmo gairdneri leucocytes.

Rainbow trout head kidney and blood leucocytes are shown to be capable of secreting a soluble macrophage-activating factor (MAF) after stimulation with concanavalin A (Con A). The presence of phorbol myristate acetate (PMA) as a co-stimulant increased the production of MAF. Both respiratory burst activity (nitroblue tetrazolium, NBT, reduction and H2O2 production) and bactericidal activity were enhanced after incubation of resident or elicited macrophages with the MAF-containing supernatants for 48-72 hr. The target culture period before the addition of MAF did not affect their responsiveness, but a continuous presence of MAF was necessary for maximal stimulation.     

Lymphocyte heterogeneity in the trout, Salmo gairdneri, defined with monoclonal antibodies to IgM

A monoclonal antibody to trout serum IgM was tested by immunofluorescence analysis with lymphocytes from thymus, spleen and head kidney. By visual examination, the antibody reacted with only a subpopulation of lymphocytes. The mean values +/- SE for positive cells were 5.2 +/- 2.3% in the thymus, 30.3 +/- 7.9% in the spleen and 12.4 +/- 3.0% in the head kidney. Flow cytofluorometric analysis revealed evidence of heterogeneity by size among the membrane IgM-positive cells of the head kidney and spleen. Depletion of head kidney cells positive for surface IgM by an immune affinity adherence technique of panning, using monoclonal anti-IgM, significantly reduced the mitogenic response to lipopolysaccharide but not to concanavalin A. It is suggested that this information supports the existence of distinct subpopulations of fish lymphocytes that may be homologous in certain respects to mammalian T and B type cells.     

Photoreceptor responses to light in the isolated pineal organ of the trout, Salmo gairdneri

Photoreceptor potentials were recorded intracellularly from the isolated pineal organ of the teleost, Salmo gairdneri, maintained in tissue culture medium for 2-20 h. After electrophysiological characterization the photoreceptor cells were iontophoretically injected with Lucifer Yellow or with horseradish peroxidase for subsequent morphological identification. A brief flash of light elicited a hyperpolarization which was graded with light intensity in the dark-adapted photoreceptor. For dim flashes, the responses were purely monophasic. At higher intensities responses either remained purely monophasic or displayed an initial transient wave which became prominent for supersaturating intensities. The latency of the responses and their rise time decreased with increasing light intensity. Threshold responses showed latencies of about 600 ms, reached a maximum in about 1100 ms and returned to the dark potential in about 5 s. Saturating flashes considerably diminished the latency to 55 ms, the rise time to about 250 ms, but increased the time of recovery from peak to dark potential up to 60 s. Intracellular responses to background illumination exhibited two different response types. One type repolarized immediately, when the background light was extinguished, whereas the other type was characterized by a slow recovery of the dark potential. The spectral sensitivity of all intracellular recorded photoreceptors peaked at lambda max = 520-530 nm.     

The cellular immune response of rainbow trout (Salmo gairdneri Richardson) to sheep red blood cells

The cellular immune response of rainbow trout, Salmo gairdneri, to sheep erythrocytes was investigated. Both the primary and secondary responses were measured using the migration inhibition factor (MIF), antigen-binding (ABC), and plaque-forming cell (PFC) assays. These immune function assays provide measures of both T and B cell activity. The kinetics of these three responses at 16 degrees C were determined by sampling fish over an 18 day period for the primary response and a ten day period for the secondary response. The peak MIF response occurred two days after injection, while the primary peak PFC response was observed 14 days post-injection. Two ABC peaks were observed in the primary response, one at four days and one at ten days after injection. In the secondary response the peak ABC response was observed four days and the peak PFC response six days post-inoculation. The possible interrelationships of the various cell populations are discussed.     

The thymus of the rainbow trout (Salmo gairdneri). Light and electron microscopic study

The rainbow trout thymus is a paired organ, organized in 3 adjacent zones. The gland lying on a thick connective tissue layer, is covered by a thin epithelial capsule. Cellular components of thymus are essentially the thymocytes and the epithelial cells. Thymocytes occur mainly in the inner and outer zones but neither cortex nor medulla are clearly delimited. Septa represent a typical epithelial structure associated with thymocytes and blood vessels. Thymus is well vascularized and electron microscopy demonstrates a characteristic relationship between vascular system and lymphoid tissue.