The fine structure of the fin musculature in two teleost species with different swimming modes,the puffer,Tetraodon steindachneri,and the goldfish,Carassius auratus

Summary Puffer fish (Tetraodon steindachneri) can execute precise maneuvers due to their highly specialized mode of propulsion. In the conventional locomotion exemplified by the goldfish (Carassius auratus), the fish thrusts are generated by lateral beating of the caudal fin. In contrast, the puffer generates its propulsive force by very rapid undulating movements of the pectoral, dorsal and anal fins. The fine structure of the fin muscles is identical in the two species of fishes, despite the differences in fin movement; cytologically, the fibers are intermediate between those of red and of white muscle. On the other hand, both the fusion frequency and the number of motor endplates are considerably higher in the fin muscles of the puffer than in those of the goldfish.     

Cytoskeletal organization and collagen orientation in the fish scales

Summary Immunofluorescence and electron microscopy were used to analyze the relationships between the organization of collagen fibrils in elasmoid scales, and the orientation of microtubules and actin microfilaments in the scleroblasts producing this collagenous stroma. Attention was focused on the basal plate of the scales because of the highly ordered three-dimensional arrangement of the collagen fibrils in superimposed plies forming an acellular plywood-like structure. The collagen fibrils are synthesized by the scleroblasts forming a monolayered pseudo-epithelium, the hyposquama, at the lowest surface of the scale. Fully developed scales with a low collagen deposition rate were compared with regenerating scales active in fibrillogenesis. When an ordered array of the collagen fibrils is found, the innermost collagen fibrils are coaligned with microtubules and actin microfilaments. Thus, because of this coalignment, microtubules and actin microfilaments of the hyposquamal scleroblasts are subjected to consecutive alterations during the formation of the plies of the basal plate. The sequence of events when the collagen fibrils change their direction from one ply to the other in the basal plate is deduced from immunofluorescence and phase-contrast-microscopic observations. During the formation of the orthogonal plywood-like structure in the regenerating scales, first microtubules may change their curse with a rotating angle of about 90°; then, actin microfilaments are disorganized and reorganized by interacting mechanically with the microtubules with which they are coaligned. Collagen fibrils are synthesized in a direction that is roughly perpendicular to that of the preceding ply. The unknown signals inducing the change in direction of the cytoskeleton may be transmitted throughout the hyposquama via gap junctions.This work is dedicated to the memory of Jacques Escaig     

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain and pituitary of the goldfish

Summary The distribution of neuropeptide Y (NPY) immunoreactivity has been studied by means of immunocytochemistry and radioimmunoassay in the brain of the goldfish. It was found that NPY had a widespread distribution in the entire brain in particular in the telencephalon, diencephalon, optic tectum and rhombencephalon. In the pituitary gland, positive type-B fibers were observed in the various lobes frequently in direct contact with secretory cells, in particular the gonadotrophs, somatotrophs and MSH (melanocyte-stimulating hormone) secreting cells. When measured by radioimmunoassay, the highest NPY concentrations were found in the pituitary and telencephalon, confirming the results of immunocytochemistry. The displacement curves obtained with serial dilutions of brain extracts were parallel to that of synthetic porcine NPY. Following high performance liquid chromatography, the NPY-like material extracted from goldfish brain co-eluted as a single peak with synthetic porcine NPY. These data demonstrate the presence of an NPY-like substance widely distributed in the goldfish brain. The observation of NPY-immunoreactive fibers in the pituitary gland suggests that, among its other functions, NPY may play a role in the neuroendocrine regulation of pituitary function.     

Influence of forskolin and carbachol on intestinal absorption of horseradish peroxidase in the goldfish (Carassius auratus)

The transepithelial route for mucosa-to-serosa transport of the tracer macromolecule horseradish peroxidase (HRP; MW 40 kDa) and modulation of this transport by forskolin and carbachol have been studied in vi-tro in stripped goldfish intestinal epithelium mounted in Ussing-type chambers. Uptake and transport have been investigated by measuring the HRP flux from the muco-sal to serosal sides by an enzymatic method and by visualising HRP reaction products in the mucosa with electron-microscopical techniques. Both the cholinergic agonist carbachol (which is thought to increase intracellular Ca²⁺ and activate protein kinase C activity) and forskolin (a direct activator of adenylylcyclase) affect the amount of enzymatically active HRP in the tissue. In control tissue, HRP product is found only within the epithelial cells, the transepithelial flux reaching a constant value of about 1.5 pmoles/cm² per h. Carbachol increases the amount of HRP product in the cells, but has no significant effect on the HRP flux compared with control values. Forskolin decreases the amount of HRP product in the cells; however, in the presence of forskolin, the lateral intercellular spaces become filled with HRP product. HRP is found in the lamina propria and the transepithelial protein flux increases more than 2.5-fold. In the presence of forskolin plus carbachol, the results are no different from the control. It is concluded that carbachol increases the endocytotic uptake of HRP, whereas forskolin inhibits the uptake but increases the paracellular permeability for HRP in goldfish intestine. Received: 10 July 1995 / Accepted: 4 February 1996     

Feeding-induced changes of melanin-concentrating hormone (MCH)-like immunoreactivity in goldfish brain

Intracerebroventricular (ICV) injection of melanin-concentrating hormone (MCH) influences feeding behavior in the goldfish and exerts an anorexigenic action in goldfish brain, unlike its orexigenic action in mammals. Despite a growing body of knowledge concerning MCH function in mammals, the role of MCH in appetite has not yet been well studied in fish. The aim of the present study was to investigate the involvement of endogenous MCH in the feeding behavior of the goldfish. We examined the distribution of MCH-like immunoreactivity (MCH-LI) in the goldfish brain and the effect of feeding status upon this distribution. Neuronal cell bodies containing MCH-LI were localized specifically to four areas of the hypothalamus. Nerve fibers with MCH-LI were found mainly in the neurohypophysis, with a few in the telencephalon, mesencephalon, and diencephalon. The number of neuronal cell bodies containing MCH-LI in the dorsal area adjoining the lateral recess of the third ventricle in the posterior and inferior lobes of the hypothalamus showed a significant decrease in fasted fish compared with that in normally fed fish, although other areas showed no evident differences. We also administered an antiserum against fish MCH (anti-MCH serum) by ICV injection and examined its immunoneutralizing effect on food intake by using an automatic monitoring system. Cumulative food intake was significantly increased by ICV injection of the anti-MCH serum. These results indicate that MCH potentially functions as an anorexigenic neuropeptide in the goldfish brain, and that the further study of the evolutionary background of the MCH system and its role in appetite is warranted. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (K.M. and A.T.) and by a research grant from the Toyama Marine Biotechnology Association (K.M.).     

Developmental changes and tissue distribution of lectin in Tulipa

A sensitive enzyme-immunoassay was developed to quantify the tulip lectin and used to follow its distribution during the life cycle of tulips cv. Attila.The tulip lectin is predominantly located in the bulbs. At planting time the absolute lectin concentration is approximately the same in all bulb scales. However, as the shoot grows and the plant turns on to flowering, the lectin concentration rapidly decreases, first in the inner bulb scales but later also in the outer bulb scale. Soon after flowering the lectin rapidly accumulates in the new daughter bulbs.Lectin levels in leaves, stems and flowers are very low. The lectin in these tissues is already present before the sprout emerges. During the first two weeks after planting, there is a small increase in lectin concentration, followed by a rapid decrease as the plant turns on to flowering. By flowering time all the lectin has disappeared from the aerial parts.Abbreviations DW dry weight - ELISA enzyme-linked immunosorbent assay - FW fresh weight - PBS phosphate-buffered saline - PBSN phosphate-buffered saline containing 0.02% sodium azide - PBST phosphate-buffered saline containing 0.02% sodium azide and 0.05% Tween 20 - TL tulip lectin - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta

Summary The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however porcine brain natriuretic peptidelike immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum. Many immunoreactive fibres were present in the rostral regions of the inferior lobes of the hypothalamus and in the dorsolateral and ventrolateral aspects of the rhombencephalon. No immunoreactivity was observed in the heart and brain using rat atrial natriuretic and eel natriuretic peptide antisera. Although the chemical structure of natriuretic peptides in the heart and brain of toadfish is unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic and/or porcine C-type natriuretic peptides. The presence of natriuretic peptides in the brain suggests that they could be important neuromodulators and/or neurotransmitters.     

Innervation and gap junctions of intestinal striated and smooth muscle cells in the loach

Summary The tunica muscularis of the proximal intestine of the loach consisted of intermingling striated and smooth muscle cells without forming any distinct sublayers. Close contacts devoid of intervention by a basal lamina sometimes occurred between these different types of muscle cells. Gap junctions were occasionally found between heterologous as well as homologous muscle cells. In freeze-fracture replicas, striated muscle cells were distinguished from smooth muscle cells by numerous, evenly distributed subsurface caveolae. These were relatively rare and linearly arranged in smooth muscle cells. Variously-sized and -formed aggregations of connexon particles were found in the protoplasmic fracture-face of both muscle cells. Striated muscle cells had aggregates of connexon particles taking the form of either a small solid polygon or an annulus with a particle-free central region. In smooth muscle cells, the particles were arranged either in variously-sized patches or in straight lines. Topologically, heterologous gap junctions observed in ultrathin section were thought to correspond to the small patchy aggregations. Striated muscle cells in the gut had neuromuscular junctions, which differed morphologically from cholinergic nerve terminals at neuromuscular junctions of typical skeletal muscle cells. The smooth muscle cells had close apposition with axonal terminals containing many granular vesicles and a variable number of small, clear vesicles. Occasionally, a cholinergic-type axonal terminal with a presynaptic active site was found close to a smooth muscle cell.     

Modulation of connexon densities in gap junctions of horizontal cell perikarya and axon terminals in fish retina: effects of light/dark cycles,interruption of the optic nerve and application of dopamine

Summary In the fish retina, connexon densities of gap junctions in the outer horizontal cells are modulated in response to different light or dark adaptation times and wavelengths. We have examined whether the connexon density is a suitable parameter of gap junction coupling under in situ conditions. Short-term light adaptation evoked low connexon densities, regardless of whether white or red light was used. Short-term dark adaptation evoked high connexon densities; this was more pronounced in the axon terminal than in perikaryal gap junctions. Under a 12 h red light/12 h dark cycle, a significant difference in connexon densities between the light and the dark period could be established in the gap junctions of the perikarya and axon terminals. Under a white light/dark cycle, only the gap junctions of axon terminals showed a significant difference. Crushing of the optic nerve resulted in an increase in connexon densities; this was more pronounced in axon terminals than in perikarya. Dopamine injected into the right eye of white-light-adapted animals had no effect. However, dopamine prevented the effect of optic-nerve crushing on connexon density. The reaction of axon-terminal gap junctions to different conditions thus resembles that of perikaryal gap junctions, but is more intense. Axon terminals are therefore thought to play an important role in the adaptation process.     

Developmental changes and tissue distribution of lectin inGalanthus nivalis L. andNarcissus cv. Carlton

A sensitive immunosorbent assay was developed to quantify the lectin in different tissues ofGalanthus nivalis (snowdrop) andNarcissus cv. Carlton (daffodil) and follow the distribution of the lectin during the life cycle of the plants. The lectin in snowdrops and daffodils occurs in almost all plant tissues. Moreover, in many tissues the lectin is the most prominent protein. High lectin concentrations are found in the bulb where the lectin accounts for up to 15% of the total protein during the resting period. However, as the shoot grows and the plant turns on to flowering the lectin content rapidly decreases. Soon after flowering the lectin accumulates in the new bulb units. Whereas in daffodil the lectin concentration in the aerial plant parts is about one order of magnitude lower than in the bulb, lectin concentrations in the upper parts of snowdrop are similar to those in the bulb. The lectin in the former tissues is already present before the sprout emerges. As the shoot starts to grow lectin concentrations in leaves, stems and flower parts gradually decrease so that at flowering time virtually all lectin has disappeared from the aerial parts. The highest lectin concentrations are found in the ovary and increase, initially, as the sprout emerges from the bulb. This work was supported in part by grants from the ‘Nationale Bank’ and the National Fund for Scientific Research (Belgium). W.J.P. is a Senior Research Associate and E.J.M.V.D. Research Assistant of this fund.     

Organization of histamine-containing neurons in the brain of the crested newt,Triturus carnifex

The distribution of immunoreactivity for histamine was studied in the brain of the urodele Triturus carnifex using the indirect immunofluorescence method. Histamine-immunoreactive cell bodies were localized in the caudal hypothalamus within the dorsolateral walls of the infundibular recesses. These immunoreactive cell bodies were pear-shaped, bipolar and frequently of the cerebrospinal-fluid-contacting type. Histaminergic nerve fibers were detected in almost all parts of the brain. Dense innervation was seen in the telencephalic medial pallium and ventral striatum, the neuropil of the preoptic area, the septum, the paraventricular organ, the posterior commissure, the caudal hypothalamus, the ventral and lateral mesencephalic tegmentum. Medium density innervation was observed in the lateral mesencephalic tegmentum and optic tectum. Poor innervation was present in the telencephalic dorsal pallium and in the central gray of the medulla oblongata. Few fibers occurred in the olfactory bulbs and in the telencephalic lateral pallium. Double immunofluorescence staining, using an antibody against tyrosine hydroxylase, showed that histamine-immunostained somata and those containing tyrosine-hydroxylase-like immunoreactivity were co-distributed in the tuberal hypothalamus. No co-occurrence of histamine-like and tyrosine hydroxylase-like immunostaining was seen in the same neuron. The pattern of histamine-immunoreactive neurons in the newt was similar to that described in other vertebrates. Our observations, carried out on the apparently simplified brain of the newt confirm that the basic histaminergic system is well conserved throughout vertebrates.     

The distribution of GABA-like-immunoreactive neurons in the brain of the newt,Triturus cristatus carnifex,and the green frog,Rana esculenta

Summary The distribution of -aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cell bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.Dedicated to Professor Valdo Mazzi (Dipartimento di Biologia Animale, Università di Torino), in honor of his 70th birthday     

Cranial meninges of goldfish: age-related changes in morphology of meningeal cells and accumulation of surfactant-like multilamellar bodies

In the optic tectum of goldfish, the outer, middle and inner layers of the endomeninx were evident in animals ranging in age from 1 month to several years. The outer layer in young animals consisted of closely overlapping cells with intertwined processes, whereas in the older animals it contained large extracellular spaces. The intermediate layer cells were always arranged in a single continuous layer, but in young animals they overlapped extensively with one another toward their edges whereas in the oldest animals they became extremely flat and non-overlapping. The inner layer included an outer tier of cells with their bases adhering to the intermediate layer, and an inner tier of cells detached from both the intermediate layer and the basal lamina overlying the brain parenchyma. Inner layer cells contained many large vacuoles that were in continuity with the extracellular space. With age, the extracellular space and the vacuolar system expanded, and the inner layer evolved into a meshwork of attenuated cytoplasmic processes embedded in the granular extracellular matrix. Another age-related feature was the accumulation adjacent to the basal lamina of uniform disc-shaped membranous structures, resembling multilamellar bodies of lung surfactant. These disc bodies were apparently generated by the coalescence of vesicles formed at the surface of the inner layer cells, possibly as a by-product of protein secretion by these cells.     

The valvula cerebelli of the spiny eel,Macrognathus aculeatus,receives primary lateral-line afferents from the rostrum of the upper jaw

Summary In the spiny eel, Macrognathus aculeatus, anterodorsal and (to a lesser degree) anteroventral lateralline nerves project massively to the granular layer of the valvula cerebelli, throughout its rostrocaudal extent. The posterior lateral-line nerve terminates in the corpus cerebelli. Thus, valvula and corpus cerebelli are supplied with mechanosensory input of different peripheral origins. An analysis of the taxonomic distribution of experimentally determined primary lateral-line input to the three parts of the teleostean cerebellum reveals that the eminentia granularis always receives such input, and that the corpus cerebelli is the recipient of primary lateral-line input in many teleosts. The valvula, however, receives primary lateral-line afferents in only two examined species. In M. aculeatus, the massive lateral-line input to the valvula probably originates in mechanoreceptors located in the elongated rostrum of the upper jaw, a characteristic feature of mastacembeloid fishes. This projection to the valvula may therefore represent a unique specialization that arose with the evolution of the peculiar rostrum.     

Colocalization of neuropeptide Y (NPY)-like and FMRFamide-like immunoreactivities in the brain of the Atlantic salmon (Salmo salar)

Summary The colocalization of the peptides neuropeptide Y (NPY) and Phe-Met-Arg-Phe-NH₂ (FMRFamide) in the brain of the Atlantic salmon was investigated with double immunofluorescence labeling and peroxidase-antiperoxidase immunocytochemical techniques. Colocalization of NPY-like and FMRE amide-like immunoreactivities was observed in neuronal cell bodies and fibers in four brain regions: in the lateral and commissural nuclei of the area ventralis telencephali, in the nucleus ventromedialis thalami, in the laminar nucleus of the mesencephalic tegmentum, and in a group of small neurons situated among the large catecholaminergic neurons in the isthmal region of the brainstem. All cell bodies in these nuclei were immunoreactive to both NPY and FMRF. We consistently observed larger numbers of FMRF-immunoreactive than NPY-immunoreactive fibers. In the nucleus ventromedialis thalami NPY- and FMRFamide-like immunoreactivities were colocalized in cerebrospinal fluid (CSF)-contacting neurons. NPY-immunoreactive, but not FMRF-immunoreactive, neurons were found in the stratum periventriculare of the optic tectum, and at the ventral border of the nucleus habenularis (adjacent to the nucleus dorsolateralis thalami). Neurons belonging to the nucleus of the nervus terminalis were FMRF-immunoreactive but not NPY-immunoreactive. The differential labeling indicates, as do our cross-absorption experiments, that the NPY and FMRFamide antisera recognize different epitopes. Thus, it is probable that NPY-like and FMRFamide-like substances occur in the same neurons in some brain regions.     

Developmental changes in the bark lectin of Sophora japonica L.

Lectin is the major protein in the phloem tissue of S. japonica. By immunohistochemistry using anti-seed lectin antibody it was demonstrated that the lectin was localized in the ray and the axial parenchyma. Neither lectin nor other cross-reactive materials were observed in the cambium, sieve tubes and companion cells. The distribution and localization changed in relation to tissue development. Lectin content in the bark changed during the year, the average in summer being about 50% of that in winter. The distribution of lectin in the bark in winter was similar from the innermost (youngest) to the outermost (oldest) region. In contrast, in summer the innermost region hardly contained any lectin, and the outermost region contained less lectin than the middle. Lectin localization in tissues and cells differed also depending on tissue age. In new tissue, produced in the current year, lectip was absent in summer, was located in the cytoplasmic layer between cell wall and vacuole in autumn, and sequestered in the vacuoles in winter. On the other hand, lectin in old tissue (formed in the previous year) was located throughout the year mainly within the vacuoles, with only very small contents in the cytoplasmic layer in autumn. Within the outermost (oldest) region, in which the lectin content was low in summer, the cells which bordered the outer bark never contained any lectin in summer. The intracellular localization in autumn in new tissue, determined by immunogold electron microscopy, was in the lumen of the endoplasmic reticulum and vesicles, with gold particles hardly present in the cytoplasm. From these findings we conclude that lectin is synthesized on the endoplasmic reticulum and most vigorously in the new tissue in autumn, and that it is mainly consumed in the outermost bark regions, where dilatation occurs and-or where cork cambium is differentiated.Abbreviations ELISA enzyme-linked immunosorbent assay - ER endoplasmic reticulum - kDa kilodalton Retired. Anatomical terms in this paper are used according to Multilingual glossary of terms used in wood anatomy edited by the Committee on Nomenclature, International Association of Wood Anatomists; reprints may be obtained from the Office of the Secretary-Treasurer, Universitätsstrasse 2, CH-8092 Zürich 6, Switzerland.     

Gene regulation by homeobox transcription factor Prox1 in murine hepatoblasts

Protein kinase C (PKC) is a signalling enzyme critically involved in many aspects of synaptic plasticity. In cyprinid retinae, the PKC alpha isoform is localized in a subpopulation of depolarizing bipolar cells that show adaptation-related morphological changes of their axon terminals. We have studied the subcellular localization of phosphorylated PKC alpha (pPKC alpha) in retinae under various conditions by immunohistochemistry with a phosphospecific antibody. In dark-adapted retinae, pPKC alpha immunoreactivity is weak in the cytoplasm of synaptic terminals, labelling being predominantly associated with the membrane compartment. In light-adapted cells, immunoreactivity is diffusely distributed throughout the terminal. Western blot analysis has revealed a reduction of pPKC alpha immunoreactivity in cytosolic fractions of homogenized dark-adapted retinae compared with light-adapted retinae. Pharmacological experiments with the isoform-specific PKC blocker Goe6976 have shown that inhibition of the enzyme influences immunolabelling for pPKC alpha, mimicking the effects of light on the subcellular distribution of immunoreactivity. Our findings suggest that the state of adaptation modifies the subcellular localization of a signalling molecule (PKC alpha) at the ribbon-type synaptic complex. We propose that changes in the subcellular distribution of PKC alpha immunoreactivity might be one component regulating the strength of the signal transfer of the bipolar cell terminal. Uwe D. Behrens and Johannes Borde contributed equally to this work. This research was supported by an SFB-430 grant.     

Catecholaminergic cells and fibers in the brain of the lizard Anolis carolinensis identified by traditional as well as whole-mount immunohistochemistry

Summary Using traditional as well as whole-mount immunohistochemistry, we described the location of tyrosine hydroxylase-and dopamine beta hydroxylase-positive cells and fibers in the brain of the lizard Anolis carolinensis. Major catecholaminergic cell groups were in the ependyma in certain ventricular regions, alous coeruleus, anterior hypothalamic and lateral hypothalamic areas, and in the mesencephalic tegmental region, locus coeruleus, nucleus of the solitary tract, vagal motor nucleus, and rhombencephalic reticular formation. Major catecholaminergic fibers, tracts and varicosities included tuberohypophysial, mesolimbic, nigrostriatal, isthmocortical, medullohypothalamic, and coeruleospinal systems. Although the catecholaminergic systems in A. carolinensis are similar to those in the brains of other lizards studied, there are a few species differences. Our information about A. carolinensis will be used to help localize the hypothalamic asymmetry in catecholamine metabolism previously described in this lizard.     

Intermediate stages in the disassembly of synaptic ribbons in cone photoreceptors of the crucian carp,Carassius carassius

Synaptic ribbons are trilaminated plate-shaped presynaptic densities of certain types of receptor cells and neurons. In cone photoreceptors, these structures dissassemble and reassemble in response to light and to a variety of other stimuli. We used the lithium-ionenhanced disassembly and reassembly of synaptic ribbons to characterize structural intermediates in these cyclic changes. A few minutes after exposure of isolated retinas from the crucian carp (Carassius carassius) to lithium, ribbons fragmented into 50-nm-sized dense globular structures. These small spheres were concentrically surrounded by synaptic vesicles attached to them by stalk-like fine bridging filaments. Disassembly always started at the free cytoplasmic edges of the ribbons and proceeded toward the membrane-associated edges. As the disassembly process never started at the membraneanchored site, synaptic ribbons appeared to be polarized structures with functionally different ends. Spheres were subjected to further depolymerization. They disintegrated into clusters of small granular material and disappeared after ca. 45 min of lithium treatment. Spheres were not observed during the reassembly of synaptic ribbons, indicating that the assembly of synaptic ribbons proceeds via smaller subunits.     

Developmental sequence of bone-resorbing cells induced by intramuscular implantation of mineral-containing bone particles into rainbow trout,Oncorhynchus mykiss

Mineral-containing bone particles (BPs) were implanted intramuscularly into rainbow trout (Oncorhynchus mykiss) to investigate the sequence of appearance of bone-resorbing cells. A fibrous substance first surrounded the implanted BPs and was gradually replaced by connective tissue containing capillaries. Two weeks after BP implantation, relatively small multinucleated cells (type-1 cells), whose cytoplasm stained deeply with hematoxylin, appeared along the surfaces of the BPs. At later stages (after 4–8 weeks), the majority of cells which appeared to be resorbing the BPs were multinucleated cells whose cytoplasm stained deeply with eosin (type-2 cells). Type-2 cells contained more nuclei than type-1 cells. Electron-microscopical observations revealed that type-2 cells had the characteristic features of osteoclasts: the presence of numerous mitochondria, vacuoles and granules, and a differentiation of the cell membrane and cytoplasm into a ruffled border and clear zone, respectively. A tartrate-resistant acid phosphatase activity, which is an established characteristic of osteoclasts in terrestrial vertebrates, but which had not previously been examined in teleosts, was demonstrated histochemically in the type-2 cells. Development of type-2 cells was closely correlated with the development of connective tissue. These findings suggest that the development of a capillary network around the implanted BPs enables circulating osteoclast-progenitors to reach the surface of the BPs.