Dissociation of photoreceptor cells from the pineal organ of the lamprey,Lampetra japonica

Summary Photoreceptor cells, nerve cells and supporting cells were dissociated from the pineal organ of the river lamprey, Lampetra japonica, by the use of 10 U/ml papain solution at 28° C for 20 min, followed by repeated trituration. With the aid of Nomarski interferencecontrast optics, photoreceptor cells, nerve cells and supporting cells were readily identified. Electron-microscopic examination revealed that isolated photoreceptor cells display an outer segment endowed with a few lamellar disks and connected to the inner segment (ellipsoid) via a connecting cilium. The structural features of the dissociated photoreceptor and supporting cells strongly resemble the morphology of the respective cellular elements in situ. We succeeded in culturing dissociated cells for time periods up to 48 h when the procedure described in detail was applied.     

Distribution of FMRFamide-like immunoreactivity in the brain and neurohypophysis of the lamprey,Lampetra japonica

Summary Distribution of molluscan cardio-excitatory tetrapeptide Phe—Met—Arg—Phe—NH₂ (FMRFamide) was determined by means of immunohistochemistry in the brain and neurohypophysis of the lamprey, Lampetra japonica. Many FMRFamide-like immunoreactive neurons were found in the periventricular nuclear region and in a region near the mammillary recess. Neurons situated in the former region were larger. The immunoreactive cell groups were shown to be located at sites differing from those of the AF-positive cell groups. The fibers of immunoreactive neurons extended in all directions within the brain and towards the spinal cord, some reaching the third ventricle and capillaries. Thus, FMRFamide-like immunoreactive peptides appear to function as neurotransmitters or neuromodulators and possibly also as neurohormones. FMRFamide-like immunoreactive material was rarely observed in the posterior neurohypophysis (neural lobe), but was noted to be present to a limited extent in the caudal part of the anterior neurohypophysis (median eminence). It would thus follow that FMRFamide-like immunoreactive neurons may not necessarily be related to the hypothalamo-neural lobe system, but may possibly be associated with the hypothalamoadenohypophysial system. The pineal body showed no FMRFamide-like immunoreactivity.     

Occurrence of unusual tubular invaginations of the plasma membrane in smooth muscle cells of the lamprey,Lampetra japonica

Numerous tubular structures were observed in the surface region of smooth muscle cells making up the vascular walls in the lamprey, Lampetra japonica; they were designated as surface tubules. The limiting membrane of the surface tubules was connected to the plasma membrane, allowing communication of the lumen of the tubule with the extracellular space. Tannic acid reacted with osmium, serving as an extracellular marker, penetrated into the tubules but not into the intracellular organelles, such as the endoplasmic reticulum and the Golgi complex. The surface tubules were grouped in longitudinal parallel rows, separated from each other by tubule-free areas where dense plaques were present. Each tubule was fairly cylindrical (approximately 60 nm in diameter) and often ramified into two or three branches with a blind end. Occasionally, these tubules were encircled by the sarcoplasmic reticulum which was located immediately beneath the plasma membrane. Similar tubules were also observed in the surface region of vascular endothelial cells and fibroblasts in the adventitial connective tissue. The possibility that the surface tubules in the present observations are analogous to the smooth muscle caveolae or the striated muscle T-tubule is discussed.     

Stellate cells storing retinol in the liver of adult lamprey,Lampetra japonica

Summary Distribution, localization and fine structure of the stellate cells in the liver of lamprey, Lampetra japonica, were studied during the spawning migration by use of Kupffer's gold-chloride method, fluorescence microscopy for vitamin A (retinol) and electron microscopy. The stellate cells in the lamprey liver differ in some of their properties from those in mammalian livers. Stellate cells which store abundant retinol in lipid droplets, occur not only in the hepatic parenchyma, but also in the dense perivascular and capsular connective tissue of the liver and in the interstitium of pancreatic tissue. In the hepatic parenchyma these cells are located perisinusoidally or along thick bundles of collagen fibrils. The stellate cells display a number of large retinol-containing lipid droplets, granular endoplasmic reticulum, tubular structures, dense bodies, Golgi complex, microtubules, and microfilaments. In the space of Disse, the stellate cells and extracellular fibrilar components such as collagen fibrils and microfibrils (11–12 nm in diameter) are intervened between the two layers of basal laminae. Differentiation and possible functions of the stellate cells in the lamprey liver are discussed.     

Membrane differentiation in the cytoplasmic-tubule system of the intestinal absorptive cells of the lamprey,Lampetra japonica

Summary Specific membrane differentiation occurs in the cytoplasmic-tubule system of the absorptive cells lining the mucosa of the lamprey anterior intestine. The absorptive cells are characterized by the presence of abundant mitochondria and a system of well-developed cytoplasmic tubules (120 nm in diameter). The cytoplasmic tubules open on to the basolateral cell surface and contain numerous lipoprotein particles (50–100 nm diam.) in their lumina. Lipoprotein particles are also observed in the endoplasmic reticulum and the Golgi complex, and they are transfered to the lateral intercellular space and lamina propria by way of the cytoplasmic tubules. Spirally-wound parallel rows of particles are found in the luminal surface of the cytoplasmic tubules. The rows are 17 nm apart and are wound spirally at a pitch of 210 nm. Freeze-fracture images of the tubule membranes also show spiral arrays of particles (9 nm in diameter) on the P-face, and complementary shallow grooves on the E-face. From these observations, it is suggested that the cytoplasmic-tubule system of the intestinal absorptive cells serves as a channel for the transport of synthesized lipoprotein into the interstitium, and is also the site of the ion and water exchange essential for the maintenance of ionic homeostasis.     

Demonstration by lectin cytochemistry of rod and cone photoreceptors in the lamprey retina

Summary Lectin cytochemical analysis was undertaken to examine the distribution of glycoconjugates associated with the short and long photoreceptor cells in the lamprey retina. Concanavalin A bound preferentially to the outer segment region of the short cells. Wheat germ agglutinin bound weakly to both long and short cells. The outer segment regions of the long cells were stained intensely with peanut agglutinin. Pretreatment with neuraminidase to remove sialic acid resulted in decreased binding of wheat germ agglutinin throughout the retina and increased binding of peanut agglutinin to the outer segment region of the short cells and the region of myoid process of the long cells. These results suggest that there is a difference in the distribution of glycoconjugate residues between the long and short cells. A rod-like character of the short cell and a cone-like character of the long are tentatively discussed. Lectin-binding patterns in other retinal regions is also examined.     

Photoreceptor cells and neural elements with long axonal processes in the pineal organ of the lamprey,Lampetra japonica,identified by use of the horseradish peroxidase method

Summary Horseradish peroxidase (HRP) was applied to the transected end of the pineal tract of the lamprey, Lampetra japonica. Distinct reaction products of HRP were observed in 2 types of cell other than ganglion cells. The first type of cell protrudes a knob-like process into the pineal lumen. This type of cell was clearly identified by electron microscopy as a photoreceptor cell; its outer segment was connected to the ellipsoid through a sensory cilium. The other type of cell was located among photoreceptor and supporting cells. The processes of these cells were thin and slender, and they obviously did not represent photoreceptor, supporting, or conventional ganglion cells. The present results indicate that, in the lamprey, some of the photoreceptor cells of the pineal organ project their axon-like processes toward the posterior commissure, but that there is also another type of cell displaying long axonal projections. HRP-containing cells were distributed randomly over the pineal organ and were occasionally also observed in the parapineal organ.     

Fine structural and volumetric changes of lamprey photoreceptor cells during light and dark periods

Summary Short photoreceptor cells of the lamprey retina exhibited a 30% increase in the width of the myoid process and a 20% increase in that of the axonal process during a 12-h light period, compared to the measurements obtained during a 12-h dark period. An increase in the amount of cytoplasm, dilation of ER cisterns, and swelling of the nucleus appeared to cause the enlargement of the myoid parts. Accumulation of synaptic vesicles occurred concurrently with a thickening of the axonal process. These morphological changes presumably represent a phase of the diurnal cycle and current synaptic activity of the short cell. By contrast, the long photorecpetor cell showed neither measurable changes nor any indication of retinomotor movement.     

Anti-lamprey retinal antibodies: immunohistochemistry on the retinas of several species of vertebrates

Summary Two monoclonal antibodies, H16 and B11, which were raised against lamprey retinal homogenate, were found to react with both short and long photoreceptor outer segments. On Western blotting of the retinal homogenate, both antibodies recognized a 40000 Da and a 80000 Da band. H16 antibody stained rod outer segments of all examined vertebrates, all cone outer segments of the turtle and chicken, and certain cone outer segments of the macaque. B11 antibody stained sub-mammalian rod outer segments and some mammalian cone outer segments, leaving all mammalian rod outer segments unstained. The epitope recognized by H16 antibody is considered to be located in a conserved or commonly inherited element of an outer segment-bound molecule, presumably rhodopsin. B11 antibody, on the other hand, seems to recognize a reactive group which has failed to be inherited by mammalian rod cells; why it recognizes all cone outer segments in the turtle and chicken and only a part of them in the cow, cat, and macaque, meanwhile ignoring all of them in the frog and fish, is subject to further study.A portion of this work is contained in a dissertation submitted by the first author, M.Y., in application for the Ph.D. degree at Yamagata University School of Medicine.     

Immunocytochemical localization of serotonin and photoreceptor-specific proteins (rod-opsin,S-antigen) in the pineal complex of the river lamprey,Lampetra japonica,with special reference to photoneuroendocrine cells

Summary The pineal complex of the river lamprey, Lampetra japonica, was examined by means of immunocytochemistry with antisera against serotonin, the precursor of melatonin, and two photoreceptor proteins, rod-opsin (the apoprotein of the photopigment rhodopsin) and S-antigen. Serotonin-immunoreactive cells were observed in both the pineal and the parapineal organ. The proximal portion of the pineal organ (atrium) comprised numerous serotonin-immunoreactive cells displaying spherical somata. In the distal end-vesicle of the pineal organ, the serotonin-immunoreactive elements resembled photoreceptors in their size and shape. These cells projecting into the pineal lumen and toward the basal lamina were especially conspicuous in the ventral portion of the end-vesicle. In addition, single serotonin-immunoreactive nerve cells were found in this location. Retinal photoreceptors were never seen to contain immunoreactive serotonin; amacrine cells were the only retinal elements exhibiting serotonin immunoreaction. Strong S-antigen immunoreactivity was found in numerous photoreceptors located in the pineal end-vesicle. In contrast, the S-antigen immunoreactivity was weak in the spherical cells of the atrium. Thus, the pattern of S-antigen immunoreactivity was roughly opposite to that of serotonin. Similar findings were obtained in the parapineal organ. The rod-opsin immunoreaction was restricted to the outer segments of photoreceptors in the pineal end-vesicle and parapineal organ. No rodopsin immunoreactive outer segments occurred in the proximal portion of the atrium. Double immunostaining was employed to investigate whether immunoreactive opsin and serotonin are colocalized in one and the same cell. This approach revealed that (i) most of the rodopsin-immunoreactive outer segments in the end-vesicle belonged to serotonin-immunonegative photoreceptors; (ii) nearly all serotonin-immunoreactive cells in the end-vesicle bore short rod-opsin-immunoreactive outer segments protruding into the pineal lumen; and (iii) the spherical serotonin-immunoreactive cells in the pineal stalk lacked rod-opsin immunoreaction and an outer segment. These results support the concept that multiple cell lines of the photoreceptor type exist in the pineal complex at an early evolutionary stage.     

Gonadotropin-like and adrenocorticotropin-like cells in the pituitary gland of hagfish, Paramyxine atami; immunohistochemistry in combination with lectin histochemistry

The pituitary system of the hagfish remains an enigma. The present study has aimed to detect possible adenohypophysial hormones in the pituitary gland of the brown hagfish, Paramyxine atami, by means of immunohistochemistry in combination with lectin histochemistry. Rabbit antisera raised against ovine luteinizing hormone (LH)β, proopiomelanocortin (POMC)-related peptides, and the growth hormone/prolactin family of tetrapod and fish species were used, and 25 kinds of lectins were tested. Three different types of adenohypophysial cells were revealed in the pituitary of brown hagfish. The first was stained with both anti-ovine LHβ and several D-mannose-binding lectins, such as Lens culinaris agglutinin and Pisum sativum agglutinin. This cell type predominated in the adenohypophysis in adults with developing gonads and thus appeared to be involved in the regulation of gonadal functions. The second was negative for anti-ovine LHβ but was stained with several N-acetylglucosamine-binding lectins, such as wheat germ agglutinin and Lycopersicon esculentum lectin. This cell type exhibited a weak positive reaction with anti-lamprey adrenocorticotropin (ACTH) and thus appeared to be related to POMC-like cells. The second cell type was found in the adenohypophysis regardless of the developmental state of the gonads. The third cell type was negative for both antisera and lectins. Since this cell type was numerous in juveniles and adults without developing gonads, most cells of this type were probably undifferentiated. These findings suggest that GTH and ACTH are major adenohypophysial hormones in the hagfish. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture (to M.N.).     

Secretion of lipoprotein particles by the cells of the kidney proximal segment in the migrating arctic lamprey,Lampetra japonica

Summary The cells of the kidney proximal segment of the migrating arctic lamprey, Lampetra japonica, contain particles of the same size, electron-density and intracellular location as particles identified by others as very low-density lipoproteins (VLDL) in the liver and intestine of teleost fishes and lampreys. These particles are synthesized within the cisternae of the smooth endoplasmic reticulum and elements of the Golgi complex. They are transferred to the lateral intercellular space and lamina propria by way of the Golgi vesicles and an intracellular channel system. Some particles are discharged into the lumina of the sinusoidal capillaries of the lamina propria. Although the physiological role of lipoprotein secretion in the renal proximal segment cells is unknown, the present observations provide morphological evidence that the kidney of the arctic lampreys synthesizes lipoproteins and releases them into the circulation at the time when they are undertaking their anadromous migration.     

Iodine distribution in the thyroid follicles of the hagfish,Eptatretus burgeri and lamprey,Lampetra japonica: Electron-probe X-ray microanalysis

Summary In the thyroid follicles of species of cyclostomes, a hagfish and a lamprey, the distribution of stable iodine was examined by electron-probe X-ray microanalysis. A high concentration of stable iodine, heterogeneously distributed, was observed in the follicular cells of hagfish thyroid follicles. In the lamprey a low concentration of iodine was seen in the follicular lumina. The relative values for stable iodine determined in this way corresponded to values obtained by a chemical analytical method.     

Evidence for the existence of serotonin-, dopamine-, and noradrenaline-containing neurons in the gut of Lampetra fluviatilis

Summary Monoamine-containing neurons in the gut of Lampetra fluviatilis are characterized by histochemical, electron microscopical and biochemical methods. Strongly yellow fluorescent, probably serotonin-containing intrinsic neurons are found along the entire length of the intestine. Their processes aggregate to form large bundles of mainly non-terminal axons, constituting a subepithelial fibre plexus. This subepithelial, ganglion cell comprising plexus is connected to a wide-meshed subserosal plexus which has ganglion cells of different size and few varicose, single axons. Intermingled with both plexus there occur — in the anterior and middle but not in the preanal portion of the lamprey intestine — scattered green fluorescent intrinsic perikarya, emanating faintly green fluorescent, poorly varicosed axons.The formaldehyde-induced neuronal fluorophores conform to serotonin (yellow fluorescent compound), noradrenaline, and dopamine (green fluorescent substance), as revealed in microspectrofluorimetric recordings. The electron microscopical analysis of the yellow fluorescent intrinsic neurons in the terminal hindgut shows nerve cell pericarya and axons equipped with a typical population of occasional small granular and many large granular vesicles (750–1600 Å). The number and opacity of cores of the small and the osmiophilia of the cores of the large granular vesicles are significantly increased following short-term treatment with 5,6-dihydroxytryptamine. Long-term treatment with 5,6- or 5,7-dihydroxytryptamine provokes severe signs of ultrastructure impairment and eventual degeneration in the supposed serotonin-containing axons, besides indications of piling-up of organelles in the non-terminal axons due to arrest of axonal transport.Chromatography of acid extracts from the lamprey intestine, gills and kidney reveals the presence of serotonin (besides another unidentified indoleamine) and dopamine and noradrenaline in the gut, but only dopamine in the brain. The detection of serotonin, noradrenaline and dopamine in the lamprey gut is confirmed by chemical determinations.The occurrence of intrinsic serotonin-, noradrenaline- and dopamine-containing neurons in the gut of Lampetra fluviatilis deviates from the established pattern of innervation of the vertebrate intestine and is considered to be a remnant of an autonomic innervation principle common in invertebrates.Supported by grants from the Deutsche Forschungsgemeinschaft.Supported by grants from the Swedish Medical Research Council (No. 14X-712 and 14X-56.The authors are indebted to Lilan Bengtsson, Gertrude Stridsberg, Eva Svensson and Rolf Frank for skilful technical assistance.     

Fine structure of the lateral-line organ of the common eel,Anguilla japonica

Summary The sensory epithelium of the lateral line organ of the common eel consists of two types of cells, (sensory and supporting). The sensory cell bears a kinocilium together with about 40 to 60 stereocilia on its surface. The kinocilium is situated either at rostral or at caudal margin of this cilial group. Such polarity of the cilial group of one cell is inverse to that of an adjacent cell.Two types of crystal-like inclusions exist in the sensory cells, consisting of granules 100 Å in diameter. Granules in one type are arranged regularly whereas those in the other rather irregularly.Two types of nerve endings exist at the base of sensory cells: one is predominant in number and contains few vesicles, accompanied by a dense spherical body surrounded by small vesicles in the sensory cell and the other is rare in number and contains many vesicles, accompanied by a small flat sac just beneath the plasma membrane of the sensory cell.The supporting cells contain numerous mitochondria, a well developed Golgi apparatus and rough-surfaced endoplasmic reticulum, and surround a sensory cell completely. Physiologic significance of some of these components is discussed.     

Studies on the surface coat of Paramecium aurelia

Summary The plasma membrane of Paramecium aurelia is covered with a ruthenium red stainable surface coat. Results obtained after digestion with pronase, trypsin and neuraminidase suggest the glycoprotein nature of this structure. Lipid extraction also affects the surface coat forming material. The results are consistent with the model proposed by Ginsburg and Kobata dealing with spatial configuration of the surface coat components.Authors are grateful to Mrs. D. Kucharczyk for very efficient technical assistence, to Mrs. Z. Kaminska for sectioning the material and Mr. A. Renski for help with the electron microscope service.     

The pronephros of the early ammocoete larva of lampreys (Cyclostomata,Petromyzontes): Fine structure of the renal tubules

Summary The renal tubules of the paired pronephros in early larvae (ammocoetes) of two lamprey species, Lampetra fluviatilis and Petromyzon marinus, were studied by use of light-, scanning- and transmission electron microscopy. They consist of (1) a variable number of pronephric tubules (3 to 6), and (2) an excretory duct. By fine-structural criteria, the renal tubules can be divided into 6 segments. Each pronephric tubule is divided into (1) the nephrostome and (2) the proximal tubule, the excretory duct consisting of (3) a common proximal tubule followed by (4) a short intermediate segment, and then by a pronephric duct composed of (5) a cranial and (6) a caudal section. The epithelium of the nephrostome displays bundles of cilia. The cells of the proximal tubule possess a brush border, many endocytotic organelles and a system of canaliculi (tubular invaginations of the basolateral plasmalemma). The same characteristics are encountered in the epithelium of the common proximal tubule; however, the number of these specific organelles decreases along the course of this segment in a posterior direction. In the intermediate segment, the epithelium appears structurally nonspecialized. The cells of the cranial pronephric duct lack a brush border; they have an extensive system of canaliculi and numerous mitochondria. The caudal pronephric duct is lined by an epithelium composed of light and dark cells; the latter are filled with mitochondria and the former contain mucus granules beneath the luminal plasmalemma. The tubular segments found in the pronephros are the same in structure and sequence as in the lamprey opisthonephroi. However, only the nephrostomes and proximal tubules occur serially in the pronephros, while the common proximal tubule, the intermediate segment and the cranial pronephric duct form portions of a single excretory duct.This paper is dedicated to the memory of Professor W. Bargmann, long-time editor of Cell and Tissue Research, the author of a splendid review on the structure of the vertebrate kidney and a master of German scientific writing.     

Intercellular junctions in the water-blood barrier of the gill lamella in the adult lamprey (Geotria australis,Lampetra fluviatilis)

Thin sections and freeze-fracture replicas of the water-blood barrier in the gill lamellae of adult lampreys (Geotria australis, Lampetra fluviatilis) demonstrate that the occluding junctions between epithelial pavement cells differ markedly from those between endothelial pillar cells in the structure and arrangement of their strands. The zonulae occludentes between pavement cells typically consist of complex networks of 4–6 strands, the mean number of which undergoes a small but significant decline when the animal is acclimated to seawater. In comparison, the occluding junctions between pillar cells are less elaborate and may represent maculae or fasciae, rather than zonulae occludentes. They do not apparently undergo a change when the animal enters saltwater. The results indicate that the main part of the paracellular diffusion barrier to proteins and ions is located in the epithelium rather than the endothelium. Communicating (gap) junctions are present between adjacent pavement cells, between pavement and basal cells and between pillar cells. These findings suggest that the epithelial cells and the pillar cells in the water-blood barrier of lampreys both form functional syncytia. The results are discussed in the context of ion-transporting epithelia in other aquatic vertebrates.This paper is dedicated to Professor H. Leonhardt on the occasion of his 75^th birthday     

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.     

Ultrastructure of the presumed ion-transporting cells in the gills of ammocoete lampreys,Lampetra fluviatilis (L.) and Lampetra planeri (Bloch)

Summary Mitochondria-rich cells were located in the interplatelet area of gill filaments from ammocoete Lampetra fluviatilis and L. planeri. The ultrastructure of this cell type differs from typical teleost chloride cells by the absence of a tubular, smooth endoplasmic reticulum (SER). This difference is discussed in relation to the presumed functions of the cell and to the evolutionary histories of lampreys and teleosts. It is concluded that the mitochondria-rich cell is responsible for the active uptake of ions by the ammocoete gill.