Immunocytochemical demonstration of the hypothalamo-hypophysial vasotocinergic system of Lampetra fluviatilis

Summary With the use of the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level, it was shown that the preoptico-hypophysial neurosecretory system of the adult migrating Lampetra fluviatilis is a vasotocinergic system. It does not synthesize vasopressin. The results are entirely consistent with earlier chromatographic and pharmacological indications that it produces little or no oxytocin-like peptide hormone. In the adenohypophysis, immunoreactive neurohypophysial peptidergic fibres are absent.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

The hypothalamo-hypophysial system of the lamprey,Lampetra fluviatilis L.

The proximal neurosecretory contact region (PNCR) of the lamprey, a homologue of the median eminence of tetrapods, was studied by light, fluorescence and electron microscopy. Paraldehyde fuchsin-positive neurosecretory fibers are seen mainly in the central part of the rostral subdivision of the PNCR. The Falck-Hillarp technique reveals a weak, mainly diffuse yellow-green fluorescence in the PNCR. The ultrastructure of the tanycyte layer of the PNCR is very similar to that in the neurohypophysis of the same species, although the funnel-shaped protrusions of the third ventricle in the rostral part of the PNCR are more frequent than in the neurophypophysis. Peptidergic A1 and A2 neurosecretory fibers are characterized by neurosecretory granules of 120-200 nm and 100-150 nm in diameter, respectively. Monoaminergic B type fibers contain granules 80-100 nm in diameter. Neurosecretory terminals and the vascular endfeet of tanycytes make contact with the basement membrane of the avascular connective tissue layer separating the PNCR from the hypophysial pars distalis. It is suggested that both peptide and monoamine neurohormones diffuse through the thick connective tissue septa into the underlying blood vessels which supply the pars distalis and thus affect the function of its glandular cells.     

The hypothalamo-hypophysial system of the lamprey,Lampetra fluviatilis L.

The distribution of monoaminergic structures was studied in the proximal neurosecretory contact region and neurohypophysis of the lamprey by light and electron microscopic radioautography. Only weak radioautographic reactions were found in the proximal neurosecretory contact region 1 h after injection of 3H-dopamine. High-resolution radioautography revealed some labeled neurosecretory terminals mainly in contact with the basement membrane of the connective tissue layer separating the proximal neurosecretory contact region from the hypophysial pars distalis. The number of silver grains as well as the number of neurosecretory terminals marked by the presence of labeled dopamine was much higher in the neurohypophysis of the same species. In the latter, labeled neurosecretory terminals were found in contact with the connective tissue layer containing blood vessels of the general circulation. Some neurosecretory terminals make synaptoid contacts with tanycyte perikarya and their basal processes. According to their ultrastructure and the size of their granules, the labeled neurosecretory terminals are identical with the B type terminals described in both neurohemal regions (transmission electron microscopy). No labeled neurosecretory terminals were observed in the proximal neurosecretory contact region and the neurohypophysis of lampreys treated with the serotonin precursor, 3H-5-hydroxytryptophan.     

Extraocular muscles in the lamprey, Lampetra fluviatilis L. I. Muscle fibres

Six extraocular muscles of the river lamprey, Lampetra fluviatilis L., were studied with the light and electron microscope. On the basis of morphology and histochemistry three types of muscle fibres were distinguished: thin, thick mitochondria-rich and thick multifibrillar fibres. In the thin fibres, 2.8-22.4 microns in diameter, myofibrils are distributed peripherally and show strong ATPase activity. The mitochondria are located paraxially. In the thick mitochondria-rich fibres, 19.4-31.0 microns in diameter, myofibrils are also located peripherally, whereas the central part of the fibre is densely packed with very numerous mitochondria possessing tubular cristae. Thick multifibrillar fibres, with a diameter similar to that of the former type, contain thin myofibrils scattered over the entire cross-section of the fibre. The activity of myofibrillar ATPase is lower in both types of thick fibres than in the thin ones. The tubules of the T system were observed frequently only in the thick multifibrillar fibres. The extraocular muscles of the lamprey are composed of large quantities of muscle fibres. Thin and thick fibres do not form separate layers, but are more or less uniformly distributed throughout the muscle. Many muscle fibres show structural features suggesting their degeneration.     

Visual cells and visual pigments of the lamprey,Lampetra fluviatilis

Summary The long and short photoreceptors in the lamprey retina possess similar cone-like outer segments where many disks are infoldings of the outer plasmic membrane. Following the treatment by the Hartwig's (1967) method, outer segments of the long receptors are stained red, and those of the short receptor are stained blue, like the cones and rods in higher vertebrates, resp. (Fig. 1). Microspectrophotometry has shown that the short cells contain P517₁ whereas the long receptors possess P555₁ (Fig. 3). Spectral sensitivity of the dark-adapted retina measured by electroretinographic b-wave and aspartate-isolated receptor potential, corresponds to P517 (Figs. 5, 8). Judging from the receptor potential, the short receptors do not saturate at high illuminances and contribute to the retinal function in photopic conditions as well (Fig. 7). Photopic ERG is of a typical cone-dominant shape (Fig. 4).It is concluded that the long photoreceptors of the lamprey retina are cones whereas the short cells should be regarded as a peculiar kind of rods which possess cone ultrastructure and can operate in scotopic as well as in photopic conditions.Abbreviation LRP late receptor potential     

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.     

Larval growth in the river lamprey, Lampetra fluviatilis

A series of 987 ammocoetes from the rivers Towy, Teme, and Taw have been identified as mainly L. fluviatilis (L.) on the basis of oocyte counts on female ammocoetes. The length frequency distributions for this material differs from either L. planeri or P. marinus in showing only three modes in addition to the young of the year and the length distribution of the final mode coincides with the length range for 119 metamorphosing and macrophthalmia stages of L. fluviatilis that have been found at the same sites. These animals measured from 80–117 mm in length and weights varied from 0.76–2.28 g. Metamorphosis is believed to take place in late summer and early autumn when in the majority of cases, the ammocoetes are four and a half years old. The evidence that the non-parasitic L. planeri has a longer larval life than the closely related parasitic L. fluviatilis is thought to have some significance in relation to the evolution of the brook lamprey species.     

Distribution of catecholamine-containing nerve cells in the walls of the major vessels of the lamprey, Lampetra fluviatilis

Using histochemical fluorescence technique, two types of adrenergic nervous cells, i.e. small with intense fluorescence and large with weak fluorescence, have been detected in tissues and walls of the main blood vessels in the lamprey Lampetra fluviatilis. The first type of cells is distributed along the whole vascular net concentrating mainly in branching nodes. The second type of cells was found only in the adventitial layer of the dorsal aorta and intestinal artery. Adrenergic nervous cells are also present in the walls of the main veins. Possibility of presence of catecholamines of different nature in the bodies of the nerve cells is discussed.     

The lamprey (Lampetra fluviatilis) erythrocyte; morphology,ultrastructure, major plasma membrane proteins and phospholipids,and cytoskeletal organization

The aim of this study was to characterize the erythrocyte of the lamprey (Lampetra fluviatilis), a primitive vertebrate. The lamprey erythrocyte predominantly has a non-axisymmetric stomatocytelike shape. It has a nucleus and a haemoglobin-filled cytosol with a few organelles and vesicular structures. Surprisingly, there is no marginal band of microtubules. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of isolated plasma membranes revealed a single band at the level of the human spectrin doublet. Major bands also occurred at approximately 175 kDa and comigrating with human erythrocyte actin (~ 45 kDa). The presence of spectrin, actin and vimentin was shown by immunoblotting. Band 3 protein, the anion exchanger in higher vertebrates, seemed to be highly deficient or lacking, as was also the case with ankyrin. Confocal laser scanning microscopy combined with immunocytochemical methods showed spectrin, actin and vimentin mainly to be localized around the nucleus, from where actin- and vimentinstrands extended out into the cytoplasm. Actin also seemed to be present at the plasma membrane. Phospholipid analyses of plasma membrane preparations showed the presence of the same four major phospholipid groups as in the human erythrocyte, although with higher and lower amounts of phospatidylcholine and sphingomyelin, respectively. The low fluorescein isothiocyanate conjugated annexin V binding, as monitored by flow cytometry, indicated that phosphatidylserine is mainly confined to the inner membrane leaflet in the lamprey erythrocyte plasma membrane.     

The intestinal blood circulation in the River lamprey, Lampetra fluviatilis

Serial sections, injections with india ink and latex, and observations on fresh material, have been used to determine the pattern of blood circulation within the intestine of larval and adult lampreys. Attention has also been paid to resolving the variable terms previously applied to many of the blood vessels, and to the possible functional significance of the differences found between the two life cycle stages. In the larva, the main arterial supply to the intestine consists of a typhlosolar artery, while the venous return is comprised of a posterior and a left and right anterior intestinal vein that usually unite before entering the liver. Although a typhlosolar artery is also present in the adult, the main venous return of the ammocoete is replaced at metamorphosis by a newly formed typhlosolar vein. Moreover, in the ammocoete a considerable amount of blood is discharged into the haemopoietic sponge-work of the typhlosole and the arterial supply to the intestine is poorly developed. By contrast, the typhlosolar sponge-work is lost in the adult and a more efficient arterial supply is developed within the lamina propria of the various intestinal regions. Furthermore, vascular couples are developed in the adult which facilitate the flow of blood in opposite directions in the intestinal wall. Since, during both life cycle stages, the arterial blood passes into tissue spaces, there is no true capillary network in the intestine and no evidence was found for the presence of a lymphatic system. It is suggested that the changes which take place in the intestinal blood supply and the internal structure of the gut during metamorphosis result in improvements both to the vascular system and to the assimilation efficiency.     

Cell division in the olfactory epithelium of the lamprey,Lampetra fluviatilis

Summary The turnover of cells within the olfactory epithelium of the lamprey Lampetra fluviatilis was investigated using tritiated thymidine followed by autoradiography. It was found that cell division occurred in three distinct regions of the olfactory lamellae. Two of these regions — a distal lamellar region and a proximal lamellar region occurred outside the sensory area proper, but appeared to contribute cells to the sensory area as well as giving rise to secretory or ciliated cells outside the sensory area. A third region of division occurrred at the base of the sensory area. Division of specialised basal or blastema cells in this region gives rise to cells that are confined to the sensory region of the lamellae. These findings are discussed in the light of previous studies on cell replacement within the olfactory epithelium.     

A golgi study on tanycytes and liquor-contacting cells in the posterior hypothalamus of the newt

Summary Golgi methods were employed to study neurons and ependymal tanycytes in the posterior hypothalamus of the newt. The tanycytes send a few coarse, spiny or barbed processes towards the pia mater. In the periventricular grey, the neurohistological methods show common neurons, ranging from a multipolar to a plumed organization, and abundant liquor-contacting cells. These cells, possibly neurons, give rise to a process that reaches the cerebro-spinal fluid, and terminates in a spindle-shaped swelling, with a thin thread at its tip. In other cells, the intraventricular endings are bulbous or finger-like. The occurrence of: (1) branches of the liquor-contacting process, running parallel to the infundibular surface; (2) infundibular processes which end at the base or between the ependymal cell bodies; and (3) axons coursing in the same position, all indicates that the subependymal layer is a site for complex intercellular relationships. The significance of liquor-contacting cells and tanycytes is discussed, in view of the possibility that they may represent part of a system for hypothalamic regulation in response to changes in the CSF.     

Population genetics of a cyclostome species pair, river lamprey (Lampetra fluviatilis L.) and brook lamprey (Lampetra planeri Bloch)

Horizontal agarose gel electrophoresis of 24 allozyme loci in four species of Central European lampreys (321 Lampetra planeri , 83 L. fluviatilis , 11 Eudontomyzon mariae and nine Petromyzon marinus ) was used to study the 'paired species' L. fluviatilis and L. planeri . The genetic differentiation of the anadromous river lamprey ( L. fluviatilis ) from the stationary brook lamprey ( L. planeri ) was within the range of ingroup differentiation of the latter, but L. fluviatilis exhibited much greater population cohesion over a more extended geographic range: G _ST = 0.0537 versus G _ST = 0.3398, N _em = 4.402 versus N _em = 0.4856, mean genetic among-stock distances D = 0.0047 versus D = 0.0257. L. planeri populations coexisting geographically with L. fluviatilis in the Rhine and Elbe river systems were genetically more cohesive than L. planeri stocks from the Danubian basin where L. fluviatilis is absent. Danubian L. planeri populations exhibit a lower degree of heterozygosity than brook lampreys from the Rhine river system, but comprise deeper genetic lineages ( G _ST = 0.4629 versus G _ST = 0.2434), despite being sampled from a much more restricted area. Isolation-by-distance is observed for L. planeri from the Danubian but not from the Atlantic drainage basins. Transspecific gene flow between L. planeri from Atlantic drainage basins and the long-distant migrating L. fluviatilis is inferred, raising doubt on the validity of two separate biospecies. E. mariae and P. marinus are clearly differentiated from Lampetra spp. at several allozyme loci.     

Locomotor activity of river lamprey Lampetra fluviatilis (L.) during the spawning season

At high latitudes (62–64°N) the river lamprey Lampetra fluviatilis (L.) is shown to have a 24-hr locomotor activity during its spawning period just before midsummer. By keeping lampreys in cold water, the spawning was delayed about one month. Analysis of event recorder graphs indicate that the 24-hr activity is caused by addition of diurnal spawning activity to the basic nocturnal activity and is not caused by weak Zeitgeber conditions during midsummer.Water temperature might indirectly determine time of spawning of the river lamprey.