Cerebrospinal fluid contacting neurons in the reduced brain ventricular system of the atlantic hagfish,Myxine glutinosa

Cerebrospinal fluid (CSF)-contacting neurons are sensory-type cells sending ciliated dendritic process into the CSF. Some of the prosencephalic CSF-contacting neurons of higher vertebrates were postulated to be chemoreceptors detecting the chemical composition of the CSF, other cells may percieve light as "deep encephalic photoreceptors". In our earlier works, CSF-contacting neurons of the mechanoreceptor-type were described around the central canal of the hagfish spinal cord. It was supposed that perceiving the flow of the CSF they are involved in vasoregulatory mechanisms of the nervous tissue. In the present work, we examined the brain ventricular system of the Atlantic hagfish with special reference to the presence and fine structure of CSF-contacting neurons. Myxinoids have an ontogenetically reduced brain ventricular system. In the adult hagfish (Myxine glutinosa) the lumen of the lateral ventricle is closed, the third ventricle has a preoptic-, infundibular and subhabenular part that are not connected to each other. The choroid plexus is absent. The infundibular part of the third ventricle has a medial hypophyseal recess and, more caudally, a paired lateral recess. We found CSF-contacting neurons in the lower part of the third ventricle, in the preoptic and infundibular recess as well as in the lateral infundibular recesses. No CSF-contacting neurons were found in the cerebral aqueduct connecting the subhabenular recess to the fourth ventricle. There is a pineal recess and a well-developed subcommissural organ at the rostral end of the aqueduct. Extending from the caudal part of the fourth ventricle in the medulla to the caudal end of the spinal cord, the central canal has a dorsal and ventral part. Dendrites of CSF-contacting neurons are protruding into the ventral lumen. Corroborating the supposed choroid plexus-like function of the wall of the dorsal central canal, segmental vessels reach a thin area on both sides of the ependymal lining. The perikarya of the CSF-contacting neurons found in the brain ventricles are mainly bipolar and contain granular vesicles of various size. The bulb-like terminal of their ventricular dendrites bears several stereocilia and contains basal bodies as well as mitochondria. Basal bodies emit cilia of the 9+0-type. Cilia may arise from the basal body and accessory basal body as well. The axons run ependymofugally and enter--partially cross--the periventricular synaptic zones. No neurohemal terminals similar to those formed by spinal CSF-contacting neurons of higher vertebrates have been found in the hagfish. We suppose that CSF-contacting neurons transform CSF-mediated non-synaptic information taken up by their ventricular dendrites to synaptic one. A light-sensitive role for some (preoptic?) groups of CSF-contacting neurons cannot be excluded.     

Plasma cells in adult Atlantic hagfish,Myxine glutinosa

Summary Hagfishes, the most primitive vertebrates, are of special interest for the evolution of immune responses. Eptatretus stoutii, the Pacific hagfish, is able to mount cellular and humoral immune responses but all attempts to demonstrate in them the presence of plasma cells have failed. In the present study we demonstrate for the first time plasma cells identifiable by ultrastructural criteria in the pronephros, a primitive lymphohaemopoietic organ, of Myxine glutinosa, the Atlantic hagfish.     

Ultrastructure of four types of striated muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary Four types of striated muscle fibers with distinctive ultrastructure were defined in the Atlantic hagfish (Myxine glutinosa, L.): white, intermediate, and red fibers of m. parietalis, and red fibers of m. craniovelaris.White fibers are thick, contain very few mitochondria and fat vacuoles, and possess distinct and separate myofibrils with thin Z-disks and distinct M-lines. Intermediate fibers are thinner, possess largely similar myofibrils that often are even better separated due to a higher content of fat vacuoles and especially mitochondria and glycogen granules. Red fibers of m. parietalis contain large amounts of mitochondria, fat vacuoles, and glycogen granules. Their myofibrils possess M-lines, and although branching more, the myofibrils of red fibers conform with a Fibrillenstruktur pattern like those of white and intermediate fibers. Red fibers of m. craniovelaris are very thin, possess many smaller fat vacuoles, and large amounts of mitochondria and glycogen granules. The myofibrils are significantly thinner than in m. parietalis fibers, run as quite independent well separated units, possess thicker Z-disks, and lack M-lines. Large amounts of myosatellite cells are associated with these red fibers.Triads are located near A/I-junctions in all four fiber types and occur irregularly, the density of triads being different in the various fiber types.We are indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and we also wish to thank Dr. Jan K. S. Jansen, Institute of Physiology, University of Oslo, for valuable suggestions during this study.     

Presence of a sodium-dependentd-glucose transport system in the kidney of the atlantic hagfish (Myxine glutinosa)

Summary A membrane fraction, rich in brushborder membranes, was prepared from the archinephric duct of the atlantic hagfish (Myxine glutinosa) and the uptake ofd-glucose and other sugars into the membrane vesicles was investigated by a rapid filtration technique. Uptake ofd-glucose was found to be sodium-dependent, phloridzin-inhibitable and osmotically sensitive. A sodium gradient dependent overshoot was demonstrated at 25° C as well as at the more physiological temperature of 4°C. The sodium dependentd-glucose transport was inhibited by -methyl-d-glucoside, but not by 2-deoxy-d-glucose. Furthermore at the same concentration of sugars the initial uptake ofd-glucose was 7.2-fold higher thanl-glucose uptake.d-glucose transport across the membrane in the presence of a sodium gradient was stimulated when SCN^– replaced Cl^– and inhibited when gluconate replaced Cl^–.d-glucose uptake in the presence of a sodium- and potassium gradient was decreased by the addition of valinomycin. In addition, the presence of ad-glucose gradient enhanced sodium uptake into the vesicles as compared to a mannitolgradient. Phloridzin inhibited thed-glucose dependent sodium flux. Thus an electrogenic stereospecific sodium glucose co-transport system, with properties similar to that found in the kidney of higher vertebrates is present in this primitive vertebrate and might participate in secondary-active sugar reabsorption in the archinephric duct.     

The semicircular canals of the hagfish Myxine glutinosa

The internal radius (r) and radius of curvature (R) of the single semicircular canals of Myxine glutinosa have unusual dimensions. In mammals and fish the increase in dimension of r and R with respect to body weight is small; in fish r is larger than in mammals of equivalent weight in order to increase the sensitivity of the canals to angular rotation and R increases correspondingly (Jones & Spells, 1963). In Myxine r is larger than in fish or mammals yet R is smaller. It is argued that the large internal radius is the result of the need to increase the sensitivity of a single canal which has to signal rotation in three planes while the small radius of curvature follows from the absence of a cupula. In order to verify that the cristae of the canals do respond to rotational velocity, recordings were obtained from the nerves serving the canals during rotation in the horizontal plane. The frequency response of several afferents recorded simultaneously at sinusoidal rotations between 0.25 and 2.0 Hz was in the form of a sine wave 90 in advance of head position, as would be expected of a velocity transducer. The gain of single afferents was an order of magnitude less than those reported for other vertebrates.     

Vascular bed-specific endothelium-dependent vasomomotor relaxation in the hagfish, Myxine glutinosa

The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates that lived some time more than 500 million years ago. Features that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. We recently reported that hagfish endothelium displays phenotypic heterogeneity in ultrastructure, lectin binding, and mechanisms of leukocyte adhesion. Thus, phenotypic cell heterogeneity evolved as an early feature of the endothelium. In the present study, we wanted to extend these observations by determining whether hagfish endothelium plays a role in mediating vasomotor tone. Response of mesenteric and skeletal muscle arteries to a variety of mediators was assayed by videomicroscopy. Phenylephrine and acetylcholine induced vasoconstriction of mesenteric and skeletal muscle arteries. Bradykinin (BK) and ADP promoted vasorelaxation in precontracted mesenteric arteries but not those from skeletal muscle. BK- and ADP-mediated vasorelaxation of the mesenteric artery was abrogated by mechanical denudation of the endothelium but was unaffected by N(G)-nitro-L-arginine methyl ester. Indomethacin significantly inhibited the vasodilatory response to ADP but not BK. The nitric oxide donor sodium nitroprusside resulted in endothelium-independent relaxation of both mesenteric and skeletal muscle arteries. Together, these data suggest that site-specific endothelium-dependent vasorelaxation is an evolutionarily conserved property of this cell lineage.     

Immunolocalization of Na+/K(+)-ATPase in mitochondrion-rich cells of the atlantic hagfish (Myxine glutinosa) gill

Using a monoclonal antibody for the alpha-subunit of the Na+/K(+)-ATPase, DASPEI (a vital mitochondria dye), and confocal laser scanning microscopy, the presence of Na+/K(+)-ATPase in mitochondrion-rich cells of the hagfish gill was confirmed. In addition, the level of Na+/K(+)-ATPase expression in the hagfish gill was compared to that of fishes with different osmoregulatory strategies (little skate, Raja erinacea and mummichog, Fundulus heteroclitus). Immunocytochemistry detected a high density of columnar cells expressing Na+/K(+)-ATPase in the afferent filamental epithelium. Positive cells were also found in the lamellar epithelium but at a much lower density. The distribution of DASPEI staining was similar to that of the Na+/K(+)-ATPase antibody, indicating that the enzyme is expressed in mitochondrion-rich cells. Immunoblot analysis confirmed the specificity of the antibody for the 97 kDa alpha-subunit of the enzyme. The immunoreactive band intensity for the Atlantic hagfish was similar to that of the little skate, but less than half that of the full-strength seawater mummichog. These results are discussed in relation to gill function in early craniates.     

Ultrastructure of motor nerve terminals on different types of muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary White and intermediate parietal muscle fibers of Myxine are innervated focally at one end. Most synaptic vesicles are empty. These terminals also contain 1–2% large 800–1.100 Å dense-core vesicles. Red fibers of parietal and craniovelar muscle are innervated in a distributed fashion, and the presynaptic profiles contain a higher number of large dense-core vesicles (averaging 9% and 15%, respectively; up to 37%). For all terminals the synaptic gap is 450–600 Å wide, and postsynaptic folds are absent.Empty synaptic vesicles exist as round or elongated profiles. The proportion of elongated profiles increases by formation from round ones when increasing the molarity of the buffer in the aldehyde fixative. Furthermore, the proportion of elongated vesicle profiles in terminals on Myxine white fibers at different buffer molarities, is identical with that in mammalian motor terminals at similar molarities. On this basis the significance and mode of formation of elongated vesicle profiles is discussed. The conclusion is made that the susceptibility of flattening depends on the osmotic pressure of the vesicle contents once the aldehyde has influenced the vesicle membrane.The different vesicle populations in terminals on different types of muscle fibers are significant. Terminals on red fibers probably contain serotonin (5-HT) either as sole transmitter or in addition to acetylcholine.The author is indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and to Mrs. Jorunn Line Vaaland for expert technical assistance.     

Intercellular junctions in the gill epithelium of the Atlantic hagfish,Myxine glutinosa

Summary The intramembrane organization of the occluding junctions in the gill epithelium of the Atlantic hagfish, Myxine glutinosa, was studied by means of freeze-fracture electron microscopy. Mitochondria-rich cells, characterized by assemblies of rod-shaped particles in the luminal plasma membrane and by an extensive intracellular amplification of the basolateral plasma membrane, are singly distributed between the pavement cells in the gill epithelium of this marine and stenohaline cyclostome. The occluding junctions between mitochondria-rich cells and pavement cells do not differ from those between adjacent pavement cells, concerning the number of superimposed strands (median 6, range 4–9) and their geometrical organization. These observations suggest that, in contrast to marine teleosts, the paracellular pathway plays a minor role in transepithelial ion movements in the hagfish gill epithelium. The findings are in agreement with the absence of hypoosmoregulatory mechanisms in hagfish, as have been evolved in various marine vertebrates. In addition, small communicating junctions are demonstrated between pavement cells; they possibly serve for a coordinated synthesis and secretion of mucus by the pavement cells.Dedicated to Prof. H. Leonhardt on the occasion of his 70th birthday     

Glomerular bypass shunts in the kidney of the Atlantic hagfish,Myxine glutinosa

Summary The vascular pathways associated with the glomerulus of the Atlantic hagfish, Myxine glutinosa have been studied by scanning electron microscopy of corroded resin casts of the vasculature. Although the overall pattern of the renal vasculature did not differ from earlier reports, a previously unreported vascular pathway which arose from the renal artery and bypassed the glomerular capillaries in 28% of glomeruli was clearly demonstrated. Glomerular bypass shunts either ran to join the loose capillary network around Bowman's capsule and thereby drain into the network of vessels associated with the mesonephric duct (ureter), or ran directly into the ureteral system of vessels and subsequently into the posterior cardinal veins. Glomerular bypass shunts which theoretically permit renal arterial blood to bypass the process of filtration may play a role in the regulation of body fluid volume.     

Angiotensin I-converting enzyme-like activity in tissues from the Atlantic hagfish (Myxine glutinosa) and detection of immunoreactive plasma angiotensins

Using a highly sensitive fluorimetric assay, significant levels of angiotensin I -converting enzyme-like activity (ACELA) were detected in a range of tissues (branchial heart, gill, kidney with associated vasculature and archinephric duct, liver, whole brain and gut) from the Atlantic hagfish (Myxine glutinosa). The highest ACELA occurred in heart and gill (1.8 and 1.5 nmol His–Leu min⁻¹ mg protein⁻¹, respectively). The mammalian angiotensin I-converting enzyme (ACE) inhibitor, captopril, at 10⁻⁵ M was a potent inhibitor of the ACELA found in all hagfish tissues. Radioimmunoassay showed that immunoreactive angiotensins (251.8±11.8 pM) were detectable in hagfish plasma. The validity of the assay for measurement of hagfish angiotensins was indicated by the parallelism of the angiotensin II standard curve against serially diluted hagfish plasma. Measurement of immunoreactive plasma angiotensins and detection of significant levels of ACELA in a wide range of tissues gives indirect evidence for the presence of a renin–angiotensin system in hagfishes, the earliest evolved group of craniates.     

Cell types in the adenohypophysis of the hagfish,Myxine glutinosa (Cyclostomata)

Summary The anterior pituitary of the Atlantic hagfish, Myxine glutinosa, was investigated by transmission electron microscopy. The cells of the adenohypophysis are arranged in follicles surrounded by connective tissue. Five cell types containing granules and one agranular cell type were identified. At the present state of the study the function of these cells remains open to discussion.This study was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (Grant no. 4070) and the Stiftungs- und Förderungsgesellschaft der Paris Lodron-Universität in Salzburg. The authors are grateful to the Director of the Biological Station in Drøbak (Norway), Amanuensis Finn Walvig, for procuring the material     

Seasonal concentrations of reproductive steroids in the gonads of the Atlantic hagfish, Myxine glutinosa

Changes in gonadal morphology, gonadal estradiol, and progesterone were examined in Atlantic hagfish, Myxine glutinosa, during a period of 17 months, beginning in April, 2001. Atlantic hagfish were captured from the ocean on a monthly basis. A total of 60 hagfish were divided into three different size classes of twenty hagfish each (small 20-35 cm, medium 35-45 cm, large 45-55+cm) and transported to the University of New Hampshire for sampling. Overall, in the medium and large size hagfish, estradiol and progesterone had significantly elevated peaks in January, 2001. There were significant increases in estradiol concentrations in January, with relatively low fluctuations in levels for the rest of the sampling period. Progesterone concentrations increased significantly in January, 2002, in medium and large hagfish, and remained elevated until June and April, 2002, for the two size classes respectively. The majority of hagfish sampled were females or hermaphrodites; few true males were identified in any of the samples. The number of females with large eggs increased following the estradiol peak in January and hermaphrodites with mature sperm were identified in the July, 2002, sample. These data represent the first evidence for a seasonal reproductive cycle in M. glutinosa and only the second seasonal reproductive cycle identified in any hagfish species.     

Orthogonal arrays of particles in the gill epithelium of the Atlantic hagfish,Myxine glutinosa

Summary Freeze-fracture replicas of hagfish gill epithelium revealed orthogonal arrays of 6-nm particles in the basolateral plasma membrane of pavement cells. The arrays consisted of 4–16 particles and occupied an area of 340±170 nm². These particle arrays, which are considered to be sites of ionic leakage, possibly contribute to the regulation of the pericellular micromilieu of adjacent mitochondria-rich cells.     

Immunoreactive endozepine-like peptides in the brain and pituitary of the Atlantic hagfish, Myxine glutinosa

Endozepines are a family of peptides capable of displacing benzodiazepines from their specific binding sites, to which belong the diazepam-binding inhibitor and the octadecaneuropeptide (ODN). This paper reports the distribution of ODN-related peptides, investigated for the first time by immunocytochemistry, in different brain and pituitary regions of the Atlantic hagfish, Myxine glutinosa. Immunoreactive ODN-like material was found in the telencephalon at the level of bundles of different olfactory nerve fibres. Moreover, at the level of the pallium, immunoreactive multipolar neurons were observed in the pars parvocellularis of the stratum griseum superficialis. Similar immunopositive nerve cell bodies were found in the nucleus medialis of the central prosencephalic complex. In the mesencephalon, few immunoreactive neurons lining and contacting the mesencephalic ventricle were detected; such nerve cells could be involved in the regulation of cerebrospinal fluid homeostasis. Dorsally in the mesencephalon, numerous ODN-containing cell bodies were present in the area praetectalis. The rhomboencephalon was immunostained only in the octavolateral area and in the nucleus motorius magnocellularis of the trigeminal nerve. Furthermore, ODN immunoreactivity was also present in the nerve cells of ganglia of the ophthalmic division of the trigeminal nerve complex. The immunocytochemical patterns described here in the brain of M. glutinosa suggest an involvement of ODN-like peptides as neuromodulators in sensory pathways, such as olfactory and visual. Finally, ODN-like substances were localized in discrete populations of adenohypophysial cells and in tanycytes lining the neurohypophyseal walls, suggesting for endozepines a paracrine and/or endocrine control of pituitary hormones release and a neurohormone role respectively. These results could give new insights into the chemioarchitecture of the brain of myxinoids.     

Natriuretic peptide binding sites in the brain of the Atlantic hagfish, Myxine glutinosa.

We have previously used immunohistochemistry to show that the brain of the hagfish, Myxine glutinosa, contains a rich distribution of natriuretic peptide-immunoreactive elements with the densest distribution occurring in the telencephalon and the diencephalon. In this study, the distribution of (125)I-rat ANP and (125)I-porcine CNP binding sites was determined in the brain of M. glutinosa. The binding pattern of (125)I-rat ANP and (125)I-porcine CNP showed similarities; however, some differences were observed in the olfactory bulb and the caudal brain regions. Specific (125)I-rat ANP and (125)I-porcine CNP binding was observed in the olfactory bulb, outer layers of the pallium, and in regions of the diencephalon. Very little specific binding was observed in the habenula and the primordium hippocampi. In the diencephalon, a distinct zone of specific (125)I-rANP binding separated a region of moderate binding in the lateral regions of the diencephalon from the thalamic and hypothalamic nuclei. Moderate levels of specific (125)I-rANP binding were observed in the mesencephalon and medulla oblongata; little or no (125)I-porcine CNP binding was observed in these regions. The data, in combination with previous immunohistochemical studies, show that the natriuretic peptide system of the hagfish brain is well-developed and suggest that natriuretic peptides have a long evolutionary history as neurotransmitters and/or neuromodulators in the vertebrate brain. J. Exp. Zool. 284:407-413, 1999.     

Velocity and displacement receptors in the skin of the hagfish Myxine glutinosa.

The skin of Myxine glutinosn contains both velocity-sensitive and displacement-sensitive mechanoreceptors. These can be distinguished from each other by the different time course of their response to skin indentation. Velocity receptors gave a brief response, consisting usually of three to four action potentials which could only be elicited by movement of the skin. No velocity receptor was spontaneously active. Displacement receptors, some of which were spontaneously active, gave a prolonged discharge which continued, with only slight adaptation, while the skin was indented. Velocity receptors code for velocity of indentation and continue to respond to repeated stimuli. Displacement receptor action potentials occur irregularly, with a Poisson-like distribution of interspike interval. The mean frequency of displacement receptor discharge increases with depth of skin indentation, while the interspike interval distribution becomes progressively biased towards shorter intervals. The threshold of displacement receptors is related to displacement velocity; sinusoidal indentations of the skin produce a minimum threshold around 60 Hz. These receptors fail to respond to oscillations in excess of 120 Hz. The structure of the skin is described. Stress-strain curves are given both for static and dynamic compression of the skin and these are discussed in relation to the properties of the skin mechanoreceptors.