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.     

The fine structure of the kidney of the hagfish (Myxine glutinosa L.)

Summary The fine structure of the kidney (glomerulus and archinephric duct) of the hagfish, Myxine glutinosa (L.) was studied in thin sections and by freeze-fracture technique.The glomerular filtration barrier is similar to that of mammalian kidneys. However, endothelial fenestrations are relatively scanty and the basement membranes of endothelial cells and podocytes always appear separated by a layer of collagen fibrils and microfibrils often surrounding numerous and extended mesangial cells. Between podocytes and their processes maculae occludentes and peculiar junctions of another type occur.The zonulae occludentes between epithelial cells of the archinephric duct are composed of five or more strands, occasionally of only one or two.Supported in part by Deutsche Forschungsgemeinschaft (SFB 146, STO B4) and NIH (ISOI-RR 05764).We are grateful to Dipl.-Ing. G. Wermbter for her helpful criticism and to Mr. H. Heidreich for his excellent technical assistance.     

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.     

Endocytosis in the ureteric duct epithelium of the hagfish (Myxine glutinosa L.)

Summary Solutions containing ferritin or thorotrast particles were microperfused through the ureteric duct of the hagfish. The markers were taken up by the epithelial cells by way of endocytosis and were transported in bulk in apical vesicles. Newly formed apical vesicles containing marker showed bristle coating on the cytoplasmic side of their limiting membrane. This coating appeared to be lost during the movement of vesicles deeper into the cytoplasm.The findings indicate that the epithelial cells in the ureteric duct have capablity for extensive bulk uptake of macromolecules from the luminal fluid. The mechanisms involved in absorption appear to be similar to those in proximal convoluted tubules of mammals.The apical dense tubules observed with some fixation techniques appear to represent collapsed endocytotic vesicles.The authors are indebted to Finn Walwig, Cand. real., Marine Biology Station, University of Oslo, Dröbak, Norway for kindly supplying the hagfishes used in this study. The technical assistance of Miss Signe Fjeldsenden and Miss Britt-Marie Pettersson is gratefully acknowledged.This work has been supported by grants from the Karolinska Institutet Medical School, Stockholm, Sweden (Therese och Johan Anderssons Minne).     

Myosatellite cells associated with different muscle fibre types in the Atlantic hagfish (Myxine glutinosa,L.)

Summary The incidence of myosatellite cells associated with white and red muscle fibres of the parietal muscle and red fibres of the craniovelar muscle was estimated by quantitative electron microscopy in the Atlantic hagfish (Myxine glutinosa, L.). Myosatellite cell nuclei constitute 3, 11 and 23 % of the total number of nuclei inside the basal lamina of the three types of muscle fibres, respectively. However, the total number of nuclei is highest in white fibres, most of the nuclei belonging to striated muscle cells. Myosatellite cell profiles in transverse sections constitute 23, 41 and 61 % of the number of muscle fibre profiles of the three types, respectively. The intervals between adjacent myosatellite cells are 135 m in white fibres, 55 m in red parietal fibres, and only 25 m in craniovelar fibres. Since craniovelar fibres are also comparatively thin, myosatellite cells constitute a significant fraction of the volume inside the basal lamina in these fibres. The myosatellite cells are 30–50 m long and up to 5 m thick. Some myosatellite cells possess few organelles, whereas others appear to contain many free ribosomes, granular endoplasmic reticulum, prominent Golgi apparatus and lysosome-like bodies.This investigation was supported by the Norwegian Research Council for Science and the Humanities (NAVF grant No. C20.30–37). The authors are indebted to Jorunn Line Vaaland and Berit Branil for technical assistance, and to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supplying the hagfish     

Fine structure of ureteric duct epithelium in the North Atlantic hagfish (Myxine glutinosa L.)

Summary The epithelial cells lining the ureteric duct in the cyclostome, Myxine glutinosa, have a brush border and show specializations of their apical cytoplasm similar to those observed in absorptive proximal tubule cells in higher vertebrate species. These features and the presence of large and numerous cytosomes, presumed to contain lysosomal enzymes, indicate that the ureteric epithelium has taken over some of the functions of the proximal tubule in the atubular kidney of Myxine. Sparsity of basal cytoplasmic processes and mitochondria in the ureteric duct cells appears to correlate with an inability for active, energy-dependent secretory and ion transport functions.This study has been supported by a grant from the Karolinska Institutet Medical School, Stockholm, Sweden (Therese och Johan Anderssons Minne). The author is indebted to Doctor Bertil Swedmark for permission to use the facilities of Kristineberg Marine Biology Station, Fiskebäckskil, Sweden, where hagfish were caught.     

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.     

Deiodination activity in extrathyroidal tissues of the atlantic hagfish, Myxine glutinosa

We measured low substrate (<1 nM) thyroid hormone (TH) deiodination activities in liver, muscle, intestine, and brain microsomes of Atlantic hagfish fasted for 2 weeks and found extremely low thyroxine (T(4)) outer-ring deiodination (T(4)ORD) and inner-ring deiodination (T(4)IRD) as well as 3,5,3'-triiodothyronine (T(3)) IRD activities. T(3)ORD, 3',5'-triiodothyronine (rT(3)) ORD and rT(3)IRD activities were undetectable. Hagfish deiodinating pathways resembled those of teleosts in requiring a thiol cofactor (dithiothreitol, DTT) and in their inhibition by established deiodinase inhibitors and by TH analogues. However, under optimal pH and DTT conditions intestinal T(4)ORD activity exceeded that of liver about 10-fold. This contrasts with the situation in teleosts but resembles that reported recently in larval and adult lampreys, suggesting the intestine as a primary site of TH deiodination in lower craniates.     

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.     

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     

Myomuscular junctions in the gill-sac muscle of the Atlantic hagfish,Myxine glutinosa: analogy with myotendinous junctions

Summary Myomuscular junctions between muscle fibers in the gill sacs of the Atlantic hagfish, Myxine glutinosa, were examined by electron microscopy. According to the presence of sarcolemmal differentiations typical of myotendinous junctions, the myomuscular junctions can be described as a symmetric myotendinous junction     

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.     

Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.)

Hagfish (Myxine glutinosa) secrete normal epidermal mucus and extruded slime. The epidermal mucus is produced continuously to prevent pathogen adherence while the extruded slime is observed predominantly during feeding, provocation or stress. To date little is known about the involvement of extruded slime in the physiological functions of hagfish. In this preliminary study, innate immune enzymes and the protein composition of hagfish normal epidermal mucus and extruded slime were analysed and compared. The lysozyme specific activity of hagfish was observed approximately two-fold higher in extruded slime than that of epidermal mucus. The extruded slime had approximately 3.5-5.0 fold increased levels of alkaline phosphatase, cathepsin B and proteases in comparison to epidermal mucus. Protease characterization using specific inhibitors showed that the extruded slime had higher levels of serine trypsin-like proteases compared to metalloproteases whereas epidermal mucus showed equal proportion of both serine and metalloproteases. SDS-PAGE analysis showed high levels of three proteins with molecular masses in the range of 13-16kDa in the extruded slime. The LC/MS/MS analysis of protein bands 1, 2 and 3 showed closest matches to hemoglobulin-3, histone H3 and H2B proteins, respectively. The observation of elevated levels of innate immune parameters in the extruded slime suggested that the extruded slime has a significant role in innate immunity of hagfish against infectious pathogens.     

Confocal imaging and phylogenetic considerations of the subcutaneous neurons in the Atlantic hagfish Myxine glutinosa

We used confocal microscopy and immunohistochemistry to characterize the morphology of the subcutaneous neurons and the innervation of the slime glands and striated muscles in the hagfish Myxine glutinosa. A rich plexus of 5HT‐, ChAT‐ and TH‐positive neurons is described in the capsule of the slime glands. These neurons, like those of the subcutaneous plexus, receive pericellular terminations from the axons of central cells. Capsular neurons receive innervation from 5HT‐positive and nNOS‐positive nerve fibres. Other nerve endings belonging to two separate nerve populations are identified in the striated muscles. They contain TH and nNOS immunoreactivity. Due to the lack of any topographical labelling, the cell origin and the projections of the neurons into the cranial and spinal nerves are unknown. This study provides anatomical evidence of multiple (5HT and nNOS) peripheral innervation of the neurons. However, it does not provide information about the function of these neurons in the hagfish. We suggest that hagfish neurons have a phylogenetic relationship with the spinal group of the dorsal cells of lampreys and the supramedullary cells of teleosts.     

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     

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.