Raja Erinacea Research Articles

Short-latency interneurons in the dorsal nucleus of the little skate, Raja erinacea

The elasmobranch electrosensory system is the most thoroughly understood of the non-teleost electrosensory systems and is useful for studying central nervous system mechanisms for the separation of behaviorally relevant signals from self-generated noise. In the little skate, Raja erinacea, the electrosensory primary afferents are responsive to electrical potentials created during the animal's own ventilation, while second-order neurons in the dorsal nucleus of the medulla suppress responses to ventilatory potentials (self-generated noise) but retain their extreme sensitivity to electric signals in the environment. The selective suppression of ventilatory noise in second-order cells is due in part to the fact that ventilatory potentials stimulate all receptors equally and simultaneously. The neuronal circuitry mediating rejection of such `common mode' signals in the dorsal nucleus likely includes inhibitory interneurons. This study describes physiological and anatomical characteristics of a group of dorsal nucleus interneurons that are distinguished from previously described interneurons by their shorter orthodromic activation latencies and higher spontaneous firing rates. The interneurons show sustained responses to an external dipole stimulus and respond well during simultaneous activation of all afferents by a `common mode' stimulus. Intracellular labeling indicates that the short-latency interneurons are located in the central and peripheral zones of the dorsal nucleus and the extent of their labeled processes is limited to the projection area of afferents from a single ampullary cluster. These features are consistent with a hypothesized role for these interneurons in inhibiting second-order cells, including inhibition which contributes to common mode rejection.

Characteristics of renal Na(+)-D-glucose cotransport in the skate (Raja erinacea) and shark (Squalus acanthias).

We have investigated the properties of the skate (Raja erinacea) and shark (Squalus acanthias) kidney Na(+)-D-glucose cotransporters (SGLT) in uptake studies of radiolabeled substrates into isolated renal brush-border membrane vesicles (BBMV). Scatchard plot analysis of the substrate dependence revealed that the Na(+)-D-glucose cotransporter population is homogenous within each species. Skate BBMV showed a relatively high affinity for D-glucose [Michaelis constant (K(m)) = 0.12 mM] with an apparent coupling ratio of approximately 2 Na+ to 1 D-glucose, whereas the shark transporter was much lower in affinity (K(m) = 1.90 mM) and had a lower coupling ratio, more like 1 Na+ to 1 D-glucose. These characteristics resemble the properties of SGLT1 and SGLT2, which are known to coexist in the mammalian kidney. Inhibitor studies using sugar analogs and glucosides suggested structural differences of the D-glucose binding site among these transporters, whereas the hydrophobic transporter domains in the vicinity of the D-glucose binding site appeared to be similar. In the high-affinity skate system, D-glucose was recognized by hydrogen bonds to the hydroxy groups at C-2, C-3, and C-4 and by hydrophobic interaction with the C-6 methylene group. In contrast, the low-affinity shark system seemed to lack the hydrophobic recognition motif for the C-6 methylene group of D-glucose.

Molecular cloning of a rod opsin cDNA from the skate retina

Skates ( Raja erinacea and R. ocellata) are among the few animals that have an exclusively rod retina. However, skate rods are unusual in that they are capable of adapting to extremely high levels of illumination that initially saturate the rod photocurrent. This adaptive process restores the ability of the visual cells to respond to incremental photic stimuli and enables them to function under ambient conditions that are subserved by the cone mechanism in mixed (rod/cone) retinae. As a first step towards exploring the molecular basis of visual adaptation in the skate retina, we have cloned and analyzed the opsin cDNA from a skate retina library. The cDNA codes for a protein 354 amino acids (aa) long and 39.7 kDa predicted molecular mass, and labels a single abundant transcript of 1.7 kb in retinal RNA. Amino acid alignments and a parsimony analysis of nucleotide alignments show the skate opsin to be homologous to other rod opsins. An analysis of the aa sequence reveals a high degree of conservation of those residues thought to be important for most aspects of rhodopsin function. However, a few critical aa replacements may indicate alterations in the interactions of skate rhodopsin with other proteins in the phototransduction cascade. In particular, replacements of Glu 150 with serine and Cys 323 with leucine are in cytoplasmic domains thought to interact with transducin and rhodopsin kinase. The latter change eliminates one of the conserved acylation sites in the carboxyl terminal tail. These substitutions increase the similarity of the cytoplasmic domains of skate opsin to those of blue-sensitive visual pigments.

Redescription of Ellobiophrya brevipes (Laird, 1959) N. Comb. (Ciliophora, Peritrichia) and the Fine Structure of its Pellicle and Cinctum

. A species of peritrich that attaches to gills of the skate, Raja erinacea, was identified by its original describer as a member of Caliperia, a genus characterized by having a noncontractile skeletal rod within the arms of its cinctum and by not having the cinctal arms bonded to one another at their tips. Our observations of the living ciliates confirmed by protargol impregnation and electron microscopy revealed that their cinctal arms are linked by a bouton and that the cytoskeletal structure within them has the fine structure of a myoneme. These characteristics place this peritrich unequivocally in the genus Ellobiophrya and it is thus renamed Ellobiophrya brevipes (Laird, 1959) n. comb. Clumps of epithelial cells clasped by the cincta of E. brevipes show damage at their bases but not on their luminal surfaces. The known species of Ellobiophrya are compared for significant structural differences that separate species of this genus.

Effect of MS-222 on response to light and rate of metabolism of the little skate Raja erinacea

The effect of tricaine methanesulfonate (MS-222) on the standard (SMR) and routine (RMR) metabolic rates of Raja erinacea was estimated from oxygen-consumption measurements. Data were gathered from a computerized, flow-through respirometry system. Individual trials were run for 96 h at 10 °C on anesthetized and untreated fish. The resulting rates, SMR = 20.1 ± 1.99 SE and RMR = 48.3± 2.5 SE mg O2 kg−1 h−1, for an unanesthetized skate of 0.5 kg standardized weight, are the lowest reported for any elasmobranch. Periodogram analysis revealed a significant light-response component to the oxygen consumption of these fish, indicating a pattern of nocturnal and crepuscular activity. This activity pattern was disrupted in skates subjected to anesthesia. The use of low-dosage MS-222 in conjunction with the respirometry trials provides positive preliminary evidence that this technique may be useful in giving quick, accurate estimates of SMR in the more intractable elasmobranch fishes.

Urea transport across the cell membrane of skate erythrocytes

The aim of this study was to determine the mechanism of urea transport by the erythrocyte cell membrane of the little skate, Raja erinacea. Radioisotope tracer studies demonstrated that urea transport is bi-directional and is not saturable. Experiments using urea homologues and analogue indicated that urea transport was not subject to competitive inhibition. Phloretin and p-chloromercuric phenylsulfonic acid (pCMBS), two inhibitors of facilitated diffusion, did not block urea transport. All these studies support the hypothesis that urea traverses the membrane via simple diffusion. Therefore, we determined whether urea enters the cell through a channel/pore or through the lipid bilayer. Studies were done using a series of urea derivatives of different molecular size and lipid solubility. Two techniques which determined hemolysis time and extent of hemolysis indicated that despite differences in size, the limiting factor in the transport of urea (and its homologues and analogues) is lipid solubility. These studies suggest that urea is transported by simple diffusion through the lipid phase in the skate erythrocyte membrane. J. Exp. Zool. 277:275–282, 1997. © 1997 Wiley-Liss, Inc.

Distinct but overlapping populations of commissural and GABAergic neurons in the dorsal nucleus of the little skate, Raja erinacea.

In the little skate, Raja erinacea, the electrosensory primary afferents are responsive to electrical potentials created during the animal's own ventilation, while second-order electrosensory cells in the dorsal nucleus of the medulla suppress the responses to ventilatory potentials but retain their extreme sensitivity to important environmental electric signals. Previous electrophysiological studies indicate a role for a commissural pathway between the bilateral dorsal nuclei in ventilatory noise suppression. In the present study, retrograde tracers were used to label dorsal nucleus commissural cells. Large round or triangular and thin elongate commissural cells occur in the central zone of the dorsal nucleus where the primary afferent fibers terminate. Elongate commissural cells also occur in the peripheral zone which is the cell body area of the major efferents of the dorsal nucleus. Immunohistochemical studies indicate that stellate cells of the molecular layer and round or triangular cells of the central zone comprise the GABA-immunoreactive cell groups of the dorsal nucleus. A subpopulation of the round commissural cells in the central zone are GABA-immunoreactive and may be candidates for mediators of common-mode noise rejection in the dorsal nucleus of skates. The non-GABAergic commissural cells may mediate crossed inhibition through an inhibitory transmitter other than GABA or may supply crossed excitation to the dorsal nucleus.

A role for GABAergic inhibition in electrosensory processing and common mode rejection in the dorsal nucleus of the little skate, Raja erinacea.

The electrosensory primary afferents in elasmobranchs are responsive to electric potentials created by the animal's own ventilation, while the second-order neurons (AENs) which receive this afferent input in the medulla suppress responses to ventilatory potentials but retain their extreme sensitivity to electric signals in the environment. Ventilatory potentials are common mode signals in elasmobranchs and a common mode rejection mechanism is one way the AENs suppress ventilatory noise. By pressure injecting the GABA-A receptor antagonist SR95531 while extracellularly recording from AENs, we tested the hypothesis that the subtractive circuitry that selectively reduces common mode signals in AENs utilizes GABA, and that a GABAergic component of the dorsal nucleus commissural pathway mediates crossed inhibition of AENs. Local application of SR95531 increased the spontaneous activity and the responsiveness of AENs to electrosensory stimuli. AEN responses to a common mode stimulus were selectively increased compared to responses to a localized stimulus due to SR95531 application. Contralateral inhibition of AENs was blocked by SR95531, indicating that GABAergic commissural cells may inhibit AENs when the contralateral side of the body is stimulated, as with common mode stimulation. We conclude that GABAergic inhibition contributes significantly to the shaping of AEN responses including common mode rejection.

Chloride and taurine effluxes occur by different pathways in skate erythrocytes.

The aim of this study was to determine whether volume-activated taurine and Cl- effluxes occur via the same system in skate (Raja erinacea) red blood cells (RBC). The effluxes were measured in isotonic and hypotonic elasmobranch Ringer solutions, in which NaCl was replaced by mannitol and the remaining exchangeable anions with gluconate. Methazolamide (0.1 mM) was added to minimize HCO3- formation. RBC Cl- content fell approximately 50%/h in both isotonic and hypotonic media, with no detectable K- loss in either medium. The observed Cl- loss was accompanied by an increase in pH. Both the Cl- loss and pH rise were inhibited by 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM), suggesting that Cl- efflux was due to H(+)-Cl- cotransport. 36Cl- effluxes in isotonic and hypotonic media were (means +/- SE, n = 11) 2.8 +/- 0.6 and 3.5 +/- 0.9 mumol.g dry wt RBC-1.min-1, respectively, whereas [3H]taurine effluxes in the same media were 0.045 +/- 0.02 and 2.1 +/- 0.05 mumol.g dry wt RBC-1.min-1, respectively (n = 6). These results indicate that taurine and Cl- effluxes occur via different pathways in skate RBC. In addition, the swelling-activated Cl- channel reported in epithelial cells does not appear to be present in skate RBC. This conclusion was confirmed by Western blots with an antibody to swelling-activated Cl- channels. Taurine and Cl- fluxes are apparently under different pathway influences in these RBC: taurine diffuses via a channel, whereas Cl- is transported by cotransporters.

Culture of ocular lens cells of the little skate,Raja erinacea

Culture of ocular lens cells of the little skate,Raja erinacea

Disruption of Cell Volume Regulation by Mercuric Chloride Is Mediated by an Increase in Sodium Permeability and Inhibition of an Osmolyte Channel in Skate Hepatocytes

The mechanism by which mercury leads to cell swelling and impairs the normal regulatory volume decrease (RVD) in cells swollen in hypotonic media was examined in hepatocytes isolated from the little skate,Raja erinacea,an osmoconforming marine elasmobranch. Skate hepatocytes treated with 50 μMHgCl2in isotonic medium swelled to volumes double those of control cells, and this was associated with an increase in Na+and K+permeability. The gain in intracellular Na+exceeded the K+loss by 0.27 μEq/mg protein, accounting in large part for the observed cell swelling. The effects of mercury were blunted when hepatocytes were incubated in medium in which the Na+was replaced with K+, and were essentially absent when Na+was replaced with choline+, indicating an important role of Na+influx in mediating mercury's effects on cell volume regulation. The inhibition of RVD by mercury was prevented if the metal was administered as a mercaptide with dithiothreitol or glutathione. However, when these chelating agents were added after the mercury, only the membrane permeant dithiothreitol was able to reverse the inhibition of RVD, suggesting an intracellular site of action. Mercuric chloride also produced a concentration-dependent inhibition of the ATP-sensitive volume-regulatory osmolyte channel in skate hepatocytes, as assessed by inhibition of swelling-activated [14C]taurine efflux. [14C]Taurine efflux was inhibited at mercury concentrations (20–40 μM) that had no effect on intracellular ATP levels or ATP/ADP ratios, consistent with a direct interaction with the channel. These findings indicate that mercury impairs cell volume regulation in skate hepatocytes at multiple sites, including the volume-regulatory osmolyte channels, and Na+and K+permeability pathways. The combined effects of increased Na+influx and the inability to extrude organic osmolytes may account for the inhibition of RVD.

High Affinity Binding of Ankyrin Induced by Volume Expansion in Skate Erythrocytes

Volume expansion of little skate (Raja erinacea) erythrocytes increases the affinity of ankyrin binding without altering in the number of binding sites. Potassium iodide-stripped inside-out vesicles (KI-IOV) were used to assess ankyrin binding under volume-expanded conditions. Under isoosmotic conditions, ankyrin binds nearly exclusively to a single class of sites (Bmax, 52 +/- 12 microg/mg; Kd, 150 +/- 28 nM). KI-IOV from volume-expanded cells (either with one-half osmolarity medium or with inclusion of the permeant solute ethylene glycol) demonstrate two ankyrin-binding populations. A high affinity population occurs transiently under volume-expanded conditions. This population has a Bmax of 18 +/- 7 microg/mg and a Kd of 25 +/- 9 nM. Total binding of high and low affinity sites is 57 +/- 17 microg/mg. This change in ankyrin affinity is reversible on volume regulatory decrease. A major target protein in the KI-IOV was identified as the skate homolog of the mammalian red cell anion exchanger band 3. Inclusion of the purified cytoplasmic domain of band 3 competes away more than 80% of the ankyrin binding. To determine whether increased ankyrin affinity is due to band 3 tetramer formation that occurs in volume expansion, cells were treated with pyridoxal 5-phosphate or 4,4'-dinitrostilbene-2,2'-disulfonic acid, two agents that increase tetramer formation under isoosmotic conditions. Both treatments altered the binding affinity with a shift toward higher affinity binding without significant alteration in the number of binding sites.

Organic osmolyte channels: Transport characteristics and regulation

Erythrocytes of the skate (Raja erinacea) exposed to hypotonic stress swell and then undergo a volume regulatory decrease by releasing taurine and other osmolytes. Previous studies showed that taurine release occurs via a volume-activated, Na(+)-independent, bi-directional transporter that has the properties of a size-limited channel. We now report on the transport characteristics of this channel and its regulation. Kinetic, competition and inhibitor studies indicate that polyols (myo-inositol) and trimethylamines (betaine) are transported by the same channel as taurine. Although the identity of the channel is still unknown a variety of evidence suggests that band 3 is involved in either channel formation or regulation. Hypotonicity causes phosphorylation and structural changes in band 3. Under isotonic conditions band 3 is predominantly in the dimeric form. Hypotonicity causes a shift to tetrameric band 3. We hypothesize that the band 3 tetramer either forms or regulates an osmolyte channel. The finding that expression of band 3 protein increases osmolyte channel activity in Xenopus oocytes supports this hypothesis.

Active microtubule-dependent secretion of a fluorescent bile salt derivative in skate hepatocyte clusters.

Fluorescence microscopy and video image analysis were used to study the transport of a fluorescent bile acid derivative [N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl-2-aminoethanesulfonate (NBD-TC)] in isolated clusters of hepatocytes from the little skate Raja erinacea. Analysis of images of hepatocyte clusters that were incubated in medium with 0.5-1 microM NBD-TC showed that the fluorescent derivative accumulated in the cells and that the clusters retained a patent canalicular lumen as well as the ability to actively transport the bile acid derivative from the cells into the lumen; i.e., the lumen-to-cell fluorescence ratio greatly exceeded unity. NBD-TC uptake by hepatocytes was inhibited by several organic anions, of which taurocholate was the most effective. Uptake was also blocked by metabolic inhibitors and by incubation in the cold. Neither Na replacement nor increased medium K, which depolarizes the membrane electrical potential [potential difference (PD)], affected NBD-TC accumulation by hepatocytes. Transport of NBD-TC into the canalicular lumen was inhibited by incubation in the cold and was substantially reduced by high-K medium; these blocks were removed by warming and transfer to normal-K medium, respectively. Treatment of hepatocytes with 20-40 microM nocodazole, a drug that reversibly depolymerizes microtubules, reduced cellular NBD-TC accumulation and blocked its secretion into the canalicular lumen; nocodazole effects were reversed by washing the hepatocyte clusters in drug-free medium. Thus uptake of NBD-TC by skate hepatocytes is active and carrier mediated but not dependent on the PD or Na gradient. NBD-TC secretion from cell to canalicular lumen also appears to be active and carrier mediated. Canalicular secretion appears to be driven at least in part by the PD and is highly dependent on an intact microtubular system in this marine species.

Characterization and function of ATP receptors on hepatocytes from the little skate Raja erinacea.

Hormonal regulation of hepatocytes, via cytosolic Ca2+ signaling, is well established in higher life forms but has not been investigated in elasmobranchs. We therefore examined Ca2+ signaling in hepatocytes isolated from the little skate, Raja erinacea. In hepatocyte populations, ATP induced a rapid, biphasic increase in Ca2+, as it does in mammalian hepatocytes. Other hormones that act on mammalian hepatocytes, such as vasopressin, angiotensin, and phenylephrine, induced no such Ca2+ increase. The initial phase of the ATP-induced Ca2+ increase was seen even in Ca(2+)-free medium, whereas the late sustained phase of the increase was not. Similar dose-response curves were obtained by stimulation with ATP, ADP, UTP, and 2-methylthio-ATP. In contrast, AMP, adenosine, beta, gamma-methyl-ATP, CTP, and GTP induced little or no Ca2+ increase. In single hepatocytes, ATP, ADP, UTP, and 2-methylthio-ATP each induced a sustained increase in Ca2+ at high concentrations, but at low concentrations induced Ca2+ oscillations. A maximal concentration of ATP (100 microM) caused a marked, transient increase in bile flow in the isolated perfused skate liver, whereas 100 microM adenosine had no such effect. These findings demonstrate that skate hepatocytes possess P2 nucleotide receptors that link to intracellular plus extracellular Ca2+ mobilization, which in turn regulates bile secretion. The broad specificity of the response to ATP and related compounds suggests either that multiple types of P2 receptors are expressed by skate hepatocytes or else that these cells possess a single primitive nucleotide receptor from which other P2 subtypes subsequently evolved.

Motor corollary discharge activity and sensory responses related to ventilation in the skate vestibulolateral cerebellum: implications for electrosensory processing

The dorsal granular ridge (DGR) of the elasmobranch vestibulolateral cerebellum is the source of a parallel fiber projection to the electrosensory dorsal nucleus. We report that the DGR in Raja erinacea contains a large percentage of units with activity modulated by the animal's own ventilation. These include propriosensory and electrosensory units, responding to either ventilatory movements or the resulting electroreceptive reafference, and an additional population of units in which activity is phase-locked to the ventilatory motor commands even in animals paralyzed to block all ventilatory movements. A principal function of processing in the dorsal nucleus is the elimination of ventilatory noise in second-order electrosensory neurons. The existence of these ventilatory motor corollary discharge units, along with other DGR units responsive to ventilatory movements, suggests that the parallel fiber projection is involved in the noise cancellation mechanisms.

Two classes of bipolar cell in the retina of the skate Raja erinacea.

We have used immunoreactions against serotonin and protein kinase C to visualize two distinct classes of bipolar cell in the all-rod retina of the skate, Raja erinacea. To enhance the immunoreaction in serotonin-accumulating bipolar cells, prior to fixation, some retinas were incubated in Ringer's solution containing serotonin and pargyline. We found the somata of serotonin-accumulating bipolar cells to be located slightly distal to the midline of the inner nuclear layer. With increasing eccentricity from the visual streak, the size of the perikarya increases, concomitant with a decline in density of their distribution. Dendrites emanate from stout primary stalks and branch out before reaching the outer plexiform layer. Axons are bistratified within the inner plexiform layer with ramifications at the border of strata 1 and 2 and in stratum 4. The overall morphology of serotonin-accumulating bipolar cells is similar to that of serotonin-accumulating OFF bipolar cells of other non-mammalian vertebrates. Protein kinase C immunoreactive cells display the typical appearance of rod bipolar cells. Somata of protein kinase C immunoreactive bipolar cells are spindle-shaped and located distal to the serotonin-accumulating bipolar cells. Dendrites of these bipolars do not ramify before reaching the outer plexiform layer. Thin axons of protein kinase C immunoreactive bipolar cells end in large, club-shaped terminals in stratum 5 of the inner plexiform layer, bearing a striking similarity to axon terminals of mammalian ON rod bipolar cells. Our findings suggest that the all-rod retina of the skate contains at least two distinct vertical pathways including an OFF bipolar cell pathway in addition to a classical rod ON bipolar pathway.

Swelling-activated anion conductance in skate hepatocytes: regulation by cell Cl- and ATP.

Cell swelling activates an outwardly rectifying anion conductance in mammalian cells. The channel responsible for this conductance mediates volume-regulatory efflux of organic osmolytes such as taurine. We observed a similar conductance in hepatocytes from the skate Raja erinacea. Whole cell Cl- conductance was increased > 100-fold by a 2-fold increase in hepatocyte volume. The conductance was outwardly rectifying and had a relative cation permeability of approximately 0.2. Cation permeability was increased by reductions in patch pipette CsCl concentration, suggesting that the channel pore contains saturable anion and cation binding sites with different anion and cation affinities. The conductance had a broad anion selectivity and a relative taurine permeability of 0.17. Activation of the conductance required intracellular ATP or a nonhydrolyzable ATP analogue. Elevation of intracellular Cl- from 20 to 155 mM reduced current activation while the rate and extent of cell swelling were unaffected. Reduction of intracellular Cl- concentration to 5-10 mM caused spontaneous current activation without cell swelling. These results suggest that increases in cell Cl- levels increase the volume set point of the channel. We propose that the main function of the outwardly rectifying anion channel is nonselective transport of organic solutes.

Carrier mediated uptake of Lucifer Yellow in liver of Raja erinacea, the small skate and the rat: A fluid phase marker revisited

Carrier mediated uptake of Lucifer Yellow in liver of Raja erinacea, the small skate and the rat: A fluid phase marker revisited

Gastric evacuation in little skate

The effects of prey type and prey preparation on the mathematical forms and rates describing gastric evacuation in little skate Raja erinacea were examined. Linear and square–root models best described the gastric evacuation of whole, thin–shelled krill Meganyctiphanes norvegica, clam feet/muscle Spisula solidissima/Placopecten magellanicus, polychaetes Glycera spp. and sand lance Ammodytes dubius. Evacuation of krill and clams was faster than polychaetes and sand lance. A logistic model best described the evacuation data of thick–shelled benthic shrimp Crangon septemspinosa/Palaemonetes spp. Cut polychaetes Nereis spp. were digested at an exponential rate and were evacuated faster than would be predicted based upon comparison with live polychaetes and previously published evacuation–temperature relationships. The results of this study suggest that a single equation does not describe the evacuation process for all prey, and that whole prey should be used if laboratory–derived rates of gastric evacuation are to reflect what might occur in wild fishes.