RESUMEN
Many species of electric fish show diurnal or socially elicited variation in electric organ discharge amplitude. In Sternopygus macrurus, activation of protein kinase A by 8-bromo-cAMP increases electrocyte sodium current magnitude. To determine whether the behavioral plasticity in electric organ discharge amplitude is controlled by electrocyte biophysical properties, we examined whether the effects of phosphorylation on ion currents in the electric organ translate directly into electric organ discharge changes. We injected the electric organ of restrained fish with 8-bromo-cAMP and monitored the electric organ discharge. The effect of protein kinase A activation on electrocyte action potentials was examined in isolated electric organ using two-electrode current clamp. Electric organ discharge and action potential amplitude and pulse duration increased in response to 8-bromo-cAMP. Pulse and action potential duration both increased by about 25%. However, the increase in electric organ discharge amplitude (approximately 400%) was several-fold greater than the action potential amplitude increase (approximately 40%). Resting membrane resistance decreased in electrocytes exposed to 8-bromo-cAMP. We propose that in the Thevenin equivalent circuit of the electric organ a moderate increase in action potential amplitude combined with a decrease in internal resistance produces a greater voltage drop across the external resistance (the water around the fish), accounting for the large increase in the externally recorded electric organ discharge.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Órgano Eléctrico/efectos de los fármacos , Órgano Eléctrico/fisiología , Animales , AMP Cíclico/farmacología , Pez Eléctrico/fisiología , Técnicas In Vitro , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Canales de Sodio/efectos de los fármacos , Cloruro de Sodio/farmacologíaRESUMEN
Female communication behaviors are often overlooked by researchers in favor of male behaviors, which are usually more overt and easier to elicit. Very little is known about female electrocommunication behaviors in brown ghost knifefish, a weakly electric wavetype Gymnotiform fish. Most behavioral studies have focused on males, and fish are usually restrained and played a stimulus near their own electric organ discharge frequency to evoke chirps (abrupt short-term frequency rises) or the jamming avoidance response. Our study focuses on categorizing and describing spontaneous and evoked electric organ discharge modulations in free-swimming female fish that were either electrically coupled to tanks containing a conspecific (male or female), or left isolated. Cluster analysis of signals produced under isolated and social conditions revealed three categories of rises: short rise, medium rise and long rise; and one category of frequency decrease (dip). Females produce significantly more short rises when electrically coupled to tanks containing lower-frequency females, and produce more long rises when electrically coupled to tanks containing males. Short rises may have an intrasexual aggressive function, while long rises may serve as an advertisement of status or reproductive condition in intersexual interactions.
Asunto(s)
Comunicación Animal , Pez Eléctrico/fisiología , Órgano Eléctrico/fisiología , Conducta Social , Animales , Conducta Animal/fisiología , Análisis por Conglomerados , Electrofisiología , Femenino , Masculino , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores SexualesRESUMEN
Weakly electric fish in the genus Sternopygus emit a sinusoidal, individually distinct, and sexually dimorphic electric organ discharge (EOD) that is used in electrolocation and communication. Systemically applied androgens decrease EOD frequency, which is set by a medullary pacemaker nucleus, and increase pulse duration, which is determined by the cells of the electric organ (the electrocytes), in a coordinated fashion. One possibility is that androgens broaden the EOD pulse duration by acting on the pacemaker neurons, thereby effecting a change in pacemaker firing frequency, and that the change in EOD pulse duration is due to an activity-dependent process. To determine whether androgens can alter pulse duration despite a stable pacemaker nucleus firing frequency, we implanted small doses of dihydrotestosterone in the electric organ. We found that androgen implants increased EOD pulse duration, but did not influence EOD frequency. In addition, using immunocytochemistry, we found that electrocytes label positively with an androgen receptor antibody. While it is not known on which cells androgens act directly, together these experiments suggest that they likely act on the electrocytes to increase EOD pulse duration. Since pulse duration is determined by electrocyte action potential duration and ionic current kinetics, androgens may therefore play a causative role in influencing individual variation and sexual dimorphism in electrocyte electrical excitability, an important component of electrocommunicatory behavior.
Asunto(s)
Andrógenos/farmacología , Pez Eléctrico/fisiología , Órgano Eléctrico/efectos de los fármacos , Andrógenos/administración & dosificación , Animales , Dihidrotestosterona/administración & dosificación , Dihidrotestosterona/farmacología , Implantes de Medicamentos , Electrofisiología , Inmunohistoquímica , Receptores Androgénicos/efectos de los fármacosRESUMEN
Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than [D[A(BC)]].
Asunto(s)
Evolución Molecular , Variación Genética , Filogenia , Canales de Sodio/genética , Vertebrados/genética , Animales , Mapeo Cromosómico , Peces , Genes Duplicados , Genes Homeobox , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Especificidad de Órganos , Estructura Secundaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salamandridae , Canales de Sodio/química , Vertebrados/clasificaciónRESUMEN
Gymnotiform weakly electric fish produce electric organ discharges (EODs) that function in electrolocation and communication. The command signal for the EOD is produced by the medullary pacemaker nucleus, which contains two well-characterized neuron types: pacemaker cells and relay cells. In this study, we characterized a third neuron type in the pacemaker nucleus. These neurons, which we have named parvocells, were smaller (7-15 microm in diameter) than relay and pacemaker cells. The parvocells were labeled with an antibody against the neuronal calcium-binding protein, parvalbumin, and were not labeled with several glial-specific antibodies. Parvocells had one to three fine processes that often terminated at the periphery of relay and pacemaker cell bodies. The parvalbumin-positive terminals of the parvocells colocalized with immunoreactivity for SV-2, suggesting that the parvocells form chemical synapses on the relay and pacemaker cells. Parvalbumin-positive neurons are frequently gamma-aminobutyric acid (GABA)ergic or glycinergic, and the cytoplasm of the parvocell somata was immunoreactive with a glycine antibody. Antibodies against glycine receptors and gephyrin, however, did not label any cells in the pacemaker nucleus, suggesting that the pacemaker nucleus does not contain glycine or GABA((A)) receptors. Electron microscopy revealed gap junctions between the membranes of parvocells and adjacent terminal-like structures. Furthermore, neurobiotin injected into individual pacemaker or relay cells labeled parvocells as well as other pacemaker and relay cells, demonstrating that the parvocells are dye-coupled to the other neuron types in the pacemaker nucleus. These findings indicate that the parvocells are histochemically distinct from relay and pacemaker cells and that they receive electrotonic inputs from and make chemical synapses back onto pacemaker and relay cells. Further study is needed to investigate the function of these neurons in regulating the EOD.
Asunto(s)
Relojes Biológicos/fisiología , Pez Eléctrico/anatomía & histología , Interneuronas/clasificación , Interneuronas/citología , Parvalbúminas/análisis , Animales , Biotina/análogos & derivados , Calbindinas , Proteínas Portadoras/análisis , Colorantes , Uniones Comunicantes/química , Interneuronas/química , Proteínas de la Membrana/análisis , Microscopía Electrónica , Neuroglía/química , Receptores de GABA-A/análisis , Receptores de Glicina/análisis , Proteína G de Unión al Calcio S100/análisis , Sinapsis/química , Sinapsis/ultraestructura , Cloruro de TolonioRESUMEN
The electric organ cells of Sternopygus generate action potentials whose durations vary over a fourfold range. This variation in action potential duration is the basis for individual variation in a communication signal. Thus, action potential duration must be precisely regulated in these cells. We had observed previously that the inactivation kinetics of the electrocyte Na(+) current show systematic individual variation. In this study, using a two-electrode voltage clamp, we found that the voltage-dependent activation and deactivation kinetics of the delayed rectifying K(+) current in these cells covary in a graded and predictable manner across fish. Furthermore, when Na(+) and K(+) currents were recorded in the same cell, their voltage-dependent kinetics were highly correlated. This finding illustrates an unprecedented degree of coregulation of voltage-dependent properties in two molecularly distinct ionic channels. Such a coregulation of ionic channels is uniquely observable in a cell specialized to generate individual differences in electrical activity and in which the results of biophysical control mechanisms are evident in behaving animals. We propose that the precise coregulation of the voltage-dependent kinetics of multiple ionic currents may be a general mechanism for regulation of membrane excitability.
Asunto(s)
Relojes Biológicos/fisiología , Pez Eléctrico/fisiología , Órgano Eléctrico/fisiología , Canales de Potasio/fisiología , Canales de Sodio/fisiología , Potenciales de Acción/fisiología , AnimalesRESUMEN
Electric organ discharge (EOD) frequency in the brown ghost knifefish (Apteronotus leptorhynchus) is sexually dimorphic, steroid-regulated, and determined by the discharge rates of neurons in the medullary pacemaker nucleus (Pn). We pharmacologically characterized ionic currents that regulate the firing frequency of Pn neurons to determine which currents contribute to spontaneous oscillations of these neurons and to identify putative targets of steroid action in regulating sexually dimorphic EOD frequency. Tetrodotoxin (TTX) initially reduced spike frequency, and then reduced spike amplitude and stopped pacemaker activity. The sodium channel blocker muO-conotoxin MrVIA also reduced spike frequency, but did not affect spike amplitude or production. Two potassium channel blockers, 4-aminopyridine (4AP) and kappaA-conotoxin SIVA, increased pacemaker firing rates by approximately 20% and then stopped pacemaker firing. Other potassium channel blockers (tetraethylammonium, cesium, alpha-dendrotoxin, and agitoxin-2) did not affect the pacemaker rhythm. The nonspecific calcium channel blockers nickel and cadmium reduced pacemaker firing rates by approximately 15-20%. Specific blockers of L-, N-, P-, and Q-type calcium currents, however, were ineffective. These results indicate that at least three ionic currents-a TTX- and muO-conotoxin MrVIA-sensitive sodium current; a 4AP- and kappaA-conotoxin SIVA-sensitive potassium current; and a T- or R-type calcium current-contribute to the pacemaker rhythm. The pharmacological profiles of these currents are similar to those of currents that are known to regulate firing rates in other spontaneously oscillating neural circuits.
Asunto(s)
Relojes Biológicos/fisiología , Canales de Calcio/metabolismo , Pez Eléctrico/fisiología , Neuronas/metabolismo , Canales de Potasio/metabolismo , Canales de Sodio/metabolismo , 4-Aminopiridina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Relojes Biológicos/efectos de los fármacos , Cadmio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Células Cultivadas , Conotoxinas/farmacología , Relación Dosis-Respuesta a Droga , Órgano Eléctrico/inervación , Técnicas In Vitro , Neuronas/citología , Neuronas/efectos de los fármacos , Níquel/farmacología , Nifedipino/farmacología , Bloqueadores de los Canales de Potasio , Bloqueadores de los Canales de Sodio , Tetraetilamonio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Weakly electric fish are good model animals to study the evolution of interspecific and sexual differences in communication signals. This is because the neural circuits producing these signals are simple and conserved among related species while the signals are highly species-specific, sexually-dimorphic, and under hormonal control. Here we focus on two related species of weakly electric gymnotiform fish that emit a wave-type discharge. These species differ in the direction of the sexual dimorphism of their electric organ discharge (EOD) frequencies and their propensity to produce aggressive communication signals called 'chirps'. Brown ghost (Apteronotus leptorhynchus) males produce high frequency EODs while females produce low frequency EODs. When presented with an EOD mimic, males chirp frequently, while females seldom chirp. By contrast, black ghost (A. albifrons) males discharge at lower EOD frequencies than females, and there is no sex difference in chirping in this species. Accordingly, non-aromatizable androgens raise EOD frequency in brown ghosts, but lower it in black ghosts. Androgens induce chirping in female brown ghosts, but do not increase the propensity to chirp in female black ghosts. Thus, the difference in sexually-dimorphic communication signals between these two species can be explained by differences in their responses to sex steroids. Future studies will elucidate how the neural circuits generating these signals are differentially sensitive to steroids in these species.
Asunto(s)
Comunicación Animal , Diferenciación Sexual/fisiología , Agresión/psicología , Animales , Conducta Animal/fisiología , Estrógenos/fisiología , Femenino , Peces , Gónadas/fisiología , Masculino , Caracteres Sexuales , Conducta Social , Testosterona/fisiologíaRESUMEN
The electric organ has evolved independently from muscle in at least six lineages of fish. How does a differentiated muscle cell change its fate to become an electrocyte? Is the process by which this occurs similar in different lineages? We have begun to answer these questions by studying the formation and maintenance of electrocytes in the genus Sternopygus, a weakly electric teleost. Electrocytes arise from the fusion of fully differentiated muscle fibers, mainly those expressing fast isoforms of myosin. Electrocytes briefly co-express sarcomeric proteins, such as myosin and tropomyosin, and keratin, a protein not found in mature muscle. The sarcomeric proteins are subsequently down-regulated, but keratin expression persists. We investigated whether the maintenance of the electrocyte phenotype depends on innervation. We found that, after spinal cord transection, which silences the electromotor neurons that innervate the electrocytes, or destruction of the spinal cord, which denervates the electrocytes, mature electrocytes re-express sarcomeric myosin and tropomyosin, although keratin expression persists. Ultrastructural examination of denervated electrocytes revealed nascent sarcomeres. Thus, the maintenance of the electrocyte phenotype depends on neural activity.
Asunto(s)
Pez Eléctrico/crecimiento & desarrollo , Órgano Eléctrico/crecimiento & desarrollo , Regeneración , Animales , Pez Eléctrico/fisiología , Órgano Eléctrico/anatomía & histología , Órgano Eléctrico/fisiología , Músculos/fisiología , Especificidad de la EspecieRESUMEN
The electric organ (EO) of the weakly electric fish Sternopygus macrurus derives from striated myofibers that fuse and suppress many muscle properties. Mature electrocytes are larger than muscle fibers, do not contain sarcomeres, or express myosin heavy chain (MHC) or tropomyosin. Furthermore, electrocytes express keratin, a protein not expressed in muscle. In S. macrurus the EO is driven continuously at frequencies higher than those of the intermittently active skeletal muscle. The extent to which differences in EO and muscle phenotype are accounted for by activity patterns, or innervation per se, was determined by assessing the expression of MHC, tropomyosin, and keratin 2 and 5 weeks after the elimination of (1) activity patterns by spinal transection or (2) all synaptic input by denervation. Immunohistochemical analyses showed no changes in muscle fiber phenotypes after either experimental treatment. In contrast, the keratin-positive electrocytes revealed an upregulation of MHC and tropomyosin. Nearly one-third of all electrocytes expressed MHC (35%) and tropomyosin (25%) 2 weeks after spinal transection, whereas approximately two-thirds (61%) expressed MHC 2 weeks after denervation. After 5 weeks of denervation or spinal transection, all electrocytes contained MHC and tropomyosin. Newly formed sarcomere clusters also were observed in denervated electrocytes. The MHC expressed in electrocytes corresponded to that present in a select population of muscle fibers, i.e., type II fibers. Thus, the elimination of electrical activity or all synaptic input resulted in a partial reversal of the electrocyte phenotype to an earlier developmental stage of its myogenic lineage.
Asunto(s)
Pez Eléctrico/fisiología , Órgano Eléctrico/química , Órgano Eléctrico/inervación , Cadenas Pesadas de Miosina/análisis , Tropomiosina/análisis , Animales , Atrofia , División Celular/fisiología , Células Cultivadas , Órgano Eléctrico/patología , Femenino , Expresión Génica/fisiología , Queratinas/análisis , Queratinas/genética , Masculino , Desnervación Muscular , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/química , Músculo Esquelético/inervación , Músculo Esquelético/patología , Cadenas Pesadas de Miosina/genética , Fenotipo , Sarcómeros/química , Médula Espinal/cirugía , Tropomiosina/genéticaRESUMEN
Gymnotiform electric fish emit an electric organ discharge (EOD) that, in many species, is sexually dimorphic and used in gender recognition. The glass knife fish Eigenmannia has been a major neuroethological model system, and there have been reports that its EOD is sexually dimorphic. However, no study has examined the role of steroids in establishing this dimorphism. We tested the effect of three androgens, testosterone, dihydrotestosterone, and 11-ketotestosterone (11kT), on the EOD of Eigenmannia virescens. Implants of any of these three androgens induced a decrease in EOD frequency, consistent with several studies showing that males have a lower EOD frequency than females. In a separate experiment, we found that 11kT treatment also increases EOD pulse duration, but has no effect on the duty cycle, the relative duration of the positive and negative phases of the wave, or the harmonic content of the EOD. Using immunocytochemistry, we found that the cells of the electric organ (the electrocytes) that produce the EOD and control its pulse duration label positively with an antibody to the androgen receptor. These experiments indicate that androgens decrease the firing frequency of the medullary pacemaker and alter the ion current kinetics in the electrocytes. Moreover, the presence of nuclear androgen receptors in electrocytes suggests that androgens act directly on the electric organ to modify EOD pulse duration.
Asunto(s)
Andrógenos/farmacología , Comunicación Animal , Conducta Animal/fisiología , Pez Eléctrico/fisiología , Receptores Androgénicos/fisiología , Animales , Dihidrotestosterona/farmacología , Órgano Eléctrico/química , Órgano Eléctrico/fisiología , Femenino , Masculino , Receptores Androgénicos/análisis , Caracteres Sexuales , Testosterona/análogos & derivados , Testosterona/sangre , Testosterona/farmacologíaRESUMEN
In most groups of electric fish, the electric organ (EO) derives from striated muscle cells that suppress many muscle phenotypic properties. This phenotypic conversion is recapitulated during regeneration of the tail in the weakly electric fish Sternopygus macrurus. Mature electrocytes, the cells of the electric organ, are considerably larger than the muscle fibers from which they derive, and it is not known whether this is a result of muscle fiber hypertrophy and/or fiber fusion. In this study, electron micrographs revealed fusion of differentiated muscle fibers during the formation of electrocytes. There was no evidence of hypertrophy of muscle fibers during their phenotypic conversion. Furthermore, although fish possess distinct muscle phenotypes, the extent to which each fiber population contributes to the formation of the EO has not been determined. By using myosin ATPase histochemistry and anti-myosin heavy chain (MHC) monoclonal antibodies (mAbs), different fiber types were identified in fascicles of muscle in the adult tail. Mature electrocytes were not stained by the ATPase reaction, nor were they labeled by any of the anti-MHC mAbs. In contrast, mature muscle fibers exhibited four staining patterns. The four fiber types were spatially arranged in distinct compartments with little intermixing; peripherally were two populations of type I fibers with small cross-sectional areas, whereas more centrally were two populations of type II fibers with larger cross-sectional areas. In 2- and 3-week regenerating blastema, three fiber types were clearly discerned. Most (> 95%) early-forming electrocytes had an MHC phenotype similar to that of type II fibers. In contrast, fusion among type I fibers was rare. Together, ultrastructural and immunohistochemical analyses revealed that the fusion of muscle fibers gives rise to electrocytes and that this fusion occurs primarily among the population of type II fibers in regenerating blastema.
Asunto(s)
Pez Eléctrico/fisiología , Órgano Eléctrico/citología , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/citología , Cadenas Pesadas de Miosina/análisis , Adenosina Trifosfatasas/análisis , Animales , Diferenciación Celular/fisiología , Femenino , Masculino , Microscopía Electrónica , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/química , Músculo Esquelético/citología , Músculo Esquelético/enzimología , Cadenas Pesadas de Miosina/genética , Fenotipo , Regeneración/fisiología , Cola (estructura animal)/fisiologíaRESUMEN
Gymnotiform electric fish emit an electric organ discharge that, in several species, is sexually dimorphic and functions in gender recognition. In addition, some species produce frequency modulations of the electric organ discharge, known as chirps, that are displayed during aggression and courtship. We report that two congeneric species (Apteronotus leptorhynchus and A. albifrons) differ in the expression of sexual dimorphism in these signals. In A. leptorhynchus, males chirp more than females, but in A. albifrons chirping is monomorphic. The gonadosomatic index and plasma levels of 11-ketotestosterone were equivalent in both species, suggesting that they were in similar reproductive condition. Corresponding to this difference in dimorphism, A. leptorhynchus increases chirping in response to androgens, but chirping in A. albifrons is insensitive to implants of testosterone, dihydrotestosterone or 11-ketotestosterone. Species also differ in the sexual dimorphism and androgen sensitivity of electric organ discharge frequency. In A. leptorhynchus, male discharge at higher frequencies than females, and androgens increase electric organ discharge frequency. In A. albifrons, males discharge at lower frequencies than females, and androgens decrease electronic organ discharge frequency. Thus, in both chirping and electric organ discharge frequency, evolutionary changes in the presence or direction of sexual dimorphism have been accompanied and perhaps caused by changes in the androgen regulation of the electric organ discharge.
Asunto(s)
Andrógenos/fisiología , Comunicación Animal , Pez Eléctrico/fisiología , Órgano Eléctrico/fisiología , Andrógenos/farmacología , Animales , Peso Corporal/fisiología , Dihidrotestosterona/farmacología , Órgano Eléctrico/efectos de los fármacos , Electrofisiología , Femenino , Masculino , Orquiectomía , Ovariectomía , Caracteres Sexuales , Conducta Sexual Animal/efectos de los fármacos , Conducta Sexual Animal/fisiología , Testosterona/análogos & derivados , Testosterona/farmacologíaRESUMEN
Steroid hormones influence the electrical activity of many neurons and effectors by regulating the transcription of their ion channels and neurotransmitter receptors, or by modulating the activity of their channels and receptors through second messenger-coupled membrane receptors, or both. In this article, four cell types with known functions and distinct electrical activities are focused on to illustrate how different steroids act synergistically with, or in opposition to, each other to modulate specific electrical phenomena such as spontaneous regular firing (GH3 cells, a pituitary cell line), action potential duration (electric organ cells), and intrinsic excitability and sensitivity to neurotransmitters (GnRH and opioidergic neurons).These examples illustrate how steroids might influence electrical activity in neurons involved in more complex central circuits.
Asunto(s)
Hormonas/fisiología , Neuronas/fisiología , Esteroides/fisiología , Animales , Órgano Eléctrico/fisiología , Electrofisiología , Endorfinas/fisiología , Genoma , Hormona Liberadora de Gonadotropina/fisiología , Humanos , Inhibición Neural/fisiologíaRESUMEN
Transdifferentiation is the conversion of one differentiated cell type into another. The electric organ of fishes transdifferentiates from muscle but little is known about how this occurs. To begin to address this question, we studied the expression of muscle- and electrocyte-specific proteins with immunohistochemistry during regeneration of the electric organ. In the early stages of regeneration, a blastema forms. Blastemal cells cluster, express desmin, fuse into myotubes, and then express alpha-actinin, tropomyosin, and myosin. Myotubes in the periphery of the blastema continue to differentiate as muscle; those in the center grow in size, probably by fusing with each other, and lose their sarcomeres as they become electrocytes. Tropomyosin is rapidly down-regulated while desmin, alpha-actinin, and myosin continue to be diffusely expressed in newly formed electrocytes despite the absence of organized sarcomeres. During this time an isoform of keratin that is a marker for mature electrocytes is expressed. One week later, the immunoreactivities of myosin disappears and alpha-actinin weakens, while that of desmin and keratin remain strong. Since nerve fibers grow into the blastema preceding the appearance of any differentiated cells, we tested whether the highly rhythmic nerve activity associated with electromotor input plays a role in transdifferentiation and found that electrocytes develop normally in the absence of electromotor neuron activity.
Asunto(s)
Órgano Eléctrico/fisiología , Músculo Esquelético/fisiología , Factores Reguladores Miogénicos/metabolismo , Regeneración/fisiología , Animales , Diferenciación Celular , Órgano Eléctrico/citología , Órgano Eléctrico/patología , Peces , Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/citología , Músculo Esquelético/embriología , Fibras Nerviosas , Receptores Colinérgicos/análisis , Sarcómeros/metabolismo , Médula Espinal/fisiologíaRESUMEN
Many species of electric fish emit sexually dimorphic electrical signals that are used in gender recognition. In Sternopygus, mature females produce an electric organ discharge (EOD) that is higher in frequency and shorter in pulse duration than that of mature males. EOD pulse duration is determined by ion currents in the electrocytes, and androgens influence EOD pulse duration by altering the inactivation kinetics of the electrocyte sodium current. We examined whether estrogen modulates the female-specific EOD and, if so, whether it regulates EOD pulse duration by acting on the same androgen-sensitive ion current in the electrocytes. We implanted gonadectomized Sternopygus with either empty SILASTIC capsules (control), one capsule filled with estradiol-17beta (E2; low dose), or three capsules of E2 (high dose). Twelve days after implantation, E2-treated fish had plasma E2 levels approximately 3.3-fold (low dose) or approximately 7.1-fold (high dose) higher than controls. After implantation, both E2-treated groups had higher EOD frequency and shorter EOD pulse duration than controls and their own preimplantation values. Through immunocytochemistry, we identified immunoreactive estrogen receptors in the nuclei of electrocytes, indicating that these cells are directly responsive to estrogen. In addition, voltage-clamp studies showed that E2 affected the electrocyte ion currents kinetics: the sodium inactivation time constant was significantly lower in E2-treated fish than in controls. Thus, sexual dimorphism in the electrocommunication signal results, at least in part, from estrogens and androgens acting in opposite directions on the same ion current in the electrocytes.
Asunto(s)
Órgano Eléctrico/fisiología , Estradiol/farmacología , Transducción de Señal/fisiología , Canales de Sodio/fisiología , Animales , Implantes de Medicamentos , Pez Eléctrico , Órgano Eléctrico/efectos de los fármacos , Estradiol/administración & dosificación , Estradiol/sangre , Femenino , Cinética , Masculino , Orquiectomía , Ovariectomía , Receptores de Estrógenos/análisis , Receptores de Estrógenos/fisiología , Transducción de Señal/efectos de los fármacos , Canales de Sodio/efectos de los fármacosRESUMEN
The inactivation kinetics of the Na+ current of the weakly electric fish Sternopygus are modified by treatment with androgens. To determine whether phosphorylation could play a role in this effect, we examined whether activation of protein kinase A by 8 bromo cyclic AMP (8 Br cAMP) altered voltage-dependent properties of the current. Using a two-electrode voltage-clamp procedure, we found no effect of 8 Br cAMP on inactivation kinetics or other voltage-dependent properties of the Na+ current of the electrocytes. However, treatment with 8 Br cAMP did produce a dose-dependent increase in the Na+ current compared with saline controls: 17.6% at 100 microM, 42.4% at 1 mM, and 43.1% at 5 mM. This effect was blocked by 30 microM H89, a PKA inhibitor, indicating that the observed effect was attributable to 8 Br cAMP activation of PKA. We conclude that androgen-induced changes in Na+ current inactivation are not mediated by PKA and suggest that PKA-mediated increases in Na+ current underlie increases in the amplitude of the electric organ discharge observed in social interactions or with changes in water conductance.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Órgano Eléctrico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Animales , AMP Cíclico/farmacología , Peces , Factores de TiempoRESUMEN
The electric organ of electric fish develops from a myogenic lineage. We have used immunohistochemistry and immunoblotting to determine which features of the muscle phenotype are retained and whether any new ones are expressed in mature electrocytes of the electric fish Sternopygus. The muscle-specific intermediate filament desmin was found throughout the electrocytes, and different desmin antibodies detected molecules with different subcellular distributions. Western blots confirm that these antibodies recognize a protein of MW = 53 kD, the molecular weight of desmin. Other muscle proteins were also present within electrocytes: Actin and sarcomeric alpha-actinin were found within the subsynaptic membrane beneath the plasmalemma of the electrocytes, and talin and acetylcholine receptors were detected both at the innervated posterior face and at the non-innervated anterior face. This was confirmed using rhodamine-conjugated alpha-bungarotoxin. Neither myosin heavy chain nor tropomyosin was present in electrocytes. Finally, we detected within electrocytes a type I acidic keratin that forms a filamentous meshwork within each cell. Immunoblots corroborate this result: A keratin-positive doublet of MW = 50 kD and 57 kD was found in both electrocytes and skin. Myosin, actin, talin, tropomyosin, desmin, alpha-actinin, and acetylcholine receptor, but not keratin, were all expressed in fish skeletal muscle fibers. Thus, electrocytes retain some muscle-specific proteins, do not express others, and in addition, express a non-muscle protein.
Asunto(s)
Pez Eléctrico/metabolismo , Órgano Eléctrico/química , Proteínas Musculares/análisis , Ratas/metabolismo , Actinina/análisis , Actinas/análisis , Animales , Linaje de la Célula , Desmina/análisis , Femenino , Secciones por Congelación , Immunoblotting , Inmunohistoquímica , Masculino , Cadenas Pesadas de Miosina/análisis , Especificidad de Órganos/fisiología , Receptores Colinérgicos/análisisRESUMEN
Weakly electric fish use tuberous electroreceptor organs to detect their own electric fields. We investigated the role of innervation upon regeneration and differentiation of tuberous electroreceptor organs. The left, infraorbital, anterior lateral line nerve of brown ghosts (Apteronotus leptorhynchus) was sectioned, and the proximal stump was dipped in ricin to prevent regrowth. Immediately after denervation, a piece of cheek skin (approximately 0.5 cm2) was removed bilaterally to induce skin regeneration. After survival periods of 3, 4, or 5 weeks, regenerated skin from the left (denervated) and the right (reinnervated) sides was removed and processed for immunocytochemistry or electron microscopy. Tuberous electroreceptor organs were present in regenerated reinnervated, as well as regenerated denervated skin patches at all survival times. With increased time after skin removal, the number of fully differentiated organs increased in the reinnervated regenerated skin while the number of organs with degenerating receptor cells or entirely devoid of receptor cells increased in the denervated regenerated skin. These results suggest that innervation is not essential for tuberous electroreceptor organ development, but that it is necessary for complete sensory cell differentiation and long-term survival.
Asunto(s)
Pez Eléctrico/fisiología , Neuronas Aferentes/fisiología , Regeneración/fisiología , Células Receptoras Sensoriales/fisiología , Piel/inervación , Animales , Diferenciación Celular , Desnervación , Inmunohistoquímica , Microscopía Electrónica , Neuronas Aferentes/ultraestructura , Células Receptoras Sensoriales/ultraestructura , Piel/ultraestructura , Fenómenos Fisiológicos de la PielRESUMEN
The South American knifefish (Apteronotus leptorhynchus), or brown ghost, produces a high-frequency (600-1000 Hz) sinusoidal electric organ discharge (EOD) with males discharging at higher frequencies than females. In addition, each fish has a unique EOD frequency within the frequency range of its gender. The electromotor circuit responsible for EOD production consists of a medullary pacemaker nucleus (PMN) and spinal electromotor neurons (EMNs). In vitro spinal slice recording showed that, similar to the PMN, EMNs fire spontaneously at rates near the EOD frequency of each fish. The persistence of firing 2 weeks after high spinal transaction demonstrated that spontaneous firing rate was intrinsic to the EMNs and was not dependent on presynaptic input. We confirmed that 11-ketotestosterone (11 kT) raised and 17-beta-estradiol (E2) lowered the EOD frequency of intact fish. Because electromotor cells fire spontaneously near EOD, frequency, we investigated whether these steroids affect endogenous firing rates. Steroid implants were made in normal or spinally transected fish. Two weeks later, PMNs of normal fish and EMNs of transected fish were recorded in vitro. 11 kT increased and E2 decreased the intrinsic firing rate of neurons in the PMN and the EMNS. Hormones shifted the intrinsic firing rates of EMNS, although they were synaptically isolated during the hormone exposure.