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PubMed2006

Distortion of symmetrical introgression in a hybrid zone: evidence for locus-specific selection and uni-directional range expansion.

The fate of species integrity upon natural hybridization depends on the interaction between selection and dispersal. The relative significance of these processes may be studied in the initial phase of contact before selection and gene flow reach equilibrium. Here we study a hybrid zone of two salamander species, Lyciasalamandra antalyana and Lyciasalamandra billae, at the initial phase of hybridization. We quantify the degree and mode of introgression using nuclear and mtDNA markers. The hybrid zone can be characterized as an abrupt transition zone, the central hybrid zone being only c. 400 m, but introgressed genes were traced up to 3 km. Introgression was traced in both sexes but gene flow may be slightly male-biased. Indirect evidence suggests that hybrid males are less viable than females. Introgression occurred at two levels: (1) locus-specific selection led to different allelic introgression patterns independent of species, while (2) asymmetrical species-level introgression occurred predominately from L. antalyana to L. billae due to range expansion of the former. This indicates that foreign genes can be incorporated into novel genomic environments, which in turn may contribute to the great diversity of morphological variants in Lyciasalamandra.

Amphibians↗
PubMed1984

Are limb development and limb regeneration both initiated by an integumentary wounding? A hypothesis.

It is proposed that, whereas an actual wound to a salamander limb may initiate limb regeneration, a local and developmentally programmed integumentary wound may initiate limb development. The electrophysiological changes induced by these lesions of the skin may be a common denominator linking limb regeneration and limb development. Such early electrical events are considered to initiate or guide the early accumulation of cells, and to help to produce the local environment in which a limb will arise. This scheme provides a self-limiting positive-feedback mechanism for the production of a localized area where other developmental mechanisms act in concert with endogenous electrical fields (or in their complete absence), thereby leading to limb differentiation. This hypothesis may not be restricted to limb formation; it may be of more general significance, i.e. in the process of organogenesis in embryos. One might reasonably suggest that, by such a mechanism, any developing placode (for example, auditory or olfactory placodes) might form and localize.

Amphibians↗
PubMed1988

Hyaluronate accumulation and nerve-dependent growth during regeneration of larval Ambystoma limbs.

Hyaluronate-mediated expansion of the extracellular matrix has been suggested as an important element of growth and morphogenesis in several developing systems. In vitro, various growth factors have been shown to stimulate hyaluronate synthesis as well as cell proliferation. A similar link between proliferation and hyaluronate production during in vivo growth is difficult to demonstrate, because in most systems the source of growth-promoting factors is either not known or not amenable to experimental manipulation. During amphibian limb regeneration, cell proliferation depends upon paracrine release of factors from axons in the limb stump, and the nerve supply can be eliminated or augmented experimentally for study of growth in this system. Denervated and amputated limbs of larval salamanders do not begin to regenerate until distal areas of the limb stumps are reinnervated. We have used such limbs to examine the effect exerted by the reappearance of nerves on the amount of hyaluronate in the tissue undergoing the growth response. Hyaluronate was demonstrated by the metachromatic dye Ethyl Stains-all, which stains hyaluronate blue while sulfated glycosaminoglycans (GAGs) and proteins in the extracellular matrix stain various shades of violet, and by microspectrophotometry of alcian-blue-stained GAGs in serial sections pretreated with buffer or with Streptomyces hyaluronidase (SH) to remove hyaluronate specifically. Both methods showed little hyaluronate in the distal region of limb stumps prior to reinnervation, while reinnervated stumps had amounts of hyaluronate similar to those of control blastemas. Autoradiography of 3H-glucosamine-labeled limbs indicated that hyaluronate in the blastemas of reinnervated limb stumps included material newly synthesized by cells throughout the growing tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Ambystoma↗
PubMed1980

The role of early sensory experience in the prey catching responses of Salamandra salamandra to stationary prey.

Two groups of newly metamorphosed Salamandra salamandra (L.) were raised with living and with stationary prey, respectively. One year later, the group which had experience exclusively with stationary prey was significantly better in responding to stationary prey than the group which only had experience with moving prey. This was the case both under light and dark conditions. The experiments show that prey-catching behavior in salamanders can be considerably modified by experience.

Animals↗
PubMed2006

Calcium-induced calcium release in rod photoreceptor terminals boosts synaptic transmission during maintained depolarization.

We examined the contribution of calcium-induced calcium release (CICR) to synaptic transmission from rod photoreceptor terminals. Whole-cell recording and confocal calcium imaging experiments were conducted on rods with intact synaptic terminals in a retinal slice preparation from salamander. Low concentrations of ryanodine stimulated calcium increases in rod terminals, consistent with the presence of ryanodine receptors. Application of strong depolarizing steps (-70 to -10 mV) exceeding 200 ms or longer in duration evoked a wave of calcium that spread across the synaptic terminals of voltage-clamped rods. This secondary calcium increase was blocked by high concentrations of ryanodine, indicating it was due to CICR. Ryanodine (50 microm) had no significant effect on rod calcium current (I(ca)) although it slightly diminished rod light-evoked voltage responses. Bath application of 50 microm ryanodine strongly inhibited light-evoked currents in horizontal cells. Whether applied extracellularly or delivered into the rod cell through the patch pipette, ryanodine (50 microm) also inhibited excitatory post-synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to rods. Ryanodine caused a preferential reduction in the later portions of EPSCs evoked by depolarizing steps of 200 ms or longer. These results indicate that CICR enhances calcium increases in rod terminals evoked by sustained depolarization, which in turn acts to boost synaptic exocytosis from rods.

Animals↗
PubMed2006

How to build and rebuild a lens.

Evolution has used many different strategies to build eyes and lenses. However, the genetic regulation involved seems to be quite conserved. Likewise, the regeneration of eye structures is remarkable, especially in salamanders. This review outlines the basic mechanisms of lens regeneration and its induction and the possibility of creating lenses by transdifferentiation of the pigment epithelial cells, by stem cells or by bioengineering.

Animals↗
PubMed1999

Modulation of extracellular glutamate concentration in rat brain slices by cystine-glutamate exchange.

1. The role of cystine-glutamate exchange in controlling the extracellular glutamate concentration in the central nervous system was examined by whole-cell clamping neurons in rat brain slices, and using their glutamate receptors as sensors of extracellular glutamate concentration. 2. Applying cystine to cerebellar slices generated a membrane current in Purkinje cells which was abolished by glutamate receptor blockers. Similar cystine-evoked currents were seen in pyramidal cells of frontal cortex slices. 3. Control experiments on non-N-methyl-D-aspartate (non-NMDA) receptors in enzymatically isolated Purkinje cells showed that cystine did not produce a current in slice Purkinje cells by directly activating glutamate receptors, nor by potentiating the action of background levels of glutamate on receptors. Experiments on isolated salamander Muller cells showed that cystine did not block Na+-dependent GLAST glutamate transporters (homologous to the transporters in the Bergmann glia ensheathing the Purkinje cells), nor did it block the current produced by EAAT4 and EAAC1 glutamate transporters in Purkinje cells. Thus the cystine-evoked current in Purkinje cells is not due to a rise in extracellular glutamate concentration caused by block of Na+-dependent uptake. 4. The dependence of cystine-evoked current on cystine concentration in slice Purkinje cells could be fitted by a Michaelis-Menten relation with a Km of 250 microM. The Km predicted from this for cystine activating glutamate efflux is less than 140 microM, because of the non-linear dependence on glutamate concentration of the Purkinje cell current. The current evoked by 1 mM cystine was little affected by removal of extracellular chloride or addition of 1 mM furosemide (frusemide), but was potentiated by 1 mM 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS). 5. These data suggest that external cystine generates a current in slice Purkinje cells by activating cystine-glutamate exchange in cells of the slice, releasing glutamate which activates non-NMDA receptors in the Purkinje cell membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2004

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells.

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.

Animals↗
PubMed2006

Remarkable amphibian biomass and abundance in an isolated wetland: implications for wetland conservation.

Despite the continuing loss of wetland habitats and associated declines in amphibian populations, attempts to translate wetland losses into measurable losses to ecosystems have been lacking. We estimated the potential productivity from the amphibian community that would be compromised by the loss of a single isolated wetland that has been protected from most industrial, agricultural, and urban impacts for the past 54 years. We used a continuous drift fence at Ellenton Bay, a 10-ha freshwater wetland on the Savannah River Site, near Aiken, South Carolina (U.S.A.), to sample all amphibians for 1 year following a prolonged drought. Despite intensive agricultural use of the land surrounding Ellenton Bay prior to 1951, we documented 24 species and remarkably high numbers and biomass of juvenile amphibians (>360,000 individuals; >1,400 kg) produced during one breeding season. Anurans (17 species) were more abundant than salamanders (7 species), comprising 96.4% of individual captures. Most (95.9%) of the amphibian biomass came from 232095 individuals of a single species of anuran (southern leopard frog[Rana sphenocephala]). Our results revealed the resilience of an amphibian community to natural stressors and historical habitat alteration and the potential magnitude of biomass and energy transfer from isolated wetlands to surrounding terrestrial habitat. We attributed the postdrought success of amphibians to a combination of adult longevity (often >5 years), a reduction in predator abundance, and an abundance of larval food resources. Likewise, the increase of forest cover around Ellenton Bay from <20% in 1951 to >60% in 2001 probably contributed to the long-term persistence of amphibians at this site. Our findings provide an optimistic counterpoint to the issue of the global decline of biological diversity by demonstrating that conservation efforts can mitigate historical habitat degradation.

Amphibians↗
PubMedDate not supplied

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus.

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.

Animals↗
PubMed1975

[Ultrastructure of the genus Enteromonas da Fonseca (Zoomastigophorea) and revision of the order of Diplomonadida Wenyon].

Fine structure of 2 species of Enteromonas, one from the intestine of the salamander, Triturus vulgairs, and another from the feces of domestic rabbit, Oryctolagus cuniculi, is described. The pyriform cell has an anteriorly located nucleus. The 4 flagella originate from an area near the anterior end of the nucleus. The recurrent flagellum (R) is lodged in a ventral depression or cytostome. The kinetosomes, arranged into 2 pairs, anterior (no. 1, no. 2) and posterior ("3, R), are interconnected by microfibrils. One microtubular fiber, connected to kinetosome "1, is situated near the anterior surface of the nucleus. Another, subnuclear, microtubular fiber is homologous to the "crossed" fiber found in Diplozoa. The cytostome is bordered by 2 lips: the preeminent left lip is equipped with several rows of microtubules, while the right lip contains only a thin microtubular fiber associated with microfibrils. The cytostome occupies 2/3 of the ventral surface. The recurrent flagellum passos over the anterior surface of the cell and then comes to lie in the cytostome. The bacteria are phagocytosed in the bottom part of the cytostome between the 2 distended lips. They are digested in numerous vacuoles. The undigested residual bodies are evacuated by a rupture of the cell membrane. The ergastoplasm is concentrated near the cell periphery. Mitochondria and the Golgi apparatus are absent. In the cyst stage, the multinucleate cell is enclosed in a microfibrillar membrane; the axonemes lie free in the cytoplasm. Diplomonad forms of Enteromonas resembling Hexamita are numerous, except that the cytostome is different in these 2 genera. In such forms, the arrangement of the 2 individuals often has binary axial symmetry, but on occasion they are associated in a more anarchic fashion. The mastigont of Enteromonas is organized like that of a single zooid of a diplozoon. It is possible that the genus Enteromonas is ancestral to Diplomonadida and that the diplomonad state, transitory in Enteromonas, became permanently established in Diplomonadida. Enteromonas appears to be more primitive than the other genera of Diplomonadida. Thus we propose 2 suborders: Enteromonadina, subord. nov. with the genus Enteromonas, and Diplomonadina Wenyon, emend., with the genera Trepomonas, Trigonomonas, Hexamita, Spironucleus, Octomitus, Giardia. The arrangement of the kinetosomes and the existence of a cytostome are the principal characters common to Enteromonas and Retortamonadida, while their "accessory" fibers are not homologous. A more complete study of division of the 2 zooflagellate orders is necessary for the presentation of a more detailed evolutionary scheme of these groups.

Animals↗
PubMed1986

Regional specialization of the membrane of retinal glial cells and its importance to K+ spatial buffering.

Electrophysiological experiments, obtained primarily from dissociated salamander cells, demonstrate that the K+ conductance of Müller cells is distributed in a highly nonuniform manner over the cell surface. A large fraction of the total cell conductance is localized to that portion of the endfoot process that faces the vitreous humor. Along the remainder of the cell, specific K+ conductance is larger in the outer plexiform layer than in neighboring regions. High-endfoot conductance directs K+ spatial buffering currents preferentially through the endfoot process, leading to an efficient form of spatial buffering termed K+ siphoning. Preliminary experiments suggest that the endfeet of astrocytes also have high K+ conductance.

Animals↗
PubMed1998

The role of innervation in the development of taste buds: insights from studies of amphibian embryos.

Amphibian embryos have long been model organisms for studies of development because of their hardiness and large size, as well as the ease with which they can be experimentally manipulated. These particular advantages have allowed us recently to test the role of innervation in the development of vertebrate taste buds using embryos of an aquatic salamander, the axolotl. The predominant model of taste bud genesis has been one of neural induction, in which ingrowing sensory neurites induce taste bud differentiation in the epithelium that lines the mouth and pharynx. However, when we prevented embryonic sensory neurons from contacting the oropharyngeal epithelium by using transplantation or tissue culture techniques, we found that taste bud differentiation was independent of nerve contact. Additionally, using similar types of experimental manipulations, we have recently shown that taste bud differentiation is not a result of interactions of the oropharyngeal epithelium with craniofacial mesenchyme. Surprisingly, we found that although taste bud genesis occurs very late in embryonic development, it is an intrinsic feature of the presumptive oropharyngeal epithelium extremely early, in fact as early as the completion of gastrulation. These data have prompted us to propose a new model for the development of amphibian taste buds: (i) The presumptive oropharyngeal epithelium is specified by the time gastrulation is complete; (ii) Subsequently, a distributed population of taste bud progenitors is set up within this epithelium via local cell-cell interactions. These progenitor cells give rise to taste buds, which are distributed throughout the mouth and pharynx. How widely applicable this model might be for the genesis of taste buds in other vertebrates remains to be seen. However, since it is likely that the taste system of axolotls more closely resembles the ancestral state from which both the amphibian and mammalian taste systems have evolved, it is possible that many of the same developmental mechanisms that give rise to amphibian taste buds are also used to generate the receptor organs in mammals.

Ambystoma↗
PubMed2002

Epizootiology of sixty-four amphibian morbidity and mortality events in the USA, 1996-2001.

A total of 44 amphibian mortality events and 20 morbidity events were reviewed retrospectively. The most common cause of amphibian mortality events was infection by ranaviruses (Family: Iridoviridae). Ranavirus epizootics have abrupt onset and affect late-stage larvae and recent metamorphs. Mortality events due to ranavirus infections affected only widespread and abundant amphibian species, and there was a clear association with high population densities. Chytrid fungal infections accounted for seven mortality events in postmetamorphic anurans only. Chytrid epizootics are insidious and easily overlooked in the field. While both ranavirus and chytrid fungal epizootics were associated with > 90% mortality rates at affected sites, only the chytrid fungal infections were linked to multiple amphibian population declines. Three primitive fungal organisms in the newly erected clade, Mesomycetozoa, caused morbidities and mortalities in anurans and salamanders.

Amphibians↗
PubMed1978

Adaptive properties of olfactory receptors analysed with odour pulses of varying durations.

1. The adaptive properties of salamander of olfactory receptors have been analysed in extracellular unit recordings. Stimulation has been by step pulses of odour of varying durations for 1--10 sec. 2. The most common response was a prolonged impulse discharge that continued throughout the duration of the pulse and terminated abruptly within 1 sec of the end of the pulse. The interval for termination was relatively independent of the pulse duration. Pulses were frequently followed by a period of impulse inactivity lasting 1--3 sec, usually independent of previous pulse duration. 3. The impulse discharges were typically slowly adapting. Initially, during the first 1--2 sec, the frequency rose to 5--10 impulses/sec, at threshold concentration. In some cases, the initial level was maintained throughout the pulse, with little or no adaptation. More commonly, there was a distinct initial phasic peak, followed by decay to a lower level of 4--8 impulses/sec, which was maintained during the pulse. It was concluded that most olfactory receptors are slowly adapting, with variable phasic responsiveness dependent on odour concentration and other factors. 4. Reductions in impulse activity, compared with background, during a pulse were rarely seen. Methods for increasing the level of background activity and the use of very long duration pulses were necessary in order to bring out this type of response. Uniformly reduced activity throughout a pulse was seen clearly in only one case. A pattern consisting of a waning and then recovery of impulse frequency during a pulse was also observed in rare cases. 5. The results have shown that olfactory receptor discharges characteristically have a relatively precise relation to step pulses of odour of varying duration. The properties of the response have implications for the steps involved in the overall processes of activation and inactivation of receptor mechanisms at the olfactory mucosa.

Action Potentials↗
PubMed1984

Control of the generator current in solitary rods of the Ambystoma tigrinum retina.

THe current suppressed by light, the generator current, was studied in solitary salamander (Ambystoma tigrinum) rod photoreceptors with the single-micropipette voltage-clamp technique. The effects of Ca, cyclic GMP, and voltage were measured while voltage- and Ca-activated currents of the inner segment were blocked with Co, Cs, and TEA (tetraethylammonium). The generator current was increased more than 5-fold by lowering the external Ca concentration from 1.5 mM to 10 microM. The generator current could be decreased approximately 1/2 by injecting Ca into an outer segment. Injection of EGTA quickly increased the generator current approximately 2-fold. After injection ceased, the increase was quickly reversed. The generator current could be increased more than 5-fold by injecting cyclic GMP or 8Br-cyclic GMP. Injection of protons, the pH buffer bicine (N,N-bis[2-hydroxyethyl]glycine), or GMP did not produce a change in the generator current. The current-voltage curve for the generator current was influenced by external Co: in 3 mM-Co the current-voltage curve had a negative resistance between -45 and -90 mV; in 0.1 mM-Co the current-voltage curve paralleled the voltage axis between -45 and -90 mV. The difference is attributed to a voltage-dependent block by Co. Susceptibility to the blocking action of Co was reduced by lowering internal or external Ca concentration, or by injecting cyclic GMP. When rods were bathed in a medium containing 7-100 microM-Ca, a step depolarization produced a time-dependent decline in current. Because the reversal potential remained constant, the decline is attributed to an inactivation. The extent of inactivation was reduced by increasing the concentration of external Ca or injecting cyclic GMP.

Ambystoma↗
PubMed1986

Synaptic transmission in amphibian retinae during conditions unfavourable for calcium entry into presynaptic terminals.

Toad (Bufo marinus) retinae were peeled from the pigment epithelium and superfused over the photoreceptor surface with a calcium-poor, cobalt-rich medium. The shape of the electroretinogram indicated that post-synaptic neurones received synaptic input. Adding the putative transmitters glutamate and N-acetylhistidine changed the shape of the electroretinogram. The change suggests that an excess of the putative transmitters blocked a component of synaptic transmission that persisted when a retina was bathed in cobalt. Salamander (Ambystomatigrinum) retinae in hemisected eye cups were superfused over their vitreal surface. Intracellular responses were recorded from photoreceptors. Reducing the calcium concentration in the superfusing medium from 1 mM to less than 10 microM slowly changed responses produced by light. The change indicates that the calcium concentration in the extracellular space surrounding photoreceptors fell to less than 100 microM. When retinae were superfused with a medium containing 1 mM-calcium, 3 mM-barium, and 10 mM-tetraethylammonium (TEA), rods produced action potentials that were later blocked by adding 1 mM-cobalt. Blocking calcium channels with cobalt and lowering the extracellular calcium concentration should together block calcium-dependent synaptic transmission. Intracellular responses were recorded from horizontal cells. After replacing external calcium with cobalt the membrane potential hyperpolarized and responses produced by light became smaller but did not entirely disappear. The responses that remained were less sensitive to light and had an altered shape. The change was reversible. Similar responses could be recorded after prolonged (30-120 min) exposure to cobalt. Electrical synapses between horizontal cells were uncoupled by adding 10 microM-forskolin to the cobalt medium. The polarity of a response could then be reversed if a cell was depolarized by injecting current. The observation of a reversal potential demonstrates that the response was produced by a conductance change. Intracellular responses were recorded from depolarizing and hyperpolarizing bipolar cells while retinae were superfused with cobalt-rich medium. After changing to a cobalt-free medium containing 1 mM-calcium, responses produced by light were slightly smaller. Large responses were recorded after superfusing with cobalt-rich, calcium-poor medium for 30-120 min. The results indicate that synaptic transmission by photoreceptors continues during conditions expected to block the entry of calcium into their presynaptic terminals.

Ambystoma↗
PubMed1990

Rod phototransduction modulated by bicarbonate in the frog retina: roles of carbonic anhydrase and bicarbonate exchange.

1. Effects on rod phototransduction following manipulation of retinal CO2-HCO3- and H+ fluxes were studied in dark-adapted retinas of the frog and the tiger salamander. 2. Rod photoresponses to brief flashes of light were recorded from the isolated sensory retina as electroretinogram mass receptor potentials and from isolated rods by the suction-pipette technique. The experimental treatments were: (1) varying [CO2] + [HCO3-] in the perfusion fluid: (2) applying acetazolamide (AAA), which inhibits the enzyme carbonic anhydrase (CA); and (3) applying 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) which blocks exchange mechanisms transporting HCO3- across cell membranes. 3. The concentration of the internal transmitter of the rods, cyclic GMP, was biochemically determined from the rod outer segment layer of retinas that had been incubated in the same solutions as were used for perfusion in the electrophysiological experiments. 4. The introduction of 6 mM-sodium bicarbonate to replace half the buffer of a nominally CO2-HCO3(-)-free (12 mM-phosphate or HEPES, [Na+] constant) Ringer solution doubled the cyclic GMP concentration in the rod outer segment layer and increased the saturating response amplitude and the relative sensitivity of rods in the intact retina. 5. The introduction of 0.5 mM-AAA into bicarbonate-containing Ringer solution accelerated the growth of saturated responses and sensitivity. Incubation of the retina in AAA-bicarbonate Ringer solution elevated the concentration of cyclic GMP ninefold compared with the phosphate control. 6. No effects of switching to bicarbonate-AAA Ringer solution were observed in the photocurrent of isolated rods drawn into suction pipettes with only the outer segment protruding into the perfusion fluid. The target of AAA is probably the CA-containing Müller cell. 7. The introduction of DIDS into the perfusate (at normal pH 7.5) set off a continuous decay of photoresponses which finally abolished light sensitivity completely. The decay proceeded regardless of whether bicarbonate and AAA were present or not. 8. Rods that had lost their photosensitivity in DIDS recovered almost fully when the pH of the DIDS perfusate was raised to 8.5. They also recovered when DIDS was washed out with bicarbonate Ringer solution at constant pH (7.5). 9. It is proposed that all our treatments ultimately modulate the intracellular pH of the rods which is determined by the relative rates of H+ leakage and HCO3- transport into the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗