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Effects of fatigue on the catchlike property in a turtle hindlimb muscle.

The purpose of this report was to test for the possibility that a catchlike, force-enhancing property, attributable to a particular stimulation pattern, could be evoked in non-mammalian turtle muscle, just as it has been shown in mammalian muscle. We tested for the presence of this property in dynamic lengthening and shortening contractions, as well as in the more commonly studied isometric contractions. A second aim was to note the effects of fatigue on the catchlike property, if the latter was present. The force response of the external gastrocnemius muscle in the adult turtle, Pseudemys ( Trachemys) scripta elegans, was compared for a control, constant-frequency 10 Hz, 1-s duration stimulation pattern using 0.1-ms pulses vs. the same pattern, but with two additional pulses within the first 100-ms interspike interval of the control stimulus train. This latter train produced a pronounced and prolonged enhancement of muscle force, which was attributed to a catchlike effect. It was greatly increased when the muscle was in a fatigued state. The extent of this force enhancement was significantly different for the three contraction types, and generally in the order: isometric>lengthening>shortening contraction. These differences were greater in fatigued vs. fresh muscle. Comparative aspects and potential mechanisms underlying the catchlike effect are discussed.

Action Potentials↗

Resistance to disuse atrophy in a turtle hindlimb muscle.

The purpose of this study was to characterize the changes in a turtle hindlimb muscle (external gastrocnemius) after exposure to three conditions of disuse: immobilization, tenotomy, and spinalization. Histochemical analysis and measurement of muscle fiber cross-sectional area and weighted cross-sectional area were used to assess the potential conversion of muscle fiber types and changes in fiber size. It was found that unlike its counterpart in mammalian endotherms, the external gastrocnemius muscle of the adult turtle, Trachemys scripta elegans, was remarkably resistant to each model of reduced muscle function. It is suggested that such resistance to disuse is due to intrinsic mechanisms that enable heterothermic mammals and ectothermic vertebrates to tolerate an unfavorable climate and food and water shortages by using hypometabolic states.

Adenosine Triphosphatases↗

Afterhyperpolarization-firing rate relation of turtle spinal neurons.

This study addressed the afterhyperploarization-firing rate relationship of unanesthetized turtle spinal motoneurons and interneurons. The afterhyperploarization of their solitary action potential at rheobase was compared to that during the cells' minimum and maximum firing rates. Like previous mammalian findings, afterhyperpolarization duration and area at rheobase were 32 and 19% less for high- versus low-threshold motoneurons. Contrariwise, maximum firing rate was two times less for the high-threshold group. Other new findings were that for high- versus low-threshold interneurons, afterhyperpolarization duration and area were 25 and 95% less, and maximum firing rate 21% higher for the high-threshold group. For combined motoneurons versus interneurons, there were no differences in afterhyperpolarization duration and area at rheobase, whereas maximum firing rate was 265% higher for the interneurons. For high-threshold motoneurons alone, there were significant associations between minimum firing rate and afterhyperpolarization duration and area measured at rheobase. In summary, this study showed that (1) the afterhyperploarization values of both turtle spinal motoneurons and interneurons at rheobase provided little indication of their corresponding values at the cells' minimum and maximum firing states, and (2) the evolution of afterhyperploarization from rheobase to maximum firing state differed both qualitatively and quantitatively for motoneurons versus interneurons.

Action Potentials↗

Adaptations to terrestrial overwintering of hatchling northern map turtles, Graptemys geographica.

We conducted a 3-year field and laboratory study of winter biology in hatchlings of the northern map turtle ( Graptemys geographica). At our study area in northern Indiana, hatchlings routinely overwintered in their natal nests, emerging after the weather warmed in spring. Winter survival was excellent despite the fact that hatchlings were exposed frequently to subfreezing temperatures (to -5.4 degrees C). In the laboratory, cold-acclimated hatchlings exhibited low rates of evaporative water loss (mean=2.0 mg g(-1) day(-1)), which would enable them to conserve body water during winter. Laboratory-reared hatchlings were intolerant of freezing at -2.5 degrees C for 24 h, conditions that are readily survived by freeze-tolerant species of turtles. Winter survival of hatchling G. geographica probably depended on their extensive capacity for supercooling (to -14.8 degrees C) and their well-developed resistance to inoculative freezing, which may occur when hatchlings contact ice and ice-nucleating agents present in nesting soil. Supercooled hatchlings survived a brief exposure to -8 degrees C. Others, held at -6 degrees C for 5 days, maintained ATP concentrations at control levels, although they did accumulate lactate and glucose, probably in response to tissue hypoxia. Therefore, anoxia tolerance, as evidenced by the viability of hatchlings exposed to N(2) gas for 8 days, may promote survival during exposure to subfreezing temperatures.

Acclimatization↗

Blood-respiratory and acid-base changes during extended diving in the bimodally respiring freshwater turtle Rheodytes leukops.

Changes in blood-gas, acid-base, and plasma-ion status were investigated in the bimodally respiring turtle, Rheodytes leukops, during prolonged dives of up to 12 h. Given that R. leukops routinely submerges for several hours, the objective of this study was to determine whether voluntarily diving turtles remain aerobic and simultaneously avoid hypercapnic conditions over increasing dive lengths. Blood PO(2), PCO(2), and pH, as well as plasma concentrations of lactate, glucose, Na(+), K(+), Cl(-), total Ca, and total Mg were determined in venous blood collected from the occipital sinus. Blood PO(2) declined significantly with dive length; however, oxy-haemoglobin saturation remained greater than 30% for all R. leukops sampled. No changes were observed in blood PCO(2), pH, [HCO(3)(-)], or plasma glucose, with increasing dive length. Despite repeated dives lasting more than 2 h, plasma lactate remained less than 3 mmol l(-1) for all R. leukops sampled, indicating the absence of anaerobiosis. Compensatory acid-base adjustments associated with anaerobiosis (e.g. declining [Cl(-)], increasing total [Ca] and [Mg]) were likewise absent, with plasma-ion concentrations remaining stable with increasing dive length. Results indicate that R. leukops utilises aquatic respiration to remain aerobic during prolonged dives, thus effectively avoiding the development of a metabolic and respiratory acidosis.

Acid-Base Equilibrium↗

Response of heart rate and cloacal ventilation in the bimodally respiring freshwater turtle, Rheodytes leukops, to experimental changes in aquatic PO2.

Changes in heart rate (f(H)) and cloacal ventilation frequency (f(C)) were investigated in the Fitzroy turtle, Rheodytes leukops, under normoxic (17.85 kPa) and hypoxic (3.79 kPa) conditions at 25 degrees C. Given R. leukops' high reliance on aquatic respiration via the cloacal bursae, the objective of this study was to examine the effect of varying aquatic PO(2) levels upon the expression of a bradycardia in a freely diving, bimodally respiring turtle. In normoxia, mean diving f(H) and f(C) for R. leukops remained constant with increasing submergence length, indicating that a bradycardia failed to develop during extended dives of up to 3 days. Alternatively, exposure to aquatic hypoxia resulted in the expression of a bradycardia as recorded by a decreasing mean diving f(H) with increasing dive duration. The observed bradycardia is attributed to a hypoxic-induced metabolic depression, possibly facilitated by a concurrent decrease in f(C). Results suggest that R. leukops alters its strategy from aquatic O(2) extraction via cloacal respiration in normoxia to O(2) conservation when exposed to aquatic hypoxia for the purpose of extending dive duration. Upon surfacing, a significant tachycardia was observed for R. leukops regardless of aquatic PO(2), presumably functioning to rapidly equilibrate blood and tissue gas tensions with alveolar gas to reduce surfacing duration.

Animals↗

Accumulation of lactate by supercooled hatchlings of the painted turtle (Chrysemys picta): implications for overwinter survival.

Hatchlings of the North American painted turtle (Chrysemys picta) typically spend their first winter of life inside the shallow, subterranean nest where they completed embryogenesis the preceding summer. Neonates at northern localities consequently may be exposed during winter to subzero temperatures and frozen soil. Hatchlings apparently survive exposure to such conditions by supercooling, but the physiological consequences of this adaptive strategy have not been examined. We measured lactate in hatchling painted turtles after exposure to each of three temperatures (0 degree C, -4 degrees C, and -8 degrees C) for three time periods (5 days, 15 days, and 25 days) to determine the extent to which overwintering hatchlings might rely on anaerobic metabolism to regenerate ATP. Whole-body lactate increased with increasing duration of exposure and decreasing temperature, and the highest levels were associated with the group that experienced the highest mortality. These results indicate that animals may develop a considerable lactic acidosis during a winter in which temperatures fall below 0 degree C for weeks or months and that accumulation of lactate may contribute to mortality of overwintering animals.

Acidosis, Lactic↗

Discordant responses of mitogen-activated protein kinases to anoxia and freezing exposures in hatchling turtles.

The role of two vertebrate mitogen-activated protein kinases (MAPKs) in mediating responses to in vivo anoxia or freezing exposures was examined in four organs (liver, heart, kidney and brain) of hatchling red-eared turtles, Trachemys scripta elegans, which are naturally tolerant of these stresses. The extracellular signal-regulated kinases were not stress-activated except in brain of frozen turtles. The c-Jun NH2-terminal kinases (JNKs) were transiently activated by anoxia exposure in all four organs (after 1 h in brain or 5 h in other organs) but activity was suppressed during freezing except in brain which showed a transient activation of JNK after 1 h. Changes in the concentrations of the transcription factors, c-Fos and c-Myc, were also stress- and organ-specific. The patterns of MAPK activation in a stress-tolerant animal suggest the relative importance of these kinase pathways in cellular adaptation to oxygen deprivation or freezing and identify novel natural activators of MAPKs in vivo. The specificity of the signaling pathways is also emphasized here as the general whole-body stresses, anoxia and freezing, activated individual MAPKs in a tissue-, time-, and stress-dependent manner.

Animals↗

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina.

The histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and immunoreactivity of neuronal nitric oxide synthase (nNOS-IR) can be demonstrated in various cell types of the vertebrate retina. In this study, we have focused on characterizing the different NADPH-d-positive amacrine cell types in turtle retina. Cryostat sections were examined by confocal laser scanning microscopy for double immunofluorescence with antibodies against nNOS and either GABA or glycine, or by combining histochemistry with immunocytochemistry to obtain triple labeling with NADPH-d, GABA, and glycine. Forty-eight percent of the NADPH-d-labeled amacrine cells colocalized GABA, 52% glycine. Here we show that two morphologically different types of amacrine cell are nNOS/glycine-IR and three types are nNOS/GABA-IR. Antibodies against calretinin, parvalbumin, somatostatin, tyrosine hydroxylase, and choline acetyltransferase did not colocalize with nNOS-IR or NADPH-d-labeled amacrine cells, but 15% of the NOS-labeled amacrine cells showed immunoreactivity against calbindin. Only GABA has been seen to colocalize with NADPH-d in amacrine cells in previous reports in other species. The finding here of glycine colocalizing with NO-containing cells is novel. We suggest that NO, apart from its well known function in gap junction regulation, can also modulate the release of both GABA and glycine in the turtle retina.

Animals↗

Molecular and cytogenetic characterization of site-specific repetitive DNA sequences in the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae).

A novel family of repetitive DNA sequences that are components of constitutive heterochromatin were cloned from BglI-digested genomic DNA of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae), and characterized by filter hybridization and chromosome in-situ hybridization. The BglI-family of repetitive sequences were classified into four types by their genome organization and chromosomal distribution as follows: the repeated sequences located on (1) two pairs of microchromosomes, (2) four pairs of microchromosomes,(3) about half the number of microchromosomes and (4) the interstitial region of the short arm of chromosome 2. The presence of microchromosome-specific repetitive sequences has also been reported in the Struthioniformes and Galliformes, suggesting that turtle chromosomes retain some similarity to the chromosome structure as well as the karyotypes of avian species.

Animals↗

Purification and characterization of a low molecular weight basic protein from marine turtle egg white.

The egg white of marine turtle (Caretta caretta Linn.) and one species of tortoise (Geomyda trijuga trijuga Schariggar) contain a low molecular weight basic protein. It has been purified to homogeneity from the egg white of marine turtle and characterized in terms of its major physicochemical and chemical properties. The molecular weight of this protein calculated from gel filtration, sodium dodecyl sulfate-gel electrophoresis in the presence of urea, sedimentation-diffusion data, and amino acid composition is 4300. Its isoelectric point is at pH 11.1 and intrinsic viscosity is 0.038 dl g-1 in 0.2 M NaCl. It has a Stokes radius of 12.6 A and a diffusion coefficient of 16.50 x 10(-7) cm2 s-1. Analysis of the far-ultraviolet circular dichroic spectrum has shown that the basic protein contains 27% beta-pleated sheet and little or no alpha-helix. It possesses a single polypeptide chain of 40 amino acid residues with three disulfide bonds. It lacks serine, methionine, phenylalanine and carbohydrate moiety. It binds to DNA and stimulates ATPase activity due to its strong basicity. The complex of DNA-basis protein is partially resistant to the action of DNase.

Amino Acids↗

Effects of 4-acetamido-4'-isothiocyano-2,2-disulfonic stilbene on ion transport in turtle bladders.

The disulfonic stilbene (4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO-3,Cl-) moiety of the short-circuiting current is eliminated by 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene, but only after its addition to the serosal bathing fluid. Whereas 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene has no effect on Na+ transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na+ + K+)-ATPase of both turtle bladder and eel electric organ.

Acetazolamide↗

Localization and characterization of transport-related elements in the plasma membrane of turtle bladder epithelial cells.

A mixed membrane preparation obtained from turtle bladder epithelial cells contains (Na+ + K+)-ATPase, adenylate cyclase and protein kinase, which interact with ouabain, norepinephrine and cyclic AMP, respectively. When such a preparation is obtained from bladders which had been preexposed to serosal fluids containing the tritiated form of 4,4'-diisothiocyano-2,2'-disulfonic stilbene, the subsequently isolated membrane proteins are enriched in tritium as well as in the afore-mentioned enzymes, none of which is inhibited. Free-flow electrophoresis separates the mixed membrane preparation into two distinguishable groups: one, construed as apical membranes, is enriched in norepinephrine-sensitive adenylate cyclase and cyclic AMP-sensitive protein kinase; the other, construed as basal-lateral membranes, is enriched in ouabain-sensitive ATPase and 4,4'-diisothiocyano-2,2'-disulfonic stilbene-binding proteins. The physiological counterparts of these enzymatically defined membrane markers are the mucosal sidedness of the transport effects of norepinephrine and cyclic AMP derivatives and the serosal sidedness of the transport effects of ouabain and disulfonic stilbenes in the intact turtle bladder. The discreteness and ion selectivity of each membrane-bound, transport-related element are discussed in relation to the corresponding characteristics of each transport process in vivo; the possibility of regulation of anion transport by adenylate cyclase-protein kinase system is also discussed.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

Effect of furosemide on ion transport in the turtle bladder: evidence for direct inhibition of active acid-base transport.

The diuretic furosemide inhibits acid-base transport in the short-circuited turtle bladder. It inhibits luminal acidification when present in either mucosal or serosal bathing fluids, but decreases alkalinization only from the serosal side of the tissue. The inhibition of both acid-base transport processes is independent of ambient Cl-; and the disulfonic stilbene, SITS, an inhibitor of Cl--HCO3- exchange, fails to prevent the furosemide-elicited inhibition of alkalinization. These results preclude an absolute requirement of a furosemide-sensitive Cl--HCO3- exchange by these transport processes. The drug also interferes with the CO2-induced stimulation of acidification and alkalinization. The inhibition of the residual acidification in acetazolamide-treated, acidotic bladders, however, suggests an action at sites other than cytosolic carbonic anhydrase. Although active Na+ and Cl- reabsorption and tissue oxygen uptake are also decreased by furosemide, the rate of oxygen consumption uncoupled by 2,4-dinitrophenol is not diminished, indicating a primary inhibition of the various ion transport processes, not of metabolism. It is proposed that inhibition of transepithelial acid-base transport by furosemide in the turtle bladder includes inhibition of the acid-base pumps.

2,4-Dinitrophenol↗

ATP-dependent H+ transport by the turtle bladder: NBD-C1 preferentially inhibits the vanadate-insensitive component in isolated membranes.

We investigated the inhibitory effects of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) on ATP-dependent H+ accumulation by membrane vesicles prepared from the turtle urinary bladder epithelium. NBD-Cl at 30 microM was found to completely inhibit the vanadate-insensitive component of H+ transport, with half-maximal inhibition occurring at 4.2 to 5.4 microM. In contrast, the vanadate-inhibitable component was unaffected by 30 microM NBD-Cl. At high concentrations (300 microM), both components were fully inhibited. The results confirm the presence of two distinct H+ transport processes in turtle bladder membranes and identify selective inhibitors, NBD-Cl and vanadate, for each process.

4-Chloro-7-nitrobenzofurazan↗

The effects of extensive forebrain lesions on visual discriminative performance in turtles (Chrysemys picta picta).

Though anatomical research has demonstrated major ascending telecephalically directed visual channels in reptiles, little behavioral research has examined reptilian forebrain visual functions. The present study reports the effects of extensive forebrain lesions, involving either severe destruction of dorsal thalamus or disruption of the fibers of the lateral forebrain bundle (by lesions of the basolateral telecephalon), upon visual discriminative performance in the turtle. Such lesions, which extensively damage the ascending visual pathways, rendered turtles incapable of relearning preoperatively acquired visual discriminative problems. The magnitude of the visual impairments observed following such forebrain lesions suggest a major role on the part of the forebrain in visual processing in reptiles.

Animals↗

Evoked potential and single unit responses to olfactory nerve volleys in the isolated turtle olfactory bulb.

An in vitro preparation of the whole olfactory bulb of the turtle has been investigated using extracellular evoked potentials and unitary spikes. The compound action potential has a slow conduction velocity (0.15 mm/msec), brief relatively refractory period (10 msec) and prolonged supernormality (several hundred msec). The evoked potentials undergo prolonged periods of partial and complete suppression, as tested by the responses to paired volleys in the olfactory nerves. The suppression has a complex temporal pattern, and lasts more than 4 sec. Similar periods were revealed by the unitary spike responses of presumed mitral cells to olfactory nerve volleys. The results are similar to those reported in in vivo studies of turtle and other species. They suggest that the basic properties of mitral cells and their synaptic interactions with local interneurons are maintained in this isolated olfactory bulb preparation.

Animals↗