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Choline acetyltransferase is found in terminals of horizontal cells that label with GABA, nitric oxide synthase and calcium binding proteins in the turtle retina.

In this study, we discriminated the various types of horizontal cell in the turtle retina on their content of neuroactive substances. Double label immunocytochemistry was performed on sectioned and wholemount retina using antisera to neural- and endothelial-nitric oxide synthase (nNOS, and eNOS), calretinin (CR), calbindin (CB), gamma-aminobutyric acid (GABA) and choline acetyltransferase (ChAT). H1 cells and their axon terminals label with CR, CB and GABA. Only H1 axon terminals label with eNOS. H2 cells contain CB, CR, nNOS and GABA maybe in their dendrites. H3 cells label only with nNOS. The localization of nNOS in the H2 and H3 cells is a novel finding. None of these antibodies labels H4 cells. The photoreceptor subtypes have been differentiated by different intensity of labeling with CB. The accessory member of the double cone is less intensely labeled with CB than the principal member and rods and blue cones do not appear to label at all. ChAT-IR is located in terminal boutons of H1 and H2 horizontal cells and H1 axon terminals and these boutons contact rods and all spectral types of cones. Clearly, GABA is present in H1 horizontal cells and may be used in neurotransmission between horizontal cells and possibly for feedback pathways to photoreceptors. The evidence of nNOS immunoreactivity in H2 and H3 horizontal cells, combined with available physiological evidence, suggests that NO may be involved in electrical coupling and/or modulation of synaptic input to these types of cells. Furthermore, our results raise the possibility that cholinergic synaptic transmission may occur from horizontal cell processes to photoreceptors in the outer plexiform layer of the turtle retina.

Animals↗

Homo- and heterosynaptic long-term potentiation in the medial cortex of the turtle brain in vitro.

Long-term potentiation of excitatory synaptic transmission was studied in an in vitro turtle brain that manifests spontaneous electrocorticogram activity. We show that in the turtle medial cortex, there is evidence of homosynaptic long-term potentiation in the septum-medial cortex pathway. This potentiation has two components, one dependent on NMDA receptor activation and the other independent of this receptor and suppressed by nifedipine, an antagonist of voltage-dependent Ca2+ channels (VDCCs), as occurs in the CA1 of rat hippocampus. Heterosynaptic long-term potentiation was also found in the medial cortex, as a tetanus in the septum also increased the cortico medial-cortico medial response. The intracellular response of pyramidal cells showed that the medial cortex EPSP increased its amplitude paripassu with an increase in the response of evoked field potential after tetanic stimulation in the septum.

Animals↗

Cloning and expression of aromatase in a turtle with temperature-dependent sex determination.

It has been hypothesized that estrogen production may play a pivotal role in the sex determination of reptiles with temperature-dependent sex determination (TSD). This hypothesis has been furthered by studies that have shown higher aromatase activity in the developing ovaries in some reptiles. However, other studies have not consistently supported this hypothesis. In the current study we addressed this issue by cloning P450 aromatase cDNA in the turtle, Trachemys scripta, and developing a quantitative competitive RT-PCR for aromatase. This assay was then used to quantify aromatase mRNA levels in adrenal-kidney-gonad complexes (AKG) during TSD. Aromatase mRNA was detected in the AKGs at both male- and female-producing temperatures from the earliest stage of development sampled (stage 15), through hatching (stage 26). However, levels remained relatively constant during the thermosensitive period of TSD. Further, no significant difference was detected between male- and female-producing temperatures during the thermosensitive period. After the thermosensitive period, aromatase mRNA levels increased in females (this coincides with the period during which the ovaries are differentiating). These results are consistent with those of several previous studies of certain reptiles with TSD. The current results suggest that the expression of aromatase may not be a pivotal regulatory step in the sex determination cascade of this turtle.

Adrenal Glands↗

Diurnal changes in synaptic ribbons of rod cells of the turtle.

Diurnal morphological changes in synaptic ribbons of the rod cells of the turtle were revealed by electron microscopy with serial ultrathin sections. Freshwater turtles (Pseudemys) were maintained under the light-dark cycle with lights on from 0600 to 1800 hr. Retinas around the posterior pole of the eyeball were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide. In total, 124 rod cells and several hundred cone cells were observed. At 0000 (midnight), ribbons are situated close to and perpendicular to the presynaptic membrane. They show single stick-shaped profiles on thin section. From midnight toward noon, the stick-shaped ribbons grow into large multilayered ribbon complexes composed of several sticks arranged parallel to each other. Then, the ribbon complexes begin to disintegrate into irregular fragments from noon toward night. At 1900, aggregates of rounded or club-shaped ribbons are predominant.

Animals↗

Organochlorine contaminants in sea turtles from the Eastern Pacific.

We measured organochlorine residues in three species of sea turtles from the Baja California peninsula, Mexico. Seventeen of 21 organochlorine pesticides analyzed were detected, with heptachlor epoxide and gamma-hexachlorocyclohexane the most prevalent (14 (40%) and 11 (31%) of the 35 tissue samples, respectively). PCBs were detected in all but one of the 9 turtles studied, with congener 18 the most commonly detected (8 (23%) of the samples). The dioxin-like congeners 118 and 180 were detected in 4 (11%) and 3 (9%) of the samples, respectively. Percent contribution of congeners was negatively correlated to their octanol-water partition coefficients, with kidney and muscle containing more lower-chlorinated congeners than liver or adipose samples. Levels of organochlorines detected in the present study were low, potentially attributable to the feeding habits of the predominant species collected in this study (herbivorous) and/or the samples obtained in an unindustrialized region.

Adipose Tissue↗

Autonomous choice in the learning process of a turtle Chinemys reevesii.

We studied animal's learning of spatial discrimination in an experimental environment. Turtles, Chinemys reevesii, were employed for the study. We focused on two independent aspects: (1) turtle's success rate in the task, which is the most common criterion to estimate the ability of the animals, and (2) the statistical properties of the time interval of the task, which is independent on the spatial criterion. For a statistical analysis, we employed the scheme of power law distributions which was recently used to estimate animal behaviors in relation to the idea of the fractal. We addressed the basic problem of whether these two criteria, or any other criteria for this matter, could or could not exclude an observer who studies the animal behavior. To demonstrate inseparability of an observer and the object, we conducted three different learning experiments: (1) complete spatial discrimination, (2) incomplete spatial discrimination, (3) another, different, complete discrimination, in this order. The incomplete one was taken to mean incomplete only for an observer. Our experiments reveal that the same result (success rate) was perceived differently by the animal if the attitude of the observer to the experiments differed. This observation comes to suggest that the notion of autonomous choice on the part of an animal is contingent upon the inseparability between an observer and the object.

Animals↗

A whole-cell clamp study of dendrodendritic synaptic activities in mitral cells of turtle olfactory bulb slices.

A procedure for preparing slices from the turtle olfactory bulb is described in this report. Individual layers of the bulb could be identified in the slices which enabled visual identification of cell types. Mitral cells retained extensive dendritic arborizations in slices of typical thickness, 300-400 microm. The presence of extensive dendritic processes was consistent with the difficulties we encountered in our attempt to achieve adequate space clamp. On the few occasions where an adequate space clamp of a mitral cell was achieved, calcium current exhibited a threshold of - 50 mV and reached its maximal level at - 10 mV. In all cases where calcium current was analysed (n=46), the current exhibited little inactivation. Depolarizing steps in 50% of the mitral cells triggered a burst of feedback synaptic activity after termination of the step. The intensity of feedback activity correlated closely with the amplitude of the depolarizing step, reaching its maximal level at - 10 mV and declining with further depolarization. The bell-shaped relationship between the feedback activity and mitral cell depolarization is consistent with the hypothesis that the feedback activity is mediated by reciprocal synapses on the mitral cell dendrite. This hypothesis is further supported by the inhibitory nature of the feedback synaptic activity: (i) the polarity of the feedback synaptic current could be inverted at the predicted chloride equilibrium potential, (ii) the feedback activity could be completely blocked by 10 microM bicuculline. The analysis of spontaneous synaptic activity showed that it was mostly inhibitory because its polarity could be reversed at the predicted chloride equilibrium potential. In some mitral cells, the frequency of spontaneous activity was noticeably increased when the holding potential was depolarized. This correlation could be attributed to the activation of dendrodendritic synapses. Results shown in this report demonstrate that dendrodendritic synapses are viable in turtle olfactory bulb slices. In addition, the suppression of feedback inhibition by large depolarizing steps of mitral cells suggests that the control of mitral cell dendritic potential is adequate to suppress calcium influx during large depolarizing steps.

Animals↗

Specific binding sites for atrial natriuretic peptide in the freshwater turtle, Amyda japonica.

Specific binding sites for atrial natriuretic peptide (ANP) were investigated by in vitro autoradiographic techniques in various tissues of the freshwater turtle, Amyda japonica. A high density of binding sites for 200 pM of 125I-labelled rANP(1-28) was located in the glomeruli of the kidney and the cortical portion of the adrenal gland. A moderate density of binding sites was seen in the arachnoid matter and choroid plexus of the third and lateral ventricles of the brain and the epididymis. A low density of binding sites was revealed in lamina propria of the mucosa of stomach and intestine, the seminiferous tubules of testes, and the epithelial layer of oviduct. In the presence of excess unlabelled rANP(1-28) (1 microM), binding to these structures were completely displaced. Therefore, specific ANP receptors exist in the kidney, adrenal gland, stomach, intestine, oviduct, epididymis, seminiferous tubules and brain. The ANP system may be involved in physiological regulatory function in the freshwater turtle, Amyda japonica.

Adrenal Glands↗

The cerebral hemisphere of the turtle in vitro. An experimental model with spontaneous interictal-like spikes for the study of epilepsy.

Slice in vitro preparations have been useful to study the cellular basis of some epilepsy related phenomena. However, the cellular mechanisms that generate ictal activity remain poorly understood. Therefore, an experimental in vitro model capable of generating seizure-like activity might contribute to the study of the cellular basis of seizures. The outstanding resistance to hypoxia of turtles enabled us to develop an in vitro preparation that keeps all the cortical neural circuitry intact. A whole cerebral hemisphere of the turtle Chrysemys d'orbigny was isolated (n = 45) and simultaneous electrographic and intracellular recordings were performed in the medial cortex. The electrographic activity was composed by a non-rhythmic, low-voltage (10-20 microV) activity interrupted by spontaneous large (50-700 microV) sharp waves (LSWs). The cellular counterpart of the LSWs was often a burst of action potentials that resembled the paroxysmal depolarisation shift (PDS). Bicuculline (20-40 microM, n = 20) increased the interictal-like activity and in some preparations (3 out of 20) provoked seizure-like events. Complex bursting activity and a slow afterhyperpolarisation were cellular events observed during seizures. We propose that this model might be a valuable tool for the study the cellular mechanisms involved in the transition from the interictal to the ictal activities.

Animals↗

Electrophysiological and morphological properties of neurons in the ventral horn of the turtle spinal cord.

In this report, we present recent findings on the electrophysiological and morphological properties of spinal motoneurons (MNs) and interneurons (INs) of the adult turtle which were studied in slices of the spinal cord. The range of values for the measured electrophysiological parameters in 96 tested cells included: resting potential, -57 to -83 mV; input resistance, 2.5-344 M omega; time constant, 2.5-63 ms; rheobase current, 0.04-5.3 nA; after-hyperpolarization (AHP) duration, 72-426 ms; AHP half-decay time; 11-212 ms; and, slope of the stimulus current-spike frequency relationship, 3.4-235 Hz/nA. For another 20 cells, we made both morphological and electrophysiological measurements (the latter values within the above ranges). Their ranges in morphological properties included: soma diameter, 20-54 microm; soma surface area, 299-2045 microm2; soma volume, 2.3-45 microm3 x 10(4); rostro-caudal dendritic projection distance, 150-1200 microm; and, sum of dendritic lengths, 1.5-16 microm x 10(3). The emphasized findings include: 1) the quality and robustness of the intracellular recordings, which enabled accurate measurement of the action potential's shape parameters (spike, afterhyperpolarization [AHP]); 2) the substantial AHP of the INs' AP; 3) no single action-potential shape parameter (nor combination of parameters) being cardinal for its (or their combined) changes matching the profile of the initial and later phases of spike-frequency adaptation; 4) the utility and flexibility of a cluster analysis (using varying combinations of passive, transitional and active cell properties) for providing a provisional classification of low (like cat S) and high (like cat F) threshold MNs, and groups of INs with non-spontaneous versus spontaneous discharge; 5) the clear-cut morphological confirmation of the provisional classification strategy; 6) the basis for testing the possibility that one of the provisionally classified MN types innervates non-twitch muscle fibers; and 7) the heuristic value of comparing the properties of MNs versus INs across vertebrate species, with an emphasis on the lamprey, turtle, and cat.

Action Potentials↗

Fictive hindlimb motor patterns evoked by AMPA and NMDA in turtle spinal cord-hindlimb nerve preparations.

Application of the glutamate agonists alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA, 5-10 microM), or N-methyl-D-aspartate (NMDA, 50-100 microM) to the turtle spinal cord produced fictive hindlimb motor patterns in low-spinal immobilized animals (in vivo) and in isolated spinal cord-hindlimb nerve preparations (in vitro). For in vivo experiments, drugs were applied onto the dorsal surface of 2-4 adjacent spinal cord segments in and near the anterior hindlimb enlargement. Motor output was recorded unilaterally or bilaterally from hindlimb muscle nerves. AMPA elicited vigorous motor patterns in vivo that included strict hip flexor-extensor and right-left alternation. In most turtles, the monoarticular knee extensor nerve FT-KE was active during the HE phase of AMPA evoked burst cycles, similar to the timing of pocket scratch motor patterns. NMDA was less effective in vivo, typically producing only weak and irregular bursting from hip nerves and little or no knee extensor (KE) discharge. Sensory stimulation of a rostral scratch reflex in vivo could reset an ongoing AMPA-evoked motor rhythm, indicating that cutaneous reflex pathways interact centrally with the chemically activated rhythm generator. Most in vitro preparations consisted of six segments of spinal cord, including the entire 5-segment hindlimb enlargement (D8-S2) and the segment immediately anterior to the enlargement (D7), with attached hindlimb nerves. In contrast to in vivo experiments, in vitro preparations exhibited highly regular, long-lasting motor rhythms when NMDA was superfused over the spinal cord. AMPA also produced rhythmic motor patterns in vitro, but these lasted only a few minutes before they were replaced with tonic discharge. FT-KE timing during in vitro chemically elicited activity was similar to that of sensory-evoked pocket scratch motor patterns. Some NMDA-evoked rhythmicity persisted even in 3-segment (D6-D8) and 1-segment (D8) in vitro preparations, demonstrating that neural mechanisms for chemically activated rhythmogenesis reside even in a single segment of the hindlimb enlargement.

Animals↗

Intestinal candidiasis in a loggerhead sea turtle (Caretta caretta): an immunohistochemical study.

Post mortem examination of a juvenile loggerhead sea turtle (Caretta caretta) stranded in the Canary Islands revealed a fishing-line in the small intestine. Histologically, severe necrotic enteritis, multiple haemorrhages, and marked oedema of the intestinal submucosa were observed. Yeast cells and fungal hyphae were seen in the lamina propria of the intestinal mucosa and in the connective tissue of the submucosa. Because fungal cultures were not taken at the time of necropsy, an immunohistochemical study was performed in order to identify the fungus involved. Specific monoclonal and heterologously absorbed polyclonal antibodies served as the primary reagents for identification of aspergillosis, candidiasis, fusariosis, geotricosis, scedosporiosis, and zygomycosis, using an indirect immunofluorescence staining technique. The fungal elements were strongly stained only by a polyclonal antibody against Candida albicans and a monoclonal antibody against C. albicans. There are no known previous reports of Candida sp. causing skin disease or systemic mycotic infection in sea turtles.

Animals↗

Bimodal respiration and ventilatory behavior in two species of central American turtles: effects of forced submergence.

Respiratory gas exchange in both air and water was measured at rest and during recovery from forced submergence in the giant Mexican musk turtle (Staurotypus triporcatus) and the white-lipped mud turtle (Kinosternon leucostomum). Diving and ventilatory behavior were also measured in unrestrained animals of each species. Despite large differences in cutaneous surface area, both species exhibited an aquatic V(O(2)) and V(CO(2)) of approximately 16 and 45%, respectively, with the remainder explained by aerial gas exchange. Aquatic V(O(2)) and V(CO(2)) did not significantly change during forced submergence or during the recovery period. Aerial V(O(2)) and V(CO(2)), however, profoundly increased after forced submergence in both species and were not significantly different from resting values until approximately 60 min following the treatment. At rest, K. leucostomum took significantly more breaths per breathing bout than S. triporcatus. This inherent ventilation pattern in each species remained unaltered following forced submergence. Cutaneous surface area, therefore, remains a minor component for these two species which rely heavily on pulmonary gas exchange to recover from forced submergence.

Animals↗

Wetness of the nest environment influences cardiac development in pre- and post-natal snapping turtles (Chelydra serpentina).

We dissected hearts from near-term embryos and hatchlings of common snapping turtles (Chelydridae: Chelydra serpentina) whose eggs had incubated on wet or dry substrates, and then dried and individually weighed the heart and yolk-free carcass from each animal. Hearts and carcasses of prenatal and neonatal animals grew at different rates, and the patterns of growth by both heart and carcass differed between wet and dry environments. Hearts grew faster, both in actual mass and in mass adjusted for variation in body size, in embryos and hatchlings whose eggs were incubated on dry substrates than in animals whose eggs were held on wet media. This finding is consistent with a hypothesis that embryos incubating in dry settings experience hypovolemia secondary to dehydration and that enlargement of the heart compensates, in part, for the associated increase in viscosity of the blood. Embryonic turtles seemingly exhibit the same plasticity and response that would be expected from other vertebrate ectotherms subjected to the physiological challenges associated with desiccation and an associated reduction in blood volume.

Animals↗

Thermal independence of muscle tissue metabolism in the leatherback turtle, Dermochelys coriacea.

Metabolic rates of animal tissues typically increase with increasing temperature and thermoregulatory control in an animal is a regional or whole body process. Here we report that metabolic rates of isolated leatherback turtle (Dermochelys coriacea) pectoralis muscle are independent of temperature from 5-38 degrees C (Q10 = 1). Conversely, metabolic rates of green turtle (Chelonia mydas) pectoralis muscle exhibit a typical vertebrate response and increase with increasing temperature (Q10 = 1.3-3.0). Leatherbacks traverse oceanic waters with dramatic temperature differences during their migrations from sub-polar to equatorial regions. The metabolic stability of leatherback muscle effectively uncouples resting muscle metabolism from thermal constraints typical of other vertebrate tissues. Unique muscle physiology of leatherbacks has important implications for understanding vertebrate muscle function, and is another strong argument for preservation of this endangered species.

Acclimatization↗

Hatchling painted turtles (Chrysemys picta) survive only brief freezing of their bodily fluids.

Neonatal painted turtles (Chrysemys picta) spend their first winter inside the shallow, subterranean nest cavity where they completed embryogenesis. Consequently, hatchlings at high latitudes may be exposed to ice and cold during the winter. This study was undertaken to determine how long hatchlings withstand freezing at temperatures slightly below 0 degree C because tolerance for freezing has been proposed to be the key to survival by overwintering animals. A thermocouple was glued to the carapace of each hatchling. The animal was dipped in water to provide a site of nucleation of ice and was then placed into a glass jar that was partially immersed in a circulating bath at -2 degrees C. Carapace temperature was monitored throughout the procedure. When a freezing exotherm was detected, timing of the freezing event began. Animals were maintained in a frozen state for 12-48 h prior to being warmed to room temperature. Of the 39 hatchlings, 22 did not survive, and mortality increased as the duration of freezing increased. Logistic regression indicates that no turtle would have survived in a frozen state for more than 54 h. These results indicate that hatchlings can survive only brief exposure to freezing of the body fluids. Thus, hatchlings cannot tolerate freezing during prolonged periods of cold.

Acclimatization↗

Calcium metabolism in olive ridley turtle eggs during embryonic development.

Analyses of calcium, magnesium, sulphur, potassium and phosphorus content of the eggshell, yolk-albumen and embryos of olive ridley turtle, Lepidochelys olivacea, have been carried out at various stages of embryonic development. Calcium is the major inorganic constituent in the egg (shell and yolk-albumen) and embryos. Other elements are present either in trace or in minute trace amounts. The egg contents (yolk and albumen) provide only 40% of the embryonic calcium requirement of the hatchling. The remaining 60% is provided by the eggshell. The eggshell also undergoes a similar reduction in its calcium content from laying to hatching. Elements other than calcium present in the yolk-albumen are sufficient for normal embryonic development. The movement of calcium from the eggshell to the embryo starts at about the 40th day of development at 29.5 degrees C. Birds, turtles and crocodiles use their eggshell as the secondary source of embryonic calcium requirement. This dependence on the eggshell varies in different groups which is highest in birds and lowest in crocodiles.

Albumins↗

Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart.

We compared responses of turtle heart at 20 degrees C to an anoxic lactic acidosis solution (LA) containing 35 mM lactic acid in an otherwise normal turtle Ringers equilibrated with 3% CO2/97% N2 at pH 7.0) to a solution simulating in vivo anoxic acidosis (VA), with elevated concentrations of lactate, Ca2+, Mg2+, and K+, and decreased Cl-, equilibrated with 10.8% CO2/89.2% N2 at pH 7.0. We examined mechanical properties on cardiac muscle strips and determined intracellular pH (pHi) and high energy phosphates on perfused hearts using 31P-NMR. Maximum active force (Fmax) and the maximum rate of force development (dF/dtmax) of muscle strips were significantly higher during VA than during LA superfusion. An elevation of Ca2+ alone (to 6 mM) in LA significantly increased both Fmax and dF/dtmax but the effects diminished toward the end of the exposure; however, hypercapnic anoxic lactic acidosis (addition of 20 mM HCO3- to LA, equilibrated with 10.8% CO2/89.2% N2, pH 7.0) did not significantly affect Fmax or dF/dtmax. During VA perfusion, pHi (6.73 +/- 0.01) was significantly higher than that during LA perfusion (pHi 6.69 +/- 0.013), but the difference is probably too small to have physiological significance. ATP, creatine phosphate, and inorganic phosphate were not significantly different in the two anoxic solutions. We conclude that the reduction of cardiac mechanical function in vivo is minimized by the integrated effects of changes of ionic concentrations, but the observed changes in Ca2+ and pHi cannot fully explain the effect.

Acidosis, Lactic↗