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Excitatory amino acid receptors mediate slow synaptic transmission in turtle cerebellum.

In the isolated turtle cerebellum intracellular recordings from Purkinje cell dendrites and somata reveal novel slow excitatory synaptic potentials evoked by activation of climbing fiber (CF) or parallel fiber (PF) inputs. Classical fast excitatory synaptic responses to CF and PF stimulation are followed by large, slow excitatory postsynaptic potentials (sEPSPs) which are associated with an increase in conductance and are enhanced by hyperpolarization. Both sEPSPs are blocked by the excitatory amino acid (EAA) antagonist kynurenate, but not by DL-2-amino-5-phosphonovalerate (AP-5). The EAA receptor antagonist L-amino-4-phosphonobutyric acid (L-AP-4) reversibly blocked the PF-sEPSP without affecting the CF-sEPSP. Two novel slow synaptic potentials mediated by excitatory amino acid receptors can therefore be observed in turtle cerebellum which may play an important role in synaptic integration.

Action Potentials↗

Intracellular injection of inositol 1,4,5-trisphosphate increases a conductance in membranes of turtle vomeronasal receptor neurons in the slice preparation.

Inositol 1,4,5-trisphosphate (IP3) was injected into turtle vomeronasal receptor neurons in the slice preparation under a whole-cell patch clamp, and the evoked current was measured. Application of 0.1 mM IP3 evoked a prolonged, inward current (52 of 98 neurons) with an average peak amplitude of 89.9 +/- 10.9 pA. The reversal potential of the response induced by IP3 was estimated to be -32.3 +/- 1.5 mV (6 neurons). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP3 evoked inward current to 18.0 +/- 4.6 pA (5 neurons). This is the first study to demonstrate that the membranes of the turtle vomeronasal neurons carry IP3-activated conductance.

Animals↗

Odor responses after complete desensitization of the cAMP-dependent pathway in turtle olfactory cells.

The degree of contribution of the cAMP signal transduction pathway to odor responses was examined by recording current responses from isolated turtle olfactory cells under the whole-cell voltage clamp conditions. The cAMP signal transduction pathway was desensitized by dialyzing 1 mM cAMP and 0.5 mM IBMX from the patch pipette into the cells. Extracellular application of 3 mM cpt-cAMP, a membrane-permeable cAMP analogue elicited no response, indicating that the cAMP pathway was completely desensitized. Application of an odorant cocktail induced a large inward current under these conditions, suggesting that the cAMP-independent signal transduction contributes significantly to generation of odor responses in the turtle.

1-Methyl-3-isobutylxanthine↗

Effects of sustained oxygen depletion on tissue pyruvate kinase activities in the freshwater turtle, Pseudemys scripta elegans.

1. The effect of sustained (48 hr) oxygen depletion on tissue (brain, heart, skeletal muscle) pyruvate kinase (PyKi) activities was examined in the pond turtle (Pseudemys scripta elegans). 2. PyKi activities in skeletal muscle are significantly increased (from 322 +/- 84 to 450 +/- 95) following 48 hr of tissue hypoxia. PyKi activities in brain may already be elevated under ambient conditions but do not change following prolonged submersion (334 +/- 75 vs 325 +/- 77). Cardiac muscle PyKi is actually decreased (from 135 +/- 35 to 94 +/- 18) under anaerobic conditions. 3. The data suggest that prolonged O2 depletion may increase PyKi biosynthesis in turtle skeletal muscle (subserving enhanced glycolysis), but also demonstrate that factors other than oxygen availability are involved in the regulation of tissue PyKi activities in intact vertebrates.

Animals↗

Glycoproteins in Rathke's gland secretions of loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempi) sea turtles.

1. The Rathke's gland secretions of loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempi) sea turtles contain 20 and 10 mg of protein/ml, respectively. The proteins of each species were separated by gel filtration into two major fractions, one (35%) in the excluded volume, and one (50%) with a molecular mass of approximately 55 kDA. 2. The 55 kDa fraction from each species' secretions exhibits a single band on SDS-PAGE (Mr approximately equal to 55,000) and a single amino-terminal sequence. 3. The amino acid compositions of the two 55 kDa proteins are similar, and the first 15 residues of their amino terminus are identical. Both proteins contain glucosamine. 4. Analyses of the amino acid and amino sugar composition of the high molecular weight fractions from the two turtle species also indicate similarities; there are distinct differences between them and their 55 kDa proteins.

Amino Acid Sequence↗

Characterization of gamma-glutamyl transpeptidase from the Rathke's gland secretions of Kemp's ridley sea turtles (Lepidochelys kempi).

The secretion produced by Rathke's glands of Kemp's ridley sea turtles (Lepidochelys kempi) contains the enzyme gamma-glutamyl transpeptidase. The approximately 200 kDa enzyme contains two different subunits, alpha (54 kDa) and beta (21 kDa), in an unknown stoichiometry. The enzyme transfers gamma-Glu from a number of different donors, such as glutamine, glutathione, S-Me-glutathione, N epsilon(gamma-Glu)-Lys, gamma-Glu-Ala, and other gamma-glutamyl amino acids, either to water or to a variety of acceptor substrates. It appears that a free alpha-amino group is the preferred acceptor. The enzyme is not inhibited by typical sulfhydryl reagents such as N-ethyl-maleimide, p-(chloro)mercuri-benzoate or 5,5'-dithio-bis-(2-nitrobenzoate) or by the active Ser reagent tosyl fluoride. Maleate stimulates the activity of the enzyme, and in the presence of 100 mM maleate 2 mM tosyl fluoride becomes an inactivator of the enzyme. The catalytic and molecular properties of the turtle gamma-glutamyl transpeptidase are similar to those established for mammalian gamma-glutamyl transpeptidase. Neither the physiological role of the enzyme nor the biological function of the secretion in which it occurs is understood at this time.

Amino Acid Sequence↗

Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum).

Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum, Kinosternidae) were analyzed by gas chromatography-mass spectrometry (GC-MS) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Analysis by GC-MS indicates 2,3-dihydroxypropanal and C3-C24 free or esterified fatty acids. Analysis by SDS-PAGE indicates a major protein component with an approximate molecular mass of 60 kDa and minor components ranging from ca. 23 to 34 kDa. The major component of K. subrubrum glandular secretions exhibits a mobility that matches that of the Kemp's ridley sea turtle (Lepidochelys kempi, Cheloniidae), suggesting that these proteins are evolutionarily conserved.

Animals↗

Climbing fiber projection to the turtle cerebellum: longitudinally oriented terminal zones within the basal third of the molecular layer.

Injections of various radiolabeled amino acids were made into the caudal rhombencephalic tegmentum in the turtle, Pseudemys scripta elegans. In animals in which injections encompassed the entire area previously identified as the possible source of cerebellar climbing fibers, the basal portion of the molecular layer was labeled almost throughout the contralateral cerebellum. In cases of restricted injections, labeled climbing fibers terminated in quite distinct longitudinally oriented zones. Control injections immediately caudal and rostral to the mentioned rhombencephalic region resulted in labeling of only the granular layer. The organization of the climbing fiber projection in the turtle is compared with the organization of the climbing fiber projection in representatives of birds and mammals as described in the literature. Reptiles, birds and mammals appear to be similar to each other in that they all have climbing fiber projections organized in longitudinal zones; whereas they differ with respect to the extent of climbing fiber penetration into the superficial portions of the molecular layer, mammals having climbing fibers that project significantly more superficially than those of birds or reptiles.

Animals↗

Cytochrome P-450 mediated metabolism of progesterone by adrenal microsomes of PCB-treated and untreated barn owl (Tyto alba) and marsh turtle (Mauremys caspica) in comparison with the guinea-pig.

1. Significant differences between species were observed in the profile of steroids produced from progesterone by adrenal microsomes as well as in the effects elicited by Aroclor 1254 on cytochrome P-450-mediated activities. 2. In the guinea-pig, the major metabolites were products of the corticosteroid pathway but products of the androgenic pathway were also detected; in the barn owl products of both pathways were also formed while in the marsh turtle only products of the corticosteroid pathway were detected. 3. The effect of Aroclor 1254 on P-450C21 activity in the turtle and barn owl was inductive in contrast to the inhibitory effect observed in the guinea-pig.

17-alpha-Hydroxyprogesterone↗

Comparative investigation of retinal responses to brief light stimuli: 2-amino-4-phosphonobutyrate studies--II. Turtle retina (Emys orbicularis).

1. Electroretinogram (ERG) and responses of single ganglion cells to 75 microseconds light flashes, presented on two different backgrounds, were studied. Additionally, stimulation with long lasting stimuli (ordinarily 5 sec ON-, 15 sec OFF-) was used. 2. 2-amino-4-phosphonobutyrate (APB) at a concentration of 450 microM on average was used to separate OFF- from ON- channels. It is known, that in other species APB selectively blocks the activity of ON- channel only. 3. The existence of APB- sensitive membrane receptors was demonstrated in the turtle retina. As in other species, APB abolished the ERG b-wave and enhanced the d-wave, when long lasting stimulation was used. 4. By means of APB, two components were shown to exist in the ERG positive wave in response to a flash. APB abolished the first component and did not influence or enhanced the second one. 5. By means of APB, one or two components in the impulse activity in response to flash were demonstrated, depending on the type of ganglion cell (ON-, OFF- or ON-OFF). The latency of the first component was 70 msec and the latency of the second one 210 msec on average. APB abolished the first component, and enhanced the second one. 6. The data obtained show that both ON- and OFF- channels take part in the generation of the turtle retinal responses to brief stimuli. 7. Based on the results obtained, some peculiarities of the network organization of the ganglion cells' receptive fields are discussed.

Action Potentials↗

Forskolin enhanced off-response in the turtle olfactory system.

Olfactory responses appear not only at the onset of odor stimulation, but also at the termination of the stimulation. It is widely considered that the cAMP-pathway is involved in the generation of odor responses. We examined how cAMP affects the generation of off-responses. To explore the role of the cAMP-pathway, odorants were applied to the turtle olfactory epithelium after forskolin at high concentrations which saturated the olfactory response to forskolin. Various odorants induced off-responses after 50 microM forskolin, indicating that off-responses are not induced via the cAMP-dependent pathway. However, the magnitude of the off-response after forskolin varied from 100 to 1400% of those of off-responses in its absence, indicating that forskolin greatly enhanced the off-responses to some odorants. The off-response after 0.1 mM citralva was also enhanced by 3 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (cpt-cAMP). These results suggested that cAMP modulates off-responses in the turtle olfactory system.

1-Methyl-3-isobutylxanthine↗

Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs.

In embryos of the European pond turtle, sexual differentiation of gonads is temperature-dependent. Production of oestrogens appears to play a key role in this phenomenon. Gonadal aromatase activity was measured in embryos incubated at 25 degrees C (masculinizing temperature) and at 30 degrees C (feminizing temperature). At the beginning of the thermosensitive period, the aromatase activity was low at both temperatures but was somewhat higher at 30 than at 25 degrees C. Afterwards, it remained low in differentiating testes at 25 degrees C, whereas it increased in differentiating ovaries at 30 degrees C to form a marked peak when germ cells underwent meiotic prophase. Eggs were shifted either from 25 to 30 degrees C (highly feminizing) or from 30 to 35 degrees C for 6 days at different stages of embryonic development. The 25-35 degrees C shifts performed during the thermosensitive period strongly increased the aromatase activity but were ineffective after this period. The 30-35 degrees C shifts increased the aromatase activity at all stages. Altogether, results indicate that, in differentiating gonads of turtle embryos, temperature acts on the regulation of synthesis (and therefore activity) of cytochrome P-450 aromatase (P-450-aro). The expression of the P-450-aro gene itself could be temperature-dependent. However, temperature could also act upon the expression of another gene involved in P-450-aro regulation.

Animals↗

Localization of GABA (gamma-aminobutyric acid) markers in the turtle's basal optic nucleus.

Recent physiological data have demonstrated that retinal slip, the sensory code of global visual pattern motion, results from complex interactions of excitatory and inhibitory visual inputs to neurons in the turtle's accessory optic system (the basal optic nucleus, BON). In the present study, the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), its synthetic enzyme, glutamic acid decarboxylase (GAD-67) and its receptor subtypes GABA(A) and GABA(B) receptors were localized within the BON. GABA antibodies revealed cell bodies and processes, whereas antibodies against GAD revealed a moderate density of immunoreactive puncta throughout the BON. GAD in situ hybridization labeled BON cell bodies, indicating a possible source of inhibition intrinsic to the nucleus. Ultrastructural analysis revealed terminals positive for GAD that exhibit symmetric synaptic specializations, mainly at neuronal processes having small diameters. Neurons exhibiting immunoreactivity for GABA(A) receptors were diffusely labeled throughout the BON, with neuronal processes exhibiting more labeling than cell bodies. In contrast, GABA(B)-receptor-immunoreactive neurons exhibited strong labeling at the cell body and proximal neuronal processes. Both these receptor subtypes are functional, as evidenced by changes of visual responses of BON neurons during application to the brainstem of selective receptor agonists and antagonists. Therefore, GABA may be synthesized by BON neurons, released by terminals within its neuropil and stimulate both receptor subtypes, supporting its role in mediating visually evoked inhibition contributing to modulation of the retinal slip signals in the turtle accessory optic system.

Animals↗

Developmental changes in the fibre population of the optic nerve follow an avian/mammalian-like pattern in the turtle Mauremys leprosa.

The changes in the axon and growth cone numbers in the optic nerve of the freshwater turtle Mauremys leprosa were studied by electron microscopy from the embryonic day 14 (E14) to E80, when the animals normally hatch, and from the first postnatal day (P0) to adulthood (5 years on). At E16, the first axons appeared in the optic nerve and were added slowly until E21. From E21, the fibre number increased rapidly, peaking at E34 (570,000 fibres). Thereafter, the axon number decreased sharply, and from E47 declined steadily until reaching the mature number (about 330,000). These observations indicated that during development of the retina there was an overproduction and later elimination of retinal ganglion cells. Growth cones were first observed in the optic nerve at as early as E16. Their number increased rapidly until E21 and continued to be high through E23 and E26. After E26, the number declined steeply and by E40 the optic nerve was devoid of growth cones. These results indicated that differentiation of the retinal ganglion cells occurred during the first half of the embryonic life. To examine the correlation between the loss of the fibres from the optic nerve and loss of the parent retinal ganglion cells, retinal sections were processed with the TUNEL technique. Apoptotic nuclei were detected in the ganglion cell layer throughout the period of loss of the optic fibres. Our results showed that the time course of the numbers of the fibres in the developing turtle optic nerve was similar to those found in birds and mammals.

Age Factors↗

Egg components, egg size, and hatchling size in leatherback turtles.

Relationships between egg size, egg components, and neonate size have been investigated across a wide range of oviparous taxa. Differences in egg traits among taxa reflect not only phylogenetic differences, but also interactions between biotic (i.e., maternal resource allocation) and abiotic (i.e. nest environment conditions) factors. We examined relationships between egg mass, egg composition, and hatchling size in leatherback turtles (Dermochelys coriacea) because of the unique egg and reproductive characteristics of this species and of sea turtles in general. Albumen comprised 63.0%+/-2.8% (mean+/-S.D.) of egg mass and explained most of the variation in egg mass, whereas yolk comprised only 33.0%+/-2.7%. Additionally, leatherback albumen dry mass was approximately 16% of albumen wet mass. Whereas hatchling mass increased significantly with egg mass (n = 218 clutches), hatchling mass increased by only approximately 2 g for each 10 g increase in egg mass and was approximately 10-20 g greater than yolk mass. Taken together, our results indicate that albumen might play a particularly significant role in leatherback embryonic development, and that leatherback eggs are both capable of water uptake from the nest substrate and also possess a large reservoir of water in the albumen. Relationships between egg mass and egg components, such as variation in egg mass being largely explained by variation in albumen mass and egg mass containing a relatively high proportion of albumen solids, are more similar to bird eggs than to eggs of other non-avian reptiles. However, hatchling mass correlates more with yolk mass than with albumen mass, unlike patterns observed in bird eggs of similar composition.

Animals↗

Beyond anoxia: the physiology of metabolic downregulation and recovery in the anoxia-tolerant turtle.

The freshwater turtle Trachemys scripta is among the most anoxia-tolerant of vertebrates, a true facultative anaerobe able to survive without oxygen for days at room temperature to weeks or months during winter hibernation. Our good friend and colleague Peter Lutz devoted nearly 25 years to the study of the physiology of anoxia tolerance in these and other model organisms, promoting not just the basic science but also the idea that understanding the physiology and molecular mechanisms behind anoxia tolerance provides insights into critical survival pathways that may be applicable to the hypoxic/ischemic mammalian brain. Work by Peter and his colleagues focused on the factors which enable the turtle to enter a deep hypometabolic state, including decreases in ion flux ("channel arrest"), increases in inhibitory neuromodulators like adenosine and GABA, and the maintenance of low extracellular levels of excitatory compounds such as dopamine and glutamate. Our attention has recently turned to molecular mechanisms of anoxia tolerance, including the upregulation of such protective factors as heat shock proteins (Hsp72, Hsc73), the reversible downregulation of voltage gated potassium channels, and the modulation of MAP kinase pathways. In this review we discuss three phases of anoxia tolerance, including the initial metabolic downregulation over the first several hours, the long-term maintenance of neuronal function over days to weeks of anoxia, and finally recovery upon reoxygenation, with necessary defenses against reactive oxygen stress.

Adaptation, Physiological↗

Effects of slope, substrate, and temperature on forces associated with locomotion of the ornate box turtle, Terrapene ornata.

In spite of several studies of the locomotor performances of reptiles, we know as yet relatively little about the mechanical forces involved. The present investigation examined the effects of substrate, slope and temperature on the pulling forces exerted by ornate box turtles tethered to a force transducer. These forces increased with body mass in a nearly isometric manner. The forces exerted during the initial effort were greatest on a styrofoam substrate, whereas maximum forces generated were greatest on pea gravel. Both forces progressively increased as slope decreased from +30 degrees to -30 degrees. The rate of force generation (N/s) was greatest at intermediate slopes. Contact force tended to increase as normal force increased, and was strongly influenced by slope, increasing from -30 degrees to +30 degrees. Surprisingly, we found no significant effects of temperature on tether forces, contraction times, or rates. This evaluation of pulling forces associated with box turtle locomotion revealed some interesting, and at times unexpected, relationships.

Animals↗

Secretory proteins in the reproductive tract of the snapping turtle, Chelhydra serpentina.

SDS-polyacrylamide gel electrophoresis was used to separate the secretory proteins produced by the epithelial and endometrial glands of the uterine tube and uterus in the snapping turtle Chelydra serpentina. The proteins were analyzed throughout the phases of the reproductive cycle from May to August, including preovulatory, ovulatory, postovulatory or luteal, and vitellogenic phases. The pattern of secretory proteins is quite uniform along the length of the uterine tube, and the same is true of the uterus, but the patterns for uterine tube and uterus are clearly different. We identify 13 major proteins in C. serpentina egg albumen. Bands co-migrating with 11 of these are found in the uterine tube, but at most 4 are found in the uterus, suggesting that the majority of the albumen proteins are most likely secreted in the uterine tube, not in the uterus. Although some of the egg albumen proteins are present in the uterine tube only at the time of ovulation, most of the bands corresponding to albumen proteins are present throughout the breeding season even though the snapping turtle is a monoclutch species. These results suggest that the glandular secretory phase in the uterine tube is active and quite homogeneous in function regardless of location or phase of the reproductive cycle.

Albumins↗