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Monoclonal antibodies to the turtle cortex reveal neuronal subsets, antigenic cross-reactivity with the mammalian neocortex, and forebrain structures sharing a pallial derivation.

The dorsal cortex of the pond turtle (Pseudemys scripta) is a relatively simple structure consisting of two principal classes of neurons that occupy three distinct layers. Morphological, pharmacological, and physiological data suggest many similarities to the mammalian neocortex, rendering it an interesting preparation for comparative studies. We prepared monoclonal antibodies to the turtle dorsal cortex by immunizing mice with cortical tissue from adult turtles. Twelve antibodies were generated that recognize specific components of the turtle cortex. Among these, eight antibodies label only neurons and four label only ependymal glial cells. Differences in tissue staining pattern and immunoglobulin class suggest a heterogeneity of antigenic specificity among the antibodies. The staining patterns of three of our antibodies are described. TC3, like all other neuron-marking antibodies generated, labels a subset of both pyramidal and stellate cell types. It also cross-reacts with a subset of mammalian cortical neurons and labels them with a pattern similar to that observed in the turtle cortex. TC5 stains ependymal cells and their glial processes in the turtle cortex, and cross-reacts with fibrous astrocytelike processes in mammalian neocortical white matter. TC9 appears to recognize antigens of neurons sharing a pallial derivation in turtle.

Animals↗

Immunohistochemical and biochemical studies on Lys8-Asn9-neurotensin8-13 (LANT6)-related peptides in the basal ganglia of pigeons, turtles, and hamsters.

The distribution of the neurotensin-related hexapeptide LANT6 within the basal ganglia and its projection targets was studied in turtles, pigeons, and hamsters by using immunohistochemical techniques, radioimmunoassay (RIA), gel chromatography, and high performance liquid chromatography (HPLC). The results in turtles and pigeons were fundamentally similar. Within the basal ganglia, LANT6-like immunoreactivity (LLI) was observed in a population of large striatal neurons (comprising 1-5% of the total number of striatal neurons) and in essentially all of the medium-large pallidal neurons. In addition, LLI was observed in neurons of such other "striatal" and "pallidal" cell groups as the olfactory tubercle and ventral pallidum, respectively. Within the dopaminergic cell fields of the tegmentum, to which the pallidal cell groups project, LLI-containing fibers were abundant. Knife-cut studies confirmed that the majority of these LLI-containing fibers arise from telencephalic levels. Biochemical studies with RIA and HPLC showed large amounts of immunoreactive LANT6 (iLANT6) in the basal telencephalon (477 pmol/g) and tegmentum of pigeons (259 pmol/g), and this material was indistinguishable from the synthetic peptide. Lower levels of iLANT6 were demonstrated in the basal telencephalon (82 pmol/g) and tegmentum (156 pmol/g) of turtles, and the majority of this activity appeared to be associated with larger molecular forms of LANT6 or a peptide related to LANT6. In addition, one or more substances resembling Neuromedin N (NMN), a mammalian counterpart to LANT6, were detected in the turtle nervous system. The labeling patterns in hamsters were similar to those in pigeons and turtles, except that in hamsters fewer neurons were labeled and the labeling was generally lighter. The lighter level of labeling may reflect a difference between the LANT6-like material present in hamster nervous system and authentic LANT6. Biochemical studies revealed that a Neuromedin N-like substance, as well as high molecular weight forms of a LANT6-like substance, are present in hamster brain. In hamsters, neurons within globus pallidus, the entopeduncular nucleus, the ventral pallidum, and the polymorph layer of the olfactory tubercle were labeled for the presence of LANT6. Fiber labeling for LANT6 in the dopaminergic tegmental cell groups that receive pallidal input was, however, light. Thus, the present results establish that LANT6 in pigeons and LANT6-related peptides in turtles and hamsters are present within many pallidal neurons. In pigeons and turtles, these pallidal neurons give rise to a major LLI-containing projection to the dopaminergic cell groups of the tegmentum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Major differences in CNS sulfonylurea receptor distribution between the rat (newborn, adult) and turtle.

Our previous results have shown that KATP channels play an important role in K+ efflux and extracellular K+ accumulation in the rat brain, and this role was quantitatively more important in the adult than in the newborn brain. The purpose of this study was to localize by autoradiographic techniques the binding sites of glibenclamide, a potent sulfonylurea ligand that targets KATP channels, in the adult and newborn rat central nervous system (CNS). Since the adult turtle is resistant to anoxia, we also compared the rat to the turtle brain sulfonylurea receptor distribution. In all three animal groups (newborn rat, adult rat, adult turtle), specific glibenclamide binding was saturable. Scatchard plots were curvilinear in the rat, thus suggesting that glibenclamide binds to two types of sites, i.e., high and low affinity sites. Scatchard analysis on turtle brain tissue showed evidence of one binding site only. We also found that the distribution of glibenclamide binding sites was heterogeneous in the adult rat CNS with a higher density in rostral than in caudal regions. The highest binding densities were seen in the cortex, hippocampus, cerebellum, substantia nigra, and a few thalamic nuclei; intermediate densities were observed in the basal ganglia, septum, thalamus, and the hypoglossal nucleus. There was a low density in most areas of the hypothalamus, midbrain, brainstem, and spinal cord. Compared with the adult rat, the newborn had a very homogeneous distribution of binding sites and densities were very low throughout the CNS; the level of binding density was even lower in some regions undetectable in the adult turtle. Our results indicate that (1) there are high and low affinity sulfonylurea receptors in the rat CNS, (2) there is a striking heterogeneity in the distribution and density of sulfonylurea receptors in the adult rat CNS and this is in sharp contrast to the homogeneous distribution and low density in both newborn rat and adult turtle; (3) sulfonylurea receptors increase in number postnatally in the rat since binding density increases and the Kd in the newborn rat is similar to that in the adult rat. We speculate that KATP channels and sulfonylurea receptors are poorly developed in the turtle and develop mostly after birth in the rat, reaching highest density in adulthood.

ATP-Binding Cassette Transporters↗

Endocrine and behavioral response to a decline in habitat quality: effects of pond drying on the slider turtle, Trachemys scripta.

The effect of the simulated drying of a pond on the behavior and corticosterone secretion of Trachemys scripta was measured in a field situation. Slider turtles were held in experimental and control ponds (12 x 15 m) enclosed with a drift fence integrated with spring-triggered livetraps. The experimental pond water level was dropped 10 cm per day for 8 d, until water was completely drained. Slider turtles responded to the draining of the pond by the emigration of the majority (75%) of the experimental population. Emigrating turtles had significantly elevated corticosterone at Time 0 (blood sample within 10 min of handling=4.48 ng/mL+/-0.503SE) when compared with turtles captured in a control pond (Time 0=0.954 ng/mL+/-0.121SE), where conditions were held constant. Turtles emigrated during the final 72 hr of pond draining when ponds reached 30 cm depth and lower and water temperature was at least 30.8 degrees C or higher. Additionally, the effect of trapping using spring-activated livetraps was tested. Turtles held in livetraps (n=6) for 45-110 min showed a characteristic corticosterone response (Time 0=0.957 ng/mL+/-0.091SE; Time 30=2.85 ng/mL+/-0.131SE), indicating that this trapping technique alone does not stimulate corticosterone secretion. The findings of the study met our predictions that turtles would respond to the draining of the pond behaviorally by emigrating from the habitat concurrent with an elevated corticosterone concentration. This supports the view that corticosterone is involved in stress avoidance mechanisms that allow organisms to respond to environmental perturbations.

Animals↗

Seasonal changes in gonadal activity and the effects of stress on reproductive hormones in the common snapping turtle, Chelydra serpentina.

The seasonal gonadal cycle (including gonadal histology, sex steroids, and gonadotropins) was studied in freshly captured common snapping turtles, Chelydra serpentina, from Wisconsin, and the effects of capture stress were evaluated. The ovarian and testicular cycles are shorter than those reported in other freshwater turtles; the cycles commence in mid-May and terminate in early September, immediately after the completion of gonadal growth and maturation. In the female, testosterone (T), 17beta-estradiol (E2), and progesterone (Pro) were highly correlated with follicular growth and vitellogenesis. Ovulation in captivity and under natural conditions occurred after mid-May. In captivity, ovulation was a rapid process (24-48 hr); as the follicles descended into the uterine horns there was a significant increase in E2 and Pro and eggs were retained in the uterine horns for about 2 weeks before oviposition. In the male, T was significantly correlated with testicular growth and spermiation. Follicle-stimulating hormone (FSH) reached significantly higher levels (P < 0.01) in males (8.99 +/- 0.38 ng/ml) than in females (2.66 +/- 0.22 ng/ml), but luteinizing hormone was undetectable in both sexes. FSH was not correlated with the steroids in either sex. Sex steroids and FSH began to rise before spermiation and vitellogenesis and remained elevated until completion of gonadal growth and maturation. Leydig cells, the main source of plasma androgen in this species, became active shortly after emergence from hibernation and remained steroidogenically active for the rest of the cycle. Sertoli cells became active only after spermatogenesis was under way but also stayed active for the rest of the summer. Courtship and mating behaviors were observed in spring, summer, and fall. The snapping turtle is strictly aquatic with no basking behavior and limited behavioral thermoregulation so there is little daily fluctuation in body temperature. Environmental correlates indicate that the snapping turtle is temperature dependent: recrudesence occurs with a slight increase in water temperature during spring and early summer, while a dramatic drop in gonadal activity accompanies a slight decrease in temperature in fall. Changes in temperature may underlie changes in gonadal activity in the face of relatively stable FSH. Male turtles subjected to captivity and periodic blood sampling show a significant decline in T. The hormonal levels continued to decline whether the turtles are exposed to optimum or extreme temperatures. However, there is more rapid decline in T values in animals with regressed testes (June) than in those with well-developed testes (July). Male and female turtles kept in captivity at different phases of the cycle exhibit different patterns and degrees of response to stress, possibly related to the hormonal levels and the condition of the gonads.

Animals↗

Nocturnal activity in the green sea turtle alters daily profiles of melatonin and corticosterone.

In nature, green turtles (Chelonia mydas) can exhibit nocturnal activity in addition to their typically diurnal activity cycle. We examined whether nocturnal activity in captive and free-living green turtles altered daily plasma profiles of melatonin (MEL) and corticosterone (CORT). In captivity, diurnally active green turtles expressed distinct diel cycles in MEL and CORT; a nocturnal rise was observed in MEL and a diurnal rise was observed in CORT. However, when induced to perform both low- and high-intensity nocturnal activity, captive green turtles exhibited a significant decrease in MEL, compared to inactive controls. In contrast, plasma CORT increased significantly with nocturnal activity, and further, the relative increase in CORT was correlated with the intensity of the nocturnal behavior. In free-living green turtles that performed nocturnal activity including: nesting, mate searching, and feeding/swimming behaviors, plasma profiles in MEL and CORT exhibited relatively little, or no, daily fluctuation. Our findings demonstrate that nocturnal activity in green turtles is often associated with MEL and CORT profiles that resemble those measured during the day. We speculate that these conspicuous changes in MEL and CORT during nocturnal activity could either support or promote behaviors that enable acquisition of transient resources important to the survival and reproductive success of green turtles.

Animals↗

Stream and riparian management for freshwater turtles.

The regulation and management of stream ecosystems worldwide have led to irreversible loss of wildlife species. Due to recent scrutiny of water policy and dam feasibility, there is an urgent need for fundamental research on the biotic integrity of streams and riparian zones. Although riverine turtles rely on stream and riparian zones to complete their life cycle, are vital producers and consumers, and are declining worldwide, they have received relatively little attention. I review the literature on the impacts of contemporary stream management on freshwater turtles. Specifically, I summarize and discuss 10 distinct practices that produce five potential biological repercussions. I then focus on the often-overlooked use of riparian zones by freshwater turtles, calculate a biologically determined riparian width, and offer recommendations for ecosystem management. Migration data were summarized on 10 species from eight US states and four countries. A riparian zone encompassing the majority of freshwater turtle migrations would need to span 150 m from the stream edge. Freshwater turtles primarily chose high, open sandy habitats to nest. Nests in North America contained eggs and hatchlings during April through September and often through the winter. In addition, freshwater turtles utilized diverse riparian habitats for feeding, nesting, and overwintering. Additional documentation of stream and riparian habitat use by turtles is needed.

Animals↗

The isthmus-tegmentum complex in the turtle and rat: a comparative analysis of its interconnections with the optic tectum.

Injections of horseradish peroxidase, wheat germ agglutinin and various amino acids into the optic tectum in both the turtle and rat, and into the nucleus isthmi magnocellularis (IsM) in the turtle were used to analyse the connections of the isthmus-tegmentum complex. The connectivities and the selective retrograde transport properties in certain tectal pathways were taken as a basis to define more accurately isthmus-tegmentum complex subdivisions. There were several main findings. In the turtle the projection from the tectum to the IsM originated in the stratum griseum periventriculare, whereas the projection from the IsM to the tectum terminated in the superficial tectal layers (both projections homolateral). The terminations of the pathway from IsM to tectum were not uniformly distributed throughout the tectal surface; rather, alternating zones of high and low termination density along the lateral dimension were observed. The turtle nucleus isthmi parvocellularis, receiving a few tectal fibers and afferents from the ipsilateral IsM, gave rise to a bilateral tectal projection. Evidence was obtained for a crossed collicular projection to the rat parabigeminal nucleus (Pbg) in addition to the established uncrossed one. GABA was retrogradely transported from the optic tectum to the Pbg in the rat, and to the dorsolateral mesencephalic tegmentum and the IsM in the turtle. After glycine injections into the optic tectum, the dorsomedial peri-parabigeminal tegmentum was retrogradely labeled in the rat, and the IsM in the turtle. An attempt was made to outline the parallelism between the organizations of the isthmus-tegmentum complexes in the turtle, pigeon and rat. It was concluded that some basic features in the inter-connectivity of the isthmus-tegmentum complex and other parts of the visual system have been preserved in evolution, despite the apparent loss of the isthmo-retinal projection in mammals.

Animals↗

Protein sequences indicate that turtles branched off from the amniote tree after mammals.

The phylogenetic relationships among the major groups of amniote vertebrates remain a matter of controversy. Various alternatives for the position of the turtles have been proposed, branching off either before or after the mammals. To discover the phylogenetic position of turtles in relation to mammals and birds, we have determined cDNA sequences for the eye lens proteins alpha A- and alpha B-crystallin of the red-eared slider turtle (Trachemys scripta elegans). In addition, databases were searched for turtle protein sequences, for which mammalian, avian, and outgroup orthologs were available. All sequences were analyzed by three phylogenetic tree reconstruction methods (neighbor-joining, maximum parsimony, and maximum likelihood). Including the alpha-crystallins, 7 out of 12 proteins support a sister-group relation of turtles and birds with all 3 methods. For each of the other five proteins no topology was consistently preferred by the three approaches. Analyses of the combined amino acid data (1,695 aligned sites) also give extremely strong evidence that turtles are nearer to birds, indicating that mammals branched off before the divergence between turtles and birds occurred.

Animals↗

Organochlorine contaminants in loggerhead sea turtle blood: extraction techniques and distribution among plasma and red blood cells.

Few studies have described the organochlorine (OC) contaminant concentrations found in sea turtle tissues. These studies have relied on the opportunistic sampling of either eggs or tissues from stranded carcasses. In this study, the use of whole blood samples as well as both blood components (plasma and red blood cells) were examined as a non-destructive alternative for monitoring OCs in free-ranging loggerhead sea turtles (Caretta caretta). Blood samples were collected from juvenile loggerhead sea turtles (n = 12) captured in Core Sound, North Carolina, USA and analyzed for 55 polychlorinated biphenyl (PCB) congeners and 24 OC pesticides by gas chromatography with electron capture detection and mass spectrometry. Using pooled loggerhead sea turtle whole blood, three different liquid:liquid extraction techniques were compared. Results were similar in terms of recovery of internal standards, lipids, and OC concentrations. An extraction technique, employing formic acid and 1:1 methyl-tert-butyl-ether: hexane, was found to be satisfactory. This method was applied to the extraction of OCs from whole blood, plasma, and red blood cell (RBC) samples from five loggerhead sea turtles. Plasma contained the highest OC concentrations on a wet mass basis, followed by whole blood and RBCs. The majority of each OC compound was found in the plasma rather than the RBCs, suggesting that OC compounds preferentially partition into the plasma. On average (SD), 89.4% (3.1 %) of total PCBs, 83.4% (11.9%) of total chlordanes, 74.3% (15.1%) of mirex, 72.6% (4.8%) of total DDTs, and 80.1% (16.6%) of dieldrin were found in the plasma. The concentrations of total PCBs, mirex, total chlordanes, and total DDTs measured in both components of the blood significantly correlated to those in whole blood. These are the first reported OC concentrations in sea turtle blood. They were found to be similar to previously reported levels in blood components of humans and of reptiles from relatively clean sites, but lower than those measured in blood of fish-eating birds and marine mammals. The results indicate that blood, preferably plasma, can be used to detect and monitor OC contaminants in loggerhead sea turtles.

Animals↗

Seed dispersal by the Florida box turtle (Terrapene carolina bauri) in pine rockland forests of the lower Florida Keys, United States.

Seed dispersal by animals is one of the most important plant-animal mutualisms, but saurochory, the dispersal of seeds by reptiles, has received little attention. We investigated the role of the Florida box turtle (Terrapene carolina bauri) as a seed dispersal agent in pine rockland forests of the lower Florida Keys and examined the effect of turtle digestion on seed germination. We obtained seeds of 11 species with fleshy fruits and 2 species with non-fleshy fruits (a grass and legume) from the feces of 145 box turtles collected on Key Deer National Wildlife Refuge from 1999 to 2000. We planted the seeds of nine species and germination percentage (percentage of seeds that germinated during the experiment) varied from 10% to 80%. Comparative germination experiments were conducted with Thrinax morrissii, Serenoa repens, and Byrsonima lucida. We compared the germination percentage and germination rate (number of days from planting to seedling emergence) of seeds from three treatments (seeds recovered from feces, control seeds with pulp, and control seeds without pulp) and continued these experiments for up to 2 years. Passage through the box turtle digestive tract greatly enhanced the germination percentage and germination rate of S. repens, but decreased the germination percentage of B. lucida and T. morrissii, and decreased germination rate for T. morrissii. Subsequent destructive seed viability tests revealed that many ungerminated T. morrissii seeds remained viable, suggesting long-term seed dormancy may occur, even after passage through the turtle digestive system. In addition, the proportion of ungerminated seeds which remained viable was greater for seeds recovered from turtle feces than from control seeds with pulp. Furthermore, removal of fleshy pulp either manually or by the turtle digestive system may allow T. morrissii to escape insect predation.

Animals↗

Chloride-induced increment in short-circuiting current of the turtle bladder. Effects of in-vivo acid-base state.

Evidence for the participation of conductive and non-conductive (exchange) transmembrane anion pathways in the luminal acidification, alkalinization, and chloride-reabsorptive functions of the turtle bladder is provided from the pattern of Cl- -induced changes in transepithelial electrical parameters of isolated urinary bladders from three groups of donor turtles: control or post-absorptive turtles (those killed 5 days after feeding); acidotic turtles (NH4Cl-loaded); and alkalotic turtles (NaHCO3-loaded). The predominance of each of the three aforementioned transport functions as well as the response to Cl- -addition is altered by the in-vivo electrolyte balance of the turtle. In post-absorptive bladders, which are poised for acidification and Cl- reabsorption, the mucosal and serosal addition of Cl- to Na+-free, (HCO3- + CO2)-containing media increases the negative short-circuiting current (Isc). In acidotic bladders, which are poised for acidification but not Cl- reabsorption, mucosal Cl- addition has no effect on this Isc whereas serosal Cl- addition increases the negative Isc in a manner identical to that observed in the post-absorptive bladders. Alkalotic bladders do not possess an acidification function but instead are poised for Cl- reabsorption and cAMP-dependent electrogenic alkali secretion (positive Isc). In these bladders, serosal Cl- addition is without effect while mucosal Cl- addition produces transient changes in this positive Isc. It is found that these results can be replicated by a model of the turtle bladder in which transmembrane Cl- and HCO3- conductive and exchange paths mediate transepithelial acidification, alkalinization and Cl- reabsorption.

1-Methyl-3-isobutylxanthine↗

Measurement of plasma renin activity in the freshwater turtle.

Components of the renin angiotensin system have been identified in many nonmammalian vertebrates. However, in many of these animals, including reptiles, the physiological functions and importance of the system remain unclear. To aid in the study of the system in a reptile we modified a commercially available radioimmunoassay (RIA) kit containing antibody against human angiotensin I (ANG I) for use in the freshwater turtle, Pseudemys scripta. Cross-reactivity between anti-human ANG I antibodies (Rainen Angiotensin I RIA Kit, New England Nuclear) and turtle ANG I was demonstrated. Cross-reactivity with the antibody in two other human ANG I assay kits (Travenol-Genentech and Biotecx) was very limited. Blood for assay was collected from conscious turtles in EDTA, centrifuged, and the plasma frozen at -20 degrees. Turtle ANG I was generated by incubation at 0.5 ml plasma at pH 5.5 for 2 hr at 30 degrees with addition of dimercaprol and 8-hydroxyquinoline. Angiotensin generation increased with temperature and with generation time. The recovery of turtle ANG I added to turtle plasma prior to incubation was 92-97%. The assay procedure was used to measure plasma renin activity (ng/ml/hr incubation) from unstimulated turtles.

Angiotensin I↗

Studies on cardiovascular fluke (Digenea: Spirorchiidae) infections in sea turtles from the Great Barrier Reef, Queensland, Australia.

Twenty-seven sea turtles (23 Chelonia mydas and four Eretmochelys imbricata) from northeast Queensland were found to be infected with cardiovascular flukes and/or their eggs. Five had originated from turtle farms in the Torres Strait, five from an oceanarium on Magnetic Island (146 degrees 56'E, 19 degrees S) and the remainder from coral reefs in the Torres Strait or near Townsville. The incidence of flukes and/or eggs in the groups was 4.8 per cent (5 of 104), 33.3 per cent (5 of 15) and 72.2 per cent (17 of 22), respectively. Affected animals ranged in size from 18 to 108 cm (curved carapace length) and weighed between 0.5 and 77 kg. The average number of flukes per host was 47. Flukes were recovered from the three chambers of the heart and major vessels (right aortic arch and brachiocephalic artery), where they were attached to the walls or free in the lumen. They were subsequently identified as Haplotrema spp. and/or Learedius spp. In 59.2 per cent (16 of 27) of turtles, flukes were not found, although their eggs were detected microscopically. Gross pathological changes associated with the presence of flukes included thickening and hardening of arterial walls (four turtles), thrombus formation (three), chronic pneumonia (two) and an excess of pericardial or peritoneal fluid (four). Microscopically, the essential changes was that of chronic inflammation, as evidenced by the proliferation of epithelial cells, reticulo-endothelial cells and fibroblasts in areas accessible to flukes and/or eggs. Multiple diffuse egg granulomas were a prominent feature of most organs, the spleen and lungs being predilection sites. Proliferative changes had occurred in the endocardium and in the endothelium of vessels supplying the spleen, stomach, intestine and pancreas (18 turtles). The walls of major arteries, lungs, liver, brain, crop and stomach were also acutely inflamed (eight turtles). Haemorrhage was recorded in the lungs and/or brain of eight turtles with heavy fluke infestations. Other vascular changes, viz. congestion, oedema and hypertrophy of arterial/arteriolar walls, resulted from the inhibition of blood flow by parasitic emboli.

Animals↗

Cerebral resistance to anoxia in the marine turtle.

The extraordinary ability of the turtle to withstand prolonged anoxia was examined in cerebral cortex in situ by recording changes in the reduction/oxidation ratio of cytochrome a,a3 by reflection spectrophotometry. Inspiration of 100% oxygen increased the oxidation of cytochrome a,a3 beyond that of the air breathing control, suggesting that cytochrome a,a3 is not fully oxidized under normoxic conditions in turtle brain. A similar response was seen also in cerebral cortex of the rat. The significance of the cytochrome a,a3 reduction in these intact tissues is discussed. Both severe hypoxia (100% N2) and asphyxia produced increasing levels of reduced cytochrome a,a3 in turtle and rat brains. The rat of change produced by N2 inspiration was greater than that produced by asphyxia in both species. This is interpreted as demonstrating an open pulmonary blood circulation during anoxia. In turtles, levels of reduced cytochrome a,a3 were maintained for over 3 h of continual N2 inspiration. Subsequent inspiration of room air resulted in a full restoration of turtle brain cytochrome a,a3 redox state within 30 sex. In the rat, continued N2 inspiration resulted in a rapid reduction of cytochrome a,a3 to a plateau (3 min) which became irreversible within a short period. An extended tolerance of N2 inspiration found in rats cooled to temperatures approximating that of the turtle was inadequate to account for the wide species of difference. We suggest that special adaptations, not related to the redox state of cytochrome oxidase under normoxic conditions, are responsible for maintaining the functional integrity and the capacity for cytochrome oxidase re-oxidation of turtle brain mitochondria under prolonged anoxia.

Animals↗

Effects of anoxia and graded acidosis on the levels of circulating catecholamines in turtles.

We measured circulating levels of catecholamines in painted turtles subjected to anoxia with different degrees of concomitant acidosis at 20 degrees C and in turtles subjected to long-term submergence at 3 and 10 degrees C. Blood levels of both epinephrine (E) and norepinephrine (NE) increased during N2-breathing, N2/CO2 breathing and submergence, with NE generally being present in higher concentrations than E. During submergence at 20 degrees C, anoxic turtles experienced an extreme acidosis and NE levels exceeded 18,000 pg/ml. The greater the degree of acidosis in anoxic turtles the higher were the levels of plasma NE (log [NE; pg/ml] = 1.640 x pHa + 15.776, r = -0.826). Elevation of plasma E under anoxic conditions was more modest and the correlation between plasma E and pHa was less pronounced (log [E; pg/ml] = -0.329 x pHa + 6.069, r = -0.285). Submergence at lower temperatures also resulted in increases in plasma levels of NE, but while plasma E generally increased during anoxia, this elevation was less dramatic than that observed for NE. Exposure of turtles to either mild (6.5% CO2) or severe (14.5% CO2) normoxic hypercapnia resulted in no increase in E and only modest increases in NE. Upon resumption of air-breathing in all of the 20 degrees C protocols, turtles rapidly restored E and NE to control levels. The function of elevated plasma catecholamines during anoxia and acidemia in turtles is unknown but may be important in stimulating respiratory and cardiovascular recovery once air-breathing is resumed. Catecholamines may also play a role in mediating the rise in blood glucose we observed in this study, which may be an important factor in maintaining tissue viability during anoxic stress.

Acidosis↗

Occurrence of hydroxysteroid oxidoreductases in liver of turtles.

1. Hydroxysteroid oxidoreductases have been partially purified from the cytosol fraction (105,000 g supernatant) of liver from a fresh-water turtle (Podocnemis expansa) and a sea-water turtle (Chelonia mydas mydas) by precipitation with ammonium sulphate (AS, 10-80% saturation). 2. The following enzymes were detected (substrates in brackets): 3 alpha-hydroxysteroid oxidoreductase (androsterone), 3 beta-hydroxysteroid oxidoreductase (DHEA) and 17 beta-hydroxysteroid oxidoreductase (testosterone, oestradiol-17 beta). NAD as well as NADP were effective as cofactors. 3. In fresh-water turtle, highest activities of the 3 alpha-enzyme were measured in the 20% AS fraction (cofactor NAD), of the 3 beta-enzyme in the 60% AS fraction (cofactor NAD) and of the 17 beta-enzyme in the 40% AS fraction (cofactor NADP). 4. In sea-water turtle, highest activities were observed for all three enzymes in the 60% AS fraction. 5. Generally, enzyme activities were higher in sea-water turtles than in fresh-water turtles. The most active enzyme in both turtles was found to be the 3 alpha-hydroxysteroid oxidoreductase, followed by the 17 beta- and the 3 beta-hydroxysteroid oxidoreductases.

17-Hydroxysteroid Dehydrogenases↗

The distribution of cholecystokinin-8 in the central nervous system of turtles: an immunohistochemical and biochemical study.

Immunohistochemical techniques, radioimmunoassay (RIA) and high performance liquid chromatography (HPLC) were used to: (1) determine the regional distribution and amounts of cholecystokinin-8 (CCK8)-like immunoreactivity in the turtle central nervous system, and (2) chemically characterize the CCK8-like material present in the turtle central nervous system. High levels of CCK8-like immunoreactivity were found in the turtle central nervous system, with the highest levels being present in the hypothalamus and neurohypophysis. Moderate levels of the CCK8-like material were found in all other regions of the turtle nervous system except the cerebellum, the olfactory bulbs and the dorsal ventricular ridge of the telencephalon, which contained low levels. The bulk (87%) of the CCK8-like material in turtle central nervous system co-eluted with CCK8-sulfate in gradient elution HPLC. The distribution of CCK8-like immunoreactivity (CCK8LI) observed using immunohistochemistry was consistent with the results of the RIA studies. Numerous CCK8LI-containing neurons and fibers were observed in the hypothalamus and neurohypophysis. Neurons and fibers containing CCK8 were, however, more sparsely distributed outside the hypothalamus. The immunohistochemical data provided evidence for the existence of two major CCK8-containing pathways in turtles that have been previously described in mammals: a pathway from the supraoptic and paraventricular magnocellular nuclei to the external zone of the median eminence and neurohypophysis and a pathway from dorsal root ganglia to the dorsal horn of the spinal cord. Overall, the present results, in conjunction with several previous studies, indicate that CCK8 has had a relatively stable evolutionary history as a CNS neuropeptide among land vertebrates. The molecular structure of CCK8 appears to have been largely (if not entirely) conserved, as has its concentration in many brain regions. A noteworthy exception to such conservatism in the localization of CCK8 is that the concentration of CCK8 in the telencephalon, particularly in the telencephalic cortex, is much lower in turtles than in mammals. The present results therefore suggest that CCK8 may not have become a prominent peptide in the telencephalic cortex (or its anatomical equivalents) until the evolution of neocortex in the mammalian lineage.

Animals↗