neuronal apoptosis and degeneration during forced submergence in painted turtles at 20 °c

can degrade NO through a non-enzymatic reaction with superoxideanion (O2

−). In this work, we analyzed the O2− sources for NO de-

gradation by mitochondria isolated from rat liver. NO concentrationin the reaction medium was followed using an electrochemicalsensor connected to a free radical analyzer. Malate-energizedmitochondria consumed NO at a slow rate that was stimulated byantimycin-A and inhibited by myxothiazol, suggesting that electronleakage from complex III favored NO degradation. When succinatewas used as substrate, a rotenone-sensitive NO consumption wasobserved, which was attributed to O2

− generated by reverse electrontransport from succinate dehydrogenase to complex I. Mitochondriaincubated with exogenous NAD(P)H presented very high O2

−-dependent NO degradation rates that were insensitive to inhibitorsof mitochondrial respiratory chain. This NAD(P)H-mediated NOdegradation was abolished after incubation of mitochondria withproteinase K, while the activity of respiratory chain enzymes was notaffected by this treatment. Peroxide production assessed withAmplex Red confirmed that larger amounts of O2

− were generatedby mitochondria in the presence of NAD(P)H, when compared tosuccinate- or malate-energized mitochondria. NAD(P)H-dependentO2− production and NO degradation were not affected in mitochon-

drial suspensions purified with a Percoll gradient, eliminating thepossibility that these activities could result from contaminantorganelles. Overall, these results suggest the existence of an NAD(P)H oxidase activity in rat liver mitochondria, non-related to therespiratory chain, that promotes O2

−-dependent NO degradation.Supported by FAPESP.

doi:10.1016/j.cbpa.2008.04.237

A5.48Neuronal apoptosis and degeneration during forced submergencein painted turtles at 20 °C

D. Warren, T. Robertson, P. Bickler (University of California, SanFrancisco)

The painted turtles, Chrysemys picta, of North America areregarded as the most anoxia-tolerant air-breathing vertebrates. Ifand when neuronal injury occurs during anoxic submergence hasnot yet been determined, however. In the present study, paintedturtles were forcibly submerged for 60 h at 20 °C and, aftertranscardial paraformaldehyde fixation, the extent of neuronalinjury assessed in 15 μm cerebrocortical sections using twohistological methods: 1) staining with Fluoro-Jade C, a marker thatspecifically labels degenerating neurons and 2) co-immunolabelingof activated caspase-3, a protein found in apoptotic cells, with NeuN,a nuclear protein expressed only in adult neurons. Plasma lactateincreased steadily during submergence and reached 100 mmol l−1

after 60 h. Fluoro-Jade C and activated caspase-3 staining werepresent throughout neurons of the medial and dorsal cortices,indicating apoptosis-mediated neuronal injury resulting from long-term anoxia. Despite this, several animals showed visible signs of lifeand required anesthesia prior to fixation. All of the animals' heartswere visibly contracting at the time of sampling. In addition, we alsopresent preliminary data from a study involving bromodeoxyuridinepulse labeling following 36 and 48 h of anoxia that addresses thepotential for reactive neurogenesis following anoxia-induced neu-ronal injury.

doi:10.1016/j.cbpa.2008.04.238

A5.49The effect of salinity on resting metabolism in Eurasian perch(Perca fluviatilis L.)

R. Ern (Aarhus University); N. Cong (Can Tho University); D. Houng, J.Overton (Can Tho University); T. Wang (Aarhus University); M. Bayley(Aarhus University)

While the Eurasian perch (Perca fluviatilis L.) is normally consideredto be a freshwater fish, populations also exist for their full life cycle inthe brackish-water of the Baltic Sea. Overton et al. (2008) recentlyshowed that long-term exposure to brackish-water markedly reducesgrowth in a freshwater population of P. fluviatilis. Food intake wasunaffected andwe, therefore,measured resting oxygenuptake (VO2) infish maintained for 21 days in either fresh or brackish (10 ppt) waterusing intermittent closed respirometry. VO2 of all fish reached stablelevels within 5 h of entering respirometers. Resting VO2 was signifi-cantly higher in fish acclimated to brackish-water compared to fresh-water controls (39.3±5.2 versus 33.1±3.1 µmol/kg/min). It might beexpected that osmoregulatory costs should follow the osmotic gradientbetween extracellular fluid and the environment. This is not born out bythe present data that do, however, agree with the previously performedgrowth experiment. Blood osmolality was significantly higher inbrackish-water than freshwater (393.9±22.4 and 345.6±20.1 mOsm,respectively) indicating that this freshwater population of P. fluviatilis isunable to properly maintain ion balance even with minor increases inthe environmental salinity. The elevated resting VO2 in 10 ppt is equi-valent to an increased energy consumption of 975 extra calories per day,and since food intakewas unaffected, it is reasonable to assume that thereduced growth seen in this population in 4 and 10 ppt saltwater ispartly the result of increased energy consumption in brackish-water.

doi:10.1016/j.cbpa.2008.04.239

A5.50Biochemical and genetic aspects linked to osmoregulation andswimming capacities in brook charr (Salvelinus fontinalis)

A. Dupont-Prinet (ISEM); A. Crespel (ISEM-QAR); G. Claireaux,(UBO); D. McKenzie (ISEM); L. Bernatchez (Université Laval); C.Audet (ISMER-UQAR)

Osmoregulation and swimming capacity are essential processes forthe migratory life-cycle of salmonids. The aim of this study was tocompare osmoregulatory and exercise performance in brook charrstrains with different life cycles, including to reveal a geneticcomponent. Two strains of wild brook charr were used to makepure and hybrid crosses. The Laval strain originates froman anadromous brook charr population from the Laval River (Québec).The Rupert strain originates from a resident freshwater populationfrom the Rupert River (Québec). In both cases, broodstock were thirdgeneration in captivity. Four crosses were performed: ♀Laval × ♂Laval,♀Rupert × ♂Rupert, or their hybrids, hence ♀Laval × ♂Rupert and♀Rupert × ♂Laval. The progeny were submitted to an incrementalswimming performance test in either fresh or brackishwater, and theircritical swimming speed was calculated individually. Each fish wassampled immediately after fatigue, they were removed from the swimflume and rapidly anaesthetized (MS-222). Aftermeasurement of bodylength and weight, blood was drawn by caudal puncture forhaematocrit, haemoglobin and cortisol level measurements. Meancellular haemoglobin concentration was calculated. The percentageof water in white muscle content was calculated. Gill Na+K+ATPase

S110 Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S97–S114