Alterations in gill structure in tropical reef fishes as a result of elevated temperatures.

Tropical regions are expected to be some of the most affected by rising sea surface temperatures (SSTs) because seasonal temperature variations are minimal. As temperatures rise, less oxygen dissolves in water, but metabolic requirements of fish and thus, the demand for effective oxygen uptake, increase. Gill remodelling is an acclimation strategy well documented in freshwater cyprinids experiencing large seasonal variations in temperature and oxygen as well as an amphibious killifish upon air exposure. However, no study has investigated whether tropical reef fishes remodel their gills to allow for increased oxygen demands at elevated temperatures. We tested for gill remodelling in five coral reef species (Acanthochromis polyacanthus, Chromis atripectoralis, Pomacentrus moluccensis, Dascyllus melanurus and Cheilodipterus quinquelineatus) from populations in northern Papua New Guinea (2° 35.765' S; 150° 46.193' E). Fishes were acclimated for 12-14 days to 29 and 31°C (representing their seasonal range) and 33 and 34°C to account for end-of-century predicted temperatures. We measured lamellar perimeter, cross-sectional area, base thickness, and length for five filaments on the 2nd gill arches and qualitatively assessed 3rd gill arches via scanning electron microscopy (SEM). All species exhibited significant differences in the quantitative measurements made on the lamellae, but no consistent trends with temperature were observed. SEM only revealed alterations in gill morphology in P. moluccensis. The overall lack of changes in gill morphology with increasing temperature suggests that these near-equatorial reef fishes may fail to maintain adequate O2 uptake under future climate scenarios unless other adaptive mechanisms are employed.

Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts.

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability (R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, ß-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1-50.1%). When ß-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

Assessment of myocardial perfusion in the empty beating porcine heart with laser Doppler flowmetry.

Laser Doppler flowmetry was applied to the empty beating heart of six pigs. Cardiopulmonary bypass was instituted and the preparation allowed continuous and simultaneous measurement of coronary sinus blood flow and local tissue perfusion. An epicardial and an intramuscular probe were used. Significant linear correlation coefficients were obtained between changes of laser Doppler signal and coronary sinus blood flow changes in all seven experiments in four animals (r from 0.71 to 0.94, p less than 0.005) and between laser Doppler signal and changes of extracorporeal bypass in five out of six experiments in four animals (r from 0.82 to 0.99, p less than 0.001). The correlation between coronary sinus flow and bypass flow of four pigs was significant (r from 0.81 to 0.98, p less than 0.001), the coronary flow being about 10% of bypass flow. Muscular activity of the heart contributed to the laser Doppler signal, the magnitude of this "noise level" varying between different experiments even in the same animal. A key question for the applicability of laser Doppler flowmetry to the beating heart is whether it will become possible to discriminate between flow and muscular contribution to the laser Doppler output.

Evidence that acetylcholine mediates increased cerebral blood flow velocity in crucian carp through a nitric oxide-dependent mechanism.

Nitric oxide (NO)-dependent regulation of brain blood flow has not been proved to exist in fish or other ectothermic vertebrates. Using epi-illumination microscopy on the brain surface (optic lobes) of crucian carp (Carassius carassius), we show that superfusing the brain with acetylcholine (ACh) induces an increase in cerebral blood flow velocity that can be completely blocked by the NO synthase inhibitors NG-nitro-L-arginine methylester (L-NAME) and NG-nitro-L-arginine. Also, sodium nitroprusside, which decomposes to liberate NO, causes an increase in cerebral blood flow velocity. By contrast, L-NAME does not block the increase in blood flow velocity caused by anoxia. The results suggest that NO is an endogenous vasodilator in crucian carp brain that mediates the effects of ACh. Because teleost fish deviated from other vertebrates 400 million years ago, these results suggest that NO-dependent brain blood flow regulation was an early event in vertebrate evolution.

Time course of anoxia-induced increase in cerebral blood flow rate in turtles: evidence for a role of adenosine.

The exceptional ability of the turtle brain to survive prolonged anoxia makes it a unique model for studying anoxic survival mechanisms. We have used epi-illumination microscopy to record blood flow rate in venules on the cortical surface of turtles (Trachemys scripta). During anoxia, blood flow rate increased 1.7 times after 45-75 min, whereupon it fell back, reaching preanoxic values after 115 min of anoxia. Topical superfusion with adenosine (50 microM) during normoxia caused a 3.8-fold increase in flow rate. Superfusing the brain with the adenosine receptor blocker aminophylline (250 microM) totally inhibited the effects of both adenosine and anoxia, while aminophylline had no effect on normoxic flow rate. None of the treatments affected systemic blood pressure. These results indicate an initial adenosine-mediated increase in cerebral blood flow rate during anoxia, probably representing an emergency response before deep metabolic depression sets in.

Role of nitric oxide in the elevation of cerebral blood flow induced by acetylcholine and anoxia in the turtle.

Nitric oxide (NO)-dependent regulation of brain blood flow has hitherto not been studied in reptiles. By observing the brain surface (telencephalon) of the freshwater turtle (Trachemys scripta) with epiillumination microscopy, we show that topical application of acetylcholine (ACh) induces an increase in CBF velocity that can be completely blocked by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). The effect of L-NAME was reversed by L-arginine. Also, sodium nitroprusside (SNP), which decomposes to liberate NO, caused an increase in CBF velocity. By contrast, L-NAME could not block the increase in blood flow velocity caused by anoxia. Interestingly, superfusing the brain with ACh or SNP during anoxia had no effect on the blood flow velocity. The results suggest that NO is an endogenous vasodilator in the turtle brain, mediating the effects of ACh during normoxia. By contrast, anoxia does not rely on NO as a vasodilator.

Relationships between sex and the size and number of forebrain gonadotropin-releasing hormone-immunoreactive neurones in the ballan wrasse (Labrus berggylta), a protogynous hermaphrodite.

This study is the first to examine the brain gonadotropin-releasing hormone (GnRH) cell population phenotype in a protogynous and monandric sequentially hermaphroditic fish. Male ballan wrasse (Labrus berggylta) had on average higher numbers of GnRH-immunoreactive (GnRH-ir) cells within the brain preoptic area (POA) than females, a difference not found in GnRH-ir cells in other brain regions. Furthermore, in males, but not females, the number of these POA GnRH-ir cells correlated with body size. Maturational state (prespawning or postspawning) had marked effects on mean profile sizes (but not numbers) of both GnRH-ir cell bodies and cell nuclei, even when existing differences in body size and allometric relationships had been taken into account. Postspawning males tended to have larger GnRH-ir profiles in all brain regions relative to both prespawning males and females. Moreover, the GnRH-ir cell number in POA, and the cell body profile size in both POA and at the level of the anterior commissure, correlated with gonad size in spermiated prespawning males, indicating a relationship between both size and number of GnRH cells and male gonadal development. These results suggest that temporary changes in the size of brain GnRH-ir neurones are coupled to the male spawning cycle, and that permanent POA GnRH-ir cell number changes are involved in the process of sex change in sequential hermaphrodites. However, smaller males had no more preoptic GnRH-ir cells than equally sized females, which may argue against a proximate inducing role of GnRH cell number changes in naturally occurring sex reversal.

Low mass-specific brain Na+/K+-ATPase activity in elasmobranch compared to teleost fishes: implications for the large brain size of elasmobranchs.

Elasmobranch fishes have long been noted for having unusually large brains for ectotherms, and therefore may be exceptions to the rule that vertebrates in general devote less than 8% of their resting metabolic rate to the central nervous system. The brain mass of sharks, skates and rays is often several times larger than that of teleost fishes of the same size. Still, the underlying reasons for this have remained unclear. Ion pumping by the Na+/K+-ATPase is the single most energy consuming process in the brain. In this study, Na+/K+-ATPase activity was measured in the brain of four species of elasmobranchs and 11 species of teleosts. While the average brain mass of the elasmobranchs examined was approximately three times that of the teleosts, the mean specific Na+/K+-ATPase activity was only about one-third of that of the teleosts. Thus, the total brain Na+/K+-ATPase activity was similar in elasmobranchs and teleosts. This suggests that the large brain size of elasmobranchs is at least partly related to a low mass-specific rate of brain energy use.

Induction of social dominance by L-dopa treatment in Arctic charr.

The effect of L-dopa on social dominance was studied in the juvenile Arctic charr (Salvelinus alpinus). L-dopa is the immediate precursor of dopamine, a neurotransmitter that has been connected with aggressive behaviour in fish as well as mammals. Arctic charr were placed in pairs. One individual in each pair was given L-dopa orally, while the other was given vehicle. The results showed that 18 out of 22 fish given 10 mg L-dopa kg-1 became dominant (p = 0.004, binomial test). A higher dose of L-dopa (200 mg kg-1) induced dyskinesia. L-dopa was found to cause a dose-dependent increase in the brain levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC, a major dopamine metabolite), as well as an increase in the DOPAC/dopamine ratio (an index of dopaminergic activity).

Predator exposure alters brain serotonin metabolism in bicolour damselfish.

The effect of predator exposure on brain serotonin utilization was studied in bicolour damselfish (Pomacentrus partitus). Predator exposure (lasting 2 h), which took place in an aquarium where a transparent wall separated the damselfish from the predator (a graysby, Epinephelus cruentatus), resulted in increased concentrations of 5-hydroxyindoleacetic acid (5-HIAA, the main serotonin metabolite) and 1.6-1.8 fold elevations of 5-HIAA/serotonin ratios (an index of serotonergic activity) in telencephalon, hypothalamus and brain stem. The results show that predator exposure, like intraspecific social stress, induces increased brain serotonergic activity in fish. Different types of stress also elevate brain serotonergic activity in mammals, indicating that this is a phylogenetically very old stress response, possibly helping the animal's coping response.

Effects of biogenic aldehydes and aldehyde dehydrogenase inhibitors on rat brain tryptophan hydroxylase activity in vitro.

The effect of indole-3-acetaldehyde, 5-hydroxyindole-3-acetaldehyde, disulfiram, diethyldithiocarbamate, coprine, and 1-amino-cyclopropanol on tryptophan hydroxylase activity was studied in vitro using high performance liquid chromatography with electro-chemical detection. With the analytical method developed, 5-hydroxytryptophan, serotonin, and 5-hydroxyindole-3-acetic acid could be measured simultaneously. Indole-3-acetaldehyde (12-1200 microM) was found to cause a 6-33% inhibition of the enzyme. Dependent upon the nature of the sulfhydryl- or reducing-agent (dithiotreitol, glutathione, or ascorbate) present in the incubates, the degree of inhibition by disulfiram varied, probably due to the formation of various mixed disulfides. Also the presence of diethyldithiocarbamate (160-1600 microM) was found to inhibit tryptophan hydroxylase (28-91%), while 5-hydroxyindole-3-acetaldehyde, coprine, or 1-aminocyclopropanol appeared to have no effect on the enzyme activity.

Brain blood flow during hypercapnia in fish: no role of nitric oxide.

Very little is known about the regulation of cerebral blood flow (CBF) in lower vertebrates, especially fish. In mammals, hypercapnia causes cerebral vasodilation and increased CBF through mechanisms that involve the production of nitric oxide (NO). We have used epi-illumination microscopy in vivo to observe effects of hypercapnia on venular erythrocyte velocity, used as an index of CBF velocity, in rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius). Rainbow trout exposed to a pCO(2) of 7.5 mmHg displayed a small increase of CBF velocity in two out of five fishes, while dorsal aortic blood pressure (P(DA)) did not change. Exposing trout to a pCO(2) of 22.5 mmHg, resulted in an 80% increase in CBF velocity and a 21% increase in P(DA). Trout exposed to a pCO(2) of 75 mmHg showed an additional increase in blood pressure, while no further increase was seen in CBF velocity compared to a pCO(2) of 22. 5 mmHg. By contrast, no change in CBF velocity was seen in crucian carp, even at a pCO(2) of 75 mmHg. None of the circulatory changes seen in the trout could be blocked by superfusing the brain surface with the NO synthase blocker N(G)-nitro-L-arginine. The results point at striking species differences in the responses of CBF and P(DA) to hypercapnia in fish, and that the hypercapnia induced increase in CBF velocity seen in rainbow trout is independent of NO production.

Extracellular levels of amino acid neurotransmitters during anoxia and forced energy deficiency in crucian carp brain.

The crucian carp is one of the few vertebrates that has the ability to survive long periods of anoxia. A devastating event in the anoxic mammalian brain is a massive release of excitatory neurotransmitters, particularly glutamate. Using microdialysis to measure extracellular levels of several amino acid neurotransmitters and related compounds in the telencephalon of crucian carp in vivo, we show here that this species avoids a release of glutamate during anoxia, which is probably related to its ability to maintain energy charge. Instead, 6 h of anoxia produced a doubling of the extracellular level of GABA, the major inhibitory neurotransmitter in brain. The release of GABA may be a mechanism for lowering neuronal activity and energy use, thereby facilitating the maintenance of energy charge. Perfusing the microdialysis probe with a high-K+ Ringer showed that the telencephalon had the ability to release both glutamate and GABA. Moreover, if energy deficiency was produced during anoxia, by inhibiting glycolysis with iodoacetate (IAA), the resulting release of GABA was more rapid and profound than that of glutamate, possibly reflecting a second line of anoxia defence aimed at minimising the effect of a temporary energy failure.

Effects of aldehyde dehydrogenase inhibitors on hexobarbital sensitivity and neuroamine metabolism in rat brain.

Several authors have found that the aldehyde dehydrogenase (ALDH) inhibitor, disulfiram, prolongs hexobarbital-induced anaesthesia. It was suggested that this effect was caused by an alteration of the serotonergic system in brain, mediated by elevated levels of biogenic aldehydes. In the present study, disulfiram (300 mg/kg) was found to cause a 4-fold prolongation of hexobarbital-induced anaesthesia, while coprine (another potent ALDH-inhibitor) had no effect. This strongly suggested that biogenic aldehydes were not involved in this effect of disulfiram. The hexobarbital concentration in brain, at the electroencephalogram (EEG) criteria used for measuring hexobarbital sensitivity, was unaffected in rats given 75-300 mg/kg disulfiram, indicating that factors other than an increased brain hexobarbital sensitivity were responsible for the prolonged anaesthesia. Also 10-100 mg/kg coprine did not affect hexobarbital sensitivity measured this way. No alteration of the dopamine level in brain was found in rats given disulfiram, and in both disulfiram- and coprine-treated rats, similar changes in the serotonergic system were found. However, the level of norepinephrine was decreased in brains of disulfiram-treated rats, but it was unaffected by coprine. Thus, norepinephrine may have been involved in the prolongation of hexobarbital-induced anaesthesia caused by disulfiram.

Relationship between ion gradients and neurotransmitter release in the newborn rat striatum during anoxia.

It has been well documented that mammalian newborns are more resistant to hypoxia than adults. The mechanisms for this tolerance has attracted considerable attention due to its clinical implications. Recently, there has been great interest in comparing the mechanisms involved in such tolerance with those of turtle brain, which has shown a remarkable tolerance to anoxia. In the latter, much attention has been paid to the role of neurotransmitters in regulating brain metabolic rate. In order to investigate this phenomenon in the mammalian neonate the pattern of neurotransmitter release with respect to pre- and postdepolarization stages was determined. Microdialysis was used to ascertain levels of neurotransmitters in the striatum of 5-day-old rats. Ion homeostasis was determined with a potassium-selective microelectrode. We report here that during anoxia at the predepolarization stage purines (inosine, hypoxanthine, xanthine and adenosine) were significantly released. However, amino acids (glutamate, gamma-amino butyric acid (GABA), aspartate and taurine) remained low during the first 30 min, but were released during anoxic depolarization. It was concluded that mammalian neonate brain differs from that of the turtle in hypoxic adaptations, which may be consequence of its comparatively undifferentiated state.

Nitric oxide synthase inhibitor blocks acetylcholine induced increase in brain blood flow in rainbow trout.

Nitric oxide (NO) dependent regulation of blood flow has hitherto not been demonstrated in rainbow trout or other salmonid fish. Through in vivo observations of the brain surface (optic lobes) of rainbow trout (Oncorhynchus mykiss) with epi-illumination microscopy, we show that application of acetylcholine (ACh) to the brain surface induces an increase in cerebral blood flow velocity that can be completely blocked by the NO synthase inhibitor NG-nitro-L-arginine. Also sodium nitroprusside, which decomposes to form NO, stimulated cerebral blood flow velocity. The results indicate that NO is a vasodilator in rainbow trout brain, mediating the effect of ACh.

Anoxic depression of light-evoked potentials in retina and optic tectum of crucian carp.

The crucian carp is an exceptionally anoxia-tolerant vertebrate. For the brain, with its very high rate of ATP use, depression of energy use is likely to be an important strategy for anoxic survival. This study shows that the light-evoked response of the retina and the corresponding evoked potential in optic tectum decrease in amplitude by 69 and 75%, respectively, during 38 min of anoxia, and by about 90% after 1 h in anoxia. Both responses were restored upon reoxygenation. The length of light exposure (5 s or 100 ms) did not affect the degree of anoxic depression. These results are the first to show an anoxia-induced depression of central nervous system (CNS) activity in vivo in this species, and indicate that the crucian carp temporarily turns off its visual sense in order to reduce neural energy use during anoxic condition.

Serotonin as a regulator of hypothalamic-pituitary-interrenal activity in teleost fish.

Evidence for the presence of a serotonin1A (5-HT1A) receptor subtype in the salmonid fish brain has recently been presented. In the present study the potent 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) was tested for its effect on plasma cortisol concentrations in rainbow trout (Oncorhynchus mykiss). Blood was sampled and 8-OH-DPAT administered through a catheter in the dorsal aorta. Thirty minutes after the injection of 40 microg of 8-OH-DPAT/kg, plasma cortisol levels had increased from 12 to 149 ng/ml, whereupon they fell, reaching baseline levels after 4 h. The effect of 1-40 microg 8-OH-DPAT/kg on plasma cortisol concentrations was dose-dependent. The results lends further support to the hypothesis that the brain serotonergic system plays a key role in integrating autonomic, behavioral and neuroendocrine stress-responses in fish as well as mammals, suggesting that not only the structural and biochemical organization, but also the function of the serotonergic system has been conserved during vertebrate evolution.

Hypoxia stimulates cerebral blood flow in the estuarine crocodile (Crocodylus porosus).

The effect of N2 respiration on cerebral blood flow (CBF) velocity on the dorsal surface of cerebellum was examined in the estuarine crocodile, Crocodylus porosus, using epi-illumination microscopy. Twelve minutes of N2 respiration resulted in a 126% increase in CBF velocity. N2 respiration had no effect on blood pressure, indicating an underlying cerebral vasodilation. In addition, heart rate increased significantly. Systemic injections of aminophylline and the NO synthase (NOS) inhibitor nitro-L-arginine (L-NA) did not affect the hypoxia induced increase in CBF. We conclude that C. porosus responds to hypoxia with adenosine and nitric oxide (NO) independent cerebral vasodilation, and that this is likely to be a mechanism protecting the brain from energy deficiency during prolonged dives.

Effect of anoxia and adenosine on cerebral blood flow in the leopard frog (Rana pipiens).

The effect of anoxia on cerebral blood velocity (CBV) on the dorsal surface of telencephalon was examined in the leopard frog, Rana pipiens, using a stereomicroscope. During exposure to anoxia, a transient 228% increase in CBV velocity was seen after 20 min, but CBV fell back to basal values after a further 20 min of anoxia. Topical application of 50 microM adenosine during normoxia caused a 52% increase in CBV, while 250 microM adenosine caused no further increase. At both concentrations, the effect was completely inhibited by the adenosine receptor blocker aminophylline (250 microM). Superfusing the brain with aminophylline during anoxia did not affect the anoxia-induced increase in CBV. We conclude that adenosine can stimulate CBV in R. pipiens. However, unlike in other anoxia-tolerant animals, adenosine seems not to be a main mediator of the anoxia induced increase in CBV in the frog.