KATP-channels play a minor role in the protective hypoxic shut-down of cerebellar activity in eider ducks (Somateria mollissima).

Eider duck (Somateria mollissima) cerebellar neurons are highly tolerant toward hypoxia in vitro, which in part is due to a hypoxia-induced depression of their spontaneous activity. We have studied whether this response involves ATP-sensitive potassium (KATP) channels, which are known to be involved in the hypoxic/ischemic defense of mammalian neural and muscular tissues, by causing hyperpolarization and reduced ATP demand. Extracellular recordings in the Purkinje layer of isolated normoxic eider duck cerebellar slices showed that their spontaneous neuronal activity decreased significantly compared to in control slices when the KATP channel opener diazoxide (600 µM) was added (F1,70=92.781, p<0.001). Adding the KATP channel blocker tolbutamide (400 µM) 5 min prior to diazoxide completely abolished its effect (F1,55=39.639, p<0.001), strongly suggesting that these drugs have a similar mode of action in this avian species as in mammals. The spontaneous activity of slices treated with tolbutamide in combined hypoxia/chemical anoxia (95% N2-5% CO2 and 2 mM NaCN) was not significantly different from that of control slices (F1,203=0.071, p=0.791). Recovery from hypoxia/anoxia was, however, slightly but significantly weaker in tolbutamide-treated slices than in control slices (F1,137=15.539, p<0.001). We conclude that KATP channels are present in eider duck cerebellar neurons and are activated in hypoxia/anoxia, but that they do not play a key role in the protective shut-down response to hypoxia/anoxia.

The role of glycogen, glucose and lactate in neuronal activity during hypoxia in the hooded seal (Cystophora cristata) brain.

The brains of diving mammals are repeatedly exposed to hypoxic conditions during diving. Brain neurons of the hooded seal (Cystophora cristata) have been shown to be more hypoxia tolerant than those of mice, but the underlying mechanisms are not clear. Here we investigated the roles of different metabolic substrates for maintenance of neuronal activity and integrity, by comparing the in vitro spontaneous neuronal activity of brain slices from layer V of the visual cortex of hooded seals with those in mice (Mus musculus). Studies were conducted by manipulating the composition of the artificial cerebrospinal fluid (aCSF), containing either 10 mM glucose, or 20 mM lactate, or no external carbohydrate supply (aglycemia). Normoxic, hypoxic and ischemic conditions were applied. The lack of glucose or the application of lactate in the aCSF containing no glucose had little effect on the neuronal activity of seal neurons in either normoxia or hypoxia, while neurons from mice survived in hypoxia only few minutes regardless of the composition of the aCSF. We propose that seal neurons have higher intrinsic energy stores. Indeed, we found about three times higher glycogen stores in the seal brain (∼4.1 ng per µg total protein in the seal cerebrum) than in the mouse brain. Notably, in aCSF containing no glucose, seal neurons can tolerate 20 mM lactate while in mouse neuronal activity vanished after few minutes even in normoxia. This can be considered as an adaptation to long dives, during which lactate accumulates in the blood.

Neuroglobin of seals and whales: evidence for a divergent role in the diving brain.

Although many physiological adaptations of diving mammals have been reported, little is known about how their brains sustain the high demands for metabolic energy and thus O(2) when submerged. A recent study revealed in the deep-diving hooded seal (Cystophora cristata) a unique shift of the oxidative energy metabolism and neuroglobin, a respiratory protein that is involved in neuronal hypoxia tolerance, from neurons to astrocytes. Here we have investigated neuroglobin in another pinniped species, the harp seal (Pagophilus groenlandicus), and in two cetaceans, the harbor porpoise (Phocoena phocoena) and the minke whale (Balaenoptera acutorostrata). Neuroglobin sequences, expression levels and patterns were compared with those of terrestrial relatives, the ferret (Mustela putorius furo) and the cattle (Bos taurus), respectively. Neuroglobin sequences of whales and seals only differ in two or three amino acids from those of cattle and ferret, and are unlikely to confer functional differences, e.g. in O(2) affinity. Neuroglobin is expressed in the astrocytes also of P. groenlandicus, suggesting that the shift of neuroglobin and oxidative metabolism is a common adaptation in the brains of deep-diving phocid seals. In the cetacean brain neuroglobin resides in neurons, like in terrestrial mammals. However, neuroglobin mRNA expression levels were 4-15 times higher in the brains of harbor porpoises and minke whales than in terrestrial mammals or in seals. Thus neuroglobin appears to play a specific role in diving mammals, but seals and whales have evolved divergent strategies to cope with cerebral hypoxia. The specific function of neuroglobin that conveys hypoxia tolerance may either relate to oxygen supply or protection from reactive oxygen species. The different strategies in seals and whales resulted from a divergent evolution and an independent adaptation to diving.

Evidence for energy savings from aerial running in the Svalbard rock ptarmigan (Lagopus muta hyperborea).

Svalbard rock ptarmigans were walked and run upon a treadmill and their energy expenditure measured using respirometry. The ptarmigan used three different gaits: a walking gait at slow speeds (less than or equal to 0.75 m s(-1)), grounded running at intermediate speeds (0.75 m s(-1) < U < 1.67 m s(-1)) and aerial running at high speeds (greater than or equal to 1.67 m s(-1)). Changes of gait were associated with reductions in the gross cost of transport (COT; J kg(-1) m(-1)), providing the first evidence for energy savings with gait change in a small crouched-postured vertebrate. In addition, for the first time (excluding humans) a decrease in absolute metabolic energy expenditure (rate of O(2) consumption) in aerial running when compared with grounded running was identified. The COT versus U curve varies between species and the COT was cheaper during aerial running than grounded running, posing the question of why grounded running should be used at all. Existing explanations (e.g. stability during running over rocky terrain) amount to just so stories with no current evidence to support them. It may be that grounded running is just an artefact of treadmill studies. Research investigating the speeds used by animals in the field is sorely needed.

Slow intrinsic oscillations in thick neocortical slices of hypoxia tolerant deep diving seals.

Direct evidence that the mammalian neocortex is an important generator of intrinsic activity comes from isolated neocortical slices that spontaneously generate multiple rhythms including those in the beta, delta and gamma range. These oscillations are also seen in intact animals where they interact with other areas including the hippocampus, thalamus and basal ganglia. Here we show that thick isolated neocortical slices from hooded seals intrinsically generate persistent spontaneous activities, both repetitive non-rhythmic activity with activity states lasting for several minutes, and oscillating activity with rhythms that are much slower (<0.1 Hz) than the rhythms previously described in vitro. These intrinsic activities were very robust and persisted for up to 1 h even in severely hypoxic conditions. We hypothesize that the remarkable hypoxia tolerance of the hooded seal nervous system made it possible to maintain functional integrity in slices thick enough to preserve intact neuronal networks capable of generating these slow oscillations. The observed activities in seal neocortical slices support the notion that mammalian cortical networks intrinsically generate multiple states of activity that include oscillatory activity all the way down to <0.1 Hz. This intrinsic neocortical excitability is an important contributor not only to sleep but also to the default awake state of the neocortex.

Development of myoglobin concentration and acid buffering capacity in harp (Pagophilus groenlandicus) and hooded (Cystophora cristata) seals from birth to maturity.

Pinnipeds rely on muscle oxygen stores to help support aerobic diving, therefore muscle maturation may influence the behavioral ecology of young pinnipeds. To investigate the pattern of muscle development, myoglobin concentration ([Mb]) and acid buffering ability (beta) was measured in ten muscles from 23 harp and 40 hooded seals of various ages. Adult [Mb] ranged from 28-97 to 35-104 mg g tissue(-1) in harp and hooded seals, respectively, with values increasing from the cervical, non-swimming muscles to the main swimming muscles of the lumbar region. Neonatal and weaned pup muscles exhibited lower (approximately 30% adult values) and less variable [Mb] across the body than adults. In contrast, adult beta showed little regional variation (60-90 slykes), while high pup values (approximately 75% adult values) indicate significant in utero development. These findings suggest that intra-uterine conditions are sufficiently hypoxic to stimulate prenatal beta development, but that [Mb] development requires additional postnatal signal such as exercise, and/or growth factors. However, because of limited development in both beta and [Mb] during the nursing period, pups are weaned with muscles with lower aerobic and anaerobic capacities than those of adults.

When the brain goes diving: glial oxidative metabolism may confer hypoxia tolerance to the seal brain.

Deep diving mammals have developed strategies to cope with limited oxygen availability when submerged. These adaptations are associated with an increased neuronal hypoxia tolerance. Brain neurons of the hooded seal Cystophora cristata remain much longer active in hypoxic conditions than those of mice. To understand the cellular basis of neuronal hypoxia tolerance, we studied neuroglobin and cytochrome c in C. cristata brain. Neuroglobin, a respiratory protein typically found in vertebrate neurons, displays three unique amino acid substitutions in hooded seal. However, these substitutions unlikely contribute to a modulation of O(2) affinity. Moreover, there is no significant difference in total neuroglobin protein levels in mouse, rat and seal brains. However, in terrestrial mammals neuroglobin resided exclusively in neurons, whereas in seals neuroglobin is mainly located in astrocytes. This unusual localization of neuroglobin is accompanied by a shift in the distribution of cytochrome c. In seals, this marker for oxidative metabolism is mainly localized in astrocytes, whereas in terrestrial mammals it is essentially found in neurons. Our results indicate that in seals aerobic ATP production depends significantly on astrocytes, while neurons rely less on aerobic energy metabolism. This adaptation may imbue seal neurons with an increased tolerance to hypoxia and potentially also to reactive oxygen species, and may explain in part the ability of deep diving mammals to sustain neuronal activity during prolonged dives.

Size and distribution of oxygen stores in harp and hooded seals from birth to maturity.

Pinnipeds rely primarily on oxygen stores in blood and muscles to support aerobic diving; therefore rapid development of body oxygen stores (TBO(2)) is crucial for pups to transition from nursing to independent foraging. Here, we investigate TBO(2) development in 45 harp (Pagophilus groenlandicus) and 46 hooded (Cystophora cristata) seals ranging in age from neonates to adult females. We found that hooded seal adults have the largest TBO(2) stores yet reported (89.5 ml kg(-1)), while harp seal adults have values more similar to other phocids (71.6 ml kg(-1)). In adults, large TBO(2) stores resulted from large blood volume (harp169, hood 194 ml kg(-1)) and high muscle Mb content (harp 86.0, hood 94.8 mg g(-1)). In contrast, pups of both species had significantly lower mass-specific TBO(2 )stores than adults, and stores declined rather than increased during the nursing period. This decline was due to a reduction in mass-specific blood volume and the absence of an increase in the low Mb levels (harp 21.0, hood 31.5 mg g(-1)). Comparisons with other phocid species suggests that the pattern of blood and muscle development in the pre- and post-natal periods varies with terrestrial period, and that muscle maturation rates may influence the length of the postweaning fast. However, final maturation of TBO(2) stores does not take place until after foraging begins.

Cutaneous heat flux models do not reliably predict metabolic rates of marine mammals.

Heat flux models have been used to predict metabolic rates of marine mammals, generally by estimating conductive heat transfer through their blubber layer. Recently, Kvadsheim et al. (1997) found that such models tend to overestimate metabolic rates, and that such errors probably result from the asymmetrical distribution of blubber. This problem may be avoided if reliable estimates of heat flux through the skin of the animals are obtained by using models that combine calculations of conductive heat flux through the skin and fur, and convective heat flux from the surface of the animal to the environment. We evaluated this approach based on simultaneous measurements of metabolic rates and of input parameters necessary for heat flux calculations, as obtained from four harp seals (Phoca groenlandica) resting in cold water. Heat flux estimates were made using two free convection models (double-flat-plate and cylindrical geometry) and one forced convection model (single-flat-plate geometry). We found that heat flux estimates generally underestimated metabolic rates, on average by 26-58%, and that small variations in input parameters caused large variations in these estimates. We conclude that cutaneous heat flux models are too inaccurate and sensitive to small errors in input parameters to provide reliable estimates of metabolic rates of marine mammals.

Panting in reindeer (Rangifer tarandus).

Two winter-insulated Norwegian reindeer (Rangifer tarandus tarandus) were exposed to air temperatures of 10, 20, 30, and 38 degrees C while standing at rest in a climatic chamber. The direction of airflow through nose and mouth, and the total and the nasal minute volumes, respectively, were determined during both closed- and open-mouth panting. The animals alternated between closed- and open-mouth panting, but the proportion of open-mouth panting increased with increasing heat load. The shifts from closed- to open-mouth panting were abrupt and always associated with a rise in respiratory frequency and respiratory minute volume. During open-mouth panting, the direction of airflow was bidirectional in both nose and mouth, but only 2.4 +/- (SD) 1.1% of the air was routed through the nose. Estimates suggest that the potential for selective brain cooling is markedly reduced during open-mouth panting in reindeer as a consequence of this airflow pattern.

On the direction and velocity of blood flow in the extradural intravertebral vein of harp seals (Phoca groenlandica) during simulated diving.

Ronald et al. (1977) suggested that blood flow in the caudal/lumbar sections of the extradural intravertebral vein (EIV) of seals changes direction from running towards the head before diving, to the opposite during diving. The possible advantage would be that the oxygen-depleted venous effluent from the brain is routed via the EIV to the posterior parts of the hepatic sinuses and the inferior caval vein and, hence, is prevented from mixing with the more oxygen-rich venous blood in their anterior parts. We have re-examined this hypothesis by use of Doppler flowmetry. A catheter-tip flow probe was introduced into the EIV of two similar-sized juvenile harp seals, and flow direction and rate determined before, during and after simulated dives lasting for 5 min, at three positions (caudal, lumbar and thoracic) along the EIV. Regardless of probe position, blood was mainly flowing towards the head in 11 of 13 experiments prior to diving, in 8 of 13 experiments during diving and in 11 of 13 experiments during recovery after diving (and away from the head in the remaining experiments). Flow direction was most variable in the caudal position. Mean blood velocity in the EIV was substantially lower during diving (0.10 +/- 0.22 cm s-1 (n=5) in thoracic position) than in the pre-dive (3.98 +/- 3.32 cm s-1 [n=5]) and post-dive (5.75 +/- 4.07 cm s-1 [n=5]) situations. Thus, the direction and rate of flow in the EIV was variable, particularly during diving, as is to be expected in a system of anastomosing, valveless veins. We conclude that the hypothesis of Ronald et al. (1977) most likely is false.

Simulations of the effect of currently used grenade harpoons for the killing of whales using a pig-model.

Physical model experiments, as well as simulations of the effects of grenade harpooning on anaesthetized pigs fully immersed in water suggest that the shock effect of the blast from the currently used grenades is relatively minor. Also the animals are not stunned to death, but loose consciousness and subsequently die from hemorrhage. Survival time is therefore very short if the animals are hit in the thorax, and is likely to be further reduced if the charge which is currently used is increased, or, even better, if shrapnel (fragment scattering) grenades are used instead of blast grenades.

Changes in fibrinolytic activity in diving grey seals.

In order to test the hypothesis that enhanced fibrinolytic activity is a factor which prevents the blood of diving seals from clotting, we instrumented two female grey seals (Halichoerus grypus) with subcutaneous electrodes for measurements of heart rate (HR) and an extradural intravertebral venous catheter for collection of blood samples before, during and after simulated dives of 10 min duration. Blood samples were used for in vitro determination of clot lysis time (CLT), which is a measure of the level of fibrinolytic activity, and for analyses of plasma levels of cortisol, noradrenaline and adrenaline (A). The seals displayed profound diving bradycardia indicative of a substantial reduction in blood flow rates (pre-dive HR: 78 (63-98) bpm; dive HR: 8 (7-10) bpm; (median (range); n = 2)) and elevated catecholamine levels (pre-dive A: 121 (98-184) pg.ml-1; peak dive/post-dive A: 3510 (447-6181) pg.ml-1), both of which are factors which promote blood coagulation. Nevertheless, we found that CLT always increased in connection with diving (pre-dive CLT: 436 (356-568) min; peak CLT during diving: 1380 (640-1800) min), which implies a reduced, rather than enhanced, fibrinolytic activity in this situation. These results show that enhanced fibrinolytic activity is not part of the defence system which prevents fatal clotting from occurring in diving grey seals.

Effects of adrenergic and cholinergic drugs on splenic arteries and veins from hooded seals (Cystophora cristata).

Isolated ring preparations of arteries and veins from hooded seal spleens were subjected in vitro to adrenaline (A), noradrenaline (NA), isoprenaline (Iso), and acetylcholine (ACh), alone or in combination with the blockers phentolamine (Phe), propranolol (Pro), and atropine (Atr). Both arteries and veins constricted in response to A (the estimated effective dose required for half-maximal response (ED50) was 3.3 and 0.2 microM, for arteries and veins, respectively) and NA (estimated ED50 was 1.5 and 0.6 microM, for arteries and veins, respectively), but these effects were abolished when the drugs were given in combination with the alpha-adrenoceptor blocker Phe. The responses of arteries and veins to ACh and the beta-adrenoceptor agonist Iso were minor and inconsistent, and were completely abolished when combined with their respective blockers (Atr and Pro, respectively). The ED50 for both A and NA are quite high in relation to normal plasma levels of A and NA in seals. This implies that these vessels (and, hence, the supply of blood to the spleen) primarily are subjected to neurogenic, rather than humoral physiological control.

Blubber and flipper heat transfer in harp seals.

The trunk of marine mammals is encased in a blubber layer which provides thermal insulation that can be changed by circulatory adjustments. The extremities, on the other hand, are poorly insulated but have vascular arrangements constructed for prevention or promotion of heat loss depending on the thermal state of the animal. We have studied the importance of different body parts as sites for heat dissipation and also assessed the effect of circulatory adjustments on heat transfer through blubber, by combining direct measurements of heat flux from the flippers and trunk with simultaneous recordings of temperature gradients through the blubber and metabolic rates of harp seals (Phoca groenlandica) subjected to water temperatures between 1 and 24 degrees C. We also determined the thermal conductivity of blubber samples from the same animals after death, and compared this with the insulative properties of live blubber. At the lowest water temperatures, the insulative properties of live blubber were similar to those of dead blubber, and heat loss from the flippers only accounted for 2-6% of the metabolic heat production. As heat load increased with increasing water temperatures, the fraction of heat lost from the flippers increased, to 19-48% at 24 degrees C, while the fraction lost from the trunk decreased, despite an increase in the convective (circulatory) heat transfer through the blubber layer.

Volume capacity and contraction control of the seal spleen.

Volume changes in the spleens of hooded seals (Cystophora cristata) and harp seals (Phoca groenlandica) were measured plethysmographically in vitro in response to epinephrine, norepinephrine, isoprenaline, phentolamine, and acetylcholine. Dilated spleens contracted forcefully within 1-3 min of alpha-adrenoceptor activation with 1.0-5.0 micrograms epinephrine/kg body mass, whereas stimulation of beta-adrenoceptors and cholinergic receptors had little effect. The mass of dilated hooded seal spleens corresponded to 2-4% (n = 7) of body mass, with volume (V; ml) relating to body mass (M; kg) as follows: V = 12.0M + 910 (r2 = 0.96, n = 4). Thus the spleen of a 250-kg hooded seal maximally expels 3.9 liters, or 13%, of its estimated total blood volume. Average hematocrit in splenic venous outflow from dilated spleens was 90 +/- 3% (n = 3) in hooded seals and 85% (n = 2) in harp seals. From these data we have estimated that the aerobic diving limit of a 250-kg hooded seal increases only 105 s, at the most, if complete emptying of the spleen occurs during diving, while the corresponding estimate for a 112-kg harp seal is 80 s.

Pineal and thyroid functions in newborn seals.

Daily variations of pineal and plasma melatonin and plasma thyroid hormones were measured in harp seals (Phoca groenlandica), grey seals (Halichoerus grypus), and hooded seals (Cystophora cristata), ranging in age from newborn to 14 days. In newborn harp seals the mean mass of the pineal gland was 273 mg (+/- 45 SEM, n = 11), containing 49 ng (median) melatonin. In newborn, 4- and 10-day-old grey seals, the pineal mass was similar, weighing on average 337 mg (+/- 74, n = 6) and containing 90 ng melatonin. Two newborn hooded seal pups had pineals weighing 520 and 1289 mg, with 254 and 7600 ng melatonin, respectively. There were no day-night differences in the pineal contents of melatonin or in the number of pineal beta-adrenergic receptors measured in newborn harp seals, and, in newborn, 4- and 10-day-old grey seals, there were no day-night or age differences in pineal melatonin content. Plasma melatonin levels were 10 times higher in newborn seals than in two 10-day-old grey seals and one 14-day-old harp seal pup. In all seal pups, the levels exhibited a 24-hr rhythmicity, with increasing night- and decreasing daytime concentrations. Plasma levels of thyroxine (T4) and triiodothyronine (T3) were generally higher in newborn seals than in 10- and 14-day-old seals or in adult females. There was no apparent 24-hr rhythmicity, but the thyroid hormone levels generally declined throughout each sampling sequence. High pineal and thyroid activities may play a thermoregulatory role in newborn seals, but the results do not indicate a stimulatory action of melatonin in the peripheral conversion of T4 to T3. It is speculated that the large and active pineal gland, particularly in newborn seals, may be related to aspects of their diving habit.

Daily energy expenditure in free living minke whales.

Six minke whales (Balaenoptera acutorostrata) were instrumented with VHF-radio transmitters and four with sonic speed-depth transmitters off the west coast of northern Norway and Svalbard and followed within view for up to 24 h. During such periods their respiratory rate was continuously recorded and their energy expenditure estimated according to Folkow & Blix (1992) at different swimming speeds and types of activity. We found that cost of swimming is remarkably low in these large animals and that their estimated daily energy expenditure on average only amounts to 80 kJ kg-1 day-1.

Nasal heat and water exchange is not an effector mechanism for water balance regulation in grey seals.

Phocid seals may effectively restrict respiratory heat and water loss by nasal heat and water exchange (NHE), and respiratory heat loss is, in fact, subject to thermoregulatory control. We have investigated whether phocid seals also control NHE and respiratory water loss to regulate water balance. Three resting juvenile female grey seals (Halichoerus grypus) were subjected to: (i) 5 days of food and water deprivation, (ii) intravenous infusion of 1000 ml of a hypersomotic (930 mM) solution of the diuretic mannitol, and (iii) oral injection of 1500 ml distilled water. During these experiments in air of 0 degree C, expired air temperature (T(ex)) and respiratory frequency (f) were recorded, and urine and blood samples collected. The results were compared with results from control experiments. Five days of food and water deprivation caused an average 10.5% and 20.8% increase in plasma (PO) and urine (UO) osmolality, respectively. Mannitol infusion induced excessive diuresis and caused an average 2.45% reduction of the estimated body water pool. Water loading caused an average 4.5% and 60% reduction in PO and UO, respectively, while urine production increased by 365%, on average. However, in no case did either T(ex) or f change significantly from mean control levels of 22.4 (range: 20.7-25.2) degrees C and 7.3 (range: 6.6-8.4) breaths min-1, respectively. Thus, water balance disturbances that initiate renal compensatory mechanisms fail to affect NHE in grey seals. This suggests that control of NHE is not an effector mechanism for regulation of water balance in grey seals.