AJP - Regu Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 291: R1482-R1489, 2006. First published June 1, 2006; doi:10.1152/ajpregu.00172.2006
0363-6119/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/5/R1482    most recent
00172.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vornanen, M.
Right arrow Articles by Paajanen, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vornanen, M.
Right arrow Articles by Paajanen, V.

COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Seasonal changes in glycogen content and Na+-K+-ATPase activity in the brain of crucian carp

Matti Vornanen and Vesa Paajanen

University of Joensuu, Department of Biology, Joensuu, Finland

Submitted 13 March 2006 ; accepted in final form 29 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Changes in the number of Na+-K+-ATPase {alpha}-subunits, Na+-K+-ATPase activity and glycogen content of the crucian carp (Carassius carassius) brain were examined to elucidate relative roles of energy demand and supply in adaptation to seasonal anoxia. Fish were collected monthly around the year from the wild for immediate laboratory assays. Equilibrium dissociation constant and Hill coefficient of [3H]ouabain binding to brain homogenates were 12.87 ± 2.86 nM and –1.18 ± 0.07 in June and 11.93 ± 2.81 nM and –1.17 ± 0.06 in February (P > 0.05), respectively, suggesting little changes in Na+-K+-ATPase {alpha}-subunit composition of the brain between summer and winter. The number of [3H]ouabain binding sites and Na-K-ATPase activity varied seasonally (P < 0.001) but did not show clear connection to seasonal changes in oxygen content of the fish habitat. Six weeks’ exposure of fish to anoxia in the laboratory did not affect Na+-K+-ATPase activity (P > 0.05) confirming the anoxia resistance of the carp brain Na pump. Although anoxia did not suppress the Na pump, direct Q10 effect on Na+-K+-ATPase at low temperatures resulted in 10 times lower catalytic activity in winter than in summer. Brain glycogen content showed clear seasonal cycling with the peak value of 203.7 ± 16.1 µM/g in February and a 15 times lower minimum (12.9 ± 1.2) in July. In winter glycogen stores are 15 times larger and ATP requirements of Na+-K+-ATPase at least 10 times less than in summer. Accordingly, brain glycogen stores are sufficient to fuel brain function for about 8 min in summer and 16 h in winter, meaning about 150-fold extension of brain anoxia tolerance by seasonal changes in energy supply-demand ratio.

anoxia; anoxia tolerance; [3H]ouabain binding; sodium pump; phenotypic plasticity

CRUCIAN CARP IS A WIDELY DISTRIBUTED cyprinid species that lives in ponds and lakes in Europe and central Asia. It is highly vulnerable to predation and uses two distinct "strategies" to reduce or avoid predation. When crucian carp coexist with piscivorous fish in lakes, the presence of predators induces a change in body morphology from a shallow and long-bodied fish to much deeper bodied form, thereby decreasing predation efficiency of the gape-limited piscivores (5, 13). An alternative survival strategy of the crucian carp is based on an equally prominent trait to completely avoid predation: crucian carp inhabit small, seasonally anoxic ponds where other vertebrate species are unable to survive. This requires exceptional physiological traits to tolerate oxygen shortage (4, 10). Although the anoxia tolerance of crucian carp greatly surpasses that of most other vertebrates, it is not a totally fixed characteristic but strongly varies according to the seasons. In May–July, crucian carp tolerate complete anoxia for about a day at 5–18°C, while in December–January, the mean anoxia survival time is about 40 days at 5°C and more than 120 days at 2°C (25). It is not, however, clear to what extent the improved anoxia tolerance in winter is an outcome of changing seasonal conditions, e.g., low temperature, or due to the seasonally bound changes in the physiology of the animal, i.e., phenotypic plasticity of the carp genome.

Anoxia survival requires that vital organs have enough energy to maintain body homeostasis. In this regard, metabolically active and hypoxia-sensitive organs, particularly brain and heart, must have adequate physiological mechanisms to cope with oxygen shortage (16, 24, 30). Because anoxic ATP production from glucose is only 7% of its aerobic value, anoxia tolerance is basically a matter of demand and supply, i.e., balancing energy requirements of the vital organs to match the reduced rate of anoxic energy supply. In the vertebrate brain, 50–80% of the total energy is used to maintain cellular ion gradients by the sodium pump (7), which, as the major energy sink, is a putative target for metabolic downregulation (12). It is not known, however, whether the brain Na+-K+-ATPase is depressed in the anoxic season and to what extent low temperature directly reduces the energy demand of the Na-K-ATPase.

On the supply side, the size of glycogen stores in the liver and other organs are probably decisive for anoxia tolerance (15, 29). Glycogen stores of the vertebrate brain tissue are generally regarded as insignificant, but the recent findings indicate that even the limited glycogen stores of the mammalian brain play a physiologically significant role in brain function (6, 9). Brain glycogen might be even more useful for species that regularly face oxygen shortage in their natural habitat. However, the significance of brain’s own glycogen stores for anoxia tolerance of neurons and glia has not yet been assessed in anoxia-resistant species. Therefore, the present study was designed to examine whether crucian carp brain shows seasonal plasticity in regard to Na+-K+-ATPase activity and brain glycogen stores, that is, how the energy supply-demand is regulated in brain tissue of fish living under natural environmental conditions. It is shown that, in winter, energy stores of the brain glycogen are 15 times larger and ATP requirements of Na+-K+-ATPase 10 times less than in summer. Accordingly, crucian carp brain’s glycogen stores are sufficient to fuel brain function for about 8 min in summer and ~16 h in winter, meaning about 150-fold extension of inherent brain anoxia tolerance by seasonal changes in energy supply-demand ratio.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Collection of animals. Crucian carp (Carassius carassius L.), 7–266 g in body mass (n = 255), were captured regularly from a local pond around the year beginning in May 2002 and ending in June 2003. The fish traps were kept in the same locations throughout the year and were checked once or twice each month. Temperature and oxygen content of the pond water were measured each time at the site of traps with a battery-operated analyzer (Cellox 325 with WTW Multiline P4, Weilheim, Germany). The fish were brought immediately into the lab and held in 500-liter stainless-steel tanks with circulating groundwater at the temperature of the pond water until used for sample preparation, which happened within 24 h from the time the fish were captured. All experiments were made with the consent of the local committee for animal experimentation.

Anoxia experiments. Crucian carp (n = 57; body mass 20.7 ± 1.26 g) caught in February were allowed several weeks’ habituation to laboratory conditions at constant photoperiod (9:15-h light-dark cycle) at 4°C before the experiments. Anoxia experiments were conducted at 4°C and included three animal groups: 1) normoxic control animals, 2) anoxic animals, and 3) a recovery group. For anoxic exposure, 4–6 fish (body mass 12–35 g) were put into water-filled Erlenmeyer bottles (3 liter), and oxygen was driven out by N2-gasing until O2 tension was close to the detection limit of the oxygen analyzer and below the threshold (0.3 mg/l) for anaerobic energy production (22). Bottles were quickly sealed with rubber stoppers and placed on the bottom of the metal tank. Animals of the recovery group (body mass 12–25 g) were exposed to the same anoxic treatment as the anoxic group but were allowed to recover at normoxic conditions (O2 11 mg/l) for 1 wk. Control animals were maintained for 6 wk in Erlenmeyer bottles (body mass 16–44 g) covered with tissue that allowed free oxygen exchange with normoxic tank water (O2 11 mg/l).

Preparation of brain samples. Dilute brain homogenates (5%) were prepared for [3H]ouabain binding and Na+-K+-ATPase assays. Fish were stunned by a blow to the head and killed by cutting the spine. Whole brains were carefully excised, blotted dry, and weighed to the nearest 0.1 mg. Brains from 2–6 fish were pooled for each sample and homogenized using a Teflon-glass homogenizer with four 20-s bursts at the maximum speed (2,200 rpm) in 20 volumes of ice-cold Tris·HCl (50 mM, pH 7.4 at 20°C) buffer. The homogenate was divided into four equal portions and stored frozen at –40°C for [3H]ouabain binding and Na+-K+-ATPase assays.

[3H]ouabain binding. [3H]ouabain (Amersham, Little Chalfont, UK) binding was performed in a total volume of 0.5 ml of solution containing 0.5 mg of wet tissue, in 4 mM H3PO4, 4 mM MgCl2, 50 mM Tris·HCl at pH 7.4 (20). All binding experiments were conducted at 20°C. Nonspecific binding was determined by measuring [3H]ouabain binding in the presence of 100 µM unlabeled ouabain (Sigma, Poole, UK). The reaction was terminated with 6 ml of ice-cold wash buffer containing: 4 mM MgCl2, 50 mM Tris·HCl at pH 7.4 at 20°C, and the suspensions were immediately filtered through Whatman GF/B filters (Merck, Poole, UK) with three 6-ml washes of cold buffer. Filters were soaked in 10 ml of scintillant (Ready Protein+, Beckman), and [3H]ouabain bound to the filter was quantified by liquid scintillation counting (Wallac 1414 WinSpectral, Wallac, Finland). This basic procedure of [3H]ouabain binding was modified for different determinations as described below.

Association binding of [3H]ouabain. The binding rate of [3H]ouabain to brain receptors was measured by incubating membrane preparations with 4, 20, 40, and 60 nM [3H]ouabain and quenching the reaction with 6 ml of the ice-cold wash buffer at times ranging from 10 to 1,440 min. Unspecific binding in the presence of 100-µM unlabeled ouabain was subtracted from the total binding to obtain specific binding. Specific binding was plotted as a function of time, and the observed association rate constant (Kobs) was calculated from exponential fits to the data

Formula

where y is specific binding (pmol·min–1·g wet weight–1), ymax maximal specific binding, Kobs = observed association rate constant (min–1) and t time is in min. Next, the Kobs values were plotted as a function of [3H]ouabain concentration and fitted to linear regression equation to obtain association rate constant (K+1, the slope of the line), dissociation rate constant (K–1, the y-axis intercept of the line), and the equilibrium dissociation constant (Kd = K–1/K+1) (see Fig. 1, A and B).


Figure 1
View larger version (22K):
[in this window]
[in a new window]
  		Fig. 1. Association binding kinetics and equilibrium binding of [3H]ouabain to the brain homogenates of the crucian carp. A: time dependence of [3H]ouabain (4 and 40 nM) binding in preparations from fish captured in June and February. The data were fit with an exponential equation to obtain observed association rate constants (Kobs). The results are means ± SE of 4 preparations. B: Kobs values are plotted as a function of [3H]ouabain concentration (4, 20, 40, and 60 nM) to obtain association rate constant (K+1, the slope of the line), dissociation rate constant (K-1, the intercept of the regression line with the y-axis), and equilibrium dissociation constant (Kd = K–1/K+1). The results are means ± SE of 4 or 5 preparations. C: equilibrium binding of [3H]ouabain (4 nM) in the presence of 10–10–10–4 M unlabeled ouabain to brain preparations of the crucian carp captured in June and February. The results are means ± SE of 7 preparations for both groups. Kd and Hill slope values of the fits are shown within the figure.

 
Equilibrium binding of [3H]ouabain. The affinity of the crucian carp brain preparations from summer and winter fish for ouabain was determined by equilibrium binding of [3H]ouabain. Total ouabain concentration was varied by increasing the concentrations of unlabeled ouabain, while maintaining a low constant level of [3H]ouabain (4 nM). Unspecific binding in the presence of 10–4 unlabeled ouabain accounted for less than 1% of the total binding and was subtracted to obtain specific binding. The data were fitted to the Hill equation

Formula

where B is the amount of bound [3H]ouabain at the concentration of c, y0 is the unexplained part of [3H]ouabain binding, Bmax is the maximum binding, Kd is the equilibrium dissociation constant, and H is the Hill slope.

Determination of Na+-K+-ATPase activity. Na+-K+-ATPase activity was determined from the release of inorganic phosphate (2) at 15°C, 25°C, and 35°C. Maximal Na+-K+-ATPase activity was obtained as the difference in inorganic phosphate liberated in the presence and absence of 3 mM ouabain in a final volume of 1.0 ml of the ATPase medium containing (in mM) 5 Na2ATP, 5 MgCl2, 20 KCl, 100 NaCl, 50 Tris·HCl, 1 EGTA, and 5 NaN3, at pH 7.2. After 10 min preincubation of the sample in the medium, the reaction was started with the addition of 25 µl of 100 mM ATP stock solution, and the incubation was continued for 120, 60, and 30 min at 15°C, 25°C, and 35°C, respectively, to give similar total breakdown of ATP. Under these assay conditions, the release of inorganic phosphate was linear with time up to the end points of the incubation. Activity of the background ATPase was obtained as ouabain-resistant ATPase activity corrected with the time-dependent breakdown of ATP in the absence of homogenate. Thermal dependence of the ATPase activity as a Q10 value, was obtained from the equation

Formula

where T1 and T2 are the temperatures that produce the ATPase rates of R1 and R2, respectively. Q10 was separately calculated for the two temperature ranges (15–25°C and 25–35°C). Specific ATPase activities are given as micromoles of inorganic phosphate liberated in 1 min/g tissue wet weight. The cycling rate of Na+-K+-ATPase (cycles/min) was obtained by dividing the specific activity with the number of ATPase alpha subunits in 1 gram of wet tissue, as determined in the [3H]ouabain binding assays.

Determination of glycogen. Glycogen content of whole brains from individual fish was determined within 24 h from the capture of the animals. Glycogen was digested in hot 30% KOH, and the released glucose was determined with phenol-sulfuric acid technique (19). The alkaline treatment dissolves both proglycogen and macroglycogen and thus gives the total glycogen content of the brain. Glycogen content is expressed in glucosyl units/g tissue wet weight.

Statistical analyses. Differences in [3H]ouabain binding and brain glycogen content were tested with one-way ANOVA followed by Tukey’s post hoc test for pairwise comparisons between any two seasonal sampling dates (SPSS, Jandel Scientific, Erkrath, Germany). ATPase activities were analyzed with repeated-measures ANOVA and Tukey’s test. Q10 values of the Na+-K+-ATPase at the two temperature ranges were compared with paired t-test. P < 0.05 was regarded as the level of statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of [3H]ouabain binding. To justify determination of [3H]ouabain binding in seasonally acclimatized fish, ouabain receptors were characterized in fish capture in winter (February) and summer (June). There were no differences in the observed rate of association binding (Kobs) between fish captured in February and June at any of the four [3H]ouabain concentrations. Accordingly, association (K+1) and dissociation (K–1) rate constants and the equilibrium dissociation constant (Kd) were similar in winter and summer fish (Figs. 1, A and B). Competitive binding experiments using a low concentration (4 nM) of [3H]ouabain and different concentrations of nonlabeled ouabain, covering six orders of magnitude, gave almost identical Kd and Hill slope values for summer and winter fish (Fig. 1C and Table 1). Collectively, these experiments indicate that high-affinity [3H]ouabain binding is qualitatively similar in fish acclimatized to summer and winter.


View this table:
[in this window]
[in a new window]
  		Table 1. Comparison of [3H]ouabain binding to brain preparations of crucian carp captured in June and February

 
Ouabain receptors and Na+-K+-ATPase activity. The number (Bmax) of Na+-K+-ATPase molecules was determined by high-affinity binding of [3H]ouabain to the alpha subunit of the ATPase (Fig. 2). Although highly statistically significant (P < 0.001), seasonal changes in the number of brain ouabain receptors are small, and the seasonal pattern of [3H]ouabain binding does not follow as clear of a pattern as brain glycogen content (Fig. 2). There is, however, apparent depressions of ouabain receptors in February before the anoxic period and in late April following the anoxic bout.


Figure 2
View larger version (49K):
[in this window]
[in a new window]
  		Fig. 2. Seasonality of [3H]ouabain (10 nM) binding (top) in crucian carp brain homogenates and seasonal changes in brain glycogen content of the fish (bottom). Changes in oxygen content, temperature, and day length are also shown. The results are means ± SE from 7–12 preparations. For each preparation, brains from 4–10 fish were pooled. Columns marked with different letters of the letter pairs a-b, c-d, e-f, and g-h differ significantly (P < 0.05) from each other. The number of time points for [3H]ouabain binding is only 12 due to the small number of fish caught in October.

 
The seasonality of ouabain-sensitive Na+-K+-ATPase activity closely resembled that of [3H]ouabain binding (Fig. 3). The peak activity occurred in midwinter (November–January), when waters were already hypoxic, with transient declines in February and April (P < 0.001). Temperature-dependence (Q10 value) of the Na+-K+-ATPase activity was about 2 between 25°C and 35°C and close to 4 between 15°C and 25°C (P < 0.001) (Fig. 4). It should be noted, however, that in winter the Q10 value between 15°C and 25°C was significantly less than in summer. To get physiologically more realistic insight into the seasonality of Na+-K+-ATPase activity, the enzyme activity at 15°C and monthly Q10 values (between 15° and 25°C) were used to calculate Na+-K+-ATPase activity at more seasonally relevant temperatures. Temperature corrected values indicate that throughout winter the activity of the Na+-K+-ATPase is only 10–15% of its activity in midsummer because of reduced cycling rate of the enzyme. It is noteworthy that ATPase activity is depressed by seasonal temperature changes before the hypoxia sets in. This analysis indicates that temperature decline causes a drastic depression of the brain Na+-K+-ATPase in winter.


Figure 3
View larger version (38K):
[in this window]
[in a new window]
  		Fig. 3. Seasonal dynamics of Na+-K+-ATPase and background ATPase activities of the crucian carp brain. The results are means ± SE of 7–12 preparations. For each preparation, brains from 4–10 fish were pooled. Columns marked with different letters of the letter pairs a-b, c-d, and e-f differ significantly (P < 0.05) from each other.

 

Figure 4
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 4. Temperature dependence of Na+-K+-ATPase activity of the crucian carp brain. Top: Q10 values at two temperature ranges (15°–25°C and 25°–35°C). Bottom: calculated Na+-K+-ATPase activity at different times of the year. The values were obtained by extrapolation of the measured Na+-K+-ATPase activity at 15°C to different seasonal temperatures by using the Q10 value between 15° and 25°C. The activity of Na+-K+-ATPase is also given as ATPase cycles per minute. Seasonal changes in oxygen content are also shown (dashed line). *Statistically significant difference (P < 0.05) in Q10 value between the two temperature ranges.

 
The activity of background ATPase was maximal in summer with few changes in other times of the year (Fig. 3). The Q10 of the background ATPase was much less than that of the Na+-K+-ATPase: 1.3 between 35°C and 25°C, and 1.8 between 25°C and 15°C.

Effect of anoxia on Na+-K+-ATPase activity. To see whether Na+-K+-ATPase activity would be further depressed by anoxia, relative to that caused by low temperature, winter-acclimatized fish were exposed to anoxia (O2 <0.3 mg/l) for 6 wk in the lab. Anoxia depleted brain glycogen from 197.2 ± 44.1 to 68.1 ± 18.7 µmol/g (P < 0.001) but did not have any effect on the activity of either Na+-K+-ATPase or background ATPase (P > 0.05) (Fig. 5). Interestingly, 1 wk of normoxic recovery seemed to specifically depress Na+-K+-ATPase activity (about 30% at 35°C) without effect on the background ATPase. The difference was not, however, statistically significant (P = 0.113).


Figure 5
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5. Effects of 6 wk anoxia (O2 < 0.3 mg/l) at 4°C on Na+-K+-ATPase activity of the crucian carp brain. Animals of the recovery group were maintained at normoxic water (O2 11 mg/l) for 1 wk after the anoxic exposure. The activity of the background ATPase and the effect of anoxia on brain glycogen are also given. The results are means ± SE of 7 or 8 preparations. *Statistically significant difference (P < 0.05) between control and anoxic group.

 
Glycogen content. Glycogen content of the crucian carp brain is characterized by strong seasonal changes (Fig. 2A). Brain glycogen level is relatively low in summer when plenty of oxygen is available but starts to increase when the hypoxic winter period sets in (P < 0.001). In spite of continuously declining oxygen content, brain glycogen stores remain high in midwinter with a peak value of 203.7 ± 16.1 µmol/g wet weight in February. However, after the long anaerobic period (a minimum of 6 wk) from February onward, glycogen stores of the brain are depleted in late April. A minimum level of 12.9 ± 1.2 µmol/g wet weight is attained in June. Taken together, these findings show that in midwinter glycogen content is 15 times higher than in midsummer and suggest that long-lasting seasonal anoxia, but not moderate hypoxia, partially depletes the glycogen stores.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the natural habitat of crucian carp, the anoxic period occurs in late winter at temperatures below 4°C. Furthermore, anoxia does not begin abruptly but sets in gradually via a deepening hypoxic period and is preceded by shortening day length and a graded decrease of water temperature, that is, anoxia is a regularly occurring seasonal condition associated with clear environmental cues. Theoretical analysis indicates that under highly predictable and repeatedly occurring conditions, reversible physiological plasticity, that is, the ability of a genotype to express different phenotypes depending on environmental conditions, provides selective advantage over phenotypes that constitutively express physiological traits sufficient to cope with the harsh environmental conditions (8). Although a number of constitutively expressed mechanisms, which are permanently active or can be recruited with a minimum delay under acute anoxia, have been well described in the brain function of the best vertebrate anaerobes, including the crucian carp (for reviews, see Refs. 16, 23, 24), the phenotypic plasticity of the brains has received little attention. This study is the first to elucidate seasonality of the brain function in crucian carp and examines the supply-demand relationship between the main energy store, glycogen, and the major energy sink, the Na+-K+-ATPase at the tissue level. The results indicate a substantial increase in brain glycogen stores and emphasize the importance of low temperature in reducing brain energy demands during winter anoxia.

Brain Na+-K+-ATPase. Na+-K+-ATPase is responsible for establishing the electrochemical gradient of Na+ and K+ across the plasma membrane, which is necessary for maintaining resting membrane potential and electrical activity of neurons for neurotransmitter uptake and osmotic balance of neuronal cells. Anoxia experiments under controlled laboratory conditions indicate that long anoxia exposure does not cause any depression in the number of Na+-K+-ATPase units or their molecular activity in the crucian carp brain. The excellent anoxia resistance of the brain Na+ pump in winter-acclimatized crucian carp completely accords with the results of a study of short-term (12 h) anoxia on brain Na+-K+-ATPase activity measured with p-nitrophenylphosphate as substrate in crucian carp reared at 12°C (14). Maintenance of Na+-K+-ATPase activity in the anoxic carp brain contrasts with the situation in mammals and in anoxia-tolerant turtles, where anoxic insults cause depression of Na+-K+-ATPase activity or Na+-pump alpha subunits or both. In mammals, the anoxic depression of brain Na+-K+-ATPase is a sign of irreversible neuronal damage, whereas in turtles it is assumed to reflect reduced recruitment of neuronal ion channels, that is, "channel arrest," which allows energy savings in active ion pumping and results in strong depression of brain activity (14, 27). As Na+-K+-ATPase activity and ion channel function are tightly coupled, anoxia resistance of the carp Na+-K+-ATPase suggests that anoxia-induced channel arrest is not invoked in the crucian carp brain. This result is in good agreement with those experiments that have failed to show any type of channel arrest in crucian carp brain (24).

In contrast to the findings of the controlled anoxia experiments, there seems to be transient reduction(s) of Na+-K+-ATPase units and activity during and after winter anoxia in the natural habitat of the fish. As laboratory experiments demonstrate a definite resistance of the brain Na+-K+-ATPase to anoxia, we have to anticipate that seasonal depression of Na+-K+-ATPase is not directly related to anoxia. It should be noted, however, that 1 wk recovery from anoxia was associated with a 30% decrease of Na+-K+-ATPase activity, suggesting that the Na+-K+-ATPase might be damaged by the relief of anoxia. In the mammalian brain, reoxygenation after hypoxia causes lipid peroxidation and decreases the number and activity of Na+-K+-ATPase more than the hypoxic insult itself (3, 21). Therefore, the transient depressions of Na+-K+-ATPase after the anoxic winter period might be due to reoxygenation-related free radical damage. This might have been better resolved if the recovery from anoxia had been followed by more frequent sampling.

Although anoxia did not affect the Na+ pump of the crucian carp brain, strong seasonal changes in Na+-K+-ATPase activity were caused by direct temperature effect on enzyme catalysis. Similar to most biological processes, thermal sensitivity of the Na+-K+-ATPase strongly increased at low temperatures, and as an outcome the activity of the brain Na+-K+-ATPase in winter was depressed to only about 10% of its activity in summer. Although temperature-dependent depression of the Na-K-ATPase produces large energy savings in ion pumping, the inhibition is so extensive that it will inevitably limit electrical activity of the brain. Temperature-dependent depression of ion pumping will slow down the replenishment of electrochemical ion gradients, and therefore the frequency of action potentials generated by passive ion flow through ion channels will be strongly depressed. This may be the reason why the acute anoxia-induced channel arrest is not needed in the crucian carp brain. In fact, a positive thermal compensation of Na+-K+-ATPase activity is evident in November–January as a consequence of reduced Q10 values, which may be necessary to prevent excessive downregulation of nervous function. Collectively, these findings indicate that the number of brain Na+-K+-ATPase units remain relatively steady around the year, but the activity of the pump is drastically depressed in winter as a result of the direct temperature effect on the catalytic rate.

Brain glycogen. The glycogen content (204 µM glucosyl units/g or 3.3% of tissue wet weight) of the winter-acclimatized crucian carp highly surpasses brain glycogen stores reported for any other vertebrate species. Schmidt and Wegener (28) reported a glycogen content of 19.4 µM/g for crucian carp brain, which is close to the value (12.9 µM/g) measured here for the summer-acclimatized carp. In frogs and reptiles, including anoxia-tolerant turtles, the concentration of brain glycogen varies between 8 and 16 µmol/g, and in mammalian and avian brains between 3 and 12 µmol/g (6, 11, 18). Thus crucian carp brain contains 10–70 times more glycogen than the brains of other vertebrates. As the glycolytic breakdown of one glucose molecule produces two ATP molecules (and 2 lactates), the 204 µM glucose/g is enough to fuel the brain Na+-K+-ATPase at its maximum rate (0.8 µmol ATP·g–1·min–1) for about 8.6 h, whereas the brain glycogen stores of the summer carp are sufficient to maintain the maximum activity of the Na+-K+-ATPase (8.6 µM ATP·g–1·min–1) only for 3 min. Because it is unlikely that Na+-K+-ATPase functions at its maximum rate in vivo, the above calculation may underestimate glycogen’s importance for anoxic brain function. On the other hand, Na+-K+-ATPase is not the sole energy sink of the brain, and therefore the sufficiency of glycogen might have been overstated. A more direct estimate of glycogen’s importance can be deduced from the rate of anaerobic glycolysis of the crucian carp brain slices, which is 0.43 µM lactate·g–1·min–1 at 12°C (17). Extrapolation of this value to 2°C (Q10 = 2), assuming that the in vivo rate of glycolysis is double the rate of excised slices, gives a value of 0.43 µmol ATP·g–1·min–1. At that rate, brain glycogen stores alone were sufficient to support brain function for almost 16 h in winter-acclimatized crucian carp, whereas in summer-acclimatized carp, glycogen would be sufficient for only 8 min. This means almost 150 times extension in brain anoxia tolerance by a change in supply-demand ratio.

The 15-fold seasonal change of the crucian carp brain glycogen content strongly suggests that brain glycogen is a physiologically important carbohydrate store for the anoxia survival of the brain. It is not, however, clear whether brain glycogen serves as an immediate energy source when anoxia sets in or whether it is an emergency reservoir that is recruited under prolonged anoxia if the circulation fails to provide sufficient glucose to meet the brain’s demands. It is important to realize that anoxia does not start abruptly but is preceded by gradual hypoxia and other environmental cues that enable preconditioning of the brain to anoxia, that is, glycogen might not be needed as an energy buffer to win time for the activation of anoxia-induced defense mechanisms. In this respect, it is also notable that brain glycogen was not used during moderately hypoxic conditions but only under total oxygen shortage. In mammals, glycogen is depleted by intense brain activity, even if exogenous glucose is available, suggesting that glycogen is not a mere energy buffer but serves a more specific physiological role (6, 9). As Na+-K+-ATPase is preferentially fueled by ATP produced from glycolysis (26), glycogen might protect neurons against anoxic depolarization (1). Whether glycogen serves some specific brain function in anoxia-tolerant vertebrates and how the metabolism of glycogen is organized between neurons and glia remain interesting future topics of research.

Limitations of the study. To understand how organisms physiologically respond to multiple environmental changes, it is important to study the acclimatization process of animals in their natural habitat, even though the complicated experimental setting makes interpretation of the results difficult. In the present study, changes in the number of brain Na+-K+-ATPase alpha subunits and their enzymatic activity were found, but they did not correlate with oxygen shortage, which the animals were facing, suggesting the changes were not directly associated with anoxic stress. This conclusion was supported by the controlled laboratory experiments. It might be argued, that anoxic depression of Na+-K+-ATPase activity was not noticed in the laboratory experiments, because ATPase activity of the fish captured in February was already depressed from its peak value in November. This is, however, unlikely since the Na+-K+-ATPase activity of the fish used in anoxia experiments was as high as in the normoxic summer fish in May–August. Furthermore, before anoxic exposure, the fish were allowed over a 2-wk recovery period in normoxia, which should be sufficient to restore cellular machinery from possible anoxic wear.

The transient changes in the number and activity of Na+-K+-ATPase in spring time were suggested to be due to reoxygenation damage of the brain membranes. It should be noted, however, that the results might have been biased due to selective sampling. First, if there is significant anoxic mortality in the fish population, the results in late winter and spring time would represent only the physiology of the survivors. Second, animals caught in the traps are not free to seek the most favorable conditions in the anoxic pond, and the captivity might have affected their physiology.

Because Na+-K+-ATPase activity of crude brain homogenates cannot be reliably measured at temperatures below 15°C, the temperature-related depression of Na+-K+-ATPase activity in winter is based on extrapolation of the results at 15 to 25°C to lower temperatures. As the Q10 value of most enzymatic reactions tends to increase at low temperatures, the extrapolated Na+-K+-ATPase activity, based on a Q10 value between 15 and 25°C, is probably an overestimate of the real activity. Accordingly, the energy demand by the Na+-K+-ATPase might be smaller and the extension of brain anoxia tolerance larger than estimated. It should be noted, however, that these limitations do not affect the central tenets of the paper, the prominent seasonal change in the energy supply-demand ratio in the brain tissue, and its contribution to anoxia tolerance.

Taken together, the present results show that strong seasonal changes occur in supply and demand of the brain energy metabolism. In winter, the increase in energy supply is provided by the 15-fold extension of brain glycogen stores, while the decrease in energy demand is produced by at least 10-fold depression of Na+-K+-ATPase activity due to the direct Q10 effect on enzyme activity at low temperatures. As an outcome, about 150-fold extension of brain anoxia tolerance is attained with tissue-level mechanisms, underscoring the importance of seasonally bound changes in physiology for survival under natural environmental conditions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was financed by a research grant from the Academy of Finland to M. Vornanen (project no. 210400).


   ACKNOWLEDGMENTS
 
Anita Kervinen is appreciated for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Matti Vornanen, Univ. of Joensuu, Dept. of Biology, P.O. Box 111, 80101 Joensuu, Finland (E-mail: matti.vornanen{at}joensuu.fi)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Allen NJ, Karadottir R, and Attwell D. A preferential role for glycolysis in preventing the anoxic depolarization of rat hippocampal area CA1 pyramidal cells. J Neurosci 25: 848–859, 2005.[Abstract/Free Full Text]
  2. Atkinson A, Gatenby AD, and Lowe AG. The determination of inorganic orthophosphate in biological systems. Biochim Biophys Acta 320: 195–204, 1973.[Medline]
  3. Balestrino M, Cupello A, Morelli A, Musante L, and Panfoli I. Measurements of (Na+, K+)ATPase after in vitro hypoxia and reoxygenation are affected by methods of membrane preparation. J Neurosci Methods 79: 201–206, 1998.[CrossRef][ISI][Medline]
  4. Blazka P. The anaerobic metabolism of fish. Physiol Zool 31: 117–128, 1958.[Medline]
  5. Brönmark C and Milner JG. Predator-induced phenotypical change in body morphology in crucian carp. Science 258: 1348–1350, 1992.[Abstract/Free Full Text]
  6. Cruz NF and Dienel GA. High glycogen levels in brains of rats with minimal environmental stimuli: implications for metabolic contributions of working astrocytes. J Cereb Blood Flow Metab 22: 1476–1489, 2002.[CrossRef][ISI][Medline]
  7. Erecinska M, Cherian S, and Silver IA. Energy metabolism in mammalian brain during development. Prog Neurobiol 73: 397–445, 2004.[CrossRef][ISI][Medline]
  8. Gabreil W. How stress selects for reversible phenotypic plasticity. J Evolution Biol 18: 873–883, 2005.
  9. Gruetter R. Glycogen: the forgotten cerebral energy store. J Neurosci Res 74: 179–183, 2003.[CrossRef][ISI][Medline]
  10. Guppy M and Withers P. Metabolic depression in animals: physiological perspectives and biochemical generalizations. Biol Rev 74: 1–40, 1999.[Medline]
  11. Hevor TK, Lehr PR, and Gayet J. Amphetamine-induced changes in body temperature and glycogen content of the encephalon in the chicken. Experientia 31: 674–676, 1975.[CrossRef][ISI][Medline]
  12. Hochachka PW. Defense strategies against hypoxia and hypothermia. Science 231: 234–241, 1986.[Abstract/Free Full Text]
  13. Holopainen IJ, Aho J, Vornanen M, and Huuskonen H. Phenotypic plasticity and predator effects on morphology and physiology of crucian carp in nature and in the laboratory. J Fish Biol 50: 781–798, 1997.[CrossRef]
  14. Hylland P, Milton S, Pek M, Nilsson GE, and Lutz PL. Brain Na+/K+-ATPase activity in two anoxia tolerant vertebrates: crucian carp and freshwater turtle. Neurosci Lett 235: 89–92, 1997.[CrossRef][ISI][Medline]
  15. Hyvärinen H, Holopainen IJ, and Piironen J. Anaerobic wintering of crucian carp (Carassius carassius L.) - I. Annual dynamics of glycogen reserves in nature. Comp Biochem Physiol 82A: 797–803, 1985.[CrossRef]
  16. Jackson DC. Hibernating without oxygen: physiological adaptations of the painted turtle. J Physiol 15: 731–737, 2002.
  17. Johansson D, Nilsson GE, and Törnblom E. Effects of anoxia on energy metabolism in crucian carp brain slices studied with microcalorimetry. J Exp Biol 198: 853–859, 1995.[Abstract]
  18. L’vova SP and Plotnikov VP. Content of glycogen and glucose in tissues of the frog and some reptiles. Zh Evol Biokhim Fiziol 14: 400–402, 1978.[Medline]
  19. Lo S, Russell JC, and Taylor AW. Determination of glycogen in small tissue samples. J Appl Physiol 28: 234–236, 1970.[Free Full Text]
  20. Matsui H and Schwartz A. Mechanism of cardiac glycoside inhibition of the (Na+-K+-)-dependent ATPase from cardiac muscle. Biochim Biophys Acta 151: 655–663, 1968.[Medline]
  21. Mishra OP, and Delivoria-Papadopoulos M. Cellular mechanisms of hypoxic injury in the developing brain. Brain Res Bull 48: 233–238, 1999.[CrossRef][ISI][Medline]
  22. Nilsson GE. Evidence for a role of GABA in metabolic depression during anoxia in crucian carp (Carassius carassius). J Exp Biol 164: 243–259, 1992.[Abstract/Free Full Text]
  23. Nilsson GE. Surviving anoxia with the brain turned on. News Physiol Sci 16: 217–221, 2001.[Abstract/Free Full Text]
  24. Nilsson GE and Lutz PL. Anoxia tolerant brains. J Cereb Blood Flow Metab 24: 475–486, 2004.[CrossRef][ISI][Medline]
  25. Piironen J and Holopainen IJ. A note on seasonality in anoxia tolerance of crucian carp (Carassius carssius L.) in the laboratory. Ann Zool Fennici 23: 335–338, 1986.
  26. Raffin CN, Rosenthal M, Busto R, and Sick TJ. Glycolysis, oxidative metabolism, and brain potassium ion clearance. J Cereb Blood Flow Metab 12: 118–123, 1992.
  27. Rosenkrantz TS, Kubin J, Mishra OP, Smith D, and Delivoria-Papadopoulos M. Brain cell membrane Na+,K+-ATPase activity following severe hypoxic injury in the newborn piglet. Brain Res 730: 52–57, 1996.[ISI][Medline]
  28. Schmidt H and Wegener G. Glycogen phosphorylase in fish brain (Carassius carassius) during hypoxia. Biochem Soc Trans 16: 621–622, 1988.
  29. Vornanen M. Seasonal adaptation of crucian carp (Carassius carassius L.) heart: glycogen stores and lactate dehydrogenase activity. Can J Zool 72: 433–442, 1994.
  30. Vornanen M and Paajanen V. Seasonality of dihydropyridine receptor binding in the heart of an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius L.). Am J Physiol Regul Integr Comp Physiol 287: R1263–R1269, 2004.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
R. Kakela, M. Mattila, M. Hermansson, P. Haimi, A. Uphoff, V. Paajanen, P. Somerharju, and M. Vornanen
Seasonal acclimatization of brain lipidome in a eurythermal fish (Carassius carassius) is mainly determined by temperature
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1716 - R1728.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. A. W. Stecyk, V. Paajanen, A. P. Farrell, and M. Vornanen
Effect of temperature and prolonged anoxia exposure on electrophysiological properties of the turtle (Trachemys scripta) heart
Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R421 - R437.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/5/R1482    most recent
00172.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vornanen, M.
Right arrow Articles by Paajanen, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vornanen, M.
Right arrow Articles by Paajanen, V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.