Am J Physiol Regul Integr Comp Physiol 291: R1482–R1489, 2006.
First published June 1, 2006; doi:10.1152/ajpregu.00172.2006.
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
anoxia; anoxia tolerance; [3H]ouabain binding; sodium pump; phenotypic plasticity
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
CRUCIAN CARP IS A WIDELY DISTRIBUTED
Address for reprint requests and other correspondence: Matti Vornanen,
Univ. of Joensuu, Dept. of Biology, P.O. Box 111, 80101 Joensuu, Finland
(E-mail: [email protected]).
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(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
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.
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Vornanen, Matti, and Vesa Paajanen. Seasonal changes in glycogen
content and Na⫹-K⫹-ATPase activity in the brain of crucian carp. Am J
Physiol Regul Integr Comp Physiol 291: R1482–R1489, 2006. First
published June 1, 2006; doi:10.1152/ajpregu.00172.2006.—Changes in
the number of Na⫹-K⫹-ATPase ␣-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 ␣-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 ␮M/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.
GLYCOGEN CONTENT AND NA⫹-K⫹-ATPASE OF CRUCIAN CARP BRAIN
y ⫽ y max共1 ⫺ eKobs 䡠 t兲
where y is specific binding (pmol 䡠 min⫺1 䡠 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
AJP-Regul Integr Comp Physiol • VOL
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).
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
B ⫽ y0 ⫹ B maxⴱcH/KdH ⫹ cH
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 䡠 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 ␮l 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
Q 10 ⫽ 共R 2 /R 1 兲 共10/共T 2⫺T 1兲兲
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
Characterization of [3H]ouabain binding. To justify determination of [3H]ouabain binding in seasonally acclimatized
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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 䡠 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 䡠 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 ␮M unlabeled ouabain
(Sigma, Poole, UK). The reaction was terminated with 6 ml of
ice-cold wash buffer containing: 4 mM MgCl2, 50 mM Tris 䡠 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
3
[ H]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-␮M 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
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GLYCOGEN CONTENT AND NA⫹-K⫹-ATPASE OF CRUCIAN CARP BRAIN
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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.
Table 1. Comparison of [3H]ouabain binding to brain
preparations of crucian carp captured in June and February
Kd, nM
Hill slope
June
February
12.87⫾2.86
⫺1.18⫾0.07
11.93⫾2.81
⫺1.17⫾0.06
Values are expressed as means ⫾ SE of seven preparations for both animal
groups. Kd is the equilibrium dissociation constant for [3H]ouabain binding.
AJP-Regul Integr Comp Physiol • VOL
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.
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
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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.
GLYCOGEN CONTENT AND NA⫹-K⫹-ATPASE OF CRUCIAN CARP BRAIN
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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.
AJP-Regul Integr Comp Physiol • VOL
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.
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, winteracclimatized 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 ␮mol/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).
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
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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.
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GLYCOGEN CONTENT AND NA⫹-K⫹-ATPASE OF CRUCIAN CARP BRAIN
declining oxygen content, brain glycogen stores remain high in
midwinter with a peak value of 203.7 ⫾ 16.1 ␮mol/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 ␮mol/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
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.
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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.
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⫹
GLYCOGEN CONTENT AND NA⫹-K⫹-ATPASE OF CRUCIAN CARP BRAIN
AJP-Regul Integr Comp Physiol • VOL
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 ␮M 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 ␮M/g for crucian carp
brain, which is close to the value (12.9 ␮M/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 ␮mol/g, and in mammalian and avian
brains between 3 and 12 ␮mol/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 ␮M glucose/g is enough to fuel the brain Na⫹-K⫹-ATPase
at its maximum rate (0.8 ␮mol ATP䡠g⫺1 䡠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 ␮M ATP䡠g⫺1 䡠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 ␮M lactate䡠g⫺1 䡠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 ␮mol ATP䡠g⫺1 䡠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 supplydemand 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 glyco291 • NOVEMBER 2006 •
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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 reoxygenationrelated 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
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GLYCOGEN CONTENT AND NA⫹-K⫹-ATPASE OF CRUCIAN CARP BRAIN
AJP-Regul Integr Comp Physiol • VOL
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.
ACKNOWLEDGMENTS
Anita Kervinen is appreciated for technical assistance.
GRANTS
This work was financed by a research grant from the Academy of Finland
to M. Vornanen (project no. 210400).
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Limitations of the study. To understand how organisms
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animals in their natural habitat, even though the complicated
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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
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The transient changes in the number and activity of Na⫹⫹
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the decrease in energy demand is produced by at least 10-fold
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