From the Department of Molecular Genetics,
Biochemistry, and Microbiology, the ** Department of
Pharmacology and Cell Biophysics, the *** Department of
Molecular and Cellular Physiology, and the
Department of Pathology and Laboratory
Medicine, University of Cincinnati, College of Medicine, Cincinnati,
Ohio 45267, the ¶ Laboratory of Membrane Biology, Neuroscience
Center, Massachusetts General Hospital, Charlestown, Massachusetts
02129, the
Department of Zoology, Miami University, Oxford, Ohio
45056, the § Committee on Neurobiology and the
¶¶ Department of Organismal Biology and Anatomy, The
University of Chicago, Chicago, Illinois 60637, and the
§§ Department of Pathology, Children's
Hospital Medical Center, Cincinnati, Ohio 45229
Received for publication, November 5, 2002
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ABSTRACT |
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Na,K-ATPase is an ion transporter that
impacts neural and glial physiology by direct electrogenic activity and
the modulation of ion gradients. Its three isoforms in brain have
cell-type and development-specific expression patterns. Interestingly,
our studies demonstrate that in late gestation, the Na,K-ATPase is a plasma membrane enzyme necessary for maintaining
the sodium and potassium ion gradients in the cell, and it drives the
sodium-dependent transport of calcium and amino acids as
well as the reuptake of neurotransmitters. The ion gradients generated
by Na,K-ATPase are also used to regulate the volume of the cell and to
support and modulate electrical activity through direct (electrogenic)
and indirect effects on membrane potential.
Na,K-ATPase is a heteromeric protein composed of an To understand further the specific roles of individual Na,K-ATPase
isoforms, we have analyzed mice in which the Genotyping and Blood Analysis--
Mice heterozygous for the
Na,K-ATPase Lung Histology--
Within 15-30 min after birth, newborn mouse
pups were sacrificed, and the lungs were carefully removed and immersed
in 10% formalin. The tissues were then embedded in paraffin and
sectioned at 5 µm. Sections were stained with hematoxylin and eosin,
and digital pictures were taken using a microscope setting on ×10 magnification.
Microsome Preparation and Western Blot Analysis--
Tissues
from at least four embryonic day 18.5 pups of the same genotype
(
Approximately 5-10 µg of microsomal membrane protein was loaded per
lane. The primary antibodies used were a Diaphragm Immunohistochemistry--
For whole mount diaphragm
preparations, diaphragms from embryonic day 18.5 mice were prepared as
described previously (19). Diaphragms were fixed in 2%
paraformaldehyde in phosphate-buffered saline
(PBS),2 blocked in 0.1 M glycine in PBS, and then permeabilized in 2% bovine
serum albumin, 1% Triton X-100 in PBS (TPBS) overnight. The muscles
were then incubated with rabbit antibodies to synaptophysin (Zymed Laboratories, San Francisco) and then incubated
simultaneously with fluorescein-conjugated donkey anti-rabbit IgG
(Jackson Immunochemicals, West Grove, PA) and
tetramethylrhodamine-conjugated Diaphragm Contractility--
Diaphragms with ribs attached were
removed from embryos (day 18.5) and placed in Krebs solution containing
(in mM) 118 NaCl, 4.7 KCl, 25 NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 0.026 EDTA, and 11 glucose,
equilibrated with 95% CO2 and 5%O2. The
diaphragm was cut to obtain a muscle strip from the central region with ribs attached to the tendon at each end. Triangular clips were attached
at each end of the muscle strip, and the muscle was held in the clip
against the ribs. Muscles were mounted in a constant temperature,
sealed chamber and fixed to a stainless steel post at one end with the
clip and the other end fixed to an isometric force transducer (Kistler
Morse, Redmond, WA). The muscle length was adjusted to produce a
resting tension of 3 millinewtons. The muscles were electrically
field stimulated using two platinum electrodes positioned along either
side of the muscle. Supramaximal voltage and frequency were determined
empirically using a series of short (1-s) tetani and subsequently
increasing the voltage or the frequency for each tetanus. The stimuli
employed capacitor discharges of equal but alternating polarity (60 Hz
at 15 V) with three to five instances of tetani or twitches at a
duration of 2 ms. Digital recordings of force production were obtained
with the BioPac data acquisition system (BioPac System, Inc., Goleta, CA) and evaluated to determine maximal twitch tension.
In Situ Hybridization--
A pregnant wild-type mouse of the
same genetic background as the Immunofluorescence--
Slices used for immunofluorescence were
prepared in an ice-cold artificial cerebrospinal fluid containing
(mM) 118 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 25 HEPES, and 30 D-glucose, pH 7.4, which was not bubbled with gas. Most slices were transferred
immediately into fixative, consisting of 2% paraformaldehyde in
periodate-lysine buffer (PLP fixative) (22). A few slices were
maintained in ice-cold artificial cerebrospinal fluid for up to 40 min
before fixing. Slices were immersed in 30% sucrose in PBS overnight
and then embedded and frozen in TBS tissue freezing medium (Triangle Biomedical Sciences, Durham, NC) in aluminum boats. Cryostat sections (12-14 µm) were picked up on ProbeOn Plus positively charged
microscope slides (Fisher Scientific) and stored at
Because we were using an anti-mouse secondary antibody on mouse tissue,
there was light nonspecific staining of large cells and blood vessels.
We tried using different blocking solutions, secondary antibodies
(different host species, and different fluorophores), and using
immunohistochemistry with horseradish
peroxidase/diaminobenzidine (not shown), but the nonspecific
stain was always visible in control sections (treated only with
secondary antibodies, no primary antibody). Nonetheless, the cellular
nonspecific stain could be differentiated easily from positive stain
because it was very light and was only seen in the cytoplasm. Positive
Central Nervous System
Electrophysiology--
600-700-µm-thick transverse medullary slices
were obtained from 31 embryonic (day 18.5) mice according to procedures
for neonatal mice described in detail elsewhere (24). Tail samples were
frozen for later genotyping. Slices used in physiology were prepared in
an ice-cold artificial cerebrospinal fluid containing (in
mM) 118 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1 NaH2PO4 and 30 D-glucose and
equilibrated with carbogen (95% O2 and 5%
CO2, pH 7.4). KCl was elevated to 8 mM over a
span of 30 min before commencing recordings. All chemicals were
obtained from Sigma. Extracellular population activity was recorded and
integrated as described previously (25). The data were digitized with a Digidata acquisition board (Axon Instruments, Foster City, CA), stored
on an IBM compatible PC with the software program Axotape (Axon), and
analyzed offline using Igor Pro (WaveMetrics, Lake Oswego, OR) and
Prism (GraphPad, San Diego, CA). All recordings had a signal:noise
ratio sufficient for quantitative evaluation. Statistical comparisons
among all three genotypes were performed using analysis of variance.
Comparisons among groups were performed subsequently using the Tukey
post-test. These post-tests sometimes yielded a significant difference
between the Na,K-ATPase
Lungs removed from pups ~15-30 min after birth as well as from day
18.5 embryos were fixed and stained with hematoxylin and eosin. As
shown in Fig. 1, lungs from the
We examined tissues from Diaphragm Functional Analysis--
Because the
To test whether the absence of the Na,K-ATPase
Fig. 6 shows immunostain for the Na,K-ATPase
Spontaneous neural activity was observed in all genotypes including the
Although the absence of the Na,K-ATPase Mice deficient in the Na,K-ATPase We show in this study by in situ hybridization that large
diameter neuronal cell bodies expressed the The lack of Interestingly, the fictive sigh rhythm was still prominent, despite its
apparent origination from the same neural network (25). This suggests
that sighs alone may not be sufficient to sustain life, even in the
relatively hypoxia-resistant newborn. Alternatively, the apparent
failure of the knockouts to take even one breath, based on the lack of
alveolar expansion, suggests that the first breath of life
is not a sigh, but might instead be a gasp. Bursts corresponding to
gasps are manifested in slices but are distinct from sighs in that
gasps are found only in anoxia and are not intermixed with eupneic
breaths (25). The decreased regularity and the decreased amplitude of
the eupnea respiratory rhythm, combined with an apparent lack of
breathing in the intact animal, indicates that the population level
activity we observed in slices is essentially failed central breaths.
The individual cells retain some bursting ability but do not
synchronize sufficiently, the amplitude being a measure of synchronous
activity, to generate a regular rhythm or to give rise to respiratory
movements. Fictive sighs are observed in the brain slice preparations
of Because the Another function of Na,K-ATPase in the brain is that the glutamate
transporter works in concert with the Na,K-ATPase has been proposed to regulate intracellular calcium via the
Na/Ca exchanger. It has been proposed that the Na,K-ATPase Last, it is also possible that the 2 isoform is
widely expressed in neurons, unlike in the adult brain, in which
2
has been shown to be expressed primarily in astrocytes. This unexpected
distribution of
2 isoform expression in neurons is interesting in
light of our examination of mice lacking the
2 isoform which fail to
survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a
primary neuronal defect and electrophysiological recordings in the
pre-Bötzinger complex, the brainstem breathing center, showed
reduction of respiratory rhythm activity, with less regular and smaller
population bursts. These data demonstrate that the Na,K-ATPase
2
isoform could be important in the modulation of neuronal activity in
the neonate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
catalytic
subunit that binds sodium and potassium ions, ATP, and cardiac glycosides, and
and
(FXYD) subunits that can
modulate substrate affinity. There are different genes that code for
multiple
,
, and
isoforms. Four
isoforms (
1,
2,
3, and
4) have been identified, and all but
4 are expressed in
the brain (1, 2). Examination of the enzymatic properties of the
and
isoforms in different expression systems revealed that the
isoforms have differences in substrate affinity and kinetic properties
(3-7). In most adult mammals, the
2 isoform is expressed most
abundantly in skeletal muscle and brain and in lower abundance in
heart, adipocytes, and eye (8-11). In situ hybridization
performed on sections of embryonic days 9.5-16.5 mouse brain revealed
that the Na,K-ATPase
2 isoform is expressed throughout most regions of the brain (12). In the adult brain it has been found in astrocytes, pia/arachnoid, and a few types of neurons (13-15).
2 isoform gene has been
knocked out. The animals died shortly after birth but did not display
obvious gross morphological defects in any tissue, including the brain.
Lack of motor activity was significant, but muscle contractility was
not found to be critically impaired. Consequently we investigated the
cellular distribution of
2 in the newborn brain and the function of
an intrinsic neuronal circuit that could contribute directly to
immediate death: generation of the breathing rhythm.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 isoform were generated as described previously (16).
Heterozygous females were mated with heterozygous males, and the
resulting offspring were genotyped by Southern blot as described
previously (16). Blood was taken from decapitated mouse pups
immediately after birth, collected in capillary tubes, and measured for
carbon dioxide and oxygen content using a Chiron blood gas analyzer
(Norwood, MA model 384). Blood glucose levels were analyzed using an
Accudata GTS Glucose Test Station (Roche Molecular Biochemicals).
2+/+,
2+/
, or
2
/
)
were pooled and microsomes prepared as described (16). The pellet was
resuspended in 1 mM imidazole, 1 mM EDTA, pH
7.4, then aliquoted and stored at
80 °C. Protein concentration was
determined using the BCA assay (Pierce Chemical Co.). The microsomal
membranes were used for Western blot analysis. SDS-PAGE was performed
as described (17). The Western blot procedure was performed as described (18).
1 isoform-specific monoclonal antibody,
6F,1
an
2 isoform-specific monoclonal antibody, McB2, and an
3
isoform-specific monoclonal antibody, MA3-915 (Affinity BioReagents,
Golden, CO). The signal was detected using an ECL system (Amersham Biosciences).
-bungarotoxin (Molecular Probes,
Eugene, OR) in 2% bovine serum albumin in TPBS overnight at 4 °C.
After washing in TPBS, diaphragms were mounted on coverslips in
glycerol-paraphenylenediamine to retard fading, viewed with
epifluorescence and filters that were selective for rhodamine or
fluorescein, and evaluated with an Axionplan2 microscope (Zeiss,
Thornwood, NY). Images were captured with a digital camera (Hamamatsu,
Bridgewater, NJ) and imaging software (QED Imaging, Pittsburgh, PA).
2
/
mice was
euthanized by carbon dioxide inhalation, and the embryonic day 18.5 pups were removed, decapitated, and the neck and head were immediately
perfused and fixed in 4% paraformaldehyde (w/v) in PBS overnight. The
tissues were cryoprotected and embedded and then cryosectioned in
6-8-µm-thick sections. The sections were dried on slides and then
postfixed, prehybridized, hybridized, and developed as described
previously (20, 21). Antisense and sense RNA probes were synthesized
with 35S-labeled rUTP from plasmids that contain either
1 or
2 Na,K-ATPase isoform-specific sequences (9).
20 °C until
use. Unstained slides were warmed to room temperature, and a PAP pen
(Kiyota International, Elk Grove, IL) was used to draw a hydrophobic
ring around the sections. Slides were rinsed in PBS for 5 min,
transferred to 95 °C 10 mM sodium citrate, pH 6.0, in a
Coplin jar standing in a boiling water bath, and incubated for 20 min.
This antigen retrieval method enhanced the detection and specificity of
the stain. The Coplin jar containing the slides was then removed from the bath and allowed to cool for 20 min. Slides were reequilibrated in
several changes of room temperature PBS over 30 min. For all subsequent
incubations, the slides were laid flat in a dark moist box. The
sections were covered (~50 µl/section) with 5% normal goat serum
in PBS with 0.3% Triton X-100 (PBSt) for 1 h at room temperature.
This blocking solution was removed with an aspirator, and primary
antibody McB2 was applied (1:4 dilution). The specificity of McB2, a
monoclonal antibody specific for the
2 isoform of Na,K-ATPase, has
been described previously (23). The sections were incubated overnight
at 4 °C in the primary antibody, rinsed in PBS (three times at 10 min each time), and then incubated in Cy3-conjugated goat anti-mouse
IgG (1:300; Accurate, Westbury, NY) in PBSt for 2 h. Finally, they
were rinsed in PBS and coverslipped in Vectashield fluorescence
mounting medium (Vector Laboratories, Burlingame, CA). Slides were
examined and images were collected on a Nikon TE300 fluorescence
microscope equipped with a Bio-Rad MRC 1024 scanning laser confocal
system, version 3.2.
2 stain, on the other hand, was much brighter and was only seen on
the plasma membrane.
2
/
group and only one of the
2+/
or wild-type groups. Because in no case did the
2+/
and wild-type groups differ significantly, we
grouped the
2+/
and wild-type recordings together to
perform the t tests and nonparametric tests reported in the text.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
/
Mice Appear Normal but Do Not
Breathe--
Mice lacking the
2 isoform are born and display no
gross anatomical or histological abnormalities. However, these mice
appear limp and do not respond to pinch. By opening the chest cavity of
newborn pups immediately after birth we established that the hearts
from the
2
/
pups were beating, indicating that the
mice were alive when born. Several minutes after birth the wild-type
and
2+/
mice breathe and turn pink, but the
2
/
animals do not appear to breathe. Therefore we
measured blood gas levels in newborn mouse pups within 15-30 min after
birth. Both the wild-type and
2+/
mice showed normal
levels of oxygen and carbon dioxide (26), whereas the
2
/
newborn pups displayed very low oxygen and high
carbon dioxide levels consistent with failure to breathe (Table
I). Blood glucose levels and body weight
were normal for the
2
/
mice (Table I).
Body weight, blood gas and blood glucose levels from Na,K-ATPase 2
homozygous (
/
),
2 heterozygous (+/
), and
wild-type (+/+) mice
2
/
mice appeared developmentally normal at both
embryonic day 18.5 and at birth compared with wild-type. Saccules of
the
2
/
mice at embryonic day 18.5 were less
expanded, however, consistent with a failure of normal prenatal
breathing motions that exchange lung fluid with amniotic fluid and
assist in the maturation of the lung (27). Postnatally, the saccules
contained more cellular debris because of hemorrhage in the lungs,
which most likely represents a secondary shock lesion in the dying pup.
The
2
/
lungs at birth also showed less postnatal
dilation of saccules, confirming that the animals did not breathe after
birth. In contrast, the lungs from the wild-type pups show dilated
respiratory bronchioles and saccules consistent with partially expanded
lungs, indicating there has been some breathing activity.
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Fig. 1.
Hematoxylin and eosin staining of
lungs of Na,K-ATPase
2
/
and wild-type mice at embryonic day 18.5 and 15-30 min after
birth. Note that the alveoli in the homozygous mice
(
2
/
) (B and D) are not as
dilated compared with wild-type (wt) (A and
C). Scale bar, 1 mm.
2
/
mice to check for any
alteration in Na,K-ATPase isoform expression. Western blot analysis of
seven tissues from embryonic day 18.5 mice shows that in wild-type
animals, the
2 isoform was detected with similar abundance in brain
and diaphragm, and a faint signal was found in heart (Fig.
2).3
In the
2
/
pups, there did not appear to be a
significant change in abundance of the
1 or
3 isoform in any
tissue compensating for the loss of the
2 isoform.
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Fig. 2.
The Na,K-ATPase 2
isoform is expressed in brain and diaphragm at embryonic day 18.5. The expression of each of the
isoforms of Na,K-ATPase was examined
in microsomes prepared from wild-type (wt) and Na,K-ATPase
2 homozygous (
/
) mice in brain (Br), heart
(Ht), skeletal muscle (Sm), diaphragm
(Dia), kidney (K), liver (Liv), and
lung (Lu). 10 µg of protein was loaded in each
lane for each antibody blot except for the kidney sample,
which contained 5 µg of protein for the
1 blot.
2 isoform was
expressed primarily in muscle and brain around the time of birth we
examined these tissues further for abnormalities resulting from the
absence of the
2 isoform. If the diaphragm were not functioning
properly it could be caused by either a defect in the neuromuscular
junction (NMJ) in which the signal from nerve to muscle is defective or
the diaphragm muscle itself could be unable to contract. In
agrin-deficient mice, for example, acetylcholine receptors are reduced
in number and density at the NMJ, and these mice die at birth from an
inability to breathe (19). We used rhodamine-labeled bungarotoxin to
detect acetylcholine receptors as a marker for NMJ development.
Synaptophysin, a synaptic vesicle-specific membrane protein, is
expressed abundantly in nerve terminal synaptic vesicle boutons, and we
used synaptophysin antibody and fluorescein isothiocyanate-labeled
secondary antibody to detect this protein as a marker for the nerve
terminal. Whole mount immunohistochemistry revealed normal NMJ
development in
2
/
mice (Fig.
3). We then tested whether the NMJ was
functional by electrically stimulating the phrenic nerve. The diaphragm
was able to contract, indicating that the synaptic connection between muscle and nerve was functional.
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Fig. 3.
Pre- and postsynaptic differentiation of
diaphragm is normal in Na,K-ATPase
2
/
mice. Whole mounts of diaphragm muscle from a wild-type
(A-C) and Na,K-ATPase
2
/
mutant
(D-F) mouse were stained simultaneously with antibodies
against synaptophysin (Syn) to label vesicles in presynaptic
nerve terminals and
-bungarotoxin (
BGT) to label
acetylcholine receptors on the postsynaptic muscle membrane. The whole
mounts were viewed with optics selective for either fluorescein
(A and D) or rhodamine (B and
E) or both fluorescein and rhodamine (C and
F). Presynaptic sites in wild-type muscle (A and
B) are located adjacent to the main intramuscular nerve
(arrow in A), and the nerve terminals are
characterized by an accumulation of clustered acetylcholine receptors
(B) underlying the terminals (C). Na,K-ATPase
2
/
mice exhibited normal synaptic differentiation
with presynaptic sites (D) localized adjacent to the nerve
(arrow in D) and the nerve terminals showing
clustered acetylcholine receptors (E) underlying the
terminals (F).
2 isoform in diaphragm altered
contractility we developed a method of electrically stimulating and
measuring isometric contractility in muscle preparations from day 18.5 embryos. Because of the small size of the diaphragm muscle as well as
the presence of the attached ribs, accurate weights were difficult to
obtain. Thus, assuming that the thickness of each diaphragm was the
same in all preparations we normalized the tension data to muscle area
(length times width) of the diaphragm strips. No significant
differences in maximum twitch force of contraction were observed
between wild-type and
2
/
mice (Fig.
4). Two other normalization routines were
evaluated: force normalized to length and force normalized to width. In
all cases of normalization (Fig. 4A) as well as the raw
tension data (Fig. 4B), a similar trend was observed, with
the
2
/
muscle producing a force within 10% of the
wild-type muscle with no statistically significant difference
(p > 0.05, Student's t test). Together,
these results demonstrate that embryonic diaphragm muscle without the
2 isoform is able to contract both by direct electrical stimulation
and by stimulation via the phrenic nerve with a force similar to that
of wild-type. Therefore the brain, which also showed expression of the
2 isoform at embyronic day 18.5, was examined further for
physiological defects associated with the absence of the
2
isoform.
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Fig. 4.
Maximal twitch force of contraction in
diaphragm of
2
/
mice is similar to wild-type mice. Diaphragm muscle strips from
embryonic day 18.5 mice were used for isometric force of contraction
measurements. A, maximal twitch force was normalized to
muscle length, muscle width, or to area (length times width) for both
wild-type (wt) and
2
/
(
/
) mice.
B, maximal twitch force without normalization. Values are
the means ± S.E. (n = 7 in the wild-type group,
n = 6 in the
2
/
group).
2 Isoform Is Expressed in Neurons at Embryonic Day
18.5--
Previous reports on Na,K-ATPase
2 isoform expression have
shown that it is expressed primarily in astrocytes of adults (for review, see Ref. 28); however, little data exist on the expression of
2 at the time of birth in mice. Therefore, as an initial step toward
the evaluation of the
2
/
mice, we determined the
expression profile of the
2 isoform from embyronic day 18.5 wild-type mice by in situ hybridization and
immunofluorescence analysis of mRNA and protein, respectively, to
determine the cell type and the regions of the brain that express it.
Fig. 5 shows in situ
hybridization of sagittal brain sections in which signals for
1 and
2 isoforms can be compared. In Fig. 5, A and
B, the choroid plexus is shown. This highly elaborated secretory epithelium that emerges from the ventricular lining shows
strong hybridization for
1 isoform mRNA with the antisense probe, but little or no hybridization above background with the
2
antisense probe. In contrast, in Fig. 5D, it can be seen
that the
2 isoform antisense probe showed extremely heavy
hybridization over the pia mater, a tissue that is known to express the
2 isoform, whereas the
1 isoform antisense probe showed less
(Fig. 5C). Neither the
1 isoform antisense nor the
2
isoform sense probes labeled the pia. These data validate the methods
by confirming the known distribution of the
1 isoform in choroid
plexus and the
2 isoform in pia. In the cortical layer, the
1
probe showed a more uniform pattern of diffuse signal in all of the
cells, small and large. In contrast, the
2 probe showed much more
cell to cell variability, with some neuronal somas showing strong
expression. Fig. 5, E and F, shows hybridization
in a region of the brainstem just ventral and caudal to the position of
the choroid plexus in the floor of the fourth ventricle. Similar
results were seen deeper in the brainstem and also in the cerebral
cortex. It can be seen that large diameter neural cell bodies were
sometimes labeled for the
1 isoform but more heavily for the
2
isoform. There was also signal above background over regions between
neurons, particularly for the
2 isoform, which is presumably in
glia, which have a less localized cytoplasm. The sense probe controls (Fig. 5, G and H) showed scattered background
grains that were not localized to any structure.
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Fig. 5.
In situ mRNA hybridization
analysis revealed that both the Na,K-ATPase 1
and
2 isoforms are expressed throughout the
brain in wild-type embryonic day 18.5 mice. Embryonic day 18.5 brain sections from wild-type mice were incubated with antisense
1
and
2 isoform-specific probes. A, C, and
E represent darkfield photomicrographs of sections
hybridized with the
1 antisense probe. B, D,
and F represent sections hybridized with the
2 antisense
probe. The
1 sense control is shown in G, and the
2
sense control is shown in H. A and B,
sections through the hippocampus. A, the dentate gyrus shows
uniform expression (bright white grains) of the
1 isoform, and the
star designates robust expression for the
1 isoform but
not the
2 isoform in the choroid plexus. The adjacent layers also
show uniform expression but overall less intensity because of the
cellularity differences. B, in contrast, the
2 pattern is
punctate although not uniform across all cells in that region. The
ependymal lining is also more intense (arrow) for the
2
isoform. C and D, sections through the cortex.
C, the
1 signal shows a mostly uniform pattern throughout
the cortex but no expression in the pia mater. D, in
contrast to C, the
2 isoform pattern is much more
nonuniform in the neuronal layers of the cortex because some neurons
are more intense than others in this layer. The pia mater shows strong
2 isoform expression (arrow). E and
F, brainstem near the ventral respiratory group; the
inset shows a brightfield view of hematoxylin and eosin
staining with the larger (neuronal) nuclei purple, and the
hybridization signal is the small black grains. The
2
isoform shows stronger expression than the
1 isoform. Scale
bar, 100 µm.
2
isoform in wild type and
2
/
mice. Unlike the
sagittal sections used for in situ hybridization, these
sections were from tissue slices like those used for
electrophysiological recording below, i.e. brainstem cut at
an angle that includes the cellular elements required for respiratory
rhythm generation. The images shown in Fig. 6, A and
B, were from the region of the pre-Bötzinger complex,
but a similar stain was seen in most of the section. Stain appeared to
be present in both neurons and glia, most prominently in stained somas
and fine processes characteristic of neurons. Fig. 6B is a
portion of Fig. 6A at higher magnification and with fewer
stacked optical sections to show more cellular detail. Fig.
6C, which shows a section through the midline raphe, shows
stain in bundles of fibers on either side of the midline raphe. In Fig.
6, D and E, are the controls, showing light stain of neurons and blood vessels with the anti-mouse secondary antibody used to detect the
2-specific antibody (Fig. 6D) and a
lack of specific anti-
2 stain in the
2
/
mouse
(Fig. 6E). These results show that neurons throughout
different regions of the brain contain abundant levels of both mRNA
and protein for the Na,K-ATPase
2 isoform at the time of birth.
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Fig. 6.
The Na,K-ATPase 2
isoform protein is localized predominantly in neurons in the
respiratory center of the brain. Immunofluorescent localization of
the Na,K-ATPase
2 subunit in wild type (A-D) and
2
/
(E) embryonic day 18.5 mouse brains is
shown. A,
2 stain in the ventral respiratory group area.
B, higher magnification showing the
2 stain in large
presumptive neuronal cell bodies (arrows). C,
2-labeled processes (arrowheads) crossing the midline in
the region of the raphe pallidus nucleus. D, control section
(primary antibody omitted) showing nonspecific label. E, the
2
/
animals were devoid of
2 stain (nonspecific
stain was caused by secondary antibody; compare with D).
Scale bar, 50 µm.
2
/
Mice Display Altered Neural
Firing in the Respiratory Network--
The electrochemical ion
gradients generated by Na,K-ATPase are essential for the electrical
excitability of cells. Because the
2 isoform was expressed
abundantly in neurons of wild-type mice, we tested the possibility that
the
2 isoform may be required for the integrated function of an
essential neural circuit: the generation of respiratory rhythm. The
respiratory center of the brain was examined in embryonic day 18.5 mice. The respiratory rhythm network in the normal brain is well
established before birth, and breathing motions occur in
utero. At birth, the brain is already transmitting the appropriate
signals for the newborn to breathe on its own. Respiratory neural
activity was recorded in vitro from the ventral respiratory
group using transverse medullary slices obtained from day 18.5 embryos
from wild-type,
2+/
, and
2
/
mice.
This slice preparation has been described previously for neonatal and
juvenile mice and rats, and it generates respiratory rhythms
corresponding to both normal breathing ("eupnea") and sighs
("augmented breaths") (25). The outcome was that rhythmic activity
showed abnormal properties.
2
/
mice, indicating that the absence of the
Na,K-ATPase
2 isoform does not result in a complete loss of neuronal
function. Rhythmic activity was observed in the ventral respiratory
group in 26 of 31 preparations examined. Of the five preparations from
which activity could not be recorded, two were from
2
/
, three from wild-type animals, and none from
2+/
. Of the 26 active slices, 19 produced two clearly
distinct patterns of activity (Fig. 7,
A and B). Larger amplitude bursts occurring at
intervals of about 40 s to several minutes represent fictive sighs, whereas smaller amplitude bursts occurring at intervals of a few
seconds represent fictive eupnea as identified previously in neonatal
preparations of outbred CD-1 mice (25). There was no correlation
between genotype and the presence or absence of rhythmic activity in
the slice (p = 0.2793, 2 × 3 chi square; p = 1.000 by Fisher's exact test with +/
and +/+
pooled).
View larger version (21K):
[in a new window]
Fig. 7.
Disrupted respiratory rhythm in
Na,K-ATPase
2
/
mice. Representative tracings of population activity recordings
from transverse slices of embryonic day 18.5 brain containing the
ventral respiratory group,
2
/
(A) and
wild-type (B) are shown. The raw data appear on the
lower traces and integrated data on the upper
traces. C, regularity of the respiratory rhythm, as
defined by the eupnea period, was lower in the
2
/
mice. D, eupnea amplitude was lower in the
2
/
mice. Asterisks designate the sigh
peaks, and the shorter peaks represent the eupnea. (n = 9 for the
2+/
and wild-type groups, and
n = 10 for the
2
/
group).
2 isoform did not result in
a complete loss of neural activity, the regularity and the amplitude of
the respiratory rhythm of normal breathing (eupnea) were affected
profoundly (Table
, Fig. 7).
Table
shows that neither the burst duration nor the mean frequency
was significantly different (p = 0.1659 and
p = 0.8225, respectively). However, the regularity of
the eupnea rhythm was significantly lower in the
2
/
mice compared with
2+/
and wild-type. This is
reflected by the coefficient of variation of cycle periods of eupnea
(designated as period CV in Table
and Fig. 7C) from 0.43 for the
2+/+ and
2+/
group to 0.66 for
the
2
/
group (p = 0.0011). Note that
in this case, a higher number means lower regularity of rhythm. We also
examined the amplitude of the eupnea rhythm. Generally, the amplitude
of the integrated population burst is not directly comparable between
preparations because of differences in recording quality and the lack
of an absolute measurement scale. However, sighs apparently reflect a
maximal activation of inspiratory cells within the respiratory network
(25), and this enabled us to assess the amplitude of the eupneic burst
as a percentage of the sigh amplitude for each individual recording.
The amplitude of the eupneic burst was significantly lower in slices
obtained from
2
/
embryos (38%) than in slices from
2+/
and wild-type animals (59%) (p = 0.0056) (Table
and Fig. 7, A and D). This
appears to be a major factor leading to the failure of the animals to
breathe effectively.
Summary of respiratory rhythm patterns measured in brain slices from
Na,K-ATPase 2 homozygous (
/
),
2 heterozygous
(+/
), and wild type (+/+)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 isoform are born but die
soon after birth. By gross examination all organs appear normal; however, the mice are akinesic. This study shows for the first time
that the
2 isoform is highly expressed in neurons throughout the
brain at the time of birth, with lower levels of expression in glia.
This would not have been predicted from the literature. A previous
study showed that
2 mRNA is present quite early in brain
development, but without cellular resolution it could not be certain
whether the signal was in neurons or glia (12). In another study,
embryonic stem cells and aggregates of undifferentiated cells expressed
1 and
2 isoform mRNA, but only
1 protein. In vitro neuronal induction of embryonic stem cells was accompanied by induction of the
3 isoform at late stages of differentiation, but
2 isoform mRNA or protein was not induced at any stage (29). Embryonic fetal forebrain examined after several days in culture either
as aggregates or as plated cells expressed the
3 isoform in neurons
and
2 isoform in glia, as assessed either by selective elimination
of cell types by toxic agents (30) or by immunofluorescence microscopy
(31). Different results have been reported for cultures of hippocampal
neurons: one paper reported finding only the
1 and
3 isoforms by
immunofluorescence (32), whereas two papers reported finding the
2
and
3 isoforms or all three isoforms by Western blots (33, 34),
although the latter result could have reflected contamination with
astrocytes (35). Cerebellar granule neurons have been reported by Soga
et al. (36) to express all three isoforms in culture,
although the presence of the
2 isoform was found exclusively in
cerebellar astrocytes in prior studies both in situ and in
granule neuron cultures (13, 14, 28), raising the possibility that the
commercial antibodies used by Soga et al. were
cross-reactive. Inspection of the published stain for the
2 isoform
in the developing mouse retina, however, suggests that it may have been
present at birth in the nascent inner nuclear layer, but subsequently
lost over the next few days (37).
2 isoform at all levels in the brain, and diffuse signal characteristic of glia was also observed. By immunofluorescence, most of the
2 isoform stain had a
reticular pattern, with outlining of presumptive neuronal somas in
places and many fine processes characteristic of neurons. At this age,
the brainstem lacks the bundles of ascending and descending myelinated
axons and well developed neuronal nuclei of the adult, but the
distribution of stain clearly differs from the purely astrocytic
pattern for the
2 isoform in the adult rat brainstem (13). Taken
together with the published evidence, it seems likely that the
2
isoform is expressed transiently in many central nervous system neurons
during development but that with phenotypic maturation either in
vivo or in culture, the expression is lost. At the time of birth
it is expressed mainly in neurons, whereas in adult it is expressed
mainly in astrocytes, which proliferate and differentiate mostly postnatally.
2 isoform expression in mice results in a gross defect
in that the
2
/
mice are unresponsive to pinch and do
not breathe. Although the
2
/
mice may have
widespread functional disturbances throughout the brain, we were able
to document the effect of the absence of
2 on the integration of
neuronal activity in the brainstem respiratory center. We observed some
basic neuronal activity in the
2
/
mice; however, the
fictive eupnea breathing rhythm recorded in vitro was
disrupted significantly in the
2
/
mice compared with
2+/
and wild-type littermates. Population bursts were
both lower in amplitude and much less regular, and in some
2
/
animals this was altered to such an extent that
this rhythm was nearly absent (Fig. 7). To obtain rhythmic activity in
slice preparations it is necessary to raise the extracellular potassium
concentration (25). Thus it is conceivable that the centrally generated
rhythm would be even weaker in vivo and insufficient to
support a normal motor output under physiological conditions.
2
/
animals. However, the data we present here
show that the likelihood of observing fictive sighs was actually higher
in
2
/
mice (10 of 12, or 83%) compared with
2+/
mice (4 of 8, or 50%) or wild-type mice (5 of 11, or 45%). The genesis of sighs might be stimulated by the failure of
the eupneic rhythm in the knockouts; perhaps the eupneic rhythm
ordinarily exerts a negative modulatory influence on the frequency of sighs.
2 isoform was found to be expressed throughout neurons
in the brain of day 18.5 embryos, it is quite possible that the altered
neuronal activity we observed in respiratory center neurons may be
representative of a more global defect in neurons throughout the rest
of the brain in the
2
/
mice. This aberrant neuronal
activity could be a result of one or more functional disturbances. A
specific role for Na,K-ATPase has been proposed to be in clearance of
potassium from the extracellular space to prevent depolarization of
neurons during high neuronal activity (38, 39). It was proposed that
glial Na,K-ATPase (
2 isoform) contributed to the initial fast uptake
of extracellular potassium K, whereas the axonal Na,K-ATPase (
3
isoform) participated in the slower poststimulus recovery from elevated
extracellular potassium (39). We would predict that in our homozygous
knockout mice, the absence of the
2 isoform might result in delayed
removal of extracellular potassium in both neurons and glia and thus
affect neuronal excitability.
3 or
2 Na,K-ATPase isoform
to clear glutamate from the extracellular space (40-42). Glutamate is
transported in a sodium- and potassium-dependent fashion
utilizing the sodium and potassium gradients set by Na,K-ATPase. It has
been shown that inhibition of Na,K-ATPase by ouabain will reduce the
amplitude of neuronal compound action potentials (41), and this
diminished neuronal activity was shown to be a result of toxicity
mediated in part by glutamate release through reverse sodium-dependent glutamate transport. Interestingly, a
recent report by Rozzo et al. (43) demonstrated that the
spontaneous rhythmicity of network spinal cord neurons is disrupted
with Na,K-ATPase inhibition by strophanthidin, a cardiotonic steroid
related to ouabain (43). Specifically they found a smaller peak
amplitude as well as irregular bursting intervals, which supports the
present data in which we observed the same phenomena in the respiratory rhythm center of the
2
/
mice. They further showed
this was a glutamate-mediated response because the altered rhythmicity
could be blocked using a glutamate receptor antagonist. The
concentration of strophanthidin used by Rozzo et al. (4 µM) would inhibit both the Na,K-ATPase
2 and
3
isoforms (43). Therefore it is intriguing to speculate that perhaps the
2 isoform works with the glutamate receptor to modulate neuronal
excitability. To test the specific contribution by the
2 isoform in
maintaining neuronal excitability versus the contribution from the
3 isoform, however, would require the specific inhibition of the
3 isoform either by pharmacological means (no specific inhibitor exists yet) or by genetic knockout (that animal has not been produced).
2 isoform
and the Na/Ca exchanger are colocalized in microdomains in the plasma
membrane of astrocytes and mesenteric artery smooth muscle cells to
work as a functional unit (34). During neuronal activity, sodium must
be pumped out of the cell to maintain excitability. If the capacity of
the neuron to pump out intracellular sodium is reduced, intracellular
sodium would rise, which then would change the driving force for Na/Ca
exchange, resulting in elevated [Ca2+]i. A recent report by
Golovina et al. (44) supports this idea in which it was
shown that in our
2
/
mice, astrocytes have elevated
levels of intracellular calcium as well as elevated stores of calcium
in the endoplasmic reticulum. We show in this study that in wild-type
animals the
2 isoform is expressed more abundantly in neurons than
in astrocytes at birth. We would predict even higher levels of
intracellular calcium in neurons than that shown for astrocytes in the
2
/
mice (44). Tang et al. (45) have
reported that a rise in intracellular calcium caused by inhibition of
the Na/Ca exchanger can enhance neurotransmitter release in chromaffin
cells. It is possible that a similar mechanism could occur in the
central nervous system of our
2
/
mice in which
glutamate release would be enhanced as a result of Na/Ca exchange
reduction. As discussed above, glutamate has been shown to alter
neuronal activity as a secondary response to inhibition of Na,K-ATPase.
In sum, it is clear from the present work that the absence of the
2
isoform results in altered integration of neuronal activity, and
further work will be required to sort out whether the mechanism entails neurotransmitters.
2 isoform directly provides a
significant contribution to the total Na,K-ATPase activity in the cell
and that with its removal, the activity of the other Na,K-ATPase
isoforms is simply not sufficient to handle the demand for ion
transport in the cell, resulting in membrane depolarization. Because
respiratory rhythm generation was not abolished, some basic synaptic
and neuronal membrane properties were still intact. Given that the
absence of the Na,K-ATPase
2 isoform could lead to a number of
different functional defects as proposed above, this work provides a
foundation to investigate further specific roles for the
2 isoform
in maintaining neuronal excitability. The present work provides a new
area of study of the
2 isoform in neurons as this cell type was not
recognized previously to express this Na,K-ATPase isoform.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Adam Redden, Pam Groen, Kathy Saalfeld, and Lisa McMillin for technical assistance; Chris Woods for photography; Dr. Bruce Patton for help with the whole mount immunohistochemistry; and Drs. Nancy Ratner and Joshua Sanes for helpful discussions and suggestions at the outset of this study.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL28573 and HL66062 (to J. B L.), NS27653 (to K. J. S.), and HL 60120 (to J. M. R.).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.
To whom correspondence should be addressed: Dept. of
Molecular Genetics, Biochemistry, and Microbiology, University of
Cincinnati, College of Medicine, 231 Albert Sabin Way, MSB Bldg., M.L.
0524, Cincinnati, OH 45267. Tel.: 513-558-5324; Fax:
513-558-8474; E-mail: jerry.lingrel@uc.edu.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M211315200
1
The 6F monoclonal antibody developed by Dr.
Fambrough was obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the NICHD and maintained by Dept. of
Biological Sciences, the University of Iowa, Iowa City, IA 52242.
3
It has been reported that 2 is expressed in
alveolar cells when their phenotype changes from ATII-like to ATI-like
in culture (46). However, there have been several reports that
2 mRNA is lacking in lung (9, 47-49), consistent with the
absence of the protein reported here.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PBS, phosphate-buffered saline; NMJ, neuromuscular junction.
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