The Na,K-ATPase α2 Isoform Is Expressed in Neurons, and Its Absence Disrupts Neuronal Activity in Newborn Mice
2003; Elsevier BV; Volume: 278; Issue: 7 Linguagem: Inglês
10.1074/jbc.m211315200
ISSN1083-351X
AutoresAmy E. Moseley, Steve P. Lieske, Randall K. Wetzel, Paul F. James, Suiwen He, Daniel A. Shelly, Richard J. Paul, Gregory P. Boivin, David P. Witte, Jan‐Marino Ramirez, Kathleen J. Sweadner, Jerry B. Lingrel,
Tópico(s)Neonatal Respiratory Health Research
ResumoNa,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 α2 isoform is widely expressed in neurons, unlike in the adult brain, in which α2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of α2 isoform expression in neurons is interesting in light of our examination of mice lacking the α2 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 α2 isoform could be important in the modulation of neuronal activity in the neonate. 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 α2 isoform is widely expressed in neurons, unlike in the adult brain, in which α2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of α2 isoform expression in neurons is interesting in light of our examination of mice lacking the α2 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 α2 isoform could be important in the modulation of neuronal activity in the neonate. 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 α catalytic subunit that binds sodium and potassium ions, ATP, and cardiac glycosides, and β and γ (FXYD) subunits that can modulate substrate affinity. There are different genes that code for multiple α, β, and γ isoforms. Four α isoforms (α1, α2, α3, and α4) have been identified, and all but α4 are expressed in the brain (1Shamraj O.I. Lingrel J.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12952-12956Crossref PubMed Scopus (219) Google Scholar, 2Shull G.E. Greeb J. Lingrel J.B. Biochemistry. 1986; 25: 8125-8132Crossref PubMed Scopus (549) Google Scholar). Examination of the enzymatic properties of the α and β isoforms in different expression systems revealed that the α isoforms have differences in substrate affinity and kinetic properties (3Blanco G. Mercer R.W. Am. J. Physiol. 1998; 275: F633-F650PubMed Google Scholar, 4Crambert G. Hasler U. Beggah A.T. Yu C. Modyanov N.N. Horisberger J.D. Lelievre L. Geering K. J. Biol. 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Acta. 1989; 988: 185-220Crossref PubMed Scopus (863) Google Scholar, 11Wetzel R.K. Sweadner K.J. Invest. Ophthalmol. Visual Sci. 2001; 43: 763-769Google Scholar). In situ hybridization performed on sections of embryonic days 9.5–16.5 mouse brain revealed that the Na,K-ATPase α2 isoform is expressed throughout most regions of the brain (12Herrera V.L. Cova T. Sassoon D. Ruiz-Opazo N. Am. J. Physiol. 1994; 266: C1301-C1312Crossref PubMed Google Scholar). In the adult brain it has been found in astrocytes, pia/arachnoid, and a few types of neurons (13McGrail K.M. Phillips J.M. Sweadner K.J. J. Neurosci. 1991; 11: 381-391Crossref PubMed Google Scholar, 14Peng L. Martin-Vasallo P. Sweadner K.J. J. Neurosci. 1997; 17: 3488-3502Crossref PubMed Google Scholar, 15Watts A.G. Sanchez-Watts G. Emanuel J.R. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7425-7429Crossref PubMed Scopus (165) Google Scholar). To understand further the specific roles of individual Na,K-ATPase isoforms, we have analyzed mice in which the α2 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 α2 in the newborn brain and the function of an intrinsic neuronal circuit that could contribute directly to immediate death: generation of the breathing rhythm. Mice heterozygous for the Na,K-ATPase α2 isoform were generated as described previously (16James P.F. Grupp I.L. Grupp G. Woo A.L. Askew G.R. Croyle M.L. Walsh R.A. Lingrel J.B. Mol. Cell. 1999; 3: 555-563Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Heterozygous females were mated with heterozygous males, and the resulting offspring were genotyped by Southern blot as described previously (16James P.F. Grupp I.L. Grupp G. Woo A.L. Askew G.R. Croyle M.L. Walsh R.A. Lingrel J.B. Mol. Cell. 1999; 3: 555-563Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). 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). 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. Tissues from at least four embryonic day 18.5 pups of the same genotype (α2+/+, α2+/−, or α2−/−) were pooled and microsomes prepared as described (16James P.F. Grupp I.L. Grupp G. Woo A.L. Askew G.R. Croyle M.L. Walsh R.A. Lingrel J.B. Mol. Cell. 1999; 3: 555-563Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). 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 (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205522) Google Scholar). The Western blot procedure was performed as described (18He S. Shelly D.A. Moseley A.E. James P.F. James J.H. Paul R.J. Lingrel J.B. Am. J. Physiol. 2001; 281: R917-R925Crossref PubMed Google Scholar). Approximately 5–10 μg of microsomal membrane protein was loaded per lane. The primary antibodies used were a α1 isoform-specific monoclonal antibody, α6F, 1The α6F 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. an α2 isoform-specific monoclonal antibody, McB2, and an α3 isoform-specific monoclonal antibody, MΑ3-915 (Affinity BioReagents, Golden, CO). The signal was detected using an ECL system (AmershamBiosciences). For whole mount diaphragm preparations, diaphragms from embryonic day 18.5 mice were prepared as described previously (19Gautam M. Noakes P.G. Moscoso L. Rupp F. Scheller R.H. Merlie J.P. Sanes J.R. Cell. 1996; 85: 525-535Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar). Diaphragms were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS), 2The abbreviations used are: PBS, phosphate-buffered saline; NMJ, neuromuscular junction blocked in 0.1m 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 α-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). 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. A pregnant wild-type mouse of the same genetic background as the α2−/− 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 (20Aronow B.J. Lund S.D. Brown T.L. Harmony J.A. Witte D.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 725-729Crossref PubMed Scopus (172) Google Scholar, 21Harper M.E. Marselle L.M. Gallo R.C. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 772-776Crossref PubMed Scopus (530) Google Scholar). Antisense and sense RNA probes were synthesized with 35S-labeled rUTP from plasmids that contain either α1 or α2 Na,K-ATPase isoform-specific sequences (9Orlowski J. Lingrel J.B. J. Biol. Chem. 1988; 263: 10436-10442Abstract Full Text PDF PubMed Google Scholar). 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) (22McLean I.W. Nakane P.K. J. Histochem. Cytochem. 1974; 22: 1077-1083Crossref PubMed Scopus (3173) Google Scholar). 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 −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 α2 isoform of Na,K-ATPase, has been described previously (23Pacholczyk T. Sweadner K.J. Protein Sci. 1997; 6: 1537-1548Crossref PubMed Scopus (21) Google Scholar). 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. 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 α2 stain, on the other hand, was much brighter and was only seen on the plasma membrane. 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 (24Telgkamp P. Ramirez J.M. J. Neurophysiol. 1999; 82: 2163-2170Crossref PubMed Scopus (69) Google Scholar). 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 (25Lieske S.P. Thoby-Brisson M. Telgkamp P. Ramirez J.M. Nat. Neurosci. 2000; 3: 600-607Crossref PubMed Scopus (362) Google Scholar). 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 α2−/− group and only one of the α2+/− or wild-type groups. Because in no case did the α2+/− and wild-type groups differ significantly, we grouped the α2+/− and wild-type recordings together to perform the t tests and nonparametric tests reported in the text. Mice lacking the α2 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 α2−/− pups were beating, indicating that the mice were alive when born. Several minutes after birth the wild-type and α2+/− mice breathe and turn pink, but the α2−/− 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 α2+/− mice showed normal levels of oxygen and carbon dioxide (26Vaillancourt C. Berger N. Boksa P. Exp. Neurol. 1999; 160: 142-150Crossref PubMed Scopus (25) Google Scholar), whereas the α2−/− newborn pups displayed very low oxygen and high carbon dioxide levels consistent with failure to breathe (Table I, Table II). Blood glucose levels and body weight were normal for the α2−/− mice (Table I).Table IBody weight, blood gas and blood glucose levels from Na,K-ATPase α2 homozygous (−/−), α2 heterozygous (+/−), and wild-type (+/+) miceGenotypeBody weightBlood gasBlood glucosePCO2pO2gmm Hgmm Hgmg/dlα2+/+1.38 ± 0.09 (4)46.1 (1)87.7 ± 42 (4)31 ± 17 (8)α2+/−1.53 ± 0.17 (8)63.9 ± 28 (7)68.8 ± 16 (9)62 ± 20 (16)α2−/−1.48 ± 0.01 (4)138.9 ± 4.8 (3)19.4 ± 1.8 (3)38 ± 24 (8)Values are given for newborns within 30 min of birth. The standard deviation is given, and numbers in parentheses indicate the number of animals examined. Open table in a new tab Table IISummary of respiratory rhythm patterns measured in brain slices from Na,K-ATPase α2 homozygous (−/−), α2 heterozygous (+/−), and wild type (+/+)GenotypeSighsEupneaFrequency ± S.E.Period CVFrequency ± S.E.Period CVBurst durationAmplitudemin−1min−1s% of sighα2−/−0.67 ± 0.160.1812.02 ± 2.510.662-aDenotes significant difference from pooled control (p < 0.01).0.42 ± 0.0338.5 ± 5.32-aDenotes significant difference from pooled control (p < 0.01).α2+/−0.59 ± 0.160.1510.29 ± 1.720.370.35 ± 0.0255.3 ± 2.5α2+/+1.08 ± 0.170.2612.57 ± 2.170.480.40 ± 0.0261.9 ± 5.8α2+/− and α2+/+0.86 ± 0.140.2111.43 ± 1.370.430.37 ± 0.0258.9 ± 3.4a Denotes significant difference from pooled control (p < 0.01). Open table in a new tab Values are given for newborns within 30 min of birth. The standard deviation is given, and numbers in parentheses indicate the number of animals examined. 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 α2−/− mice appeared developmentally normal at both embryonic day 18.5 and at birth compared with wild-type. Saccules of the α2−/− 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 (27Harding R. Hooper S.B. J. Appl. Physiol. 1996; 81: 209-224Crossref PubMed Scopus (294) Google Scholar). 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 α2−/− 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. We examined tissues from α2−/− 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 α2 isoform was detected with similar abundance in brain and diaphragm, and a faint signal was found in heart (Fig. 2). 3It has been reported that α2 is expressed in alveolar cells when their phenotype changes from ATII-like to ATI-like in culture (46Ridge K.M. Rutschman D.H. Factor P. Katz A.I. Bertorello A.M. Sznajder J.L. Am. J. Physiol. 1997; 273: L246-L255Crossref PubMed Google Scholar). However, there have been several reports that α2 mRNA is lacking in lung (9Orlowski J. Lingrel J.B. J. Biol. Chem. 1988; 263: 10436-10442Abstract Full Text PDF PubMed Google Scholar, 47O'Brodovich H. Staub O. Rossier B.C. Geering K. Kraehenbuhl J.P. Am. J. Physiol. 1993; 264: C1137-C1143Crossref PubMed Google Scholar, 48Crump R.G. Askew G.R. Wert S.E. Lingrel J.B. Joiner C.H. Am. J. Physiol. 1995; 269: L299-L308PubMed Google Scholar, 49Ingbar D.H. Weeks C.B. Gilmore-Hebert M. Jacobsen E. Duvick S. Dowin R. Savik S.K. Jamieson J.D. Am. J. Physiol. 1996; 270: L619-L629Crossref PubMed Google Scholar), consistent with the absence of the protein reported here. In the α2−/− pups, there did not appear to be a significant change in abundance of the α1 or α3 isoform in any tissue compensating for the loss of the α2 isoform. Because the α2 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 α2 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 (19Gautam M. Noakes P.G. Moscoso L. Rupp F. Scheller R.H. Merlie J.P. Sanes J.R. Cell. 1996; 85: 525-535Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar). 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 α2−/− 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. To test whether the absence of the α2 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 α2−/− mice (Fig. 4). Two other normalization routines were evaluated: force normalized to length and force normalized to width. In all cases of normalization (Fig. 4 A) as well as the raw tension data (Fig. 4 B), a similar trend was observed, with the α2−/− 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 α2 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 α2 isoform at embyronic day 18.5, was examined further for physiological defects associated with the absence of the α2 isoform. Previous reports on Na,K-ATPase α2 isoform expression have shown that it is expressed primarily in astrocytes of adults (for review, see Ref. 28Peng L. Arystarkhova E. Sweadner K.J. Glia. 1998; 24: 257-271Crossref PubMed Scopus (36) Google Scholar); however, little data exist on the expression of α2 at the time of birth in mice. Therefore, as an initial step toward the evaluation of the α2−/− mice, we determined the expression profile of the α2 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 situhybridization of sagittal brain sections in which signals for α1 and α2 isoforms can be compared. In Fig. 5, A andB, the choroid plexus is shown. This highly elaborated secretory epithelium that emerges from the ventricular lining shows strong hybridization for α1 isoform mRNA with the antisense probe, but little or no hybridization above background with the α2 antisense probe. In contrast, in Fig. 5 D, it can be seen that the α2 isoform antisense probe showed extremely heavy hybridization over the pia mater, a tissue that is known to express the α2 isoform, whereas the α1 isoform antisense probe showed less (Fig. 5 C). Neither the α1 isoform antisense nor the α2 isoform sense probes labeled the pia. These data validate the methods by confirming the known distribution of the α1 isoform in choroid plexus and the α2 isoform in pia. In the cortical layer, the α1 probe showed a more uniform pattern of diffuse signal in all of the cells, small and large. In contrast, the α2 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 α1 isoform but more heavily for the α2 isoform. There was also signal above background over regions between neurons, particularly for the α2 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. Fig. 6 shows immunostain for the α2 isoform in wild type and α2−/− 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 andB, 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. 6 B is a portion of Fig. 6 A at higher magnification and with fewer stacked optical sections to show more cellular detail. Fig. 6 C, 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 α2-specific antibody (Fig. 6 D) and a lack of specific anti-α2 stain in the α2−/− mouse (Fig. 6 E). These results show that neurons throughout different regions of the brain contain abundant levels of both mRNA and protein for the Na,K-ATPase α2 isoform at the time of birth. The electrochemical ion gradients generated by Na,K-ATPase are essential for the electrical excitability of cells. Because the α2 isoform was expressed abundantly in neurons of wild-type mice, we tested the possibility that the α2 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
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