ARTICLE |
Correspondence to: Nobuteru Usuda, Dept. of Anatomy II, Fujita Health Univ. School of Medicine, Toyoake, Aichi 470-1192, Japan. E-mail: n-usuda@fujita-hu.ac.jp
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Summary |
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We studied the localization of calcineurin by immunoblotting analysis and immunohistochemistry as a first step in clarifying the role of calcineurin in the retina. Rat, bovine, and human retinal tissues were examined with subtype-nonspecific and subtype-specific antibodies for the A and Aß isoforms of its catalytic subunit. In mature retinas of the three species, calcineurin was localized mainly in the cell bodies of ganglion cells and the cells in the inner nuclear layer, in which amacrine cells were distinctively positive. The calcineurin A
and Aß isoforms were differentially localized in the nucleus and the cytoplasm of the ganglion cell, respectively. Calcineurin was also present in developing rat retinas, in which the ganglion cells were consistently positive for it. The presence of calcineurin across mammalian species and regardless of age shown in the present study may reflect its importance in visual function and retinal development, although its function in the retina has not yet been clarified. (J Histochem Cytochem 49:187195, 2001)
Key Words: calcineurin, phosphatase, retina, immunohistochemistry
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Introduction |
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Calcineurin (protein phosphatase 2B) (
Although the presence of calcineurin has been reported in chick retina (
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Materials and Methods |
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Antibodies
Three kinds of polyclonal antibodies against peptides of the deduced amino acid sequences of the N-terminal regions of the human and murine calcineurin catalytic subunit A, A, and Aß isoforms were prepared (
(DPKLSTTDRVV: amino acids 818) and Aß (PPPPGADRVVK: amino acids 1828) were designated Amino X antibody, ACN-2, and ACN-3. The latter two have been employed in a previous immunohistochemical study (
Processing of the Tissues
Retinal tissues of Wistar rats at various developmental ages [embryonic Day 18 (E18), postnatal Days 1 (P1), 4 (P4), 7 (P7), and 14 (P14)], retinal tissues of male Wistar rats at 4 and 12 weeks, and mature bovine retinal tissues obtained from a local slaughterhouse were used. The rats were sacrificed under deep anesthesia with diethylether. Tissues were fixed in 4% paraformaldehyde/100 mM sodium phosphate, pH 7.4, for 24 hr at 4C by immersion, after washing for 3 hr with 100 mM lysine/100 mM sodium phosphate, (pH 7.4)/150 mM sodium chloride to quench free aldehyde groups. Human retinal tissues were obtained at autopsy from three patients with no clinical evidence of neurological disease. These cases had acute respiratory failure (n=1, age 85, male) and liver cirrhosis (n=2, ages 53 and 63, male). These human specimens were fixed in 10% neural buffered formalin for 2448 hr. After dehydration in ethanol and xylene, they were embedded in paraffin.
Light Microscopic Immunohistochemistry
The tissues were dehydrated in an ethanol and xylene series, embedded in paraffin, and sectioned at 5 µm. After deparaffinization in xylene/ethanol series and rehydration, sections were treated in a microwave oven (MR-M201; Hitachi, Tokyo, Japan) for antigen retrieval in 10 mM sodium citrate, pH 6.0, for 10 min ( or Aß isoform to the primary antibody, and by omitting the primary antibody.
Immunoblotting Analysis
Tissues were frozen at -80C until analysis. Rat, bovine, and human retinal tissues and whole rat brain tissue were homogenized in 10 volumes of 10 mM potassium phosphate, (pH 7.5)/150 mM sodium chloride containing the protease inhibitors pepstatin (4 µg/ml), PMSF (3.5 µg/ml), leupeptin (4 µg/ml), and E-64 (4 µg/ml). These homogenates were dissolved in a sample buffer solution containing 1% sodium dodecyl sulfate (SDS) and 200 mM 2-mercaptoethanol, subjected to SDS-polyacrylamide slab gel electrophoresis, transferred to nitrocellulose membranes, and stained with each of the primary antibodies for calcineurin (Amino X, ACN-2, and ACN-3). Goat anti-rabbit IgG antibody conjugated with alkaline phosphatase and bromochloro-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) were sequentially applied to each membrane for visualization. For analysis of retinal development, whole eyeballs were subjected to electrophoresis, because the retina could not be isolated from the eyes of embryos or newborn animals. This electrophoresis was done with samples of the same amount of protein or of the amount of protein corrected by the volume ratio of retina:total eyeball measured on light microscopic specimens. The negative controls for Amino X antibody were made by adding the peptide of Amino X or recombinant proteins of the calcineurin A or Aß isoform to the primary antibody. The amount of protein was measured by protein assay CBB solution (Nakarai; Kyoto, Japan) with bovine serum albumin as a standard.
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Results |
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Immunoblotting Analysis
Amino X antibody detected signals in all tissues examined (Fig 1A). The mobility of each signal for rat brain, rat, bovine, and human retinas was the same as that of the corresponding recombinant proteins of calcineurin A subunit, both A and Aß isoforms. The mobilities of the two isoforms were very close and their mixture showed a broad signal. In the staining controls, the addition of recombinant proteins or synthetic polypeptides to the Amino X antibody rendered the staining weak or negative. The presence of the calcineurin A subunit, both A
and Aß isoforms in E18, P4, P7, P14, and mature rat eyeballs was detected with Amino X, ACN-2, and ACN-3 antibodies (Fig 1B1D). The signals for Amino X antibody were weaker in the samples from postnatal animals than in embryonic samples in both cases when the same amounts of protein were analyzed (Fig 1B) or the amounts of protein corrected by the volume ratios of retina:total eyeballs were analyzed (data not shown). ACN-2 and ACN-3 antibodies detected signals for A
and Aß isoforms in all samples of developmental rat eyes (Fig 1C and Fig 1D). The signals for ACN-3 were more easily detectable than ACN-2 when signals were developed (data not shown). These data indicated the presence of two isoforms in the retina of the three mammalians and showed that the two are present in rat eyes through all developmental stages, in amounts that do not increase with increasing development.
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Immunohistochemistry of the Mature Retina
The localization of the calcineurin A subunit in the mature retina is shown in Fig 2. The ganglion cell layer, inner plexiform layer, and inner nuclear layer of the mature rat retina were strongly positive for the reaction of Amino X antibody (Fig 2A). The ganglion cells and amacrine cells in the inner nuclear layer were the most strongly immunoreactive. Other kinds of cells in the inner nuclear layer, bipolar and horizontal cells, and the outer plexiform layer were also immunopositive. In the rat inner plexiform layer, laminae 1, 2, and 3 were visible as well-delineated bands, and the inner segment of photoreceptor cells was weakly positive. Similar to the immunoreaction of the mature rat retina, the ganglion cell, inner plexiform, and inner nuclear layers in the bovine and human retinas reacted strongly to the Amino X antibody (Fig 2B and Fig 2C). The ganglion cells and both the amacrine and bipolar cells of the inner nuclear layer were strongly immunoreactive, with the horizontal cells somewhat less so. The inner plexiform layer showed diffuse positive reactivity without well-delineated bands. The inner segment of the photoreceptor cells was weakly positive. In the staining controls, recombinant proteins or synthetic polypeptides reduced the staining of Amino X antibody to weak or negative (Fig 2D).
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The reaction products for A and Aß isoforms detected by ACN-2 and ACN-3 were observed in ganglion cells and the inner nuclear layer. The staining of ganglion cells was distinctive, in that subcellular localization in these cells differed between ACN-2 and ACN-3. A
and Aß isoforms were localized in the nucleus for ACN-2 (Fig 3A, Fig 3C, and Fig 3E) and in the cytoplasm for ACN-3 (Fig 3B, Fig 3D, and Fig 3F) in rat, bovine, and human retinas. The staining in the inner nuclear layer was weak for ACN-2 antibody (data not shown). The control for the staining employing recombinant proteins or synthetic polypeptides made the staining weak or negative (data not shown).
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Immunohistochemistry of Calcineurin in the Developing Rat Retina
Developmental changes in the location of the calcineurin A subunit were examined in rat retinas using the Amino X antibody. At E18 the reaction appeared rather diffuse overall, preferentially localized in the inner half of the retina (Fig 4A), the region separated from the neuroblastic layer by the inner plexiform layer. The reaction products become concentrated in specific kinds of cells thereafter. With P4, the reaction products observed in the ganglion cell and the inner plexiform layers were strongly positive, and the cells in the neuroblastic layer above the inner plexiform layer (which appeared to be amacrine cells) were positive (Fig 4B). With P7, the ganglion cell, inner plexiform, inner nuclear, and outer plexiform layers all become positive. Ganglion cells and the amacrine and horizontal cells in the inner nuclear layer were strongly positive, and their immunopositive nerve cells possessed processes extending into the inner plexiform layer. Two intensely immunoreactive bands were observed in the inner plexiform layer (Fig 4C). With P14, the ganglion cell, inner plexiform, inner nuclear, and outer plexiform layers were immunopositive. Ganglion cells and the amacrine cells in the inner nuclear layer were strongly positive, and the bipolar cells were also positive. The horizontal cells were weakly labeled, although the labeling may have been masked by the intense labeling of other kinds of cells in the inner nuclear layer. Three immunoreactive bands were observed in the inner plexiform layer. At this stage, both inner and outer segments are present in the retina and were weakly immunopositive (Fig 4D). The reaction product for the calcineurin A subunit was observed in the ganglion cell layer through these developmental stages from the embryonic stage. Other kinds of cells became positive later.
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Discussion |
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In the preliminary experiment for localizing calcineurin in the retina, the antibody was raised against purified protein of its A subunit. This antibody was not suitable for the immunostaining because the reaction was not inhibited by adding the recombinant proteins of the A subunit. Amino X antibody was made for the peptide of the common sequence near the N-termini of A and Aß isoforms of mouse calcineurin (
isoform of rat, bovine, human, and mouse enzymes (
and Aß isoforms of human calcineurin. It detected signals in rat brain and in rat, bovine, and human retinal tissues by immunoblotting analysis. The Amino X antibody, in addition to the subtype-specific antibodies ACN-2 and ACN-3 (
and Aß isoforms and by the synthetic antigen polypeptide. This Amino X antibody was shown to be useful in detecting calcineurin or its A subunit of both isoforms by immunoblotting analysis and immunohistochemistry. The Amino X antibody was also employed on chick retina by immunoblotting analysis and immunohistochemistry (data not shown) as preliminary experiments. Strong immunoreactivity was observed in ganglion cells, amacrine cells, and the inner plexiform layer. The outer plexiform layer and inner segment of photoreceptor cells were weakly positive. These data agreed with the previous report by
In the tissues examined, the presence of calcineurin was confined to retinal neurons and was not detected in Müller cells. This preferential localization in the neuron was similar to that in the brain, as shown by in situ hybridization and immunocytochemistry ( and Aß isoforms in the nucleus and in the cytoplasm of the ganglion cells also reflects the differential distribution of isoforms observed in the nerve cells of the brain (
isoform than for the Aß isoform in the brain (
using ACN-2 and ACN-3 antibodies, both by immunoblotting analysis and immunohistochemistry. The relative abundance of the Aß isoform compared with A
in the retina might explain the stronger staining of the cytoplasm by Amino X and ACN-3 antibodies than by ACN-2 antibody.
All three antibodies to calcineurin A subunit detected signals in the developing eye tissues of the rat by immunoblotting analysis. The intensity of the signals decreased with development. However, by immunohistochemistry, the localization of calcineurin changed during development, from a rather diffuse pattern limited to the inner half of the retina at E18, before neural layers are distinguishable, to concentration in the ganglion cells and amacrine cells in the inner nuclear layer after birth. In the blotting analysis there was a tendency for antibody labeling to decrease, whereas in the sections there may be an opposite trend. These results from two different procedures are apparent but contradictory. The reason for this is not clear, but the different pattern of localization (a diffuse pattern in the immature retina and specifically localized in particular areas in the mature retina) may explain this contradiction. Summarizing these observations: (a) calcineurin is present in the retina of three mammalian species, including humans; (b) calcineurin is most abundant in the ganglion cells and cells in the inner nuclear layer; (c) A and Aß isoforms are differentially located in the nucleus and in the cytoplasm of ganglion cells; and (d) both isoforms, A
and Aß, are already present in the eye of the developing rat, but their rather diffuse presence in the immature retina becomes confined to ganglion cells and amacrine cells after the developmental period.
In our study, calcineurin was shown to be present in the retinal neurons. The functions of Ca++ in the retina have been studied extensively in the photoreceptor cells (for review see
In the present study, the calcineurin isoforms A and Aß were localized mainly in the nucleus and the cytoplasm, respectively, the same as neurons of the central nervous system (
and Aß has not been clarified yet.
To understand the presence of calcineurin in the retina, it is also important to consider its localization in relation to the presence and the function of other Ca++-binding proteins in the retina. Calcineurin comprises two subunits, of which the catalytic subunit (A) has serine/threonine activity, while the regulatory subunit (B) binds the A subunit in a Ca++-dependent manner involving CaM. Therefore, calcineurin not only binds calcium but is regulated by a most representative Ca++-binding protein, CaM. Among the Ca++-binding proteins whose presence has been shown by immunohistochemistry in various retinal cells (ganglion and amacrine but, specifically, neither photoreceptor nor Müller cells) are CaM (
The developmental changes of these proteins in the retina have been examined for CaM, calbindin D-28K, parvalbumin, protein kinase C, and CaM kinase IV. CaM appears in the embryonic mouse retina before the formation of neural layers, and is confined to the ganglion cells and cells in the inner nuclear layer during development (
The present study demonstrated the presence of calcineurin in mammalian retinas, especially in limited kinds of retinal cells. Despite the presence of calcineurin in the retina throughout development and the presence of calcineurin, together with other Ca++-binding proteins and Ca++/CaM-dependent kinases, in similar kinds of cells of the retina, information on this Ca++/CaM-dependent protein phosphorylation and dephosphorylation system in the retina is very limited. The physiological role of this major Ca++/CaM-dependent protein phosphatase in the retina, in morphogenesis, and in visual function needs to be elucidated.
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Acknowledgments |
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We thank Dr Randall L. Kincaid (Department of Pharmacology, Pennsylvania State University College of Medicine) for kindly providing the calcineurin antibodies and Dr Yasuo Watanabe (Department of Pharmacology, Nagoya University) for helpful discussion.
Received for publication April 7, 2000; accepted September 6, 2000.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akopian A, Gabriel R, Witkovsky P (1998) Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina. J Neurophysiol 80:1105-1115
Bastianelli E, Takamatsu K, Okazaki K, Hidaka H, Pochet R (1995) Hippocalcin in rat retina. Comparison with calbindin-D28k, calretinin and neurocalcin. Exp Eye Res 60:257-266[Medline]
Bito H, Deisseroth K, Tsien RW (1996) CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87:1203-1214[Medline]
Chow CW, Rincon M, Cavanagh J, Dickens M, Davis RJ (1997) Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway. Science 278:1638-1641
Cohen P (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem 58:453-508[Medline]
Cooper NG, McLaughlin BJ, Tallant EA, Cheung WY (1985) Calmodulin-dependent protein phosphatase: immunocytochemical localization in chick retina. J Cell Biol 101:1212-1218[Abstract]
Ellis DZ, Edwards SC (1994) Characterization of a calcium/calmodulin-dependent protein phosphatase in the Limulus nervous tissue and its light regulation in the lateral eye. Vis Neurosci 11:851-860[Medline]
Endo T, Kobayashi S, Onaya T (1985) Parvalbumin in rat cerebrum, cerebellum and retina during postnatal development. Neurosci Lett 60:279-282[Medline]
Giri PR, Higuchi S, Kincaid RL (1991) Chromosomal mapping of the human genes for the calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit. Biochem Biophys Res Commun 181:252-258[Medline]
Giri PR, Marietta CA, Higuchi S, Kincaid RL (1992) Molecular and phylogenetic analysis of calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit genes. DNA Cell Biol 11:415-424[Medline]
Goto S, Matsukado Y, Mihara Y, Inoue N, Miyamoto E (1986) The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay. Brain Res 397:161-172[Medline]
Griffith JP, Kim JL, Kim EE, Sintchak MD, Thomson JA, Fitzgibbon MJ, Fleming MA, Caron PR, Hsiao K, Navia MA (1995) X-ray structure of calcineurin inhibited by the immunophilin-immunosupressant FKBP12-FK506 complex. Cell 82:507-522[Medline]
Guerini D, Klee CB (1989) Cloning of human calcineurin A: evidence for two isozymes and identification of a polyproline structural domain. Proc Natl Acad Sci USA 86:9183-9187[Abstract]
GünhanAgar E, Kahn D, Chalupa LM (2000) Segregation of ON and OFF bipolar cell axonal arbors in the absence of retinal ganglion cells. J Neurosci 20:306-314
Hashimoto Y, King MM, Soderling TR (1988) Regulatory interactions of calmodulin-binding proteins: phosphorylation of calcineurin by autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Proc Natl Acad Sci USA 85:7001-7005[Abstract]
Hsu S, Raine L, Fanger H (1981) Use of avidinbiotinperoxidase complex (ABC) in immunoperoxidase techniques: comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580[Abstract]
Ito A, Hashimoto T, Hirai M, Takada T, Shuntoh H, Kuno T, Tanaka C (1989) The complete primary structure of calcineurin A, a calmodulin binding protein homologous with protein phosphatases 1 and 2A. Biochem Biophys Res Commun 163:1492-1497[Medline]
Kasahara J, Fukunaga K, Miyamoto E (1999) Differential effects of a calcineurin inhibitor on glutamate-induced phosphorylation of a Ca2+/calmodulin-dependent protein kinase in cultured rat hippocampal neurons. J Biol Chem 274:9061-9067
Kikuchi M, Kashii S, Mandai M, Yasuyoshi H, Honda Y, Kaneda K, Akaike A (1998) Protective effects of FK506 against glutamate-induced neurotoxicity in retinal cell culture. Invest Ophthalmol Vis Sci 39:1227-1232[Abstract]
Kincaid RL, Giri PR, Higuchi S, Tamura J, Dixon SC, Marietta CA, Amorese DA, Martin BM (1990) Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods. J Biol Chem 265:11312-11319
Kincaid RL, Nightingale MS, Martin BM (1988) Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin. Proc Natl Acad Sci USA 85:8983-8987[Abstract]
Kitagawa H, Hotta Y, Fujiki K, Kanai A (1996) Cloning and high expression of rabbit FKBP25 in cornea. Jpn J Ophthalmol 40:133-141[Medline]
Klauck TM, Faux MC, Labudda K, Langeberg LK, Jaken S, Scott JD (1996) Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science 271:1589-1592[Abstract]
Klee CB, Crouch TH, Krinks MH (1979) Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Natl Acad Sci USA 76:6270-6273[Abstract]
Klee CB, Ren H, Wang X (1998) Regulation of the calmodulin stimulated protein phosphatase, calcineurin. J Biol Chem 273:13367-13370
Korf HW, White BH, Schaad NC, Klein DC (1992) Recoverin in pineal organs and retinae of various vertebrate species including man. Brain Res 595:57-66[Medline]
Kuno T, Takeda T, Hirai M, Ito A, Mukai H, Tanaka C (1989) Evidence of a second isoform of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A). Biochem Biophys Res Commun 165:1352-1358[Medline]
Mansuy IM, Mayford M, Jacob B, Kandel ER, Bach ME (1998) Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92:39-49[Medline]
Mulkey RM, Endo S, Shenolikar S, Malenka RC (1994) Involvement of a calcineurin/inhibitor-1 phosphatase cascade in hippocampal long-term depression. Nature 369:486-488[Medline]
Muramatsu T, Kincaid RL (1992) Molecular cloning and chromosomal mapping of the human gene for the testis-specific catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A). Biochem Biophys Res Commun 188:265-271[Medline]
Muramatsu T, Kincaid RL (1993) Molecular cloning of a full-length cDNA encoding the catalytic subunit of human calmodulin-dependent protein phosphatase (Calcineurin A). Biochim Biophys Acta 1178:117-120[Medline]
Muramatsu T, Kincaid RL (1996) Inhibition of NF-AT signal transduction events by a dominant-negative form of calcineurin. Biochem Biophys Res Commun 218:466-472[Medline]
Nakano A, Tories M, Watanabe M, Usuda N, Merritt T, Haddock H (1992) Neurocalcin, a novel calcium binding protein with three EF-hand domains, expressed in retinal amacrine cells and ganglion cells. Biochem Biophys Res Commun 186:1207-1211[Medline]
Ochiishi T, Terashima T, Sugiura H, Yamauchi T (1994) Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase II in the rat retina. Brain Res 634:257-265[Medline]
Oguni M, Setogawa T, Shinohara H, Kato K (1998) Calbindin-D 28 kD and parvalbumin in the horizontal cells of rat retina during development. Curr Eye Res 17:617-622[Medline]
O'Keefe SJ, Tamura J, Kincaid RL, Tocci MJ, O'Neil EA (1992) FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature 357:692-694[Medline]
Parsons JN, Wiederrecht GJ, Salowe S, Burbaum JJ, Rokosz LL, Kincaid RL, O'Keefe SJ (1994) Regulation of calcineurin phosphatase activity and interaction with the FK-506 FK-506 binding complex. J Biol Chem 269:19610-19616
Pochet R, Pasteels B, SetoOhshima A, Bastianelli E, Kitajima S, Van Eldik LJ (1991) Calmodulin and calbindin localization in retina from six vertebrate species. J Comp Neurol 314:750-762[Medline]
Sakagami H, Kondo H (1996) Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase type IV in the mature and developing rat retina. Brain Res 719:154-160[Medline]
SetoOhshima A, Yamazaki Y, Kawamura N, Kitajima S, Sano M, Mizutani A (1987) The early expression of immunoreactivity for calmodulin in the nervous system of mouse embryos. Histochemistry 86:337-343[Medline]
Shi S-R, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem 41:1599-1604
Shibasaki F, Kondo E, Akagi T, Mckeon F (1997) Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature 386:728-731[Medline]
Snyder SH, Lai MM, Burnett PE (1998) Immunophilins in the nervous system. Neuron 21:283-294[Medline]
Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A, Hvalby O, Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF, Sun W, Webstar LC, Grant SGN, Eilers J, Konnerth A, Li J, McNamara JO, Seeburg PH (1998) Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell 92:279-289[Medline]
Steiner JP, Dawson TM, Fotuhi M, Glatt CE, Snowman AM, Cohen N, Snyder SH (1992) High brain densities of the immunophilin FKBP colocalized with calcineurin. Nature 358:584-587[Medline]
Takaishi T, Saito N, Kuno T, Tanaka C (1991) Differential distribution of the mRNA encoding two isoforms of the catalytic subunit of calcineurin in the brain. Biochem Biophys Res Commun 174:393-398[Medline]
Uesugi R, Yamada M, Mizuguchi M, Baimbridge KG, Kim SU (1992) Calbindin D-28k and parvalbumin immunohistochemistry in developing rat retina. Exp Eye Res 54:491-499[Medline]
Usuda N, Arai H, Sasaki H, Hanai T, Nagata T, Muramatsu T, Kincaid RL, Higuchi S (1996) Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation. J Histochem Cytochem 44:13-18
Usuda N, Kong Y, Hagiwara M, Uchida C, Terasawa M, Nagata T, Hidaka H (1991) Differential localization of protein kinase C isozymes in retinal neurons. J Cell Biol 112:1241-1247[Abstract]
Watanabe Y, Perrino BA, Chang BH, Soderling TR (1995) Identification in the calcineurin A subunit of the domain that binds the regulatory B subunit. J Biol Chem 270:456-460
Yarfitz S, Hurley JB (1994) Transduction mechanisms of vertebrate and invertebrate photoreceptors. J Biol Chem 269:14329-14332
Yin JPC, Vecchio MD, Tully T (1995) CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila. Cell 81:107-115[Medline]
Yoshida K, Imaki J, Fujisawa H, Harada T, Ohki K, Matsuda H, Hagiwara M (1996) Differential distribution of CaM kinases and induction of c-fos expression by flashing and sustained light in rat retinal cells. Invest Ophthalmol Vis Sci 37:174-179[Abstract]
Zhang DR, Yeh HH (1991) Protein kinase C-like immunoreactivity in rod bipolar cells of the rat retina: a developmental study. Vis Neurosci 6:429-437[Medline]