- Email: [email protected]
Expression of aquaporin-1 immunoreactivity by photoreceptor cells in the mouse retina
Neuroscience Letters 388 (2005) 96–99
Expression of aquaporin-1 immunoreactivity by photoreceptor cells in the mouse retina Ianors Iandiev a,b , Thomas Pannicke b , Martin B. Reichel a , Peter Wiedemann a , Andreas Reichenbach b , Andreas Bringmann a,∗ a
Department of Ophthalmology and Eye Clinic, University of Leipzig Medical Faculty, Liebigstrasse 10-14, D-04103 Leipzig, Germany b Paul Flechsig Institute of Brain Research, University of Leipzig Medical Faculty, D-04109 Leipzig, Germany Received 31 May 2005; received in revised form 17 June 2005; accepted 21 June 2005
Abstract Aquaporin water channels play a crucial role in the maintenance of ionic and osmotic homeostasis in the neural tissue. In the sensory retina, aquaporin-4 is expressed by M¨uller glial cells, predominantly in the inner retina, while aquaporin-1 is expressed mainly in the outer retina. However, it is unknown whether aquaporin-1 expression occurs in M¨uller cells or photoreceptor cells. By using immunohistochemical staining of retinal slices from rds mice, we show that the immunoreactivity for aquaporin-1 disappears along with the photoreceptor cell degeneration. In suspensions of dissociated retinal cells from control mice, photoreceptor cells expressed aquaporin-1 immunoreactivity while M¨uller cells were largely devoid of staining. The data suggest that photoreceptor cells, but not M¨uller cells, express aquaporin-1 in the murine retina. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Aquaporin; Immunohistochemistry; M¨uller glia cells; Photoreceptor cells; Retina; rds Mouse
Aquaporin water channels are critically involved in the maintenance of ionic and osmotic balance in the central nervous system [15]. There are at least 13 different members of the aquaporin protein family that mediate bidirectional movement of water across membranes, in response to osmotic gradients and differences in hydrostatic pressure [16]. Though the mammalian sensory retina may express gene transcripts for a variety of aquaporins [14], the immunoreactivities of only two aquaporins have hitherto been demonstrated in the retina [4,9,10]. Aquaporins are suggested to contribute to the fluid-absorbing function of retinal glial and pigment epithelial cells [8,13]. Normally, the subretinal space is dehydrated by the pigment epithelium while the inner retinal tissue is apparently dehydrated by transcellular water movements through M¨uller glial cells [2,8]. The transglial water transport is facilitated by aquaporin-4 water channels [4,9,10]. In addition to aquaporin-4 which is expressed predominantly in the inner retina, the retinal expression of aquaporin-1 has been described. Aquaporin-1 ∗
Corresponding author. Tel.: +49 341 9721557; fax: +49 341 9721659. E-mail address: [email protected] (A. Bringmann).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.06.046
is expressed by pigment epithelial cells [13], by a subpopulation of glycinergic amacrine cells [5], and within the outer retina [6,8]. It has been argued that the aquaporin-1 in the outer retina may be involved in the light-dependent hydration of the space surrounding the photoreceptors [6,7]. However, it is unknown which cell type expresses aquaporin-1 in the outer retina, i.e., photoreceptor cells or M¨uller glial cells. In order to solve this question, we made two attempts; (i) we immunocytochemically stained acutely dispersed cells from the mouse retina, and (ii) we investigated the expression of aquaporin-1 immunoreactivity in slices of the rds mutant mouse retina which shows a nearly complete loss of photoreceptor cells after several months [11]. Albino mice of the strain Balb/c carrying the rds (Prph2Rd2 ) mutation (5 months or 1-year old) and wildtype Balb/c animals (6 months old) were used, and treated in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and with the EC Directive 86/609/EEC. The rds mice carry a null mutation in the Prph2 gene encoding the structural protein, peripherin/rds, which is essential for the formation of photoreceptor outer segments [3]. The mice display a degeneration of
I. Iandiev et al. / Neuroscience Letters 388 (2005) 96–99
photoreceptor cells of early-onset and slow progression [11] and are widely used as a model for retinitis pigmentosa. Mice were euthanized with CO2 , and the eyes were enucleated. For immunohistochemical staining, isolated retinas were kept immersed in fixative solution containing 4% paraformaldehyde (PFA) for 2 h. After washing in phosphatebuffered saline, the tissue was embedded in saline containing 3% agarose (w/v), and 70 m-thick slices were cut by using a vibratome. Retinal sections were incubated in 5% normal goat serum plus 0.3% Triton X-100 in saline for 2 h and, subsequently, in primary antibody for 15 h at 4 ◦ C. After washing in 1% bovine serum albumin, the secondary antibodies were applied for 2 h at room temperature. Cell nuclei were counterstained using Hoechst 33258. To prepare suspensions of dissociated cells, isolated retinas were stored for 30 min in saline containing 0.4 mg/ml papain (Boehringer, Mannheim, Germany) at 37 ◦ C. After fixation with 4% PFA for 10 min and washing with saline, the tissues were triturated with a pipette until single cells were dissociated. The cells were blocked and permeabilized for 30 min with 5% normal goat serum and 0.3% Triton-X 100, followed by incubation with primary antibodies for 6 h at 4 ◦ C. Images were taken by using a confocal laser scanning microscope LSM 510 Meta (Zeiss, Oberkochen, Germany). The following antibodies were used: rabbit anti-aquaporin-1 (1:200; Chemicon, Hofheim, Germany), rabbit anti-rat aquaporin-4 (1:200; Sigma–Aldrich, Taufkirchen, Germany), mouse antivimentin (1:250; 13.2 clone, Sigma–Aldrich), and mouse anti-glutamine synthetase (1:250; Chemicon). The secondary antibodies were Cy3-conjugated goat anti-rabbit IgG (1:400; Dianova, Hamburg, Germany) and Cy2-coupled goat antimouse IgG (1:400; Dianova). The slow degeneration of rds mice retinas causes a reduction of the retinal thickness, particularly of the outer nuclear layer. As shown in Fig. 1, the thickness of the outer nuclear layer is reduced to 1–3 rows of photoreceptor nuclei in retinas of 5 months old rds mice when compared to the 7–10 rows of photoreceptor nuclei normally present in retinas of age-matched control mice. In retinas of control and rds mice, vimentin immunoreactivity is expressed by astrocytes as well as by M¨uller cell fibers predominantly in the inner retinal layers, i.e., from the nerve fiber layer up to the outer plexiform layer (Fig. 1A). In addition, the outer limiting membrane was
97
observed to be faintly stained against vimentin in some slices (Fig. 1B). In the sensory retina, aquaporin-4 is expressed by M¨uller cells [9]. In control murine retinas, the immunoreactivity for aquaporin-4 was expressed predominantly in the inner retinal layers, with most pronounced expression around blood vessels and ganglion cells in the nerve fiber/ganglion cell layers, within distinct sublayers of the inner plexiform layer, and around vessels at both sides of the inner nuclear layer (Fig. 1A). In addition, a faint staining was observed at the outer limiting membrane. There was a substantial colocalization of aquaporin-4 and vimentin proteins in the mouse retina, as indicated by the yellow color in the images of Fig. 1A (right), suggesting a glial expression of aquaporin4. This expression pattern of aquaporin-4 immunoreactivity was unchanged in retinal slices from 5 months (Fig. 1A) and 1-year old rds mice (not shown), with the exception of an apparent upregulation of expression surrounding cell nuclei in the outer half of the inner nuclear layer (Fig. 1A). In control retinas, aquaporin-1 immunoreactivity was expressed prominantly in the outer retina, i.e., from the outer plexiform layer up to the photoreceptor segments (Fig. 1B). The aquaporin-1 immunoreactivity enclosed the photoreceptor cell nuclei in the outer nuclear layer. There was no overlay of the aquaporin-1 and vimentin immunoreactivities (Fig. 1B, right). In the outer plexiform layer, both immunoreactivities were sharply segregated, with aquaporin-1 immunoreactivity expressed only at the outer border of this layer, surrounding the photoreceptor cell nuclei (inset in Fig. 1B). Along with the degeneration of photoreceptor cells in retinas of rds mice, the expression of aquaporin-1 immunoreactivity disappeared. Fig. 1B shows two examples of retinal slices from 5 months old rds mice at different stages of degeneration. In the example shown in Fig. 1B (middle row), the receptor segments are missing while a certain number of photoreceptor cell bodies is still present, showing immunoreactivity for aquaporin-1. In the example shown in Fig. 1B (bottom row), only few photoreceptor cell nuclei are present which are surrounded by aquaporin-1 immunoreactivity. Other parts of the rds retinas are fully devoid of aquaporin-1 immunoreactivity. Retinas of 1-year old rds mice showed a full absence of photoreceptor cells and aquaporin-1 immunoreactivity (not shown). The disappearance of aquaporin-1 immunoreactivity along with the photoreceptor cell degeneration in rds mice
Fig. 1. Aquaporin-1 and aquaporin-4 immunoreactivities in retinas of control and rds mice. (A) Co-immunostaining against aquaporin-4 and vimentin. Arrows indicate perivascular staining for aquaporin-4. The inset shows aquaporin-4 staining around a vessel at the border between the IPL and INL at higher magnification. (B) Immunoreactivities for aquaporin-1 and vimentin. Aquaporin-1 immunoreactivity is expressed by photoreceptor cells in the mouse retina and disappears during retinal degeneration in rds mice. The insets show the OPL (above) and single aquaporin-1-positive photoreceptor cell bodies (below) at higher magnification. Arrows indicate single photoreceptor nuclei which are surrounded by aquaporin-1 immunoreactivity. Arrowheads mark aquaporin-1 immunoreactivity expressed by red blood cells. (C) Co-immunostainings for aquaporin-4 and aquaporin-1 (red), respectively, and glutamine synthetase (green) in acutely isolated cells of retinas from a control mouse and a rds mouse. The left images show the aquaporin immunoreactivities; the right images show the overlays of aquaporin and glutamine synthetase immunoreactivities. Large arrowheads indicate the endfoot region of M¨uller glial cells; small arrowheads mark the soma of M¨uller cells. The large arrow indicates photoreceptor cells which are attached to the distal end of the M¨uller cell; small arrows mark isolated photoreceptor cell bodies and debris of photoreceptor cells. Cell nuclei were counterstained with Hoechst 33258 (blue). Scale bars, 20 and 5 m (insets), respectively. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PRS, photoreceptor segments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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I. Iandiev et al. / Neuroscience Letters 388 (2005) 96–99
I. Iandiev et al. / Neuroscience Letters 388 (2005) 96–99
retinas suggests that photoreceptor cells, but not M¨uller glial cells, express aquaporin-1. To support this assumption, we stained dissociated retinal cells for aquaporins and glutamine synthetase, as a specific marker of M¨uller glial cells. As shown in Fig. 1C, the glutamine synthetase-positive cell bodies of single M¨uller cells did not express aquaporin-1 immunoreactivity (including the stem processes, passing through the outer nuclear layer in situ) while they express strong immunoreactivity for aquaporin-4, as indicated by the yellow color in the overlays of both images. However, photoreceptor cells which are attached to the distal end of isolated M¨uller cells, as well as isolated photoreceptor cell bodies, expressed immunoreactivity for aquaporin-1 (Fig. 1C). The data suggest that in the mouse retina, aquaporin-1 is expressed by photoreceptor cells but not by M¨uller glial cells. Our data support the view that in the sensory retina, there is a striking segregation of the expression of two aquaporins. Aquaporin-4 is expressed by glial cells, predominantly in the inner retina, while aquaporin-1 is expressed, in addition to a subpopulation of glycinergic amacrine cells [5], predominantly in the outer retina, i.e., by photoreceptor and pigment epithelial cells [6,9,13]. The reason(s) for this molecular segregation of retinal aquaporin expression which includes also other molecules such as vimentin (Fig. 1B) remains to be determined. Noteworthy, this molecular segregation reflects the two fluid-absorption areas in the retina, i.e., the inner retina which is dehydrated by M¨uller cells [2,8], and the outer retina that is dehydrated by the pigment epithelium. The functional role of aquaporin-1 in photoreceptor cells is unclear. It may be assumed that this water channel is involved in the maintenance of the osmotic balance necessary for the phototransduction process. Interestingly, in contrast to aquaporin-4 which is a pure water channel, aquaporin-1 is a channel for both water and cations [17] that can be gated by cyclic GMP [1,12]. However, it remains to be proven whether aquaporin1 plays a role in the regulation of photoreceptor cell excitability.
Acknowledgements This work was supported by grants from the Interdisziplin¨ares Zentrum f¨ur Klinische Forschung (IZKF) at the Faculty of Medicine of the University of Leipzig (project C21), and from the Deutsche Forschungsgemeinschaft (BR 1249/2-1).
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References [1] T. Anthony, H. Brooks, D. Boassa, S. Leonov, G.M. Yanochko, J.W. Regan, A.J. Yool, Cloned aquaporin-1 is a cyclic GMP-gated ion channel, Mol. Pharmacol. 57 (2000) 576–588. [2] A. Bringmann, A. Reichenbach, P. Wiedemann, Pathomechanisms of cystoid macular edema, Ophthalmic Res. 36 (2004) 241–249. [3] G. Connell, R. Bascom, L. Molday, D. Reid, R.R. McInnes, R.S. Molday, Photoreceptor peripherin is the normal product of the gene responsible for retinal degeneration in the rds mouse, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 723–726. [4] A. Frigeri, M.A. Gropper, C.W. Turck, A.S. Verkman, Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 4328–4331. [5] I.B. Kim, E.J. Lee, S.J. Oh, C.B. Park, D.V. Pow, M.H. Chun, Light and electron microscopic analysis of aquaporin 1-likeimmunoreactive amacrine cells in the rat retina, J. Comp. Neurol. 452 (2002) 178–191. [6] I.B. Kim, S.J. Oh, S. Nielsen, M.H. Chun, Immunocytochemical localization of aquaporin 1 in the rat retina, Neurosci. Lett. 244 (1998) 52–54. [7] J.D. Li, B.K. Govardovskii, R.H. Steinberg, Light-dependent hydration of the space surrounding photoreceptors in the cat retina, Vis. Neurosci. 11 (1994) 743–752. [8] E.A. Nagelhus, Y. Horio, A. Inanobe, A. Fujita, F.M. Haug, S. Nielsen, Y. Kurachi, O.P. Ottersen, Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains, Glia 26 (1999) 47–54. [9] E.A. Nagelhus, M.L. Veruki, R. Torp, F.M. Haug, J.H. Laake, S. Nielsen, P. Agre, O.P. Ottersen, Aquaporin-4 water channel protein in the rat retina and optic nerve: polarized expression in M¨uller cells and fibrous astrocytes, J. Neurosci. 18 (1998) 2506–2519. [10] R.V. Patil, I. Saito, X. Yang, M.B. Wax, Expression of aquaporins in the rat ocular tissue, Exp. Eye Res. 64 (1997) 203–209. [11] S. Sanyal, A. De Ruiter, R.K. Hawkins, Development and degeneration of retina in rds mutant mice: light microscopy, J. Comp. Neurol. 194 (1980) 193–207. [12] S.M. Saparov, D. Kozono, U. Rothe, P. Agre, P. Pohl, Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry, J. Biol. Chem. 276 (2001) 31515–31520. [13] W.D. Stamer, D. Bok, J. Hu, G.J. Jaffe, B.S. McKay, Aquaporin-1 channels in human retinal pigment epithelium: role in transepithelial water movement, Invest. Ophthalmol. Vis. Sci. 44 (2003) 2803–2808. [14] S. Tenckhoff, M. Hollborn, L. Kohen, S. Wolf, P. Wiedemann, A. Bringmann, Diversity of aquaporin mRNA expressed by rat and human retinas, NeuroReport 16 (2005) 53–56. [15] A.S. Verkman, Role of aquaporin water channels in eye function, Exp. Eye Res. 76 (2003) 137–143. [16] A.S. Verkman, A.K. Mitra, Structure and function of aquaporin water channels, Am. J. Physiol. 278 (2000) F13–F28. [17] A. Yool, W. Stamer, J. Regan, Forskolin stimulation of water and cation permeability in aquaporin 1 water channels, Science 273 (1996) 1216–1218.
Expression of aquaporin-1 immunoreactivity by photoreceptor cells in the mouse retina Ianors Iandiev a,b , Thomas Pannicke b , Martin B. Reichel a , Peter Wiedemann a , Andreas Reichenbach b , Andreas Bringmann a,∗ a
Department of Ophthalmology and Eye Clinic, University of Leipzig Medical Faculty, Liebigstrasse 10-14, D-04103 Leipzig, Germany b Paul Flechsig Institute of Brain Research, University of Leipzig Medical Faculty, D-04109 Leipzig, Germany Received 31 May 2005; received in revised form 17 June 2005; accepted 21 June 2005
Abstract Aquaporin water channels play a crucial role in the maintenance of ionic and osmotic homeostasis in the neural tissue. In the sensory retina, aquaporin-4 is expressed by M¨uller glial cells, predominantly in the inner retina, while aquaporin-1 is expressed mainly in the outer retina. However, it is unknown whether aquaporin-1 expression occurs in M¨uller cells or photoreceptor cells. By using immunohistochemical staining of retinal slices from rds mice, we show that the immunoreactivity for aquaporin-1 disappears along with the photoreceptor cell degeneration. In suspensions of dissociated retinal cells from control mice, photoreceptor cells expressed aquaporin-1 immunoreactivity while M¨uller cells were largely devoid of staining. The data suggest that photoreceptor cells, but not M¨uller cells, express aquaporin-1 in the murine retina. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Aquaporin; Immunohistochemistry; M¨uller glia cells; Photoreceptor cells; Retina; rds Mouse
Aquaporin water channels are critically involved in the maintenance of ionic and osmotic balance in the central nervous system [15]. There are at least 13 different members of the aquaporin protein family that mediate bidirectional movement of water across membranes, in response to osmotic gradients and differences in hydrostatic pressure [16]. Though the mammalian sensory retina may express gene transcripts for a variety of aquaporins [14], the immunoreactivities of only two aquaporins have hitherto been demonstrated in the retina [4,9,10]. Aquaporins are suggested to contribute to the fluid-absorbing function of retinal glial and pigment epithelial cells [8,13]. Normally, the subretinal space is dehydrated by the pigment epithelium while the inner retinal tissue is apparently dehydrated by transcellular water movements through M¨uller glial cells [2,8]. The transglial water transport is facilitated by aquaporin-4 water channels [4,9,10]. In addition to aquaporin-4 which is expressed predominantly in the inner retina, the retinal expression of aquaporin-1 has been described. Aquaporin-1 ∗
Corresponding author. Tel.: +49 341 9721557; fax: +49 341 9721659. E-mail address: [email protected] (A. Bringmann).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.06.046
is expressed by pigment epithelial cells [13], by a subpopulation of glycinergic amacrine cells [5], and within the outer retina [6,8]. It has been argued that the aquaporin-1 in the outer retina may be involved in the light-dependent hydration of the space surrounding the photoreceptors [6,7]. However, it is unknown which cell type expresses aquaporin-1 in the outer retina, i.e., photoreceptor cells or M¨uller glial cells. In order to solve this question, we made two attempts; (i) we immunocytochemically stained acutely dispersed cells from the mouse retina, and (ii) we investigated the expression of aquaporin-1 immunoreactivity in slices of the rds mutant mouse retina which shows a nearly complete loss of photoreceptor cells after several months [11]. Albino mice of the strain Balb/c carrying the rds (Prph2Rd2 ) mutation (5 months or 1-year old) and wildtype Balb/c animals (6 months old) were used, and treated in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and with the EC Directive 86/609/EEC. The rds mice carry a null mutation in the Prph2 gene encoding the structural protein, peripherin/rds, which is essential for the formation of photoreceptor outer segments [3]. The mice display a degeneration of
I. Iandiev et al. / Neuroscience Letters 388 (2005) 96–99
photoreceptor cells of early-onset and slow progression [11] and are widely used as a model for retinitis pigmentosa. Mice were euthanized with CO2 , and the eyes were enucleated. For immunohistochemical staining, isolated retinas were kept immersed in fixative solution containing 4% paraformaldehyde (PFA) for 2 h. After washing in phosphatebuffered saline, the tissue was embedded in saline containing 3% agarose (w/v), and 70 m-thick slices were cut by using a vibratome. Retinal sections were incubated in 5% normal goat serum plus 0.3% Triton X-100 in saline for 2 h and, subsequently, in primary antibody for 15 h at 4 ◦ C. After washing in 1% bovine serum albumin, the secondary antibodies were applied for 2 h at room temperature. Cell nuclei were counterstained using Hoechst 33258. To prepare suspensions of dissociated cells, isolated retinas were stored for 30 min in saline containing 0.4 mg/ml papain (Boehringer, Mannheim, Germany) at 37 ◦ C. After fixation with 4% PFA for 10 min and washing with saline, the tissues were triturated with a pipette until single cells were dissociated. The cells were blocked and permeabilized for 30 min with 5% normal goat serum and 0.3% Triton-X 100, followed by incubation with primary antibodies for 6 h at 4 ◦ C. Images were taken by using a confocal laser scanning microscope LSM 510 Meta (Zeiss, Oberkochen, Germany). The following antibodies were used: rabbit anti-aquaporin-1 (1:200; Chemicon, Hofheim, Germany), rabbit anti-rat aquaporin-4 (1:200; Sigma–Aldrich, Taufkirchen, Germany), mouse antivimentin (1:250; 13.2 clone, Sigma–Aldrich), and mouse anti-glutamine synthetase (1:250; Chemicon). The secondary antibodies were Cy3-conjugated goat anti-rabbit IgG (1:400; Dianova, Hamburg, Germany) and Cy2-coupled goat antimouse IgG (1:400; Dianova). The slow degeneration of rds mice retinas causes a reduction of the retinal thickness, particularly of the outer nuclear layer. As shown in Fig. 1, the thickness of the outer nuclear layer is reduced to 1–3 rows of photoreceptor nuclei in retinas of 5 months old rds mice when compared to the 7–10 rows of photoreceptor nuclei normally present in retinas of age-matched control mice. In retinas of control and rds mice, vimentin immunoreactivity is expressed by astrocytes as well as by M¨uller cell fibers predominantly in the inner retinal layers, i.e., from the nerve fiber layer up to the outer plexiform layer (Fig. 1A). In addition, the outer limiting membrane was
97
observed to be faintly stained against vimentin in some slices (Fig. 1B). In the sensory retina, aquaporin-4 is expressed by M¨uller cells [9]. In control murine retinas, the immunoreactivity for aquaporin-4 was expressed predominantly in the inner retinal layers, with most pronounced expression around blood vessels and ganglion cells in the nerve fiber/ganglion cell layers, within distinct sublayers of the inner plexiform layer, and around vessels at both sides of the inner nuclear layer (Fig. 1A). In addition, a faint staining was observed at the outer limiting membrane. There was a substantial colocalization of aquaporin-4 and vimentin proteins in the mouse retina, as indicated by the yellow color in the images of Fig. 1A (right), suggesting a glial expression of aquaporin4. This expression pattern of aquaporin-4 immunoreactivity was unchanged in retinal slices from 5 months (Fig. 1A) and 1-year old rds mice (not shown), with the exception of an apparent upregulation of expression surrounding cell nuclei in the outer half of the inner nuclear layer (Fig. 1A). In control retinas, aquaporin-1 immunoreactivity was expressed prominantly in the outer retina, i.e., from the outer plexiform layer up to the photoreceptor segments (Fig. 1B). The aquaporin-1 immunoreactivity enclosed the photoreceptor cell nuclei in the outer nuclear layer. There was no overlay of the aquaporin-1 and vimentin immunoreactivities (Fig. 1B, right). In the outer plexiform layer, both immunoreactivities were sharply segregated, with aquaporin-1 immunoreactivity expressed only at the outer border of this layer, surrounding the photoreceptor cell nuclei (inset in Fig. 1B). Along with the degeneration of photoreceptor cells in retinas of rds mice, the expression of aquaporin-1 immunoreactivity disappeared. Fig. 1B shows two examples of retinal slices from 5 months old rds mice at different stages of degeneration. In the example shown in Fig. 1B (middle row), the receptor segments are missing while a certain number of photoreceptor cell bodies is still present, showing immunoreactivity for aquaporin-1. In the example shown in Fig. 1B (bottom row), only few photoreceptor cell nuclei are present which are surrounded by aquaporin-1 immunoreactivity. Other parts of the rds retinas are fully devoid of aquaporin-1 immunoreactivity. Retinas of 1-year old rds mice showed a full absence of photoreceptor cells and aquaporin-1 immunoreactivity (not shown). The disappearance of aquaporin-1 immunoreactivity along with the photoreceptor cell degeneration in rds mice
Fig. 1. Aquaporin-1 and aquaporin-4 immunoreactivities in retinas of control and rds mice. (A) Co-immunostaining against aquaporin-4 and vimentin. Arrows indicate perivascular staining for aquaporin-4. The inset shows aquaporin-4 staining around a vessel at the border between the IPL and INL at higher magnification. (B) Immunoreactivities for aquaporin-1 and vimentin. Aquaporin-1 immunoreactivity is expressed by photoreceptor cells in the mouse retina and disappears during retinal degeneration in rds mice. The insets show the OPL (above) and single aquaporin-1-positive photoreceptor cell bodies (below) at higher magnification. Arrows indicate single photoreceptor nuclei which are surrounded by aquaporin-1 immunoreactivity. Arrowheads mark aquaporin-1 immunoreactivity expressed by red blood cells. (C) Co-immunostainings for aquaporin-4 and aquaporin-1 (red), respectively, and glutamine synthetase (green) in acutely isolated cells of retinas from a control mouse and a rds mouse. The left images show the aquaporin immunoreactivities; the right images show the overlays of aquaporin and glutamine synthetase immunoreactivities. Large arrowheads indicate the endfoot region of M¨uller glial cells; small arrowheads mark the soma of M¨uller cells. The large arrow indicates photoreceptor cells which are attached to the distal end of the M¨uller cell; small arrows mark isolated photoreceptor cell bodies and debris of photoreceptor cells. Cell nuclei were counterstained with Hoechst 33258 (blue). Scale bars, 20 and 5 m (insets), respectively. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PRS, photoreceptor segments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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I. Iandiev et al. / Neuroscience Letters 388 (2005) 96–99
I. Iandiev et al. / Neuroscience Letters 388 (2005) 96–99
retinas suggests that photoreceptor cells, but not M¨uller glial cells, express aquaporin-1. To support this assumption, we stained dissociated retinal cells for aquaporins and glutamine synthetase, as a specific marker of M¨uller glial cells. As shown in Fig. 1C, the glutamine synthetase-positive cell bodies of single M¨uller cells did not express aquaporin-1 immunoreactivity (including the stem processes, passing through the outer nuclear layer in situ) while they express strong immunoreactivity for aquaporin-4, as indicated by the yellow color in the overlays of both images. However, photoreceptor cells which are attached to the distal end of isolated M¨uller cells, as well as isolated photoreceptor cell bodies, expressed immunoreactivity for aquaporin-1 (Fig. 1C). The data suggest that in the mouse retina, aquaporin-1 is expressed by photoreceptor cells but not by M¨uller glial cells. Our data support the view that in the sensory retina, there is a striking segregation of the expression of two aquaporins. Aquaporin-4 is expressed by glial cells, predominantly in the inner retina, while aquaporin-1 is expressed, in addition to a subpopulation of glycinergic amacrine cells [5], predominantly in the outer retina, i.e., by photoreceptor and pigment epithelial cells [6,9,13]. The reason(s) for this molecular segregation of retinal aquaporin expression which includes also other molecules such as vimentin (Fig. 1B) remains to be determined. Noteworthy, this molecular segregation reflects the two fluid-absorption areas in the retina, i.e., the inner retina which is dehydrated by M¨uller cells [2,8], and the outer retina that is dehydrated by the pigment epithelium. The functional role of aquaporin-1 in photoreceptor cells is unclear. It may be assumed that this water channel is involved in the maintenance of the osmotic balance necessary for the phototransduction process. Interestingly, in contrast to aquaporin-4 which is a pure water channel, aquaporin-1 is a channel for both water and cations [17] that can be gated by cyclic GMP [1,12]. However, it remains to be proven whether aquaporin1 plays a role in the regulation of photoreceptor cell excitability.
Acknowledgements This work was supported by grants from the Interdisziplin¨ares Zentrum f¨ur Klinische Forschung (IZKF) at the Faculty of Medicine of the University of Leipzig (project C21), and from the Deutsche Forschungsgemeinschaft (BR 1249/2-1).
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