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(Investigative Ophthalmology and Visual Science. 2002;43:573-579.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Mildly Abnormal Retinal Function in Transgenic Mice without Müller Cell Aquaporin-4 Water Channels

Jiang Li1, Rajkumar V. Patil2 and A. S. Verkman1

1 From the Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California; and the 2 Departments of Ophthalmology and Visual Sciences and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. Immunocytochemistry showed strong aquaporin (AQP)-4 water channel expression in Müller cells in mouse retina and fibrous astrocytes in optic nerve. This study was designed to test the hypothesis that AQP4 is required for vision by comparing electroretinograms and retinal morphology in wild-type mice and transgenic knockout mice with no AQP4.

METHODS. Electroretinograms were recorded over a 105-fold range of flash intensities in dark-adapted mice and analyzed for a- and b-wave amplitude and latency, a-wave normalized slope, and oscillatory potential amplitude and latency. AQP4 protein was localized in mouse retina by immunocytochemistry, and retinal morphology was studied by light and electron microscopy.

RESULTS. Significantly reduced electroretinogram b-wave potentials were recorded in 10-month-old null mice with smaller changes in 1-month-old mice. Immunocytochemistry showed strong AQP4 protein expression in retina of wild-type mice. Morphologic analysis of retina by light and electron microscopy showed no differences in retinal ultrastructure.

CONCLUSIONS. Retinal function is mildly impaired in AQP4-null mice, suggesting a role for AQP4 in Müller cell fluid balance. These results support the paradigm that AQP4 expression in supportive cells in the nervous system facilitates neural signal transduction in nearby electrically excitable cells.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The aquaporins (AQPs) are a family of water-transporting channels that are expressed widely in mammalian fluid-transporting epithelia and endothelia. The eye expresses at least four AQPs: AQP1 in corneal endothelium, ciliary epithelium, and lens epithelium; AQP3 in conjunctiva; AQP4 in ciliary epithelium and retina; and AQP5 in corneal epithelium and lacrimal gland.1 2 3 4 5 6 Because of this expression pattern, it has been proposed that AQPs play a role in intraocular pressure regulation, corneal and lens transparency, and vision.

Recently our laboratory has generated transgenic mice without each of the four eye AQPs, individually and in pairs. The mice have been informative in defining the role of AQPs in extraocular functions (reviewed in Ref. 7 ). For example, the deletion of AQP1 produced defects in urinary concentrating ability,8 9 10 lung water transport,11 and dietary fat processing.12 Deletion of AQP3 resulted in nephrogenic diabetes insipidus,13 and deletion of AQP5 gave reduced saliva secretion,14 airway submucosal gland secretion,15 and alveolar epithelial water permeability.16 However, the tissue-specific expression of an AQP does not always indicate physiological significance. For example, AQP5 deletion does not affect tear secretion by lacrimal glands,17 and AQP4 deletion does not produce demonstrable abnormalities in skeletal muscle function18 or gastric acid secretion,19 despite its expression in muscle cell plasmalemma and gastric parietal cells.

Indirect evidence has suggested a role for AQP4 in retinal function. AQP4 was first localized in the eye in ciliary epithelium and in glial cells in the inner nuclear layer of the retina.6 High-resolution morphology showed AQP4 protein expression in Müller cells in the retina and fibrous astrocytes in the optic nerve.3 Studies of brain edema in AQP4-null mice implied an important role for AQP4 in fluid balance,20 supporting the possibility that AQP4 may participate in the maintenance of retinal water balance during synaptic transmission and retinal edema. During neurophysiological activity, action potentials and osmotic gradients are generated by ion fluxes from ion-solute pumps and exchangers. AQP4 has been shown to be the orthogonal array protein (OAP) by the absence of OAPs in AQP4-null mice,21 the creation of OAPs in AQP4-transfected cells,22 and label-fracture studies in brain tissue.23 Based on the colocalization of Kir4.1 potassium channels and AQP4-containing OAPs in specific membrane domains of retinal Müller cells, it was proposed that AQP4 is important in retinal signal transduction involving interactions between Müller and bipolar cells.24 25 Similar interactions occur between AQP4-expressing glial cells in the central nervous system and adjacent neurons,26 27 as well as in AQP4-expressing supportive cells (Claudius, Hensen, and inner sulcus cells) in cochlea and adjacent sensory hair cells,28 where they are proposed to play an important role in acoustic signal transduction.29

The purpose of this study was to test the hypothesis that AQP4 plays a role in retinal function. We compared electroretinograms (ERGs) in wild-type and AQP4-null mice and performed morphologic analysis of retina and optic nerve. We found significantly reduced ERG b-wave amplitude and latency in AQP4-null mice without ultrastructural abnormalities, providing the first direct evidence for a functional role of an AQP in the eye.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Transgenic Mice
Transgenic knockout mice deficient in AQP4 were generated by targeted gene disruption, as originally described.30 Measurements were performed in litter-matched wild-type and knockout mice of specified age produced by intercrossing of heterozygous mice. For all studies the investigators were blinded to genotype information until completion of the analysis. Protocols were approved by the University of California San Francisco’s Committee on Animal Research and are in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Retinal Immunocytochemistry and Morphology
For immunocytochemistry and light microscopy, mice were perfused through the aorta with 4% heparin in PBS and then with freshly prepared 4% paraformaldehyde in PBS for immunocytochemistry and 2% paraformaldehyde with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for light microscopy. Eyes were enucleated, and a small cut was made in the cornea. Eyes were bisected through the optic nerve head, using as markers the superior and inferior rectus muscles and the long posterior ciliary arteries,31 and immersed in the respective fixative solution for 2 hours at room temperature. After fixation, eyes were dehydrated and embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Miles Laboratories, Elkhart, IN), for cutting 3- to 4-µm cryostat sections, and in glycol methacrylate for cutting 3-µm plastic sections. Immunocytochemistry was performed with a polyclonal anti-AQP4 antibody raised against the AQP4 C-terminus, as described previously.6 For light microscopy, eyes were infiltrated with JB-4 monomer (Polysciences Inc., Niles, IL), embedded under vacuum at room temperature, sectioned on a microtome (Sorvall, Newtown, CT), and stained with toluidine blue. For electron microscopy, mice were perfused with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. Tissues were postfixed in PBS containing 1% osmium tetroxide for 1 hour at room temperature. Fixed tissues were dehydrated in a graded series of ethanol (30%–100%) and embedded in Epon 812. Thin (80–90 nm) sections were placed on 2 x 1-mm copper grids, stained with uranyl acetate and lead citrate, and examined by transmission electron microscopy (EM 902A; Carl Zeiss, Oberkochen, Germany). Investigators were blinded to genotype information until completion of the morphologic evaluation.

Electroretinography
As shown schematically in Figure 1A , the instrument consisted of a flashlamp with focusing and filtering optics and amplifiers (Biopac System, Inc., Goleta, CA) and recording hardware and software. Optics were constructed to control light intensity and deliver the light to the eye using a fiber-optic bundle and Lucite (DuPont, Wilmington, DE) coupler that conformed to the curvature of the globe (coupled with optical gel). As described by Lyubarsky and Pugh,32 this configuration provides uniform full-field retinal illumination. The stimulus consisted of single white-light pulses of 20-µsec duration produced by a stroboscope (NovaStrobe; Monarch Instruments, Amherst, NH). The light was collected, focused, and filtered onto a 7-mm diameter fiber-optic bundle (Edmund Scientific, Barrington, NJ). The bundle was brought into a Faraday cage and inserted into the tapered Lucite coupler, which was held by a 4-axis micromanipulator (Narashige Instruments, Greenvale, NY). ERGs were recorded using a 0.15-mm diameter platinum wire coil that was secured to the rim of the rod tip, making electrical contact with the cornea through a layer of methylcellulose solution (Gonisol; Iolab Pharmaceuticals, Claremont, CA). Reference and ground subdermal needle electrodes were placed on the forehead and right front leg, respectively. Responses were differentially amplified at a gain of 10,000, bandpassed at 0.1–1000 Hz, digitized at a sampling rate of 4000 Hz, and recorded using a workstation (model MP100; Biopac, Inc.) equipped with differential amplifier (model ERS100B) and stimulator modules (model STM100A; both from Biopac, Inc.).



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  		Figure 1. (A) Schematic of apparatus for measurement of ERGs in mice. The mouse was placed on a heating pad and light flashes were delivered using a fiber-optic bundle and a coupler. Light from a flashlamp was attenuated by neutral-density filters and focused onto the fiber-optic. (B) Analysis of ERG waveforms in a 10-month-old wild-type CD1 mouse, measured in response to a single 20-µsec flash of 1.5 x 1011 photons (top). The same ERG was filtered using a 100- to 300-Hz band-pass filter to visualize the oscillatory potentials (bottom). Some deduced parameters included a- and b-wave amplitude and latency, oscillatory potential amplitude and latency, and a-wave slope.

 
Mice were dark adapted overnight and anesthetized under dim red light, with ketamine (80 mg/kg body weight) and xylazine (16 mg/kg body weight). Their pupils were dilated and anesthetized with 2% phenylephrine and 0.5% proparacaine hydrochloride. Mice were placed on a heating pad (Braintree Scientific, Inc., Braintree, MA), and body temperature was strictly maintained at 37.0 ± 0.5°C, as measured by an indwelling rectal thermistor. The mouse, heating pad, and micromanipulator controlling the Lucite coupler were enclosed in a copper Faraday cage and a light-tight, sound-insulated black box. ERG waveforms were generally recorded in triplicate and averaged, in order of increasing light intensity. A filter wheel containing neutral density filters was used to set illumination intensity. The time between flashes was 30 seconds at low flash intensities and 60 seconds at the highest flash intensity. Control studies were performed to confirm that ERG waveforms were not affected by repeated flashes.

Data Analysis
Statistical significance in data comparing wild-type with AQP4-null mice was taken as P < 0.05, using the Student’s t-test (InStat 2.03 software; GraphPad, San Diego, CA). ERG waveforms were analyzed for a- and b-wave amplitude and latency, the leading edge of a-wave, and oscillatory potential amplitude and latency, as described in the Results section. Data are reported as mean ± SE for measurements in 10 mice in each group. Averaged amplitudes and latencies were compared between wild-type and AQP4-null mice at each light intensity.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Electroretinography
ERGs were recorded over a 105-fold range of flash intensities by monocular full-field illumination using the apparatus in Figure 1A . Figure 1B shows a representative ERG waveform elicited by a single light flash. Averaged ERG waveforms from triplicate measurements in each mouse at each flash intensity were analyzed to obtain the depicted parameters. The amplitudes of the a- and b-waves were deduced from peak-to-peak analysis. The latencies of the a- and b-waves were determined as the time between the flash stimulus and the a-wave minimum and b-wave maximum (neglecting oscillations). In addition, normalized a-wave downward deflections were compared (described later). The amplitude and latency of oscillations were measured after applying a 100- to 300-Hz band-pass filter (bottom curve) to suppress the broad a- and b-waves.

Figure 2 shows representative series of ERG recordings from 1-month-old (Fig. 2A) and 10-month-old (Fig. 2B) wild-type and AQP4-null mice. The timing and waveforms are similar to those reported previously.32 33 34 The amplitudes of the waveforms increased with increasing flash intensity, and the a-wave (generated by photoreceptors) and the oscillations became more prominent. The oscillatory potentials have been proposed to originate in the vicinity of the inner plexiform layer of retina.35 The b-wave, produced by bipolar cell depolarization, possibly accompanying Müller cell activation,36 37 38 39 40 also becomes more prominent with increasing flash intensity. ERGs were very reproducible in the same mice on different days, and, in general, the individual waveforms from triplicate determinations were superimposable. It was noted qualitatively that b-wave amplitudes were reduced with age and AQP4 deletion.



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  		Figure 2. Representative ERG waveforms measured in (A) 1-month-old and (B) 10-month-old litter-matched wild-type and AQP4-null mice in a CD1 genetic background. Each waveform is the average of triplicate determinations. Relative flash intensities are indicated with a value of 1.0 corresponding to 1.5 x 1011 photons per flash delivered to the left eye by full-field illumination.

 
The results of ERG waveform analysis for a series of young and older mice are summarized in Figure 3 . Each point is the mean and SE of measurements in 10 mice, where statistical significance (P < 0.05) for measurements at individual flash intensities is denoted by asterisks. There was little effect of AQP4 deletion in the young mice (Fig. 3A) . Although differences in the seven parameters in most cases were not statistically significant for any individual flash intensity, paired analysis showed reductions (P < 0.05) in b-wave and oscillatory potential amplitudes in the null mice. In the older mice (Fig. 3B) , there were significant reductions in b-wave amplitudes and latencies at the highest flash intensities, as well as in the amplitude of the third oscillatory potential at the highest flash intensity.



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  		Figure 3. Summary of ERG waveform analysis in (A) 1-month-old and (B) 10-month-old wild-type and AQP4-null CD1 mice. Each point is the mean ± SE of data for 10 mice in each group. *Significant difference (P < 0.05).

 
The leading edge of the a-wave in response to the highest flash intensity was further analyzed based on methods reported by Hood and Birch41 42 43 and Ren et al.44 Figure 4A shows expanded a-waves from a series of wild-type (solid curves) and AQP4-null (dashed curves) mice after amplitude normalization. There was substantial overlap in the a-waves’ downward deflections. Figure 4B summarizes normalized initial slopes and latencies, showing only minor effects of AQP4 deletion. Thus AQP4 deletion has little effect on the a-wave’s downward slope—the major parameter of rod phototransduction.



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  		Figure 4. Analysis of a-waves at highest flash intensities for wild-type and AQP4-null mice. (A) Comparison of the leading edge of the dark-adapted ERG a-wave in six wild-type and six AQP4-null mice at the ages of 1 month (top) and 10 months (bottom). The response of each mouse has been normalized to the amplitude of the a-wave. (B) Summary of normalized slope (left) and time to minimum (right) in ERG a-waves shown in (A). Data are the mean ± SE of six mice in each group. *Significant difference (P < 0.05).

 
Immunocytochemistry and Tissue Morphology
Immunocytochemistry was performed to confirm AQP4 protein expression in mouse retina and optic nerve. Figure 5 (top and middle left) shows AQP4 immunolabeling in wild-type mice extending from the ganglion cell layer (GCL) to the outer plexiform layer (OPL). There was no labeling of the retinal pigment epithelium (RPE). The optic nerve head (ONH) showed weak labeling compared with retina and optic nerve (bottom, left and middle). These findings are in agreement with reported AQP4 localization in rat and human eye.3 4 6 There was no AQP4 immunoreactivity in eye tissues from AQP4-null mice (middle and bottom, right).



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  		Figure 5. Immunolocalization of AQP4 protein in mouse retina and optic nerve. Section of retina viewed by bright-field (top) and fluorescence (middle) microscopy showing AQP4 immunoreactivity extending from GCL to OPL and concentrated around vessels. Specific immunostaining not seen in retina from AQP4-null mouse (middle right). Bottom: Bright-field (left) and immunofluorescence (middle) of optic nerve from wild-type mouse, with immunofluorescence of AQP4-null mouse shown on the right. PhL, photoreceptor cell layer; RE, retina; OH, optic nerve. Scale bar, 50 µm.

 
Light and electron microscopy of retina were performed to determine whether structural differences could account for the differences in ERGs. Figure 6 shows light microscopy of the central part of retina of three wild-type (top) and three AQP4-null (bottom) mice at the ages of 1 month and 10 months. The cellular morphology and depths of the retinal layers where AQP4 is expressed (GCL, inner plexiform layer [IPL], outer nuclear layer [ONL], and OPL; see legend to Fig. 6 ) did not differ qualitatively between wild-type and AQP4-null mice. Also, there were no differences in morphology of the optic nerve head and body (not shown). Table 1 summarizes the thicknesses of retinal layers in three different regions of the retina of three wild-type and three AQP4-null mice at age of 10 months (0.48 and 1.25 mm from the edge of the optic nerve and 0.45 mm from the peripheral edge of the retina). No significant differences were found, except for small decreases in the thicknesses of the IPL and OPL at 1.25 mm, and the inner nuclear layer (INL) at 0.45 mm.



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  		Figure 6. Morphologic analysis of mouse retina by light microscopy. Stained thin, plastic-embedded sections of the central portion of retinas from two wild-type (top) and two AQP4-null (bottom) mice at the ages of 1 month (A) and 10 months (B). Ch, choroid; PhL, photoreceptor cell layer. Scale bar, 10 µm.

 

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  		Table 1. Average Thickness of Retinal Layers in Wild-Type and AQP4 Null Mice at the Age of 10 Months

 
The ultrastructure of retina and ONH of wild-type and AQP4-null mice were compared by electron microscopy. Eyes from five wild-type and seven AQP4-null mice were examined. Representative electron micrographs are shown in Figure 7 . Photoreceptor cells appeared normal in wild-type and AQP4-null mice (Fig. 7A , left and right). The ONL (Fig. 7B) , INL (Fig. 7C) , and GCL (Fig. 7D) also appeared normal and did not differ between wild-type and AQP4-null mice. Müller cells seen in the INL (confirmed by vimentin staining) were normal in wild-type and AQP4-null mice (Fig. 7C) . In the ONH, the axonal profile was preserved with normal mitochondria in wild-type and AQP4-null mice (Fig. 7E) . Together, these results suggest that structural differences do not account for the ERG change in AQP4-null mice.



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  		Figure 7. Electron microscopy of retina and optic nerve of (left) wild-type mice (right) AQP4-null mice. Electron micrographs of outer segments (A), ONL (B), INL (C), GCL (D), and ONH (E). OS, outer segments; Mu, Müller cell; n, nucleus; m, mitochondria. (E, arrowheads) Axonal profile. Scale bar, 1 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The rationale for this study was the strong expression of AQP4 in retinal Müller cells and its proposed functional association with a K+ channel.25 It was postulated that Müller cell–bipolar cell interactions might be similar to astroglia–neuronal interactions in the central nervous system and supportive cell–hair cell interactions in the cochlea. In the central nervous system, AQP4 in astroglial cells appears to be important in brain water balance and neural excitability.20 In the ear, AQP4 in supportive Hensen, Claudius, and inner sulcus cells is important for acoustic signal transduction by sensory hair cells.29 Although the detailed mechanisms of these cell–cell interactions remain to be established, local osmotic gradients created during neural transmission may be dissipated by the highly water-permeable Müller cells in retina, astroglial cells in brain, and supportive cells in cochlea. In addition, local coupling (siphoning)25 of water and K+ movement may occur near AQP4-containing cell membranes. Because inhibitors of AQP4 are not yet available, we tested the hypothesis that AQP4 plays a functional role in retina by comparison of ERG waveforms in wild-type and transgenic AQP4-null mice. The principal finding was a modest but significant reduction in ERG b-wave amplitude and latency in the AQP4-null mice, without demonstrable abnormalities in retinal or optic nerve morphology.

The principal abnormality in ERG waveforms in AQP4-null mice was in b-wave amplitude and latency. Current source–density analysis has suggested that the b-wave source is in IPL and sink in the OPL,45 where AQP4 is expressed. The b-wave has been thought to be created by bipolar cell depolarization, in close association with K+ spatial buffering in Müller cells39 40 ; however, the relationship between b-wave generation and Müller cell activity has been questioned by several groups.46 47 48 49 50 Using 2-amino-4-phosphonobutyrate to selectively eliminate the ON bipolar response in the retina, a strong correlation between the b-wave and ON bipolar waveforms supported the possibility that the ERG b-wave is the direct result of ON bipolar activity. Our results showing abnormal b-waves in AQP4-null mice are most easily explained by involvement of Müller cells, where AQP4 is expressed, in b-wave generation. Low Müller cell water permeability in AQP4-null mice may alter the light-induced changes in retinal hydration and [K+]. The AQP4-dependent enhancement of K+ siphoning25 may further contribute to the differences in ERG waveforms in the AQP4-null mice. Measurement of K+ currents and water transport across Müller cells in intact retina are needed for further mechanistic evaluation and to understand the basis of the relatively mild changes in ERGs in AQP4-null mice.

In summary, the current results provide the first functional evidence for a role of an AQP in eye physiology. AQP4 may also be involved in retinal water balance, as it may in cerebral and inner ear water balance. Experimental animal models of retinal edema, such as that after laser-induced venous thrombosis51 and retinal vein occlusion52 might be informative in this regard. It remains to be determined whether AQP4 plays a role in human retinal diseases associated with retinal edema, such as retinal vein thrombosis, cystoid macular edema, and central serous chorioretinopathy.53


   Acknowledgements
 
The authors thank Liman Qian for transgenic mouse breeding and genotype analysis, Belinda McMahan and Jean Jones for technical assistance in light and electron microscopy, Matthew M. LaVail (University of California San Francisco) for help in the interpretation of light microscopy, and Gülgün Tezel and Martin B. Wax (Washington University, St. Louis) for help in the interpretation of electron microscopy.


   Footnotes
 
Supported by National Institutes of Health Grants DK35124, HL59198, HL60288, and DK43840 and Grant R613 from the Cystic Fibrosis Foundation (ASV); National Eye Institute Grant EY12506 (RVP); and funds from Research to Prevent Blindness (RVP).

Submitted for publication June 20, 2001; revised October 2, 2001; accepted November 5, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: A. S. Verkman, Cardiovascular Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, San Francisco, CA 94143-0521;


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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