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Am J Physiol Cell Physiol 295: C451-C457, 2008. First published June 4, 2008; doi:10.1152/ajpcell.00124.2008
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Altered visual function in monocarboxylate transporter 3 (Slc16a8) knockout mice

Lauren L. Daniele,1 Brian Sauer,2 Shannon M. Gallagher,2 Edward N. Pugh, Jr,1 and Nancy J. Philp2

1F. M. Kirby Center for Molecular Ophthalmology, Department of Ophthalmology, School of Medicine, University of Pennsylvania; and 2Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 26 February 2008 ; accepted in final form 2 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To meet the high-energy demands of photoreceptor cells, the outer retina metabolizes glucose through glycolytic and oxidative pathways, resulting in large-scale production of lactate and CO2. Mct3, a proton-coupled monocarboxylate transporter, is critically positioned to facilitate transport of lactate and H+ out of the retina and could therefore play a role in pH and ion homeostasis of the outer retina. Mct3 is preferentially expressed in the basolateral membrane of the retinal pigment epithelium and forms a heteromeric complex with the accessory protein CD147. To examine the physiological role of Mct3 in the retina, we generated mice with a targeted deletion in Mct3 (slc16A8). The overall retinal histology of 4- to 36-wk-old Mct3–/– mice appeared normal. In the absence of Mct3, expression of CD147 was lost from the basolateral but not apical RPE. The saturated a-wave amplitude (amax) of the scotopic electroretinogram (ERG) was reduced by approximately twofold in Mct3–/– mice relative to wild-type mice. A fourfold increase in lactate in the retina suggested a decrease in outer-retinal pH. In single-cell recordings from superfused retinal slices, saturating amplitudes of single rod photocurrents (Jmax) were comparable indicating that Mct3–/– mouse photoreceptor cells were inherently healthy. Based on these data, we hypothesize that disruption of Mct3 leads to a potentially reversible decrease in subretinal space pH, thereby reducing the magnitude of the light suppressible photoreceptor current.

monocarboxylate transporter 3; lactate transport; retinal pigment epithleium; pH regulation; photoreceptor; electroretinogram

THE RETINAL PIGMENT EPITHELIUM (RPE) forms the outer-blood retinal barrier and performs many functions essential for maintaining the health and functional activity of photoreceptors. Interposed between the choroidal vessels and the photoreceptor cells, the RPE regulates the transport of glucose to the avascular outer retina. Eighty percent of the glucose transported into the outer retina is metabolized via glycolysis producing large amounts of lactate both in the light and the dark (41, 42, 44). Lactate produced by glycolysis in Müller glia is then used to fuel oxidative phosphorylation in photoreceptor cells (35). The transfer of lactate from glia to neurons is facilitated by proton-coupled monocarboxylate transporters and has been referred to as the "lactate shuttle" (27). Inhibition of monocarboxylate transport or glycolysis results in attenuation of light-stimulated responses of photoreceptor cells, demonstrating the importance of this metabolic pathway in the retina (8, 9).

The supposed pathway for removal of excess lactate out of the SRS is across the RPE where it can be cleared from the eye by the choroidal circulation. In vivo and in vitro studies have shown that there is a net efflux of lactate from the SRS to the choroidal circulation thus indicating a role for the RPE in regulating lactate levels in the outer retina (1, 24, 42). Lactate is transported across cell membranes by monocarboxylate transporters (Mcts), which are members of the slc16a family of transporters. Mcts, like other solute transporters, have 12 predicted transmembrane domains with the NH2- and COOH-terminals being cytoplasmic. There are at least 14 members in this family, but only Mct1–4 have been shown to transport lactate (14a).

The RPE expresses two Mcts, which could facilitate the transepithelial transport of lactate, H+, and H2O from the retina to the choroid; Mct1 in the apical and Mct3 in the basolateral membrane (34). Whereas Mct1 (slc16A1) is expressed in many tissues, Mct3 (slc16A8) has only been detected in RPE, where it is highly expressed, and choroid plexus epithelium, where it is much less abundant (34). Mct1, Mct3, and Mct4 form heteromeric complexes with CD147, an immunoglobulin superfamily protein (12, 20). The complex is assembled in the endoplasmic reticulum and in the absence of one subunit, the other is targeted for degradation (12, 14). Mice with a targeted deletion of the gene encoding CD147 (Basigin;Bsg) have abnormal electroretinographic (ERG) responses that reflect functional deficits in photoreceptor cells. We found that in these mice there is a loss of expression of the Mcts in the RPE and retina due to a trafficking defect. These findings support the hypothesis that Mcts play an essential role in maintenance of metabolic and ionic homeostasis of the outer retina.

Mct3 is in a pivotal position to control the transport of lactate out of the retina. Changes in expression or distribution of Mct3 would be predicted to affect the pH and ionic composition and lead to suppression of photoreceptor cell function. In the present study, the functional role of Mct3 was examined by disruption of the Mct3 gene in mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Experiments were performed in compliance with National Institutes of Health guidelines, as approved by the Institutional Animal Care and Use Committees of Thomas Jefferson University and the University of Pennsylvania.

Targeted disruption of Mct3. Murine embryonic stem (ES) cells were electroporated with the Mct3 knockout vector (supplemental fig. 1) and selected for resistance to G418. Recombination of the targeting vector ablates the Mct3 gene by deleting 2.2 kb of DNA spanning the entire coding portion of exons 2-4. Southern blot analysis of both the 5' and 3' junction fragments of the Mct3 gene was used to detect positive ES cell clones. Correctly targeted ES cells (line IG3) were injected into C57Bl/6J blastocysts and resulting male chimeric pups were backcrossed with C57Bl/6J for >5 generations. Heterozygous progeny from the C57Bl/6J matings were intercrossed to establish homozygous Mct3–/–. For PCR genotyping of pups the disrupted Mct3 gene was detected using neospecific primers and the wild-type gene was detected using primers lying within the deleted region of Mct3.

Chemicals and antibodies. Antibodies specific for Mct1, Mct3, and Mct4 were produced for us and characterized in our lab (32). Anti-Glut1 antibody was purchased from Sigma; anti-ZO-1 and Alexa-Fluor conjugate of phalloidin, avidin, and secondary antibodies were obtained from Molecular Probes (Eugene, OR). Horseradish peroxidase-conjugated secondary antibodies were obtained from Bio-Rad Laboratories (Hercules, CA) and Molecular Probes.

Histology. Mouse eyes were enucleated and fixed in 4% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.4, with 8.0 mM CaCl2 for 2 h on ice and postfixed with 1% OsO4 for 1 h. After dehydration in a graded ethanol series followed by propylene oxide, the eyes were infiltrated and embedded in Epon (EM Sciences, Fort Washington, PA). Thick sections (1 µm) were cut and stained with methylene blue-azure blue for light microscopic examination. Thin sections were cut using a Reichert UCT ultramicrotome and poststained with aqueous uranyl acetate followed by phosphotungstic acid or lead citrate. Sections were examined at 80 kV using a Tecnai12 transmission electron microscope equipped with a Gatan Ultrascan US1000 2 K digital camera.

Immunohistochemical analysis. Frozen sections of paraformaldehyde-fixed mouse eyes were prepared for immunohistochemical analysis and confocal imaging as previously described (11). Immunofluorescence labeling of tissue was visualized with a Zeiss LSM 510 confocal microscope (Zeiss Microscope Imaging, Thornwood, NY). Images were exported in "tagged image file" format using LSM Image Browser software (version 3, 5, 0, 376, copyright Carl Zeiss Jena 1997–2005; Zeiss Microscope Imaging, Thornwood, NY). Adjustments were made to brightness and contrast only.

Tissue lysates and Western blot analysis. Eyes were enucleated from wild-type and Mct3–/– mice, and RPE-choroid and retinal lysates were prepared for immunoblot analysis as previously described (11, 34).

Lactate assay. Extracellular lactate was measured in wild-type and Mct3–/– mouse retinas using a lactic acid dehydrogenase based kit (Trinity Biotech, St. Louis, MO) as previously described (13). Briefly, dark-adapted mice were euthanized in the light by lethal injection and the eyes enucleated. The anterior portion of the eye was discarded, and the retina was removed from the posterior eye cup with a pair of fine forceps. Retinas were then placed in a microfuge tube containing 50 µl of PBS. The samples were centrifuged at 14,000 g for 1 min, and the supernatant was removed for lactate assay. The retinas were then solubilized in 100 µl of lysis buffer [25 mM HEPES buffer, pH 7.4, 150 mM NaCl, 5 mM MgCl2, and protease inhibitors (Complete Mini, Roche, Indianapolis, IN) containing 1% Triton X-100], and the protein concentration of the detergent soluble lysate was determined. The lactate concentration in the retinal wash was normalized to detergent soluble protein for each sample.

Electroretinogram recording. Electroretinograms (ERGs) were recorded as previously described (25, 26). Briefly, dark-adapted mice were anesthetized with an intraperitoneal injection (20 µl/g mouse) of ketamine (1.0 mg/ml), xylazine (0.4 mg/ml), and urethane (40 mg/ml). Mice were then placed on a platform (heated to 37°C) and stabilized with an aluminum bite bar serving as reference electrode. The pupils were dilated with tropicamide (1% solution; Alcon Laboratories, Fort Worth, TX). ERG stimuli consisted of brief monochromatic flashes (~1 ms) from Xenon flash lamps presented through calibrated filters into a multiport customized ganzfeld. An unfiltered flash was used to elicit a-wave responses of saturating magnitude. Responses to light flashes were measured with corneal electrodes that made contact through saline. Light calibrations were performed as described previously (26).

Single cell recordings. Mice were euthanized by CO2 inhalation, eyes enucleated, and retinas removed from eyecups under infra-red illumination. Anterior segments of eyes were removed with fine surgical scissors, and the retina was chopped into fine pieces in a drop of Locke's solution (28). Retina pieces were transferred to a heated chamber and were perfused continuously with bicarbonate-buffered Locke's solution (pH 7.4). Rod outer segments were drawn into a suction electrode and responses to light were amplified and collected. Light stimulation and data acquisition were performed as described previously (28).

Sampling of single-rod circulating current magnitudes. To compare the photoreceptor function in Mct3–/– and wild-type mice, it was important to randomly sample the circulating current magnitudes without bias. To do this, rod photoreceptors were selected using a single criterion, the presence of straight outer segment morphology. Occasionally rods from wild-type (1 of 27 attempts) and Mct3–/– mice (1 of 18) had little or no responses to light, likely due to shearing from the pipette. These were omitted from analysis. Typically light responses were recorded from rods of Mct3–/– and wild-type mice in the same session with the same electrode.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mct3 (Slc16A8)-deficient mice. To determine whether Mct3 plays a role in maintaining normal visual function, we generated a mouse model with a targeted disruption of Mct3 (supplemental Fig. 1). The targeting strategy for generation of the Mct3–/– mice resulted in the deletion of exons 2-4, which encode the first 11 predicted transmembrane domains (supplemental Fig. 1, A and B). Southern blot analysis confirmed the homologous recombination (supplemental Fig. 1C). Heterozygous mating of mice produced the expected Mendelian ratio of wild-type, hemi-, and homozygous animals. Slc16A8+/– (Mct3+/–) and Slc16A8–/– (Mct3–/–) mice could not be distinguished from wild-type based on size and overall appearance. This was the expected phenotype since Mct3 mRNA and protein are preferentially expressed in the RPE.

Mct3 expression is undetectable in the RPE of Mct3–/– mice. RT-PCR (Fig. 1A) and Western blot analysis (Fig. 1B) showed a total absence of Mct3 expression in the RPE from Mct3–/– mice. Reduction in levels of Mct3 mRNA and protein was observed in heterozygous mice, indicating a gene dosage effect (Fig. 1, A and B). Cryosections of eyes from 3-mo-old wild-type and Mct3–/– mice were colabeled with anti-Mct3 and anti-CD147 antibodies. Mct3 was detected in the basolateral membrane of RPE of wild-type mice but was not detected in sections from Mct3–/– mice (Fig. 1C). CD147 antibody labeled both the apical and basolateral membranes of the RPE of wild-type mice but was only detected in the apical membrane of RPE of Mct3–/– mice. This finding shows that in the absence of Mct3, the accessory protein CD147 is not trafficked to the basolateral membrane.


Figure 1
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  		Fig. 1. Expression of Mct3 mRNA and protein in Mct3+/+, Mct+/–, and Mct3–/– mice. A: images of ethidium bromide-stained gels showing results of RT-PCR analysis applied to total RNA of retinal pigment epithelium (RPE)-choroid with Mct3- and Mct1-specific primers. B: Western blot analysis of protein lysates of RPE-choroid probed with antibodies to Mct3 and β-actin as a loading control. Equal amounts of protein (10 µg) were loaded in each lane. C: cryosections of fixed wild-type (top) and Mct3–/– (bottom) eyes from 3- to 4-mo-old mice labeled with anti-Mct3 and anti-CD147 antibodies. The differential interference contrast (DIC) image shows the basal surface of the RPE is adjacent to Bruch's membrane (BM), and the apical surface is in intimate contact with the outer segments (OS) of the photoreceptor cells. Arrowheads point to the basal membrane of the RPE. Scale bar = 10 µm.

 
Scotopic ERG responses are reduced in Mct3–/– mice. Visual function in Mct3–/– mouse retinas was assessed with flash ERGs performed at 4, 9, and 40 wk. A set of averaged responses recorded from dark-adapted wild-type and Mct3–/– mice to brief flashes of light of the indicated intensity are shown (Fig. 2A). As described elsewhere (36a), the initial negative deflection in each trace (a-wave) arises from light suppression of photoreceptor current, whereas the subsequent positive deflection (b-wave) arises from the light-stimulated depolarization of on type-bipolar cells. The magnitudes of photoreceptor-derived a-wave and bipolar cell-derived b-wave were reduced by ~50% in Mct3–/–. The ERG records from mice at 9 wk of age were representative of all ages tested (9 to 40 wk).


Figure 2
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  		Fig. 2. Morphology and function of Mct3–/– mouse retinas. A: scotopic electroretinogram (ERG) responses of wild-type (WT, left) and Mct3–/– mice (right) to 500-nm flashes of intensities estimated to produce the indicated photoisomerizations ({Phi}) in WT rods. B: images of methylene blue-azure blue-stained 1-µm thick sections of epon-embedded eyes from 9-mo-old WT and Mct3–/– mice. C: electron micrograph of a thin section from the same eye as in B showing the lamella of RPE cell processes (arrowheads) contacting rod outer segments (ROS). Several dark pigment granules can be seen inside the RPE cell. Scale bars: 20 µm (B), 1 µm (C).

 
Histology of the RPE and retina in Mct3–/– mice. To assess whether the reduced amplitudes of the light-evoked scotopic a-waves were due to a loss of photoreceptor cells or disruption of the outer-blood retinal barrier, plastic sections of eyes from Mct3–/– and wild-type mice were examined. There was no evidence that the reduced ERG responses reflected retinal cell degeneration in the Mct3–/– mice. The overall histology and lamination of the retinas remained normal at all ages. Sections from 9-mo-old Mct3–/– mice showed that the RPE monolayer was intact and there was close apposition between the RPE and photoreceptors (Fig. 2, B and C). The length and diameter of photoreceptor inner and outer segments were indistinguishable from that of wild-type (Fig. 2B). Electron micrographs of thin sections from the same Mct3–/– mouse eye confirm the integrity of the apical processes of RPE and photoreceptor outer segments (Fig. 2C).

Normal polarization and tight-junction integrity is retained in Mct3–/– mouse RPE. To determine whether expression or distribution of other Mcts was altered in Mct3–/– mice, cryosections of eyes were immunolabeled with anti-Mct1, Mct4, and Glut1 antibodies. Glut1 was detected in the apical and basolateral membranes of the RPE in Mct3–/– mice as is observed in wild-type mice (Fig. 3A) (32). Mct1 remained polarized to the apical membrane and was not redirected to the basolateral membrane (Fig. 3B). Mct4, an isoform expressed in nascent RPE cells and upregulated in cultured human RPE, was not detected in the RPE of wild-type or Mct3–/– mouse (data not shown) (6, 33).


Figure 3
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  		Fig. 3. Increased levels Mct1 and lactate in Mct3–/– mouse retina. Immunofluorescence localization of Glut1 (A), Mct1 (B), and zonula occluden-1 (ZO-1) (C) in cryosections of 6-mo-old mouse Mct3–/– mouse eyes. Each panel includes a partial merge of immunofluorescence with DIC views of the section. Scale bar = 5 µm. Arrows indicate the basolateral membrane, arrowheads indicate tight junctions. D: Western blot analysis of Mct1 expression in RPE lysates prepared from WT and Mct3–/– mice. Equal amounts of total protein were loaded in each lane. E: extracellular lactate levels in retinas isolated from WT and Mct3–/– mice. Data are means ± SE of individual retinas. *P <= 0.05, Student's t-test.

 
Tight junctions of the RPE form the outer blood-retinal barrier (BRB) (37). Despite normal RPE morphology, deletion of Mct3 could lead to disrupted RPE tight junctions. To determine whether RPE tight junctions were intact in the Mct3–/– mouse, sections of eyes from 12-mo-old mice were labeled with an antibody to zonulae occludens-1 (ZO-1), a scaffolding protein found at tight junctions. ZO-1 was detected at the apical tight junctions of RPE cells in sections from wild-type and Mct3–/– mice at 3 mo, 6 mo, and 1 year of age, suggesting that the outer BRB was intact (Fig. 3C and data not shown).

Increased retinal lactate and Mct1 expression in Mct3–/– mice. If Mct3 is a key transporter of lactate across the basolateral RPE, deletion of Mct3 would be expected to lead to increased SRS lactate. Increased extracellular lactate was reported to upregulate expression of Mct1 in muscle cells (16), therefore, we examined the levels of Mct1 in RPE lysates prepared from eyes of 3- to 4-mo-old wild-type and Mct3–/– mice. Immunoblot analysis of RPE lysates showed an increase in Mct1 in the RPE of Mct3–/– mice (Fig. 3D). Consistent with the immunofluorescence data, Mct4 was not detected in RPE lysates by immunoblot analysis. The lactate concentration was measured in isotonic washes of retinas isolated from wild-type and Mct3–/– mice. As shown in Fig. 3E, the amount of lactate recovered in retinal washes from Mct3–/– mice was approximately four times higher than that from wild-type retinas.

Light responses of Mct3–/– mouse rods were normal in superfused retinal slices. Despite the marked effect of Mct3 deletion on the response to light measured in vivo, there was little or no change in cell morphology. The absence of Mct3 could result in altered SRS ionic composition, affecting light stimulated activity of photoreceptor cells without altering their intrinsic health. The accumulation of lactate in the Mct3–/– retina (Fig. 3E) supports this interpretation. To further address the basis of the diminished ERG response in the Mct3–/– mouse retina, suction electrode recordings were made from rod photoreceptors in isolated retinal slices superfused with standard bicarbonate-buffered physiological solution. The responses of single-rod photoreceptors of Mct3–/– mice were indistinguishable from those obtained from wild-type mice. The average maximal light suppressible circulating current Jmax = 12.6 pA ± 0.6 (Mct3–/–, n = 17; means ± SE) and 13.1 pA ± 0.7 (wild-type, n = 26) (shaded bars; Fig. 4C). The values for Jmax are not significantly different. The responses recorded from superfused rods contrast with the scotopic ERG responses (Fig. 4C) where the mean rod amax was 187.4 ± 14 µV (Mct3–/–, n = 5) and 362 ± 20 µV (wild-type, n = 4).


Figure 4
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  		Fig. 4. Light responses of single Mct3–/– mouse rods in perfused retinal slices. Light response families of a representative WT (A) and Mct3–/– rod (B) elicited by 500-nm light flashes ranging in intensity from 6 to 2,400 photons/µm2. C: magnitude of the maximal a-wave amplitudes (amax, open bars) and light-suppressible currents (Jmax, shaded bars) for Mct3–/– and WT mice over a population. Mean amax = 187.4 ± 14 µV (Mct3–/–, n = 5) and 362 ± 20 µV (wt, n = 4). Mean Jmax = 12.6 pA ± 0.6 (Mct3–/–, n = 17) and 13.1 pA ± 0.7 (wt, n = 26). Error bars represent means ± SE. *Significantly different values for amax (P < 0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study show that disruption of the Mct3 gene reduced visual function as a consequence of changes in the microenvironment of the outer retina and not from intrinsic changes in photoreceptor cells. Despite reduced photocurrents in vivo as revealed in the amplitudes (amax) of the scotopic ERG a-wave, rods of Mct3–/– mice had normal amplitude photocurrents (Jmax) when recorded in isolated, superfused retinal slices (Fig. 4). The latter evidence, along with the normal ultrastructure, indicates that photoreceptor cells of Mct3–/– mice are intrinsically healthy and suggests that Mct3 plays an essential role in regulating the ionic composition of the outer retina.

Previous studies from our lab identified Mct3 as a lactate transporter that is preferentially expressed at high levels in the basolateral membrane of the RPE (30, 45). Like Mct1 and Mct4, Mct3 is a heteromeric transporter and forms a stable complex with the accessory protein CD147. CD147 was originally identified by Schlosshauer and Herzog (38) as neurothelin, a blood-retinal barrier antigen. At that time, they proposed a transport function for CD147 based on their observation that the expression of this protein in the retina increased during embryogenesis and postnatal development. This was well before CD147 was identified as an accessory protein for lactate transporters. Our findings in Mct3–/– mice show Schlosshauer and Herzog's instincts were correct since, in the absence of Mct3, CD147 is not detected in the basolateral membrane of the RPE (Fig. 1C). This shows for the first time in vivo that trafficking of CD147 to the plasma membrane is regulated through its association with Mcts.

The level of expression of both Mct1 and Mct3 in the RPE is increased during postnatal development and is correlated with the structural and functional differentiation of the neural retina. It has been demonstrated that in the dark there is a net flux of lactate from retina to choroid (42). The finding that Mct1 is expressed in the apical membrane of the RPE and Mct3 is expressed basolaterally suggested that these two proton-coupled symporters could regulate the transepithelial transport of lactate from the SRS to the choroidal circulation (15, 19, 21). Deletion of Mct3 would be predicted to increase lactate and decrease pH in the SRS. Consistent with this hypothesis we found that lactate levels were increased in the retina. Whereas methods are not currently available for measuring pH in the retina, an increase in lactate would be expected to decrease pH, since lactate transport is coordinated with transport of H+ and H2O.

A decrease in SRS pH could account for the functional deficits in the ERGs of Mct3–/– mice. It has been well established in the literature that changes in extracellular pH can influence the light responses of photoreceptors. Reductions in the pH of the superfusate bathing isolated retinas leads to suppression of the photoreceptor dark current (23). The accepted explanation of this suppression is that extracellular protons reversibly lower the turnover number of the photoreceptor Na+/Ca2+, K+ exchanger, inhibiting Ca2+ extrusion that normally balances the influx of Ca2+ ions in the dark (36). The higher [Ca2+]i would lead to partial closure of cGMP-activated channels and reduction of the dark current (36). Decreased pH also affects the gating of photoreceptor presynaptic voltage-gated Ca2+ channels (5) and is likely to have effects on other ion channels in the photoreceptor as well.

Tight regulation of retinal pH has been shown to be essential for maintaining normal visual function (18, 23). pH homeostasis in the outer retina is maintained by the coordinated activities of electrogenic Na+/HCO3– cotransporters, Na+/H+ exchangers, Na+/HCO3– exchangers, and carbonic anhydrases (CA). Mutations in genes that contribute to acid-base regulation in the retina are known to cause ocular phenotypes with varying degrees of severity (2, 3, 7, 17, 29). CA XIV is an integral membrane protein with an extracellular catalytic domain that is expressed in the apical processes of both Müller and RPE cells. Like other members of this family, it is a zinc-binding enzyme that catalyzes the hydration of carbon dioxide, which is important for pH homeostasis and lactate transport out of the retina. The ocular phenotype of the CA XIV–/– mouse is very similar to the phenotype of Mct3–/– mouse reported in the current study. In CA XIV–/– mice, there was a nonprogressive reduction in the scotopic a-wave (35%) and b-wave (26%) of the ERG and normal retinal morphology (29). As with Mct3–/– mice, CA XIV–/– mice would be expected to have decreased SRS pH due to a reduction in SRS buffering capacity. These two mutations in Müller cell and/or RPE specific genes are unique in that they have a greater impact on photoreceptor cell function than viability.

Our results reveal a limitation in the use of ERG as the sole functional assay for assessing the health of photoreceptor cells in the investigation of eye disease. The early visual impairment in Mct3–/– mice resulted from the altered SRS microenvironment and not from cell degeneration or irreversible changes in cell function. Mct3 belongs to a class of proteins that regulate metabolic and pH homeostasis in the outer retina. Mutations in genes of this class result in early loss of retinal function and delayed onset retinal degeneration similar to what is observed in Mct3–/– mice (29, 40). In animal models of retinal disease where photoreceptors appear structurally sound, single-cell recording in isolated tissue can help to clarify the nature of a functional deficit revealed in the ERG. Patients with visual deficits in the absence of structural changes may have mutations in genes that alter the microenvironment that eventually cause late onset degeneration. The development of noninvasive optical methods for imaging the retina should help to identify those patients with visual deficits whose retinal structure and lamination is intact, and who would likely benefit from early therapeutic intervention (10, 22, 39).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants EY-012042 (to N. J. Philp) and EY-02660 (to E. N. Pugh).


   ACKNOWLEDGMENTS
 
The authors thank Evelyn F. Grollman for helpful discussions and careful reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. J. Philp, Dept. of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson Univ., 1020 Locust St., Philadelphia, PA 19107 (e-mail: nancy.philp{at}jefferson.edu)

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


   REFERENCES
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 GRANTS
 REFERENCES
 
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