|Molecular Vision 2005;
Received 20 January 2005 | Accepted 29 July 2005 | Published 6 September 2005
Early markers of retinal degeneration in rd/rd mice
Monica L. Acosta,1 Erica L. Fletcher,2 Serap
Azizoglu,3 Lisa E. Foster,2 Debora B. Farber,4
1Department of Optometry and Vision Science, University of Auckland, Auckland, New Zealand; Departments of 2Anatomy and Cell Biology and 3Optometry and Vision Sciences, The University of Melbourne, Victoria, Australia; 4Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA
Correspondence to: Professor Michael Kalloniatis, Department of Optometry and Vision Science, University of Auckland, Private Bag 92019, Auckland, New Zealand; Phone: +64 9 373 7599, ext. 82977; FAX: +64 9 373 7058; email: firstname.lastname@example.org
Purpose: In the rd/rd mouse, the cell death of rod photoreceptors has been correlated to abnormal levels of the cyclic nucleotide cGMP within photoreceptors. Given that cGMP is required for opening of the cationic channels, there is the possibility that a high cGMP concentration would maintain these channels open, at a high energy cost for the retina.
Methods: We investigated whether cation channels were maintained in an open state in the rd/rd mouse retina by determining the labeling pattern of an organic cationic probe (agmatine, AGB) which selectively enters cells through open cationic channels. The metabolic activity of the rd/rd mice was measured by assaying lactate dehydrogenase (LDH) activity in several tissues and Na+/K+ATPase activity was measured as a function of development and degeneration of the retina.
Results: AGB neuronal labeling showed a systematic increase consistent with the known neuronal functional maturation in the normal retina. There was a significant higher AGB labeling of photoreceptors in the rd/rd mouse retina from P6 supporting the possibility of open cationic channels from an early age. There were no changes in the LDH activity of tissues that contain PDE6 or that have a similar LDH distribution as the retina. However, LDH activity was significantly higher in the rd/rd mouse retina than in those of control mice from birth to P6, and it dramatically decreased from P9 as the photoreceptors degenerated. The predominant LDH isoenzyme changes and loss after degeneration appeared to be LDH5. ATPase activity increased with age, reaching adult levels by P16. Unlike LDH activity, there was no significant difference in Na+/K+ATPase activity between control and rd/rd mice at any age examined.
Conclusions: We conclude that AGB is a useful marker of photoreceptors destined to degenerate. We discard the possibility of a generalized metabolic effect in the rd/rd mice. However, the elevated LDH activity present before photoreceptor differentiation indicated altered retinal metabolic activity that could not be associated with open cationic channels alone. Therefore, altered metabolic activity as indicated by LDH measurements in the retina appeared to be the earliest sensitive sign of future photoreceptor dysfunction in the rd/rd mice.
Retinitis pigmentosa (RP) refers to a family of hereditary disorders that are manifested as a gradual loss of photoreceptors and consequently vision. Many genes identified as causally linked to the onset of RP are required for different functions, including phototransduction, maintenance of photoreceptor outer segment integrity, and retinal pigment epithelium retinoid chemistry [1-4]. Despite the identification of possible genetic causes of RP, little information as to the cellular mechanisms that lead to the RP phenotype is available.
A common feature in some forms of autosomal recessive RP is an elevation in the retinal levels of the cyclic nucleotide cGMP, due to specific defects in the cGMP degradation pathway in photoreceptors [5-12]. As cGMP gates cationic channels, elevation of cGMP concentration may contribute to the reduced light sensitivity of rod cells and increased dark current [13-16]. However, an increase in retinal cGMP concentration seems to have a more deleterious effect, as it appears to precede both photoreceptor and inner retina degeneration . Although retarded growth of photoreceptor inner segments is observed after postnatal day 4 [17,18], and swelling of the mitochondria of inner segments is seen by 8 days of life in rd/rd mice, pyknotic nuclei are first seen at postnatal day 10 [10,19]. Despite such studies and the established elevation of cGMP in some animal models of RP [5,8,10], identifying early biochemical events in the degenerating process may provide useful insights regarding the underlying mechanisms of photoreceptor cell death.
We addressed the issue of identifying the onset of cationic channels opening in the rd/rd degenerating retinas by examining the entry of agmatine (AGB), a channel-permeable organic cationic probe. AGB enters nonselective cationic channels and has been used as a suitable marker for permeability of ligand-gated channels [20-25]. In the RCS rat retina, photoreceptors destined to degenerate were labeled with AGB before apoptotic markers [26-29], presumably because of the expression of, or the abnormal functioning of cationic channels.
The consequence of elevated cGMP levels in the outer segment of photoreceptors is that these cells would be in a state of constant activity to maintain the influx of sodium and calcium through the cGMP-gated channels; this means that photoreceptors would be maintaining the dark current at a considerable energy cost . This requirement for extraordinary energy would be reflected in the activity of the Na+/K+ATPase pump that maintains the ionic gradient, and metabolic enzymes that provide substrates for the tricarboxylic acid cycle.
Another potential marker of metabolic demand is lactate dehydrogenase (LDH). There is a dramatic alteration in the distribution of LDH isoenzymes in retinal degeneration models , with a marked reduction of LDH5 at P7 in the rat retina , an age where photoreceptors are not developed and well before anatomical alterations are evident in the RCS rat [33-35]. In addition, a significant decrease in LDH activity has been shown to occur secondary to photoreceptor degeneration in the RCS rat [36,37]. In the rd/rd mouse model, metabolic anomalies have been demonstrated before anatomical differences. Noell  showed an increase in oxygen uptake, glucose utilization and lactic acid production (aerobic) detectable at P8 in the rd/rd mouse retina, followed by a rapid decrease from P12. The implication of altered metabolic changes at this age is that rd/rd photoreceptors may have changed their metabolic demand secondary to abnormal cation entry caused by elevated cGMP levels.
The primary lesion of the rd/rd mice is on the PDE6B gene. PDE6 is found in the retina, and in the brain [39,40]. Studies on the dentate gyrus region of the rd/rd mice showed a decrease in number of neurons  although synaptic plasticity has been related to the activity of PDE5 instead of PDE6 . In another model of retinal degeneration, (the RCS rat), the cause of the retinal degeneration has been associated to a defect in the retinal pigment epithelium (RPE) in a receptor tyrosine kinase that is required for phagocytosis of shed photoreceptor outer segments. However, several studies have shown that there is the possibility of a systemic defect in carbohydrate metabolism in the RCS rats [43,44]. The animals experience difficulty in breeding and a poor muscle reflex .
As part of this study we sought to determine if animal models of retinal degeneration also have a systemic metabolic defect and identify early markers in the retina of impending photoreceptor degeneration. Metabolic activity in the rd/rd mice was probed by determining enzyme activity of LDH in several tissues and Na+/K+ATPase in the retina. Photoreceptor cation channel permeability was assessed using an organic cation channel probe (AGB).
All procedures were approved by a University of Melbourne or University of Auckland ethics committee and comply with the Association for Research in Vision and Ophthalmology statement for the use of animals in Ophthalmic and Vision research. Groups of C57BL/6 and rd/rd mice of several ages were killed by decapitation or cervical dislocation, their eyes removed and the retinas dissected free from the eyecup. In addition to the retina, samples were also taken from the cerebral cortex (somatosensory area), ventricular heart, and skeletal muscle. Tissue samples were homogenized in ice-cold 0.9% (w/v) NaCl in a glass-teflon homogenizer and centrifuged at 5000x g for 5 min at 4 °C. Supernatants were removed, stored on ice, and used for the assays within 30 min post-dissection.
Lactate dehydrogenase activity
The retinas, brain, heart, and skeletal muscle were collected from C57BL/6 and rd/rd mice at P1, P3, P5, P6, P7, P8, P9, P11, P15, P20, and adult (>P50). Retina samples were prepared by pooling ten retinas from age P3 or younger, eight retinas from ages P5 to P7 and six retinas from all other ages. The heart muscle and skeletal muscle from the hind leg were weighed out, to ensure equal amounts of tissue were used from each animal. Each retinal LDH datum point is the mean value derived from at least six samples. The mean of individual tissue assays was the average of six absorbance measurements. LDH activity was determined using the LDH reagent (Trace Scientific, Victoria, Australia). Tissue samples were diluted to 0.05 mg/ml in 0.9% NaCl and added to the LDH reagent containing: pyruvate (0.6 mM), NADH (0.23 mM) in phosphate buffer (55 mM) at pH 7.5. The change in NADH absorbance over time ΔA/min was measured in a spectrophotometer (Shimadzu UV-2501PC) at 340 nm. The activity of LDH in each sample was calculated and expressed in μmoles/min/mg protein.
Measurement of protein concentration
Protein concentration was measured in a colorimetric reaction using a BCA Protein assay reagent (Pierce, Rockford, IL) and detected in a microplate reader (ELx800, Bio-Tek Instruments Inc., Winooski, VT).
LDH isoenzyme separation
LDH isoenzymes are formed from homo- or heterotetramers of the LDHA and LDHB gene products to generate five isoenzymes. The polypeptides subunits are referred as H and M, which combine to form two pure types of isoenzymes, LDH-1 (H4), and LDH-5 (M4), and three hybrids LDH-2 (H3M), LDH-3 (H2M2), and LDH-4 (HM3) . Native gel electrophoresis was used to separate the five known LDH isoenzymes. This protocol is based on the procedure of Nissen and Schousboe  using 1.5% agarose gels (Agarose-1000, Life Technologies) in 25 mM Tris/250 mM glycine (pH 9.5). Electrophoresis was conducted for 90 min at 100 V in a 5 mM Tris, 40 mM glycine, pH 9.5 running buffer. Each sample was run in triplicate with a minimum of five samples per point. Upon completion of electrophoresis, the gel was washed in 0.1 M Tris (pH 8.5) and an LDH staining solution containing lactate (3.24 mg/ml, Sigma, St. Louis, MO), nicotinamide adenine dinucleotide (NAD, 0.3 mg/ml, Sigma), nitroblue tetrazolium (0.8 mg/ml, Sigma) and phenazine methosulfate (0.167 mg/ml, Sigma) dissolved in 0.01 M Tris (pH 8.5) was applied. The Kodak Imaging 1D system was used for visualization and analysis of the gel (Eastman Kodak Company, Rochester, NY). The relative amount of each isoenzyme (LDH1 through LDH5) was determined by quantification of the density of pixels in each isoenzyme band. The cumulative density of the five bands in each sample was calculated and the fraction of individual bands was used to determine the amount of each isoenzyme as a percentage of the whole sample.
Na+/K+ATPase activity assay
Retinas from C57BL/6 control mice and rd/rd mice were used at P4, P8, P16, and adult. Four retinas were pooled for P4 assays, and two retinas were pooled for all other age groups. ATPase activity was measured using a protocol modified from Else and coworkers . Experiments were repeated a minimum of five times. The samples were homogenized in 100 μl 250 mM sucrose, 5 mM EDTA, 20 mM imidazole, (pH 7.4) with a glass-teflon homogenizer followed by brief sonication. A similar volume of SDS detergent (0.75 mg/ml) was added stepwise under constant mixing and incubated at room temperature for 15 min. ATPase activity was assayed in a solution containing 30 mM L-Histidine, 128 mM NaCl, 4 mM MgCl2, and 20 mM KCl. The ATPase reaction was started by the addition of 25 μl of 30 mM ATP (disodium salt) to 225 μl volume to the other assay constituents, incubating at 37 °C, and was stopped exactly 5 min later with the addition of 0.8 N ice-cold perchloric acid. ATPase activity was also assayed in the presence of 1 mM ouabain to inhibit Na+/K+ATPase activity. After centrifugation at 10,000x g for 15 min, the supernatants were reacted with a coloring reagent (10 mg/ml of (NH4)6MO7O24·4H2O, 3.3% H2SO4, and 78 mM FeSO4·7H2O) that reacts with the inorganic phosphate liberated in the ATPase reaction . Na+/K+ATPase activity was calculated as the difference in inorganic phosphate liberated in the presence (Mg2+/Ca2+ ATPase) and absence (total ATPases) of ouabain and expressed as μmoles PO4 released per mg protein per hour. Both, Na+/K+ATPase and Mg2+/Ca2+ATPase activities were plotted as a function of age.
AGB enter the cells in a temperature, concentration, and time dependent fashion [49,50], and is a suitable marker of permeability of cationic channels in the vertebrate retina . Incubating the isolated retina under basal conditions (no ligand activation), allows for the assessment of endogenous excitatory drive [23,51] and determination of the activity of cation channels . Retinas from control and rd/rd mice were dissected and mounted on filter membranes (0.8 μm pore size, Gelman Sciences, Ann Arbor, MI) using a method previously described [23-25,29]. The filter-mounted retinas were incubated in a modified physiological medium containing 100 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 10 mM dextrose, 2 mM CaCl2, 1 mM MgCl2 , to which 25 mM AGB was added. The incubation times varied from 5 to 45 min with optimal detection achieved at 15 min. After the AGB incubation procedure, retinas were fixed in 1% paraformaldehyde/2.5% glutaraldehyde at 4 °C for 1 h. The tissue was dehydrated and embedded in epon resin following procedures previously described in Kalloniatis and Fletcher . Semi-thin (250 nm) serial sections were probed with the AGB antibody provided by Dr. Robert E. Marc (University of Utah, Salt Lake city, UT) and silver-intensified immunogold was used for detection of antibody reactivity. Digital images were acquired with a LEICA DC 500 camera, Twain model V184.108.40.206 mounted on a LEICA DC 500 microscope (Leica Microsystems Ltd., Wetzlar, Germany).
We quantified AGB labeled photoreceptors as a function of age in the outer nuclear layer of retinas drawn with the aid of a camera lucida. Only the central about 2x2 mm area was used for all the quantification to minimize any variation in eccentricity-dependent photoreceptor degeneration. The drawings were also restricted to areas with no signs of conspicuous AGB labeling in the inner retina [23,29]. AGB positive photoreceptor cells were sampled in three separated areas of central retina, which accounted, as closely as possible, for approximately 100,000 μm2 per retina. The area over which counting was performed was calculated using a calibrated grid placed over the camera lucida drawings. A person blind to the purpose of the experiment performed the drawings and counting. Table 1 outlines the number and age of animals used in the quantification and total area examined in control and rd/rd mice retinas.
Statistical analysis was performed using one-way analysis of variance (ANOVA) with an α of 0.05 followed by pairwise comparison using Tukey Honestly Significant difference test. All assay data are presented as mean and standard deviation; p<0.05 was considered statistically significant.
AGB labeling patterns in the developing retina
We determined the AGB labeling patterns of different age groups in the mouse retina (Figure 1). At P4, no cellular AGB labeling was detected in control (C57BL/6) or rd/rd retinas, with little labeling of the developing inner plexiform layer. Light labeling of ganglion cells and the inner plexiform layer was observed in the control P6 retina, with rare labeling of amacrine cell somata and the developing photoreceptor cells (Figure 1C). In the rd/rd mouse, a similar AGB labeling pattern was observed, except for an increased number of AGB labeled photoreceptor cells (Figure 1D, Figure 2). At P8 and in older retinas, AGB immunoreactivity increased in amount and number of cells labeled. There was occasional labeling of horizontal cells and light labeling of bipolar cells and the plexiform layers (Figure 1).
The AGB labeling pattern markedly changed by P10 in both control and rd/rd retinas. In the control retina there were no major changes in the labeling of the inner retina, except for horizontal cells. In contrast, there was an increase in the number of labeled cells in the rd/rd retina. At this age, there was dense AGB labeling in the photoreceptors area (Figure 1H). Conspicuous AGB labeling of the inner retina was evident by P12 in both control and rd/rd retinas (Figure 1I,J). There was an increase in the number of labeled amacrine cell somata. Horizontal cells were intensely labeled, as were the cells in the ganglion cell layer. By P15, the morphology of the control retina was similar to the adult stage although conspicuous labeling of bipolar cells was not evident (Figure 1K). In the adult, the most outstanding differences between control and rd/rd mice was the absence of the photoreceptor layer and labeling of the inner retina in the rd/rd mice, consistent with other reports [51,54]. At this stage, the control retina showed an AGB labeling pattern similar to that in the adult rat .
Figure 2 shows the proportion of AGB-labeled photoreceptors as a function of age, with the total area sample outlined in Table 1. Although the number of AGB-labeled photoreceptors steadily increased in control retina, the rd/rd retinas had at all ages a significantly higher number of these labeled cells than the control retinas (p<0.01). In the rd/rd retina, a rapid increase in number of AGB-labeled photoreceptors per unit area was observed after P8. By P15, the reduction of the outer nuclear layer width resulted in a ratio of AGB-labeled photoreceptors approximately three times higher than control retina.
LDH in the control mouse
Mammalian LDH exists as five tetrameric isoenzymes referred to as LDH1 through LDH5. The distribution of LDH isoenzymes has been suggested to correlate with different metabolic environments and considering previous work on generalized metabolic dysfunction in models of retinal degeneration, we examined the isoenzyme distribution in mouse heart, skeletal muscle, retina, and brain. As shown in Figure 3, skeletal muscle, which is thought to have high levels of anaerobic metabolism, expressed predominantly the LDH5 isoenzyme. By contrast, heart tissue, which has high levels of aerobic metabolism, expressed predominantly isoenzymes LDH1-3. Although the retina is part of the CNS, it displayed an LDH distribution more akin to muscle than brain. In the retina, LDH5 is the predominant isoenzyme expressed, with decreasing amounts of LDH4 to LDH1. The isoenzyme distribution of brain was similar to heart. These findings are consistent with previous findings , in particular that the retina has a greater rate of anaerobic metabolism than any other tissue in the CNS and contains more than three times the lactate per kilogram of tissue compared to brain .
Changes in LDH activity during degeneration
To test our hypothesis that the rd/rd mouse sustain higher metabolic levels than those in control mice, we evaluated their LDH activity as a function of development. As shown in Figure 4A, LDH activity increased in a biphasic manner during development in the control mouse retina. There was a steady increase in LDH activity in the control rat retina, particularly between P6 and P9, a time when photoreceptors begin to form their outer segments, followed by a further increase around eye opening (about P13) and photoreceptor maturation (P20). LDH activity had reached adult levels by P20. In contrast, LDH activity in the rd/rd mouse was higher than control from P1 to P6 (p<0.001, Figure 4A), remained stable between P7 and P8 with values not significantly different from control and decreased thereafter as the photoreceptor cells degenerated reaching LDH activity values encountered in very young retinas.
Figure 4B-D reflects the total LDH activity as a function of age for three other different rd/rd tissues that either contain PDE6 (brain) and therefore could be affected in the rd/rd mouse or tissues that have a high content of LDH (heart, muscle). The rd/rd retina is the only tissue that displayed a consistent increased activity early in development followed by a marked reduction relative to the control retina.
Changes in LDH isoenzyme distribution during degeneration
Changes in LDH activity are often correlated with a shift in expression of LDH isoenzymes. We examined their distribution and quantified the activity of two of the most abundant LDH isoenzymes at five stages of development (Figure 5). There was very little change in the distribution of LDH5 or LDH3 during early developmental ages of the control mouse retina (Figure 5C). In contrast, following eye opening there was a significant decrease in LDH5 content in the rd/rd mouse (Figure 5A,C). The relative distribution of the LDH5 isoenzyme appeared to parallel the loss of photoreceptors. On the other hand, quantification of LDH3 activity resulted in a small but significant increase (p<0.01) in rd/rd adult retina compared with control values (Figure 5B). We conclude that the overall loss of LDH activity is due to a greater selective loss of the LDH5 isoenzyme.
Changes in Na+/K+ATPase during degeneration
It is well known that photoreceptor function has a high metabolic demand. A key enzyme that has been associated with consuming much of this energy is Na+/K+ATPase, which is said to account for 50% of energy required by photoreceptors . We evaluated Na+/K+ATPase activity in the mouse retina during development and in the degenerating rd/rd retina. As shown in Figure 6, total ATPase, Mg2+/Ca2+ATPase and Na+/K+ATPase activities steadily increased during development. To our surprise, however, there was no difference in activity in degenerating retinas compared with controls at any of the ages examined (Figure 6A-C). The relative change during development of Mg2+/Ca2+ATPase and Na+/K+ATPase appeared to be different. In order to emphasize the relative change in the two ATPases, we calculated a ratio plot. Figure 6D shows that Na+/K+ATPase underwent a relatively larger change in the early developmental ages, compared with Mg2+/Ca2+ATPase.
The results of this study demonstrated elevated cationic channel permeability by P6, well before apoptotic markers and morphological changes are observed at the microscopic level [10,56]. In addition, LDH activity was elevated in degenerating mouse retinas prior to the onset of loss of photoreceptors. Changes in LDH activity were not significantly different in non-retinal tissue. The elevated LDH activity present at birth in the retina was not related to a change in LDH isoenzyme distribution. A sharp decrease in overall LDH activity with a selective reduction of LDH5 was observed after eye opening, well after the onset of photoreceptor death.
AGB labeling occurs earlier than apoptotic markers in the rd/rd mouse retina
In other models of photoreceptor degeneration, such as RCS rat retina, AGB labeling of photoreceptors is observed before the visualization of apoptotic cells [26-28], indicating that cationic channel permeability precedes the onset of cell death. In a similar way, we found a significant increase of AGB labeled photoreceptors from P6 in the rd/rd mouse retina. Normally in phototransduction, activated rhodopsin triggers the activation of transducin, which in turn activates phosphodiesterase (PDE), lowering the cGMP levels to close the cationic channels. However, in the rd/rd mouse, there is a mutation in the gene encoding the β-subunit of rod PDE, resulting in higher cGMP levels in the rod outer segment [7,57]. The increased cGMP content causes the cationic channels in the outer segment to remain open, leading to continued influx of Na+ and Ca2+ ions and induces a sustained dark current that utilizes a high metabolic load . In order to preserve the photoreceptor electrochemical gradient of Na+, ATP is required. ATP fuels the removal of Na+ from the photoreceptor, via the action of the Na+/K+ATPase. The AGB labeling of photoreceptors destined to degenerate in the rd/rd mouse retina may indicate open cationic channels (that is, open cyclic nucleotide gated channels), or possibly the expression of other cation permeable channels.
Changes in the pattern of AGB labeling in the rd/rd retina were also observed in the inner retina, suggesting that altered photoreceptor function is reflected in postneuronal activation. Studies on the permeability of AGB entry in the vertebrate retina [23,24] indicate that AGB labeling of post-receptoral neurons occurs secondary to glutamate receptors activation [23-25] and basal activation [23,24,29,58]. The basal AGB activation pattern reflects overall AGB gating by endogenous activity. It is characterized by a prominent labeling of the outer part of the inner nuclear layer [25,29,58], with occasionally labeled amacrine or ganglion cells. Our observation of altered AGB labeling of the rd/rd inner retina is in agreement with recent reports that the degenerating retina remodels and establishes excitatory drive [51,54,59-61]. We established that in basal conditions, using 25 mM AGB for longer periods (for example, 15 min) resulted in the labeling of depolarizing bipolar cells; this can be blocked with L-AP4 . The prominent labeling of the outer part of the inner nuclear layer (non-horizontal cells) in the adult control retina is not present in the rd/rd retina. Such a finding would be expected if "ON" bipolar cell labeling is reduced, as predicted by the finding of the reduced metabotropic glutamate receptor mGluR6 distribution in the outer plexiform layer of rd/rd retinas . However, the presumed demand to maintain excitatory drive in the remodelled amacrine and ganglion cell layer , likely leads to diffuse AGB labeling of the inner nuclear and ganglion cell layers.
There is a higher level of metabolism prior to degeneration in the rd/rd mouse retina
Our results indicated that in the control retina, LDH activity increased during development in two phases. There was a steady increase in activity in the control retina between birth and P9, followed by a slow, progressive increase until adulthood. The change in activity, especially between birth and P9 most likely reflects the extent of maturation of retinal structures that occurs prior to eye opening. The mouse retina is relatively immature at birth, consisting of a ganglion cell layer, inner plexiform layer and large neuroblastic layer . The outer plexiform and inner nuclear layers become clearly separated by P4 and photoreceptor outer segments can be observed by P8. The increase in LDH activity observed in the rd/rd mouse during this early phase could be related to the elevated and increasing cGMP levels relative to protein levels observed from P6 in the rd/rd retina , and decreased cGMP-PDE activity that starts to manifest at P5 . Previous studies [63,64], have shown that synaptogenesis is linked with major changes in metabolism in other regions of the CNS. In particular, LDH activity has been shown to increase during periods of synaptic reorganization in the dentate gyrus following deafferentation .
Although the AGB labeling would predict altered metabolic demand from P6, we found a small but significant increase in LDH activity from birth through to P6 in the rd/rd retina. The increase in LDH activity prior to P6 is consistent with an increase in retinal metabolism prior to the onset of photoreceptor metabolism, and although the activity is not totally consistent with the increase being due to altered photoreceptor metabolic demand alone, is indicative of early altered metabolism in the rd/rd retina. Rhodopsin is first detected at about P4 with subsequent photoreceptor development and differentiation rapidly occurring in the next few days [10,65-67]. The earliest anatomical difference between the rd/rd and control mouse is abnormal synaptogenesis at P4  but the first signs of degeneration are observed at P8.
It has not been established if altered energetic metabolism leads to degeneration of the retina. However, the altered LDH enzyme activity is not new to models of retinal degeneration. Graymore  published the first reported alteration in LDH distribution in the RCS rat compared to control rat retina. He showed an alteration in the distribution of LDH isoenzymes with a marked reduction of LDH5 at P7, an age where photoreceptors have not attained adult-like morphology in the rat retina and well before anatomical alterations are evident in the RCS rat [31,33-35]. In the rd/rd mouse model, metabolic anomalies have also been demonstrated before degeneration, and at a similar time to those reported in this study. Several studies [38,68,69], have shown an increase in oxygen uptake, glucose utilization and lactic acid production (aerobic) detectable at P8 in the rd/rd mouse retina, followed by a rapid decrease from P12. Metabolic substrate concentrations and high energy phosphate compounds do not show major differences between control and rd/rd mice [70,71], especially between P15 and P20. The changes in oxygen consumption, glucose utilization, and lactic acid production  are consistent with altered LDH activity data, and identify an early metabolic dysfunction in the rd/rd mouse, well before photoreceptor cells die through apoptosis at about P10-P12 in the rd/rd mouse . The implication of altered metabolic changes at birth is that the genetic mutation leads to altered retinal metabolic function. The chief conclusion that can be drawn from such work is that in two disparate models of retinal degeneration, the RCS rat, caused by a mutation of the tyrosine kinase gene Mertk [72,73] and the rd/rd mouse (caused by a mutation of PDE) , both display metabolic anomalies and increased photoreceptor AGB labeling  before anatomical or apoptotic markers of impending photoreceptor degeneration.
Is the LDH5 isoenzyme localized within photoreceptors?
It has been proposed that LDH5 is the predominant isoenzyme within glia of the CNS, whereas LDH1 is predominantly found in neurons [74-76]. This proposition lead to the suggestion that LDH1 is predominantly located within photoreceptors, known to have high use of pyruvate . Consequently, a model was developed for the retina where glucose would be converted to lactate via LDH5 within Müller cells, the lactate transported to photoreceptors where it would be converted to pyruvate via LDH1 and utilized in aerobic metabolism: known as the "lactate shuttle" [75,78,79]. The "lactate shuttle" hypothesis remains controversial [80,81]; however, our previous work has indicated that monocarboxylate transport is important for longer-term retinal function and a range of amino acids are likely shunted into metabolic pools to sustain retinal function [82,83]. Studies on the behavior of LDH isoenzymes at near physiological concentration [84,85] suggest that the activity of LDH isoenzymes may not be as proposed in the lactate shuttle hypothesis. Wuntch's experiments demonstrated that pyruvate inhibition of LDH1 does not occur at concentrations higher than 3.5x10-7 M, such as those encountered in physiological conditions. Moreover, it has been recently shown that neurons and glia are both net producers of lactate . These observations do not contradict the studies of Bui et al. , who observed that when monocarboxylate transporters are blocked, there is a decrease in retinal function as measured by the electroretinogram. Lactate transport inhibition occurs both at the plasma membrane and mitochondrial membrane, opening the possibility that intracellular lactate traffic could be affected. Despite the controversy on the cellular producers of lactate, these studies [87,88] suggest that lactate is a key energy metabolite for retinal neurons, and LDH activity is required for retinal function.
Our observation that the LDH isoenzymes varied considerably between retinal and brain tissue poses the question regarding how the lactate shuttle would operate within the retina. In particular, we observed substantial levels of LDH5 within the retina, and little if any LDH1. It should be noted however, that LDH5 has an equilibrium constant of 1, suggesting that this isoenzyme is just as efficient in producing pyruvate under aerobic conditions as LDH1. Since the oxygen tension in the outer retina is very high [89,90], it is highly probable that the LDH5 equilibrium will be in the direction of pyruvate production. Moreover, the LDH isoenzyme directionality does not seem to apply to physiological concentrations of the enzyme [84,85]. Consequently, in contrast to neurons of the brain that contain LDH1, production of pyruvate or lactate within photoreceptors and Müller cells could be catalyzed by LDH5 under normal metabolic conditions. This suggestion is supported by the distribution pattern of the total LDH activity in the outer retinal layers in both monkey and rabbit retinas . Lowry's work  showed that LDH activity in retinal layers is higher in the outer nuclear layer than in the inner retina. It is unlikely that LDH1 alone determines the high activity observed in this layer. In fact, it appears that regulation of LDH5 accounts for the LDH activity of the retina. Several independent studies corroborate this statement: (1) the significant reduction in LDH5 activity in a rat model of photoreceptor degeneration [32,36,37] and (2) LDH5 activity increased in a retinal preparation where the glial metabolism has been impeded , suggesting that altered metabolite availability from Müller cells leads to upregulation within neurons. Since LDH5 is the predominant isoenzyme of the mouse retina, it is likely that photoreceptors express this isoform, and that blocking glial metabolism enhances LDH5 activity in the photoreceptors. Further analysis of LDH isoenzyme distribution in the retina is required to support this hypothesis.
ATPase activity in the retina
Na+/K+ATPase restores Na+ and K+ gradients within photoreceptors and is integral to the maintenance of the photoreceptor dark current. Ames et al.  indicated that 41% of oxygen uptake and 58% of glycolysis was consumed by photoreceptor Na+/K+ATPase. Moreover, several studies have demonstrated that knockout mice that lack the β2-subunit of Na+/K+ATPase, develop photoreceptor degeneration, and that replacing the β2-subunit with a β1-subunit in a "knock-in" experiment ameliorated the degeneration [92-94]. These studies suggest that normal function of Na+/K+ATPase is crucial for photoreceptor survival.
We examined Na+/K+ATPase activity as a function of development in control and rd/rd retinas. Following eye-opening, there was a substantial increase in Na+/K+ATPase activity in control and rd/rd retinas. This most likely reflects the increase in retinal function that occurs at this time. The electroretinogram is a gross retinal potential that provides a measure of the response of the retina to light. It is first detected at P12 in the control mouse retina . At this time the rates of glycolysis and oxygen consumption increase by 2.5 fold [96,97]. This roughly coincides with the greatest increase in Na+/K+ATPase activity observed.
However, the activity of Mg2+/Ca2+ATPase appeared to be affected early in the development of the rd/rd mouse retina. The close functional association between PDE activity and Ca2+ levels suggest that upregulation of Ca2+ ATPases would accompany the degeneration process. Yet, Mg2+/Ca2+ATPase activity was only increased early in the development. It is unlikely that the high Mg2+/Ca2+ATPase activity of the rd/rd mice at P4 was a consequence of the onset of the degeneration because PDE activity and cGMP levels remain normal at this age [62,98]. Increased cGMP levels and concomitant reduction in PDE activity were first observed at P6 [62,98]. Although there is expanding evidence on the role of the family of plasma membrane Ca2+ ATPases (PMCAs) as important participants in maintaining Ca2+ export during normal and pathological conditions [99-102], the role of Mg2+/Ca2+ATPase in the rd/rd retina development remains unclear.
It is intriguing that we found no difference in Na+/K+ATPase activity in rd/rd retinas compared with controls. In particular, Na+/K+ATPase activity was unchanged even when photoreceptors were significantly reduced in number in the rd/rd mouse . It is well known that shifts in metabolism and deafferentation cause major alterations in LDH activity. It is less clear what the relationship is between LDH and levels of aerobic or anaerobic metabolism. We propose that LDH activity is an excellent indicator of photoreceptor function, rather than Na+/K+ATPase activity. Wetzel et al.  demonstrated that Na+/K+ATPase subunits are expressed throughout the retina, and functional Na+/K+ATPase is required for maintaining ion gradients in the inner retina. Our results indicated that there was no preferential higher distribution of ATPase activity in the outer retina.
In summary, we evaluated the use of AGB as a marker of early retinal degeneration, LDH isoenzyme distribution, and activity in control and rd/rd mouse retinas and other tissues and found that between birth and P8, LDH activity was higher in the rd/rd mouse retinas than in controls. Together with the observation of high AGB permeation into photoreceptors, this supported the hypothesis of altered metabolism from birth and early dysfunction in cation channel gating of photoreceptors.
This work was supported by grants from the National Health and Medical Research Council (numbers 145735 and 208950), Retina Australia, and the University of Auckland Staff Research Fund. DBF is the recipient of an RPB Senior Scientist Research Award, and MK holds a professorship funded by the Robert G. Leitl estate. We thank Kerry King for conducting the AGB labeling density counts, Professor Robert Marc for the gift of antibodies and Associate Professor P. Else for providing advice and useful discussion.
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