Rod phototransduction and light signal transmission during type 2 diabetes Running Title: Outer Retina Function in Diabetes

Diabetic retinopathy is a major complication of diabetes recently associated with attenuated photoreceptor function. Multiple stressors in diabetes, such as hyperglycemia, oxidative stress and inflammatory factors, have been identified, but systemic effects of diabetes on outer retina function are incompletely understood. Ex vivo ERG presents the unique opportunity to test rod photoreceptor light signal transduction and transmission and determine whether they are permanently compromised and/or adapt to hyperglycemia in chronic type 2 diabetes. Lightevoked rod photoreceptor signals in control and diabetic mice in vivo were compared to those recorded ex vivo under normal or elevated extracellular glucose. Transduction and transmission of light signals were compromised in 6 mo. diabetic mice in vivo. In contrast, ex vivo rod signaling was similar in isolated retinas from 6 mo. control and diabetic mice under normoglycemic conditions. Acutely elevated glucose ex vivo increased light-evoked photoreceptor responses in control mice, but did not affect light responses in diabetic mice. In summary, our data suggest that long-term diabetes does not irreversibly change the ability of rod photoreceptors to transduce and mediate light signals. However, type 2 diabetes appears to induce adaptational changes in the rods that render them less sensitive to increased availability of glucose. Diabetic retinopathy, one of the leading causes of irreversible vision loss worldwide (1), has traditionally been characterized by retinal vascular abnormalities. Recent studies in human patients with diabetes suggest, however, that retinal diabetic neuropathy with neuroglial dysfunction and degeneration frequently precedes visible microvasculopathy (2-4). Even in the absence of or with minimal signs of retinal vascular abnormalities, patients with diabetes show dysfunction and loss of retinal neuronal cells. In fact, impairment of the neurovascular unit has been suggested to lead to or accelerate retinal vascular disease progression, since implicit time delay of the multifocal electroretinogram (ERG) predicts onset of visible vascular changes (5). Damage to nerve fiber, ganglion cell and inner plexiform layers in the human eye has been demonstrated in diabetes by impaired pattern ERG and oscillatory potentials (6) and thinning of inner retinal layers (4). Reduced amplitudes and longer implicit times of the scotopic and photopic bwaves (6) also implicate dysfunction of rod and cone bipolar cells in retinal diabetic neuropathy of human patients. These findings are not universal, however, and reduced scotopic aand b-wave amplitudes have not been shown in all studies (7). Murine models of diabetes share many features of retinal dysfunction and cell loss with those in patients with diabetes. The positive component of the scotopic threshold response, as a measure of retinal ganglion cell function, is attenuated (8) and nerve fiber and ganglion cell layer thickness and retinal ganglion cell density are reduced (4) in streptozotocin (STZ)-induced diabetes in the rat and mouse, a type 1 diabetes model, although this is not universal in all mouse strains (9). Loss of amacrine cells observed in type 1 diabetes models (10; 11) has been suggested to underlie reduced amplitude and delayed implicit time of oscillatory potentials (8). Similarly to human studies, outer retinal dysfunction and cell loss in diabetic animal models remain controversial. Rod photoreceptor and bipolar cell dysfunction, based on reduced amplitudes and delayed implicit times of the scotopic aand b-waves, were reported in the db/db mouse, a type 2 diabetes model (12). Photoreceptor death was also reported in a rat STZ model (13), however, other studies did not observe loss of photoreceptors (14; 15) and reported no or only minor changes in the scotopic ERG (8), or even increased photoreceptor function in Zucker Diabetic Fatty (ZDF) rats, a type 2 diabetes model (16). Pigment regeneration and dark adaptation have not been extensively studied in diabetic animal models or human patients, but some studies have suggested deficits in pigment regeneration or dark adaptation caused by diabetes (17; 18). Functional discrepancies in previous human and animal studies remain unexplained. It is possible that outer retinal cells are not permanently damaged, but that their function is modulated by systemic factors. Transient hyperglycemia in patients with diabetes was associated with increased scotopic band photopic aand b-wave amplitudes (19). While the multifocal ERG waveform was delayed in normoglycemic patients with diabetes compared to healthy volunteers, this was partly reversed by elevation of blood glucose to their usual level of (habitual) hyperglycemia (20; 21). These findings indicate that both acute hyperglycemia and chronic diabetes modulate retinal signaling. We provide evidence for our hypothesis that long-term uncontrolled type 2 diabetes in db/db mice does not permanently alter intrinsic mechanisms in rod photoreceptors responsible for transducing and transmitting light signals. However, we find that rod photoreceptors in the ex vivo isolated diabetic retina are less sensitive to acute elevation of extracellular glucose concentration. We suggest that attenuation of the in vivo ERG in diabetic mice is likely due to diabetes-induced systemic factors influencing rod photoreceptors, which once removed, can no longer mediate suppression of photoreceptor responses in the context of the ex vivo testing. RESEARCH DESIGN AND METHODS