Elsevier

Experimental Eye Research

Volume 85, Issue 3, September 2007, Pages 413-422
Experimental Eye Research

Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons

https://doi.org/10.1016/j.exer.2007.06.005Get rights and content

Abstract

Neural deficits suggestive of involvement of the GABA signaling pathway can often be detected early in the course of diabetic retinopathy, a leading cause of blindness in the United States. To examine in greater detail the nature of the neuronal changes associated with hyperglycemia, we investigated GABA receptor activity on retinal bipolar cells in streptozotocin-induced diabetic rats; cells from age-matched normal rats served as controls. Patch-clamp recordings from isolated rod-bipolar cells revealed that diabetes enhanced the whole cell currents elicited by GABA. Responses of the GABAC receptor, the predominant GABA receptor on rat rod bipolar cells, exhibited a greater sensitivity to GABA, larger maximum current responses, slower response kinetics, and a smaller single channel conductance among diabetic cells relative to those recorded from normal controls. Compared with the properties of homomeric ρ1 and heteromeric ρ1ρ2 receptors formed in a heterologous expression system, these results suggested that there was a greater contribution from the ρ1 subunit in the GABAC receptor-mediated response of diabetic cells. The levels of mRNA, measured with real-time RT-PCR, were consistent with this finding. There was a significant enhancement in the ratio of ρ1/ρ2 subunit expression in the retina of diabetic animals, although the levels of GABA ρ1 subunit expression were comparable in diabetic and normal retinas. Taken together, the results suggest that diabetes modifies the subunit composition of the GABAC receptor on retinal neurons, most likely through its effect on the efficacy of gene transcription.

Introduction

Diabetic retinopathy is a common complication of diabetes and is a leading cause of blindness in the United States (EDIC, 1999). Although diabetic retinopathy is typically diagnosed by abnormalities in the retinal microvasculature after prolonged hyperglycemia, there is well-documented evidence to indicate that neural deficits occur early in the course of the disease (see Fletcher et al., 2005; Barber, 2003 for review). Particularly relevant to this study is that diabetes alters the GABA signaling pathway of the inner retina. For example, electroretinographic recordings from diabetic patients often exhibit reductions in the amplitude and implicit time of the oscillatory potentials (Tzekov and Arden, 1999), a GABA-sensitive component of the flash ERG (Wachtmeister, 1998, Tzekov and Arden, 1999, Wachtmeister, 2001). An altered GABA inhibitory signal in the diabetic retina is also supported by evidence of disrupted processing of lateral inhibition (Davies and Morland, 2002), and the fact that GABA levels are elevated in the vitreous of patients with proliferative diabetic retinopathy (Ambati et al., 1997).

Studies of diabetic animal models also reveal aberrations in GABA activity. Changes in the content and localization of GABA are evident in the diabetic retina, with both amacrine and Müller cells accumulating high concentration of GABA (Ishikawa et al., 1996a, Ishikawa et al., 1996b, Takeo-Goto et al., 2002). In addition, the activities of metabolic enzymes for GABA are also modified (Ishikawa et al., 1996a, Vilchis and Salceda, 1996, Honda et al., 1998), and the diabetic rat retina exhibits an enhanced capacity to transport GABA (Vilchis and Salceda, 1996). However, despite the substantial evidence indicating that GABA signaling within the inner retina is affected in diabetes, there is little information about the activity of GABA receptors in response to hyperglycemia.

In a previous study (Ramsey et al., 2006), we established a streptozotocin-induced diabetes model in pigmented (Long Evans) rats, and characterized the global changes in neuronal activity in the retina by means of electroretinography. The results indicated that pathways mediating inhibitory signaling in the inner retina are affected by the disease. In addition, our results provided preliminary evidence that the GABA responses of rod-bipolar cells were altered in diabetes. In the present study, we investigated the modulatory effects of hyperglycemia on the GABAC receptors present on retinal neurons, with a focus on the GABAC receptor activity of rod-driven bipolar cells. The GABAC receptors on retinal bipolar cells have a simple molecular composition that primarily consists of GABA ρ1 and ρ2 subunits (Qian and Ripps, 2001, Zhang et al., 2001). These receptors are expressed predominately on the axon terminals of retinal bipolar cells to control signal outflow from the second order neuron in the retina. Patch-clamp recordings from isolated retinal neurons revealed a number of changes in GABAC receptor activity in the diabetic retina; in particular, an enhanced sensitivity to GABA, slower response kinetics, and a smaller single channel conductance. These results, and those obtained by real time RT-PCR, suggest that hyperglycemia affects the GABA ρ subunit composition of GABAC receptors on retinal neurons.

Section snippets

Materials and methods

All experimental procedures conformed to the statement on animal care of the Association for Research in Vision and Ophthalmology, and adhered to the guidelines for the Care and Use of Laboratory Animals formulated by the Animal Committee of the University of Illinois, College of Medicine.

GABA-mediated responses of bipolar cells

To extend our previous observations on the diabetic modification of the GABAC receptor activity on retinal bipolar cells (Ramsey et al., 2006), we examined the property of GABA-elicited responses from retinal bipolar cells derived from STZ-induced diabetic and normal rats. Rod bipolar cells in the rat retina display a characteristic morphology, i.e., a rounded cell body from which extends several dendrites and a long axon that terminates in a prominent expansion (Fig. 1A). In agreement with

Discussion

GABAC receptors are prominently expressed in the vertebrate retina, and they constitute the majority of the GABA receptor population on rod bipolar cells in the rat retina (Feigenspan et al., 1993, Euler and Wassle, 1998, Lukasiewicz et al., 2004). The GABAC receptors are thought to be formed by GABA ρ subunits, and each of the three ρ subunits cloned thus far is expressed in retina (Zhang et al., 1995, Ogurusu and Shingai, 1996). The GABA ρ1 and ρ2 subunits have been detected on bipolar cells,

Acknowledgments

The authors would like to thank Dr Ralph Enz for a generous gift of the GABAC receptor antibody, Dr Stuart Lipton for providing rat GABA ρ1 and ρ2 subunits and Ms Ruth Zelkha for her expert assistance with imaging and fluorescence analysis. This work was supported by grants from the National Eye Institute (EY-12028, EY-06516, and EY-01792), an unrestricted departmental award from Research To Prevent Blindness (RPB) to the Department of Ophthalmology and Visual Sciences, and a Medical Student

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