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Eldred Lab Research Interests:
The retina is a massively parallel analog visual processing computer that uses a wide variety of neurotransmitters and millions of synaptic connections. A large percentage of these connections involve conventional neurotransmitter release at anatomically defined synaptic connections onto well characterized and localized receptors to produce postsynaptic changes in electrical activity at the millisecond time scale. However, many of these same transmitters and receptors can also activate biochemical signal transduction systems which process this same information at the millisecond to minute's time scales. Several of these biochemical signal transduction systems utilize gases like nitric oxide, carbon monoxide or hydrogen sulfide as signaling molecules. The focus of the research in the Eldred lab is how each of these gasses is used in retinal signaling processing.
In recent years, it has become apparent that the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway is one of the most widespread in the retina (Eldred, 2000). This pathway is distinguished by having NO produced on demand, that NO is not released using synaptic vesicles, and that NO does not bind to specific receptors on the postsynaptic membrane. Basically any synaptic mechanism that can increase intracellular calcium either directly through receptor channels or by release from intracellular stores can potentially activate this pathway. This increased calcium can activate calmodulin which in turn activates either endothelial nitric oxide synthase (eNOS) or neuronal nitric oxide synthase (nNOS) to synthesize NO.
NO has been shown to influence the physiology of all neuronal types in the retina and every cell type in the retina has been shown to make NO. The most clearly characterized downstream signaling pathway for NO has been the activation of soluble guanylate cyclase (sGC) to synthesize cGMP. For instance, NO has been shown to increase the gain and extend the voltage range of exocytosis in cone photoreceptors. In bipolar cells, NO donor produces an inward current accompanied by a rise in dim and bright flash response amplitudes, and an increase in membrane conductance. Cyclic GMP has recently been shown to selectively enhance responses to dim, but not bright, stimuli through a purely postsynaptic mechanism that is blocked by inhibitors of cGMP dependent kinase.
Perhaps the most comprehensive studies have examined the role of NO in horizontal cells. Injection of the NO precursor, L arginine, into H1 luminosity type horizontal cells in turtle retina reduces their light responses, dramatically increases their input resistance, decreases their response to a surround, and increases their response to stimulation of their receptive field center. Bath application of L arginine also decreases the kainate responses in H1 cells in a manner similar to cGMP and NO. Thus it is likely that the H1 horizontal cells can serve as their own source and target of NO to negatively modulate the gain at photoreceptor horizontal cell synapses. Finally, Yu and Eldred (2003) have shown that GABAA and GABAC receptor antagonists increase retinal cGMP levels through the activation of nitric oxide synthase (NOS) and that NO stimulates GABA release and inhibits glycine release in retina (Yu and Eldred, 2005). The NO stimulated GABA release from horizontal cells was shown to be due to the reversal of the GABA uptake transporter. In amacrine cells, by working through cGMP, the NO released by light stimulation decreases the rod input and increases the cone input during light adaptation by uncoupling the AII amacrine cells from the cone bipolars. NO also modulates cGMP gated channels by activating a NO sensitive sGC to increase levels of cGMP in photoreceptors, bipolar cells, and ganglion cells. Finally, bath application of L arginine or NO donor usually reduces the peak discharge rates of ON responses in ganglion cells by about 40%, and completely blocks the OFF responses in most ganglion cells.
Clearly the NO/cGMP signaling system is critical for many aspects of retinal function and it is important to understand its role in specific retinal cell types. In particular, we are determining which cells contain specific NOS isoforms, what stimuli can activate NO production in identified cells, and what downstream signaling pathways are activated by the NO that is produced.
We have several ongoing projects focusing on the NO/cGMP signaling pathways in retina. These studies are being done using salamanders, turtles, mice, or rats as model systems. Our results indicate that there are reciprocal relationships between NO and the retinal neurotransmitters dopamine, glycine, and GABA in that NO modulates the release of each of these transmitters and each of these transmitters modulate the production of NO. We are actively investigating the cellular and molecular mechanisms involved in these interactions. In addition we are exploring the modulation of NO/cGMP by the cholinergic system as well. Using NO imaging methods we have shown that although every retinal cell type can make NO, it is not always freely diffusible and is in fact retained within some retinal neurons. We are also investigating the cellular basis for these differences in diffusion of NO.
Nitric oxide (NO) has normal physiological functions in every retinal cell type, and every retinal cell type can potentially make NO. NO is also involved in many ocular pathologies including diabetic retinopathy and inhibiting NO is often beneficial. NO signaling is regulated by many factors, both in normal retinal function and pathology, making it desirable to target just the pathological pathways. Our research focuses on how NO can be selectively targeted to decrease the neuronal and vascular pathology in diabetic retinopathy. We are testing the following two hypotheses. 1) Diabetes increases the retinal levels of adrenomedullin (ADM), which in turn activates neuronal nitric oxide synthase (nNOS) to increase retinal NO production to pathological levels. These studies are providing the first demonstration of the ADM/nNOS/NO signaling pathways in retina and how they are modulated in the neuronal and vascular pathology in diabetic retinopathy. 2) Neuronal and vascular pathology in diabetic retinopathy share similar molecular pathways and are amenable to similar pharmacological interventions. Our results are clarifying the role of specific NOS isoforms and ADM in diabetic retinopathy and how these pathways can be optimally targeted to treat the pathology. Upregulation of ADM is also found in proliferative vitreoretinopathy, uveitis, vitreoretinal disorders, primary open angle glaucoma, and retinitis pigmentosa. A clearer understanding of the ADM/NOS/NO signaling pathways and how they can be manipulated in retina may have broad implications for much ocular pathology.
Our studies have biochemically detected HO-2 in turtle and rat retinas. HO-2-like immunoreactivity (LI) is present in the inner segment of photoreceptors, in the somata of several types of amacrine, some bipolar and ganglion cells, in some cells resembling astrocytes, and in the processes in the IPL in turtle retina. HO-2-LI is also colocalized with cGMP-LI in the somata of some bipolar, amacrine, and ganglion cells. These results suggest that HO-2 is widespread in the retina and that CO could be produced at many sites. The colocalization of HO-2 with cGMP strongly supports that CO could function as an endogenous modulator of the sGC/GMP signal transduction system in the retina. We have also found HO-2 and nNOS to be colocalized in many retinal neurons which strongly suggest that interactions between CO and the NO may occur. This interaction is supported by our studies demonstrating that CO can amplify the NO-induced levels of cGMP-LI in large numbers and types of retinal neurons. Application of NOS inhibitors blocked the increases in cGMP-LI induced by CO. These results support that CO is a modulator of the sGC/GMP signalling pathway, and that there are interactions between CO and NO on cGMP levels in the retina.
Hydrogen sulfide has been recently proposed as the third gaseous neurotransmitter, following NO and CO. Cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CGL) are responsible for the endogenous production of hydrogen sulfide in mammalian tissues, with CBS as the predominant enzyme in the brain and nervous system. CGL is mainly expressed in the liver, kidney, arteries and veins only traces found in the brain. CBS is strongly expressed in the whole neural tube and primary brain vesicles. CBS mRNA is found in the neuroblastic layer of the retina and lens at all developmental stages. Relatively high levels of H2S have been found in rat, human, and bovine brains (50-160µM).
Physiological concentrations of H2S are found to facilitate LTP induction in the hippocampus of rats and regulate release of corticotropin-releasing hormone from the hypothalamus, which further supports the role of H2S as a neuromodulator in the brain. In astrocytes, hydrogen sulfide is found to increase intracellular calcium and induce calcium waves, while in hippocampal slices, it enhances the responses of neurons to glutamate. Another role hydrogen sulfide is to increase levels of the antioxidant glutathione through enhancing gamma-glutamylcysteine synthetase activity and upregulating cysteine transport, thereby protecting neurons in primary cultures from oxidative stress.
There have been no previous studies of H2S in retina. Our biochemical results indicate that both CBS and CGL proteins and enzyme activity are present in retina. CBS is often found in the synaptic plexiform layers, while in salamander retina CGL is found in Müller cells. Several projects are currently underway examining the modulation of H2S production in response to neural activity and the effects of H2S on neurotransmitter release and uptake.