Fletcher Lab - Techniques
Techniques Used in the Laboratory
We use rats and mice to examine the structure and function of the normal retina, and transgenic or chemically induced rodent models of diabetes and retinal degeneration. In addition we have an animal model of Age-Related Macular Degeneration. We use a variety of tools to assess the structure and function of the retina with and without treatment.
1. Testing the function of the visual system in living animals.
(i) Eletroretinogram recording: The electroretinogram (ERG) is a measure of the electrical signals emminating from the retina. Like the ECG or EEG, activity from neurons within the retina generate a signal that can be detected from electrodes on the surface of the eye. We use this clinical tool to assess the gross retinal function of control and disease animals to determine whether neurons are functioning abnormally.
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Figure: A) Structure of the rat retina showing the retinal layers and neuronal subtypes. (B) An ERG waveform showing the major components including the a-wave, b-wave and oscillatory potentials. The a-wave reflects photoreceptor function, the b-wave ON bipolar cell function and the Oscillatory potentials are derived from amacrine cells. Therefore, analyzing each waveform individually provides information about how cohorts of neurons are functioning within the retina. (C) Examples of how the raw data is analysed. |
(ii) Visual acuity
We are currently developing ways of assessing the visual acuity of rodents by evaluating the head movements an animal makes in response to moving stimulus.
(iii) Assessment of the integrity of the retina.
We are developing ways of visualizing the retinal fundus and assessing the integrity of the blood retinal barrier with fluorescein angiography.
2. Examination of the structural integrity of the retina.
(i) Histological and immunocytochemical analysis
We evaluate the structure of mammalian retinae, using a variety of high performance, sensitive immunocytochemical tools. This includes indirect immunofluorescence for detecting changes in synaptic proteins and neurotransmitter receptors, immunoelectron microscopy .using preembedding and postembedding techniques. Using post-embedding amino acid immunocytochemistry, we are also able to track the uptake and degradation of neurotransmitters in the retina. This provides a novel way of assessing function using anatomical tools, and complements other measures of retinal function.
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Figure: (A) Transmission electron micrograph of a cone photoreceptor synapse from the rabbit retina. (B) A vertical section of the rat retina that has been incubated with the glutamate analogue, D-aspartate. D-aspartate immunoreactivity can be seen in the retinal Müller cells (the major glial cell type in the retina), because these cells express the glutamate transporter, GLAST. |
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(ii) Analysis of gene expression using real time PCR.
We use a variety of molecular biological tools to assess the change in expression of particular genes within the retina. In the broadest sense this involves quantifying differences in mRNA expression using real-time PCR. We are also developing Laser Capture Microscopy (using the Zeiss Palm system) to dissect particular regions of the retina to then analyse.
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Figure: A PCR gel showing the expression of the five glutamate transporters (EAAT1-EAAT5) in rat retina and brain. |
3. Analysis of functional changes in specific retinal cell types.
We examine the interaction of glia with the retinal vasculature using calcium imaging in cultured glia or retinal wholemounts.
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Figure: flatmounted retina that was preloaded with the calcium sensitive dye, Fluo 4AM. Retinal astrocytes show changes in calcium over time. |