Characterisation of Ganglion Cells in Human and Non-Human Primate Retinas

Abstract

Background: Ganglion cells are the output neurons of the retina, sending visual signals from the eye to the brain. In total 17 types of retinal ganglion cell have been identified, but only a low number is well characterized. This is because reliable markers for less well characterized, “wide-field” ganglion cell types are hard to come by. Antibodies against calcium binding proteins have been recognized as markers for specific neurons in the retina. Here, immunohistochemistry and intracellular injections were used to identify and characterise the calretinin and calbindin expressing cell types in the ganglion cell layer of human and non-human primate retinas. The main focus of this study is to investigate some of the low density ganglion cell types in human and marmoset retinas. This is a significant cause for investigation because dysfunction in ganglion cells can cause irreversible blindness. Therefore, the more that is known about these important cells, and what methods can be used to study them, the more can be contributed to future clinical research into treatable blinding diseases. Aim: To characterise low-density ganglion cell types in the retina. Hypothesis: Calcium binding proteins calretinin and calbindin are markers for sub-populations of retinal ganglion cells. Chapter 1 introduces the anatomical and morphological characteristics of retinal neurons in human and non-human primates. Chapter 2 investigates the use of antibodies against calretinin to characterise cells in the ganglion cell layer of marmoset monkey retina. Retinas were fixed, cut into quadrants and sectioned vertically using a vibratome, or processed as whole mounts. Double labelling was performed using antibodies against calretinin and RBPMS (RNA binding protein with multiple splicing, expressed in ganglion cell bodies). The proportion of calretinin positive cells were quantified. Cells from additional retinas were pre-labelled and intracellularly injected with dye to analysetheir morphology. The dendritic field size, soma size stratification and eccentricity of the cells were measured and compared with previous data. The majority of calretinin positive ganglion cells were characterised into narrow thorny and broad thorny ganglion cell types. Chapter 3 investigates the use of antibodies against calbindin to characterise cells in the ganglion cell layer of human retina. Retinas were fixed, cut and processed as sections or whole mounts with antibodies as described in the previous chapter, including double and triple labelling using antibodies against calbindin, RBPMS, melanopsin and ChAT (choline\ acetyl transferase, expressed in starburst amacrine cells). Additional retinas were again pre labelled and intracellularly injected with dye and cells were morphologically analysed. It was found that calbindin positive cells have large dendritic fields and a morphology corresponding to intrinsically photosensitive melanopsin-expressing ganglion cells. Double labelling confirmed melanopsin-expressing cells are calbindin positive. Chapter 4 explores the implications of these findings for future research into the use of calcium binding proteins as markers for subpopulations of less well characterised ganglion cell types.