Calcium Signalling in Cultured Human Retinal Neurons

Abstract

Abstract: : Purpose: Ca₂₊ overload has been implicated in neurodegeneration, and Ca₂₊ channel blockers have been proposed as potential neuroprotective agents in prevention of retinal ganglion cell death in glaucoma. We have investigated the role of the L-type voltage-operated Ca₂₊ channel in shaping Ca₂₊ signalling events in cultured human retinal neurons. Methods: Human retinas were obtained from donor eyes within 24 hours post mortem. The neural retina was dissected from the globe and dissociated using papain. Cells were plated onto glass coverslips and maintained in Ham/F12 medium with 10% FCS and 10ng/ml bFGF. Following 1 week in culture, neuronal cells were distinguishable by morphological characteristics. Following Fura-2 incorporation (3µM for 45 minutes at 35°C), changes in cytosolic Ca₂₊ concentration were monitored in retinal neurons using real-time epifluorescence techniques. Experiments were carried out in the absence of external Mg₂₊. Glutamate and glutamate agonists were added in the presence of glycine (100µM). Results: Depolarization of the retinal neurons by addition of KCl (40mM) resulted in an increase in cytosolic Ca₂₊. Application of the L-type Ca₂₊ channel blocker nifedipine (10µM) in the presence of KCl returned cytosolic Ca₂₊ to resting levels. A subset of neurons responded to a 30 second pulse of glutamate (1mM) by a sustained rise in cytosolic Ca₂₊ lasting up to 15 minutes in duration. Kainate (20µM) and NMDA (100µM) also increased cytosolic Ca₂₊ within these cells. Furthermore, pre-treatment with thapsigargin, which specifically depletes the InsP₃-sensitive Ca₂₊ store, had no effect on the glutamate or the kainate response, indicating that these responses are via ionotropic rather than metabotropic receptors. The Ca₂₊ response to glutamate was attenuated by nifedipine (10µM). Inhibition of the initial rate of increase as well as the amplitude of the response was observed. Conclusion: Cultures of retinal neurons derived from adult human donor material provide an excellent model for examining receptor-mediated Ca₂₊ dynamics. These models may prove useful in investigating the mechanisms involved in neuronal cell death and in neuroprotection with respect to glaucoma.

Keywords: 560 retinal culture • 334 calcium • 401 excitatory amino acid receptors