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S.I. Firth, M.B. Feller; Cellular basis of periodic bursts of spontaneous activity in cultured retinal neurons . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5311.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Before vision, spontaneous activity in developing retinal ganglion cells (RGCs) is critical for the refinement of retinal projections. Spontaneous bursts of action potentials are rhythmic, with a period on the order of minutes. To study the cellular basis of this slow pacemaker, we developed a culture system that generates bursts of activity with a similar temporal pattern. Methods: Dissociated neuronal cultures from P1 rat retinas were plated at 2x104cells/cm2. After 7–10 days in vitro, spontaneous activity was measured using both whole–cell recordings and calcium imaging of the indicator Oregon–green–BAPTA–AM (10 µg/mL). To isolate amacrine–RGC networks, all experiments were conducted in the presence of the glutamate receptor antagonists CNQX (20 µM) and APV (50 µM). Results: Whole–cell voltage clamp recordings from presumptive RGCs revealed bursts of postsynaptic currents (PSCs) with a frequency of 94.0±33.2s, similar to those recorded in the intact retina. Bath application of GABA–A receptor antagonists (SR95531 5µM or picrotoxin 100µM) blocked all compound PSCs. However, low concentrations of SR95531 (0.3 µM) did not alter the interburst interval (97.6±24.0 %, % control±SD), duration (109.3±32.8 %), or interevent interval (91.1±10.5 %); despite significant reduction of PSC amplitude (17.5±7.6 %). These findings indicate pacemaker activity is not driven by a GABA–A receptor mediated network. Calcium imaging in the presence of blockers of fast neurotransmitter receptors was used to identify cell–autonomous pacemakers. We are currently performing current clamp recordings from putative pacemakers to determine whether cells have slow oscillations in their membrane potential. Conclusion: Our findings indicate that a cell–autonomous rather than a network–based pacemaker determines the temporal pattern of spontaneous activity in the developing retina.
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