1. High-voltage-activated Ca2+ currents [ICa(HVA)] were studied in immunolabeled mouse retinal ganglion neurons (RGNs) to elucidate channel-specific components and their developmental changes in vitro. 2. Neurons were dissociated at postnatal day 5. RGNs were selected for electrophysiological measurements by vital labeling with an antibody against Thy-1.2. ICa(HVA) were recorded with patch electrodes in the whole cell configuration at a holding voltage (Vh) of -90 mV. 3. A total of 111 neurons was studied. On average, 13% of ICa(HVA) was reversibly blocked by 10 microM nifedipine, approximately 30% of the compound current displayed an irreversible block by 2.5 microM omega-conotoxin (omega-CTX) GVIA. The remainder current was resistant to both drugs, suggesting that the total ICa(HVA) was a mixture of at least three different components. 4. Developmental analysis revealed a significant increase of the omega-CTX-GVIA/nifedipine-resistant component of ICa(HVA) (31% at day in vitro (DIV) 0–2, 70% at DIV 18–26) mainly at the expense of the omega-CTX-GVIA-sensitive current. No significant change was found in the nifedipine-sensitive component of ICa(HVA). 5. To characterize the Ca2+ current component that was resistant to both omega-CTX-GVIA and nifedipine at Vh -90 mV, three tests were performed. The P channel antagonist omega-agatoxin IVA (omega-Aga-IVA, 200 nM) completely failed to block ICa(HVA) in mouse RGNs. The novel Ca2+ channel blocker omega-CTX-MVIIC (5 microM) decreased the ICa(HVA) remaining after omega-CTX-GVIA treatment by only approximately 10%.(ABSTRACT TRUNCATED AT 250 WORDS)
Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration
