Voltage-dependent conductances of solitary ganglion cells dissociated from the rat retina

Abstract

1. Ganglion cells were dissociated from the enzyme-treated rat retina, identified with specific fluorescent labels, and maintained in vitro. Electrophysiological properties of solitary retinal ganglion cells were investigated with both conventional intracellular and patch-clamp recordings. Although comparable results were obtained for most measurements some important differences were noted. 2. The input resistance of solitary retinal ganglion cells was considerably higher when measured with 'giga-seal' suction pipettes than with conventional intracellular electrodes. Under current-clamp conditions with both intracellular and patch pipettes, these central mammalian neurones maintained resting potentials of about -60 mV and displayed action potentials followed by an after-hyperpolarization in response to small depolarizations. The membrane currents during this activity, analysed under voltage clamp with patch pipettes, consisted of five components: Na+ current (INa), Ca2+ current (ICa), and currents with properties similar to the delayed outward, the transient (A-type), and the Ca2+-activated K+ currents (IK, IA and IK(Ca), respectively). 3. Ionic substitution, pharmacological agents, and voltage-clamp experiments revealed that the regenerative currents were carried by both Na+ and Ca2+. 100 nM-1 microM-tetradotoxin (TTX) reversibly blocked the fast spikes carried by the presumptive INa, which under voltage-clamp analysis had classical Hodgkin-Huxley-type activation and inactivation. 4. Single-channel recordings of the Na+ current (iNa) permitted comparison of these 'microscopic' events with the 'macroscopic' whole-cell current (INa). The inactivation time constant (tau h) fitted to the averaged single-channel recordings of iNa in outside-out patches was slower than the tau h obtained during whole-cell recordings of INa. 5. In the presence of 1-40 microM-TTX and 20 mM-TEA, slow action potentials appeared in intracellular recordings and were probably mediated by Ca2+. The potentials were abrogated by 3 mM-Co2+ or 200 microM-Cd2+; conversely, increasing the extracellular Ca2+ concentration from 2.5 to 10-25 mM or substitution of 1 mM-Ba2+ for 2.5 mM-Ca2+ enhanced their amplitude. ICa was measured directly in whole-cell recordings with patch pipettes after blocking INa with extracellular 1 microM-TTX and K+ currents with intracellular 120-mM Cs+ and 20 mM-TEA. 6. During whole-cell recordings with patch electrodes, extracellular 20 mM-TEA suppressed IK and, to a lesser extent, IA. Extracellular 5 mM-4-AP or a pre-pulse of the membrane potential to -40 mV prior to stronger depolarization completely blocked IA.(ABSTRACT TRUNCATED AT 400 WORDS)