Patch-clamp recording from identified rat ciliary ganglion neurons in primary culture

Adult rat parasympathetic ciliary ganglion (CG) neurons were retrogradely labelled by intraocular injection of the carbocyanine fluorescent dye 1,1-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI). Whole-cell and nystatin perforated patch recording techniques were then used to examine the electrophysiological properties of labelled CG neurons growing in primary culture. The resting membrane potential of CG neurons in dissociated cell culture was -50 +/- 8 mV, and isolated neurons fired overshooting action potentials in response to depolarizing current injection. Voltage-clamp recordings of membrane currents revealed a transient tetrodotoxin-sensitive Na+ inward current and both sustained and transient outward K+ currents. Sustained outward K+ current was reduced (55-77%) by 5 mM tetraethylammonium and to a lesser extent (42-46%) by superfusion with nominally Ca2+ free external solution. Transient outward current was blocked by 100 microns 4-aminopyridine and exhibited steady-state inactivation at potentials depolarized to -50 mV. These data demonstrate that identified adult mammalian CG neurons can be successfully maintained in culture. Cultured CG neurons retain electrical excitability, with voltage-sensitive Na+ and K+ currents giving rise to action potentials.