Cryopreservation of postnatal rat retinal ganglion cells: persistence of voltage- and ligand-gated ionic currents.
Established methods for cryopreservation of living cells were modified for freeze-storage of postnatal retinal ganglion cells from rat. Retinal cell suspensions containing fluorescently labeled ganglion cells were frozen after addition of 8% dimethyl sulfoxide and stored at -80 degrees C for up to 66 days. Viability of identified retinal ganglion cells was assessed by their ability to take up and cleave fluorescein diacetate to fluorescein. No significant difference was found in the number of living retinal ganglion cells when cells obtained from the same dissociation were counted before and after freezing (6.65 +/- 2.37 x 10(4) vs 7.05 +/- 3.67 x 10(4) retinal ganglion cells per ml, respectively; mean +/- S.D., n = 4). In culture following cryopreservation, the cells appeared morphologically normal, and developed neurites and growth cones similar to their freshly dissociated counterparts. Since very little is known about the electrophysiology and membrane properties of neurons after cryopreservation, we used the whole-cell configuration of the patch-clamp technique to study voltage- and ligand-gated conductances in cryopreserved retinal ganglion cells. The cryopreserved retinal ganglion cells studied under current-clamp maintained resting potentials of -60.9 +/- 6.6 mV (n = 10) and upon depolarization fired action potentials. During voltage-clamp in the whole-cell mode, depolarizing voltage steps activated Na(+)-(INa), Ca(2+)-(ICa), and K(+)-currents in all cells tested (n = 122). INa could be reversibly blocked by 1 microM tetrodotoxin added to the external solution. ICa was blocked by external 250 microM Cd2+ or 3 mM Co2+. In some cells, ICa consisted of both a transient and prolonged component. The outward K(+)-current consisted of Ca(2+)-dependent and -independent components. The Ca(2+)-insensitive portion of the K+ outward current was separated into four distinct components based upon pharmacological sensitivity and biophysical properties. In many cells, a rapidly inactivating current similar to the A-type K(+)-current (IA) observed in freshly cultured retinal ganglion cells was isolated by its greater sensitivity to 4-aminopyridine (5 mM) than to tetraethylammonium (20 mM). A tetraethylammonium-sensitive current with a more prolonged time course reminiscent of IK, the delayed rectifier, was also found. When the 4-aminopyridine- and tetraethylammonium-insensitive portions of the outward current were further analysed with voltage protocols, an additional slowly decaying potassium current became apparent. The inhibitory amino acids, GABA (20 microM) and glycine (100 microM), activated chloride-selective currents that were selectively blocked by bicuculline methiodide (10 microM) and strychnine (5 microM), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Sucher
N J
NJ
Department of Neurology, Children's Hospital, Boston, MA.
Cheng
T P
TP
Lipton
S A
SA
D000109:Acetylcholine / Q000494:pharmacology; D000596:Amino Acids / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D015925:Cryopreservation; D004121:Dimethyl Sulfoxide; D004566:Electrodes; D004594:Electrophysiology; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008024:Ligands; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D051381:Rats; D012165:Retinal Ganglion Cells / Q000378:metabolism*
D000596:Amino Acids; D007473:Ion Channels; D008024:Ligands; D015221:Potassium Channels; D000109:Acetylcholine; D004121:Dimethyl Sulfoxide