Cryopreservation of postnatal rat retinal ganglion cells: persistence of voltage- and ligand-gated ionic currents.

Abstract 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°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 × 10 4 vs 7.05 ± 3.67 × 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 neurones 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 ± 6.6mV(n = 10) and upon depolarization fired action potentials. During voltage-clamp in the whole-cell mode, depolarizing voltage steps activated Na + -(I Na ),Ca 2+ -(I Ca ) , and K + -currents in all cells tested (n = 122) . I Na could be reversibly blocked by 1 μM tetrodotoxin added to the external solution. I Ca was blocked by external 250 μM Cd 2+ or 3 mM Co 2+ . In some cells, I Ca consisted of both a transient and prolonged component. The outward K + -current consisted of Ca 2+ -dependent and -independent components. The Ca 2+ -intensitive 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 (I A ) 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 I K , the delayed rectifier, was also found. When the 4-aminopyridine- and tetraethylammonium-insensitive portions of the outward current were further analysed with voltage protocols, and additional slowly decaying potassium current became apparent. The inhibitory aminom acids, GABA (20 μM) and glycine (100 μM), activated chloride-selective currents that were selectively blocked by bicuculline methiodide (10 μM) and strychnine (5 μM), respectively. The excitatory amino acids glutamate (100 μM), N-methyl- d -aspartate (200 μM), and kinate (125 μM) activated currents that were seletively blocked by 2-amino-5-phosphonovalerate (100 μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM), respectively. Acetylcholine-(20 μM) induced currents were blocked by hexamethonium (20 μM), but not atropine (10 μM). The integrity of these ligand receptors and ionic channels in cryopreserved neurons is of critical importance for their normal function. In addition to the traditional role of ion channels in mediating fast cellular information signalling, these channels are also important in neuronal outgrowth and survival. Most importantly, cryopreservation can help reduce the number of animals used for research purposes; by collecting large numbers of cells from a single animal, multiple, temporally-separated electrophysiological experiments can be performed. Also, the capability of storing cells may facilitate the application of both molecular biology and electrophysiology techniques to defined subsets of CNS neurons and the exchange of specific batches of cells between laboratories.