SEPARATION OF CALCIUM CURRENTS IN RETINAL GANGLION-CELLS FROM POSTNATAL RAT

A culture system of the postnatal rat retina was established to invest igate Ca2+ currents and synaptic transmission in identified neurons. M ethods are described that allowed us to select retinal ganglion neuron s (RGNs) in short term cultures (up to 48 h in vitro) and in long-term cultures (3 to 21 days in vitro). The specific aim of the present stu dy was to identify channel specific components in whore-cell Ca2+ curr ents of RGNs and to clarify the potential use of the lanthanide Gd3+ a s a selective Ca2+ channel blocker. About one third of freshly dissoci ated RGNs generated both low voltage activated Ca2+ currents (I-Ca(LVA )) and high voltage activated Ca2+ currents (I-Ca(HVA)). The remaining 2/3 of RGNs in short term culture and most RGNs in long-term culture displayed only I-Ca(HVA). The latter comprised at least three differen t components that were functionally rather similar, but could be separ ated pharmacologically. A significant portion (about 40%) of I-Ca(HVA) was irreversibly blocked by the N channel antagonist omega-CgTx(5 mu M). The L channel antagonist nifedipine (10 mu M) eliminated about 25% of I-Ca(HVA). Thus, about 1/3 of the HVA Ca2+ or Ba2+ current remaine d unaffected by either omega-CgTx or nifedipine. omega-AgaTx (200 nM) completely failed to block HVA Ca2+ or Ba2+ currents in RGNs. Gd3+ exe rted contrasting actions on LVA and HVA Ca2+ currents. While I-Ca(LVA) consistently increased in the presence of Gd3+ (0.32-3.2 mu M), I-Ca( HVA)) always decreased, especially when using higher concentrations of Gd3+ (10-32 mu M) The blocking action of Gd3+ was not restricted to t he omega-CgTx-sensitive HVA current component, but also concerned omeg a-CgTx- and nifedipine-resistant components. The decay of Ca2+ current s was accelerated in the presence of Gd3+. Even in RGNs lacking I-Ca(L VA), application of 3.2 mu M Gd3+ significantly reduced the time const ant of decay from an average of 64 ms to 36 ms (voltage steps from -90 to O mV; 10 mM [Ca2+](o);26 degrees C). This is in contrast to what h ad to be expected if an N-type HVA current component was selectively s uppressed by Gd3+. Gd3+ diminished glutamatergic spontaneous synaptic activity in retinal cultures tested during the 3rd week in vitro. Both frequency and amplitude were reduced. Occasionally, the application w as followed by a rebound increase of EPSC frequency. A stimulatory eff ect during application of Gd3+ has never been observed. These experime nts indicate that RGNs express at least 4 different types of Ca2+ curr ents, that resemble in some aspects T, N and L channel currents. A sig nificant component of the HVA Ca2+ current was resistant to the availa ble HVA channel blockers suggesting the presence of a pharmacologicall y distinct type of HVA Ca2+ channel type in RGNs. Our experiments also show that Gd3+ is not suitable for isolation of HVA subcomponents in RGNs, but it can be used to distinguish between LVA and HVA Ca2+ curre nts, as these currents reacted to Gd3+ in an opposite way. The purely depressive effect of this lanthanide on spontaneous synaptic activity is consistent with the assumption that in retinal neurons LVA Ca2+ cha nnels are not involved in the regulation of glutamate release.