Separation of calcium currents in retinal ganglion cells from postnatal rat

A culture system of the postnatal rat retina was established to investigate Ca²⁺ currents and synaptic transmission in identified neurons. Methods are described that allowed us to select retinal ganglion neurons (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 study was to identify channel specific components in whole-cell Ca²⁺ currents of RGNs and to clarify the potential use of the lanthanide Gd³⁺ as a selective Ca²⁺ channel blocker. About one third of freshly dissociated 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 different components that were functionally rather similar, but could be separated pharmacologically. A significant portion (about 40%) of I_Ca(HVA) was irreversible blocked by the N channel antagonist ω-CgTx (5 μM). The L channel antagonist nifedipine (10 μM) eliminated about 25% of I_Ca(HVA). Thus, about 1/3 of the HVA Ca²⁺ or Ba²⁺ current remained unaffected by either ω-CgTx or nifedipine. ω-AgaTx (200 nM) completely failed to block HVA Ca²⁺ or Ba²⁺ currents in RGNs. Gd³⁺ exerted contrasting actions on LVA and HVA Ca²⁺ currents. While I_Ca(LVA) consistently increased in the presence of Gd³⁺ (0.32–3.2 μM), I_Ca(HVA) always decreased, especially when using higher concentrations of Gd³⁺ (10–32 μM). The blocking action of Gd³⁺ was not restricted to the ω-CgTx-sensitive HVA current component, but also concerned ω-CgTx- and nifedipine-resistant components. The decay of Ca²⁺ currents was accelerated in the presence of Gd³⁺. Even in RGNs lacking I_Ca(LVA), application of 3.2 μM Gd³⁺ significantly reduced the time constant of decay from an average of 64 ms to 36 ms (voltage steps from −90 to 0 mV; 10 mM [Ca²⁺]₀; 26°C). This is in contrast to what had to be expected if an N-type HVA current component was selectively suppressed 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 was followed by a rebound increase of EPSC frequency. A stimulatory effect during application of Gd³⁺ has never been observed. These experiments indicate that RGNs express at least 4 different types of Ca²⁺ currents, that resemble in some aspects T, N and L channel currents. A significant component of the HVA Ca²⁺ current was resistant to the available HVA channel blockers suggesting the presence of a pharmacologically 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⁺ currents, 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⁺ channels are not involved in the regulation of glutamate release.