Patch Clamp Analysis of Voltage–Gated Na+ Currents in Human Retinal Bipolar Cells

Abstract

Purpose: : The photoreceptors and retinal bipolar cells were considered non–spiking neurons. However, recently we showed that voltage–gated Na⁺ channels are expressed in human photoreceptors (Neuron, 2001; IOVS, 2005). We performed the patch–clamp recording of human retinal bipolar cells to examine whether voltage–gated Na⁺ currents are observed in human bipolar cells as well as photoreceptors. Whether Na⁺ channel transcripts could be detected in bipolar cells was also examined by using RT–PCR.

Methods: : Voltage–gated currents were recorded using the whole–cell patch–clamp technique on bipolar cells in slice preparations and isolated bipolar cells, obtained by surgically excised human retina. To examine whether Na⁺ channel transcripts are detected in bipolar cells, single–cell RT–PCR analysis was performed.

Results: : The whole–cell patch–clamp recording was performed at inner nuclear layer. The recorded cell was identified based on the characteristic morphology by injected Lucifer yellow. Under voltage–clamp conditions, depolarizing voltage steps induced a transient inward current in some bipolar cells. This current began to be activated at –60 mV and was maximal at –40 mV. This current also decayed within 30 ms. Therefore, it seemed transient calcium current. However, fast transient inward current was observed in several bipolar cells. This current was activated from –60 mV to +10 mV (maximal at –20 mV) and inactivated within 5 ms. And the current was blocked by TTX. These results suggest that human retinal bipolar cells express voltage–gated Na⁺ channels. The transient potassium current and delayed rectifier outward currents were observed in most of bipolar cells. However, the hyperpolarization–activated cation current (h current) was not observed in human retinal bipolar cells (n = 0/16), while h currents were observed in human photoreceptors. Next, we performed a single–cell RT–PCR analysis to test whether bipolar cells express the Na⁺ channel transcript. Na_v1.2 channels identified from retinal mRNA were observed in 5 of 6 bipolar cells. This suggests that human bipolar cells express Na_v1.2 channels as well as human photoreceptors.

Conclusions: : Voltage–gated Na⁺ channels are expressed in human retinal bipolar cells as well as human photoreceptors.

Keywords: bipolar cells • electrophysiology: non-clinical • ion channels