L-type Calcium Currents In The Rod Bipolar Cells Of SD Rat

Retinal bipolar cells are glutamatergic interneurons that transmit light-induced photoreceptor signals to amacrine cells and ganglion cells in the inner plexiform layer via chemical synapses. And bipolar cells also are restrained by amacrine cells and ganglion cells, so bipolar cells are key in the process of transmission of signals. On the basis of morphological and physiological criteria, two different types of bipolar cells can be distinguished: the cone bipolar cells(CBCs), which are subdivided into several classes, and the rod bipolar cells (RBCs). Rod bipolar cells of rat are easily identified, but cone bipolar cells are divided into nine types by morphology.Because calcium is very important for the neurotransmitter, many scientists are working on the calcium channels. And they discovered that the dilivery of neuro-transmitter of rod bipolar cells were based on the L-type Calcium currents. Thus the disorder of the L-type Calcium currents would be bad for signal transmission of rod bipolar cells.Two kinds of Spontaneous mutation rat were report by our department, con-genital stationary night blindness (CSNB) and retinal cone degeneration (RCD) .Now wo find the virulence gene of CSNB is cacna1f,which is the gene of the L-type calcium channel. So wo need to find the method to research the chan-nels and find the characteristic of the L-type calcium channel, it would help us research two kind of the special rats in our department.To know how to research the channels, our used the whole cell patch clamp technique on the rod bipolar cells of the SD rat. The first way to getting the cells is dissociating retina mechanically, and the second way is making retina slice. We find the second way is good for research.Material and methodWe got the bipolar cells from 4-7-wk-old SD rats.The first method, Retinas were removed and placed in a Hanks' solution with papain (30,000USP u/mg,Calbiochem-Novabiochem), and the retinas were mechanically dissociated by triturating with a glass pipette. Then we observed the cells by the inverted microscope(IX70,Olympus,Japan),and recorded the currents by EPC10(Heka,Germany)and Pulse 8.6(Heka,Germany).The second method, we prepared the retina slices by hand microtome(ST-20, Narishige, Tokyo, Japan).Then we observed the cells by the microscope (Heka,Germany) and recorded the currents by EPC10(Heka,Germany)and Pulse 8.6(Heka,Germany).result1. By the first method, we could observe many healthy rod bipolar cells, but we could not record the L-type calcium channel currents except one. However, we had recorded the GABA currents2. By the second method, we observed the healthy rod bipolar cells, and re-corded the currents. Slow capacitance :3.2±0.3pF(n=11),L-type calcium currents were recorded,voltage-step:31.1±3.3 pA(n=11),voltage-ramp:32.5±3.1pA(n=11); 3. After record, we put the nifedinpine into the solution(5μM),the currents were blocked by nifedipine.Conclusion1. The first method: we could not record the L-type calcium currents. The reason may be the injury of the axon of the rod bipolar cells.2. The second method: the characteristic of the currents were according to the L-type calcium currents. And the nifedipine could block the currents. The results were according to the articles of others. So the currents re-corded in the experiment were L-type calcium currents.It is feasible that We can record the L-type calcium currents using the whole cell patch clamp technique in the rod bipolar cells.