| X-linked congenital stationary night blindness (X-CSNB) is a congenital non-progressive retinal disorder characterized by defective in rod signal system. It can be divided into complete and incomplete form depending on electroretino-gram (ERG) and X-link or autosomal forms depending on genetic mode. The pa-thology mechanism of CSNB is not very clear now and there have no effective therapeutic methods for treatment. Model animal is an essential element for the investigations of hereditary diseases. The spontaneous CSNB model rats devel-oped by our laboratory will improve those investigations as following: the pa-thopoiesis gene of these rats has been identified--Cacna1f gene, so their genetic trait is as X-linked and incomplete form CSNB (XL-iCSNB);electrophysiology and behavioral research demonstrated that they have overcome shortcomings re-flected by genetic engineering model animals, such as obvious discrepancy from clinical circumstance. Therefore they would be a useful model for investigations of CSNB's pathology mechanism and the circuit of retinal signal transmission. Bipolar cells (BCs) play a critical role in the process of retinal signal trans-mission, especially in the interaction between retinal rod and cone signal trans-mission path. The L-type calcium channel encoded by Cacna1f gene has been considered as a key point in triggering off neurotransmitter vesicular release of retinal neuron. The dysfunction of L-type calcium channel might cause distur-bances of retinal signal transmission. In order to test the hypothesis, whole-cell patch clamp technique was adopt to investigate the electrophysiological and pharmacological properties of L-type calcium channel of RBCs in iCSNB rats in this study. As the anatomic structure and physiological function of vertebrate re-tina have great resemblances, the research might provide proof for elucidation of CSNB's pathology mechanism and subsequent invention of effective therapeutic measures.Methods and materialThe CSNB rats identified and breed by our laboratory were adopted in this re-search (4-8weeks old,F20/F21). Wild SD rats as control were provided by La-boratory Animal Center of Fourth Military Medical University. According to the reference, eyeball was extracted from rats which were anesthetized and decapi-tated and 200μm retinal vertical slices were prepared by hand microtome. Under infrared microscope,the RBCs from retinal slices were identified by their special shape and established a tight-seal whole-cell recording configurations for calcium channel currents recording. Resting membrane capacitance (restCm) and the in-crease of restCm(△Cm) after protocol activation(depolarization from -60 mV to 0 mV, time course 200ms) were used to calculate the exocytotic index(EI). L-type calcium channel blocker and GABA-receptor blocking pharmacon PTX were used for observing the pharmacology properties of L-type calcium channel in RBCs. Data were analyzed off-line using pClamp8.1 (Axon, America) and Origin Programs (Microcal software, UK).Results1. Inward currents were recorded both in SD and CSNB rat group. The electro-physiology characteristic of these inward currents were consistent with L-type calcium currents': activated at high depolarize potential; very slow inactivation; channel ion carrier was Ca2+ ----similar currents could not be recorded if Ca2+ was replaced with Mg2+ in the extracellular perfusate.2. Pharmacology properties of these inward currents : At low concentration (10μm) ,L-type specified calcium channel blocker nifedipine could obviously and effectively block these currents both in SD group and CSNB group. Combining with electrophysiology characteristic of these currents, it might derive the con-clusion that the currents recorded in experiment were L-type calcium currents.3. The L-type calcium currents in SD group activated at -40mv, and reached their peak amplitude between the -20 and -30mV with an average of -33.2±2.5pA dur-ing ramp depolarizing (n=20); whereas CSNB group activated between the -20 and -30mV, and reached their peak amplitude around the of -15mV with an aver-age of 12.2±2.3pA during ramp depolarizing (n=23).4. Outwards currents overlaying on calcium current during depolarization at higher degrees (-20 and -30mV) were observed in SD group( n=11) .The ampli-tude of the currents were closely related to the position and depth of RBCs in re-tinal vertical slices, and they could be effectively eliminated by GABA-receptor blocker PTX(100μm). But no similar current was observed in RBCs of CSNB group during depolarization.5.After 200ms depolarization from -60mv to 0mv, the restCm of both groups increased(△Cm). The EI (Exocytotic index )was calculated based on△Cm. The mean restCm,△Cm and EI were 4146.600±544.202 fF, 29.600±7.472 fF and 0.723±0.199 in SD rat group (n=20) and 4356.957±549.493, 12.348±6.919 and 0.291±0.173 in CSNB group (n=23), respectively. Compared with SD group, the△Cm and EI were significantly lower in CSNB rat group ( p<0.01).Conclusion1. L-type calcium channel currents could be recorded both in RBCs of SD and CSNB rat groups. The peak amplitude of L-type calcium channel currents in CSNB rat group is significantly lower than SD rat group. It suggests that the Ca2+ concentration in synapse terminals of RBCs of CSNB rat might be too low to trigger off the neurotransmitter vesicular release at normal velocity and might arise disturbances of retinal signal transmission to third order neurons.2. The I-V curve shift to right in RBCs of CSNB might deteriorate the depolariza-tion of RBCs at physiology dark adaptation potentials(-70mV to -20mV).3. Inhibitory postsynaptic currents (IPSCs) could not be recorded in CSNB rat suggests that disruption of neurotransmitter release might cause dysfunction of negative feedback from rod-amacrine cells. This dysfunction of negative feed-back might interrupt the amplification mechanism and flexibility of visual signal transmission circuit.4. The EI is decreased in RBCs of CSNB rat than SD rat. It may provide more direct proof for the argument of neurotransmitter vesicularr release disruption in CSNB group's RBCs.In summary, the alterations of L-type calcium channel property in rod bipolar cell of CSNB led to disruption of neurotransmitter release and visual signal transmission, which might be one of pathophysiological mechanisms for CSNB.
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