Study On The Unique Functional Properties And Mechanisms Of Retinal Ganglion Cells In The Mouse Retinal On-/Off-Pathways

ObjectiveThe retina senses visual signals, codes visual information into different spike trains, and then sends them into the brain. Distinct signaling pathways within the retina are involved in coding different visual information. The cone pathways mediate coding of visual information for photopic vision at relatively high light intensities. By contrast, the rod pathways underlie the coding for scotopic vision at relatively low light intensities. Light increments and decrements are processed within different cone and rod signaling pathways. The pathways for signaling light increments are called On-pathways, the pathways for signaling light decrements are referred to as Off-pathways.Two APB sensitive rod pathways convey the light increment signal in scotopic vision:①rods→rod bipolar cells→All amacrine cells→gap junctions→On cone bipolar cells→On ganglion cells.②rods→gap junctions→cones→On cone bipolar cells→On ganglion cells. APB blocks these two pathways. Three distinct APB sensitive and APB insensitive rod Off-pathways process the light decrement signals in the dark-adapted retina:①rods→rod bipolar cells→All amacrine cells→glycinergic synapses→Off cone bipolar cells→Off ganglion cells.②rods→gap junctions→cones→Off cone bipolar cells→Off ganglion cells.③rods→directly contacting Off cone bipolar cells→Off ganglion cells. In these three rod Off-pathways,①is APB sensitive, while②and③are APB insensitive. Under photopic conditions, signaling light incre-ments pathway:cones→On cone bipolar cells→On ganglion cells, signaling light decrements pathway:cones→Off cone bipolar cells→Off ganglion cells.It has been found that the light threshold of APB sensitive rod Off-pathways is lower than that of APB insensitive rod Off-pathways. Interestingly, human psychophysical studies suggest two rod pathways:one thransferring slow and low-threshold signals, and another conveying faster and high-threshold signals. NO selectively blocks Off responses in the APB sensitive rod Off-pathway in the ferret retina. Additionally, the laboratory demonstrated that lack of nNOS reduced the sensibivity of RGCs to light under dark adaptation. Thus it can be seen there are different functional properties and mechanisms in distinct On-/Off-pathways.As we all know, rod and cone pathways converge onto individual RGC. To investigate the response properties, the stimulus/response, the effects of NO on sensitivity of RGC to light and the mechanisms, whole-cell patch-clamp recordings were made from RGCs.Methods1. C57BL/6 mouse retinal preparation.2. Light-evoked responses were obtained by delivering spots of light to the retina from a computer monitor, with a green phosphor.3. Under scotopic condition, current-clamp records light responses of RGCs in APB sensitive and insensitive rod Off-pathways, and measure the peak firing rate and latency of those light responses.4. Current-clamp recordings were made to investigate the frequency following properties of RGCs to flash light under both scotopic and photopic conditions.5. Under scotopic conditions, nitric oxide donor, SNAP is applicated to test the effects on the light responses of RGCs in the APB sensitive and insensitive rod Off-pathways.6. Spontaneous and light-evoked excitatory and inhibitory synaptic current and voltage were recorded from RGCs to investigate the effects and mechanisms of L-NAME on the sensitivities of RGCs to light under photopic conditions.7. Images of the labled cells were taken by an confocal microscope.Results1. Under scotopic conditions, the latencies of Off responses of RGCs in the APB sensitive rod Off-pathways are significantly longer than those in the APB insensitive rod Off-pathways, whereas there is no significant difference for peak firing rate in those two pathways.2. Under scotopic conditions, the frequency following properties of RGCs in the APB sensitive rod Off-pathway were lower than those of RGCs in the APB insensitive rod Off-pathways. RGCs in the On-pathways could follow a relatively high stimulus frequency. After switching to photopic conditions, the frequency following properties of RGCs in the APB sensitive and insentive rod Off-pathwyas were increased.3. Under scotopic conditions, NO selectively blocks light-evoked responses in the APB sensitive rod Off-pathway in the mouse retina.4. Under photopic conditions, L-NAME decreased the sensitivity of RGCs to light.5. Under photopic conditions, spontaneous and light-evoked IPSCs of RGCs in the On-/Off-pathways were reduced by L-NAME. L-NAME signifycantly reduced the amplitude and frequency of spontaneous and light-evoked EPSCs, compared with STR and PTX alone.6. Under photopic conditions, L-NAME did not change sEPSCs of RGCs after photobleaching cones by HA. L-NAME did not affect the sEPSCs of RGCs after APB treatment reducing glutamate release onto RGCs.DiscussionOur research proved that there are different functional properties in the distinct On-/Off-pathways. Under scotopic conditions, the latencies of Off responses of RGCs in the APB sensitive rod Off-pathways are significantly longer than those in the APB insensitive rod Off-pathways. There are two possible reasons:①The retinal circuits of these two pathways are different. There are more synapses in the APB sensitive rod Off-pathways than in the APB insensitive rod Off-pathways.②The efficiency of APB sensitive rod Off-pathways are slower than that of APB insensitive rod Off-pathways. We also found that the light-evoked reponses of RGCs in the APB insensitive rod Off-pathways could follow much higher stimulus frequencies than those in the APB sensitive rod Off-pathways. Volgyi found that APB sensitive rod Off-pathways has been shown to respond at a lower thresholds of light inten-sity than APB insensitive rod Off-pathways. Collectively, APB sensitive rod Off-pathway processes the light decrement signal with low frequency and intensity, and transfers the information to the brain slowly. In contrast, APB insensitive rod Off-pathways mediate the light decrement signal at relatively high frequencies and intensities, and convey this information to the brain rapidly. Our previous data and the results in this study demonstrated NO specifically modulates the APB sensitive rod Off-pathway, which suggests the specific modulation of NO on this pathway is a common mechanism across different animal species.By switching recordings of each RGC from scotopic to photopic conditions, we could dissect the frequency following properities of different components within these visual pathways. The RGCs in the APB sensitive rod Off-pathway under scotopic conditions could follow much higher stimulus frequencies after switching to photopic conditions, while the frequency following properties of On-pathways are significantly higher than the APB sensitive rod Off-pathway. These results indicate rods, rod bipolar cells, All amacrine cells, Off cone bipolar cells and Off RGCs can follow high stimulus frequencies. Collectively, the glycinergic synapse between All amacrine cells and Off cone bipolar cells may limit the frequency following through the APB sensitive rod Off-pathways. When the light turns on All amacrine cells release glycine to inhibit Off cone bipolar cells, and thus the synaptic inputs from Off cone bipolar cells to Off RGCs are reduced. When the light turns off, the inhibition from glycine to Off cone bipolar cells is removed, allowing Off cone bipolar cells to release glutamate to Off GRCs, which generate Off responses. The removal of glycine inhibition from Off cone bipolar cells is the trigger for Off responses in the APB sensitive rod Off-pathway. Under high frequency stimuli, each On and Off cycles are short, which affects the generation and removal of glycine inhibition to Off cone bipolar cells, thereby limiting the high frequency stimuli following through the APB sensitive rod Off-pathway. We also tested the frequency following properties of APB insensitive rod Off-pathways by using the same protocol. The results showed the Off cone bipolar cells and Off GRCs in this pathway could follow high frequency stimuli. So we inferred that rods or cones maybe the sites which limit the frequency following properties of this pathway. It has been documented that cones have faster rates of responses than rods. In the signaling pathways, if a component has the lowest frequency following property,this component serves as the limiting mechanism that controls the high stimulus frequencies flowing through the signaling pathway. Thus, in this pathway only rods can not follow high frequencies, therefore, rods serve as the mechanism that limits the frequency following through the APB insensitive Off-pathway. Our previous data showed that NO selectively blocked the APB sensitive rod Off-pathway and reduced the sensitivity of RGCs to light under scotopic conditions. In the current study we found that L-NAME significantly de-creased the amplitude of spontaneous and light-evoked EPSPs and frequency of sEPSCs of RGCs after blocking inhibitory synaptic currents. These results support that NO stimulates excitatory neurotransmitter release. So we inferred that L-NAME reduces endogenous NO in the retina to reduce the excitatory synaptic inputs to RGCs and consequently reduces theirs sensitivity to light. We also investigated the possible mechanisms for inhibition of NOS by L-NAME to diminish excitatory synaptic currents of RGCs. The results demonstrate that L-NAME did not reduce the frequency of sEPSCs with or without STR and PTX after photobleaching, thereby suggesting that L-NAME acts on cones to reduce presynaptic glutamate release under light adaptation, which decreases the sEPSCs of RGCs. This may account for the desensitization of RGCs to light stimuli. Under photopic conditions, more possible mechanisms that L-NAME reduced the sensitivity of RGCs to light remains to be established.Conclusions1. APB sensitive rod Off-pathway processes the light decrement signal with low frequency and intensity, and transfers the information to the brain slowly. APB insensitive rod Off-pathways mediate the light decrement signal at relatively high frequencies and intensities, and convey this information to the brain rapidly.2. NO specifically modulates the APB sensitive rod Off-pathway in mice retina.3. The glycinergic synapse between All amacrine cells and Off cone bipolar cells may limit the frequency following through the APB sensitive rod Off-pathways.4. Rods serve as the mechanism that limits the frequency following through the APB insensitive Off-pathway.5. L-NAME inhibits NOS in cones to reduce NO level, which decreased excitatory synaptic currents and the sensitivity of RGCs to light under photopic conditions.