T three intensity levels (0.00003, 0.0001 and 0.0003 cd*s/m2) ten flashes, presented at 0.5 Hz, were averaged. At the next three intensity levels (0.001, 0.003, and 0.01 cd*s/m2), four 10781694 flashes, presented at 0.2 Hz, were averaged. At the next five intensity levels (0.03, 0.1, 0.3, 1, and 3 cd*s/m2), four flashes, presented at 0.1 Hz, were averaged and at the last two intensity levels (10 and 25 cd*s/m2), one flash was presented. Next, photopic flash ERG responses were recorded after 10 minutes of light adaptation (30 cd/m2) using the HMsERG background light. Responses to standard and highintensity flashes (3 and 10 cd*s/m2, an average of 32 flashes at 2 Hz) were recorded. Finally, cone flicker responses to the standard and high-intensity flashes were recorded (3 and 10 cd*s/m2, an average of 128 flashes at 31.25 Hz). A- and b-wave amplitudes were measured from baseline to the first trough and from that trough to the next positive peak, respectively. The kinetics of the responses was measured in terms of implicit times (IT), which is the corresponding time interval between the stimulus onset to the trough or to the positive peak.Statistical AnalysisValues are presented as mean 6 SEM. Student’s t-tests were performed to compare mean values of the apoE3 and apoE4 mice. Significant difference between groups was set at P,0.05.ResultsHistological examination revealed that the general retinal morphology and thickness were similar in the apoE3 and apoE4 mice. Furthermore, the thickness of the outer and inner nuclear layers in which the photoreceptor and bipolar cells reside and of the synaptic outer and inner plexiform layers were also unaffected by the apoE genotype (Figure 1). Next, we examined the possibility that the levels of distinct subpopulations of retinal neurons are affected by apoE4. This was performed immunohistochemically utilizing cell-specific markers, as described in Materials and Methods. As shown in figure 2, thelevels of the photoreceptor and ganglion cells and of the bipolar cells that link them were the same in the apoE4 and apoE3 mice. The levels of the modulatory horizontal and amacrine cells were also not significantly affected by apoE4. We next Gracillin chemical information considered the possibility that retinal synapses, unlike their corresponding perikarya, are affected by apoE4. Immunohistochemical measurements of the synaptic density, utilizing the synaptic protein synaptophysin as a marker revealed, as previously described, pronounced staining in the IPL and OPL [35]. Quantification of this staining in the IPL and OPL revealed that the levels of synaptophysin staining were significantly lower in the apoE4 than in the apoE3 mice (Figure 3A, left panels). Similar results were obtained by immunoblot experiments, which revealed that the levels of the 38 kDa synaptophysin immunoblot band were lower in the apoE4 than in the corresponding apoE3 mice (Figure 3B). The neuronal specificity of this synaptic effect was next assessed both pre-synapticly and post-synapticly. The presynaptic measurements were performed utilizing the pre-synaptic vesicular transporters VGluT1, VGaT and VAChT. The Nal.pone.0066676.gIntegrated miRNA-mRNA Analysis of Chordomasfindings [25]. However, these genes were former is a marker of glutamatergic nerve terminals whereas VGaT resides in both GABAaergic and 1676428 Glycinergic nerve terminals and VAChT resides in chiolinergic nerve terminals. This revealed, in accordance with the literature [36?8], that IPL was stained by VGluT1, VGaT and VAChT (strata s2 and s4) and that the OPL was stained by VGluT1, very weakly by VGaT and not by.T three intensity levels (0.00003, 0.0001 and 0.0003 cd*s/m2) ten flashes, presented at 0.5 Hz, were averaged. At the next three intensity levels (0.001, 0.003, and 0.01 cd*s/m2), four 10781694 flashes, presented at 0.2 Hz, were averaged. At the next five intensity levels (0.03, 0.1, 0.3, 1, and 3 cd*s/m2), four flashes, presented at 0.1 Hz, were averaged and at the last two intensity levels (10 and 25 cd*s/m2), one flash was presented. Next, photopic flash ERG responses were recorded after 10 minutes of light adaptation (30 cd/m2) using the HMsERG background light. Responses to standard and highintensity flashes (3 and 10 cd*s/m2, an average of 32 flashes at 2 Hz) were recorded. Finally, cone flicker responses to the standard and high-intensity flashes were recorded (3 and 10 cd*s/m2, an average of 128 flashes at 31.25 Hz). A- and b-wave amplitudes were measured from baseline to the first trough and from that trough to the next positive peak, respectively. The kinetics of the responses was measured in terms of implicit times (IT), which is the corresponding time interval between the stimulus onset to the trough or to the positive peak.Statistical AnalysisValues are presented as mean 6 SEM. Student’s t-tests were performed to compare mean values of the apoE3 and apoE4 mice. Significant difference between groups was set at P,0.05.ResultsHistological examination revealed that the general retinal morphology and thickness were similar in the apoE3 and apoE4 mice. Furthermore, the thickness of the outer and inner nuclear layers in which the photoreceptor and bipolar cells reside and of the synaptic outer and inner plexiform layers were also unaffected by the apoE genotype (Figure 1). Next, we examined the possibility that the levels of distinct subpopulations of retinal neurons are affected by apoE4. This was performed immunohistochemically utilizing cell-specific markers, as described in Materials and Methods. As shown in figure 2, thelevels of the photoreceptor and ganglion cells and of the bipolar cells that link them were the same in the apoE4 and apoE3 mice. The levels of the modulatory horizontal and amacrine cells were also not significantly affected by apoE4. We next considered the possibility that retinal synapses, unlike their corresponding perikarya, are affected by apoE4. Immunohistochemical measurements of the synaptic density, utilizing the synaptic protein synaptophysin as a marker revealed, as previously described, pronounced staining in the IPL and OPL [35]. Quantification of this staining in the IPL and OPL revealed that the levels of synaptophysin staining were significantly lower in the apoE4 than in the apoE3 mice (Figure 3A, left panels). Similar results were obtained by immunoblot experiments, which revealed that the levels of the 38 kDa synaptophysin immunoblot band were lower in the apoE4 than in the corresponding apoE3 mice (Figure 3B). The neuronal specificity of this synaptic effect was next assessed both pre-synapticly and post-synapticly. The presynaptic measurements were performed utilizing the pre-synaptic vesicular transporters VGluT1, VGaT and VAChT. The former is a marker of glutamatergic nerve terminals whereas VGaT resides in both GABAaergic and 1676428 Glycinergic nerve terminals and VAChT resides in chiolinergic nerve terminals. This revealed, in accordance with the literature [36?8], that IPL was stained by VGluT1, VGaT and VAChT (strata s2 and s4) and that the OPL was stained by VGluT1, very weakly by VGaT and not by.
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