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NEURAL REGENERATION RESEARCH Volume 7,Issue 16,June 2012 Cite this article as:Neural Regen Res.2012;7(16):1234-1240.1234 Jufang Huang,M.D.,Researcher,Department of Anatomy and Neurobiology,Xiangya School of Medicine,Central South University,Changsha 410013,Hunan Province,China Corresponding author:Dan Chen,M.D.,Lecturer,Department of Anatomy and Neurobiology,Xiangya School of Medicine,Central South University,Changsha 410013,Hunan Province,China Received:2012-02-22 Accepted:2012-05-03(N20111028002/H)Huang JF,Zhou LH,Wang H,Luo J,Xiong K,Zeng LP,Chen D.Spatiotemporal alterations of presynaptic elements in the retina after high intraocular pressure.Neural Regen Res.2012;7(16):1234- www.nrronline.org doi:10.3969/j.issn.1673-5374.2012.16.005 Spatiotemporal alterations of presynaptic elements in the retina after high intraocular pressure*Jufang Huang,Lihong Zhou,Hui Wang,Jia Luo,Kun Xiong,Leping Zeng,Dan Chen Department of Anatomy and Neurobiology,Xiangya School of Medicine,Central South University,Changsha 410013,Hunan Province,China Abstract A rat model of acute high intraocular pressure was established by injecting saline into the anterior chamber of the left eye.Synaptophysin expression was increased in the inner plexiform layer at 2 hours following injury,and was widely distributed in the outer plexiform layer at 37 days,and then decreased to the normal level at 14 days.This suggests that expression of this presynaptic functional protein experienced spatiotemporal alterations after elevation of intraocular pressure.There was no significant change in the fluorescence intensity and distribution pattern for synapse-associated protein 102 following elevated intraocular pressure.Synapse-associated protein 102 immunoreactivity was confined to the outer plexiform layer,while synaptophysin immunoreactivity spread into the outer plexiform layer and the outer nuclear layer at 3 and 7 days following injury.These alterations in presynaptic elements were not accompanied by changes in postsynaptic components.Key Words synaptophysin;synapse-associated protein 102;synaptic plasticity;elevated intraocular pressure;retina;neural regeneration Abbreviations SYN,synaptophysin;SAP102,synapse-associated protein 102;IPL,inner plexiform layer;OPL,outer plexiform layer;HIOP,high intraocular pressure INTRODUCTION The morphologic changes in synapses and the modulation of the strength or efficacy of synaptic signaling are commonly known as synaptic plasticity.Synaptic plasticity,which plays an important role in the development of synaptic connections and in the functioning of the mature nervous system,is dependent on presynaptic as well as postsynaptic changes.Synaptophysin(SYN),also known as P38,is an acidic calcium binding glycoprotein closely associated with synaptic structure and function,and is an integral membrane protein of synaptic vesicles1.SYN is widely used as a marker of synaptogenesis and presynaptic terminals2-3.Synapse-associated protein 102(SAP102)is a member of the membrane-associated guanylate kinase protein family,and is enriched in postsynaptic densities and is involved in receptor-mediated synaptic transmission.SAP102 is crucial for the regulation of synaptic signaling and plasticity4-5.In the rat retina,synapses in the outer and inner plexiform layers(OPL and IPL)play an important role in visual signal transmission6.Studies on synaptic changes in these regions following injury may provide significant insight into pathogenetic and protective mechanisms in eye diseases such as glaucoma.Elevation of intraocular pressure(IOP)is a risk factor for glaucoma.Previous studies have shown that acute high intraocular pressure(HIOP)causes thinning of the inner part of the retina and selective loss of cells in the ganglion cell www.nrronline.org Huang JF,et al./Neural Regeneration Research.2012;7(16):1234-1240.1235 layer;thus visual function suffers irreversible damage7.Does elevated IOP impact on synapses in the retina?We previously investigated the expression of SYN between 1 and 14 days after acute HIOP,and found that changes in protein expression mainly occurred within the OPL and outer nuclear layer.SYN expression was transiently up-regulated,with broadened distribution,returning to a normal pattern by day 148.Unfortunately,we were unable to investigate alterations in synaptic structure and function because of significant damage in the IPL after 1 day following acute HIOP.The formation and maintenance of normal synaptic structure and function needs the mutual participation of presynaptic and postsynaptic elements.However,the changes in postsynaptic elements in the retina after elevated IOP are still unclear.In the present study,we investigate the expression and relationship of presynaptic and postsynaptic markers SYN and SAP102 in the OPL and IPL within 1 day(at which stage there is no obvious structural loss in the IPL)and after 1 day following acute elevated IOP to understand synaptic changes in the rat retina.RESULTS Quantitative analysis of experimental animals A total of 108 healthy adult Sprague-Dawley rats were included in this experiment.Two animals died after anesthesia,four died after induction of HIOP,and six rats were deemed invalid due to unsuccessful induction of HIOP.The remaining 96 rats were equally and randomly divided into sham surgery,HIOP 2 hours,6 hours,12 hours,1 day,3 days,7 days and 14 days groups.Six rats from each group were used for immunofluorescence detection,and the remaining six rats for immunoblotting.Acute HIOP model was established by injecting saline in the anterior chamber of the left eye in animals in the HIOP groups.The right eye served as the normal control.A total of 96 rats were included in the final analysis.SYN expression in the retina of rats with acute HIOP The expression of SYN protein in the retina of normal and acute HIOP rats was examined using immunofluorescence staining(Figure 1).Figure 1 Fluorescence immunoreactivity of synaptophysin(SYN)and synapse-associated protein 102(SAP102)following acute high intraocular pressure(HIOP)in the rat retina under a fluorescence microscope.Panels A-I depict SYN(red)antibody labeling in the normal control,sham surgery,and HIOP 2 hours,6 hours,12 hours,1 day,3 days,7 days and 14 days groups,respectively.Panels A-I indicate SAP102(green)antibody labeling in the normal control,sham surgery,and HIOP 2 hours,6 hours,12 hours,1 day,3 days,7 days and 14 days groups,respectively.Panels A*-I*show double-labeling of SYN(red)and SAP102(green)antibody in the normal control,sham surgery,and HIOP 2 hours,6 hours,12 hours,1 day,3 days,7 days and 14 days groups,respectively.Nuclei of the cells were marked with Hoechst in A*(blue).The rectangle below in H*is a higher magnification of the upper selected region.ONL:Outer nuclear layer;OPL:outer plexiform layer;INL:inner nuclear layer;IPL:inner plexiform layer;GCL:ganglion cell layer.Scale bar represents 25 m.Huang JF,et al./Neural Regeneration Research.2012;7(16):1234-1240.1236 Immunofluorescence for SYN was most prominent in the OPL and IPL in the normal retina,with a punctate appearance(Figure 1A).The expression and distribution of SYN were stable in the OPL within 1 day of HIOP induction.However,in the IPL within 1 day,SYN immunofluoresence appeared slightly more intense than in the normal eye(Figures 1CF).After 1 day,SYN immunoreactivity gradually broadened in the OPL and extended into the inner part of the outer nuclear layer where there was no SYN immunostaining normally,reaching maximum distribution in the OPL and outer nuclear layer on day 7 post injury.On day 14,the distribution of SYN in the OPL and outer nuclear layer narrowed,gradually returning to normal.In this period,SYN immunoreactivity in the IPL was still present in the entire layer,but IPL structure was markedly lost and the IPL became progressively thinner,as reported previously7,9-10(Figures 1GI).SYN immunoreactivity in the sham surgery group was similar to the normal control group(Figure 1B).Western blot analysis was employed to examine the expression of SYN protein in the retina following acute HIOP.SYN expression was easily detectable in the normal retina as a single 38 kDa band.Expression began to increase and the band became black and thick in the acute HIOP rats at 2 hours compared with the normal control group,and then decreased gradually after 6 hours(Figure 2).Statistical analysis showed that expression was significantly upregulated at 2 hours compared with the normal control group,and its relative integrated absorbance value was higher than in the normal control group(P 0.05,vs.normal control group;Figure 2).From 3 to 14 days,the SYN expression reached the lowest level,which was significantly lower than in the normal control group(P 0.01;Figure 2).Double labeling for SYN and SAP102 in the retina of rats with acute HIOP In the normal retina,immunofluorescence for SAP102 was distinctly located in the IPL and OPL,and was similar to the distribution of SYN,with a punctate appearance in the inner part of the IPL(Figure 1A).In the OPL,SAP102 immunoreactivity mainly overlapped with that of SYN,although the distribution of SAP102 in the OPL was closer to the inner nuclear layer,while SYN was closer to the outer nuclear layer(Figure 1A*).In the IPL,SAP102 immunoreactivity mostly overlapped with that for SYN.Furthermore,weak labeling was also present in the inner nuclear layer.There was no significant change in the fluorescence intensity or distribution pattern for SAP102 within 1 day of elevated IOP(Figures 1AI).Both SAP102 and SYN expression in the IPL was decreased,accompanied with a marked reduction in the thickness of the IPL,after 1 day,but their distribution pattern did not change remarkably.It is worth noting that while SAP102 immunoreactivity did not extend beyond the OPL,SYN labeling gradually broadened in the OPL from the third day after injury and spread to the inner part of the outer nuclear layer,where there was no SYN immunostaining under normal conditions.Only SYN labeling was detected in the outer nuclear layer,and there was no double labeling of SAP102 and SYN.Immunofluorescence for SYN and SAP102 in the sham surgery group was similar to the normal control group(Figures 1A*I*).DISCUSSION SYN participates in synaptic vesicle formation and exocytosis,playing an important role in neurotransmitter release2,11.SYN participates in multiple crucial aspects of synaptic vesicle trafficking,including the initiation of Ca2+-dependent neurotransmitter release,recycling of synaptic vesicles,synaptogenesis,as well as in synaptic plasticity associated with short-term depression and long-term potentiation12-14.SYN immunoreactivity has been used to label synaptic densities in many studies,Figure 2 Western blot assay for expression of synaptophysin(SYN)protein in rats with acute high intraocular pressure(HIOP).Data are presented as mean SD.aP 0.05,vs.normal control group(one-way analysis of variance).To quantify the temporal change in SYN protein,six eyes were evaluated at each survival time point,and all experiments were performed three times.N:normal control group;sham:sham surgery group;2 h,6 h,12 h,1 d,3 d,7 d,14 d:survival time of 2,6,12 hours,1,3,7 and 14 days after operation.Fold change of SYN/GAPDH a a a a N sham 2 h 6 h 12 h 1 d 3 d 7 d 14 d Survival time 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 N sham 2 h 6 h 12 h 1 d 3 d 7 d 14 d SYN GAPDH 38 kDa 36 kDa Huang JF,et al./Neural Regeneration Research.2012;7(16):1234-1240.1237 and its expression is a reliable indicator of synaptogenesis15-16.In the present study,we found that the expression of SYN in the rat retina following acute elevated IOP had a distinct spatiotemporal pattern.The results of SYN expression in the OPL after 1 day were in accordance with our preliminary work8,suggesting that synapses in the retinal OPL might undergo functional enhancement or synaptogenesis following elevated IOP determined by western blot method.The reduction in SYN expression after 3 days of elevated IOP might be due to extensive loss of SYN protein in the IPL.SYN expression increased in the IPL in the early stage,with a widened distribution in the OPL in the middle stage of injury,and recovered to normal in the later period.The spatiotemporal alterations indicate that synapses in the retina might undergo plastic changes,internally to externally,following acute IOP elevation,which may be due to enhanced synaptic activity or new synapse formation.Changes in synaptic function or formation and maturation of new synapses require the coordinated participation of both presynaptic and postsynaptic elements.We sought to clarify whether retinal synaptic plasticity was accompanied by changes in the expression of the presynaptic protein SYN,and if this was linked to alterations of postsynaptic elements as well.We also examined expression of the postsynaptic marker SAP102 in rats with elevated IOP.SAP102 is a member of the membrane-associated guanylate kinase protein family,which are essential proteins at the postsynaptic density,clustering and anchoring glutamate receptors and other proteins at synapses,including postsynaptic density 95(SAP90),postsynaptic density 93(Chapsyn-110),SAP102 and SAP9717-18.These proteins are required for the proper localization and function of glutamate receptors and K1 channels,and are involved in neurotransmitter release and nerve growth and development and also play crucial roles in synaptic organization and plasticity19-22.SAP102 is highly expressed at postsynaptic sites of excitatory synapses in both young and mature neurons as well as in dendrites and axons,and mediates receptor trafficking during synaptogenesis4-5,18.SAP102 is concentrated in the IPL with a punctate appearance in processes,which are postsynaptic at bipolar cell ribbon synapses(dyads).Furthermore,distinct SAP102 labeling was also present in horizontal cell processes in the OPL,which is inserted as lateral elements into photoreceptor ribbon synapses(triads)23.We detected SAP102 immunoreactivity in the entire IPL and OPL.After elevated IOP,the fluorescence intensity and distribution pattern for SAP102 did not show substantial changes.In the IPL,SAP102 expression mostly overlapped with SYN,and their expression gradually decreased with time of injury and with the thinning of the IPL.However,SAP102 immunoreactivity remained confined to the OPL,whereas SYN distribution broadened and extended into the outer nuclear layer.These results indicate that presynaptic changes might not be accompanied by changes in postsynaptic components.Studies on retinal damage and repair by elevated IOP have mainly concentrated on the protection of retinal ganglion cells(RGCs),such as promoting RGC survival and axon regeneration24-27.However,these protective measures have had limited efficacy,suggesting that not only RGCs,but also other retinal neurons and their connections on the visual pathway upstream of RGCs impact on recovery following
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