长时间光谱剥夺对豚鼠光感受器细胞超微结构的影响

目的研究长时间光谱剥夺后豚鼠光感受器超微结构的变化,探讨色觉发育的可塑性。方法将出生3d的30只豚鼠随机分为3组,分别置于与两种视锥细胞吸收光谱波峰值相适应的绿光（530nm）、紫光（400nm）及白色混合光下照射8周后,电镜观察各组背侧、腹侧视网膜光感受器的超微结构改变。结果绿光照射组视网膜背侧与腹侧相比,与对照组背侧相比,紫光照射组视网膜腹侧与背侧相比,及与对照组腹侧相比,光感受器细胞体长度无明显变化（P〉0.05）;外节长度明显减少（P〈0.01）,膜盘部分空泡化;内节长度增加（P〈0.01）,线粒体变丰富;光感受器细胞核变大（P〈0.01）。结论绿光照射组背侧视网膜和紫光照射组腹侧视网膜光感受器超微结构变化一致,可能是光感受器细胞对光谱剥夺的一种适应性反应,这为色觉发育的可塑性提供了解剖学证据。     

rd11小鼠视锥细胞变性过程中视蛋白的变化特点

背景视网膜变性11（rd11）小鼠是近年来新发现的一种自发突变的视网膜变性小鼠。研究证实,rd11小鼠出生后随着鼠龄的增长出现快速的光感受器变性,且视杆细胞变性早于视锥细胞变性,但对于视网膜不同区域视锥细胞变性的特点还不十分清楚。目的应用视网膜铺片免疫荧光染色技术观察不同鼠龄rd11小鼠M-视蛋白和S-视蛋白在视网膜的表达分布及变化特点,为相关疾病的基因治疗研究提供实验依据。方法取出生后14、28、42d的rd11小鼠各5只,制备视网膜铺片,采用免疫荧光组织化学法分别标记小鼠视网膜后极部颞上、颞下、鼻上和鼻下象限M-视蛋白和S-视蛋白的表达,观察随rd11小鼠鼠龄的变化视网膜各区域M-视蛋白和S-视蛋白的荧光形态和密度,并与相应鼠龄的C57BL／6J小鼠进行比较。结果出生14d的rd11小鼠视网膜M-视蛋白和S-视蛋白的红色荧光形态和密度与C57BL／6J小鼠接近,但出生28d的rd11小鼠视网膜后极部颞上、颞下、鼻上、鼻下4个区域M-视蛋白和S-视蛋白表达密度均明显降低,荧光形态由纺锤形逐渐变为点状,出生42d的rd11小鼠视网膜部分区域M-视蛋白和S-视蛋白表达消失。出生28d的rd11小鼠视网膜后极部颞上、颞下、鼻上、鼻下区域M-视蛋白的表达密度分别为（414±32）、（300±8）、（324±22）和（250±20）个／0．037mm^2,明显低于同龄C57BL／6J小鼠的（484±21）、（442±19）、（459±34）和（436±12）个／0．037mm^2,差异均有统计学意义（t=4．114、15．225、7．505、17．990,均P〈0．05）;上述4个区域S-视蛋白的表达密度下降更明显,分别为（8±4）、（175±16）、（74±13）、（315±20）个／0．037mm^2,明显低于C57BL／6J小鼠的（73±16）、（436±30）、（393±30）和（480±19）个／0．037mm^2,差异均有统计学意义（t=8．555、17．076、21．637、13．498,均P〈0．05）。结论rd11小鼠视锥细胞中的M-视蛋白和S-视蛋白均随着鼠龄的增长而急剧减少,以S-视蛋白更为明显。随着鼠龄的增长,rd11小鼠M-视蛋白变性从视神经周围区向鼻下方,再向颞上方逐渐进展,而S-视蛋白变性由颞上方向鼻下方逐渐进展。     

外源性全反视黄酸对出生后豚鼠锥细胞视蛋白表达和方向性的影响

目的观察一定浓度的外源性全反视黄酸（all-trans retinoic acid,ATRA）对豚鼠锥细胞视蛋白表达和方向性的影响。方法出生1周豚鼠28只，随机分为ATRA组（n=14）和正常对照组（n=14）。ATRA组随机选取一眼于暗红光下球周注射0．4mg／ml浓度ATRA 0．05ml，对照组随机选择一眼球周注射稀释ATRA用的相应浓度二甲基亚砜（0．001ml/L）和注射用水共0．05ml/L。4周后取眼球，行免疫组织化学方法和RT-PCR技术观察M-视蛋白和S-视蛋白的分布和表达密度，并检测两种视蛋白的mRNA表达，方向性以Leica光学显微镜进行观察。结果ATRA处理眼S-视蛋白的表达密度：下方为（499．4±147．6）MM^-2，上方为（87．8±44．9）MM^-2，中央为（968．4±210．2）MM^-2；正常组S-视蛋白的表达密度：下方为（805．0±203．3）mm^-2，上方为（100．0±57．7）mm^-2；中央为（1637．2±314．1）mm^-2。ATRA处理眼M-视蛋白的表达密度：上方为（1326．1±267．0）mm^-2，中央为（2984．0±613．4）mm^-2，下方为（232．9±173．6）mm^-2；正常组M-视蛋白的表达密度：上方为（946．2±388．5）mm^-2，中央为（1666．7±137．8）mm^-2，下方为（175．0±100．9）mm^-2。0．4mg／ml浓度的ATRA0．05ml局部球周注射后使豚鼠视网膜的M-视蛋白的表达密度较正常对照组增加,S-视蛋白的表达密度减少：RT-PCR检测示M-视蛋白的相对光密度值分别为1．25±0．11（ATRA处理眼）和0．51±0．10（对照眼）,S-视蛋白的相对光密度值分别为0．61±0．09（ATRA处理眼）和1．25±0．06（对照眼）。ATRA处理眼与对照眼相比,M-视蛋白的mRNA表达增加，S-视蛋白的mRNA表达下降．两因素方差分析示两组差异均有统计学意义（P〈0．05）。铺片的免疫组化结果经光学显微镜观察提示，正常豚鼠视网膜M-视蛋白有一较小的偏斜角，朝向较垂直,ATRA作用后可见M-视蛋白偏斜角明显,朝向水平     

形觉剥夺对豚鼠锥视蛋白表达的影响研究

目的探讨形觉剥夺对豚鼠锥细胞视蛋白表达的影响。方法出生1周的豚鼠28只,分为正常对照组和形觉剥夺组,各14只,按自然亮暗周期饲养。形觉剥夺组随机选取一只眼以半透明橡胶遮盖,正常对照组随机选择一眼作为对照。4周后取眼球行RT-PCR检测神经视网膜M-视蛋白和S-视蛋白的mRNA表达,并进行豚鼠视网膜铺片后免疫组织化学方法观察两种视蛋白的分布和表达密度,一抗为兔抗鼠红／绿锥视蛋白抗体或蓝锥视蛋白抗体,二抗为羊抗兔IgG带488荧光抗体。以Leica光学显微镜和Leica共聚焦显微镜观察豚鼠视网膜腹侧（下方）和背侧（上方）及中央区的锥细胞视蛋白的表达。结果实验前屈光状态为：对照眼（5．63±0．87）D,形觉剥夺处理眼（5．47±1．02）D,4周后屈光状态分别为：对照眼（4．38±1．02）D,形觉剥夺处理眼（-3．03±0．78）D;屈光度的改变分别为：（-1．25±0．53）和（-8．38±1．44）D。正常豚鼠的视网膜短波敏感锥细胞分布为腹侧（下方）多于背侧（上方）;中央密度最高,密度变化为突变。豚鼠的中波敏感锥细胞分布在背部（上方）多于腹侧（下方）,中央密度最高,密度变化为渐变;正常组短波敏感蛋白的表达密度：下方为（805．0±203．3）mm^-2,上方为（100．0±57．7）mm^-2;中央为（1637．2±314．1）mm^-2。形觉剥夺组：下方为（640．9±196．8）mm^-2;中央为：（1016．7±144．6）mm^-2,上方为（70．9±30．8）mm^-2;正常组M-视蛋白的表达密度：上方为（946．2±388．5）mm^-2,中心为（1666．7±137．8）mm^-2,下方为（175．0±100．9）mm^-2;形觉剥夺组：上方为（1436．7±366．0）mm^-2,中心为：（2780．0±180．5）mm^-2,下方为（318．2±172．7）mm^-2。RT-PCR检测示形觉剥夺眼和对照眼的M-视蛋白的相对吸光度值分别为1．06±0．07和0．51±0．10,S-视蛋白的吸光度值分别为0．70±0．07和1．25±0．06。形觉剥夺性近视眼视网膜3个观察区域M-视蛋白的表达均较正常对照增加,S-视蛋白的表达减少,两因素方差分析示差别均有统计学意义（P〈0．05）。结论形觉剥夺性近视眼M-视蛋白的表达增加,S-视蛋白的表达减少,提示感光细胞锥细胞的视蛋白表达有可能在近视的发生发展中起了作用。     

自主感光视网膜神经节细胞与彩色光瞳孔测量

自主感光视网膜神经节细胞（ipRGCs）是除视杆细胞、视锥细胞以外的第三类光感受器细胞,位于视网膜内层,由于其内含黑视蛋白,故具备自主感光能力。瞳孔对光反应（PLR）主要由ipRGCs介导产生。ipRGCs可通过黑视蛋白直接感受光信号产生PLR,也可被来自视杆、视锥细胞的信号激活产生PLR。由于视杆细胞、视锥细胞和黑视蛋白产生的PLR各具特点,可采用不同强度和波长的光信号选择性刺激视杆细胞、视锥细胞和黑视蛋白,通过对产生的PLR进行分析可间接反映视杆细胞、视锥细胞和含黑视蛋白的ipRGCs的功能,这一方法称为彩色光瞳孔测量。现主要对ipRGCs介导PLR的通路、视杆/视锥细胞和黑视蛋白引起的PLR特点、彩色光瞳孔测量及其临床应用作一综述,希冀为相关眼科疾病的诊断及鉴别诊断提供新思路。     

黄芪多糖对急性高眼压诱导大鼠视网膜神经节细胞的保护作用

背景青光眼患者视神经保护的问题日益引起关注。黄芪多糖（APS）是黄芪的主要活性成分,可增加再生神经蛋白的表达并促进损伤的周围神经修复,但其对视网膜神经节细胞（RGCs）再生作用的研究少见报道。目的探讨APS对急性高眼压状态下RGCs的保护作用。方法采用抽签法将40只SPF级sD大鼠随机分为正常对照组、模型对照组、低剂量APS组和高剂量APS组,每组各10只。低剂量APS组和高剂量APS组大鼠自实验开始每日分别给予APS500mg／kg、2000mg／kg（均溶于2．5ml生理盐水）灌胃,模型对照组仅给予2．5ml生理盐水灌胃,正常对照组不做任何处理。用药2周后,除正常对照组外,其余3个组均抽取0．2ml房水继而单眼前房注射等体积甲基纤维素使眼压升高至22mmHg（1mmHg=0．133kPa）以上制作急性高眼压模型。造模后5d,过量麻醉法处死动物,摘除该眼球制作视网膜石蜡切片,常规组织病理学观察视网膜的形态结构变化,采用免疫组织化学法检测caspase-3蛋白在大鼠视网膜中的表达,采用TUNEL染色法观察和计算各组大鼠RGCs的凋亡率。ImageProPlus5．1软件测量各组大鼠视网膜厚度和神经纤维层厚度。结果大鼠成模后5d,模型对照组、低剂量APS组和高剂量APS组大鼠的眼压均明显高于正常对照组,差异均有统计学意义（t=-8．900、-10．700、-11．300,P〈0．01）。正常对照组大鼠视网膜形态正常;模型对照组大鼠视网膜水肿,细胞排列紊乱;低剂量APS组视网膜可见空泡样变性,但视网膜细胞排列较模型对照组整齐,视网膜水肿减轻;高剂量APS组视网膜水肿较明显。低剂量APS组视网膜厚度、外颗粒层及视神经纤维层厚度值均明显低于模型对照组,差异均有统计学意义（t=-23．700、-14．770、-11．640,P〈0．01）,但高剂量APS组外颗粒层及视神经纤维层厚度与模型对照组比较差异均无统计学意义（t=-0．780、-0．460,P〉0．05）。低剂量APS组大鼠caspase-3蛋白阳性RGCs百分比及RGCs凋亡百分率均明显低于模型对照组（caspase-3蛋白：F=87．710,P=0．001;RGCs凋亡：F=272．840,P〈0．01）,差异均有统计学意义（t=-11．700、-8．600,P〈0．01）,高剂量APS组与模型对照组比较caspase-3蛋白阳性RGCs百分比及RGCs凋亡百分率的差异均有统计学意义（t=-7．900、-6．400,P〈0．05）。结论500mg／kgAPS可有效抑制急性高眼压模型大鼠的视网膜水肿及RGCs的凋亡率,对急性高眼压大鼠的RGCs有保护作用。     

蛋白激酶C活性调节剂对豚鼠近视眼视网膜Mtiller细胞的作用及意义

目的研究蛋白激酶C（PKC）激活剂PMA和抑制剂GF109203X对豚鼠近视眼视网膜Mailer细胞生长状态及PKC蛋白表达的影响。方法眼罩遮盖诱导近视模型,酶消化法培养其视网膜Mailer细胞,并鉴定。PMA、GF109203X分别干预近视眼Mailer细胞。MTT测细胞活力,TUNEL染色检测凋亡细胞,Westren blotting测PKC蛋白表达。结果95％以上的培养细胞阳性表达胶质纤维酸性蛋白（GFAP）和波形蛋白（Vimentin）。近视组PKC蛋白表达较正常组上调（P〈0．05）。PMA、GF109203X均能引起近视眼Mailer细胞的抑制率升高和诱导其凋亡（P〈0．05）。PMA诱导近视眼Mailer细胞PKC蛋白表达进一步上调,100nmol／L组和1000nmol／L组表达量高于10nmol／L组（P〈0．05）。1μmol／L和10μmol／L GF109203X均能引起近视眼Mailer细胞PKC蛋白表达下调,10μmol／L的抑制作用大于1μmol／L（P〈0．05）。结论PKC蛋白在豚鼠近视眼视网膜Mailer细胞表达升高,且其表达受PMA和GF109203X调控。     

蛋白激酶C活性调节剂对豚鼠近视眼视网膜Mller细胞的作用及意义

目的研究蛋白激酶C（PKC）激活剂PMA和抑制剂GF109203X对豚鼠近视眼视网膜Mailer细胞生长状态及PKC蛋白表达的影响。方法眼罩遮盖诱导近视模型,酶消化法培养其视网膜Mailer细胞,并鉴定。PMA、GF109203X分别干预近视眼Mailer细胞。MTT测细胞活力,TUNEL染色检测凋亡细胞,Westren blotting测PKC蛋白表达。结果95％以上的培养细胞阳性表达胶质纤维酸性蛋白（GFAP）和波形蛋白（Vimentin）。近视组PKC蛋白表达较正常组上调（P〈0．05）。PMA、GF109203X均能引起近视眼Mailer细胞的抑制率升高和诱导其凋亡（P〈0．05）。PMA诱导近视眼Mailer细胞PKC蛋白表达进一步上调,100nmol／L组和1000nmol／L组表达量高于10nmol／L组（P〈0．05）。1μmol／L和10μmol／L GF109203X均能引起近视眼Mailer细胞PKC蛋白表达下调,10μmol／L的抑制作用大于1μmol／L（P〈0．05）。结论PKC蛋白在豚鼠近视眼视网膜Mailer细胞表达升高,且其表达受PMA和GF109203X调控。     

高糖对体外培养的视网膜Mǖller细胞活性的影响

背景视网膜Mǖiler细胞具有为视网膜组织提供营养、维持视网膜的正常结构等多种生理功能,研究发现Mǖiler细胞的病变会导致视网膜血管发生相应的改变。探讨高糖对视网膜Mǖiler细胞的影响对于糖尿病视网膜病变（DR）的发病机制研究具有重要意义。目的研究不同浓度的葡萄糖对体外培养的视网膜Mǖiler细胞活性的影响。方法取出生后10d清洁级SD大鼠的视网膜组织,用组织块培养法在含质量分数20％胎牛血清的DMEM培养液中体外原代培养Mǖiler细胞并传代,取第3代细胞用免疫组织化学法对细胞进行鉴定。将不同浓度（5．5、30．0、40．0mmol／L）的葡萄糖加入培养基中培养4d,MTT比色法测定各组波长570nm处Mǖller细胞的吸光度（A570）值,计算各组细胞的相对存活率;采用流式细胞仪检测各组Mǖller细胞的凋亡率。结果培养的细胞贴壁生长,呈长梭形;95％以上细胞神经胶质纤维酸性蛋白（GFAP）反应阳性。MTT比色法检测显示,正常葡萄糖组及30．0mmo／L、40．0mmol／L葡萄糖处理组Mǖller细胞A570值分别为0．24±0．01、0．21±0．03和0．20±0．02,总体差异有统计学意义（F=6．755,P〈0．05）。与正常葡萄糖组比较,30．0mmol／L、40．0mmol／L葡萄糖处理组A570值均明显降低,差异有统计学意义（q=0．645、0．486,P〈0．05）。流式细胞仪检测结果表明,正常葡萄糖组、30．0mmol／L和40．0mmol／L葡萄糖处理组Mǖller细胞凋亡率分别为（26．40±0．25）％、（30．19±0．16）％和（36．23±0．19）％,总体差异有统计学意义（F=294．530,P〈0．05）,与正常葡萄糖组比较,30．0mmol／L、40．0mmol／L葡萄糖组凋亡率均明显升高,差异有统计学意义（q=0．754、0．484,P〈0．05）。结论高浓度葡萄糖可抑制视网膜Mǖller细胞的生长并增加其凋亡率,葡萄糖的上述作用呈浓度依赖性。     

一种改良的光诱导视网膜分级损伤大鼠模型

背景大鼠视网膜光损伤动物模型因诸多影响因素较难复制,使不同因子（如药物）对视网膜光损伤生物学效应的研究受到影响。目的改良大鼠视网膜光损伤动物模型的制作方法,建立大鼠视网膜光损伤程度分级模型。方法选用8～10同龄雄性SD大鼠24只,根据强光暴露时间的不同将动物按随机数字表法分为正常对照组和光损伤1、2、3h组,每组6只大鼠。为获得眼球的全视野光辐照,采用环形光照箱将LED灯集中分布在光照箱的上下和光照箱四周共8个方向,保证大鼠在光照箱内做单向环形活动时其右眼接受约5000lx的光照。实验中每只动物单独接受光辐照,以避免动物之间的相互干扰。光辐照后5d进行全视野视网膜电图（ERG）记录,并进行常规组织病理学观察,评价视网膜外核层（ONL）的厚度变化。结果5000lx强光暴露1、2、3h后暗适应条件下的ERG视杆细胞反应、标准光最大混合光反应的b波幅值分别下降了26．2％、52．5％、70．7％和24．4％、39．3％、58．1％,OPs波、视锥细胞反应、20Hz闪烁光反应也均有明显下降,不同光照时间组间各波平均振幅值的总体差异均有统计学意义（视杆：F=71．690,P=0．000;混合光反应：F=56．250,P=0．000;OPs：F=23．610,P=0．000;视锥：F=27．130,P=0．000;20Hz：F=27．030,P=0．000）;不同光照时间组间各波平均隐含时的总体差异均有统计学意义（视杆：F=1．370,P=0．282;混合光反应：a波：F=0．800,P=0．508;b波：F=11．840,P=0．000;视锥：F=2．080,P=0．136）。振幅下降及隐含时延长的程度均与光暴露时间有关。视网膜的光损伤以视网膜颞上区域明显,光辐照1、2、3h后该区域ONL厚度较正常对照组分别减少了11．3％、25．6％、72．5％,各组的平均ONL厚度随光照时间的延长逐渐变薄,差异有统计学意义（F=410．270,P=0．000）。结论光强度为5000lx连续光辐照后可以造成大鼠视网膜损伤,光辐照1、2、3h后损伤程度分为轻、中、重3级,主要损伤部位在大鼠视网膜颞上区域。研究提出了一种视网膜光损伤程度标准的建议。     

Guinea pigs reared in a monochromatic environment exhibit changes in cone density and opsin expression

This study aimed to determine if a monochromatic environment will affect the development of cones in a guinea pig model. Thirty 3-day-old guinea pigs were randomized into three groups and exposed to green, violet, and white light (control) for 8 weeks. The animals were sacrificed and the density of middle-wavelength cones (M cones) and short-wavelength sensitive (S cones) and expression of M-opsin and S-opsin were determined. The density of M cones was increased in the green light group as compared to the control group, and decreased in the violet light group as compared to the control group (both, p < 0.05). There was no significant difference in the density of the S cones among the groups (all, p > 0.05). The density of coexpressing cones in the middle retina was significantly increased in the green light group in comparison to the violet light group (p < 0.01). In addition, there was a significant increase in the level of M-opsin as determined by Western blotting and M-opsin mRNA expression as determined by PCR analysis in the green light group as compared to the control group and a significant decrease in violet light group as compared to the control group (all, p < 0.05). No significant difference in S-opsin level or S-opsin mRNA expression was noted among the groups. We concluded that monochromatic lighting affected the density of cones and expression of opsins in a guinea pig model, and this indicates that the retinal color visual system of the guinea pig possess developmental plasticity.     

Photoreceptor distribution in the retina of adult Pacific salmon: corner cones express blue opsin

The retina of salmonid fishes has two types of cone photoreceptors: single and double cones. At the nuclear level, these cones are distributed in a square mosaic such that the double cones form the sides of the square and the single cones occupy positions at the centre and at the corners of the square. Double cones consist of two members, one having visual pigment protein maximally sensitive to green light (RH2 opsin), the other maximally sensitive to red light (LWS opsin). Single cones can have opsins maximally sensitive to ultraviolet (UV) or blue light (SWS1 and SWS2 opsins, respectively). In Pacific salmonids, all single cones express UV opsin at hatching. Around the time of yolk sac absorption, single cones start switching opsin expression from UV to blue, in an event that proceeds from the ventral to the dorsal retina. This transformation is accompanied by a loss of single corner cones such that the large juvenile shows corner cones and UV opsin expression in the dorsal retina only. Previous research has shown that adult Pacific salmon have corner cones over large areas of retina suggesting that these cones may be regenerated and that they may express UV opsin. Here we used in-situ hybridization with cRNA probes and RT-PCR to show that: (1) all single cones in non-growth zone areas of the retina express blue opsin and (2) double cone opsin expression alternates around the square mosaic unit. Our results indicate that single cone driven UV sensitivity in adult salmon must emanate from stimulation of growth zone areas.     

Different sensations from cones with the same photopigment

We used adaptive optics to study color fluctuation in the appearance of tiny flashes of light. For five subjects, near threshold, monochromatic stimuli with full widths at half maximum of 1/3 arcmin were delivered throughout a patch of retina near 1 deg in which we also determined the locations of L, M, and S cones. Subjects reported a wide variety of color sensations, even for long-wavelength stimuli, and all subjects reported blue or purple sensations at wavelengths for which S cones are insensitive. Subjects with more L cones reported more red sensations, and those with more M cones tended to report more green sensations. White responses increased linearly with the asymmetry in L to M cone ratio. The diversity in the color response could not be completely explained by combined L and M cone excitation, implying that photoreceptors within the same class can elicit more than one color sensation.     

Differences in the pharmacological activation of visual opsins

Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of "pigment adaptation" with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, beta-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of beta-ionone. To test the two hypotheses, we applied beta-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of transducin, whereas all cones contain another. beta-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod transducin upon the addition of beta-ionone. Thus opsin structure determines the identity of beta-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.     

Monoclonal antibody labels both rod and cone outer segments of turtle photoreceptors.

We have studied the immunoreactivity of turtle photoreceptors to a monoclonal antibody (MAb 15-18) which binds to the external loop connecting bovine rhodopsin helices IV-V. Three chromatic types of cone photoreceptors were identified by the presence and color of oil droplets. MAb 15-18 intensely labeled the outer segments of both rods and green cones. In addition, a weak cross-reactivity was also found in the outer segments of red cones having a pale-green oil droplet, and of blue cones. Other morphological subtypes of red cones, cones with a red oil droplet and both members of double cones, showed no labeling. Our results indicate that rhodopsin and green cone opsin have a similar antigenic determinant, and that two different structural forms of red cone opsin may be present in the turtle retina.     

Immunocytochemical reactivity of rod and cone visual pigments in the sturgeon retina.

Microspectrophotometry and immunocytochemistry with several antivisual pigment antibodies were used to study visual cells of the Siberian sturgeon, Acipenser baeri Brandt. The retina contained rods and three morphological types of cones: large cones with oil drops, small cones with oil drops, and cone-like cells without oil drops. Rods and cone-like drop-free cells were found to possess porphyropsin-549, while the large oil drop-bearing cones contained red-sensitive (P613), green-sensitive (P542), and blue-sensitive (P462) visual pigments. The immunocytochemical staining pattern with three antibodies to visual pigment proteins also revealed one visual pigment in rods and three visual pigments in cones. Rods were labeled with all three antibodies, while the majority of large cones (type I), presumably the red-sensitive ones, were negative with the polyclonal serum AO against bovine opsin. A less-frequently occurring large cone type (type II) was stained by all three antibodies including mAb COS-1 specific to middle-to-long-wave visual pigments in birds and mammals, and is thought to be green-sensitive. An even less-frequent large cone type (type III, probably the blue-sensitive one) did not bind COS-1. The small cones with oil droplets showed immunoreactivities similar to either type II or type III cones. The oil drop-free small photoreceptor exhibited a staining pattern identical with that of rods. These results indicate that the immunocytochemical approach can be used to reveal photoreceptor-specific neural connections in the sturgeon retina.     

The role of opsin expression and apoptosis in determination of cone types in human retina

In primates, short wavelength sensitive cones (S cones) and medium- or long-wavelength-sensitive cones (L/M cones) are two separate populations. Each cone type has a different developmental timecourse, contributes to different intra-retinal circuits, and transmits different types of information to the brain. However, in fetal human retina a significant population of cones express both S and L/M opsin (S+L/M cones), raising questions about whether S+L/M cones die or change opsin expression during development. We have utilized fetal, postnatal and adult human retinae to study the immunohistochemical distribution and morphology of S+L/M cones during development. Because S cones appear to be at higher density in fetal compared to adult retinae, we used antibodies to S opsin and alpha-transducin to estimate the proportion of S-cones, and TUNEL labelling to detect apoptotic death in the L/M, S or S+L/M population during development. S cones were present in central retina from fetal week (Fwk)11 and covered the retina by Fwk20. L/M cones appeared in the foveal cone mosaic 3-4 weeks after S-opsin was first detected, and covered the retina by birth. S+L/M cones were detected in all retinae older than Fwk14. They were most numerous at the retinal eccentricity where L/M opsin was just appearing; i.e. at the 'front' of L/M opsin expression. In this region, five morphological types of cones were present. (1) Heavily labelled S cones had thick cell bodies, a thick basal axon and pedicle, and a nucleus at any level of the outer nuclear layer (ONL). (2) Heavily labelled L/M cones were wine goblet shaped with a small round cell body, a large nucleus at the outer ONL edge, and a thin axon with a prominent synaptic pedicle. (3) Goblet-shaped S+L/M cones. (4) Goblet-shaped cones lightly labelled for S-opsin. (5) Cones that were not immunoreactive to either opsin. Only type 1 S cones were present peripheral to the L/M expression front, and their labelling intensity, morphology and distribution indicates that these are the 'true blue' cones of the adult mosaic. Only type 2 L/M cones were present in the foveal cone mosaic. Types 3 and 4 were most numerous within 500-750 microm of the L/M expression front, but type 3 S+L/M cones were also scattered throughout more central regions in fetal, infant and adult retinae. S+L/M cones comprised 5-10% of opsin immunoreactive cones at the L/M front in fetal and early postnatal retinas but 0.01-0.03% throughout P8mo and adult retinae. We found no evidence of significant levels of apoptosis in L/M cones at the expression front, suggesting that this decrease was not due to cell death. The findings suggest that goblet-shaped cones destined to express L or M opsin may initially and transiently express S opsin. Near the optic disc, at Fwk17 S cone density was around 2000 cells mm(-2), which dropped 50% by Fwk20 and stabilized at around 500 cells mm(-2) by birth. Double labelling with alpha-transducin showed that throughout this period 8-10% of all cones expressed S opsin. TUNEL labelling found no significant apoptosis in the S cone population. The decrease in S cone density near the optic disc occurs in the absence of apoptosis, and is likely due to other developmental events acting on the photoreceptor layer, including displacement of cones towards the fovea.