Structure And Function In Retinal Neurons

在眼睛里，神经回路处理图像。这些电路在从星光到正午太阳的各种照明下保持图像的稳定性。视杆进化为夜间视觉，视锥进化为白天视觉。独立的中间神经元处理这些杆状或锥体信号。猫、兔子和啮齿动物等哺乳动物是人类杆状回路的合理模型。在灵长类动物中，视锥类型和视锥回路进一步进化为色觉，但在普通实验室哺乳动物中，视锥密度低，色觉弱。与东半球灵长类动物一样，斑马鱼通过大量可调光谱的锥体进化出了色觉。不同的是，东半球灵长类动物发育出三种锥体类型，而斑马鱼则使用八种。然而，用于处理色度信息和控制锥体显影的电路策略可能是相似的。斑马鱼基因操作的简便性是有利的，而且有大量的视觉系统突变和转基因文库。出于这些原因，该实验室和其他实验室致力于将斑马鱼作为光感受器和视觉系统发育、回路和功能的电生理和神经解剖学研究的模型。 甲状腺激素β2核受体(Trb2)是最近的一个研究热点。该基因是红色球果分化和发育所必需的。来自Rachel Wong实验室的转基因株系(CRX：mYFP-2A-trb2和gnat2：mYFP-2A-trb2)是功能获得系，要么在错误的视锥类型(gnat2：trb2)中表达trb2，要么在视网膜发育过程中过早地在视网膜祖细胞中表达trb2。对于CRX：trb2，分化的视锥细胞对红色视蛋白的免疫反应密度增加了一倍，而对绿色、蓝色或紫外视蛋白的视锥细胞的免疫反应显著降低。与此同时，幼虫红色视锥信号的幅度大大增加，与基因复制的LWS2红光产生信号，也发生在对照幼虫的眼睛中。绿色、蓝色和紫外线视锥在CRX：trb2幼虫体内产生的信号幅度极大地减弱。对于GNAT：trb2，红视蛋白免疫反应锥体的密度也增加了一倍，但早期幼虫红色锥体的信号幅度增加了不到10%，而其他类型的锥体的信号幅度保持不变。不同的是，trb2正在诱导已经合成了天然绿色、蓝色或紫外线视蛋白的视锥细胞产生红色视蛋白，因此大多数视锥细胞对两种视蛋白产生免疫反应，一种是原始的，另一种是新的。随着红色视蛋白在发育的后期引入，转化为红色视锥的过程需要更长的时间，因为最初的视蛋白会通过圆盘脱落慢慢去除。在UV视锥中，这种转换必须发生，因为UV视蛋白基因的活动报告在gnat2：trb2视网膜中被沉默。到成年时，只有或几乎只有在两个转基因个体中，红色视锥的幅度被测量，而不像对照组，继续表达5到6个不同的视锥视蛋白信号。另一种转变发生了。在成人中，对照红锥的幅度在lws2和lws1红色视蛋白的幅度之间平均分配，而在转基因的lws1视锥中，lws1视锥的幅度更大。在幼虫中，我们也注意到锥体形态被功能转基因的trb2增益所扭曲，导致内节更宽，轴突比对照锥体更短。似乎trb2在视网膜发育中有非常广泛的作用，除了作为红锥体规范的必要条件之外。 通过对trb2阅读框中第一外显子的Crispr破坏，建立了一个没有幼虫或成虫红锥信号和增强的UV锥体信号的trb2-/-突变体。我们先前发现该品系缺乏对红色/黑色和绿色/黑色对比的幼虫视动或视动反应，并且缺乏对长波刺激的ERG反应。通常在红绿双锥体中表达的arrestin 3a抗原(ZPR-1抗体)丢失。

In the eye, neural circuits process images. These circuits maintain image constancy over illuminations ranging from starlight to noontime sun. Rods evolved for nocturnal vision, and cones for diurnal vision. Separate interneurons process these rod or cone signals. Mammals such as cats, rabbits, and rodents, are reasonable models for human rod circuitry. In primates further cone types and cone circuits evolved for color vision, but in common laboratory mammals cone density is low, and color sense is weak. Zebrafish, like old-world primates, evolved color vision through a proliferation of spectrally tuned cone types. The difference is that old-world primates developed three cone types, but zebrafish employ eight. Nonetheless the circuitry strategies for processing chromatic information and controlling cone development may be similar. The ease of genetic manipulation in zebrafish is advantageous, and there are extensive libraries of visual-system mutants and transgenics. For these reasons, this lab and others have worked to develop zebrafish as a model for electrophysiological and neuroanatomical studies of photoreceptor and visual system development, circuitry, and function. The thyroxin beta 2 nuclear receptor (trb2) has been a recent focus. This gene is required for differentiation and development of red cones. Transgenic lines from the Rachel Wong lab (crx:mYFP-2A-trb2 and gnat2:mYFP-2A-trb2) are gain-of-function lines, either expressing trb2 in the wrong cone types (gnat2:trb2), or in retinal progenitors, prematurely in retinal development. For crx:trb2, the resultant density of differentiated larval cones immunoreactive for red opsin doubles, while immunoreactivity for cones with green, blue or UV opsin is greatly reduced. Concomitantly larval red cone signals greatly increase in amplitude, with the gene-duplicated LWS2 red opsin generating the signals, as also occurs in control larval eyes. Green, blue and UV opsin cones generate greatly diminished signal amplitudes in crx:trb2 larvae. For gnat:trb2 the density of red-opsin immunoreactive cones also doubles, but early larval signal amplitudes of red cones increase less than 10%, and the signal amplitudes of other cone types remains unchanged. The difference is that trb2 is inducing red opsin production in cones already synthesizing the native green, blue or UV opsins so that most cones become immunoreactive for two opsins, an original and a newcomer. With the introduction of red opsin this late in development, the process of transformation into a red cone takes longer, as original opsins are slowly removed by disk shedding. In UV cones, this turnover must occur, as activity reporters for the UV opsin gene are silenced in the gnat2:trb2 retinas. By adulthood in both transgenics only, or nearly only, red cone amplitudes are measured, unlike controls, which continue to express 5 to 6 different cone opsin signals. A further transformation occurs. Whereas control red cone amplitudes in adults are evenly split between amplitudes for LWS2 and LWS1 red opsins, LWS1 opsins amplitudes are larger in the transgenics. In larvae we have also noted that cone morphology is distorted by the trb2 gain-of function transgenes, bringing about wider inner segments and shorter axons than control cones. It appears that trb2 has a very wide range of actions in retinal development, in addition to being a requirement for red-cone specification. A trb2 -/- mutant line with no larval or adult red-cone signals, and enhanced UV-cone signals was established by Crispr corruption of the first-exon in the trb2 reading frame. We previously found this line lacked larval optomotor or optokinetic responses for both red/black and green/black color contrasts, and lacked ERG responses to long-wavelength stimuli. The arrestin 3a antigen, (zpr-1 antibody), normally expressed in red-green double cones, was lost.