干擾哺乳類視網膜外層神經功能對無軸突細胞網絡之影響

Effects of disrupted outer retinal function on amacrine cell circuits in mammalian retinas

涂宏雅(Hung-Ya Tu)

指導教授 ： 焦傳金

國立清華大學/生命科學院/分子醫學研究所/博士(2016年)

摘要

哺乳類動物的視網膜被認為是中樞神經系統最容易取得以進行研究的部分。其中位於視網膜外層的感光細胞將視覺刺激轉化成膜電位變化並釋放神經傳導物質，經由位於外突觸層的神經突觸將外界來的視覺訊號從少數幾種感光細胞（視錐及視桿細胞）分散至數種雙極細胞，再傳遞給下游為數眾多的無軸突細胞和節細胞。截至目前為止已有許多研究力圖闡明，在視網膜發育過程中出現的視網膜波此一自發神經活性對於後端視覺神經系統的影響；至於當源自視網膜外層的訊號無法正常傳遞時，對於內層神經細胞在功能上的影響則相對了解得較少。此篇博士學位論文描述了當在幼兔及成體小鼠上分別以施予藥物及轉殖基因的方式阻斷視網膜內外層的訊號傳遞時，對於AII無軸突細胞和星狀無軸突細胞所造成的影響。藉由這兩個彼此獨立的研究，本論文說明了在哺乳類視網膜中，神經網絡對於不等程度的神經活性變化具有不同的適應機制。
過去在成體動物中，已有諸多研究描述了多條視桿細胞傳訊路徑，包含參與其中的細胞種類及調節機制，其中由Cx36蛋白構成的間隙通道在多條路徑裡皆扮演了關鍵的角色，尤以AII無軸突細胞主導的路徑最廣為人知；然而，在該路徑的發育過程中，Cx36 間隙通道的參與程度及調控則尚未明瞭。在此論文第一部分的研究中，藉由免疫染色首先說明了幼兔視網膜裡Cx36蛋白在不同發育階段的空間分布情形，並且利用顯微注射染劑的方式發現，在出生後十天大的幼兔視網膜上，即便AII無軸突細胞已表達了少量Cx36蛋白，這些細胞仍未形成穩定、可讓特定染劑通透的間隙通道結構，反之需至出生後第三週方具有和成兔視網膜相仿的通透能力；此外，「全暗養殖」及「藥物注射」這兩種可在發育過程中阻斷神經活性的方式都導致AII無軸突細胞的Cx36表現量提高。上述的發現表示此路徑可能不如過去所猜測的，在開眼時已具有高度成熟的訊息傳遞功能，並且其中的間隙通道可能受到視網膜外層神經活性的調控。
另一方面，在許多成體小鼠的研究中發現，感光細胞退化（如rd1和rd10）會導致視網膜節細胞產生自發、有節律的神經活性，近期數篇文獻則指出，這些神經活性是由AII無軸突細胞自發產生、並藉由其間隙通道網絡傳遞至雙極細胞而後至節細胞。在第二部分的研究中，使用了轉殖基因小鼠rhoΔCTA作為感光細胞退化的動物模型進行研究，此種小鼠的退化病程與rd1相似，故可藉此了解在不同退化模型上是否具有相似的自發神經活性及機制。在rhoΔCTA視網膜中發現除了部分節細胞之外，兩種亞群的星狀無軸突細胞亦有類似於rd1節細胞的神經活性，並且是受到以麩胺酸作為神經傳導物質的雙極細胞驅動；當阻斷快鈉通道(NaV)及間隙通道時，皆會導致這些神經活性消失。當利用多巴胺阻斷AII細胞的間隙通道網絡以及利用flupirtine阻斷AII細胞上的M型鉀通道時，則只會抑制ON類型的星狀細胞神經活性，對OFF類型的星狀細胞則沒有顯著影響。上述的結果表示可能有第二種不同於AII假說的細胞機制，在感光細胞退化時與AII無軸突細胞並行引發視網膜內層的高度神經活性。
總結而言，此篇論文研究分別在幼兔及成鼠視網膜上觀察到阻斷視網膜外層神經活性所導致的內層神經細胞改變，不論是在蛋白質表現量或神經突觸傳遞活動上，皆說明了哺乳類視網膜神經網絡中的縱向調控對於不等程度的神經活性變化有不同的適應機制。

關鍵字

AII無軸突細胞 ； 星狀無軸突細胞 ； 間隙通道蛋白36 ； 神經活性 ； 視桿細胞傳訊路徑 ； 感光細胞退化 ； 全暗養殖

並列摘要

The mammalian retina is considered as the most accessible part of the central nervous system. Visual stimuli are detected by photoreceptors in the outer retina, and then transmitted to the inner retinal neurons including bipolar cells, amacrine cells, and retinal ganglion cells. The first synapses are between the photoreceptors and bipolar cells at the outer plexiform layer, where the visual signals are diverged into parallel pathways via diverse bipolar cell types. While many studies have focused on the effects of spontaneous retinal activities, such as retinal waves, on the development of visual system, fewer efforts have been put to characterize the functional changes of inner retinal neurons in the absence of outer retinal activities. In this dissertation, I examined two most important amacrine cell types – AII amacrine cells (AII-ACs) in developing rabbit retina and starburst amacrine cells (SACs) in adult mouse retina under the conditions of pharmacologically and genetically altered signal transmission from the outer retina to the inner retina, respectively. These two independent studies are described separately here, and they represent a coherent effort in elucidating the cellular mechanisms of activity dependent circuitry adaptation in mammalian retinas.
The rod photoreceptor signaling pathways in adult retina have been extensively investigated and characterized in terms of the distinctive components and light-adaptive properties. Gap junctions are composed of connexin 36 (Cx36) and play critical roles in most of these pathways, despite little is known about the contribution and regulation of gap junctions to the development of the AII-AC mediated primary rod pathway. Using immunohistochemistry and microinjection, the first study demonstrates a steady increase in relative Cx36 protein expression in both plexiform layers of the rabbit retina at around the time of eye opening. However, immediately after eye opening, most Cx36 immunoreactive AII-ACs show no gap junction coupling pattern to neighboring cells and it is not until the third postnatal week that AII cells begin to exhibit an adult-like tracer coupling pattern. Moreover, studies using dark-rearing and AMPA receptor blockade during postnatal development both revealed that relative levels of Cx36 immunoreactivity in AII-ACs were increased when neural activity was inhibited. These findings suggest that Cx36 expression in the AII-mediated rod pathway is activity dependent in the developing rabbit retina.
It has been shown in rd1 and rd10 models of photoreceptor degeneration (PD) that inner retinal neurons display spontaneous and rhythmic activities. An autosomal dominant PD model called rhoΔCTA, whose rods overexpress a C-terminally truncated mutant rhodopsin and degenerate with a rate similar to that of rd1, was used to investigate the generality and mechanisms of heightened inner retinal activity following PD. Excitatory postsynaptic current (EPSC) oscillations and non-rhythmic inhibitory postsynaptic currents (IPSCs) were observed in both ON- and OFF-SACs. Similar to reported RGC oscillation in rd1 mice, EPSC oscillation was synaptically driven by glutamate and sensitive to blockades of NaV channels and gap junctions, suggesting that akin to rd1 mice, AII-AC is a prominent oscillator in rhoΔCTA mice. However, weakening the AII-AC gap junction network by activating retinal dopamine receptors abolished oscillations in ON-SACs but not in OFF-SACs. The latter persisted in the presence of flupirtine, an M-type potassium channel activator recently reported to dampen the intrinsic AII-AC bursting. These data suggest the existence of a novel oscillation mechanism in mice with PD.
In conclusion, disruption of outer retinal activities in either developing rabbit retina or mature mouse retina results in changes of protein expression and synaptic transmission in inner retinal neurons, respectively. These two studies demonstrate a vertically modulated circuitry adaptation in mammalian retinas.

並列關鍵字

AII amacrine cell ； Starburst amacrine cell ； Connexin 36 ； Neural activity ； Rod photoreceptor pathway ； Photoreceptor degeneration ； Dark-rearing

參考文獻

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