Thangaraj, Gopenath (2012)
On Roles of Cholinergic Amacrine and Müller Glial Cells in the Development of Networks in the Inner Plexiform Layer of the Chick Retina.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

The inner plexiform layer (IPL) of the Vertebrate retina is a highly organized synaptic region amassed with a myriad of processes from different cell types, like ganglion cells (GCs), amacrine cells (ACs) and bipolar cells (BCs). Their dendrites stratify at different levels within this cell-free zone, e.g. so-called subbands. Further, Müller glial cells (MCs) span radially through all retinal layers. In this study, I have focused on cholinergic ACs and MCs, and their possible roles in the formation of the IPL in the embryonic chicken retina. In the first chapter I have analysed the in vivo development and differentiation of starburst amacrine cells (SACs) and their role in cholinergic stratification. Type-I and type-II SACs are mirror-symmetrically arranged ACs on both sides of the IPL, which send their processes into the latter, where they stratify as subbands ‘a’ and ‘d’. Acetylcholine is the predominant neurotransmitter of these cells, e.g. they are cholinergic. Using the cholinergic markers choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), I show here immunohistochemically that an early postmitotic pool of cells in the inner half retina expresses first AChE, and then also ChAT. SACs and GCs are derived from this pool. By embryonic stage E6, two narrow rows of ChAT+ cells appeared in the future IPL, separated by a narrow band of AChE. Thus, AChE might be playing a guiding role for ChAT+ processes. Type-I SACs retained their high ChAT+, but not AChE expression, which was less pronounced in type-II SACs. Remarkably, type-I SACs in the INL were neighbored by strongly AChE-reactive cells which were ChAT-, a finding whose functional implications needs further attention. In chapter II, I show that the earliest ChAT+ cells also co-expressed the ganglion cell-specific marker Brn3a and CRABP. By E6, one set of cells down-regulated both ChAT and CRABP, but up-regulated Brn3a, marking the specification of GCs, while another set of cells reacted to the contrary, marking the specification of the two future types of SACs. By E8, type-I cells at the IPL/INL border lost their expression of CRABP, while type-II SACs in the IPL retained this protein. A co-localization of the early GC marker Brn3a with ChAT+ cells revealed that early GCs transiently express ChAT. Throughout these developmental stages, both the GCs and the ACs expressed AChE. Now following, I have performed in vitro experiments to analyze the role of SACs and Müller glial cells during IPL development, using both 3D retinal spheroids and retinal explants. In chapter III, I describe the establishment of a novel explant culture system, presenting the development of all retinal layers of an in vivo retina, except the GCL. Although being used here for studies of the inner retina, it is worthwhile to mention that with these explants the initial formation of photoreceptor outer segments can be followed. In continuation to the above mentioned in vivo studies, in chapter IV, I have similarly analyzed IPL formation by using both in vitro systems. Since type-II SACs are closely associated with GCs, I postulated that GCs could influence their differentiation, and in turn the development of the IPL as well. To test this hypothesis, I investigated the development of the IPL in explants, in which GCs are completely lost due to the absence of an optic nerve. In addition, 3D retinospheroids were used, where cells are completely dissociated and allowed to form histotypical structures, but again mostly in absence of GCs. Interestingly, in both in vitro culture models the IPL still developed, including both cholinergic subbands and migration of type-II SACs. This is strong evidence that major parts of the IPL develop in absence of GCs. Besides SACs, early Müller glial cells (MCs) have been suggested to provide guiding cues to establish the neuritic plexus of the IPL. Therefore, in the last chapter V, I have used the specific gliotoxin DL-alpha aminoadipate (AAA) in retinal spheroids to target MCs and thereby analyze the role of MCs during IPL development. At a sub-toxic concentration of 0.4 mM AAA, though the IPL was developed, the cholinergic stratification was severely disturbed, indeed suggesting an organizing role of MCs during IPL development. Moreover, thereby the numbers of inner retinal cells increased, while photoreceptor differentiation was inhibited, indicating further roles of MCs in cell lineage regulation.

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