Abstract
GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABAA receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABAA receptor system. To quantify the effects on proliferation by GABAA receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABAA receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABAA receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl–transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABAA receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABAA receptors. This supported the depolarising role for the GABAA receptors. Inhibition of L-type voltage-gated Ca2+ channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABAA receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27KIP1, along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27KIP1 after inhibition of either the GABAA receptors or the L-type VGCCs suggests a link between the GABAA receptors, membrane potential, and intracellular Ca2+ in regulating the cell cycle.
Highlights
In many vertebrates, the development of the retina is not complete after the embryonic period
This study focuses on the regulation of the proliferation of retinal progenitor cells and of cells from the non-pigmented epithelium of the chicken ciliary body
This study shows that chicken non-pigmented ciliary epithelial (NPE) cells express extrasynapticlike GABAA receptors that are involved in regulating the proliferation of the cells
Summary
The development of the retina is not complete after the embryonic period. This study focuses on the regulation of the proliferation of retinal progenitor cells and of cells from the non-pigmented epithelium of the chicken ciliary body. Chicken non-pigmented ciliary epithelial (NPE) cells are derived from the optic cup neuroepithelium and share similarities with early retinal progenitors such as the expression of Pax and Chx10 [4]. Neurons generated from the chicken NPE do not integrate into the neural retina [4] and are probably not an endogenous source for retinal regeneration. They have the potential to be harvested, cultured and transplanted back into injured retinas to replace lost neurons [12]
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