Investigating the Expression and Functionality of Mechanosensitive Channels in Zebrafish Retina
Open Access
- Author:
- Friemel, Taylor
- Graduate Program:
- Anatomy
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 03, 2020
- Committee Members:
- Salvatore Lucia Stella, Jr., Dissertation Advisor/Co-Advisor
Salvatore Lucia Stella, Jr., Committee Chair/Co-Chair
Alistair J Barber, Committee Member
Amy Christine Arnold, Committee Member
Christopher Martin Yengo, Outside Member
Patricia Mclaughlin, Committee Member
Patricia Mclaughlin, Program Head/Chair - Keywords:
- Vision
Retina
Mechanosensitive Channel
Retinal Ganglion Cells
Zebrafish
Piezo
Immunohistochemistry - Abstract:
- Retinal ganglion cells (RGCs) are the output cells of the retina and provide the image forming conduit to the brain. Most optic neuropathies (e.g., glaucoma, ocular trauma, etc.) damage RGCs and their axons, which can lead to apoptosis (cell death) and blindness. For many years, studies have centered on a variety of deleterious mechanisms that can damage RGCs in pathological conditions, including mitochondrial dysfunction, calcium overload, glial remodeling, synaptic pruning, and excitotoxicity; however, identifying a potential trigger that could initiate these pathological processes has been largely overlooked. For example, in glaucoma, elevated intraocular pressure (IOP) initiates a cascade of events that ultimately leads to RGC death, however, the trigger is unknown. Recently, the ability of RGCs to sense pressure in the surrounding environment has shifted focus to mechanosensitive channels or channels that possess mechanosensing attributes. Mechanosensitive channels are ion channels that can sense mechanical stimuli (stretch, tension, force, compression, etc.) that lead to channel opening and subsequent influx of ions, in particular, Ca2+ into the cell. This dissertation highlights the impact of four important Ca2+ permeable channels with mechanosensitive properties (Piezo1, P2X7, TRPV1, and TRPV4) and their functional expression on zebrafish RGCs. To study the functional aspects of these channels, we used Ca2+ imaging in flat mount retinal preparations with a transgenic zebrafish expressing a heritable Ca2+ indicator, GCaMP6f, targeted to RGCs [Tg(elavl3:GCaMP6f)] in the zebrafish retina. Selective agonists and antagonists to Piezo1, P2X7, TRPV1, and TRPV4 were used for each channel to determine whether zebrafish RGCs possessed these mechanosensitive channels. In addition, we mapped the expression profile for each of these channels using immunohistochemistry with specific antibodies targeted to Piezo1, P2X7, TRPV1, and TRPV4 channels in both zebrafish and rodent retina. The Piezo family of mechanosensitive channels, Piezo1 (FAM38A) and Piezo2 (FAM38B), are unique mechanosensitive channels in that they are the only known mechanosensitive channels that respond solely to mechanical stimulation. In this dissertation, we explore the functional expression and physiological responses of Piezo1 channels in the zebrafish retina, using Yoda1, the Piezo1 selective agonist, which evoked Ca2+ increases in zebrafish RGCs. Piezo1 expression was robust in both adult zebrafish and rodent RGCs and their axons, with other retinal neurons showing immunoreactivity. Yoda1-evoked dose-dependent Ca2+ increases in zebrafish RGCs that were inhibited by several mechanosensitive channel antagonists. Here, we provide functional physiological evidence of Piezo1 channel activity in adult vertebrate RGCs, and a possible therapeutic target for treating optic neuropathies and RGC diseases in the retina. P2X7 receptors are unique ion channels that function as larger pores and possess indirect mechanosensitive properties that can be linked to pannexin-1 hemichannels. P2X7 channels have previously been characterized on mammalian RGCs but have not been identified in zebrafish RGCs. To assess whether P2X7 receptors are expressed and functional on zebrafish RGCs, Ca2+ imaging and immunohistochemistry were used to map the functional expression of these channels in zebrafish. Like mammals, we uncovered the expression of P2X7 receptors and measured functional responses in zebrafish RGCs. These findings in zebrafish are similar to previous reports in mammalian retina and serve as the first evidence of functional P2X7 receptor expression on zebrafish RGCs. TRPV1 and TRPV4 channels have been shown to be expressed on RGCs in a variety of different species, including mice and humans. However, only TRPV4 has been identified in the zebrafish retina. Stimulation of TRPV1 and TRPV4 has been shown to lead to an increase in intracellular calcium in RGCs, and this can contribute to RGC death. We used immunolabeling techniques and calcium imaging to identify the functional expression pattern of TRPV1 and TRPV4 channels in the zebrafish retina. In this dissertation, we revealed the presence of functional TRPV1 and TRPV4 Ca2+ responses and expression in zebrafish RGCs. These findings indicate that both functional TRPV1 and TRPV4 channels are expressed by zebrafish, however, more studies are needed to parse out the mechanosensitive aspects of these channels. The identification of these ion channels in zebrafish RGCs has provided a step towards deciphering how mechanosensitive channel properties (Piezo1, P2X7, TRPV1, and TRPV4) can serve as an initial trigger leading to a cascade of lethal events and RGC death. More importantly, zebrafish serve as a powerful model system to explore the consequences of these mechanosensitive channels expressed on RGCs in health and disease.