Research
Current Projects
I. Dynamics of Calcium Signals Control Neurotransmitter Release in Retinal Ribbon Synapses
Rapid and high local calcium (Ca2+) signals, termed Ca2+ transients, are essential for triggering neurotransmitter release from presynaptic terminals. In specialized bipolar ribbon synapses in the retina, these local Ca2+ signals control multiple processes, including the priming, docking, and translocation of vesicles on the ribbon before exocytosis, and the replenishment of release-ready vesicles to the fusion sites to sustain neurotransmission. However, the temporal and spatial characteristics of Ca2+ transients along the axis of the ribbon active zone, which are critical for regulating the processes aforementioned, remain largely unknown. In this project, we use fast confocal quantitative dual-color ratiometric line-scan imaging of a fluorescently labeled ribbon binding peptide and Ca2+ indicators to monitor the spatial and temporal aspects of Ca2+ transients of individual ribbon active zones in zebrafish retinal rod bipolar cells.
II. CaMKII Regulation of Synaptic Vesicle Release and Replenishment in Retinal Rod Bipolar Cell Ribbon Synapses
Calmodulin (CaM) is activated by submicromolar Ca2+ and seems to mediate Ca2+-dependent vesicle recruitment at conventional synapses and cone photoreceptors, being, therefore, a Ca2+-binding protein involved in intracellular signaling. The protein is an important activator of downstream Ca2+/calmodulin-dependent protein kinases, including CaMKII, a Ca2+ signaling and synaptic transmission moderator protein. CaMKII is enriched at presynaptic terminals, including ribbon synapses, where it associates with synaptic vesicles. Syntaxin-3B, a synaptic vesicle fusion protein, appears to be a substrate for CaMKII, suggesting a role for CaMKII in synaptic vesicle dynamics. However, various studies have suggested that CaMKII is capable of either enhancing or inhibiting neurotransmitter release and/or synaptic plasticity, depending on experimental conditions and the synaptic parameters investigated. Therefore, the mechanisms by which CaMKII regulates retinal ribbon synapse function in rod bipolar cell ribbon synapses remain unclear. With this project, we aim to identify the roles of CaMKII in regulating exocytosis and vesicle replenishment in retinal rod bipolar ribbon synapses of zebrafish by negatively and positively regulating CaMKII and using the confluence of state-of-the-art imaging, electrophysiological, and pharmacological tools.
III. AMPK Regulation of Synaptic Transmission in Retinal Rod Bipolar Cells
Adenosine monophosphate-activated protein kinase (AMPK) is a key energy sensor that regulates cellular energy homeostasis and has been implicated in modulating neuronal functions. In retinal rod bipolar cells (RBCs), where energy demand is high to sustain synaptic transmission, AMPK may play a critical role in facilitating synaptic dynamics. This project investigates the mechanisms by which AMPK modulates neurotransmitter release and synaptic vesicle dynamics at ribbon synapses. Modulating AMPK activity, we examine its effects on synaptic release rates, local Ca2+ signals at ribbon sites, and the voltage dependence of CaV channel activation. Additionally, we explore how AMPK influences the frequency of spontaneous Ca2+ action potentials in dissociated RBCs. By integrating advanced imaging, electrophysiology, and pharmacological approaches, this study aims to elucidate the role of AMPK in energy-dependent regulation of synaptic transmission in retinal ribbon synapses.
IV. Single Vesicle Imaging Reveals Novel Insights into Synaptic Vesicle Transport in Retinal Rod Bipolar Cell Ribbon Synapses
Our research explores how retinal bipolar cells (RBCs) sustain neurotransmission by efficiently transporting synaptic vesicles to the active zone. Using super-resolution imaging and a transgenic zebrafish model expressing photoactivatable fluorescent proteins, we track single vesicles in living RBC terminals. Our findings reveal distinct vesicle movement patterns—continuous and stepwise—regulated by local calcium levels. Additionally, we identified vesicles that exhibit jittery movements without participating in release. These discoveries highlight the diversity in vesicle transport and neurotransmitter release, providing new insights into synaptic function at RBC ribbon synapses. Building on these findings, future research can investigate the molecular mechanisms governing vesicle movement, particularly the role of calcium signaling and associated proteins. Exploring how disease-related mutations or pharmacological interventions influence vesicle transport could yield critical insights into retinal disorders and potential therapeutic strategies. Furthermore, utilizing advanced imaging techniques and computational modeling could provide a more comprehensive understanding of vesicle trafficking dynamics at the ribbon synapse, offering deeper insights into synaptic function and plasticity.
V. The Role of Light Entrainment on Zebrafish Eye Development
Environmental factors, such as light exposure, play a crucial role in the development of the visual system. While extensive research has examined the impact of light entrainment on the primary visual cortex, little is known about the effects of normal light-dark cycles on development on the complex nature of retinal development. Zebrafish, with their cone-dominant vision and rapid retinal development, serves as an ideal model for studying these processes due to their similarities to human visual systems. While numerous studies have explored light entrainment's impact on the visual system, most focus on the primary visual cortex in mice. Little is known about the effects on retinal synaptic development under different light environments in zebrafish addressing a critical knowledge gap. We have previously demonstrated that embryonic hyperglycemia disrupts retinal synapses by altering the development of the synaptic ribbon, which can lead to visual defects. Here, we characterize the light entrainment on retinal ribbon synapses during zebrafish embryonic development under a normal light-dark cycle (12 hours light/12 hours dark), constant light, or darkness over five days. Our approach utilized immunofluorescence labeling for synaptic proteins and confocal imaging to compare expression patterns of synaptic proteins in developing zebrafish embryos under distinct light exposures. This study highlights the critical role of light as a sensory input in shaping retinal synaptic development. It underscores the importance of light exposure in establishing functional visual circuits and demonstrates how visual deprivation affects synaptic plasticity in the retina. The findings have significant implications for biology and medicine, offering insights into mechanisms underlying visual system development and plasticity. This knowledge could inform therapeutic strategies for managing developmental and degenerative retinal disorders, advancing approaches to preserving and restoring vision. Further investigations using zebrafish models will help elucidate the molecular pathways governing retinal synapse formation and their role in visual function.
Past Projects
VI. The effect of aging on rod bipolar cells
We applied the knowledge obtained from studying calcium dynamics in rod bipolar cell synaptic transmission to determine whether defects with local calcium signal homeostasis are a prelude to disease. Visual impairment represents a significant global health concern that affects millions of people. The underlying causes of aging are intricate and involve factors such as abnormal mitochondria, epigenetic alterations, elevated levels of reactive oxygen species, and reduced length of chromosomal telomeres. Age-related changes are particularly evident in the gradual loss of sensory systems such as hearing and vision. In the auditory system, age-related hearing loss is linked to the decline of inner hair cell ribbon synapses and decreased hearing sensitivity, leading to decreased speech comprehension. Hallmark changes to the visual system that specifically affect the retina include neuronal loss in the macula, tissue thinning, increased retinal pigment epithelium, and reduced visual function. Of note, the decline in scotopic vision is much more evident than in cone-mediated photopic vision, indicating that the rod pathway is more susceptible to the effects of aging than cone-mediated vision. Rod-generated signals pass to the inner retina via the rod bipolar cell, and in diurnal Chilean Degu (Octodon degus), significant age-related degeneration was observed in the number of rod bipolar cells, dendrites, and terminals than in those of younger rodents. Despite the extensive characterization of age-related changes in the retina and sensory systems, functional changes in rod bipolar cells and their ribbon synapses have not been deeply explored. To better define the impact of aging on vision, we investigated the changes in the function and morphology of rod bipolar cell ribbon synapses in the zebrafish (Danio rerio). Zebrafish are highly effective model organisms because they are easily maintained and cost-effective, attain sexual maturity after 3-4 months, and yield 200-300 offspring on a weekly basis. Most importantly, when the protein-coding genes of zebrafish and humans are compared, 71% of human genes have at least one orthologue in the zebrafish genome, with 82% of human disease-related genes exhibiting homology with at least one zebrafish gene. The short life span of zebrafish provides a unique advantage to study the progression of aging using markers comparable to those used in humans. For example, aged zebrafish often display spinal curvature, cognitive impairment, and visual impairments such as cataracts. The primary aim of the study is to investigate age-related changes in the function and morphology of rod bipolar cell ribbon synapses synaptic ribbons from zebrafish retinas.
VII. Dysregulation of calcium signaling on the glaucoma model
Glaucoma is a complex, multifactorial, polygenetic disease that is the leading cause of irreversible blindness worldwide. Trends predict that by 2040, as many as 111.8 million people worldwide will have glaucoma 2, and many of those will be legally blind due to optic nerve damage caused by the disease. Various subtypes of adult-onset glaucoma share the common clinical pathologies of retinal ganglion cell and optic nerve axonal damage, as well as subsequent visual field defects. Several risk factors are known for this disease 6-8, with elevated intraocular pressure being the major factor targeted by current glaucoma treatments. Previous work from Dr. Jablonski’s lab established that a novel gene that modulates intraocular pressure, α2δ1subunit (aka Cacna2d1), is voltage-dependent and that pregabalin, an antagonist with high specificity for Cacna2d1, lowers intraocular pressure in a dose-dependent manner. However, the mechanism of action by which PRG could function as a neuroprotective agent to retinal ganglion cells is unknown, which is the question my lab will be addressing by testing the hypothesis that the first step of the mechanism of action by which pregabalin is neuroprotective is by attenuating the influx of ionic Ca2+ across the cell membrane. We were able to successfully establish approaches to measure calcium signaling in retinal ganglion cells through the loading of ratiometric calcium dyes via optic stump, as well as ciliary body and trabecular meshwork by optimizing recording solution for calcium imaging with ratiometric calcium dyes.