Humans hear through the conversion of pressure waves vibrating in the inner ear into chemical signals released by activated hair cells. Loss of function of hair cells due to damage can result in permanent hearing loss. Zebrafish are one of the model organisms used to study hair cells, since zebrafish have hair cells similar to humans. Unlike humans, zebrafish can regenerate their hair cells after damage. Regeneration occurs when support cells differentiate into hair cells. Determining how zebrafish support cells differentiate into hair cells is important to understand if human support cells can be induced to differentiate in a similar way. This requires comparing the structural differences between support cells and hair cells. One important aspect of neuronal signaling is the release of calcium ions from the cell’s endoplasmic reticulum (ER). This experiment looked to answer whether the ER in zebrafish hair and support cells was quantifiably different. Since structure mediates function and support cells do not signal to neurons, their ER structure should not be equivalent to the hair cells’. To test this, I manually segmented and reconstructed serial block-face scanning electron microscope (SBF-SEM) images of both cell types’ ER into 3D. Using SBF-SEM as a reconstruction method allowed for more detailed visualization of both the cell volume and the ER, in contrast to other methods such as confocal microscopy. Support cells had an ER volume of 50.51 µm3, while hair cells had an ER volume of 29.09 µm3. Support cells had a higher ER to cell volume and ER surface area to volume ratio than hair cells. It can be concluded that ER structures of support and hair cells are quantifiably different. Quantifying ER differences between support and hair cells is an important step toward discovering solutions to deafness caused by damage to human hair cells.

The primate brain effortlessly segments objects from background stimuli, however, the neuronal underpinnings of this process are largely unknown. Past studies in V1, the primary visual cortex, have demonstrated that neurons encode visual information within the receptive field through edge detection, by which the greatest local contrast evokes the largest response. These studies have typically focused on luminance contrast, but edge detection and object segmentation can rely on a variety of other factors including contrasts in color, texture and blur. The primary goal of my research project is to investigate how variances in contrasts of luminance, color saturation, orientation and blur within a textural image affect neuronal response in primary visual cortex V1 and in subsequent stages V2 and V4. I have created a novel stimulus set that individually incorporates these varying types of contrast. I will record the responses of neurons in the visual cortex of a macaque monkey to these stimuli using Neuropixels, which will allow us to determine whether neurons are selective to object form and whether the strength and latency of form selectivity depends on the stimulus dimensions that define object boundary. We expect that different subgroups of neurons in V1 and V2 may be sensitive to different types of stimulus contrasts, while the same neuron in V4 may be sensitive to object shape regardless of the stimulus modality but the different modalities may be associated with different latencies. Data analysis and discussion of results will be completed and prepared for presentation by May. Our research will build upon and contribute to the greater understanding of visual information processing within the primary visual cortex. 

Loss of neurons in the retina underlie many blinding diseases, such as macular degeneration, glaucoma, and diabetic retinopathy. These retinal neurons are not replaceable. The Reh Lab has developed a strategy to achieve functional regeneration of neurons in the adult mammalian retina, but not enough regeneration to restore vision loss. One of the potential limitations to restoring vision is the inflammation that occurs during retinal injury or disease. All mammalian retinas contain microglia, which are the primary immune cells of the nervous system that respond to pathogens, injury, and disease. My goal is to determine how microglia respond to damage-induced regeneration in the retina. After damaging mice retinas with an excitotoxin (NMDA), we induced neuronal regeneration with a proneural transcription factor called Ascl1. Using immunohistochemistry and confocal fluorescence microscopy, I stained various markers that represent different inflammation states of microglia. I quantified microglia and analyzed their response to neural regeneration. Data from confocal fluorescence microscopy revealed that microglia surround newly regenerated neurons and display a variety of subtypes during regeneration. Follow up single-cell sequencing experiments confirmed that these immune cells show a molecular heterogeneity of states. This study provides a better understanding about how microglia function during retinal regeneration. Results from this study can help reveal targets for manipulation to improve regeneration of the retina. Further work can be done to analyze how these microglia behaviors impact regeneration of lost retinal neurons.

In the Reh Lab, our research team focuses on neurodegenerative diseases that affect the retina by causing the death of neurons which result in irreversible blindness. In working towards treatments for retinal diseases we have engineered Muller glia to serve as a source for functional regeneration in the mammalian retina, but not enough to restore lost vision. While this is remarkable progress, limitations to this regeneration strategy still exist in regards to the amount of neurons generated. Recently, we have found that the endogenous immune cell of the retina, the microglia, responds to dead neurons by causing inflammation, which restricts the regenerative capacity of Muller glia. However, microglia are not the only immune cell present during regeneration and we currently do not have an understanding of the diversity of cell types that contribute to this regeneration restricting inflammation. My research project aimed to fill this gap by revealing which immune cells infiltrate the retina after injury and thereby revealing immunomodulation targets to improve retinal regeneration. In order to begin to understand the interaction of the peripheral immune system and the nervous system and how it impacts neurogenesis, I will present on my examination of monocyte and neutrophil invasion after retinal injury. For this study I have processed retinas using cryosectioning, immunohistochemistry, fluorescent antibody labeling, and confocal microscopy. Using this data I visualized and quantified the infiltration dynamics of monocytes and neutrophils in the damaged and regenerating retina. Data from this proposal will determine the types of immune cells and their window of infiltration for cell-type specific immunomodulation strategies to improve the regenerative capacity of the mammalian retina.This understanding of the immune component of regeneration is critical in moving forward to develop therapies for not only retinal diseases such as glaucoma but all neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

Induced pluripotent stem cells (iPSCs) can be directed to create 3D mini retinas in vitro known as retinal organoids. Retinal organoids recapitulate the developmental timeline of the human fetal retina and have the potential to serve as disease models for a multitude of retinopathies. However, we have observed that retinal organoids contain a disorganized inner nuclear layer and discontinuous lamination hindering their ability to be considered as a comprehensive disease model. The protein β-catenin is produced from the canonical WNT signaling pathway and has been shown to be essential for retinal lamination in mice due to its role in cellular adhesion. I investigated the effects of a canonical WNT signaling pathway agonist, CHIR,  in order to improve the disorganization and lamination of retinal organoids so that we can develop a comprehensive disease model for different retinopathies. We have used three different stem cell lines to construct retinal organoids that I cultured with the addition of the WNT pathway agonist, CHIR. I performed immunohistochemistry staining followed by microscopy analysis and have obtained data that shows an increase in lamination and cellular organization with the addition of the WNT signaling pathway agonist. Our data suggests that the WNT signaling pathway plays a role in maintaining organization and lamination in the developing human fetal retina.