3 million Americans and 65 million people worldwide have been diagnosed with epilepsy. In two-thirds of these patients, the causes of epilepsy, and therefore the therapeutic targets, are unknown. Recently, de novo mutations in the EEF1A2 gene, which encodes for a protein translation regulator, were found in several individuals with pediatric epilepsy and intellectual disability. In order to study how a mutation in this gene causes the neurological defects, we generated mutant eef1a2 zebrafish using the CRISPR/Cas9 method. Zebrafish models to study diseases have gained momentum due to their genetic similarity to humans, large amount of offspring in each generation, easy genetic manipulation and amenability to large-scale drug screens. Our initial in situ hybridization studies showed that eef1a2 has a limited expression pattern in the developing zebrafish that includes neurons involved in movement and escape behavior. To understand whether the mutation causes observable behavioral dysfunction or seizures, we tracked wild type (WT) zebrafish and heterozygous and homozygous mutants in various behavioral assays and genotyped tracked fish using Sanger sequencing. We are expecting that the mutants to have defect in movement and escape response. In a future study, the electrophysiology of the forebrain will be recorded to determine whether eef1a2 mutants reveal abnormal electrical discharge characteristic of a seizure. These studies with the zebrafish model will provide more insight about the EEF1A2 protein and its neurological function, and may lead to a better understanding of epilepsy etiology and the design of targeted therapeutic approaches.

Hair cells are mechanosensory receptors in the inner ear used for hearing and balance. When hair cells are damaged or die, hearing is lost or impaired. Many studies have also shown that genetic mutations affecting mitochondrial function are another cause of hearing loss. However, it is unknown which cells in the auditory system are affected. In this study, we sought to determine whether or not these deafness-causing mutations impact hair cells. To do so, we took advantage of zebrafish as a model system and studied hair cells of the lateral line. To generate mutant fish, we used the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas system, a genome editing tool that allows for gene-specific changes to be made to DNA. If these mutations do affect hair cells, we predict to see changes in mitochondrial and hair cell function. Characterizing these mutations in zebrafish will inform our understanding of the cellular mechanisms of hearing loss.

Retinitis Pigmentosa, a progressive neurodegenerative disease, is a significant contributor to loss of vision due to retinal degeneration. The retina is made up of light sensing cells called rods (for night vision) and cones (for day vision). In patients afflicted with this disease, there is a degeneration of rod photoreceptors over time, which leads to a decreased visual field and night blindness. At a certain point, the loss of rod cells leads to the breakdown and loss of cone cells, which are responsible for color vision. Mutations in the rod gene Rhodopsin have been linked to the development of this disease. By down regulating this defective gene, the degeneration of rods would likely be prevented. Subsequently, cone degeneration would also be prevented, limiting the loss of vision in afflicted individuals. The expression of Rhodopsin is controlled by a nuclear hormone-like receptor transcription factor called Nr2e3. In collaboration with another lab (Dr. Sheng Ding, UCSF), we have identified small drug-like molecules that can block Nr2e3, and we have called them CA88, CA95, and CA97. We predict these antagonists will lower the expression of rod genes. Wildtype mouse retinas were treated in culture with CA88, CA95, or CA97. The use of these small molecules resulted in the significant down regulation of a number of rod genes. These genes include Rhodopsin, NRL, NR2E3, and GNAT1.  Stained tissue sections further confirmed a decrease in Rhodopsin with treated retinas.  When retinal ex plants were done using mice with a P23H mutation in the Rhodopsin gene, treated retinas were found to have a significantly thicker photoreceptor layer than non-treated, indicating the ability for these compounds to prevent retinal degeneration. With a way to reliably down regulate problematic genes such as Rhodopsin, a potential avenue for treatment of humans affected by Retinitis Pigmentosa is opened.

Calcium is an ubiquitous and critical signaling molecule that mediates a wide range of cellular processes. Cytosolic calcium is maintained at concentrations that are orders of magnitude below both the extracellular space and intracellular organelles. Even small, sustained increases in cytosolic calcium can have harmful effects on the cell. As such, calcium homeostasis mediated in large part by the endoplasmic reticulum (ER) and mitochondria that serve to sequester calcium enable normal functioning of the cell. To examine cellular mechanisms that mediate calcium homeostasis, we are using mechanosensory hair cells of the zebrafish lateral line. Mechanosensory hair cells can be stimulated via a mechanical stimulus or drug-mediated calcium release from intracellular stores. The hair cells' abilities to easily uptake drugs and respond to physical stimuli make them an ideal tool to examine calcium homeostasis. Preliminary studies using transgenic zebrafish in which the calcium indicator GCaMP3 is targeted to the cytosol vs. the mitochondrial matrix demonstrates that stimulation of hair cells causes an immediate increase in cytosolic calcium in contrast to a gradual increase in mitochondria calcium. The question is whether the calcium in the ER is changing in response to the changes in mitochondrial and cytosolic calcium. As a first step to investigating the role of ER in calcium homeostasis, we will use gateway cloning to generate transgenic zebrafish lines in which calcium indicators are targeted to the ER. We predict that stimulation of mechanosensory hair cells will cause changes in ER calcium levels. These studies may provide valuable insight in how cells maintain health by buffering intracellular calcium levels in response to activity. 

Area V4 is a cortical region that carries out intermediate visual processing along the ventral ("what") visual pathway. It integrates information about boundary and surface features from the primary visual cortex and sends signals downstream to the inferior temporal cortex, which supports complex object recognition. Neurons in area V4 have been shown to encode the boundary curvature of 2-dimensional (2D) shapes in an object-centered coordinate system. For example, a neuron may prefer a wide variety of stimuli that have a protrusion (convexity) to the upper right. Our goal is to determine whether this type of boundary representation along 2D boundaries also applies in three dimensions (3D). We also aim to determine whether the same V4 neurons that encode 2D shapes also encode 3D form, or whether there might be separate populations activated by 2D and 3D form. To address this, we created a set of images of spheres that are deformed by the addition of a dent or protrusion. We varied the pose and the direction of lighting. The images were based on wire-frame models created in Blender. We recorded responses from single neurons in the fixating macaque monkey using a novel set of 156 3D images and a set of 230 2D images have been studied in previous experiments. We found that our 3D stimulus set elicited robust differential responses from V4 neurons. Some neurons responded better to 2D shapes without surface shading, while others preferred 3D shapes. Our next steps are to determine whether responses 2D and 3D shapes can be explained with one unified model. Ultimately, we want to understand how neurons encode natural scenes, and studying responses to such parametric, naturalistic stimuli brings us closer to achieving that goal.

Disorders and diseases of chronic pain have placed a tremendous burden on modern society, imposing myriad consequences on many individuals and populations. Despite extensive investment to date, therapies to treat chronic and acute pain remain limited; the most-widely used, researched, and prescribed drugs often act generally upon the nervous system. This characteristic of most existing therapies carries with it many unintended consequences and unwanted effects for pain patients. The limitations of these therapies are often traceable to our limited understanding of the neuronal circuitry responsible for the sensation and transduction of noxious or other pain-inducing stimuli. We utilize zebrafish in our forward genetic screen to identify mutations that disrupt the ability to perceive pain. Behavioral tracking of larval zebrafish, specifically the progeny of mutated genetic lines, allows us to screen for a large number of potential phenotypic mutations. By identifying mutant lineages in this screen, we hope to further elucidate and characterize the neuron cell populations responsible for nociception. Our genetic screen has identified several lineage of fish that lacked the behavioral “escape” response typical of embryonic zebrafish when immersed in a noxious chemical environment, in this case, 10µM allyl isothiocyanate (AITC), the pungent compound in mustard oil. Subsequent complementation to known mutants suggested that one of these families carried a mutation in a previously identified nociceptive ion channel, TRPA1, which is specifically expressed in somatosensory neurons, and has been shown to be required for the pain response induced by noxious chemicals, including AITC. We have crossed mutants into well-characterized genetic backgrounds and used RNA-seq based mapping to narrow these lesions to a region of Linkage Group 9. Following fine-mapping, we will subsequently study the role these gene(s) play in the sensation of pain. Our findings suggest that our screening strategy is likely to be successful and is feasible for identifying genes involved in nociception.

Recognizing partially occluded objects is a major challenge for even the most cutting edge computer vision systems even though humans recognize partially occluded objects effortlessly. To begin to understand the neural basis, past research studies have investigated the correlation between animal behavior and neural activity during recognition under occlusion. These studies have been done on stable occluded objects even though it is well known that stimulus motion can attract attention to objects and can facilitate segmentation of objects in clutter, for example, in breaking camouflage. We investigated whether motion of an occluded object facilitates its recognition. To address this question, we asked human subjects to report whether two stimuli were same or different by pressing keys, M or N, for match or non-match. We moved the second stimulus at different speeds behind a field of occluding dots and asked how the percent correct performance and reaction time were influenced by the speed of movement of the visual stimulus. We found that 4 of the 6 subjects we tested exhibited a decreased reaction time and increased percent correct performance with stimulus motion. To verify that this difference was not simply because motion revealed parts of objects that were previously occluded by dots, we designed a control where the occluded shape underwent random jumps as opposed to coherent motion. In this case we did not observe the benefit of motion on performance. Thus, our results support the hypothesis that motion of an occluded stimulus enhances its recognition and discriminability.

Retinal degeneration is one of the leading causes for blindness in working-age people. It results from the loss of one or more of the seven different cell types in the retina which eventually disrupts the structure and signaling between the cells, ultimately leading to irreversible vision loss. Unfortunately, mammalian retinas cannot regenerate lost neurons, and current therapies only slow down disease progression. Alternatively, cell therapy using self-renewing, pluripotent embryonic stem cells (ESCs) could provide an unlimited source of replacement retinal cells. Many groups, including our own have developed protocols to derive retinal neurons from ESCs. While these can generate photoreceptors well, the generation of complete retinas from ESCs in a consistent and more efficient manner has remained elusive. Our research thus focused on optimizing the existing retinal differentiation protocols for mouse ESCs, with the goal of producing more laminated retinas consisting of all retinal cell types. In the current study, we used the three-dimensional method to produce embryoid bodies (EB) from our ESCs. We tested the effectiveness of Fetal Bovine Serum (FBS) and hyperoxia to enhance retinal differentiation by supplying them to our EBs. The samples were collected every week for five weeks and analyzed by immunohistochemistry. We found that both conditions increased production of inner retinal neurons such as ganglion cells and amacrine cells, and EBs grown under hyperoxia were healthier looking. We then compared the ESC-derived retinas to age-matured mouse retinas and found that both retinas were similar in structure with the different cell types organized in the correct arrangement. These results suggest that FBS and oxygen are additional factors that can be integrated into existing protocols to improve the differentiation of mouse and possibly also human ESCs into retinal neurons.