Tag: Philip Washbourne

The Gut Microbiota Influences Synapse Formation during Zebrafish Development

Presenter: Collette Goode

Faculty Mentor: Philip Washbourne, Alexandra Tallafuss

Presentation Type: Poster 64

Primary Research Area: Science

Major: Biology

The central nervous system depends on the appropriate formation of synapses between neurons to enable communication throughout neural circuits, which generates behavioral and cognitive functions. Recent studies have shown that some individuals with Autism Spectrum Disorder (ASD) have an abnormal composition of the bacterial community resident, referred to as microbiota, within their intestine. Studies in mice suggest that the microbiota can signal to the developing brain, indicating that changes in the intestinal microbiota may underlie some of the deficits seen in ASD. We began to test this hypothesis by using zebrafish as a model to learn how the microbiota affect synapse formation. We used immunohistochemistry and confocal microscopy to compare synaptic protein distribution in the brains of conventional and germ-free zebrafish. We focused on the forebrain, which is speculated to correlate with complex behavior in zebrafish. We labeled the pre-synaptic proteins SV2 and Synapsin1/2 to allow us to image and quantify synapse density in the telencephalon of germ-free and conventional zebrafish larvae at 6 days post-fertilization. We found a significant increase in the number of synapses expressing Synapsin1/2, but no difference in synapses expressing SV2 in germ-free compared to conventionalized zebrafish. Further study of synapse density, function and behavior of germ-free fish will promote our understanding of the correlation between the microbiome, synapse formation, and prevalent neurodevelopmental disorders.

Synapse Formation Is Modulated by a Molecular Hitch

Presenter: Erik Burlingame

Faculty Mentor: Philip Washbourne

Presentation Type: Poster 50

Primary Research Area: Science

Major: Biochemistry

Funding Source: Undergraduate Research Fellowship, Center on Teaching and Learning, $9369; UROP Mini-grant, Undergraduate Research Opportunity Program, $1000; McNair Scholars Program, TRiO, $13221; Swayne Family Scholarship, Swayne Family Foundation, $8800

Synapses are functional units of connectivity that permit the exchange of information between cells in the nervous system. As such, aberrant synapse formation is implicated in a host of neurodevelopmental disorders. Normal formation of synapses requires the transport, recruitment, and stabilization of the synaptic vesicle-regulating protein synapsin to nascent synapses. Recruitment of synapsin to nascent synapses is regulated by cyclin-dependent
kinase 5 (Cdk5), but the downstream effectors of Cdk5 that enable this recruitment remain elusive. Using a zebrafish model, our research examines a putative role of the scaffolding protein calcium/calmodulin-dependent serine kinase a (CASKa) in synapsin recruitment. The mammalian ortholog CASK participates in multipartite transport complexes and is localized to presynaptic terminals by Cdk5-mediated phosphorylation. These observations spurred our hypothesis that Cdk5 phosphorylates CASKa to recruit synapsin to presynaptic terminals. Using a stereotypical touch-evoked behavior to assess synapse function, we found that embryos misexpressing non-phosphorylatable CASK protein exhibit a significant reduction in touch response when compared to embryos expressing either endogenous CASKa or exogenous mammalian CASK protein. Immunofluorescent characterization of synapses from touch-sensitive neurons in these touch-insensitive embryos shows deficits in synapsin localization, further supporting a physical intersection of CASK and Cdk5 during synaptic development. With human CASK dysfunction being linked to defects in synapses, microcephaly, and X-linked intellectual disability, this examination may help establish a novel target for CASK-associated disorder remediation.

Exploring Neuronal Cell Ablation & the Social Behavior Network in Zebrafish

Presenter(s): Allison Hoslett

Faculty Mentor(s): Alexandra Tallafuss & Phil Washbourne

Poster 71

Session: Sciences

Research for neurodevelopmental disorders characterized by social deficits, such as Autism Spectrum Disorder, has helped increase the quality of life in individuals and families afflicted by these diagnoses. This research aims to understand the neuronal network underlying social behavior in the developing brains of zebrafish (Danio rerio), an experimental animal model that shares relevant cellular pathways and social behaviors that are conserved between vertebrate animals. The neuronal circuit involved in social behavior is poorly understood. We are using genetic and behavioral research techniques to identify populations of neurons that are necessary to recognize biological motion, an important part of social behavior. We are combining behavioral assays and genetic tools that allow for cell tracking and targeted cell death using the nitroreductase/metronidazole system. After targeted ablation of neurons in different areas of the brain, we measure the social behavior of individual zebrafish larvae. We do this by projecting dots that imitate the presence and movement of another fish, tracking the fish’s reaction with these dots, which is then calculated into a social index. We were able to identify neuronal populations that, after ablation, severely reduce social behavior. Using this approach will allow us to identify a more complete picture of how the social circuit works and which neuronal populations are involved. Unraveling the social circuit will allow early identification and more targeted treatment of patients with neurodevelopmental disorders that are characterized by impairments in typical social behaviors.

Influence of Sensory Systems on Social Behavior

Presenter(s): Adeline Fecker

Faculty Mentor(s): Phil Washbourne & Sarah Stednitz

Poster 75

Session: Sciences

Disruption in social behavior is characteristic of Autism Spectrum Disorder, which is a neurodevelopmental disorder that appears in early childhood. Previous papers measured social orienting behavior in zebrafish in a dyad assay and showed lesioning of the ventral forebrain reduced social orienting specifically (Stednitz, 2018). These specific neurons may be evolutionarily conserved and may be found in humans. This study aims to identify other parts of the forebrain that may be implicated in social behavior, to understand which senses contribute to social behavior, and to understand how brain activity patterns relate to sensory conditions and behavior. Measuring behavior in an open field allows us to qualify more complex social behavior like orienting, following and dispersing. The Stednitz (2018) paper suggested subjects must be able to see each other to demonstrate orienting behavior and show activation of the ventral forebrain. However, in an open field, subjects are able to interact without visual stimulus. A deeper investigation into the importance of sensory systems in social behavior can be achieved through olfactory and mechanosensory ablation. Early results suggest the visual system is not required for social interaction, as zebrafish can still follow each other in the dark. Whole brain immunolabeling with pERK and ERK allows for an unbiased approach to identifying important brain regions in social orienting. The neurons in the ventral forebrain (marked “ y321 ” with GFP) acts as our landmark, and our results confirm the importance of the ventral forebrain in social behavior. However, other regions of the forebrain vary in activity in different experimental sensory conditions. Our analysis of behavior and corresponding brain activity will shed light onto more regions that may be implicated in social behavior.

Visual Input Principally Drives Zebrafish Social Behavior

Presenter(s): Adeline Fecker—Biology

Faculty Mentor(s): Phil Washbourne

Session 6: Interact & React

Disruption in social behavior is characteristic of Autism Spectrum Disorder, a neurodevelopmental disorder that appears in early childhood . Previous experiments in zebrafish showed lesioning of the ventral forebrain reduced social engagement . Results also suggested subjects must be able to see each other to socialize (Stednitz, 2018) . Subsequent experiments demonstrated that zebrafish can interact without vision, perhaps using their other senses like the water-pressure mechanosensory and olfactory system . Our study investigates how sensory modalities contribute to social behavior . Measuring behavior in an open field allows for quantification of complex social behaviors like orienting, following, and dispersing . We manipulated sensory modalities by recording behavior in the dark and lateral line ablated conditions . Our results show the loss of the visual input causes a 20% reduction in orienting behaviors but no reduction in following behavior . When we ablate visual input and mechanosensation, we do not observe a reduction in orienting or following behaviors . Another outstanding question is which brain regions are activated during social behavior by the contributing senses . We use whole brain immunolabeling with neuronal activity markers as an unbiased approach to identifying and quantifying active brain regions in social and alone conditions . We found the anterior dorsal pallium of the forebrain is significantly more active in social than alone conditions . Further analysis will reveal regions of activity due to manipulated sensory modalities . Our study of behavior and corresponding brain activity sheds light on the importance of sensory modalities and the anterior dorsal pallium in social behavior of zebrafish .

Cell Specific Ablation: An Examination of Zebrafish Social Circuitry

Presenter(s): Matthew Dawson—Biology/Psychology

Faculty Mentor(s): Philip Washbourne

Session 5.5: McNair Scholars Presentations

The zebrafish has been long used in research to examine the effects of manipulating genes using tools such as the CRISPR/Cas9 system . Nitroreductase is an enzyme, native to E . coli, commonly used in biology for cell specific ablation . By inserting the gene for producing nitroreductase, as well as Gal4/UAS system, one can achieve temporal and spatial selectivity for ablation . After cell ablation, fish were placed into a virtual social behavior assay to look for differences in social interactions . Fish were then terminally sampled and stained using immunohistochemistry and imaged using the Spinning Disk Confocal microscope . Using these images, an average brain was constructed allowing for mapping of the neurons necessary for social behavior . This study aims to begin characterizing the neural circuitry for social behavior in zebrafish .

Identifying Neurons Necessary for Social Behavior

Presenter(s): Matthew Dawson—Biology and Psychology

Faculty Mentor(s): Philip Washbourne

Session 5.5: McNair Scholars Presentations

Humans have a long history of working in social groups with a plethora of research supporting the benefits of positive human interaction . While these behaviors may seem intuitive, they can be argued as the most intricate behaviors displayed by animals . To generate a situationally relevant response to social others, an animal must be able to track dynamic systems . Given the inherent complexity of social behaviors, it is expected that this system can be perturbed by a multitude of neurological disorders . Using animal models, we can begin to construct the neuronal circuitry necessary for social behaviors . This circuitry can be used to understand social behavior deficits and may reveal possible interventions for disorders . Zebrafish, Danio rerio, perform stereotyped social behaviors, such as shoaling, which can be used to explore neuronal changes associated with deficits in performing the behavior . Through chemo-genetic ablations, we were able to cause cell death to select neurons due to variations in gene expression . Chemically treated fish were put into a virtual social assay that untreated zebrafish respond to . Zebrafish exhibiting decreased social response would indicate that the ablated neurons are necessary for social behavior . Our results indicate a population of neurons in the forebrain that are necessary for correct social behavior . This insight will help us construct the circuitry underlying social interactions .