Tag: Adam Miller

The Role of Synaptic Adhesion Molecules in Electrical Synapse Formation in Zebrafish

Presenter: Margaret Grivette − Neuroscience

Faculty Mentor(s): Adam Miller, Jen Michel

Session: (In-Person) Poster Presentation

Chemical and electrical synapses work together to shape brain function but little is known about the regulation of electrical synapse formation4. Chemical synapses are junctions that send neurotransmitters across the gap to the receiving neuron. Electrical synapses are physically connected by connexin proteins which are supported by scaffolding proteins that allow charged ions to diffuse between neural cells1. ZO1b is a MAGUK scaffolding protein required for the formation and function of electrical synapses. It contains three PDZ binding domains that bring other proteins together to organize multiple interactions 1,2. Kirrel proteins belong to the transmembrane immunoglobulin superfamily of cell adhesion proteins and have a cytoplasmic PDZ binding domain (PBD). Kirrel 3 is particularly important because alterations in the gene are associated with intellectual disability and the Kirrel 3 protein has recently been found to help build synapses in the mouse hippocampus 3. To determine whether Kirrels may play a role in electrical synapse formation in zebrafish, we tested whether the predicted Kirrel 3 PDB binds to ZO1b PDZ domains using an in vitro binding assay. We found that the Kirrel 3 C-terminus binds to PDZ1 of ZO1b. This interaction is dependent upon the predicted PDB since removal of the last 6 amino acids of the Kirrel 3 tail abolished the interaction.

Utilizing the Optomotor Response to Measure the Effect of Cadaverine on Larval Zebrafish Behavior

Presenter(s): Laura Reich − Biology

Faculty Mentor(s): Adam Miller, Matt Smear

Poster 41

Research Area: Natural Science

Zebrafish behavior is strongly influenced by environmental stimuli, and olfaction (sense of smell) is a powerful driver of behavioral responses. Our overarching goal is to measure an odorant’s effect on a behavioral response and to understand the sensorimotor transformations that occur within the brain as the animal smells its world and reacts accordingly. As a first step towards this goal, we used a repetitive visual stimulus to induce the optomotor response, causing the zebrafish to swim in the direction of perceived motion. This method allows us to orient the larval zebrafish towards a region of water with an odorant of interest. This research specifically focuses on the impact of cadaverine, an odorant known to stimulate an aversive response, on larval zebrafish. We hypothesize that while a repetitive visual stimulus is in use, the distribution of larval zebrafish in a petri dish will differ when cadaverine is administered to a section of the water. Rather than moving with the visual stimulus, we predict that the fish will avoid regions with cadaverine, halting the optomotor response. This research serves to demonstrate that olfaction, the sense that is often forgotten and taken for granted, plays an important role in zebrafish and can potentially overcome visually-directed behavior.

The genetic basis of the first connections in the brain

Presenter(s): Anisha Adke – Biology

Faculty Mentor(s): Adam Miller

Poster 46

Research Area: Biology

Funding: Vice President for Research and Innovation (VPRI) Undergraduate Fellowship, UROP Mini-grant

An estimated 100 billion neurons form the human brain, equal to the number of stars in our galaxy. Nervous system function emerges from connections, or synapses, between these neurons, which are either electrical or chemical. The synapses form a wired circuit that emerges over development as directed by an organism’s genetic code. The first synapses that form are critical to normal circuit wiring, as they lay the foundation upon which mature circuits are built. Research has shown that these first synapses are electrical, but it is unknown which genes are responsible for the connections. This project aims to identify the genes required for the first synapses and investigate their roles from a molecular, circuit, and behavioral standpoint. This will provide a critical understanding of nervous system wiring, as genetic defects that alter normal circuit wiring are linked to neurodevelopmental disorders such as autism and schizophrenia.
To explore the genes responsible for electrical synapses, we examined the first spinal cord circuits that form in zebrafish. We identified genes-of-interest using RNAseq and are finding when, where, and how these genes control the formation of the first synapses, analyzing them for neural network and behavioral changes. Preliminary behavioral analysis suggests that the gene Cx46.8 is involved in early circuit formation, while mutations in Cx35.1, Cx34.1, Cx35.5, Cx34.7, and Cx43.4 show no visible disruption. Future work using RNA in situ hybridization will characterize Cx46.8 localization, allowing for a better understanding of where the gene is at work. We are also using fluorescent calcium indicators to characterize the mutations and the resulting disruptions to circuit wiring in order to understand the roles of the genes in early electrical synapse formation.

Connexins are not responsible for specification of the electrical synapse

Presenter(s): Elisa Trujillo

Faculty Mentor(s): Adam Miller & Abagael Lasseigne

Poster 56

 Session: Sciences

In order to initiate synaptogenesis two cells must come together and undergo intracellular communication; both can be done through a protein with cell adhesive properties. At chemical synapses, extracellular cell adhesion molecules allow two neurons to communicate in order to recruit compatible pre- and postsynaptic machinery. By contrast little is known about electrical synapses, where gap junction channels physically couple neurons. Transmembrane gap junction proteins at the electrical synapse, Connexins, have adhesive properties. We hypothesized that Connexins are required to initiate electrical synapse formation. To investigate this we created Connexin mutant animals and assessed whether or not a highly stereotyped electrical circuit containing Mauthner neurons was still morphologically normal. We used the localization of the required scaffolding protein, Tjp1b, as an indicator for electrical synapse specification. Connexin proteins are co-dependent; without one Connexin the other is unable to localize to the synapse. I tested the requirement of the pre- and postsynaptically required Connexin proteins for normal neuron morphology and Tjp1b localization by selecting fish with green fluorescent protein (GFP) positive Mauthner neurons and immunostaining zebrafish larvae for Tjp1b, and GFP in animals with non-functional Connexin proteins. Despite the loss of Connexins, Tjp1b still localized at the potential electrical synapse site and the morphology of the Mauthner neuron remained normal. Thus, Connexins do not appear to be the proteins responsible for electrical synapse initiation. My future work will aim to identify the protein with cell adhesion properties necessary for electrical synaptogenesis.

An Investigation into the Genetic Basis of Spinal Cord Regeneration

Presenter(s): Tova Kruss

Faculty Mentor(s): Dylan Farnsworth & Adam Miller

Poster 5

Session: Sciences

Unlike mammals, Zebrafish (Danio Rerio) are unique in that they can regenerate their nervous systems. The genetic expression involved in Zebrafish nervous system regeneration remains widely uninvestigated, however with potential applications in mammalian cell therapy and injury recovery, it remains a critical area of ongoing research. Throughout the following experiments, the spinal cord of Zebrafish was used as a simplified model for the entire connectome, with regeneration initiated through mechanical lesioning. To narrow the scope of the inquiry gene expression exploration focused on the neural progenitor lineage of olig2 expressing cells, a subset of neurons including motor neurons and glia. Through the utilization of single cell RNA sequencing gene expression during regeneration was assessed on a cell by cell basis, and Vimentin was found as an exciting candidate gene for further study due to the high degree of specific expression of this gene in regenerating cells.

Utilizing Behavioral and Molecular Techniques to Study Gap Junction Channels in Developing Zebrafish

Presenter(s): Laura Reich—Biology

Faculty Mentor(s): Rachel Lukowicz, Adam Miller

Session 5: The Bonds that Make Us

Animal behavior requires coordination between the nervous and muscular systems . These systems communicate at specialized subcellular structures, found within and between systems, that allow the cells to coordinate their activity to achieve movement . One type of communication arises from gap junction channels (GJCs), which are built by the Connexin (Cx) family of proteins that allow for direct ionic and small molecule exchange between interconnected cells . The GJC family is large with up to 20 individual genes encoded in the human genome . Given this complexity, it is unknown how individual Cxs contribute to behavior . We are using the embryonic zebrafish to address this question due to its rapid development, genetic access, and its first behavior, spontaneous coiling, which requires GJCs . We first identified Cxs that were likely to contribute to coiling using a combination of RNA-seq and RNA detection in vivo and found a previously uncharacterized Cx, Cx46 .8, expressed in slow muscle fibers . To understand Cx46 .8’s involvement in coiling, we developed an automated behavioral tracking system, using DeepLabCuts (DLC), to track movement during spontaneous coiling . Using this system, we found that animals lacking Cx46 .8 have defects in coiling, indicating that we have identified a novel Cx that contributes to behavior . Further experimentation will utilize DLC, in addition to molecular techniques, to unravel the molecular and functional mechanisms of Cx46 .8 and other Cxs that contribute to GJC communication in behavior .