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Neuroscience [clear filter]
Tuesday, April 24
 

9:00am PDT

Inducing Gene Expression Of Major Urinary Proteins In The Female Murine Liver Cell Line Hepa1-6
Mice, Mus musculus, are a primarily nocturnal species that rely heavily on their olfactory system to detect changes in their environment. Specifically, mice rely on protein pheromones, the Major Urinary Proteins (MUPs), non-volatile molecules which are detected by the vomeronasal organ (VNO). MUPs are synthesized in the liver, excreted in the urine, and serve as genetically encoded pheromones which direct social behaviors such as countermarking, aggression, or mate preference. Mice can also use MUPs as a way to detect sex, status, and identity of the emitting individual. MUP expression is thought to be controlled by a set of hormonal axes consisting of testosterone, growth hormone, and thyroxine. The mouse genome encodes 21 MUPs, yet, each adult male mouse will express a unique set of 4-12 MUPs. The mechanism by which MUPs are chosen for expression is non-random but not well understood. This study looks to understand how individual MUPs are chosen for expression by utilizing a cell culture model system. The female murine liver cell line Hepa1-6, is being used because it does not endogenously show expression of any MUPs, but previous studies have shown that female mice are capable of producing MUPs at male levels if they are exposed to testosterone. A combination of hormonal and drug treatments consisting of methylation inhibitors and deacetylation inhibitors are being used to induce MUP expression in these cultured cells. Following treatment of cells, they are harvested for RNA isolation, and the resulting cDNA library is examined for MUP expression. The results of this study using the chosen concentrations and treatment periods are not sufficient to induce MUP expression. As such, a working protocol for the induction of MUP expression is yet to be established. The creation of a working protocol will in the future contribute to the greater understanding of gene expression.

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Tuesday April 24, 2018 9:00am - 9:20am PDT
014 Zeis Hall

9:20am PDT

Cloning Of Vomeronasal Type-2 Receptor For Deorphanization
The vomeronasal organ (VNO) is a chemosensory organ present in amphibians, reptiles, and non-primate mammals. In mice, vomeronasal neurons express vomeronasal-1 receptors (V1R) or vomeronasal-2 receptors (V2R), both of which are G protein-coupled receptors involved in pheromone detection. V2Rs are expressed by the basal neurons of the VNO. They are of special interest because they are used to detect protein pheromones, the Major Urinary Proteins (MUPs), which induce intermale aggression, female responsiveness to mating, and territory marking behaviors. Because V2Rs use combinatorial coding instead of a labelled line coding strategy, linking pheromone responses to the correct V2R has so far been difficult. We designed primers for each V2R to amplify separate individual sequences from a cDNA library, which could then be transfected into cells using vectors. This cell culture method would allow for deorphanization of V2Rs by creating entire cell populations which only express a single V2R. With V2Rs deorphanized, mapping of pathways can begin from a bottom-up method, instead of the more difficult top-down approach. Here we show how to isolate and clone V2Rs for individual eventual expression in mammalian cells using DNA purification, Zero Blunt TOPO cloning PCR kit and ligation into mammalian vector. V2R 121 was successfully cloned into a bacterial vector with a complete sequence. The blunt end cloning did result in correct directionality when the V2R was transferred into a mammalian vector. These results demonstrate progress towards setting up a cell culture based V2Rs expression system. This system will allow for further research to deorphanize individual V2Rs by matching them to their corresponding ligands. Stimulation of V2Rs reproducibly activate specific neural circuits in mouse brains, allowing for reliable study of how external environmental cues can direct behaviors. Cloning V2Rs is the first step to identify receptor-ligand interactions, which can be utilized for future research.

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Tuesday April 24, 2018 9:20am - 9:40am PDT
014 Zeis Hall

9:40am PDT

Developing a Model System to Establish Electrophysiological Protocols Necessary for the Deorphanization of Vomeronasal Sensory Receptors (VNSRs)
Chemical signaling mediates many complex behavioral interactions such as mating and the establishment of hierarchy for a variety of animal species. A key form of chemical signaling in rodents is facilitated by the production and detection of intraspecific pheromones known as major urinary proteins (MUPs). Pheromones such as MUPs convey specific information which is innately recognized by members of a species. In rodents, MUPs are detected by receptors on specialized sensory neurons in the vomeronasal organ (VNO) known as vomeronasal sensory receptors (VNSRs). Once a MUP binds to a VNSR, the sensory neuron initiates neural circuits extending to other sections of the brain such as the amygdala and surrounding limbic structures leading to a behavioral response. Sensory neurons expressing different VNSRs will activate different neural circuits, thus allowing different MUPs to evoke specific behaviors. The exact behavioral response to a certain MUP is predictable for all members of the species and indicates common neural circuitry associated with MUP communication. This uniformity of neural circuitry in mice allows for MUP communication to serve as a reliable model system for studying how chemical stimuli code for behavioral outputs. This study aims to determine which MUPs activate a given VNSR in order to elucidate the neural circuits responsible for specific behavioral reactions. To achieve this understanding, patch clamp analysis will be used to deorphanize VNSRs by monitoring electrophysiological changes in cells expressing VNSRs upon exposure to specific MUPs. If the MUP being introduced is able to bind the specific VNSRs expressed by the cells, a measurable change in the cells voltage will occur. Currently, a model system using CHO cells transfected to express kir2.2 channels is being established to yield preliminary methodologies that will be used to complete this study.

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Tuesday April 24, 2018 9:40am - 10:00am PDT
014 Zeis Hall

11:20am PDT

Cloning Vomeronasal Type-2 Receptors For Expression And Analysis In A Cell Culture Model System
The vomeronasal organ (VNO) is an olfactory sense organ in the nose of mice that detects pheromone signals through ligand binding to G-protein coupled receptors. There are three families of VNO receptors, V1R, V2R, and FPR. V2Rs in mice primarily serve to bind large molecules like the major urinary proteins (MUPs), proteins secreted in urine that trigger contextual behaviors in the recipient. Through combinatorial coding, multiple combinations of MUPs can activate multiple V2Rs in different ways, leading to complex signals based on a small library of ligands. However, VNO receptors are orphaned, it is not known which MUP ligand binds with which VNO receptor. This research set out to deorphanize V2Rs and pair them with their cognate ligands to create a library of receptor-ligand pairings. Receptor deorphanization will involve cloning V2Rs into mammalian cells, then analyzing them using patch clamp to measure membrane voltage changes when exposed to MUP ligands. Sequences coding for V2Rs were amplified through PCR, visualized on a gel, relevant bands were extracted and purified, then TOPO cloned into bacterial plasmids and transformed into JM109 E. coli cells for mass growth. Plasmids from E. coli were restriction digested to verify insert sequence length, then ligated into mammalian pEGFP vectors for eventual transfection into eukaryotic cells. To date, receptors have been cloned, visualized, extracted, purified, and transformed for V2Rs 34, 60, 92, 121, 122, 81, and 83. One sample (122-1) indicated a full length sequence, and has been ligated and sent for sequencing. If the results indicate a full length sequence inside the mammalian vector, the plasmid will be transfected for surface expression. This experiment is an important first step to being able to better understand and map the exact neural pathways activated by an environmental chemical stimulus, and how it produces a response in the host.

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Tuesday April 24, 2018 11:20am - 11:40am PDT
123 Zeis Hall