Recent Submissions

  • EFFECT OF GABAERGIC NEURONS IN SUSCEPTIBLE VERSUS RESILIENT MALE RATS TO PTSD

    Dixon, Rachel; Mandavilli, Rohan; Department of Phychological Sciences; Department of Biological Sciences; Departmetn of Pharmacology & Toxicology; Bunting, Kristopher M; Alexander, Khadijah; Vazdarjanova, Almira; Augusta University (2019-02-13)
    Post-traumatic stress disorder (PTSD) is a psychological disorder that can occur after a traumatic event. Individuals with PTSD exhibit extreme anxiety and learning and memory difficulties. Once exposed, 12-35% develop PTSD with women twice as likely to be affected than men. Our goal is to discover underlying mechanisms to prevent PTSD, as we investigate the relevance of glutamic acid decarboxylase positive (GAD+) neurons on susceptible (SUS) and resilient (RES) male rats. SUS and RES phenotypes were assessed using the highly advanced RISP protocol to reveal susceptibility to a PTSD-like phenotype. The increase of GAD+ cells in the medial prefrontal cortex (mPFC) informs us that more GABAergic neurons are present, which can cause inappropriate recall. We will be examining if there is a difference in the number of GAD+ cells in the RES versus SUS male rats. To investigate, we used cryosectioned brains from SUS or RES rats. The brains were stained using immunohistochemistry to isolate the GAD+ neurons in the mPFC and were counted. The results of this experiment will be determined and examined at a later date closer to our presentation. We expect to see a SUS male rats to have a higher number of GAD+ neurons.
  • THE MECHANISM OF INVERSE AGONISTS ON HISTAMINE RECEPTORS, HISTAMINE RECEPTOR H1, AND HISTAMINE RECEPTOR H2

    Patel, Shrey P; Department of Phychological Sciences; Department of Pharmacology & Toxicology; Lambert, Nevin; Augusta University (2019-02-13)
    The experiment discusses the role of inverse agonist binding to receptors and how its effect cell signaling. The specific receptors that was focused on in the project was histamine receptor H1 (HRH1) and histamine receptor H2 (HRH2) which are types of G-protein coupled receptors (GPCR). Both receptors are activated when a ligand, specifically a histamine molecule, which binds to the receptor and activates the signaling pathway within the cell. The main protein within the signaling pathway is the G-protein which helps the cascade effect of the signal to other molecules. G-proteins are activated through GTP. An inverse agonist works like an agonist but will have an opposite end effect within the cell. It was originally thought that inverse agonist works the same way as an agonist to recruit a GTP and activate a G-protein for signaling. The experiment being tests tries to explain the opposite that the inverse agonist could activate the protein without GTP and continue to have its effect on the cell. Human embryonic cells were transfected with plasmids that contain sequences for the receptors and the G-protein, which were also tagged with a fluorophore to measure any bioluminescence with interaction of G-protein and the receptor when the ligands binds. From collecting data from the bioluminescence effect, it shows that there is an interaction a receptor and G-protein complex when the inverse agonist is bound.
  • The Study of 5ht-1d and 5ht-1f Receptor Interactions with Mini G Proteins via Bret Analysis

    Trang, Amy; Department of Chemistry & Physics; Department of Pharmacology and Toxicology; Spencer, Angela; Lambert, Nevin; Augusta University (2019-02-13)
    G protein-coupled receptors (GPCRs) are receptors involved in signal transduction, a process for converting extracellular signals into internal messages to elicit a cellular response. Signal transduction pathways involve activating various G protein subtypes (Gs, Gi/o, Gq/11�and G12/13) which typically lead to second messenger production. Traditionally, second messenger concentration assays are used to identify GPCR coupling with G protein(s), but they are not efficient in profiling GPCRs since they compare the concentrations from different downstream signals. Instead, novel tools, such as Bioluminescence Resonance Energy Transfer (BRET) and mini G (mG) proteins, can be used to profile GPCRs. BRET is a technique that provides quantitative data when protein-protein interaction occurs and requires the proteins of interest to be fused with either a bioluminescent protein or fluorescent protein. In this study, we used mG proteins representing each G protein subtype to identify 5-hydroxytryptamine (5-HT; serotonin) receptor coupling upon serotonin stimulation. Through BRET assays, we determined that both the 5-HT1D�and 5-HT1F�receptors couple primarily with the mGsiand mGo�classes of mG proteins. This supports previous studies that these receptors couple to Gi/o�proteins and suggests that the use of mG proteins in BRET assays is an effective tool for GPCR profiling.
  • Unconventional Coupling of 5HT7 receptors to Gs heterotrimers

    Adams, Elizabeth; Department of Pharmacology and Toxicology; Lambert, Nevin; Augusta University (2019-02-13)
    GPCRs play a major role in cell signaling through their interactions with heterotrimeric G proteins. In conventional models of GPCR-G protein coupling, agonist binding promotes a conformational change within the receptor, which then associates with G proteins, facilitating the exchange of GDP for GTP. GTP-bound G proteins dissociate from the receptor and exert their effects on downstream signaling molecules. Previous studies suggest that serotonin 5HT7 receptors associate with Gs�heterotrimers prior to agonist binding, and that 5HT7-Gs�complexes dissociate after the G protein is activated. Here we study this unconventional mode of coupling using bioluminescence resonance energy transfer (BRET) between luciferase-tagged 5HT7 receptors and Gs�heterotrimers labeled with Venus. Our results confirm that 5HT7 receptors interact with inactive (GDP-bound) Gs�heterotrimers in the absence of an agonist, and that this interaction is stabilized by the inverse agonist methiothepin. Stimulation with the endogenous agonist serotonin (5HT) decreased BRET between 5HT7 receptors and Gs, indicating that the activation of the receptor leads to 5HT7-Gscomplex dissociation. Interestingly, Gs�activation was not required for complex dissociation. These results are consistent with the hypothesis that 5HT7 receptors couple to Gs�heterotrimers via an unconventional mechanism involving ligand-sensitive complexes of receptors and inactive Gs.