Neurosteroids are a group of endogenous compounds locally produced from cholesterol by neurons and glial cells of the central and peripherial nervous system without the aid of any peripherial nervous system. These compounds have numerous effects on neuronal function and physiology with a variety of potential neuropharmacological utilities. Pregnenolone sulfate (PREGS) is one of the most abundant neurosteroids in the mammalian CNS, which shows a role in memory and cognition The definitive characterization of the endogenous role and mechanism of action of this neurosteroid has not yet been elucidated. There is evidence supporting that the enchancement of cognition observed with PREGS is due to its modulator effects on neuronal ion channels and metabotropic receptors. PREGS have been shown to potentiate AMPAR mediated miniature excitory postsynaptic currents (mEPCs) frequency but not amplitude and decrease paired pulse facilitation of autaptic EPSCs caused by depolarization. We hypothesize that PREGS-induced increase of glutamate release induces postsynaptic upregulation of AMPA receptor subunits. The goal of this research is to determine if PREGS increases cell surface expression of GluR1, GluR2/3, and/or Glur4 subunits of AMPA receptors. We will use an approach based on cross-linking of extracellular proteins followed by Western immunoblotting to assess the surface expression of these subunits. The effects of PREGS will be demonstrated in Adult Sprague-Dawley rat pup at P3–4, handled according to protocol currently approved by the UNMHSC Institional Animal Care and USE Committee. We will demonstrate these effects through differential expression of intracellular to extracellular pools of AMPA receptors subunits in PREG treated vs control hippocampal neurons. Our initial studies have shown a difference in receptor pools expression. Comparison of receptor levels will be performed using paired student’s t-test. Statistical significance will be set at a p≤0.05, B=0.8. Our research alone will not be able to distinguish the effects of PREGS on localization of AMPAR subunits from extrasynaptic sites mediating observed biological effects or through protein phosphorylation and activation. We are conducting separate eletrophysiological experiments to provide further evidence that in combination with this research may prove our current hypthopesis.
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