Fragile X Syndrome (FXS) is the leading cause of inherited intellectual disability. The disorder is caused by a trinucleotide expansion that silences the Fragile X Mental Retardation 1 (Fmr1) gene resulting in the loss of its protein product, the Fragile X Mental Retardation Protein (FMRP). FXS patients show broad clinical phenotypes including intellectual disability, as well as a number of cognitive and behavioral problems. The lack of FMRP is believed to be the direct cause of the deficits seen in FXS patients. FMRP is an RNA-binding protein that is expressed in the brain and testes. This protein is believed to form a messenger ribonucleoprotein complex with mRNAs in the nucleus and subsequently export them to polyribosomes in the cytoplasm, therefore influencing translation of its bound mRNAs. Importantly, FMRP has long been suspected to be involved in synaptic plasticity due to its ability to bind several mRNAs that encode for proteins important in synaptic plasticity. Such proteins include the GluN1, GluN2A and GluN2B subunits of the N-methyl-D- aspartate receptor (NMDAR). FMRP is expressed in the hippocampus, a region of the brain involved in learning and memory processes. Recently, impaired NMDAR functioning in the dentate gyrus (DG) subregion of the hippocampus has been observed in Fmr1 knockout (-/y) mice. This impairment also resulted in reduction in long-term potentiation (LTP) and long-term depression (LTD) of synaptic efficacy, two biological models of learning and memory. In the present study, I focused on the levels of the NMDAR GluN1, GluN2B and Glu2B subunits in order to determine the synaptic plasticity alterations seen in the DG of Fmr1-/y mice. Using Western blotting, I found that there is a decrease in the GluN1, GluN2A and GluN2B subunits in the DG of young adult Fmr1-/y mice, indicating that these mice have significantly lower amounts of total NMDARs. These results could explain the altered LTP and LTD seen in Fmr1-/y mice at the molecular level and might contribute to the intellectual impairments seen in these KO mice. NMDARs appear to be important in the development and maturation of synapses. The GluN2A and GluN2B subunits are developmentally regulated, where GluN2B is predominantly expressed early in development and GluN2A in the adult brain. A dysregulation of GluN2A and GluN2B subunits has been proposed to affect the maturation and formation of synapses. Intriguingly, FMRP is also believed to play a functional role in early brain development. Thus, this study also focused on the developmental expression of the GluN1, GluN2A and GluN2B subunits in the DG, Cornu Ammonis, prefrontal cortex and cerebellum of Fmr1-/y mice, all of which are brain regions implicated in FXS. We found that the developmental expression of these subunits is altered in Fmr1-/y mice in specific brain regions. Together, these results demonstrate that the loss of FMRP differentially affects GluN1, GluN2A and GluN2B subunit expression both developmentally and spatially, further implicating NMDARs in the pathophysiology of FXS. / Graduate

Long-term potentiation (LTP) and depression (LTD) are important forms of activity-dependent synaptic plasticity believed to play a role in memory at the cellular level. It has previously been described that synthesis of new proteins is needed to maintain LTP longer than a few hours. Other reports argue that sufficient proteins for stable LTP are already available. The present study aims to examine the role of protein synthesis in LTD, the presumed mirror mechanism of LTP. Experiments were carried out in hippocampal slices from young (12-45 days) and old (12-18 weeks) Sprague-Dawley rats. Extracellular techniques were used to study synaptic responses in the Schaffer-collateral-commissural pathway. Plasticity was induced electrically by low frequency stimulation (2-3 trains at 1 Hz for 15 min) or chemically by brief exposure to certain glutamate receptor agonists (NMDA at 20 µM for 3 min or DHPG at 100 µM for 10 min). Whole slice protein synthesis was quantified by assessing 3H-leucine incorporation. Stable LTD (> 8 h) was be obtained by either electrical or chemical activation. Protein synthesis inhibitors anisomycin (40 uM) and cycloheximide (100 uM) both failed to influence the magnitude of LTD. Moreover, no age difference was found, in terms of stable LTD in both young and old rats under inhibition of protein synthesis. The potency of the inhibitors was found to be high, depressing synthesis down to a few percent. It is concluded that sufficient proteins for generating stable LTD are normally present in the brain, implying a large safety-margin for cellular memory.

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Mild traumatic brain injury (mTBI), often referred to as concussion, has become increasingly recognized as a serious health issue in the general population. The prevalence of mTBI in athletes, particularly repeated injuries in young athletes, is of great concern as injuries to the developing brain can have long-term detrimental effects. In this study we used a novel awake closed-head injury (ACHI) model in rodents to examine repeated mTBI (rmTBI), to determine if repeated injuries produced the neurological and molecular changes evident with human concussion. Animals were administered 4, 8, and 16 rmTBIs and acute neurological assessments were performed after the injuries. Changes in glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) levels were assessed using Western blot analysis at one day following rmTBI in the ipsilateral dentate gyrus (DG) and the cornu ammonis (CA) regions of the hippocampus and the cortex (CX) indicative of astrocyte and microglial cell reactivity. Results indicated that the ACHI model produces neurological deficits immediately after the injuries, with the most deficits arising in the rmTBI16 group. Despite deficits in all injury groups, histological staining with cresyl violet revealed no significant morphological tissue damage to the brain. Western blot analysis, however, showed a significant increase in DG and CX GFAP expression in the rmTBI16 group with no changes in Iba-1 levels. This suggests an acute activation of astrocytes in response to injury, with a delay or absence of microglial activation. Our findings show that with repetitive concussions, we are able to detect acute neurological and molecular changes in the juvenile female brain. However, further investigation is necessary to determine if these are transient changes. / Graduate