Lori L. McMahon - US grants

An inhibitory role of strychnine-sensitive glycine-gated chloride channels (GlyRs) in the brain has largely been ignored, despite the known expression of these receptors throughout the CNS. Interestingly, in some animal models of epilepsy, increasing the glycine levels in cerebral spinal fluid (CSF) depresses repetitive neuronal firing in hippocampus and cortex, implicated an important function of GlyRs in modulating neuronal excitability. GlyRs are expressed in hippocampus, a brain region highly dependent upon effective neuronal inhibition for normal function, however, the physiological role of these receptors is simply not known. The major hypothesis of this proposal is that in hippocampus, activation of GlyRs provides an undescribed, fundamental inhibitory mechanism that depresses the activity of excitatory pyramidal cells and inhibitory interneurons via direct activation of post-synaptic receptors and furthermore, that GlyR activation limits the of the neuronal network by depressing synaptic transmission. The long-term goal of this proposal is to establish a new role for GlyRs in hippocampus and to understand the cellular mechanisms mediating the effects of GlyR activation on neuronal activity. Extracellular popspike recordings of pyramidal cells and whole-cell and perforated patch recordings of pyramidal cells and interneurons in rat hippocampal slices will be combined with pharmacological tools to examined the functional expression of post- synaptic GlyRs in hippocampus throughout development and determine how activation of these receptors affects the synaptic network. We will carefully address the following Specific Aims: 1) To test the hypothesis that functional GlyR expression by pyramidal cells and interneurons continues throughout development and that the physiological and pharmacological properties of GlyRs expressed by these two cell types are the same and 2) To test the hypothesis that GlyR activation limits the activity of the synaptic network under basal and hyperexcitable conditions through a post-synaptically-mediated depression of transmission. The use of the hippocampal slice preparation in this study has the advantage of allowing direct glycine effects on individual neurons and synaptic circuits to be examined in a well-characterized system, where recorded neurons remain in their native synaptic environment. Our approach is anticipated to yield novel information that will pave the way for a new area of investigation into the inhibitory role of GlyRs. Furthermore, it is expected that the results from this study will provide insight into the design of novel therapeutic strategies for the prevention of seizure activity.

DESCRIPTION (provided by applicant): The Women's Health Initiative (WHI) reported in 2003-2004 that hormone replacement therapy, either progesterone and estrogen in combination or estrogen alone, provided no cardiovascular or cognitive benefit in postmenopausal women. These conclusions lead millions of women to withdraw from hormone replacement therapy. However, clear benefits observed in epidemiological studies have initiated a critical reevaluation of the WHI study. Potential conflicts include the use of equine conjugated hormones, the duration of hormone deficiency, and the advanced age of the subjects. It is becoming increasingly obvious that there is a "window of opportunity" in which hormone replacement is beneficial for maintaining both cardiovascular and cognitive health. Unfortunately, our understanding of why E2 replacement may or may not rescue cognitive deficit is hindered by insufficient mechanistic information at the cellular level regarding how E2 modulates synaptic function. Thus, the discrepancy in the clinical data will only be resolved by further investigation into the basic mechanisms through which E2 acts. Because all women undergo menopause and spend nearly 1/3 of their life in this state, intensive research effort must be dedicated to obtaining new knowledge that will provide insight for interventions to sustain mental and cognitive health. At hippocampal CA3-CA1 synapses, estradiol (E2) increases spine density, NMDAR transmission, and LTP5-8, mechanisms believed to underlie the enhanced memory. However, it remains unknown whether there is a functional relationship between the increased spine density and LTP. This is important because in aged animals, E2 increases NMDAR expression but not spine density, which may explain the age related decrease in cognitive benefit of E2 replacement therapy. On the other hand, aged animals may be able to increase plasticity without the growth of new synapse. It is also not known whether the cortical input onto CA1 cells from the entorhinal cortex is modulated by E2 similarly to CA3 Schaffer collateral synapses. This is critical to know because these synapses drive CA1 cells during spatial exploration and estradiol increases spatial memory. It is known that E2 requires cholinergic innervation to enhance memory, but is it not known whether the same is true for the increase in synaptic function. Because E2 protects cholinergic cells from degeneration, loss of E2 in menopause could lead to increased cholinergic cell death. This is significant because post menopausal women are at greater risk of developing Alzheimer's disease than men and E2 replacement decreases this risk. Therefore insufficient cholinergic transmission would limit the ability of E2 to cause cognitive benefit. Finally, no study has investigated the impact of prolonged hormone loss on the ability of E2 to induce changes in synaptic function. Only one study has investigated potential alterations in NMDAR mRNA levels, but no significant results were observed. This is of high clinical importance because determining the window of opportunity and how this relates to chronological age is absolutely essential to further our understanding of the effectiveness of hormone replacement therapy. In this proposal, we will use extracellular dendritic field potential and whole-cell patch clamp recording techniques in area CA1 of acute slices from young adult, middle aged and aged ovariectomized (OVX) female rats treated with estradiol or vehicle to pursue the following Specific Aims: AIM 1 will test the hypothesis that E2 increases the magnitude of LTP by increasing the density of silent synapses which express NMDARs containing NR2B subunits;AIM 2 will test the hypothesis that E2 mediated effects on spine density and synaptic function are not limited to the Schaffer collateral pathway but also include synapses between the entorhinal cortex and the distal dendrites of CA1 pyramidal cells;AIM3 will test the hypothesis that cholinergic denervation will prevent the E2 induced increase in spine density, NMDAR transmission, and LTP magnitude but that sympathetic sprouting from the superior cervical ganglia will rescue these deficits;AIM 4 will test the hypothesis that prolonged hormone loss combined with normal aging prevents the ability of E2 replacement to induce morphological and functional changes at CA3-CA1 synapses.

DESCRIPTION (provided by applicant): Under basal conditions, the majority of glucose is metabolized through the glycolytic pathway, but 2-4 % is metabolized via the hexosamine biosynthetic pathway (HBP). The HBP modifies glucose to produce an O-linked N-acetylglucosamine (O-GlcNAc) moiety that can be added to serine/threonine residues of proteins in a highly dynamic and reversible reaction. Flux through the HBP is increased when glucose levels are in excess, which can lead to a pathological increase of O-GlcNAcylated proteins. Two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase are responsible for adding and removing, respectively, the O-GlcNAc moiety to serine/threonine residues in proteins. Hippocampal neurons have the highest expression of O- GlcNAc transferase (OGT) and O-GlcNAcase in forebrain. Long-term changes in the efficacy of CA3-CA1 synapses underlie hippocampal dependent learning. The high expression of OGT and O-GlcNAcase in hippocampus suggests that normal synaptic function in this brain region is modulated by O-GlcNAc turnover of synaptic proteins. However little to nothing is known regarding how O-GlcNAcylation modulates synaptic function. Furthermore, the possibility exists that abnormal addition of O-GlcNAc on synaptic proteins could interfere with the ability of synapses to express long-term plasticity required for memory processing and could explain deficits in hippocampal synaptic function and learning known to occur in animal models of diabetes, where O-GlcNAcylation is pathologically elevated. No study to date has investigated the effects of O-GlcNAcylation on memory formation, either under physiological or pathological conditions. In recent studies, we find that OGT and O-GlcNAcase are tonically active and bidirectionally modulate the strength of basal synaptic transmission, suggesting the natural flux through the HBP sets the level of excitability in the circuit. Furthermore, we find that an increase in O-GlcNAcylation limits the ability of synapses to express normal LTP, with no effect on LTD. In this proposal we will investigate the cellular and molecular mechanisms mediating the synaptic depression induced by increased O-GlcNAcylation and determine whether chronic increases in O-GlcNAcylation causes synaptic dysfunction and learning deficits. Thus, the successful demonstration of a physiological role of O-GlcNAcylation in modulating synaptic transmission and plasticity could be the next major discovery in the field of learning and memory. The results obtained will launch a new area of investigation aimed at understanding how fluctuations in glucose metabolism by the HBP can directly affect synaptic function in physiological and pathological conditions.

? DESCRIPTION (provided by applicant): How fluctuations in ovarian hormones contribute to the development of major depressive disorder (MDD), and how they impact antidepressant efficacy continues to be understudied and ill-defined, despite women being diagnosed with MDD twice as often as men. This is unacceptable given that women make up half of the world's population and all undergo loss of ovarian hormones in menopause, increasing risk of depression and cognitive deficits. Memory impairment, the most commonly reported cognitive symptom associated with MDD, is linked with decreased hippocampal volume and neurogenesis, implicating hippocampal dysfunction. Importantly, 17? estradiol (E2), the major ovarian hormone which has both antidepressant and cognitive-enhancing effects, directly opposes the negative consequences of stress and glucocorticoids by increasing neurogenesis, dendritic spine density, synaptic plasticity, and learning and memory. Therefore, significant fluctuations in E2 increase vulnerability of hippocampal circuits to stress, potentially contributig to the greater incidence of MDD in women. Unfortunately, currently available treatments strictly target improving mood, with much less focus on therapeutic development to reverse the associated cognitive dysfunction. Thus, identifying therapeutic strategies and cellular mechanisms that target both mood and cognitive deficits are key to improving treatment of depression in women. Using surgically menopausal rats, we previously reported that E2 replacement increases hippocampal synaptic function and learning, and decreases acquisition of depression-like behavior following inescapable shock, confirming its beneficial effects on cognition and resilience to stress. Remarkably, a striking overlap exists between the plasticity related molecules required for the E2-induced increase in memory with those required for the rapid antidepressant effects of acute ketamine. Even more striking is that the E2-induced increase in GluN2B current, which is required for increasing LTP and novel object recognition, also occurs following acute ketamine treatment, suggesting that ketamine may transiently improve cognitive function. Thus, these shared mechanisms indicate critical interactions exist between ovarian hormones and ketamine in improving depression symptoms. Here, using behavior and brain slice electrophysiology we will investigate shared mechanisms between E2 and ketamine, with a focus on excitatory circuits and GABAergic interneurons in hippocampus and mechanisms that contribute to learning and memory. Clearly, this is an area of research that is urgently needed to improve treatment efficacy in women. Importantly, results obtained herein will define the mechanisms driving the antidepressant and cognitive effects of E2 and ketamine that are critical to advancing their effective therapeutic use either alone or together in treating depression and associated cognitive deficits in women over the lifespan.

Hippocampal synaptic function and learning and memory are vulnerable to alterations in protein O- GlcNAcylation, the O-linked attachment of ?-N-acetylglucosamine (GlcNAc) to serine/threonine (ser/thr) residues. O-GlcNAcylation is now recognized as a possible therapeutic target for cognitive dysfunction, particularly in the treatment of Alzheimer's disease (AD), where decreased O-GlcNAc may be permissive for pathological tau hyperphosphorylation. Systemic administration of the OGA inhibitor, thiamet-G, reversed the increase in tau phosphorylation and improved spatial learning and memory in transgenic AD mice. Obviously, determining how O-GlcNAcylation modulates neuronal and synaptic function under physiological and pathophysiological conditions is imperative to understanding its impact on learning and memory, and the risks and benefits of therapeutic intervention. Our lab has made significant contributions to this new area of research by showing that acute and selective increase in O-GlcNAcylation of AMPAR GluA2 subunits underlies expression of a novel form of LTD at CA3- CA1 synapses (O-GlcNAc LTD), as well as the dampening pathological hyperexcitability in seizure models. We also find that acute increases in O-GlcNAc interferes with some forms of hippocampus-dependent learning and memory. Because excitation/inhibition balance in memory circuits governs normal learning and memory, and GABAAR function and trafficking is modified by serine phosphorylation, we have used our expertise to investigate how rapid changes in O-GlcNAcylation occurring under physiological conditions modulates the efficacy of GABAergic inhibition. Importantly, because not all GABAergic interneurons express GluA2 subunits, O-GlcNAc LTD will only occur at glutamatergic synapses on a subset of interneurons, which will alter circuit dynamics when O-GlcNAcylation is high. In preliminary experiments, we found that acutely increasing protein O-GlcNAcylation decreases the amplitude and frequency of sIPSCs and the amplitude of mIPSCs recorded from CA1 pyramidal cells in rat hippocampal slices. In this exploratory proposal, we test the hypothesis that O- GlcNAcylation directly modulates the strength of synaptic inhibition via postsynaptic GABAARs and receptor internalization, and indirectly via expression of O-GlcNAc LTD at excitatory synapses onto specific interneurons possessing GluA2-containing AMPARs. The results of these exploratory studies will establish an entirely novel fundamental mechanism that directly and indirectly controls GABAergic inhibition, thereby providing a framework for future studies targeting O-GlcNAc in neurodegenerative diseases, such as Alzheimer's disease, and in neurodevelopmental disorders such as autism and Down syndrome, where imbalances in excitatory and inhibitory circuits underlie cognitive dysfunction. The results of these studies will make a huge advance in a field that is in its infancy.

SPAD at Auburn University at Montgomery Like STEM faculty at Primarily Undergraduate Institutions (PUIs) everywhere, Auburn University at Montgomery (AUM) faculty must overcome a daunting array of disincentives to pursue external funding. Heavy teaching loads, lack of dedicated research space or time for research, an unfamiliarity with grant programs and procedures, a perception that the likelihood of success was remote, even the ready availability of summer teaching opportunities, all acted to create a faculty culture of low expectations with regards to grant seeking. But with a range of new initiatives designed to better serve the needs of our diverse student body, AUM is experiencing a cultural change, where expectations for faculty research and engagement are escalating rapidly. At this critical juncture, AUM has crucial need for an expanded and reorganized OSPR. In support of new initiatives and a changing campus culture focusing on research and engagement, AUM is seeking SPAD funding to reorganize and expand its Office of Sponsored Programs (OSP) to keep pace with the rapidly growing faculty interest in pursuing external grant funding. The specific aims of the project are to: 1. Increase the capabilities of the OSP by hiring and training new staff and providing them with the competency- based training and professional development that includes training and building an infrastructure that supports continuing education in this area. 2. Establish new OSP services, policies, and procedures for the development and submission of applications and provide training and support to faculty in these new services, policies, and procedures; 3. Develop a faculty reward structure that emphasizes cost sharing and workload management to promote research and creative activity; 4. Expand collaborative research partnerships and funding opportunities for faculty and students, especially with Auburn University and the University of Alabama at Birmingham. 5. Offer students the opportunity to work with faculty and the OSP to acquire grant writing skills; and 6. Partner with the AUM Office of Diversity to offer scaffolded workshops in cultural competency for faculty to facilitate creating effective grant writing teams for grants targeted at PUIs. The SPAD grant would enable AUM to create an OSPR that would play an important role in fostering this new culture of research and scholarship, that would encourage grant-assisted faculty training and curriculum development, and help create new programs for research training and mentorship of underprivileged African- American students, who comprise 42% of our student population. The success of the project will be determined by measuring increases in faculty interactions with the OSP, in proposal submissions, in the creation of new research collaborations, in the expanded mentoring of student researchers, and in the volume of publications.