Neuroscience
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Item Intracellular Chloride Regulation of Supraoptic Vasopressin Neurons during Salt Loading(2019-03-05) Farmer, George; Little, Joel; Bachelor, Martha; Cunningham, J. Thomas; Balapattabi, KirthikaaPurpose Salt loading (SL) increases intracellular chloride concentration [Cl]i, impairing GABAA inhibition of arginine vasopressin (AVP) neurons in the supraoptic nucleus (SON) of hypothalamus. But the regulatory mechanisms leading to increased [Cl]i is not completely understood. Based on previous studies, we hypothesize that SL activates tyrosine receptor kinase B (TrkB) and downregulates K+/Cl- co-transporter 2 (KCC2) membrane expression. Downregulation of KCC2 decreases the efflux of chloride, Cl ion causing increase in [Cl]i in SON AVP neurons. In this study, we combined virally mediated ClopHensorN, a relatively new ratiometric Cl imaging technique with capillary based Simple Wes to record changes in [Cl]i and specifically detect KCC2 protein expression in individual SON AVP neurons. Methods Adult male Sprague Dawley rats were bilaterally injected in the SON with rAAV2-0VP1-ClopHensorN. The ClopHensorN (Addgene Plasmid #50758) was packaged in an AAV2 vector with an AVP promotor (Addgene Plasmid #40868). After 2 weeks, the rats were given either water or 2% NaCl to drink for 7 days. At the end of the protocol, the rats were anesthetized with inactin and their SONs were dissociated. The cells were plated on coverslips and placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. ClopHensorN positive neurons were tested for decrease or increase in [Cl]i to focal application of GABAA agonist muscimol (100uM). After imaging, individual neurons were collected by aspirating into a patch pipette to verify KCC2 and ß-Actin protein expression. Protein Simple Wes (12-230kDa matrix) was used to identify and quantitate very low concentration of protein from single neuron. Data were analyzed by Chi-squared test and one-way ANOVA with Bonferroni comparisons. Results Muscimol application to SL SONs either significantly increased Cl efflux (p Conclusion Salt loading increases [Cl]i in SON AVP neurons through TrKB-KCC2 mechanism.Item Caloric restriction attenuates motor function decline in rats: Evidence from an advanced middle-aged cohort(2019-03-05) Kelley, Parker; Venable, Katy; Terrebonne, Jennifer; Ingram, Donald; Salvatore, Michael; Kasanga, EllaPurpose: The maintenance of physical function is a hallmark of successful aging. This is, however, not achievable for every individual. In the rapidly aging elderly population, between 30-50% may suffer from locomotor impairment as a function of aging, a phenomenon known as aging-related parkinsonism. This motor impairment is a serious public health concern as it greatly compromises the ability to perform daily life activities, further contributing to loss of independent living, frailty, and mortality. In this population, lifestyle strategies could prove beneficial for reducing aging-related parkinsonism. Caloric restriction (CR) can reduce aging-related locomotor decline when instituted either as a lifelong intervention or during middle age in rats. This study seeks to determine if there is an aging-related limit of caloric restriction efficacy to attenuate motor decline and if nigrostriatal dopamine regulation is associated with motor effects. Method: Male Brown-Norway/Fischer 344 F1 hybrid (BNF) rats (18 months old) previously maintained on a lifelong ad libitum (AL) diet were grouped into CR and AL groups. CR was gradually introduced with a 30% restriction being achieved 3 weeks after initiation of the study. This was maintained for 6 months with open-field locomotor assessments conducted every 6 weeks. Results: CR prevented decreased motor function as a function of aging; an effect that was observed in the AL group. There was an increase in movement number and horizontal activity which are both indices of the ability to initiate movement. Significant decline in motor function was observed in the AL group 12 weeks after initiation of the study and this effect was sustained till the end of the study. Conclusion: Caloric restriction initiated in advanced middle-aged rats leads to a preservation of motor function suggesting that there may be no aging limit to its beneficial effect on motor performance. Identifying the molecular mechanisms can reveal targets for pharmacological or genetic approaches to mitigate motor impairment in individuals where CR would be contraindicated.Item Development of a serum free astrocyte culture method that mimic resting in vivo astrocyte phenotype(2019-03-08) Winters, Ali; Chaudari, Kiran; Hersh, Jessica; Liu, Ran; Yang, Shaohua; Prah, JudePurpose Primary astrocyte cultures have been extensively used for characterization of astrocytes functions in physiological and pathological conditions. The current primary astrocytes are mostly maintained in fetal bovine serum (FBS) containing medium. Although FBS contains growth elements that fulfills many metabolic needs of cultured astrocytes, it alters the genotypic and morphological profiles of primary astrocytes as well as induces astrocyte activation. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state with phenotypes that mimic in vivo astrocytes. Methods Primary astrocytes were isolated from the cerebral cortex of postnatal day 1 C57BL/6 mice and cultured in serum-free astrocyte basal medium containing FGF2 and EGF (ABM-FGF2-EGF). The phenotype of primary astrocytes cultured in ABM-FGF2-EGF were compared with astrocytes cultured in FBS supplemented DMEM medium (MD-10% FBS). Growth assays, immunostaining, Western blot, quantitative polymerase chain reaction, and metabolic assays were used to access the growth rates, metabolic phenotype, mRNA expression profiles and quiescent or reactive states of astrocytes. Results and Conclusions We demonstrated that the novel serum free ABM-FGF2-EGF medium supports astrocytes growth and enhanced glycolytic metabolism with higher glycogen content, lower GFAP and vimentin expression, and increased glutamate transporter mRNA levels as compared to astrocytes cultured in the MD-10% FBS medium. Our study suggests that our serum free culture method produces astrocytes with a biosynthetic phenotype and morphology similar to in vivo resting astrocytes. Additionally ABM- FGF2-EGF cultured primary astrocytes could be activated by various pathological conditions. The developed serum-free and EGF/FGF2-containing astrocyte basal medium will provide a critical tool for defining the precise function of astrocytes under physiological and pathological conditions.Item A rare case of primary germinoma in corpus callosum.(2019-03-05) Anderson, Jenna; Ellis, Thomas; Kata, KarolinaBackground Primary intracranial germinoma is a rare lesion which accounts for approximately 0.5–2% of all central nervous system (CNS) tumors. Generally, this neoplasm occurs in the midline structures with the majority located in the pineal and suprasellar regions. Germinoma presenting primarily in the corpus callosum is highly unusual and reportings of similar cases in scientific literature are limited. The aim of this case report is to describe clinical features, imaging findings, and management of a primary germinoma uniquely presenting in the corpus callosum. Case Information 21-year-old man with Parinaud syndrome, gait instability, altered mood, and remote history of orbital trauma presented initially for an ophthalmology evaluation of vision change. These symptoms prompted intracranial imaging, including MRI which revealed an enhancing lesion in the corpus callosum, evidence of obstructive hydrocephalus, and an arachnoid band in the region of the aqueduct of Sylvius. The lesion was believed to potentially represent either a primary CNS lymphoma, glioblastoma, anaplastic astrocytoma, or tumefactive demyelinating disease. The patient underwent a right frontal stereotactic brain biopsy and third ventriculostomy. Pathologic evaluation with immunohistochemistry and tumor marker analysis confirmed a diagnosis of primary germinoma. Post-operative plan included oncology consultation to establish chemotherapy and radiation treatment. Additional imaging showed corpus callosum mass with evidence of diffuse extension in the surrounding structures. Interestingly, in contrast to pre-operative imaging, enhancement of the pineal gland area was noted and provided additional rationale for development of obstructive hydrocephalus. Conclusion Primary germinoma of corpus callosum has not yet been extensively described in literature. The present case demonstrates that primary germinoma can occur in uncommon midline structures and present with unique imaging findings. This report contributes to improving recognition and further understanding of clinical presentation and course and potentially to optimize the treatment in similar future case.Item IMPACT OF T-CELLS ON ASTROCYTES IN VIVO & IN VITRO: IMPLICATIONS POST-ISCHEMIC STROKE(2019-03-05) Xie, Luokun; Li, Wenjun; Liu, Ran; Yang, Shaohua; Hersh, JessicaPurpose: Post-ischemic stroke, T-lymphocytes enter the brain. The role of T-cells in the progression of cerebral infarction or repair mechanisms is unclear. We hypothesized that T-cells interact with astrocytes directly leading to an anti-inflammatory response. Methods: In vivo, ischemic stroke was induced by middle cerebral artery occlusion in young adult C57/B6 male mice. Mice were sacrificed at 3 days or 1-month post-ischemic stroke. Paraffin-embedded brain sections demonstrated co-localization of astrocytes and CD4+ and CD8+ T-cells in the ischemic region 1 month after stroke. T-cells were harvested from the brain by digestion; percoll enriched, and incubated with anti-CD3 and CD25 antibodies. T-cells were sorted via flow cytometry. The cytokine profile of brain infiltrated CD4+ and CD8+ T-cells were compared to spleen T-cells using QT-PCR. In vitro, C8-S murine astrocyte type II clone cell line (ATCC® CRL-2535™), and T-cells extracted from the spleens of 3-month-old C57/B6 female mice were placed in co-culture at a 1:1 for 48 and 72 hours and compared to individual cell cultures. Anti-CTLA-4 antibodies were added to each culture condition as another experimental group. Astrocytes and T-cells were collected separately for QT-PCR analysis. Results: In vivo, the following cytokine gene expressions poststroke, were found to be elevated: IFNγ, IL-10, IL-17, TNFα, and perforin. In vitro, IL-10 gene expression was elevated in astrocytes and T-cells individually harvested from 1:1 co-cultures compared to astrocytes and T-cells alone at 48 and 72 hours respectively. IL-10 was produced primarily by T-cells stimulated by direct contact with astrocytes. Anti-CTLA-4 antibodies blocked the direct cell-to-cell interaction by reducing IL-10 gene expression in both astrocytes and T-cells. Conclusions: Our data suggests that T-cells release pro- and anti- inflammatory cytokines while in close proximity to astrocytes after ischemic stroke. In co-cultures, astrocytes directly interact with T-cells increasing their IL-10 gene expression by 72 h., implying a neuroprotective mechanism exists via astrocyte stimulation of T-cell IL-10 production.Item Differential Effects of Young and Old Serum Exosomes on Ischemic Stroke Outcomes in Aged Rats(2019-03-05) Jin, kunlin; Zhang, HongxiaBackground: Aging is associated with striking increases in the incidences of stroke and neurodegenerative diseases, both of which are major causes of disability among those age 70 years and older in the United States. Despite progress in understanding molecular mechanisms of neuronal cell death after stroke, effective treatment remains elusive. Recent studies showed that systemic factors in the blood can profoundly reverse aging-related impairments, and our study show that aging systemic milieu could worse outcome after ischemic stroke in rats. However, the underlying mechanism remain unclear. Exosomes are extra-cellular microvesicles that play important roles in intercellular signaling and in regulating various physiological and pathological conditions. Here, we explore the role of young and old serum-derived exosomes on ischemic outcome in aged rats. Method: The exosomes were isolated from serum of young or old rats, and then were intravenously injected into aged ischemic rats via tail for 3 days, respectively. Infarct volume was determined with triphenyltetrazolium chloride (TTC) staining and motor function was assessed with neurobehavioral tests including running ladder and cylinder tests. To elucidate the potential mechanism underlying the functional improvement or deterioration, neuroplasticity was examined after treatment of young and old serum exosomes using Golgi-Cox staining and data were analyzed using Imaris software. Results: We found that injection of young serum exosomes into aged ischemic rats reduced infarct volume and improved motor functional deficits. On the contrary, injection of old serum exosomes increased infarct volume and worsened motor function. We also found that the dendritic length and spine numbers were significantly increased after injection of young exosomes, while decreased after injection of old exosomes. Conclusion: Our data suggest that young and old serum exosomes differentially affect functional outcome in aged rats after ischemic stroke, which potentially be translated into novel therapeutic intervention by minimizing the destructive potential of detrimental molecules and enhancing the beneficial contributions to repair the damaged brain.Item What makes subtypes of dopamine receptor different?(2019-03-05) Luedtke, Robert R.; Liu, Jin; Hayatshahi, HamedPurpose: Selective targeting of different subtypes of dopamine receptors is a strategy for fighting many neurological disorders such as Parkinson’s disease and drug addiction. It has been shown that simultaneous targeting of the dopamine orthosteric binding site and a nearby allosteric site with “bitropic” ligands can enhance the selectivity of ligands for a specific receptor subtype. We hypothesize that there are regions and residues in each receptor structure, which makes it targetable for selective compounds. We aim to identify these regions and residues with molecular dynamics (MD) simulations. Methods: We have generated over 360 microseconds of MD simulations of free D2 and D3 receptor subtypes and their complex with five different compounds. The structure and dynamics of the receptor and ligands, and their interactions are analyzed in atomistic level to highlight the differences. Results: The MD simulations highlighted some differences in dynamics of the receptor subtypes in free form. Also, it is shown that the binding affinities of the bitropic ligands are enhanced compared with their orthosteric counterparts with both 1) excluding more solvent from the binding sites, and 2) making interaction with more residues in the allosteric site. The first extracellular loop (ECL1) is a very important site of allosteric interaction in D2 subtype vs. the second extracellular loop (ECL2) for D3 subtype. A specific interaction that makes ligands selective for D2 receptor is the pi-stacking interaction and hydrogen bonding with a tryptophan residue located in the ECL2, whereas in D3, hydrogen bonding with a cysteine and a serine in ECL1 is important. Conclusions: We have identified important residues and regions in D2 and D3 that make these receptor subtypes respond differently to various ligands. We have also shown how two very closely related and similarly structures GPCR receptors have different dynamics in free form and in complex with drug-like compounds.Item Astrocyte HIV-1 Proviral Reservoirs in HAND(2019-03-05) Borgmann, Kathleen; Ghorpade, Anuja; Edara, Venkata ViswanadhPurpose: Even though astrocytes are restrictively infected with HIV-1, they are capable of producing neurotoxic viral proteins and proinflammatory mediators. This can significantly aggravate the pathogenesis of HIV-associated neurocognitive disorders (HAND). Thus, there is a great need to identify latently infected astrocytes and develop strategies to target this elusive population. We hypothesize that harboring HIV-1 proviral reservoirs alters astrocyte function in conjunction with unique gene expression patterns that could serve as biomarkers and facilitate targeted therapy. Methods: Red/Green-HIV-1 (R/G-HIV-1) was used to visualize viral promoter (LTR) activity in primary human astrocytes. Astrocytes with active (R+/G+) and silent (R+/G-) LTRs were enriched using FACS. Results: Nested Alu-gag PCR confirmed the presence of integrated R/G-HIV-1 provirus in transduced astrocytes. Astrocytes with silent promoter activity were devoid of late viral proteins such as p24, indicating a functionally silent HIV-1 LTR. However, interluekin-1β (IL-1β) and Vorinostat, a histone deacetylase inhibitor (HDACi), reactivated silent HIV-1 LTR in R/G-HIV-1+ astrocytes. Glutamate clearance ability and cell proliferation were significantly impaired in astrocytes with either silent (R+/G-) or active (R+/G+) HIV LTRs when compared to integrase deficient R/G-HIV-1 (D116A) transduced cells. Conclusions: Our data suggest that harboring HIV-1 provirus, either active or silent, interfered with astrocyte function and growth. Hence, we propose that identifying biomarkers for astrocytes harboring HIV provirus, and therapeutic gene editing to eliminate proviral gene expression will improve physiological function compared to HIV-1 infected cells.Item Influence of ovarian hormone deprivation length on the neuroprotective effects of genistein in stroke(2019-03-05) Metzger, Daniel; Sun, Fen; Sumien, Nathalie; Schreihofer, Derek; Oppong-Gyebi, AnthonyPURPOSE: Advancing age increases women’s susceptibility to stroke compared to men, especially after the menopausal transition. Among the reasons proposed for high stroke incidence in postmenopausal women is a significant decrease in estrogen (E2) concentration, based on well-established evidence that E2 is neuroprotective during ischemia in animal studies. While E2 treatment can be beneficial, extended delays in its replacement can result in detrimental actions on the brain which contributes to widespread mistrust of menopausal hormone therapy. Interest in the beneficial effects of soy isoflavones has grown as a viable alternative for E2. However, results from clinical trials have been inconsistent as there seems to be no consensus on the benefits of soy isoflavones in menopausal women. Notwithstanding, evidence suggests a time-dependent benefit of soy isoflavones, even though there is no systematic assessment in preclinical studies to identify the window of opportunity for their proposed optimal benefits. Hypothesis: After long-term hormone deprivation, the soy isoflavone genistein will maintain the ability to provide neuroprotection in the brain following aging and the loss of endogenous E2 in an experimental stroke model. METHODS: Young adult and retired proven breeder Sprague-Dawley rats ( [greater than] 9 mo) were bilaterally ovariectomized, divided into 2 post-ovariectomized time points (2 and 12 weeks) and fed with an isoflavone free (IF) diet. At the end of each time point, rats were continued on IF diet or switched to genistein diet. Two weeks later, rats underwent transient middle cerebral artery occlusion for 60 mins. After stroke rats were subjected to a series of behavioral tests including neurological function, cylinder test, rotarod, and the Morris Water Maze (MWM). RESULTS:Our results demonstrated a significant effect (p CONCLUSION: Dietary genistein had little effect on the sensorimotor outcomes but holds a promise in improving cognitive function post-stroke in the long term.Item Brain-Targeting Prodrug Design for Thyrotropin Releasing Hormone(2019-03-05) Nguyen, Vien; Prokai, Laszlo; Prokai-Tatrai, Katalin; Del La Cruz, DanielPurpose: Thyrotropin releasing hormone (TRH) has many neuromodulatory effects throughout the brain, however, treatment using this peptide induces unwanted peripheral side-effects. Based on our novel prodrug design that synergistically employs lipoamino acid residues (LAAs) and a brain-enzyme sensitive linker for prolyl oligopeptidase (POP), we have developed a set of lead compounds in silico. Computationally assessing lipophilicity and POP-binding affinity of our virtual prodrugs led to the selection of a representative, termed Prodrug (1), for membrane affinity studies to predict brain access from circulation. Method: Prodrugs with different LAAs and POP-sensitive linkers were designed in silico for docking with POP’s binding site using SCIGRESS and AutoDock Vina software. The prodrugs’ calculated logP (clogP) and POP-binding affinity were determined with built-in SCIGRESS applications. AutoDock results were represented as Gibbs free energy of binding (ΔG). The molecules with the most negative ΔG and adequate clogP were selected for synthesis. Prodrug (1) was compared to TRH in membrane affinity studies via immobilized artificial membrane chromatography (IAMC), an established method to predict membrane affinity (i.e., BBB permeability) with a chromatographic column comprised of immobilized synthetic lipids that mimic biological membranes. A high IAMC retention time correlates to a greater membrane affinity, with IAM Chromatographic Hydrophobicity Index (CHIIAM) values being the quantitative measure of retention. A range of reference compounds with known CHIIAM values were selected; plotting these values against their experimentally determined gradient retention times through the IAM column provided the linear relationship equation used to convert Prodrug (1)’s gradient retention time into its CHIIAM value. Results: A virtual library of prodrugs having various LAAs and POP-sensitive linkers were designed and docked to POP’s binding site. The binding of Prodrug (1) was comparable to the calculated ΔG of POP’s published, co-crystalized ligand. This prodrug also showed favorable clogP for transport into the brain and a significantly increased CHIIAM compared to TRH. Conclusion: Based on the prediction of POP-binding, using in silico docking and the favorable membrane affinity of Prodrug (1), we expect this prodrug to efficiently deliver TRH into the brain and serve as a template for fine-tuning future prodrug constructs for the efficacious brain-delivery of TRH.Item Analyzing the Sex-Dependent Effects of Intranasal Insulin on Memory Impairment Secondary to High-Fat Diet(2019-03-05) Siraj, Sohail; dos Santos, Natalia; Thompson, Lucien PhD; Ahmed, NadiaPurpose: Insulin can improve memory by enhancing the intrinsic excitability of hippocampal CA1 pyramidal neurons during memory consolidation. Chronic high-fat diet (HFD), however, can significantly impair spatial memory via reduction in excitability of these same neurons in both male and female rodent models. Interestingly, sex-dependent experimentation in these models has also shown that CA1 neurons from HFD females retain insulin-sensitivity while those from HFD males do not. Combining these findings from previous studies, it can be hypothesized that insulin therapy would improve memory deficits in females but not males fed a HFD. The following study aims to explore these sex-dependent responses to insulin therapy, as well as the use of intranasal insulin as an alternative and novel method of insulin administration that could potentially eliminate the harmful peripheral side effects of insulin via injection. Methods: Spatial memory of male and female Long-Evans rats fed control vs high-fat diet (HFD) was assessed in a spontaneous alternation task (SAT) using a four-arm radial maze (plus maze). Normally, rats will remember which arm of the maze they last visited and will attempt to sequentially explore new arms. The ability of the rats to do this is scored, with a low score indicating hippocampal impairment. Following behavioral experimentation, insulin tolerance testing was performed in order to rule-out peripheral presence of elevated insulin. Results: Intranasal delivery of insulin reversed memory impairments secondary to high-fat diet in both male and female rats. This was demonstrated by improvement in SAT scores of HFD rats treated with intranasal insulin therapy vs saline. These results were not as expected, but may be explained by the lack of a significant difference in fasting blood glucose levels of control vs high-fat diet animals. This indicates that high-fat diet animals were not showing symptoms of diabetes. It is possible then, that the animals in this experiment were in a pre-disease state where impairments were more readily reversible in both sexes and not just females. Additionally, lack of a decrease in peripheral glucose levels following intranasal insulin administration indicates that intranasal insulin did not have peripheral effects. Conclusions: This study highlights the possibility of the intranasal route as a novel method of insulin administration. Further studies should be conducted to explore the viability of this option compared to the current method of injecting insulin. For example, CSF extraction could be performed to confirm the presence of elevated insulin levels in the brain following intranasal insulin administration, as well as studies that provide further evidence that intranasal delivery bypasses the harmful peripheral side-effects of injected insulin. Lastly, although this study failed to reproduce diabetic responses, future experimentation in aged animals that have been on a HFD for [greater than] 12 weeks could better elucidate whether sex-dependent responses to chronic HFD would have an effect on insulin therapy and reversal of memory impairment.Item Analysis of Acetylcholine in Cerebrospinal Fluids by Liquid Chromatography–Tandem Mass Spectrometry(2019-03-05) Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo; Ochoa, SamanthaAnalysis of Acetylcholine in Cerebrospinal Fluids by Liquid Chromatography–Tandem Mass Spectrometry Samantha M. Brewer, Vien Nguyen, Katalin Prokai-Tatrai and Laszlo Prokai Department of Pharmacology and Neuroscience, Graduate School of Biomedical Sciences Purpose: Acetylcholine (ACh) is a neurotransmitter whose decreased levels have been associated with several diseases impacting the central nervous system. Quantification of extracellular ACh in cerebrospinal fluid (CSF) is difficult due to its low concentration (owing to its rapid hydrolysis to choline) and matric effects upon employing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification. The purpose of this study was to develop and validate an LC-MS/MS method for the high-throughput determination of ACh in rat brain microdialysates and possibly in clinical samples in the future. Methods: All analyses were performed on a TSQ Quantum Ultra mass spectrometer connected to a Surveyor MS high-performance liquid chromatography system (Thermo Scientific, San Jose, CA). Atmospheric spray ionization was used, and analytes were detected after fragmenting their intact cations through collision-induced dissociation. Deuterium-labeled ACh (ACh-d4) was used as an internal standard for quantification. Several solvent systems were compared to identify the best conditions for the preparation of ACh and ACh-d4 stock solutions to calibrate the assay. The optimized method was used to determine the extracellular ACh levels in rat brain microdialysates using the calibration curve obtained in aCSF. Results: In order to set-up a reliable assay, the MS signals for ACh and deuterium-labeled ACh (ACh-d4) used as an internal standard had to be optimized, along with optimizing the solvent systems for the preparation of their stock solutions. The optimal solvent system afforded linear calibration and resisted signal suppression by artificial cerebrospinal fluid (aCSF). Artificial cerebrospinal fluid is used as perfusion fluid upon microdialysis as it matches the composition of actual CSF. We have established that the use of purely aqueous stock solutions of ACh and ACh-d4 afforded the best calibration in aCSF. With these measures, reliable analyses of ACh in aCSF were achieved. The obtained linear calibration could be used for quantitation of low (physiological) ACh levels in samples obtained upon in vivo intracranial microdialysis from rat cortex. Conclusions: The method presented here allows for reliable quantification of ACh in cerebrospinal fluids, and may be used to evaluate the efficiency and mode of action of potential cholinergic agents. Acknowledgment: This research has been supported in part by The Welch Foundation (endowment BK-0031). IACUC Number: 2018-0006Item Automated Diagnostic Testing for Detection of Alzheimer’s Disease(2019-03-05) Conger, Tori BS; Hall, James PhD; O'Bryant, Sid PhD; Julovich, David BSPurpose: Alzheimer’s disease (AD) historically has been detected with brain imaging scans by magnetic resonance imaging (MRI) or positron emission tomography (PET) and by measurement of protein levels in cerebrospinal fluid (CSF). These tests pose limitations due to high cost and invasiveness of the procedures. Our automated blood-based biomarker screening utilizes ELISA based detection and provides a realistic, cost-effective approach for detection of AD and mild cognitive impairment (MCI). Methods: MesoScale Discovery (MSD) multiplex biomarker platform, integrated with a Hamilton StarPlus liquid handler and Quanterix single molecule array (Simoa) HD-1 fully automated analyzer are the platforms used to conduct our multiplexed biomarker screening. Several experiments to assess the effectiveness of an automated system were conducted. Precision and accuracy of hand pipetting verses automated pipetting was tested using aliquots of control materials. Assays were conducted over a three-day period by separate individuals and by the automation. Plate washing and ECL addition was held constant and carried out by the automation for both groups. Covered verses uncovered incubation determined if evaporative loss caused differences on biomarker detection levels. Sample incubation times were tested by shortening the interval from the recommended maximum to determine if less time had effect on detection levels. Automation liquid classes were verified using a Mettler Toledo balance and Hamilton software. Gravimetric measurements were made under control conditions (room temperature, humidity and reagent temperature). Different sample volumes were tested to determine the effect of freeze thaw on detection levels. Results: Although interestingly similar the automation pipetting profile was slightly different than the hand pipetting; automation was more precise. Covered verses uncovered incubation showed virtually no difference in detection levels. Shorting sample incubations times had little effect on detection levels. Gravimetric measurements of liquid classes showed excellent precision and accuracy. Sample volume size did influence detection levels, lower volumes showed a decrease in biomarker levels. Conclusions: Although differences are seen screening with automation verses hand processing, the significance is negligible. The automated diagnostic screening for detection of AD would be a cost effect, easily implemented solution and could become the first in line test.