Neuroscience
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Item INVOLVEMENT OF P38 MAPK IN REACTIVE ASTROGLIOSIS INDUCED BY ISCHEMIC STROKE(2013-04-12) Roy Choudhury, GouravPurpose: Reactive astrogliosis is an important response during early phase of cerebral ischemia but as time progresses; these activated astrocytes develop into a glial scar. During acute phase of ischemia, astrocyte activation is beneficial as it limits the spread of infarct however in long term the glial scar developed becomes a barrier to migrating axons and hinder the recovery process. The hallmark of astrogliosis is an increase in the expression of GFAP (Glial fibrillary acidic protein). However the signaling events underlying these phenomenons have not been yet clearly elucidated. p38MAPK, a stress signaling Mitogen activate protein kinase plays an essential role in inflammation and has been widely reported to be activated during ischemic injury. Since inflammation is major determinant of events after ischemic injury in brain, we hypothesize that p38 MAPK plays a critical role in reactive astrogliosis after ischemic injury and its inhibition attenuates glial scar formation Methods: For in vitro studies rodent primary astrocytes were used and were subjected to Oxygen-glucose deprivation (OGD) for 3h. p38MAPK's role in astrocyte activation was determined pharmacologically with a p38 inhibitor (SB 239063) and genetically by an astrocyte specific p38 knockout. 24 hours following insult and the expression of GFAP was determined using Western blots. Wound healing and Transwell assay were used to determine role of P38 in astrocyte migration. For in vivo studies astrocyte specific conditional p38 knockout mice (p38KO) were used. Transient or Permanent middle cerebral artery occlusion (tMCAO or pMCAO) was performed to model ischemic stroke. Walk initiation, Negative geotaxis and Ladder rung walking test were used to determine motor dysfunction after MCAO. Astrogliosis morphometric analysis was to quantify glial scar in GFAP stained brain sections Results: In primary astrocyte cultures, hypoxia and scratch injury-induced astrogliosis was attenuated by both p38 inhibition and knockout of p38 MAPK. In vivo studies showed that p38KO mice after permanent MCAO had a significantly smaller glial scar compared to their wild type (WT) littermates. Results also indicated that p38KO mice performed significantly better in behavioral studies compared to their WT only in tMCAO Conclusions: p38MAPK plays an essential role in evolution of glial scar after ischemia and its inhibition attenuates glial scar formation. P38 inhibition significantly improves motor dysfunction in tMCAO but the protection is lost in pMCAOItem P38MAPK PROMOTES ASTROGLIOSIS AFTER FOCAL ISCHEMIC STROKE IN MICE(2013-04-12) Roy Choudhury, GouravPurpose: Patients surviving ischemic stroke are often left with long term functional disabilities suggesting that the ischemia associated pathology continues to persist in long term and limits recovery. One such reason for limited functional recovery is formation of glial scar. Glial scar is formed in response to ischemic injury and may protect the cerebral tissue from further injury during early phase of injury. But in long term, this glial scar establishes a physical and chemical barrier to the axonal migration and growth and hinders recovery. The events of a post ischemic brain are prominently influenced by inflammation and p38MAPK is an important signal transducer of cell's response to inflammatory mediators. So in our current study we hypothesize that astrocyte activation and glial scar formation after stroke is regulated by astrocytic p38MAPK and its inhibition will attenuate the glial scar formation Methods: Primary astrocyte cultures isolated from GFAP/p38 knockout mice and wild type littermates were used for in vitro studies. In vitro hypoxic condition (0.5% O2 and 5%CO2) was simulated by depriving Oxygen and glucose (OGD) for 3 hours. Wound healing and Transwell assay were used to determine role of p38MPAK in astrocyte migration. For in vivo studies, permanent middle cerebral artery occlusion (pMCAO) was conducted to induce ischemic stroke. Astrogliosis morphometric analysis was performed using nucleator method to quantify glial scar in GFAP stained brain sections Results: Results from in vitro studies indicated that p38MAPK knockout significantly attenuated OGD induced increase in glial fibrillary acidic protein (GFAP) expression and reduced astrocyte migration. In vivo studies showed that conditional GFAP/p38 knockout mice after MCAO had a significantly smaller glial scar compared to their wild type litter mates Conclusions: Astrocyte reactivation and glial scar formation in brain after ischemic injury is mediated p38MAPK and its inhibition attenuates glial scarItem GLUCOCORTICOID SUPPRESSION OF COX-2 EXPRESSION IN HYPOTHALAMIC NEURONAL CELL LINE(2013-04-12) Stacey, WinfredPurpose: Glucocorticoids have long been administered as potent anti-inflammatory agents. They are secreted in response to activation of the hypothalamic-pituitary-adrenal (HPA) axis and act as regulators of the immune responses, preventing overshoot of inflammatory processes. Dysfunction of this regulatory feedback leads to inflammatory processes unchecked and has been implicated in various chronic inflammatory disorders such as rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis, all of which are associated with increased expression of pro-inflammatory genes. One pro-inflammatory gene regulated by glucocorticoids is cyclooxygenase-2 (cox-2), a key and rate-limiting enzyme in the production of prostaglandins (PGs). The mechanisms by which glucocorticoids suppress this enzyme's activity have been well studied outside of the central nervous system. However, there is limited knowledge on how glucocorticoids suppress cox-2 expression in the paraventricular nucleus of the hypothalamus (PVH). In the PVH, studies have shown that in response to inflammatory stimuli, cox-2 expression and prostaglandins levels increase which stimulate parvocellular neurons to release corticotropin-releasing hormone (CRH). CRH release ultimately results in inflammation-induced activation of the HPA axis. Uninhibited, cox-2 activity would lead to excessive levels of PGs and a hyperactive HPA response. The hypothesis for this study is, glucocorticoids suppress cox-2 expression by inhibiting Nuclear Factor-𝛋B(NF-kB) transcriptional activity in hypothalamic IVB neuronal cell line Methods: To characterize the model, a hypothalamic IVB cell line was analyzed for cox-2 expression following stimulation with Phorbol 12-Myristate 13-Acetate (PMA) for 30m, 60m, and 120m. A dose response curve was generated using 1µm, 0.3µm, 0.1µm, 30nm, 10nm, and 1nm of PMA. COX-2 mRNA expression was analyzed using Real time polymerase chain reaction (RT-PCR). COX-2 protein levels were probed by western blot. COX-2 immunoreactive cells were analyzed using Immunocytochemistry (ICC). Cells were treated with 10-7M dexamethasone and COX-2 mRNA, protein levels and immunoreactive cells were analyzed. Results: COX-2 mRNA, protein levels and immunoreactivity increased following PMA treatment of hypothalamic IVB neuronal cells. Dexamethasone (Dex) treatment decreased COX-2 mRNA, protein levels as well as COX-2ir cells Conclusions: PMA treatment induces cox-2 expression and dex treatment has an inhibitory effect on its expression.Item EFFECT OF CURCUMIN ON BODY WEIGHT AND COGNITIVE FUNCTION(2013-04-12) Sarker, MarjanaPurpose: Curcumin (CURC,) a widely-consumed phytochemical, has been reported to attenuate inflammation and improve cognition. The current study addressed the hypothesis that curcumin produces these effects by attenuating adipose tissue. In this study, curcumin-fed, middle aged (15 months) C57BL/6 male mice were used as an experimental model. Methods: Three groups were used in the study: Ad libitum (AL), Caloric restriction (30%) and CURC (1000mg/kg of diet). The mice underwent different cognitive tests (n=19) after 8 weeks of dietary treatment, which tested spatial function (Morris water maze, MWM) and cognitive flexibility (Discriminated active avoidance, DA). Adipose tissue, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) was collected at the end of the 12 week treatment. Results: Calorically restricted mice (30.33+0.86) weighed significantly less than mice consuming CURC (34.89+0.72) or the AL mice (34.09+0.64), beginning from week 2 of treatment. Food intake of CURC (3.60+0.12) was significantly higher than AL (3.26+0.09) mice. Caloric restriction (8.53+0.70) and CURC (8.16+0.45) took fewer trials to reach criterion in session 3 of DA compared to AL (10.19+0.83). On average, CR (12.33+0.78) took fewer trials to reach criterion in DA. No significant difference between the groups in learning index of MWM. Caloric restriction mice has decreased VAT(0.31+0.03) and SAT (0.26+ 0.02) compared to CURC, VAT(1.09+0.09) and SAT (0.54+0.07), and AL, VAT (1.16+0.11) and SAT (0.61+0.08). Conclusions: Results from this study indicate that curcumin supplementation has positive effects on specific domains of cognition independent of adiposity. Curcumin supplementation may also be responsible for blunting weight gain since CURC mice have increased food intake compared to AL but there is no significant difference in weight between CURC and AL mice.Item EFFECT OF WATER DEPRIVATION ON KCC2 EXPRESSION IN HYPOTHALAMIC VASOPRESSIN NEURONS IN RAT(2013-04-12) Knapp, BlaynePurpose: Argininge Vasopressin (AVP) is a neurohypophyseal hormone that contributes to body fluid homeostasis by regulating plasma fluid and electrolyte composition. It is released from the posterior pituitary (PP) by magnocellular neurosecretory cells (MNCs) located within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. The regulation of AVP release is critical for maintaining blood volume and blood pressure, and although the molecular mechanisms of AVP regulation are not fully understood, we do know that it is partially due to the vital balance of synaptic excitatory and inhibitory inputs that determine the relationship between plasma osmolality and AVP release. Dysregulation of this system and the resulting disturbances of water and electrolyte balance can lead to increased morbidity associated with disease states such as heart and liver failure. This is why it is critical for us to elucidate the molecular mechanisms involved with its physiological control and its inappropriate release in disease states. Models of water deprivation represent a physiological challenge that requires sustained release of AVP from the PP into circulation as a means of maintaining body fluid homeostasis. We attempt to address the gaps that remain controversial or unexplained in our current understanding of AVP regulation by measuring changes in the expression of extruder KCC2 in water deprived and euhydrated adult male rats and measuring its effects on inhibitory GABAergic neurotransmission in AVP MNCs. Methods: Using laser capture microdissection (LCM), AVP MNCs were harvested from the SON and PVN of euhydrated and 48 hour water deprived adult male rats and reverse transcriptase polymerase chain reaction (RT-PCR) studies were used to test for changes in KCC2 message. Western blot protocols were used to measure changes in protein expression. Results: We observed a significant elevation in KCC2 mRNA expression in AVP cells of the SON (WD 2.5 ± 0.52; Control 1.0 ± 0.06, p<0.05) but not in the PVN (WD 3.5 ± 1.7; Control 1.0 ± 0.1). Immunofluorescence demonstrated the colocalization of KCC2 and AVP in the SON. Conclusions: Increased expression of KCC2 could be associated with decreased intracellular Cl- in AVP neurons in the SON, thereby serving to maintain or enhance the inhibitory tone of AVP neurons in the SON but not the PVN during WD.Item EFFECTS OF METABOLIC ACIDOSIS ON MOTOR AND COGNITIVE FUNCTION IN YOUNG AND AGED MICE(2013-04-12) Kindle, RobbynPurpose: Metabolic acidosis is defined as a reduction of serum pH below 7.4 due to the inability of the kidneys to remove an adequate proportion of hydrogen protons (H+), and can be a symptom of diabetes, kidney disease, and aging. The effects of acidosis on the nervous system are not well understood, and little research has been done to establish those effects and their potential role in cognitive decline. Methods: Four- and twenty-month-old male C57BL/6J mice (n=8) were fed either a control diet or the control diet supplemented with 2% ammonium chloride (NH4Cl) to induce metabolic acidosis. Urine pH was measured weekly to insure inducement of acidosis in the NH4Cl supplemented diet groups. After 4 weeks of pre-treatment, the mice were tested for various functional tasks and remained on their respective diet during behavioral testing. The tests were done in the following order: locomotor activity, reflexes, wire suspension, bridge walking, active avoidance (T-maze), and Morris water maze. Results: Overall, urine pH of the mice on the NH4Cl diet was decreased compared to control mice. Though the NH4Cl diet did not seem to affect the weights of the young mice, a significant decrease was seen in the aged mice. Aged mice on the NH4Cl diet had increased spontaneous activity and better performance on motor tasks. On spatial learning and memory, the NH4Cl mice learned to locate the platform more efficiently than their age-matched controls. In general, the NH4Cl mice took fewer trials to reach the criteria of avoidance and discrimination. Conclusions: Interestingly, the data suggest a potential benefit on motor and cognitive function of a mild acidosis in young and aged mice. More studies will solidify these observed effects and identify a potential mechanism by investigating acid sensing ion channel concentration in various brain regions through immunoblot analysis.Item MIRROR THERAPY'S EFFICACY AND MECHANISM IN THE PROMOTION OF MOTOR RECOVERY IN HEMIPARETIC POST STROKE POPULATIONS(2013-04-12) Guild, JeffreyPurpose: Mirror therapy (MT) has been thought to be affective for treating complex regional pain syndrome, hemiparesis, and phantom limb pain. This treatment uses imagery to reflect movements of the unaffected side off a mirror as if it were the affected side. The purpose of this review is to highlight the advances in MT research as a treatment option for patients suffering from hemiparesis post stroke. Methods: Five authors searched all accessible databases from a university. All significant randomized controlled trials (RCT's) and history of mirror therapy were obtained. For context, cortical neuroplasticity post stroke, bilateral training, imagery training, electrical cortical stimulation, and constraint-induced movement therapy (CIMT) were also reviewed. Results: Over the past decade RCT's and reviews have supported the efficacy for treating motor impairments due to stroke in acute, subacute, and chronic conditions. Functional outcome measures to support these findings were the Fugl-Meyer Assessment, Brunnstrom scale, and the Motor Assessment Scale. The mechanism for MT is based on fMRI studies which show MT promotes cortical reorganization of the ipsilesional primary motor cortex (M1) and may even prevent over dominance of the contralesional M1 region. CIMT and electrical stimulation use the same mechanism of preventing inhibition of the ipsilesional M1 region by the contralesional M1 region by stimulating the ipsilesional side. While the MT literature addresses the upper extremities the most, some RCT's have shown similar outcomes with the lower extremities. Outcomes for treating sensory recovery has been supported with some evidence, however, there are stronger arguments and evidence that mirror therapy is most effective with motor recovery in patients suffering from hemi-paresis post stroke. Conclusions: By providing patients with a mirror at home or in the clinic, MT provides a simple, inexpensive, and effective way to improve functional motor recovery in patients suffering from hemiparesis. Other methods have been used consistently in the literature along with MT including standard physical and occupational therapy, bilateral arm training, strength training, and neuromuscular electrical stimulation. CIMT has not been studied with MT. Since a functional extremity is required with CIMT and MT has promoted motor recovery in acute populations, MT may be the beginning segway into motor recovery before transitioning to CIMT along with other treatment methods.Item EFFECT OF GLUCOCORTICOIDS ON MITOCHONDRIAL GENE EXPRESSION IN AMYGDALAR AND HYPOTHALAMIC NEURONAL CELL LINES(2013-04-12) Dalwadi, DhwanilPurpose: To determine the effect of glucocorticoids on mitochondrial gene expression in amygdalar AR-5 and hypothalamic IVB cell lines. Methods: Microarray was used to identify differentially expressed genes between AR-5 and IVB cell lines at the basal level. The RNA samples were analyzed on the Affymetrix Rat Genome 230 2.0 array using the UTSW microarray core facility. Microarray data was verified by real time PCR (RT-PCR). To determine the effect of glucocorticoids on mitochondrial gene expression, the cells were treated with 100 nM dexamethasone (dex) for 2 hours. RNA was extracted and mRNA levels were measured by RT-PCR. Results: 30,000 genes were analyzed on the array, of which 1710 genes were found to be differentially expressed. Mitochondrial-encoded genes NADH-ubiquinone oxidoreductase chain 1, 2, 3 (ND1, ND2, ND3), cytochrome C oxidase subunit 1, 2 (COX1, COX2), and Cytochrome b (CYTB) were relatively abundant in AR-5 compared to the IVB cells. The mRNA expression of these genes was 400-fold or greater in the AR-5 relative to IVB cells. Following 2 hr, 100 nM dex treatment, there was no difference in expression of mitochondrial encoded genes. Conclusions: The amygdalar AR-5 cell line have significantly greater abundance of mitochondrial encoded message than the hypothalamic cell line. Dexamethasone does not appear to regulate mitochondrial encoded genes, atleast when treated with 100 nM dex for 2 hours.Item TIME COURSE OF CHANGES OF FOSB IMMUNOREACTIVITY IN NUCLEUS OF SOLITARY TRACT DURING CHRONIC INTERMITTENT HYPOXIA(2013-04-12) Wu, QiongPurpose: To determine the time course of the onset of FosB expression during 7 days of exposure to chronic intermittent hypoxia (CIH), a widely used model of the arterial hypoxemia that occurs during sleep apnea. FosB is a member of the transcription factor Activator Protein-1 (AP-1) family. FosB is not constitutively expressed in the central nervous system (CNS) and is induced following chronic stimulation. A previous study from our group found that the number of FosB immunoreactive neurons in the nucleus of solitary tract (NTS) is increased after 7 days of exposure to CIH (alternating 3min periods of 10% O2 with 3min 21% O2 from 8am-4pm). Methods: We measured the number of FosB immunoreactive neurons in male Sprague Dawley rats exposed to either room air (control) or to CIH. On the day following exposure to CIH for 1 day, 3 days, 5 days or 7 days (n= 6 for each CIH group and 14 for control group), rats were perfused with paraformaldehyde and brains processed for FosB immunohistochemistry using an antibody (Santa Cruz) that does not distinguish FosB from ΔFosB. Results: The number of FosB immunoreactive neurons in caudal NTS in the control group was 11± 2 cells/section (c/s). After 1 day CIH the number of FosB immunoreactive neurons increased to 40± 11 c/s, 3 days CIH 35±8 c/s, 5 days CIH 36±5 c/s and 7 days CIH 29±4 c/s. The differences between the control group and the CIH groups were significant. Three days after 7 days exposure to CIH the number of FosB immunoreactive neurons in NTS had returned to 10 ± 3 c/s (n=2). Conclusions: This study indicates that FosB expression develops rapidly during exposure to CIH and remains elevated during a 7 day exposure.Item P38-KNOCKOUT CREATES A SEX DIFFERENCE IN IN MITOCHONDRIAL FUNCTIONS.(2013-04-12) Metzger, DanielPurpose: to determine sex differences and P38 expression among transgenic mice Methods: Isolated mitochondria from male and female transgenic mice were used. XF Seahorse was used to monitor cell respiration in real time Results: These data suggest that P38 is required for the mitochondrial respiration of male mice. When P38 is depleted below a normal level, females maintain mitochondrial respiration perhaps through the estrogen-mediated COX upregulation. Conclusions: This mouse model may be useful for studying mechanisms involving Purkinje neuronal P38 and mitochondrial disorders.Item ASTROCYTE-ELEVATED GENE-1 PROTECTS HUMAN ASTROCYTES FROM OXIDATIVE STRESS-INDUCED CELL DEATH: A POTENTIAL SURVIVAL MECHANISM IN HAND AND GLIOMA PATHOGENESIS(2013-04-12) Vartak, NehaPurpose: Astrocyte elevated gene-1 (AEG-1), a novel human immunodeficiency virus (HIV-1)- and tumor necrosis factor-ɑ-inducible oncogene, has generated significant interest in the field of cancer research as a therapeutic target for many metastatic cancers. However, little is known of its role in astrocyte behavior and function during HIV-1 central nervous system (CNS) infection, and whether it contributes towards the development of HIV-1-associated neurocognitive disorders. Based on its putative role in cancer as a chemotherapy resistance marker, here, we investigated whether AEG-1 induction in astrocytes alters their responses to oxidative stress, a hallmark feature of neuroinflammatory disorders. Methods: Oxidative stress induced changes in AEG-1 mRNA and protein were assessed by real-time PCR, immunoblotting and confocal microscopy. Astrocyte responses to oxidative stress were assayed by measuring the changes in catalase activity, protein carbonylation and glutathion production. AEG-1 mediated protection against oxidative damage was assessed by measuring the anti-oxidant mechanisms upon AEG-1 knockdown. Results: Analysis of AEG-1 mRNA levels in an aging cohort of HIV-1 seropositive and seronegative human brain tissues showed a significant positive correlation to aging. A dose-dependent increase in AEG-1 astrocyte nucleolar localization was noted following treatment with oxidative stress stimuli, hydrogen peroxide, as assayed by immunostaining and confocal microscopy. Cell death and cell survival assays to quantify apoptotic nuclei, mitochondrial depolarization and activity, and cell membrane permeability demonstrated a novel role of AEG-1 in protecting astrocytes from oxidative-stress-induced damage. Conclusions: Together, findings from this study suggest that AEG-1 may play a role in protecting astrocytes from oxidative stress-induced DNA damage, a plausible mechanism of astrocyte survival during HIV-1 CNS infection-induced toxicity.Item DEXAMETHASONE INDUCES FORMATION OF A PUTATIVE REPRESSOR COMPLEX AND ASSOCIATED CHROMATIN MODIFICATIONS IN THE REGION OF THE CORTICOTROPIN-RELEASING HORMONE GENE PROMOTER(2013-04-12) Sharma, DharmendraPurpose: Glucocorticoids down-regulate expression of hypothalamic CRH; however, mechanisms by which they do so are not fully understood. The proximal promoter cAMP response element (CRE), negative GRE (nGRE), and methylated CpG islands all play a role in crh down-regulation. Here we are reporting the involvement of DNA and Histone methyltion in the regulation of crh gene expression. Methods: Real time PCR to measure the levels of mRNA. Immunocytochemistry to localize immunoreactive proteins. Western Blotting to check the expression of Proteins. Coimmunoprecipitation to look for the interaction of various proteins involved. Chromatin immunoprecipitation to analyze the recruitmentment of various regulatory molecules at the promoter. Promoter methylation analysis by sodium bisulphite sequencing Results: Previously, we showed that Dex-repressed crh expression is associated with GR and histone deacetylase 1 (HDAC1) recruitment to the crh promoter region. Given that HDAC1 may be present in methylated CpG binding protein 2 (MeCP2) complexes, and that MeCP2 is known to play a role in regulating crh expression, we sought to determine whether or not HDAC and/or MeCP2 could interact with the GR. Dex enhanced GR interactions with both proteins.Glucocorticoid regulation of crh has also been associated with CpG methylation, thus we assessed whether GR could interact with a DNA Methyltransferase (DnMT). Indeed, the GR interacted with DnMT3b but not DnMT3a. In addition, Dex also induced occupancy of crh by, HDAC1, MeCP2, and DnMT3b in the promoter region and consequently it led to an increase in the level of promoter methylation that appeared to be CpG site specific. Lastly, to extend previous assessment of chromatin modifications in this promoter region, the degree of histone methylation was measured. Dex increased tri-methylation of histone 3 lysine 9 (H3K9), a marker of gene suppression, however levels of di and tri methylated H3K4 were not significantly changed. Conclusions: The data indicate that a GR:HDAC1:MeCP2 repressor complex participates in Dex mediated crh down regulation. Furthermore, recruitment of this complex occurs in parallel with GR interaction with DnMT3b and DnMT3b recruitment to the crh proximal promoter and simultaneous increase in site specific promoter methylation. Lastly, specific histone methylation appears to play a part in crh downregulation, as well, in that Dex treatment leads to a co-temporaneous increase in tri- methylated H3-K9.Item METHYL CPG BINDING PROTEIN 2 (MECP2) IS NECESSARY FOR GLUCOCORTICOID RECEPTORS (GR) MEDIATED CORTICOTROPIN RELEASING HORMONE (CRH) GENE REPRESSION IN RAT HYPOTHALAMIC CELL LINE.(2013-04-12) Bhave, Shreyas A.Purpose: Methylation of CpG islands in CRH gene (crh) promoter region plays an important role in crh regulation. MeCp2 binds to methylated CpG islands and act as a transcriptional repressor. Increased crh promoter activity and expression following mutations in MeCP2 suggests its role in crh regulation. In hypothalamus glucocorticoids down-regulate CRH gene expression as part of negative feedback mechanism. The molecular mechanism and role of different co-regulators in this process is poorly understood. The purpose of this study is to elucidate the role of MeCP2 in the dexamethasone- a synthetic glucocrticoid (Dex) induced GR mediated crh repression in rat hypothalamic cell line. Methods: We used small hairpin RNA (shRNA) induced MeCP2 knockdown to understand the importance of MeCP2 in GR mediated crh regulation. We first screened the shRNA plasmids against rat MeCP2 for knock down efficiency using western blotting. Scrambled sequence was used as negative control. The IVB- rat hypothalamic immortalized cell line was transfected with shRNA or scrambled DNA containing vector with GFP. The transfection efficiency was monitored by fluorescent imaging using EVOS digital microscope. The transfected cells were incubated in stripped serum media for 48hrs prior to treatment with Dex 10-7M for 2hrs. Total RNA was extracted using Tri reagent and cDNA was synthesized. qPCR was performed using gene specific primers for gapdh, crh and heteronuclear RNA (hnRNA) of crh (hncrh). ΔΔCT method was used for calculation of relative gene expression. Results: After 72 hrs, following the shRNA transfection Mecp2 protein expression was knocked down to significant level in one out of four plasmids as compared to scrambled control. This shRNA plasmid was used for further experiments. Following Dex 10-7M treatment for 2 hrs there was considerable decrease in CRH mRNA as well as hnRNA. This effect of Dex was lost following MeCP2 Knock down. Conclusions: From the preliminary data we conclude that MeCP2 plays an important role in crh regulation. Furthermore, presence of MeCP2 is necessary for the GR mediated down regulation of crh.Item STIMULATION OF THE AMPA RECEPTOR IN RETINAL GANGLION CELLS INCREASES PHOSPHORYLATION OF CREB(2013-04-12) Park, YongPurpose: Activation of the ɑ-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor through glutamate or other agonists allows influx of cations including calcium, potassium and sodium. The purpose of this study was to investigate the neuroprotective role of the AMPA receptor in-vitro, in purified retinal ganglion cells (RGCs) and retinal mixed culture lacking RGCs (mixed retinal culture), by assessing the phosphorylation of cAMP response element-binding protein (p-CREB) stimulated by calcium influx. Methods: Purified rat RGCs were isolated from P3-P7 Sprague-Dawley rats and cultured by a double immunopanning technique using an antibody to Thy 1.1. The residual neurons in the retina following RGC isolation (supernates of the panning procedure) were used as the mixed retinal culture (lacking RGCs). Calcium imaging was used to identify the functionality of the AMPA receptors and selectivity of the AMPA agonist. RGCs and mixed retinal neurons were cultured for 7 days before AMPA treatment. Following treatment with AMPA for 6 hours, proteins were extracted and western blot analysis was carried out to determine changes in expression of the p-CREB and p-ERK1/2, which were normalized to total CREB, total ERK1/2, and beta tubulin. Results: AMPA receptors were stimulated through administration of AMPA (100μM), which depolarized the purified RGCs and increased intracellular calcium. The AMPA mediated calcium ion influx was significantly attenuated by approximately 87.8% (p<0.001) following pre-treatment with 20μM of NBQX (AMPA receptor antagonist). Pretreatment with a NMDA receptor (I μM MK801) or Kainate receptor (50μM UBP301) antagonists simultaneous with AMPA administration did not significantly decrease calcium influx. AMPA increased p-CREB by 4.3 ± 1.1 fold in purified RGCs (p=0.004), however, in mixed culture p-CREB did not change appreciably compared to control. ERK1/2 phosphorylation was significantly decreased in mixed retina culture (p<0.05), but not in purified RGC culture treated with AMPA. Conclusions: The data demonstrate distinct differences in the response to AMPA between RGCs and other neuronal populations in the retina. In particular, the lack of significant changes in the phosphorylation of ERK1/2 in purified RGCs following AMPA treatments suggests that an alternative pathway for phosphorylation of CREB maybe more important in RGC cell survival.Item ANTIOXIDANT COMPOUNDS INHIBIT COCAINE-CONDITIONED LOCOMOTION(2013-04-12) Nguyen, JacquesPurpose: Cocaine, a potent psychostimulant, produces various neuroadaptations that further contribute to its overall abuse. Although there currently are no known pharmacological treatments for cocaine addiction, N-acetylcysteine, a thiol-containing antioxidant, has been shown to prevent relapse in animal models of cocaine-addiction and to effect drug reward value following psychostimulant toxicity. This study evaluates the mechanisms that underlie this compound's ability to affect cocaine-conditioned locomotion and compares its efficacy to other potential intervention(s) for cocaine-induced effects. Methods: Cocaine (40mg/kg) and 0.9% saline were administered to Swiss-Webster mice via intraperitoneal injection (i.p.), in either the testing apparatus or the home cage, separated by a 2-hour time interval. When the animals were placed into the testing chambers, they were given 30 minutes to explore freely and spontaneous locomotion was monitored using Digiscan animal tracking software. On the following day, N-acetylcysteine (25, 50, 100 mg/kg), dimethylthiourea (5, 10, 25, 50 mg/kg), or vehicle control were administered prior to placement into the activity chambers. Cocaine conditioned effect was inferred by a change in horizontal activity counts, respective to controls. Results: N-acetylcysteine (100mg/kg) and dimethylthiourea (25 and 50mg/kg) successfully inhibited cocaine-conditioned locomotion. Conclusions: N-acetylcysteine and dimethylthiourea's common ability to inhibit the cocaine-conditioned locomotion suggests that pro-oxidating cellular redox state may underlie acute neuroadaptations that occur during cocaine exposure. Understanding the nature of cocaine-conditioned effects, using various pharmacological interventions, may further provide insight into the molecular mechanisms underlying these drug-induced behaviors.Item SIGMA 1 RECEPTOR SELECTIVE LIGAND ATTENUATES SCOPOLAMINE INDUCED IMPAIRMENT IN LEARNING AND MEMORY(2013-04-12) Malik, ManinderPurpose: Cognitive deficit is seen in patients with Alzheimer's disease, Parkinson's disease, after brain traumatic injury and stroke. Cognitive deficit is mainly due to the alteration in the cholinergic pathway. All currently prescribed therapeutic drugs provide only symptomatic relief and become ineffective as the disease progresses. Therefore, additional novel therapeutic agents need to be developed to slow or stop the progressive loss of memory forming cells. In this project, we will focus on characterizing a ligand selective at sigma 1 receptor for neuroprotection. Methods: A filtration-binding assay was used to characterize the binding properties of novel sigma compound at D2 like dopamine receptors and at sigma receptors. C57BL/6J mice injected with scopolamine were used as our experimental model to evaluate the cognitive properties of test drug. The neuroprotective properties were evaluated using water maze, active avoidance test and novel objet recognition tests. Results: Scopolamine treated animals exhibited impaired learning and memory in water maze and active avoidance test. Animals pre-treated with test drug attenuated the scopolamine-mediated effect. Conclusions: Test drug was able to attenuate the scopolamine-induced impairment in learning and memory.Item MEASUREMENTS OF BIOACTIVE ESTROGENS IN THE RAT BRAIN AND SERUM AFTER PREMARIN TREATMENT(2013-04-12) Szarka, SzabolcsPurpose: To investigate estrogen uptake into brain tissues after the administration of conjugated equine estrogens (Premarin®) using an animal model. Methods: Ovariectomized Spague-Dawley rats were treated with a single dose of Premarin® (1 mg/kg body weight, i.v.). The blood was collected by cardiac puncture, clotted on ice and centrifuged in order to obtain serum samples. Tissue homogenates were prepared in pH 7.4 phosphate buffer. Samples were spiked with internal standards, and estrogens were extracted with ethyl acetate. The organic layer collected was dried under a nitrogen stream, and the residue was derivatized with dansyl chloride. Quantification of bioactive estrogens was performed by isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: To assess biodistribution of selected bioactive estrogens formed after Premarin® injection, we developed an LC-MS/MS assay for the simultaneous detection and quantification of estrone (E1), 17ɑ-estradiol (ɑE2), and 17β-estradiol (βE2). We also validated the method according to US FDA guidelines. The assay revealed that all three endogenous estrogens appeared in the serum. As expected, E1 was present at the highest concentration, 1892±73 pg/mL when measured 30 min after drug administration, since E1-sulfate is the major constituent of Premarin®. The corresponding ɑE2 and βE2 concentrations were 82±13 pg/mL and 1134±75 pg/mL, respectively. At the same time, Premarin® treatment not only produced significant serum estrogen levels, but 3554 ± 109 pg/g of E1, 237 ± 25 pg/g of ɑE2, and 633 ± 25 pg/g of βE2 were also measured in rat brain samples 30 min after drug treatment. Conclusions: The injection of Premarin® not only increased circulating serum estrogen levels, but significant brain uptakes of bioactive estrogens have also been demonstrated. However, our study is the first to show that serum estrogen concentrations are not indicative of brain levels thereby implicating the role of localized steroid-metabolizing enzymes.Item SOY PHYTOESTROGENS ENHANCE NEUROGENESIS IN THE RAT SUB VENTRICULAR ZONE(2013-04-12) Waters, RebeccaPurpose: The purpose of this study was to test the hypothesis that soy phytoestrogens enhance neuronal stem cell proliferation in the brain following ischemic injury similar to estrogen. Methods: Female Sprague-Dawley rats were randomly assigned to one of 3 groups soy-free diet + vehicle (Veh), soy-free diet + estrogen (E), or high soy diet (Soy). Rats were bilaterally ovariectomized and, after 4 weeks of treatment, underwent 90 minutes of middle cerebral artery occlusion (MCAO) or sham MCAO. Twenty-four hours after MCAO, rats were euthanized and brain sections were prepared for histology. Coronal brain sections encompassing the subventricular-proliferating zone (SVZ) were stained with Fluorojade B (FJB) to detect dying neurons. In alternate sections, immuno fluorescence staining for Ki67 was used to identify proliferation, and staining for doublecortin (DCX) was used to detect early neurogenesis. Microscopic images were digitally captured and analyzed manually. Results: Fluoro-Jade B staining revealed reduced injury in the E and Soy treated groups compared to Veh, in agreement with previous studies. In both the MCAO and sham groups, E and Soy increased proliferation (Ki67 staining) in the SVZ more than 2-fold compared to Veh. DCX double labeling revealed that the majority of these cells were of neuronal lineage. Conclusions: Because our results demonstrated higher amounts of Ki67 & DCX in the Soy and E groups compared to Veh, we conclude that a soy diet induces neuroproliferation in the SVZ both in the presence and absence of injury. Thus, in addition to its neuroprotective role, dietary soy may mimic the neurorestorative action of estrogen.