Schizophrenia is a debilitating neurodevelopmental disorder that generates a broad symptomology. These symptoms include positive, negative, and cognitive dysfunctions, and many of these symptoms are well-replicated in animal models. One such model involves sub-chronic administration of phencyclidine (PCP) to non-human primates. The action of PCP results in the blockade of NMDA receptors, leading to glutamatergic dysfunction, which has become a basis for study of schizophrenia-like pathologies. The prefrontal cortex is an area of the brain involved in higher cognitive abilities, such as working memory and executive function. Impairments within the dorsolateral prefrontal cortex (dlPFC) have shown to mimic the cognitive symptoms of schizophrenia. The current study investigates the effect of the PCP model on the cells of the dlPFC. Layer V/VI pyramidal neurons of the dlPFC were analyzed in female juvenile, male juvenile, and male adolescent non-human primates after PCP administration. The pyramidal cells from female juvenile monkeys administered PCP did not demonstrate any statistical difference in dendrite morphology compared to age-matched controls (saline-injected). The cells from male juveniles administered PCP contained greater dendritic length in a spatial analysis of the apical arbor relative to controls. Finally, the cells from the male adolescents administered PCP demonstrated reduced dendritic length in certain values in the basal arbor compared to age-matched controls. The apical dendrites of the cells from the male adolescent subjects also had a reduced number of segments, branch points, branch tips, and dendritic length close to the soma. These findings suggest age and gender may affect the cell morphology in the dlPFC in schizophrenia-like pathologies. / acase@tulane.edu

Opioids acting at the mu (morphine) receptor represent the vast majority of clinically used opioids and remain the most effective analgesics for treating moderate to severe pain. The use of morphine and similar compounds for the management of pain is limited by adverse side effects including respiratory depression, abuse potential, motor impairment and cognitive deficits. Novel mu opioid receptor agonists developed in our laboratory are based on the structure of the endogenous ligands (endomorphins) and provide potent antinociception. Studies from our laboratory indicate that these endomorphin (EM) analogs produce fewer adverse side-effects in rodents than morphine, including reduced respiratory depression, motor impairment, tolerance, and abuse potential. Recent studies have indicated that repeated injection of morphine, for as little as a few days, can induce glial activation, an inflammatory response that can led to a “paradoxical” morphine-induced pain (Watkins et al, 2005, 2007). Morphine is recognized by glia cells similarly to a foreign antigen via toll-like receptor 4 (Hutchinson et al., 2010; Watkins et al., 2009). Since endomorphin analogs are more similar in structure to endogenous peptides, we hypothesized that they would not be recognized by the immune system as pathogenic. In support of this hypothesis, our laboratory showed activation of microglia in the spinal cord after treatment with morphine, but not endomorphin analogs (Zadina et al., 2012). This may be of particular importance in the treatment of pain in patients already vulnerable to inflammation-induced pathologies, including during older adulthood and after traumatic brain injury (TBI). The focus of this project was to evaluate the cognitive effects of morphine and endomorphin analogs using aging and TBI models. Morphine impaired cognition after both acute and chronic drug administration but EM analogs did not, despite equal or greater duration of antinociception relative to morphine. Chronic studies using young, middle-aged, and old rats revealed that middle-aged rats were more susceptible to cognitive deficits caused by morphine than younger or older rats. Likewise, morphine exacerbated cognitive deficits produced by TBI. Electrophysiology experiments revealed that morphine altered hippocampal long-term potentiation to a greater extent than an EM analog. Immunohistochemistry of the hippocampus indicated that astrocytes are activated after treatment with morphine, but not an EM analog. In TBI studies, morphine treatment led to a reduction in the number of neurons in the dentate gyrus despite an increase in volume, which may be due to increased glial activation. Thus, EM analogs may serve as safer analgesics as indicated by several models, and differential effects of morphine and EM analogs are likely mediated, in part, by alterations in glial activation. / acase@tulane.edu

Rodents rely on spatial learning and memory to efficiently navigate a complex environment. Distinct brain areas mediate different types of spatial learning strategies. The hippocampus-dependent place strategy utilizes spatial cues in the environment to guide the rodent to a goal, while striatum-dependent response and stimulus-response strategies rely on proprioceptive cues or cues proximal to a goal, respectively, to guide the rodent to a goal. The spatial strategy employed to learn a task is influenced by a range of factors including biological sex, age, and anxiety. Previous reports have found that high levels of natural, or trait, anxiety tend to bias prepubertal male rats towards a stimulus-response learning strategy. The effect of reduced transient, or state, anxiety on spatial strategy preference in prepubertal rats is yet to be determined. In the present study, an anxiolytic agent, diazepam, was administered to prepubertal male rats to determine its effect on expression of anxiety-like behaviors on an open field test and spatial learning strategy preference on a visible platform water maze (VPWM) task. Prepubertal male rats treated with a low dose of diazepam (2.5 mg/kg) displayed reduced anxiety in comparison to prepubertal male rats treated with vehicle or a high dose of diazepam (5 mg/kg). A separate sample of prepubertal male rats treated with vehicle displayed a significant preference for a stimulus-response learning strategy as reported previously, while males treated with either a low or high dose of diazepam displayed no spatial learning strategy preference. Taken together, these results support previous findings that higher levels of anxiety bias prepubertal male rats towards a stimulus-response learning strategy, indicating that reduction of anxiety shifts strategy preference away from a stimulus-response learning strategy. / acase@tulane.edu

The blood and lymphatic vascular systems coordinate to play critical roles in tissue fluid homeostasis and immune function and are fundamentally associated with diseases including inflammation, wound healing, edema, and tumor progression and metastasis. Their coordination during vascular growth and remodeling represents an under-investigated area of research in which a better understanding will provide insights into future therapeutic approaches. Angiogenesis and lymphangiogenesis are the growth of new blood or lymphatic vessels, respectively, from pre-existing vessels. The comprehensive goal of this work was to provide a new perspective on the relationships between angiogenesis and lymphangiogenesis in microvascular networks and develop tools needed to probe the mechanisms involved in lymphatic/blood vessel patterning and identity. The first aim of this study was to characterize the spatiotemporal relationships between lymphatic and blood vessel growth in response to an inflammatory stimulus. We found that lymphangiogenesis temporally lagged angiogenesis during inflammation and that the presence of lymphatic vessels attenuated angiogenesis. We also identified increased lymphatic/blood endothelial cells connections and a novel lymphatic marker. These results motivated the need for a system to probe the multicellular and multisystem interactions suggested by these findings. Our lab recently developed the rat mesentery culture model as an ex vivo model for investigating angiogenesis in the context of intact microvascular networks. The second aim was to determine whether this model can be used to study lymphangiogenesis. We found that vascular endothelial growth factor C stimulated lymphatic sprout formation in the rat mesentery culture model and confirmed the ability to observe angiogenesis and lymphangiogenesis simultaneously in an ex vivo environment. This suggests the rat mesentery culture model can be used to investigate the dynamics of lymphatic/blood vessel patterning and plasticity motivated by our in vivo work. The final aim of this work was to investigate the ability to induce phenotypic plasticity in intact vasculature by using lysophosphatidic acid, an agonist suggested to cause blood-to-lymphatic endothelial cell transition. We demonstrated that while lysophosphatidic acid stimulated angiogenesis, it was not sufficient to reprogram blood vessels to acquire a lymphatic phenotype. These results underscore the necessity of investigating these multisystem relationships in the context of intact microvascular networks. These studies as a whole demonstrate the coordination that exists between blood and lymphatic vessels and reinforce the need for novel models that incorporate the complexities of the entire microvasculature. / acase@tulane.edu

There is a clinical need for large numbers of phenotypes which are suitable for tissue engineering and cell therapy applications. Pluripotent stem cells (PSCs) are readily expanded in vitro and can differentiate into any somatic phenotype, making them a potential cell source. However, generating clinically-relevant numbers of phenotypes requires culture in stir-based bioreactor systems which expose cells to shear stress. Here we use a parallel plate bioreactor as a surrogate model system to better understand the effects of shear stress on pluripotent embryonic stem cells (ESCs). Initial studies examined the impact of cell deformation by shear stress during early ESC differentiation. Shear-treatment regulated specification into the three germ lineages and promoted mesodermal differentiation. Next we examined the response to shear stress during later specification events. The application of shear stress was found to promote mesodermal differentiation towards both definitive hematopoietic and mature endothelial phenotypes, although delayed applications were less effective at promoting hematopoietic specification. The next studies used low oxygen treatment to study the impact of another differentiation cue in the presence and absence of shear stress. Hypoxia promoted mesodermal phenotypes but the response was highly dependent on the physical microenvironment such as the culture method and the presence of shear stress. The next group of studies examined the impact of shear stress on ESC expansion. ESCs expanded under flow conditions maintained pluripotency but mesodermal specification was regulated in a manner that was dependent on the presence or absence of a ROCK inhibitor. The final studies used small molecule inhibitors to determine the role of specific signaling molecules during the shear-mediated differentiation of ESCs. Although inhibition of ROCK had little effect, inhibition c-SRC, JNK, or ERK modulated the shear-response. These studies highlight the important effects of shear stress during PSC culture and increase the basic science understanding of stem cell regulation by the physical microenvironment. The systematic approach to analyze multiple parameters allows for an improved translation of techniques from the bench-top into large-scale bioprocessing systems. Altogether these studies can inform large-scale differentiation techniques and bioreactor design in order to help establish the cell banks needed for clinical applications. / acase@tulane.edu

acase@tulane.edu

Parent-child conversations in science museums may support children's interest and understanding of science. Researchers have been investigating programs to optimize parental guidance and deepen families' scientific exploration in museums. Inquiry is an approach to science teaching science that mirrors the scientific method, allowing participants to raise questions about a scientific phenomenon and conduct investigations to answer those questions. In this study, an experimental design was used to test the impact of participation in an inquiry-based activity on families' conversations at six science museum exhibits. Half of the families participated in an inquiry activity before exploring exhibits, and half of the families participated in an inquiry activity after exploring exhibits. Two sets of variables found to be important contributors to parent-child conversation at science museums (i.e., exhibit qualities and individual characteristics) were considered. Results indicated that participation in a guided inquiry activity significantly increased parents' high-quality learning talk, specifically providing explanations and making connections to prior experience. Exhibit qualities and individual characteristics are important considerations for designing and implementing an inquiry intervention. Families talked about the inquiry activity after the visit and applied what they had learned to real-life situations at home. Inquiry activities have the potential to empower parents with tools to help them scaffold children's emerging scientific knowledge during shared activity in a science museum. Implications and future directions are discussed. / acase@tulane.edu

Understanding angiogenesis increases comprehension of pathological conditions such as tumor growth and peripheral artery disease. Angiogenesis is the growth of new vessels from pre-existing vessels and commonly associated with two modes: capillary sprouting and capillary splitting. Past observations in our laboratory provide evidence of vascular islands in the adult microcirculation. Vascular islands are defined as endothelial cell segments disconnected from nearby networks. The two objectives of this specific aim were to (1) determine if vascular islands are involved in angiogenesis during microvascular network growth, and (2) determine whether vascular islands associated with microvascular regression are involved in microvascular remodeling. Mesenteric tissues were harvested from adult male Wistar rats according to the experimental groups: unstimulated, post stimulation (3, 10 and 70 days), and 70 days post stimulation + restimulation (3 and 10 days). Stimulation was induced by mast cell degranulation via intraperitoneal injections of compound 48/80. Tissues were immunolabeled for PECAM (endothelial cells), NG2 (pericytes), collagen IV (basement membrane), and BrdU (proliferation). On day 3, the percentage of islands with at least one BrdU-positive cell increased compared to the unstimulated level and was equal to the percentage of capillary sprouts with at least one BrdU-positive cell. At day 10, the number of vascular islands per vascular area dramatically decreased compared to unstimulated and day 3 levels. Data collected independently for both aims showed that percent vascular area per tissue area and length density increased by day 10 post stimulation compared to the unstimulated group. At day 70, vascular area and length density were then decreased, indicating vascular regression compared to the day 10 levels. During regression at day 70, the number of islands increased. The disconnected endothelial cells were commonly bridged to surrounding networks by collagen IV labeling. NG2-positive pericytes were observed both along the islands and the collagen IV tracks. At 3 days post restimulation, vascular islands contained BrdU-positive cells. By day 10 post restimulation, when vascular area and length density were again increased, and the number of vascular islands was dramatically reduced. The result of this study suggest that (i) segment proliferation correlates with sprouting and network remodeling, (ii) blood vessel segments could provide a novel mode of endothelial cell presence in angiogenesis, (iii) blood vessel segments may be a reserve to connect with existing vasculature, and (iv) vascular islands originating during microvascular regression are capable of undergoing proliferation and incorporation into nearby networks during network regrowth. / acase@tulane.edu

The project examines hypermasculine attitudes and depressive symptoms in emerging adult males. Recent research has suggested that although males have historically reported lower rates of depressive symptoms than females (Boticello, 2009), emerging adulthood may be a time when males are at an increased risk of developing depressive symptoms due to fear of failing to fulfill traditional masculine roles (e.g., breadwinner) and failure to achieve intimate romantic partnerships as a result (Oliffe et al., 2010). Some males may attempt to cope with these negative feelings by adopting maladaptive and exaggerated hypermasculine attitudes. Hypermasculine attitudes are associated with a variety of negative outcomes including violence toward women and substance abuse (Mosher & Sirkin, 1984). Substance use is also associated with depressive symptoms as a form of self-medication (Joiner et al., 1992) and masculinity in the college social context (Iwamoto et al., 2011). Hypermasculinity was originally conceptualized as a personality trait, but more recent research has examined it as a reactive coping strategy (Cunningham & Meunier, 2004). Furthermore, there is also evidence that hypermasculine attitudes may be more multidimensional and that different profiles of hypermasculine attitudes may be associated with different behavioral and psychological outcomes (Burke, Burkhart, & Sikorski, 2004). 328 males ages 18-25 who attend college completed the survey. The results do not support the hypothesized profile of hypermasculine attitudes. However, analysis of demographic characteristics did yield one large homogeneous cluster (n =213) for whom hypermasculine attitudes may be serving as a reactive coping strategy for depressive symptoms, and another large heterogeneous cluster (n = 115) for whom hypermasculine attitudes may not be serving as a coping strategy for depressive symptoms. / acase@tulane.edu

Objectives. Experimental stroke studies have shown that endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms exhibit opposite effects on brain injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to identify the nNOS in brain microvascular endothelial cells (BMECs) and characterize its functional role. Methods and Results. Primary BMECs from humans (hBMECs) and rats (rBMECs) were used in our studies. Immunocytochemistry identified von Willebrand factor, eNOS, and nNOS in hBMECs. Western blot analysis using antibodies targeting N-terminal domain of nNOS revealed an approximately 130 kD immunoband of a potential nNOS splice variant in hBMECs as opposed to 160 kD nNOS specific immunoband in cultured rat cortical neurons. In contrast, antibodies targeting C-terminal domain of nNOS failed to show nNOS specific immunoband. PCR experiments using the species specific primers identified the mRNA of nNOS in hBMECs. Electron Spin Resonance (ESR) spectroscopy revealed reduction of superoxide levels in hBMECs by two structurally different nNOS inhibitors, (N-ω-Propyl-L-arginine; NPA and ARL-17477), compared with vehicle (ethanol) treated cells. In contrast, treatment with eNOS inhibitor (L-N5-(1-Iminoethyl) ornithine; NIO) significantly enhanced the superoxide levels in hBMECs compared with vehicle (DMSO) treated cells. Supplementation of tetrahydrobiopterin (BH4) resulted in reduced superoxide levels in the hBMECs whereas BH4 co-treatment had no effect on the superoxide levels in the NPA or ARL-17477 treated hBMECs. NO measurements in hBMECs by ESR spectroscopy showed greatly diminished magnitude of the NO signal by the treatment with NIO, compared to vehicle (DMSO) treatment, and whereas, treatment with NPA enhanced NO signal intensity compared to vehicle (ethanol) treated cells. Conclusions. We identified a constitutively active nNOS splice variant in hBMECs that is distinct from the nNOS expressed in neurons. In addition, we found that uncoupled nNOS, significantly contributes to basal superoxide generation in hBMECs that reduces the NO bioavailability. In contrast, eNOS is the only source of NO produced by the hBMECs. We conclude that hBMECs express a unique nNOS isoform distinct from the nNOS expressed in neurons and also exhibiting effects which are distinctly opposite of eNOS. / acase@tulane.edu