Although neural plasticity has traditionally been studied within the brain, evidence indicates that the spinal cord is quite plastic as well. Spinal neurons can even support a simple form of instrumental learning (Grau et al., 1998), as indicated by spinally transected rats’ ability to exhibit an increase in hind limb flexion duration when limb extension is associated with shock (controllable shock). If limb extension is not associated with shock (uncontrollable shock), a learning deficit develops. Recent research indicates that other forms of plasticity, such as long-term potentiation and central sensitization, do not depend on neural activity alone, but also on glial cells. I examined whether glial cells are also necessary in spinal instrumental learning and the learning deficit. Therefore, two glial inhibitors were selected: minocycline and fluorocitrate. To examine the role of glial cells in spinal instrumental learning, rats received intrathecal minocycline, fluorocitrate, or saline prior to testing with 30-minutes of controllable leg-shock. Results indicate that both drugs dose-dependently reduced acquisition, with higher doses resulting in shorter response durations. Once the response was acquired, fluorocitrate did not alter response maintenance. This suggests that glial cells are involved in the acquisition, but not the maintenance, of spinal learning. To examine the role of glial cells in the spinal learning deficit rats were given intrathecal minocycline, fluorocitrate, or saline prior to testing with 6-minutes of uncontrollable tail shock or no shock. Twenty-four hours later all rats were tested with 30-minutes of controllable leg-shock. Results indicated the learning deficit induced by uncontrollable shock was prevented by prior administration of fluorocitrate. Minocycline did not prevent the deficit; moreover, it appears that even in the absence of shock, minocycline caused a learning deficit. Overall, this data indicate that glial cells are necessary for the acquisition of spinal instrumental learning and the learning deficit. Furthermore, it provides further evidence for the role of glial cells in plasticity.

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs.

In vertebrate and invertebrate systems, the role of glia-neuron interactions during development and behavior is becoming apparent. Recent studies have been aimed at characterizing glial-expressed proteins that affect the modulation of activities traditionally thought to be regulated by the neuron itself. The soil nematode Caenorhabditis elegans has recently emerged as an important invertebrate model to study glial roles in nervous system function and development. My dissertation work focuses on the characterization of HLH-17, a C. elegans basic helix-loop-helix transcription factor that is strongly and constitutively expressed in the glial cells that associate with four of the cephalic (CEP) neurons in the head of the animal. The CEP neurons are four of eight dopaminergic neurons with well characterized roles in the modulation of a number of behavioral activities in the worm. Although HLH-17 is required for neither the specification nor the development of the CEPsh glia or the CEP neurons, it does have a defined role during dopamine responses. We show that HLH-17 functions upstream of the dopamine receptors DOP-1, DOP-3 and the dopamine transporter DAT-1 to affect DA-dependent behaviors. Also, our microarray analyses provide preliminary evidence that HLH-17 targets factors responsible for receiving and transducing signaling molecules that are involved in the modulation of synaptic events in the worm nervous system. Together these results point to a role for HLH-17 in glia-neuron interactions in C. elegans. My dissertation studies therefore provide further support for the role of glial-expressed proteins in the regulation of activities mediated by the nervous system.

Taste is detected by cells of taste buds in the oral cavity. Mammalian taste buds contain three types of cells: receptor, presynaptic, and glial-like. Of these three, glial-like cells are the least studied. Their only known function is that they clear neurotransmitters from the extracellular space. The present work describes two previously undocumented properties of glial-like cells. First, Oxytocin receptor (OXTR) mRNA was detected by RT-PCR in taste tissue of mice. In the taste buds of Oxtr-YFP knockin mice, YFP was seen in glial-like taste cells and other cells immediately outside the taste bud, but no other cells in oral epithelium. Oxytocin (OXT) elicited Ca2+ responses from cells that resemble glial-like taste cells (by criteria including gene expression and lack of excitability). The EC50 for OXT in these cells was 33 nM, and responses saturated at 1 µM. 500 nM L-371,257 (an OXTR antagonist) significantly inhihited the responses to OXT. In a semi-intact preparation of lingual slices, OXT did not alter bitter tastant-evoked Ca2+ responses. Further, in behavioral studies, OXT (10 mg/kg i.p.) did not alter the responses of mice to aversive salty (NaCl), bitter (quinine), or sour (citric acid) solutions. In contrast, OXT (0.1 mg/kg i.p.) significantly decreased taste behavioral responses to low-to-intermediate concentrations of sucrose. My data suggest that OXT may modulate sweet taste sensitivity in vivo by acting on glial-like cells in taste buds. Second, Renal Outer Medullary K channel (ROMK) mRNA was also detected by RT-PCR in taste buds . Immunostaining revealed that ROMK is localized to the apical tips of glial-like taste cells. In the kidney, ROMK, apically localized in nephron epithelium facilitates a unidirectional flow (i.e. excretion) of K+. I suggest that, analogous to glia in the central nervous system, glial-like taste cells homeostatically redistribute extracellular [K+ ] within taste buds to maintain their sensitivity. The results of this study reveal that glial-like taste cells resemble nervous system glia in more ways than simply clearing neurotransmitters. They may also modulate the sensory output of the taste bud and buffer the extracellular [K+]. A more active role for glial-like cells in the functioning of the taste bud should be investigated.

Ulm, Univ., Diss., 2001.

Microglia, the innate immune cells of the brain, are not only immune surveyors, but also play important roles in neural development and maintenance. Microglial aberrations, including changes in morphology, gene expression, and phagocytic activity, have been observed in humans and animal models of pathologies involving cognitive and behavioral consequences. However, the precise contribution of microglial biology is not well characterized. Expression profiling of microglia and neural stem cells, co-culture assays, and transgenic mice were used to identify microglial micro-RNAs and genes, and study their roles in neural development. The results show that a specific micro-RNA, miR-155, participates in the neurogenic deficits induced by inflammation, and microglia-derived Wnt5a is essential for neural differentiation and maturation. This indicates the potential involvement of abnormal microglia in neurodevelopmental disorders such as autism spectrum disorders (ASDs). ASDs are group of debilitating disorders characterized by behavioral symptoms, including social and communication deficits and repetitive or restricted behaviors. I hypothesize that aberrant microglial biology plays a role in neurogenic and behavioral deficits in a mouse model of ASD. I performed a time-course study of microglial gene expression profiling, neural and microglial morphology, neurophysiology, and behavior in the maternal immune activation (MIA) model of ASD induced by the innate immunity ligand polyinosinic:polycytidylic acid. Microglia in MIA offspring displayed altered expression of 22 genes including 14 involved in cell-cell interaction, increased complexity of branching, and increased interactions with dendritic spines of cortical layer V pyramidal neurons. Microglial abnormalities were associated with neurophysiological alterations, measured by whole-cell patch clamp recordings, increased neuronal spine density, and ASD-like behaviors. MIA offspring treated with a colony stimulating factor -1 receptor inhibitor, to deplete and replenish microglia, showed correction of specific behaviors, microglial gene expression and branching, microglia-spine interactions, and spine density, and partial correction of neurophysiology. The data presented here shed new insight into the functional effects of microglia gene and microRNA expression in neurodevelopment. Furthermore, inflammation induces microglial aberrations that lead to altered neurodevelopment; this strongly supports the idea that targeting specific microglial genes and miRNAs will be a worthwhile approach to pursue for molecular intervention in ASD and related disorders. / 2018-11-02T00:00:00Z

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs. / Medicine, Faculty of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / Transforming growth factor β-activated kinase 1 (TAK1), a hub kinase at the convergence of multiple signaling pathways, is critical to the development of the central nervous system and has been found to play a role in cell death and apoptosis. TAK1 may have the potential to elucidate mechanisms of cell cycle and neurodegeneration. The Belecky-Adams laboratory has aimed to study TAK1 and its potential roles in cell cycle by studying its role in chick retinal development as well as its possible implication in the progression of diabetic retinopathy (DR). Chapter 3 includes studies that explore TAK1 in a study in chick retinal development and TAK1 in in vitro studies in retinal microglia. Using the embryonic chick, immunohistochemistry for the activated form of TAK1 (pTAK1) showed localization of pTAK1 in differentiated and progenitor cells of the retina. Using an inhibitor or TAK1 activite, (5Z)-7-Oxozeaenol, in chick eye development showed an increase in progenitor cells and a decrease in differentiated cells. This study in chick suggests TAK1 may be a critical player in the regulation of the cell cycle during retinal development. Results from experimentation in chick led to studying the potential role of TAK1 in inflammation and neurodegeneration. TAK1 has previously been implicated in cell death and apoptosis suggesting that TAK1 may be a critical player in inflammatory pathways. TAK1 has been implicated in the regulation of inflammatory factors in different parts of the CNS but has not yet been studied specifically in retina or in specific retinal cells. Chapter 2 includes studies from the Belecky-Adams laboratory of in vitro work with retinal microglia. Retinal microglia were treated with activators and the translocation to the nucleus of a downstream factor of TAK1 was determined: NF-kB. Treatment of retinal microglia in the presence of activators with TAKinib, an inhibitor of TAK1 activation, revealed that TAK1 inhibition reduces the activation of downstream NF-kB. Together this data suggests that TAK1 may be implicated in various systems of the body and further studies on its mechanisms may help elucidate potential therapeutic roles of the kinase.

Great strides have been made with regard to neurogenesis in the enteric nervous system (ENS), rapidly following in the wake of recent revelations about the neurogenic properties of the central nervous system (CNS). As the ENS is more exposed, highly complex, and vulnerable to a variety of developmental, acquired, and multisystemic disorders, there is a sense of urgency for studies to address the potential within the gut to restore neurons that are injured or lost. It is the intricacies of the ENS and yet unclear relationships between agonists and embryonic precursors that have made demonstrating the arrival of new neurons in mature gut difficult under steady-state conditions. This thesis demonstrates that inducible models of a wide range of insults to the gut have yielded crucial information about ENS neurogenesis, while in vivo experimentation under steady-state conditions has proven inconsistent. Specifically, the signaling pathways of Ret and PTEN have revealed the existence of a ‘natural block’ that normally regulates neurogenesis and keeps proliferation well controlled. Furthermore, the overwhelming effects of serotonin agonism on neuron density in response to injury have uncovered an essential role of neuronal transdifferentiation by enteric glial cells that extends beyond what was once understood to be a largely homeostatic role. The influence of extrinsic innervation of the gut will also be explored, physiology of which is important for both the utility of gut microbiota and the role of Schwann cell progenitors in the development of the ENS. Therefore, this thesis will outline ENS organization and function, as well as describe common pathologies that serve as the foundation upon which neurogenesis is investigated. Critical inducible models to which chemical and molecular agonists are applied will be examined in detail, as it is through these models that therapeutics and biomedical engineering can be optimized in order to correct the pathophysiology of enteric neuropathies that as of now are still treated with surgical intervention.

Introduction Neurogenesis, the development of new neurons, starts soon after the formation of the neural tube and is largely completed by birth. Development of the brain after birth is mainly reliant on the formation of new connections between surviving neurons. However, adult neurogenesis does continue in the subgranular zone of the hippocampus from quiescent adult neural stem cells. Traditionally neural stem cells were cultured as neurospheres, a heterogeneous agglomeration of neural cells at various stages of differentiation. This heterogeneity prevented accurate quantitative analysis. In 2008 Sun et al produced the first non-immortalised human foetal neural stem (NS) cell line from nine week old human foetal cortex. These cells are cultured as monolayers, have a radial glia like appearance, self renew and form all three neural cell types, neurons, astrocytes and oligodendrocytes upon differentiation. More recently human foetal neuroepithelial like (NES) stem cells have been produced from five week old human foetal hind-brain, they resemble neuroepithelial cells, with characteristic rosettes, upon differentiation they appear to form a pure population of neurons. These homogeneous monolayer cultures enable quantitative proteomic analysis, to increase our understanding of early brain development Methods Three NES and two NS cell lines were available for analysis. They proliferate by stimulation from FGF and EGF, removal of these growth factors results in spontaneous differentiation. Proliferating NES and NS cells were compared using SILAC labelling. In addition, each cell line was differentiated for 12 days, 6 timepoints were taken and compared using label free quantitation. Results 4677 proteins were quantitated with 473 differentially expressed, revealing fundamental differences between NES and NS cells. NES cells are less differentiated, expressing SOX2 and LIN28, have active cell cycle processes, DNA elongation, histone modification and miRNA mediated gene silencing. Whereas NS cells are more developmentally defined, express multiple membrane proteins, have activated focal adhesion, thereby increasing their binding and interaction with their environment. NS metabolism is more oxidative, utilises lipid metabolism, the pentose phosphate pathway and produces creatine phosphate. Upon differentiation the cell cycle processes are downregulated and neurogenic and gliogenic processes increased. Conclusion This work represent a detailed in vitro characterisation of non immortalised human foetal neural stem cells, it describes the regulatory, metabolic and structural changes occurring within neural stem cells in early brain development. The information herein points towards de-differentiation potentially through LIN28-let7, as a means to produce more neurogenic neural stem cells in vitro thus aiding regenerative therapies, as well as provides a wealth of information for better understanding neurological developmental disorders.