Fragile X Syndrome (FXS) is the most common form of inherited mental retardation, affecting approximately 1/4000 males and 1/6000 females worldwide. FXS is caused by loss of FMR1 gene expression, resulting in the lack of the protein product, Fragile X protein (FMRP). FMRP is an RNA-binding protein thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs in neurons. To determine whether FMRP is also required in early brain development we examined the distribution of cell cycle markers in Drosophila FMR1 (dFmr1) mutant brains compared to wild-type brains. Our results indicate that the loss of dFmr1 leads to a significant increase in the number of mitotic neuroblasts and BrdU incorporation in the brain, consistent with the notion that FMRP controls proliferation in neural stem cells. To determine the role of FMRP in neuroblast division and differentiation, we used Mosaic Analysis with a Repressible Marker (MARCM) approaches in the developing larval brain and found that single dFmr1 neuroblasts generate significantly more neurons than controls. Developmental studies suggest that FMRP also inhibits neuroblast exit from quiescence, or reactivation, in early larval brains, as indicated by misexpression of the G1 to S phase transition marker Cyclin E. We have also identified a novel role for FMRP in the glia surrounding the neuroblasts, indicating that FMRP in these cells contributes to the regulation of neuroblast reactivation via signaling from the supporting glial cells. Our results demonstrate that FMRP is required during brain development to control the exit from quiescence and proliferative capacity of neuroblasts as well as neuron production, which may provide insights into Fragile X Syndrome and other Autism-Spectrum disorders.

The neocortex is the most recently evolved part of the mammalian brain which is involved in a repertoire of higher order brain functions, including those that separate humans from other animals. Humans have evolved an expanded neocortex over the course of evolution through a massive increase in neuron number (compared to our close relatives-­‐‑ the chimpanzees) in spite of sharing similar gestation time frames. So what do humans do differently compared to chimpanzees within the same time frame during their development? This dissertation addresses this question by comparing the developmental progression of neurogenesis between humans and chimpanzees using cerebral organoids as the model system. The usage of cerebral organoids, has enabled us to compare the development of both the human neocortex, and the chimpanzee neocortex from the very initiation of the neural phase of embryogenesis until very long periods of time. The results obtained so far suggest that the genetic programs underlying the development of the chimpanzee neocortex and the human neocortex are not very different, but rather the difference lies in the timing of the developmental progression. These results show that the chimpanzee neocortex spends lesser time in its proliferation phase, and allots lesser time to the generation of its neurons than the human neocortex. In more scientific terms, the neurogenic phase of the neocortex is shorter in chimpanzees than it is in humans. This conclusion is supported by (1) an earlier onset of gliogenesis in chimpanzees compared to humans which is indicative of a declining neurogenic phase, (2) an earlier increase in the chimpanzee neurogenic progenitors during development, compared to humans, (3) a higher number of stem cell– like progenitors in human cortices compared to chimpanzees, (4) a decline in neurogenic areas within the chimpanzee cerebral organoids over time compared to human cerebral organoids.

Morphine has been and continues to be one of the most potent and widely used drugs for the treatment of pain. Clinical and animal models investigating sex differences in pain and analgesia demonstrate that morphine is a more potent analgesic in males than in females; indeed, we report the effective dose of morphine for female rats is twice that of male rats. In addition to binding to the neuronal mu opioid receptor, morphine binds to the innate immune receptor toll-like receptor 4 (TLR4) on microglia. Morphine action at TLR4 initiates a neuroinflammatory response and directly opposes morphine analgesia. Our recent studies demonstrate that administration of chronic morphine activates microglia within the ventrolateral periaqueductal gray (vlPAG), a critical brain region for the antinociceptive effects of morphine, while blockade of vlPAG microglia increases morphine analgesia and suppresses the development of tolerance in male rats. Despite increasing evidence of the involvement of microglia in altering morphine efficacy, no studies have examined sex differences in microglia within the PAG. The present experiments seek to characterize the distribution and activity of vlPAG microglia in males and females using behavioral, immunohistochemical and molecular techniques, while demonstrating the sufficiency and necessity of vlPAG microglia to produce sex differences in morphine analgesia using site-specific pharmacological manipulation of TLR4. We also investigate a novel pharmacokinetic mechanism underlying the sexually dimorphic effects of morphine administration on microglial activity. Here, we address a fundamental gap in our current understanding of sex differences in morphine analgesia and establish a mechanistic understanding of how the activation of vlPAG microglia sex-specifically influences morphine analgesia.

Astrocytes are the most abundant non-neuronal cells in vertebrate brains. Although Drosophila melanogaster has fewer astrocytic cells relative to neuronal and other glial cell populations, they, like vertebrate astrocytes, are located in synaptic regions, organized into exclusive, minimally-overlapping domains, and play developmental roles in synaptogenesis. But, do Drosophila astrocytes have parallel roles in the regulation of synaptic signaling? Preliminary electron microscopic (EM) data indicates that astrocytic processes are located at a greater distance, on average, from Drosophila synapses than they are from vertebrate synapses, thus raising questions about their capacity to alter synaptic signals. Do astrocytic cells and processes occupy stereotyped synaptic regions across repeating segmental structures and across individuals? In the studies presented here, we have addressed these questions directly in the ventral nerve cord (VNC) of the third-instar larva. We collected the first whole-cell patch-clamp recordings from Drosophila astrocytes. These indicate that intrinsic membrane properties, such as low membrane resistance, high capacitance, a hyperpolarized resting potential relative to neurons, a passive current-voltage relationship, coupling to other astrocytic cells, and an absence of voltage-gated currents, are shared between astrocytes of highly divergent species. Next, we optogenetically activated of a group of glutamatergic pre-motor neurons and showed that astrocytes respond with a glutamate transporter current that is mediated by Eaat1, and that acute, pharmacological and chronic, genetic blockades of this transporter have subsequent effects on the decay of post-synaptic motor neuron currents. Then, we used three-dimensional EM to locate the pre-motor glutamatergic neurons that were activated in the physiological study and measured the distance from each presynaptic site to the nearest astrocytic process. We found that these distances vary 100-fold even along a single neurite and that these structures are rarely in direct contact, but that no synapse is positioned greater than one micron from an astrocytic process. Thus, it is in this anatomical configuration that the regulation of post-synaptic currents by Eaat1 occurs. Finally, we generated a library of single, fluorescently-labeled astrocytes that were co-labeled with fiduciary landmarks, and used this library to compare the placement of astrocyte cell bodies and arbors across VNC segments and individuals. We found substantial variation in the gross shape, size, and territory covered by astrocytes, and conclude that their neuropil domains are not reliably stereotyped. Given the consistent placement of neuronal connectome elements, this indicates that signals of a specific synapse are not regulated by a designated astrocyte. Together, these findings reveal new functional parallels between Drosophila and vertebrate astrocytes. These findings argue for the relevance and applicability of mechanistic discovery in Drosophila astrocytes, and set the stage for further inquiry into the genetic determinants of astrocyte morphology and physiology.

Morphine is one of the most widely used drugs for the treatment of pain but its clinical efficacy is limited by adverse effects including persistent constipation and colonic inflammation. Morphine-induced colonic inflammation is facilitated by microbial dysbiosis and bacterial translocation. In this study, we demonstrate that secondary inflammation and persistent constipation are modulated by enteric glia. In chronic morphine treated mice (75 mg morphine pellet/5 days), ATP-induced currents were significantly enhanced in enteric glia isolated from the mouse colon myenteric plexus. Chronic morphine resulted in significant disruption of the colonic epithelium and increased Il-1β in the myenteric plexus. The increase in ATP-induced currents, IL-1β expression and ATP release were also observed in isolated glia treated with lipopolysaccharide (LPS) consistent with bacterial translocation as a potential mediator of chronic morphine-induced inflammation. These effects of LPS were reversed by carbenoxolone, a connexin43 hemichannel blocker. In-vivo treatment with carbenoxolone (25 mg/kg) prevented 1) ATP-induced currents in enteric glia, 2)the decrease in neuronal density, and 3) colonic inflammation in chronic morphine treated mice. Inhibition of connexin43 in enteric glia also reversed morphine mediated decrease in gastrointestinal transit. These findings indicate that bacterial translocation-induced enteric glial activation and inflammation is a significant modulator of morphine-related constipation.

Approximately 16 million people in America are diagnosed with Alcohol Use Disorder (AUD) but no efficacious medical treatments exist. Alcohol-related behaviors can be studied in model organisms, and changes in these behaviors can be correlated with either (i) a risk for alcohol dependence or (ii) a symptom/feature of AUD itself. Although AUD is a disease of the central nervous system, a majority of research has focused on the neuronal underpinnings, leaving glial contributions largely undescribed. We used Drosophila melanogaster (fruit fly) to identify genes whose expression in glia regulates alcohol sedation. Mammals and Drosophila have conserved behavioral responses to alcohol and functionally similar adult glial cells, especially astrocytes. Since previous research in mammals and flies has demonstrated that glia respond to alcohol administration, we hypothesized that glia are important regulators of alcohol-related behaviors. To pursue this, we characterized a pan-glial steroid-inducible GeneSwitch transgenic fly, which allows gene manipulation within glia during adulthood. We performed a targeted screen and manipulated genes that were known to be expressed within Drosophila glia and measured their alcohol sedation sensitivity using the ethanol sedation assay. We identified the genes Cysteine proteinase 1 (Cp1) and Tyramine decarboxylase 2 (Tdc2). Knocking down Cp1 in cortex glia, as well as all glia during adulthood, increased alcohol sedation sensitivity and may also enhance rapid tolerance development. We could not identify what pathway Cp1 was functioning within to mediate this response, suggesting that Cp1 may have a unique function within glia. Knockdown or overexpression of Tdc2 in glia increased or decreased alcohol sedation sensitivity, respectively. Tdc2 functions upstream of the vesicular monoamine transporter (VMAT) and the SNARE complex to regulate alcohol sedation. These results were specific to astrocytes, as well as all glia during adulthood. These results suggest that tyramine synthesis via Tdc2 and its release via vesicular exocytosis regulates alcohol sedation. Taken together, these results suggest that glia are important regulators of alcohol-related behaviors in flies. Interestingly, fly cortex glia and astrocytes are functionally similar to mammalian astrocytes, indicating that these results may be translatable to mammals.

Retinal degeneration is characterized by the irreversible loss of photoreceptors. A key research question is the identification and characterization of photoreceptor protective factors that prevent or delay vision loss. The Wnt pathway is a critical cellular communication pathway involved in development and diseases of the central nervous system (CNS). Recently, we discovered that multiple components of the Wnt pathway were differentially expressed in the rd1 mouse model of retinal degeneration. One of the most highly upregulated genes was Dickkopf3 (Dkk3), a secreted Wnt pathway protein of unknown function. Additionally, we demonstrated that Wnt signaling is neuroprotective in primary retinal culture (Yi et al., 2007). These data led to the hypothesis that Dkk3 is a regulator of Wnt-mediated neuroprotection during retinal degeneration. The role of Dkk3 in the retina and its activity in the Wnt pathway was identified in this dissertation project using a series of biochemical, molecular and cell biology methodologies. First, Dkk3 was shown to be expressed and secreted from Muller glia in mouse retinal tissue and primary Muller glia culture. I then demonstrated that Muller glia are a Wnt-responsive cell type and that Dkk3 potentiates Wnt3a-mediated signaling. Interestingly, the latter effect was not observed in other cell types in the retina such as retinal ganglion cells and retinal pigmented epithelial cells. Thus, Dkk3 may act on Muller glia to positively modulate Wnt signaling during retinal degeneration, which could potentially amplify the neuroprotective activity of the Wnt pathway. Next, the role of Dkk3 in cellular viability was explored. HEK293 cells stably expressing Dkk3 were shown to be significantly protected from staurosporine-induced apoptosis compared with vector control. This result suggests that Dkk3 may mediate a direct pro-survival effect onto photoreceptors during retinal degeneration. Protein interaction experiments demonstrated that Dkk3 formed a complex with the single pass transmembrane proteins Krm1 and Krm2 in the membrane, potentially in the endoplasmic reticulum (ER). Furthermore, Wnt signaling luciferase reporter assays demonstrated that Krm2, but not Krm1, abolished Dkk3-mediated Wnt3a potentiation. These data suggest that Dkk3 modulates Wnt signaling by antagonizing Dkk1-Krm dependent Wnt inhibition. Further studies will determine whether this activity is sufficient for the potentiation of Wnt signaling by Dkk3. Lastly, co-immunoprecipitation followed by mass spectrometry analysis was used to identify a novel interacting protein of Dkk3. Dkk3 was shown to interact with glucose response protein 78 (GRP78), an ER-resident chaperone. This suggested that Dkk3 protein is part of the unfolded protein response through GRP78 in the ER. In conclusion, these studies identified two novel functions of Dkk3 in regulating Wnt signaling pathway and cellular viability and suggest a physiological role for Dkk3 and Wnt signaling during retinal degeneration. Future studies will explore the significance of Dkk3-Krm and Dkk3-GRP78 interactions in the retina. Further, elucidation of the regulation of Dkk3 and other Wnt ligands in the ER and the consequence of ER stress on the biological activity of Wnt signaling will provide a better understanding of the role of the Wnt pathway during retinal degeneration.

Research into the mechanisms of hyperalgesia is ongoing with the goal of improving clinical management of chronic pain. One animal model of chronic musculoskeletal pain uses two injections of acidic saline into a lateral gastrocnemius muscle to induce a long-lasting bilateral decrease in paw withdrawal thresholds. This study tested whether the two injections need to occur in the same muscle. Male Sprague-Dawley rats were injected with acidic saline (pH 4.0) in either the lateral or medial head of gastrocnemius or the contralateral gastrocnemius (lateral head). All animals received a second injection in the ipsilateral gastrocnemius (lateral head). Mechanical withdrawal thresholds were reduced in all groups when tested 24 hours after the second injection. Animals in which the first muscle injection was substituted with a non-specific treatment (intraperitoneal injection of lipopolysaccharide) developed bilateral hyperalgesia after a single acidic saline injection. Thus, the mechanism of hyperalgesia in this model is not restricted to the injected tissues and may include central nervous system structures. Consistent with this, an inhibitor of glia cell activation (minocycline) blocked the development of bilateral hyperalgesia. These data indicate that the central nervous system may play a large role in mediating chronic musculoskeletal pain. / October 2007

Research into the mechanisms of hyperalgesia is ongoing with the goal of improving clinical management of chronic pain. One animal model of chronic musculoskeletal pain uses two injections of acidic saline into a lateral gastrocnemius muscle to induce a long-lasting bilateral decrease in paw withdrawal thresholds. This study tested whether the two injections need to occur in the same muscle. Male Sprague-Dawley rats were injected with acidic saline (pH 4.0) in either the lateral or medial head of gastrocnemius or the contralateral gastrocnemius (lateral head). All animals received a second injection in the ipsilateral gastrocnemius (lateral head). Mechanical withdrawal thresholds were reduced in all groups when tested 24 hours after the second injection. Animals in which the first muscle injection was substituted with a non-specific treatment (intraperitoneal injection of lipopolysaccharide) developed bilateral hyperalgesia after a single acidic saline injection. Thus, the mechanism of hyperalgesia in this model is not restricted to the injected tissues and may include central nervous system structures. Consistent with this, an inhibitor of glia cell activation (minocycline) blocked the development of bilateral hyperalgesia. These data indicate that the central nervous system may play a large role in mediating chronic musculoskeletal pain.

Research into the mechanisms of hyperalgesia is ongoing with the goal of improving clinical management of chronic pain. One animal model of chronic musculoskeletal pain uses two injections of acidic saline into a lateral gastrocnemius muscle to induce a long-lasting bilateral decrease in paw withdrawal thresholds. This study tested whether the two injections need to occur in the same muscle. Male Sprague-Dawley rats were injected with acidic saline (pH 4.0) in either the lateral or medial head of gastrocnemius or the contralateral gastrocnemius (lateral head). All animals received a second injection in the ipsilateral gastrocnemius (lateral head). Mechanical withdrawal thresholds were reduced in all groups when tested 24 hours after the second injection. Animals in which the first muscle injection was substituted with a non-specific treatment (intraperitoneal injection of lipopolysaccharide) developed bilateral hyperalgesia after a single acidic saline injection. Thus, the mechanism of hyperalgesia in this model is not restricted to the injected tissues and may include central nervous system structures. Consistent with this, an inhibitor of glia cell activation (minocycline) blocked the development of bilateral hyperalgesia. These data indicate that the central nervous system may play a large role in mediating chronic musculoskeletal pain.