Synapses are specialized points of contact between neurons allowing for rapid transfer of signals in electrical or chemical form. Synaptic transmission and plasticity are integral to information processing and memory functions in any nervous system, regardless of its level of complexity. Chemical synapses can be broadly classified as excitatory or inhibitory, depending on how their activity affects the membrane potential of the postsynaptic neuron. The majority of excitatory synapses are made onto small protrusions in dendrite membrane termed dendritic spines, while inhibitory synaptic terminals contact smooth membrane of dendritic shafts and cell bodies. The initial process of synapse formation, or synaptogenesis, occurs early in animal development and in humans as well as in mice it takes place postnatally over an extended period of time. Many neurodevelopmental as well as neurodegenerative disorders involve synapse and spine abnormalities, suggesting that undisturbed synaptogenesis is important to development of a healthy brain. Astrocytes and microglia are non-neuronal cells which play supportive and protective roles and thus shape the environment within the nervous system. Although their function is critical to proper neuronal operation, under pathological conditions their activities can have deleterious effects on neurons and their synapses. The purpose of the presented research was to elucidate the potentially detrimental influence of glial activation on developing synapses. Using the lipopolysaccharide model of systemic inflammation we induced glial inflammatory response in mice at several points of postnatal development. Hippocampus, one of the best described brain structures was used as the region of interest. We observed varying levels of microglial activation depending on the age of the animal and similar levels of astrocyte reactivity at all ages. Morphometric analysis of dendritic spines identified a period of vulnerability, manifested as a decrease in spine density in response to inflammation. The density of presynaptic excitatory terminals was similarly affected. When the systemic inflammation was extended from 24h to 8 days, the negative effects on the excitatory terminals were more pronounced and suggested a reduced excitatory drive. The improvement of seizure outcomes confirmed this hypothesis. We also investigated synaptic development in the mouse model of Nasu-Hakola disease, a genetic neurodegenerative disorder characterized by dementia and microglial activation. We found that the mice failed to develop normal levels of excitatory presynaptic terminals, while exhibiting reduced susceptibility to seizures. Furthermore, inducing a systemic inflammation in these mice resulted in a decrease in inhibitory terminal density and higher seizure susceptibility. The results of this study demonstrate a period in postnatal development with elevated sensitivity to immune inflammatory responses. Using a mouse disease model we confirmed the impact of inflammation on synaptic development.

Fragile X Syndrome (FXS) is a neurodevelopmental disorder and the leading known genetic cause of autism spectrum disorder (ASD). Approximately 96% of individuals with ASD and FXS experience improper processing of sensory stimuli. Possible mechanisms attributed to this hyperarousal state include altered excitatory and inhibitory balance and impaired synaptic development. How these developmental changes impair sensory processing, neural responses and functions remain unclear. The major goal of my dissertation project is to identify the developmental mechanisms underlying the pathophysiology of FXS. Our previous studies have shown that elevated levels of Matrix Metalloproteinase-9 (MMP-9) contribute to the hyper-responsiveness of auditory cortex in Fmr1 KO mice by affecting perineuronal net (PNN) formation around parvalbumin (PV)-expressing inhibitory interneurons. Abnormal development of PV neurons most likely contributes to abnormal electroencephalography (EEG)-based phenotypes of auditory hypersensitivity in the Fmr1 KO mice that are remarkably similar to those seen in humans with FXS. However, how the expression of Fmr1 in different cell types shapes normal cortical responses during circuit development is not known.Therefore, I investigated whether the disruption of communication between excitatory neurons and inhibitory PV cells was sufficient to trigger abnormal phenotypes using several mouse models. In the first part of this study, cell-specific deletion of Fmr1 was achieved in cortical excitatory neurons during early embryonic development. I demonstrated that embryonic deletion of Fmr1 from cortical excitatory neurons did indeed trigger PV cell loss, abnormal cortical responses, and behavioral phenotypes in the auditory cortex of Fmr1 KO mice. In the second part of this study, I examined whether conditional deletion (cOFF) or re-expression (cON) of Fmr1 in excitatory neurons during the critical postnatal day (P)14-P21 period of PV cell development is sufficient to trigger or prevent abnormal phenotypes. Our results indicate that postnatal deletion or re-expression of FMRP in excitatory neurons is sufficient to elicit or ameliorate structural and functional cortical deficits as well as abnormal behavioral phenotypes in mice, informing future gene re-expression studies about appropriate treatment window and providing a new insight into the mechanism of cortical circuit dysfunctions in FXS. Lastly, with the discovery of FMRP in astrocytes, coupled with a role of astrocytes in synaptic function and inhibition in particular, it is possible that astrocytes contribute, in some capacity, to the impaired inhibition and circuit hyperexcitability seen in FXS. Therefore, in the third part of this study, I aimed to determine whether astrocyte-specific deletion of Fmr1 during critical developmental period of inhibitory circuit maturation would alter GABAergic signaling and PV cell development leading to cortical hyperexcitability and behavioral alterations. Our results demonstrate a profound role of astrocytic FMRP in the development of inhibitory circuits and shaping normal inhibitory responses.

The hippocampus is a region of the brain required for associative memory formation, and hippocampal neurons retain a high level of synaptic plasticity in the adult that is thought to contribute to life-long learning in adults. Astrocytes play a critical role in synaptic plasticity and maintenance in the adult hippocampus and astrocyte dysfunctions are implicated in neurodevelopmental disorders associated with impaired learning and memory. In addition, many astrocyte-secreted factors are found to promote synaptogenesis and synaptic maturation, the close proximity of astrocytic processes to synapses suggests contact mediated factors contribute to synapse dynamics. This research investigates the role of astrocytic ephrin-B1 regulation in CA1 hippocampal synapses during memory formation. EphB receptors are expressed in both presynaptic CA1 and postsynaptic CA1 neurons, and trans-synaptic Eph/ephrin interactions have been shown to promote synaptic maturation by the clustering and recruitment of synaptic NMDA and AMPA receptors. These studies indicate that astrocytic ephrin-B1 mediates the elimination of immature hippocampal synapses possibly through competitive binding

with neuronal EphB receptors triggering the trans-phagocytosis of synapses inhibiting memory formation. Ablation of astrocytic ephrin-B1 increased the number of immature glutamatergic synapses in the SR of the CA1 hippocampus. In contrast, overexpression of ephrin-B1 decreased synapse number and remaining synapses were more mature than tdTomato expressing WT mice. Excess immature synapses in KO mice contributed to enhanced contextual recall by activity dependent maturation. KO mice had a significant increase in vGlut1/PSD95 co-localization after fear conditioning and deficits in synaptic AMPAR found in naïve mice were no longer seen after fear conditioning. In addition, OE mice had deficits in contextual recall. Diminished recall may be attributed to a reduction in synaptic connection after fear conditioning, as OE of astrocytic ephrin-B1 also inhibited activity dependent dendritic spine formation. Activity dependent AMPAR recruitment is also not observed in OE mice possibly due to a lack of immature synapses available for maturation prior to fear conditioning. In vitro studies suggest astrocytes eliminate synapses by trans-phagocytosis induced by neuronal EphB stimulated activation of ephrin-B1 reverse signaling.

Fragile X Syndrome (FXS) is a leading inherited cause of autism and intellectual disability. FXS occurs as a result Fmr1 gene hypermethylation, which leads to inactivation and loss of Fragile X Mental Retardation Protein (FMRP). Structural and functional studies of humans with FXS indicate auditory cortical deficits. Most notable is hypersensitivity to sounds. The Fmr1 knockout (KO) mouse is a well characterized animal model for FXS which also shows signs of auditory hypersensitivity, including increased acoustic startle and susceptibility to audiogenic seizures. In vivo electrophysiology data obtained from KO mouse primary auditory cortex (A1) indicate that neurons are hyperexcitable when presented with sounds and exhibit altered spectrotemporal processing. Fmr1 KO mice also exhibit EEG measures of abnormal network function, including baseline and stimulus-evoked cortical hyperexcitability. However, the developmental trajectory of these phenotypes has not been studied. Moreover, the molecular mechanisms underlying this altered excitatory/inhibitory balance are unknown. In this study, we characterized developmental changes in perineuronal net expression around GABAergic interneurons, neuronal response magnitudes, and network excitability in Fmr1 KO auditory cortex. In developing Fmr1 KO auditory cortex, there is a transient reduction in perineuronal net expression around GABAergic interneurons which coincides with single neuron hyperexcitability. In addition, there is increased oscillatory gamma during rest. These findings suggest that FXS is associated with developmental impairments in cortical processing. Future therapeutics may be more beneficial if administered during a developmental critical period.

Although once thought of as simply supportive cells of the brain, astrocytes are now understood to be active participants in maintaining neuronal homeostasis through their glial-neuronal and glial-glial interactions. Indeed, astrocytes are exquisitely equipped to sense and respond to their environment, allowing them to perform a diverse array of essential activities such as ion and neurotransmitter homeostasis, and regulation of synaptogenesis, synapse elimination, and synapse plasticity. Developing and maintaining the proper number of synapses as well as an appropriate balance of excitatory and inhibitory synapses is critical for proper brain function. Indeed, excitatory-inhibitory (E/I) imbalance and impaired inhibition are hypothesized to underlie the development of hyperactive neuronal networks, driving the pathological phenotypes and behavioral impairments observed in neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD) and epilepsy. Dysfunctions in parvalbumin (PV) expressing inhibitory interneurons including reduced numbers and hypofunction of PV interneurons have been observed in several NDDs, prompting investigation into the mechanisms involved in regulating the formation, maintenance, and function of PV connections with excitatory pyramidal cells (PCs). The work presented in this thesis investigates the role of astrocytic ephrin-B1 in glial-neuronal interactions, with a particular focus on the development of inhibitory PV->PC synapses and glial-glial interactions, investigating the role of astrocytic ephrin-B1 in astrocyte-oligodendrocyte communications. Ephrin-B/EphB trans-synaptic signaling at excitatory synapses is well studied and has been shown to be critical for regulating excitatory synapse development and maturation, however the role of ephrin-B/EphB signaling in inhibitory synapses is less clear. Astrocytic ephrin-B1 was previously demonstrated to negatively regulate excitatory synapse development and positively regulate inhibitory synapse development, however the mechanism behind this regulation was unclear. My studies delineate a mechanism in which astrocytic ephrin-B1 facilitates the development of PV->PC inhibitory synapses through regulating the expression of its EphB receptor in PV boutons. My work also identifies a previously undescribed negative regulatory role of trans-synaptic EphB2/ephrin-B signaling at PV->PC inhibitory synapses. My work demonstrates that EphB2 expression in PV interneurons disrupts PV->PC inhibitory synapse development and that astrocytic ephrin-B1 positively regulates PV->PC synapse formation by interfering with EphB receptor signaling in PV boutons. Disruption of this regulatory mechanism by removal of astrocytic ephrin-B1 during development led to impaired inhibition in vivo as evidenced by increased seizure susceptibility and repetitive behaviors. My work also investigated astrocyte-oligodendrocyte interactions during development and suggests that astrocytic ephrin-B/EphB receptor signaling positively regulates oligodendrocyte development and myelination. Loss of astrocytic ephrin-B1 during development impaired oligodendrocyte and myelin gene expression, leading to impaired myelination and reduced numbers of oligodendrocytes in the corpus callosum, as well as the presence of a clasping phenotype. Together, these studies implicate astrocytic ephrin-B1 as an important regulator of several neurodevelopmental processes, including inhibitory PV->PC synaptogenesis and oligodendrocyte development.

Fragile X syndrome (FXS) is the most common single-gene inherited form of intellectual disability, with behaviors characteristic of autism. FXS is caused by silencing of the Fragile X mental retardation gene (FMR1). Individuals with FXS display childhood seizures, hyperactivity, anxiety, developmental delay, attention deficits, and visual-spatial memory impairment, as well as repetitive and perseverative behavior. Many of these behavioral deficits are also associated with mice that lack the Fmr1 gene (Fmr1^-/-). Furthermore, Fmr1^-/- neurons, similar to the human condition, exhibit defects in dendritic spine maturation which may underlie the cognitive and behavioral abnormalities in FXS. Dendritic spines are small protrusions on the surface of the dendrite that receive the majority of excitatory synapses in the brain and changes in their morphology affect synaptic efficacy. Much research has focused on treatment options to improve the deficits associated with FXS to ultimately improve the quality of life for the individuals afflicted with this syndrome. The basis of my research examined how minocycline improved behavioral deficits, such as a reduction in hyperactivity, anxiety, obsessive compulsive tendencies and susceptibility to audiogenic seizures as well as promoted dendritic spine maturation in the Fmr1^-/-. Minocycline is a semi-synthetic antibiotic that also has many non-microbial targets, such as inhibiting the enzyme matrix metalloproteinase-9 (MMP-9). MMP-9 has been implicated in many neurological disorders, including multiple sclerosis, cerebral ischemia and mild cognitive impairment. The concurrent focus of my project was on the involvement of MMP-9 in FXS. We discovered that active MMP-9 promoted an immature dendritic spine profile in cultured hippocampal neurons, and that MMP-9 expression and activity were upregulated within the hippocampus of the Fmr1^-/-. To further expound upon the role of MMP-9, we also analyzed MMP-9's expression levels within the blood and brain of individuals with FXS and found a decrease in relative activity in the blood, but an increase in total expression within postmortem hippocampal and neocortical tissue. Overall, this study elucidated a novel mechanism for MMP-9 that appears to play a causative role in the pathology of FXS as well as provided a new therapeutic option for FXS treatment.

Neurons communicate with each other through synaptic connections. During development, it is essential that synapses undergo structural and functional modification. Improper development of neural connections can lead to neurodevelopmental disorders such as autism and intellectual disabilities. The synapse development process is comprised of three major steps: synaptogenesis, synapse pruning, and synapse stabilization. This refined process involves several key players, including astrocytes that come into close association with synapses, forming a tripartite complex with the pre- and postsynaptic structures. This association allows astrocytes to monitor and alter synaptic functions. Astrocytes communicate with synapses through either secreted factors or contact-mediated interactions. Release of specific astrocyte-derived gliotransmitters can affect both the structure and function of neurons. Besides affecting synaptogenesis and function, astrocytes are also involved in pruning of unnecessary synapses through contact-mediated phagocytosis; however, the exact “eat me” signal is still not known. My research investigates the role of astrocytic ephrin-B1 regulation of hippocampal synapses during early postnatal development, adulthood, and learning and memory consolidation. Ephrin-B1 is a membrane bound protein that acts as a ligand for EphB receptors, allowing for bi-directional signaling through cell-cell interactions. Astrocyte-neuronal interactions may allow astrocytes expressing ephrin-B1 to find and engulf synapses that are marked for removal by targeting unoccupied EphB receptors. Therefore, hippocampal circuitry may be modulated through the Eph/ephrin interaction between neuron and astrocytes. My studies indicate that astrocytic ephrin-B1 is a negative regulator of synapse formation. During early postnatal development, astrocytic ephrin-B1 is essential for proper excitatory/inhibitory (E/I) circuit formation, as loss of astrocytic ephrin-B1 resulted in enhanced excitatory function of CA1 pyramidal cells and diminished inhibitory function. Dysregulation of E/I balance impaired sociability and increased repetitive behaviors. In contrast, in adulthood astrocytic ephrin-B1 maintains synapse numbers. Ablation of astrocytic ephrin-B1 in adulthood resulted in increased excitatory synaptogenesis, particularly of immature synapses. Overabundance of immature synapses reduced CA1 pyramidal cell excitatory function. Interestingly, astrocytic ephrin-B1 functions in an activity dependent manner, specifically modulating synapse formation on activated neurons during learning and memory consolidation and recall. Together, these results suggest that astrocytic ephrin-B1 influences hippocampal circuits by restricting synapse formation.

Dendritic spines are the post-synaptic sites of most excitatory synapses in the brain, and changes in their morphology are implicated in synaptic plasticity and long-term memory. F-actin dynamics are thought to be a basis for both the formation of dendritic spines during development and their structural plasticity (Ethell and Pasquale, 2005; Pontrello and Ethell, 2009). We have shown that the F-actin-severing protein cofilin, which is regulated by phosphorylation, can induce remodeling of mature dendritic spines in hippocampal neurons (Shi et al., 2009). We also demonstrate that β-Arrestins play an important role in spatial control over cofilin activity in dendritic spines, which underlies NMDA-mediated dendritic spine remodeling. Cofilin activity in dendritic spines is regulated through CaMKII-mediated suppression of cofilin activity by phosphorylation, and calcineurin-dependent cofilin activation through its dephosphorylation. In addition, while the EphB receptor promotes spine stabilization through cofilin inactivation, the NMDA receptor prevents EphB-mediated cofilin inactivation through calcineurin. NMDAR activation also promotes the translocation of cofilin to dendritic spines, an event that requires cofilin dephosphorylation and is also dependent on β-Arrestins, which have recently been shown to scaffold cofilin with its regulators, LIM kinase and slingshot phosphatase (Zoudilova et al., 2010). Our studies demonstrate that cofilin clustering in the spines is affected in both β-Arrestin1- and β-Arrestin2- deficient neurons under normal synaptic activity, and a constitutively-active cofilin^S3A mutant fails to translocate to spines in response to NMDA in β-Arrestin2 KO neurons. Moreover, while wt neurons display dendritic spine remodeling in response to NMDA or with over-expression of cofilin^S3A, β-Arrestin2-deficient neurons are resistant to both NMDA-induced and cofilin^S3A-induced spine remodeling. In contrast, dominant-negative cofilin^S3D prevents NMDA-induced dendritic spine remodeling in wt neurons, and also rescues a mature spine phenotype that is lost in β-Arrestin1 KO neurons. In addition, over-expression of β-Arrestin in the KO neurons rescues spine abnormalities. β-Arrestin1-deficient neurons also develop immature spines in vivo, whereas hippocampal neurons lacking β-Arrestin2 develop normal mature spines, but fail to remodel in response to NMDA. Our studies demonstrate novel functions of β-Arrestin1 in the development of mature dendritic spines, and β-Arrestin2 in NMDAR-mediated dendritic spine plasticity through spatial control over cofilin activity.

Fragile X syndrome (FXS), the most common single gene cause of inherited intellectual disability, is caused by a trinucleotide CGG repeat expansion in the 5' untranslated region of the Fragile X Mental Retardation gene (FMR1), which results in promoter hypermethylation and gene silencing. This loss or reduction of the gene product expression, Fragile X Mental Retardation Protein (FMRP), results in the translational dysregulation of specific target mRNAs. Patients with FXS and Fragile X mental retardation gene knockout (ko) mice, an animal model for FXS, exhibit defects in dendritic spine maturation that may underlie cognitive and behavioral abnormalities in FXS, which tend to be at the extreme of the autistic spectrum. Dendritic spines are small protrusions on the surface of dendrites that receive the majority of excitatory synapses in the brain, and changes in their morphology affect synaptic strength. Previous studies have shown that minocycline promotes dendritic spine maturation in primary cultures of hippocampal neurons and in the developing hippocampus of Fmr1 ko mice, which is accompanied by improvements in behavioral performance. Here I confirm the phenotypes previously reported for the Fmr1 ko mouse and investigate a possible link between matrix metalloproteinase-9 (MMP-9) and FMRP by characterizing double Fmr1/Mmp9 ko mice. I show that the deletion of mmp-9 in Fmr1 ko mice rescues the spine development both in vitro and in vivo, and that this deletion also returns mGluR-dependent LTD to the normal levels in Fmr1 ko mice. LTP however remains affected. I also investigated if Fmr1 ko mice exhibit certain behavioral problems, and whether a deletion of mmp-9 can ameliorate these behavioral deficits. The Fmr1 ko mice exhibit increased anxiety and hyperactivity in the open field and defects in social novelty discrimination, most of which are also improved by the removal of mmp-9. Further, I also examined the role of MMP-9 activity in physical traits that are observed in FXS, such as macroorchidism and found that those physical traits are also ameliorated by the genetic removal of mmp-9. Additionally, previous studies have shown the rates of basal protein synthesis to be higher in Fmr1 ko mice. I confirm this and show that the deletion of mmp-9 reduces both the basal levels of protein synthesis and the levels of intracellular signaling as the levels of phosphorylation of Akt, mTOR and eIF4e are reduced to wild type (wt) levels. The levels of MMP-9 and MMP-2 are also upregulated in the hippocampi of the Fmr1 ko mice and since there no differences in the mRNA levels of these two transcripts between Fmr1 ko and wt animals, this upregulation must be due to a translational dysregulation following the loss of FMRP. The fact that most of the FXS phenotypes are reversed by the genetic removal of mmp-9, strongly suggests that increased MMP-9 protein levels underlie these phenotypes and validates the use of specific MMP-9 inhibitors as possible therapeutic avenues for FXS.