Changes in the number, size, and shape of dendritic spines are associated with synaptic plasticity, which underlies cognitive functions such as learning and memory. This plasticity is attributed to reorganization of actin, but the molecular signals that regulate this process are poorly understood. In this study, we show that neural Wiskott-Aldrich syndrome protein (N-WASP) regulates the formation of dendritic spines and synapses in hippocampal neurons. N-WASP localized to spines and active, functional synapses as shown by loading with FM4-64 dye. Knockdown of endogenous N-WASP expression by RNAi or inhibition of its activity by treatment with a specific inhibitor, wiskostatin, caused a significant decrease in the number of spines and excitatory synapses. Deletion of the C-terminal VCA region of N-WASP, which binds and activates the Arp2/3 complex, dramatically decreased the number of spines and synapses, suggesting that activation of the Arp2/3 complex is critical for spine and synapse formation. Consistent with this, Arp3, like N-WASP, was enriched in spines and excitatory synapses and knockdown of Arp3 expression impaired spine and synapse formation. A similar defect in spine and synapse formation was observed when expression of an N-WASP activator, Cdc42, was knocked down. Thus, activation of N-WASP and subsequently the Arp2/3 complex appears to be an important molecular signal for regulating spines and synapses. Arp2/3 mediated branching of actin could be a mechanism by which dendritic spine heads enlarge and subsequently mature. Collectively, our results point to a critical role for N-WASP and the Arp2/3 complex in spine and synapse formation.

The primate visual system is highly interconnected and hierarchically organized. Three separate pathways, originating in the layers of the lateral geniculate nucleus (LGN), carry retinal information to primary visual cortex (V1) which, in turn, sends information into the ventral and dorsal streams. These higher areas also send massive feedback to V1. The contribution of these feedforward and feedback inputs to the functional organization of V1 remains unclear. The broad aim of this thesis is to address this question. The specific aims were as follows: 1) To obtain a more complete picture of V1 functional maps by characterizing the distribution of temporal frequency selectivity in bush baby V1. 2) To tease out the contributions of magno- (M) and parvocellular (P) feedforward LGN inputs to the functional organization of V1. 3) To investigate the functional role of feedback from middle temporal area (MT) to V1. As part of the latter aim, the retinotopic organization of MT was investigated. We first demonstrated that, unlike spatial frequency and orientation, temporal frequency selectivity is not organized into domains but is distributed uniformly across V1. Inactivating either P or M channels reduced activity in V1. Blocking the P input left the map of orientation preference unchanged but altered spatial frequency map. M block resulted in greater reduction in activity than P block, suggesting that in bush baby, the M pathway may play a major role in the function of V1. Next, we found that MT has both a local and global retinotopic organization in owl monkeys. The cardinal axes of visual space are represented anisotropically. As part of a collaborative effort, we also showed that MT depends on V1 for its main drive. Additionally, we showed that feedback from MT influences the overall activity and the temporal frequency responses in V1. This thesis provided new information about the organization of primate V1 by showing that functional maps in V1 depend on contributions of both feedforward LGN channels and feedback from MT.

The neuron-specific electroneutral potassium (K+) and chloride (Cl-) cotransporter, KCC2, is a key regulator of neuronal Cl-. KCC2 has been shown to play a critical role in controlling neuronal excitability, yet little is known about its regulation. Protein-protein interactions are generally well known to provide insight into membrane transporter regulation. Therefore, I have chosen to identify novel KCC2 protein-protein interactions, with the goal of studying how these interactions affect KCC2 activity. I performed a yeast-two hybrid screen of a mouse brain cDNA library and identified several novel binding partners of the carboxyl terminus of KCC2 (KCC2-CT). One of these identified binding partners is Protein Associated with Myc, or PAM. Binding between KCC2 and the RCC1 domain of PAM (RCC1/PAM) was demonstrated using yeast two-hybrid, GST-pull-down assay, and coimmunoprecipitation. In order to study the functional role of PAM binding to KCC2, I identified the binding site of RCC1/PAM on the KCC2-CT, and within this site created a point mutant which disrupts RCC1/PAM binding. This point mutation was then transferred into full-length KCC2 and compared with wild-type KCC2 in 86Rb+/K+ uptake experiments to assess differences in transport activity. These flux experiments, along with experiments to measure changes in RNA and protein levels, lead to the conclusion that PAM binding to KCC2 does not primarily affect K-Cl transport by altering cell-surface expression of the cotransporter. Rather, PAM binding to the carboxyl terminus of KCC2 likely participates in the net dephosphorylation of KCC2, which in turn leads to activation of KCC2-mediated ion transport.

Neuroscience Dissertation under the direction of Professor Randy D. Blakely This dissertation is focused on the function and regulation of the choline transporter (CHT). I used CHT heterozygous and homozygous mice, and combined biochemical, pharmacological, and behavioral approaches to test the hypothesis that CHT is essential for acetylcholine (ACh) turnover and behaviors dependent on cholinergic signaling in vivo. My work demonstrates that CHT homozygous knockout mice are born with wild type levels of ACh, but are unable to sustain ACh synthesis and release under high demand for ACh turnover. CHT heterozygosity, on the other hand, results in diminished pools of ACh. Consequenly, focused behavioral and pharmacological challenges reveal that CHT heterozygous mice are vulnerable to sustained demands on cholinergically-supported behaviors. In summary, my thesis work provides new insights into the role of CHT in the maintenance of ACh turnover and cholinergic neurotransmission. The CHT heterozygous mice constitute a new animal model of cholinergic dysfunction, and offer opportunities for further studies of CHT function and regulation, with the eventual goal of manipulating CHT in disorders of cholinergic origin. Approved: Professor Randy D. Blakely, May 2008

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) may provide a novel approach that could be useful in the treatment of certain central nervous system disorders. However, their physiological effects and pharmacological properties, such as sites of action, are not fully understood. It has been suggested that two distinct mGluR5 PAMs, CDPPB and CPPHA, might act by binding to two distinct allosteric sites. We synthesized a series of CDPPB analogs and reported that these compounds bind to an allosteric antagonist (MPEP) site with affinities that are closely related to their potencies as mGluR5 PAMs. Furthermore, their allosteric potentiation is antagonized by a neutral ligand at the MPEP site called 5MPEP and reduced by a mutation that eliminates MPEP binding. Additionally, we have also reported that CPPHA potentiates mGluR5 responses by a mechanism that is distinct from that of VU-29, a CDPPB analog. CPPHA-induced potentiation of mGluR5 responses is also blocked by 5MPEP. However, 5MPEP and MPEP inhibit CPPHA in a non competitive manner compared with its competitive inhibition of the effect of VU-29. Consistent with this, the mutation that eliminates the effect of VU-29 has no effect on the response to CPPHA. Conversely, a distinct mutation that eliminates the effect of CPPHA does not alter the response to VU-29. Together, these data suggest that CPPHA and VU-29 act at two distinct sites. mGluR5 has been implicated as playing an important role in hippocampal synaptic plasticity. However, the precise roles of mGluR5 in different forms of hippocampal synaptic plasticity remain unclear. We found VU-29 significantly enhanced threshold theta burst stimulation (TBS)-induced long-term potentiation (LTP), which was completely blocked by 5MPEP and was mimicked by a structurally distinct mGluR5-selective PAM. In addition, VU-29 potentiated LTP was blocked by the NMDA receptor antagonist or src kinase inhibitor, suggesting that it shares a common mechanism with suprathreshold TBS-induced LTP. These raise the possibility that mGluR5 PAMs could be used as potential cognition enhancing agents.

Mutations in the human K-Cl cotransporter-3 (KCC3) gene lead to a severe neurological disorder called peripheral neuropathy associated with agenesis of the corpus callosum (ACCPN). At the same time as that discovery, the KCC3 knockout mouse was created by our laboratory and was found to drag its hind limbs. My dissertation research focused on the KCC3 knockout mouse as a model for the disease. First, through behavioral characterization of the KCC3 knockout mouse, I show that it is a relevant model of ACCPN. Then, I concentrated on the peripheral neuropathy component of KCC3 loss. In order to assess the role of KCC3 in peripheral axon and/or myelin development and maintenance, I determined its expression in wild-type sciatic nerves. This work is the first to show that sciatic nerves do express KCC3. It is expressed in sciatic nerves of juvenile, but not adult, wild-type mice, specifically by Schwann cells. I performed a detailed morphometric analysis of sciatic nerves at different ages to determine the underlying pathophysiology of the peripheral neuropathy and answer whether the lack of KCC3 leads to a developmental or neurodegenerative disorder. In the knockout, Schwann cell and myelin development appears normal at P3, but axons are swollen. At P8 and P30, abnormal periaxonal swelling occurs in some myelinated fibers. These initial swelling pathologies are followed by Wallerian-like degeneration in adult KCC3 knockout nerves. To test whether these changes in the adult mouse lead to neurophysiologial deficits, I assessed nerve conduction velocity and pain sensitivity in wild-type and knockout mice. Mutant mice exhibit a reduction in nerve conduction velocity and sensitivity to noxious pain. The evidence I show here for fluid-related axonopathy, which ultimately results in neurodegeneration, implicates cell volume regulation, likely in conjunction with potassium buffering, as a critical component of peripheral nerve maintenance.

The claustrum is a telencephalic grey matter nucleus of unclear structure and function, although it has been recently proposed to be a generator of conscious percepts. Our studies redefine the structural boundaries and connections of the claustrum in the rat, as well as provide evidence for an equivalent redefinition of claustral anatomy in all therian mammal brains, including human. An analysis of cortical representations in the claustrum are also presented and the findings are considered in a functional context. Finally, G protein gamma-2 is identified as a novel and discrete neuroanatomical marker of the claustrum whose expression pattern is in register with the our anatomical definitions. The results of this work alter long-held tenets of claustral anatomy and allow for an assessment of claustral function through molecular lesioning techniques targeting G protein gamma-2.

Frontoparietal cortex, consisting of motor and early somatosensory areas, is the first cortical region to receive inputs from the spinal cord and the last to convey outputs to the spinal cord. This dissertation investigated the functioning of frontoparietal cortex and the spinal cord in three parts. First, the organization of motor cortex in marmosets, which has not previously been examined, was investigated. The physiologic and architectonic results suggest that marmoset motor cortex is organized similarly to that of other primates. Second, the organization of somatosensory cortex after a lesion of the dorsal columns of the spinal cord was investigated in marmosets. The physiologic, behavioral, and architectonic results suggest that marmoset somatosensory cortex is capable of cortical plasticity similar to that of other New World Primates, and that the behavioral deficit and the extent of cortical reorganization may be linked. Third, the cellular composition of the spinal cord was compared across several primate species and suggests that in larger brains, disproportionately more neurons are added to the cortex and cerebellum than to the rest of the brain or the spinal cord.

RNA transcripts encoding the 2C-subtype of the serotonin (5HT2C) receptor are modified by adenosine-to-inosine editing events to generate as many as twenty-four 5HT2C receptor protein isoforms. These modified receptors are expressed in a region-specific manner in the central nervous system (CNS) and demonstrate differences in their constitutive activity and efficacy to interact with specific G-proteins. To determine the physiologic relevance of 5HT2C RNA editing, I generated mutant mice solely expressing the non-edited isoform (5HT2C-INI) of the receptor. Heterozygous mutant dams display profound behavioral deficits in maternal care including poor nest formation and altered pup retrieval that affect growth and anxiety-related behavior in both wild-type and mutant offspring. Treatment with a selective 5HT2C inverse agonist rescues deficits in pup retrieval, indicating that altered 5HT2C signaling underlies the observed alterations in maternal behavior. These studies not only indicate a role for 5HT2C signaling in maternal care, but also demonstrate the importance of normal patterns of 5HT2C RNA editing in vivo.

In the spinal cord, most oligodendrocytes, the myelinating cell type of the CNS, and motor neurons arise from common, ventral, olig2-expressing precursors called pMN cells. In the hindbrain, the degree to which motor neurons and oligodendrocytes share common origins and, consequently, are specified by similar molecular mechanisms is not clear. This ambiguity results, at least in part, from the fact that both oligodendrocyte progenitor cells (OPCs) and some motor neurons migrate, obscuring whether these cells have common or distinct origins. Therefore, we used zebrafish as a model system to investigate hindbrain motor neuron and oligodendrocyte development. We found, using cell specific markers, transgenic reporter lines and time-lapse microscopy, that a subset of OPCs and abducens motor neurons, which control eye movement, arise from common olig2-expressing precursors in rhombomeres (r) 5 and 6. To investigate the role of olig2, we reduced its function using translation-blocking morpholino oligonucleotides. This produced deficits of OPCs and abducens motor neurons, but not other motor neuron populations. However, we also found that facial motor neurons failed to complete their migration from r4 into r6 and r7. This raises the possibility that the olig2+ precursors provide a positional cue necessary for facial motor neuron migration. A better understanding of proper OPC and neuronal specification and migration in the hindbrain will bring increased insight to motor neuron disorders such as Mobius Syndrome and Duane Syndrome, which affect the facial and abducens motor neurons, respectively, as well as demyelinating disorders, such as Multiple Sclerosis.