The non-receptor protein tyrosine kinase Src is expressed throughout the central nervous system and is involved in diverse biological functions like cell growth, differentiation, and postsynaptic signalling. Despite the well-documented functions of Src in hippocampal synaptic plasticity, roles in social behaviours, motor function, cognition, and synaptic signalling in other brain regions remain largely untested. This work investigates the neurocellular and behavioural effects of in-vivo inhibition and disinhibition of the Src tyrosine kinase pathway in mice. To this end, we employed a cell permeant Src inhibitory peptide, mutant mice harbouring a loss-of-function point mutation in Src, and a mutant mouse with deficits in the inhibitory C-terminal Src kinase (Csk). Alterations in Src signalling were associated with profound changes in NMDA receptor signalling, synaptic plasticity, motor function, cued fear conditioning, and a variety of social behaviours, underscoring the ubiquity and importance of Src signalling in the mammalian central nervous system. Blocking the interaction of the Src tyrosine kinase with the NMDAR complex impaired auditory conditioned fear memory and social recognition. Inhibition of Src-NMDAR interactions also attenuated NR2B phosphorylation and decreased NR2B surface expression in the amygdala. Furthermore, at the lateral to basolateral nucleus pathway (LA-BLA), inhibition of Src impaired long-term potentiation. Mice harbouring a Src point mutation (Src(thl/thl) mice) exhibited behavioural abnormalities and growth retardation. We also observed differences in behaviour phenotypes analogous to mouse models of Williams-Beuren syndrome (WBS) and humans with WBS . WBS is neurodevelopmental disorder characterized by distinctive facial features, hypersociability, mild to moderate mental retardation, and a unique cognitive disability (Meyer-Lindenberg et al., 2006). Sociability and social vocalization were increased in three different social affiliation tasks in Src(thl/thl) mice. Mutant mice exhibited hyperactivity in the open field and spent significantly less time in the centre of the open field. Also, motor function was impaired in three different motor performance tasks. The Src(thl/thl) mice showed an enhanced startle response to loud stimuli, impaired cued fear conditioning, and deficient visiospatial memory in the Morris water maze. Furthermore, Src(thl/thl) mice were not able to learn a visual object recognition task. These results underscore the importance of Src in an array of behavioural, motor, and cognitive functions in mice. The C-terminal Src kinase (Csk) acts to suppress Src activity, so Csk(+/-) mice were employed to examine the behavioural impact of enhanced Src signalling. Decreased Csk expression led to enhanced long-term and short-term social olfactory recognition and social transmission of food preference. We also found elevated NR2B phosphorylation in the olfactory bulb and amygdala, two brain regions critical for the behavioural expression of anxiety and social recognition in mice. Deficiencies in the Src tyrosine kinase pathway were associated with impaired synaptic plasticity in the amygdala and behaviour disturbances that are relevant to WBS. Conversely, up-regulation of the Src tyrosine kinase pathway by reducing Csk expression increased social olfactory cognition. A more detailed understanding of the Src pathway could facilitate the development of new treatments for diseases characterized by aberrant social behaviours.

The non-receptor protein tyrosine kinase Src is expressed throughout the central nervous system and is involved in diverse biological functions like cell growth, differentiation, and postsynaptic signalling. Despite the well-documented functions of Src in hippocampal synaptic plasticity, roles in social behaviours, motor function, cognition, and synaptic signalling in other brain regions remain largely untested. This work investigates the neurocellular and behavioural effects of in-vivo inhibition and disinhibition of the Src tyrosine kinase pathway in mice. To this end, we employed a cell permeant Src inhibitory peptide, mutant mice harbouring a loss-of-function point mutation in Src, and a mutant mouse with deficits in the inhibitory C-terminal Src kinase (Csk). Alterations in Src signalling were associated with profound changes in NMDA receptor signalling, synaptic plasticity, motor function, cued fear conditioning, and a variety of social behaviours, underscoring the ubiquity and importance of Src signalling in the mammalian central nervous system. Blocking the interaction of the Src tyrosine kinase with the NMDAR complex impaired auditory conditioned fear memory and social recognition. Inhibition of Src-NMDAR interactions also attenuated NR2B phosphorylation and decreased NR2B surface expression in the amygdala. Furthermore, at the lateral to basolateral nucleus pathway (LA-BLA), inhibition of Src impaired long-term potentiation. Mice harbouring a Src point mutation (Src(thl/thl) mice) exhibited behavioural abnormalities and growth retardation. We also observed differences in behaviour phenotypes analogous to mouse models of Williams-Beuren syndrome (WBS) and humans with WBS . WBS is neurodevelopmental disorder characterized by distinctive facial features, hypersociability, mild to moderate mental retardation, and a unique cognitive disability (Meyer-Lindenberg et al., 2006). Sociability and social vocalization were increased in three different social affiliation tasks in Src(thl/thl) mice. Mutant mice exhibited hyperactivity in the open field and spent significantly less time in the centre of the open field. Also, motor function was impaired in three different motor performance tasks. The Src(thl/thl) mice showed an enhanced startle response to loud stimuli, impaired cued fear conditioning, and deficient visiospatial memory in the Morris water maze. Furthermore, Src(thl/thl) mice were not able to learn a visual object recognition task. These results underscore the importance of Src in an array of behavioural, motor, and cognitive functions in mice. The C-terminal Src kinase (Csk) acts to suppress Src activity, so Csk(+/-) mice were employed to examine the behavioural impact of enhanced Src signalling. Decreased Csk expression led to enhanced long-term and short-term social olfactory recognition and social transmission of food preference. We also found elevated NR2B phosphorylation in the olfactory bulb and amygdala, two brain regions critical for the behavioural expression of anxiety and social recognition in mice. Deficiencies in the Src tyrosine kinase pathway were associated with impaired synaptic plasticity in the amygdala and behaviour disturbances that are relevant to WBS. Conversely, up-regulation of the Src tyrosine kinase pathway by reducing Csk expression increased social olfactory cognition. A more detailed understanding of the Src pathway could facilitate the development of new treatments for diseases characterized by aberrant social behaviours.

The role of hypocretin neurons in modulating somatic motoneuron excitability and hence muscle tone is poorly understood. We investigated whether hypocretin neurons influence the hypoglossal and trigeminal motor pools that innervate the genioglossus and masseter muscles respectively, both of which function to maintain upper airway patency. We hypothesized hypocretin neurons facilitate motor outflow at the motor pools. We pharmacologically manipulated hypocretin neuron activity in anaesthetized mice to determine their role in somatic motoneuron excitability. We also antagonized hypocretin receptors in the hypoglossal motor pool to determine the pathway through which hypocretin neurons influence motoneuron excitability. We demonstrated that hypocretin neurons potently excite somatic motoneurons and hence facilitate genioglossus and masseter muscle tone. Furthermore, we demonstrated that an endogenous hypocretinergic drive on somatic motoneurons facilitated muscle tone under anaesthesia. These studies demonstrate that hypocretin is an excitatory neuromodulator of muscle tone and contributes to the excitatory regulation of somatic motoneurons.

NMDA-type glutamate receptors (NMDAR) regulate neurotransmission and excitotoxicity. NMDAR signaling is believed to be dependent on NR2 subunit (A-D) composition and interactions with intracellular proteins. To determine the role of individual NR2 subunits in NMDAR signaling, I examined the biochemical interactions and colocalization of NR2A and NR2B NMDAR subunits with PSD-95 and CaMKII. Immunofluorescent colocalization revealed that by perturbing PSD-95 PDZ interactions using a targeted peptide (TAT-NR2B9c) increased association of PSD-95 with NR2A and CaMKII with NR2B; furthermore, decreases in association of CaMKII with NR2A and PSD-95 with NR2B were observed. The effects of TAT-NR2B9c were dependent upon NMDAR stimulation with 10μM NMDA and were not observed in untreated cells or at toxic doses of NMDA (40 μM). Thus, disrupting PSD-95 PDZ interactions produced activity-dependent differences in the co-localization of NR2A and NR2B with key signaling proteins, providing evidence that individual NR2 subunits may confer differential signaling to NMDARs.

The role of hypocretin neurons in modulating somatic motoneuron excitability and hence muscle tone is poorly understood. We investigated whether hypocretin neurons influence the hypoglossal and trigeminal motor pools that innervate the genioglossus and masseter muscles respectively, both of which function to maintain upper airway patency. We hypothesized hypocretin neurons facilitate motor outflow at the motor pools. We pharmacologically manipulated hypocretin neuron activity in anaesthetized mice to determine their role in somatic motoneuron excitability. We also antagonized hypocretin receptors in the hypoglossal motor pool to determine the pathway through which hypocretin neurons influence motoneuron excitability. We demonstrated that hypocretin neurons potently excite somatic motoneurons and hence facilitate genioglossus and masseter muscle tone. Furthermore, we demonstrated that an endogenous hypocretinergic drive on somatic motoneurons facilitated muscle tone under anaesthesia. These studies demonstrate that hypocretin is an excitatory neuromodulator of muscle tone and contributes to the excitatory regulation of somatic motoneurons.

NMDA-type glutamate receptors (NMDAR) regulate neurotransmission and excitotoxicity. NMDAR signaling is believed to be dependent on NR2 subunit (A-D) composition and interactions with intracellular proteins. To determine the role of individual NR2 subunits in NMDAR signaling, I examined the biochemical interactions and colocalization of NR2A and NR2B NMDAR subunits with PSD-95 and CaMKII. Immunofluorescent colocalization revealed that by perturbing PSD-95 PDZ interactions using a targeted peptide (TAT-NR2B9c) increased association of PSD-95 with NR2A and CaMKII with NR2B; furthermore, decreases in association of CaMKII with NR2A and PSD-95 with NR2B were observed. The effects of TAT-NR2B9c were dependent upon NMDAR stimulation with 10μM NMDA and were not observed in untreated cells or at toxic doses of NMDA (40 μM). Thus, disrupting PSD-95 PDZ interactions produced activity-dependent differences in the co-localization of NR2A and NR2B with key signaling proteins, providing evidence that individual NR2 subunits may confer differential signaling to NMDARs.

The neuronal transmembrane proteins Neto1 and Neto2, along with different protein partners, perform multiple roles in diverse processes including axon guidance in the developing nervous system and synaptic plasticity in the adult brain. My project focuses on identifying the membrane bound interacting partners of Neto2 using Membrane Yeast Two Hybrid (MYTH). By performing MYTH screens for the Neto2 molecule using human adult and embryonic whole brain cDNA libraries, I have identified several novel membrane bound putative interacting partners including, VAMP associated protein B (VAPB) and Glutamate transporter EAAT3-associated protein (GTRAP3-18), which play diverse functions during the glutamatergic neurotransmission. Initial studies to validate these interactions in vivo are currently underway by co-immunoprecipitation approach using mouse brain tissue. If these two candidate proteins are confirmed to be true interactors, it will open important avenues of research for the Neto2 protein during excitatory neurotransmission in the mammalian central nervous system.

The neuronal transmembrane proteins Neto1 and Neto2, along with different protein partners, perform multiple roles in diverse processes including axon guidance in the developing nervous system and synaptic plasticity in the adult brain. My project focuses on identifying the membrane bound interacting partners of Neto2 using Membrane Yeast Two Hybrid (MYTH). By performing MYTH screens for the Neto2 molecule using human adult and embryonic whole brain cDNA libraries, I have identified several novel membrane bound putative interacting partners including, VAMP associated protein B (VAPB) and Glutamate transporter EAAT3-associated protein (GTRAP3-18), which play diverse functions during the glutamatergic neurotransmission. Initial studies to validate these interactions in vivo are currently underway by co-immunoprecipitation approach using mouse brain tissue. If these two candidate proteins are confirmed to be true interactors, it will open important avenues of research for the Neto2 protein during excitatory neurotransmission in the mammalian central nervous system.

Neural function depends on precise wiring of axon during development. Previous studies have demonstrated that the erythropoietin producing hepatocellular carcinoma (Eph) family of tyrosine receptor kinases is crucial for the proper development of a number of neural circuits in the mammalian central nervous system (CNS). Mice lacking Eph receptors have been shown to exhibit deficits in pathways which include the thalamocortical, callosal, retinal and corticospinal tract. Due to the large number of Eph family members, the relative contribution of each receptor to axon pathfinding and neural function remains elusive. In this thesis, I have addressed the function of EphA4, EphB2 and EphB3 in the regulating the formation of interhemispheric projections within the forebrain and motor axon connections within the spinal cord using EphA4, EphB2 and EphB3and combinatorial null mice. To perform a detailed examination of the process of axon guidance regulated by these receptors within the forebrain, high resolution magnetic resonance imaging (MRI), immunofluorescence and in vivo stereotactic fluorescent labeling were performed. This work resulted in the development and validation of MRI-based analytic tools performed using EphB2 mutants which we have previously shown to exhibit specific morphologic defects in the anterior commissure (AC). Analysis of EphA4 null mice using high resolution MRI revealed for the first time that in addition to errors of midline crossing, loss of EphA4 activity results in a positional reorganization of the rostral AC. Results demonstrate that while EphB2 and A4 each regulate distinct aspects of guidance within ACpp, these receptors also operate cooperatively to control the guidance of axons in the pars anterior of the AC, a pathway not been previously implicated in Eph-mediated guidance. With respect to the spinal cord, mice deficient in EphB2 and EphA4 display prominent axon guidance errors in the medial subsets of the lateral motor column (LMCm); neurons which normally innervate ventral limb musculature. Finally, I have addressed the functional effect which Eph mutants exhibit with respect to motor behavior by examining a detailed set of motor coordination parameters

The chemical synapse is an asymmetric structure consisting of presynaptic and postsynaptic terminals in direct apposition to each other. Synapses function to mediate the transmission of signals between neurons and their targets. The formation of synapses is a tightly regulated process requiring the interaction of many genes and molecular pathways. I am interested in identifying genes and signaling pathways that are required for proper synapse formation. Using the GABAergic neuromuscular junctions of C. elegans as a model system, I have identified fsn-1 (F-box synaptic protein), a gene required for the control of synaptic growth. fsn-1 mutants exhibit a synaptic defect characterized by both synaptic over differentiation and under differentiation. FSN-1 is an F-box protein with a SPRY (SPla and RYanodine receptor) domain that functions cell-autonomously in neurons to regulate synaptic growth. I have shown that it functions in an E3 ubiquitin ligase-like complex with the RING-H2 finger protein RPM-1 (Regulator of presynaptic morphology), SKR-1 and Cullin. The composition of this complex is similar to SCF (Skp1, Cullin, F-box) E3 ubiquitin ligases. We hypothesize that this complex controls synapse formation by down regulating synapse promoting factors through an ubiquitin mediated process. We have identified two receptor tyrosine kinases that genetically interact with fsn-1, the Anaplastic Lymphoma Kinase homolog SCD-2 (Suppressor of Constitutive Dauer) and the C. elegans insulin receptor DAF-2 (abnormal DAuer Formation). Loss of function mutations in scd-2 or daf-2 partially suppress the synaptic differentiation defects of fsn-1 mutants, suggesting that they participate in signaling pathways whose activities are normally inhibited by FSN-1 during synapse formation. Unlike FSN-1 that functions in GABAergic neurons, I found that SCD-2 and DAF-2 have cell non-autonomous functions at GABAergic neuromuscular junctions. SCD-2 is required in the nervous system in the RID interneuron where it could modulate synapse formation through ligands present on the motoneuron cell surface. The DAF-2/insulin pathway functions in postsynaptic muscle cells to regulate FSN-1 dependent presynaptic growth likely through a retrograde or feedback mechanism. I propose a model where FSN-1 regulates synapse formation by attenuating signals that converge upon the presynaptic terminal to stimulate or inhibit synaptic growth.