Matthew Dalva, PhD - US grants

DESCRIPTION (provided by applicant): During development, maps of stimuli in the world are established in the cortex. Understanding the role neuronal activity plays in this process has implications for the effects of abnormal sleep, and of toxins and drugs, during pre- and postnatal development in humans. While understanding the underlying cortical circuitry is fundamental to understanding certain neurological pathologies, including mental retardation. Motivated by both experimental and theoretical studies, a prevailing computational model for how maps are formed has been that afferent activity, either spontaneous or environmentally evoked, drives their development. This model assumes that intracortical connections play a subservient role to feedforward ones, that intrinsically generated spontaneous activity does not significantly influence cortical plasticity, and that intraconnections are more or less fixed during development. However, recent data undermine all of these assumptions: Modeling studies have shown that maps can arise without patterned feedforward connections. Electrophysiological records of spontaneous activity during initial map formation reveal correlations that may arise from intraconnections patterned by molecular cues. And, photo-stimulation experiments suggest that extensive changes occur in intraconnections during subsequent map development. To test the hypothesis that intrinsic spontaneous activity reinforces pre-existing, rudimentary cortical circuits during early map formation, and reinforces changes in cortical maps during subsequent periods of heightened plasticity, this study will: (1) Characterize activity patterns in sleep and wake during initial map formation and subsequent map remodeling in vivo. (2) Map the extent of excitatory and inhibitory intraconnections in vitro. (3) Develop computational models that reproduce the patterns of cortical activity imaged in vivo based on the patterns of lateral connectivity mapped in vitro, and that predict the time-course of map formation and interactions between sleep and wake during map remodeling

Formation and plasticity of synapses are essential for normal functioning of the brain and key events for learning and adaptive plasticity. Diseases such as addiction, epilepsy, and Alzheimer's that involve maladaptive plasticity appear to highjack these same mechanisms controlling these events leading to disease states. The area of addition is particular pressing given the devastating impact the disease has on society and the clear like between addictive behaviors and the formation of new synapses and/or maladaptive plastic changes in the brain. Therefore, understanding the mechanisms that regulate normal develop and plasticity in the brain are likely to be critical for any advances in treatment of these diseases. The majority of synaptic contacts that form are made on dendritic spines, which are also a key site of synaptic plasticity. Dendritic spines contain specialized structures called postsynaptic densities (PSDs) that are directly apposed to pre-synaptic neurotransmitter release sites and which scale in size with changes in synaptic strength. Despite having understood this relationship for many years, the molecular dynamics of the translocation and accumulation of PSD proteins and presynaptic proteins following structural plasticity remain poorly understood. Our preliminary data indicate that pre- and postsynaptic proteins for scale in a modular fashion with dendritic spine size. We will determine the synaptic molecular architecture and address how the molecular architecture of the spine synapse responds to structural plasticity in three aims: 1) Determine the nanoarchitecture of glutamate receptors at spine synapses. 2) Determine the nanoscale organization of synchronous and asynchronous synaptic release sites. 3) Determine how PSD-95 nanomodule number and plasticity are regulated. Collectively these studies will advance our understand of basic mechanisms that impact the ability of the nervous system to grow and change, events that are likely central to disease of maladaptive plasticity such as addiction and Alzheimer's.

DESCRIPTION (provided by applicant): During the past decade stunning advances have been made in imaging, molecular biology and biochemistry that enable the visualization of the behavior of single proteins in vivo. Here, I propose to develop and visualize the temporal and spatial dynamics of intracellular signaling within living neurons. Much as early work in calcium imaging redefined our understanding of the importance of calcium influx by defining its spatial and temporal characteristics, I believe visualizing the spatial and temporal dynamics of intracellular signaling will have similar benefits to our understanding of the nervous system. Initially we have developed indicators that enable visualization of one of the key first steps in many intracellular signaling cascades: tyrosine phosphorylation. During the past several years, we have developed a system that relies on ratiometric imaging of changes in a genetically encoded fluorescent indicator of phosphorylation. We now propose three specific aims to develop these tools into a system for monitoring signaling during neuronal plasticity and development. We propose to: 1) Develop a library of indicators targeted to report activity of specific kinases;2) Develop indicators that localize to specific cellular compartments;3) Develop indicators to report activity of multiple signaling molecules simultaneously. Using our indicators, workers will be able to elucidate the dynamics of signals that underlie synaptic plasticity. Thus, our tools will enable novel insights into essential mechanisms that underlie neuronal plasticity. PUBLIC HEALTH RELEVANCE: Project Narrative Neuronal plasticity underlies many fundamental functions within the brain, while abnormal neuronal plasticity is associated with disease. Excessive plasticity may underlie diseases like epilepsy and addiction, while defects in plasticity could play important roles in epilepsy, neurodegenerative, and autism spectrum disorders. Our research will have broad impacts across all these levels by developing new tools to visualize dynamic neuronal signaling with subcellular resolution.

DESCRIPTION (provided by applicant): During the past decade stunning advances have been made in imaging, molecular biology and biochemistry that enable the visualization of the behavior of single proteins in vivo. Here, I propose to develop and visualize the temporal and spatial dynamics of intracellular signaling within living neurons. Much as early work in calcium imaging redefined our understanding of the importance of calcium influx by defining its spatial and temporal characteristics, I believe visualizing the spatial and temporal dynamics of intracellular signaling will have similar benefits to our understanding of the nervous system. Initially we have developed indicators that enable visualization of one of the key first steps in many intracellular signaling cascades: tyrosine phosphorylation. During the past several years, we have developed a system that relies on ratiometric imaging of changes in a genetically encoded fluorescent indicator of phosphorylation. We now propose three specific aims to develop these tools into a system for monitoring signaling during neuronal plasticity and development. We propose to: 1) Develop a library of indicators targeted to report activity of specific kinases; 2) Develop indicators that localize to specific cellular compartments; 3) Develop indicators to report activity of multiple signaling molecules simultaneously. Using our indicators, workers will be able to elucidate the dynamics of signals that underlie synaptic plasticity. Thus, our tools will enable novel insights into essential mechanisms that underlie neuronal plasticity. PUBLIC HEALTH RELEVANCE: Project Narrative Neuronal plasticity underlies many fundamental functions within the brain, while abnormal neuronal plasticity is associated with disease. Excessive plasticity may underlie diseases like epilepsy and addiction, while defects in plasticity could play important roles in epilepsy, neurodegenerative, and autism spectrum disorders. Our research will have broad impacts across all these levels by developing new tools to visualize dynamic neuronal signaling with subcellular resolution.

DESCRIPTION (provided by applicant): Neural progenitor cells originating in the subventricular zone (SVZ) integrate into the neural circuitry that mediates olfactory learning and impacts habit formation. The SVZ produces new neurons throughout development and adulthood that are in part responsible for the life-long elaboration and refinement of the olfactor bulb cytoarchitecture. These new neurons migrate from the SVZ to the olfactory bulb via a pathway called the rostral migratory stream. However, the molecular mechanisms that guide these migrating cells to the olfactory bulb are poorly defined. Building on strong initial findings implicating ephrinB and EphB proteins in control of migration of cells within the rostral migratory steam, we have established novel assay systems that allow us to begin to define the exact role of astrocytes in adult migration. We propose three specific aims: Determine whether expression of ephrin-B2 in the astrocyte sheath constrains migration of adult neural progenitor cells within the rostral migratory steam. Determine whether EphB2 is required for astrocyte dependent-migration of adult neural progenitor cells in the rostral migratory steam. Determine whether astrocytes lining the rostral migratory steam constrain migration by activation of EphB2 in migrating neural progenitor cells. Because previous results have demonstrated progenitor cell proliferation is reduced in depression and EphB and ephrinB expression is altered by changes in neuronal activity our proposed studies will have board impact on the understanding of the basic biology of progenitor cell migration and the cellular-molecular mechanisms controlling pathophysiology.

Abstract NLGNs are known to play crucial roles in synaptogenesis and the maturation of the postsynaptic density through recruitment of and interactions with scaffolding proteins, neurotransmitter receptors, and ion channels. It has been shown that NRXN binding is crucial for NLGN function, as NRXN is capable of clustering NLGNs at the postsynaptic density (PSD) and initiating recruitment of downstream interacting proteins necessary for synapse stabilization and maturation. Most vertebrates possess four NLGN genes: NLGN1, NLGN2, NLGN3, and NLGN4. In most mammals, NLGN4 is located on the X chromosome, however in higher order primates such as Homo sapiens and Pan troglodytes NLGN4X is complemented on the Y chromosome (sometimes referred to as NLGN5) with nearly 98% homology to NLGN4X. It is believed that this NLGN4Y isoform originated from a duplication event that occurred relatively recently during evolution Unlike almost all vertebrates, humans possess X chromosome and Y chromosome isoforms of NLGN4, however the function of NLGN4 at synapses remains unknown. We intend to investigate the functional properties of NLGN4X and NLGN4Y such as NRXN binding, synaptogenesis, and localization. We plan on addressing these questions with the following three specific aims: 1) Determine whether NLGN4X and NLGN4Y are localized to human synaptic sites. 2) Determine the affinities of NLGN4X and NLGN4Y for NRXN. 3) Determine the molecular mechanism responsible for differences in the synaptogenic activity of NLGN4X and NLGN4Y. We have conducted experiments to demonstrate that we can achieve the goals of each of the three ambitious aims we proposed. For specific aim 1, we established approaches to study NLGN function in human brain tissue and cells. For Specific aim 2, we have established an assay and begun experiments to determine the affinity of NLGN4 in biological sex-specific combinations. For specific aim 3, we have begun experiments using a synapse assay system and expressed NLGN4X and NLGN4Y and generated constructs to begin structure function analysis. Together these preliminary data indicate that we are poised to make rapid progress on our understanding of these sexually dimorphic genes. Results are likely to shed light on the matter of synaptic dysfunction in disorders such as ASD and learning disabilities and propose a potential difference in synaptic function between males and females.

Project Summary / Abstract: In a rodent model of cervical spinal cord injury (SCI), we propose to examine the contribution of altered EphB2 receptor-NMDA receptor (NMDAR) interaction to both excitatory synaptic neurotransmission in the superficial dorsal horn (DH) and persistent neuropathic pain (NP). The development of NP occurs in a significant portion of individuals affected by SCI, resulting in debilitating and often chronic physical and psychological burdens. Importantly, this pathological pain is particularly refractory to treatment, urgently calling for the identification of mechanistic targets that both robustly regulate pathological pain and avoid the devastating effects of opioid- based interventions. Hyperexcitability of DH circuitry (?central sensitization?) is a major substrate for NP after SCI. Studies have shown that NP is linked to EphB/ephrinB signaling through potentiation of NMDAR function, suggesting that the EphB-NMDAR interaction may be an important target for control of SCI-induced NP. We recently discovered that the EphB2-NMDAR interaction is regulated by a single extracellular amino acid of EphB2 (Y504). We demonstrated in vitro that EphB2-Y504 phosphorylation is required in spinal cord neurons for EphB-NMDAR interaction, NMDAR synaptic localization, and excitatory synapse function. We also found that transduction of DH neurons in vivo with EphB2 that constitutively interacts with the NMDAR results in long- lasting allodynia. We hypothesize that modulating the EphB2-NMDAR interaction in superficial dorsal horn (DH) neurons will impact synaptic localization and function of NMDARs, excitatory synaptic transmission between primary sensory afferents and DH neurons, and NP-related behaviors after cervical contusion SCI. Aim 1. Determine whether interaction with EphB2 drives NMDA receptors to synapses between primary nociceptive afferents and superficial DH neurons following cervical SCI. We will determine whether knocking down EphB2 in both uninjured and cervical contusion SCI mice using DH neuron subtype- specific expression of EphB2-shRNA reduces the localization of NMDAR subunits to excitatory synapses. Aim 2. Determine whether EphB2 regulates excitatory synaptic transmission in DH and NP-related behaviors after cervical SCI. We will determine whether DH neuron subtype-specific knockdown of EphB2 impacts: (2a) synaptic transmission between primary afferents and laminae I-II neurons using whole-cell patch clamp recording in an intact ex vivo preparation; and (2b) initiation and/or persistence of NP-related behaviors. Aim 3. Determine whether EphB2-Y504 phosphorylation regulates EphB2-NMDAR synaptic interaction in the DH and NP-related behaviors after cervical SCI. By expressing wild-type EphB2-Y504 or constitutively-phosphorylated (Y504E) or non-phosphorylatable (Y504F) mutants in a DH neuron subtype- specific manner, we will determine whether modulating EphB2-Y504 phosphorylation impacts: (3a) EphB2- NMDAR interaction, (3a) NMDAR levels at excitatory synapses, (3c) excitatory synaptic transmission between primary sensory afferents and DH neurons, and (3d) NP-related behaviors after cervical contusion SCI.

Abstract: As much as 20% of the population will suffer from chronic pain lasting for more than 6 months. Chronic pain and its underlying pathophysiology, can result in depression and other debilitating neurological effects and although there are effective treatments for acute pain chronic pain is resistant to most current treatments requiring the development of novel therapeutics that target molecular events underlying these pain states. Neuropathic and persistent post-surgical pain occurs, at least in part, due to long lasting changes in the function of excitatory synaptic transmission in the spinal dorsal horn resulting in enhanced pain signalling (hyperalgesia) and innocuous stimuli evoking pain (allodynia). These synaptic events share many features of neuronal plasticity that has been studied in higher CNS areas. Many of these changes are NMDAR dependent resulting in increased synaptic strength. One mechanism that has emerged underlying these changes in synaptic function is the potentiation of NMDAR function by a direct molecular interaction with the EphB receptor tyrosine kinase. Building on our published work, we will test the hypothesis that an EphB-NMDAR interaction is responsible for the development of a chronic pain state by directing NMDARs to synapses by expressing wild type or mutant EphB2 receptors in vitro and in mice. To test this hypothesis, we will determine the mechanism mediating the EphB-NDMAR interaction, characterize molecules and other tools to disrupt this interaction, and determine whether preventing the EphB-NMDAR interaction will alleviate chronic pain. To address these questions we will undertake three specific aims: 1. Determine the domain on the NMDAR responsible for the EphB-NMDAR interaction. 2. Test the hypothesis that VLK directs phosphorylation of Y504 on EphB2. 3. Determine the functional significance of VLK in pain plasticity. Collectively these aims will create a new knowledge that will provide a deeper understanding of the role of EphB-NMDAR interaction in pain and enable progress toward understanding the basic mechanisms behind chronic pain states.

Project Abstract As much as 20% of the population will suffer from chronic pain lasting for more than 6 months. Chronic pain and its underlying pathophysiology, can result in depression and other debilitating neurological effects and although there are effective treatments for acute pain chronic pain is resistant to most current treatments requiring the development of novel therapeutics that target molecular events underlying these pain states. Neuropathic and persistent post-surgical pain occurs, at least in part, due to long lasting changes in the function of excitatory synaptic transmission in the spinal dorsal horn resulting in enhanced pain signaling (hyperalgesia) and innocuous stimuli evoking pain (allodynia). These synaptic events share many features of neuronal plasticity that has been studied in higher CNS areas. Many of these changes are NMDAR dependent resulting in increased synaptic strength. One mechanism that has emerged underlying these changes in synaptic function is the potentiation of NMDAR function by a direct molecular interaction with the EphB receptor tyrosine kinase. Building on our published work, we will test the hypothesis that an EphB-NMDAR interaction is responsible for the development of a chronic pain state by directing NMDARs to synapses by expressing wild type or mutant EphB2 receptors in vitro and in mice. To test this hypothesis, we will determine the mechanism mediating the EphB-NDMAR interaction, characterize molecules and other tools to disrupt this interaction, and determine whether preventing the EphB-NMDAR interaction will alleviate chronic pain. To address these questions we will undertake three specific aims: 1. Determine the domain on the NMDAR responsible for the EphB-NMDAR interaction. 2. Test the hypothesis that VLK directs phosphorylation of Y504 on EphB2. 3. Determine the functional significance of VLK in pain plasticity. Collectively these aims will create a new knowledge that will provide a deeper understanding of the role of EphB-NMDAR interaction in pain and enable progress toward understanding the basic mechanisms behind chronic pain states.