Shashank Dravid, PhD - US grants

DESCRIPTION (provided by applicant): In the US alone, an estimated 40 million adults suffer from anxiety disorders which may have debilitating consequences. Common forms of anxiety disorders include social anxiety, specific phobias and post-traumatic stress disorders (PTSD). Current therapy for anxiety disorders have unpleasant side effects and may fail. Thus, there is an urgent need to develop new therapeutic interventions. The treatment of choice for a number of anxiety disorders is exposure-based psychotherapy. Pilot studies in human show that D-cycloserine (DCS), an agonist for N-methyl-D-aspartate (NMDA) receptors, augments the effects of exposure therapy for simple and social phobia, obsessive compulsive disorder (OCD) and panic disorder. Despite very promising translational results demonstrating a robust effect of DCS in enhancing exposure therapy, the molecular mechanism of DCS action is unknown. In this proposal using behavioral, electrophysiological and biochemical techniques we will assess the effect of DCS on synaptic strengthening or depotentiation of cortico-amygdala circuits. The long- term goal of this proposal is to understand NMDA receptor mediated mechanisms of learning in the amygdala. PUBLIC HEALTH RELEVANCE: The treatment of choice for a number of anxiety disorders is exposure-based psychotherapy. D-cycloserine (DCS) augments the effects of exposure therapy for simple and social phobia, obsessive compulsive disorder (OCD) and panic disorder. This study will assess the molecular pathway of DCS action.

DESCRIPTION (provided by applicant): It is estimated that ~1 in every 110 children is affected by autism spectrum disorders (ASDs) with devastating consequence to the individual and family along with being an economic burden. There is currently no effective therapy available for ASD and understanding the underlying mechanisms may lead to identification of novel therapeutic targets. Human genome-wide association studies have identified genetic variations that may contribute to ASDs. These studies have established that ASD-associated genes are part of a large functional network involved in synapse formation and signaling. However, the precise physiological role of several of the newly identified candidate genes is still unknown. GRID1 (Glutamate Receptor Ionotropic Delta-1) gene, which codes for the glutamate delta-1 (GluD1) subunit, is one such gene identified as a susceptibility gene for ASDs and schizoaffective disorders. Lower GluD1 expression has also been reported in ASD and schizoaffective disorder patients. Moreover, recent studies have identified a potential role of GluD1 in synapse formation via interaction with presynaptic Neurexin1, which itself is an autism susceptibility gene. These converging findings suggest that GluD1 may be a part of the functional synaptic network that is dysregulated in ASD. Our preliminary data strongly indicates that deletion of GluD1 leads to several behavioral and molecular abnormalities that resemble deficits in human ASD patients. The goal of this proposal is to specifically understand the neural basis of these abnormal behaviors in GluD1 knockout mice. Towards this end we will determine the effect of GluD1 deletion in mouse on spine morphology and synaptic function in the medial prefrontal cortex, which regulates social behaviors and cognitive abilities known to be affected in ASD patients. We have also found that an NMDA receptor agonist D-cycloserine is able to rescue social deficits in GluD1 knockout mouse. We will therefore test whether reversal of synaptic abnormalities in the medial prefrontal cortex underlie the efficacy of D-cycloserine in social deficits. These studies will provide the first evidence for a crucial role of GluD1 in the regulation of synaptic structure and function that eventually modulate behavior and potentially identify a novel therapeutic target for ASD. Additionally, our studies will support the hypothesis that facilitation of activity-dependent mechanisms may have therapeutic value in alleviating ASD phenotype.

Neurons in the brain communicate with each other through proteins that are present on specialized structures called spines that extend out from the neuron cell membrane. During neuron activity, the number and shape of spines change. Understanding how these changes occur is important because the changes are associated with learning and memory. The PI will study one particular protein that is highly abundant in spines and known to be critical for learning. The goal of the project is to determine how this protein and the proteins that it interacts with control spine shape and number. Undergraduate and graduate students, including underrepresented minorities, will participate in the research. The PI will present seminars on this area of research to the Health Science-Multicultural and Community Affairs (HS-MACA) Postbaccalaureate Program that is offered at his institution.

Glutamate delta-1 receptors are orphan receptors that, unlike other members of the ionotropic glutamate receptor family, do not function as typical ligand-gated ion channels. Understanding their mechanism of action is important because the receptors are crucial for normal dendritic spine pruning, associative and reversal learning, and execution of complex behaviors. This project will investigate the potential interaction of glutamate delta-1 receptor with metabotropic glutamate receptors, which are also enriched at glutamatergic synapses. The working hypothesis for the proposed studies is that the glutamate delta-1 receptor regulates downstream metabotropic glutamate receptor signaling, including the mammalian target of rapamycin (mTOR) pathway, which regulates spine morphology. A multidisciplinary approach will be carried out that will make use of genetic and pharmacological tools, functional assays, diolistic labeling, and three dimensional reconstruction techniques. Outcomes are directed at developing a mechanistic understanding of glutamatergic synapse structure and function in the central nervous system.

The delta family of ionotropic glutamate receptors consisting of glutamate delta 1 (GluD1) and glutamate delta 2 (GluD2) are unusual since they do not exhibit typical ligand-gated ionic currents. Instead, they are endowed with synaptogenic property by forming a trans-synaptic GluD-Cerebellin1 (Cbln1)-Neurexin complex and inducing synapse formation. In addition, the GluD receptors have C-terminal interactions which may stabilize postsynaptic density machinery and contribute to synaptic plasticity. Although the function of GluD2 subunit in the formation and plasticity of parallel fiber- Purkinje cell synapse in the cerebellum is well established the role of GluD1 enriched in the forebrain remains largely unknown. GluD1 is enriched in the striatum which receives strong excitatory inputs from the cortex and thalamus. Our preliminary results demonstrate a critical role of GluD1 in excitatory neurotransmission in medium spiny neurons in the striatum. Our goal is to address potential cell-type and synapse-selectivity in this effect upon loss of GluD1 which will support its role as a synaptic organizer. We will pursue the following specific aims; (i) Determine the localization of GluD1 in the striatum and the effect of GluD1 loss on synaptic structure. We will use a range of complementary electron microscopy, immunohistochemistry and biochemistry methods to analyze distribution of GluD1 in the striatum and impact of GluD1 loss on striatal synapses and potential reorganization of synaptic components. (ii) Determine the role of GluD1 in synaptic neurotransmission and plasticity. We will use conventional electrophysiology together with ex vivo optogenetics to stimulate specific synapses to address potential synapse-specific roles of GluD1. (iii) Determine the role of striatal GluD1 in cognitive and behavioral control. We will address the impact of changes in synaptic function upon loss of striatal GluD1 on emotional, cognitive and motor behaviors that are regulated by striatal circuits. These studies will be complemented with DREADD technique to manipulate specific striatal pathways. Together, the proposed studies will systematically address the synaptic organizational principle of GluD1 in the striatum and address its role in synaptic and behavioral phenotypes relevant to neuropsychiatric and neurological disorders.

Globus pallidus externus (GPe) is a nucleus in the indirect pathway of basal ganglia circuitry which was originally considered to be a group of homogenous GABAergic projection neurons producing downstream inhibition of subthalamic nuclei (STN). However, recent evidence using genetic reporter lines have revealed that there are three classes of neurons in this region which have different electrophysiology properties and different projections. These cell types include parvalbumin neurons, arkypallidal neurons and Lhx6 neurons. This knowledge has important implications for our basic understanding of GPe physiology and to the GPe dysfunction observed in movement disorders including Parkinson's disease and Huntington's disease. However, there remains a critical gap in our knowledge of glutamatergic system in GPe which can potentially be used to modulate dysfunctional basal ganglia circuitry. In particular the function of various NMDA receptor subtypes in the different classes of GPe neurons is unknown. We propose to address the expression and function of GluN2C/GluN2D subunits in GPe since these receptors have many unusual properties including lower sensitivity to Mg2+-block, higher glutamate/glycine affinity and lack of desensitization which are optimal for tonic activity observed in GPe neurons. Our preliminary data and previous reports suggest that GluN2C/GluN2D may express in a cell-specific manner in GPe. Therefore, activation of GluN2C/GluN2D receptors may produce unique changes in GPe function and downstream inhibition which may allow fine tuning of disrupted basal ganglia circuitry in movement disorders. We will address the functional expression of the GluN2C and/or GluN2D subunits in GPe in a cell-type specific manner and elucidate their roles in cellular excitability and inhibitory tone in STN and potential relevance to behavioral deficits and cortical oscillation abnormalities in Parkinson's disease. We will use a multidisciplinary approach involving electrophysiological and behavioral studies together with pharmacological and DREADD tools as well as genetic and reporter lines and parkinsonian mouse models to address our goals.

Summary: Pain is a serious clinical problem that affects more than 100 million Americans. The economic costs of pain have been estimated to be more than several hundred billion dollars including healthcare costs and lost productivity. Persistent pain may produce long-term disability and lead to precipitation of depression, anxiety and cognitive impairment. Currently used medications for chronic pain are not always effective and have limitations in terms of tolerance and abuse liability. Thus, identifying novel therapeutic targets is essential to address this clinical burden. Peripheral and central pathways that encode, transmit, and amplify or reduce pain signals have been identified, including the spinothalamic and spinoparabrachial pathways. Plasticity of glutamatergic synapses along key nodes in the spinoparabrachial-amygdala pathway plays an important role in pain modulation and in the transition from subacute to chronic pain. However, the mechanisms governing the development, maintenance and plasticity of this system and their role in persistence of pain behaviors remain poorly understood. The proposed research will advance the concept that the trans-synaptic signaling complex centered on glutamate delta 1 receptor regulates function of synapses in the laterocapsular region of central amygdala also known as ?nociceptive amygdala? and contributes to persistent pain mechanisms. Specific Aim1 will define the cell type- and projection-specific distribution of these receptors and their role in regulating amygdala circuitry and nocifensive and averse-affective behavior under normal conditions. Specific Aim 2 will determine persistent/chronic pain-related changes in glutamate delta 1 signaling using inflammatory and neuropathic pain models and test the effect of a rescue strategy on synaptic neuroplasticity in pain models. Changes in ultrastructure of amygdala synapses in pain models will be evaluated using 3D-electron microscopy. Specific Aim 3 will determine the effect of restoring trans- synaptic signaling through the glutamate delta 1 receptor in mitigating nocifensive and averse-affective behaviors in pain models. Complementary experiments will address the effect of cell-type specific manipulation of central amygdala circuitry in mitigating pain. To accomplish these aims we will utilize a combination of brain slice electrophysiology, behavior, chemo- and opto-genetics, confocal and electron microscopy (immuno and 3D), and genetic approaches to determine the functional and structural mechanisms through which the glutamate delta 1 signaling complex regulates pain-related neuroplasticity and behaviors. This project is significant because it would identify a novel brain mechanism of pain that could be targeted for pain management. Scientific rigor of research design is established by the use of multiple methods and approaches, replication of experiments in independent laboratories, use of validated models and reagents, consideration of blinding, biological variables and sex in addition to other aspects of experimental design.