Sreekanth H. Chalasani - US grants

DESCRIPTION (provided by applicant): All organisms possess an intrinsic ability to detect and respond to threats in their environments, but the underlying molecular mechanisms are poorly understood. A complete understanding of this process requires knowledge of the underlying neural circuits along with an ability to measure and, most importantly, perturb their activity. This is difficult to obtain in complex vertebrate circuits. However, invertebrate circuit with their well-defined neuroanatomy and quantitative behaviors are ideally placed to decipher the underlying machinery guiding complex outputs. This proposal aims to understand the neural mechanisms that code threat responses (both behavioral and physiological) in an invertebrate brain model. The nematode, Caenorhabditis elegans, provides a unique opportunity to analyze, using a multi- scale approach, genes, cells and circuits that regulate complex behaviors. The Chalasani lab has developed a novel model of threat behaviors using the interactions between C. elegans and a second nematode, Pristionchus pacificus. A starving Pristionchus will attack and devour C. elegans in 30 minutes. C. elegans in turn, will avoid both Pristionchus and its secretions. Apart from this behavioral response, C. elegans also activates mitochondrial stress upon exposure to Pristionchus. The goals of the proposed research program are to define the cellular and molecular mechanisms regulating avoidance behavior in this model system. It has already been determined that a novel neural circuit including three new sensory neurons (ASJ, ASK and ASI) drive avoidance behavior and physiological stress responses. Specific aim 1 will identify this neuronal circuit and the associated neurotransmitters and receptors that regulate predator avoidance and mitochondrial stress responses. Aim 2 will optimize an automated behavioral platform to rapidly analyze behaviors from large numbers of worms and perform a large screen for genes affecting avoidance behavior. A pilot screen has identified 4 interesting genes as required for regulating avoidance behavior. These include a TRPV channel (might be part of the Pristionchus sensing machinery), glutamate transporters and serotonin biosynthesis enzyme and serotonin re-uptake transporter. Aim 3 is focused on validating these and other candidates from the genetic screen. These studies will clarify how neural circuits process information about environmental threats at the level of synapses, neural circuits and whole organisms. Moreover, we will identify basic principles and conserved mechanisms of how neural circuits integrate glutamate and serotonin signaling to generate complex behaviors.

Summary A central challenge in neuroscience is to develop methods to manipulate specific cell types within the mammalian brain. Recent developments in optogenetics have revolutionized our ability to control the activity of both neurons and non-neuronal cells. However, this approach suffers from one drawback, the difficulty in delivery light stimulus to target cells that are located deep within the brain or the body. The Chalasani lab has recently demonstrated a noninvasive method to control the activity of neurons. They have identified a pore- forming subunit of a mechanosensitive channel (TRP-4) that responds to low-intensity ultrasound. Further, they showed that expressing this channel is specific cells renders those target cells sensitive to mechanical deformations generated by noninvasive ultrasound waves. This proposal aims to develop this approach (they have termed ?sonogenetics?) to control specific cells within the mouse brain. Further, they find that this approach can be used to control the activity of mammalian neurons in vitro. They plan on using a high- throughput assay system to test whether other members of the TRP-N family are sensitive to ultrasound pulses. Additionally, they will also analyze whether altering the number of ankyrin repeats affects the ultrasound responsiveness of these channels (consistent with a recent study showing similar results in the Drosophila TRP-N channel) (Aim 1). They also plan on developing a new head device with a slot for a tiny, lightweight ultrasound transducer to deliver ultrasound stimulus to the mouse brain (Aim 2). Finally, they will test the efficacy of the sonogenetic approach in vivo using electrophysiological and behavioral analysis. They will express TRP-4 or other mechanosensitive channels in cortical PV interneurons, striatal D1 or D2 medium spiny projection neurons and control their activity in vivo. Optogenetic methods have been previously used to control these cell populations providing benchmarks for comparison. These studies will develop a noninvasive method to manipulate the activity of specific cells within the rodent brain or its body. Further, these methods can be translated into the human to target specific cell populations for therapeutic purposes.

Summary Atypical sensory-based behaviors are a common feature of a number of human conditions, including autism spectrum disorder, schizophrenia, fragile X, etc. Despite this, little is known about how the genes associated with these conditions affect sensory behavior. A complete understanding of this process requires a thorough characterization of the underlying neural circuitry, along with the ability to measure and perturb the activity of these circuits. The nematode, Caenorhabditis elegans, provides a unique opportunity to analyze genes, cells, and circuits regulating complex behaviors, as its nervous system consists of just 302 neurons interconnected via identified synapses that utilize highly conserved synaptic machineries. The Chalasani lab has shown that C. elegans homologs of the human autism-associated genes (neurexin (NRX) and neuroligin (NLG)) affect sensitivity to specific sensory stimuli, and that mutations in these genes result in hyposensitivity to a repellent copper stimulus. They propose to identify the specific C. elegans synapses where these two synaptic proteins function to modify sensory behaviors. Additionally, they plan to identify the developmental time window in which these genes are required to generate a typically behaving young adult (Aim 1). Moreover, they have shown that sensory defects associated with neuroligin mutants (nlg-1) are rescued by mutations in the gene npr-1, a gene that when mutated alone results in a ?social? aggregation behavior. They propose to identify the neural mechanisms that underlie this interaction and reveal components of the NPR-1 signaling pathway that act to suppress nlg-1 behavioral defects (Aim 2). Finally, they have identified Nipecotic acid and CGP-13501 as candidate small molecules that suppress nlg-1 behavioral deficits. They plan to map the cellular and molecular targets of these drugs in C. elegans, analyzing the genetic pathways modifying NRX-1/NLG-1 signaling in this model (Aim 3). These studies will reveal mechanisms by which NRX-NLG signaling modifies sensory behavior at the level of genes, synapses, circuits, and whole animals, providing a solid foundation for further analyses in vertebrate models. As both NLG and NRX have been implicated in autism spectrum disorder, results may shed light on molecular and circuit mechanisms underlying human disorders that have been linked to abnormalities in sensory processing.

Abstract A key challenge in neuroscience is the development of methods to non-invasively manipulate specific neuronal cell types in vivo. While recent opto-, chemo- and magneto-genetic approaches have revolutionized our ability to control both neuronal and non-neuronal cell types, they each suffer from critical drawbacks, including the inability to deliver light to targets deep within the brain or to large volumes of the brain (opto-), and the lack of precise temporal control for both chemo- and magneto-genetic approaches. The Chalasani lab has recently demonstrated a noninvasive method for controlling the activity of neurons using ultrasound, a system they call sonogenetics. They have demonstrated that mechanosensitive TRP-N channel homologs from C. elegans, Hydractinia, and Hydra magnipapillata can be used to non-invasively activate mammalian cells both in vitro and in vivo. They hypothesize that target cells expressing these TRP-N channels are rendered sensitive to mechanical deformations generated by non-invasive ultrasound waves. This proposal aims to extend the sonogenetic approach to control specific neuronal populations throughout large volumes of the mouse brain, a system that would be useful for reversing electrophysiological and behavioral deficits seen in epilepsy, for example. They will identify channels with non-overlapping ultrasound stimulus ranges by testing variants and chimeras of the Hydra TRP-N channels in high-throughput imaging and slice culture electrophysiology assays in vitro, as well as in feeding and electromyography assays in vivo (Aim 1). They also plan to develop a new lithium niobate-based transducer that will deliver ultrasound throughout the mouse brain. Specifically, they will use Schroeder?s optimal diffuser design in a device that will generate spatiotemporally incoherent ultrasound that upon reflection, avoids interference and localized spikes in ultrasound (Aim 2). Finally, they plan to activate GABAergic inhibitory interneurons broadly throughout the brain to alleviate behavioral and electrophysiological deficits in mouse models of epilepsy and Rhett?s syndrome. Optogenetic, chemogenetic, and pharmacological methods have been previously used to control these cell populations, providing benchmarks for comparison. These studies will develop a noninvasive method for manipulating the activity of specific cells within large volumes of the rodent brain or body. Further, these methods can be translated into the human system to target specific cell populations for therapeutic purposes.