Petr Tvrdik, Ph.D. - US grants

DESCRIPTION (provided by applicant): Glia play many critical yet poorly understood roles in normal brain physiology, as well as in a wide range of neuropathologies. Far from being neutral glue of the nervous system, glia are heterogeneous, active players in axonal pathfinding, synapse formation and signal transmission efficacy. Serious conditions arise when these functions become defective. Astrocytes, for example, have been implicated in neurodevelopmental diseases such as Rett syndrome, fragile X mental retardation and in epileptogenesis. Microglia are involved in the initiation and progression of neurological disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, as well as obsessive compulsive disorder and schizophrenia. Progress in understanding the causes of these diseases would be much faster if better tools were available for monitoring and manipulating glial cell activity. Intracellular calcium transients represent such convenient, measurable signals that strongly correlate with cellular activity. Here we propose to implement the latest generation of genetically encoded calcium indicators (GECI) into mouse reporters, using specific gene knock-in techniques. We will also explore the potential of using an alternative site-specific recombination system in the mouse, based on Dre, a tyrosine recombinase similar to Cre but possessing a distinct DNA specificity. Availability of this alternative genetic labeling technology will enable experiments investigating the relationship between two distinct cell populations, such as specific neurons and glia, without any confounding issues of dye loading efficiency, viral injections, etc., because all the required tools can be brought together by breeding. This technology will make it possible to stimulate, for example, a specific population of neurons with channel rhodopsin and monitor surrounding glia with calcium indicators, and vice versa. We will evaluate these experiments in acute brain slices, with ultrafast two-photon imaging technology in a raster scanning mode or using a powerful new approach termed targeted path scanning for rapid investigation of neural networks. The proposed tools will allow us to study reactive astrocytes in mouse models of temporal lobe epilepsy and to investigate Hoxb8 mutant microglia, which have been implicated in obsessive compulsive-like behavior in mice. Temporal lobe epilepsy is the most common type of epilepsy in humans. Only recently, astrocytes have been shown to play an important role in the disease progress. Dramatic changes occur in the expression of neurotransmitter receptors, voltage gated ion channels, inflammatory cytokines, and a variety of other proteins in response to seizure activity. Astrocytic networks will be studie in normal and epileptic brain slices, establishing an experimental framework for drug development and testing. Microglia arise from several developmental origins, including one in the Hoxb8 expression domain. Microglia are sensors of neuronal activity. We will be able to determine if Hoxb8 mutant microglia respond to neural stimuli correctly. Taken together, the proposed work will advance the set of tools available to address the underlying causes of glial and other disorders.

Genetically-encoded reporters of neural activity are a transformative tool for understanding brain function because they allow for the simultaneous measurement of activity across many neurons defined by genetic and anatomical criteria. The current generation of such reporters use light to signal activity, which limits their ability to be used deep in brain tissue and across the full range of neuronal activity. The goals of the project are to overcome these limitations by developing reporter proteins that can be engineered to emit unique electrical or magnetic signals in response to neural activity. The project will also develop sensors that are optimized for detecting these signals from individual neurons in intact brain tissue in the freely-behaving animal. The proposed 'electrogenetic' toolbox will allow neural activity to be recorded with high fidelity from defined cell types across the entire physiological range of neuronal firing rates, from any location in the mammalian brain, and in the freely-behaving animal. This strategy leverages existing and widely available technology for recording electromagnetic signals in the brain, and thus has the potential to be rapidly adopted for a wide range of neuroscience applications.

This project will develop a new strategy for measuring neural activity from genetically-targeted neurons in the intact brain. An interdisciplinary team of investigators will first use gene therapy techniques to express candidate proteins in particular neurons, then will screen for electrical or magnetic signals using conventional electrodes or nanoscale magnetometer probes. Understanding how neurons of a particular type are activated in the behaving animal is crucial for understanding the neural basis of sensation, cognition and behavior. Indeed, the lack of tools for interrogating identified neurons while they are in action is a major impediment to understanding functional neural circuits in the brain. In addition to breaking this impasse in basic science, a potential broader impact of this project is that the tools to be developed in this proposal may provide information leading to improved diagnosis and treatment of nervous system disorders including mental illness, autism, addiction and epilepsy.

Abstract Ischemic stroke is a leading cause of death and severe disability in US and worldwide. Stroke-induced hypoxia promotes a cascade of pathophysiological responses that lead to necrosis in the ischemic core, and apoptosis in the hypo-perfused tissue known as penumbra. Cell death triggers an inflammatory response that contributes to secondary injury and potentially harms the neurons surviving the initial insult. Microglia are the principal immune cells in the brain parenchyma but their specific roles in secondary injury and the underlying mechanisms of induction remain unclear. We hypothesize that increased calcium signaling is a key mechanism in the acute stroke-induced microglial activation, possibly leading to increased release of proinflammatory cytokines. We have developed a mouse reporter that indicates intracellular calcium in microglial cells. In this system, we use 2- photon imaging and middle cerebral artery occlusion (MCAo) to study microglial responses to ischemic injury in vivo. We have demonstrated periodical waves of calcium activity in cortical microglia following intraarterial occlusion, consistent with patterns of cortical spreading depolarizations (CSD). We propose to test the role of these calcium transients by pharmacological inhibition of calcium influx, mediated by the calcium release- activated calcium (CRAC) channels, and by genetic ablation of CRAC channel subunits. In Aim 1, we will directly test whether the novel CRAC channel inhibitors developed by CalciMedica can reduce microglial activation, neuro-inflammation and ultimately infarct size in the mouse model of MCAo. Pharmacological effects of these blockers will be characterized with 2-photon imaging and their immune-protective effects in vivo will be evaluated by cytokine profiling. In Aim2, the CRAC channel subunits Stim1 and Stim2 will be genetically ablated in brain microglia and behavioral outcomes and infarct size after MCAo stroke will be evaluated. Stroke kills almost 130,000 Americans each year. If successful, clinical translation of this approach could help to reduce the burden of this disease. Our overreaching objective is to apply the tools and techniques assembled under this pilot study to a broader R01 project investigating CRAC-mediated calcium overload in all other brain cells during ischemic injury.