John Patrick Card - US grants

Neurotropic viruses produce specific patterns of infection in the nervous system by virtue of their ability to invade neurons, replicate in a predictable fashion, and then pass transneuronally through sites of synaptic contact. The goal of this proposal is to exploit the neurotropic properties of alpha herpesviruses for two independent but closely related purposes. The first is to use a well characterized virus, the swine herpesvirus known as pseudorabies virus (PRV), to analyze patterns and mechanisms of viral infection in the mammalian nervous system. The second is to utilize this information to develop viral infectivity as a reliable transneuronal tracing methodology and to apply this method in an analysis of the synaptic organization of functionally distinct components of the visual system. Thr proposal includes two specific aims. The first is to define the neuroinvasive properties of wild type and well characterized mutant strains of PRV introduced into the neuraxis by injection into a variety of central and peripheral sites. The objectives of these studies are to determine the patterns of primary neuronal infection and transynaptic spread produced by inoculation of skin, cranial musculature, vasculature, olfactory mucosa, basal ganglia, and components of the limbic circuitry associated with the hippocampal formation. These areas were chosen because of their functional and anatomical diversity as well as their clinical relevance. In addition, the well characterized gene deletions in these isogenic strains will allow us to identify properties of the virus required for viral invasion and dissemination. The analysis will determine the temporal course of infection produced y each strain, whether infection travels through routes of established connections, if all components of a circuit are equally susceptible to infection, the direction of viral transport through each circuit, and whether susceptibility to infection is influenced by the type of neuron (e.g. sensory vs. motor), its chemical phenotype, or other properties. The second specific aim is to capitalize upon the specific affinity of mutant strains of PRV for functionally distinct components of the visual neuraxis to characterize the synaptic organization of components of the central visual projection systems involved in the photic modulation of hypothalamic function. These strains of virus will be used in combination with classical neuronal tracers and other antigenically- distinct strains of PRV to define multisynaptic circuits devoted to circadian, autonomic and neuroendocrine function. The collective intent of the program is to provide insight into mechanisms underlying viral invasion of clinically relevant neural circuits and to use this information to gain further insight into the functional organization of the nervous system.

DESCRIPTION (provided by applicant): Cardiovascular disease is the leading cause of death in the United States and hypertension is a major risk factor for the development of cardiovascular disease. Alarmingly, statistics indicate that more than a quarter of the US population had hypertension in 2004. Contemporary research has demonstrated that the caudal brain stem exerts a profound influence over cardiovascular function. In particular, neurons of the rostroventrolateral medulla (RVLM) are known to be an important node in a central network that contributes to this regulation and malfunction of the network in experimental animals can produce hypertension. This network involves multiple cell groups in the brain stem, midbrain, diencephalon and forebrain. We recently developed a novel technology that allowed us to map the projections of C1 catecholamine neurons in RVLM. The data from that investigation confirmed the projection of C1 neurons to the sympathetic column in thoracic spinal cord but also revealed dense projections to a number of supraspinal cell groups with demonstrated influences upon cardiovascular function. In this application we hypothesize that collateralization of the C1 population to cardiovascular regulatory cell groups provides the neural substrate to coordinate activity within this distributed network. Experiments in three Specific Aims are advanced to test this hypothesis. Aim 1 will use our novel technology (lentivirus mediated anterograde tracing of C1 projections) and electron microscopy to test the hypothesis that recurrent collaterals of C1 neurons synapse upon projection specific and phenotypically distinct populations of RVLM neurons. Lentivirus mediated reporter gene (EGFP) expression will be used to label C1 axon terminals and ultrastructural analysis will characterize the synaptology of these afferents with subpopulations of neurons that differ in phenotype, projection patterns and topography within RVLM. Aim 2 will test the hypothesis that reticulospinal C1 neurons provide a substrate for coordination of network activity through projections that collateralize diffusely to supraspinal targets. Although surprisingly few studies have addressed this issue it is widely accepted that reticulospinal C1 neurons do not contribute substantially to supraspinal projections. Nevertheless, our data and that derived from a small number of studies examining C1 efferents challenge this conclusion. Experiments in this aim will employ dual retrograde labeling combined with immunocytochemical characterization of neuronal phenotype to address this question in a comprehensive fashion. Aim 3 will utilize conditional replication of pseudorabies virus and transneuronal labeling to test the hypothesis that reticulospinal C1 neurons receive synaptic input from all sources of RVLM afferents. Importantly, we hypothesize that neuronal circuits synaptically linked to reticulospinal C1 neurons will constitute a subset of the neurons that project to RVLM. These experiments will exploit a novel technology for conditional replication of PRV. Lentivirus vectors will be injected into RVLM to achieve targeted expression of cre recombinase (CRE) in C1 neurons or in neurons throughout RVLM. A strain of pseudorabies virus (PRV- 2001) whose replication is conditional upon the presence of CRE will be injected into the projection targets of reticulospinal RVLM neurons in thoracic spinal cord. Retrograde transport of PRV-2001 to RVLM will lead to restricted replication of the virus in CRE-containing neurons and retrograde transneuronal labeling of synaptically linked neurons. Collectively these data promise to provide important functional insights into the way in which C1 and the RVLM contribute to cardiovascular homeostasis as well as providing a wiring diagram that should improve understanding of the neural basis of neurogenic hypertension. PUBLIC HEALTH RELEVANCE: A distributed network of neurons in the central nervous system is known to exert a regulatory influence over cardiovascular function and malfunction of the network can produce hypertension, a major risk factor for the development of cardiovascular disease. However, the way in which neuronal activity is coordinated within the central cardiovascular network is not known. This proposal tests the hypothesis that collateralized projections of C1 catecholamine neurons in the rostroventrolateral medulla provides the neural substrate for this integration.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, 06-NS-106: Validating new methods to study brain connectivity. We propose to test a new method that provides substantial improvement over previous Cre-conditional viral tracers. The technology combines the Brainbow multicolor cell marking technology with the retrograde, circuit tracing properties of pseudorabies virus (PRV), a neuroinvasive alpha herpesvirus. We have constructed a prototype PRV Brainbow virus called PRV263 that we propose will enable simultaneous identification of distinct chains of neurons projecting to a phenotypically defined population of neurons, and promises to provide predictive data on the strength of different connections among those neurons. Importantly, these PRV Brainbow tracers will have distinct advantages over present tracers. Our concept takes advantage of conditional, site-specific recombination of the genome of a DNA virus to produce multiple reporters so that neurons upstream (presynaptic) of a Cre recombinase (Cre) expressing neuron will be a different color from the Cre-expressing neuron. This novel concept will be expanded to produce second generation prototypes of PRV Brainbow tracers that do not rely on Cre-transgenic mice and can be used in the many mammalian species susceptible to PRV infection. A third generation prototype will be constructed that not only marks circuits, but also reports on neuronal activity. In this latter concept, the PRV Brainbow virus also will include a genetically encoded calcium indicator that fluoresces when calcium is bound. As viral tracing of neural circuitry has become an essential tool in the neuroscience community, our new tracers will be immediately applicable for many ongoing fundamental research projects in neuroscience in a variety of animals. These new tools have promise to reveal detailed functional insights into neural circuit organization that have not been possible to achieve in the past. Viral tracing of neural circuitry has become an essential tool in the neuroscience community. The new, robust viral tracers that will result from our work have promise to reveal detailed functional insights into trans-neuronal spread of herpesviruses, as well as neural circuit organization that have not been possible to achieve in the past. These neural tracers would be powerful tools to elucidate brain micro-circuitry, providing a better understanding of nervous system functions.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
The nervous system exerts profound regulatory influence over peripheral physiology and this control is essential for homeostasis and coordinating adaptive changes across physiological systems. The regulatory oversight exerted by the brain necessary for the integrated action within and among these systems is considerable, and the brain meets this challenge through regulation of the autonomic nervous system and neuroendocrine control over the pituitary gland; the general goal of this research program is to understand the organization of this regulation. During the past 25 years much has been learned about how the brain controls the autonomic nervous system and this project continues that work by addressing two fundamental questions that remain unanswered. First, what is the nature of the brain circuitry that allows both selective control of specific tissues and also global control involved in activating all sympathetic outflows as occurs in response to certain stressors. Second, what are the brain circuits that provide for the coordinated control of sympathetic and parasympathetic outflows such that when sympathetic activity is increased parasympathetic activity decreases. This project examines these issues using novel neuroanatomical techniques relying on a genetically engineered virus that gets passed among connected nerve cells thereby allowing the visualization of neural circuits in rats. Understanding the nature of these neural circuits is essential for understanding of physiological regulation in mammals. In addition to addressing key questions pertaining to the organization of central autonomic circuits, these studies will provide strong foundational support for this methodology, which can be generally applied to complex neuroanatomical issues. Also, undergraduate student researchers are incorporated into all aspects of this research program, and this project provides an excellent opportunity to immerse undergraduate students in basic neurobiological research. One component of this is the development of a new course in autonomic neuroscience, in which students will be given their own component of this project to develop, analyze, and present their findings.

Project Summary/Abstract Cardiovascular disease is the leading cause of death in the United States and hypertension is a major risk factor for the development of cardiovascular disease. Alarmingly, statistics indicate that more than a quarter of the US population had hypertension in 2004. Contemporary research has demonstrated that the caudal brain stem exerts a profound influence over cardiovascular function. In particular, neurons of the rostroventrolateral medulla (RVLM) are known to be an important node in a central network that contributes to this regulation and malfunction of the network in experimental animals can produce hypertension. This network involves multiple cell groups in the brain stem, midbrain, diencephalon and forebrain. We recently developed a novel technology that allowed us to map the projections of C1 catecholamine neurons in RVLM. The data from that investigation confirmed the projection of C1 neurons to the sympathetic column in thoracic spinal cord but also revealed dense projections to a number of supraspinal cell groups with demonstrated influences upon cardiovascular function. In this application we hypothesize that collateralization of the C1 population to cardiovascular regulatory cell groups provides the neural substrate to coordinate activity within this distributed network. Experiments in three Specific Aims are advanced to test this hypothesis. Aim 1 will use our novel technology (lentivirus mediated anterograde tracing of C1 projections) and electron microscopy to test the hypothesis that recurrent collaterals of C1 neurons synapse upon projection specific and phenotypically distinct populations of RVLM neurons. Lentivirus mediated reporter gene (EGFP) expression will be used to label C1 axon terminals and ultrastructural analysis will characterize the synaptology of these afferents with subpopulations of neurons that differ in phenotype, projection patterns and topography within RVLM. Aim 2 will test the hypothesis that reticulospinal C1 neurons provide a substrate for coordination of network activity through projections that collateralize diffusely to supraspinal targets. Although surprisingly few studies have addressed this issue it is widely accepted that reticulospinal C1 neurons do not contribute substantially to supraspinal projections. Nevertheless, our data and that derived from a small number of studies examining C1 efferents challenge this conclusion. Experiments in this aim will employ dual retrograde labeling combined with immunocytochemical characterization of neuronal phenotype to address this question in a comprehensive fashion. Aim 3 will utilize conditional replication of pseudorabies virus and transneuronal labeling to test the hypothesis that reticulospinal C1 neurons receive synaptic input from all sources of RVLM afferents. Importantly, we hypothesize that neuronal circuits synaptically linked to reticulospinal C1 neurons will constitute a subset of the neurons that project to RVLM. These experiments will exploit a novel technology for conditional replication of PRV. Lentivirus vectors will be injected into RVLM to achieve targeted expression of cre recombinase (CRE) in C1 neurons or in neurons throughout RVLM. A strain of pseudorabies virus (PRV- 2001) whose replication is conditional upon the presence of CRE will be injected into the projection targets of reticulospinal RVLM neurons in thoracic spinal cord. Retrograde transport of PRV-2001 to RVLM will lead to restricted replication of the virus in CRE-containing neurons and retrograde transneuronal labeling of synaptically linked neurons. Collectively these data promise to provide important functional insights into the way in which C1 and the RVLM contribute to cardiovascular homeostasis as well as providing a wiring diagram that should improve understanding of the neural basis of neurogenic hypertension.