Clifford G. Kentros - US grants

[unreadable] DESCRIPTION (provided by applicant): We propose a methodology to create lines of mice that are capable of the robust expression of any transgene in an anatomically specific manner. We call this method "subtractive transgenics", because it involves (by a combination of two proven transgenic technologies) the subtraction of the anatomical specificity of one expression pattern from that of another. We plan to use these mice to dissect out the functional circuitry of the central nervous system (CNS) by expressing "silencers" (constructs which turn neurons off) with unprecedented anatomical specificity. This should allow us and others to analyze the neural circuits of the CNS in a way analagous to how an engineer analyzes an electronic circuit: short out one element, and then record what happens to downstream elements. The first few specific constructs we propose should result in transgene expression in various parts of the forebrain that have been implicated in neuropathologies as diverse as Alzheimer's disease and other dementiae (the hippocampal formation) to Parkinson's disease and other disorders of the basal ganglia (striatum). The heightened understanding of the functional circuitry of the CNS the resulting mice will enable will lead to a better understanding of the etiology of its pathological states, and allow for the generation of better mouse models of these human disorders. However, it should be stressed that the method can increase the anatomical specificity of transgene expression in any tissue type, and should therefore be useful to biomedical research in general. PUBLIC HEALTH RELEVANCE. The mammalian brain is the most anatomically-complex structure in nature (and we have the most complex mammalian brain), composed of innumerable electrical interactions between literally thousands of different cell types. It is an incredibly complex biological circuit, in essence, and the cell types are its component parts. Many neurological and psychiatric disorders can be thought of as imbalances in different parts of this central circuitry. We propose a method to create genetically-modified lines of mice that can express transgenes in different specific areas of these central circuits, to enable researchers to try to understand what the different parts do. The transgenes that can be expressed range from things that just turn the cells off, as we propose to do, to specific genetic disease models. The first few specific constructs we propose should result in transgene expression in various parts of the forebrain that have been implicated in neuropathologies as diverse as as Alzheimer's disease and other dementiae (the hippocampal formation) to Parkinson's disease and other disorders of the basal ganglia (striatum). The heightened understanding of the functional circuitry of the mammalian brain the resulting mice will enable will lead to a better understanding of its pathological states, and allow for the generation of better mouse models of human disorders.

DESCRIPTION (provided by applicant): The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. Understanding how these networks function both in health and disease is dependent on understanding the precise connectivity between specific neurons types. It is therefore apparent that, in order to have an adequate understanding of the nervous system, it is necessary to have detailed descriptions of neuronal connectivity with the same level of precision at which these systems operate. The research proposed here is aimed at the development, refinement, and validation of a novel set of tools that will allow researchers to readily and systematically uncover neural circuits with cell type-specific resolution. These tools build on previous work in one of the PIs lab, developing and validating the potential for use of genetically modified rabies viruses, in combination with other genetic and viral technologies, to probe neural circuits. The new tools to be developed and tested include mouse lines and helper viruses which can be used to achieve cell type specific expression of genes that interface with the rabies tracing system. This will allow the modified rabies viruses to selectively infect specific cell types and to label the direct inputs to those cells. These new tools will be tested and protocols developed for their use in a broad range of nervous system structures, whose function is relevant to understanding disease states. New variants of rabies virus will also be generated in order to interface with the newly developed mouse lines. These variants will express genes to drive conditional expression of genes encoded in the genomes of the transgenic mice, such that inputs to specific cell types targeted for infection by the rabies virus can be identified. These new rabies viruses will also be tested and protocols developed for assaying neural circuits in a broad range of relevant structures. Overall, this project will result in the generation and validation of very valuable new tools which will then be available to the neuroscience research community. PUBLIC HEALTH RELEVANCE: Understanding neural circuits with increasingly sophisticated and higher resolution tools is crucial to understanding diseases that are caused by neural circuit disorders, including Parkinson's, neuromuscular disorders, paralysis, schizophrenia, depression, autism and attention disorders, among many others. The development of new tools for revealing circuits will therefore have a large impact on these diseases.

DESCRIPTION (provided by applicant): This application describes experiments that analyze the neural circuitry of the hippocampal formation (HF), the brain area most clearly implicated in memory and disorders such as Alzheimer's and related dementias. It combines neuron-specific transgene expression with electrophysiological techniques to study the transformation of information in the mammalian hippocampal formation at the cellular and network level. The approach is similar to the nodal analysis performed by engineers to analyze electronic circuits. Here, however, the nodes are neural cell types rather than electrical components. The nodes are specified by transgenic driver lines which direct the expression of various transgenes (depending upon which transgenic payload lines they are crossed to) enabling cell type specific changes in activity. Performing this nodal analysis, we manipulate the activity of one node while recording from others, much as when a potentiometer controlling the impedance of an amplifier circuit changes audio output when manipulated. We do so by driving the expression of transgenes which increase or decrease the membrane potential of neurons when activated either by specific drugs or wavelengths of light. We can then manipulate the activity of an identifiable population of primary neurons in the HF circuit while recording the activity of others gaining a functional understanding of how information is transformed from one region to the next.