Advances in molecular genetic techniques are revealing new details of the neuroanatomical organization of brain circuitry and the functional role of these circuits in behavior. Engineered viral vector constructs have been developed to label axonal projections of targeted neurons with unprecedented clarity, while others allow for retrograde transynaptic labeling of neurons providing inputs or anterograde transynaptic labeling of post-synatpic targets of axonal projections. Development of optogenetic and DREADD techniques provide the ability to functionally manipulate neural circuits to study their role in behavior while calcium indicators provide the ability to analyze the physiologic activity in targeted neuron populations. Together these approaches are providing new insights into the functional organization of neural circuits. For example, optogenetic studies, using light activation of Channel Rhodopsin (ChR), have demonstrated the ability to functionally manipulate specific neural pathways to determine their role in behaviors including fear memory, anxiety, feeding, and movement. The analytic potential of these approaches is enhanced by the ability to target specific neuron populations, which are defined components of neural circuits. One approach involves the use of transgenic Cre-driver mouse lines in which Cre-recombinase is expressed under the control of gene-specific promoters. In recent years we characterized BAC-Cre driver lines from the GENSAT project that allow for targeting components of the neural circuits of the cerebral cortex and basal ganglia. Of particular significance lines were characterized with selective labeling in cortical layer 2/3, in layer 4, in layer 5 and in layer 6. Each of these cortical layers contain neuron subtypes with distinct axonal projection patterns. Those in layer 2/3 providing the major pathways through which information is integrated between functionally distinct cortical areas, such as sensory and motor areas. Neurons layer 5 are composed of two main subtypes, those that are similar to layer 2/3 neurons with cortical axonal projections, and those that project to subcortical circuits involved in the generation of movement, including the thalamus, superior colliculus and pontine nuclei. Both of these layer 5 neuron subtypes provide the major input to the input structure of the basal ganglia, the striatum. Neurons in layer 6 provide projections to the thalamus. These BAC-Cre lines provide unprecedented ability to study the specific function of these cortical subtypes in behavior. In the past year we collaborated with investigators at Janelia Farms, Harvard, the Salk Institute and Cold Spring Harbor in studies that used the BAC-Cre lines to determine the relationship between the organization of information transfer between sensory, motor and association cortical areas and the planning and initiation of movements. During this year we used the GENSAT BAC-Cre driver lines that were characterized in a number of studies to analyze the functional organization of the relationship between the cerebral cortex and basal ganglia. In a collaborative study with the Allen Institute for Brain Science, the GENSAT BAC-Cre lines were used to generate a comprehensive database of the connections of the mouse brain ( Oh et al., 2014, A mesoscale connectome of the mouse brain, Nature 508:207). In this study over 1000 cases of axonal tracings with Cre dependent AAV EGFP injections are mapped into a reference atlas framework, which provides the ability to analyze the axonal projections of specific neuron subtypes. Ongoing collaborative studies are analyzing the relationship between the organization of cortico-cortical connections, which are involved in the integration of sensory and motor functions, with subcortical circuits through the basal ganglia that are responsible for the initiation of motor behavior. In a collaborative study with Bernardo Sabatini at Harvard, GENSAT BAC-Cre lines were used distinguish between cholinergic and GABAergic projections of the globus pallidus to the cerebral cortex (Saunders et al., 2015). While the excitatory cholinergic projection has been well studied, the identification of an inhibitory GABAergic projection from the globus pallidus was novel and provides an important new component of the relationship between the basal ganglia and the cerebral cortex. In a collaborative study with Karel Svoboda's group at the Howard Hughes Janelia Farms Institute, GENSAT BAC Cre lines were used to analyze the function of a motor cortical area involved in the generation of voluntary movement (Li et al, 2014). This study used GENSAT BAC-Cre driver lines that allow analysis of two distinct subtypes of cortical layer 5 neurons, those that have only cortico-cortical connections and those that project to subcortical circuits. Results determined that following the sensory detection phase of the behavior, preparatory activity during which the movement response is planned involved cortico-cortical circuits, whereas the initiation of the motor response involved cortical neurons projecting to subcortical circuits. This study demonstrates the specific cortical circuits involved in the sensory cue, planning and execution of directed motor behavior. In a collaborative study with Loren Looger's group at Janelia Farms a technique was developed that greatly expands the research tools available for axonal tract tracing ((Viswanathan, 2015). In recent years the injection of AAV viral vectors that are incorporated into neurons and express fluorescent proteins such as EGFP or dTomato have become the standard for tracing axonal projections in the brain. Looger's group developed additional AAV constructs that express other proteins that may be detected with immuno-histochemical techniques. The use of these AAV vectors allows for injections into 4 to 6 brain sites to analyze the axonal projections from multiple brain areas, which allow for detailed analysis of the topographic organization of projections from cortical areas or the convergence of inputs from multiple areas into a give brain site. Our contribution to these studies was to provide experimental paradigms to analyze the efficacy of the technique as well as to develop immuno-histochemical and brain imaging techniques to analyze 4 to 6 axonal tracers in a single brain.
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