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 trans-synaptic labeling of neurons providing inputs or anterograde trans-synaptic labeling of post-synaptic 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 provide 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. 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 NIDA, Howard Hughes Medical Institute Janelia Farms Research, and the Salk Institute 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 neuroanatomical techniques were developed to analyze the functional organization of the relationship between the cerebral cortex and basal ganglia. Our work focuses on the organization of neural circuits responsible for integrating different modes of sensory and experiential information that is utilized in the planning and execution of behavior. To do this we use viral vectors that label the axons of specific cortical neuron subtypes in GENSAT BAC-Cre mouse lines. Viral vectors injected into different cortical areas in BAC-Cre lines expressing in cortical layers 2/3 and 5a map the connections between functional cortical areas, which are responsible for integration of information, while injections in BAC-Cre lines expressing in layers 5b and 6 label axonal projections to subcortical systems, which are involved in turning cortical activity into behavior. The labeled axons are visualized using immunohistochemical techniques in 50m sections of the brain and imaged to reveal multiple injections in different cortical areas reveal specific patterns of connectivity between functionally distinct cortical areas and subcortical systems. To analyze these complex patterns of connectivity we developed an efficient process for reconstructing the images through the whole mouse brain using the NIH ImageJ program (Paletzki and Gerfen, 2015). These fully reconstructed whole mouse brain image sets displaying the axonal projections of specific neuron subtypes in multiple cortical cortical areas are registered to a common mouse brain atlas. The ability to register patterns of axonal projections obtained from many brains provides the ability to analyze the complex organization of the neural circuits integrating information between functional cortical areas and how it is transmitted to subcortical circuits responsible for behavior. Work with the Janelia MouseLight Project provides an exciting advance in analysis of the connectivity of cortical neural circuits (Economo et al., 2016). The MouseLight Project has developed a platform for tracing the axonal projections of individual cortical neurons through the whole brain The axonal projections of individual cortical neurons project collaterals to as many as 15 cortical or subcortical areas. In some cases the total axonal length of an individual neurons axon collaterals has been shown to be 10 to 15 cm, which is quite remarkable considering that the mouse brain is approximately 1.5 cm in length from the frontal cortical pole to the cerebellum. The MouseLight Project goal is to provide the axonal projections of 1000 individual neurons over a 2 year period, which will provide a major resource for understanding the organization of brain circuits. We collaborated with the Karpova and Looger labs at Janelia Farms on their development of Adeno-associated viral (AAV) vector variants engineered as efficient retrograde axonal transport markers (Tervo et al., 2016). Viral vectors are versatile tools for tracing axonal connections as they may be combined with other genetic tools such as Cre-driver lines or GCamp to analyze neuronal activity. While AAV vectors are most efficiently taken up by the neuronal soma, the Janelia group developed variants that are transported retrogradely from axonal terminals. This provides an important new research tool. In addition, with the Janelia groups we published a paper reviewing the recent advances in neuroanatomical tract tracing (Gerfen et al., 2017). In a collaboration with the Svoboda lab at Janelia Farms to study brain circuits responsible for movement, we demonstrated that a circuit between the anterior lateral motor cortex (ALM) and the thalamus is responsible for the persistent activity that is preparatory to the initiation of specific behavior (Guo et al., 2017). In two studies analyzing the organization of basal ganglia circuits advantage was taken of BAC-Cre reporter lines we developed for the GENSAT project (Gerfen et al., 2013) that express Cre selectively in either the patch or matrix compartments of the striatum. In one study, modified rabies virus was used to trans-synaptically label the inputs to striatal patch or matrix neurons from the cerebral cortex (Smith et al. 2016). This study extended my work (Gerfen, 1989) that showed cortical inputs to the striatal compartments are related to their laminar distribution. In another study using AAV vectors to trace axonal projections, we demonstrated that striatal patch neurons selectively target dopamine neuron dendrites in the midbrain (Crittenden et al., 2017). A collaboration with the Lin lab at NIDA analyzed the activity of striatal direct and indirect pathway neurons during movement (Barbera et al., 2016). A long standing model of the basal ganglia suggests that activity in the direct pathway facilitates movement while activity in the indirect pathway inhibits movement. Using an innovative approach to visualize the activity of striatal neurons using a molecular marker that is loaded selectively into direct or indirect pathway neurons using BAC-Cre reporter lines, it was possible to monitor the activity in a population of striatal neurons while an animal was awake and moving. Results showed that both direct and indirect pathway neurons are active during movement, but that clusters of neurons of similar neuron type are active at distinct times during the movement. This finding demonstrates that the striatal pathway that inhibits movement plays an important role during movement.
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