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Linking glutamatergic spinal cord and brainstem neuronal circuits to the control of locomotor behavior

Mid-Term Report Summary - NEURONSINMOTION (Linking glutamatergic spinal cord and brainstem neuronal circuits to the control of locomotor behavior)

The major goal of this proposal is to understand the functional organization of the excitatory spinal neuron networks that generate rhythmic locomotion in mammals, including the organization of commands signals from the brain and their integration in the network. In the grant period we have addressed a number of specific research objectives related to this general goal.

To determine the specific molecular coding of rhythm generating glutamatergic neurons in the locomotor region of the ventral spinal cord we have used sensitive fluorescence activated cell-sorting of different populations of GFP labeled glutamatergic neurons, followed by RNA sequencing to capture their transcriptomes. The comparative analysis of the transcriptomes from these populations was hold against bioinformatics databases and immunohistochemical/in situ hybridization expression patterns to identify new molecular markers that may delineate rhythm generating populations. To create tools to link the glutamatergic neuron populations to behavior, we have crated mouse strains that allow acute optogenetic activation and inactivation of neurons using either the cre-loxP recombination system alone or in combination with the Flp-Frt recombination system. This intersectional approach provides us with the degree of specificity needed to target specific neuronal populations for physiological experiments.

In physiological experiments we have used optogenetics in transgene mice to show that activation of glutamatergic neurons is both sufficient and necessary for generating locomotor-like activity. Using this genetically driven functional dichotomy, we demonstrate that the mammalian hindlimb locomotor network can be segregated into multiple intrinsically rhythmogenic modules. These experiments differentiate among several proposed models for rhythm generation in vertebrates and define the most basic units of the locomotor network in rodents as independent rhythmogenic modules (Hägglund…Kiehn 2013, PNAS). Using a suite of molecular techniques to identify, chronically silence, and optogenetically control glutamatergic neurons expressing the transcription factor Shox2, we show that a subpopulation of these neurons constituent neurons of the locomotor rhythm generator (Dougherty et al. 2013). These experiments are the first of the their kind to molecularly identify rhythm generating neurons in mammalian spinal cord. The study defines two other populations of excitatory neurons involved in pattern generation. In a separate study we have functionally defined spinal EphA4 signaling in excitatory neurons as required for apposite organization of the spinal locomotor circuitry (Borgius… Kiehn 2014 Journal of Neuroscience). The study suggests a role for the glutamatergic EphA4 expressing neurons in rhythm generating in the mammalian locomotor network. In a model study we have tested the potential connectivity of these cells and provided predictive schemes for the connectivity matrix (Rybak…Kiehn 2013, J. Physiol.)

As part of the grant we also project the excitatory network structures onto the left-right and flexor extensor coordinating circuitries previously defined by us and others to provide an understanding of the network structure. We show that a group of transcriptional defined commissural neurons the V0 population is necessary for sustaining left-right alternation during locomotion (Talpalar…Kiehn 2013, Nature). The V0 population is composed of inhibitory V0 neurons that are important for securing alternation at low frequencies of locomotion while the excitatory V0 population is essential for securing left-right alteration and high frequencies of locomotion. The study shows that the central controls of locomotion incorporates a modular organization and suggest that the different excitatory interneurons are driving these modules at different speeds of locomotion (Talpalar et al. 2013; Rybak et al. 2013).

Finally, we have mapped the distribution of glutamatergic neurons in the brain stem, with focused optogenetic stimulation and revealed the expression pattern of different populations of molecular defined glutamatergic neurons in the brain stem. Using transsynaptic labeling and anterograde labeling we start to define the connectivity matrix of these descending fiber systems. These studies start to delineated the molecular code of the descending activation system and its integration in the locomotor network.