Periodic Report Summary 1 - MODULATIONSPINALCORD (Modulating motor output in the mammalian spinal cord) Project Context and Objectives:Motor control circuitry of the central nervous system must be flexible so that motor behaviors can be adapted to suit the varying demands of different states, developmental stages, and different environment. Flexibility in motor control is largely provided by neuromodulatory systems which adjust the output of motor circuits by modulating the properties of neurons within them. The spinal circuitry which controls locomotion is subjected to a range of neuromodulatory influences, including some intrinsic to the spinal cord. One such intrinsic neuromodulatory system is the C bouton system. C boutons are large, cholinergic inputs to motor neurons which were first described over 40 years ago. A small cluster of spinal interneurons (V0C) was identified as the sole source of C boutons; a neuron to synapse divergence of 1:1000. We have identified En1 (V1) and Chx10 (V2a) as additional targets of V0C neurons. The V0C subset represents a modulatory inter¬neuro¬nal system designed to fine-tune motor neuron firing and muscle activation according to the demands of particular locomotor tasks. Genetic inactivation of the cholinergic output of the V0C subset, leads to partial loss of the ability to increase muscle activation in a task dependent manner. Because of the abundance of the C boutons on motor neurons, a more severe phenotype was expected, suggesting i) compensatory mechanisms, ii) the presence of a second neurotransmitter in the synapse or iii) the contribution of other modulatory systems. To overcome compensation we set up a virus based system in order to acutely inactivate or force activate V0C neurons by light and examine the impact on locomotion. The observation that the C bouton synapse persisted after the inactivation of the cholinergic output points to the existence of a second functional neurotransmitter. Indeed, our recent data indicate that the neurotransmitter Cart (cocaine amphetamine regulated transcript) is present in somata and terminals of V0C neurons. Confocal microscopy and the use of Volocity software confirm Cart presence in the presynaptic space. Finally, we consider the V0G subset as a candidate for a “similar function” modulatory system. V0C and V0G, the smallest homogeneous subsets identified so far, share a p0 domain origin, as well as the expression of the transcription factor Pitx2, indicating a possible common function. We are exploring the hypothesis that the two subsets together constitute a modulatory operon for motor control. Analysis of their connectivity revealed that V0C form synapses on V0G somata and proximal processes and vice versa, providing the first evidence of their communication. In parallel, we are thoroughly analyzing the anatomy and function of the V0G subset. Motor neurons were already excluded as targets of this particular subset. We have recently found that V0G neurons contact Chx10 (V2a) interneurons and preliminary data suggest that V2a neurons send projections to V0G neurons as well. We are currently analyzing the effects of inactivation of V0G neurons in locomotion.Understanding organization of this circuit and its role in locomotion may provide far-reaching insights into the logic of neuronal circuit assembly in other regions of the CNS.