This proposal will study the evolution of neural networks that drive locomotion in vertebrates. To understand how animals locomote, it is necessary to identify and study the neurons in the networks that control locomotion. Homologs of a set of excitatory interneurons that participate in locomotor control in fish and tadpoles have now been identified in the mouse spinal cord, based on their expression of the Chx10 transcription factor gene. This proposal will determine whether the function of these Chx10 cells has been conserved in mammals. Optical imaging using calcium-sensitive dyes will be used to study the activity of the Chx10 neurons during the locomotor activity evoked by tail stimulation or transmitter application to the isolated spinal cord. The principal investigator of the collaborative proposal has generated transgenic mice lacking the Chx10 neurons, which will be used to study the effects of this loss on locomotor activity in the isolated spinal cord. Finally, the firing properties of Chx10 neurons will be studied as well as their modulation by serotonin, which helps to initiate locomotion. It is expected that these neurons are essential for normal locomotion, though altered movements (such as synchronous galloping activity) may still occur in the absence of these neurons. Serotonin should modify these neurons to prepare them for participation in the locomotor network. This project will help train a postdoctoral fellow and a minority undergraduate to study the organization and function of neural networks that generate simple rhythmic behaviors.
This grant supported work on how the nervous system organizes locomotor behavior, such as walking and running. The neural networks that control locomotion are located in the spinal cord; the brain sends commands to start and stop, and to walk with a particular gait, but the spinal "Central Pattern Generator", or CPG networks convert these commands into a pattern of muscle contractions to move the legs appropriately. We are studying the neural composition and organization of the locomotor CPG in the mouse spinal cord. Genetic tools now make it possible to label specific types of neurons in mice with fluorescent tags, or to manipulate their activity. We studied the V2a interneurons, which are excitatory neurons that drive other neurons on the same side of the spinal cord. The first project was to study the consequences of genetically deleting these neurons. Mice without V2a interneurons can walk normally at low speeds, but become unstable at intermediate speeds. However, at high speeds, they switch to a stable galloping gait, with synchronous left-right limb movements like a horse or cheetah. Normal mice never do this. The same pattern of left-right alternation at low speeds shifting to left-right synchrony at high speeds can be seen during "fictive locomotion", the activation of the locomotor pattern in the isolated spinal cord upon addition of activating neurotransmitters or electrical stimulation of sensory inputs. These experiments suggest that the V2a interneurons play a role to prevent the gait switch to galloping in normal animals. Why would this be advantageous? Perhaps mice have evolved to be maximally agile, and in their niche it may be more important to be able to turn quickly to dodge and weave, rather than running fast in a straight line. The second project was to study the properties and activity of the V2a interneurons during fictive locomotion. We used transgenic mice where the V2a interneurons were tagged with a fluorescent marker, so we could find them and record their activity. About half of the V2a interneurons become rhythmically active when the locomotor CPG is activated, firing in phase with either the flexor or extensor motoneurons on the same side of the spinal cord. As was predicted from our studies in the intact mutant mouse, the V2a interneurons were weakly active or silent at low fictive locomotor speeds, and became much more active at higher speeds. Thus, they may act to assure left-right movements at high speeds, but not at low speeds, when other, as yet unidentified, interneurons play this role. How can the V2a interneurons control left-right alternation during walking? Our collaborator, Dr. Kamal Sharma, showed that the V2a interneurons make connections with and drive a set of commissural interneurons, which send their axons to the other side of the cord and inhibit the corresponding contralateral neurons. Thus, only one side is active at a time, allowing alternation of the left and right legs. We also studied the intrinsic properties of these neurons. The V2a class turns out to be heterogeneous, with different V2a interneurons firing in different ways to stimulation, and showing different synaptic interactions within each class. We have just completed a project to study the activity of the V2a interneurons during spontaneous mistakes of fictive locomotion, when one set of motoneurons (for example, flexors on the right side) will fail to fire for one or more cycles, but then return at the same phase as before. The V2a interneurons fell into two groups. One group fell silent along with the motoneurons, and lost its synaptic drive, indicating that it is on the output side of the CPG network. The other group continued to fire rhythmically through a motor deletion, showing that it is inside the CPG and associated with the neurons that set the locomotor rhythm. These experiments also allowed us to train two postdoctoral fellows and four undergraduate students in the methods of neuroscientific research. It is important to train the next generation of scientists to carry on research in the future. This work has important consequences not only for understanding how animals and humans walk, but also for future studies of spinal cord injury. Now that the V2a interneurons are known to participate in the locomotor CPG, we can proceed to study the consequences of a spinal cord lesion on their activity and their connections. This may lead to future treatments to keep the CPG in a healthy state after spinal cord injury, so when eventually we understand how to restore the connections from the brain to the spinal cord, the CPG network will be ready to generate locomotion once again.
