This collaborative project aims to study the neurons in the spinal cord that regulate how animals walk. A genetic marker for a small group of neurons has recently been identified. This marker gene has allowed the use genetic manipulations in the mice to test the function(s) of identified neurons. A series of mice have been generated using gene targeting. Preliminary studies suggest that these neurons are critical for controlling the movement of left and right legs as the mice walk. This project will investigate how these neurons function and explore the nature of the neuronal circuit that controls walking in mice. These basic questions will be addressed using transgenic mice that have been generated. Analysis of these mice will include a variety of anatomical and functional assays. The anatomical studies will reveal how these neurons connect to motor circuits in the spinal cord. The functional assays are designed to test the physiological significance of these neurons for motor control. This collaboration will involve students at both institutions, and will help both groups learn to combine molecular genetics and electrophysiological approaches to the study of neural circuits.
Project Title: Collaborative Research: Excitatory Interneurons in the Mouse Locomotor PI: Kamal Sharma In this collaborative research project we have investigated neural networks in the spinal cord that control locomotion in mammals. Locomotion is a complex behavior that requires coordinated movement of multiple joints in each leg. For many decades we have known that neural networks in the spinal cord produce this coordinated movement. However, individual neurons that make up these neural networks have not been identified. In this project we focused on one excitatory class of neurons called the V2a interneurons. In the mouse spinal cord these neurons uniquely express a gene called chx10. By targeting a transgene that encodes a fluorescent protein marker (CFP, cyan fluorescent protein) to the chx10 gene locus, we were able to label V2a neurons in the spinal cord. (1) V2a neurons control gait: In this study we asked whether V2a neurons play a role in neural control of gait. Many animals are capable of changing gait with speed of locomotion. The neural basis of gait control and its dependence on speed are not fully understood. Mice normally use a single "trotting" gait while running at all speeds, either over ground or on a treadmill. Transgenic mouse mutants in which the trotting is replaced by hopping also lack a speed-dependent change in gait. Here we describe a transgenic mouse model in which the V2a interneurons have been ablated by targeted expression of diphtheria toxin A chain (DTA) under the control of the Chx10 gene promoter (Chx10::DTA mice). Chx10::DTA mice show normal trotting gait at slow speeds but transition to a galloping gait as speed increases. Although left–right limb coordination is altered in Chx10::DTA mice at fast speed, alternation of forelegs and hind legs and the relative duration of swing and stance phases for individual limbs is unchanged compared with wild-type mice. These results show that spinal V2a interneurons are required for maintaining left–right alternation at high speeds. Whether animals that generate galloping or hopping gaits, characterized by synchronous movement of left and right forelegs and hindlegs, have lost or modified the function of V2a interneurons is an intriguing question. In Mice Lacking V2a Interneurons, Gait Depends on Speed of Locomotion. Crone, S.A., Zhong, Guisheng, Harris-Warrick Ronald and Sharma, K. (2009), J Neurosci 29, 7098-7109. (2) How V2a neurons control locomotion: In this study we asked how V2a neurons behave during locomotion and correlated their activity with the locomotor rhythm. We used the Chx10-CFP mouse line to investigate the properties and locomotion-related activity of V2a interneurons in the mouse spinal cord. We found that V2a interneurons can be divided into three classes, based on their tonic, phasic or delayed-onset responses to step depolarization. Electrical coupling is found only between neurons of same class, and helps to synchronize neuronal activity within the class. We found that about half of V2a interneurons fire rhythmically with ventral root-recorded motor activity; the rhythmic V2a interneurons fired during one half of the cycle, in phase with either the ipsilateral or contralateral L2 ventral root bursts. The percentage of rhythmically firing V2a interneurons increases during higher frequency fictive locomotion, and they become significantly more rhythmic in their firing during the locomotor cycle; this may help to explain the frequency-dependent shift in left-right coupling in Chx10::DTA mice which lack these neurons. These results reinforce the proposal that the V2a interneurons are components of the hindlimb CPG, helping to organize left-right locomotor coordination in the neonatal mouse spinal cord. Electrophysiological characterization of V2a interneurons and their locomotor-related activity in the neonatal mouse spinal cord. Zhong, G., Droho, S., Crone, S.A., Dietz, S., Kwan, A.C., Webb, W.W., Sharma, K., and Harris-Warrick, R.M. (2010) J Neurosci 30, 170-182. (3) How V2a neurons change their activity depending on the speed of locomotion: How does the nervous system control leg movement when animals walk or run at different speeds? The principles governing the recruitment of interneurons during acceleration in vertebrate locomotion are unknown. The V2a spinal interneurons are dispensable for left–right coordination at low locomotor frequencies, but their function is essential for maintaining left–right coordination at high frequencies. In this study we explored the mechanisms driving this frequency-dependent role using four methods to determine how V2a interneurons are recruited at different locomotor frequencies. We showed that half of the V2a interneurons receive rhythmic locomotor synaptic drive, which increases with cycle frequency, recruiting more of the neurons to fire at higher frequencies. The increased role of V2a interneurons at higher locomotor frequencies arises from increased synaptic drive to recruit subthreshold oscillating V2a neurons, and not from recruitment of a second set of silent V2a interneurons. Frequency-dependent recruitment of V2a interneurons during fictive locomotion in the mouse spinal cord. (2011) Zhong, G., Sharma, K. and Harris-Warrick, R.M. Nature Communications ;2:274.
