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Imaging the Spinal Neural Circuits During Fictive Locomotion
Ronald M. Harris-Warrick, Professor of Neurobiology and Behavior, Cornell University.
Funding: NINDS R01 R37NS017323 Neurotransmitters, Neuromodulators and Motor Systems (4/81-12/09)

Central pattern generators (CPGs) are neural circuits that generate rhythmic outputs and underlie many physiological behaviors, including respiration, locomotion, and mastication. For locomotion, the CPGs are formed by groups of interneurons located within the ventral spinal cord, yet the genetic identities and functions of these neurons remain undefined (Diaz et al., 2007). Recent electrophysiology studies have shown that two groups of interneurons, the Hb9 and Chx10 neurons, may be key players in the circuit. Little is known about their coordinated neural activities during fictive locomotion in the intact spinal cord, and this project is ideal for multiphoton calcium imaging (Wilson et al., 2006; Stosiek et al., 2003). Using a combination of genetics, imaging, and electrophysiology, we aim to record the spatiotemporal activation patterns of identified subpopulations of interneurons and elucidate how these interneurons contribute to the initiation and maintenance of fictive locomotion.

To identify neuronal subpopulations, we used a transgenic mouse line where GFP expression is driven by the Hb9 promoter. In these Hb9-GFP mice, transgene expression is limited to motor neurons and a subset of interneurons in the ventromedial region in the spinal cord. Spinal cords are dissected from neonatal mice and the motor output recorded from the spinal cord using glass suction electrodes to lumbar ventral roots containing the axons of motoneurons that innervate hindlimb muscles. To elicit fictive locomotion, we either bath apply neurotransmitters or electrically stimulate the tail dorsal roots. In the same cord, we bulk-loaded cells with the calcium indicators Fluo-3 AM by bolus injection at a depth of ~100 μm near the midline. Using MPM we identify Hb9 interneurons by their GFP fluorescence and dye-loaded cells by basal fluorescence of Fluo-3 (Fig. 1).  We monitored the fluorescence changes that result from calcium transients in multiple Hb9-GFP interneurons and other non-specific cells. During elicited fictive locomotion episodes, Hb9-GFP interneurons showed activation patterns that were phase-locked with the motor output (Fig. 1). Interestingly, the evoked activity contained a variable number of burst that was different among cells and among episodes. Further work is required to understand the observed activation patterns and how they relate to fictive locomotion.

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Improved signal-to-noise ratios would lead to better identification of when calcium influxes and activities occur, and this project relies heavily on the detector sensitively advances proposed in Core R&D project I. It may be possible to deconvolve the fluorescence signal to obtain instantaneous firing rates. Other cell types, such as Chx10 interneurons, may also be integral components of the CPG. The Harris-Warrick lab is collaborating with Prof. Kamal Sharma at University of Chicago to develop transgenic mouse lines in which these interneurons will be labeled, and our technique can be easily adapted to study these new mouse models. This project continues the Resource’s long-standing interest in combining electrophysiology with deep tissue imaging.

References

Diaz, M.E., D.A. Dombeck, W.W. Webb, R.M. Harris-Warrick. (2007). Serotonin modulates dendritic calcium influx in commissural interneurons in the mouse spinal locomotor network. J. Neurophysiol. in press.

Stosiek, C., O. Garaschuk, K. Holthoff, and A. Konnerth (2003) In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. 100:7319-7324.

Wilson, J.M., D.A. Dombeck, Diaz-Rios, M., R.M. Harris-Warrick, and R.M. Brownstone. (2007). Two-photon calcium imaging of network activity in XFP-expressing neurons in the mouse. J. Neurophysiol. 97:3118-3125.

 

 

 

 

 



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