Society for Neuroscience Chicago: Methods: Electrophysiology Physiological Methods Support: Hope Center for Neurological Disorders 2014 Pilot Award
Authors: *J. R. GAMBLE, S. SAKIYAMA-ELBERT, D. L. BARBOUR; Biomed. Engin., Washington Univ. In St Louis, Saint Louis, MO
Disclosures: J.R. Gamble: None. S. Sakiyama-Elbert: None. D.L. Barbour: None.
Recent stem cell transplantation therapies for spinal cord injury (SCI) have elicited encouraging though limited improvement in motor and sensory function using spinal cord neural progenitors and embryonic stem cells (ES). This recovery appears to be promoted through various mechanisms, including the creation of new spinal circuits 1) externally, between grafted neuronal populations and the endogenous host circuit, and 2) internally, within the transplant. Current assays of transplant efficacy in animal models of SCI include gross measures of electrophysiological input/output, as well as molecular quantifications of graft neurite outgrowth and synapse formation. Missing is any assessment of the functional connectivity within the graft network, owing to the insurmountable challenges making these measurements in vivo. This shortcoming poses a significant barrier to designing effective SCI therapies because functional interactions involving transplanted ES-derived neuronal populations will greatly affect the transmission of activity through the transplant. We have developed a novel high-throughput in vitro assay combining ES technology and microelectrode array (MEA) platforms to study functional connectivity of candidate neuronal populations directly for the first time. To induce transplant candidate populations enriched for Chx10-expressing V2a interneurons, which are excitatory and ipsilaterally projecting spinal cord neurons in vivo, mouse RW4 ESs were subjected to an appropriate 2-/4+ protocol in vitro prior to being dissociated and cultured on 60channel MEAs. Recorded spike waveforms were sorted offline using thresholding, principal components analysis and k-means clustering. After two weeks in culture, neural network activity revealed two regimes of activity: synchronized network bursting and spontaneous random firing. Different patterns of putative neuron-neuron connectivity, including monosynaptic and disynaptic connections, were inferred from spike-sorted activity using spike-time cross correlation histograms.