Due to the complexity of the CNS, the connectivity and communications among individual neurons within pattern generating networks remain undefined. The proposed work will probe the fundamentals of motor rhythms by isolating the smallest possible spinal region still capable of pattern generation. This situation will be approached by explanting a portion of the neonatal mouse spinal cord (with or without the hindlimbs) into an isolated support chamber. Motor activity will be recorded extracellularly from: 1) ventral roots; 2) laminae VII - IX of the spinal cord; and 3) selected muscle nerves. This will include spontaneous activity as well as activity evoked by electrical and pharmacological stimulation. Slice cultures of lumbar spinal segments will also be analyzed for pattern generation. All data will be compared to what is known for intact vertebrates as well as to rhythms that develop in dissociated spinal cell culture. Intracellular recordings will be obtained from neurons identified as either motoneurons or interneurons based on antidromic and dorsal root stimulation tests. Information can then be gathered regarding the synaptic input to identified spinal neurons during rhythmicity. The neurite domains of all intracellularly recorded neurons will be labeled using HRP and mapped in relation to their surrounding circuitry using computerized image processing. This will establish a database of characteristic morphological features that can be correlated with the activity patterns produced under a variety of electrical and pharmacological manipulations. In summary, this proposal is to conduct systematic electrophysiological, pharamacological and morphological analyses of the smallest possible mammalian spinal explants, still capable of generating locomotor-like patterns.
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