Project 5: Peripheral nerve injury causes major alterations in neuronal activity, excitability, and synaptic organization in spinal locomotor circuits and motoneurons. The intrinsic membrane and integrative properties of neurons in the central nervous system depend on highly regulated expression and subcellular distribution of specific ion channels. Membrane ion channel clustering possibly represents an important and functionally significant mechanism for sequestering channels in specific membrane microdomains, presumably co- localized with other key signaling molecules, receptors, and ion channels. Voltage-gated Kv2.1 (delayed rectifier) channels are highly clustered at specific postsynaptic sites in motoneurons and the goal of this project is to elucidate mechanisms by which axon injury alters the subcellular distribution and clustering of membrane Kv2.1 channels, and to examine the functional relationships between altered channel distribution and intrinsic neuronal membrane properties. By studying the dynamics of Kv2.1 clustering in detail the first Specific Aim will test the hypothesis that motor axon damage, in vivo, causes Kv2.1 ion channels to become diffusely distributed in the somatic and proximal dendritic membrane following their dissociation from well defined large clusters. The second Specific Aim will consider the possible functional implications of any changes in Kv2.1 channel distribution, using electrophysiological analysis of intrinsic membrane properties in vivo. In parallel with PPG Projects 1 and 2, the quantitative confocal microscopic immunohistochemical analyses will extend over the complete time course of the motoneuron's response to axon injury, regeneration, muscle reinnervation (or lack thereof), and the loss and subsequent remodeling of identified excitatory and inhibitory synapses on the motoneuron soma. Complementary studies of channel localization and physiological role will also be performed for calcium-activated (SK2 and SK3) potassium channels and hyperpolarization-activated mixed cation channels (HCN1) that are abundantly expressed in spinal motoneurons and that underlie critical membrane properties that are altered after axotomy. This project will address fundamental gaps in our knowledge of ion channel organization in motoneurons and provide novel insight regarding the regulation of key membrane ion channels selected from the large number of channels that potentially make important contributions to cell excitability and integrative properties in normal and injured motoneurons (MNs). Importantly, this project helps to tie together two themes of this PPG, namely, the effects of injury per se and the activity-dependent regulation of cell excitability and will thus provide key insights into how postsynaptic excitability changes may contribute to altered network function following nerve injury.
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