This proposal aims to investigate the electrophysiological/ pharmacological characteristics and subunit composition of GABA receptors located on human primary afferent neurons. The proposed research stems from intriguing results of recent investigations which establish that cultured human primary afferent neurons express GABA receptors with unique, and hitherto undescribed, pharmacological properties. An association with both a large transplant center and an internationally recognized spinal cord trauma unit has provided the applicants with access to human neurons. Three separate laboratories with expertise in: (1) neuronal cell culturing; (2) electrophysiology and pharmacology of GABA receptors; and (3) molecular biology of neurotransmitter receptors will participate in these studies. The overall intent is to clarify the properties of these novel human GABA receptors by using a combination of sophisticated electrophysiological techniques, pharmacological probes, and molecular biological methods so as to define the channel/molecular properties and to determine the GABA receptor subunits in human sensory neurons. The proposed investigations are important to neurobiology, implicated in disease states (e.g., spinal spasticity, pathophysiology of pain). The results will be relevant to understanding how spinal neuron excitability is controlled by therapeutic agents that alter afferent transmission by acting at GABA receptors, and hence should foster eventual development of more efficacious pharmacological treatment of spinal cord injuries, pain states, and other pathologies. Whole-cell, cell-attached, and outside-out patch clamp recordings from cultured embryonic and adult human dorsal root ganglion (DRG) neurons will be used to define the electrophysiological/pharmacological properties of GABA receptors. The results of these investigations will be compared with data from molecular biological experiments designed to determine the subset of GABA receptor subunit-encoding mRNAs expressed by cultured embryonic and adult human DRG neurons. RT-PCR on cultures of DRG neurons will reveal the potential array of involved subunits. Based on conclusions drawn from these data, the candidate subunit combinations reproducing the properties of the novel GABA receptor will be identified using exogenous expression systems and antisense oligonucleotides. The reduced number of subunits potentially responsible for the observed properties reduced in this manner to a manageable number will allow performance of single-neuron RT-PCR to correlate expression of functional properties with expression of particular subunit mRNAs. These investigations will allow the applicants to specify the properties of a defined human GABA receptor with unique pharmacological properties within a native neuronal environment.
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