The major goals of this proposal are: (1) to characterize the ionic currents affected by neurotransmitters in sympathetic neurons and (2) to determine the intracellular biochemical mechanisms involved in the transduction of neurotransmitter signals between membrane receptors and ion channels in these cells. The lumbar paravertebral sympathetic ganglia from bullfrogs will be used because they are discrete, easily accessible, and remain viable in vitro for hours. Furthermore, bullfrog sympathetic neurons exhibit the same types of synaptic responses as do their mammalian counterparts. The sympathetic ganglia have been used as a model for the study of synaptic potentials and have proven to be valuable in understanding the mechanisms of synaptic events occurring in less accessible areas of the nervous system, such as the brain and spinal cord. Several recent technical innovations will be applied to the study of these ganglia. First, the ganglia will be enzymatically dissociated to single cells to avoid the complications which arise when studying neurons in intact ganglia due to ion accumulation in the narrow intercellular spaces and due to the release of neurotransmitters or metabolites from surrounding cells. Second, a suction electrode in the whole cell recording configuration will be used to control membrane voltage and record ionic currents, allowing a more precise control of membrane potential over a wider voltage range and with better time resolution than has previously been possible in studies of these cells. Third, the inside of the recording electrode will be perfused with a fine tube placed near the tip of the electrode. By perfusing the inside of the electrode it will be possible to change intracellular constituents. Fourth, a single cell perfusion system will be used for controlling the extracellular medium which will allow the external medium to be changed rapidly and will permit precise application of neurotransmitters. The effects of neurotransmitters on ionic currents will be quantified. The biochemical pathways involved in the neurotransmitter responses will be determined by manipulating the intracellular constituents. Second messenger pathways to be exa- mined include the cyclic nucleotides, cAMP and cGMP, and the pathways activated by G proteins, including diacylglycerol, protein kinase C and inositol trisphosphate.
