Our long-term goal is to understand intracellular signalling by cyclic nucleotides (cyclic AMP and cyclic GMP) in neurons. Cyclic nucleotides are crucial regulators of cellular processes including metabolism, cell growth and synaptic transmission. Cyclic AMP plays a major role in triggering changes in synaptic structure and function that occur during short and long term learning. Disruption of cyclic nucleotide signalling is likely to contribute to many clinical disorders, and cyclic nucleotide signalling cascades are the targets of many drugs. While much is known about the biochemistry of cyclic nucleotide signalling cascades, we know little about the dynamics and spatial distribution of these signal in neurons. This proposal is focused on a newly-discovered class of ion channels in olfactory receptor cells that are gated directly by cyclic nucleotides. These channels are crucial for converting a change in cyclic nucleotide concentration into an electrical signal during olfactory signal transduction. In addition to studying the channels per se, we shall use the channels as novel tools for detecting cyclic nucleotides in other cell types. Membrane patches containing the channels will be obtained using the patch clamp technique, and these patches will inserted into molluscan (Aplysia and Helix) neurons, where the activity of the detector channels will reflect the local intracellular concentrations of cyclic AMP and cyclic GMP. We will measure the kinetics of elevation and decay of cyclic nucleotides during neurotransmitter responses that modulate electrical activity and synaptic transmission. In addition we will examine changes in cyclic nucleotides when transmitter is applied paired with action potentials in the neuron. Such pairing is thought to alter cyclic nucleotide metabolism, resulting in long-term synaptic plasticity which contributes to associative conditioning. The ability to monitor cyclic nucleotides with olfactory patches, combined with the use of pharmacological agents, will enable us to determine which steps in the cyclic nucleotide cascade are rate-limiting for the onset and decay of transmitter responses, which steps persist during long-lasting responses, and which steps are altered during pairing. In addition a cloned gene encoding the olfactory cyclic nucleotide-gated channel will be expressed in these neurons, essentially directing the neurons to synthesize their own directly-gated cyclic nucleotide channel detectors. Activation of the expressed channels will be assessed with patch clamp measurements, and alternatively using Ca2+ influx through the channels in cells loaded with Ca2+, indicator dyes. Expression of the cloned channel by the neurons will allow the investigation of the spatial distribution and intracellular spread of cyclic nucleotide signals in response to neurotransmitters.
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