Joseph Farley, Ph.D. - US grants

The general goal of this proposal is to determine the molecular and biochemical mechanisms by which a family of calcium- and phospholipid- dependent protein kinases (PKC) contribute to memory-related changes in neural excitability of Type B photoreceptors in the eyes of the nudibranch mollusc Hermissenda crassicornis. The amino acid sequences of PKC isozymes will be deduced through nucleotide sequencing of cDNA and genomic DNA clones. This sequence information will be used to construct oligonucleotide probes for in situ hybridization localization of mRNA transcripts for the different isozymes and nay changes in expression levels of these transcripts produced by learning. Recombinant PKC isozymes, as well as authentic proteins purified from the nervous system, will be biochemically characterized. Several post-translational modifications of PKC that might be produced by learning and increase the activity of the enzyme will be assessed. These include the generation of: 1) a catalytic fragment of PKC by limited proteolysis, 2) a membrane-inserted form of PKC which is constitutively active, or 3) a phosphorylated form of PKC with increased activity. Monoclonal and polyclonal antibodies against different PKC isozymes will be generated and used to map the distribution of the isozymes, as well as in functional studies. Learning-produced increases in PKC concentration within Type B cell membranes will be studied in situ using laser confocal scanning optical microscopy. Electrophysiological experiments will determine whether PKC's reductions in K channel activities in Type B photoreceptors reflect a direct phosphorylation of the ion channel complex, or are instead mediated via a recently described putative G-protein. These studies will contribute to our understanding of the molecular bases of a simple form of associative learning and memory.

DESCRIPTION (provided by applicant): We will study cellular processes underlying non-coincidence learning and conditioned inhibition in the mollusk Hermissenda (H.c.). This fundamental, but poorly understood category of associative learning can be readily produced in H.c., by exposing animals to a classical conditioning procedure where light (the CS) signals the absence of rotation (the US). Explicitly unpaired (EU) presentations of light and rotation increase animals' phototactic behavior. We will continue to characterize the persistent excitability changes that are produced in Type B and A photoreceptors within the CS-pathway. We have found that ocular Type B cells from EU-trained, but not control-condition, animals exhibit persistent decreases in their steady-state depolarizing generator potentials (SSGPs), and decreases in light-evoked spike frequencies. We will complete our studies demonstrating that Type A cells show complementary changes with EU-training: increases in SSGPs and light-elicited spike frequency. The ionic bases of the changes in photoreceptor excitability will be studied using voltage-clamp, ion substitution, and pharmacological methods. We will complete our studies that indicate that the decreased excitability of B cells is due to persistent increases in somatic K+ currents (IA and IK-Ca), and further characterize the mechanisms responsible. The contribution of calcium-dependent processes to expression of B cell photoresponse changes will be studied, as well as the contribution of changes in IA to the EU decreases in spike frequency. We will also determine the ionic conductance changes responsible for the increased excitability of Type A cells. We will test for the occurrence of EU-produced changes in synaptic transmission between B-B and B-A photoreceptors, through simultaneous intracellular recordings from pre- and postsynaptic cells on retention days following learning. We will continue our studies of the induction of decreases in B cell excitability, through the use of an in vitro conditioning protocol, and will test for the involvement of calcium, protein phosphatase 1 (PP1), and arachidonic acid (AA) in the production of the EU-associated decreases in B cell excitability. Because temporally-varying [Ca2+]i levels may be a crucial factor as to whether B cells exhibit excitability decreases or increases due to different stimulation regimens involving light and rotation, we will measure B cell [Ca2+]i levels at different time points following light. Ratiometric fura-2 imaging methods will be used to see if the interstimulus intervals that result in successful EU-conditioning match a specific [Ca2+]i level.