Patch clamp recording will be used to study the properties of single channel and whole cell events activated by glutamate (the probable neurotransmitter mediating fast synaptic excitation) and to compare these to the properties of fast synaptic excitation in cultures of hippocampal and spinal cord neurons. Conditions have been found in which a phenomenon similar to long term potentiation can be induced between two neurons in dissociated cultures of hippocampus. this presents the first opportunity where the molecular mechanisms which produce long term potentiation can be examined in both pre- and postsynaptic neurons simultaneously using dual whole cell recordings. In addition, a new but simple technique of patch pipette perfusion will permit intracellular injection of compounds and enzyme systems thought to modulate or mediate synaptic plasticity. Material can be independently injected into either neuron of synaptically coupled pairs after a period of recording in control conditions thus limiting the action of drugs and enzymes to either pre- or postsynaptic elements. This will permit detailed examination of the loci of actions of drugs which previously could only be applied globally. Three protein kinase systems will be investigated for effects on synaptic efficacy and plasticity. Calcium/calmodulin type II protein kinase, protein kinase C and protein kinase A have all been implicated as factors which control or modulate synaptic plasticity. Intracellular injections of both pre- and postsynaptic neurons with the activated kinases and co-factors as well as their activators and inhibitors will be used to identify the loci of action of the kinases. The effects of these systems on single channel events will also be studied using cell-attached and excised patches in order to determine their mechanisms of action at the molecular level. The information sought by the proposed research will increase our understanding of the molecular mechanisms of chemically mediated synaptic transmission and synaptic plasticity. Since long term potentiation is one of the best cellular models for long term information storage in the central nervous system, determining its basic mechanism could aid our understanding of learning and memory and therefore deficits in these functions in pathological states.
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