PRE AND POSTSYNAPTIC CELLULAR MECHANISMS OF LTP

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Understanding the mechanisms of learning and memory is a fundamental problem in neurobiology and cellular modifications of synaptic transmission have been implicated as an underlying substrate of learning. Fast synaptic transmission in the vertebrate central nervous system (CNS) is mediated largely by the release of amino acids. This transmission is subject to many modifying influences, including long term potentiation (LTP). LTP is defined as an increase in synaptic efficacy lasting for hours or longer, initiated by a brief period of high frequency stimulation (tetanus) to the synapse. This phenomenon is widespread at glutaminergic synapses in the vertebrate brain including the lamprey rhombencephalon. This from of LTP shares much in common with that seen in the mammalian hippocampus, including the role of glutamate receptor and a post-synaptic calcium flux. For the study of the cellular aspects of LTP the lamprey preparation offers distinct advantages. The large size of lamprey pre- synaptic terminals makes them available to direct intracellular electrophysiological and optical measurement, whilst the unprecedented optical clarity of the tissue affords excellent visibility for in vitro microfluorimetric techniques. The accessibility of the presynaptic terminal will, in particular, be used to investigate the role of this structure, its receptors and putative retrograde messengers in the induction and maintenance of LTP. It is intended to utilize the lamprey CNS to study cellular aspects of LTP in detail. This preparation has distinct advantages for this investigation. Presynaptic axons and terminals are large and may be directly recorded with patch pipettes or microelectrodes. The post- synaptic neurones are very large (up to 400 micromoles) and may be recorded stably for many hours. Additionally the tissue in which the cells are located is optically clear, providing excellent opportunities for imaging and photo-release of caged compounds within an intact preparation. Experiments will be performed using microelectrode or whole-cell patch recordings from both pre- and post-synaptic sides of the synapse. Second messenger pathways involved in induction and maintenance of LTP will be investigated by electrophysiological recording and imaging of Ca2+- sensitive dyes in axons and post-synaptic soma. Intracellular pathways will be experimentally manipulated by precise release of photoreleasable caged compounds (eg caged nucleotides and Ca2+) in precisely defined cellular compartments. This will be achieved by a combination of micro injection of agents into particular cellular structures and targeting of uv radiation to known cellular compartments using a confocal microscope scanner. Micro injection of enzyme inhibitors will also be used to interrupt pathways believed to be involved in LTP (eg CaM kinase II, protein kinase C, guanylate cyclase).