More than 90% of the excitatory synapses in the central nervous system occur on dendritic spines. Changes in the structure of these tiny protrusions have long been implicated in learning and memory, though a clear delineation of such morphological changes has awaited the magnification and resolution provided by serial electron microscopy (EM). Long term potentiation (LTP) is an enduring change in synaptic efficacy that is widely studied as a cellular memory mechanism. Numerous anatomical studies have searched for anatomical correlates of LTP. These studies have lacked sufficient resolution to determine accurately the magnitude of the reported changes in the number of morphology of synapses and dendritic spines. A complete morphometric study is proposed to delineate the anatomical alterations at synapses and dendritic spines, to define their duration, and to relate these to various stages of LTP.
The specific aims are: 1) To delineate the changes in synaptic and dendritic spine morphology that are associated with early and late phases of LTP in the hippocampus of postnatal day 15 (P15). This age is chosen because LTP is robust and enduring, even though the spines, synapses and dendrites have not attained their mature numbers or morphology. Because vigorous synaptogenesis is occurring at this age, multiple candidates exist for morphological plasticity that could subserve LTP. 2) To test whether mature synapses express morphological correlates of LTP that are similar to or different from those delineated at P15. It is essential for the understanding of mature memory mechanisms to establish whether mature synapses are similar to, differ only in degree from, or are categorically different from P15 synapses in their morphological plasticity. Methods: Standard procedures will be used to maintain in vitro hippocampal slices and induce and measure LTP extracellularly. Control hippocampal slices will receive the same stimulation paradigms as the LTP slices, But in the presence of DL-2-amino-phosphono-valeric acid (APV), a known blocker of LTP. Rapid microwave-enhanced fixation and routine processing will be used to prepare slices for electron microscopy. The irregularity in shape of dendritic spines and their synapses makes it impossible to extrapolate from partial measurements to their complete dimensions. Thus, the technically demanding approach of three-dimensional reconstruction from serial EM is required. Historically, the main drawback of serial EM has been the small sample sizes utilized because of the labor-intensive nature of the work. A new unbiased approach for sampling large areas of neutrophil in combination with serial EM is proposed to obtain accurate characterization of synaptic, spine, and dendritic morphologies and to allow for complete quantification of their ultrastructure in three dimensions. Health Relatedness: Improved understanding about the cell biology of learning and memory will help to clarify the cellular mechanisms underlying mental retardation or cognitive deterioration that occurs with many disorders of the central nervous system.
Showing the most recent 10 out of 23 publications
