NOVEL APPROACHES TO THE STUDY OF SYNAPTIC PLASTICITY IN THE RAT HIPPOCAMPUS
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Synaptic plasticity refers to changes in the strength of connections between neurons in response to previous activity. Its experimental correlates, i.e., long-term-potentiation (LTP), a persistent increase in synaptic efficacy, and long-term-depression (LTD), a persistent decrease in synaptic efficacy, are the leading cellular models of learning and memory. The observed changes in LTP and LTD are detected by electrophysiological methods as lasting modifications of synaptic transmission, but they may be accompanied by morphological changes in the synapses undergoing plasticity. However, published accounts of the relationship between functional and structural changes are incomplete and often inconsistent. This is partially due to the lack of available tools and technologies allowing direct observations and interpretations of obtained results. In this thesis I explore potential technological advances for investigating neuronal activity, and examine the relationship of functional and structural changes at synapses undergoing plasticity. First, I evaluate a number of novel genetically-encoded calcium indicators (GECIs) for optical monitoring of neuronal activity in rat hippocampal slices. The results indicate that most GECIs are too slow and/or insensitive to follow individual action potentials at high frequencies necessary for some intended uses. Next, I demonstrate that recently-developed ultrafast GECIs provide vastly improved kinetics, but at the expense of weaker fluorescent signals. Further improvements in these indicators may be highly valuable for future applications. Finally, I present a multifaceted approach combining double intracellular electrophysiological recordings from pairs of synaptically connected hippocampal neurons with two-photon-excitation fluorescence microscopy and calcium imaging to study structural plasticity of individually identified synapses undergoing plasticity. My results suggest that physiological stimulation and plasticity induction do not cause significant persistent changes in size of either pre- or postsynaptic elements. In this context, I also describe how optimizing a variety of recording variables (e.g., days in vitro, sub-regional localization, distance between cell pairs) contribute to the success of paired recordings in rat organotypic hippocampal slices, and how it can aid our studies on structural and functional plasticity.