Abstract

The ability to adapt to environmental changes, to learn and to memorize information is one of the brain’s most extraordinary features. One important process underlying this ability is considered to be synaptic plasticity, i.e. the structural and functional modification of synaptic connections. Synaptic plasticity can occur either by genesis or elimination of synaptic connections, or at existing connections by modifications in the strength of synaptic transmission. Synaptic connections are complex entities consisting of different functional structures: The majority of hippocampal and cortical excitatory synapses are made up of a postsynaptic compartment called dendritic spine and a presynaptic compartment called bouton. Within the spine and the bouton dense molecular structures, which serve the synaptic transmission between pre‐ and postsynapse, exist, namely the postsynaptic density (PSD) in the spine, and the active zone (AZ) in the bouton. All these structures are correlated in size and with synaptic strength. The function of this correlation serves the efficient and fast transmission of neuronal signals. During synaptic plasticity, a coordinated change in the size of all synaptic structures is expected, for the maintenance of their correlation. However, to date, such coordinated modifications have not been examined in detail. Furthermore, the mechanisms underlying the maintenance of structural and functional changes after synaptic plasticity remain poorly understood. The aim of this thesis was to explore these questions. To achieve this I carried out two complementing experimental approaches: In a first set of experiments, I studied changes in spine and PSD size by twophoton time‐lapse imaging to explore correlated modifications in these two synaptic structures. To induce structural spine plasticity I stimulated single dendritic spines of Schaffer collateral synapses in cultured hippocampal slices by two‐photon glutamate uncaging. This was shown previously to be accompanied by an increase in spine size and synaptic strength. To visualize structural plasticity of spines and their PSD, the cytosolic marker tdTomato and EGFP‐tagged structural proteins of the PSD, namely PSD‐95 and Homer1c, were co‐expressed. PSD‐95 and Homer1c are important and abundant scaffolding proteins of the PSD, which have been used previously as markers for PSD size. I found that both PSD‐95 and Homer1c levels increased after spine stimulation. Homer1c increased rather rapidly whereas PSD‐95 did so in a delayed manner relative to the increase in spine volume. Thus, the naïve correlation between PSD protein level and spine volume was only transiently disrupted after plasticity induction, but was reestablished over a time course of 3 hours. Furthermore, PSD‐95 level only increased significantly in spines with persistent enlargement, but not in spines with non‐persistent enlargement. On the other hand, Homer1c level initially increased both in spines with and without persistent enlargement, and then decayed back to original level in spines with non‐persistent enlargement. Because the increase in PSD‐95 level was delayed, I investigated whether the application of the PKA activator forskolin, which supports an increased and persistent enlargement of spines after glutamate uncaging, might promote and therefore accelerate an increase in PSD‐95 level. However, these experiments led to unexpected results: forskolin application neither had an effect on spine volume nor on PSD‐95 level increase. Although PSD‐95 and Homer1c are important and abundant PSD scaffolding proteins, they represent only two out of a multitude of proteins which form the PSD. Consequently, an increase in the PSD marker proteins does not necessarily represent an increase of the PSD as a whole. Therefore, in a second experimental approach, I applied electron microscopy to stimulated spines which displayed a stable enlargement over 3 hours after stimulation. Hereby, I was able not only to reconstruct the spine and the entire PSD, but also the bouton at the stimulated spine: I found that spine, PSD and bouton displayed matching dimensions 3 hours after stimulation, similar to naïve, unstimulated spines. In summary, by combining two‐photon glutamate uncaging with time‐lapse imaging and electron microscopy, I found that spine, the PSD and bouton increase during structural plasticity, and that the correlation between these structures is reestablished after stimulation on a time scale of 3 hours. Furthermore, an increase of synaptic structures correlates with the stabilization of synaptic modifications after plasticity. This suggests a model where the balancing of synaptic structures is a hallmark for the stabilization of structural modifications during synaptic plasticity.