UC Davis

Learning and memory are crucial cognitive processes that promote an organism’s survival, allowing it to rapidly modify its behavior in response to changes in the environment and repeat patterns of behavior that enhance survivability and reproduction. At a cellular level, the phenomena of learning and memory are facilitated by the restructuring of plastic neural circuits that encode behaviors. The dynamic nature of dendritic spines, small, actin-rich, membranous protrusions on the dendrites of neurons, underlies this plasticity and the neural capacity for modification. Dendritic spine are the sites where neurons receive most of their excitatory inputs and several studies have linked the dynamic changes dendritic spines can undergo to learning and lasting memory. Amongst these changes are the spontaneous outgrowth and subsequent persistence of new dendritic spines. New spine outgrowth increases during learning paradigms, and the fraction of these new spines that are retained correlates with improved memory of a learned experience. Little is known about the mechanisms that determine whether a new spine stabilizes and persists, but recent work has demonstrated that strong synaptic stimulation, particularly that which induces long-lasting potentiation (LTP) of spine volume and synaptic strength at mature spines, enhances the survivorship of nascent dendritic spines. This phenomenon can be induced absent of presynaptic input using two-photon (2p) glutamate uncaging of 4-methoxy-7-nitroindolinyl (MNI)-caged glutamate at a high-frequency (HFU) that allows for the spatially restricted release of glutamate at a single dendritic spine. LTP-inducing glutamatergic stimulation initiates several molecular signaling cascades that occur within theiii spine; which of these mechanisms exist in new spines and play a role in enhancing new spine stability remains a mystery. The interaction between the GluN2B subunit of the NMDA-type glutamate receptor (NMDAR) and CaMKII that is activated downstream of NMDAR-mediated Ca2+ influx is one molecular mechanism induced during LTP that has been implicated in new spine survivorship. This interaction was found to be necessary for activity-dependent new spine stabilization and genetically disrupting GluN2B-CaMKII binding blocks the stabilizing effects of glutamatergic stimulation. The work described in this dissertation follows up on these results by further defining the role that CaMKII plays in activity-dependent new spine stabilization. Chapter 1 of this dissertation is a review of the current understanding of new spines, including their role in the dynamic restructuring of neural circuits to facilitate learning and memory, the mechanisms that govern their formation, and the current understanding of the mechanisms that promote their stabilization. Chapter 1 also reviews what is known about the signaling cascades that are involved in nascent dendritic spine stability and the role that CaMKII and its myriad functions and interactions may play in enhancing new spine stabilization. Chapter 2 of this dissertation describes experiments which further define the roles that CaMKII structural and enzymatic activity play in activity-dependent new spine stabilization. 2p imaging and HFU of MNI-caged glutamate was used to stimulate spontaneously formed nascent dendritic spines shortly after outgrowth on CA1 neurons in cultured organotypic hippocampal slices where CaMKIIα enzymatic and structural activities were altered. These experiments provided direct evidence that CaMKIIα structural and scaffolding interactions, but not its kinase activity, are required for the previously reported enhancement of new spine stability following 2p glutamate uncaging. Chapter 3 of this dissertation contains additional analysis performed on the data described in Chapter 2. These analyses corroborate previous results that HFU at a new spine, which enhances survivorship, often results in new spine enlargement, but new evidence described in this chapter suggests that this enlargement is not necessary to stabilize new spines. Also shown in Chapter 3 is evidence that CaMKIIα kinase activity inhibits spine enlargement at new spines, iv despite it being necessary for spine enlargement at mature spines. Chapter 4 describes preliminary experiments that further explore the non-enzymatic role that CaMKIIα performs at new spines by targeting two binding partners that do not require CaMKIIα to function: the 26S proteasome and synGAP-1α. The first section of Chapter 4 describes experiments that aim to determine the role of the 26S proteasome in activity-dependent new spine stabilization. Pharmacological inhibition of the 26S proteasome was utilized to determine if the proteasome had a role in enhancing new spine survivorship rates following HFU. The preliminary results of these experiments show that inhibition of the 26S proteasome increased basal rates of new spine survivorship such that any changes or deficits in activity-induced stabilization could not be determined. The second section of Chapter 4 focuses on the role of synGAP-1α, a negative regulator of synaptic stability, plasticity, and maturation, in activity-dependent new spine stabilization. Green fluorescent protein (GFP)-tagged synGAP-1α was overexpressed in CA1 neurons in cultured organotypic hippocampal slices and HFU of MNI-caged glutamate was used to stimulate individual new spines that had formed on these cells. The preliminary findings of these experiments show that synGAP-1α, which is enriched at lower levels in new spines compared to their mature counterparts, undergoes activity-dependent evacuation from new spines following HFU. This evacuation does not require CaMKIIα kinase activity, as preliminary results show that synGAP-1α dispersion from new spines still occurs during pharmacological inhibition of CaMKIIα. Together, my results demonstrate that non-enzymatic scaffolding activities of CaMKIIα, such as the interactions between CaMKII and the 26S proteasome or synGAP-1α, regulate new spine survivorship and volume changes, providing further insight into the molecular mechanisms that govern new spine stability and maturation.