Mapping Memory Encoding and Recall Using Translation-Dependent Reporters of Synaptic Potentiation in Physiological and Pathological Conditions

The mechanisms underlying the formation and retention of memory have been long sought after questions in Neuroscience. These central topics in research on the brain are tightly linked with the postulated existence of an “engram”, defined as an enduring modification of the neural tissue, which can remain latent, reactivated only when necessary to obtain the recall of a specific memory. This field has been recently revolutionized by a series of seminal studies in rodents, that have provided strong evidence that specific cell assemblies in the hippocampus, amygdala and cortex are necessary and sufficient for specific episodic memories to be recalled and behaviorally expressed. These findings support the existence of a memory engram at the cellular level. However, parallel studies on the basic mechanisms of information storage in neurons have demonstrated that selected subsets of the synaptic array of a single neuron – possibly belonging to identifiable neural circuits – undergo plastic modifications in response to stimuli inducing learning and memory. Linking these two aspects, namely memory engrams and plasticity at the synaptic resolution, has been largely hindered by a limitation in appropriate tools. This impediment has been recently overcome through the development of systems to label neurons and synapses activated by external inputs. Among these, ‘SynActive’ is a genetic toolbox which utilizes the 5’ and 3’ untranslated regions (UTRs) from the mRNA for the immediate-early gene Arc to confer to recombinant proteins (i.) transport to the base of dendritic spines in a dormant state and (ii.) activity- dependent translation upon synaptic potentiation. The focus of my work has been the in vivo use of gene constructs from the SynActive toolbox, expressed into the mouse hippocampus using adeno-associated viral vectors (AAVs). I first combined the SynActive tool with GFP Reconstitution Across Synaptic Partners (eGRASP) to obtain SA-eGRASP. eGRASP is based on splitting the GFP into two halves, which are expressed pre- and post-synaptically, respectively. Only when these two moieties are facing each other, i.e. are localized at the two sides of a synapse, reconstitution of fully functional, fluorescent GFP occurs. Therefore, SA-eGRASP allows circuit-specific labeling of potentiated synapses. This tool is based on four AAVs, allowing the expression of (i.) presynaptic neuronal label (Turquoise-2 blue fluorescent protein) and rtTA; TetON-controlled presynaptic half of eGRASP; (ii.) TetON-controlled presynaptic half of eGRASP; (iii.) postsynaptic neuronal label (tdTomato) and rtTA and (iv.) a TetON- and Synactive-controlled postsynaptic half of GRASP. By injecting AAVs (i.) and (ii.) within the CA3, and (iii.) and (iv.) to the CA1, SA-eGRASP was employed to identify and map both learning and memory-related synaptic potentiation in the Schaffer collateral pathway. Furthermore, this same method is being used to investigate learning- and memory-dependent synaptic potentiation in a transgenic mouse model of Alzheimer’s disease providing an opportunity to identify possible variations in the cartography of synaptic potentiation throughout disease progression. Moreover, I employed the SynActive approach to drive the expression of an optogenetic probe (channelrhodopsin-XXM; ChRXXM) combined with a fluorescent protein (mVenus) in a time and activity-dependent fashion. In addition, the TetON system was employed to control the time window of transcription of this construct. After careful optimization of the conditions for achieving optimal expression and for preventing ectopic localization of ChRXXM-mVenus in the CA1 region, I could identify the repertoire of synapses that undergo potentiation following induction of the formation of an associative episodic memory via exposure to contextual fear conditioning (CxFC). Notably, Notably, with further characterization, this tool has the potential to provide a method, not only for the identification and mapping of potentiated synapses following a learning- or memory-related protocol, but may allow for their subsequent manipulation through optogenetics, thus representing a stepping stone towards the demonstration of the existence of memory engram at the synaptic level.Taken together, the different sub-projects of the present thesis constitute a coordinated effort to create, validate and employ genetically encoded tools to contribute to shifting the resolution of studies on the physical substrates of memory from the whole neuron to specific subsets of its synapses.