As the population ages, memory dysfunction is an increasingly prevalent issue resulting from neurodegenerative diseases such as Alzheimer’s disease and dementia. Despite the resulting decline in quality of life, current treatment options remain limited for this patient population. These treatment options do not target specific neural circuits, remaining agnostic to the cognitive processes underlying memory. To increase therapeutic specificity for neurodegenerative diseases, a fundamental understanding of memory processes is required. Memory recall is traditionally induced by external stimuli, though recent advances have allowed for memory reactivation through neural stimulation. The network effects of stimulation induced memory reactivation remain unknown because the dynamics resultant of such stimulation have not been well studied. In this work I first increase the fidelity of recording from multiple neuronal populations before developing novel methods for stimulating memory associated neurons and apply these techniques to investigate the downstream dynamics during induced memory reactivation. Investigation of expressing multiple fluorophores resulted in significantly improved co-expression over previously established methods, thus enabling high fidelity dual color imaging. After optimizing fluorophore expression for imaging, I next developed a novel method to conduct induced memory recall. Previously, only the blue-light activated channelrhodopsin had been used for induced memory reactivation, thus limiting experimental designs to those compatible with blue light stimulation. This work demonstrates the potential of ChrimsonR, a red-shifted opsin, to induce memory recall and shows that ChrimsonR induced memory recall can take place in both open field and head-fixed experimental paradigms. These results enable calcium imaging during induced memory recall, allowing for all-optical stimulation and recording of memory-associated neuron activity. Having developed the tools for observing neural activity during induced memory recall, I then applied this approach to observe the downstream network dynamics during upstream memory-associated neuron stimulation. This investigation uncovered the inhibitory mechanism responsible for reducing the firing of downstream non memory-associated cells during stimulation. This mechanism provides novel insight into the neural basis of induced memory recall. Additionally, this demonstration of calcium imaging during induced memory recall provides a proof of concept for future investigation of the dynamics of individual memories throughout the brain. / 2025-01-17T00:00:00Z

In the thesis is described the development of two platforms to optogenetically induce single neuron excitation and formation of memory in biological in-vitro neuronal networks.One platform is a photonic chip, with aperiodic grating scatterers able to create a specific light distribution on the surface of the chip. The second platform is a digital light processor device integrated in a microscopy setup, able to create on the sample plane whatever pattern with 3 um resolution. Stimulating simultaneously many neurons, it was demonstrated the ability of this system to induce potentiation on the illuminated cells, and thus engram formation.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Kristýna Ludínova Supervisor: PharmDr. Jan Zitko, Ph.D. External supervisors: Dr. Manfred Oswald, Prof. Dr. Rohini Kuner Title of diploma thesis: Delineating Pain and Fear Engrams in the Prefrontal Cortex Pain is a complex process associated with activation of various brain centres. According to evidence of imaging studies in humans and rodents the medial prefrontal cortex (mPFC) ranks amongst the brain area consistently activated during painful perception. The mPFC circuits underlies functionally-distinct processes, such as pain, emotional response, decision-making, attention amongst others. However, the precise contribution of mPFC in pain related function remains to be unknown. The aim of the study was to delineate how pain and fear are manifested at the cellular level within the regions of PFC. By employing activity dependent neuronal labelling we tested if cellular ensembles activated in pain and fear behaviours within the mPFC are distinct. We investigated a potential use of activity-dependent DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) expression in order to test for the functional role of PFC ensembles in pain and fear behaviour. Our...

Animals learn from past experience to guide future behavior and improve survival. This ability relies in part on specific episodic memories of past events encoded by neuronal activity and stored by updated connectivity between neurons. The unique architecture and activity of the hippocampus and related cortical regions are crucial for supporting these episodic memories. Hippocampal models propose the need for a pattern separation function to disambiguate similar memories and a pattern completion function to recall the full breadth of an experience from a partial cue. Past work suggests that neuronal activity in the dentate gyrus (DG) of hippocampus contributes to memory-guided navigation and plays a role in pattern separation. We tested the role of specific DG neuronal ensembles (i.e. engrams) in supporting the pattern separation function and altering downstream neural activity and, ultimately, behavior. To that end, we used an activity-dependent labeling paradigm to identify and manipulate engram ensembles during navigational and contextual fear conditioning (CFC) tasks. The results of our first experiment revealed that the DG partially disambiguates specific maze trajectories while still exhibiting greater overlap than chance levels. These findings suggest that the DG contributes to memory-guided navigation by both pattern separation and completion. Our second experiment manipulated nonspecific memory-related DG populations to assess the functional role of these cells in task generalization across contexts and ongoing spatial working memory. Optogenetic activation of these ensembles disrupted performance accuracy and exhibited a time-dependent impairment effect suggesting a role of the DG in task generalization between contexts. The final experiments investigated the physiological ramifications of artificial memory ensemble reactivation during ongoing navigation behavior. We recorded local field potential (LFP) and single unit responses in mouse DG and CA1 during artificial reactivation of a DG-mediated CFC memory engram. Stimulation of the DG entrained LFP and individual cell spiking in a subpopulation of CA1 pyramidal cells. Their spatial information was disrupted by stimulation despite stable navigational representation before and after the manipulation. Further, the presence of stimulation could be reliably decoded by the firing rate of the network, suggesting that engram reactivation forced the CA1 to adopt a repeatable state, perhaps to support behavioral expression of memories. In summary, my dissertation work presents empirical and theoretical evidence for the role of the dentate gyrus as a single node of an extended separation/completion circuit distributed anatomically and temporally as a neural mechanism supporting episodic memory.