Periodic Reporting for period 2 - HighMemory (Beyond classical conditioning: Hippocampal circuits in higher-order memory processes)
Période du rapport: 2022-11-01 au 2024-04-30
1. Odor-Taste Sensory Preconditioning. We have set up a odor-taste sensory preconditioning where animals show clear mediated and direct conditioning responses. We have slightly modified the protocol due to technical challenges, but we have now an interesting paradigm to study the brain circuits involved in these complex cognitive processes. This behavioral model has been presented in the IBRO meeting (González-Parra JA. et al. September 2023).
2. Light-Tone Sensory Preconditioning. We have also set up a light-tone Sensory preconditioning to be able to study if the brain circuits involved in higher-order conditioning are shared between different sensory modalities. This behavioral model has been presented in several meetings such as Neuronus (Pinho J et al. 2022), FENS (Pinho J et al. July 2022) and IBRO (Pinho J et al. September 2023).
3. Light-Tone Second-Order Conditioning. We have set up a light-tone second-order conditioning to explore the brain mechanisms and potential differences between different behavioral paradigms used to study higher-order conditioning. This behavioral model was presented in the IBRO meeting (Canela M. et al., September 2023).
After setting up all these behavioral protocols we have mainly developed Aim 1 and 2:
Aim 1. Characterizing hippocampo-cortical circuits in sensory preconditioning.
In this aim, we wanted to combine two approaches to characterize the activity of hippocampo-cortical projections engaged during higher-order conditioning paradigms. These two approaches proposed where based on (1) cFOS-related experiments and (2) the fiber photometry approaches.
(1) cFOS-related experiments. In this sub-aim we have modified the tools used although the main aim of using cFOS-dependent approaches to identify brain circuits involved in higher-order conditioning has been maintained. In this regard, we have acquired the (TRAP2) mice, which allow us to induce Cre recombination in Fos-expressing cells and/or brain circuits engaged by specific stimuli or behavioral experiences. In addition, we have crossed this mouse line with the Ai14 mice, which express tdtomato (red fluorescent protein) in a cre-dependent manner. With this transgenic mice we can identify the brain regions (or brain cells) activated in two different behavioral phases in the same animal. For example, during the preconditioning phase through the injection of 4-Hydroxi-Tamoxifen (4-OHT) and the induction of tdtomato expression and during the probe test with the classical cFOS analysis. Overall, we are able to identify the memory engrams created during associations between low-salience stimuli (e.g. light-tone or odor-taste) in the preconditioning and compare them with the cells activated during the mediated learning test. We have applied this approach with the 3 different behavioral protocols presented above and we are now analyzing the brain circuits involved in light-tone sensory preconditioning and the light-tone second-order conditioning. However, in the odor-taste sensory preconditioning we are more advanced as the results using the TRAP2 mice have identified the hippocampal sub-region Dentate Gyrus and the amygdala as key brain regions engaged during the preconditioning phase and tests.
(2) Fiber Photometry. We have acquired all the equipment, set up the surgical approaches and the photometry recordings in the lab and we are also developing some python scripts to analyze the calcium imaging data and couple it with behavior. All this setting up steps were required to have a functional fiber photometry technique to explore activity-dependent changes in particular phases of higher-order conditioning paradigms. In this sense, we have analyzed the activity of neurons and astrocytes in dorsal and ventral hippocampus during the different phases of light-tone sensory preconditioning and we have obtained interesting results that suggest the involvement of hippocampal astrocytes in the encoding of associations between low-salience stimuli. However, all this part of the project will have to be confirmed in the next months.
Aim 2. Modulating hippocampo-cortical circuits in sensory preconditioning.
In this aim, we wanted to establish firm causal relationships between the activity of hippocampo-cortical projections and higher-order conditioning. As a result of our TRAP2 data, we decided to inhibit the activity of the central and/or basolateral amygdala using the infusion of inhibitory DREADDS into this brain region. Interestingly, the chemogenetic inhibition of the amygdala during the preconditioning phase of the odor-taste sensory preconditioning, prevent the formation of mediated learning. Thus, we are identifying a new brain circuit, where the amygdala plays a crucial role, involved in higher-order conditioning. In future experiments, we will perform similar experiments but modulating specific projections from or to the amygdala or other brain regions such as the hippocampus.