Periodic Reporting for period 2 - HighMemory (Beyond classical conditioning: Hippocampal circuits in higher-order memory processes)
Período documentado: 2022-11-01 hasta 2024-04-30
Animals and humans adapt to changes in the environment through the encoding and storage of previous experiences. Although associative learning involving a reinforcer has been the major focus in the field of cognition, other forms of learning are gaining popularity as they are likely more relevant and frequent in human daily choices. Indeed, associations between non-reinforcing stimuli represent the most evolutionarily advanced way to increase the chances of predicting future events and adapting individuals’ behavior. Animals are also able to form these higher-order conditioning processes, but more research is needed to understand how the brain encode and store these complex cognitive processes. The HighMemory project (Figure 1) proposes to study the role of hippocampo-cortical circuits in higherorder conditioning processes. These processes explain why individuals are very often repulsed or attracted by stimuli (persons, places, sounds), which do not have intrinsic repellent or appealing value and were never explicitly paired with negative or positive outcomes. A possible explanation of these “ungrounded” aversions or repulsions is that these stimuli were incidentally associated with other cues directly reinforced. This is called higher-order conditioning or mediated learning (ML). However, with increased number of incidental associations, the subjects acquire more information, allowing the separation between the real saliences of two different stimuli. Therefore, with the increase of training, ML evolves into what researchers define as “reality testing” (RT). Importantly, these behavioral processes involve the hippocampus, are characterized by defined and accessible phases and involve several brain regions, making them perfect models to study the tight regulation of behavior by hippocampo-cortical projections. By using cutting edge genetic (viral and chemogenetic techniques), Ca2+ imaging and mouse behavioral (sensory preconditioning) approaches, the aim of HighMemory is to dissect and characterize, at macro- (brain regions), meso- (cell sub-types) and micro-scale (activity changes), the causal involvement of hippocampo-cortical projections in higher-order cognitive processes, from the formation of ML, the transition to RT and the expression of these distinct mental states. Notably, HighMemory will provide important information to better understand and tackle the physiology of complex cognitive processes and mental disorders such as psychotic-like states.
The first months of the Highmemory project were dedicated to set up the behavioral paradigms required for the research aims proposed. It has been challenging to obtain reliable behavioral models to study higher-order conditioning processes although we have now 3 different mouse behavioral paradigms:
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.
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.
Until the end of the project, we will provide a better understanding of the brain circuits involved in complex cognitive processes using different behavioral tasks to assess higher-order conditioning. In addition, we are generating new behavioral paradigms to assess higher-order conditioning that are not present in the literature and that can be considered new tools to study these complex cognitive functions. In addition, we are also generating new computational tools to assess these higher-order cognitive processes that will be novel and useful tools for the cognitive field. Finally, we have a couple of preliminary results that are promising to provide a new concept in the study of higher-order conditioning, which will be to use social cues and/or stimuli in the animal protocols used. This has been never explored and if it works will provide very interesting findings for the field increasing the translational aspect of studying higher-order conditioning. Moreover, we are also exploring the possibility to try to build a new behavioral set up to evaluate fear memories.