Investigating the neural ensembles underlying the encoding of memory in zebrafish

Project Information

ZebraHipNetwork

Grant agreement ID: 101066743

DOI

10.3030/101066743

Project closed

EC signature date 27 June 2022

Start date 1 December 2022

End date 30 November 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 226 751,04

Coordinated by

NORGES TEKNISK-NATURVITENSKAPELIGE UNIVERSITET NTNU
Norway

Periodic Reporting for period 1 - ZebraHipNetwork (Investigating the neural ensembles underlying the encoding of memory in zebrafish)

Reporting period: 2022-12-01 to 2024-11-30

Summary of the context and overall objectives of the project

Learning and the formation of new memories is a fundamental mechanism that allows animals and human alike to adapt and survive in their respective environments. In mammals, the hippocampus is responsible for the formation of new memories. This structure is highly interconnected with other cortical and limbic brain regions forming multiple subnetworks within the mammalian brain. Given the overlapping nature of these subnetworks, we still do not fully understand how these inter-regional connections affect the formation of new memories. To investigate this, we would have to record the neural activity from the entire brain of an awake animal. But given the large size and complexity of the mammalian brain, this makes it extremely difficult to record the neural activity of the hippocampal network at a single-cell resolution in awake animals. Instead, other animal models, such as the semi-transparent zebrafish gives us the opportunity to record the neural activity of the entire telencephalon (thousands of cells) at a single-cell resolution. Unlike mammals, zebrafish do not have a properly defined cortex, however, many past studies have shown that its dorsal telencephalon (forebrain) contains many cortical-like structures including a hippocampal-like region, the dorsal lateral telencephalon (DL).
The overall aim of this project was to investigate how does the network features of the zebrafish hippocampal analogue (DL) facilitates the encoding of new memories. To do so, I first examined the molecular and biophysical characteristics of the zebrafish DL neurons. Afterwards, to characterize the connectivity of the DL network, I have combined neuroanatomical tracing with electrophysiology to map the anatomical connections between the DL and its adjacent brain regions. More importantly, this allowed me to characterize the main inputs and outputs of the DL. Next, to understand how the zebrafish DL process sensory information prior to learning, I used two-photon calcium imaging to record the neural activity of populations of neurons while presenting the head-restrained fish with different sensory modalities. Finally, I then used a classical conditioning paradigm to investigate how learning affects the DL network. The results from this ZebraHipNetwork project will therefore allow us to unveil the key neural mechanisms underlying the formation of new memories that are conserved across species.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

First, to characterize the major cell types of the hippocampal analogue (DL) in the zebrafish telencephalon, I collaborated with my colleagues to identify the molecular identity of the different cell types using spatial transcriptomics. Results from these analyses have revealed that hippocampal-like cells were highly dispersed into different regions in the zebrafish dorsal telencephalon. Next, to understand how sensory information is processed by the hippocampal-like cells, I first used anatomical tracing methods in combination with electrophysiological recordings to map out the main connectivity patterns entering and existing the telencephalon as well as the interregional connectivity between the DL and adjacent regions. These results allowed me to create a connectivity map of the dorsal telencephalon and interestingly, I found that the major input arriving to the dorsal telencephalon originate from the preglomerular nucleus (PG), which is believed to be a thalamic-like structure in fish. Afterwards, I performed in vivo whole-brain recordings using Ca2+ imaging in head-restrained fish while simultaneously presenting the fish with different sensory modalities (red light vs mechanical vibrations). I found that different sensory modalities were encoded by different cells within the PG further supporting its analogous role as a thalamic-like structure. In contrast, specific regions in the zebrafish dorsal telencephalon preferentially encoded the red light (DL), while another preferentially encoded the mechanical vibration. Additionally, there were population of neurons in the dorsal central telencephalic area that would invariantly encode either tested sensory modalities and other neurons in the anterior telencephalon that could integrate i.e. combine both sensory modalities. These results therefore revealed that sensory information is processed in a hierarchical manner in the zebrafish dorsal telencephalon, a network architecture that is reminiscent of the mammalian system.
Now that we know how sensory information is processed in the zebrafish telencephalon and in DL, I then proceeded to record the neural activity of the zebrafish telencephalon while the head-restrained fish underwent a classical conditioning paradigm. This learning paradigm was designed to teach the fish to associate a neutral stimulus (red light) to an aversive stimulus (mechanical vibration). By recording the neural activity of thousands of neurons throughout this paradigm, I found cells that responded to distinct phases of the behavioral paradigm in distinct regions of the dorsal telencephalon. In particular, I found cells in the anterior telencephalon that would respond to the expected aversive stimulus while other cells in the adjacent areas that would strengthen their connections throughout the learning process.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Recent work has revealed that memory formation and consolidation require a constant dialogue between the cortex and the hippocampus. However, technical challenges have so far limited our ability to study these interactions in awake animals and therefore, ZebraHipNetwork has sought to address this. First, we have shown that the hippocampal-like cells were highly dispersed across the zebrafish telencephalon. Next, to establish how sensory information is processed in the DL, I have mapped the major connections within the zebrafish dorsal telencephalon while also characterizing the major input to the DL. This connectivity map will then be very useful for other research groups interested in studying the zebrafish telencephalon. Using whole-brain recording techniques, I have then described how sensory information is processed in a topographically selective manner from the thalamic-like PG to the dorsal telencephalon, including the DL. These experiments have further revealed, for the first time in any fish species, that sensory information is processed in a hierarchical manner in the fish dorsal telencephalon similarly to other vertebrates. These novel findings will hopefully inspired other research groups to consider zebrafish as an alternative animal model to study the neural basis of sensory-cortical computations in the brain. Finally, while recording the DL neurons during a learning paradigm, I have also unveiled distinct populations of cells that can change during the course of learning. These results will therefore be used as a foundation for my future independent research group aimed at unveiling the mechanisms underlying sensory processing and memory formation in ancestral vertebrates.
I have already presented the work performed in ZebraHipNetwork at multiple international conferences, including SFN 2023, the CSHL Zebrafish neurobiology meeting 2023, the Thalamocortical Interactions GRC 2024 and finally the FENS forum meeting 2024. Currently I am writing my manuscript which I aim to submit very soon, first on BioRxiv and then to a well-known open-access peer-reviewed journal.