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Funding
Funding
HFSP supports novel, innovative and interdisciplinary basic research focused on the complex mechanisms of living organisms. A clear emphasis is placed on novel collaborations that bring biologists together to focus on problems at the frontier of the life sciences.
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Awardees
Awardees
In this section you find information about the awardees in the HFSP scientific programs. This includes a searchable database of HFSP awardees, information on the HFSP Nakasone Award and other achievements by HFSP awardees.
- News & Impact
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Events
Events
HFSP promotes and participates in several events every year. We are committed to bringing together the scientific community and forging new opportunities to network and have scientific discussions that help to create bridges and partnerships for the future of frontier science.
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About
About
The Human Frontier Science Program is a program of funding for frontier research in the life sciences. It is implemented by the International Human Frontier Science Program Organization (HFSPO) with its office in Strasbourg. Here you can find all you need to know about the HFSPO.
Awardees
2024
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Long-Term Fellowships - LTF
Unraveling spatial landscape of Mycobacterium tuberculosis transcriptome & its role in TB pathology
MEHDIRATTA Kritee (INDIA)
Immunology and Infectious Diseases - President and Fellows of Harvard College, Harvard T. H. Chan School of Public Health - Boston - United States
RUBIN Eric (Host supervisor)
Despite the advent of BCG vaccination and antibiotics, the global burden of tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains high. Previously regarded as non-compartmentalised cellular system due to lack of well defined intracellular organelles, recent studies have shown Mycobacteria to possess defined cellular organisation with distinctive spatiotemporal localisation of proteins. In eukaryotes, it is well reported that targeting mRNA transcript to specific cellular locations is critical for spatial protein synthesis thereby highlighting RNA localisation as a driver of sub cellular organisation. Fascinatingly, only recently similar phenomenon of RNA localisation has been demonstrated in bacteria like E. coli. Bacterial mRNA can localise to nucleoid, cytoplasm, poles, and inner membrane. However, the underlying molecular mechanisms dictating their localisation is not completely understood. Interestingly, the transcriptome organisation of Mtb remains elusive so far. Moreover, the biological relevance of defined RNA localisation patterns in bacteria needs to be ascertained. Hypothesis- RNA localisation in eukaryotes is an important post transcriptional regulatory mechanism. However, the scope of RNA localisation in pathogens like Mtb remains unknown. The Mtb transcriptome might be spatiotemporally organised to maximise gene function and to provide fast and efficient regulation in response to changing host environmental stimuli. Aims- 1. Delineating spatiotemporal organisation of Mtb transcriptome under homeostasis & if there is reconfiguration in response to different physiological stresses. 2. Characterising molecular mechanisms driving Mtb RNA localisation & enabling tethering to specified location. 3. Investigating whether spatial information regulates transcriptional activity & if disruption of spatial organisation introduces vulnerabilities in Mtb growth and survival. To address these aims we will take advantage of methods recently developed in the Rubin lab to perform fluorescence in situ localisation (FISH) to localise transcripts in viable Mtb and to recreate pseudotemporal localisation in fixed bacteria. We will use cellular fractionation and RNA sequencing techniques to spatially map the complete Mtb transcriptome & immunoprecipitation studies to identify any interacting motor and cytoskeleton proteins. We will define sequence determinants that could direct transcripts to subcellular compartments and identify proteins that might play a role in localisation. Finally, will generate mutants that mislocalise RNAs and determine their physiological effects. Conclusion- RNA localisation adds a layer of complexity to prokaryotic cellular architecture. Understanding the physiological relevance of such dynamic spatial organisation of molecular components especially in clinically relevant pathogens like Mtb, might provide a new paradigm of gene regulation and function that can be therapeutically exploited in future.
2024
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Grant Awardees - Program Grants
Hormone-like Bacterial Signaling Molecules as Mediators of Gut-Brain Dialogues
XAVIER Karina (PORTUGAL)
- Fundação GIMM - Gulbenkian Institute for Molecular Medicine - Lisbon - PORTUGAL
MEIJLER Michael (ISRAEL)
- Ben-Gurion University of the Negev - Be'er-Sheva - ISRAEL
NEEDHAM Brittany (United States)
- Indiana University - Indianapolis - United States
Microbial symbionts inhabit all animals, with constant exchange of molecular cues between microbes and host. These communities of microbes, referred to as microbiota, influence many aspects of host physiology, including the brain and behavior. Specific signals and host receptors involved in these interactions remain largely unknown. To understand the permeating effects of interbacterial chemical crosstalk within the microbiota community, as well as signaling between the microbiota and the host, we will focus on the mammalian intestine, which houses a dense bacterial community and is ideal for elucidating these pathways. Recently, a novel family of bacterial signaling molecules, named pyrazinones, was found to regulate communication and virulence in bacterial intestinal pathogens. We predict that these pyrazinones are also produced by commensal members of a healthy gut microbiota. Intriguingly, bacterial pyrazinone receptors can sense human-derived neurotransmitters due to structural similarities between the compounds. This shared ligand profile between pyrazinones and host neurotransmitters, along with pyrazinone production by known (and potentially many unknown) microbes within the gut, offers a compelling new avenue of microbe-host connection. Pyrazinones are likely perceived by host neurotransmitter receptors, modulating the enteric, peripheral and/or central nervous systems. Thus, our proposal offers the transformative and unexplored notion that pyrazinones can serve as microbial-made neurotransmitters, impacting both animal behaviors and bacterial group behaviors. To test this, we will apply advance metabolomic methods to identify and characterize the pyrazinones produced by gut microbiota and synthesize novel chemical probes to identify host receptors and their spatial distribution in mice. We will genetically modify pyrazinone biosynthetic pathways in gut bacteria, allowing us to compare their effects in colonized mice. Once host receptors are characterized, we will use mutant mouse lines to confirm their involvement. Using these tools, we will study microbe-dependent neurological alterations and behavioral phenotypes to assess interplay with the nervous systems. Overall, we will determine how host monitoring of these microbial signals leads to altered host functions, establishing a new avenue of crosstalk between host and microbial symbionts.
2024
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Long-Term Fellowships - LTF
Reward-based learning of assemblies in olfaction
MIEHL Christoph (AUSTRIA)
Department of Neurobiology - The University of Chicago - Chicago - United States
DOIRON Brent (Host supervisor)
OSWALD Anne-Marie (Host supervisor)
It is widely accepted that groups of pyramidal neurons (PNs) that display synchronous activity and strong recurrent synaptic connectivity, often referred to as an ‘assembly’; represent a specific stimulus or concept, hence acting as a fundamental unit of memory storage. Understanding the mechanics of assembly formation is needed to link the biology of the nervous system to its function. In previous models, assemblies have formed from unsupervised learning of sensory stimuli via synaptic plasticity (see e.g. my PhD work). However, learning certainly depends on supervisory signals such as reward. While reward-based learning is widely used in machine learning applications like reinforcement learning, it is not clear how it is incorporated in biological circuits. In addition, unpublished data from the Oswald lab challenges the current working model of assembly learning: They present two odors to freely behaving mice, one rewarded and one unrewarded. Based on a combination of activity-dependent labeling, optogenetic stimulation, and in vitro (soon in vivo) recordings of neurons in the piriform cortex they find that, unintuitively, an assembly only forms for neurons selective to the rewarded odor. But how is reward-based learning implemented in a biological circuit? Published data from the Oswald lab suggests that activation of VIP inhibitory neurons (INs) can gate the synaptic plasticity essential for assembly formation (Canto-Bustos et al., 2022). Therefore, the following hypothesis of reward-based assembly learning emerges: Presenting an odor together with a reward activates VIP neurons, which gates synaptic plasticity among odor-specific PNs. Based on a close collaboration with the Oswald lab, my host supervisor’s experience in modeling circuits with multiple INs, and my prior knowledge in modeling plastic excitatory/inhibitory networks, I aim to uncover how reward-based learning is implemented in biologically plausible circuits. First, my aim is to build an experimentally constrained computational model of the piriform cortex circuitry, including PNs and the major INs (PV, SST, and VIP) with plastic synapses, to test the above-described hypothesis of reward-based assembly learning gated via VIP neurons. My second aim is to show that only rewarded odor-specific PNs form an assembly; here preliminary data suggests that SSTs play a pivot role. Third, I aim to extend the model to flexibly allow to encode multiple odor assemblies with overlapping chemical components in order to study how multiple components are combined to form a meaningful odor perception. My work will lead to a better understanding of 1) olfactory processing, 2) how cortical circuits are able to support reward-based learning paradigms, 3) be the biological basis of understanding more complex reward-based learning paradigms (e.g. reinforcement learning), and 4) my results will be generalizable to other brain regions involved with associative learning problems (such as hippocampus).
2024
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Long-Term Fellowships - LTF
Exploring the viral influence on the human gut microbiome
MILLMAN Adi (ISRAEL)
Department of Biology - Massachusetts Institute of Technology - MIT - Cambridge - United States
LAUB Michael (Host supervisor)
"Bacteriophages can either directly kill bacteria or integrate into bacterial genomes affecting their gene content. As a result, phages have a profound impact on their environments. This impact likely extends to bacterial communities within the human microbiome, and better understanding of phage-bacteria dynamics can shed light on the ecological and evolutionary forces shaping the microbiome. In this study I aim to elucidate how phages influence the bacterial composition of the gut by using a large longitudinal dataset of shotgun microbiome sequencing.
Studies show that phage ""kill-the-winner"" dynamics observed in the ocean do not apply in the gut microbiome. It is likely that phage-bacteria coexistence in the gut is achieved by the temperate phage lifestyle. To test that hypothesis, I plan to follow phage dynamics in the microbiome. By comparing the coverage of prophages to the coverage of the host genomes over time, I can estimate the amount of lytic replication in the microbiome. This ratio could also be indicative of the phage host range, as phages that outnumber the coverage on the host reference genome might be residing in multiple hosts. To test this, I plan to search for reads that represent new prophage-bacteria junctions.
Phages play a major role in horizontal gene transfer of the gut microbiome, equipping bacteria with various traits such as virulence, pathogenicity, antibiotic resistance, and antiphage resistance. To assess the contribution of phage transduction to bacterial adaptation in the gut, I plan to track the gene flow between phages and bacteria in patients and healthy individuals. Understanding what is the genetic cargo phages carry and transfer between genomes can shed light on the advantageous traits necessary to thrive in the gut. To measure the contribution of phage transduction to bacterial adaptation in the gut, I will monitor the rate of these events in the microbiome over a prolonged period of time.
To study whether bacteria have adapted to take advantage of phage transduction, I will analyze the gene content bacterial genomes encode in the vicinity of packaging (pac) sites that allow generalized transduction, and attachment (att) sites that allow specialized transduction. These genomic positions are likely enriched with genes contributing to bacterial fitness, as the genes in these positions have higher probability of being packaged and transduced by phages.
While most microbiome studies focus on the bacterial species composition in the gut, the prophages of these species are mostly overlooked. Analysis of prophage dynamics, their host range, and the cargo genes they carry can better illuminate the forces shaping the gut microbiome and expose the functions that help various bacterial species in the gut adapt to different conditions.".
2024
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Grant Awardees - Program Grants
From nano to organismal scale: structural regulation of regenerating jellyfish
SINIGAGLIA Chiara (ITALY)
- CNRS Languedoc-Roussillon - Montpellier - FRANCE
MODES Carl (United States)
- Max Planck Institute of Molecular Cell Biology and Genetics (Max-Planck-Institut für Molekulare Zellbiologie und Genetik) - Dresden - GERMANY
SHIMANOVICH Ulyana (ISRAEL)
- Weizmann Institute of Science - Rehovot - ISRAEL
Diverse species can recover lost body parts, but why not all injuries lead to regeneration, and how animals re-organise their bodies remain key open questions. Mechanical features, notably of the extracellular matrix (ECM), are known critical regulators of development - however they are only rarely considered by regeneration studies. New models are needed to understand how shapes re-emerge, able to address different cues across the molecular, cellular and organismal scales.
The small jellyfish Clytia hemisphaerica boasts excellent repair capacities: wounds heal within 12 hours, and organs regenerate in a week. Recovery is regulated by both transcriptional and mechanical cues, and the simple anatomy, wherein a thick ECM (mesoglea) is covered by contractile epithelia, offers an unexplored experimental paradigm. We propose a unified vision of regeneration, where mechano-chemical features of cells and tissues robustly modulate Clytia repair. Our central hypothesis is that processes related to organ regeneration in Clytia, including recruitment of progenitor cells, regulation of transcriptional responses and orchestration of cell differentiation, are linked to the mechanisms of tissue-matrix interaction, and to the mechanical and topological features of the injured tissues.
A purely in vivo investigation remains challenging, due to the complexity of living tissues and the ECM dynamics. Our solution is to combine in vivo manipulations with the power of analytical tools. We will thus be able to link the structural properties of the mesoglea (fibrillar network of structural and biologically active protein fibrils) with the forces generated within contractile epithelia (regenerative biology, in vivo – Sinigaglia). We will build topologically-informed mechanical models coupling the mesoglea network to the aligned patterns of actomyosin, enabling us to capture, in simple predictive rules, how different classes of injuries – to actomyosin pattern or mesogleal fidelity – affect regeneration (theoretical biophysics and computational biology, in silico – Modes). We will combine these manipulations with biomaterials engineering approaches to build synthetic analogues of self-repairing jellyfish-inspired biomaterial machinery (soft matter, biochemistry, in vitro – Shimanovich). Our regenerative platform, modular and cross-scale, will be adaptable to other contexts.
2024
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Long-Term Fellowships - LTF
Neural dynamics of social role-taking and role-reversal in the medial prefrontal cortex
MUGNAINI Matias (ARGENTINA)
Animal Physiology/ Systems Neurobiology and Neural Computation - Humboldt-Universität zu Berlin - Berlin - GERMANY
BRECHT Michael (Host supervisor)
Most current knowledge about the neural representations of social role-taking stems from the study of dominance-subordination hierarchies, mostly in the tube test (TT). This knowledge has only recently expanded to different social contexts with the rat-human version of hide-and-seek (HS). Unlike social status, social play tends to be pleasurable for all involved individuals and relies more on cooperative behaviors that allow animals to pretend to occupy roles that they usually do not. Despite these differences in the social context and in the freedom animals have to reverse roles, recent studies of the medial prefrontal cortex (mPFC) activity hint at the existence of a common neural code that pervades social roles in all contexts. Anecdotal evidence from these studies suggests that, although mPFC neurons encode specific behaviors in the TT and in HS, respectively, subsets of these neurons may underlie the abstract representation of social role itself. For example, in HS, where rats learn that the starting-box opening indicates them to be hiders (open) or seekers (closed), nearly 30% of mPFC neurons exhibit responses to the starting box closure during “seek” trials that are not explained by other variables, suggesting an influence on role definition. The current understanding of the high-level representation of social roles (i.e., independent of specific social contexts) is limited to a few results and has not been systematically addressed. The goal of this project is to study the neuronal representation of social roles across role-taking situations (i.e. playful or not) to disentangle the common neural code that may underlie them. The hypothesis is that abstract features associated with similar roles (i.e., dominance-seeking, subordinance-hiding) are redundantly encoded by mPFC neuronal subsets. Specifically, we aim to: 1) explore the high-dimensional neuronal space for common codes between tasks; 2) identify role-defining neurons to study their dynamics during task transitions and role-reversal; 3) use decoding tools to test the hypothesised code redundancies for similar roles; and 4) optogenetically manipulate role-taking by targeting mPFC neurons. To this end, Long-Evans rats will be chronically implanted with multiple-shank Neuropixels 2.0 or optic fibers in the Cingulate, Prelimbic and Infralimbic cortices, trained to play HS and tested in the TT to identify social ranks. Contrary to previous studies, this will allow the trial-by-trial simultaneous recording of hundreds of units across cortices and layers in the same animal and the comparison of activity across tasks and neural subpopulations. Unlike other role-plays (e.g., play fighting), HS does not involve direct aggression that could damage implants or produce confounding data related to effortful behaviors. Overall, this project will start to fill the gap in our knowledge of the neural underpinnings of social roles and the cortical dynamics by which they are adopted and reversed.
2024
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Cross Disciplinary Fellowships - CDF
Synthesis, characterization, and in vivo identification of novel oxyendocannabinoidome metabolites
MULLER chante (USA)
Medicine - Laval University (Université Laval, Québec) - Quebec - CANADA
DI MARZO Vincenzo (Host supervisor)
FLAMAND Nicolas (Host supervisor)
The endocannabinoidome (eCBome) is a signaling system that heavily regulates many physiological processes and disorders via multiple signaling molecules, catabolic/metabolic enzymes, and receptors, including cannabinoid receptors 1 and 2 (CB1; CB2). CB1 and CB2 regulate numerous functions, notably appetite, adipogenesis, inflammation, and inflammatory pain, and are activated by two primary ligands stemming from arachidonic acid: the N-acyl-ethanolamine (NAE) anandamide (AEA) and the monoacylglycerol (MAG) 2-arachidonoyl-glycerol (2-AG). There is well-documented connection between the eCBome and the gut microbiome. Thus, there is evidence of their co-modulation. E.g., compounds produced by commensal bacteria can modulate eCBome receptors, while other bacteria-derived metabolites produced by the oxidation of linoleic acid have proven beneficial against obesity and related disorders. Metabolites, provided from the intestinal flora, may yield new eCBome modulators, some with unknown physiological effects. Thus, we believe that eCBome and/or gut metabolites are the primary link between the two. Cyclooxygenase-2 and lipoxygenases metabolize 2-AG and AEA into their oxidated counterparts, except 5-lipoxygenase. Due to steric hindrance, the MAG and NAE headgroups cannot enter the 5-lipoxygenase active site and therefore 5-oxygenated metabolites of MAGs and NAEs should not exist. However, leukocytes have been found to produce novel 5-oxygenated MAG and NAE congeners of fatty acids utilizing a novel biosynthetic pathway. Therefore, we hypothesize that leukocytes and possibly other cells can produce novel oxygenated metabolites that modulate eCBome receptors and have modulatory effects on inflammation, some of which arising from the interaction of host cells with the gut microbiota. The aims of this project are to provide evidence of this pathway via the 1) biosynthesis, characterization, and 2) in vivo identification of novel oxygenated metabolites, and 3) define the cellular receptors by which they exert their biological effects. Suspected metabolites will be synthesized using previously published methods (PMID 33915294). Gut microbiota-derived metabolites will be used to synthesize an array of MAG and NAE congeners, and we will validate their existence in human blood and tissue samples from the IUCPQ tissue bank. Cellular targets of novel metabolites will be screened at GPCRs using the PRESTO-Tango assay, at TRP channels using the Fluo-4 AM assay, and at PPARs using luciferase reporter assays. The expected outcome of this project is to validate the existence of a novel biosynthetic pathway in leukocytes and possibly other cells, identify the scope of the metabolites it can generate, and obtain evidence of their physiological functions via their cellular targets. These data will provide basis for, and shape, the direction of future projects including investigation of host-gut interactions and metabolite variation between obese and non-obese samples.
2024
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Long-Term Fellowships - LTF
Dissecting long-range gene regulation of cell differentiation through biophysical approaches
NAGANO Masahiro (JAPAN)
Department of Biological Engineering - Massachusetts Institute of Technology - MIT - Cambridge - United States
HANSEN Anders (Host supervisor)
Proper regulation of gene expression is critical to biological function, and dysregulation is widely implicated in disease. Typical genes are controlled by transcription factors that bind both at promoter-proximal elements as well as distal enhancers as far as hundreds of kilobases away and tend to be highly cell-type specific. Improper enhancer-promoter interactions can cause devastating effects, with a prime example being oncogenes’ hijacking of other genes’ enhancers in tumorigenesis. While enhancer-promoter associations were traditionally thought to involve stable long-range looping of chromatin to physically bridge regulatory elements, convincing alternatives have emerged from recent studies on loop-extrusion and biomolecular condensates. Therefore, the precise molecular mechanism governing enhancer-promoter interactions remains unsettled. The major roadblocks hindering our understanding of enhancer-promoter interaction mechanisms are technical. First, the close spatial proximity of many enhancers to their cognate promoters cannot be resolved by typical methods (e.g., Hi-C or FISH). Second, dynamic mechanisms cannot be resolved with static methods (e.g., Hi-C or FISH). Here, to overcome these limitations, we will combine two cutting-edge techniques developed in the host lab: region capture Micro-C (RC-MC) and Super-Resolution Live Cell Imaging (SRLCI). Whereas RC-MC enables the visualization of the local genome architecture around select target loci with unprecedentedly high resolution in space to catalog all potential enhancer-promoter pairs, SRLCI complementarily tracks defined enhancer-promoter interactions over time to chronicle dynamic reorganization. As a novel addition, we aim to further visualize nascent transcription simultaneously by using PP7 technology. Finally, in collaboration with biophysicist Dr. Leonid Mirny (also at MIT), we will build a 3D polymer-simulation model recapitulating dynamic enhancer-promoter contact to reconcile experimental results with competing hypotheses. These experiments will be carried out using a differentiation system producing three different lineages. By focusing on a master transcription factor for one specific lineage, we will dissect the cis-regulatory circuits promoting nascent transcription and ultimately lineage specification– a central open question in developmental biology. In terms of perturbations to this system, we will modulate the composition of loop-extruding cohesin complex and transcription proteins. My project integrating bioengineering, biophysics, computational modeling, genomics, and genetics will therefore answer two major questions: “how do enhancers mechanistically regulate transcription” and “how does dynamic 3D rewiring of enhancer-promoter contacts at a key transcription factor locus drive lineage specification.” Consequently, these fundamental results will advance our understanding of physiology and disease, impacting the broader scientific community.
2024
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Long-Term Fellowships - LTF
Visualizing specific lipid processing and uncovering molecular mechanisms using gut organoids
NAITOU Yuki (JAPAN)
Biotechnology Centre (BIOTEC) - TU Dresden (Technische Universität Dresden) - Dresden - GERMANY
HONIGMANN Alf (Host supervisor)
Lipids are one of the most essential nutrients for biological activity, playing a variety of roles such as energy storage, construction of cell membranes, and intracellular signal transduction including developmental biology and cancer. Depending on their chemical structures, there are more than 1000 different types of lipids in a typical eukaryotic cell. However, it is still not well known “how the structural diversity of lipids function differently in vivo context?” Although lipid research has made progress in the identification of lipid species using mass spectrometry, it falls behind protein research, especially in terms of imaging. This is because, unlike proteins, the observation of lipid species under light microscopy at high resolution using gene editing or chemical reactions without perturbing their structures and functions has not been achieved. Thus, the high-resolution visualization of lipids with their native structures and functions should develop this lipid field and pioneer a new field in biology. Recently, a technique called bifunctional lipid probes has been developed using photo-activation and click chemistry techniques. This technique enables us to observe the localization of near-native lipid species in cells with high resolution fluorescence microscopy. This study aims to investigate the biological significance of the structural diversity of lipids in terms of how they function and are involved in biological contexts by combining a novel lipid visualization technique with super-resolution microscopy observations and mass spectrometry. To demonstrate the feasibility of this approach in monitoring lipid dynamics, I will apply the visualization system to the small intestinal organoids model. The small intestine is one of the major lipid processing organs such as uptake, transport, subcellular conversion, and secretion (lipid flux). This organoid model exhibits intracellular transportation of lipids and offers convenient imaging accessibility. I will test the hypothesis that lipid structure, e.g. head group, chain length, and saturation, determines trafficking routes and kinetics within intestinal cells. This study will reveal the spatiotemporal dynamics and hidden biological functions of structurally diverse lipids with unprecedented resolution. It will provide a novel platform for the analysis of lipid flux at the intercellular and tissue level and an important resource to reveal the function of specific lipids in homeostasis and disease. After this proof-of-concept experiment, I will practically apply the imaging system to the mouse primordial germ cell (PGC) culture system, where I established an imaging system. PGC development has unique and interesting dynamics of lipids. For example, PGCs have transiently conspicuous lipid droplets during the migration to future gonads, however, their function is still unknown. Thus, this future study will provide novel insights into lipid function during germ cell development.
2024
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Long-Term Fellowships - LTF
Closing the neural and behavioural feedback loop of social interactions in freely-behaving primates
NAKAHASHI Ayuno (JAPAN)
Cognitive Neuroscience - Deutsches Primatenzentrum GmbH - Goettingen - GERMANY
GAIL Alexander (Host supervisor)
I aspire to study the neural basis of social cognition, especially the processes that implement Theory of Mind (ToM) in humans, which likely exist in other species to varying extents. ToM is the ability to put oneself in someone else’s shoes. It lets us adapt our behaviour based on norms, read between lines and keep unity in a complex society. People with autism spectrum disorder often struggle with this, which may lead to constant social stress and higher comorbidity risks. Thus, understanding the neural basis of social cognition is imperative. I propose to study the neurocognitive processes of social decisions in rhesus macaques using an innovative closed-loop approach in free-foraging individuals. Macaques are evolutionary much closer to humans than rodents and the best studied species for higher neurocognitive functions, making them the most advanced animal model for theoretical and clinical neuroscience. Still, to what extent their cooperative behaviour compares to that of humans remains debated. Also, due to technical limitations, most neurophysiological data come from experiments using highly restrained behaviours. In contrast, natural social interactions are dynamic mutual exchanges between agents. I aim to fill these gaps by recording neural data from macaque pairs during free social foraging in a novel and unique environment with multiple experimentally controlled dyadic feeders. I hypothesize that for macaques to behave cooperatively, there must be dynamic interactions between the foraging reward distribution and the pairs’ social and spatial states (e.g., hierarchy, past interactions, how far the feeders/peers are). I predict, 1) the level of cooperation will vary based on these states and reward distribution, and 2) the neural activity in social, spatial, and reward-oriented brain areas will distinctly reflect respective states. I will combine wireless neural recording, AI-based action recognition, and cortical microstimulation to analyse and causally manipulate the activity of target areas and behaviour. I expect to 1) establish the link between cingulate areas and social states, retrosplenial cortex and spatial states, and prefrontal areas and reward states, and 2) test their causality by stimulating in one area while recording in the rest. My project will explore the fundamental yet previously inaccessible decision variables in an ethologically relevant setup with rigorous experimental control, closing the loop of brain and behaviour as an organic unit. With my PhD on value-based motor decisions, I gained a solid foundation on decision neuroscience. Through this project, I will expand my electrophysiology skills to wireless recording in multiple unrestrained agents, learn the latest multi-agent tracking methods, and grow new collaborative networks, while contributing my computational modeling expertise and perspectives of the evolution of organisms as control systems. With that, I am advancing toward my ambition: to study ToM.
2024
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Long-Term Fellowships - LTF
Memory trace in a neuron: dissecting dendritic mechanisms of learning
NOGUCHI Asako (JAPAN)
Zuckerman Institute - The Trustees of Columbia University in the City of New York - New York - United States
LOSONCZY Attila (Host supervisor)
Dendrites, the elaborate tree-like structure of neurons, are the key sites of neuronal computations by processing synaptic inputs and transmitting information to the cell body of neurons. Their significance is underscored by their observed degeneration and dysfunction in numerous neurodegenerative diseases. While interdependent somatic and dendritic activity has been suggested to implement functions that determine the input-output relationships in individual neurons, it is still technically impossible to capture dendritic computations in isolation from the influence of somatic activity in the intact brain. Therefore, we still do not know how soma and dendrites operate independently or collaboratively to achieve cognitive functions in behaving animals. To address this question, it is necessary to monitor and manipulate the activity of soma separately from dendrites in behaving animals. In my post-doctoral work, I will combine in vivo dendritic voltage imaging, intracellular recordings, and manipulation of somatic membrane potentials in hippocampal pyramidal neurons to elucidate the potential roles of dendritic computations in neuronal plasticity underlying learning and memory. My research will leverage the formation and subsequent stabilization of spatial representations in hippocampal CA1 pyramidal neurons as a model of neuronal plasticity associated with spatial learning. The plasticity rule behind this process, known as behavioral timescale plasticity (BTSP), is characterized by dendritic plateau potentials and somatic burst spiking and suggested to involve somatic and dendritic plasticity, the precise roles of which remain enigmatic in vivo. I will first reveal the relationships between dendritic computations, somatic spikes, and dendritic plateau potentials throughout spontaneous place field formation and subsequent refinement of the spatial information associated with sharp-wave ripples (SWRs), the high-frequency oscillations in the hippocampal local field potentials (LFP). To achieve this, I will conduct two-photon voltage imaging of dendritic activity with ultrafast acousto-optical microscopy and simultaneous whole-cell patch-clamp recordings from their soma, together with hippocampal LFP recordings while mice explore a novel environment. Next, I will manipulate somatic membrane potential to prevent soma from emitting spikes and study dendritic activity. This revolutionary approach will clarify for the first time whether and how somatic spikes and plateau potentials are causally related to dendritic computation and propose a potential role of dendrites in spatial memory formation. The innovative combination of technologies that I am pioneering will open up a whole new world in understanding soma-dendrite computation in general and may provide novel insights into the pathologies caused by its abnormalities.
2024
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Long-Term Fellowships - LTF
Molecular and functional dissection of anti-polarity signal transmission in dopamine neurons
NOZAWA Kazuya (JAPAN)
Department of Neurobiology - Harvard Medical School - Boston - United States
KAESER Pascal (Host supervisor)
Signal transmission in a neuron has a defined polarity as it proceeds from dendrites and the cell body to the axon. Santiago Ramon y Cajal proposed this principle, the “law of dynamic polarization”, over a century ago. It has been validated by functional, anatomical, and molecular studies across various types of neurons. Surprisingly, the host laboratory has recently discovered that midbrain dopamine neurons have an action potential initiation mechanism in distal dopamine axons in the striatum. Axonal firing is triggered by local cholinergic interneurons and leads to dopamine release independent of ascending activities from the cell bodies in the substantia nigra pars compacta (SNc). These findings go against core principles of the dynamic polarization law, and establish that dopamine neurons contain specialized machinery for initiating signal transmission in distal axons. The architecture of this machinery, and how far axonally-induced action potentials can propagate, has remained unexplored. Remarkably, dopamine neurons release dopamine not only from axons in the striatum, but also from somatodendritic compartments in the SNc. Building on these features, I hypothesize that there is a reverse information flow in dopamine neurons: action potentials initiated in distal dopamine axons in the striatum can backpropagate to the SNc to trigger somatodendritic dopamine release. Alternatively, these action potentials might selectively regulate striatal activities without backpropagation, accounting for different spatiotemporal signaling dynamics between axonal and somatodendritic compartments. I propose to dissect the directionality of information flow in dopamine neurons and the underlying signaling structures with a three-pronged approach. In aim 1, I will determine the molecular and functional architecture of somatodendritic dopamine release sites. I will combine electrophysiological recordings and mouse genetics with newly developed morphological methodologies that combine cryo-fixation and expansion microscopy. In aim 2, I will dissect the protein machineries for action potential initiation in striatal dopamine axons. I will assess whether there are non-uniform and co-organized distributions of three key components: vesicular release sites of cholinergic interneurons, nicotinic acetylcholine receptors on dopamine axons which depolarize their membrane, and voltage-gated sodium channels on these axons which initiate and propagate action potentials. In aim 3, I will test whether action potentials induced in distal dopamine axons reach the soma and trigger somatodendritic dopamine release in vivo. I will utilize optogenetics, in vivo imaging with genetically encoded dopamine sensors and electrophysiology in mice. My work is likely to revise fundamental principles on the directionality of information flow in neurons and will define the molecular machineries for signaling polarity reversal.
2024
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Long-Term Fellowships - LTF
Illuminating orphan GPCR signaling by novel biochemical approaches
OKAMOTO Hiroyuki (JAPAN)
Pharmaceutical Chemistry - The Regents of the University of California, San Francisco - San Francisco - United States
MANGLIK Aashish (Host supervisor)
G protein-coupled receptors (GPCRs) are important for cells to sense their external environment. Plenty of studies on their structure, pharmacology and so on has led to successful therapies of GPCR-related diseases. However, there are still hundreds of "orphan" GPCRs whose function remains unknown. Revealing the function and ligands of orphan GPCRs, called “deorphanization”. Deorphanization of these receptors could have significant implications for human physiology and disease treatment, but efforts to find their activating molecules have slowed down over the past decade. The lack of an activator makes it difficult to study downstream signaling pathways and to develop assays for probe molecule discovery of orphan GPCRs. My research aims to develop new approaches to illuminate the function of orphan GPCRs, particularly those expressed in the central nervous system. To overcome these challenges, I propose a new approach to purify orphan GPCRs and use them as ”bait” to find endogenous hormones or surrogate activators for orphan GPCRs, combining biochemistry, native mass spectrometry, antibody development and structural biology. As a prototype of screening of orphan GPCR activators, I chose GPR85, the most conserved GPCR in all vertebrates. Knock out of GPR85 leads to a 20% increase in brain mass, while overexpression studies implicate it as a negative regulator of hippocampal neurogenesis and spatial learning. But the lack of ligand of GPR85 hinders understanding of the precise function and signaling of GPR85. My research has two aims. The first aim is to identify endogenous ligands for GPR85 using mass spectrometry and NMR techniques. And I will use extracts from the hippocampus to identify other molecules that bind tightly to GPR85. The identified molecules will be used to determine the cellular signaling pathways modulated by GPR85. The second aim is to use combinatorial antibody libraries to find new surrogate ligands for GPR85. The approach will identify molecules that bind to GPR85 in a specific conformation and have the potential to induce or inhibit GPR85-mediated signaling. The candidate molecules will be screened for activity against a panel of cellular signaling pathways. Orphan GPCRs represent a huge opportunity for drug discovery, but progress has been slow. A new approach could shed light on the function of these mysterious receptors and pave the way for similar approaches to tackle other orphan receptors. The ultimate goal is to illuminate the function of one of the most intriguing orphan GPCRs in the genome and pave the way for similar approaches to numerous other orphan receptors. This approach could lead to the discovery of new therapies for recalcitrant diseases. The support from the Human Frontier Science Program would enable a high-risk, high-reward approach to a longstanding problem in sensory biology.
2024
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Long-Term Fellowships - LTF
Parasite immune evasion in a murine microcosm of the human immune system
PARIKH Shivang (INDIA)
Ragon Institute of Mass General, MIT, and Harvard - Massachusetts General Hospital (Mass General) - Boston - United States
BATISTA Facundo (Host supervisor)
Background: Hosts and parasites exist in a delicate balance: many of these associations are ancient, the result of an equilibrium of adaptation and counter-adaptation in the Red Queen's Race. The host-parasite interface is thus the site of rapid evolution, and of nature's most elaborate weapons and ruses. The shapeshifting of Plasmodium falciparum, the most lethal malaria parasite, provides an example: infection with sporozoites (PfSPZ), the stage of which moves from bite through blood to the liver does not induce sterilizing immunity. Vaccines and therapeutics for this stage must contend with its immune evasion. PfSPZ's surface is heavily covered with circumsporozoite protein (PfCSP), which is critical for mammalian host cell invasion. PfCSP includes a central region comprised of repeating tetrapeptides: these copious repeats have been proposed as an immune-evasive feature, drawing unproductive responses from the host. Aims of the proposal: To determine whether the central repeat or other PfCSP epitopes are immune-distracting and whether immuno-focused approaches can teach a host to evade the parasite's own evasive maneuvers. 1. Develop preclinical models expressing human PfCSP antibodies. Antibodies will either bind only a single PfCSP epitope (in/out of the repeat region), or will be dual-specific to the central repeat and at least one other PfCSP epitope. 2. Investigate which epitopes impart a robust response. Humanized mice will be vaccinated with immunogens containing various segments of PfCSP. 3. Generate mini-repertoires to mimic the host environment. B cells are in competition for antigen: to determine whether responses to the central repeat crowd out other responses by B cell competition, we will generate mice with multiple humanized B cell lineages, expressing precursors to antibodies to various parts of PfCSP. Central hypothesis: Humoral immune responses to the central repeat will not be protective: furthermore, immuno-focused responses excluding the central repeat will outperform broad responses. The experimental system: The Batista lab has developed an approach to rapidly generate mice with B cells bearing fully humanized B cell receptors (BCRs), as well as state-of-the-art immunocharacterization approaches to trace of a single B cell lineage. This proposal advances those methods by generating models capable of expressing multiple PfCSP antibodies, recreating the competitive environment in humans in miniature. Conclusions: Whether the central repeat performs an immune-evasive function will be determined by both direct measurement of the humoral responses (identifying which cells are activated, the amount and specificity of the circulating antibody response), and, perhaps more tellingly, how vaccinated mice respond to a malaria challenge. This will provide either confirmation or refutation of this proposed mechanism by which parasites overcome acquired immunity not by avoiding a response, but by eliciting a harmless one.
2024
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Cross Disciplinary Fellowships - CDF
Ultrasound neuromodulation: search for the physical origin
PAVLIC Alen (SLOVENIA)
Division of Chemistry and Chemical Engineering - California Institute of Technology - Pasadena - United States
SHAPIRO Mikhail (Host supervisor)
Ultrasound (US) is an exciting tool for biomedical purposes due to its ability to non-invasively interact with matter deep inside our body with relatively high precision. These interactions can be exploited at low intensities for imaging, or at high intensities for destruction of tissue. The intermediate US intensity range, however, presents a grand challenge, since it affects biological cells through a complex interplay of several forces, such as the acoustic radiation force (ARF), acoustic interaction force (AIF), forces from acoustic streaming (AS) and cavitation. These US-induced forces can lead to significant biological responses, enabling for example neuromodulation, biomarker release, sonoporation, and angiogenesis; however, despite their enormous potential these forces remain poorly understood due to the entanglement of the underlying mechanisms, as well as the fast time- and small length-scales making experimental analysis challenging. Neuromodulation with US (USNM) has been demonstrated in vitro and in vivo and is particularly interesting, as it could yield significant breakthroughs in neuroscience and neurological treatments. Potential uses include suppression of epileptic seizures or symptoms of psychiatric disorders. In the proposed project, I plan to first discover which acoustic forcing mechanisms are responsible for USNM and to what extent, and then determine the magnitude and nature of the involved forces. Currently, several mechanisms are hypothesized to be responsible for USNM, specifically: pressure and velocity variations on the microsecond time-scale, ARF, AIF and AS. These mechanisms are in the typical focused US acting concurrently. However, standing-wave US fields, combined with varying the position of neurons, media properties and US parameters, could enable a decoupling of the mechanisms. The experiments developed in the project will be based on standing-wave lab-on-a-chip acoustofluidics and will provide well-defined acoustic environments for neurons to interact with, each promoting a different isolated mechanism of acoustic forcing and thus revealing its role in USNM. The forces on neurons at the cellular level will be investigated through particle image velocimetry, traction force microscopy, and fluorescence resonance energy transfer force sensors. Computational models of a neuron in US for quantification of forces will be developed and calibrated through extensive material characterization of neurons and the surrounding media. The project aims at discovering the main underlying mechanisms of USNM and could therefore provide the missing foundation, which would have an enormous impact on the development of USNM and its translation to clinics and neuroscience research. Furthermore, the developed methodology could pave the way towards understanding how US interacts with biological cells in the context of other, equally promising biological responses, such as angiogenesis or release of cancer biomarkers.
2024
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Long-Term Fellowships - LTF
Islands of Calleja: a novel neural substrate linking maternal cannabis use with mental disorders
PROKOP Susanne (HUNGARY)
Gill Center for Biomolecular Science - Indiana University - Bloomington - United States
MACKIE Kenneth (Host supervisor)
GUO Feng (Host supervisor)
Prenatal exposure to psychotropic substances, such as cannabis, greatly increases offspring vulnerability to psychiatric disorders. However, the molecular, cellular, and circuit level mechanisms that predispose exposed children to psychiatric disorders are poorly understood. I recently found that cariprazine, a novel antipsychotic and antidepressant medicine, primarily binds to a subregion of the ventral striatum called the "Islands of Calleja" (IoC). By developing a fluorescent-ligand-based labeling approach for super-resolution microscopy, I observed that cariprazine preferentially binds to D3 dopamine receptors on IoC neurons, suggesting this understudied brain area has substantial psychiatric significance. The IoC has also been linked to neurodevelopmental processes, but the impact of early drug exposure on IoC development and how molecular processes in the IoC increase susceptibility to psychiatric disorders remain unknown. My specific hypothesis is that maternal substance abuse elicits maladaptive changes in the dopaminergic system within the IoC, which may contribute to offspring vulnerability. Importantly, the prevalence of maternal cannabis use is steadily increasing with its worldwide trend for legalization. However, prenatal exposure to ?9-tetrahydrocannabinol (THC), the psychoactive substance in cannabis, leads to a hyperdopaminergic state in offspring in preclinical models, and longitudinal studies demonstrate a strong association between maternal cannabis use and the risk of psychiatric disorders. Thus, prenatal THC (pTHC) treatment is a suitable model with timely translational relevance to address my hypothesis. Prof. Ken Mackie is one of the leaders in cannabinoid research. His group has recently established in vivo pTHC mouse models and developed an innovative microfluidic system in collaboration with Dr. Feng Guo, enabling high-throughput pharmacological tests on human brain organoids in a standardized manner. His state-of-the-art approaches, combined with my prior experience in super-resolution imaging, offer a unique opportunity to investigate drug effects within the microenvironment of the developing brain, providing testable hypotheses for in vivo studies. My proposed strategy is to integrate microfluidic technology with IoC-containing striatal organoids and use fluorescent ligand-based super-resolution imaging of the dopaminergic system to determine the molecular and cellular effects of THC exposure on IoC circuit maturation in organoids. I will test if the most important findings can be replicated in the developing brain in vivo and use interrogation experiments to elucidate which molecular and cellular changes lead to behavioral abnormalities. The proposed research will not only lead to a better understanding of the adverse effects of maternal cannabis use but will also provide a conceptual and methodical framework for future studies on a wide range of perinatal stressors and their causal link to psychiatric disorders.
2024
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Long-Term Fellowships - LTF
Unravelling the molecular mechanisms of thyroglobulin endocytosis mediated by the R2 receptor
RAMANADANE Karthik (FRANCE)
Department of structural biology - Human Technopole - Milano - ITALY
COSCIA Francesca (Host supervisor)
Thyroid hormones (TH) are essential for development and metabolism in all vertebrates. TH imbalance severely compromises the well-being of about 10% of the world population, and represents a yearly financial burden of over several billion dollars for the health system in a country like the United States of America. However, the molecular mechanism behind their regulation is far from being understood, as so their tuning in vivo. TH are synthesized via iodination of their protein precursor thyroglobulin (TG) within the extracellular lumen of thyroid follicles, then released into the bloodstream to reach target tissues in the organism (e.g., brain, muscles, liver). TG endocytosis is a key step for TH release and a membrane protein receptor (TGR) has been shown to mainly promote TG internalization from the extracellular lumen. How the TG recruitment and internalization machinery works in different cellular conditions remains an outstanding question in the field. We propose to fill this knowledge gap, unraveling the mechanistic aspects of TG endocytosis by TGR via an integrative structural biology approach.
First, using single particle cryo-electron microscopy (cryo-EM), we aim to unravel the first structure of human TG receptor in isolation and in complex with TG, identifying the determinants of iodinated TG recognition by TGR, mapping the effects of disease mutations. Secondly, using a combination of proteomics, gene editing and cellular endocytosis assays, we aim to unravel the complete machinery of the TG internalization complex. Finally, we seek to visualize it in situ, recapitulating the internalization process directly in cells.
We expect to decipher the rules of TG endocytosis in the thyroid, opening a new front for targeted TH control in organisms. Since TGR is also expressed in many other tissues and mediates the transport of other organ-specific cargoes this study will have ample application to understand key trafficking routes in other tissues.
2024
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Grant Awardees - Early Career
A novel approach to tropical dendroclimatology using hyperspectral imaging and deep learning [ATHIL]
RANNESTAD Meley (NORWAY)
- Norwegian University of Life Sciences - Ås - NORWAY
SIYUM Zenebe (ETHIOPIA)
- Mekelle University, Institute of Climate and Society - Mekelle - ETHIOPIA
Dendroclimatology, the science of extracting past climate records from the distinct properties of annual tree-rings plays a critical role in climate research. The science has been widely applied in the temperate and boreal regions, where the strong yearly cycles in temperature and light availability induces annual ring formation in trees. In the tropics, however, its application has been limited as tree-ring formation in many tropical tree species can be complex due to mainly the low seasonal climatic changes and the high climatic fluctuations within seasons in the region. Consequently, paleo-climate records in tropical countries, particularly in Africa are scarce and the continent’s climate science is currently the least advanced in the world. Nevertheless, tree-rings are the widely available climate-proxy data sources in Africa, indicating the need for improved tree-ring detecting and characterizing technologies. Hyperspectral imaging (HSI) provides an efficient method for the characterization of physical and chemical features in objects at the molecular level. HSI systems capture vast amounts of spatially resolved spectral data and provide rich information about the composition and characteristics of objects in an image. On the other hand, deep learning (DL) provides an effective HIS data processing technique owing to its ability to automatically learn and extract complex patterns and features from images. In this context, the aim of the proposed project is to test a novel approach to dendroclimatology using a combination of these cutting-edge analytical technologies on two selected tropical tree species. To achieve the aim, we will collaborate between a fully equipped HSI in Norway with an emerging tropical dendroclimatology lab in Ethiopia. The proposed project will involve research in both HSI and DL techniques. Different HSI systems, sample preparation and instrumental setup techniques, and light conditions appropriate for detecting and characterizing tree-rings from different sample types will be explored in laboratory environment. Subsequently, advanced DL data processing algorithms will be explored to obtain good spectral signal from the samples and quality data for robust prediction models. Finally, a hyperspectral camera with relevant wavelength bands will be applied outdoors for field analysis of wooden samples.
2024
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Long-Term Fellowships - LTF
Understanding the formation of schema representations in prefrontal cortex
REINERT Sandra (GERMANY)
Sainsbury Wellcome Center - University College London (UCL) - London - United Kingdom
MRSIC-FLOGEL Thomas (Host supervisor)
BEHRENS Timothy (Host supervisor)
Humans and animals learn schemas – common structures in prior experiences – and apply them to novel problems to efficiently guide their decisions. Prefrontal cortex (PFC) has been identified as a key brain area that encodes schemas. However, how such schemas are implemented in neural circuits is unclear yet fundamental for a mechanistic understanding of flexible behavior. Emerging research in the host laboratories has begun to provide insight into this question; in a sequence learning task in mice, they revealed how PFC neurons encode the learned schema and map it to new tasks. Critically, the data suggest a mechanism for building the schema representation that, if true, would generalize to complex real-life schemata and reveal a novel general mechanism for structuring behavior. Testing this requires tracking individual neurons in PFC from the onset of learning to expert behavior – exactly the skillset I developed during my PhD. In this project, I aim to characterize how prefrontal cortex forms schema representations with learning. First, I will develop a head-fixed version of the sequence learning task to enable simultaneous two-photon calcium imaging. I will use this design to follow the activity of PFC neurons in task across learning to identify how and when the schema representation arises. Finally, I will investigate the role of inputs from key areas including entorhinal cortex (EC) and hippocampus (HC) and dopamine signals in the formation of schema representations. I hypothesize that: 1) at the start of learning, PFC neurons map progress through individual goal-directed actions (termed phase cells); 2) these cells split into groups tracking the progress of each action in the schema (i.e. subgoals), allowing generalization across tasks; 3) this learning and subsequent generalization relies on inputs from EC and HC (providing schema content), and dopamine signals (enabling remapping). I will use virtual reality technology to create a spatial sequence learning task for mice that are head-fixed on a track ball. I will then make medial PFC accessible for two-photon imaging with a microprism implant and transduce neurons in PFC with a fluorescent calcium indicator, utilizing the expertise from my PhD. This will enable chronic recording of cellular and subcellular calcium activity in behaving animals throughout learning. Next, two-photon imaging of a dopamine sensor will allow me to follow dopamine signals in PFC during learning and generalization. Lastly, by expressing a calcium indicator in EC and HC neurons, I will image their axon terminals in PFC and record the information communicated to PFC before, during and after learning. With this, I plan to observe what inputs PFC neurons integrate during learning to elucidate what changes in neuronal computations lead to schema representations. In conclusion, my project will shed light on how prefrontal cortex forms models of tasks to efficiently and flexibly guide behavior in novel situations.
2024
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Cross Disciplinary Fellowships - CDF
Mechano-Chemical Coupling in Cytoplasmic Flows: The competition between Self-Organization and Chaos
RIEDL Michael (AUSTRIA)
Excellence Cluster Physics of Life - TU Dresden (Technische Universität Dresden) - Dresden - GERMANY
BRUGUES Jan (Host supervisor)
During embryonic development the cytoplasm separates into distinct compartments prior to cell division. This partitioning occurs spontaneously and in the absence of external stimuli or physical boundaries (i.e., the plasma membrane). Regulated by microtubules, the partitioning forces can lead to large-scale cytoplasmic flows. These flows, in turn, redistribute cytoplasm and nuclei, which define the new cell centers. Meanwhile, the constituents of the cytoplasm are free to move. Yet, they must relocate to a suitable position before the subsequent cell division. Reliant on the capacity of the cytoplasm for self-organization, the coordination of this redistribution results from the interplay between chemical and physical processes. While spatially synchronized chemical oscillators determine the timing of the cell cycle, active processes facilitate flow patterns that robustly redistribute the constituents within the cytoplasm. The potential for chaos has been hypothesized to pose an inherent restriction to development, which relies on maintaining order in the chemical oscillations as well as cytoplasmic flows. While simulations of the cell cycle oscillator confirm its potential for chaos, experiments have yet to confirm this possibility. Additionally, the consequences of a perturbed cell cycle oscillator on the spatially reorganization of the cytoplasm are unknown. Recent studies showed that active fluids can transition to turbulent flows even in the absence of inertial forces. Unpublished results in the host lab confirmed this for flows in reconstituted cytoplasm of Xenopus laevis. In this proposal, I aim to experimentally investigate the role of both cellularization and coupling of active flows with the cell cycle oscillator as strategies to avoid chaos. I hypothesize that the mechano-chemical coupling between the cytoplasm and the cell cycle actively regulates the transition to chaos. First (i), using microfluidics, I will generate droplets containing cycling cytoplasmic extract. By varying droplet size and boundary properties (e.g., stiffness), I will explore the effects of boundary interactions on internal flows. Second (ii), I will extend my investigation to a multiple droplet system to explore the effects of direct or indirect coupling (e.g., chemical or mechanical) of adjacent droplets and its effect on internal flows. Third (iii), I will perturb timing and spatial coupling with the cell cycle in (i) and (ii). I will evaluate the robustness of these systems by analyzing their response to external perturbations (e.g., mechanical deformations or localized temperature increases). In summary, this proposal aims to investigate the competition between the mechanisms of self-organization and the antagonizing transition to chaos in cytoplasmic flows. This research has the potential to explain why some embryos develop as syncytial masses while others divide into individual cells.