California Institute Of Technology

NIH Research Projects · FY 2026 · 2023-07

Project Summary / Abstract Research in the Robb group is focused on expanding the frontiers of the emergent field of polymer mechanochemistry, where mechanical force is harnessed to selectively activate productive chemical transformations in stress-sensitive molecules known as mechanophores. Our expertise is in the molecular design and development of new mechanophores and reaction strategies, enabling access to stimuli-responsive polymers that address challenges in a variety of areas including stress sensing and mechanically triggered molecular release. Our research advances the fundamental understanding of mechanochemical reactivity through the development of structure–activity relationships and novel molecular design principles, providing a foundation for creating innovative materials. Nevertheless, critical gaps remain that have limited the translation of polymer mechanochemistry to applications in biology and medicine. In this proposal, we outline a multifaceted approach for the development of systems that enable acoustically controlled molecular delivery from mechanochemically active polymers using biocompatible focused ultrasound, specifically targeting biological applications that have thus far remained out of reach. In the five-year period of this MIRA grant, we will build on a powerful mechanophore platform developed in our group for the mechanically triggered release of diverse small molecule payloads that leverages the mechanochemical activation of masked 2-furylcarbinol derivatives. While ultrasonication is routinely used in the laboratory for the mechanochemical activation of polymers, the strong acoustic cavitation of dissolved gases at these acoustic pressures is highly destructive to tissues, making it incompatible for most biological applications. Complementing our development of novel chemistries, we propose to develop unprecedented systems for achieving remote control of mechanochemical reactions using focused ultrasound under physiological conditions with spatial and temporal precision. The unique synergy provided by novel materials design and biocompatible acoustic activation strategies will realize the translational potential of polymer mechanochemistry and establish mechanophores for triggered release as an untapped biomedical tool. Our research will target the delivery of a wide range of payloads useful for theranostics to bioimaging that demonstrate the power of this approach and pave the way toward diverse applications in biology, medicine, and human health.

NIH Research Projects · FY 2026 · 2023-07

The enteric nervous system (ENS), the largest portion of the peripheral nervous system, is derived from neural crest populations referred to as “vagal” and “sacral”, arising from the neck and tail regions, respectively. However, much less has been published about sacral than vagal neural crest. To rectify this knowledge gap, we propose to: 1. characterize the temporal sequence of migration and differentiation of sacral neural crest cells into neuronal subtypes in the hindgut; 2. transcriptionally profile the sacral neural crest as at multiple time points and compare with that of vagal neural crest; 3. test function of transcription factors that may drive sacral neural crest cell fate choice. The results will enable us to test whether vagal and sacral neural crest cells give rise to similar or distinct types of enteric neurons and elucidate the influence of the intestinal environment on their differentiation. We will characterize the chick sacral neural crest using a novel lineage labeling technique of Replication Incompetent Avian (RIA) retroviruses that enables us to specifically target and isolate by FACS the sacral neural crest-derived population to perform the following aims: Specific Aim 1: Retrovirus-mediated lineage analysis of the chick sacral compared with vagal neural crest: We will label the neural tube caudal to somite 27 with RIA retroviruses that permanently label sacral neural crest cells in order to follow their long term fate. Preliminary results suggest that sacral neural crest-derived cells populate the post-umbilical gut and differentiate into cholinergic motor neurons as well as tyrosine hydroxylase positive cells. Sacral neural crest will be compared with vagal neural crest and interactions between the two populations examined. Finally, clonal relationships between sacral crest cells will be characterized. Specific Aim 2: Single cell RNA-seq of sacral neural crest-derived cells in the post-umbilical gut. To elucidate gene regulatory programs controlling progressive differentiation of sacral neural crest cells into neurons and glia in the hindgut, we propose to characterize the transcriptional profile of sacral crest-derived cells FACS sorted from the post-umbilical gut and processed by single cell (sc) RNA-seq at embryonic days (E) 2.5, E6, E8, 10, 15, 21 (prehatching); these will be compared with vagal crest-derived cells at comparable stages. Preliminary scRNA-seq data on E10 suggest that there are differences in neuronal subtypes produced by sacral vs vagal crest. scRNA-seq will enable us to sample different neuronal subtypes and infer developmental trajectories. Specific Aim 3: Role of transcriptional regulators into differentiation of sacral neural crest into neuronal and glial subtypes. Focusing on transcription factors that are present in the enteric precursor cluster (e.g. Nfatc1, Foxn2, Sox4, Sox21, Elf2, Znf536), we will test whether sacral neural crest-enriched transcription factors are critical for mediating proliferation, migration within the gut, and/or cell fate decisions. To this end, we will perform targeted loss of function in the sacral neural crest using a single-plasmid CRISPR-Cas9 strategy and examine subsequent effects on distribution and/or differentiation of sacral neural crest-derived cells.

NIH Research Projects · FY 2024 · 2023-07

Project Summary/Abstract Innate behaviors, also known as instincts, are essential for the survival of all species in the animal kingdom, including humans. Of great interest is understanding how the experience-induced behavioral plasticity observed in innate behaviors can lead to maladaptive behavioral expressions, including uncontrolled aggression, sex offenses, and generalized anxiety. Such mental disorders carry a substantial emotional and financial burden to families and society, rendering the development of effective therapeutic approaches of high significance. The neural substrates of innate behaviors are distributed across numerous brain regions. An essential step towards understanding how the brain orchestrates behavior is to identify how inter-region interactions of neural activity influence behavior in freely moving animals. However, methodological limitations have largely restricted such investigations to single nuclei, while the deep brain remains to date inaccessible to in vivo mesoscale neural recordings in freely moving animals. The research proposed here uses custom-designed technology to break this methodological barrier, making possible recordings with single-unit resolution across twenty subregions of the deep brain (hypothalamus) in freely moving, innate behavior-expressing mice. In Aim 1, using this technology, I will investigate the presence of behavior-specific mesoscale hypothalamic neural features in the innate and learned expressions of social and fear-related behaviors. I will test whether the activity of specific brain regions can predict specific behaviors, and I will identify the spatial distribution of hypothalamic clusters exhibiting behavior-tuning or mixed selectivity. In Aim 2, I will dissect how aggression- or fear-conditioning alters mesoscale neural activity dynamics and test the effect of chronic administration of pharmacological reagents known to reduce the expression of maladaptive social and fear behaviors. Lastly, in Aim 3 – the independent phase of the award, I will expand the use of these tools to investigate mesoscale neural activity dynamics across diverse areas of the corticolimbic system, seeking to identify local and global principles that guide the expression of the physiological and maladaptive forms of innate behaviors. Collectively, the above approaches target the development of an experimental and analytical platform to address fundamental questions of systems neuroscience on the mesoscale mechanisms that govern the expression of innate behaviors. Following these initial steps, this work will be complemented by experimentation aiming to test whether the identified neural features in the behaviors of interest have a causal role. Through the mentored phase of this award, my vision is to build a highly complementary skillset to the one I acquired during my graduate training, and create a unique, state-of-the-art independent research program. By pursuing work spanning diverse disciplines, including molecular, cellular, systems neuroscience, behavioral and computational approaches, I am committed to developing a mechanistic understanding of the neural basis of innate behaviors, with the potential to lead to advanced therapeutic solutions.

NIH Research Projects · FY 2025 · 2023-06

Project Abstract The fundamental objective of this proposal is to fractionate human defensive circuits and behaviors. Our approach addresses three key issues. These include the knowledge that: (i) experimental paradigms have failed to fractionate the diverse subjective emotions and neural representations across different levels of threat imminence; (ii) a failure to create richer behavioral assays in humans to address the growing measurement gap between rodent and human studies of emotion; and (iii) a lack of focus on how these neural representations and behaviors diverge as a function of sex, therefore providing little or no insight in the sex disparity in anxiety disorders. To answer these questions, a critical first step is to understand how the human brain responds to different levels of threat imminence. In this proposal, we will create a new set of paradigms that provide a comprehensive fractionation of the human defensive system that include the emotions of fear and anxiety, and link them to a rich set of covert behavioral assays and overt decisions that vary along changing modes of threat imminence. We address these challenges via four specific aims: Aim 1. Provide a comprehensive fractionation of human defensive circuits, bodily emotions, and behaviors. Aim 1 has three key goals. This includes fractionating the defensive circuits involved in pre- and post-encounter (potential threat) and circa-strike (acute threat) modes of threat using a single task. We will identify novel behavioral measures for each mode of threat. Finally, we will measure subjective bodily emotional states for each level of threat imminence and correlate with Expts. 1-3. Aim 2. What brain regions direct the switching between offensive and defensive states? Here we aim to validate Aim 1 by examining the defensive behaviors and neural basis of post-encounter and circa-strike threat. Further, we extend on Aim 1 by investigating the role of the hypothalamus, amygdala, and vmPFC during switching between offensive and defensive survival states. Aim 3. What parts of the defensive circuit coordinate avoidance decisions? Extends on Aims 1 and 2 by assessing which parts of the defensive circuitry are involved in explicit, pre-emptive avoidance decisions. Further, we will examine the effects of attack predictability on risky or safe decisions and learning, thereby extending on the more implicit behaviors measured in Aims 1-2. Aim 4. Investigate how different parts of the defensive circuits differ in response to threats in males and females. Given that females demonstrate higher rates of anxiety disorders, we will investigate how sex is associated with brain activity, subjective emotions, defense behaviors, and decisions measured in Aims 1-3. We will cross-validate the sex difference findings across Aims 1-3 by comparing overlapping circuits and behaviors. At conclusion, this work will provide fundamental insights into how different parts of the human defensive systems operate under well-defined modes of threat imminence. The close links between our aims also provides several advantages, including a cross-validation of our results, the ability to examine how behaviors and brain systems diverge across the sexes, provide closer links with contemporary animal models.

NIH Research Projects · FY 2026 · 2023-06

Project Abstract The fundamental objective of this proposal is to fractionate human defensive circuits and behaviors. Our approach addresses three key issues. These include the knowledge that: (i) experimental paradigms have failed to fractionate the diverse subjective emotions and neural representations across different levels of threat imminence; (ii) a failure to create richer behavioral assays in humans to address the growing measurement gap between rodent and human studies of emotion; and (iii) a lack of focus on how these neural representations and behaviors diverge as a function of sex, therefore providing little or no insight in the sex disparity in anxiety disorders. To answer these questions, a critical first step is to understand how the human brain responds to different levels of threat imminence. In this proposal, we will create a new set of paradigms that provide a comprehensive fractionation of the human defensive system that include the emotions of fear and anxiety, and link them to a rich set of covert behavioral assays and overt decisions that vary along changing modes of threat imminence. We address these challenges via four specific aims: Aim 1. Provide a comprehensive fractionation of human defensive circuits, bodily emotions, and behaviors. Aim 1 has three key goals. This includes fractionating the defensive circuits involved in pre- and post-encounter (potential threat) and circa-strike (acute threat) modes of threat using a single task. We will identify novel behavioral measures for each mode of threat. Finally, we will measure subjective bodily emotional states for each level of threat imminence and correlate with Expts. 1-3. Aim 2. What brain regions direct the switching between offensive and defensive states? Here we aim to validate Aim 1 by examining the defensive behaviors and neural basis of post-encounter and circa-strike threat. Further, we extend on Aim 1 by investigating the role of the hypothalamus, amygdala, and vmPFC during switching between offensive and defensive survival states. Aim 3. What parts of the defensive circuit coordinate avoidance decisions? Extends on Aims 1 and 2 by assessing which parts of the defensive circuitry are involved in explicit, pre-emptive avoidance decisions. Further, we will examine the effects of attack predictability on risky or safe decisions and learning, thereby extending on the more implicit behaviors measured in Aims 1-2. Aim 4. Investigate how different parts of the defensive circuits differ in response to threats in males and females. Given that females demonstrate higher rates of anxiety disorders, we will investigate how sex is associated with brain activity, subjective emotions, defense behaviors, and decisions measured in Aims 1-3. We will cross-validate the sex difference findings across Aims 1-3 by comparing overlapping circuits and behaviors. At conclusion, this work will provide fundamental insights into how different parts of the human defensive systems operate under well-defined modes of threat imminence. The close links between our aims also provides several advantages, including a cross-validation of our results, the ability to examine how behaviors and brain systems diverge across the sexes, provide closer links with contemporary animal models.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Global histone deacetylation is linked to many types of cancer and is controlled by histone deacetylases (HDACs). Although HDAC inhibitors are widely used in cancer treatment, their activity does not target specific HDAC isoforms nor specific genes and therefore results in significant side effects for patients. Thus, there is a pressing need to target HDAC activity in a highly precise, gene-specific manner to develop safer and more effective treatments. The overall objective of this proposal is to understand how HDACs are recruited to specific genome regions. Because HDACs do not possess intrinsic DNA binding activity, they are thought to be recruited to chromatin through interactions with DNA binding proteins, though this mechanism has not been fully explored. Recently, our lab and others identified that the SHARP RNA-binding protein directly interacts with the Xist long noncoding RNA (lncRNA) to specifically recruit HDAC3 to the future inactive X chromosome (Xi). In this way, the Xist-SHARP/SMRT/HDAC3 repressive complex deacetylates histones and silences gene expression on the Xi. Our lab has also demonstrated that SHARP localizes to many nuclear sites (beyond the Xi) in an RNA-dependent manner, raising the question of which additional RNAs recruit it (along with HDAC3) and to what specific genomic locations. Notably, nearly all human HDACs associate within multi-protein complexes containing SHARP and other RNA-binding proteins, suggesting that this mechanism of RNA-guidance may extend beyond SHARP and Xist. I hypothesize that HDAC complexes are recruited by RNAs to achieve specificity to their various regulatory targets throughout the nucleus. First, I will determine which SHARP-RNA interactions are functionally important by genetically perturbing the SHARP binding sites of candidate RNAs. I will then measure effects on gene expression and if HDAC3 activity is required for these effects (Aim 1). Second, I will comprehensively identify RNA-binding proteins associated with other cancer-associated HDACs and define their in vivo RNA binding sites. I will then determine which protein-bound candidate RNAs from this screen are regulated in an HDAC-dependent manner (Aim 2). The proposed research has the potential to transform our understanding of RNAs and RNA- binding proteins as central regulators in organizing chromatin modifications by HDACs in both normal and cancerous cell states.

NIH Research Projects · FY 2025 · 2023-04

Project Summary Significant progress in human cancer therapy in the last decade has been driven by conceptionally new approaches to treating cancer, including cancer immunotherapy, cancer nanotherapy (e.g. liposomal doxorubicin or mRNA vaccines), or new types of biologics and small molecules. I propose to develop new approaches to targeting protein-membrane interactions that could yield unprecedented methods to modulate cancer signaling and generate useful paradigms for pharmacology at large. The role of protein-membrane and drug-membrane interactions will be explored and targeted on various levels, each highly relevant to cancer pharmacology: 1. Cancer signaling, a hallmark of cancer, is largely dependent on the recruitment of kinases (e.g. PI3K or PKC) and GTPases (e.g. RAS) to hotspots localized at the inner plasma membrane leaflet. I aim to develop bifunctional inhibitors with the capacity to modulate these interactions as a new approach to target cancer signaling. 2a. Membrane-integrated receptors are also key players in cellular signaling (e.g. enzyme-linked receptors or GPCRs). Recently, a number of pharmacophores have been discovered that target these proteins directly from the intramembrane space. These `Intramembrane pharmacophores' first partition into the membrane and then engage their target through lateral diffusion and entry. I aim to systematically modulate membrane exposed pharmacophore sites to explore principles governing the action of these pharmacophores which in turn will aid in the discoveries of new intramembrane pharmacophores. 2b. The majority of bioactive molecules acts on membrane proteins or intracellular targets and therefore needs to partition into or cross biological membranes. I propose to use combinatorial chemistry to discover new principles and chemical structures that modulate and privilege pharmacophores for cellular uptake. These will be tested in live cells using high throughput assays as a holistic approach to covering all possible uptake mechanisms on the first level of screening (e.g. endocytosis, transporters, passive diffusion). Combined, the proposed research will provide important insights into the functional role of protein- membrane interactions in cancer signaling and their vulnerability to small molecule-based modulation. Each of the proposed directions has the potential to yield fundamentally new and unprecedented approaches to targeting cancer and other diseases. My mentor and collaborators have extensive experience in cancer pharmacology, drug discovery, and the biophysical characterization of protein-membrane interfaces and will provide the training needed to conduct the proposed research. They will also provide the mentorship needed to acquire all skills and preliminary data needed for a successful transition to an independent career in cancer research.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT The challenge addressed by this proposal is to generate a map, the global human cell-surface interactome, that defines in vitro interactions among the extracellular domains of human cellsurface proteins (CSPs) and secreted proteins. This map will have a major impact on biomedical research, because cell-cell interactions mediated by CSPs are central to human physiology, controlling almost every biological process that is affected by disease. CSPs and secreted ligands comprise the majority of the therapeutic targets that have been successfully developed in recent years. Knowledge of interaction partners is essential for assessing the therapeutic potential of a CSP, since this knowledge defines the biological processes that it controls. For example, PD-1 was identified as a negative regulator of T cell function in 1992, but its value as a target for cancer immunotherapy only became clear much later, when its ligand PD-L1 was identified and found to be expressed on tumor cells. We will not only generate a complete map of in vitro interactions among human CSPs and secreted proteins, but also assess the functions of these interactions in cells of the human immune and nervous systems. This is a huge project, because there are about 2000 human single-transmembrane domain CSPs and 200 “orphan” secreted factors. Creation of a map of pairwise interactions among all of these proteins requires testing 4.8 million interactions. This is beyond the capacity of current screening methods, so execution of this screen at an academic institution will require the development of new technologies. This project is too large to be supported by a traditional RO1, but is perfectly suited to the transformative research award mechanism. Here we propose new ways to multiplex both in vitro biochemical screens and in vivo functional screens, so as to make it possible to define all in vitro interactions among CSPs and secreted ligands and to assess the functions of many of these within a 5-year funding period. To do this, we will first multiplex and sensitize in vitro interactome screens using color-coded beads and high-avidity nanoparticles. We will then develop methods to convert in vitro protein interaction screens into high-throughput DNA sequencing screens, which have a huge multiplexing capacity. For the functional screens, multiplexing single- cell analysis of cell fate perturbations can allow us to assess the effects of many different ligands on single immune system and neural cells in a single experiment. The rationale for the overall approach described here is that it defines a stepwise process in which we systematically develop and optimize screen technologies, then use the technology that performs best for execution of the actual screens.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Profiling kinases and their activities in single cells will enable exploration of cell type specific functional and biochemical pathways. While we have learned a great deal about transcriptional states of different cell types in the past decade through single cell genomics, we still understand little about proteins and in particular kinases networks in diverse cell populations. This is largely because single-cell kinome analysis faces major technical challenges. Unlike nucleic acids, proteins cannot be amplified, making detection of minute quantities from single cells difficult. We recently demonstrated a proof-of-principle experiment to accurately detect protein PTM isoforms in single cells. Here we propose to scale this method up to profile kinases at the global level in single cells in both cell culture and in tissues. We will integrate the single cell kinome analysis with RNA measurements to identify cell type specific kinase profiles. Ultimately, we will map the kinome with spatial context in the mouse and human brain to examine the kinase pathways involved in aging and neurodegeneration which could identify druggable targets. This project is a major departure from our current research. Its goals are ambitious but achievable. The project will leverage our expertise in single cell technology development and in application of new technology to diverse biological systems.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Imaging and genomics are becoming increasingly intertwined, as multiplexed RNA FISH and multiplexed immunohistochemistry now make it possible to perform “omic” measurements while preserving spatial information. These new technologies are allowing us to create a new, descriptive understanding of normal and diseased tissues. For cell culture models, they offer the promise of measuring multiple facets of cellular behavior – ranging from cell shape to gene expression – all in the same cell. This can be done by pairing dynamic live-cell imaging data with end-point spatial genomics measurements. Such measurements could even be performed in the setting of perturbations, creating a powerful tool for mapping biological networks. In this proposal, I seek to make these methods accessible to the life science community by using large-scale data annotation, deep learning, and cloud computing to solve several outstanding cellular image analysis problems facing the spatial genomics field. I also propose to develop a simple, scalable approach to performing perturbations in imaging-based experiments. The work proposed here is three-fold. First, we will develop deep learning methods for performing whole cell segmentation in tissues as well as segmentation and lineage construction in live-cell imaging movies. To ensure these models generalize across tissues, cell lines, and imaging platforms we will undertake a large-scale data annotation effort to create a standardized collection of images that have been annotated with single cell resolution. Second, we will also develop new deep learning methods for unsupervised learning of cellular behaviors. Third, we will create a new approach to imaging-based reverse genetic screens. In this approach, we will use CRISPR-Display to create multi-color spatial patterns in cell nuclei. This will allow us to link cells and perturbations in images while minimizing the number of collected images. Libraries with 100’s of thousands of perturbations would be interpretable with only 1-2 rounds of low-magnification 4 color imaging. Achieving these high-risk, high-reward goals will constitute a transformative advance as it will empower researchers studying living systems with imaging at the resolution of a single cell with both ease and scale. Once finished, this work will place the microscope back at the center of the biologist’s toolkit and enable images to become a universal datatype for biology.

NIH Research Projects · FY 2024 · 2022-09

Abstract Naphthalenones are bicyclic scaffolds found in many natural and synthetic compounds which display diverse biological activities and serve as building blocks to access more complex molecules. Naphthalenones have been successfully synthesized via dearomatization of naphthols with simultaneous installation of new C–C or C–X bonds. Formation of these new quaternary centers with stereoselective control has proven to be a synthetic challenge and commonly requires use of expensive, rare, or toxic transition metals. In few cases where earth abundant iron or organic catalysts are utilized, enantioselective control and/or yield are compromised. Biocata- lysts provide a great alternative for a more sustainable synthetic route which operate under mild conditions, generate enantioenriched products, reduce side products, and achieve high substrate selectivity. Through itera- tive rounds of genetic mutation and natural selection, enzymes can evolve to catalyze new-to-nature reactions. Herein, I propose to engineer an alternative catalytic route to synthesize chiral naphthalenones utilizing the di- verse library of hemoproteins developed by the Arnold group. This work will be achieved through engineering and evolving hemoproteins for (i) dearomative amination of 2-naphthols by nitrene insertion, (ii) dearomatization of 2-naphthols by carbene insertion, and (iii) further functionalization of naphthalenone core scaffolds by stereo- specific biocatalytic reactions. These reactions will be the first of its kind to be characterized in hemoproteins. The products of these reactions will feature new quaternary carbon centers and will expand current mechanistic knowledge of the robust hemoproteins and their abilities to tame reactive carbenes and nitrenes for highly en- antioselective reactions. This work will explore new methods to access substituted polycyclic structures in a facile and sustainable approach expanding the synthetic feasibility of biologically relevant naphthalenones. The biocatalytic method generated in this proposal will begin to elucidate new strategies for dearomatization and allow for transformation of many readily available 2-dimensional aromatic feedstocks into intricate pharmaco- phores.

NIH Research Projects · FY 2025 · 2022-08

SUMMARY Tight regulation of gene expression in space and time is necessary for development and homeostasis in multicellular organisms. Regions of the genome outside gene coding sequences (cis-regulatory modules, or CRMs/enhancers) serve as assembly platforms for transcription factors that facilitate gene expression in response to cellular or environmental cues. Although many genes are regulated by multiple CRMs, mechanisms that coordinate the action of these CRMs and that regulate their local chromatin dynamics are poorly understood. In the fruit fly Drosophila melanogaster, expression of the transcription factor Brinker (Brk) is activated in the early embryo by two distal CRMs, one 5’ and one 3’ to the brk gene. Initially thought to be redundant, these CRMs were found to drive sequential, partially overlapping patterns of expression along the embryonic dorso-ventral axis. Further, these CRMs depend on a promoter-proximal element (PPE) that appears to facilitate the sequential, long-range interaction of each CRM with the promoter. We have additional evidence that the brk PPE is required for brk expression in several other tissues. We hypothesize that this PPE located upstream of the brk gene in Drosophila represents a general mechanism for coordinating multiple cis-regulatory modules’ (CRMs’) interaction with the promoter, and that this coordination of local chromatin dynamics is important for proper gene expression, development and maintenance of homeostasis. To test this hypothesis, we propose the following experimental directions: Aim 1 will test the idea that the brk PPE manages chromatin conformation at the brk gene locus; Aim 2 will identify molecular effectors supporting brk PPE action; and Aim 3 will investigate a role for one PPE-binding protein Odd paired (Opa) in supporting global CRM-promoter interactions at other loci in addition to brk. Many genes across diverse taxa are regulated by multiple CRMs – including so-called super, stretch or shadow enhancers – yet we know very little about how these various regulatory contributions are coordinated during normal development. Insights will come from the study of already well-characterized genes such as brk, whose expression depends on CRM coordination by a promoter proximal element; as well as through whole genome assays of chromatin conformation to uncover the mechanisms regulating CRM/enhancer-promoter interactions, in general. New experimental approaches, which permit targeted manipulation and direct observation of chromatin in live, differentiated cells of an intact organism, combined with tried-and-true techniques for the analysis of genetic and developmental phenomena in Drosophila can provide a link between CRM-promoter interaction and the contributions of transcription factor binding to the regulation of gene expression. Because many genes, pathways and regulatory mechanisms are shared between Drosophila and higher organisms, improved understanding of how gene regulation is coordinated at complex loci in flies is likely to inform new approaches to understand these phenomena in wild-type as well as disease-relevant human contexts.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY The AAV BRAIN Safe and Effective Neuromodulator and Sensor Utilization across Species (SENSUS, led by Caltech) of the NIH BRAIN Initiative Armamentarium Project will develop, validate, and disseminate integrated (capsid and genome) engineered adeno-associated virus (AAV) tools to monitor and manipulate molecularly defined neuronal cell types across vertebrate species. To broaden access to these reagents within the neuroscientific community, we propose a close partnership between the AAV BRAIN SENSUS and California State Polytechnic University, Pomona (CPP), which will host a new Armamentarium Vector Core (ArmVC). This collaboration will also increase access to the research community itself for underrepresented minorities (URM) and minority-serving institutions (MSI), as CPP is a designated Hispanic-Serving Institution that also serves large populations of Pell-eligible, first-generation, and African American students. This partnership will foster close interfacing between experts in AAV engineering for neuroscience (PI Gradinaru), AAV production innovations and dissemination (PI Miles), and URM undergraduate research and mentoring (Co-I Steele) to build the physical and human infrastructure for AAV production, validation, and dissemination at CPP while comprehensively integrating MSI undergraduates. Caltech’s CLOVER Center (directed by PI Miles) will utilize their experience in running a successful AAV dissemination center to work with Co-I Steele and Damien Wolfe, M.S., a CPP and Steele lab alum and CLOVER technician who will work at ArmVC, to establish robust AAV production and dissemination capabilities in the ArmVC at CPP (Aim 1). CLOVER and ArmVC will cooperatively run a new AAV BRAIN SENSUS Program for Inclusive Research Experience (ASPIRE) wherein CPP undergraduate students chosen as ASPIRE Scientists will produce validated AAV for dissemination to the neuroscience community as part of the new ArmVC (Aim 2) and work in Co-I Steele’s lab to independently validate, using immunohistochemistry, the novel AAV identified via high-throughput methods in PI Gradinaru’s lab (Aim 3). ASPIRE Scientists will benefit from activities including workshops, poster presentations, research seminars, and career-focused mentoring meetings with PI Gradinaru at CPP and Caltech. Yearly training (and re-training of returning staff and undergraduates) in AAV production by CLOVER staff will ensure that methodological advances in AAV production, purification, and characterization developed or adopted by the CLOVER center are quickly transferred between sites. ArmVC offerings will be publicized and disseminated through a stand-alone website while also being integrated into AAV BRAIN SENSUS electronic resources for seamless reagent cataloguing and feedback. With strong quality control and quality assurance processes in place and overseen by CLOVER, the ArmVC at CPP will broaden access to modern neuroscientific tools and help bridge the gap in URM career progression in research science.

NIH Research Projects · FY 2026 · 2022-06

The vertebrate enteric nervous system (ENS), the largest portion of the peripheral nervous system, mostly derives from the vagal neural crest which arises in the caudal hindbrain, migrates to the foregut and along the entire length of the gut, differentiating into many different neuronal subtypes. In humans, defects in ENS formation cause Hirschsprung’s Disease, or colonic agangliogenesis. While ENS neurons play critical roles in regulating gastrointestinal motility, surprisingly little is known about how or what controls neuronal lineage specification in the ENS. The recent advent of single-cell technologies promises to help elucidate identification of neuronal cell types and molecular mechanisms underlying enteric neuronal differentiation. Zebrafish offer several advantages for tackling important questions in ENS development due to their simplified enteric nervous system, accessibility to genetic manipulation and facility of imaging. Similar to amniotes, the zebrafish gut contains neural crest-derived neuronal subtypes, ranging from serotonergic, cholinergic and dopaminergic neurons to VIP, Substance P and Nitric Oxide (NO)-containing neurons. Here, we propose to perform single cell RNA-seq of individual enteric precursors and neurons at different developmental stages (2-6 dpf) within the developing ENS. The function of candidate transcription and signaling factors in ENS neuronal specification will be tested by CRISPR-Cas9 perturbation experiments in both zebrafish and chick. Finally, single cell ATAC-seq will be used to identify and then dissect enteric enhancers to build an ENS gene regulatory network. We propose to perform the following aims: Aim 1: Transcriptional profiling of the enteric neural crest-derived cells at individual cell resolution using single cell RNA-seq and multiplex fluorescent in situ hybridization. We will perform single cell RNA-seq on thousands of cells per time point (2-6 days post-fertilization) of enteric precursors and neurons dissected and sorted from the zebrafish embryonic gut. We will validate expression of genes of interest, in particular transcription factors and signaling molecules, using hybridization chain reaction (HCR) and infer developmental trajectories from progenitor to neuronal differentiation. Aim 2: Role of transcription factors in differentiation of ENS neuronal subtypes in zebrafish and chick. We will mine the scRNA-seq to identify transcription factors whose expression correlates with the progenitor state (e.g. hey1a) and various neuronal subtype markers (e.g. ebf1a, etv1, Klf6a, Insm1a) for functional validation using CRISPR-Cas9 mediated knock-out in zebrafish and in chick. Aim 3: Identifying active enhancers associated with neuronal differentiation in the ENS using single cell ATAC-seq. We will use single cell ATAC-seq to identify and test putative regulatory elements functioning in neuronal precursor and differentiating neurons in the developing zebrafish ENS. Putative enhancing regions will be tested for their ability to drive ENS expression in zebrafish, mutated and tested for conservation with amniotes.

NIH Research Projects · FY 2025 · 2022-06

Project Summary Gout affects 8.3 million of US adults. Gout and hyperuricemia are associated with hypertension, progression of renal disease, cardiovascular disease, and dyslipidemia. Dietary modifications can lower SU by 1 mg/dL. Urate lowering therapy (ULT) has demonstrated improvement in clinical outcomes. Yet despite these findings and national guideline recommendations for the management of gout, knowledge about dietary purine content is poor and adherence with gout medications is the lowest among 7 chronic diseases. Our group has developed a cutaneous sensor patch that can detect uric acid (UA) in sweat. Sweat UA has strong correlation with serum urate (SU) levels making it an ideal non-invasive method to frequently sample subject’s uric acid levels. We postulate that providing patients with gout their pre- and post-prandial UA results will result in better dietary and medication adherence decisions. To better understand the impact of urate control on gout and other metabolic conditions, we seek to expand the breadth of metabolites and nutrients monitored by this system. We seek to extend the duration of use for the skin patch to include morning and evening meals. We seek to develop a friendly, easy to use interface for data collection and patient reports. We will evaluate the impact of the URic AcId + metabolite Monitoring System (UR+AIMS) enhancements on gout and other metabolic clinical outcomes though a 10-week randomized trial for subjects with gout either on or off urate lowering treatments (4 arms). Specifically, we will test whether the use of UR+AIMS with patient pre- and post-prandial uric acid reports results in improved serum urate control as measured by proportion of patients with SU < 6 mg/dL. Since urate is intertwined with other metabolic pathways, we will also evaluate whether UR+AIMS intervention results in improved blood pressure, blood sugar and lipid control. With the detailed (almost continuous) prospective data on urate and other metabolites, we will evaluate the changes in metabolites prior to a gout flare. These observations may lead to new understanding about the triggering factors preceding a gout flare. In addition to purine metabolites, we will be measuring the allopurinol (most common urate lowering medication) metabolite, oxypurinol. Effective dosing of allopurinol has not been achieved at population level. Confusion arises from conflicting dosing recommendations over the years and current dosing recommendations (start low and titrate up slowly to target dose that lowers SU < 6 mg/dL). Furthermore, impact of renal disease, body size and diuretics that all impact effective dose needed to achieve SU goal. With continuous oxypurinol measures, we will evaluate if the initial steady oxypurinol along with change in UA can predict the ultimate dose at the end of titration required to achieve SU < 6 mg/dL. This prediction rule would simplify future allopurinol dosing schedules, reducing the number of lab visits and provider interactions.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY / ABSTRACT The discovery of powerful new methods for the synthesis of organic compounds can be enabling for biomedical research, e.g., by providing more ready access to known families of target molecules or access for the first time to new classes of molecules. Catalytic and enantioselective methods for carbon–carbon, carbon– nitrogen, and carbon–oxygen formation are of particular interest, due to issues including sustainability, the potentially divergent bioactivity of the two enantiomers of a compound, and the predominance of such bonds in the backbone of organic molecules, respectively. The substitution reaction of an alkyl electrophile by a nucleophile is a particularly straightforward approach to the assembly of organic molecules. Classical pathways for substitution, such as the SN1 and the SN2 reactions, are limited in scope with respect to both the electrophile and the nucleophile. Furthermore, these pathways almost never provide access to highly enantioenriched products from readily available racemic starting materials. Through the use of transition-metal catalysis, wherein the electrophile is converted into an organic radical, it is possible to begin to address both of the key challenges in nucleophilic substitution reactions of alkyl electrophiles–broader scope and control of enantioselectivity. For example, chiral nickel and copper complexes can catalyze the enantioconvergent coupling of a number of racemic secondary and tertiary alkyl electrophiles with a variety of nucleophiles. To date, only a small fraction of the conceivable permutations of electrophilic and nucleophilic partners for metal-catalyzed substitution reactions of alkyl electrophiles have been explored, and still fewer such processes have been rendered enantioselective. The goal of this research program is to address the many unsolved challenges in this area. Efforts will focus on the development of mild and versatile methods to couple families of electrophiles and nucleophiles that have not previously been shown to be suitable reaction partners in aliphatic substitution reactions, including highly hindered substrates, while controlling stereoselectivity at the same time (at up to two stereocenters), including with racemic electrophiles and nucleophiles that lack directing groups. Success in this endeavor will substantially facilitate the synthesis of enantioenriched molecules. Mechanistic studies will be pursued in order to provide insight into the pathways by which the new metal- catalyzed substitution reactions proceed. The mechanistic investigations will facilitate reaction development, as well as enhance the community’s understanding of fundamental chemical reactivity.

NIH Research Projects · FY 2026 · 2022-04

Project Summary/Abstract: The objective of this research program is to discover and develop new reaction methodology en route to the synthesis of complex bioactive molecules. Our proposed studies will focus on the investigation and optimization of technologies that enable the synthesis of core structural and stereochemical subunits prevalent in many bioactive, polycyclic natural products. The processes that we develop will find utility in the synthesis of a wide variety of structures for which there are currently no efficient synthetic roadmaps. Importantly, the methods presented in this application will be useful outside of the contexts described herein and will arm practitioners of synthetic chemistry (in academic, government, and industrial laboratories) with a new set of important tools to access enantioenriched and functionally diverse chemical building blocks for synthesis. The research proposed in this grant application is focused on a) the development of new stereoselective reactions that produce densely substituted building blocks for synthesis, b) the development of transition metal catalyzed reactions for asymmetric alkylation, acylation, and hydrogenation processes, c) the development of these novel methods specifically for the preparation of building blocks containing all-carbon quaternary stereocenters and arrays of stereocenters, and d) the implementation of these new tactics in the syntheses of highly complex, bioactive natural products. These molecules are not only important from a biological standpoint, they also serve as a testing ground for our new technologies. As a consequence of this approach, we will have access to a) novel, medicinally relevant structures, b) a general platform for their synthesis, and c) new synthetic methodology that will impact a host of diverse applications.

NIH Research Projects · FY 2026 · 2022-01

Addiction involves brain systems mediating internal states of motivation, arousal and reward, as well as emotions. Such internal states influence goal-directed behaviors and decision-making. A common feature of such internal states is their valence and their persistence: they can have a positive or negative valence, and can outlast their triggering stimulus for many minutes. However the neurobiological mechanisms that underlie the persistence of internal states, and their relationship to the encoding of valence, are poorly understood. Drosophila provides a tractable genetic model organism for studying how neuromodulators act on neural circuits to control persistent internal states that govern goal-directed behavior and decision-making. We have discovered that P1 interneurons, which control male courtship behavior, can when activated promote a persistent internal state of social arousal or motivation, which can last for minutes. In a publication supported by the base grant, we have obtained evidence of a link between P1 interneurons and neurons that respond to octopamine (OA), an insect homolog of norepinephrine (NE), which is known to facilitate psychostimulant self- administration in rodents. We have also identified a downstream target of P1 neurons, called pCd cells, which appear to play a key requisite role in determining the persistence of an internal state of social arousal. During the extension period, we will continue our studies of how P1 neurons promote a rewarding internal state, and the relationship of these mechanisms to the positive valence, or rewarding nature, of P1 stimulation. In the first 2 years, we will focus on pursuing Aims 3 and 4 of the base grant. These aims were: Aim 3) to test the hypothesis that P1 neuron activation is positively valenced and rewarding; Aim 4) to investigate neuromodulatory mechanisms involved in P1 reward learning. In unpublished experiments, we have discovered that activation of P1 neurons can produce a real-time place preference (RTPP), and that it can also serve as an unconditional stimulus (US) for conditioned olfactory preference (COP). Both of these findings indicate that P1 activation is positively valenced, and that it can be rewarding. We plan to investigate whether plasticity during COP occurs at or downstream of P1 neurons, and whether P1 neurons are necessary for expression of the COP (Aim 3). Preliminary experiments suggest that dopamine (DA) may play a role in modulating the effects effects of P1 stimulation. We will confirm and extend these findings, and also investigate the role(s) of other neuromodulators including biogenic amines such as octopamine (OA), which we have shown to modulate the effect of P1 stimulation to activate aSP2 neurons that control social behavior9. Furthermore, we will investigate whether mushroom body (MB) neurons involved in reward learning are also involved in P1-mediated odor conditioning (Aim 4). Given previous data, we expect to find a role for the MB, but precisely which subset of MB neurons are involved is not clear. In Merit Extension Aim 5, we will investigate the role of other neuromodulators in P1-induced persistent social arousal and reward learning. Candidate neuromodulatory targets of P1 neurons include serotonergic (Trh+) neurons, a subset of which is activated in response to P1 stimulation (preliminary results), and neuropeptide F (NPF), which has been implicated in reward in other contexts. We will approach this problem using functional connectomics, in which optogenetic activation of P1 neurons is combined with calcium imaging in populations containing putative neuromodulatory targets of these cells. Target neurons can be “filled” using photo-activatable GFP (PA-GFP), and their morphology used as a “search image” to identify specific genetic drivers that label that subset of cells. Using these drivers, activation and silencing of these neurons can be performed in the context of both P1-mediated reward (RTPP and COP assays), and persistent social arousal. We have successfully established this approach and used it to identify pCd neurons, which are persistently activated by P1 neurons and required for persistent social behaviors triggered by P1 activation. As a complementary approach, we will take advantage of recent advances that we have made in techniques for whole-mount fluorescent in situ hybridization (FISH) in the adult brain, which allow identification of candidate follower cells activated by optogenetic stimulation of P1 neurons using FISH probes for hr38, an immediate early gene (analogous to c-fos) in Drosophila. Double-label FISH can be performed using hr38 and probes for neurotransmitter biosynthetic enzymes or neuropeptides, to identify neuromodulators expressed in P1 targets. A fundamental question is whether the mechanism mediating P1-induced persistent activity is also involved in reward. To address this question, in Merit Extension Aim 6 we will investigate the role of pCd neurons in P1- mediated reward learning, using functional manipulations of these cells. Preliminary data suggest that persistent activation of pCd neurons by P1 cells is modulated by DA, and we will investigate how this modulatory influence is exerted. We anticipate that these experiments will yield general principles of how persistent internal reward states are encoded by brains, with potentially broad relevance across phylogeny.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY The discovery and development of fluorescent proteins and optogenetics revolutionized biology by making it possible to image and control specific cellular processes with visible light. While these tools have enabled countless biological discoveries, the poor penetration of light into living tissue makes it difficult to use optical techniques in intact animals. As a result, biological phenomena ranging from the signaling of neurons in deep- brain regions, to the infiltration of immune cells into tumors, to the microbial colonization of the GI tract, are challenging to study within their natural in vivo context. If instead of light it were possible to visualize and manipulate cellular function using a more penetrant form of energy such as ultrasound, this would open previously inaccessible domains of in vivo biology to direct investigation. In addition, it would enhance the development of cell-based therapies by allowing cellular agents to be seen and controlled after administration into the human body. The physics of ultrasound make it an ideal modality for deep-tissue cellular communication. Sound waves in the MHz range are weakly scattered by tissue and can therefore penetrate several cm into the body. With wavelengths on the order of 100 µm and travel times < 1 ms, ultrasound can access many key structures and processes. When focused, sound waves can deliver mechanical and thermal energy to precise anatomical locations. These properties have already made ultrasound one of the world’s most widely used technologies for medical imaging and non-invasive surgery. However, the potential of ultrasound to serve as a tool for cellular imaging and control has been relatively untapped due to a lack of methods to connect it to the function of specific cells and biomolecules. In previous work, the Shapiro lab has pioneered the use of ultrasound in cellular and molecular imaging by developing the first acoustic reporter genes and biosensors for ultrasound, aiming to “do for ultrasound what fluorescent proteins have done for fluorescence microscopy”. The major goal of our proposed new research direction is to “do for ultrasound what optogenetics has done for light” by giving sound waves the ability to control specific cellular functions such as neuronal excitation, gene expression and intracellular signaling in vivo. The basic principle of our approach is to (1) use focused ultrasound to deposit acoustic energy at a specific location in tissue, (2) use genetically encoded “acoustic antennae” to convert this energy into local mechanical force, and (3) use this force to actuate mechanosensitive receptors to produce specific cellular signals. We will implement this approach in neurons and immune cells to enable unique neuroscience and cell therapy applications. If successful, this work will help establish the new field of sonogenetics by providing researchers and clinicians with the unprecedented ability to “point and click” on cells deep within the body and tell them what to do.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY How does the brain balance the need to preserve prior knowledge with the necessity to continuously learn new information? The tradeoff between stability and plasticity is inherent in both biological and artificial learning systems constrained by finite resources and capacity. The hippocampus is a brain region critical for memory formation and spatial learning, which can provide a powerful experimental system for characterizing this tradeoff. The role of the hippocampus in spatial cognition is supported by the finding that pyramidal neurons in this area (place cells) fire in specific locations in an environment (place fields). The population of place cells active in an environment is believed to form a neural representation or cognitive map of that environment. Spatial learning is critical for survival and involves two competing constraints: representations of space must be plastic to enable fast learning of new environments and changes in behavioral contingencies, and stable over time to enable recognition of familiar environments, reliable navigation, and leveraging of previous learning. How do these competing constraints affect the stability of place fields across time? The experimental characterization of the long-term stability of spatial representations in the hippocampus has been challenging as it requires tracking the activity of multiple place cells across extended periods of time (days to weeks). We propose to use novel approaches in large-scale electrophysiology and imaging in behaving rodents to characterize which neurons change their spatial tuning and how these changes depend on behavior. Furthermore, we will use recordings and circuit perturbations to characterize the activity patterns that predict changes in tuning stability. Our analysis will be carried out in the context of a theoretical framework for understanding the interplay between plasticity and stability of hippocampal representations. Characterizing the evolution of neural representations is of fundamental importance in understanding how information is maintained across brain circuits and how such maintenance is perturbed in brain disorders.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract The overarching goal of this proposal is to understand how the stereotypical structure of retina forms during embryonic development. Towards this goal, this proposal seeks to develop novel molecular recording technologies that allow the reconstruction of lineage history based on endpoint measurements. This method leverages genome editing techniques to stochastically create heritable mutations within synthetic barcode arrays that accumulate edits over time. Because readout of these arrays is compatible with spatial transcriptomics technologies, these methods when combined will allow the simultaneous capturing of transcriptional cell state, lineage relationships, and spatial position of single cells within retina tissue. The resulting lineage tree datasets will then be analyzed using a novel statistical tool termed Lineage Motif Analysis, a computational approach to identify all significantly over- or under-represented cellular patterns. This approach systematically enumerates all possible arrangements of observed fates on progressively larger subtrees and then compares their frequencies to those expected in a null model based on uncorrelated cell fate between cell divisions. Lineage trees will reveal how birth-order of cell types is regulated on the clonal level and lineage motifs will reveal the extent to which lineage in the retina is stochastic or preprogrammed. Furthermore, lineage motifs represent direct insight into the “rules” that govern how the retina forms during development, and provide a way to describe how such “rules” change in different contexts like disease or pharmacological perturbation. The datasets and analysis proposed here will inform the development of new therapeutic strategies for regenerative medicine to treat retinal diseases that lead to blindness. My training program outlined here will equip me with the necessary tools and knowledge to (1) carry out the aims of this proposal and gain novel biological understanding, and (2) advance me towards my career goal of leading a research team focused on studying the fundamental principles that underlie embryonic development and disease progression. I will work with Dr. Long Cai, a pioneer in spatial transcriptomics, to learn imaging and image processing techniques, as well as Dr. Carlos Lois, an expert in neurobiology, to learn mouse manipulation and surgical procedures. Dr. Elowitz and I will meet regularly to discuss my research progress, writing plans for paper publications and grants, teaching/mentoring students, and opportunities to present my research at Caltech and national conferences. As a PhD student at Caltech, I will have access to leaders well-versed in applying quantitative approaches to study developmental biology, state-of-the-art core facilities, and cutting-edge coursework in both biology and statistics. By funding the rest of my PhD research, this fellowship will enable me to uncover the fundamental principles that underlie retina development and set me up for independence as I transition towards becoming an independent investigator in my later career.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Metabolic syndrome is on the rise as the leading cause of morbidity and mortality, affecting more than a third of all U.S. adults. If untreated, patients who develop type 2 diabetes mellitus (T2D) are at high-risk for major adverse cardiovascular events, including stroke, myocardial infarction, and cardiovascular related deaths. Despite chronic screening and monitoring for patient-specific prediction and prevention for cardiometabolic disease, there remains a bottleneck to detect and monitor the metabolic risk factors underlying the rising epidemic of obesity-associated with hyperlipidemia, hypertension, and diabetes. For these reasons, developing wearable molecular sensors, which allow for seamless screening, monitoring, and potentially enables timely intervention, is clinically significant to confront the rising endemic of cardiometabolic disorders. In this project, we propose to continuously monitor a panel of key metabolic biomarkers including glucose, uric acid, branched-chain amino acids (BCAAs: leucine, isoleucine, and valine), and insulin using an integrated Molecular Sensing System (iMSS). We hypothesize that seamless detection of cardiometabolic biomarkers accelerates our capacity to identify metabolic risk factors in our prediabetic patients with obesity for early nutrition intervention to reduce health disparities in the U.S. In addition to integrating with our existing glucose and uric acid sensors, we propose to develop novel laser-engraved wearable sensors for continuous monitoring of BCAAs and insulin based on a novel nanobiosensing approach that combines high-throughput laser-fabricated graphene, molecular imprinting based ‘artificial antibody’, and in situ sensor regeneration technique. This approach will enable large-scale, low-cost fabrication of highly sensitive and selective sensors for continuous monitoring of clinical meaningful cardiometabolic analytes in human sweat at ultralow concentrations (such as BCAAs). The use of laser-induced microfluidics and numerical simulation-guided design optimization enables efficient fluid sampling with minimized effects from the sensing delay and fluid evaporation. Harnessing the power of concurrent multiplexed cardiometabolic sensing, adjusted electrochemical measurements based on pH, electrolytes, temperature, and sweat rate calibration minimize the systematic uncertainties persisted in the current generation of wearable sensing systems. We will validate the correlation of the sweat/blood biomarkers in healthy subjects using the iMSS and deploy these epidermal sensors to the high-risk patients. We envision that the iMSS system will provide an entry point to identify pre- diabetes and obesity at risk for conversion to T2D, and will have translational significance to mitigate clinical manifestation of major adverse cardiovascular events.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract This proposal responds to an FOA (RFA-NS-18-030) calling for 1) “novel approaches to understand neural circuitry associated with well-defined social behaviors;” 2) Distributed circuits that contribute to the coordination of motivational states and reward behavior;” 3) “Empirical and analytical approaches to understand how behavioral states are emergent properties of the interaction of neurons, circuits and networks.” The study of subcortical circuits that control conserved, naturalistic behaviors is crucial to understanding brain function. We aim to understand how dynamic interactions between different circuit nodes in the Hypothalamic-Extended Amygdala Decision (“HEAD”) network control innate social behavior decisions, e.g., between aggressive and reproductive behaviors. We propose an integrated approach to this problem that combines microendoscopic imaging (MEI) of genetically identified neuronal subpopulations with automated, machine learning-based classification of social behavior in freely moving mice, together with functional perturbations of neuronal activity in vivo. Our broad, long-term objective is to understand how distributed activity among interconnected structures in the HEAD network controls moment-to-moment decisions between competing goal-directed behaviors that are crucial for the survival of animals and humans. The central objective of this proposal is to understand how information flows through this network during social interactions, and is decoded to control the decision to engage in reproductive vs. aggressive social behaviors. To understand how activity in “upstream” nodes controls neural representations in “downstream” nodes, we will implement a novel approach combining reversible chemogenetic inhibition of the former with concurrent imaging of neuronal population activity in the latter. The rationale for this approach is that an understanding of the system requires characterizing the effects of functional manipulations on both behavioral and circuit-level phenotypes. To achieve our objective, we will first characterize the neural coding of behavior and conspecific sex identity in multiple nodes of the extended amygdala, using single-site microendoscopic imaging and computational analytic approaches (Aim 1); determine how perturbations in the activity of such nodes influence representations in hypothalamic nodes (Aim 2); investigate the roles of intra- and inter-nuclear interactions in determining the balance of activity between aggression and reproduction-promoting hypothalamic nodes (Aim 3); determine how this balance is decoded by downstream mid-brain structures to determine the type of social behavior to express (Aim 4). This contribution is significant because it represents a systems-level approach to understanding how a subcortical network controls behavioral decision-making. The contribution is innovative because it integrates analysis of neuronal population activity, quantitative measurement of naturalistic social behavior and functional perturbations of activity in specific neuronal subpopulations to gain insight into how distributed neural circuits control survival behaviors, in a context that is relevant to maladaptations causing human psychiatric disorders.

NIH Research Projects · FY 2025 · 2021-08

Abstract: The long-term objective of this application is to understand cortical processing of sensory to motor transformations within the human cerebral cortex. A vast number of computations must be performed to achieve sensory-guided motor control. Standing out among these computations, visual information of the goals of action must be transformed from the coordinates of the retina to the coordinates of effectors used for movement, for instance limb coordinates for reaching under visual guidance and to world coordinates for interactions in the environment. Once an object is grasped, somatosensory signals from the hand are required for dexterous manipulation of grasped objects. Internal models within the sensory motor pathway are essential for estimating the current state of the body and the external environment, accounting for lags in sensory feedback, and calibrating the body to the environment. We will use the rare opportunity of being able to record from populations of single neurons in a clinical study designed to develop neural prosthetics for tetraplegic participants paralyzed by spinal cord injuries. Cortical implants of microelectrode arrays will be made within three key locations in the sensorimotor system: primary motor cortex, primary somatosensory cortex, and posterior parietal cortex. These microelectrode arrays enable both recording and intracortical microstimulation. We will test the hypothesis that somatosensory and motor cortex represent imagined reaches in hand coordinates, but posterior parietal cortex is task dependent, and its population neural activity can flexibly change coordinate frames to enable encoding of the spatial relations within the body (arm and eyes), between the body and world (arm and reach targets; objects relative to self), and within the world (relative position of objects in the world) as required by task demands. Percepts evoked by intracortical microstimulation and imagined sensations will be used to understand the representation of cutaneous and proprioceptive information within primary somatosensory cortex and posterior parietal cortex. The hypothesis to be tested is that imagined sensation and electrically evoked sensations are highly overlapping—not just in primary somatosensory cortex but also in posterior parietal cortex. Lastly, we hypothesize that the posterior parietal cortex contains in humans an internal model of state estimation that shows plasticity for both natural and brain-control behaviors and transfers this learning to motor cortex. These studies will not only greatly advance our understanding of the human sensorimotor cortical circuit, but also will provide basic knowledge for the design of future neural prosthetics.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Internal sodium balance is critical for many physiological functions, including osmoregulation and action potentials. Deciphering the mechanisms that control sodium intake is essential for understanding the principles of appetite regulation and sodium homeostasis in the body. Our understanding of central sodium appetite regulation is still lacking compared to other appetite circuits such as thirst and hunger. I propose to study this fundamental brain circuit that controls our internal ion balance using transcriptomic and molecular genetic tools. Our preliminary and published results have identified specific neural populations in the mouse hindbrain and forebrain that acutely regulate sodium ingestion. However, it is currently unknown how these distinct neural nodes contribute to sodium appetite. Our central hypothesis is that distinct neural circuits regulate sodium appetite and tolerance. We will test this idea through three specific aims. In Aim 1, we will use gain- and loss-of-function manipulations to examine if individual neural populations control behavioral aversion and/or attraction toward sodium. This study is expected to identify the functional roles of each genetically defined neural population in sodium ingestion. In Aim 2, we propose to use genetics, virus tracing, and physiological recording to dissect the circuit organization underlying sodium ingestion. Once the anatomical map is identified, we will use projection specific neural perturbation to examine the function of the individual downstream regions. In Aim 3, we propose to identify cell types from downstream areas of sodium appetite neurons. We will achieve this by combining activity-dependent high-throughput single-cell transcriptomics and neural perturbation in the upstream population. This approach is expected to dissect specific cell types that receive sodium appetite signals from upstream neurons. We will then use genetic information of the identified downstream cell types to examine how signals from distinct nuclei interact to drive sodium appetite. Together, this proposal will provide critical insights into the brain-wide regulatory mechanisms underlying sodium ingestion.