1997 — 2000 |
Zemelman, Boris V |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Dissecting Vesicular Transport in Vivo @ Sloan-Kettering Institute For Cancer Res
steroid hormone receptor; membrane fusion; alleles; vesicle /vacuole; gene expression; intracellular transport; stress proteins; synaptic vesicles; tissue /cell culture; HeLa cells;
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0.898 |
2015 — 2016 |
Ellington, Andrew D [⬀] Zemelman, Boris V (co-PI) |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A Robust Ionotropic Activator For Brain-Wide Manipulation of Neuronal Function @ University of Texas, Austin
? DESCRIPTION (provided by applicant): This proposal embodies the rational design, high throughput screening, and in vitro characterization of novel neuronal actuators. The starting point for our endeavor is an ionotropic channel that launched the optogenetic revolution. We are confident that the highly original and comprehensive development scheme we have outlined will yield a new set of transformative tools for functional brain analysis. For nearly a decade, functional analysis of brain circuitry has relied on methods that allow neuronal activity to be perturbed in an intact brain with cell type-specificity. Genetically-encoded neuron actuators have ranged from chimeric G-protein coupled receptors (GPCRs) with orthogonal ligands to light-gated ionotropic channels. While these tools have helped uncover cellular substrates of cognitive and behavioral states, significant limitations remain. Optical fiber implantation is destructive, and illumination is limited by mechanical constraints and the requirement that the target site be identified in advance. GPCRs are often inefficient, display poor temporal control, and can produce long-term functional changes in neurons. We propose to develop and test a neuronal activator that embodies the strongest features of existing approaches. Based on the purinergic P2X receptor, this ionotropic channel will display high unitary conductance, negligible desensitization, and tunable gating. Its small molecule ligand will readily cross the blood-brain barrier. Complementary modifications in channel and ligand structure will help generate a family of orthogonal receptor-ligand pairs for independent control over multiple cell populations within the brain while eliminating crosstalk with endogenous factors. The strength of this and other pharmacogenetic approaches is that the locations of target neurons need not be known a priori; however, should precise temporal regulation be needed, the ligands can be chemically disabled (caged), enabling brief localized photoactivation. We are confident that our novel synthetic purinergic activator (SPArk) will advance functional brain mapping, providing robust control over discrete neuronal populations that represent known neurochemical classes or are selected using pioneering activity-based molecular-genetic methods. SPArk is a timely, highly efficient and flexible alternative to existing approaches; it is essential for continued progress in in vivo mechanistic interrogation of neuronal signaling pathways. We envision a panoply of tools that will be deployed brain-wide across species to control distinct ensembles of neurons, uncovering circuit connectivity and signaling hierarchies. However, just as with existing technologies, much work remains to be done not only to engineer the synthetic receptors, but also to synthesize and screen orthogonal ligands that are well-tolerated, easy to administer, and that readily reach target sites in the brain. We will work across experimental systems, in yeast and fibroblasts, to identify the most promising actuator candidates, to be subjected to extensive testing and optimization in vitro prior to deployment in animals.
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1 |
2015 — 2016 |
Drew, Michael R (co-PI) [⬀] Martin, Stephen (co-PI) [⬀] Zemelman, Boris V |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A Viral System For Light-Dependent Trapping of Activated Neurons @ University of Texas, Austin
? DESCRIPTION (provided by applicant): A central goal in neuroscience is to identify cellular ensembles supporting mental and behavioral states, but these ensembles cannot be defined a priori. The dentate gyrus (DG), for example, contains more than 1M granule cells, which are essentially indistinguishable from each other, but less than 5% of these seemingly identical neurons are active during any one behavioral event, suggesting that the associated mental states are each mediated by a small subset of neurons. We propose to develop a novel method for identifying and gaining genetic access to such transient, behaviorally-relevant assemblies of neurons in awake animals. The key unique features of our approach are (1) its temporal precision is unprecedented because it is the first neuronal tagging technique that matches the timescale of naturalistic behavior; and (2) its ability to label multiple cell populations in the sme animal enables the comparison of state-specific cell ensembles. Our novel molecular-genetic technique first identifies activated neurons on the basis of elevated intracellular calcium and then tags them using light. Light application is especially attractive because it is temporally precise: just as other optogenetic methods have aided neuronal circuit analysis by approximating the timescale of cell activity, so too will a light-dependent labeling technique illuminate functional cell assemblies. The technique will be entirely virus-based, so it is usable across species without relying on transgenic animals. Under this award we will establish the technique by developing and testing two critical innovations: (1) a synthetic bidirectional promoter system, and (2) caging chemistry for multi-wavelength visible light regulation of promoter function. Ultimately this technique will be used to elucidate the neuronal substrates of diverse mental states, such as fear, hunger, depression, anxiety, and addiction, thereby advancing the exploration of critical brain networks. This high-risk, high-reward project comprises multiple innovative features. Elements of the nascent reporter system described here, such as promoter strength, mechanism for regulating gene expression, choice of activating ligand, caging chemistry, and in vivo ligand and light delivery represent starting point that will benefit from extensive optimization. Once existing reporter components have been sufficiently refined, we envision replacing fluorescent reporters with recombinases, so that actuators can be expressed in identified cells for testing neuronal function. Other features, including the development of novel caged ligands, as well as additional methods for brain-wide activity reporting will also be addressed following achievement of our Aims. Despite the inherent risks, we are confident that our proposed system represents a fundamental and much-needed departure from existing techniques. We believe that our approach will evolve from its present status as a promising endeavor into a widely-used tool with the support of the BRAIN Initiative.
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1 |
2016 — 2018 |
Geisler, Wilson S (co-PI) [⬀] Seidemann, Eyal J [⬀] Zemelman, Boris V (co-PI) |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
An Optical-Genetic Toolbox For Reading and Writing Neural Population Codes in Functional Maps @ University of Texas, Austin
The overarching goal of this proposal is to develop an optical-genetic toolbox for reading and writing neural population codes in functional maps of awake, higher mammals. Such tools could ultimately be used to restore perceptual capabilities in patients with damage to peripheral sensory pathways by direct stimulation of early sensory cortex. Advanced optical methods for reading and writing neural codes using genetically-encoded reporters and actuators have become powerful tools for studying neural circuits in rodents. However, rodents are a suboptimal model for human perception because of their vastly different sensory representations and perceptual capabilities. For example, rodents' primary visual cortex (V1) lacks the functional columnar organization which is a hallmark of primate vision. In contrast to rodents, the macaque monkeys' sensory representations and perceptual capabilities are highly similar to those of humans. Furthermore, the behaving macaque provides a unique opportunity to develop and test tools for reading and writing neural codes at the level of functional domains such as the orientation columns in V1. However, multiple technical hurdles remain before the optical-genetic methods currently available in rodents could be readily applied in larger, non- transgenic mammals. Here we propose to take advantage of the unique expertise of our team members to develop optical techniques that utilize virally delivered transgenes for monitoring and manipulating neural population codes in behaving macaques. Specifically, we will address three technical goals. First, we will develop and test new genetic methods that will provide long-term expression of transgenes in primates with cell-type and activity- dependent specificity. Second, we will develop a two-photon microscope for behaving monkeys that will allow one to monitor these signals with cellular resolution and complement current imaging techniques with larger coverage but coarser resolution. Finally, we will develop methods for writing neural population codes in functional maps by combining patterned light stimulation that target specific functional domains and selective expression of actuators. We will validate and optimize these techniques by linking V1 responses (elicited by both visual and direct patterned optogenetic stimulation) and monkeys' behavior in visual discrimination tasks. The tools that we will develop will enable a deeper understanding of the neural code and a better characterization of the capabilities and limitations of methods for reading and writing neural population codes in functional maps in humans.
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1 |
2017 — 2018 |
Golding, Nace L [⬀] Zemelman, Boris V (co-PI) |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Discovery of Functional Cell Types in the Inferior Colliculus With Combined Molecular-Genetic and Electrophysiological Approaches @ University of Texas, Austin
Project Summary The central nucleus of the inferior colliculus (ICC) is a midbrain nucleus that serves as a pivotal point of convergence for a large number of ascending and descending auditory pathways. Understanding how computations carried out in the ICC relate to the underlying circuitry has been unusually difficult, in large part due to the lack of clear definitions of cell types. Indeed, there appears to be no correlations between intrinsic firing properties and cell morphology, even across excitatory and inhibitory cells. We propose that functionally distinct classes of neurons in the ICC can be identified and genetically accessed using viruses with promoters to different neurochemical markers. To address this hypothesis, we will combine electrophysiological and anatomical approaches with interdependent recombinant adeno-associated viruses (rAAV) targeting excitatory and inhibitory classes of calbindin D-28k ICC neurons (CB-excitatory and CB- inhibitory). CB is a calcium binding protein expressed in subsets of neurons of the ICC and in many other brain areas. Our preliminary results show that viruses carrying a CB promoter element target ICC neurons that exhibit a single firing phenotype, and that these neurons may comprise both excitatory and inhibitory subclasses. In Aim 1, we will combine in vivo labeling of ICC neurons with rAAV viruses, fluorescence guided patch recordings in IC slices, and anatomical analyses to fully characterize the two pools of CB neurons, including their physiology, morphology and axonal projections. In parallel, labeled neurons will be characterized according to their complement of expressed neurochemical markers. In Aim 2, we will use our molecular-genetic tools to drive channelrhodopsin-2 selectively in CB-excitatory or CB-inhibitory neurons. We will use structured illumination to map CB neurons' local inputs and targets in the ICC as well as to facilitate paired whole-cell patch recordings from connected neuron pairs, enabling characterization of synaptic properties between physiologically identified network partners. These experiments will serve as a blueprint for identifying and characterizing other functionally distinct circuits in the ICC. Our goal is to establish a foundation for future studies involving cell-specific optogenetic manipulations in vivo, where we can assess the roles of CB neurons and other defined ICC cell types in auditory computations.
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