Alan Jasanoff, Ph.D. - US grants

DESCRIPTION (provided by applicant): The overall goal of this project is the development and in vivo testing of a set of contrast agents that will allow noninvasive imaging of neuronal calcium levels by MRI. These reagents will provide a rapid, direct, and potentially cellular-resolution readout of dynamic brain activity. The new MRI calcium sensors will have a great impact on brain research, both through applications to the study of neurological disease in animal models, and as tools for the analysis of neural network function in basic neuroscience. The long-term objectives of this laboratory include applying the new sensors in transgenic rodents to dissect neural circuitry involved in learning and memory. Work performed under this proposal will establish a methodological platform for future biological studies. The contrast agents we will synthesize and test take advantage of the unique potency of superparamagnetic iron oxide nanoparticles (SPIOs) as imaging agents in MRI. Sensors are formed by conjugating calcium sensor proteins to the SPIOs; in the presence of calcium, the particles aggregate and produce large MRI intensity changes in T2-weighted images. A prototype sensor has been formed by conjugating calmodulin (CaM) and its substrate peptide M13 to two populations of nanoparticles; calcium-dependent aggregation with an EC50 of 0.8 mu/M Ca was observed along with large MRI signal changes, but some modifications of this sensor are required for calcium sensing in cells. In Specific Aim 1, we propose computational modeling and site-directed mutagenesis of the CaM/M13 interaction interface to reduce the sensor's potential for cross-reactivity with cellular proteins. In Specific Aim 2, we propose synthesis of new ultrasmall iron oxide nanoparticle conjugates (diameter<< 20 nm) which will respond quickly to changes in calcium concentration. A revision of our prototype calcium sensor that incorporates results of Aims 1 and 2 will be ideal for further studies in vivo. In Specific Aim 3, we propose to test the calcium responses of new nanoparticle sensors in cells, first by injection into Xenopus oocytes, and then by injection into blowfly neurons-previous work from our laboratory showed that the blowfly is a good test system for neuroimaging agents because of its ease of handling, large neurons, and absence of hemodynamic effects. Noninvasive delivery and applications of these proposed MRI calcium sensors in mammalian brains are beyond the scope of this proposal, but constitute a further step in our trajectory.

NIH Roadmap Initiative tag; brain imaging /visualization /scanning; technology /technique development

DESCRIPTION (provided by applicant): The overall goal of this project is the development of next-generation functional imaging methods and novel contrast agents that will allow noninvasive measurement of neuroplasticity-related signaling molecules by magnetic resonance imaging (MRI). The work fits into our laboratory's long term agenda of analyzing the function of neural circuitry at a whole-brain level in awake, behaviorally active animals. Our aims will also help establish a broadly significant new generation of functional imaging (fMRI) techniques that combine noninvasiveness and high spatial resolution with readouts specific to neural physiology at the molecular and cellular level, and that may be applied in small animals, primates, and perhaps humans. The proposed research builds on our laboratory's recent introduction of a family of protein-based MRI contrast agents sensitive to an important neuroplasticity-related signaling molecule, the neurotransmitter dopamine (DA). In collaboration with Frances Arnold's group at Caltech, we applied advanced protein engineering methods to create sensors based the heme-binding domain of the bacterial cytochrome P450-BM3 (BM3h). We then used MRI with BM3h-based sensors to detect DA transport in vitro (in PC12 cells) and in vivo (in injected rat brains). Our preliminary results constitute one of the first demonstrations of real-time fMRI with a molecular reporter in vivo, and justify the Specific Aims we propose here. In Specific Aim 1, we will improve on our pilot injection studies, by developing minimally invasive methods for delivery of BM3h-based sensors to large regions of the rodent brain. We will implement two methods for blood brain barrier disruption, in conjunction with intravascular contrast agent delivery. We will also explore gene-directed expression of our protein sensors from cells as a constitutive, potentially targetable delivery strategy. In Specific Aim 2, we will establish robust functional imaging techniques for use with our contrast agents. We will combine MRI pulse sequences for rapid acquisition with flow suppression techniques for removal of hemodynamic artifacts. These methods will improve the sensitivity and confidence with which we can study DA function and its relationship to learning and plasticity in vivo. Sensitivity enhancement will also be achieved by modifying our existing sensors, as part of Specific Aim 3. In Specific Aim 4, we plan to create further BM3h-based sensors for critical extracellular and intracellular signaling molecules related to plasticity in neural systems, exploiting the remarkable versatility of the molecular engineering approach we used to create MRI DA sensors. The work we propose to perform is primarily relevant to two aspects of public health: First, experiments our new methods and reagents will facilitate in animal models will inform our understanding of neuroplasticity processes in humans, and may contribute to the development of treatments for neurological diseases. Second, the reagents we create may eventually be useful as diagnostic imaging tools in clinical practice, once they have proven safe and effective for measuring neural signaling molecules in animals. PUBLIC HEALTH RELEVANCE: Analysis of plasticity in neural systems could be dramatically accelerated using new methods that permit noninvasive measurement of specific brain signaling molecules in intact animals and patients. Here we propose strategies for using and improving a new class of designer proteins we have engineered to sense neuroplasticity-related signaling molecules in conjunction with magnetic resonance imaging. Our initial work involves a sensor for dopamine (a neurotransmitter closely associated with learning and drug addiction), and we also propose extension of our methods to create sensors for a variety of additional targets, with both basic science and clinical significance.

DESCRIPTION (provided by applicant): The goal of this project is to establish a strategy that will make neuronal electrical signaling detectable via magnetic resonance imaging (MRI) at a whole-brain level. Our approach is built on the novel concept of using cell-adhesive micron-scale electronic devices to transduce neuronal potentials across the brain into magnetic field fluctuations. As part of our validation of these voltage-sensing microprobes, we also propose to implement a new, scalable method for simultaneous recording of MRI and electrophysiological data. The methods we propose to develop will be broadly applicable to problems in neurobiology, and will transform neuroscientists'ability to study integrative functions of the brain. Our microprobe approach will also help establish a new paradigm in diagnostic medicine and molecular imaging, where tiny machines, rather than conventional chemical contrast agents, will report on aspects of cellular physiology. Recent work has dem- onstrated that micron-scale electrodes, coated with cell-adhesive molecules and juxtaposed against cultured cells allow recording of millivolt-scale action potentials, comparable to intracellular recordings. The current induced in a microelectrode can be converted into a modest, transient magnetic field if it is channeled into an inductor. In Specific Aim 1, we will model the magnetic fields produced by feasible currents in spiral or solenoidal microcoils of defined geometry, compute predicted effects on MRI signal amplitude and phase as a function of microprobe distribution, and fabricate the microprobes themselves. Preliminary calculations indicate that localized, transient fields of about 10 nT could be produced in individual 10-turn microcoils of 1 5m diameter. Magnetic fields of this order are greater than endogenous neuronal fields detected in tech- nologies like magnetoencephalography, and have been shown previously to be measurable by MRI in some contexts. In Specific Aim 2, we will test the ability of our microprobes to report action potentials from neu- ronal populations in MRI. The microdevices wil first be applied to cultured neurons or neural tissue slices and placed in an MRI scanner. Data series will be obtained using multiple protocols to detect variations of MRI signal due to variations in neuronal activity. If experiments in culture are successful, microprobes will be site-specifically injected into the cerebral cortex of anesthetized rats, and tested in an somatosensory stimu- lation paradigm. In Specific Aim 3, we will establish a simultaneous MRI and conventional electrophysiology approach to validate the novel MRI voltage probes directly. Performing electrophysiology in an MRI scanner is complicated by artifacts induced by the scanning hardware, in particular due to switched gradient fields. To circumvent this problem, we will measure neuronal potentials using differential recording from pairs of channels on tetrodes or modified tetrodes. Once the in-scanner recording method has been refined, MRI- based and conventional electrophysiology data will be obtained and compared to assess performance of the voltage-sensing microprobes, and to guide further improvements, if necessary. PUBLIC HEALTH RELEVANCE: Noninvasive MRI-based electrophysiology using microelectronic devices will have high impact in biology, and specifically in brain research, both through applications to the study of neurological disease and as tools for the analysis of neural network function in basic neuroscience. The microprobes we propose to develop represent a new paradigm in diagnostic medicine, where tiny machines, rather than conventional chemical contrast agents, will report on aspects of cellular physiology.

DESCRIPTION (provided by applicant): Analysis of neural mechanisms involved in normal brain function and disease could be dramatically accelerated using novel measurement methods that report neural processing events with molecular specificity, noninvasively, and across the entire brain. Here we propose to develop a molecular sensor platform for imaging the two most important amino acid neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), by noninvasive magnetic resonance imaging (MRI). Sensors will be formed by modifying a family of amino acid binding proteins with gadolinium chelating groups. The resulting probes will enable neurochemical imaging with behaviorally-relevant temporal resolution, on the scale of entire brain regions or intact brains. This qualitatively new capabilit will enable us and others to answer specific questions about how the dominant excitatory and inhibitory neurotransmitters in the vertebrate central nervous system participate in multiple facets of brain physiology. Our sensor design is based on the large ligand-dependent structural changes induced in so-called venus flytrap domains (VFDs). By conjugating gadolinium-containing groups to VFDs at strategically chosen amino acid positions, we expect to generate MRI contrast agents with sensitivity to the ligands that naturally bind to each corresponding VFD. In Aim 1, we will use this strategy to generate glutamate-sensitive MRI contrast agents based on VFDs from the bacterial periplasmic binding protein YbeJ. In preliminary work on this Aim, we have already observed large MRI changes due to glutamate binding to gadolinium-derivatized YbeJ variants, indicating the overall promise of our approach. In Aim 2, we will apply our glutamate sensors in rat brains and use them to map glutamate release patterns during a representative somatosensory stimulus. We will also compare glutamate and conventional functional MRI (fMRI) activation maps to examine the hypothesis that hemodynamic fMRI measures closely reflect glutamatergic signaling. Our recent detection of dopamine release using a less potent form of MRI sensor in rat brains represents a precedent for the proposed in vivo work, and again indicates promise. In Aim 3, we will extend our glutamate sensor design strategy to target GABA, the dominant inhibitory neurotransmitter in the brain. The proposed research has

DESCRIPTION (provided by applicant): Noninvasive measurement and manipulation of the nervous system could be possible using magnetic techniques, but a missing link is the availability of highly magnetic handles on cellular or molecular function. We therefore take on the challenge of engineering proteins that spontaneously become highly magnetic-so-called hypermagnetic ferritins (hFts)-and incorporating them into genetically encodable sensors for comprehensive brain activity mapping studies using magnetic resonance imaging (MRI). The magnetic proteins we produce will have impact in three broad areas: First and foremost, they will serve as building blocks for functional molecular neuroimaging probes, our principal interest and the application we focus on in this proposal. Second, magnetic proteins we produce will be uniquely powerful reporters for additional magnetic neuroimaging approaches. Third, highly magnetic proteins will be tools for magnetic manipulation of cells in the nervous system. These applications relate to nearly all areas of neuroscience, so the engineered proteins are likely to have very general significance. Our approach focuses on high throughput directed evolution of ferritin iron storage proteins for enhanced iron loading (Aim 1) and magnetization (Aim 2). Selected proteins will then be incorporated into sensors. This approach is justified because preliminary results already show that wild type or minimally engineered ferritins can be used as imaging agents, MRI sensors, and even magnetic manipulation approaches. Aim 1 makes use of a novel cytosolic iron reporting system to screen for hFt variants with abnormally high iron accumulation. Our preliminary results already show efficacy, and since brain Ft is normally only ~15% iron loaded, a considerable dynamic range should be accessible by applying this screening method over repeated cycles. Aim 2 applies a magnetic column based screen to identify even more highly magnetic hFt variants from yeast expressing mutant libraries. The ideal outcome of this approach will be an hFt variant that spontaneously acquires a superparamagnetic iron oxide core with specific magnetization and resulting magnetic properties up to 100-fold greater than the ferrihydrite core of wild type ferritins. In Aim 3, we wll incorporate hFt variants into calcium sensitive genetically encoded MRI contrast agents. Potentiating the magnetic moment of Ft-based calcium sensors by incorporating hypermagnetic building blocks will be make a critical difference to the sensors' ability to support robust functional imaging in vivo, and to applications in which these probes are genetically targeted to discrete cell types or circuit elements. The proposal is consistent on multiple levels with the EUREKA program goals. Although the proposed research is high risk, it builds on promising preliminary results, as well as the PI's record in developing novel neuroimaging probes. We believe that it is feasible to consider completely the proposed research within the four year EUREKA grant period.

? DESCRIPTION (provided by applicant): The development of minimally invasive direct readouts of neural activity is one of the greatest challenges facing neuroscience today. Our recent work has shown that it is possible to perform high resolution functional magnetic resonance imaging (fMRI) of molecular-level phenomena using MRI contrast agents sensitive to hallmarks of neurotransmitter release. An even more valuable contribution would be the creation of calcium sensors suitable for molecular fMRI of intracellular neural signaling processes. Functional imaging performed with these sensors would combine the noninvasiveness and whole-brain coverage of MRI with the molecular specificity and broad applicability of established optical calcium neuroimaging techniques. Calcium-dependent fMRI will be a breakthrough technique for analysis of neural circuits in animals, with potential longer term applications in humans. The technique could achieve cellular resolution in conjunction with ultrahigh field MRI scanners and cell labeling techniques. A major hurdle in realizing this advance is the creation of effective calcium-dependent MRI contrast agents, however. This proposal describes strategies for creating novel MRI calcium probes suitable for molecular fMRI, as well as initial experiments that validate the approach in animals. Innovations include the rational design of membrane permeable probes themselves as well as approaches for in vivo calcium imaging and genetically targeted applications. In Aim 1, we synthesize MRI calcium sensors based on paramagnetic cell-permeable aromatic chelates and characterize them in vitro. In Aim 2, we form acetomethoxy derivatives of the calcium probes and validate them first in cell culture and then in rats, using a somatosensory stimulation paradigm. Results of Aim 2 will direct further refinement of the probes, if necessary. In Aim 3, we adapt the calcium sensors for intracellular trapping by selective esterases that will promote probe accumulation in genetically targeted cells. This technique will provide a means for cell type-specific and in some cases individual cell-specific functional imaging of dynamic calcium levels in the brain. Potential impact of the project and preliminary achievements of the research team make this next-generation neuroimaging project particularly suitable for BRAIN Initiative funding under RFA-NS-14-007.

? DESCRIPTION (provided by applicant): An improved understanding of dopamine signaling patterns in the brain's reward processing systems will lead to mechanistic descriptions of reward-related behavior, and also to the discovery of new biomarkers and therapeutic intervention points for treatment of addiction. Here we propose to use a unique new molecular-level functional brain imaging technique to characterize dopamine signaling evoked by some of the most widely studied and broadly significant stimuli in addiction research. The technique uses magnetic resonance imaging (MRI) in conjunction with contrast agents that bind and report dopamine concentrations, reversibly, as a function of time. In Specific Aim 1, we will use this molecular fMRI technique to map the structure and dynamics of dopamine release patterns in the striatum, a key target of dopamine signaling related to reward and addiction. We will measure dopamine release in response to an important addictive drug, amphetamine, as well as to reward-related brain stimulation. We will also push the spatial coverage and resolution of the imaging itself in order to characterize details of the dopamine response, such as specificity to anatomically and neurochemically defined striatal subregions, and we will for the first time conduct dopamine MRI studies in awake animals. Data will be obtained at a spatial resolution of 100-200 µm and over temporal scales ranging from seconds to minutes, sufficient for resolving both phasic and longer-lasting dopamine changes. In Specific Aim 2, we will establish a quantitative, spatially-resolved correspondence between molecular fMRI readings and conventional hemodynamic fMRI signals, which broadly reflect neuronal population activi- ty. These experiments will provide an empirical description of the relationship between BOLD signal and dopamine release, and also facilitate circuit level description of dopaminergic function at a neural population level across the striatum and beyond. In Specific Aim 3, we propose to develop an alternative MRI sensor that will detect dopamine concentrations of 0.1-1 µM, 10-100 times better than our current sensors, and in the range of levels evoked by naturalistic rewarding stimuli. The improved sensors will be formed from na- noscale arrays of magnetic particles that change configuration in the presence of target ligands, bringing about MRI contrast changes. In additional to enabling sensitive dopamine detection, the new design will in the future be generalizable to other neural targets. These Aims will have broad impact on the study of do- paminergic neural systems important in addiction, and will also address goals of the federal BRAIN Initiative.

? DESCRIPTION (provided by applicant): Reverse engineering the brain is one of the greatest scientific and engineering challenges of our time, and will depend on numerous breakthroughs in the technology for studying the brain. Achieving these breakthroughs will require training successive generations of neurotechnology experts who are highly knowledgeable and adept in both neurobiological and engineering fields. Recognizing this, the interim report of the NIH BRAIN Initiative advisory panel recommended that training be a key component of how innovative neurotechnologies are established and disseminated. Here we propose to create a new Neurobiological Engineering Training Program (NBETP) which addresses the need for advanced, state-of-the-art predoctoral training in neurotechnology at the Massachusetts Institute of Technology (MIT). The objective of the program is to educate a set of high quality students, who will emerge from the NBETP with outstanding expertise and leadership ability at the intersection of basic neuroscience and engineering. These students will be selected from existing MIT graduate programs according to their interests and research potential, as well as diversity criteria. They will be given a grounding via coursework and training in the responsible conduct of research. They will also be offered opportunities to lead activities and network with faculty and other students at the top of their field. Students admitted into the program will be in or entering their second year of graduate school, when they are just beginning to specialize and choose a research project, and they will be supported for two years each. MIT's NBETP will be characterized by two salient and innovative features. The first is its extreme multidisciplinarity, reflected in the breadth of faculty involved in the initiative. The program will extend in particulr to newer engineering fields, such as bioengineering and materials science, which will be central to the development of next generation neurotechnologies, but which are omitted from more classical conceptions of neuroengineering. A second hallmark of the NBETP will be its primary focus on technology development for basic neuroscientific research, as well as extensions to medical applications. This feature specifically addresses the currently pressing need to foster the development of tools for understanding brain function. As a first-of-its kind program, the NBETP will directly benefit students and faculty engaged in neurotechnology research at MIT. The program will serve as a powerful catalyst to interactions across departments and disciplines at MIT, and will produce highly trained graduates positioned to have global impact on academic, industrial, and clinical research throughout the country.

? DESCRIPTION (provided by applicant): The establishment of new and precise strategies for mapping brain activity in human subjects is one of the highest priorities of the BRAIN Initiative. Here we propose to meet this challenge by developing a transformative noninvasive molecular neuroimaging approach capable of mapping molecular events in the brain; the method will offer a combination of sensitivity and resolution that could ultimately revolutionize neuroscientific investigation of human subjects. Our strategy is based on a fundamentally new type of chemical imaging probe designed to produce noninvasive neuroimaging readouts by purposefully manipulating endogenous hemodynamic contrast in the brain-in effect hijacking the blood oxygen level dependent (BOLD) effect to perform neural target-specific molecular imaging. This unprecedented concept combines three key advantages: First, by providing time-dependent sensitivity to molecular species such as neurotransmitters, our strategy will enable well-defined neurobiological phenomena to be mapped dynamically across the brain, dramatically surpassing today's functional imaging approaches. Second, by influencing an endogenous contrast source detectable by virtually any noninvasive imaging modality, the new approach will be compatible with a formidable existing infrastructure for experimentation and analysis over multiple spatial and temporal scales and applicable in the lab, field, or clinic. Third, by circumventing limitations of established optical, magnetic, and radioactive probe designs, the new vasoactive imaging probes will combine exquisite sensitivity approaching that of positron emission tomography (PET) with spatiotemporal resolution comparable to functional magnetic resonance imaging (fMRI), while at the same time avoiding toxicity associated with existing imaging agents. The sensitivity afforded by vasoactive probes will also permit minimal doses of these agents to be delivered noninvasively past the blood-brain barrier (BBB), an essential requirement for human functional molecular neuroimaging. In the spirit of this planning grant, we will complete three Specific Aims that set the stage for next- generation functional neuroimaging in humans. In Aim 1 we will build on our preliminary work with vasoactive imaging probes to create new sensors for detecting the neurotransmitters dopamine and glutamate in the brain. In Aim 2, we will modify vasoactive probes to enable noninvasive trans-BBB delivery by means of receptor mediated transcytosis. In Aim 3, we will adapt our vasoactive probes for neuroimaging experiments in primates-a key stepping stone toward imaging trials in people. To these Aims, we bring a collaborative research team with unparalleled expertise in a combination of molecular engineering and imaging, fMRI in rodents and nonhuman primates, and human functional brain imaging. This strength in all aspects of our research trajectory will optimize our ability within eight years to validate the new technology in human subjects, where we expect it to become a powerful approach for functional imaging in both scientific and medical contexts.

The dominant techniques for brain-wide functional imaging in humans and opaque mammals make use of he- modynamic contrast that results from coupling between neural activity and changes in blood flow and can be detected by noninvasive imaging methods including functional magnetic resonance imaging (fMRI), functional photoacoustic tomography (fPAT), functional ultrasound imaging (fUS), and others. Although hemodynamic functional imaging in both humans and animals has been used to make some of the most important discover- ies in neuroscience, the techniques are limited by their lack of specificity to mechanistically-distinct compo- nents of brain activity. Our group has helped lead efforts to bypass limitations of hemodynamic functional imag- ing by developing molecular probes that report physiologically-specific hallmarks of neural activity via noninva- sive imaging. Here we propose the complementary and hitherto unprecedented strategy of working ?within the system? to improve the specificity intrinsic hemodynamic readouts themselves. Specifically, we propose to ge- netically enhance a signaling pathway that directly couples the activity of targeted neurons or glia to blood ves- sels, thus introducing artificial hemodynamic functional signals that can be attributed to distinct cell type- or circuit-specific cellular processes in the brain. This approach will harness the amplification afforded by hemo- dynamic imaging while bypassing numerous complexities of endogenous neurovascular mechanisms, in effect hijacking hemodynamic signals to report on circuit- or cell type-specific activity. Compared with efforts based on more conventional imaging methods, the engineered hemodynamic imaging approach will offer key ad- vantages: (1) capability for selective imaging of genetically-targeted brain circuits and cell types, (2) compatibil- ity with a variety of genetic tools, in particular including viral transduction and tracing vectors, (3) applicability to brain-wide deep tissue imaging in multiple species, (4) compatibility with many established modalities for func- tional neuroimaging, (5) potential sensitivity to relatively low activity levels and sparse neuronal populations. Our work on this novel strategy will be organized into two Aims: In Aim 1 we will engineer a new family of genetically encodable neural activity reporters that will underlie the new engineered hemodynamic imaging approach. The reporters will be derived from naturally occurring nitric oxide synthase enzymes and are re- ferred to as NOSTICs. In Aim 2, we will deliver NOSTIC genes to rat brains using viral vectors, and perform an extensive series of imaging and histological investigations to validate and optimize our new imaging in vivo. We anticipate that completion of these Aims will introduce a potent new approach for multimodal imaging- based investigations of circuitry and cell type-specific contributions to neural function in diverse species and behavioral contexts.

Analysis of neural mechanisms involved in normal brain function and addiction-related perturbations to these functions could be dramatically accelerated using novel noninvasive measurement methods to report neural processing events mediated by multiple neurotransmitters with molecular specificity and across the entire brain. Recent work from our lab has shown that functional molecular neuroimaging can be performed using neurotransmitter-sensitive magnetic resonance imaging (MRI) contrast agents. However, current pro- tein-based sensors have only micromolar sensitivity, necessitating the use of strong stimuli and restricting our study to areas of the brain where neurotransmitter concentrations are unusually high. In addition, our current sensors are not readily adaptable to neurotransmitters beyond dopamine (DA) or serotonin, like glu- tamate and ?-aminobutyric acid (GABA), that mediate important processes relevant to motivation, reward, and addiction. Here we propose to develop a suite of nanoscale molecular MRI sensors, based on a novel, generalizable contrast mechanism, which will enable detection of the neurotransmitters dopamine, gluta- mate, GABA, and serotonin in vivo with nanomolar sensitivity. These highly-sensitive contrast agents will facilitate the measurement of mesoscale topological maps of neurotransmitter signaling throughout the brain. Such an approach will enable ?noninvasive neurochemical dissection? of addiction-related changes in neurotransmission, resulting in a more complete understanding of altered brain function in addiction which will ultimately guide the development of behaviorally-effective treatments. Our work on the project will ad- dress three Aims. In Aim 1, we will establish a novel sensing mechanism based on paramagnetic nanoscale liposomes whose efficacy as contrast agents is regulated by binding to target neurotransmitters. We will pilot the design and explore design parameters in the context of dopamine sensors, where our earlier work establishes a precedent on which we plan to improve. In Aim 2, we will extend the new liposome-based sensing mechanism to address the additional neurotransmitters glutamate, GABA, and serotonin. In Aim 3, we will begin validating the new sensor design in vivo, focusing on the dopamine-sensitive nanoprobes and employing a stimulation and imaging paradigm we have used successfully in previous work. Completion of this work will open the way for detection of naturalistic signaling levels of four neurotransmitters and the mapping of their spatial relationships across large regions of the brain in experimental animals.

Primate brains contain cortical areas that exhibit selective engagement in high-level sensory or behavioral operations. The functional specialization of these regions is thought to be central to primate-specific cognitive faculties and to associated disorders. Deciphering the origins of functional specialization in primate brain regions has been an enormously challenging task, however, due in large part to the absence of suitable experimental tools. To address this problem, we will develop a method for measuring the activity of inputs to specialized areas from throughout the brain, permitting systematic analyses of information flow in the multiregional neural circuitry that gives rise to high-level functions. Our method will employ a conceptually new family of genetically encoded imaging probes called NOSTICs, which transduce the calcium signaling of NOSTIC-expressing neurons into localized hemodynamic signals that can be dynamically monitored using brain-wide measurement techniques like functional magnetic resonance imaging (fMRI). When delivered using retrogradely transported viral vectors, NOSTICs can permit targeted fMRI-based recording of neural activity in distributed cell populations that provide monosynaptic input to any injection target in the brain. In our preliminary work, we have created first-generation NOSTIC probes and used them to demonstrate genetically targeted functional imaging in rodents. In Aim 1 of this project, we will take two steps that adapt this tool for use in nonhuman primates. We will create second- generation NOSTICs that display improved performance for circuit-specific functional imaging, while also devel- oping viral vectors that allow expression of these probes to be tracked longitudinally in primate brains. We will also adapt the NOSTIC probes for incorporating into adeno-associated viruses, which provide extended capa- bility compared with the herpes viruses we currently use. In Aim 2, we will perform pilot experiments to investigate whether NOSTICs can provide circuit-specific readouts in nonhuman primates. These tests will already be pos- sible using our currently available probes and vectors, and new variants from Aim 1 will also be tested when available. Successful demonstration of NOSTIC functionality for circuit imaging in marmosets constitutes our proposed go/no-go criterion for entry into the UH3 stage of this project. Then in Aim 3 (UH3 stage), we will validate NOSTIC probes in two paradigms that explore their performance across brain regions, experimental contexts, and primate species. In the first paradigm, we will apply NOSTICs to examine origins of functional specialization in face-selective regions of the marmoset brain. In the second paradigm, we will apply NOSTICs to investigate brain-wide contributions to object selective responses in the ventral stream of the macaque visual cortex. These experiments will be performed as multi-laboratory collaborations that both harness and dissemi- nate the NOSTIC technology; this work will therefore establish a broadly applicable transformative approach for mechanistic analysis of primate brain function.

A major goal of the BRAIN Initiative is to promote the development of neural activity measurement tools that bridge between spatial scales, so that the processing roles of individual neurons and microcircuits can be related to broader regional or brain-wide dynamics. Here we propose to create novel chemical probes of neuronal cal- cium signaling that will enable cross-modal comparison of readouts obtained at multiple scales, using both inva- sive and noninvasive imaging methods. Using these multifunctional probes, investigators will be able to record wide-field neural activity dynamics at varying depths and spatiotemporal resolutions from well-defined molecular sources that permit precise interpretation, without the potential for artifacts associated with parallel application of disparate probe modalities. This will be particularly valuable for validation and use of the probes in noninvasive imaging modalities, for which probe technologies are still relatively rudimentary and untested, and relating wide- field signals to micron-resolution optical results could be especially informative. The new probes we will create are derived from a cell-permeable aromatic chelator called texaphryin (Tex). Complexes of Tex variants with different metal ions function as potent fluorophores, photoacoustic reporters, and T1-weighted contrast agents for magnetic resonance imaging (MRI). We recently discovered that combining paramagnetic Tex complexes with calcium-responsive moieties such as 1,2-bis(o-aminophenoxy)ethane-N,N,N?,N?-tetraacetic acid (BAPTA) results in strong calcium-dependent contrast changes, thus providing a promising basis for synthesis of sensors suitable for simultaneous or parallel measurement by MRI, optical, and photoacoustic readouts. In this project, we will synthesize and optimize the multimodal sensors, evaluate their optical and magnetic imaging capabilities, and begin in vivo validation studies that directly exploit the unique advantages these novel probes offer. These experiments will establish a first-of-its-kind molecular platform with potentially powerful capability for multimodal analysis of neural activity dynamics across spatial and temporal scales in a variety of species and behavioral contexts.

The large-scale dynamics of neural circuitry depend on interactions among numerous neurochemical spe- cies that play functionally distinct roles throughout the brain. Understanding the spatial and temporal character- istics of chemical signaling is thus crucial for building mechanistic models of brain function. Our laboratory has introduced paramagnetic neurotransmitter sensors that enable functional analysis of neurochemical phenomena over large fields of view by molecular-level functional magnetic resonance imaging (molecular fMRI). We have published applications of these sensors to spatiotemporal mapping of neurochemical phenomena in a series of substantial papers. The scope of such experiments has however been limited by the modest sensitivity provided by the existing probes, which must be applied at concentrations that substantially exceed physiological neuro- transmitter levels. The goal of this proposal is to establish a platform technology for noninvasive neurochemical imaging with substantially higher sensitivity, focusing initially on monoamine transmitters. Our approach is based on a novel principle for biochemical sensing in MRI that uses paramagnetic liposomes as responsive contrast agents. In this mechanism, the presence of neurotransmitter targets gates large contrast effects afforded by the liposomes, giving rise to a formidable amplification factor with respect to previous probes. Using this design, we predict that sensitivity to behaviorally relevant low-micromolar or submicromolar neurotransmitter concentrations will be achieved, with minimal potential for buffering effects. In addition, our preliminary studies suggest that wide-field brain delivery with these probes is achievable, and we also predict that perisynaptic cell type-specific readouts can be obtained by targeting the liposomes. Our work will address three Aims: In Aim 1, we will establish our liposome-based nanosensor (LBN) plat- form by combining lipid, polypeptide, and small molecular components to establish the new sensing mechanism we seek to exploit. We will use a variety of synthetic and molecular engineering methods to optimize this mech- anism for detection of behaviorally relevant interstitial dopamine and serotonin concentrations, with the goal of achieving sensitivity in the 0.1-1 µM range. In Aim 2, we will optimize strategies for brain-wide delivery of these probes, exploiting chemically-mediated blood-brain barrier disruption and infusion into cerebrospinal fluid. We will also implement a perisynaptic targeting approach. In Aim 3, we will validate liposome-based dopamine and serotonin LBNs by molecular fMRI in live rat brains, with reference to parallel neurochemical and hemodynamic fMRI measurements. In addition to establishing the novel neurochemical imaging platform we propose, these experiments will yield first-of-their-kind data about the wide-field distribution of dopamine and serotonin signaling in response to stimuli, as well as the relationship of these neurochemicals to conventional brain activity readouts. Although the technology we will develop will initially be applied in sedated rodents, we expect it to be applicable to many additional species and behavioral contexts.

We propose to develop a probe technology for monitoring human brain function with molecular precision; in conjunction with magnetic resonance imaging (MRI) or other imaging modalities, the probes will provide a combination of sensitivity and resolution that could permit unprecedented noninvasive studies of dynamic neu- rophysiological processes in people. Our strategy is based on a fundamentally new type of chemical imaging probe designed to produce neuroimaging readouts by purposefully manipulating endogenous hemodynamic contrast in the brain?repurposing the blood oxygen level dependent (BOLD) effect that underlies conventional functional MRI (fMRI). This new ?vasoprobe? concept offers three key advantages: First, by providing time-de- pendent sensitivity to dilute molecular species such as neurotransmitters, the probes can enable well-defined neurobiological phenomena to be mapped dynamically across the entire brain, dramatically surpassing existing nonspecific fMRI approaches. Second, because of the endogenous contrast source they influence, the probes are detectable on a variety of spatiotemporal scales by noninvasive imaging modalities complementary to fMRI, such as diffuse optical or ultrasound-based methods. Third, by circumventing limitations of established optical, magnetic, and radioactive probe designs, vasoprobes combine exquisite sensitivity approaching that of positron emission tomography (PET) with the resolution and versatility of MRI. In this project, we will build on our recent proof-of-concept work with vasoprobes to establish noninvasive brain-wide delivery strategies and to develop robust neurochemical sensors that function in primates. The technology we establish will address multiple goals in basic and applied neuroscience, and we expect it to yield molecular probes that will be appropriate for clinical evaluation in human subjects by the end of the project period. In Aim 1, we will create vasoprobe variants that can be delivered to the brain via intravenous injection and spontaneous permeation through the blood-brain barrier (BBB). We will form conjugates of vasoprobe-based sensors with ?brain shuttle? antibodies that have previously been shown to enable brain import via receptor- mediated transcytosis. Demonstration of brain-permeable vasoprobes will establish a clinically viable path for facile, noninvasive applications of vasoprobes throughout the brain. In Aim 2, we will optimize vasoprobes to sense the key neurotransmitters dopamine and glutamate; we will then apply them on a brain-wide scale for molecular-level fMRI in rodent brains. These experiments, in conjunction with outcome of Aim 1, will set the stage for applications of neurotransmitter-sensitive vasoprobes and related sensors in primate brains. Accord- ingly, in Aim 3, we will adapt neurotransmitter-sensitive vasoprobe technology for functional molecular neuroim- aging in marmosets, a tractable primate species with which we have previous experience. Successful completion of validation experiments in marmosets will therefore establish groundbreaking imaging agents suitable for trans- lation to humans, as well as for adaptation to many further neurophysiological targets.