Venkatesh N. Murthy - US grants

DESCRIPTION (provided by applicant):The reliability and efficacy of communication between neurons is governed by the fundamental properties of synapses. In addition, modification of synaptic properties is likely to be a crucial component of learning and formation of memories. The long-term objectives of this research program are to understand the elementary steps in release of neurotransmitter from synaptic terminals, and how they can be modulated. Although many of the molecules in the presynaptic terminal have been identified, their precise roles in various steps in release and recycling of vesicles are incompletely understood. Synapses contain functionally heterogeneous populations of vesicles. Release is thought to occur from a small pool of vesicles termed the readily releasable pool, and when this pool is fully or partially depleted, it is filled with vesicles from the reserve pool. This work will focus on characterizing the different pool of synaptic vesicles, the movement of vesicles between them and activity-dependent changes in the characteristics of the vesicle pools. Experiments are designed to use the fluorescent dyes and optical microscopy, combined with molecular biology and electrophysiology, to study synaptic vesicle release and trafficking at visualized individual synapses. The specific objectives are to determine (i) the mechanisms regulating the size and accessibility of the recycling vesicle pool, (ii) the roles of peripherally associated synaptic vesicle proteins in trafficking of vesicles between the different pools, and (iii) the mechanisms in activity-dependent, long-term changes in the size of the vesicle pools. Since synaptic transmission is adversely affected in many disorders of the nervous system, a better understanding of synaptic release mechanisms will provide a rational basis for treatment.

Lay abstract

CAREER: Long-term changes in synaptic function induced by selective
suppression of activity in single neurons

The adaptation of the brain to environmental changes is achieved in part
through modifications in the strength of synapses, which are specialized
connections between neurons. The precise rules governing synapse
formation, maturation and subsequent modification in the central nervous
system are not fully understood. Using electrophysiology, fluorescence
microscopy and molecular biological tools, Dr. Murthy will investigate
long-term modification of identified synapses in a simple neural network
in vitro. The overall research objectives of the proposal are (i) to
determine the consequences of selective, long-term reduction of activity
in a single neuron within an active network, and (ii) to understand the
locus and mechanisms in synaptic modification resulting from this
manipulation.

Two general classes of results might be anticipated - homeostatic
modifications that allow neuron-wide increase in synaptic strength that
will restore the overall activity of the 'quiet' cell to normal levels,
and competitive modifications that reduce or eliminate synaptic input and
output of the 'quiet' cell. This experimental system will offer
unparalleled access to detailed cell biological investigations of synaptic
modification - from the level of gene expression to physiological analysis
at single synapse resolution. The investigator will also integrate
education with his research program by developing up-to-date instructional
material for an advanced course in neuronal cell biology. Since optical
microscopy has become an indispensable tool in cellular-level research,
Dr. Murthy will organize short departmental workshops on advanced imaging
techniques. In addition to these instructional elements, the investigator
will also mentor undergraduate and graduate students, including those from
underrepresented groups.

DESCRIPTION (provided by applicant): The function of a neural circuit is constrained by the properties of individual neurons and their wiring. In many sensory systems, the responses of the circuit elements vary systematically with physical position, leading to a topographic representation of the stimulus space. Sensory representation in the olfactory system has been harder to decipher, in part due to the difficulty in finding appropriate metrics to characterize the odor space and in sampling this space densely. Progress has also been slowed by technological limitations in probing and controlling individual circuit elements in early olfactory circuits. In this proposal, we aim to develop new methods that will greatly aid the dissection of functional neural circuits in the olfactory system in mice. Mice rely on olfaction to find food, choose mates and avoid predators. In mammals, olfactory sensory neurons send their axons to the olfactory bulb (OB), where there is a characteristic physical layout of inputs in the glomerular layer. Each glomerulus receives convergent afferents from a large number of olfactory sensory neurons expressing the same odorant receptor, so each point on the surface of the OB has a specific chemical response spectrum. The principal neurons in the OB, the mitral and tufted (M/T) cells, typically have a single primary dendrite that projects to a single glomerulus. M/T cells also receive lateral GABAergic inputs from a variety of interneurons in the glomerular and external plexiform layers, thus allowing them to sample information from several functionally diverse glomeruli. Odor processing in the OB is also strongly modulated by feedback from the cortex as well as brainstem neuromodulatory centers. Here, we propose to develop new reagents and methods that will accelerate the pace of research into mammalian olfaction. Our experiments will be guided by three specific aims. Aim 1: To generate transgenic mouse lines that express the light-activated ion channel channelrhodopsin specifically in olfactory sensory neurons, rendering the input layer of the olfactory bulb (glomeruli) optically excitable. Aim 2: To demonstrate the feasibility of using this mouse model to study functional connectivity in the OB and its downstream target areas using in vitro slice preparation and digital mirror device technology. Aim 3: To demonstrate the feasibility of constructing glomerular receptive fields of neurons in the OB and its target brain areas in the intact, freely breathing mouse. Tools developed here will help advance our understanding of odor coding. In addition, since the olfaction is often used as a sensory gateway to study higher brain function such as decision making, our tools will also have broader use. Finally, by crossing these "opto-olfactory" mice with other mouse models of disease, we can catalyze studies of sensory dysfunction in brain disorders such as autism and Alzheimer's disease. PUBLIC HEALTH RELEVANCE: Our studies will produce novel reagents - transgenic mouse lines and optical stimulation devices - that will aid future investigation of how sensory information processing occurs normally in the brain and how it is modulated. More generally, our studies also have direct implications for human health, since olfactory deficits have also been strongly associated with aging and some neurological diseases such as Alzheimer's.

DESCRIPTION (provided by applicant): The excitement about neural stem cells arises in large part from the hope that they can be harnessed therapeutically to repair diseased brains. Very little is known, however, about how these new neurons integrate into existing brain circuits by making appropriate connections - this process can be compared to changing the wheels of a car while it is running. In addition, major questions of how adult neurogenesis and functional integration are regulated by local factors as well as by an animal's experience remain largely unanswered. In this proposal, we will conduct innovative experiments to investigate how new neurons are integrated into synaptic circuits in the mouse olfactory bulb. The rodent olfactory bulb is an excellent model system because of the high rate of neurogenesis, its accessibility, modular organization and behavioral relevance. We will describe the natural history of adult-born neurons in their native environment by imaging their morphology and function at high resolution in the intact brains of living mice using multiphoton laser scanning microscopy. We will also begin to uncover the cellular and molecular processes involved in the functional integration of new neurons into existing circuits using genetic perturbations. To achieve our goals, we will use stereotaxic viral injections, genetically-encoded calcium indicators and chronic multiphoton microscopy to examine in real time how newborn granule cells develop their morphological and functional properties. By tracking identified neurons over several weeks using time-lapse imaging in vivo, we will be able to uncover structural and functional changes that are not visible to conventional methods that obtain single snapshots in each animal. We will alter the odor experience of mice in a critical period during which labeled newborn cells are integrated into the bulb and investigate how this alters their functional properties and their survival. Experiments in this project will be guided by three Aims. Aim 1: To determine the time evolution of sensory responses of identified adult-born neurons over their development using multiphoton microscopy. Aim 2: To determine how sensory experience affects the functional properties of adult-born neurons cells and their survival. Aim 3: To determine the cellular and molecular mechanisms in the refinement of functional properties of adult-born neurons. The research proposed here will provide a deeper understanding about how newborn cells find appropriate synaptic partners and integrate into the adult brain. Insights gained from this study will inform efforts to treat human brain disorders using neuron replacement therapies.

? DESCRIPTION (provided by applicant): The midbrain serotonergic system has been implicated in a diverse range of brain functions, including modulation of sensory processing, motor output, respiration and mood. This diversity presumably arises from diffuse projections from a small number of neurons in the raphe nucleus to nearly all regions of the forebrain, and the large number of cellular signaling pathways. Sensory systems offer significant advantages when linking cellular effects of serotonin to systems-level phenomena because of their circumscribed function, better understood circuitry and the easier control over their stimulus-output properties in vivo. In this project, we will conduct innovative experiments to understand how the selective and natural activation of the serotonergic system modulates the function of identified neurons in an ethologically relevant sense, in a genetically-accessible mammalian model, the mouse. We will investigate serotonergic modulation in the olfactory bulb (OB), since it has rich innervation from the raphe nucleus and many serotonin receptors are expressed there. We will use optogenetic and physiological methods to test the hypothesis that serotonergic neurons affect principal neurons in the OB in a rapid and temporally diverse manner using multiple neurotransmitters. To achieve our goals, we will use optogenetic activators to selectively activate serotonergic neurons and trigger natural secretion of neurotransmitters. Then using a suite of methods already established in our group (genetically-encoded calcium indicators, multiphoton microscopy and patch-clamp electrophysiology), we will dissect the cellular mechanisms of serotonergic modulation in principal neurons in the OB. Experiments in this project will be guided by three Aims. Aim 1: To determine the effects of dorsal raphe nucleus activation on the activity of different classes of identified neurons in the OB. Aim 2: To determine the effects of selective optogenetic activation of raphe neurons on the fast time-scale activity of individual output neurons of the OB. Aim 3: To determine the cellular mechanisms underlying the complex effects of serotonergic neuron activation on the OB output neurons. The research proposed here will provide a mechanistic understanding of how natural activation of the serotonergic system alters the functional properties of a sensory circuit. Insighs gained from this study will may help in strategies aimed at correcting dysfunctions involving the serotonergic system.

The NSF Physics of Living Systems (PoLS) Student Research Network (SRN) strives to unite students and faculty working at the interface of physics and biology at different institutions ("nodes") within the US and internationally. A well functioning virtual network could give students at local nodes the ability to take advantage of global educational and research opportunities in PoLS. PoLS is a diverse field, and is composed of researchers and students from varied backgrounds. No single institution can offer (1) the breadth and depth of research and (2) courses that both cover the relevant intellectual landscape and provide in-depth training for students. Such training is critical to create the next generation of researchers who can contribute quantitatively to biophysics, with the ability to move between biology, physics, mathematics, and engineering; PoLS students have important roles to play in this next generation. In addition, no single institution has the range of equipment needed to study PoLS on the enormous range of time and length scales encountered in biological systems. Finally, few single institutions can fruitfully integrate science and engineering to inspire biomedical, robotic and prosthetic devices that will result from basic PoLS research. The HF-SRN will create an environment for students in which they can work among various disciplines while maintaining the physics mindset (simplified systems, few parameter predictive models) and developing new physics. This network will train students (paraphrasing Philip Nelson in his 2008 Biological Physics textbook) "who can switch fluidly between both kinds of brain: the `developmental/historical/complex' sciences and the 'universal/ahistorical/reductionist'." As significant collaborative and educational flux develops within the HF-SRN, successful activities will be broadened to the other US nodes (ultimately with the expectation to engage PoLS SRN international partners). The evaluation plan will help guide aspects of the HF-SRN that could increase flux in other programs in the NSF Science Across Virtual Institutes initiative. More broadly, PoLS SRN students can be leaders in the next generation of researchers who blend biology and physics research seamlessly. Such students will create materials which will seed future K-12 as well as university PoLS curricula. Efforts will be made to extend the educational and research efforts developed within the HF-SRN (and entire SRN) to a broader community including local minority serving institutions. Advances in PoLS can lead to advances in applications such as genome editing, cancer dynamics, robotics and human-assist devices, among others.

During the last period of funding as part of the SRN, the Georgia Tech, Harvard and Maryland nodes have advanced their respective PoLS programs, developing cohesive local communities. The goal of this project is to further develop opportunities for students (and their ideas) to "flow" more easily within the SRN and thereby discover working principles of increased human network flux that can be transferred into the larger SRN. To do so, significant interactions (and evaluations of those interactions) will be developed among three existing SRN nodes (adding Emory as a subcontract to Georgia Tech), forming a "High Flux SRN" (HF-SRN). The HF-SRN will engage in activities such as 1) Collaborative Focused Research Projects, which span nodes and are "built to succeed" by leveraging student and faculty expertise in current projects; 2) Student-Led Dynamic Working Groups (e.g., in biomolecular, microbial, cellular and organismal physics) leveraging faculty research strengths and student interest to develop cross-node communities for these topics. 3) Student-Led Bootcamps: intense 2-3 day tutorials (e.g., microscopy, robophysics, image analysis) with cross-subgroup cutting themes, open to HF-SRN members and held at a particular node; 4) Student-Led Workshops: composed of talks, poster and discussion sessions, inviting the entire PoLS SRN; 5) Curriculum development via open-source course materials, integrating complementary expertise across nodes. All activities will be evaluated and assessed by a Council composed of the lead PIs at each node. This project is being jointly supported by the Physics of Living Systems program in the Division of Physics, the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences, and the Modulation Program in the Division of Integrative Organismal Systems.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

A key problem in sensory neuroscience is how nervous systems can detect objects of interest in noisy and cluttered sensory scenes, which then guide further action. This ability to pick out important information from the array of mixed stimuli is thought to involve extensive top-down modulation that interacts with feedforward processing of information. We propose to investigate this important function in the olfactory system of behaving mice using optogenetic and optophysiological methods. Olfaction is critical for the survival of many animals, which use this sense for to find food and mates, and to avoid predators. Information about volatile odorants sensed by olfactory sensory neurons is passed on to the olfactory bulb (OB), whose output reaches many cortical directly areas. This feedforward architecture is disrupted by massive feedback connections throughout the olfactory system, which are thought to provide context and learning-related signals to aid in odor perception. We recently developed an odor-guided behavioral task in which mice are required to parse complex odorous stimuli. In this project, we will use this exciting new behavioral assay to examine the role of cortical feedback to the OB in the genetically-accessible mouse model. To achieve our goals, we will first characterize how complex odor mixtures are represented in the OB, and then examine the effects of selective optogenetic perturbation of cortical feedback projections on the coding of complex odor stimuli in the OB. Then using a suite of cutting-edge methods already established in our group, we will uncover how the feedback signals conveyed to the OB evolve as mice learn to perform this task. Experiments in this project will be guided by three Aims. Aim 1: To determine how the output neurons of the OB represent odorant mixtures and how they are altered after mice learn the mixture task. Aim 2: To determine the effects of selective optogenetic perturbation of olfactory cortical neuron activity on odor coding in OB output neurons. Aim 3: To determine the features of stimulus-related signals carried by cortical feedback axons to the OB in naïve mice and in mice that have learnt the mixture task. The research proposed has broad relevance for neuroscience because it will shed light on how brains interpret ambiguous sensory stimuli in cluttered environments, an ability that is thought to involve top-down feedback. This ability is thought to be impaired with aging, as well as in some mental disorders. Therefore, understanding this process in the normal brain could help devise specific and efficacious treatments in abnormal conditions.

Summary The ability to learn associations between a specific sensory stimulus and an outcome such as re- ward or punishment is a basic requirement for flexible behaviors. Malfunctions of this associative process may underlie various disorders such as drug addiction and binge eating. Rodents can learn novel stimulus-response associations after only a few repetitions, but the circuits that are modified during learning are largely unknown. The olfactory tubercle (OT), a part of the ventral stri- atum, is located at the interface between sensory and reward centers, receiving strong olfactory sensory input as well as dopaminergic innervation from the ventral tegmental area (VTA). It has been implicated in reward and is a recognized ?hot spot? for cocaine self-administration. These ob- servations suggest that the OT is the site of heterosynaptic plasticity to establish valence represen- tation associated with odors. The PIs have developed a behavioral paradigm in mice that allows rapid and flexible association of arbitrary odor cues with reward or aversion. Using this behavior, they have found evidence for an explicit representation of reward in the OT. In this project, the PIs will test the hypothesis that neural activity in the OT is modified during learning to reflect the va- lence of stimuli, and that dopaminergic signals from the VTA play a key role in this learning. Aim 1: To determine whether OT neurons signal explicit (odor-independent) valence signals after learn- ing. Mice will be trained to learn the arbitrarily assigned valence of a panel of odors and record spiking activity using tetrodes from the OT in behaving animals. The hypothesis tested is that there is an explicit valence representation in the activity of OT neurons and this representation emerges rapidly when novel odor associations are learned. Aim 2: To determine how reward and aversion are represented in the OT. The PIs will use aversive and rewarding stimuli to ask whether OT neu- rons represent true valence signals, or if they signal motivational salience. The hypothesis is that OT activity will be modulated in opposite directions for rewarding and aversive cues, signaling ex- plicit valence, with potential heterogeneity across OT cell types and subregions. Aim 3: To deter- mine whether dopaminergic axons targeting OT carry valence related signals that evolve during learning. The PIs will use fiber photometry and microendoscopy to record valence-related activity of dopaminergic axons in the OT and optogenetics to stimulate these axons in behaving mice. The hypothesis is that the OT receives dopamine inputs that represent value prediction errors that can shape the valence-related activity of OT neurons. The research proposed has broad relevance for neuroscience because it will shed light on how reward-predicting signals are learnt and represent- ed in the brain, which could help devise treatments in abnormal conditions such as addiction.

7. Project Summary/ Abstract The Molecular Biophysics Training Grant at Harvard University supports a predoctoral training program focused at the interface of physical and biological sciences. The goal of the program is to provide students with strong undergraduate backgrounds in quantitative sciences (especially physics and mathematics) with broad training in the biophysical, chemical and molecular concepts and techniques that are required to address outstanding problems in biology and biomedical sciences. The training program links a highly interactive group of 51 faculty members from four departments in Harvard's Faculty of Arts and Sciences, the School of Engineering and Applied Sciences, six departments at Harvard Medical School, and five affiliated hospitals. The training program offers a flexible curriculum drawn from courses offered at Harvard, Harvard Medical School and Massachusetts Institute of Technology (MIT), and research opportunities in a variety of disciplines relevant to molecular biophysics with particular strengths in the areas of structural biology, computational biology, quantitative cell biology, single molecule biophysics, neuroscience, and imaging. In addition to coursework and research activities, the training program sponsors seminars and guest lectures; a student-run research seminar series; a yearly offsite research retreat in the Fall that includes student and faculty research talks, a plenary lecture and a poster session featuring research of program students; a mini-symposium featuring talks by program faculty as well as a student poster session during the Biophysics Program recruitment weekend in the Spring term; and social events for all trainees. Over the past 29 years, this training program has helped foster a number of new initiatives in graduate training, and has been remarkably successful in promoting collaborative research among its faculty and interdisciplinary training for its students spanning all of Harvard and some of MIT. In this competing renewal, we request support for 16 training slots for students who are affiliated with Harvard's Biophysics Program or who are jointly affiliated with the Harvard Biophysics Program and Medical Engineering and Medical Physics Ph.D. program in the joint Harvard/ MIT Health Sciences and Technology initiative. Students will be preferentially funded in their first and second year of graduate studies.

Project Summary/Abstract In order to control specific behavioral responses, transcriptionally distinct cell types assembled into dynamic brain circuits integrate environmental information with internal states and generate purposeful motor actions. While tools have been developed to independently measure the activity dynamics, connectivity and transcriptional profiles of individual neurons, it remains challenging to integrate this diverse information into a coherent model of behavior. To address this challenge, we aim to uncover the sensorimotor transformations leading to a complex naturalistic behavior, male and female parenting, by developing innovative molecular, imaging and systems-level approaches and by integrating multimodal information obtained from single neurons in behaving animals. In aim 1, we will develop new tools to uncover the activity and the transcriptional identity of neuronal cell types involved in infant-mediated behavior. In aim 2, we will explore in molecular, functional, and behavioral terms how olfactory and other sensory modalities underlie parenting behavior in males and females and in virgin versus mated states. In aim 3, we will investigate how specific hypothalamic cell types process information reflecting behavioral outcomes using a combination of spatial transcriptomic, functional imaging, circuit tracing and behavioral methods. In aim 4, we will combine cell-type based neuronal architecture with activity patterns across key brain regions to formulate predictive models underlying the neural control of parenting.