Academic Appointments

Honors & Awards

  • United States Physics Team, International Physics Olympiad, Bad Ischl, Austria (1988)
  • Detur Scholar, Harvard University (1989)
  • John Harvard Scholarships, John Harvard Scholarships (1989-1991)
  • Barry Goldwater Fellowship for Excellence in Science, United States, Barry Goldwater Fellowship for Excellence in Science, United States (1990)
  • Junior Phi Beta Kappa for top 12 Junior men, Harvard University (1991)
  • Winston Churchill Fellowship, Winston Churchill Foundation of the United States (1992-1993)
  • Predoctoral Fellowship, NSF (1993-1996)
  • Predoctoral Fellowship, American Heart Association (1996-1998)
  • Charlotte Elizabeth Procter Honorific Fellowship, Princeton Univeristy (1997-1998)
  • Burroughs Wellcome Fellowship, Program in Mathematics and Molecular Biology (1998-1999)
  • McKnight Technological Innovations in Neuroscience Award, McKnight Foundation (2000)
  • Young Investigator Award (with #1 world ranking), Human Frontiers in Science Program (2002)
  • Cutting Edge Basic Research Award (CEBRA) Science, National Institutes of Health (2003)
  • Member of TR100,World's Top 100 Innovators under age 35, Technology Review Magazine (2003)
  • Young Investigator Award, Office of Naval Research, Cognitive & Neural Division (2004)
  • Klingenstein Fellowship in the Neurosciences, Klingenstein Foundation (2004)
  • Young Investigator Award, Beckman Foundation (2004)
  • Presidential Early Career Award in Science and Engineering 2004, Presented at the White House on June 13, 2005 (2004)
  • Alfred P. Sloan Foundation Research Fellow, Alfred P. Sloan Foundation (2005)
  • Fellowship in Science & Engineering, David & Lucille Packard Foundation (2005)
  • Beckman Interdisciplinary Translational Research Program Award, Stanford University (2005)
  • Terman Fellow, Stanford University (2006)
  • NIH Director's Pioneer Award, National Institutes of Health (2007)
  • The Brilliant 10, Top ten brilliant scientists under age 40, Popular Science Magazine (2007)
  • W.M. Keck Foundation, Medical Research Program grant, W.M. Keck Foundation (2007)
  • Best Techniques Paper, Co-Author, American Society of Biomechanics (2007)
  • HHMI Investigator, Howard Hughes Medical Institute (2008)
  • Michael & Kate Bárány Young Investigator Award, Biophysical Society (2010)
  • Allen Distinguished Investigator Award, Paul G. Allen Family Foundation (2010)
  • National Academy Keck Futures Initiative Award, W.M. Keck Foundation (2011)
  • Ellison Senior Scholar Award, Ellison Foundation (2012)

Current Research and Scholarly Interests

The long-term goal of our research is to advance experimental paradigms for understanding normal cognitive and disease processes at the level of neural circuits, with emphasis on learning and memory processes. By contrast, much current research on learning and memory concentrates on levels of organization in the nervous system that are either more macroscopic (e.g. in cognitive psychology) or more microscopic (e.g. in synaptic physiology).

Our approach combines behavioral, electrophysiological, and computational methodologies with high-resolution fluorescence optical imaging that is capable of resolving individual neurons and dendrites. By necessity, we aim to advance imaging methods so that we can examine dynamics of neuronal populations or of dendritic compartments in behaving animals. En route, we are also performing experiments on circuit properties in anesthetized animals, such as the studies that use our newly invented fluorescence endoscopes for examining hippocampal cells and dendrites in vivo.

We seek explanations that span different levels of organization, from cells to entire circuits. We work with both genetic model organisms, mice and fruit flies, and human subjects. Our research emphasizes understanding the control and learning of motor behaviors, as well as the potential application of our newly developed imaging techniques to clinical use in humans.

2018-19 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • Fast, in vivo voltage imaging using a red fluorescent indicator. Nature methods Kannan, M., Vasan, G., Huang, C., Haziza, S., Li, J. Z., Inan, H., Schnitzer, M. J., Pieribone, V. A. 2018


    Genetically encoded voltage indicators (GEVIs) are emerging optical tools for acquiring brain-wide cell-type-specific functional data at unparalleled temporal resolution. To broaden the application of GEVIs in high-speed multispectral imaging, we used a high-throughput strategy to develop voltage-activated red neuronal activity monitor (VARNAM), a fusion of the fast Acetabularia opsin and the bright red fluorophore mRuby3. Imageable under the modest illumination intensities required by bright green probes (<50mWmm-2), VARNAM is readily usable in vivo. VARNAM can be combined with blue-shifted optical tools to enable cell-type-specific all-optical electrophysiology and dual-color spike imaging in acute brain slices and live Drosophila. With enhanced sensitivity to subthreshold voltages, VARNAM resolves postsynaptic potentials in slices and cortical and hippocampal rhythms in freely behaving mice. Together, VARNAM lends a new hue to the optical toolbox, opening the door to high-speed in vivo multispectral functional imaging.

    View details for DOI 10.1038/s41592-018-0188-7

    View details for PubMedID 30420685

  • Long-Term Consolidation of Ensemble Neural Plasticity Patterns in Hippocampal Area CA1. Cell reports Attardo, A., Lu, J., Kawashima, T., Okuno, H., Fitzgerald, J. E., Bito, H., Schnitzer, M. J. 2018; 25 (3): 640


    Neural network remodeling underpins the ability to remember life experiences, but little is known about the long-term plasticity of neural populations. To study how the brain encodes episodic events, we used time-lapse two-photon microscopy and a fluorescent reporter of neural plasticity based on an enhanced form of the synaptic activity-responsive element (E-SARE) within the Arc promoter to track thousands of CA1 hippocampal pyramidal cells over weeks in mice that repeatedly encountered different environments. Each environment evokes characteristic patterns of ensemble neural plasticity, but with each encounter, the set of activated cells gradually evolves. After repeated exposures, the plasticity patterns evoked by an individual environment progressively stabilize. Compared with young adults, plasticity patterns in aged mice are less specific to individual environments and less stable across repeat experiences. Long-term consolidation of hippocampal plasticity patterns may support long-term memory formation, whereas weaker consolidation in aged subjects might reflect declining memory function.

    View details for DOI 10.1016/j.celrep.2018.09.064

    View details for PubMedID 30332644

  • Three-photon imaging of mouse brain structure and function through the intact skull NATURE METHODS Wang, T., Ouzounov, D. G., Wu, C., Horton, N. G., Zhang, B., Wu, C., Zhang, Y., Schnitzer, M. J., Xu, C. 2018; 15 (10): 789-+


    Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-μm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.

    View details for DOI 10.1038/s41592-018-0115-y

    View details for Web of Science ID 000448820100025

    View details for PubMedID 30202059

    View details for PubMedCentralID PMC6188644

  • Long-term optical brain imaging in live adult fruit flies NATURE COMMUNICATIONS Huang, C., Maxey, J. R., Sinha, S., Savall, J., Gong, Y., Schnitzer, M. J. 2018; 9: 872


    Time-lapse in vivo microscopy studies of cellular morphology and physiology are crucial toward understanding brain function but have been infeasible in the fruit fly, a key model species. Here we use laser microsurgery to create a chronic fly preparation for repeated imaging of neural architecture and dynamics for up to 50 days. In fly mushroom body neurons, we track axonal boutons for 10 days and record odor-evoked calcium transients over 7 weeks. Further, by using voltage imaging to resolve individual action potentials, we monitor spiking plasticity in dopamine neurons of flies undergoing mechanical stress. After 24 h of stress, PPL1-α'3 but not PPL1-α'2α2 dopamine neurons have elevated spike rates. Overall, our chronic preparation is compatible with a broad range of optical techniques and enables longitudinal studies of many biological questions that could not be addressed before in live flies.

    View details for DOI 10.1038/s41467-018-02873-1

    View details for Web of Science ID 000426275300003

    View details for PubMedID 29491443

    View details for PubMedCentralID PMC5830414

  • Unsupervised Discovery of Demixed, Low-Dimensional Neural Dynamics across Multiple Timescales through Tensor Component Analysis. Neuron Williams, A. H., Kim, T. H., Wang, F., Vyas, S., Ryu, S. I., Shenoy, K. V., Schnitzer, M., Kolda, T. G., Ganguli, S. 2018


    Perceptions, thoughts, and actions unfold over millisecond timescales, while learned behaviors can require many days to mature. While recent experimental advances enable large-scale and long-term neural recordings with high temporal fidelity, it remains a formidable challenge to extract unbiased and interpretable descriptions of how rapid single-trial circuit dynamics change slowly over many trials to mediate learning. We demonstrate a simple tensor component analysis (TCA) can meet this challenge by extracting three interconnected, low-dimensional descriptions of neural data: neuron factors, reflecting cell assemblies; temporal factors, reflecting rapid circuit dynamics mediating perceptions, thoughts, and actions within each trial; and trial factors, describing both long-term learning and trial-to-trial changes in cognitive state. We demonstrate the broad applicability of TCA by revealing insights into diverse datasets derived from artificial neural networks, large-scale calcium imaging of rodent prefrontal cortex during maze navigation, and multielectrode recordings of macaque motor cortex during brain machine interface learning.

    View details for DOI 10.1016/j.neuron.2018.05.015

    View details for PubMedID 29887338

  • Diametric neural ensemble dynamics in parkinsonian and dyskinetic states. Nature Parker, J. G., Marshall, J. D., Ahanonu, B., Wu, Y. W., Kim, T. H., Grewe, B. F., Zhang, Y., Li, J. Z., Ding, J. B., Ehlers, M. D., Schnitzer, M. J. 2018


    Loss of dopamine in Parkinson's disease is hypothesized to impede movement by inducing hypo- and hyperactivity in striatal spiny projection neurons (SPNs) of the direct (dSPNs) and indirect (iSPNs) pathways in the basal ganglia, respectively. The opposite imbalance might underlie hyperkinetic abnormalities, such as dyskinesia caused by treatment of Parkinson's disease with the dopamine precursor L-DOPA. Here we monitored thousands of SPNs in behaving mice, before and after dopamine depletion and during L-DOPA-induced dyskinesia. Normally, intermingled clusters of dSPNs and iSPNs coactivated before movement. Dopamine depletion unbalanced SPN activity rates and disrupted the movement-encoding iSPN clusters. Matching their clinical efficacy, L-DOPA or agonism of the D2 dopamine receptor reversed these abnormalities more effectively than agonism of the D1 dopamine receptor. The opposite pathophysiology arose in L-DOPA-induced dyskinesia, during which iSPNs showed hypoactivity and dSPNs showed unclustered hyperactivity. Therefore, both the spatiotemporal profiles and rates of SPN activity appear crucial to striatal function, and next-generation treatments for basal ganglia disorders should target both facets of striatal activity.

    View details for DOI 10.1038/s41586-018-0090-6

    View details for PubMedID 29720658

  • Neuronal Representation of Social Information in the Medial Amygdala of Awake Behaving Mice CELL Li, Y., Mathis, A., Grewe, B. F., Osterhout, J. A., Ahanonu, B., Schnitzer, M. J., Murthy, V. N., Dulac, C. 2017; 171 (5): 1176-+


    The medial amygdala (MeA) plays a critical role in processing species- and sex-specific signals that trigger social and defensive behaviors. However, the principles by which this deep brain structure encodes social information is poorly understood. We used a miniature microscope to image the Ca2+ dynamics of large neural ensembles in awake behaving mice and tracked the responses of MeA neurons over several months. These recordings revealed spatially intermingled subsets of MeA neurons with distinct temporal dynamics. The encoding of social information in the MeA differed between males and females and relied on information from both individual cells and neuronal populations. By performing long-term Ca2+ imaging across different social contexts, we found that sexual experience triggers lasting and sex-specific changes in MeA activity, which, in males, involve signaling by oxytocin. These findings reveal basic principles underlying the brain's representation of social information and its modulation by intrinsic and extrinsic factors.

    View details for DOI 10.1016/j.cell.2017.10.015

    View details for Web of Science ID 000415317000019

    View details for PubMedID 29107332

    View details for PubMedCentralID PMC5731476

  • Social behaviour shapes hypothalamic neural ensemble representations of conspecific sex NATURE Remedios, R., Kennedy, A., Zelikowsky, M., Grewe, B. F., Schnitzer, M. J., Anderson, D. J. 2017; 550 (7676): 388-+


    All animals possess a repertoire of innate (or instinctive) behaviours, which can be performed without training. Whether such behaviours are mediated by anatomically distinct and/or genetically specified neural pathways remains unknown. Here we report that neural representations within the mouse hypothalamus, that underlie innate social behaviours, are shaped by social experience. Oestrogen receptor 1-expressing (Esr1+) neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) control mating and fighting in rodents. We used microendoscopy to image Esr1+ neuronal activity in the VMHvl of male mice engaged in these social behaviours. In sexually and socially experienced adult males, divergent and characteristic neural ensembles represented male versus female conspecifics. However, in inexperienced adult males, male and female intruders activated overlapping neuronal populations. Sex-specific neuronal ensembles gradually separated as the mice acquired social and sexual experience. In mice permitted to investigate but not to mount or attack conspecifics, ensemble divergence did not occur. However, 30 minutes of sexual experience with a female was sufficient to promote the separation of male and female ensembles and to induce an attack response 24 h later. These observations uncover an unexpected social experience-dependent component to the formation of hypothalamic neural assemblies controlling innate social behaviours. More generally, they reveal plasticity and dynamic coding in an evolutionarily ancient deep subcortical structure that is traditionally viewed as a 'hard-wired' system.

    View details for DOI 10.1038/nature23885

    View details for Web of Science ID 000413247900059

    View details for PubMedID 29052632

    View details for PubMedCentralID PMC5674977

  • Cerebellar granule cells encode the expectation of reward NATURE Wagner, M. J., Kim, T. H., Savall, J., Schnitzer, M. J., Luo, L. 2017; 544 (7648): 96-?


    The human brain contains approximately 60 billion cerebellar granule cells, which outnumber all other brain neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes. Although evidence suggests a role for the cerebellum in cognition, granule cells are known to encode only sensory and motor context. Here, using two-photon calcium imaging in behaving mice, we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed sugar-water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward-omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from the current understanding of these neurons and markedly enriches the contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum.

    View details for DOI 10.1038/nature21726

    View details for Web of Science ID 000398323300040

    View details for PubMedID 28321129

  • Neural ensemble dynamics underlying a long-term associative memory NATURE Grewe, B. F., Grundemann, J., Kitch, L. J., Lecoq, J. A., Parker, J. G., Marshall, J. D., Larkin, M. C., Jercog, P. E., Grenier, F., Li, J. Z., Luthi, A., Schnitzer, M. J. 2017; 543 (7647): 670-?


    The brain's ability to associate different stimuli is vital for long-term memory, but how neural ensembles encode associative memories is unknown. Here we studied how cell ensembles in the basal and lateral amygdala encode associations between conditioned and unconditioned stimuli (CS and US, respectively). Using a miniature fluorescence microscope, we tracked the Ca(2+) dynamics of ensembles of amygdalar neurons during fear learning and extinction over 6 days in behaving mice. Fear conditioning induced both up- and down-regulation of individual cells' CS-evoked responses. This bi-directional plasticity mainly occurred after conditioning, and reshaped the neural ensemble representation of the CS to become more similar to the US representation. During extinction training with repetitive CS presentations, the CS representation became more distinctive without reverting to its original form. Throughout the experiments, the strength of the ensemble-encoded CS-US association predicted the level of behavioural conditioning in each mouse. These findings support a supervised learning model in which activation of the US representation guides the transformation of the CS representation.

    View details for DOI 10.1038/nature21682

    View details for Web of Science ID 000397619700046

    View details for PubMedID 28329757

  • Long-Term Optical Access to an Estimated One Million Neurons in the Live Mouse Cortex CELL REPORTS Kim, T. H., Zhang, Y., Lecoq, J., Jung, J. C., Li, J., Zeng, H., Niell, C. M., Schnitzer, M. J. 2016; 17 (12): 3385-3394


    A major technological goal in neuroscience is to enable the interrogation of individual cells across the live brain. By creating a curved glass replacement to the dorsal cranium and surgical methods for its installation, we developed a chronic mouse preparation providing optical access to an estimated 800,000-1,100,000 individual neurons across the dorsal surface of neocortex. Post-surgical histological studies revealed comparable glial activation as in control mice. In behaving mice expressing a Ca(2+) indicator in cortical pyramidal neurons, we performed Ca(2+) imaging across neocortex using an epi-fluorescence macroscope and estimated that 25,000-50,000 individual neurons were accessible per mouse across multiple focal planes. Two-photon microscopy revealed dendritic morphologies throughout neocortex, allowed time-lapse imaging of individual cells, and yielded estimates of >1 million accessible neurons per mouse by serial tiling. This approach supports a variety of optical techniques and enables studies of cells across >30 neocortical areas in behaving mice.

    View details for DOI 10.1016/j.celrep.2016.12.004

    View details for Web of Science ID 000390895600026

    View details for PubMedID 28009304

  • Cell-Type-Specific Optical Recording of Membrane Voltage Dynamics in Freely Moving Mice CELL Marshall, J. D., Li, J. Z., Zhang, Y., Gong, Y., St-Pierre, F., Lin, M. Z., Schnitzer, M. J. 2016; 167 (6): 1650-?


    Electrophysiological field potential dynamics are of fundamental interest in basic and clinical neuroscience, but how specific cell types shape these dynamics in the live brain is poorly understood. To empower mechanistic studies, we created an optical technique, TEMPO, that records the aggregate trans-membrane voltage dynamics of genetically specified neurons in freely behaving mice. TEMPO has >10-fold greater sensitivity than prior fiber-optic techniques and attains the noise minimum set by quantum mechanical photon shot noise. After validating TEMPO's capacity to track established oscillations in the delta, theta, and gamma frequency bands, we compared the D1- and D2-dopamine-receptor-expressing striatal medium spiny neurons (MSNs), which are interspersed and electrically indistinguishable. Unexpectedly, MSN population dynamics exhibited two distinct coherent states that were commonly indiscernible in electrical recordings and involved synchronized hyperpolarizations across both MSN subtypes. Overall, TEMPO allows the deconstruction of normal and pathologic neurophysiological states into trans-membrane voltage activity patterns of specific cell types.

    View details for DOI 10.1016/j.cell.2016.11.021

    View details for Web of Science ID 000389470500024

    View details for PubMedID 27912066

    View details for PubMedCentralID PMC5382987

  • Distinct Hippocampal Pathways Mediate Dissociable Roles of Context in Memory Retrieval. Cell Xu, C., Krabbe, S., Gründemann, J., Botta, P., Fadok, J. P., Osakada, F., Saur, D., Grewe, B. F., Schnitzer, M. J., Callaway, E. M., Lüthi, A. 2016; 167 (4): 961-972 e16


    Memories about sensory experiences are tightly linked to the context in which they were formed. Memory contextualization is fundamental for the selection of appropriate behavioral reactions needed for survival, yet the underlying neuronal circuits are poorly understood. By combining trans-synaptic viral tracing and optogenetic manipulation, we found that the ventral hippocampus (vHC) and the amygdala, two key brain structures encoding context and emotional experiences, interact via multiple parallel pathways. A projection from the vHC to the basal amygdala mediates fear behavior elicited by a conditioned context, whereas a parallel projection from a distinct subset of vHC neurons onto midbrain-projecting neurons in the central amygdala is necessary for context-dependent retrieval of cued fear memories. Our findings demonstrate that two fundamentally distinct roles of context in fear memory retrieval are processed by distinct vHC output pathways, thereby allowing for the formation of robust contextual fear memories while preserving context-dependent behavioral flexibility.

    View details for DOI 10.1016/j.cell.2016.09.051

    View details for PubMedID 27773481

  • Changes in sarcomere lengths of the human vastus lateralis muscle with knee flexion measured using in vivo microendoscopy JOURNAL OF BIOMECHANICS Chen, X., Sanchez, G. N., Schnitzer, M. J., Delp, S. L. 2016; 49 (13): 2989-2994


    Sarcomeres are the basic contractile units of muscle, and their lengths influence muscle force-generating capacity. Despite their importance, in vivo sarcomere lengths remain unknown for many human muscles. Second harmonic generation (SHG) microendoscopy is a minimally invasive technique for imaging sarcomeres in vivo and measuring their lengths. In this study, we used SHG microendoscopy to visualize sarcomeres of the human vastus lateralis, a large knee extensor muscle important for mobility, to examine how sarcomere lengths change with knee flexion and thus affect the muscle׳s force-generating capacity. We acquired in vivo sarcomere images of several muscle fibers of the resting vastus lateralis in six healthy individuals. Mean sarcomere lengths increased (p=0.031) from 2.84±0.16μm at 50° of knee flexion to 3.17±0.13μm at 110° of knee flexion. The standard deviation of sarcomere lengths among different fibers within a muscle was 0.21±0.09μm. Our results suggest that the sarcomeres of the resting vastus lateralis at 50° of knee flexion are near optimal length. At a knee flexion angle of 110° the resting sarcomeres of vastus lateralis are longer than optimal length. These results show a smaller sarcomere length change and greater conservation of force-generating capacity with knee flexion than estimated in previous studies.

    View details for DOI 10.1016/j.jbiomech.2016.07.013

    View details for Web of Science ID 000385472300057

    View details for PubMedID 27481293

  • Genetically encoded indicators of neuronal activity. Nature neuroscience Lin, M. Z., Schnitzer, M. J. 2016; 19 (9): 1142-1153


    Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.

    View details for DOI 10.1038/nn.4359

    View details for PubMedID 27571193

  • Large-Scale Fluorescence Calcium-Imaging Methods for Studies of Long-Term Memory in Behaving Mammals COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Jercog, P., Rogerson, T., Schnitzer, M. J. 2016; 8 (5)


    During long-term memory formation, cellular and molecular processes reshape how individual neurons respond to specific patterns of synaptic input. It remains poorly understood how such changes impact information processing across networks of mammalian neurons. To observe how networks encode, store, and retrieve information, neuroscientists must track the dynamics of large ensembles of individual cells in behaving animals, over timescales commensurate with long-term memory. Fluorescence Ca(2+)-imaging techniques can monitor hundreds of neurons in behaving mice, opening exciting avenues for studies of learning and memory at the network level. Genetically encoded Ca(2+) indicators allow neurons to be targeted by genetic type or connectivity. Chronic animal preparations permit repeated imaging of neural Ca(2+) dynamics over multiple weeks. Together, these capabilities should enable unprecedented analyses of how ensemble neural codes evolve throughout memory processing and provide new insights into how memories are organized in the brain.

    View details for DOI 10.1101/cshperspect.a021824

    View details for Web of Science ID 000377084600005

    View details for PubMedID 27048190

  • High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor SCIENCE Gong, Y., Huang, C., Li, J. Z., Grewe, B. F., Zhang, Y., Eismann, S., Schnitzer, M. J. 2015; 350 (6266): 1361-1366


    Genetically encoded voltage indicators (GEVIs) are a promising technology for fluorescence readout of millisecond-scale neuronal dynamics. Previous GEVIs had insufficient signaling speed and dynamic range to resolve action potentials in live animals. We coupled fast voltage-sensing domains from a rhodopsin protein to bright fluorophores through resonance energy transfer. The resulting GEVIs are sufficiently bright and fast to report neuronal action potentials and membrane voltage dynamics in awake mice and flies, resolving fast spike trains with 0.2-millisecond timing precision at spike detection error rates orders of magnitude better than previous GEVIs. In vivo imaging revealed sensory-evoked responses, including somatic spiking, dendritic dynamics, and intracellular voltage propagation. These results empower in vivo optical studies of neuronal electrophysiology and coding and motivate further advancements in high-speed microscopy.

    View details for DOI 10.1126/science.aab0810

    View details for Web of Science ID 000366162400039

    View details for PubMedID 26586188

    View details for PubMedCentralID PMC4904846

  • In Vivo Imaging of Human Sarcomere Twitch Dynamics in Individual Motor Units NEURON Sanchez, G. N., Sinha, S., Liske, H., Chen, X., Viet Nguyen, V., Delp, S. L., Schnitzer, M. J. 2015; 88 (6): 1109-1120


    Motor units comprise a pre-synaptic motor neuron and multiple post-synaptic muscle fibers. Many movement disorders disrupt motor unit contractile dynamics and the structure of sarcomeres, skeletal muscle's contractile units. Despite the motor unit's centrality to neuromuscular physiology, no extant technology can image sarcomere twitch dynamics in live humans. We created a wearable microscope equipped with a microendoscope for minimally invasive observation of sarcomere lengths and contractile dynamics in any major skeletal muscle. By electrically stimulating twitches via the microendoscope and visualizing the sarcomere displacements, we monitored single motor unit contractions in soleus and vastus lateralis muscles of healthy individuals. Control experiments verified that these evoked twitches involved neuromuscular transmission and faithfully reported muscle force generation. In post-stroke patients with spasticity of the biceps brachii, we found involuntary microscopic contractions and sarcomere length abnormalities. The wearable microscope facilitates exploration of many basic and disease-related neuromuscular phenomena never visualized before in live humans. VIDEO ABSTRACT.

    View details for DOI 10.1016/j.neuron.2015.11.022

    View details for Web of Science ID 000368443900008

  • Entorhinal Cortical Ocean Cells Encode Specific Contexts and Drive Context-Specific Fear Memory. Neuron Kitamura, T., Sun, C., Martin, J., Kitch, L. J., Schnitzer, M. J., Tonegawa, S. 2015; 87 (6): 1317-1331

    View details for DOI 10.1016/j.neuron.2015.08.036

    View details for PubMedID 26402611

  • Impermanence of dendritic spines in live adult CA1 hippocampus. Nature Attardo, A., Fitzgerald, J. E., Schnitzer, M. J. 2015; 523 (7562): 592-596


    The mammalian hippocampus is crucial for episodic memory formation and transiently retains information for about 3-4 weeks in adult mice and longer in humans. Although neuroscientists widely believe that neural synapses are elemental sites of information storage, there has been no direct evidence that hippocampal synapses persist for time intervals commensurate with the duration of hippocampal-dependent memory. Here we tested the prediction that the lifetimes of hippocampal synapses match the longevity of hippocampal memory. By using time-lapse two-photon microendoscopy in the CA1 hippocampal area of live mice, we monitored the turnover dynamics of the pyramidal neurons' basal dendritic spines, postsynaptic structures whose turnover dynamics are thought to reflect those of excitatory synaptic connections. Strikingly, CA1 spine turnover dynamics differed sharply from those seen previously in the neocortex. Mathematical modelling revealed that the data best matched kinetic models with a single population of spines with a mean lifetime of approximately 1-2 weeks. This implies ∼100% turnover in ∼2-3 times this interval, a near full erasure of the synaptic connectivity pattern. Although N-methyl-d-aspartate (NMDA) receptor blockade stabilizes spines in the neocortex, in CA1 it transiently increased the rate of spine loss and thus lowered spine density. These results reveal that adult neocortical and hippocampal pyramidal neurons have divergent patterns of spine regulation and quantitatively support the idea that the transience of hippocampal-dependent memory directly reflects the turnover dynamics of hippocampal synapses.

    View details for DOI 10.1038/nature14467

    View details for PubMedID 26098371

  • Impermanence of dendritic spines in live adult CA1 hippocampus NATURE Attardo, A., Fitzgerald, J. E., Schnitzer, M. J. 2015; 523 (7562): 592-?
  • Distinct speed dependence of entorhinal island and ocean cells, including respective grid cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sun, C., Kitamura, T., Yamamoto, J., Martin, J., Pignatelli, M., Kitch, L. J., Schnitzer, M. J., Tonegawa, S. 2015; 112 (30): 9466-9471


    Entorhinal-hippocampal circuits in the mammalian brain are crucial for an animal's spatial and episodic experience, but the neural basis for different spatial computations remain unknown. Medial entorhinal cortex layer II contains pyramidal island and stellate ocean cells. Here, we performed cell type-specific Ca(2+) imaging in freely exploring mice using cellular markers and a miniature head-mounted fluorescence microscope. We found that both oceans and islands contain grid cells in similar proportions, but island cell activity, including activity in a proportion of grid cells, is significantly more speed modulated than ocean cell activity. We speculate that this differential property reflects island cells' and ocean cells' contribution to different downstream functions: island cells may contribute more to spatial path integration, whereas ocean cells may facilitate contextual representation in downstream circuits.

    View details for DOI 10.1073/pnas.1511668112

    View details for Web of Science ID 000358656500084

    View details for PubMedCentralID PMC4522738

  • Dexterous robotic manipulation of alert adult Drosophila for high-content experimentation. Nature methods Savall, J., Ho, E. T., Huang, C., Maxey, J. R., Schnitzer, M. J. 2015; 12 (7): 657-660


    We present a robot that enables high-content studies of alert adult Drosophila by combining operations including gentle picking; translations and rotations; characterizations of fly phenotypes and behaviors; microdissection; or release. To illustrate, we assessed fly morphology, tracked odor-evoked locomotion, sorted flies by sex, and dissected the cuticle to image neural activity. The robot's tireless capacity for precise manipulations enables a scalable platform for screening flies' complex attributes and behavioral patterns.

    View details for DOI 10.1038/nmeth.3410

    View details for PubMedID 26005812

  • Dexterous robotic manipulation of alert adult Drosophila for high-content experimentation. Nature methods Savall, J., Ho, E. T., Huang, C., Maxey, J. R., Schnitzer, M. J. 2015; 12 (7): 657-660

    View details for DOI 10.1038/nmeth.3410

    View details for PubMedID 26005812

  • The BRAIN Initiative: developing technology to catalyse neuroscience discovery PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Jorgenson, L. A., Newsome, W. T., Anderson, D. J., Bargmann, C. I., Brown, E. N., Deisseroth, K., Donoghue, J. P., Hudson, K. L., Ling, G. S., MacLeish, P. R., Marder, E., Normann, R. A., Sanes, J. R., Schnitzer, M. J., Sejnowski, T. J., Tank, D. W., Tsien, R. Y., Ugurbil, K., Wingfield, J. C. 2015; 370 (1668): 8-19


    The evolution of the field of neuroscience has been propelled by the advent of novel technological capabilities, and the pace at which these capabilities are being developed has accelerated dramatically in the past decade. Capitalizing on this momentum, the United States launched the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative to develop and apply new tools and technologies for revolutionizing our understanding of the brain. In this article, we review the scientific vision for this initiative set forth by the National Institutes of Health and discuss its implications for the future of neuroscience research. Particular emphasis is given to its potential impact on the mapping and study of neural circuits, and how this knowledge will transform our understanding of the complexity of the human brain and its diverse array of behaviours, perceptions, thoughts and emotions.

    View details for DOI 10.1098/rstb.2014.0164

    View details for Web of Science ID 000354071400002

    View details for PubMedID 25823863

    View details for PubMedCentralID PMC4387507

  • Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach NEURON Hamel, E. J., Grewe, B. F., Parker, J. G., Schnitzer, M. J. 2015; 86 (1): 140-159


    Fluorescence imaging offers expanding capabilities for recording neural dynamics in behaving mammals, including the means to monitor hundreds of cells targeted by genetic type or connectivity, track cells over weeks, densely sample neurons within local microcircuits, study cells too inactive to isolate in extracellular electrical recordings, and visualize activity in dendrites, axons, or dendritic spines. We discuss recent progress and future directions for imaging in behaving mammals from a systems engineering perspective, which seeks holistic consideration of fluorescent indicators, optical instrumentation, and computational analyses. Today, genetically encoded indicators of neural Ca(2+) dynamics are widely used, and those of trans-membrane voltage are rapidly improving. Two complementary imaging paradigms involve conventional microscopes for studying head-restrained animals and head-mounted miniature microscopes for imaging in freely behaving animals. Overall, the field has attained sufficient sophistication that increased cooperation between those designing new indicators, light sources, microscopes, and computational analyses would greatly benefit future progress.

    View details for DOI 10.1016/j.neuron.2015.03.055

    View details for Web of Science ID 000352552900018

    View details for PubMedID 25856491

  • The neural representation of taste quality at the periphery NATURE Barretto, R. P., Gillis-Smith, S., Chandrashekar, J., Yarmolinsky, D. A., Schnitzer, M. J., Ryba, N. J., Zuker, C. S. 2015; 517 (7534): 373-U511


    The mammalian taste system is responsible for sensing and responding to the five basic taste qualities: sweet, sour, bitter, salty and umami. Previously, we showed that each taste is detected by dedicated taste receptor cells (TRCs) on the tongue and palate epithelium. To understand how TRCs transmit information to higher neural centres, we examined the tuning properties of large ensembles of neurons in the first neural station of the gustatory system. Here, we generated and characterized a collection of transgenic mice expressing a genetically encoded calcium indicator in central and peripheral neurons, and used a gradient refractive index microendoscope combined with high-resolution two-photon microscopy to image taste responses from ganglion neurons buried deep at the base of the brain. Our results reveal fine selectivity in the taste preference of ganglion neurons; demonstrate a strong match between TRCs in the tongue and the principal neural afferents relaying taste information to the brain; and expose the highly specific transfer of taste information between taste cells and the central nervous system.

    View details for DOI 10.1038/nature13873

    View details for Web of Science ID 000347810300047

    View details for PubMedID 25383521

  • Visualizing mammalian brain area interactions by dual-axis two-photon calcium imaging NATURE NEUROSCIENCE Lecoq, J., Savall, J., Vucinic, D., Grewe, B. F., Kim, H., Li, T. Z., Kitch, L. J., Schnitzer, M. J. 2014; 17 (12): 1825-1829

    View details for DOI 10.1038/nn.3867

    View details for Web of Science ID 000345484000032

  • High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nature neuroscience St-Pierre, F., Marshall, J. D., Yang, Y., Gong, Y., Schnitzer, M. J., Lin, M. Z. 2014; 17 (6): 884-889


    Accurate optical reporting of electrical activity in genetically defined neuronal populations is a long-standing goal in neuroscience. We developed Accelerated Sensor of Action Potentials 1 (ASAP1), a voltage sensor design in which a circularly permuted green fluorescent protein is inserted in an extracellular loop of a voltage-sensing domain, rendering fluorescence responsive to membrane potential. ASAP1 demonstrated on and off kinetics of ∼2 ms, reliably detected single action potentials and subthreshold potential changes, and tracked trains of action potential waveforms up to 200 Hz in single trials. With a favorable combination of brightness, dynamic range and speed, ASAP1 enables continuous monitoring of membrane potential in neurons at kilohertz frame rates using standard epifluorescence microscopy.

    View details for DOI 10.1038/nn.3709

    View details for PubMedID 24755780

  • Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors NATURE COMMUNICATIONS Gong, Y., Wagner, M. J., Li, J. Z., Schnitzer, M. J. 2014; 5

    View details for DOI 10.1038/ncomms4674

    View details for Web of Science ID 000335221800005

    View details for PubMedID 24755708

  • Bidirectional plasticity of purkinje cells matches temporal features of learning. journal of neuroscience Wetmore, D. Z., Jirenhed, D., Rasmussen, A., Johansson, F., Schnitzer, M. J., Hesslow, G. 2014; 34 (5): 1731-1737


    Many forms of learning require temporally ordered stimuli. In Pavlovian eyeblink conditioning, a conditioned stimulus (CS) must precede the unconditioned stimulus (US) by at least about 100 ms for learning to occur. Conditioned responses are learned and generated by the cerebellum. Recordings from the cerebellar cortex during conditioning have revealed CS-triggered pauses in the firing of Purkinje cells that likely drive the conditioned blinks. The predominant view of the learning mechanism in conditioning is that long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses underlies the Purkinje cell pauses. This raises a serious conceptual challenge because LTD is most effectively induced at short CS-US intervals, which do not support acquisition of eyeblinks. To resolve this discrepancy, we recorded Purkinje cells during conditioning with short or long CS-US intervals. Decerebrated ferrets trained with CS-US intervals ≥150 ms reliably developed Purkinje cell pauses, but training with an interval of 50 ms unexpectedly induced increases in CS-evoked spiking. This bidirectional modulation of Purkinje cell activity offers a basis for the requirement of a minimum CS-US interval for conditioning, but we argue that it cannot be fully explained by LTD, even when previous in vitro studies of stimulus-timing-dependent LTD are taken into account.

    View details for DOI 10.1523/JNEUROSCI.2883-13.2014

    View details for PubMedID 24478355

  • Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nature communications Gong, Y., Wagner, M. J., Zhong Li, J., Schnitzer, M. J. 2014; 5: 3674-?


    Genetically encoded fluorescence voltage sensors offer the possibility of directly visualizing neural spiking dynamics in cells targeted by their genetic class or connectivity. Sensors of this class have generally suffered performance-limiting tradeoffs between modest brightness, sluggish kinetics and limited signalling dynamic range in response to action potentials. Here we describe sensors that use fluorescence resonance energy transfer (FRET) to combine the rapid kinetics and substantial voltage-dependence of rhodopsin family voltage-sensing domains with the brightness of genetically engineered protein fluorophores. These FRET-opsin sensors significantly improve upon the spike detection fidelity offered by the genetically encoded voltage sensor, Arclight, while offering faster kinetics and higher brightness. Using FRET-opsin sensors we imaged neural spiking and sub-threshold membrane voltage dynamics in cultured neurons and in pyramidal cells within neocortical tissue slices. In live mice, rates and optical waveforms of cerebellar Purkinje neurons' dendritic voltage transients matched expectations for these cells' dendritic spikes.

    View details for DOI 10.1038/ncomms4674

    View details for PubMedID 24755708

  • High-speed laser microsurgery of alert fruit flies for fluorescence imaging of neural activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sinha, S., Liang, L., Ho, E. T., Urbanek, K. E., Luo, L., Baer, T. M., Schnitzer, M. J. 2013; 110 (46): 18374-18379


    Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-µm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12- to 350-µm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca(2+) signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5-20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation.

    View details for DOI 10.1073/pnas.1216287110

    View details for Web of Science ID 000326830900029

    View details for PubMedID 24167298

    View details for PubMedCentralID PMC3832030

  • Engineering Approaches to Illuminating Brain Structure and Dynamics NEURON Deisseroth, K., Schnitzer, M. J. 2013; 80 (3): 568-577


    Historical milestones in neuroscience have come in diverse forms, ranging from the resolution of specific biological mysteries via creative experimentation to broad technological advances allowing neuroscientists to ask new kinds of questions. The continuous development of tools is driven with a special necessity by the complexity, fragility, and inaccessibility of intact nervous systems, such that inventive technique development and application drawing upon engineering and the applied sciences has long been essential to neuroscience. Here we highlight recent technological directions in neuroscience spurred by progress in optical, electrical, mechanical, chemical, and biological engineering. These research areas are poised for rapid growth and will likely be central to the practice of neuroscience well into the future.

    View details for DOI 10.1016/j.neuron.2013.10.032

    View details for Web of Science ID 000326609900004

    View details for PubMedID 24183010

  • Sarcomere lengths in human extensor carpi radialis brevis measured by microendoscopy MUSCLE & NERVE Cromie, M. J., Sanchez, G. N., Schnitzer, M. J., Delp, S. L. 2013; 48 (2): 286-292


    Second-harmonic generation microendoscopy is a minimally invasive technique to image sarcomeres and measure their lengths in humans, but motion artifact and low signal have limited the use of this novel technique.We discovered that an excitation wavelength of 960 nm maximized image signal; this enabled an image acquisition rate of 3 frames/s, which decreased motion artifact. We then used microendoscopy to measure sarcomere lengths in the human extensor carpi radialis brevis with the wrist at 45° extension and 45° flexion in 7 subjects. We also measured the variability in sarcomere lengths within single fibers.Average sarcomere lengths in 45° extension were 2.93±0.29 μm (±SD) and increased to 3.58±0.19 μm in 45° flexion. Within single fibers the standard deviation of sarcomere lengths in series was 0.20 μm.Microendoscopy can be used to measure sarcomere lengths at different body postures. Lengths of sarcomeres in series within a fiber vary substantially. Muscle Nerve, 48: 286-292, 2013.

    View details for DOI 10.1002/mus.23760

    View details for Web of Science ID 000322158500019

    View details for PubMedID 23813625

  • GABAergic Lateral Interactions Tune the Early Stages of Visual Processing in Drosophila NEURON Freifeld, L., Clark, D. A., Schnitzer, M. J., Horowitz, M. A., Clandinin, T. R. 2013; 78 (6): 1075-1089


    Early stages of visual processing must capture complex, dynamic inputs. While peripheral neurons often implement efficient encoding by exploiting natural stimulus statistics, downstream neurons are specialized to extract behaviorally relevant features. How do these specializations arise? We use two-photon imaging in Drosophila to characterize a first-order interneuron, L2, that provides input to a pathway specialized for detecting moving dark edges. GABAergic interactions, mediated in part presynaptically, create an antagonistic and anisotropic center-surround receptive field. This receptive field is spatiotemporally coupled, applying differential temporal processing to large and small dark objects, achieving significant specialization. GABAergic circuits also mediate OFF responses and balance these with responses to ON stimuli. Remarkably, the functional properties of L2 are strikingly similar to those of bipolar cells, yet emerge through different molecular and circuit mechanisms. Thus, evolution appears to have converged on a common strategy for processing visual information at the first synapse.

    View details for DOI 10.1016/j.neuron.2013.04.024

    View details for Web of Science ID 000321026900013

    View details for PubMedID 23791198

  • Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators PLOS ONE Gong, Y., Li, J. Z., Schnitzer, M. J. 2013; 8 (6)
  • Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals ACS NANO Marshall, J. D., Schnitzer, M. J. 2013; 7 (5): 4601-4609


    Biophysicists have long sought optical methods capable of reporting the electrophysiological dynamics of large-scale neural networks with millisecond-scale temporal resolution. Existing fluorescent sensors of cell membrane voltage can report action potentials in individual cultured neurons, but limitations in brightness and dynamic range of both synthetic organic and genetically encoded voltage sensors have prevented concurrent monitoring of spiking activity across large populations of individual neurons. Here we propose a novel, inorganic class of fluorescent voltage sensors: semiconductor nanoparticles, such as ultrabright quantum dots (qdots). Our calculations revealed that transmembrane electric fields characteristic of neuronal spiking (~10 mV/nm) modulate a qdot's electronic structure and can induce ~5% changes in its fluorescence intensity and ~1 nm shifts in its emission wavelength, depending on the qdot's size, composition, and dielectric environment. Moreover, tailored qdot sensors composed of two different materials can exhibit substantial (~30%) changes in fluorescence intensity during neuronal spiking. Using signal detection theory, we show that conventional qdots should be capable of reporting voltage dynamics with millisecond precision across several tens or more individual neurons over a range of optical and neurophysiological conditions. These results unveil promising avenues for imaging spiking dynamics in neural networks and merit in-depth experimental investigation.

    View details for DOI 10.1021/nn401410k

    View details for Web of Science ID 000319856300102

    View details for PubMedID 23614672

  • Long-term dynamics of CA1 hippocampal place codes NATURE NEUROSCIENCE Ziv, Y., Burns, L. D., Cocker, E. D., Hamel, E. O., Ghosh, K. K., Kitch, L. J., El Gamal, A., Schnitzer, M. J. 2013; 16 (3): 264-266


    Using Ca(2+) imaging in freely behaving mice that repeatedly explored a familiar environment, we tracked thousands of CA1 pyramidal cells' place fields over weeks. Place coding was dynamic, as each day the ensemble representation of this environment involved a unique subset of cells. However, cells in the ∼15-25% overlap between any two of these subsets retained the same place fields, which sufficed to preserve an accurate spatial representation across weeks.

    View details for DOI 10.1038/nn.3329

    View details for Web of Science ID 000315474800006

    View details for PubMedID 23396101

  • Nanotools for Neuroscience and Brain Activity Mapping ACS NANO Alivisatos, A. P., Andrews, A. M., Boyden, E. S., Chun, M., Church, G. M., Deisseroth, K., Donoghue, J. P., Fraser, S. E., Lippincott-Schwartz, J., Looger, L. L., Masmanidis, S., McEuen, P. L., Nurmikko, A. V., Park, H., Peterka, D. S., Reid, C., Roukes, M. L., Scherer, A., Schnitzer, M., Sejnowski, T. J., Shepard, K. L., Tsao, D., Turrigiano, G., Weiss, P. S., Xu, C., Yuste, R., Zhuang, X. 2013; 7 (3): 1850-1866


    Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.

    View details for DOI 10.1021/nn4012847

    View details for Web of Science ID 000316846700005

    View details for PubMedID 23514423

  • Improving FRET Dynamic Range with Bright Green and Red Fluorescent Proteins 57th Annual Meeting of the Biophysical-Society Lam, A. J., St-Pierre, F., Gong, Y., Marshall, J. D., McKeown, M. R., Schnitzer, M. J., Tsien, R. Y., Lin, M. Z. CELL PRESS. 2013: 683A–683A
  • Photon Shot Noise Limits on Optical Detection of Neuronal Spikes and Estimation of Spike Timing BIOPHYSICAL JOURNAL Wilt, B. A., Fitzgerald, J. E., Schnitzer, M. J. 2013; 104 (1): 51-62


    Optical approaches for tracking neural dynamics are of widespread interest, but a theoretical framework quantifying the physical limits of these techniques has been lacking. We formulate such a framework by using signal detection and estimation theory to obtain physical bounds on the detection of neural spikes and the estimation of their occurrence times as set by photon counting statistics (shot noise). These bounds are succinctly expressed via a discriminability index that depends on the kinetics of the optical indicator and the relative fluxes of signal and background photons. This approach facilitates quantitative evaluations of different indicators, detector technologies, and data analyses. Our treatment also provides optimal filtering techniques for optical detection of spikes. We compare various types of Ca(2+) indicators and show that background photons are a chief impediment to voltage sensing. Thus, voltage indicators that change color in response to membrane depolarization may offer a key advantage over those that change intensity. We also examine fluorescence resonance energy transfer indicators and identify the regimes in which the widely used ratiometric analysis of signals is substantially suboptimal. Overall, by showing how different optical factors interact to affect signal quality, our treatment offers a valuable guide to experimental design and provides measures of confidence to assess optically extracted traces of neural activity.

    View details for DOI 10.1016/j.bpj.2012.07.058

    View details for Web of Science ID 000313541200008

    View details for PubMedID 23332058

    View details for PubMedCentralID PMC3540268

  • Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators. PloS one Gong, Y., Li, J. Z., Schnitzer, M. J. 2013; 8 (6): e66959


    A longstanding goal in neuroscience has been to develop techniques for imaging the voltage dynamics of genetically defined subsets of neurons. Optical sensors of transmembrane voltage would enhance studies of neural activity in contexts ranging from individual neurons cultured in vitro to neuronal populations in awake-behaving animals. Recent progress has identified Archaerhodopsin (Arch) based sensors as a promising, genetically encoded class of fluorescent voltage indicators that can report single action potentials. Wild-type Arch exhibits sub-millisecond fluorescence responses to trans-membrane voltage, but its light-activated proton pump also responds to the imaging illumination. An Arch mutant (Arch-D95N) exhibits no photocurrent, but has a slower, ~40 ms response to voltage transients. Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane. We also describe Arch mutant sensors (Arch-EEN and -EEQ) that exhibit faster kinetics and greater fluorescence dynamic range than Arch-D95N, and no photocurrent at the illumination intensities normally used for imaging. We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials. This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.

    View details for DOI 10.1371/journal.pone.0066959

    View details for PubMedID 23840563

    View details for PubMedCentralID PMC3686764

  • Towards a Photonic Crystal Mode-Locked Laser Conference on Novel In-Plane Semiconductor Lasers XII Leedle, K., Janjua, A., Paik, S., Schnitzer, M. J., Harris, J. S. SPIE-INT SOC OPTICAL ENGINEERING. 2013

    View details for DOI 10.1117/12.2005418

    View details for Web of Science ID 000322737200016

  • Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation NATURE METHODS Prakash, R., Yizhar, O., Grewe, B., Ramakrishnan, C., Wang, N., Goshen, I., Packer, A. M., Peterka, D. S., Yuste, R., Schnitzer, M. J., Deisseroth, K. 2012; 9 (12): 1171-U132


    Optogenetics with microbial opsin genes has enabled high-speed control of genetically specified cell populations in intact tissue. However, it remains a challenge to independently control subsets of cells within the genetically targeted population. Although spatially precise excitation of target molecules can be achieved using two-photon laser-scanning microscopy (TPLSM) hardware, the integration of two-photon excitation with optogenetics has thus far required specialized equipment or scanning and has not yet been widely adopted. Here we take a complementary approach, developing opsins with custom kinetic, expression and spectral properties uniquely suited to scan times typical of the raster approach that is ubiquitous in TPLSMlaboratories. We use a range of culture, slice and mammalian in vivo preparations to demonstrate the versatility of this toolbox, and we quantitatively map parameter space for fast excitation, inhibition and bistable control. Together these advances may help enable broad adoption of integrated optogenetic and TPLSMtechnologies across experimental fields and systems.

    View details for DOI 10.1038/NMETH.2215

    View details for Web of Science ID 000312093500018

    View details for PubMedID 23169303

  • Unified Resolution Bounds for Conventional and Stochastic Localization Fluorescence Microscopy PHYSICAL REVIEW LETTERS Mukamel, E. A., Schnitzer, M. J. 2012; 109 (16)


    Superresolution microscopy enables imaging in the optical far field with ~20 nm-scale resolution. However, classical concepts of resolution using point-spread and modulation-transfer functions fail to describe the physical limits of superresolution techniques based on stochastic localization of single emitters. Prior treatments of stochastic localization microscopy have defined how accurately a single emitter's position can be determined, but these bounds are restricted to sparse emitters, do not describe conventional microscopy, and fail to provide unified concepts of resolution for all optical methods. Here we introduce a measure of resolution, the information transfer function (ITF), that gives physical limits for conventional and stochastic localization techniques. The ITF bounds the accuracy of image determination as a function of spatial frequency and for conventional microscopy is proportional to the square of the modulation-transfer function. We use the ITF to describe how emitter density and photon counts affect imaging performance across the continuum from conventional to superresolution microscopy, without assuming emitters are sparse. This unified physical description of resolution facilitates experimental choices and designs of image reconstruction algorithms.

    View details for DOI 10.1103/PhysRevLett.109.168102

    View details for Web of Science ID 000309905400027

    View details for PubMedID 23215134

  • Improving FRET dynamic range with bright green and red fluorescent proteins NATURE METHODS Lam, A. J., St-Pierre, F., Gong, Y., Marshall, J. D., Cranfill, P. J., Baird, M. A., McKeown, M. R., Wiedenmann, J., Davidson, M. W., Schnitzer, M. J., Tsien, R. Y., Lin, M. Z. 2012; 9 (10): 1005-?


    A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

    View details for DOI 10.1038/NMETH.2171

    View details for Web of Science ID 000309519300023

    View details for PubMedID 22961245

    View details for PubMedCentralID PMC3461113

  • In vivo optical microendoscopy for imaging cells lying deep within live tissue. Cold Spring Harbor protocols Barretto, R. P., Schnitzer, M. J. 2012; 2012 (10): 1029-1034


    Although in vivo microscopy has been pivotal in enabling studies of neuronal structure and function in the intact mammalian brain, conventional intravital microscopy has generally been limited to superficial brain areas such as the olfactory bulb, the neocortex, or the cerebellar cortex. For imaging cells in deeper areas, this article discusses in vivo optical microendoscopy using gradient refractive index (GRIN) microlenses that can be inserted into tissue. Our general methodology is broadly applicable to many deep brain regions and areas of the body. Microendoscopes are available in a wide variety of optical designs, allowing imaging across a range of spatial scales and with spatial resolution that can now closely approach that offered by standard water-immersion microscope objectives. The incorporation of microendoscope probes into portable miniaturized microscopes allows imaging in freely behaving animals. When combined with the broad sets of available fluorescent markers, animal preparations, and genetically modified mice, microendoscopic methods enable sophisticated experimental designs for probing how cellular characteristics may underlie or reflect animal behavior and life experience, in healthy animals and animal models of disease.

    View details for DOI 10.1101/pdb.top071464

    View details for PubMedID 23028071

  • In vivo microendoscopy of the hippocampus. Cold Spring Harbor protocols Barretto, R. P., Schnitzer, M. J. 2012; 2012 (10): 1092-1099


    Conventional intravital microscopy has generally been limited to superficial brain areas such as the olfactory bulb, the neocortex, or the cerebellar cortex. In vivo optical microendoscopy uses gradient refractive index (GRIN) microlenses that can be inserted into tissue to image cells in deeper areas. This protocol describes in vivo microendoscopy of the mouse hippocampus. The general methodology can be applied to many deep brain regions and other areas of the body.

    View details for DOI 10.1101/pdb.prot071472

    View details for PubMedID 23028072

  • Estimation Theoretic Measure of Resolution for Stochastic Localization Microscopy PHYSICAL REVIEW LETTERS Fitzgerald, J. E., Lu, J., Schnitzer, M. J. 2012; 109 (4)


    One approach to super-resolution fluorescence microscopy, termed stochastic localization microscopy, relies on the nanometer scale spatial localization of individual fluorescent emitters that stochastically label specific features of the specimen. The precision of emitter localization is an important determinant of the resulting image resolution but is insufficient to specify how well the derived images capture the structure of the specimen. We address this deficiency by considering the inference of specimen structure based on the estimated emitter locations. By using estimation theory, we develop a measure of spatial resolution that jointly depends on the density of the emitter labels, the precision of emitter localization, and prior information regarding the spatial frequency content of the labeled object. The Nyquist criterion does not set the scaling of this measure with emitter number. Given prior information and a fixed emitter labeling density, our resolution measure asymptotes to a finite value as the precision of emitter localization improves. By considering the present experimental capabilities, this asymptotic behavior implies that further resolution improvements require increases in labeling density above typical current values. Our treatment also yields algorithms to enhance reliable image features. Overall, our formalism facilitates the rigorous statistical interpretation of the data produced by stochastic localization imaging techniques.

    View details for DOI 10.1103/PhysRevLett.109.048102

    View details for Web of Science ID 000306690700012

    View details for PubMedID 23006110

    View details for PubMedCentralID PMC3478896

  • Miniaturized integration of a fluorescence microscope NATURE METHODS Ghosh, K. K., Burns, L. D., Cocker, E. D., Nimmerjahn, A., Ziv, Y., El Gamal, A., Schnitzer, M. J. 2011; 8 (10): 871-U147


    The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.

    View details for DOI 10.1038/NMETH.1694

    View details for Web of Science ID 000295358000024

    View details for PubMedID 21909102

  • Symmetries in stimulus statistics shape the form of visual motion estimators PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fitzgerald, J. E., Katsov, A. Y., Clandinin, T. R., Schnitzer, M. J. 2011; 108 (31): 12909-12914


    The estimation of visual motion has long been studied as a paradigmatic neural computation, and multiple models have been advanced to explain behavioral and neural responses to motion signals. A broad class of models, originating with the Reichardt correlator model, proposes that animals estimate motion by computing a temporal cross-correlation of light intensities from two neighboring points in visual space. These models provide a good description of experimental data in specific contexts but cannot explain motion percepts in stimuli lacking pairwise correlations. Here, we develop a theoretical formalism that can accommodate diverse stimuli and behavioral goals. To achieve this, we treat motion estimation as a problem of Bayesian inference. Pairwise models emerge as one component of the generalized strategy for motion estimation. However, correlation functions beyond second order enable more accurate motion estimation. Prior expectations that are asymmetric with respect to bright and dark contrast use correlations of both even and odd orders, and we show that psychophysical experiments using visual stimuli with symmetric probability distributions for contrast cannot reveal whether the subject uses odd-order correlators for motion estimation. This result highlights a gap in previous experiments, which have largely relied on symmetric contrast distributions. Our theoretical treatment provides a natural interpretation of many visual motion percepts, indicates that motion estimation should be revisited using a broader class of stimuli, demonstrates how correlation-based motion estimation is related to stimulus statistics, and provides multiple experimentally testable predictions.

    View details for DOI 10.1073/pnas.1015680108

    View details for Web of Science ID 000293385700073

    View details for PubMedID 21768376

    View details for PubMedCentralID PMC3150910

  • An infrared fluorescent protein for deeper imaging NATURE BIOTECHNOLOGY Lecoq, J., Schnitzer, M. J. 2011; 29 (8): 715-716

    View details for DOI 10.1038/nbt.1941

    View details for Web of Science ID 000293696500021

    View details for PubMedID 21822247

  • Defining the Computational Structure of the Motion Detector in Drosophila NEURON Clark, D. A., Bursztyn, L., Horowitz, M. A., Schnitzer, M. J., Clandinin, T. R. 2011; 70 (6): 1165-1177


    Many animals rely on visual motion detection for survival. Motion information is extracted from spatiotemporal intensity patterns on the retina, a paradigmatic neural computation. A phenomenological model, the Hassenstein-Reichardt correlator (HRC), relates visual inputs to neural activity and behavioral responses to motion, but the circuits that implement this computation remain unknown. By using cell-type specific genetic silencing, minimal motion stimuli, and in vivo calcium imaging, we examine two critical HRC inputs. These two pathways respond preferentially to light and dark moving edges. We demonstrate that these pathways perform overlapping but complementary subsets of the computations underlying the HRC. A numerical model implementing differential weighting of these operations displays the observed edge preferences. Intriguingly, these pathways are distinguished by their sensitivities to a stimulus correlation that corresponds to an illusory percept, "reverse phi," that affects many species. Thus, this computational architecture may be widely used to achieve edge selectivity in motion detection.

    View details for DOI 10.1016/j.neuron.2011.05.023

    View details for Web of Science ID 000292410700014

    View details for PubMedID 21689602

  • Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy NATURE MEDICINE Barretto, R. P., Ko, T. H., Jung, J. C., Wang, T. J., Capps, G., Waters, A. C., Ziv, Y., Attardo, A., Recht, L., Schnitzer, M. J. 2011; 17 (2): 223-U120


    The combination of intravital microscopy and animal models of disease has propelled studies of disease mechanisms and treatments. However, many disorders afflict tissues inaccessible to light microscopy in live subjects. Here we introduce cellular-level time-lapse imaging deep within the live mammalian brain by one- and two-photon fluorescence microendoscopy over multiple weeks. Bilateral imaging sites allowed longitudinal comparisons within individual subjects, including of normal and diseased tissues. Using this approach, we tracked CA1 hippocampal pyramidal neuron dendrites in adult mice, revealing these dendrites' extreme stability and rare examples of their structural alterations. To illustrate disease studies, we tracked deep lying gliomas by observing tumor growth, visualizing three-dimensional vasculature structure and determining microcirculatory speeds. Average erythrocyte speeds in gliomas declined markedly as the disease advanced, notwithstanding significant increases in capillary diameters. Time-lapse microendoscopy will be applicable to studies of numerous disorders, including neurovascular, neurological, cancerous and trauma-induced conditions.

    View details for DOI 10.1038/nm.2292

    View details for Web of Science ID 000286969900032

    View details for PubMedID 21240263

  • Journal club. A neuroscientist learns about algorithms for motor learning. Nature Schnitzer, M. J. 2010; 463 (7279): 273-?

    View details for DOI 10.1038/463273e

    View details for PubMedID 20090712

  • Automated Analysis of Cellular Signals from Large-Scale Calcium Imaging Data NEURON Mukamel, E. A., Nimmerjahn, A., Schnitzer, M. J. 2009; 63 (6): 747-760


    Recent advances in fluorescence imaging permit studies of Ca(2+) dynamics in large numbers of cells, in anesthetized and awake behaving animals. However, unlike for electrophysiological signals, standardized algorithms for assigning optically recorded signals to individual cells have not yet emerged. Here, we describe an automated sorting procedure that combines independent component analysis and image segmentation for extracting cells' locations and their dynamics with minimal human supervision. In validation studies using simulated data, automated sorting significantly improved estimation of cellular signals compared to conventional analysis based on image regions of interest. We used automated procedures to analyze data recorded by two-photon Ca(2+) imaging in the cerebellar vermis of awake behaving mice. Our analysis yielded simultaneous Ca(2+) activity traces for up to >100 Purkinje cells and Bergmann glia from single recordings. Using this approach, we found microzones of Purkinje cells that were stable across behavioral states and in which synchronous Ca(2+) spiking rose significantly during locomotion.

    View details for DOI 10.1016/j.neuron.2009.08.009

    View details for Web of Science ID 000270569700009

    View details for PubMedID 19778505

  • In vivo brain imaging using a portable 2.9 g two-photon microscope based on a microelectromechanical systems scanning mirror OPTICS LETTERS Piyawattanametha, W., Cocker, E. D., Burns, L. D., Barretto, R. P., Jung, J. C., Ra, H., Solgaard, O., Schnitzer, M. J. 2009; 34 (15): 2309-2311


    We present a two-photon microscope that is approximately 2.9 g in mass and 2.0 x 1.9 x 1.1 cm(3) in size and based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic microprism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this microscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice.

    View details for Web of Science ID 000269405900022

    View details for PubMedID 19649080

  • In vivo fluorescence imaging with high-resolution microlenses NATURE METHODS Barretto, R. P., Messerschmidt, B., Schnitzer, M. J. 2009; 6 (7): 511-U61


    Micro-optics are increasingly used for minimally invasive in vivo imaging, in miniaturized microscopes and in lab-on-a-chip devices. Owing to optical aberrations and lower numerical apertures, a main class of microlens, gradient refractive index lenses, has not achieved resolution comparable to conventional microscopy. Here we describe high-resolution microlenses, and illustrate two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice.

    View details for DOI 10.1038/nmeth.1339

    View details for Web of Science ID 000267442900019

    View details for PubMedID 19525959

  • Motor Behavior Activates Bergmann Glial Networks NEURON Nimmerjahn, A., Mukamel, E. A., Schnitzer, M. J. 2009; 62 (3): 400-412


    Although it is firmly established that neuronal activity is a prime determinant of animal behavior, relationships between astrocytic excitation and animal behavior have remained opaque. Cerebellar Bergmann glia are radial astrocytes that are implicated in motor behavior and exhibit Ca(2+) excitation. However, Ca(2+) excitation in these cells has not previously been studied in behaving animals. Using two-photon microscopy we found that Bergmann glia exhibit three forms of Ca(2+) excitation in awake, behaving mice. Two of these are ongoing within the cerebellar vermis. During locomotor performance concerted Ca(2+) excitation arises in networks of at least hundreds of Bergmann glia extending across several hundred microns or more. Concerted Ca(2+) excitation was abolished by anesthesia or blockade of either neural activity or glutamatergic transmission. Thus, large networks of Bergmann glia can be activated by specific animal behaviors and undergo excitation of sufficient magnitude to potentially initiate macroscopic changes in brain dynamics or blood flow.

    View details for DOI 10.1016/j.neuron.2009.03.019

    View details for Web of Science ID 000266146100011

    View details for PubMedID 19447095

  • Advances in Light Microscopy for Neuroscience ANNUAL REVIEW OF NEUROSCIENCE Wilt, B. A., Burns, L. D., Ho, E. T., Ghosh, K. K., Mukamel, E. A., Schnitzer, M. J. 2009; 32: 435-506


    Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists.

    View details for DOI 10.1146/annurev.neuro.051508.135540

    View details for Web of Science ID 000268504100018

    View details for PubMedID 19555292

  • High-speed, miniaturized fluorescence microscopy in freely moving mice NATURE METHODS Flusberg, B. A., Nimmerjahn, A., Cocker, E. D., Mukamel, E. A., Barretto, R. P., Ko, T. H., Burns, L. D., Jung, J. C., Schnitzer, M. J. 2008; 5 (11): 935-938


    A central goal in biomedicine is to explain organismic behavior in terms of causal cellular processes. However, concurrent observation of mammalian behavior and underlying cellular dynamics has been a longstanding challenge. We describe a miniaturized (1.1 g mass) epifluorescence microscope for cellular-level brain imaging in freely moving mice, and its application to imaging microcirculation and neuronal Ca(2+) dynamics.

    View details for DOI 10.1038/nmeth.1256

    View details for Web of Science ID 000260532500010

    View details for PubMedID 18836457

  • Lock-and-key mechanisms of cerebellar memory recall based on rebound currents JOURNAL OF NEUROPHYSIOLOGY Wetmore, D. Z., Mukamel, E. A., Schnitzer, M. J. 2008; 100 (4): 2328-2347


    A basic question for theories of learning and memory is whether neuronal plasticity suffices to guide proper memory recall. Alternatively, information processing that is additional to readout of stored memories might occur during recall. We formulate a "lock-and-key" hypothesis regarding cerebellum-dependent motor memory in which successful learning shapes neural activity to match a temporal filter that prevents expression of stored but inappropriate motor responses. Thus, neuronal plasticity by itself is necessary but not sufficient to modify motor behavior. We explored this idea through computational studies of two cerebellar behaviors and examined whether deep cerebellar and vestibular nuclei neurons can filter signals from Purkinje cells that would otherwise drive inappropriate motor responses. In eyeblink conditioning, reflex acquisition requires the conditioned stimulus (CS) to precede the unconditioned stimulus (US) by >100 ms. In our biophysical models of cerebellar nuclei neurons this requirement arises through the phenomenon of postinhibitory rebound depolarization and matches longstanding behavioral data on conditioned reflex timing and reliability. Although CS-US intervals<100 ms may induce Purkinje cell plasticity, cerebellar nuclei neurons drive conditioned responses only if the CS-US training interval was >100 ms. This bound reflects the minimum time for deinactivation of rebound currents such as T-type Ca2+. In vestibulo-ocular reflex adaptation, hyperpolarization-activated currents in vestibular nuclei neurons may underlie analogous dependence of adaptation magnitude on the timing of visual and vestibular stimuli. Thus, the proposed lock-and-key mechanisms link channel kinetics to recall performance and yield specific predictions of how perturbations to rebound depolarization affect motor expression.

    View details for DOI 10.1152/jn.00344.2007

    View details for Web of Science ID 000259967000055

    View details for PubMedID 17671105

  • Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans NATURE Llewellyn, M. E., Barretto, R. P., Delp, S. L., Schnitzer, M. J. 2008; 454 (7205): 784-788


    Sarcomeres are the basic contractile units of striated muscle. Our knowledge about sarcomere dynamics has primarily come from in vitro studies of muscle fibres and analysis of optical diffraction patterns obtained from living muscles. Both approaches involve highly invasive procedures and neither allows examination of individual sarcomeres in live subjects. Here we report direct visualization of individual sarcomeres and their dynamical length variations using minimally invasive optical microendoscopy to observe second-harmonic frequencies of light generated in the muscle fibres of live mice and humans. Using microendoscopes as small as 350 microm in diameter, we imaged individual sarcomeres in both passive and activated muscle. Our measurements permit in vivo characterization of sarcomere length changes that occur with alterations in body posture and visualization of local variations in sarcomere length not apparent in aggregate length determinations. High-speed data acquisition enabled observation of sarcomere contractile dynamics with millisecond-scale resolution. These experiments point the way to in vivo imaging studies demonstrating how sarcomere performance varies with physical conditioning and physiological state, as well as imaging diagnostics revealing how neuromuscular diseases affect contractile dynamics.

    View details for DOI 10.1038/nature07104

    View details for Web of Science ID 000258228000049

    View details for PubMedID 18600262

  • A portable two-photon fluorescence microendoscope based on a two-dimensional scanning mirror IEEE/LEOS International Conference on Optical MEMS and Nanophotonics Piyawattanametha, W., Cocker, E. D., Barretto, R. P., Jung, J. C., Flusberg, B. A., Ra, H., Solgaard, O., Schnitzer, M. J. IEEE. 2007: 6–7
  • Long-term cellular level imaging of deep brain areas using one- and two-photon fluorescence microendoscopy 51st Annual Meeting of the Biophysical-Society Ko, T. H., Jung, J. C., Barretto, R. P., Wang, T. J., Capps, G., Recht, L., Schnitzer, M. J. CELL PRESS. 2007: 155A–155A
  • Next-generation optical technologies for illuminating genetically targeted brain circuits JOURNAL OF NEUROSCIENCE Deisseroth, K., Feng, G., Majewska, A. K., Miesenbock, G., Ting, A., Schnitzer, M. J. 2006; 26 (41): 10380-10386


    Emerging technologies from optics, genetics, and bioengineering are being combined for studies of intact neural circuits. The rapid progression of such interdisciplinary "optogenetic" approaches has expanded capabilities for optical imaging and genetic targeting of specific cell types. Here we explore key recent advances that unite optical and genetic approaches, focusing on promising techniques that either allow novel studies of neural dynamics and behavior or provide fresh perspectives on classic model systems.

    View details for DOI 10.1523/JNEUROSCI.3863-06.2006

    View details for Web of Science ID 000241192800010

    View details for PubMedID 17035522

  • Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two-dimensional scanning mirror OPTICS LETTERS Piyawattanametha, W., Barretto, R. P., Ko, T. H., Flusberg, B. A., Cocker, E. D., Ra, H., Lee, D., Solgaard, O., Schnitzer, M. J. 2006; 31 (13): 2018-2020


    Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.

    View details for Web of Science ID 000238494600026

    View details for PubMedID 16770418

  • In vivo Imaging of mammalian cochlear blood flow using fluorescence microendoscopy Annual Meeting of the American-Neurotology-Society Monfared, A., Blevins, N. H., Cheung, E. L., Jung, J. C., Popelka, G., Schnitzer, M. J. LIPPINCOTT WILLIAMS & WILKINS. 2006: 144–52


    We sought to develop techniques for visualizing cochlear blood flow in live mammalian subjects using fluorescence microendoscopy.Inner ear microcirculation appears to be intimately involved in cochlear function. Blood velocity measurements suggest that intense sounds can alter cochlear blood flow. Disruption of cochlear blood flow may be a significant cause of hearing impairment, including sudden sensorineural hearing loss. However, inability to image cochlear blood flow in a nondestructive manner has limited investigation of the role of inner ear microcirculation in hearing function. Present techniques for imaging cochlear microcirculation using intravital light microscopy involve extensive perturbations to cochlear structure, precluding application in human patients. The few previous endoscopy studies of the cochlea have suffered from optical resolution insufficient for visualizing cochlear microvasculature. Fluorescence microendoscopy is an emerging minimally invasive imaging modality that provides micron-scale resolution in tissues inaccessible to light microscopy. In this article, we describe the use of fluorescence microendoscopy in live guinea pigs to image capillary blood flow and movements of individual red blood cells within the basal turn of the cochlea.We anesthetized eight adult guinea pigs and accessed the inner ear through the mastoid bulla. After intravenous injection of fluorescein dye, we made a limited cochleostomy and introduced a compound doublet gradient refractive index endoscope probe 1 mm in diameter into the inner ear. We then imaged cochlear blood flow within individual vessels in an epifluorescence configuration using one-photon fluorescence microendoscopy.We observed single red blood cells passing through individual capillaries in several cochlear structures, including the round window membrane, spiral ligament, osseous spiral lamina, and basilar membrane. Blood flow velocities within inner ear capillaries varied widely, with observed speeds reaching up to approximately 500 microm/s.Fluorescence microendoscopy permits visualization of cochlear microcirculation with micron-scale optical resolution and determination of blood flow velocities through analysis of video sequences.

    View details for Web of Science ID 000235346400003

    View details for PubMedID 16436982

  • Fiber-optic fluorescence imaging NATURE METHODS Flusberg, B. A., Cocker, E. D., Piyawattanametha, W., Jung, J. C., Cheung, E. L., Schnitzer, M. J. 2005; 2 (12): 941-950


    Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components.

    View details for DOI 10.1038/NMETH820

    View details for Web of Science ID 000233767700016

    View details for PubMedID 16299479

  • Statistical kinetics of macromolecular dynamics BIOPHYSICAL JOURNAL Shaevitz, J. W., Block, S. M., Schnitzer, M. J. 2005; 89 (4): 2277-2285


    Fluctuations in biochemical processes can provide insights into the underlying kinetics beyond what can be gleaned from studies of average rates alone. Historically, analysis of fluctuating transmembrane currents supplied information about ion channel conductance states and lifetimes before single-channel recording techniques emerged. More recently, fluctuation analysis has helped to define mechanochemical pathways and coupling ratios for the motor protein kinesin as well as to probe the contributions of static and dynamic disorder to the kinetics of single enzymes. As growing numbers of assays are developed for enzymatic or folding behaviors of single macromolecules, the range of applications for fluctuation analysis increases. To evaluate specific biochemical models against experimental data, one needs to predict analytically the distribution of times required for completion of each reaction pathway. Unfortunately, using traditional methods, such calculations can be challenging for pathways of even modest complexity. Here, we derive an exact expression for the distribution of completion times for an arbitrary pathway with a finite number of states, using a recursive method to solve algebraically for the appropriate moment-generating function. To facilitate comparisons with experiments on processive motor proteins, we develop a theoretical formalism for the randomness parameter, a dimensionless measure of the variance in motor output. We derive the randomness for motors that take steps of variable sizes or that move on heterogeneous substrates, and then discuss possible applications to enzymes such as RNA polymerase, which transcribes varying DNA sequences, and to myosin V and cytoplasmic dynein, which may advance by variable increments.

    View details for DOI 10.1529/biophysj.105.064295

    View details for Web of Science ID 000232147600011

    View details for PubMedID 16040752

  • In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope OPTICS LETTERS Flusberg, B. A., Lung, J. C., Cocker, E. D., Anderson, E. P., Schnitzer, M. J. 2005; 30 (17): 2272-2274


    We introduce a compact two-photon fluorescence microendoscope based on a compound gradient refractive index endoscope probe, a DC micromotor for remote adjustment of the image plane, and a flexible photonic bandgap fiber for near distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9 g and provides micrometer-scale resolution. We used portable two-photon microendoscopy to visualize hippocampal blood vessels in the brains of live mice.

    View details for Web of Science ID 000231436900028

    View details for PubMedID 16190441

  • Retinal coding of visual scenes - Repetitive and redundant too? NEURON Mukamel, E. A., Schnitzer, M. J. 2005; 46 (3): 357-359


    Visual information reaches the brain by way of a fine cable, the optic nerve. The million or so axons in the optic nerve represent an information bottleneck in the visual pathway-where the fewest number of neurons convey the visual scene. It has long been thought that to make the most of the optic nerve's limited capacity the retina may encode visual information in an optimally efficient manner. In this issue of Neuron, Puchalla et al. report a test of this hypothesis using multielectrode recordings from retinal ganglion cells stimulated with movies of natural scenes. The authors find substantial redundancy in the retinal code and estimate that there is an approximately 10-fold overrepresentation of visual information.

    View details for DOI 10.1016/j.neuron.2005.04.018

    View details for Web of Science ID 000229011400002

    View details for PubMedID 15882630

  • Fiber optic two-photon fluorescence microendoscopy: Towards brain imaging in freely moving mice Conference on Lasers and Electro-Optics (CLEO) Flusberg, B. A., Jung, J. C., Cocker, E. D., Anderson, E. P., Schnitzer, M. J. OPTICAL SOC AMERICA. 2005: 2233–2235
  • In vivo mammalian brain Imaging using one- and two-photon fluorescence microendoscopy JOURNAL OF NEUROPHYSIOLOGY Jung, J. C., Mehta, A. D., Aksay, E., Stepnoski, R., Schnitzer, M. J. 2004; 92 (5): 3121-3133


    One of the major limitations in the current set of techniques available to neuroscientists is a dearth of methods for imaging individual cells deep within the brains of live animals. To overcome this limitation, we developed two forms of minimally invasive fluorescence microendoscopy and tested their abilities to image cells in vivo. Both one- and two-photon fluorescence microendoscopy are based on compound gradient refractive index (GRIN) lenses that are 350-1,000 microm in diameter and provide micron-scale resolution. One-photon microendoscopy allows full-frame images to be viewed by eye or with a camera, and is well suited to fast frame-rate imaging. Two-photon microendoscopy is a laser-scanning modality that provides optical sectioning deep within tissue. Using in vivo microendoscopy we acquired video-rate movies of thalamic and CA1 hippocampal red blood cell dynamics and still-frame images of CA1 neurons and dendrites in anesthetized rats and mice. Microendoscopy will help meet the growing demand for in vivo cellular imaging created by the rapid emergence of new synthetic and genetically encoded fluorophores that can be used to label specific brain areas or cell classes.

    View details for DOI 10.1152/jn.00234.2004

    View details for Web of Science ID 000224475000044

    View details for PubMedID 15128753

  • Fiber optic in vivo imaging in the mammalian nervous system CURRENT OPINION IN NEUROBIOLOGY Mehta, A. D., Jung, J. C., Flusberg, B. A., Schnitzer, M. J. 2004; 14 (5): 617-628


    The compact size, mechanical flexibility, and growing functionality of optical fiber and fiber optic devices are enabling several new modalities for imaging the mammalian nervous system in vivo. Fluorescence microendoscopy is a minimally invasive fiber modality that provides cellular resolution in deep brain areas. Diffuse optical tomography is a non-invasive modality that uses assemblies of fiber optic emitters and detectors on the cranium for volumetric imaging of brain activation. Optical coherence tomography is a sensitive interferometric imaging technique that can be implemented in a variety of fiber based formats and that might allow intrinsic optical detection of brain activity at a high resolution. Miniaturized fiber optic microscopy permits cellular level imaging in the brains of behaving animals. Together, these modalities will enable new uses of imaging in the intact nervous system for both research and clinical applications.

    View details for DOI 10.1016/j.conb.2004.08.017

    View details for Web of Science ID 000224721200014

    View details for PubMedID 15464896

  • Multiphoton endoscopy OPTICS LETTERS Jung, J. C., Schnitzer, M. J. 2003; 28 (11): 902-904


    Despite widespread use of multiphoton fluorescence microscopy, development of endoscopes for nonlinear optical imaging has been stymied by the degradation of ultrashort excitation pulses that occurs within optical fiber as a result of the combined effects of group-velocity dispersion and self-phase modulation. We introduce microendoscopes (350-1000 microm in diameter) based on gradient-index microlenses that effectively eliminate self-phase modulation within the endoscope. Laser-scanning multiphoton fluorescence endoscopy exhibits micrometer-scale resolution. We used multiphoton endoscopes to image fluorescently labeled neurons and dendrites.

    View details for Web of Science ID 000182927700012

    View details for PubMedID 12816240

  • Multineuronal firing patterns in the signal from eye to brain NEURON Schnitzer, M. J., Meister, M. 2003; 37 (3): 499-511


    Population codes in the brain have generally been characterized by recording responses from one neuron at a time. This approach will miss codes that rely on concerted patterns of action potentials from many cells. Here we analyze visual signaling in populations of ganglion cells recorded from the isolated salamander retina. These neurons tend to fire synchronously far more frequently than expected by chance. We present an efficient algorithm to identify what groups of cells cooperate in this way. Such groups can include up to seven or more neurons and may account for more than 50% of all the spikes recorded from the retina. These firing patterns represent specific messages about the visual stimulus that differ significantly from what one would derive by single-cell analysis.

    View details for Web of Science ID 000181081600014

    View details for PubMedID 12575956

  • Biological computation: Amazing algorithms NATURE Schnitzer, M. J. 2002; 416 (6882): 683-683

    View details for Web of Science ID 000175033500022

    View details for PubMedID 11961533

  • Molecular motors - Doing a rotary two-step NATURE Schnitzer, M. J. 2001; 410 (6831): 878-?

    View details for Web of Science ID 000168152300028

    View details for PubMedID 11309597

  • Force production by single kinesin motors NATURE CELL BIOLOGY Schnitzer, M. J., Visscher, K., Block, S. M. 2000; 2 (10): 718-723


    Motor proteins such as kinesin, myosin and polymerase convert chemical energy into work through a cycle that involves nucleotide hydrolysis. Kinetic rates in the cycle that depend upon load identify transitions at which structural changes, such as power strokes or diffusive motions, are likely to occur. Here we show, by modelling data obtained with a molecular force clamp, that kinesin mechanochemistry can be characterized by a mechanism in which a load-dependent isomerization follows ATP binding. This model quantitatively accounts for velocity data over a wide range of loads and ATP levels, and indicates that movement may be accomplished through two sequential 4-nm substeps. Similar considerations account for kinesin processivity, which is found to obey a load-dependent Michaelis-Menten relationship.

    View details for Web of Science ID 000089697000017

    View details for PubMedID 11025662

  • Single kinesin molecules studied with a molecular force clamp NATURE Visscher, K., Schnitzer, M. J., Block, S. M. 1999; 400 (6740): 184-189


    Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.

    View details for Web of Science ID 000081324900059

    View details for PubMedID 10408448

  • Force and velocity measured for single molecules of RNA polymerase SCIENCE Wang, M. D., Schnitzer, M. J., Yin, H., Landick, R., Gelles, J., Block, S. M. 1998; 282 (5390): 902-907


    RNA polymerase (RNAP) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.

    View details for Web of Science ID 000076727300038

    View details for PubMedID 9794753

  • Kinesin hydrolyses one ATP per 8-nm step NATURE Schnitzer, M. J., Block, S. M. 1997; 388 (6640): 386-390


    Kinesin is a two-headed, ATP-dependent motor protein that moves along microtubules in discrete steps of 8 nm. In vitro, single molecules produce processive movement; motors typically take approximately 100 steps before releasing from a microtubule. A central question relates to mechanochemical coupling in this enzyme: how many molecules of ATP are consumed per step? For the actomyosin system, experimental approaches to this issue have generated considerable controversy. Here we take advantage of the processivity of kinesin to determine the coupling ratio without recourse to direct measurements of ATPase activity, which are subject to large experimental uncertainties. Beads carrying single molecules of kinesin moving on microtubules were tracked with high spatial and temporal resolution by interferometry. Statistical analysis of the intervals between steps at limiting ATP, and studies of fluctuations in motor speed as a function of ATP concentration, allow the coupling ratio to be determined. At near-zero load, kinesin molecules hydrolyse a single ATP molecule per 8-nm advance. This finding excludes various one-to-many and many-to-one coupling schemes, analogous to those advanced for myosin, and places severe constraints on models for movement.

    View details for Web of Science ID A1997XM52800053

    View details for PubMedID 9237757

  • Statistical kinetics of processive enzymes Cold Spring Harbor Symposia on Quantitative Biology - Protein Kinesis: The Dynamics of Protein Trafficking and Stability Schnitzer, M. J., Block, S. M. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1995: 793–802

    View details for Web of Science ID A1995VA12500084

    View details for PubMedID 8824454

  • Theory of continuum random walks and application to chemotaxis. Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics Schnitzer, M. J. 1993; 48 (4): 2553–68

    View details for PubMedID 9960890