@article {45852, title = {Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins.}, journal = {Journal of Neuroscience}, volume = {34}, year = {2014}, month = {2014 Dec 10}, pages = {16698-712}, abstract = {

Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.3888-14.2014}, author = {Fortin, Dale A and Tillo, Shane E and Yang, Guang and Rah, Jong-Cheol and Melander, Joshua B and Bai, Suxia and Soler-Cede{\~n}o, Omar and Qin, Maozhen and Zemelman, Boris V and Guo, Caiying and Mao, Tianyi and Zhong, Haining} } @article {6295, title = {Organization of cortical and thalamic input to pyramidal neurons in mouse motor cortex.}, journal = {The Journal of Neuroscience}, volume = {33}, year = {2013}, month = {2013 Jan 9}, pages = {748-60}, abstract = {

Determining how long-range synaptic inputs engage pyramidal neurons in primary motor cortex (M1) is important for understanding circuit mechanisms involved in regulating movement. We used channelrhodopsin-2-assisted circuit mapping to characterize the long-range excitatory synaptic connections made by multiple cortical and thalamic areas onto pyramidal neurons in mouse vibrissal motor cortex (vM1). Each projection innervated vM1 pyramidal neurons with a unique laminar profile. Collectively, the profiles for different sources of input partially overlapped and spanned all cortical layers. Specifically, orbital cortex (OC) inputs primarily targeted neurons in L6. Secondary motor cortex (M2) inputs excited neurons mainly in L5B, including pyramidal tract neurons. In contrast, thalamocortical inputs from anterior motor-related thalamic regions, including VA/VL (ventral anterior thalamic nucleus/ventrolateral thalamic nucleus), targeted neurons in L2/3 through L5B, but avoided L6. Inputs from posterior sensory-related thalamic areas, including POm (posterior thalamic nuclear group), targeted neurons only in the upper layers (L2/3 and L5A), similar to inputs from somatosensory (barrel) cortex. Our results show that long-range excitatory inputs target vM1 pyramidal neurons in a layer-specific manner. Inputs from sensory-related cortical and thalamic areas preferentially target the upper-layer pyramidal neurons in vM1. In contrast, inputs from OC and M2, areas associated with volitional and cognitive aspects of movements, bypass local circuitry and have direct monosynaptic access to neurons projecting to brainstem and thalamus.

}, keywords = {Animals, Cerebral Cortex, Data Interpretation, Efferent Pathways, Electric Stimulation, Electrophysiological Phenomena, Female, Inbred C57BL, Male, Mice, Motor Cortex, Neurons, Patch-Clamp Techniques, Pons, Pyramidal Tracts, Statistical, Thalamus}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.4338-12.2013}, author = {Hooks, Bryan M and Mao, Tianyi and Gutnisky, Diego A and Yamawaki, Naoki and Svoboda, Karel and Shepherd, Gordon M G} } @article {6301, title = {A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.}, journal = {Nature Neuroscience}, volume = {15}, year = {2012}, month = {2012 May}, pages = {793-802}, abstract = {

Cell type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of four knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0 and archaerhodopsin Arch-ER2. All four transgenes mediated Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent and inducible nature of our ChR2 mice represents a significant advance over previous lines, and the Arch-ER2 and eNpHR3.0 mice are to our knowledge the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.

}, keywords = {Action Potentials, Animals, Archaeal Proteins, Brain, Electroporation, Halorhodopsins, Integrases, Light, Luminescent Proteins, Mice, Neurogenesis, Neurons, Optics and Photonics, Proteins, Rhodopsin, Transgenic, Wakefulness}, issn = {1546-1726}, doi = {10.1038/nn.3078}, author = {Madisen, Linda and Mao, Tianyi and Koch, Henner and Zhuo, Jia-min and Berenyi, Antal and Fujisawa, Shigeyoshi and Hsu, Yun-Wei A and Garcia, Alfredo J and Gu, Xuan and Zanella, Sebastien and Kidney, Jolene and Gu, Hong and Mao, Yimei and Hooks, Bryan M and Boyden, Edward S and Buzs{\'a}ki, Gy{\"o}rgy and Ramirez, Jan Marino and Jones, Allan R and Svoboda, Karel and Han, Xue and Turner, Eric E and Zeng, Hongkui} } @article {2311, title = {Inputs to the dorsal striatum of the mouse reflect the parallel circuit architecture of the forebrain.}, journal = {Frontiers in Neuroanatomy}, volume = {4}, year = {2011}, month = {2011}, pages = {147}, abstract = {

The basal ganglia play a critical role in the regulation of voluntary action in vertebrates. Our understanding of the function of the basal ganglia relies heavily upon anatomical information, but continued progress will require an understanding of the specific functional roles played by diverse cell types and their connectivity. An increasing number of mouse lines allow extensive identification, characterization, and manipulation of specified cell types in the basal ganglia. Despite the promise of genetically modified mice for elucidating the functional roles of diverse cell types, there is relatively little anatomical data obtained directly in the mouse. Here we have characterized the retrograde labeling obtained from a series of tracer injections throughout the dorsal striatum of adult mice. We found systematic variations in input along both the medial-lateral and anterior-posterior neuraxes in close agreement with canonical features of basal ganglia anatomy in the rat. In addition to the canonical features we have provided experimental support for the importance of non-canonical inputs to the striatum from the raphe nuclei and the amygdala. To look for organization at a finer scale we have analyzed the correlation structure of labeling intensity across our entire dataset. Using this analysis we found substantial local heterogeneity within the large-scale order. From this analysis we conclude that individual striatal sites receive varied combinations of cortical and thalamic input from multiple functional areas, consistent with some earlier studies in the rat that have suggested the presence of a combinatorial map.

}, issn = {1662-5129}, doi = {10.3389/fnana.2010.00147}, author = {Pan, Weixing X and Mao, Tianyi and Dudman, Joshua T} } @article {5283, title = {Long-range neuronal circuits underlying the interaction between sensory and motor cortex.}, journal = {Neuron}, volume = {72}, year = {2011}, month = {2011 Oct 6}, pages = {111-23}, abstract = {

In the rodent vibrissal system, active sensation and sensorimotor integration are mediated in part by connections between barrel cortex and vibrissal motor cortex. Little is known about how these structures interact at the level of neurons. We used Channelrhodopsin-2 (ChR2) expression, combined with anterograde and retrograde labeling, to map connections between barrel cortex and pyramidal neurons in mouse motor cortex. Barrel cortex axons preferentially targeted upper layer (L2/3, L5A) neurons in motor cortex; input to neurons projecting back to barrel cortex was particularly strong. Barrel cortex input to deeper layers (L5B, L6) of motor cortex, including neurons projecting to the brainstem, was weak, despite pronounced geometric overlap of dendrites with axons from barrel cortex. Neurons in different layers received barrel cortex input within stereotyped dendritic domains. The cortico-cortical neurons in superficial layers of motor cortex thus couple motor and sensory signals and might mediate sensorimotor integration and motor learning.

}, keywords = {Animals, Inbred C57BL, Membrane Potentials, Mice, Molecular Imaging, Motor Cortex, Neural Pathways, Neuroanatomical Tract-Tracing Techniques, Neurons, Somatosensory Cortex, Vibrissae}, issn = {1097-4199}, doi = {10.1016/j.neuron.2011.07.029}, author = {Mao, Tianyi and Kusefoglu, Deniz and Hooks, Bryan M and Huber, Daniel and Petreanu, Leopoldo and Svoboda, Karel} } @article {1646, title = {Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.}, journal = {Nature Methods}, volume = {6}, year = {2009}, month = {2009 Dec}, pages = {875-81}, abstract = {

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.

}, keywords = {Animals, Brain, Caenorhabditis elegans, Calcium, Cell Line, Drosophila melanogaster, Fluorescence Resonance Energy Transfer, Humans, Mice, Neurons}, issn = {1548-7105}, doi = {10.1038/nmeth.1398}, author = {Tian, Lin and Hires, S Andrew and Mao, Tianyi and Huber, Daniel and Chiappe, M Eugenia and Chalasani, Sreekanth H and Petreanu, Leopoldo and Akerboom, Jasper and McKinney, Sean A and Schreiter, Eric R and Bargmann, Cornelia I and Jayaraman, Vivek and Svoboda, Karel and Looger, Loren L} } @article {4273, title = {Myosin-dependent targeting of transmembrane proteins to neuronal dendrites.}, journal = {Nature Neuroscience}, volume = {12}, year = {2009}, month = {2009 May}, pages = {568-76}, abstract = {

The distinct electrical properties of axonal and dendritic membranes are largely a result of specific transport of vesicle-bound membrane proteins to each compartment. How this specificity arises is unclear because kinesin motors that transport vesicles cannot autonomously distinguish dendritically projecting microtubules from those projecting axonally. We hypothesized that interaction with a second motor might enable vesicles containing dendritic proteins to preferentially associate with dendritically projecting microtubules and avoid those that project to the axon. Here we show that in rat cortical neurons, localization of several distinct transmembrane proteins to dendrites is dependent on specific myosin motors and an intact actin network. Moreover, fusion with a myosin-binding domain from Melanophilin targeted Channelrhodopsin-2 specifically to the somatodendritic compartment of neurons in mice in vivo. Together, our results suggest that dendritic transmembrane proteins direct the vesicles in which they are transported to avoid the axonal compartment through interaction with myosin motors.

}, keywords = {Adaptor Proteins, Animals, Cell Compartmentation, Cell Polarity, Cerebral Cortex, Cytoskeleton, Dendrites, Membrane Proteins, Mice, Microfilaments, Microtubules, Molecular Motor Proteins, Myosins, Protein Transport, Rats, Recombinant Fusion Proteins, Rhodopsin, Signal Transducing, Transport Vesicles}, issn = {1546-1726}, doi = {10.1038/nn.2318}, author = {Lewis, Tommy L and Mao, Tianyi and Svoboda, Karel and Arnold, Don B} } @article {5251, title = {Subcellular dynamics of type II PKA in neurons.}, journal = {Neuron}, volume = {62}, year = {2009}, month = {2009 May 14}, pages = {363-74}, abstract = {

Protein kinase A (PKA) plays multiple roles in neurons. The localization and specificity of PKA are largely controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA in neurons and the roles of specific AKAPs are poorly understood. We imaged the distribution of type II PKA in hippocampal and cortical layer 2/3 pyramidal neurons in vitro and in vivo. PKA was concentrated in dendritic shafts compared to the soma, axons, and dendritic spines. This spatial distribution was imposed by the microtubule-binding protein MAP2, indicating that MAP2 is the dominant AKAP in neurons. Following cAMP elevation, catalytic subunits dissociated from the MAP2-tethered regulatory subunits and rapidly became enriched in nearby spines. The spatial gradient of type II PKA between dendritic shafts and spines was critical for the regulation of synaptic strength and long-term potentiation. Therefore, the localization and activity-dependent translocation of type II PKA are important determinants of PKA function.

}, keywords = {A Kinase Anchor Proteins, Animals, Cerebral Cortex, Cyclic AMP-Dependent Protein Kinases, Dendritic Spines, Hippocampus, Mice, Microtubule-Associated Proteins, Pyramidal Cells, Rats, Subcellular Fractions, Tissue Distribution, Transgenic}, issn = {1097-4199}, doi = {10.1016/j.neuron.2009.03.013}, author = {Zhong, Haining and Sia, Gek-Ming and Sato, Takashi R and Gray, Noah W and Mao, Tianyi and Khuchua, Zaza and Huganir, Richard L and Svoboda, Karel} } @article {2185, title = {The subcellular organization of neocortical excitatory connections.}, journal = {Nature}, volume = {457}, year = {2009}, month = {2009 Feb 26}, pages = {1142-5}, abstract = {

Understanding cortical circuits will require mapping the connections between specific populations of neurons, as well as determining the dendritic locations where the synapses occur. The dendrites of individual cortical neurons overlap with numerous types of local and long-range excitatory axons, but axodendritic overlap is not always a good predictor of actual connection strength. Here we developed an efficient channelrhodopsin-2 (ChR2)-assisted method to map the spatial distribution of synaptic inputs, defined by presynaptic ChR2 expression, within the dendritic arborizations of recorded neurons. We expressed ChR2 in two thalamic nuclei, the whisker motor cortex and local excitatory neurons and mapped their synapses with pyramidal neurons in layers 3, 5A and 5B (L3, L5A and L5B) in the mouse barrel cortex. Within the dendritic arborizations of L3 cells, individual inputs impinged onto distinct single domains. These domains were arrayed in an orderly, monotonic pattern along the apical axis: axons from more central origins targeted progressively higher regions of the apical dendrites. In L5 arborizations, different inputs targeted separate basal and apical domains. Input to L3 and L5 dendrites in L1 was related to whisker movement and position, suggesting that these signals have a role in controlling the gain of their target neurons. Our experiments reveal high specificity in the subcellular organization of excitatory circuits.

}, keywords = {Animals, Axons, Dendrites, Mice, Neocortex, Neural Pathways, Pyramidal Cells, Rhodopsin, Thalamic Nuclei}, issn = {1476-4687}, doi = {10.1038/nature07709}, author = {Petreanu, Leopoldo and Mao, Tianyi and Sternson, Scott M and Svoboda, Karel} } @article {4258, title = {Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators.}, journal = {PLoS One}, volume = {3}, year = {2008}, month = {2008 Mar 19}, pages = {e1796}, abstract = {

Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10\% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50\% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37\%-264\%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.

}, keywords = {Action Potentials, Animals, Calcium, Hippocampus, Humans, Rats, Subcellular Fractions}, issn = {1932-6203}, doi = {10.1371/journal.pone.0001796}, author = {Mao, Tianyi and O{\textquoteright}Connor, Daniel H and Scheuss, Volker and Nakai, Junichi and Svoboda, Karel} }