Inhibitory mechanisms that generate centre and surround properties in ON and OFF brisk-sustained ganglion cells in the rabbit retina.
AuthorsBuldyrev I, Taylor WR,
JournalJ Physiol
PubMed ID23045347
Lateral inhibition produces the centre-surround organization of retinal receptive fields, in which inhibition driven by the mean luminance enhances the sensitivity of ganglion cells to spatial and temporal contrast. Surround inhibition is generated in both synaptic layers; however, the synaptic mechanisms within the inner plexiform layer are not well characterized ... More
Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging.
AuthorsYasuda R, Harvey CD, Zhong H, Sobczyk A, van Aelst L, Svoboda K
JournalNat Neurosci
PubMed ID16429133
'To understand the biochemical signals regulated by neural activity, it is necessary to measure protein-protein interactions and enzymatic activity in neuronal microcompartments such as axons, dendrites and their spines. We combined two-photon excitation laser scanning with fluorescence lifetime imaging to measure fluorescence resonance energy transfer at high resolutions in brain ... More
Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.
AuthorsPanchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung WY, Haugland RP
JournalJ Histochem Cytochem
PubMed ID10449539
'Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the ... More
Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons.
AuthorsKarpova AY, Tervo DG, Gray NW, Svoboda K
JournalNeuron
PubMed ID16337911
'Inducible and reversible silencing of selected neurons in vivo is critical to understanding the structure and dynamics of brain circuits. We have developed Molecules for Inactivation of Synaptic Transmission (MISTs) that can be genetically targeted to allow the reversible inactivation of neurotransmitter release. MISTs consist of modified presynaptic proteins that ... More
The kinetics of phagosome maturation as a function of phagosome/lysosome fusion and acquisition of hydrolytic activity.
AuthorsYates RM, Hermetter A, Russell DG
JournalTraffic
PubMed ID15813751
'Professional phagocytes function at the hinge of innate and acquired immune responses by internalizing particulate material that is digested and sampled within the phagosome of the cell. Despite intense interest, assays to measure phagosome maturation remain insensitive and few in number. In this current study, we describe three novel assays ... More
A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid ß.
AuthorsIliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, Nagelhus EA, Nedergaard M,
JournalSci Transl Med
PubMed ID22896675
'Because it lacks a lymphatic circulation, the brain must clear extracellular proteins by an alternative mechanism. The cerebrospinal fluid (CSF) functions as a sink for brain extracellular solutes, but it is not clear how solutes from the brain interstitium move from the parenchyma to the CSF. We demonstrate that a ... More
Microcircuits of excitatory and inhibitory neurons in layer 2/3 of mouse barrel cortex.
AuthorsAvermann M, Tomm C, Mateo C, Gerstner W, Petersen CC,
JournalJ Neurophysiol
PubMed ID22402650
'Synaptic interactions between nearby excitatory and inhibitory neurons in the neocortex are thought to play fundamental roles in sensory processing. Here, we have combined optogenetic stimulation, whole cell recordings, and computational modeling to define key functional microcircuits within layer 2/3 of mouse primary somatosensory barrel cortex. In vitro optogenetic stimulation ... More
LIM domain only 4 (LMO4) regulates calcium-induced calcium release and synaptic plasticity in the hippocampus.
AuthorsQin Z, Zhou X, Gomez-Smith M, Pandey NR, Lee KF, Lagace DC, Béïque JC, Chen HH,
JournalJ Neurosci
PubMed ID22442089
'The LIM domain only 4 (LMO4) transcription cofactor activates gene expression in neurons and regulates key aspects of network formation, but the mechanisms are poorly understood. Here, we show that LMO4 positively regulates ryanodine receptor type 2 (RyR2) expression, thereby suggesting that LMO4 regulates calcium-induced calcium release (CICR) in central ... More
Intracellular fluid flow in rapidly moving cells.
AuthorsKeren K, Yam PT, Kinkhabwala A, Mogilner A, Theriot JA,
JournalNat Cell Biol
PubMed ID19767741
'Cytosolic fluid dynamics have been implicated in cell motility because of the hydrodynamic forces they induce and because of their influence on transport of components of the actin machinery to the leading edge. To investigate the existence and the direction of fluid flow in rapidly moving cells, we introduced inert ... More
Dendritic spine dynamics are regulated by monocular deprivation and extracellular matrix degradation.
AuthorsOray S, Majewska A, Sur M
JournalNeuron
PubMed ID15603744
'The mammalian primary visual cortex (V1) is especially susceptible to changes in visual input over a well-defined critical period, during which closing one eye leads to a loss of responsiveness of neurons to the deprived eye and a shift in response toward the open eye. This functional plasticity can occur ... More
Carbohydrate oxidation acidifies endosomes, regulating antigen processing and TLR9 signaling.
AuthorsLewis CJ, Cobb BA,
JournalJ Immunol
PubMed ID20200279
'Phagocytes kill encapsulated microbes through oxidative cleavage of surface carbohydrates, releasing glycan fragments and microbial contents that serve as ligands for immune receptors, which tailor the immune response against the offending pathogen. The glycan fragments serve as MHC class II (MHC II) ligands and innate receptor agonists, whereas microbial proteins ... More
Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser.
AuthorsLiu TM, Chu SW, Sun CK, Lin BL, Cheng PC, Johnson I
JournalScanning
PubMed ID11534811
'With its output wavelength covering the infrared penetrating window of most biological tissues at 1,200-1,250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers ... More
Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy.
AuthorsDing JB, Takasaki KT, Sabatini BL,
JournalNeuron
PubMed ID19709626
'Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescence imaging of neuronal structure and function within neural tissue. However, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here, we demonstrate supraresolution 2PLSM within brain slices. Imaging beyond the diffraction limit is accomplished by using ... More
P2X7 receptor inhibition improves recovery after spinal cord injury.
AuthorsWang X, Arcuino G, Takano T, Lin J, Peng WG, Wan P, Li P, Xu Q, Liu QS, Goldman SA, Nedergaard M
JournalNat Med
PubMed ID15258577
'Secondary injury exacerbates the extent of spinal cord insults, yet the mechanistic basis of this phenomenon has largely been unexplored. Here we report that broad regions of the peritraumatic zone are characterized by a sustained process of pathologic, high ATP release. Spinal cord neurons expressed P2X7 purine receptors (P2X7R), and ... More
Activity-dependent long-term potentiation of intrinsic excitability in hippocampal CA1 pyramidal neurons.
AuthorsXu J, Kang N, Jiang L, Nedergaard M, Kang J
JournalJ Neurosci
PubMed ID15716411
'The efficiency of neural circuits is enhanced not only by increasing synaptic strength but also by increasing intrinsic excitability. In contrast to the detailed analysis of long-term potentiation (LTP), less attention has been given to activity-dependent changes in the intrinsic neuronal excitability. By stimulating hippocampal CA1 pyramidal neurons with synaptic ... More
Nonlinear regulation of unitary synaptic signals by CaV(2.3) voltage-sensitive calcium channels located in dendritic spines.
AuthorsBloodgood BL, Sabatini BL
JournalNeuron
PubMed ID17224406
'The roles of voltage-sensitive sodium (Na) and calcium (Ca) channels located on dendrites and spines in regulating synaptic signals are largely unknown. Here we use 2-photon glutamate uncaging to stimulate individual spines while monitoring uncaging-evoked excitatory postsynaptic potentials (uEPSPs) and Ca transients. We find that, in CA1 pyramidal neurons in ... More
'Although action potentials are typically generated in the axon initial segment (AIS), the timing and pattern of action potentials are thought to depend on inward current originating in somatodendritic compartments. Using two-photon imaging, we show that T- and R-type voltage-gated Ca(2+) channels are colocalized with Na(+) channels in the AIS ... More
Targeted single-cell electroporation of mammalian neurons in vivo.
AuthorsJudkewitz B, Rizzi M, Kitamura K, Häusser M,
JournalNat Protoc
PubMed ID19444243
'In order to link our knowledge of single neurons with theories of network function, it has been a long-standing goal to manipulate the activity and gene expression of identified subsets of mammalian neurons within the intact brain in vivo. This protocol describes a method for delivering plasmid DNA into single ... More
Nonlinear [Ca2+] signaling in dendrites and spines caused by activity-dependent depression of Ca2+ extrusion.
AuthorsScheuss V, Yasuda R, Sobczyk A, Svoboda K
JournalJ Neurosci
PubMed ID16885232
'Spine Ca2+ triggers the induction of synaptic plasticity and other adaptive neuronal responses. The amplitude and time course of Ca2+ signals specify the activation of the signaling pathways that trigger different forms of plasticity such as long-term potentiation and depression. The shapes of Ca2+ signals are determined by the dynamics ... More
Activity regulates functional connectivity from the vomeronasal organ to the accessory olfactory bulb.
'The mammalian accessory olfactory system is specialized for the detection of chemicals that identify kin and conspecifics. Vomeronasal sensory neurons (VSNs) residing in the vomeronasal organ project axons to the accessory olfactory bulb (AOB), where they form synapses with principal neurons known as mitral cells. The organization of this projection ... More
A sensitive, versatile microfluidic assay for bacterial chemotaxis.
AuthorsMao H, Cremer PS, Manson MD
JournalProc Natl Acad Sci U S A
PubMed ID12704234
'We have developed a microfluidic assay for bacterial chemotaxis in which a gradient of chemoeffectors is established inside a microchannel via diffusion between parallel streams of liquid in laminar flow. The random motility and chemotactic responses to L-aspartate, L-serine, L-leucine, and Ni(2+) of WT and chemotactic-mutant strains of Escherichia coli ... More
Extracellular diffusivity determines contribution of high-versus low-affinity receptors to neural signaling.
AuthorsSavtchenko LP, Rusakov DA
JournalNeuroimage
PubMed ID15734347
'Diffusion-weighted magnetic resonance imaging detects physiological changes in the human brain by highlighting alterations in local diffusivity. However, the causal link between brain tissue diffusivity and neural activity is poorly understood. Synaptic physiology studies in vitro coupled with biophysical modeling have suggested that extracellular diffusion affects the spatial profile of ... More
Compartmentalized and binary behavior of terminal dendrites in hippocampal pyramidal neurons.
AuthorsWei DS, Mei YA, Bagal A, Kao JP, Thompson SM, Tang CM
JournalScience
PubMed ID11567143
'The dendritic arbor of pyramidal neurons is not a monolithic structure. We show here that the excitability of terminal apical dendrites differs from that of the apical trunk. In response to fluorescence-guided focal photolysis of caged glutamate, individual terminal apical dendrites generated cadmium-sensitive all-or-none responses that were subthreshold for somatic ... More
Enhanced neurite outgrowth in PC12 cells mediated by connexin hemichannels and ATP.
AuthorsBelliveau DJ, Bani-Yaghoub M, McGirr B, Naus CC, Rushlow WJ
JournalJ Biol Chem
PubMed ID16731531
'Gap junctions have traditionally been described as transmembrane channels that facilitate intercellular communication via the passage of small molecules. Connexins, the basic building blocks of gap junctions, are expressed in most mammalian tissues including the developing and adult central nervous system. During brain development, connexins are temporally and spatially regulated ... More
Electroporation of neurons and growth cones in Aplysia californica.
AuthorsLovell P, Jezzini SH, Moroz LL
JournalJ Neurosci Methods
PubMed ID16174534
'Specific labeling of individual neurons and neuronal processes is virtually an everyday task for neuroscientists. Many traditional ways for delivery of intracellular dyes have limitations in terms of speed, efficiency and reproducibility. Electroporation is a fast, reliable and efficient method to deliver microscopic amounts of polar and charged molecules into ... More
Imaging in vivo: watching the brain in action.
AuthorsKerr JN, Denk W,
JournalNat Rev Neurosci
PubMed ID18270513
'The appeal of in vivo cellular imaging to any neuroscientist is not hard to understand: it is almost impossible to isolate individual neurons while keeping them and their complex interactions with surrounding tissue intact. These interactions lead to the complex network dynamics that underlie neural computation which, in turn, forms ... More
Ca(2+) regulation of gap junctional coupling in lens epithelial cells.
AuthorsChurchill GC, Lurtz MM, Louis CF
JournalAm J Physiol Cell Physiol
PubMed ID11502574
'The quantitative effects of Ca(2+) signaling on gap junctional coupling in lens epithelial cells have been determined using either the spread of Mn(2+) that is imaged by its ability to quench the fluorescence of fura 2 or the spread of the fluorescent dye Alexa Fluor 594. Gap junctional coupling was ... More
Modulation of presynaptic Ca2+ entry by AMPA receptors at individual GABAergic synapses in the cerebellum.
AuthorsRusakov DA, Saitow F, Lehre KP, Konishi S
JournalJ Neurosci
PubMed ID15901774
'Cerebellar Purkinje cells (PCs) receive GABAergic input that undergoes powerful retrograde modulation by presynaptic cannabinoid and glutamate receptors. Here we examine a distinct modulatory mechanism at these synapses, which does not require postsynaptic depolarization and acts via presynaptic AMPA receptors. We find that this mechanism operates mainly in the somatic ... More
Genetic evidence for a protein-kinase-A-mediated presynaptic component in NMDA-receptor-dependent forms of long-term synaptic potentiation.
AuthorsHuang YY, Zakharenko SS, Schoch S, Kaeser PS, Janz R, Südhof TC, Siegelbaum SA, Kandel ER
JournalProc Natl Acad Sci U S A
PubMed ID15967982
'The synaptic vesicle protein Rab3A is a small GTP-binding protein that interacts with rabphilin and RIM1alpha, two presynaptic substrates of protein kinase A (PKA). Mice lacking RIM1alpha and Rab3A have a defect in PKA-dependent and NMDA receptor (NMDAR)-independent presynaptic long-term potentiation (LTP) at hippocampal mossy-fiber and cerebellar parallel-fiber synapses. In ... More
Synapse-specific plasticity and compartmentalized signaling in cerebellar stellate cells.
AuthorsSoler-Llavina GJ, Sabatini BL
JournalNat Neurosci
PubMed ID16680164
'Here we demonstrate that cerebellar stellate cells diffusionally isolate synaptically evoked signals in dendrites and are capable of input-specific synaptic plasticity. Sustained activity of parallel fibers induces a form of long-term depression that requires opening of calcium (Ca(2+))-permeable AMPA-type glutamate receptors (CP-AMPARs) and signaling through class 1 metabotropic glutamate receptors ... More
Target cell-dependent normalization of transmitter release at neocortical synapses.
AuthorsKoester HJ, Johnston D
JournalScience
PubMed ID15774725
'The efficacy and short-term modification of neocortical synaptic connections vary with the type of target neuron. We investigated presynaptic Ca2+ and release probability at single synaptic contacts between pairs of neurons in layer 2/3 of the rat neocortex. The amplitude of Ca2+ signals in boutons of pyramids contacting bitufted or ... More
Neuronal activity regulates diffusion across the neck of dendritic spines.
AuthorsBloodgood BL, Sabatini BL
JournalScience
PubMed ID16272125
'In mammalian excitatory neurons, dendritic spines are separated from dendrites by thin necks. Diffusion across the neck limits the chemical and electrical isolation of each spine. We found that spine/dendrite diffusional coupling is heterogeneous and uncovered a class of diffusionally isolated spines. The barrier to diffusion posed by the neck ... More
Selective labeling of retinal ganglion cells with calcium indicators by retrograde loading in vitro.
Here we present a retrograde loading technique that makes it possible for the first time to rapidly load a calcium indicator in the majority of retinal ganglion cells (RGCs) in salamander retina, and then to observe physiological activity of these dye-loaded cells. Dextran-conjugated calcium indicator, dissolved in water, was applied ... More
Optical induction of synaptic plasticity using a light-sensitive channel.
AuthorsZhang YP, Oertner TG
JournalNat Methods
PubMed ID17195846
We have combined millisecond activation of channelrhodopsin-2 (ChR2), a light-gated ion channel, with two-photon calcium imaging to investigate active synaptic contacts in rat hippocampal slice cultures. Calcium influx was larger during light-induced action potentials than during action potentials induced by somatic current injection, leading to highly reproducible synaptic transmission. Pairing ... More
Bipolar cells use kainate and AMPA receptors to filter visual information into separate channels.
AuthorsDeVries SH
JournalNeuron
PubMed ID11163271
Unlike cone photoreceptors, whose light responses have a uniform time course, retinal ganglion cells are tuned to respond to different temporal components in a changing visual scene. The signals in a mammalian cone flow to three to five morphologically distinct "OFF" bipolar cells at a sign-conserving, glutamatergic synapse. By recording ... More
Control of firing patterns through modulation of axon initial segment T-type calcium channels.
Spontaneously active neurons typically fire either in a regular pattern or in bursts. While much is known about the subcellular location and biophysical properties of conductances that underlie regular spontaneous activity, less is known about those that underlie bursts. Here, we show that T-type Ca(2+) channels localized to the site ... More
Mechanisms of quenching of Alexa fluorophores by natural amino acids.
Quenching of fluorophores by the same proteins that they covalently label is a phenomenon that is neither well-known nor well-characterized. It is often assumed that fluorophores are unperturbed by their target proteins. However, it has been observed that attached fluorophores can be quenched by contact with amino acids within the ... More
The life cycle of Ca(2+) ions in dendritic spines.
AuthorsSabatini BL, Oertner TG, Svoboda K
JournalNeuron
PubMed ID11832230
Spine Ca(2+) is critical for the induction of synaptic plasticity, but the factors that control Ca(2+) handling in dendritic spines under physiological conditions are largely unknown. We studied [Ca(2+)] signaling in dendritic spines of CA1 pyramidal neurons and find that spines are specialized structures with low endogenous Ca(2+) buffer capacity ... More
Hindered diffusion through an aqueous pore describes invariant dye selectivity of Cx43 junctions.
AuthorsHeyman NS, Burt JM,
JournalBiophys J
PubMed ID17921206
The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size ... More
eGFP Expression under UCHL1 Promoter Genetically Labels Corticospinal Motor Neurons and a Subpopulation of Degeneration-Resistant Spinal Motor Neurons in an ALS Mouse Model.
AuthorsYasvoina MV, Genç B, Jara JH, Sheets PL, Quinlan KA, Milosevic A, Shepherd GM, Heckman CJ, Ozdinler PH,
JournalJ Neurosci
PubMed ID23637180
Understanding mechanisms that lead to selective motor neuron degeneration requires visualization and cellular identification of vulnerable neurons. Here we report generation and characterization of UCHL1-eGFP and hSOD1(G93A)-UeGFP mice, novel reporter lines for cortical and spinal motor neurons. Corticospinal motor neurons (CSMN) and a subset of spinal motor neurons (SMN) are ... More
Changing the responses of cortical neurons from sub- to suprathreshold using single spikes in vivo.
Action Potential (APs) patterns of sensory cortex neurons encode a variety of stimulus features, but how can a neuron change the feature to which it responds? Here, we show that in vivo a spike-timing-dependent plasticity (STDP) protocol-consisting of pairing a postsynaptic AP with visually driven presynaptic inputs-modifies a neurons' AP-response ... More
Transfer of small interfering RNA by single-cell electroporation in cerebellar cell cultures.
AuthorsTanaka M, Yanagawa Y, Hirashima N,
JournalJ Neurosci Methods
PubMed ID19114056
RNA interference (RNAi) is a powerful means to investigate functions of genes involved in neuronal differentiation and degeneration. In contrast to widely used methods for introducing small interfering RNA (siRNA) into cells, recently developed single-cell electroporation has enabled transfer of siRNA into single and identified cells. To explore the availability ... More
Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo.
AuthorsKitamura K, Judkewitz B, Kano M, Denk W, Häusser M,
JournalNat Methods
PubMed ID18157136
Here we describe an approach for making targeted patch-clamp recordings from single neurons in vivo, visualized by two-photon microscopy. A patch electrode is used to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus enabling the neuron to be visualized as a negative image ('shadow') ... More
Characterizing the conductance underlying depolarization-induced slow current in cerebellar Purkinje cells.
AuthorsKim YS, Kang E, Makino Y, Park S, Shin JH, Song H, Launay P, Linden DJ,
JournalJ Neurophysiol
PubMed ID23197456
Brief strong depolarization of cerebellar Purkinje cells produces a slow inward cation current [depolarization-induced slow current (DISC)]. Previous work has shown that DISC is triggered by voltage-sensitive Ca influx in the Purkinje cell and is attenuated by blockers of vesicular loading and fusion. Here, we have sought to characterize the ... More
Monitoring Dynamic GPCR Signaling Events Using Fluorescence Microscopy, FRET Imaging, and Single-Molecule Imaging.
AuthorsXu X, Brzostowski JA, Jin T,
JournalMethods Mol Biol
PubMed ID19763980
How a eukaryotic cell translates a small concentration difference of a chemoattractant across the length of its surface into highly polarized intracellular responses is a fundamental question in chemotaxis. Chemoattractants are detected by G-protein-coupled receptors (GPCRs). Binding of chemoattractants to GPCRs induces the dissociation of heterotrimeric G-proteins into Galpha and ... More
High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision.
Two-photon calcium imaging of neuronal populations enables optical recording of spiking activity in living animals, but standard laser scanners are too slow to accurately determine spike times. Here we report in vivo imaging in mouse neocortex with greatly improved temporal resolution using random-access scanning with acousto-optic deflectors. We obtained fluorescence ... More
A simple method for quantitative calcium imaging in unperturbed developing neurons.
AuthorsAlbantakis L, Lohmann C,
JournalJ Neurosci Methods
PubMed ID19682493
Calcium imaging has been widely used to address questions of neuronal function and development. To gain deeper insights into the actions of calcium as a second messenger, but also to measure synaptic function, it is necessary to quantify the level of calcium at rest and during calcium transients. While quantification ... More
Regional differences in hippocampal calcium handling provide a cellular mechanism for limiting plasticity.
AuthorsSimons SB, Escobedo Y, Yasuda R, Dudek SM,
JournalProc Natl Acad Sci U S A
PubMed ID19666491
Although much is known about the mechanisms underlying synaptic plasticity, the cellular mechanisms that negatively regulate plasticity in some brain regions are considerably less studied. One region where neurons do not reliably express long-term potentiation (LTP) is the CA2 subfield of the hippocampus. Given the connection between synaptic plasticity and ... More
Somatic spikes regulate dendritic signaling in small neurons in the absence of backpropagating action potentials.
AuthorsMyoga MH, Beierlein M, Regehr WG,
JournalJ Neurosci
PubMed ID19535592
Somatic spiking is known to regulate dendritic signaling and associative synaptic plasticity in many types of large neurons, but it is unclear whether somatic action potentials play similar roles in small neurons. Here we ask whether somatic action potentials can also influence dendritic signaling in an electrically compact neuron, the ... More
Radially expanding transglial calcium waves in the intact cerebellum.
Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent ... More
Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity.
The dynamic ability of neuronal dendrites to shape and integrate synaptic responses is the hallmark of information processing in the brain. Effectively studying this phenomenon requires concurrent measurements at multiple sites on live neurons. Substantial progress has been made by optical imaging systems that combine confocal and multiphoton microscopy with ... More
Real-time spectrofluorometric assays for the lumenal environment of the maturing phagosome.
AuthorsYates RM, Russell DG,
JournalMethods Mol Biol
PubMed ID18425459
The ultimate goal of phagosomal maturation is the delivery of internalized, particulate cargo to acidic, hydrolytically competent compartments capable of mediating its degradation. Here we outline in detail three fluorometric techniques that allow the study of phagosomal maturation in macrophages by quantifying functionally important features of the lumenal environment of ... More
Live-cell superresolution imaging by pulsed STED two-photon excitation microscopy.
AuthorsTakasaki KT, Ding JB, Sabatini BL,
JournalBiophys J
PubMed ID23442955
Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of ... More
Dye loading with patch pipettes.
AuthorsEilers J, Konnerth A,
JournalCSH Protoc
PubMed ID20147143
This protocol describes the loading of individual cells with fluorescent probes via patch pipettes. The patch-clamp methodology has been successfully used for single-cell dye labeling in cultured neurons, brain slices, and in vivo preparations. A broad range of dyes can be used with this loading technique. Markers for morphological reconstruction ... More
The number of glutamate receptors opened by synaptic stimulation in single hippocampal spines.
AuthorsNimchinsky EA, Yasuda R, Oertner TG, Svoboda K
JournalJ Neurosci
PubMed ID14985448
The number of receptors opening after glutamate release is critical for understanding the sources of noise and the dynamic range of synaptic transmission. We imaged [Ca2+] transients mediated by synaptically activated NMDA receptors (NMDA-Rs) in individual spines in rat brain slices. We show that Ca2+ influx through single NMDA-Rs can ... More
Hypocretin and nicotine excite the same thalamocortical synapses in prefrontal cortex: correlation with improved attention in rat.
AuthorsLambe EK, Olausson P, Horst NK, Taylor JR, Aghajanian GK
JournalJ Neurosci
PubMed ID15917462
Thalamic projections to prefrontal cortex are important for executive aspects of attention. Using two-photon imaging in prefrontal brain slices, we show that nicotine and the wakefulness neuropeptide hypocretin (orexin) excite the same identified synapses of the thalamocortical arousal pathway within the prefrontal cortex. Although it is known that attention can ... More
SK channels and NMDA receptors form a Ca2+-mediated feedback loop in dendritic spines.
AuthorsNgo-Anh TJ, Bloodgood BL, Lin M, Sabatini BL, Maylie J, Adelman JP
JournalNat Neurosci
PubMed ID15852011
Small-conductance Ca(2+)-activated K(+) channels (SK channels) influence the induction of synaptic plasticity at hippocampal CA3-CA1 synapses. We find that in mice, SK channels are localized to dendritic spines, and their activity reduces the amplitude of evoked synaptic potentials in an NMDA receptor (NMDAR)-dependent manner. Using combined two-photon laser scanning microscopy ... More
Facilitation at single synapses probed with optical quantal analysis.
AuthorsOertner TG, Sabatini BL, Nimchinsky EA, Svoboda K
JournalNat Neurosci
PubMed ID12055631
Many synapses can change their strength rapidly in a use-dependent manner, but the mechanisms of such short-term plasticity remain unknown. To understand these mechanisms, measurements of neurotransmitter release at single synapses are required. We probed transmitter release by imaging transient increases in [Ca(2+)] mediated by synaptic N-methyl-D-aspartate receptors (NMDARs) in ... More
In vivo calcium imaging of circuit activity in cerebellar cortex.
AuthorsSullivan MR, Nimmerjahn A, Sarkisov DV, Helmchen F, Wang SS
JournalJ Neurophysiol
PubMed ID16079125
In vivo two-photon calcium imaging provides the opportunity to monitor activity in multiple components of neural circuitry at once. Here we report the use of bulk-loading of fluorescent calcium indicators to record from axons, dendrites, and neuronal cell bodies in cerebellar cortex in vivo. In cerebellar folium crus IIa of ... More
Postsynaptic excitability is necessary for strengthening of cortical sensory responses during experience-dependent development.
AuthorsKomai S, Licznerski P, Cetin A, Waters J, Denk W, Brecht M, Osten P
JournalNat Neurosci
PubMed ID16921372
Sensory experience is necessary for normal cortical development. This has been shown by sensory deprivation and pharmacological perturbation of the cortex. Because these manipulations affect the cortical network as a whole, the role of postsynaptic cellular properties during experience-dependent development is unclear. Here we addressed the developmental role of somatodendritic ... More
Presynaptic activation of silent synapses and growth of new synapses contribute to intermediate and long-term facilitation in Aplysia.
AuthorsKim JH, Udo H, Li HL, Youn TY, Chen M, Kandel ER, Bailey CH
JournalNeuron
PubMed ID14527440
The time course and functional significance of the structural changes associated with long-term facilitation of Aplysia sensory to motor neuron synaptic connections in culture were examined by time-lapse confocal imaging of individual sensory neuron varicosities labeled with three different fluorescent markers: the whole-cell marker Alexa-594 and two presynaptic marker proteins-synaptophysin-eGFP ... More
Highly ordered arrangement of single neurons in orientation pinwheels.
AuthorsOhki K, Chung S, Kara P, Hübener M, Bonhoeffer T, Reid RC
JournalNature
PubMed ID16906137
In the visual cortex of higher mammals, neurons are arranged across the cortical surface in an orderly map of preferred stimulus orientations. This map contains 'orientation pinwheels', structures that are arranged like the spokes of a wheel such that orientation changes continuously around a centre. Conventional optical imaging first demonstrated ... More
Differential requirements for actin polymerization, calmodulin, and Ca2+ define distinct stages of lysosome/phagosome targeting.
AuthorsStockinger W, Zhang SC, Trivedi V, Jarzylo LA, Shieh EC, Lane WS, Castoreno AB, Nohturfft A
JournalMol Biol Cell
PubMed ID16452628
Fusion of phagosomes with late endocytic organelles is essential for cellular digestion of microbial pathogens, senescent cells, apoptotic bodies, and retinal outer segment fragments. To further elucidate the biochemistry of the targeting process, we developed a scintillation proximity assay to study the stepwise association of lysosomes and phagosomes in vitro. ... More
Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex.
AuthorsOhki K, Chung S, Ch'ng YH, Kara P, Reid RC
JournalNature
PubMed ID15660108
Neurons in the cerebral cortex are organized into anatomical columns, with ensembles of cells arranged from the surface to the white matter. Within a column, neurons often share functional properties, such as selectivity for stimulus orientation; columns with distinct properties, such as different preferred orientations, tile the cortical surface in ... More
Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores.
AuthorsCsutora P, Su Z, Kim HY, Bugrim A, Cunningham KW, Nuccitelli R, Keizer JE, Hanley MR, Blalock JE, Marchase RB
JournalProc Natl Acad Sci U S A
PubMed ID9874782
Depletion of endoplasmic reticulum Ca2+ stores leads to the entry of extracellular Ca2+ into the cytoplasm, a process termed capacitative or store-operated Ca2+ entry. Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca2+ stores were depleted by chemical and genetic means, ... More
Initiation of sodium spikelets in basal dendrites of neocortical pyramidal neurons.
Cortical information processing relies critically on the processing of electrical signals in pyramidal neurons. Electrical transients mainly arise when excitatory synaptic inputs impinge upon distal dendritic regions. To study the dendritic aspect of synaptic integration one must record electrical signals in distal dendrites. Since thin dendritic branches, such as oblique ... More
The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities.
Gap junctions have traditionally been characterized as nonspecific pores between cells passing molecules up to 1 kDa in molecular mass. Nonetheless, it has become increasingly evident that different members of the connexin (Cx) family mediate quite distinct physiological processes and are often not interchangeable. Consistent with this observation, differences in ... More
A transient diffusion model yields unitary gap junctional permeabilities from images of cell-to-cell fluorescent dye transfer between Xenopus oocytes.
As ubiquitous conduits for intercellular transport and communication, gap junctional pores have been the subject of numerous investigations aimed at elucidating the molecular mechanisms underlying permeability and selectivity. Dye transfer studies provide a broadly useful means of detecting coupling and assessing these properties. However, given evidence for selective permeability of ... More
Imaging input and output of neocortical networks in vivo.
AuthorsKerr JN, Greenberg D, Helmchen F
JournalProc Natl Acad Sci U S A
PubMed ID16157876
Neural activity manifests itself as complex spatiotemporal activation patterns in cell populations. Even for local neural circuits, a comprehensive description of network activity has been impossible so far. Here we demonstrate that two-photon calcium imaging of bulk-labeled tissue permits dissection of local input and output activities in rat neocortex in ... More
Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells.
AuthorsBeltramello M, Bicego M, Piazza V, Ciubotaru CD, Mammano F, D'Andrea P
JournalBiochem Biophys Res Commun
PubMed ID12767933
Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26-pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of ... More
Cytosolic phospholipase A(2) alpha mediates electrophysiologic responses of hippocampal pyramidal neurons to neurotoxic NMDA treatment.
AuthorsShen Y, Kishimoto K, Linden DJ, Sapirstein A
JournalProc Natl Acad Sci U S A
PubMed ID17389392
The arachidonic acid-generating enzyme cytosolic phospholipase A(2) alpha (cPLA(2)alpha) has been implicated in the progression of excitotoxic neuronal injury. However, the mechanisms of cPLA(2)alpha toxicity have yet to be determined. Here, we used a model system exposing mouse hippocampal slices to NMDA as an excitotoxic injury, in combination with simultaneous ... More