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106 Publications

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    05/15/07 | Dendritic D-type potassium currents inhibit the spike afterdepolarization in rat hippocampal CA1 pyramidal neurons.
    Metz AE, Spruston N, Martina M
    The Journal of Physiology. 2007 May 15;581(Pt 1):175-87. doi: 10.1113/jphysiol.2006.127068

    In CA1 pyramidal neurons, burst firing is correlated with hippocampally dependent behaviours and modulation of synaptic strength. One of the mechanisms underlying burst firing in these cells is the afterdepolarization (ADP) that follows each action potential. Previous work has shown that the ADP results from the interaction of several depolarizing and hyperpolarizing conductances located in the soma and the dendrites. By using patch-clamp recordings from acute rat hippocampal slices we show that D-type potassium current modulates the size of the ADP and the bursting of CA1 pyramidal neurons. Sensitivity to alpha-dendrotoxin suggests that Kv1-containing potassium channels mediate this current. Dual somato-dendritic recording, outside-out dendritic recordings, and focal application of dendrotoxin together indicate that the channels mediating this current are located in the apical dendrites. Thus, our data present evidence for a dendritic segregation of Kv1-like channels in CA1 pyramidal neurons and identify a novel action for these channels, showing that they inhibit action potential bursting by restricting the size of the ADP.

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    05/15/07 | Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta.
    Tanaka K, Truman JW
    Developmental Biology. 2007 May 15;305(2):539-50. doi: 10.1016/j.ydbio.2007.02.042

    The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.

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    Magee Lab
    05/01/07 | Associative pairing enhances action potential back-propagation in radial oblique branches of CA1 pyramidal neurons.
    Gasparini S, Losonczy A, Chen X, Johnston D, Magee JC
    The Journal of Physiology. 2007 May 1;580(Pt.3):787-800. doi: 10.1002/cbic.201000254

    Back-propagating action potentials (bAPs) are involved in associative synaptic plasticity and the modulation of dendritic excitability. We have used high-speed confocal and two-photon imaging to measure calcium and voltage signals associated with action potential propagation into oblique branches of CA1 pyramidal neurons in adult hippocampal slices. The spatial profile of the bAP-associated Ca(2+) influx was biphasic, with an initial increase in the proximity of the branch point followed by a progressive decrease. Voltage imaging in the branches showed that bAP amplitude was initially constant and then steadily declined with distance from the soma. To determine the role of transient K(+) channels in this profile, we used external Ba(2+) (150 microm) as a channel blocker, after characterizing its effect on A-type K(+) channels in the apical trunk. Bath application of Ba(2+) significantly reduced the A-type K(+) current in outside-out patches and nearly eliminated the distance-dependent decrease in bAP amplitude and its associated Ca(2+) signal. Finally, small amplitude bAPs at more distal oblique branch locations could be boosted by simultaneous branch depolarization, such that the paired Ca(2+) signal became nearly the same for proximal and distal oblique dendrites. These data suggest that dendritic K(+) channels regulate the amplitude of bAPs to create a dendritic Ca(2+) signal whose magnitude is inversely related to the electrotonic distance from the soma when bAPs are not associated with a significant amount of localized synaptic input. This distance-dependent Ca(2+) signal from bAPs, however, can be amplified and a strong associative signal is produced once the proper correlation between synaptic activation and AP output is achieved. We hypothesize that these two signals may be involved in the regulation of the expression and activity of dendritic voltage- and ligand-gated ion channels.

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    05/01/07 | Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases.
    Wolterink-van Loo S, van Eerde A, Siemerink MA, Akerboom J, Dijkstra BW, van der Oost J
    The Biochemical Journal. 2007 May 1;403(3):421-30. doi: 10.1042/BJ20061419

    Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.

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    Svoboda Lab
    05/01/07 | Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections.
    Petreanu L, Huber D, Sobczyk A, Svoboda K
    Nature Neuroscience. 2007 May;10:663-8. doi: 10.1038/nn1891

    The functions of cortical areas depend on their inputs and outputs, but the detailed circuits made by long-range projections are unknown. We show that the light-gated channel channelrhodopsin-2 (ChR2) is delivered to axons in pyramidal neurons in vivo. In brain slices from ChR2-expressing mice, photostimulation of ChR2-positive axons can be transduced reliably into single action potentials. Combining photostimulation with whole-cell recordings of synaptic currents makes it possible to map circuits between presynaptic neurons, defined by ChR2 expression, and postsynaptic neurons, defined by targeted patching. We applied this technique, ChR2-assisted circuit mapping (CRACM), to map long-range callosal projections from layer (L) 2/3 of the somatosensory cortex. L2/3 axons connect with neurons in L5, L2/3 and L6, but not L4, in both ipsilateral and contralateral cortex. In both hemispheres the L2/3-to-L5 projection is stronger than the L2/3-to-L2/3 projection. Our results suggest that laminar specificity may be identical for local and long-range cortical projections.

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    05/01/07 | Flies at the farm: Drosophila at Janelia.
    Moses K
    Fly. 2007 May-Jun;1(3):139-41

    On August 1, 2006 the Howard Hughes Medical Institute's first stand-alone research campus opened at Janelia Farm, near Washington DC. Our mission at Janelia is to do exceptional fundamental research. Our two scientific foci are to understand the function of neural circuits and to develop synergistic imaging technologies. To achieve this we have changed many of the conventions of academic and/or industrial science. The founding director at Janelia is the well-known Drosophilist Gerry Rubin, who has been a central figure in fly molecular, developmental and genomic biology in recent decades. Not coincidentally, we at Janelia fully appreciate the potential of flies to contribute to an understanding of neuronal circuits. Our objectives are ambitious, and in the first ten months of operations at Janelia we have made some good beginnings.

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    04/25/07 | Differential regulation of action potential- and metabotropic glutamate receptor-induced Ca2+ signals by inositol 1,4,5-trisphosphate in dopaminergic neurons.
    Cui G, Bernier BE, Harnett MT, Morikawa H
    The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2007 Apr 25;27(17):4776-85. doi: 10.1523/JNEUROSCI.0139-07.2007

    Ca2+ signals associated with action potentials (APs) and metabotropic glutamate receptor (mGluR) activation exert distinct influences on neuronal activity and synaptic plasticity. However, it is not clear how these two types of Ca2+ signals are differentially regulated by neurotransmitter inputs in a single neuron. We investigated this issue in dopaminergic neurons of the ventral midbrain using brain slices. Intracellular Ca2+ was assessed by measuring Ca2+-sensitive K+ currents or imaging the fluorescence of Ca2+ indicator dyes. Tonic activation of metabotropic neurotransmitter receptors (mGluRs, alpha1 adrenergic receptors, and muscarinic acetylcholine receptors), attained by superfusion of agonists or weak, sustained (approximately 1 s) synaptic stimulation, augmented AP-induced Ca2+ transients. In contrast, Ca2+ signals elicited by strong, transient (50-200 ms) activation of mGluRs with aspartate iontophoresis were suppressed by superfusion of agonists. These opposing effects on Ca2+ signals were both mediated by an increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels, because they were blocked by heparin, an IP3 receptor antagonist, and reproduced by photolytic application of IP3. Evoking APs repetitively at low frequency (2 Hz) caused inactivation of IP3 receptors and abolished IP3 facilitation of single AP-induced Ca2+ signals, whereas facilitation of Ca2+ signals triggered by bursts of APs (five at 20 Hz) was attenuated by less than half. We further obtained evidence suggesting that the psychostimulant amphetamine may augment burst-induced Ca2+ signals via both depression of basal firing and production of IP3. We propose that intracellular IP3 tone provides a mechanism to selectively amplify burst-induced Ca2+ signals in dopaminergic neurons.

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    04/21/07 | Comparative analysis of spatial patterns of gene expression in Drosophila Melanogaster imaginal discs.
    Harmon C, Ahammad P, Hammonds AS, Weiszmann R, Celniker SE, Sastry S, Rubin GM
    International Conference on Research in Computational Molecular Biology. 2007 Apr 21:

    Determining the precise spatial extent of expression of genes across different tissues, along with knowledge of the biochemical function of the genes is critical for understanding the roles of various genes in the development of metazoan organisms. To address this problem, we have developed high-throughput methods for generating images of gene expression in Drosophila melanogaster imaginal discs and for the automated analysis of these images. Our method automatically learns tissue shapes from a small number of manually segmented training examples and automatically aligns, extracts and scores new images, which are analyzed to generate gene expression maps for each gene. We have developed a reverse lookup procedure that enables us to identify genes that have spatial expression patterns most similar to a given gene of interest. Our methods enable us to cluster both the genes and the pixels that of the maps, thereby identifying sets of genes that have similar patterns, and regions of the tissues of interest that have similar gene expression profiles across a large number of genes.

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    04/07/07 | Developing photo activated localization microscopy
    George H. Patterson , Eric Betzig , Jennifer Lippincott-Schwartz , Harald F. Hess
    4th IEEE International Symposium on Biomedical Imaging: From Nano to Macro. 2007 Apr 15:. doi: 10.1109/isbi.2007.357008

    In conventional biological imaging, diffraction places a limit on the minimal xy distance at which two marked objects can be discerned. Consequently, resolution of target molecules within cells is typically coarser by two orders of magnitude than the molecular scale at which the proteins are spatially distributed. Photoactivated localization microscopy (PALM) optically resolves selected subsets of protect fluorescent probes within cells at mean separations of <25 nanometers. It involves serial photoactivation and subsequent photobleaching of numerous sparse subsets of photoactivated fluorescent protein molecules. Individual molecules are localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities. In this paper, some of the limitations for PALM imaging under current experimental conditions are discussed.

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    04/12/07 | Automatic segmentation of nuclei in 3D microscopy images of C. elegans.
    Long F, Peng H, Myers E
    2007 4TH IEEE International Symposium on Biomedical Imaging: Macro to Nano, VOLS 1-3. 2007 Apr 12-15:536-9

    Automatic segmentation of nuclei in 3D microscopy images is essential for many biological studies including high throughput analysis of gene expression level, morphology, and phenotypes in single cell level. The complexity and variability of the microscopy images present many difficulties to the traditional image segmentation methods. In this paper, we present a new method based on 3D watershed algorithm to segment such images. By using both the intensity information of the image and the geometry information of the appropriately detected foreground mask, our method is robust to intensity fluctuation within nuclei and at the same time sensitive to the intensity and geometrical cues between nuclei. Besides, the method can automatically correct potential segmentation errors by using several post-processing steps. We tested this algorithm on the 3D confocal images of C.elegans, an organism that has been widely used in biological studies. Our results show that the algorithm can segment nuclei in high accuracy despite the non-uniform background, tightly clustered nuclei with different sizes and shapes, fluctuated intensities, and hollow-shaped staining patterns in the images.

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