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23 Publications
Showing 1-10 of 23 resultsWe describe an approach for automation of the process of reconstruction of neural tissue from serial section transmission electron micrographs. Such reconstructions require 3D segmentation of individual neuronal processes (axons and dendrites) performed in densely packed neuropil. We first detect neuronal cell profiles in each image in a stack of serial micrographs with multi-scale ridge detector. Short breaks in detected boundaries are interpolated using anisotropic contour completion formulated in fuzzy-logic framework. Detected profiles from adjacent sections are linked together based on cues such as shape similarity and image texture. Thus obtained 3D segmentation is validated by human operators in computer-guided proofreading process. Our approach makes possible reconstructions of neural tissue at final rate of about 5 microm3/manh, as determined primarily by the speed of proofreading. To date we have applied this approach to reconstruct few blocks of neural tissue from different regions of rat brain totaling over 1000microm3, and used these to evaluate reconstruction speed, quality, error rates, and presence of ambiguous locations in neuropil ssTEM imaging data.
Subiculum, the primary efferent pathway of hippocampus, participates in memory for spatial tasks, relapse to drug abuse, and temporal lobe seizures. Subicular pyramidal neurons exhibit low-threshold burst firing driven by a spike afterdepolarization. Here we report that burst firing can be regulated by stimulation of afferent projections to subiculum. Unlike synaptic plasticity, burst plasticity did not require synaptic depolarization, activation of AMPA or NMDA receptors, or action potential firing. Rather, enhancement of burst firing required synergistic activation of group I, subtype 1 metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChR). When either of these receptors was blocked, a suppression of bursting was revealed, which in turn was blocked by antagonists of group I, subtype 5 mGluRs. These results indicate that the output of subiculum can be strongly and bidirectionally regulated by activation of glutamatergic inputs within the hippocampus and cholinergic afferents from the medial septum.
Spine growth and retraction with synapse formation and elimination plays an important role in shaping brain circuits during development and in the adult brain, yet the temporal relationship between spine morphogenesis and the formation of functional synapses remains poorly defined. We imaged hippocampal pyramidal neurons to identify spines of different ages. We then used two-photon glutamate uncaging, whole-cell recording, and Ca(2+) imaging to analyze the properties of nascent spines and their older neighbors. New spines expressed glutamate-sensitive currents that were indistinguishable from mature spines of comparable volumes. Some spines exhibited negligible AMPA receptor-mediated responses, but the occurrence of these "silent" spines was uncorrelated with spine age. In contrast, NMDA receptor-mediated Ca(2+) accumulations were significantly lower in new spines. New spines reconstructed using electron microscopy made synapses. Our data support a model in which outgrowth and enlargement of nascent spines is tightly coupled to formation and maturation of glutamatergic synapses.
We present a reconstruction of the dynamics of flight initiation from kinematic data extracted from high-speed video recordings of the fruit fly Drosophila melanogaster. The dichotomy observed in this insect’s flight initiation sequences, generated by the presence or absence of visual stimuli, clearly generates two contrasting sets of dynamics once the flies become airborne. By calculating reaction forces and moments using the unconstrained motion formulation for a rigid body, we assess the fly’s responses amidst these two dynamic patterns as a step towards refining our understanding of insect flight control.
The neuronal immediate early gene Arc/Arg-3.1 is widely used as one of the most reliable molecular markers for intense synaptic activity in vivo. However, the cis-acting elements responsible for such stringent activity dependence have not been firmly identified. Here we combined luciferase reporter assays in cultured cortical neurons and comparative genome mapping to identify the critical synaptic activity-responsive elements (SARE) of the Arc/Arg-3.1 gene. A major SARE was found as a unique approximately 100-bp element located at >5 kb upstream of the Arc/Arg-3.1 transcription initiation site in the mouse genome. This single element, when positioned immediately upstream of a minimal promoter, was necessary and sufficient to replicate crucial properties of endogenous Arc/Arg-3.1’s transcriptional regulation, including rapid onset of transcription triggered by synaptic activity and low basal expression during synaptic inactivity. We identified the major determinants of SARE as a unique cluster of neuronal activity-dependent cis-regulatory elements consisting of closely localized binding sites for CREB, MEF2, and SRF. Consistently, a SARE reporter could readily trace and mark an ensemble of cells that have experienced intense activity in the recent past in vivo. Taken together, our work uncovers a novel transcriptional mechanism by which a critical 100-bp element, SARE, mediates a predominant component of the synapse-to-nucleus signaling in ensembles of Arc/Arg-3.1-positive activated neurons.
Understanding the diverse activities of the multisubunit core promoter recognition complex TFIID in vivo requires knowledge of how individual subunits contribute to overall functions of this TATA box-binding protein (TBP)/TBP-associated factor (TAF) complex. By generating altered holo-TFIID complexes in Drosophila we identify the ETO domain of TAF4 as a coactivator domain likely targeted by Pygopus, a protein that is required for Wingless-induced transcription of naked cuticle. These results establish a coactivator function of TAF4 and provide a strategy to dissect mechanisms of TFIID function in vivo.
Phenotypic plasticity allows organisms to quickly adapt in response to changing environments. Little is known of the genetic, environmental and epigenetic contribution to the expression of alternative adaptive developmental outcomes. We study aphid polyphenisms, which offer a unique, compelling opportunity to study multiple levels of biological organization, especially insect epigenetics. The pea aphid, Acyrthosiphon pisum, exhibits an adaptive reproductive polyphenism whereby genetically identical individuals reproduce either sexually (meiosis) or asexually (parthenogenesis) depending on environmental conditions during maternal development (short or long photoperiod, respectively). To understand how facultative asexuality evolved in aphids, we first determined meiosis gene activity in sexuals and asexuals. I determined that the pea aphid genome encodes single copies of homologs for the majority of the core meiotic machinery, suggesting that meiotic plasticity is not due simply to gene loss or expansion. Next, we determined if these core meiosis genes are expressed using PCR spanning across at least one intron from cDNA isolated from asexual and sexual ovaries. Surprisingly, meiosis specific genes (e.g., Spo11, Msh4, Msh5, Hop2 and Mnd1) are expressed in not only in asexual ovaries but also in somatic tissue and an obligately asexual aphid strain. Interestingly, the Spo11 PCR product contained intronic sequence, thus representing unspliced mRNA. Germline expression of Spo11, Mnd1 and Hop2 was confirmed by in situ analysis. Preliminary results identified candidate methylation sites in the Spo11 locus, indicating an epigenetic basis for this expression difference. Further characterization will help us better understand the molecular and epigenetic mechanisms underlying this adaptive facultative plasticity.
SmY RNAs are a family of approximately 70-90 nt small nuclear RNAs found in nematodes. In C. elegans, SmY RNAs copurify in a small ribonucleoprotein (snRNP) complex related to the SL1 and SL2 snRNPs that are involved in nematode mRNA trans-splicing. Here we describe a comprehensive computational analysis of SmY RNA homologs found in the currently available genome sequences. We identify homologs in all sequenced nematode genomes in class Chromadorea. We are unable to identify homologs in a more distantly related nematode species, Trichinella spiralis (class: Dorylaimia), and in representatives of non-nematode phyla that use trans-splicing. Using comparative RNA sequence analysis, we infer a conserved consensus SmY RNA secondary structure consisting of two stems flanking a consensus Sm protein binding site. A representative seed alignment of the SmY RNA family, annotated with the inferred consensus secondary structure, has been deposited with the Rfam RNA families database.
We introduce an efficient search strategy to substantially accelerate feature based registration. Previous feature based registration algorithms often use truncated search strategies in order to achieve small computation times. Our new accelerated search strategy is based on the realization that the search for corresponding features can be dramatically accelerated by utilizing Johnson-Lindenstrauss dimension reduction. Order of magnitude calculations for the search strategy we propose here indicate that the algorithm proposed is more than a million times faster than previously utilized naive search strategies, and this advantage in speed is directly translated into an advantage in accuracy as the fast speed enables more comparisons to be made in the same amount of time. We describe the accelerated scheme together with a full complexity analysis. The registration algorithm was applied to large transmission electron microscopy (TEM) images of neural ultrastructure. Our experiments demonstrate that our algorithm enables alignment of TEM images with increased accuracy and efficiency compared to previous algorithms.