Genetic dissection of an amygdala microcircuit that gates conditioned fear

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ^+ neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ^− neurons in CEl. Electrical silencing of PKC-δ^+ neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called Cel_(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing

RESPONSE OF A POROUS SEABED UNDER RANDOM WAVE LOADING

ABSTRACT Th

Random Wave-Induced Seabed Response (No. R864)

In this report, unlike the most previous investigations for wave-induced soil response, a simple analytical model for the random wave-induced soil response is established for an infinite or finite thickness. Two different wave spectra, B-M and JONSWAP spectra, are considered in the new model. The influence of random wave loading on the soil response is investigated by comparing with the corresponding representative regular wave results through a parametric study, which includes the effect of the degree of saturation, soil permeability, wave height, wave period and seabed thickness. The maximum liquefaction depth under the random waves is also examined. The difference on the soil response under the two random wave types, B-M and JONSWAP frequency spectra, is also discussed in the present work

Mislocalization of Rieske Protein PetA Predominantly Accounts for the Aerobic Growth Defect of <i>tat</i> Mutants in <i>Shewanella oneidensis</i>

<div><p><i>Shewanella oneidensis</i> exhibits a remarkable versatility in respiration, which largely relies on its various respiratory pathways. Most of these pathways are composed of secretory terminal reductases and multiple associated electron transport proteins that contain cofactors such as Fe-S, molybdopterin, and NiFe. The majority of these cofactors are inserted enzymatically in the cytoplasm, and thus are substrates of the twin-arginine translocation (Tat) protein export system, which transports fully folded proteins. Using genomic array footprinting, we discovered that loss of TatA or TatC caused a reduction in the growth rate of <i>S. oneidensis</i> under aerobic conditions. Mutational analysis of the predicted Tat substrates revealed that PetA, the Rieske Fe-S subunit of the ubiquinol-cytochrome <i>c</i> reductase, predominantly dictates the aerobic growth defect of <i>tat</i> mutants in <i>S. oneidensis</i>. In addition, evidence is presented that the signal sequence in PetA appears to be resistant to cleavage after the protein is inserted into the cytoplasmic membrane.</p> </div

Growth of <i>S. oneidensis tat</i> mutants under aerobic conditions.

<p>Fresh M1 defined medium was inoculated with overnight cultures grown from a single colony by 1∶100 dilution, incubated on a shaker at 250 rpm at 30°C under aerobic conditions, and growth was measured by recording cell densities of cultures at 600 nm. For clarity, mutants were presented in two panels, A and B. Δ<i>tat</i>^c represents Δ<i>tat</i> (Δ<i>tatABC</i>) containing complementation vector pHG101-<i>tatABC</i>. Δ<i>tatA</i>^c and Δ<i>tatC</i>^c represent Δ<i>tatA</i> and Δ<i>tatC</i> containing complementation vectors pHG101-<i>tatA</i> and pHG102-<i>tatC</i>, respectively. Growth assays were performed at least three times, with the error bars representing the standard deviation.</p

PetA is a Tat substrate in <i>S. oneidensis</i>.

<p>Fluorescence images of the wild type strain, Δ<i>tat</i> and its complemented strains carrying IPTG-inducible plasmid expressing hybrid protein PetA-GFP or PetA^KK-GFP. Cells at exponential phase (∼0.4 of OD₆₀₀) were induced by 0.1 mM IPTG for 2 hours, and then sampled and visualized under a Zeiss LSM-510 confocal laser scanning microscope. The images were taken 1 h after the end of induction.</p

Refined putative Tat substrates in <i>S. oneidensis</i>.

a<p>P: periplasmic protein; OM: outer membrane protein; IM: inner membrane protein; E: extracellular protein.</p

Characterization of mutants lacking one of Tat substrates in <i>S. oneidensis</i>.

<p><b>A.</b> Growth of the wild type, Δ<i>tat</i>, Δ<i>nrfA</i>, and Δ<i>dmsE</i> with DMSO (D) or nitrite (N) as the sole electron acceptor under anaerobic conditions. <b>B.</b> Growth of <i>S. oneidensis</i> mutants lacking one of Tat substrates. Generation time of each strain was deduced from the early exponential phase (∼0.1 to 0.4 of OD₆₀₀) and normalized to that of the wild type. Generation time of wild type is defined as 1.00. The single asterisk (<i>p</i><0.05) and double asterisk (<i>p</i><0.01) indicate significant difference in growth rate when compared to the wild type. The consensus motif in signal peptides of the 24 predicted Tat substrates prepared with WEBLOGO version 2.8.2 (<a href="http://weblogo.berkeley.edu" target="_blank">http://weblogo.berkeley.edu</a>) was shown. <b>C.</b> Growth of the wild type, Δ<i>tat</i>, Δ<i>petA</i>, and Δ<i>petA</i> complemented by pHG101 containing the wild type <i>petA</i> (Δ<i>petA</i>^c) or mutant <i>petA</i> (Δ<i>petA</i>^KK) under aerobic conditions. <i>petA</i>^KK encodes a protein whose conserved RR within the consensus motif of signal peptides was replaced by KK. Growth assays were performed at least three times, with the error bars representing the standard deviation.</p

<i>S. oneidensis</i> Tat system recognizes <i>E. coli</i> Tat signal peptide.

<p>Fluorescence images of the wild type, <i>tat</i> mutant and their complemented strains carrying IPTG-inducible plasmid expressing hybrid protein TorA_Ec-GFP (the signal peptide from the <i>E. coli</i> TorA fused to GFP). Cells at exponential phase (∼0.4 of OD₆₀₀) were induced by 0.1 mM IPTG for 2 hours, and then sampled and visualized under a Zeiss LSM-510 confocal laser scanning microscope. The images were taken 1 h after the end of induction.</p