Directional Theta Coherence in Prefrontal Cortical to Amygdalo-Hippocampal Pathways Signals Fear Extinction

Theta oscillations are considered crucial mechanisms in neuronal communication across brain areas, required for consolidation and retrieval of fear memories. One form of inhibitory learning allowing adaptive control of fear memory is extinction, a deficit of which leads to maladaptive fear expression potentially leading to anxiety disorders. Behavioral responses after extinction training are thought to reflect a balance of recall from extinction memory and initial fear memory traces. Therefore, we hypothesized that the initial fear memory circuits impact behavioral fear after extinction, and more specifically, that the dynamics of theta synchrony in these pathways signal the individual fear response. Simultaneous multi-channel local field and unit recordings were obtained from the infralimbic prefrontal cortex, the hippocampal CA1 and the lateral amygdala in mice. Data revealed that the pattern of theta coherence and directionality within and across regions correlated with individual behavioral responses. Upon conditioned freezing, units were phase-locked to synchronized theta oscillations in these pathways, characterizing states of fear memory retrieval. When the conditioned stimulus evoked no fear during extinction recall, theta interactions were directional with prefrontal cortical spike firing leading hippocampal and amygdalar theta oscillations. These results indicate that the directional dynamics of theta-entrained activity across these areas guide changes in appraisal of threatening stimuli during fear memory and extinction retrieval. Given that exposure therapy involves procedures and pathways similar to those during extinction of conditioned fear, one therapeutical extension might be useful that imposes artificial theta activity to prefrontal cortical-amygdalo-hippocampal pathways that mimics the directionality signaling successful extinction recall.     

The Common Ancestor Process Revisited

We consider the Moran model in continuous time with two types, mutation, and selection. We concentrate on the ancestral line and its stationary type distribution. Building on work by Fearnhead (J. Appl. Probab. 39 (2002), 38–54) and Taylor (Electron. J. Probab. 12 (2007), 808–847), we characterise this distribution via the fixation probability of the offspring of all individuals of favourable type (regardless of the offspring’s types). We concentrate on a finite population and stay with the resulting discrete setting all the way through. This way, we extend previous results and gain new insight into the underlying particle picture.     

Maternal eNOS deficiency determines a fatty liver phenotype of the offspring in a sex dependent manner

Maternal environmental factors can impact on the phenotype of the offspring via the induction of epigenetic adaptive mechanisms. The advanced fetal programming hypothesis proposes that maternal genetic variants may influence the offspring's phenotype indirectly via epigenetic modification, despite the absence of a primary genetic defect. To test this hypothesis, heterozygous female eNOS knockout mice and wild type mice were bred with male wild type mice. We then assessed the impact of maternal eNOS deficiency on the liver phenotype of wild type offspring. Birth weight of male wild type offspring born to female heterozygous eNOS knockout mice was reduced compared to offspring of wild type mice. Moreover, the offspring displayed a sex specific liver phenotype, with an increased liver weight, due to steatosis. This was accompanied by sex specific differences in expression and DNA methylation of distinct genes. Liver global DNA methylation was significantly enhanced in both male and female offspring. Also, hepatic parameters of carbohydrate metabolism were reduced in male and female offspring. In addition, male mice displayed reductions in various amino acids in the liver. Maternal genetic alterations, such as partial deletion of the eNOS gene, can affect liver metabolism of wild type offspring without transmission of the intrinsic defect. This occurs in a sex specific way, with more detrimental effects in females. This finding demonstrates that a maternal genetic defect can epigenetically alter the phenotype of the offspring, without inheritance of the defect itself. Importantly, these acquired epigenetic phenotypic changes can persist into adulthood.     

Improved Hidden Markov Model training for multiple sequence alignment by a particle swarm optimization-evolutionary algorithm hybrid

Multiple sequence alignment (MSA) is one of the basic problems in computational biology. Realistic problem instances of MSA are computationally intractable for exact algorithms. One way to tackle MSA is to use Hidden Markov Models (HMMs), which are known to be very powerful in the related problem domain of speech recognition. However, the training of HMMs is computationally hard and there is no known exact method that can guarantee optimal training within reasonable computing time. Perhaps the most powerful training method is the Baum-Welch algorithm, which is fast, but bears the problem of stagnation at local optima. In the study reported in this paper, we used a hybrid algorithm combining particle swarm optimization with evolutionary algorithms to train HMMs for the alignment of protein sequences. Our experiments show that our approach yields better alignments for a set of benchmark protein sequences than the most commonly applied HMM training methods, such as Baum-Welch and Simulated Annealing.     

Triplet Imaging of Oxygen Consumption during the Contraction of a Single Smooth Muscle Cell (A7r5)

The measurement of tissue and cell oxygenation is important for understanding cell metabolism. We have addressed this problem with a novel optical technique, called triplet imaging, that exploits oxygen-induced triplet lifetime changes and is compatible with a variety of fluorophores. A modulated excitation of varying pulse widths allows the extraction of the lifetime of the essentially dark triplet state using a high-fluorescence signal intensity. This enables the monitoring of fast kinetics of oxygen concentration in living cells combined with high temporal and spatial resolution. First, the oxygen-dependent triplet-state quenching of tetramethylrhodamine is validated and then calibrated in an L-ascorbic acid titration experiment demonstrating the linear relation between triplet lifetime and oxygen concentration according to the Stern-Volmer equation. Second, the method is applied to a biological cell system, employing as reporter a cytosolic fusion protein of β-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg⁸]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays monoexponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and the subcellular level.     

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation.     

Urinary protein profiling with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry in ETB receptor-deficient rats

The pathways leading to salt-sensitive hypertension and renal damage in rescued ETB receptor-deficient (ETBRd) rats are still unknown. The objective of the study was therefore to identify modifications of urinary peptide and protein expression in ETBRd rats (n = 9) and wild-type controls (n = 6) using SDS - polyacrylamide gel electrophoresis (SDS-PAGE) and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. Glomerular filtration rate, glomerulosclerosis, and tubulointerstitial fibrosis did not differ between the groups. ETBRd rats showed slightly higher blood pressure (p < 0.001), media/lumen ratio of intrarenal arteries (p < 0.01), and albuminuria (p < 0.01). SDS-PAGE confirmed albuminuria, but showed no differences in the urinary excretion of low molecular weight proteins (<60 kDa). SELDI-TOF-MS profiling revealed 9 proteomic features at molecular masses (Da) of 2720, 2980, 3130, 3345, 6466, 6682, 8550, 18 729, and 37 492, which were significantly elevated (p < 0.02) in urine of ETBRd rats. The results demonstrate that, independent of structural changes in the kidneys, ETB-receptor deficiency causes specific differences in urinary peptide and protein excretion. SELDI-TOF-MS may be a valuable tool for the characterization of urinary biomarkers helping to uncover the mechanism of ETBR action in the kidney.     

Glycoprotein Capture and Quantitative Phosphoproteomics Indicate Coordinated Regulation of Cell Migration upon Lysophosphatidic Acid Stimulation

The lipid mediator lysophosphatidic acid (LPA) is a serum component that regulates cellular functions such as proliferation, migration, and survival via specific G protein-coupled receptors. The underlying signaling mechanisms are still incompletely understood, including those that operate at the plasma membrane to modulate cell-cell and cell-matrix interactions in LPA-promoted cell migration. To explore LPA-evoked phosphoregulation with a focus on cell surface proteins, we combined glycoproteome enrichment by immobilized lectins with SILAC-based quantitative phosphoproteomics. We performed biological replicate analyses in SCC-9 squamous cell carcinoma cells and repeatedly quantified the effect of 1.5- and 5-min LPA treatment on more than 700 distinct phosphorylations in lectin-purified proteins. We detected many regulated phosphorylation events on various types of plasma membrane proteins such as cell adhesion molecules constituting adherens junctions, desmosomes, and hemidesmosomes. Several of these LPA-regulated phosphorylation sites have been characterized in a biological context other than G protein-coupled receptor signaling, and the transfer of this functional information suggests coordinated and multifactorial cell adhesion control in LPA-induced cell migration. Additionally, we identified LPA-mediated activation loop phosphorylation of the serine/threonine kinase Wnk1 and verified a role of Wnk1 for LPA-induced cell migration in knock-down experiments. In conclusion, the glycoproteome phosphoproteomics strategy described here sheds light on incompletely understood mechanisms in LPA-induced cell migratory behavior.The plasma membrane separates the interior of a mammalian cell from the environment. To respond to external signals such as growth factors, cells possess various types of plasma membrane-spanning receptors that communicate to the intracellular signaling machinery in a ligand-regulated manner. G protein-coupled receptors (GPCRs),1 which are integral membrane proteins with seven transmembrane helices, constitute the largest superfamily of cell surface receptors. GPCRs mediate intracellular activation of heterotrimeric G proteins in response to extracellular ligand binding. A plethora of different factors are known to act on GPCRs, including peptide ligands, proteases, nucleotides as well as bioactive lipid molecules such as lysophosphatidic acid (LPA). LPA induces various biological responses including proliferation and migration in a wide range of mammalian cell types and has been implicated in the progression of several human cancers (1, 2). Upon LPA binding to its cognate receptors, heterotrimeric G proteins from the G_i, G_q, and G_12/13 families are activated by guanine nucleotide exchange factors resulting in their dissociation into activated Gα and Gβγ subunits. Activated G protein subunits interact with various effector proteins including phospholipase C and adenylate cyclase isoforms as well as guanine nucleotide exchange factors for Rho family GTPases, which either directly or via second messenger production communicate to cellular kinase signaling. GPCR activation by LPA is also known to trigger the proteolytic activity of ADAM transmembrane metalloproteases, such as ADAM17, which processes epidermal growth factor receptor (EGFR) ligand precursors on the extracellular side to release mature growth factors triggering EGFR activation (3–8). The molecular mechanisms involved in the control of ADAM metalloprotease activity are not clear yet. The resulting transactivation of the EGFR tyrosine kinase provides a link to signaling modules such as mitogen-activated protein kinase cascades and has been implicated in the control of cell proliferation and migration upon LPA treatment (9). Regarding the induction of cell motility upon LPA, previous studies have reported several signaling elements in addition to EGFR transactivation that contribute to this complex cellular behavior. In particular, RhoGTPase-dependent signals that activate downstream effectors such as Rho kinase and focal adhesion kinase are involved in the control of cytoskeletal organization and cell attachment to the surrounding extracellular matrix (ECM) (10, 11). The coordinated regulation of such integrin-mediated interactions is required to enable cell movement and occurs in dedicated macromolecular assemblies such as focal adhesion complexes and hemidesmosomes (12, 13). Despite the key role of integrins, the molecular mechanisms that underlie their functional modulation upon GPCR activation are poorly understood. Moreover, cell-cell contacts such as adherens junctions and desmosomes have to dissociate prior to cell migration. Likewise, it is unclear how the components of these structures, such as members of the cadherin family, might be regulated by GPCR-mediated signaling pathways. Both LPA levels and LPA_1–3 receptor expression are often elevated in cancer patients, and the bioactive lipid acts as a potent inducer of cell migration and invasion in vitro. Due to the key role of protein phosphorylation in LPA-induced signal transmission, comprehensive phosphorylation analysis of regulated proteins might generate new insights into pro-migratory signaling mechanisms in cancer cells. Mass spectrometry (MS)-based analysis has emerged as the key method for unbiased protein phosphorylation studies due to various technological advances in recent years (14, 15). Because of the substoichiometric nature of many site-specific phosphorylation events, phosphopeptides constitute only a small fraction in total peptide samples. Therefore, they need to be efficiently enriched prior to MS analysis, which has become routinely possible by phosphate group-selective purification strategies employing capture reagents such as immobilized metal ion affinity chromatography or titanium dioxide beads (16). Moreover, phosphopeptide analysis has benefited enormously from the availability of hybrid mass spectrometers that combine the sensitivity and speed of linear ion traps with the high resolution and accuracy of orbitrap mass analyzers (17). These advances together with quantitative approaches such as stable isotope labeling by amino acids in cell cultures (SILAC) (18, 19) and substantial progress in computational proteomics (20) now allow for concomitant identification and quantification of several thousand phosphorylation sites from single cellular extracts (21–23).We previously analyzed cell signaling responses in A498 kidney carcinoma cells upon LPA and heparin-binding EGF-like growth factor treatment by monitoring phosphorylation changes in total cell lysate and protein kinase-enriched fractions (24). In a complementary approach, we now aimed for a systematic survey of LPA-induced phosphorylation changes on plasma membrane proteins and their interaction partners. Furthermore, we were interested in time-resolved analysis of LPA-induced phosphorylation changes on ADAM17 and the EGFR to gain further insights into possible mechanisms underlying the still enigmatic EGFR transactivation process. In our present study, we therefore analyzed SCC-9 squamous carcinoma cells due to their pronounced EGFR transactivation response upon LPA. As plasma membrane proteins usually contain covalently attached carbohydrate structures, we performed lectin affinity enrichment of glycosylated proteins prior to SILAC-based quantitative phosphoproteomics (25). This experimental strategy enabled us to acquire in-depth data about LPA regulation of diverse glycoproteins and revealed coordinated phosphoregulation of cell adhesion proteins as likely mechanism underlying cell migratory behavior.     

Mechanoreception and synaptic transmission of hydrozoan nematocytes

Mechanoelectric transduction and its ultrastuctural basis were studied in the cnidocil apparatus of stenotele nematocytes of marine and freshwater Hydrozoa (Capitata and Hydra) as a paradigm for invertebrate hair cells with concentric hair bundles. The nematocytes respond to selective deflection of their cnidocil with phasic-tonic receptor currents and potentials, similar to vertebrate hair cells but without directional dependence of sensitivity. Ultrastructural studies and the use of monoclonal antibodies allowed correlating the mechanoelectric transduction with structural components of the hair bundle. Two other types of depolarising current and voltage changes in nematocytes are postsynaptic, as concluded from their ionic and pharmacological characteristics. One of these types is induced by mechanical stimulation of distant nematocytes and sensory hair cells. It is graded in amplitude and duration, but different from the presynaptic receptor potential. Adequate chemical stimulation of the stenoteles strongly increases the probability of discharge of their cnidocyst, if the chemical stimulus precedes the mechanical one. Simultaneously, the probability of synaptic signalling induced by mechanical stimulation is increased, reaching nearly 100%. The chemoreception of the phospholipids used could be localized in the shaft of the cnidocil, because of the water-insolubility of the stimulant. This chemical stimulation itself does not cause a receptor potential; its action is classified as a modulatory process. Electron microscopy of serial sections of the tentacular spheres of Coryne revealed synapses that are efferent to nematocytes and hair cells besides neurite–neurite synapses, each containing 3–10 clear and/or dense-core vesicles of 70–150 nm diameter. The only candidates to explain the graded afferent signal transmission of nematocytes and hair cells are regularly occurring cell contacts associated with 1(–4) clear vesicles of 160–1100 nm diameter. Transient fusion and partial depletion of stationary vesicles are discussed as mechanisms to reconcile functional and structural data of many cnidarian synapses. Review contributed to the Symposium on Neuro-Anatomy and -Physiology of Coelenterates; 7th International Conference on Coelenterate Biology, Lawrence, Kansas, USA; July 6–11, 2003.