Modulation of Network Excitability by Persistent Activity: How Working Memory Affects the Response to Incoming Stimuli

Persistent activity and match effects are widely regarded as neuronal correlates of short-term storage and manipulation of information, with the first serving active maintenance and the latter supporting the comparison between memory contents and incoming sensory information. The mechanistic and functional relationship between these two basic neurophysiological signatures of working memory remains elusive. We propose that match signals are generated as a result of transient changes in local network excitability brought about by persistent activity. Neurons more active will be more excitable, and thus more responsive to external inputs. Accordingly, network responses are jointly determined by the incoming stimulus and the ongoing pattern of persistent activity. Using a spiking model network, we show that this mechanism is able to reproduce most of the experimental phenomenology of match effects as exposed by single-cell recordings during delayed-response tasks. The model provides a unified, parsimonious mechanistic account of the main neuronal correlates of working memory, makes several experimentally testable predictions, and demonstrates a new functional role for persistent activity.     

Cytokine expression and ultrastructural alterations in fresh-frozen,freeze-dried and γ-irradiated human amniotic membranes

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.     

Structural study of the ovary,oogenesis and brooding in Tonicia lebruni (Polyplacophora Chitonidae) from Patagonia

Tonicia lebruni, a common, lower intertidal and subtidal chiton inhabiting Patagonian rocky shores, is a gonochoristic iteroparous species producing large eggs (≈ 400 μm in diameter), which are fertilized and brooded within the pallial groves until released as juveniles. A free larval stage is absent, despite this, T. lebruni is widely distributed along the south‐western Atlantic. At Puerto Deseado, T. lebruni has a marked seasonality in the reproductive cycle, reproducing only once a year. The reproductive period is quite short and defined in time: spawning and brooding take place during the late austral winter and beginning of spring. Recovery of the female gonad starts very soon after spawning. Oogenesis takes about 10–11 months for completion. Brood size is correlated with length of maternal individual. The number of embryos per brood varied between 785 and 5945. Extensive resorption of abortive eggs is viewed as related to limitation of space available for brooding. The egg hull is formed by a large number of minute pentagonal or hexagonal plates each one bearing a short spine bent onto the egg surface. The morphology and the surface of the hull could contribute to the cohesiveness of the brooded egg mass within the pallial grooves.     

The Parkinson’s disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson’s disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.     

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed.Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms.We found that the 15th exon encodes for the distal β-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner.As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.     

Multisensory Interactions between Vestibular,Visual and Somatosensory Signals

Vestibular inputs are constantly processed and integrated with signals from other sensory modalities, such as vision and touch. The multiply-connected nature of vestibular cortical anatomy led us to investigate whether vestibular signals could participate in a multi-way interaction with visual and somatosensory perception. We used signal detection methods to identify whether vestibular stimulation might interact with both visual and somatosensory events in a detection task. Participants were instructed to detect near-threshold somatosensory stimuli that were delivered to the left index finger in one half of experimental trials. A visual signal occurred close to the finger in half of the trials, independent of somatosensory stimuli. A novel Near infrared caloric vestibular stimulus (NirCVS) was used to artificially activate the vestibular organs. Sham stimulations were used to control for non-specific effects of NirCVS. We found that both visual and vestibular events increased somatosensory sensitivity. Critically, we found no evidence for supra-additive multisensory enhancement when both visual and vestibular signals were administered together: in fact, we found a trend towards sub-additive interaction. The results are compatible with a vestibular role in somatosensory gain regulation.     

TNFA Haplotype Genetic Testing Improves HLA in Estimating the Risk of Celiac Disease in Children

Background

TNF-α and IFN-γ play a role in the development of mucosal damage in celiac disease (CD). Polymorphisms of TNFA and IFNG genes, as well as of the TNFRSF1A gene, encoding the TNF-α receptor 1, might underlie different inter-individual disease susceptibility over a common HLA risk background. The aims of this study were to ascertain whether five SNPs in the TNFA promoter (-1031T>C,-857C>T,-376G>A,-308G>A,-238G>A), sequence variants of the TNFRSF1A gene and IFNG +874A>T polymorphism are associated with CD in a HLA independent manner.

Methods

511 children (244 CD, 267 controls) were genotyped for HLA, TNFA and INFG (Real Time PCR). TNFRSF1A variants were studied (DHPLC and sequence).

Results

Only the rare TNFA-1031C (OR=0.65, 95% CI:0.44-0.95), -857T (OR=0.42, 95% CI:0.27-0.65), -376A (OR=2.25, 95% CI:1.12-4.51) and -308A (OR=4.76, 95% CI:3.12-7.26) alleles were significantly associated with CD. One TNFRSF1A variant was identified (c.625+10A>G, rs1800693), but not associated with CD. The CD-correlated TNFA SNPs resulted in six haplotypes. Two haplotypes were control-associated (CCGG and TTGG) and three were CD-associated (CCAG, TCGA and CCGA). The seventeen inferred haplotype combinations were grouped (A to E) based on their frequencies among CD. Binary logistic regression analysis documented a strong association between CD and HLA (OR for intermediate risk haplotypes=178; 95% CI:24-1317; OR for high risk haplotypes=2752; 95% CI:287-26387), but also an HLA-independent correlation between CD and TNFA haplotype combination groups. The CD risk for patients carrying an intermediate risk HLA haplotype could be sub-stratified by TNFA haplotype combinations.

Conclusion

TNFA promoter haplotypes associate with CD independently from HLA. We suggest that their evaluation might enhance the accuracy in estimating the CD genetic risk.     

Effects of the Infusion of 4% or 20% Human Serum Albumin on the Skeletal Muscle Microcirculation in Endotoxemic Rats

Background

Sepsis-induced microcirculatory alterations contribute to tissue hypoxia and organ dysfunction. In addition to its plasma volume expanding activity, human serum albumin (HSA) has anti-oxidant and anti-inflammatory properties and may have a protective role in the microcirculation during sepsis. The concentration of HSA infused may influence these effects. We compared the microcirculatory effects of the infusion of 4% and 20% HSA in an experimental model of sepsis.

Methods

Adult male Wistar rats were equipped with arterial and venous catheters and received an intravenous infusion of lipopolysaccharide (LPS, serotype O127:B8, 10 mg/kg over 30 minutes) or vehicle (SHAM, n = 6). Two hours later, endotoxemic animals were randomized to receive 10 mL/kg of either 4% HSA (LPS+4%HSA, n = 6), 20% HSA (LPS+20%HSA, n = 6) or 0.9% NaCl (LPS+0.9%NaCl, n = 6). No fluids were given to an additional 6 animals (LPS). Vessel density and perfusion were assessed in the skeletal muscle microcirculation with sidestream dark field videomicroscopy at baseline (t0), 2 hours after LPS injection (t1), after HSA infusion (t2) and 1 hour later (t3). The mean arterial pressure (MAP) and heart rate were recorded. Serum endothelin-1 was measured at t2.

Results

MAP was stable over time in all groups. The microcirculatory parameters were significantly altered in endotoxemic animals at t1. The infusion of both 4% and 20% HSA similarly increased the perfused vessel density and blood flow velocity and decreased the flow heterogeneity to control values. Microvascular perfusion was preserved in the LPS+20%HSA group at t3, whereas alterations reappeared in the LPS+4%HSA group.

Conclusions

In a rat model of normotensive endotoxemia, the infusion of 4% or 20% HSA produced a similar acute improvement in the microvascular perfusion in otherwise unresuscitated animals.     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

SMG6 is the catalytic endonuclease that cleaves mRNAs containing nonsense codons in metazoan

Messenger RNAs harboring nonsense codons (or premature translation termination codons [PTCs]) are degraded by a conserved quality-control mechanism known as nonsense-mediated mRNA decay (NMD), which prevents the accumulation of truncated and potentially harmful proteins. In Drosophila melanogaster, degradation of PTC-containing messages is initiated by endonucleolytic cleavage in the vicinity of the nonsense codon. The endonuclease responsible for this cleavage has not been identified. Here, we show that SMG6 is the long sought NMD endonuclease. First, cells expressing an SMG6 protein mutated at catalytic residues fail to degrade PTC-containing messages. Moreover, the SMG6-PIN domain can be replaced with the active PIN domain of an unrelated protein, indicating that its sole function is to provide endonuclease activity for NMD. Unexpectedly, we found that the catalytic activity of SMG6 contributes to the degradation of PTC-containing mRNAs in human cells. Thus, SMG6 is a conserved endonuclease that degrades mRNAs terminating translation prematurely in metazoa.