Towards advancing scientific knowledge of climate change impacts on short-duration rainfall extremes.

A large number of recent studies have aimed at understanding short-duration rainfall extremes, due to their impacts on flash floods, landslides and debris flows and potential for these to worsen with global warming. This has been led in a concerted international effort by the INTENSE Crosscutting Project of the GEWEX (Global Energy and Water Exchanges) Hydroclimatology Panel. Here, we summarize the main findings so far and suggest future directions for research, including: the benefits of convection-permitting climate modelling; towards understanding mechanisms of change; the usefulness of temperature-scaling relations; towards detecting and attributing extreme rainfall change; and the need for international coordination and collaboration. Evidence suggests that the intensity of long-duration (1 day+) heavy precipitation increases with climate warming close to the Clausius-Clapeyron (CC) rate (6-7% K-1), although large-scale circulation changes affect this response regionally. However, rare events can scale at higher rates, and localized heavy short-duration (hourly and sub-hourly) intensities can respond more strongly (e.g. 2 × CC instead of CC). Day-to-day scaling of short-duration intensities supports a higher scaling, with mechanisms proposed for this related to local-scale dynamics of convective storms, but its relevance to climate change is not clear. Uncertainty in changes to precipitation extremes remains and is influenced by many factors, including large-scale circulation, convective storm dynamics andstratification. Despite this, recent research has increased confidence in both the detectability and understanding of changes in various aspects of intense short-duration rainfall. To make further progress, the international coordination of datasets, model experiments and evaluations will be required, with consistent and standardized comparison methods and metrics, and recommendations are made for these frameworks. This article is part of a discussion meeting issue 'Intensification of short-duration rainfall extremes and implications for flash flood risks'.

The directed migration of gonadal distal tip cells in Caenorhabditis elegans requires NGAT-1, a ß1,4-N-acetylgalactosaminyltransferase enzyme.

Glycoproteins such as growth factor receptors and extracellular matrix have well-known functions in development and cancer progression, however, the glycans at sites of modification are often heterogeneous molecular populations which makes their functional characterization challenging. Here we provide evidence for a specific, discrete, well-defined glycan modification and regulation of a stage-specific cell migration in Caenorhabditis elegans. We show that a chain-terminating, putative null mutation in the gene encoding a predicted ß1,4-N-acetylgalactosaminyltransferase, named ngat-1, causes a maternally rescued temperature sensitive (ts) defect in the second phase of the three phase migration pattern of the posterior, but not the anterior, hermaphrodite Distal Tip Cell (DTC). An amino-terminal partial deletion of ngat-1 causes a similar but lower penetrance ts phenotype. The existence of multiple ts alleles with distinctly different molecular DNA lesions, neither of which is likely to encode a ts protein, indicates that NGAT-1 normally prevents innate temperature sensitivity for phase 2 DTC pathfinding. Temperature shift analyses indicate that the ts period for the ngat-1 mutant defect ends by the beginning of post-embryonic development-nearly 3 full larval stages prior to the defective phase 2 migration affected by ngat-1 mutations. NGAT-1 homologs generate glycan-terminal GalNAc-ß1-4GlcNAc, referred to as LacdiNAc modifications, on glycoproteins and glycolipids. We also found that the absence of the GnT1/Mgat1 activity [UDP-N-acetyl-D-glucosamine:α-3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase 1 (encoded by C. elegans gly-12, gly-13, and gly-14 and homologous to vertebrate GnT1/Mgat1)], causes a similar spectrum of DTC phenotypes as ngat-1 mutations-primarily affecting posterior DTC phase 2 migration and preventing manifestation of the same innate ts period as ngat-1. GnT1/Mgat1 is a medial Golgi enzyme known to modify mannose residues and initiate N-glycan branching, an essential step in the biosynthesis of hybrid, paucimannose and complex-type N-glycans. Quadruple mutant animals bearing putative null mutations in ngat-1 and the three GnT genes (gly-12, gly-13, gly-14) were not enhanced for DTC migration defects, suggesting NGAT-1 and GnT1 act in the same pathway. These findings suggest that GnTI generates an N-glycan substrate for NGAT-1 modification, which is required at restrictive temperature (25°C) to prevent, stabilize, reverse or compensate a perinatal thermo-labile process (or structure) causing late larval stage DTC phase 2 migration errors.

Sensitivity and specificity of three-point compression ultrasonography performed by emergency physicians for proximal lower extremity deep venous thrombosis.

OBJECTIVE: The present study aims to quantify the sensitivity and specificity of three-point compression ultrasonography for diagnosing proximal lower extremity deep venous thrombosis when performed by Australian consultant emergency physicians with limited specific training. Secondary aims included quantifying rapidity, technical adequacy, predictability of equivocal results and relationships between emergency physician experience and proficiency. METHODS: This prospective diagnostic study enrolled a convenience sample of adult patients presenting to a major ED with suspected lower extremity deep venous thrombosis. The index test was abbreviated compression ultrasonography examining three points: common femoral, proximal great saphenous and popliteal veins. Emergency physicians received specific training. The reference test was full-leg duplex ultrasonography in the Radiology Department. RESULTS: A total of 15 emergency physicians participated, enrolling 178 subjects. Sensitivity of the index test was 77.8% (95% confidence interval: 54.8-91.0%), specificity was 91.4% (95% confidence interval: 84.9%-95.3%) and accuracy was 89.6% (95% confidence interval: 83.1-94.2%). Median duration of the index test was 10 min 34 s (interquartile range: 6 min 31 s) and ED diagnosis occurred significantly before Radiology Department diagnosis. The only statistically significant relationship between emergency physician experience and proficiency related to rapidity, which increased from the 36th scan. Equivocal index tests occurred in 9.2% of examinations and emergency physicians predicted equivocal assessments with specificity of 86.1% (95% confidence interval: 78.8-91.1%). CONCLUSIONS: Abbreviated ultrasonography performed by emergency physicians for proximal lower extremity deep venous thrombosis could be valuable. However, more precise estimates for sensitivity and greater understanding of relationships between emergency physician experience and proficiency are required.

Distribution of muscarinic acetylcholine receptor mRNA in the brain of the weakly electric fish Apteronotus leptorhynchus.

Various neuromodulators have been shown to be involved in shaping the sensory information available to the brain. Acetylcholine (ACh) modulation, through muscarinic receptors, is a particularly widespread mechanism of controlling sensory information transmission. The precise effects of ACh modulation depend on the subtype of muscarinic ACh receptors that are activated. In weakly electric fish, previous work suggested a role of ACh, via muscarinic receptors, in the modulation of information transmission in the electrosensory lateral line lobe (ELL) of the hindbrain. In this study, we determined which muscarinic receptor (mAChR) subtypes are present in the brain of Apteronotus leptorhynchus as well as their spatial distribution. We partially cloned three subtypes of muscarinic receptors (mAChR2, -3, and -4) from brain tissue of A. leptorhynchus and used in situ hybridization in transverse sections of the brain to determine their distributions. Sites labeled for the three muscarinic receptor mRNAs were found in various brain regions devoted to the processing of different sensory modalities. The mRNA probes for the three receptor types showed differential distribution but also overlapping presence of two or more receptors in particular nuclei. In addition to the presence of mAChR3 in the ELL region, electrosensory nuclei including the nucleus praeeminentialis, dorsal torus semicircularis and optic tectum showed expression of one or more mAChRs. Thus, the overall pattern of mAChR expression found is in agreement with mAChR expression in other species, with additional presence evident in specialized regions of the electrosensory system, which suggests an important modulating role of ACh in this sensory modality.

Ionic and neuromodulatory regulation of burst discharge controls frequency tuning.

Sensory neurons encode natural stimuli by changes in firing rate or by generating specific firing patterns, such as bursts. Many neural computations rely on the fact that neurons can be tuned to specific stimulus frequencies. It is thus important to understand the mechanisms underlying frequency tuning. In the electrosensory system of the weakly electric fish, Apteronotus leptorhynchus, the primary processing of behaviourally relevant sensory signals occurs in pyramidal neurons of the electrosensory lateral line lobe (ELL). These cells encode low frequency prey stimuli with bursts of spikes and high frequency communication signals with single spikes. We describe here how bursting in pyramidal neurons can be regulated by intrinsic conductances in a cell subtype specific fashion across the sensory maps found within the ELL, thereby regulating their frequency tuning. Further, the neuromodulatory regulation of such conductances within individual cells and the consequences to frequency tuning are highlighted. Such alterations in the tuning of the pyramidal neurons may allow weakly electric fish to preferentially select for certain stimuli under various behaviourally relevant circumstances.

Localization of the Diaphanous-related formin Daam1 to neuronal dendrites.

The Rho family of small GTPase proteins are involved in the formation and maintenance of neuronal dendrites. In this study, we show that Daam1, a member of the Diaphanous-related formin protein family and a downstream effector for RhoA, is localized to the dendrites of hippocampal neurons. Immunoblot analysis showed that Daam1 is enriched in the mouse hippocampus and co-fractionates in brain lysates with dendritic and synaptic proteins. Immunohistochemical analysis revealed that Daam1 protein distributes in a punctate pattern throughout the cell body and dendritic shafts of dissociated hippocampal neurons and organotypic hippocampal cultures. Although Daam1 is mostly expressed in the shaft of dendrites, co-stainings with SV2 or PSD95 revealed that Daam1 is also present at some synapses. In addition, viral directed expression of a fluorescently tagged Daam1 fusion protein in hippocampal slices resulted in targeted delivery to the dendrites of pyramidal neurons, leading to a reduction in the density of spines.

Differential distribution of SK channel subtypes in the brain of the weakly electric fish Apteronotus leptorhynchus.

Calcium signals in vertebrate neurons can induce hyperpolarizing membrane responses through the activation of Ca(2+)-activated potassium channels. Of these, small conductance (SK) channels regulate neuronal responses through the generation of the medium after-hyperpolarization (mAHP). We have previously shown that an SK channel (AptSK2) contributes to signal processing in the electrosensory system of Apteronotus leptorhynchus. It was shown that for pyramidal neurons in the electrosensory lateral line lobe (ELL), AptSK2 expression selectively decreases responses to low-frequency signals. The localization of all the SK subunits throughout the brain of Apteronotus then became of substantial interest. We have now cloned two additional SK channel subunits from Apteronotus and determined the expression patterns of all three AptSK subunits throughout the brain. In situ hybridization experiments have revealed that, as in mammalian systems, the AptSK1 and 2 channels showed a partially overlapping expression pattern, whereas the AptSK3 channel was expressed in different brain areas. The AptSK1 and 2 channels were the primary subunits found in the major electrosensory processing areas. Immunohistochemistry further revealed distinct compartmentalization of the AptSK1 and 2 channels in the ELL. AptSK1 was localized to the apical dendrites of pyramidal neurons, whereas AptSK2 channels are primarily somatic. The distinct expression patterns of all three AptSK channels may reflect subtype-specific contributions to neuronal function, and the high homology between subtypes from a number of species suggests that the functional roles for each channel subtype are conserved from early vertebrate evolution.

Regulated expression of N-methyl-D-aspartate receptors and associated proteins in teleost electrosensory system and telencephalon.

Several types of N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity are characterized by differences in polarity, induction parameters, and duration, which depend on the interactions of NMDARs with intracellular synaptic and signaling proteins. Here, we examine the NMDAR signaling components in the brain of the weakly electric fish Apteronotus leptorhynchus. Compared with mammalian orthologs, high levels of sequence conservation for known functional sites in both NMDAR subunits (NR1, NR2A-C) and signaling proteins (fyn tyrosine kinase, RasGRF-1 and -2) were found. In situ hybridization analysis demonstrated that, similar to the case in the adult mammal brain, NR2A and NR2B are expressed at moderate levels in most brain regions and at very high levels in the dorsal telencephalon. RasGRF-1 and fyn have a similar distribution and appear to be coexpressed with NR2B in telencephalic regions known to support learning and long-term memory. Both NR2A and NR2B are highly expressed in pyramidal cells of the electrosensory lateral line lobe (ELL) known to exhibit the short-term synaptic plasticity that underlies adaptive feedback cancellation of redundant sensory input. In contrast, nonplastic pyramidal cells expressed only the NR2A subunit. Furthermore, field recordings show that ifenprodil-sensitive NR2B-containing NMDARs predominate for the plastic feedback input to ELL pyramidal cells. However, RasGRF-1 and fyn are expressed only at low levels in a subset of these pyramidal cells. Our data suggest that NMDAR functions are highly conserved between fish and mammals and that synaptic plasticity dynamics in different brain regions are related to the expression patterns of the synaptic signaling proteins interacting with NMDARs.

SK channels provide a novel mechanism for the control of frequency tuning in electrosensory neurons.

One important characteristic of sensory input is frequency, with sensory neurons often tuned to narrow stimulus frequency ranges. Although vital for many neural computations, the cellular basis of such frequency tuning remains mostly unknown. In the electrosensory system of Apteronotus leptorhynchus, the primary processing of important environmental and communication signals occurs in pyramidal neurons of the electrosensory lateral line lobe. Spike trains transmitted by these cells can encode low-frequency prey stimuli with bursts of spikes and high-frequency communication signals with single spikes. Here, we demonstrate that the selective expression of SK2 channels in a subset of pyramidal neurons reduces their response to low-frequency stimuli by opposing their burst responses. Apamin block of the SK2 current in this subset of cells induced bursting and increased their response to low-frequency inputs. SK channel expression thus provides an intrinsic mechanism that predisposes a neuron to respond to higher frequencies and thus specific, behaviorally relevant stimuli.

Muscarinic receptors control frequency tuning through the downregulation of an A-type potassium current.

The functional role of cholinergic input in the modulation of sensory responses was studied using a combination of in vivo and in vitro electrophysiology supplemented by mathematical modeling. The electrosensory system of weakly electric fish recognizes different environmental stimuli by their unique alteration of a self-generated electric field. Variations in the patterns of stimuli are primarily distinguished based on their frequency. Pyramidal neurons in the electrosensory lateral line lobe (ELL) are often tuned to respond to specific input frequencies. Alterations in the tuning of the pyramidal neurons may allow weakly electric fish to preferentially select for certain stimuli. Here we show that muscarinic receptor activation in vivo enhances the excitability, burst firing, and subsequently the response of pyramidal cells to naturalistic sensory input. Through a combination of in vitro electrophysiology and mathematical modeling, we reveal that this enhanced excitability and bursting likely results from the down-regulation of an A-type potassium current. Further, we provide an explanation of the mechanism by which these currents can mediate frequency tuning.

A C-terminal domain directs Kv3.3 channels to dendrites.

Pyramidal neurons of the electrosensory lateral line lobe (ELL) of Apteronotus leptorhynchus express Kv3-type voltage-gated potassium channels that give rise to high-threshold currents at the somatic and dendritic levels. Two members of the Kv3 channel family, AptKv3.1 and AptKv3.3, are coexpressed in these neurons. AptKv3.3 channels are expressed at uniformly high levels in each of four ELL segments, whereas AptKv3.1 channels appear to be expressed in a graded manner with higher levels of expression in segments that process high-frequency electrosensory signals. Immunohistochemical and recombinant channel expression studies show a differential distribution of these two channels in the dendrites of ELL pyramidal neurons. AptKv3.1 is concentrated in somas and proximal dendrites, whereas AptKv3.3 is distributed throughout the full extent of the large dendritic tree. Recombinant channel expression of AptKv3 channels through in vivo viral injections allowed directed retargeting of AptKv3 subtypes over the somadendritic axis, revealing that the sequence responsible for targeting channels to distal dendrites lies within the C-terminal domain of the AptKv3.3 protein. The targeting domain includes a consensus sequence predicted to bind to a PDZ (postsynaptic density-95/Discs large/zona occludens-1)-type protein-protein interaction motif. These findings reveal that different functional roles for Kv3 potassium channels at the somatic and dendritic level of a sensory neuron are attained through specific targeting that selectively distributes Kv3.3 channels to the dendritic compartment.

Inactivation of Kv3.3 potassium channels in heterologous expression systems.

Kv3.3 K+ channels are believed to incorporate an NH2-terminal domain to produce an intermediate rate of inactivation relative to the fast inactivating K+ channels Kv3.4 and Kv1.4. The rate of Kv3.3 inactivation has, however, been difficult to establish given problems in obtaining consistent rates of inactivation in expression systems. This study characterized the properties of AptKv3.3, the teleost homologue of Kv3.3, when expressed in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. We show that the properties of AptKv3.3 differ significantly between CHO and HEK cells, with the largest difference occurring in the rate and voltage dependence of inactivation. While AptKv3.3 in CHO cells showed a fast and voltage-dependent rate of inactivation consistent with N-type inactivation, currents in HEK cells showed rates of inactivation that were voltage-independent and more consistent with a slower C-type inactivation. Examination of the mRNA sequence revealed that the first methionine start site had a weak Kozak consensus sequence, suggesting that the lack of inactivation in HEK cells could be due to translation at a second methionine start site downstream of the NH2-terminal coding region. Mutating the nucleotide sequence surrounding the first methionine start site to one more closely resembling a Kozak consensus sequence produced currents that inactivated with a fast and voltage-dependent rate of inactivation in both CHO and HEK cells. These results indicate that under the appropriate conditions Kv3.3 channels can exhibit fast and reliable inactivation that approaches that more typically expected of "A"-type K+ currents.

Functional characterization of the D188V mutation in neuronal voltage-gated sodium channel causing generalized epilepsy with febrile seizures plus (GEFS).

Mutations in the alpha 1 subunit of the voltage-gated sodium channel (SCN1A) have been increasingly recognized as an important cause of familial epilepsy in humans. However, the functional consequences of these mutations remain largely unknown. We identified a mutation (D188V) in SCN1A segregating with generalized epilepsy with febrile seizures (GEFS) in a large kindred. Compared to wild-type sodium channels, in vitro expression of channels harboring the D188V mutation were found to be more resistant to the decline in amplitude that is normally observed over the course of high frequency pulse trains. This small change on a single aspect of channel function is compatible with an increase in membrane excitability, such as during sustained and uncontrolled neuronal discharges. These data suggest that this specific effect on sodium channel function could be a general mechanism in the pathophysiology of epilepsies caused by mutations in sodium channels in humans.

Excitatory amino acid receptors of the electrosensory system: the NR1/NR2B N-methyl-D-aspartate receptor.

The amino acid sequence of the N-methyl-D-aspartate (NMDA) receptor subunit NR2B from the brown ghost knife fish Apteronotus leptorhynchus has been determined and compared with the sequence of the murine NR2B. This comparison revealed high levels of sequence conservation throughout the ligand binding and membrane spanning segments. The functional properties of the NR1 and NR2B receptor complex were examined by coexpression in HEK cells. The recombinant AptNR1/NR2B receptors produced robust currents after stimulation with glutamate or NMDA in the presence of glycine. Measurements of the concentration dependencies for these agonists indicated that the agonist binding sites on the apteronotid receptor are highly conserved, with nearly identical agonist affinities to those of the murine NR1/NR2B receptor. The kinetic responses of the fish receptor were also highly conserved, with deactivation rates for the AptNR2B receptor matching those of the murine NR2B containing receptor. Evidently, most of the unique functional properties that reside in the NR2B receptor subunit have been well conserved in teleost NMDA receptors. On the other hand, the apteronitid receptor displayed a lowered sensitivity to voltage-dependent Mg(2+) block and a reduced affinity for the NR2B-specific noncompetitive antagonist ifenprodil. We conclude that the functional properties that result from the incorporation of the NR2B receptor in the NMDA receptor complex have been maintained since the evolutionary divergence of teleost and mammalian organisms.

Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav.

The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites, several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptors Gads and Grb2, as well as to phospholipase Cgamma1 (PLCgamma1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y(132), Y(171), Y(191), and Y(226) have been shown previously to be critical for binding to Gads, Grb2, and PLCgamma1. In this report, we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y(171) and potentially Y(226) are docking sites for the Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.

Ceruloplasmin regulates iron levels in the CNS and prevents free radical injury.

Ceruloplasmin is a ferroxidase that oxidizes toxic ferrous iron to its nontoxic ferric form. We have previously reported that a glycosylphosphatidylinositol-anchored form of ceruloplasmin is expressed in the mammalian CNS. To better understand the role of ceruloplasmin in iron homeostasis in the CNS, we generated a ceruloplasmin gene-deficient (Cp(-/-)) mouse. Adult Cp(-/-) mice showed increased iron deposition in several regions of the CNS such as the cerebellum and brainstem. Increased lipid peroxidation was also seen in some CNS regions. Cerebellar cells from neonatal Cp(-/-) mice were also more susceptible to oxidative stress in vitro. Cp(-/-) mice showed deficits in motor coordination that were associated with a loss of brainstem dopaminergic neurons. These results indicate that ceruloplasmin plays an important role in maintaining iron homeostasis in the CNS and in protecting the CNS from iron-mediated free radical injury. Therefore, the antioxidant effects of ceruloplasmin could have important implications for various neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease in which iron deposition is known to occur.

Oscillatory burst discharge generated through conditional backpropagation of dendritic spikes.

Gamma frequencies of burst discharge (>40 Hz) have become recognized in select cortical and non-cortical regions as being important in feature extraction, neural synchrony and oscillatory discharge. Pyramidal cells of the electrosensory lateral line lobe (ELL) of Apteronotus leptorhynchus generate burst discharge in relation to specific features of sensory input in vivo that resemble those recognized as gamma frequency discharge when examined in vitro. We have shown that these bursts are generated by an entirely novel mechanism termed conditional backpropagation that involves an intermittent failure of dendritic Na+ spike conduction. Conditional backpropagation arises from a frequency-dependent broadening of dendritic spikes during repetitive discharge, and a mismatch between the refractory periods of somatic and dendritic spikes. A high threshold class of K+ channel, AptKv3.3, is expressed at high levels and distributed over the entire soma-dendritic axis of pyramidal cells. AptKv3.3 channels are shown to contribute to the repolarization of both somatic and dendritic spikes, with pharmacological blockade of dendritic Kv3 channels revealing an important role in controlling the threshold for burst discharge. The entire process of conditional back-propagation and burst output is successfully simulated using a new compartmental model of pyramidal cells that incorporates a cumulative inactivation of dendritic K+ channels during repetitive discharge. This work is important in demonstrating how the success of spike backpropagation can control the output of a principle sensory neuron, and how this process is regulated by the distribution and properties of voltage-dependent K+ channels.