Über den Einfluss von Neuropeptid Y auf die Amplitude und die Entladungsrate axonterminaler Aktionspotenziale retinaler Ganglienzellen der Klasse R2 und R3 im Tectum opticum während visueller Stimulation der Rotbauchunke Bombina orientalis

Neuropeptid Y (NPY) ist ein potenter Neurotransmitter im zentralen und peripheren Nervensystem der Mammalia. Es ist an der Regulation einer Vielzahl von physiologischen Prozessen beteiligt und scheint auch im retino-tectalen Transfer des visuellen Systems von Anuren eine zentrale Funktion einzunehmen. Die Retina bildet die erste Funktionseinheit bei der Verarbeitung visuellen Inputs. Für die Weiterverarbeitung sind primär das Tectum opticum (TO) und das Praetectum verantwortlich. Es gilt als wahrscheinlich, dass der praetecto-tectale Transfer durch NPY inhibitorisch moduliert wird und damit wesentlichen Einfluss auf die visuelle Mustererkennung und die Dämpfung der tectalen Erregungsausbreitung hat. Die Applikation von NPY auf die Tectumoberfläche schwächt die anfängliche Erregungswelle visuell evozierter Feldpotenziale stark ab und NPY könnte somit Einfluss auf die Axonendknoten retinaler Ganglienzellen des Typs R2, R3 und auch R4 haben. Es können jedoch keine detaillierten Aussagen gemacht werden welche Neuronen in welchem Umfang daran beteiligt sind. Im Rahmen meiner Arbeit, sollte der Einfluss von NPY auf die Spike-Amplitude und die Spike-Rate retinaler Ganglienzellen R2 und R3 bei Bombina orientalis analysiert werden, da diese den größten Input bei der visuellen Mustererkennung liefern und unterschiedliche Funktionen in diesem neuronalen Netzwerk haben. Hierzu wurden visuell evozierte Aktionspotenziale von R2 und R3 Neuronen im TO von Bombina orientalis abgeleitet und mit Hilfe der Analysesoftware Spike 2 bearbeitet und analysiert. Es konnte nachgewiesen werden, dass die Spike-Amplituden der R2 Neuronen 20 min nach NPY Applikation auf die Tectumoberfläche reduziert werden. Nach einer Erholungsphase 10 min nach Beenden der NPY-Applikation konnte ein Wiederanstieg der Spike-Amplituden gemessen werden, 20 min nach Beenden der NPY-Applikation kam es zu einem Abfall der Spike-Amplituden dessen Ursache unbekannt ist. Ob es ein Artefakt ist oder ob es sich hierbei um einen spezifischen Effekt von R2 Neuronen handelt muss noch geklärt werden. Die Spike-Amplituden der R3 Neuronen waren bereits 10 min nach NPY-Applikation reduziert, ein weitere Abfall der Spike-Amplituden konnte nicht verzeichnet werden. 10 min nach Beenden der NPY-Applikation konnte ein Anstieg der Spike-Amplituden verzeichnet werden, der sich stetig fortsetzte. Bei beiden Neuronentypen wurden 20 min nach Beenden der NPY-Applikation Spike-Amplituden nahe der Ausgangsamplitudenhöhe gemessen. Aufgrund des Verlaufes der R3 Neuronen ist davon auszugehen, dass die Feldpotenziale eher durch R3 Neuronen als durch R2 Neuronen beeinflusst werden, da er dem der Feldpotenziale gleicht. Auch bei der Untersuchung der Spike-Raten konnte eine Beeinflussung durch NPY nachgewiesen werden. Die R2 Neuronen zeigten 10 min nach NPY-Applikation einen Abfall der Spike-Raten der sich nach 20 min weiter fortsetzte. 10 min nach Beenden der NPY-Applikation konnte ein Wiederanstieg der Spike-Raten verzeichnet werden der sich stetig fortsetzte, die Werte blieben jedoch deutlich unter den gemessenen Ausgangswerten ohne eine NPY-Beeinflussung. Bei den R3 Neuronen konnte ein Abfall der Spike-Raten deutlich zeitverzögert nachgewiesen werden. 20 min nach Beenden der NPY-Applikation konnte ein Anstieg der Spike-Rate verzeichnet werden, jedoch gab es keine signifikanten Unterschiede der Spike-Raten zu den Werten ohne NPY-Beeinflussung. Der Vergleich der R2 und R3 Neuronen zeigt, dass bei den der R2 Neuronen ein schnellerer Effekt von NPY nachweisbar ist als die den R3 Neuronen. Aufgrund der von mir nachgewiesene NPY-induzierte Spike-Amplitudenabnahme retinaler R2 und R3 Neuronen muss davon ausgegangen werden, dass die Reduktion der Feldpotential durch NPY eher auf den Einfluss anderer Neuronen als R2 und R3 Neuronen zurückzuführen ist. Weder bei den R2 noch bei den R3 Neuronen konnte eine so schnelle und so starke Beeinflussung der Spike- Amplituden verzeichnet werden. Weiterhin zeigen meine Ergebnisse neuronale Bestätigung der von Funke 2005 beschrieben geringeren Strahlungsintensität sowie der geringeren Glukosemetabolisierung bei der 14C-2-Desoxyglukose Technik. Dies ist in der Form nur auf den Einfluss von R2 und R3 Neuronen zurückzuführen. Die von mir erzielten Ergebnisse stützen die Hypothese, dass NPY den retino-tectalen Signaltransfer inhibitorisch steuert einhergehend mit einer reduzierten Ausschüttung des praetectotectalen Transmitters Glutamat und weisen darauf hin, dass NPY über zwei verschiedene second-messenger vermittelte Prozesse diesen Signaltransfer steuert. Interessant ist in diesem Zusammenhang, dass diese nachhaltige Beeinflussung der visuellen Informationsverarbeitung durch NPY bei Bombina orientalis einem phylogenetisch basalen Vertreter der Anuren nachgewiesen werden konnte. Dies lässt den Schluss zu, dass solche grundlegenden neurochemischen Effekte des retino-tectalen Informationsgefüges evolutionär konserviert sind.

Egyptian wheat

Wheat as the major cereal crop in Egypt is the core of the government's food security policy. But there are rapid losses of the genetic resources of the country as a result of ongoing modernization and development. Thus we compiled the largest possible number of Egyptian accessions preserved in the world gene banks. In the present study we collected nearly 1000 Egyptian wheat accessions. A part from the Triticum species of the Egyptian flora four species have been found, which were recorded for the first time T. turanicum, T. compactum, T. polonicum and T. aethiopicum. To classify the Egyptian wheat species using morphological studies, 108 accessions were selected. Thereafter, these accessions were cultivated and evaluated morphologically to confirm the validity of the classified species. During the morphological evaluation study, a new case was noticed for the number of glumes in one of the Egyptian wheat accessions. Three glumes per spikelet were observed in a branched spike. This led us to assess the phenomenon in all varieties with branching spikes within the genus Triticum. All varieties which have branching spikes at least in some spikletes have three glumes. We considered the case of the third glume as indicator for the domestication syndrome. Also, a new case of other forms of branching in the genus Triticum was investigated, which was a compromise between true and false-branching. We called it true-false branching. Comparative anatomical studies were carried out between Egyptian Triticum species to investigate the possibility of using anatomical features to classify the Egyptian wheat species. It was concluded that it is difficult to use anatomical features alone to differentiate between two Triticum species, especially when they belong to the same ploidy level. A key for the identification of Egyptian Triticum taxa was established.

Vesicle motion during sustained exocytosis in chromaffin cells

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles’ arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that including the fusion-time statistics in our model does not produce any significant changes on the results. These findings indicate that the motion of the whole ensemble of vesicles towards the membrane is directed and reflected in the amperometric signals. Our results confirm the conclusions of previous imaging studies performed on single vesicles that vesicles’ motion underneath plasma membranes is not purely random, but biased towards the membrane.

The phytocannabinoid Delta(9)-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

Background and purposeThe phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS.Experimental approachEffects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro.Key resultsThe cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB(1) receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide.Conclusions and implicationsWe show for the first time that Delta(9)-THCV acts as a functional CB(1) receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB(1) receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV- and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.British Journal of Pharmacology advance online publication, 3 March 2008; doi:10.1038/bjp.2008.57.

Monte Carlo phase diagram for diblock copolymer melts

The phase diagram for diblock copolymer melts is evaluated from lattice-based Monte Carlo simulations using parallel tempering, improving upon earlier simulations that used sequential temperature scans. This new approach locates the order-disorder transition (ODT) far more accurately by the occurrence of a sharp spike in the heat capacity. The present study also performs a more thorough investigation of finite-size effects, which reveals that the gyroid (G) morphology spontaneously forms in place of the perforated-lamellar (PL) phase identified in the earlier study. Nevertheless, there still remains a small region where the PL phase appears to be stable. Interestingly, the lamellar (L) phase next to this region exhibits a small population of transient perforations, which may explain previous scattering experiments suggesting a modulated-lamellar (ML) phase.

Simple and surprisingly effective one-step extraction-cleanup by Soxflo for DDT and its metabolites from environmental samples

A pilot study found that DDT breakdown at the GC inlet was extensive in extracts from some-but not all-samples with high organic carbon contents. However, DDT losses could be prevented with a one-step extraction-cleanup in the Soxflo instrument with dichloromethane and charcoal. This dry-column procedure took 1 h at room temperature. It was tested on spiked soil and peat samples and validated with certified soil and sediment reference materials. Spike recoveries from freshly spiked samples ranged from 79 to 111% at 20-4000 mug/kg concentrations. Recoveries from the real-world CRMs were 99.7-100.2% of DDT, 89.7-90.4% of DDD and 89.6-107.9% of DDE. It was concluded that charcoal cleanups should be used routinely during surveys for environmental DDX pollution in order to mitigate against unpredictable matrix-enhanced breakdown in the GC. (C) 2004 Elsevier B.V. All rights reserved.

Ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the N-terminal domain of N protein

Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 A (monoclinic) and at 1.85 A (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Age-related effects of Ginkgo biloba extract on synaptic plasticity and excitability

EGb 761 is a standardized extract from the Ginkgo biloba leaf and is purported to improve age-related memory impairment. The acute and chronic effect of EGb 761 on synaptic transmission and plasticity in hippocampal slices from young adult (8-12 weeks) and aged (18-24 months) C57B1/6 mice was tested because hippocampal plasticity is believed to be a key component of memory. Acutely applied EGb 761 significantly increased neuronal excitability in slices from aged mice by reducing the population spike threshold and increased the early phase of long-term potentiation, though there was no effect in slices from young adults. In chronically treated mice fed for 30 days with an EGb 761-supplemented diet, EGb 761 significantly increased the population spike threshold and long-term potentiation in slices from aged animals, but had no effect on slices from young adults. The rapid effects of EGb 761 on plasticity indicate a direct interaction with the glutamatergic system and raise interesting implications with respect to a mechanism explaining its effect on cognitive enhancement in human subjects experiencing dementia. (C) 2003 Elsevier Inc. All rights reserved.

The phytocannabinoid Δ9-tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum

Background and purpose: The phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) has been reported to exhibit a diverse pharmacology; here, we investigate functional effects of Delta(9)-THCV, extracted from Cannabis sativa, using electrophysiological techniques to define its mechanism of action in the CNS. Experimental approach: Effects of Delta(9)-THCV and synthetic cannabinoid agents on inhibitory neurotransmission at interneurone-Purkinje cell (IN-PC) synapses were correlated with effects on spontaneous PC output using single-cell and multi-electrode array (MEA) electrophysiological recordings respectively, in mouse cerebellar brain slices in vitro. Key results: The cannabinoid receptor agonist WIN 55,212-2 (WIN55) decreased miniature inhibitory postsynaptic current (mIPSC) frequency at IN-PC synapses. WIN55-induced inhibition was reversed by Delta(9)-THCV, and also by the CB1 receptor antagonist AM251; Delta(9)-THCV or AM251 acted to increase mIPSC frequency beyond basal values. When applied alone, Delta(9)-THCV, AM251 or rimonabant increased mIPSC frequency. Pre-incubation with Delta(9)-THCV blocked WIN55-induced inhibition. In MEA recordings, WIN55 increased PC spike firing rate; Delta(9)-THCV and AM251 acted in the opposite direction to decrease spike firing. The effects of Delta(9)-THCV and WIN55 were attenuated by the GABA(A) receptor antagonist bicuculline methiodide. Conclusions and implications: We show for the first time that Delta(9)-THCV acts as a functional CB1 receptor antagonist in the CNS to modulate inhibitory neurotransmission at IN-PC synapses and spontaneous PC output. Delta(9)-THCV- and AM251-induced increases in mIPSC frequency beyond basal levels were consistent with basal CB1 receptor activity. WIN55-induced increases in PC spike firing rate were consistent with synaptic disinhibition; whilst Delta(9)-THCV-and AM251-induced decreases in spike firing suggest a mechanism of PC inhibition.

Methods for comparing and constraining models of dendritic spines

Physiological evidence using Infrared Video Microscopy during the uncaging of glutamate has proven the existence of excitable calcium ion channels in spine heads, highlighting the need for reliable models of spines. In this study we compare the three main methods of simulating excitable spines: Baer & Rinzel's Continuum (B&R) model, Coombes' Spike-Diffuse-Spike (SDS) model and paired cable and ion channel equations (Cable model). Tests are done to determine how well the models approximate each other in terms of speed and heights of travelling waves. Significant quantitative differences are found between the models: travelling waves in the SDS model in particular are found to travel at much lower speeds and sometimes much higher voltages than in the Cable or B&R models. Meanwhile qualitative differences are found between the B&R and SDS models over realistic parameter ranges. The cause of these differences is investigated and potential solutions proposed.

A structural analysis of M protein in coronavirus assembly and morphology

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.

Algorithmic statistical analysis of electrophysiological data for the investigation of structure-activity relationship in single neurons

We are developing computational tools supporting the detailed analysis of the dependence of neural electrophysiological response on dendritic morphology. We approach this problem by combining simulations of faithful models of neurons (experimental real life morphological data with known models of channel kinetics) with algorithmic extraction of morphological and physiological parameters and statistical analysis. In this paper, we present the novel method for an automatic recognition of spike trains in voltage traces, which eliminates the need for human intervention. This enables classification of waveforms with consistent criteria across all the analyzed traces and so it amounts to reduction of the noise in the data. This method allows for an automatic extraction of relevant physiological parameters necessary for further statistical analysis. In order to illustrate the usefulness of this procedure to analyze voltage traces, we characterized the influence of the somatic current injection level on several electrophysiological parameters in a set of modeled neurons. This application suggests that such an algorithmic processing of physiological data extracts parameters in a suitable form for further investigation of structure-activity relationship in single neurons.

Effects of dendritic morphology on CA3 pyramidal cell electrophysiology: a simulation study

We investigated the effect of morphological differences on neuronal firing behavior within the hippocampal CA3 pyramidal cell family by using three-dimensional reconstructions of dendritic morphology in computational simulations of electrophysiology. In this paper, we report for the first time that differences in dendritic structure within the same morphological class can have a dramatic influence on the firing rate and firing mode (spiking versus bursting and type of bursting). Our method consisted of converting morphological measurements from three-dimensional neuroanatomical data of CA3 pyramidal cells into a computational simulator format. In the simulation, active channels were distributed evenly across the cells so that the electrophysiological differences observed in the neurons would only be due to morphological differences. We found that differences in the size of the dendritic tree of CA3 pyramidal cells had a significant qualitative and quantitative effect on the electrophysiological response. Cells with larger dendritic trees: (1) had a lower burst rate, but a higher spike rate within a burst, (2) had higher thresholds for transitions from quiescent to bursting and from bursting to regular spiking and (3) tended to burst with a plateau. Dendritic tree size alone did not account for all the differences in electrophysiological responses. Differences in apical branching, such as the distribution of branch points and terminations per branch order, appear to effect the duration of a burst. These results highlight the importance of considering the contribution of morphology in electrophysiological and simulation studies.

Revealing ensemble state transition patterns in multi-electrode neuronal recordings using hidden Markov models

In order to harness the computational capacity of dissociated cultured neuronal networks, it is necessary to understand neuronal dynamics and connectivity on a mesoscopic scale. To this end, this paper uncovers dynamic spatiotemporal patterns emerging from electrically stimulated neuronal cultures using hidden Markov models (HMMs) to characterize multi-channel spike trains as a progression of patterns of underlying states of neuronal activity. However, experimentation aimed at optimal choice of parameters for such models is essential and results are reported in detail. Results derived from ensemble neuronal data revealed highly repeatable patterns of state transitions in the order of milliseconds in response to probing stimuli.

Asynchronous flowering and within-plant flowering diversity in wheat and the implications for crop resilience to heat

Self-pollination dominates in wheat , with a small level of out-crossing due to flowering asynchrony and male sterility. However, the timing and synchrony of male and female flowering in wheat is a crucial determinant of seed set and may be an important factor affecting gene flow and resilience to climate change. Here, a methodology is presented for assessing the timing and synchrony of flowering in wheat. From the onset of flowering until the end of anthesis, the anther and stigma activity of each floret was assessed on the first five developing ears in potted plants grown under ambient conditions and originating from cv Paragon or cvs Spark-Rialto backgrounds. At harvest maturity, seed presence, size and weight was recorded for each floret scored. The synchrony between pollen dehiscence and stigma collapse within a flower was dependent on its relative position in a spike and within a floret. Determined on the basis of synchrony within each flower, the level of pollination by pollen originating from other flowers reached approximately 30% and did not change throughout the duration of flowering. A modelling exercise parameterised by flowering observations indicated that the temporal and spatial variability of anther activity within and between spikes may influence the relative resilience of wheat to sudden, extreme climatic events which has direct relevance to predicted future climate scenarios in the UK.