Voltage-sensitive Dye Recording from Axons,Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (V_m-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes ². Many aspects of the initial technology have been continuously improved over several decades ^{3, 5, 11}. Additionally, previous work documented two essential characteristics of V_m-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; ^{10, 14, 16}). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible ^{4, 7}. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects ^{4, 6, 12, 13}. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the V_m-imaging technique. The current sensitivity permits multiple site optical recordings of V_m transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.     

When is a native species invasive? Incursion of a novel predatory marsupial detected using molecular and historical data

Aim

Range expansions facilitated by humans or in response to local biotic or abiotic stressors provide the opportunity for species to occupy novel environments. Classifying the status of newly expanded populations can be difficult, particularly when the timing and nature of the range expansion are unclear. Should native species in new habitats be considered invasive pests or actively conserved? Here, we present an analytical framework applied to an Australian marsupial, the sugar glider (Petaurus breviceps), a species that preys upon on an endangered parrot in Tasmania, and whose provenance was uncertain.

Location

Tasmania, Australia.

Methods

We conducted an extensive search of historical records for sugar glider occurrences in Tasmania. Source material included museum collection data, early European expedition logs, community observation records, and peer‐reviewed and grey literature. To determine the provenance of the Tasmanian population, we sequenced two mitochondrial genes and one nuclear gene in Tasmanian animals (n = 27) and in individuals across the species' native range. We then estimated divergence times between Tasmania and southern Australian populations using phylogenetic and Bayesian analyses.

Results

We found no historical evidence of sugar gliders occurring in Tasmania prior to 1835. All Tasmanian individuals (n = 27) were genetically identical at the three genes surveyed here with those individuals being 0.125% divergent from individuals from a population in Victoria. Bayesian analysis of divergence between Tasmanian individuals and southern Australian individuals suggested a recent introduction of sugar gliders into Tasmania from southern Australia.

Main conclusions

Molecular and historical data demonstrate that Tasmanian sugar gliders are a recent, post‐European, anthropogenic introduction from mainland Victoria. This result has implications for the management of the species in relation to their impact on an endangered parrot. The analytical framework outlined here can assist environmental managers with the complex task of assessing the status of recently expanded or introduced native species.

     

Methanol Extracts of 28 Hieracium Species from the Balkan Peninsula – Comparative LC–MS Analysis,Chemosystematic Evaluation of their Flavonoid and Phenolic Acid Profiles and Antioxidant Potentials

Introduction

Hieracium s. str. represents one of the largest and most complex genera of flowering plants. As molecular genetics seems unlikely to disentangle intricate relationships within this reticulate species complex, analysis of flavonoids and phenolic acids, known as good chemosystematic markers, promise to be more reliable. Data about pharmacological activity of Hieracium species are scarce.

Objective

Evaluation of the chemosystematic significance of flavonoids and phenolic acids of methanol extracts of aerial flowering parts of 28 Hieracium species from the Balkans. Additionally, investigation of antioxidant potentials of the extracts.

Methods

Comparative qualitative and quantitative analysis of flavonoids and phenolic acids was performed by LC–MS. Multivariate statistical data analysis included non‐metric multidimensional scaling (nMDS), unweighted pair‐group arithmetic averages (UPGMA) and principal component analysis (PCA). Antioxidant activity was evaluated using three colorimetric tests.

Results

Dominant phenolics in almost all species were luteolin type flavonoids, followed by phenolic acids. Although the investigated Hieracium species share many compounds, the current classification of the genus was supported by nMDS and UPGMA analyses with a good resolution to the group level. Hieracium naegelianum was clearly separated from the other investigated species. Spatial and ecological distances of the samples were likely to influence unexpected differentiation of some groups within H. sect. Pannosa. The vast majority of dominant compounds significantly contributed to differences between taxa. The antioxidant potential of the extracts was satisfactory and in accordance with their phenolics composition.

Conclusions

Comparative LC–MS analysis demonstrated that flavonoids and phenolic acids are good indicators of chemosystematic relationships within Hieracium, particularly between non‐hybrid species and groups from the same location. Copyright © 2017 John Wiley & Sons, Ltd.     

Den 1, Den2 and Den3, ATP-inhibited deoxyribonucleases from Dropsophila embryonic nuclei

Three Drosophila embryonic deoxyribonucleases, designated den1, den2 and den3, are identified in nuclear extracts separated by glycerol density gradient centrifugation. Den1, removes short products from the 5-ends of single-stranded DNA or double-stranded DNA with either blunt or 5-recessed termini. Den2 is inactive with single-stranded DNA and acts as 3-exonuclease with double-stranded DNA possessing either blunt or 3-recessed termini. Den3 preferentially uses partial duplex DNA containing single-stranded gap and it catalyzes hydrolysis, in 3-5 direction, of one of the shorter strands that flank the gap. Nucleolytic activities of den1, den2 and den3 are inhibited with ATP.     

Binding of transition metals to monosilicic acid in aqueous and xylem (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) solutions: a low-T electron paramagnetic resonance study

The supplementation of monosilicic acid [Si(OH)₄] to the root growing medium is known to protect plants from toxic levels of iron (Fe), copper (Cu) and manganese (Mn), but also to mitigate deficiency of Fe and Mn. However, the physicochemical bases of these alleviating mechanisms are not fully understood. Here we applied low-T electron paramagnetic resonance (EPR) spectroscopy to examine the formation of complexes of Si(OH)₄ with Mn²⁺, Fe³⁺, and Cu²⁺ in water and in xylem sap of cucumber (Cucumis sativus L.) grown without or with supply of Si(OH)₄. EPR, which is also useful in establishing the redox state of these metals, was combined with measurements of total concentrations of metals in xylem sap by inductive coupled plasma. Our results show that Si(OH)₄ forms coordination bonds with all three metals. The strongest interactions of Si(OH)₄ appear to be with Cu²⁺ (1/1 stoichiometry) which might lead to Cu precipitation. In line with this in vitro findings, Si(OH)₄ supply to cucumber resulted in dramatically lower concentration of this metal in the xylem sap. Further, it was demonstrated that Si(OH)₄ supplementation causes pro-reductive changes that contribute to the maintenance of Fe and, in particular, Mn in the xylem sap in bioavailable 2+ form. Our results shed more light on the intertwined reactions between Si(OH)₄ and transition metals in plant fluids (e.g. xylem sap).     

Myoglobin with chlorophyllous chromophores: influence on protein stability

The stabilities of myoglobin, apo-myoglobin, and of two myoglobins with chlorophyllous chromophores (Zn-pheophorbide a and Zn-bacteriopheophorbide a), have been studied by thermal and chemical denaturation. With guanidinium chloride, the stability order is myoglobin>Zn-pheophorbide-myoglobin>Zn-bacteriopheophorbide-myoglobin approximately apo-myoglobin. The thermal behavior is more complex. The transition temperature of thermal unfolding of the apoprotein (62.4 degrees C) is increased by Zn-pheophorbide a (83.9 degrees C) and Zn-bacteriopheophorbide a (82.6 degrees C) to a similar degree as by the native chromophore, heme (83.5 degrees C). The recovery with Zn-pheophorbide (92-98%) is even higher than with heme (74-76%), while with Zn-bacteriopheophorbide (40%) it is as low as with the apoprotein (42%). Recovery also depends on the rates of heating, and in particular the time spent at high temperatures. It is concluded that irreversibility of unfolding is related to loss of the chromophores, which are required for proper re-folding.     

Silicon increases phosphorus (P) uptake by wheat under low P acid soil conditions

Plant and Soil - Although silicon (Si) is known to improve plant growth under low phosphorus (P) conditions, the in planta mechanisms responsible for this effect are still unknown. Here, we...