Impact of human alpha-synuclein overexpression on the nigrostriatal dopaminergic neurotransmission

Alpha-synuclein is a small 140 amino acid presynaptic protein associated with everal neurodegenerative disorders such as Parkinson’s disease (PD). In idiopathic PD, abnormally misfolded wild-type proteins aggregate in the cytosol of certain neurons, forming what is the main component of Lewy bodies (LBs) - a major PD hallmark. Besides its role in regulating synaptic vesicle functions, alpha-synuclein appears to modulate dopamine (DA), a neurotransmitter particularly important in PD symptoms manifestation. DA can be found sparsely distributed in the brain and is predominantly expressed in midbrain neurons. From the midbrain, two main dopaminergic (DAergic) pathways densely innervate the dorsal portion of the striatum. Degeneration of such DAergic terminals is known to dysregulate DA homeostasis in the striatum. Despite several decades of PD research, the physiological influence of alpha-synuclein on DAergic neurotransmission in the two main areas of the dorsal striatum is still poorly understood. These areas are the dorsomedial striatum (DMS), mainly receiving innervation from the ventral tegmental area, and the dorsolateral striatum (DLS), which receives input from the substantia nigra pars compacta and is more susceptible to neurodegeneration. To clarify how alpha-synuclein may interfere with DAergic neurotransmission in either area, fast-scan cyclic voltammetry experiments were carried out in the DMS and DLS of transgenic mice overexpressing human alpha-synuclein (Tg) at twelve, six and three months of age. Additionally, pharmacological assays, behavioural tasks, and ex vivo immunofluorescence staining were performed to support the electrophysiological ex vivo results. In this thesis, it is hypothesized that due to its intrinsic characteristics, alpha-synuclein preferentially interferes with DA neurotransmission dynamics in the dorsolateral region of the striatum. Data analysis confirmed that overexpression of human alpha-synuclein differentially interfered with normal DA release in the DLS in an age‐dependent manner. At older ages (twelve-month-old Tg mice), decreased evoked DA release and slower DA uptake kinetics were observed. In addition, alterations in dopamine transporter (DAT) distribution, which appeared as increasing amounts of DAT-positive clumps, were found only in the DLS. Moreover, at pre-symptomatic stages (six-month-old) DA neurotransmission appeared to be stabilised before DA disruption. Surprisingly, at younger ages (three-month-old) increased electrically evoked DA release was also recorded in the DLS. Such changes were in line with the motor learning enhancement observed in their behavioural phenotype. In addition, DA uptake appeared to be impaired as evidenced by reduced extracellular DA withdrawal. Further pharmacological experiments demonstrated that such alterations were mediated by DAT. In summary, the present findings indicate that abnormal DAergic neurotransmission and function of DLS can be identified before the onset of structural pathologies in a model of transgenic expression of human alpha-synuclein. Depending on the progression of the pathology, human alpha-synuclein has different impacts on neurotransmission, initially enhancing it but impairing it at later stages. It is here proposed that assessment of DLS function by non-invasive brain imaging and neuropsychological techniques might be relevant in early PD diagnosis and help design appropriate therapeutic intervention

Potentiostatic infrared titration of 11-Mercaptoundecanoic acid monolayers

Acknowledgment This work was supported by the Spanish DGICYT under grant CTQ2008-00371 and by the Junta de Andalucía under grant P07-FQM-02492.Peer reviewedPostprin

Extraction of wood compounds by use of subcritical fluids

A study of the extraction of oak wood compounds with subcritical water-ethanol mixtures as extractant, with an ethanol content between 0-60%, is reported. Identification and characterisation of the extracted compounds have been made by spectrophotometry and gas chromatography with either flame ionisation or mass detectors. Extraction was performed statically manner by use of a single cycle or repeated cycles. All variables affecting the extraction process were studied and optimised. Extraction time and temperature are 60 min and 200ºC, respectively. Comparison of the extract thus obtained with commercial extracts showed the former to be rich in compounds characteristic of the commercial extracts. The method enables manipulation of the extract composition by changing the temperature and water/ethanol ratio used. It is faster than the traditional procedures for obtaining wood extracts

Two-parameter determination in vinegar by a flow injection-pervaporation system

A flow-injection method (FI) for the sequential determination of ethanol and acetic acid in vinegar is reported. The determination of ethanol is based on the oxidation of the pervaporated ethanol by Cr2O7K2. The acetic acid is determined by an acid-base reaction with Thymol Blue as the indicator. Both reactions are monitored photometrically at 600 nm using a single detector. Optimisation studies and assessment of the sequential FI method are also reported. The linear determination range is between 0-12 % (v/v) for ethanol and 0-10 % (grams of acetic acid in 100 ml) for acetic acid. The sample throughput of the sequential manifold is 7 per hour. The new method has been applied to vinegar samples and the results obtained are in excellent agreement with those from the reference methods used in Spain

Semiautomatic flow-injection method for the determination of volatile acidity in wines

A flow-injection (FI) method based on analytical pervaporation was assessed for its routine use in the determination of volatile acidity in winery laboratories. The new method was compared with both the official method and the Mathieu method, which is most often used in Spanish wineries, by testing 30 different wines, including young and aged, and sweet and dry wines from Montilla-Moriles appellation d´origine. The robustness of the new method was established and then all three methods were studied in terms of range of linearity and regression of the calibration curve, repeatability, reproducibility, sensitivity, detection and quantification limits (LOD and LQ, respectively) and time of analysis. The FI method surpasses the Mathieu method in reproducibility and both the Mathieu and official methods in LOD and LQ and sensitivity; it also requires less personnel involvement and shorter analysis time. The statistical criteria established by the “Office International de la Vigne et du Vin” were applied to the data and the results obtained indicated that the differences between the analytical parameters of the 3 methods are not significant and can be applied indistinctly. The correlation of the methods was studied by taking them 2 by 2, and the corresponding equations, coefficients and deviations confirmed the statistical results. Thus, the new method can be used in winery laboratories with clear advantages over its 2 counterparts (the routine and official methods)

Determination of ethanol in beverages by flow injection, pervaporation and density measurements

A fast, clean and easy to automate flow injection-pervaporation method for the determination of ethanol in different beverages using density measurements is proposed. The method is based on separation of the ethanol from the sample using a pervaporation module, thus obtaining in the acceptor chamber of the pervaporator a water-alcohol mixture, the density of which is measured. After optimisation by either the univariate or multivariate approach as required, a linear range between 0-40% was established. Then, the assessment of the method versus a reference one was studied in terms of repeatability (0.12% v/v), reproducibility (0.32% v/v), detection limit (0.11% v/v) and traceability. The sample throughput was 15 samples h-1. The method was in agreement with the reference methods used in the European Union

Superheated liquids for the extraction of solid residues from winemaking processes

Solid residues from winemaking process have been subjected to extraction with superheated water-ethanol mixtures. Identification and characterisation of the extracted compounds have been made by spectrophotometry, gas chromatography with either flame ionisation or mass detectors and high performance liquid chromatography with UV detection. The extraction has been performed in a static manner by single or repetitive cycles. All variables affecting the extraction process have been studied and optimised. The extraction time and temperature are 65 min and 210ºC, respectively. Two phases constitute the extract: an aqueous phase, which is rich in phenol compounds and an oily phase, mainly formed by fatty acids. The method allows manipulation of the extract composition by changing the applied pressure, temperature, water-ethanol ratio and pH. The method is faster than the traditional extraction procedures for obtaining valuable compounds from these residues

Sequential Spectrophotometric determination of methanol and iron in vinegar by a flow injection-pervaporation method

An easily automatable sequential flow-injection-pervaporation method is proposed for the photometric determination of methanol and iron in vinegar. The method is based on separation of the methanol from the sample matrix by pervaporation followed by its oxidation to formaldehyde with permanganate, decolouration of the latter with S2O5-2 and subsequent reaction of formaldehyde with p-rosaniline to yield a violet reaction product with maximum absorption at 567 nm. Iron is determined by an existing method based on reaction with thiocyanate in acidic medium and monitoring at 508 nm. After optimisation by either the univariate or multivariate approaches, as required, the linear range was established for methanol (4-1000 mg L-1) and iron (0.18-20 mg L-1); The proposed method was then compared with reference methods for methanol and iron in terms of repeatability (2.452 mg L-1 and 0.245 mg L-1, respectively), reproducibility (4.435 mg L-1 and 0.356 mg L-1, respectively), detection limit (LOD=82 and 0.234 mg L-1, respectively) and traceability. The throughput was nine samples per hour

Method for the simultaneous determination of total polyphenol and anthocyan indexes in red wines using a flow injection approach

A simultaneous and fast method for the determination of total polyphenol index (t.p.i.) and total anthocyan index (t.a.i.) has been developed by a flow injection approach and a diode array spectrophotometer for monitoring at 280 nm and 520 nm, respectively. Linear ranges were obtained from 20 to 70 index units and from 20 to 500 mg l-1 for t.p.i. and t.a.i., respectively. The results provided by the proposed method agree with those obtained using the polyphenol index at 280 nm and the Ribereau-Gayon method for the determination of total anthocyans. The sample throughput was 25-30 samples per hour. Analytical features such as repeatability, reproducibility and detection and quantification limits as well as the results of a robustness study based on the Steiner-Younden procedure are also given

“Living on the edge” : the role of field margins for common vole (Microtus arvalis) populations in recently colonised Mediterranean farmland

Acknowledgments RRP was supported by a PhD-studentship from the University of Valladolid (co-funded by Banco Santander, RR 30/04/2014). Financial support was provided by ECOCYCLES (BIODIVERSA 2008, Era-net European project, EUI2008-03658 and NERC NE/G002045/1 to XL) and ECOVOLE projects (CGL2012-35348; Ministerio de Economía y Competitividad of Spain). The article also contributes to project ECOTULA (CGL2015-66962-C2-1-R). We held all the necessary licenses and permits for conducting this work (JJLL, FM and RRP held animal experimentation permits of level B for Spain, and a capture permit was provided by the Consejería de Fomento y Medio Ambiente, Junta de Castilla y León (Expte: EP/CYL/665/2014)). We thank two anonymous reviewers for providing and constructive comments to improve the manuscript.Peer reviewedPublisher PD