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ABSTRACT: Until today, brain tumors especially glioblastoma are difficult to treat and therefore, results in a poor survival rate of 0-14% over five years. To overcome this problem, the development of novel therapeutics as well as optimization of neurosurgical procedures to remove the tumor tissue are subject of intensive research. The main problem of the tumor excision, as the primary clinical intervention is the diffuse infiltration of the tumor cells in unaltered brain tissue that complicates the complete removal of residual tumor cells. In this context, we are developing novel approaches for the label-free discrimination between tumor tissue and unaltered brain tissue in real-time during the surgical process. Using our impedance spectroscopy-based measurement system in combination with flexible microelectrode arrays we could successfully demonstrate the discrimination between a C6-glioma and unaltered brain tissue in an in vivo rat model. The analysis of the impedance spectra revealed specific impedance spectrum shape characteristics of physiologic neuronal tissue in the frequency range of 10-500kHz that were significantly different from the tumor tissue. Moreover, we used an adapted equivalent circuit model to get a deeper understanding for the nature of the observed effects. The impedimetric label-free and real-time discrimination of tumor from unaltered brain tissue offers the possibility for the implementation in surgical instruments to support surgeons to decide, which tissue areas should be removed and which should be remained.
Biosensors & bioelectronics 02/2013; 46C:8-14. · 5.43 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) and other tauopathies comprise death of cell bodies, synapses and neurites but there is surprising little knowledge of the temporal sequence and the causal relationships among these events. Here, we present a novel biosensoric approach to monitor retrograde neurite degeneration before cell death occurs. We induced tau hyperphosphorylation in organotypic hippocampal slice cultures (OHSC) and applied marker-independent real-time electrical impedance spectroscopy (EIS) for cellular real-time pathology monitoring. Using this approach, we were able to define two distinct phases of neurite degeneration, first a rapid swelling of axonal processes that manifests itself in relative impedance above control levels followed by a slower phase of collapse and subsequent fragmentation indicated by decreased relative impedance below control levels. Initial axon swelling is strictly dose-dependent and swelling intensity correlates with second phase impedance decrease implicating a causative link between both degenerative mechanisms. Moreover, suppressing tau hyperphosphorylation by kinase inhibition nearly prevented both phases of axon degeneration. Our findings demonstrate that the temporal sequence of tau-triggered neurite degeneration can be directly visualized by EIS-based, non-invasive and label-free monitoring. We therefore suggest this approach as a powerful extension of high content applications to study mechanisms of neurite degeneration and to exploit therapeutic options against AD and tau-related disorders.
Biosensors & bioelectronics 02/2012; 32(1):250-8. · 5.43 Impact Factor
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ABSTRACT: Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.
PLoS ONE 01/2012; 7(2):e31669. · 4.09 Impact Factor
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ABSTRACT: Electrochemical biosensors allow simple, fast and sensitive analyte detection for various analytical problems. Especially immunosensors are favourable due to specificity and affinity of antigen recognition by the associated antibody. We present a novel electrode array qualified for parallel analysis and increased sample throughput. The chip has nine independent sample chambers. Each chamber contains a circular gold working electrode with a diameter of 1.9 mm that is surrounded by a ring-shaped auxiliary electrode with a platinum surface. The corresponding silver/silver chloride reference electrodes are embedded in a sealing lid. The chip is open to the full range of electrochemical real-time detection methods. Among these techniques, impedance spectroscopy is an attractive tool to detect fast and label-free interfacial changes originating from the biorecognition event at the electrode surface. The capabilities of the novel electrode array are demonstrated using the example of tumour marker tenascin C detection. This glycoprotein of the extracellular matrix is expressed in cancerous tissues, especially in solid tumours such as glioma or breast carcinoma. Electrodes covered with specific antibodies were exposed to tenascin C containing samples. Non-occupied binding sites were identified using a secondary peroxidase-conjugated antibody that generated an insoluble precipitate on the electrode in a subsequent amplification procedure. The charge transfer resistance obtained from impedimetric analysis of ferri-/ferrocyanide conversion at the electrode served as analytic parameter. This assay detected 14 ng (48 fmol) tenascin C that is sufficient for clinical diagnostics. The electrode surface could be regenerated at least 20-fold without loss of its analytical performance.
Lab on a Chip 09/2011; 11(17):2884-92. · 5.67 Impact Factor
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ABSTRACT: Cardiovascular diseases represent the most common cause of death in industrialized countries. In this context vascular smooth muscle cells (SMCs) are a major key player that is involved in pathological processes like hypertension and atherosclerosis. Therefore the pharmaceutical industry is intensively investigated in developing non-destructive and label-free monitoring techniques for a quantitative detection of SMC characteristics in the field of active pharmaceutical development as well as clinical diagnostics. Hence, we developed a novel multiwell interdigital electrode sensor-array in standardized ANSI 96-well layout. Through optimization of electrode geometry and material as well as passivation/adhesion-layer we obtained a novel biohybrid chip for the sensitive and quantitative detection of SMC contractility as well as relaxation via impedance spectroscopy. For the validation of our multiwell sensor-array we established a SMC culture model derived from primary cells that is switchable from a non-contractile pathological to a functional contractile phenotype. Using the reference compounds acetylcholine (ACh) and amlodipine, we could quantify SMC contraction by an impedance decrease to 40% while SMC relaxation was detectable by an impedance increase to 110%. More strikingly we could monitor aging of the isolated SMC which arose by an attenuated contractility over successive passaging. Demonstrating the performance of our self-developed multiwell sensor-array based impedance measurement setup we provide a suitable sensor-array coupled cell model to study the mechanisms that activated SMCs undergo in response to inflammatory mediators or vessel injury.
Lab on a Chip 11/2010; 10(21):2965-71. · 5.67 Impact Factor
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ABSTRACT: Transient receptor potential (TRP) channels are non-selective ion channels permeable to cations including Na(+), Ca(2+) and Mg(2+). They play a unique role as cellular sensors and are involved in many Ca(2+)-mediated cell functions. Failure in channel gating can contribute to complex pathophysiological mechanisms. Dysfunctions of TRP channels cause diseases but are also involved in the progress of diseases. We present a novel method to analyse chemical compounds as potential activators or inhibitors of TRP channels to provide pharmaceutical tools to regulate channel activity for disease control. Compared to common methods such as patch clamp or Ca(2+) imaging, the presented impedance assay is automatable, experimental less demanding and not restricted to Ca(2+) flow. We have chosen TRPA1 from the TRPA ('ankyrin') family as a model channel which was stimulated by allyl isothiocyanate (AITC). HEK293 cells stably transfected with human TRPA1 cDNA were grown on microelectrode arrays. Confluent cell layers of high density were analysed. Impedance spectra of cell-covered and non-covered electrodes yielded a cell-specific signal at frequencies between 70 and 120 kHz. Therefore, 100 kHz was chosen to monitor TRPA1 activity thereupon. An average impedance decrease to about 70% of its original value was observed after application of 10 μM AITC indicating an increased conductance of the cell layer mediated by TRPA1. Transfected cells pretreated with 10 μM of inhibitor ruthenium red to prevent channel conductance, as well as control cells lacking TRPA1, showed no impedance changes upon AITC stimuli demonstrating the specificity of the novel impedance assay.
Biosensors & bioelectronics 10/2010; 26(5):2376-82. · 5.43 Impact Factor
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ABSTRACT: Herewith we developed a novel 3D in vitro Alzheimer's disease (AD) model, based on the human neuroblastoma cell line SH-SY5Y, which is well differentiated without the application of any agents. Furthermore AD-like pathological neurodegeneration can be induced by okadaic acid (OA) mediated hyperphosphorylation of the microtubule associated protein tau. Moreover, we established stable "rapid tauopathy cell lines" expressing additional EGFP-fused (enhanced green fluorescent protein) wildtype or a pathology-promoting mutant tau variant (P301L) by lentiviral transduction. For the sensitive and feasible quantitative detection of pathological effects on neuronal 3D-cultures by electrochemical impedance spectroscopy (EIS) we optimized and redesigned a microcavity array (MCA). The cellular contribution to impedance could be increased by the factor of 2.5 and the variance decreased by 40%. Using our optimized MCA and impedance measurement setup we were able to detect quantitatively an OA concentration- and time-dependent decrease of the impedance in 3D SH-SY5Y cultures. Moreover, we were able to detect and quantify distinct, AD-related effects triggered by tau-mutant (P301L) expression and hyperphosphorylation in our organotypic 3D-cultures with the help of impedance spectroscopy.
Biosensors & bioelectronics 09/2010; 26(1):162-8. · 5.43 Impact Factor
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ABSTRACT: Crucial roles of netrins and semaphorins and their receptors in neuronal development have been reported. For further research
without using animal models it is important to know if well established neuronal cell lines express such guiding cues and
their receptors. Here we focused on netrin-1, netrin G1, G2, semaphorin 3A, 3F, 7A, and their receptors, respectively. We
screened the neuroblastoma cell line SH-SY5Y before and after differentiation with 20 nM staurosporine via Reverse Transcription
Polymerase Chain Reaction and qualitative analysis by agarose gel electrophoresis. Additionally, we determined the breast
cancer cell line T47D for identifying potential neuronal markers and representing other functions of the selected neuronal
guiding cues outside the nervous system in an in-vitro model. Our results showed no expression of netrin-1 and its receptor DCC in SH-SY5Y cells. Interestingly netrin-1 gene expression
could be detected in the breast cancer cells T47Ds. The netrin-1 receptors Unc5A, B and C were expressed in both, treated
and untreated SH-SY5Y cells, whereas Unc5D was only expressed in treated cells. Semaphorin 3A and 3F as well as the receptors
neuropilin-2, plexinA2, A3 and plexinC1 could be detected in differentiated and undifferentiated SH-SY5Y cells. Neuropilin-
1 was only synthesized in differentiated neuroblastoma cells in contrast to plexinA1, which was present in undifferentiated
SH-SY5Y. Therefore, Unc5C, neuropilin-1, and plexinC1 could be identified as potential neuronal markers.
03/2010: pages 98-101;
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ABSTRACT: An overall objective of pharmaceutical research is the controlled release or delivery of drugs at the biological target site in a therapeutically and pharmacodynamically optimal amount. In relation to "intelligent" drug delivery, several basic aspects are important, i.e., release of active pharmaceutical ingredients from the formulation, transport to and penetration across biological barriers, and subsequent biotransformation depending on a controlled release process. Future development of advanced and/or controlled drug releasing systems, e.g. polymeric or particulate drug targeting systems, nano-carbon tube related and/or nano-pillar based drug release, or electronically mediated molecule delivery, is expected to take advantage of progress in molecular cell biology, cell and tissue engineering, membrane nano-biophysics, and bioelectronic properties (Bramstedt et al. 2005; Gardner et al. 2006). In this chapter novel aspects of the development of innovative drug delivery systems described and are categorized into polymeric, lipid-based or electronically mediated delivery systems (De la Heras et al. 2004).
Handbook of experimental pharmacology 01/2010;
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ABSTRACT: The book chapter “Biosensing and Drug Delivery at the Microscale: Novel Devices for a Controlled and Responsive Drug Delivery”
published in the Handbook of Experimental Pharmacology vol. 197: Drug Delivery has been retracted. For further details see Erratum.
12/2009: pages 87-112;
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ABSTRACT: Tauopathies such as Alzheimer's disease (AD) belong to the group of neurodegenerative diseases that are characterised by hyperphosphorylation of the protein tau. Hyperphosphorylation of tau is one of the salient events leading to neuronal cytotoxicity and cognitive impairments. In this context, inhibition of tau hyperphosphorylation by specific tau kinase inhibitors can provide an excellent drug target for the treatment of AD and other tau-related neurodegenerative diseases. To improve the identification, optimisation and validation during the high-cost hit-to-lead cycle of AD drugs, we established a fast and sensitive label-free technique for testing the efficacy of tau kinase inhibitors in vitro. Here, we report for the first time that microelectrode-based impedance spectroscopy can be used to detect the pathological risk potential of hyperphosphorylated tau in the human neuroblastoma cell line SH-SY5Y. Our findings provide a novel real-time recording technique for testing the efficiency of tau kinase inhibitors or other lead structures directed to tau hyperphosphorylation on differentiated SH-SY5Y cells.
Lab on a Chip 06/2009; 9(10):1422-8. · 5.67 Impact Factor
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ABSTRACT: Heart diseases represent the most common cause of death in industrialised countries. For this reason target identification and development of novel anti-target drugs are in the focus of pharmaceutical industry. Especially cardiac infarct is a topical field of research. A bottleneck in today's long-duration and high-cost drug development is the lack of fast, label-free and cell-based high throughput/high content screening (HTS/HCS) assays for bridging the gap between cell-free screening and animal experiments. Here, we report for the first time on an in vitro cardiac ischemic model, where pathological consequences of simulated cardiac infarct can be detected quantitatively by microelectrode array-based impedance spectroscopy. Using the contractile HL-1 cell line and defined ischemic conditions we were able to develop a standardised and reproducible pathologic model. We characterised and verified the HL-1 based ischemic model by apoptosis and proliferation assays as well as immunochemical analysis of cell-cell junctions. We showed that the observed cell and biomolecular effects correspond with results obtained by impedance spectroscopy. Functionality of the impedimetric assay was demonstrated by real-time detection of reduced pathological effects due to application of the selective Rac1 inhibitor NCS23766. Numerical analysis by means of an equivalent circuit allowed the quantification of changes in resistance and capacitance of the adherent cell layer after ischemic treatment and application of NSC23766 as drug model. Our findings provide a novel cell-based real-time screening system for testing drug candidates against cardiac infarct and its implications.
Biosensors & bioelectronics 03/2009; 24(9):2798-803. · 5.43 Impact Factor
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ABSTRACT: We describe the use of rotary cultures (72 rpm) as an excellent method for generating spheroids from dispersed bovine granulosa cells (GC). The GC spheroids were symmetrical (diameter between 100 and 200 microm), easily accessible, and could be obtained at high yields. On day one, the spheroids showed a two-layered outer zone of cells that stained lighter than the inner zone in semi-thin sections. Bromodeoxyuridine (BrdU) uptake was frequent and randomly distributed. By day two, a striking decrease in BrdU uptake was noted. Apoptotic bodies appeared up to day four, as did TUNEL and propidium iodide labelled dead cells. At that time, the inner zone contained cells with large-sized vacuoles and the core was amorphous. The large-sized vacuoles were identified at the ultrastructural level and represented autophagosomes and autophagolysosomes that were in different stages of development. Surprisingly, conspicuous signs of cell death were accompanied by an increase in spontaneous luteinization compared to conventional stationary cultures. We detected high levels of progesterone (immunoassay) accompanied by high levels of the proteins and enzymes relevant for steroidogenesis (StAR, P450scc, 3beta-HSD by immunoblot and immunohistochemistry, respectively). Conclusions: Concomitant to cell death, GC spheroids augment progesterone synthesis. The GC spheroids provide an ideal model for studying steroidogenesis coupled to programmed cell death at the level of the mitochondria.
Differentiation 02/2009; 77(1):60-9. · 2.81 Impact Factor
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ABSTRACT: The glial cell line-derived neurotrophic factor (GDNF) family consists of the four ligands GDNF, neurturin (NRTN), artemin and persephin, which bind to the four co-receptors GDNF family receptor alpha1-4 and control through the activation of the receptor tyrosin kinase Ret several developmental processes. The purpose of this study was to analyse the expression and the influence of NRTN in the developing retina. We used retinospheres, a three-dimensional model system of the developing chicken retina. The expression of NRTN and the GDNF family receptor alpha 2 increased during development. Furthermore, expression was comparable in retinae and retinospheres. Analysis of signalling pathways influenced by NRTN in retinospheres showed activation of phosphatidylinositol-3 kinase and mitogen-activated protein kinase (MAPK). Activation of MAPK could be localised in cells of the innermost rows of the inner nuclear layer which were predominantly acetylcholinesterase-positive cells. Exogenous application of NRTN increased the amount of acetylcholinesterase-positive cells within the retinospheres at late culture stages. Additionally, we could show that Müller glia cells did not express the GFRalpha2 receptor and were probably not involved in NRTN signalling. Therefore, we conclude that NRTN directly participates in regulatory processes concerning the differentiation of acetylcholinesterase-positive cells in the chicken retina.
Journal of Neurochemistry 09/2008; 107(1):96-104. · 4.06 Impact Factor
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ABSTRACT: Close to realistic responses to anti-cancer drugs are not adequately provided in monolayer or single cells assays. 3-dimensional multicellular cultures (spheroids) mimicking in vivo-like conditions are established as cell biological models for microtumors/metastases. For a non-invasive real-time monitoring of the electrical parameters of such spheroid cultures we designed, fabricated and tested a 3D multifunctional electrode-based microcavity array. In a non-adherent assay acute tests with tumor spheroids were done maintaining their spherical shape and cellular arrangement. The sensor chip with 15 individual square microcavities containing four gold electrodes each was used for impedance spectroscopy to analyze the tissue models in terms of morphological and structural changes. Cell type specific differences in the spectra and varying responses to several anti-tumor drugs were found. Further development of the prototype will provide a promising tool for the use in pharmacological high-throughput studies.
Lab on a Chip 07/2008; 8(6):879-84. · 5.67 Impact Factor
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ABSTRACT: Persephin (PSPN), a member of the glial cell line-derived neurotrophic factor family, and its implication in the retina is not well understood but might be an interesting therapeutic target for degenerative diseases. Although, PSPN is lost in the chicken during evolution, its target, the GDNF family receptor alpha 4 (GFRalpha4), is still expressed in a temporal and spatial pattern in the developing retina. We used this "knockout-precondition" to study the bioactivity and the effect of exogenous PSPN application and subsequent GFRalpha activation during retinal development in vitro without impairments of endogenous PSPN. Retinospheres, derived from dissociated chicken retina of embryonic day 6, were treated with PSPN and intracellular signalling was monitored. Additionally, PSPN was added during cultivation of the retinospheres and immunhistochemical stainings and Western blotting were performed to evaluate changes in proliferation, apoptosis and differentiation. Exogenous applied PSPN enhanced phosphatidylinositol-3-kinase (PI-3K) signalling and decreased signalling of mitogen-activated protein kinases (MAPK). Most importantly early retinal proliferation was enhanced and glutamine synthetase expression was decreased whereas differentiation of major retinal cell types was not changed. In contrast to GDNF, PSPN is exclusively influencing early progenitors whereas differentiation is not effected and seems to be regulated through PSPN-independent mechanisms. Since the binding site of PSPN and therefore the target of potential therapeuticals, is well conserved among species and is with high probability not able to bind other members of the GDNF-family, these results might be assigned to other species including mammals and humans.
Neuroscience Letters 07/2008; 442(1):10-4. · 2.11 Impact Factor
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ABSTRACT: Multicellular tumour spheroids that mimic a native cellular environment are widely used as model systems for drug testing. To study drug effects on three-dimensional cultures in real-time we designed and fabricated a novel type of sensor chip for fast, non-destructive impedance spectroscopy and extracellular recording. Precultured spheroids are trapped between four gold electrodes. Fifteen individual 100microm deep square microcavities with sizes from 200 to 400microm allow an optimised positioning during the measurement. Although apoptosis was induced in human melanoma spheroids by Camptothecin (CTT), treated cultures did not show disintegration but displayed increased impedance magnitudes compared to controls after 8h resulting from an altered morphology of the outer cells. Contractions in cardiomyocyte spheroids were monitored when the innovative chip was used for recording of extracellular potentials. The silicon-based electrode array is used as an acute test system for the monitoring of any kind of 3D cell cultures. Since no adherence of cells or labelling is necessary the multifunctional sensor chip provides a basis for improved drug development by high content screenings with reduced costs and assay times. Additional improvements for parallel testing of different substances on one chip are presented.
Biosensors and Bioelectronics 06/2008; 23(10):1473-80. · 5.60 Impact Factor
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ABSTRACT: Sensorchip based impedance spectroscopy can detect inhibitory effects of human neuropeptide Y (hNPY) on living cells in a non-invasive labelling free way in real time without the need of supporting reagents. Since the discovery that neoplasmatic transformations in breast cancer are correlated with a change of the receptor subtype expression of hNPY in the affected tissue, the hNPY receptor-ligand system has come to the fore of cancer research. Today there are different methods detecting hNPY receptor interactions like fluorescent and radioactive labelling or detecting hNPY-pathway activation like cyclic adenosine monophosphate (cAMP) and G protein-coupled receptor (GPCR)-assays. For all these assays it is necessary to either label related proteins with additional substances, which can affect the nature state of the cell, or the need of producing cell lysate which allows only a snapshot of the investigated cells. To overcome these problems we established a new method to detect hNPY-receptor interactions. Therefore, we monitor the complex electric resistance (impedance) of cells attached to a microelectrode over a wide frequency range. Cell alterations are detected as changes in the impedance spectra. After application of the adenylyl cyclase-stimulating reagent forskolin, impedance is decreased at 5 kHz frequency within minutes. This effect can be inhibited by preincubating the cells with hNPY for a time range of 20 min. The inhibitory effect of hNPY can be washed out and the same cells can be stimulated by forskolin again.
Biosensors and Bioelectronics 05/2008; 24(2):253-9. · 5.60 Impact Factor
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ABSTRACT: Glycogen is the major energy reserve in neural tissues including the retina. A key-enzyme in glycogen metabolism is glycogen phosphorylase (GP) which exists in three differentially regulated isoforms. By applying isozyme-specific antibodies it could be demonstrated that the GP BB (brain), but not the GP MM (muscle) isoform is expressed in the chicken retina in neuronal and glial (Müller) cells. In the embryonic chicken retina, GP showed a development-dependent expression pattern. Double-labeling experiments with cell type-specific antibodies revealed that GP is expressed in various layers of the retina some of which, e.g., the photoreceptor inner segments, are known to be sites of high energy consumption. This suggests important roles of GP BB, and therefore glycogen, in early differentiation, spontaneous wave generation and in formation and stabilization of synapses.
Neurochemical Research 03/2008; 33(2):336-47. · 2.24 Impact Factor
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11/2006: pages 79 - 101; , ISBN: 9783527609611
