ACS Chemical Neuroscience

Publications in this journal

• Article: Two-photon optical interrogation of single dendritic spines with caged dopamine.

  Roberto Araya, Victoria Andino-Pavlovsky, Rafael Yuste, Roberto Etchenique
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  ABSTRACT: We introduce a novel caged dopamine compound (RuBi-Dopa) based on ruthenium photochemistry. RuBi-Dopa has a high uncaging efficiency and can be released with visible light (blue-green) and IR light in a two-photon regime. We combine two-photon photorelease of RuBi-Dopa with two-photon calcium imaging for an optical imaging and manipulation of individual dendritic spines in living brain slices, demonstrating that cortical dendritic spines can express functional dopamine receptors. This novel compound allows for the mapping of functional dopamine receptors in living brain tissue with exquisite spatial resolution.
  ACS Chemical Neuroscience 05/2013;
• Article: Brain Glucose-6-phosphate Dehydrogenase Protects against Endogenous Oxidative DNA Damage and Neurodegeneration in Aged Mice.

  Winnie Jeng, Margaret M Loniewska, Peter G Wells
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  ABSTRACT: Glucose-6-phosphate dehydrogenase (G6PD) protects the embryo from endogenous and xenobiotic-enhanced oxidative DNA damage and embryopathies. Here we show in aged mice that G6PD similarly protects against endogenous reactive oxygen species (ROS)-mediated neurodegeneration. In G6PD-normal (G6PD(+/+)) and heterozygous (G6PD(+/def)) and homozygous (G6PD(def/def)) G6PD-deficient male and female mice at about 2 years of age, oxidative DNA damage in various brain regions was assessed by 8-oxo-2'-deoxyguanosine formation using high-performance liquid chromatography and immunohistochemistry. Morphological changes in brain sections were assessed by H&E staining. DNA oxidation was increased in G6PD(def/def) mice in the cortex (p < 0.02), hippocampus (p < 0.01) and cerebellum (p < 0.006) compared to G6PD(+/+) mice, and was localized to distinct cell types. Histologically, in G6PD(+/def) mice, enhanced regionally and cellularly specific neurodegenerative changes were observed in those brain regions exhibiting elevated DNA oxidation, with a 53% reduction in the Purkinje cell count. These results show G6PD is important in protecting against the neurodegenerative effects of endogenous ROS in aging, and suggest that common hereditary G6PD deficiencies may constitute a risk factor for some neurodegenerative diseases.
  ACS Chemical Neuroscience 05/2013;
• Article: Identification of a novel indoline derivative for in vivo fluorescent imaging of blood-brain barrier disruption in animal models.

  Yuhei Nishimura, Kenichiro Yata, Tsuyoshi Nomoto, Tomoaki Ogiwara, Kohei Watanabe, Taichi Shintou, Akira Tsuboyama, Mie Okano, Noriko Umemoto, Zi Zhang, Miko Kawabata, Beibei Zhang, Junya Kuroyanagi, Yasuhito Shimada, Takeshi Miyazaki, Takeshi Imamura, Hidekazu Tomimoto, Toshio Tanaka
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  ABSTRACT: Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741.
  ACS Chemical Neuroscience 05/2013;
• Article: First Experimental Evidence of Dopamine Interactions with Negatively Charged Model Biomembranes.

  Katarzyna Jodko-Piorecka, Grzegorz Litwinienko
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  ABSTRACT: Dopamine is essential for receptor-related signal transduction in mammalian central and peripheral nervous systems. Weak interactions between the neurotransmitter and neuronal membranes have been suggested to modulate synaptic transmission; however, binding forces between dopamine and neuronal membranes have not yet been quantitatively described. Herein, for the first time, we have explained the nature of dopamine interactions with model lipid membranes assembled from neutral 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), and the mixture of these two lipids using isothermal titration calorimetry and differential scanning calorimetry. Dopamine binding to anionic membranes is a thermodynamically favored process with negative enthalpy and positive entropy, quantitatively described by the mole ratio partition coefficient, K. K increases with membrane charge to reach its maximal value, 705.4 ± 60.4 M(-1), for membrane composed from pure DMPG. The contribution of hydrophobic effects to the binding process is expressed by the intrinsic partition coefficient, K(0). The value of K(0) = 74.7 ± 6.4 M(-1) for dopamine/DMPG interactions clearly indicates that hydrophobic effects are 10 times weaker than electrostatic forces in this system. The presence of dopamine decreases the main transition temperature of DMPG, but no similar effect has been observed for DMPC. Basing on these results, we propose a simple electrostatic model of dopamine interactions with anionic membranes with the hydrophobic contribution expressed by K(0). We suggest that dopamine interacts superficially with phospholipid membranes without penetrating into the bilayer hydrocarbon core. The model is physiologically important, since neuronal membranes contain a large (even 20%) fraction of anionic lipids.
  ACS Chemical Neuroscience 05/2013;
• Article: Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits.

  Briana J Davie, Arthur Christopoulos, Peter J Scammells
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  ABSTRACT: Since the cholinergic hypothesis of memory dysfunction was first reported, extensive research efforts have focused on elucidating the mechanisms by which this intricate system contributes to the regulation of processes such as learning, memory and higher executive function. Several cholinergic therapeutic targets for the treatment of cognitive deficits, psychotic symptoms and the underlying pathophysiology of neurodegenerative disorders, such as Alzheimer's disease and schizophrenia, have since emerged. Clinically-approved drugs now exist for some of these targets, however they all may be considered suboptimal therapeutics in that they produce undesirable off-target activity leading to side effects, fail to address the wide variety of symptoms and underlying pathophysiology that characterize these disorders and/or afford little to no therapeutic effect in subsets of patient populations. A promising target for which there are presently no approved therapies is the M1 muscarinic acetylcholine receptor (M1 mAChR). Despite avid investigation, development of agents that selectively activate this receptor via the orthosteric site has been hampered by the high sequence homology of the binding site between the five muscarinic receptor subtypes and the wide distribution of this receptor family in both the central nervous system (CNS) and the periphery. Hence, a plethora of ligands targeting less structurally-conserved allosteric sites of the M1 mAChR have been investigated. This review aims to explain the rationale behind allosterically targeting the M1 mAChR, comprehensively summarize and critically evaluate the M1 mAChR allosteric ligand literature to date, highlight the challenges inherent in allosteric ligand investigation that are impeding their clinical advancement, and discuss potential methods for resolving these issues.
  ACS Chemical Neuroscience 05/2013;
• Article: Glutaraldehyde Cross-Linked Glutamate Oxidase Coated Microelectrode Arrays: Selectivity and Resting Levels of Glutamate in the CNS.

  Jason J Burmeister, Verda A Davis, Jorge E Quintero, Francois Pomerleau, Peter Huettl, Greg A Gerhardt
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  ABSTRACT: Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both non-enzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observed comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific wet coating procedures.
  ACS Chemical Neuroscience 05/2013;
• Article: Preparation and evaluation at the delta opioid receptor of a series of linear Leu-enkephalin analogs obtained by systematic replacement of the amides.

  Kristina Rochon, Arnaud Proteau-Gagné, Philippe Bourassa, Jean-François Nadon, Jérome Côté, Véronique Bournival, Fernand Jr Gobeil, Brigitte Guérin, Yves L Dory, Louis Gendron
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  ABSTRACT: Leu-enkephalin analogs, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD7.4) and plasma stability of the active analogs were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogs when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogs with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds.
  ACS Chemical Neuroscience 05/2013;
• Article: APP+, a Fluorescent Analogue of the Neurotoxin MPP+, Is a Marker of Catecholamine Neurons in Brain Tissue, but Not a Fluorescent False Neurotransmitter.

  Richard J Karpowicz, Matthew Dunn, David Sulzer, Dalibor Sames
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  ABSTRACT: We have previously introduced fluorescent false neurotransmitters (FFNs) as optical reporters that enable visualization of individual dopaminergic presynaptic terminals and their activity in the brain. In this context, we examined the fluorescent pyridinium dye 4-(4-dimethylamino)phenyl-1-methylpyridinium (APP+), a fluorescent analogue of the dopaminergic neurotoxin MPP+, in acute mouse brain tissue. APP+ is a substrate for the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT), and as such represented a candidate for the development of new FFN probes. Here we report that APP+ labels cell bodies of catecholaminergic neurons in the midbrain in a DAT- and NET-dependent manner, as well as fine dopaminergic axonal processes in the dorsal striatum. APP+ destaining from presynaptic terminals in the dorsal striatum was also examined under the conditions inducing depolarization and exocytotic neurotransmitter release. Application of KCl led to a small but significant degree of destaining (approximately 15% compared to control), which stands in contrast to a nearly complete destaining of the new generation FFN agent, FFN102. Electrical stimulation of brain slices at 10 Hz afforded no significant change in the APP+ signal. These results indicate that the majority of the APP+ signal in axonal processes originates from labeled organelles including mitochondria, whereas only a minor component of the APP+ signal represents the releasable synaptic vesicular pool. These results also show that APP+ may serve as a useful probe for identifying catecholaminergic innervations in the brain, although it is a poor candidate for the development of FFNs.
  ACS Chemical Neuroscience 05/2013;
• Article: Analysis of Glutamate, GABA, Noradrenaline, Dopamine, Serotonin and Metabolites using microbore UHPLC with Electrochemical Detection.

  Nico J Reinhoud, Hendrik-Jan Brouwer, Lusi M van Heerwaarden, Gerdien A H Korte-Bouws
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  ABSTRACT: The applicability of microbore ultra-high performance liquid chromatography (UHPLC) with electrochemical detection for offline analysis of a number of well-known neurotransmitters in less than 10 µL microdialysis fractions is described. Two methods are presented for the analysis of monoamine or amino acid neurotransmitters, using the same UHPLC instrument. Speed of analysis of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and the metabolites homovanillic acid (HVA), 5-hydroxyindole aceticacid (5-HIAA), and 3,4-dihydroxyphenylacetic acid (DOPAC) was predominated by the retention behavior of NA, the non-ideal behavior of matrix components, and the loss in signal of 5-HT. This method was optimized to meet the requirements for detection sensitivity and minimizing the size of collected fractions, which determines temporal resolution in microdialysis. The amino acid neurotransmitters glutamate (Glu) and gamma-aminobutyric acid (GABA) were analyzed after an automated derivatization procedure. Under optimized conditions, Glu was resolved from a number of early eluting system peaks, while the total runtime was decreased to 15 min by a fourfold increase of the flow rate under UHPLC conditions. The detection limit for Glu and GABA was 10 nmol/L (15 fmol in 1.5 µL); the monoamine neurotransmitters had a detection limit between 32 - 83 pmol/L (0.16 - 0.42 fmol in 5 µL) in standard solutions. Using UHPLC, the analysis times varied from 15 min to less than 2 min depending on the complexity of the samples and the substances to be analyzed.
  ACS Chemical Neuroscience 05/2013;
• Article: Probing the ability of presynaptic tyrosine kinase receptors to regulate striatal dopamine dynamics.

  Aaron K Apawu, Francis K Maina, James R Taylor, Tiffany A Mathews
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  ABSTRACT: Brain-derived neurotrophic factor (BDNF) modulates the synaptic transmission of several monoaminergic neuronal systems. Molecular techniques using synapatosomes in previous studies have suggested that BDNF's receptor, tyrosine kinases (Trk), can quickly regulate dopamine release and transporter dynamics. Our main objective in this study is to determine whether slice fast scan cyclic voltammetry can be used to investigate the role of the TrkB receptor on dopamine release and uptake processes in the caudate-putamen. Fast scan cyclic voltammetry measured dopamine release and uptake rates in the presence of BDNF, or its agonist 7,8-dihydroxyflavone, or a TrkB inhibitor K252a. Superfusion of BDNF led to partial recovery of the electrically-stimulated dopamine release response in BDNF+/- mice which is blunted compared to wildtype mice, with no effect in wildtype mice. Conversely, infusion of 7,8-dihydroxyflavone increased electrically-stimulated dopamine release in wildtype mice with no difference in BDNF+/- mice. Overall, BDNF and 7,8-dihydroxyflavone had no effect on dopamine uptake rates. Concentrations greater than 3 μM 7,8-dihydroxyflavone affected DA uptake rates in BDNF+/- mice only. To demonstrate that BDNF and 7,8-dihydroxyflavone modulate DA release by activating the TrkB receptor, both genotypes were pretreated with K252a. K252a was able to block BDNF and 7,8-DHF induced increases during stimulated dopamine release in BDNF+/- and wildtype mice, respectively. Fast scan cyclic voltammetry demonstrates that acute TrkB activation potentiates dopamine release in both genotypes.
  ACS Chemical Neuroscience 05/2013;
• Article: A Sub-Cellular Biochemical Investigation of Purkinje Neurons Using Synchrotron Radiation Fourier Transform Infrared Spectroscopic Imaging with a Focal Plane Array Detector.

  Mark J Hackett, Ferenc Borondics, Devin Brown, Carol J Hirschmugl, Shari E Smith, Phyllis G Paterson, Helen Nichol, Ingrid Jane Pickering, Graham Neil George
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  ABSTRACT: Coupling Fourier transform infrared spectroscopy with focal plane array detectors at synchrotron radiation sources (SR-FTIR-FPA) has provided a rapid method to simultaneously image numerous biochemical markers in situ at diffraction limited resolution. Since cells and nuclei are well resolved at this spatial resolution, a direct comparison can be made between FTIR functional group images and the histology of the same section. To allow histological analysis of the same section analyzed with infrared imaging, unfixed air dried tissue sections are typically fixed (after infrared spectroscopic analysis is completed) via immersion fixation. This post fixation process is essential to allow histological staining of the tissue section. Although immersion fixation is a common practice in this filed the initial rehydration of the dehydrated unfixed tissue can result in distortion of sub-cellular morphology and confound correlation between infrared images and histology. In this study, vapor fixation, a common choice in other research fields where post-fixation of un-fixed tissue sections is required was employed in place of immersion fixation post spectroscopic analysis to provide more accurate histology with reduced distortions as the dehydrated tissue section is fixed in vapor rather than during rehydration in an aqueous fixation medium. With this approach, accurate correlation between infrared images and histology of the same section revealed that Purkinje neurons in the cerebellum are rich in cytosolic proteins and not depleted as once thought. In addition, we provide the first direct evidence of intracellular lactate within Purkinje neurons. This highlights the significant potential for future applications of SR-FTIR-FPA imaging to investigate cellular lactate under conditions of altered metabolic demand such as increased brain activity and hypoxia or ischemia.
  ACS Chemical Neuroscience 05/2013;
• Article: Assessment of Tissue Viability Following Electroosmotic Push-Pull Perfusion from Organotypic Hippocampal Slice Cultures.

  Amy E Rupert, Y Ou, M Sandberg, S G Weber
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  ABSTRACT: We have developed a novel sampling technique that allows both introduction and removal of fluid from the extracellular space of living tissue. This method is based on the fluidics of push-pull perfusion but flow is driven by electroosmosis. We have applied this method to organotypic hippocampal cultures. A source capillary is inserted into the tissue and a collection capillary is in contact with the tissue surface through a thin layer of fluid. A voltage is applied across the proximal ends of source and collection capillary. In the applied field, fluid will move from source, into the tissue, and then be collected. In this process, damage to cells may occur. To understand better what sampling conditions influence damage most, we tested various sampling geometries and applied voltages, quantifying damage 16-24 h later using propidium iodide as a cell death marker. We found that damage correlates with both voltage drop and power dissipated in the tissue, but that voltage drop is a better indicator of damage when comparing models in which capillary arrangement and length are different.
  ACS Chemical Neuroscience 05/2013;
• Article: Age-related changes in melatonin release in the murine distal colon.

  Lucy Diss, Stephen Robinson, Yukyee Wu, Sara Fidalgo, Mark S Yeoman, Bhavik Anil Patel
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  ABSTRACT: Constipation and fecal impaction are conditions of the bowel whose prevalence increases with age. Limited information is known about how these conditions manifest, however functional deficits are likely to be due to changes in signalling within the bowel. This study investigated the effects of age on colonic mucosal melatonin (MEL) release and the consequences this had on colonic motility. Electrochemical measurements of MEL overflow demonstrated that both basal and mechanically stimulated MEL release decreased with age. The MEL:serotonin also decreased with increasing age and the trend was similar to that of MEL overflow, suggestive that age-related changes were primarily due to a reduction in MEL levels. Levels of N-acetylserotonin and the N-acetylserotonin:serotonin ratio were reduced with age providing an explanation for the reduction in MEL release. Decreases in colonic motility were observed between 3 and 24 month old animals. Exogenous application of MEL could reverse this deficit in aged colon. In summary, we propose that the age-related decline in MEL release may be due to either decreases or alterations in mechanosensory channels and/or a loss in levels/activity of the N-acetyltransferase enzyme responsible for the synthesis of N-acetylserotonin. Decreases in MEL release may explain the decreases in colonic motility observed in 24 month old animals and could offer a new potential therapeutic treatment for age-related constipation.
  ACS Chemical Neuroscience 04/2013;
• Article: A Comparison of the Subsecond Dynamics of Neurotransmission of Dopamine and Serotonin.

  Katie A Jennings
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  ABSTRACT: The neuromodulators dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT) are similar in a number of ways. Both monoamines can act by volume transmission at metabotropic receptors to modulate synaptic transmission in brain circuits. Presynaptic regulation of 5-HT and DA is governed by parallel processes, and behaviorally, both exert control over emotional processing. However, differences are also apparent: more than twice as many 5-HT receptor subtypes mediate postsynaptic effects than DA receptors and different presynaptic regulation is also emerging. Monoamines are amenable to real-time electrochemical detection using fast scan cyclic voltammetry (FSCV), which allows resolution of the subsecond dynamics of release and reuptake in response to a single action potential. This approach has greatly enriched understanding of DA transmission and has facilitated an integrated view of how DA mediates behavioral control. However, technical challenges are associated with FSCV measurement of 5-HT and understanding of 5-HT transmission at subsecond resolution has not advanced at the same rate. As a result, how the actions of 5-HT at the level of the synapse translate into behavior is poorly understood. Recent technical advances may aid the study of 5-HT in real-time. It is timely, therefore, to compare and contrast what is currently understood of the subsecond characteristics of transmission for DA and 5-HT. In doing so, a number of areas are highlighted as being worthy of exploration for 5-HT.
  ACS Chemical Neuroscience 04/2013;
• Article: Electroosmotic Push-pull Perfusion: Description and Application to Qualitative Analysis of the Hydrolysis of Exogenous Galanin in Organotypic Hippocampal Slice Cultures.

  Amy E Rupert, Yangguang Ou, Mats Sandberg, Stephen G Weber
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  ABSTRACT: We demonstrate here a method that perfuses a small region of an organotypic hippocampal culture with a solution containing an enzyme substrate - a neuropeptide. Perfusate containing hydrolysis products is continually collected and subsequently analyzed for the products of the enzymatic degradation of the peptide substrate. The driving force for perfusion is an electric field. The fused silica capillaries used as "push" and "pull" or "source" and "collection" capillaries have a ζ-potential that is negative and greater in magnitude than the tissue's ζ-potential. Thus, depending on the magnitudes of particular dimensions, the electroosmotic flow in the capillaries augments the fluid velocity in the tissue. The flow rate is not directly measured, however we determine it using a finite-element approach. We have determined the collection efficiency of the system using an all D-amino acid internal standard. The flow rates are low, in the nL/min range, and adjustable by controlling the current or voltage in the system. The collection efficiency of the D-amino acid peptide internal standard is variable, increasing with increased current and thus electroosmotic flow rate. The collection efficiency can be rationalized in the context of a Peclet number. Electroosmotic push-pull perfusion of the neuropeptide galanin (gal1-29) through the extracellular space of an organotypic hippocampal culture results in its hydrolysis by ectopeptidase reactions occurring in the extracellular space. The products of hydrolysis were identified by MALDI-MS. Experiments at two levels of current (8-12 μA and 19-40 μA) show that the probability of seeing hydrolysis products (apparently from aminopeptidases) is greater in the Cornu Ammonis area 3 (CA3) than in the Cornu Ammonis area 1 (CA1) in the higher current experiments. In the lower current experiments, shorter peptide products of aminopeptidases (gal13-29 to gal20-19) are seen with greater frequency in CA3 than in CA1 but there is no statistically significant difference for longer peptides (gal3-29 to gal12-29).
  ACS Chemical Neuroscience 04/2013;
• Article: Physiologically Relevant Changes in Serotonin Resolved by Fast Microdialysis.

  Hongyan Yang, Andrew B Thompson, Bryan J McIntosh, Stefanie C Altieri, Anne M Andrews
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  ABSTRACT: Online microdialysis is a sampling and detection method that enables continuous interrogation of extracellular molecules in freely moving subjects under behaviorally relevant conditions. A majority of recent publications using brain microdialysis in rodents report sample collection times of 20-30 min. These long sampling times are due, in part, to limitations in the detection sensitivity of high performance liquid chromatography (HPLC). By optimizing separation and detection conditions, we decreased the retention time of serotonin to 2.5 min and the detection threshold to 0.8 fmol. Sampling times were consequently reduced from 20 to 3 min per sample for online detection of serotonin (and dopamine) in brain dialysates using a commercial HPLC system. We developed a strategy to collect and to analyze dialysate samples continuously from two animals in tandem using the same instrument. Improvements in temporal resolution enabled elucidation of rapid changes in extracellular serotonin levels associated with mild stress and circadian rhythms. These dynamics would be difficult or impossible to differentiate using conventional microdialysis sampling rates.
  ACS Chemical Neuroscience 04/2013;
• Article: Two cell circuits of oriented adult hippocampal neurons on self-assembled monolayers for use in the study of neuronal communication in a defined system.

  Darin Edwards, Maria Stancescu, Peter Molnar, James J Hickman
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  ABSTRACT: In this study, we demonstrate the directed formation of small circuits of electrically active, synaptically connected neurons derived from the hippocampus of adult rats through the use of engineered chemically modified culture surfaces that orient the polarity of the neuronal processes. Although synaptogenesis, synaptic communication, synaptic plasticity, and brain disease pathophysiology can be studied using brain slice or dissociated embryonic neuronal culture systems, the complex elements found in neuronal synapses makes specific studies difficult in these random cultures. The study of synaptic transmission in mature adult neurons and factors affecting synaptic transmission are generally studied in organotypic cultures, in brain slices, or in vivo. However, engineered neuronal networks would allow these studies to be performed instead on simple functional neuronal circuits derived from adult brain tissue. Photolithographic patterned self-assembled monolayers (SAMs) were used to create the two-cell "bidirectional polarity" circuit patterns. This pattern consisted of a cell permissive SAM, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA), and was composed of two 25 µm somal adhesion sites connected with 5 µm lines acting as surface cues for guided axonal and dendritic regeneration. Surrounding the DETA pattern was a background of a non-cell permissive poly(ethylene glycol) (PEG) SAM. Adult hippocampal neurons were first cultured on cover slips coated with DETA monolayers and were later passaged onto the PEG-DETA bidirectional polarity patterns in serum-free medium. These neurons followed surface cues, attaching and regenerating only along the DETA substrate to form small engineered neuronal circuits. These circuits were stable for more than 21 days in vitro (DIV) during which synaptic connectivity was evaluated using basic electrophysiological methods.
  ACS Chemical Neuroscience 04/2013;
• Article: GluN2A and GluN2B NMDA Receptor Subunits Differentially Modulate Striatal Output Pathways and Contribute to Levodopa-Induced Abnormal Involuntary Movements in Dyskinetic Rats.

  Omar S Mabrouk, Flora Mela, Mariangela Calcagno, Mirco Budri, Riccardo Viaro, Andrzej Dekundy, Christopher G Parsons, Yves P Auberson, Michele Morari
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  ABSTRACT: Dual probe microdialysis was used to investigate whether GluN2A and GluN2B NMDA receptor subunits regulate striatal output pathways under dyskinetic conditions. The preferential GluN2A antagonist NVP-AAM077 perfused in the dopamine-depleted striatum of 6-hydroxydopamine hemilesioned dyskinetic rats reduced GABA and glutamate levels in globus pallidus whereas the selective GluN2B antagonist Ro 25-6981 elevated glutamate without affecting pallidal GABA. Moreover, intrastriatal NVP-AAM077 did not affect GABA but elevated glutamate levels in substantia nigra reticulata whereas Ro 25-6981 elevated GABA and reduced nigral glutamate. To investigate whether GluN2A and GluN2B NMDA receptor subunits are involved in motor pathways underlying dyskinesia expression, systemic NVP-AAM077 and Ro 25-6981 were tested for their ability to attenuate levodopa-induced abnormal involuntary movements. NVP-AAM077 failed to prevent dyskinesia while Ro 25-6981 mildly attenuated it. We conclude that in the dyskinetic striatum, striatal GluN2A subunits tonically stimulate the striato-pallidal pathway whereas striatal GluN2B subunits tonically inhibit striato-nigral projections. Moreover, GluN2A subunits are not involved in dyskinesia expression whereas GluN2B subunits minimally contribute to it.
  ACS Chemical Neuroscience 04/2013;
• Article: Structures and Free Energy Landscapes of the A53T Mutant-Type α-Synuclein Protein and Impact of A53T Mutation on the Structures of the Wild-Type α-Synuclein Protein with Dynamics

  Orkid Coskuner, Olivia Wise-Scira
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  ABSTRACT: The A53T genetic missense mutation of the wild-type α-synuclein protein was initially identified in Greek and Italian families with familial Parkinson’s disease. Detailed understanding of the structures and the changes induced in the wild-type αS structure by the A53T mutation as well as establishing the direct relationships between the rapid conformational changes and free energy landscapes of these intrinsically disordered fibrillogenic proteins helps to enhance our fundamental knowledge and to gain insights into the pathogenic mechanism of Parkinson’s disease. We employed extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations to determine the secondary and tertiary structural properties as well as the conformational free energy surfaces of the wild-type and A53T mutant-type αS proteins in an aqueous solution medium using both implicit and explicit water models. The confined aqueous volume effect in the simulations of disordered proteins using an explicit model for water is addressed for a model disordered protein. We also assessed the stabilities of the residual secondary structure component interconversions in αS based on free energy calculations at the atomic level with dynamics using our recently developed theoretical strategy. To the best of our knowledge, this study presents the first detailed comparison of the structural properties linked directly to the conformational free energy landscapes of the monomeric wild-type and A53T mutant-type α-synuclein proteins in an aqueous solution environment. Results present that the β-sheet structure is significantly more altered than the helical structure upon A53T mutation of the monomeric wild-type αS protein in aqueous solution. The β-sheet content close to the mutation site in the N-terminal region is more abundant while the NAC and C-terminal regions show a decrease in β-sheet abundance upon A53T mutation. Obtained results utilizing our new theoretical strategy show that the residual secondary structure conversion stabilities resulting in α-helix formation are not significantly affected by the mutation. Interestingly, the residual secondary structure conversion stabilities show that secondary structure conversions resulting in β-sheet formation are influenced by the A53T mutation and the most stable residual transition yielding β-sheet occurs directly from the coil structure. Long-range interactions detected between the NAC region and the N- or C-terminal regions of the wild-type αS disappear upon A53T mutation. The A53T mutant-type αS structures are thermodynamically more stable than those of the wild-type αS protein structures in aqueous solution. Overall, the higher propensity of the A53T mutant-type αS protein to aggregate in comparison to the wild-type αS protein is related to the increased β-sheet formation and lack of strong intra-molecular long-range interactions in the N-terminal region in comparison to its wild-type form. The specific residual secondary structure component stabilities reported herein provide information helpful for designing and synthesizing small organic molecules that can block the β-sheet forming residues, which are reactive towards aggregation.
  ACS Chemical Neuroscience 04/2013;
• Article: Visualizing neurotransmitters and metabolites in central nervous systems by high resolution and high accuracy mass spectrometric imaging.

  Hui Ye, Jingxin Wang, Tyler Greer, Kerstin Strupat, Lingjun Li
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  ABSTRACT: The spatial localization and molecular distribution of metabolites and neurotransmitters within biological organisms is of tremendous interest to neuroscientists. In comparison to conventional imaging techniques like immunohistochemistry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has demonstrated its unique advantage by directly localizing the distribution of a wide range of biomolecules simultaneously from a tissue specimen. Although MALDI-MSI of metabolites and neurotransmitters is hindered by numerous matrix-derived peaks, high-resolution and high-accuracy mass spectrometers (HRMS) allow differentiation of endogenous analytes from matrix peaks, unambiguously obtaining biomolecular distributions. In this study, we presented MSI of metabolites and neurotransmitters in rodent and crustacean central nervous systems acquired on HRMS. Results were compared with those obtained from a medium-resolution mass spectrometer (MRMS), tandem time-of-flight instrument, to demonstrate the power and unique advantages of HRMSI and reveal how this new tool would benefit molecular imaging applications in neuroscience.
  ACS Chemical Neuroscience 04/2013;