Toward determining amyloid fibril structures using experimental constraints from Raman spectroscopy.

We present structural models for three different amyloid fibril polymorphs prepared from amylin20-29 (sequence SNNFGAILSS) and amyloid-ß25-35 (Aß25-35) (sequence GSNKGAIIGLM) peptides. These models are based on the amide C=O bond and Ramachandran ψ-dihedral angle data from Raman spectroscopy, which were used as structural constraints to guide molecular dynamics (MD) simulations. The resulting structural models indicate that the basic structural motif of amylin20-29 and Aß25-35 fibrils is extended ß-strands. Our data indicate that amylin20-29 forms both antiparallel and parallel ß-sheet fibril polymorphs, while Aß25-35 forms a parallel ß-sheet fibril structure. Overall, our work lays the foundation for using Raman spectroscopy in conjunction with MD simulations to determine detailed molecular-level structural models of amyloid fibrils in a manner that complements gold-standard techniques, such as solid-state nuclear magnetic resonance and cryogenic electron microscopy.

A facile approach to create sensitive and selective Cu(ii) sensors on carbon fiber microelectrodes.

A facile platform derived from deposition of ethynyl linkers on carbon fiber microelectrodes has been developed for sensitive and selective sensing of Cu(ii). This study is the first to demonstrate the successful anodic deposition of ethynyl linkers, specifically 1,4-diethynylbenzene, onto carbon fiber microelectrodes. Multi-scan deposition of DEB on these microelectrodes resulted in an increased sensitivity and selectivity towards Cu(ii) that persists amidst other divalent interferents and displays sustained performance over four days while stored at room temperature. This method can be extended to other ethynyl terminal moieties, thereby creating a versatile chemical platform that will enable improved sensitivity and selectivity for a new frontier of biomarker measurement.

Measuring tryptophan dynamics using fast scan cyclic voltammetry at carbon fiber microelectrodes with improved sensitivity and selectivity.

Despite the fact that tryptophan (Trp) is an essential amino acid that humans typically obtain through diet, there are several interesting tryptophan dynamics at play in the body. Quantifying and understanding these dynamics are crucial in studies of depression, autism spectrum disorder, and other disorders that involve neurotransmitters directly synthesized from tryptophan. Here we detail the optimization of waveform parameters in fast scan cyclic voltammetry at carbon fiber microelectrodes to yield four-fold higher sensitivity and six-fold higher selectivity compared to previously reported methods. We demonstrate the utility of our method in measuring (1) exogenous Trp dynamics from administration of Trp to PC-12 cells with and without overexpression of tryptophan hydroxylase-2 and (2) endogenous Trp dynamics in pinealocyte cultures with and without stimulation via norepinephrine. We observed interesting differences in Trp dynamics in both model systems, which demonstrate that our method is indeed sensitive to Trp dynamics in different applications.

A tale of two transmitters: serotonin and histamine as in vivo biomarkers of chronic stress in mice.

BACKGROUND: Stress-induced mental illnesses (mediated by neuroinflammation) pose one of the world's most urgent public health challenges. A reliable in vivo chemical biomarker of stress would significantly improve the clinical communities' diagnostic and therapeutic approaches to illnesses, such as depression. METHODS: Male and female C57BL/6J mice underwent a chronic stress paradigm. We paired innovative in vivo serotonin and histamine voltammetric measurement technologies, behavioral testing, and cutting-edge mathematical methods to correlate chemistry to stress and behavior. RESULTS: Inflammation-induced increases in hypothalamic histamine were co-measured with decreased in vivo extracellular hippocampal serotonin in mice that underwent a chronic stress paradigm, regardless of behavioral phenotype. In animals with depression phenotypes, correlations were found between serotonin and the extent of behavioral indices of depression. We created a high accuracy algorithm that could predict whether animals had been exposed to stress or not based solely on the serotonin measurement. We next developed a model of serotonin and histamine modulation, which predicted that stress-induced neuroinflammation increases histaminergic activity, serving to inhibit serotonin. Finally, we created a mathematical index of stress, Si and predicted that during chronic stress, where Si is high, simultaneously increasing serotonin and decreasing histamine is the most effective chemical strategy to restoring serotonin to pre-stress levels. When we pursued this idea pharmacologically, our experiments were nearly identical to the model's predictions. CONCLUSIONS: This work shines the light on two biomarkers of chronic stress, histamine and serotonin, and implies that both may be important in our future investigations of the pathology and treatment of inflammation-induced depression.

Low-Frequency Oscillations of In Vivo Ambient Extracellular Brain Serotonin.

Serotonin is an important neurotransmitter that plays a major role in many aspects of neuroscience. Fast-scan cyclic voltammetry measures fast in vivo serotonin dynamics using carbon fiber microelectrodes. More recently, fast-scan controlled-adsorption voltammetry (FSCAV) has been developed to measure slower, minute-to-minute changes in ambient extracellular serotonin. We have previously demonstrated that FSCAV measurements of basal serotonin levels give critical information regarding brain physiology and disease. In this work, we revealed the presence of low-periodicity fluctuations in serotonin levels in mouse hippocampi, measured in vivo with FSCAV. Using correlation analyses, we found robust evidence of oscillations in the basal serotonin levels, which had a period of 10 min and were not present in vitro. Under control conditions, the oscillations did not differ between male and female mice, nor do they differ between mice that underwent a chronic stress paradigm and those in the control group. After the acute administration of a selective serotonin reuptake inhibitor, we observed a shift in the frequency of the oscillations, leading us to hypothesize that the newly observed fluctuations were transporter regulated. Finally, we optimized the experimental parameters of the FSCAV to measure at a higher temporal resolution and found more pronounced shifts in the oscillation frequency, along with a decreased oscillation amplitude. We postulate that this work may serve as a potential bridge for studying serotonin/endocrine interactions that occur on the same time scale.

Frontiers in Electrochemical Sensors for Neurotransmitter Detection: Towards Measuring Neurotransmitters as Chemical Diagnostics for Brain Disorders.

It is extremely challenging to chemically diagnose disorders of the brain. There is hence great interest in designing and optimizing tools for direct detection of chemical biomarkers implicated in neurological disorders to improve diagnosis and treatment. Tools that are capable of monitoring brain chemicals, neurotransmitters in particular, need to be biocompatible, perform with high spatiotemporal resolution, and ensure high selectivity and sensitivity. Recent advances in electrochemical methods are addressing these criteria; the resulting devices demonstrate great promise for in vivo neurotransmitter detection. None of these devices are currently used for diagnostic purposes, however these cutting-edge technologies are promising more sensitive, selective, faster, and less invasive measurements. Via this review we highlight significant technical advances and in vivo studies, performed in the last 5 years, that we believe will facilitate the development of diagnostic tools for brain disorders.

Analysis of Electrochemically Elusive Trace Metals with Carbon Fiber Microelectrodes.

There is great interest in rapidly monitoring metals of biological and environmental interest. Electrochemistry is traditionally a powerful tool for metal analysis but can be limited by its scope and low temporal resolution. The scope is limited by the potential window of the working electrode and rapid analysis is limited, in part, by the need for nucleation/growth for preconcentration. In prior work, we showed that a rapid equilibrium mediated preconcentration process facilitated fast scan cyclic voltammetry (FSCV) responses to Cu(II) and Pb(II) at carbon fiber microelectrodes (CFMs). In this manuscript, we apply this same principle to Ca(II), Al(III), Mg(II), and Zn(II), metal ions that are traditionally difficult to electroanalyze. We demonstrate FSCV reduction peaks for these four metals whose positions and amplitudes are dependent on scan rate. The adsorption profiles of these ions onto CFMs follow Langmuir's theory for monolayer coverage. We calculate the thermodynamic equilibrium constant of metal adsorption onto CFMs and find that these constants follow the same order as those previously reported by other groups on other activated carbon materials. Finally, a real-time complexation study is performed with ligands that have preference for divalent or multivalent ions to probe the selectivity of the real-time signal. We observe a linear relationship between formation constant ( kf) and % change in the FSCV signal and use this correlation to, for the first time, report the kf of an Al(III)-complex. This work demonstrates the versatility of FSCV as a method with capacity to extend the scope of rapid electroanalysis.

Methods of Measuring Enzyme Activity Ex Vivo and In Vivo.

Enzymes catalyze a variety of biochemical reactions in the body and, in conjunction with transporters and receptors, control virtually all physiological processes. There is great value in measuring enzyme activity ex vivo and in vivo. Spatial and temporal differences or changes in enzyme activity can be related to a variety of natural and pathological processes. Several analytical approaches have been developed to meet this need. They can be classified broadly as methods either based on artificial substrates, with the goal of creating images of diseased tissue, or based on natural substrates, with the goal of understanding natural processes. This review covers a selection of these methods, including optical, magnetic resonance, mass spectrometry, and physical sampling approaches, with a focus on creative chemistry and method development that make ex vivo and in vivo measurements of enzyme activity possible.

Higher Aminopeptidase Activity Determined by Electroosmotic Push-Pull Perfusion Contributes to Selective Vulnerability of the Hippocampal CA1 Region to Oxygen Glucose Deprivation.

It has been known for over a century that the hippocampus, the center for learning and memory in the brain, is selectively vulnerable to ischemic damage, with the CA1 being more vulnerable than the CA3. It is also known that leucine enkephalin, or YGGFL, is neuroprotective. We hypothesized that the extracellular hydrolysis of YGGFL may be greater in the CA1 than the CA3, which would lead to the observed difference in susceptibility to ischemia. In rat organotypic hippocampal slice cultures, we estimated the Michaelis constant and the maximum velocity for membrane-bound aminopeptidase activity in the CA1 and CA3 regions. Using electroosmotic push-pull perfusion and offline capillary liquid chromatography, we inferred enzyme activity based on the production rate of GGFL, a natural and inactive product of the enzymatic hydrolysis of YGGFL. We found nearly 3-fold higher aminopeptidase activity in the CA1 than the CA3. The aminopeptidase inhibitor bestatin significantly reduced hydrolysis of YGGFL in both regions by increasing apparent Km. Based on propidium iodide cell death measurements 24 h after oxygen-glucose deprivation, we demonstrate that inhibition of aminopeptidase activity using bestatin selectively protected CA1 against delayed cell death due to oxygen-glucose deprivation and that this neuroprotection occurs through enkephalin-dependent pathways.

Numerical Modeling of Electroosmotic Push-Pull Perfusion and Assessment of Its Application to Quantitative Determination of Enzymatic Activity in the Extracellular Space of Mammalian Tissue.

Many sampling methods have been developed to measure the extracellular concentrations of solutes in the extracellular space of mammalian tissue, e.g., brain. However, few have been used to quantitatively study the various processes, such as enzymatic degradation, that determines the fate of these solutes. For a method to be useful in this pursuit, it must be able to (1) perfuse tissue and collect the perfusate for quantitative analysis of the solutes introduced and reaction products produced, (2) control the average residence time of the active solutes, and (3) have the appropriate spatial resolution for the process of interest. Our lab previously developed a perfusion technique based on electroosmosis (EO), called EO push-pull perfusion (EOPPP), that is in principle suitable to meet these needs. However, much like the case for other sampling methods that came before, there are parameters that are needed for quantitative interpretation of data but that cannot be measured easily (or at all). In this paper, we present a robust finite element model that provides a deep understanding of fluid dynamics and mass transport in the EOPPP method, assesses the general applicability of EOPPP to studying enzyme activity in the ECS, and grants a simple approach to data treatment and interpretation to obtain, for example, Vmax and Km for an enzymatic reaction in the extracellular space of the tissue. This model is a valuable tool in optimizing and planning experiments without the need for costly experiments.

Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures.

This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push-pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push-pull perfusion can distinguish ectoenzyme activity with a ~100 µm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus.