To Fluoresce or Not to Fluoresce: Investigation of Structural and Fluorescence Characteristics of CBI-Dopamine, CBI-Serotonin, and Their Structural Analogs.

Dopamine (DA) and serotonin (5-HT) are neurotransmitters that are vital for proper brain function and are implicated in a wide variety of diseases and disorders. Unfortunately, quantitative analysis of DA and 5-HT is difficult, as they are present at low concentrations in complex biological matrices. The fluorogenic reaction of napththalene-2,3-dicarboxaldehyde (NDA) with a primary amine in the presence of cyanide (CN) creates an N-substituted 1-cyanobenz[f]isoindole (CBI) derivative, whose fluorescence can be sensitively monitored in biological matrices. Given their biological importance, there are surprisingly few reports showing fluorescence of CBI-DA and no prior publications concerning CBI-5-HT. In this work, nuclear magnetic resonance spectroscopy (NMR) was employed to determine the atom connectivity of over 10 CBI-products, including CBI-DA and CBI-5-HT. NMR and fluorescence spectroscopy were applied to CBI-DA, CBI-5-HT, and select structural analogs to determine structural correlations with the observed lack of fluorescence. Experiments with CBI-DA and structural analogs indicated fluorescence was rapidly quenched due to both complexation with the historically employed buffer and oxidation in solution. Fluorescence of CBI-DA was recovered by modifying the derivatization background to prevent complexation and oxidation. In contrast, fluorescence characterization of CBI-5-HT and its structural analogs indicated that 5-HT was acting as a quencher of the CBI-ring. The addition of acid to protonate 5-HT was found to disrupt this interaction and enable the first reported fluorescence detection of CBI-5-HT. In the future, this work will be applied to detect DA and 5-HT in biological systems to gain insight into neurobiological disease states and disorders.

Optimized derivatization of primary amines with the fluorogenic reagent naphthalene-2,3-dicarboxaldehyde toward reproducible quantitative analysis in biological systems.

Primary amines are the target of many bioanalytical analyses, as they are ubiquitous in biological systems and responsible for numerous important processes including neurotransmission and cell signaling. Primary amines can be sensitively detected via fluorescence after their reaction with the fluorogenic reagent naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide through the formation of fluorescent N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives. While fluorogenic reagents such as NDA can be advantageous for sensitive detection, improvements in both long-term stability and speed of reaction are necessary to enable practical and reproducible quantitative analysis. In this work, various CBI-amines were interrogated for their fluorescence characteristics over time under previously reported conditions (75:25 aqueous buffer:acetonitrile). An amine-specific decline in fluorescence and delay to reach maximum fluorescence were observed. Based on structural characteristics, we hypothesized that these effects were due to the solvents employed enabling analyte intermolecular interactions that resulted in fluorescence quenching over time. To mitigate fluorescence-quenching intermolecular interactions, we developed two strategies to improve the fluorescence of the CBI-product over long time periods: (1) the addition of the complexation reagent ß-cyclodextrin to the reaction matrix and (2) the substitution of acetonitrile with dimethyl sulfoxide. Both strategies improved fluorescence stability over time, and the incorporation of dimethyl sulfoxide also enabled more rapid attainment of maximum fluorescence and a higher absolute fluorescence when compared to initial conditions. When employed in combination, these two approaches further improve fluorescence stability over time for the most hydrophobic analytes. In the future, these strategies can be employed for the practical and reproducible quantitative analysis of primary amines in biological systems, including those related to neurological disorders and disease states.

Fast serotonin voltammetry as a versatile tool for mapping dynamic tissue architecture: I. Responses at carbon fibers describe local tissue physiology.

It is important to monitor serotonin neurochemistry in the context of brain disorders. Specifically, a better understanding of biophysical alterations and associated biochemical functionality within subregions of the brain will enable better of understanding of diseases such as depression. Fast voltammetric tools at carbon fiber microelectrodes provide an opportunity to make direct evoked and ambient serotonin measurements in vivo in mice. In this study, we characterize novel stimulation and measurement circuitries for serotonin analyses in brain regions relevant to psychiatric disease. Evoked and ambient serotonin in these brain areas, the CA2 region of the hippocampus and the medial prefrontal cortex, are compared to ambient and evoked serotonin in the substantia nigra pars reticulata, an area well established previously for serotonin measurements with fast voltammetry. Stimulation of a common axonal location evoked serotonin in all three brain regions. Differences are observed in the serotonin release and reuptake profiles between these three brain areas which we hypothesize to arise from tissue physiology heterogeneity around the carbon fiber microelectrodes. We validate this hypothesis mathematically and via confocal imaging. We thereby show that fast voltammetric methods can provide accurate information about local physiology and highlight implications for chemical mapping. Cover Image for this issue: doi: 10.1111/jnc.14739.

Corrigendum: In vivo Hippocampal Serotonin Dynamics in Male and Female Mice: Determining Effects of Acute Escitalopram Using Fast Scan Cyclic Voltammetry.

[This corrects the article DOI: 10.3389/fnins.2019.00362.].

In vivo Hippocampal Serotonin Dynamics in Male and Female Mice: Determining Effects of Acute Escitalopram Using Fast Scan Cyclic Voltammetry.

Depression is a highly prevalent psychiatric disorder, impacting females at a rate roughly twice that of males. This disparity has become the focus of many studies which are working to determine if there are environmental or biological underpinnings to depression pathology. The biology of depression is not well understood, but experts agree that a key neurotransmitter of interest is serotonin. Most research on basic serotonin neurochemistry, by us and others, has predominantly focused on male models. Thus, it is now critical to include female models to decipher possible fundamental differences between the sexes that may underlie this disorder. In this paper, we seek to determine any such differences using fast-scan cyclic voltammetry (FSCV) and fast-scan controlled adsorption voltammetry. These techniques allow us to probe the serotonergic system via measurement of evoked and ambient serotonin at carbon fiber microelectrodes (CFMs). Our data reveal no statistical differences, in the hippocampus, in female serotonin chemistry during the different stages of the estrous cycle compared to the mean female response. Furthermore, no difference was observed in evoked serotonin release and reuptake, nor ambient extracellular serotonin levels between male and female mice. We applied a previously developed mathematical model that fits our serotonin signals as a function of several synaptic processes that control the extracellular levels of this transmitter. We used the model to study potential system differences between males and females. One hypothesis brought fourth, that female mice exhibit tighter autoreceptor control of serotonin, is validated via literature and methiothepin challenge. We postulate that this tight regulation may act as a control mechanism against changes in the serotonin signal mediated by estrogen spikes. Importantly, this safety mechanism has no consequence for acutely administered escitalopram's (ESCIT's) ability to increase extracellular serotonin between the sexes. This work demonstrates little fundamental differences in in vivo hippocampal serotonin between the sexes, bar control mechanisms in female mice that can be observed under extraneous circumstances. We thus highlight the importance of considering sex as a biological factor in determining pharmacodynamics for personalized medical treatments that involve targeting serotonin receptors.

PDMS/glass hybrid device with a reusable carbon electrode for on-line monitoring of catecholamines using microdialysis sampling coupled to microchip electrophoresis with electrochemical detection.

On-line separations-based sensors employing microdialysis (MD) coupled to microchip electrophoresis (ME) enable the continuous monitoring of multiple analytes simultaneously. Electrochemical detection (EC) is especially amenable to on-animal systems employing MD-ME due to its ease of miniaturization. However, one of the difficulties in fabricating MD-ME-EC systems is incorporating carbon working electrodes into the device. In this paper, a novel fabrication procedure is described for the production of a PDMS/glass hybrid device that is capable of integrating hydrodynamic MD flow with ME-EC using a flow-gated interface and a pyrolyzed photoresist film carbon electrode. This fabrication method enables the reuse of carbon electrodes on a glass substrate, while still maintaining a good seal between the PDMS and glass to allow for pressure-driven MD flow. The on-line MD-ME-EC device was characterized in vitro and in vivo for monitoring analytes in the dopamine metabolic pathway. The ultimate goal is to use this device and associated instrumentation to perform on-animal, near-real time in vivo monitoring of catecholamines.

Microchip electrophoresis with electrochemical detection for the determination of analytes in the dopamine metabolic pathway.

A method for the separation and detection of analytes in the dopamine metabolic pathway was developed using microchip electrophoresis with electrochemical detection. The microchip consisted of a 5 cm PDMS separation channel in a simple-t configuration. Analytes in the dopamine metabolic pathway were separated using a background electrolyte composed of 15 mM phosphate at pH 7.4, 15 mM SDS, and 2.5 mM boric acid. Two different microchip substrates using different electrode materials were compared for the analysis: a PDMS/PDMS device with a carbon fiber electrode and a PDMS/glass hybrid device with a pyrolyzed photoresist film carbon electrode. While the PDMS/PDMS device generated high separation efficiencies and good resolution, more reproducible migration times were obtained with the PDMS/glass hybrid device, making it a better choice for biological applications. Lastly, the optimized method was used to monitor l-DOPA metabolism in a rat brain slice.

A review of microdialysis coupled to microchip electrophoresis for monitoring biological events.

Microdialysis is a powerful sampling technique that enables monitoring of dynamic processes in vitro and in vivo. The combination of microdialysis with chromatographic or electrophoretic methods with selective detection yields a "separation-based sensor" capable of monitoring multiple analytes in near real time. For monitoring biological events, analysis of microdialysis samples often requires techniques that are fast (<1 min), have low volume requirements (nL-pL), and, ideally, can be employed on-line. Microchip electrophoresis fulfills these requirements and also permits the possibility of integrating sample preparation and manipulation with detection strategies directly on-chip. Microdialysis coupled to microchip electrophoresis has been employed for monitoring biological events in vivo and in vitro. This review discusses technical considerations for coupling microdialysis sampling and microchip electrophoresis, including various interface designs, and current applications in the field.