Histamine is a neurotransmitter crucial to the visual processing of Drosophila melanogaster. It is inactivated by metabolism to carcinine, a β-alanyl derivative, and the same enzyme that controls that process also converts dopamine to N-β-alanyl-dopamine. Direct detection of histamine and carcinine has not been reported in single Drosophila brains. Here, we quantify histamine, carcinine, dopamine, and N-β-alanyl-dopamine in Drosophila tissues by capillary electrophoresis coupled to fast-scan cyclic voltammetry (CE-FSCV). Limits of detection were low, 4 ± 1 pg for histamine, 10 ± 4 pg for carcinine, 2.8 ± 0.3 pg for dopamine, and 9 ± 3 pg for N-β-alanyl-dopamine. Tissue content was compared in the brain, eyes, and cuticle from wild-type (Canton S) and mutant (tan3 and ebony1) strains. In tan3 mutants, the enzyme that produces histamine from carcinine is nonfunctional, whereas in ebony1 mutants, the enzyme that produces carcinine from histamine is nonfunctional. In all fly strains, the neurotransmitter content was highest in the eyes and there were no strain differences for tissue content in the cuticle. The main finding was that carcinine levels changed significantly in the mutant flies, whereas histamine levels did not. In particular, tan3 flies had significantly higher carcinine levels in the eyes and brain than Canton S or ebony1 flies. N-β-Alanyl-dopamine was detected in tan3 mutants but not in other strains. These results show the utility of CE-FSCV for sensitive detection of histamine and carcinine, which allows a better understanding of their content and metabolism in different types of tissues to be obtained.Keywords: capillary electrophoresis; carcinine; D. melanogaster; dopamine; fast-scan cyclic voltammetry; histamine

Small, robust, sensitive electrodes are desired for in vivo neurotransmitter measurements. Carbon nanopipettes have been previously manufactured and used for single-cell drug delivery and electrophysiological measurements. Here, a modified fabrication procedure was developed to produce batches of solid carbon nanopipette electrodes (CNPEs) with ∼250 nm diameter tips, and controllable lengths of exposed carbon, ranging from 5 to 175 μm. The electrochemical properties of CNPEs were characterized with fast-scan cyclic voltammetry (FSCV) for the first time. CNPEs were used to detect the electroactive neurotransmitters dopamine, serotonin, and octopamine. CNPEs were significantly more sensitive for serotonin detection than traditional carbon-fiber microelectrodes (CFMEs). Similar to CFMEs, CNPEs have a linear response for dopamine concentrations ranging from 0.1 to 10 μM and a limit of detection of 25 ± 5 nM. Recordings with CNPEs were stable for over 3 h when the applied triangle waveform was scanned between −0.4 and +1.3 V vs Ag/AgCl/Cl– at 400 V/s. CNPEs were used to detect endogenous dopamine release in Drosophila larvae using optogenetics, which verified the utility of CNPEs for in vivo neuroscience studies. CNPEs are advantageous because they are 1 order of magnitude smaller in diameter than typical CFMEs and have a sharp, tunable geometry that facilitates penetration and implantation for localized measurements in distinct regions of small organisms, such as the Drosophila brain.

Drosophila melanogaster is a widely used model organism for studying neurological diseases with similar neurotransmission to mammals. While both larva and adult Drosophila have central nervous systems, not much is known about how neurotransmitter tissue content changes through development. In this study, we quantified tyramine, serotonin, octopamine, and dopamine in larval, pupal, and adult fly brains using capillary electrophoresis coupled to fast-scan cyclic voltammetry. Tyramine and octopamine content varied between life stages, with almost no octopamine being present in the pupa, while tyramine levels in the pupa were very high. Adult females had significantly higher dopamine content than males, but no other neurotransmitters were dependent on sex in the adult. Understanding the tissue content of different life stages will be beneficial for future work comparing the effects of diseases on tissue content throughout development.Keywords: capillary electrophoresis; development; Drosophila melanogaster; neurotransmitters; tissue content

Fast-scan cyclic voltammetry (FSCV) can detect small changes in dopamine concentration; however, measurements are typically limited to scan repetition frequencies of 10 Hz. Dopamine oxidation at carbon-fiber microelectrodes (CFMEs) is dependent on dopamine adsorption, and increasing the frequency of FSCV scan repetitions decreases the oxidation current, because the time for adsorption is decreased. Using a commercially available carbon nanotube yarn, we characterized carbon nanotube yarn microelectrodes (CNTYMEs) for high-speed measurements with FSCV. For dopamine, CNTYMEs have a significantly lower ΔEp than CFMEs, a limit of detection of 10 ± 0.8 nM, and a linear response to 25 μM. Unlike CFMEs, the oxidation current of dopamine at CNTYMEs is independent of scan repetition frequency. At a scan rate of 2000 V/s, dopamine can be detected, without any loss in sensitivity, with scan frequencies up to 500 Hz, resulting in a temporal response that is four times faster than CFMEs. While the oxidation current is adsorption-controlled at both CFMEs and CNTYMEs, the adsorption and desorption kinetics differ. The desorption coefficient of dopamine-o-quinone (DOQ), the oxidation product of dopamine, is an order of magnitude larger than that of dopamine at CFMEs; thus, DOQ desorbs from the electrode and can diffuse away. At CNTYMEs, the rates of desorption for dopamine and dopamine-o-quinone are about equal, resulting in current that is independent of scan repetition frequency. Thus, there is no compromise with CNTYMEs: high sensitivity, high sampling frequency, and high temporal resolution can be achieved simultaneously. Therefore, CNTYMEs are attractive for high-speed applications.

Carbon nanotube (CNT)-based microelectrodes have been investigated as alternatives to carbon-fiber microelectrodes for the detection of neurotransmitters because they are sensitive, exhibit fast electron transfer kinetics, and are more resistant to surface fouling. Wet spinning CNTs into fibers using a coagulating polymer produces a thin, uniform fiber that can be fabricated into an electrode. CNT fibers formed in poly(vinyl alcohol) (PVA) have been used as microelectrodes to detect dopamine, serotonin, and hydrogen peroxide. In this study, we characterize microelectrodes with CNT fibers made in polyethylenimine (PEI), which have much higher conductivity than PVA-CNT fibers. PEI-CNT fibers have lower overpotentials and higher sensitivities than PVA-CNT fiber microelectrodes, with a limit of detection of 5 nM for dopamine. The currents for dopamine were adsorption controlled at PEI-CNT fiber microelectrodes, independent of scan repetition frequency, and stable for over 10 h. PEI-CNT fiber microelectrodes were resistant to surface fouling by serotonin and the metabolite interferant 5-hydroxyindoleacetic acid (5-HIAA). No change in sensitivity was observed for detection of serotonin after 30 flow injection experiments or after 2 h in 5-HIAA for PEI-CNT electrodes. The antifouling properties were maintained in brain slices when serotonin was exogenously applied multiple times or after bathing the slice in 5-HIAA. Thus, PEI-CNT fiber electrodes could be useful for the in vivo monitoring of neurochemicals.

The surface properties of carbon-based electrodes are critically important for the detection of biomolecules and can modulate electrostatic interactions, adsorption and electrocatalysis. Carbon nanotube (CNT) modified electrodes have previously been shown to have increased oxidative sensitivity and reduced overpotential for catecholamine neurotransmitters, but the effect of surface functionalities on these properties has not been characterized. In this study, we modified carbon-fiber microelectrodes (CFMEs) with three differently functionalized single-wall carbon nanotubes and measured their response to serotonin, dopamine, and ascorbic acid using fast-scan cyclic voltammetry. Both carboxylic acid functionalized and amide functionalized CNTs increased the oxidative current of CFMEs by approximately 2–6 fold for the cationic neurotransmitters serotonin and dopamine, but octadecylamine functionalized CNTs resulted in no significant signal change. Similarly, electron transfer was faster for both amide and carboxylic acid functionalized CNT modified electrodes but slower for octadecylamine CNT modified electrodes. Oxidation of ascorbic acid was only increased with carboxylic acid functionalized CNTs although all CNT-modified electrodes showed a trend towards increased reversibility for ascorbic acid. Carboxylic acid-CNT modified disk electrodes were then tested for detection of serotonin in the ventral nerve cord of a Drosophila melanogaster larva, and the increase in sensitivity was maintained in biological tissue. The functional groups of CNTs therefore modulate the electrochemical properties, and the increase in sensitivity from CNT modification facilitates measurements in biological samples.

Carbon nanotubes (CNTs) have been incorporated in electrochemical sensors to decrease overpotential and improve sensitivity. In this review, we focus on recent literature that describes how CNT-based electrochemical sensors are being developed to detect neurotransmitters, proteins, small molecules such as glucose, and DNA. Different types of electrochemical methods are used in these sensors including direct electrochemical detection with amperometry or voltammetry, indirect detection of an oxidation product using enzyme sensors, and detection of conductivity changes using CNT-field effect transistors (FETs). Future challenges for the field include miniaturizing sensors, developing methods to use only a specific nanotube allotrope, and simplifying manufacturing.

Adenosine is an important neuromodulator in the brain. Traditionally, adenosine is thought to arise in the extracellular space by either an extracellular mechanism, where it is formed outside the cell by the breakdown of released ATP, or an intracellular mechanism, where adenosine made inside the cell is transported out. Recently, a third mechanism of activity dependent adenosine release has also been proposed. Here, we used fast-scan cyclic voltammetry to compare the time course and mechanism of adenosine formation evoked by either low- or high-frequency stimulations in striatal rat brain slices. Low-frequency stimulations (5 pulses at 10 Hz) resulted in an average adenosine efflux of 0.22 ± 0.02 μM, while high-frequency stimulations (5 pulses, 60 Hz) evoked 0.36 ± 0.04 μM. Blocking intracellular formation by inhibiting adenosine transporters with S-(4-nitrobenzyl)-6-thioinosine (NBTI) or propentofylline did not decrease release for either frequency, indicating that the release was not due to the intracellular mechanism. Blocking extracellular formation with ARL-67156 reduced low-frequency release about 60%, but did not affect high-frequency release. Both low- and high-frequency stimulated release were almost completely blocked by the removal of calcium, indicating activity dependence. Reducing dopamine efflux did not affect adenosine release but inhibiting ionotropic glutamate receptors did, indicating that adenosine release is dependent on downstream effects of glutamate. Therefore, adenosine release after short, high-frequency physiological stimulations is independent of transporter activity or ATP metabolism and may be due to the direct release of adenosine after glutamate receptor activation.Keywords (keywords): ARL 67156; carbon-fiber microelectrode; Caudate-putamen; electrical stimulation; propentofylline; reserpine

Carbon-fiber microelectrodes (CFMEs) have been a useful tool for measuring rapid changes in neurotransmitters because of their small size, sensitivity, and good electrochemical properties. In this article, we highlight recent advances using CFMEs for measuring neurotransmitters in vivo. Dopamine has been a primary neurotransmitter of interest but direct electrochemical detection of other neurochemicals including nitric oxide and adenosine has also been investigated. Surface treatments have been studied to enhance electrode sensitivity, such as covalent modification or the addition of a layer of carbon nanotubes. Enzyme-modified microelectrodes that detect non-electroactive compounds further extend the usefulness of CFMEs beyond the traditional monoamines. CFMEs continue to be used in vivo to understand basic neurobiological mechanisms and the actions of pharmacological agents, including drugs of abuse. Advances in sensitivity and instrumentation now allow CFMEs to be used for measurements of natural dopamine release that occur during behavioral experiments. A new technique combining electrochemistry with electrophysiology at a single microelectrode facilitates a better understanding of neurotransmitter concentrations and their effects on cell firing. Future research in this field will likely concentrate on fabricating smaller electrodes and electrode arrays, as well as expanding the use of CFMEs in neuroscience beyond dopamine.

Dopamine and serotonin are important neurotransmitters that interact in the brain. While dopamine is easily detected with electrochemical sensors, the detection of serotonin is more difficult because reactive species formed after oxidation can adsorb to the electrode, reducing sensitivity. Carbon nanotube treatments of electrodes have been used to increase the sensitivity, promote electron transfer, and reduce fouling. Most methods have focused on nanotube coatings of large electrodes and slower electrochemical techniques that are not conducive to measurements in vivo. In this study, we investigated carbon-fiber microelectrodes modified with single-walled carbon nanotubes for the co-detection of dopamine and serotoninin vivo. Using fast-scan cyclic voltammetry, S/N ratios for the neurotransmitters increased after nanotube coating. Electrocatalytic effects of nanotubes were not apparent at fast scan rates but faster kinetics were observed with slower scanning. Nanotube-modified microelectrodes showed significantly less fouling after exposure to serotonin than bare electrodes. The nanotube-modified electrodes were used to monitor stimulated dopamine and serotonin changes simultaneously in the striatum of anesthetized rat after administration of a serotonin synthetic precursor. These studies show that nanotube-coated microelectrodes can be used with fast scanning techniques and are advantageous for in vivo measurements of neurotransmitters because of their greater sensitivity and resistance to fouling.

Serotonin signaling plays a key role in the regulation of development, mood and behavior. Drosophila is well suited for the study of the basic mechanisms of serotonergic signaling, but the small size of its nervous system has previously precluded the direct measurements of neurotransmitters. This study demonstrates the first real-time measurements of changes in extracellular monoamine concentrations in a single larval Drosophila ventral nerve cord. Channelrhodopsin-2-mediated, neuronal type-specific stimulation is used to elicit endogenous serotonin release, which is detected using fast-scan cyclic voltammetry at an implanted microelectrode. Release is decreased when serotonin synthesis or packaging are pharmacologically inhibited, confirming that the detected substance is serotonin. Similar to tetanus-evoked serotonin release in mammals, evoked serotonin concentrations are 280–640 nM in the fly, depending on the stimulation length. Extracellular serotonin signaling is prolonged after administering cocaine or fluoxetine, showing that transport regulates the clearance of serotonin from the extracellular space. When ChR2 is targeted to dopaminergic neurons, dopamine release is measured demonstrating that this method is broadly applicable to other neurotransmitter systems. This study shows that the dynamics of serotonin release and reuptake in Drosophila are analogous to those in mammals, making this simple organism more useful for the study of the basic physiological mechanisms of serotonergic signaling.