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Selective serotonin reuptake inhibitor (SSRI) antidepressants are commonly prescribed treatments for depression, but their effects on serotonin reuptake and release are not well understood. Drosophila melanogaster, the fruit fly, expresses the serotonin transporter (dSERT), the major target of SSRIs, but real-time serotonin changes after SSRIs have not been characterized in this model. The goal of this study was to characterize effects of SSRIs on serotonin concentration and reuptake in Drosophila larvae. We applied various doses (0.1-100 μM) of fluoxetine (Prozac), escitalopram (Lexapro), citalopram (Celexa), and paroxetine (Paxil), to ventral nerve cord (VNC) tissue and measured optogenetically-stimulated serotonin release with fast-scan cyclic voltammetry (FSCV). Fluoxetine increased reuptake from 1 to 100 μM, but serotonin concentration only increased at 100 μM. Thus, fluoxetine occupies dSERT and slows clearance but does not affect concentration. Escitalopram and paroxetine increased serotonin concentrations at all doses, but escitalopram increased reuptake more. Citalopram showed lower concentration changes and faster reuptake profiles compared with escitalopram, so the racemic mixture of citalopram does not change reuptake as much as the S-isomer. Dose response curves were constructed to compare dSERT affinities and paroxetine showed the highest affinity and fluoxetine the lowest. These data demonstrate SSRI mechanisms are complex, with separate effects on reuptake or release. Furthermore, dynamic serotonin changes in Drosophila are similar to previous studies in mammals. This work establishes how antidepressants affect serotonin in real-time, which is useful for future studies that will investigate pharmacological effects of SSRIs with different genetic mutations in Drosophila.

Rapid adenosine signaling has been detected spontaneously or after mechanical stimulation in the brain, providing rapid neuromodulation in a local area. To measure rapid adenosine signaling, a single carbon-fiber microelectrode has traditionally been used, which limits spatial resolution and an understanding of regional coordination. In this study, we utilized dual-channel fast-scan cyclic voltammetry to measure the spontaneous or mechanically stimulated adenosine release at two electrodes placed at different spacings in hippocampal CA1 mouse brain slices. For mechanically stimulated adenosine release, adenosine can be detected up to 150 μm away from where it was stimulated, although the signal is smaller and delayed. While spontaneous adenosine transients were detected at both electrodes, only 10 percent of the events were detected concurrently, and that number was similar at 50 and 200 μm electrode spacings. Thus, most adenosine transients were not caused by the widespread coordination of release. There was no evidence of diffusion of spontaneous transients to a second electrode 50-200 μm away. This study shows that spontaneous adenosine events are very localized and thus provide only local neuromodulation. Injury, such as mechanical stimulation, allows adenosine to diffuse farther, but the neuroprotective effects are still regional. These results provide a better understanding of the spatial and temporal profiles of adenosine available to act at receptors, which is crucial for future studies that design neuroprotective treatments based on rapid adenosine signaling.

Carbon nanoelectrodes enable the detection of neurotransmitters at the level of single cells, vesicles, synapses and small brain structures. Previously, the etching of carbon fibers and 3D printing based on direct laser writing have been used to fabricate carbon nanoelectrodes, but these methods lack the ability of mass manufacturing. In this paper, we mass fabricate carbon nanoelectrodes by growing carbon nanospikes (CNSs) on metal wires. CNSs have a short, dense and defect-rich surface that produces remarkable electrochemical properties, and they can be mass fabricated on almost any substrate without using catalysts. Tungsten wires and niobium wires were electrochemically etched in batch to form sub micrometer sized tips, and a layer of CNSs was grown on the metal wires using plasma-enhanced chemical vapor deposition (PE-CVD). The thickness of the CNS layer was controlled by the deposition time, and a thin layer of CNSs can effectively cover the entire metal surface while maintaining the tip size within the sub micrometer scale. The etched tungsten wires produced tapered conical nanotips, while the etched niobium wires were long and thin. Both showed excellent sensitivity for the detection of outer sphere ruthenium hexamine and the inner sphere test compound ferricyanide. The CNS nanosensors were used for the measurement of dopamine, serotonin, ascorbic acid and DOPAC with fast-scan cyclic voltammetry. The CNS nanoelectrodes had a large surface area and numerous defect sites, which improved the sensitivity, electron transfer kinetics and adsorption. Finally, the CNS nanoelectrodes were compared with other nanoelectrode fabrication methods, including flame etching, 3D printing, and nanopipettes, which are slower to make and more difficult for mass fabrication. Thus, CNS nanoelectrodes are a promising strategy for the mass fabrication of nanoelectrode sensors for neurotransmitters.

Carbon nanotube yarn microelectrodes (CNTYMEs) have micron-scale surface crevices that momentarily trap molecules. CNTYMEs improve selectivity among cationic catecholamines because secondary reactions are enhanced, but no anions have been studied. Here, we compared fast-scan cyclic voltammetry (FSCV) of dopamine and anionic interferents 3,4 dihydroxyphenylacetic acid (DOPAC) and L-ascorbic acid (AA) at CNTYMEs and carbon fiber microelectrodes (CFMEs). At CFMEs, dopamine current decreases with increasing FSCV repetition frequency at pH 7.4, whereas DOPAC and AA have increasing currents with increasing frequency, because of less repulsion at the negative holding potential. Both DOPAC and AA have side reactions after being oxidized, which are enhanced by trapping. At pH 4, the current increases for DOPAC and AA because they are not repelled. In addition, AA has a different oxidation pathway at pH 4, and an extra peak in the CV is enhanced by trapping effects at CNTYMEs. At pH 8.5, co-detection of dopamine in the presence of DOPAC and AA is enhanced at 100 Hz frequency because of differences in secondary peaks. Thus, the trapping effects at CNTYMEs affects anions differently than cations and secondary peaks can be used to identify dopamine in mixture of AA and DOPAC with FSCV.

Transient changes in adenosine and dopamine have been measured in vivo, but no studies have examined if these transient changes occur simultaneously. In this study, we characterized spontaneous adenosine and dopamine transients in anesthetized mice, examining coincident release in the caudate-putamen for the first time. We found that in C57B mice, most of the dopamine transients (77%) were coincident with adenosine, but fewer adenosine transients (12%) were coincident with a dopamine transient. On average, the dopamine transient started 200 ms before its coincident adenosine transient, so they occurred concurrently. There was a positive correlation (r=0.7292) of adenosine and dopamine concentrations during coincident release. ATP is quickly broken down to adenosine in the extracellular space and the coincident events may be due to co-release, where dopaminergic vesicles are packaged with ATP, or co-transmission, where ATP is packaged in different vesicles released simultaneously with dopamine. The high frequency of adenosine transients compared to dopamine transients suggests adenosine is also released from non-dopaminergic vesicles. We investigated how A(1) and A(2A) adenosine receptors regulate adenosine and dopamine transients using A(1) and A(2A) KO mice. In A(1)KO mice, the frequency of adenosine and dopamine transients increased, while in A(2A)KO mice, the frequency of adenosine alone increased. Adenosine receptors modulate coincident transients and could be drug targets to modulate both dopamine and adenosine release. Many spontaneous dopamine transients have coincident adenosine release and regulating adenosine and dopamine co-transmission could be important for designing treatments for dopamine diseases, such as Parkinson’s or addiction.

Intercellular communication mediated by a large number of neuromodulators diversifies physiological actions, yet neuromodulation remains poorly understood despite the recent upsurge of genetically encoded transmitter sensors. Here, we report the development of a versatile genetically encoded sensor-based image analysis program (GESIAP) that utilizes MATLAB-based algorithms to achieve high-throughput, high-resolution processing of sensor-based functional imaging data. GESIAP enables delineation of fundamental properties (e.g., transmitter spatial diffusion extent, quantal size, quantal content, release probability, pool size, and refilling rate at single release sites) of transmission mediated by various transmitters (i.e., monoamines, acetylcholine, neuropeptides, and glutamate) at various cell types (i.e., neurons, astrocytes, and other non-neuronal cells) of various animal species (i.e., mouse, rat, and human). Our analysis appraises a dozen of newly developed transmitter sensors, validates a conserved model of restricted non-volume neuromodulatory synaptic transmission, and accentuates a broad spectrum of presynaptic release properties that variegate neuromodulation.

Serotonin (5-HT) is a phylogenetically conserved monoamine neurotransmitter modulating important processes in the brain. To directly visualize the release of 5-HT, we developed a genetically encoded G-protein-coupled receptor (GPCR)-activation-based 5-HT (GRAB5-HT) sensor with high sensitivity, high selectivity, subsecond kinetics and subcellular resolution. GRAB5-HT detects 5-HT release in multiple physiological and pathological conditions in both flies and mice and provides new insights into the dynamics and mechanisms of 5-HT signaling.

The geometric structure of carbon electrodes affects their electrochemical behavior, and large-scale surface roughness leads to thin layer electrochemistry when analyte is trapped in pores. However, the current response is always a mixture of both thin layer and diffusion processes. Here, we systematically explore the effects of thin layer electrochemistry and diffusion at carbon fiber (CF), carbon nanospike (CNS), and carbon nanotube yarn (CNTY) electrodes. The cyclic voltammetry (CV) response to the surface-insensitive redox couple Ru(NH3)63+/2+ is tested, so the geometric structure is the only factor. At CFs, the reaction is diffusion-controlled because the surface is smooth. CNTY electrodes have gaps between nanotubes that are about 10 μm deep, comparable with the diffusion layer thickness. CNTY electrodes show clear thin layer behavior due to trapping effects, with more symmetrical peaks and ΔEp closer to zero. CNS electrodes have submicrometer scale roughness, so their CV shape is mostly due to diffusion, not thin layer effects. However, even the 10% contribution of thin layer behavior reduces the peak separation by 30 mV, indicating ΔEp is influenced not only by electron transfer kinetics but also by surface geometry. A new simulation model is developed to quantitate the thin layer and diffusion contributions that explains the CV shape and peak separation for CNS and CNTY electrodes, providing insight on the impact of scan rate and surface structure size. Thus, this study provides key understanding of thin layer and diffusion processes at different surface structures and will enable rational design of electrodes with thin layer electrochemistry.

Benefits Students are more likely to come to class and participate Students vote with their attendance, and remote classes with an active learning component are better attended than remote lectures This contribution is part of a series featuring teaching analytical science during the pandemic in order to support instructors in preparing their courses Dividing the students into small groups and giving them a problem to work on first, followed by a recap with the whole class, leads to higher student attendance, more student engagement, and more discussion Typical class format with active learning in groups For most class sessions, students submit a short, easy pre-class assignment that orients them to the day's topic [Extracted from the article] Copyright of Analytical & Bioanalytical Chemistry is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission However, users may print, download, or email articles for individual use This abstract may be abridged No warranty is given about the accuracy of the copy Users should refer to the original published version of the material for the full abstract (Copyright applies to all Abstracts )

Recent work indicates a role for RING finger protein 11 (RNF11) in Parkinson disease (PD) pathology, which involves the loss of dopaminergic neurons. However, the role of RNF11 in regulating dopamine neurotransmission has not been studied. In this work, we tested the effect of RNF11 RNAi knockdown or overexpression on stimulated dopamine release in the larval Drosophila central nervous system. Dopamine release was stimulated using optogenetics and monitored in real-time using fast-scan cyclic voltammetry at an electrode implanted in an isolated ventral nerve cord. RNF11 knockdown doubled dopamine release, but there was no decrease in dopamine from RNF11 overexpression. RNF11 knockdown did not significantly increase stimulated serotonin or octopamine release, indicating the effect is dopamine specific. Dopamine clearance was also changed, as RNF11 RNAi flies had a higher V(max) and RNF11 overexpressing flies had a lower V(max) than control flies. RNF11 RNAi flies had increased mRNA levels of dopamine transporter (DAT) in RNF11, confirming changes in DAT. In RNF11 RNAi flies, release was maintained better for stimulations repeated at short intervals, indicating increases in the recycled releasable pool of dopamine. Nisoxetine, a DAT inhibitor, and flupenthixol, a D2 antagonist, did not affect RNF11 RNAi or overexpressing flies differently than control. Thus, RNF11 knockdown causes early changes in dopamine neurotransmission, and this is the first work to demonstrate that RNF11 affects both dopamine release and uptake. RNF11 expression decreases in human dopaminergic neurons during PD, and that decrease may be protective by increasing dopamine neurotransmission in the surviving dopaminergic neurons.

Drosophila melanogaster, the fruit fly, is an excellent model organism for studying dopaminergic mechanisms and simple behaviors, but methods to measure dopamine during behavior are needed. Here, we developed fast-scan cyclic voltammetry (FSCV) to track in vivo dopamine during sugar feeding. First, we employed acetylcholine stimulation to evaluate the feasibility of in vivo measurements in an awake fly. Next, we tested sugar feeding by placing sucrose solution near the fly proboscis. In the mushroom body medial tip, 1 pmol acetylcholine and sugar feeding released 0.49±0.04 μM and 0.31±0.06 μM dopamine, respectively but sugar-evoked release lasted longer than with acetylcholine. Administering the dopamine transporter inhibitor nisoxetine or D2 receptor antagonist flupentixol significantly increased sugar-evoked dopamine. This study develops FSCV to measure behaviorally evoked release in fly, enabling Drosophila studies of neurochemical control of reward, learning, and memory behaviors.

Carbon is a popular electrode material for neurotransmitter detection due to its good electrochemical properties, high biocompatibility, and inert chemistry. Traditional carbon electrodes, such as carbon fibers, have smooth surfaces and fixed shapes. However, newer studies customize the shape and nanostructure the surface to enhance electrochemistry for different applications. In this review, we show how changing the structure of carbon electrodes with methods such as chemical vapor deposition (CVD), wet-etching, direct laser writing (DLW), and 3D printing leads to different electrochemical properties. The customized shapes include nanotips, complex 3D structures, porous structures, arrays, and flexible sensors with patterns. Nanostructuring enhances sensitivity and selectivity, depending on the carbon nanomaterial used. Carbon nanoparticle modifications enhance electron transfer kinetics and prevent fouling for neurochemicals that are easily polymerized. Porous electrodes trap analyte momentarily on the scale of an electrochemistry experiment, leading to thin layer electrochemical behavior that enhances secondary peaks from chemical reactions. Similar thin layer cell behavior is observed at cavity carbon nanopipette electrodes. Nanotip electrodes facilitate implantation closer to the synapse with reduced tissue damage. Carbon electrode arrays are used to measure from multiple neurotransmitter release sites simultaneously. Custom-shaped carbon electrodes are enabling new applications in neuroscience, such as distinguishing different catecholamines by secondary peaks, detection of vesicular release in single cells, and multi-region measurements in vivo.

Adenosine is a neuromodulator and rapid increases in adenosine in the brain occur spontaneously or after mechanical stimulation. However, the regulation of rapid adenosine by adenosine receptors is unclear, and understanding it would allow better manipulation of neuromodulation. The two main adenosine receptors in the brain are A(1) receptors, which are inhibitory, and A(2A) receptors, which are excitatory. Here, we investigated the regulation of spontaneous adenosine and mechanically-stimulated adenosine by adenosine receptors, using global A(1) or A(2A) knockout mice. Results were compared in vivo and in brain slices models. A(1) KO mice have increased frequency of spontaneous adenosine events, but no change in the average concentration of an event, while A(2A) KO mice had no change in frequency but increased average event concentration. Thus, both A(1) and A(2A) self-regulate spontaneous adenosine release, but A(1) acts on the frequency of events, while A(2A) receptors regulate concentration. The trends are similar both in vivo and slices, so brain slices are a good model system to study spontaneous adenosine release. For mechanically-stimulated adenosine, there was no effect of A(1) or A(2A) KO in vivo, but in brain slices there was a significant increase in concentration evoked in A(1)KO mice. Mechanically-stimulated release was largely unregulated by A(1) and A(2A) receptors, likely because of a different release mechanism than spontaneous adenosine. Thus, A(1) receptors affect the frequency of spontaneous adenosine transients and A(2A) receptors affect the concentration, so future studies could probe drug treatments targeting A(1) and A(2A) receptors to increase rapid adenosine neuromodulation.

Adenosine is a ubiquitous neuromodulator that plays a role in sleep, vasodilation, and immune response and manipulating the adenosine system could be therapeutic for Parkinson's disease or ischemic stroke. Spontaneous transient adenosine release provides rapid neuromodulation; however, little is known about the effect of sex as a biological variable on adenosine signaling and this is vital information for designing therapeutics. Here, we investigate sex differences in spontaneous, transient adenosine release using fast-scan cyclic voltammetry to measure adenosine in vivo in the hippocampus CA1, basolateral amygdala, and prefrontal cortex. The frequency and concentration of transient adenosine release were compared by sex and brain region, and in females, the stage of estrous. Females had larger concentration transients in the hippocampus (0.161 ± 0.003 µM) and the amygdala (0.182 ± 0.006 µM) than males (hippocampus: 0.134 ± 0.003, amygdala: 0.115 ± 0.002 µM), but the males had a higher frequency of events. In the prefrontal cortex, the trends were reversed. Males had higher concentrations (0.189 ± 0.003 µM) than females (0.170 ± 0.002 µM), but females had higher frequencies. Examining stages of the estrous cycle, in the hippocampus, adenosine transients are higher concentration during proestrus and diestrus. In the cortex, adenosine transients were higher in concentration during proestrus, but were lower during all other stages. Thus, sex and estrous cycle differences in spontaneous adenosine are complex, and not completely consistent from region to region. Understanding these complex differences in spontaneous adenosine between the sexes and during different stages of estrous is important for designing effective treatments manipulating adenosine as a neuromodulator.

Colonies of the red harvester ant, Pogonomyrmex barbatus, regulate foraging activity based on food availability and local conditions. Colony variation in foraging behavior is thought to be linked to biogenic amine signaling and metabolism. Measurements of differences in neurotransmitters have not been made among ant colonies in a natural environment. Here, for the first time, we quantified tissue content of 4 biogenic amines (dopamine, serotonin, octopamine, and tyramine) in single forager brains from 9 red harvester ant colonies collected in the field. Capillary electrophoresis coupled with fast-scan cyclic voltammetry (CE-FSCV) was used to separate and detect the amines in individual ant brains. Low levels of biogenic amines were detected using field-amplified sample stacking by preparing a single brain tissue sample in acetonitrile and perchloric acid. The method provides low detection limits: 1 nM for dopamine, 2 nM for serotonin, 5 nM for octopamine, and 4 nM for tyramine. Overall, the content of dopamine (47 ± 9 pg/brain) was highest, followed by octopamine (36 ± 10 pg/brain), serotonin (20 ± 4 pg/brain), and tyramine (14 ± 3 pg/brain). Relative standard deviations were high, but there was less variation within a colony than among colonies, so the neurotransmitter content of each colony might change with environmental conditions. This study demonstrates that CE-FSCV is a useful method for investigating natural variation in neurotransmitter content in single ant brains and could be useful for future studies correlating tissue content with colony behavior such as foraging. Graphical abstract.

Serotonin is a neuromodulator implicated in depression that is often measured in real-time by fast-scan cyclic voltammetry (FSCV). A specialized "Jackson" waveform (JW, 0.2, 1.0 V, -0.1 V, 0.2 V, 1000 V s-1) was developed to reduce serotonin fouling, but the 1.0 V switching potential limits sensitivity and electrodes still foul. The goal of this study was to test the effects of extending the FSCV switching potential to increase serotonin sensitivity and decrease fouling. We compared the Jackson waveform, the dopamine waveform (DA, -0.4 V, 1.3 V, 400 V s-1), and two new waveforms: the extended serotonin waveform (ESW, 0.2, 1.3, -0.1, 0.2, 1000 V s-1) and extended hold serotonin waveform (EHSW, 0.2, 1.3 (hold 1 ms), -0.1, 0.2, 400 V s-1). The EHSW was the most sensitive (LOD = 0.6 nM), and the JW the least sensitive (LOD = 2.4 nM). With the Jackson waveform, electrode fouling was significant with repeated injections of serotonin or exposure to its metabolite, 5-hydroxyindoleacetic acid (5-HIAA). Using the extended waveforms, electrodes fouled 50% less than with the Jackson waveform for both analytes. No electrode fouling was observed with the dopamine waveform because of the negative holding potential. The Jackson waveform was the most selective for serotonin over dopamine (800×), and the ESW was also highly selective. All waveforms were useful for measuring serotonin with optogenetic stimulation in Drosophila larvae. These results provide new FSCV waveforms to measure dynamic serotonin changes with different experimental requirements, like high sensitivity (EHSW), high selectivity (ESW, JW), or eliminating electrode fouling (DA).

Graphene oxide (GO) is a carbon-based material that is easily obtained from graphite or graphite oxide. GO has been used broadly for electrochemistry applications and our hypothesis is that GO coating a carbon-fiber microelectrode (CFME) will increase the sensitivity for dopamine by providing more adsorption sites due to the enhancement of oxygen functional groups. Here, we compared drop casting, dip coating, and electrodeposition methods to directly coat commercial GO on CFME surfaces. Dip coating did not result in much GO coating and drop casting resulted in large agglomerations that produced noisy signals and slow rise times. Electrodeposition method with cyclic voltammetry increase the current for dopamine and this method was the most reproducible and had the least noise compared to the other two coating methods. The optimized method used a triangular waveform scanned from −1.2 V to 1.5 V at 100 mV/s for 5 cycles in 0.2 mg/mL GO in water. With fast-scan cyclic voltammetry (FSCV), the optimized GO/CFME enhanced the dopamine oxidation peak two-fold. The sensitivity of the modified electrode is 41±2 nA/μM with a linear range from 25 nM to 1 μM, and a limit of detection of 11 nM. The optimized electrodes were used to detect electrically-stimulated dopamine in brain slices to demonstrate their performance in tissue. Thus, GO can be used to enhance the sensitivity of electrodes for dopamine and improve biological measurements.

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes (CFMEs) is a versatile electrochemical technique to probe neurochemical dynamics in vivo. Progress in FSCV methodology continues to address analytical challenges arising from biological needs to measure low concentrations of neurotransmitters at specific sites. This review summarizes recent advances in FSCV method development in three areas: (1) waveform optimization, (2) electrode development, and (3) data analysis. First, FSCV waveform parameters such as holding potential, switching potential, and scan rate have been optimized to monitor new neurochemicals. The new waveform shapes introduce better selectivity toward specific molecules such as serotonin, histamine, hydrogen peroxide, octopamine, adenosine, guanosine, and neuropeptides. Second, CFMEs have been modified with nanomaterials such as carbon nanotubes or replaced with conducting polymers to enhance sensitivity, selectivity, and antifouling properties. Different geometries can be obtained by 3D-printing, manufacturing arrays, or fabricating carbon nanopipettes. Third, data analysis is important to sort through the thousands of CVs obtained. Recent developments in data analysis include preprocessing by digital filtering, principal components analysis for distinguishing analytes, and developing automated algorithms to detect peaks. Future challenges include multisite measurements, machine learning, and integration with other techniques. Advances in FSCV will accelerate research in neurochemistry to answer new biological questions about dynamics of signaling in the brain.

Carbon microelectrodes enable in vivo detection of neurotransmitters, and new electrodes are being developed to optimize the carbon surface. However, the work is mainly empirical and there have not been corresponding theoretical studies using molecular-level simulations of the diffusion and orientation of neurotransmitters near these surfaces. Here, we employ molecular dynamics simulations to investigate in atomistic detail the surface diffusion of dopamine (DA), its oxidation product dopamine-o-quinone (DOQ), and their protonated forms on the pristine basal plane of flat graphene. All DA species rapidly adsorb to the surface and remain adsorbed for the full length of the equilibrium simulations, even without a holding potential or graphene surface defects. The diffusivities of the adsorbed and the fully solvated DA are similar, and all molecular diffusion on the surface is slower than that of an adatom of comparable molecular weight. The protonated species diffuse more slowly than their corresponding neutral forms, and the oxidized species diffuse more rapidly. The underlying hexagonal graphene structure has little influence over the molecular adsorbate's lateral position. The vertical placement of the amine group on dopamine is highly dependent upon its charge, and the protonated amine prefers to be above the surface near the solvating waters. Solvation has a large effect on surface diffusivities when diffusion is compared to that in a vacuum. These are the first results of molecular dynamics simulations of dopamine diffusion at the aqueous-graphene interface, and they show that dopamine diffuses quickly on graphene surfaces, even without an applied potential. These calculations provide a basis for future simulations to predict the behavior of neurotransmitter diffusion on advanced carbon materials electrodes.

Ischemic stroke causes damage in the brain, and a slow buildup of adenosine is neuroprotective during ischemic injury. Spontaneous, transient adenosine signaling, lasting only 3 s per event, has been discovered that increases in frequency in the caudate-putamen during early stages of mild ischemia-reperfusion injury. However, spontaneous adenosine changes have not been studied in the hippocampus during ischemia, an area highly susceptible to stroke. Here, we investigated changes of spontaneous, transient adenosine in the CA1 region of rat hippocampus during three different models of the varied intensity of ischemia. During the early stages of the milder bilateral common carotid artery occlusion (BCCAO) model, there were fewer spontaneous, transient adenosine, but no change in the concentration of individual events. In contrast, during the moderate 2 vertebral artery occlusion (2VAO) and severe 4 vessel occlusion (4VO) models, both the frequency of spontaneous, transient adenosine and the average event adenosine concentration decreased. Blood flow measurements validate that the ischemia models decreased blood flow, and corresponding pathological changes were observed by transmission electron microscopy (TEM). 4VO occlusion showed the most severe damage in histology and BCCAO showed the least. Overall, our data suggest that there is no enhanced spontaneous adenosine release in the hippocampus during moderate and severe ischemia, which could be due to depletion of the rapidly releasable adenosine pool. Thus, during ischemic stroke, there are fewer spontaneous adenosine events that could inhibit neurotransmission, which might lead to more damage and less neuroprotection in the hippocampus CA1 region. Read the Editorial Highlight for this article on page 800.

Carbon fiber microelectrodes (CFMEs) are the standard electrodes for fast-scan cyclic voltammetry (FSCV) detection of neurotransmitters. CFMEs are generally used untreated but the surface can be activated with different treatments to improve electrochemical performance. In this work, we explored electrochemical treatments to clean and activate the CFME surface. We used different solution conditions for electrochemical treatment and found that electrochemical pretreatment in KOH outperforms treatment in KCl, H2O2, or HCl by accelerating the surface renewal process. The etching rate of carbon with electrochemical treatment in KOH is 37 nm/min, which is 10 times faster than that in the other solutions. Electrochemical treatment in KOH for several minutes regenerates a new carbon surface, which introduces more oxygen functional groups beneficial for adsorption and electron transfer. The KOH-treated CFMEs improved the limit of detection (LOD) to 9 ± 2 nM from 14 ± 4 nM for untreated CFMEs, and they successfully detected stimulated dopamine release in rat brain slices, demonstrating that they are stable and sensitive enough to use in biological systems. Electrochemical treatment in KOH completely restores the electrode sensitivity after biofouling. The proposed electrochemical treatment is simple and fast and can be applied prior to using CFMEs or after use to restore the surface. Thus, the method has potential to be a standard step to clean the carbon surface, or restore the sensitivity of electrodes from biofouling.

Carbon nanomaterials are used to improve electrodes for neurotransmitter detection, but what properties are important for maximizing those effects? In this work, we compare a newer form of graphene, carbon nanospikes (CNSs), with carbon nanotubes (CNTs) grown on wires and carbon fibers (CFs). CNS electrodes have a short, dense, defect-filled surface that produces remarkable electrochemical properties, much better than CNTs or CFs. The CNS surface roughness is 5.5 times greater than glassy carbon, while CNTs enhance roughness only 1.8-fold. D/G ratios are higher for CNS electrodes than CNT electrodes, an indication of more defect sites. For cyclic voltammetry of dopamine and ferricyanide, CNSs have both higher currents and smaller ΔEp values than CNTs and CFs. CNS electrodes also have a very low resistance to charge transfer. With fast-scan cyclic voltammetry (FSCV), CNS electrodes have enhanced current density for dopamine and cationic neurotransmitters due to increased adsorption to edge plane sites. This study establishes that not all carbon nanomaterials are equally advantageous for dopamine electrochemistry, but that short, dense nanomaterials that add defect sites provide improved current and electron transfer. CNSs are simple to mass fabricate on a variety of substrates and thus could be a favorable material for neurotransmitter sensing.

Electrochemical measurements of neurotransmitters provide insight into the dynamics of neurotransmission. In this review, we describe the development of electrochemical measurements of neurotransmitters and how they started with extrasynaptic measurements, but now are pushing to synaptic measurements. Traditionally, behavioral measurements with biosensors or fast-scan cyclic voltammetry have monitored extrasynaptic levels of neurotransmitters, such as dopamine, serotonin, adenosine, glutamate, and acetylcholine. Amperometry and electrochemical cytometry techniques have revealed mechanisms of exocytosis, suggesting partial release. Advances in nanoelectrodes now allow spatially resolved, electrochemical measurements in a synapse, which is only 20–100 nm wide. Synaptic measurements of dopamine and acetylcholine have been made. In this article, electrochemical measurements are also compared to optical imaging and mass spectrometry measurements, and while these other techniques provide enhanced spatial or chemical information, electrochemistry is best at monitoring real-time neurotransmission. Future challenges include combining electrochemistry with these other techniques, in order to facilitate multisite and multianalyte monitoring.

L- Glutamate is the main excitatory neurotransmitter in the central nervous system and hyperglutamatergic signaling is implicated in neurological and neurodegenerative diseases. Monitoring glutamate with a glutamate oxidase-based amperometric biosensor offers advantages such as high spatial and high temporal resolution. However, commercially-available glutamate biosensors are expensive and larger in size. Here, we report the development of 50 03BCm diameter biosensor for real-time monitoring of L-glutamate in vivo. A polymer, poly-o-phenylenediamine (PPD) layer was electropolymerized onto a 50 μm Pt wire to act as a permselective membrane. Then, glutamate oxidase entrapped in a biocompatible chitosan matrix was cast onto the microelectrode surface. Finally, ascorbate oxidase was coated to eliminate interferences from high levels of extracellular ascorbic acid present in brain tissue. L-glutamate measurements were performed amperometrically at an applied potential of 0.6 V vs Ag/AgCl. The biosensor exhibited a linear range from 5 to 150 μM, with a high sensitivity of 0.097 ± 0.001 nA/μM and one-week storage stability. The biosensor also showed a rapid steady state response to L-glutamate within 2 s, with a limit of detection of 0.044 μM. The biosensor was used successfully to detect stimulated glutamate in the subthalamic nucleus in brain slices and in vivo. Thus, this biosensor is appropriate for future neuroscience applications.

Rapid adenosine signaling, on the time frame of seconds, has been discovered in the brain that can modulate neurotransmission or blood flow. Rapid adenosine release can occur spontaneously or be evoked after a mechanical stimulation, but these two modes of adenosine have not been compared. Here, we compared spontaneous and mechanically-stimulated adenosine release in the prefrontal cortex, striatum, and hippocampus of anesthetized mice. For spontaneous adenosine, the number of adenosine events in the prefrontal cortex (40 ± 4 per hour) was significantly lower than in the striatum (54 ± 3) or hippocampus (56 ± 3). Similarly, the concentration per transient was lower in the prefrontal cortex but highest in the striatum. For mechanically-stimulated adenosine, the peak concentration in the prefrontal cortex (8 ± 2 μM) and striatum (8 ± 1 μM) were significantly lower than in the hippocampus (16 ± 2 μM). Comparing the two modes, the hippocampus had high mechanically-stimulated concentration and high spontaneous frequency, while the prefrontal cortex had lower spontaneous frequency and mechanically-stimulated release. However, there is no pattern with the striatum and thus no direct correlations between spontaneous and mechanically-stimulated adenosine. Thus, there may be different pools of adenosine or mechanisms of formation for these two modes. Because of the high frequency of spontaneous events and high concentration of mechanically-stimulated release in the hippocampus, there may be some areas that have stronger adenosine signaling and thus stronger neuromodulatory control by adenosine.

Microelectrodes are typically used for neurotransmitter detection, but nanoelectrodes are not because there is a trade-off between spatial resolution and sensitivity that is dependent on surface area. Cavity carbon-nanopipette electrodes (CNPEs), with tip diameters of a few hundred nanometers, have been developed for nanoscale electrochemistry. Here, we characterize the electrochemical performance of CNPEs with fast-scan cyclic voltammetry (FSCV) for the first time. Dopamine detection using cavity CNPEs, with a depth equivalent to a few radii, is compared with that using open-tube CNPEs, an essentially infinite geometry. Open-tube CNPEs have very slow temporal responses that change over time as the liquid rises in the CNPE. However, a cavity CNPE has a fast temporal response to a bolus of dopamine that is not different from that of a traditional carbon-fiber microelectrode. Cavity CNPEs, with tip diameters of 200-400 nm, have high currents because the small cavity traps and increases the local dopamine concentration. The trapping also leads to an FSCV frequency-independent response and the appearance of cyclization peaks that are normally observed only with large concentrations of dopamine. CNPEs have high dopamine selectivity over ascorbic acid (AA) because of the repulsion of AA by the negative electric field at the holding potential and the irreversible redox reaction. In mouse-brain slices, cavity CNPEs detected exogenously applied dopamine, showing they do not clog in tissue. Thus, cavity CNPEs are promising neurochemical sensors that provide spatial resolution on the scale of hundreds of nanometers, which is useful for small model organisms or for locations near specific cells.

Dopamine is a neuromodulator that is secreted to the synapse to relay chemical signals to target neurons. Abnormal levels of dopamine release leads to various neurodegenerative diseases. Therefore, measuring dopamine is essential to understand how dopamine is regulated under normal and pathological conditions. Drosophila melanogaster, the fruit fly, is an ideal model system for studying fundamental neurological processes and diseases because of the availability of sophisticated genetic tools and well conserved neurological processes between mammals and flies. Majority of neuroscience studies involved in modifying a gene and measure the effect of genetic mutation on output behaviors. However, dopamine release is highly dynamic because of the complex activity of dopamine transporters and autoreceptors. Therefore, to understand how dopamine signaling controls the behavior, a direct measurement of changes in dopamine release is necessary. Fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode is an electrochemical technique that trace concentration changes in dopamine release on the sub-second time scale. Our lab pioneered directly measuring various endogenous neuromodulators in the fly central nervous system with FSCV. Initially, these studies were performed in ex vivo preparations, where brains were isolated from larvae and adult fly, and thus could not monitor neuromodulators during behavior. In this study, we developed in vivo FSCV method to measure phasic dopamine in the mushroom body (MB) during behavior for the first time. The MB in fly has been extensively studied as an associative center for regulating olfactory learning and memory. First, acetylcholine stimulation was applied to the MB heel and medial tip to characterize dopamine signaling and to demonstrate the feasibility of in vivo FSCV in intact fly brain. Application of 0.2 pmol acetylcholine released 0.36 ± 0.06 µM dopamine in the medial tip, which is slightly higher than 0.22 ± 0.06 µM dopamine in the heel. Compartmental differences in evoked release suggest heterogeneity of dopamine regulation in the MB. Nisoxetine, a dopamine transporter inhibitor, and flupentixol, a D2 antagonist, increased stimulated dopamine release. We then applied the in vivo method to monitor changes in behaviorally evoked dopamine release during sugar feeding. Sugar feeding evoked 0.31 ± 0.09 µM dopamine in the medial tip of MB. Flupentixol significantly increased sugar evoked release implying D2 receptor acts as autoreceptor and regulates dopamine signaling during sugar feeding. Therefore, this developed in vivo FSCV method is a great addition to the existing tools to measure endogenous neuromodulators in the fly and valuable for studying real-time dopamine signaling during behavior. This in vivo method also can be further extended to better understand how dopamine and other neuromodulators regulate complex behaviors, such as reward associated learning and memory formation.

Carbon-fiber microelectrodes (CFMEs) are typically constructed from glass capillaries pulled to a fine taper or from a polyimide-coated capillary that is 90 μm in outer diameter. Here, a new fabrication method is developed to insulate carbon-fiber microelectrodes with a thin epoxy coating. A polytetrafluoroethylene (Teflon) mold was laser etched with channels 30–40 μm deep and wide and each channel filled with Armstrong C7 epoxy. A carbon fiber was laid into each channel so that the fiber extended past the mold, and the epoxy cured in an oven. One end of the fiber was trimmed to about 100 μm to form a cylindrical carbon-fiber microelectrode, while the other end was attached to a pin and connected to a potentiostat. Epoxy-insulated electrodes were tested with fast-scan cyclic voltammetry. For dopamine, the sensitivity is similar to glass and polyimide-coated capillary electrodes with a linear range of 0.1–10 μM and a LOD of 24 nM. SU-8 epoxy was tested as an alternative insulator because it cures at a lower temperature using light, but it was more brittle. Carbon nanotube fibers were also successfully insulated with epoxy. Epoxy-insulated CFMEs were used to detect stimulated dopamine release in vivo. Epoxy-insulated electrodes are smaller in diameter than polyimide-coated capillary electrodes and amenable to mass production. They are advantageous for use in higher order mammals, where glass is not permitted, and with alternative electrode materials, such as carbon nanotube fibers, that cannot be fabricated in a capillary puller.

Advanced carbon microelectrodes are being developed for the in vivo detection of neurotransmitters, such as dopamine (DA), including many carbon nanotube (CNT) based electrodes. Our prior simulations of DA and dopamine-o-quinone (DOQ) on pristine, flat graphene showed rapid surface diffusion for all adsorbed species, but it is not known how CNT surfaces affect dopamine adsorption and surface diffusivity. In this work, we use molecular dynamics simulations to investigate the adsorbed structures and surface diffusion dynamics of DA and DOQ on CNTs of varying curvature and helicity. In addition, we study DA dynamics in a groove between two aligned CNTs to model the spatial constraints at the junctions within CNT assemblies. We find that the adsorbate diffusion on a solvated CNT surface depends upon curvature. However, this effect cannot be attributed to changes in the surface energy roughness because the lateral distributions of the molecular adsorbates are similar across curvatures, diffusivities on zigzag and armchair CNTs are indistinguishable, and the curvature dependence disappears in the absence of solvent. Instead, adsorbate diffusivities correlate with the vertical placement of the adsorbate's moieties, its tilt angle, its orientation along the CNT axis, and the number of waters in its first hydration shell -- all of which will influence its effective hydrodynamic radius. Finally, DA diffuses into and remains in the groove between a pair of aligned and solvated CNTs, enhancing diffusivity along the CNT axis. These first studies of surface diffusion on a CNT electrode surface are important for understanding the changes in diffusion dynamics of dopamine on nanostructured carbon electrode surfaces.

Carbon nanotube yarn microelectrodes (CNTYMEs) are an alternative to carbon-fiber microelectrodes (CFMEs) with interesting electrochemical properties because analyte is momentarily trapped in cavities between the CNTs. Here, we compare fast-scan cyclic voltammetry (FSCV) detection of catecholamines, including dopamine, norepinephrine, and epinephrine, at CNTYMEs, CFMEs, as well as cavity carbon nanopipette electrodes (CNPEs). At CFMEs, current decreases dramatically at high FSCV repetition frequencies. At CNTYMEs, current is almost independent of FSCV repetition frequency because the analytes are trapped in the crevices between CNTs, and thus the electrode acts like a thin-layer cell. At CFMEs, small cyclization product peaks are observed due to an intramolecular cyclization reaction to form leucocatecholamine, which is electroactive, and these peaks are largest for the secondary amine epinephrine. At CNTYMEs, more of the leucocatecholamine cyclization product is detected for all catecholamines because of the enhanced trapping effects, particularly at higher repetition rates where the reaction occurs more frequently and more product is accumulated. For epinephrine, the secondary peaks have larger currents than the primary oxidation peaks at 100 Hz, and similar trends are observed with faster scan rates and 500 Hz repetition frequencies. Finally, we examined CNPEs, which also momentarily trap neurotransmitters. Similar to CNTYMEs, at CNPEs, catecholamines have robust cyclization peaks, particularly at high repetition rates. Thus, CNTYMEs and CNPEs have thin layer cell behavior that facilitates high temporal resolution measurements, but catecholamines CVs are complicated by cyclization reactions. However, those additional peaks could be useful in discriminating the analytes, particularly epinephrine and norepinephrine.

Direct laser writing, a nano 3D-printing approach, has enabled fabrication of customized carbon microelectrode sensors for neurochemical detection. However, to detect neurotransmitters in tiny biological organisms or synapses, submicrometer nanoelectrodes are required. In this work, we used 3D printing to fabricate carbon nanoelectrode sensors. Customized structures were 3D printed and then pyrolyzed, resulting in free-standing carbon electrodes with nanotips. The nanoelectrodes were insulated with atomic layer deposition of Al2O3 and the nanotips were polished by a focused ion beam to form 600 nm disks. Using fast-scan cyclic voltammetry, the electrodes successfully detected stimulated dopamine in the adult fly brain, demonstrating that they are robust and sensitive enough to use in tiny biological systems. This work is the first demonstration of 3D printing to fabricate free-standing carbon nanoelectrode sensors and will enable batch fabrication of customized nanoelectrode sensors with precise control and excellent reproducibility.

Fast-scan cyclic voltammetry (FSCV) is widely used for in vivo detection of neurotransmitters, but identifying analytes, particularly mixtures, is difficult. Data analysis has focused on identifying dopamine from cyclic voltammograms, but it would be better to analyze all the data in the three dimensional FSCV color plot. Here, the goal was to use image analysis-based analysis of FSCV color plots for the first time, specifically the structural similarity index (SSIM), to identify rapid neurochemical events. Initially, we focused on identifying spontaneous adenosine events, as adenosine cyclic voltammograms have a primary oxidation at 1.3 V and a secondary oxidation peak that grows in over time. Using SSIM, sample FSCV color plots were compared with reference color plots, and the SSIM cutoff score was optimized to distinguish adenosine. High-pass digital filtering was also applied to remove the background drift and lower the noise, which produced a better LOD. The SSIM algorithm detected more adenosine events than a previous algorithm based on current vs time traces, with 99.5 ± 0.6% precision, 95 ± 3% recall, and 97 ± 2% F(1) score (n = 15 experiments from three researchers). For selectivity, it successfully rejected signals from pH changes, histamine, and H(2)O(2). To prove it is a broad strategy useful beyond adenosine, SSIM analysis was optimized for dopamine detection and is able to detect simultaneous events with dopamine and adenosine. Thus, SSIM is a general strategy for FSCV data analysis that uses three dimensional data to detect multiple analytes in an efficient, automated analysis.

Drosophila melanogaster, the fruit fly, is an exquisite model organism to understand neurotransmission. Dopaminergic signaling in the Drosophila mushroom body (MB) is involved in olfactory learning and memory, with different compartments controlling aversive learning (corner) vs appetitive learning (medial tip). Here, the goal was to develop techniques to measure endogenous dopamine in compartments of the MB for the first time. We compared three stimulation methods: acetylcholine (natural stimulus), P2X2 (chemogenetics), and CsChrimson (optogenetics). Evoked dopamine release was measured with fast-scan cyclic voltammetry in isolated adult Drosophila brains. Acetylcholine stimulated the largest dopamine release (0.40 μM), followed by P2X2 (0.14 μM), and CsChrimson (0.07 μM). With the larger acetylcholine and P2X2 stimulations, there were no regional or sex differences in dopamine release. However, with CsChrimson, dopamine release was significantly higher in the corner than the medial tip, and females had more dopamine than males. Michaelis-Menten modeling of the single-light pulse revealed no significant regional differences in Km, but the corner had a significantly lower Vmax (0.12 μM/s vs. 0.19 μM/s) and higher dopamine release (0.05 μM vs. 0.03 μM). Optogenetic experiments are challenging because CsChrimson is also sensitive to blue light used to activate green fluorescent protein, and thus, light exposure during brain dissection must be minimized. These experiments expand the toolkit for measuring endogenous dopamine release in Drosophila, introducing chemogenetic and optogenetic experiments for the first time. With a variety of stimulations, different experiments will help improve our understanding of neurochemical signaling in Drosophila.

Adenosine is important for local neuromodulation, and rapid adenosine signaling can occur spontaneously or after mechanical stimulation, but little is known about how adenosine is formed in the extracellular space for those stimulations. Here, we studied mechanically stimulated and spontaneous adenosine to determine if rapid adenosine is formed by extracellular breakdown of adenosine triphosphate (ATP) using mice globally deficient in extracellular breakdown enzymes, either CD39 (nucleoside triphosphate diphosphohydrolase 1, NTPDase1) or CD73 (ecto-5'-nucleotidase). CD39 knockout (KO) mice have a lower frequency of spontaneous adenosine events than wild-type (WT, C57BL/6). Surprisingly, CD73KO mice demonstrate sex differences in spontaneous adenosine; males maintain similar event frequencies as WT, but females have significantly fewer events and lower concentrations. Examining the mRNA expression of other enzymes that metabolize ATP revealed tissue nonspecific alkaline phosphatase (TNAP) was upregulated in male CD73KO mice, but not secreted prostatic acid phosphatase (PAP) or transmembrane PAP. Thus, TNAP upregulation compensates for CD73 loss in males but not in females. These sex differences highlight that spontaneous adenosine is formed by metabolism of extracellular ATP by many enzymes. For mechanically stimulated adenosine, CD39KO or CD73KO did not change stimulation frequency, concentration, or t1/2. Thus, the mechanism of formation for mechanically stimulated adenosine is likely direct release of adenosine, different than spontaneous adenosine. Understanding these different mechanisms of rapid adenosine formation will help to develop pharmacological treatments that differentially target modes of rapid adenosine signaling, and all treatments should be studied in both sexes, given possible differences in extracellular ATP degradation.

Fast-scan cyclic voltammetry (FSCV) is used with carbon-fiber microelectrodes for the real-time detection of neurotransmitters on the subsecond time scale. With FSCV, the potential is ramped up from a holding potential to a switching potential and back, usually at a 400 V s−1 scan rate and a frequency of 10 Hz. The plot of current vs. applied potential, the cyclic voltammogram (CV), has a very different shape for FSCV than for traditional cyclic voltammetry collected at scan rates which are 1000-fold slower. Here, we explore the theory of FSCV, with a focus on dopamine detection. First, we examine the shape of the CVs. Background currents, which are 100-fold higher than faradaic currents, are subtracted out. Peak separation is primarily due to slow electron transfer kinetics, while the symmetrical peak shape is due to exhaustive electrolysis of all the adsorbed neurotransmitters. Second, we explain the origins of the dopamine waveform, and the factors that limit the holding potential (oxygen reduction), switching potential (water oxidation), scan rate (electrode instability), and repetition rate (adsorption). Third, we discuss data analysis, from data visualization with color plots, to the automated algorithms like principal components regression that distinguish dopamine from pH changes. Finally, newer applications are discussed, including optimization of waveforms for analyte selectivity, carbon nanomaterial electrodes that trap dopamine, and basal level measurements that facilitate neurotransmitter measurements on a longer time scale. FSCV theory is complex, but understanding it enables better development of new techniques to monitor neurotransmitters in vivo.

Adenosine is an endogenous neuroprotectant that modulates vasodilation in the central nervous system. Oxygen changes occur when there is an increase in local cerebral blood flow and thus are a measure of vasodilation. Transient oxygen events following rapid adenosine events have been recently discovered, but the relationship between adenosine and blood flow change during ischemia/reperfusion (I/R) has not been characterized. Caffeine is a nonselective adenosine receptor antagonist that can modulate the effects of adenosine in the brain, but how it affects adenosine and oxygen levels during I/R is also unknown. In this study, extracellular changes in adenosine and oxygen were simultaneously monitored using fast-scan cyclic voltammetry during bilateral common carotid artery occlusion (BCCAO) and the effects of a specific A(2A) antagonist, SCH 442416, or general antagonist, caffeine, were studied. Measurements were made in the caudate-putamen for one hour of normoxia, followed by 30 min of BCCAO and 30 min of reperfusion. The frequency and number of both adenosine and oxygen transient events significantly increased during I/R. The specific A(2A) antagonist, SCH 442416 (3 mg/kg, i.p.), eliminated the increase in adenosine and oxygen events caused by I/R. The general adenosine receptor antagonist, caffeine (100 mg/kg, i.p.), decreased the frequency of adenosine and oxygen transient events during I/R. These results demonstrate that during BCCAO, there are more rapid release events of the neuromodulator adenosine and correlated local oxygen changes, and these rapid, local effects are dampened by caffeine and other A(2A) antagonists.

University of Virginia, like many other universities, has developed a chemistry outreach program (called Chemistry Learning through Experimentation, Awareness, and Demonstration; i.e., Chemistry LEAD) that visits K–5 schools and teaches inquiry-based chemistry lessons in order to engage local students with science. Chemistry LEAD is graduate student organized and led. The number of classrooms the group can visit in any year is limited; consequently, to extend their impact, the group decided to organize a summer, 1 day professional workshop for teachers. The goal was to give teachers the knowledge, confidence, and materials necessary to implement inquiry-based chemistry lessons. The graduate students worked as a team, with different students leading different activities, to decrease the burden of workshop planning and implementation. The workshop was repeated for two summers; a total of 52 teachers from six school districts participated. The teachers enjoyed learning from the graduate students, and follow-...

The fruit fly, Drosophila melanogaster, is a popular model organism for studying neurological processes and diseases due to the availability of sophisticated genetic tools. While endogenous neurotransmitter release has been characterized in Drosophila larvae, here, we measured endogenous dopamine release in isolated adult Drosophila brains for the first time. Dopamine was measured with fast-scan cyclic voltammetry (FSCV), and acetylcholine or nicotine were used as the stimulus, as both interact with nicotinic acetylcholine receptors (nAChRs) to evoke endogenous dopamine release. Stimulations with 10 pmol of acetylcholine elicited 0.26 ± 0.05 μM dopamine, while 70 fmol nicotine stimulations evoked 0.29 ± 0.03 μM in the central complex. Nicotine-stimulated dopamine release lasted much longer than acetylcholine-stimulated release. Dopamine release is reduced in the presence of nAChR antagonist α-bungarotoxin and the sodium channel blocker tetrodotoxin, indicating release is mediated by nAChRs and exocytosis. The identity of dopamine was confirmed by using 3-iodotyrosine, a dopamine synthesis inhibitor, and by confirming that release was not changed in octopamine synthesis mutant flies, Tdc2 RO54. Additionally, the half-decay time ( t50) in fumin (67 ± 15 s), dopamine transporter mutant flies, was larger than in wild-type flies (16 ± 3.7 s) further proving that acetylcholine stimulation evokes dopamine release. This study demonstrates that stimulation of nAChRs can be used to elicit endogenous dopamine release in adult fly brains, which will be a useful technique for future studies probing dopamine changes during aging or in neurodegenerative diseases.

Acetylcholine is an excitatory neurotransmitter in the central nervous system of insects and the nicotinic acetylcholine receptor (nAChR) is a target for neonicotinoid insecticides. Functional insect nAChRs are difficult to express in host cells, and hence difficult to study. In mammals, acetylcholine and nicotine evoke dopamine release, but the extent to which this mechanism is conserved in insects is unknown. In intact larval ventral nerve cords (VNCs), we studied dopamine evoked by acetylcholine, nicotine, or neonicotinoids. Using fast-scan cyclic voltammetry, we confirmed dopamine was measured by its cyclic voltammogram and also by feeding Drosophila the synthesis inhibitor, 3-iodotyrosine, which lowered the evoked dopamine response. Acetylcholine (1.8 pmol) evoked on average 0.43 ± 0.04 μM dopamine. Dopamine release significantly decreased after incubation with α-bungarotoxin, demonstrating the release is mediated by nAChR, but atropine, a muscarinic AChR antagonist, had no effect. Nicotine (t1/2 = 71 s) and the neonicotinoids nitenpyram and imidacloprid (t1/2 = 86 s, 121 s respectively) also evoked dopamine release, which lasted longer than acetylcholine-stimulated release (t1/2 = 19 s). Nicotine-stimulated dopamine was significantly lower in the presence of sodium channel blocker, tetrodotoxin, showing that the release is exocytotic. Drosophila that have mutations in the nAChR subunit α1 or β2 have significantly lower neonicotinoid-stimulated release but no changes in nicotine-stimulated release. This work demonstrates that nAChR agonists mediate dopamine release in Drosophila larval VNC and that mutations in nAChR subunits affect how insecticides stimulate dopamine release.

Fibers made of CNTs are attractive microelectrode sensors because they can be directly fabricated into microelectrodes. Different protocols for making CNT fibers have been developed, but differences in surface structure and therefore electrochemical properties that result have not been studied. In this study, we correlated the surface and electrochemical properties for neurochemical detection at 3 types of materials: CNT fibers produced by wet spinning with (1) polyethylenimine (PEI/CNT) or (2) chlorosulfonic acid (CA/CNT), and (3) CNT yarns made by solid-based CNT drawing. CNT yarns had well-aligned, high purity CNTs, abundant oxygen functional groups, and moderate surface roughness which led to the highest dopamine current density (290 ± 65 pA/cm 2 ) and fastest electron transfer kinetics. The crevices of the CNT yarn and PEI/CNT fiber microelectrodes allow dopamine to be momentarily trapped during fast-scan cyclic voltammetry detection, leading to thin-layer cell conditions and a response that was independent of applied waveform frequency. The larger crevices on the PEI/CNT fibers led to a slower time response, showing too much roughness is detrimental to fast detection. CA/CNT fibers have a smoother surface and lower currents, but their negative surface charge results in high selectivity for dopamine over uric acid or ascorbic acid. Overall, small crevices, high conductivity, and abundant oxygen groups led to high sensitivity for amine neurotransmitters, such as dopamine and serotonin. Thus, different surfaces of CNT fibers result in altered electrochemical properties and could be used in the future to predict and control electrochemical performance.

Carbon nanotube (CNT) based microelectrodes exhibit rapid and selective detection of neurotransmitters. While different fabrication strategies and geometries of CNT microelectrodes have been characterized, relatively little research has investigated ways to selectively enhance their electrochemical properties. In this work, we introduce two simple, reproducible, low-cost, and efficient surface modification methods for carbon nanotube yarn microelectrodes (CNTYMEs): O2 plasma etching and antistatic gun treatment. O2 plasma etching was performed by a microwave plasma system with oxygen gas flow and the optimized time for treatment was 1 min. The antistatic gun treatment flows ions by the electrode surface; two triggers of the antistatic gun was the optimized number on the CNTYME surface. Current for dopamine at CNTYMEs increased 3-fold after O2 plasma etching and 4-fold after antistatic gun treatment. When the two treatments were combined, the current increased 12-fold, showing the two effects are due to in...