Analysis of biogenic amines in a single Drosophila larva brain by capillary electrophoresis with fast-scan cyclic voltammetry detection.

Drosophila, the fruit fly, is a common model organism in biology; however, quantifying neurotransmitters in Drosophila is challenging because of the small size of the central nervous system (CNS). Here, we develop neurotransmitter quantification by capillary electrophoresis with fast-scan cyclic voltammetry (CE-FSCV) detection, which allows peak identification by both migration time and the cyclic voltammogram, in contrast to traditional amperometric detection which provides no chemical identification. Tissue content of biogenic amine neurotransmitters was determined in a single CNS dissected from a Drosophila larva. Low detection limits, 1 nM for dopamine and serotonin, 2.5 nM for tyramine, and 4 nM for octopamine, were achieved using field-amplified sample stacking by diluting the homogenized tissue with percholoric acid and acetonitrile. Two different strains of wild-type flies, Oregon R and Canton S, have similar dopamine and serotonin levels but different octopamine content. When flies are fed NSD-1015, which inhibits dopamine decarboxylase (Ddc) a synthesis enzyme in the dopamine and serotonin pathways, dopamine significantly decreases by 52%. A genetically altered driver line, Ddc-GAL4, had lower serotonin and dopamine content as did w(118) flies. When the Ddc-GAL4 line was used to produce flies overexpressing the serotonin synthesis enzyme tryptophan hydroxylase (Ddc-GAL4;UAS-Trh), the serotonin tissue content was greater than for Ddc-GAL4 but not significantly different than the wild-type. These results show that CE-FSCV is useful for monitoring the impact of genetic and pharmacological manipulations on the content of multiple neurotransmitters in a CNS from a Drosophila larva.

Functional groups modulate the sensitivity and electron transfer kinetics of neurochemicals at carbon nanotube modified microelectrodes.

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.

Detection of endogenous dopamine changes in Drosophila melanogaster using fast-scan cyclic voltammetry.

Drosophila melanogaster, the fruit fly, is a commonly used model organism because of its homology to mammals and facile genetic manipulations. However, the size of the nervous system is very small. We report a method to evoke and detect rapid changes in extracellular dopamine in a single nerve cord isolated from a Drosophila larva. Flies were genetically modified to express Channelrhodopsin-2, a blue-light activated cation channel, in only dopaminergic neurons. Extracellular dopamine changes were measured with fast-scan cyclic voltammetry at an implanted carbon-fiber microelectrode. Stimulations of 7 s with blue light result in an average peak dopamine concentration of 810 +/- 60 nM, similar to electrically-stimulated release in mammals. Stimulations repeated at 15 min intervals are stable for 65 min, allowing pharmacological experiments in the same sample. Peak duration is extended after cocaine or nisoxetine, inhibitors of the dopamine transporter (DAT). Release was reduced upon exposure to reserpine, which inhibits vesicular packaging. Chronic administration of NSD-1015, a dopamine synthesis inhibitor, decreased dopamine release and inhibited pupation, showing a link between neurotransmission and physiology. This is the first method to measure endogenous dopamine in an intact larval Drosophila nervous system and will allow studies of genetic and pharmacological manipulations of dopamine release and uptake.

Assessing Anthocyanin Biosynthesis in Solanaceae as a Model Pathway for Secondary Metabolism.

Solanaceae have played an important role in elucidating how flower color is specified by the flavonoid biosynthesis pathway (FBP), which produces anthocyanins and other secondary metabolites. With well-established reverse genetics tools and rich genomic resources, Solanaceae provide a robust framework to examine the diversification of this well-studied pathway over short evolutionary timescales and to evaluate the predictability of genetic perturbation on pathway flux. Genomes of eight Solanaceae species, nine related asterids, and four rosids were mined to evaluate variation in copy number of the suite of FBP enzymes involved in anthocyanin biosynthesis. Comparison of annotation sources indicated that the NCBI annotation pipeline generated more and longer FBP annotations on average than genome-specific annotation pipelines. The pattern of diversification of each enzyme among asterids was assessed by phylogenetic analysis, showing that the CHS superfamily encompasses a large paralogous family of ancient and recent duplicates, whereas other FBP enzymes have diversified via recent duplications in particular lineages. Heterologous expression of a pansy F3'5'H gene in tobacco changed flower color from pink to dark purple, demonstrating that anthocyanin production can be predictably modified using reverse genetics. These results suggest that the Solanaceae FBP could be an ideal system to model genotype-to-phenotype interactions for secondary metabolism.

Kinetics of the dopamine transporter in Drosophila larva.

Dopamine transporters (DAT) regulate neurotransmission and are important in diseases such as addiction and attention deficit hyperactivity disorder. The Drosophila dopamine transporter (dDAT) is analogous to the mammalian DAT, but Michaelis-Menten kinetic parameters have not been characterized in vivo. In this study, dopamine clearance kinetics were measured in a Drosophila larval CNS using an implanted carbon-fiber microelectrode and fast-scan cyclic voltammetry. Dopamine was pressure ejected from a micropipet implanted 15-20 µm from the microelectrode. Clearance of exogenously applied dopamine was significantly reduced in dDAT null (fumin) mutants, and kinetic constants in these mutants were used to determine clearance by other mechanisms including diffusion. After correction for diffusion, the maximal rate of uptake, Vmax, was estimated to be 0.11 ± 0.02 µM/s and Km was 1.3 ± 0.6 µM in wild-type flies. The clearance rate was significantly reduced following treatment with the DAT inhibitor cocaine in wild-type flies, but not in fumin mutants, which indicates that serotonin transporter is not contributing significantly to dopamine clearance in these larvae. Clearance of endogenous dopamine, evoked by optical stimulation in flies expressing Channelrhodopsin2, was similar to clearance of exogenous dopamine, but it was not possible to evoke concentrations that were close to saturation. The ability to quickly assess the role of the dopamine transporter in any Drosophila larva will be useful for future studies of how transporters regulate neurotransmission and to understand the underlying mechanisms of drug addiction.

Drosophila Dopamine2-like receptors function as autoreceptors.

Dopaminergic signaling pathways are conserved between mammals and Drosophila and D2 receptors have been identified in Drosophila. However, it has not been demonstrated whether Drosophila D2 receptors function as autoreceptors and regulate the release of dopamine. The goal of this study was to determine if Drosophila D2 receptors act as autoreceptors by probing the extent to which D2 agonists and antagonists affect evoked dopamine release. Fast-scan cyclic voltammetry was used to measure stimulated dopamine release at a carbon-fiber microelectrode implanted in an intact, larval Drosophila nervous system. Dopamine release was evoked using 5-second blue light stimulations that open Channelrhodopsin-2, a blue light activated cation channel that was specifically expressed in dopaminergic neurons. In mammals, administration of a D2 agonist decreases evoked dopamine release by increasing autoreceptor feedback. Similarly, we found that the D2 agonists bromocriptine and quinpirole decreased stimulated dopamine release in Drosophila. D2 antagonists were expected to increase dopamine release and the D2 antagonists flupenthixol, butaclamol, and haloperidol did increase stimulated release. Agonists did not significantly modulate dopamine uptake although the modulatory effects of D2 drugs on release were affected by prior administration of the uptake inhibitor nisoxetine. These results demonstrate that the D2 receptor functions as an autoreceptor in Drosophila. The similarities in dopamine regulation validate Drosophila as a model system for studying the basic neurobiology of dopaminergic signaling.

Intrinsic conformational preferences of substituted cyclohexanes and tetrahydropyrans evaluated at the CCSD(T) complete basis set limit: implications for the anomeric effect.

A series of MP2 and CCSD(T) computations have been carried out with correlation consistent basis sets as large as aug-cc-pV5Z to determine the intrinsic equatorial-axial conformational preference of CH(3)-, F-, OCH(3)-, and OH-substituted cyclohexane and tetrahydropyran rings. The high-accuracy relative electronic energies reported here shed new light on the intrinsic energetics of these cyclic prototypes for the anomeric effect. At the CCSD(T) complete basis set (CBS) limit, the energy of the equatorial conformation relative to the axial position (DeltaE (CBS)(CCSD(T))) is -1.75, -0.20, -0.21, and -0.56 kcal mol(-1) in methyl-, fluoro-, methoxy-, and hydroxycyclohexane, respectively, while DeltaE(CBS)(CCSD(T) is -2.83, +2.45, +1.27, and +0.86 kcal mol(-1) for 2-methyl-, 2-fluoro-, 2-methoxy-, and 2-hydroxytetrahydropyran, respectively. Note that the equatorial and axial conformers are nearly electronically isoenergetic in both fluoro- and methoxycyclohexane. For all eight cyclic species, a zero-point vibrational energy correction decreases Delta by a few tenths of a kilocalorie per mole. Relative energies obtained with popular methods and basis sets are unreliable, including Hartree-Fock theory, the B3LYP density functional, and the 6-31G and 6-311G families of split-valence basis sets. Even with the massive pentuple-zeta basis sets, the HF and B3LYP methods substantially overestimate the stability of the equatorial conformers (by as much as 0.99 and 0.73 kcal mol(-1), respectively, for 2-methoxytetrahydropyran). Only because of a consistent cancellation of errors do these popular approaches sometimes provide reasonable estimates of the anomeric effect.