Alexander G. Zestos

High Temporal Resolution Measurements of Dopamine with Carbon Nanotube Yarn Microelectrodes

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 Nanotubes Grown on Metal Microelectrodes for the Detection of Dopamine

Microelectrodes modified with carbon nanotubes (CNTs) are useful for the detection of neurotransmitters because the CNTs enhance sensitivity and have electrocatalytic effects. CNTs can be grown on carbon fiber microelectrodes (CFMEs) but the intrinsic electrochemical activity of carbon fibers makes evaluating the effect of CNT enhancement difficult. Metal wires are highly conductive and many metals have no intrinsic electrochemical activity for dopamine, so we investigated CNTs grown on metal wires as microelectrodes for neurotransmitter detection. In this work, we successfully grew CNTs on niobium substrates for the first time. Instead of planar metal surfaces, metal wires with a diameter of only 25 μm were used as CNT substrates; these have potential in tissue applications due to their minimal tissue damage and high spatial resolution. Scanning electron microscopy shows that aligned CNTs are grown on metal wires after chemical vapor deposition. By use of fast-scan cyclic voltammetry, CNT-coated niobium (CNT-Nb) microelectrodes exhibit higher sensitivity and lower ΔEp value compared to CNTs grown on carbon fibers or other metal wires. The limit of detection for dopamine at CNT-Nb microelectrodes is 11 ± 1 nM, which is approximately 2-fold lower than that of bare CFMEs. Adsorption processes were modeled with a Langmuir isotherm, and detection of other neurochemicals was also characterized, including ascorbic acid, 3,4-dihydroxyphenylacetic acid, serotonin, adenosine, and histamine. CNT-Nb microelectrodes were used to monitor stimulated dopamine release in anesthetized rats with high sensitivity. This study demonstrates that CNT-grown metal microelectrodes, especially CNTs grown on Nb microelectrodes, are useful for monitoring neurotransmitters.

Polyethylenimine carbon nanotube fiber electrodes for enhanced detection of neurotransmitters.

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.

PKCβ Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux

Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCβ) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCβ inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum in vitro. In this study, we utilized in vivo microdialysis in live, behaving rats to assess the effect of the PKCβ inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 μM enzastaurin or 1 μM ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCβ inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKCβ inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKCβ inhibitors to reduce the effects of amphetamine.

An Easy Method To Monitor Lactide Polymerization with a Boron Fluorescent Probe

Fluorescence spectroscopy has been widely used to monitor different polymer processes such as polymerization kinetics, chain entanglements, and thermal transitions. The solvent-free controlled ring-opening polymerization (ROP) of lactide is significant both commercially and for research; thus, monitoring this process with a simple fluorescence method can be very useful. Here, a fluorescent dye, difluoroboron 4-methoxydibenzoylmethane (BF(2)dbmOMe) is employed to probe lactide bulk ROP by measuring the emission from solidified reaction aliquots at room temperature. It was found that, through the course of polymerization, the fluorescence of BF(2)dbmOMe in the solid-state aliquots exhibited a systematic shift from yellow to green and then to blue, accompanied by a gradual reduction in the decay lifetime. The fluorescence color change is sensitive to the monomer percent conversion, not the polymer molecular weight. On the basis of these observations and experimental data, we propose that the long-wavelength emission with perceivably longer lifetimes arises from BF(2)dbmOMe dye aggregates (ground and/or excited states), while the dissolved individual dye molecules are responsible for the blue fluorescence with a shorter lifetime. This demonstration of the utility of BF(2)dbmOMe as a fluorescent probe for lactide polymerization could have important practical implications.

Microdialysis Coupled with LC-MS/MS for In Vivo Neurochemical Monitoring

Microdialysis is a powerful sampling technique used to monitor small molecules in vivo. Despite the many applications of microdialysis sampling, it is limited by the method of analyzing the resulting samples. An emerging technique for analysis of microdialysis samples is liquid chromatography-tandem mass spectrometry (LC-MS/MS). This technique is highly versatile, allowing multiplexed analysis of neurotransmitters, metabolites, and neuropeptides. Using LC-MS/MS for polar neurotransmitters is hampered by weak retention reverse phase LC columns. Several derivatization reagents have been utilized to enhance separation and resolution of neurochemicals in dialysate samples including benzoyl chloride (BzCl), dansyl chloride, formaldehyde, ethylchloroformate, and propionic anhydride. BzCl reacts with amine and phenol groups so that many neurotransmitters can be labeled. Besides improving separation on reverse phase columns, this reagent also increases sensitivity. It is available in a heavy form so that it can be used to make stable-isotope labeled internal standard for improved quantification. Using BzCl with LC-MS/MS has allowed for measuring as many as 70 neurochemicals in a single assay. With slightly different conditions, LC-MS/MS has also been used for monitoring endocannabinoids. LC-MS/MS is also useful for neuropeptide assay because it allows for highly sensitive, sequence specific measurement of most peptides. These advances have allowed for multiplexed neurotransmitter measurements in behavioral, circuit analysis, and drug effect studies.

An immune-beige adipocyte communication via nicotinic acetylcholine receptor signaling

Beige adipocytes have recently been shown to regulate energy dissipation when activated and help organisms defend against hypothermia and obesity. Prior reports indicate that beige-like adipocytes exist in adult humans and that they may present novel opportunities to curb the global epidemic in obesity and metabolic illnesses. In an effort to identify unique features of activated beige adipocytes, we found that expression of the cholinergic receptor nicotinic alpha 2 subunit (Chrna2) was induced in subcutaneous fat during the activation of these cells and that acetylcholine-producing immune cells within this tissue regulated this signaling pathway via paracrine mechanisms. CHRNA2 functioned selectively in uncoupling protein 1 (Ucp1)-positive beige adipocytes, increasing thermogenesis through a cAMP- and protein kinase A-dependent pathway. Furthermore, this signaling via CHRNA2 was conserved and present in human subcutaneous adipocytes. Inactivation of Chrna2 in mice compromised the cold-induced thermogenic response selectively in subcutaneous fat and exacerbated high-fat diet-induced obesity and associated metabolic disorders, indicating that even partial loss of beige fat regulation in vivo had detrimental consequences. Our results reveal a beige-selective immune–adipose interaction mediated through CHRNA2 and identify a novel function of nicotinic acetylcholine receptors in energy metabolism. These findings may lead to identification of therapeutic targets to counteract human obesity.Inhibition of immune cell–derived acetylcholine synthesis or of its signaling via CHRNA2 in beige adipocytes reduces thermogenesis and exacerbates diet-induced obesity, suggesting a new mode of immuno–fat communication in energy metabolism.

Direct and Systemic Administration of a CNS-Permeant Tamoxifen Analog Reduces Amphetamine-Induced Dopamine Release and Reinforcing Effects

Amphetamines (AMPHs) are globally abused. With no effective treatment for AMPH addiction to date, there is urgent need for the identification of druggable targets that mediate the reinforcing action of this stimulant class. AMPH-stimulated dopamine efflux is modulated by protein kinase C (PKC) activation. Inhibition of PKC reduces AMPH-stimulated dopamine efflux and locomotor activity. The only known CNS-permeant PKC inhibitor is the selective estrogen receptor modulator tamoxifen. In this study, we demonstrate that a tamoxifen analog, 6c, which more potently inhibits PKC than tamoxifen but lacks affinity for the estrogen receptor, reduces AMPH-stimulated increases in extracellular dopamine and reinforcement-related behavior. In rat striatal synaptosomes, 6c was almost fivefold more potent at inhibiting AMPH-stimulated dopamine efflux than [3H]dopamine uptake through the dopamine transporter (DAT). The compound did not compete with [3H]WIN 35,428 binding or affect surface DAT levels. Using microdialysis, direct accumbal administration of 1 μM 6c reduced dopamine overflow in freely moving rats. Using LC-MS, we demonstrate that 6c is CNS-permeant. Systemic treatment of rats with 6 mg/kg 6c either simultaneously or 18 h prior to systemic AMPH administration reduced both AMPH-stimulated dopamine overflow and AMPH-induced locomotor effects. Finally, 18 h pretreatment of rats with 6 mg/kg 6c s.c. reduces AMPH-self administration but not food self-administration. These results demonstrate the utility of tamoxifen analogs in reducing AMPH effects on dopamine and reinforcement-related behaviors and suggest a new avenue of development for therapeutics to reduce AMPH abuse.

Chemical biomarkers of epileptogenesis and ictogenesis in experimental epilepsy

Epilepsy produces chronic chemical changes induced by altered cellular structures, and acute ones produced by conditions leading into individual seizures. Here, we aim to quantify 24 molecules simultaneously at baseline and during periods of lowered seizure threshold in rats. Using serial hippocampal microdialysis collections starting two weeks after the pilocarpine-induced status epilepticus, we evaluated how this chronic epilepsy model affects molecule levels and their interactions. Then, we quantified the changes occurring when the brain moves into a pro-seizure state using a novel model of physiological ictogenesis. Compared with controls, pilocarpine animals had significantly decreased baseline levels of adenosine, homovanillic acid, and serotonin, but significantly increased levels of choline, glutamate, phenylalanine, and tyrosine. Step-wise linear regression identified that choline, homovanillic acid, adenosine, and serotonin are the most important features to characterize the difference in the extracellular milieu between pilocarpine and control animals. When increasing the hippocampal seizure risk, the concentrations of normetanephrine, serine, aspartate, and 5-hydroxyindoleacetic acid were the most prominent; however, there were no specific, consistent changes prior to individual seizures.

Communication—Carbon Nanotube Fiber Microelectrodes for High Temporal Measurements of Dopamine

Carbon nanotube (CNT) yarn and fiber-microelectrodes were developed for neurotransmitter detection using fast scan cyclic voltammetry (FSCV). Fibers were made by suspending CNTs in acid/surfactant and extruding into acetone/polyethyleneimine (PEI) and compared to a CNT yarn. They were FSCV frequency independent for dopamine up to 100 Hz. With faster frequencies, up to 500 Hz, high currents are maintained, which allows a 2 ms sampling rate for FSCV, compared to 100 ms. CNT fibers have rough surfaces which trap dopamine and dopamine-o-quinone (DOQ), creating more reversible CVs. CNT yarns and fibers are beneficial for high sensitivity, rapid measurements of neurotransmitters.