The impact of thiamethoxam on the feeding and behavior of 2 soybean herbivore feeding guilds.

Over the past few decades, inadvertent consequences have stemmed from the intensified use of neonicotinoids in agroecosystems. Neonicotinoid applications can result in both positive (e.g., reduced persistent virus transmission) and negative (e.g., increased host susceptibility) repercussions exhibiting ambiguity for their use in crop production. In soybean, aspects of neonicotinoid usage such as the impact on nonpersistent virus transmission and efficacy against nontarget herbivores have not been addressed. This study evaluated the interaction between the neonicotinoid thiamethoxam and soybean variety and the impact on different pest feeding guilds. Feeding and behavioral bioassays were conducted in the laboratory to assess the effect of thiamethoxam on the mortality and weight gain of the defoliator, Chrysodeixis includens (Walker). Bioassays evaluated impacts dependent and independent of soybean tissue, in addition to both localized and systemic efficacy within the soybean plant. Additionally, using the electrical penetration graph technique (EPG), the probing behavior of 2 piercing-sucking pests, Aphis gossypii Glover and Myzus persicae (Sulzer), was observed. Results from defoliator bioassays revealed thiamethoxam had insecticidal activity against C. includens. Distinctions in thiamethoxam-related mortality between bioassays dependent and independent of soybean tissue (~98% versus ~30% mortality) indicate a contribution of the plant towards defoliator-related toxicity. Observations of defoliator feeding behavior showed a preference for untreated soybean tissue relative to thiamethoxam-treated tissue, suggesting a deterrent effect of thiamethoxam. EPG monitoring of probing behavior exhibited a minimal effect of thiamethoxam on piercing-sucking herbivores. Findings from this study suggest neonicotinoids like thiamethoxam may provide some benefit via insecticidal activity against nontarget defoliators.

Pannexin1 channels regulate mechanically stimulated but not spontaneous adenosine release.

Fast-scan cyclic voltammetry (FSCV) is a rapid technique to measure neuromodulators, and using FSCV, two modes of rapid adenosine have been discovered. Spontaneous transients occur randomly in the brain, while mechanical stimulation also causes a rapid adenosine event. Pannexin1 channels are membrane channels that transport ions, including ATP, out of the cell where it is rapidly broken down into adenosine. Pannexin 1 channels (Panx1) have a flickering mode of rapid opening and are also mechanically stimulated. Here, we test the extent to which pannexin channels, specifically pannexin1 (Panx1) channels, are responsible for rapid adenosine events. Spontaneous adenosine release or mechanosensitive adenosine release were measured using fast-scan cyclic voltammetry in hippocampal (CA1) brain slices. In global Panx1KO mice, there is no significant difference in the frequency or concentration of spontaneous adenosine release, indicating Panx1 is not a release mechanism for spontaneous adenosine. Spontaneous adenosine frequency decreased slightly after administration of a large (100 µM) dose of carbenoxolone, a nonspecific inhibitor of many pannexin and connexin channels, suggesting other hemichannels only play a small role at most. For mechanically stimulated adenosine release, the concentration of each adenosine event significantly decreased 30% in Panx1KO mice and the frequency of stimulations that evoked adenosine also decreased. The response was similar in WT mice with carbenoxolone. Thus, Panx1 is a release mechanism for mechanically stimulated adenosine release, but not the only mechanism. These results demonstrate that pannexin channels differentially regulate rapid adenosine release and could be targeted to differentially affect mechanically stimulated adenosine due to brain damage.

Predator-Pest Dynamics of Arthropods Residing in Louisiana Soybean Agroecosystems.

Over the past two decades, management practices within Louisiana soybean production have shifted. Successful application of an integrated pest management (IPM) strategy requires an understanding of how these changes have affected predator-pest dynamics. Surveys monitoring foliage-foraging arthropod populations in soybean took place across six locations within Louisiana over six years (2012-2014 and 2015-2018). Temporal associations of pest groups, defoliating and piercing-sucking, and predator groups relating to soybean phenology were observed. Additionally, soybean maturity groups (III, IV, and V) were also evaluated to delineate potential differences. Results indicated higher abundances of piercing-sucking pests compared to defoliating pests across both datasets (2012-2014 and 2015-2018). Pest groups were more abundant in later soybean reproductive periods, mainly attributed to Chrysodeixis includens and Piezodorus guildinii. Predator populations were mainly comprised of Araneae and Geocoridae throughout the survey periods. From 2015 to 2018, soybean growth had a significant effect on total predator abundance with more predators present at the pod-fill and soybean maturity stage. Correlations between total pest abundance and total predators exhibited a moderate positive linear relationship. Soybean maturity groups only influenced piercing-sucking pest abundance, with later maturing groups (IV and V) having higher numbers. Thus, control tools and tactics aimed at controlling late season pests should be modified to avoid reducing predator populations.

Mechanism of Histamine Oxidation and Electropolymerization at Carbon Electrodes.

Histamine plays an important role in neuromodulation and the biological immune response. Although many electrochemical methods have been developed for histamine detection, the mechanism of its redox reaction has not been directly investigated. Here, we studied the mechanism of histamine oxidation at carbon electrodes and used that mechanistic information to design better fast-scan cyclic voltammetry (FSCV) methods for histamine. Using amperometry, cyclic voltammetry (CV), and X-ray photoelectron spectroscopy (XPS), we demonstrate that histamine oxidation requires a potential of at least +1.1 V vs Ag/AgCl. We propose that histamine undergoes one-electron oxidation on an imidazole nitrogen that produces a radical. The radical species dimerize and continue to undergo oxidation, leading to electropolymerization, which fouls the electrode. CV shows a peak at 1.3 V that is pH dependent, consistent with a one-proton, one-electron oxidation reaction. This mechanism is confirmed using 1- and 3-methylhistamine, which do not electropolymerize, compared to Nα-methylhistamine, which does. XPS also revealed a nitrogen-containing product adsorbed on the electrode surface after histamine oxidation. For FSCV detection of histamine at carbon-fiber microelectrodes, histamine oxidation was adsorption-controlled, and the anodic peak was observed at +1.2 V on the backward scan because of the rapid scan rate. However, the oxidation fouled the electrode and convoluted the FSCV temporal response; therefore, we implemented Nafion coating to alleviate the electrode fouling and preserve the time response of FSCV. Knowing the mechanism of histamine oxidation will facilitate design of better electrochemical methods for real-time monitoring of histamine.

Cavity Carbon-Nanopipette Electrodes for Dopamine Detection.

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.

Development of a novel micro biosensor for in vivo monitoring of glutamate release in the brain.

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 µm 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 ±â€¯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.

3D-Printed Carbon Electrodes for Neurotransmitter Detection.

Implantable neural microsensors have significantly advanced neuroscience research, but the geometry of most probes is limited by the fabrication methods. Therefore, new methods are needed for batch-manufacturing with high reproducibility. Herein, a novel method is developed using two-photon nanolithography followed by pyrolysis for fabrication of free-standing microelectrodes with a carbon electroactive surface. 3D-printed spherical and conical electrodes were characterized with slow scan cyclic voltammetry (CV). With fast-scan CV, the electrodes showed low dopamine LODs of 11±1 nm (sphere) and 10±2 nm (cone), high sensitivity to multiple neurochemicals, and high reproducibility. Spherical microelectrodes were used to detect dopamine in a brain slice and in vivo, demonstrating they are robust enough for tissue implantation. This work is the first demonstration of 3D-printing of free-standing carbon electrodes; and the method is promising for batch fabrication of customized, implantable neural sensors.

Regional Variations of Spontaneous, Transient Adenosine Release in Brain Slices.

Transient adenosine signaling has been recently discovered in vivo, where the concentration is on average 180 nM and the duration only 3-4 s. In order to rapidly screen different brain regions and mechanisms of formation and regulation, here we develop a rat brain slice model to study adenosine transients. The frequency, concentration, and duration of transient adenosine events were compared in the prefrontal cortex (PFC), hippocampus (CA1), and thalamus. Adenosine transients in the PFC were similar to those in vivo, with a concentration of 160 ± 10 nM, and occurred frequently, averaging one every 50 ± 5 s. In the thalamus, transients were infrequent, occurring every 280 ± 40 s, and lower concentration (110 ± 10 nM), but lasted twice as long as in the PFC. In the hippocampus, adenosine transients were less frequent than those in the PFC, occurring every 79 ± 7 s, but the average concentration (240 ± 20 nM) was significantly higher. Adenosine transients are largely maintained after applying 200 nM tetrodotoxin, implying they are not activity dependent. The response to adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) differed by region; DPCPX had no significant effects in the PFC, but increased the average transient concentration in the thalamus and both the transient frequency and concentration in the hippocampus. Thus, the amount of adenosine available to activate receptors, and the ability to upregulate adenosine signaling with DPCPX, varies by brain region. This is an important consideration for designing treatments that modulate adenosine in order to cause neuroprotective effects.

Automated Algorithm for Detection of Transient Adenosine Release.

Spontaneous adenosine release events have been discovered in the brain that last only a few seconds. The identification of these adenosine events from fast-scan cyclic voltammetry (FSCV) data is difficult due to the random nature of adenosine release. In this study, we develop an algorithm that automatically identifies and characterizes adenosine transient features, including event time, concentration, and duration. Automating the data analysis reduces analysis time from 10 to 18 h to about 40 min per experiment. The algorithm identifies adenosine based on its two oxidation peaks, the time delay between them, and their current vs time peak ratios. In order to validate the program, four data sets from three independent researchers were analyzed by the algorithm and then compared to manual identification by an analyst. The algorithm resulted in 10 ± 4% false negatives and 9 ± 3% false positives. The specificity of the algorithm was verified by comparing calibration data for adenosine triphosphate (ATP), histamine, hydrogen peroxide, and pH changes and these analytes were not identified as adenosine. Stimulated histamine release in vivo was also not identified as adenosine. The code is modular in design and could be easily adjusted to detect features of spontaneous dopamine or other neurochemical transients in FSCV data.

Clearance of rapid adenosine release is regulated by nucleoside transporters and metabolism.

Adenosine is a neuromodulator that regulates neurotransmission in the brain and central nervous system. Recently, spontaneous adenosine release that is cleared in 3-4 sec was discovered in mouse spinal cord slices and anesthetized rat brains. Here, we examined the clearance of spontaneous adenosine in the rat caudate-putamen and exogenously applied adenosine in caudate brain slices. The V max for clearance of exogenously applied adenosine in brain slices was 1.4 ± 0.1 µmol/L/sec. In vivo, the equilibrative nucleoside transport 1 (ENT1) inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI) (1 mg/kg, i.p.) significantly increased the duration of adenosine, while the ENT1/2 inhibitor, dipyridamole (10 mg/kg, i.p.), did not affect duration. 5-(3-Bromophenyl)-7-[6-(4-morpholinyl)-3-pyrido[2,3-d]byrimidin-4-amine dihydrochloride (ABT-702), an adenosine kinase inhibitor (5 mg/kg, i.p.), increased the duration of spontaneous adenosine release. The adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (10 mg/kg, i.p.), also increased the duration in vivo. Similarly, NBTI (10 µmol/L), ABT-702 (100 nmol/L), or EHNA (20 µmol/L) also decreased the clearance rate of exogenously applied adenosine in brain slices. The increases in duration for blocking ENT1, adenosine kinase, or adenosine deaminase individually were similar, about 0.4 sec in vivo; thus, the removal of adenosine on a rapid time scale occurs through three mechanisms that have comparable effects. A cocktail of ABT-702, NBTI, and EHNA significantly increased the duration by 0.7 sec, so the mechanisms are not additive and there may be additional mechanisms clearing adenosine on a rapid time scale. The presence of multiple mechanisms for adenosine clearance on a time scale of seconds demonstrates that adenosine is tightly regulated in the extracellular space.

Characterization of spontaneous, transient adenosine release in the caudate-putamen and prefrontal cortex.

Adenosine is a neuroprotective agent that inhibits neuronal activity and modulates neurotransmission. Previous research has shown adenosine gradually accumulates during pathologies such as stroke and regulates neurotransmission on the minute-to-hour time scale. Our lab developed a method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale with fast-scan cyclic voltammetry (FSCV). Recently, adenosine release lasting a couple of seconds has been found in murine spinal cord slices. In this study, we characterized spontaneous, transient adenosine release in vivo, in the caudate-putamen and prefrontal cortex of anesthetized rats. The average concentration of adenosine release was 0.17±0.01 µM in the caudate and 0.19±0.01 µM in the prefrontal cortex, although the range was large, from 0.04 to 3.2 µM. The average duration of spontaneous adenosine release was 2.9±0.1 seconds and 2.8±0.1 seconds in the caudate and prefrontal cortex, respectively. The concentration and number of transients detected do not change over a four hour period, suggesting spontaneous events are not caused by electrode implantation. The frequency of adenosine transients was higher in the prefrontal cortex than the caudate-putamen and was modulated by A1 receptors. The A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine, 6 mg/kg i.p.) increased the frequency of spontaneous adenosine release, while the A1 agonist CPA (N(6)-cyclopentyladenosine, 1 mg/kg i.p.) decreased the frequency. These findings are a paradigm shift for understanding the time course of adenosine signaling, demonstrating that there is a rapid mode of adenosine signaling that could cause transient, local neuromodulation.