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Zinc (Zn(II)) is a divalent cation involved in regulating intracellular signal transduction and gene expression through transcription factor activity, and can act as a metal neurotransmitter by modulating synaptic activity and neuronal plasticity. Previous research has demonstrated spatial heterogeneity of Zn(II) in the brain, has estimated extracellular concentrations of Zn(II) across various brain regions, and has measured rapid intracellular changes in Zn(II) concentration during glutamate flux. Despite this work, quantification of rapid extracellular Zn(II) release from neurons, on a millisecond time scale, in real time has remained difficult with existing technologies. Here, we have developed an electrochemical waveform, called the "extended sawhorse waveform (ESW)," for fast-scan cyclic voltammetry detection at carbon-fiber microelectrodes which enabled rapid and stable Zn(II) monitoring over time. This waveform was developed to overcome existing challenges in monitoring metallotransmitters stably over time electrochemically by introducing a brief cleaning step to facilitate rapid cleaning of the electrode surface in between scans. The ESW scans from 0.5 V down to -1.0 V, up to 1.45 V for 3 ms (cleaning step), and back to 0.5 V at a scan rate of 400 V/s. Repeated introductions of Zn(II) at the electrode using a traditional waveform cause plating which ultimately deteriorates the sensitivity over time; however, using the ESW, significant improvements in stability were observed. Overall, we provide a unique approach to monitor and quantitate rapid Zn(II) signaling in the brain at carbon electrodes which will impact our ability to advance fundamental knowledge of Zn(II) involvement in extracellular signaling pathways in the brain.
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Guanosine and adenosine are important neuromodulators in the brain and work in cooperation to mitigate the effects of stroke, traumatic injury, and other neurological events. Both purines can act on slow (minutes to hours) and rapid (milliseconds to seconds) time scales. A guanosine-adenosine interaction has been proposed in which guanosine modulates adenosine levels, and the two work together to control glutamate neurotransmission. Traditional methods to codetect purines, such as HPLC with microdialysis, are robust but lack the temporal resolution necessary to quantify release in real time. Fast-scan cyclic voltammetry (FSCV) has been used to detect guanosine and adenosine independently, but codetection has not been possible. Here, we developed a novel "scalene waveform" to codetect guanosine and adenosine with nanomolar limits of detection in real time with FSCV. The scalene waveform uses a slow rate (100 V/s) on the forward scan and the conventional rate (400 V/s) on the back scan; potentials go from -0.4 to 1.45 V and back to -0.4 V. The scan rates were optimized to increase the separation of the oxidative peaks for guanosine and adenosine. The temporal separation of the primary peaks was increased (4.6 ± 0.1)-fold at the scalene waveform compared to the traditional waveform. Both exogenously applied guanosine and adenosine and endogenous transient release were detected at the scalene waveform in rat-brain slices. We show the first method for codetecting guanosine and adenosine using FSCV, which can be used to study the guanosine-adenosine interaction and better understand their cooperative therapeutic effects.
Assuntos
Adenosina/análise , Encéfalo/metabolismo , Guanosina/análise , Microeletrodos , Potenciometria/métodos , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Guanosine is an important neuromodulator and neuroprotector in the brain and is involved in many pathological conditions, including ischemia and neuroinflammation. Traditional methods to detect guanosine in the brain, like HPLC, offer low limits of detection and are robust; however, subsecond detection is not possible. Here, we present a method for detecting rapid fluctuations of guanosine concentration in real-time using fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. The optimized waveform scanned from -0.4 V to 1.3 V and back at a rate of 400 V s-1 and application frequency of 10 Hz. Potential limits were chosen to increase selectivity of guanosine over the structurally similar interferent adenosine. Two oxidation peaks were detected with the optimized waveform: the primary oxidation reaction occurred at 1.3 V and the secondary oxidation at 0.8 V. Guanosine detection was stable over time with a limit of detection of 30 ± 10 nM, which permits its use to monitor low nanomolar fluctuations in the brain. To demonstrate the feasibility of the method for in-tissue detection, guanosine was exogenously applied and detected within live rat brain slices. This paper demonstrates the first characterization of guanosine using FSCV, and will be a valuable method for measuring signaling dynamics during guanosine neuromodulation and protection.
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Técnicas Eletroquímicas/métodos , Guanosina/análise , Animais , Carbono/química , Núcleo Caudado/metabolismo , Técnicas Eletroquímicas/instrumentação , Feminino , Guanosina/química , Guanosina/metabolismo , Limite de Detecção , Camundongos Endogâmicos C57BL , Microeletrodos , Monitorização Fisiológica/métodos , Oxirredução , Putamen/metabolismoRESUMO
Guanosine acts in both neuroprotective and neurosignaling pathways in the central nervous system; in this paper, we present the first fast voltammetric measurements of endogenous guanosine release during pre- and post-ischemic conditions. We discuss the metric of our measurements via analysis of event concentration, duration, and interevent time of rapid guanosine release. We observe changes across all three metrics from our normoxic to ischemic conditions. Pharmacological studies were performed to confirm that guanosine release is a calcium-dependent process and that the signaling observed is purinergic. Finally, we show the validity of our ischemic model via staining and fluorescent imaging. Overall, this paper sets the tone for rapid monitoring of guanosine and provides a platform to investigate the extent to which guanosine accumulates at the site of brain injury, i.e., ischemia.
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Lesões Encefálicas , Isquemia Encefálica , Humanos , Isquemia , Cálcio , GuanosinaRESUMO
Local stimulation of tissue can occur naturally in events like immune-mediated inflammation and focal ischemic injuries in brain and is confined to specific regions within tissue, occurring on various timescales. Making chemical measurements at the exact site of stimulation with current technologies is difficult yet important for understanding tissue response. We have developed a microfluidic device capable of local stimulation of brain slices with minimal lateral spread over time and submillimeter, tunable spatial resolution. This device is compatible with electrochemical measurements to monitor signaling at the site of stimulation over time. The PDMS-based device is three layers and contains a culture well, channel layer, and exit port layer for the channels. Channels with exit ports straddling the stimulus channels and ports were specifically fabricated to focus the stimulus over time. We demonstrated that the device is compatible with fast-scan cyclic voltammetry (FSCV) recording of neurotransmitter release. Localized hypoxia in tissue was verified using Image-iT Green Hypoxia Reagent and coupling this device with FSCV enabled measurement of local dopamine changes at the site of focal ischemia for the first time. This work provides a significant advance in knowledge of local neurochemical fluctuations during sustained tissue injury. Overall, the unique capabilities of the device to deliver sustained localized stimulation combined with real-time sensing provide an innovative platform to answer significant biological questions about how tissues respond at the site of controlled, localized injury and chemical stimulation.
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Encéfalo , Dopamina , Técnicas Eletroquímicas , Humanos , Hipóxia , Isquemia , Transmissão SinápticaRESUMO
Adenosine triphosphate (ATP) is an important rapid signaling molecule involved in a host of pathologies in the body. Historically, ATP is difficult to directly detect electrochemically with fast-scan cyclic voltammetry (FSCV) due to limited interactions at bare carbon-fibers. Systematic investigations of how ATP interacts at electrode surfaces is necessary for developing more sensitive electrochemical detection methods. Here, we have developed gold nanoparticle (AuNP), and platinum nanoparticle (PtNP) modified carbon-fiber microelectrodes coupled to FSCV to measure the extent to which ATP interacts at metal nanoparticle-modified surfaces and to improve the sensitivity of direct electrochemical detection. AuNP and PtNPs were electrodeposited on the carbon-fiber surface by scanning from -1.2 to 1.5 V for 30 s in 0.5 mg/mL HAuCl4 or 0.5 mg/mLK2PtCl6. Overall, we demonstrate an average 4.1 ± 1.0-fold increase in oxidative ATP current at AuNP-modified and a 3.5 ± 0.3-fold increase at PtNP-modified electrodes. Metal nanoparticle-modified surfaces promoted improved electrocatalytic conversion of ATP oxidation products at the surface, facilitated enhanced adsorption strength and surface coverage, and significantly improved sensitivity. ATP was successfully detected within living murine lymph node tissue following exogenous application. Overall, this study demonstrates a detailed characterization of ATP oxidation at metal nanoparticle surfaces and a significantly improved method for direct electrochemical detection of ATP in tissue.
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We present a method to modify carbon-fiber microelectrodes (CFME) with porous carbon nanofibers (PCFs) to improve detection and to investigate the impact of porous geometry for dopamine detection with fast-scan cyclic voltammetry (FSCV). PCFs were fabricated by electrospinning, carbonizing, and pyrolyzing poly(acrylonitrile)-b-poly(methyl methacrylate) (PAN-b-PMMA) block copolymer nanofiber frameworks. Commonly, porous nanofibers are used for energy storage applications, but we present an application of these materials for biosensing which has not been previously studied. This modification impacted the topology and enhanced redox cycling at the surface. PCF modifications increased the oxidative current for dopamine 2.0 ± 0.1-fold (n = 33) with significant increases in detection sensitivity. PCF are known to have more edge plane sites which we speculate lead to the two-fold increase in electroactive surface area. Capacitive current changes were negligible providing evidence that improvements in detection are due to faradaic processes at the electrode. The ΔEp for dopamine decreased significantly at modified CFMEs. Only a 2.2 ± 2.2 % change in dopamine current was observed after repeated measurements and only 10.5 ± 2.8% after 4 hours demonstrating the stability of the modification over time. We show significant improvements in norepinephrine, ascorbic acid, adenosine, serotonin, and hydrogen peroxide detection. Lastly, we demonstrate that the modified electrodes can detect endogenous, unstimulated release of dopamine in living slices of rat striatum. Overall, we provide evidence that porous nanostructures significantly improve neurochemical detection with FSCV and echo the necessity for investigating the extent to which geometry impacts electrochemical detection.
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Here, we provide evidence that functionalizing the carbon-fiber surface with amines significantly improves direct electrochemical adenosine triphosphate (ATP) detection with fast-scan cyclic voltammetry (FSCV). ATP is an important extracellular signaling molecule throughout the body and can function as a neurotransmitter in the brain. Several methods have been developed over the years to monitor and quantitate ATP signaling in cells and tissues; however, many of them are limited in temporal resolution or are not capable of measuring ATP directly. FSCV at carbon-fiber microelectrodes is a widely used technique to measure neurotransmitters in real-time. Many electrode treatments have been developed to study the interaction of cationic compounds like dopamine at the carbon surface yet studies investigating how to improve anionic compounds, like ATP, at the carbon fiber surface are lacking. In this work, carbon-fibers were treated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) which reacts with carboxylic acid groups on the carbon surface followed by reaction with ethylenediamine (EDA) to produce NH2-functionalized carbon surfaces. Overall, we a 5.2 ± 2.5-fold increase in ATP current with an approximately 9-fold increase in amine functionality, as analyzed by X-ray Photoelectron Spectroscopy, on the carbon surface was observed after modification with EDC-EDA. This provides evidence that amine-rich surfaces improve interactions with ATP on the surface. This study provides a detailed analysis of ATP interaction at carbon surfaces and ultimately a method to improve direct and rapid neurological ATP detection in the future.
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Trifosfato de Adenosina , Aminas , Carbono , Fibra de Carbono , MicroeletrodosRESUMO
Latrophilin-3 (LPHN3) is an adhesion G protein coupled receptor involved in regulating neuroplasticity. Variants of LPHN3 are associated with increased risk of attention-deficit hyperactivity disorder. Data from mouse, zebrafish, Drosophila, and rat show that disruption of LPHN3 results in hyperactivity, and in the Sprague-Dawley Lphn3 knockout rat, exhibit deficits in learning and memory and changes in dopamine (DA) markers in the neostriatum. To determine the effects of Lphn3 deletion on DA neurotransmission, we compared the concentration, duration, and frequency of DA transients in KO and wild-type rats using fast-scan cyclic voltammetry in brain slices. Lphn3 KO rats showed higher release of DA, and the duration and interevent time were markedly decreased compared with wild-type rats. The data demonstrate that LPHN3 plays a heretofore unrecognized role in DA signaling and may represent a new target for small molecule regulation of DA neurotransmission with translational implications.