Modified Sawhorse Waveform for the Voltammetric Detection of Oxytocin

Carbon fiber microelectrodes (CFMEs) have been used to detect neurotransmitters and other biomolecules using fast-scan cyclic voltammetry (FSCV) for the past few decades. This technique measures neurotransmitters such as dopamine and, more recently, physiologically relevant neuropeptides. Oxytocin, a pleiotropic peptide hormone, is physiologically important for adaptation, development, reproduction, and social behavior. This neuropeptide functions as a stress-coping molecule, an anti-inflammatory agent, and serves as an antioxidant with protective effects especially during adversity or trauma. Here, we measure tyrosine using the Modified Sawhorse Waveform (MSW), enabling enhanced electrode sensitivity for the amino acid and oxytocin peptide. Applying the MSW, decreased surface fouling and enabled codetection with other monoamines. As oxytocin contains tyrosine, the MSW was also used to detect oxytocin. The sensitivity of oxytocin detection was found to be 3.99 ± 0.49 nA/µM, (n=5). Additionally, we demonstrate that applying the MSW on CFMEs allows for real time measurements of exogenously applied oxytocin on rat brain slices. These studies may serve as novel assays for oxytocin detection in a fast, sub-second timescale with possible implications for in vivo measurements and further understanding of the physiological role of oxytocin.

Sub-second striatal dopamine dynamics assessed by simultaneous fast-scan cyclic voltammetry and fluorescence biosensor

Fast-scan cyclic voltammetry (FSCV) is an electrochemical method used to detect dopamine on a subsecond time scale. Recordings using FSCV in freely behaving animals revolutionized the study of behaviors associated with motivation and learning. Despite this advance, FSCV cannot distinguish between catecholamines, which limits its use to brain regions where dopamine is the predominant neurotransmitter. It has also been difficult to detect dopamine in vivo in some striatal subregions with FSCV. Recently, fluorescent biosensors for dopamine were developed, allowing for discrimination between catecholamines. However, the performance of these biosensors relative to FSCV has not been determined. Thus, we compared fluorescent photometry responses of the dopamine biosensor, dLight, with FSCV. We also used dLight photometry to assess changes in tonic and phasic dopamine, which has not been possible with FSCV. Finally, we examined dopamine dynamics during Pavlovian conditioning in striatal subregions, including the dorsolateral striatum where dopamine measurements are challenging with FSCV.

Terminal Deoxynucleotidyl Transferase Extension-Dominated In Situ Signal Attenuation-Free Electrochemical Platform and Its Logic Gate Manipulation

Signal generation of traditional electrochemical biosensors suffers from the random diffusion of electroactive probes in a electrolyte solution, which is accompanied by poor reaction kinetics and low signal stability from complex biological systems. Herein, a novel circuit system with autonomous compensation solution ohmic drop (noted as “fast-scan cyclic voltammetry (FSCV)”) is developed to solve the above problems, and employed to achieve terminal deoxynucleotide transferase (TdT) and its small molecule inhibitor analysis. At first, a typical TdT-mediated catalytic polymerization in the conditions of original DNA, deoxythymine triphosphate (dTTP) and Hg2+ is applied for the electrode assembly. The novel electrochemical method can provide some unattenuated signals due to in-situ Hg redox reaction, thus improving reaction kinetics and signal stability. This approach is mainly dependent on TdT-mediated reaction, so it can be applied properly for TdT investigation, and a detection limit of 0.067 U/mL (S/N=3) is achieved successfully. More interesting, we also mimic the function of TdT-related signal communication in various logic gates such as YES, NOT, AND, N-IMPLY, and AND-AND-N-IMPLY cascade circuit. This study provides a new method for the detection of TdT biomarkers in many types of diseases and the construction of a signal attenuation-free logic gate.

Cocaine increases stimulation-evoked serotonin efflux in the nucleus accumbens

Cocaine is one of the most common illicit drugs globally, but the role of serotonin in its mechanism of action is insufficiently characterised. Consequently, we investigated the acute effects of the psychomotor stimulant cocaine on electrical stimulation-evoked serotonin (phasic) release in the nucleus accumbens core (NAcc) of urethane-anesthetized (1.5 g/kg i.p.) male Sprague-Dawley rats using N-shaped fast-scan cyclic voltammetry (N-FSCV). A single carbon fiber microelectrode was first implanted in the NAcc. Stimulation was applied to the medial forebrain bundle using 60 Hz, 2 ms, 0.2 mA, 2 s biphasic pulses before and after cocaine (2 mg/kg i.v.) was administered. Stimulation-evoked serotonin release significantly increased 5 minutes after cocaine injection compared to baseline (153±21 nM vs 257±12 nM; p = 0.0042; n = 5) but was unaffected by saline injection (1 ml/kg i.v.; n = 5). N-FSCV's selective measurement of serotonin release in vivo was confirmed pharmacologically via administration of the selective serotonin reuptake inhibitor escitalopram (10 mg/kg i.p.) which effectively increased the signal in a separate group of rats (n = 5). Selectivity to serotonin was further confirmed in vitro in which dopamine was minimally detected by N-FSCV with a serotonin to dopamine response ratio of 1:0.04 (200 nM of serotonin:1 mM dopamine ratio; p = 0.0048; n = 5 electrodes). This study demonstrates a noteworthy influence of cocaine on serotonin dynamics, and confirms that N-FSCV can effectively and selectively measure phasic serotonin release in the NAcc.

Effectiveness and relationship between biased and unbiased measures of dopamine release and clearance

Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine (DA) release and clearance throughout the brain, including the ventral and dorsal striatum. Striatal DA terminals are abundant with signals heavily regulated by release machinery and the dopamine transporter (DAT). Peak height is a common method for measuring release but can be affected by changes in clearance. The Michaelis-Menten model has been a standard in measuring DA clearance, but requires experimenter fitted modeling subject to experimenter bias. The current study presents the use of the first derivative (velocity) of evoked DA signals as an alternative approach for measuring dopamine release and clearance and can be used to distinguish the two measures. Maximal upwards velocity predicts reductions in DA peak height due to D2 and GABAB receptor stimulation and by alterations in calcium concentrations. The Michaelis-Menten maximal velocity (Vmax) measure, an approximation for DAT numbers, predicted maximal downward velocity in slices and in vivo. Dopamine peak height and upward velocity were similar between wildtype C57 (WT) and DAT knock out (DATKO) mice. In contrast, downward velocity was considerably reduced and exponential decay (tau) was increased in DATKO mice, supporting use of both measures for changes in DAT activity. In slices, the competitive DAT inhibitors cocaine, PTT and WF23 increased peak height and upward velocity differentially across increasing concentrations, with PTT and cocaine reducing these measures at high concentrations. Downward velocity and tau values decreased and increased respectively across concentrations, with greater potency and efficacy observed with WF23 and PTT. In vivo recordings demonstrated similar effects of WF23 and PTT on measures of release and clearance. Tau was a more sensitive measure at low concentrations, supporting its use as a surrogate for the Michaelis-Menten measure of apparent affinity (Km). Together, these results inform on the use of these measures for DA release and clearance.

Characterization of Electroactive Amino Acids with Fast-Scan Cyclic Voltammetry

Small molecules and signaling peptides are extensively involved in controlling basic brain function. While classical neurotransmitters can be detected with a variety of techniques, methods for measurement of rapidly-released neuropeptides remain underdeveloped. Fast-scan cyclic voltammetry (FSCV) is an electrochemical technique often used for subsecond detection of small molecule neurotransmitters, in vivo. A few peptides have been detected with FSCV; however, a detailed analysis of the electrochemical signature of all electroactive amino acids with FSCV has not been fully investigated. Because the mechanisms, locations, and timescales for signaling peptide release in the brain are relatively unexplored, developing sensitive and selective tools capable of quantitating neuropeptide signaling is essential. To bridge this gap, we used FSCV to characterize the electroactive amino acids: cysteine, methionine, histidine, tryptophan, and tyrosine. We show that tyrosine, tryptophan, and histidine are easily oxidized on carbon fiber surfaces with FSCV, while detection of the sulfur-containing amino acids is more difficult. This study provides critical information for electrochemical waveform design and optimization for detection of peptides containing these amino acids.

Defining a Path Toward the Use of Fast-Scan Cyclic Voltammetry in Human Studies

Fast Scan Cyclic Voltammetry (FSCV) has been used for decades as a neurochemical tool for in vivo detection of phasic changes in electroactive neurotransmitters in animal models. Recently, multiple research groups have initiated human neurochemical studies using FSCV or demonstrated interest in bringing FSCV into clinical use. However, there remain technical challenges that limit clinical implementation of FSCV by creating barriers to appropriate scientific rigor and patient safety. In order to progress with clinical FSCV, these limitations must be first addressed through (1) appropriate pre-clinical studies to ensure accurate measurement of neurotransmitters and (2) the application of a risk management framework to assess patient safety. The intent of this work is to bring awareness of the current issues associated with FSCV to the scientific, engineering, and clinical communities and encourage them to seek solutions or alternatives that ensure data accuracy, rigor and reproducibility, and patient safety.