Electrochemical measurements of neurotransmitters provide insight into the dynamics of neurotransmission. In this review, we describe the development of electrochemical measurements of neurotransmitters and how they started with extrasynaptic measurements but now are pushing toward synaptic measurements. Traditionally, biosensors or fast-scan cyclic voltammetry have monitored extrasynaptic levels of neurotransmitters, such as dopamine, serotonin, adenosine, glutamate, and acetylcholine. Amperometry and electrochemical cytometry techniques have revealed mechanisms of exocytosis, suggesting partial release. Advances in nanoelectrodes now allow spatially resolved, electrochemical measurements in a synapse, which is only 20–100 nm wide. Synaptic measurements of dopamine and acetylcholine have been made. In this article, electrochemical measurements are also compared to optical imaging and mass spectrometry measurements, and while these other techniques provide enhanced spatial or chemical information, electrochemistry is best at monitoring real-time neurotransmission. Future challenges include combining electrochemistry with these other techniques in order to facilitate multisite and multianalyte monitoring.

Adenosine is a ubiquitous neuromodulator that plays a role in sleep, vasodilation, and immune response and manipulating the adenosine system could be therapeutic for Parkinson's disease or ischemic stroke. Spontaneous transient adenosine release provides rapid neuromodulation; however, little is known about the effect of sex as a biological variable on adenosine signaling and this is vital information for designing therapeutics. Here, we investigate sex differences in spontaneous, transient adenosine release using fast‐scan cyclic voltammetry to measure adenosine in vivo in the hippocampus CA1, basolateral amygdala, and prefrontal cortex. The frequency and concentration of transient adenosine release were compared by sex and brain region, and in females, the stage of estrous. Females had larger concentration transients in the hippocampus (0.161 ± 0.003 µM) and the amygdala (0.182 ± 0.006 µM) than males (hippocampus: 0.134 ± 0.003, amygdala: 0.115 ± 0.002 µM), but the males had a higher frequency of events. In the prefrontal cortex, the trends were reversed. Males had higher concentrations (0.189 ± 0.003 µM) than females (0.170 ± 0.002 µM), but females had higher frequencies. Examining stages of the estrous cycle, in the hippocampus, adenosine transients are higher concentration during proestrus and diestrus. In the cortex, adenosine transients were higher in concentration during proestrus, but were lower during all other stages. Thus, sex and estrous cycle differences in spontaneous adenosine are complex, and not completely consistent from region to region. Understanding these complex differences in spontaneous adenosine between the sexes and during different stages of estrous is important for designing effective treatments manipulating adenosine as a neuromodulator.

Fast-scan cyclic voltammetry (FSCV) is widely used for in vivo detection of neurotransmitters, but identifying analytes, particularly mixtures, is difficult. Data analysis has focused on identifying dopamine from cyclic voltammograms, but it would be better to analyze all the data in the three-dimensional FSCV color plot. Here, the goal was to use image analysis-based analysis of FSCV color plots for the first time, specifically the structural similarity index (SSIM), to identify rapid neurochemical events. Initially, we focused on identifying spontaneous adenosine events, as adenosine cyclic voltammograms have a primary oxidation at 1.3 V and a secondary oxidation peak that grows in over time. Using SSIM, sample FSCV color plots were compared with reference color plots, and the SSIM cutoff score was optimized to distinguish adenosine. High-pass digital filtering was also applied to remove the background drift and lower the noise, which produced a better LOD. The SSIM algorithm detected more adenosine events than a previous algorithm based on current versus time traces, with 99.5 ± 0.6% precision, 95 ± 3% recall, and 97 ± 2% F1 score (n = 15 experiments from three researchers). For selectivity, it successfully rejected signals from pH changes, histamine, and H2O2. To prove it is a broad strategy useful beyond adenosine, SSIM analysis was optimized for dopamine detection and is able to detect simultaneous events with dopamine and adenosine. Thus, SSIM is a general strategy for FSCV data analysis that uses three-dimensional data to detect multiple analytes in an efficient, automated analysis.

Transient changes in adenosine and dopamine have been measured in vivo, but no studies have examined if these transient changes occur simultaneously. In this study, we characterized spontaneous adenosine and dopamine transients in anesthetized mice, examining coincident release in the caudateputamen for the first time. We found that in C57B mice, most of the dopamine transients (77%) were coincident with adenosine, but fewer adenosine transients (12%) were coincident with a dopamine transient. On average, the dopamine transient started 200 ms before its coincident adenosine transient, so they occurred concurrently. There was a positive correlation (r = 0.7292) of adenosine and dopamine concentrations during coincident release. ATP is quickly broken down to adenosine in the extracellular space, and the coincident events may be due to corelease, where dopaminergic vesicles are packaged with ATP, or cotransmission, where ATP is packaged in different vesicles released simultaneously with dopamine. The high frequency of adenosine transients compared to that of dopamine transients suggests that adenosine is also released from nondopaminergic vesicles. We investigated how A 1 and A 2A adenosine receptors regulate adenosine and dopamine transients using A 1 and A 2A KO mice. In A 1 KO mice, the frequency of adenosine and dopamine transients increased, while in A 2A KO mice, the frequency of adenosine alone increased. Adenosine receptors modulate coincident transients and could be drug targets to modulate both dopamine and adenosine release. Many spontaneous dopamine transients have coincident adenosine release, and regulating adenosine and dopamine cotransmission could be important for designing treatments for dopamine diseases, such as Parkinson's or addiction.