This paper reports the fabrication of an insertable amperometric dual microsensor and its application for the simultaneous and fast sensing of NO and CO during acutely induced seizures of living rat brain cortex. NO and CO are important signaling mediators, controlling cerebrovascular tone. The dual NO/CO sensor is prepared based on a dual microelectrode having Au-deposited Pt microdisk (WE1, 76 μm diameter) and Pt black-deposited Pt disk (WE2, 50 μm diameter). The different deposited metals for WE1 and WE2 allow the selective anodic detection of CO at WE1 (+0.2 V vs Ag/AgCl) and that of NO at WE2 (+0.75 V vs Ag/AgCl) with sufficient sensitivity. Fluorinated xerogel coating on this dual electrode provides exclusive selectivity over common biological interferents, along with fast response time. The miniaturized size (end plane diameter < 300 μm) and tapered needle-like sensor geometry make the sensor become insertable into biological tissues. The sensor is applied to simultaneously monitor dynamic changes of NO and CO levels in a living rat brain under acute seizure condition induced by 4-aminopyridine in cortical tissue near the area of seizure induction. In-tissue measurement shows clearly defined patterns of NO/CO changes, directly correlated with observed LFP signal. Current study verifies the feasibility of a newly developed NO/CO dual sensor for real-time fast monitoring of intimately connected NO and CO dynamics.

Monoamine oxidase (MAO) is believed to mediate the degradation of monoamine neurotransmitters, including dopamine, in the brain. Between the two types of MAO, MAO-B has been believed to be involved in dopamine degradation, which supports the idea that the therapeutic efficacy of MAO-B inhibitors in Parkinson’s disease can be attributed to an increase in extracellular dopamine concentration. However, this belief has been controversial. Here, by utilizing in vivo phasic and basal electrochemical monitoring of extracellular dopamine with fast-scan cyclic voltammetry and multiple-cyclic square wave voltammetry and ex vivo fluorescence imaging of dopamine with GRABDA2m, we demonstrate that MAO-A, but not MAO-B, mainly contributes to striatal dopamine degradation. In contrast, our whole-cell patch-clamp results demonstrated that MAO-B, but not MAO-A, was responsible for astrocytic GABA-mediated tonic inhibitory currents in the rat striatum. We conclude that, in contrast to the traditional belief, MAO-A and MAO-B have profoundly different roles: MAO-A regulates dopamine levels, whereas MAO-B controls tonic GABA levels.

In this work, we developed a dual amperometric/potentiometric microsensor for sensing nitric oxide (NO) and potassium ion (K(+)). The dual NO/K(+) sensor was prepared based on a dual recessed electrode possessing Pt (diameter, 50 μm) and Ag (diameter, 76.2 μm) microdisks. The Pt disk surface (WE1) was modified with electroplatinization and the following coating with fluorinated xerogel; and the Ag disk surface (WE2) was oxidized to AgCl on which K(+) ion selective membrane was loaded subsequent to the silanization. WE1 and WE2 of a dual microsensor were used for amperometric sensing of NO (106 ± 28 pA μM(-1), n = 10, at +0.85 V applied vs Ag/AgCl) and for potentiometric sensing of K(+) (51.6 ± 1.9 mV pK(-1), n = 10), respectively, with high sensitivity. In addition, the sensor showed good selectivity over common biological interferents, sufficiently fast response time and relevant stability (within 6 h in vivo experiment). The sensor had a small dimension (end plane diameter, 428 ± 97 μm, n = 20) and needle-like sharp geometry which allowed the sensor to be inserted in biological tissues. Taking advantage of this insertability, the sensor was applied for the simultaneous monitoring of NO and K(+) changes in a living rat brain cortex at a depth of 1.19 ± 0.039 mm and near the spontaneous epileptic seizure focus. The seizures were induced with 4-aminopyridine injection onto the rat brain cortex. NO and K(+) levels were dynamically changed in clear correlation with the electrophysiological recording of seizures. This indicates that the dual NO/K(+) sensor's measurements well reflect membrane potential changes of neurons and associated cellular components of neurovascular coupling. The newly developed NO/K(+) dual microsensor showed the feasibility of real-time fast monitoring of dynamic changes of closely linked NO and K(+) in vivo.

Although fast-scan cyclic voltammetry (FSCV) has been widely used for in vivo neurochemical detection, the sensitivity and selectivity of the technique can be further improved. In this study, we develop fast cyclic square-wave voltammetry (FCSWV) as a novel voltammetric technique that combines large-amplitude cyclic square-wave voltammetry (CSWV) with background subtraction. A large-amplitude, square-shaped potential was applied to induce cycling through multiple redox reactions within a square pulse to increase sensitivity and selectivity when combined with a twodimensional voltammogram. As a result, FCSWV was significantly more sensitive than FSCV (n = 5 electrodes, two-way ANOVA, p = 0.0002). In addition, FCSWV could differentiate dopamine from other catecholamines (e.g., epinephrine and norepinephrine) and serotonin better than conventional FSCV. With the confirmation that FCSWV did not influence local neuronal activity, despite the large amplitude of the square waveform, it could monitor electrically induced phasic changes in dopamine release in rat striatum before and after injecting nomifensine, a dopamine reuptake inhibitor.

This study reports real-time, in vivo functional measurement of nitric oxide (NO) and carbon monoxide (CO), two gaseous mediators in controlling cerebral blood flow. A dual electrochemical NO/CO microsensor enables us to probe the complex relationship between NO and CO in regulating cerebrovascular tone. Utilizing this dual sensor, we monitor in vivo change of NO and CO simultaneously during direct epidural electrical stimulation of a living rat brain cortex. Both NO and CO respond quickly to meet physiological needs. The neural system instantaneously increases the released amounts of NO and CO to compensate the abrupt, yet transient hypoxia that results from epidural electrical stimulation. Intrinsic-signal optical imaging confirms that direct electrical stimulation elicits robust, dynamic changes in cerebral blood flow, which must accompany NO and CO signaling. The addition of l-arginine (a substrate for NO synthase, NOS) results in increased NO generation and decreased CO production compared to control stimulation. On the other hand, application of the NOS inhibitor, l-N(G)-nitroarginine methyl ester (l-NAME), results in decreased NO release but increased CO production of greater magnitude. This observation suggests that the interaction between NO and CO release is likely not linear and yet, they are tightly linked vasodilators.