PROBING NEUROCHEMISTRY WITH FAST-SCAN CYCLIC VOLTAMMETRY

Fast-scan cyclic voltammetry (FSCV) with carbon-fiber microelectrodes is a prominent analytical technique for rapid and sensitive detection of electrochemically active analytes in mammalian brain. In recent years this technique became very popular among neuroscientists. However, many improvements for FSCV are possible. Chapter 1 introduces the technique and provides brief description of recent improvements in fast-scan cyclic voltammetry. During voltammetric experiments, potential applied to electrodes changes carbon surface. Chapter 2 describes investigation of the changes induced by waveforms with anodic potential limits of 1.0 V, 1.3 V and 1.4 V. Instrumental methods of analysis such as XPS, AFM and SEM together with electrochemical studies were used. It was observed, that for waveforms with high anodic potential (1.3 V and 1.4 V) carbon electrode surface continuously oxidizes and etches away. Thus, the electrode surface which has surface groups that promote adsorption of catechols is constantly renewed. A benefit of surface renewal is sustainability to chemical fouling. Carbon electrode surface has electrochemically active chemical groups which are oxidized and reduced during voltammetric potential ramps. Electrochemical reactions for these groups involve protons, thus changes in pH of solution generate characteristic cyclic voltammograms. Hence, FSCV can be used to sample rapid pH fluctuations in the brain that are associated with metabolism and changes in the cerebral blood flow. However, cyclic voltammograms for pH changes recorded in brain in vivo and in the flow cell have different shapes, which compromises the identity of pH signal. Chapter 3 describes investigation of the peaks in cyclic voltammogram for pH which led to the conclusion that adsorption of electrochemically inert species to electrode surface is responsible for the interference and the mismatch. Identity of pH signal in brain in vivo was confirmed by inducing acidosis by increasing concentration of carbon dioxide in breathing mixture (hypercapnia). Acidic pH shift with characteristic cyclic voltammogram was recorded with FSCV in hypercapnia. Traditionally, FSCV experiments are done with a single carbon-fiber microelectrode. These electrodes are produced manually and they are very fragile. Chapter 4 describes alternative microfabricated microelectrode arrays (MEAs) which are more robust than glass-encased carbon fibers and can be produced using batch fabrication methods. Microelectrodes in MEAs are distant from each other, thus heterogeneity in analyte concentration such as difference in dopamine release in the brain can be studied. Also, microelectrodes in the array are independently addressable which means that multiplexed detection of different analytes in brain can be performed simultaneously. Instrumentation for FSCV experiments in freely moving animals is custom made which limits the dissemination of this technique. Chapter 5 describes instrumentation for FSCV experiments for combined electrochemical and electrophysiological measurements in details. All electronic components are documented and layouts of electronic circuits are provided in this chapter. Recording of brain functions with multiple electrodes is beneficial in providing information about interconnections of different brain regions as well as synchronization of their activity. This approach was limited to electrophysiological recordings. Chapter 6 describes recordings of endogenous and pharmacologically induced activity of dopaminergic neurons in separate brain hemispheres of anesthetized rat. Synchronization of activity of dopaminergic neurons between two separate and symmetrical systems is observed. Possible link to slow wave oscillations that occur in brain cortex during sleep is discussed.Doctor of Philosoph

In vivo comparison of norepinephrine and dopamine release in rat brain by simultaneous measurements with fast-scan cyclic voltammetry: Comparison of norepinephrine and dopamine release

Brain norepinephrine and dopamine regulate a variety of critical behaviors such as stress, learning, memory, and drug addiction. Here, we demonstrate differences in the regulation of in vivo neurotransmission for dopamine in the anterior nucleus accumbens (NAc) and norepinephrine in the ventral bed nucleus of the stria terminalis (vBNST) of the anesthetized rat. Release of the two catecholamines was measured simultaneously using fast-scan cyclic voltammetry (FSCV) at two different carbon-fiber microelectrodes, each implanted in the brain region of interest. Simultaneous dopamine and norepinephrine release was evoked by electrical stimulation of a region where the ventral noradrenergic bundle (VNB), the pathway of noradrenergic neurons, courses through the ventral tegmental area/substantia nigra (VTA/SN), the origin of dopaminergic cell bodies. The release and uptake of norepinephrine in the vBNST were both significantly slower than for dopamine in the NAc. Pharmacological manipulations in the same animal demonstrated that the two catecholamines are differently regulated. The combination of a dopamine autoreceptor antagonist and amphetamine significantly increased basal extracellular dopamine whereas a norepinephrine autoreceptor antagonist and amphetamine did not change basal norepinephrine concentration. α-Methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, decreased electrically evoked dopamine release faster than norepinephrine. The dual-microelectrode FSCV technique along with anatomical and pharmacological evidence confirms that dopamine in the NAc and norepinephrine in the vBNST can be monitored selectively and simultaneously in the same animal. The high temporal and spatial resolution of the technique enabled us to examine differences in the dynamics of extracellular norepinephrine and dopamine concurrently in two different limbic structures

Simultaneous Decoupled Detection of Dopamine and Oxygen Using Pyrolyzed Carbon Microarrays and Fast-Scan Cyclic Voltammetry

Microfabricated structures utilizing pyrolyzed photoresist have been shown to be a useful for monitoring electrochemical processes. These previous studies, however, were limited to constant potential measurements and slow scan voltammetry. Work described in this report utilizes microfabrication processes to produce devices that enable multiple fast-scan cyclic voltammetry (FSCV) waveforms to be applied to different electrodes on a single substrate. This enabled the simultaneous, decoupled, detection of dopamine and oxygen. This paper describes the fabrication process of these arrays and shows that pyrolyzed photoresist electrodes possess comparable surface chemistry and electrochemical properties to PAN type, T-650, carbon fiber microelectrodes using background-subtracted FSCV. The functionality of the array is discussed in terms of the degree of crosstalk in response to flow injections of physiologically relevant concentrations of dopamine and oxygen. Finally, other applications of pyrolyzed photoresist microelectrode arrays are shown, including: spatially resolved detection of analytes and combining FSCV with amperometry for the detection of dopamine

Microfabricated FSCV-compatible microelectrode array for real-time monitoring of heterogeneous dopamine release

Fast scan cyclic voltammetry (FSCV) has been used previously to detect neurotransmitter release and reuptake in vivo. An advantage that FSCV has over other electrochemical techniques is its ability to distinguish neurotransmitters of interest (i.e. monoamines) from their metabolites using their respective characteristic cyclic voltammogram. While much has been learned with this technique, it has generally only been used in a single working electrode arrangement. Additionally, traditional electrode fabrication techniques tend to be difficult and somewhat irreproducible. Described in this report is a fabrication method for a FSCV compatible microelectrode array (FSCV-MEA) that is capable of functioning in vivo. The microfabrication techniques employed here allow for better reproducibility than traditional fabrication methods of carbon fiber microelectrodes, and enable batch fabrication of electrode arrays. The reproducibility and electrochemical qualities of the probes were assessed along with cross talk in vitro. Heterogeneous release of electrically stimulated dopamine was observed in real-time in the striatum of an anesthetized rat using the FSCV-MEA. The heterogeneous effects of pharmacology on the striatum was also observed and shown to be consistent across multiple animals

Cross-hemispheric dopamine projections have functional significance

Decades of research have described dopamine’s importance in reward-seeking behavior and motor control. Although numerous investigations have focused on dopamine’s mechanisms in modulating behavior, the long-standing belief that dopamine neurons project solely unilaterally has limited the exploration of interhemispheric dopamine signaling. Here we resolve disparate descriptions of unilateral vs. bilateral projections by reporting that dopamine neurons can release dopamine in the contralateral hemisphere. Using voltammetry in awake and anesthetized rats, we reveal an unprecedented synchrony of dopamine fluctuations between hemispheres. Via stimulation with amphetamine, we demonstrate functional cross-hemispheric projections in a hemiparkinsonian model. This previously undescribed capacity for interhemispheric dopamine signaling can precipitate new areas of inquiry. Future work may exploit properties of bilateral dopamine release to improve treatments for Parkinson’s disease, including deep brain stimulation

Simultaneous monitoring of dopamine concentration at spatially different brain locations in vivo

When coupled with a microelectrode, background-subtracted fast scan cyclic voltammetry (FSCV) allows fast, sensitive and selective determination of analytes within a small spatial location. For the past 30 years experiments using this technique have been largely confined to recordings at a single microelectrode. Arrays with closely separated microelectrodes would allow researchers to gain more informative data as well as probe regions in close spatial proximity. This work presents one of the first FSCV microelectrode arrays (MEA) implemented in vivo with the ability to sample from different regions in close spatial proximity (equidistant within 1 mm). The array is manufactured from fused silica capillaries and a microfabricated electrode spacer. The functionality of the array is assessed by simultaneously monitoring electrically stimulated dopamine (DA) release in the striatum of anesthetized rat. As expected, heterogeneous dopamine release was simultaneously observed. Additionally, the pharmacological effect of raclopride (D2 receptor antagonist) and cocaine (monoamine uptake blocker) on the heterogeneity of DA release, in spatially different brain regions was shown to alter neurotransmitter release at all four electrode sites

Characterization of Local pH Changes in Brain Using Fast-Scan Cyclic Voltammetry with Carbon Microelectrodes

Transient local pH changes in the brain are important markers of neural activity that can be used to follow metabolic processes that underlie the biological basis of behavior, learning and memory. There are few methods that can measure pH fluctuations with sufficient time resolution in freely moving animals. Previously, fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for the measurement of such pH transients. However, the origin of the potential dependent current in the cyclic voltammograms for pH changes recorded in vivo was unclear. The current work explored the nature of these peaks and established the origin for some of them. A peak relating to the capacitive nature of the pH CV was identified. Adsorption of electrochemically inert species, such as aromatic amines and calcium could suppress this peak, and is the origin for inconsistencies regarding in vivo and in vitro data. Also, we identified an extra peak in the in vivo pH CV relating to the presence of 3,4-dihydroxyacetic acid (DOPAC) in the brain extracellular fluid. To evaluate the in vivo performance of the carbon-fiber sensor, carbon dioxide inhalation by an anesthetized rat was used to induce brain acidosis induced by hypercapnia. Hypercapnia is demonstrated to be a useful tool to induce robust in vivo pH changes, allowing confirmation of the pH signal observed with FSCV

Chronically Implanted, Nafion-Coated Ag/AgCl Reference Electrodes for Neurochemical Applications

Fast-scan cyclic voltammetry at carbon-fiber microelectrodes can be used to measure behaviorally correlated dopamine changes in the extracellular fluid of the brain of freely moving rats. These experiments employ a chronically implanted Ag/AgCl reference electrode. When dopamine measurements are taken 4 days after implantation, there is often a potential shift, typically greater than +0.2 V, in the anodic and cathodic peaks in the cyclic voltammogram for dopamine. In this work, we optimized a method to coat sintered Ag/AgCl reference electrodes with the perfluorinated polymer, Nafion, to prevent this shift. We find that we can stabilize reference electrodes for up to 28 days. Immunohistochemistry of the tissue around the implant site shows extensive glial encapsulation around both bare and Nafion-coated devices. However, the lesion around bare electrodes has a rough texture implying that these cells are strongly adsorbed onto the bare reference electrode, while the lesion around a Nafion-coated electrode shows that cells are more intact implying that they adsorb less strongly. Energy dispersive X-ray spectroscopy and scanning electron microscopy analysis of the surface of the electrodes confirms this by visualizing a heavy buildup of plaques, organic in nature, only on bare electrodes. Impedance spectroscopy indicates no difference between the impedance of bare and Nafion-coated Ag/AgCl electrodes, indicating that glial encapsulation does not lead to an increase in uncompensated resistance between the working and reference electrodes. The electrochemical shift therefore must be due to the unique chemical microenvironment around the reference electrode that alters the chloride equilibrium, a process that the Nafion coating prevents

Higher Sensitivity Dopamine Measurements with Faster-Scan Cyclic Voltammetry

Fast-scan cyclic voltammetry with carbon-fiber microelectrodes has been successfully used to detect catecholamine release in vivo. Generally, waveforms with anodic voltage limits of 1.0 V or 1.3 V (vs. Ag/AgCl) are used for detection. The 1.0 V excursion provides good temporal resolution, but suffers from a lack of sensitivity. The 1.3 V excursion increases sensitivity, but also increases response time which can blur the detection of neurochemical events. Here, the scan rate was increased to improve the sensitivity of the 1.0 V excursion while maintaining the rapid temporal response. However, increasing scan rate increases both the desired faradaic current response and the already large charging current associated with the voltage sweep. Analog background subtraction was used to prevent the analog-to-digital converter from saturating from the high currents generated with increasing scan rate by neutralizing some of the charging current. In vitro results with the 1.0 V waveform showed approximately a four-fold increase in signal to noise ratio with maintenance of the desired faster response time by increasing scan rate up to 2400 V/s. In vivo, stable stimulated release was detected with an approximate four-fold increase in peak current. The scan rate of the 1.3 V waveform was also increased, but the signal was unstable with time in vitro and in vivo. Adapting the 1.3 V triangular wave into a sawhorse design prevented signal decay and increased the faradaic response. The use of the 1.3 V sawhorse waveform decreased the detection limit of dopamine with FSCV to 0.96 ± 0.08 nM in vitro and showed improved performance in vivo without affecting the neuronal environment. Electron microscopy showed dopamine sensitivity is in a quasi-steady state with carbon-fiber microelectrodes scanned to potentials above 1.0 V

Carbon Microelectrodes with a Renewable Surface

Electrode fouling decreases sensitivity and can be a substantial limitation in electrochemical experiments. In this work we describe an electrochemical procedure that constantly renews the surface of a carbon microelectrode using periodic triangle voltage excursions to an extended anodic potential at a scan rate of 400 Vs−1. This methodology allows for the regeneration of an electrochemically active surface and restores electrode sensitivity degraded by irreversible adsorption of chemical species. We show that repeated voltammetric sweeps to moderate potentials in aqueous solution causes oxidative etching of carbon thereby constantly renewing the electrochemically active surface. Oxidative etching was established by tracking surface-localized fluorine atoms with XPS, by monitoring changes in carbon surface morphology with AFM on pyrolyzed photoresist films, and also by optical and electron microscopy. The use of waveforms with extended anodic potentials showed substantial increases in sensitivity towards the detection of catechols. This enhancement arose from the adsorption of the catechol moiety that could be maintained with a constant regeneration of the electrode surface. We also demonstrate that application of the extended waveform could restore the sensitivity of carbon microelectrodes diminished by irreversible adsorption (electrode fouling) of byproducts resulting from the electrooxidation and polymerization of tyramine. Overall, this work brings new insight into the factors that affect electrochemical processes at carbon electrodes and provides a simple method to remove or reduce fouling problems associated with many electrochemical experiments