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Michael AC, Borland LM, editors. Electrochemical Methods for Neuroscience. Boca Raton (FL): CRC Press/Taylor & Francis; 2007.

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Electrochemical Methods for Neuroscience.

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Chapter 4Fast Scan Cyclic Voltammetry of Dopamine and Serotonin in Mouse Brain Slices

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Introduction

Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that can be used to monitor release and uptake dynamics of endogenous monoamine levels both in vitro [1–3] and in vivo [4–6]. Fast scan cyclic voltammetry is able to measure the three major monoamine neurotransmitters, dopamine (DA), serotonin (5-HT), and norepinephrine (NE), because these substances can be oxidized at low voltages, providing selective electrochemical detection based on voltage-dependent oxidation and reduction processes.

Briefly, a voltage that is sufficient to oxidize monoamines is applied to a carbon fiber microelectrode. The oxidation process results in current flow at the electrode surface; the amount of current is converted into concentration of monoamine in the vicinity of the electrode tip by means of calibration in a flow injection system. The current is measured rapidly, every 100 ms, and when plotted against time, can provide information as to how, for example, stimulation parameters and pharmacological agents can alter the dynamics of the monoamine released near the surface of the electrode. Ultimately, these measurements make it possible to distinguish kinetic parameters for release and uptake processes, a distinguishing feature of electrochemical techniques like FSCV. This chapter is designed to describe the use of FSCV to measure DA and 5-HT in mouse brain slices.

Advantages of the Use of Mouse Brain Slices

Guinea pigs and rats have been most commonly used to explore monoamine dynamics, particularly in brain slices [1,2]. It was nearly 10 years ago with the advent of the DA transporter (DAT) knockout (DAT-KO) mouse [7] that FSCV was first routinely used in mouse brain slices. Recent increases in the use of genetically defined mouse lines have made mice an important animal model for monoamine research. Thus far, FSCV research in mice has focused almost exclusively on DA dynamics in striatal brain slices. While species generality does seem to apply to striatal DA detection, it is important to note that species variability in midbrain monoamine recording has been shown between guinea pig and rat [8], and we have recently documented further variations in the mouse midbrain [9]. Therefore, the reasoned choice of animal models can aid in obtaining desired information about specific monoamines in specific brain areas. This fact highlights the importance of characterizing mouse neurochemistry in its own right and suggests that direct translation of knowledge gained from one species is not necessarily applicable to another species.

Brain slices were chosen as the in vitro assay in which to assess monoamine dynamics because several slices can be made from each region of interest to provide the opportunity to test one pharmacological agent on several slices to reduce statistical variability or to test several pharmacological agents in a single mouse brain. Additionally, one animal can be used to test slices from multiple brain regions. These facts have made brain slice voltammetry an exemplary tool to study DA dynamics in ex vivo studies; we have had the unique opportunity to study changes in the DA system (release, uptake, autoreceptor control, and drug effects) in hard-to-get brain tissue from rats and monkeys that had long histories of drug or alcohol self-administration [10,11]. Brain slices are also convenient because anatomical structures within the slice are visible and electrode placement can be made vs. anatomical landmarks, rather than by stereotaxic coordinates, as is the case of in vivo experiments. Due to the small size of the carbon fiber microelectrode (7 μm diameter, 50–200 μm in length), it is possible to differentiate structures that are spatially closely associated, even in the mouse brain, for example: nucleus accumbens (NAc) core vs. NAc shell or ventral tegmental area (VTA) vs. substantia nigra (SN). The use of brain slices also allows greater control of the physiological environment (e.g., temperature), and experimentally manipulated stimulation parameters can greatly reduce interference from such contaminants as pH changes, which are not possible to control in vivo. Furthermore, brain slice work does not suffer from the presence of anesthetic drugs that are necessary in an anesthetized in vivo preparation. Finally, the slice perfusion system makes drug application simple and reproducible, and cumulative drug concentration–effect curves can be collected within a single slice. We have determined that cumulative concentrations of pharmacological agents (most notably DA uptake inhibitors) applied to brain slices to not differ in their ability to alter uptake or release when compared to the same pharmacological agent applied at a single concentration. Furthermore, an advantage of brain slices is that the pharmacological action of drugs can be localized to the brain region of interest, as there are no contributions of pharmacological actions in the periphery or at other central locations. These features of brain slice voltammetry allow for an efficient way to assess monoamine dynamics in a variety of brain areas and carry out several pharmacological manipulations in each region, while reducing the number of animals necessary to collect large data sets.

Methodology for Fast Scan Cyclic Voltammetry in Mouse Brain Slices

Electrode Fabrication and Calibration

A triad of reference, auxiliary and working electrodes are typically used in brain slice FSCV recordings. We use a silver/silver chloride reference electrode made from silver wire (A-M Systems, Inc., Carlsborg, WA) that is coated with a thin layer of silver chloride by electroplating in a solution of 1 M HCl. A 22 AWG tinned copper wire (Belden, Parkville, Victoria, Australia) is used for the auxiliary electrode.

To prepare the cylindrical carbon fiber microelectrodes [12], a single carbon fiber (7 μm radius, type P-450, Amoco, Greenville, SC) is aspirated into a glass capillary with a microfilament (1.6 mm inside diameter, 1.2 mm outside diameter, A-M Systems, Inc., Carlsborg, WA). Each capillary is pulled into two electrodes using a micropipette puller (Model PE-2, Narashige, Tokyo, Japan or Model P-97, Sutter Instrument Co., Novato, CA) so that carbon fiber is exposed at the tapered (pulled) end. The exposed carbon fiber is trimmed to a length of approximately 50–200 μm with the aid of a non-inverting microscope at 40× magnification (Model BH-2, Olympus Corp., Lake Success, NY). Before use, the electrodes are backfilled with 150 mM potassium chloride using a spinal needle (25 gauge, 3.5 in., Becton–Dickinson and Co., Franklin Lakes, NJ), and a 4.5 in Kynar UL 1422 28/1 g wire (Squires Electronics, Inc., Cornelius, OR) is inserted in the blunt end to make an electrical connection with the carbon fiber.

When the carbon-fiber aspirated capillaries are pulled, it is important that the taper of the glass make a tight seal around the carbon fiber. If a good seal between the glass and the carbon is not obtained, the electrode can be sealed with epoxy; however, we have moved away from this practice because it is not necessary for the majority of electrodes produced. Traditionally, carbon fiber microelectrodes have been fabricated on vertical pullers, for example, Model PE-2 from Narashige Inc. On this style of puller, two settings, magnet and heat, are adjusted to create the desired type of glass seal on the carbon fiber. Recently, we have begun to fabricate electrodes on a horizontal puller, the Sutter Instruments Model P-97, similar to those used to fabricate electrophysiology electrodes. This style of puller requires four settings—heat, pull, velocity, and time—to be adjusted. In this case, the electrodes are heated and pulled in a manner controlled by a program entered into the puller’s computerized controller. This allows a greater amount of flexibility and control over working electrode fabrication. On either style puller, it is initially necessary to adjust the puller settings to obtain the best seal of glass to carbon fiber and desired shape. However, very few adjustments are necessary after this initial determination of optimal settings.

A flow injection system [13] is used to calibrate electrode responses to DA and 5-HT. The calibration buffer consists of (in mM) NaCl (126), KCl (2.5), NaH2PO4 (1.2), CaCl2 (2.4), MgCl2 (1.2), NaHCO3 (25), and HEPES acid (20), pH 7.4 at 23°C (room temperature). It is ideal to calibrate electrodes in a solution that is identical to that in which brain slice recording are obtained, i.e. artificial cerebrospinal fluid (aCSF), however, we have determined that the inclusion of ascorbate and glucose in aCSF used in electrode calibration did not change the sensitivity of our electrodes to DA or 5-HT [9]. It is important to note that calibration of electrodes must occur with concentrations of monoamines that are relevant to the biological sample, as decreased sensitivity of electrodes is evident with high monoamine concentrations [9]. In our hands, monoamine release in brain slices is generally ≤1 μM, therefore we use calibration factors obtained with concentrations of DA or 5-HT that are between 0.1 and 1 μM.

FSCV Data Acquisition

The commercially available EI-400 (Cypress Systems, Inc., Lawrence, KS) is both a waveform generator and a biopotentiostat which measures and amplifies the current at the working electrode relative to the reference and sends that information to the National Instruments (Austin, TX) interface boards in the computer for analog-to-digital conversion and subsequent analysis with locally written data analysis programs. For FSCV, an EI-400 is used in B-channel and two- or three electrode mode (three-electrode mode if an auxiliary reference electrode is utilized). In this configuration, the EI-400 uses the reference electrode as ground, and alters the potential of the carbon fiber microelectrode relative to that ground. For DA recording, the potential of the carbon fiber microelectrode is linearly scanned from −400 to 1200 mV and back to −400 mV vs. Ag/AgCl (Figure 4.1a, triangle waveform); for 5-HT recording, the potential is scanned from 0 to 1200 to −600 mV and back to 0 vs. Ag/AgCl (Figure 4.1b, N waveform). The difference in applied waveforms is due to the difference in the adsorption properties of DA and 5-HT. Both DA and 5-HT are positively charged molecules and are attracted to the carbon surface when sitting at negative potentials. Dopamine and 5-HT are both adsorbed onto the electrode surface when a negative resting potential is applied between scans. However, adsorption occurs to a much greater extent with 5-HT phenolic and hydroxyindole oxidation products, blocking portions of the electrode surface, thereby greatly reducing electrode sensitivity and also causing slow electrode response times. To combat this problem, 5-HT recording uses an applied waveform that is maintained at 0 mV vs. Ag/AgCl between scans, preventing 5-HT accumulation around the surface of the electrode; the electrode is scanned positively to oxidize 5-HT, then negatively to reduce 5-HT, and returned to a 0 mV potential vs. Ag/AgCl. For both DA and 5-HT waveforms, each is applied at 300 V/s, lasts 9–10 ms and occurs every 100 ms. While it is not possible to directly distinguish the catecholamines DA and NE with FSCV, catecholamines can be distinguished from the indolamines, including 5-HT. Catecholamines and indolamines produce distinct current vs. voltage curves (voltammograms), and as seen in Figure 4.2, DA and 5-HT can be distinguished based on their reduction current peaks. Typically, DA and 5-HT oxidation peaks are both at approximately 500–700 mV vs. Ag/AgCl, while the single DA reduction peak is around −200 mV and the double 5-HT reduction peaks are around 0 and −500 vs. Ag/AgCl.

FIGURE 4.1. Voltages applied to carbon fiber electrodes during voltammetric recordings of dopamine (DA) and serotonin (5-HT).

FIGURE 4.1

Voltages applied to carbon fiber electrodes during voltammetric recordings of dopamine (DA) and serotonin (5-HT). (a) The triangle waveform, −400 to 1200 and back to −400 mV vs. Ag/AgCl, is used for DA recording. (b) The N waveform, 0–1200 (more...)

FIGURE 4.2. Dopamine and 5-HT voltammograms.

FIGURE 4.2

Dopamine and 5-HT voltammograms. Dopamine (1 μM) voltammograms (current vs. voltage curves) show one oxidation and one reduction peak. Serotonin (1 μM) voltammograms show one oxidation and two reduction peaks. Voltammograms were obtained (more...)

We utilize background-subtraction techniques in order to create voltammograms. This is because the fast scan rate generates a large background current which can obscure the voltammetric current due to monoamines. Fortunately, the background current is fairly stable. Therefore, to obtain voltammograms, the background current obtained before electrical stimulation of the slice is digitally subtracted from that of the voltammograms of interest (generally the peak of monoamine release). In this work, five scans are averaged together to obtain both the background and stimulation-induced signals. To obtain current vs. time plots, the current at the oxidation potential (typically 500–700 mV vs. Ag/AgCl for both DA and 5-HT) is converted to concentration based on post-calibration of the electrode in 1 μM DA or 5-HT.

Brain Slice Preparation

Mice between 2 and 4 months of age are asphyxiated with CO2. Once the animal has stopped breathing, it is removed and quickly decapitated. To remove the brain, the skin and fur on the top of the head are removed with large dissecting scissors, cuting from the nape of the neck to the nose. Then, any remaining spinal column and membranous tissue from the posterior part of the skull over the cerebellum is removed with large dissecting scissors. The skull is removed by cutting the lateral aspects of both the occipital and interparietal bones to the lambdoid suture and then removing the occipital and interparietal bones. Then, the remaining skull removed by cutting along the line of the lateral sutures of the parietal and frontal bones, to the nasal septum. Then, a flat spatula is inserted at the base of the skull along the rostral–caudal axis. The brain is lifted up slightly, any remaining muscles or nerves connecting the brain to the skull are cut, and then the brain is fully removed and placed in approximately 100 mL of ice cold oxygenated (95% O2/5% CO2) aCSF. The aCSF consists of (in mM) NaCl (126), KCl (2.5), NaH2PO4 (1.2), CaCl2 (2.4), MgCl2 (1.2), NaHCO3 (25), glucose (11), HEPES (20), L-ascorbic acid (0.4), pH 7.4.

After the brain chills for 1 min in ice-cold aCSF, it is placed on a Petri dish containing frozen aCSF that is covered with a paper towel soaked with ice-cold aCSF. There, a single-edged carbon steel blade (Electron Microscopy Sciences, Hatfield, PA) is used to remove the olfactory bulbs and cerebellum; the brain is then cut in half along the coronal midline with a single-edged blade. The cut end of the brain is glued to the cutting stage of the specimen chamber (which is prechilled in a −20°C freezer) of the vibratome (Leica VT1000S, Vashaw Scientific Inc., Norcross, GA) with cyanoacrylate glue (Instant Krazy Glue, Advanced Formula, Columbus, OH). The specimen chamber is then mounted within the vibratome, a small cube of frozen aCSF is added to the specimen chamber to keep the buffer cold, and the chamber is then filled with ice-cold oxygenated aCSF. The area surrounding the chamber is then filled with crushed ice and ice-cold water, with attention paid to avoiding getting ice or water within the chamber. One half of a double-edged stainless steel razor blade (Electron Microscopy Sciences, Hatfield, PA) is placed in the blade holder of the vibratome to cut slices 400 μm thick. All slices are made within 20 min of brain removal. Using a spatula and small horse-hair paint brush, slices are transferred to a 100 mL beaker containing room temperature aCSF that is constantly oxygenated, where they incubate for at least 1 h.

Electrically Stimulated Monoamine Release

Electrical Stimulation

Slices used for FSCV are placed in a locally constructed slice perfusion chamber, weighted down with 2–3 mm sections of platinum wire (0.5 mm diameter, Alfa Aesar, Ward Hill, MA) and superfused with oxygenated aCSF (either 23 or 32°C) at 1 mL/min. We have carried out brain slice voltammetry experiments in both room temperature baths and those closer to physiological temperature. While evoked monoamine release and subsequent uptake can be monitored at room temperature, monoamine transporters act in a temperature-dependent manner [14] and uptake kinetics are slower at room temperature, compared to physiological temperature.

The electrical stimulation is generated by the computer (Dell Optiplex GX280, Dell Inc., Round Rock, TX) and sent to digital to analog and input–output interface boards (National Instruments, Austin, TX), which sends the stimulus to optical isolators (Neuro Log NL-800, Medical Systems, Greenvale, NY). Optical isolators are used to separate the electrical stimulation from the electrochemical measurements. After optical isolation, the stimulus is sent to the bipolar stimulating electrodes (MS303/3, Plastics One, Roanoke, VA). Monophasic stimulation pulses, 1 or 4 ms wide, 350 μA, are generated by locally constructed software. The stimulating electrode is mounted on a micromanipulator (Model MM3, Narishige International U.S.A. represented by Pacer Scientific, Los Angeles, CA) and positioned so that it rests on the surface of the slice. The carbon-fiber microelectrode, mounted on a separate but identical micromanipulator, is positioned 75 μm into the slice, 100 μm from the center of the tips of the bipolar stimulating electrode, creating an equilateral triangle. Electrode placements are made with the aid of a stereo-microscope (Sterozoom 6, Leica, Vashaw Scientific, Norcross, GA). Stimulation parameters differ between each brain region of interest. Generally, a 1-pulse stimulation (4 ms wide) is used to elicit DA from the caudate-putamen (CPu), NAc core and NAc shell. 30-pulse, 30 Hz stimulations (1 ms wide) are used to elicit monoamine release from VTA, SN pars compacta (SNc), SN pars reticulata (SNr) and dorsal raphe nuclei (DRN). Occasionally, stimulus artifacts (noise) are generated by the stimulation. Reducing the duration (width) or amplitude of the stimulus pulse(s) is usually effective in reducing or eliminating the artifacts.

Advantages of the Use of Electrical Stimulation

Electrical stimulation is used to elicit the release of endogenous monoamines from the brain slice and then uptake kinetics can be monitored. This offers a great advantage over exogenously applied monoamine studies. While transporter uptake rates can be reasonably estimated from low concentrations of exogenously applied monoamines, the high concentrations of often used exogenously applied monoamines can complicate the interpretation of uptake data. For example, high concentrations of DAT substrates, such as DA or amphetamine, can cause substrate transport both into (forward direction) and out of (reverse direction) the cytoplasm. The kinetics of transporters that are dually functioning to take up substrate, as well as release substrate, are complicated and transporter kinetics under these conditions cannot be attributed to uptake alone.

Experimental Design

In a typical brain slice FSCV experiment, evoked overflow is first determined by obtaining responses to electrical stimulation in aCSF. Interstimulus intervals vary in each brain region and we generally stimulate every 5 min in the CPu, NAc core, and NAc shell and every 10 min in the VTA, SNc, SNr, and DRN. It was noted in midbrain regions that interstimulus intervals of less than 10 min caused depletion of the releasable pool of monoamine. This is presumably due to the slower rates of uptake and lower synthesis enzyme levels of these regions, compared to the striatum. Responses usually stabilize within 1 h and are required to be stable for at least 30 min before pharmacological agents are applied. Pharmacological agents are applied to the slices via the superfusate, and responses are recorded throughout the drug application. It is important to determine the time for the maximal effect of acute superfusion of a pharmacological agent for each individual drug, as factors such as lipophilicty are important determinants of the ability of the drug to act at the recording site.

Data Analysis

Uptake Rate Determination

The extracellular concentration of monoamines primarily reflects a balance between release and uptake; pharmacological agents can change one or both of these processes and these drug effects are of neurobiological interest. Generally, a Michaelis–Menten based kinetic model is used to evaluate release and uptake kinetics [15].

d[DA]dt=f[DA]p-Vmax(Km/[DA])+1
4.1

[DA] is the instantaneous extracellular concentration of DA, f is the pulse frequency, [DA]p is the concentration of DA elicited per stimulus pulse, and Vmax and Km are Michaelis–Menten uptake parameters. For this type of modeling, three assumptions are made: (1) each stimulus pulse releases a fixed quantity of monoamine into the extracellular space, [DA]p or [5-HT]p, (2) uptake is a saturable process, and (3) uptake is the primary mechanism-clearing, released monoamine. Furthermore, this modeling assumes a control Km value (inversely related to the affinity of the transporter for its monoamine) of 0.16 μM for DA at DAT and 0.17 μM for 5-HT and the 5-HT transporter (SERT). An additional kinetic parameter, Vmax, which is related to the number and turnover rate of the monoamine transporters, is also determined. These assumptions are best suited for evaluating release and uptake in striatal regions that use 1-pulse stimulations and have rapid uptake, but can also be used with multiple pulse stimulations in areas with low uptake rates, such as the midbrain [9].

Michaelis–Menten kinetic determination of uptake parameters would be inaccurate to describe DA clearance in DAT-KO mice, with genetically deleted DAT, because, as stated above, there must be the assumption that uptake is the primary mechanism clearing released monoamine. This is because Michaelis–Menten kinetics assumes two processes: (1) monoamine binding to the transporter (with the affinity described by Km), and (2) subsequent “catalysis” of DA, i.e. transport into the intracellular space, which is maximal (described by Vmax) under saturating conditions. Because DAT-KO mice are genetically lacking DAT, the first step of this process is impossible. Previously in DAT-KO mice, DA clearance was estimated using a Michaelis–Menten kinetic modeling program with the Km value set at a high concentration. Then a pseudo first-order rate constant, k, was calculated by dividing Vmax by Km [16]. Jones et al [16]. reported that the k value for DA clearance in CPu slices from DAT-KO mice was 0.05 s−1. When using the Michaelis–Menten kinetic model in monoamine transporter knockout mice, one must assume a Km of greater than 10 μM. This makes intuitive sense, given that the absence of the transporter implies no affinity of the transporter for its monoamine. Figure 4.3 illustrates an example of k values obtained using various presumed Km values in the Michaelis–Menten kinetic model in a single CPu slice from a DAT-KO mouse. As is apparent, the Km value assumed in wild type animals (approximately 0.2 μM) would greatly overestimate the k value, which should be approximately 0.05 s−1, reflective of the first-order elimination process of diffusion.

FIGURE 4.3. Pseudo first-order rate constant k values, using the equation k = Vmax/Km, obtained using various assumed Km values in the Michaelis–Menten kinetic model (obtained from modeling a single DA transporter knockout (DAT-KO) CPu slice).

FIGURE 4.3

Pseudo first-order rate constant k values, using the equation k = Vmax/Km, obtained using various assumed Km values in the Michaelis–Menten kinetic model (obtained from modeling a single DA transporter knockout (DAT-KO) CPu slice). It is necessary (more...)

Due to the differences in stimulation parameters and uptake kinetics between regions of the striatum and regions of the midbrain, and the fact that there is no active uptake in monoamine transporter knockout animals, a different method of modeling data has proven useful for the midbrain as well as knockout mice. In areas where release is in the low micromolar range, uptake can be simplified to fit first order kinetics, as described by Cragg et al [17]. In our analyses, a one-phase exponential decay fit well. Figure 4.4 shows examples of VTA uptake modeled with both Michaelis–Menten kinetics and one-phase exponential decay, both of which determine fits of the data with r2≥0.90. Additionally, this method can also be used to determine uptake rates in monoamine transporter knockout mice. We have independently determined the uptake rate constant k using slices from midbrain and from animals with a genetic deletion of monoamine transporters by fitting the clearance portion of the current vs. time curves to a one-phase exponential decay. In the VTA of DAT-KO mice, we have found k values of 0.06±0.02 [18] and 0.07±0.01 [9] s−1 utilizing Michaelis–Menten modeling (k = Vmax/Km) and one-phase exponential decay, respectively (Figure 4.5), which are both in agreement with the 0.05 s−1 published for DAT-KO CPu [16].

FIGURE 4.4. Representative efflux curves measured by fast scan cyclic voltammetry (FSCV) in response to 30-pulse, 30-Hz (350 μA, 1 ms, indicated by the line under the efflux curves) stimulation in an individual wildtype mouse ventral tegmental area (VTA) slice.

FIGURE 4.4

Representative efflux curves measured by fast scan cyclic voltammetry (FSCV) in response to 30-pulse, 30-Hz (350 μA, 1 ms, indicated by the line under the efflux curves) stimulation in an individual wildtype mouse ventral tegmental area (VTA) (more...)

FIGURE 4.5. Two methods to determine rate constant k values in averaged data from VTA slices from DATKO mice.

FIGURE 4.5

Two methods to determine rate constant k values in averaged data from VTA slices from DATKO mice. Michaelis–Menten modeling (left bar, from Mateo, Y. et al., Proc. Natl Acad. Sci. U.S.A., 101, 372, 2004) and one-phase exponential decay modeling (more...)

Regional Variation in Uptake Rates

As suggested above, the uptake rate of a given transmitter is brain-region specific. Striatal DA uptake rates are faster in the CPu than in the NAc, which are in turn faster than in amygdala, prefrontal cortex or somatodendritic regions like the VTA and SN [2,19–21]. This suggests that DA signaling is regionally distinct, and has led to the evolution of the term “volume transmission” to describe extracellular diffusion to reach sites of action distant from the synapse [17,22–24]. It is important to understand that active uptake and volume transmission are not mutually exclusive concepts. Diffusion to sites of action and clearance by active uptake both occur during DA volume transmission [25]. In fact, it is well established that DATs are located outside the synapse [26,27], as are the majority of DA receptors [28,29], making extrasynaptic diffusion necessary and traditional synaptic neurotransmission unlikely. Furthermore, quantitative evaluation of diffusion and uptake in midbrain suggests that diffusion is the most important determinant of the lifetime of DA [17]. Even in the dorsal striatum, where DA uptake is very rapid and predominately determines DA time course, diffusion is an important component of DA transmission [30]. Even so, the relative contribution of uptake mechanisms is a factor in determining the role of diffusion [25], which can vary greatly between brain regions, and the role of uptake can contribute to envisioning transmission in a particular brain area as synaptic-like or paracrine-like.

Monoamine Uptake Inhibitors and Releasers

Mechanisms of Action

Psychostimulants, which interact with monoamine transporters to increase extracellular monoamine levels, come in two varieties: those that are pure uptake inhibitors and those that function as releasers. Uptake inhibitors bind to transporter proteins and inhibit transporter function, slowing the uptake of monoamines from the extrasynaptic space and thereby increasing the extracellular concentration of monoamines [31–34]. Releasers, like amphetamine, increase extracellular monoamine levels primarily by acting as false substrates, competitively inhibiting neurotransmitter reuptake and promoting reverse transport [31,35–39]. In addition, releasers displace monoamines from synaptic vesicles [38,40] and act as monoamine oxidase inhibitors; [41,42] both of these effects aid to increase the cytoplasmic monoamine concentration that drives reverse transport. Binding sites on monoamine transporters likely differ for uptake inhibitors and releasers, in agreement with their different mechanisms of action [43–45]. Ultimately, uptake inhibitors enhance activity-dependent monoamine transmission, while releasers primarily promote activity-independent efflux of monoamines.

Using FSCV to Evaluate Changes in Monoamine Dynamics in Response to Uptake Inhibitors and Releasers

Here, we use an uptake inhibitor, cocaine, and a releaser, methamphetamine, to demonstrate the utility of FSCV to measure changes in DA and 5-HT dynamics. Uptake inhibitors and releasers have very different effects on the time course of DA overflow curves measured by FSCV. However, both are effective at inhibiting monoamine uptake, which is evidenced by the widening of the clearance portion of the current vs. time curves with increasing concentrations of cocaine and methamphetamine (Figure 4.6).

FIGURE 4.6. The effects of an uptake inhibitor, cocaine, and a releaser, methamphetamine, on DA and 5-HT voltammetric signals in wild-type mouse brain slices.

FIGURE 4.6

The effects of an uptake inhibitor, cocaine, and a releaser, methamphetamine, on DA and 5-HT voltammetric signals in wild-type mouse brain slices. Dopamine is measured in caudate-putamen (CPu) (1-pulse, 4 ms stimulations) and 5-HT is measured in SN pars (more...)

In addition to preventing monoamine reuptake, uptake inhibitors typically cause an increase in monoamine signal peak height at low concentrations (Figure 4.6, cocaine) [46,47], due to uptake inhibition during stimulation trains and/or diffusion of the monoamine to the electrode from distant sites. The increased peak height of voltammetric signals in response to the diffusion increasing aspects of uptake inhibition is important because the voltammetric electrode is sampling from a larger anatomical area after the drug than before. The increase in sampling area is generally thought of as a larger-diameter sphere. We generally assume that these increases are not due to actual enhancement of DA release from presynaptic terminals, although a minor contribution cannot be ruled out. At higher concentrations of uptake inhibitors, there is an apparent decrease in release resulting from the increased extracellular monoamine concentrations activating presynaptic autoreceptors (Figure 4.6, cocaine) [48] and obscuring diffusion-related increases in peak height. This effect can be prevented with an autoreceptor antagonist.

The magnitude of change following uptake inhibition is highly correlated with the baseline rate of uptake. For example, in the CPu, where DA uptake is rapid and diffusion is restricted, a large dynamic range of uptake inhibitor effects can be seen; an uptake inhibitor will greatly increase the diffusion distance of DA in the CPu. In contrast, in areas with lower uptake rates, such as the VTA, diffusion is already substantial and uptake inhibition results in a smaller-magnitude effect. For 5-HT the latter case is most common, as there are no brain areas with as great a SERT density as that of DAT in CPu. In the case of cocaine acting at the DAT and SERT (Figure 4.6), the apparent lesser effect of cocaine on 5-HT uptake is also related to the greater affinity of cocaine for the DAT than the SERT [49]. While psychostimulants induce a proportionally smaller absolute change in uptake rate (and dialysate monoamine levels) in non-striatal brain regions, there are, nevertheless, still substantial physiological effects of uptake inhibition in these regions.

Current vs. time plots usually return to the baseline established before electrical stimulation after uptake inhibition, albeit slowly. In contrast, after the application of releasers, monoamine levels do not always return to baseline and may appear to “hang-up” or establish a new baseline (Figure 4.6, methamphetamine effects on DA in the CPu). This is because releasers cause the transporter to operate in two directions, and electrically-stimulated release of monoamine shifts the extracellular concentration of substrate toward a new equilibrium. Amphetamine and its derivatives function as releasers, but these effects can also be caused by endogenous substrates, including phenylethylamine, tyramine, DA, NE, and 5-HT. One must use caution in interpreting the effects of releasers on monoamine clearance curves, as discussed above with regard to exogenous monoamine application (see “Advantages of the Use of Electrical Stimulation”). Michaelis–Menten modeling is useful for amphetamine-altered current vs. time curves that do return to baseline. However, it would be more appropriate to use first-order kinetic decay modeling for those current vs. time plots that do not return to baseline. Also, it should be noted that signals that do not return to baseline (either hanging, drifting, or dropping below baseline) in the absence of transporter substrate are likely being affected by pH, Ca2+, or oxygen changes [50,51] and should not be included in data analysis unless subtraction techniques are available [51]. Also, in contrast to uptake inhibitors, the release decreasing effects of releasers is only partially attributable to autoreceptor activation [52–54]. Electrically evoked release is dependent on vesicular storage of monoamines; releasers cause displacement of monoamines from vesicles [38,40], thereby decreasing the releasable pool of monoamines and decreasing evoked release.

Concluding Remarks

Here, we have described the use of FSCV to measure DA and 5-HT in mouse brain slices. The growing use of genetically defined mouse lines have made mice an important animal model for monoamine research. The utility of FSCV comes primarily from its superb temporal (ms) and spatial (micron) resolution, as well as its reasonable chemical specificity and sensitivity for monoamines. FSCV is a powerful tool that can measure fast changes in extracellular monoamine concentrations in discrete brain regions, both in vitro and in vivo. FSCV in brain slices is particularly useful for characterizing acute, local drug effects; the effects of chronic in vivo drug administration on monoamine systems can be evaluated as well. While brain slice FSCV provides useful information on monoamine dynamics (release, uptake, and diffusion), coupling both in vitro and in vivo data, as well as microdialysis and electrophysiological studies, will provide the most complete description of monoamine functioning in the brain.

Acknowledgments

We would like to thank Dr Marc G. Caron for the gift of DAT-KO mice. This work was supported by AA11997 and DA016498 to CEJ, and AAO14091, AA013900 and DA018815 to SRJ.

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