Chapter 6Using High-Speed Chronoamperometry with Local Dopamine Application to Assess Dopamine Transporter Function

Gulley JM, Larson GA, Zahniser NR.

Publication Details

Introduction

Importance of Dopamine and the Dopamine Transporter (DAT)

The catecholamine dopamine (DA) is a neurotransmitter in the central nervous system [1], where it contributes to normal physiological functions such as motor planning and movement, affect, and reward. Accordingly, DA also plays an important role in diseases such as Parkinson’s disease, schizophrenia, and drug addiction. Thus, there are many reasons why neuroscientists and clinicians are interested in understanding DA neurotransmission and how it is regulated.

DA is released from axon varicosities (terminals) of DA neurons and from their dendrites. DA release occurs at low levels spontaneously and at higher levels upon neuronal depolarization, in particular when DA neurons “burst fire.” Following diffusion across the synaptic cleft, DA produces its effects by activating two families of postsynaptic DA receptors, D1-like (D1, D5) and D2-like (D2, D3, D4) receptors. DA can also activate presynaptic D2-like receptors localized on the DA neurons themselves (DA autoreceptors), thereby regulating DA neuronal firing rate, DA synthesis, DA release and DA reuptake. All DA receptors are Guanosine triphosphate (GTP) binding protein-coupled receptors that signal through heterotrimeric G proteins to activate or inhibit particular enzymes and ion channels. Because of the receptor signaling cascades involved, DA is considered to be a relatively slower acting neurotransmitter, or even a neuromodulator, as compared to the fast neurotransmitters whose receptors are ligand-gated ion channels.

Despite that DA is a relatively slow acting neurotransmitter, similar to all neurotransmitters, it is critical to limit its lifetime in the synapse and its extent of diffusion. In this way, DA neurotransmission can be tightly controlled in both time and space, preventing prolonged receptor stimulation and desensitization. The DA transporter (DAT) is the primary mechanism for clearing extracellular DA. The DAT not only terminates DA neurotransmission by transporting DA back into DA neurons (thus eliminating the possibility for DA to interact with its receptors), but also conserves DA by recycling it into the neuronal cytoplasm, from which it can be taken up into synaptic vesicles for re-release. Additional mechanisms also contribute to eliminating extracellular DA; these include diffusion, intraneuronal metabolism by monoamine oxidase (MAO), and extraneuronal metabolism by catechol-O-metyl-transferase (COMT).

DA uptake was first reported more than 30 years ago when it was shown that tritium-labeled DA ([3H]DA) was accumulated in a Na+- and energy-dependent manner by homogenates of DA-rich brain regions such as striatum [2,3]. Synaptosomes are an in vitro preparation of isolated nerve endings made by a sequence of precise homogenization and centrifugation steps. This preparation is most often used for in vitro uptake studies. When made from striatum, synaptosomes include a relatively high proportion of DA nigrostriatal neuronal terminals; thus, uptake of DA is readily observed in this preparation. DA uptake by DAT, rather than reflecting the activity of a plasma membrane energy-driven pump per se, is dependent upon the electrochemical gradient set up across cellular membranes by the Na+/K+-ATPase. The co-transport of 2 Na+ and 1 Cl ions is required to translocate 1 DA+ molecule (positively charged at physiological pH) [4,5]. DAT normally transports DA in an inward direction, i.e., from the extracellular space into DA neuronal dendrites and terminals, from which DA is subsequently either transported into synaptic vesicles by the vesicular monoamine transporter 2 (VMAT2), a H+-antiporter, or broken down by MAO. However, if the neuronal concentration of free cytoplasmic DA and Na+ ions becomes locally high in the proximity of DAT, as happens with exposure to amphetamine or tyramine, the DAT can mediate reverse transport of DA out of the DA neuron. This is the basis for amphetamine-induced DA release [6,7].

Drugs such as cocaine, nomifensine, and mazindol bind to DAT and block [3H]DA uptake with micromolar to nanomolar affinities [8,9]. Although these drugs are all reasonably high affinity inhibitors of DAT, it is important to note that they also have similar (cocaine) or higher (nomifensine and mazindol) affinity for inhibiting the norepinephrine transporter (NET) [10]. Cocaine also inhibits the serotonin transporter (SERT or 5-HTT) with an affinity similar to its inhibition of DAT and NET. GBR 12909 is the drug with the highest selectivity between DAT and NET (about fifty fold) [11] most commonly used. When injected in vivo, all of these DAT inhibitors increase extracellular concentrations of DA, resulting in psychomotor stimulation. Similar to amphetamine and its derivatives, these drugs are self-administered by laboratory animals and have a high abuse and addiction liability in humans [12]. Thus, understanding the function and regulation of DAT is of particular interest to drug abuse researchers.

In the early 1990s, the cloning of DAT revealed that it is a member of the plasma membrane Na+/Cl-dependent transporter family, more recently defined as the solute carrier 6 (SLC6) gene family, which includes NET and SERT, as well as transporters for the inhibitory amino acid neurotransmitters GABA (GAT) and glycine (GYT) [13,14]. The human gene for DAT is named SLC6A3. Based on primary sequence, DAT and NET are most closely related; thus, some overlap among inhibitors of these two transporters is not surprising, although there are several highly selective (greater than thousand fold) NET inhibitors. Deletion of the DAT gene (DAT knockout; DAT KO) in mice in 1996 provided some of the strongest evidence for the importance of DAT in terminating normal DA neurotransmission [15,16]. DAT KO mice are hyperactive, particularly in a novel environment, despite marked decreases in DA tissue levels, release and receptors— compensatory changes for the elevated extracellular DA concentration. In fact, DA persists 100 times longer in striatum of DAT KO mice than in wild-type mice. Neither cocaine nor amphetamine activates DAT KO mice. However, it came as a surprise that DAT KO mice still self-administer cocaine and exhibit cocaine conditioned place preference. In these mice, SERT inhibition by cocaine is sufficient to mediate cocaine reward [17].

DAT is found exclusively in DA neurons [18,19], in which it is functional only when expressed at the plasma membrane. Electron microscopic studies revealed that plasma membrane DAT is localized both near and distant from synaptic junctions [19]. Based on its “perisynaptic/extrasynaptic” localization, one might think that DAT is not all that important. However, the marked behavioral effects of DAT inhibitors and the results with the DAT KO mice argue otherwise. Also, in release assays using rat striatal slices, inclusion of a DAT inhibitor such as nomifensine in the superfusion buffer is required to detect endogenous DA overflow following mild electrical field stimulation [20].

In the past ten years, it has been appreciated that transporters such as DAT are highly regulated [21–25]. This regulation provides additional evidence for their importance. Regulation may occur rapidly (minutes) or long-term (days to weeks). Mechanisms for rapid regulation include altered DAT kinetics, presumably because of post-translational modifications and conformational changes in the transporter molecule, and DAT trafficking, because of changes in the number of DATs expressed at the plasma membrane. At least in model expression systems, DAT constitutively traffics in a clathrin-mediated manner away from and back to the cell surface, providing an ideal mechanism for rapid regulation [26]. Substrates, inhibitors, ligands acting through presynaptic receptors, and other signaling molecules also rapidly regulate DAT; this often occurs via altered trafficking. One of the main challenges for the future is to develop methods for studying rapid regulation of DAT function in individual neurons, as well as in the intact brain. Disease states and chronic drug administration can also regulate DAT activity over days to weeks. This type of regulation is most often associated with changes in the number of functional DATs; however, whether this is because of alterations in transporter trafficking, turnover (synthesis/degradation rates), or both remains to be elucidated.

Because DA is electroactive, electrochemical methods provide valuable ways of measuring real-time DAT function, most often by determining the clearance of DA by DAT. Before we discuss our electrochemical approach for measuring DA clearance, however, we first mention briefly some of the non-electrochemical approaches used to measure DAT number and function.

Non-Electrochemical Methods Used to Measure DAT Number and Function

Radioligand Binding and Antibodies

The number of DATs in tissue or cell samples is most often measured using radioligand binding studies. DAT binding is determined by calculating specific binding (total binding–nonspecific binding) measured at steady state with a radioligand that has high affinity and reasonable selectivity for DAT, such as the cocaine congener [3H]WIN 35,428 (or [3H]CFT) [27]. Binding can be measured in vitro by incubating the radioligand with brain membranes, brain slices, and intact cells or in vivo by injecting the radioligand systemically. Using DAT radioligands adapted to positron emission tomography (PET) or single photon emission tomography (SPET), one can also visualize changes in DAT in the brains of living humans [28]. Radioligands are generally lipophylic and bind to the total population of transporters, viz., transporters associated not only with plasma membranes, but also with intracellular membranes. However, because the binding of [3H]WIN 35,428 is strongly Na+- dependent and the intracellular concentration of Na+ is low, in intact cells [3H]WIN 35,428 can selectively detect cell surface binding, which represents the population of functional transporters [29]. When full saturation binding experiments are conducted, the affinity of DAT for the radioligand (Kd) and the maximal number of binding sites (Bmax) are determined. Comparison of Bmax values is valuable as an indicator of whether the number of DATs has been altered by a particular manipulation. The advantage of radioligand binding is that it is highly quantitative and, when coupled with image analysis, can yield precise anatomical resolution as well. However, the drawback to this approach is that it measures only the binding interaction of the ligand with the transporter, not transporter function.

DAT antibodies are useful for determining relative amounts of DAT protein and identifying DAT-protein interactions in homogenized tissue or membranes, as well as for localizing DAT protein in fixed tissue and live cell preparations. Although antibody-based methods are less quantitative than radioligand binding, they offer other advantages. For example, assuming equal amounts of protein are loaded in each lane on a gel, detection of bands on a western blot with specific antibodies allows comparison among samples of the relative amounts of DAT protein (not just binding sites). Western blots also reveal potentially different molecular weight forms of DAT, such as dimers and higher weight oligomers [30].

To assess the number of functional transporters and for trafficking studies, DATs at the cell surface need to be measured. Unfortunately, however, few antibodies are available that interact with the extracellular loop of DAT, thereby detecting exclusively surface DATs, particularly in live cells. Thus, surface DATs have been measured most commonly by chemical cross-linking methods, e.g., with a cell-impermeant biotin reagent in synaptosomes before western blotting [31].

DATs containing a fluorescent protein-tag have been extremely useful for visualizing DAT trafficking in live cells. New photoswitchable fluorescent-protein derivatives are being developed for use in trafficking and kinetic studies [32]. In general, the drawback has been that these constructs must be overexpressed acutely in cells or neurons. Thus, this approach does not allow native DATs to be studied. In the future, mice expressing fluorescent DATs could be generated. Transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter have already been generated, thus allowing visualization of live DA neurons [33].

DA Uptake Assays

The oldest, and still an extremely useful, way to assess DAT function is to measure the accumulation of DA by DAT. This approach is limited to in vitro tissue preparations and cells. Both [3H]DA and unlabeled DA have been used to monitor uptake. Rotating disk electrode voltammetry is a highly useful method for studying DA uptake mechanisms in vitro [34], although [3H]DA is more commonly used to simply measure uptake because of its ease of detection. As with radio ligand binding, specific uptake by DAT can only be determined indirectly, by subtracting nonspecific uptake from total uptake. In terms of brain tissue, either synaptosomes or slices can be used. Synaptosomes have lower nonspecific uptake and fewer tissue barriers than slices; however, slices tend to be more viable during longer incubations. Because the rate of uptake is determined, DA accumulation is measured under conditions where it increases linearly with time (minutes). Specific uptake of a single concentration of [3H]DA provides a snapshot comparison whereas a full kinetic curve must be generated to determine if an observed difference between samples is because of a change in affinity (Km) or maximal velocity (Vmax) of DAT.

DAT-Associated Currents

As uptake occurs, the inward translocation of 2 Na+ ions: 1 Cl ion: 1 DA+ molecule with each transport cycle would be expected to generate a small net inward current. Thus, about ten years ago, two-electrode voltage-clamp recording was used to test for such currents in Xenopus laevis oocytes expressing the cloned human DAT (hDAT) [35]. As predicted, this work revealed small, inward DA-induced currents in oocytes expressing hDATs, but not in control oocytes. The transport associated currents were concentration-dependent, blocked by pre-exposure to DAT inhibitors, such as cocaine, and magnified at more hyperpolarized potentials. Furthermore, the voltage-dependency of these currents established that DAT is an electrogenic transporter. The results suggested that uptake would be reduced upon depolarization and enhanced upon hyperpolarization. This was confirmed by measuring DA uptake in voltage-clamped hDAT-expressing oocytes. Thus, transport associated currents provide an alternative real-time (msec) method to assess DAT function and regulation in vitro.

The surprising finding regarding the hDAT currents was that in addition to the transport associated current, several channel-like leak currents were also associated with DAT expression [35]. One of these conductances was because of a constitutive, nonselective cation leak current; both DAT substrates and inhibitors blocked this leak current, resulting in an outward current. The fact that substrates both induced the transport current and blocked the leak current, whereas inhibitors only blocked the leak current, provided a way to distinguish unequivocally between DAT substrates and blockers. Thus, for example, amphetamine and tyramine were confirmed to be substrates, and cocaine and methylphenidate were confirmed as blockers.

These currents also provide additional approaches for evaluating DAT trafficking and function. For example, capacitance measurements are useful for studying DAT internalization in oocytes [36]. Patch-clamp recording can be used to measure transport-associated currents in mammalian cells, and this has been combined with microamperometric measurements of monoamine flux to provide a more comprehensive picture of NET and DAT function/regulation [37,38]. An interesting, new insight from these studies is that unlike DA, amphetamine has a unique ability to cause DA efflux through a bursting, channel-like mode [38]. On the other hand, whole cell patch clamp and perforated-patch recordings in primary cultures of midbrain DA neurons have revealed that DAT mediated conductances induced by both DA and amphetamine increase the excitability of DA neurons [39].

In Vivo Microdialysis

Although samples collected during in vivo microdialysis experiments are often analyzed for monoamines such as DA using high performance liquid chromatography (HPLC) and electrochemical detection, we mention this approach in this section on non-electrochemical methods because it does not use direct electrochemical recording in the brain. Microdialysis experiments are most often conducted in awake, behaving animals—from mice to nonhuman primates. Initially, the animal is anesthetized so that a guide cannula can be implanted under stereotaxic control into the brain region of interest; and this, along with a swivel device for later attachment of the sample tubing to a tether allowing freedom of movement, is then attached securely to the animal’s skull.

Following a recovery period, the microdialysis probe is inserted into the guide cannula and slowly perfused with an artificial cerebrospinal fluid (aCSF) buffer. Small molecules such as neurotransmitters, which are present in the extracellular milieu of the brain but not the perfusion buffer, freely diffuse across the dialysis membrane and equilibrate in the perfusion buffer according to their extracellular concentrations. Thus, conventional DA microdialysis experiments measure the extracellular concentration of DA in five to twenty-minute samples; this represents the net result of primarily DA release and uptake processes. To determine absolute levels of basal DA and to quantify changes in DA release and uptake, the “no-net flux” or “zero-net flux” method of quantitative microdialysis is used. Although not without caveats [40,41], this quantitative microdialysis method has been powerful, e.g., revealing altered basal and cocaine-evoked DAT activity during abstinence from cocaine and how behavioral responsiveness and basal DAT function are related [42,43]. Another in vivo microdialysis method used to assess DAT function selectively relies on the cellular extraction of [3H]MPP+ by DAT and [14C]mannitol as a reference [44].

Issues with the in vivo microdialysis technique include the amount of tissue damage resulting from relatively large probes (~200 um diameter) and the need for in vivo calibration of the probe recovery [45]. In general, as noted above, this approach uses relatively long sampling periods, but has the appropriate sensitivity for determining low nanomolar levels of basal extracellular DA. It is also ideal for measuring the relatively slow effects of drugs such as DAT inhibitors on extracellular levels of DA and relating drug-induced changes in DAT function with behavioral changes in freely behaving animals.

Technological advances involving more rapid sampling and detection methods continue to improve the temporal and spatial resolution of the in vivo microdialysis approach. Analytical methods, such as automated capillary liquid chromatography, also allow simultaneous determination of multiple neurotransmitters in the dialysate. For example, this method has been used to elucidate the effect of the DAT blocker nomifensine on multiple neuroactive amines and amino acids (multi-analyte capability) in a particular brain region [46].

Electrochemical Methods Used to Measure in Vitro and in Vivo DAT Function

Electrochemical detection of brain catecholamines such as DA, which was initially described more than thirty years ago (for a review, see Adams) [47], has been used extensively by neuroscience researchers interested in elucidating the function of these neurotransmitter systems. Multiple variations of electrochemical detection methods exist, but they all use the facts that electroactive species oxidize in response to an applied voltage and that the resulting movement of electrons can be measured as a change in current. Two commonly used methods are high-speed chronoamperometry (HSC) and fast cyclic voltammetry (FSV). Both HSC and FSV offer high sensitivity with excellent temporal and spatial resolution. Using small diameter (10–30 μm) carbon fibers, detection limits are in the low nanomolar concentration range and measurements can be made within 100 ms. These advantages make electrochemical techniques appealing to those studying the function of neurotransmitters, such as DA, that are characterized by their relatively rapid, synaptic actions.

The primary difference between HSC and FCV is the method of delivering the applied voltage. With HSC, the potential is held constant (e.g., 0.55 V) for a short period of time (typically 100 ms) during which DA at the electrode is oxidized, producing an oxidation (OX) current. This is followed by a return to a resting voltage (typically 0 V) during which the DA is reduced, producing a reduction (RED) current. In most cases, this square-wave pulse is repeated multiple times per second with measurements made at the same time (steady state) during each of the voltage pulses. Because the applied voltage is held at a constant potential and is instantaneously ramped up from the resting value, the measured current is directly proportional to the concentration of the species electrolyzed, and the electrode is sensitive to rapid changes in concentration.

FCV uses a triangular potential, typically with a range of −0.4 to +1.0 V, that is applied rapidly (300 V/sec) to the carbon fiber, with the resulting OX and RED currents measured every 100 ms. An advantage of this voltage-sweep method is that current changes can be resolved over the time and voltage domains, thereby providing an electrochemical “fingerprint” that can be used to aid the identification of the various electroactive species (e.g., DA) that are found in the microenvironment surrounding the electrode. Chemical identification (or verification) is provided, to some degree, with HSC by analysis of the ratio between the RED and OX currents.

Use of High-Speed Chronoamperometry (HSC) with Local DA Application to Measure in Vivo DAT Function in Discrete Brain Regions of Anesthetized Rats and Mice

In the sections that follow, we describe our typical setup and protocols for measuring DAT function in vivo. We initially chose to measure the clearance of locally applied exogenous DA as an “uncomplicated” indicator of DAT function [48] because this approach eliminates the potential confound of altered DA release. In Summary of Evidence that this Approach Measures DAT Function, we summarize the data showing that this measure does reflect primarily DAT activity. Nonetheless, as the rapid regulation of DAT began to be appreciated, it became apparent that our application of DA could regulate DAT function. This potential confound is discussed in Rapid Regulation of DAT Can Influence Measures of Exogenous DA Clearance. In our experiments, HSC measurements are made using an IVEC-10/FAST-12 system (Quanteon, Nicholasville, Kentucky) that interfaces with an electrode/micropipette assembly. Assemblies are positioned in the brains of rats or mice that are anesthetized and positioned in a stereotaxic apparatus. In Additional Applications of High-speed Chronoamperometry with Local DA Application to Measure DAT Function, we describe modifications that allow us to measure DAT function in brain slices maintained for several hours in a perfusion chamber or in awake, freely behaving rats.

Methods

HSC

The IVEC-10/FAST-12 system consists of three main components. The first is a low-noise preamplifier, sometimes referred to as a headstage, that interfaces directly with the electrode implanted in the brain. The headstage provides conversion of the measured current into voltage and provides gains of up to 20 picoamps/mV. The second component is a control box that contains secondary amplifiers and a potentiostat for controlling the applied potential. For experiments in which we are interested in measuring the oxidation of DA, we apply square-wave pulses of 0.00–0.55 V (with respect to reference) at a frequency of 5 Hz. The last component is a DOS- or Windows-based computer that contains a National Instruments A/D board. The computer provides a user interface with the control hardware and software and is used for data acquisition, analysis, and storage.

Electrode Construction, Calibration, and Assembly with Micropipettes

Carbon-fiber recording electrodes can be purchased from a number of commercial sources or they can be fabricated easily in the laboratory. We choose the latter approach. For recordings in anesthetized animals, we construct batches of about twenty-five electrodes in three stages, each involving several steps. In the first stage, a 5 cm piece of 4-mm diameter glass tubing (Schott, Elmsford, New York) is pulled to a long, tapered point so that the shank is approximately 2.5 cm long. A carbon fiber (30–33 μm; Textron Systems, Wilmington, Massachusetts) is then inserted through the larger opening of the glass rod and fed as far as possible into the tapered point. The pointed end of the glass rod is then broken by carefully touching it to the surface of a rotating cutting wheel that is powered by a variable speed rotary tool (Dremel, Racine, Wisconsin). When done correctly, the tip will be broken to a diameter a little more than 30 μm and the carbon fiber will protrude through the tip. The fiber is then gently pulled through the small opening so that several millimeters extend outward; a small amount of Epoxylite (The Epoxylite Corp., Irvine, California) is injected into the tip via the larger opening in the glass rod. The electrodes are then cured in a 125°C-oven overnight. Note that if the tip of the glass rod is broken to a diameter much larger than 30 μm, a tight seal will not form between the protruding carbon fiber and the wall of the glass rod. This will result in leakage of the relatively viscous Epoxylite, which will adhere to the surface of the carbon fiber and render it unusable for electrochemical detection.

In the second stage of construction, the cured electrodes are allowed to cool to room temperature and are then loaded with a graphite epoxy paste (Graphpoxy; Dylon Industries, Inc., Cleveland, Ohio). A beveled wooden applicator stick is used to densely pack the paste into the barrel of the glass rod through the larger opening. The tip of the glass rod should be completely filled with paste and contain no pockets or air bubbles. The final step of this stage is to insert an insulated copper wire, with its bottom 5–10 mm scraped bare, into the paste at a sufficient depth so that it forms a continuous contact with the carbon fiber (via the graphite in the paste). The electrodes are then cured in a 125°C-oven overnight.

The final stage of electrode construction is to cut the carbon fiber with a scalpel blade so that 100–200 μm of it are exposed outside the tip of the glass rod. This step is best performed with the assistance of a stereomicroscope. Lastly, and before they are prepared for use in experiments, we typically clean the electrode tips by swirling them for several minutes in a beaker containing an isopropyl alcohol solution. Note that for recording in awake, freely behaving animals, several modifications to the electrode design are necessary, notably substituting fused silica tubing for the glass, and these are described elsewhere [49] (see also In Vivo Chronic Recording in Freely Behaving Rats).

On the day of experimentation, electrodes are initially prepared by coating the exposed carbon fibers with Nafion (5% w/v solution; Sigma–Aldrich, St. Louis, Missouri), which is a perfluorosulfinated polymer that blocks the absorption of potentially interfering anionic species, such as ascorbic acid (AA) and the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) [50]. The coating consists of dipping the electrode tips into the Nafion solution, swirling them for several seconds, and then drying them in a 200°C-oven for five minutes. High-temperature drying is an important step that helps ensure electrodes will posses optimal selectivity and response times [51]. Electrodes are then tested for both their selectivity for and response to DA. This is accomplished by placing the electrode in a beaker of 100 mM phosphate buffered saline and adding known concentrations of both AA and DA. We typically test responses to 250 μM AA, followed by 2–12 μM DA in 2 μM-increments. Only those electrodes exhibiting a high selectivity for DA over AA (greater than or equal to 1000:1) and a linear response to DA (r2 ≥0.997) are used. Electrodes with a lower selectivity for DA are recoated with Nafion, and the in vitro calibration procedure is repeated, whereas those with an undesirable linear response to DA are not useable (a likely result of a misstep in the construction process). Note that the current response to incremental DA challenges is saved in the computer and later used to convert in vivo measurements of OX current to concentration values.

After the animal has been surgically prepared for the experiment (see below), an electrode/- micropipette assembly is constructed by attaching either a single- or multibarrel pipette to the electrode using sticky wax (Patterson Dental Supply, Aurora, Colorado) that has been liquefied by touching it to a hot metal microspatula. The micropipettes (A–M Systems, Carlsborg, Washington), which are first pulled and broken to a tip diameter of 10–15 μm, are attached so that the tips are parallel and are separated by 150–300 μm (Figure 6.1). The electrode and the micropipette should also be in the same plane as one another (i.e., the tips should be simultaneously in focus under the microscope). Assemblies that do not possess these characteristics typically fail to yield consistent, or even measurable, electrochemical signals once they are implanted in the brain and exogenous DA is applied. A syringe fitted with a flexible microfilament (MicroFil; World Precision Instruments, Sarasota, Florida) is used to fill the single-barrel micropipettes, or one barrel of multibarrel micropipettes, with a DA solution (typically 200 μM in either 154 mM NaCl or 100 mM phosphate buffered saline; pH 7.4, adjusted with NaOH) that also contains 100 μM AA (added to prevent the oxidation of DA in the pipette barrel).

FIGURE 6.1. A picture (a) and schematic diagram (b) of an electrode/micropipette assembly with the micropipette on the left and the carbon fiber electrode on the right.

FIGURE 6.1

A picture (a) and schematic diagram (b) of an electrode/micropipette assembly with the micropipette on the left and the carbon fiber electrode on the right. (a) The picture shows both the assembly (left) and its shadow (right). The micropipette, which (more...)

Surgical Procedures

With the exception of survival surgeries in animals fitted with chronic electrochemical recording electrode implants [52], rats (200–450 g) or mice (15–30 g) are anesthetized with urethane (1.25–1.5 g/kg, i.p.) to a deep surgical level and placed in a stereotaxic frame. We have shown that urethane, unlike chloral hydrate, does not alter exogenous DA clearance, compared to that in an awake, freely behaving rat [53]. Body temperature is maintained at 37°C via a heating pad coupled to a rectal thermometer. A 2-cm incision is made in the scalp under aseptic conditions, which is retracted to expose the surface of the skull. A variable speed rotary tool (Dremel, Racine, Wisconsin) is then used to drill a hole in the skull overlying the brain areas of interest. In our case, these are usually the dorsal striatum (dSTR) and nucleus accumbens (NAc), both of which can be accessed through the single hole in the skull (0.7–1.5 mm anterior and 1.5–2.2 mm lateral to bregma for rats) [54]. An additional hole is drilled just anterior to the interaural line for insertion and attachment (via dental acrylic) of a Ag/AgCl reference electrode. The reference electrode is prepared fresh daily by plating a silver wire (A–M Systems, Carlsborg, Washington) with chloride, which can be done with a 12-V power supply and a plating bath of 1 M HCl saturated with NaCl. An opening is then made in the dura with a sharp probe or needle, and the electrode/micropipette assembly is slowly lowered into the brain under stereotaxic control. For the duration of the experiment, the skull and exposed brain surface are bathed in saline.

Experimental Details

After its initial insertion into the brain, the electrode/micropipette assembly is left undisturbed, at a position above the brain area of interest, for about fifteen minutes to allow the electrode and surrounding brain tissue to stabilize. During this time and continuing throughout the experiment, chronoamperometric measurements are made continuously at 5 Hz, with the +0.55 V OX potential applied for 100 ms followed by a 0.00 V resting potential for 100 ms. The resulting OX and RED currents are digitally integrated during the last 70–90 ms (steady state) of each 100-ms pulse. OX current changes are converted to DA concentrations based on the in vitro calibration of the electrode.

When recordings are made in the rat dSTR and NAc, electrode/micropipette assemblies are lowered 4.0–5.0 mm and 6.5–8.0 mm ventral to the skull, respectively [54]. For each recording, a stable background current is established and set to zero before pressure-ejecting DA (5–20 psi for 0.1–5.0 s) at calibrated volumes (10–300 nl). The pressure ejection is controlled by a Picospritzer II (General Valve Corp., Fairfield, New Jersey), and injection volume is verified through a stereomicroscope fitted with an eyepiece reticule to note the movement of fluid in the pipette barrel. Generally, it is desirable to inject as small an amount of DA as possible that will yield signal amplitudes of between 0.75–4 μM. The volume required to achieve this will vary depending upon a number of factors, including the distance between the tips of the electrode and the micropipette, the sensitivity of the electrode, and the number of DATs present in the brain tissue located near the exposed carbon fiber. For most experiments, DA ejections are made at five-minute intervals, with signals used to determine “baseline” established when signal parameters (see Data Analysis) vary by less than or equal to 15% for two consecutive applications. The importance of a five-minute separation between DA ejections is not entirely clear, but we have observed that it leads to DA signals that are stable for long time periods (Figure 6.2). Following baseline measurements, pharmacological manipulations of DAT function are usually performed, with recordings maintained for an additional thirty minutes or more.

FIGURE 6.2. DA signals measured with HSC in the dSTR of a urethane-anesthetized rat after local pressure ejection of DA (90 nL) every five minutes (solid arrows) are relatively stable after multiple DA ejections.

FIGURE 6.2

DA signals measured with HSC in the dSTR of a urethane-anesthetized rat after local pressure ejection of DA (90 nL) every five minutes (solid arrows) are relatively stable after multiple DA ejections. OX currents were converted to DA concentrations based (more...)

After experiments are completed, and while the animal is still deeply anesthetized, a small current is passed for several seconds through the recording electrode to produce a marking lesion at its tip. This can be done by setting both the applied and resting voltages to 2 V, turning off the headstage gain, and then applying the voltage to the carbon fiber. The animal is then euthanized, and the brain is subsequently removed and stored in buffered formalin (4% w/v) for several days. Coronal sections (40 μm) at the level of the brain area of interest are then cut using a microtome, mounted to glass slides, stained with Cresyl Violet, and analyzed under a light microscope to verify the location of the recording site(s).

Data Analysis

A number of electrochemical signal parameters can be analyzed and used to determine in vivo DAT function. We have commonly used two measures that are obtained from DA OX currents: maximal amplitude (Amax) and signal decay time (T80; Figure 6.3). Amax reflects the maximal extracellular DA concentration detected, whereas T80 is the time for the signal to rise to Amax and decay by 80%. The T80 is chosen because it is in the curvilinear portion of the DA signal curve, takes into account changes in the “tail” of the decay curve where DA concentrations are lower, and is more sensitive to changes induced by DAT uptake inhibitors.

FIGURE 6.3. The baseline capacity of DATs for clearing exogenous DA in dSTR and NAc is reduced in Lewis rats, compared to F344 rats.

FIGURE 6.3

The baseline capacity of DATs for clearing exogenous DA in dSTR and NAc is reduced in Lewis rats, compared to F344 rats. (a) Electrochemical signals measured with HSC in dSTR of a representative Lewis and F344 rat following the local pressure ejection (more...)

Other options for measuring DA clearance that we have used in previous studies include analyses of the pseudolinear portion of the decay curve (T20–60 or T40–60) [55,56] and fitting the curve to a first-order exponential decay function [52]. The latter approach allows one to determine the rate constant (k) for DA clearance by approximating from each signal the initial velocity of DA clearance (v) by setting v = k*Amax. Following an experiment in which increasing amounts of DA are applied, a plot of v versus the amount DA applied can be generated; and an apparent Km and Vmax can be determined from curve fitting of the rectangular hyperbola [52]. Importantly, all of the signal parameters mentioned in this section are affected, to some extent, by DAT inhibitors [52,57] and reflect changes in DA clearance (see Summary of Evidence that this Approach Measures DAT Function). Several other investigators have used similar approaches, while also highlighting other analysis options [58–62].

Because of individual differences in baseline DAT function measures, we typically normalize data when comparing groups of rats by first obtaining a mean value for parameters during a two- or three-ejection baseline period, setting this value as 100% and expressing all data (including the baseline data points) as a percentage of baseline. When suitable, or when individual differences are minimal (e.g., studies in inbred rats), non-normalized data are also presented. Appropriate statistical tests are then conducted (e.g., ANOVA with post-hoc Holm-Sidak tests for between-group comparisons at particular data points).

Results

We have used the techniques described above to analyze baseline differences in DAT function, as well, as the effects of drugs that alter DAT activity. For example, in a recent set of experiments, we compared DAT function in the dSTR and NAc of inbred Lewis and Fischer 344 (F344) rat strains, which are known to exhibit differential behavioral responsiveness to psychostimulants such as cocaine [63]. Consistent with a report in the literature that used quantitative autoradiography to reveal Lewis rats had fewer DAT uptake sites in multiple brain regions [64], we observed that Lewis rats cleared exogenous DA at a slower rate compared to F344 rats (Figure 6.3, panel A). The mean differences in T80 values between the two strains were about 70% in dSTR and about 40% in NAc (Figure 6.3, panel B). In these studies, signal amplitudes were maintained at similar levels (1–2 mm) from one experiment to the next by adjusting the amount of DA ejected once every five minutes from the micropipette within each experiment; thus, there were no group differences in Amax. We also found no group differences in DA ejection volume. We recently reported a similar finding of baseline strain differences in DAT function in the dSTR of inbred long- and short-sleep mice, which exhibit differential responsiveness to the sedative–hypnotic effects of alcohol and also exhibit diverse behavioral responses to cocaine [65].

After measures of basal DAT function were obtained, we assessed the effects of systemically administered d-amphetamine sulfate (AMPH) on exogenous DA clearance in the dSTR of Lewis and F344 rats (Figure 6.4). We determined that the baseline clearance responses were stable before a control saline injection was given three minutes before the third DA ejection (t = 10 min). The saline was injected intravenously (i.v.) through catheters implanted in either the lateral tail vein or the jugular vein. After two additional DA ejections, rats were injected with 0.5 mg/kg AMPH (i.v.) three minutes before the sixth DA ejection (t = 25 min). DA ejections were continued at five minute intervals for fifteen minutes, followed by a second AMPH injection (0.5 mg/kg; i.v.) three minutes before the tenth DA ejection (t = 45 min). DA ejections were then continued at five-minute intervals for fifteen minutes, when the experiment was terminated. Although the i.v. route of drug administration was used here, more convenient systemic routes of drug administration (e.g., subcutaneous or intraperitoneal) can also be used. Furthermore, if assessment of local drug effects is desired, the electrode/micropipette assembly can be constructed with a multibarrel micropipette [57]. DA is loaded into one barrel and the drug solution(s) (pH adjusted to 7.4) is(are) loaded into one or more of the other barrels. Note, however, that local application of drugs must be done with the appropriate controls because particular drugs can alter electrode sensitivity [66].

FIGURE 6.4. Prolongation of the DA signal decay time in dSTR of F344 rats following i.

FIGURE 6.4

Prolongation of the DA signal decay time in dSTR of F344 rats following i.v. d-amphetamine (AMPH). DA was ejected once every five minutes, and OX currents were measured with HSC as in Figure 6.3. There was no significant change in the T80 after saline (more...)

Summary of Evidence That This Approach Measures DAT Function

Two fundamental assumptions of the technique described in this chapter are as follows: (1) the changes in the electrochemical OX signal that are observed after local pressure ejection of a DA solution are the result of a change in local DA concentration and (2) the rise and decay of this electrochemical signal represents the clearance of the applied DA solution by DAT. There are several empirical findings that support the validity of these assumptions.

Questions about the first assumption may seem, at least initially, to be easily addressed given that the change in electrochemical signal results within one second after the time of pressure ejection of the DA solution (Figure 6.3). However, because our system simply measures changes in the OX current that result from an applied potential set to a particular voltage (0.55 V in our experiments), any substance that oxidizes at this voltage has the potential to contribute to current changes. Two substances that fit this profile, AA and DOPAC, are found in abundance in brain areas (e.g., dSTR or NAc) where one might assess DAT function. In fact, AA is a component of the DA solution that we pressure eject from our micropipettes. A common solution to this problem is to coat carbon fibers with Nafion, which significantly reduces the OX and/or RED of AA and DOPAC at the electrode surface, so that their influence on the measured signal is greatly attenuated. Through various means, including local infusions of AA or DOPAC via multibarrel micropipettes, we and others have demonstrated that putative DA signals that are measured with Nafion-coated carbon fiber electrodes are, in fact, primarily attributable to DA [50,56,67–69]. Another strategy for identification of the species that compose the electrochemical signal measured with HSC is to monitor the ratio of integrated RED:OX currents [70]. This ratio, which is calculated at the peak of the electrochemical response, is approximately 0.6–0.8 for DA. It can be distinguished from AA, which has a ratio of 0.0 because it lacks a reduction current, and DOPAC (ratio of ~1.0).

With respect to the second assumption (i.e., the rise and decay of the electrochemical signal represents the clearance of the applied DA solution), there are several empirical reasons to be confident that it is also reasonable. First, a range of detailed kinetic analyses, performed both in vitro and in vivo, have demonstrated that signal amplitude and decay time primarily reflect transporter activity [56,60,71–76]. Diffusion of applied DA does contribute to certain aspects of the signal parameters [58,77], but its contribution, in particular in the dSTR where a large number of DATs are present, appears to be relatively minimal [15,56,78,79]. Second, inhibiting DAT activity has consistently been shown to produce changes in these parameters that are indicative of a reduction in DAT function. For example, Amax, and even more typically T80, are increased by systemic injection or local infusion of DA uptake inhibitors such as mazindol, nomifensine, and cocaine [55,57,71,74,80]. However, structurally similar compounds that lack significant DAT antagonist properties (e.g., lidocaine) have no effect on Amax or T80 [56]. When DAT sites are removed from the brain by 6-OHDA or MPTP lesions of DA cell bodies and nerve terminals in the substantia nigra and STR, Amax [81] and especially T80 [80,81] are increased by up to 130%.

Rapid Regulation of DAT Can Influence Measures of Exogenous DA Clearance

It has been estimated that DATs can be regulated rather rapidly, on the order of several minutes to several hours, upon exposure to both substrates and blockers [22]. In particular, DA has been reported to reduce DAT activity in measures of DAT-associated currents [69] and [3H]DA uptake [82]. D2 receptor agonists have been shown to increase DA uptake by the DAT [83], whereas D2 receptor antagonists have the opposite effect [84,85]. Indeed, D2 receptor ko mice exhibit reduced exogenous DA clearance in dSTR, suggesting that the locally applied DA normally activates D2 autoreceptors and, thereby, increases DAT surface expression and velocity [29,86]. Potentially, however, local application of DA via micropipettes might alter DAT function either directly, through substrate induced down-regulation, or indirectly through its action on D2 autoreceptors. In fact, we have reported [69] that frequent (e.g., every two minutes) applications of DA decreases exogenous DA clearance by the DAT, consistent with substrate-induced DAT down-regulation. Although the regulation of DAT is complex, it is noteworthy that DA applications every five minutes do not appear to change DAT function over time (see Figure 6.2).

Additional Applications of HSC with Local Da Application to Measure DAT Function

In Vitro Recording in Acutely Prepared Brain Slices

The analysis of DAT function in vivo provides the important advantage of measurement in the intact organism, but it leads one to give up some aspects of experimental control. Studies in brain slices [87], which incorporate the same electrode/micropipette assemblies and HSC techniques that we employ in our in vivo studies, provide an in vitro option that regains some of that control. For example, this approach allows for precise anatomical localization of recording electrodes in brain regions of interest. It is relatively easy, using a stereomicroscope, to place electrodes in subregions of the NAc (e.g., the core and shell) that may have different functional populations of DATs. Dose– response analyses of drugs with activity at the DAT are also more practical with in vitro studies because compounds can be bath applied and washed out (at least theoretically) with a relatively rapid time course. Furthermore, multiple brain slices can be tested in a relatively short period of time. For example, the results of an experiment performed in our laboratory demonstrate the effects of different concentrations of cocaine on DAT function in dSTR slices (Figure 6.5).

FIGURE 6.5. Consistent with DAT inhibition, in vitro exposure to cocaine increases DA signal decay time in a concentration-dependent manner in slices containing rat dSTR.

FIGURE 6.5

Consistent with DAT inhibition, in vitro exposure to cocaine increases DA signal decay time in a concentration-dependent manner in slices containing rat dSTR. DA was locally applied at five-minute intervals, and HSC was used to measure OX currents. Cocaine (more...)

For in vitro studies, rats are sacrificed by decapitation; their brains are rapidly removed and mounted on a chilled metal block within a small chamber. This chamber, which is filled with cold aCSF, is then attached to a Vibratome (Series 1000; Ted Pella, Inc., Redding, California) that is used to cut 400 μm-thick coronal sections at the level of the dSTR and NAc. The brain slices are allowed to recover their energy stores and membrane potentials by incubating them at 22°C in oxygenated aCSF for at least one hour before they are used for HSC. The issue of slice recovery and viability is key to the success of this type of experiment. After recovery, the slices are moved to a small electrophysiological- type slice recording chamber in which they are superfused (2 ml/min) with aCSF and maintained at 32°C–33°C. The electrode/micropipette assembly is then lowered into the desired recording area and, after reproducible baseline signals are obtained, drugs (e.g., DAT inhibitors) are introduced into the chamber. We typically administer drugs at one hundred times the desired final concentration, directly into the aCSF flow via a syringe pump (Model A-99; Razel Scientific Instruments, Stanford, Connecticut) set at 1/100 the normal flow rate (i.e., 20 μl/min). Using this technique, we are able to obtain fairly long lasting (greater than or equal to forty-five minutes) and reproducible HSC signals in response to repeated applications of exogenous DA (Figure 6.5).

In Vivo Chronic Recording in Freely Behaving Rats

Along with our collaborators, we have adapted methods of assessing in vivo DAT function with local DA applications and HSC to studies in awake, freely behaving rats [49,52]. This approach, while technically challenging, offers the opportunity to correlate changes in DAT function with behavior, which is especially useful in studies of chronic drug effects. It also removes potential effects of anesthesia on brain function, although urethane does not appear to influence exogenous DA clearance [53]. Several modifications to the general technique described in this chapter are made, including adjustments to the electrode construction, electrode/micropipette assembly, and analysis of the data. They are described in detail elsewhere [49,52] and are summarized in brief below.

To provide the durability necessary for chronic implantation, recording electrodes are constructed from 30 μm carbon fibers sealed in fused silica tubing. Stainless steel guide cannula are then attached to the electrode assembly; these guide the insertion of fused silica tubing used for rapid infusion of exogenous DA at the recording site, which replaces the glass barrel pipettes used in acute experiments with anesthetized rats. The entire assembly is then lowered via stereotaxic control and cemented to the skull with dental acrylic. After a five- to seven-day recovery period, electrochemical recordings are obtained by inserting the injector and attaching to the electrode assembly a miniature potentiostat/headstage, which interfaces with the IVEC-10 system via a tether cable connected to an electrical commutator. Random movement-generated signal artifacts are eliminated with a low-pass filter (cut-off frequency greater than 0.028 Hz) during fast Fourier transformation analysis [52].

We have used this technique to study the effects of both acute and repeated administration of cocaine on in vivo DAT function in the dSTR and NAc [88,89]. The effects of cocaine on the clearance of exogenous DA by the DAT depended on the animal’s behavioral response to the drug. Following an injection of 10 mg/kg cocaine, a reduction in baseline exogenous DA clearance in dSTR and NAc was observed in outbred male Sprague–Dawley rats with an elevated locomotor response to the drug (i.e., high cocaine responders, or HCRs), but not in those with a reduced locomotor response (i.e., low cocaine responders, or LCRs). After six additional injections with 10 mg/kg cocaine on successive days and a seven-day withdrawal period, LCRs exhibited a sensitized behavioral response to a challenge cocaine injection, whereas HCRs exhibited a response similar to the one they had after the first drug injection. In parallel with the behavioral findings in LCRs was an increased ability of cocaine to inhibit exogenous DA clearance by the DAT in dSTR and NAc. Cocaine-induced inhibition of DAT function was unchanged in HCRs from the last cocaine injection compared to the first. These findings, which were interpreted as evidence for an important role of the DAT in individual differences in cocaine responses, highlight the utility of combining in vivo measures of DAT function with studies in behaving animals.

Future Directions in Using HSC to Measure DAT Function

The approach of using HSC combined with local ejection of DA has provided a number of insights about DAT function and its regulation. One need for this approach in the future is further miniaturization of the electrochemical electrode/micropipette assemblies. This would facilitate recording in mice (genetic models), as well as in even more circumscribed brain sub-regions. Obviously, any improvements that enhance recording in freely behaving animals will also be important because these will allow simultaneous behavioral and DAT clearance measurements to be compared. This approach should also be useful in helping to understand the physiological and pharmacological relevance of rapid DAT regulation by allowing us to measure transient changes in in vivo DAT function in the brain.

Acknowledgments

We thank Ms. Melissa Adams for her help in preparing this chapter and acknowledge our grant support from NIDA (DA016485, DA015050, DA004216, DA014204).

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