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

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Chapter 7Determining Serotonin and Dopamine Uptake Rates in Synaptosomes Using High-Speed Chronoamperometry

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Introduction: History of Investigating Monoamine Neurotransmitter Uptake

The use of electroanalytical methods to investigate brain chemistry originated in the laboratory of Ralph N. Adams in 1967, with specific reports on the use of chronoamperometry for neurochemical applications beginning to emerge in the literature in the early 1980s [1–6]. Today, the voltammetry techniques most widely used to investigate various aspects of neuronal intercellular signaling, in addition to chronoamperometry, include constant potential amperometry, differential pulse voltammetry, rotating disk electrode voltammetry and fast cyclic voltammetry (for recent reviews on these techniques see [7–13]). All of these methods have most often been applied to the study of the catecholamine neurotransmitter dopamine, due to the importance of dopamine in locomotor and reward-associated behaviors [14–17]. In addition, dense dopamine projections ramify in neuroanatomically discrete brain regions making this electroactive neurotransmitter highly amenable to investigation by voltammetric methods. Serotonin (5-hydroxytryptamine, 5-HT), like dopamine, is oxidized at potentials less than 1 V at the surface of carbon fiber microelectrodes (Figure 7.1a). However, far fewer investigations have focused on serotonergic neurotransmission, partly because sparse serotonin innervation is highly overlapping with other electrochemically active neurotransmitters, complicating analysis by voltammetry [18–24].

FIGURE 7.1. Chronoamperometry in neuronal synaptosomes.

FIGURE 7.1

Chronoamperometry in neuronal synaptosomes. Delayed-mode high-speed chronoamperometry was used to measure uptake rates in synaptosomes. (a) Changes in current due to serotonin oxidation were recorded in response to a 1 Hz square wave step potential. The (more...)

Serotonin neurotransmitter signaling is integral to many types of complex central nervous system function including the regulation of mood and anxiety states, reward-related behavior, and cognitive function [25–30]. The serotonin transporter (SERT) is a presynaptic 68 kD transmembrane protein that regulates the concentration of serotonin in the extraneuronal space by taking serotonin back up into presynaptic neurons after it has been released [31]. SERT is the primary molecular target for many important therapeutics used in the treatment of depression and anxiety disorders (i.e., the tricyclic antidepressants and the serotonin-selective reuptake inhibitors or SRIs) [32–35], as well as recreationally abused substances including cocaine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) [36–39]. SRIs constitute the most widely prescribed class of antidepressant/anti-anxiety drugs and these agents prevent serotonin inward transport from the extracellular space by blocking the transporter [40–42]. Long-term treatment with SRIs (weeks to months) causes sustained reductions in serotonin uptake that likely lead to prolonged increases in extraneuronal serotonin concentrations [40,43,44]. This fundamental homeostatic alteration in serotonin neurotransmission is thought to cause adaptive responses in pre- and postsynaptic neurons, including long-term changes in gene expression, that ultimately underlie the therapeutic efficacy of the SRIs [29,45,46].

In addition to pharmacologic modulation of the serotonin transporter, genetically controlled alterations in serotonin reuptake occur. A polymorphic element (alternate DNA base sequence) was discovered in the promoter region of the human SERT gene (SLC6A4) [47] that results in 40% reductions in SERT expression in approximately 70% of the normal population [48]. These decreases in SERT have been correlated with increases in anxiety-related personality traits, particularly neuroticism in men and women [48–52] and increased risk for stress-associated depression [53].

Genetically modified mice have become indispensable tools in biomedical research, increasing our understanding of the contributions of various genes to normal behavior, disease states, and their treatment. Mice have been engineered to express reduced amounts of the serotonin transporter [54], dopamine transporter [15, 55], or norepinephrine transporter [56]. Like their human counterparts, mice with reduced SERT expression display an increase in anxiety-like behavior [57– 59]. Thus, SERT deficient mice, particularly those having 50% reductions in SERT expression, appear to model closely the genetic variability in SERT expression widely occurring in the human population [60].

Chronoamperometry has been used to investigate changes in serotonin uptake in SERT deficient mice in the hippocampus in vivo [61]. The results of this study showed that serotonin uptake is decreased in mice lacking one copy of the SERT gene (SERT+/− mice) compared to wild-type mice (SERT+/+ mice). However, these findings contradicted the original report on SERT deficient mice where no differences in synaptosomal serotonin uptake rates between SERT+/− and SERT+/+ mice were found using classic radiochemical methods [38]. Based on these discrepancies, we developed chronoamperometry methods specifically aimed at determining serotonin, and later dopamine, uptake rates in synaptosomes. Synaptosomes are neuronal liposomes prepared from brain tissue that typically contain one or more mitochondria, and they retain glycolytic capabilities [62–64]. They also possess active ion and neurotransmitter transport systems across the plasma membrane.

The advantages of using synaptosomes as a simplified model system to investigate serotonin or dopamine uptake kinetics are twofold. First, numerous transport experiments described in the literature have been performed in synaptosomes; thus, data obtained from electroanalytical investigation of synaptosomal uptake can be directly compared to results using radiochemical methods. Second, by employing synaptosomes, the uptake process can be characterized independently of neurotransmitter release and diffusion, processes that also contribute to changes in neurotransmitter concentrations detected at microelectrodes in intact tissue. Under the conditions described here, the chemical identity and concentration of the neurotransmitter under study can be closely controlled while electrochemically monitoring the uptake process. As a result, chronoamperometry studies in synaptosomes lead to straightforward determinations of the kinetics of serotonin or dopamine uptake as they relate to changes in transporter expression or other genetic or pharmacologic manipulations.

The methodology for using chronoamperometry to measure transporter-mediated uptake into synaptosomes is described here. Alterations in uptake in brain tissue from mice lacking one or both (SERT−/−) intact copies of the SERT gene are detailed and compared with previous studies using either radiometric methods in synaptosomes [38] or chronoamperometry in vivo [61]. The factors underlying the discrepancies between the results of radiochemical and chronoamperometric serotonin uptake experiments are also explored. We show that oxygen saturation of the assay buffer greatly influences clearance rates and that a clear incremental relationship exists between serotonin transporter gene expression and serotonin uptake rates [65,66]. We have discovered that mechanical disruption of synaptosomes by stirring or filtration leads to significant loss of transported serotonin resulting in misleadingly low maximal uptake rates (Vmax) and transporter substrate affinities (Km), as well as a loss of sensitivity for determining biologically important alterations in uptake rates [66]. Finally, we show how chronoamperometry has been used to determine decreases in dopamine uptake in striatal synaptosomes prepared from a transgenic mouse model expressing a mutant form of human α-synuclein, which is associated with a hyperactive phenotype [16].

Chronoamperometry Methods for Determining Uptake in Synaptosomes

Mouse Models of Altered Uptake

SERT deficient mice intact were generated at the National Institute of Mental Health (Bethesda, Maryland) on a mixed CD-1×129S6/SVev background [38,67]. Animals used in the present studies were 4- to 6-month-old male mice bred and genotyped at The Pennsylvania State University and were the product of eight to fifteen generations of SERT+/− brother–sister matings.

α-Synuclein transgenic mice expressing mutant human A53T α-synuclein (A53T-tg) or wild-type human (WT-tg) α-synuclein and nontransgenic littermates (non-tg) were generated as previously described at the Johns Hopkins Medical Institutions (Baltimore, Maryland) [68]. These mice were derived in a C3H/C57BL6-F1 hybrid, and the established lines were maintained by back-crossing to a C57BL/6 background. Male and female transgenic mice and nontransgenic littermates used for the experiments described here were bred and genotyped at Johns Hopkins and transferred to The Pennsylvania State University. These animals were matched for similar low levels of overexpression of the two forms of human α-synuclein [16].

All mice were housed in single sex groups of two to five mice per cage on a 12 h light/dark cycle (0600 h lights on) with food and water ad libitum. The Pennsylvania State University or Johns Hopkins Medical Institutions Institutional Animal Care and Use Committees approved all animal procedures, which were conducted in strict accordance with National Institutes of Health Animal Care Guidelines.

Carbon Fiber Microelectrode Construction

Cylindrical electrodes were prepared according to the method of Gerhardt and coworkers [69,70] with minor modifications. Microelectrodes were constructed by aspirating 30 μm carbon fibers (Textron Specialty Materials, Lowell, Massachusetts) into 1.2 mm×0.68 mm, 4″ borosilicate glass capillaries. An automatic micropipette puller was used to pull the glass capillaries to create two electrodes. Electrodes were dipped in epoxy (Polysciences Inc., Warrington, Pennsylvania) for five to seven minutes, followed by acetone for five to ten seconds, and heated overnight at 100°C. Carbon fiber tips were cut to a final length of ~100 μm (Figure 7.1c). Nafion (Sigma–Aldrich, St. Louis, Missouri), an anion exchange resin, was coated onto carbon fiber surfaces to increase selectivity and sensitivity for cationic neurotransmitters [71,72]. Electrodes were dipped in Nafion for 10 s and then dried for five minutes at 150°C. This was repeated three to four times with the numbers of Nafion coatings being periodically adjusted to obtain optimal selectivity. Each electrode was filled with 4 M potassium acetate/150 mM potassium chloride to make electrical contact between the carbon fiber and a silver wire to which a gold pin connector (Newark Electronics, Long Island, New York) was soldered.

Preparation of Synaptosomes

Synaptosomes were prepared using a modification of the technique reported by Hyde and Bennett [73]. Brain stem (including tissue containing the dorsal and median raphe nuclei), frontal cortex, and bilateral striata from two mice per genotype were quickly dissected over ice. Tissues were pooled by brain region and genotype, and homogenized by hand in 10 volumes (ml/mg wet tissue weight) Tris-sucrose buffer (0.5 mM Tris–HCl, 0.32 mM sucrose, pH 7.4) with a glass mortar and Teflon pestle. Homogenates were centrifuged at 2000 g for 10 min. The supernatants were carefully removed and centrifuged again at 16,000 g for 10 min. The pellets were then resuspended in 2 mL of assay buffer (124 mM NaCl, 1.80 mM KCl, 1.24 mM KH2PO4, 1.40 mM MgSO4, 2.50 mM CaCl2, 26.0 mM NaHCO3, 10.0 mM glucose, saturated with 95% O2/5% CO2, pH 7.4 with phosphoric acid). A 50 μL aliquot of each homogenate was reserved for protein analysis according to the method of Lowry et al. [74]. Chronoamperometric experiments were conducted using protein concentrations ranging from 0.4 to 0.9 mg/mL protein.

Precalibrated microelectrodes and reference electrodes were placed in synaptosomes in a final volume of 2 mL. A+0.55 V pulsed potential was applied and the current was recorded until a stable baseline was obtained (Figure 7.1b). Serotonin or dopamine (20 μL) was added to each synaptosomal solution and the change in current with respect to time was recorded. Where indicated, synaptosomal preparations from SERT+/+ mice were preincubated with paroxetine (1.0 μM final concentration) or fluoxetine (100 nM final concentration) for 30 min. Before the addition of serotonin for uptake measurements, synaptosomes were centrifuged briefly at 16,000 g and brought up in fresh oxygenated assay buffer.

Chronoamperometry and Electrode Calibration

High-speed chronoamperometry was performed in the delayed-pulse mode [70]. Changes in current were recorded in response to a 1 Hz square wave potential step generated by an IVEC-10/FAST-12 potentiostat (Quanteon Center for Sensor Technology, Lexington, Kentucky). A 100 ms oxidative pulse at +0.55 V was followed by a 100 ms reductive pulse at 0 V (Figure 7.1a). The resulting current was integrated over the last 80 ms of each oxidative and reductive phase. The potential was maintained at 0 V for an additional 800 ms (delayed-mode) to prevent fouling of the electrode [72,75]. Voltage at the carbon fiber working electrode was applied with respect to a Ag/AgCl glass reference electrode (BAS, West Lafayette, Indiana).

Microelectrodes were calibrated over a concentration range of 0.25–1.5 μM for serotonin or 0.25–1.25 μM for dopamine in phosphate buffered saline (pH 7.4) to determine linear responses. They were challenged with 0.5 μM ascorbate before calibration with serotonin or 0.25 μM 3,4-dihydroxyphenylalanine (DOPAC) prior to dopamine calibration to determine the sensitivity and selectivity of the Nafion coating for cations. Redox ratios between 0.1 and 0.3 for serotonin or 0.5 and 0.7 for dopamine were obtained. Only electrodes with high linear responses (r2>0.99) and cation selectivity ratios greater than 700:1 were used in subsequent experiments.

Because electrode sensitivity decreases rapidly by ~60% and then stabilizes as a result of exposure of electrodes to synaptosomes [65], precalibration was performed to evaluate the sensitivity and linear response of each electrode prior to use in experiments. After uptake experiments were complete, all electrodes were recalibrated and data analysis was performed using postcalibration standard curves. Uptake rates are expressed as pmol 5-HT or DA taken up/mg protein-min. Uptake rates are reported as “not detectable” when TC (defined below) was less than 0.1 nM/s over the course of 20 min.

HPLC Analysis of Synaptosomal Filtrates

To determine the amount of serotonin lost from synaptosomes during filtration, synaptosomal filtrates were analyzed by high performance liquid chromatography with electrochemical detection (HPLC-ED) using an ESA Coulochem II electrochemical detector (E1 = −175 mV and E2 = +220 mV; Chelmsford, Massachusetts) with minor modifications [76]. After observing complete uptake of 1 μM serotonin into brain stem synaptosomes by chronoamperometry (currents returned to baseline after ten to 20 min depending on SERT genotype), synaptosomal suspensions (2 mL) were filtered (300 mm Hg) through Whatman GF/C filters presoaked in 2% polyethyleneimine using a Millipore sampling manifold (Bedford, Massachusetts) so that filtrates from individual samples could be collected. Samples were then washed with 500 μL of assay buffer. Aliquots of initial filtrates and washes (20 μL each) were injected onto a 100×4.6 mm Spherisorb 3 μm octadecylsulfate reversed-phase chromatography column (Thomson Instruments, Springfield, Virginia) in a mobile phase containing 0.1 M monochloroacetic acid, 8% acetonitrile, 0.5 g/L octanesulfonic acid, 0.3% triethylamine, and 10 μM EDTA at a flow rate of 0.7 ml/min. Serotonin concentrations were quantified by comparing peak areas to a standard curve (0–10 μM 5-HT), and the total amount of serotonin detected in the filtrate from each sample was obtained by adding the amount of serotonin detected in the initial filtrate to that detected in the wash.

Data Analysis and Statistics

For chronoamperometric recordings, the rate of uptake is estimated from TC, the slope of the electrochemical signal between T20 and T60 (times needed to transport 20% and 60% of the total neurotransmitter added, respectively; Figure 7.1d) [70]. To determine Vmax and Km values for 5-HT uptake, data were fit to a one-site hyperbolic function for uptake rates versus initial 5-HT concentrations using Equation 7.1 (GraphPad Prism, San Diego, California). Here, uptake rate is the concentration-dependent TC, Vmax is the maximal rate of uptake, Km is the affinity of the transporter for 5-HT, and [5-HT] is the initial concentration added to synaptosomal suspensions.

Uptake rate=Vmax×[5-HT]Km+[5-HT]
7.1

Data were analyzed statistically by a student’s t-test or one-way analysis of variance (ANOVA) with genotype as the independent variable using the Statistical Analysis System (SAS Institute, Carey, North Carolina). A priori differences between groups are indicated by t-test probabilities.

Characterization of Microelectrode Responses

We assessed carbon fiber microelectrode responses to serotonin over a concentration range of 0.25–1.5 μM. Both oxidative and reductive responses were found to be highly linear before and after the exposure of electrodes to tissue [65]. However, contact of electrodes with solutions containing synaptosomes reduced electrode responsiveness [65]. Upon initial exposure to synaptosomes, baseline currents dropped steadily for about 30 min, after which they soon stabilized. Uptake measurements were made only after baseline currents had stabilized, and data was collected with a particular electrode only as long as the baseline current remained relatively stable. The response of a large group of electrodes (n = 41) to tissue exposure was systematically evaluated. The average decrease in the response factor for serotonin was 57±2% and the mean reduction in the redox ratio was 23±2%. As a result, all electrodes were recalibrated at the end of each experiment, and data analysis was performed using postcalibration values to account for reductions in electrode sensitivity occurring as a result of tissue exposure.

Effects of Oxygen on Synaptosomal Uptake of Serotonin

The ability of SERT to transport serotonin actively from the extracellular space depends on the co-transport of Na+, K+ and Cl ions down their respective concentration gradients [31]. The Na+/K+-ATPase, which uses the chemical energy stored in ATP to fuel the extrusion of Na+ from and the accumulation of K+ into cells, produces these ion gradients. Neurons rely heavily on oxidative respiration to produce large amounts of ATP, and thus, to maintain ion gradients, which are essential for the reuptake of neurotransmitters, as well as for the generation of action potentials. Therefore, normal function of SERT is energetically coupled to the Na+/K+-ATPase and depends on mitochondrial respiration and the presence of O2 to produce high levels of ATP. Synaptosomes, which are sealed presynaptic structures, maintain the ability to take up oxygen and glucose, as well as the capacity to respire and maintain normal membrane potentials [77–79]. Based on this, we evaluated the effects of assay buffer oxygen saturation on uptake rates using chronoamperometry. Serotonin uptake rates were measured in unstirred synaptosomal solutions prepared from SERT+/+ mice [65]. Each sample was divided and half was incubated in nonoxygenated assay buffer, while the other half was incubated in assay buffer saturated for 30 min with 95% O2/5% CO2. In the absence of O2, uptake of 1 μM 5-HT was detected in only three of six samples (mean uptake rate 21±4 pmol/mg protein-min). In the remaining three samples, uptake was not detectable over a twenty-minute period. In the corresponding oxygenated samples, the mean 5-HT uptake rate was 126±8 pmol/mg protein-min (n = 6), demonstrating a tenfold increase in uptake rates. These data support the conclusion that oxygen is an essential component in synaptosomal preparations for optimal function of monoamine transporters.

Effects of Stirring on Synaptosomal Uptake of Serotonin

Rotating disk electrode voltammetry has been used to study neurotransmitter transport kinetics in minced tissue and cultured cells [80–84]. One of the main advantages of this technique is that uptake can be determined under conditions in which the effects of diffusion and mass transport are minimized by the rapid rotation of the electrode [84]. To reduce the effects of mass transport in our system, synaptosomal solutions were stirred and the effects on uptake were determined. However, in contrast to chopped tissue or whole cells, stirring synaptosomal suspensions (2 mL total volume) with a miniature magnetic stirring bar (6 mm length, 2 mm diameter) at the slowest possible stirring plate speed while measuring serotonin clearance by chronoamperometry eliminated measurable uptake [65]. In fact, uptake was not detectable for as long as 90 min after the addition of 5-HT, even in the presence of O2 in stirred synaptosomes (n = 3). We hypothesize that this is because of leakage of transported 5-HT from synaptosomal structures caused by stirring. Unlike intact tissue or cells, synaptosomes have disrupted intracellular cytoskeletal and extracellular matrix elements caused by the tissue homogenization process.

Effects of Uptake Inhibitors on Synaptosomal Uptake of Serotonin

Many drugs that selectively inhibit the serotonin transporter are commonly used in the treatment of mood and anxiety disorders [85]. Paroxetine (trade name Paxil®) [86–88], a compound that belongs to this class of drugs, was used to test the hypothesis that time-dependent reductions in the chronoamperometric signal in response to the addition of 5-HT to synaptosomes occurs specifically because of serotonin transporter activity. For these experiments, synaptosomal samples were divided. Half of each sample was used to measure serotonin uptake in the absence of paroxetine (saline preincubation), while the other half was preincubated with 1 μM paroxetine for 30 min. After preincubation, both sets of samples were briefly centrifuged and brought up in fresh oxygenated assay buffer. Evaluation of serotonin uptake in saline pretreated striatal synaptosomes from SERT+/+ mice yielded a mean uptake rate of 153±12 pmol/mg protein/min (n = 6). By contrast, uptake was not detectable in striatal synaptosomes preincubated with paroxetine (n = 6) [65].

Uptake was also determined in brain stem synaptosomes preincubated with 100 nM fluoxetine (trade name Prozac®), another selective serotonin uptake inhibitor [66]. No uptake of 5-HT was detected by chronoamperometry in synaptosomes pretreated with fluoxetine (Figure 7.2a) [66]. After 20 min of chronoamperometric recording, synaptosomes were filtered to separate them from the surrounding solution and 95% of the serotonin added was detected in the filtrate using HPLC (Figure 7.2b). This further indicates that serotonin is not taken up in synaptosomes pretreated with fluoxetine. These results demonstrate that the reduction in the electrochemical signal with respect to time after addition of 5-HT to synaptosomes is attributable to uptake of serotonin by SERT and is not the result of baseline drift, 5-HT oxidation by the microelectrode or uptake by other monoamine neurotransmitters co-expressed in the tissues studied.

FIGURE 7.2. Effects of fluoxetine and filtration.

FIGURE 7.2

Effects of fluoxetine and filtration. Synaptosome samples from brain stem (n = 3) were divided, and half of each sample was preincubated with either 100 nM fluoxetine in assay buffer or assay buffer alone for 30 min. Samples were then briefly centrifuged (more...)

Inactivation of Serotonin Transporter Expression Results in a Gene Dose-Dependent Reduction in Serotonin Uptake

Serotonin-transporter deficient mice lack either one or both copies of the SERT gene constitutively (from conception throughout life). Previous studies have demonstrated that SERT+/− mice have ~50% reductions in serotonin transporter protein levels throughout the brain, including in the striatum, frontal cortex, and brain stem. SERT−/− mice fail to express the serotonin transporter in all brain regions [54,66]. Gene dose-dependent increases in extracellular serotonin levels occur in striatum and frontal cortex in SERT+/− and SERT−/− mice [89]. Furthermore, locomotor responses to the psychomotor stimulant MDMA are attenuated in SERT+/− mice and absent in SERT−/− mice [38]. These observations, and particularly those in SERT+/− mice, indicate that mice lacking one intact copy of the SERT gene are suitable models for investigating alterations in neurochemistry and behavior as they relate to the human SERT promoter polymorphism.

Uptake rates in synaptosomes prepared from the three different genotypes of SERT knockout mice were evaluated using chronoamperometry in synaptosomes prepared from the striatum, frontal cortex, and brain stem [65,66]. Striatum is a brain region that is important for the control of movement, and degeneration of neurons projecting to this brain area is the hallmark of Parkinson’s disease [25]. Additionally, ventral striatum (nucleus accumbens) has long been associated with reward-related behavior and drug addiction [17,90]. The frontal cortex is thought to be important in arousal, cognition and emotional states, and the modulation of personality [91,92]. The serotonin neuronal cell bodies are located in the brain stem, and high levels of SERT are also expressed in this brain region [66]. We hypothesized that gene dose-dependent decreases in SERT gene expression in these three brain regions would lead to parallel reductions in serotonin uptake rates and that these changes would be detectable by chronoamperometry.

Representative recordings from brain stem synaptosomes after the addition of serotonin are shown in Figure 7.3. The data indicate that 1 μM (2 nmol) 5-HT is taken up by brain stem synaptosomes from SERT+/+ mice in about 10 min (Figure 7.3a). By contrast, it took about 17 min to take up the same amount of 5-HT into brain stem synaptosomes prepared from SERT+/− mice (Figure 7.3b). No uptake of 5-HT occurred in brain stem synaptosomes from SERT−/− mice (Figure 7.3c).

FIGURE 7.3. Serotonin uptake in brain stem synaptosomes from SERT deficient mice.

FIGURE 7.3

Serotonin uptake in brain stem synaptosomes from SERT deficient mice. Representative oxidative traces after the addition of 1 μM serotonin to brain stem synaptosomes from (a) SERT+/+ mice, (b) SERT+/− mice and (c) SERT−/− (more...)

Table 7.1 shows mean serotonin clearance rates across the three genotypes of SERT deficient mice in different brain regions. In SERT+/− mice, serotonin uptake was significantly reduced by ~60% in the striatum [t(16) = 9.6; p<0.0001], frontal cortex [t(7) = 7.5; p<0.0001], and brain stem [t(8) = 5.8; p<0.001] compared to SERT+/+ mice. Serotonin uptake was not detectable in any brain region in SERT−/− mice. These data support the theory that gene-related reductions in SERT expression result in similar magnitude decreases in the rates of transporter-mediated serotonin clearance.

TABLE 7.1

TABLE 7.1

Serotonin Uptake Rates Determined by Chronoamperometry in Synaptosomes

Uptake Kinetics of Serotonin by Chronoamperometry

Concentrations of 1 μM serotonin were chosen for the uptake experiments described above because this concentration was originally thought to be at least tenfold higher than the Km (~0.05 μM) of the serotonin transporter for 5-HT based on our previous radiochemical studies of serotonin uptake [54]. However, uptake rates measured by chronoamperometry ended up being much higher than those determined by radiometric assay [54,65,66]. For example, Vmax values reported for radiochemical analysis of serotonin uptake in synaptosomes typically range from 0.2 to 5 pmol/mg protein-min [54,93–95]. Serotonin uptake rates determined by chronoamperometry are ~150 pmol/mg protein-min (Table 7.1). In light of this, the kinetic parameters Km and Vmax for serotonin uptake in striatal synaptosomes were reevaluated by high-speed chronoamperometry using concentrations of 5-HT ranging from 0.25 to 1.25 μM.

Figure 7.4 shows the mean kinetic curves for SERT+/+ and SERT+/− mice. Maximal uptake rates determined by nonlinear curve fitting were 198±16 and 118±9.4 pmol/mg protein-min in SERT+/+ and SERT+/− mice, respectively, and these were significantly different from each other [t(16) = 3.60; p<0.01]. Affinity constants were 1.1±0.1 and 0.8±0.1 μM for SERT+/+ and SERT+/− mice, respectively, and these were not significantly different [t(16) = 1.83; p<0.09]. Kinetic analysis of serotonin uptake in SERT−/− mice was not carried out because it had been demonstrated previously that striatal synaptosomes derived from these mice do not take up serotonin [65]. These data further substantiate that inactivation of one copy of the SERT gene results in a 50% reduction in maximal uptake rates, while affinity of SERT for 5-HT is unchanged. Moreover, they further point to large discrepancies between serotonin uptake rates measured by voltammetric versus radiometric methods.

FIGURE 7.4. Kinetic analysis of serotonin uptake in striatal synaptosomes from SERT deficient mice.

FIGURE 7.4

Kinetic analysis of serotonin uptake in striatal synaptosomes from SERT deficient mice. Data represent mean uptake rates ±SEM from eight to eleven determinations per data point. Data were analyzed by nonlinear curve fitting to a one-site hyperbolic (more...)

Chronoamperometry versus Radiochemical Methods: Why Such Discrepancies in Uptake Rates?

One of our major impetuses for developing chronoamperometry methods to measure uptake rates in synaptosomes was to facilitate direct comparisons between kinetic parameters determined by voltammetric versus radiochemical methods using the same tissue preparation. Thus, we attempted to determine why uptake rates determined by radioassay are so much lower than those obtained by chronoamperometry. The first point of consideration is that there are minor differences in the assay conditions between the two types of analyses. The assay buffer that we use for the radiometric assay is similar to that used for chronoamperometry studies with the exception that ascorbic acid and pargyline are included in the former [54,66]. In addition, higher assay temperatures are typically used for radiometric assay (37°C versus 24°C). However, these differences in conditions are expected to maximize rates of uptake in the radiochemical assay by minimizing the oxidation of serotonin, preventing its metabolism by monoamine oxidase, and taking advantage of the temperature dependence of transport rates, respectively. Thus, these minor methodological differences would not account for the greatly lower uptake rates determined by the radiochemical method (compare uptake rates in Table 7.1 with those in Table 7.2).

TABLE 7.2

TABLE 7.2

Effects of Oxygen Saturation of the Assay Buffer on the Kinetics of [3H]5-HT Uptake

The second point to consider is that oxygenation of the assay buffer in chronoamperometry experiments increases the rates of serotonin uptake by sevenfold [65]. We assessed whether the same magnitude of effect is observed in radiometric uptake experiments. As discussed above, oxygen is essential for optimal functioning of the monoamine transporters, which depend on the Na+/K+ ATPase to maintain the ion gradients necessary for neurotransmitter transport [96,97]. As such, we systematically reevaluated uptake of [3H]5-HT in synaptosomes from SERT+/+ mice using an oxygen saturated assay buffer and standard radiochemical assay conditions [66]. The results of these studies showed that Vmax values were only slightly increased in frontal cortex and brain stem synaptosomes, while Vmax in striatal synaptosomes was not significantly altered (Table 7.2). Oxygenation of the assay buffer had no effect on Km values, which were still much lower than those measured in chronoamperometry experiments. Thus, despite the fact that the maximal uptake rates determined by the radiometric assay were slightly increased in the presence of oxygen, the magnitude of this increase was considerably smaller than that observed in chronoamperometry experiments. Furthermore, it did not account for the much larger uptake rates calculated by the latter method.

The third point that we considered was the hypothesis that loss of 5-HT during the filtration process used in the radiometric assay accounts for the substantially lower maximal uptake rates and affinity constants measured by this method. As discussed above, stirring of synaptosomal suspensions has detrimental consequences on 5-HT uptake into synaptosomes. Similarly, filtration through a cell harvester or filter manifold requires the use of vacuum pressure to separate synaptosomes and the neurotransmitter they have taken up from the neurotransmitter remaining in the extrasynaptosomal solution. Synaptosomes are further subjected to three to four washes under vacuum pressure with assay buffer before scintillation counting of the filter paper onto which they have been retained.

We have carried out experiments to determine the concentration of serotonin in filtrates (extrasynaptosomal solution) following uptake of 1 μM 5-HT into synaptosomes [66]. Once complete uptake of 5-HT was observed by chronoamperometry (Figure 7.5a), synaptosomal suspensions were filtered through Whatman GF/C filters using a filter manifold (Figure 7.5b). This apparatus permits collection of individual filtrates, which is not feasible using a cell harvester, which is typically used for radiochemical experiments. We hypothesized that if filtration disrupted synaptosomes similar to what we had observed with stirring, then the 5-HT that had been taken up would be subsequently detected in the extrasynaptosomal filtrates (Figure 7.5c). Filtrates were analyzed by HPLC-ED and this confirmed that 75% of the serotonin that had been taken up into synaptosomes was later detected outside of the synaptosomes in the filtrate (see also Figure 7.2b). These observations have lead us to conclude that loss of transported 5-HT during the filtration process used in the radiochemical method leads to: (1) significantly lower uptake rates calculated by this method compared to chronoamperometry; (2) an underestimation of the potency of the effects of oxygen in the radiochemical assay; and (3) a relative lack of sensitivity of the radiometric assay for determining subtle but important alterations in uptake rates predicted by the biology of the system, such as those occurring between SERT+/+ and SERT+/− mice.

FIGURE 7.5. Measuring serotonin in extrasynaptosomal filtrates.

FIGURE 7.5

Measuring serotonin in extrasynaptosomal filtrates. (a) Serotonin (1 μM) was added to each sample and uptake was monitored for 20 min by chronoamperometry. (b) Once uptake was complete, samples were filtered individually using aMillipore filtration (more...)

Determination of Dopamine Uptake in α-Synuclein Transgenic Mice by Chronoamperometry

Genetic and biochemical abnormalities associated with the abundantly expressed synaptic protein α-synuclein have been linked to the etiology of Parkinson’s disease (PD) [98]. Two point mutations in α-synuclein have been discovered in rare heritable forms of PD: an alanine to proline substitution at position 30 (A30P), and an alanine to threonine mutation at amino acid 53 (A53T) [99–102]. Mutant and normal α-synuclein proteins have been shown to accumulate in Lewy bodies and Lewy neurites, which are characteristic brain pathologies of inherited, as well as the much more commonly occurring forms of sporadic PD. Mice have been genetically engineered to produce normal and mutant forms of human α-synuclein [68], and expression of A53T mutant human α-synuclein results in a striking adult-onset hyperactive phenotype [16] that is later followed by a fatal motoric syndrome in mice expressing high levels of A53T α-synuclein. Based on this complex phenotype and our observations of reduced striatal dopamine transporter expression in A53T-tg mice, we investigated whether concomitant changes in dopamine uptake occur in these mice.

Dopamine uptake rates in synaptosomes prepared from striatum from A53T-tg mice, WT-tg mice, and the corresponding non-tg littermate mice were evaluated by high-speed chronoamperometry using the same conditions as those developed for analyzing serotonin uptake. Similar to serotonin, when 1 μM dopamine is added to synaptosomes, the current rises quickly as dopamine is detected at the carbon fiber microelectrode surface. As dopamine is taken up by the dopamine transporter the current decreases and the slope of the current decay curve between the time needed to clear 20% and 60% of the added dopamine is calculated and reported as the dopamine uptake rate in pmol/mg protein-min. One-way ANOVA of uptake data gleaned from α-synuclein transgenic mice demonstrated a significant effect of genotype [F(3,17) Z 3.31, p<0.05]. In A53T-tg mice, dopamine clearance rates were 40% lower than those measured in A53T non-tg mice (Figure 7.6). Mean uptake rates were 32±6 and 53±5 pmol/mg protein/min for A53T-tg and A53T non-tg mice, respectively. By contrast, no significant differences in dopamine uptake were detected between WT-tg mice and the corresponding non-tg littermates (49±5 and 42±4 pmol/mg protein/min, respectively). Additionally, uptake rates in A53T-tg mice were significantly decreased compared to WT-tg mice. These data show that the expression of A53T mutant human α-synuclein in mice and the resulting hyperlocomotive phenotype is associated with reduced dopamine uptake in striatum [16]. By contrast, expression of human wild-type α-synuclein does not increase locomotor activity and has no discernable effect on striatal dopamine clearance. From a more global perspective, the results of this study demonstrate that chronoamperometry can be used to measure dopamine, in addition to serotonin, uptake rates and that this technique is sufficiently sensitive to detect modest changes in monoamine neurotransmission in animal models in which the primary genetic alteration may not directly impact transporter expression but in which the behavioral phenotype is indicative of alterations in neurotransmission.

FIGURE 7.6. Dopamine uptake rates measured by chronoamperometry in striatal synaptosomes from α-synuclein transgenic mice.

FIGURE 7.6

Dopamine uptake rates measured by chronoamperometry in striatal synaptosomes from α-synuclein transgenic mice. Uptake rates were determined after the addition of 1 μM dopamine to synaptosomes prepared from striatum from A53T-tg, WT-tg, (more...)

Conclusions

Traditional radiochemical methods have been used by neurobiologists to measure uptake kinetics for fifty years. These methods require the accumulation of radiolabeled neurotransmitter over the course of minutes. Subsequent determination of uptake rates is based on a single measurement per sample. As such, these types of methods do not offer high temporal resolution of the time course of uptake and, thus, provide significantly less information about transport kinetics. Chronoamperometry, by contrast, yields information on changes in the concentrations of serotonin or dopamine resulting from uptake on a per second basis leading to excellent temporal resolution of uptake rates over the total time period under investigation. The result is improved sensitivity to changes in uptake rates.

The data from the chronoamperometry studies described above support the conclusion that active uptake of serotonin is completely absent in the brains of SERT−/− mice, which concurs with the radiochemical results and SERT protein expression levels published by Bengel et al. [38]. In in vivo experiments by Montanez and co-workers, clearance of exogenously applied serotonin was significantly reduced but not abolished in SERT−/− mice [61]. Clearance rates increased linearly with increasing concentrations of 5-HT in SERT−/− mice, in contrast to saturable uptake rates as a function of concentration of 5-HT in SERT+/+ and SERT+/− mice. These data suggest that under normal conditions involving intact tissue, serotonin clearance is a combination of transporter-mediated (saturable) and diffusional (nonsaturable) processes. They also highlight an advantage of using synaptosomes to differentiate between these two processes. Because synaptosomal uptake is measured in finite volumes, changes in the concentration of serotonin can be attributed to active transport, as the studies described above demonstrate, particularly those involving uptake inhibitors or SERT−/− mice. Furthermore, the lack of change in the electrochemical signals in synaptosomes derived from SERT−/− mice after the addition of serotonin is a strong indication that SERT-mediated uptake of serotonin is completely absent in these mice.

In addition to greatly improved temporal resolution, electrochemical studies have revealed that the filtration process used during radiochemical analysis disrupts synaptosomal membranes leading to a loss of transported neurotransmitter. This finding is significant because it explains a number of major discrepancies between uptake rates determined by radiometric versus chronoamperometric methods. Namely, imprecise determination of the amount of neurotransmitter taken up as a function of time accounts for the inability of the radiochemical method to accurately detect reductions in serotonin uptakes rates in with intermediate reductions in SERT expression. Furthermore, it leads to a gross underestimation of the effects of oxygen to increase uptake rates. Finally, these findings explain the considerably lower maximal uptake rates and affinity constants determined radiochemically compared to those measured by chronoamperometry. Specifically, chronoamperometry experiments employing full kinetic analysis indicate that serotonin uptake rates in synaptosomes are approximately 100-fold higher than those commonly reported from radiochemical studies (e.g.~200 pmol/mg protein-min versus ~2 pmol/mg protein-min, respectively) [54,65,66,93–95]. Moreover, the affinity of SERT for 5-HT is calculated to be ~1 μM in the present chronoamperometry experiments in synaptosomes, as well as by in vivo chronoamperometry studies, as opposed to ~0.05 μM reported in radiometric studies [54,61,66].

In addition to determining serotonin or dopamine uptake as described above, chronoamperometry will also be useful for measuring norepinephrine transport into synaptosomes. We anticipate that future studies aimed at directly comparing kinetic parameters determined by chronoamperometry versus radiochemical methods for dopamine and norepinephrine will yield similar results to those reported for serotonin in that maximal uptake rates and transporter affinities are expected to be greatly underestimated by radiometric analysis.

The methods developed here can also be applied to the investigation of transporter-mediated monoamine efflux or “reverse transport” by preloading synaptosomes with serotonin, dopamine or norepinephrine and monitoring increases in their concentrations with respect to time at carbon fiber microelectrodes upon addition of monoamine releasers, such as the substituted amphetamines [103]. Chronoamperometry methods are also highly amenable to transport studies in cultured cells. The use of smaller Ag/AgCl wire reference electrodes and carbon fiber working electrodes presents further possibilities for studying uptake in reduced volumes and for multiplexing. Microelectrode arrays could be used for the simultaneous determination of uptake in multiple samples in well-plate type assays for high throughput drug screening applications. In all cases, the use of chronoamperometry to investigate monoamine transporter kinetics obviates the need for radioactivity and is expected to lead to more accurate determinations of the kinetic parameters of monoamine transport and to improved sensitivity to changes in Vmax and/or Km occurring in response to genetic or pharmacologic manipulations.

Acknowledgments

This research was supported by a grant from the National Institute of Mental Health (MH64756). Many individuals contributed to the work presented here and the authors would like to express sincere appreciation to Dr. Erica Unger, Dr. Francesca Coppelli, Laura Bianco, Drew Kiraly, Dawn Reichenbach, Dawn Rupp, and Christian Squallanti for their technical assistance; Dr. Dennis Murphy for his generous gift of the SERT deficient mice breeding pairs; Dr. Michael Lee for the α-synuclein transgenic mice; Dr. Jim Schenk for his expert technical advice regarding electrochemical methods; and Drs. Lyn Daws, Andrew Ewing, Greg Gerhardt, and Paul Weiss for their insightful discussions regarding related manuscripts.

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