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

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Electrochemical Methods for Neuroscience.

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Chapter 5High-Speed Chronoamperometry to Study Kinetics and Mechanisms for Serotonin Clearance In Vivo

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Introduction: Serotonin and the Serotonin Transporter

Serotonin (5-HT) regulates many complex behaviors and physiological functions. These include mood, sleep, feeding and thermoregulation, to name but a few. As a result, dysregulation of 5-HT neurotransmission can have severe consequences. For example, reduced serotonin neurotransmission is thought to underlie such affective disorders as depression and anxiety (Owens and Nemeroff 1994; Nemeroff and Owens 2004) and predispose to addictive disorders such as alcoholism (McBride et al. 1993; Virkkunen and Linnoila 1997). In addition, the hyperserotonergic state resulting from high doses of substituted amphetamines such as 3,4- methylenedioxymethamphetamine (MDMA, “Ecstasy”) contributes to the “serotonin syndrome,” symptoms of which include agitation, high body temperature, tachycardia, convulsions, coma, and in some cases death (Hegadoren et al. 1999; Green et al. 2003). Given the clear importance of maintaining homeostatic 5-HT neurotransmission, the regulation of extracellular concentration of 5-HT has been the focus of intense study for the past several decades.

The principle mechanism for terminating the neurochemical actions of 5-HT released from nerve terminals is its high affinity uptake from extracellular fluid by the serotonin transporter (5-HTT; SERT). Consequently, since this transporter was first identified in the brain (Shaskan and Snyder 1970; Kuhar et al. 1972), it has been studied extensively (Fuller and Wong 1990; Blakely et al. 1991; Rudnick and Wall 1993; Bengel et al. 1998; Blakely et al. 1998; Kilic et al. 2003). Until relatively recently, active uptake of 5-HT by its transporter has been studied using in vitro techniques such as [3H]5-HT uptake into brain synaptosomes (Shaskan and Snyder 1970; Kokoshka et al. 1998) or into cells transfected with the 5-HTT (Wall et al. 1995; Ramamoorthy and Blakely 1999; Zhu et al. 2005). However, modern voltammetric technologies that stemmed from the pioneering work of Ralph Adams in the 1970s (Kissinger et al. 1973) have allowed 5-HTT function to be measured in vivo. Herein we describe applications of high-speed chronoamperometry, an amperometric technique, to study 5-HTT function in vivo as well as to identify alternative mechanisms for 5-HT clearance from extracellular fluid.

Voltammetric Techniques Used to Study Kinetics of Serotonin Clearance in Brain

As described throughout this book, many forms of voltammetry/amperometry exist (see also Kawagoe et al. 1992; Michael and Wightman 1999; Gerhardt and Burmeister 2000). In simplest terms, they are distinguished by the manner in which a potential is applied to the recording electrode. Two commonly used variations are fast cyclic voltammetry and high-speed chronoamperometry. Applications of fast cyclic voltammetry to measure dopamine and serotonin release and clearance are described elsewhere in this book (see Chapter 4 by Carrie, C. E. and Jones, S. R.; Chapter 8 by Threlfell, S. and Cragg, S. J. Chapter 12 by Garris, P. A. et al.). This chapter details the utility of high-speed chronoamperometry to study mechanisms for 5-HT clearance in the rodent brain.

High-Speed Chronoamperometry

The principle of high-speed chronoamperometric recording is based on methods originally described by Cottrell (1902), but modern electronics and digital data acquisition have allowed measurements to be made more rapidly. For our studies of 5-HT clearance, we have adopted and modified methods developed by the Gerhardt group for monitoring the extracellular concentration of dopamine in vivo (see Gerhardt et al. 1984; Gerhardt and Hoffman 2001). To measure 5-HT, a carbon fiber electrode (CFE) coated with Nafion (see below) is positioned in suitable buffer solution, tissue section or slice or, as will be detailed in this chapter, in brain in vivo. Figure 5.1 shows the parameters for the square wave potential applied to the CFE. The CFE is held at a zero resting potential vs. a Ag/AgCl reference electrode. Then the potential is stepped to +0.55 V. This voltage step is selected to exceed the peak 5-HT oxidation potential by 0.15 V. The square wave voltage step is held at +0.55 V for 100 ms and this produces a rapid change in the current (typically in a range of 1–3 nA) recorded by the CFE that decays as a function of time. The initial current charges the electrode capacitance and, based on the CFE time constant, is permitted to decay for 20 ms before the oxidation current is sampled and integrated for 80 ms. The square wave voltage step is then returned to 0.0 V and held at this potential for 900 ms. This return to the resting potential gives rise to a reduction current, which for 5-HT and its oxidation products is close to zero. For other neurotransmitters such as dopamine, the applied and resting potentials are typically each 100 ms in duration. However, unlike dopamine, which is readily oxidized and reduced, serotonin is “reluctantly” reduced.

FIGURE 5.1. Square wave voltage pulses applied to the carbon fiber electrode for chronoamperometric recording of 5-HT.

FIGURE 5.1

Square wave voltage pulses applied to the carbon fiber electrode for chronoamperometric recording of 5-HT. B. Ohmic current recorded in the absence of exogenously applied 5-HT (a) is set as “zero” and the change in current produced by oxidation (more...)

One important implication of this is that the lack of sizable “reverse” current can cause “fouling” of the CFE. Electrogenerated compounds produced from the oxidation of 5-HT can adsorb to the CFE, thereby reducing the active recording area of the CFE and the subsequent ability to detect 5-HT (see also Jackson et al. 1995). We find that leaving a delay of 900 ms between each applied potential greatly increases the lifetime of our CFEs, presumably by decreasing the rate at which adsorptive products are created. CFE potential is therefore stepped at 1 Hz. In theory then, one of the advantages of chronoamperometry is that the high frequency at which recordings are made enhances signal-to-noise because small changes in current due to the detection of 5-HT are not masked by large and potentially varying background current. We presently use the FAST-12 and FAST-16 systems (Quanteon, Lexington, KY) for our chronoamperometric recordings.

CFE Calibration

CFEs are available commercially (e.g., from Quanteon, Lexington, KY) or can be custom made. Details for electrode fabrication can be found in Perez and Andrews (2005) and references therein (Gerhardt 1995; Hoffman and Gerhardt 1998; Gerhardt and Hoffman 2001). Prior to using CFEs for in vivo recordings, they are coated with Nafion and calibrated for 5-HT. Nafion is a perfluronated ion exchange resin that excludes the passage of anions to the CFE (Gerhardt et al. 1984). It has been used with great success to increase the selectivity of CFEs for cations, such as 5-HT, and prevent oxidation of anionic “contaminants” such as the metabolite of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), uric acid and ascorbic acid, all of which are present in brain in a much higher concentration than 5-HT and have oxidation potentials that overlap that of 5-HT (Crespi et al. 1983; Cespuglio et al. 1986; Crespi et al. 1990; Rivot et al. 1995). The electrode is simply dipped and swirled in Nafion (1–2 s) and then dried in an oven at 200°C. This process is repeated 2–5 times and results in a CFE that is highly selective for 5-HT (see also Gerhardt and Hoffman 2001 for methodological considerations). Our criteria is that the CFE must be 500 times more selective for 5-HT than for its metabolite 5-HIAA. To determine this, the CFE is placed in a beaker containing either phosphate buffered saline (PBS) or artificial cerebral spinal fluid (aCSF). Once the current response to stepping the CFE potential stabilizes (within a few minutes), 5-HIAA (250 μM) is added followed by increasing concentrations of 5-HT (typically 0.5 μM increments, ranging from 0.0 to 3.0 μM, see Figure 5.2). In addition to having a 5-HT:5-HIAA selectivity greater than 500:1, electrodes used for our in vivo experiments must have a linear response to 5-HT (r2>0.90). We typically find electrodes prepared in this way have a detection limit in the range of 30–60 nM. Figure 5.2 shows the effectiveness of Nafion to prevent detection of 5-HIAA and the excellent linearity of response to increasing concentrations of 5-HT over the range of 0.0–3.0 μM (r2 = 0.9956). Also shown is the response to much higher concentrations of 5-HT (6 and 12 μM). At these higher concentrations, the calibration clearly becomes curvilinear, likely due to adsorption of electrogenerated products to the electrode (Jackson et al. 1995), however, regression analysis continues to yield strong r2 values (in the range of 0.90–0.98) showing the utility of chronoamperometry to measure relatively high concentrations of 5-HT. We find that if we limit exposure of the electrode to these higher concentrations of 5-HT (1–2 exposures), we can effectively establish values for maximal rates of 5-HT clearance (Vmax) in various brain regions in vivo (Daws et al. 2005). Some of these data are discussed in Section 5.3.2.

FIGURE 5.2. Calibration of a Nafion-coated carbon fiber electrode in room temperature (22°C) phosphate buffered saline (PBS).

FIGURE 5.2

Calibration of a Nafion-coated carbon fiber electrode in room temperature (22°C) phosphate buffered saline (PBS). To determine the selectivity produced by the Nafion film, the electrode was challenged with 250 μM 5-HIAA before the first (more...)

High-Speed Chronoamperometry Coupled to Microejection of Serotonin

We have taken the technique of high-speed chronoamperometric recording and coupled it to microinjection of 5-HT and other compounds into discrete brain regions. We developed the use of multibarrelled glass micropipettes (typically 4 or 7 barrels) so as to take full advantage of the excellent spatial resolution afforded by the small size of the active recording area of the CFE (Daws et al. 1997). For our experiments, the CFE electrode (diameter 30 μm) is cut such that 150 μm extends from the pulled glass capillary in which it is housed. By using multibarrel pipettes, we can test the effect of several drugs and/or establish dose-relationships within the same discrete location of brain within each animal. This greatly reduces the number of animals required and importantly, the need to record from multiple sites within a given brain region. The excellent spatial resolution of electrodes used for voltammetric recordings can be a double-edged sword depending on the question being asked. For example, they provide an excellent approach to investigating heterogeneity of response within a given brain structure (e.g., Cass et al. 1993; Cass and Gerhardt 1995; Daws et al. 1998, 2005), but if the research question requires restriction to a confined region (e.g., a distinct set of stereotaxic coordinates), then use of multibarrel pipettes allows considerably greater flexibility in the nature and amount of data that can be obtained from that site without the need to move and/or withdraw the micropipette-electrode assembly (i.e., to exchange or refill the micro-pipette). Of course, this is a distinct advantage when recording from multiple sites as well. We obtain our 4- and 7-barrel pipettes from Frederick Haer Corp. Inc. (Bowdoinham, ME).

First the CFE is coated with Nafion and calibrated to 5-HT as described above. Then the barrels of the micropipette are filled with the desired solutions, typically 5-HT, vehicle and drugs of interest and a length of PE tubing (~30 cm) glued with cyanoacrylate adhesive into each of the barrel openings. This tubing serves as the connector to a picospritzer, which is used to deliver solutions into brain by pressure-ejection. The amount of 5-HT or drug delivered can be very accurately quantified by measuring the amount of fluid displaced from the micropipette using a dissection microscope fitted with an eyepiece reticule. This can be simply calculated using the equation V = πr2×l, where V is the volume displaced, r is the inside radius of the pipette barrel and l is the distance the meniscus falls within the barrel during pressure application. Given that the concentration of each solution contained within the barrel is known, and the volume ejected is readily measured, the number of moles of 5-HT or drug delivered can be easily calculated. A cartoon showing the typical arrangement of our multi-barrel micropipette and electrode assembly is shown in Figure 5.3. The multi-barrel micropipette and CFE are attached just above the taper in the micropipette using sticky wax (Kerr Brand, Emoryville, CA). This is done by clamping the CFE and microelectrode into separate arms of a micromanipulator and carefully aligning the tips in the same plane and separated by a set distance: 200 μm for experiments in mice and 300 μm for experiments in rats. Tip alignment and separation is confirmed using a dissecting microscope fitted with an eyepiece micrometer. This assembly is implanted, using stereotaxic apparatus, into the desired brain region.

FIGURE 5.3. Cartoon showing only the tips of the carbon fiber electrode and 7-barrel micropipette.

FIGURE 5.3

Cartoon showing only the tips of the carbon fiber electrode and 7-barrel micropipette. Serotonin is pressure-ejected into the brain and diffuses across to the CFE where it is oxidized. This generates a current, which is converted into a micromolar value (more...)

Surgical Procedures

Experiments are routinely performed in anesthetized mice and rats. The procedures are essentially identical with a few minor modifications for the mouse. Rats are anesthetized with an intraperitoneal (ip) injection of chloralose (85 mg/kg) and urethane (850 mg/kg). For mice a mixture of chloralose (35 mg/kg) and urethane (350 mg/kg) is injected at 2 ml/kg body weight to more accurately titrate dose. A stainless steel tube is inserted into the trachea to facilitate breathing and the animal is then placed into the stereotaxic frame. The animal is kept warm by means of a water-circulated heating pad and body temperature monitored using a rectal probe. Body temperature is maintained at 37±1°C. The scalp is then incised and reflected and the skull overlying the brain region of interest is removed. Once the dura mater is removed, the electrode–micropipette assembly is lowered into the region of interest. Applications of high-speed chronoamperometry coupled to microinjection of 5-HT are described in the following sections.

Kinetics of Serotonin Clearance

One of the greatest advantages of locally applying 5-HT to brain is that the source and identity of the neurotransmitter analyte is known. Certainly there are many applications where evoking release of endogenous 5-HT is desirable, such as those described by Carrie John and Sara Jones in the previous chapter. However, when the desired measure is that of transporter function, specifically in terms of its ability to remove neurotransmitter from the extracellular milieu, measuring clearance of exogenously applied 5-HT provides a straightforward and readily interpretable approach.

Serotonin Signal Parameters

Figure 5.4 shows the reproducibility of the signal produced by pressure-ejection of 5-HT into the CA3 region of hippocampus. A minimum of 3 min is allowed to elapse between ejections. We routinely find that 3–5 ejections of serotonin into hippocampus are needed before the signal becomes reproducible. Before this stable baseline is achieved, the signal amplitude for the same pmol amount of serotonin declines most markedly, but also signal time course decreases modestly. Part of this is no doubt attributable to initial disruption of the extracellular matrix by the bolus ejection of serotonin that could change factors known to influence diffusion (e.g., tortuosity, volume fraction) and hence, clearance. In addition, we believe that trafficking of the 5-HTT to the plasma membrane in response to the rapid increase in extracellular 5-HT also contributes. This phenomenon has been very well characterized by Randy Blakely and his group using in vitro models (Ramamoorthy and Blakely 1999). While experiments are ongoing in our laboratory to address 5-HTT trafficking in vivo, we find that once we have attained stability in the signal produced by repeated pressure-ejection of serotonin into hippocampus, the signal remains remarkably stable for very long periods (h). This provides an excellent preparation for studying the ability of drugs to impact various signal parameters. These are shown in Figure 5.4 and include the peak signal amplitude, rise time (the time for the signal to reach its peak amplitude) the time for the signal to decay by 20, 50, 60, and 80 percent of its peak amplitude (T20, T50, T60, and T80), area under the curve and clearance rate. We and others (e.g., Sabeti et al. 2002) calculate clearance rate in two ways. One method is by calculating the slope of the most linear portion of descending phase of the signal, Tc, defined as the slope between T20 and T60. The other is by calculating the velocity, V, for 5-HT clearance by fitting the entire descending portion of the signal to an equation describing one-phase exponential decay. Y = [5-HT]o,i. e k t+[5-HT]o,t, where Y is the concentration of 5-HT (μM), [5-HT]o,i is the initial maximal extracellular concentration of 5-HT (μM), [5-HT]o,t is the extracellular concentration of 5-HT (μM) at baseline and k is the rate constant (s−1). The half-life of decay is 0.6932/k. Note that because the concentration of 5-HT at baseline (i.e., [5-HT]o,t) equals “zero” (the value given to represent the baseline electrochemical signal prior to addition of exogenous 5-HT), this term of the equation reduces to zero and is not used in the analyses. The calculated k value from this equation is then multiplied by the amplitude of the signal to give the velocity of transport (V). These calculations are made for signals of increasing amplitude until an apparent Vmax is reached.

FIGURE 5.4. Signal parameters and reproducibility of electrochemical signals produced by local application of 5-HT in the CA3 region of the anesthetized mouse.

FIGURE 5.4

Signal parameters and reproducibility of electrochemical signals produced by local application of 5-HT in the CA3 region of the anesthetized mouse. Serotonin (2 pmol, indicated by arrows along the lower abscissa) was applied at ~3 min intervals by pressure-ejection. (more...)

Brain Region Dependency

This approach has been used to demonstrate the relationship between 5-HTT expression and 5-HT clearance (Montañez et al. 2003; Daws et al. 2005). For example, [3H]cyanoimpramine binding to the 5-HTT was carried in the dorsal raphe nucleus (DRN), a cell body region of the 5-HT system, which contains the highest density of 5-HTT in brain (Hensler et al. 1994); the CA3 region of hippocampus, a terminal field region (Hensler et al. 1994), and the corpus callosum, a fiber tract with very few 5-HTTs (Reyes-Haro et al. 2003). Shown in Figure 5.5 are the [3H]cyanoimipramine binding data (Figure 5.5a) and 5-HT clearance profiles (Figure 5.5b). Figure 5.5c shows the remarkable positive correlation between 5-HTT expression and Vmax for 5-HT clearance. The greater the density of 5-HTT, the greater the rate of 5-HT clearance (details can be found in Daws et al. 2005). We have carried out similar experiments in the CA3 region of hippocampus of 5-HTT mutant mice (Montañez et al. 2003). As anticipated, apparent Vmax values for 5-HT clearance in heterozygote mice, which express 50% fewer 5-HTTs than their wild-type counterpart, were approximately half that of wild-type mice. Serotonin clearance rates were substantially reduced in mice lacking the 5-HTT. Indeed, the rate for 5-HT clearance in these mice was comparable to that observed in corpus callosum of rat (see Figure 5.5b); thus, two situations where 5-HTT expression is minimal or absent yield similar profiles (but see also text below and Figure 5.7).

FIGURE 5.5. Relationship between 5-HTT expression and maximal rate (Vmax) of 5-HT clearance in rat brain.

FIGURE 5.5

Relationship between 5-HTT expression and maximal rate (Vmax) of 5-HT clearance in rat brain. (a) Specific binding of [3H]cyanoimipramine (CN-IMI) to 5-HTTs in the dorsal raphe nucleus (DRN), CA3 region of hippocampus and corpus callosum (CC). (b) Velocity (more...)

FIGURE 5.7. Evidence for alternative mechanisms for 5-HT clearance in 5-HTT KO mice.

FIGURE 5.7

Evidence for alternative mechanisms for 5-HT clearance in 5-HTT KO mice. Velocity of 5-HT clearance in CA3 region of hippocampus is shown for wild-type (closed circles, n = 3) and 5-HTT−/− (open circles, n = 3) mice as a function of increasing (more...)

Transporter Promiscuity

The clearance profiles obtained in corpus callosum and 5-HTT KO mice at first glance appear indicative of simple diffusion of 5-HT away from the recording electrode. While this reasonably accounts in large part for the shape of 5-HT clearance profiles in regions of low or no 5-HTT expression, is diffusion alone the only contributor? Our studies in rats treated with 5,7-dihydroxytryptamine (5,7-DHT) to destroy 5-HT neurons (Daws et al. 1998; Daws et al. 2005) and 5-HTT KO mice (Daws et al. 2006) suggest that other processes contribute to 5-HT clearance in the absence of 5-HTTs.

Figure 5.6 shows the clearance profile for 5-HT locally applied to the CA3 region of hippo-campus of male Sprague–Dawley rats that had been previously treated with 5,7-DHT (200 μg total; 100 μg in PBS delivered at 10 μL/min bilaterally into the lateral ventricles) or sham operated (for experimental details, see Daws et al. 2005). The success of lesion was confirmed by quantitative autoradiography for [3H]CN–IMI binding to the 5-HTT. Likewise, the success of pretreatment with nomifensine (30 mg/kg, ip) to protect norepinephrine transporters (NET) was also confirmed by [3H]nisoxetine binding to the NET (see Daws et al. 2005). Our expectation was that the Vmax for 5-HT clearance in 5,7-DHT treated rats would be drastically reduced. It was not. As is clear in Figure 5.6, rather than a change in Vmax, there was a clear shift to the right of the concentration effect curve showing that the in vivo affinity value (referred to as K T ) for “active” clearance mechanisms in the CA3 region of hippocampus had increased (i.e., decreased affinity for 5-HT uptake). These results harkened the early work of Shaskan and Synder (1970) showing that 5-HT uptake was mediated by at least two processes: one high affinity/low capacity the other low affinity but with a high capacity to transporter 5-HT. Because NET is also present in the CA3 region of hippocampus, albeit with expression levels approximately 25% that of 5-HTT, we thought it possible that when 5-HTT was eliminated, the NET may represent another key mechanism for removal of 5-HT from the extracellular milieu. Further investigations in rats where noradrenergic terminals had been destroyed by pre-treatment with 6-hydroxydopamine revealed that the NET is indeed capable of clearing 5-HT from ECF in hippocampus (see Daws et al. 1998, 2005).

FIGURE 5.6. Maximal velocity of 5-HT clearance in CA3 region of hippocampus is not altered by neurotoxic destruction of 5-HTTs.

FIGURE 5.6

Maximal velocity of 5-HT clearance in CA3 region of hippocampus is not altered by neurotoxic destruction of 5-HTTs. Velocity of 5-HT clearance in the CA3 region of hippocampus is shown for rats treated with 5,7-DHT (closed circles, dashed line), or vehicle (more...)

One puzzling observation has been why the apparent Vmax for 5-HT clearance in the hippo-campus of 5-HTT KO mice is markedly lower than that of wild-type mice. If the NET and/or other transporter is able to maintain Vmax for 5-HT clearance in 5,7-DHT treated rats (i.e., rats with no 5-HTT) at rates similar to those in vehicle-treated rats, why do we not see the same phenomenon in 5-HTT KO mice? One possibility is that in mice, we have simply not yet attained a sufficiently high extracellular concentration of 5-HT to reveal clearance by transporters other than the 5-HTT. Our studies in rat have extended the concentration range to as high as 14 μM (Daws et al. 2005), whereas in mouse, extracellular concentrations of exogenously applied 5-HT have rarely exceeded 8 μM (Montañez et al. 2003). We have begun to study a higher concentration range in 5-HTT mutant mice and we have found, as illustrated in Figure 5.7, that when signal amplitudes produced by exogenous application of 5-HT exceed ~ 15 μM, Vmax for 5-HT, clearance between wild-type and 5-HTT KO mice becomes indistinguishable (unpublished observations). The black solid lines in Figure 5.7 represent sigmoid fits with variable slope to all data within a genotype. These fits yielded Vmax values of 231±25 nM/s and 254±2 nM/s for 5-HTT+/+ and 5-HTT−/−, respectively. KT values (corrected for volume fraction, α = 0.2) were 0.58±0.06 μM and 3.10±0.01 μM for 5-HTT+/+ and 5-HTT−/− mice, respectively. These fits are consistent with 5-HT clearance in wild-type mice being mediated primarily by the 5-HTT, a high affinity, low capacity transporter for 5-HT, whereas in the 5-HTT KO mice, clearance of 5-HT is mediated by a low affinity but high capacity transporter. The grey dashed lines represent a hypothetical fit revealing two sigmoid functions. The lower plateau “Vmax1” in 5-HTT KO mice (at around 50 nM/s) may reflect the diffusion component for 5-HT clearance away from the CFE. The higher plateau “Vmax2” (at approximately 250 nM/s) may reflect a low affinity, but high capacity transporter for 5-HT such as the NET.

Compelling evidence for the existence of alternative mechanisms for 5-HT clearance in 5-HTT mutant mice come from our studies investigating the effect of ethanol on 5-HT clearance in these mice. We found that ethanol inhibits 5-HT clearance in wild-type mice. Remarkably, however, the ability of ethanol to inhibit 5-HT clearance was significantly potentiated in a 5-HTT genotype-dependent manner, where inhibition was greatest in mice lacking the 5-HTT and intermediate in 5-HTT± mice, which express 50% fewer 5-HTTs than wild-type mice (Daws et al., J. Neuroscience, 2006). These data clearly demonstrate that (1) while the 5-HTT may be a site of action for ethanol, it is not necessary for ethanol-induced inhibition of 5-HT clearance and (2), that genetic inactivation of the 5-HTT causes a marked upregulation of the principle mechanisms involved. Investigations are ongoing to identify these mechanisms.

Physiological Relevance of Applying Micromolar Concentrations of 5-HT

Of fundamental importance is whether exogenous application of micromolar concentrations of 5-HT or its uptake by catecholaminergic neurons is of physiological relevance. Although studies do not yet provide a complete answer, we believe this approach can yield important insight into the normal physiology and pathophysiology of 5-HT neurotransmission. The current estimate for synaptic concentrations of 5-HT in brain is 6 mM (Bunin and Wightman 1999). This value is consistent with that reported for other neurotransmitters such as glutamate and dopamine (Clements 1996; Cragg and Rice 2004). It has been estimated that 5-HT can diffuse greater than 20 μm away from the synaptic cleft, where the concentration of 5-HT falls to the micromolar and nanomolar range (Bunin and Wightman 1998, 1999). Thus, the micromolar amounts of 5-HT required to reach Vmax for 5-HT clearance in the studies described herein fit well within the current framework of our knowledge. Our data are also in good agreement with those of Bunin and co-workers, in that they support paracrine (or extrasynaptic) transmission as a mode for 5-HT neurotransmission. Ultrastructural studies show that the 5-HTT is located primarily extrasynaptically (on axons and dendrites) (Miner et al. 2000) and also at a distance from potential receptor target sites (Huang and Pickel 2002). The same is true for NET labeling and of particular significance to our studies is evidence that noradrenergic neurons arborize extensively within the hippocampus (Schroeter et al. 2000). Thus, both anatomical and functional data provide strong support that the range of concentrations of 5-HT used in our experiments are physiologically realistic and relevant and further support the involvement of multiple transport mechanisms in regulating 5-HT neurotransmission.

Biological Significance of Transporter Promiscuity

While there is no doubt that the 5-HTT is the “prime-mover” of 5-HT from the extracellular fluid back into the nerve terminal, substantial evidence for uptake of 5-HT by other transporters, including the NET and DAT has been amassed over the past 5 decades (Shaskan and Snyder 1970; Jackson and Wightman 1995; Daws et al. 1998; Pan et al. 2001; Zhou et al. 2002; Callaghan et al. 2005; Daws et al. 2005; Zhou et al. 2005). The data discussed herein suggest that these alternative mechanisms for 5-HT transport play a larger role when extracellular levels of 5-HT are very high and/or when the 5-HTT is compromised or eliminated (genetically or pharmacologically). These findings are particularly intriguing given growing evidence that a low-functioning variant in the promoter region of the human 5-HTT gene is positively associated with risk for emotional disorders and alcoholism. Moreover, they raise important considerations for the use of SSRIs and other therapeutics used in the treatment of disorders such as depression, anxiety and alcoholism. For example, patients who do not respond well to SSRIs may have a greater relative amount of functioning NET than do responders. One could speculate that greater NET function would serve to reduce the overall ability of SSRI treatment to elevate extracellular 5-HT, presumably the first critical step in achieving therapeutic benefits. Taken together with the rapidly expanding body of literature describing interactions between genetically defined expression of the 5-HTT (e.g., 5-HTTLPR polymorphism) (Collier et al. 1996; Smits et al. 2004), environment (Caspi et al. 2003) and response to drug treatment (Eichhammer et al. 2003; Smits et al. 2004), the design of a biological system with built-in functional redundancies makes evolutionary sense in order to maintain homeostasis of serotonergic neurotransmission. Consideration of these alternative mechanisms for 5-HT uptake may lead to the development of improved therapeutics for numerous disorders, in particular those where a hyposerotonergic state is thought to be the key predisposing or exacerbating factor.

Receptor Regulation of the Serotonin Transporter

Another way in which neurotransmitter uptake can be regulated is by receptor control of the neurotransmitter transporter itself. For example, it has been well established that the dopamine2 (D2) autoreceptor can regulate activity of the DAT (Meiergerd et al. 1993; Parsons et al. 1993; Cass and Gerhardt 1994; Wieczorek and Kruk 1995; Dickinson et al. 1999; Mayfield and Zahniser 2001; Gulley and Zahniser 2003). Described in the following section are examples of our application of high-speed chronoamperometry to studying regulation of the 5-HTT by auto- and hetero- receptors.

5-HT1B Receptor

Reports that the D2 autoreceptor could regulate DAT activity turned our attention to the possibility that the 5-HTT could also be under autoreceptor control. The 5-HT1B receptor is located both post-synaptically and also on the pre-synaptic terminal where it functions as an autoreceptor to regulate 5-HT release from the terminal. To test the hypothesis that it also serves as a regulator of 5-HT clearance by influencing the activity of the 5-HTT, we used a pharmacological approach. Cyano-pindolol, a 5-HT1B receptor antagonist, was loaded into a 7-barrel glass micropipette. Coupled with a CFE, these multibarrel micropipettes permit the study of dose–response relationships of a locally applied drug on 5-HT clearance at a discrete site within brain. The key advantage is that a different concentration of drug can be loaded into 5 of the 7 barrels, with 5-HT and vehicle loaded into the remaining 2 barrels. In this way, different amounts (typically pmol amounts) of drug can be delivered in equivalent volumes by pressure-ejection. This eliminates any potential confound that might result from pressure-ejection of different volumes of the same barrel concentration of drug to deliver a given pmol amount. It also reduces variability resulting from local heterogeneity of 5-HTT expression.

First, 5-HT was pressure-ejected into the CA3 region of hippocampus until the signal produced became reproducible. This serves as the baseline signal. A given pmol amount of cyanopindolol or vehicle was then pressure-ejected into the same region and 60–90 s. permitted to elapse before again delivering 5-HT. Once all 5-HT was cleared (or 3 min later, whichever was the longer interval), another ejection of 5-HT was made. Pressure-ejection of 5-HT continued according to this schedule until the signal returned to its pre-drug baseline. Once at least two reproducible 5-HT signals were obtained, the next dose of cyanopindolol was locally applied and the protocol repeated.

Cyanopindolol dose dependently inhibited clearance of exogenously applied 5-HT in the CA3 region of hippocampus. Remarkably, the maximal effect (Emax) and the concentration of cyanopindolol producing half Emax (EC50) (42% increase in T80 time course and 3.2 pmol respectively) were comparable to that of the SSRI, fluvoxamine (Emax = 38% increase in T80 and EC50 = 3.6 pmol) (see Daws et al. 2000). Similar results were obtained in rats pretreated with parachlorophenylalanine (PCPA) to deplete endogenous stores of 5-HT. The rationale for these experiments was based on the possibility that exogenously applied 5-HT might inhibit release of endogenous 5-HT by stimulating the 5-HT1B autoreceptor. If the autoreceptor is blocked by an antagonist such as cyanopindolol, then this would prevent inhibition of 5-HT release and lead to greater extracellular 5-HT. This in turn could manifest as an increase in signal amplitude and/or time course of the signal produced by exogenous application of 5-HT. The finding that cyanopindolol continued to prolong the time course for clearance of exogenously applied 5-HT in PCPA-treated rats rules out any contribution of endogenously released 5-HT to the signal. In addition, competition binding assays confirmed that inhibition of 5-HT clearance by cyanopindolol was not likely mediated by a direct action at the 5-HTT (Ki value>35,000 nM). Together with data showing that 5-HT moduline and methiothepin, antagonists more selective for the 5-HT1B receptor than cyanopindolol, also inhibited 5-HT clearance (Daws et al. 1999, 2000), these data provide compelling evidence that the 5-HT1B autoreceptor regulates the activity of 5-HTT.

Alpha2-Adrenoceptor

The 5-HT1B autoreceptor is not the only modulator of 5-HT release. Activation of presynaptic alpha2 (α 2)-adrenoceptors have also been demonstrated to regulate 5-HT release (Maura et al. 1992; Gobbi et al. 1993; Numazawa et al. 1995; Gobert et al. 1998; Schneiber et al. 2001). In collaboration with Randy Blakely’s group, we explored the possibility that, like the 5-HT1B autoreceptor, the α 2-adrenoceptor may also regulate 5-HTT function. Using a similar approach to that described above, we found that in rat parietal cortex, the α 2-adrenoceptor agonist, 5-bromo-N- [4,5-dihyro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304), inhibited clearance of exogenously applied 5-HT (Ansah et al. 2003). In vitro, yohimbine, an α 2 antagonist, augmented [3H]5-HT uptake into mouse forebrain synaptosomes. The augmentation of basal 5-HTT activity by yohimbine may suggest tonic inhibition of 5-HTT by α 2-adrenoceptor agonists. In vivo, however, no effect of yohimbine on 5-HT clearance was observed. This apparent lack of effect may be related to dose; as to date, only one dose has been tested in vivo (52 pmol). Alternatively, this discrepancy may be related to the preparations themselves—in vitro versus in vivo. Norepinephrine (NE) was detected by high performance liquid chromatography (HPLC) analysis in the synaptosomal preparation (Ansah et al. 2003); therefore yohimbine could be blocking leaked or released NE in this preparation. In vivo, leakage of NE might be less likely. While studies investigating the mechanism by which this cross-talk between the α 2-adrenoceptor and 5-HTT occurs are ongoing, these data provide another example of the utility of in vivo chronoamperometric approaches to identifying the existence of receptor regulation of the 5-HTT outside of the “dish.”

Adenosine Receptor

Using a rat basophilic leukemia cell line (RBL-2H3), Miller and Hoffman (1994) provided the first evidence of adenosine receptor (AR) regulation of the 5-HTT via a cGMP-dependent mechanism. More recent studies from Randy Blakely’s group (e.g, Zhu et al. 2004) have extended these initial observations to reveal the existence of two protein kinase G (PKG)-dependent pathways that support rapid regulation of 5-HTT by the adenosine 3 (A3) receptor. One augments 5-HTT surface trafficking and the other, a p38 mitogen-activated protein kinase (MAPK)-dependent process, increases 5-HTT intrinsic activity (Zhu et al. 2004, 2005). However, to date all of these studies have been carried out in cell lines stably transfected with 5-HTT or synaptosomal preparations. In collaboration with Randy Blakely, we have begun to study regulation of the 5-HTT by these pathways using in vivo chronoamperometric approaches.

First we investigated the dose-dependency for modulation of 5-HT clearance in rat brain by the non-selective AR agonist, 5′-N-ethylcarboxamidoadenosine (NECA). Similar approaches to those described above were used. A reproducible baseline 5-HT signal was established and then NECA was locally applied by pressure-ejection 90 s. prior to the next ejection of 5-HT. Consistent with in vitro studies, NECA enhanced clearance of exogenously applied 5-HT in CA3 region of hippo-campus, but within a very narrow dose range (Figure 5.8). To investigate a role for PKG and p38 MAPK in mediating this effect of NECA, we then applied either the PKG inhibitor, N-[2-(methyamino) ethyl]-5-isoquinoline-sulfonamide (H8), the p38 MAPK inhibitor, 4-(4-fluorophenyl)-2- (4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazol (SB203580) or vehicle, 2 min prior to NECA. Pretreatment with either H8 or SB203580 blocked NECA-induced enhancement of 5-HT clearance (Figure 5.9). These studies remain in their very early stages but clearly demonstrate the utility of chronoamperometry coupled to pressure-ejection of exogenous 5-HT to not only identify phenomenological changes in 5-HT clearance, but importantly, to investigate the mechanisms through which these receptor-mediated changes in 5-HT clearance occur in vivo.

FIGURE 5.8. Dose-dependent effects of the AR agonist, NECA, on 5-HT clearance in rat hippocampus.

FIGURE 5.8

Dose-dependent effects of the AR agonist, NECA, on 5-HT clearance in rat hippocampus. Data are mean ± SEM percent change in T80 time course parameter 5 min after local application of NECA. Number of rats per “dose” is shown in (more...)

FIGURE 5.9. Inhibitors of PKG and p38 MAPK block AR agonist stimulated clearance of 5-HT.

FIGURE 5.9

Inhibitors of PKG and p38 MAPK block AR agonist stimulated clearance of 5-HT. H8 (14 pmol), SB203580 (14 pmol) or equivalent volume of PBS were given either alone, or 2 min. prior to NECA (55 pmol). Data are mean±SEM percent change in T80 time (more...)

Significance of Receptor-Mediated Regulation of 5-HTT

Based on a rapidly growing body of in vitro studies, it is now well established that the 5-HTT can be regulated by multiple signaling pathways (Blakely and Bauman 2000; Blakely et al. 2005). The studies described in the previous sections provide initial but convincing evidence that the 5-HTT is also subject to this kind of regulation in the intact brain in vivo. As such, receptor regulation of 5-HTT expression and/or activity presents a new approach to drug development for the treatment of psychiatric disorders linked to dysregulation of 5-HT signaling (e.g., depression and addiction). For example, phosphorylation and sequestration of the 5-HTT is dependent on ligand occupancy (Ramamoorthy and Blakely 1999). Therefore, certain ligands may have superior therapeutic utility by not only preventing 5-HT reuptake but also by allowing kinase-linked signaling pathways to shift the cellular distribution of the 5-HTT. These findings become particularly important given the recent report that individuals with naturally occurring 5-HTT coding variants show altered sensitivity to drugs that target PKG and p38 MAPK (Prasad et al. 2005). In turn, altered 5-HTT regulation by means of PKG and p38 MAPK-linked pathways could influence risk for disorders related to compromised 5-HT neurotransmission.

Concluding Remarks

Chronoamperometry used to measure clearance of exogenously applied 5-HT offers a unique approach for examining functional changes in 5-HTT activity, in vivo, as they occur in response to genetic aberrations, environmental stimuli and acute or chronic drug treatment. Although in vitro approaches will no doubt continue to provide valuable insights into novel regulatory signaling pathways as well as transport mechanisms for 5-HT uptake, the importance of demonstrating their existence in the whole animal will always remain of paramount importance. In vivo electro-chemical approaches, such as those described here, are currently among the most robust methods available to reveal how transporters function in their native environment.

Acknowledgments

Studies described herein were funded in part by NIH grants MH64489 and DA18992, NARSAD Young Investigator Awards and the Texas Higher Education Coordinating Board Advanced Research Program Award to LCD and NIH grant HL071645 to GMT. The authors gratefully acknowledge the following individuals for their significant contributions toward generating the data contained within: Nicole L. Baganz, Dr. Randy D. Blakely, Alfred S. Calderon, Paul D. Callaghan, Douglas J. Davis, Dr. Alan Frazer, Dr. Greg A. Gerhardt, Dr. Georgianna G. Gould, Rebecca E. Horton, Dr. Sylvia Montañez, Jaclyn L. Munn, W. Anthony Owens, and Susan D. Teicher.

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