J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07407.x
High doses of amphetamine (AMPH) are thought to disrupt normal patterns of action potential-dependent dopaminergic neurotransmission by depleting vesicular stores of dopamine (DA) and inducing robust non-exocytotic DA release or efflux via dopamine transporter (DAT) reversal. However, these cardinal AMPH actions have been difficult to establish definitively in vivo. Here, we use fast-scan cyclic voltammetry (FSCV) in the urethane-anesthetized rat to evaluate the effects of 10 and 20 mg/kg AMPH on vesicular DA release and DAT function in dorsal and ventral striata. An equivalent high dose of cocaine (40 mg/kg) was also examined for comparison to psychostimulants acting preferentially by DAT inhibition. Parameters describing exocytotic DA release and neuronal DA uptake were determined from dynamic DA signals evoked by mild electrical stimulation previously established to be reinforcing. High-sensitivity FSCV with nanomolar detection was used to monitor changes in the background voltammetric signal as an index of DA efflux. Both doses of AMPH and cocaine markedly elevated evoked DA levels over the entire 2-h time course in the dorsal and ventral striatum. These increases were mediated by augmented vesicular DA release and diminished DA uptake typically acting concurrently. AMPH, but not cocaine, induced a slow, DA-like rise in some baseline recordings. However, this effect was highly variable in amplitude and duration, modest, and generally not present at all. These data thus describe a mechanistically similar activation of action potential-dependent dopaminergic neurotransmission by AMPH and cocaine in vivo. Moreover, DA efflux appears to be a unique, but secondary, AMPH action.
maximal evoked concentration of dopamine
concentration of dopamine released per stimulus pulse
fast-scan cyclic voltammetry
vesicular monoamine transporter
Although the psychostimulant AMPH acts on multiple sites of pre-synaptic dopaminergic neurotransmission, the three primary targets are considered to be competitive blockade of the DAT, depletion of vesicular DA stores, and reversal of DAT function driving a non-exocytotic form of DA release called efflux (Kuczenski and Segal 1994; Fleckenstein et al. 2007). All three drug effects can occur concurrently (Jones et al. 1998; Schmitz et al. 2001). Vesicular actions are mediated in part by AMPH directly interacting with the vesicular monoamine transporter (VMAT) as a substrate, resulting in the reverse transport of DA similar to AMPH effects on DAT (Rothman and Baumann 2003; Rothman et al. 2006). The mechanism of AMPH is also dose-dependent. Blockade of DAT predominates at low doses, with DA efflux playing a minor role because of limited cytosolic DA levels, whereas DA efflux predominates at moderate to high doses, with AMPH redistributing DA from vesicular to cytosolic pools (Seiden et al. 1993; Sulzer et al. 2005). Robust DA efflux is proposed to mediate the comparatively greater increases in extracellular DA concentration elicited by AMPH compared with other psychostimulants that preferentially inhibit DA uptake (Kuczenski et al. 1991). Moreover, although most drugs of abuse activate action potential-dependent dopaminergic neurotransmission, AMPH is in contrast thought to disrupt normal patterns of signaling by the combined outcomes of compromised exocytotic DA release and unregulated DA efflux (Hyman 2005; Sulzer 2011).
Despite ample supporting evidence obtained from in vitro preparations (Seiden et al. 1993; Kuczenski and Segal 1994; Sulzer et al. 2005; Fleckenstein et al. 2007; Sulzer 2011), cardinal AMPH targets have been difficult to establish definitively in vivo. For example, some studies report decreased levels of electrically evoked DA with AMPH (Ewing et al. 1983; Kuhr et al. 1985, 1986), presumably consistent with vesicular depletion, but others actually show an enhancement of unknown origin (May et al. 1988; Suaud-Chagny et al. 1989; Dugast et al. 1994). Voltammetric analysis has been additionally unable to demonstrate AMPH-induced DA efflux with a moderate drug dose (Wiedemann et al. 1990). This latter result is particularly striking compared with the micromolar DA efflux recorded with the same technique in brain slices (Jones et al. 1998; Schmitz et al. 2001). The large increases in dialysate DA elicited by AMPH in vivo (Carboni et al. 1989; Kuczenski et al. 1991, 1997) are more difficult to interpret in terms of DA efflux because probe-tissue interactions render quantitative determinations with microdialysis very challenging (Bungay et al. 2003; Borland et al. 2005). More recently, we have used voltammetry to characterize the effects of a wide range of AMPH doses in the anesthetized rat. Although robust increases in electrically evoked DA levels and DAT inhibition were documented, neither a significant decrease in exocytotic DA release nor any evidence for DA efflux was observed at any dose (Ramsson et al. 2011). Clearly, there is a great need to better understand the mechanism of AMPH action in the intact brain of the whole animal and to reconcile discrepant results for this important psychostimulant.
The goal of the present study was thus to investigate further the effects of AMPH on pre-synaptic dopaminergic signaling in vivo. High doses of AMPH (10 and 20 mg/kg) were administered to assess its vesicular DA-depleting and DA-efflux promoting actions. FSCV at a carbon-fiber microelectrode was used to monitor extracellular DA in the dorsal and ventral striatum of the urethane-anesthetized rat. Exocytotic DA release and neuronal DA uptake were determined from electrically evoked DA dynamics, and background voltammetric recordings indexed DA efflux. As AMPH effects may be related to stimulus intensity (Ramsson et al. 2011), stimulation parameters previously established for reinforcing and to elicit responses mimicking endogenous DA signals recorded in awake rats (Cheer et al. 2005) were employed. High-sensitivity FSCV was also selected (Heien et al. 2005) to address the concern that prior voltammetric analysis of DA efflux lacked sufficient detection limits. Finally, a high dose of cocaine (40 mg/kg), a psychostimulant that has been shown to competitively inhibit DAT and up-regulate exocytotic DA release (Jones et al. 1995; Venton et al. 2006), served as a comparison drug. We show here that similar to cocaine, AMPH blocks DA uptake and augments vesicular DA release thereby activating action potential-dependent dopaminergic neurotransmission in vivo. AMPH, in contrast to cocaine, induced DA efflux as well, but this effect was modest and highly variable.
Male Sprague-Dawely rats (300–500 g) were purchased from Harlan Industries (Indianapolis, IN, USA). Food and water were provided ad libitum, and animals were housed in a temperature-controlled facility with a 12-h light/dark cycle. Animal care was approved by the Institutional Animal Care and Use Committee of Illinois State University in accordance with NIH guidelines (Institute of Laboratory Animal Resources 1996).
For most animals, FSCV was performed simultaneously in the dorsal and ventral striatum. After voltammetric responses stabilized, a time course of evoked and non-evoked signals was collected with electrical stimulation applied every 5 min. Pre- and post-drug periods were 25 and 120 min, respectively. The three drug groups were 10 mg/kg AMPH (n = 8 and 5 in the dorsal and ventral striatum, respectively), 20 mg/kg AMPH (n = 5 and 6 in the dorsal and ventral striatum, respectively), and 40 mg/kg cocaine (n = 6 and 5 in the dorsal and ventral striatum, respectively). All drugs were injected intraperitoneally.
The surgical procedure was according to Ramsson et al. (2011). Rats were anesthetized with urethane (1.5 g/kg i.p.) and placed in a stereotaxic frame (Kopf Instrumentation; Tujunga, CA, USA). After exposing the skull, holes were drilled for lowering electrodes. All stereotaxic manipulations were according to flat-skull coordinates (Paxinos and Watson 1986), using bregma and top of brain as landmarks. Deltaphase Isothermal Pads (Braintree Scientific, Braintree, MA, USA) maintained core body temperature. The stimulating electrode was placed initially just dorsal to the medial forebrain bundle (−4.6 AP, +1.4 ML, −6.5 DV). Recording electrodes were positioned in the dorsal (+1.2 AP, +2.0 or +3.0 ML, −4.5 DV initially) or ventral striatum (+1.2 AP, +2.0 or +3.0 ML, −6.5 DV initially). Electrodes positioned at +3.0 ML were angled at 6°. A Ag/AgCl reference electrode (i.e. chloridized silver wire) was placed contralaterally in superficial cortex. The DV location of stimulating and recording electrodes was optimized so that DA release was robust and fast. After optimization, their positions were not changed for the duration of the experiment.
Fast-scan cyclic voltammetry
FSCV was recorded at a glass-sealed carbon-fiber microelectrode (Cahill et al. 1996). Individual carbon fibers (3.5-μm radius; Cytec Engineering Materials, West Patterson, NJ, USA) were aspirated into borosilicate capillary tubes (1.0 or 1.2 mm o.d.; Sutter Instrument, Novato, CA, USA) and pulled in a micropipette puller (Narishige, Tokyo, Japan). The carbon fiber was cut so that ∼100 μm extended beyond the glass/carbon-fiber seal. Electrical connection between the carbon fiber and 26-gage lead wire (Newark, Palatine, IL, USA) was provided by a low-melting point bismuth alloy (Small Parts, Inc., Miramar, FL, USA), which was melted with a heat gun (Master Appliance Corp., Racine, WI, USA) and allowed to cool. A triangle waveform (−0.4 to 1.3 V, 400 V/s) was applied at 10 Hz, with a bias potential of −0.4 V. Electrochemistry was performed by an EI400 (Ensman Instrumentation, Bloomington, IN, USA) or UEI bipotentiostat (Department of Chemistry Electronics Facility, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA) and controlled by TH-1 software (ESA, Chelmsford, MA, USA). Background subtracted cyclic voltammograms were used for electrochemical identification. Time-dependent changes in DA were obtained by monitoring current at the DA oxidation peak (∼0.6 V) and converting to concentration based on post-calibration (Wu et al. 2001b).
Biphasic stimulus pulses were applied to a twisted bipolar electrode (Plastics One, Roanoke, VA, USA), with tips separated by ∼1 mm. The current and duration of each phase were 125 μA and 2 ms. Trains were applied at a frequency of 60 Hz for 0.4 s (24 pulses). Electrical stimulation was computer generated and passed through an optical isolator and constant-current generator (Neurolog NL800; Digitimer Limited, Letchworth Garden City, UK).
Non-electrically evoked changes in the background current were measured at the DA-oxidation potential. These responses were used as an index of DA efflux (Jones et al. 1998; Schmitz et al. 2001; Ramsson et al. 2011) and to assess spontaneous DA release events during phasic signaling (Stuber et al. 2005). The amplitude of electrically evoked DA signals was reported as the maximal DA concentration ([DA]max). These same dynamic responses were analyzed to determine parameters for exocytotic DA release and neuronal DA uptake according to (Wightman et al. 1988):
where [DA]p is the concentration of DA released per stimulus pulse, f is the frequency of stimulation, and k is the first-order DA uptake term. First-order kinetics was selected to describe DA uptake to avoid the complexities of determining whether AMPH at long time points alters Vmax perhaps via DAT internalization (Fleckenstein et al. 1999) in addition to Km. Resolving competitive from uncompetitive DAT inhibition is difficult with in vivo voltammetry (Wu et al. 2001b). However, Michaelis–Menten kinetics can be approximated to first order when Km ≥ [DA] (Garris and Wightman 1994). This condition is met by the low DA concentrations evoked by the reinforcing stimulation (Cheer et al. 2005) and the expected large drug-induced values for Km following AMPH and cocaine administration (Wu et al. 2001b; Ramsson et al. 2011). Curve fitting to determine [DA]p and k employed a non-linear regression based on a simplex-minimization algorithm (Wu et al. 2001b).
When appropriate, data are presented as the mean ± SEM. Statistical analysis was performed with a repeated-measures MANOVA using SAS version 9.1.3 (SAS Institute Inc. 2004). Significance was set at p < 0.05.
Urethane, cocaine hydrochloride, and d-amphetamine sulfate were purchased from Sigma (St. Louis, MO, USA). All drugs were dissolved in 150 mM saline prepared with nanopure water (Barnstead/Thermolyne Coporation, Dubuque, IA, USA) prior to injection. d-AMPH and cocaine dose were determined by base weight.
Effects of AMPH and cocaine on evoked DA dynamics
Figure 1 shows representative evoked responses measured by FSCV in the dorsal (Panel a) and ventral striatum (Panel b) for the three drug treatment groups. All changes in the recorded signals were identified as DA by voltammograms (data not shown). Both 10 and 20 mg/kg AMPH and 40 mg/kg cocaine elicited increases in electrically evoked DA levels in the two striatal subregions. Psychostimulants broadened the responses, indicative of a slowed clearance of released DA because of DAT inhibition. Increases in signal amplitude could be a result of either reduced neuronal DA uptake and/or enhanced exocytotic DA release. Both mechanisms regulating striatal levels of extracellular DA operate concurrently during the stimulus train (Wightman et al. 1988; Wu et al. 2001b). Psychostimulant effects appear to be time dependent.
Effects of AMPH and cocaine on evoked [DA]max
Figure 2 shows the time course of evoked [DA]max in the dorsal (Panel a) and ventral striatum (Panel b) for the three drug treatment groups. Both 10 and 20 mg/kg AMPH and 40 mg/kg cocaine robustly enhanced [DA]max for the duration of the 120-min post-drug recording period. All three drug treatments rapidly increased [DA]max to a plateau in the dorsal striatum. Cocaine acted similarly in the ventral striatum, although the rise was somewhat delayed. However, the response to AMPH in this striatal subregion initially reached a peak followed by a slow decay. [DA]max eventually reached a plateau for 20 mg/kg but continued to drop for 10 mg/kg. The repeated-measures MANOVA revealed a significant effect of time on [DA]max in both dorsal (F6,8 = 5.32, p = 0.0171) and ventral (F6,8 = 3.61, p = 0.0489) striatum. The effects of drug treatment on [DA]max (F2,13 = 0.01, p = 0.9856, dorsal striatum; F2,13 = 1.25, p = 0.3197, ventral striatum) and interaction (F12,18 = 0.65, p = 0.7735, dorsal striatum; F12,18 = 0.82, p = 0.6294, ventral striatum) were not significant in either striatal subregion. As a result of the limitations with degrees of freedom, only seven time points, or every 20 min beginning at time 0 min, were analyzed with the MANOVA.
Effects of AMPH and cocaine on neuronal DA uptake
Figure 3 shows effects of the three drug treatment groups on neuronal DA uptake in the dorsal (Panel a) and ventral striatum (Panel b) for the three drug treatment groups. Uptake was determined at selected time points from the evoked DA responses averaged in Fig. 2. Both 10 and 20 mg/kg AMPH and 40 mg/kg cocaine reduced DA uptake in both striatal subregions. This decrease emerged at 20 min and was sustained for the duration of the time course. DAT inhibition by psychostimulants appeared more robust in the dorsal compared with the ventral striatum, and AMPH appeared more potent than cocaine overall. The repeated-measures MANOVA revealed a significant effect of time on DA uptake in both dorsal (F5,10 = 21.24, p < 0.0001) and ventral (F5,9 = 63.78, p < 0.0001) striatum, a significant effect of drug treatment on DA uptake in the ventral striatum (F2,13 = 9.63, p = 0.0027), and a significant interaction in the dorsal striatum (F10,22 = 2.45, p = 0.0383). There was no significant effects of drug treatment on DA uptake (F2,14 = 1.53, p = 0.2511) in the dorsal striatum and no significant interaction in the ventral striatum (F10,20 = 1.68, p = 0.1550).
Effects of AMPH and cocaine on exocytotic DA release
Figure 4 shows effects of the three drug treatment groups on exocytotic DA release in the dorsal (Panel a) and ventral striatum (Panel b). Like uptake, release was determined at selected time points from the evoked DA responses averaged in Fig. 2. Both 10 and 20 mg/kg AMPH and 40 mg/kg cocaine elevated exocytotic DA release in both striatal subregions. In contrast to psychostimulant effects on DA uptake, this increase emerged earlier at 5 min, but it was similarly sustained for the duration of the time course. Overall, the up-regulation of exocytotic DA release appeared more robust in the dorsal compared with the ventral striatum, with the exception for 10 mg/kg AMPH, which appeared to elicit greater effects in the ventral subregion. The repeated-measures MANOVA revealed a significant effect of time on exocytotic DA release (F5,10 = 3.74, p = 0.036) in the dorsal striatum and a trend for a significant effect of time on exocytotic DA release (F5,9 = 3.10, p = 0.0667) in the ventral striatum. The effects of drug treatment on exocytotic DA release (F2,14 = 0.17, p = 0.8479, dorsal striatum; F2,13 = 0.98, p = 0.3999, ventral striatum) and interaction (F10,22 = 1.15, p = 0.3753, dorsal striatum; F10,20 = 1.65, p = 0.1647, ventral striatum) were not significant in either striatal subregion.
Effects of AMPH and cocaine on non-electrically evoked responses
Figure 5 shows representative traces of continuous voltammetric recordings during the first 35 min of the drug time course. All responses were collected in the dorsal striatum of different animals. Current was monitored at the peak oxidative potential for DA on each voltammogram. Rapid current deflections characterize the DA response to electrical stimulation at each 5-min interval, whereas the intervening segments of background current could reflect changes in basal DA levels. Drugs were administered shortly after the evoked response was collected at time 0 min. Consistent with individual signals shown in Fig. 1 and average results shown in Fig. 2, an increase in the amplitude of the evoked signal is evident for 10 mg/kg AMPH (Panels a, b, and c) and for 40 mg/kg cocaine (Panel d).
As also depicted in Fig. 5, AMPH elicited three types of slow changes in the background current measured between the evoked DA deflections. These changes did not seem to be biased towards a dose or striatal subregion. The first type, shown in Panel (a), consisted of a small, short-lived increase in background current emerging ∼4 min after drug injection. This increase could be attributed to DA according to the individual voltammogram (Baur et al. 1988) and the serial display of voltammograms in the color plot (Michael et al. 1998). In the expanded time scale, the concentration increase of the signal if all is attributed to DA is 21 nM. Four of 23 responses showed this type of background change. The second type, shown in Panel (b), is considerably larger and longer. The concentration increase in the expanded time scale if the signal was DA is 44 nM. However, although DA-like voltammograms are present, the color plot shows additional analytes that may interfere with the DA determination. Two of 23 responses showed this type of background change. The third type, show in Panel (c), consisted of no discernable increase in background current attributed to DA and occurred in 17 of 23 responses. Some, but not all, recordings exhibited marked decreases in the signal because of unknown electrochemistry. In contrast to AMPH, cocaine did not appreciably alter background current in either dorsal or ventral striatum as shown representatively in Panel (d).
Figure 6 displays representative traces depicting more rapid changes in background current elicited by 20 mg/kg AMPH in the ventral striatum. These current spikes also demonstrated no bias for dose or striatal subregion but were not observed for cocaine. The left side of Panel (a) shows these subsecond signals riding on top of an AMPH-induced slow change in background current emerging ∼ 2 min after drug injection. Both slow and fast changes can be attributed to DA according to voltammograms and would register a concentration of ∼60 nM and ∼20 nM, respectively. The right side of Panel (a) shows an expanded time scale of two clearly demarcated concentrations spikes in this same record. Panel (b) shows similar subsecond DA signals elicited 70 min after administration of AMPH but recorded on a stable baseline. Overall, 6 of 23 responses exhibited these fast changes in DA concentration.
AMPH activates action potential-dependent dopaminergic neurotransmission
The present study used stimulus parameters that were previously shown to be reinforcing in the paradigm of intracranial self-stimulation and to elicit responses mimicking the amplitude and duration of phasic DA signals associated with goal-directed behavior (Phillips et al. 2003; Cheer et al. 2005). To our knowledge, this stimulation is the mildest in terms of train duration and current intensity used to study AMPH effects in vivo. Consistent with the notion recently postulated by us that AMPH effects vary with stimulation (Ramsson et al. 2011), we show increased levels of electrically evoked DA in agreement with previous work using a moderate stimulus (May et al. 1988; Suaud-Chagny et al. 1989; Dugast et al. 1994), but opposing studies reporting decreases using more robust parameters (Ewing et al. 1983; Kuhr et al. 1985, 1986).
Stimulus parameters do not appear to alter the effects of AMPH on DA uptake. We show robust AMPH-induced DAT inhibition in agreement with our previous voltammetric analysis in vivo (Ramsson et al. 2011), in vitro voltammetry (Jones et al. 1998; Schmitz et al. 2001; John and Jones 2007), and classical uptake measurements using 3H-dopamine and synaptosomes or homogenates (Harris and Baldessarini 1973; Richelson and Pfenning 1984; Krueger 1990). Although this inhibition has been identified as competitive for AMPH, such a determination was not possible in the present work because first-order, not Michaelis–Menten, kinetics were employed. It should also be mentioned that apparent reduced rates for DA uptake in response to AMPH could also reflect a component of reverse transport. However, the analysis of evoked DA responses used in the present study could not distinguish between DAT inhibition and DA efflux.
The selection of stimulus parameters has profound implications for assessing the effects of AMPH on exocytotic DA release. Indeed, using reinforcing stimulation we show for the first time an up-regulation in this key mechanism of action potential-dependent dopaminergic signaling. By comparison, our previous in vivo voltammetry study using moderate stimulus parameters found no significant changes with AMPH doses up to 10 mg/kg (Ramsson et al. 2011). Interestingly, nearly complete elimination of exocytotic DA release (∼70 to 100%) by AMPH is observed by voltammetry in slices made from the dorsal striatum (Jones et al. 1998; Schmitz et al. 2001; Patel et al. 2003). It is difficult to compare stimulus parameters between in vivo and in vitro designs, because electrical stimulation in the former elicits action potentials in ascending dopaminergic axons, whereas in the latter dopaminergic terminals are directly stimulated. Thus, other factors besides stimulation may also contribute to discrepant results between preparations.
AMPH-induced changes in non-electrically evoked DA signals
AMPH-induced slow rises in the baseline recording in both striatal subregions. Ostensibly, this result opposes previous voltammetric analyses in the anesthetized rats, which failed to detect increases in basal DA levels (Wiedemann et al. 1990; Ramsson et al. 2011). However, the majority of recording sites did not support AMPH-enhanced signals and of those that did, they were modest in amplitude and of short duration. Thus, most recordings collected in the present study with high-sensitivity FSCV would likely have yielded a negative result with the more conventional FSCV measurements used previously. One caveat is that some voltammograms did not always indicate a clean DA signal, but rather suggested the presence of additional analytes. It is for these reasons that AMPH-induced baseline changes should be considered ‘DA-like’, and their concentrations be stated cautiously. The technique of chemometrics using principle component regression may better resolve the DA component of these baseline changes (Heien et al. 2005).
Taken together, voltammetry suggests that DA efflux is a secondary mechanism of AMPH action in the anesthetized rat compared with the primary targets of exocytotic DA release and DA inhibition (Ramsson et al. 2011; Wiedemann et al. 1990; present study). This conclusion is clearly at variance with the robust, ∼4-μM DA efflux reported with similar voltammetric analysis in striatal slices (Jones et al. 1998; Schmitz et al. 2001) and the ∼30-fold increase in dialysate DA with moderate to high doses of AMPH (Kuczenski et al. 1991, 1997). Discrepant results between in vivo and in vitro voltammetry could be because of the loss of feedback circuits in the reduced preparation, which counter the elevation of striatal levels of extracellular DA in response to AMPH (Forster et al. 2002; Miller et al. 2002). The microdialysis measurements are also difficult to interpret fully, as DAT inhibition drives DA flux to the probe leading to greater recovery independent of alterations in extracellular DA concentration (Bungay et al. 2003; Borland et al. 2005). Similar probe-tissue interactions prevent determining an absolute concentration for the increase in dialysate DA, further complicating comparisons to the present study.
Our result demonstrating modest AMPH-induced DA efflux is similarly at odds with voltammetric analysis of non-exocytotic DA release in single cells expressing DAT (Kahlig et al. 2005) and non-voltammetric measurements in striatal synaptosomes using a protocol that distinguishes psychostimulants as ‘releasers’ versus DAT inhibitors (Rothman and Baumann 2003). AMPH and related analogs have additionally been shown to release DA potently from isolated synaptic vesicles, using a similar protocol but in this case resolving reverse transport from inhibition of VMAT (Rothman et al. 2006). Collectively, these in vitro results are difficult to fully rationalize with the present in vivo study, as we found no evidence for AMPH depleting vesicular DA stores and for AMPH-induced DA efflux in most recordings. Clearly, AMPH is acting quite differently in the intact brain than in reduced preparations. Discordant results between in vitro and in vivo studies have also been reported for methamphetamine (Rothman and Baumann 2003).
Concentration spikes of DA were also observed in the voltammetric record after AMPH administration. These sub-second responses resemble the phasic DA transients elicited by burst firing of dopaminergic neurons that occur spontaneously in the awake animal or are evoked by rewards and their predictive cues (Carelli and Wightman 2004; Schultz 2007). To our knowledge, this is the first report of AMPH alone activating phasic DA signaling. AMPH induces DA burst firing via an adrenergic mechanism, but only when administrated with a DA D2 antagonist (Shi et al. 2000) to overcome the activation of somatodendritic DA autoreceptors by DA released in response to the psychostimulant (Mercuri et al. 1989). AMPH may also promote burst firing by attenuating other inhibitory components such as metabotropic glutamate receptors (Paladini et al. 2001). Nonetheless, the importance of enhanced phasic DA signaling to AMPH action remains to be determined. At least in the present study, only a few recording sites supported such a result, but this low proportion of responses could be an artifact of urethane anesthesia suppressing burst firing (Kelland et al. 1990). As DA release events were also observed on the slow rising envelop, it is tempting to speculate that a component of AMPH-induced increases in basal DA levels is because of phasic signaling.
Comparisons to cocaine
As a result of presumed different actions on pre-synaptic dopaminergic neurotransmission, cocaine was selected as a model drug in the present study. Fostering comparisons to AMPH, cocaine has also been investigated using similar approaches, including 3H-DA uptake (Richelson and Pfenning 1984; Krueger 1990), microdialysis (Carboni et al. 1989; Kuczenski et al. 1991), in vitro voltammetry (Jones et al. 1995; John and Jones 2007), and in vivo voltammetry (Wu et al. 2001a). A consensus from these studies is that competitive DA uptake blockade is a key mechanism mediating cocaine-induced increases in striatal extracellular DA. Robust DAT inhibition may also play a similarly important role for AMPH action in vivo when mild to moderate stimulation is used to evoke DA levels (Dugast et al. 1994; May et al. 1988; Ramsson et al. 2011; Suaud-Chagny et al. 1989; present study).
Cocaine has more recently been shown to up-regulate exocytotic DA release in striatal slices (Jones et al. 1995; Lee et al. 2001). This increase was not altered by blockade of DA receptors and was not observed for two other DAT inhibitors, WIN 35428 and nomifensine. Although a similar result is shown in the present work, we did not report enhanced release with cocaine in a previous study in anesthetized rats (Wu et al. 2001a). However, more intense stimulus parameters were used, further supporting the idea the stimulation is an important variable in assessing psychostimulant action. A cocaine-mediated, synapsin-dependent up-regulation of exocytotic DA release has also been observed in vivo after first depleting readily releasable DA stores by inhibiting DA synthesis (Venton et al. 2006). Another psychostimulant, amfonelic acid, appears to increase exocytotic DA release in vivo by mobilizing a reserve storage pool as well (Ewing et al. 1983). The present results suggest that AMPH shares with some but not all psychostimulants the ability to augment exocytotic DA release.
Whether AMPH shares a common mechanism with cocaine and amfonelic acid for up-regulating exocytotic DA release is not known. AMPH promotes exocytosis from chromaffin cells by liberating vesicular Ca2+ (Mundorf et al. 1999). If storage pools are differentially sensitive to AMPH (Chen and Ewing 1995; Anderson et al. 1998), then Ca2+ from depleted vesicles could drive exocytosis from intact ones. Differentially sensitive storage pools may also link the effects of psychostimulants on exocytotic DA release and stimulus parameters because intense stimulations are thought to mobilize reserve pools (Rizzoli and Betz 2005). AMPH could additionally increase vesicular content by providing more cytosolic DA for packaging via inhibition of monoamine oxidase and activation of DA synthesis, two other actions of the psychostimulant on pre-synaptic dopaminergic neurotransmission (Seiden et al. 1993; Kuczenski and Segal 1994). In support of this idea, L-DOPA, the immediate biosynthetic precursor to DA, increases DA quantal size in PC12 cells (Pothos et al. 1998).
Another recently identified target of cocaine on dopaminergic neurotransmission is phasic signaling. In awake rats, cocaine increases the amplitude and frequency of spontaneously occurring phasic DA release events (Stuber et al. 2005). Similar activation of these so-called DA transients is observed in the anesthetized rat, but only after co-administering a DA D2 antagonist (Park et al. 2010). In agreement, we did not observe phasic DA release after administration of cocaine alone in the present study. Rather surprisingly, we observed DA transients in some recording sites in response to AMPH, which may suggest that this psychostimulant activates phasic DA signaling by a different mechanism than cocaine and other DAT inhibitors such as nomifensine and GBR 12909 (Venton and Wightman 2007; Park et al. 2010). Nevertheless, this novel AMPH action is consistent with intact exocytotic DA release.
Our results also need to be placed in the context of the established finding that AMPH elicits comparatively greater increases in dialysate DA than other psychostimulants such as cocaine, whose primary action is inhibition of DAT (Kuczenski et al. 1991; Kuczenski and Segal 1992). These more potent effects on basal levels of extracellular DA have been attributed to the unique ability of AMPH to elicit DA efflux (Kuczenski and Segal 1994; Rothman et al. 2006). As we observed AMPH-induced DA efflux at least in a subset of recordings, it is entirely possible that this mechanism could still contribute to greater increases in dialysate DA. Other potential origins are more potent inhibition of DAT in vivo (Ramsson et al. 2011), and perhaps greater activation of vesicular DA release and/or phasic DA signaling (vide supra). Future work should consider these latter two possibilities, by comparing the dose-dependence of AMPH and cocaine on vesicular DA release using the stimulation protocol established here and investigating the effects of the two psychostimulants on spontaneous DA transients in anesthetized animals in the presence of a DA D2 antagonist (Venton and Wightman 2007; Park et al. 2010) or in awake animals (Stuber et al. 2005).
The most important, and yet surprising, finding of the present study was that AMPH augments, rather than disrupts, exocytotic DA release in the anesthetized rat. This result potentially has several implications not only for AMPH specifically but also for drugs of abuse and addiction in general. For example, the difference between the mechanism of AMPH-like and cocaine-like psychostimulants may not be as great as initially thought. Indeed, in our hands, both drugs robustly increased electrically evoked levels of striatal extracellular DA by a common action: up-regulated exocytotic DA release and DAT inhibition. As such, AMPH may share with other drugs of abuse the ability to enhance action potential-dependent dopaminergic neurotransmission during the addiction process. Current thinking excludes AMPH from this overall effect elicited by most other abused drugs (Hyman 2005; Sulzer 2011). It remains to be determined how the present results relate to the intense stereotypy in rats and toxic psychosis in humans induced by high doses of AMPH (Seiden et al. 1993). At a minimum, however, the present results raise the intriguing question whether these behaviors result from massive drug-induced DA efflux. Future work should also be directed at determining whether similar AMPH actions manifest in the awake animal to rule out anesthesia artifacts confounding the present study and at reconciling the robust effects of AMPH and related analogs on DAT and VMAT to reverse DA transport and measured in isolated preparations with the modest DA efflux observed herein in the intact brain.
This research was funded by NIH DA 021770 (PAG). The authors declare no conflict of interest.