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Keywords:

  • degradation;
  • exocytosis;
  • rat;
  • synthesis;
  • voltammetry

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Amphetamine has well-established actions on pre-synaptic dopamine signaling, such as inhibiting uptake and degradation, activating synthesis, depleting vesicular stores, and promoting dopamine-transporter reversal and non-exocytotic release. Recent in vivo studies have identified an additional mechanism: augmenting vesicular release. In this study, we investigated how amphetamine elicits this effect. Our hypothesis was that amphetamine enhances vesicular dopamine release in dorsal and ventral striata by differentially targeting dopamine synthesis and degradation. In urethane-anesthetized rats, we employed voltammetry to monitor dopamine, electrical stimulation to deplete stores or assess vesicular release and uptake, and pharmacology to isolate degradation and synthesis. While amphetamine increased electrically evoked dopamine levels, inhibited uptake, and up-regulated vesicular release in both striatal sub-regions in controls, this psychostimulant elicited region-specific effects on evoked levels and vesicular release but not uptake in drug treatments. Evoked levels better correlated with vesicular release compared with uptake, supporting enhanced vesicular release as an important amphetamine mechanism. Taken together, these results suggested that amphetamine enhances vesicular release in the dorsal striatum by activating dopamine synthesis and inhibiting dopamine degradation, but targeting an alternative mechanism in the ventral striatum. Region-distinct activation of vesicular dopamine release highlights complex cellular actions of amphetamine and may have implications for its behavioral effects.

Abbreviations used
αMpT

alpha-methyl-para-tyrosine

AMPH

amphetamine

DA

dopamine

DAT

DA transporter

Substantive evidence indicates that amphetamine (AMPH), a highly addictive drug of abuse exhibiting clinical efficacy for treating narcolepsy and attention deficit hyperactivity disorder (Heal et al. 2009; Peacock and Benca 2010), targets pre-synaptic dopamine (DA) signaling. Effects include inhibiting the DA transporter (DAT) and monoamine oxidase and activating tyrosine hydroxylase, but depleting vesicular DA stores and promoting non-exocytotic DA release via DAT reversal are considered primary (Fleckenstein et al. 2007; Sulzer 2011). More recently, AMPH has been shown to augment vesicular DA release in both dorsal and ventral striata in vivo (Ramsson et al. 2011b; Daberkow et al. 2013). While the significance of this unexpected finding to overall drug effect remains to be determined, enhanced vesicular DA release may drive AMPH-induced increases in phasic DA signaling (Ramsson et al. 2011b; Daberkow et al. 2013), which is important for reinforcement-learning in goal-directed behavior and addiction (Hyman 2005; Wanat et al. 2009). Several other DAT inhibitors have also been shown to increase vesicular DA release (Ewing et al. 1983; Kuhr et al. 1986; Jones et al. 1995; Lee et al. 2001; Venton et al. 2006; Oleson et al. 2009; Kile et al. 2010; Chadchankar et al. 2012), suggesting a common action for a major psychostimulant class.

How AMPH augments vesicular DA release is unknown, but potential mechanisms are suggested by other DAT inhibitors. Cocaine and methylphenidate act on DA storage pools associated with synapsin (Venton et al. 2006; Kile et al. 2010) and α-synuclein (Chadchankar et al. 2012), respectively. Several DAT inhibitors re-distribute cytosolic and membrane-bound vesicles (Riddle et al. 2002, 2007; Volz et al. 2007) and increase vesicular DA uptake (Brown et al. 2001; Volz et al. 2008). As a drug with complex actions, AMPH could exert additional, unique effects, including the inhibition of DA degradation (Scorza et al. 1997) and activation of DA synthesis (Kuczenski 1975) leading to elevated cytosolic DA levels and vesicular packaging, promoting exocytosis by liberating intracellular Ca2+ stores (Mundorf et al. 1999), and increasing membrane excitability as a DAT substrate (Ingram et al. 2002).

This study used voltammetry and electrical stimulation to investigate the mechanism by which AMPH augments vesicular DA release in dorsal and ventral striata in vivo. We employed an experimental design previously used to demonstrate that amfonelic acid and cocaine reinstate vesicular DA release after its near-complete depletion (Ewing et al. 1983; Kuhr et al. 1986; Venton et al. 2006). These results were interpreted as the two psychostimulants mobilizing the reserve DA pool to replenish the readily releasable DA pool independently of an action on DA synthesis, because tyrosine hydroxylase was pharmacologically blocked. However, vesicular mobilization was not directly assessed and thus not proven. We selected this design, because the robust response serves as a gauge of AMPH's effectiveness and because amfonelic acid and cocaine are perhaps the best-established DAT inhibitors for up-regulating vesicular DA release. Indeed, amfonelic acid has been recognized for decades as an archetypal enhancer of vesicular release (Aceto et al. 1970; Shore 1976), and this cocaine effect manifests across brain-slice (Jones et al. 1995; Lee et al. 2001; Kile et al. 2010), anesthetized (Ramsson et al. 2011b), and awake (Oleson et al. 2009) preparations. Because AMPH could conceivably act by inhibiting DA degradation, in addition to activating DA synthesis, we modified the design to also incorporate pharmacological blockade of monoamine oxidase, in order to assess the respective contributions of both pre-synaptic mechanisms. The experimental design also permitted resolving the respective contributions of vesicular DA release and DA uptake to observed AMPH-induced changes in electrically evoked DA levels. The hypothesis tested was that AMPH distinctly up-regulates vesicular DA release in striatal sub-regions by differentially targeting DA synthesis and degradation. Our results are consistent with a mechanism of AMPH action characterized by generalized uptake inhibition and up-regulation of vesicular release across striatal sub-regions, but a specific degradation- and synthesis-sensitive enhancement of vesicular release in the dorsal striatum only.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Animals

Adult, male Sprague–Dawley rats (281–443 g) were purchased from Harlan Industries (Indianapolis, IN, USA). Animals were housed in a temperature-controlled vivarium, with a 12-h light/dark cycle and ad libitum food and water. All procedures in this study follow the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Illinois State University Institutional Animal Care and Use Committee.

Drugs

Urethane, alpha-methyl-dl-tyrosine-methyl ester hydrochloride (αMpT), pargyline hydrochloride, and d-amphetamine sulfate were purchased from Sigma-Aldrich (St Louis, MO, USA) and dissolved in 0.9% sterile saline.

Surgery

Rats were anesthetized with urethane (1.5 g/kg i.p.) and immobilized in a stereotaxic frame (Kopf Instrumentation, Tujunga, CA, USA) (Ramsson et al. 2011a,b). Holes were drilled in the skull to position recording, stimulating and reference electrodes. Recording electrodes targeted the dorsal (AP +1.2, ML −2.0, DV −4.5; 0° angle) and ventral (AP +1.4, ML +3.0, DV −6.6; 12° angle) striatum. The stimulating electrode was directed towards the medial forebrain bundle (AP −4.6, ML +1.4, DV −7.0; 0° angle) ipsilateral to recording electrodes. The silver/silver chloride reference electrode was positioned in contralateral superficial cortex. All coordinates are from a flat-skull brain atlas (Paxinos and Watson 1986). After the initial placement, the stimulating and recording electrodes were incrementally lowered to locate sites supporting robust levels of electrically evoked DA. After this optimization, electrode positions were not changed for the duration of the experiment.

Fast-scan cyclic voltammetry

Fast-scan cyclic voltammetry recorded DA at a carbon-fiber microelectrode (Ramsson et al. 2011a,b). This recording electrode was fabricated by aspirating a single carbon fiber (3.5 μm radius; Cytec Engineering Materials, West Patterson, NJ, USA) into a borosilicate capillary tube (1.2 mm o.d.; Sutter Instrument, Novato, CA, USA) and pulling to a taper using a micropipette puller (Narishige, Tokyo, Japan). The end of the carbon fiber protruding beyond the glass insulation was cut to a length of ~100 μm. Fast-scan cyclic voltammetry (−0.4 to 1.3 V, 400 V/s, 10 Hz) was performed by a 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). Current recorded at the peak oxidative potential for DA (~0.6 V) was converted to DA concentration using a post-calibration factor obtained from flow injection analysis (Logman et al. 2000). DA was identified by the background-subtracted cyclic voltammogram (Michael et al. 1998).

Electrical stimulation

Electrical stimulation was computer generated and consisted of optically isolated, constant-current (Neurolog NL800; Digitimer, Letchworth Garden City, UK) biphasic pulses (300 μA and 2 ms each phase). Tips of the twisted bipolar stimulating electrode (Plastics One, Roanoke, VA, USA) were separated by ~1 mm. Two types of stimulus trains were applied (Venton et al. 2006). The supraphysiological depleting stimulation (60 Hz, 10 s) was used to disrupt the readily releasable pool for DA. A more-physiological stimulation (60 Hz, 0.4 s) was used to assess the effects of AMPH on vesicular DA release. This stimulation is referred to here as reinforcing-like, because frequency, duration, and pulse width are identical to trains that animals will lever press to obtain during the operant paradigm of intracranial self-stimulation (Cheer et al. 2007). However, a higher current intensity (±300 versus 125 μA) was used in this study, because anesthesia blunts the amplitude of electrically evoked DA levels (Garris et al. 1997). This reinforcing-like stimulation is also highly efficacious in revealing the augmentation of vesicular DA release by cocaine and AMPH in vivo (Venton et al. 2006; Oleson et al. 2009; Ramsson et al. 2011b; Daberkow et al. 2013).

Experimental design

The experimental design, modified from Venton et al. (2006), is shown in Fig. 1. There were three periods, pre-treatment, treatment, and post-treatment. The pre-treatment period (−10 to 0 min) established baseline measurements of DA to evaluate treatment effects. A total of five reinforcing-like trains were applied every 2 min during this period. The treatment period (0–100 min) used the depleting stimulation to disrupt vesicular DA release and drugs to block DA synthesis (αMpT; 200 mg/kg i.p.) and degradation (pargyline; 75 mg/kg i.p.). A total of five trains were applied every 20 min during this period. Drugs were injected immediately following the first stimulation. The post-treatment period (100–160 min) established post-treatment baseline measurements and examined the effects of AMPH (10 mg/kg i.p.; calculated as free base). A total of 30 reinforcing trains were applied every 2 min during this period. AMPH was administered at 118 min in this period. A total 52 rats were used in this study. There were five treatment groups: (i) no-depleting stimulation control (= 8); (ii) depleting stimulation-only control (= 12); (iii) depleting stimulation with αMpT (= 7); (iv) depleting stimulation with pargyline (= 13); and (v) depleting stimulation with αMpT and pargyline (= 12). Although not always successful, simultaneous DA recording was attempted in both the dorsal and ventral striatum of all rats. The AMPH dose was selected, because 10 mg/kg increases vesicular DA release in both the dorsal and ventral striatum of anesthetized animals (Ramsson et al. 2011b). While both 1 and 10 mg/kg also elevate vesicular DA release in both striatal sub-regions of awake animals, the low dose is only effective in the ventral striatum under anesthesia (unpublished observations; Daberkow et al. 2013). Additionally, the intense depleting stimulation is contraindicated in awake animals.

image

Figure 1. Experimental design. See Materials and methods section for details.

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Data analysis

DA signals evoked by the reinforcing-like stimulation were analyzed to determine maximal amplitude ([DA]max), vesicular DA release, and DA uptake. Analysis of vesicular release and uptake permits resolving the respective contributions of these mechanisms to observed AMPH-induced changes in [DA]max. Vesicular DA release was calculated as the concentration of DA elicited per stimulus pulse ([DA]p), and DA uptake was calculated as a first-order rate constant (k) according to:

  • display math(1)

where f is stimulation frequency (Wightman et al. 1988). Absolute values for [DA]max, [DA]p, and k are reported in Results section for the combined pre-treatment data set. To minimize heterogeneity of evoked DA responses recorded in the dorsal and ventral striatum (May and Wightman 1989; Garris et al. 1994), data for comparing treatment effects were normalized to a percent of pre-treatment (% pre-treatment) in each animal. We have previously used first-order kinetics to analyze DA uptake in AMPH responses evoked by the same reinforcing-like stimulation (Ramsson et al. 2011b; Daberkow et al. 2013). The use of first-order kinetics has been previously justified on the basis that the effects of AMPH are uncompetitive (i.e. both Km and Vmax change)(Fleckenstein et al. 1999; Ramsson et al. 2011b), which is difficult to assess using Michaelis–Menten kinetics and in vivo voltammetry (Wu et al. 2001b), and that evoked DA levels are near or below Km for DA uptake, which establishes pseudo-first-order conditions (Ramsson et al. 2011b). This approach has also been shown, under conditions in which uptake inhibition by AMPH should be competitive (i.e. change in Km), to provide similar determinations of vesicular DA release and simulated curves that well describe data compared with Michaelis–Menten kinetics (Daberkow et al. 2013). A non-linear regression with a simplex minimization algorithm was used to determine best-fit parameters for vesicular DA release and DA uptake (Wu et al. 2001b). Responses calculated from eqn (1) were temporally distorted equivalently before comparing to measured responses.

Statistical analysis

Repeated measures multivariate anova in SAS (SAS Institute Inc., Cary, NC, USA) was used to assess significant differences in [DA]max, [DA]p, and k over time within each treatment group (Figs 26). One-way anova with contrasts in SAS was used to assess significant differences in [DA]max across treatment groups during the post-treatment period (Fig. 7). Post hoc testing used a sequential Bonferroni correction for contrasts. Correlations (Fig. 8) were performed by Sigma Plot 12.0 (Systat Software Inc., San Jose, CA, USA). Two-way anova with contrasts in SAS was used to assess significant differences in pre-AMPH baseline [DA]max (Fig. 9). Post hoc testing used Dunnett's test with the no-depleting stimulation treatment serving as the control. Striatal sub-regional differences in the absolute values for [DA]max, [DA]p, and k for the pre-treatment condition and described in Results section were analyzed in Excel (Microsoft, Redmond, WA, USA) using a two-tailed t-test. The significance level for all comparisons was set at p ≤ 0.05.

image

Figure 2. Stimulation-only control. Individual recordings evoked by the reinforcing-like stimulation are shown in panel (a) for the dorsal (top) and ventral (bottom) striatum. Time −2 min (black) was collected during the pre-treatment baseline period, time 118 min (red) was collected during the post-treatment baseline period, and time 138 min (blue) was collected during the post-treatment, AMPH-test period. The short line underneath the recordings demarcates the stimulus pulse train. Averaged results for [DA]max are shown in panel (b). Green symbols and arrow demarcate the time of AMPH injection. For the pre- and post-treatment periods, data were calculated as a percent of the average [DA]max evoked by the reinforcing-like stimulation collected during the pre-treatment period; for the treatment period, data were calculated as a percent of the [DA]max evoked by the first depleting stimulation (% pre-treatment). Data for statistical analysis are shown in panel (c) for [DA]max (left), vesicular DA release or [DA]p (middle), and DA uptake or k (right). Time 118 min was collected immediately before AMPH administration, time 128 min was collected 10 min after AMPH administration and time 138 min was collected 20 min after AMPH administration. Data were calculated as a percent of the average responses collected during the pre-treatment period (% pre-treatment). In panels (b) and (c), data are expressed as mean ± SEM.

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Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Controls

Although we have previously demonstrated that AMPH inhibits DA uptake and augments vesicular DA release in both dorsal and ventral striata (Ramsson et al. 2011b; Daberkow et al. 2013), this study employed a longer and more complex experimental design with treatments incorporating a depleting stimulation and pharmacological manipulation of DA synthesis and degradation. We thus first needed to establish appropriate controls examining the effects of the supraphysiological depleting stimulation on the AMPH response in the absence of drugs. These controls are shown in Figs 2 and 3.

image

Figure 3. Control treatment without the depleting stimulation. See legend to Fig. 2 for details, with the exception that no depleting stimulations were applied and no drug was injected during the treatment period.

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Figure 2 shows the depleting stimulation-only control. Individual recordings evoked by the reinforcing-like stimulation reflect a decrement after the depleting stimulation and an augmentation after AMPH in both striatal sub-regions (Fig. 2a). Averaged results (Fig. 2b) show similar phenomena. The depleting stimulation alone caused a ~25% decrease in maximal amplitude ([DA]max) in both the dorsal and ventral striatum, although the time courses appeared to differ. AMPH-induced increases in [DA]max evoked by the reinforcing-like stimulation were comparable in both striatal sub-regions. Across all animals in this study, [DA]max of the pre-treatment response was 261±37 nM (= 36) in the dorsal striatum and 253±36 nM (= 28) in the ventral striatum. These concentrations, which are not significantly different (= 0.88; t-test), fall within the range of naturally occurring phasic DA signals recorded in awake, freely behaving animals (Robinson and Wightman 2007). This result further supports our contention that the reinforcing-like stimulation is physiological.

Data for statistical analysis are displayed in Fig. 2c for [DA]max (left), vesicular DA release ([DA]p, middle), and DA uptake (k, right), for three time points of the post-treatment period. These time points were selected to capture the initial AMPH effect. Parameters for vesicular DA release and DA uptake were calculated from the same responses evoked by the reinforcing-like stimulation from which [DA]max was determined. For the pre-treatment responses across all animals, k was significantly (p < 0.0001; t-test) higher in the dorsal compared with the ventral striatum (1.15 ± 0.08 and 0.69 ± 0.07 s−1, respectively) but [DA]p was not different significantly (= 0.77; t-test) between the striatal sub-regions (14.4 ± 2.2 and 13.5 ± 2.5 nM, respectively). For the depleting stimulation-only control, AMPH increased [DA]max and vesicular DA release but decreased DA uptake in both striatal sub-regions. Statistical analysis revealed a significant effect of time for [DA]max (F2,14 = 10.54, p = 0.0016), vesicular DA release (F2,14 = 7.03, p = 0.0077), and DA uptake (F2,14 = 10.80, p = 0.0015). There was a significant effect of region on DA uptake (F1,4 = 13.96, p = 0.020) but not for [DA]max or vesicular DA release. All region × time interactions were not significant. The sample size was = 7 in the dorsal striatum and = 6 in the ventral striatum.

Figure 3 shows the no-depleting stimulation control. Individual (Fig. 3a) and averaged (Fig. 3b) responses demonstrate signal maintenance across the 100-min interval when the various treatments of the depleting stimulation and drugs would be applied and an increase in [DA]max evoked by the reinforcing-like stimulation following AMPH administration. This increase was more rapid compared with the depleting stimulation-only control (Fig. 2). In both the dorsal and ventral striatum for the no-depleting stimulation control, [DA]max and vesicular DA release increased and DA uptake decreased after AMPH administration (Fig. 3c). Statistical analysis revealed a significant effect of time for [DA]max (F2,8 = 7.85, p = 0.013), vesicular DA release (F2,8 = 5.43, p = 0.032), and DA uptake (F2,8 = 64.77, p < 0.0001). There was a significant effect of region on DA uptake (F1,3 = 12.97, p = 0.037) but not for [DA]max or vesicular DA release. All region × time interactions were not significant. The sample size was = 5 in the dorsal striatum and = 4 in the ventral striatum. Overall, the control experiments demonstrated that AMPH elevated electrically evoked DA levels, inhibited DA uptake, and increased vesicular DA release in both striatal sub-regions whether the depleting stimulation was applied or not and that the depleting stimulation delayed the AMPH response.

Blockade of DA synthesis

Figure 4 shows the effects of the depleting stimulation and αMpT (200 mg/kg i.p.), a tyrosine hydroxylase blocker. This experiment thus examines whether AMPH activation of DA synthesis underlies the AMPH-induced increase in vesicular DA release observed in the depleting stimulation-only control (Fig. 2). This experiment is also identical to the original design of Venton et al. (2006) demonstrating the ability of cocaine to reinstate vesicular DA release after near-complete depletion of the readily releasable DA pool. Individual (Fig. 4a) and averaged (Fig. 4b) responses show that the depleting stimulation and αMpT caused a marked decrease in [DA]max during the treatment period. AMPH elicited a robust increase in [DA]max evoked by the reinforcing-like stimulation in the ventral striatum after this treatment. By comparison, the increase was considerably blunted in the dorsal striatum. Data for statistical analysis (Fig. 4c) demonstrated that AMPH caused a greater increase in [DA]max and vesicular DA release in the ventral versus the dorsal striatum but a similar decrease in DA uptake in both sub-regions. Statistical analysis revealed a significant effect of region and time for both [DA]max (F1,5 = 11.86, p = 0.018 and F2,10 = 5.86, p = 0.021, respectively) and vesicular DA release (F1,5 = 6.62, p = 0.050, and F2,10 = 5.73, p = 0.022, respectively) but only a significant effect of time for DA uptake (F2,10 = 10.80, p = 0.0032). All region × time interactions were not significant. The sample size was = 6 in the dorsal striatum and = 6 in the ventral striatum. Overall, this experiment suggests that, while AMPH similarly inhibited DA uptake in both striatal sub-regions after the αMpT treatment, the AMPH-induced augmentation of electrically evoked DA levels and vesicular DA release is more dependent on DA synthesis in the dorsal compared with the ventral striatum.

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Figure 4. Pharmacological treatment with αMpT. See legend to Fig. 2 for details, with the exception that αMpT was injected during the treatment period (blue symbols and left arrow).

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Blockade of DA degradation

Figure 5 shows the effects of the depleting stimulation and pargyline (75 mg/kg i.p.), a monoamine oxidase blocker. This experiment thus examines whether AMPH inhibition of DA degradation underlies the AMPH-induced increase in vesicular DA release observed in the depleting stimulation-only control (Fig. 2). Individual (Fig. 5a) and averaged (Fig. 5b) responses show that the depleting stimulation and pargyline caused a small decrease in [DA]max during the treatment period. AMPH increased [DA]max evoked by the reinforcing-like stimulation in the ventral striatum only under these conditions. Data for statistical analysis (Fig. 5c) demonstrate that AMPH increased [DA]max and vesicular DA release in the ventral but not the dorsal striatum, and decreased DA uptake to a similar degree in both striatal sub-regions. Statistical analysis revealed a significant effect of region for both [DA]max (F1,4 = 45.73, p = 0.0025) and vesicular DA release (F1,4 = 45.89, p = 0.0025) but of time for DA uptake (F2,14 = 9.33, p = 0.0027). All region × time interactions were not significant. The sample size was = 7 in the dorsal striatum and = 6 in the ventral striatum. Overall, this experiment suggests that, while AMPH similarly inhibited DA uptake in both striatal sub-regions after the pargyline treatment, the AMPH-induced augmentation of electrically evoked DA levels and vesicular DA release is more dependent on DA degradation in the dorsal compared with the ventral striatum.

image

Figure 5. Pharmacological treatment with pargyline. See legend to Fig. 2 for details, with the exception that pargyline was injected during the treatment period (blue symbols and left arrow).

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Combined blockade of DA synthesis and degradation

Figure 6 shows the effects of the depleting stimulation and blockade of DA synthesis with αMpT (200 mg/kg, i.p.) and DA degradation with pargyline (75 mg/kg, i.p.). This experiment thus further examines whether AMPH activation of DA synthesis and inhibition of DA degradation underlie the AMPH-induced increase in vesicular DA release observed in the depleting stimulation-only control (Fig. 2). Individual (Fig. 6a) and averaged (Fig. 6b) responses show that the depleting stimulation with αMpT and pargyline caused a modest decrease in [DA]max during the treatment period. AMPH increased [DA]max evoked by the reinforcing-like stimulation in the ventral striatum only under these conditions. Data for statistical analysis (Fig. 6c) demonstrate that AMPH increased [DA]max and vesicular DA release in the ventral but not the dorsal striatum, and decreased DA uptake to a similar degree in both regions. Statistical analysis revealed a significant effect of region (F1,5 = 64.79, p = 0.0005), time (F2,16 = 5.45, p = 0.0157), and region × time (F2,10 = 6.50, p = 0.0155) on [DA]max, a significant effect of region (F1,5 = 49.24, p = 0.0009) and region × time (F2,10 = 4.23, p = 0.0465) on vesicular DA release, and a significant effect of time (F2,16 = 6.39, p = 0.0091) on DA uptake. The sample size was = 10 in the dorsal striatum and = 6 in the ventral striatum. Overall, this experiment suggests that, while AMPH similarly inhibited DA uptake in both striatal sub-regions after the combined αMpT and pargyline treatment, the AMPH-induced augmentation of electrically evoked DA levels and vesicular DA release is more dependent on DA synthesis and degradation in the dorsal compared with the ventral striatum.

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Figure 6. Pharmacological treatment with αMpT and pargyline. See legend to Fig. 2 for details, with the exception that αMpT and pargyline were injected during the treatment period (blue symbols and left arrow).

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Across-treatment analysis of AMPH effects on [DA]max

The previous experiments examined individual treatment effects of AMPH between regions. The goal of this analysis was to compare these effects across treatments. Figure 7 shows [DA]max evoked by the reinforcing-like stimulation during the post-treatment period. Data are from Figs 2 to 6 and were normalized to the baseline value at time 0 min, which is equivalent to time 118 min of the previous figures. This manipulation was performed to minimize treatment effects on [DA]max. Data collected in the dorsal striatum segregated into two groups, with AMPH eliciting a robust increase in [DA]max in controls but having minimal to no effect in the pharmacological treatments (Fig. 7a). Statistical analysis of the last time point (40 min) revealed a significant effect of treatment (F4,30 = 6.34, p = 0.0008) and a significant difference between the pooled controls and each of the pharmacological treatments (p < 0.004). In the ventral striatum by contrast, data appeared to segregate into a single group, and AMPH elicited a robust increase across treatments (Fig. 7b). Although individual treatments with αMpT and pargyline did not achieve the same AMPH-induced increase in [DA]max as the two controls or combined drug cocktail, statistical analysis revealed no significant (F4,23 = 1.42, p = 0.26) effect of treatment. Figure 7 also showed that the depleting stimulation delayed the AMPH response but did not alter its magnitude in both striatal sub-regions. Overall, this across-treatment analysis suggests that AMPH-induced increases in electrically evoked DA levels are more dependent on DA synthesis and degradation in the dorsal compared with the ventral striatum.

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Figure 7. Effects of AMPH on [DA]max across all treatment groups. Panels (a) and (b) show [DA]max evoked by the reinforcing-like stimulation during the post-treatment period and collected in the dorsal and ventral striatum, respectively. Data are from Figs 2 to 6 and were normalized by subtracting the value at time 0 min (equivalent of time 118 min) from values at all of the time points in each animal and then averaging across groups. ‘Δ% pre-AMPH’ thus represents the absolute percent increase in [DA]max during the post-treatment period. The black square demarcates the time of AMPH injection. Data are expressed as the mean ± SEM. Treatment key: no-stimulation control, no-depleting stimulation control; stimulation control, depleting stimulation-only control; αMpT, depleting stimulation with αMpT; pargyline, depleting stimulation with pargyline; αMpT, pargyline, depleting stimulating with αMpT and pargyline. *Significantly different from the pooled control group at p < 0.05. NS, no significant main effect of treatment.

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Relationships between [DA]max and vesicular DA release or DA uptake

Figure 8 shows correlations between vesicular DA release or DA uptake and [DA]max. This analysis was performed, because Figs 2 to 6 suggested that AMPH reduced DA uptake consistently across treatments but elicited variable increases in vesicular DA release that mirrored [DA]max. If [DA]max better correlates with vesicular DA release than DA uptake, then the pronounced striatal sub-regional differences with [DA]max shown in Fig. 7 also relate more to differences in vesicular DA release than DA uptake. Determining [DA]p and k for all of the responses in Fig. 7 would be very time consuming and is thus prohibitive. This correlation analysis could additionally reveal insight into the relative importance of the pre-synaptic mechanisms of vesicular DA release and DA uptake in mediating AMPH-induced alterations in extracellular DA levels. Data in Fig. 8 are the averaged values displayed in panel (c) of Figs 26 and combined across treatment and region. Vesicular DA release was tightly associated with the trend line and significantly correlated (r = 0.990; p < 0.01) with [DA]max (Fig. 8a). In contrast, although a significant correlation was found for DA uptake and [DA]max (r = 0.484; p < 0.05), data were more diffusely scattered around the trend line (Fig. 8b). Overall, these results suggest that augmented vesicular DA release plays a greater role in increasing electrically evoked DA levels than DA uptake inhibition after AMPH administration.

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Figure 8. Relationships between vesicular DA release (a) or DA uptake (b) and [DA]max. Data are from panel (c) of Figs 26. All treatment groups in both dorsal and ventral striata were combined. The solid line is the best-fit line for linear regression. r, regression coefficient.

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Treatment effects on pre-AMPH baseline [DA]max

Figure 9 compares treatment effects on the pre-AMPH baseline responses to the reinforcing-like stimulation. This analysis was performed, because the analysis of individual treatments in Figs 26 suggested that treatments elicited variable effects on [DA]max prior to AMPH administration, which may have altered the AMPH responses shown in Fig. 7. Overall, pre-AMPH baseline [DA]max was altered in a region- and treatment-specific manner. Statistical analysis revealed a significant effect of region (F1,53 = 8.80, p = 0.0045) and treatment (F4,53 = 18.69, p < 0.0001) but no significant interaction (F4,53 = 1.49, p = 0.22). Dunnett's test of the main treatment effects using the no-depleting stimulation group (−stim) as the control revealed a significant decrease by αMpT (αMT; p < 0.0001) and increase by pargyline (P; p = 0.026). The increase in [DA]max with pargyline could be due to increased cytosolic DA levels after monoamine oxidase inhibition leading to greater vesicular packaging and enhanced DA release, consistent with previous studies (Kuhr et al. 1986; Garris and Wightman 1995; Heien et al. 1995). There was also a trend for a decrease with depleting stimulation (+stim; p = 0.098) but not for the combined treatment of αMpT and pargyline (αMT, P; p = 0.91). The lower [DA]max in the depleting stimulating-only compared with no-depleting stimulation control may thus be responsible for the delayed AMPH response in the former treatment (Figs 2 and 3). Treatments with pargyline (P and αMT, P) also appeared to elicit the most prominent differential effects between striatal sub-regions, with greater increases in the ventral compared with the dorsal striatum (P: 152.8±17.0 and 97.7±13.6% pre-treatment, respectively; αMT, P: 98.1±14.4 and 62.1±8.9% pre-treatment, respectively), but these comparisons were not statistically analyzed due to the non-significant interaction. Overall, these results suggest that treatments elicited variable effects on [DA]max, a lower [DA]max may be responsible for the delayed AMPH response in the depleting-stimulation-only control, and pargyline may be more effective in the ventral compared with the dorsal striatum.

image

Figure 9. Treatments effects on pre-AMPH baseline [DA]max. Data are from Figs 2 to 6 and show [DA]max evoked by the reinforcing-like stimulation and collected at time 118 min during the post-treatment period in the dorsal and ventral striata. This time point, identified as time 0 min, is immediately before AMPH administration. Data are expressed as the mean ± SEM. Abbreviations: −stim, no-depleting stimulation control; +stim, depleting stimulation-only control; αMT, αMpT; P, pargyline; αMTP, αMpT and pargyline.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

This study investigated how AMPH augments vesicular DA release, a recently identified target of this psychostimulant on pre-synaptic DA signaling. We showed that AMPH effects on extracellular DA levels evoked by reinforcing-like stimulation are altered by drugs manipulating DA synthesis and degradation in a striatal sub-region-specific manner and that these effects were mediated predominately by changes in vesicular DA release and not DA uptake. Our results are consistent with AMPH enhancing vesicular DA release by activating DA synthesis and inhibiting DA degradation in the dorsal striatum but by targeting an alternative mechanism in the ventral striatum. Overall, this study advances our understanding of the complex cellular actions of AMPH and the hypothesis that effects of abused drugs are regionally distinct in the striatum.

Experimental design

Two controls, with and without the depleting stimulation, were incorporated into the experimental design. Both controls showed that AMPH increased evoked DA levels, inhibited DA uptake, and up-regulated vesicular DA release in the dorsal and ventral striatum, similar to our previous work (Ramsson et al. 2011a,b; Daberkow et al. 2013). The depleting stimulation delayed the response of [DA]max to AMPH but not its magnitude (Fig. 7). This delay could be due to compromised releasable DA stores, because pre-AMPH baseline [DA]max had not completely rebounded (Figs 2, 3 and 9). Given a wait time of 5 s per stimulus pulse (Wightman et al. 1988), 50 min is required for the releasable pool to recover fully after the 10-s depleting stimulation, and AMPH was injected 40 min after the last train of the treatment period. Also, reinforcing-like trains were applied during the post-treatment period, which would have additionally taxed the releasable pool.

The experimental design also incorporated kinetic analysis that resolved the respective contributions of vesicular DA release and DA uptake to observed AMPH-induced changes in [DA]max. This capability permitted us to identify vesicular release as the predominant contributor to observed treatment effects on AMPH responses. The basis for this conclusion is two-fold. First, AMPH effects on [DA]max varied by striatal sub-region, as shown by the analysis of individual drug treatments (Figs 46). However, only vesicular release, but not uptake, exhibited a similar, region-distinct variation. Moreover, while AMPH significantly slowed DA uptake in all treatments, we did not observe a significant effect of striatal sub-region on this inhibition for any drug treatment. Second, this same issue was addressed across treatments in Fig. 8. The x-axis clearly shows the variation of treatment effects on the response of [DA]max to AMPH, ranging from < 20% to > 200% pre-treatment. However, vesicular release better correlated with this variation in [DA]max than uptake. The poorer correlation with uptake was not due to limited dynamic range, as uptake varied from pre-AMPH baseline levels near 100% to near 25% after amphetamine.

The veracity of the kinetic analysis is defended by the demonstration that in both control treatments AMPH decreased DA uptake and increased DA vesicular release in the dorsal and ventral striatum, which we have previously established. AMPH also slowed DA uptake in all treatments, that is, two controls and three drug treatments, as expected for a DA uptake inhibitor. Moreover, analysis of the combined pre-treatment data set showed significantly higher DA uptake in the dorsal compared with the ventral striatum, which is also consistent with our previous work (Garris and Wightman 1994; Garris et al. 1994; Wu et al. 2001a,b). Interestingly, we did not observe greater vesicular DA release and lower evoked DA levels in the dorsal striatum, as shown before, but these differences could be due to the shorter train duration used in this study (0.4 versus 2.0 s). Longer train durations interrogate different storage pools (Rizzoli and Betz 2005) and allow more time for uptake to exert control over evoked DA levels during the stimulation compared with shorter train durations. This latter point is buttressed by the better correlation between vesicular release and [DA]max evoked by the 0.4-s, reinforcing-like stimulation than for uptake (Fig. 8).

One important caveat of the experimental design was the differential effects of treatments on pre-AMPH baseline [DA]max (Fig. 9). By reflecting the status of releasable DA stores, these treatment effects may have altered AMPH responses to some degree. For example, the AMPH-induced increase in [DA]max after αMpT in the ventral striatum may not be as great as either control, because the releasable pool was reduced by inhibiting DA synthesis. This αMpT-induced decrease in the releasable pool was offset by pargyline in the combined drug cocktail, where the response to AMPH was similar to controls. Restoring [DA]max alone is not sufficient to return the AMPH response to control levels, because AMPH elicited minimal to no change in the dorsal striatum after the cocktail of αMpT and pargyline, suggestive of a different AMPH mechanism in this striatal sub-region. A ceiling effect may conversely explain why the AMPH-induced increase in [DA]max in the ventral striatum after pargyline was not as great as controls and the combined drug treatment. Indeed, baseline [DA]max after pargyline in this striatal region was the highest of any treatment. There may thus be a limit as to how much AMPH can increase [DA]max after pargyline when vesicular release is already activated. Clearly, more work needs to be done to better understand the nuances of these treatment effects.

Mechanism of AMPH action on vesicular DA release

The AMPH-induced increase in vesicular DA release was prevented in the dorsal striatum by the drug cocktail of αMpT and pargyline. This result is consistent with a mechanism of AMPH inhibiting monoamine oxidase (Scorza et al. 1997) and/or activating tyrosine hydroxylase (Kuczenski 1975) leading to elevated cytosolic DA levels, greater packaging of DA into vesicles, and ultimately increased quantal size. At this time, it is not possible to determine the respective contributions of DA degradation and synthesis, because both αMpT and pargyline alone largely prevented the AMPH-induced enhancement of vesicular DA release. Nevertheless, our results are consistent with DA degradation and synthesis as the predominant targets for AMPH augmenting vesicular DA release in the dorsal striatum.

In contrast, the AMPH-induced augmentation of vesicular DA release in the ventral striatum was insensitive to the combination of αMpT and pargyline. This result suggests an alternative mechanism to activating DA synthesis and inhibiting DA degradation in this striatal sub-region, perhaps mobilizing the reserve DA pool, as has previously been proposed albeit not proven for cocaine and amfonelic acid in studies with a similar experimental design as used here (Ewing et al. 1983; Kuhr et al. 1986; Venton et al. 2006). Transgenic synapsin-knockout mice show that cocaine-induced increases in vesicular DA release are mediated by synapsin (Venton et al. 2006; Kile et al. 2010), a protein that segregates vesicles into the reserve pool (Greengard et al. 1993; Pieribone et al. 1995). It is thus interesting to speculate that AMPH similarly acts on a synapsin-dependent reserve DA pool to enhance vesicular DA release. α-Synuclein, another synaptic protein involved in vesicular storage and trafficking and implicated in methylphenidate-induced up-regulation of vesicular DA release (Chadchankar et al. 2012), should also be considered. Other potential targets for AMPH in the ventral striatum are identified in Introduction section.

Although lower doses of AMPH also inhibit DA degradation and activate DA synthesis (Kuczenski 1983), whether they similarly contribute to increases in vesicular DA release in the dorsal striatum as observed here with 10 mg/kg requires further study. Nevertheless, at a minimum, our results demonstrate a robust enhancement of vesicular DA release in the ventral striatum mimicking the actions of cocaine and amfonelic acid, and are relevant to high-dose behavioral effects of AMPH, such as stereotypy and toxic psychosis (Seiden et al. 1993). Moreover, because of the controversial nature of AMPH mechanism, our results of augmented vesicular DA release are contrasted with the numerous reports of AMPH depleting vesicular DA stores (vide infra).

DAT Inhibitors and vesicular DA release

Several DAT inhibitors have been shown to augment vesicular DA release, including cocaine (Jones et al. 1995; Lee et al. 2001; Venton et al. 2006; Oleson et al. 2009; Kile et al. 2010), methylphenidate (Volz et al. 2008; Chadchankar et al. 2012), amfonelic acid (Ewing et al. 1983), nomifensine (Lee et al. 2001), and WIN 35428 (Lee et al. 2001). There is now emerging evidence to suggest that AMPH also acts similarly (this study; Ramsson et al. 2011b; Daberkow et al. 2013). It remains to be determined whether a mechanism of enhancing vesicular DA release generalizes to all DAT inhibitors and to what extent, and the relevance of this mechanism to overall drug effect. Because of its complex actions, including effects on tyrosine hydroxylase, monoamine oxidase, and membrane excitability, AMPH may prove to be more efficacious in enhancing vesicular DA release than psychostimulants predominantly acting via DAT inhibition.

One complication in assessing the hypothesis that DAT inhibitors augment vesicular DA release is that these effects are stimulus-dependent and differ between preparations (Ramsson et al. 2011a). Indeed, cocaine and amphetamine both robustly inhibit DA uptake and enhance vesicular DA release in vivo when evaluated using short-duration pulse trains as we have used here (Venton et al. 2006; Oleson et al. 2009; Ramsson et al. 2011b; Daberkow et al. 2013), whereas the predominant effect using pulse trains of intermediate duration is uptake inhibition (May et al. 1988; Wu et al. 2001a; Ramsson et al. 2011a). Moreover, AMPH decreases vesicular DA release in brain slices (Jones et al. 1998; Schmitz et al. 2001; Patel et al. 2003) and single cells (Chen and Ewing 1995; Anderson et al. 1998), but only long-duration trains reveal this effect in vivo (Ewing et al. 1983; Kuhr et al. 1985, 1986). It will thus be necessary to compare DAT inhibitors using uniform experimental conditions that support enhanced vesicular DA release.

Another important issue is the respective contribution of altered DA release versus uptake in mediating the effects of DAT inhibitors on DA function. Uptake is clearly an important site of action, because drug potencies for DAT binding and self-administration correlate well (Ritz et al. 1987), and DAT inhibitors can increase striatal levels of extracellular DA in the absence of effects on vesicular DA release (Wu et al. 2001a; Ramsson et al. 2011a). Although AMPH both robustly inhibits DA uptake and augments vesicular DA release in awake animals, DA release is better correlated with [DA]max (Daberkow et al. 2013). We extend this finding in this study to canvass a broader range of values to include conditions of disrupted vesicular DA release. Taken together, these results suggest that augmented vesicular DA release plays the more predominant role in mediating the AMPH-induced augmentation of [DA]max evoked by the reinforcing-like stimulation.

The DAT inhibitors, cocaine (Stuber et al. 2005) and nomifensine (Robinson and Wightman 2004), have additionally been shown to augment naturally occurring phasic DA transients. These sub-second increases in extracellular DA are generated by burst firing, occur spontaneously or in response to rewards and their predictive cues, and are important learning signals in goal-directed behavior (Wanat et al. 2009). Their hyper-activation by drugs of abuse is proposed to usurp reward-related circuits during addiction (Hyman 2005). We have also demonstrated an up-regulation of phasic DA transients with AMPH (Ramsson et al. 2011b; Daberkow et al. 2013). The DA responses evoked by the reinforcing-like stimulation resemble these physiological DA signals in amplitude and dynamics (Robinson and Wightman 2007) and as described above, are enhanced by AMPH to a greater extent by increases in vesicular DA release compared with decreases in DA uptake. It can thus be argued that AMPH-induced increases in vesicular DA release may play a more prominent role in activating phasic DA signaling than decreased DA uptake.

Regional effects of abused drugs

Drugs of abuse elicit regionally distinct actions in the striatum, with primary reinforcing and conditional effects mediated by the ventral portion and instrumental learning and habit formation mediated by the dorsal portion (Everitt and Robbins 2005). Abused drugs, such as cocaine and AMPH, also preferentially increase extracellular DA levels in the ventral versus dorsal striatum (Di and Imperato 1988; Carboni et al. 1989; Cass et al. 1992; Kuczenski and Segal 1992; Wu et al. 2001a; Ramsson et al. 2011b). The findings of this study extend regional effects of addictive drugs in the striatum to distinct mechanisms for AMPH augmenting vesicular DA release. Although the functional significance of our findings is not established, one possibility is that distinct mechanisms confer a differential ability to maintain activated DA signaling. For example, extracellular DA levels electrically evoked by long-duration stimulation are less sensitive to the depleting effects of AMPH in the ventral compared with the dorsal striatum (Ewing et al. 1983; Kuhr et al. 1985, 1986; Ramsson et al. 2011a). It may thus be that the mechanism by which AMPH increases vesicular DA release in the ventral striatum is better capable of sustaining higher rates of release than in the dorsal striatum, which is more dependent upon DA degradation and synthesis. Regionally distinct effects of AMPH on vesicular DA release may additionally contribute to the preferential increases in extracellular DA levels in the ventral striatum.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

We show that AMPH uniquely augments vesicular DA release in the dorsal and ventral striatum and that these effects contribute more to the amplitude of phasic-like DA signals than DA uptake inhibition. The results of this study thus advance our understanding of the complex cellular mechanisms targeted by this psychostimulant and further highlight vesicular DA release as an important site of action for DAT inhibitors. Future studies should explore the role of DA synthesis, degradation and storage pools in mediating the effects of AMPH on behaviorally relevant DA signals under physiological conditions and further characterize the effects of monoamine inhibition on DA signaling in striatal sub-regions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

This research was supported by grant NIH DA 021770 and NSF DBI-0754615. The authors declare no conflict of interest. AJA performed all experiments and analyzed all data. PAG conceived and designed the study, and wrote the manuscript with AJA. SAJ designed and interpreted statistical analyses. All authors approved the submitted version of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References
  • Aceto M. D., Botton I., Martin R., Levitt M., Bentley H. C. and Speight P. T. (1970) Pharmacologic properties and mechanism of action of amfonelic acid. Eur. J. Pharmacol. 10, 344354.
  • Anderson B. B., Chen G., Gutman D. A. and Ewing A. G. (1998) Dopamine levels of two classes of vesicles are differentially depleted by amphetamine. Brain Res. 788, 294301.
  • Brown J. M., Hanson G. R. and Fleckenstein A. E. (2001) Cocaine-induced increases in vesicular dopamine uptake: role of dopamine receptors. J. Pharmacol. Exp. Ther. 298, 11501153.
  • Carboni E., Imperato A., Perezzani L. and Di Chiara G. (1989) Amphetamine, cocaine, phencyclidine and nomifensine increase extracellular dopamine concentrations preferentially in the nucleus accumbens of freely moving rats. Neuroscience 28, 653661.
  • Cass W. A., Gerhardt G. A., Mayfield R. D., Curella P. and Zahniser N. R. (1992) Differences in dopamine clearance and diffusion in rat striatum and nucleus accumbens following systemic cocaine administration. J. Neurochem. 59, 259266.
  • Chadchankar H., Ihalainen J., Tanila H. and Yavich L. (2012) Methylphenidate modifies overflow and presynaptic compartmentalization of dopamine via an alpha-synuclein-dependent mechanism. J. Pharmacol. Exp. Ther. 341, 484492.
  • Cheer J. F., Aragona B. J., Heien M. L., Seipel A. T., Carelli R. M. and Wightman R. M. (2007) Coordinated accumbal dopamine release and neural activity drive goal-directed behavior. Neuron 54, 237244.
  • Chen G. and Ewing A. G. (1995) Multiple classes of catecholamine vesicles observed during exocytosis from the Planorbis cell body. Brain Res. 701, 167174.
  • Daberkow D. P., Brown H. D., Bunner K. D., Kraniotis S. A., Doellman M. A., Ragozino M. E., Garris P. A. and Roitman M. F. (2013) Amphetamine paradoxically augments exocytotic dopamine release and phasic dopamine signals. J. Neurosci. 33, 452463.
  • Di C. G. and Imperato A. (1988) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Natl Acad. Sci. U. S. A. 85, 52745278.
  • Everitt B. J. and Robbins T. W. (2005) Neural systems of reinforcement for drug addiction: from actions to habits to compulsion. Nat. Neurosci. 8, 14811489.
  • Ewing A. G., Bigelow J. C. and Wightman R. M. (1983) Direct in vivo monitoring of dopamine released from two striatal compartments in the rat. Science 221, 169171.
  • Fleckenstein A. E., Haughey H. M., Metzger R. R., Kokoshka J. M., Riddle E. L., Hanson J. E., Gibb J. W. and Hanson G. R. (1999) Differential effects of psychostimulants and related agents on dopaminergic and serotonergic transporter function. Eur. J. Pharmacol. 382, 4549.
  • Fleckenstein A. E., Volz T. J., Riddle E. L., Gibb J. W. and Hanson G. R. (2007) New insights into the mechanism of action of amphetamines. Annu. Rev. Pharmacol. Toxicol. 47, 681698.
  • Garris P. A. and Wightman R. M. (1994) Different kinetics govern dopaminergic transmission in the amygdala, prefrontal cortex, and striatum: an in vivo voltammetric study. J. Neurosci. 14, 442450.
  • Garris P. A. and Wightman R. M. (1995) Distinct pharmacological regulation of evoked dopamine efflux in the amygdala and striatum of the rat in vivo. Synapse 20, 269279.
  • Garris P. A., Ciolkowski E. L. and Wightman R. M. (1994) Heterogeneity of evoked dopamine overflow within the striatal and striatoamygdaloid regions. Neuroscience 59, 417427.
  • Garris P. A., Christensen J. R., Rebec G. V. and Wightman R. M. (1997) Real-time measurement of electrically evoked extracellular dopamine in the striatum of freely moving rats. J. Neurochem. 68, 152161.
  • Greengard P., Valtorta F., Czernik A. J. and Benfenati F. (1993) Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 259, 780785.
  • Heal D. J., Cheetham S. C. and Smith S. L. (2009) The neuropharmacology of ADHD drugs in vivo: insights on efficacy and safety. Neuropharmacology 57, 608618.
  • Heien M. L., Phillips P. E., Stuber G. D., Seipel A. T. and Wightman R. M. (2003) Overoxidation of carbon-fiber microelectrodes enhances dopamine adsorption and increases sensitivity. Analyst. 128, 14131419.
  • Hyman S. E. (2005) Addiction: a disease of learning and memory. Am. J. Psychiatry 162, 14141422.
  • Ingram S. L., Prasad B. M. and Amara S. G. (2002) Dopamine transporter-mediated conductances increase excitability of midbrain dopamine neurons. Nat. Neurosci. 5, 971978.
  • Jones S. R., Garris P. A. and Wightman R. M. (1995) Different effects of cocaine and nomifensine on dopamine uptake in the caudate-putamen and nucleus accumbens. J. Pharmacol. Exp. Ther. 274, 396403.
  • Jones S. R., Gainetdinov R. R., Wightman R. M. and Caron M. G. (1998) Mechanisms of amphetamine action revealed in mice lacking the dopamine transporter. J. Neurosci. 18, 19791986.
  • Kile B. M., Guillot T. S., Venton B. J., Wetsel W. C., Augustine G. J. and Wightman R. M. (2010) Synapsins differentially control dopamine and serotonin release. J. Neurosci. 30, 97629770.
  • Kuczenski R. (1975) Effects of catecholamine releasing agents on synaptosomal dopamine biosynthesis: multiple pools of dopamine or multiple forms of tyrosine hydroxylase. Neuropharmacology 14, 110.
  • Kuczenski R. (1983) Biochemical actions of amphetamine and other stimulants, in Stimulants: Neurochemical, Behavioral and Clinical Perspectives (Creese I., ed.), pp. 3161. Raven Press, New York.
  • Kuczenski R. and Segal D. S. (1992) Differential effects of amphetamine and dopamine uptake blockers (cocaine, nomifensine) on caudate and accumbens dialysate dopamine and 3-methoxytyramine. J. Pharmacol. Exp. Ther. 262, 10851094.
  • Kuhr W. G., Ewing A. G., Near J. A. and Wightman R. M. (1985) Amphetamine attenuates the stimulated release of dopamine in vivo. J. Pharmacol. Exp. Ther. 232, 388394.
  • Kuhr W. G., Bigelow J. C. and Wightman R. M. (1986) In vivo comparison of the regulation of releasable dopamine in the caudate nucleus and the nucleus accumbens of the rat brain. J. Neurosci. 6, 974982.
  • Lee T. H., Balu R., Davidson C. and Ellinwood E. H. (2001) Differential time-course profiles of dopamine release and uptake changes induced by three dopamine uptake inhibitors. Synapse 41, 301310.
  • Logman M. J., Budygin E. A., Gainetdinov R. R. and Wightman R. M. (2000) Quantitation of in vivo measurements with carbon fiber microelectrodes. J. Neurosci. Methods 95, 95102.
  • May L. J. and Wightman R. M. (1989) Heterogeneity of stimulated dopamine overflow within rat striatum as observed with in vivo voltammetry. Brain Res. 487, 311320.
  • May L. J., Kuhr W. G. and Wightman R. M. (1988) Differentiation of dopamine overflow and uptake processes in the extracellular fluid of the rat caudate nucleus with fast-scan in vivo voltammetry. J. Neurochem. 51, 10601069.
  • Michael D., Travis E. R. and Wightman R. M. (1998) Color images for fast-scan CV measurements in biological systems. Anal. Chem. 70, 586A592A.
  • Mundorf M. L., Hochstetler S. E. and Wightman R. M. (1999) Amine weak bases disrupt vesicular storage and promote exocytosis in chromaffin cells. J. Neurochem. 73, 23972405.
  • Oleson E. B., Salek J., Bonin K. D., Jones S. R. and Budygin E. A. (2009) Real-time voltammetric detection of cocaine-induced dopamine changes in the striatum of freely moving mice. Neurosci. Lett. 467, 144146.
  • Patel J., Mooslehner K. A., Chan P. M., Emson P. C. and Stamford J. A. (2003) Presynaptic control of striatal dopamine neurotransmission in adult vesicular monoamine transporter 2 (VMAT2) mutant mice. J. Neurochem. 85, 898910.
  • Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic Press, New York.
  • Peacock J. and Benca R. M. (2010) Narcolepsy: clinical features, co-morbidities & treatment. Indian J. Med. Res. 131, 338349.
  • Pieribone V. A., Shupliakov O., Brodin L., Hilfiker-Rothenfluh S., Czernik A. J. and Greengard P. (1995) Distinct pools of synaptic vesicles in neurotransmitter release. Nature 375, 493497.
  • Ramsson E. S., Covey D. P., Daberkow D. P., Litherland M. T., Juliano S. A. and Garris P. A. (2011a) Amphetamine augments action potential-dependent dopaminergic signaling in the striatum in vivo. J. Neurochem. 117, 937948.
  • Ramsson E. S., Howard C. D., Covey D. P. and Garris P. A. (2011b) High doses of amphetamine augment, rather than disrupt, exocytotic dopamine release in the dorsal and ventral striatum of the anesthetized rat. J. Neurochem. 119, 11621172.
  • Riddle E. L., Topham M. K., Haycock J. W., Hanson G. R. and Fleckenstein A. E. (2002) Differential trafficking of the vesicular monoamine transporter-2 by methamphetamine and cocaine. Eur. J. Pharmacol. 449, 7174.
  • Riddle E. L., Hanson G. R. and Fleckenstein A. E. (2007) Therapeutic doses of amphetamine and methylphenidate selectively redistribute the vesicular monoamine transporter-2. Eur. J. Pharmacol. 571, 2528.
  • Ritz M. C., Lamb R. J., Goldberg S. R. and Kuhar M. J. (1987) Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science 237, 12191223.
  • Rizzoli S. O. and Betz W. J. (2005) Synaptic vesicle pools. Nat. Rev. Neurosci. 6, 5769.
  • Robinson D. L. and Wightman R. M. (2004) Nomifensine amplifies subsecond dopamine signals in the ventral striatum of freely-moving rats. J. Neurochem. 90, 894903.
  • Robinson D. L. and Wightman R. M. (2007) Rapid dopamine release in freely moving rats, in Electrochemical Methods for Neuroscience (Michael A. C. and Borland L. M., eds), pp. 1734. CRC Press, Boca Raton.
  • Schmitz Y., Lee C. J., Schmauss C., Gonon F. and Sulzer D. (2001) Amphetamine distorts stimulation-dependent dopamine overflow: effects on D2 autoreceptors, transporters, and synaptic vesicle stores. J. Neurosci. 21, 59165924.
  • Scorza M. C., Carrau C., Silveira R., Zapata-Torres G., Cassels B. K. and Reyes-Parada M. (1997) Monoamine oxidase inhibitory properties of some methoxylated and alkylthio amphetamine derivatives: structure–activity relationships. Biochem. Pharmacol. 54, 13611369.
  • Seiden L. S., Sabol K. E. and Ricaurte G. A. (1993) Amphetamine: effects on catecholamine systems and behavior. Annu. Rev. Pharmacol. Toxicol. 33, 639677.
  • Shore P. A. (1976) Actions of amfonelic acid and other non-amphetamine stimulants on the dopamine neuron. J. Pharm. Pharmacol. 28, 855857.
  • Stuber G. D., Wightman R. M. and Carelli R. M. (2005) Extinction of cocaine self-administration reveals functionally and temporally distinct dopaminergic signals in the nucleus accumbens. Neuron 46, 661669.
  • Sulzer D. (2011) How addictive drugs disrupt presynaptic dopamine neurotransmission. Neuron 69, 628649.
  • Venton B. J., Seipel A. T., Phillips P. E., Wetsel W. C., Gitler D., Greengard P., Augustine G. J. and Wightman R. M. (2006) Cocaine increases dopamine release by mobilization of a synapsin-dependent reserve pool. J. Neurosci. 26, 32063209.
  • Volz T. J., Farnsworth S. J., King J. L., Riddle E. L., Hanson G. R. and Fleckenstein A. E. (2007) Methylphenidate administration alters vesicular monoamine transporter-2 function in cytoplasmic and membrane-associated vesicles. J. Pharmacol. Exp. Ther. 323, 738745.
  • Volz T. J., Farnsworth S. J., Rowley S. D., Hanson G. R. and Fleckenstein A. E. (2008) Methylphenidate-induced increases in vesicular dopamine sequestration and dopamine release in the striatum: the role of muscarinic and dopamine D2 receptors. J. Pharmacol. Exp. Ther. 327, 161167.
  • Wanat M. J., Willuhn I., Clark J. J. and Phillips P. E. (2009) Phasic dopamine release in appetitive behaviors and drug addiction. Curr. Drug Abuse Rev. 2, 195213.
  • Wightman R. M., Amatore C., Engstrom R. C., Hale P. D., Kristensen E. W., Kuhr W. G. and May L. J. (1988) Real-time characterization of dopamine overflow and uptake in the rat striatum. Neuroscience 25, 513523.
  • Wu Q., Reith M. E., Kuhar M. J., Carroll F. I. and Garris P. A. (2001a) Preferential increases in nucleus accumbens dopamine after systemic cocaine administration are caused by unique characteristics of dopamine neurotransmission. J. Neurosci. 21, 63386347.
  • Wu Q., Reith M. E., Wightman R. M., Kawagoe K. T. and Garris P. A. (2001b) Determination of release and uptake parameters from electrically evoked dopamine dynamics measured by real-time voltammetry. J. Neurosci. Methods 112, 119133.