Amphetamine and Methamphetamine Reduce Striatal Dopamine Transporter Function Without Concurrent Dopamine Transporter Relocalization

Amphetamine and Methamphetamine Reduce Striatal Dopamine Tran…ction Without Concurrent Dopamine Transporter Relocalization 2018/4/16, 3+57 PM
J Neurochem. Author manuscript; available in PMC 2014 Mar 21. PMCID: PMC3962019
Published in final edited form as: NIHMSID: NIHMS393937
J Neurochem. 2012 Oct; 123(2): 288–297. PMID: 22804716
Published online 2012 Aug 23. doi: 10.1111/j.1471-4159.2012.07875.x
Amphetamine and Methamphetamine Reduce Striatal Dopamine

Transporter Function Without Concurrent Dopamine Transporter
Relocalization
Christopher L. German, Glen R. Hanson, and Annette E. Fleckenstein
Department of Pharmacology & Toxicology, University of Utah, Salt Lake City, UT 84112
Corresponding Author: Annette E. Fleckenstein, Ph.D., University of Utah, Department of Pharmacology & Toxicology, 30 South 2000
East, Room 201, Salt Lake City, UT 84112, fleckenstein@hsc.utah.edu, Telephone: 801-585-7474
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The publisher's final edited version of this article is available free at J Neurochem
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Abstract Go to:
Amphetamine (AMPH) and methamphetamine (METH) alter dopamine (DA) transporter (DAT)
function. In vitro heterologous cell line and synaptosome studies demonstrate AMPH-induced DAT
internalization, implicating relocalization in reduced DAT uptake following drug exposure. However,
few studies have evaluated DAT localization following in vivo AMPH/METH administration. To
determine DAT subcellular localization following drug administration, a centrifugation technique was
developed to isolate striatal synaptosomal membrane and vesicle fractions. DAT was distributed
between the synaptosomal membrane (60%) and endosomal vesicles (40%), and in vitro application of
the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) to striatal synaptosomes caused
DAT internalization into the vesicle fractions. In contrast, neither single nor repeated in vivo AMPH
and/or METH administrations altered DAT localization 5, 15, 30 or 60 min post-treatment, despite
reduced DAT uptake. Importantly, repeated METH injections uniformly decreased total DAT
immunoreactivity within all fractions 7 d post-treatment. These findings suggest factors other than
internalization can contribute to the observed acute and persistent DAT dysfunction and dopaminergic
deficits following in vivo AMPH or METH administration.
Keywords: Dopamine Transporter, Amphetamine, Methamphetamine, Striatum, Internalization,

Relocalization
Introduction Go to:
The dopamine (DA) transporter (DAT) is responsible for moving extracellular DA into presynaptic
terminals following neurotransmitter release, and disruption of DAT function profoundly alters
intracellular and extracellular DA concentration. A single, high-dose administration of
methamphetamine (METH) or amphetamine (AMPH) reversibly reduces DAT function within the
striatum (Fleckenstein et al. 1997b) – an effect not due to residual drug from in vivo treatment within
the synaptosomal preparation (Fleckenstein et al. 1997b, Kokoshka et al. 1998). In contrast, repeated
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962019/ Page 1 of 18
high-dose METH administrations lead to long-term (greater than 7 d) striatal dopaminergic deficits
characterized by loss of DAT immunoreactivity, decreased DA uptake, reduced DA content, loss of
tyrosine hydroxlyase activity, and the formation of DAT oligomeric complexes (for review see
(Fleckenstein et al. 2007).
AMPH and its analog METH disrupt dopaminergic terminals through several simultaneous
mechanisms of action. At low concentrations, amphetamines function as DAT substrates, driving DA
efflux through a shift in substrate concentration within the synaptic cleft (Kahlig et al. 2005, Connor &
Kuczenski 1986). Higher concentrations of amphetamines diffuse through the plasma membrane and
prevent vesicular sequestration of cytoplasmic DA (Fleckenstein et al. 2007, Mack & Bonisch 1979,
Kahlig et al. 2005, Sulzer & Rayport 1990), which is rapidly transported into the synaptic cleft through
DAT in channel-like fashion (Kahlig et al. 2005). The DAT transport reversal and reduction in activity
caused by amphetamines are both regulated by protein kinase C (PKC) phosphorylation of the
transporter (Johnson et al. 2005b). Pharmacological PKC inhibition prior to administration of
amphetamines preserves DAT function, while PKC activation reduces synaptosomal DAT function
(Sandoval et al. 2001, Vaughan et al. 1997). PKC phosphorylation also drives DAT internalization
(Melikian & Buckley 1999), suggesting transporter endocytosis may underlie reduced DAT function
following exposure to AMPH. Indeed, administration of AMPH to cell lines expressing DAT results in
DAT internalization within minutes (Saunders et al. 2000). Subsequent studies, however, demonstrate
DAT trafficking as biphasic (Johnson et al. 2005a). Large amounts of DAT are rapidly trafficked to the
cell surface within the first minute of AMPH treatment and then internalized as drug exposure persists.
AMPH-induced DAT internalization has been primarily demonstrated in vitro (Melikian & Buckley
1999, Saunders et al. 2000, Chen et al. 2009, Richards & Zahniser 2009, Johnson et al. 2005a), and few
studies have investigated in vivo AMPH or METH treatment upon DAT localization. Consequently, this
study evaluated terminal DAT localization following single and multiple in vivo AMPH and METH
treatment paradigms. To detect drug-induced changes in DAT distribution, plasma membrane and
vesicular fractions were isolated from striatal synaptosomes using differential centrifugation combined
with equilibrium ultracentrifugation across linear sucrose gradients. Using these techniques, this study
suggests in vivo AMPH and METH treatments can decrease DAT function without altering DAT
distribution between the membrane and endosomal compartments. Therefore, mechanisms other than
internalization are likely responsible for the acute and persistent dopaminergic deficits caused by
amphetamines and reported herein.
Materials & Methods Go to:
Reagents and Antibodies

All chemicals, unless otherwise noted, were purchased from Sigma Aldrich (St. Louis, MO).
Antibodies against DAT (1:500; sc-1433) and the DAT peptide (DAT P; sc-1433P) were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against Na+/K+ ATPase (1:500; 63958)
and Rab11 (1:500; 610656) were purchased from BD Biosciences (San Jose, CA). Antibody against
lysosomal-associate membrane protein 2 (LAMP2, 1:500; 51-2200) was purchased from Zymed
Laboratories (San Francisco, CA). HRP-conjugated secondary antibody against rabbit (1:10000; 711-
035-152) and goat (1:10000; 705-035-147) was purchased from Jackson Immuno Research (West
Grove, PA).
Animals and Drug Treatment Paradigms

Male Sprague-Dawley rats (300 – 400 g; Charles River Laboratories, Raleigh, NC) were housed with
food and water provided ad libidum in a temperature- (22 °C) and light-controlled (14/10 light/dark
cycle) environment. Rats were kept in a warm environment (approximately 25 °C) during drug
treatment to permit increases in core body temperature. All experiments were performed in accordance
with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals, approved
by the University of Utah Animal Care and Use Committee, and care was taken to ensure the
minimization of animal pain and distress throughout treatment. d/l-METH HCl was generously donated
by the National Institute of Drug Abuse and synthesized by Research Triangle Institute (Research
Triangle Park, NC), and d-AMPH sulfate was acquired from Sigma-Aldrich (St. Louis, MO). Dosages
of both drugs were calculated as free-base and dissolved to a 1 ml/kg concentration in sterile 0.9%
saline solution. Rats were sacrificed by decapitation. Striata were dissected and quickly placed in ice-
cold 0.32M sucrose buffer (0.32 M sucrose, 3.75 mM NaH2PO4, 12.7 mM Na2HPO4, 1 mM
phenylmethanesulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 1 mM Na3VO4, 1 mM NaF, pH 7.4); an
environment in which the endocytic pathway, endosomal sorting, intracellular signaling and the vast
majority of enzymatic and phosphatase/protease activity is halted. Two drug treatment paradigms,
consisting of either single or multiple drug injections, were used in this study and paired with various
assessment time points. The single treatment paradigm involved a single, subcutaneous (s.c.) injection
of AMPH (15 mg/kg), METH (15 mg/kg) or 0.9% sterile saline solution (1 ml/kg) and decapitation 5,
15, 30 or 60 min later. The multiple treatment paradigm involved four METH (7.5 mg/kg/injection,
s.c.) or 0.9% sterile saline solution (1 ml/kg/injection) injections administered at two-hour intervals
with decapitation 1 h or 7 d after the last injection.
Synaptosomal Isolation, Subcellular Fractionation and Continuous Sucrose Gradients

To isolate striatal synaptosomes, tissues were homogenized in 0.32M sucrose buffer with 25 strokes of
a Dounce homogenizer. Figure 1 outlines the subsequent isolation steps employed within this study.
Briefly, homogenate was then centrifuged at 800 × g for 12 minutes at 4 °C to pellet cellular debris.
The supernatant was transferred to new spin tubes and centrifuged at 22000 × g for 15 minutes at 4 °C.
The microsomal supernatant was discarded and the resulting synaptosomal pellet was either
resuspended in assay buffer (126 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.4 mM MgSO4, 11 mM
glucose, 1 mM ascorbic acid, 3.75 mM NaH2PO4, 12.7 mM Na2HPO4, pH 7.4) for [3H]DA uptake
assays or lysed in ice-cold ddH20 for further subcellular fractionation.
Open in a separate window

Figure 1
Illustration of the isolation protocol employed in this study
To isolate subcellular structures, synaptosomes were lysed in 450 µl ddH20, triturated 25 times with a
glass pipette and disrupted with 5 strokes of a Dounce homogenizer. Samples were then mixed with 50
µl 1M potassium tartrate (KT) and 0.25M HEPES pH 7.4 to yield a 100 mM KT and 25 mM HEPES
buffer (final concentration) and halt osmotic lysis. Samples were then centrifuged at 1500 × g for 15
minutes at 4 °C to separate the synaptosomal membrane (pellet, SM) from synaptosomal vesicular
structures (supernatant). The SM pellet was resuspended in 1M KT and 0.25M HEPES buffer, the
vesicular supernatant was transferred to new spin tubes and all samples were spun again at 1500 × g for
15 minutes at 4 °C to remove contaminants. Two fractions, the SM (pellet) and vesicle (supernatant),
were isolated at this stage. The SM pellet was resuspended with 500 µl RIPA lysis buffer (1% Nonidet-
P40 (v/v), 24 mM sodium deoxycholate, 3.47 mM sodium dodecyl sulfate, 2 mM
ethylenediaminetetraacetic acid, 1X tris-buffered saline (TBS; 20 mM Tris, 137 mM NaCl, pH 8.0), 1
mM PMSF, 10 µg/ml aprotinin, 1 mM Na3VO4, 1 mM NaF) and stored at -80 °C until further use. The
vesicle fraction supernatant was either stored at -80 °C until further use or further fractioned across
continuous sucrose gradients.
Synaptosomal vesicles were further separated by overlaying the 500 µl of 1500 × g supernatant (vesicle
fraction) on 4.0 ml 0.6M – 1.6M continuous sucrose gradients (sucrose mixed in buffer containing 150
mM NaCl, 10 mM HEPES, 1 mM EGTA, 0.1 mM MgCl2, 1 mM PMSF, 10 µg/ml aprotinin, 1 mM
Na3VO4, 1 mM NaF) with a 500 µl 2.0M sucrose pad followed by equilibrium centrifugation using a
Beckman (Brea, CA) SW55Ti rotor and Optima L-100XP centrifuge to spin at 115000 × g for 18 hours
at 4 °C. Twenty-five 200 µl fractions were collected from the top of each sample with a probe
(Labconco Auto Densi-flow; Kansas City, MO) and fraction collector (Gilson FC203B; Middleton,
WI) then stored at -80 °C until further use.
Synaptosomal [3H]DA Uptake
Uptake of [3H]DA into striatal synaptosomes was performed as previously described (Hadlock et al.
2009). Briefly, samples containing striatal synaptosomes prepared in assay buffer and 1 µM pargyline
were incubated at 37 °C for 10 minutes followed by the addition of 0.5 nM [3H]DA for three minutes.
Samples with the addition of 50 µM cocaine were included to determine nonspecific uptake. Following
incubation samples were passed through 0.05% polyethylenimine-soaked GF/B filter paper (Whatman,
Clifton, NJ) and washed three times with ice-cold 0.32M sucrose. Filters were cut and radioactivity
trapped within bound synaptosomes was counted using a liquid scintillation counter. Protein
concentrations were determined by Bradford assay (Bradford 1976).
SDS-PAGE and Western Blotting

Protein concentration of all samples was determined by bicinchoninic acid protein assay according to
manufacturer’s instruction (Pierce Biotechnology). All samples were mixed with Laemmli sample
buffer containing 20% β-mercaptoethanol, loaded onto 4-12.5% Tris-HCl Polyacrylamide gels with
SeeBlue Plus2 protein ladder (Invitrogen; Carlsbad, CA) and run with constant voltage. Gels were then
transferred to polyvinyl difluoride membranes (Whatman) with 6 amps total current, blocked with 5%
non-fat dry milk in Tris-buffered saline with Tween-20 (TBS-T; 20 mM Tris, 137 mM NaCl, 0.2%
Tween-20, pH 7.6) then probed with primary antibody overnight at 4 °C. Following primary antibody
incubation, membranes were washed with TBS-T for 30 minutes, probed with secondary antibody for 1
hour, washed again with TBS-T for 30 minutes, then developed using Super Signal West Pico
Chemiluminescent substrate (Thermo Scientific) and imaged with an Alpha Innotech FluorChem HD2
imager.
Equivalent protein from each sample was loaded onto gels when evaluating the synaptosomal DA
uptake (20 µg) experiments and the synaptosomal membrane fractions (20 µg). For continuous sucrose
gradient experiments, protein equivalents for all fractions of all samples within a given experiment
were normalized to 75 µl volume of each fraction of saline 1. For example, if fraction 3 of saline 1
contained 15 µg protein in 75 µl volume then 15 µg protein from fraction 3 of each subsequent sample
was loaded. To normalize across membranes, two samples containing 2.5 µg DAT peptide were loaded
onto each gel. For all other experiments, 20 µg protein from each sample was analyzed.
Data Analysis
All data were analyzed with GraphPad Prism 5 (GraphPad Software; La Jolla, CA). DA uptake data
were analyzed by one-way ANOVA with Newman-Keuls post-hoc tests. Gradient DAT
immunoreactivity data were analyzed by two-way ANOVA with Bonferroni post-hoc tests.
Results Go to:
A single, high-dose (15 mg/kg, s.c.) administration of AMPH or METH reduced [3H]DA uptake within
striatal synaptosomes prepared 1 h following treatment (Fig. 2; one-way ANOVA Saline vs. AMPH = p
< 0.001; Saline vs. METH = p < 0.001). No change in total DAT immunoreactivity, however, was
observed among striatal synaptosomes prepared from AMPH-, METH- or saline-treated rats (data not
shown).
Figure 2
A single in vivo AMPH or METH treatment reduced synaptosomal DA uptake
Rats were treated with a single saline (1 ml/kg, s.c.), AMPH (15 mg/kg, s.c.) or METH (15 mg/kg, s.c.)
injection and sacrificed 1 h later (n = 8 per treatment group). Striatal synaptosomal DA uptake was calculated
as described in Materials and Methods. *** Different than saline (p ≤ 0.001).
To evaluate the effects of AMPH and METH on DAT localization, plasma membrane and intracellular
vesicle fractions were isolated from striatal synaptosomes (Figure 1 and Materials & Methods) and
DAT distribution within these fractions was determined. Osmotic lysis of untreated synaptosomes
followed by differential centrifugation allowed separation of synaptosomal vesicles, a heterogeneous
population of synaptic vesicles and endosomal components, from the synaptosomal membrane. These
fractions demonstrated 61.1% of the DAT resided at the plasma membrane (i.e., in the 1500 × g pellet;
Fig. 3A) and 38.9% of the DAT resided within synaptosomal vesicles (i.e., in the 1500 × g supernatant;
Fig. 3A). Equilibrium ultracentrifugation (115000 × g 18 h) of the synaptosomal vesicles fraction
(1500 × g supernatant) over 0.6M to 1.6M continuous sucrose gradients provided further
discrimination among the vesicle populations. Within the vesicle fraction, DAT immunoreactivity
predominantly localized across fractions 3 through 11 with the highest protein concentration found in
fraction 5 (Fig. 3B). Endosomal structures were highly enriched within these fractions, as indicated by
the recycling endosome marker rab11 within fractions 3 through 7 and the lysosomal-associated
membrane protein 2 (LAMP2) marker within fractions 5 through 11 (Fig. 3B). Importantly, the plasma
membrane protein Na+/K+-ATPase was highly enriched within the synaptosomal membrane fraction
but not within the vesicle fractions, indicating little plasma membrane contamination within the vesicle
fractions (Fig. 3B). While nearly all synaptosomal membranes are removed from vesicles, small
amounts of vesicular protein (LAMP2, rab11) remain within the synaptosomal membrane fraction.

Figure 3
Distribution of DAT within striatal synaptosomal membrane and vesicle fractions
(A) Synaptosomal membrane and total vesicle fractions were isolated from striatal synaptosomes, as
described in Materials & Methods, and DAT immunoreactivity was measured by densitometry (n = 3 per
group). (B) Distribution of DAT within synaptosomal vesicle fractions isolated across 0.6M – 1.6M
continuous sucrose gradients and the synaptosomal membrane fraction. The recycling endosome marker
rab11 and lysosome marker LAMP2 localized to DAT within the vesicle fractions, whereas the plasma
membrane protein Na+/K+ ATPase did not. ’SM’ indicates synaptosomal membrane fraction.
To demonstrate that DAT internalization can be detected within the synaptosomal membrane and
sucrose gradient fractions described above, striatal synaptosomes were treated with the PKC activator
phorbol 12-myristate 13-acetate (PMA). Application of 10 µM PMA to striatal synaptosomes for 30
min (37 °C) increased DAT immunoreactivity within the vesicle fractions as compared to dimethyl
sulfoxide (DMSO) vehicle-treated control fractions (Fig. 4A; two-way ANOVA, p = 0.0035 between
treatments), indicating movement of DAT into the vesicle fractions (Fraction 3 = 39.8% increase;
Fraction 5 = 68.7% increase). This concentration of PMA and time point was selected due to its
capacity to phosphorylate striatal synaptosomal PKC within 30 min (Vaughan et al. 1997). Treatment
of synaptosomes with concentrations of PMA lower than 10 µM yielded no change in DAT localization
(data not shown). PMA treatment did not, however, change synaptosomal membrane DAT
immunoreactivity (Fig. 4B), likely due to differences in sensitivity of detection between the vesicle and
synaptosomal membrane fractions. Since vesicular proteins are spread across the continuous sucrose
gradients, the signal to noise ratio within individual synaptosomal vesicle fractions will be greater than
the synaptosomal membrane fraction.
Figure 4
PMA increased DAT immunoreactivity within the synaptosomal vesicle fractions
Striatal synaptosomes were treated with 10 µM phorbol 12-myristate 13-acetate (PMA) for 30 min at 37 °C (n
= 6 per group). Following treatment, synaptosomal membrane and vesicle fractions were isolated and DAT
immunoreactivity was measured by densitometry. (A) DAT immunoreactivity was increased within PMA-
treated vesicles as compared to saline-treated vesicles. (B) No difference in DAT immunoreactivity was
observed between PMA- and saline-treated synaptosomal membranes. ** Treatment across fractions different
than saline-treated fractions (p ≤ 0.01). ‘DAT P’ indicates DAT peptide control lane.
A paradigm previously shown to reduce total DAT immunoreactivity within the striatum (4 × 7.5
mg/kg/injection METH, 2-h intervals, s.c., sacrifice 7 d later) was used to demonstrate the capacity of
the synaptosomal membrane and sucrose gradient vesicle fractions to detect loss of DAT (Hadlock et
al. 2010). In agreement with previous work (Hadlock et al. 2010), repeated METH administration
reduced DAT immunoreactivity within both synaptosomal membrane (Fig. 5B; 55% decrease METH
vs. control, t-test p = 0.0030) and vesicle fractions (Fig. 5A; two-way ANOVA, p = 0.0068 between
treatments) as compared to saline controls. Importantly, those fractions with the greatest DAT
immunoreactivity within saline-treated animals, fractions 3 and 5, had the greatest decrease in DAT
immunoreactivity following repeated METH treatment (Fig. 5A; Fraction 3 = 59.0% decrease, p <
0.001; Fraction 5 = 41.4% decrease, p < 0.05). METH-treated rats experienced hyperthermia, as
assessed by average core body temperature throughout treatment, as compared to saline controls
(METH = 40.0 °C ± 0.2 °C; Saline = 37.4 °C ± 0.1 °C; over the course of repeated METH injections),
consistent with previous studies (Metzger et al. 2000).
Figure 5
Repeated METH administrations resulted in persistent loss of striatal DAT immunoreactivity
Rats were treated with repeated saline (4 × 1 ml/kg/injection, 2-h intervals, s.c.) or METH (4 × 7.5
mg/kg/injection, 2-h intervals, s.c.) injections and DAT immunoreactivity within synaptosomal membrane and
vesicle fractions was determined 7 d later (n = 6 per group). (A) DAT immunoreactivity was reduced within
METH-treated fractions as compared to saline-treated control fractions. (B) DAT immunoreactivity was
reduced within METH-treated synaptosomal membranes as compared to saline-treated controls. ## Treatment
across fractions different than saline-treated fractions (p ≤ 0.01). * Fraction different than comparable saline-
treated fraction (p ≤ 0.05). ** Fraction different than comparable saline-treated fraction (p ≤ 0.01). ***
Fraction different than comparable saline-treated fraction (p ≤ 0.001). ‘DAT P’ indicates DAT peptide control
lane.
A single AMPH or METH injection (15 mg/kg, s.c.; sacrifice 60 min later), regimens that decreased
DAT function (Fig. 2), did not alter DAT localization (Figs. 6A, 6C, 8A, 8B). To determine if DAT
localization was altered more acutely following AMPH exposure, DAT localization was evaluated in
rats given a single AMPH (15 mg/kg, s.c.) injection and sacrificed 5 min, 15 min, or 30 min later (
Fig. 6B). No difference in DAT localization was observed between AMPH-treated and saline-treated
control rats within the vesicle fractions at any time point (Figs. 6A, 6B). Accordingly, no difference in
DAT localization within the striatal synaptosomal membrane fractions was observed between AMPH
or saline-treated rats evaluated 5 min, 15 min, 30 min (data not shown), or 60 min (Fig. 6C) following
drug exposure.

Figure 6
Single in vivo AMPH treatments did not alter striatal DAT localization 5 - 60 min after drug exposure
Rats received a single saline (1 ml/kg, s.c.) or AMPH (15 mg/kg, s.c.) injection and were sacrificed 5 min, 15
min, 30 min or 60 min following treatment (n = 6 per group). No difference in DAT immunoreactivity within
the vesicle (A, B) or synaptosomal membrane (C) fractions was observed. ‘DAT P’ indicates DAT peptide
control lane.
Previous studies have demonstrated that repeated METH administrations (4 × 7.5 mg/kg/injection, 2-h
intervals, s.c.) decreased DAT activity by approximately 75% 1 h following treatment (Chu et al.
2008), and that these acute deficits may lead to the persistent dopaminergic deficits caused by METH
(e.g., Fig. 5). This treatment paradigm (4 × 7.5 mg/kg/injection, 2-h intervals, s.c., sacrifice 1 h after
last injection) did not, however, alter DAT localization or total quantity, within the striatal
synaptosomal membrane or vesicle fractions when compared to saline-treated controls (Fig. 8C, 8D).
METH-treated rats experienced hyperthermia with an average core temperature of 40.2 °C ± 0.3 °C as
compared to 37.4 °C ± 0.1 °C within saline-treated controls.
Discussion Go to:
Extensive in vitro work suggests amphetamines cause biphasic DAT relocalization that coincides with
reduced extracellular DA uptake (Robertson et al. 2009). When cell lines stably expressing DAT were
treated with physiologically relevant concentrations of AMPH (e.g., 2 µM; (Clausing et al. 1995)),
DAT moved from endosomal stores to the membrane within the first 2 min and then internalized within
15 min following treatment (Saunders et al. 2000, Johnson et al. 2005a). Similar DAT relocalization
occured in striatal synaptosomes treated with AMPH in vitro, but over a longer time period. Treatment
of mouse striatal synaptosomes with AMPH increased DAT expression on the plasma membrane
within the first 2 min of treatment, and then DAT internalization occurred 1 h following treatment
(Chen et al. 2009, Johnson et al. 2005a). Similarly, rat striatal synaptosomal DAT internalization was
not observed until 1 h following a large (20 µM) AMPH application (Richards & Zahniser 2009).
This study evaluated the effects of in vivo (rather than in vitro) AMPH and METH upon DAT function
and localization, demonstrating in vivo drug treatment acutely decreases DAT function in striatal
synaptic terminals without concurrent DAT relocalization (Figs. 2, 6, 7, 8). Importantly, thorough
washing of synaptosomes in the process of isolation paired with previous analysis of METH levels in
synaptosomal preparations suggests this acute loss of DAT function is not due to residual drug within
the synaptosomal preparation (Kokoshka et al. 1998, Fleckenstein et al. 1997b). These findings are
consistent previous work by Richards and Zahniser (2009), in which no change in DAT localization
was observed within striatal synaptosomes prepared 45 min following AMPH treatment (2 mg/kg).
Since no change in DAT localization was observed and the amount of total synaptosomal DAT
immunoreactivity remained consistent between AMPH- METH- and saline-treated animals 1 h
following treatment, DAT was neither transported out of synaptic terminals nor degraded via
proteosomal or lysosomal means. Instead, mechanisms other than internalization and relocalization are
likely responsible for reduced DAT function at the plasma membrane. However, the possibility of a
brief DAT relocalization on a time frame shorter than evaluated within the present work (less than 5
min) cannot be excluded.

Figure 7
A single or repeated METH administration did not alter striatal DAT localization 60 min following
treatment
Rats received either single or multiple injections of saline (single = 1 ml/kg, s.c.; multiple = 4 × 1
ml/kg/injection, 2-h intervals, s.c.) or METH (single = 15 mg/kg, s.c.; multiple = 4 × 7.5 mg/kg/injection, 2-h
intervals, s.c.) and sacrificed 60 min following treatment (n = 6 per group). No changes in DAT
immunoreactivity within the synaptosomal membrane (A, C) or vesicle (B, D) fractions were observed with
either treatment paradigm. ‘DAT P’ indicates DAT peptide control lane.
To detect drug-induced changes in synaptosomal DAT localization, a technique was developed to

separate the synaptosomal membrane from discrete subcellular fractions using differential
centrifugation, with further separation of vesicles using equilibrium ultracentrifugation across
continuous sucrose gradients (Fig. 1). Isolation of synaptosomal membrane (P2) from synaptosomal
vesicles (S2) revealed approximately 60% of DAT was located at the synaptosomal (plasma)
membrane (P2) and 40% of DAT was located within vesicular structures (S2; Fig. 2A) of untreated
animals. The synaptosomal vesicles were further separated across 0.6M - 1.6M continuous sucrose
gradients using equilibrium ultracentrifugation. These synaptosomal vesicle fractions were enriched
with endocytic structures (rab11 and LAMP2) and were not contaminated with plasma membrane,
indicated by the lack of Na+/K+ ATPase (Fig. 3). Consistent with other studies (Rao et al. 2010, Chen
et al. 2009), DAT localized to those fractions enriched with endocytic vesicles (rab11 and LAMP2).
The presence of DAT within mobile endocytic stores supports a proposed model of constitutive DAT
cycling between recycling endosomes (rab11) and the plasma membrane (Chen et al. 2009).
The sensitivity of the isolation method to detect movement between and change within the
synaptosomal membrane and vesicle fractions was demonstrated through two experiments. First,
internalization was detected through increased DAT immunoreactivity within the vesicle fractions of
PMA-treated striatal synaptosomes (Fig. 3A). PMA has been previously shown to induce clathrin-
mediated DAT endocytosis through phosphorylation by PKC (Melikian & Buckley 1999, Foster et al.
2008, Sorkina et al. 2005, Vaughan et al. 1997), though recent work suggests PKC alone cannot drive
substantial DAT internalization (Boudanova et al. 2008, Rao et al. 2010). Our data support the latter
assertion, since only a small amount of DAT internalization was observed despite treating
synaptosomes with a relatively high concentration of PMA (10 µM; Fig. 3A). Second, DAT
immunoreactivity was lost from both synaptosomal membrane and vesicle fractions 7 d following
repeated METH injections (Fig. 4), demonstrating a net, uniform subcellular loss of DAT from the
striatum and the capacity of this isolation method to detect well-characterized, persistent dopaminergic
deficits (Hadlock et al. 2010, Fumagalli et al. 1998).
AMPH- and METH-induced long-term dopaminergic deficits have been associated with acute
alteration of DAT function (Fumagalli et al. 1998, Hadlock et al. 2010). Since administration of a
METH regimen well documented to cause acute and persistent dopaminergic deficits (4 × 7.5
mg/kg/injection, 2-h intervals, s.c.) did not relocalize DAT (Fig. 4, 7), internalization is not likely a
causative factor linking acute and persistent stimulant-induced dopaminergic deficits.
Differences in environmental context may explain the disparity in DAT localization observed between
in vivo and in vitro tissue culture/synaptosome models following AMPH and METH administration.
Proteins involved in DAT regulation and internalization are not shared between cell lines that
exogenously express DAT and dopaminergic neurons. For example, EM4 cells, a derivation of
HEK293 cells first used to characterize AMPH-induced DAT internalization (Saunders et al. 2000),
lack endogenous expression of calcium-calmodulin dependent protein kinase II (CaMKII), a protein
kinase known to phosphorylate and alter DAT function following AMPH treatment (Xu et al. 2008).
Since heterologous cell lines do not express DA receptors, they cannot respond to a primary
environmental consequence of exposure to amphetamines – elevated extracellular DA – in the same
manner as neurons (Zhou et al. 1990). Activation of DA receptors is required for well-characterized
METH-induced dopaminergic deficits to develop (Granado et al. 2011). Finally, AMPH and METH
impact non-dopaminergic neurons outside of the striatum that connect to and directly influence
dopaminergic projections within the striatum. High-dose METH administration enhances the activity of
striatonigral GABAergic projections, which increases the release of glutamate within the striatum and
leads to long-term striatal DA depletion (Mark et al. 2004). Individually or together, these differences
may underlie observed discrepencies in DAT localization between in vivo and in vitro models following
AMPH and METH treatment.
While internalization is unlikely to account for AMPH- and METH-induced DAT dysfunction observed
herein, a large body of evidence suggests DAT may be structurally altered by drug exposure. A number
of interacting protein partners are known to cause post-translational modifications of DAT following
exposure to amphetamines, which likely occur at the membrane. PKC, syntaxin1A and CaMKII have
all been implicated in changing DAT transport rate or initiating DA efflux through interaction with
DAT, and are probable candidates responsible for reduced in vivo transport activity (Binda et al. 2008,
Johnson et al. 2005b, Fog et al. 2006). DAT may also be oxidatively inactivated (Fleckenstein et al.
1997a, Berman et al. 1996). Oxidation of excessive terminal DA into reactive oxygen species and
quinone formations reduce DA uptake through DAT (Berman et al. 1996, Fleckenstein et al. 1997a,
LaVoie & Hastings 1999), which may occur at the membrane.
In summary, this study demonstrates that while in vivo AMPH or METH exposure reduce DAT activity
within striatal synaptic terminals, no change in DAT localization is observed at the time points
assessed. These studies contrast with previous in vitro AMPH and METH studies, presumably due to
differences between in vitro and in vivo models. Mechanisms other than internalization likely cause
reduced DAT function and persistent dopaminergic deficits following in vivo AMPH and METH
exposure. Substantial evidence suggests modification of the DAT protein itself, through post-
translational modification or structural alteration, may underlie drug-induced dysfunction.
Consequently, further investigation into the post-translational modifications of striatal DAT and the
signaling mechanism involved during AMPH and METH treatment is warranted.
Acknowledgments Go to:
This work was supported by grants DA00869, DA04222, DA13367, DA11389, DA019447, and
DA00378 from the National Institute on Drug Abuse. We thank Dr. James W. Gibb for his critical
reading of this manuscript.
Abbreviations Go to:
METH methamphetamine
AMPH amphetamine
DA dopamine
DAT dopamine transporter
PMA phorbol 12-myristate 13-acetate
Footnotes Go to:
The authors have no conflicts of interest to declare.
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Amphetamine and Methamphetamine Reduce Striatal Dopamine Transporter Function Without Concurrent Dopamine Transporter Relocalization

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Amphetamine and Methamphetamine Reduce Striatal Dopamine Transporter Function Without Concurrent Dopamine Transporter Relocalization

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Amphetamine and Methamphetamine Reduce Striatal Dopamine Tran…ction Without Concurrent Dopamine Transporter Relocalization 2018/4/16, 3+57 PM

Amphetamine and Methamphetamine Reduce Striatal Dopamine

Keywords: Dopamine Transporter, Amphetamine, Methamphetamine, Striatum, Internalization,

Materials & Methods Go to:

Reagents and Antibodies

Animals and Drug Treatment Paradigms

Synaptosomal Isolation, Subcellular Fractionation and Continuous Sucrose Gradients

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Synaptosomal [3H]DA Uptake

SDS-PAGE and Western Blotting

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compared to 37.4 °C ± 0.1 °C within saline-treated controls.

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To detect drug-induced changes in synaptosomal DAT localization, a technique was developed to

DAT dopamine transporter

PMA phorbol 12-myristate 13-acetate

The authors have no conflicts of interest to declare.

2008;54:605–612. [PMC free article] [PubMed]

dopaminergic neurons and chromaffin granules: a mechanism of action. Neuron. 1990;5:797–

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