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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 296:876–883, 2001
Vol. 296, No. 3
3300/887799
Printed in U.S.A.
Locomotor Effects of Acute and Repeated Threshold Doses of
Amphetamine and Methylphenidate: Relative Roles of
Dopamine and Norepinephrine
RONALD KUCZENSKI and DAVID S. SEGAL
Department of Psychiatry, School of Medicine, University of California at San Diego, La Jolla, California
Received September 6, 2000; accepted November 29, 2000
This paper is available online at http://jpet.aspetjournals.org
Stimulant medications were first introduced in the treatment
of children with attention deficit hyperactivity disorder (ADHD)
in 1937. With the exception that methylphenidate (MP) has
replaced amphetamine (AMPH) as the primary stimulant, the
clinical use of these drugs has remained essentially unchanged.
In recent years, however, the prescribed use of MP has risen
dramatically, prompting heightened concerns of potential adverse long-term consequences arising from the prolonged exposure to this drug in children with ADHD.
In this regard, one long-term consequence of the repeated administration of AMPH-like stimulants is an augmentation of some stimulant-induced behaviors in response to subsequent drug challenge (Robinson and
Becker, 1986; Segal and Kuczenski, 1994). This augmentation, portions of which persist after prolonged periods of
drug abstinence, has generally been referred to as behavioral sensitization, and it has been suggested that this
process may be implicated in the development of stimulant
addiction/abuse (Robinson and Berridge, 1993). Sensitization has been extensively documented following repeated
administration of moderate-to-high doses of AMPH and
This work was supported by U.S. Public Health Service Grant DA-01568.
longed exposure to this drug, we examined whether changes in
the locomotor response occurred with repeated administration
of these stimulant doses. Threshold doses of methylphenidate
(0.5–1.0 mg/kg) or amphetamine (0.1– 0.25 mg/kg) were administered twice daily, and then animals were tested in response to
2.5 mg/kg methylphenidate or 0.5 mg/kg amphetamine. Our
results provide evidence that low-dose stimulant administration
can result in the development of behavioral sensitization, which
is evident in the subsequent behavioral response to the drug. The
relevance of these data to the therapeutic uses of these drugs is
discussed within the context of the many variables that can affect
the behavioral and neurochemical responses to stimulants.
cocaine (for reviews, see Robinson and Becker, 1986; Segal
and Kuczenski, 1994; White and Kalivas, 1998). In contrast, fewer studies have characterized the effects of repeated MP, and results have been inconsistent. For example, some investigators have reported an increased
locomotor response (Gaytan et al., 1997a; Sripada et al.,
1998), whereas others have not (Crawford et al., 1998;
Izenwasser et al., 1999). Likewise, an augmented stereotypy response has been observed by some (Browne and
Segal, 1977; Crawford et al., 1998) but not others (McNamara et al., 1993; Izenwasser et al., 1999). Therefore,
based on the available animal data it is not possible to
conclude that repeated MP results in the same pattern of
behavioral augmentation that has been well documented
for the other amphetamine-like stimulants.
Furthermore, in assessing the possible consequences of
long-term MP treatment in children, it is important to note
that a variety of factors, including drug dose, route, and
pattern of administration, and chronicity can influence both
the qualitative and quantitative features of the development
and/or expression of chronic stimulant-induced behavioral
and neurochemical alterations (for reviews, see Robinson and
Becker, 1986; Segal and Kuczenski, 1994; White and Kalivas,
ABBREVIATIONS: ADHD, attention deficit hyperactivity disorder; MP, methylphenidate; AMPH, amphetamine; DA, dopamine; NE, norepinephrine; 5HT, serotonin; AUC, area under the curve.
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ABSTRACT
The prescribed use of methylphenidate (Ritalin) in the treatment
of attention deficit hyperactivity disorder has risen dramatically
in recent years. The relative roles of dopamine, norepinephrine,
and serotonin in the therapeutic action of these drugs was
assessed by comparing the responses of extracellular nucleus
accumbens dopamine and serotonin and hippocampus norepinephrine to the acute administration of low methylphenidate
and amphetamine doses. The comparative neurochemical profiles in response to methylphenidate and amphetamine suggest
that the norepinephrine effects may play an important role in
the therapeutic effects of low doses of psychostimulants. In
addition, to assess possible long-term consequences of pro-
Psychostimulant Threshold Doses: Behavior and Neurochemistry
Experimental Procedures
Subjects. Male Sprague-Dawley rats, weighing 325 to 350 g at
the beginning of drug treatment, were housed for at least 1 week
before experimental manipulation in groups of two or three in wire
mesh cages, with ad libitum access to food and water, in a temperature- and humidity-controlled room, maintained on a 14-h light
(5:00 AM–7:00 PM), 10-h dark cycle. Animals were obtained from
Simonsen Labs (Gilroy, CA). All studies adhered to animal welfare
guidelines (Principles of Laboratory Animal Care, National Institutes of Health Publication 85-23).
Apparatus. Behavior was monitored in custom-designed activity
chambers (Segal and Kuczenski, 1987). Briefly, each of the chambers
was located in a sound-attenuated cabinet maintained on a 14-h/10-h
light/dark cycle with constant temperature (20°C) and humidity
(55 ⫾ 5%). Each chamber consisted of two compartments: an activity/
exploratory compartment (30 ⫻ 20 ⫻ 38 cm) and a smaller “home”
compartment (14 ⫻ 14 ⫻ 10 cm) in which food and water were
available ad libitum. Movements of the animal between quadrants
within the activity/exploratory compartment (i.e., crossovers) and
rearings against the wall, as well as eating and drinking and other
vertical (e.g., contact with a hanging stimulus) and horizontal movements (e.g., intercompartment crossings) were monitored continuously by computer. In addition to the computer-monitored behaviors,
representative animals (n values ⫽ 5–7) were simultaneously videotaped for 60 s at successive 5-min intervals throughout the response
to assess the qualitative features of the response. The appearance of
responses or behavior patterns, undetectable by our automated
methods, was noted by the rater after each sampling interval.
Drugs. Drugs were dissolved in saline. Methylphenidate HCl
(National Institute on Drub Abuse, Rockville, MD) was administered
i.p., and amphetamine SO4 (Sigma, St. Louis, MO) was administered
s.c. Doses represent the free base.
Methods (See Results for Specific Details). Three days before
the beginning of drug treatment, animals were placed in individual
experimental chambers where they remained for the duration of the
experiment. For all experiments, n values ⫽ 9 to 11/group. To facilitate habituation to the chambers and procedures, animals were
handled and injected with saline at least once a day. During the
remainder of the day and night, animals were not disturbed and
their behavior was continuously monitored. Throughout the remaining phases of each study, control animals were administered daily
saline injections equivalent in number of injections to the experimental groups.
For dialysis studies, animals were stereotaxically implanted with
guide cannulae using procedures previously described in detail (Kuczenski and Segal, 1989). Guide cannulae extended 2.6 mm below the
surface of the skull and were aimed at the dorsal hippocampus (3.8
mm posterior to bregma, 2.0 mm lateral, and 4.0 mm below dura)
and the nucleus accumbens (2.2 mm anterior, 1.5 mm lateral, 7.8
mm below dura). Following surgery, animals were housed individually and allowed at least 1 week to recover before receiving any
treatment.
On the day before the experimental day (3:00 – 4:00 PM), each rat
was placed in the dialysis chamber and the dialysis probes were
inserted to allow for acclimation to the test environment and for
adequate equilibration of the dialysis probes. The dialysis chambers
were essentially identical to the behavioral chambers described
above, with the exceptions that the home compartment and hanging
stimulus were removed to prevent interferences introduced by the
dialysis methodology. Concentric microdialysis probes were constructed of Spectra/Por hollow fiber (mol. wt. cut-off 6000, 250-␮m
o.d.) according to the method of Robinson and Whishaw (1988) with
modifications (Kuczenski and Segal, 1989). The length of the active
probe membrane was 2 mm for hippocampus and 1.5 mm for nucleus
accumbens. Probes were perfused with artificial cerebrospinal fluid
(147 mM NaCl, 1.2 mM CaCl2, 0.9 mM MgCl2, 4.0 mM KCl) delivered
by a microinfusion pump (1.5 ␮l/min) via 50 cm of Micro-line ethyl
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1998). In this regard, the daily doses of stimulants commonly
used to treat children with ADHD, 0.5 mg/kg MP and 0.25
mg/kg AMPH (Patrick and Markowitz, 1997), typically
administered orally twice daily, are near threshold for the
induction of locomotor activation in rodents, and are substantially below conventional preclinical treatment doses used in
studies of sensitization. Two published reports used stimulant doses within these clinical ranges: McNamara et al.
(1993) (1 mg/kg MP) and West et al. (1999) (0.25 mg/kg
AMPH) and both reported the absence of sensitization when
the test and pretreatment doses were identical. However, the
dose of drug used to test for the expression of a sensitized
response might be a significant factor, and neuronal adaptations to repeated low-dose pretreatment may become evident
in the response to a higher challenge dose. Within the context
of a sensitization model for addiction, augmentation of higher
dose effects would still be consistent with an increase in the
incentive value of the stimulant (Robinson and Berridge,
1993), thus perhaps increasing the likelihood of stimulant
abuse.
With regard to underlying mechanisms, most evidence implicates the effects of psychostimulants on dopamine (DA)
pathways in the treatment of ADHD (Pliszka et al., 1996). In
addition, stimulant-induced effects on both norepinephrine
(Florin et al., 1994) and serotonin (Segal, 1976; Jacobs and
Fornal, 1997) have been implicated in the expression of stimulant-induced behaviors, and some evidence suggests possible roles for both these transmitters in the therapeutic efficacy of stimulants in the treatment of ADHD (Biederman
and Spencer, 1999). One approach to assessing the possible
role of the various stimulant-induced neurochemical changes
is to compare and contrast the effects of AMPH and MP at
therapeutically relevant doses. In this regard, our previous
comparisons of the behavioral and neurochemical responses
to moderate doses of AMPH and MP indicated that both
drugs promoted profound effects on DA and NE systems
(Florin et al., 1994; Kuczenski and Segal, 1997), although
differential drug-specific dose-response relationships for the
two transmitters were observed. In contrast, although we
showed that moderate-to-high doses of AMPH can affect regional extracellular serotonin (5HT) (Kuczenski and Segal,
1989), no acute or chronic dose of MP altered extracellular
concentrations of this transmitter (Kuczenski and Segal,
1997; Segal and Kuczenski, 1999), consistent with the relatively low affinity of MP for the 5HT transporter (Gatley et
al., 1996).
To gain insight into the relative roles of DA and NE in the
therapeutic action of these drugs, we compared the nucleus
accumbens DA and hippocampus NE responses to the acute
administration of threshold MP and AMPH doses. In addition, studies were designed to further assess possible changes
in behavioral response associated with repeated exposure to
more therapeutically relevant stimulant doses and administration patterns: threshold doses of MP (0.5–1.0 mg/kg) or
AMPH (0.1– 0.25 mg/kg) were administered i.p. or s.c. twice
daily, and then animals were tested in response to 2.5 mg/kg
MP or 0.5 mg/kg AMPH. Our results suggest that stimulantinduced effects on NE transmission may play a critical role in
the therapeutic actions of MP and AMPH, and, in addition,
provide evidence for low-dose stimulant-induced sensitization.
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Kuczenski and Segal
Results
AMPH Pretreatment/AMPH Challenge. In the initial
study, groups of animals received saline or AMPH (0.1 or
0.25 mg/kg) twice daily (10:00 AM and 2:00 PM) for 5 days.
Four days after the last treatment, animals were challenged
with AMPH (0.5 mg/kg). The pattern of locomotor activity
produced by the AMPH challenge is summarized in Fig. 1.
This response exhibited a graded increase [ANOVA,
F(2,27) ⫽ 3.42, p ⬍ 0.05] as a function of the pretreatment
dose, but only the behavior of the higher dose group achieved
statistical significance.
MP Pretreatment/AMPH Challenge. To determine
whether repeated exposure to MP would also result in an
altered response to subsequent AMPH challenge (cross-sensitization), groups of animals received twice daily injections
of saline or MP (0.5 or 1.0 mg/kg), and 4 days after the last
pretreatment were challenged with AMPH (0.5 mg/kg). The
locomotor response profiles to the challenge injection are
summarized in Fig. 2. As with AMPH pretreatment, there
was a graded response [ANOVA, F(2,27) ⫽ 3.48, p ⬍ 0.05]
dependent on the pretreatment dose, but only the higher
pretreatment dose resulted in a significant cross-sensitization to AMPH.
MP Pretreatment/MP Challenge. Because the locomotor response following MP pretreatment was only marginally
increased, we sought to determine whether a longer MP
pretreatment would result in a more robust increase in locomotion. In this study, animals were pretreated with saline or
MP (1.0 mg/kg) twice daily for 11 days, and 4 days later, were
challenged with 2.5 mg/kg MP (Fig. 3). The locomotor response was significantly elevated in the MP-pretreated
group.
Extracellular DA in Nucleus Accumbens and NE in
Hippocampus in Response to Threshold Doses of MP
and AMPH. Our previous results comparing the extracellular DA and NE responses to MP (Kuczenski and Segal, 1997)
were consistent with some data in the literature that MP
exhibits a higher affinity for the NE compared with the DA
transporter (Ferris et al., 1972; Andersen, 1987). To determine whether a similar relationship could be observed at
more clinically relevant threshold doses, the nucleus accum-
Fig. 1. Effects of AMPH pretreatment
on the temporal pattern of horizontal
locomotion in response to an AMPH
(0.5 mg/kg) challenge. Groups of animals were administered saline or
AMPH (0.1 or 0.25 mg/kg) twice daily
for 5 days. Four days after the last
pretreatment injection, animals were
challenged with AMPH (0.5 mg/kg).
Values represent the mean ⫾ S.E.M.
Bar graphs represent the cumulated
response during the initial 120 min
following
drug
administration.
ANOVA revealed a significant effect of
pretreatment [F(2,27) ⫽ 3.42; p ⬍
0.05]. *p ⬍ 0.05 compared with saline
pretreated group (t ⫽ 2.54, df ⫽ 27).
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vinyl acetate tubing connected to a fluid swivel. Dialysate was collected through glass capillary tubing into vials containing 20 ␮l of
25% methanol, 0.2 M sodium citrate, pH 3.8. Under these conditions,
dialysate NE, DA, and 5HT and metabolites were stable throughout
the collection and analysis interval. Samples were collected outside
the experimental chamber to avoid disturbing the animal. Individual
probe recoveries were estimated by sampling a standard DA solution
in vitro. Preliminary studies indicated that individual probe recoveries for DA and NE were similar. At the end of the experiment, each
animal was perfused with formalin for histological verification of
probe placements.
Dialysate samples were collected every 20 min. Nucleus accumbens samples were assayed for DA, 3,4-dihydroxyphenylacetic acid,
homovanillic acid, 3-methoxytyramine, 5-hydroxyindoleacetic acid,
and 5HT. In all experiments, solutions of standards revealed a clean
separation between 3-methoxytyramine and 5HT. The high pressure
liquid chromatography-electrochemical detector consisted of a 100 ⫻
4.6 mm ODS-C18 3-␮m column (Regis, Morton Grove, IL) maintained at 40°C. Mobile phase (0.05 M citric acid, 7% methanol, 0.1
mM Na2EDTA, and 0.2 mM octane sulfonate adjusted to pH 4.0 – 4.5)
was delivered at 0.6 to 0.8 ml/min by a Waters model 510 pump. In
hippocampus samples, NE was separated using a similar mobile
phase containing 4% methanol and 1.5 mM octane sulfonate. Amines
were detected with a Waters 460 detector with a glassy carbon
electrode maintained at ⫹0.65 V relative to a Ag/AgCl reference
electrode. Concentrations were estimated from peak heights using a
Waters Maxima 820 data station. Substances in the dialysates were
corrected for individual probe recoveries to account for this source of
variability, and, although the exact relationship between dialysate
concentration and actual extracellular transmitter content is not
clear, values are presented as dialysate concentration to allow for
meaningful comparisons to other data in the literature.
Data Analysis. Behavioral and neurochemical data were statistically analyzed using repeated measures ANOVA and t tests with
Bonferroni corrections for specific group/time comparisons.
Psychostimulant Threshold Doses: Behavior and Neurochemistry
879
Fig. 3. Effects of MP (1.0 mg/kg) pretreatment on the temporal pattern of
horizontal locomotion in response to
a MP (2.5 mg/kg) challenge. Groups
of animals were administered saline
or MP twice daily for 11 days. Four
days after the last pretreatment injection, animals were challenged
with MP (2.5 mg/kg). Values represent the mean ⫾ S.E.M. The bar
graph represents the cumulated response during the 90-min interval
following drug administration. *p ⬍
0.05 compared with saline-pretreated group (t ⫽ 2.15, df ⫽ 18).
bens DA and hippocampus NE responses were characterized
at various MP doses (0.5–2.5 mg/kg), and compared with a
low dose of AMPH (0.25 mg/kg). All of these doses are within
the therapeutic range, at least on a milligram per kilogram
basis. The results are summarized in Figs. 4 and 5. MP
produced a dose-dependent increase in extracellular DA,
both in peak and AUC, although the lowest dose (0.5 mg/kg)
failed to significantly increase extracellular DA concentrations. However, the magnitude of the highest MP-induced DA
response was significantly less than for AMPH (Fig. 4). In
contrast, in the same animals, all the doses of MP increased
extracellular concentrations of NE, and unlike the relative
effects of the two drugs on DA, the two highest MP doses
increased NE significantly more than did AMPH (Fig. 5).
Concomitant evaluation of extracellular 5HT concentrations
in nucleus accumbens revealed no significant effects in response to any of the doses tested (data not shown).
To determine whether a significant accumbens DA response to 0.5 mg/kg MP would emerge with repeated MP
pretreatment, animals received twice daily injections of sa-
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Fig. 2. Effects of MP pretreatment on
the temporal pattern of horizontal locomotion in response to an AMPH (0.5
mg/kg) challenge. Groups of animals
were administered saline or MP (0.5
or 1.0 mg/kg) twice daily for 5 days.
Four days after the last pretreatment
injection, animals were challenged
with AMPH (0.5 mg/kg). Values represent the mean ⫾ S.E.M. Bar graphs
represent the cumulated response
during the indicated interval following drug administration. ANOVA revealed a significant effect of pretreatment [F(2,27) ⫽ 3.48; p ⬍ 0.05]. *p ⬍
0.05 compared with saline pretreated
group (t ⫽ 2.38, df ⫽ 13).
880
Kuczenski and Segal
Fig. 5. Left, temporal profile of the
hippocampus extracellular NE response to an acute injection of AMPH
(0.25 mg/kg) or MP (0.5, 1.0, 2.5 mg/
kg). *p ⬍ 0.05 compared with the sample collected immediately before drug
administration. Right, bar graphs represent the peak response and AUC
during the 120-min interval following
drug administration. Peak response
ANOVA: F(3,40) ⫽ 7.83, p ⬍ 0.0001;
⫹⫹
p ⬍ 0.01 compared with response to
0.25 mg/kg AMPH (t ⫽ 2.64, df ⫽ 8.9).
AUC ANOVA: F(3,40) ⫽ 7.83, p ⬍
0.0001; ⫹p ⬍ 0.05, ⫹⫹⫹p ⬍ 0.001 compared with 0.25 mg/kg AMPH; *p ⬍
0.05 compared with 0.5 mg/kg MP response (all t values ⬎2.36).
line or MP (0.5 mg/kg) for 5 days, and, 48 h after the last
pretreatment injection, were challenged with MP (0.5 mg/
kg). Repeated MP pretreatment did not alter the hippocampus NE response (Fig. 6, left), nor was a significant nucleus
accumbens DA response produced (Fig. 6, right).
Discussion
The results of these studies indicate that the repeated
administration of MP and AMPH at doses near threshold for
locomotor activation, and within the therapeutic range for
ADHD treatment, at least on a milligram per kilogram basis,
can produce sensitization-like effects on the locomotor response to a subsequent stimulant exposure. That we observed an increased locomotor response to stimulant challenge following repeated MP treatment is consistent with
some (Gaytan et al., 1997a; Sripada et al., 1998) but not other
data in the literature (Crawford et al., 1998; Izenwasser et
al., 1999). Although a variety of experimental variables likely
account for these divergent observations, at least two characteristics of the treatment protocols may be responsible for
this discrepancy. First, an apparent sensitization to MP was
observed only in experiments in which the home cage served
as the test chamber (present results; Gaytan et al., 1997a;
Sripada et al., 1998). This observation suggests that contextdependent conditioning might play an important role in the
development/expression of an augmented locomotor response
to these low-dose MP treatments. However, in the present
study although we observed a brief augmented response to
the first saline injection after drug pretreatment, this effect
was not apparent in response to the last saline injection
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Fig. 4. Left, temporal profile of the
nucleus accumbens extracellular DA
response to an acute injection of
AMPH (0.25 mg/kg) or MP (0.5, 1.0,
2.5 mg/kg). Values represent the
mean ⫾ S.E.M. Baseline values are
the median of the three samples collected immediately before drug administration. *p ⬍ 0.05 compared with
the sample collected immediately before drug administration. Right, bar
graphs represent the peak response
and AUC during the 120-min interval
following drug administration. Peak
response ANOVA: F(3,36) ⫽ 7.92, p ⬍
0.001; ⫹p ⬍ 0.05; ⫹⫹⫹p ⬍ 0.001 compared with response to 0.25 mg/kg
AMPH (all t values ⬎2.37). AUC
ANOVA: F(3,36) ⫽ 13.14, p ⬍ 0.0001;
⫹
p ⬍ 0.05, ⫹⫹⫹p ⬍ 0.001 compared
with 0.25 mg/kg AMPH response; *p ⬍
0.05 compared with 0.5 mg/kg MP response (all t values ⬎2.23).
Psychostimulant Threshold Doses: Behavior and Neurochemistry
881
Fig. 6. Left, temporal profile of the
hippocampus extracellular NE. Right,
nucleus accumbens extracellular DA
responses to 0.5 mg/kg MP in groups
of animals pretreated with repeated
saline or MP (0.5 mg/kg b.i.d.). Values
represent the mean ⫾ S.E.M. Baseline
values are the median of the three
samples collected immediately before
drug administration. Inset, bar graph
represents the AUC during the initial
60 min following drug administration.
There was no significant effect of MP
pretreatment (t ⫽ 1.05, df ⫽ 15, p ⬎
0.3).
1983; Patrick et al., 1984; Cho et al., 1999; Thai et al., 1999)
compared with 15 to 30 min following the oral route (Wargin
et al., 1983; Patrick et al., 1984); and in ADHD subjects, peak
plasma concentrations [as well as therapeutic efficacy (Swanson et al., 1978)] of these drugs are not achieved until 90 to
180 min after oral administration (Wargin et al., 1983). Rate
of drug accumulation is an important variable that has been
significantly linked to abuse liability (Balster and Schuster,
1973). Furthermore, in rats peak plasma concentrations are
2- to 6-fold higher after intraperitoneal compared with oral
administration (Wargin et al., 1983; Patrick et al., 1984). A
similar relationship is evident in corresponding behavioral
measures (Foltin, 1982). Thus, the doses we used in our
studies would likely have produced smaller behavioral and
neurochemical responses following oral/intragastric administration.
Other important variables that impact on the interpretation of our results include species differences in rate and
pattern of drug metabolism, age differences, and time of day
at which drug was administered. For example, AMPH exhibits a half-life of 6 to 8 h in humans (Brown et al., 1979),
compared with about 60 min in rats (Melega et al., 1995). MP
is also more slowly metabolized in humans compared with
rats (2.5 versus 1 h, respectively) (Wargin et al., 1983; Srinivas et al., 1992). Species differences in exposure time to these
drugs may be very important, especially with respect to repeated, long-term administration. Likewise, evidence suggests that the response of adults may differ from that of
younger subjects (Roffman and Raskin, 1997), a potentially
important distinction for animal models of ADHD treatment.
Furthermore, our injection of drug during the light phase,
i.e., the period of normal inactivity/sleep for the rat, may also
have significantly influenced the response to these stimulants (Gaytan et al., 1997b, 1999). Thus, caution is appropriate in extrapolating from the conditions used in our studies
to human exposure patterns.
Although these concerns apply equally to interpretation of
our neurochemical data, comparison of the pattern of neurotransmitter responses promoted by the acute administration
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before challenge (data not shown). Thus, it seems unlikely
that context-dependent factors alone can account for the augmented challenge response in our studies. Another variable
distinguishing the divergent sets of data is the MP dose. An
augmented locomotor response to stimulant challenge was
previously reported when both the pretreatment and challenge doses were ⱕ2.5 mg/kg MP (present results; Gaytan et
al., 1997a; Sripada et al., 1998), but not when either dose was
ⱖ5 mg/kg MP (Gaytan et al., 1997a; Crawford et al., 1998;
Izenwasser et al., 1999). One interpretation for this observation is that higher MP doses introduce effects that inhibit
and/or obscure increases in the locomotor response to subsequent drug exposure. In this regard, an altered responsivity
may be expressed in other behaviors that are not readily
detected using automated measures. We have previously observed that significant stereotypy can emerge with repeated
administration of stimulant doses that acutely produce predominantly locomotor activation (Segal and Kuczenski,
1987). Clearly, additional studies, including detailed observations of the animals, will be necessary to further elucidate
the consequences of repeated low-dose MP.
Several important factors, in addition to dose, impact on
the potential relevance of the sensitization data to the therapeutic use of stimulants. For one, a variety of pharmacokinetic variables must be considered. Although, on a milligram
per kilogram basis, we used doses of MP and AMPH within
the therapeutic range, route of administration and species
differences in drug metabolism also significantly affect
plasma and brain levels of the drug, and correspondingly the
qualitative and quantitative features of the behavioral response. In this regard, it is important to note that whereas
we injected AMPH and MP s.c. or i.p., as in most animal
studies, the clinical use of psychostimulants typically involves oral administration. These differences in route of administration affect both the rate of accumulation of the drug
in plasma/brain as well as the peak drug concentrations that
are achieved. Thus, for example, in rats, peak stimulant
concentrations are achieved within 10 min following intraperitoneal or subcutaneous administration (Wargin et al.,
882
Kuczenski and Segal
of the drug in plasma/brain and peak drug concentrations
that are achieved, both of which play critical roles in the
qualitative and quantitative features of the behavioral response. In fact, the interpretation of previous animal studies
is limited to the degree that these studies fail to simulate the
treatment conditions associated with long-term stimulant
use in humans. Therefore, further research incorporating
those factors that more closely approximate the therapeutic
conditions will be required before more definitive conclusions
can be reached regarding the effects of stimulant treatment
in children with ADHD.
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of AMPH and MP within the context of the generality of
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specific neurochemical changes. For example, the doses of
AMPH and MP most relevant to therapeutic efficacy, 0.25
mg/kg AMPH and 0.5 mg/kg MP, produced markedly divergent DA responses. These DA effects are consistent with our
previous higher dose comparisons between psychostimulants
with different mechanisms of interaction, i.e., DA releasers
like AMPH typically promote severalfold higher extracellular
DA responses than behaviorally similar doses of DA uptake
blockers such as MP. In contrast to the divergent DA responses, we observed similar NE effects to the low doses of
AMPH (0.25 mg/kg) and MP (0.5 mg/kg). In fact, in contrast
to its effect on NE, this dose of MP did not affect accumbens
DA even after repeated administration. Although our results
do not take into account possible regional differences in neurotransmitter responsivity, they are consistent with a potentially important role for NE in the behavioral effects of low
doses of psychostimulants.
Within this same context, we did not observe an effect of
these doses of AMPH and MP on extracellular 5HT in nucleus accumbens. We have previously shown that moderate
doses of AMPH (⬃2 mg/kg) can increase extracellular serotonin levels in a variety of brain regions (Kuczenski and
Segal, 1989; Segal and Kuczenski, 1997). In contrast, even
chronic administration of high doses of MP (up to 30 mg/kg)
failed to affect extracellular 5HT levels (Segal and Kuczenski, 1999), consistent with the relatively low affinity of this
stimulant for the 5HT transporter (Gatley et al., 1996).
Taken together with the present data, our results suggest
that, in contrast to recent speculations (Gainetdinov et al.,
1999), a stimulant-induced increase in extracellular 5HT
likely does not play a significant role in the therapeutic
effects of low doses of these drugs.
Finally, the present neurochemical and behavioral results
may also provide some insight with regard to mechanisms
contributing to low-dose stimulant-induced sensitization.
Our data showed that repeated administration of the lowest
dose of MP we used (0.5 mg/kg) failed to result in the development of an augmented behavioral response to subsequent
stimulant challenge. This treatment also had no effect on
extracellular DA in nucleus accumbens, an effect that appears to be important to the development of stimulant abuse.
In this regard it is also important to recognize that the oral
administration of the doses we used in these studies would be
expected to produce significantly lesser effects than we observed following intraperitoneal and subcutaneous administration. Thus, the absence of a significant DA response may
have important implications for the abuse liability of lowdose, long-term MP treatment.
In summary, the comparative neurochemical profiles in
response to MP and AMPH suggest that the NE effects may
play an important role in the therapeutic effects of these
drugs. In addition, repeated administration of AMPH and
MP in the milligram per kilogram therapeutic dose range for
treatment of ADHD can result in the development of behavioral sensitization, which is evident in the response to subsequent stimulant challenge. However, even with doses that
appear to be relevant to clinical usage, interpretation of our
results is limited because of other factors that significantly
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Psychostimulant Threshold Doses: Behavior and Neurochemistry
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Send reprint requests to: Ronald Kuczenski, Ph.D., Psychiatry Department
(0603), University of California San Diego School of Medicine, 9500 Gilman
Dr., La Jolla, CA 92093. E-mail: [email protected]
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