Thousands of children receive methylphenidate (MPH; Ritalin) for attention deficit/hyperactivity disorder (ADHD), yet the long-term
neurochemical consequences of MPH treatment are unknown. To mimic clinical Ritalin treatment in children, male rats were injected
greater immunoreactivity (-ir) for the catecholamine marker tyrosine hydroxylase (TH), 60% more Nissl-stained cells, and 40% less
norepinephrine transporter (NET)-ir density. In hippocampal dentate gyrus, MPH-receiving rats showed a 51% decrease in NET-ir
density and a 61% expanded distribution of the new-cell marker PSA-NCAM (polysialylated form of neural cell adhesion molecule). In
PND135 rats showed major structural differences with MPH exposure. These findings suggest that developmental exposure to high
Attention deficit/hyperactivity disorder (ADHD) affects 2–18%
of children in the United States, with higher prevalence among
boys (Silver, 1999; Marshall, 2000; Hechtman, 2006). ADHD is
characterized by developmentally inappropriate symptoms of
impulsivity, inattentiveness, and overactivity (Sagvolden and
Sergeant, 1998). Methylphenidate (MPH; Ritalin) is one of the
most commonly prescribed drugs for alleviating ADHD symp-
toms and is efficacious in reducing hyperactivity and improving
attentiveness (Popper, 1997; Angold et al., 2000). Although the
benefits of MPH for ADHD symptoms are well documented,
little is known about the long-term impacts of MPH on brain
years, because the number of MPH prescriptions has risen dra-
matically, children are beginning MPH treatment as young as 2
years of age (Zito et al., 2000), and the duration of MPH treat-
ment has been lengthening, sometimes lasting into adulthood
(Solanto, 1998; Angold et al., 2000).
The acute and short-term actions of MPH in the brain are
relatively well established. MPH treatment blocks the dopamine
seen in dopamine D2receptors and in dopaminergic transmis-
sion in at least the striatum and prefrontal cortex (Gatley et al.,
1996; Kuczenski and Segal, 1997; Volkow et al., 1997, 1998,
1999a,b, 2001; Russell et al., 1998; Izenwasser et al., 1999). Addi-
tionally, MPH affects noradrenergic and serotonergic systems by
blocking the norepinephrine and serotonin transporters, al-
though affinity is lower than for the dopamine transporter (Gat-
inergic transmission have been observed in the nucleus
Correspondence should be addressed to Dr. Teresa A. Milner, Division of Neurobiology, Weill-Cornell Medical
7196 • TheJournalofNeuroscience,July4,2007 • 27(27):7196–7207
accumbens, temporal cortex (including the hippocampus), cere-
Persistent alteration of monoaminergic transmission trig-
gered by chronic MPH during development can potentially have
cytoarchitectural and neurochemical consequences. Normal
processes occurring in a temporally and regionally dependent
manner (Barone et al., 2000; Rice and Barone, 2000). Mono-
amines are important regulators of these processes, and altering
one et al., 2000; Rice and Barone, 2000). The effects of MPH are
potentially most pronounced in regions associated with cogni-
are at their peak of synaptogenesis during childhood and adoles-
cence in humans and rats (Bayer et al., 1993; Klintsova and
Greenough, 1999; Rice and Barone, 2000).
To study these issues, we used a rat model that mimics thera-
peutic doses of MPH given to children and adolescents with
ADHD. Brain regions implicated in ADHD, cognition, stress,
appetite, and/or attentional processes were immunocytochemi-
ters in male rats immediately after 4 weeks of MPH administra-
tion and 3 months after MPH administration. Neurotransmitter
systems examined included dopamine, norepinephrine, seroto-
nin, and acetylcholine. Additionally, weight gain and long-term
effects on anxiety behavior were measured to confirm the rele-
vance of the model (Rapport and Moffitt, 2002; McFadyen-
Leussis et al., 2004).
approved by the Weill-Cornell Medical College Institutional Animal
Care and Use Committee. Timed-pregnant Sprague Dawley female rats
(n ? 12) were obtained from Taconic Farms (Chatham, NY). Mother
rats were housed individually in free-standing “shoe-box” cages (model
6; dimensions, 30.80 ? 59.37 ? 22.86 cm; Thoren Caging Systems,
Hazleton, PA) and kept in a reversed light cycle room (lights off at 11:00
A.M. and lights on at 11:00 P.M.) equipped with red (GBX-2) darkroom
safelights (Eastman Kodak, Rochester, NY) and kept at an ambient tem-
perature of 21–24°C. After weaning [postnatal day 21 (PND21)] and
(n ? 80) were pair-housed in cages stacked in ventilator racks (cage
model 4; dimensions, 30.80 ? 30.80 ? 18.72 cm; Thoren Caging Sys-
tems). The air exchange rate inside the cages during normal operation
was 80 changes/h.
Choice of experimental model
The most common medication for the treatment of ADHD is MPH
of ADHD is usually made before the age of 7 years or in the first years of
ages 3–5 years have demonstrated that MPH is effective in reducing
weight loss, and decreased appetite (Wigal et al., 2006). In general, the
sequence of regional brain development in humans and rodents is simi-
lar; however, regional development of the rodent proceeds on a timeline
of days compared with months in humans (Bayer et al., 1993; Rice and
Barone, 2000). We chose to start administering MPH to rats at PND7
because at this time point the first adult-like neurons are observed in the
rat hippocampal dentate gyrus (Jones et al., 2003), and synaptogenesis
and neuronal growth are actively occurring (Bayer et al., 1993). MPH
administration continued until PND35 because at this age the matura-
tion level of the brain telencephalon corresponds to that observed in
et al., 2000). Male rats were the focus of the analysis, because boys have
higher rates of ADHD than girls (Angold et al., 2000).
MPH is metabolized into the less-active p-hydroxylated-MPH more
readily in rats than in humans (15% in rats compared with 1–4% in
humans) (Faraj et al., 1974; Wargin et al., 1983; Torres-Reveron and
Dow-Edwards, 2005); some of this may be attributable to the route of
administration because the oral route reduces the percentage of metab-
administration is twice as potent as oral administration in terms of in-
creasing extracellular dopamine and stimulating locomotion (Gerasi-
mov et al., 2000). MPH was given during the active cycle (dark phase of
the photo period) because rats are nocturnal animals, and MPH is given
to control symptoms during the active (diurnal) period in humans (An-
gold et al., 2000). In addition, rats were assessed at two time points
occur from MPH exposure during development persist later into
On PND3, the litters were sexed and culled to 10 pups per litter. Males
and females were included in this phase of the experiment to keep litters
could be distinguished by the experimenters but not by the mother rat
of rat pups were weighed and administered either 5 mg/kg D,L-threo-
MPH (obtained from National Institute on Drug Abuse, Research
Triangle Park, NC) in saline or an equal volume of saline intraperitone-
ally twice daily, 6 h apart during their dark (i.e., active) cycle. At the end
were divided into two groups. Group 1 was examined immediately after
after cessation of MPH drug administration (PND135). During the ces-
sation period, PND135 rats were weighed every 3 or 4 d. Repeated mea-
sures of weight were evaluated for systematic variation by litter and sex,
using repeated-measures ANOVA (RMANOVA). Differences were con-
sidered significant at p ? 0.05.
To assess the long-term effects of developmental exposure to MPH on
between PND130 and PND134 using previously described procedures
(Ramos and Mormede, 1998). For both behavioral tests, the experi-
Open-field test. On the day of testing, each rat was transferred to the
open-field testing room. Tests were conducted between 1:00 and 4:00
P.M. The floor of the apparatus was a 14 ? 14 cm grid composed of 25
squares (16 outer and 9 inner). A 150 W light bulb and the video camera
were positioned above the apparatus, illuminating the open field and
recording the rat’s behavior. Each rat was placed in the same corner
square of the open field at the beginning of the 5 min test. All four paws
had to cross the line of a center square to be considered an entry into the
center of the open field. Time spent in the center of the open field com-
pared with the squares closer to the walls (wall hugging) also was mea-
open-field apparatus was thoroughly cleaned with 30% ethanol.
Elevated plus-maze. Elevated plus-maze tests were conducted under
dim red light illumination between 1:00 and 4:00 P.M. The elevated
and two open arms. A video camera was positioned above the apparatus
to record the behavior of the rat. Each rat was placed in the center of the
elevated plus-maze (facing an open arm) at the beginning of the 5 min
test. Similar to the open-field test, all four paws had to cross the entry of
the open or closed arm to be considered an entry. Amount of time spent
the end of the test, the rat was returned to its home cage, and the plus-
maze was thoroughly cleaned with 30% ethanol.
Grayetal.•DevelopmentalMPHAltersBrainJ.Neurosci.,July4,2007 • 27(27):7196–7207 • 7197
Statistical analyses. Latency, frequency data, and time spent in center
or wall of the open-field test or open versus closed arms on the elevated
plus-maze were analyzed by two-tailed Student’s t tests. Data were ex-
pressed as averages ? SEM. Differences were considered significant at
p ? 0.05.
Section preparation. On PND35 (group 1) or PND135 (group 2), six
littermate pairs of male rats, each consisting of an MPH-administered
and a saline-administered rat, were deeply anesthetized with sodium
pentobarbital (150 mg/kg), and their brains fixed by aortic arch perfu-
sion sequentially with the following: (1) 10–15 ml of saline (0.9%) con-
taining 1000 U of heparin; (2) 50 ml of 3.75% acrolein and 2% parafor-
paraformaldehyde in PB (Milner and Veznedaroglu, 1992). After the
perfusion, the brains were removed and cut into 5 mm coronal blocks
30 min in the latter fixative. The brains were sectioned on a Leica (Ban-
nockburn, IL) VT1000X Vibratome (40 ?m thick) and stored at ?30°C
in cryoprotectant (30% sucrose and 30% ethylene glycol in PB) until
immunocytochemical processing. Before immunocytochemistry, sec-
tions from littermate rat pairs were rinsed in PB, coded with hole
taken to match rostrocaudal levels of the paired sections so that near-
treated with 1% sodium borohydride in PB for 30 min to neutralize free
aldehydes and rinsed thoroughly in PB. Sections then were rinsed in 0.1
M Tris-buffered saline (TS; pH 7.6) and incubated in 0.5% bovine serum
albumin (BSA) in TS for 30 min.
Brain regions analyzed and corresponding atlas levels. Coronal sections
containing brain regions implicated in ADHD, animal models of hyper-
activity (Grund et al., 2006), and/or learning and attentional processes
(Castellanos et al., 2006) were selected for analysis using the Swanson
and basolateral nuclei; levels 27 and 28; anteroposterior (AP), ?2.00 to
mm from bregma), globus pallidus (levels 20–22; ?0.46 to ?4.60 mm
from bregma), hippocampal formation (CA1, CA3, and dentate gyrus;
levels 28–38; ?2.45 to ?5.65 mm from bregma), dorsomedial (DMH)
and paraventricular (PVN) hypothalamic nuclei (levels 23–30; ?1.08 to
mm from bregma), nucleus accumbens (core and shell; levels 12–14;
?1.08 mm from bregma), prefrontal cortex (dorsal and medial; levels
to ?9.80 mm from bregma)], septal nuclei (medial septal nucleus and
somatosensory cortex (primary; levels 27–29; ?2.00 to ?2.85 mm from
bregma), striatum (13–16; ?1.20 to ?0.10 mm from bregma), and sub-
stantia nigra and ventral tegmental area (levels 37–39; ?5.25 to ?6.06
mm from bregma).
rotransmitter systems implicated in ADHD and/or targeted by MPH
(e.g., monoamines) and to identify particular cell populations known to
develop postnatally (e.g., newly born cells in the dentate gyrus) or to be
important for development (e.g., glial cells). All antisera have been well
characterized and tested for specificity previously. More details for anti-
neurons and processes, a mouse monoclonal antibody to tyrosine hy-
droxylase (TH; diluted 1:10,000), the catecholamine-synthesizing en-
zyme, was obtained from Incstar (Stillwater, MN) (Milner et al., 1999).
Dopaminergic neurons and processes were identified by using a poly-
obtained from Millipore (Billerica, MA) (Sesack et al., 1998). Noradren-
ergic neurons and processes were identified with a rabbit polyclonal
antiserum to dopamine-?-hydroxylase (DBH; diluted 1:10,000), sup-
plied by Dr. Cory Abate (University of Medicine and Dentistry of New
Jersey, Newark, NJ) (Joh and Ross, 1983), and a rabbit polyclonal anti-
serum to the norepinephrine transporter (NET; diluted 1:20,000), sup-
plied by Dr. Randy Blakely (Vanderbilt University School of Medicine,
Nashville, TN) (Miner et al., 2003). Serotonin neurons and processes
were identified with a goat polyclonal antiserum to the serotonin trans-
porter (SERT; diluted 1:5000), obtained from Santa Cruz (Brown and
Molliver, 2000). Cholinergic neurons were identified by using a goat
polyclonal antibody to vesicular acetylcholine transporter (VAChT; di-
luted 1:20,000), obtained from DiaSorin (Stillwater, MN) (Arvidsson et
al., 1997). Selected neuropeptides were labeled using (1) a monoclonal
to neuropeptide Y (NPY; diluted 1:2000) obtained from Peninsula (Bel-
mont, CA) (Milner and Veznedaroglu, 1992). Cell-specific markers also
were used in this study. Thionin was used to visualize Nissl substance in
to glial fibrillary acidic protein (GFAP; diluted 1:6000) obtained from
Millipore (Brezun and Daszuta, 2000) was used. To identify granule cell
nuclei, a rabbit polyclonal antibody to the divergent homeobox protein
PROX1 (diluted 1:10,000) obtained from Millipore was used (Elliott et
clonal antibody to calbindin (diluted 1:500; Sigma-Aldrich, St. Louis,
MO) was used (Scharfman et al., 2000). A mouse monoclonal antibody
to polysialylated form of neural cell adhesion molecule (PSA-NCAM;
doublecortin (DCX; diluted 1:2000) obtained from Santa Cruz Biotech-
nology (Santa Cruz, CA) were used to label newly born cells in the den-
tate gyrus (Francis et al., 1999; Brezun and Daszuta, 2000).
Immunocytochemical processing. Matched coronal sections containing
the desired brain areas were processed for the immunocytochemical lo-
temperature and for 24 h at 4°C in the specified dilution of antiserum in
0.1% BSA in TS, pH 7.6. All sections except those incubated in PSA-
NCAM were incubated in a 1:400 dilution of biotinylated anti-Ig (IgG)
against the species in which the primary antiserum was raised (Jackson
for PSA-NCAM were incubated in biotinylated anti-mouse IgM (Cym-
bus Biotechnology, Flanders, NJ). After incubation in the secondary an-
tibody, the sections were rinsed in TS and incubated in peroxidase–
avidin complex (at twice the recommended dilution; Vector
Laboratories, Burlingame, CA) for 30 min followed by development in
3,3?-diaminobenzidine (Sigma-Aldrich) and H2O2in TBS for 6 min.
Sections were mounted on slides previously coated with 1% gelatin and
dried in a desiccator. The following day, the slides were dehydrated
through a graded series of ethanols and coverslipped from xylene with
(Tokyo, Japan) E800 or 80i light microscope equipped with bright-field
camera (Q Imaging, Barnaby, British Columbia, Canada). For the qual-
itative analysis, low-magnification (2? or 4?) photomicrographs were
taken of the brain region of interest (ROI) from matched areas in each
littermate pair of sections. Photographs were laid side by side to deter-
mine whether MPH administration apparently altered the pattern or
density of particular markers in the ROI. If differences were observed in
the pattern of labeled fibers, higher-magnification photographs (10? or
20?) were taken of the ROI. If apparent differences in the density of a
densitometry was performed on the ROI using previously described
methods (Pierce et al., 1999). For this, additional sections through the
using optimal, empirically derived dilutions of antisera appropriate for
1999; Chang et al., 2000). ROIs then were captured using a Dage CCD
C72 camera and NIH Image 1.50 software on a Nikon 80i microscope
from the background and ROI were taken and averaged before analysis.
7198 • J.Neurosci.,July4,2007 • 27(27):7196–7207Grayetal.•DevelopmentalMPHAltersBrain
For the striatum, subdivisions were delimited using criteria previously
defined by Yano and Steiner (2005a).
To compensate for background staining and control for variations in
illumination level between images, the average pixel density for three
regions that lacked labeling was subtracted. An observer blind to the
experimental group read each ROI twice. ROI readings were averaged
and analyzed using a two-tailed Student’s t test. When marginal signifi-
cance values were obtained, or a parametric test was not appropriate,
ranks test. Differences were considered significant at p ? 0.05.
Volume of the hilus of the dentate gyrus was calculated using the
Cavalieri principle (Severi et al., 2005). For this, a regularly spaced ran-
rostrocaudal extent of the dentate gyrus. Volume for each section was
the hilus by section thickness (40 ?m) and by sampling interval (24
analyzed using a two-tailed Student’s t test with significance at p ? 0.05.
The density of Nissl-labeled cells in the medial prefrontal cortex
(mPFC) was determined using the “Grain counting” menu in MCID
analysis software (Imaging Research, St. Catharines, Ontario, Canada)
on a PC. For this, grayscale images of Nissl sections were converted to
binary images, using a threshold that was consistent across images. An
oval of equal dimensions for all pictures was drawn using the corpus
callosum as an anatomical landmark. The density of Nissl-stained cell
Student’s t test was used to determine significance ( p ? 0.05).
Final photomicrographs were generated from digital images adjusted
for levels, brightness, and contrast in Adobe (San Jose, CA) Photoshop
7.0 on a Macintosh G5 computer (Apple, Cupertino, CA). Graphs were
prepared with GraphPad Prism 4.01 (GraphPad Software, San Diego
CA); quantitative data are represented as average ? SEM. Images and
graphs were assembled into the final figures using Quark (Denver, CO)
V.11.0.1 (SPSS, Chicago, IL).
Repeated measures of weight were evaluated for systematic vari-
ation by litter and gender, using RMANOVA. In all analyses, the
rates of weight gain across litters. Thus, litter was entered as a
covariate in analyses of between-groups effects.
receiving MPH gained weight at a significantly slower rate than
control littermates of the same sex, as evidenced by a significant
interaction between weight and administration status (F(4,46)?
4.437; p ? 0.004) for weight gain analyzed in blocks of 3 d. Also,
analysis of weight gained on each individ-
ual day from PND10 to PND18 revealed a
significant interaction of weight and ad-
exposed animals gained less weight than
matched controls (F(8,24)? 2.459; p ?
the MPH-administered rats gained signif-
icantly less weight on PND16–PND18
( p ? 0.05), and a similar trend was ob-
finding is consistent with clinical data on
humans; reduced appetite and weight loss
are recognized side effects of MPH treat-
ment (Rapport and Moffitt, 2002).
After weaning on PND21, for the next
11 d, rats receiving MPH continued to gain weight at a signifi-
by a significant weight by administration group interaction on
RMANOVA (F(3,47)? 3.809; p ? 0.016) on data analyzed in
blocks of 3 d. Additional analysis showed that MPH-exposed
animals gained weight at a slower rate from PND25 to PND27
( p ? 0.05) (Fig. 1B).
weight gain was not different between MPH-administered and
of weight by administration was not significant (F(3,35)? 2.701;
as all animals continued to gain weight in a linear manner over
this period (F(3,35)? 399.28; p ? 0.0001) (Fig. 1C).
Some studies show decreases (McFadyen-Leussis et al., 2004),
and others show increases (Carrey et al., 2000; McFadyen et al.,
2002; Bolanos et al., 2003) in anxiety-like behaviors after MPH
assess anxiety levels in the male PND135 rats, which received
MPH or saline from PND7–PND35 and then were drug-free for
MPH-administered rats showed a trend toward crossing fewer
total lines in the open field compared with controls, but this did
not reach statistical significance ( p ? 0.08). There were no dif-
ferences between MPH-administered and control rats in their
latency to enter the center of the open field, number of center
amount of time in the squares close to the wall versus the center
with periodic episodes of grooming and rearing. Although stan-
dard habituation time (Ramos and Mormede, 1998) was used in
this study, a longer test period may have revealed results similar
to those observed in the elevated plus-maze.
times anxiety may increase activity, whereas in other cases it may
cause freezing behavior (Ramos and Mormede, 1998). Because
our results suggested an effect of MPH on anxiety, the elevated
significantly less than control littermates. B, After weaning and during MPH administration, the weight difference was still
Grayetal.•DevelopmentalMPHAltersBrainJ.Neurosci.,July4,2007 • 27(27):7196–7207 • 7199
plus-maze test was used to more specifi-
cally assess anxiety. In the elevated plus-
maze, MPH-administered rats spent a sig-
nificantly larger amount of time in the
open arms compared with controls [con-
6.37 s (n ? 9); t(16)? 2.35; p ? 0.05].
ferences in locomotor activity because
MPH-administered rats had a total num-
ber of crossings similar to the control
group ( p ? 0.12). These results suggest
that 3 months after MPH administration
during development, male rats exhibited
less anxiety-like behaviors as adults.
velopment, quantitative differences were
identified in four brain regions that have
previously been implicated in the etiology
of ADHD, learning and attentional pro-
cesses, and homeostatic regulation: the
prefrontal cortex, striatum, hippocampal
formation, and hypothalamus. No differ-
ences were apparent between control and
MPH-administered rats in the other 12
brain regions investigated at either time
point after developmental administration
of MPH (Table 1).
The PFC is thought to be a crucial regula-
tor of executive functions and decision
making whose proper functioning de-
dopamine and norepinephrine activity
(Dalley et al., 2004; Arnsten and Li, 2005).
dopamine innervation from ventral teg-
mental area, and MPH administration has been shown to signif-
icantly increase available dopamine in this region while simulta-
neously improving performance on cognitive tasks in ADHD
(Sesack et al., 1998; Miner et al., 2003), almost no DAT-
immunoreactive processes were observed in the rat PFC. In con-
trol rats assessed at either time point, TH-labeled fibers were
concentration in layer 6 (Fig. 2A1). At the end of administration
(PND35), MPH-administered rats had a significant increase
(55%) in the density of TH-immunoreactive fibers in layer 6 in
the mPFC (Fig. 2A2,D) (t(10)? 2.39; p ? 0.05). This increase in
density of TH-containing fibers tended to remain at PND135, 3
months after cessation of MPH exposure; however, although a
(t(10)? 1.09; p ? 0.05). To investigate the possibility that differ-
ences in TH density at PND35 correlated with changes in cell
distribution and/or number, Nissl staining of the mPFC was as-
sessed. At PND35, the density of Nissl stained cells was signifi-
cantly greater (60%) in layer 6 of MPH-exposed rats (Fig. 2C,D)
(t(10)? 2.28; p ? 0.05). No significant difference in the distribu-
time point (Fig. 2D) (t(10)? 1.31; p ? 0.05).
and the reports that MPH can inhibit norepinephrine and sero-
Kuczenski and Segal, 2002), labeling for NET, DBH, and SERT
was examined. NET-immunoreactive fibers were less abundant
2B1). Quantitative analysis revealed that the number of NET-
immunoreactive fibers in the mPFC was significantly smaller
2B2,D) (t(10)? 2.25; p ? 0.05). This MPH-linked difference in
NET-ir was not observed in PND135 rats (Fig. 2D) (t(4)? 0.266;
p ? 0.05). Despite the difference observed with NET at PND35,
DBH at either time point (PND35, t(10)? 0.61, p ? 0.05;
PND135, t(10)? 0.81, p ? 0.05). Similarly, no differences were
fibers in the mPFC at either PND35 (t(6)? 0.23; p ? 0.05) or
PND135 (Table 1).
7200 • J.Neurosci.,July4,2007 • 27(27):7196–7207 Grayetal.•DevelopmentalMPHAltersBrain
The distribution of cholinergic fibers in the PFC was assessed
with VAChT because of the suggested role of the basal forebrain
system in learning and memory (Muir, 1997) and our previous
findings that cholinergic basal forebrain neurons receive numer-
ous inputs from monoamine terminals (Milner, 1991; Milner
and Veznedaroglu, 1993). No differences were observed in
at either PND35 or PND135 (Table 1).
The striatum is important for the coordination of motor move-
ments and, like the PFC, is rich in dopamine terminals and has
been implicated in ADHD (Solanto, 1998; Izenwasser et al.,
1999). Previous animal studies have shown that metabolism and
dopamine D2receptor availability are altered in the striatum in
response to MPH administration (Volkow et al., 1997). The nu-
cleus accumbens (also known as ventral striatum) is another
structure that has been implicated in reinforcement (Nestler,
2001) and is a site for the stimulant actions of MPH (Gerasimov
et al., 2000). In control rats from either time point, TH labeling
of the internal capsule and was densest in the dorsal, medial, and
ventral subregions (Fig. 3A). Quantitative densitometry revealed
in the density of TH-immunoreactive fibers immediately after
MPH administration (PND35) (Fig. 3B) (Wilcoxon Z(5)?
?2.02; p ? 0.05). This difference was not present 3 months after
tern of TH or DAT immunoreactivity in the striatum did not
appear different in response to MPH administration at either
PND35 or PND135. No significant differences in the density of
DAT-immunoreactive processes were observed in any region of
the striatum at either PND35 or PND135 (Fig. 3C).
No difference in the density of TH-ir was seen in the caudal
separate) at PND35 (t(10)? ?0.15; p ? 0.05).
ory processes (McGaugh, 2000) and is one of the few sites of
ongoing neurogenesis in the adult brain (Gould et al., 2000).
ally, the hippocampal formation, especially the ventral portion,
the hippocampal formation, immunoreactivities to TH, NET,
and DBH were localized. In control PND35 and PND135 rats,
TH-, NET-, and DBH-immunoreactive fibers were diffusely dis-
tributed throughout all hippocampal lamina and were most
dense in the hilus of the dentate gyrus (Fig. 4A1,B1) (DBH not
shown) and stratum lucidum of region CA3. A significant de-
crease (51%) in the number of NET-labeled fibers was observed
decrease at PND135 (Fig. 4D) (subgranular zone, t(10)? 0.04,
p ? 0.05; central hilus, t(10)? 0.94, p ? 0.05). TH-labeled fiber
density was not different at either time point (Fig. 4A,D)
(PND35, t(10)? 0.06, p ? 0.05; PND135, t(10)? 0.19, p ? 0.05).
Likewise, DBH-labeled fiber density in the dentate hilus was not
at PND35 (t(10)? 0.11; p ? 0.05) or PND135 (t(10)? 0.44; p ?
or DBH-ir were observed in the CA1 or CA3 regions of MPH-
administered rats at either PND35 or PND135.
of the mPFC of MPH-administered rats (A2) compared with controls (A1). B, At PND35, the
(B2) relative to controls (B1). C, At PND35, the density of Nissl-stained cell bodies (arrows)
increased in mPFC layer 6 in MPH-administered rats (C2) compared with controls (C1). Atlas
level 9 is shown (AP, ?2.80 mm from bregma) (Swanson, 1992). cc, Corpus callosum. Scale
bars, 100 ?m. D, Comparisons of the density of TH- and NET-immunoreactive fibers (optical
density and a significant decrease in NET-labeled fibers in MPH-exposed rats. No differences
Grayetal.•DevelopmentalMPHAltersBrainJ.Neurosci.,July4,2007 • 27(27):7196–7207 • 7201
PSA-NCAM and DCX were used (Francis
et al., 1999; Brezun and Daszuta, 2000).
PSA-NCAM and DCX primarily identify
late neuronal progenitors (Seki and Arai,
1993; Jessberger et al., 2005). At PND35,
PSA-NCAM and DCX immunoreactivi-
ties in control rats were observed in cell
throughout the subgranular zone of the
dentate gyrus (Fig. 4C1). At the end of the
PSA-NCAM labeling in the medial blade
of the subgranular zone of the dentate gy-
rus extended over a 61% wider area in
MPH-administered rats than in controls
(Fig. 4C1,C2) (t(10)? ?3.20; p ? 0.009).
Additionally, the hippocampi of MPH-
administered rats appeared slightly mis-
shapen in two of six cases (Fig. 4A2). This
was unlikely to be caused by mismatched
sections, because great care was taken to
other studies of adult rats (Brown et al.,
2003), at PND135 PSA-NCAM- or DCX-
immunoreactive cell bodies were found in
sporadically distributed clusters in the
subgranular zone of the dentate gyrus in
in either the lamination or number of
PSA-NCAM- or DCX-immunoreactive
cells (Table 1) [controls (n ? 3), 10.3 ? 1.0 DCX cells/mm of
granule cell layer (gcl) length; MPH (n ? 3), 10.4 ? 0.3 DCX
cells/mm of gcl length; t(4)? 0.06; p ? 0.05].
In addition to the difference in lamination of PSA-NCAM
seen in the present study, other studies have revealed that MPH
administration significantly decreases the survival of newly born
affect the hippocampal volume, the number of ectopic cells,
and/or lamination, and thus these parameters were evaluated.
The total volume of the dentate hilar region of MPH-
administered and control rats was assessed using immunoreac-
tivity for L-Enk. In agreement with previous studies (Gall et al.,
lineated the hilus of the dentate gyrus. The hilar volume was not
significantly different at PND35 (t(10)? 0.31; p ? 0.05) or at
ber of ectopic cells in the dentate hilar region was altered after
MPH exposure, sections were labeled with PROX1. At both the
numbers of PROX1-labeled nuclei in the hilus were found
(PND35, t(10)? 0.53, p ? 0.05; PND135, t(14)? 0.15, p ? 0.05).
The lamination pattern of dentate gyrus was assessed with calbi-
hilus), and the distribution of astroglia in the dentate gyrus was
assessed using GFAP. Neither the calbindin nor GFAP staining
patterns appeared different between the two groups at either
PND35 or PND135 (Table 1).
Given the changes in weight observed with MPH administration
regulation (Fu et al., 2004; Kishi et al., 2005), NPY-ir was exam-
In control rats at either PND35 or PND135, dense NPY-ir was
found in the PVN (especially the medial parvocellular part) and
the posterior region of the DMH (Fig. 5A1,B1). In MPH-
distribution of NPY-immunoreactive fibers was more wide-
spread in MPH-exposed rats at PND35 (Fig. 5B2,C), although
there was considerable variability between MPH rats, and the
density of NPY-labeled fibers in the central DMH did not attain
significant difference (Wilcoxon Z(5)? ?1.36; p ? 0.08). No
were observed between the MPH group and the control group at
the DMH (Wilcoxon Z(5)? ?0.734; p ? 0.05).
groups at either time point, with the exception of one MPH rat
from the PND35 group. This rat had an enlarged rostral lateral
not shown). Sections through this region were excluded from
minergic neurons in the substantia nigra and ventral tegmental
area innervate the striatum and nucleus accumbens, respectively
(Heimer et al., 1995). Consistent with previous studies (Fallon
and Loughlin, 1987), TH-immunoreactive neurons were partic-
ularly dense in the pars compacta of the substantia nigra (SNc)
and in the parabrachial (PBP) and paranigral (PN) subdivisions
the MPH group. C, The density of DAT-immunoreactive fibers was similar in MPH-administered and control rats at both time
7202 • J.Neurosci.,July4,2007 • 27(27):7196–7207Grayetal.•DevelopmentalMPHAltersBrain
of the ventral tegmental area. No significant differences were
MPH-administered and control rats at PND35 (SNc, t(10)? 1.1,
p ? 0.05; PN, t(10)? ?0.62, p ? 0.05; PBP, t(10)? ?0.53, p ?
0.05) or PND135 (SNc, t(10)? 0.35, p ? 0.05; PN, t(10)? 1.26,
p ? 0.05; PBP, t(10)? ?0.15, p ? 0.05).
This study reveals that prolonged developmental exposure to
high therapeutic doses of MPH has short-term effects on several
neurotransmitter systems in the rat mPFC, striatum, hippocam-
pus, and hypothalamus and few long-term effects. These MPH-
induced changes may contribute to our observed alterations in
brain regions that may be involved in the therapeutic and acces-
sory actions of MPH in ADHD treatment.
echolamines in this region and complements previous findings
(see Introduction). The trend toward redistribution of TH-ir
istration (PND35). A, Controls (A1) and MPH-administered rats (A2) had similar densities of
A decrease in the density of NET-immunoreactive fibers in the central hilus was observed in
Comparison of the density of TH and NET fibers and the width of PSA-NCAM labeling in the
cant decrease in NET-labeled fibers (*) and a significant increase in the width of PSA-NCAM
MPH administration (PND35). A, At PND35, increases in the density of NPY-immunoreactive
fibers than controls (B1). Atlas levels 26 (AP, ?1.78 mm from bregma; A1, A2) and 30 (AP,
DMH in control and MPH-exposed rats at PND35 and PND135. A significant (*) increase in
Grayetal.•DevelopmentalMPHAltersBrainJ.Neurosci.,July4,2007 • 27(27):7196–7207 • 7203
unchanged, suggesting either that the TH enzyme is selectively
sensitive to MPH exposure or perhaps that a subset of the cat-
echolaminergic fibers (e.g., dopaminergic) were affected. An ac-
cumulation of TH-immunoreactive fibers in deep mPFC layers
in MPH-administered rats resembled the gathered catechol-
amine fibers present in normal immature cortex (Berger et al.,
1985), suggesting that MPH may delay developmental processes
in the mPFC. Consistent with this idea, MPH-administered rats
had more Nissl-stained cells in deep cortical layers, where the
early postmitotic neurons collect (Kriegstein and Noctor, 2004).
The altered distribution of cells and catecholamine fibers at
dal cells (Krimer et al., 1997) and, to a lesser extent, GABAergic
interneurons (Sesack et al., 1995). Catecholaminergic innerva-
tion of pyramidal cells matures gradually postnatally, whereas
that of interneurons remains stable (Lambe et al., 2000). Devel-
opmental exposure to other psychostimulants alters dendritic
morphology of PFC pyramidal cells and diminishes certain
GABAergic interneuron populations (Robinson and Kolb, 1999;
Morrow et al., 2005). Similarly, MPH exposure during the con-
struction of catecholaminergic connections to excitatory mPFC
MPH-exposed rats. The present immunolabeling conditions fa-
vored detection of cytosolic NET, because the use of detergents
can dissolve membranes containing plasmalemmal NET; NET is
primarily cytosolic under resting conditions but redistributes to
the plasmalemma with certain stimuli (Miner et al., 2003, 2006).
Because dopamine is the preferred substrate for NET and can be
taken up by noradrenergic PFC axons (Sesack et al., 1998), NET
may have moved to the plasmalemma in response to MPH-
induced dopamine increases. Alternatively, NET may have been
downregulated in response to elevated extracellular dopamine
and/or norepinephrine after chronic MPH administration.
A decrease in dopaminergic fibers, identified by TH, was seen in
medial striatum with MPH administration (PND35). These ob-
servations are consistent with the findings that acute MPH ad-
ministration induces changes in striatal transcription factors (c-
Fos, zif268), genes that regulate synaptic plasticity (Homer1a),
and substance P (in striatonigral outputs) (Yano and Steiner,
2005a,b). Moreover, these MPH-induced effects occur primarily
through a dopaminergic mechanism, because they are inhibited
by blocking striatal D1receptors (Yano et al., 2006). The medial
striatum receives excitatory inputs from the anterior cingulate
cortex and prelimbic PFC (Berendse et al., 1992; Willuhn et al.,
2003), suggesting that chronic MPH specifically targets corticos-
triatal circuits that are involved in sensorimotor functions.
TH-ir decreased in the striatum but increased in the mPFC
with MPH administration. Several differences in dopaminergic
transmission between the two regions may contribute to these
opposite responses. The striatum and mPFC are innervated by
striatum contains abundant DAT, whereas the mPFC contains
almost none (Sesack et al., 1998). Moreover, in striatum, dopa-
mine is primarily taken up by DAT at synaptic release sites,
whereas in mPFC, dopamine uptake involves NET on nearby
norepinephrine terminals or, to a lesser extent, DAT on preter-
minal dopaminergic axons (Sesack et al., 1998; Miner et al.,
immunoreactive fibers, but not TH-labeled fibers, in the dentate
hilus. No differences were observed at PND135. The decrease in
change in NET fiber density is probably not caused by shrinkage
or expansion of the dentate gyrus, because no volume changes
were found. TH is almost exclusively in noradrenergic fibers in
the dentate gyrus and labels nearly the identical population of
fibers identified by DBH (Milner and Bacon, 1989). Thus, the
lack of change in TH-ir in the dentate gyrus is similar to the lack
of change in DBH-ir in the mPFC; both suggest stability of affer-
ents carrying norepinephrine.
zone is the last portion of the dentate to mature (Bayer et al.,
1993), and it is possible that its maturation is delayed in young
rats exposed to MPH, producing less restricted distribution of
new cells. Consistent with this possibility, new-cell markers have
a wider distribution in younger rat hilus (Herrick et al., 2006).
(PND135), nor was there a difference in the number of new cells
developmental exposure to MPH does not alter new-cell prolif-
the functional implications of juvenile MPH exposure, it is im-
portant to note that long-term survival of newly born dentate
cells was diminished in adults (Lagace et al., 2006).
Rats that received MPH showed increased NPY-ir in the hypo-
thalamic PVN and a redistribution of NPY-ir in the DMH at
PND35. NPY-ir was normal in distribution and density in both
1999), the increase in NPY density likely reflects an increase in
a concentration of NPY fibers in a smaller area. Regardless, our
results are consistent with observations that NPY systems in hy-
pothalamus and other brain areas are responsive to dopamine-
altering psychostimulant drugs and other stimuli (Lewis et al.,
1993; Westwood and Hanson, 1999; D’Este et al., 2006). Simi-
larly, NPY induction of the stress hormone corticotrophin-
releasing factor (CRF) is regulated by catecholamines (Haas and
MPH-induced changes in PVN and DMH likely affect several
autonomic functions and may have contributed to the observed
those containing CRF, are reciprocally connected with
cardiovascular-regulating medullary neurons (Milner et al.,
echolamine responses to stress (Dayas et al., 2004) through a
mechanism involving the mPFC (Spencer et al., 2005). DMH
and the expression of food-entrainable circadian rhythms (Bell-
inger and Bernardis, 2002; Gooley et al., 2006). Increased NPY
levels have been correlated with weight gain (Bellinger and Ber-
7204 • J.Neurosci.,July4,2007 • 27(27):7196–7207Grayetal.•DevelopmentalMPHAltersBrain
nardis, 2002), and our observed increases in NPY may reflect
compensatory responses to MPH exposure.
Three months after administration (PND135), MPH-exposed
plus-maze, suggesting that MPH reduced anxiety-like behaviors.
Consistent with our results, 2 mg/kg MPH administered to mice
the elevated plus-maze as adults (Bolanos et al., 2003), whereas
opposite effect (McFadyen-Leussis et al., 2004). Together, the
findings suggest that exposure to MPH during earlier develop-
mental periods, when synaptogenesis is still actively occurring
(Bayer et al., 1993), might render animals less susceptible to
anxiety-like behaviors in adulthood.
This study provides novel anatomical evidence that therapeutic
doses of MPH chronically administered to normal juvenile rats
results in short-term changes in four brain areas involved in mo-
tivated behaviors, cognition, appetite, and stress. These changes
were mirrored by differences in weight gain and anxiety. No sig-
although a trend toward persistent differences in mPFC suggests
tent with the views that MPH targets brain regions beyond those
lanos et al., 2006) and that the duration and timing of MPH
treatment are important variables affecting cognitive and neural
functions. This study also suggests regions of interest for future
functional neuroimaging studies and clinical investigations.
Moreover, these results suggest that although the observed neu-
tion of young brains with MPH may exert effects on brain neu-
rochemistry that modify some behaviors in adulthood.
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