Chronic low dose Adderall XR® down-regulates cfos expression in infantile and prepubertal rat striatum and cortex

Neuroscience 169 (2010) 1901–1912

CHRONIC LOW DOSE ADDERALL XR® DOWN-REGULATES cfos EXPRESSION IN INFANTILE AND PREPUBERTAL RAT STRIATUM AND CORTEX J. K. ALLEN,a M. WILKINSON,a,b,c E. C. SOO,d J. P. M. HUI,d T. D. CHASEb AND N. CARREYb,e*

FOS-ir was observed in the cerebral cortex following doses lower than the threshold dose necessary to increase FOS-ir in the striatum. This was not the case in the PD10 rats. In conclusion, our efforts to calibrate biological responses, such as immediate early gene expression, to clinically relevant blood levels of stimulants confirmed that expression of cfos is very sensitive to repeated low doses of Adderall XR®. It is now feasible to examine whether other genes are also affected in these young rats and if the changes we report are reversible. The implications of such studies should be relevant to the putative effects of psychostimulant treatment of very young children. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Obstetrics and Gynaecology, IWK Health Centre, Halifax, Nova Scotia, Canada B3K 6R8 b Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 c Division of Endocrinology and Metabolism, VG Hospital, Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 2Y9 d Institute for Marine Biosciences, National Research Council of Canada, Halifax, Nova Scotia, Canada B3H 3Z1 e Department of Psychiatry, IWK Health Centre, Halifax, Nova Scotia, Canada B3K 6R8

Key words: Amphetamine, methylphenidate, development, liquid chromatography–mass spectrometry.

Abstract—We previously reported that treatment of prepubertal male rats with low, injected or oral, doses of methylphenidate stimulated cfos, fosB and arc expression in many areas of the developing brain. In the present study our objective was to determine whether the widely prescribed psychostimulant Adderall XR® (ADD) exerted similar effects in infantile and prepubertal rat brain. We report here, for the first time, that low threshold doses of oral ADD, an extendedrelease mixture of amphetamine salts, now routinely used for the treatment of Attention Deficit Hyperactivity Disorder (ADHD), also increased cfos expression in infantile (postnatal day 10; PD10) and prepubertal (PD24) rat brain. These threshold doses were correlated with blood levels of amphetamine determined by liquid chromatography–mass spectrometry. Moreover, we observed that chronic treatment with oral ADD (1.6 mg/kg; ⴛ14 days) not only significantly down-regulated cfos expression following a final challenge dose of ADD in prepubertal (PD24) rat striatum and cortex, quantified in terms of FOS immunoreactivity (FOS-ir), but did so at a daily dose that was without effect with methylphenidate (MPH); that is a much higher oral dose of MPH (7.5 mg/kg; ⴛ14 days) failed to induce down-regulation of cfos expression. Similar experiments in infantile rats (PD10), but using a threshold injected dose of ADD (1.25 mg/kg sc) also significantly reduced striatal and cingulate cortical FOS-ir. An additional finding in the prepubertal rats was that oral ADD-induced

Stimulants have been prescribed for many years for the treatment of attention deficit hyperactivity disorder (ADHD), and the last two decades have witnessed increased rates for stimulant prescriptions overall, for longer periods of time and in children younger than 3 years (Zito et al., 2000; Mayes et al., 2008; Greenhill et al., 2008). This transformation in clinical practice took place largely in the absence of long-term safety studies, and the possible impact of these drugs on the developing nervous system remains a critical research focus (Andersen, 2005; Carlezon and Konradi, 2004). This is especially relevant given that growth and maturity of the human brain continues well into the second and third decades of life (Sowell et al., 2003; Rice and Barone, 2000). Difficulty in diagnosis of ADHD, including overly inclusive criteria and non-specificity in younger children, may result in inadvertent adverse consequences of these drugs (Teicher et al., 1996). Exposure of the immature brain to psychostimulants such as cocaine, amphetamine (Adderall XR®) and methylphenidate (Ritalin) could alter gene expression and lead to irreversible changes in cellular responsiveness and synaptic connectivity (Robinson and Kolb, 2004; Stanwood and Levitt, 2004; Andersen, 2005). Expression of immediate early genes in brain tissue from experimental animals is routinely used as a probe to localize the effects of stimulant drugs (Hughes and Dragunow, 1995; Yano and Steiner, 2007). For example we demonstrated an acute stimulatory effect of injected methylphenidate (MPH) on cfos expression in the prepubertal mouse (Penner et al., 2002; Hawken et al., 2004) and rat striatum (Chase et al., 2003, 2005a,b), which was attenuated by repeated treatment. In contrast repeated MPH elevates FOSB-ir in the prepubertal rat striatum (Chase et

*Correspondence to: N. Carrey, Department of Psychiatry, IWK Health Centre, PO Box 9700, Halifax, Nova Scotia, Canada B3K 6R8. Tel: ⫹1-902-470-8375. E-mail address: [email protected] (N. Carrey). Abbreviations: ADD, Adderall XR®; ADHD, attention deficit hyperactivity disorder; AMPH, amphetamine; arc, activity regulated cytoskeletal associated; bdnf, brain derived neurotrophic factor; CC, corpus callosum; DAT, dopamine transporter; ⌬FOSB, delta FOSB; FOS-ir, FOS immunoreactivity; FOS⫹, cells positive for FOS staining; FrC, frontal/cingulate cortex; LC–MS, liquid chromatography–mass spectrometry; LV, lateral ventricle; MPH, methylphenidate; MRM, multiple reaction monitoring; Par, parietal cortex; PBS, Dulbecco’s phosphatebuffered saline; PD, postnatal day; PFA, paraformaldehyde; PFC, prefrontal cortex; Pir, piriform cortex; SAL, saline; SPE, Solid phase extraction; Str, striatum; VMAT2, vesicular monoamine transporter 2.

0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.06.029

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al., 2005a,b) and in adolescent mice (Kim et al., 2009). Subsequent studies revealed that MPH also regulates genes that are implicated in neuroplasticity, such as arc (Chase et al., 2007), homer 1 (Yano and Steiner, 2007) and bdnf (Banerjee et al., 2009). In total the data suggest that MPH targets many regions of the immature rodent brain in addition to its presumed target, the striatum (Chase et al., 2005a; Devilbiss and Berridge, 2008). An alternative stimulant prescribed for ADHD is Adderall XR® (ADD), a long-acting mixture of approximately 24% L-amphetamine and 76% D-amphetamine salts (Joyce et al., 2007). There has been a remarkable increase in the number of prescriptions for this drug at the same time as a striking decrease in the number for Ritalin (Swanson et al., 2007). Although the effects of amphetamine on cfos gene expression in adult rodent brain is well-described (e.g., Graybiel et al., 1990; Simpson et al., 1995), little attention has been paid to the possible long-term influence of amphetamine on gene expression in the immature brain, though differences in expression through development were observed in response to acute amphetamine (AMPH) (Snyder-Keller and Keller, 1998; Andersen et al., 2001). These studies indicated that the degree and pattern of expression depends on age and developmental period of the animal. Similar investigations with Adderall, and in particular its extended release form, Adderall XR®, are urgently needed. There are two additional reasons for such a study: (1) the mechanism of action of amphetamine is different to that of MPH; that is, MPH increases synaptic dopamine levels by blocking reuptake of dopamine into nerve terminals, whereas AMPH induces an increase in the secretion of dopamine from both vesicular stores and from nerve terminals (Fleckenstein and Hanson, 2003), and (2) ADD provides increased dopamine release, over a prolonged time course, when compared to amphetamine or MPH alone (Joyce et al., 2007; Schiffer et al., 2006). We reasoned that treatment of immature rats with Adderall XR® would provide a quantitative, and qualitative, difference in gene expression profile when compared to MPH. Thus, one objective of the present work was to determine the influence of ADD on gene expression in immature rat brain. A second goal was to use a clinically relevant method of administering ADD to immature rats to simulate the therapeutic treatment of children and to determine the subsequent serum levels of ADD. A shortcoming of most of the existing experimental work on the neurochemical or behavioral effects of stimulants is the use of high, injected doses of the drugs (Carrey et al., 2009). Also, there is limited information available on the blood levels of amphetamine in immature rats. Diaz Heijtz et al. (2003) reported that a single injection of D,L-amphetamine (0.5 mg/kg s.c.) gave peak plasma levels of 85⫾4 ng/mL at 5–15 min post-injection, with an estimated half-life of 1 h in prepubertal male rats. This is in the same range as blood amphetamine levels seen in children (6 –12 years) following oral boluses of Adderall XR® (10 –30 mg; 29 – 89 ng/mL; McGough et al., 2003; Greenhill et al., 2003; Carrey et al., 2009). In the present study we successfully treated immature, peripubertal, rats with oral ADD and established the

corresponding blood levels of the drug by mass spectrometry. In addition we extended our experiments to include much younger rats. As noted already, children as young as 3 years are now being prescribed psychostimulants (Zito et al., 2000; Mayes et al., 2008; Greenhill et al., 2008). We estimated, based on the work of Andersen (2003), that rats at PD10 should developmentally approximate children aged 3 years. In previous work we established an optimal oral dose of MPH using the localization of MPH-induced gene expression (Chase et al., 2003, 2005a,b). These data indicated that in prepubertal rats the threshold dose of oral MPH required to elicit an acute biological response in the brain is in the range 7.5–10.0 mg/kg. The rationale for the present studies was therefore to: (1) use the localization of FOS immunoreactivity (FOS-ir) as a means to determine an optimal dose of ADD that could be used to investigate the long-term effects of this drug in the immature striatum and cortex; (2) determine the effects of repeated, daily, oral doses of ADD on FOS-ir in striatum and cortex, and (3) use liquid chromatography–mass spectrometry to determine the time course and blood levels of amphetamine enantiomers in serum obtained from infantile (PD10) rats and immature (PD24) rats treated with low doses of ADD.

EXPERIMENTAL PROCEDURES Rats Litters of male Sprague–Dawley rats were shipped with their mothers at PD 15–17, or on PD 4, from Charles River Laboratories (Montreal, QC, Canada). They were housed on a 12:12 hour light:dark cycle (lights on from 0600 h to 1800 h), in plastic cages (28⫻12⫻16 cm3), with free access to food (Lab Diet 5P00, Prolab RMH 3000, PMI Nutrition Int., Brentwood, MO, USA) and reverse osmosis drinking water. The Dalhousie University Committee on Laboratory Animals approved all procedures and protocols for handling of laboratory animals. Rats were weaned on PD21, housed two or three per cage, and were weighed every 2 or 3 days throughout the experiments.

Adderall treatment of prepubertal rats Oral treatment of PD24 rats. To simulate oral medication in children, we previously developed a non-stressful method of administering stimulants orally without the need for restraint or food deprivation (Chase et al., 2007). In brief, prepubertal rats (postnatal day 18 –23) were trained to consume a palatable chocolate drink (Ensure®) from a needle-free syringe presented to them in their home cage (see Fig. 1). Rats quickly (1–2 days) learned to consume the drink and did so within 30 s. To establish an ADD dose-response relationship, using FOS-ir as an endpoint, prepubertal (PD24) male rats were administered a single oral dose (0, 0.4, 1.6, 3.0 and 6.0 mg/kg) of ADD (Adderall XR®; Office of Controlled Substances, Health Canada). The contents of a single capsule were pulverized by hand in a mortar and pestle, in physiological saline (pH 5.5; 3 ml), and then centrifuged (2000 rpm; 15 min) to yield a supernatant that contained the ADD components. This centrifugation step was included because in preliminary experiments we noted that some of the rats found the pulverized capsule contents unpalatable. We therefore elected to use the supernatant, free of the drug carrier, for oral treatment of the rats after mixing it with the chocolate drink (0.1 ml supernatant plus 0.4 ml chocolate drink), or saline (choc-

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(3) ADD (14 days; ADD/ADD); (4) saline (14 days; SAL/SAL), and sacrificed 2 h post-treatment on PD24 (see below).

Immunohistochemistry

Fig. 1. Oral administration of drug. Rats were trained to consume Adderall XR® contained in a palatable chocolate drink presented to them, without restraint, in a needle-free syringe in their home cage. Rats usually drank the chocolate within 30 s.

olate drink only). The precise composition of the oral dose, in terms of D- and L-amphetamine, was determined using liquid chromatography–mass spectrometry (see below). Rats were sacrificed by perfusion/fixation 2 h post-treatment (see below). To investigate the long-term effects of oral ADD in PD24 rats, we chose the lowest dose of oral ADD (1.6 mg/kg) that robustly increased FOS-ir in striatum and that also induced a reproducible behavioural response; that is at this dose an increase in their locomotion and general activity was easily seen. This response was not quantified but was seen reproducibly by two independent observers. Thus prepubertal rats were treated once daily from PD24 –37 with 1.6 mg/kg ADD as follows: (1) ADD and saline (13 days ADD ⫹ saline day 14; ADD/SAL); (2) saline plus ADD (13 days saline ⫹ challenge dose of ADD day 14; SAL/ADD); (3) ADD (14 days; ADD/ADD); (4) saline (14 days; SAL/SAL), and sacrificed 2 h post-treatment on PD37 (see below). In addition we compared the effect of oral ADD on FOS-ir with that of oral MPH. In previous experiments (Chase et al., 2007; Chase, 2005) we demonstrated that the minimum oral dose of MPH required to increase the expression of striatal arc and cfos in young rats was 7.5 mg/kg. Thus we repeated the above experiment and treated PD24 rats for 14 days with oral MPH (7.5 mg/kg; Office of Controlled Substances, Health Canada). Treatment of PD10 rats with ADD. These experiments were designed to replicate the above experiments but using younger (PD10) rats in order to approximate the age at which very young children are first given ADD or Ritalin (Zito et al., 2000; Mayes et al., 2008; Greenhill et al., 2008). We were unsuccessful in training infantile pups to accept oral ADD on a daily basis, and therefore elected to inject ADD s.c. However, unlike the experiments with PD24 rats, it was difficult to observe a behavioural response to the injected ADD because the pups were almost always suckling and became agitated following removal from the mother. We therefore determined the lowest dose of ADD that elicited a robust increase in striatal FOS-ir; that is, we established a FOS-ir dose/ response for injected ADD. For these experiments with PD10 pups we injected the ADD as a fine suspension obtained as described above, but omitting the centrifugation step. In this way we could confidently calculate the dose based on the known capsule contents (30 mg). After determining the optimal dose of ADD, we performed 14-day experiments in the same way as for the older (PD24) pups. PD10 rats were treated once daily from PD10 –23 with injections of ADD (1.25 mg/kg sc): (1) ADD and saline (13 days ADD ⫹ saline day 14; ADD/SAL); (2) saline plus ADD (13 days saline ⫹ challenge dose of ADD day 14; SAL/ADD);

Rats were deeply anaesthetized 2 h following the final drug treatment (Somnotol, 65 mg/kg i.p.) on PD10, 24 or 38, and were perfused intracardially with ice-cold Dulbecco’s phosphate-buffered saline (PBS; GIBCO BRL) followed by 4% paraformaldehyde (PFA; pH 7.4) fixation. Brains were collected, post-fixed in 4% PFA for 6 days at 4 °C, and then cryoprotected in 30% sucrose solution for 4 days at 4 °C. Coronal sections (40 ␮m) were cut through the striatum and processed for FOS-ir. Four serial sections per rat, representing the rostral to caudal extent of the striatum, were chosen according to stereotaxic landmarks (Paxinos and Watson, 1986) and were processed for FOS-ir as follows: Four sections per rat were incubated with a polyclonal cFOS antiserum (Santa Cruz; # SC-52; 1:40,000) in 3% goat serum/ PBS-TritonX 100 (SIGMA; 0.2%; pH 7.4) for 48 h at 4 °C, and were subsequently incubated in biotinylated goat anti-rabbit secondary antibody (Vector; 1:500; in 1% goat serum/PBS-TritonX (0.2%)) for 90 min. Sections were incubated with avidin/biotin reagent (1:250 Vectastain Standard ABC Elite Kit, Vector; in PBS) for 90min, and were finally reacted with diaminobenzidine (Sigma; 10mg/50mls) plus nickel ion enhancement (ammonium nickel sulfate; 300mg/50mls), in the presence of hydrogen peroxide (0.006%) for visualization of the dark blue reaction product. Sections were mounted on gelatin-subbed glass slides and coverslipped using Entellan.

Quantification of FOS-ir Sections (four per rat; left and right hemispheres) immunoreacted for FOS-ir were photographed (63⫻) using a Leitz microscope coupled to a digital Q-Imaging camera (Retiga 1300) which transferred the greyscale images to an Apple G4 computer running Openlab (v. 3.5.1) software for image capture, and NIH Image (v. 1.6) for analysis of FOS staining. Cells that were the darkest black to medium grey were included in the count. These cells represented the population that was visible above background levels. In the striatum we confined our quantification to the dorsal area only, since this was the only place that we saw elevations in FOS-ir at the low doses of ADD used in these experiments. Such a localization following amphetamine challenge has been reported previously (Simpson et al., 1995). The total number of FOS⫹ (positive; stained) cells from four different brain regions: striatum, frontal/cingulate cortex, parietal cortex and piriform cortex (see Fig. 2), in each of the four sections, was combined to give a total “profile” for each region in each animal. Profiles were then averaged across the number of rats used for each treatment group. Immunoreactive profiles for FOS-ir were expressed as a percentage of their corresponding saline control for each experiment. This facilitated comparison across different treatments.

Determination of serum amphetamine levels by liquid chromatography–mass spectrometry (LC–MS) following treatment with ADD Groups of rats (PD24; n⫽5 per time point) were treated with oral ADD (1.6 mg/kg) and killed by decapitation at 10, 20, 30, 60, 120, 240 and 360 min post-treatment. Trunk blood was collected, stored on ice until centrifugation (2000 rpm; 15 min; 4 °C) and the resulting serum then frozen at ⫺20 °C until analyzed for amphetamine content (see below). A similar experiment was performed in PD10 rats injected with ADD (1.25 mg/kg sc), and blood samples obtained at 10, 15, 20, 30, 40, 60, 120 and 190 min post-treatment.

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J. K. Allen et al. / Neuroscience 169 (2010) 1901–1912 Determination of amphetamine concentration. Quantitation of amphetamine in serum samples was performed by integrating the peak area using QuanLynxTM. The internal standard was used to correct for variations between injections and each sample was injected in triplicate. A separate dilution series for D- and L-enantiomers of amphetamine standards was prepared from 0.25 ng/mL to 1000 ng/mL. The resulting linearity curve was used to calculate the level of D- or L-amphetamine in each plasma sample collected at various time points. Amphetamine levels in supernatant obtained from ADD capsules. In the initial oral treatment experiments we utilized a suspension of the contents of Adderall XR capsules (30 mg), that is, the contents of a single capsule were pulverized by hand in a mortar and pestle, in saline (2 ml), and the resulting fine suspension was added to the chocolate drink. With this technique we sometimes encountered a small group of rats that refused to take the drink and we assumed that a component of the pulverized delayed-release beads (Weisler, 2005) was unpalatable. In subsequent studies with PD24 rats we prepared a suspension as described, and then centrifuged it (2000 rpm; 15 min) to yield a supernatant that contained the ADD components. The concentration of D- and L-enantiomers of amphetamine in the supernatant was determined by LC–MS. Suspensions from Adderall capsules were diluted 1000-fold prior to injection and a linearity curve, ranging from 250 ng/mL to 10 ␮g/mL, was used for determination of the concentrations of D- and L-amphetamine.

Fig. 2. Schematic of brain regions selected for densitometric analysis of FOS-ir. Boxes represent approximate area photographed and counted. CC, corpus callosum; FrC, frontal/cingulate cortex; Par, parietal cortex; LV, lateral ventricle; Str, striatum; Pir, piriform cortex. Diagram modified from Plate 13 in Paxinos and Watson (1986).

Solid phase extraction (SPE) of amphetamine from rat serum. Extraction of amphetamine from serum was carried out using Oasis MCX cartridges (30 mg; Waters, Milford, MA, USA). The cartridge was first conditioned with methanol (1 mL; Caledon, Georgetown, ON, Canada) and de-ionized water (2 mL). Prior to loading the sample, each serum sample was spiked with an internal standard, (DL)-amphetamine– d6 (Cerilliant, Round Rock, TX, USA), at a concentration of 2 ␮g/mL. The cartridge was washed with 1 mL of 0.05 M acetic acid (Sigma-Aldrich, St. Louis, MO, USA) and elution of amphetamine was carried out using 1 mL of 5% ammonium hydroxide (Caledon) in methanol. The eluted fraction was then mixed with an equal volume of 0.1% formic acid (EMD, Darmstadt, Germany) for LC–MS analysis. LC–MS analysis. Chiral separation of the enantiomers of amphetamine was achieved on a CYCLOBONDTM I 2000 column (4.6 mm ID⫻150 mm, 5 ␮m, Supelco, Bellefonte, PA, USA) using an isocratic gradient that consisted of ammonium formate (30 mM; pH 4; Sigma-Aldrich): acetonitrile (Caledon; 98:2 v/v) delivered at a flow-rate of 0.5 mL/min. A Waters Quattro PremierTM XE tandem quadrupole mass spectrometer (Q1q2Q3) equipped with an electrospray ionization (ESI) source was used in the positive mode. The MS parameters were as follows: 3 kV capillary voltage; 20 V cone voltage; 120 °C source temperature; 450 °C desolvation temperature; 50 L/h cone gas; 650 L/h desolvation gas; 15 V for both LM1 and HM1 resolutions and a collision energy of 15 V. To selectively detect and quantify the amphetamine and its internal standard ((DL)-amphetamine– d6), acquisitions were made in the multiple reaction monitoring (MRM) mode such that Q1 detects for a specific precursor ion while Q3 for a major fragment ion. Accordingly, two MRM transitions were set up to monitor for amphetamine (m/z 136.1¡91.1) and amphetamine-d6 (m/z 142.1¡93.1), each at a dwell time of 0.5 s. All data were acquired using MassLynxTM version 4.1.

Data analysis Statistical analysis of FOS-ir (profiles), and serum levels of amphetamine enantiomers, was performed using ANOVA followed by Tukey’s post hoc test. Significance was set at P⬍0.05.

RESULTS Adderall-induced FOS-ir: dose-responses in PD24 and PD10 rats The results of the mass-spectrometric determination of amphetamine levels in the supernatants from capsule extraction revealed that the concentration of D-amphetamine was 4.8⫾0.4 mg/mL and of L-amphetamine 1.9⫾0.2 mg/mL (n⫽4). Note that these values are significantly less than the expected amounts contained in the capsules, that is, the capsules of ADD contain 30 mg of a mixture of Dand L-amphetamine salts (approximately 24% L-amphetamine and 76% D-amphetamine; Joyce et al., 2007). The theoretical yield of extracted amphetamine salts should therefore be 2.4 mg/mL of the L-form and 7.6 mg/mL D-form. The reason for the reduced yield following saline extraction is unclear, but may reflect the complex structure of the drug-containing Microtrol® beads that are used in the ADD capsules (Weisler, 2005). The capsules are designed to release pulses of amphetamine from pH-sensitive immediate- and delayed-release beads. Thus our technique of room-temperature extraction of the beads, in pH 5.5 saline, provided an incomplete release of amphetamine salts. Nonetheless the ratio of D-form to L-form in the capsules (approx. 3:1) was essentially maintained at 2.5:1 in the saline extract. With this information in hand we observed that a single oral treatment with ADD (1.6 mg/kg) in PD24 rats reliably increased general activity of the pups and significantly elevated FOS-ir in striatum (Fig. 3A). However,

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Fig. 3. Adderall XR®-induced FOS-ir in immature rat brain. Representative photomicrographs of striatal FOS-IR in prepubertal (PD24; A) rats and in infantile rats (PD10; B) treated with oral ADD (1.6 mg/kg; PD 24) or with injected ADD (1.25 mg/kg sc; PD10). Insets illustrate the lack of effect on FOS-ir of a dose of saline. Scale bar: 100 ␮m.

when we compared the striatal response with those in different brain regions we observed that cells in cingulate and parietal cortex were more responsive to ADD than those in striatum, that is, significant increases in FOS-ir were seen in cortex with oral doses as low as 0.4 mg/kg (Fig. 4B). As noted in Experimental Procedures, we were unsuccessful in training infantile pups (PD10) to accept oral ADD on a daily basis. Most of the pups refused to swallow even a single dose. These experiments were therefore conducted with a subcutaneously injected suspension of ADD, that is, the contents of the ADD capsules were finely pulverized into a slurry in saline, and this was used for the injections. Thus the rats were given doses calculated according to the known content of the capsules (30 mg). As with the experiments on the PD24 pups, we first determined the threshold dose of ADD required to increase FOS-ir in striatum. Fig. 4C indicates that small doses of ADD (0.3 and 0.6 mg/kg) did increase striatal FOS-ir, particularly in the medial striatum, though the levels were very low. However at 1.25 mg/kg there was a large increase in expression and patches of FOS-ir were seen throughout the striatum (Fig. 3B). Note that there is a marked difference in the distribution of striatal FOS-ir in the two ages of rats (PD10 vs. PD24; Fig. 3A, B). The patchy appearance in the younger pups, and its disappearance by PD24, is identical to that shown previously reported by Snyder-Keller and Keller (1998). This dose of 1.25 mg/kg was chosen as the threshold dose to examine possible ADD-induced down-regulation (see below). Unlike the effects seen in PD24 rats, there was no effect of ADD (0.3 and 0.6 mg/kg) in the parietal cortex until a dose of 1.25 mg/kg (⫹210%; P⬍0.001) (data not shown). A similar response was seen in frontal/cingulate cortex (P⬍0.01 @ 1.5 mg/kg; ⫹260%) and in piriform cortex (P⬍0.01 @ 1.25 mg/kg; ⫹270%) (not shown).

Effect of repeated treatment of PD24 pups with oral ADD on FOS-ir in striatum, and comparison with oral MPH We observed no effect of ADD treatment (14 days) on the normal gain in body weight in these immature rats. In a typical experiment control rats weighed 161.8⫾2.3 g (n⫽12) and the treated rats weighed 164.2⫾2.4 g (n⫽12) at the end of the treatment period. The response of cfos to a challenge dose of oral ADD (1.6 mg/kg) was significantly reduced in the dorsal striatum following repeated daily treatments with oral ADD (1.6 mg/kg; Fig. 5A). Although the response to the challenge was attenuated (⫺74%; P⬍0.0001; SAL/ADD vs. ADD/ADD), striatal cells still retained some response to stimulation with oral ADD (i.e., SAL/SAL vs. ADD/ADD; fourfold increase; P⬍0.05). Note also that 24 h after the final treatment with oral ADD, striatal FOS-ir remained slightly but significantly higher than in saline treated controls (SAL/SAL vs. ADD/SAL; P⬍0.05). Note also that the percentage increase in FOS-ir in the SAL/ADD group (approx 1350%) is strikingly less than that seen in the dose-response experiment (Fig. 4A) where the increase was fivefold higher. In marked contrast to the effects of oral ADD, repeated higher doses of oral MPH (7.5 mg/kg) did not down-regulate striatal cfos expression (Fig. 5B). Reductions in FOS-ir were also recorded in cerebral cortex (Fig. 6). Moderate but significant reductions were seen in cingulate cortex (Fig. 6C, D; ⫺40%; P⬍0.05) and in piriform cortex (Fig. 6E, F; ⫺27%; P⬍0.05) whereas a large inhibition was noted in parietal cortex (Fig. 6A, B; ⫺81%; P⬍0.0001). The reductions in FOS-ir levels in cingulate and piriform cortices occurred uniformly across the thickness of the cortex, whereas in parietal cortex (Fig. 6A, B) cfos expression was attenuated largely in layer IV (see LaHoste et al., 1996).

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Fig. 4. Dose-response relationships for Adderall XR®-induced FOS-ir in immature rat brain: Densitometric analysis of the effects of low doses of ADD on: (A) striatal FOS-ir in prepubertal (PD24) rats given oral ADD; and (B) striatal FOS-ir in infantile (PD10) rats injected with ADD; (C) FOS-ir in several brain regions in PD24 rats given low dose oral ADD. Graphs shows mean number of FOS⫹ nuclear profiles (⫾ SEM) per treatment group. Values are expressed as a percentage of values in the saline group (n⫽3– 4 per group). * P⬍0.05; ** P⬍0.01; P⬍0.001 versus saline groups.

Effect of repeated treatment of PD10 pups with injected ADD There was no effect on body weight of 14 days of treatment with ADD. In a typical experiment control rats weighed 63.1⫾1.9 g (n⫽10) compared to treated rats: 62.4⫾1.1 g (n⫽11). Fig. 7 shows that daily injections of a low, threshold dose of ADD (1.25 mg/kg sc; 14 days) significantly reduced striatal FOS-ir when these pups were subsequently challenged with the same dose of ADD (⫺74%; SAL/ADD vs. ADD/ADD; P⬍0.0001). Nevertheless, striatal cells previously exposed to ADD (13 days) still responded

to ADD with a small increase in FOS-ir (SAL/SAL vs. ADD/ADD; P⬍0.05). However, unlike our observations in older rats treated with oral ADD (see above), we saw no accumulation of FOS-ir 24 h after the final injection of ADD (i.e., ADD/SAL rats). In contrast to the effects of repeated ADD on striatal cells, down-regulation of FOS-ir in cerebral cortex was only seen in the cingulate, but not in parietal or piriform, cortex. Thus a single injection of ADD (1.25 mg/kg sc), following 13 daily injections of saline, significantly increased FOS-ir in cingulate cortex (⫹ 302⫾31%; P⬍0.01). However, in

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trast, a single injection of ADD suspension gave a much faster response in infantile pups (PD10; 1.25 mg/kg). Damphetamine levels peaked at 10 min post-treatment (Fig. 8B; 170.0⫾15.8 ng/mL) but remained elevated (35.2⫾3.5 ng/mL) until at least 190 min post-treatment. The L-enantiomer reached peak levels at 10 min (46.4⫾2.8 ng/mL) and these fell to 9.4⫾0.7 ng/mL by 190 min post-injection.

DISCUSSION Oral Adderall-induced cfos expression in prepubertal (PD24) rats

Fig. 5. Oral Adderall XR®-induced down-regulation of cfos expression in prepubertal rats; comparison with oral MPH. Densitometric analysis of chronic oral ADD-induced (A), or MPH-induced (B), FOS-ir in prepubertal rat striatum. Rats were treated daily with one dose of drug (PD24 –PD37). Bars represent mean (⫾ SEM) numbers of FOS⫹ nuclear profiles per treatment group, expressed as a percentage of values in the saline group (n⫽4 – 6 per group). (A) Graph shows significant down-regulation of FOS-ir following repeated treatment with oral ADD (1.6 mg/kg). *** P⬍0.001 versus saline (SAL/SAL vs. SAL/ ADD); *** P⬍0.001 versus an acute dose (SAL/ADD vs. ADD/ADD). (B) Graph shows chronic treatment with oral MPH (7.5 mg/kg) does not down-regulate striatal FOS-ir; * P⬍0.05 versus saline.

rats previously exposed to 13 injections of ADD, the response to a final injection of ADD was reduced to 195⫾21% (P⬍0.05; ADD/ADD vs. SAL/ADD). No reduction in FOS-ir was seen in parietal and piriform cortices. Serum levels of D- and L-amphetamine Quantification of serum levels of the D- and L-forms of amphetamine in PD24 rats revealed that oral ADD (1.6 mg/kg) yielded peak values of D-AMPH (212.3⫾27.9 ng/ mL) by 60 min post-treatment, with an approximate half-life of 120 min; the L-enantiomer also reached a peak at 60 min (40.6⫾5.5 ng/mL), with a similar half-life (Fig. 8A). In con-

Our data suggest that the immature rat brain is especially sensitive to stimulation with low doses of amphetamine; a low dose of oral ADD (0.4 mg/kg) significantly increased FOS-ir, especially in parietal and cingulate cortex of prepubertal rats (PD24), in the absence of a significant change in striatal gene expression. It seems possible that the effects of low dose oral Adderall XR® in children with ADHD could conceivably be mediated, at least partially, via a non-striatal mechanism, or through a process that does not involve an increase in cfos expression in striatum. Berridge et al. (2006) demonstrated that low doses of oral MPH (2.0 mg/kg), devoid of behavioural effects, preferentially increased dopamine and norepinephrine outflow in rat prefrontal cortex (PFC) when compared to nucleus accumbens. Much lower doses of MPH (0.5 mg/kg ip) enhanced working memory and increased PFC neuronal responsiveness (Devilbiss and Berridge, 2008). Our present data suggest that ADD may also have preferential effects in the cortex. The important question of what constitutes a low dose of amphetamine in experimental rats was carefully reviewed by Grilly and Loveland (2001). Their conclusion was that the low dose range was 0.1– 0.4 mg/kg (s.c. or i.p.). It is significant, therefore, that in further work from Berridge and Stalnaker (2002), low doses of injected amphetamine (0.15 and 0.25 mg/kg sc) provoked wakefulness and also increased dopamine and norepinephrine efflux from PFC. Our results, using an oral dose of 0.4 mg/kg, are consistent with these data. Chronic low dose oral ADD, but not MPH, downregulates cfos expression Our demonstration that repeated treatment with low doses of Adderall XR® down-regulated cfos expression in the immature rat striatum and cortex has not previously been described, though a similar phenomenon is known to occur in adult rats treated with repeated doses of amphetamine (Renthal et al., 2008). However in contrast to our present work, the earlier studies used much larger, injected doses of amphetamine (4 –10 mg/kg; Renthal et al., 2008; Graybiel et al., 1990; Simpson et al., 1995) to investigate cfos expression. A higher oral dose of MPH (7.5 mg/kg; 14 days) did not downregulate striatal cfos expression. This is in keeping with the work of Russell et al. (1998) who demonstrated that D-amphetamine was 7–17 times more potent than MPH in releasing dopamine from slices of rat cortex and striatum. Similar results were obtained by Schiffer et al.

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Fig. 6. Oral Adderall XR®-induced down-regulation of cfos expression in cerebral cortex of prepubertal rats. Representative photomicrographs of cortical FOS-ir in rats treated daily with either vehicle or oral ADD (1.6 mg/kg; PD24 –PD37). The vehicle-treated rats received a single dose of ADD on PD37. (A), (C), and (E) illustrate the response of cfos to a single stimulation with ADD on PD37 in parietal (A), cingulate (C) and piriform (E) cortex. (B), (D) and (F) show reduced FOS-ir following 13 days of oral ADD, followed by a single oral challenge dose (1.6 mg/kg). Scale bar: 100 ␮m.

(2006) who measured a fourfold increase in extracellular dopamine in freely moving rats, when comparing the effects of amphetamine and MPH. As noted earlier, amphetamine and MPH exert contrasting effects at the nerve terminal (Fleckenstein and Hanson, 2003) and this is likely due to their different influence on the vesicular monoamine transporter, VMAT2 (Riddle et al., 2007). In addition amphetamine and MPH exhibit different affinities for other amine transporters, that is, they have comparable affinities

at the dopamine and norepinephrine transporters (approx. 0.1 ␮M) but differ by approximately three to fivefold at the serotonin transporter (20 –30 ␮M vs. 100 ␮M; AMPH vs. MPH). This indicates a possible serotonin-mediated component of low-dose amphetamine action that may be absent following MPH treatment. The increased effectiveness of repeated low-dose ADD in reducing striatal FOS-ir levels may also be due to the combination of amphetamine salts that constitute Adderall XR®. Joyce et al. (2007)

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levels of amphetamine, emphasize that our data are relevant to the possible adverse effects of extended ADD treatment in children. There is surprisingly little information available on blood levels of amphetamine in children or in young rats, particularly following oral intake of drug (Carrey et al., 2009). For example, children and adolescents— variously treated with oral AMPH, mixed amphetamine salts or ADD— had blood levels of D-AMPH in the range of

Fig. 7. Effect of repeated injections of Adderall XR® on FOS-ir in infantile rats. Densitometric analysis of chronic ADD-induced FOS-ir in infantile rat striatum. Rats were treated daily (PD10 –PD23) with a single dose of ADD (1.25 mg/kg sc). Bars represent mean (⫾ SEM; n⫽4 – 6 per group) numbers of FOS⫹ nuclear profiles per treatment group, expressed as a percentage of values in the saline group. Graph shows significant down-regulation of FOS-ir following repeated treatment with injected ADD (1.25 mg/kg). *** P⬍0.0001 versus saline (SAL/SAL vs. SAL/ADD); *** P⬍0.0001 versus an acute dose (SAL/ ADD vs. ADD/ADD).

showed that ADD, microinjected into adult rat striatum, increased dopamine secretion over a prolonged timecourse when compared to D- and to D, L-amphetamine. It remains unclear why this particular ratio of amphetamine salts, present in the Adderall mixture, should have such a prolonged effect, although there is evidence that L-amphetamine, like D-amphetamine, may have stimulant effects and has been used to treat ADHD in children (Arnold et al., 1976). Down-regulation of cfos expression in infantile (PD10) rats Our lack of success in training infantile (PD10) pups to accept oral ADD was unexpected. In an attempt to circumvent this we also used the method described by Wheeler et al. (2007), but even with this technique our pups resolutely refused to drink the chocolate or the ADD-spiked chocolate. We concluded that these experiments may require a longer training period. Nevertheless a repeated low dose of injected ADD, close to the threshold dose required for cfos activation, and sufficient to provide therapeutic blood levels of D-AMPH, significantly attenuated the increase in FOS-ir in striatum and cingulate cortex normally seen after a single challenge injection of ADD. This result is notable for at least two reasons: first, because the pronounced effects on gene expression were induced with serum levels of amphetamine comparable to the therapeutic range reported in children; and second because the clearance of amphetamine is much faster in the rat than in children (see below). Blood levels of amphetamine in immature rats Our efforts to use low, minimally-effective, doses of ADD in our experiments, together with the determination of blood

Fig. 8. Serum concentrations of amphetamine enantiomers in Adderall XR®-treated rat pups. Groups of rat pups were treated with either a single oral dose of ADD (A, 1.6 mg/kg; PD24; n⫽5 per group) or a single s.c. injection of ADD (B, 1.25 mg/kg sc; PD10; n⫽5 per group) and killed by decapitation at various times post-injection. Blood samples were collected, serum obtained via centrifugation and frozen at –20 °C, and then analyzed by LC–MS to provide levels of D- and L-amphetamine. Values are means⫾SEM.

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18 –140 ng/mL (Brown et al., 1980; Greenhill et al., 2003; McGough et al., 2003; Kramer et al., 2005; Borcherding et al., 1989). A study on prepubertal rats (PD25), injected with a low dose of D, L-AMPH (0.5 mg/kg sc), reported a maximum blood level of 85 ng/mL, with an approximate half-life of 60 min (Diaz Heijtz et al., 2003). Our results indicate that a low, oral dose of ADD (1.6 mg/kg) given to prepubertal rats, produced peak serum levels of D-AMPH at 60 min (212.3⫾27.9 ng/mL) that remained within the clinical range for up to 240 min (38.6⫾13.9 ng/mL). These are slightly higher than blood levels seen in children, but the shape of the time/concentration curve is similar to that for D-AMPH levels in children treated with oral ADD (McGough et al., 2003). Note however that it is of much shorter duration (i.e., 3 h vs. ⬎20 h in children) and indicates that AMPH clearance in the rat is much faster than in children. Nonetheless, a comparison of the area under the serum concentration-time curves revealed that the value for PD24 rats (585 h⫻ng/mL) was within the range reported for children (333– 667 h⫻ng/mL; Kramer et al., 2005). Injection of an ADD suspension in infantile pups (PD10; 1.25 mg/kg) gave a peak D-amphetamine level at 10 min post-injection (170.0⫾15.8 ng/mL) but D-AMPH remained elevated (35.2⫾3.5 ng/mL) until at least 190 min post-treatment. Thus in PD10 pups, although we saw a significant down-regulation of cfos expression in striatum, the area under the curve (215 h⫻ng/mL) was less than that seen in children. Implications for enduring behavioural effects of early stimulant medication Controversy surrounds the concern that stimulant treatment of children might predispose them to later substance abuse (for a detailed review see Berman et al., 2008). Thus some, but not all, studies indicate that early stimulant treatment for ADHD might reduce the risk for subsequent substance abuse. Nevertheless, as pointed out by Berman et al (2008), “. . . no controlled studies have explored adverse behavioral . . . . . . . consequences of years, much less decades, of chronic amphetamine treatment”. They also point out that even though stimulant medication of children with ADHD might reduce the frequency of later substance abuse, there are no data on the influence of initiating stimulant treatment in adolescence, or later. We have previously reviewed this issue with respect to experiments in rodents (Carrey et al., 2009) and concluded that the degree of brain maturation at the time of initial stimulant treatment may dictate subsequent behavioural actions of psychostimulants such as cocaine. As noted elsewhere in the present paper, much of the animal literature devoted to the effects of stimulants on brain maturation are compromised by the high doses of MPH and amphetamine used in these investigations. In our experiments treatment of prepubertal and infantile rats with low doses of ADD was able to reproduce blood levels of D-AMPH close to those previously seen in children. Thus it should now be possible to investigate the long-term influence of such treatment on subsequent behavioural outcomes in adults.

CONCLUSION Our data indicate that it is possible to use infantile and prepubertal rats, given clinically relevant doses of both MPH and AMPH, as experimental animal models in ADHD. Moreover we suggest that even a relatively brief exposure of the immature rat brain to clinically-relevant blood levels of D-AMPH is sufficient to cause down-regulation of cfos expression. Whether this is a general phenomenon applicable to other genes remains unknown, but our data imply that such changes may possibly occur in the immature human brain. Complementary information on blood levels of AMPH obtained from adults has also been published, and overall the data parallel those reported for children, with values of D-AMPH between 30 and 106 ng/mL (Carrey et al., 2009; Tulloch et al., 2002; Auiler et al., 2002; Clausen et al., 2005; Ermer et al., 2007). A significant additional study on adult non-human primates reported that low oral doses of an amphetamine mixture (0.12–1.00 mg/kg), that approximated ADD, gave blood levels that varied from 60 –200 ng/ml over a period of 4 weeks (Ricaurte et al., 2005). This treatment appeared to be neurotoxic and striatal levels of dopamine, dopamine transporters (DAT) and VMAT2 (vesicular monoamine transporter 2) were all attenuated. Taken together with our own data, these results suggest that the possible adverse neural effects of Adderall, especially in its extended release form, should be investigated in young animals. In contrast to this view however, Diaz Heijtz et al. (2003) demonstrated that low doses of amphetamine (0.5 mg/kg sc; ⫻2 per day; PD22–34) induced increases in dendritic length and branches of prefrontal cortical neurons. These authors suggested that this effect constituted a trophic influence of AMPH on the immature brain. In a related report, a much higher dose of amphetamine also increased dendritic length in neurons of the ventral tegmental area (2.0 mg/kg sc; PD10,12 and 14; Mueller et al., 2006). Thus AMPH, even at low doses, exerts structural changes in brain tissue from young rats. These data raise interesting questions: (a) what is the connection, if any, between ADD-induced down-regulation of cfos and the structural changes in neurons reported by Diaz Heijtz et al. (2003)?; (b) would a longer exposure to ADD (weeks) induce neurotoxic effects in young rat pups?; (c) is the ADD-induced down-regulation of cfos expression reversible? Answers to these questions might be forthcoming with a clearer understanding of the functional nature of the observed downregulation of cfos expression. For example, the lack of response of cfos to a challenge dose of ADD, following 13 days of treatment with ADD, might be because the dopamine receptors normally responsive to ADD are somehow desensitized, or downregulated. Amphetamine is known to produce a downregulation of D1/D2 stimulated adenylate cyclase activity, though this usually occurs following high doses (2.5 and 7.5 mg/kg; Roberts-Lewis et al., 1986; Roseboom and Gnegy, 1989). However microarray studies indicated that D1 and D2 gene expression was unaltered by MPH in young rats (2 mg/kg ip; Adriani et al., 2006; Carrey et al., 2009), suggesting that downregulation may not occur in

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young rats given low doses of MPH. The effects of ADD need to be examined. A recent report describes a more direct downregulation of cfos expression via an amphetamine-induced ⌬FosB-mediated epigenetic desensitization (Renthal et al., 2008). It is relevant that we have detected an oral ADD-induced increase in histone phosphorylation in striatum of prepubertal rats (Carrey et al., 2009), as well as an increase in fosB expression in young MPH-treated rats (Chase et al., 2005a,b). These observations appear to be consistent with the data reported by Renthal et al. (2008). Nonetheless, whether a reduction in cfos expression is deleterious to the developing brain remains to be determined, though evidence exists that cfos may be protective against methamphetamine toxicity (Deng et al., 1999). Our efforts to calibrate biological responses, such as immediate early gene expression, to clinically relevant blood levels of stimulants confirmed that expression of cfos is very sensitive to repeated low doses of Adderall XR®. It will be instructive to repeat these experiments with a view to determining whether low oral doses of ADD might also modify dendritic structure. The implications of such studies should be relevant to the putative effects of psychostimulant treatment of very young children. Acknowledgments—Financial support for these studies was obtained from the IWK Health Centre, NSHRF, CIHR, the Atlee Endowment and NRC-Marine Biosciences. The authors are indebted to D.A. Wilkinson and P.M.H. Wilkinson for their invaluable technical assistance.

REFERENCES Adriani W, Leo D, Greco D, Rea M, di Porzio U, Laviola G, PerroneCapano C (2006) Methylphenidate administration to adolescent rats determines plastic changes on reward-related behavior and striatal gene expression. Neuropsychopharmacology 31:1946 –1956. Andersen SL (2003) Trajectories of brain development: point of vulnerability or window of opportunity? Neurosci Biobehav Rev 27:3–18. Andersen SL (2005) Stimulants and the developing brain. Trends Pharmacol Sci 26:237–243. Andersen SL, LeBlanc CJ, Lyss PJ (2001) Maturational increases in c-fos expression in the ascending dopamine systems. Synapse 41:345–350. Arnold LE, Huestis RD, Smeltzer DJ, Scheib J, Wemmer D, Colner G (1976) Levoamphetamine vs dextroamphetamine in minimal brain dysfunction. Replication, time response, and differential effect by diagnostic group and family rating. Arch Gen Psychiatry 33: 292–301. Auiler JF, Liu K, Lynch JM, Gelotte CK (2002) Effect of food on early drug exposure from extended-release stimulants: results from the Concerta, Adderall XR Food Evaluation (CAFE) Study. Curr Med Res Opin 18:311–316. Banerjee PS, Aston J, Khundakar AA, Zetterstrom TS (2009) Differential regulation of psychostimulant-induced gene expression of brain derived neurotrophic factor and the immediate-early gene Arc in the juvenile and adult brain. Eur J Neurosci 29:465– 476. Berman SM, Kuczenski R, McCracken JT, London ED (2008) Potential adverse effects of amphetamine treatment on brain and behavior: a review. Mol Psychiatry 14:123–142. Berridge CW, Stalnaker TA (2002) Relationship between low-dose amphetamine-induced arousal and extracellular norepinephrine

1911

and dopamine levels within prefrontal cortex. Synapse 46:140 – 149. Berridge CW, Devilbiss DM, Andrzejewski ME, Arnsten AF, Kelley AE, Schmeichel B, Hamilton C, Spencer RC (2006) Methylphenidate preferentially increases catecholamine neurotransmission within the prefrontal cortex at low doses that enhance cognitive function. Biol Psychiatry 60:1111–1120. Borcherding BG, Keysor CS, Cooper TB, Rapoport JL (1989) Differential effects of methylphenidate and dextroamphetamine on the motor activity level of hyperactive children. Neuropsychopharmacology 2:255–263. Brown GL, Ebert MH, Mikkelsen EJ, Hunt RD (1980) Behavior and motor activity response in hyperactive children and plasma amphetamine levels following a sustained release preparation. J Am Acad Child Psychiatry 19:225–239. Carlezon WA Jr, Konradi C (2004) Understanding the neurobiological consequences of early exposure to psychotropic drugs: linking behavior with molecules. Neuropharmacology 47(Suppl 1):47– 60. Carrey N, Chase T, Allen J, Wilkinson M (2009) Psychostimulantinduced developmental neuroadaptation: implications for the treatment of ADHD. In: Attention deficit hyperactivity disorder (ADHD). New York, NY: Nova Science Publishers. Chase T (2005) PhD Dissertation, Dalhousie University, Halifax, Nova Scotia, Canada. Chase T, Carrey N, Soo E, Wilkinson M (2007) Methylphenidate regulates activity regulated cytoskeletal associated but not brainderived neurotrophic factor gene expression in the developing rat striatum. Neuroscience 144:969 –984. Chase TD, Brown RE, Carrey N, Wilkinson M (2003) Repeated methylphenidate attenuates cfos expression in the striatum of prepubertal rats. Neuroreport 14:769 –772. Chase TD, Carrey N, Brown RE, Wilkinson M (2005a) Methylphenidate regulates c-fos and fosB expression in multiple regions of the immature brain. Brain Res Dev Brain Res 156:1–12. Chase TD, Carrey N, Brown RE, Wilkinson M (2005b) Methylphenidate differentially regulates c-fos and fosB expression in the developing rat striatum. Brain Res Dev Brain Res 157:181–191. Clausen SB, Read SC, Tulloch SJ (2005) Single- and multiple-dose pharmacokinetics of an oral mixed amphetamine salts extendedrelease formulation in adults. CNS Spectr 10:6 –15. Deng X, Ladenheim B, Tsao LI, Cadet JL (1999) Null mutation of c-fos causes exacerbation of methamphetamine-induced neurotoxicity. J Neurosci 19:10107–10115. Devilbiss DM, Berridge CW (2008) Cognition-enhancing doses of methylphenidate preferentially increase prefrontal cortex neuronal responsiveness. Biol Psychiatry 64:626 – 635. Diaz Heijtz R, Kolb B, Forssberg H (2003) Can a therapeutic dose of amphetamine during pre-adolescence modify the pattern of synaptic organization in the brain? Eur J Neurosci 18:3394 –3399. Ermer JC, Shojaei A, Pennick M, Anderson CS, Silverberg A, Youcha SH (2007) Bioavailability of triple-bead mixed amphetamine salts compared with a dose-augmentation strategy of mixed amphetamine salts extended release plus mixed amphetamine salts immediate release. Curr Med Res Opin 23:1067–1075. Fleckenstein AE, Hanson GR (2003) Impact of psychostimulants on vesicular monoamine transporter function. Eur J Pharmacol 479: 283–289. Graybiel AM, Moratall R, Robertson HA (1990) Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum. Proc Natl Acad Sci U S A 87:6912– 6916. Greenhill LL, Posner K, Vaughan BS, Kratochvil CJ (2008) Attention deficit hyperactivity disorder in preschool children. Child Adolesc Psychiatr Clin N Am 17:347–366. Greenhill LL, Swanson JM, Steinhoff K, Fried J, Posner K, Lerner M, Wigal S, Clausen SB, Zhang Y, Tulloch S (2003) A pharmacokinetic/pharmacodynamic study comparing a single morning dose of

1912

J. K. Allen et al. / Neuroscience 169 (2010) 1901–1912

adderall to twice-daily dosing in children with ADHD. J Am Acad Child Adolesc Psychiatry 42:1234 –1241. Grilly DM, Loveland A (2001) What is a “low dose” of d-amphetamine for inducing behavioral effects in laboratory rats? Psychopharmacology (Berl) 153:155–169. Hawken CM, Brown RE, Carrey N, Wilkinson M (2004) Long-term methylphenidate treatment down-regulates c-fos in the striatum of male CD-1 mice. Neuroreport 15:1045–1048. Hughes P, Dragunow M (1995) Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmacol Rev 47:133–178. Joyce BM, Glaser PE, Gerhardt GA (2007) Adderall produces increased striatal dopamine release and a prolonged time course compared to amphetamine isomers. Psychopharmacology (Berl) 191:669 – 677. Kim Y, Teylan MA, Baron M, Sands A, Nairn AC, Greengard P (2009) Methylphenidate-induced dendritic spine formation and DeltaFosB expression in nucleus accumbens. Proc Natl Acad Sci U S A 106:2915–2920. Kramer WG, Read SC, Tran BV, Zhang Y, Tulloch SJ (2005) Pharmacokinetics of mixed amphetamine salts extended release in adolescents with ADHD. CNS Spectr 10:6 –13. LaHoste GJ, Ruskin DN, Marshall JF (1996) Cerebrocortical Fos expression following dopaminergic stimulation: D1/D2 synergism and its breakdown. Brain Res 728: 97–104. Mayes R, Bagwell C, Erkulwater J (2008) ADHD and the rise in stimulant use among children. Harv Rev Psychiatry 16:151–166. McGough JJ, Biederman J, Greenhill LL, McCracken JT, Spencer TJ, Posner K, Wigal S, Gornbein J, Tulloch S, Swanson JM (2003) Pharmacokinetics of SLI381 (ADDERALL XR), an extended-release formulation of Adderall. J Am Acad Child Adolesc Psychiatry 42:684 – 691. Mueller D, Chapman CA, Stewart J (2006) Amphetamine induces dendritic growth in ventral tegmental area dopaminergic neurons in vivo via basic fibroblast growth factor. Neuroscience 137:727–735. Paxinos G, Watson C (1986) The Rat Brain in stereotaxic coordinates. New York, NY: Academic Press. Penner MR, McFadyen MP, Pinaud R, Carrey N, Robertson HA, Brown RE (2002) Age-related distribution of c-fos expression in the striatum of CD-1 mice after acute methylphenidate administration. Brain Res Dev Brain Res 135:71–77. Renthal W, Carle TL, Maze I, Covington HE, Truong HT, Alibhai I, Kumar A, Montgomery RL, Olson EL, Nestler EJ (2008) ⌬FosB mediates epigenetic desensitization of the c-fos gene after chronic amphetamine exposure. J Neurosci 28:7344 –7349. Ricaurte GA, Mechan AO, Yuan J, Hatzidimitriou G, Xie T, Mayne AH, McCann UD (2005) Amphetamine treatment similar to that used in the treatment of adult attention-deficit/hyperactivity disorder damages dopaminergic nerve endings in the striatum of adult nonhuman primates. J Pharmacol Exp Ther 315:91–98. Rice D, Barone S Jr (2000) Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect 108(Suppl 3):511–533. Riddle EL, Hanson GR, Fleckenstein AE (2007) Therapeutic doses of amphetamine and methylphenidate selectively redistribute the vesicular monoamine transporter-2. Eur J Pharmacol 57:25–28. Roberts-Lewis JM, Roseboom PH, Iwaniec LM, Gnegy ME (1986) Differential downregulation of D1-stimulated adenylate cyclase ac-

tivity in rat forebrain after in vivo amphetamine treatments. J Neurosci 6:2245–2251. Robinson TE, Kolb B (2004) Structural plasticity associated with exposure to drugs of abuse. Neuropharmacology 47(Suppl 1):33– 46. Roseboom PH, Gnegy ME (1989) Acute in vivo amphetamine produces a homologous desensitization of dopamine receptor-coupled adenylate cyclase activities and decreases agonist binding to the D1 site. Mol Pharmacol 35:139 –147. Russell V, de Villiers A, Sagvolden T, Lamm M, Taljaard J (1998) Differences between electrically-, ritalin- and D-amphetaminestimulated release of [3H]dopamine from brain slices suggest impaired vesicular storage of dopamine in an animal model of Attention-Deficit Hyperactivity Disorder. Behav Brain Res 94:163–171. Schiffer WK, Volkow ND, Fowler JS, Alexoff DL, Logan J, Dewey SL (2006) Therapeutic doses of amphetamine or methylphenidate differentially increase synaptic and extracellular dopamine. Synapse 59:243–251. Simpson JN, Wang JQ, McGinty JF (1995) Repeated amphetamine administration induces a prolonged augmentation of phosphorylated cyclase response element-binding protein and Fos-related antigen immunoreactivity in rat striatum. Neuroscience 69:441– 457. Snyder-Keller A, Keller RW Jr (1998) Stimulant-mediated c-fos induction in striatum as a function of age, sex, and prenatal cocaine exposure. Brain Res 794:88 –95. Sowell ER, Peterson BS, Thompson PM, Welcome SE, Henkenius AL, Toga AW (2003) Mapping cortical change across the human life span. Nat Neurosci 6:309 –315. Stanwood GD, Levitt P (2004) Drug exposure early in life: functional repercussions of changing neuropharmacology during sensitive periods of brain development. Curr Opin Pharmacol 4:65–71. Swanson JM, Kinsbourne M, Nigg J, Lanphear B, Stefanatos GA, Volkow N, Taylor E, Casey BJ, Castellanos FX, Wadhwa PD (2007) Etiologic subtypes of attention-deficit/hyperactivity disorder: brain imaging, molecular genetic and environmental factors and the dopamine hypothesis. Neuropsychol Rev 17:39 –59. Teicher MH, Ito Y, Glod CA, Barber NI (1996) Objective measurement of hyperactivity and attentional problems in ADHD. J Am Acad Child Adolesc Psychiatry 35:334 –342. Tulloch SJ, Zhang Y, McLean A, Wolf KN (2002) SLI381 (Adderall XR), a two-component, extended-release formulation of mixed amphetamine salts: bioavailability of three test formulations and comparison of fasted, fed, and sprinkled administration. Pharmacotherapy 22:1405–1415. Weisler RH (2005) Safety, efficacy and extended duration of action of mixed amphetamine salts extended-release capsules for the treatment of ADHD. Expert Opin Pharmacother 6:1003–1018. Wheeler TL, Eppolito AK, Smith LN, Huff TB, Smith RF (2007) A novel method for oral stimulant administration in the neonate rat and similar species. J Neurosci Methods 159:282–285. Yano M, Steiner H (2007) Methylphenidate and cocaine: the same effects on gene regulation? Trends Pharmacol Sci 28:588 – 596. Zito JM, Safer DJ, dosReis S, Gardner JF, Boles M, Lynch F (2000) Trends in the prescribing of psychotropic medications to preschoolers. JAMA 283:1025–1030.

(Accepted 12 June 2010) (Available online 19 June 2010)