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The effect of chronic methylphenidate administration on presynaptic dopaminergic parameters in a rat model for ADHD
European Neuropsychopharmacology (2010) 20, 714–720
www.elsevier.com/locate/euroneuro
The effect of chronic methylphenidate administration on presynaptic dopaminergic parameters in a rat model for ADHD Y. Simchon a , A. Weizman a,b,c , M. Rehavi a,d,⁎ a
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Israel Research Unit, Geha Mental Health Center, Petah-Tikva, Israel c Research Unit, Felsenstein Medical Research Center, Petah-Tikva, Israel d The Dr. Miriam and Sheldon G. Adelson Chair in the Biology of Addictive Diseases, Tel-Aviv University, Israel b
Received 28 January 2010; received in revised form 14 April 2010; accepted 17 April 2010
KEYWORDS Attention deficit hyperactivity disorder (ADHD); Spontaneous hypertensive rats (SHR); Wistar Kyoto rats (WKY); Dopamine transporter (DAT); Vesicular monoamine transporter 2 (VMAT2); Methylphenidate (MPH)
Abstract Dysregulations in monoaminergic systems have been implicated in attention deficit/hyperactivity disorder. The spontaneous hypertensive rats (SHR) are used as an animal model for ADHD. Juvenile SHR rats exhibited low dopamine transporter (DAT) density, low vesicular monoamine transporter 2 (VMAT2) density and lower unstimulated dopamine (DA) release in comparison to their corresponding WKY controls. Chronic methylphenidate treatment of the young SHR rats was associated with lower DAT density and lower unstimulated basal dopamine release but with enhanced potassium- and amphetamine-induced dopamine release. These neurochemical alterations might be relevant to the pathophysiology and to the beneficial effect of methylphenidate in ADHD. © 2010 Elsevier B.V. and ECNP. All rights reserved.
1. Introduction Attention deficit/hyperactivity disorder (ADHD) is a neurobehavioral disorder most common in youth. A recent report has indicated that the prevalence of ADHD in school aged children is 6–8% worldwide (Biederman, 2005).
⁎ Corresponding author. Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel. Tel.: +972 3 6408759; fax: +972 3 6409113. E-mail address: [email protected] (M. Rehavi).
Even though the disorder was introduced over 100 years ago, its neurobiology is still not completely understood. Fronto-subcortical pathways (lateral prefrontal cortex, dorsal anterior cingulate cortex, caudate and putamen), which are rich in catecholamines, were suggested to play a major role in this disorder. These amines, especially dopamine (DA) and norepinephrine (NE), are involved in the mechanism of action of the stimulant medications that are used to treat the disorder (Biederman, 2005; Curatolo et al., 2009). This led to the hypothesis of catecholamine dysfunction in ADHD (Pliszka, 2005); inefficient function of the posterior cortical attentional system is suggested to be
0924-977X/$ - see front matter © 2010 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2010.04.007
The effect of chronic MPH administration on presynaptic dopaminergic parameters for ADHD due to dysregulation of the norepinephrine system, while the impaired function of the anterior executive system is related to dopamine dysregulation (Curatolo et al., 2009). Goto et al. (2007) hypothesized that the tonic DA release is reduced in ADHD. As a consequence, the phasic dopamine release is increased. This excessive phasic release overstimulates reward centers, which leads to excessive sensation seeking behavior (Pliszka, 2005). A strong validity to this hypothesis, or a weak validity to the dopamine deficiency hypothesis, is the fact that drugs with dopaminergic effect are ineffective for treating ADHD. Drugs for the treatment of ADHD selectively potentiate dopaminergic and noradrenergic neurotransmission in the brain, especially in the striatum. Stimulant drugs, such as methylphenidate, raise extracellular dopamine levels supposedly by blockade of the dopamine transporter (DAT). This mechanism prevents reuptake of dopamine into the neuron, which results in higher extracellular dopamine levels (Madras et al., 2005; Volz et al., 2005). Additionally, according to Volz and Fleckenstein (Fleckenstein et al., 2009; Volz et al., 2008), acute administration of methylphenidate redistributes vesicular monoamine transporter 2 (VMAT2) protein from membrane associated vesicles fraction to the cytoplasmic vesicles fraction, which results in an increase in dopamine content in both fractions. Imaging studies using fMRI showed reduced striatal activation in ADHD children in comparison to controls. These differences disappeared after methylphenidate treatment (Wilens, 2008). Methylphenidate treatment was associated with improved spatial working memory and response inhibition in the prefrontal cortex in ADHD children and adults (Arnsten, 2006). It was shown that age at the time of drug treatment and pharmacokinetic differences in absorption, distribution and metabolism could influence both the acute and chronic effect of psychostimulants (Yang et al., 2006). As ADHD is highly heterogeneous disorder, both genetically and environmentally, using animal models can simplify and promote the understanding of this disorder. The spontaneously hypertensive rats (SHR) strain was developed in Japan by selective inbreeding of rats from the Wistar Kyoto strain (WKY) that exhibited high systolic blood pressure. During the selective inbreeding, the new strain was more active in comparison to its progenitor, the normotensive WKY strain (Kantak et al., 2008). The SHR strain fulfills the validation criteria which make it an adequate animal model for ADHD; face validity — SHRs mimic the behavioral characteristics of ADHD described in children. Impulsiveness, which is absent initially and develop gradually over time, sustained attention a deficit that is demonstrated only when stimuli are widely spaced in time, hyperactivity, which is not observed in a novel non-treating environment and develop over time. Construct, validity — the two main behavioral processes that are proposed to be major contributory factors in the etiology of ADHD, altered reinforcement of novel behavior and deficient extinction of previously reinforced behavior, are demonstrated. These processes are associated with hypofunction of the dopamine system. Predictive validity — predictions are currently focused on the behavioral level, although the usefulness of the model might become apparent in areas like genetics, neurobiology and pharmacology (Sagvolden et al., 2005). A dilemma concerning the usage of SHR rats revolves around their development of hypertension. It is unclear whether the neurocognitive deficits are result of hypertension. Since the hypertension develops around weeks 12–14 and is not present
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in young hyperactive rats, it is accepted that the neurocognitive alterations appear prior to the hypertension (Kantak et al., 2008; Russell, 2002; Sagvolden et al., 2005). Studies on methylphenidate and its neurochemical effects have been done before, but the majority of them were conducted in juvenile or adult rats without ADHD-like symptoms. In the first part of this study, we assessed the differences in dopaminergic parameters between juvenile SHR and their corresponding controls, juvenile WKY rats. Secondly, we examined the effect of chronic methylphenidate treatment on the same dopaminergic parameters in juvenile SHR rats.
2. Materials and methods 2.1. Animals Male Wistar Kyoto rats (WKY strain) and spontaneous hypertensive rats (SHR strain) at the age of 4 weeks (80–100 g) were purchased from Harlan laboratories. The rats were housed 4 per cage at 22± 2 °C and a 12 light:12 dark cycle (lights on at 05:00 h) with unlimited access to commercial pellet food and tap water.Animal procedures were approved by the Animal Care Committees of Tel-Aviv University (approval numbers: M-08-087 and M-09-034).
2.2. Materials [3H]GBR12935 (specific activity: 38.5 Ci/mmol) and [3H]dopamine (specific activity: 55.1 Ci/mmol) were purchased from Perkin-Elmer (Boston, MA, USA). [3H]TBZOH (specific activity: 20 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Mazindol was purchased from Sandoz Pharmaceuticals (New Jersey, USA). TBZOH was purchased from Fluka (Buch, Switzerland). D-Amphetamine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methylphenidate hydrochloride (Ritalin®) was synthesized by Novartis Pharma AG (Basel, Switzerland). All other chemicals were of highest purity obtainable through regular commercial sources.
2.3. Methylphenidate treatment protocol Two weeks old male SHR rats were treated (i.p.) every morning (around 10 AM) for 21 days with 3 mg/kg methylphenidate (Russell et al., 2000) or saline. On the last day, 2 h after administration, the rats were sacrificed (decapitation by guillotine) and their brains were dissected on ice.
2.4. [3H]GBR12935 binding to rat brain membranes [3H]GBR12935 is a specific antagonist of the dopamine transporter and its binding was carried out as described previously (Janowsky et al., 1986). Rat brain striatal tissue was dissected over ice and placed into a chilled Potter–Elvehjem glass homogenizing vessel containing 10 volumes of ice cold 0.32 M sucrose. The tissue was homogenized 8 times with a teflon pestle and centrifuged for 10 min at 1000g. The supernatant was centrifuged for 15 min at 26,890g. The resulting pellet was resuspended in 40 volumes of 50 mM Tris HCl (pH 7.4) containing 120 mM NaCl, using Brinkman Polytron. Using the same buffer containing 0.01% BSA, the tissue was diluted 1:5. The incubation mixture contained 100 μl striatal membranes, 50 μl [3H] GBR12935 (0.5–8 nM) and 350 μl buffer (50 mM Tris HCl, pH 7.4, containing 120 mM NaCl and 0.01% BSA). Following a 45-minute incubation period (at 25 °C), the incubate was quickly diluted in 3 ml ice cold buffer and filtered under vacuum through glass fiber filters (Whatman GF/C). The filters were washed 4 times with 3 ml ice cold buffer and counted in a liquid scintillation (Opti flour Packard) using
716 a β counter (Packard 2100 TR). The specific binding was defined as the difference between the total [3H]GBR12935 binding and the nonspecific binding, measured using excess of unlabeled antagonist (50 μM mazindol). Scatchard analyses were used to calculate the transporter density (Bmax) and the ligand affinity to the transporter (Kd).
2.5. [3H]Dopamine uptake to rat brain synaptosomes The [3H]dopamine uptake was performed as described before (Gordon et al., 1996; Coyle and Snyder, 1969). Forebrains (whole brain minus brain stem and cerebellum) of male rats were homogenized using Potter–Elvehjem homogenizer (8 strokes) in 10 volumes of ice cold 0.32 M sucrose using teflon pestle. Homogenates were centrifuged at 1000g for 10 min and the resulting supernatant containing the synaptosomal membranes was used for the uptake studies. A standard assay contained: 50 μl homogenate, 900 μl Krebs Ringer phosphate buffer (119 mM NaCl, 3.9 mM KCl, 0.65 mM MgSO4, 0.51 mM CaCl2, 0.19 mM phosphate buffer pH 7.4, 11.1 mM glucose, 1.13 mM ascorbic acid, 0.16 mM EDTA, and 0.1 mM pargyline). The buffer solution was prepared fresh daily and equilibrated with 95% O2–5% CO2 for 10 min prior to use. The tubes were preincubated at 37 °C for 10 min. Thereafter 50 μl of [3H]dopamine (1 × 10− 8–5 × 10− 7 M) was added and the incubation was continued for another 4 min. The reaction was stopped by rapidly cooling the tubes on ice. Non-specific uptake was measured by incubating identically prepared tubes at 0–4 °C. The incubate was filtered under vacuum through glass fiber filters (Whatman GF/C). The filters were washed 4 times with 3 ml of ice cold buffer and counted. Specific uptake per minute was defined as the difference between the total uptake and non-specific uptake divided by 4 min. Km and Vmax values were obtained from Lineweaver Burk charts.
2.6. [3H]TBZOH binding to rat brain membranes [3H]TBZOH is a specific antagonist of the vesicular monoamine transporter 2 and its binding was carried out as described previously (Scherman, 1986). Brain striatal tissue was homogenized in 10 volumes of 0.32 M sucrose in a Potter–Elvehjem homogenizer (8 strokes), using a teflon pestle. Homogenate was centrifuged at 1000g for 10 min. The supernatant was diluted 1:5 in HEPES buffer 20 mM, pH 8.0 containing 300 mM sucrose, and centrifuged 10 min at 26,890g. The pellet was resuspended in 50 volumes of Hepes sucrose buffer and centrifuged 10 min at 26,890g. Finally, the pellet was disrupted with Brinkman polytron and resuspended in HEPES sucrose buffer to yield a homogenate concentration of 20 mg/ml. The assay included 150 μl buffer, 50 μl membranes and 50 μl of various concentrations of [3H]TBZOH (0.5–8 nM). Non-specific binding was determined using excess unlabeled TBZOH (10 μM). After 30 min of incubation at 25 °C, the mixture was filtered with vacuum on glass fiber filters (Whatman GF/C). The filters were washed 4 times with ice cold 50 mM Tris buffer, pH 8, and the radioactivity was counted as mentioned before. Scatchard analysis was used to assess the density of the binding sites (Bmax) and the ligand affinity to VMAT2 (Kd).
Y. Simchon et al. the slices were washed for another 40 min. Aliquots of the tissue were added to baskets with a polypropylene mesh bottom (100 μm pore size) and placed in a warm bubbled buffer for another 20 min prior to the release assay. For the release assay, baskets were transferred at 1 min intervals through a series of vials containing bubbled buffer (37 °C). For stimulated release, the baskets were transferred to vials containing 25 mM K+, 1 μM amphetamine or 50 μM methylphenidate. At the end of the transfers, the baskets were placed in 1 M NaOH overnight to solubilize the tissue. On the following day, 2 M acetic acid was added to neutralize the solution. Aliquots of 0.5 ml were taken to scintillation vials, and the radioactivity was determined as mentioned before. The percent of [3H]dopamine overflow was calculated as the amount of radioactivity released into the medium during a given period, divided by the sum of radioactivity released in that time and in subsequent periods plus the radioactivity remaining in the slices.
2.8. Protein assays Protein concentration was determined by Lowry et al. (1951) and Bradford (1976) methods.
2.9. Statistical analysis The independent samples t-test, using SPSS software (SPSS Inc. Chicago, Illinois), was used as appropriate. Results represent individual rat tissues and are expressed as means ± S.D.
3. Results 3.1. Dopaminergic parameters in SHR vs. WKY 3.1.1. [3H]GBR12935 binding The density of [3H]GBR12935 binding sites was significantly lower in the striatum of SHR rats in comparison to WKY rats (915 ±87 vs. 1120± 91 fmol/mg protein; t = 3.8, df= 9, p = 0.004) (Fig. 1). There was no difference between the two strains in the affinity of [3H] GBR12935 to its binding site in the striatal membranes (2.8 ±0.7 vs. 2.8 ± 0.6 nM; t = −0.13, df = 9, p = 0.89).
2.7. [3H]TBZOH binding to rat brain membranes The [3H]dopamine release was performed as described by Gordon et al. (1995). Striatal tissue was dissected on ice and was cut in two perpendicular directions using a razor blade. The slices were washed with Krebs Ringer buffer in 37 °C, containing 124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 1.2 mM CaCl2, 10 mM glucose, 5.68 mM ascorbic acid, and 10 μM pargyline, bubbled continuously with 95% O2 (containing 5% CO2), which gave pH of 7.4. The tissue was washed in the buffer with continuous bubbling for 40 min, buffer changes every 10 min. The slices were then incubated with 5 × 10− 8 M [3H]dopamine for 30 min. After the uptake period,
Figure 1 Brain striatal dopamine transporter density in juvenile SHR rats (n = 5) compared to juvenile WKY rats (n = 6). [3H]GBR12935 binding (0.5–8 nM) was measured in brain striatal membranes of 4 week old rats. Bmax values were evaluated using Scatchard analysis. Non-specific binding was determined in the presence of 50 μM mazindol. Results are expressed as means ± S.D. ***p b 0.005 vs. WKY.
The effect of chronic MPH administration on presynaptic dopaminergic parameters for ADHD
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3.1.2. [3H]Dopamine uptake No difference was observed between the two strains in the maximal [3H]dopamine uptake rate (Vmax) to brain synaptosomes or in the Km values (6940± 1080 vs. 6940± 1680 DPM/min mg protein; t = 0.001, df = 9, p = 0.99; 1.01 × 10− 7 ± 0.23 vs. 1.41 × 10− 7 ± 0.65 M; t = 1.29, df= 9, p = 0.22, respectively).
3.1.3. [3H]TBZOH binding The density of [3H]TBZOH binding sites in striatal membranes of SHR rats was significantly lower in comparison to the WKY controls (820 ± 89 vs. 1040 ± 130 fmol/mg protein; t = 3.37, df = 10, p = 0.007) (Fig. 2). A modest but significant difference was found in the affinity of [3H]TBZOH to the binding site, in SHR vs. WKY (0.96 ± 0.15 vs. 1.23± 0.11 nM; t = 3.135, df= 8, p = 0.014) but most Kd values are in the low nanomolar range.
3.1.4. [3H]Dopamine release The basal release of [3H]dopamine was significantly lower in striatal slices of SHR strain in comparison to the WKY strain (73 ± 13 vs. 106 ± 14 mm2; t = 4.2, df = 10, p = 0.002). Stimulated [3H] dopamine release evoked by high potassium, amphetamine or methylphenidate did not reveal significant differences between the two strains (54 ± 8 vs. 56 ± 6; t = 0.463, df = 10, p = 0.65; 57 ± 14 vs. 55 ± 12; t = −0.22, df = 7, p = 0.82 and 31 ± 4 vs. 34 ± 7 mm2; t = 0.84, df = 8, p = 0.42, respectively) (Fig. 3).
3.2. The effect of chronic methylphenidate treatment on dopaminergic parameters in juvenile SHR rats 3.2.1. [3H]GBR12935 binding The methylphenidate treatment induced a significant decrease in the density of [3H]GBR12935 binding sites (805 ± 240 vs. 1020 ± 102 fmol/mg protein; t = 2.198, df= 12, p = 0.048) (Fig. 4). The treatment had no effect on the affinity of [3H]GBR12935 to the
Figure 3 Basal and stimulated [3H]dopamine release from striatal slices of juvenile SHR rats (n = 6) as compared to juvenile WKY rats (n = 6). Brain striatal slices of 4 week old rat were loaded with [3H]dopamine at 37°c for 30 min. Release was assessed by calculating the area under the curve. Stimulated [3H]dopamine release was induced by 25 mM K+, 1 μM amphetamine or 50 μM methylphenidate. Results are expressed as means ± S.D. ***p b 0.005 vs. WKY.
striatal membranes (1.73 ± 0.27 vs. 1.93 ± 0.38 nM; t = 1.11, df= 12, p = 0.28).
3.2.2. [3H]Dopamine uptake The methylphenidate treatment did not affect [3H]dopamine uptake into striatal synaptosomes (6160± 1030 vs. 5750 ±810 DPM/ min mg protein; t=−0.83, df =12, p =0.42) or the Km values (7.71 × 10− 8 ± 0.71 vs. 7.41 × 10− 8 ± 1.4 M; t=−0.48, df = 12, p=0.63).
3.2.3. [3H]TBZOH binding Chronic treatment with methylphenidate did not affect the density of [3H]TBZOH binding sites (832 ± 110 vs. 851 ± 109 fmol/ mg protein; t = 0.34, df = 14, p = 0.73). The chronic treatment had
Figure 2 Brain vesicular monoamine transporter 2 density in juvenile SHR rats (n = 6) compared to juvenile WKY rats (n = 6). Vesicular monoamine transporter 2 density was determined using [3H]TBZOH binding (0.5–8nM) that was measured in brain striatal membranes of 4 week old rats. Bmax values were evaluated using Scatchard analysis. Non-specific binding was determined in the presence of 10 μM TBZOH. Results are expressed as means ± S.D. **p b 0.01 vs. WKY.
Figure 4 Brain striatal dopamine transporter density in juvenile (2 week old) SHR rats (n = 7) treated for 21 days with methylphenidate (3 mg/kg, i.p.). [3H]GBR12935 binding (0.5–8 nM) was measured in rat brain striatal membranes. Results are expressed as means ± S.D. *p b 0.05 vs. untreated SHR rats (n = 7).
718 no effect on the affinity of [3H]TBZOH to the striatal membranes (0.93 ± 0.11 vs. 0.99 ± 0.17 nM; t = 0.80, df= 14, p = 0.43).
3.2.4. [3H]Dopamine release The methylphenidate treatment induced a significant reduction in the basal [3H]dopamine release from striatal slices (54 ± 9 vs. 72 ± 4 mm2; t = 3.98, df = 8, p = 0.004). Potassium-induced [3H]dopamine release was increased by 29% after 21 days of methylphenidate treatment (55±9 vs. 43± 10 mm2; t=−2.25, df =10, p= 0.048). Amphetamine-induced [3H]dopamine release was also enhanced (29%) by the chronic methylphenidate treatment (77±4 vs. 59±10 mm2; t=−3.72, df=8, p= 0.006) (Fig. 5).
4. Discussion In this study, we used spontaneous hypertensive rats (SHR) as an animal model for ADHD. We characterized several key synaptic dopaminergic parameters; dopamine transporter level, dopamine uptake, vesicular monoamine transporter 2 level and dopamine release in brains of SHR rats in comparison to their controls, normotensive Wistar Kyoto rats (WKY). The dopaminergic parameters in SHR rats were also studied before and after chronic methylphenidate administration. We found that the density of the striatal DAT was lower in juvenile SHR rats (4 weeks old) in comparison to juvenile WKY rats. Previous studies reported both increased and decreased levels of DAT in SHR in comparison to WKY rats. Leo et al. (2003) studied the DAT mRNA levels at different post-natal days. They found that the mRNA levels were significantly lower in SHR rats at 27–49 post-natal days (which is equivalent to 4–7 week old rats) in comparison to WKY rats. In contrast to Leo et al. findings, using autoradiography with the ligand [125I]β-CIT, Watanabe et al. (1997) found higher level of binding sites in the caudate– putamen of SHR in comparison to WKY rats. They evaluated
Figure 5 Basal and stimulated [3H]dopamine release from striatal slices of juvenile (2 weeks old) SHR rats treated for 21 days with methylphenidate (3 mg/kg, i.p.). Rat brain striatal slices were loaded with [3H]dopamine at 37°c for 30 min. Release was assessed by calculating the area under the curve. Stimulated [3H]dopamine release was induced by 25 mM K+ or 1 μM amphetamine. Results are expressed as means ± S.D. *p b 0.05, **p b 0.01, ***p b 0.005 vs. untreated SHR rats. (n = 5, n = 6, n = 5 vs. n = 5, n = 6, n = 5 respectively).
Y. Simchon et al. the density in 2 week old rats, and they also reported that the higher density was kept during maturity to adult SHR rats (15 weeks old) in comparison to the adult WKY rats. In our study, no statistical difference could be detected in the Vmax and Km values of [3H]dopamine uptake between the two strains. In contrast, Leo et al. described a significant decrease in striatal [3H]dopamine uptake in SHR rats in comparison to WKY rats. They suggested that the difference could be related to reduced DAT density, or variation in the affinity of dopamine to the transporter (Leo et al., 2003). It has been suggested (Goto et al., 2007) that the higher dopamine phasic release in ADHD patients is due to the lower dopamine basal tonic release. In accordance with this hypothesis, we found that the basal dopamine release from striatal slices of juvenile SHR rats was lower than the release from juvenile WKY striatal slices. Our results in SHR juvenile rats are also in agreement with another study (Russell et al., 1995) that reported on lower release of dopamine in striatal slices of adult SHR rats in comparison to adult WKY rats. According to Russell et al., the reduced dopamine release is due to overexpression of dopamine inhibitory presynaptic autoreceptors. They found that high potassium concentration induced less dopamine release from striatal slices of adult SHR rats in comparison to WKY rats while the amphetamine-induced dopamine release was higher in striatal slices of SHR rats in comparison to WKY rats. In our study, we observed a non-significant reduction in dopamine release induced by high potassium concentration and increased dopamine release induced by amphetamine in striatal slices of juvenile SHR rats. It is of note that high potassium induces release from vesicular stores while amphetamine induces release from cytoplasmic stores. As the stimulated dopamine release from vesicular stores (high potassium concentration induced) was reduced, while the dopamine release from cytoplasmic stores (amphetamine induced) was enhanced, it is possible that impaired vesicular storage capability is accompanied by leakage of dopamine from the vesicles to the cytoplasm (Russell et al., 1998; Russell, 2002; Russell et al., 2000). It is known that the synaptic dopamine transmission depends on several components. We suggest that the lower expression of DAT in the striatum of the juvenile SHR rats is due to a compensatory mechanism aimed to overcome the decreased striatal tonic release of dopamine leading to reduced dopamine concentration in the synaptic cleft. This assumption is supported by our finding that dopamine uptake via DAT is similar between the strains. The lack in change in DA uptake may indicate that sufficient amount of DA transporters is available to cope with the changes in synaptic DA concentrations despite the changes in DA release and DAT expression. Alternatively, it is possible that alterations in the presynaptic DA autoreceptors are responsible for the lack of change in DA uptake between the two strains. Unfortunately, in the present study we did not assess the DA autoreceptors. The role of VMAT2 is to protect monoamines, including DA, from mitochondrial MAO oxidation in the cytoplasm by transporting the monoamines from the cytoplasm into the synaptic vesicles. This process is responsible for maintaining high concentration of these monoamines in the presynaptic neuron and enabling further exocytosis. There are no reports on the expression of VMAT2 in juvenile SHR or WKY rats.
The effect of chronic MPH administration on presynaptic dopaminergic parameters for ADHD We found that the density of striatal VMAT2 in juvenile WKY rats was higher than in juvenile SHR rats, although the ligand affinity to VMAT2 was modestly higher in juvenile SHR rats. These findings correlate with our previous results; as the dopamine tonic release is lower in the striatum of young SHR rats, less dopamine is taken into the neuron and the need for VMAT2 to protect the dopamine from oxidation is attenuated. Thus it appears that there is a parallel decrease in both; the DAT and VMAT2 densities. In the second part of our research, we studied the effect of chronic methylphenidate treatment on the dopaminergic parameters in juvenile SHR rats. We expected that after 21 days of methylphenidate administration to the 2 week old SHR rats, their dopaminergic parameters will be similar to the dopaminergic parameters of their corresponding controls, WKY rats. In contrast to our expectations, the density of the dopamine transporter in the striatum of young SHR rats decreased after the chronic treatment. Yet, our results are in accordance with the radiolabeled imaging studies in brains of ADHD adults and children treated with methylphenidate (Wilens, 2008). In the latter human research a significant decrease in the DAT binding signal was demonstrated. As the time of the imaging analysis relative to the drug administration in not completely defined, it is not clear whether the low imaging signal is related to the occupation of DAT by methylphenidate (Wilens, 2008). In our hands, the density of the striatal DAT in young SHR rats decreased following the chronic methylphenidate treatment. Moll et al. (2001) showed a decline in the density of striatal DAT after 14 days of methylphenidate administration in juvenile Wistar rats. According to Moll et al., maturation of the rats is accompanied by a decrease in DAT density, regardless of the methylphenidate treatment. Since methylphenidate treatment induced a decrease in DAT density, he suggested that the treatment causes an additional decrease in the DAT density. Our data indicates that after 21 days of chronic methylphenidate administration, the basal dopamine release from SHR striatal brain slices was significantly lower in comparison to the release of the saline treated SHR rats. We mentioned previously that the tonic release in SHR rats is lower in comparison to that of WKY rats. It is possible that methylphenidate blockade of the DAT leads to excess dopamine presence in the synaptic cleft and to the activation of inhibitory presynaptic autoreceptors. Such putative activation might lead to a further reduction of tonic DA release (Seeman and Madras, 1998). The lower tonic release means that more dopamine stays in the neuron (cytoplasmic and vesicular stores). Induced release, either by high potassium concentration or by amphetamine, empties the elevated dopamine stores, which results in a higher amount of dopamine release. It is possible that methylphenidate promotes DAT density downregulation, as a compensatory mechanism to the reduced dopamine release (Madras et al., 2005; Moll et al., 2001). The density of VMAT2, as assessed by high affinity [3H] TBZOH binding, did not change after the chronic methylphenidate treatment. A preliminary study in our laboratory (data not shown) failed to demonstrate an effect of chronic methylphenidate treatment on striatal VMAT2 density in adult Sprague Dawley rats. Using different methods, Sandoval et al., (2002) described an increase in [3H]TBZOH binding to VMAT2 as well as an increase in vesicular [3H]
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dopamine uptake after acute methylphenidate administration (40 mg/kg) to adult Sprague Dawley rats. These authors suggested the existence of two types of synaptic vesicles: membrane associated fraction and cytoplasmic fraction. According to these investigators, the VMAT2 protein redistributes from the membrane associated fraction to the cytoplasmic fraction as a response to methylphenidate treatment leading to an increase in VMAT2 density and an increase in vesicular dopamine uptake (Volz et al., 2008; Volz et al., 2007). It is possible that if we would have separated the vesicles into these fractions, we would have also seen an increased density of VMAT2 in the cytoplasmic fraction due to such putative redistribution. The fact that the VMAT2 density remained unaltered, in spite of the chronic methylphenidate treatment, can actually strengthen the redistribution hypothesis. Namely, there is no new synthesis of VMAT2 but the transporter redistributes. Methylphenidate treatment is currently the drug of choice for treating ADHD children and adolescence. Chronic methylphenidate treatment of the young SHR rats was associated with lower DAT density and lower unstimulated basal dopamine release but with enhanced potassium- and amphetamine- induced dopamine releases. These dopaminergic changes might be relevant to the beneficial effect of the methylphenidate treatment in ADHD.
Role of the funding source There is no involvement of the funding source.
Contributors Yaarit Simchon performed the entire research as part of her MSc thesis. She participated in designing the experiments, treating the rats, performing all of the biochemical assays. Abraham Weizman participated in writing the final draft and the statistics. Moshe Rehavi designed the project and was Yaarit Simchon mentor in the project, he wrote the first draft. All authors contributed to and have approved the final manuscript.
Conflict of interests There are no conflicts of interest.
Acknowledgment The study was supported by a grant from the Dr. Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Tel-Aviv University, Israel.
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720 Coyle, J.T., Snyder, S.H., 1969. Catecholamine uptake by synaptosomes in homogenates of rat brain: stereospecificity in different areas. J Pharmacol Exp Ther 170 (2), 221–231. Curatolo, P., Paloscia, C., D'Agati, E., Moavero, R., Pasini, A., 2009. The neurobiology of attention deficit/hyperactivity disorder. Eur J Paediatr Neurol 13 (4), 299–304. Fleckenstein, A.E., Volz, T.J., Hanson, G.R., 2009. Psychostimulantinduced alterations in vesicular monoamine transporter-2 function: neurotoxic and therapeutic implications. Neuropharmacology 56 (Suppl 1), 133–138. Gordon, I., Weizman, R., Rosenne, E., Rehavi, M., 1995. Developmental and age-related alterations in rat brain presynaptic dopaminergic mechanisms. Brain Res Dev Brain Res 85 (2), 225–228. Gordon, I., Weizman, R., Rehavi, M., 1996. Modulatory effect of agents active in the presynaptic dopaminergic system on the striatal dopamine transporter. Eur J Pharmacol 298 (1), 27–30. Goto, Y., Otani, S., Grace, A.A., 2007. The Yin and Yang of dopamine release: a new perspective. Neuropharmacology 53 (5), 583–587. Janowsky, A., Berger, P., Vocci, F., Labarca, R., Skolnick, P., Paul, S.M., 1986. Characterization of sodium-dependent [3H]GBR12935 binding in brain: a radioligand for selective labelling of the dopamine transport complex. J Neurochem 46 (4), 1272–1276. Kantak, K.M., Singh, T., Kerstetter, K.A., Dembro, K.A., Mutebi, M.M., Harvey, R.C., et al., 2008. Advancing the spontaneous hypertensive rat model of attention deficit/hyperactivity disorder. Behav Neurosci 122 (2), 340–357. Leo, D., Sorrentino, E., Volpicelli, F., Eyman, M., Greco, D., Viggiano, D., et al., 2003. Altered midbrain dopaminergic neurotransmission during development in an animal model of ADHD. Neurosci Biobehav Rev 27 (7), 661–669. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193 (1), 265–275. Madras, B.K., Miller, G.M., Fischman, A.J., 2005. The dopamine transporter and attention-deficit/hyperactivity disorder. Biol Psychiatry 57 (11), 1397–1409. Moll, G.H., Hause, S., Ruther, E., Rothenberger, A., Huether, G., 2001. Early methylphenidate administration to young rats causes a persistent reduction in the density of striatal dopamine transporters. J Child Adolesc Psychopharmacol 11 (1), 15–24. Pliszka, S.R., 2005. The neuropsychopharmacology of attentiondeficit/hyperactivity disorder. Biol Psychiatry 57 (11), 1385–1390. Russell, V., de Villiers, A., Sagvolden, T., Lamm, M., Taljaard, J., 1995. Altered dopaminergic function in the prefrontal cortex, nucleus accumbens and caudate–putamen of an animal model of attention-deficit hyperactivity disorder — the spontaneously hypertensive rat. Brain Res 676 (2), 343–351.
Y. Simchon et al. Russell, V., de Villiers, A., Sagvolden, T., Lamm, M., Taljaard, J., 1998. Differences between electrically-, ritalin- and D-amphetamine-stimulated 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 (1), 163–171. Russell, V.A., 2002. Hypodopaminergic and hypernoradrenergic activity in prefrontal cortex slices of an animal model for attention-deficit hyperactivity disorder — the spontaneously hypertensive rat. Behav Brain Res 130 (1–2), 191–196. Russell, V.A., de Villiers, A.S., Sagvolden, T., Lamm, M.C., Taljaard, J.J., 2000. Methylphenidate affects striatal dopamine differently in an animal model for attention-deficit/hyperactivity disorder — the spontaneously hypertensive rat. Brain Res Bull 53 (2), 187–192. Sagvolden, T., Russell, V.A., Aase, H., Johansen, E.B., Farshbaf, M., 2005. Rodent models of attention-deficit/hyperactivity disorder. Biol Psychiatry 57 (11), 1239–1247. Sandoval, V., Riddle, E.L., Hanson, G.R., Fleckenstein, A.E., 2002. Methylphenidate redistributes vesicular monoamine transporter-2: role of dopamine receptors. J Neurosci 22 (19), 8705–8710. Scherman, D., 1986. Dihydrotetrabenazine binding and monoamine uptake in mouse brain regions. J Neurochem 47 (2), 331–339. Seeman, P., Madras, B.K., 1998. Anti-hyperactivity medication: methylphenidate and amphetamine. Mol Psychiatry 3 (5), 386–396. Volz, T.J., Bjorklund, N.L., Schenk, J.O., 2005. Methylphenidate analogs with behavioral differences interact differently with arginine residues on the dopamine transporter in rat striatum. Synapse 57 (3), 175–178. Volz, T.J., Farnsworth, S.J., Hanson, G.R., Fleckenstein, A.E., 2008. Methylphenidate-induced alterations in synaptic vesicle trafficking and activity. Ann N Y Acad Sci 1139, 285–290. Volz, T.J., Farnsworth, S.J., King, J.L., Riddle, E.L., Hanson, G.R., Fleckenstein, A.E., 2007. Methylphenidate administration alters vesicular monoamine transporter-2 function in cytoplasmic and membrane-associated vesicles. J Pharmacol Exp Ther 323 (2), 738–745. Watanabe, Y., Fujita, M., Ito, Y., Okada, T., Kusuoka, H., Nishimura, T., 1997. Brain dopamine transporter in spontaneously hypertensive rats. J Nucl Med 38 (3), 470–474. Wilens, T.E., 2008. Effects of methylphenidate on the catecholaminergic system in attention-deficit/hyperactivity disorder. J Clin Psychopharmacol 28 (3 Suppl 2), S46–53. Yang, P.B., Swann, A.C., Dafny, N., 2006. Acute and chronic methylphenidate dose–response assessment on three adolescent male rat strains. Brain Res Bull 71 (1–3), 301–310.
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The effect of chronic methylphenidate administration on presynaptic dopaminergic parameters in a rat model for ADHD Y. Simchon a , A. Weizman a,b,c , M. Rehavi a,d,⁎ a
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Israel Research Unit, Geha Mental Health Center, Petah-Tikva, Israel c Research Unit, Felsenstein Medical Research Center, Petah-Tikva, Israel d The Dr. Miriam and Sheldon G. Adelson Chair in the Biology of Addictive Diseases, Tel-Aviv University, Israel b
Received 28 January 2010; received in revised form 14 April 2010; accepted 17 April 2010
KEYWORDS Attention deficit hyperactivity disorder (ADHD); Spontaneous hypertensive rats (SHR); Wistar Kyoto rats (WKY); Dopamine transporter (DAT); Vesicular monoamine transporter 2 (VMAT2); Methylphenidate (MPH)
Abstract Dysregulations in monoaminergic systems have been implicated in attention deficit/hyperactivity disorder. The spontaneous hypertensive rats (SHR) are used as an animal model for ADHD. Juvenile SHR rats exhibited low dopamine transporter (DAT) density, low vesicular monoamine transporter 2 (VMAT2) density and lower unstimulated dopamine (DA) release in comparison to their corresponding WKY controls. Chronic methylphenidate treatment of the young SHR rats was associated with lower DAT density and lower unstimulated basal dopamine release but with enhanced potassium- and amphetamine-induced dopamine release. These neurochemical alterations might be relevant to the pathophysiology and to the beneficial effect of methylphenidate in ADHD. © 2010 Elsevier B.V. and ECNP. All rights reserved.
1. Introduction Attention deficit/hyperactivity disorder (ADHD) is a neurobehavioral disorder most common in youth. A recent report has indicated that the prevalence of ADHD in school aged children is 6–8% worldwide (Biederman, 2005).
⁎ Corresponding author. Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel. Tel.: +972 3 6408759; fax: +972 3 6409113. E-mail address: [email protected] (M. Rehavi).
Even though the disorder was introduced over 100 years ago, its neurobiology is still not completely understood. Fronto-subcortical pathways (lateral prefrontal cortex, dorsal anterior cingulate cortex, caudate and putamen), which are rich in catecholamines, were suggested to play a major role in this disorder. These amines, especially dopamine (DA) and norepinephrine (NE), are involved in the mechanism of action of the stimulant medications that are used to treat the disorder (Biederman, 2005; Curatolo et al., 2009). This led to the hypothesis of catecholamine dysfunction in ADHD (Pliszka, 2005); inefficient function of the posterior cortical attentional system is suggested to be
0924-977X/$ - see front matter © 2010 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2010.04.007
The effect of chronic MPH administration on presynaptic dopaminergic parameters for ADHD due to dysregulation of the norepinephrine system, while the impaired function of the anterior executive system is related to dopamine dysregulation (Curatolo et al., 2009). Goto et al. (2007) hypothesized that the tonic DA release is reduced in ADHD. As a consequence, the phasic dopamine release is increased. This excessive phasic release overstimulates reward centers, which leads to excessive sensation seeking behavior (Pliszka, 2005). A strong validity to this hypothesis, or a weak validity to the dopamine deficiency hypothesis, is the fact that drugs with dopaminergic effect are ineffective for treating ADHD. Drugs for the treatment of ADHD selectively potentiate dopaminergic and noradrenergic neurotransmission in the brain, especially in the striatum. Stimulant drugs, such as methylphenidate, raise extracellular dopamine levels supposedly by blockade of the dopamine transporter (DAT). This mechanism prevents reuptake of dopamine into the neuron, which results in higher extracellular dopamine levels (Madras et al., 2005; Volz et al., 2005). Additionally, according to Volz and Fleckenstein (Fleckenstein et al., 2009; Volz et al., 2008), acute administration of methylphenidate redistributes vesicular monoamine transporter 2 (VMAT2) protein from membrane associated vesicles fraction to the cytoplasmic vesicles fraction, which results in an increase in dopamine content in both fractions. Imaging studies using fMRI showed reduced striatal activation in ADHD children in comparison to controls. These differences disappeared after methylphenidate treatment (Wilens, 2008). Methylphenidate treatment was associated with improved spatial working memory and response inhibition in the prefrontal cortex in ADHD children and adults (Arnsten, 2006). It was shown that age at the time of drug treatment and pharmacokinetic differences in absorption, distribution and metabolism could influence both the acute and chronic effect of psychostimulants (Yang et al., 2006). As ADHD is highly heterogeneous disorder, both genetically and environmentally, using animal models can simplify and promote the understanding of this disorder. The spontaneously hypertensive rats (SHR) strain was developed in Japan by selective inbreeding of rats from the Wistar Kyoto strain (WKY) that exhibited high systolic blood pressure. During the selective inbreeding, the new strain was more active in comparison to its progenitor, the normotensive WKY strain (Kantak et al., 2008). The SHR strain fulfills the validation criteria which make it an adequate animal model for ADHD; face validity — SHRs mimic the behavioral characteristics of ADHD described in children. Impulsiveness, which is absent initially and develop gradually over time, sustained attention a deficit that is demonstrated only when stimuli are widely spaced in time, hyperactivity, which is not observed in a novel non-treating environment and develop over time. Construct, validity — the two main behavioral processes that are proposed to be major contributory factors in the etiology of ADHD, altered reinforcement of novel behavior and deficient extinction of previously reinforced behavior, are demonstrated. These processes are associated with hypofunction of the dopamine system. Predictive validity — predictions are currently focused on the behavioral level, although the usefulness of the model might become apparent in areas like genetics, neurobiology and pharmacology (Sagvolden et al., 2005). A dilemma concerning the usage of SHR rats revolves around their development of hypertension. It is unclear whether the neurocognitive deficits are result of hypertension. Since the hypertension develops around weeks 12–14 and is not present
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in young hyperactive rats, it is accepted that the neurocognitive alterations appear prior to the hypertension (Kantak et al., 2008; Russell, 2002; Sagvolden et al., 2005). Studies on methylphenidate and its neurochemical effects have been done before, but the majority of them were conducted in juvenile or adult rats without ADHD-like symptoms. In the first part of this study, we assessed the differences in dopaminergic parameters between juvenile SHR and their corresponding controls, juvenile WKY rats. Secondly, we examined the effect of chronic methylphenidate treatment on the same dopaminergic parameters in juvenile SHR rats.
2. Materials and methods 2.1. Animals Male Wistar Kyoto rats (WKY strain) and spontaneous hypertensive rats (SHR strain) at the age of 4 weeks (80–100 g) were purchased from Harlan laboratories. The rats were housed 4 per cage at 22± 2 °C and a 12 light:12 dark cycle (lights on at 05:00 h) with unlimited access to commercial pellet food and tap water.Animal procedures were approved by the Animal Care Committees of Tel-Aviv University (approval numbers: M-08-087 and M-09-034).
2.2. Materials [3H]GBR12935 (specific activity: 38.5 Ci/mmol) and [3H]dopamine (specific activity: 55.1 Ci/mmol) were purchased from Perkin-Elmer (Boston, MA, USA). [3H]TBZOH (specific activity: 20 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Mazindol was purchased from Sandoz Pharmaceuticals (New Jersey, USA). TBZOH was purchased from Fluka (Buch, Switzerland). D-Amphetamine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methylphenidate hydrochloride (Ritalin®) was synthesized by Novartis Pharma AG (Basel, Switzerland). All other chemicals were of highest purity obtainable through regular commercial sources.
2.3. Methylphenidate treatment protocol Two weeks old male SHR rats were treated (i.p.) every morning (around 10 AM) for 21 days with 3 mg/kg methylphenidate (Russell et al., 2000) or saline. On the last day, 2 h after administration, the rats were sacrificed (decapitation by guillotine) and their brains were dissected on ice.
2.4. [3H]GBR12935 binding to rat brain membranes [3H]GBR12935 is a specific antagonist of the dopamine transporter and its binding was carried out as described previously (Janowsky et al., 1986). Rat brain striatal tissue was dissected over ice and placed into a chilled Potter–Elvehjem glass homogenizing vessel containing 10 volumes of ice cold 0.32 M sucrose. The tissue was homogenized 8 times with a teflon pestle and centrifuged for 10 min at 1000g. The supernatant was centrifuged for 15 min at 26,890g. The resulting pellet was resuspended in 40 volumes of 50 mM Tris HCl (pH 7.4) containing 120 mM NaCl, using Brinkman Polytron. Using the same buffer containing 0.01% BSA, the tissue was diluted 1:5. The incubation mixture contained 100 μl striatal membranes, 50 μl [3H] GBR12935 (0.5–8 nM) and 350 μl buffer (50 mM Tris HCl, pH 7.4, containing 120 mM NaCl and 0.01% BSA). Following a 45-minute incubation period (at 25 °C), the incubate was quickly diluted in 3 ml ice cold buffer and filtered under vacuum through glass fiber filters (Whatman GF/C). The filters were washed 4 times with 3 ml ice cold buffer and counted in a liquid scintillation (Opti flour Packard) using
716 a β counter (Packard 2100 TR). The specific binding was defined as the difference between the total [3H]GBR12935 binding and the nonspecific binding, measured using excess of unlabeled antagonist (50 μM mazindol). Scatchard analyses were used to calculate the transporter density (Bmax) and the ligand affinity to the transporter (Kd).
2.5. [3H]Dopamine uptake to rat brain synaptosomes The [3H]dopamine uptake was performed as described before (Gordon et al., 1996; Coyle and Snyder, 1969). Forebrains (whole brain minus brain stem and cerebellum) of male rats were homogenized using Potter–Elvehjem homogenizer (8 strokes) in 10 volumes of ice cold 0.32 M sucrose using teflon pestle. Homogenates were centrifuged at 1000g for 10 min and the resulting supernatant containing the synaptosomal membranes was used for the uptake studies. A standard assay contained: 50 μl homogenate, 900 μl Krebs Ringer phosphate buffer (119 mM NaCl, 3.9 mM KCl, 0.65 mM MgSO4, 0.51 mM CaCl2, 0.19 mM phosphate buffer pH 7.4, 11.1 mM glucose, 1.13 mM ascorbic acid, 0.16 mM EDTA, and 0.1 mM pargyline). The buffer solution was prepared fresh daily and equilibrated with 95% O2–5% CO2 for 10 min prior to use. The tubes were preincubated at 37 °C for 10 min. Thereafter 50 μl of [3H]dopamine (1 × 10− 8–5 × 10− 7 M) was added and the incubation was continued for another 4 min. The reaction was stopped by rapidly cooling the tubes on ice. Non-specific uptake was measured by incubating identically prepared tubes at 0–4 °C. The incubate was filtered under vacuum through glass fiber filters (Whatman GF/C). The filters were washed 4 times with 3 ml of ice cold buffer and counted. Specific uptake per minute was defined as the difference between the total uptake and non-specific uptake divided by 4 min. Km and Vmax values were obtained from Lineweaver Burk charts.
2.6. [3H]TBZOH binding to rat brain membranes [3H]TBZOH is a specific antagonist of the vesicular monoamine transporter 2 and its binding was carried out as described previously (Scherman, 1986). Brain striatal tissue was homogenized in 10 volumes of 0.32 M sucrose in a Potter–Elvehjem homogenizer (8 strokes), using a teflon pestle. Homogenate was centrifuged at 1000g for 10 min. The supernatant was diluted 1:5 in HEPES buffer 20 mM, pH 8.0 containing 300 mM sucrose, and centrifuged 10 min at 26,890g. The pellet was resuspended in 50 volumes of Hepes sucrose buffer and centrifuged 10 min at 26,890g. Finally, the pellet was disrupted with Brinkman polytron and resuspended in HEPES sucrose buffer to yield a homogenate concentration of 20 mg/ml. The assay included 150 μl buffer, 50 μl membranes and 50 μl of various concentrations of [3H]TBZOH (0.5–8 nM). Non-specific binding was determined using excess unlabeled TBZOH (10 μM). After 30 min of incubation at 25 °C, the mixture was filtered with vacuum on glass fiber filters (Whatman GF/C). The filters were washed 4 times with ice cold 50 mM Tris buffer, pH 8, and the radioactivity was counted as mentioned before. Scatchard analysis was used to assess the density of the binding sites (Bmax) and the ligand affinity to VMAT2 (Kd).
Y. Simchon et al. the slices were washed for another 40 min. Aliquots of the tissue were added to baskets with a polypropylene mesh bottom (100 μm pore size) and placed in a warm bubbled buffer for another 20 min prior to the release assay. For the release assay, baskets were transferred at 1 min intervals through a series of vials containing bubbled buffer (37 °C). For stimulated release, the baskets were transferred to vials containing 25 mM K+, 1 μM amphetamine or 50 μM methylphenidate. At the end of the transfers, the baskets were placed in 1 M NaOH overnight to solubilize the tissue. On the following day, 2 M acetic acid was added to neutralize the solution. Aliquots of 0.5 ml were taken to scintillation vials, and the radioactivity was determined as mentioned before. The percent of [3H]dopamine overflow was calculated as the amount of radioactivity released into the medium during a given period, divided by the sum of radioactivity released in that time and in subsequent periods plus the radioactivity remaining in the slices.
2.8. Protein assays Protein concentration was determined by Lowry et al. (1951) and Bradford (1976) methods.
2.9. Statistical analysis The independent samples t-test, using SPSS software (SPSS Inc. Chicago, Illinois), was used as appropriate. Results represent individual rat tissues and are expressed as means ± S.D.
3. Results 3.1. Dopaminergic parameters in SHR vs. WKY 3.1.1. [3H]GBR12935 binding The density of [3H]GBR12935 binding sites was significantly lower in the striatum of SHR rats in comparison to WKY rats (915 ±87 vs. 1120± 91 fmol/mg protein; t = 3.8, df= 9, p = 0.004) (Fig. 1). There was no difference between the two strains in the affinity of [3H] GBR12935 to its binding site in the striatal membranes (2.8 ±0.7 vs. 2.8 ± 0.6 nM; t = −0.13, df = 9, p = 0.89).
2.7. [3H]TBZOH binding to rat brain membranes The [3H]dopamine release was performed as described by Gordon et al. (1995). Striatal tissue was dissected on ice and was cut in two perpendicular directions using a razor blade. The slices were washed with Krebs Ringer buffer in 37 °C, containing 124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 1.2 mM CaCl2, 10 mM glucose, 5.68 mM ascorbic acid, and 10 μM pargyline, bubbled continuously with 95% O2 (containing 5% CO2), which gave pH of 7.4. The tissue was washed in the buffer with continuous bubbling for 40 min, buffer changes every 10 min. The slices were then incubated with 5 × 10− 8 M [3H]dopamine for 30 min. After the uptake period,
Figure 1 Brain striatal dopamine transporter density in juvenile SHR rats (n = 5) compared to juvenile WKY rats (n = 6). [3H]GBR12935 binding (0.5–8 nM) was measured in brain striatal membranes of 4 week old rats. Bmax values were evaluated using Scatchard analysis. Non-specific binding was determined in the presence of 50 μM mazindol. Results are expressed as means ± S.D. ***p b 0.005 vs. WKY.
The effect of chronic MPH administration on presynaptic dopaminergic parameters for ADHD
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3.1.2. [3H]Dopamine uptake No difference was observed between the two strains in the maximal [3H]dopamine uptake rate (Vmax) to brain synaptosomes or in the Km values (6940± 1080 vs. 6940± 1680 DPM/min mg protein; t = 0.001, df = 9, p = 0.99; 1.01 × 10− 7 ± 0.23 vs. 1.41 × 10− 7 ± 0.65 M; t = 1.29, df= 9, p = 0.22, respectively).
3.1.3. [3H]TBZOH binding The density of [3H]TBZOH binding sites in striatal membranes of SHR rats was significantly lower in comparison to the WKY controls (820 ± 89 vs. 1040 ± 130 fmol/mg protein; t = 3.37, df = 10, p = 0.007) (Fig. 2). A modest but significant difference was found in the affinity of [3H]TBZOH to the binding site, in SHR vs. WKY (0.96 ± 0.15 vs. 1.23± 0.11 nM; t = 3.135, df= 8, p = 0.014) but most Kd values are in the low nanomolar range.
3.1.4. [3H]Dopamine release The basal release of [3H]dopamine was significantly lower in striatal slices of SHR strain in comparison to the WKY strain (73 ± 13 vs. 106 ± 14 mm2; t = 4.2, df = 10, p = 0.002). Stimulated [3H] dopamine release evoked by high potassium, amphetamine or methylphenidate did not reveal significant differences between the two strains (54 ± 8 vs. 56 ± 6; t = 0.463, df = 10, p = 0.65; 57 ± 14 vs. 55 ± 12; t = −0.22, df = 7, p = 0.82 and 31 ± 4 vs. 34 ± 7 mm2; t = 0.84, df = 8, p = 0.42, respectively) (Fig. 3).
3.2. The effect of chronic methylphenidate treatment on dopaminergic parameters in juvenile SHR rats 3.2.1. [3H]GBR12935 binding The methylphenidate treatment induced a significant decrease in the density of [3H]GBR12935 binding sites (805 ± 240 vs. 1020 ± 102 fmol/mg protein; t = 2.198, df= 12, p = 0.048) (Fig. 4). The treatment had no effect on the affinity of [3H]GBR12935 to the
Figure 3 Basal and stimulated [3H]dopamine release from striatal slices of juvenile SHR rats (n = 6) as compared to juvenile WKY rats (n = 6). Brain striatal slices of 4 week old rat were loaded with [3H]dopamine at 37°c for 30 min. Release was assessed by calculating the area under the curve. Stimulated [3H]dopamine release was induced by 25 mM K+, 1 μM amphetamine or 50 μM methylphenidate. Results are expressed as means ± S.D. ***p b 0.005 vs. WKY.
striatal membranes (1.73 ± 0.27 vs. 1.93 ± 0.38 nM; t = 1.11, df= 12, p = 0.28).
3.2.2. [3H]Dopamine uptake The methylphenidate treatment did not affect [3H]dopamine uptake into striatal synaptosomes (6160± 1030 vs. 5750 ±810 DPM/ min mg protein; t=−0.83, df =12, p =0.42) or the Km values (7.71 × 10− 8 ± 0.71 vs. 7.41 × 10− 8 ± 1.4 M; t=−0.48, df = 12, p=0.63).
3.2.3. [3H]TBZOH binding Chronic treatment with methylphenidate did not affect the density of [3H]TBZOH binding sites (832 ± 110 vs. 851 ± 109 fmol/ mg protein; t = 0.34, df = 14, p = 0.73). The chronic treatment had
Figure 2 Brain vesicular monoamine transporter 2 density in juvenile SHR rats (n = 6) compared to juvenile WKY rats (n = 6). Vesicular monoamine transporter 2 density was determined using [3H]TBZOH binding (0.5–8nM) that was measured in brain striatal membranes of 4 week old rats. Bmax values were evaluated using Scatchard analysis. Non-specific binding was determined in the presence of 10 μM TBZOH. Results are expressed as means ± S.D. **p b 0.01 vs. WKY.
Figure 4 Brain striatal dopamine transporter density in juvenile (2 week old) SHR rats (n = 7) treated for 21 days with methylphenidate (3 mg/kg, i.p.). [3H]GBR12935 binding (0.5–8 nM) was measured in rat brain striatal membranes. Results are expressed as means ± S.D. *p b 0.05 vs. untreated SHR rats (n = 7).
718 no effect on the affinity of [3H]TBZOH to the striatal membranes (0.93 ± 0.11 vs. 0.99 ± 0.17 nM; t = 0.80, df= 14, p = 0.43).
3.2.4. [3H]Dopamine release The methylphenidate treatment induced a significant reduction in the basal [3H]dopamine release from striatal slices (54 ± 9 vs. 72 ± 4 mm2; t = 3.98, df = 8, p = 0.004). Potassium-induced [3H]dopamine release was increased by 29% after 21 days of methylphenidate treatment (55±9 vs. 43± 10 mm2; t=−2.25, df =10, p= 0.048). Amphetamine-induced [3H]dopamine release was also enhanced (29%) by the chronic methylphenidate treatment (77±4 vs. 59±10 mm2; t=−3.72, df=8, p= 0.006) (Fig. 5).
4. Discussion In this study, we used spontaneous hypertensive rats (SHR) as an animal model for ADHD. We characterized several key synaptic dopaminergic parameters; dopamine transporter level, dopamine uptake, vesicular monoamine transporter 2 level and dopamine release in brains of SHR rats in comparison to their controls, normotensive Wistar Kyoto rats (WKY). The dopaminergic parameters in SHR rats were also studied before and after chronic methylphenidate administration. We found that the density of the striatal DAT was lower in juvenile SHR rats (4 weeks old) in comparison to juvenile WKY rats. Previous studies reported both increased and decreased levels of DAT in SHR in comparison to WKY rats. Leo et al. (2003) studied the DAT mRNA levels at different post-natal days. They found that the mRNA levels were significantly lower in SHR rats at 27–49 post-natal days (which is equivalent to 4–7 week old rats) in comparison to WKY rats. In contrast to Leo et al. findings, using autoradiography with the ligand [125I]β-CIT, Watanabe et al. (1997) found higher level of binding sites in the caudate– putamen of SHR in comparison to WKY rats. They evaluated
Figure 5 Basal and stimulated [3H]dopamine release from striatal slices of juvenile (2 weeks old) SHR rats treated for 21 days with methylphenidate (3 mg/kg, i.p.). Rat brain striatal slices were loaded with [3H]dopamine at 37°c for 30 min. Release was assessed by calculating the area under the curve. Stimulated [3H]dopamine release was induced by 25 mM K+ or 1 μM amphetamine. Results are expressed as means ± S.D. *p b 0.05, **p b 0.01, ***p b 0.005 vs. untreated SHR rats. (n = 5, n = 6, n = 5 vs. n = 5, n = 6, n = 5 respectively).
Y. Simchon et al. the density in 2 week old rats, and they also reported that the higher density was kept during maturity to adult SHR rats (15 weeks old) in comparison to the adult WKY rats. In our study, no statistical difference could be detected in the Vmax and Km values of [3H]dopamine uptake between the two strains. In contrast, Leo et al. described a significant decrease in striatal [3H]dopamine uptake in SHR rats in comparison to WKY rats. They suggested that the difference could be related to reduced DAT density, or variation in the affinity of dopamine to the transporter (Leo et al., 2003). It has been suggested (Goto et al., 2007) that the higher dopamine phasic release in ADHD patients is due to the lower dopamine basal tonic release. In accordance with this hypothesis, we found that the basal dopamine release from striatal slices of juvenile SHR rats was lower than the release from juvenile WKY striatal slices. Our results in SHR juvenile rats are also in agreement with another study (Russell et al., 1995) that reported on lower release of dopamine in striatal slices of adult SHR rats in comparison to adult WKY rats. According to Russell et al., the reduced dopamine release is due to overexpression of dopamine inhibitory presynaptic autoreceptors. They found that high potassium concentration induced less dopamine release from striatal slices of adult SHR rats in comparison to WKY rats while the amphetamine-induced dopamine release was higher in striatal slices of SHR rats in comparison to WKY rats. In our study, we observed a non-significant reduction in dopamine release induced by high potassium concentration and increased dopamine release induced by amphetamine in striatal slices of juvenile SHR rats. It is of note that high potassium induces release from vesicular stores while amphetamine induces release from cytoplasmic stores. As the stimulated dopamine release from vesicular stores (high potassium concentration induced) was reduced, while the dopamine release from cytoplasmic stores (amphetamine induced) was enhanced, it is possible that impaired vesicular storage capability is accompanied by leakage of dopamine from the vesicles to the cytoplasm (Russell et al., 1998; Russell, 2002; Russell et al., 2000). It is known that the synaptic dopamine transmission depends on several components. We suggest that the lower expression of DAT in the striatum of the juvenile SHR rats is due to a compensatory mechanism aimed to overcome the decreased striatal tonic release of dopamine leading to reduced dopamine concentration in the synaptic cleft. This assumption is supported by our finding that dopamine uptake via DAT is similar between the strains. The lack in change in DA uptake may indicate that sufficient amount of DA transporters is available to cope with the changes in synaptic DA concentrations despite the changes in DA release and DAT expression. Alternatively, it is possible that alterations in the presynaptic DA autoreceptors are responsible for the lack of change in DA uptake between the two strains. Unfortunately, in the present study we did not assess the DA autoreceptors. The role of VMAT2 is to protect monoamines, including DA, from mitochondrial MAO oxidation in the cytoplasm by transporting the monoamines from the cytoplasm into the synaptic vesicles. This process is responsible for maintaining high concentration of these monoamines in the presynaptic neuron and enabling further exocytosis. There are no reports on the expression of VMAT2 in juvenile SHR or WKY rats.
The effect of chronic MPH administration on presynaptic dopaminergic parameters for ADHD We found that the density of striatal VMAT2 in juvenile WKY rats was higher than in juvenile SHR rats, although the ligand affinity to VMAT2 was modestly higher in juvenile SHR rats. These findings correlate with our previous results; as the dopamine tonic release is lower in the striatum of young SHR rats, less dopamine is taken into the neuron and the need for VMAT2 to protect the dopamine from oxidation is attenuated. Thus it appears that there is a parallel decrease in both; the DAT and VMAT2 densities. In the second part of our research, we studied the effect of chronic methylphenidate treatment on the dopaminergic parameters in juvenile SHR rats. We expected that after 21 days of methylphenidate administration to the 2 week old SHR rats, their dopaminergic parameters will be similar to the dopaminergic parameters of their corresponding controls, WKY rats. In contrast to our expectations, the density of the dopamine transporter in the striatum of young SHR rats decreased after the chronic treatment. Yet, our results are in accordance with the radiolabeled imaging studies in brains of ADHD adults and children treated with methylphenidate (Wilens, 2008). In the latter human research a significant decrease in the DAT binding signal was demonstrated. As the time of the imaging analysis relative to the drug administration in not completely defined, it is not clear whether the low imaging signal is related to the occupation of DAT by methylphenidate (Wilens, 2008). In our hands, the density of the striatal DAT in young SHR rats decreased following the chronic methylphenidate treatment. Moll et al. (2001) showed a decline in the density of striatal DAT after 14 days of methylphenidate administration in juvenile Wistar rats. According to Moll et al., maturation of the rats is accompanied by a decrease in DAT density, regardless of the methylphenidate treatment. Since methylphenidate treatment induced a decrease in DAT density, he suggested that the treatment causes an additional decrease in the DAT density. Our data indicates that after 21 days of chronic methylphenidate administration, the basal dopamine release from SHR striatal brain slices was significantly lower in comparison to the release of the saline treated SHR rats. We mentioned previously that the tonic release in SHR rats is lower in comparison to that of WKY rats. It is possible that methylphenidate blockade of the DAT leads to excess dopamine presence in the synaptic cleft and to the activation of inhibitory presynaptic autoreceptors. Such putative activation might lead to a further reduction of tonic DA release (Seeman and Madras, 1998). The lower tonic release means that more dopamine stays in the neuron (cytoplasmic and vesicular stores). Induced release, either by high potassium concentration or by amphetamine, empties the elevated dopamine stores, which results in a higher amount of dopamine release. It is possible that methylphenidate promotes DAT density downregulation, as a compensatory mechanism to the reduced dopamine release (Madras et al., 2005; Moll et al., 2001). The density of VMAT2, as assessed by high affinity [3H] TBZOH binding, did not change after the chronic methylphenidate treatment. A preliminary study in our laboratory (data not shown) failed to demonstrate an effect of chronic methylphenidate treatment on striatal VMAT2 density in adult Sprague Dawley rats. Using different methods, Sandoval et al., (2002) described an increase in [3H]TBZOH binding to VMAT2 as well as an increase in vesicular [3H]
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dopamine uptake after acute methylphenidate administration (40 mg/kg) to adult Sprague Dawley rats. These authors suggested the existence of two types of synaptic vesicles: membrane associated fraction and cytoplasmic fraction. According to these investigators, the VMAT2 protein redistributes from the membrane associated fraction to the cytoplasmic fraction as a response to methylphenidate treatment leading to an increase in VMAT2 density and an increase in vesicular dopamine uptake (Volz et al., 2008; Volz et al., 2007). It is possible that if we would have separated the vesicles into these fractions, we would have also seen an increased density of VMAT2 in the cytoplasmic fraction due to such putative redistribution. The fact that the VMAT2 density remained unaltered, in spite of the chronic methylphenidate treatment, can actually strengthen the redistribution hypothesis. Namely, there is no new synthesis of VMAT2 but the transporter redistributes. Methylphenidate treatment is currently the drug of choice for treating ADHD children and adolescence. Chronic methylphenidate treatment of the young SHR rats was associated with lower DAT density and lower unstimulated basal dopamine release but with enhanced potassium- and amphetamine- induced dopamine releases. These dopaminergic changes might be relevant to the beneficial effect of the methylphenidate treatment in ADHD.
Role of the funding source There is no involvement of the funding source.
Contributors Yaarit Simchon performed the entire research as part of her MSc thesis. She participated in designing the experiments, treating the rats, performing all of the biochemical assays. Abraham Weizman participated in writing the final draft and the statistics. Moshe Rehavi designed the project and was Yaarit Simchon mentor in the project, he wrote the first draft. All authors contributed to and have approved the final manuscript.
Conflict of interests There are no conflicts of interest.
Acknowledgment The study was supported by a grant from the Dr. Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Tel-Aviv University, Israel.
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