The usefulness of the spontaneously hypertensive rat to model attention-deficit/hyperactivity disorder (ADHD) may be explained by the differential expression of dopamine-related genes in the brain

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Abstract

Spontaneously hypertensive rats (SHR) are considered to represent a genetic animal model for attention-deficit hyperactivity disorder (ADHD). In the present studies, we compared the locomotor activity, learning and memory functions of juvenile male SHR, with age- and gender-matched genetic control Wistar–Kyoto rats (WKY). In addition, we investigated potential differences in brain morphology by magnetic resonance imaging (MRI). In other complimentary studies of the central nervous system, we used real-time PCR to examine the levels of several dopaminergic-related genes, including those coding for the five major subtypes of dopamine receptor (D1, D2, D3, D4 and D5), those coding for enzymes responsible for synthesizing tyrosine hydroxylase and dopamine-β-hydroxylase, and those coding for the dopamine transporter. Our data revealed that SHR were more active than WKY in the open field (OF) test. Also, SHR appeared less attentive, exhibiting inhibition deficit, but in the absence of memory deficits relative to spatial learning. The MRI studies revealed that SHR had a significantly smaller vermis cerebelli and caudate–putamen (CPu), and there was also a significantly lower level of dopamine D4 receptor gene expression and protein synthesis in the prefrontal cortex (PFC) of SHR. However, there were no significant differences between the expression of other dopaminergic-related genes in the midbrain, prefrontal cortex, temporal cortex, striatum, or amygdala of SHR and WKY. The data are similar to the situation seen in ADHD patients, relative to normal volunteers, and it is possible that the hypo-dopaminergic state involves a down regulation of dopamine D4 receptors, rather than a general down-regulation of catecholamine synthesis. In conclusion, the molecular and behavioural data that we obtained provide new information that may be relevant to understanding ADHD in man.

Introduction

Attention-deficit hyperactivity disorder (ADHD) is characterized by age-inappropriate inattention, impulsiveness, and hyperactivity (Wilens et al., 2002). Approximately 5–10% of school-aged children worldwide have ADHD, with the incidence being three times higher in boys than girls (Barkley and Biederman, 1997, Brown et al., 2001, Hinshaw, 1992, Scahill and Schwab-Stone, 2000). Unfortunately, the disease continues into adulthood in 30–70% of patients (Silver, 2000). It has been proposed that ADHD may lead to memory deficits, delinquency, substance abuse, and problematic personality disorders, in addition to constituting one of the highest risk factors for other mental illnesses (Taylor, 1998). Although the precise etiology and pathological mechanisms underlying ADHD are poorly understood, accumulating data indicates that genetics may influence its incidence. For example, studies focusing on twins showed that the heritability of ADHD was 0.80, indicating a strong genetic predisposition (Faraone and Biederman, 1998). Other case-controlled and/or family-based association studies have targeted several specific candidate genes. This approach has identified the potential involvement of dopamine D2, D4 and D5 receptor genes, and serotonin 2A genes, as well as the genes coding the dopamine and serotonin transporters (LaHoste et al., 1996). Indeed, the dopamine D4 receptor gene has been one of the most studied genes in ADHD, with a recent quantitative trait study showing that a 7-repeat allele may be linked to specific neuropsychological behaviours to give rise to a new phenotype of ADHD (Swanson et al., 1998).

In addition to the molecular studies, magnetic resonance imaging (MRI) of the brains of ADHD patients has revealed a smaller sized basal ganglia, corpus callosum, prefrontal cortex (PFC), and cerebellum compared to normal individuals (Castellanos et al., 1996, Filipek et al., 1997). These observations link with the molecular findings, since dopaminergic projections from midbrain ventral tegmental area (VTA) to the striatal and prefrontal cortical areas, play a major role in motor control, and attention and impulsion (Eells, 2003).

Adult spontaneously hypertensive rats (SHR) are commonly used in cardiovascular research. However, juvenile (4–6-week old) SHR are being used increasingly to model ADHD because they are hyperactive (Castanon et al., 1993, Sagvolden et al., 1992, Whitehorn et al., 1983), inattentive (Berger and Sagvolden, 1998) and impulsive (Boix et al., 1998). These three behavioral characteristics correlate with the classical symptoms exhibited by children with ADHD (Hendley, 2000, Sagvolden, 2000), and are seen prior to the development of hypertension in these animals (Sagvolden et al., 2005b). Notwithstanding these important behavioral correlates, SHR also have a lower turnover of dopamine in the VTA, striatum, and frontal cortex that is relevant to the clinical situation (de Villiers et al., 1995, Linthorst et al., 1994).

The level and function of particular transmitter in the neuro-effector junction is controlled by a variety of factors. In the case of dopamine, this includes the level of activity of tyrosine hydroxyls (TH), which catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA); dopa decarboxylase (DDC) which catalyzes the conversion of DOPA to dopamine; dopamine-β-hydroxylase (DβH) which catalyzes the formation of noradrenaline from dopamine and there are also metabolic enzymes that can deactivate dopamine, including monoamine oxidase.

Dopamine itself interacts with five major dopamine receptors and is removed from the synaptic cleft by a specific dopamine transporter (Missale et al., 1998). However, no studies have investigated the possibility that genes coding the synthetic/metabolic pathways of dopamine, or its receptors, are differentially expressed in SHR compared to its genetic control. In the present studies, therefore, we decided to compare the locomotors activity, attention, and learning and memory functions of juvenile male SHR with age- and gender-matched genetic control Wistar–Kyoto rats (WKY), with particular emphasis on the potential differences of gene expression relevant to the dopaminergic system in different brain areas. MRI was also employed to probe for potential morphological differences between brain areas of SHR and WKY. It was hoped that the results of the study would help address issues relating to the appropriateness of the use of juvenile SHR to model ADHD.

Section snippets

Animals

Juvenile male SHR aged 4–6-week old, and age- and gender-matched genetic control WKY, were obtained from the Laboratory Animal Services Centre, The Chinese University of Hong Kong (CUHK). The original colony originated from Harlan Olac, UK. The rats had free access to standard laboratory rodent chow and water and were housed in a room with 12 h light–dark cycle; temperature and humidity were maintained at 22 ± 1 °C and 45–55%, respectively. The experiments were approved by the Animal

Open field test

SHR were more active than WKY during the 5 min locomotor test (SHR distance travelled, 1708.13 ± 70.44 cm; WKY distance travelled, 1089.13 ± 82.21 cm; p < 0.0001) (Fig. 1). The average velocity of movement of SHR was faster than WKY (SHR = 5.7 ± 0.24 cm/s; WKY = 3.63 ± 0.27 cm/s; p < 0.0001) (Fig. 1).

Experiment 1: special learning and memory

On the 1st day of water maze testing, there were significant differences (p < 0.05) between SHR and WKY. Thus, the latencies to locate the hidden platform for SHR and WKY were 28 ± 2.94 and 77.18 ± 5.3 s, respectively. On the

Discussion

In the present studies, we assessed the behavior of SHR with age and gender matched WKY using established open field, Morris water maze tests, water finding task and prepulse inhibition. This was done for the first time in conjunction with comparative studies of the central nervous system by MRI and molecular techniques targeted to the dopaminergic system. SHR were clearly more spontaneously active than WKY in the open field test, and they also had a higher level of activity and attention

Acknowledgements

We would like to thank the staff of Department of Diagnostic Radiology & Organ Imaging for their valuable assistance. Also we thank Prof. Shaw and Mr. Kelvin of the Department of Biochemistry for their helpful comments on the water maze experiments. We appreciate the help from Prof. Yeung of the Department of Surgery for the use of the startle box to do the prepulse inhibition task.

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