Sequence analysis of Drd2, Drd4, and Dat1 in SHR and WKY rat strains
© Mill et al; licensee BioMed Central Ltd. 2005
Received: 23 August 2005
Accepted: 15 December 2005
Published: 15 December 2005
The Spontaneously Hypertensive Rat (SHR) shows a number of behaviours that closely parallel those seen in children with attention-deficit hyperactivity disorder. These include motor hyperactivity, excessive responses under a fixed-interval/extinction schedule, difficulty in acquiring operant tasks and increased sensitivity to immediate behavioural reinforcement. As in children with ADHD, the behavioural and cognitive deficits in the SHR are responsive to stimulants, including d-amphetamine and d,l-methylphenidate. The non-hyperactive Wistar Kyoto (WKY) rat strain is often used as a control in behavioural studies of the SHR, and WKY itself has been suggested to be a useful animal model of depression. Numerous studies have shown that dopaminergic neurotransmission is altered between the two strains. Human genetic studies have found associations between several dopaminergic genes and both ADHD and depression.
We sequenced three candidate dopaminergic genes (Drd2, Drd4, and Dat1) in the SHR and WKY to identify between-strain sequence differences.
No between-strain sequence differences were found in either Drd2 or Drd4, but several variations were found in the Dat1 gene that encodes the dopamine transporter.
It is plausible that DNA sequence changes in the Dat1 gene account for some of the behavioural differences observed between the SHR and WKY strains. Future work will focus on elucidating the functional effects of the observed polymorphisms.
Attention-deficit hyperactivity disorder (ADHD) is a common neurobehavioural disorder defined by symptoms of developmentally inappropriate inattention, impulsivity and hyperactivity. It is estimated that between 3–6% of school age children are diagnosed with ADHD, making it the most prevalent disorder of childhood. While the precise aetiology of ADHD is yet to be ascertained, it is clear from numerous family, twin and adoption studies that genetic factors play in a key role in susceptibility to the disorder. Polymorphisms in several genes have been associated with ADHD, with a particular focus on genes implicated in monoamine neurotransmission .
A number of animal models have been proposed for ADHD and these have helped inform research into the biological basis of the clinical disorder. The spontaneously hypertensive rat (SHR) is one of the most widely validated animal models of ADHD [2, 3]. The SHR shows a number of behaviours that closely parallel those seen in children with ADHD including motor hyperactivity, increased impulsiveness and deficient sustained attention [3, 4]. Furthermore, like children with ADHD, the SHR is more sensitive to immediate behavioural reinforcement and less sensitive to delayed reinforcement than non-hypertensive WKY control rats [3, 4]. The behavioural and cognitive deficits in the SHR are responsive to stimulants, including d-amphetamine and d,l-methylphenidate . Finally, several studies have shown that dopaminergic and noradrenergic neurotransmission is altered in the SHR compared to the WKY, strongly implicating these systems in the aetiology of ADHD [2, 6, 7].
The WKY strain, from which the SHR was initially derived by selective outbreeding , is itself proposed to be a model of another psychiatric condition – depression. As for ADHD, the aetiology of depression has been shown to be strongly influenced by genetic factors  and dysregulation of the dopaminergic system has been strongly implicated . WKY rats have been shown to exhibit exaggerated neuroendocrine and behavioral responses to stress that exceed normal controls and are especially prone to develop stress-induced depressive disorder [11, 12]. A recent study by Will et al found that selectively bred WKY rats were a particularly good animal model of depression and hyper-responsiveness to anti-depressants . Interestingly, the dopamine neurotransmitter pathway has been strongly implicated in the depression-like behaviours exhibited by WKY rats. Jiao et al observed differences in the density and distribution of dopamine transporter sites in WKY rats that may lead to altered modulation of synaptic dopamine levels in the cell body and mesolimbic regions .
In this study we have sequenced three dopaminergic candidate genes (Drd2, Drd4, and Dat1) in the SHR and WKY rat strains to identify potential genetic variants that may explain some of the behavioural differences observed between the two strains.
Blood was obtained from animals housed at the University of Oslo and DNA was extracted using a standard protocol . Bioinformatic analyses were performed to identify regions in the rat genome containing homologues of human ADHD candidate genes using sequence data deposited in the Rat Genome Database http://rgd.mcw.edu/. Where no annotated rat genome sequence was available, BLAST searches were performed on raw sequence data to identify the relevant regions http://www.ncbi.nlm.nih.gov/BLAST/. Primers were designed to span the promoter and exonic regions of three candidate genes (Drd2, Drd4, and Dat1) using Primer Express software (Applied Biosystems, Foster City, CA, USA). To conserve space oligo sequences are not given in this manuscript, but are available from the authors on request. The chromosomal location, amino-acid length and human-rat homology for the three genes sequenced in this study can be seen in Table 1.
The chromosomal location, amino-acid length and human-rat homology for the three genes sequenced in this study.
The rat Drd2 gene is located on chromosome 8q23 and is 95% homologous with the human DRD2 gene. The putative promoter and exonic regions of Drd2 were sequenced. No differences were observed between the SHR and WKY strains, and both sequences were identical to those in the Rat Genome Database http://rgd.mcw.edu/.
Variants noted in the Drd4 gene in both WKY and SHR strains compared to the sequence deposited in the Rat Genome Database http://rgd.mcw.edu/.
GGCGCG (not present in WKY/SHR)
CACA (2 extra CA motifs in SHR/WKY)
In this study we sequenced three dopaminergic genes to examine differences between SHR and WKY rat strains. No between-strain sequence differences were found in genes encoding either the dopamine D2 receptor (Drd2) or the dopamine D4 receptor (Drd4), although for Drd4 both the WKY and SHR strains were found to differ from the sequence available in the Rat Genome Database http://rgd.mcw.edu/. In contrast, several between-strain variations were found in the dopamine transporter gene (Dat1). Although none of the sequence changes results in a direct coding change to the DAT protein, it is plausible that they may alter expression-related processes such as transcription or splicing efficiency. Alternatively, it is possible that these changes are markers of other linked mutations carried on the same chromosome. These results are interesting given that the SHR and WKY strains are considered to be valid models of ADHD and depression respectively, and the postulated role of disrupted dopaminergic neurotransmission in both disorders.
It is pertinent that abnormalities in DAT expression and functioning have been noted in both rat strains. SHR strains have been shown to exhibit elevated DAT expression in mesocortical projections [16, 17]. It appears that excess DAT expression in the SHR may not be directly genetic in origin, but is in fact a response to excess mesocortical dopamine during early development resulting from hypofunctioning DAT protein that is presumably genetic [2, 16]. The WKY strain also appears to have an unusual DAT profile compared to non-depressive control strains. Jiao et al report lower DAT density in the nucleus accumbens, amygdala, ventral tegmental area, and the reticular part of the substantia nigra of these animals, but higher expression in the hippocampus and hypothalamus .
It is interesting that these findings are partially mirrored in studies on human psychiatric patients. Whilst individuals with ADHD have been shown to exhibit increased DAT density in the brain [18, 19], depressive patients were found to have overall decreased levels of DAT . Furthermore, genetic association studies suggests an association between a polymorphism in the human dopamine transporter gene (DAT1) and ADHD , although to date there is no evidence linking this polymorphism to the aetiology of depression.
In this study we have sequenced three dopaminergic genes in two inbred rat strains considered to be good models of human psychiatric illness. No between strain differences were observed in either the Drd2 or Drd4 genes, suggesting that neither gene is likely to mediate the behavioural differences observed between the WKY and SHR strains, although a number of polymorphisms common to both strains were detected in Drd4. In contrast, WKY/SHR differences were observed in the 3rd exon of Dat1. Whilst these mutations do not result in direct amino-acid changes to the DAT protein, it is possible that they mediate some other process that explains the differences in DAT expression and function observed between the two strains. Future work should focus on further characterizing the genetic differences between these two strains, and investigating the functional consequences of the observed polymorphisms and how they relate to the putative depressive and hyperactive behaviours observed in the two strains.
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