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/lp/springer-journals/sequence-analysis-of-drd2-drd4-and-dat1-in-shr-and-wky-rat-strains-26WcNS374t
- Publisher
- Springer Journals
- Copyright
- Copyright © 2005 by Mill et al; licensee BioMed Central Ltd.
- Subject
- Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
- eISSN
- 1744-9081
- DOI
- 10.1186/1744-9081-1-24
- pmid
- 16356184
- Publisher site
- See Article on Publisher Site
Abstract
Background: 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. Methods: We sequenced three candidate dopaminergic genes (Drd2, Drd4, and Dat1) in the SHR and WKY to identify between-strain sequence differences. Results: 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. Conclusion: 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. Background genetic factors play in a key role in susceptibility to the Attention-deficit hyperactivity disorder (ADHD) is a com- disorder. Polymorphisms in several genes have been asso- mon neurobehavioural disorder defined by symptoms of ciated with ADHD, with a particular focus on genes impli- developmentally inappropriate inattention, impulsivity cated in monoamine neurotransmission [1]. and hyperactivity. It is estimated that between 3–6% of school age children are diagnosed with ADHD, making it A number of animal models have been proposed for the most prevalent disorder of childhood. While the pre- ADHD and these have helped inform research into the cise aetiology of ADHD is yet to be ascertained, it is clear biological basis of the clinical disorder. The spontane- from numerous family, twin and adoption studies that ously hypertensive rat (SHR) is one of the most widely Page 1 of 5 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:24 http://www.behavioralandbrainfunctions.com/content/1/1/24 Table 1: The chromosomal location, amino-acid length and human-rat homology for the three genes sequenced in this study. Gene Human Rat Homology ID Location AA Location AA (%) DRD2 11q23.2 443 8q23 444 95% DRD4 11q15.5 387 1q41 385 75% DAT1 5p15.33 620 1p11 619 94% validated animal models of ADHD [2,3]. The SHR shows identify regions in the rat genome containing homo- a number of behaviours that closely parallel those seen in logues of human ADHD candidate genes using sequence children with ADHD including motor hyperactivity, data deposited in the Rat Genome Database http:// increased impulsiveness and deficient sustained attention rgd.mcw.edu/. Where no annotated rat genome sequence [3,4]. Furthermore, like children with ADHD, the SHR is was available, BLAST searches were performed on raw more sensitive to immediate behavioural reinforcement sequence data to identify the relevant regions http:// and less sensitive to delayed reinforcement than non- www.ncbi.nlm.nih.gov/BLAST/. Primers were designed to hypertensive WKY control rats [3,4]. The behavioural and span the promoter and exonic regions of three candidate cognitive deficits in the SHR are responsive to stimulants, genes (Drd2, Drd4, and Dat1) using Primer Express soft- including d-amphetamine and d,l-methylphenidate [5]. ware (Applied Biosystems, Foster City, CA, USA). To con- Finally, several studies have shown that dopaminergic and serve space oligo sequences are not given in this noradrenergic neurotransmission is altered in the SHR manuscript, but are available from the authors on request. compared to the WKY, strongly implicating these systems The chromosomal location, amino-acid length and in the aetiology of ADHD [2,6,7]. human-rat homology for the three genes sequenced in this study can be seen in Table 1. The WKY strain, from which the SHR was initially derived by selective outbreeding [8], is itself proposed to be a The exonic regions were amplified on an MJ PTC-225 model of another psychiatric condition – depression. As thermal cycler (MJ Research) with an initial 9-min dena- for ADHD, the aetiology of depression has been shown to turing step at 95°C, followed by 35 cycles of 93°C for 1 be strongly influenced by genetic factors [9] and dysregu- min, 55°C for 1 min and 72°C for 1 min, and a final lation of the dopaminergic system has been strongly extension phase of 72°C for 10 min. Reactions were per- implicated [10]. WKY rats have been shown to exhibit formed in 22 ul volumes and included 50 ng of genomic exaggerated neuroendocrine and behavioral responses to DNA, 1.5 mM MgCl , 0.2 mM dNTP's, 10 mM GeneAmp stress that exceed normal controls and are especially 10× PCR Gold Buffer (PE Applied Biosystems, Foster City, prone to develop stress-induced depressive disorder US) and 1 unit of AmpliTaq Gold (PE Applied Biosystems, [11,12]. A recent study by Will et al found that selectively Foster City, US). PCR products were run out on a 2% aga- bred WKY rats were a particularly good animal model of rose gel stained with ethidium bromide and analysed depression and hyper-responsiveness to anti-depressants under UV light. PCR products were purified using Qiagen [13]. Interestingly, the dopamine neurotransmitter path- Gel Extraction Columns (Qiagen, Crawley, UK). Follow- way has been strongly implicated in the depression-like ing purification, forward and reverse dye terminator behaviours exhibited by WKY rats. Jiao et al observed dif- sequencing was carried out using ABI BigDye V3.0 and ferences in the density and distribution of dopamine samples run on either an ABI 377 or 3100 machine transporter sites in WKY rats that may lead to altered mod- (Applied Biosystems, Foster City, CA, USA). Sequencing ulation of synaptic dopamine levels in the cell body and traces were analysed using Sequencher software (Gene- mesolimbic regions [14]. Codes Corporation, Ann Arbor, MI, USA), and multiple sequences aligned to aid mutation detection. In this study we have sequenced three dopaminergic can- didate genes (Drd2, Drd4, and Dat1) in the SHR and WKY Results rat strains to identify potential genetic variants that may Drd2 explain some of the behavioural differences observed The rat Drd2 gene is located on chromosome 8q23 and is between the two strains. 95% homologous with the human DRD2 gene. The puta- tive promoter and exonic regions of Drd2 were sequenced. No differences were observed between the Methods Blood was obtained from animals housed at the Univer- SHR and WKY strains, and both sequences were identical sity of Oslo and DNA was extracted using a standard pro- to those in the Rat Genome Database http://rgd.mcw.edu/ tocol [15]. Bioinformatic analyses were performed to . Page 2 of 5 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:24 http://www.behavioralandbrainfunctions.com/content/1/1/24 Table 2: Variants noted in the Drd4 gene in both WKY and SHR amplification using more distal primers suggested the strains compared to the sequence deposited in the Rat Genome presence of extra sequence in the SHR compared to the Database http://rgd.mcw.edu/. WKY (see Figures 1b and 1c). Sequencing of this region highlighted a synonymous single base change (T→C) Location Variation within the coding sequence of exon 3 (see Figure 2), and Promoter region GATGAA[G/T]AGTGAG* a 160 bp section of sequence immediately upstream of Intron 1 GGCGCG (not present in WKY/SHR) exon 3 present in SHR but not WKY. Bioinformatic analy- Intron 2 CACA (2 extra CA motifs in SHR/WKY) sis of the public rat genome database demonstrated that Intron 2 GAATGG[A/G]GACATA* this 160 bp sequence is also present ~1000 bp upstream (in intron 4 of Dat1). *WKY/SHR allele given second. Drd4 Discussion The rat Drd4 gene is located on chromosome 1q41 and In this study we sequenced three dopaminergic genes to shares 75% homology with the human DRD4 gene. The examine differences between SHR and WKY rat strains. No putative promoter, exons, and introns of Drd4 were between-strain sequence differences were found in genes sequenced. No differences were found between the SHR encoding either the dopamine D2 receptor (Drd2) or the and WKY strains, although both were found to contain dopamine D4 receptor (Drd4), although for Drd4 both non-coding sequence differences from the Brown Norway the WKY and SHR strains were found to differ from the Rat sequence available in the Rat Genome Database (see sequence available in the Rat Genome Database http:// Table 2). These differences included two single nucleotide rgd.mcw.edu/. In contrast, several between-strain varia- polymorphisms (SNPs), one located in the putative 5' tions were found in the dopamine transporter gene promoter region of the gene and the other in intron 2, (Dat1). Although none of the sequence changes results in along with length variation at a CA microsatellite repeat in a direct coding change to the DAT protein, it is plausible intron 2. In addition, neither the WKY or SHR strains were that they may alter expression-related processes such as found to have the short first intron (GGCGCG) present transcription or splicing efficiency. Alternatively, it is pos- between exon 1 and 2 in the sequence available in the Rat sible that these changes are markers of other linked muta- Genome Database. tions carried on the same chromosome. These results are interesting given that the SHR and WKY strains are consid- Dat1 ered to be valid models of ADHD and depression respec- The rat Dat1 gene is located on chromosome 1p11 and is tively, and the postulated role of disrupted dopaminergic 94% homologous with the human DAT1 gene. The puta- neurotransmission in both disorders. tive promoter and exonic regions of Dat1 were sequenced. The exon 3 amplicon could not initially be amplified by It is pertinent that abnormalities in DAT expression and PCR in the WKY strain (see Figure 1a). Subsequent PCR functioning have been noted in both rat strains. SHR PCR ampl Figure 1 ification of Dat1 exon 3 using three alternative primer pairs on SHR (S) and WKY (W) DNA PCR amplification of Dat1 exon 3 using three alternative primer pairs on SHR (S) and WKY (W) DNA. On WKY DNA the reaction did not work using primer set (a) and resulted in a smaller PCR product than seen using SHR DNA with sets (b) and (c). Page 3 of 5 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:24 http://www.behavioralandbrainfunctions.com/content/1/1/24 Sing Figure 2 le base polymorphism in exon 3 of Dat1: WKY (T) → SHR (C) Single base polymorphism in exon 3 of Dat1: WKY (T) → SHR (C). The polymorphism is silent, and in both strains results in anasparagine amino-acid. strains have been shown to exhibit elevated DAT expres- Conclusion sion in mesocortical projections [16,17]. It appears that In this study we have sequenced three dopaminergic genes excess DAT expression in the SHR may not be directly in two inbred rat strains considered to be good models of genetic in origin, but is in fact a response to excess meso- human psychiatric illness. No between strain differences cortical dopamine during early development resulting were observed in either the Drd2 or Drd4 genes, suggesting from hypofunctioning DAT protein that is presumably that neither gene is likely to mediate the behavioural dif- genetic [2,16]. The WKY strain also appears to have an ferences observed between the WKY and SHR strains, unusual DAT profile compared to non-depressive control although a number of polymorphisms common to both strains. Jiao et al report lower DAT density in the nucleus strains were detected in Drd4. In contrast, WKY/SHR dif- rd accumbens, amygdala, ventral tegmental area, and the ferences were observed in the 3 exon of Dat1. Whilst these mutations do not result in direct amino-acid reticular part of the substantia nigra of these animals, but higher expression in the hippocampus and hypothalamus changes to the DAT protein, it is possible that they medi- [14]. ate some other process that explains the differences in DAT expression and function observed between the two It is interesting that these findings are partially mirrored in strains. Future work should focus on further characteriz- studies on human psychiatric patients. Whilst individuals ing the genetic differences between these two strains, and with ADHD have been shown to exhibit increased DAT investigating the functional consequences of the observed density in the brain [18,19], depressive patients were polymorphisms and how they relate to the putative found to have overall decreased levels of DAT [20]. Fur- depressive and hyperactive behaviours observed in the thermore, genetic association studies suggests an associa- two strains. tion between a polymorphism in the human dopamine transporter gene (DAT1) and ADHD [21], although to Competing interests date there is no evidence linking this polymorphism to The author(s) declare they have no competing interests. the aetiology of depression. Authors' contributions JM carried out the molecular genetic work and drafted the manuscript. TS provided the animal tissue used in this Page 4 of 5 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:24 http://www.behavioralandbrainfunctions.com/content/1/1/24 21. Madras BK, Miller GM, Fischman AJ: The Dopamine Transporter: study and participated in the overall study design. PA Relevance to Attention Deficit Hyperactivity Disorder supervised the project and helped draft the manuscript. (ADHD). 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Dresel S, Krause J, Krause KH, LaFougere C, Brinkbaumer K, Kung available free of charge to the entire biomedical community HF, Hahn K, Tatsch K: Attention Deficit Hyperactivity Disor- der: Binding of [99mTc]TRODAT-1 to the Dopamine Trans- peer reviewed and published immediately upon acceptance porter Before and After Methylphenidate Treatment. Eur J cited in PubMed and archived on PubMed Central Nucl Med 2000, 27(10):1518-24. 20. Meyer JH, Kruger S, Wilson AA, Christensen BK, Goulding VS, Schaf- yours — you keep the copyright fer A, Minifie C, Houle S, Hussey D, Kennedy SH: Lower Dopamine BioMedcentral Transporter Binding Potential in Striatum During Depres- Submit your manuscript here: sion. Neuroreport 12(18):4121-5. 12-21-2001 http://www.biomedcentral.com/info/publishing_adv.asp Page 5 of 5 (page number not for citation purposes)
Journal
Behavioral and Brain Functions – Springer Journals
Published: Dec 15, 2005