Decreased behavioral activation following caffeine, amphetamine and darkness in A3 adenosine receptor knock-out mice
Introduction
Adenosine A3 receptors (A3R) are the most recently discovered of the adenosine receptors, which belong to the G protein-coupled receptor (GPCR) family, see [1]. Adenosine A3R are Gi coupled and their activation leads to both inhibition of adenyl cyclase and stimulation of inositide-dependent phospholipase C [2], [3]. They show large differences between species [2] and although valuable pharmacological tools are available for the human receptors there is a lack of really specific ligands to study the function of A3R in rodents [1]. Therefore, a recently developed A3R null mouse [4] has been useful to elucidate functions of these receptors.
Adenosine A3R mRNA is found in several regions of the brain using PCR albeit at low density. Using in situ hybridization and radioligand binding it is difficult to detect a clear distribution pattern and it was even questioned if A3 receptors are expressed in brain [5]. It is now clear that functional A3 receptors are present on microglial cells [6] and astrocytes [7], [8], [9], [10]. The presence in neurons was demonstrated by PCR in laser dissected neurons and by western blotting [11], but the functional role of A3 receptors in neurons is unclear. It has been speculated that these receptors react only to high concentrations of adenosine and that they therefore may play a role mostly during pathological conditions [12]. However, at human A3 receptors adenosine is as potent as it is at A1 and A2A receptors [13], [14].
Despite the fact that some prior studies have been performed [15] we wanted to further examine consequences of eliminating adenosine A3 receptor. Previous investigations from this [16], [17], [18] and other laboratories [19] on the functional consequences of eliminating A1R and A2AR have shown that major changes are seen only when the animals are challenged. Therefore, we first examined the behavioral effects of the non-selective adenosine receptor antagonist, caffeine, in A3R wild type (WT) and A3R knock-out (KO) mice.
Caffeine is known to be a powerful antagonist at A1, A2A and A2B receptors, but more than 10 times higher concentrations are needed to afford significant inhibition of A3 receptors [14]. Thus, it is generally assumed that at behaviorally relevant doses blockade of A3 receptors does not contribute significantly to the actions of caffeine. We were therefore surprised when, as a part of characterization of several adenosine receptor knock-out mice, we found that caffeine was behaviorally less active in A3R KO mice than in the corresponding wild type mice.
This unexpected result made us examine another stimulatory drug, amphetamine, to see if the response to this psychostimulant would as well be affected, indicating a more general mechanism. We also analysed animals at different ages and at different times of light–dark cycle. In addition, attempts were made to determine if major attenuation in dopaminergic pathways might be responsible for these effects.
Next, a possible A3 receptor function in modulating the brain response to challenge with a developmental toxicant was evaluated. We compared cerebellar functions and locomotor activity in A3R KO and WT mice exposed to the environmental pollutant methylmercury (MeHg). The brain under development is especially sensitive to the toxic effects of MeHg that reaches the fetus by crossing the placenta [20]. Some of the changes induced by MeHg were found to correlate with neurochemical alterations in the dopaminergic system [21]. We have recently shown that even with very low doses of MeHg administered it is possible to demonstrate interaction with adenosine receptor mechanisms [16].
Our tentative conclusion is that A3 receptors are important in development and their lack leads to behavioral attenuation lasting into adult life.
Section snippets
Animals
Two types of mice were used: adenosine A3 receptor deficient mice (A3R KO mice) generated by Merck Research Laboratories and wild type control mice (C57Bl/6). It has previously been described that the A3R KO mice are deficient in the adenosine A3 receptor by Southern blotting [4] which we have also verified by genotyping done using PCR. These animals were bred at the Department of Physiology and Pharmacology, Karolinska Institutet, Sweden. The A3R KO mice were backcrossed for more than 10
Adenosine receptors
We have shown that the A3 receptor gene has been knocked out from the genomic DNA in A3R KO mice by PCR (Fig. 1A) as well as that the A3R mRNA is absent in the A3R KO animals by RT-PCR (Fig. 1B).
The mRNA expression of other adenosine receptor subtypes was examined by real-time RT-PCR. As seen in Fig. 1C, there was no difference in the expression of A1, A2A or A2B receptor mRNA in the mouse striatum taken from A3R KO and WT mice, suggesting that there were no compensatory changes due to genetic
Discussion
In agreement with previous data [15], [28] we found that adult male mice carrying a genetic deletion of A3 receptors differ from the wild type male mice in that the overall locomotor activity was higher. In particular, we found that the rearing activity was considerably enhanced, but there was also an increase in locomotor activity that was evident when the arousal level was decreased in habituated mice. No genotype effect was observed in adult female mice, but this is perhaps due to the fact
Acknowledgments
We thank Eva Lindgren for help with the RT-PCR measurements. This work was supported by Karolinska Institutet, the Swedish Brain Foundation, the Tore Nilson Foundation, the Swedish Science Council (project No. 2553), European Commission (LSHM-CT2005-518189), the Swedish Society of Medicine, the Åke Wiberg Foundation and the General Maternity Hospital foundation. The funding agencies do not take any responsibility for the contents of the article.
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