Since the discovery of receptor cloning and heterologous expression, novel compounds are evaluated at human receptors (the ultimate drug target); however, early in vivo studies are performed in rodents, generally rats (a common laboratory species) [1]. It is said that laboratory rats were already in use by 1850 and are most probably the first mammalian species bred specially for biological testing, given that a rat resembles the human body’s physiology (notably, rat and human neural networks are comparable) [2, 3, 4].
In our search for novel adenosine receptor ligands, our group determines a test compound’s binding affinities at rat adenosine A1 and A2A receptors – more specifically at male rat adenosine receptors. This is also the case with other researchers studying adenosine receptors [5, 6, 7]; although, many do not state whether male or female rat brain membranes were used [8, 9, 10, 11, 12].
The rat is genetically well-characterized: In both humans and rats, the adenosine A1 receptor subtype contains 326 amino acids and amino acid sequence homology is 95%. The adenosine A2A receptor subtype is the largest subtype and contains 412 amino acids in humans and 410 in rats with 82% amino acid sequence homology [1].
It is vital to account for sex as a biological variable to ensure reproducibility in biomedical research [13, 14, 15]; yet a reason for using male (instead of or in addition to female) rodents are seldom given. Indeed, most behavioral studies using rodents use male rodents only, seeing as researchers fear that hormonal changes during the oestrous cycle cause greater variability [16] (as well as increased costs) [13, 17]. Beery (2018) found that the ratio of male to female test subjects was 5:1 in neuroscience rodent studies [18]; furthermore, male-only studies seem to be increasing [19]. Seeing as only male rats are used, female rats are more often than not culled, though the justification for culling is controversial [20]. Female rodents are occasionally used because of ethical or economic reasons [16].
Behavioral studies have reported that female rodents are not more variable than male rodents across diverse biological traits [21, 22, 23]. In a meta-analysis of neuroscience studies, Becker et al. (2016) found that even when female rats are used in neuroscience experiments (without regard for their oestrous cycle), their data is not less consistent than their male counterparts [13]. As stated similar results have been obtained for gene expression in humans versus rats [24].
Interestingly, neurological conditions such as Parkinson’s disease, depression, and dementia, among others (notably, adenosine receptors are associated with the potential treatment of these diseases), affect women and men differently; therefore, it is reasonable that rodent models of these diseases include both male and female subjects [13, 21].
In the drug discovery process, in vivo animal studies follow adenosine receptor affinity and selectivity determination (if a promising drug candidate is identified). Considering the latter, in vitro radioligand binding assays utilizing rat membranes expressing adenosine receptors are relevant. This pilot study aims to determine the binding affinities of four well-known adenosine receptor ligands (often used as reference standards) at rat adenosine A1 and A2A receptors using male and female rat whole brain (expressing A1) and striatal (expressing A2A) membranes. To the best of our knowledge, we compare and document for the first time the variance in the inhibition constant (Ki) values of the reference standards at male and female rat adenosine A1 and A2A receptors. Based on these results, we may provide evidence for the use of both male and female rats for in vitro testing of adenosine receptor ligands.