Oxytocin and Opioid Receptor Gene Polymorphisms Associated with Greeting Behavior in Dogs

Meeting humans is an everyday experience for most companion dogs, and their behavior in these situations and its genetic background is of major interest. Previous research in our laboratory reported that in German shepherd dogs the lack of G allele, and in Border collies the lack of A allele, of the oxytocin receptor gene (OXTR) 19208A/G single nucleotide polymorphism (SNP) was linked to increased friendliness, which suggests that although broad traits are affected by genetic variability, the specific links between alleles and behavioral variables might be breed-specific. In the current study, we found that Siberian huskies with the A allele approached a friendly unfamiliar woman less frequently in a greeting test, which indicates that certain polymorphisms are related to human directed behavior, but that the relationship patterns between polymorphisms and behavioral phenotypes differ between populations. This finding was further supported by our next investigation. According to primate studies, endogenous opioid peptide (e.g., endorphins) receptor genes have also been implicated in social relationships. Therefore, we examined the rs21912990 of the OPRM1 gene. Firstly, we found that the allele frequencies of Siberian huskies and gray wolves were similar, but differed from that of Border collies and German shepherd dogs, which might reflect their genetic relationship. Secondly, we detected significant associations between the OPRM1 SNP and greeting behavior among German shepherd dogs and a trend in Border collies, but we could not detect an association in Siberian huskies. Although our results with OXTR and OPRM1 gene variants should be regarded as preliminary due to the relatively low sample size, they suggest that (1) OXTR and OPRM1 gene variants in dogs affect human-directed social behavior and (2) their effects differ between breeds.


Introduction
The three major types of opioid receptors are distributed throughout the central nervous system and periphery. Each type belongs to a subfamily of G protein-coupled receptors (Lutz and Kieffer, 2013). Mu and delta opioid receptors functionally interact in vivo. The two receptors are co-expressed in neurons from brain networks related to water and food consumption, sexual behavior, perception and responses to aversive stimuli (Erbs et al., 2014) . The distribution and/or function of κ receptor may differ between sexes (Chartoff and Mavrikaki, 2015). κ receptor is involved in stress, depression, anxiety, dysphoria (Land et al., 2008), and substance dependence in humans (Gerra et al., 2007), as well as voluntary alcohol-drinking in mice (Vadasz et al., 2000).

Subject
For the pilot study brain samples from one male beagle dog euthanized on the owner's request were obtained at the Department of Anatomy and Histology, Faculty of Veterinary Science, Szent István University, Budapest, Hungary.

Reverse transcriptase (RT)-PCR analysis and real-time PCR
Total RNA samples from three brain regions (prefrontal cortex, amygdala and hippocampus) of one male beagle dog were isolated by RNeasy kit (Qiagen, Valencia, CA). The isolated RNA was treated with DNaseI enzyme (Thermo Fisher Scientific, Waltham, MA, USA). 1 µg RNA was reverse-transcribed with M-MLV reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) using random primers in 20 µL volume. Negative control was prepared without reverse transcriptase.
For OPRK1 mRNA (NCBI, XM_544080.3) 5' CGC ACA CCC ATG AAG GCA AAG A 3' forward and 5'TGG GAA TTG CAA GGA GCA CTC GAT 3' reverse primers were used. The primers were designed to span different exons resulting in 100-150 bp PCR products. Expression levels of the dog HPRT mRNA were detected with TaqMan assay in 25 µL final volume containing 0.2 µL cDNA, 1× ABI PCR master mix, gene-specific TaqMan primers and FAM-labeled probe (Thermo Fisher Scientific, Waltham, MA, USA). Amplification and signal detection were performed using an ABI 7300 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Denaturation at 95°C for 10 min was followed by 40 thermocycles (65°C, 15 sec and 60°C, 1 min). Reactions were performed in triplicate using RNase-free water as negative control. Expression levels of opiate receptors were first normalized to the HPRT internal control gene and then to expression levels measured in the prefrontal cortex. Results were expressed as fold changes calculated with the formula 2-ΔΔCT as in Bence et al. (2016).

Results and Discussion
Relative expression of OPRM1 gene in different brain areas PCR product quality check by agarose gel electrophoresis showed no aspecific band or genomic DNA contamination. In accordance with previous results (Peckys and Landwehrmeyer, 1999), the expression patterns varied at the different brain regions ( Figure S1). According to our raph estimates, the highest expression of OPRM1 was found in the amygdala, and a lower expression was found in the hippocampus, while the mRNA level in the canine cortex was very low. These preliminary results might support the role of this receptor in emotional reactions and social behavior (Adolphs, 2003). Figure S1. Relative gene expression levels of the different opiate receptor subtypes in various brain regions. Gene expression was measured by real-time quantitative PCR, and data were analyzed using the CT-method. Expression level of the HGPRT gene was investigated as an internal control for relative quantification. All measurements were carried out in triplicates. The figure shows the relative gene expression value of each individual measurement.