Table 2.

Animal models for the 118A>G SNP

Author	Animal model	Phenotypes	Results of either ex vivo or in vitro-related analyses
Miller et al35	770G Rhesus macaque. These non-human primates show the substitution of a C with a G at position 77 resulting in a substitution of an arginine with a proline in the orthologue μ-opioid receptor	• 77G macaques showed lower basal and ACTH-stimulated plasma Cortisol levels • 77G macaques had higher aggressive threat scores than 77C macaques	The expression of 77G-containing rhesus monkey μ-opioid receptor clones in HEK293 cells was related to a ~3.5 fold increase of μ-opioid receptor affinity for β-endorphin but not for exogenous opioid ligands
Barr et al40	77C>G Rhesus macaque. These non-human primates show the substitution of a C with a G at position 77 resulting in a substitution of an arginine with a proline in the orthologue μ-opioid receptor	• 77G macaques had higher alcohol preference than 77C homozygous subjects • After naltrexone administration 77G carriers decreased their preference compared to vehicle and no longer differed from 77C homozygous subjects
Mague et al41	112A>G mice: knock-in mouse model in which a point mutation (substitution of a A with a G in position 112) has been inserted in the mouse μ-opioid receptor gene. The 112A>G leads to the substitution of an asparagine with aspartic acid at position 38 of the amino acid sequence of the mouse receptor protein and causes the elimination of a N- glycosylation site, similarly to human 118A>G SNP	• In contrast to 112AA mice, homozygous 112G mice did not exhibit hyperactivity following acute morphine administration. In contrast to 112AA mice, 112GG mice did not develop locomotor sensitization following repeated morphine administration • Hot-plate assay: 112GG mice showed lower analgesic effect of morphine than 112AA mice. At high temperatures 112GG mice showed both higher sensitivity to pain and lower analgesic effect of morphine than 112AA mice. Following 7 days of repeated morphine injections all animals showed the occurrence of tolerance • A sex x genotype interaction is evident in behavioral responses associated to hedonia, with female mice homozygous for the 112G allele showing a reduction in both the rewarding properties of morphine and in the aversive components of naloxone-precipitated morphine withdrawal	• Ex vivo analyses of μ-opioid receptor expression and levels: mRNA was reduced in 112GG mice in several brain regions related to pain, stress and reward (PAG, hypothalamus, VTA NAc and cortex). Receptor protein levels were reduced in 112GG animals compared to 112AA mice, particularly in the thalamus • Variant receptors showed binding affinity for β-endorphin, morphine and naloxone comparable to the wild-type
Ramchandani et al43	Humanized mouse genes for the μ-opioid receptor: in these mice the first exon of the mouse μ-opioid receptor gene has been replaced by the corresponding human sequence carrying either 118A or 118G allele	• Microdialysis on humanized mice: 118GG mice showed a 4-fold increase in DA release in striatum in response to alcohol compared to 118AA mice • PET study on humans using [11C]-raclopride (an antagonist of D2 receptors): 118AG subjects showed greater DA release than 118AA individuals in striatum after an intravenous alcohol challenge • This mouse model shares a specific neurochemical pattern with humans	• Expression of humanized μ-opioid receptor mouse gene in CHO cells: variant receptors showed binding affinity for β-endorphin comparable to the wild-type • Electrophysiological analyses in isolated trigeminal ganglion neurons: there were no genotype differences in Ca++ currents in response to the μ-opioid receptor agonists DAMGO and β-endorphin • [3H]-DAMGO binding on mouse brain sections did not show genotype differences in receptor densities across a number of brain regions examined (ventral and dorsal striatum and VTA)
Mahmoud et al48	Humanized mouse genes for the μ-opioid receptor: in these mice the first exon of the mouse μ-opioid receptor gene has been replaced by the corresponding human sequence carrying either 118A or 118G allele	118AA mice had a greater analgesic response to morphine compared to 118GG mice	• Sensory neurons isolated from 118G homozygous humanized mice show a fivefold reduced potency of morphine, (but not of fentanyl) in inhibiting voltage-gated calcium channels downstream μ-opioid receptors compared with neurons isolated from 118A homozygous mice • Biophysical parameters (cell size, current density and peak current amplitude potential) were the same in both groups of neurons

Abbreviations: ACTH, adrenocorticotropic hormone; HEK293, human 293 embryonic kidney cells; PAG, periaqueductal gray; VTA, ventral tegmental area; NAc, nucleus accumbens; DA, dopamine; PET, positron emission tomography; CHO, Chinese hamster ovary; DAMGO, [D-ala2,MePhe4,Gly(ol)5]enkephalin.