Oxytocin receptor is not required for social attachment in prairie voles

Abstract

Prairie voles are among a small group of mammals that display long-term social attachment between mating partners. Many pharmacological studies show that signaling via the oxytocin receptor (Oxtr) is critical for the display of social monogamy in these animals. We used CRISPR mutagenesis to generate three different Oxtr-null mutant prairie vole lines. Oxtr mutants displayed social attachment such that males and females showed a behavioral preference for their mating partners over a stranger of the opposite sex, even when assayed using different experimental setups. Mothers lacking Oxtr delivered viable pups, and parents displayed care for their young and raised them to the weanling stage. Together, our studies unexpectedly reveal that social attachment, parturition, and parental behavior can occur in the absence of Oxtr signaling in prairie voles.

Keywords: CRISPR; monogamy; nursing; oxytocin receptor; pair-bonding; parental behavior; partner preference; prairie vole; social attachment.

Figure 1.. CRISPR mutagenesis yields multiple null alleles of Oxtr in prairie voles

A. Schematic of CRISPR-based targeting to generate Oxtr mutant prairie voles. Single cell embryos injected with Cas9-sgRNA ribonucleoprotein were cultured (1) to the blastocyst stage and transferred (2) to pseudopregnant recipient females who carried the embryos to term (3). B. Schematic (top) of Oxtr locus encompassing first two exons. DNA sequence of WT and targeted Oxtr alleles. Dash/missing nucleotide represents a deletion (Oxtr⁴, Oxtr⁵) and red highlighted “C” (Oxtr¹) is an insertion. PAM, protospacer adjacent motif. C. Predicted amino acid sequence of WT Oxtr, Oxtr¹, Oxtr⁴, and Oxtr⁵ (only first 100 amino acids shown). See also Figure S1.

Figure 2.. Oxtr mutant voles lack functional, ligand-binding Oxtr

A. Loss of binding with the competitive agonist 125I-OVTA visualized in coronal sections through the rostral telencephalon: Left: Labeling in PFC (prefrontal cortex), NAcc (nucleus accumbens), and CeA (central amygdala) of WT sibling controls; Middle: Labeling in equivalent sections through PFC, Nacc, and CeA from Oxtr¹ homozygous mutant voles; Right: The same mutant sections as in the middle panels, stained for Acetylcholine esterase to demonstrate equivalence of sections chosen for WT and mutants. B. Non-specific binding (NSB) control shows no off-target binding in WT or Oxtr¹ mutant sections. C-E. Optical density-based quantification of binding to 125I-OVTA shows that binding is essentially undetectable in mutants null for Oxtr¹ in PFC (C), NAcc (D), or CeA (E). Scale bar =5 mm; boxplot depicts max-minScale bar =5 mm; boxplot depicts max-min, midline denotes mean; n=3 for WT and mutant males and females (C-E). Scale bar =5 mm; boxplot depicts max-minSee also Figure S2.

Figure 3.. Female and male prairie voles lacking Oxtr exhibit pair bonding Scale bar =5 mm; boxplot depicts max-min

A-B. Schematic of partner preference test performed in a branched (A) or linear chamber with two partitions (B). Experimental vole is free to move between chambers (A) or partitioned space (B) whereas stimulus voles (opposite sex partner and stranger) are restrained by tethers. Scale bar =5 mm; boxplot depicts max-min C-E. WT, Oxtr^4−/− (C), Oxtr^5−/− (D), and Oxtr^1−/− (E) voles spent more time with their partner than a stranger of the opposite sex. F-H. WT, Oxtr^4−/− (F), and Oxtr^5−/− (G) voles attacked the stranger more frequently than their partner. WT Oxtr¹ males attacked the stranger more frequently (H). Mean ± SEM; n = 25 WT and 21 mutant males, 19 WT and 21 mutant females (C); 27 WT and mutant males each, 19 WT and 21 mutant females (D); 8 WT and 8 mutant males, 8 WT and 8 mutant females each (E); 15 WT and 14 mutant males, 9 WT and 11 mutant females (F); 18 WT and mutant males each, 9 WT and 12 mutant females 15 (G); 8 WT and mutant males and females each (H); *p<0.05, **p<0.01, ***p<0.001; N.S., not significant. See also Figure S3.

Figure 4.. Prairie voles lacking Oxtr display bi-parental care and can raise pups to weaning

A-F. WT, Oxtr^4–/–, and Oxtr^5–/– mothers and fathers exhibit equivalent nest occupancy (A, D), pup-directed contacts (B, E), and nursing behavior (C, F) with their pups. G, J. Oxtr^4–/– mothers wean fewer litters compared to WT mothers. H, K. Litters from Oxtr^4−/− and Oxtr^5−/− mothers have fewer pups surviving to weaning compared to WT mothers I, L. Oxtr^4−/− mothers and fathers and Oxtr^5−/− mothers weaned pups with significantly lower body weight compared to WT parents. Only litters with ≥1 pup surviving to weaning were analyzed. Mean ± SEM; n = 9 WT and mutant males each, 10 WT and 5 mutant females (A-C); 10 WT and mutant males each, 10 WT and 9 mutant females (D-F); 30 WT and 25 mutant males, 30 WT and 23 mutant females (G,H); 29 WT and 25 mutant males, 30 WT and 11 mutant females (I); 17 WT and 16 mutant males, 13 WT and mutant females each (J,K); 17 WT and 16 mutant males, 12 WT and 8 mutant females (L);*p<0.05, **p<0.01, ***p<0.001; N.S., not significant. See also Figure S4.