Neonatal melanocortin receptor agonist treatment reduces play fighting and promotes adult attachment in prairie voles in a sex-dependent manner

Abstract

The melanocortin receptor (MCR) system has been studied extensively for its role in feeding and sexual behavior, but effects on social behavior have received little attention. α-MSH interacts with neural systems involved in sociality, including oxytocin, dopamine, and opioid systems. Acute melanotan-II (MTII), an MC3/4R agonist, potentiates brain oxytocin (OT) release and facilitates OT-dependent partner preference formation in socially monogamous prairie voles. Here we examined the long-term impact of early-life MCR stimulation on hypothalamic neuronal activity and social development in prairie voles. Male and female voles were given daily subcutaneous injections of 10 mg/kg MTII or saline between postnatal days (PND) 1-7. Neonatally-treated males displayed a reduction in initiated play fighting bouts as juveniles compared to control males. Neonatal exposure to MTII facilitated partner preference formation in adult females, but not males, after a brief cohabitation with an opposite-sex partner. Acute MTII injection elicited a significant burst of the immediate early gene EGR-1 immunoreactivity in hypothalamic OT, vasopressin, and corticotrophin releasing factor neurons, when tested in PND 6-7 animals. Daily neonatal treatment with 1 mg/kg of a more selective, brain penetrant MC4R agonist, PF44687, promoted adult partner preferences in both females and males compared with vehicle controls. Thus, developmental exposure to MCR agonists lead to a persistent change in social behavior, suggestive of structural or functional changes in the neural circuits involved in the formation of social relationships.

Keywords: Early experience; Melanocortin receptor; Melanotan-II; Oxytocin; Prairie voles; Social behavior.

Figure 1. Experimental design

In Experiment 1, neonates were injected daily from PND1-7 with 10 mg/kg of melanotan-II (MTII) and either tested for juvenile play behavior (Sal, n = 11f, 16m; MTII, n = 11f, 15m) or adult partner preference (Sal, n = 15f, 10m; MTII, n = 18f, 12m). In Experiment 2, PND6-7 neonates were sacrificed 1-hr after an acute injection of 10mg/kg MTII or saline, or handling only (H, handled). Either immunohistochemistry (IHC) for hypothalamic neuropeptide activation (n = 6H, 4 sal, 4MTII) was performed or plasma corticosterone was assayed (n = 8H, 8sal, 9MTII). In experiment 3, neonates were injected daily with a melanocortin-4 agonist (MC4R-A, PF-446687, Pfizer) and tested for partner preference in adulthood (Vehicle, n = 9f, 9m; 0.1 mg/kg, n = 10f, 15m; 1 mg/kg, n = 9f, 15m). Animal numbers are outlined in tables on the right. F, female; M, male. PPT, Partner preference test.

Figure 2. Daily neonatal MTII treatment reduced weight gain and induced darkened pigmentation

Prairie voles injected with MTII from PND1-7 weighed less than saline controls on PND2-7 (A), which was normalized by weaning at PND21 (B). Although not empirically quantified, daily MTII led to fur darkened pigmentation in neonates (C, left Sal, right MTII). Darkened pigmentation from early MTII injections was still observed at weaning (D, left Sal, right MTII). Asterisks in A indicate post-hoc Student’s t-tests with p<0.05

Figure 3. Daily neonatal MTII reduced male juvenile play bouts

Between PND24-29, juvenile prairie voles injected with MTII or saline from PND1-7 were singly housed and tested for play behaviors for 10min with a novel untreated stimulus animal over a 3d period. Bouts of initiated play increased over time in saline males, but remained stable in MTII males (A). On the third testing day, MTII male play was significantly lower than saline male play (B), which was driven by a decrease in boxing and wrestling/tackling (C). MTII treated females displayed greater levels of sniffing than saline females on the third day of testing (D). Asterisks in A-B indicate post-hoc Student’s t-tests with p<0.05 and in C-D indicate Mann-Whitney U tests with p<0.05. AG, aggressive grooming; B, boxing; C, chasing; Pi, Pinning; Po, pouncing; PB, pulling/biting; Su, supine; WT, wrestling/tackling; Allo, allogrooming; GI, genital investigation; H, huddling; Sn, sniffing; SC, social contact.

Figure 4. Daily neonatal MTII facilitated adult female partner preference

In females, early MTII treatment facilitated adult partner preference after an abbreviated 6 hr cohabitation without mating (A). MTII treatment had no effect on male partner preference after 24- or 48-hr of cohabitation with an unprimed female (B). Asterisk indicates planned Student’s t-tests with p<0.025.

Figure 5. MTII-induced neuropeptide activation in the PVN

PND6-7 neonates were euthanized 1hr after an acute peripheral injection of 10mg/kg MTII and representative images of brains processed for immunohistochemistry for OT-EGR1 (A,B,C), AVP-EGR1 (D,E,F), and CRF-EGR1 (G,H,I) are shown. The left panel was handled (A,D,G), the middle was injected with saline (B,E,H), and the right received MTII (E,F,I). In fluorescent images, OT and AVP positive cell bodies are green and EGR1-labeled nuclei are red. In DAB processed slices, CRF cell bodies are brown, and EGR1 nuclei are black. MTII elicited significant activation in OT, AVP, and CRF neurons, whereas saline only activated CRF cells in comparison to handled controls (J). MTII injected neonates mounted a corticosterone response (K). H, handled. Color channels were adjusted in Image J (NIH) for fluorescent images. Asterisks indicate Student’s t-tests with p< 0.05. Scale bar represents 50 μM.

Figure 6. Daily neonatal treatment with the specific MC4R agonist PF446687 facilitated partner preference in both sexes

Daily injection with either 0.1mg/kg or 1mg/kg PF446687 did not lead to a significant weight loss in neonates (A) or juveniles (B) in comparison to vehicle (Veh)-injected controls. Prairie vole females injected with the either dose of PF446687 from PND1-7 spent significantly more time huddling with the partner over the stranger after an abbreviated 6hr cohabitation (C). Only those males injected with the high dose of PF446687 spent more time with the partner at both 24 and 48hr of cohabitation with an unprimed female (D). Asterisks indicate planned Student’s t-tests with p < 0.017.