Analysis of the Effect of Oxytocin Analogues on a Mouse Contextual Fear-Conditioning Behavior

Background Oxytocin, a reproductive peptide hormone, has been studied for decades due to its pleiotropic actions involving the central nervous system, but the effects of oxytocin analogues on fear conditioning are not well understood. Aims & Objectives In the present study, we examined whether oxytocin and its analog (carbetocin and atosiban) could reduce fear-conditioning behavior of mice that is often applied in the studies of dental phobia. Method Male C57BL/6J mice were used at 8 weeks after birth. Fear-conditioning experiment was conducted according to the methods in previous study (Bouchekioua et al., 2022 Bio-protocol). In the 6th day, mice were administered intranasally with 5 μL of 100 μM oxytocin, 10 mM carbetocin, 10 mM atosiban, or saline after electrical shock session. The effect of fear-conditioning was evaluated as lick-suppression ratio by following equation; (Baseline−Performance)/Baseline, where Baseline = averaged lick number on 4th and 5th day, Performance = lick number on 7th – 10th day. For immunohistologial analysis, mice brains were dissected and formalin-fixed at 30 min. after drug administration. StatPlus software (AnalystSoft Inc., CA, USA) was used to determine statistical difference. Results Lick-suppression ratio of saline-treated group (fear control) was significantly higher than that of non-conditioned group (naïve control) at 7th day (F(4,27)=5.72, p=0.002 in one-way ANOVA; p<0.05 in Tukey’s test). It was also determined that lick-suppression ratio of carbetocin-treated group was significantly lower than saline-treated group (p<0.05 in Tukey’s test). Statistically significant difference between groups was not detected at 8th – 10th day (F(4,26)=2.71, p=0.052; F(4,26)=2.21, p=0.10, and F(4,22)=0.76, p=0.56). Since amygdala and hippocampus are involved in fear memory formation in contextual fear-conditioning test, we examined the number of c-Fos-positive cells in amygdala. It was found that number of c-Fos-positive cells in lateral amygdala of fear-conditioned group statistically significantly increased compared with that of non-conditioned group (F(4,37)=6.49, p=0.0005 in one-way ANOVA; p=0.0011 in Tukey’s test). It was also found that the number of c-Fos-positive cells in lateral amygdala of oxytocin- or carbetocin-treated group statistically significantly decreased compared with that of saline-treated group (p=0.03 and 0.03 respectively in Tukey’s test). It was further found that the number of c-Fos-positive cells in basolateral amygdala of saline-, oxytocin-, carbetocin-, or atosiban-treated group statistically significantly increased compared with non-conditioned group (F(4,37)=32.03, p<0.0005 in one-way ANOVA; p<0.001). It was also found that the number of c-Fos-positive cells in basolateral amygdala of oxytocin- or carbetocin-treated group were statistically significantly decreased compared with saline-treated group (p=0.046 and 0.017 in Tukey’s test). Discussion & Conclusions The results of this study suggest that intranasal administration of carbetocin immediately after electric shock session effectively reduces fear memory formation in mice. Carbetocin selectively activates the Gq protein-related response of the oxytocin receptor, suggesting that selective responses downstream of the oxytocin receptor may contribute to the reduction of the fear conditioning effect. However, further analysis is needed to clarify which neural nuclei and neural circuits carbetocin targets.