Intranasal oxytocin compensates for estrus cycle-specific reduction of conditioned safety memory in rats: Implications for psychiatric disorders

Abstract

Stress and anxiety disorder patients frequently fail to benefit from psychotherapies which often consist of inhibitory fear learning paradigms. One option to improve the therapy outcome is medication-enhanced psychotherapy. Research in humans and laboratory rodents has demonstrated that oxytocin (OT) reduces fear and facilitates fear extinction. However, the role of OT in conditioned safety learning, an understudied but highly suitable type of inhibitory fear learning, remains to be investigated. The present study aimed at investigating the effect of intranasal OT on conditioned safety. To test this, Sprague Dawley rats (♂n = 57; ♀n = 72) were safety conditioned. The effects of pre-training or pre-testing intranasal OT on conditioned safety and contextual fear, both measured by the acoustic startle response, and on corticosterone plasma levels were assessed. Furthermore, the involvement of the estrous cycle was analyzed. The present data show that intranasal OT administration before the acquisition or recall sessions enhanced conditioned safety memory in female rats while OT had no effects in male rats. Further analysis of the estrus cycle revealed that vehicle-treated female rats in the metestrus showed reduced safety memory which was compensated by OT-treatment. Moreover, all vehicle-treated rats, regardless of sex, expressed robust contextual fear following conditioning. Intranasal OT-treated rats showed a decrease in contextual fear, along with reduced plasma corticosterone levels. The present data demonstrate that intranasal OT has the capacity to compensate deficits in safety learning, along with a reduction in contextual fear and corticosterone levels. Therefore, add-on treatment with intranasal OT could optimize the therapy of anxiety disorders.

Keywords: Anxiety disorder; Corticosterone; Fear; Fear inhibition; Oxytocin; Safety learning.

Fig. 1

Experimental Design of the Study and Intranasal Application Procedure. Upon one week of handling to the intranasal application procedure, the first blood sample (−3 Day) was drawn. On Day 1 and 2, rats were submitted to startle baseline sessions for habituation. 24 h later, rats underwent the pre-test (Day 3), followed by two safety conditioning sessions (Day 4 and 5). On the last day, the rats' memory for conditioned safety was tested in the recall session (Post-Test, Day 6). Intranasal administration of oxytocin either took place before each of the two conditioning sessions (pre-conditioning, A) or before the recall session (post-conditioning, B). Blood samples were drawn as indicated. (C) Holding position of the rat during the intranasal administration procedure.

Fig. 2

Pre- or post-conditioning intranasal administration of oxytocin does not facilitate conditioned safety memory in male rats but reduces plasma corticosterone levels along with diminished contextual fear-potentiated startle. In male rats, intranasally administered oxytocin (OT) before safety conditioning (A-C) or before the recall session (D-F) did not facilitate conditioned safety memory but reduced contextual fear and plasma corticosterone. Context startle was measured by assessing the baseline startle of the Pre-Test and Post-Test (A and D). Intranasal applications of VEH or OT pre- (A) or post-conditioning (D) revealed that while there was no difference between treatment groups in startle magnitude during the baseline before safety conditioning (Pre-Conditioning), while context startle in the recall session (Post-Conditioning) was increased in VEH-treated rats only, suggesting contextual fear conditioning (A and D) (*p < 0.05, comparison to Pre-Conditioning). Male rats that received intranasal OT pre-conditioning or post-conditioning did not display a significant increase in context startle (A and D). Pre-conditioning, the to-be-learned safety CS had no effect on the startle response, neither in VEH nor in OT-treated male rats (B, left panel; E, left panel). In the recall session (Post-Conditioning), both treatment groups significantly attenuated their startle magnitude upon presentation of the safety CS (B, right panel; E, right panel) (**p < 0.01, comparison to Startle Alone). There was no effect of treatment on the effect of the safety CS. Intranasal administration of OT pre- and post-conditioning significantly reduced plasma corticosterone (CORT) levels (C and F) (*p < 0.05, **p < 0.01, comparison to VEH-treated rats; #p < 0.05, ##p < 0.01, within-group comparison of OT-treated rats to Pre-Test CORT levels; §§p < 0.01, within-group comparison of VEH-treated rats to Pre-Test CORT levels). Data are represented as group averages +SEM. Numbers depicted in the bars represent the n of each group.

Fig. 3

Intranasal oxytocin facilitates the acquisition and recall of conditioned safety memory in female rats and reduces plasma corticosterone levels along with diminished contextual fear-potentiated startle. In female rats, intranasally administered oxytocin (OT) before safety conditioning (A-C) or before the recall session (D-F) enhanced conditioned safety memory and reduced contextual fear and plasma corticosterone. Context startle was measured by assessing the baseline startle of the Pre-Test and Post-Test (A and D). Intranasal applications of vehicle (VEH) or OT pre- (A) and post-conditioning (D) revealed that while there was no difference between treatment groups in context startle before safety conditioning (Pre-Conditioning), context startle in the recall session (Post-Conditioning) was increased in VEH-treated rats only, suggesting contextual fear conditioning (A and D) (**p < 0.01, comparison to Pre-Conditioning). Female rats that received intranasal OT pre-conditioning or post-conditioning did not display such a significant increase in context startle (A and D) (##p < 0.01 comparison to VEH-treated rats). Pre-conditioning, the to-be-learned safety CS had no effect on the startle response, neither in vehicle (VEH) nor in OT-treated male rats (B, left panel; E, left panel). In the recall session (Post-Conditioning), both treatment groups significantly attenuated their startle magnitude upon presentation of the safety CS (B, right panel; E, right panel) (**p < 0.01, comparison to Startle Alone). Notably, OT-treated females showed an enhanced recall of safety memory as compared to VEH-treated females (#p < 0.05, ##p < 0.01, comparison to VEH treatment). Alongside, intranasal administration of OT pre- and post-conditioning significantly reduced plasma corticosterone (CORT) levels in female rats (C and F) (*p < 0.05, **p < 0.01, comparison to VEH-treated rats; #p < 0.05, ##p < 0.01, within-group comparison of OT-treated rats to Pre-Test CORT levels; §§p < 0.01, within-group comparison of VEH-treated rats to Pre-Test CORT levels). Data are represented as group averages +SEM. Numbers depicted in the bars represent the n of each group.

Fig. 4

Estrus cycle influences the recall of safety memory in vehicle-treated rats but has no effect on plasma corticosterone levels. (A) The effect of the phase of the estrus cycle at the time point of the recall session was investigated in all females, regardless of when IN OT was administered (A, left panel). While VEH-treated females in the Pro/Est showed enhanced recall of safety memory, this effect was not apparent in VEH-treated females in the Met/Die. In OT-treated female rats, phase of the estrus cycle did not seem to matter as both groups showed enhanced safety memory (A, left panel) (**p < 0.01, between-group comparison; ##p < 0.01, within-group comparison). Splitting the groups based on the time point of IN OT treatment showed a similar pattern (A, right panel). (B) The influence of cycle phase or OT-treatment on plasma corticosterone (CORT) levels was further assessed in female rats (B, left panel). While there was a small trend in VEH-treated females showing less CORT when in the Pro/Est, this effect was not significant. OT-treated female rats, in turn, showed reduced plasma CORT, regardless of estrus cycle phase (B, left panel) (**p < 0.01, between-group comparison). Splitting the groups based on the time point of IN OT treatment showed a similar pattern in CORT release (B, right panel), with rats receiving IN OT pre-conditioning showed higher CORT levels in general, possibly due to undergoing a conditioning session prior to blood sampling. Data are represented as group averages +SEM. Numbers depicted in the bars represent the n of each group.