Induction of reversible bidirectional social approach bias by olfactory conditioning in male mice

Abstract

Social avoidance is a common component of neuropsychiatric disorders that confers substantial functional impairment. An unbiased approach to identify brain regions and neuronal circuits that regulate social avoidance might enable development of novel therapeutics. However, most paradigms that alter social avoidance are irreversible and accompanied by multiple behavioral confounds. Here we report a straightforward behavioral paradigm in male mice enabling the reversible induction of social avoidance or approach with temporal control. C57BL/6J mice repeatedly participated in both negative and positive social experiences. Negative social experience was induced by brief social defeat by an aggressive male CD-1 mouse, while positive social experience was induced by exposure to a female mouse, each conducted daily for five days. Each social experience valence was conducted in a specific odorant context (i.e. negative experience in odorant A, positive experience in odorant B). Odorants were equally preferred pre-conditioning. However, after conditioning, mice sniffed positive experience-paired odorants more than negative experience-paired odorants. Furthermore, positive- or negative-conditioned odorant contexts increased or decreased, respectively, the approach behavior of conditioned mice toward conspecifics. Because individual mice undergo both positive and negative conditioning, this paradigm may be useful to examine neural representations of social approach or avoidance within the same subject.

Keywords: Social interaction; associative learning; olfaction; olfactory conditioning; social approach; social avoidance.

Figure 1.

C57BL/6J mice discriminate almond from banana odor but do not demonstrate an odor preference. A. Mice (n = 15) were repeatedly and sequentially presented with cotton swabs dipped in water, almond extract, or banana extract (1:100 dilutions), and the amount of time spent sniffing was quantified. Mice habituate to repeated presentations of the same odor, and dishabituate when presented with a novel odorant, demonstrating intact discrimination. B. Mice (n = 15) were given a choice between swabs dipped in almond and banana extracts (1:100 dilutions) and time spent sniffing each swab was quantified. No significant differences were observed in time spent sniffing either odorant. Data are expressed as mean ± s.e.m. ****p < 0.0001.

Figure 2.

Conditioning paradigm and testing schematic. Prior to conditioning, mice are tested in the 3-chamber maze to assess pre-conditioning odorant sniffing preference. The olfactory conditioning paradigm is then conducted daily for 5 days. C57BL/6J mice experience a negative valence interaction (interaction with an aggressive CD-1 male mouse) and a positive valence interaction (interaction with a C57BL/6J female mouse) for 10 mins each, in the presence of either almond or banana scent. Post-tests are conducted beginning in the afternoon of day 5 following the final conditioning paradigm, and were continued up until day 9. In addition to counterbalancing the odorant-interaction pairings and the order in which interactions occurred each day (depicted in the schematic), we also alternated the order of interactions experienced by any individual mouse (example for an individual mouse: day 1: female interaction followed by CD-1 interaction, day 2: CD-1 interaction followed by female, and so forth).

Figure 3.

Associative learning of odorant-social interaction pairings of positive or negative valence induced differential valuation of conditioned odors, without general locomotor or exploratory changes. A. Subject mice were tested in a three-chamber apparatus. The lateral chambers contained a porous cup with gauze soaked in diluted almond or banana extract, while the middle chamber was empty. Mice were tested prior to the conditioning paradigm (“pre-conditioning”), and then following conditioning (“post-conditioning”). The percent of time spent sniffing the odorant that was positively-conditioned was increased following the conditioning paradigm as compared to before the conditioning paradigm. Furthermore, mice sniffed the positively-conditioned odorant at a percentage greater than chance only after conditioning. B. Conditioning did not induce a significant preference in time spent in the positively-conditioned odorant chamber, nor did it increase the time spent in the chamber to greater than chance. C. Locomotion, quantified by distance traveled during three-chamber maze exploration, did not change significantly after conditioning. Data are expressed as mean ± s.e.m. n = 11 mice for all panels. #p < 0.05 one-sample t test versus 50%.

Figure 4.

Olfactory conditioning differentially modifies social approach behavior. A. Schematic of post-conditioning social interaction test. Pairs of mice with opposite conditioning experiences interacted in a clean, neutral mouse cage in the presence of almond odorant, banana odorant, or no odorant. B. The number of approaches made by each mouse during the interactions depicted in (A) were quantified during a 10-min period (n = 7 mice per each conditioning group). C. The number of approaches made by a conditioned subject mouse toward an unconditioned male conspecific was quantified during a 10-min period (n = 5 for banana-positive/almond-negative and n = 10 for almond-positive/banana-negative). D. The number of approaches shown in (C) was collapsed across odorants into interaction contexts. “Positive context” denotes that the interaction occurs in the presence of the positive experience-paired odor for the subject mouse. “Neutral context” denotes interaction in the absence of an odorant. “Negative context” denotes that the interaction occurs in the presence of the negative-experience-paired odor. There was a significant difference between number of approaches in the presence of the positive- and negative-interaction-paired odors, as well as a significant linear test for trend (p = 0.0023) across the three ordered contexts (n = 15 per context). E. The number of approaches made by the unconditioned male conspecific mice toward conditioned mice do not differ across the three odorant contexts. Data are expressed as mean ± s.e.m. **p < 0.01.

Figure 5.

Olfactory conditioning does not result in changes in immobility in a measure sensitive to chronic stress. Mice underwent olfactory conditioning and were then tested in the tail suspension test in the presence of the odor paired with positive social experience (n = 7) or paired with negative social experience (n = 8). The time spent immobile during the 6-min test was quantified, and there was no significant difference between the two groups. Data are expressed as mean ± s.e.m.