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We studied three lines of oxytocin (Oxt) and oxytocin receptor (Oxtr) knockout (KO) male mice [Oxt−/−, total Oxtr−/− and partial forebrain Oxtr (OxtrFB/FB)] with established deficits in social recognition to further refine our understanding of their deficits with regard to stimulus female's strain. We used a modified social discrimination paradigm in which subjects are singly housed only for the duration of the test. Additionally, stimulus females are singly housed throughout testing and are presented within corrals for rapid comparison of investigation by subject males. Wild-type (WT) males from all three lines discriminated between familiar and novel females of three different strains (C57BL/6, BALB/c and Swiss-Webster). No KO males discriminated between familiar and novel BALB/c or C57BL/6 females. Male Oxt−/− and Oxtr−/− mice, but not OxtrFB/FB mice, discriminated between familiar and novel Swiss-Webster females. As this might indicate a global deficit in individual recognition for OxtrFB/FB males, we examined their ability to discriminate between females from different strains and compared performance with Oxtr−/− males. WT and KO males from both lines were able to distinguish between familiar and novel females from different strains, indicating the social recognition deficit is not universal. Instead, we hypothesize that the Oxtr is involved in ‘fine’ intrastrain recognition, but is less important in ‘broad’ interstrain recognition. We also present the novel finding of decreased investigation across tests, which is likely an artifact of repeated testing and not because of stimulus female's strain or age of subject males.
Social recognition, the ability to distinguish familiar from novel conspecifics, is imperative for the display of appropriate social behaviors (Ferguson et al. 2002; Winslow & Insel 2002). Oxytocin (Oxt) administration facilitates social recognition (Benelli et al. 1995; Dluzen et al. 1998; Popik & van Ree 1991; Popik et al. 1996) and the development of knockout (KO) mice provided further evidence of Oxt's role in social recognition. Oxt KO mice (Oxt−/−) fail to distinguish between familiar and novel conspecifics (Ferguson et al. 2000, 2001). Two independently derived lines of Oxt receptor KO (Oxtr−/−) mice also have social recognition deficits (Lee et al. 2008; Takayanagi et al. 2005), as does a relatively forebrain-specific Oxtr KO (OxtrFB/FB) (Lee et al. 2008). However, the social recognition deficit differs between Oxtr−/− and OxtrFB/FB males when given the same social recognition task (two trial), whereas OxtrFB/FB males show no social recognition deficit on the five-trial habituation/dishabituation task (Lee et al. 2008). These results indicate a need for further examination of the social recognition deficits of these two Oxtr KO mouse lines.
To do so, we have developed a modified version of the two-trial social discrimination task previously used by Engelmann et al. (1995). However, here we use group-housed subject males, which show better social memory than singly housed subject males (Kogan et al. 2000). We also use singly housed stimulus females to prevent transfer of major urinary proteins between females, which could reduce recognition of individuals by a confusion of odors (see Cheetham et al. 2007). Stimulus females are presented within corrals, which reduces direct contact between the subject and stimulus animals while still eliciting high interest and investigation by the subject (Choleris et al. 2003; Kudryavtseva et al. 2002). As males cannot directly contact corralled females there is no need to extinguish sex behavior before the test, as with other social recognition paradigms (see Ferguson et al. 2001; Lee et al. 2008). Finally, presenting females in corrals allows the use of gonadally intact females, which elicit higher interest from males than ovariectomized females when contact is prevented (Muroi et al. 2006).
The modified two-trial social discrimination task was used to examine social recognition in male Oxt, Oxtr and OxtrFB/FB KOs and their male wild-type (WT) siblings. The ability of WT and KO males from each line to discriminate between females of the same strain was examined to determine whether using stimulus females from inbred (C57BL/6 or BALB/c) or outbred (Swiss-Webster: SW) strains affects recognition abilities. WT males from all three lines distinguish between individual females of the same strain. Oxt−/− and Oxtr−/− males distinguish between SW females, but not females from the inbred strains. However, OxtrFB/FB males have a complete inability to discriminate between females of the same strain. Therefore, the ability of OxtrFB/FB males to distinguish between females from different strains is assessed and compared with Oxtr−/− males. We find that the discrimination deficit is not universal, but is instead specific to intrastrain recognition.
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We used a modified version of a social discrimination task to assess social recognition memory in WT and KO males from Oxt, Oxtr and partial forebrain-specific Oxtr mouse lines. In agreement with previous studies using other social recognition tasks (Ferguson et al. 2000; Lee et al. 2008; Takayanagi et al. 2005), WT males from all three lines discriminated between familiar and novel stimulus females of the same strains (Figs 2–7). This study is the first to show intact social recognition in WT males independent of stimulus female's strain, but impaired social recognition in KO males dependent upon stimulus female's strain. Oxt−/− and Oxtr−/− males did not discriminate between familiar and novel BALB/c or C57BL/6 females (as shown previously in Oxtr lines: Lee et al. 2008; Takayanagi et al. 2005), but did discriminate between familiar and novel outbred SW females (contrary to Choleris et al. 2003 in a different paradigm; Figs 2 and 3). The ability to discriminate between SW females may be specific to the strain and not generalizable to all outbred strains, as Oxt KO mice cannot distinguish in another paradigm between CD-1 mice (Ferguson et al. 2000, 2001).
Feral female mice are more likely to mate within their own strain than with a male from another strain. Oxt−/− females are unable to successfully discriminate between odors from healthy males and those infected with a parasite (Kavaliers et al. 2003). Oxt is therefore implicated in the ability to choose a mate who is parasite-free, more fit and is less likely to infect the female during mating (see Kavaliers et al. 2005), consistent with an increased emphasis on the role of Oxt in intrastrain recognition.
Unlike the Oxt and Oxtr KO males, OxtrFB/FB males did not discriminate between familiar and novel females regardless of stimulus strain, including SW (Fig. 4). OxtrFB/FB males spent only 60% of their time investigating the novel SW females compared with 69% in Oxtr−/− males and 80% in Oxt−/− males. We have previously reported a general decrease in investigation of all stimulus animals (whether familiar or novel) in OxtrFB/FB males, but a maintained recognition memory on the habituation-dishabituation social recognition task (Lee et al. 2008). The current results refine those obtained with the two-trial social recognition task (Lee et al. 2008), clarifying the deficit in social memory in the OxtrFB/FB line.
The targeted mutation is the same in the Oxtr−/− and OxtrFB/FB mice and the three strains differ slightly in genetic background (see Methods). However, as performances on the social discrimination task by WT males from the Oxt and Oxtr lines are identical, it is unlikely that background strain plays a large role. Both total Oxt−/− and Oxtr−/− males show the same social discrimination deficit, indicating that 129Sv flanking DNA is unlikely to be responsible. The presence of Cre recombinase allele in the OxtrFB/FB males could account for the inability of OxtrFB/FB males to discriminate between SW females. However, in Experiment 2 heterozygous mice (for both Cre recombinase and forebrain Oxtr inactivation) had nearly identical performances to the WT males, and discriminated between familiar and novel SW females. Therefore, Experiment 2 indicates that the Cre recombinase transgene does not account for the social recognition defect in OxtrFB/FB males.
Instead, the results from Experiments 1 and 2 indicated that OxtrFB/FB males may have a general inability to recognize individuals. To test this hypothesis, we examined the ability of OxtrFB/FB and Oxtr−/− males to discriminate between females from different strains. We hypothesized that OxtrFB/FB males would not make this discrimination, but WT males and Oxtr−/− males would. Contrary to the hypothesis, WT and KO males from both lines were equally able to discriminate between familiar and novel females of different strains (Fig. 6). Why should OxtrFB/FB males be unable to distinguish between females of the same strain, but able to distinguish between females of different strains? One possibility may be that the postnatal loss of the Oxtr in the OxtrFB/FB in contrast to never expressing Oxtr in the total Oxtr KO leads to the difference. Another possibility may lie with the distribution of the Oxtr within the brain. Deletion of the Oxtr in the OxtrFB/FB conditional line is not complete, with Oxtr expression remaining within the medial amygdala, olfactory bulb and neocortex, but lacking in the hippocampal formation (Lee et al. 2008). As both the amygdala and the hippocampal formation (including the entorhinal cortex) are important for individual recognition (Ferguson et al. 2001; Petrulis 2009, respectively), the altered Oxtr expression pattern in the OxtrFB/FB conditional line may prevent OxtrFB/FB males from discriminating between females of the same strain, even if outbred, but allow discrimination between the greatly different odors of females from different strains. In other words, lack of and/or presence of Oxtr in the above-mentioned regions interferes with ability to make ‘difficult’ discriminations (i.e., between females of the same strain), but is not involved in making ‘easy’ discriminations (i.e., between females of different strains). Results from Oxtr KO males indicate that in the complete absence of Oxtr mice are able to distinguish between different strains. Combined, our data indicates that Oxt acting at the Oxtr may be involved in more detailed individual recognition, but some other peptide or hormone may underlie broad recognition of individuals. For example, follow-up studies using this social discrimination paradigm in vasopressin 1a and 1b receptor KO males are planned to determine whether the closely related peptide may be involved in more general individual recognition.
WT males from all three lines were very consistent in their investigation times, with all spending approximately 75% of the time with the novel females on test 1; 63–68% of the time investigating the novel females on test 2 and 65–70% of the time investigating the novel females on test 3. In contrast, the two-trial social recognition tasks may result in greatly different investigation times of familiar females (compared to novel) (e.g., only ∼ 40% in Lee et al. 2008, but ∼ 75% in Ferguson et al. 2000). Inconsistent performance across KO lines when utilizing the same task makes it difficult to compare results directly. We hope that the current task, which uses group-housed subjects and singly housed gonadally intact stimulus animals contained within corrals, will result in consistent performance across labs using C57BL/6 and C57BL/129 background lines. We have found highly correlated scores from two different intralab investigators (r = 0.991; data not shown), indicating that with appropriate training and exposure to the task, scoring is consistent across investigators.
There is a possible confound of estrous state influencing male investigation with use of gonadally intact females, which were chosen as they elicit higher interest from males than ovariectomized females. However, the day of estrous cycle has previously not been shown to greatly influence male investigation (Muroi et al. 2006). Females in all states of estrous elicit higher investigation from males than do ovariectomized females (Ingersoll & Weinhold 1987). Sexually naïve males (as are the subjects in the current experiments) indicate no preference between receptive and non-receptive females' odors (Carr et al. 1965). Furthermore, subjects of both genotypes would experience any effect of estrous state, thereby eliminating possible genotype × estrous condition interaction.
We believe this study is the first to repeatedly test males' social recognition memory. We find that repeated testing does not interfere with discrimination ability, but does generally result in decreased investigation times by test 3. A previous study indicates that mice from C57BL/6 and 129S2/SvHad strains decrease exploration and activity with repeated testing and handling (Voikar et al. 2004). Males were 2–3 months older at the third test compared with the first. However, we found no overall effect of age on investigation times for both Oxtr+/+ and OxtrFB/FB males (Fig. 7). It is therefore likely that the decrease in investigation in the current study is an artifact of repeated testing. Furthermore, as in the third test in Experiment 1, Experiment 2 (Fig. 5) shows that WT males discriminated in a single test between familiar and novel SW females, while OxtrFB/FB males did not. These results indicate that the social recognition deficit observed in Experiment 1 is true, and not an artifact of repeated testing. Thus, Experiment 2 suggests that order of presentation of stimulus female's strain does not affect social recognition ability in OxtrFB/FB males. These results do underscore the importance of limiting testing history for animals used in social recognition tests, as previous testing can impact willingness to investigate.
The current study expands upon earlier findings that Oxt and the Oxtr are important in social recognition. Our results indicate that Oxt and the Oxtr are less important for ‘broad’ social recognition (interstrain), but are seemingly more important for ‘fine’ social recognition (intrastrain). These results were obtained utilizing a modified social discrimination paradigm that generates very similar investigation times by WT males of stimulus females across all lines tested.