Comprehensive behavioral phenotyping of multiple cohorts of neuroligin-3 R451C knockin mice and their littermate controls revealed that the NL3 mutation did not significantly affect reciprocal social interactions in juveniles or social approach in adults. No perseveration was detected in any genotype on ability to reverse a spatial position habit in the water maze. The great majority of measures of developmental milestones, pup separation vocalizations, general health, home cage behaviors, reflexes, sensory abilities including olfaction and nociception, and motor functions including locomotion, balance, and gait, were normal in NL3 male and female mice. Taken together, these corroborating results do not support the hypothesis that an R451C point mutation in NL3 produces behaviors in mice that are analogous to the first and third diagnostic symptoms of autism, abnormal reciprocal social interactions and repetitive restricted interests. Furthermore, generally similar scores in NL3 and WT on the initial acquisition of the location of a hidden platform using distal environmental cues in the Morris water maze spatial task, along with similar scores on emotional learning and memory in contextual and cued fear conditioning, do not support the hypothesis that the R451C NL3 mutation directly affects learning and memory.
Our findings of generally normal developmental milestones, physical attributes, simple procedural abilities, and complex behavioral traits in NL3 may be viewed in the broader context of the multiple neurexin and neuroligin proteins and the specific binding of their splice variants during synaptic formation (Chih et al., 2005
; Chubykin et al., 2005
; Varoqueaux et al., 2006
; Budreck and Scheiffele, 2007
; Chubykin et al., 2007
; Conroy et al., 2007
; Craig and Kang, 2007
; Levinson and El-Husseini, 2007
; Sheng and Hoogenraad, 2007
). Reduction in the cell surface expression of the neuroligin-3 protein may not be sufficient to affect a large number of synapses relevant to social and cognitive abilities, if other neuroligins remain available. However, this does not appear to be the case for neuroligin-4, as an elegant study of behavioral phenotypes in neuroligin-4 knockout mice reported reduced social interactions and ultrasonic vocalizations (Jamain et al., 2008
The present findings are at variance with some of the results and interpretations of behavioral phenotypes in another line of NL3 R451C mice (Tabuchi et al., 2007
). Both lines were normal on measures of general health, motor abilities, and anxiety-related behaviors. However, social deficits and improved learning were reported in the other line (Tabuchi et al., 2007
). Careful examination of the social behavior results shown in the previous report (, Tabuchi et al., 2007
), reveals that the NL3 R451C mutant did not differ from their WT controls on most measures of social behaviors. The first social task reported, shown in Tabuchi et al. , employed separate sequential test sessions. The subject mouse was first given five minutes to explore a novel object, and then given five minutes to explore a novel mouse. Unlike the many excellent and well-validated assays of social interaction used routinely by behavioral neuroscientists and behavioral neuroendocrinologists, this design of separate sequential opportunities is not a validated measure of interest in a social partner. Too many other factors could influence time spent with the novel mouse, such as generally increased exploration or heightened interest in the wire mesh container during the second five minutes.
Tabuchi et al. employed the three-chambered social approach task which we originally developed and validated (Moy et al., 2004; Nadler et al., 2004; Yang et al., 2007
; McFarlane et al. 2008
). The habituation and test sessions were performed using methods consistent with the ones in this manuscript. However, the statistical analyses were improperly conducted, using only pairwise t-tests. In fact, the data in of Tabuchi et al., 2007
appear to show that both genotypes spent more time with the novel mouse than with the novel object. It seems likely that both strains exhibited significant sociability, if properly compared with ANOVA statistics. This evidence for significant sociability in the NL3 group argues against a social deficit in the NL3 line tested in Tabuchi et al.
Further, in the conventional test for initial reciprocal social interactions between unfamiliar pairs of mice, there was no difference between WT and NL3 in the previous report ( panel D, Tabuchi et al., 2007
), consistent with the lack of genotype differences between WT and NL3 on reciprocal social interactions during the juvenile test session in the present findings. The test of direct social interaction in Tabuchi et al. employed only a 2 minute session, which is considerably shorter than social interaction tests used in the great majority of behavioral neuroscience assays. Further, the single parameter shown in , time spent in interaction, is a very gross composite measure. Most behavioral neuroscientists use a large number of specific parameters to characterize direct social interaction, as shown in our and 1S
. The data shown in Tabuchi et al. , left bars, clearly demonstrate that both genotypes showed the same amount of social interaction. This lack of genotype difference in their best measure of social behaviors supports an interpretation that their R451C NL3 knockins display normal social behaviors.
The data shown in Tabuchi et al. , right bars, represents a social memory task. Again, the statistical analysis was incorrectly conducted, using a pairwise t-test. As a result, a misleading interpretation was given. Given the standard error bars of , it appears unlikely that there was a genotype difference in social memory if the proper ANOVA statistical analysis had been conducted.
Faster learning curves on both the initial acquisition and reversal learning in the Morris water maze were reported for one cohort of NL3 in Tabuchi et al. (2007)
but not seen in the two large cohorts tested in the present studies, although distance traveled during acquisition was somewhat shorter in NL3 during the initial acquisition in both the present study and in Tabuchi et al. (2007)
. Different techniques for generating the knockin mutation, and different genetic backgrounds for the two NL3 lines, C57BL/6J in the present line from the Heintz laboratory versus a mixed C57BL/6J and 129/ImJ in the line from the Sudhof laboratory, along with methodological differences between the two laboratories in conducting the cognitive assays, may have contributed to the divergent results on spatial learning.
In the present studies, NL3 differed from their WT littermates on several minor but potentially interesting developmental measures. NL3 mice displayed slower righting reflexes during the first few days after birth, emitted fewer ultrasonic vocalizations at postnatal day 8, varied from WT in body weights, engaged in less vertical activity in the open field, had longer latencies to fall from the rotarod, and spent less time near the walls in the Morris water maze. Females displayed more homing behaviors and a wider stance in the footprint test. Both males and females responded less strongly to acoustic startle stimuli. The lower startle response is not necessarily indicative of impaired hearing, as acoustic thresholds were similar across genotypes, and electrophysiological recordings during the startle response indicate no direct correlation between hearing and amplitude of startle response (Willott et al., 1984
). It remains possible that the few significant differences detected were simply the outcome of a large number of statistical comparisons. However, it seems likely that the several minor genotype differences, taken together, represent small developmental abnormalities, and/or decreased arousal levels in response to sensory and motor stimuli, caused by the NL3 mutation.