The current study assessed the contributions of both the maternal VIP genotype, and the VIP genotype of offspring, to general health measures, social behavior and cognitive function. Adult WT and VIP deficient offspring of both WT and VIP deficient mothers were compared, the most salient comparison being between the VIP +/− offspring of WT and VIP deficient mothers. The most important discoveries were: 1) the VIP genotype of the mother was a greater predictor of deficiencies in her offspring than their own VIP genotype, and 2) the offspring of VIP deficient mothers exhibited deficiencies in social behavior and reversal learning that were more prevalent in male offspring.
The VIP deficient (+/−) offspring of WT mothers did not differ in a single measure from their WT littermates. The level of intrauterine VIP maintained by a WT mother during the period before the fetuses began making their own VIP apparently initiated the normal patterns of growth and development such that the subsequent deficiency in her VIP deficient offspring had no impact on the measures evaluated here. The results were more complex among the offspring of VIP deficient mothers. In some measures, for example, open field activity, all offspring of VIP deficient mothers showed the same increased activity, regardless of their own sex or genotype. However, in social approach and reversal training in the Morris water maze, sex differences were revealed. All male offspring of VIP deficient mothers had equal deficits in these measures, regardless of their own genotype. The female offspring of VIP deficient mothers had lesser deficits in these behaviors than their male littermates, with the greatest deficiencies seen in the VIP null females. Moderate deficiencies were seen in VIP +/− females, and the behaviors of WT female offspring of VIP deficient mothers were normal in most measures. Nonetheless, all VIP +/− offspring of VIP deficient mothers had deficiencies; none of the VIP +/− offspring of WT mothers had deficiencies.
While this study did not reveal major defects in general health measures in the offspring of VIP deficient mothers, these mice were more active in the open field, and the male null mutants (VIP −/−) appeared to have significant muscle weakness, confirming the previous findings of Girard et al. (2006)
. The significantly reduced grooming and lack of positional passivity of the offspring of VIP deficient mothers may be linked to the motor hyperactivity of these mice. Although none of the offspring of VIP deficient mothers vocalized during the general health examination, vocalization was not common among the offspring of WT mothers under these conditions. The general health and reflex data presented here are consistent with the attainment of developmental milestones of the VIP knockout mice (Lim et al., 2008
) in which all offspring of VIP deficient mothers exhibited only delays in the appearance of developmental milestones and were eventually able to perform all tasks.
Mice are a highly social species, and in the automated three chamber social approach apparatus used here, normal C57BL/6 mice exhibit high levels of sociability as measured by frequent approaches and interactions with an unfamiliar mouse (Moy et al., 2004
; Nadler et al., 2004
). Compared with WT mice, the male offspring of VIP deficient mothers showed severe deficits in sociability, regardless of their own genotype, in all measures of the social approach test. The deficits were less severe in the female offspring of VIP deficient mothers where all genotypes expressed some sociability in both the sociability and preference for social novelty phases of the study. However, the degree of deficit in social behavior appeared to be related to the genotype of the female mouse with the VIP null females exhibiting the greatest reductions in all phases of the test and the WT females expressing normal behavior in the of sociability phase of the test. These results were similar to those obtained from mice that had been exposed to VIP antagonist treatment during the period of E8-E10 (Hill et al., 2007a
). While male adults in these experiments exhibited significantly reduced sociability in the sociability phase of the social approach task, and no preference for social novelty, female littermates did not show these deficiencies (Hill et all, 2007a
). Furthermore, similarly treated male mice have been shown to have abnormal reactions in the social recognition task in which, despite having the capacity to differentiate between unfamiliar mice, they did not react to the introduction of a novel mouse by increased sniffing (Hill et al., 2007b
). The results of these and the current experiments support the suggestion that there was critical period in the establishment of normal social responses, and that in the male mouse it was related to the action of VIP during embryogenesis. The social responses of female mice were less affected by maternal VIP deficits and were influenced by their own VIP deficiency. Since the olfactory habituation/dishabituation test showed that the mice in the previous (Hill et al., 2007a
) and current experiments were capable of distinguishing between individual mice, their deficits appeared to be in their motivation to interact with members of their own species.
The offspring of VIP deficient mothers expressed normal learning in cued and contextual fear conditioning. And, although the offspring of VIP deficient mothers had longer latencies than the offspring of WT mothers during visible platform training in the Morris water maze, perhaps reflecting a different response to swimming or the use of a different strategy, these mice expressed no deficits in learning in the acquisition phase of spatial discrimination training, nor in memory in the probe test following acquisition training. However, the offspring of VIP deficient mothers exhibited impaired performance in the reversal phase of the Morris water maze, in which the position of the hidden platform was changed. During the first, but not subsequent days of reversal training, all male offspring of VIP deficient mothers swam over the previously correct platform position significantly more than WT males of WT mothers. On subsequent days of reversal training, the male mice did not swim significantly more often over the original platform position and appeared able to learn the new position. However, in the probe test following reversal training, male offspring of VIP deficient mothers did not prefer the new platform location over the old platform location, indicating an impaired memory for the new platform location. Among the female offspring of VIP deficient mothers the WT females were normal and showed normal reversal training and spent more time in the new target quadrant than in any of the other quadrants in the probe test. However, like their male littermates, the VIP deficient (+/−, and −/−) female offspring of VIP deficient mothers exhibited continued preference for the original platform position on day 1 of reversal training and in the probe test they did not spend more time in the new platform location than in the old platform location. The errors in performance by offspring of VIP deficient mothers in reversal learning, which examines cognitive flexibility (Coldren and Halloran, 2003
; McAlonan and Brown, 200; White, 2004
), may have been due to a slower learning process or the use of a different strategy in this situation. However, they appear to be a perseveration of the previously learned strategy (perseveration errors) rather than the inability to learn a new strategy (regressive errors) and indicate a cognitive inflexibility, or a resistance to change, in these mice (Ragozzini et al., 2002a; Ragozzini et al., 2002b; Palencia and Ragozzino, 2004
; Chadman et al., 2006
We have shown that maternal VIP deficiency had more severe outcomes in social behavior and spatial reversal learning in the male offspring than in their female littermates. The results indicate that for the normal expression of these adult behaviors, the male mouse required normal maternal levels of VIP during embryogenesis, whereas the female mouse was more dependent upon normal fetal expression of VIP. The reasons for the sex difference in the behaviors examined here are not clear. However, in the developing rodent testes, testosterone secretion is stimulated by VIP (Romanelli et al., 1997
: El-Gehani et al., 1998a
; El-Gehani et al., 1998b
), and reduced maternal VIP during embryogenesis might have influenced levels of circulating testosterone, the rate of sexual differentiation, and the organization of the fetal male brain.
The mechanisms through which reduced VIP might influence prenatal development may occur through direct VIP actions on neurogenesis, (Brenneman et al., 1990
; Pincus et al., 1990
; Pincus et al., 1994
; Okumura et al., 1994
; Waschek, 1995
; Lu et al., 1996
; Iwasaki et al., 2001
); however, the indirect actions of VIP provide a mechanism for its mediation of diverse developmental processes (Brenneman et al., 1990
). VIP is known to regulate numerous neurotrophic and neuroprotective factors including several chemokines (Brenneman et al., 1999
), cytokines (Brenneman et al., 1995
; Brenneman et al., 2003
), insulin-like growth factor 1 (IGF-1) (Servoss et al, 2001
), nerve growth factor (Hill et al. 2002
), activity dependent neurotrophic factor (Brennemen and Gozes, 1996; Glazner et al., 1999
), and activity dependant neuroprotective protein (ADNP) (Bassan et al., 1999
; Furman 2004
). ADNP is necessary for embryonic neural tube closure (Bassan et al., 1999
; Pinhasov et al., 2003
), and recent evidence from the VIP knockout mouse indicated that the maternal VIP genotype influenced the ADNP expression in the brains of her offspring (Giladi et al., 2007
). Although the mechanisms underlying the abnormalities are not fully understood, the behavior deficits of the offspring of VIP deficient mothers appear to be the result of insufficient VIP to accomplish the normal actions of this neuropeptide on neurogenesis, or that insufficient VIP interfered with the expression, secretion or timing of developmentally important downstream effectors.
The current study has shown that intrauterine events had a greater influence on behavioral outcome than did genetic inheritance. These results are consistent with an increasing awareness that suboptimal intrauterine conditions maybe related to an increased susceptibility to a wide range of chronic diseases (Gluckman and Hanson, 2004
), including the evidence that damage caused by infections early in gestation may be linked to the incidence of psychiatric disorders with apparent developmental origins, such as schizophrenia and autism (Meyer et al., 2007
). Higher than normal concentrations of VIP were found in the blood of newborns, that were later shown to have Down syndrome and autism (Nelson et al., 2001
), and evidence indicates that autism has its origins during the neural tube closure period of early embryogenesis (see review, Arndt et al., 2005
), the period during which VIP regulates embryogenesis in the mouse (Gressens et al., 1993
: Gressens et al., 1994
; Hill et al., 1994
; Hill et al., 1996
; Hill et al., 1999
; Spong et al., 1999
). Autism has a male to female ratio of about 4:1 and is characterized by deficits in social behavior and communication, and restricted and repetitive behaviors, sometimes characterized as a resistance to change (Kanner, 1943
; American Psychiatric Association, 1994
, Lord et al., 2000
). In the current study the deficits in social behavior and resistance to change were more prevalent in male mice. In previous studies, reduced and abnormal social behaviors were apparent only in the male mice that had experienced VIP blockage during embryogenesis (Hill et al., 2007a
). Although it is not known whether intrauterine VIP is involved in the phenotype expressed by autistic persons, the deficits in social behavior, a resistance to change, and an increased prevalence of these deficits in male mice born to VIP deficient mothers indicate a potential usefulness of the VIP knockout mouse as a mouse model for some aspects of autism.