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. 2016 Dec 8;11(12):e0167652.
doi: 10.1371/journal.pone.0167652. eCollection 2016.

A Time to Wean? Impact of Weaning Age on Anxiety-Like Behaviour and Stability of Behavioural Traits in Full Adulthood

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A Time to Wean? Impact of Weaning Age on Anxiety-Like Behaviour and Stability of Behavioural Traits in Full Adulthood

S Helene Richter et al. PLoS One. .
Free PMC article

Abstract

In mammals, weaning constitutes an important phase in the progression to adulthood. It comprises the termination of suckling and is characterized by several changes in the behaviour of both mother and offspring. Furthermore, numerous studies in rodents have shown that the time point of weaning shapes the behavioural profile of the young. Most of these studies, however, have focused on 'early weaning', while relatively little work has been done to study 'late weaning' effects. The aim of the present study was therefore to explore behavioural effects of 'late weaning', and furthermore to gain insights into modulating effects of weaning age on the consistency of behavioural expressions over time. In total, 25 male and 20 female C57BL/6J mice, weaned after three (W3) or four (W4) weeks of age, were subjected to a series of behavioural paradigms widely used to assess anxiety-like behaviour, exploratory locomotion, and nest building performance. Behavioural testing took place with the mice reaching an age of 20 weeks and was repeated eight weeks later to investigate the stability of behavioural expressions over time. At the group level, W4 mice behaved less anxious and more explorative than W3 animals in the Open Field and Novel Cage, while anxiety-like behaviour on the Elevated Plus Maze was modulated by a weaning-age-by-sex interaction. Furthermore, weaning age shaped the degree of behavioural stability over time in a sex-specific way. While W3 females and W4 males displayed a remarkable degree of behavioural stability over time, no such patterns were observed in W3 males and W4 females. Adding to the existing literature, we could thus confirm that effects of weaning age do indeed exist when prolonging this phase, and were furthermore able to provide first evidence for the impact of weaning age and sex on the consistency of behavioural expressions over time.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Experimental design.
The experiment was split into two phases: 1) Observation of maternal behaviour during weeks 3 and 4, and 2) behavioural testing of the offspring in full adulthood. Male and female C57BL/6J mice were weaned after either three (W3) or four (W4) weeks of age and housed in same-sex groups of two to five siblings per cage. To account for litter effects in the experimental design, only one or two subjects per sex and litter were included in the experiment. At the age of 20 weeks, mice were subjected to the first round of behavioural tests, including Elevated Plus Maze (EPM), Open Field (OF), Novel Cage (NC), and Nest Test (NT). To investigate behavioural stability over time, the same tests were repeated at an interval of eight weeks with the animals reaching an age of about 28 weeks.
Fig 2
Fig 2. Maternal behaviour in C57BL/6J dams from postnatal days (PND) 15 to 28.
(A) The frequency of nursing decreased over time, while the frequency of non-nursing contact increased steadily. (B) The frequencies of eating and drinking displayed by the dam decreased as pups started to switch to eating solid food. (C) Frequencies of nest building and licking/grooming remained constant throughout this period, while the frequency of self-grooming appeared to be slightly increased in the second half of the observation period. Data are presented as means ± SEM. Samples sizes PNDs 15–21 (W3 and W4 dams): n = 21, PNDs 22–28 (W4 dams only): n = 10.
Fig 3
Fig 3. Body weight development in male and female C57BL/6J mice weaned after either three (W3) or four weeks of age (W4).
Besides a significant effect of sex and a sex-by-time interaction, a significant interaction between weaning age and time indicated a differential body weight development in W3 and W4 offspring over the course of the experiment. Data are presented as means ± SEM. Statistics: Repeated measures ANCOVA (including only data from week 6 to week 30). Sample sizes: Nmales_W3 = 12, Nfemales_W3 = 10, Nmales_W4 = 13, Nfemales_W4 = 10.
Fig 4
Fig 4. Performance of C57BL/6J mice weaned after either three weeks (W3) or four weeks (W4) of age in a battery of behavioural tests conducted at the age of about 20 weeks.
(A) Relative time on open arms of the Elevated Plus Maze (EPM), (B) time spent in the centre of the Open Field (OF), (C) total path travelled in the OF, and (D) number of rearings displayed in the Novel Cage (NC). Data are presented as means + SEM. Statistics: 2-way ANOVA, tP < 0.1, *P < 0.05, **P < 0.01, sample sizes: NW3 = 22, NW4 = 23.
Fig 5
Fig 5. Performance of male and female C57BL/6J mice weaned after either three weeks (W3) or four weeks (W4) of age on the Elevated Plus Maze (EPM) at the age of about 20 weeks.
(A) Relative number of open arm entries, and (B) distance travelled on the open arms. While W4 males displayed less anxiety-like behaviour than W3 males, it was the other way round in females. Data are presented as means + SEM. Statistics: 2-way ANOVA, sample sizes: Nmales_W3 = 12, Nfemales_W3 = 10, Nmales_W4 = 13, Nfemales_W4 = 10.
Fig 6
Fig 6. Behavioural stability over time in the time spent on the open arms of the Elevated Plus Maze (EPM) in male and female mice weaned after either three (W3) or four (W4) weeks.
Correlations between test round I (20 weeks of age) and test round II (28 weeks of age) are separately presented for all four groups: (A) W3 males, (B) W3 females, (C) W4 males, and (D) W4 females. While there were significant correlations in W3 females and W4 males, no correlations were found in W3 males and W4 females. Statistics: Spearman’s rank correlation coefficient (1-tailed). Sample sizes: Nmales_W3 = 12, Nfemales_W3 = 10, Nmales_W4 = 13, Nfemales_W4 = 10.

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Grant support

This study was supplied by a grant from the German Research Foundation to NS (SFB/TRR58, Project A01, http://sfbtrr58.uni-muenster.de/a01.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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