Corrigendum to: Maternal antibiotic administration during a critical developmental window has enduring neurobehavioural effects in offspring mice

It has come to our attention that there may be errors in the reporting of certain descriptive statistics in this manuscript. Following a careful re-examination of the original data, we identified inadvertent transcription errors associated with the transfer of values from the SPSS output. The corrected statistics are presented and highlighted in bold in the revised Results section below. Importantly, these corrections do not alter the statistical significance of the reported findings, with one minor exception. Specifically, a secondary outcome measure in the light–dark box -the number of exploratory entries, which was not even discussed in the original manuscript-now reaches statistical significance, whereas it was previously reported as non-significant. All other results remain unchanged, and the overall conclusions of the study remain robust. We apologize for these errors and any confusion they may have caused. 3. Results 3.1 The effects of antibiotic administration on maternal body weight Mothers exposed to the antibiotic cocktail one day following the birth of their pups experienced weight-loss in the days after initiation of treatment. (Fig. 1A) (Repeated-measured two-way ANOVA, F (14, 84) = 3.516: P = 0.0002 (interaction between treatment and time), F (7, 84) = 35.89, P < 0.0001 (time), F (2, 12) = 2.283: P = 0.1445 (treatment). Post hoc assessment found a significant difference in body weight between vehicle and cocktail groups (P<0.05) at P1-4 only, and between cocktail and Penicilln V group at P1 only (P<0.05) after which no differences were observed between the groups. Offspring weight was observed throughout the course of the experiment, however no differences were observed (Fig. 1B) (repeated measured two-way ANOVA: F (2, 37) = 2.128: P = 0.1334 (treatment), F (16, 296) = 1.720: P = 0.0422 (interaction between treatment and time) 3.2 Early-life behaviours are altered following antibiotic administration As the disruption to the maternal microbiota through the administration of antibiotics in the drinking water occurs during a critical point of development in early life, the effects at this early stage are measured by observing behaviour in the days following cessation of antibiotics to the mothers. It was observed that in early-life (P9) there was no difference in either the total number of vocalisations (Fig. 2a) (F_(2,36)=0.9887,P=0.3877) or the total duration of vocalisations (Fig. 2b) (F_(2.36)=2.413,P=0.111) between any of the groups. Offspring attachment to maternal bedding was also analysed, with the time spent in maternal bedding being significantly reduced in animals whose mother had received a cocktail of antibiotics compared to those whose mothers received vehicle (Fig. 2c) (F_(2,36)=7.29,P=<0.002). Indicating that these animals may be exhibiting early social recognition and maternal attachment deficits compared to the other groups tested. 3.3 Anxiety-like behaviours are altered by early-life maternal antibiotic administration When mice are exposed to an unfamiliar environment, the increased potential for threat means that there may be an associated increase in anxiety. It is possible to measure this in rodents via several behavioural tests. In the marble burying test, increased levels of marble burying correspond to an increased in repetitive behaviours and is regarded as neophobic behaviour [39]. Here, the offspring of mothers administered both low-dose penicillin V, or an antibiotic cocktail buried a significantly greater number of marbles (Fig. 3) (F_(2,36)=5.24,P=<0.01). The elevated plus maze provides an additional measure of anxiety-like behaviour in these experimental animals. As rodents generally display an aversion to open spaces, an increased amount of time in the ‘open’ arms of the maze represents an anxiolytic-like behaviour [40]. The EPM results (Fig. 4a) correspond with that seen in the marble burying test (Fig 3) in some measures of the experiment. As well as measuring durations in each area, the number of entries to the open arms also serves as a measure of anxiolytic-like behaviour [41], and is significantly reduced in both groups whose mothers received antibiotic treatment (Fig. 4a) (F _(2,29)=5.9,P=<0.007); whereas, the number of entries to the closed arms remains unaffected (Fig. 4b) (F_(2,29)=0.5219,P=0.402). When the amount of time in the open arms is assessed, no changes are observed following any of the antibiotic treatments (Fig 4c) (F _(2,29)=0.09,P=0.9152) The number of head dips were similarly unaffected (Fig 4d) (F_(2,29)=0.166,P=0.4311). The light-dark box test acts as another measure for rodent anxiety, taking into account the natural aversion of mice to brightly illuminated areas as well as well as their spontaneous exploratory behaviour in novel environments [42]. Here, the offspring of mice treated with an antibiotic cocktail (but not penicillin V) spent significantly less time in the dark than the control group (Fig. 5a) (F_(2,36)=6.66,P<0.003). The number of transitions (Fig. 5b), (F_(2,36)=2.51,P=0.0956), however, and the number of exploratory rearing's (Fig. 5c) (F_(2,36)=3.58,P=0.038) was significantly different between the control group and the penicillin group. 3.4 Antidepressant-sensitive behaviour remains unaffected The mouse FST is used as a measure of antidepressant-sensitive behaviour in animals and regularly serves to determine the efficacy of novel antidepressant compounds [43]. It has also been used as a measure of the effect of microbiota manipulation on these behaviours [4,22]. Antibiotic administration has been shown to increase immobility time in the FST in rodent models [4,22]. These effects were not observed in either of the antibiotic treatment groups. (Fig 6) (F_(2,36)=1.54,P=0.2293). 3.5. Maternal penicillin administration induces deficits in social recognition The three-chamber test measures various aspects of social behaviour in rodents. Social preference is assessed by giving mice the choice of interacting with either a novel mouse, or an object. When given the choice, all groups exhibited normal social behaviour, (Fig 7a) (Two-way ANOVA, F(2,66)=7.123, P=0.0016 (treatment), F(2,66)=0.7064, P=0.4971 (interaction between treatment and mouse/object)). Post-hoc tests confirm that they spend more time with the mouse than the object (Vehicle object vs Vehicle mouse: P<0.001, Penicillin V object Vs Penicillin V mouse: P<0.001, Cocktail object vs cocktail mouse: P<0.001). When the test was repeated, with the object being replaced with an unfamiliar mouse, the test can be used to measure social recognition. In this case, different effects are seen following three treatments (Fig 7b) (Two-way ANOVA F (2,62) = 0.2414 P=0.7863 (treatment), F (2,62)=3.661, P=0.0314 (interaction between treatment and mouse/object). Post-hoc tests show a greater amount of time spent with the novel mouse in the vehicle group (P<0.001) and the antibiotic cocktail group (P<0.01) but not in the penicillin V group. 3.6 Maternal antibiotic cocktail administration induces cognitive deficits in offspring The novel object recognition test measures hippocampal-dependent memory in rodents. Harnessing the inherent preference of mice for novelty, this test determines the memory of previously encountered objects in these animals. Differences were observed between the groups (Fig 8a) (Two-way ANOVA, F (2, 72)=2.087: P=0.1315 (treatment), F(2, 72)=4.141: P = 0.0198 (interaction between treatment and Novel/familiar object), while post hoc comparison confirmed that while the vehicle treated group (P<0.05) and the penicillin V treated group (P<0.05) spent a greater time with the novel over the familiar object, this was not the case in the cocktail-treated group. Furthermore, when the percentage time spent with the novel object is assessed, there is a significant decrease in the interaction time in the antibiotic cocktail group only (Fig 8b) (One Way ANOVA F_(2,36)=3.87,P<0.03). 3.7 Plasma corticosterone levels are unaffected Plasma corticosterone acts as a measure of HPA axis activation following an acute stressor. Increased levels of circulating corticosterone following a stressor indicate that there is an abnormal response to the event. Here, no differences were observed in HPA axis response between any of the groups (Fig 9.) (Repeated-measures Two-way ANOVA: F (2, 28) = 1.182: P = 0.3217 (Treatment), F (8, 112) = 1.724: P = 0.1005 (interaction between treatment and time). 3.8 Physiological alterations As well as behavioural assessments, physiological differences were measured both during the experiment, as well as following the completion of behavioural assessments. Intestinal transit was assessed using the inert dye carmine red (Fig 10a) (F_(2,33) =2.59,P=0.0903). Previous studies have shown that this measure can be affected by gut microbiota composition [44]; however, we did not observe any differences in transit time in antibiotic treated animals. In addition to this, fat masses (Fig 10b) (F_(2,36)=2.73,P=0.0784) (Fig 10c) (F_(2,36)=1.56,P=0.2240) (Fig 10d) (F_(2,36)=2.77,P=0.0761) (Fig 10e) (F_(2,36)=1.2,P=0.3141) and cecum weight (Fig 10f) (F_(2,36)=2.29,P=0.1160) were assessed when the animals were culled. The only difference that was observed between the groups was spleen weight, which was significantly increased in the group treated with an antibiotic cocktail (Fig 10g) (F_(2,36)=3.47,P<0.42). 3.9 Hippocampal PCR analysis To determine whether any of the treatment groups were able to affect the expression of plasticity related genes in the hippocampus, expression measured in the hippocampus. A reduction in hippocampal expression of Bdnf was observed in the penicillin V group compared to control (Fig 11a) (F_(2,21)=2.496,P=0.0393). None of the other genes were affected, however including GluN2A (Fig 11b) (F_(2,22)=0.01173,P=0.9883), GluN2B (Fig 11c) (F_(2,22)=0.04879,P=0.9525), PSD-95 (Fig 11D) (F_(2,21)=0.6620,P=0.5262), CamKii (Fig 11D) (F_(2,21)=0.4272,P=0.6579), fos (Fig 11E) (F_(2,22)=0.9823,P=0.3903).