24 Development of the cerebral cortex may be influenced by the composition of the maternal gut 25 microbiota. To test this possibility, we administered probiotic Lactococcus lactis in the drinking 26 water to mouse dams from day 10.5 of gestation and until pups reached postnatal day 1 (P1).27 Pups were assessed in a battery of behavioral tests starting at 10 weeks old. We found that 28 females, but not males, exposed to probiotic during prenatal development spent more time in the 29 center of the open field and also displayed decreased freezing time in cue associated learning, 30 compared to controls. Furthermore, we found that probiotic exposure changes the densities of 31 cortical neurons and increases the density of blood vessels in the cortical plate of P1 pups. Sex-32 specific differences were observed in the numbers of mitotic neural progenitor cells, which were 33 increased in probiotic exposed female pups. In addition, we found that probiotics treatment 34 throughout pregnancy significantly increased plasma oxytocin levels in mouse dams, but not in 35 the offspring. These results suggest that exposure of naïve, unstressed dams to probiotic may 36 exert sex-specific long-term effects on cortical development and anxiety related behavior in the 37 offspring. 38 39 40 41 Introduction 42 Neocortical development is influenced by a variety of environmental factors, including maternal 43 nutrition, stress and exposure to pathogens. In terms of cortical patterning and growth, the impact 3 44 of essential micronutrients, such as choline, has been well-characterized; yet less is known on 45 how food-derived microbiota, e.g. probiotics, may be altering cortical development at different 46 stages [1] . While the fetal gut is sterile before birth, it is increasingly clear that factors from the 47 maternal microbiota can induce specific patterns of gene expression in the fetal gut, as well as in 48 the brain [1][2][3] . After birth, microbial colonization of the infant gastrointestinal tract has been 49 shown to have widespread influence on brain development and behavior [4] . For example, mice 50 whose gut microbiota were depleted via antibiotics or who were raised in a germ-free facility 51 demonstrate exaggerated responses to stress and social stimuli, and display neurochemical and 52 brain structural abnormalities [5, 6] . Recently, neonatal exposure to probiotics, such as Bifidus 53 longum and Lactobacillus rhamnosous, have been shown to reverse maladaptive learning 54 behaviors in innately anxious mice [7] . Probiotics have also alleviated behavioral effects in other 55 models of anxiety disorders, such as those induced by maternal separation, social defeat or other 56 types of early life stress [8][9][10] . Mounting data on brain-gut axis has revealed several pathways 57 where probiotic treatment can affect microbiota populations, brain signaling, and subsequently 58 anxiety in mouse models of disease. However, there is less consensus on the behavioral effects 59 of probiotics in naïve, wild type mice which h...