Hidden Markov Model Analysis of Maternal Behavior Patterns in Inbred and Reciprocal Hybrid Mice Valeria Carola1,2, Olivier Mirabeau1¤, Cornelius T. Gross1* 1 Mouse Biology Unit, European Molecular Biology Laboratory (EMBL), Monterotondo, Italy, 2 Santa Lucia Foundation, European Centre for Brain Research (CERC), Rome, Italy Abstract Individual variation in maternal care in mammals shows a significant heritable component, with the maternal behavior of daughters resembling that of their mothers. In laboratory mice, genetically distinct inbred strains show stable differences in maternal care during the first postnatal week. Moreover, cross fostering and reciprocal breeding studies demonstrate that differences in maternal care between inbred strains persist in the absence of genetic differences, demonstrating a non- genetic or epigenetic contribution to maternal behavior. In this study we applied a mathematical tool, called hidden Markov model (HMM), to analyze the behavior of female mice in the presence of their young. The frequency of several maternal behaviors in mice has been previously described, including nursing/grooming pups and tending to the nest. However, the ordering, clustering, and transitions between these behaviors have not been systematically described and thus a global description of maternal behavior is lacking. Here we used HMM to describe maternal behavior patterns in two genetically distinct mouse strains, C57BL/6 and BALB/c, and their genetically identical reciprocal hybrid female offspring. HMM analysis is a powerful tool to identify patterns of events that cluster in time and to determine transitions between these clusters, or hidden states. For the HMM analysis we defined seven states: arched-backed nursing, blanket nursing, licking/grooming pups, grooming, activity, eating, and sleeping. By quantifying the frequency, duration, composition, and transition probabilities of these states we were able to describe the pattern of maternal behavior in mouse and identify aspects of these patterns that are under genetic and nongenetic inheritance. Differences in these patterns observed in the experimental groups (inbred and hybrid females) were detected only after the application of HMM analysis whereas classical statistical methods and analyses were not able to highlight them. Citation: Carola V, Mirabeau O, Gross CT (2011) Hidden Markov Model Analysis of Maternal Behavior Patterns in Inbred and Reciprocal Hybrid Mice. PLoS ONE 6(3): e14753. doi:10.1371/journal.pone.0014753 Editor: Thomas Burne, University of Queensland, Australia Received July 22, 2010; Accepted January 31, 2011; Published March 8, 2011 Copyright: 2011 Carola et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Santa Lucia Foundation (V.C.), Fritz Thyssen Stiftung (C.G.), NARSAD (C.G.), and funds from European Molecular Biology Laboratory (O.M. and C.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gross@embl.it and carola@embl.it ¤ Current address: Institute of Neurobiology Alfred Fessard, CNRS, Gif-sur-Yvette, France Introduction Natural variation in the amount and type of maternal care received is associated with differences in adult behavioral traits in a wide range of mammalian species. Although much of this association depends on the fact that mothers and their offspring share significant genetic variation, cross fostering and reciprocal breeding experi- ments in rodents have shown that non-genetic or epigenetic mechanisms also play an important part in this association [1,2]. In mice, the study of maternal effects on the offspring has been aided by the existence of inbred strains that show large and stable differences in maternal care. Mothers of the C57BL/6 inbred strain, for example, exhibit more licking and grooming of pups and arched- back nursing compared to the BALB/c strain [3–11] and offspring of these two strains show differences in adult behavior that can at least partially be traced back to their differences in maternal care [10,12– 14]. Maternal non-genetic or epigenetic mechanisms also contrib- utes to maternal behavior itself, with female offspring of cross fostering or reciprocal breeding between C57BL/6 and BALB/c parents showing maternal behavior that reflects the behavior of their mother [3,4,6,10,15,16]. The frequency of several maternal behaviors in laboratory mice has been previously described, including nursing and grooming pups, tending to the nest, and feeding [7,9,10,11]. However, the ordering, clustering, and transitions between these behaviors have not been systematically described and thus a global description of maternal behavior is lacking. A powerful tool for the analysis of sequential patterns of events is the hidden Markov model (HMM) formalism [17]. Although used exten- sively for the analysis of speech and hand gestures, as well as DNA and protein sequences [18–21] HMM has been rarely used to analyse behavioral mouse data. It has been applied to the analysis of locomotor patterns in several behavioral tests [22,23]. HMM can provide information both about which observed behavioral variables are sequentially associated as well as the frequencies of transitions between these associated behaviors, or ‘‘states’’. Here, we used HMM to define and characterize states that des- cribe the maternal behavior patterns of C57BL/6 and BALB/c mice and their reciprocal hybrid offspring. Our analysis iden- tified gross differences in behavioral repertoires and trajec- tories that included differences in the frequency, composition, PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e14753