HMM of Maternal Behavior Figure 5. Maternal behavior strategy of BALB/c mothers. Graphical representation of composition, duration, frequency, and transition probabilities of HMM states for BALB/c mothers. Most states are composed of a single dominant behavior and multiple minor behaviors. State frequency and mean duration are indicated in site each circle. The area of each circle is proportional to state frequency. Arrows indicate absolute transition probabilities between states (transitions/minute). doi:10.1371/journal.pone.0014753.g005 female offspring. Subsequently, we subjected sequences of maternal behavior to HMM analysis to extract and describe maternal behavioral states. An analysis and comparison of the frequency, composition, and transitions of behavioral states provided a global assessment of maternal behavior in laboratory mice and revealed key differences in behavioral strategy between mouse pedigrees. Our initial analysis of frequencies of observed maternal behaviors (Table 1) was consistent with previous reports demonstrating increased maternal licking and grooming of pups and arched back nursing in C57BL/6 mice when compared to BALB/c mice [4–6,10,11]. C57BL/6 mothers also spent more time in the nest, and performed more sniffing nest and carrying pup while BALB/c mothers spent more time in blanket nursing, eating, moving pups, rearing, digging, and sleeping (Figure 1A–C and Table 1). These data suggest that C57BL/6 mothers favoured pup-oriented behavior while BALB/c mothers favoured exploration of their environment and self-oriented behavior. Finally, C57BL/6 mothers showed more persistent maternal care, continuing to provide high levels of nursing behavior throughout postnatal period, while nursing behaviors of BALB/c mothers declined sharply after postnatal day 10 (Figure 1B–C). Strikingly, differences in frequencies of behavior between reciprocal hybrid strains were restricted to licking/grooming pups and sniffing pups, with the former greater in B6xC and the latter greater in CxB6 mothers during all postnatal period. These results are different from the ones obtained from previous paper [15] where no overall strain differences were observed between these reciprocal hybrid mothers, but only a drastic drop of the maternal care in CxB6 at postnatal day 10. Our findings are instead in line with previous reported data [3,10,16,26] suggesting the existence of non-genetic inheritance of licking and grooming behavior within these pedigrees. However it is not possible to completely rule out that imprinted genes partially contribute to the differences we observe between reciprocal hybrid strains. Our data also reveal that the transmission from the mothers to their female offspring of maternal behavior is limited to subsets of the maternal behavior repertoire with special adaptive function. It may be, for example, that this mode of inheritance helps the mother to ensure consistent levels of offspring care and foraging over generations in a way that is resistant to the introduction of genetic variation into her pedigree. This hypothesis is supported by the finding that offspring of B6xC and CxB6 reciprocal hybrid mothers show significant differences in anxiety and exploratory behaviour [10,15,16]. HMM analysis allowed us to go beyond a simple assessment of frequency to assess temporal associations and transitions between behaviors. The HMM formalism has been extensively used to analyse sequences of speech and gestures [17,21] and also the analysis of protein and DNA sequences [18,20]. Markovian models have been rarely used to analyse behavioral mouse data [22,23]. Hidden Markov models are particularly powerful at identifying events that are clustered in time and the precise transition points between these clusters, or states. Such an analysis is particularly adapted to describe behavioral sequences in which the animal shifts between modes of activity that are dominated by one behavior but frequently interrupted by minor behaviors. By clustering frequently associated behaviors precisely determining their transitions, HMM states give a more accurate description of behavioral patterns. PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e14753