2026 ECNP Congress

PS04-4015

Optimising the maternal separation paradigm for early-life stress research: from behaviour to lipidomics

Johanna Diehl — M. Davis · Clinic of Psychiatry- Psychosomatic Medicine and Psychotherapy- University Hospital Frankfurt, Laboratory of Translational Psychiatry, Frankfurt am Main, Germany

2026 ECNP Congress

Early-life stress is a major environmental risk factor for psychiatric disorders [1, 2], yet preclinical models such as the maternal separation (MS) paradigm show considerable variability in behavioural outcomes and limited reproducibility across studies [3, 4]. This variability may arise from differences in experimental parameters. Establishing a stable and reproducible model is therefore essential to investigate downstream mechanisms, including metabolic and lipid signalling pathways implicated in stress-related psychopathology [5].

In preliminary experiments with MS for 3 h daily from postnatal day (P)2–P15 at ~37 °C with continued littermate contact, behavioural effects were limited (see below). To enhance model sensitivity and validity, the MS paradigm was systematically refined by maintaining pups at ~32 °C during separation to better mimic the absence of maternal warmth. Moreover, pups were physically separated using partitioned compartments to reduce social buffering.

Control animals were reared under standard conditions without separation. Behavioural phenotyping used a comprehensive test battery assessing anxiety-like behaviour, locomotor activity, cognition, and social interaction. Data were analysed using two-way ANOVA with the factors sex (female, male) and treatment group (MS, CTRL).

In the first part of the study, pilot data from the standard MS protocol were analysed to determine whether the initial conditions induced robust behavioural alterations. In the open field, no significant main effect of treatment was observed for locomotor or anxiety-related parameters (distance travelled: F(1,20) = 1.206, p = 0.285), whereas females displayed higher locomotor activity than males (F(1,20) = 7.671, p = 0.012). Centre entries were increased in females (F(1,20) = 5.23, p = 0.033), with a trend for a sex × group interaction (F(1,20) = 3.002, p = 0.099); post-hoc testing indicated higher centre entries in female CTRL than male CTRL mice (pTukey = 0.046), while no sex difference was detected within MS mice.

Object exploration showed only a trend towards reduced interaction time in MS animals (F(1,20) = 4.273, p = 0.052), while object recognition did not reveal a robust MS-related deficit (treatment: F(1,20) = 0.315, p = 0.581; treatment × sex: F(1,20) = 0.245, p = 0.626).

In the Barnes maze, treatment effects were more pronounced, with MS animals spending more time in the target zone during training (F(1,20) = 9.097, p = 0.007) and during the second probe trial (F(1,20) = 5.166, p = 0.034), indicating altered spatial search behaviour under standard MS conditions.

The refined MS paradigm is being evaluated to determine whether increased stress intensity leads to more robust and reproducible behavioural phenotypes. Samples from these mice will be analysed using metabolomic and lipidomic profiling to determine molecular signatures associated with early-life stress and to link behavioural phenotypes to downstream biological alterations. These adjustments are expected to provide a more reliable basis for investigating molecular mechanisms underlying stress-related phenotypes.

Taken together, this study identifies critical experimental parameters influencing MS efficacy and establishes a refined protocol for modelling early-life stress. By linking behavioural validation with molecular profiling, this work contributes to improving reproducibility and translational relevance of preclinical models in psychiatric research.

References

  1. Halldorsdottir, T., & Binder, E. B. (2017). Gene x environment interactions: from molecular mechanisms to behaviour. Annual Review of Psychology, 68, 215–241.
  2. Nemeroff, C. B. (2016). Paradise lost: the neurobiological and clinical consequences of child abuse and neglect. Neuron, 89, 892–909.
  3. Candemir, E. et al. (2019). Limited effects of early life manipulations on sex-specific gene expression and behaviour in adulthood. Behavioural Brain Research, 369, 111927.
  4. Millstein, R. A., & Holmes, A. (2007). Effects of repeated maternal separation on anxiety- and depression-related phenotypes in different mouse strains. Neuroscience & Biobehavioural Reviews, 31, 3–17.
  5. Schneider, M. et al. (2017). Lipids in psychiatric disorders and preventive medicine. Neuroscience & Biobehavioural Reviews, 76, 336–362.