Background

In the 1950s, Hall and Hanford first reported that rats placed on a restricted feeding schedule and given access to a running wheel exhibit a paradoxical increase in running and a decrease in food intake, leading to extreme weight loss (Hall et al., 1953; Hall and Hanford, 1954). The extent to which these responses are life-threatening was established in a later study, in which all the tested rats effectively ran themselves to death (Routtenberg and Kuznesof, 1967). Collectively, these findings formed the basis for the most commonly used animal model of anorexia nervosa, activity-based anorexia (ABA). ABA shares several characteristics with the human disorder, including extreme weight loss, increased voluntary physical activity, self-starvation, loss of estrus (Dixon et al., 2003), and greater vulnerability during adolescence (Woods and Routtenberg, 1971). As an animal model, it enables examination of how caloric restriction in combination with physical activity leads to neural and endocrine adaptations that may contribute to the development of the disorder. While most ABA studies have used rats, an increasing number are using mice, with results that vary based on strain, sex, age, the amount of time food is available, the number of days of food restriction, and how wheel-running is analyzed (Gelegen et al., 2007 and 2008; Klenotich et al., 2012). Given the number of variables affecting susceptibility to ABA, consistency across studies with respect to the parameters used and how the data are analyzed would enhance utility of the model.

A critical question in the field has been what factors promote vulnerability or resilience to anorexia nervosa. This has been difficult to model in ABA since most studies restrict food for 5 days or less, during which all animals show significant weight loss. Weight loss is particularly rapid in adolescent animals. Some investigators categorize animals as ‘vulnerable’ or ‘resilient’ based on how quickly they lose weight during this time period, either by evaluating individual differences within a group (Barbarich-Marsteller et al., 2013; Chowdhury et al., 2013) or by making strain comparisons (Gelegen et al., 2007). However, until recently no implementation of ABA has demonstrated a truly resilient phenotype, defined as adaptation and weight stabilization in the face of food restriction and exercise. We recently published our ABA study in which roughly half of the animals exhibit resilience by decreasing total running, increasing food intake, and reaching a stable bodyweight (Beeler et al., 2020). The other half of the animals are vulnerable to ABA, as demonstrated by increased wheel running, a failure to adapt food intake to increased energy expenditure, and excessive weight loss. Notably, we also detected vulnerability and resilience in food-restricted mice housed with a locked running wheel; although, wheel running does appear to accelerate weight loss. Here, we present the detailed protocol that we used to obtain distinct vulnerable and resilient phenotypes, which involves the use of young adult C57/BL6 female mice instead of adolescent mice, extending the number of days of food restriction from 5 to 10, and conducting a thorough analysis of individual differences.

This protocol will be useful for future studies aimed at characterizing vulnerability and resilience to anorexia nervosa. By generating both phenotypes, this protocol allows studies to examine the physiological, neural, and endocrine factors that differentiate the two, facilitating greater insight into the biological basis of this eating disorder. This protocol also provides an opportunity to evaluate the extent to which interventions decrease vulnerability and favor adaptation and resilience.