Repeated testing and housing conditions impact complex mouse behaviors independently of surgical interventions

Behavioral testing in rodents constitute crucial methods for assessing phenotypic outcomes associated with diseases, therapeutic interventions and surgical procedures. These tests provide valuable insights into neurological, psychological, and physiological alterations, enabling comprehensive evaluation of cognitive function, memory, motor activity and pain perception. However, the validity of behavioral research is often compromised by high variability arising from factors, such as genetic background and possibly study designs that often require repeated animal testing, usually before and after interventions, as well as social isolation (single-caging) to avoid injuries after a surgery. Since the above may impact on the behaviors themselves and makes data interpretation and cross-study comparisons less reliable, we investigated how repeated behavioral testing and single housing influence anxiety-, compulsive-, and depressive-like/passive coping behaviors in mice. We exposed group- and single-caged mice with or without surgical intervention to the behavioral box, marble burying, and forced swim tests at different time-points. Repeated exposure significantly reduced exploratory activity in the behavioral boxes and increased immobility in the forced swim test, indicating habituation or adaptation rather than pain-induced comorbidities. Short-term single housing did not affect the tested behaviors, however, long-term isolation reduced exploratory drive and altered digging behavior. Our findings provide novel evidence that repeated testing and housing conditions shape rodent behavior, emphasizing that non-intervention variables substantially affect behavioral performance and need to be taken into considerations when designing test paradigms to improve reproducibility and interpretation, especially in preclinical models exploring interventional effects on pain-like behaviors.