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Play behavior is a fundamental and intrinsic neurobehavioral process in the mammalian brain. Using rough-and-tumble play in the juvenile rat as a model system to study mammalian playfulness, some of the relevant neurobiological substrates for this behavior have been identified, and in this review this progress. A primary-process executive circuit for play in the rat that includes thalamic intralaminar nuclei, frontal cortex and striatum can be gleaned from these data. Other neural areas that may interact with this putative circuit include amygdala, ventral hypothalamus, periaqueductal gray (PAG), and deep tectum, as well as ascending dopamine systems which participate in all types of seeking urges At the neurochemical level, considerable evidence points to specific cholinergic and dopaminergic controls, but also endogenous opioids and cannabinoids as having a positive modulatory influence over playfulness, with all europeptides known to have aversive effects to reduce play. Monoamines such as norepinephrine and serotonin certainly modulate play, but they influence all psychobehavioral systems, suggesting non-specific effects. We proceed to discuss how increased insights into the neurobiological mechanisms of play can inform our understanding of normal and abnormal childhood development.