Early-life experience influences neuroendocrine and behavioral responses to stress long-term, with profound implications for emotional health throughout life, but the molecular mechanisms of this effect remain unclear. Studies during the current award period found that reduced expression of the stress neuromodulator corticotrophin releasing hormone (CRH) in hypothalamus was an early event in experience-evoked 'programming' of the hypothalamic pituitary adrenal (HPA) system: CRH mRNA was downregulated already on postnatal day 9 in daily handled pups, preceding the onset of attenuated stress responses and the life-long enhancement of hippocampal glucocorticoid receptor (GR) mRNA and stress-'coping' behaviors. The goal of the current proposal is to investigate the mechanisms of this stress-response neuroplasticity after handling-evoked alterations of maternal care, by testing systematically several mechanistic hypotheses: (1) An early phase of 'handling'-evoked HPA axis neuroplasticity in rat pups involves coordinated transient activation of neuronal transcription factors in stress-regulating sensory integration regions. This effect is evoked by a 'burst' of maternal care elicited by the return of 'handled' pups to the cage. (2) Recurrence of this neuronal activation, particularly in 'stress-memory storage regions' is required for enduring downregulation of hypothalamic CRH gene transcription and steady state expression. (3) Reduction of hypothalamic CRH expression is necessary and sufficient to promote the attenuated neuroendocrine stress responses and enduring elevation of hippocampal GR expression and 'coping' behaviors characteristic of high maternal care early in life. (4) CRH acts via the CRF1, receptor to 'program' HPA responses long-term, so that selective CRF1 antagonists will convert hippocampal GR expression and long-term stress responses to those seen in early-life 'handled' rats. (5) The mechanisms by which reduced CRH levels and decreased activation of CRH receptors alter hippocampal GR expression and stress responses long-term involves modulation of plasma glucocorticoid levels, so that 'clamping' these levels will prevent handling-evoked HPA axis programming. The proposed experiments will delineate the spatiotemporal onset and progression of the protein effects of early life experience (and maternal care) on the immature HPA system, and use molecular and pharmacological means to define the responsible mechanisms. These studies should help clarify the molecular and neuroanatomical basis for early-life plasticity of the HPA system. In addition, they should yield mechanistic, targeted therapy for reversing the HPA hyperactivity consequent to deficient early-life experience that characterizes, and perhaps underlies, certain human affective and anxiety disorders.