MOLECULAR PROJECT: SUMMARY Adolescent administration of Δ9-tetrahydrocannabinol (THC) causes long-lasting neurobehavioral impairments in mice and rats. The Molecular Project will test the hypothesis that these enduring effects result from excessive activation of CB1-type cannabinoid receptor (CB1R), which triggers epigenetic processes resulting in region- and circuit-specific down-regulation of endocannabinoid (ECB) signaling. We have two specific aims. In Aim 1, we will examine whether acute or prolonged exposure to THC during adolescence alters molecular components of the ECB system, and/or the ability of this system to be engaged by environmental stimuli. We will test, in mice and rats of both sexes, acute and prolonged THC regimens designed to mimic occasional or daily cannabis use in teenagers. Outcomes will be assessed at three stages of the animals’ lifespan: end of treatment (24 h after last THC injection), adulthood (postnatal day, PND 70) and middle age (PND 300). To determine the age-dependence of the effects of THC, adult animals (PND 70) will be subjected to an identical protocol. We will determine the effects of acute or prolonged adolescent THC exposure on (i) lipid, protein and gene constituents of the ECB complex; (ii) epigenetic modifications; and (iii) stimulus- dependent ECB signaling in vitro and in vivo. Aim 2 will identify molecular mechanisms responsible for the induction and maintenance of persistent cannabinoid-dependent alterations in ECB signaling. Focusing on the prolonged THC protocol – which we expect will have the strongest impact on ECB system, synaptic activity and behavior – we will use loss-of-function and/or gain-of-function strategies to define the CBR subtype(s) mediating the long-lasting effects of adolescent THC treatment; and describe epigenetic and biochemical mechanisms potentially responsible for the transition to persistent changes in ECB signaling. We anticipate that (i) CB1R, not CB2R, mediates the long-lasting effects of adolescent THC exposure and (ii) genetic manipulations targeting specific genes or histone modifications affected by THC will correct molecular, synaptic and behavioral deficits observed in THC-treated animals. A deep understanding of the enduring actions of THC at the genetic, epigenetic and biochemical level will have several applications: first, it will guide the research activities of all other ICAL projects: second, it will help interpret the datasets generated by prospective studies that assess the long-term health impact of adolescent substance use (e.g, the ABCD study); and, third, it will guide the future discovery of predictive biomarkers of exposure outcomes as well as for the rational development of medications aimed at correcting the neurobehavioral consequences of teenage cannabis use.