Adolescence is a critical phase of active brain development often characterized by the initiation of marijuana (Cannabis sativa) use. Limited information is known regarding the endogenous cannabinoid system of the adolescent brain as well as related neurotransmitters that appear sensitive to cannabis exposure. We recently observed that adult rats pre-exposed to Δ-9-tetrahydrocannabinol (THC) during adolescence self-administered higher amounts of heroin and had selective impairments of the enkephalin opioid system within the nucleus accumbens (NAc) implicated in reward-related behavior. To explore the ontogeny of the cannabinoid and opioid neuronal systems in association with adolescence THC exposure, rats were examined at different adolescent stages during an intermittent THC paradigm (1.5 mg/kg i.p. every third day) from postnatal days (PNDs) 28-49. Rat brains were examined 24 hours after injection at PND 29 (early adolescence), PND 38 (mid adolescence) and PND 50 (late adolescence) and analyzed for endocannabinoids (anandamide and 2-arachidonoylglycerol), Met-enkephalin, cannabinoid CB 1 receptors and µ opioid receptors (µOR) in the NAc, caudate-putamen and prefrontal cortex (PFC). Of the markers studied, the endocannabinoid levels had the most robust alterations throughout adolescence and were specific to the PFC and NAc. Normal correlations between anandamide and 2-arachidonoylglycerol concentrations in the NAc (positive) and PFC (negative) were reversed by THC. Other significant THC-induced effects were confined to the NAc -increased anandamide, decreased Met-enkephalin and decreased µORs. These findings emphasize the dynamic nature of the mesocorticolimbic endocannabinoid system during adolescence and the selective mesocorticolimbic disturbance as a consequence of adolescent cannabis exposure.

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of -opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of receptors undergoes conformational changes following receptor activation that can be selectively detected by these regionspecific antibodies. We used these antibodies to characterize receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G␣ i -coupled (␦-opioid, CB1 cannabinoid, ␣ 2A -adrenergic) as well as G␣ s -(␤ 2 -adrenergic) and G␣ q -coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.Family A GPCRs 2 play a critical role in normal cell function and are the focus of intense studies and targets for drug development. A tremendous effort has been put toward understanding the mechanism of activation of family A GPCRs at a molecular level. Spin label studies with rhodopsin have shown that exposure to light leads to a movement of helices relative to one another that is important for activation of transducin (1-4). Studies using a variety of techniques suggest that small agonists bind to a pocket formed by the surrounding transmembrane helices, and in addition peptide ligands contact additional determinants in extracellular loops and possibly the N-terminal tail (5-10). Binding of agonists, but not antagonists, leads to the stabilization of the helical bundle into a conformation, which, in turn, leads to the uncovering of molecular determinants at the bottom of the core required for G-protein binding and activation (11). Although a comprehensive mechanism for receptor activation, including the N-and C-terminal regions, is not yet available, accumulating evidence suggests that these regions undergo strong structural pertu...

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