To directly address whether regulating mRNA localization can influence animal behavior, we created transgenic mice that conditionally express Zipcode Binding Protein 1 (ZBP1) in a subset of neurons in the brain. ZBP1 is an RNA-binding protein that regulates the localization, as well as translation and stability of target mRNAs in the cytoplasm. We took advantage of the absence of ZBP1 expression in the mature brain to examine the effect of expressing ZBP1 on animal behavior. We constructed a transgene conditionally expressing a GFP-ZBP1 fusion protein in a subset of forebrain neurons and compared cocaine-cued place conditioning in these mice versus noninduced littermates. Transgenic ZBP1 expression resulted in impaired place conditioning relative to nonexpressing littermates, and acutely repressing expression of the transgene restored normal cocaine conditioning. To gain insight into the molecular changes that accounted for this change in behavior, we identified mRNAs that specifically immunoprecipitated with transgenic ZBP1 protein from the brains of these mice. These data suggest that RNA-binding proteins can be used as a tool to identify the post-transcriptional regulation of gene expression in the establishment and function of neural circuits involved in addiction behaviors.

BACKGROUND:Cue reactivity, the ability of cues associated with addictive substances to induce seeking and withdrawal, is a major contributor to addiction. Although human imaging studies show that cigarette-associated cues simultaneously activate the insula and the orbitofrontal cortex and evoke craving, how these activities functionally contribute to distinct elements of cue reactivity remains unclear. Moreover, it remains unclear whether the simultaneous activation of these cortical regions reflects coordinated functional connectivity or parallel processing. METHODS:We selectively lesioned the insula or orbitofrontal cortex with the excitotoxin ibotenic acid in mice, and their approach to nicotine-associated cues (n = 6-13/group) and avoidance of withdrawal-associated cues (n = 5-12/group) were separately examined in place conditioning paradigms. We additionally tested the role of these two cortical structures in approach to food-associated cues (n = 6-7/group) and avoidance of lithium chloride-associated cues (n = 6-7/group). RESULTS:Our data show a double dissociation in which excitotoxic lesions of the insula and orbitofrontal cortex selectively disrupted nicotine-induced cue approach and withdrawal-induced cue avoidance, respectively. These effects were not entirely generalized to approach to food-associated cues or avoidance of lithium chloride-associated cues. CONCLUSIONS:Our data provide functional evidence that cue reactivity seen in addiction includes unique neuroanatomically dissociable elements and suggest that the simultaneous activation of these two cortical regions in response to smoking-related cues does not necessarily indicate functional connectivity.

Background: Nicotine is one of the determinants for the development of persistent smoking, and this maladaptive behavior is characterized by many symptoms, including withdrawal and nicotine seeking. The process by which withdrawal affects nicotine seeking is poorly understood.Method: The impact of a withdrawal-associated cue on nicotine (.2 mg/kg)-conditioned place preference was assessed in male C57BL/6J mice (n = 8-17/group). To establish a cue selectively associated with withdrawal distinct from those associated with nicotine, a tone was paired with withdrawal in their home cages; mice were chronically exposed to nicotine (200 mu g/mL for 15 days) from drinking water in their home cages and received the nicotinic acetylcholine receptor antagonist mecamylamine (2.5 mg/kg) to precipitate withdrawal in the presence of a tone. The effect of the withdrawal-associated tone on nicotine-conditioned place preference was then evaluated in the place-conditioning apparatus after a delay, when nicotine-conditioned place preference spontaneously disappeared.Results: A cue associated with precipitated withdrawal reactivated the dormant effect of nicotine-associated cues on conditioned place preference. This effect occurred during continuous exposure to nicotine but not during abstinence.Conclusions: A conditioned withdrawal cue could directly amplify the incentive properties of cues associated with nicotine. This observation extends the contemporary incentive account of the role of withdrawal in addiction to cue-cue interaction.

The core nature of nicotine dependence is evident in wide variations in how individuals become and remain smokers. Individuals with pre-existing behavioral traits are more likely to develop nicotine dependence and experience difficulty when attempting to quit. Many molecular factors likely contribute to individual variations in the development of nicotine dependence and behavioral traits in complex manners. However, the identification of such molecules has been hampered by the phenotypic complexity of nicotine dependence and the complex ways molecules affect elements of nicotine dependence. We hypothesize that nicotine dependence is, in part, a result of interactions between nicotine and pre-existing behavioral traits. This perspective suggests that the identification of the molecular bases of such pre-existing behavioral traits will contribute to the development of effective methods for reducing smoking dependence and for helping smokers to quit.

BACKGROUND:The addictive properties of nicotine are mediated via dopaminergic pathways and their post-synaptic neurons in the striatum. Because post-synaptic neurons within the striatum contain high levels of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), we hypothesized that DARPP-32 may functionally contribute to the behavioral effects of nicotine. METHODS:We examined the behavioral effects of nicotine and the phosphorylation state of DARPP-32 in wild-type (WT) and DARPP-32 knockout (KO) mice. In one experiment, we assessed voluntary nicotine intake (0-50 microg/ml) of WT and KO mice in a two-bottle choice paradigm. In a separate experiment, the motor-depressant effects of acute and repeated nicotine injections (0-.8 mg/kg, subcutaneously [SC]) were assessed. The phosphorylation of DARPP-32 at threonine34 and threonine75 were examined using Western blotting. RESULTS:A heightened responsiveness to nicotine was seen in KO mice when compared with WT mice in oral intake and motor depression. The enhanced responsiveness in KO mice was not due to alterations in taste sensations, fluid intake, or blood nicotine or cotinine levels. Systemic injections of nicotine resulted in increased striatal DARPP-32 phosphorylation at threonine34 and threonine75. CONCLUSIONS:DARPP-32 opposes the behavioral effects of nicotine possibly via concurrent phosphorylation at the two threonine sites.