Deconstructing nicotine cue reactivity: cortical control, PKG-dependent maintenance, and incentive-withdrawal interaction

Abstract

Tobacco smoking represents a major health crisis worldwide. Nicotine is a major component of tobacco that induces dependence. In dependent smokers, cues associated with smoking contribute to persisted smoking, craving, and relapse, a process termed cue reactivity. Cue-induced craving includes the desire for acute nicotine and the desire to avoid nicotine withdrawal. Despite the importance of cue reactivity to nicotine dependence, its neural and molecular mechanisms are poorly understood.;Cue reactivity can be modeled in laboratory animals, distinct elements of cue reactivity can be independently assessed. The purpose of this work is to deconstruct cue reactivity into its elements, determine the anatomical and molecular substrates of these components, and to determine how- these elements interact. In order to address these questions, we hypothesized that 1) nicotine- and withdrawal-cue reactivity are neuroanatomically distinct processes, 2) prolonged nicotine- and/or withdrawal-cue reactivity require specific molecular substrates, and 3) there exists a functional interaction between the two elements of cue reactivity.;To test the first hypothesis, we lesioned two cortical regions associated with smoking cue reactivity, the insula and the orbitofrontal cortex, with ibotenic acid in C57BL/6J mice. We then tested these mice for nicotine cue approach and withdrawal cue avoidance using a place conditioning paradigm. Our results demonstrate that lesion of the insula completely disrupts nicotine-cue approach without affecting withdrawal-cue avoidance. Conversely, lesion of the orbitofrontal cortex abolishes withdrawal-cue avoidance, but not nicotine-cue approach. These results suggest that nicotine- and withdrawal-cue reactivity are anatomically dissociable within the prefrontal cortex.;We next tested the hypothesis that maintained nicotine- and withdrawal-cue reactivity have distinct molecular substrates. Because nitric oxide (NO) is induced in the brain by nicotine, and this pathway has been implicated in nicotine cue approach, we targeted the cGMP-dependent protein kinases (PKG), a downstream signaling molecule of NO. Our results show that a single nicotine injection upregulates PKG activity in the caudate-putamen of 5 - 6 week-old male C57BL/6J mice. Because PKG type II (PKG-II), but not PKG-I is abundantly expressed in this region, we hypothesized that this isoform may play a functional role in nicotine cue reactivity. We examined the role of PKG-II in nicotine cue approach using congenic PKG-II knockout (KO), heterozygous (HT), wild-type (WT) mice. Our data show that nicotine induces cue approach in all genotypes on the first testing day. However, while KO mice failed to maintain this behavior thereafter, WT and HT mice maintained it for several days. By contrast, PKG-II KO mice were normal in withdrawal-cue avoidance.;In order to determine the anatomical substrate of the action of PKG-II in the maintenance of nicotine cue reactivity, we selectively expressed PKG-II in the caudate-putamen using a lentiviral vector carrying Prkg2 in KO mice. Our data demonstrate that expression of PKG-II in the caudate-putamen is sufficient to restore expression of nicotine-cue approach in KO mice. Taken together, our data suggest that sustained maintenance of nicotine cue reactivity requires PKG-II in the caudate-putamen.;To test our hypothesis that these two processes interact, we assessed the impact of a cue selectively associated with withdrawal on the expression of nicotine-cue approach. Our results demonstrate a cue associated with precipitated withdrawal reactivates nicotine cue approach at a time when this behavior is normally absent. These data suggest that nicotine and withdrawal cue reactivity interact, and a conditioned withdrawal cue could directly amplify the incentive properties of cues associated with nicotine.;Taken together, our data suggest that cue reactivity is a dual process, with dissociable neuroanatomical and molecular bases. Moreover, these dissociable elements interact in nicotine addiction. In sum, this thesis work has identified hitherto unknown neuroanatomical and molecular bases of nicotine cue reactivity.