These studies demonstrate that activation of CREB in the NAc shell is critical for nicotine CPP, a form of appetitive contextual conditioning. We further show that a nicotine-paired context increases levels of pCREB in the NAc shell in the absence of overt nicotine seeking or locomotor activation, suggesting that exposure to an environment associated with nicotine can result in activation of the transcription factor CREB. Context-associated elevations of pCREB were specific to the NAc shell and not observed in the NAc core or the hippocampus using these parameters. Under place conditioning training conditions where animals were given a choice of chamber, disruption of CREB activation in the NAc shell blocked nicotine CPP, suggesting that induction of pCREB by exposure to contextual cues may support nicotine reward learning.
These data expand on previous studies of CREBαδ knockout mice (Walters et al., 2005
) suggesting that regulation of CREB is critical for nicotine CPP. We show that in animals that develop with normal CREB function, inactivation of CREB in the NAc shell in adulthood is sufficient to disrupt nicotine CPP. Previous reports have shown that HSV-mCREB infusion into the NAc shell enhances cocaine and morphine CPP (Carlezon et al., 1998
; Walters and Blendy, 2001
; Barrot et al., 2002
). In contrast, the current study shows that nicotine CPP cannot occur without CREB activity in the NAc shell. This suggests that although various drugs of abuse converge on the NAc in terms of DA release (Di Chiara and Imperato, 1988
), the circuit level adaptations that underlie nicotine reward differ from those of other drugs of abuse (Nestler, 2005
). Interestingly, chronic nicotine results in decreases in pCREB in the NAc (Brunzell et al., 2003
), and thus may contribute to the ability of cigarette smoking to promote the transition to use of other drugs of abuse (Lai et al, 2000
). Other studies, however, show that elevated pCREB is associated with cocaine CPP in the NAc (Miller and Marshall, 2005
; Walters et al., 2005
) and antisense-induced reductions of NAc CREB in shell or core attenuate the primary reinforcing efficacy of cocaine as well as its ability to act as primer during reinstatement (Choi et al, 2006
), perhaps due to more chronic dosing regimens of cocaine self administration (McClung and Nestler, 2003
) Results obtained with CPP do not always translate onto intravenous self administration data, but unfortunately, reliable nicotine self-administration is difficult to establish in mice.
Place conditioning provides a valuable tool for assessing Pavolovian contributions to changes in signaling and consequent reward learning. The present studies expand upon earlier data, which did not discriminate between subdivisions of the NAc (Pandey et al., 2001
; Brunzell et al., 2003
; Walters et al., 2005
), to show a dichotomy between the NAc core and shell in nicotine-associated regulation of pCREB. Previous data shows that CPP testing, but not a similar regimen of nicotine exposure and withdrawal in the homecage, results in elevations of NAc pCREB (Walters et al., 2005
). In the current study mice showed an increase in NAc shell levels of pCREB while isolated to a nicotine-paired chamber 22 h after the last of 3 daily pairings of nicotine with that chamber. Hence, Pavlovian nicotine place conditioning resulted in elevations of pCREB in the absence of overt nicotine seeking. Microdialysis studies have shown that elevated DA release, which ought to lead to D1-associated activation of CREB, occurs in the NAc shell but not the core during early acquisition sessions (fewer than 7), indicating that CREB in the NAc shell may contribute to initiation of nicotine use (Spina et al, 2006
). These Pavlovian conditioning data support the hypothesis that the CS properties of the context are sufficient to activate CREB in the NAc shell.
At the molecular level, activation of pCREB in the NAc follows a pattern that is similar to that seen for DA neuron activity during stimulus reward learning. Studies in primates and rodents show that after repeated pairing of a rewarding stimulus with a cue, DA neuron firing shifts from responding to the primary rewarding stimulus, becoming dependent instead on the conditioned cue (Schultz et al., 1997
; Day et al., 2007
). Similarly, elevations of pCREB were observed in the NAc in response to acute nicotine exposure, but not following 4 days of exposure to nicotine in the homecage (Walters et al., 2005
) or during Pavlovian training on the 3rd
day of nicotine exposure in these studies. Instead, NAc pCREB levels were elevated in response to the nicotine-paired chamber, suggesting that the CS properties of the context and not the rewarding properties of nicotine regulate pCREB in the NAc shell after training that is sufficient to lead to nicotine CPP.
CREB signaling is necessary for neural plasticity associated with learning and addiction (Silva et al, 1998
; Carlezon et al, 2005
). Genes such as fos and BDNF that are regulated by nicotine and thought to be important for tobacco addiction (Schroeder et al., 2001
; Li et al, 2008
) are upregulated as a result of CREB overexpression and are inhibited by expression of mCREB in the NAc of transgenic mice (McClung and Nestler, 2003
). Disruption of CREB activity could interfere with expression of many genes that have CRE sequences in their promoters. For example, CREB activity is necessary for the regulation of mu opiate receptors in response to nicotine administration, and this upregulation contributes to nicotine CPP (Walters et al., 2005
). Thus, CRE-mediated neuroadaptations that normally occur in response to nicotine may lead to lasting changes in systems that regulate incentive salience and drug reward.
There was no effect of nicotine or a nicotine-paired chamber on regulation of pCREB in the hippocampus in the current study, as was observed using a 4 day regimen of CPP training in previous studies (Walters et al., 2005
). One potential reason for this difference may be that animals were not actively seeking the nicotine-paired chamber in the current study, but were rather confined to that chamber by the experimenter. Some evidence suggests that regulation of CREB in the dorsal hippocampus is more critical for spatial navigation than context conditioning (Pittenger et al, 2002
). Although CREB was not regulated in the NAc core or hippocampus during exposure to a nicotine-paired chamber on the 3rd
day of training, we cannot conclude that CREB in these brain areas is not critical for learning during earlier trials or for consolidation processes related to nicotine CPP (Josselyn and Nguyen, 2005
). Human studies show that the dorsal striatum and orbitofrontal cortex may also play a role in cue reward for drugs of abuse (David et al., 2007
; Franklin et al., 2007
; McClernon et al., 2008
; Volkow et al., 2008
). Whereas guide canulae prevented expression of mCREB along the canulae tracks, damage to these overlying structures could have impaired cue reward. LacZ control animals showed levels of nicotine CPP similar to untreated controls, however, suggesting that potential unilateral damage during infusion did not affect nicotine CPP.
In summary, these studies show that activation of CREB in the NAc shell is critical for nicotine CPP. The phosphorylation state of CREB may therefore regulate the motivational valence for nicotine or the conditioned rewarding effects of nicotine-associated cues, such as a context associated with nicotine. A nicotine-paired environment can elicit CREB phosphorylation in the NAc shell, suggesting that contextual cues may drive changes in CREB that promote nicotine reward learning. We conclude from the current study that pharmacological agents that decrease CREB phosphorylation might be novel targets for smoking cessation.