Although nicotine has long been known to enhance synaptic plasticity (Fujii et al, 1999
), the manner by which the drug alters consolidation processes underlying learning and memory has not been explored until now. Changes in gene transcription and protein synthesis are believed to be vital factors of long-term memory consolidation (Athos et al, 2002
; Hernandez and Abel, 2008
; Alberini, 2009
), and previous work has found that nicotine can alter gene transcription (Greenberg et al, 1986
; Bartel et al, 1989
). However, this is the first demonstration that nicotine and learning interact to alter gene transcription and that these changes in gene transcription likely underlie the consolidation of nicotine-enhanced learning.
Nicotine administration and training in contextual FC interact to upregulate JNK1 mRNA in the hippocampus 30
min after training. Neither nicotine administration alone nor FC alone resulted in the upregulation of JNK1 mRNA. This upregulation of JNK1 mRNA was dependent on the effects of nicotine at β
2-containing nAChRs, as the upregulation was not found in β
2 KO mice that were administered nicotine before conditioning. At 2
h after training, JNK1 mRNA was downregulated in the hippocampus of mice trained with nicotine, suggesting that the upregulation seen at 30
min after training could lead to a later period of suppressed JNK1 mRNA expression. The interactive effects of nicotine and learning were specific to JNK1 mRNA expression as other members of the MAPK family did not show the same pattern of transcriptional regulation. The nicotine-associated increase in JNK1 signaling may contribute to enhanced consolidation of contextual memories; during consolidation, hippocampal infusion of a dose of a JNK inhibitor subthreshold for impairing conditioning blocked the nicotine-induced enhancement, and pJNK1 levels in the dorsal hippocampus were increased after learning in the presence of nicotine. Given that pJNK1 is the active form of JNK1, an increase in pJNK1 suggests that the downstream effectors of JNK1 are also being activated and likely contribute to the nicotine-induced enhancement of learning and memory. Taken together, these data suggest that JNK1 signaling mediates the nicotine-induced enhancement of hippocampal synaptic plasticity that underlies the enhanced contextual fear memory.
Interestingly, JNK may also have a role in the consolidation of contextual fear memories in the absence of drug, as post-training hippocampal administration of a higher dose of a JNK inhibitor that inactivates all three JNK isoforms produced a deficit; an effect only previously observed with passive avoidance (Bevilaqua et al, 2003
). Given that a subthreshold dose of a JNK inhibitor in the hippocampus blocked the consolidation of a nicotine-enhanced contextual fear memory, nicotine may increase the contribution of JNK to FC through an upregulation of the JNK1 gene. What is particularly intriguing is that the upregulation of JNK1 mRNA was unique to the enhancing effect of nicotine on contextual FC, as a stronger training protocol that produced similar levels of conditioning as seen in nicotine-treated mice did not result in such an upregulation. Thus, the increased JNK1 mRNA after learning in the presence of nicotine may be a mechanism through which nicotine usurps the neural substrates of learning and memory.
Although the increase in JNK1 mRNA in the hippocampus 30
min after training in the presence of nicotine might suggest that this treatment should also result in an increase in the JNK1 protein, no increase in the JNK1 protein was found at the 1-h post-training time point. This may be because of the fact that qRT-PCR is more sensitive in detecting small changes in mRNA levels than is western blotting at detecting small changes in protein levels. Alternatively, it may be the case that the 1-h time point does not coincide with the peak of protein production after an increase in JNK1 mRNA levels. Although JNK1 mRNA levels decrease between 30
min and 2
h after training in the presence of nicotine, an increase in the JNK1 protein may occur later than 1
h after training because mRNA half-life tends to be shorter than protein half-life, although there is considerable variability in these parameters across proteins (Hargrove and Schmidt, 1989
). An extensive time course for changes in JNK1 mRNA and JNK1 protein levels after training in the presence of nicotine could further elucidate this issue.
The nicotine-induced enhancement of contextual learning (Kenney and Gould, 2008b
) may have an important role in the high addictive liability of nicotine. Contextual stimuli have an integral role in the development and maintenance of nicotine self-administration (Caggiula et al, 2002
); for example, exposure to contextual cues previously associated with nicotine administration increased drug seeking (Crombag et al, 2008
; Diergaarde et al, 2008
), and such contextual stimuli both delay the extinction of nicotine self-administration and are involved in reinstatement of self-administration in rats (Wing and Shoaib, 2008
). Furthermore, smokers reported greater amelioration of cravings when nicotine-associated cues were given along with nicotine than when either nicotine or cues were presented alone (Rose et al, 2000
). Thus, the ability of nicotine to increase the strength of contextual memories may facilitate the development of nicotine addiction and increase the likelihood of context-induced relapse during quit attempts.
It has been established that both the hippocampus and β
2-containing nAChRs are critically involved in the effects of nicotine on contextual FC (Wehner et al, 2004
; Davis and Gould, 2007
; Davis et al, 2007
). Mechanisms mediating the memory-enhancing effect downstream from nAChRs are only now beginning to be elucidated (). The effects of nicotine at nAChRs may interact with NMDA receptor-mediated processes to alter contextual FC (Gould and Lewis, 2005
). In support of nicotinic and glutamatergic interaction, glutamate receptors and nAChRs colocalize to pyramidal cells in the cortex (Levy and Aoki, 2002
); hippocampal pyramidal cells have yet to be examined, but nAChRs have been found to functionally alter glutamate sensitive neurons in the hippocampus (Ji et al, 2001
; Alkondon et al, 2003
). Activation of nicotinic receptors may directly lead to post-synaptic depolarization and removal of the NMDA magnesium block, thereby allowing an influx of calcium. Alternatively, β
2-containing nAChRs are themselves capable of gating calcium, the extent of which is dependent on the specific stoichiometry of the receptor (Tapia et al, 2007
). Calcium influx can lead to an increase in the activation of cell signaling molecules such as protein kinase C, cAMP-dependent protein kinase (PKA), and/or exchange protein activated by cAMP, which are capable of phosphorylating ERK (Roberson et al, 1999
; Gelinas et al, 2008
); a kinase implicated in contextual FC (Sweatt, 2004
) and the enhancement of contextual FC by nicotine (Raybuck and Gould, 2007
), and activated by nicotine (Valjent et al, 2004
). Relevant to the present findings, ERK activates various transcription factors such as CREB, Sp1, and c-myc (Turjanski et al, 2007
) that may regulate JNK1 mRNA transcription. Thus, the effects of nicotine on high-affinity hippocampal nAChRs could lead to an ERK-mediated increase in JNK1 mRNA expression. The suggestion that ERK mediates JNK1 activity is not unprecedented, as ERK is known to indirectly increase JNK1 activation in epithelial cells through interactions with kinases upstream from JNK1 (Pedram et al, 1998
). Alternatively, nAChR activation could lead to the stimulation of a yet unidentified cascade that leads to increased JNK1 mRNA expression.
Figure 5 A proposed cell-signaling cascade underlying the enhancement of contextual fear conditioning by nicotine. Nicotine acts at high-affinity β2-subunit containing nAChRs to directly or indirectly gate calcium through NMDA receptors. Increased intracellular (more ...)
There are a number of downstream effectors of JNK that may contribute to the effects of nicotine on contextual FC. In particular, pJNK1 activates a number of transcription factors, such as the jun family, ATF-2, and Elk-1 among others (Gupta et al, 1996
; Bogoyevitch and Kobe, 2006
). Interestingly, JNK1 contributes to hippocampal long-term depression and short-term synaptic plasticity in the CA1 region, possibly through ATF-2- and c-jun-mediated processes (Li et al, 2007
). Thus, changes in ATF-2 or c-jun activation could contribute to the enhancement of contextual FC by nicotine. In addition, an increase in activated hippocampal Elk-1 and junB has been observed after contextual FC (Sananbenesi et al, 2002
; Strekalova et al, 2003
) and nicotine administration (Nisell et al, 1997
; Nuutinen et al, 2007
). Increased levels of pJNK1 after FC paired with nicotine administration could lead to the activation of transcription factors involved in synaptic plasticity and thus an enhanced memory.
Of the various JNK isoforms, JNK1 is the least potent at activating various transcription factors (Gupta et al, 1996
; Coffey et al, 2002
) but most effective in phosphorylating another downstream effector, microtubule-associated proteins (MAPs) (Chang et al, 2003
; Bjorkblom et al, 2005
). Although the role of JNK in the activation of MAPs has primarily been studied with regard to neuronal development, MAPs are also important for the synaptic remodeling believed to underlie plasticity in the hippocampus (Muller et al, 2002
) and for the consolidation of contextual fear memories (Woolf et al, 1999
). MAPs also act as A-kinase anchoring proteins (Diviani and Scott, 2001
), which aid in the localization of PKA, a kinase extensively implicated in long-term synaptic plasticity and contextual FC (Abel et al, 1997
; Nguyen and Woo, 2003
). This suggests that another mechanism by which nicotine could enhance contextual FC and synaptic plasticity is by altering PKA signaling through JNK1 phosphorylation of MAPs.
Overall, we show that the effects of nicotine on learning and memory are mediated, at least in part, by JNK1. This is the first demonstration that nicotine and learning interact at the level of gene transcription to produce unique patterns of expression. This effect of nicotine is dependent on β2-containing nAChRs and involves changes in hippocampal function that may enhance consolidation of contextual memories. The implication of JNK1 in the effects of nicotine on cognition suggests a novel avenue of research for understanding the effects of nicotine on learning and synaptic plasticity and the manner by which nAChRs may contribute to addiction and various mental disorders.