The findings of the present study indicate that nicotine and learning interact to alter CREB phosphorylation at the jnk1
promoter region in the hippocampus. The increase in pCREB binding to the jnk1
promoter is not due to an increase in CREB binding or accompanied by an increase in histone acetylation. Furthermore, as we have demonstrated for the enhancement of learning by nicotine and the nicotine/learning-associated increase in hippocampal jnk1
, the increase in pCREB binding to the jnk1
promoter following learning in the presence of nicotine requires β2-subunit containing nAChRs. In addition, the increase in pCREB binding to the jnk1
promoter does not occur throughout the brain, as it is absent in the cerebellum, although this does not rule out the possibility that it is occurring in other brain regions. Taken together, these data suggest that the signaling events that lead to an increase in CREB phosphorylation at the jnk1
promoter in the hippocampus are important for the mnemonic effects of nicotine on hippocampus dependent learning and memory.
The finding that nicotine may modulate contextual fear learning via CREB phosphorylation is consistent with previous work that has found that CREB is involved in learning, memory and the rewarding properties of nicotine. Genetic disruption of CREB function consistently results in learning and memory deficits in a variety of hippocampus dependent tasks 
. Furthermore, nicotine administration results in an increase in CREB phosphorylation in the ventral tegmental area (VTA) and nucleus accumbens and the rewarding effects of the drug have been found to require CREB activation in the nucleus accumbens (Walters et al., 2005; Brunzell et al., 2009). In vitro
, nicotine has been found to result in an increase in pCREB via ERK signaling in both dissociated hippocampal neurons and PC12 cells (Nakayama et al., 2001; Hu et al., 2002). In ciliary ganglion neurons, nicotine administration results in an increase in pCREB via both ERK and calmodulin kinase II/IV (CaMK) signaling pathways (Chang and Berg, 2001). Thus, the findings from the present study are in strong agreement with the previous literature and significantly extend it by being the first study to find a specific gene that may be regulated by nicotine through a change in CREB phosphorylation.
Implicating CREB phosphorylation in the effects of nicotine on learning and memory suggests the involvement of various signaling molecules given that CREB is known to be phosphorylated via the ERK, PKA, CaMKIV and p38 MAPK pathways 
. Thus, there are two possibilities for how learning and nicotine may interact to increase CREB phosphorylation at the jnk1
promoter: 1) nicotine and fear conditioning are acting on the same pathway or 2) nicotine and fear conditioning are acting on two separate pathways that converge on CREB and both must be activated for the memory enhancing effects of nicotine. In support of the first interpretation, the enhancement of contextual fear conditioning by nicotine has been found to be dependent upon ERK 
and the action of nicotine at high affinity nAChRs in the hippocampus is able to reverse learning deficits induced by NMDA glutamate receptor antagonists 
. The action of glutamate at NMDARs in the hippocampus and ERK activation are both known to be integral to contextual fear learning 
and blocking NMDARs also prevents ERK activation in the hippocampus during learning, in hippocampal slices during LTP stimulation, and in primary cell culture in response to glutamate 
. ERK can phosphorylate CREB via the activation of p90 ribosomal S6 kinase (RSK) and mitogen and stress activated protein kinase (MSK) 
. It may be the case that learning and nicotine both independently activate the ERK pathway and are additive in their eventual effect on CREB phosphorylation at the jnk1
promoter. Alternatively, in support of the second interpretation that nicotine and fear conditioning may be acting via parallel pathways, the fact that neither nicotine alone nor fear conditioning alone are sufficient to alter either jnk1
or CREB phosphorylation at the jnk1
promoter, suggests that nicotine may be acting on a hitherto yet unidentified pathway or one that is not typically recruited during fear conditioning. It may be the case that nicotine acts in a permissive fashion that allows learning to engage additional mechanisms resulting in a greater overall response. Delineating between these two interpretations of how nicotine modulates learning is an important goal of future research.
Changes in gene transcription due to CREB activity are largely thought to be regulated via phosphorylation at Ser133 as CREB is considered to be constitutively bound to CRE sites throughout the genome, although there is some conflicting evidence. Binding studies performed in PC12 cells suggest that increasing CREB phosphorylation at Ser133 does not alter its affinity for DNA 
, however, data from hepatoma cells using DNA footprinting and bandshift assays suggest that protein kinase A (PKA) activation of CREB alters binding at various half and palindromic CRE sites 
. In the present study, there was an increase in pCREB, but not total CREB, binding to the jnk1
promoter in response to learning and nicotine administration, suggesting that CREB phosphorylation is not regulating the binding of CREB to the jnk1
promoter in this context. In contrast, Walters and colleagues 
found that a single dose of nicotine by itself results in an increase in CREB binding to the μ -opiod receptor gene promoter in the ventral tegmental area. While it is not altogether clear what specifically may be responsible for whether or not CREB is constitutively bound at any particular promoter region, differences in magnesium ion concentration 
, basal levels of PKA 
or DNA methylation 
may play a role. Thus, there appears to be considerable diversity in the regulation of CREB binding to various promoter regions, perhaps reflecting the particular composition of the cellular milieu at any given time.
One important implication of the present work is that the identification of CREB binding sites in the jnk1
promoter may provide candidate regions for identifying polymorphisms that contribute to the cholinergic contribution of the pathology of various cognitive disorders that involve nAChR function such as Alzheimer's disease, schizophrenia and addiction 
. The information obtained in the present study using ChIP allows for the approximate determination of the likely CREB binding site in the jnk1
promoter. Using evolutionary conservation analysis (Ovcharenko et al., 2004), two highly conserved half-CRE sites immediately upstream from the 5′UTR in the jnk1
promoter were identified. CREB binding was greatest, and the increase in pCREB binding due to learning and nicotine was significant, at the region covered by the jnk1b primer set. The jnk1b primer set was the closest of the sets used in the present study to the conserved CRE sites (see ), which strongly suggests that these conserved binding sites are the most likely candidates for the potential regulation of jnk1
transcription via increases in CREB phosphorylation. Thus, it may be the case that polymorphisms at these conserved CREB binding sites may play a role in mediating the cognitive effects of nicotine given that genetic variability is known to modulate the effects of nicotine in both mice and humans 
Taken together, the findings from the present study implicate CREB phosphorylation in the regulation of jnk1 transcription in the hippocampus following learning in the presence of nicotine. The increase in CREB phosphorylation is not accompanied by an increase in CREB binding to the jnk1 promoter or an increase in histone acetylation in the promoter region, suggesting that the chromatin at the jnk1 promoter is likely in an open state and poised for initiating increased transcription. Further work is required to determine how nicotine may be modulating upstream signaling cascades to interact with those stimulated by contextual fear conditioning. The CREB regulated jnk1 transcription in response to learning and nicotine may be important for the effects of nicotine on cognition and the modulation of cholinergic deficits observed in various disease states.