Effects of protracted nicotine exposure and ... - Wiley Online Library

Journal of Neurochemistry, 2001, 77, 943±952

Effects of protracted nicotine exposure and withdrawal on the expression and phosphorylation of the CREB gene transcription factor in rat brain Subhash C. Pandey, Adip Roy, Tiejun Xu and Navdha Mittal The Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois at Chicago and Veterans Affairs Chicago Health Care System (West Side Division), Chicago, USA

Abstract Addiction to nicotine may result in molecular adaptations in the neurocircuitry of speci®c brain structures via changes in the cyclic AMP-responsive element binding protein (CREB)dependent gene transcription program. We therefore investigated the effects of chronic nicotine exposure and its withdrawal on CREB and phosphorylated CREB (p-CREB) protein levels in the rat brain. We report here that chronic nicotine exposure (1-h withdrawal) had no effect on the expression of CREB and p-CREB in the rat cortex and amygdala. On the other hand, decreases in the expression of CREB protein and phosphorylation of CREB occur in the cingulate gyrus, and in the parietal and the piriform but not in the frontal cortex during nicotine withdrawal (18 h) after nicotine exposure. It was also observed that CREB and p-CREB protein levels were signi®cantly decreased in the

medial and basolateral, but not in the central amygdala during nicotine withdrawal (18 h) after chronic nicotine exposure. Furthermore, it was found that nicotine withdrawal (18 h) after chronic nicotine exposure leads to decreased CRE-DNA binding without modulating cAMP-dependent protein kinase A activity in the cortex and the amygdala of rats. In addition, chronic nicotine treatment produced anxiolytic effects whereas nicotine withdrawal (18 h) produced anxiety in rats as measured by the elevated plus-maze test. These results provide the ®rst evidence that decreased CREB activity and/or expression in speci®c cortical and amygdaloid brain structures may be involved in the underlying molecular mechanisms of nicotine dependence. Keywords: anxiety, CREB, CRE-DNA binding, protein kinase A, nicotine dependence, rat brain. J. Neurochem. (2001) 77, 943±952.

Tobacco smoking is a major public health concern worldwide (Surgeon General 1988; Malarcher et al. 1997). There is evidence that chronic exposure to nicotine, the major constituent of cigarette smoking, leads to addiction (Stolerman and Jarvis 1995; Dani and Heinemann 1996). The mechanisms associated with nicotine addiction are not well understood, but an up-regulation of nicotinic receptors may be involved in this process (Benwell et al. 1988; Flores et al. 1992; Mark et al. 1992). Another possible mechanism for neuroadaptation to long-term nicotine exposure may be alterations in gene transcription via changes in the activity of transcription factors (Pich et al. 1997; Koob et al. 1998; Pandey et al. 1999b). Recent studies suggest the involvement of cAMPresponsive element binding protein (CREB)-dependent transcription in the molecular mechanisms of cocaine, morphine or ethanol dependence (Nestler 1992; Maldonado et al. 1996; Nestler and Aghajanian 1997; Carlezon et al. 1998; Pandey et al. 1999c); however, the adaptational

changes of CREB expression in the neurocircuitry of speci®c brain structures during nicotine dependence are unknown. CREB is regulated by cAMP-dependent protein kinase A (PKA) via phosphorylation (Brindle and Montminy 1992). In addition, other Ca21-dependent protein kinases are involved in the regulation of CREB phosphorylation (Gonzales and Montminy 1989; Dash et al. 1992). The induction of CREB Received January 18, 2001; revised manuscript received February 9, 2001; accepted February 12, 2001. Address correspondence and reprint requests to Dr Subhash C. Pandey, Department of Psychiatry, University of Illinois and, VA Chicago Health Care System (West Side Division; M/C 151), 820 South Damen Avenue, Chicago, IL 60612, USA. E-mail: [email protected] Abbreviations used: CRE, cyclic AMP-responsive elements; CREB, cyclic AMP-responsive element binding protein; p-CREB, phosphorylated CREB; DTT, dithiothreitol; EPM, elevated plus-maze; PMSF, phenylmethylsulfonyl ¯uoride; PKA, protein kinase A.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 943±952

943

944 S. C. Pandey et al.

activity leads to modulation of the expression of genes with CRE-elements in their promoters. Signal cascade changes that are triggered by Ca21- in¯ux as a result of nicotineinduced stimulation of nicotinic receptors may lead to alterations in gene transcription programs via CREB, which could be associated with nicotine dependence. To increase our understanding of the underlying molecular mechanisms associated with the long-term effects of nicotine exposure, we investigated the consequences of protracted nicotine exposure and its withdrawal on CREB expression and phosphorylation in cortical and amygdaloid brain structures. Our ®ndings demonstrate that the neuroadaptational changes that occur during nicotine dependence may involve decreased expression and phosphorylation of CREB transcription factor in speci®c brain structures. Materials and methods Animals and nicotine treatment Adult male Sprague-Dawley rats (225±250 g) were used in this study. All rats were housed under a 12-h light/dark cycle and had free access to water and food. Rats were injected subcutaneously with vehicle (n-saline) or nicotine hydrogen tartrate salt (Sigma, St Louis, MO, USA) 2 mg/kg twice a day for 10 days. Following 1 and 18 h after the last injection of nicotine or vehicle, rats were used for behavior and neurochemical studies. The duration and dose of nicotine treatment were based on previous studies (Flores et al. 1992; Ulrich et al. 1997). All animal use procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Animals and were approved by the local Animal Care Committee. Elevated plus-maze test for the measurement of anxiety The nicotine dependence phenomenon was demonstrated by measuring the anxiety levels in rats during nicotine treatment and its withdrawal using the elevated plus-maze (EPM) test. The test procedure was the same as that previously described by us (Pandey et al. 1999c). The EPM apparatus consisted of two open and two closed arms arranged directly opposite each other and connected to a central platform. After a 5-min habituation period in a testing room, the test rat was placed on the central platform facing an open arm. Each rat was observed for a 5-min test period while it explored both the arms of the EPM. The number of entries and time spent on each type of arm (open or closed) were recorded. EPM test results were expressed as the mean ^ SEM of the percentage of open-arm entries and the mean percentage of the time spent on the open arms (open-arm activity). The general activity of rats was represented by the number of entries onto the closed arms of the elevated plusmaze test, as determined by other investigators (Shulteis et al. 1998). Gold-immunolabeling of CREB and p-CREB protein in rat brain The gold-immunolabeling procedure for CREB and p-CREB was performed according to the method recently described by us (Pandey et al. 2001). The CREB and p-CREB antibodies used here were well characterized by us (Pandey et al. 1999a; 2001) and other investigators (Moore et al. 1996; Tanaka et al. 1999). Rats

were anesthetized and then perfused intracardially with n-saline (100 mL), followed by 400 mL of 4% ice-cold paraformaldehyde ®xative. Brains were dissected out and placed in ®xative for 20 h at 48C. After ®xation, brains were soaked in 10%, followed by 20% and then 30% sucrose (prepared in 0.1 m phosphate buffer, pH 7.4). Brains were then frozen and 20 mm coronal sections, corresponding to cortical and amygdaloid structures, were prepared using a cryostat. These sections were placed in 0.01 m phosphatebuffered saline (PBS) at 48C. Sections were washed with PBS (2 10 min) and then blocked with RPMI medium 1640 with l-glutamine (Life Technologies, Gaitherburg, MD, USA) for 30 min, followed by 10% normal goat serum (diluted in PBS containing 0.25% Triton X-100) for 30 min at room temperature (258C). Sections were then incubated with 1% bovine serum albumin (BSA prepared in PBS containing 0.25% Triton X-100) for 30 min at room temperature. Sections were further incubated with CREB or p-CREB antibodies (1 : 500 dilution in 1% BSA prepared in PBS containing 0.25% Triton X-100) for 18 h at room temperature. (The primary antibodies for CREB and p-CREB were purchased from Upstate Biotechnology, Lake Placid, NY, USA). Following two 10-min washes with PBS and two 10-min washes with 1% BSA in PBS, sections were incubated with gold particles (1 nm) and conjugated anti-rabbit secondary antibody (1 : 200 dilution in 1% BSA in PBS) for 1 h at room temperature. Sections were further rinsed several times with 1% BSA in PBS, followed by rinsing in double-distilled water. The gold particles were then silver enhanced (Ted Pella Inc., Redding, CA, USA) and sections were washed several times with doubledistilled water. Sections were then mounted on slides and examined under a light microscope. For the negative control sections, an identical protocol was used, except that 1% BSA in PBS was substituted for the primary antibody. The quanti®cation of goldimmunolabeled CREB or p-CREB particles was performed by using the Loats Image Analysis System (Loats Associates Inc., Westminster, MD, USA), connected to a light microscope, which calculated the number of gold particles/100 mm2 area of de®ned brain structures in these tissue sections. The threshold of each image was set up so that areas without staining should have given zero counts. Under this condition, gold particles in the de®ned areas of three adjacent brain sections of each rat were counted and then values were averaged for each rat. The serial brain sections of the same groups of rats were used for CREB and p-CREB immunolabeling. Electrophoretic gel-mobility shift assay Electrophoretic gel-mobility shift assays were performed according to the procedures described by Pandey et al. (1999c). Cerebral cortices or amygdalae were homogenized in 2.0 mL of homogenizing buffer I (10 mm HEPES, pH 7.9; 1.5 mm MgCl2; 10 mm KCl; 1 mm DTT; 0.5 mm PMSF; 10 mg/mL aprotinin; 10 mg/mL leupeptin; 1 mg/mL pepstatin) and centrifuged at 100 000 g for 30 min. The supernatant was carefully removed and discarded and the pellet was suspended in homogenizing buffer II (20 mm HEPES, pH 7.9; 0.84 m NaCl; 1.5 mm MgCl2; 0.4 mm EDTA; 0.5 mm DTT; 50% glycerol; protease inhibitors as in buffer I). The homogenates were incubated at 48C for 15 min with frequent agitation and then centrifuged at 20 000 g for 15 min. The nuclear extracts (supernatant) were carefully collected and protein content was determined by the method of Lowry et al. (1951).


CREB and nicotine dependence 945

Binding reactions were carried out by pre-incubating 10 mg of nuclear extract protein with 1 mg of poly-(dI-dc) and 6 mg of BSA in a reaction mixture (20 mm HEPES, pH 7.9; 1 mm DTT; 0.3 mm EDTA; 0.2 mm EGTA; 80 mm NaCl; 10% glycerol; and 0.2 mm PMSF) for 15 min at room temperature. After this, 32 P-labeled CRE-oligonucleotide (5 0 GATTGGCTGACGTCAGA GAGCT3 0 ) at approximately 40 000 disintegrations per minute (DPM) was added to each tube and incubated for 30 min at room temperature. The DNA-protein complexes were separated by electrophoresis using a 4% non-denaturing polyacrylamide gel in a buffer containing 25 mm Tris borate (pH 8.2) and 0.5 mm EDTA. The gel was dried and exposed to Kodak ®lm (Eastman Kodak Company, Rochester, NY, USA) at 2808C and the ®lm was then developed. The optical densities of the CRE-DNA binding complexes on the auto radiogram were measured by Loats Image Analysis using the inquiry program. The results are expressed as a percentage of the control. For the super shift, 10mg of nuclear extract protein from cortex and amygdala of rats was incubated with 1mg of CREB, p-CREB (Upstate Biotechnology, Lake Placid, NY, USA), or CREM-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibody for 20 h at 48C and then subjected to electrophoretic gel-mobility shift assay as described above.

undergoing withdrawal experienced a high level of anxiety, whereas nicotine per se is anxiolytic. Gold-immunolabeling of total CREB- and p-CREB protein in rat brain structures during nicotine exposure and withdrawal We next examined the protein levels of total CREB and p-CREB in various brain structures, namely, the cortex (cingulate gyrus, frontal, parietal and piriform) and the amygdala (basolateral, medial and central) structures of rats during nicotine treatment and its withdrawal. We counted the number of gold particles/100 mm2 area for CREB and

cAMP-dependent protein kinase A activity The activity of cAMP-dependent protein kinase A in nuclear extracts was determined using a cAMP-dependent protein kinase assay kit (Upstate Biotechnology, Lake Placid, NY, USA), as recently reported by us (Pandey et al. 1999a). The assay is based on the phosphorylation of the kemptide (125 mm) substrate with PKA stimulated by 2.5 mm cAMP. The phosphorylated substrate was separated from the residual [g-32P]ATP by spotting the 20 mL reaction mixture onto P81 phosphocellulose papers, washing with 0.75% phosphoric acid, and ®nally, washing with acetone. The results are expressed as picomoles of phosphate incorporated into kemptide per min per mg protein. Statistical analysis Differences among groups were evaluated using the Kruskel±Wallis test. Speci®c subgroup comparisons were performed using the MannWhitney U-test. A value of p , 0.05 was considered signi®cant.

Results Effects of nicotine exposure and its withdrawal on anxiety levels in rats We established whether the proposed treatment paradigm produced a nicotine dependence phenomenon, such as development of anxiety. For this purpose, anxiety levels in rats during nicotine treatment and withdrawal were measured using the elevated plus-maze test. It was observed that nicotine treatment per se produced anxiolytic effects, as demonstrated by increased activity on the open arms of the EPM. On the other hand, nicotine-withdrawn (18 h) rats spent signi®cantly less time on the open arms of EPM (Fig. 1). The activity of rats on the EPM was not signi®cantly altered by chronic nicotine treatment or its withdrawal, as demonstrated by measuring the total number of closed-arm entries (Fig. 1). These results indicate that rats

Fig. 1 Effects of nicotine exposure and withdrawal on the open and closed arm activity in the elevated plus-maze (EPM) test of rats. Rats were treated with nicotine (2 mg/kg twice daily) or n-saline for 10 days. Nicotine-treated rats were withdrawn for 1 and 18 h after the last injection of nicotine and used for measurement of anxiety levels. Values are the mean ^ SEM from 6 rats in the 18- h group and from 11 rats in the 1-h group. *Signi®cantly different from the control groups ( p , 0.05).



Fig. 2 Area marked with black squares on the cortical and amygdaloid maps represent the anatomical areas chosen for quantitations of the number of gold particles and for locus for the microphotographs. 1 cingulate gyrus; 2 frontal cortex; 3 parietal cortex; 4 piriform cortex; 5 central amygdala; 6 medial amygdala; 7 basolateral amygdala.

p-CREB immunolabeling in the de®ned brain structures of nicotine and vehicle-treated rats (Fig. 2). The immunolabeling of CREB and p-CREB was speci®c because we did not observe any labeling in the negative brain sections (data not

Fig. 3 Effect of chronic nicotine exposure (1 h after last injection of nicotine) on CREB and p-CREB protein levels (number of gold particles/100 mm2 area) in various structures of the rat cortex (a) and

shown). It was observed that chronic nicotine exposure had no effect on the protein levels of CREB and p-CREB in various structures of the cortex (Fig. 3) and the amygdala (Fig. 3). On the other hand, the protein levels of total CREB and p-CREB were signi®cantly decreased in the parietal and the piriform cortex and in the cingulate gyrus, but not in the frontal cortex during nicotine withdrawal (Figs 4 and 6). It was also observed that withdrawal (18 h) after chronic nicotine treatment produced a signi®cant reduction in the protein levels of total CREB and p-CREB in the medial and basolateral amygdala, but not in the central amygdala (Figs 5 and 6). These results indicate that nicotine withdrawal, but not nicotine exposure, decreased CREB protein expression and phosphorylation in speci®c neurocircuitry of the cortex and the amygdala. Effects of nicotine withdrawal after chronic nicotine exposure on CRE-DNA binding in the rat brain We determined if decreased protein levels of CREB and p-CREB were associated with decreased CRE-DNA binding activity in the cortex and the amygdala of rats during nicotine withdrawal. In the present study, the pattern of the CRE-DNA protein complex revealed by electrophoretic mobility gel shift assay is similar to observations previously

amygdala (b). Values are the mean ^ SEM from 5 rats. *Signi®cantly different from control groups ( p , 0.05).



Fig. 4 Low-magni®cation views of CREB and p-CREB goldimmunolabeling in various structures of the cortex of control and nicotine-withdrawn (18 h) rats. (a, c and e) Indicate the CREBpositive nuclei in the cingulate gyrus (CG), the parietal cortex (Par), and the piriform cortex (Piri) of vehicle-treated rats, respectively. (b and d and f ) Indicate the CREB-positive nuclei in CG, Par, and

Piri structures of the cortex, respectively, in nicotine-withdrawn (18 h) rats. (g and i and k) Indicate the p-CREB-positive nuclei in CG, Par, and Piri structures of vehicle-treated rats, respectively. (h and j and l) Indicate the p-CREB-positive nuclei in CG, Par, and Piri structures of the cortex, respectively, in nicotine-withdrawn rats. Scale bar 40 mm in (a±l).

Fig. 5 Low-magni®cation views of CREB and p-CREB gold-immunolabeling in medial, and basolateral amygdaloid structures of control and nicotine- withdrawn (18 h) rats. (a and c) Indicate the CREBpositive nuclei in the medial (MePD), and the basolateral amygdala (BLA) of vehicle-treated rats, respectively. (b and d) Indicate the

CREB-positive nuclei MePD, and BLA of nicotine-withdrawn rats, respectively. (e and g) Indicate the p-CREB-positive nuclei in the MePD, and BLA of vehicle-treated rats, respectively. (f and h) Indicate the p-CREB-positive nuclei in MePD, and BLA of nicotinewithdrawn rats, respectively. Scale bar 40 mm in (a±h).



Fig. 6 Effect of nicotine withdrawal (18 h) after chronic nicotine exposure (10 days) on CREB and p-CREB protein levels (number of gold particles/100mm2 of area) in various structures of the rat cortex

(a) and amygdala (b). Values are mean ^ SEM from 5 to 8 rats. *Signi®cantly different from control groups ( p , 0.05).

reported by our group (Pandey et al. 1999a,c) and other investigators (Ishige et al. 1996). We characterized the nature of the CRE-DNA binding complex and found that, in fact, this complex contains CREB, p-CREB and cyclic AMP-responsive modulatory (CREM) proteins in the rat cortex and amygdala (Fig. 7). By using the super shift assays with a CREB antibody and competitive experiments with normal and mutated CREB oligonucleotides, we have also previously shown that CRE-DNA binding activity in the rat cortex and amygdala is speci®c to CRE sites and consists of CREB protein (Pandey et al. 1999a,c). In Fig. 8(a and b), we show that withdrawal (18 h) after chronic nicotine exposure leads to a signi®cant reduction in CRE-DNA binding activity in the cortex and the amygdala. These results thus indicate that nicotine withdrawal (18 h) after 10 days of repeated injections of nicotine caused decreased CRE-DNA binding activity in rat cortex and amygdala.

regions of nicotine-withdrawn and control rats. No changes were found in cAMP-dependent PKA activity in the nuclear extracts of the cortex or the amygdala of nicotine-withdrawn (18 h) rats compared with vehicle-treated rats (Fig. 9). These results indicate that the decreased phosphorylation of p-CREB during nicotine withdrawal in these rat brain structures was not caused by changes in the cAMPdependent PKA activity but may be related to the decreased expression of CREB.

cAMP-dependent PKA activity in cortex and amygdala during withdrawal after chronic nicotine exposure To examine if the decrease in phosphorylation of CREB in the cortex and the amygdala is related to a decrease in PKA activity during nicotine withdrawal, we determined cAMPdependent PKA activity in nuclear extracts of these brain

Discussion The novel ®ndings of this study are that nicotine withdrawal, but not chronic nicotine treatment, caused a signi®cant reduction in protein levels of CREB and p-CREB in the neurocircuitry of speci®c structures of the cortex and the amygdala. The decreased expression of CREB and p-CREB leads to decreased CRE-DNA binding in the nuclear extracts of the cortex and amygdala during nicotine withdrawal. The mechanism by which nicotine withdrawal after chronic nicotine exposure regulates the CREB signaling cascade in the cortex and amygdala is not clear, but present result suggests that withdrawal after repeated nicotine exposure decreases the expression of CREB and p-CREB in the



Fig. 7 An autoradiogram showing the super shift assay of CRE-DNA binding in the presence of CREB, p-CREB, and CREM-1, antibodies in the rat cortex and amygdala. Nuclear extract protein (10 mg) was incubated with 1 mg of anti-CREB, anti-p-CREB, or anti-CREM-1, antibodies at 48C for 20 h and then subjected to a gelmobility shift assay as described in the Materials and methods section.

cingulate gyrus, in the parietal and piriform cortex, and also in the medial and basolateral amygdala. Furthermore, a nicotine injection may allow this reduction to a normal level in the above-mentioned neurocircuitry of nicotine-withdrawn rats. It is possible that smokers undergoing withdrawal may experience decreased function of CREB in these brain structures and that they smoke again to maintain the normal activity of the CREB gene transcription factor. Thus, it appears that decreased CREB activity and/or expression in speci®c structures of the cortex and amygdala may be a crucial factor in the molecular mechanisms of nicotine dependence. Because CRE-DNA binding activity is decreased in the rat cortex and amygdala during withdrawal after chronic nicotine exposure, and as this binding complex consists of CREB and p-CREB protein, we tested the possibility that this decrease was related to decreased protein levels of CREB and/or to the phosphorylation of CREB. In cortical (parietal and piriform cortex, and cingulate gyrus) and amygdaloid (medial and basolateral) structures, nicotine

withdrawal after chronic exposure did cause decreased levels of total CREB and p-CREB proteins. The mechanisms by which CREB regulates gene transcription are somewhat controversial. Wol¯ et al. (1999) observed that CREB interacts with gene promoters in a phosphorylation-dependent manner, whereas other investigators (Usukura et al. 2000) have shown that CREB constitutively bound to gene promoters and that CREB phosphorylation leads to conformational changes, which then recruits CREB binding proteins (CBP) as a cofactor and thus regulates gene transcription. Regardless of the mechanisms involved, the present studies suggest that the decreased CRE-DNA binding activity in the above-mentioned brain structures during nicotine withdrawal may be related to the decreased phosphorylation and expression of CREB proteins. We also investigated the possibility that the decreased CRE-DNA binding and p-CREB levels could be caused by decreased catalytic activity of PKA in the cortex and the amygdala. Measurement of PKA activity in nuclear extracts provides information about the functional status of PKA, as



Fig. 8 (a) Representative autoradiogram of the gel mobility shift assay showing the changes in nuclear CRE-DNA binding activity in the cortex, and the amygdala during nicotine withdrawal after chronic exposure. Rats were treated with nicotine for 10 days and sacri®ced 18 h after the last injection of nicotine. The various brain structures of these rats were used for the measurement of CRE-DNA binding activity. Ten micrograms of nuclear extract protein were incubated with 32P-labeled CRE oligonucleotides, and CRE-DNA protein complexes were separated out by gel electrophoresis as described in the Materials and Methods section. (b) The effect of nicotine withdrawal (18 h) after chronic nicotine exposure (10 days) on CRE-DNA binding activity in the rat cortex and amygdala. Values are the means ^ SEM of 6±8 experiments and are represented as a percentage of the normal controls. *Signi®cantly different from the vehicle-treated rats ( p , 0.05).

after activation the catalytic unit of PKA translocates into the nucleus, where it phosphorylates CREB and thus modulates CRE-DNA binding activity (Dash et al. 1992). It was observed that withdrawal after chronic nicotine treatment decreased the levels of p-CREB without modulating PKA activity. Taken together, these results suggest that decreased CRE-DNA binding activity during nicotine withdrawal may be due to decreased p-CREB levels in the rat cortex and amygdala. Furthermore, decreased p-CREB levels in the cortical or amygdaloid structures are due to the decreased expression of CREB and not to alterations in PKA activity. The present results also suggest the possibility that nicotine withdrawal may directly modulate CREB gene

expression in cortical and amygdaloid brain structures. CREB can also be phosphorylated by Ca21 and calmodulindependent protein kinase IV (Soderling 1999) and by ribosomal S6 kinase via the mitogen-activated protein kinase signaling cascade (Impey et al. 1999). It is possible that decreased CREB phosphorylation during nicotine withdrawal may be related to decreased activity of these protein kinases and future studies will explore these possibilities. It is important to note that nicotine withdrawal does not produce generalized changes in cortical and amygdaloid brain structures because the nuclei of frontal cortex and central amygdaloid brain structures showed no changes in CREB or p-CREB protein levels.



The data presented here provides the ®rst evidence that decreased CREB activity and expression may be associated with the neuroadaptational mechanisms underlying nicotine dependence. Interestingly, CREB has been shown to be involved in the neuroadaptational mechanisms of chronic exposure to morphine, cocaine, or ethanol (Nestler 1992; Maldonado et al. 1996; Lane-Ladd et al. 1997; Nestler and Aghajanian 1997; Pandey et al. 2001). Thus, it appears that changes in neuronal plasticity via the CREB gene transcription factor may be a common cellular target for the action of the long-term effects of these addictive drugs. Acknowledgements Fig. 9 Effect of nicotine withdrawal (18 h) after chronic nicotine exposure (10 days) on cAMP-dependent PKA activity in rat cortex and amygdala. Values are the mean ^ SEM of six experiments and are represented as a percent of the normal controls.

Nicotine addiction is a multifaceted phenomenon, possibly caused by alterations in nicotinic receptor binding sites or to alterations in the Ca21 signaling cascade that controls synaptic plasticity. Decreased CRE-DNA binding activity and decreased CREB and p-CREB protein levels during nicotine withdrawal may lead to decreased expression of cAMPinducible genes, permitting long-term expression of this altered neuroplasticity, and thus modulating the behavioral consequences of nicotine withdrawal. Cessation of smoking results in the development of withdrawal symptoms in chronic smokers, including anxiety and depressed mood (Surgeon General 1988; Hughes et al. 1991). It has recently been shown that ¯uoxetine (antidepressant) treatment prevents smoking behaviors in smokers (Aubin et al. 1996; Cornelius et al. 1997). Fluoxetine treatment has been shown to increase CREB expression and CRE-mediated gene transcription in rodent brain structures (Nibuya et al. 1996; Duman et al. 1997; Thome et al. 2000). Here, we observed that nicotine treatment per se is anxiolytic, whereas withdrawal produces anxiety in rats. Nicotine withdrawal produced decreased expression and phosphorylation of CREB in the cortex and amygdala, and nicotine treatment maintained the normal level of expression and phosphorylation of CREB in these structures. It is possible that ¯uoxetine is effective in the treatment of nicotine dependence because it has the capability of increasing the expression of CREB in the brain and thereby prevents the development of nicotine withdrawal symptoms. Although it remains speculative, it is possible that decreased CREB activity during nicotine withdrawal in the speci®c amygdaloid and/or cortical structures may be associated with nicotine withdrawal symptoms, such as anxiety and depressed mood. Further studies are needed to establish the cause and/or effect relationship of decreased CREB function to nicotine withdrawal symptoms.

This work was supported in part by the National Institute of Alcohol Abuse and Alcoholism (Grant AA 10005) and by the Department of Veterans Affairs Merit Grant awarded to Dr S. C. Pandey. We would like to thank Dr Shihong Li for her technical help in the initial stages of the study.

References Aubin H. J., Tilkete S. and Barrucand D. (1996) Depression and smoking. Encephale 22, 17±22. Benwell M. E. M., Balfour D. J. K. and Anderson J. M. (1988) Evidence that tobacco smoking increases the density of (-) [3H]nicotine binding sites in human brain. J. Neurochem. 50, 1243±1247. Brindle P. K. and Montminy M. R. (1992) The CREB family of transcription activators. Curr. Opin. Genet. Dev. 2, 199±204. Carlezon W.A. Jr., Thome J., Olson V. G., Lane-Ladd S. B., Brodkin E. S., Hiroi N., Duman R. S. and Nestler E. J. (1998) Regulation of cocaine reward by CREB. Science 282, 2272±2275. Cornelius J. R., Salloum I. M., Ehler J. G., Jarrett P. J., Cornelius M. D., Black A., Perel J. M. and Thase M. E. (1997) Double-blind ¯uoxetine in depressed alcoholic smokers. Psychopharmacol. Bull. 33, 165±170. Dani J. A. and Heinemann S. (1996) Molecular and cellular aspects of nicotine abuse. Neuron 16, 905±908. Dash P. K., Karl K. A., Colicos M. A., Prywes R. and Kandel E. R. (1992) cAMP response element-binding protein is activated by Ca21 calmodulin as well as cAMP-dependent protein kinase. Proc. Natl Acad. Sci. USA 88, 5061±5065. Duman R. S., Heninger G. R. and Nestler E. J. (1997) A molecular and cellular theory of depression. Arch. Gen. Psychiatry 54, 597±606. Flores C. M., Rogers S. W., Pabreza L. A., Wolfe B. B. and Kellar K. J. (1992) A subtype of nicotinic cholinergic receptor in rat brain is composed of a4 and b2 subunits and is upregulated by chronic nicotine treatment. Mol. Pharmacol. 41, 31±37. Gonzales G. A. and Montminy M. R. (1989) Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59, 675±680. Hughes J. R., Gust S. W., Skoog K., Keenan R. M. and Fenwick J. W. (1991) Symptoms of tobacco withdrawal: a replication and extension. Arch. Gen Psychiatry 48, 52±59. Impey S., Obrietan K. and Storm D. R. (1999) Making new connections: role of ERK/MAP kinase signaling in neuronal plasticity. Neuron 23, 11±14. Ishige K., Aizawa M., Ito Y. and Fukuda H. (1996) g-Butyrolactoneinduced absence-like seizures increase nuclear CRE- and AP-1 DNA binding activity in mouse brain. Neuropharmacology 35, 45±55.



Koob G. F., Sanna P. P. and Bloom F. E. (1998) Neuroscience of addiction. Neuron 21, 467±476. Lane-Ladd S. B., Pineda J., Boundy V. A., Pfeuffer T., Krupinski J., Aghajanian G. K. and Nestler E. J. (1997) CREB (cAMP response element-binding protein) in the locus coeruleus: biochemical, physiological and behavioral evidence for a role in opiate dependence. J. Neurosci. 17, 7890±7901. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265±275. Malarcher A. M., Chrismon J. H., Giovino G. A. and Erikson M. P. (1997) Smoking-attributable mortality and years of potential life lost-United States, 1984. Morb. Mortal. Wkly Rep. 46, 444±451. Maldonado R., Blendy J. A., Tzavara E., Gass P., Roques B. P., Hanoune J. and Schutz G. (1996) Reduction of morphine abstinence in mice with a mutation in the gene encoding CREB. Science 273, 657±659. Mark M. J., Pauly J. R., Gross S. D., Deneris E. S., Hermans-Borgmeyer I., Heinemann S. F. and Collins A. C. (1992) Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment. J. Neurosci. 12, 2765±2784. Moore A. N., Waxham M. N. and Dash P. K. (1996) Neuronal activity increases the phosphorylation of the transcription factor cAMP response element-binding protein (CREB) in rat hippocampus and cortex. J.Biol. Chem. 271, 14214±14220. Nestler E. J. (1992) Molecular mechanism of drug addition. J. Neurosci. 12, 2439±2450. Nestler E. J. and Aghajanian G. K. (1997) Molecular and cellular basis of addiction. Science 278, 58±63. Nibuya M., Nestler E. J. and Duman R. S. (1996) Chronic antidepressant administration increases the expression of cAMP response element-binding protein (CREB) in rat hippocampus. J. Neurosci. 16, 2365±2372. Pandey S. C., Mittal N., Lumeng L. and Li T.-K. (1999a) Involvement of the cyclic AMP-responsive element binding protein gene transcription factor in genetic preference for alcohol drinking behavior. Alcohol Clin. Exp Res. 23, 1425±1434. Pandey S. C., Xu T., and Zhang D. (1999b) Regulation of AP-1 gene transcription factor binding activity in rat brain during nicotine dependence. Neurosci Lett, 264, 21±24. Pandey S. C., Zhang D., Mittal N. and Nayyar D. (1999c) Potential role of the gene transcription factor cyclic AMP-responsive element

binding protein in ethanol withdrawal-related anxiety. J. Pharmacol. Exp. Ther. 288, 866±878. Pandey S. C., Roy A., and Mittal N. (2001) Effects of chronic ethanol intake and its withdrawal on the expression and phosphorylation of the CREB gene transcription factor in rat cortex. J. Pharmacol. Exp. Ther. 296, 857±868. Pich M. E., Pagliusi S. R., Tessari M., Talabot-Ayer D., Hooft Van Huijsduijnen R. and Chiamulera C. (1997) Common neural substrates for the addictive properties of nicotine and cocaine. Science 275, 83±86. Shulteis G., Yackey M., Risbrough V. and Koob G. F. (1998) Anxiogenic-like effects of spontaneous and naloxone-precipitated opiate withdrawal in the elevated-plus maze. Biochem. Pharmacol. Behav. 60, 727±731. Soderling T. R. (1999) The Ca21-calmodulin-dependent protein kinase cascade. Trends Biochem. Sci. 24, 232±236. Stolerman I. P. and Jarvis M. J. (1995) The scienti®c case that nicotine is addictive. Psychopharmacology 117, 2±10. Surgeon General, US Department of Health and Human Services (1988) The health consequences of smoking: nicotine addiction, A Report of the Surgeon General (DHHS Publication Number CDC 88± 8406), Public Health Service Of®ce on Smoking and Health, Rockville, MD. Tanaka K., Nagata E., Suzuki S., Dembo T., Nogawra S. and Fukuuchi Y. (1999) Immunohistochemical analysis of cyclic AMP response element-binding protein phosphorylation in focal cerebral ischemia in rats. Brain Res. 818, 520±526. Thome J., Sakai N., Shin K.-H., Steffen C., Zhang Y.-J., Impey S., Storm D. and Duman R. S. (2000) cAMP response elementmediated gene transcription is upregulated by chronic antidepressant treatment. J. Neurosci. 20, 4030±4036. Ulrich Y. M., Hargreaves K. M. and Flores C. M. (1997) A comparison of multiple injections versus continuous infusion of nicotine for producing up-regulation of neuronal [3H]epibatidine binding sites. Neuropharmacology 36, 1119±1125. Usukura J., Nischizawa Y., Shimomura A., Kobayashi K., Nagatsu T. and Hagiwara M. (2000) Direct imaging of phosphorylationdependent conformational change and DNA binding of CREB by electron microscopy. Gene Cells 5, 515±522. Wol¯ S., Martinez C. and Majzoub J. A. (1999) Inducible binding of cyclic adenosine 3 0 , 5 0 -monophosphate (cAMP)-responsive element binding protein (CREB) to a cAMP-responsive promoter in vivo. Mol. Endocrinol. 13, 659±669.
