Experimental Procedures

Materials

Chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and Bio-Rad Laboratories (Richmond, CA). Total CREB antibody and p-CREB antibody were purchased from Upstate Biotechnology (Lake Placid, NY). PKA α-isoforms of catalytic subunit antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies for CaM kinase IV and for PKA RII-β were obtained from Transduction Laboratories (Lexington, KY). β-Actin antibody was purchased from Sigma Chemical Co.

Ethanol Administration to Rats

Male Sprague-Dawley rats weighing 300 to 350 g were used in all experiments. Rats were given control or ethanol Lieber-DeCarli liquid diet as described previously (Pandey et al., 1999a). Rats were housed individually and received 80.0 ml of Lieber-DeCarli control diet (BioServe, Frenchtown, NJ) as their sole source of food and liquid. One group of rats continued to receive the control liquid diet and another group was gradually introduced to ethanol and was then maintained on the Lieber-DeCarli diet-containing ethanol (9% v/v) for 15 days. To maintain similar caloric intakes between the groups, the control rats were pair-fed, i.e., they were offered an amount of diet equal to the mean of the previous day's consumption by the ethanol group. All rats were weighed twice a week. The ethanol-fed rats were withdrawn for 0 or 24 h after 15 days of ethanol treatment. There were no significant differences in body weight between the control diet-fed and the ethanol-fed or ethanol-withdrawn rats. We chose 24 h of withdrawal because previously we had shown that peak anxiety and peak reduction in CRE-DNA binding occur at this time point of withdrawal after 15 days of ethanol treatment (Pandey et al., 1999a). Pair-fed control and ethanol-withdrawn (0 and 24 h) rats were used for histochemical and biochemical experiments as described below. All animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care Committee of the University of Illinois at Chicago and VA Chicago Health Care System (West Side Division), Chicago, IL.

Gold Immunolabeling of CREB, p-CREB, and CaM Kinase IV in Rat Brain

Rats were anesthetized and then perfused intracardially with n-saline (100 ml), followed by 400 ml of 4% ice-cold paraformaldehyde fixative. Brains were dissected out and placed in fixative for 20 h at 4°C. After postfixation, 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-μm coronal sections were prepared using a cryostat. These sections were placed in 0.01 M phosphate-buffered saline (PBS) at 4°C.

Sections were washed with PBS (2 × 10 min) and then blocked with RPMI medium 1640 with l-glutamine (Life Technologies, Grand Island, NY) for 30 min, followed by 10% normal goat serum (diluted in PBS containing 0.25% Triton X-100) for 30 min at room temperature. Sections were then incubated with 1% 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 and/or CaM kinase IV antibody (1:500 dilution for CREB and p-CREB and 1:200 for CaM kinase IV) in 1% BSA prepared in PBS containing 0.25% Triton X-100) for 18 h at room temperature. Following 2 × 10-min washes with PBS and 2 × 10-min washes with 1% BSA in PBS, sections were incubated with gold particles (1 nm) conjugated anti-rabbit secondary antibody for CREB and p-CREB and anti-mouse secondary antibody for CaM kinase IV (1:200 dilution in 1% BSA in PBS) for 1 h at room temperature. Sections were further rinsed several times in 1% BSA in PBS, followed by rinsing in double distilled water. The gold particles were then silver enhanced (Ted Pella Inc., Redding, CA) for 12 to 20 min and washed several times with double distilled 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 quantification of gold-immunolabeled particles of CREB, p-CREB, and CaM kinase IV was performed by using the Loats Image Analysis System connected to a light microscope that calculated the number of gold particles/100 μm² area of defined cortical structures. The threshold of each image was set up in such a way that areas without staining should give zero counts. Under this condition, gold particles in the defined 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 animals were used for CREB, p-CREB, and CaM kinase IV gold immunolabeling.

Western Blotting of Protein Kinase A, CaM Kinase IV, p-CREB, and Calcineurin in Rat Cortex

For preparation of nuclear extracts, cerebral cortices were homogenized in 5.0 ml of buffer 1 (10 mM HEPES, pH = 7.9; 1.5 mM MgCl₂; 10 mM KCl; 1 mM dithiothreitol; 0.5 mM phenylmethylsulfonyl fluoride; 10 μg/ml aprotinin; 10 μg/ml leupeptin; and 1 μg/ml pepstatin) and centrifuged at 100,000g for 30 min. The resulting pellet was suspended in 500 μl to 1.0 ml of buffer 2 (20 mM HEPES, pH = 7.9; 0.84 M NaCl; 1.5 mM MgCl₂; 0.4 mM EDTA; 0.5 mM dithiothreitol; 50% glycerol; and protease inhibitors as in buffer 1). After 15 min of incubation on ice with frequent agitation, the nuclear extracts were separated by centrifugation at 20,000g for 15 min, and then used for Western blotting. For preparations of membranal and cytosolic fractions, cortices were homogenized in 2 ml of homogenization buffer (40 mM Tris HCl, 4 mM EGTA, 9 mM EDTA, and 500 mM sucrose, pH = 7.5) containing protease inhibitors (2 mM dithiothreitol, 44 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin, and 8.7 mg/ml phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 100,000g for 1 h at 4°C. The resultant supernatant (cytosolic fraction) was used for immunoblotting. The pellet (membranal fraction) was dissolved in homogenizing buffer (described above) and used for immunoblotting as described below. The protein content in nuclear, membranal, and cytosolic fractions was determined by the method of Lowry et al. (1951).

The proteins were separated by sodium SDS-polyacrylamide gel electrophoresis (PAGE). Protein was then electrophoretically transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL) using transfer buffer (25 mM Tris base; 192 mM glycine; 20% v/v methanol, pH = 8.4). Nonspecific binding was blocked overnight at 4°C using blotto buffer [5% nonfat milk in Tris-buffered saline (10 mM Tris pH = 7.5, 100 mM NaCl) (TBS) containing 0.05% Tween 20)]. The nitrocellulose membrane was then incubated with primary antibodies of the PKA catalytic α-subunit, the PKA regulatory RII-β-subunit, CaM kinase IV, and/or calcineurin (dilution 1:1000 in blotto buffer except calcineurin, which was diluted 1:250 in blotto buffer) for 1 h at room temperature and washed with TBS (containing 0.1% Tween 20) for 3 × 10 min and then incubated with a horseradish-peroxide-linked secondary anti-rabbit antibody for PKA-Cα (1:2000 dilution in TBS containing 0.05% Tween 20) and with secondary anti-mouse antibody for CaM kinase IV, calcineurin, and PKA RII-β-subunit (1:2000 dilution in TBS containing 0.05% Tween 20) for 1 h at room temperature. The nitrocellulose membranes were washed as described above (4 × 10 min) and bound antibody was detected by the enhanced chemiluminescence method (Amersham). The p-CREB immunoblotting in rat cortex nuclear extract (40 μg) was performed according to the procedures described previously (Pandey et al., 1999b).

For immunoblotting of β-actin, the blots were stripped with stripping buffer (62.5 mM Tris-HCl; 100 mM 2-mercaptoethanol; 2% SDS, pH = 6.7) and then blocked and incubated with β-actin primary antibody (1:1000 dilution) and then secondary anti-mouse antibody (1:2000 dilution) according to the procedure described above. The bands on the autoradiograms were quantified by using the Loats Image Analysis System, and values were normalized to β-actin immunoreactivity in each sample and expressed as a percentage of the control.

Determination of cAMP-Dependent PKA Activity

cAMP-dependent PKA activity in the nuclear extracts was determined using kemptide as the substrate according to the manufacturer's instructions (cAMP-Dependent Kinase Assay kit; Upstate Biotechnology) and also as described by our group previously (Pandey et al., 1999b). The procedure is based on the phosphorylation of kemptide (125 μM) by cAMP (2.5 μM)-stimulated PKA. In brief, the nuclear extract (2 μg of protein) is incubated with and without cAMP along with 125 μM kemptide and 125 μM ATP (cold ATP) and 0.25 μCi of [γ-³²P]ATP for 10 min at 30°C. The phosphorylated substrate is separated from the residual [γ-³²P]ATP by spotting the reaction mixture (20 μl) onto P81 phosphocellulose paper, washing with 0.75% phosphoric acid, and finally, washing with acetone. The papers are transferred into vials and then scintillation cocktail is added. Bound radioactivity is determined in a scintillation counter. Basal and cAMP-dependent PKA activity is expressed as picomoles of phosphate incorporated into kemptide per minute per milligram of protein.

Determination of CREB mRNA Levels by Quantitative RT-PCR

The mRNA levels of CREB were determined by RT-PCR using internal standards. Briefly, the procedure is described below.

Preparation of Internal Standards.

mRNAs from normal rat cortex were isolated using an mRNA isolation kit (MiniRiboSap, Bedford, MA) and used for making cDNA clones for CREB. Isolated mRNA was reverse transcribed with reverse transcriptase in the presence of oligo (dt) and then PCR was performed with Taq DNA polymerase and 1 μM each of primer (forward primer 5′ AGG GCC TGC AGA CAT TAA CCA TGA CCA AT 3′ and backward primer 5′ GGT TTT CAA GCA CTG CCA CTC TGT TCT CTA A 3′) and 1 mM each of NTP in a final volume of 20 μl of the reaction mixture (PCR conditions: 94°C for 2 min; 94°C for 30 s; 60°C for 30 s; 72°C for 45 s; total 30 cycles and then 72°C for 10 min). PCR products were separated on a 2% agarose gel by electrophoresis, and DNA bands were visualized by ethidium bromide. The DNA bands were cut out and purified using the gene clean kit. Fragments of CREB were cloned into vector PCR 2.1 using the TA Cloning kit (Invitrogen, San Diego, CA). The plasmids were purified and the resulting cDNA was sequenced using the Sequenase DNA Sequencing kit (USD, Cleveland, OH). The sequences were compared with published sequences of CREB (Gonzalez et al., 1989) and demonstrated 100% homology. Internal standard templates of CREB were generated by site-directed mutagenesis using the PCR-overlap extension. Using the internal mutating primer, a restriction site for the XhoI enzyme was introduced into the middle of the DNA fragment of CREB. The cRNA internal standard was synthesized by in vitro transcription using CREB templates and T7 RNA polymerase according to the manufacturer's instructions (Promega, Madison, WI). The cRNA with the XhoI restriction site was used for the RT-PCR along with the test RNA sample.

Competitive RT-PCR.

RNA was extracted from the cortex using the CsCl₂ ultracentrifugation technique. The RNA pellet was suspended in nuclease-free water and then extracted by a phenol/chloroform mixture (1:1, v/v), and finally precipitated by ethanol and 5 M ammonium acetate. The final RNA pellet was suspended in 20 μl of diethyl pyrocarbonate-treated water and quantitated by measuring the optical density. One microgram of total RNA from each sample, along with various concentrations (10, 50, 100, and 200 pg) of internal standard, was reverse transcribed with reverse transcriptase in the presence of random hexamers and then competitively amplified using Taq DNA polymerase and CREB primers (as described above) in the presence of [³²P]dCTP. Following PCR, the DNA products were digested by XhoI restriction endonuclease enzyme and separated out by gel electrophoresis using 2% agarose gel and were stained in ethidium bromide.

For quantitation of mRNA levels of CREB, the ethidium bromide-stained bands were removed and incorporation of [³²P]dCTP into endogenous CREB and mutated CREB was determined by counting the gel-containing bands. For the blank count, the same procedures were performed without using the template. The blank count (cpm) was subtracted from the counts of both endogenous CREB and mutated CREB. The ratio between the counts incorporated into the internal standard (CREB mutated) and the counts incorporated into the CREB (endogenous) were plotted against each concentration of internal standard added to test sample. The concentration of the internal standard at which the ratio of the counts equaled one was taken as the concentration of the CREB mRNA. The mRNA levels were represented as attomoles per microgram of RNA.

Statistical Analyses

Differences among control, ethanol-fed, and ethanol-withdrawn rats were evaluated by using nonparametric Kruskal-Wallis test. Specific subgroup comparisons (control versus ethanol or control versus withdrawal) were performed using the Mann-Whitney U test. A value of p < 0.05 was considered significant.

Results

Effects of Chronic Ethanol Exposure and Its Withdrawal on p-CREB Protein Levels in Rat Cortex.

Since we had shown previously that CRE-DNA binding was decreased in the nuclear extract of rat cortex during ethanol withdrawal but not during ethanol treatment (Pandey et al., 1999a), we examined whether this was related to decreased phosphorylation of CREB in the rat cortex. We measured p-CREB immunoreactivity in the nuclear extract of the cortex of control-fed, ethanol-fed, and ethanol-withdrawn rats using the Western blot technique. Representative Western blots show that p-CREB antibody recognized a major protein of about 43 kDa (Fig.1A), and this is consistent with those reported in the literature (Moore et al., 1996; Yang et al., 1998;Tanaka et al., 1999; Davis et al., 2000). As described underExperimental Procedures, the optical density of the p-CREB was normalized with the optical density of the β-actin in each lane. It was observed that ethanol withdrawal but not ethanol treatment produced small but significant reductions in the immunolabeling of p-CREB in the nuclear extracts of the rat cortex (Fig. 1B).

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Figure 1

A, representative Western blots of the p-CREB immunoreactivity along with its respective β-actin in the nuclear extracts of the cortex of control, ethanol-treated, and ethanol-withdrawn rats. Lane 1 of the blot indicates the immunoreactivity to p-CREB protein in the nuclear extracts (10 μg of protein) of HeLa cells used as the positive cell lysate (Upstate Biotechnology, Lake Placid, NY). Nuclear proteins (40 μg) from the rat cortex were resolved on SDS-PAGE (7.5%) and were transferred to nitrocellulose membranes using electrophoresis. Detection and quantification of protein was performed as described underExperimental Procedures. B, effects of ethanol treatment and its withdrawal on protein levels of p-CREB in the nuclear extracts of the rat cortex. Values are the mean ± S.E.M. of six rats in each group and are represented as a percentage of the control. *Significantly different from their respective control groups (p < 0.05; U value = 3.00).

Neuroanatomical Localization of Changes in p-CREB and CREB Protein Levels in Cortical Structures during Ethanol Treatment and Its Withdrawal.

We examined the subcellular localization of CREB and p-CREB protein expression in various structures of the cortex; namely, the cingulate gyrus, and the frontal, parietal, and piriform cortex of control-fed, ethanol-fed, and ethanol-withdrawn rats using gold-immunolabeling histochemistry. The immunolabeling of CREB and p-CREB was specific because we did not observe any labeling in the negative brain sections (Fig. 2). It was found that CREB and p-CREB protein levels were not modified by ethanol treatment in the frontal, parietal, or piriform cortex (Figs. 3 and4). On the other hand, CREB protein levels were not changed during ethanol withdrawal, but p-CREB protein levels were significantly decreased in the frontal (layer IV/V), parietal (layer IV/V), and piriform (layer II) cortex during ethanol withdrawal after chronic ethanol intake (Figs.3-5). In contrast, CREB and p-CREB levels were regulated differentially in the cingulate gyrus (layer IV/V) during ethanol treatment and withdrawal. It was found that CREB and p-CREB protein levels were decreased during ethanol treatment but that CREB protein levels increased significantly during withdrawal, and that p-CREB levels in cingulate gyrus, although lower than those of controls, were higher than of ethanol-treated rats (Figs. 3-5). The changes in the p-CREB protein levels were specific to layer IV/V of frontal cortex, parietal cortex, and cingulate gyrus because protein levels of CREB and p-CREB were not altered in layers II/III of these structures during withdrawal after chronic ethanol intake (data not shown). These results suggest that the phosphorylation status of CREB is decreased in the specific neurocircuitry of frontal, parietal, and piriform cortex during ethanol withdrawal after chronic ethanol intake.

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Figure 2

Low-magnification views of negative sections of the frontal cortex that were incubated with 1% BSA in PBS containing 0.25 Triton X-100 without primary antibodies for CREB/p-CREB (A) and CaM kinase IV (B). Scale bar, 40 μm in A and B.

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Figure 3

Low-magnification views of CREB immunolabeling in various structures of the cortex of ethanol-withdrawn (0 and 24 h) and control rats. A–D, CREB-positive nuclei in the cingulate gyrus (CG), the frontal cortex (Fr), the parietal cortex (Par), and the piriform (Piri) cortex, respectively, of control-diet fed rats. E–H, CREB-positive nuclei in CG, Fr, Par, and Piri structures, respectively, of ethanol-treated rats. I–L, CREB-positive nuclei in CG, Fr, Par, and Piri structures, respectively, of ethanol-withdrawn rats. Scale bar, 40 μm in A–L.

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Figure 4

Low-magnification views of p-CREB immunolabeling in various structures of the cortex of ethanol-withdrawn (0 and 24 h) and control rats. A–D, p-CREB-positive nuclei in the cingulate gyrus (CG), the frontal cortex (Fr), the parietal cortex (Par), and the piriform (Piri) cortex, respectively, of control-diet fed rats. E–H, p-CREB-positive nuclei in CG, Fr, Par, and Piri structures, respectively, of ethanol-treated rats. I–L, p-CREB-positive nuclei in CG, Fr, Par, and Piri structures, respectively, of ethanol-withdrawn rats. Scale bar, 40 μm in A–L.

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Figure 5

Effects of protracted ethanol treatment and its withdrawal (24 h) on CREB and p-CREB protein levels (number of gold particles per 100-μm² area) in various structures of rat cortex. Control, ■; ethanol, ▨; withdrawal, ▪. Values are the means ± S.E.M. of five rats in each group. *Significantly different from their respective control groups (p< 0.05).

Effects of Ethanol Treatment and Withdrawal on the Levels of CREB mRNA in Rat Cortex.

We examined whether ethanol treatment and its withdrawal modified CREB gene expression in the rat cortex. Figure6A shows the site-directed mutagenesis scheme (G → C) for the CREB-cDNA template to introduce anXhoI restriction site in the middle of the sequence. Figure6B shows a representative agarose gel electrophoresis of competitive RT-PCR of endogenous and mutated (internal standard) CREB products at different concentrations of internal standards. It was found that ethanol withdrawal but not treatment significantly increased (54%) the mRNA levels of CREB in the rat cortex (Fig. 6C). These results indicate that the gene expression of CREB was up-regulated in the rat cortex during ethanol withdrawal after chronic ethanol intake.

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Figure 6

A, site-directed mutagenesis scheme for the CREB-cDNA template to introduce an XhoI restriction site (G → C) in the middle of the sequence. B, representative agarose gel electrophoresis of competitive RT-PCR of endogenous and mutated CREB amplified products. Total RNA was isolated from the cortex of control, ethanol-treated, and ethanol-withdrawn rats and quantitative RT-PCR was performed as described under Experimental Procedures. C, effects of ethanol treatment and withdrawal on the levels of CREB mRNA in the rat cortex. Values are the mean ± S.E.M. of six rats in each group and are represented as attomoles of mRNA per microgram of total RNA. *Significantly different from their respective control groups (p < 0.05; U value = 5.00).

Effects of Chronic Ethanol Exposure and Its Withdrawal on PKA Activity and on Expression of PKA Subunits in Rat Cortex.

We examined whether the decrease in p-CREB levels during ethanol withdrawal in cortical structures was related to a decrease in PKA activity and/or expression of the regulatory RII-β- or the catalytic Cα subunit of PKA in the rat cortex. No changes were found in cAMP-dependent PKA activity in the nuclear extracts of the cortex during ethanol treatment and withdrawal (Fig.7). Figure8A shows representative Western blots of the α-subunit of the PKA catalytic (PKA-Cα) protein and its respective β-actin protein levels and also shows representative Western blots of the PKA regulatory subunit (RII-β-subunit) protein and its respective β-actin protein levels. The upper band in the PKA-Cα subunit blot is nonspecific and the lower band is specific (Pandey et al., 1999b). The optical densities of the lower band of PKA-Cα and the optical densities of RII-β-subunit bands were normalized with the optical densities of β-actin in their respective lanes. It was found that ethanol treatment and its withdrawal had no effect on levels of PKA-Cα protein and RII-β protein (Fig. 8B) in the rat cortex. These results suggest that the decreased p-CREB levels in the rat cortex during ethanol withdrawal are not related to alterations in cAMP-dependent PKA activity or to alterations in the protein levels of the α-isoform of the PKA catalytic or RII-β regulatory subunits.

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Figure 7

Basal and cAMP-dependent PKA activity in nuclear extracts obtained from the cortex of control, ethanol-treated, and ethanol-withdrawn rats. Data are the mean ± S.E.M. of six rats in each group. Values are represented as picomoles of phosphate incorporated into kemptide per minute per milligram of protein.

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Figure 8

A, representative Western blots of the α-isoform of the catalytic subunit of PKA (PKA-Cα) along with its respective β-actin, and also blots of the regulatory RII-β-subunit of PKA along with its respective β-actin, in nuclear extracts from the cortex of control, ethanol-treated, and ethanol-withdrawn rats. Proteins (40 μg) were resolved on SDS-PAGE (12.5%) and were transferred to nitrocellulose membranes using electrophoresis. Detection and quantification of proteins was performed as described under Experimental Procedures. B, effects of ethanol treatment and its withdrawal on protein levels of the α-isoform of the catalytic subunit of PKA (PKA-Cα) and on the RII-β-regulatory subunit of PKA in nuclear extracts of the rat cortex. Values are the mean ± S.E.M. of nine rats in each group and are represented as a percentage of the control.

CaM Kinase IV Expression in Rat Cortex during Ethanol Treatment and Withdrawal.

We investigated whether the decreased phosphorylation of CREB during ethanol withdrawal is related to decreased expression of CaM kinase IV in the rat cortex. The protein levels of CaM kinase IV were determined by the Western blot technique. Figure9A shows representative Western blots of CaM kinase IV in membranal, cytosolic, and nuclear fractions, along with their respective β-actin protein levels in the rat cortex. The optical densities of the CaM kinase IV bands were normalized with the optical densities of the β-actin in their respective lanes. It was found that ethanol withdrawal produced a significant reduction in the protein levels of CaM kinase IV in the nuclear fraction but not in the membranal or cytosolic fractions of the cortex (Fig. 9B). On the other hand, protracted ethanol treatment has no effect on the protein expression of CaM kinase IV in nuclear, cytosolic, or membranal fractions of the rat cortex. These results indicate that CaM kinase IV expression is decreased in the nuclear fraction of the rat cortex during ethanol withdrawal but not during ethanol treatment.

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Figure 9

A, representative Western blots of CaM kinase IV along with its β-actin in the nuclear, membranal, and cytosolic fractions of the cortex of control, ethanol-treated, and ethanol-withdrawn rats. Proteins (20–40 μg) were resolved on SDS-PAGE (12.5%) and were transferred to nitrocellulose membranes using electrophoresis. Detection and quantification of proteins were performed as described under Experimental Procedures. B, effects of ethanol treatment and its withdrawal on CaM kinase IV protein levels in nuclear, membranal, and cytosolic fractions of the rat cortex. Values are the mean ± S.E.M. of six to nine rats in each group and are represented as a percentage of the control. *Significantly different from their respective control groups (p < 0.05; U value = 5.5).

Neuroanatomical Localization of Changes in CaM Kinase IV Protein Expression in Rat Cortex during Ethanol Treatment and Its Withdrawal.

We examined the subcellular expression of CaM kinase IV in cortical structures of rat brain during ethanol treatment and withdrawal using a gold-immunolabeling histochemical procedure (Fig.10). It was found that the protein expression of CaM kinase IV was significantly decreased in the frontal (layer IV/V), parietal (layer IV/V), and piriform cortex (layer II) during ethanol withdrawal after 15 days of treatment (Figs. 10 and 11); however, ethanol treatment had no effect on the expression of CaM kinase IV in these layers of frontal, parietal, or piriform cortex. On the other hand, CaM kinase IV protein levels were significantly decreased in the cingulate gyrus (layer IV/V) during ethanol treatment but reverted to normal levels during ethanol withdrawal (Fig.11). The changes in CaM kinase IV expression were restricted to layer IV/V of the frontal and parietal cortex and of the cingulate gyrus because CaM kinase IV expression was not modified in layer II/III of these cortical structures during ethanol withdrawal after chronic ethanol intake (data not shown). These results indicate that CaM kinase IV protein expression is decreased in specific neurocircuitry of the frontal, parietal, and piriform cortex during ethanol withdrawal.

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Figure 10

Low-magnification views of CaM kinase IV immunolabeling in various structures of the cortex of ethanol-withdrawn (0 and 24 h) and control rats. A–D, CaM kinase IV-positive nuclei in the cingulate gyrus (CG), the frontal cortex (Fr), the parietal cortex (Par), and the piriform (Piri) cortex, respectively, of control diet-fed rats. E–H, CaM kinase IV-positive nuclei in CG, Fr, Par, and Piri structures, respectively, of ethanol-treated rats. I–L, CaM kinase IV-positive nuclei in CG, Fr, Par, and Piri structures, respectively, of ethanol-withdrawn rats. Scale bar, 40 μm in A–L.

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Figure 11

Effects of protracted ethanol treatment and its withdrawal on CaM kinase IV protein levels (number of gold particles per 100-μm² area in various structures of rat cortex. Control, ■; ethanol, ▨; withdrawal, ▪.Values are the mean ± S.E.M of five rats in each group. *Significantly different from their respective control groups (p < 0.05).

Effects of Ethanol Treatment and Withdrawal on Expression of Calcineurin in Rat Cortex.

We examined whether the decreased phosphorylation of CREB may be related to dephosphorylation due to increased expression of calcineurin in the rat cortex during ethanol withdrawal after chronic ethanol intake. Figure12A shows representative Western blots of calcineurin and its respective β-actin protein levels in the nuclear fractions of rat cortex during ethanol treatment and withdrawal. It was observed that calcineurin protein levels were not changed during ethanol treatment and its withdrawal (Fig. 12B). These results indicate that decreased p-CREB levels in rat cortical structures may not be due to changes in calcineurin protein expression during ethanol withdrawal after 15 days of ethanol treatment.

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Figure 12

A, representative Western blots of calcineurin along with its β-actin protein in the nuclear fractions of the cortex of control, ethanol-treated, and ethanol-withdrawn rats. Proteins were resolved on SDS-PAGE and were transferred to nitrocellulose membranes using electrophoresis. Detection and quantification of protein were performed as described under Experimental Procedures. B, effects of ethanol treatment and its withdrawal on calcineurin protein levels in the nuclear fractions of the rat cortex. Values are the mean ± S.E.M. of six rats in each group and are represented as a percentage of the control.

Discussion

The novel observation of the present investigation is that the phosphorylation of CREB is decreased in the specific neurocircuitry of several cortical structures of rats during ethanol withdrawal after protracted ethanol intake. This decrease in CREB phosphorylation correlates neuroanatomically with the decreased expression of CaM kinase IV in these cortical structures during ethanol withdrawal. Interestingly, CREB gene expression is increased in the rat cortex during ethanol withdrawal, and this increase in mRNA levels of CREB is associated with increased protein levels in the cingulate gyrus but not in the frontal, parietal, or piriform cortex. The pattern of CREB and p-CREB gold immunolabeling in rat cortical structures is similar to the findings reported in the literature using diaminobenzidine immunostaining (Tanaka et al., 1999, 2000). Since phosphorylation of CREB is decreased in the frontal, parietal, and piriform cortex during ethanol withdrawal but not during ethanol treatment, this suggests the possibility that decreased expression of CREB-dependent genes in these cortical structures may be involved in the phenomenon of alcohol dependence. Both CREB expression and phosphorylation were decreased in the cingulate gyrus during ethanol treatment but returned to normal levels during ethanol withdrawal. This suggests that the decreased CREB-dependent gene expression in the cingulate gyrus during ethanol treatment may be involved in the process of alcohol tolerance.

Regulation of CREB Phosphorylation during Ethanol Treatment and Withdrawal.

Phosphorylation of CREB can be regulated by protein kinase A and by Ca²⁺ and CaM-dependent protein kinases, specifically CaM kinase IV (Gonzalez et al., 1989; Hagiwara et al., 1993; Soderling, 1999). The protein expression of total CREB is not modified, but only p-CREB protein levels are decreased during ethanol withdrawal, suggesting that kinases that regulate the phosphorylation status of CREB may be altered in the frontal, parietal, or piriform cortex. Data collected here indicate that expressions of catalytic (PKA-Cα) and regulatory subunit (RII-β) of PKA as well as PKA activity in the rat cortex are not modified by chronic ethanol treatment and withdrawal. However, ethanol withdrawal but not treatment significantly decreased the expression of CaM kinase IV in the frontal, parietal, and piriform cortex and this decrease is correlated with the decreased phosphorylation of CREB. Our findings that chronic ethanol treatment did not modify the expression of the catalytic α-subunit of PKA and CaM kinase IV in the nuclear extracts of rat cortex are similar to the studies of Yang et al. (1998), who reported similar findings in the nuclear extracts of rat cerebellum. Also, it has been shown that Ca²⁺-calmodulin-dependent protein phosphorylation in the synaptosomes of rat cortex is not modified during ethanol treatment (Rius et al., 1986). It is important to mention that, similar to CaM kinase IV, changes in PKA protein levels in the rat cortex may be layer-specific, therefore future immunohistochemical studies are needed to investigate focal changes in PKA protein expression in the cortex during ethanol withdrawal.

CaM kinase IV is localized in the cytoplasm and the nucleus, and nuclear CaM kinase IV regulates the phosphorylation of CREB (Soderling, 1999). Since we found that CaM kinase IV is decreased in the nuclear fractions but not in cytosolic or membranal fraction of the cortex during ethanol withdrawal, this suggests the possibility that the decreased nuclear CaM kinase IV expression may lead to decreased phosphorylation of CREB during ethanol withdrawal. This possibility is supported by a recent observation that CaM kinase IV-deficient (−/−) mice have lower levels of p-CREB in brain structures compared with wild-type mice (Ho et al., 2000). It has been shown that the promoter of CaM kinase IV contains CRE elements (Sun et al., 1995). Thus, it is also possible that decreased CaM kinase IV in the neurocircuitry of frontal, parietal, and piriform cortex may be related to the decreased phosphorylation of CREB during ethanol withdrawal. Nonetheless, the data presented here provide neuroanatomical evidence to suggest that CaM kinase IV may be a key-regulating enzyme of CREB phosphorylation, and thereby CREB-dependent gene expression, in cortical structures during ethanol withdrawal after chronic ethanol intake.

The state of phosphorylation of CREB is also regulated by Ca²⁺-dependent calcineurin via dephosphorylation (Tokuda and Hatase, 1998). We examined whether the decrease in p-CREB levels in the rat cortex during ethanol withdrawal was related to the decreased expression of calcineurin. It was found that the protein levels of calcineurin in the rat cortex are not modulated by chronic ethanol intake or its withdrawal. Yang et al. (1998) demonstrated that expression of protein phosphatase-1 enzyme is not modified in the rat cerebellum by chronic ethanol exposure. These results suggest the possibility that only the phosphorylation but not the dephosphorylation mechanism of CREB is altered during withdrawal after chronic ethanol intake.

CREB Gene Expression during Ethanol Treatment and Withdrawal.

Interestingly, it was found that mRNA levels of CREB are increased in the cortex during ethanol withdrawal after chronic ethanol intake. This increase in mRNA levels of CREB is associated with a small but significant increase in CREB protein levels in the cingulate gyrus but not in the other cortical structures investigated. It is possible that increased CREB gene expression may be due to the neuronal hyperexcitability (Koob and Bloom, 1988; Harris and Buck, 1990) occurring during ethanol withdrawal, but that due to post-translational modification such as decreased phosphorylation, CREB function is decreased during ethanol withdrawal. It has been shown that CREB regulates its own gene expression because of the presence of the CRE sequence in the promotor region of CREB (Meyer and Habener, 1993; Meyer et al., 1993). Thus, it is also possible that the decreased phosphorylation of CREB may lead to a decreased expression of downstream CREB-related genes, but that CREB may increase its own gene expression to compensate for the decreased CREB function during ethanol withdrawal. It has been shown that regulation of CREB gene expression occurred via different mechanisms in different cell types, which suggests differential regulation of the CREB promoter (Coven et al., 1998). Some studies have shown that CREB interacts with gene promoters in a phosphorylation-dependent manner (Wolfl et al., 1999) and other studies have shown that CREB is constitutively bound to gene promoters and that CREB phosphorylation causes conformational changes and consequently, recruits CREB binding proteins and regulates gene transcription (Andrisani, 1999; Usukura et al., 2000). Although, the mechanisms by which CREB regulates gene transcription are less clear, the present results suggest the possibility that, as reported previously, decreased CRE-DNA binding in the rat cortex (Pandey et al., 1999a) during ethanol withdrawal may be related to decreased phosphorylation of CREB in the frontal, parietal, or piriform cortex.

Brain-derived neurotrophic factor is one of the CREB-regulated genes (Duman et al., 1997; Xu et al., 1998). We have also shown previously that protein expression of brain-derived neurotrophic factor is decreased in the rat cortex during ethanol withdrawal after chronic ethanol treatment (Pandey et al., 1999a). Regardless of the mechanisms involved, these results suggest that CREB-dependent events may be decreased in the cortex during ethanol withdrawal. Decreased CRE-DNA binding and p-CREB protein levels during ethanol withdrawal may lead to decreased cAMP-inducible genes, thus permitting long-term expression of altered neuroplasticity, and thereby, the development of ethanol withdrawal symptoms.

Implications of Findings in Relation to Ethanol Withdrawal Symptoms.

Abrupt cessation of long-term ethanol intake leads to the development of ethanol withdrawal symptoms (Koob and Bloom, 1988;Harris and Buck, 1990). Symptoms such as anxiety develop at early stages of alcohol withdrawal and play an important role in the continued use of alcohol by alcoholics (Wilson, 1988; Kushner et al., 1990). We previously showed that there is a temporal correlation between the development of anxiety and the reduction of CRE-DNA binding in the rat cortex during ethanol withdrawal (Pandey et al., 1999a). As mentioned before, it is possible that decreased CRE-DNA binding may be related to the decreased phosphorylation of CREB in the frontal, parietal, and piriform cortex during ethanol withdrawal. Fluoxetine (5-hydroxytryptamine uptake blocker) treatment has been shown to increase CREB mRNA levels and CRE-DNA binding in the rodent brain (Nibuya et al., 1996; Thome et al., 2000). It was also found that treatment with fluoxetine normalizes the decrease in CRE-DNA binding activity in the cortex and also prevents the development of anxiety during ethanol withdrawal (Pandey et al., 1999a). Although speculative, taken together, these results suggest the possibility that decreased CREB function in the neurocircuitry of cortical structures may be associated with the development of some of the ethanol withdrawal symptoms, e.g., anxiety. Further studies are underway to establish the cause and/or effect relationship of decreased CREB function in specific brain regions to ethanol-withdrawal related anxiety.

Conclusions.

The data provided here present the first evidence that decreased phosphorylation of CREB and decreased expression of CaM kinase IV in cortical structures may be associated with the neuroadaptational mechanisms underlying alcohol dependence. CREB has been shown to be involved in the long-term effects of other drugs of abuse, e.g., morphine and cocaine (Lane-Ladd et al., 1997; Nestler and Aghajanian, 1997; Carlezon et al., 1998), which suggests that CREB may be a common molecular target for the cellular mechanisms of drug addiction.