Regulation of CART peptide expression by CREB in the rat nucleus accumbens in vivo

Abstract

Production of mRNA from the cocaine- and amphetamine-regulated transcript (CART) gene is regulated by cocaine and other drugs of abuse in the nucleus accumbens (NAc), a brain reward region. Current hypotheses postulate that CART peptides there oppose the rewarding actions of cocaine by opposing the effects of dopaminergic transmission. Since over expression of CREB was shown to decrease cocaine-mediated reward, we hypothesized that CART could be a target gene for CREB in the NAc and that over expression of CREB would increase CART peptide levels. Transcription factor (TF) binding to DNA is influenced by sequences adjacent to consensus TF binding sites and other factors. We thus examined CREB binding to a 27mer oligonucleotide containing the CRE sequence from the CART gene proximal promoter. Using electrophoretic mobility shift assays and TF-antibody super shift assays, CREB was found to bind to the CRE sequence from the CART promoter. To test if over expression of CREB in the NAc affected CART peptide levels, Herpes simplex virus-1 vectors over expressing CREB (HSV-CREB), or a vector that expressed LacZ (HSV-LacZ) as a control, were injected into the NAc of rats. Western blotting and in situ hybridization showed that HSV-CREB injections increased CART mRNA and peptide levels. Injections of a dominant negative CREB mutant (HSV-mCREB) did not alter either CART mRNA or peptide levels. The finding that CREB can regulate the levels of CART mRNA and peptides in vivo in the NAc supports a role for CART peptides in psychostimulant-induced reward and reinforcement.

Fig. 1

Oligonucleotides containing the CART promoter CRE cis-regulatory element bind to CREB and phospho-CREB from the rat NAc. EMSA and super shift analyses were performed using nuclear proteins from the NAc as described in the Experimental procedures (the gel shown is representative of the data from at least five other animals assayed separately in independent experiments). The sequence of the regions of the CART promoter containing the CRE cis-element (the 27mer is referred to as the “CRE”) is given below the picture of the gel in the box, and the core binding sequence is underlined. ³²P-CRE+NAc transcription factor (TF) complexes were visualized by incubating proteins extracted from cell nuclei with a ³²P-radiolabeled oligonucleotide identical in sequence to the CART gene CRE cis-regulatory element along with its flanking sequence, and separating free ³²P-CRE (see figure's bottom right) from the ³²P-CRE+NAc TF complex on a polyacrylamide gel (complex is denoted by boxed arrow at the figure's top left). The identity of the protein in the ³²P-CRE+NAc TF complex was determined by super shift analysis using either a CREB- or ser133 phospho-CREB-specific antibody (see box at top right denoting the ³²P-CRE+NAc TF+antibody complex). See text for additional details.

Fig. 2

Nissl-stained hemisection in the coronal plane shows an example of the site of injection. HSV vectors were injected into the NAc as described in the Experimental procedures. Stereotaxic coordinates were determined from the Rat Atlas (Paxinos and Watson, 2002). The section is at A/V +1.5, M/L±1.6 and D/V−7.6 and 1.6×1000 magnification. The anterior commissure, ventricles and injection site are delineated and the accumbens region has been circled to orient the reader.

Fig. 3

HSV-CREB injections into the rat NAc increase CREB protein levels. The levels of CREB protein (approximately 45 kDa) in NAc from an HSV-CREB treated animal was determined by Western blot and compared to CREB levels in a control animal treated with HSV-LacZ (ratio of 1.42±052, p<0.01). The two lanes on the left show duplicate repeats using tissue from one of a pair of animals, and the two lanes on the right are duplicate repeats from the other, control animal of the pair. All lanes were loaded with the same amount of tissue protein as measured by Bradford assay previous to loading the gel. Quantification and statistical analysis of immunoreactive bands were performed using the Scion Image software (NIH, Bethesda, MD) as described in the Experimental procedures. This result is representative for a total of five pairs of animals prepared and assayed separately in independent experiments. See text for details.

Fig. 4

HSV-CREB injections into the rat NAc increase CART peptide levels. The levels of CART peptide (approximately 6.5 kDa) in NAc from an HSV-CREB treated animal were determined by Western blot and compared to CART peptide levels in a control animal treated with HSV-LacZ (ratio of 1.32±0.073, p<0.01). The three lanes on the left show triplicate repeats using tissue from one of a pair of animals, and the three lanes on the right are triplicate repeats from the other, control animal of the pair. Each lane is normalized to whole tissue actin (approximately 50 kDa) as measured by Western blot. After normalization, the ratio of the pair of values were calculated and analyzed as described in the Experimental procedures. This result is representative of a total of six pairs of animals prepared and assayed separately in independent experiments. See text for details.

Fig. 5

Over-expression of CREB increases CART mRNA in the rat NAc. CART mRNA levels were measured by in situ hybridization 36 h following viral infection with either HSV-CREB or mCREB. Animals received HSV-CREB or HSV-mCREB in one hemisphere and a control (sham injection or HSV-LacZ vehicle) in the contralateral hemisphere thus allowing each animal to serve as its own control. (A) Representative autoradiogram showing increased radioactive CART mRNA signal in the HSV-CREB treated hemisphere. (B) Quantitative analysis was conducted by measuring the relative OD of the radioactive signal in the NAc. Data is expressed as the mean± SEM and significance was tested with a one-way ANOVA and Newman–Keuls post hoc test. HSV-infected animals had significantly higher CART mRNA levels compared to all controls and HSV-mCREB-infected animals (*p<0.01). CART mRNA levels in the NAc of HSV-mCREB treated rats did not differ from those in rats treated with sham injections or HSV-LacZ alone.

Fig. 6

HSV-mCREB injections into the rat NAc trend towards decreasing CREB protein levels. The levels of CREB protein (approximately 45 kDa) in NAc from an HSV-mCREB treated animal was determined by Western blot and compared to CREB levels in an HSV-LacZ treated control animal (ratio of 0.73±0.148, p>0.05). The two lanes on the left show duplicate repeats using tissue from one of a pair of animals, and the two lanes on the right are duplicate repeats from the other, control animal of the pair. All lanes were normalized to its histone 2B content using an antibody specific to H2B. After normalization, the ratio of the pair of values were calculated and analyzed as described in the Experimental procedures. Quantification and statistical analysis of immunoreactive bands were performed using the Scion Image software (NIH, Bethesda, MD) as described in the Experimental procedures. This result is representative of a total of four pairs of animals prepared and assayed in independent experiments. See text for additional details.

Fig. 7

HSV-mCREB injections into the rat NAc decrease phospho-CREB levels. The levels of ser133 phospho-CREB (approximately 45 kDa) in NAc from an HSV-mCREB treated animal was determined by Western blot and compared to phospho-CREB levels in an HSV-LacZ treated control animal (ratio of 0.64±0.145, p<0.05). In this representative blot showing phospho-CREB levels in a pair of animals, all lanes were normalized to the histone 2B content. After normalization, the ratio of the pair of values were calculated and analyzed as described in Methods. The two lanes on the left show duplicate repeats using tissue from one of a pair of animals, and the two lanes on the right are duplicate repeats from the other, control animal of the pair. Quantification and statistical analysis of immunoreactive bands were performed using the Scion Image software (NIH, Bethesda, MD) as described in the Experimental procedures. This result is representative for a total of seven pairs of animals prepared and assayed separately in independent experiments. See text for additional details.

Fig. 8

HSV-mCREB injections into the rat NAc do not change CART peptide levels. The levels of CART peptide (approximately 6.5 kDa) in tissue from an HSV-mCREB treated animal were determined by Western blot and compared to CART peptide levels in an HSV-LacZ treated control animal (ratio of 1.02±0.071, P=0.842). In this representative blot showing CART levels in a pair of animals, each lane is normalized to whole tissue actin (approximately 50 kDa). After normalization, the ratio of the pair of values were calculated and analyzed as described in the Experimental procedures. This result is representative of a total of seven pairs of animals prepared and assayed separately in independent experiments. See text for additional details.