Regulation of drug reward by cAMP response element-binding protein: evidence for two functionally distinct subregions of the ventral tegmental area

Abstract

The transcription factor cAMP response element binding protein (CREB) is implicated in the actions of drugs of abuse in several brain areas, but little information is available about a role for CREB in the ventral tegmental area (VTA), one of the key reward regions of the brain. Here, we demonstrate that chronic exposure to drugs of abuse induces CREB activity throughout the VTA. Using viral-mediated gene transfer, we expressed green fluorescent protein (GFP)-tagged CREB or mCREB (a dominant-negative form of CREB) in the VTA and, using a conditioned place-preference paradigm, found that CREB activation within the rostral versus caudal subregions of the VTA produces opposite effects on drug reward. We identified VTA subregion-specific differences in the proportion of dopaminergic and GABAergic neurons and in the dopaminergic projections to the nucleus accumbens, another brain region implicated in drug reward, and suggest that this may contribute to behavioral differences in this study. We also measured expression levels of tyrosine hydroxylase and the AMPA glutamate receptor subunit GluR1, both of which are known to contribute to drug reward in the VTA, and found that both of these genes are upregulated following the expression of CREB-GFP and downregulated following expression of mCREB-GFP, raising the possibility that CREB may exert its effects on drug reward, in part, via regulation of these genes. These results suggest a novel role for CREB in mediating drug-induced plasticity in the VTA and establish two functionally distinct subregions of the VTA in which CREB differentially regulates drug reward.

Figure 1.

Chronic morphine upregulates CRE-mediated transcription in the VTA. A, CRE-LacZ expression is upregulated by chronic morphine in rostral and caudal VTA relative to saline-treated controls. B, CRE-LacZ expression in dopaminergic neurons (identified by colabeling for TH) is upregulated by chronic morphine in both rostral and caudal VTA. C, CRE-LacZ expression is also upregulated by chronic morphine in nondopaminergic neurons but in rostral VTA only. This rostrocaudal difference was significant (p < 0.05) by ANOVA. Data are expressed as cell numbers per section ± SEM (n = 8 animals per group). ^*p < 0.05; ^**p < 0.01; t test.

Figure 2.

CREB differentially regulates cocaine reward in rostral versus caudal VTA. Animals received injections of HSV-CREB-GFP or HSV-mCREB-GFP throughout the rostrocaudal axis of the VTA and then were analyzed for conditioned place preference to a threshold dose of cocaine (1.25 mg/kg, i.p.). Data reported are mean change in time spent on the drug-paired side minus the saline-paired side before and after conditioning (± SEM), which provides a measure of place conditioning. Sham injection controls and HSV-Cre-GFP and HSV-LacZ viral controls were not significantly different and thus were pooled into a single control group. A, Place-preference scores (mean ± SEM) are plotted as a function of the HSV injection site, revealing an inflection point of approximately -5.5 mm bregma. B, Rostral to this inflection point, HSV-CREB makes a subthreshold dose of cocaine rewarding, whereas HSV-mCREB makes it aversive. Conversely, caudal to this inflection point, HSV-CREB makes the subthreshold dose of cocaine aversive, whereas HSV-mCREB has the opposite effect. ^*p < 0.05; t test; n = 6-20. C, G, Schematic representations of coronal sections through rostral and caudal VTA, respectively. These images are from Paxinos (1995), reprinted with permission. D, E, H, I, Representative coronal sections through the VTA of rats injected with HSV-CREB-GFP into the rostral or caudal VTA and immunohistochemically labeled with an antibody to GFP. F, J, Adjacent sections labeled with an antibody to TH.

Figure 3.

HSV-CREB-GFP and HSV-mCREB-GFP regulate CRE-mediated transcription in the VTA. Animals received injections of HSV-CREB-GFP, HSV-mCREB-GFP, or HSV-Cre-GFP (a control protein) throughout the rostrocaudal axis of the VTA, and sections throughout the VTA were analyzed for CRE-LacZ expression levels. In both rostral and caudal VTA, HSV-CREB-GFP increased, whereas HSV-mCREB-GFP decreased, CRE-LacZ expression levels relative to control animals injected with HSV-Cre-recombinase-GFP. Data are expressed as cell numbers per section ± SEM (n = 3-4). ^*p < 0.05; t test.

Figure 4.

CREB differentially regulates morphine reward in rostral versus caudal VTA. Animals received injections of either HSV-CREB-GFP or HSV-mCREB-GFP throughout the rostrocaudal axis of the VTA and were then trained for conditioned place preference to a threshold dose of morphine (0.125 mg/kg, s.c.) (A) or a dose range (0.125, 0.25, or 0.5 mg/kg, s.c.) (B, C). Data reported are mean change in time spent on the drug-paired minus the saline-paired side before and after conditioning (±SEM), which provides a measure of place conditioning. Control animals were injected with HSV-LacZ. Note the opposite effects of CREB and mCREB in the rostral versus caudal VTA (A). The effect of mCREB in rostral VTA (B) and the effect of CREB incaudal VTA (C) were overcome by higher doses of morphine. ^*p < 0.01; ANOVA and t test (n = 3-8).

Figure 5.

Regulation of GluR1 and TH expression in the VTA by CREB. A, GluR1 and TH mRNA expression levels in the VTA of rats injected with HSV-CREB-GFP (n = 11) or HSV-mCREB-GFP (n = 12) as measured by real-time RT-PCR. Data are displayed as a percentage of expression relative to the control group (HSV-Cre-GFP; n = 13) and are normalized to levels of β-actin, which is not regulated by these treatments. B, GluR1 and TH immunoreactivity in the VTA of rats injected with HSV-CREB-GFP (n = 7) or HSV-Cre-GFP (control; n = 7) was measured by Western blot analysis and normalized to levels of β-tubulin, which is not regulated by these treatments. Data are displayed as a percentage of expression in the control group ± SEM. ^*p < 0.05; t test.

Figure 6.

HSV vectors infect different populations of neurons in rostral versus caudal VTA. A, Percentage of infected neurons colabeled with TH or GAD varies as a function of bregma coordinates. Data points are taken from sections with a minimum of 20 infected neurons. B, In rostral VTA, similar proportions of HSV-infected neurons were colabeled with TH and GAD, respectively. However, in caudal VTA, far more infected neurons were colabeled with TH than GAD (^**p < 0.001; t test). C-E, Representative section taken from an animal injected with HSV-LacZ into rostral VTA, labeled immunohistochemically for β-gal (C) or GAD (D); a merged image is shown in E. F-H, Representative section taken from an animal injected with HSV-CREB-GFP injected into caudal VTA, labeled immunohistochemically for GFP (F) or TH (G); a merged image is shown in H.

Figure 7.

A large portion of neurons infected in caudal VTA project to the NAc and are dopaminergic. The number of β-gal-positive neurons did not significantly differ between rostral and caudal VTA. However, more neurons were retrogradely labeled in caudal VTA than in rostral VTA after injection of HS in to the NAc. More neurons in caudal VTA than in rostral VTA also were colabeled with β-gal and HS. Most of the HS- and β-gal-positive neurons in rostral and caudal VTA were also TH positive. B-G, A representative section from caudal VTA, triple labeled for HS (B), β-gal (C), and TH (D). E, Merged image from B and C showing neurons colabeled with HS and β-gal. F, Merged image from B and D showing neurons colabeled with HS and TH. G, Merged image from B-D showing triple-labeled neurons (arrows). Data are expressed as cell numbers per section ± SEM (n = 3). ^*p < 0.05; ^**p < 0.01.