Inhibition of cAMP response element-binding protein or dynorphin in the nucleus accumbens produces an antidepressant-like effect

Abstract

The cAMP response element-binding protein (CREB) is a critical integrator of neural plasticity that is responsive in a brain region-specific manner to a variety of environmental and pharmacological stimuli, including widely prescribed antidepressant medications. We developed inducible transgenic lines of mice that express either CREB or a dominant-negative mutant of CREB (mCREB) in forebrain regions and used these mice to determine the functional significance of this transcription factor in the learned helplessness paradigm, a behavioral model of depression. We also use a complementary viral-mediated gene transfer approach to directly test the effect of mCREB in the nucleus accumbens, a brain region important for motivation and reward. The results demonstrate that blockade of CREB by overexpression of mCREB in transgenic mice or by viral expression of mCREB in the nucleus accumbens produces an antidepressant-like effect, whereas overexpression of CREB in transgenic mice results in the opposite phenotype. In addition, mCREB expression was colocalized with and decreased the expression of prodynorphin in nucleus accumbens medium spiny neurons, and antagonism of dynorphin in the nucleus accumbens was sufficient to produce an antidepressant-like effect similar to that observed after blockade of CREB. Together, the results demonstrate that nucleus accumbens CREB-dynorphin influence behavior in the learned helplessness model and suggest that this signaling cascade may contribute to symptoms of depression.

Fig. 1.

Pattern of FLAG–mCREB overexpression in the forebrain of bitransgenic mice. FLAG–mCREB expression was determined by immunohistochemistry in bitransgenic (tTA⁺/mCREB⁺) mice. Doxycycline was administered to bitransgenic mice until weaning, and brains were harvested at 10 weeks of age for immunohistochemical analysis of FLAG–mCREB. In bitransgenic mice, intense staining of FLAG–mCREB is observed in many cells throughout the striatum, particularly the dorsal and medial aspects, and in the nucleus accumbens. A cross section at the level of the dorsal hippocampus also demonstrates FLAG–mCREB staining in deep layers of cerebral cortex and in the CA1 pyramidal cell layers. Higher magnification demonstrates that FLAG–mCREB is localized in the nucleus. Results are representative of the analysis of at least three animals in each group. No FLAG–mCREB was observed in wild-type or single transgenic (tTA⁺/mCREB⁻) mice (data not shown).

Fig. 2.

Transgenic expression of mCREB or CREB produces opposite effects in the learned helplessness model of depression. Single (tTA⁺/mCREB⁻ or tTA⁺/CREB⁻) or bitransgenic (tTA⁺/mCREB⁺ or tTA⁺/CREB⁺) mice were exposed to sham treatment or IES and subsequently tested in an active avoidance paradigm (30 trials of 30 sec duration). The results demonstrate that there is a significant reduction in the number of escape failures and the latency to escape in the mCREB bitransgenic mice and that the opposite phenotype is observed in the CREB bitransgenic mice. There was no difference in escape failures or latency between the sham-treated single or bitransgenic animals, indicating that the alteration in active avoidance behavior is a specific response to IES exposure. The results are presented as the mean ± SEM for each group (n = 8–15 per group). *p < 0.05 compared with single transgenic mice (ANOVA and Fisher's post hoc test).

Fig. 3.

Viral-mediated expression of mCREB in the nucleus accumbens produces an antidepressant-like effect in the learned helplessness paradigm. HSV–LacZ (control) or HSV–mCREB was infused into the nucleus accumbens of rats as described in Materials and Methods. Three days later, when levels of viral expression are maximal, rats were exposed to IES and subsequently tested in an active avoidance paradigm (30 trials, 30 sec duration). Rats receiving intra-accumbens infusions of HSV–mCREB displayed a significant decrease in the number of escape failures and latency to escape. The results are expressed as the mean ± SEM (n = 10 animals per group). *p< 0.05 compared with HSV–LacZ controls (Student's ttest). NAc, Nucleus accumbens; ac, anterior commissure; β-gal; β-galactosidase.

Fig. 4.

FLAG–mCREB in bitransgenic mice is colocalized with prodynorphin- and proenkephalin-positive neurons in the striatum. FLAG–mCREB immunohistochemistry was combined with³⁵S-prodynorphin or ³⁵S-proenkephalin in situ hybridization to characterize the subpopulations of neurons that express mCREB. Emulsion grains for both prodynorphin and proenkephalin were localized in the FLAG–mCREB immunoperoxidase-stained cells in the nucleus accumbens (A, B) and dorsal striatum (data not shown). The total number of FLAG–mCREB cells that were positive for either prodynorphin or proenkephalin were counted (331 and 502 for prodynorphin or proenkephalin, in 3 or 4 animals, respectively). The percentage ± SEM of FLAG–mCREB and double-labeled prodynorphin or proenkephalin cells is shown in C. The level of prodynorphin expression in the FLAG–mCREB-immunopositive cells was also determined. The number of ³⁵S-prodynorphin emulsion grains over FLAG–mCREB-positive cells or adjacent FLAG–mCREB-negative cells in the same sections (50 of each, from 3 different animals) was determined. The results (D) are presented as percentage of control and are the mean ± SEM. *p < 0.05 compared with mCREB-negative controls (Student's t test). NAc, Nucleus accumbens.

Fig. 5.

Antagonism of dynorphin-κ-opioid receptors produces an antidepressant-like effect in the learned helplessness paradigm. Rats were exposed to IES as described in Materials and Methods. One day later, the κ-opioid receptor antagonist norBNI was microinfused into the lateral ventricles, the nucleus accumbens, or the dentate gyrus of the dorsal hippocampus, and, 3 d later, the animals were tested in an active avoidance paradigm (30 trials, 30 sec duration). The results demonstrate that intracerebroventricular or intra-accumbens, but not intrahippocampal, microinfusions of norBNI significantly decrease the number of escape failures and the latency to escape. The results are expressed as means ± SEM (n = 8–10 animals per group for intracerebroventricular and intra-accumbens infusions;n = 4 for intrahippocampus infusions). *p < 0.05 compared with saline controls (Student's t test).