Desipramine reduces stress-activated dynorphin expression and CREB phosphorylation in NAc tissue

Abstract

The nucleus accumbens (NAc) is a critical brain area for reward and motivated behavior. Accumulating evidence suggests that altered function of the transcription factor cAMP response element binding protein (CREB) within the NAc is involved in depressive behavior. In rats, stress activates CREB within the NAc, and elevation of CREB expression in this region produces depressive-like behaviors that are accompanied by activation of CREB-regulated target genes. The depressive-like behaviors seem to be due, at least in part, to CREB-mediated increases in dynorphin function, because they are mimicked by kappa-opioid receptor (KOR) agonists and attenuated by KOR antagonists. We hypothesized that if CREB-mediated dynorphin expression in the NAc contributes to depressive behavior, then antidepressants might reduce dynorphin function in this region. Here, we demonstrate that desipramine (DMI), a norepinephrine reuptake inhibitor that has been used for decades to treat clinical depression, blocks swim stress-induced activation of prodynorphin (encodes dynorphin) in the NAc. In primary cultures of NAc and striatum, DMI decreases basal and stimulated CREB phosphorylation by causing reductions in intracellular calcium (Ca(2+)) availability that are independent of norepinephrine or other monoaminergic inputs, identifying a potential mechanism for alterations in CREB-mediated gene expression. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, has similar effects in culture, suggesting a common intracellular effect of these antidepressants. These findings raise the possibility that a therapeutically relevant mechanism of action of DMI occurs through attenuation of CREB-mediated gene transcription, which is mediated via previously uncharacterized mechanisms that occur directly within the NAc.

Fig. 1.

DMI regulates molecular consequences of CREB activity within the NAc. A, prodynorphin mRNA levels within the NAc shell were significantly elevated 24 h after exposure to forced swimming. A treatment regimen of DMI (10 mg/kg) that produces antidepressant-like effects in the FST completely blocked the induction of prodynorphin mRNA but had no effect in rats exposed to a sham swim session (“Sham”; see Materials and Methods). Data are expressed as relative levels of prodynorphin mRNA, corrected for β-actin mRNA content. ^*, P < 0.05 compared with sham-treated rats; ψ, P < 0.05 comparing FST and DMI + FST, Fisher's protected t tests, 6 to 13 rats per group. B, visualization of prodynorphin and β-actin amplicons from Q-PCR.

Fig. 2.

DMI affects basal CREB phosphorylation in primary cultures of NAc and striatal tissue. Levels of CREB (□) and P-CREB (•) were quantified by Western blot (representative blots at right). The ratios of CREB/β-actin or P-CREB/β-actin were determined for each sample and normalized to the control group ratio to yield a -fold induction. A, 1-h pretreatment with DMI altered P-CREB but not CREB expression. B, 24-h pretreatment with DMI altered both P-CREB and CREB expression. ^**, P < 0.01 compared with the corresponding vehicle (Veh) control, Fisher's protected t tests, n = 2 to 4 experiments per dose of DMI with treatments given in triplicate. C, 24-h pretreatment of primary cultures with DMI (3 μM) does not affect cell viability, whereas a 20-min treatment with 70% methanol results in 100% cell death. Representative images are shown from NAc/striatal cultures treated with the respective drugs. Dead cells fluoresce red with ethidium homodimer-1 and live cells fluoresce green with calcein. The number of green plus red cells was used as the total cell population, and data are expressed as the percentage of live cells (± S.E.M.). ^**, P < 0.01 compared with vehicle-treated cells, Fisher's protected t tests.

Fig. 3.

DMI reduces KCl-stimulated CREB phosphorylation in primary cultures of NAc and striatal tissue. The ratio of P-CREB/β-actin was determined for each sample and normalized to the control group ratio to yield a -fold induction. Representative Western blots are shown to the right of each panel. A, a 3-h treatment with KCl (40 mM) significantly increased P-CREB; a 1-h pretreatment with DMI dose dependently decreased KCl-stimulated P-CREB. B, likewise, a 24-h pretreatment with DMI decreased KCl-stimulated P-CREB. ^*, P < 0.05; ^**, P < 0.01 compared with vehicle (Veh). #, P < 0.05; ##, P < 0.01 compared with KCl alone. Fisher's protected t tests, n = 3 to 4 experiments per dose of DMI with treatments given in triplicate.

Fig. 4.

Effects of DMI on Ca²⁺-mediated signaling in primary cultures of NAc and striatal tissue. The ratio of P-CREB/β-actin was determined for each sample and normalized to the control group ratio to yield a -fold induction. Representative Western blots are shown below each panel. A, a 15-min treatment with thapsigargin (100 nM) significantly increased P-CREB; a 1-h pretreatment with DMI dose dependently blocked thapsigargin-induced P-CREB. B, 24-h pretreatment with DMI dose dependently decreased, but not significantly, thapsigargin-induced P-CREB. ^**, P < 0.01 compared with vehicle (Veh); ##, P < 0.01 compared with thapsigargin alone (Thaps). Fisher's protected t tests, n = 2 to 3 experiments with treatments given in triplicate.

Fig. 5.

Effects of FLX on KCl- and thapsigargin-induced P-CREB in primary cultures of NAc and striatal tissue. The ratio of P-CREB/β-actin was determined for each sample and normalized to the control group ratio to yield a -fold induction. Representative Western blots are shown below each panel. A, a 3-h treatment with KCl (40 mM) significantly increased P-CREB; a 1-h pretreatment with FLX dose-dependently decreased KCl-stimulated P-CREB. B, likewise, a 24-h pretreatment with FLX decreased KCl-stimulated P-CREB. ^**, P < 0.01 compared with vehicle (Veh); ##, P < 0.01 compared with KCl alone. Fisher's protected t tests, n = 2 to 3 experiments with treatments given in triplicate. C, a 15-min treatment with thapsigargin (100 nM) significantly increased P-CREB; a 1-h pretreatment with FLX blocked thapsigargin-induced P-CREB. A 1-h pretreatment with FLX 1 h before Veh dose-dependently decreased basal P-CREB (inset). B, 24-h pretreatment with FLX also decreased thapsigargininduced P-CREB. A 24-h pretreatment with FLX 24 h before Veh had no significant effect on basal P-CREB (inset). ^**, P < 0.01 compared with vehicle (Veh); #, P < 0.05; ##, P < 0.01 compared with thapsigargin alone (Thaps), Fisher's protected t tests, n = 2 to 3 experiments with treatments given in triplicate.