EJ Nestler

The present study demonstrates that chronic, but not acute, adminstration of several different classes of antidepressants, including serotonin- and norepinephrine-selective reuptake inhibitors, increases the expression of cAMP response element binding protein (CREB) mRNA in rat hippocampus. In contrast, chronic administration of several nonantidepressant psychotropic drugs did not influence expression of CREB mRNA, demonstrating the pharmacological specificity of this effect. In situ hybridization analysis demonstrates that antidepressant administration increases expression of CREB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cell layers of the hippocampus. In addition, levels of CRE immunoreactivity and of CRE binding activity were increased by chronic antidepressant administration, which indicates that expression and function of CREB protein are increased along with its mRNA. Chronic administration of the phosphodiesterase (PDE) inhibitors rolipram or papaverine also increased expression of CREB mRNA in hippocampus, demonstrating a role for the cAMP cascade. Moreover, coadministration of rolipram with imipramine resulted in a more rapid induction of CREB than with either treatment alone. Increased expression and function of CREB suggest that specific target genes may be regulated by these treatments. We have found that levels of brain-derived neurotrophic factor (BDNF) and trkB mRNA are also increased by administration of antidepressants or PDE inhibitors. These findings indicate that upregulation of CREB is a common action of chronic antidepressant treatments that may lead to regulation of specific target genes, such as BDNF and trkB, and to the long-term effects of these treatments on brain function.

Behavioral and electrophysiological evidence suggests that glutamatergic neurotransmission plays an important role in some of the long-term effects of cocaine and other drugs of abuse on brain function. We therefore examined the effect of repeated cocaine treatment on glutamate receptor subunit expression in central dopamine (DA) pathways implicated in many of cocaine's behavioral actions. By immunoblotting procedures using subunit-specific antibodies, we found that repeated, but not acute, cocaine treatment increased the levels of immunoreactivity of GluR1 (an AMPA receptor subunit) and NMDAR1 (an NMDA receptor subunit) in the ventral tegmental area (VTA), a nucleus containing mesolimbic DA neurons. In contrast, chronic cocaine treatment did not alter levels of GluR2 (an AMPA receptor subunit), NMDA2A/B (NMDA receptor subunits), or GluR6/7 (kainate receptor subunits) in this brain region. Moreover, GluR1 and NMDAR1 levels were not regulated in other regions of the mesolimbic or nigrostriatal DA pathways, including the substantia nigra. Because several drugs of abuse and stress can elicit common and cross-sensitizing effects on mesolimbic DA function, we next examined whether repeated morphine and stress treatments would regulate these proteins similarly in the VTA. Although morphine delivered by subcutaneous pellet implantation had no significant effect on subunit levels, morphine delivered intermittently by subcutaneous injections of escalating doses elevated GluR1 levels in the VTA. Repeated restraint stress also paradigm (2 stressors/d under variable conditions) increased both GluR1 and NMDAR1 levels in this brain region. Unlike cocaine, morphine, and stress, repeated treatment with other psychotropic drugs (haloperidol, raclopride, sertraline, and desipramine) that lack reinforcing or sensitizing properties did not regulate GluR1 or NMDAR1 subunit levels in the VTA. Increased glutamate receptor subunit expression in the VTA may represent an important molecular mechanism by which drugs of abuse and stress exert common, long-term effects on mesolimbic DA function.

We have compared the time course of the behavioral manifestations of opiate withdrawal to the in vivo activity of locus coeruleus (LC) neurons and to increases in the levels of G-proteins, adenylate cyclase, and cAMP-dependent protein kinase known to occur in the LC in opiate-dependent animals. Rats were given morphine by daily subcutaneous implantation of morphine pellets for 5 d. On the sixth day, morphine withdrawal was induced by subcutaneous administration of naltrexone, an opiate receptor antagonist, with additional doses given 6 and 24 hr later, conditions that resulted in sustained, maximal levels of withdrawal over the duration of the experiment. We found a striking parallel between the time courses of the behavioral signs and the increased activity of LC neurons during withdrawal, both of which appeared to follow 2 phases. There was an early, rapid phase, during which withdrawal signs and increased LC activity became most pronounced within 15-30 min after naltrexone administration, and then recovered rapidly by over 50% within 4 hr of withdrawal. Subsequently, there was a slower phase, during which the persisting withdrawal signs and elevated LC activity remained roughly constant from 4 to 24 hr and did not recover completely until after 72 hr of continuous withdrawal. Adenylate cyclase and cAMP-dependent protein kinase activities in isolated LC subcellular fractions, both elevated in dependent animals, recovered to control levels after 6 hr of withdrawal, in parallel with the rapid phase of withdrawal. Levels of G1 and Go, also elevated in dependent animals, remained only slightly elevated at 6 hr and returned to normal by 24 hr. Taken together, these data suggest that increased neuronal activity in the LC is associated temporally with the behavioral morphine withdrawal syndrome and that increased levels of G-proteins and an up-regulated cAMP system may contribute to the early withdrawal activation of these neurons.

Quantitative blot immunolabeling techniques were used to determine the concentrations of ERK1 (M(r) 44 kDa) and ERK2 (M(r) 42 kDa), the two major extracellular signal-regulated protein kinases, in different regions of rat brain. The aggregate ERK concentrations (ERK1 and ERK2) were relatively high in each of the brain regions studied, ranging from approximately 0.35 ng/microgram protein in cerebellum to approximately 1.2 ng/microgram protein in nucleus accumbens. However, differences in the regional distributions of ERK1 and ERK2 resulted in ratios of their relative abundance that differed by close to 10-fold among the regions studied. The ratios of ERK1 protein to ERK2 protein varied along a rostral-caudal gradient from a low of 0.16 in frontal cortex to a high of 1.5 in pons/medulla. In hypotonic homogenates from regions at either extreme of the gradient, ERK1 and ERK2 were both found to be predominantly (> 80%) soluble. In subcellular fractions prepared from sucrose homogenates of frontal cortex and pons/medulla, both ERK1 and ERK2 were enriched in the synaptosomal and cytosolic fractions, whereas ERK2 was also enriched in the microsomal fraction. By contrast, in subfractions containing purified nuclei, levels of ERK1 and ERK2 were about one-third of those seen in homogenates and, in subfractions enriched in mitochondria, both ERK1 and ERK2 were barely detectable. The catalytic activity of the ERKs paralleled their protein levels in all of the brain regions except the hippocampus, in which the activity and phosphotyrosine content were disproportionately high. As a possible explanation for this apparent disparity, the regional distribution of ERK kinase (MEK), which phosphorylates and activates the ERKs, was also investigated. The levels of immunoreactivity of the M(r) 45 kDa ERK kinase band differed by about threefold among the brain regions, with the highest levels being present in nucleus accumbens, hippocampus, substantia nigra, and caudate/putamen. Therefore, a higher concentration of ERK kinase immunoreactivity did not appear to account for the disproportionate levels of ERK activity and phosphotyrosine content in the hippocampus. Potential regulation of ERK and ERK kinase levels was also investigated in rats subjected to chronic morphine treatment. ERK1 and ERK2 levels were increased selectively in locus coeruleus and caudate/putamen after chronic morphine treatment, whereas ERK kinase immunoreactivity remained unchanged in all of the brain regions analyzed. In summary, the regional differences in ERK and ERK kinase expression and the region-specific regulation of ERK expression suggest that ERK-related signaling may play an important role in CNS function and its adaptive responses.