Beta-2 adrenergic receptors mediate stress-evoked reinstatement of cocaine-induced conditioned place preference and increases in CRF mRNA in the bed nucleus of the stria terminalis in mice.

RATIONALE: Understanding the mechanisms responsible for stress-induced relapse is important for guiding treatment strategies aimed at minimizing the contribution of stress to addiction. Evidence suggests that these mechanisms involve interactions between noradrenergic systems and the neuropeptide corticotropin-releasing factor (CRF). OBJECTIVES: The interaction between ß-adrenergic receptors (ARs) and CRF as it relates to the reinstatement of cocaine-conditioned reward in response to a stressor was examined in mice. We hypothesized that ß2-ARs are required for stress-induced activation of CRF pathways responsible for reinstatement. METHODS: Stress-induced relapse was examined based on the re-establishment of cocaine-induced conditioned place preference (CPP; 4 × 15 mg/kg cocaine, i.p.) after extinction using forced swim (6 min at 22 °C) or an injection of the ß2-AR agonist, clenbuterol (4 mg/kg, i.p.). The CRF-R1 antagonist antalarmin (10 mg/kg, i.p.) or the ß2-AR antagonist ICI-118,551 (1 mg/kg, i.p.) were given 30 min prior to reinstating stimuli. Quantitative PCR was conducted in dissected bed nucleus of the stria terminalis (BNST) and amygdala, putative sources of CRF that contribute to reinstatement, to examine the effects of ICI-118,551 on swim-induced increases in CRF messenger RNA (mRNA) in mice with a cocaine history. RESULTS: Pretreatment with ICI-118,551 or antalarmin blocked swim-induced reinstatement of CPP. Reinstatement by clenbuterol was also blocked by antalarmin. ICI-118,551 pretreatment prevented swim-induced increases in CRF mRNA in the BNST. Effects in the amygdala were not observed. CONCLUSIONS: These findings indicate that, during stress, norepinephrine, via ß2-ARs, either directly or indirectly activates CRF-releasing neurons in the BNST that interface with motivational neurocircuitry to induce reinstatement of cocaine-conditioned reward.

Natural and synthetic corticosteroids inhibit uptake 2-mediated transport in CNS neurons.

In addition to exerting actions via mineralocorticoid and glucocorticoid receptors, corticosteroids also act by inhibiting uptake(2), a high-capacity monoamine transport system originally described in peripheral tissues. Recent studies have demonstrated that uptake(2) transporters are expressed in the brain and play roles in monoamine clearance, suggesting that they mediate some corticosteroid effects on physiological and behavioral processes. However, the sensitivity of brain uptake(2) to many natural and synthetic corticosteroids has not been characterized. Cultured rat cerebellar granule neurons (CGNs) were previously shown to exhibit corticosterone-sensitive accumulation of the uptake(2) substrate 1-methyl-4-phenylpyridinium (MPP(+)). We examined the expression of uptake(1) and uptake(2) transporters in CGNs, and tested the effects of a variety of natural and synthetic corticosteroids on accumulation of [(3)H]-MPP(+) by these cells. Cultured rat CGNs expressed mRNA for three uptake(2)-like transporters: organic cation transporters 1 and 3, and the plasma membrane monoamine transporter. They did not express mRNA for the dopamine or norepinephrine transporters, and expressed very little mRNA for the serotonin reuptake transporter. Accumulation of [(3)H]-MPP(+) by CGNs was dose-dependently inhibited by corticosterone and decynium-22, known inhibitors of uptake(2). Accumulation of MPP(+) was also dose-dependently inhibited, with varying efficacies, by aldosterone, 11-deoxycorticosterone, cortisol, and cortisone, and by the synthetic glucocorticoids betamethasone, dexamethasone and prednisolone, and the glucocorticoid receptor antagonist RU38486. These studies demonstrate that uptake(2) in the CNS is inhibited by a variety of natural and synthetic corticosteroids, and suggest that inhibition of uptake(2)-mediated monoamine clearance may underlie some behavioral and physiological effects of these hormones.

Dynamic Lkb1-TORC1 signaling as a possible mechanism for regulating the endoderm-intestine transition.

The intestinal epithelium arises from undifferentiated endoderm via a developmental program known as the endoderm-intestine transition (EIT). Previously we found that the target of rapamycin complex 1 (TORC1) regulates intestinal growth and differentiation during the EIT in zebrafish. Here we address a possible role for the tumor-suppressor kinase Lkb1 in regulating TORC1 in this context. We find that TORC1 activity is transiently upregulated during the EIT in both zebrafish and mouse. Concomitantly, Lkb1 becomes transiently localized to the nucleus, suggesting that these two phenomena may be linked. Morpholino-mediated knockdown of lkb1 stimulated intestinal growth via upregulation of TORC1, and also induced precocious intestine-specific gene expression in the zebrafish gut epithelium. Knockdown of tsc2, which acts downstream of lkb1, likewise induced early expression of intestine-specific genes. These data suggest that programmed localization of Lkb1 could represent a novel mechanism for regulating the EIT during intestinal development in vertebrates.

A whole-animal microplate assay for metabolic rate using zebrafish.

Regulation of whole-body metabolism and energy homeostasis has been shown to require signaling between multiple organs. To identify genetic programs that determine metabolic rate, and compounds that can modify it, a whole-animal assay amenable to large-scale screening was developed. The direct correlation of acid production with metabolic rate was exploited to use a noninvasive colorimetric assay for acid secretion by individual zebrafish larvae in a 96-well plate format. A 3-fold increase in metabolic rate was detected that accompanied development between 24 and 96 h postfertilization. Dynamic changes in metabolic rate were also detected in response to different conditions such as temperature and drug treatments, in general agreement with the rate of oxygen consumption measured concomitantly. This assay was used to measure metabolic rate in the progeny of fish known to carry a recessive mutation in a gene required for ribosome biogenesis ( npo(fW07-g)), which would be expected to reduce energy consumption. A strong correlation was found (p < 10(-6) ) between reduced metabolic rate and genotype even before the developmental defect was visually evident. These studies support the conclusion that whole-animal acid secretion can be used as a readout for energy metabolism, thus enabling large-scale screening for genetic and chemical regulators of metabolic rate in a vertebrate.

Target of rapamycin (TOR) signaling controls epithelial morphogenesis in the vertebrate intestine.

The target of rapamycin (TOR) signaling pathway regulates cell growth and proliferation, however the extent to which TOR signaling mediates particular organogenesis programs remains to be determined. Here we report an examination of TOR signaling during zebrafish development, using a combination of small molecule treatment and morpholino-mediated gene knockdown. First, we amplified and sequenced the full-length cDNA for the zebrafish TOR ortholog (ztor). By in situ hybridization, we found that ztor is expressed ubiquitously in the early embryo, but displays a dynamic pattern in the gut between 48 and 72 h post-fertilization (hpf). Treatment of zebrafish embryos with rapamycin induced only a mild general developmental delay up to 72 hpf, but digestive tract development became arrested at the primitive gut tube stage. Rapamycin inhibited intestinal epithelial growth, morphogenesis and differentiation. Using morpholino-mediated gene knockdown of TOR pathway components, we show that this effect is mediated specifically by the rapamycin-sensitive TOR complex 1 (TORC1). Thus, in addition to regulating cell growth and proliferation, TOR signaling controls the developmental program guiding epithelial morphogenesis in the vertebrate intestine.

Zebrafish Offers New Perspective on Developmental Role of TOR Signaling.

Genetic studies on the molecular basis of growth control have converged on the target of rapamycin (TOR) pathway as a key regulator.1 When stimulated by nutrients (i.e. amino acids) or growth factors (i.e. insulin), TOR activates protein synthesis and other anabolic pathways to promote cell growth.1 Our knowledge of TOR's function in vivo is still rudimentary, particularly in the setting of vertebrate development. An important question is whether TOR functions as a constitutive regulator of growth in all cell types, or as a stage and organ specific regulator. Recently we employed the zebrafish as a vertebrate model system to study the developmental role of TOR signaling. We found that TOR signaling was required for a discrete step prior to epithelial differentiation. The results support the view that different organs may be reliant on TOR activity to differing degrees. In the case of the zebrafish, the digestive tract exhibits the greatest sensitivity to rapamycin, which may reflect its reliance on TOR signaling for normal growth. We suggest the hypothesis that TOR signaling may regulate the size of the intestine's absorptive surface area in response to systemic nutrient demand.

Glutathione S-transferase pull-down assays using dehydrated immobilized glutathione resin.

We have developed an affinity-precipitation technique to facilitate conducting glutathione S-transferase (GST) pull-down assays. The dehydrated immobilized glutathione resin format, when combined with microcentrifuge spin columns, is a powerful tool that enables the simultaneous performance of resin hydration, the binding of the GST fusion protein, and the pull-down step with the appropriate protein partner in a semihigh-throughput fashion (multiple samples processed at the same time). The entire assay process is shortened and recovery is enhanced when coupled with a spin-column format, providing a convenient way to study protein-protein interactions. We successfully tested the resin format/technique in three common pull-down applications utilizing radiolabeled, overexpressed, and activated endogenous interacting protein partners.