The zebrafish (Danio rerio) is emerging as a promising model organism for experimental studies of stress and anxiety. Here we further validate zebrafish models of stress by analyzing how environmental and pharmacological manipulations affect their behavioral and physiological phenotypes. Experimental manipulations included exposure to alarm pheromone, chronic exposure to fluoxetine, acute exposure to caffeine, as well as acute and chronic exposure to ethanol. Acute (but not chronic) alarm pheromone and acute caffeine produced robust anxiogenic effects, including reduced exploration, increased erratic movements and freezing behavior in zebrafish tested in the novel tank diving test. In contrast, ethanol and fluoxetine had robust anxiolytic effects, including increased exploration and reduced erratic movements. The behavior of several zebrafish strains was also quantified to ascertain differences in their behavioral profiles, revealing high-anxiety (leopard, albino) and low-anxiety (wild type) strains. We also used LocoScan (CleverSys Inc.) video-tracking tool to quantify anxiety-related behaviors in zebrafish, and dissect anxiety-related phenotypes from locomotor activity. Finally, we developed a simple and effective method of measuring zebrafish physiological stress responses (based on a human salivary cortisol assay), and showed that alterations in whole-body cortisol levels in zebrafish parallel behavioral indices of anxiety. Collectively, our results confirm zebrafish as a valid, reliable, and high-throughput model of stress and affective disorders.

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To evaluate the role of the mitochondrial peripheraltype benzodiazepine receptor (PBR) in steroidogenesis, we developed a molecular approach based on the disruption of the PBR gene, by homologous recombination, in the constitutive steroid producing R2C rat Leydig tumor cell line. Inactivation of one allele of the PBR gene resulted in the suppression of PBR mRNA and ligand binding expression. Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PBR protein in the selected clone. Although mitochondria from the PBR-negative cells contained high levels of the constitutively expressed 30-kDa steroidogenic activity regulator protein, these cells produced minimal amounts of steroids compared with normal cells (5%). Moreover, mitochondria from PBR-negative cells failed to produce pregnenolone when supplied with exogenous cholesterol. Addition of the hydrosoluble cholesterol derivative, 22R-hydroxycholesterol, increased steroid production by the PBRnegative R2C cells, indicating that the cholesterol transport mechanism was impaired. Stable transfection of the PBR-negative R2C Leydig cells with a vector containing the PBR cDNA resulted in the recovery of the steroidogenic function of the cells. These data demonstrate that PBR is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P-450.

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Background-This research examines student evaluations of their experience and attitudes in an 11 week mind-body skills course for first year medical students.Aims-The aim is to understand the impact of this course on students' self-awareness, selfreflection, and self-care as part of their medical education experience.Methods-This study uses a qualitative content analysis approach to data analysis. The data are 492 verbatim responses from 82 students to six open-ended questions about the students' experiences and attitudes after a mind-body skills course. These questions queried students' attitudes about mind-body medicine, complementary medicine, and their future as physicians using these approaches.Results-The data revealed five central themes in students' responses: connections, self discovery, stress relief, learning, and medical education.Conclusions-Mind-body skills groups represent an experiential approach to teaching mindbody techniques that can enable students to achieve self-awareness and self-reflection in order to engage in self-care and to gain exposure to mind-body medicine while in medical school. IntroductionMedical school is a challenging environment in which students are confronted with multiple psychological and physical stressors; teaching students how to deal with these stressors is important for their health and well-being. Training physicians to focus on self-awareness, self-reflection and self-care may produce more reflective and well balanced doctors who may provide better patient care. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptOne approach to helping students to deal with the stressors of medical school focuses on teaching mind-body skills (MBS) that promote self-awareness, self-reflection and self-care. While medical teaching on self-reflection and professionalism is more wide spread in the UK (Maudsley & Fryer-Edwards 2003), curricula focusing on MBS and the promotion of self-awareness and self-reflection is less well established in medical schools in the United States (Dannoff & Corbet 2005). The learning objectives of self-awareness and reflection are often challenging to integrate into a medical school curriculum and equally difficult to assess (Boenink et al. 2004). There have been a variety of interventions employed to help students alleviate stress, promote well-being and self-reflection, this paper describes the results of an experiential intervention focusing on MBS techniques to achieve these outcomes.Medical students have been surveyed about the physical and psychological challenges of medical school since the early 1970s (Pitts et al. 1961;Linn & Zeppa 1984;Firth 1986;Wolf 1989;Mosley et al. 1994;Stewart et al. 1997). Findings of increased stress levels have motivated a variety of interventions designed to provide sensitivity training (Dashef et al. 1974;Hilberman et al. 1975), self-awareness (Cadden et al. 1969), self-reflection (Killion & Todnem 1991;Maudsley & Fryer-Edwards 2003;Boenink et al. 2004), sharing of feeli...

Glucocorticoid excess has broad pathogenic potential including neurotoxicity, neuroendangerment, and immunosuppression. Glucocorticoid synthesis is regulated by ACTH, which acts by accelerating the transport of the precursor cholesterol to the mitochondria where steroidogenesis begins. Ginkgo biloba is one of the most ancient trees, and extracts from its leaves have been used in traditional medicine. A standardized extract of Ginkgo biloba leaves, termed EGb 761 (EGb), has been shown to have neuroprotective and antistress effects. In vivo treatment of rats with EGb, and its bioactive components ginkgolide A and B, specifically reduces the ligand binding capacity, protein, and messenger RNA expression of the adrenocortical mitochondrial peripheral-type benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased corticosteroid synthesis. As expected, the ginkgolide-induced decrease in glucocorticoid levels resulted in increased ACTH release, which in turn induced the expression of the steroidogenic acute regulatory protein. Because ginkgolides reduced the adrenal PBR expression and corticosterone synthesis despite the presence of high levels of steroidogenic acute regulatory protein, these data demonstrate that PBR is indispensable for normal adrenal function. In addition, these results suggest that manipulation of PBR expression could control circulating glucocorticoid levels, and that the antistress and neuroprotective effects of EGb are caused by to its effect on glucocorticoid biosynthesis.

Di-(2-ethylhexyl) phthalate, a widely used plasticizer, and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to exert adverse effects on the reproductive tract in developing and adult animals. As yet, however, the molecular mechanisms by which they act are uncertain. In the present study, we address the molecular and cellular mechanisms underlying the effects of MEHP on basal and human chorionic gonadotropin (hCG)-stimulated steroid production by MA-10 Leydig cells, using a systems biology approach. MEHP induced dose-dependent decreases in hCG-stimulated steroid formation. Changes in mRNA and protein expression in cells treated with increasing concentrations of MEHP in the presence or absence of hCG were measured by gene microarray and protein high-throughput immunoblotting analyses, respectively. Expression profiling indicated that low concentrations of MEHP induced the expression of a number of genes that also were expressed after hCG stimulation. Cross-comparisons between the hCG and MEHP treatments revealed two genes, Anxa1 and AR1. We suggest that these genes may be involved in a new self-regulatory mechanism of steroidogenesis. The MEHP-induced decreases in hCG-stimulated steroid formation were paralleled by increases in reactive oxygen species generation, with the latter mediated by the Cyp1a1 gene and its network. A model for the mechanism of MEHP action on MA-10 Leydig cell steroidogenesis is proposed.

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