Hakima Amri

Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish

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.

Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line.

To evaluate the role of the mitochondrial peripheral-type 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 PBR-negative 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.

Modeling withdrawal syndrome in zebrafish

The zebrafish (Danio rerio) is rapidly becoming a popular model species in behavioral neuroscience research. Zebrafish behavior is robustly affected by environmental and pharmacological manipulations, and can be examined using exploration-based paradigms, paralleled by analysis of endocrine (cortisol) stress responses. Discontinuation of various psychotropic drugs evokes withdrawal in both humans and rodents, characterized by increased anxiety. Sensitivity of zebrafish to drugs of abuse has been recently reported in the literature. Here we examine the effects of ethanol, diazepam, morphine and caffeine withdrawal on zebrafish behavior. Overall, discontinuation of ethanol, diazepam and morphine produced anxiogenic-like behavioral or endocrine responses, demonstrating the utility of zebrafish in translational research of withdrawal syndrome.

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis ☆

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.

Molecular mechanisms mediating the effect of mono-(2-ethylhexyl) phthalate on hormone-stimulated steroidogenesis in MA-10 mouse tumor Leydig cells.

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.

Transcriptional suppression of the adrenal cortical peripheral-type benzodiazepine receptor gene and inhibition of steroid synthesis by ginkgolide B☆

Treatment of rats and adrenocortical cells with ginkgolide B (GKB), a purified component of Ginkgo biloba leaf extracts, reduces the mRNA, protein, and ligand-binding levels of the adrenal peripheral-type benzodiazepine receptor (PBR), a mitochondrial cholesterol-binding protein, leading to decreased corticosteroid synthesis. In the Y1 adrenocortical cell line, GKB reduced both PBR levels and cyclic AMP-induced steroid formation. In these cells, GKB, but not various steroids and vitamins, reduced the expression of a reporter gene driven by the DNA sequence -624/-513 relative to the transcription start site of the PBR encoding gene. GKB treatment did not affect the SV40 promoter and increased the cytochrome P450 17alpha-hydroxylase gene promoter driven expression of the reporter gene. Electrophoretic mobility shift assays (EMSAs) indicated the presence of a functional transcriptional element bound to the -624/-513 DNA fragment. This GKB-induced inhibition of PBR was mediated by an interaction with a transcription factor that binds to the -636/-616 PBR-promoter region. Deletion or mutation of this sequence eliminated the DNA-protein interaction and the inhibitory effect of GKB on PBR gene transcription. This DNA-binding protein could be detected in nuclear extracts of rat brain, liver, and testis, but not kidney. It is also present in the human adrenal glands. However, the inhibitory effect following GKB treatment could be seen only in the adrenal glands. These results demonstrate that the GKB-activated inhibition of glucocorticoid production is due to a specific transcriptional suppression of the adrenal PBR gene and suggest that GKB might serve as a pharmacological tool to control excess glucocorticoid formation.

Stress Biomarkers in Medical Students Participating in a Mind Body Medicine Skills Program

Georgetown University School of Medicine offers an elective Mind-Body Medicine Skills (MBMS) course to medical students to promote self-care and self-awareness. Participating medical students reported better management of academic stress and well-being than non-participants. In this study, we sought to assess the stress-reducing effects of MBMS by measuring physiological changes in first-year medical students. Saliva samples were collected before (January, time 1 (T1)-pre-intervention) and upon completion of the course (May, time 2 (T2p)-post-intervention), as well as from non-participating medical students (May, time 2 (T2c)-control). The T2p and T2c collections coincided with the period of final examinations. Cortisol, dehydroepiandrosterone-sulfate (DHEA-S), testosterone and secretory immunoglobulin A (sIgA) were measured. The mean morning salivary cortisol at T2p was 97% of the mean at baseline T1 which was significantly lower than for T2c (2.4) (95% confidence interval (CI) 0.57–1.60, P =  .001); DHEA-S showed similar pattern as cortisol where the T2p levels were significantly lower than T2c (P <  .001) in both morning and evening collections. Testosterone ratio at T2p (0.85) was also lower than T2c (1.6) (95% CI 0.53–1.3, P =  .01). sIgA levels were not statistically different. On direct comparison, the T2c and T2p means were significantly different for all cortisol, DHEA-S and testosterone values. Participants maintained their hormonal balance within the normal range throughout the academic semester while the control group showed significantly increased levels, probably exacerbated by the end of the semester exam stress. To our knowledge, this is the first study to assess the physiologic benefits of a MBMS program in medical students.

Multischool, international survey of medical students' attitudes toward "holism".

Purpose Core and optional courses of study in complementary and alternative medicine (CAM) are being incorporated into medical curricula. The authors carried out this study to validate a tool to examine students’ attitudes toward holism and CAM and explore the relationships between their attitudes and other demographic and education-related characteristics in a large, multischool, international sample of medical students. Method In 2003 the authors used a modified version of the Integrated Medicine Attitude Questionnaire (IMAQ) to survey students at a total of six medical schools in the United Kingdom, New Zealand, Canada, the United States, and Hong Kong, China. A three-factor model was tested using confirmatory factor analysis, and the internal consistency of the factors were identified using Cronbach’s alpha coefficients. A multiple-indicator multiple-cause (MIMIC) analysis was carried out to determine the relationship between IMAQ factors and student characteristics. Results The authors validated a three-factor model for the IMAQ: (1) attitudes toward holism, (2) attitudes toward the effectiveness of CAM, and (3) attitudes toward introspection and the doctor–patient relationship. Cronbach’s α coefficients ranged from .41 to .71. The MIMIC model indicated that various background variables were associated with IMAQ factors (gender, race/ethnicity, and school), depending on whether students had previously visited a CAM practitioner and whether students were willing to undertake a special study module in CAM. Conclusions Further development work on the IMAQ is required and qualitative research to verify and examine the reasons behind the relationships found in this study between students’ attitudes to holism and their demographic and education-related characteristics.

Peripheral-Type Benzodiazepine Receptor

Eukaryotic steroid hormones, derived from cholesterol, are involved in the maintenance of the organism’s homeostasis, adaptability to the environment, and developmental and reproductive functions. In addition to the well-defined actions in peripheral tissues, steroids have pleiotropic actions on the central nervous system (CNS), where they control a number of neuroendocrine and behavioral functions. Thus, comprehension of the molecular systems underlying the control of steroid hormone biosynthesis is essential for the study and treatment of a multitude of physiological disorders.