Assignment Task

Background

The physiological stress response is a highly conserved adaptation among vertebrates used to maintain internal homeostasis in response to a variable environment, or ‘stressors’. The physiological stress response is characterized by the activation of the hypothalamic-pituitary–interrenal (HPI) axis, leading to the release of stress hormones which induce a cascade of physiological responses which are critical for mediating adaptive metabolic, physiological, and behavioral adjustments. The HPI axis is one of the best examples of neuroendocrine-immune interactions critical to an individual’s survival. If this pathway is disrupted, it inhibits an organism’s ability to cope with stress by altering or even inhibiting the cascade of physiological responses.

In response to a stressor, the pathway operates by first releasing corticotropin-releasing factor protein (CRF) from the hypothalamus, this stimulates the pituitary gland to release pro-opiomelanocortin hormone protein (POMC) culminating in the rapid production, release of, and circulation of glucocorticoids from interrenal cells. Glucocorticoids, predominantly cortisol, and catecholamines released into the blood and tissues, including gonads, brain, immune cells, and gills, bind and activate corticosteroid receptors, namely the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) which signals molecular programming changes in target tissues by regulating the transcription of specific genes. These receptors are highly expressed on immune cells, thereby chronic or repeated stress exposure has a potent effect that is classically considered to suppress immune function.

Experimental Set-Up

In fish, chronic exposure to Monocrotophos (MCP), an organophosphorus pesticide, in their environment is associated with impaired health and mortality. You decide to investigate the effect of MCP pesticide exposure on the physiological stress response in fish using a model organism, Zebrafish (Danio rerio). To do this, you expose a group of fish (n=50) to MCP for 21 days and maintain a control group (n=50) under the same conditions without pesticide exposure for 21 d. After 21 days you euthanize the fish; you keep half of the individuals from each group (experiment & control; n=25) as whole fish samples which are immediately frozen at -80°C for analysis. For the remaining individuals (experimental & control, n=25), you excise the brain in an RNA-free environment, place it directly in an RNase-free tube and store the samples at −80 °C for analysis.

Describe the materials and methods you would use to answer the following research questions:

1. How is the stress response pathway in fish affected by the MCP pesticide (Experiment 1)?
2. Which tissues are targeted by glucocorticoids in response to MCP exposure stress (Experiment 2)?

Your proposed experiments should be based on the techniques covered in module 1. Describe the equipment and facilities you would use and outline all the experimental steps for your methodology in a logical order. Describe how you would interpret your data to answer your research question. Discuss the possible advantages and drawbacks of your chosen methods.