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You are asked to review a paper in which one of the experiments is testing the effects of stress on brain activation in the paraventricular nucleus of the hypothalamus (PVN). Specifically, the researchers kept mice in a 12:12 light dark cycle (lights on 6 am, lights off 6pm) and exposed the experimental group to a 15-minute swim stress at 11 am. An hour later (12:15 pm), the researchers collected the brains of these animals to analyze Fos expression in the PVN. The control (unstressed) group was collected at 12:15 am. The results show that the control group and the experimental group have similar levels of Fos expression in the PVN, and the researchers concluded that swim stress does not cause overt neural activation in the PVN. 1. What feedback would you give to the authors of this paper? Do you agree with the experimental design and why? Make sure you include in your response the reason why the researchers use Fos in this experiment

Explanation / Answer

The hypothalamus pituitary adrenal (HPA) axis and glucocorticoid secretion undergoes diurnal changes that are responsible for the metabolic balance in an organism.

C-fos is an immediate early gene that is rapidly expressed in the areas of brain in response to external stimuli. Hence, it is a marker for neuronal activation as well as how neuronal circuits are affected by external stimuli. The c-fos gene is known to undergo diurnal variation in the HPA axis.

Forced swimming is known to trigger c-fos expression in the hypothalamic paraventricular nucleus (PVN). C-fos mRNA increases in the day but decreases in the night. It is lower after 2 hours after the lights are switched on and are higher 1 hour before lights are switched off. Hence, under no stress conditions, c-fos mRNA should be higher in the night. This is what happens in the control mice.

In the experiment designed by the authors, the expression at 12.15 am corresponds to night (PM) expression of c-fos, which will be higher. The c-fos expression in the day (12.15 pm) after the swim text will also increase, as c-fos will be up regulated by swim stress. Hence, when the experimental and control group of mice will show same level of c-fos expression.

The authors have not designed the experiment properly. The control group should have been mice that were not subjected to swim test with their brain collected at 12.15 pm. The control group used (brains collected at 12.15 am) is not appropriate for the experiment as the c-fos is regulated diurnally.

Another experimental group, wherein the mice were subjected to 15 min swim at 11 pm and brains collected at 12.15 am should have been included. Comparison of all these groups would indicate whether the swim stress caused any change in c-fos expression in PVN and whether it activates neuronal activity in PVN.

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