1. What behavioral observation do the authors make? What question do they investigate regarding this observation? How could you state it as a hypothesis?

2. What is meant by neuroplasticity in general? What is neurotransmitter switching (hint: a citation would be good)? How do these terms relate?

3. The first figure legend includes “Statistical significance *p < 0.05, **p < 0.01, ***p < 0.001 was assessed by two-sided paired t-test (b) or two-sided Welch’s t-test”.
Describe in your own words what is meant by “statistical significance”, and what a t-test is.

4. Briefly summarize figure 2. Part 2c compares observations of runner and non-runner mice. What do you notice about the bar graph? Why do you think it was included?

5. What specific neurotransmitter switching is relevant to this article? What methods are used to look for it? What is the significance of NeuN, c-fos, ChAT, and GAD1?

6. Why do you think the authors were interested in the pedunculopontine nucleus (PPN)? What is the role of the PPN understood to be? The researchers further divide this nucleus into two sections – what are they, what do the names mean, and in what ways are they functionally different (don’t just tell me what it says in this paper though).

7. Briefly describe the ChAT-IRES-Cre mouse. What does the AAV-DIO-mRuby2 construct enable researchers to identify? What do they use this ability to do with the experimental mice?

8. The authors use sensitive fluorescence in situ hybridization. What (briefly) is this, and what does it enable them to look at in the mice? What do they find?

9. Having established a correlation between a behavioral phenomenon and a cellular phenotype, what intervention(s) do the authors make? What do you think is the purpose? What are the methods? What is the result of this intervention?

10. What relevance can you envision these findings having to human health?