1. Introducing Hippocampal UCP2
Advanced Seminars in Behavioral Neuroendocrinology : Exercise & Anxiety
Introducing Hippocampal UCP2
T.M. Hanna

3. The question is not whether exercise reduces anxiety, but instead in whom, to what degree, and by what mechanisms.

4. Tie-in Schoenfeld et al.
Objectives 1
Tie-in Schoenfeld et al. 2 3
Hippocampal UCP2
Wang et al.
Advancing the Story
Hare et al.
Something Good Cometh!

5. Where We Left Off Physical Exercise Prevents Stress-Induced Activation of Granule Neurons and Enhances Local Inhibitory Mechanisms in the Dentate Gyrus 1
Yufeng’s take home messages

6. Rodent: vHipp > Emotions > Stress & Anxiety Regulation
Problem, RQ, & Ha
Rodent: vHipp > Emotions > Stress & Anxiety Regulation
Long-term running =  anxiety
Running  no. excitatory neurons in DG
However,  activity in vHipp  anxious behavior
RQ: What gives?
Ha: Running changes inhib. activation in Hipp
Schoenfeld et al.

7. GABA is alles Aug. inhibitory signaling by  vGAT in vDG
GABAergic mediation of excitatory signaling associated with stress response

8. Exercise-Associated Changes in the Corticosterone Response to Acute Restraint Stress and Reduced Corticosterone Response Duration 2
ADDING TO THE STORY

9. Habituation to Acoustic Startle Response Test

12. Hare et al. Startle Test CORT Response – Restraint CORT Suppression – GR Agonist CORT Response – Exogenous ACTH Challenge
Hare et al. ZT=Zeitgeber Time ZT0=End of Dawn Trans ZT12=Lights Off Under LD Cond CT=Circadian Time CT0=End of Subjective Dawn DD=Constant Dark

13. D1: ASR Baseline D2: Access to Wheel D14: ASR w/ CORT Measurement (n=16; n=8 ZT12, n=8 ZT13) D28: ASR w/ CORT Measurement (ditto)

14. D29 Running Mice n=62 (ad lib Wheel) Sedentary Mice n=62 (no access) Restraint= 30 min in 50ml tube Tail 0, 10, 20, & 30 and post stress 60, 90, 120 (minutes)
Experiment 1: CORT response to restraint

15. Baseline ASRT sedentary (n=50) and running (n=50) groups established
ip Dexamethasone
Part 1-Periph Dex Suppression
Part 2-Central Dex Suppression
Part 3-Periph & Central Dex w/ Intermediate Dose
Part 1: 3 randomly assigned doses (0.0, 0.01, 0.05 mg/kg) of dex/sigma-aldrich dissolved in 2% etoh then diluted dH2O2 given to three cages over five days (running n=20, sedentary n=19)
Part 2: (running n=30, sedentary n=31). Dex injected into central cerebral ventricles . Infused over 10s and blood collected 4 hours post infusion and then sacrifice
Part 3: n=16 io, n=16 ICV n=8 per group at intermediate dose mg/kg ip & 1.5 ng ICV
Experiment 2: CORT suppression following administration of synth GR agonist dexamethasone

16. Experiment 3: CORT Response to ACTH challenge
Running n=16, Sedentary n=16
Colony acclimatization
ASRT baseline and 28 day ASRT
D29:
Base CORT taken 0800
Dexamethasone (0.05 mg/kg) (lmt endo ACTH)
4hrs post Dex: blood draw then…
ACTH Challenge
Experiment 3: CORT Response to ACTH challenge

17. CRH & Vasopressin  ACTH  Glucocorticoids (Cortisol etc.)
Why Does ACTH Matter?

18. Results

19. Figure 1: Mean Startle Amplitude
Figure 1 Mean startle amplitude (arbitrary unit±SEM) during the 4-week wheel-running period in Experiment 1. Running mice demonstrated reduced startle amplitude compared with baseline on days 14 and 28 of wheel access. ***p < in comparison with respective test day 0.
Figure 1: Mean Startle Amplitude

20. Figure 2 Mean plasma corticosterone concentrations (ng/ml±SEM) during restraint stress procedure. Running mice demonstrated an elevated response compared with sedentary animals at 20min, and also returned to baseline more rapidly. **p<0.01 running in comparison with sedentary at time 20. ##p<0.01 sedentary time 120 in comparison with sedentary time 0.
Figure 2: Mean plasma corticosterone concentrations (ng/ml±SEM) during restraint stress procedure.

21. Figure 3 (a) Adrenal and (b) thymus weights following 28 days of wheel running (mg±SEM). Total and right adrenal weights (normalized to body weight) were elevated in running mice compared with sedentary controls. Thymus weights were similar in running and sedentary mice. *p<0.05 in comparison with sedentary animals.
Figure 3: (a) Adrenal and (b) thymus weights following 28 days of wheel running (mg±SEM).

22. Figure 4: (a) ZT12 and (b) ZT13 corticosterone samples following 14
Figure 4 (a) ZT12 and (b) ZT13 corticosterone samples following 14 and 28 days of wheel running (ng/ml±SEM). Corticosterone at the onset
of the active phase did not differ between running and sedentary mice at any sample point.
Figure 4: (a) ZT12 and (b) ZT13 corticosterone samples following 14
and 28 days of wheel running (ng/ml±SE

23. Figure 5: Mean plasma corticosterone (ng/ml±SEM) response
Figure 5 Mean plasma corticosterone (ng/ml±SEM) response following ip dexamethasone administration in running and sedentary mice following 28 days of wheel access. Plasma corticosterone was reduced similarly in running and sedentary mice at the highest dose of dexamethasone (0.05 mg/kg). Administration of an intermediate dose of dexamethasone (0.025 mg/kg) did not produce divergent results (inset). ***p<0.001 in comparison with respective 0 mg/kg dose.
Figure 5: Mean plasma corticosterone (ng/ml±SEM) response

24. Figure 6: Mean plasma corticosterone (ng/ml±SEM) response following ICV dexamethasone administration in running and sedentary mice following 28 days of wheel access. Dexamethasone 2 ng resulted in reduced corticosterone in running and sedentary mice. Administration of an intermediate dose of dexamethasone (750 ng/ml) did not produce
divergent results (inset). *p<0.05, **p<0.01 in comparison with respective 0 ng dose.
Figure 6 Mean plasma corticosterone (ng/ml±SEM) response following ICV dexamethasone administration

25. Figure 7: Mean plasma corticosterone (ng/ml±SEM) response following ACTH administration in running and sedentary mice. Adrenal sensitivity was similar following 50 mg/kg ACTH administration. In contrast, running animals demonstrated enhanced sensitivity to ACTH after administration of 5 mg/kg ACTH. *p<0.05 running in comparison with sedentary at 5 mg/kg dose.
Figure 7 Mean plasma corticosterone (ng/ml±SEM) response following ACTH administration

26. Discussion Wheel Running  anxiety-like behavior
More rapid CORT response & decay
 adrenal size
 sensitivity to ACTH
Enhanced central control over HPA-axis response to future stressors

27. Discussion Wheel Running  Resilience
Robust stress response w/  chance of overexposure to glucocorticoids during stress

28. Hippocampal UCP2 Is Essential For Cognition and Resistance to Anxiety But Not Required For the Benefits of Exercise 3
UCP2 reduces mitochondrial energy production and increase cell thermogenesis
UCP2 is critical in Hipp neuronal development & associated behaviors
Inhibition diminished neuronal number and size, dendritic growth, and synaptogenesis

29. Mitochondrial Uncoupling Protein 2 Adding to the HPA-Axis Story UPC2 deficiency may disorder structures and processes associated with cognition, mood, & behavior.

30. ASO=Antisense Oligonucleotides
4 groups
Sedentary control (SED)
Sedentary ASO Treated (SED+ASO)
Exercise control (Ex)
Exercise ASO-treated (Ex + ASO)

31. Test of UCP2 Role
UCP2 Deficient Mice
Y-Maze
T-Maze
Object Recognition Test (ORT)
Open Field Test (OFT)
Light-Dark Exploration Test (LET)
Elevated Plus Maze

32. Only LET & EPM assess anxiety

33. Fig. 1. Experimental procedures (A), running distance (B) and body weight (C). Habituation: habituation to cage, food and rodent wheel running (if any); ASO: antisense oligonucleotides; Behavioral tests order: Y-maze test, T-maze test, object recognition test (ORT), open field test (OFT), light–dark exploration test (LET), and elevated plus maze (EPM). Running distance (km) per day by exercised mice over eight weeks of running wheel access and body weight (g) per week are expressed as meanÅ}SEM (n=12). Significance is not shown in the figure.
Fig. 1. Experimental procedures (A), running distance (B) and body weight (C). Habituation

34. OFT & EPM 
T-Maze & Y-Maze 

35. ORT & LET

36. Fig. 2. Exercise reverses ASO-induced cognitive deficits in Y-maze test (A), T-maze test (B), and Object recognition test (C). Data are expressed as mean ± SEM (n=12). *p<0.05, **p<0.01 vs. SED; ##p<0.01 vs. SED+ASO.
Fig. 2: Y-maze test (A), T-maze test (B), and Object recognition test (C).

37. Fig. 3. Exercise reverses ASO-induced anxiety-like behaviors in the open field test. Data are expressed as mean ± SEM (n=12). *p<0.05,
**p<0.01 vs. SED; ##p<0.01 vs. SED+ASO.
Fig. 3. Exercise reverses ASO-induced anxiety-like behaviors in the open field test

38. Fig. 4. Exercise reverses ASO-induced anxiety-like behaviors in the light–dark exploration test. Data are expressed as mean ± SEM (n=12). **p<0.01 vs. SED; #p<0.05, vs. SED+ASO.
Fig. 4. Exercise reverses ASO-induced anxiety-like behaviors in the light–dark exploration test.

39. Fig. 5. Exercise reverses ASO-induced anxiety-like behaviors in the elevated plus maze test. Data are expressed as mean ± SEM (n=12).
*p<0.05, **p<0.01 vs. SED; #p<0.05, ##p<0.01 vs. SED+ASO.
Fig. 5. Exercise reverses ASO-induced anxiety-like behaviors in the elevated plus maze test.

40. Fig. 6. Effect of ASO administration and exercise on hippocampal monoamines. Data are expressed as mean ± SEM (n=6–8). *p<0.05, **p<0.01 vs. SED; #p<0.05, ##p<0.01 vs. SED+ASO.
Fig. 6. Effect of ASO administration and exercise on hippocampal monoamines.

41. Fig. 7. UCP2 expression in the hippocampus.
Fig. 7. UCP2 expression in the hippocampus. (A) Real-time PCR analyses demonstrate lower UCP2 mRNA levels in the hippocampus and liver of mice treated with UCP2 ASO (n=8, two-tailed unpaired t test). Data are expressed as mean ± SEM. **p<0.01 vs. SED. (B) Representative examples of Western immunoblots in hippocampal samples assessed for UCP2 protein levels.
Fig. 7. UCP2 expression in the hippocampus.

42. Discussion
Anxiolytic Effects of Exercise Are Not UCP2 Dependent—in UCP2 KO exercise recovered L&M
Exercise can not rescue UCP2 deficiency

43. WHERE DOES THIS LEAVE US?

44. Why Dam Stress May Be Critical
Simon-Areces et al  Natural BirthUCP2 induced Effects Hippocampus & Adult Behavior (see final slide for full citation)