Introducing Hippocampal UCP2 Advanced Seminars in Behavioral Neuroendocrinology 2014-15: Exercise & Anxiety Introducing Hippocampal UCP2 T.M. Hanna

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

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!

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

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.

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

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

Habituation to Acoustic Startle Response Test

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

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)

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

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. 0.025 mg/kg ip & 1.5 ng ICV Experiment 2: CORT suppression following administration of synth GR agonist dexamethasone

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

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

Results

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 < 0.001 in comparison with respective test day 0. Figure 1: Mean Startle Amplitude

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.

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).

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

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

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

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

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

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

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

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

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

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

Only LET & EPM assess anxiety

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

OFT & EPM  T-Maze & Y-Maze 

ORT & LET

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).

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

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.

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.

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.

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.

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

WHERE DOES THIS LEAVE US?

Why Dam Stress May Be Critical Simon-Areces et al. 2012  Natural BirthUCP2 induced Effects Hippocampus & Adult Behavior (see final slide for full citation)