1 An Animal Model of a Behavioral Intervention for Depression Daniela D. Pollak, Francisco J. Monje,1 Lee Zuckerman, Christine A. Denny, Michael R. Drew,1 and Eric R. Kandel Neuron,2008 Speaker: Kuo Chung-Hsun( 郭忠訓 ) Advisor: Dr. Hong Chen-Jee ( 洪成志 )

2 Outline 》 Introduction 》 Materials 》 Methods & Results 》 Conclusion 》 Comment

3 Introduction

4 Fear And Depression 》 Instinctive and learned fear are essential for survival. 》 In humans, pathological forms of learned fear are hallmarks of severe psychopathologies such as anxiety disorders, posttraumatic stress disorders, and depression

5 Learned Fear And Learned Safety 》 Learned fear - a positive correlation (pairing). 》 Learned safety - a negatively correlated (unpaired). 》 Learned safety as the learning and memory resulting from a conditioned inhibition of fear training procedure (Rogan et al.,2005). 》 The ability to identify events that afford relief from ongoing strain is thought to be crucial for the prevention of chronic stress, a precipitating factor for the development of anxiety disorders and depression (Chan et al., 2001; Davis and Shi, 1999; LeDoux,1993; Rogan et al., 2001).

6 Hypothesis 》 Learned safety, as a predictor of a break from continuously imminent, stress-producing danger, may have antidepressant effects

7 Aim 》 Antidepressant effects in animal model. 》 The molecular mechanisms of learned safety.

8 Materials

9 》 Male C57BL6/N mice (10–12 weeks old) 》 Charles River Laboratories, Willington,MA 》 Mice were kept in clear plastic cages with ad libitum food and water.

10 Methods & Results

11 Freezing 》 Freezing, defined as complete immobility except for breathing, is a common response to sudden fearful situations in many species 》 CS (cue) – Tone, 350Hz, 72dB, 20s 》 US (shock) – 0.6mA, 2S

12 Behavior protocol preCS

13 Learned Safety Induces Reduction of Contextual Fear

14 Learned Safety Retards Subsequent Fear Conditioning to the Same Stimulus Learned safety + Learned fear 1day naive + Learned fear 1day Learned safety + Learned fear 2day naive + Learned fear 2day

15 Learned Safety Exerts Anxiolytic Effects

16 Learned Safety Acts to Induce Antidepressant-like Behaviors No training Learned safety Learned safety No training

17 Unpredictable Chronic Mild Stress (UCMS) 》 Brain interleukin-1 mediates chronic stress-induced depression in mice via adrenocortical activation and hippocampal neurogenesis suppression. Mol. Psychiatry, 2007 》 The following stimuli were administered each week in a random order for four weeks 》 3hrs of 45°cage tilt, two times a week 》 Shocked cage for 12hrs, once a week 》 Water deprivation for 14hrs, once a week 》 Lights on for 9hrs during the dark phase, once a week 》 Noise in the room for 3hrs, three times a week 》 Flashing light for 30mins, three times a week

18 Sucrose Preference Test 》 Twelve hours after exposure to the last stressor of the UCMS regime 》 Animals were deprived of food and water and tested for sucrose preference 23 hr later. 》 Testing was carried out in the home cage in the form of a two bottle choice paradigm (2% sucrose versus water) in the presence of the CS for 1 hr. 》 Sucrose preference rate was calculated according to the formula: % preference = [(sucrose intake/total intake) × 100%]

19 Learned Safety Acts to Induce Antidepressant-like UCMS

20 Brain-Derived Neurotrophic Factor 》 Brain-derived neurotrophic factor – BDNF 》 BDNF is known to be induced by antidepressant treatment in the hippocampus, particularly in the dentate gyrus.(Nibuya et al., 1995; Russo-Neustadt et al., 2004) 》 BDNF could be responsible for the increase in and survival of hippocampal neurons following antidepressant drug treatment (Duman, 2004a, 2004b)

21 Learned Safety Leads to Increased Expression of BDNF in the Dentate Gyrus of the Hippocampus 》 Dentate gyrus BDNF immunohistochemistry in mice sacrificed 4 hr after the last day of behavioral training

22 BrdU 》 Bromodeoxyuridine (5-bromo- 2-deoxyuridine, BrdU) 》 BrdU is commonly used in the detection of proliferating cells in living tissues 》 BrdU can be incorporated into the newly synthesized DNA of replicating cells.

23 Learned Safety And Newborn Cells in the Hippocampal Dentate Gyrus Learned Safety Learned Fear Tone Control How many cells remain after different behavior protocol? BrdU labeled Survival Learned Safety Learned Fear Tone Control BrdU labeled How many cells born after different behavior protocol? Proliferation

24 Learned Safety Promotes the Survival of Newborn Cells in the Hippocampal Dentate Gyrus No data

25 Ablation of Hippocampal Neurogenesis Retards Safety Learning

26 Genes Differentially Expressed in the Basolateral Amygdala of Learned-Safety and Learned-Fear Mice 》 Dopamine 2 receptor 》 Substance P 》 Prodynorphin 》 Preproenkephalin 1

27 mRNA Expressed in the Basolateral Amygdala Dopamine 2 receptor Substance P

28 Dopamine 2 Receptor Is Critically Involved in Learned Safety Blockade before the memory recall test Agonist before the memory recall test Blockade Prior to the FST

29 Substance P Is Critically Involved in Learned Safety Blockade During training on the response to the CS in the memory recall test Agonist During training on the response to the CS in the memory recall test

30 5-HT1A Receptors Are Not Critically Involved in Learned Safety Blockade During training on the response to the CS in the memory recall test Blockade before the memory recall test

31 Conclusion

32 Learned Safety 》 Behavioral outcomes similar to pharmacological interventions. 》 Induce cell-biological changes known to result from antidepressant pharmacotherapy but is mediated through different molecular pathways.

33 Comment 》 Learned safety is like the protection in clinical 》 In author’s data, the learned fear is hard to be changed. 》 Learned fear is like the depression

34 THANKS FOR YOUR ATTENTION

35 Ablation of Hippocampal Neurogenesis Inactivates the Antidepressant Effect of the Safety Signal

36 X-Ray Irraditation 》 Siemens Stabilopan X-ray 》 A cumulative 15Gy dose was the minimum necessary to produce a reduction of cell proliferation greater than 85% in the dentate gyrus, which lasted for at leasted two month. 》 Used x-irradiation of the dentate gyrus to ablate hippocampal neurogenesis in mice. 》 Verified the absence of newly generated cells by doublecortin immunohistochemistry (a marker for neurons younger than 1 month of age) 6 weeks later.