Probing Neural Substrates Mediating Sex Differences in Animal Models of Depression = 우울증 동물모델을 활용한 우울증 발병 원리의 성차를 매개하는 신경생리학적 기전 연구

다국어 초록 (Multilingual Abstract)

Depression, a mood disorder characterized by a significantly higher prevalence in women, often involves internalize emotional struggles in women, while men more frequently display outward behaviors. This research aims to elucidate the mechanisms underlying these sex differences by implementing targeted animal models and examining specific brain regions.
To identify sex-specific brain regions and potential neural circuits associated with stress responses for further studies, I used c-Fos protein expression comparison after acute stress exposure. This approach highlighted areas showing differential activation by sex. Female LHb showed significantly higher activity in response to stress compared to males, while RE activation was greater in males. Additionally, bootstrap analysis suggested that interconnectivity between the IL and PL regions is likely enhanced in females by stress exposure. (Chapter 2).
The LHb, which processes negative values and emotions and mediates depressive behaviors when hyperactive, was examined for sex-specific neuronal activity. Electrophysiological comparisons indicated that LHb neurons in female mice showed distinct sex-specific characteristics and alterations influenced by the estrous cycle, suggesting that hormonal changes can affect stress-related neuronal activity. This changes in LHb activity in females are likely to be closely associated with their vulnerability to stress (Chapter 3).
Various animal models were used to study depression mechanisms, though they often overlook sex differences, complicating physiological interpretation. I used the aLH model, a widely utilized model, to investigate potential sex-specific behavioral changes associated with depression. My investigation indicated that while the acute learned helplessness (aLH) model is effective for investigating male depression, as evidenced by the observed increase in helplessness behaviors among male mice, it fails to adequately capture depressive behaviors in females (Chapter 4).
The sub-chronic variable stress (SCVS) model is a female-specific depression animal model, with increased anxiety and anhedonia-like behaviors as key features observed in females. In-depth analysis for behaviors in the TST revealed that male and female showed distinct behavioral changed upon the same SCVS experience. While females showed elevated despair, while males displayed greater movement. This observation suggested the SCVS may fulfill the need for animal models that accurately represent female-specific depressive behaviors (Chapter 5).
In exploring the electrophysiological properties of the mPFC, PL neurons in females exposed to SCVS altered their activity to respond more effectively to input signals and transmit them to the IL. In contrast, IL neurons showed diminished responsiveness to excitatory inputs. The RE neurons, which are closely connected to the mPFC and highly stress-responsive in males (Chapter 2), displayed increased excitatory transmission in SCVS males over females. These findings suggest distinct functional changes in the mPFC between males and females, with females exhibiting unique responses to stress exposure (Chapter 6).
Lastly, the effects of administering female-specific metabolites on depressive behaviors provide potential biochemical pathways that may contribute to the etiology of depression in females. Collaborating researchers and I have identified female-specific metabolites upon SCVS, and SA and PA were used among the candidates to identify their effect on depressive behaviors. Interestingly, only SA triggers depressive like behavior specifically in stress-exposed females (Chapter 7).
In conclusion, this research suggests the importance of employing appropriate animal models to understand the distinct mechanisms of depression in females, highlighting the relationship between neurobiological changes and behavioral outcomes that may inform future studies and potential therapeutic strategies.

국문 초록 (Abstract)

우울증은 여성에서 더 높은 유병률을 보이는 대표적인 기분장애이다. 여성 우울증 환자는 우울감, 무기력, 감정적 어려움을 표현하는 데 더 큰 어려움을 겪는 반면, 남성 우울증 환자는 대외적인 역할 수행을 과도하게 강화하려는 성향을 보이며, 충동적 행동을 자주 나타내는 경향이 있는 것으로 보고되고 있다. 그러나 이러한 성별 차이에도 불구하고, 대부분의 우울증 기전 연구는 수컷 개체를 중심으로 진행되었으며, 암컷의 우울증 취약성에 대한 기전은 충분히 밝혀지지 않았다. 본 연구는 암컷과 수컷 생쥐를 이용해 성별 특이적으로 스트레스에 반응하는 뇌 영역을 확인하고, 우울증의 성별 기전 차이를 규명하고자 했다.
먼저, 급성 구금 스트레스를 가한 후 c-Fos 단백질 발현을 분석하여 성별 특이적으로 스트레스에 반응하는 뇌 영역을 확인했다. 암컷 생쥐의 측유상핵(lateral habenula, LHb) 뉴런은 스트레스에 의해 활성화가 증가했지만, 시상의 결합핵(nucleus reuniens) 뉴런은 수컷에서 더 큰 활성화를 보였다. 또한, 전전두엽피질(prefrontal cortex) 하위 영역인 변연계 아래 피질(infralimbic cortex, IL)과 변연전 피질(prelimbic cortex, PL)의 연결성이 스트레스를 받은 암컷에서 강화된 것으로 나타났다.
이후, 측유상핵 뉴런의 전기생리학적 특성을 성별과 스트레스 노출 여부에 따라 비교하였다. 암컷 생쥐의 LHb 뉴런은 휴지막 전위, 전기적 흥분성, 흥분성 신호전달에서 발정 주기의 영향을 받았으며, 더 약한 흥분성 신경 신호를 보였습니다. 스트레스 노출 후 수컷의 뉴런도 암컷과 유사하게 낮은 흥분성을 나타냈습니다. 이러한 결과는 LHb의 뉴런의 전기생리학적 기능이 성호르몬 주기와 성별에 따라 서로 다르게 변화하는 것을 시사한다.
우울 행동의 성별 차이를 확인하기 위해 급성 학습된 무기력 모델(acute learned helplessness, aLH)을 사용하여 관찰한 결과, 암컷과 수컷 모두 높은 불안 행동을 보였지만, 무기력 행동은 수컷에서 더 두드러졌다. 이에 따라 성별 특이적 우울 행동을 유발하는 아만성 가변 스트레스 모델(sub-chronic variable stress, SCVS)을 사용해 추가 연구를 진행했다. 암컷과 수컷 개체 모두 공통적으로 불안 행동이 증가하는 것을 확인할 수 있었으며, 특히 SCVS를 받은 암컷은 스트레스 상황에서의 무기력한 행동이 증가하였다. 반면에 SCVS를 경험한 수컷은 짧고 빈번한 움직임으로 스트레스 상황에 대응하는 행동 변화를 보이는 것을 확인하였다.
SCVS 모델에서 전전두엽피질 하위 영역의 전기생리학적 특성을 분석한 결과, IL의 신경세포는 수컷 SCVS에서 전기적 흥분성이 감소했으며 흥분성 신경신호전달도 약화되었다. 반면, PL 신경세포는 암컷 SCVS에서 흥분성이 증가했고 억제성 신호전달은 수컷에 비해 약화되어 있었다. PL에서 IL로 전달되는 흥분성 신호 대비 억제성 신호의 비율도 암컷 SCVS에서 유의미하게 증가했다.
마지막으로, 혈장 내 대사체의 변화를 분석한 결과 스테아르산(stearic acid, SA)의 혈장 농도가 암컷 SCVS에서만 증가한 것을 토대로, SA를 주사하여 행동적인 변화를 관찰했을 때 암컷 개체에서만 우울행동 및 불안행동이 높아진 것을 확인할 수 있었다.
위의 일련의 연구 결과를 통해 이전까지 밝혀지지 않았던 우울행동의 성별에 따른 차이를 매개할 수 있는 뉴런 및 신경회로 수준의 기전을 밝혔으며, 스트레스 및 대사체 농도 변화에 대한 행동적인 변화 양상에 성별에 따른 차이가 있음을 확인하였다. 이러한 연구 결과는 우울증의 성별 특이성을 이해하는데 기여하며, 특히, 기존의 남성 중심적인 연구를 넘어 여성의 우울증 취약성을 설명할 수 있는 신경생리학적 기반을 제공할 것으로 기대한다.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• 1.1. Sex differences in depression 1
• 1.1.1. Sex differences in the prevalence and symptomatology of depression 1
• 1.1.2. Genetic and epigenetic contributions to sex differences in depression 2
• 1.1.3. Sex differences in hormones and depression 4
• 1.2. Animal models to study the depression 5
• 1.2.1. Acute learned helplessness 5
• 1.2.2. Chronic restraint stress 6
• 1.2.3. Chronic mild stress 6
• 1.2.4. Chronic social defeat stress 7
• 1.3. Animal models to study the sex differences in depression 7
• 1.3.1. Social instability stress 8
• 1.3.2. Chronic unpredictable mild stress 8
• 1.3.3. Sub-chronic variable stress 9
• 1.4. Brain areas mediating depression and showing sex differences 10
• 1.4.1. The medial prefrontal cortex 10
• 1.4.2. The lateral habenula 12
• 1.4.3. The nucleus accumbens 13
• 1.4.4. The hippocampus 13
• 1.4.5. Other brain regions 14
• 1.5. Metabolic Changes in Depression 15
• 1.6. The purpose of this study 16
• Chapter 2. Brain-wide cellular mapping of acute stress-induced activation in male and female mice 19
• 2.1. Introduction 19
• 2.2. Materials and Methods 22
• 2.2.1. Animals 22
• 2.2.2. Acute stress procedure 22
• 2.2.3. Immunohistochemistry 22
• 2.2.4. Network connectivity analysis 23
• 2.2.5. Data analysis 24
• 2.3. Results 25
• 2.3.1. Identification of common stress-responsive areas in male and female brains 25
• 2.3.2. Areas with subtle sex differences 25
• 2.3.3. Stress-responsive areas in female mice but not in male mice 26
• 2.3.4. Stress-responsive areas with sex-dependent difference 26
• 2.3.5. Sex-specific stress-responsive network 27
• 2.4. Discussion 38
• Chapter 3. Dynamic interaction of estrous cycle and stress on synaptic transmission and neuronal excitability in the lateral habenula 43
• 3.1. Introduction 43
• 3.2. Materials and Methods 46
• 3.2.1. Animals 46
• 3.2.2. Acute stress procedure 46
• 3.2.3. Slice preparation and electrophysiology 46
• 3.2.4. Experimental design and statistical analyses 47
• 3.3. Results 49
• 3.3.1. The effect of the estrous cycle on the passive and active electrical properties of LHb neurons 49
• 3.3.2. The effect of the estrous cycle in the synaptic transmission of the LHb 49
• 3.3.3. The effect of stress exposure and the estrous cycle in basal neuronal properties of LHb 50
• 3.3.4. The effect of stress exposure and the estrous cycle in spontaneous excitatory transmission of LHb 51
• 3.4. Discussion 66
• Chapter 4. Sex difference in behavioral phenotypes of acute learned helplessness animal model of depression 70
• 4.1. Introduction 70
• 4.2. Materials and Methods 72
• 4.2.1. Animals 72
• 4.2.2. Stress procedure 72
• 4.2.3. Behavioral tests 72
• 4.2.4. Data analysis 74
• 4.3. Results 75
• 4.3.1. aLH induced anxiety-like behavior in both male and female mice 75
• 4.3.2. aLH induces moderate depressive-like behavior in male mice 75
• 4.4. Discussion 81
• Chapter 5. The evaluation of the sub-chronic variable stress paradigm as a female-sensitive animal model of depression 83
• 5.1. Introduction 83
• 5.2. Materials and Methods 85
• 5.2.1. Animals 85
• 5.2.2. Stress procedure 85
• 5.2.3. Behavioral tests 85
• 5.2.4. Data analysis 87
• 5.3. Results 89
• 5.3.1. Disparate behavioral changes are observed following modified SCVS based on sex 89
• 5.3.2. Females are more susceptible to SCVS with 80 foot-sh cks than males 91
• 5.3.3. The duration of the SCVS effect in behavioral phenotypes is a week or so 93
• 5.4. Discussion 111
• Chapter 6. Sex difference on alteration of neuronal activity in the mPFC of depression animal model 113
• 6.1. Introduction 113
• 6.2. Materials and Methods 115
• 6.2.1. Animals 115
• 6.2.2. Sub-Chronic Variable Stress (SCVS) procedure 115
• 6.2.3. Tail-suspension test (TST) 115
• 6.2.4. Slice preparation and electrophysiology 115
• 6.2.5. Data analysis 116
• 6.3. Results 118
• 6.3.1. Sex-Specific functional alterations in IL neurons under SCVS 118
• 6.3.2. Sex-specific excitability changes in PL neurons following SCVS 118
• 6.3.3. Differential effects of SCVS on spontaneous transmision in IL and PL neurons 119
• 6.3.4. Intra-circuitry differences in the mPFC in SCVS male and female mice 119
• 6.3.5. Basal synaptic transmission in the two output brain egions of the mPFC in male and female 120
• 6.4. Discussion 133
• Chapter 7. Sex differences in plasma fatty acid of mice with depressive-like behavior 136
• 7.1. Introduction 136
• 7.2. Materials and Methods 138
• 7.2.1. Animals 138
• 7.2.2. Sub-chronic variable stress (SCVS) 138
• 7.2.3. Behavioral test 138
• 7.2.4. Metabolites injection 138
• 7.2.5. Statistical analysis 139
• 7.3. Results 140
• 7.3.1. Six days of SA injection is insufficient to induce d pressive-like behavior in the TST 140
• 7.3.2. Female-specific effect of SA in promoting depressive behaviors following stress exposure 140
• 7.3.3. PA fails to change depression behavior 141
• 7.4. Discussion 152
• Chapter 8. Conclusion 154
• 8.1. Sex differences in expression and behavioral patterns of individuals with depression 154
• 8.2. Genetic and hormonal contributions to sex specific depression susceptibility 156
• 8.3. Neural circuit dynamics underlying sex-specific stress responses 158
• Appendix 205
• Abstract (in Korean) 207