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Studies of Circadian Nuclear Receptor REV-ERBα in the Dorsal Raphe 5-HT Neurons on Social Interaction Behavior

Studies of Circadian Nuclear Receptor REV-ERBα in the Dorsal Raphe 5-HT Neurons on Social Interaction Behavior
Sangwon Jang
DGIST Authors
Sangwon JangHan Kyoung ChoeKyungjin Kim
Kyungjin Kim
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Awarded Date
serotonin; social interaction; social preference; dorsal raphe; Tph2; REV-ERBα; circadian rhythm; fiber-photometry; optogenetics
Social interaction among conspecifics is essential for maintaining adaptive, cooper-ative, and social behaviors, along with survival among mammals. The 5-hydroxytryptamine (5-HT) neuronal system is an important neurotransmitter for regulat-ing social behaviors, however, the circadian role of 5-HT in social interaction behaviors is unclear. To investigate whether the circadian nuclear receptor, REV-ERBα, a transcrip-tional repressor of the rate-limiting enzyme tryptophan hydroxylase 2 (Tph2) gene in 5-HT biosynthesis, may affect social interaction behaviors, I generated a conditional knock-out (cKO) mouse by targeting Rev-Erbα in the dorsal raphe (DR) 5-HT neurons (5-HTDR-specific REV-ERBα cKO) using the CRISPR/Cas9 gene editing system, and assayed social behaviors, including social preference and social recognition with a three-chamber social interaction test at two circadian time (CT) points, i.e., at dawn (CT00) and dusk (CT12). The genetic ablation of Rev-Erbα in DR 5-HTergic neurons caused impaired social inter-action behaviors, particularly social preference but not social recognition, regardless of two CT points. This deficit of social preference was induced by Rev-Erbα in 5-HTDR-specific mice is functionally associated with real-time elevated neuron activity and 5-HT levels at dusk as determined by fiber-photometry imaging sensors. Moreover, optogenetic inhibition of DR to nucleus accumbens (NAc) 5-HTergic circuit restored the impairment of social preference in 5-HTDR-specific REV-ERB α cKO mouse. These results suggest the significance of the circadian regulation of 5-HT levels by REV-ERBα in regulating social interaction behaviors.|포유류에서 동족 간의 사회적 상호작용은 적응, 협동 및 사회성 (sociability) 행동을 유지하고 종족의 생존에 필수적이다. 세로토닌 (5-hydroxytryptamine, 5-HT)은 사회적 행동을 조절하는 중요한 신경전달물질로서 일주기성을 나타내지만, 사회성 행동에서 세로토닌 뉴런에서 일주기 생체시계 유전자와의 연관성에 대해서는 많은 연구가 이루어지지 않았다. 본 연구에서는 생체 시계 유전자 중 하나인 일주기 핵 수용체 REV-ERBα가 사회성 상호작용 행동에 어떤 영향을 미치는지 연구하고자 유전자 가위 (CRISPR/Cas9) 시스템을 사용하여 세로토닌 뉴런-특이적으로 세로토닌 합성효소 유전자인Tryptophan hydroxylase2 (Tph2)의 전사 억제 인자인 REV-ERBα가 결손된 생쥐 모델을 제작하였고, 낮 (CT00)과 밤 (CT12)의 두 시간대에서 3개의 구획으로 이루어진 사회적 상호작용 행동실험 기법 (three-chamber social interaction test)을 통해 social preference (새로운 생쥐로의 접근, 선호도)와 social recognition (사회성 인지, 기억 정도)를 분석하였다. Dorsal raphe nucleus (DR, 등쪽솔기핵)에 위치한 세로토닌 뉴런에서 Rev-Erbα 유전자 제거는 사회성 행동 중 social preference를 손상시켰지만, social recognition에는 별다른 영향을 주기 않았다. 이는 fiber-photometry (광섬유 광도측정) 기법으로 실시간 세로토닌 뉴런의 활성 및 등쪽솔기핵 세로토닌 양이 증가와 깊은 연관이 있었으며, 이를 등쪽솔기핵에서 nucleus accumbens, (NAc, 측좌핵)을 잇는 신경 회로에서 광유전학적 (optogenetic) 억제로 세로토닌 뉴런 특이적 Rev-Erbα결손 생쥐에서 유도되었던 사회성 손상을 회복시키는 것을 확인하였다. 이러한 결과들은 REV-ERBα에 의한 세로토닌의 정교한 일주기적 조절이 사회성 행동을 조절하는 데 중요하며, REV-ERBα의 조절이 세로토닌 관련 질환의 치료 타겟이 될 수 있음을 시사한다.
Table Of Contents
1.1 Circadian rhythm 1
1.1.1 Master and local or peripheral clock in mammals 5
1.1.2 Molecular clockwork in mammals 6
1.1.3 Circadian regulation of physiology and behaviors 10
1.2 Serotonin (5-hydroxytryptamine, 5-HT) 11
1.2.1 Biosynthesis of serotonin 13
1.2.2 Serotonin transduction and serotonin receptors 17
1.2.3 Functions of central serotonin 18
1.3 Social behavior 19
1.3.1 Social preference and recognition in mammals 20
1.3.2 Serotonin system in social preference and recognition in mammals 21
1.4 Purpose 22
2.1 Animals 23
2.2 Molecular cloning adeno-associated virus (AAV) vectors and virus production 23
2.2.1 Single-guide RNA (sgRNA) 23
2.2.2 AAV production 24
2.2.3 Surgical procedures 24
2.3 Drug preparation and application 25
2.3.1 Local microinjection of SR8278 or GSK4112 25
2.4 RNA isolation and real-time qPCR 25
2.5 High-performance liquid chromatography-coupled electrochemical detector (HPLC-ECD) 26
2.5.1 Sample preparation 26
2.5.2 Measurements of 5-HT and its metabolites 26
2.6 Three chamber social interaction test 27
2.7 Fiber-photometry 28
2.8 Optogenetic manipulation 29
2.9 Immunohistochemistry 29
2.10 Statistical analysis 30
ⅠⅠⅠ. Results 31
3.1 5-HTDR-specific REV-ERBα cKO mouse altered the circadian rhythm of Tph2 and 5-HT levels 31
3.2 Ablation of 5-HTDR-specific REV-ERBα induced impairment of so-cial behavior, especially in social preference, not social recognition 33
3.3 Pharmacological manipulation of REV-ERBα through agonist and/or antagonist affects social behaviors 34
3.4 Changes in the real-time circadian 5-HT neuron activity together with increased 5-HT levels by fiber photometry during the social preference test 36
3.5 DR-NAc circuit-specific ablation of REV-ERBα in 5-HT neurons ex-hibited a deficit in social preference 38
3.6 Optogenetic inhibition of DR 5-HTergic projection to NAc rescued impaired social preference in REV-ERBα cKO mouse 39
ⅠV. Discussion 79
4.1 Circadian regulation of DR 5-HT 79
4.2 DR-NAc 5-HTergic innervation in social interaction 82
4.3 Conclusion 85
V. References 86
VI. Abstract in Korean 96
Department of Brain Sciences
Related Researcher
  • 최한경 Choe, Han Kyoung 뇌과학과
  • Research Interests Modulation of neural circuit; Circadian regulation of behavior and perception; Neurotechnology
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Department of Brain Sciences Theses Ph.D.


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