Minseok Jeong. (2025). The functional heterogeneity of dentate gyrus mossy cells and its implications in psychiatric disorder. doi: 10.22677/THESIS.200000839686
치아이랑 모시세포의 기능적 이형성과 정신 질환에서의 역할 적당한 스트레스는 주의력, 기억 및 기타 인지 기능을 향상시켜 일상 생활에 이로운 영향을 미칠 수 있습니다. 그러나 외상성 스트레스는 개인의 대처 능력을 압도할 수 있으며, 주요 우울 장애와 외상 후 스트레스 장애(PTSD)의 중요한 위험 요소로 작용합니다. PTSD의 주요 특징 중 하나는 두려움의 일반화로, 이는 외상성 스트레스에 대한 부적응 반응으로, 위협적인 상황과 안전한 상황을 구별하는 능력을 저하시킵니다. 그러나, 이 결핍의 신경 기전은 아직 명확하지 않습니다. 본 연구에서는 외상성 스트레스 노출유도 모델인 학습된 무기력 모델(LH 모델)에서 위협과 안전 맥락 간의 맥락 구별을 저하시킨다는 것을 발견했습니다. 외상성 스트레스에 대한 반응으로 등측 모시세포는 억제되지만, 배측 모시세포는 그렇지 않습니다. 학습된 무기력 모델에서 배측 모시세포 활동의 양방향 조작은 모시세포 억제가 맥락 구별 장애와 인과적으로 연결되어 있음을 보여줍니다. 맥락 구별 장애에 대한 기전은 모시세포 억제에 따라 주어진 맥락에서 활성화된 과립 세포의 수를 증가시켜 맥락별 앙상블이 상당히 겹치게 합니다. 외상성 스트레스 후 등측 모시세포의 부적절한 억제가 두려움의 일반화와 맥락 구별 장애의 중요한 메커니즘임을 입증하였으며, 이는 PTSD의 인지 증상에 대한 잠재적인 치료 표적이 될 수 있음을 시사합니다. 모시세포의 외상성 스트레스에 대한 차별적 반응을 바탕으로, 쥐에서 모시세포의 구조적, 분자적 및 기능적 특성이 등측-배측 위치에 따라 어떻게 다른지를 조사하였습니다. 등측 모시세포가 배측 모시세포와는 다른 축삭 연결성을 가진다는 것을 발견했습니다. 또한, 번역 리보솜 친화성 정제(TRAP)를 사용한 세포 유형 특이적 RNA 시퀀싱을 통해 등측과 배측 모시세포 간의 독특한 분자 서명을 확인했습니다. 등측 모시세포는 신경 연결성과 시냅스 전송과 관련된 유전자가 유의미하게 증가하여 발현됩니다. 반면에, 배측 모시세포는 대사 및 세포 과정과 관련된 유전자의 높은 발현을 보입니다. 또한 등측 모시세포는 배측 모시세포와는 달리 과립 세포에 대한 억제 조절 기능을 수행하며, 빠른 맥락 구별에 중요하다는 것을 발견했습니다. 이는 모시세포의 등배축 간의 이질성은 기능적 차별화의 새로운 메커니즘을 제공하고 해마의 종축 길이에서의 뚜렷한 연관성을 나타낼 수 있습니다. 해마의 긴 곡선 구조가 설치류에서 배측-복측 축을 따라, 비인간 영장류에서 후방-전방 축을 따라 기능적으로 보존되어 있다는 증거를 바탕으로, 설치류의 모시세포의 등측-배측 이질성이 비인간 영장류에서도 보존되는지를 추가로 조사했습니다. 생쥐와 원숭이의 치아이랑에서 모시세포의 신경 해부학을 비교했습니다. 모시세포 표지자인 칼레티닌은 두 가지 하위 집단인 분절축과 측두축을 구별합니다. 이 하위 집단의 축삭 투사 패턴을 비교하기 위해 이중 형광 라벨링을 사용했습니다. 생쥐와 원숭이 모두에서 분절축 및 측두축 모시세포는 같은 축의 치아이랑의 종축을 따라 축삭을 투사하여 보존된 연관 투사를 나타냅니다. 그러나, 생쥐와 달리 원숭이의 모시세포 하위 집단은 반대측 치아이랑으로 연합 투사를 하지 않습니다. 원숭이에서는 측두축 모시세포가 오직 내분자층에만 연합 섬유를 보내는 반면, 분절축 모시세포는 여러 분자층에 걸쳐 넓은 축삭 투사를 합니다. 보존된 분절측두축에 따른 이형성에도 불구하고, 세포 조직의 배측 MC에서 각 분자층의 상대 축삭 밀도와 같은 종 간 차이가 관찰됩니다. 이러한 발견은 치아이랑의 분절측두축을 따라 기능적 차별화를 이해하는 데 기여하며, 포유류의 DG 회로의 해부학적 진화에 대한 지식을 넓히는 데 도움이 됩니다.
핵심어: 해마, 이형성, 치아이랑, 모시세포, 과립세포, RNA 시컨싱, 공포 일반화, 맥락 구분, 기억 엔그렘, 패턴분리력 |Moderate stress can enhance vigilance, memory, and other cognitive functions, offering benefits in daily life. However, traumatic stress can overwhelm an individual’s capacity to cope, acting as a significant risk factor for conditions such as major depressive disorder and post-traumatic stress disorder (PTSD). Traumatic stress induces major changes in brain circuits that can lead to maladaptive responses, often involving structural, molecular, and functional alterations that become detrimental over time. Understanding the neural mechanisms underlying maladaptive consequences of traumatic stress is critical for the elucidation of PTSD pathophysiology and the identification of new treatment strategies. Fear overgeneralization is a maladaptive response to traumatic stress that is associated with the inability to discriminate between threat and safety contexts, a hallmark feature of post-traumatic stress disorder. Despite its significance, the neural mechanisms underlying this deficit remain unclear. In this study, I found that traumatic stress exposure impairs contextual discrimination between threat and safety contexts in the learned helplessness (LH) model. Dorsal mossy cells (MCs) are suppressed in response to traumatic stress, but not ventral MCs. Bidirectional manipulation of dorsal MC activity in the LH model reveals that MC inhibition is causally linked to impaired contextual discrimination. Mechanistically, MC inhibition increases the number of active granule cells in a given context, significantly overlapping context-specific ensembles. I demonstrated that maladaptive inhibition of dorsal MCs after traumatic stress is a substantial mechanism underlying fear overgeneralization with contextual discrimination deficit, suggesting a potential therapeutic target for cognitive symptoms of PTSD. The functional heterogeneity of the hippocampus can be broadly segregated along the dorsoventral axis, which has distinct characteristics and specialized functions. These differences are evident at multiple levels, including connectivity and molecular level through distinct gene expression profiles. Understanding the heterogeneity is crucial for understanding how the hippocampus processes diverse types of information, and how disruptions in these processes might lead to neurological and psychiatric disorders. In this study, I found substantial heterogeneity of MCs in mice along the dorsoventral axis of the DG, in terms of structural and molecular and functional characteristics. I found that dorsal and ventral MCs display distinct axonal projections in the molecular layers of the DG along the dorsoventral axis. Furthermore, by cell-type-specific RNA sequencing using Translating Ribosome Affinity Purification (TRAP), I identified that dorsal and ventral MCs show distinct neurobiological molecular signatures. I found that dorsal MCs, but not ventral MCs exert inhibitory control on granule cells and are critical for rapid contextual discrimination. Collectively, dorsoventral heterogeneity of MCs may provide a novel mechanism for functional differentiation as well as distinct association along the longitudinal extent of the hippocampus. The long, curved structure of the hippocampus is conserved along the dorsal-to-ventral axis in rodents and the posterior-to-anterior axis in primates. I further examined whether the dorsoventral heterogeneity of MCs in rodents is conserved in non-human primates. I compared the neuroanatomy of MCs in the DG of mice and monkeys. The MC marker calretinin distinguishes two subpopulations: septal (dorsal in rodents, posterior in primates) and temporal (ventral in rodents, anterior in primates). Dual-colored fluorescence labeling is utilized to compare the axonal projection patterns of these subpopulations. In both mice and monkeys, septal and temporal MCs project axons across the longitudinal axis of the ipsilateral DG, indicating conserved associational projections. However, unlike in mice, no MC subpopulations in monkeys make commissural projections to the contralateral DG. In monkeys, temporal MCs send associational fibers exclusively to the inner molecular layer, while septal MCs give rise to wide axonal projections spanning multiple molecular layers, akin to equivalent MC subpopulations in mice. Despite conserved septotemporal heterogeneity, interspecies differences are observed in the topological organization of septal MCs, particularly in the relative axonal density in each molecular layer along the septotemporal axis of the DG. In summary, these findings have implications for understanding functional differentiation along the septotemporal axis of the DG and contribute to our knowledge of the anatomical evolution of the DG circuit in mammals. In conclusion, my research reveals the selective roles of MCs in stress-induced psychiatric disorders. Additionally, it highlights the structural and molecular heterogeneity of MCs in rodents, with evidence that this heterogeneity extends to non-human primates. These findings suggest that MCs possess unique characteristics with significant translational potential for mental health treatments. Keywords: Hippocampus, Heterogeneity, Dentate gyrus, Mossy cells, Granule cells, TRAP-seq, PTSD, Fear generalization, Contextual discrimination, Memory ensemble, Pattern separation.
Table Of Contents
List of Contents Abstract i List of contents iii List of figures viii
Chapter 1. Background 1 1. Hippocampus 1 1.1 Trisynaptic circuitry of the hippocampus 1 1.2 Microcircuit of the dentate gyrus 1 1.3 Pattern separation and the dentate gyrus 2 2. Post traumatic stress disorder 4 2.1 Natural Course and Risk Factors 4 2.2 Core of PTSD symptom of fear overgeneralization 5 2.3 Preclinical animal models of PTSD: Learned helplessness 5
Chapter 2. Maladaptation of dentate gyrus mossy cells mediates contextual discrimination deficit after traumatic stress 7 2.1 Introduction 7 2.2 Materials and method 10 2.2.1 Animals 10 2.2.2 Viral constructs 10 2.2.3 Stereotaxic surgery 10 2.2.4 Histology and imaging 11 2.2.5 Cell counting 12 2.2.6 CNO administration 12 2.2.7 Behavioral procedures 13 2.2.8 Fluorescence in situ hybridization 17 2.2.9 catFISH assay 17 2.2.10 Quantification and statistical analysis 18 2.3 Results 19 2.3.1 Traumatic stress impairs contextual discrimination in the LH model 19 2.3.2 MCs are suppressed in response to traumatic stress exposure 23 2.3.3 Intact contextual discrimination is disabled by chemogenetic Inhibition of MCs in stress-resilient mice 29 2.3.4 Selective activation of MCs is sufficient to restore contextual fear overgeneralization in susceptible mice 39 2.3.5 MC inhibition enlarges active GC subpopulation in response to a contextual stimulus 46 2.3.6 MCs modulate non-overlapping reactivation of context-specific GC ensembles 50 2.4 Discussion 57
Chapter 3. Structural, molecular, functional heterogeneity of MCs along the dorsoventral axis of the DG 66 3.1 Introduction 66 3.2 Materials and method 67 3.2.1 Animals 68 3.2.2 Dual fluorescence labeling of dMCs and vMCs 68 3.2.3 Immunohistochemistry 69 3.2.4 Fluorescence imaging of dMCs and vMCs 70 3.2.5 Three-dimensional imaging of MC projections 70 3.2.6 Quantification of dMCs and vMCs subpopulation 71 3.2.7 Transcriptional profiling of dMCs and vMCs 71 3.2.8 Gi-DREADD-dependent MC manipulation 74 3.2.9 Behavioral procedures 75 3.2.10 Data analysis and statistics 76 3.3 Results 77 3.3.1 Dorsoventral heterogeneity within MCs in their axonal projections 77 3.3.2 Spatial distribution and quantities of two distinct MC subpopulations 85 3.3.3 Transcriptional heterogeneity of MCs along the DV axis of the DG 87 3.3.4 Differential gene expressions between dorsal and ventral MCs 91 3.3.5 Distinct neurobiological properties between dorsal and ventral MCs 94 3.3.6 Net inhibitory control of dentate GCs by dMCs, but not by vMCs 98 3.3.7 Selective roles of dMCs in pattern separation 101 3.4 Discussion 106
Chapter 4. Comparative anatomy of the dentate mossy cells in non-human primates: their spatial distributions and axonal projections compared with mouse mossy cells 110 4.1 Introduction 110 4.2 Materials and method 112 4.2.1 Animals 112 4.2.2 Adeno-associated virus (AAVs) and stereotaxic surgery 113 4.2.3 Brain tissue preparation 114 4.2.4 Immunochemistry 115 4.2.5 Confocal imaging 115 4.2.6 Quantification of confocal images 116 4.2.7 Experimental design and statistical analysis 117 4.3 Results 118 4.3.1 Spatial segregation of two distinct MC subpopulations along the septotemporal axis of the DG in mouse and monkey 118 4.3.2 Associational and commissural projections of septal and temporal MCs In the mouse DG 112 4.3.3 Associational projections of septal and temporal MCs, but Absence of commissural projections, in the monkey DG 112 4.3.4 Species difference in topological projections pattern of septal MCs In the molecular layers of the DG 128 4.4 Discussion 134
Chapter 5. Conclusion 140 Reference 141 Summary in Korean 156
List of Figures Figure 1. Traumatic stress impairs contextual discrimination in the LH model Figure 2. MCs in the dorsal DG are suppressed after traumatic stress exposure Figure 3. MCs in the dorsal DG are persistently suppressed after traumatic stress exposure Figure 4. c-Fos immunoreactivity in the ventral MCs is not different between resilient and susceptible mice in the LH mode Figure 5. Hippocampal subregion- and cell type-specific expression of control mCherry and hM4Di construct in AAV-injected Calcrl-Cre mice Figure 6. Chemogenetic inhibition of MC during either training or testing session does not alter stress-susceptibility itself in the LH model Figure 7. Intact contextual discrimination in resilient mice is disabled by chemogenetic inhibition of MCs Figure 8. Chemogenetic inhibition of MCs in resilient mice did not alter basal behaviors Figure 9. Chemogenetic inhibition of dorsal MCs does not affect contextual discrimination bet ween highly distinct context pair Figure 10. Chemogenetic activation of dorsal MCs is sufficient to restore contextual fear overgeneralization in susceptible mice Figure 11. Chemogenetic activation of Gq-DREADD expressing MCs Figure 12. Chemogenetic activation of MCs in susceptible mice did not alter basal behaviors Figure 13. Chemogenetic inhibition of dorsal MCs enlarges active GC subpopulations through PV+BC inhibition across the dorsoventral axis of the DG Figure 14. MCs respond to contextual stimuli regardless of threat or safety contexts Figure 15. Suppression of MC activation exacerbates overlap between context-specific GC ensembles Figure 16. Calcrl-Cre mice displays a high levels of specificity of MCs Figure 17. dMCs and vMCs extend distinct axonal projections along the longitudinal axis of the DG Figure 18. Axonal fibers in MML of vDG are originated from dMCs Figure 19. Holistic visualization of dMC and vMC projections in the DG Figure 20. Two distinct MC subpopulations are spatially segregated along the DV axis Figure 21. TRAP-based isolation of MC subpopulation-specific mRNA Transcripts from the hippocampus Figure 22. RNA-seq DEGs analysis of MC-TRAP dorsal and ventral MCs Figure 23. Distinct neurobiological properties between dorsal and ventral MCs Figure 24. Acute inhibition of dMCs, but not of vMCs, results in hyperexcitation of GCs across the DV axis of the DG Figure 25. Contextual fear memory acquisition is not interfered by MC inhibition along the DV axis Figure 26. dMCs are crucial for contextual pattern separation Figure 27. Two distinct MC subpopulations in mouse and monkey are spatially segregated along the septotemporal axis Figure 28. Septal and temporal MCs in monkey make associational projections in the ipsilateral DG, but not commissural projections in the contralateral DG Figure 29. Septotemporal heterogeneity of MCs in their axonal projections in the DG molecular layers in the mouse Figure 30. Septotemporal heterogeneity of MCs in their axonal projections in the DG molecular layers in the monkey
