Role of hilar mossy cells in the CA3-dentate gyrus network during sharp wave-ripple activity in vitro

Der Gyrus dentatus (DG) des Hippokampus wird als Eingangsstation für Informationen aus dem entorhinalen Kortex betrachtet. In das DG-Netzwerk sind zwei exzitatorische Zelltypen eingebettet: Körnerzellen, die Signale von dem entorhinalen Kortex empfangen, und Hilus-Mooszellen (MCs), die Signale von Körnerzellen als auch von feedback-Projektionen von CA3-Pyramidenzellen (PCs) empfangen. Postsynaptische Ziele von MC-Projektionen umfassen DG Körnerzellen und verschiedene Interneurone in der selben und in der kontralateralen Hemisphäre des Gehirns. Die Rolle von MCs während rhythmischer Populationsaktivität, und insbesondere während Sharp-Wave / Ripple-Komplexen (SWRs), ist bisher weitgehend unerforscht. SWRs sind prominente Ereignisse im Hippocampus während des Tiefschlafs (Slow wave sleep) und des ruhigen Wachzustandes, und sie sind an der Gedächtniskonsolidierung beteiligt. In der vorliegenden Arbeit, untersuchen wir mithilfe eines in-vitro-Modells von SWRs, inwieweit Mooszellen an SWRs in CA3 beteiligt sind. Mit CA3-Feldpotential-Ableitungen und gleichzeitigen ‚cell-attached‘ Messungen von einzelnen MCs konnten wir beobachten, dass ein wesentlicher Anteil von MCs (47%) während der SWRs in das aktive neuronale Netzwerk rekrutiert werden. Darüber hinaus fanden wir in MCs SWR-assoziierte synaptische Aktivität, bei denen sowohl die exzitatorischen als auch die inhibitorischen Komponenten phasenkohärent und verzögert zur Ripple Oszillation in CA3 auftreten. Simultane Patch-clamp Messungen von CA3-Pyramidenzellen und MCs zeigten längere exzitatorische und inhibitorische Latenzzeiten bei MCs, was die Hypothese einer von CA3 ausgehenden Feedback-Rekrutierung unterstützt. Unsere Daten zeigen zusätzlich, dass das Verhältnis exzitatorischer zu inhibitorischer Aktivität in MCs höher ist als in CA3-Pyramidenzellen, wodurch die MCs mit höherer Wahrscheinlichkeit während SWRs überschwellig aktiviert werden. Schließlich zeigen wir, dass ein signifikanter Anteil (66%) der getesteten Körnerzellen SWR-assoziierte exzitatorische Signale erhalten, im Vergleich zu MCs zeitlich verzögert, was auf eine indirekte Aktivierung von Körnerzellen durch CA3 PCs über MCs hinweist. Zusammengefasst zeigen unsere Daten die aktive Beteiligung von Mooszellen an SWRs und deuten auf eine funktionelle Bedeutung als Schaltstelle für das CA3- Gyrus dentatus Netzwerk in diesem wichtigen physiologischen Netzwerkzustand hin.The dentate gyrus (DG) is considered as the hippocampal input gate for the information arriving from the entorhinal cortex. Embedded into the DG network are two excitatory cell types –granule cells (GCs), which receive inputs from the entorhinal cortex, and hilar mossy cells (MCs), which receive input from GCs and feedback projections from CA3 pyramidal cells (PCs). The postsynaptic targets of MC projections are the GCs and hilar interneurons in both ipsilateral and contralateral hemispheres of the brain. The role of MCs during rhythmic population activity, and in particular during sharp-wave/ripple complexes (SWRs), has remained largely unexplored. SWRs are prominent field events in the hippocampus during slow wave sleep and quiet wakefulness, and are involved in memory consolidation and future planning. In this study, we sought to understand whether MCs participate during CA3 SWRs using an in vitro model of SWRs. With simultaneous CA3 field potential– and cell-attached recordings from MCs, we observed that a significant fraction of MCs (47%) are recruited into the active neuronal network during SWRs. Moreover, MCs receive pronounced, compound, ripple-associated synaptic input where both excitatory and inhibitory components are phase-coherent with and delayed to the CA3 ripple. Simultaneous patch recordings from CA3 pyramidal neurons and MCs revealed longer excitatory and inhibitory latencies in MCs, supporting a feedback recruitment from CA3. Our data also show that the excitatory to inhibitory charge transfer (E/I) ratio in MCs is higher than in the CA3 PCs, making the MCs more likely to spike during SWRs. Finally, we demonstrate that a significant fraction (66%) of tested GCs receive SWR-associated excitatory inputs that are delayed compared to MCs, indicating an indirect activation of GCs by CA3 PCs via MCs. Together, our data suggest the involvement of mossy cells during SWRs and their importance as a relay for CA3-dentate gyrus networks in this important physiological network state

The role of hippocampal mossy cells in antidepressant actions

Hippocampus, Mossy cells, antidepressant, p11/AnxA2/Smarca3 complexMost antidepressants, including selective serotonin reuptake inhibitors (SSRIs), initiate their drug actions by rapid elevation of serotonin, but they take several weeks to achieve ther-apeutic onset. This therapeutic delay suggests slow adaptive changes in multiple neuronal subtypes and their neural circuits over prolonged periods of drug treatment. Mossy cells are excitatory neurons in the dentate hilus that regulate dentate gyrus activity and function. Here I show that neuronal activity of hippocampal mossy cells is enhanced by chronic, but not acute, SSRI administration. Behavioral and neurogenic effects of chronic treatment with the SSRI, fluoxetine, are abolished by mossy cell-specific knockout of p11 or Smarca3 or by an inhibi-tion of the p11/AnxA2/SMARCA3 heterohexamer, an SSRI-inducible protein complex. Fur-thermore, simple chemogenetic activation of mossy cells using Gq-DREADD is sufficient to elevate the proliferation and survival of the neural stem cells. Conversely, acute chemogenetic inhibition of mossy cells using Gi-DREADD impairs behavioral and neurogenic responses to chronic administration of SSRI. In addition, modulations of mossy cell activity are influence to excitation-inhibition balance in dentate gyrus. The present data establish that mossy cells play a crucial role in mediating the effects of chronic antidepressant medication. These results indicate that compounds that target mossy cell activity would be attractive candidates for the development of newer antidepressant medications.openChapter 1. Background 1 1. Major depressive disorder 1 1.1 MDD in the hippocampus 1 2. Antidepressive therapeutics 4 Chapter 2. The role of hippocampal mossy cells in antidepressant actions. 7 1. Introduction 7 1.1 Molecular mechanism of SSRI actions 7 1.2 Mossy cells in the hippocampus 10 1.3 p11, An SSRI-inducible molecule 13 2. Materials and methods 15 2.1 Materials 15 2.1.1 Antibodies 15 2.1.2 Virus strains 15 2.1.3 Recombinant DNAs 16 2.1.4 Chemicals 16 2.1.5 Experimental models 17 2.2 Methods 18 2.2.1 Animal breeding 18 2.2.2 Drug treatment 18 2.2.3 Plasmid constructions 19 2.2.4 Immunoprecipitation of p11/AnxA2/SMARCA3 complex 19 2.2.5 Stereotaxic surgery 20 2.2.6 Behavioral assessments 21 2.2.6.1 Elevated plus maze (EPM) 21 2.2.6.2 Open field test (OF) 22 2.2.6.3 Light and dark box test (LD box) 22 2.2.6.4 Novelty suppressed feeding test (NSF) 22 2.2.6.5 Tail suspension test (TST) 23 2.2.7 Chronic unpredictable mild Stress paradigm 23 2.2.8 Immunohistochemistry 25 2.2.9 BrdU labeling and neurogenesis assay 26 2.2.10 Electrophysiological recordings of mossy cells 27 2.2.10.1 Fluorescence labeling of mossy cells 27 2.2.10.2 Slice preparation 27 2.2.10.3 Electrophysiology 28 2.2.11. Data analysis and statistics 29 3. Results 30 3.1. The role of p11/AnxA2/SMARCA3 complex in hippocampal mossy cells in an-tidepressant responses 30 3.1.1 Effects of genetic deletion of p11 or Smarca3 in hippocampal mossy cells on behavioral responses to chronic SSRI administration 30 3.1.2 Effects of mossy cell-specific inhibition of the p11/AnxA2/SMARCA3 com-plex on neurogenic and behavioral responses to chronic antidepressant treatment 38 3.1.3. Effects of cell type-specific inhibition of the p11/AnxA2/SMARCA3 complex on neuronal activity of mossy cells 50 3.2. The role of hippocampal mossy cells in antidepressant responses 59 3.2.1 Effects of selective stimulation of dentate mossy cells on adult neurogenesis in the hippocampus 59 3.2.2 Effects of selective inhibition of dentate mossy cells on antidepressant actions in the hippocampus 67 3.3. Effects of modulation of mossy cells on micro-circuits in the dentate gyrus 78 4. Discussion 82 Reference 95 Summary in Korean 103Selective serotonin reuptake inhibitors(SSRI)를 포함한 대부분의 항우울제는 약물 복용 즉시 체내 세로토닌의 양이 증가하게 된다. 반면, 항우울 약물 복용으로 인한 치료 효과가 나타나기까지는 수주 동안의 시간이 필요하다. 이러한 치료 지연 현상은 약물 복용 이후 신경세포에서 장기간에 걸쳐 일어나는 유전자 발현 및 신경회로의 변화가 동반되어 나타나는 결과라고 할 수 있다. 모시세포는 치아이랑의 hilus 구역에 존재하는 흥분성 뉴런으로, 치아이랑의 활성 및 기능을 조절하는 역할을 한다고 알려져 있다. 본 연구에서는 해마 모시세포가 항우울제 단기 복용이 아닌, 장기복용에 의해서 활성이 강화 된다는 것을 보였다. 또한, SSRI, fluoxetine 장기 복용에 따른 행동학적 그리고 신경발생학적 효과가 모시세포에 한해서 p11 또는 Smarca3 단백질을 억제하거나 혹은 p11/AnxA2/Smarca3 복합체를 억제하였을 경우 사라졌음을 보였다. 더불어, Gq-DREADD 시스템을 이용한 모시세포의 활성 강화는 신경 줄기 세포의 증식과 생존을 증진시키기에 충분함을 보였다. 반대로, Gi-DREADD 시스템을 이용한 모시세포의 활성 억제는 항우울 약물 장기 복용에 의해 나타나는 행동학적 그리고 신경발생학적 효과를 손상시킴을 보였다. 모시세포의 활성 변화는 치아이랑 내부의 흥분성 / 억제성 신호의 조화에 영향을 끼치는 것 또한 확인할 수 있었다. 따라서 본연구에서는 항우울 약물 반응을 매개하는데 있어서 모시세포가 중요한 역할을 한다는 것을 입증하였다. 해당 연구 결과는 해마 내 모시세포가 새로운 항우울 약물 개발에 중요 후보군이 될 것임을 보여준다.DoctordCollectio

Rac1 and Rac3 GTPases Regulate the Development of Hilar Mossy Cells by Affecting the Migration of Their Precursors to the Hilus

We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have addressed the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus

DEFINING THE RADIORESPONSE OF MOSSY CELLS

Clinical radiotherapy is used to treat a variety of brain tumors within the central nervous system. While effective, it can result in progressive and debilitating cognitive impairment that can diminish quality of life. These impairments have been linked to hippocampal dysfunction and corresponding deficits in spatial learning and memory. Mossy cells are a major population of excitatory neurons located within the dentate hilus and highly involved in hippocampal circuitry. They play critical roles in spatial navigation, neurogenesis, memory, and are particularly vulnerable to a variety of neurotoxic insults. However, their sensitivity to ionizing radiation has yet to be investigated in detail. I hypothesize that mossy cells are critical targets for ionizing radiation, whereby damage to these targets contributes to the mechanisms associated with radiation-induced hippocampal dysfunction. To test this idea, wild-type mice were exposed to clinically relevant doses of cranial x-ray irradiation and their hippocampi were examined 1 month and 3 months post treatment. A significant decline in both the number of mossy cells and their activity were observed. In addition, dentate granular cells demonstrated reduced levels of activity, as well as reduced proliferation within the subgranular zone. A second cohort of mice was introduced to a novel environment in order to induce the expression of immediate early genes. Analysis of c-Fos mRNA yielded a significant increase in control but not irradiated animals, suggesting that radiotherapy impaired immediate early gene expression and resultant functional behavioral outcomes. These findings support the proposition that radiation-induced damage to mossy cells contributes to hippocampal deficiencies which result in cognitive dysfunction

The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus.

One of the most distinctive and common cell types in Golgi preparations of the hilus of the rat dentate gyrus is the mossy cell. We have used a variety of techniques including the Golgi method, the combined Golgi and electron microscopic (EM) method and the retrograde transport of horseradish peroxidase (HRP) to study the development, ultrastructure and synaptic connections of this cell type. The mossy cells identified in our light microscopic preparations are characterized by: triangular or multipolar shaped somata; three to four primary dendrites that arise from the soma and bifurcate once or more to produce an extensive dendritic arborization restricted, for the most part, to the hilus; numerous thorny excrescences on their somata and proximal dendrites with typical spines on distal dendrites; and axons that bifurcate and are directed toward the fimbria and the molecular layer of the dentate gyrus. The mossy cells have an immature appearance at birth and on subsequent days their maturation appears to lag somewhat behind that of the hippocampal pyramidal cells. On postnatal day 1, many of the dendrites bear growth cones primarily at their termini and have long, thin filipodia emanating from various points along their lengths. Many of the dendrites enter the molecular layer of the dentate gyrus, though this is rarely seen in the mature brain. Typical pedunculate spines are first commonly seen on the distal dendrites around postnatal day 7 while thorny excrescences are first commonly seen between postnatal days 11 and 14. By postnatal day 21, the dendrites have attained a mature appearance although the density of both typical spines and thorny excrescences is less than that found in adults. Two different retrograde transport methods were used to confirm that mossy cells give rise to the commissural projection to the contralateral dentate gyrus. The first method combined HRP histochemistry with a silver intensification procedure and the second method combined HRP histochemistry with Golgi staining. While the majority of commissurally projecting hilar neurons had the appearance of mossy cells, there were others that were smaller and either ovoid or fusiform. In EM preparations, the somata of mature mossy cells display round nuclei that lack infoldings and intranuclear rods. The perikaryal cytoplasm contains the organelles typically found in pyramidal cells of the hippocampus. Somal spines with complex shapes and branching patterns are commonly observed. The thorny excrescences on the proximal dendrites correspond to spines with long thin stalks and complex end bulbs that may appear mushroom shaped.(ABSTRACT TRUNCATED AT 400 WORDS

Expression of c-fos in hilar mossy cells of the dentate gyrus in vivo.

Granule cells (GCs) of the dentate gyrus (DG) are considered to be quiescent--they rarely fire action potentials. In contrast, the other glutamatergic cell type in the DG, hilar mossy cells (MCs) often have a high level of spontaneous activity based on recordings in hippocampal slices. MCs project to GCs, so activity in MCs could play an important role in activating GCs. Therefore, we investigated whether MCs were active under basal conditions in vivo, using the immediate early gene c-fos as a tool. We hypothesized that MCs would exhibit c-fos expression even if rats were examined randomly, under normal housing conditions. Therefore, adult male rats were perfused shortly after removal from their home cage and transfer to the laboratory. Remarkably, most c-fos immunoreactivity (ir) was in the hilus, especially temporal hippocampus. C-fos-ir hilar cells co-expressed GluR2/3, suggesting that they were MCs. C-fos-ir MCs were robust even when the animal was habituated to the investigator and laboratory where they were euthanized. However, c-fos-ir in dorsal MCs was reduced under these circumstances, suggesting that ventral and dorsal MCs are functionally distinct. Interestingly, there was an inverse relationship between MC and GC layer c-fos expression, with little c-fos expression in the GC layer in ventral sections where MC expression was strong, and the opposite in dorsal hippocampus. The results support the hypothesis that a subset of hilar MCs are spontaneously active in vivo and provide other DG neurons with tonic depolarizing input

The role of hippocampal mossy cells in novelty detection

At the encounter with a novel environment, contextual memory formation is greatly enhanced, accompanied with increased arousal and active exploration. Although this phenomenon has been widely observed in animal and human daily life, how the novelty in the environment is detected and contributes to contextual memory formation has lately started to be unveiled. The hippocampus has been studied for many decades for its largely known roles in encoding spatial memory, and a growing body of evidence indicates a differential involvement of dorsal and ventral hippocampal divisions in novelty detection. In this brief review article, we discuss the recent findings of the role of mossy cells in the ventral hippocampal moiety in novelty detection and put them in perspective with other novelty-related pathways in the hippocampus. We propose a mechanism for novelty-driven memory acquisition in the dentate gyrus by the direct projection of ventral mossy cells to dorsal dentate granule cells. By this projection, the ventral hippocampus sends novelty signals to the dorsal hippocampus, opening a gate for memory encoding in dentate granule cells based on information coming from the entorhinal cortex. We conclude that, contrary to the presently accepted functional independence, the dorsal and ventral hippocampi cooperate to link the novelty and contextual information, and this dorso-ventral interaction is crucial for the novelty-dependent memory formation

Involvement of mossy cells in sharp wave-ripple activity in vitro

The role of mossy cells (MCs) of the hippocampal dentate area has long remained mysterious. Recent research has begun to unveil their significance in spatial computation of the hippocampus. Here, we used an in vitro model of sharp wave-ripple complexes (SWRs), which contribute to hippocampal memory formation, to investigate MC involvement in this fundamental population activity. We find that a significant fraction of MCs (~47%) is recruited into the active neuronal network during SWRs in the CA3 area. Moreover, MCs receive pronounced, ripple-coherent, excitatory and inhibitory synaptic input. Finally, we find evidence for SWR-related synaptic activity in granule cells that is mediated by MCs. Given the widespread connectivity of MCs within and between hippocampi, our data suggest a role for MCs as a hub functionally coupling the CA3 and the DG during ripple-associated computations

Hilar Mossy Cells Provide the First Glutamatergic Synapses to Adult-Born Dentate Granule Cells

Adult-generated granule cells (GCs) in the dentate gyrus must establish synapses with preexisting neurons to participate in network activity. To determine the source of early glutamatergic synapses on newborn GCs in adult mice, we examined synaptic currents at the developmental stage when NMDA receptor-mediated silent synapses are first established. We show that hilar mossy cells provide initial glutamatergic synapses as well as disynaptic GABAergic input to adult-generated dentate GCs