기억형성에 의한 엔그램 세포 사이의 시냅스 변화에 대한 연구

학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 생명과학부, 2019. 2. 강봉균.기억이 저장되는 원리와 장소를 찾기 위하여 오랜 기간 동안 많은 노력들이 이어져왔다. 최근 연구들에 의하면 기억은 엔그램 세포들에 의해 저장된다는 것이 밝혀졌다. 하지만 기억저장에 있어서 시냅스 가소성의 중요성을 고려했을 때 기억이 저장되는 장소를 세포수준이 아닌 시냅스 수준에서 밝히는 연구가 필요하다. 기술적 한계로 인하여 한 신경세포의 시냅스들을 분류하는 것이 불가능하였기 때문에 어떤 시냅스들이 특이적으로 기억형성에 의해 강화되는지 확인할 수 없었다. 본 연구는 엔그램 세포 사이의 시냅스가 특이적으로 강화되는 것을 보임으로서 엔그램을 시냅스 수준에서 찾아내고자 하였다. 이를 위해 한 수상돌기의 시냅스들을 시냅스 전 신경세포의 종류에 따라 구분해낼 수 있는 dual-eGRASP라는 기술을 개발하였다. 이를 이용하여 CA3 – CA1 사이의 가능한 네 종류의 시냅스 (엔그램 – 엔그램, 엔그램 – 비엔그램, 비엔그램 – 엔그램, 비엔그램 – 비엔그램)들 중에서 엔그램 – 엔그램 시냅스가 개수와 크기 측면에서 특이적으로 증가해있다는 것을 발견하였다. 이에 더하여 전기생리학적 실험으로 CA3 엔그램 시냅스의 분비확률증가와 CA1 엔그램 시냅스의 시냅스 후 반응 증가를 보임으로서 엔그램 세포 사이의 시냅스의 기능적 증가를 증명하였다. 이러한 결과들을 바탕으로 엔그램 세포 사이 시냅스가 구조적, 기능적 증가를 통해 기억을 저장하는 시냅스가 된다고 밝혀내었다.The specific sites responsible for memory storage has been focused for a long time. Recent studies demonstrated that memory is encoded in engram cells distributed across the brain. However, the memory substrate at synapse-level within these engram cells remains theoretical while it is generally accepted that synaptic plasticity encodes memory. Because of technical limitations, synapses between engram cells with other synapses has not yet been directly compared. To study engram in synapse-level, I developed dual-eGRASP technique to differentiate the synapses in one dendrite based on its presynaptic neuronal population. By comparing the four possible synapses (engram to engram, engram to non-engram, non-engram to engram, non-engram to non-engram) between CA3 – CA1 connections, I found the increased number and size of spines on CA1 engram cells received input from CA3 engram cells than other synapses. In addition, electrophysiological experiments revealed the functional enhancement of synapses between engram cells by showing CA3 engram synapses exhibit increased release probability, while CA1 engram synapses exhibit enhanced postsynaptic responses. These results strongly suggest that increased structural and functional connectivity between engram cells across two directly connected brain regions forms the synaptic correlate of memory.CONTENTS Abstract 1 List of Figures 5 Chapter I. Introduction Background 8 Purpose of this study 14 Chapter II. Development of dual-eGRASP and its application in mouse brain. Introduction 17 Experimental Procedures 19 Results 24 Discussion 38 Chapter III. Increased synapse number and spine size between CA3 engram and CA1 engram cells after memory formation. Introduction 41 Experimental Procedures 42 Results 47 Discussion 63 Chapter IV. Enhanced synaptic transmission between CA3 engram and CA1 engram cells through pre- and post-synaptic mechanisms. Introduction 66 Experimental Procedures 67 Results 72 Discussion 79 Chapter V. Conclusion 81 References 85 국문초록 94Docto

Rôle de deux groupes de vésicules dans la transmission synaptique

Les synapses formées par les fibres moussues (FM) sur les cellules principales de la région CA3 (FM-CA3) jouent un rôle crucial pour la formation de la mémoire spatiale dans l’hippocampe. Une caractéristique des FM est la grande quantité de zinc localisée avec le glutamate dans les vésicules synaptiques recyclées par la voie d’endocytose dépendante de l’AP3. En combinant l’imagerie calcique et l’électrophysiologie, nous avons étudié le rôle des vésicules contenant le zinc dans la neurotransmission aux synapses FM-CA3. Contrairement aux études précédentes, nous n’avons pas observé de rôle pour le zinc dans l’induction des vagues calciques. Nos expériences ont révélé que les vagues calciques sont dépendantes de l’activation des récepteurs métabotropiques et ionotropiques du glutamate. D’autre part, nos données indiquent que les vésicules dérivées de la voie dépendante de l’AP3 forment un groupe de vésicules possédant des propriétés spécifiques. Elles contribuent principalement au relâchement asynchrone du glutamate. Ainsi, les cellules principales du CA3 de souris n’exprimant pas la protéine AP3 avaient une probabilité inférieure de décharge et une réduction de la synchronie des potentiels d’action lors de la stimulation à fréquences physiologiques. Cette diminution de la synchronie n’était pas associée avec un changement des paramètres quantiques ou de la taille des groupes de vésicules. Ces résultats supportent l’hypothèse que deux groupes de vésicules sont présents dans le même bouton synaptique. Le premier groupe est composé de vésicules recyclées par la voie d’endocytose utilisant la clathrine et participe au relâchement synchrone du glutamate. Le second groupe est constitué de vésicules ayant été recyclées par la voie d’endocytose dépendante de l’AP3 et contribue au relâchement asynchrone du glutamate. Ces deux groupes de vésicules sont nécessaires pour l’encodage de l’information et pourraient être importants pour la formation de la mémoire. Ainsi, les décharges de courte durée à haute fréquence observées lorsque les animaux pénètrent dans les places fields pourraient causer le relâchement asynchrone de glutamate. Finalement, les résultats de mon projet de doctorat valident l’existence et l’importance de deux groupes de vésicules dans les MF qui sont recyclées par des voies d’endocytoses distinctes et relâchées durant différents types d’activités.Mossy fiber-CA3 pyramidal cell synapses play a crucial role in the hippocampal formation of spatial memories. These synaptic connections possess a number of unique features substantial for its role in the information processing and coding. One of these features is presence of zinc co-localized with glutamate within a subpopulation of synaptic vesicles recycling through AP3-dependent bulk endocytosis. Using Ca2+ imaging and electrophysiological recordings we investigated role of these zinc containing vesicles in the neurotransmission. In contrast to previous reports, we did not observe any significant role of vesicular zinc in the induction of large postsynaptic Ca2+ waves triggered by burst stimulation. Moreover, our experiments revealed that Ca2+ waves mediated by Ca2+ release from internal stores are dependent not only on the activation of metabotropic, but also ionotropic glutamate receptors. Nevertheless, subsequent experiments unveiled that the vesicles derived via AP3-dependent endocytosis primary contribute to the asynchronous, but not synchronous mode of glutamate release. Futhermore, knockout mice lacking adaptor protein AP3 had a reduced synchronization of postsynaptic action potentials and impaired information transfer; this was not associated with any changes in the synchronous release quantal parameters and vesicle pool size. These findings strongly support the idea that within a single presynaptic bouton two heterogeneous pools of releasable vesicles are present. One pool of readily releasable vesicles forms via clathrin mediated endocytosis and mainly participates in the synchronous release; a second pool forms through bulk endocytosis and primarily supplies asynchronous release. The existence of two specialized pools is essential for the information coding and transfer within hippocampus. It also might be important for hippocampal memory formation. In contrast to low firing rates at rest, dentate gyrus granule cells tend to fire high frequency bursts once an animal enters a place field. These burst activities, embedded in the lower gamma frequency, should be especially efficient in the triggering of substantial asynchronous glutamate release. Therefore, the results of my PhD project for the first time provide strong evidence for the presence and physiological importance of two vesicle pools with heterogeneous release and recycling properties via separate endocytic pathways within the same mossy fiber bouton

From the Cover: 7,8-Dihydroxyflavone Rescues Lead-Induced Impairment of Vesicular Release: A Novel Therapeutic Approach for Lead Intoxicated Children

Childhood lead (Pb2+) intoxication is a public health problem of global proportion. Lead exposure during development produces multiple effects on the central nervous system including impaired synapse formation, altered synaptic plasticity, and learning deficits. In primary hippocampal neurons in culture and hippocampal slices, Pb2+ exposure inhibits vesicular release and reduces the number of fast-releasing sites, an effect associated with Pb2+ inhibition of NMDA receptor-mediated trans-synaptic Brain-Derived Neurotrophic Factor (BDNF) signaling. The objective of this study was to determine if activation of TrkB, the cognate receptor for BDNF, would rescue Pb2+-induced impairments of vesicular release. Rats were chronically exposed to Pb2+ prenatally and postnatally until 50 days of age. This chronic Pb2+ exposure paradigm enhanced paired-pulse facilitation of synaptic potentials in Schaffer collateral-CA1 synapses in the hippocampus, a phenomenon indicative of reduced vesicular release probability. Decreased vesicular release probability was confirmed by both mean-variance analysis and direct 2-photon imaging of vesicular release from hippocampal slices of rats exposed to Pb2+in vivo. We also found a Pb2+-induced impairment of calcium influx in Schaffer collateral-CA1 synaptic terminals. Intraperitoneal injections of Pb2+ rats with the TrkB receptor agonist 7,8-dihydroxyflavone (5 mg/kg) for 14-15 days starting at postnatal day 35, reversed all Pb2+-induced impairments of presynaptic transmitter release at Schaffer collateral-CA1 synapses. This study demonstrates for the first time that in vivo pharmacological activation of TrkB receptors by small molecules such as 7,8-dihydroxyflavone can reverse long-term effects of chronic Pb2+ exposure on presynaptic terminals, pointing to TrkB receptor activation as a promising therapeutic intervention in Pb2+-intoxicated children

Neurotrophins Role in Depression Neurobiology: A Review of Basic and Clinical Evidence

Depression is a neuropsychiatric disorder affecting a huge percentage of the active population especially in developed countries. Research has devoted much of its attention to this problematic and many drugs have been developed and are currently prescribed to treat this pathology. Yet, many patients are refractory to the available therapeutic drugs, which mainly act by increasing the levels of the monoamines serotonin and noradrenaline in the synaptic cleft. Even in the cases antidepressants are effective, it is usually observed a delay of a few weeks between the onset of treatment and remission of the clinical symptoms. Additionally, many of these patients who show remission with antidepressant therapy present a relapse of depression upon treatment cessation. Thus research has focused on other possible molecular targets, besides monoamines, underlying depression. Both basic and clinical evidence indicates that depression is associated with several structural and neurochemical changes where the levels of neurotrophins, particularly of brain-derived neurotrophic factor (BDNF), are altered. Antidepressants, as well as other therapeutic strategies, seem to restore these levels. Neuronal atrophy, mostly detected in limbic structures that regulate mood and cognition, like the hippocampus, is observed in depressed patients and in animal behavioural paradigms for depression. Moreover, chronic antidepressant treatment enhances adult hippocampal neurogenesis, supporting the notion that this event underlies antidepressants effects. Here we review some of the preclinical and clinical studies, aimed at disclosing the role of neurotrophins in the pathophysiological mechanisms of depression and the mode of action of antidepressants, which favour the neurotrophic/neurogenic hypothesis

Optical analysis of synaptic plasticity in rat hippocampus

Long-term potentiation (LTP) in the CA1 region of the hippocampus is dependent on NMDA receptor activation. Downstream of NMDA receptor signaling, the activation of α-calcium/calmodulin-dependent protein kinase II (αCaMKII) is both necessary and sufficient for the induction of this form of LTP. It has been shown that αCaMKII accumulates in spines after glutamate application or ‘chemical LTP’. This postsynaptic accumulation of αCaMKII could be a key step for the induction of LTP, because it localizes the activated kinase close to the substrates of synaptic potentiation. It is not clear, however, what the threshold, time course of αCaMKII translocation are, and whether it is specific to the stimulated synapses only. To address these three questions, I combined optical stimulation techniques (Channelrhodopsin-2 stimulation and two-photon glutamate uncaging) with optical measurements of calcium transients and αCaMKII concentration. This ‘all-optical’ approach made it possible to investigate synapse-specific changes during the induction of LTP. I could show that coincident activity of pre- and postsynaptic cells was needed to trigger the translocation of αCaMKII. Functional potentiation could be measured immediately after stimulation, whereas αCaMKII accumulation reached its peak ~10 min later. This points to an additional structural role of αCaMKII at the postsynaptic density. Both αCaMKII fractions, the cytoplasmic fraction and postsynaptic bound αCaMKII, increased after optical LTP induction. These changes were restricted to stimulated spines. In spines that showed a persistent volume increase, the amount of bound αCaMKII was increased by a factor of two after 30-40 minutes. A second very interesting finding was the close correlation between spine volume changes and LTP, in terms of the time course, induction threshold and specificity. The optical LTP protocol led to a lasting volume increase only in the stimulated spines, but not in directly neighboring spines on the same dendrite. Spine volume reached its maximum immediately after stimulation. Since my all-optical approach relied heavily on the use of a newly identified light-gated cation channel (Channelrhodopsin-2, ChR2), I finally also characterized light activation of ChR2 in hippocampal pyramidal cells in detail. Neuronal activity could be controlled by blue light with millisecond precision. No direct activation of ChR2 was observed by two-photon imaging lasers, making it possible to combine the ChR2 stimulation technique with two-photon imaging. This led to a third important finding: the release probability of ChR2-expressing axonal terminals was increased if the action potential was induced by light. As a result, pairing of light stimulation with postsynaptic depolarization induced reliable long-term potentiation at CA1 synapses. In summary, the new all-optical approach that combines ChR2 stimulation, two-photon glutamate uncaging, and optical measurements of calcium transients and protein concentration, provides a new avenue for investigating plasticity at the level of single synapses. The induction of LTP in single synapses revealed that accumulation of αCaMKII is input specific thus validating Hebb’s postulate on a micrometer scale

An investigation of the processes underlying late-phase long-term potentiation

In this thesis I have investigated the role of RNA and protein synthesis, during the late-phase of hippocampal long-term potentiation (L-LTP). Additionally, the involvement of protein kinase C (PKC) in the induction of this late phase was addressed. Using protocols that allowed hippocampal slices to be maintained for long periods, L-LTP lasting 8 hours or more was successfully achieved. Field EPSPs were recorded from pyramidal cells in the CAl region of the stratum radiatum. Tetanically induced L-LTP was blocked by bath application of inhibitors of transcription (actinomycin-D) and translation (emetine), whilst L-LTP in slice preparations lacking presynaptic cells bodies was unaffected. In addition, bath application of bisindolylmaleimide I, a highly selective inhibitor of PKC, was found to block both E-LTP and L-LTP induction if applied within the first 15 minutes, after the tetanus. These results are consistent with a requirement for both protein synthesis and postsynaptic RNA synthesis during L-LTP induction, coupled with a requirement for a critical period of PKC activity. The locus of protein synthesis during L-LTP in CAl pyramidal cells was investigated by focally applying emetine to apical dendrites, while stimulating afferents to basal and apical dendrites. This significantly reduced L-LTP in the projection to apical dendrites, whilst leaving L-LTP unaffected in the projection to basal dendrites. Focal application of emetine to the cell bodies had no significant effect on L-LTP in either the apical or basal dendrites. Furthermore, preliminary data suggest that L-LTP in the basal dendrites is unaffected by focal emetine application. These experiments provide evidence that pyramidal cells in the CAl region of the hippocampus can support two different forms of L-LTP: type I, in the apical dendrites (stratum radiatum) that depends on protein synthesis, and a component of which relies on local dendritic protein synthesis and type II, in the basal dendrites (stratum oriens), that remains protein synthesis-independent for at least 8 hours

A General Hippocampal Computational Model Combining Episodic and Spatial Memory in a Spiking Model

Institute for Adaptive and Neural ComputationThe hippocampus, in humans and rats, plays crucial roles in spatial tasks and nonspatial tasks involving episodic-type memory. This thesis presents a novel computational model of the hippocampus (CA1, CA3 and dentate gyrus) which creates a framework where spatial memory and episodic memory are explained together. This general model follows the approach where the memory function of the rodent hippocampus is seen as a “memory space” instead of a “spatial memory”. The innovations of this novel model are centred around the fact that it follows detailed hippocampal architecture constraints and uses spiking networks to represent all hippocampal subfields. This hippocampal model does not require stable attractor states to produce a robust memory system capable of pattern separation and pattern completion. In this hippocampal theory, information is represented and processed in the form of activity patterns. That is, instead of assuming firing-rate coding, this model assumes that information is coded in the activation of specific constellations of neurons. This coding mechanism, associated with the use of spiking neurons, raises many problems on how information is transferred, processed and stored in the different hippocampal subfields. This thesis explores which mechanisms are available in the hippocampus to achieve such control, and produces a detailed model which is biologically realistic and capable of explaining how several computational components can work together to produce the emergent functional properties of the hippocampus. In this hippocampal theory, precise explanations are given to why mossy fibres are important for storage but not recall, what is the functional role of the mossy cells (excitatory interneurons) in the dentate gyrus, why firing fields can be asymmetric with the firing peak closer to the end of the field, which features are used to produce “place fields”, among others. An important property of this hippocampal model is that the memory system provided by the CA3 is a palimpsest memory: after saturation, the number of patterns that can be recalled is independent of the number of patterns engraved in the recurrent network. In parallel with the development of the hippocampal computational model, a simulation environment was created. This simulation environment was tailored for the needs and assumptions of the hippocampal model and represents an important component of this thesis