What is memory? The present state of the engram

The mechanism of memory remains one of the great unsolved problems of biology. Grappling with the question more than a hundred years ago, the German zoologist Richard Semon formulated the concept of the engram, lasting connections in the brain that result from simultaneous "excitations", whose precise physical nature and consequences were out of reach of the biology of his day. Neuroscientists now have the knowledge and tools to tackle this question, however, and this Forum brings together leading contemporary views on the mechanisms of memory and what the engram means today

PKM and the maintenance of memory.

How can memories outlast the molecules from which they are made? Answers to this fundamental question have been slow coming but are now emerging. A novel kinase, an isoform of protein kinase C (PKC), PKMzeta, has been shown to be critical to the maintenance of some types of memory. Inhibiting the catalytic properties of this kinase can erase well-established memories without altering the ability of the erased synapse to be retrained. This article provides an overview of the literature linking PKMzeta to memory maintenance and identifies some of the controversial issues that surround the bold implications of the existing data. It concludes with a discussion of the future directions of this domain

살아있는 뉴런과 동물에서 mRNA 관찰에 대한 연구

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 물리·천문학부(물리학전공), 2021.8. 이병훈.mRNA는 유전자 발현의 첫번째 산물이면서, 리보솜과 함께 단백질을 합성한다. 특히 뉴런에서, 몇몇 RNA들은 자극에 의해 만들어지고, 뉴런의 특정 부분으로 수송되어 국소적으로 단백질 양을 조절할 수 있게 한다. 최근 mRNA 표지 기술의 발전으로 살아있는 세포에서 단일 mRNA를 관찰하는 것이 가능해졌다. 이 연구에서, 우리는 RNA 이미징 기술을 이용해, 기억 형성과 상기할 때 활성화된 뉴런의 집합을 찾는 것 뿐 아니라, 뉴런의 축삭돌기에서 mRNA가 어떻게 수송되는지를 관찰했다. 이 논문의 첫 부분에서 우리는 신경 자극에 반응해서 만들어지는 것으로 알려진, Arc 유전자의 전사를 관찰하였다. 기억은 engram 혹은 기억 흔적 (memory trace)라고 불리는 뉴런들의 집합에 저장되어 있다고 생각된다. 그러나, 시간에 따라서 이런 기억 흔적세포들의 집합이 어떻게 변하고, 변화하면서도 어떻게 정보를 유지할 수 있는지 잘 알려져 있지 않다. 또한, 살아있는 동물에서, 기억 흔적세포를 긴 시간 동안 여러 번 찾아내는 것은 어려운 일이었다. 이 연구에서는 genetically-encoded RNA indicator (GERI) 기술을 사용해, 기억 흔적세포의 표식으로 널리 사용되는 Arc mRNA의 전사과정을 살아있는 쥐에서 관찰하였다. GERI를 이용함으로써, 기존 방법들의 한계점이었던 시간 제약 없이, 실시간으로 Arc를 발현하는 뉴런들을 찾아낼 수 있었다. 쥐에게 공간 공포 기억을 주고 나서 여러 번 기억을 상기시키는 행동실험 후에 Arc를 발현하는 세포를 식별했을 때, CA1에서는 Arc를 발현하는 세포가 이틀 후에는 더 이상 활성화되지 않았으나, RSC의 경우 4퍼센트의 뉴런들이 계속해서 활성화하는 것을 관찰했다. 신경활동과 유전자 발현을 같이 조사하기 위해, 쥐가 가상 환경을 탐험하고 있을 때 GERI와 칼슘 이미징을 동시에 진행하였다. 그 결과, 기억을 형성할 때와 상기시킬 때 Arc를 발현했던 뉴런들이 기억을 표상하는 것을 알아낼 수 있었다. 이처럼 GERI 기술을 이용해 살아있는 동물에서 유전자 발현된 세포를 찾아내는 방식은 다양한 학습 및 기억 과정에서 기억 흔적세포의 dynamics에 대해 알아낼 수 있을 것으로 기대된다. 이 논문의 두번째 부분에서, 우리는 세포 골격의 기본 구성 단위가 되는 β-actin의 mRNA를 축삭돌기에서 관찰하였다. mRNA의 국소화 (localization)를 통한 국소 단백질 합성은 축삭돌기 (axon)의 성장과 재생에 중요한 역할이 있다고 알려져 있다. 하지만, 아직 mRNA의 국소화가 축삭돌기에서 어떻게 조절되고 있는지 잘 알려져 있지 않다. 이 문제를 해결하기 위해서, 우리는 모든 β-actin mRNA가 형광으로 표지된 유전자 변형 쥐를 이용해, 살아있는 축삭돌기에서 β-actin mRNA를 관찰하였다. 이 쥐의 뉴런을 축삭을 구분해 줄 수 있는 미세유체 장치 (microfluidic device)에 배양한 뒤에, β-actin mRNA를 관찰하고 추적을 진행했다. 축삭은 세포 몸통으로부터 길게 자라기 때문에 mRNA가 먼 거리를 수송되어야 함에도 불구하고, 대부분의 mRNA가 수상돌기에 비해 덜 움직이고 작은 영역에서 움직이는 것을 보았다. 우리는 β-actin mRNA가 주로 축삭돌기의 가지가 될 수 있는 filopodia 근처와, 시냅스가 만들어지는 bouton 근처에 국소화되는 것을 관찰했다. Filopodia와 bouton이 actin이 풍부한 부분으로 알려져 있기 때문에, 우리는 액틴 필라멘트와 β-actin mRNA의 움직임간에 연관성을 조사했다. 흥미롭게도, 우리는 β-actin mRNA가 액틴 필라멘트와 같이 국소화 되고, β-actin mRNA가 액틴 필라멘트 안에서 sub-diffusive한 움직임을 보였으며, 먼 거리를 움직이던 mRNA도 액틴 필라멘트에 고정되는 모습도 확인할 수 있었다. 축삭에서 β-actin mRNA 움직임을 본 이번 관찰은 mRNA 수송 및 국소화에 대한 생물물리학 적 메커니즘의 기반이 될 수 있을 것이다.mRNA is the first product of the gene expression and facilitates the protein synthesis. Especially in neurons, some RNAs are transcribed in response to stimuli and transported to the specific region, altering local proteome for neurons to function normally. Recent advances of mRNA labeling techniques allowed us to observe the single mRNAs in live cells. In this thesis, we applied RNA imaging technique not only to identify the neuronal ensemble that activated during memory formation and retrieval, but also to traffic mRNAs transported to the axon. In the first part of the thesis, we observed the transcription site of Arc gene, one of the immediate-early gene, which is rapidly transcribed upon the neural stimuli. Because of the characteristic of expressing in response to stimuli, Arc is widely used as a marker for memory trace cells thought to store memories. However, little is known about the ensemble dynamics of these cells because it has been challenging to observe them repeatedly over long periods of time in vivo. To overcome this limitation, we present a genetically-encoded RNA indicator (GERI) technique for intravital chronic imaging of endogenous Arc mRNA. We used our GERI to identify Arc-positive neurons in real time without the time lag associated with reporter protein expression in conventional approaches. We found that Arc-positive neuronal populations rapidly turned over within two days in CA1, whereas ~4% of neurons in the retrosplenial cortex consistently expressed Arc upon contextual fear conditioning and repeated memory retrievals. Dual imaging of GERI and calcium indicator in CA1 of mice navigating a virtual reality environment revealed that only the overlapping population of neurons expressing Arc during encoding and retrieval exhibited relatively high calcium activity in a context-specific manner. This in vivo RNA imaging approach has potential to unravel the dynamics of engram cells underlying various learning and memory processes. In the second part of this thesis, we imaged β-actin mRNAs, which can generate a cytoskeletal protein, β-actin, through translation. Local protein synthesis has a critical role in axonal guidance and regeneration. Yet it is not clearly understood how the mRNA localization is regulated in axons. To address these questions, we investigated mRNA motion in live axons using a transgenic mouse that expresses fluorescently labeled endogenous β-actin mRNA. By culturing hippocampal neurons in a microfluidic device that allows separation of axons from dendrites, we performed single particle tracking of β-actin mRNA selectively in axons. Although axonal mRNAs need to travel a long distance, we observed that most axonal mRNAs show much less directed motion than dendritic mRNAs. We found that β-actin mRNAs frequently localize at the neck of filopodia which can grow as axon collateral branches and at varicosities where synapses typically occur. Since both filopodia and varicosities are known as actin-rich areas, we investigated the dynamics of actin filaments and β-actin mRNAs simultaneously by using high-speed dual-color imaging. We found that axonal mRNAs colocalize with actin filaments and show sub-diffusive motion within the actin-rich regions. The novel findings on the dynamics of β-actin mRNA will shed important light on the biophysical mechanisms of mRNA transport and localization in axons.1. INTRODUCTION, 1 1.1. Neuronal ensemble, 1 1.2. Immediate-early Gene (IEG), 3 1.3. Methods for IEG-positive neurons, 3 1.4. Two-photon microscope, 5 1.5. References, 7 2. IMAGING ARC mRNA TRANSCRIPTION SITES IN LIVE MICE, 9 2.1. Introduction, 9 2.2. Materials and Methods, 10 2.3. Results and Discussion, 18 2.4. References, 26 3. NEURONS EXPRESSING ARC mRNA DURING REPEATED MEMORY RETRIEVALS, 28 3.1. Introduction, 28 3.2. Results and Discussion, 28 3.3. References, 35 4. NEURAL ACTIVITIES OF ARC+ NEURONS, 36 4.1. Introduction, 36 4.2. Materials and Methods, 37 4.3. Results and Discussion, 38 4.4. References, 52 5. AXONAL mRNA DYNAMICS IN LIVE NEURONS, 54 5.1. Introduction, 54 5.2. Materials and Methods, 55 5.3. Results and Discussion, 59 5.4. References, 70 6. CONCLUSION AND OUTLOOK, 72 ABSTRACT IN KOREAN (국문초록), 76박

The elusive transcriptional memory trace

This work was supported byt he Spanish Research Agency (MICINN) under the grant PGC2018-094630-B-100toFAM, cofinanced by the European Regional Development Fund(ERDF).F.A.M.isarecipientofaRyC-2014-14961contract.B.G.-M.is a recipien to fa predoctoral fellowship,grantnumber SFPI/2020/00878(UAM).C.G.B.is a recipien to faFPU predoctoral fellowship, grantnumber FPU19/04449(MEFP).S.P.-F.is a recipien tofaJAEintro fellowship, grantnumbe rJAEINT_21_02520(CSIC

Hippocampal neurons with stable excitatory connectivity become part of neuronal representations

Experiences are represented in the brain by patterns of neuronal activity. Ensembles of neurons representing experience undergo activity-dependent plasticity and are important for learning and recall. They are thus considered cellular engrams of memory. Yet, the cellular events that bias neurons to become part of a neuronal representation are largely unknown. In rodents, turnover of structural connectivity has been proposed to underlie the turnover of neuronal representations and also to be a cellular mechanism defining the time duration for which memories are stored in the hippocampus. If these hypotheses are true, structural dynamics of connectivity should be involved in the formation of neuronal representations and concurrently important for learning and recall. To tackle these questions, we used deep-brain 2-photon (2P) time-lapse imaging in transgenic mice in which neurons expressing the Immediate Early Gene (IEG) Arc (activity-regulated cytoskeleton-associated protein) could be permanently labeled during a specific time window. This enabled us to investigate the dynamics of excitatory synaptic connectivity-using dendritic spines as proxies-of hippocampal CA1 (cornu ammonis 1) pyramidal neurons (PNs) becoming part of neuronal representations exploiting Arc as an indicator of being part of neuronal representations. We discovered that neurons that will prospectively express Arc have slower turnover of synaptic connectivity, thus suggesting that synaptic stability prior to experience can bias neurons to become part of representations or possibly engrams. We also found a negative correlation between stability of structural synaptic connectivity and the ability to recall features of a hippocampal-dependent memory, which suggests that faster structural turnover in hippocampal CA1 might be functional for memory

Shaping Memories via Stress:A Synaptic Engram Perspective

Stress modulates the activity of various memory systems and can thereby guide behavioral interaction with the environment in an adaptive or maladaptive manner. At the cellular level, a large body of evidence indicates that (nor)adrenaline and glucocorticoid release induced by acute stress exposure affects synapse function and synaptic plasticity, which are critical substrates for learning and memory. Recent evidence suggests that memories are supported in the brain by sparsely distributed neurons within networks, termed engram cell ensembles. While the physiological and molecular effects of stress on the synapse are increasingly well characterized, how these synaptic modifications shape the multiscale dynamics of engram cell ensembles is still poorly understood. In this review, we discuss and integrate recent information on how acute stress affects synapse function and how this may alter engram cell ensembles and their synaptic connectivity to shape memory strength and memory precision. We provide a mechanistic framework of a synaptic engram under stress and put forward outstanding questions that address knowledge gaps in our understanding of the mechanisms that underlie stress-induced memory modulation

Cellular and synaptic correlates of learning and memory and their impairment in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is characterized by synaptic dysfunction and progressive memory loss. The hippocampus is indispensable for memory processes and early affected by disease-associated pathology. It is still debated how in particular encoding and retrieval of memories is impaired in AD. Therefore, the current study investigated how individual neurons in the hippocampus encode a memory and whether this process is disturbed under AD-like conditions in a pre-clinical model. To achieve this goal a cutting-edge technology – two-photon in vivo imaging – was used to repetitively analyze the activity of neurons in the hippocampus throughout a hippocampus-dependent memory test. Initially, this study revealed two populations of hippocampal CA1 neurons that differ in their long-term activity: a subset of neurons was continuously active over several days, whereas another population showed variable activity. The latter provided the population that responded to memory encoding as well as retrieval and hence, formed the cellular memory trace, also known as engram. Interestingly, network activity and engram formation under AD-like conditions (APP/PS1 mice) was intact. However, a further analysis of neurons composing the "retrieval network" identified an additional neuronal ensemble in CA1 that superimposed the memory trace suggesting a causal relationship of memory trace superimposition and memory impairment. Indeed, mimicking superimposition by artificial activation of a non-related memory trace coding a different context caused reduced memory performance in healthy mice and thus, presents a potential mechanism for impaired memory retrieval in APP/PS1 mice. Furthermore, parvalbumin-expressing (PV+) interneurons in CA1 were indispensable for successful memory encoding and retrieval in healthy mice. Their functional impairment represented a potential explanation of the observed engram superimposition in APP/PS1 mice. Finally, a learning-related loss of synaptic connections was discovered on dendrites of CA1 pyramidal neurons in healthy mice suggesting a mechanism of synaptic selection important for encoding of new information. Learning-induced changes of synaptic connectivity were absent in APP/PS1 mice indicating that synaptic connectivity deficits might be causally related to memory trace superimposition and ultimately memory impairment under AD-like conditions. Summarized, the present study provides a refinement of the engram's characteristics and furthermore, identifies a novel mechanism of memory impairment on the cellular and synaptic level in a enmouse model of AD.Die Alzheimer-Krankheit (AD) ist durch synaptische Fehlfunktionen, eine Dysregulation des neuronalen Netzwerkes und darauf folgende Gedächtnisstörungen gekennzeichnet. Der Hippocampus ist für Lern- und Gedächtnisfunktionen unverzichtbar und sehr früh von der charakteristischen Pathologie der Krankheit betroffen. Wie genau die Bildung oder auch das Abrufen von Gedächtnisinhalten gestört wird, wurde noch nicht hinreichend geklärt. Die vorliegende Studie untersuchte die Beteiligung individueller Nervenzellen an der Bildung und dem Abrufen von Erinnerungen und eruiert mögliche Fehlfunktionen in einem präklinischen AD Model. Die Zwei-Photonen Intravitalmikroskopie wurde benutzt um repetitiv die Aktivität individueller Nervenzellen während einer hippocampus-abhängigen Lern- und Gedächtnisaufgabe zu verfolgen. Die untersuchte CA1-Region des Hippocampus wies hierbei zwei unterschiedliche Nervenzellpopulationen auf. Diese unterschieden sich hinsichtlich ihrer stabilen beziehungsweise wechselhaften Einbindung in das aktive Netzwerk. Nervenzellen letzterer Population wurden während des Lernens und Erinnerns in das aktive Netzwerk rekrutiert und somit als Träger der Erinnerung identifiziert, als sogenanntes Engramm. In einem Mausmodell, das Aspekte der Alzheimer-Krankheit repräsentiert (APP/PS1 Mäuse), wies die generelle Aktivität der CA1 Pyramidenzellen sowie das für den Erinnerungsprozess wichtige Engramm keine Abweichungen auf. Jedoch wurden im Erinnerungsnetzwerk der APP/PS1 Tiere zusätzlich aktivierte Nervenzellen identifiziert, die zu einer Überlagerung der Erinnerung und einer damit verbundenen Gedächtnisstörung führten. Durch künstliches Erzeugen dieser überlagernden Aktivität in experimentell gesunden Mäusen konnte ihre Gedächtnisfähigkeit vermindert und die Hypothese der Erinnerungsüberlagerung bestätigt werden. Des Weiteren wurde die Bedeutung von hippocampalen PV-positiven (PV+) Interneuronen bei Lern- und Gedächtnisprozessen bewiesen. Eine mögliche Unterfunktion ihrer hemmenden Leistung auf CA1 Pyramidenzellen könnte der vorher beschriebenen Überlagerung zugrunde liegen. Abschließend wurde ein durch Lernen induzierter Verlust von dendritischen Dornenfortsätzen im Stratum Radiatum der CA1 Pyramidenzellen gesunder Mäuse untersucht. Diese strukturelle Veränderung stellt einen potentiellen Selektionsmechanismus dar, der nicht in APP/PS1 Tieren nachgewiesen wurde. Zusammenfassend liefert die vorliegende Arbeit wichtige Erkenntnisse bezüglich der Charakteristika eines Engramms und deckt einen neuen Mechanismus der hippocampalen Gedächtnisstörung bei der Alzheimer-Krankheit auf