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1 尋找形成記憶的分子元素 Searching for Memory Molecules Yi-Shuian Huang ( 生醫所 N703, 黃怡萱 ) [email protected] 高中生認知單元 Dec. 10, 2016

2 Outlines 大綱神經系統中的細胞學簡介 Neuron/ Glia/ microglia 樹突, 軸突, 突觸 (dendrite/axon/ synapse) 神經細胞間如何傳遞訊息? Excitatory vs. inhibitory 突觸可塑性 ( Synaptic plasticity): 學習與記憶的分子機制長期記憶的分子本質如何研究記憶的實驗方法

3 Learning and Memory- Making who we are Learning 學習 : 接受新的訊息 Memory: 將接受到的訊息存於腦中, 可分為 Declarative (explicit) memory: 陳述性 ( 外顯 ) 記憶 Cognitive memory- Facts and event Nondeclarative (implicit) memory: 非陳述性 ( 內隱 ) 記憶 Procedural memory- skills, habits, behaviors

4 Memory can last for various time 記憶可因存在時間的長短而又分成長期記憶, 短期記憶及暫時記憶 Long-Term, Short-Term, and Working (temporary) Memory 但長期記憶的形成不一定要經過短期記憶的過程 Sensory information Short-term memory Consolidation Long-term memory Sensory information Short-term memory Consolidation Long-term memory Time

5 Different Phases of Memory Acquisition (learning, encoding information) Consolidation Reconsolidation Retrieval Extinguish (not equal to forgetting, more like relearning )

6 Searching for the brain regions involved in Memory Localization of Declarative Memories in the Neocortex Experiments macaque monkeys Differentiate objects based on shapes Studies in humans: The effects of temporal lobectomy (H.M. patient- work of Brenda Milner)

Searching for the brain regions involved in Memory Electrical Stimulation of the Human Temporal Lobes Dr. Penfield s experiments Electrical stimulation of the temporal lobe Complex sensations, Dr.

7 Searching for the brain regions involved in Memory Electrical Stimulation of the Human Temporal Lobes Dr. Penfield s experiments Electrical stimulation of the temporal lobe Complex sensations, Dr. Penfield s patients: Sensations like hallucinations, recall past experiences Temporal lobe: Role in memory storage Temporal lobe stimulation 額葉 Different from stimulation of other areas of neocortex 頂葉枕葉顳葉

Medial temporal lobes and memory processing Recognition memory test: DNMS: Delayed non-match to sample Medial temporal structures: Important for consolidation of memory

8 Medial temporal lobes and memory processing Recognition memory test: DNMS: Delayed non-match to sample Medial temporal structures: Important for consolidation of memory mean% of correction 教猴子學會在新的積木下有好吃的所以猴子要能記住舊的積木才知新的積木是那一個 Which curve represents normal? Which curve represents with bilateral medial temporal lobe lesions?

9 Searching for engrams (memory traces) in the brain Lashley s Studies of Maze Learning in Rats (1920) - Found the size (but not the location) of the lesion correlates with the poor memory - Concluded all cortical area contribute equally to the memory Is this true? What is your opinion?

10 Molecular basis of memory: Hebb s theory Hebb and the Cell Assembly External events are represented by cortical cells Cells reciprocally interconnected reverberation Active neurons cell assembly Consolidation by growth process Fire together, wire together Hebb and the engram Widely distributed among linked cells in the assembly Could involve neurons involved in sensation and perception Hebb s Cell Assembly and Memory Storage Memory is distributed in a circuit of cells 記憶存在一群相互連結的細胞間

11 The Neuron Doctrine Camillo Golgi( ) Santiago Ramon Cajal ( ) The neuron, even in its adult form, is one cell Neurons communicate by contact, not continuity

12 The neuron doctrine: Neuron is a function unit in the brain Cajal s neuron doctrine: Neurons communicate by contact, not continuity Which technical invention has established Cajal s theory in ~1950?

13 Nobel Prize 2014: Super-resolution microscopy (Nanoscopy) Dani et al.(2010) Neuron

14 訊息傳遞: Axon, pre-synapse, post-synpase, dendrite, axon... 樹突 (Dendrite) 軸突 (Axon) 突觸 (Synapse) 突觸囊泡 (Synaptic vesicle) 樹突起 (Dendritic Spines)

15 Hippocampal neurons

16 Neurotransmitters: excitatory & inhibitory chemical signals at presynapses 神經傳導物質

Channel Proteins ( 離子通道 ) Ion selectivity and gating Voltage-gated ion channel Ligand (neurotransmitter)-gated

17 Neurotransmitter Receptors: excitatory & inhibitory receivers at postsynapses 神經傳導物質受體 : 皆會造成膜電位的改變有些本身是離子通道, 有些會間接造成其他離子通道的開啟使膜電位更正 (more positive) 的是興奮性傳遞反之是抑制性傳遞 Lipid bilayers: 不導電 Neurotransmitter Receptors (NTRs) - Channel Proteins ( 離子通道 ) Ion selectivity and gating Voltage-gated ion channel Ligand (neurotransmitter)-gated ion channel - Receptors Ion Pumps ( 離子幫浦 ) Formed by membrane spanning proteins Uses energy from ATP breakdown

The ionic basis of the resting membrane potential Equilibrium Potentials No net movement of ions when separated by a phospholipid membrane Equilibrium reached when K + channels inserted into the

18 The ionic basis of the resting membrane potential Equilibrium Potentials No net movement of ions when separated by a phospholipid membrane Equilibrium reached when K + channels inserted into the phospholipid bilayer Four important points Large changes in V m Minuscule changes in ionic concentrations Net difference in electrical charge Inside and outside membrane surface Rate of movement of ions across membrane Proportional V m E ion Concentration difference known: Equilibrium potential can be calculated by the Nernst equation

19 Electrorecording detects the changes in membrane potential Membrane potential: Voltage across the neuronal membrane Electro-Recording Voltage-clamp: fix V, recording I Current-clamp: fix I, recording V Principle: Ohm s Law V= IR V: voltage R: resistance I= Vg I: current g: conductivity R: varies depending on channel open/close 記錄時採固定電位然後記錄電流的變化, 或固定電流的方式然後記錄電位的變化, 由此可算出細胞膜電阻力的變化細胞膜電阻力的變化取決於離子通道的開與關

20 Action Potential Conduction: Single direction/ All-or-none/ Saltatory 軸突起始段 (Axon initial segment )

21 Dendrite: information computation/ Axon: action potential (all-or-none) Axon initial segment 樹突 _ 軸突起始段 + _ + ++ _ ++ _ + + _ 突觸 from Riken + : excitatory synapses - : inhibitory synapses

22 Synaptic structure correlates with neuronal connectivity Synapses (突觸): Connections in the network 樹突軸突突觸 from Synaptosome: 生化方法分離出的突觸部分 from Wen-Biao Gan (in vivo image, 1 mon apart)

23 CPEB3 wild type (WT) CPEB3 knockout (KO) Hippocampal neurons

10 15 synapses axon Cerebral cortex ~ 20 x10 9 neurons Long-term plasticity and long-term memory

24 How to modify synapse (plasticity) for long-term dendrite inactive terminal Nerve system ~75 x10 6 neurons ~ synapses Cerebral cortex ~ 4 x10 6 neurons N Nerve system ~85 x10 9 neurons; ~ synapses axon Cerebral cortex ~ 20 x10 9 neurons Long-term plasticity and long-term memory requires de novo protein synthesis 施打蛋白質合成抑制劑, 抑制長期記憶的形成, 所以長期記憶的分子本質是蛋白質 Flexner et al (1963) Science 141:57

25 Summary: Prototypical neuron The Soma (Cell body 細胞體 : 細胞質 + 細胞核 ) Dendrites 樹突 (dendritic tree, dendritic spine) Antennae of neurons Post-synaptic site neurotransmitter receptors (receives signals from axon terminal) Axons 軸突 Electric cable of neurons, myelin sheath: as an insulator Axon hillock (beginning: Action potential- all or none) Axon terminal (end: electric to chemical transmission) Pre-synaptic terminal (signal : neurotransmitter release) Synapses 突觸 Communication loci of neurons Electric-to-chemical-to-electric transformation of neurons

26 Memory hub 記憶中樞 : Hippocampus 海馬體取出胎鼠的腦, 再分離出海馬體 Hippocampal neuron culture 海馬體神經培養 stained with lucifer yellow to reveal tiny spines rendered in artificial color

27 Hippocampal neuron development in culture morphological changes of synapses

28 High density neuron culture (bright field) Images of GFP-expressing neurons

29 MAP2 + Hoechst

30 Organotypic slice culture: Preserve the organization of neurons Organotypic slice culture e.g. hippocampal, basal forebrain cholinergic system Preserve the organization of neurons. Preserve (likely) the neuronal circuitry.

用電生理來證明長期性的突觸可塑性是需要蛋白的合成 CA1 CA3 Dentate Gyrus Recording electrode Two forms of long-term synaptic plasticity -Protein synthesis dependent -Local translation BDNF-

31 用電生理來證明長期性的突觸可塑性是需要蛋白的合成 CA1 CA3 Dentate Gyrus Recording electrode Two forms of long-term synaptic plasticity -Protein synthesis dependent -Local translation BDNF- induced LTP (long-term potentiation) Kang and Schuman (1996) Science DHPG- induced LTD (long-term depression) Huber et al., 2000, Science + BDNF-> LTP +CHX/ BDNF -> no LTP

32 Morris water maze: 用水迷宮來測試老鼠的空間記憶 Spatial cue

33 from youtube

34 Approaches Translational Regulator Cued fear conditioning Contextual fear conditioning CPEB3 Amyg Hippo 1 mm WT/KO: n=9/7 Freezing (%) WT/KO: n=7/7 KO mouse model Electro -recording fepsp slope (%baseline) 100 Hz 250 Wild CP3WT Type (n=8) 5 Hz CPEB-3 CP3KO KO (n=9) min (h) WT mock KO chemical LTD (NMDA) WT KO PRAY for something wrong with my mice LOST IN TRANSLATION

35 Morris water maze (hippocampus-dependent spatial memory): KO wins CPEB3 spatial cues WT/KO: n=8/8 Day 5: probe trial 1 Swimming speed: WT: 16.6 ± 2.4 cm/sec KO: 17.3 ± 1.4 cm/sec

36 Morris water maze : Perseverance (stubborn) Q3 Q4 Day5 Probe trial 1 Q2 Q1 Day9 Q3 Q4 Day11 Q3 Q4 Day13 Q3 Q4 4 trials 4 trials 4 trials Q2 Q1 Q2 Q1 Q2 Q1 Time (%) * Reversal test I Day10 Reversal test II Day12 Reversal test III Day14

(30 ) Freezing (%) 100 80 60 40 20 * WT KO context B

37 Context-specific cued fear conditioning and extinction: PTSD-like Context A Context B Tone: cued (30 ) Freezing (%) * WT KO context B ** A 0 E#1 E#2 (E: extinction) Context A extinction Context B

Searching for engrams (memory traces) in the

poor memory - Concluded all cortical area

38 Searching for engrams (memory traces) in the brain Lashley s Studies of Maze Learning in Rats (1920) - Found the size (but not the location) of the lesion correlates with the poor memory - Concluded all cortical area contribute equally to the memory Is this true? What is your opinion?

39 用型態分析來研究基因剔除鼠的突觸是否不同於野生鼠的突觸 CPEB3 WT mock CPEB3 KO chemical LTD (3 min NMDA+30min) CPEB3 WT CPEB3 KO cumulative probability density (#spine/ 20 µm) P=0.413 P=0.044 P=0.471 P=0.005 P=0.126 P= length of spine (µm) P=0.063 P<0.001 P<0.001 P<0.001 P<0.001 P= width of spine head (µm) P<0.001 P<0.001 P=0.192 P<0.001 P<0.001 P< WT WT+NMDA KO KO+ NMDA

用電生理來研究基因剔除鼠的突觸可塑性是否不同於野生鼠 potentiation depotentiation fepsp slope (%baseline) 100 Hz 250 Wild WT Type (n=5) CPEB-3 KO (n=5) KO 200 150 100

40 用電生理來研究基因剔除鼠的突觸可塑性是否不同於野生鼠 potentiation depotentiation fepsp slope (%baseline) 100 Hz 250 Wild WT Type (n=5) CPEB-3 KO (n=5) KO time (h) 1 fepsp slope (%baseline) 100 Hz Hz Wild WT (n=8) Type CPEB-3 KO (n=9) KO min time (h) 1

9 11 13 15 17 19 W K W K W K W K W K W K W K W K W K W

41 用生化方法來研究基因剔除鼠的神經蛋白組成是否不同於野生鼠 (DIV) axon initiation dendrite growth spine growth spine maintenance W K W K W K W K W K W K W K W K W K W K W: WT K: KO CPEB3 NR1 GluR1 PSD95 LRP130 tubulin β-actin

42 Presynapse Vesicles Ca 2+ Postsynapse GluR1 NR2a/b NR1 S831+P PSD GK S73+P 1 Ca 2+ Calcineurin/ PP1 Delivered to PSD region PKC CaMKII S295+P? CPEB3 Adenylate cyclase GluR1 JNK? camp S845+P LTD Degradation of synaptic proteins PKA PSD 95 synthesis/ degradation

43 在受刺激的神經中, 研究蛋白質體 (memory molecules) 組成的變化用化學方式來標定新合成的蛋白然後用質譜儀來分析這些新合成蛋白是什麼?

44 記憶有最大極限嗎? 過目不忘 (photographic memory) 好嗎? Stephen Wiltshire drew this sketch of New York from memory after a 20-minute helicopter flight over the city.

45 Optogenetics: 用光來影響動物體內的神經活性, 進而改變其行為 Optogenetics is a novel technique of delivering genes that encode proteins capable of conveying light sensitivity to electrical changes in target cells. Thus, it provides a precision and efficiency way to manipulate neuronal circuit at milliseond-time scale to understand brain information processing as well as potential application for individualized therapeutic clinical approaches. 用基因工程的方式, 將從藻類中找到的蛋白表達在老鼠或其他動物身上此蛋白是 light-gated ion channel proteins such as channelrhodopsins. e.g. ChR2 ( 打開, 正電流流入 ), halorhodopsin, VChR1 ( 打開, 負電流流入 ) 1. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K ( 2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8: Zhang F, Wang LP, Boyden ES, Deisseroth K (2006). Channelrhodopsin-2 and optical control of excitable cells. Nat. Methods 3 3. Nagel G, Ollig D, Fuhrmann M, et al. (2002) Channelrhodopsin-1: a light-gated proton channel in green algae. Science 296: Nagel G, Szellas T, Huhn W, et al. (2003) Channelrhodopsin-2, a directly light-gated cationselective membrane channel. Proc. Natl. Acad. Sci. U.S.A. 100:

46 Method of the year 2010: Optogenetics

Chapter 8

Chapter 8 Consciousness, The Brain and Behavior 識行 I. States of Consciousness 識狀 (I) Electroencephalogram The electroencephalogram provides one means of defining the states of consciousness. 了識狀 Electric