實驗神經概論. Membrane Potential Synaptic Transmission Sleep Physiology

實驗神經概論 Membrane Potential Synaptic Transmission Sleep Physiology ( 含 Principles of Neuroscience Ch6- Ch12) 楊靜修老師

Neuron as the functional units of nervous system Structure of a neuron 1x Neuron: carrying signals 50x Supporting cells: protect, insulate, reinforce, assist neurons Multiple sclerosis (MS): immune system-myelin sheath loss of signal conduction, muscle control, brain function

Drawing of the fluid-mosaic model of membranes, showing the phospholipid bilayer and imbedded proteins.

Ion Channels Are Important for Signaling in the Nervous System Ion channels have three important properties 1. They conduct ions 2. They recognize and select specific ions 3. They open and close in response to specific electrical, mechanical, or chemical signals. Gates: voltage-gated channels, ligand-gated channels, mechanically gated channels, non-gated channels

Ion Channels Are Proteins That Span the Cell Membrane 正離子 ion channel 內含負電可吸引 Na + d = 0.095 nm 較強之 electric field strength K + d = 0.133 nm ion 通過 channel 很快 1 ms, 非 binding Na + 去除水後可入而 K + 之直徑太大無進入 Channel selective Lipid bilayer: 6-8 nm

Several types of stimuli control the opening and closing of ion channels

Voltage-gated channel [Ca ++ ]i

Only a very thin shell of charge difference is needed to establish a membrane potential.

Resting potential: the voltage across the plasma membrane of a resting neuron

A nerve signal originates as a change in the resting potential: The action potential Any + ion 閾值 Vg Na + channel open Squid giant axons Hodgkin and Huxley, 1940s action potential (nerve signal) Vg K + channel open

One direction 原因 : refractory period (Vg Na + channel inactivation) all or none not vary in size

Chemical synapse Synapse (relay point, junction) 1. Electrical synapse 2. Chemical synapse

Action potential 發生過程 ( 簡要 ) 1. 靜止膜電位 -70 mv, K + permeability 最大, 極化狀況 2. 正離子入或負離子出細胞皆可造成極化狀況減少, 而引發電位開啟之 Na + 孔道打開, Na + 因電及濃度關係由外向內 3. 當膜電位改變大於閾值, Na + 入細胞又使細胞內正電增多因而造成更多 Na + 入細胞 ( 正迴饋作用 ) 之不可逆變化至電位變化至 +35 mv 左右 Na + 孔道不活化且電位開啟之 K + 孔道開啟,K + ( 因電及濃度關係 ) 離開細胞 4. K + 離開細胞使膜電位向極化方向, ( 因 K + 為正電離開細胞 ) 最終比靜止膜電位更負 ( 因 K + permeability 更大 ) 最後回靜止膜電位 Na + -K + pump 運作始回復

Giant squid axons Hodgkin and Huxley, 1940s action potential (nerve signal) Na + 只是其中一因子閾值 Vg Na + channel open 利用 voltage clamp 將 action potential 以分解動作來探討各膜電壓下發生之離子進出情況 V( 膜電位固定 )= I ( 電流進出 ) x R (1/R 為通透度語 I 成正比可推論 channel 是否開啟 ) Vg K + channel open

Refractory period ( 不反應期 )

Opening of voltage-gated channels is all-or-none Patch-clamp

Genes encoding the K +, Na +, and Ca 2+ channels stem from a common ancestor

Gating of the Na + channel is thought to rely on redistribution of net charge in the S4 region

Action potentials move rapidly along myelinated axons because the only parts of the neuronal membrane that undergo ion movements are the section at the Nodes of Ranvier.

Active Electrical Properties of the Neuron 1. 電位變化不隨時間及距離衰減 2. 電位變化不與刺激成正比 3. All or none 4. Vg Na + and K + channel 5. 不反應期 6. 傳導 7. Myelin sheath

Synaptic Transmission

Chemical synapse:

Chemical synapse: neuromuscular junction 1.very accessible to experimentation 2.one muscle one neurotransmitter 3.one presynaptic axon 4.simple mechanism 5.single type of ion channel

Chemical synapse: neuromuscular junction

Nicotinic receptor

Electrophysiological properties: endplate potential (EPP) in muscle cell Curare ( 箭毒 ): ACh receptor antagonist

Summary: neuromusclar junction Vg Ca 2+ channel Nicotinic receptor Na + N receptor K + depolarization Vg Na + Vg K + Channel--AP

ATP Skeletal muscle 之 AP 為起始 1. 肌肉收縮力量 Ca ++ i 2. Ca ++ 利用 Ca ++ pump 很快收回 3. Ca ++ 來自細胞內所以不影響電位變化

Central Nervous System Four primary neurons communicate to one secondary neuron. One primary neuron communicates to four secondary neurons.

IPSP and EPSP

CNS-synaptic transmission Sti 1 Sti 2 Recording 1 EPSP Recording 2 IPSP

1. Spatial summation ( 空間總合 ) 2. Temporal summation ( 時間總合 )

t = time constant

由 receptor 決定反應為何

Figure 49-6 Both acetylcholine (ACh) and a luteinizing hormone-releasing hormone (LHRH)-like peptide are released by presynaptic cells at synapses in the sympathetic chain ganglia in the bullfrog. The two transmitters produce different types of postsynaptic potentials in different postganglionic neurons because of their actions on different receptors..

Possible drug effects on synaptic effectiveness: A. release and degradation of the neurotransmitter inside the axon terminal. B. increased neurotransmitter release into the synapse. C. prevention of neurotransmitter release into the synapse. D. inhibition of synthesis of the neurotransmitter. E. reduced reuptake of the neurotransmitter from the synapse. F. reduced degradation of the neurotransmitter in the synapse. G.agonists (evoke same response as neurotransmitter) or antagonists (block response to neurotransmitter) can occupy the receptors. H. reduced biochemical response inside the dendrite.

Drugs that inhibit synthesis of neruotransmitter Drugs that interfere with storage of neurotransmitter Drugs that affect neuronal uptake Drugs that inhibit release of neruotransmitter Drugs that promote release of neruotransmitter Drugs that stimulate autonomic receptors Drugs that block autonomic receptors Drugs that inhibit metabolism of neurotransmitter

Adrenergic transmission:

Drugs that affect neuronal uptake and inhibit synthesis of neruotransmitter Drugs that promote release of neruotransmitter Drugs that inhibit metabolism of neurotransmitter Drugs that interfere with storage of neurotransmitter Drugs that inhibit release of neruotransmitter Drugs that stimulate autonomic receptors Drugs that block autonomic receptors

Cholinergic transmission:

Autonomic pharmacology Ganglionic blockers (transmission and receptor) -block sympatheric and parasympathetic nerve activity (other factor) Drugs that inhibit synthesis of neruotransmitter Drugs that inhibit release of neruotransmitter Drugs that promote release of neruotransmitter Drugs that interfere with storage of neurotransmitter Drugs that affect neuronal uptake Drugs that inhibit metabolism of neurotransmitter Drugs that block autonomic receptors Drugs that stimulate autonomic receptors Adrenoceptor-activating drugs Adrenoceptor-antagonist drugs Chlinoceptor-activating & chlinesterse-inhibiting drugs Cholinoceptor-blocking drugs

Effects of drugs: mechanism

Chemical synapse: many to one

Central Nervous System Four primary neurons communicate to one secondary neuron. One primary neuron communicates to four secondary neurons.

Sleep Physiology Speaker: 楊靜修

Outline and Summary Introduction-sleep 幾乎佔我們生命的 1/3 時間 Sleep 的原因 ( 日夜變化代謝 [ 被動 ] 學習記憶 [ 主動 ] 仍不知??) 人的最佳睡眠時間每人不同, 睡 7 h, 睡不好會產生疾病幾天 (2-3 w) 不睡會死亡 Sleep 睡眠各期腦波變化腦波是否可反應想法或被侵入竊取?frequency amplitude 德國 Hans Berger, 1929 第一位記到人的腦波圖 (1935 年始睡醒腦波 1953 年才發現 REM) 如何檢測睡眠品質? Sleep 主觀檢查 ( 問卷 ) PSQI 表 Sleep 客觀檢查 ( 生理參數變化 ) 人不動 ( 手上拿東西東西掉表示睡著 ; 床動記錄儀 ; active watch) EEG EMG EOG- 因為同時發生所以拿來作為睡眠代表 ( 不見得是對只有溫血動物記的到腦波 ) 睡眠偵測及分期標準 (R&K, 1968 訂 ) 大鼠以 K&Y lab 為例目前仍在等待新自動判讀方法客觀與主觀睡眠評估對應仍不佳原因不知?? Sleep 理論睡眠中心 ( 動物實驗 [ 解剖及行為方法 ] 及其它人的研究集合而成可解釋 ) 腦波來源研究 ( 探討 delta power REM 腦波來源 ) 睡眠疾病種類很多 ; 睡眠品質不好與許多疾病有關用 sleep/wake 當作是工具來分類以 K&Y lab 為例

Why we need sleep? Circadian rhythm Avoid danger ( 演化觀點 - 如夜行性動物白天睡 ) Conservation of metabolic energy ( 活動消耗 ) Neural maturation REM sleep in baby Cognition Memory consolidation

How much sleep do we need? 實際上 REM 多的動物並沒有較會學習? 可能有其它意義 Science (2001)

Sleep Duration Cox proportional hazard survival models 636,095 women (A) 480,841 men (B) 多因子分析以 7 h 為標準 6 年研究 >7 或 < 7 Mortality hazard ratio 皆較高 Arch Gen Psychiatry. 2002; 59:131-136

*8-10 year followed up hypertension incidence 發生率 Hypertension. 2006;47:833-839

睡眠主觀評量 PSQI > 5 或 7 表示有睡眠問題 ν ν 0, 1, 2, 3 分 0, 1, 2, 3 分

SSS 嗜睡量表 Stanford Sleepiness Scale 其它 VAS 量表

Epworth Sleepiness Scale

Cycling of Human Sleep 睡眠客觀評量 >13 Hz 8-13 Hz <4 Hz

Polysomnography in Human

Polysomnography 鼻根枕骨突窿 A2 A1

Polysomnography (PSG) and Sleep EOG EMG EEG ECG Respiratory 德國 Hans Berger, 1929 第一位記到人的腦波圖 1935 年始睡醒腦波 1953, Kleitman 年才發現 REM Rechtschaffen and Kales, 1968 (R&K) 訂出睡眠判讀標準

Polysomnographic Events Approaches: A. EEG activity and waveforms B. Eye movement C. Sleep onset and microsleep D. Arousals and awakenings --EEG, EOG, EMG: --paper speed: 10 mm/sec (30 sec /epoch)

Stages of Sleep 人 - 仍需技術員判讀 Stage W (wakefulness) Stage 1 (NREM1) Stage 2 (NREM2) Stage 3 (NREM3) NEW! ---- slow wave sleep and replaces the R&K nomenclature of stage 3 and stage 4 sleep---- Stage R (rapid eye movement, REM) 睡眠偵測及分期標準 (R&K, 1968 訂 )

δ wave: 慢波 -- < 4 Hz, 原則上 < 2 Hz -- infant, child 的睡眠大多數是 δ wave θ wave: 徐波,4~7 Hz α wave: 8~13 Hz -- 醒著閉眼會出現 α wave, 張眼時消失 β wave: 快波,> 13 Hz Frequency and amplitude

Stage W (wakefulness) 1.Alpha activity constitutes 50% or more of the recording epoch ( > 50%) 2.In non-alpha producers, eye movements, blinks and high tonic submentalis EMG activity are present O1-A2

Sleep stage 1 (0-15%) 1. Low voltage, mixed frequency EEG activity 2. Alpha less than 50% of epoch ( < 50%) 3.May contain vertex sharp waves and slow eye movements O1-A2 EOG EMG

Sleep stage 2 (>50%) 1. Contains sleep spindles, K complexes, or both 2. No more than 20% of epoch may contain delta activity (d < 20%) 3. Eye movements are absent C3-A2 O1-A2 EMG

Sleep stage 3 (10-15%) 1. High amplitude (> 75 mv) delta waves constitute 20% or more of recording epoch (d > 20%) 2. Sleep spindles may be present 3. Eye movements are absent C3-A2 EMG

Sleep stage R (REM 25%) 1. Low voltage, mixed frequency EEG activity 2. Rapid eye movements occur 3. Tonic REM: phase EMG is absent or diminished 4. Phasic REM: bursts occur EOG EMG

Sleep stage histogram

TST: total sleep time (- all W) TST/TRT=SE% WASO: wake after sleep onset Arousal no Arousal index: arousal no/ TST TRT: total recording time

Sleep latency SWS (S3+S4) SWS/TST=SWS% REM, REM% S1, S2, S3 S1%, S2%, S3%

REML (latency) REMSL: REM sleep latency

In 1916, Von Economo identify the brain area ( 嗜睡性腦炎病人腦 - hypothalamus- 睡眠中樞 ) Sleep Center Extremely tired, difficult to fall sleep, short sleep time. Brainstem, hypothalamus, basal forebrain

The First of Sleep Center Research VLPO Science 1996, 271:216~218

Research Methods Behavioral Normal sleep analysis, sleep deprivation Electrophysiology EEG, single neuron recording (in vivo, vitro) Neuroanatomy Immunohistochemistry (c-fos) Tracer (anterograde and retrograde) other Other Drug (agonists, antagonists) Lesion Gene translation (Knock out..)

C-fos protein expression in VLPO In VLPO neuron : cfos-immunostined A. Dark period (15%) B. Light period (63%) C. Sleep deprivation (83%) Fos protein: Normal sleep neuron activated SD

Retrograde tracer A. Cholera toxin B (CTB) label tuberomammillary nucleus TMN B. Cholera toxin B (CTB) injection into VLPO (1 week later) VLPO C. CTB (TMN) cell body labeled and Fos-ir (VLPO) cell doubly labeled

Summary

The ascending arousal system promotes wakefulness First pathway-thalamus-thalamocortical (yellow pathway) pedunculopontine (PPT) laterodorsal tegmental nuclei (LDT)-ACh Second pathway -hypothalamus-cortex (red) tuberomammillary nucleus (TMN)-His ventral periaqueductal gray matter (VPAG)-DA dorsal and median raphe nuclei-5-ht locus coeruleus (LC)-NA basalforebrain (BF)-ACh, GABA lateral hypothalamus (LHA)-ORX, MCH Neurotransmitters : histamine (His), dopamine (DA), serotonin (5-HT), noradrenaline (NA), orexin (ORX) melanin-concentrating hormone (MCH), aminobutyric acid (GABA) Nature (2005)

The VLPO Promotes Sleep Show the key projections of the VLPO to the main components of the ascending arousal system. tuberomammillary nucleus (TMN) A10 cell group raphe cell groups the locus coeruleus (LC) lateral hypothalamus (LHA; green), pedunculopontine (PPT) laterodorsal tegmental nuclei (LDT) Nature (2005)

Sleep-Awake Circuit 1. 2. Awake Pathway Sleep Pathway (NREM and REM sleep) LDT: laterodorsal tegmental nuclei, PPT: pedunculopontine TMN: tuberomammillary nucleus, Raphe: raphé nuclei, LC:locus coeruleus VLPO: ventrolateral preoptic nucleus REM Pericoeruleus (PC), parabranchial (PB)-Glu-basal forebrain (BF)-cortex -sublaterodorsal nucleus (SLD)-Glu-atonia

Flip-Flop Switch Model ( 正反器 all-or-none) 1. Self-reinforcing loop 2. Wake-Sleep transition quickly 3. Homeostatic forces Saper et al. Nature (2005)

Homeostatic Regulation of sleep Set point During prolonged wakefulness Accumulate: need to sleep Sleep deprivation Accumulate neurotransmitter: Adenosine

Adenosinergic modulation ATP degradation adenosine in basal forebrain-increase sleep propensity Adenosine antagonist effects of caffeine Behavioural Brain Research 2000,115 :183 204

Behavioural Brain Research 2000,115 :183 204

The Possible Roles of Autonomic Nervous System ( 自律神經系統 ) in Normal Sleep ( 睡眠 ) and Sleep-related Disorders Cheryl C. H. Yang ( 楊靜修 ) Professor, Institute of Brain Science Director, Sleep Research Center, National Yang-Ming University, Taipei, Taiwan

研究概況 :sleep & ANS 研究正常醒睡中自律神經系統之角色扮演性別 [ 性別差異月經週期停經 ] 年齡 [ 成長及老化 ] 溫度 [ 冬季低溫生日季節舒適度 ] 運動 [ 強迫運動及主動運動 ] 午睡後期睡眠 [ 與心血管疾病好發關係 ] 工作壓力限制睡眠值班 [ 輪班及長期值夜 ] 時差音樂床枕異常時如高血壓失眠中風時睡眠自律神經系統之可能病理角色及可能改善方式 [ 運動音樂呼吸調整中醫介入健康管理等 ]

ANS Sympathetic N. S. during sleep Parasympathetic N. S. during wake EEG+EMG and/or Wake late sleep in winter Sleep Bp dipping MBPS EOG Normal Sympathetic N. S. during sleep Parasympathetic N. S. during wake Hypertension Insomnia? Sleep fragmentation Arousal no. increase Unstable sleep Insomnia Difficulty falling asleep Sleep onset latency Sleep disorders

NEW ANS Sympathetic N. S. during sleep Parasympathetic N. S. during wake EEG+EMG and/or Wake late sleep in winter Sleep Bp dipping MBPS EOG Normal Sympathetic N. S. during sleep Parasympathetic N. S. during wake Prevention and Treatment Hypertension Sleep fragmentation Arousal no. increase Unstable sleep Insomnia (1,2,3,4 types) Difficulty falling asleep Sleep onset latency NEW Sleep disorders 調整呼吸 ; Exercise 中強度 ; 平甩功 Music; 針灸 ; Stress, 精油, Light, Ta NEW 儀器開發及驗證應用軟體開發及驗證及應用及服務等 NEW

Summary-ANS in Normal sleep (1) The changes of ANS functioning during normal sleep. (2) Parasympathetic activity before falling asleep and sympathetic activity before awakening both changed concomitantly with the shift in EEG activity. (3) Differential changes and interactions of autonomic functioning and sleep architecture before and after 50 years of age. (4) In rat sleep, the late-sleep structure instability is associated with higher sympathetic activity, which may account for a higher cardiovascular risk. (5) A rat model demonstrated that sleep-wake cycle was the main factor in determining BP dipping, the magnitude of which was associated with waking sympathetic activity and sleep vagal activity. (6) A temperature shift model mimicking the winter season in Taiwan revealed that low ambient temperature resulted in a rise in MBPS and sympathetic activation. (7) A sleep-in bedding system selected by manual muscle testing increased sleep related BRS in young men.

Summary-ANS in sleep-related disorders (1) SHRs have significant sympathetovgal imbalance, which exaggerated particularly during sleep as sympathetic overactivation. (2) SHRs are poor sleepers. (3) Sleep disturbance among SHR is mediated by an 1-adrenergic mechanism. (4) Electrical stimulation of the rostral ventrolateral medulla promotes wakefulness in WKY rats (5) Exercise training prevents the establishment of hypertension in SHRs: changes in sympathetic activity and sleep patterns. (6) Effect of paced breathing on vagal activity and sleep pattern in selfreported insomnia. (7) The association between prolonged sleep onset latency and heart rate dynamics among young sleep-onset insomniacs and good sleepers (8) APP

第一組 ANS 研究議題第二組第三組 Sleep Sleep-ANS 第四組 Hypertension 1-1 儀器建立 1-2 分析方法建立 AJP, 1999 1-3 AJP, 2003 1-4 ClinNeuro, 2009 1-5 居家照護硬體 ( 專利 ) 軟體 ( 專利 ) 2-1 儀器建立 - 大鼠 2-2 儀器建立 - 小鼠 2-3 大鼠睡眠分析建立 2-4 小鼠睡眠分析建立 2-5 人的睡眠分析建立, 2012 2-6 Sleep, 2004 2-7 感應充電 ( 大鼠 ), 2012 2-8 溫度控制設計 2-9 簡易睡眠偵測分析系統 2-10 腦控開關 3-1 NSL, 2002 3-2 NSL, 2003 3-3 Sleep, 2008 3-4 Nap 3-5 Endocrinology, 2010 3-6 BP dipping 3-7 cold-induce BP surge 3-8 sleepiness-ans (mice) 3-9 shift work, 2010, 2012 3-10 MCAO,2012 3-11 late sleep, 2012 4-1 AJP, 2004 4-2 Sleep, 2005 4-3 Circulation, 2005 4-4 less diurnal rhythm 4-5 cold-induced BP surge in SHR 4-6 sleep fragmentation & 1 adrenoceptor, 2012 4-7 Sleep-ANS change during 24 h 4-8 運動降血壓第五組 Estrogen 5-1 內生性 estrogenrats, 2010 5-2 大鼠去卵巢停經 5-3 大鼠自動停經 5-4 女性月經週期 5-5 停經失眠及自主神經失調第六組 Age-related 6-1 健康老化 - 人 6-2 老人 - 大鼠 6-3 老化 - 高血壓 - 人 6-4 老化 - 高血壓 - 大鼠 6-5 cold-induce BP surge in aged rats 第七組 Application 7-1 運動 NeuLett, 2008 7-2 運動 Age, 2011 7-3 滾輪運動, 2012 7-4 Bedding system 7-5 呼吸控制改善睡眠問題 7-6 耳針 7-7 Zolpidem 7-8 音樂藥物第八組失眠 & Others 8-1 失眠與 ANS 8-2 遙控電刺激 8-3 精神科用藥及治療 8-4 精神分裂活動異常 8-5 同情行為模式建立 8-6 人格特質 8-7 手機成癮

考題 :( 兩題寫在 A4 單面一頁不可超過內容宜避免直接抄講義 ) 圖一說明 action potential 過程

實驗神經概論考題 - 問答題 ( 請選兩題作答 ) 1. 睡醒中樞理論如何証明? 目前睡及醒的理論? 2. 傳統睡眠判讀的優缺點論述? 睡眠主觀及客觀評量異? 同? 為什麼? 3. 醒 (a) 及睡眠 [nrem 第一期 (b), 第二期 (c) 到第三期 (d), REM (e)] 的腦波肌電及眼動波變化? 4. 自律神經系統在醒及睡眠裡扮演的角色? (K&Y lab 的研究 )