神經(jīng)生物學(xué)教學(xué)課件ach神經(jīng)遞質(zhì)

1、Chapter 6: AcetylcholineSection I: Distribution and projection of cholinergic neurons in the CNSGeneral knowledgeHistory of Ach as a neurotransmitter:1867:Chemically synthesized.Dale (1914): the effect of Ach on the heart is similar with that of vagal stimulationLoewi(1921)、 Dale（1924): infusion exp

2、eriment of frog heart. 1936:Nobel Prize. Concept of cholinergic neurons:Neurons that use acetylcholine as the neurotransmitter. General knowledgeChemical property of acetylcholine：a.light sensitive. b. Easily hydrolyzed: stable at pH3.8-4.5；complete hydrolysis at pH10 and 100C. c. Easily decompounde

3、d by cholinesterase.General knowledgeIndirect methods：a. Measure of cholinesterase activity：Koelle, Friedenwald (1947) Acetylthiocholine (乙酰硫代膽堿法, AchE法）.b. ChAT: 1970s, ChAT multiple colonial antibody;Eckenstein (1981) ChAT monoclonal antibody. Houser (1983): Consistence of AchE method and ChATmeth

4、od.Direct methodsBiological identification;RIA; chromatogram;HPLC et al.Imunohistology + fluorescent tracing.Measure of acetylcholineGeneral knowledgeClassification of central cholinergic neurons：Projection neuron Local circuit neuronGeneral knowledge(I) 基底前腦膽堿能系統(tǒng)(basal forebrain cholinergic system)

5、Mesulam和Perry分群：ch1- ch6。Ch1、Ch2、Ch3分別相當(dāng)于隔內(nèi)側(cè)核（medial septal nucleus）、斜角帶垂直部（vertical nucleus of the diagonal band）、斜角帶水平部（horizontal limb of the diagonal band）；ch4則包含了視前大細(xì)胞區(qū)、蒼白球底板的無(wú)名質(zhì)及其向前延伸的蒼白球腹側(cè)區(qū)（紋狀體下灰質(zhì)）、Meynert基底核（nucleus basalis of Meynert ）、豆?fàn)钆屎说冉缦薏⒉缓芮宄膮^(qū)域。I. 膽堿能投射神經(jīng)元(Projection neuron)基底前腦膽堿能系統(tǒng)

6、(basal forebrain cholinergic system)的主要投射通路：它們的投射纖維主要形成以下通路：隔內(nèi)側(cè)核、斜角帶-海馬通路；斜角帶-杏仁復(fù)合體通路；隔區(qū)、視前區(qū)-僵內(nèi)側(cè)核（medial habenula）、中腦腳間核通路； Meynert基底核-大腦皮層通路。其中由Meynert基底核向皮質(zhì)額葉、顳葉、頂葉和視皮層的膽堿能投射，與學(xué)習(xí)和記憶功能密切相關(guān)。(II) 腦干膽堿能系統(tǒng)(brainstem cholinergic system) 由腦橋被蓋膽堿能系統(tǒng)（pontine tegmental cholinergic system）和延髓的膽堿能神經(jīng)元組成。它們的纖維分

7、背側(cè)被蓋束和腹側(cè)被蓋束，向頭端投射至丘腦、丘腦下部、蒼白球、尾殼核等，并與其它上行纖維一起組成網(wǎng)狀上行激活系統(tǒng)，引起覺(jué)醒和警覺(jué)。腦橋被蓋膽堿能系統(tǒng)主要包括腳間腦橋被蓋區(qū)（pedunculopontine tegmental region, ppt）和背外側(cè)或外側(cè)被蓋核（dorsolateral or laterodorsal tegmental nucleus, ldt）中的膽堿能神經(jīng)元，相當(dāng)于Mesulam和Perry分群中的Ch5、Ch6。I. 膽堿能投射神經(jīng)元(Projection neuron)中腦和腦橋內(nèi)還分布著動(dòng)眼神經(jīng)（III）、滑車神經(jīng)（IV）、外展神經(jīng)（VI）的膽堿能軀體運(yùn)動(dòng)和

8、/或內(nèi)臟運(yùn)動(dòng)胞體。 延髓中的膽堿能神經(jīng)元主要分布在舌下神經(jīng)核（XII）、迷走神經(jīng)背核與疑核（X）、面神經(jīng)核（VII）、三叉神經(jīng)脊束核（V），以及孤束核、前庭外側(cè)核、外側(cè)網(wǎng)狀核、巨細(xì)胞網(wǎng)狀核、中縫大核等。脊髓中介核I. 膽堿能投射神經(jīng)元(Projection neuron)(III) Cholinergic neurons in the spinal cord這類神經(jīng)元在核團(tuán)內(nèi)局部組成環(huán)路，不向核外發(fā)出投射，屬中間神經(jīng)元。主要位于紋狀體（尾-殼核復(fù)合體，caudate-putamen complex）、伏隔核(nucleus accumben)、嗅結(jié)節(jié)（olfactory tubercle）、海

9、馬和大腦皮質(zhì)內(nèi)-層。脊髓背角的膽堿能神經(jīng)元投射到脊髓Rexed-III層、小腦內(nèi)第層顆粒細(xì)胞投射到層細(xì)胞也形成局部環(huán)路。紋狀體內(nèi)膽堿能神經(jīng)元主要為大、中型無(wú)棘突多極神經(jīng)元，參與黑質(zhì)-紋狀體多巴胺系統(tǒng)對(duì)運(yùn)動(dòng)的調(diào)節(jié)，其異常與帕金森氏?。≒arkinsons Disease, PD）的病理過(guò)程密切相關(guān)。 II. Local circuit neuronSection II: biosynthesis, storage and release-S-(I) Biosynthesis (I) Biosynthesis ChAT, globulin, MW 68000. Synthesized in cel

10、l body, takes effect in terminals.Essential chemical groups: imidazole, -SH. Enzyme dynamics (Km)：choline 7.510-4 mol/L; Acetyl CoA 1.0 10-5 mol/L.Site of synthesis：Cholinergic terminalsEnzyme:(I) Biosynthesis 1. Acetyl CoA: located in mitochondria . Source: (1) glucose-pyruvic acid-Acetyl CoA. （2）F

11、atty acid beta-oxidation. （3）synthesized from citric acid. (4) acetate (non-neural tissues)Substrate:(I) Biosynthesis 2. Choline: neurons do not syntheses; choline in circulation cannot pass blood-brain barrier (?).Source: a. uptake and hydrolyze lecithin from circulation. b. hydrolysis of lecithin

12、of neuronal tissues. c. hydrolysis of Ach and reuptake of choline. 1/3-1/2.(I) Biosynthesis Low affinity carrier:distributed in all neurons and glia cells. Km: 40-100 micro mol/L.facilitated diffusionTransportation of choline:High affinity carrierspecifically distributed in cholinergic terminals. Km

13、: 0.4-4 micro mol/LNa+,ATP-dependent active transportation. Satured uptake. The most effective limit factor of acetylcholine synthesis (!). Choline is the limit substrate.(I) Biosynthesis Regulating factorsSupply of cholineSupply of acetyl CoARule of mass-reaction rule.Regulation of biosynthesis(I)

14、Biosynthesis Hemicholium-3 (HC-3)Mechanism：compete high affinity carrier with choline. Antagonized by choline.characteristics：not able to pass though blood-brain barrier; central administration needed. Strong, slow, lasting effect.Triethylcholine(CH3CH2) 3 N+-(CH2)2-OHMechanism：synthesis of fake neu

15、rotransmitter。4-(1-naphthylvinyl)pyridine：specific in vitro ChAT inhibitor.Agents affecting the biosynthesis of Ach(I) Biosynthesis 試述乙酰膽堿生物合成的主要底物、過(guò)程以及主要影響因素。Question 1 ATP-vesicle protein -Ach+Vesicle Acetylcholine Transporter (VAchT)(II) Storage and release Storage:Release:Phosphorylation and dep

16、hosphorylation of synapsin I in regulation of releasable and reserve pools of vesicles.Roles of calcium and CAM kinase II.(II) Storage and release Ca+synapsin IactinActive zone of synapse.(II) Storage and release 1. Electrophysiological observations:- resting neuromuscular junctions often had sponta

17、neous miniature endplate potentials (mepps) of less than 1 mV in amplitude.amplitude of stimulated epp were multiples of the mepps.Conclusion:-transmitter is released in discrete units (quanta).-smallest mepp represents the release of 1 quanta. (II) Storage and release Evidence of quata release:2. E

18、lectron microscopyEvidence of quata release:Freeze-fracture electron micrograph 3. NeurochemistryNa+K+Ca2+VSCCreleaseefflux* DH, chromograninsLactate dehydrogenase*Evidence of quata release:3. NeurochemistryNa+K+Ca2+VSCCreleaseefflux* DH, chromograninsLactate dehydrogenase*Reserpine+MAO-IEvidence of

19、 quata release:Evidence of quata release:4. Cloning of VAchT and finding its blocker visamicol什么是量子釋放？依你所知，有哪些證據(jù)證明神經(jīng)遞質(zhì)是以量子方式釋放的？Question 2 SNAP-25SYNTAXINSynaptobrevin (VAMP)Botulinum toxinTetanus toxin-bungarotoxin- bungarotoxinH+vesamicolTransmitter isGLYCINETransmitter isACETYLCHOLINESection III:

20、 Enzymatic decomposition and inactivationSpecific cholinesterase (AchE) ：mainly in neural tissues。Non- Specific cholinesterase (butyrylcholinesterase):glia cells, non-neural tissues. Chemical structure of AchEAnion site: imidazole group of histidine(組氨酸）.Site of Enzymatic decomposition: -OH of serin

21、e , nucleophilicI. Enzymatic decomposition and inactivationLife cycle of Ach（I）reversiblePhysostigmine, neostigmine: 為含季銨基團(tuán)的氨基甲酸酯類。Edrophonium: 依酚氯銨：含季銨基團(tuán)，但無(wú)氨基甲?；Ｆ鹦Э?，作用短。（II）irreversible : organic phosphorus pesticide, chemical warfare agent（VX, 沙林）II. Cholinesterase inhibitorsOrganophosphorousinse

22、cticidesAnticholinesterasesBACDA 氯磷定(2-PAMCl)B 雙復(fù)磷(LH-6)C 甲磺磷定(P2S)D 雙磷定(TMB-4)Alzheimers disease 四氫氨基吖啶（tetrohydro aminoacridine antidotes to anticholinesterasessymptomatic “antidote” - atropinepralidoxime (2-PAM)mode of action?Section IV: Cholinergic ReceptorsI. Muscarinic receptorII. Nicotinic re

23、ceptorN1N2Purification of cholinergic receptorsPurification of receptorscDNA libraryHomology screening 銀環(huán)蛇 Bungarus multicinctus銀環(huán)蛇 Bungarus multicinctus銀環(huán)蛇 Bungarus multicinctus銀環(huán)蛇 Bungarus multicinctus銀環(huán)蛇 Bungarus multicinctus銀環(huán)蛇 Bungarus multicinctus金環(huán)蛇 Bungarus fasciatus銀環(huán)蛇 Bungarus multicinctus

24、銀環(huán)蛇 Bungarus multicinctusPurification of receptorsCarb-nAchRTorpedo electric organnAchRnAchRnAchRsolubilizationHomogenizationCarbamylcholineSequence analysis of segmentsSynthesis of probe RNAa-BTXReconstruction in lipid bilayer (functional?)cDNA library screeningTorpedo electric organHomogenizationI

25、solate total RNAsIsolate mRNAsprobe RNAcDNA libraryIn vitro transcription of the cDNA libraryInjection of cRNAs into Xenopus oocytes Electrophysiological measurement of ligand-induced currentSequencing Injection of cRNAs into Xenopus oocytes cDNA library screeningLow density receptorsHomogenizationI

26、solate total RNAsIsolate mRNAsprobe RNAcDNA libraryIn vitro transcription of the cDNA libraryInjection of cRNAs into Xenopus oocytes Electrophysiological measurement of ligand-induced currentSequencing Injection of cRNAs into Xenopus oocytes PCRHomology screeningcDNA librarycDNA 1cDNA 2cDNA 3Subtype

27、 1Subtype 2Subtype 3Whole-sequence RNA probeStructure and functions：Membrane-binding glucoprotein.Five homologous subunits (, , , , ) make up the channel.Each subunits has four -helical membrane spinning domains (T1-T4), and T2 domain of each subunit make up the linen of the channel.The two subunits

28、 are the binding sites for Ach (-BGTX)Both a receptor and a channel, mediates fast signaling, no second messenger is necessary.(I) Peripheral nicotinic receptorsSubunits of AchR are 4TMHydropathy index analysisTM2 segment is the pore-lining domain of AchR試述尼古丁型乙酰膽堿受體的結(jié)構(gòu)和功能特點(diǎn) Question 3 （II）Central n

29、icotinic receptors (including ganglion)Subunit composition of central nicotinic receptors:2- 10 (the peripheral subunit as 1)2- 4 (the peripheral subunit as 1)About 2- 6, 2- 4:Do not form homomeric receptors, no receptors with only or only subunits exist.Common receptor types and functions in the CN

30、SThe heteromeric 4223 subtype: facilitate GABAergic and glycinergic neurotransmission. Insensitive to -BGTX，sensitive to Kappa or F toxin.GABAglycine42234223The homomeric 75 subtype: facilitate glutamatergic neurotransmission. sensitive to -BGTX.glutamate75Latest advancesNicotine increased the frequency and amplitude of the spontaneous EPSCs in inspiratory activatedairway vagal preganglionic neurons (IA-AVPNs)Dihydro-erythroidine (DHE), but not methyllycaconitine (MLA), prevented t