神经生物学教学课件ach神经递质

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 (乙酰硫代胆碱法, 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) 基底前脑胆碱能系统(basal forebrain cholinergic system)

5、Mesulam和Perry分群：ch1- ch6。Ch1、Ch2、Ch3分别相当于隔内侧核（medial septal nucleus）、斜角带垂直部（vertical nucleus of the diagonal band）、斜角带水平部（horizontal limb of the diagonal band）；ch4则包含了视前大细胞区、苍白球底板的无名质及其向前延伸的苍白球腹侧区（纹状体下灰质）、Meynert基底核（nucleus basalis of Meynert ）、豆状攀核等界限并不很清楚的区域。I. 胆碱能投射神经元(Projection neuron)基底前脑胆碱能系统

6、(basal forebrain cholinergic system)的主要投射通路：它们的投射纤维主要形成以下通路：隔内侧核、斜角带-海马通路；斜角带-杏仁复合体通路；隔区、视前区-僵内侧核（medial habenula）、中脑脚间核通路； Meynert基底核-大脑皮层通路。其中由Meynert基底核向皮质额叶、颞叶、顶叶和视皮层的胆碱能投射，与学习和记忆功能密切相关。(II) 脑干胆碱能系统(brainstem cholinergic system) 由脑桥被盖胆碱能系统（pontine tegmental cholinergic system）和延髓的胆碱能神经元组成。它们的纤维分

7、背侧被盖束和腹侧被盖束，向头端投射至丘脑、丘脑下部、苍白球、尾壳核等，并与其它上行纤维一起组成网状上行激活系统，引起觉醒和警觉。脑桥被盖胆碱能系统主要包括脚间脑桥被盖区（pedunculopontine tegmental region, ppt）和背外侧或外侧被盖核（dorsolateral or laterodorsal tegmental nucleus, ldt）中的胆碱能神经元，相当于Mesulam和Perry分群中的Ch5、Ch6。I. 胆碱能投射神经元(Projection neuron)中脑和脑桥内还分布着动眼神经（III）、滑车神经（IV）、外展神经（VI）的胆碱能躯体运动和

8、/或内脏运动胞体。 延髓中的胆碱能神经元主要分布在舌下神经核（XII）、迷走神经背核与疑核（X）、面神经核（VII）、三叉神经脊束核（V），以及孤束核、前庭外侧核、外侧网状核、巨细胞网状核、中缝大核等。脊髓中介核I. 胆碱能投射神经元(Projection neuron)(III) Cholinergic neurons in the spinal cord这类神经元在核团内局部组成环路，不向核外发出投射，属中间神经元。主要位于纹状体（尾-壳核复合体，caudate-putamen complex）、伏隔核(nucleus accumben)、嗅结节（olfactory tubercle）、海

9、马和大脑皮质内-层。脊髓背角的胆碱能神经元投射到脊髓Rexed-III层、小脑内第层颗粒细胞投射到层细胞也形成局部环路。纹状体内胆碱能神经元主要为大、中型无棘突多极神经元，参与黑质-纹状体多巴胺系统对运动的调节，其异常与帕金森氏病（Parkinsons Disease, PD）的病理过程密切相关。 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 试述乙酰胆碱生物合成的主要底物、过程以及主要影响因素。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什么是量子释放？依你所知，有哪些证据证明神经递质是以量子方式释放的？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(组氨酸）.Site of Enzymatic decomposition: -OH of serin

21、e , nucleophilicI. Enzymatic decomposition and inactivationLife cycle of Ach（I）reversiblePhysostigmine, neostigmine: 为含季铵基团的氨基甲酸酯类。Edrophonium: 依酚氯铵：含季铵基团，但无氨基甲酰基。起效快，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 双复磷(LH-6)C 甲磺磷定(P2S)D 双磷定(TMB-4)Alzheimers disease 四氢氨基吖啶（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 银环蛇 Bungarus multicinctus银环蛇 Bungarus multicinctus银环蛇 Bungarus multicinctus银环蛇 Bungarus multicinctus银环蛇 Bungarus multicinctus银环蛇 Bungarus multicinctus金环蛇 Bungarus fasciatus银环蛇 Bungarus multicinctus

24、银环蛇 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试述尼古丁型乙酰胆碱受体的结构和功能特点 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