神經(jīng)生物學：5-遞質(zhì)系統(tǒng)（臨床醫(yī)學5年制）

1、Neurotransmitter SystemsIntroductionStudying Neurotransmitter SystemsLocalization of transmitters and synthesizing enzymesStudying transmitter release / synaptic mimicry / receptorsNeurotransmitter ChemistryCholinergic/Catecholaminergic/Serotonergic/mino acidergic neuronsOther neurotransmitter candi

2、dates and intercellular messengersTransmitter-gated ChannelsThe basic structure of transmitter-gated channelsAmini acid-gated channelsG-Protein-Coupled Receptors And EffectorsThe basic structure of G-protein-coupled receptorsThe ubiquitous G-proteinG-protein-coupled effector systemsDivergence and Co

3、nvergence in Neurotransmitter SystemsNeurotransmitters (amino acids, amines, and peptides)PlusThe molecular machinery (for transmitter synthesis, vesicular packaging, reuptake and degradation, and transmitter action)Acetylcholine (ACh), the first NT, identified in the 1920s.The neurons producing and

4、 releasing Ach given the term cholinergic by British pharmacologist Henry Dale (shared the 1936 Nobel Prize with Loewi).The suffix ergic: noradrenergic, glutamatergic, GABAergic, peptidergic, and so on, for the various synapses, neurons and neurotransmitter systems. IntroductionIntroductionElements

5、of neurotransmitter systemsStudying Neurotransmitter SystemsCriteria to identify a neurotransmitter: 1. The molecule must be synthesized and stored in the presynaptic neuron.2. The molecule must be released by the presynaptic axon terminal upon stimulation.3. The molecule, when experimentally applie

6、d, must produce a response in the postsynaptic cell that mimics the response produced by the release of neurotransmitter from the presynaptic neuron.Localization of Transmitters and their Synthesis EnzymespWhatever the inspiration, the first step in confirming the hypothesis on a new neurotransmitte

7、r is to show that the molecule is, in fact, localized in, and synthesized by, particular neurons. pMany methods have been used to satisfy this criterion for different neurotransmitters. Two of the most important techniques used today are immunocytochemistry（免疫細胞化學）and in situ hybridization （原位雜交）.St

8、udying Neurotransmitter SystemsImmunocytochemistry. This method uses labeled antibodiesto identify the location of molecules within cells. Studying Neurotransmitter Systemsp Immunocytochemistry can be used to localize any molecule for which a specific antibody can be generated, including the neurotr

9、ansmitters themselves and the synthesizing enzymes for transmitter candidates.Immunocytochemical localization ofa peptide neurotransmitter in neuronsStudying Neurotransmitter Systems Strands of mRNA consist of nucleotides arranged in a specific sequence. Each nucleotide will stick to one other compl

10、ementary nucleotide. In the method of in situ hybridization, a synthetic probe is constructed containing a sequence of complementary nucleotides that will allow it to stick to the mRNA. If the probe is labeled, the location of cells containing the mRNA willbe revealed.In situ hybridization Studying

11、Neurotransmitter SystemsIn situ hybridization of the mRNA for a neuropeptide transmitter in hippocampus of mice (A. wild type and B. the peptide Knockout). Only neurons with the proper mRNA are labeled, with clusters of white dots.Studying Neurotransmitter SystemsStudying Transmitter Releasep To sho

12、w that a neurotransmitter candidate is actually released upon stimulation. Axon stimulation test biological activity chemical analysis(as Loewi and Dale did in identification of ACh as a transmitter at many peripheral synapses)p A diverse mixture synapses in CNS makes it impossible to stimulate a si

13、ngle population of synapses. Researchers have to collect and measure all the chemical mixture (e.g. using brain slices in a solution containing a high K+ concentration).p Also have to show Ca2+ dependency of release, and from the presynaptic axon terminal uponStudying Neurotransmitter SystemsStudyin

14、g Synaptic MimicryTo meet the third criterion, microionophoresis (離子微電泳) is often use to assess the postsynaptic actions of a transmitter candidate.The candidates in solutions in a glass pipette is ejected on in very small amounts by passing electrical current through the surface of neurons, and the

15、 membrane potential can be measured.If it mimics the effects of transmitter released at the synapse, and if the other criteria of localization, synthesis, and release have been met, then the molecule and the transmitter usually are considered to be the same chemical.Studying Neurotransmitter Systems

16、MicroionophoresisStudying Neurotransmitter SystemsStudying ReceptorsAs a rule, no two neurotransmitters bind to the same receptor; however, one neurotransmitter can bind to many different receptors, receptor subtype. e.g. two different cholinergic receptor subtypes.Three approaches to study the diff

17、erent receptor subtypes:pNeuropharmacological Analysis. For instance, cholinergic receptor subtypes respond differently to various drugs. Nicotine (煙堿), a receptor agonist in skeletal muscle, nicotinic ACh receptors (channels). Curare (筒劍毒) is its selective antagonist.Muscarine (毒蕈堿), a receptor ago

18、nist in the heart, muscarinic receptors (GPCR). Atropine is its selective antagonistNicotinic and muscarinic receptors also exist in the brain. Studying Neurotransmitter SystemsThree subtypes of glutamate receptors at the synaptic excitation in the CNS: AMPA receptors, NMDA receptors, and kainate re

19、ceptors, each named for a different chemical agonist. The neurotransmitter glutamate activates all the subtypes, but AMPA acts only at the AMPA receptor. Two subtypesthes of NE receptors, and , and of GABA receptors, GABAA and GABAB. Thus, selective drugs have been extremely useful for categorizing

20、receptor subclasses. In addition, neuropharmacological analysis has been invaluable for assessing the contributions of neurotransmitter systems to brain function.Studying Neurotransmitter SystemsThe neuropharmacology of cholinergic synaptic transmission. Sites on transmitter receptors can bind eithe

21、r the transmitter itself (ACh), an agonist that mimics the transmitter, or an antagonist that blocks the effects of the transmitter and agonists.Studying Neurotransmitter SystemsThe neuropharmacology of glutamatergic synaptic transmission Three subtypes of glutamate receptors, each of which binds gl

22、utamate, and each of which is activated selectively by a different agonist. Studying Neurotransmitter SystemsStudying Neurotransmitter SystemsLigand-Binding Methods.Selective drugs provide an opportunity to analyzereceptors directly, even before the neurotransmitteritself had been identified. Story

23、of discovery of Opiate receptorsSolomon Snyder (1938-) at Johns Hopkins UniversityOpiates effects on the brainHypothesis: opiates are agonists for specific receptors in CNS. Radioactively labeled opiate compounds labeled specific sites on some neurons in the brain.Led to the discovery of opiate rece

24、ptors, and identification of endogenous opiates, or endorphins (內(nèi)啡肽), e.g. enkephalin Opiate neurotransmitter systems eventually proved . Studying Neurotransmitter SystemsOpiate receptor binding to a slice of rat brain. Special film was exposed to a brain section that had radioactive opiate receptor

25、 ligands bound to it. The dark regions contain more receptors.Studying Neurotransmitter SystemsMolecular AnalysisEnable us to divide the neurotransmitter receptor proteins into two groups: transmitter-gated ion channels and G-protein-coupled (metabotropic) receptors The structure of receptor subunit

26、s by molecular analysis presented a broad extent of the diversity in subunit compositione.g. Each GABA receptor channel requires five subunits, from five major classes, , , , and , 1-6 isoforms, 1-4, 1-4. Theoretically, there are 151,887 possible combinations and arrangements of subunits. What this

27、means? Studying Neurotransmitter Systemsp Evolution is conservative and opportunistic, and it often puts common and familiar things to new uses.p Amino acids are essential to life. Most of the known neurotransmitter molecules are either (1) amino acids, (2) amines derived from amino acids, or (3) pe

28、ptides constructed from amino acids. p ACh is an exception; but it is derived from acetyl CoA, a ubiquitous product of cellular respiration in mitochondria, and choline, important for fat metabolism.p Amino acid and amine transmitters are generally each stored in and released by separate sets of neu

29、rons, so-called Dales principle. However, many peptide-containing neurons violate Dales principle. Co-transmitters: peptide + amino acid or peptide + amineNeurotransmitter ChemistryCholinergic Neurons Acetylcholine (ACh) p The neurotransmitter at NMJ, synthesized by all the motor neurons in the spin

30、al cord and brain stem. Other cholinergic cells contribute to the functions of specific circuits in the PNS and CNS.p ACh synthesis needs an enzyme, choline acetyltransferase (ChAT). Only cholinergic neurons contain ChAT, so this enzyme is a good marker, e.g. antibody-ICC. ChAT transfers an acetyl g

31、roup from acetyl CoA to choline .p Transport of choline into the neuron is the rate-limiting step in ACh synthesis. p Acetylcholinesterase (AChE) secreted from Cholinergic and noncholinergic neurons to degrades ACh. Inhibition of AChE disrupts transmission at cholinergic synapses on skeletal muscle

32、and heart muscle. Neurotransmitter ChemistryNeurotransmitter ChemistryThe life cycle of AChlow micromolarconcentrationsrate-limiting step with the fastest catalytic rate. the target of nerve gases and insecticidesNeurotransmitter ChemistryAcetylcholine. (a) ACh synthesis. (b) ACh degradation.Neurotr

33、ansmitter ChemistryCatecholaminergic NeuronspThe amino acid tyrosine is the precursor for three different amine neurotransmitters that contain a chemical structure catechol, collectively called catecholamines (兒茶酚胺).pInclude dopamine (DA), norepinephrine (NE), and epinephrine. Catecholaminergic neur

34、ons are found in regions of the nervous system for regulation of movement, mood, attention, and visceral function.CatecholgroupDopamine norepinephrine epinephrine (nonadrenaline) adrenaline Neurotransmitter Chemistryp All such neurons contain tyrosine hydroxylase (TH), which catalyzes the first step

35、 in catecholamine synthesis, the conversion of tyrosine to a compound called dopa (L-dihydroxyphenylalanine 二羥苯基丙氨酸).p The activity of TH is rate limiting for catecholamine synthesis, regulated by various signals in the cytosol of the axon terminal (end-product inhibition, increase when Ca2+i elevat

36、ed by a high rate release).p Dopa is converted into the neurotransmitter dopamine by the enzyme dopa decarboxylase. Parkinsons disease and dopa supplement therapy.Neurotransmitter Chemistry酪氨酸二羥苯基丙氨酸（多巴）多巴胺去甲腎上腺素腎上腺素酪氨酸羥化酶多巴脫羧基酶多巴胺羥化酶苯基乙醇胺-N-甲基轉(zhuǎn)移酶rate limiting enzyme located within the synaptic vesi

37、cles located in the cytosolNeurotransmitter Chemistryp Neurons that use NE as a neurotransmitter contain, in addition to TH and dopa decarboxylase, the enzyme dopamine -hydroxylase (DBH), which converts dopamine to norepinephrine. DA is transported from the cytosol to the synaptic vesicles, and ther

38、e it is made into NE.p The last in the line of catecholamine neurotransmitters is epinephrine (adrenaline). Adrenergic neurons contain the enzyme phentolamine Nmethyltransferase (PNMT), which converts NE to epinephrine.p In addition to serving as a neurotransmitter in the brain, epinephrine is relea

39、sed by the adrenal gland into the bloodstream. Circulating epinephrine acts at receptors throughout the body to coordinate visceral response.Neurotransmitter Chemistryp The actions of catecholamines in the synaptic cleft are terminated by selective uptake of the neurotransmitters back into the axon

40、terminal via Na+-dependent transporters. p This step is sensitive to a number of different drugs. For example, amphetamine and cocaine block catecholamine uptake. p Once inside, the axon terminal, the catecholamines may be reloaded into synaptic vesicles for reuse, or they may be enzymatically destr

41、oyed by the action of monoamine oxidase (MAO), an enzyme found on the outer membrane of mitochondria.Neurotransmitter ChemistrySerotonergic Neuronsp The amine neurotransmitter serotonin, also called 5-hydroxytryptamine and abbreviated 5-HT, is derived from the amino acid tryptophan. Serotonergic neu

42、rons are relatively few in number, but they appear to play an important role in the brain systems that regulate mood, emotional behavior, and sleep. p Serotonin synthesis appears to be limited by the availability of tryptophan in the extracellular fluid bathing neurons. The source of brain tryptopha

43、n is the blood, and the source of blood tryptophan is the diet. p 5-HT is removed from the synaptic cleft by the action of a specific transporter, which is sensitive to a number of different drugs. e.g. antidepressant drugs like fluoxetine. Once back in the cytosol, 5-HT is either reloaded to SVs or

44、 degraded by MAO.Neurotransmitter ChemistryThe synthesis of serotonin from tryptophan.色氨酸5-羥色氨酸5-羥色胺色氨酸羥化酶5-羥基色氨酸脫羧酶Neurotransmitter ChemistryAmino Acidergic Neurons glutamate (Glu) Glycine (Gly) GABAp Amino acid neurotransmitters Glu, Gly, and GABA serve as neurotransmitters at most CNS synapsesp G

45、lutamate and glycine are synthesized from glucose and other precursors by enzymes existing in all cells. Differences among neurons are quantitative rather than qualitative. p e.g. the glutamatergic terminals have about 20 mM Glu, only 2-3 times higher than nonglutamatergic cells. Importantly, in glu

46、tamatergic terminalss, but not in others, the glutamate transporter concentrates Glu in SVs to reach about 50 mM.Neurotransmitter Chemistryp GABA is not one of the 20 amino acids used to construct proteins, it is synthesized in large quantities only by the neurons that use it as a neurotransmitter.

47、p The precursor for GABA is glutamate. The key synthesizing enzyme is glutamic acid decarboxylase (GAD), a good marker for GABAergic neurons. One chemical step to convert the major excitatory into the major inhibitory neurotransmitter in the brain!p The synaptic actions of GABA are terminated by sel

48、ective uptake into the terminals and glia via specific Na+-dependent transporters. Inside the cytosol, GABA is metabolized by the enzyme GABA transaminase. GADOther Neurotransmitter Candidates and Intercellular Messengersp ATP is concentrated in vesicles at many synapses in the CNS and PNS, released

49、 into the cleft by presynaptic spikes in a Ca2+-dependent manner. p ATP is often packaged in vesicles along with another classic transmitter (e.g. catecholamine) which means they are probably co-transmitters.p ATP directly excites some neurons by gating a cation channel. ATP binds to purinergic rece

50、ptors, both transmitter-gated ion channels and a large class of G-protein coupled purinergic receptors.Neurotransmitter Chemistryp The interesting discovery in the past few years is that small lipid molecules, endocannabinoids (大麻酚,endogenous cannabinoids), can be released from postsynaptic neurons

51、and act on presynaptic terminals, called retrograde signaling; thus, endocannabinoids are retrograde messengers, a kind of feedback regulation.p Vigorous firing in the postsynaptic neuron Ca2+ influx throughvoltage-gated calcium channels of postsynaptic neurons Ca2+ i increase stimulates the synthes

52、is of endocannabinoid molecules from membrane lipids. Neurotransmitter ChemistryRetrograde signalingwith endocannabinoids p There are several unusual qualities about endocannabinoids: 1. They are not packaged in vesicles like other neurotransmitters; instead, they are produced rapidly and on-demand.

53、 2. They are small and membrane permeable; once synthesized, they can diffuse rapidly across the membrane to contact neighboring cells.3. They bind selectively to the CB1 type of cannabinoid receptor, mainly located on certain presynaptic terminals.Neurotransmitter Chemistry CannabisMarijuana大麻THC (

54、9-tetrahydrocannabinol)Short-term effects on brain?Neurotransmitter Chemistryp CB1 receptors are GPCRs, and their main effect is often to reduce the opening of presynaptic calcium channels, and inhibit release of its neurotransmitter.p Gaseous molecule, nitric oxide (NO). Carbon monoxide (CO) has al

55、so been suggested as a messenger, being extensively studied and hotly debated. p Many of the chemicals we call neurotransmitters may also be present and function in non-neural parts of the body. (Amino acids, ATP, Nitric oxide, Ach, serotonin)Transmitter-gated Channelsp The transmitter-gated ion cha

56、nnels are magnificent tiny machines. p A single channel can be a sensitive detector of chemicals and voltage, it can regulate the flow of surprisingly large currents with great precision, it can sift and select between very similar ions, and it can be regulated by other receptor systems. p Yet each

57、channel is only about 11 nm long, just barely visible with the best computer-enhanced electron microscopic methods. Transmitter-gated ChannelsThe Basic Structure of Transmitter-Gated Channels p The most thoroughly studied transmitter-gated ion channel is the nicotinic ACh receptor at NMJ. Five subun

58、its arrange like a barrel to form a single pore. Four different subunits , , , are used. There is one ACh binding site on each of the subunits. p The nicotinic ACh receptor on neurons is also a pentamer, but, unlike the muscle receptor, most of them are comprised of and subunits only.p The subunits

59、of GABA- and Glycine-gated channels have a similar primary structure to the nicotinic ACh receptor, with four hydrophobic segments to span the membrane, also thought to be pentameric complexes.Transmitter-gated ChannelsThe subunit arrangement of the nicotinic ACh receptor(a) Side view showing how th

60、e four -helices of each subunit packed together.(b) Top view showing the location of the two ACh binding sites.Transmitter-gated ChannelsSimilarities in subunit structure for different transmitter-gated ion channels(a) They have in common the four regions called M1M4, which are segments where the po