8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 1 Development of Complex Curricula for Molecular Bionics and InfobionicsPrograms within a consortial* framework** Consortium leader PETER PAZMANYCATHOLIC UNIVERSITY Consortium members SEMMELWEISUNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realisedwith the support of the European Union and has been co-financed by the European Social Fund *** **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY sote_logo.jpg dk_fejlec.gif INFOBLOKK 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 Peter Pazmany Catholic University Faculty of Information Technology BASICS OF NEUROBIOLOGY RELEASE OF NEUROTRANSMITTERS www.itk.ppke.hu Neurobiológia alapjai (Neurotranszmitter felszabadulás) ZSOLT LIPOSITS Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 www.itk.ppke.hu QUANTAL RELEASE OF NEUROTRANSMITTERS THE EXPERIMENTS PERFORMED BY HEUSER (1977) AND HEUSER AND REESE(1981) ON FROG NEUROMUSCULAR JUNCTION SPONTANEOUS RELEASE OF TRANSMITTERS FROM SYNAPTIC VESICLES OCCURS IN PACKETS (QUANTA) IN THE ABSENCE OF ACTION POTENTIAL THE RELEASED QUANTA EVOKE MINIATURE POSTSYNAPTIC POTENTIALS: EXCITATORY (mEPSPs)AND INHIBITORY (mIPSPs) TYPES THE ACTION POTENTIAL INCREASES, ACCELERATES AND SYNCHRONIZES THERELEASE OF QUANTA IN ORDER TO EVOKE A POSTSYNAPTIC POTENTIAL THE SIZE OF THE QUANTUM IS INDEPENDENT OF THE ACTION POTENTIAL AND THE CYTOPLASMIC CONCENTRATION OF THE TRANSMITTER INTERFERING WITH FILLING OF SYNAPTIC VESICLES WITH TRANSMITTERSCAUSES A REDUCTION IN THE mEPSPs THE NUMBER OF TRANSMITTER MOLECULES IN A VESICLE EQUALS THAT OF THE RELEASED QUANTUM Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 www.itk.ppke.hu RECYCLING OF SYNAPTIC VESICLES DURING TRANSMITTER RELEASE, THE SYNAPTIC VESICLES FUSE WITH THE PLASMA MEMBRANE AND EMPTY THEIR CONTENT INTO THE SYNAPTIC CLEFT IN ORDER TO ENSURE THE PROPER RELEASABLE POOL OF VESICLES IN THE TERMINAL BOTH THE SYNAPTIC VESICLE MEMBRANE AND THE TRANSMITTER SUBSTANCE UNDERGO RECYCLING THE SYNAPTIC MEMBRANE IS RETRIEVED BY ENDOCYTOSIS. THE CLATHRIN-COATED RECOVERED MEMBRANE APPEARS IN ENDOSOMES THAT ARE USED FOR THE PRODUCTION OF NEW VESICLES THE PRESYNAPTIC MEMBRANE OF THE AXON TERMINAL CONTAINS TRANSMITTER TRANSPORTERS (FOR CHOLINE, DOPAMINE, NORADRENALINE, GABA, GLUTAMATE, SEROTONIN) THAT ALLOW THE UPTAKE OF THE TRANSMITTER OR ITS BREAKDOWN PRODUCT FROM THE SYNAPTIC CLEFT FOR RECYCLING DRUGS ACTING ON MEMBRANE TRANSPORTERS ARE POWERFUL MODULATORSOF SYNAPTIC FUNCTIONS AND PERFORMANCE OF NEURONAL NETWORKS Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 www.itk.ppke.hu EXCITATION-COUPLED SECRETION OF VESICLES ACTION POTENTIALS OPEN VOLTAGE GATED CALCIUM CHANNELS IN THE TERMINALS THAT ALLOW THE INFLUX OF CALCIUM THE CALCIUM CHANNELS ARE SITUATED IN THE MEMBRANE FACING THE ACTIVE ZONE OF THE SYNAPSE WHERE THE DOCKED AND PRIMED VESICLES ARE WAITING FOR RELEASE THE ELEVATION OF THE INTRACELLULAR CALCIUM CONCENTRATION ACCELERA-TES THE QUANTAL RELEASE DECREASING THE CALCIUM CONCENTRATION OF THE EXTRACELLULAR BATH RESULTS IN AN ATTENUATED RESPONSE HIGH CALCIUM CONCENTRATION AND FUNCTIONAL CALCIUM CHANNELS CAN EVOKE TRANSMITTER RELEASE EVEN UNDER BLOCKED SODIUM ACTION POTENTIAL AND POTASSIUM CHANNELS DIVALENT CATIONS THAT BLOCK CALCIUM CHANNELS ARREST THE VESICULAR RELEASE OF TRANSMITTERS Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 www.itk.ppke.hu THE SYNAPTIC VESICLE CLASSIC NEUROTRANSMITTERS ARE PACKED INTO SMALL SIZED (40-50nm), ELECTRON LUCENT VESICLES POSSESSING EITHER ROUND OR FLATTENED SHAPE THE BIOCHEMICAL PURIFICATION OF SYNAPTIC VESICLES REVELED IMPORTANT PROTEIN STRUCTURES BUILT IN THE SYNAPTIC MEMBRANE VESICULAR TRANSMITTER TRANSPORTERS. UPTAKE AND ACCUMULATION OF TRANSMITTERS PROTON PUMP.GENERATION OF ELECTROCHEMICAL GRADIENT SYNAPTOBREVIN. VESICULAR FUSION SYNAPTOTAGMIN.BINDING OF CALCIUM IONS SYNAPSIN. BINDING TO ACTIN RAB3. REGULATION OF VESICLE TARGETING CYSTEINE STRING PROTEIN. REGULATION OF Ca CHANNELS SYNAPTOPHYSIN.FUNCTION IS UNKNOWN SV2.FUNCTION IS UNKNOWN AXON TERMINALS (1,2) FILLED WITH SYNAP-TIC VESICLES. BOUTON 1 FORMS AN ASYM-METRIC SYNAPSE (ARROWHEADS) WITH A NEIGHBORING DENDRITE (D) Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 www.itk.ppke.hu UPTAKE OF TRANSMITTERS INTO SYNAPTIC VESICLES THE SYNAPTIC VESICLES CONTAIN PROTON PUMP, A MULTI-SUBUNIT ATPase WHICH CATALYZES THE TRANSLOCATION OF PROTONS FROM CYTOPLASM TO THE ORGANELLE THE ESTABLISHED ELECTROCHEMICAL GRADIENT IS USED FOR THE UPTAKE OF NEUROTRANSMITTERS INTO THE VESICLES THE PROCESS RESULTS IN AN EXTREME ACCUMULATION OF THE TRANSMITTER IN THE VESICLES IN COMPARISON WITH ITS ORIGINAL CYTOPLASMIC CONCENTRATION THE DIFFERENCE MIGHT BE 1000-FOLD GREATER THE NEUROTRANSMITTER TRANSPORTERS ARE PROTEINS WITH 12 MEMBRANESPANNING DOMAINS VESICULAR MEMBRANE TRANSPORTERS HAVE BEEN CLONED FOR GLUTAMATE,ACETYLCHOLINE, GABA, SEROTONIN, DOPAMINE, NOREPINEPHRINE AND GLYCINE Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 www.itk.ppke.hu THE PRESYNAPTIC ACTIVE ZONE CALCIUM RUSHED IN THE TERMINAL LIBERATES THE SYNAPTIC VESICLES FROM ACTIN FILAMENTS OF THE CYTOSKELETON VESICLES GET INSERTED IN THE PRE-SYNAPTIC GRID WHICH IS A HEXAGONAL ARRAY OF ELECTRON DENSE PARTICLES ATTACHED TO THE CYTOPLASMICFACE OF THE PRESYNAPTIC MEMBRANE. IT CAN BE REVEALED BY STAINING WITH ETHANO-LIC PHOSPHOTUNGSTIC ACID THE PRESYNAPTIC GRID (WEB) CONSISTS OF 50 nmPYRAMID-SHAPED PARTICLES BEING INTERCONNECTED BY 100 nm SPACED FIBRILS THE PROCESS OF INSERTION OF SYNAPTIC VESICLES INTO THE PRESYNAPTIC GRID AND ESTABLISHING CONTACT WITH THE PRESYNAPTIC MEMBRANE IS CALLEDDOCKING SCHEMATIC ILLUSTRATION OF THE PRESYNAPTIC WEB. SYNAPTIC VESICLES ARE ATTACHED TO THEACTIVE ZONE OF THE PRESYNAPTIC MEMBRANE. THE VESICLES FIT THE HOLES OF THE PRESYNAPTIC WEB (GRID) AND GET CLOSE TO THE FUSION SITE Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 www.itk.ppke.hu MEMBRANE FUSION THE SNARE PROTEIN SUPERFAMILY (SNAP-(SOLUBLE NSF ATTACHMENT PROTEIN) RECEPTORS) IS COMPOSED OF DOZENS OF PEPTIDES A PIVOTAL ROLE OF SNARE PROTEINS IS TO ASSIST THE DOCKING, FUSING AND EMPTYING OF SYNAPTIC VESICLES IN ADDITION TO THE SYNAPTIC MEMBRANE PROTEIN SYNAPTOBREVIN, PROTEINS SYNTAXIN AND SNAP 25 CONTRIBUTE TO THE FORMATION OF THE SNARE ORPORE COMPLEX. SYNTAXIN AND SNAP-25 ARE PRIMARILY ASSOCIATED WITH THE CELL MEMBRANE THE VESICULAR COMPONENTS OF THE COMPLEX ARE REFERRED TO AS V-SNARE, WHILE THE MEMBRANE-ASSOCIATED UNIT IS CALLED T-SNARE THE TIGHT SNARE COMPLEX BRINGS TOGETHER THE TWO MEMBRANES THE CALCIUM INFLUX TRIGGERS THE FORMATION OF AN INITIAL MEMBRANE PORE SIMILAR TO A GAP JUNCTION THAT GRADUALLY ENLARGES AND LEADS TO THE COLLAPSE OF THE EXOCYTOTIC VESICLE. SYNAPTOTAGMIN SERVES ASCALCIUM SENSOR IN THE PROCESS Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 www.itk.ppke.hu SCHEMATIC ILLUSTRATION OF DOCKING AND FUSION OF SYNAPTIC VESICLES NOTE THE INTERACTION OF VESICULAR AND TARGET MEMBRANE-ASSOCIATED PROTEINS (V-SNARE AND T-SNARE). THE PROTEIN COMPLEX ATTACHES THE VESICLE TO THE MEMBRANE. NSF AND SNAP PROTEINS ALSO CONTRIBUTE TO THE FORMATION OF THE FUSION COMPLEX. THE FUSION AND FISSION OF THE VESICLE IS TRIGGERED BY CALCIUM AND THE DISASSEMBLY OF THE FUSION COMPLEX BY ATP HYDROLYSIS (NSF). THE TRANSMITTER GETS RELEASED AND THE VESICULAR MEMBRANE RECYCLED. BOTULINUM AND TETANUS TOXINS CLEAVE THE SNARE PROTEINS AND PREVENT THE RELEASE OF THE TRANSMITTERS. BOTH INFECTIONS MAY BE LETHAL COURTESY OF LUNDBECK INSTITUTE, DENMARK Basics of Neurobiology: Release of neurotransmitters 8/5/2011. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 www.itk.ppke.hu USE OF VESICULAR MEMBRANE TRANSPORTERS IN RESEARCH VESICULAR MEMBRANE TRANSPORTERS ARE GENUINE PHENOTYPICAL MARKERSOF TRANSMITTERERGIC NEURONS THE IMMNUNOCYTOCHEMICAL DETECTION OF THE SPECIFIC VESICULAR TRANSPORTERS UNAMBIGUOUSLY IDENTIFIES THE TRANSMITTER CHARACTEROF NEURONS A B IN FIG. A. VESICULAR GLUTAMATE TRANSPORTER 2 (VGLUT2)-CONTAIN- ING AXONS (ARROWS) INNERVATE A TRH NEURON. IN FIG. B. COLLOID- AL GOLD PARTICLES LABEL VGLUT 2 IMMUNOREACTIVE SYNAPTIC VESI- CLES(SV) IN A GLUTAMATERGIC AXON TERMINAL (AT)