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D1 5 6. sinapsa, hemijska neurotransmisija 2h

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  • 1. SINAPSE
  • 2.
    • Drecun – Osnovi fiziologije
  • 3.
    • Gradja hemijske sinapse
    • Procesi u presinaptickom kompleksu
    • Procesi u sinaptickoj pukotini
    • Procesi u postsinaptickom kompleksu
    • Generisanje akcionog potencijala u postsinaptickom neuronu
    • Presinapticka inhibicija
    • Facilitacija neurona
    • Neurotransmiteri
    • Neuropeptidi
    • Aksoplazmatski transport
    • Jonotropni I metabolotropni efekti sinapticke transmisije
    • Serotonin
    • Dopamin
    • Noradrenalin
    • Acetilholin
    • Gaba
    • Glicin
    • Ekscicitotoksicnost neurona
    • Kola reverberacije
    • Kola divergencije
    • Kola konvergencije
    • Dugotrajna potencijacija i depresija sinaticke transmisije
    • Retrogradna sinapticka transmisija
    • Neurotroficni faktori
  • 4. NERVNO-MIŠIĆNA SINAPSA Sinapsa je mesto funkcionalnog kontakta i komunikacije između bilo koje dve nadražljive ćelije Nervno-mišićna sinapsa ili motorna sinapsa - sinapsa između vlakna motorne nervne ćelije i mišićne ćelije hemijske sinapse -poruke se prenose posredstvom hemijskih agenasa - neurotransmitera
  • 5.  
  • 6. Električne sinapse -AP se prenosi direktno sa jedne ćelije na drugu Nervni-mišićna sinapsa je sinapsa koja se formira između završnog dugmića motorne nervne ćelije i mišićne ćelije Motorna nervna ćelija je presinaptička ćelija , a mišićna ćelija je postsinaptička ćelija
  • 7. Membrane presinaptičke i postsinaptičke ćelije nisu u direktnom kontaktu-razdvojene su sinaptičkom pukotinom ( vanćelijskim prostorom od nekoliko desetina nm) U završnim dugmićima MNĆ nalazi se veliki broj sinaptičkih vezikula od kojih svaka sadrži nekoliko hiljada molekula neurotransmitera-actilholina (Ach) -hemijskog glasnika, prenosioca poruke
  • 8. Postsinaptička membrana sadrži specifične proteine receptore za Ach - jonski kanali prohodni pretežno za jone Na koji ulaze u ćeliju Kada AP stigne do završnih dugmića motorne nervne ćelije promena potencijala presinaptičke membrane uslovljava prolazno povećanje unutarćelijske koncentracije jona Ca 2+
  • 9. Povećanje koncentracije Ca 2+ u citoplazmi završnog dugmića izaziva pomeranje sinaptičkih vezikula prema presinaptičkoj membrani , njihovo spajanje sa njom i oslobađanje Ach iz vezikula u sinaptičku pukotinu procesom egzocitoze Ach putuje kroz sinaptičku pukotinu i vezuje se za receptore na postsinaptičkoj membrani što dovodi do ulaska jona Na i depolarizaciju membrane mišićne ćelije - potencijal motorne ploče
  • 10. Potencijal motorne ploče izaziva otvarnje voltažno-zavisnih kanala za Na i nastaje AP
  • 11. CENTRALANA SINAPSA
  • 12. Centralne sinapse - sinapse koje se uspostavljaju između NĆ u CNS-u Većina NĆ gradi sinapse sa ogromnim brojem, najčešće nekoliko stotina, drugih NĆ Prenos signala sa jedne NĆ na drugu u CNS-u je posredovan različitim transmiterima od kojih svaki prepoznaje svoj specifični receptor Efekat delovanja neurotransmitera u centralnoj sinapsi zavisi od same prirode neurotransmitera , kao i od receptora za koji se vezuje
  • 13. Sinaptičke veze između NĆ su hemijske sinapse i najčešće se uspostavljaju između nervnog vlakna presinaptičke NĆ i dendrita postsinaptičke NĆ- AKSODENDRITSKE SINAPSE Presinaptička membrana – membrana terminalnih dugmića i postsinaptička membrana - dendritska membrana su razdvojene sinaptičkom pukotinom Na postsinaptičkoj membrani su receptori za neurotransmiter
  • 14.  
  • 15.  
  • 16. Različite NĆ sadrže različite neurotransmitere Neurotransmiteri se prema hemijskoj strukturi mogu svstati u četiri kategorije: amino-kiseline , amini , peptidi i purinski nukleotidi Vezivanje neurotransmitera za receptor uslovljava promenu oblika receptorskog proteina i on postaje funkcionalan
  • 17. Efekat neurotransmitera je ekscitatoran kada nakon vezivanja neurotransmitera dođe do promene oblika receptorskog proteina , što dovodi do otvaranja jonskih kanala koji su pod kontrolom ovog receptora i propuštanja jona Na pri čemu dolazi do depolarizacije postsinaptičke membrane- ekscitatorni postsianptički potencijal (EPSP)
  • 18. Efekat neurotransmitera je inhibitoran kada bezivanje neurotransmitera za receptor na postsinaptičkoj memebrani uslovljava otvaranje jona za K ili Cl , jer tada dolazi do hiperpolarizacije postsinaptičke membrane - inhibitorni postsinaptički potencijal (IPSP)
  • 19. Pojedinačni EPSP nema dovoljnu amplitudu da izazove AP, ali kako NĆ uspostavljaju sinapse sa velikim brojem drugih NĆ istovremena aktivnost većeg broja presinaptičkih NĆ može dovesti do sabiranja sinaptičkih potencijala i nastanka AP Ovaj tip sabirajućeg uticaja većeg broja presinaptičkih neurona naziva se PROSTORNO SABIRANJE Ukoliko se višestruka sinaptička transmisija dešava sa jedne na drugu NĆ na drugu NĆ takođe dolazi do sabiranja – VREMENSKOG SABIRANJA  
  • 20. SYNAPTIC TRANSMISSION I Tim Murphy NRSC 500, 2009   The definition of synaptic transmission is simply the communication between two nerve cells. Communication believed to involve specialized structures termed "synapses".   We will focus on: 1) The discovery of synaptic transmission 2) The form of transmission, i.e. chemical or electrical 3) Criteria for a chemical transmitter 4) Ionic requirement for release 5) Quantal aspects of release: vesicle theory
  • 21. Discovery of synaptic transmission
    • Cajal's golgi staining methods suggested the presence of contacts between cells that were used for communication
    • Sherrington proposed the term "synapse" meaning to clasp to describe the structure.
  • 22. Shepherd 1997 TINS Cajal’s drawings Sherrington’s insights 1890s. of golgi stain.
  • 23. Modern golgi staining, YFP mouse cortical fluorescence, can be bred to other KO’s, transgenics.
  • 24. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm
  • 25. Otto Loewi, chemical transmitter.
    • 1936 Nobel prize for Medicine
    • Showed that vagus nerve stimulation liberates a diffusible transmitter.
    • Perfusate from one stimulated Frog Heart could be transferred to another and have an effect on beat frequency.
  • 26. Debate on synaptic transmission - chemical or electrical.
    • Otto Loewi showed that acetylcholine could mimic the effect of vagal nerve stimulation.
    • What additional experiment would have been nice to prove that the vagal nerve released acetylcholine.
    • The results of Loewi's experiments sparked debate about whether chemical and electrical transmission was occurring.
    • Sub sequently shown that both chemical and electrical transmission exist.
  • 27. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm
  • 28. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm Chemical Electrical
  • 29. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm
  • 30. Electrical Synapses
    • Gap junction-type communication important for rapidly synchronizing syncytia of cells as is observed in astrocytes, heart, and developing brains. Present in invertebrates to promote rapid defensive secretions.
    • Problems with electrical: difficult to modulate gating of channels (exceptions exist cAMP, pH).
    • Can't change sign, i.e. charge always flows "down hill."
    • Electrical synaptic transmission requires that the presynaptic cell or terminal be larger than the postsynaptic cell for it to inject considerable charge, no real amplification mechanism.
  • 31. Electrical synaptic transmission . Fundamental Neuro.
  • 32.  
  • 33. Saint-Amant and Drapeau Neuron 2001 Chemical transmission inhibitors do not block transmission in developing Zebrafish.
  • 34. Saint-Amant and Drapeau Neuron 2001 Gap junction inhibitors block transmission in developing Zebrafish.
  • 35. Chemical transmission.
    • Contrary to electrical transmission multiple steps are required to release transmitter chemicals and for them to act on postsynaptic receptors, resulting in a time delay (can be as short as 0.1 msec).
    • Directional, select localization of release machinery to presynaptic terminals and receptors to postsynaptic specializations.
    • Can change sign by release of inhibitory transmitter.
    • Highly modulatable as it has many steps presynaptic terminal and at the postsynaptic sites.
  • 36. Chemical Synaptic Transmission.
    • Definition: Communication between cells which involves the rapid release and diffusion of a substance to another cell where it binds to a receptor (at a localized site) resulting in a change in the postsynaptic cells properties.
  • 37. A hall mark of chemical transmission is a delay between presynaptic Ca2+ elevation and secretion. The delay can be as short as 0.1 ms, but is usually longer due to a variety of factors. Fundamental Neuro. 2002
  • 38. Steps to chemical synaptic transmission.
    • First need to bring the presynaptic neuron to threshold at axon hillock.
    • Conduction down axon, length, R*C dependent.
    • Opening of voltage gated Ca channels.
    • Diffusion and action of Ca at release machinery.
    • Exocytosis and diffusion of transmitter in cleft.
    • Activation of postsynaptic receptors.
  • 39. Synaptic delays can be less than 0.2 ms from calcium entry (Fund. Neurosci. Chap 8) to the beginning of secretion, but are typically longer when all steps are considered. From Sudhof 2004
  • 40. Chemical synapse types.
    • Axosomatic, axoaxonic, axodendritic, and dendrodendritic.
    • Excitatory (type I) and inhibitory (type II) synapses have different structure in CNS neurons.
    • CNS synapses usually release one or small number of release sites while nerve muscle synapses have up to 300 active zones.
    See http://synapses.mcg.edu/atlas
  • 41. See http://synapses.mcg.edu/atlas Josef Spacek Type I Excitatory Type II Inhibitory
  • 42. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm
  • 43. Synapse structure like real estate location, location, location!! From Squire et al. Fund. Neurosci 2 nd ed. Multiple release sites NMJ
  • 44. Criteria for a chemical transmitter, make a case for glutamate.
    • The transmitter substance must be synthesized in the presynaptic neuron. Experiment?
    • It must be present in the presynaptic terminal and released in amounts sufficient to result in the level of response produced by the endogenous transmitter. experiment?
    • When applied exogenously the substance should mimic the effect of the endogenous transmitter. experiment?
    • A specific mechanism must exist for removing the transmitter from the synaptic cleft. experiment?
  • 45. Ionic requirement for release.
    • Calcium influx is the trigger for fast evoked transmitter release
    • An elevation in intracellular calcium concentration is an absolute requirement for transmitter release. Na+ and K+ ions not necessary for release.
    • How do you test this hypothesis?
  • 46. Ion substitution and pharmacology experiments.
    • The influx of calcium is triggered by voltage gated ion channels.
    • Depolarization itself is not needed to stimulate release, as calcium can be elevated by other means (caged calcium chelators).
    • The relationship between calcium influx and release is related to the " power (exponent)" of calcium entry and is highly nonlinear. For example for a 4th power relationship a doubling of calcium entry can produce a 16 fold increase in release.
  • 47. Slope on a log-log plot indicates power relation, a small change in calcium produces a large change in release. Slope=5.0 Slope=1.6 Release~[Ca]^3-5 from Delaney Enc. of Sci.
  • 48. G. Augustine Curr. Op. in Neurobiol. Neurotransmitter release is triggered by a locally-activated low affinity sensor since bulk cytosolic [Ca] rarely exceeds 10 -6 M, yet transmitter release requires much higher [Ca].
  • 49. Squid giant axon and release
    • Due to its large size the squid giant axon has been used to examine the calcium dependence of transmitter release.
    • Squid studies using different Ca2+ buffers indicate high concentrations of Ca2+ that are reached for less than 1 ms trigger release.
    • Most calcium entry which triggers release occurs during the falling phase of action potential. Why is this advantageous?
  • 50. Rate of calcium binding by buffer (chelator) provides insight into release machinery.
    • Fast BAPTA (k on 8x10 8 M -1 *sec -1 ) buffers block release whereas,
    • slow EGTA (k on 1x10 7 M -1 *sec -1 ) buffers do not (Adler et al. 1991 J. Neurosci.).
    • Time for equilibration of EGTA with calcium~1000  s versus 12.5  s for BAPTA.
    • To estimate the buffer equilibration time take 1/(k off +k on* [Ca]), use k off of 8x10 1 sec –1 for both buffers and 1x10 -4 for [Ca] at peak.
  • 51. Temporal requirements.
    • Calcium trigger must be able to act within 0.1 ms of presynaptic stimulation. This requirement restricts the class of chemical events that may be involved in evoked release.
    • Phosphorylation, protein synthesis, gene expression all out.
    • Everything must be ready and localized- diffusion could not move transmitter vesicles or calcium very far.
  • 52. Reviewed in EF Stanley TINS 1997 Calcium channels are clustered on the release face of the chick caylx synapse. Outside of synapse Release face of synapse Patch config.
  • 53. Diffusion time of Ca 2+ limits release latency.
    • Buffered calcium diffusion coeff. are on the order of 200  m 2 /sec (D) so calcium could only diffuse a small distance at the most (~0.2  m) during the synaptic delay (0.0001 s, 0.1 ms), so Ca 2+ channels need to be very close to the release machinery.
    • distance=Sqrt(2Dt)
    • D=diffusion coeff., t=time
  • 54. The diffusion time is dependent on the square of the distance (d).
    • t=d 2 /(2*D), where t=time, d=distance, and D=diffusion coefficient.
    • For 0.1  m the time is 0.025 ms.
    • However for 1.0  m the time jumps by the square to 2.5 ms (100 times longer!), way longer than the release
    • latency.
    • These equations are for reference.
  • 55. Calcium as a local messenger.
    • Fast channel activation, diffuses to couple excitation to synaptic chemistry.
    • Low affinity, rapid off rate, and restricted microdomains are characteristics of the calcium flux that evokes release.
    • Rapid off rate allows for re-loading of release mechanism. Affinity=k on /k off
    • high off rate = low affinity.
  • 56. Kd or Km, concentration at ½ max binding or activity. Kd=Km=kdissoc/kassoc affinity=1/km, low affinity means a big km which usually means a large kdissoc Note koff=kdissoc and kon=kassoc
  • 57. Low affinity binding gives rapid off rate.
    • Concentration for ½ max activity is ~1/affinity and is termed the Kd or Km, if a binding site has a low affinity more ligand is needed to get ½ saturation.
    • Therefore Kd=k off /k on , assuming a diffusion limited k on of 5x10 8 M –1 s –1 then k off must be 5x10 4 s -1 if the Kd is 100  M.
    • To estimate the dissociation time constant take
    • 1/(k off + k on* [Ca]) or 20  s.
    • Note after the channels close [Ca] is ~ 1x10 -7 and the k on *[Ca] is small compared to k off .
    • These equations are for reference.
  • 58. For a simple reaction. [ES] [E]+[S] k on *[S] k off The time constant will be :   k off + k on *[S]) 20  s=1/( 5x10 4 s -1 + 5x10 8 M –1 s –1 *1x10 -7 M) For reference
  • 59. Local domains of Ca 2+ near channel mouths control transmitter release. EF Stanley TINS 1997 It is unclear whether release is always triggered by a single channel or whether multiple ones cooperate.
  • 60. Transmitter release microdomains. 1.8 mM Activation of Ca channels Resting state just after channel closure, bulk Ca is up only 10%.
  • 61. Atwood & Shanker Karunanithi DIVERSIFICATION OF SYNAPTIC STRENGTH: PRESYNAPTIC ELEMENTS Nature Reviews Neuroscience 3, 497 -516 (2002). To make full use of microdomains the vesicle must be bound to the calcium channels.
  • 62. Atwood & Shanker Karunanithi DIVERSIFICATION OF SYNAPTIC STRENGTH: PRESYNAPTIC ELEMENTS Nature Reviews Neuroscience 3, 497 -516 (2002). B BAPTA, a faster buffer than EGTA more readily blocks synaptic transmission. Given 100  M Ca 2+ , BAPTA equilibrates in <20  s while EGTA takes ~1000  s.
  • 63. Calcium as a local messenger.
    • Fast channel activation, diffuses to couple excitation to synaptic chemistry.
    • Low affinity, rapid off rate, and restricted microdomains are characteristics of the calcium flux that evokes release.
    • Rapid off rate allows for re-loading of release mechanism. Affinity=k on /k off , high off rate = low affinity.
  • 64.  
  • 65. Quantal aspects of release at the neuromuscular junction.
    • At the neuromuscular junction small spontaneous potentials (depolarizations) termed miniature end plate potentials are observed.
    • The amplitude of evoked responses (due to calcium influx) is always an integer multiple of the unitary response.
    • Shown that calcium increases the probability of observing a unitary response and not its amplitude.
    • These data suggested the existence of transmitter quanta or packets.
    • Evoked transmission mediated by the release of ~ 150 quanta over a 1-3 ms period. Each quantum leads to about 0.5 mV depolarization.
  • 66. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm
  • 67. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm
  • 68. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm
  • 69. 1X 4X 2X Stimulation 1 mV 1X 2X 3X 4X mini Mini histogram. Evoked amplitudes. Squire Fund. Neurosci. 2002
  • 70.  
  • 71. CNS synapses and quanta.
    • At synapses with only a single release site, changing the probability of release (changing calcium concentration) does not effect the amplitude of the response (as only zero or one vesicle is released).
    • At synapses with multiple release sites, changing probability can change the response amplitude as more transmitter is released.
    • At the NMJ a single nerve can elicit a postsynaptic AP given multiquantal release, while at the CNS multiple synapses must cooperate, forces a network.
  • 72. From Kristin Harris Lectures. http://synapses.mcg.edu/lab/harris/lectures.htm Define the number of readily releasable vesicles a synapse has available. A consequence of having of limited number is depletion at high stimulus frequency.
  • 73. Many vesicles In the RRP. Few vesicles in the RRP, undergoes depression. Fundamental Neuro. 2002 Remember depression over short timescales can be caused by other mechanisms including desensitization and autoreceptors.
  • 74. Residual Ca can facilitate transmission if not all quanta are released on the first stimulus. If transmission is robust on the first stimulus most readily releasable vesicles will be gone and depression results. Squire Fundamental Neurosci. 2002 Short term plasticity, history dependent changes in responsiveness. Stim.
  • 75. Time Voltage Response types at single CNS synapses with different #s of release sites. failure 1 vesicle 2 ves. 1 ves. fail
  • 76. Response of CNS synapse can reflect sum behavior of individual synapses that may act in an all or none manner. electrode
  • 77. When multiple synapses (or release sites) are involved facilitation can reflect an increase in release probability (all or none secretion) at single synapses. Trial 1 Trial 2, 25 ms later electrode
  • 78. Readings
    • Neuroscience 4 th Ed. Purves Chapters 4-6 optional
    • Fundamental Neuroscience 1 st Ed. Chapters 7 and 8 (for Neurochem also), 2 nd Ed. Chapters 7 and 8, 3 rd Ed. Chapters 7,8.
    • Delaney, Kerry R (March  2000 ) Calcium and Neurotransmitter Release. In: Encyclopedia of Life Sciences , London: Nature Publishing Group, http://www.els.net/doi:10.1038/npg.els.0000027
    • Harold L. Atwood & Shanker Karunanithi Diversification of synaptic strength: presynaptic elements. Nature Reviews Neuroscience 3, 497 -516 (2002). Advanced review comprehensive.
    • For great EM pictures of synapse see Josef Spacek’s site http://synapses.mcg.edu/atlas