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Lecture 2

Lecture 2.docx

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Rutsuko Ito

Lecture 2  neurotransmission Synaptic mechanisms  presynaptic mechanisms o neurotransmitter release at CNS synapses o role of Ca2+  influx of Ca2+  release of internal Ca2+ stores  leads to release of neurotransmitter substance o binds to post-synaptic receptors o reuptake o degraded o float away from synaptic sites into other areas  'volume transmission'  postsynaptic mechanisms o excitatory neurotransmitters  glutamate and its receptors o inhibitory neurotransmitters  GABA and its receptors  glycine and its receptors Synaptic vesicle release  opening of voltage gated Ca2+ channels  neurotransmitter binding to postsynaptic receptors in less than 1 ms o action potential in nerve terminal opens Ca2+ channels o Ca2+ entry causes vesicle fusion and transmitter release o Receptor channels open o Na+ enters the postsynaptic cell and vesicles recycle Early studies on neurotransmitter release  Fatt and Katz (1952) o discovery of spontaneous miniature end plate potentials (MEPPs) of ~0.5 mV in amplitude at the neuromuscular junction of frog (during resting state) o presynaptic depolarisation increased the frequency of their occurrence, but not their amplitude   lead to the idea that 1 ACh molecule  1 ACh receptor  idea turned out to be wrong o BUT  amplitude of MEPPs could be manipulated by the administration of curare (nAChR blocker) or acetylcholinesterase (breaks down ACh)  inotophoretic application of ACh caused smooth, small depolarising responses  MEPP must reflect opening of many individual ACh receptors, rather than the one Quantal model of transmitter release  Delcastillo and Katz (1954) o under conditions of low extracellular Ca2+,  stimulation of the presynaptic motor neuron caused the magnitude of the end plate potential (EPP) to fluctuate in a stepwise manner  evoked EPP amplitudes were: o of the size of a MEPP o sizes at integral multiples of minis o quantal hypothesis of neurotransmitter release  neurotransmitters are released from the presynaptic terminal in discrete units / quanta  MEPP is response to a spontaneous release of a single quantum, and is thus a building block of EPP  EPP is a response to some 100s of these quanta  Ca2+ levels controls the probability of a given quantum being released o MEPP = quantum / EPP = quanta  EPP = multiples of MEPP Vesicle = quantum?  DeRoberts (1958) o first visualisation of synaptic vesicles as the anatomical equivalent of a quantum of neurotransmitter using electron microscopy o Do the numbers add up?  at the neuromuscular junction (NMJ), a single ACh channel opening generate ~0.5 mV MEPP, 1000 channels must open  each ACh channel requires 2 ACh molecules  some molecules will be destroyed by esterase  some will be lost in the cleft before reaching target  estimate of 5000 ACh molecules is needed o Vesicle  50 nm in diameter  4000-10000 molecules if iso-osmotic with cell Evidence of exocytosis - electron micrographs  neurotransmitter release o fusion  all or nothing release, slow  synaptic vesicle becomes part of synaptic membrane o kiss-and-run  rapid, allowing for more sustained activity  temporary / transient fusion of synaptic vesicle o Review! are the neurotransmitter released at the presynaptic plasma membrane or postsynaptic plasma membranne? or at the synapse?  the neurotransmitter is released at the presynaptic plasma membrane into the synaptic cleft Evidence for exocytosis - membrane capacitance  patch clamp of mast cells / chromaffin cells that have large granules  based on principle that exocytosis involves incorporation of vesicle membranes into cell membranes, o leading to increased in total cell surface area  capacitance is inversely proportional to membrane area multiplied my C o Cmis dependent on thickness of membrane, so likely to stay constant  C = membrane capacity m  size of steps maps nicely to membrane area of secretory vesicles  Review! -- what is the moral of this story? Exocytosis - patch amperometry  direct measure of transmitter release through oxidation / reduction currents of neurotransmitter molecules at the carbon fibre electrode o 'the foot'  dribble of neurotransmitter release reflecting pore size fluctuations o fusion pore fully opening to release remaining contents of granule  may reflect kiss and run mechanism of neurotransmitter release  carbon fiber electrode o picks up and measures amount of neurotransmitter release  serotonin  oxidation product + 2e- Combined capacitance measure and patch amperometry  experimental evidence of both mechanisms of neurotransmitter release o full fusion model  constant o kiss and run model  flickers How does the vesicle fuse with the plasma membrane and how is this regulated by Ca2+? SNARE proteins  core complex required for vesicle fusion with plasma membrane o synaptobrevin  on vesicle membrane - vSNARE o syntaxin / SNAP-25  plasma membrane - tSNARE  forms a transient fusion complex SNARE cycle  synaptic vesicle approaching membrane o high energy state  synaptobrevin binding to SNAP-25 and syntaxin o loose trans-SNARE complex  pulls vesicle closer o tight trans-SNARE complex  fusion pore opening o change in conformation of complex from trans to cis o cis-SNARE complelx  return to high energy state Ca2+ dependence of vesicular fusion  Ca2+ entry is required for vesicle fusion and neurotransmitter release  change in intracellular Ca2+ concentration has to be sensed o what and where is the sensor?  Review! Ca2+ sensor  synaptotagmin o vesicular protein that has 2 Ca2+ binding sites and phospholipid binding properties  C2A  C2B Synaptotagmin as Ca2+ sensor  Geppert et al (1994) o knockout mice with a functional disruption of the synaptotagmin 1 gene were generated  wildtype vs syt mutant  loss of synchronous Ca2+ o triggered neurotransmitter release  Review! Which model?  Probably both! o two pools of vesicles have been identified in neurons  readily releasable pool of vesicles located at or close to the active zone of the presyaptic membrane  kiss and run vesicles  a reserve pool of vesicles located away from the active zone of the presynaptic membrane  full fusion vesicles o relative numbers of two types of vesicles could depend on type of neurons and strength of stimulation  Why should we care? o currently unclear if kiss-and-run releases all neurotransmitter from vesicle, which may have impact on our understanding of transmitter release in the brain  i.e. are there other methods of neurotransmitter release other than quantal release? Post-synaptic mechanisms  many of the neurotransmitter substances can cause more than one post synaptic response o dictated mostly by pharmacologically distinct post-synaptic receptors  same neurotransmitter can have different effects in different regions of the brain, or even in neighbouring cells within the same local circuit  while acting through its own distinct class of receptors, different neurotransmitters modify the same ionic currents  Review! Post synaptic excitation  excitation o increasing probability of AP discharge  usually achieved by opening non-selective cation channels  or closing of anion / K+ channels  different time scales  fast and slow EPSC (current) / EPSP (potential) o different functions?  degree of excitation depends on:  amount and frequency of neurotransmitter release  life-time of nerotransmitter in synaptic cleft  responsiveness, number and location of receptors  excitation state of cell Glutamate  Glutamate o main excitatory neurotransmitter in CNS  ion channel associated  ionotropic (iGluR) o fast excitatory  NMDA  AMPA  Kainate  G protein coupled  metabotropic (mGluR) o slow excitatory  group I o slow inhibitory  group II  group III  Acts via two types of receptors o ionotropic (fast) and metabotropic (slow) receptors Fast EPSP (2-10ms)  glutamate acting through activation of ionotropic GLU receptors o takes 2-10 ms to go to an action potential Ionotropic glutamate receptors (iGluRs)  directly gated ion channels  glutamate binding triggers opening of non-selective cation channel (Na+ / K+ / Ca2+)  functional heterotetramers (4, non-identical subunits) o heterotetramers  4 subunits  hetero = non identical o 3 major types  named after main pharmacological agonist / antagonist  AMPA R o agonist  AMPA  quisqualic acid o antagonist  CNQX  DNQX  NMDA R o agonist  NMDA  quinolinic acid  ibotenic acid o antagonist  D-AP5  MK801  ketamine  Kainate R o agonist  kainate  domoic acid o antagonist  CNQX  DNQX  Review! o Ionotropic vs Metabotropic receptors AMPA receptors  responsible for fast EPSP o GluR 1-4 (or A-D) subunits o 2 x dimers (e.g. 2 x Gl2R + 2 x G3uR ) o 1 Glu binding per subunit  maxwith 1 mM glutamate AMPA R - splice variants  AMPA receptor subunits exist as two splice variants located in S2 domain o different in only a few amino acids  flip form  predominates within early development  implication o sustained excitatory transmission  flop form  predominates in post natal / adult life AMPA R I-V relation  linear o roughly equal permeability to Na+ and K+ ions  cation channel mixture of K+ and Na+  ohm's law o Iion gionEm- Eion GluR2subunit  Lu et al (2009) neuron o dominant subunit  has the most impact on the biophysical properties of the resulting heteromeric complexes o any combination of gene deletion involving the Glu2 subunit led to a non-linear I-R relation at more positive potentials (inward rectification)  knockout mice o 81% of AMPA receptors in the hippocampus CA p1ramidal cell synapse is of the Glu1 + GluR 2e
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