Chapter 12 and 13 notes.pdf

11 Pages
110 Views
Unlock Document

Department
Cell and Systems Biology
Course
CSB332H1
Professor
Francis Bambico
Semester
Winter

Description
Chapter  13  243-­‐248;  250-­‐252;  255-­‐256   Chapter  12  213-­‐217;  231-­‐234     Chapter  13  Release  of  Neurotransmitters   -­‐ Stimulus  to  release  NT  is  the  depolarization  of  nerve  terminal   -­‐ Release  occurs  as  result  of  calcium  into  terminal  through  voltage -­‐activated  calcium  channels   -­‐ Delay  about  0.5  ms  intervenes  between  presynaptic  depolarization  and  transmitter  release   –   due  to  time  taken  for  calcium  channels  to  open;  remainder  due  to  time  required  for  calcium   to  cause  transmitter  release   -­‐ Transmitter  secreted  in  multimolecul ar  packets  (quanta)  –  each  containing  several   thousand  transmitter  molecules   -­‐ In  response  to  AP,  1-­‐300  quanta  are  released  almost  synchronously  from  nerve  terminal   depending  on  type  of  synapse   -­‐ At  rest,  nerve  terminals  release  quanta  spontaneously  at  slow  ra te  giving  rise  to   spontaneous  miniature  synaptic  potentials   -­‐ Also,  there  is  continuous,  nonquantal  leak  of  transmitter  from  nerve  terminals   -­‐ One  quantum  of  transmitter  corresponds  to  contents  of  one  synaptic  vesicles  and  comprises   of  several  thousand  molecul es  of  low-­‐molecular-­‐weight  transmitter   -­‐ Release  occurs  by  process  of  exocytosis  which  synaptic  vesicles  membrane  fuses   w/presynaptic  membrane  and  contents  of  vesicles  are  released  into  synaptic  cleft   -­‐ Components  of  vesicles  are  retrieved  by  endocytosis,  sort ed  in  endosomes  and  recycled  into   new  synaptic  vesicles     Characteristics  of  Transmitter  Release   Axon  Terminal  Depolarization  and  Release   -­‐ Stellate  ganglion  of  squid  used  to  determine  precise  relation  btw  presynaptic  membrane   depolarization  and  amount  of  tra nsmitter  release   -­‐ Simultaneous  records  were  made  of  AP  in  presynaptic  terminal  and  response  of   postsynaptic  fiber   -­‐ When  TTX  was  applied  to  preparation,  the  presynaptic  AP  gradually  decreased  in  amplitude   over  next  15  mins   -­‐ The  postsynaptic  AP  also  decreased  i n  amplitude  but  then  abruptly  disappeared  because  of   excitatory  postsynaptic  potential  (EPSP)  failed  to  reach  threshold   -­‐ From  this  point,  size  of  synaptic  potential  could  be  used  as  a  measure  of  amount  of   transmitter  released   -­‐ When  amplitude  of  EPSP  is  plott ed  against  amplitude  of  failing  presynaptic  impulse,  the   synaptic  potential  decreases  rapidly  as  presynaptic  AP  falls  below  75mV  and  amplitude  less   than  about  45mV  –  there  are  no  postsynaptic  responses   -­‐ TTX  has  no  effect  on  sensitivity  of  postsynaptic  membr ane  to  transmitter  so  fall  in  synaptic   potential  amplitude  indicates  a  reduction  in  amount  of  transmitter  released  from   presynaptic  terminal   -­‐ Thus,  there  is  threshold  for  transmitter  release  at  about  45mV  depolarization  after  which   amount  released  and  hence  EPSP  amplitude  increases  rapidly  w/presynaptic  AP  amplitude   -­‐ Second  electrode  was  placed  in  presynaptic  terminal   –  brief  depolarization  current  pulses   applied  –  mimicking  presynaptic  AP   -­‐ Relationship  btw  amplitude  of  artificial  AP  and  that  of  synaptic  poten tial  was  same  as  the   relation  obtained  w/failing  AP  during  TTX  poisoning   -­‐ Results  indicates  that  normal  fluxes  of  sodium  and  potassium  ions  responsible  for  AP  are  not   necessary  for  transmitter  release;  only  depolarization  is  required     Synaptic  Delay   -­‐ One  characteristic  of  transmitter  release  is  there  is  lag  time  btw  onset  of  presynaptic  AP  and   beginning  of  synaptic  potential   -­‐ Lag  time  known  as  synaptic  delay   -­‐ In  these  experiments  on  squid  giant  synapse  (done  at  ~10C),  the  delay  was  ~3 -­‐4ms   -­‐ Detailed  measurements  at  frog  NMJ  show  synaptic  delay  of  0.5ms  at  room  temperature   -­‐ Time  too  long  to  be  accounted  for  by  diffusion  of  ACh  across  synaptic  cleft  (distance  50nm)   which  should  take  no  longer  than  ~50 µs   -­‐ When  ACh  applied  to  junction  ionophoretically  from   micropipette,  delays  of  as  little  as   150µs  can  be  achieved  even  though  pipette  is  much  farther  from  postsynaptic  receptors  than   are  nerve  terminals   -­‐ Synaptic  delay  much  more  sensitive  to  temperature  than  would  be  expected  if  it  were  due  to   diffusion   -­‐ Cooling  frog  nerve-­‐muscle  preparation  to  2.5C  increases  delay  to  as  long  as  7ms  whereas   delay  in  response  to  ionophoretically  applied  to  ACh  is  not  perceptibly  altered   -­‐ Thus,  delay  is  largely  in  transmitter  release  mechanism     Evidence  that  Calcium  is  Required  for   Release   -­‐ Calcium  essential  link  in  process  of  synaptic  transmission   -­‐ When  concentration  in  ECF  decreased,  release  of  ACh  at  NMJ  reduced  and  eventually   abolished   -­‐ Importance  of  calcium  for  release  has  been  generalized  further  to  other  secretory  processes   such  as  liberation  of  hormone  cells  of  pituitary  gland,  release  of  epinephrine  from  adrenal   medulla,  and  secretion  by  salivary  glands   -­‐ Evoke  transmitter  release  is  preceded  by  calcium  entry  into  terminal  and  is  antagonized  by   ions  that  block  calcium  entry  (ie  magnesium,  cadmium,  nickel,  manganese  and  cobalt)   -­‐ Transmitter  release  can  be  reduced  either  by  removing  calcium  from  bathing  solution  or  by   adding  a  block  ion   -­‐ For  transmitter  release  to  occur,  calcium  must  be  present  in  bathing  solution  at  time  of   depolarization  of  presynaptic  terminal     Measurement  of  Calcium  entry  into  presynaptic  nerve  terminals   -­‐ Entry  of  calcium  into  nerve  terminal  is  through  voltage -­‐sensitive  calcium  channels  of  Ca 2   v family  that  are  activated  upon  depolarization  by  presynaptic  AP   -­‐ Using  voltage  clamp  techniques,  Llinas  and  colleagues  measured  magnitude  and  time  course   of  calcium  current  produced  by  presynaptic  depolarization  at  squid  giant  synapse   -­‐ Sodium  and  potassium  conductances  associated  with  the  AP  were  blocked  by  TTX  and  TEA   so  that  only  voltage-­‐activated  calcium  channels  remained   -­‐ Depolarizing  the  presynaptic  terminal  to   -­‐18mV  produced  an  inward  calcium  current  in   terminal  that  increased  slowly  in  magnitude  to  about  400nA  and  large  synaptic  potential  in   postsynaptic  cell   -­‐ When  terminal  was  depolarized  to  +60mV,  approximating  calcium  equilibrium  potential,   calcium  current  was  suppressed  during  the  pulse  and  no  synaptic  potential  was  seen   –  this   demonstrates  that  depolarization  of  terminal  is  not  sufficient  on  its  own  to  trigger  release;   calcium  entry  must  occur   -­‐ On  repolarization,  there  was  brief  inward  calcium  current  through  channels  remaining  open   after  depolarization,  accompanied  by  small  postsynaptic  potential     Localization  of  Calcium  Entry  Sites   -­‐ Calcium  entering  terminal  through  singl e  channel  collects  briefly  in  small   nanodomain  with   concentration  falling  rapidly  over  a  few  tens  of  nm  from  channel  as  ion  diffuse  into  bulk   solution  or  are  bound  by  intrinsic  calcium  chelators   -­‐ Calcium  entering  through  a  group  of  closely  apposed  channels   occupies  a  microdomain  that   can  spread  over  a  distance  of  a  few  hundred  nm  from  the  channel  cluster     -­‐ Due  to  restricted  spread  of  incoming  ions,  the  spatial  relation  btw  calcium  channels  and   associated  transmitter  release  sites  is  of  critical  importance   -­‐ Experiments  on  squid  giant  synapse  using  calcium  buffers  have  provided  information  about   proximity  of  calcium  channels  to  sites  of  transmitter  secretion   -­‐ In  these  experiments,  injection  of  BAPTA  a  potent  calcium  buffer  into  presynaptic  terminal   resulted  in  severe  attenuation  of  transmitter  release  without  affecting  presynaptic  AP   -­‐ On  other  than,  EGTA  a  calcium  buffer  of  equal  potency,  had  little  effect  on  release   -­‐ Disparity  due  to  the  fact  that  calcium  is  bound  hundreds  of  times  faster  by  BAPTA  than  by   EGTA   -­‐ Thus  calcium  ions  have  little  opportunity  to  diffuse  from  sites  of  entry  before  being  bound  by   BAPTA  but  can  traverse  some  distance  before  being  captured  by  EGTA   -­‐ From  rates  of  calcium  diffusion  and  binding  to  EGTA,  it  can  be  calculated  that  calcium -­‐ binding  site  associated  with  release  process  must  lie  within  100nm  or  less  of  site  of  calcium   entry   -­‐ On  other  hand,  similar  experiments  at  some  neuronal  synapses  have  shown  effect  of  EGTA   on  release,  suggesting  that  in  these  cells  calcium  may  diffuse  some  distance  f rom  calcium   channels  to  sites  that  trigger  release  or  modulate  release     P250-­‐252   Quantal  Release   -­‐ General  scheme  for  transmitter  release  can  be  summarized  as:   Presynaptic  depolarization   à  calcium  entry  à  transmitter  release   -­‐ In  experiments  on  frog  NMJ,  ACh  can  be  released  from  terminals  in  multimolecular  packets   (quanta)   -­‐ Each  quantum  corresponds  to  approximately  7000  molecules  of  ACh   -­‐ Quantal  release  then  means  that  any  response  to  stimulation  will  consist  of  roughly  7000   molecules  or  14,000  but  not  4250  or  10,776   -­‐ At  any  given  synapse,  number  of  quanta  released  from  nerve  terminal  in  response  to  an  AP   (the  quantum  content  of  the  synaptic  potential  may  vary  considerably  from  trial  to  trial  but   the  mean  number  of  molecules  in  each  quantum  ( quantal  size)  is  fixed  (variance  ~10%)     Spontaneous  release  of  multimolecular  Quanta   -­‐ First  evidence  for  packaging  of  ACh  in  multimolecular  quanta  was  the  observation  by  Fatt   and  Katz  that  at  motor  end  plate,  but  not  elsewhere  in  muscle  fiber,  spontaneous   depolarizations  of  about  1mV  occurred  irregularly     -­‐ They  had  same  time  course  as  potential  evoked  by  nerve  stimulation   -­‐ The  spontaneous  miniature  end -­‐plate  potentials  (MEPPs)  were  decreased  in  amplitude  and   eventually  abolished  by  increasing  concentrations  of  ACh  receptor  antagonist  curare,  and   were  increased  in  amplitude  and  time  course  by  AChE  inhibitors  such  as  prostigmine   -­‐ These  two  pharmalogical  tests  indicated  that  potentials  were  produced  by  spontaneous   release  of  discrete  amounts  of  ACh  from  nerve  terminal   and  ruled  out  possibility  that  they   might  be  due  to  single  ACh  molecules   -­‐ Patch  electrode  recordings  demonstrated  directly  that  the  amount  of  current  that  flows   through  an  individual  ACh  receptor  will  produce  a  potential  change  in  muscle  fiber  of   approximately  1µV   -­‐ Thus,  spontaneous  miniature  potentials  are  indeed  due  to  multimolecular  packets  of  ACh   liberated  by  nerve  terminal   -­‐ For  example,  depolarization  of  nerve  terminal  by  passing  a  steady  current  through  it  causes   an  increase  in  frequency  of  spontaneous  a ctivity  whereas  muscle  depolarization  has  no   effect  on  frequency   -­‐ Botulinum  toxin  which  blocks  release  of  ACh  in  response  to  nerve  stimuli  also  abolishes   spontaneous  activity   -­‐ Shortly  after  denervation  of  a  muscle,  as  the  motor  nerve  terminal  degenerates,  th e   miniature  potentials  disappear   -­‐ Surprisingly,  after  an  interim  period,  spontaneous  potentials  reappear  in  denervated  frog   muscles;  these  arise  because  of  ACh  released  from  Schwann  cells  that  have  engulfed   segments  of  degenerating  nerve  terminals  by  phagoc ytosis     Fluctuation  in  the  End-­‐Plate  potential   -­‐ typical  synaptic  potential  at  skeletal  NMJ  depolarizes  postsynaptic  membrane  by  50mV  to   70mV  many  times  greater  than  depolarization  produced  by  single  quantum   -­‐ In  order  to  find  out  how  response  to  stimulation  w as  related  to  spontaneously  released   quanta,  Fatt  and  Katz  reduced  amplitude  of  evoked  synaptic  potential  by  lowering   extracellular  calcium  and  adding  extracellular  magnesium   -­‐ Under  these  conditions  the  responses  fluctuated  in  stepwise  manner   -­‐ Some  stimuli  produced  no  response  at  all  –  a  failure  of  transmission   -­‐ Some  stimuli  produced  a  response  ~1mV  in  amplitude   –  similar  in  size  and  shape  to  an   MEPP;  other  evoked  responses  that  appeared  to  be  two,  three,  or  four  times  larger   -­‐ This  remarkable  observation  led  Fa tt  and  Katz  to  propose  the  quantum  hypothesis  –  tha   single  quantal  events  observed  to  occur  spontaneously  also  represented  the  building  blocks   for  synaptic  potentials  evoked  by  stimulation   -­‐ Normally,  end-­‐plate  potential  is  made  up  of  ~200  quanta   units  and  variations  in  its  size  are   not  obvious   -­‐ In  low  calcium  concentrations,  the  quantal  size  remains  the  same  but  quantum  contents  is   shall  –  1  to  3  quanta  –  and  fluctuates  randomly  from  trial  to  trial  resulting  in  stepwise   fluctuations  in  amplitude  of  the  end-­‐plate  potential     P255-­‐256   Number  of  Molecules  in  a  Quantum   -­‐ Although  clear  from  experiments  that  at  NMJ  one  quantum  contained  more  than  one  ACh   molecule,  question  of  how  many  molecules  were  in  a  quantum  remained   -­‐ First  accurate  determination  was  made  by   Kuffler  and  Yoshikami  who  used  very  fine   pipettes  for  ionophoresis  of  ACh  onto  postsynaptic  membrane  of  snake  muscle   -­‐ By  careful  placement  of  pipette,  they  were  able  to  produce  a  response  to  brief  pulse  of  ACh   that  mimicked  almost  exactly  the  MEPP   -­‐ To  measure  number  of  molecules  released  by  pipette,  ACh  was  released  by  repetitive  pulses   into  small  (~0.5µl)  droplet  of  saline  under  oil   -­‐ Droplet  was  then  applied  to  the  end  plate  of  snake  muscle  fiber  and  resulting  depolarization   measured   -­‐ Response  was  compared  with  responses  to  droplets  of  exactly  same  size  containing  known   concentration  of  ACh   -­‐ In  this  way,  the  concentration  of  ACh  in  test  droplet  was  determined  and  number  of  ACh   molecules  released  per  pulse  was  calculated   -­‐ Pulse  of  ACh  required  to  mimic  an  MEPP  contained  approximately  7000  molecules     Number  of  Channels  Activated  by  a  Quantum   -­‐ Given  that  quantum  of  ACh  consists  of  ~7000  molecules,  one  might  expect  that  only  a  few   thousand  of  these  would  actually  combine  with  postsynaptic  recep tors  at  NMJ  –  remainder   being  lost  to  diffusion  out  of  cleft  or  hydrolysis  by  cholinesterases   –  this  expectation  is   correct   -­‐ Number  of  receptors  activated  by  a  quantum  can  be  determined  by  comparing  conductance   change  that  occurs  during  a  miniature  potentia l  with  that  produced  by  a  single  ACh-­‐ activated  channel   -­‐ Measurement  of  miniature  end -­‐plate  currents  by  voltage  clamp  in  frog  muscle  indicate  a   peak  conductance  change  on  the  order  of  40  nanosiemens  (nS)   -­‐ A  single  frog  ACh  receptor  has  a  conductance  of  about   30  picosiemens  (pS)   -­‐ Thus,  miniature  end-­‐plate  potential  is  produced  by  about  1300  open  channels     -­‐ This  is  similar  to  the  number  calculated  by  Katz  and  Miledi,  who  estimated  the  contribution   of  single  channel  to  end-­‐plate  potential  from  noise  measurements   -­‐ Similar  value  for  the  number  of  channels  opened  by  a  quantum  of  transmitter  was  obtained   at  glycine-­‐mediated  inhibitory  synapses  in  lamprey  brainstem  cells   -­‐ Lower  values  are  observed  at  other  synapses   –  for  example,  at  synapses  on  hippocampal   cells,  a  quantal  response  corresponds  to  activation  of  15 -­‐65  channels   -­‐ Why  are  there  such  differences  among  synapses?  A  little  thought  leads  to  conclusion  that  the   number  of  postsynaptic  receptors  activated  by  quantum  of  transmitter  released  from  a   single  presynaptic  bouton  must  be  tailored  to  size  of  cell   -­‐ In  large  cells  with  low  input  resistances,  such  as  skeletal  muscle  fibers  or  lamprey  Muller   cells,  a  large  number  of  receptors  must  be  activated  for  the  effect  of  a  quantum  to  be   significant   -­‐ Activation  of  same  number  of  r eceptors  on  a  very  small  cell  would  overwhelm  all  other   conductances,  depolarizing  the  cell  to  a  potential  near  zero  if  the  synapse  were  excitatory  or   locking  its  membrane  potential  firmly  at  chloride  equilibrium  potential  if  the  effect  were   inhibitory   -­‐ How  is  the  match  btw  cell  size  and  number  of  receptor  activated  by  quantum  achieved?  Is   the  number  of  molecules  in  a  quantum  reduced,  or  is  the  number  of  available  postsynaptic   receptor  lower?   -­‐ Precise  values  for  number  of  molecules  of  transmitter  in  a  CNS  syn aptic  vesicle  are  not   available   -­‐ However,  reported  estimate  for  glutamate -­‐containing  vesicles  is  4000  which  is  same  order   of  magnitude  as  number  of  ACh  molecules  in  vesicles  at  NMJ   -­‐ On  other  hand,  analysis  of  quantal  fluctuation  at  excitatory  and  inhibitory   synapses  at   hippocampal  cells  suggest  that  number  of  available  postsynaptic  receptors  is  much  lower   than  at  the  NMJ   -­‐ Amplitude  of  these  quantal  events  at  hippocampal  synapses  show  remarkably  little  variance   suggesting  that  number  of  molecules  released  in  si ngle  quantum  is  always  more  than   sufficient  to  activate  all  available  receptors   -­‐ Conversely,  at  NMJ  an  increase  in  number  of  transmitter  molecules  in  quantum  can  result  in   a  larger  quantal  event   -­‐ Difference  in  available  postsynaptic  receptors  deduced  from   quantal  fluctuations  is   consistent  with  difference  in  synaptic  morphology   -­‐ Thus,  at  NMJ,  receptors  are  packed  at  high  density  (~10,000/ µm )  throughout  a  large   expanse  of  postsynaptic  membrane,  providing  essentially  limitless  sea  of  receptors  for  each   quantum  of  transmitter   -­‐ At  typical  hippocampal  synapse,  the  estimated  postsynaptic  receptor  density  is  lower   (~2800µm )  and  area  occupied  by  postsynaptic  membrane  is  very  small  (0.04 µm )  thus,   2 fewer  than  100  postsynaptic  receptors  may  be  present     P213-­‐217   Indirect  Mechanisms  of  Synaptic  Transmission   -­‐ NT  not  only  open  ion  channels  but  bind  to  other  membrane  receptors  known  as   metabotropic  receptors   -­‐ Metabotropic  receptors  influence  on  channels  indirectly  through  membrane -­‐associated  or   cytoplasmic  secondary  messenger   -­‐ At  many  synapses  in  central  and  autonomic  nervous  systems,  excitatory  and  inhibitory   transmission  occurs  solely  by  these  indirect  mechanisms   -­‐ At  other  locations,  indirect  mechanisms  serve  to  modulate  direct  transmission   -­‐ Most  metabotropic  receptors  (G  protein-­‐coupled  receptors)  produce  their  effects  by  first   interacting  with  G  proteins  in  cell  membrane   -­‐ G  proteins  because  they  bind  guanine  nucleotides  are  trimmers  of  three  subunits   α,  β,  and  γ   -­‐ When  G  protein  activated  by  its  receptor ,  the  α-­‐  and  βγ-­‐  subunit  dissociate   -­‐ The  free  subunit  can  diffuse  and  then  bind  to  and  modulate  activity  of  intracellular  targets   -­‐ Some  G  protein  subunits  bind  to  ion  channels,  producing  relatively  brief  effects   -­‐ For  example,  when  ACh  binds  to  muscarinic  receptors  in  heart  atriu m,  a  G  protein  is   activated  and  the  freed  βγ-­‐subunit  then  opens  a  potassium  channel   –  slowing  heart  rate   -­‐ A  second  mechanism  of  G  protein  action  is  through  activation  of  enzymes  that  produce   intracellular  second  messengers   o Example  –  activation  of  β-­‐adrenergic  receptors  in  heart  by  epinephrine   -­‐ The  α-­‐subunit  of  dissociated  G  protein  stimulates  the  enzyme  adenylyl  cyclase   -­‐ Resulting  increase  in  intracellular  cyclic  adenosine  monophosphate  (AMP  or  cAMP)  activates   another  enzyme,  cAMP-­‐dependent  protein  kinase  whic h  modifies  the  activity  of  channels   and  enzymes  through  phosphorylation     -­‐ Such  responses  may  last  for  seconds,  minutes  or  hours   –  often  persisting  long  after   transmitter  interaction  with  receptors  has  stopped   -­‐ These  mechanisms  provide  both  amplification  and   radiation  of  signals   -­‐ Potassium  and  calcium  channels  are  prime  targets  for  such  indirect  transmitter  action   -­‐ Indirect  action  can  cause  channels  to  be  opened,  closed  or  changed  in  voltage  sensitivity   -­‐ Thus,  indirectly  acting  transmitter  open  potassium  channels  in  heart  atrial  cells;  inhibit  N -­‐ type  calcium  channels  and  M-­‐type  potassium  channels  in  sympathetic  neurons;  and  increase   probability  that  calcium  channels  will  open  in  response  to  depolarization   in  cardiac  muscle   cells   -­‐ Changes  in  channel  activation  in  axon  terminals  modify  transmitter  release   -­‐ In  postsynaptic  cells,  such  changes  alter  spontaneous  activity  and  responses  to  synaptic   inputs   -­‐ There  are  also  tertiary  messengers  which  are  generated  by  som e  forms  of  synaptic   activation   -­‐ These  include  endocannabinoids  (lipid  messengers)  and  a  gas  nitric  oxide  (NO)   -­‐ These  molecules  diffuse  freely  and  so  have  effect  outside  synapse  or  cell  in  which  they  are   made   -­‐ Calcium  ions  constitute  another  tertiary  messenger  and  produce  both  short-­‐term  and  long-­‐ term  changes  in  neuron  excitability  and  synaptic  function   -­‐ Long-­‐term  changes  include  effects  on  gene  transcription  and  synaptic  wiring     Direct  versus  Indirect  Transmission   -­‐ NT  released  from  presynaptic  ending  binds  to  an d  within  millisecond,  open  ion  channels   (ionotropic  receptors)  in  postsynaptic  membrane   -­‐ Thus,  direct  transmission  is  extremely  fast  and  is  required  for  high -­‐speed  integrated  motor   performance     -­‐ However,  many  of  the  essential  human  functions  require  a  more  g radual  and  longer  lasting   form  of  communication   o
More Less

Related notes for CSB332H1

Log In


OR

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit