Comparison with Modulation by NA

The  depression  observed  with  5-­HT  and  a  5-­HT2R  agonist  reminded  me  of  that  observed   with  10  µM  NA  observed  earlier  in  this  laboratory.  Before  going  any  further,  I  will  present   some  data  on  how  NA  modulated  excitatory  transmitter  release  in  these  cells.  In  4.4.3.1,   I  present  data  for  the  noradrenergic  modulation  of  spontaneous  release,  and  point  out   the   mechanism(s)   involved.   In   4.4.3.2,   I   present   data   on   how   NA   modulated   evoked   release.  The  mechanism  involved  in  the  EPSC  depression  will  be  provided  in  4.4.3.3.   Note  that  most  of  the  data  in  sections  4.4.3.1  and  4.4.3.2  was  acquired  by  Drs.  Julian  

crucial  data  as  to  the  molecular  mechanism  in  4.4.3.3.  Some  of  these  data  will  appear  in   joint  publications  (Choy  et  al.,  2017a;;  Choy  et  al.,  2017b).  

4.4.3.1.  Involvement  of  Store  Release  in  NA-­Mediated  mEPSC  Frequency  Increase   Any  receptor  that  is  capable  of  activating  the  classical  Gq  cascade,  may  release  Ca2+   from   stores   to   modulate   transmitter   release.   Some   potential   candidates,   such   as   the   group  I  metabotropic  glutamate  receptors  (Simkus  &  Stricker,  2002a)  and  α1-­AR  were   investigated  in  this  laboratory.  The  impact  of  α1-­AR  activation  on  spontaneous  release  is   shown  in  Fig.  4.4.15.  In  A,  the  individual  mEPSC  frequencies  during  the  control  period   (blue  bar)  and  after  addition  of  10  µM  NA  (red  bar)  are  presented.  The  average  mEPSC   frequency  during  control  was  39  ±  1  Hz,  and  the  amplitude  -­13.0  ±  0.1  pA.  After  addition   of   NA   at  t   =   0,   and   the   resumption   of   the   recording   after   another   5   min,   the   average   mEPSC  frequency  increased  by  34  ±  3%  to  53  ±  1  Hz  (A,  D).  Minute  averages  of  the   mEPSC  frequency  were  normalised  to  the  average  value  during  control,  and  are  shown   in   B.   Note   that,   in   contrast   to   the   case   with   5-­HT,   the   mEPSC   frequency   increase   persisted   for   the   whole   period   after   addition   of   NA.   The   mEPSC   amplitude   remained   unchanged   (-­13.0   ±   0.1   pA;;   C),   not   significantly   different   from   control   (E).   When   the   average   time   courses   of   mEPSCs   were   superimposed   (F)   there   was   no   significant   change  by  NA.  

When  all  such  experiments  were  analysed  (n  =  49),  it  was  found  that  24  (49%)  pyramidal   cells  showed  a  sustained  increase  in  the  mEPSC  frequency  as  described  above.  Like  in   Part   3,   these   cells   were   referred   to   as   responders.   On   average,   NA   increased   the   mEPSC   frequency   by   56   ±   7%   from   39   ±   2   to   59   ±   3   Hz   (ppt   <   10-­8;;   Fig.   G)   in   these   responders.  This  increase  persisted  throughout  the  recording.  The  rest  did  not  show  any   significant  increase  in  the  mEPSC  frequency  (non-­responders).  A  significantly  smaller   mEPSC   frequency   during   the   control   period   was   observed   in   responders   than   non-­ responders  (39  ±  2  vs.  53  ±  4  Hz;;  pt  <  10-­3).  In  addition,  the  NA  and  agonists  caused  a   significant  decrease  of  Rin  only  in  responders  (9  ±  7%  from  122  ±  8  to  103  ±  7  MΩ;;  ppt  =   0.01).  

The  mechanisms  causing  the  increase  in  mEPSC  frequency  were  identified  as  follows   (Choy  et  al.,  2017a).  1)  NA  acted  via  ɑ1-­ARs,  which  in  turn  2)  activated  PLCβ,  resulting   in  3)  PIP2  hydrolysis  to  produce  IP3  and  DAG.  4)  The  former  bound  to  IP3R  on  presynaptic   stores  to  cause  5)  Ca2+  _{release  from  stores,  which  then  6)  drove  vesicle  fusion,  observed}   as  an  increase  in  mEPSC  frequency.  

Figure  4.4.15.  NA  increased  the  mEPSC  persistently.  

A.  Individual  instantaneous  mEPSC  frequencies  in  a  single  pyramidal  cell  shown  during   control  (t  <  0  min),  and  after  addition  of  NA  (t  ≥  0  min).  B.  Minute  averages  of  the  data  in  A   after  normalising  the  average  control  value  to  0%,  including  the  respective  error  bars  which   were  smaller  than  the  diameter  of  the  dots.  Dashed  line  indicates  no  change.  C.  Same  as   in   B,   but   for   the   mEPSC   amplitude.   D.   cPDFs   of   the   instantaneous   frequencies   during   control  and  after  NA.  E.  Same  as  in  F,  but  for  the  mEPSC  amplitude.  F.  Superposition  of   the  average  mEPSC  time  courses  during  control  and  in  NA.  G.  Minute  averages  of  the   pooled  mEPSC  frequency  change  in  responders,  after  normalising  as  in  B.  

Consequently,   in   regard   to   spontaneous   transmitter   release,   both   NA   and   5-­HT   1)   increased   the   mEPSC   frequency   without   affecting   its   amplitude.     2)   The   increase   in   mEPSC  frequency  was  restricted  to  a  subset  of  pyramidal  cells  (responders).  3)  Both   agonists   signalled   via   a   classical   Gq   cascade   to   increase   the   mEPSC   frequency   via  

presynaptic   Ca2+   release   from   stores.   However,   in   contrast   to   NA,   the   increase   in   mEPSC   frequency   by   5-­HT   was   transient,   but   become   maintained   after   PKC   was   blocked.  The  impact  of  NA  on  evoked  transmitter  release,  and  how  it  compared  with  5-­ HT,  will  be  presented  next.  

4.4.3.2.  EPSCs  Depression  by  NA  

Different   from   what   was   expected   from   its   impact   on   spontaneous   release,   NA   also   depressed  evoked  glutamate  release  in  this  layer.  

The  results  obtained  in  a  paired-­recording  after  exposure  to  10  µM  NA  is  illustrated  in   Fig.  4.4.16.  In  A,  the  time  course  of  the  presynaptic  AP  (top)  with  the  evoked  average   EPSC  (bottom)  during  control  is  presented.  The  average  peak  EPSC  amplitude  was  -­ 47.2  ±  1.2  pA.  Addition  of  NA  depressed  the  EPSC  amplitude  by  71  ±  5%  to  -­13.7  ±  0.9   pA  (B).  Individual  peak  amplitudes  during  control  and  after  NA  are  plotted  in  C.  It  shows   that  NA  caused  a  persisting  depression  within  5  min  of  exposure.  The  time  courses  of   the   respective   EPSCs   are   given   in   D,   with   the   traces   superimposed   (left)   and   peak-­ scaled  for  comparison  (right).  

Seven  such  experiments  are  presented  in  E,  where  the  paired  values  of  the  amplitudes   in  control  and  NA  for  the  respective  pairs  are  joined  by  a  grey  line.  The  average  EPSC   amplitude  during  control  was  -­24.0  ±  5.6  pA,  but  depressed  by  62  ±  7%  to  -­8.3  ±  2.0  pA   (ppt  =  0.01)  after  addition  of  NA.  Even  though  this  depression  was  strictly  not  larger  than   the  one  caused  by  5-­HT,  it  was  close  to  significant  (62  ±  7  vs.  49  ±  3%;;  pt  =  0.06).   The  depression  by  NA  was  caused  by  ɑ1-­AR  activation  (Choy  et  al.,  2017b).  Specifically,   application  of  the  ɑ1-­AR  agonist  cirazoline  (5  µM)  caused  a  constant  EPSC  depression   by  54  ±  18%  from  -­8.2  ±  1.5  to  -­3.1  ±  0.3  pA  (n  =  4;;  ppt  <  0.05).  Compared  to  NA,  the   extent  of  the  depression  caused  by  the  ɑ1-­AR  agonist  was  indistinguishable  (pt  =  0.40).   This   depression   persisted   in   the   presence   of   ɑ2-­   and   β-­AR   blockers   yohimbine   and   propranolol,  respectively  (1  µM  in  both  cases;;  n  =  5),  further  strengthening  the  idea  that   ɑ1-­AR  caused  this  depression.  It  is  also  important  to  add  that  in  all  cases  tested  PPR   was  not  significantly  altered  (ppt  =  0.37).  

Comparing  the  impacts  of  NA  on  transmitter  release  with  those  of  5-­HT,  the  following   observations  can  be  made:  1)  Both,  NA  and  5-­HT  considerably  depressed  the  EPSC   amplitude,  but  increased  spontaneous  transmitter  release.  2)  Both,  the  depression  and   the  increase  in  m  EPSC  frequency  were  downstream  of  a  Gq-­linked  signalling  cascade.   3)  In  both  cases,  despite  a  large  EPSC  depression,  on  average  PPR  was  not  altered.