Synapse 2011 - Volume 5

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Cornell Synapse 2012 For more information email us at synapse.submisson@gmail.com

cornell

synapse Volume 5 2011

Editor-in-Chief Chong Guo ‘13

Managing Editor Yoshiko Toyoda ‘14 Jonathan Lin ‘14

Graduate Peer Editors

Design Editors

Loren Law Hsien-Wei Meng Spencer Park Shane Peace Jade Wu

Louis Hopkins ‘14 Trianna Lutchman ‘14 Lindsay Rappa ‘14

Undergraduate Peer Editors

Staff Writers

Daniel Acker ‘13 Jeanie Gribben ‘15 Shayra Kamal ‘14 Alexandra Mattei ‘14 Brian Morris ‘14 Tracy Netemeyer ‘14 Samantha Olyha ‘14 Camille Shaw ‘14 Ryan Woolley ‘14

Daniel Acker ‘13 Emily Acton ‘13 Peter Cohn ‘14 Daniel Lee ‘14 Brian Morris ‘14 Ethan Romano ‘14

Faculty Advisor Bruce Johnson

The publication of this journal was made possible with funding from the Cornell University Student Assembly Finance Commission (SAFC). Views expressed in this publication may not necessarily reflect those of the SAFC or Cornell University.

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EDITOR S LETTER Dear Readers of Synapse, I have recently encountered a book written by John A. Adam on mathematics in nature. In the introduction to the chapter on fractals, the author wrote something to the effect of “caution, do not proceed if you wish to preserve your childhood perception of clouds, you will never see them quite the same way again at the end of this chapter.”

In a sense, this facetious (or maybe completely serious) statement captures well what neurobiologists had accomplished in advancing our scientific understanding of the nervous system. From the epoch-setting doctrinal debate between Golgi and Remon y Cajal to the ingenious formulation of the Hodgkin-Huxley model, neuroscience had indeed come a long way in dispelling many of our ancient dogmas about how the mind works. While it is reassuring to know that we do not see via tentacle-like threads originating from the pupils as Greek philosophers once believed or that hydraulic affects of the ventricular system formed the basis of all cognitive functions, neuroscience as it stands today is still an on-going endeavor, one which involves researchers, clinicians and educators alike. To that end, Synapse exists as a window for students to witness this vibrant enterprise and to share their own works and ideas via an open platform with the rest of the Cornell community.

In this issue of Synapse, we will explore some interesting topics such as the relationship between mirror neurons and social behaviors, the efficacy of Omega 3 supplements, the growth of new neurons in adult brain and neurological diseases such as epilepsy, migraine, and Parkinson’s. We strived to find a good balance between basic science and translational research in our editing process and we hope that this final selection may interest as wide an audience as possible. Having said that, as much as those of us here at Synapse would like to share with you our passions and curiosities about the brain, there is a limit to what a group of twenty or so undergraduate students can modestly accomplish with a 40 page publication. We wish that we could caution our readers with the boldness of the aforementioned author that “a reading of Synapse shall forever shatter your existing belief about clouds” or, failing to do that, “how you can perceive them at all.” Nevertheless, we hope that you may walk away with a few amusing tidbits about the brain, some nerdy conversation starters that may or may not work with that girl from BIONB 2220 and ,if you are lucky, finally discovering what you do or do not want to do with the rest of your life.

The familiar readers of Synapse will note some key changes in the publication’s structure. Two entirely new sections have been added to the magazine, namely, the Featured Articles section and Neuro-in-the-News. While the rigorousness with which Synapse selects and publishes literature review and original research from Cornell undergraduate has been our biggest strength since the very inception of this journal, we wish to engage a wider audience as well as to provide opportunities for students to write about neuroscience in a more open and expressive format.

In an effort to diversify our publication in terms of its style and content, we have created a brand new publication which we are very excited to share with you.

Last but not the least, I want to extend my sincerest appreciation to our staff writers, undergraduate/graduate editors and the layout designers for all the work they have put into this issue of Synapse. I am very grateful also for having the opportunity to work with our two amazing managing editors Yoshiko Toyoda and Jonathan Lin. The publication process wouldn’t have gone nearly as smoothly without their help.

Sincerely,

Chong Guo Biometry and Statistic ‘13

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Vol 5 ¦ 2011

Cornell Synapse

TABLE OF CONTENTS News

Creativity in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

by Jegath Athinlingam The Effect of Methamphetamine on Memory in Snails . . . . . . . . . . . . . . . . . . . . . . 4

by Peter Cohn I am My Connectome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

by Chong Guo Single Cell Endoscope Opens Door to Nanoscale Interaction With Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

by Daniel Acker Stimulation of Entorhinal Cortex Improves Memory in Epileptic Patients . . 6

by Emily Acton Love Bug or Love Drug?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

by Jade Wu

Feature Articles Progeria: We’re Not All Dying Slowly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

by Daniel Acker Light Driven Memory Recall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

by Peter Wang

Research Articles Anti-Epileptic Drugs and Seizure Severity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

by Kaitlin Hardy Cloning and Characterization of slo Family Potassium Channel Isoforms in Lobster Nervous System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

by Vinay Patel

Review Articles Hippocampal Neurogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

by Rachel Bavley Omega-3 or Omega-6? Breastmilk or Formula? . . . . . . . . . . . . . . . . . . . . . . . 31

by Jonathan Lin and Yoshiko Toyoda There is More to Parkinson’s Disease than Motor Dysfunctions. . . . . . . . . . 38

by Diana Hong Migraine Prevention: Neuromodulation for the Hyperexcitable Brain . . . . 42

by Bryanna Gulotta

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Vol 5 ¦ 2011

Creativity in the Brain Jegath Athilingam , Arts and Sciences 11 Human Development and Neuroscience  What  is  creativity?  Charles  Limb  from  Johns  Hopkins  University  aims  to  answer  this  question  in  a  VFLHQWL¿F PDQQHU E\ VWXG\LQJ FUHDWLYLW\ LQ WKH EUDLQ ,Q D UHFHQW UHVHDUFK SURMHFW /LPE VWXGLHG WKH GLIIHUHQFHV in  neural  activation  when  professional  jazz  musicians  SOD\HG PHPRUL]HG PXVLF YHUVXV LPSURYL]HG PXVLF XV-­ LQJ D PDJQHWLFDOO\ VDIH NH\ERDUG WKDW DOORZHG WKH PXVL-­ FLDQV WR SOD\ ZKLOVW O\LQJ LQ D IXQFWLRQDO PDJQHWLF UHVR-­ QDQFH LPDJLQJ I05, VFDQQHU +H IRXQG DQ LQWHUHVWLQJ pattern  of  activation  in  the  frontal  lobe,  the  area  of  the  EUDLQ UHVSRQVLEOH IRU H[HFXWLYH IXQFWLRQ DQG KLJKHU RUGHU WKLQNLQJ 7KH PHGLDQ SUHIURQWDO FRUWH[ DQ DUHD DVVRFLDWHG ZLWK DXWRELRJUDSKLFDO VHOI H[SUHVVLRQ LQ-­ FUHDVHG LQ DFWLYLW\ GXULQJ LPSURYLVHG PXVLF ZKLOH WKH ODWHUDO SUHIURQWDO FRUWH[ D UHJLRQ DVVRFLDWHG ZLWK VHOI PRQLWRULQJ GUDVWLFDOO\ GHFUHDVHG LQ DFWLYLW\ /LPE SRV-­ LWV WKDW FUHDWLYLW\ FRPHV IURP WKLV GLVVRFLDWLRQ LQ WKH IURQWDO OREH ZKHUH WKH LQKLELWLRQ RI VHOI PRQLWRULQJ DO-­ ORZV VHOI H[SUHVVLRQ WR Ã&#x20AC;RZ ZLWKRXW DQ\ VXSSUHVVLRQ /LPE & 3UHVHQWHU -DQ <RXU %UDLQ RQ ,PSURY 7(' 7DONV $YDLODEOH IURP KWWS ZZZ WHG FRP WDONVFKDUOHVBOLPEB \RXUBEUDLQBRQBLPSURY KWPO

The Eï¬&#x20AC;ects of Methamphetamine on Memory in Snails

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Peter Cohn, Arts and Sciences 14, :DQJ 6 6 ³0HPRU\ *HWV -ROW LQ %UDLQ 5HVHDUFK ´ :DOO 6WUHHW Psychology -RXUQDO )HEUXDU\ :HE :KLOH VFLHQWLVWV KDYH D SDUWLDO XQGHUVWDQGLQJ RI WKH PHFKDQLVPV RI GUXJ DGGLFWLRQ WKHUH LV VWLOO PXFK XQNQRZQ DERXW WKH VSHFL¿F SURFHVVHV WKDW UHVXOW LQ WKLV FRQGLWLRQ ,Q D UHFHQW DUWLFOH IURP WKH Journal  of  Ex-­ perimental  Biology 'U %DUEDUD 6RUJ RI :DVKLQJWRQ 6WDWH 8QLYHUVLW\ H[SORUHG WKH HIIHFWV RI DQ DGGLFWLYH GUXJ PHWKDPSKHWDPLQH RQ SRQG VQDLOV

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NEWS bases  for  future  advancement  in  theoretical  neurosci ence  as  well  as  translational  research  for  disorders  that  H[SOLFLWO\ DOOHJHGO\ UHVXOW LQ ¾PLV ZLUHGœ FRQQHFWRPHV (i.e.  schizophrenia).  Seung,  Sebastian  (Presenter).  (2010,  July).  I  am  my  connectome  TED  Talks.  Available  from  http://www.ted.com/talks/sebastian_ seung.html

I am my Connectome Chong Guo, Agriculture and Life Sciences 13 Statistics and Biological Sciences  There  is  hint  of  haughtiness  in  the  air  when  the  audiences  at  July  2010â&#x20AC;&#x2122;s  Oxford  TED  Conference  chanted  out  in  unison,  â&#x20AC;&#x2DC;I  am  my  connectomeâ&#x20AC;&#x2122;  during  the  talk  given  by  Sebastian  Seung,  a  star  computational  neuroscientist  at  MIT.  A  connectome  is  an  anatomical  and  functional  mapping  of  neuronal  connections  in  our  central  nervous  system.  Since  the  introduction  of  this  term  in  2005  by  Dr.  Dlaf  Sporns  at  Indiana  University,  connectomics  had  quickly  grown  into  one  of  the  hottest  UHVHDUFK DUHDV LQ ELRORJ\ 7KH ÂżHOG JDLQHG DGGLWLRQDO momentum  from  NIHâ&#x20AC;&#x2122;s  Blue  Print  for  Neuroscience  Research,  which  announced  in  2009  their  48.5  million  GROODU ÂżYH \HDU +XPDQ &RQQHFWRPH 3URMHFW

-RLQWO\ KRVWHG E\ WKH +DUYDUG 0*+ 8&/$ &RQVRUWLXP DQG :DVKLQJWRQ 8QLYHUVLW\ 0LQQHVRWD &RQVRUWLXP WKH SURMHFW LV D FROODERUDWLRQ EHWZHHQ QHX roscientists,  physicists  and  computer  scientists  in  map ping  out  the  the  connectome  of  the  human  brain.  Many  sophisticated  imaging  and  image  processing  tools  are  HPSOR\HG 7KH +DUYDUG 0*+ 8&/$ &RQVRUWLXP XWL lizes  a  technique  known  as  diffusion  tensor  magnetic  resonance  imaging  (dtMRI),  which  tracks  the  move ment  of  water  molecule  across  the  axons  during  an  ac tion  potential,  providing  3D  data  on  the  wiring  of  nerve  bundles.  Their  sister  consortium  at  Washington  and  Minnesota  University  relies  more  on  functional  MRI  in  their  study  of  local  neural  activities  by  measuring  FKDQJHV LQ EORRG Ă&#x20AC;RZ WR VSHFLÂżF UHJLRQV LQ WKH EUDLQ during  a  prescribed  action  or  behavior.

7KH +XPDQ &RQQHFWRPH 3URMHFW DORQJ ZLWK other  related  efforts  (worth  noting  here  the  privatly  funded  Allan  Institute  for  Brain  Science)  will  spark  technological  innovations  and  provide  invaluable  data

Single Cell Endoscope Opens Door to Nanoscale Interaction With Neurons Daniel W. Acker, Agriculture and Life Sciences 13 Biological Sciences and Animal Science 5HVHDUFKHUV DW %HUNHOH\ 1DWLRQDO /DERUDWRU\ developed  a  nanoscale  endoscope,  called  a  nanoscope,  that  can  emit  or  detect  light  within  a  single  cell.   They  built  the  remarkable  instrument  by  attaching  a  nanow LUH ZDYHJXLGH WR WKH WLS RI DQ RSWLFDO ÂżEHU 7KH\ ZHUH WKHQ DEOH WR IRFXV ODVHU OLJKW WKURXJK WKH RSWLFDO ÂżEHU DQG FRQWURO LWV HPLVVLRQ ZLWKLQ D +H/D FHOO 7KH\ ZHUH also  able  to  record  wavelength  information  from  within  the  cell  by  attaching  a  spectrometer  to  the  device.  This  type  of  endoscope  is  remarkable  because  it  breaks  the  diffraction  barrier.   The  diffraction  barrier  is  a  problem  encountered  when  using  lensed  microscopes.   ,W FRQVWLWXWHV WKH GLIÂżFXOW\ PHW ZKHQ WU\LQJ WR H[DP LQH DQ REMHFW VPDOOHU WKDQ WKH ZDYHOHQJWK RI OLJKW EHLQJ used  as  illumination.   The  nanoscope  circumvents  this  issue  with  its  nanowire  component,  which  can  transmit  VXEZDYHOHQJWK OLJKW WKURXJK Ă&#x20AC;XLG PHGLD  The  researchers  have  already  demonstrated  sev eral  uses  for  their  nanoscope.   In  addition  to  imaging  and  illumination,  they  have  shown  that  it  can  be  used  in  WKH GLUHFWHG GHOLYHU\ RI D SD\ORDG WR D VSHFLÂżHG LQWUDFHO lular  area.   They  attached  quantum  dots  to  the  tip  of  the  nanowire,  and  were  able  to  release  these  particles  into  targeted  regions  by  cleaving  their  link  to  the  nanowire  with  low  intensity  ultraviolet  stimulation.  In  addition  to  its  spatial  accuracy,  this  delivery  method  was  shown  to  KDYH KLJK WHPSRUDO VSHFLÂżFLW\ DV WKH UHOHDVH WLPH RI WKH quantum  dots  could  be  controlled  to  within  a  minute.

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Stimulation of Entorhinal Cortex Improves Memory in Epileptic Patients

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additional  research  to  validate  the  result,  this  study  presents  an  exciting  new  pathway  of  exploration  for  the  future  treatment  of  human  memory  disorders.

Love Bug or Love Drug?

Emily Acton, Agriculture and Life Sciences 13 Food Sciences

Jade Wu , Arts and Sciences 11 Psychology

â&#x20AC;&#x153;Memory  Gets  Jolt  in  Brain  Research,â&#x20AC;?  a  recent  article  published  in  the  Wall  Street  Journal,  chronicles  the  implications  of  a  study  presenting  a  â&#x20AC;&#x153;new  sparkâ&#x20AC;?  in  research  on  memory  disorders.   The  referenced  study  by  Nanthia  et  al.  (2012)  recently  published  in  the  New  England  Journal  of  Medicine  presented  preliminary  GDWD VXJJHVWLQJ WKDW GHHS EUDLQ VWLPXODWLRQ LQ VSHFLÂżF DUHDV RI WKH EUDLQ PD\ KDYH IXWXUH VLJQLÂżFDQFH IRU WKH treatment  of  memory  disorders  including  Alzheimerâ&#x20AC;&#x2122;s  disease.   Deep  brain  stimulation  already  has  notable  use  as  medical  treatment,  including  approval  for  the  treat-­ ment  of  Parkinsonâ&#x20AC;&#x2122;s  disease,  dystonia  and  utilities  in  the  treatment  of  chronic  pain  and  severe  depression.1  This  study  tested  the  effects  of  deep  brain  stimulation  in  seven  epilepsy  patients  whose  baseline  memory  capacity  varied  from  normal  to  severely  lim-­ ited.   The  researchers  applied  deep  brain  stimulation,  undetectable  bursts  of  electricity  through  depth  elec-­ trodes,  to  different  regions  of  the  brain.   When  the  re-­ searchers  tested  the  patientsâ&#x20AC;&#x2122;  spatial  memory  through  a  location-­recollection  game,  it  was  found  that  stimula-­ tion  of  the  entorhinal  cortex  yielded  improved  memory  in  six  of  the  patients  implanted  with  electrodes  in  this  region,  regardless  of  their  baseline  memory  capability.  The  entorhinal  cortex  is  central  in  the  transformation  of  H[SHULHQFHV LQWR PHPRU\ DQG LV RQH RI WKH ÂżUVW VHFWLRQV of  the  brain  to  be  altered  by  Alzheimerâ&#x20AC;&#x2122;s  disease.

Prior  data  in  human  models  from  studies  published  in  2008  and  2010  provide  limited  support  of  the  poten-­ tial  for  a  correlation  between  brain  stimulation  and  im-­ proved  memory.   Further,  animal  models  examining  the  impact  of  stimulating  the  entorhinal  cortex  in  mice  sup-­ ported  the  results  of  this  study,  with  the  mice  demon-­ strating  improved  brain  cell  growth,  increased  memory  for  locations  and  spatial  knowledge  upon  stimulation.   While  the  investigators  acknowledge  the  necessity  for Â

 Love  is  a  mysterious  thing  indeed  but  it  can  take  the  role  of  both  Dr.  Jekyll  and  Mr.  Hyde.  On  the  one  hand,  research  has  shown  that  love  can  truly  be  a  drug:  an  analgesic  (the  fancy  name  for  painkiller).  Studies  that  examine  love  as  a  pain  reliever  usually  provide  pain  through  a  heat  probe  on  the  hand  while  having  par-­ ticipants  look  at  images  of  their  loved  ones  or  holding  the  hand  of  a  loved  one.  In  these  studies,  presence  or  WKRXJKWV RI D URPDQWLF SDUWQHU VLJQLÂżFDQWO\ GHFUHDVHG subjective  reports  of  pain.  Distraction  (in  the  form  of  a  math  task)  was  also  shown  to  decrease  pain  but  love-­ induced  analgesia  was  associated  with  brain  reward  centers  while  distraction-­induced  analgesia  was  not.  The  activation  of  the  brainâ&#x20AC;&#x2122;s  reward  circuitry  is  another  way  that  love  acts  as  a  drug  and  can  explain  loveâ&#x20AC;&#x2122;s  addictive  qualities.  Scientists  hypothesize  that  the  dopaminergic  reward  neurons  in  the  midbrain  may  interact  with  endorphins  (the  bodyâ&#x20AC;&#x2122;s  natural  opiods)  to  provide  pain  relief  as  well  as  that  euphoric  rush  that  lovestricken  people  often  report.  But  the  analgesia  and  high  is  only  the  Dr.  Je-­ kyll  side  of  love.  Scientists  are  debating  whether  the  condition  of  â&#x20AC;&#x2DC;lovesicknessâ&#x20AC;&#x2122;  could  be  considered  a  valid  mental  illness  as  it  can  often  be  characterized  by  symp-­ toms  such  as  mania,  depression,  stress,  and  obsessive  compulsive  disorder.  In  addition,  those  suffering  the  end  of  a  relationship  often  show  symptomssimilar  to  drug  withdrawal.  Love  can  be  painful  or  pain-­relieving,  like  a  coin  with  2  faces.

Parker-­Rope,  T.  (2010,  Oct).  Love  and  Pain  Relief.  New  York  Times  Blog.  http://  well.blogs.nytimes.com/2010/10/13/loveand-­ pain-­relief/ Tallis,  F.  (2005,Feb).  Crazy  for  you.The  Psychologist,  8,72-­74.

FEATURE

Vol 5 ÂŚ 2012

Progeria â&#x20AC;&#x2019; We re Not All Dying Slowly Daniel W. Acker, Agriculture and Life Sciences 13 Biological Sciences and Animal Science  Aging  is  a  fact  of  mammalian  life.  The  basic  life  cycle  of  birth,  growth,  maintenance,  decrepitude,  and  death  is  universal.  Humans  can  even  be  consid-­ ered  lucky  because  we  enjoy  a  particularly  long  lifes-­ pan  compared  to  some  of  our  closest  relatives.  Humans  at  retirement  age  can  often  expect  to  live  for  another  twenty  years,  enjoying  a  period  of  relative  leisure.  Because  of  this,  old  age  has  become  somewhat  glam-­ orized.  Many  suburbanites  dream  of  retiring  one  day  and  spending  their  sunset  years  living  in  Florida,  play-­ ing  golf  and  absorbing  the  sunlight.  Nevertheless,  few  would  give  up  their  young  life  to  instantly  become  old.   <RXWK LV D WLPH RI GLVFRYHU\ DQG VHOI GHÂżQLWLRQ QRW WR mention  peak  physical  health  accompanied  by  great  stamina,  strength,  and  regenerative  capability.  Youth  is  quintessential  to  the  human  experience,  and  this  may  be  why  progeria  is  so  thoroughly  unsettling.  Progeria  is  a  disease  that  affects  young  children.  Sufferers  experience  symptoms  such  as  a  lack  of  hair  and  teeth,  a  diminished  stature,  a  narrow  and  wrinkled  face,  thin,  dry  skin,  and  an  impeded  range  of  motion.  Nearly  all  of  those  affected  die  in  their  early  to  mid  teens  because  of  heart  attacks  or  strokes.  One  of  the  ORQJHVW OLYHG LQGLYLGXDOV ZLWK D YHULÂżHG FDVH RI SURJH-­ ria  was  an  artist  and  musician  named  Leon  Botha  who  died  in  June  of  2011  at  the  age  of  26.  In  short,  progeria  mimics  the  decay  portion  of  the  aging  process,  turning  children  into  seniors.  To  add  to  the  tragedy,  progeria  is  untreatable.  Those  who  receive  the  diagnosis  can,  at  best,  expect  to  live  into  their  early  twenties.  All  that  their  doctors  can  do  at  the  moment  is  prescribe  aspirin  in  hopes  of  delaying  heart  failure.  Because  of  its  unsettling  effects  and  the  similar-­ ity  of  these  symptoms  to  the  normal  aging  process,  a  great  deal  of  research  has  been  focused  on  identifying  the  mechanisms  that  underlie  progeria.  As  a  result,  we  Cornell University

now  know  a  lot  about  why  progeria  occurs.  Discover-­ ies  have  also  led  to  the  testing  of  potential  treatments,  although  none  have  made  it  to  market  as  of  yet.  The  fundamental  cause  of  progeria  was  identi-­ ÂżHG DV JHQHWLF LQ E\ D WHDP RI VFLHQWLVWV ZRUNLQJ at  the  New  York  State  Institute  for  Basic  Research  in  Developmental  Disabilities.  They  found  that,  in  19  out  of  20  subjects,  there  was  a  single  base  pair  substitu-­ tion  in  the  DNA  coding  for  the  protein  lamin  A.  The  UHVHDUFKHUV ZHQW RQ WR ÂżQG WKDW WKLV VXEVWLWXWLRQ UHVXOW-­ ed  in  a  50  base  pair  long  deletion  when  the  DNA  was  transcribed  into  RNA.   This  deletion  was  found  to  lead  to  the  production  of  a  defective  version  of  lamin  A.  Later  work  has  shown  that  many  different  mu-­ tants  of  lamin  A  can  lead  to  progeria-­like  symptoms.  A  JURXS RI )UHQFK VFLHQWLVWV IRXQG LQ WKDW VRPH RI these  mutations  led  to  the  formation  of  a  mutant  pro-­ tein  called  progerin.   Progerin  is  similar  to  lamin  A,  but  has  an  extra  farnesyl  group.  Such  groups  often  con-­ fer  membrane-­anchoring  tendencies  on  the  proteins  to  which  they  are  bound.  This  tendency  was  observed  in  progerin,  which  associates  with  the  nuclear  membrane  that  surrounds  the  nucleus.  These  discoveries  led  to  experimentation  with  farnesyltransferase  inhibitors.  Farnesyltransferase  in-­ hibitors  disable  the  farnesyltransferase  molecules  that  are  responsible  for  attaching  the  extra  farnesyl  group  to  progerin.  A  2008  collaborative  study  between  scientists  at  Emory  University,  New  York  University,  the  Nation-­ al  Human  Genome  Research  Institute,  and  the  National  Heart,  Lung,  and  Blood  Institute  found  that  administer-­ ing  farnesyltransferase  inhibitors  could  prevent  heart  disease  in  mice  with  progeria  symptoms.   Furthermore,  a  2010  study  by  researchers  at  UCLA  and  the  Univer-­ sity  of  Kentucky  at  Lexington  found  that  when  mice  were  engineered  to  express  unfarnesylated  progerin, Â

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Vol 5 ÂŚ 2012

ÂŚ Progeria ÂŚ

they  showed  no  symptoms  of  progeria.  The  farnesyl  group  on  progerin  was  so  well  implicated  as  a  cause  of  progeria,  and  preventing  its  attachment  was  so  well  implicated  with  progeria  treatment,  that  a  human  trial  with  the  farnesyltransferase  inhibiting  drug  lonafarnib  was  approved  in  2007.   The  study  was  conducted  at  Childrenâ&#x20AC;&#x2122;s  Hospital  Boston.   Unfortunately,  although  the  study  was  said  to  have  concluded  in  2009,  no  results  have  been  published  to  date.  Hopefully,  the  conductors  of  this  study  will  SXEOLVK WKHLU UHVXOWV VRRQ EHFDXVH ÂżQGLQJ DQ HIIHFWLYH treatment  would  be  a  victory  for  anyone  interested  in  alleviating  human  suffering.   However,  the  health  implications  of  progeria  research  extend  beyond  the  obvious.   Scientists  at  the  National  Human  Genome  Research  Institute  in  Bethesda  found  in  2007  that  elevated  levels  of  progerin  lead  to  defects  in  mitosis  that  are  similar  to  what  could  be  observed  in  cells  after  normal  aging.   This  connection  was  reinforced  by  research  from  the  National  Cancer  Institute  in  Bethesda  that  showed  that  the  elderly  often  exhibited  the  same  lamin  A  mutation  that  causes  progeria.  It  seems  that,  to  a  certain  extent,  everyone  is  dying  from  the  same  disease.   The  only  difference  between  those  who  are  deemed  healthy  and  those  with  progeria  seems  to  be  the  rate  of  its  progression.   A  progeria  cure  could  have  a  potentially  monumental  effect  on  how  old  age  is  thought  of.   It  may  be  audacious  to  think  that  a  cure  would  completely  halt  the  aging  process,  but  itâ&#x20AC;&#x2122;s  reasonable  to  assume  that  it  would  lead  to  treatments  for  at  least  some  of  the  undesirable  symptoms  associated  with  old  age.

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FEATURE References

Aboobaker,  S  (2011,  June,  7).  Cape  DJ  dies  of  Progeria.  Independent  Online.  Retrieved  2011,  December,  17  from  iol. co.za/  Capell  BC,  Olive  M,  Erdos  MR,  Cao  K,  Faddah  DA,  Tavarez  UL,  Conneely  KN,  Qu  X,  San  H,  Ganesh  SK,  Chen  X,  Avallone  H,  Kolodgie  FD,  Virmani  R,  Nabel  EG,  Collins  FS.  2008.  A  farnesyltransferase  inhibitor  prevents  both  the  onset  and  late  progression  of  cardiovascular  disease  in  a  progeria  mouse  model.  Proc  Natl  Acad  Sci  U  S  A.  106  (31):13143.

Eriksson  M,  Brown  WT,  Gordon  LB,  et  al.  2003.  Recurrent  de  novo  point  mutations  in  lamin  A  cause  Hutchinson-­Gilford  progeria  syndrome.  Nature  423  (6937):  293â&#x20AC;&#x201C;8.

Makar,  AB;Íž  McMartin,  KE;Íž  Palese,  M;Íž  Tephly,  TR  1975.  Phase  II  trial  of  Lonafarnib  (a  farnesyltransferase  inhibitor)  for  progeria.  Biochemical  medicine  13  (2):  117â&#x20AC;&#x201C;26.

McClintock  D,  Ratner  D,  Lokuge  M,  et  al.  2007.  Lewin,  Alfred.  ed.  The  Mutant  Form  of  Lamin  A  that  Causes  Hutchinson-­Gilford  Progeria  Is  a  Biomarker  of  Cellular  Aging  in  Human  Skin.  PLoS  ONE  2  (12):  e1269 Progeria:  Treatment.  MayoClinic.  Retrieved  2011,  December,  17  from  MayoClinic.com/ Sandre-­Giovannoli  A,  Bernard  R,  Cau  P,  Navarro  C,  Amiel  J,  Boccaccio  I,  Lyonnet  S,  Stewart  C,  Munnich  A,  Merrer  M,  Levy  N.  2003.  Lamin  A  Truncation  in  Hutchinson-­Gilford  Progeria.  Science  27,  Vol.  300  no.  5628  p.  2055 Yang  S,  Chang  S,  Ren  S,  Wang  Y,  Andres  D,  Spielmann  H,  Fong  L,  Young  S.  2010.  Absence  of  progeria-­like  disease  phenotypes  in  knock-­in  mice  expressing  a  nonfarnesylated  version  of  progerin.  Hum.  Mol.  Genet.  10.1093

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Cornell University

FEATURE

Vol 5 ÂŚ 2012

light driven

MEMORY RECALL Peter Wang, Arts and Sciences 11 College Scholar, Physics and Neurobiology  Imagine  that  with  a  toggle  of  a  switch,  someone  can  evoke  powerful  memories  in  your  mind.  This  idea,  ZKLFK KDV DOZD\V UHVLGHG LQ WKH UHDOP RI G\VWRSLDQ ÂżF-­ tions  (remember  the  Brave  New  World  or  Manchurian  Candidate?),  has  recently  been  warped  into  reality  by  a  newly  developed  toolbox  in  neuroscience:  optogenet-­ LFV 7KLV UHSUHVHQWV D KDQGIXO RI JHQHWLFDOO\ PRGLÂżHG proteins,  derived  from  those  used  in  bacteria  for  pho-­ totaxis,  that  causes  neurons  to  depolarize  or  hyperpo-­ ODUL]H LQ UHVSRQVH WR OLJKW RI D VSHFLÂżF ZDYHOHQJWK ,Q other  words,  expressing  these  proteins  in  neurons  lets  you  control  their  activity  and  hence  an  organismâ&#x20AC;&#x2122;s  be-­ havior  â&#x20AC;&#x201C;  with  light. Â

Figure  1.  A  ChR-­2  expressing  neuron  excited  by  blue  light.

6SHFL¿FDOO\ FKDQQHOUKRGRSVLQ &K5 LV DQ optogenetic  protein  that  opens  up  its  channel  pore  to  OHW LQ QRQVSHFL¿F FDWLRQV LQVLGH WKH QHXURQ LQ UHVSRQVH to  blue  light.  As  such,  it  is  popular  in  neuroscience  labs  for  its  reliable  induction  of  action  potentials  when  expressed  on  neuronal  membranes.  The  idea  behind  driving  memory  recall  is  to  selectively  express  ChR-­ 2  in  neurons  that  are  active  during  fear  conditioning.  When  the  mouse  is  older,  we  can  trigger  the  same  fear  response  by  activating  precisely  those  neurons  which  ZHUH DFWLYH ZKHQ WKH UHVSRQVH ZDV ¿UVW DFTXLUHG Cornell University

 The  approach,  developed  by  Matteo  Rizzi  in  the  Hausser  lab,  is  to  express  ChR-­2  under  control  of  an  im-­ mediate  early  gene  promoter  (c-­fos-­ChR2-­EGFP)  into  a  brain  region  important  for  memory  formation  (granule  cells  of  the  dorsal  dentate  gyrus).  Then  induce  a  strong  memory  formation  via  fear  conditioning  (auditory  tone  paired  by  foot  shock).  C-­fos  is  a  protein  whose  expres-­ sion  is  rapidly  and  transiently  induced  upon  stimulation  of  neuronal  cells.  Thus,  when  the  mouse  undergoes  fear  FRQGLWLRQLQJ WKH QHXURQV ZKLFK ¿UH WKH PRVW ZLOO SUH-­ sumably  express  the  most  C-­fos-­ChR2-­  EGFP.  This  is  an  elegant  way  to  selectively  express  ChR2  in  neurons involved  in  fear  conditioning.  To  activate  these  neurons  with  light,  an  optical  ¿EHU ZDV LPSODQWHG LQWUD FUDQLDOO\ QHDU WKH '* :KDW they  found  was  that  medium  level  of  optical  stimulation  induced  recall  of  fear  memory,  measured  as  freezing  behavior.  What  was  most  remarkable  was  that  a  small  subpopulation  of  granule  cells  (less  than  100)  was  suf-­ ¿FLHQW WR FDXVH WKLV HIIHFW $V D FRQWURO WKH\ WUDQV-­ fected  neurons  with  a  general  promoter  (pCAG-­ChR2)  into  the  same  region  and  failed  to  induce  memory  recall  after  stimulation.  The  only  caveat  is  that  c-­fos  expres-­ sion  is  indeed  transient,  which  means  that  ChR2  levels  will  taper  off,  and  memory  recall  will  be  less  effective  as  more  time  passes  from  the  initial  fear  conditioning.  However,  this  experiment  demonstrates  that  we  can  in-­ deed  trigger  memory  recall  and  all  the  behavioral  re-­ sponses  associated  with  it  by  just  a  click  of  a  button.

Reference Rizzi,  M.,  Powell,  K.,  Hefendehl,  J.,  Fernandes,  A.  &  Häusser  M.  â&#x20AC;&#x153;Memory  recall  driven  by  optical  stimulation  of  functionally  iden-­ WLÂżHG VXE SRSXODWLRQV RI QHXURQV´ 3RVWHU ** 6RFLHW\ for  Neuroscience  annual  meeting,  2009).

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RESEARCH

Vol 5 ¦ 2011

Anti-Epileptic Drugs and Seizure Severity in Bss1 and Sda Drosophila

Kaitlin Hardy Agriculture and Life Sciences 12, Biological Sciences

Methods

Abstract FACES;;  Facts,  Advocacy,  and  Control  of  Epileptic  6HL]XUHV EHJDQ DV D QRQ SUR¿W RUJDQL]DWLRQ GHGLFDW-­ ed  to  epilepsy  outreach  with  the  goal  of  erasing  any  negative  misconceptions  surrounding  seizure  disor-­ ders  and  providing  direct  assistance  to  people  of  all  ages  living  with  epilepsy.   The  FACES  lab  at  Cornell  is  the  only  research  lab  entirely  run  by  undergradu-­ ates  in  the  history  of  the  university.   Through  the  use  of  two  bang  sensitive  Drosophila  Melanogaster  mutants  bang  senseless  (bss1)  and  slamdance  (sda),  the  FACES  lab  is  investigating  the  neural  mecha-­ nisms  of  commonly  prescribed  anti-­epileptic  drugs  (AEDs)  with  a  focus  on  the  commonly  prescribed  brand  name  drugs  Lamictal  (Lamotrigine)  and  Keppra  XR  (Levitracetam).

Each  AED  is  administered  to  Drosophila  at  differ-­ ent  dosages  beginning  at  0.015  mg/ml  and  increas-­ ing  until  a  maximum  dosage  of  0.4  mg/ml  is  reached.   )OLHV DUH H[SRVHG WR WKH $(' IRU ¿YH GD\V GXULQJ which  they  are  tested  once  for  seizure  sensitivity  through  vortex  at  intervals  of  ten  seconds.   Both  EVV DQG VGD 'URVRSKLOD GLVSOD\HG WKH PRVW VLJQL¿-­ cant  reduction  in  each  measure  of  seizure  behavior  and  severity  when  tested  after  three  days  on  0.15  PJ PO .HSSUD ;5 %RWK EVV DQG VGD Ã&#x20AC;\ PXWDQWV showed  reduction  across  all  measures  of  seizure  be-­ havior  after  three  days  of  exposure  to  0.15  mg/ml  Lamictal  as  well,  though  to  a  lesser  degree.   %RWK EVV DQG VGD Ã&#x20AC;LHV UHVSRQG SRVLWLYHO\ WR WKH human  AEDs  Keppra  and  Lamictal  when  adminis-­ WHUHG DW VSHFL¿F GRVDJHV DQG ZLWKLQ D QDUURZ PDU-­ gin  of  time  elapsed  since  initial  exposure.   Future  tests  will  include  administration  of  alternate  anti-­ epileptic  drugs.

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7KH VDPH EDVH DJDU PL[ ZDV XVHG IRU HDFK IHHG-­ LQJ FRQGLWLRQ DQG LQFOXGHG 4XDNHU <HOORZ &RUQPHDO $JDU 7\SH ,, 7DWH DQG /\OH FRUQ V\UXS VROLGV /\QVLGH 1XWUL ,QDFWLYH 1XWULWLRQDO <HDVW DQG $'0 VR\ Ã&#x20AC;RZHU The  food  that  contained  AEDs  did  not  include  excess  ZDWHU DV D UHVXOW RI WKH DGGLWLRQ RI GLVVROYHG $('V DQG DOO $('V ZHUH JURXQG LQWR D XQLIRUP ¿QH SRZGHU EH-­ IRUH GLVVROYHG LQ GLVWLOOHG ZDWHU $V D UHVXOW DOO IRRG SODWHV KDG WKH VDPH RYHUDOO FRQVLVWHQF\

$ WRWDO VDPSOH VL]H RI Ã&#x20AC;LHV RI HDFK JHQR-­ W\SH DQG IRU HDFK RI WKH ¿YH GD\V RI WHVWLQJ was  ex-­ posed  WR HLWKHU RI WKH GD\V RI WHVWLQJ ZDV H[SRVHG WR HLWKHU RQH RI WKH ¿YH $(' FRQWDLQLQJ IRRG HQYLURQ-­ PHQWV RU WKH FRQWURO QRQ $(' IRRG HQYLURQPHQW ,Q WRWDO GDWD IRU DW OHDVW Ã&#x20AC;LHV RI HDFK JHQRW\SH LQ WRWDO ZDV REWDLQHG HDFK GD\ RYHU D ¿YH GD\ WHVWLQJ

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Cornell University

Vol 5 ¦ 2011

¦ FACES ¦

period for each of the six feeding conditions. In WRWDO VOLJKWO\ PRUH WKDQ ÀLHV ZHUH WHVWHG RYHU D SHULRG RI IRXU PRQWKV ÀLHV RI HDFK JHQRW\SH JHQRW\SHV GD\V RI WHVWLQJ VL[ IHHGLQJ FRQGLWLRQV

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RESEARCH

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RESEARCH

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Vol 5 ¦ 2011

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Cornell University

Vol 5 ¦ 2011

¦ FACES ¦

RESEARCH

FIGURE  2.  Therapeutic  does  of  Lamictal  and  its  ability  to  control  seizures  in  bss1  Drosophila.  N  =  150  for  each  group.   Procedures  for  the  testing  of  Lamictal  in  seizure  control  were  identical  to  the  procedures  followed  when  testing  Keppra  XR.   Like  the  Keppra  XR  JURXSV RI WKH LQGLYLGXDOV VWXGLHG DFURVV DOO JURXSV ERWK WKRVH WKDW ZHUH H[SRVHG WR $(' IRRG DQG WKRVH ZKR ZHUH H[SRVHG WR QRQ $(' IRRG DQG UHFRUGHG DQG KRXUV DIWHU H[SRVXUH GLG QRW GLVSOD\ DQ\ VHL]XUH EHKDYLRU DQG LPPHGLDWHO\ UHVXPHG QHJD WLYH JHRWD[LV EHKDYLRU XSRQ UHPRYDO IURP WKH YRUWH[ 'DWD IRU WKH KRXU H[SRVXUH JURXS LV LQFOXGHG LQ WKLV ¿JXUH GXH WR LOOXVWUDWH LWV LPSURYHPHQW RYHU WKH QRQ $(' H[SRVHG JURXS DQG DOPRVW LGHQWLFDO UHVXOWV WR WKH KRXU JURXS

6ZKHQ WKH\ EHJLQ LUUHJXODU UDSLG ¿ULQJ DQG UHSHWLWLYH HOHFWULFDO VLJQDOV %\ EORFNLQJ VRGLXP LRQV IURP QHUYH FHOOV LUUHJXODU DFWLYLW\ LV SUHYHQWLQJ IURP VSUHDGLQJ WKURXJK QHUYH FHOOV LQ WKH EUDLQ  It  was  expected  that  Lamictal,  a  drug  that  WDUJHWV VRGLXP FKDQQHO DFWLYLW\ ZRXOG EH VXFFHVVIXO in  controlling  seizures  in  Bss1  mutants,  a  strain  that  is  susceptible  to  seizures  as  a  result  of  a  sodium  channel  PXWDWLRQ FDXVHG E\ DQ DOOHOH RI WKH SDUDO\WLF YROWDJH gated  sodium  channel  gene.   The  mechanism  of  Keppra  XR  is  currently  unknown,  but  was  successful  in  seizure  VXSSUHVVLRQ LQ D PDMRULW\ RI EVV Ã&#x20AC;LHV WHVWHG PRVW notably  when  administered  a  dose  of  0.15  mg/ml  for  KRXUV 7KH UHVXOWV IRU WRWDO Ã&#x20AC;LHV SDUDO\]HG DIWHU hours  of  exposure  to  a  0.15  mg/ml  AED  agar  mixture  of  each  medication  were  statistically  identical  with  RI WKH WRWDO Ã&#x20AC;LHV WHVWHG UHVXPLQJ QHJDWLYH JHRWD[LV EHKDYLRU LPPHGLDWHO\ 5HFRYHU\ WLPH DIWHU SDUDO\VLV was  also  extremely  similar  for  each  AED.   Data  for  Lamictal  was  more  consistent  across  the  48,  72,  and  96  KRXU WLPH LQWHUYDOV KRZHYHU IRU WKH PRVW HIIHFWLYH GRVH and  duration  of  exposure  (0.15  mg/ml  for  72  hours)  EVV PXWDQWV RQ .HSSUD KDG D VOLJKWO\ IDVWHU DYHUDJH UHFRYHU\ WLPH 2Q DYHUDJH RI Ã&#x20AC;LHV LQ WKLV JURXS UHFRYHUHG E\ VHFRQGV DIWHU YRUWH[LQJ 7KH VDPH experimental  group  in  terms  of  dosage  administered  and  time  of  exposure  that  was  administered  Lamictal  LQVWHDG RI .HSSUD ;5 UHDFKHG DQ UHFRYHU\ UDWH DIWHU DQ DYHUDJH RI VHFRQGV :KLOH WKLV GLIIHUHQFH is  slight,  it  does  fall  within  the  range  of  statistical  VLJQL¿FDQFH Cornell University

 This  suggests  that  a  possible  mechanism  RI .HSSUD ;5 LQFOXGHV VRPH IRUP RI SRVLWLYH LQWHUYHQWLRQ LQ VRGLXP FKDQQHO IXQFWLRQ D SRVVLELOLW\ is  an  ion  blocking  mechanism  similar  to  that  utilized  by  Lamictal.   It  is  also  possible  that  sodium  channel  LQWHUYHQWLRQ LV RQH RI VHYHUDO PHWKRGV WKURXJK ZKLFK .HSSUD ;5 VXSSUHVVHV VHL]XUH DFWLYLW\  It  was  expected  that  bss1  mutants  would  display  greater  phenotypic  differences  than  sda  mutants  when  H[SRVHG WR WKH YRUWH[ WHVW DV EVV DUH WKH PRVW VHYHUH RI WKH EDQJ VHQVLWLYH 'URVRSKLOD PXWDQWV DQG SDVW VWXGLHV KDYH QHYHU FRQVLVWHQWO\ DFKLHYHG VXFFHVV LQ seizure  suppression.  Statistical  analysis  of  data  collected  in  this  study  ZDV IRFXVHG RQ LPPHGLDWH SDUDO\VLV DIWHU YRUWH[LQJ 1XPEHU RI LQGLYLGXDOV SDUDO\]HG LV UHSUHVHQWDWLYH RI Ã&#x20AC;LHV WKDW DYRLGHG VHL]XUHV DOWRJHWKHU DQG LV WKHUHIRUH PRVW DQDORJRXV WR $(' FRQWUROOHG VHL]XUH DFWLYLW\ LQ humans.   In  order  for  AEDs  to  be  considered  successful  in  treatment  for  people  with  epilepsy,  they  would  H[SHULHQFH QR SDUDO\VLV RU VHL]XUH DFWLYLW\ DW DOO 7KLV PHDVXUH RI Ã&#x20AC;\ DFWLYLW\ PDWFKHV WKH JXLGHOLQHV IRU successful  AED  use  in  humans.

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Cloning and characterization of slo family potassium channel isoforms in lobster nervous system Vinay Patel Arts and Sciences 10, Biological Sciences

Abstract

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PXOWLSOH YDULDQWV DQG ZHUH WKXV GH¿QHG DV DOWHU-­ In  the  nervous  system,  potassium  channels  help  QDWH VSOLFLQJ VLWHV ; ; )LQDOO\ RXU YROWDJH FODPS maintain  the  neuronâ&#x20AC;&#x2122;s  resting  potential,  decrease  H[SHULPHQWV KDYH KHOSHG SURJUHVV WRZDUGV GH¿QLQJ a  neuronâ&#x20AC;&#x2122;s  overall  excitability,  and  shape  neuronal  WKH SURSHUWLHV RI WKH VOR DOSKD VXEXQLW ¿ULQJ SDWWHUQV 2QH SDUWLFXODU VXEIDPLO\ RI SRWDV-­ sium  channels,  the  big  K  (BK)  calciumdependent  Introduction SRWDVVLXP FKDQQHOV FRQWULEXWH VLJQL¿FDQWO\ WR WKH WRWDO SRWDVVLXP FRQGXFWDQFH RI WKH QHXURQ :H  Ion  channels  are  essential  to  the  functioning  H[DPLQHG WKH PROHFXODU EDVLV IRU WKHVH LPSRUWDQW of  the  nervous  system.  They  provide  the  currents  that  FKDQQHOV LQ QHUYRXV WLVVXH IURP WKH OREVWHU 3DQXOL-­ change  the  neuronâ&#x20AC;&#x2122;s  membrane  potential,  which  under-­ UXV LQWHUUXSWXV &DOFLXP DFWLYDWHG SRWDVVLXP FXU-­ lies  its  excitability  and  ability  to  transmit  information.  UHQWV SOD\ FULWLFDO UROHV IRU H[DPSOH LQ VSLNH WHUPL-­ 3RWDVVLXP FKDQQHOV VSHFL¿FDOO\ KHOS PDLQWDLQ WKH QHX-­ nation  and  plateau  potential  termination  in  neurons  ronâ&#x20AC;&#x2122;s  resting  potential,  decrease  its  overall  excitability,  IURP WKH OREVWHU VWRPDWRJDVWULF JDQJOLRQ 67* $ DQG VKDSH QHXURQDO ¿ULQJ SDWWHUQV E\ EHLQJ ODUJHO\ UH-­ ODUJH IDPLO\ RI %. FKDQQHOV ZLWK YDULDEOH SURS-­ sponsible  for  repolarization  after  an  action  potential,  HUWLHV LV GHULYHG IURP WKH VOR JHQH DV D UHVXOW RI thus  setting  spike  frequency1.

DEXQGDQW DOWHUQDWLYH VSOLFLQJ RI LWV 51$ PRGL¿FD-­  One  particular  subfamily  of  potassium  chan-­ WLRQ E\ UHJXODWRU\ È&#x2022; VXEXQLWV DQG PHWDEROLF UHJX-­ nels,  termed  big  K  (BK)  calcium-­dependent  potassium  ODWLRQ :H XVHG VOR VHTXHQFHV IURP RWKHU VSHFLHV FKDQQHOV FRQWULEXWH VLJQL¿FDQWO\ WR WKH WRWDO SRWDV-­ GHJHQHUDWH 3&5 DQG ¶ DQG ¶ 5DSLG $PSOL¿FDWLRQ sium  conductance  of  the  neuron.  This  class  of  potas-­ RI F'1$ HQGV 5$&( PHWKRGV WR FORQH WKH VOR VH-­ sium  channel  is  well  regulated,  with  its  conductance  TXHQFH IURP OREVWHU QHUYRXV WLVVXH :H DOVR LGHQWL-­ controlled  in  a  calcium  and  voltage  dependent  manner  ¿HG DQG IXUWKHU H[DPLQHG VHYHQ DOWHUQDWH VSOLFLQJ (Salkoff  et  al.,  2006).  A  large  family  of  BK  channels  VLWHV ZLWK 3&5 )LQDOO\ ZH SUHSDUHG DQG LQMHFWHG is  derived  from  the  slo1  gene  as  a  result  of  abundant  51$ IURP VHOHFW FORQHG VHTXHQFHV LQWR ;HQRSXV DOWHUQDWLYH VSOLFLQJ RI LWV 51$ PRGL¿FDWLRQ E\ UHJX-­ RRF\WHV DQG XVHG VWDQGDUG WZR HOHFWURGH YROWDJH latory  B-­subunits,  and  metabolic  regulation.  The  pro-­ clamp  methods  to  attempt  to  characterize  the  chan-­ teins  encoded  by  this  gene,  which  was  initially  cloned  QHO SURSHUWLHV :H REWDLQHG XQLTXH DQG YLDEOH and  studied  in  Drosophila  melanogaster,  display  a  large  IXOO OHQJWK VHTXHQFHV UDQJLQJ IURP WR WR-­ versatility  in  their  characteristics  and  properties,  with  WDO DPLQR DFLGV LQ OHQJWK ZLWK DQG wide  differences  in  their  voltage  and  calcium  sensitiv-­ KRPRORJ\ WR FUDE 'URVRSKLOD DQG FRFN-­ ity,  kinetics  of  activation  and  inactivation,  and  single  URDFK QXFOHRWLGH VHTXHQFHV UHVSHFWLYHO\ $OLJQPHQW channel  currents2.

RI WKH IXOO OHQJWK VHTXHQFHV VKRZHG LQ IUDPH GLIIHU-­  The  slo1gene  is  highly  conserved  among  23spe-­ HQFHV DW VHYHQ LGHQWL¿HG ORFDWLRQV ZKLFK IHDWXUHG cies,  and  encodes  the  alpha  subunit  of  the  BK  channel.  Synapse

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Â

Figure  1:  A)  Transmembrane  organization  of  the  slo1  alpha  subunit  protein  when  inserted  into  the  cell  membrane.  The  subunit  features  an  extracellular  N-­terminus,  seven  transmembrane  segments  (S0-­S6)  and  their  associated  linker  regions,  which  include  the  voltage  sensing  (S4)  and  pore  forming  (P)  segments,  two  intracellular  regulators  of  conductance  of  po-­ tassium  domains  (RCK  1  and  2),  an  intracellular  calcium  bowl  (Ca2+  sensing)  domain,  and  intracellular  C-­terminus.  The  PDMRULW\ RI WKH VXEXQLW LV FRQVHUYHG FRPSDUHG WR WKRVH RI RWKHU VSHFLHV 7KH YDU\LQJ VSOLFH VLWHV ; ; DUH LGHQWL¿HG %

$PLQR DFLG VHTXHQFH RI FORQH / 7KLV FORQH HQFRGHV D SURWHLQ RI $$ 7KH IXQFWLRQDOO\ VLJQL¿FDQW SRUWLRQV DQG VSOLFH VLWHV DUH LGHQWL¿HG $W WKH VSOLFH VLWHV WKH RULJLQDO / VHTXHQFH LV VKRZQ DV WKH WRS EUDQFK ZKLOH DOWHUQDWLYH VHTXHQFHV LGHQWL¿HG DUH VKRZQ XQGHUQHDWK 3K\VLRORJLFDOO\ LPSRUWDQW UHJLRQV DUH ODEHOHG 5&. LV WKH ¿UVW VHJPHQW RI LWDOLFL]HG OHW-­ ters,  while  RCK2  is  the  second.

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It  contains  an  extracellular  N-­terminus,  seven  transmembrane  domains  (S0-­S6),  two  regulator  of  conductance  of  potassium  (RCK)  domains,  a  calcium  sensing  â&#x20AC;&#x153;calcium  bowl,â&#x20AC;?  four  hydrophobic  domains  (S7-­S10),  and  intracellular  C-­terminus  (Fig.  1).  These  regions  are  generally  categorized  as  the  â&#x20AC;&#x153;coreâ&#x20AC;?  consisting  of  the  transmembrane  segments,  and  the  â&#x20AC;&#x153;tailâ&#x20AC;?  region  consisting  of  the  S9  to  C-­terminus  region.  The  folded  product  results  in  a  K+  selective  pore  and  membrane  spanning  voltage  sensor  from  the  core  region,  and  intracellular  calcium  sensing  and  channel  regulating  regions  from  the  tail2.  In  addition  to  the  alpha  subunit,  BK  channels  DOVR IHDWXUH Č&#x2022; VXEXQLWV WKDW PDLQO\ VHUYH D UHJXODWRU\ purpose;Íž  they  have  intracellular  N-­  and  C-­termini  and  two  transmembrane  segments  and  interact  with  the  alpha  subunit  at  the  S0  transmembrane  segment3.  This  interaction  has  been  seen  to  cause  variable  effects.  One  study  noted  increased  Ca2+  sensitivity,  decreased  voltage  dependence,  and  slowing  of  channel  kinetics4.  Other  studies  using  smooth  muscle  showed  that  co-­ expression  of  alpha  and  beta  subunits  caused  increased  YROWDJH DQG &D VHQVLWLYLW\ ,Q DGGLWLRQ Č&#x2022; VXEXQLWV KDYH EHHQ QRWHG WR UHJXODWH %. WUDIÂżFNLQJ XOWLPDWHO\ reducing  steady-­state  BK  surface  expression  levels5.  Though  both  subunits  are  generally  co-­expressed,  only  alpha-­subunit  expression  is  necessary  to  obtain  BK  currents  in  Xenopus  oocytes6.  One  unique  characteristic  of  the  gene  is  its  large  variability  due  to  alternative  splicing.  Across  species,  slo1  has  between  two  and  seven  alternative  splicing  sites,  resulting  in  multiple  different  potential  transcripts7,  8,  9,  10,  11.  In  humans,  alternatively  spliced  transcripts  are  differentially  expressed  throughout  the  brain,  demonstrating  functional  importance  of  the  variability  on  a  physiological  level9.  The  variable  properties  of  these  alternative  BK  channels,  in  addition  to  the  fact  that  calcium-­activated  potassium  currents  play  critical  roles  in  spike  termination12  and  plateau  potential  termination13  within  the  stomatogastric  ganglion  (STG),  makes  examining  BK  channels  within  lobster  nervous  tissue  especially  interesting14.  The  spiny  lobster  (Panulirus  interruptus)  STG  is  often  used  as  a  model  system  for  studying  central  pattern  generation15.  The  14  neuron  pyloric  network  within  the  STG  is  a  simple  well  studied  network  of  six  cell  types  that  is  rhythmically  active.  BK  channels  could  play  an  important  role  in  regulating  the  pyloric  network,  as  they  have  previously  been  noted  to  set  the Â

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plateau  phase  of  oscillatory  neurons  as  well  as  help  to  terminate  oscillations16.  Furthermore,  BK  channels  have  been  seen  to  co-­localize  with  voltage-­dependent  Ca2+  channels,  and  thus  could  serve  as  an  essential  negative  feedback  mechanism  to  regulate  neurotransmitter  release17,  18.  Thus,  examining  the  molecular  and  genetic  VWUXFWXUH DQG IXQFWLRQ RI %. FKDQQHOV VSHFL¿FDOO\ those  encoded  by  the  slo1  gene  would  provide  a  direct  link  and  deeper  understanding  of  how  physiological  processes  of  the  STG  and  other  rhythmic  networks  may  be  regulated.  To  explore  the  properties  of  slo1  and  BK  channels  within  the  spiny  lobster,  we  established  three  PDLQ JRDOV 7KH ¿UVW DLP ZDV WR FORQH WKH VOR JHQH from  lobster  nervous  tissue  using  degenerate  PCR  and  œ DQG œ 5DSLG $PSOL¿FDWLRQ RI F'1$ HQGV 5$&(

methods.  The  second  aim  was  to  identify  the  alternate  splicing  sites  in  PIslo1,  and  then  identify  the  different  sequences  present  within  each  site.  Finally,  the  third  aim  was  to  characterize  channel  properties  by  injecting  Xenopus  laevis  oocytes  with  our  transcripts  and  using  standard  two-­electrode  voltage  clamp  methods  to  measure  BK  currents.

Methods RNA  Isolation  Lobster  nervous  tissue  (brain,  abdominal,  &  thoracic  ganglia)  was  dissected  from  several  lobsters  and  used  to  isolate  total  RNA  using  the  RNAqueous-­4PCR  NLW $PELRQ &RQWDPLQDWLQJ '1$ ZDV UHPRYHG XVLQJ '1DVH , WUHDWPHQW DW R IRU PLQXWHV 7KH TXDOLW\ RI WKH 51$ ZDV FRQ¿UPHG E\ 51$ JHO HOHFWURSKRUHVLV this  showed  the  presence  of  18S  rRNA  (~2kb)  and  28S  rRNA  (~5kb),  demonstrating  lack  of  degradation.

Reverse-­transcription  PCR  (RT-­PCR)  and  initial  FORQLQJ RI WKH ¿UVW 3,VOR IUDJPHQW

,VRODWHG DQG SXULÂżHG 51$ ZDV UHYHUVH WUDQVFULEHG WR F'1$ DQG DPSOLÂżHG XVLQJ 6XSHUVFULSW ,,, 57 .LW ,QYLWURJHQ 7KH WRWDO 51$ ZDV ÂżUVW UHYHUVH WUDQVFULEHG LQWR SULPDU\ F'1$ VWUDQGV XVLQJ DQ ROLJR dT  primer.  The  accuracy  of  the  reverse  transcription  was  checked  using  primers  designed  to  amplify  a  highly  conserved  â&#x20AC;&#x153;housekeepingâ&#x20AC;?  gene  rig/S15  (Platinum  TAQ  Polymerase,  Invitrogen).  Primers  pairs  Slo6/7  &  Slo3/4  (Table  1)  were  used  to  successfully  amplify  873bp  and  520bp  bands  respectively  based  on  highly  conserved  regions  of  the  gene.  All  fragments  were

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cloned  into  pGEM-­T  Easy  vector  (Promega)  and  sequenced  for  accuracy.

'HJHQHUDWH 3&5 ¶ ¶ 5DSLG $PSOL¿FDWLRQ RI F'1$ Ends  (5â&#x20AC;&#x2122;/3â&#x20AC;&#x2122;  RACE)  To  extend  the  cloned  sequence  of  PIslo1,  degenerate  primers  were  developed  against  alignedslo1sequences  from  crab,  Drosophila,  and  cockroach.  These  were  used  in  PCRs  to  obtain  varying  length  overlapping  fragments  (Platinum  TAQ  Polymerase,  Invitrogen)  which  were  cloned  into  pGEM-­T  Easy  vector  (Promega)  and  sequenced.  Overlapping  fragments  were  combined  and  aligned  with  respect  to  the  original  sequences  to  further  HQVXUH DFFXUDF\ 3,VOR VSHFL¿F SULPHUV VOR1*63 VOR1*63 7DEOH ZHUH GHYHORSHG IRU WKH ¶ DQG ¶ 5$&( UHDFWLRQV EDVHG RQ ES DQG ES IUDJPHQWV REWDLQHG IURP GHJHQHUDWH 3&5V UHVSHFWLYHO\ ¶ DQG ¶ 5$&( SURFHGXUHV ZHUH FDUULHG RXW XVLQJ SURWRFROV RXWOLQHG LQ ¶ DQG ¶ 5$&( 6\VWHPV IRU UDSLG DPSOL¿FDWLRQ RI F'1$ HQGV NLWV *LEFR %5/ 8.

(QG WR (QG 3&5

$IWHU WKH ¶ DQG ¶ WHUPLQDO VHTXHQFHV ZHUH obtained  by  the  RACE  procedures,  two  reactions  were  performed  to  obtain  the  complete  open  reading  frame  25) IRU 3,VOR 3ULPHU SDLU 6OR(( 6OR((5HY (Table  1)  was  designed  outside  the  ORF  to  amplify  our  VSHFL¿F F'1$ RI LQWHUHVW $IWHU REWDLQLQJ WKH H[SHFWHG EDQG DW a NE SULPHUV 6OR(( DQG 6OR((5HY ZHUH GHVLJQHG QHVWHG ZLWKLQ WKH ¿UVW VHTXHQFH DQG Ã&#x20AC;DQNLQJ the  ORF  (Platinum  TAQ  Polymerase,  Invitrogen).  The  products  were  cloned  into  pDrive  vector  (Invitrogen)  and  sequenced. $OWHUQDWH 6SOLFLQJ ,GHQWL¿FDWLRQ

3ULRU WR DOWHUQDWH VSOLFLQJ LGHQWL¿FDWLRQ 51$ Isolation  and  RTPCR  were  performed  on  newly  dissected  WLVVXH WR REWDLQ YLDEOH F'1$ ZKLFK ZDV FKHFNHG IRU TXDOLW\ ZLWK SULPHU SDLUV 6OR(( 6OR((5HY XQLTXH IXOO OHQJWK FORQHV ZHUH LGHQWL¿HG LQ HQGWR end  PCR.  The  amino-­acid  sequences  from  each  clone  were  aligned  using  MegAlign  8  software  (Clustal  W  method),  demonstrating  seven  sites  of  variability;;  these  ZHUH VHTXHQWLDOO\ QDPHG ; ; EDVHG RQ WKHLU UHODWLYH SUR[LPLW\ WR WKH 1 WHUPLQXV

3ULPHU SDLUV ZHUH GHVLJQHG ; ; )RUZDUG 5HYHUVH Ã&#x20AC;DQNLQJ HDFK VLWH DPSOL¿HG 3ODWLQXP 7$4 3RO\PHUDVH ,QYLWURJHQ DQG VHTXHQFHG 1R YLDEOH Cornell University

Table  1:  )RUZDUG DQG UHYHUVH SULPHUV XVHG IRU HQG WR HQG DQG VSOLFH VLWH LGHQWL¿FDWLRQ ZLWK DVVRFLDWHG DQQHDOLQJ WHPSHUDWXUHV ; 5HYHUVH SULPHU ZDV XVHG IRU ERWK ; DQG ; IRUZDUG 3&5 UHDFWLRQV

SULPHUV FRXOG LVRODWH ; LQGHSHQGHQWO\ DQG WKXV ; ZDV FRPELQHG ZLWK WKH ; VHTXHQFH WKXV UHVXOWLQJ LQ WKH K\EULG VSOLFH LGHQWL¿FDWLRQ VLWH ; ;HQRSXV RRF\WH ([SUHVVLRQ

3,VOR VSOLFH YDULDQWV & % % ZHUH OLQHDUL]HG ZLWK 6SH, DQG UHYHUVH WUDQVFULEHG LQ YLWUR ZLWK 7 0HVVDJH 0DFKLQH NLW $PELRQ 6SOLFH YDULDQWV $ ' ZHUH OLQHDUL]HG ZLWK 1RW, DQG UHYHUVH WUDQVFULEHG LQ YLWUR ZLWK 7 0HVVDJH 0DFKLQH .LW $PELRQ 7KH OLQHDUL]HG 51$ WUDQVFULSWV ZHUH SXUL¿HG ZLWK WKH 51HDV\ PLQL NLW 4LDJHQ 6WDJH 9 WR 9, RRF\WHV ZHUH VXUJLFDOO\ UHPRYHG IURP IHPDOH Xenopus  laevis  frogs  that  had  been  anesthetized  using  06 DPLQREHQ]RLF DFLG HWK\O HVWHU IRU minutes  or  until  unconscious.  The  oocytes  were  washed  DQG WUHDWHG IRU PLQXWHV ZLWK PJ P/ FROODJHQDVH W\SH ,$ LQ 2RF\WH 5LQJHU 25 VROXWLRQ P0 1D&O P0 .&O P0 0J&O P0 +(3(6 DGMXVWHG WR S+ ,VRODWHG RRF\WHV ZHUH WKHQ LQMHFWHG ZLWK YDU\LQJ DPRXQWV RI 3,VOR 51$V Q/ WKURXJK a  micropipette.  This  was  performed  using  a  homemade  SUHVVXUH LQMHFWRU DQG D SXOVH JHQHUDWRU 0DVWHU $03, -HUXVDOHP ,VUDHO WR GHOLYHU SUHVVXUH SXOVHV SVL K= PV GXUDWLRQ ,QMHFWHG RRF\WHV ZHUH WKHQ FXOWXUHG LQ 1' VROXWLRQ FRQWDLQLQJ P0 1D&O P0 .&O P0 &D&O + 2 P0 0J&O DQG P0 +(3(6 S+ VXSSOHPHQWHG ZLWK PJ / JHQWDPLFLQ P0 1D S\UXYDWH DQG KRUVH VHUXP the  solution  was  changed  every  day  until  the  end  of  the

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ÂŚ Lobster Nervous System ÂŚ

experiment.  Standard  two-­electrode  voltage  clamp  recordings  were  performed  using  ND-­96  Recording  solution  containing  100mM  NaCl,  2mM  KCl,  2mM  CaCl2(2H2O),  5mM  HEPES,  1mM  MgCl2(6H2O),  pH  7.5.  Several  recordings  were  performed  in  chloride  free  ND-­96,  composed  of  96mM  Sodium  Gluconate,  2mM  Potassium  Gluconate,  2.7mM  Cacium  Gluconate,  1mM  Magnesium  Gluconate,  5mM  HEPES,  pH  7.5.  In  some  experiments,  we  applied  the  chloride  channel  blocker  anthracene  9-­carboxylate  (2.5mM)  in  chloride-­free  ND96.  The  calcium  ionophore  A23187  was  applied  in  Ca2+-­free  ND96  (96mM  NaCl,  2mM  KCl,  1mM  MgCl2,  and  5mM  HEPES  adjusted  to  pH  7.6).

Results Cloning  and  Sequencing  of  PIslo1

2XU ÂżUVW JRDO ZDV WR REWDLQ IXOO 3,VOR RSHQ reading  frame  (ORF).  This  was  accomplished  through  a  combination  of  degenerate  PCR  and  5â&#x20AC;&#x2122;  and  3â&#x20AC;&#x2122;  RACE  procedures.  Degenerate  primers  were  designed  based  RQ FRQVHUYHG UHJLRQV RI SUHYLRXVO\ LGHQWLÂżHG VOR sequences  in  other  species  to  obtain  intermediate  overlapping  fragments  of  the  gene.  These  overlapping  fragments  were  combined  and  compared  to  the  slo1  sequences  from  other  species.  The  degenerate  PCR  also  provided  two  fragments  (839bp  and  874bp)  which  ZHUH XVHG WR GHVLJQ 3,VOR VSHFLÂżF SULPHUV VOR1*63 and  sloNGSP2  for  the  5â&#x20AC;&#x2122;  and  3â&#x20AC;&#x2122;  RACE  procedures  respectively.  The  5â&#x20AC;&#x2122;  RACE  experiment  yielded  a  1,099bp  fragment  containing  the  start  codon,  while  the  3â&#x20AC;&#x2122;  RACE  experiment  provided  a  610bp  fragment  containing  the  stop  codon.  With  this  information  two  additional  primer  pairs  (SloEE1/SloEERev1  &  SloEE2/ 6OR((5HY ZHUH GHVLJQHG Ă&#x20AC;DQNLQJ WKH HQWLUH 25) which  were  used  to  obtain  full  length  slo1  sequences.  End  to  end  cloning  of  slo1  resulted  in  an  open  UHDGLQJ IUDPH VHTXHQFH VLPLODU WR WKRVH LGHQWLÂżHG LQ other  species  (Fig  1).  The  percent  homology  of  the  PIslo1  nucleotide  sequence  to  crab,  Drosophila,  and  cockroach  is  96.6%,  82.5%,  and  82.9%  respectively.  The  hypothesized  protein  structure  should  thus  closely  UHVHPEOH WKH SUHYLRXVO\ LGHQWLÂżHG PRUSKRORJ\ RI WKH slo  alpha  subunit  (Figure  1A).

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cloned  21  full  length  ORFs  from  PIslo1  and  sequenced  DOO RI WKHP 2YHUDOO WKLV HQG WR HQG FORQLQJ LGHQWL¿HG 13  distinct,  viable  full  length  sequences.  Of  the  eight  other  clones,  two  were  copies  of  clones  A49  and  C35,  and  six  were  sequences  featuring  nonsense  mutations.  The  13  sequences  ranged  from  1107  to  1164  total  amino  acids  in  length  (3324  nucleotides  to  3495  nucleotides),  corresponding  to  a  range  of  123.5kDa  to  130.0kDa.  Alignment  of  the  amino  acid  sequences  for  the  13  clones  showed  conservation  among  six  of  the  seven  transmembrane  regions,  regulators  of  conductance  of  potassium  (RCK)  regions,  and  calcium  bowl  regions.  7KHUH ZHUH LQ IUDPH GLIIHUHQFHV DW VHYHQ LGHQWL¿HG locations,  which  showed  multiple  variants  and  were  WKXV GH¿QHG DV DOWHUQDWH VSOLFLQJ VLWHV ; ; EDVHG on  their  relative  proximity  to  the  N-­terminus  (Figure  $ DQG % )LYH RI WKH VHYHQ VSOLFH VLWHV ; ; DUH located  downstream  of  the  transmembrane  regions,  thus  representing  intracellular  portions  of  the  subunit.  The  remaining  two  sites  occur  in  the  S1-­S2  linkerregion  ; DQG LQ WKH 3 6 OLQNHU UHJLRQ DV ZHOO DV LQ WKH 6 WUDQVPHPEUDQH UHJLRQ ;

 We  further  analyzed  each  of  the  seven  alternative  splicing  sites.  The  PCR  reactions  from  experiments  XVLQJ SULPHU SDLUV Ă&#x20AC;DQNLQJ HDFK RI WKH VHYHQ VSOLFH VLWHV VKRZHG VLJQLÂżFDQW YDULDWLRQ LQ EDQG VL]H ZKHQ UXQ RQ D DJDURVH JHO DQG VRPH SULPHU SDLUV DPSOLÂżHG multiple  bands  at  a  splice  site  (Figure  2).  Based  on  the

Figure  2:  3.5%  agarose  gel  of  X1-­X7  PCR  products  using  SULPHU SDLUV Ă&#x20AC;DQNLQJ HDFK VLWH ; IHDWXUHV RQH EDQG ; Alternative  Splicing  in  PIslo1 IHDWXUHV WZR EDQGV ; IHDWXUHV WKUHH EDQGV ; IHDWXUHV

6LJQL¿FDQW DOWHUQDWLYH VSOLFLQJ LV FRPPRQ RI DOO EDQGV ; IHDWXUHV EDQGV ; IHDWXUHV EDQGV DQG slo1  genes  that  have  been  characterized  so  far.  To  begin  ; IHDWXUHV EDQGV (DFK EDQG DPRQJ WKH GLIIHUHQW VLWHV a  search  for  splice  variants  in  the  lobster  PIslo1,  we  UHSUHVHQWV RQH RU PRUH VSOLFH YDULDQW V LGHQWL¿HG

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ÂŚ Lobster Nervous System ÂŚ

variation  observed  at  each  splice  site  in  the  full  length  clones,  we  expected  to  obtain  varying  length  products  from  each  reaction.  These  products  would  vary  by  the  numbers  of  nucleotides  in  each  different  splice  sequence.  Thus,  each  band  that  appeared  represented  one  or  more  splice  variant(s)  for  that  site.  Any  sequences  that  varied  by  <10  nucleotides  generally  appeared  as  one  band  with  a  slight  smear  due  to  an  inability  to  properly  separate  them  on  the  3.5%  agarose  gel.  Since  the  bands  represented  different  splice  sequences,  each  was  isolated,  cloned  and  sequenced  to  identify  additional  sequences  not  present  in  the  full  length  clones.  In  total,  GLIIHUHQW VSOLFH VHTXHQFHV ZHUH LGHQWLÂżHG DFURVV WKH seven  sites,  seven  of  which  were  not  present  in  the  full  length  clones  (Table  2).  Ultimately  all  22  sequences  observed  in  full  length  clones  and  7  novel  sequences  were  obtained.  We  failed  to  clone  the  largest  band  in  X7  (~350bp),  the  largest  band  in  X6  (~340bp),  and  the  two  largest  bands  in  X3  (~390  &  410bp).  Similar  experimental  procedures  were  followed  but  failed Â

RESEARCH

to  yield  a  clone  with  appropriate  sized  inserts  when  checked  by  EcoRI  digestion  and  gel  electrophoresis.  Each  full  length  clone  was  composed  of  FRQVHUYHG UHJLRQV DV ZHOO DV D VSHFL¿F VHTXHQFH LQ each  splice  site.  Using  the  splice  sequences  in  Table  2,  we  outlined  the  composition  of  each  full  length  sequence  (Table  3).  Certain  splice  sequences  were  expressed  more  often  than  others  among  the  full  length  sequences.  The  most  common  splice  sequence  (mode)  LV LGHQWL¿HG DW WKH ERWWRP RI 7DEOH Attempt  to  express  PIslo1  In  other  species,  the  alpha  subunit  encoded  by  the  slo1  gene  is  adequate  to  form  a  functional  ion  channel.  Thus,  we  attempted  to  express  the  PIslo1  RNA  in  Xenopus  oocytes.  RNA  was  prepared  from  the  A27,  B45,  B63,  C45,  and  D2  full  length  clones  (contained  within  pDrive  vector),  and  varying  amounts  were  injected  into  Xenopus  oocytes.  After  2-­7  days  incubation  at  30oC,  we  attempted  to  record  IK(Ca)  by  Table  2:  Splice  variants  for  X1-­X7  LGHQWL¿HG WKURXJK IXOO OHQJWK VH-­ TXHQFLQJ DQG VSOLFH VLWH VHTXHQFLQJ experiments.  29  total  variants  were  REWDLQHG DFURVV WKH VHYHQ VLWHV 7KH YDULDQWV ZHUH DOLJQHG XVLQJ WKH 0HJ$OLJQ VRIWZDUH &OXVWDO : PHWKRG

Table  3:  Splice  site  composition  RI WKH FORQHV REWDLQHG WKURXJK IXOO OHQJWK VHTXHQFLQJ /HWWHUV IRU HDFK VSOLFLQJ VLWH FRUUHVSRQG WR WKRVH GHVFULEHG LQ 7DEOH 7KH WR-­ WDO QXFOHRWLGH OHQJWK DQG ZHLJKW N'D DUH SURYLGHG IRU HDFK FORQH 7KH PRGH RI HDFK VSOLFH VLWH ZKLFK VSOLFH YDULDQW DSSHDUV LQ JUHDWHVW IUHTXHQF\ LV SURYLGHG

Cornell University

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RESEARCH

ÂŚ Lobster Nervous System ÂŚ

two-­electrode  voltage  clamp  measurements  in  ND-­ 96  recording  solution,  using  a  series  of  10mV  steps  from  a  -­60mV  holding  voltage  to  +70mV.  This  was  unsuccessful:  a  total  of  120  oocytes  were  recorded,  with  no  induced  current  greater  than  the  endogenous  currents  in  the  oocytes.  After  these  failures,  additional  conditions  were  used  prior  to  running  the  voltage  clamp protocol.  Our  initial  hypothesis  was  that  the  external  Ca2+  failed  to  enter  the  cell  and  thus  could  not  activate  the  BK  channels,  which  are  both  voltage-­and  calcium-­ dependent.  To  mitigate  this,  injected  and  control  cells  ZHUH WUHDWHG ZLWK Č?0 $ D GLYDOHQW FDWLRQ VSHFLÂżF LRQRSKRUH LQ &D IUHH 1' VROXWLRQ minutes  prior  to  voltage  clamp  experiments.  During  the  voltage  clamp  measurements,  the  extracellular  VROXWLRQ ZDV VXSSOHPHQWHG ZLWK YDU\LQJ OHYHOV P0 WR P0 RI &D WR HQVXUH WKH SUHVHQFH RI VXIÂżFLHQW [Ca2+]i.  Second,  we  recorded  currents  in  chloride-­ free  solutions,  along  with  a  chloride  channel  blocker  DQWKUDFHQH FDUER[\ODWH P0 WR DYRLG FXUUHQWV from  Ca2+  activated  chloride  channels  which  are  endogenous  to  oocytes  and  could  mask  the  desired  ,. &D 1RQH RI WKHVH PRGLÂżFDWLRQV VKRZHG DQ\ expression  of  the  PIslo  channel.  Finally,  we  performed  positive  controls  with  injections  of  cloned  mouse  slo  51$ GRQDWHG E\ ' 0F&REE DQG RXU SUHYLRXVO\ expressed  lobster  shal  K+  channel  transcripts  to  check our  experimental  recording  procedures  for  accuracy.  %RWK RI WKHVH WUDQVFULSWV H[SUHVVHG ZHOO WKXV FRQÂżUPLQJ proper  injection,  incubation,  and  recording  techniques.  Additional  conditions  were  tested  by  Dr.  Qing  Ouyang,  with  consistently  negative  results.  Among  the  various  FRQGLWLRQV GLVFXVVHG QR UHOLDEOH ,. &D ZDV GHWHFWHG from  the  oocytes,  and  there  was  generally  no  difference  EHWZHHQ FRQWURO DQG LQMHFWHG FHOOV )LJXUH

Discussion

20

Cloning,  Sequencing,  and  Alternate  Splicing  of  PIslo1

$V SUHYLRXVO\ VHHQ LQ RWKHU VSHFLHV 3,VOR GHPRQVWUDWHV D VLJQL¿FDQW DPRXQW RI DOWHUQDWH VSOLFLQJ UHVXOWLQJ LQ DW OHDVW XQLTXH WUDQVFULSWV within  Panulirus  interruptus  nervous  tissue.  Based  RQ WKH VSOLFH YDULDQWV LGHQWL¿HG WKHUH DUH potential  transcripts  that  could  be  created,  assuming  an  equal  probability  of  each  variant  being  expressed.  7KURXJK RXU H[SHULPHQWV ZH LGHQWL¿HG GLIIHUHQW YLDEOH FORQHV ZLWK GXSOLFDWHV IRXQG IRU WZR FORQHV $

Vol 5 ÂŚ 2011

DQG & +DYLQJ LGHQWLÂżHG WZR GXSOLFDWHV DPRQJ WKH 21  sequenced  clones,  it  is  possible  that  the  transcripts  ZH LGHQWLÂżHG WKRXJK UDQGRP DUH PHUHO\ WKH PRVW common  among  all  that  are  expressed.  Furthermore,  the  high  degree  of  similarity  between  PIslo1  and  crab,  Drosophila,  and  cockroach  nucleotide  sequences  DQG UHVSHFWLYHO\ GHPRQVWUDWHV the  evolutionary  importance  of  BK  channels  among  species.  Though  not  all  of  these  organisms  feature  similar  rhythmic  networks,  BK  channels  are  highly  conserved  and  thus  probably  play  an  important  role  in  physiological  regulation  mechanisms.19   Among  the  seven  splice  sites,  there  are  a  few  interesting  trends.  The  X2  splice  site  is  located  in  the  P-­S6  linker  region  and  on  the  S6  transmembrane  region,  which  has  previously  been  observed  in  cockroach  slo1  sequences  and  may  be  very  important  IRU FKDQQHO IXQFWLRQ %DVHG RQ WKHLU SUR[LPLW\ WR WKH 5&. DQG &DOFLXP %RZO UHJLRQV ; ; PD\ FDXVH functional  changes  in  channel  kinetics  and  calcium  sensitivity.  This  trend  is  prominent  among  many  other  species  as  the  â&#x20AC;&#x153;tailâ&#x20AC;?  region  generally  shows  a  great  deal  of  variability.1  X1  and  X2  on  the  other  hand,  probably  have  a  direct  impact  on  the  functionality  and  morphology  of  the  subunit  being  located  among  the  transmembrane  segments.

; LQ SDUWLFXODU UDLVHV LQWHUHVW ZLWK LWV ¿YH LGHQWL¿HG VSOLFH YDULDQWV WZR RI ZKLFK DUH ODFNLQJ D majority  of  the  S6  transmembrane  sequence.  These  WZR KRZHYHU ZHUH LGHQWL¿HG GXULQJ WKH VSOLFH VLWH LGHQWL¿FDWLRQ SURFHVV DQG ZHUH QHYHU REVHUYHG GLUHFWO\ within  our  full-­length  clones,  most  likely  because  this  ZRXOG VLJQL¿FDQWO\ DOWHU FKDQQHO PRUSKRORJ\ DQG probably  inactivate  the  channel  function.  Though  we  never  observed  the  two  sequences  lacking  the  S6  coding  region  among  the  cloned  full-­length  se  quences,  they  were  present  among  at  least  one  transcript  since  all  PCR  reactions  utilized  our  complete  lobster  cDNA  library  as  a  template.  The  presence  of  these  splice  sequences  within  full-­length  transcripts  could  negatively  regulate  the  alpha  subunit  by  decreasing  the  number  of  functional  FKDQQHOV ZLWKLQ WKH PHPEUDQH FKDQQHO GHQVLW\ RU by  acting  as  a  dominant  negative  subunit  within  the  tetrameric  channel  structure  to  down-­regulate  the  number  of  functional  receptors.  These  transcripts  could  WKXV VHUYH DV D XVHIXO PHWKRG WR GHFUHDVH ,. &D

and  could  be  selectively  expressed  under  conditions  that  would  require  increased  excitability  or  greater  neurotransmitter  release  at  select  synapses.

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Vol 5 ÂŚ 2011

ÂŚ Lobster Nervous System ÂŚ

Of  further  interest  is  that  each  of  the  seven  splice  sites  features  one  variant  that  is  a  complete  removal  or  near  complete  removal  (1AA)  of  the  splice  sequence.  Five  RI WKHVH $$ YDULDQWV ZHUH LGHQWL¿HG LQ IXOO OHQJWK VHTXHQFHV ZLWK ; ; VWUD\LQJ IURP WKLV WUHQG :H DUH thus  able  to  hypothesize  that  the  X2  and  X3  alternative  exons  may  serve  a  more  functionally  important  role,  ZLWK WKH RWKHU VLWHV FRQWULEXWLQJ WR RYHUDOO FKDQQHO variability  in  less  essential  ways.  Finally,  the  S4  GRPDLQ SURYLGHV D YROWDJH JDWLQJ IHDWXUH DV SUHYLRXVO\ LGHQWL¿HG LQ RWKHU VSHFLHV 7KH LGHQWL¿HG 6 DPLQR DFLG sequence  was  [IGLRFLRALRLMSVPDIL],  consistent with  the  previous  trend  which  noted  the  presence  of  a  basic  amino  acid  (bold)  every  3AA  residues.20  %HFDXVH 6 LV DQ DOSKDKHOLFDO WUDQVPHPEUDQH VHJPHQW which  features  a  full  turn  every  3-­4  residues,  placement  RI WKHVH UHVLGXHV ZRXOG FRQFHQWUDWH SRVLWLYH FKDUJHV RQ RQH VLGH RI WKH DOSKD KHOL[ HQDEOLQJ WKH VWULQJ RI SRVLWLYH FKDUJHV WR LQWHUDFW ZLWK RWKHU DPLQR DFLGV to  stabilize  open  or  closed  states  of  the  channel.  Depolarization  causes  the  movement  of  the  residues  DZD\ IURP WKHLU LQLWLDO SDUWQHU DPLQR DFLGV ³VOLSSLQJ´ WKH FKDLQ RI SRVLWLYH FKDUJH RXWZDUG WR RSHQ WKH channel,  which  has  been  previously  seen  in  human  slo1  transcripts.21  Additional  characteristics  followed  H[SHFWHG DQG SUHYLRXVO\ VHHQ WUHQGV 7R FRQ¿UP WKHVH DQDO\VHV KRZHYHU SK\VLRORJLFDO H[SUHVVLRQ GDWD would  be  required.

Figure  3:  Two-­electrode  voltage  clamp  traces  from  control  (left)  and  RNA  injected  (right)  Xenopus  oocytes  using  ND-­96  recording  solution.  Top  traces:  voltage  recordings  of  10mV  voltage  steps  (800ms  in  duration)  from  a  holding  voltage  of  -­60mV  up  to  +70mV.  Bottom  traces:  Outward  currents  increased  sharply  in  response  to  each  step  up  to  a  maximum  of  +70mV  of  around  20nA  for  both  cells.

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Attempted  expression  of  PIslo1  Our  expression  experiments  in  Xenopus  oocytes  failed  to  detect  any  RNA-­evoked  BK  currents.  7\SLFDO FXUUHQWV REVHUYHG IURP PRXVH VOR H[SUHVVHG in  Xenopus  oocytes  demonstrate  a  rapid  increase  in  RXWZDUG FXUUHQWV UHDFKLQJ VHYHUDO Č?$ LQ DPSOLWXGH ZLWK JUDGXDO LQDFWLYDWLRQ 2XU LQLWLDO H[SHULPHQWV XVHG VWDQGDUG YROWDJH FODPS SURFHGXUHV XVLQJ 1' UHFRUGLQJ VROXWLRQ DQG P9 VWHSV IURP P9 WR +70mV  on  RNA  injected  and  control  cells  up  to  seven  days  after  injection.  However,  these  experiments  did  not  detect  any  difference  in  currents  between  injected  DQG FRQWURO FHOOV :H WKXV WHVWHG VHYHUDO K\SRWKHVHV WR address  this  failure.  Our  initial  hypothesis  was  that  the  external  Ca2+  failed  to  enter  the  cell  and  thus  could  not  activate  the  BK  complex.  Previous  studies  showed  that  nine  DFLGLF UHVLGXHV DPRQJ D SRUWLRQ RI WKH WDLO VHJPHQW WKH ÂłFDOFLXP ERZO ´ ZHUH KLJKO\ FRQVHUYHG DPRQJ VSHFLHV DQG FDXVHG VLJQLÂżFDQW FKDQJHV LQ FDOFLXP VHQVLWLYLW\ when  deleted.24  Additional  studies  demonstrated  that  the  tail  portion  (S9  to  C-­terminus)  of  the  alpha  VXEXQLW LQKLELWV %. FKDQQHO JDWLQJ ZKLFK KHOSV PDLQWDLQ WKH FORVHG VWDWH XQGHU UHVWLQJ FRQGLWLRQV It  was  thus  possible  that  if  the  oocytes  had  limited  or  no  permeability  to  Ca2+,  thus  successfully  expressed  DOSKD VXEXQLWV ZRXOG QRW RSHQ GXULQJ WKH YROWDJH FODPS H[SHULPHQWV 7R PLWLJDWH WKLV ZH WUHDWHG FHOOV ZLWK $ D GLYDOHQW FDWLRQ VSHFLÂżF LRQRSKRUH DQG VXSSOHPHQWHG WKH H[WUDFHOOXODU VROXWLRQ ZLWK YDU\LQJ OHYHOV RI &D GXULQJ YROWDJH FODPS UHFRUGLQJV 'HVSLWH VXIÂżFLHQW >&D @ WKHVH H[SHULPHQWV GLG QRW detect  any  difference  in  currents  between  injected  and  control  cells.  One  caveat  to  treatment  with  A23187  was  that  it  could  have  allowed  activation  of  additional  HQGRJHQRXV FDOFLXP DFWLYDWHG FXUUHQWV

$GGLWLRQDO FXUUHQWV HQGRJHQRXV WR WKH RRF\WH could  have  masked  our  BK  currents.  Previous  studies  showed  that  intracellular  injections  of  calcium  evoked  VWURQJ FKORULGH FXUUHQWV WKURXJK &D DFWLYDWHG chloride  channels  in  Xenopus  oocytes.10,  26  Our  application  of  A23187  and  supplementation  with  additional  calcium  could  have  induced  similar  inward  FKORULGH FXUUHQWV ZKLFK ZRXOG KDYH VLJQL¿FDQWO\ masked  any  BK  currents.  As  an  extra  check  to  ensure  these  chloride  currents  would  not  interfere  with  our  UHFRUGLQJV ZH SHUIRUPHG YROWDJH FODPS H[SHULPHQWV LQ FKORULGH IUHH VROXWLRQV DORQJ ZLWK D FKORULGH FKDQQHO EORFNHU DQWKUDFHQH FDUER[\ODWH 7KLV SURFHVV HQVXUHG

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¦ Lobster Nervous System ¦

that  any  outward  current  would  most  likely  be  due  to  BK  conductance.  Once  again,  however,  these  experiments  did  not  detect  any  difference  in  currents  between  injected  and  control  cells.  Finally,  we  performed  positive  controls  with  injections  of  previously  cloned  mouse  slo  RNA  (donated  by  Dr.  David  McCobb)  and  our  previously  expressed  lobster  shal  K+  transcripts  which  both  expressed  well.  We  thus  ensured  that  RNA  degradation  was  not  occurring  during  our  injection  procedures  and  that  incubation  and  voltage  clamp  experiments  were  maintaining  live  cells  that  could  express  injected  RNA.  It  is  possible  that  the  Xenopus  oocyte  may  lack  some  endogenous  factor  that  is  necessary  for  expression  of  PIslo1  RNA,  though  this  is  less  likely  because  of  the  successful  expression  of  shal  and  mslo  RNA  in  injected  oocytes.

$Q DGGLWLRQDO SRVVLELOLW\ LV WKDW WKH È&#x2022; VXEXQLWV that  are  generally  associated  with  the  alpha  subunits  may  be  essential  for  proper  functionality  of  PIslo1,  either  WKURXJK UHJXODWRU\ PHFKDQLVPV RU FKDQQHO WUDI¿FNLQJ to  the  membrane  surface,  both  previously  implicated  in  RWKHU VSHFLHV 7RUUHV HW DO 6LQFH WKH È&#x2022; VXEXQLWV have  such  diverse  roles,  one  possible  future  experiment  ZRXOG EH WR FORQH WKH OREVWHU VOR È&#x2022; ±VXEXQLW DQG FR H[SUHVV LW ZLWK RXU FORQHG Ä® VXEXQLW WUDQVFULSWV WR WHVW for  expression.  In  current  experiments  being  pursued,  Dr.  Qing  Ouyang  injects  RNA  into  lobster  STG  neurons  to  test  for  expression  of  enhanced  IK(Ca)  in  addition  to  the  cellâ&#x20AC;&#x2122;s  own  endogenous  currents.  The  ultimate  goal  will  be  to  understand  how  BK  channel  activity  regulates  the  rhythmic  patterns  of  the  STG,  and  then  further  apply  that  knowledge  to  understand  larger  and  more  complex  rhythmic  systems  such  as  those  involved  in  vertebrate  locomotion.

References

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/DWRUUH 5 %UDXFKL 6 /DUJH FRQGXFWDQFH &D activated  K+  (BK)  channel:  Activation  by  Ca2+  and  voltage. %LRO 5HV

2. Â Salkoff, Â L., Â Butler, Â A., Â Gonzalo, Â F., Â Santi, Â C. Â & Â Wei, Â A. Â +LJK FRQGXFWDQFH SRWDVVLXP FKDQQHOV RI WKH 6/2 IDPLO\ 1DWXUH

3.  Wallner,  M.,  Meera,  P.  &  Toro,  L.  Molecular  basis  of  fast  LQDFWLYDWLRQ LQ YROWDJH DQG &D DFWLYDWHG . FKDQQHOV D WUDQVPHPEUDQH % VXEXQLW KRPRORJ 3URF 1DWO $FDG 6FL 86$

%UHQQHU 5 -HJOD 7 - :LFNHQGHQ $ /LX < $OGULFK R.W.  Cloning  and  Functional  Characterization  of  Novel Â

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/DUJH &RQGXFWDQFH &DOFLXP DFWLYDWHG 3RWDVVLXP &KDQQHO % 6XEXQLWV K.&10% DQG K.&10% - %LRO &KHP

7RUUHV < 3 0RUHUD ) - &DUYDFKR , /DWRUUH 5 $ 0DUULDJH RI &RQYHQLHQFH % 6XEXQLWV DQG 9ROWDJH GHSHQGHQW . &KDQQHOV - %LRO &KHP (2007). +D 7 6 -HRQJ 6 < &KR 6 -HRQ + 5RK * 6 &KRL W.S.  &  Park,  C.  Functional  characteristics  of  two  BKCa  channel  variants  differentially  expressed  in  rat  brain  tissues.  (XU - %LRFKHP

-RQHV ( 0 & *UD\ .HOOHU 0 )HWWLSODFH 5 7KH UROH RI &D DFWLYDWHG . FKDQQHO VSOLFHG YDULDQWV LQ WKH WRQRWRSLF RUJDQL]DWLRQ RI WKH WXUWOH FRFKOHD - 3K\LVRORJ\

8.  Derst,  C.,  Messutat,  S.,  Walther,  C.,  Eckert,  M.,  Heinemann,  S.H.  &  Wicher,  D.  The  large  conductance  &D DFWLYDWHG SRWDVVLXP FKDQQHO S6OR RI WKH FRFNURDFK Periplaneta  Americana:  structure,  localization  in  neurons,  DQG HOHFWURSK\VLRORJ\ (XURSHDQ - 1HXURVFL (2003).

7VHQJ &UDQN - )RVWHU & ' .UDXVH & ' 0HUW] 5 *RGLQRW 1 'L&KLDUD 7 - 5HLQKDUW 3 + &ORQLQJ ([SUHVVLRQ DQG 'LVWULEXWLRQ RI )XQFWLRQDOO\ GLVWLQFW &D Activated  K+  Channel  Isoforms  from  Human  Brain.  Neuron. Â

$GHOPDQ - 3 6KHQ . .DYDQDXJK 0 3 :DUUHQ 5 $ :X < /DJUXWWD $ %RQG & 7 1RUWK 5 $ &DOFLXP Activated  Potassium  Channels  Expressed  from  Cloned  &RPSOHPHQWDU\ '1$V 1HXURQ

/DQJHU 3 *UXQGHU 6 5XVFK $ ([SUHVVLRQ RI &D $FWLYDWHG %. &KDQQHO P51$ DQG ,WV 6SOLFH 9DULDQWV LQ WKH 5DW &RFKOHD - &RPSDUDWLYH 1HXURORJ\

/RYHOO 3 9 0F&REE ' 3 3LWXLWDU\ FRQWURO RI %.

SRWDVVLXP FKDQQHO IXQFWLRQ DQG LQWULQVLF ¿ULQJ SURSHUWLHV RI DGUHQDO FKURPDI¿Q FHOOV 1HXURVFL

.LHKQ 2 +DUULV :DUULFN 5 0 +7 PRGXODWLRQ RI K\SHUSRODUL]DWLRQDFWLYDWHG LQZDUG FXUUHQW DQG FDOFLXP GHSHQGHQW RXWZDUG FXUUHQW LQ D FUXVWDFHDQ PRWRU QHXURQ - 1HXURSK\VLRO

7XUULJLDQR * * /H0DVVRQ * 0DUGHU ( 6HOHFWLYH regulation  of  current  densities  underlies  changes  in  the  DFWLYLW\ RI FXOWXUHG QHXURQV - 1HXURVFL (1995).

15.  Marder,  E.  &  Bucher,  D.  Understanding  Circuit  Dynamics  Using  the  Stomatogastric  Nervous  System  of  Lobsters  and  &UDEV $QQX 5HY 3K\VLRO

7VDQHYD $WDQDVRYD . 6KHUPDQ $ *RRU ) 6WRMLONRYLF 6 6 0HFKDQLVP RI 6SRQWDQHRXV DQG 5HFHSWRU Controlled  Electrical  Activity  in  Pituitary  Somatotrophs:  ([SHULPHQWV DQG 7KHRU\ - 1HXURSK\VLRO (2007).

0DUULRQ 1 9 7DYDOLQ 6 - 6HOHFWLYH DFWLYDWLRQ RI

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ÂŚ Lobster Nervous System ÂŚ

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Ca2+-­  activated  K+  channels  by  co-­localized  Ca2+channels  in  hippocampal  neurons.  Nature.  395,  900-­  904  (1998).

18.  Prakriya,  M.  &  Lingle,  C.J.  BK  channel  activation  E\ EULHI GHSRODUL]DWLRQV UHTXLUHV &D LQĂ&#x20AC;X[ WKURXJK / DQG 4 W\SH RI &D FKDQQHOV LQ UDW FKURPDIÂżQ FHOOV - Neurophysiol.  81,  2267-­2278  (1999).

19.  Harris-­Warrick,  R.M.  Ion  channels  and  receptors:  molecular  targets  for  behavioral  evolution.  J  Comp  Physiol  A.  186,  605-­616  (2000).

20.  Papazian,  D.M.,  Timpe,  L.C.,  Jan,  Y.N.  &  Jan,  L.Y.  Alteration  of  voltagedependence  of  Shaker  potassium  channel  by  mutations  in  the  S4  sequence.  Nature.  349,  305-­ 310.

21.  Stefani,  E.,  Ottolia,  M.,  Noceti,  F.,  Olcese,  R.,  Wallner,  M.,  Latorre,  R.  &  Toro,  L.  Voltage-­controlled  gating  in  a  large  conductance  Ca2+-­sensitive  K+  channel  (hslo).  Proc  Natl  Acad  Sci  USA.  94,  5427-­5431  (1997).

22.  Ghatta,  S.,  Nimmagadda,  D.,  Xu,  Xiaoping.  &  Oâ&#x20AC;&#x2122;Rourke,  S.T.  Large-­conductance,  calcium-­activated  potassium  chanels:  Structural  and  functional  implications.  Pharmacology  &  Theraputics.  110,  103-­116.

23.  Butler,  A.,  Tsunoda,  S.,  McCobb,  D.P.,  Wei,  A.  &  6DONRII / P6OR D FRPSOH[ PRXVH JHQH HQFRGLQJ ³PD[L´ calcium-­activated  potassium  channels.  Science.  261,  221-­ 224  (1993).

24.  Schreiber,  M.  &  Salkoff,  L.  A  novel  calcium-­sensing  domain  in  the  BK  channel.  Biophys  J.  73,  1355-­1363  (1997).

25.  Moss,  B.L.  &  Magleby,  K.L.  Gating  and  conductance  properties  of  BK  channels  are  modulated  by  the  S9-­S10  tail  domain  of  the  alpha  subunit.  A  study  of  mSlo1  and  mSlo3  wild-­type  and  chimeric  channels.  J.Gen.  Physiol.  115,  711  734  (2001).

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Vol 5 ¦ 2011

Migraine Prevention: Neuromodulation for the Hyperexcitable Brain

Bryanna Gulotta, Agriculture and Sciences 13, Biological Sciences Abstract Migraines associated and unassociated with au-­ ras are linked to excitation in the neural network.

Cortical spreading depression (CSD) is a phenom-­ enon characterized by sudden excitatory potentials followed by an extended period of inhibition that moves in waves across the brain. CSD may underlie the presentation of aura in migraine sufferers and is used to model aura in mice. Although the exact mechanisms governing migraine headaches are currently unknown, the presentation of neural ex-­ citation and aura during migraines and CSD makes CSD an ideal system to study neural activity po-­ WHQWLDOO\ LQÀXHQFLQJ PLJUDLQH RFFXUUHQFH 6LPSO\ studying neural excitation exhibited during CSD is not enough, however. Lamotrigine is a medication that inhibits neural excitation;; it is particularly ef-­ fective as suppressing CSD and migraine with aura.

Its mechanisms of action are well investigated. Ex-­ amining how lamotrigine obstructs generation and propagation of neural excitation offers insight into how detrimental neural excitation occurs. Explor-­ ing how neural activity is altered in CSD patterns and migraine events and how lamotrigine impedes such altered activity illuminates mechanisms of mi-­ graine pathogenesis, inhibition, and prophylaxis.

Neural Hyperexcitation

Migraines are the result of hyperexcited neural networks in the brain1. Understanding the root cause of hyperexcitability could help neurobiologists and physicians prevent and treat migraines as well as other disorders caused by neural excitation, such as epilepsy2.

Over 10% of the United States population suffers from migraines3. Generally, the debilitating condition mani-­ fests as a throbbing headache localized to one side of

42

the brain4,5. Nausea, vomiting, dizziness, visual distor-­ tion, and sensitivity to light, sound, and smell may ac-­ company the pain. These secondary symptoms, called auras, precede migraine headaches about 20% of the time. Auras are caused by a condition called Cortical Spreading Depression (CSD), which is known to begin with excessive, or hyper-­, excitation of neurons6. Al-­ though they may be seen as separate occurrences, both CSD, presenting as auras, and migraines are caused by hyperexcitability in the neural network. Additionally, CSD has been noted in some migraine patients without aura presentation, which strengthens the link between the two conditions6. While migraine genesis is still a mystery, CSD can be experimentally induced in mice7.

CSD also has a characteristic pattern of activity as it travels through groups of neurons6. For this reason, examining CSD is an ideal way to investigate hyper-­ excitability and ways to stop or prevent it.

The Model: CSD

$OWKRXJK &6' KDV EHHQ VRPHZKDW GLI¿FXOW WR detect and study in humans, ample evidence of CSD in animals and some evidence about CSD in humans have been garnered. CSD is usually localized to a particular lobe, generally in the occipital lobe in humans6. It is characterized by an initial depolarization and a result-­ ing action potential in a group of neurons, followed by an extended (inhibitory) refractory period6. During this refractory period, the neurons cannot depolarize for IXUWKHU ¿ULQJ &6' PRYHV LQ VORZ ZDYHV WKURXJK WKH brain, traveling at a rate of approximately three milli-­ meters per minute8. The waves spread from their origin in one lobe to other groups of neurons in other parts of the brain. A sudden decrease of extracellular calcium that is presumably a result of the initial depolarization distinguishes CSD9. Overall, CSD prevents communi

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Vol 5 ¦ 2011

¦ Migraine ¦

cation  among  neurons.  Thus,  sufferers  usually  experi-­ HQFH VHQVRU\ GH¿FLWV GXULQJ &6' HSLVRGHV WKDW DUH SHU-­ ceived  as  auras6.   Although  successful  treatments  for  migraine  DQG &6' VHSDUDWHO\ DUH QRW DOZD\V FURVV HIIHFWLYH recent  research  has  demonstrated  that  if  they  are  used  long-­term,  some  migraine  medications  can  reduce  the  IUHTXHQF\ RI &6' HSLVRGHV6,7.

Medicating Hyperexcitation

  A  medication  called  lamotrigine  has  beenvery  VXFFHVVIXO LQ WUHDWLQJ &6' ,Q D UHFHQW WULDO ODPRWULJLQH UHGXFHG &6' RFFXUULQJ LQ WKH DQWHULRU DQG SRVWHULRU sections  of  the  brain  in  rats  most  successfully  ZKHQ FRPSDUHG ZLWK RWKHU PLJUDLQH PHGLFDWLRQV10.  ,W DOVR GHPRQVWUDWHG WKH EHVW WUHDWPHQW RXWFRPH ZKHQ FRPSDUHG ZLWK FDUEDPD]HSLQH JDEDSHQWLQ R[FDUED]HSLQH DQG WRSLUDPDWH LQ WUHDWPHQW RI SDUWLDO RQVHW VHL]XUHV LQ HSLOHSWLF SDWLHQWV HSLOHSV\ LV another  disorder  of  neural  excitation11. 2WKHU ZRUNV demonstrate  a  neuromodulatory  function  of  lamotrigine  LQ WKH QHXURQV 6WXGLHV LQ UDWV KDYH VKRZQ WKDW lamotrigine  decreases  the  frequency  of  spontaneous  excitatory  postsynaptic  potentials  by  decreasing  the  release  frequency  of  glutamate,  an  excitatory  neurotransmitter12.  Lamotrigine  also  increased  the  amplitude  and  frequency  of  spontaneous  inhibitory  postsynaptic  potentials  by  increasing  the  release  of  GABA,  an  inhibitory  neurotransmitter12. ,Q KXPDQV lamotrigine  has  demonstrated  the  ability  to  suppress  aura  and  the  potential  to  suppress  migraines  in  patients  ZLWK FRPRUELG RU PXOWLSOH VLPXOWDQHRXV DXUD13.   Overall,  lamotrigine  may  suppress  hyperexcitability  DW ERWK WKH QHXURQDO DQG QHXUDO QHWZRUN OHYHOV 7KLV PDNHV ODPRWULJLQH DQ H[FHOOHQW FDQGLGDWH WR VWXG\ WKH pathogenesis  of  migraines.

7KH IROORZLQJ UHYLHZ DLPV WR H[SRXQG XSRQ this  research  and  discuss  means  of  neuromodulating  excitatory  and  inhibitory  postsynaptic  potentials  in  RUGHU WR LQÃ&#x20AC;XHQFH PLJUDLQH SDWKRJHQHVLV 3UHVHQWHG LQYHVWLJDWLRQV RI &6' ZLOO LOOXVWUDWH PHFKDQLVPV RI neural  excitation.  Next,  discussion  on  the  function  of  ODPRWULJLQH ZLOO FRQ¿UP KRZ QHXUDO H[FLWDWLRQ PLJKW DULVH DQG SURSDJDWH DQG KRZ LW PD\ EH VWRSSHG RU SUHVHQWHG 7KH JRDO RI WKLV UHYLHZ LV WR SURYLGH HYLGHQFH that  simultaneous  modulation  to  decrease  excitation  DQG LQFUHDVH LQKLELWLRQ LQ QHXURQV DQG QHXUDO QHWZRUNV DW GLIIHUHQW OHYHOV RI H[FLWDWLRQ LV DQ HIIHFWLYH ZD\ WR Cornell University

REVIEW

DOOHYLDWH &6' DQG LI QRW PLJUDLQHV LQ JHQHUDO DW OHDVW migraines  in  the  portion  of  patients  that  experience  comorbid  aura.

From Neural Excitation to Migraines

,QLWLDO GHSRODUL]DWLRQ RU H[FLWDWLRQ RI QHXURQV LQ &6' LV PDUNHG E\ D GHFUHDVH LQ H[WUDFHOOXODU FDOFLXP OHYHOV ,Q D UHFHQW H[SHULPHQW E\ 5LFKWHU HW DO D EORFNDGH RI KLJK WKUHVKROG YROWDJH JDWHG FDOFLXP FKDQQHOV 9*&&V WUDQVODWHG LQWR GHFUHDVHG &6' ZDYH SURSDJDWLRQ14.  Their  experiment  explicitly  GHPRQVWUDWHV WKDW &6' DQG H[FLWDWLRQ FDQ EH LQKLELWHG E\ EORFNLQJ FDOFLXP Ã&#x20AC;RZ IURP WKH H[WUDFHOOXODU VSDFH into  neurons.

7KH OLQN EHWZHHQ FDOFLXP DQG QHXUDO H[FLWDWLRQ rests  in  calciumâ&#x20AC;&#x2122;s  function  in  individual  neurons.  ,QWUDFHOOXODU FDOFLXP LV LQYROYHG LQ SUHV\QDSWLF FHOO VLJQDOOLQJ +LJK WKUHVKROG 9*&&¶V NQRZQ DV 1 RU 3 4 W\SH FKDQQHOV UHJXODWH WKH HQWU\ RI FDOFLXP into  presynaptic  terminals,  and  consequently  are  at  least  partially  responsible  for  the  amounts  of  the  neurotransmitters  glutamate  and  GABA  that  are  released  into  the  synaptic  cleft12,15,16,17. 6SHFL¿FDOO\ WKH EORFNDGH RI 1 DQG 3 4 W\SH FKDQQHOV UHVXOWHG LQ GHFUHDVHG UHSHWLWLRQ RI &6' ZDYHV DFURVV WKH UDW EUDLQ LQ WKH SUHVHQFH RI D NQRZQ &6' JHQHUDWRU SRWDVVLXP chloride  crystals14.  6LQFH &6' LV GHSHQGHQW XSRQ H[FLWDWRU\ DFWLYLW\ IROORZHG E\ LQKLELWRU\ DFWLYLW\ WKDW FKDQJHV LQ FDOFLXP FRQGXFWDQFH DOWHU &6' SDWWHUQV implies  an  alteration  in  excitatory  and  inhibitory  potentials  is  occurring.   Another  indicator  that  calcium  conductance  LV D IDFWRU LQ &6' DFWLYLW\ LV WKDW D PXWDWLRQ LQ WKH Ä® $ VXEXQLW RI SUHV\QDSWLF 3 4 W\SH 9*&&V LQ PLFH in  vivo  resulted  in  a  resistance  to  chemically  induced  &6' ZDYHV DQG RQFH HVWDEOLVKHG D VORZHU ZDYH SURSDJDWLRQ VSHHG ,Q WKLV SDUWLFXODU H[SHULPHQW WKH glutamate  release  pattern  via  microdialysis  in  these  PLFH LQGLFDWHG D JUHDWHU WKDQ WZR IROG DWWHQXDWLRQ RI the  calcium  current  and  a  decreased  probability  ofopen  3 DQG 4 FKDQQHOV FRPSDUHG ZLWK WKH FRQWURO PLFH 7KH glutamate  measurements  in  this  experiment  indicated  that  the  interruption  in  communication  impeded  the  excitation  of  postsynaptic  neurons7.  Malfunction  or  EORFNDGH RI WKH 3 4 W\SH FKDQQHOV LQKLELWV FHOO WR FHOO communication  via  reductions  in  glutamate  release  -­  this  may  be  the  actual  cause  of  disruption  in  neuronal  communication.   Generally,  the  evidence  suggests  that Â

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decreasing  calcium  conductance  reduces  the  likelihood  of  hyperexcitation  of  neurons  and  of  the  neural  network.   +XPDQ JHQHWLF VWXGLHV VLPLODUO\ ¿QG WKDW calcium  channel  mutations  affect  neural  excitation.   The  gene  CACNL1A4  encodes  for  P/Q  calcium  channels.   Members  of  several  families  with  Familial  Hemiplegic  Migraine  (FHM),  which  is  accompaniedby  aura,  were  determined  to  have  missense  mutations  in  this  gene.  In  FHM,  authors  suggested  that  the  mutations  would  lead  to  malformation  and  dysfunction  of  the  P/Q  channels  GXH WR DOWHUDWLRQ LQ WKH Ď $ VXEXQLW RI WKH FKDQQHO (Fig.1).Mutation  at  the  locus  19p13,  observed  in  the  FHM  patients,  has  also  been  implicated  in  migraines  with-­out  aura.  Interestingly,  unrelated  patients  suffering  from  Episodic  Ataxia  Type-­2  (EA-­2),  causing  uncoordinated  movement  and  sometimes  cerebellar  atrophy,  were  determined  to  have  either  mutations  or  deletions  in  this  same  gene.  In  EA-­2,  the  DNA  changes  possibly  lead  to  the  gain  of  function  due  to  changes  in  WKH FKDQQHO Ď VXEXQLW 7KLV ZRXOG H[SODLQ ZK\ LQ WKH PLFH GHVFULEHG DERYH Ď $ VXEXQLW PXWDWLRQV UHGXFH the  likelihood  of  CSD,  but  other  P/Q  channel  mutations  increase  the  likelihood  of  neural  excitation18.  A  caveat  to  work  presented  thus  far  is  the  unsuccessful  treatment  of  migraines  with  the  voltage-­ gated  calcium  channel  blocker  Nifedipine19.  However,  Nifedipine  is  an  L-­type  calcium  channel  blocker.   L-­type  calcium  channels  are  also  VGCCs,  but  are  composed  of  different  amino  acid  chains  than  the  P/Q  type  channel;͞  they  have  been  shown  to  be  largely  unrelated  to  repetitive  CSD  waves  and  to  the  effects  of  lamotrigine14,16.

Medicating Hyperexcitation: Analysis  Lamotrigine  appears  to  have  multiple  modes  RI DFWLRQ ,WV ÂżUVW GRFXPHQWHG HIIHFW ZDV UHGXFLQJ sodium  conductance  by  blocking  voltage  gated  sodium  channels20.  Lamotrigine  halted  sodium  dependent  sustained  action  potentials  in  mouse  spinal  cord  neurons20.   In  a  separate  experiment,  lamotrigine  inhibited  the  function  of  neuromodulator  veratridine,  which  opens  voltage  sensitive  sodium  channels,  and  competed  for  binding  locations  with  toxin  [3H  EDWUDFKRWR[LQLQLQ $ ÄŽ EHQ]RDWH ZKLFK SUHYHQWV voltage  gated  sodium  channelinactivation20,21,22.  These  ¿QGLQJV UHYHDO WKDW ODPRWULJLQH VSHFLÂżFDOO\ DFWV RQ WKH inactive  forms  of  the  voltage  gated  sodium  channel  and  portrays  the  drug  as  a  neuromodulator  itself.   It Â

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is  thought  that  by  reducing  sodium  conductance  and  LQKLELWLQJ GHSRODUL]DWLRQ RI WKH SUHV\QDSWLF FHOO lamotrigine  decreases  the  release  rate  of  glutamate  to  the  postsynaptic  neuron,  and  thus  the  frequency  of  excitatory  postsynaptic  potentials21.  Lamotrigine  plays  a  similar  neuromodulatory  role  in  calcium  conductance,  which,  based  on  the  CSD  and  migraine  research,  offers  insight  into  how  the  drug  could  work  to  suppress  these  disorders.   Under  lamotrigine  treatment,  the  N  and  P/Q  VGCCs  demonstrate  decreased  conductance  of  calcium16.   This  treatment  would  also  reduce  the  release  of  glutamate  from  the  presynaptic  terminal.                 Â

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Figure  1.  Map  of  the  P/Q  channel  calcium  channel  depicting  mutations  prevalent  in  Familial  Hemiplegic  Migraine,  mouse  Etaxia  Type-­2,  and  in  two  types  of  mice  used  by  Ataya  1999,  tg  and  tg1a.  Mutations  in  the  channel  DSSHDU WR LQĂ&#x20AC;XHQFH FDOFLXP FRQGXFWDQFH DQG VXEVHTXHQWO\ neural  excitability  (Terwindt  1998).

,W LV LQWHUHVWLQJ WR QRWH WKDW WKH ÄŽ $ VXEXQLW ZKLFK confers  voltage  sensitivity  which  confers  voltage  sensitivity  in  calcium  channels,  is  a  homologue  to  that  found  in  sodium  channels.  This  may  explain  why  lamotrigine  appears  to  affect  conductance  of  both  ions15.  Furthermore,  the  concentration of lamotrigine used to evoke changes in sodium conductance and calcium conductance is similar, supporting the idea that lamotrigine is multimodal17.  The  work  presented  above  contradicts  the  work  of  Cunningham  and  colleagues,  who  found  that  lamotrigineâ&#x20AC;&#x2122;s  effect  on  spontaneous  potentials  does  not  depend  on  the  activity  of  sodium  and  calcium  channels12.  They  speculated  that  lamotrigine  may  function  as  a  neuromodulator  that  interacts  with  the  vesicular  system  involved  in  releasing  neurotrans-­

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DFWLYLW\ RI ODPRWULJLQH PD\ GLIIHU DFFRUGLQJ WR WKH VWDWH RI H[FLWDWLRQ LQ FHOOV 7KH UHVXOWV IURP &XQQLQJ-­ KDP HW DO DQG *LEEV HW DO ERWK VXJJHVW WKDW ODPRWULJLQH LQFUHDVHV *$%$ UHOHDVH DQG GHFUHDVHV JOXWDPDWH UHOHDVH LQ WDQGHP 7KXV LQ LQVWDQFHV RI KLJK YROWDJH DFWLYLW\ ODPRWULJLQH PD\ DIIHFW KLJK WKUHVKROG FDOFLXP DQG KLJK WKUHVKROG VRGLXP FKDQQHOV DQG FKDQJH WKH LRQV¶ FRQGXFWDQFH ZKLFK GHFUHDVHV QHXURWUDQVPLWWHU UHOHDVH ,Q FRQGLWLRQV RI ORZ H[FLWDWLRQ ODPRWULJLQH PD\ UHGXFH ERWK WKH IUHTXHQF\ RI JOXWDPDWH UHOHDVH DQG LQFUHDVH IUHTXHQF\ DQG FRQFHQWUDWLRQ RI *$%$ UHOHDVH E\ DIIHFWLQJ SUHV\QDSWLF YHVLFXODU UHOHDVH mechanisms.12

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points  to  a  possible  mechanism  by  which  sufferers  are  predisposed  to  migraines.   It  also  proposes  a  direction  for  research  on  future  medications.   It  is  clearly  im-­ portant  in  patients  with  disorders  of  neural  excitation  to  look  at  both  brain  activity  during  periods  of  hyper-­ excitability  and  when  activity  is  supposedly  normal  in  orderto  tease   out  events  whose  inhibition  or  reversal  could  prevent  neural  excitation  altogether.  Hopefully,  future  research  will  determine  how  lamotrigine  has  its  remarkable  effect  on  cells  and  what  can  be  done  to  correct  hyperexcitability  when  it  develops.

References 1.  Dâ&#x20AC;&#x2122;Andrea,  G.,  &  Leon,  A.  2010.  Pathogenesis  of  migraine:from      neurotransmitters  to  neuromodulators  and  beyond.  Neuro-­ ORJLFDO 6FLHQFHV 2IÂżFLDO -RXUQDO RI WKH ,WDOLDQ 1HXURORJL-­ cal  Society  and  of  the  Italian  Society  of  Clinical  Neuro-­ Table  1,  2  Lamotrigine  effectively  suppressed  tonic-­clonic  physiology,  31  :  1-­7. and  generalized  absence  epileptic  seizure  activity  in  the  %LJDO 0 ( /LSWRQ 5 % &RKHQ - 6LOEHUVWHLQ 6 rat  thalamocrotical  slices.   cTBs  and  sTBs  are  neural  D.  2003.  Epilepsy  and  migraine.  Epilepsy  and  Behavior  wave  models  for  general  absence  and  tonic-­clonic  seizures  (4):  13-­24. respectively  in  rat  neurons.   This  effect  was  seen  despite  /LSWRQ 5 % 6FKHU $ , .RORGQHU . /LEHUPDQ - no  evidence  of  an  effect  of  lamotrigine  on  calcium  conduc-­ 6WHLQHU 7 - 6WHZDUW : ) -DQXDU\ 0L-­ graine  in  the  United  States:  epidemiology  and  patterns  of  tance  during  spontaneous  potentials  in  Cunningham  et  al. health  care  use.  Neurology,  58,  6,  885-­94 Discussion 2VWHUKDXV - 7 *XWWHUPDQ ' / 3ODFKHW-­ ND - 5 -XO\ +HDOWKFDUH 5HVRXUFH DQG Lost  Labour  Costs  of  Migraine  Headache  in  the   Voltage  gated  calcium  channel  conductance  US.Pharmacoeconomics,  2,  1,  67-­76. play  a  central  role  in  incidences  of  CSD.   Further-­ 6HOE\ * /DQFH - : -DQXDU\ 2EVHUYD-­ more,  due  to  the  effect  of  VGCCâ&#x20AC;&#x2122;s  on  neurotransmit-­ tions  on  500  cases  of  migraine  and  allied  vascular  head-­ ter  release,  it  appeared  that  glutamate  levels  were  DFKH -RXUQDO RI 1HXURORJ\ 1HXURVXUJHU\ DQG 3V\FKLDWU\ directly  involved  in  neural  excitation,  as  might  be  23,  23-­32.  suspected  from  an  excitatory  neurotransmitter.  These  6.  Parsons,  A.  A.  2004.  Cortical  spreading  depression:  its  experiments  on  CSD  events  demonstrated  clearly  that  role  in  migraine  pathogenesis  and  possible  therapeutic  in-­ changes  in  ion  channel  conductance  were  a  key  fac-­ tervention  strategies.  Current  Pain  and  Headache  Reports,  tor  leading  to  neural  excitation.Reports  on  lamotrigine  8  (  5)  :  410-­6.

FRQÂżUPHG WKLV ÂżQGLQJ DQG VWUHQJWKHQHG WKH QRWLRQ $\DWD & 6KLPL]X 6DVDPDWD 0 /R ( + 1RHEHOV - that  neurotransmitter  release  was  crux  of  the  propaga-­ L.,  &  Moskowitz,  M.  A.  1999.  Impaired  neurotransmitter  release  and  elevated  threshold  for  cortical  spreading  de-­ tion  of  neural  excitation.   In  addition,  the  studies  on  lamotrigine  expose  how  neural  excitation  may  actually  SUHVVLRQ LQ PLFH ZLWK PXWDWLRQV LQ WKH #ÄŽ $$ VXEXQLW RI P/Q  type  calcium  channels.  Neuroscience  95(3):  639-­645. have  many  progenitors.   While  periods  of  high  activ-­ ity,  such  as  during  hyperexcitation  may  be  stopped  or  5HLG . + 0DUUDQQHV 5 :DXTXLHU $ -DQXDU\ prevented  by  the  use  of  VGCC  blockers,  events  which  1988).  Spreading  depression  and  central  nervous  system  SKDUPDFRORJ\ -RXUQDO RI 3KDUPDFRORJLFDO 0HWKRGV precede  hyperexcitation  are  not  affected  by  such  1-­21 blockade.   In  the  hyperexcitable  brain,  there  may  be  an  /DXULW]HQ 0 -DQXDU\ 3DWKRSK\VLRORJ\ RI increased  ratio  of  spontaneous  excitatory  to  inhibitory  the  migraine  aura.  The  spreading  depression  theory.  Brain  :  potentials.   Lamotrigine  corrects  this  ratio,  possibly  by  D -RXUQDO RI 1HXURORJ\ affecting  vesicular  release  machinery.   This  discovery  10.  Bogdanov,  V.  B.,  Multon,  S.,  Chauvel,  V.,  Bogdanova, Â

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O. V., Prodanov, D., Schoenen, J., & Makarchuk, M. Y. 2011. Migraine preventive drugs differentially affect corti-­ cal spreading depression in rat. Neurobiology of Disease, 41 (2): 430-­435.

11. Marson, A. G., Al-­Kharusi, A. M., Alwaidh, M., Apple-­ ton, R., Baker, G. A., Chadwick, D. W., Cramp, C., Cocker-­ ell, O.C., Cooper, P.N., Doughty, J., Eaton, B., Gamble, C, Goulding, P.J., Howell, S.J., Hughes, A., Jackson, M., Jaco-­ by, A., Kellet, M., Lawson, G.R., Leach, J.P., Nicolaides, P., Roberts, R., Shackley, P., Shen, J., Smith, D.F., Smith, P.E., Smith, C.T., Vanoli, A., Williamson, P. R. (March 24, 2007). The SANAD study of effectiveness of carbamaze-­ pine, gabapentin, lamotrigine, oxcarbazepine, or topiramate for treatment of partial epilepsy: an unblinded randomised controlled trial. The Lancet, 369, 9566, 1000-­1015.

12. Cunningham, M. O., & Jones, R. S. 2000. The anti-­ convulsant, lamotrigine decreases spontaneous glutamate release but increases spontaneous GABA release in the rat entorhinal cortex in vitro. Neuropharmacology, 39 (11) : 2139-­2146.

13. Lampl, C., Katsarava, Z., Diener, H. C., & Limmroth, V. 2005. Lamotrigine reduces migraine aura and migraine attacks in patients with migraine with aura. Journal of Neu-­ rology, Neurosurgery, and Psychiatry, 76 (12) : 1730-­2.

14. Richter, F., Ebersberger, A., & Schaible, H.-­G. 2002.

Blockade of voltage-­gated calcium channels in rat inhibits repetitive cortical spreading depression. Neuroscience Let-­ ters, 334 (2): 123.

15. Kwan, P., Sills, G. J., & Brodie, M. J. 2001. The mech-­ anisms of action of commonly used antiepileptic drugs.

Pharmacology & Therapeutics 90(1): 21-­34 16. Stefani, A., Spadoni, F., Siniscalchi, A., & Bernardi, G. 1996. Lamotrigine inhibits Ca2^+^ currents in corti-­ cal neurons: functional implications. European Journal of Pharmacology 307(1): 113-­116.

17. Stefani, A., Spadoni, F., & Bernardi, G. 1997. Voltage-­ activated calcium channels: targets of antiepileptic drug therapy?. Epilepsia 38( 9): 959-­65 18. Ophoff, R. A., Terwindt, G. M., Vergouwe, M. N., van, E. R., Oefner, P. J., Hoffman, S. M., Lamerdin, J. E., Mohrenweiser, H.W., Bulman, D.E., Ferrari, M., Haan, J., Lindhout, D., van Ommen, G. B., Hofker, M.H., Ferrari, M.D., Frants, R. R. 1996. Familial hemiplegic migraine and episodic ataxia type-­2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 87(3): 543-­52 19. Welch KM. 1993. Drug therapy of migraine. The New England Journal of Medicine. 329(20): 1476-­83 20. Cheung, H., D. Kamp, E. Harris. 1992. An in vitro investigation of the action of lamotrigine on neuronal voltage-­activated sodium channels. Epilepsy Research 13(2): 107 21. Brodie, M. J. 1992. Lamotrigine. Lancet 339(8806): 1397-­400.

22. McNeal, E. T., Lewandowski, G. A., Daly, J. W., &

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Creveling, C. R. 1985. [3H]Batrachotoxinin A 20 alpha-­ benzoate binding to voltage-­sensitive sodium channels: a rapid and quantitative assay for local anesthetic activity in a variety of drugs. Journal of Medicinal Chemistry 28(3): 381-­8 23. Gibbs, J. W. ., Zhang, Y. F., Ahmed, H. S., & Coul-­ ter, D. A. 2002. Anticonvulsant actions of lamotrigine on spontaneous thalamocortical rhythms. Epilepsia 43 (4): 342-­349.

24. Greenhill, S. D., & Jones, R. S. G. 2010. Diverse an-­ tiepileptic drugs increase the ratio of background synaptic inhibition to excitation and decrease neuronal excitability in neurones of the rat entorhinal cortex in vitro. Neurosci-­ ence 167 (2): 456-­474.

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Hippocampal Neurogenesis: A Role for Adult Neuroplasticity in New Learning and Memory Formation Rachel Bavley, Arts and Sciences 13, Psychology Abstract

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the  answers  of  which  promise  to  have  widespread  im-­ The  hippocampus  is  one  of  the  few  areas  of  the  mam-­ plications  for  our  understanding  of  the  brainâ&#x20AC;&#x2122;s  lifelong  malian  brain  that  continues  to  generate  new  neurons  ability  to  learn,  remember,  and  adapt  to  the  environ-­ into  adulthood,  a  process  that  is  regulated  at  least  in  ment. part  by  experiences  that  engage  the  hippocampus.  Mechanisms of Neurogenesis +RZHYHU WKH VSHFLÂżF UROH WKDW WKHVH QHZ QHXURQV play  in  the  various  learning  and  memory  functions   One  of  the  hallmarks  of  the  brain  is  its  plastic-­ attributed  to  the  hippocampus  remains  unclear,  and  more  research  is  needed  before  the  true  purpose  of  ity,  or  ability  to  change  and  adapt  in  response  to  the  adult  hippocampal  neurogenesis  can  be  determined.  environment  1.  Adult  neurogenesis  is  one  example  of  Computational  models  suggest  an  important  rela-­ how  the  brain  can  continue  to  change  throughout  life  tionship  between  adult  hippocampal  neurogenesis,  by  producing  new  neurons  which  can  be  integrated  into  FRQWH[W SURFHVVLQJ DQG FRJQLWLYH Ă&#x20AC;H[LELOLW\ DQG FDQ the  brainâ&#x20AC;&#x2122;s  neural  networks.  However,  perhaps  because  help  explain  the  role  of  adult  hippocampal  neuro-­ RI WKH GLIÂżFXOWLHV DVVRFLDWHG ZLWK VXFFHVVIXOO\ LQFRU-­ genesis  in  memory  formation  and  the  neurocogni-­ porating  new  neurons  into  existing  neural  networks  2 ,  neurogenesis  is  generally  limited  to  only  two  areas  tive  pathologies  associated  with  mood  disorders.  of  the  mammalian  brain:  the  subgranular  zone  (SGZ)  of  the  hippocampal  formation,  and  the  subventricular  Introduction zone  (SVZ)  which  provides  new  neurons  to  the  olfac-­  Ever  since  the  discovery  that  brain  regions  such  tory  bulbs  3.  Adult  neurogenesis  can  be  studied  in  the  brains  as  the  hippocampus  continue  to  produce  new  neurons  into  adulthood,  there  has  been  a  growing  interest  in  of  experimental  animals  using  the  chemical  marker  the  role  that  these  new  neurons  play  in  brain  function.  bromodeoxyuridine  (BrdU),  a  synthetic  thymidine  ana-­ An  accumulating  body  of  research  has  shown  that  new  logue  that  acts  by  incorporating  into  replicating  DNA  neurons  generated  in  the  hippocampus  are  involved  in  and  can  be  subsequently  detected  using  immunohisto-­ certain  hippocampus-­dependent  learning  tasks.  Fur-­ FKHPLVWU\ WR LGHQWLI\ D VSHFLÂżF JHQHUDWLRQ RI SUROLIHUDW-­ thermore,  learning  such  tasks  can  actually  increase  ing  neurons  4.  Using  this  technique,  we  now  know  that  the  survival  rate  of  new  hippocampal  neurons,  most  of  the  SGZ  produces  about  9,000  new  neurons  each  day  which  would  otherwise  die  within  a  few  weeks.  How-­ in  rats,  many  of  which  migrate  to  the  dentate  gyrus  of  ever,  the  primary  role  that  newly-­generated  hippocam-­ the  hippocampus  and  differentiate  into  granule  neurons  pal  neurons  play  in  learning  and  memory  processes  5.  Furthermore,  evidence  suggests  that  these  new  neu-­ KDV \HW WR EH GHWHUPLQHG DQG ÂżQGLQJV UHJDUGLQJ ZKLFK rons  are  capable  of  becoming  synaptically  integrated  kinds  of  learning  tasks  require  adult  hippocampal  neu-­ into  existing  neural  networks  in  the  hippocampus.  They  rogenesis  have  been  inconclusive  and  often  contradic-­ begin  projecting  axons  to  area  CA3  of  the  hippocam-­ WRU\ 7KH VSHFLÂżF IXQFWLRQ RI KLSSRFDPSDO QHXURJHQH-­ pus  4-­10  days  after  mitosis  6  and  eventually  become  sis  within  the  adult  brain  is  currently  an  open  question,  capable  of  generating  action  potentials  and  producing Cornell University

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hippocampal neurons seem to be particularly sensitive to environmental manipulations that occur during a critical period beginning about one week after they are produced 10, which corresponds to the time at which theyare beginning to extend axons into the CA3 region 11, 12 and therefore becoming synaptically integrated.

This is evidenced by the fact that the survival rate is increased when environmental manipulations such as enrichment begin up to three weeks after BrdU administration, with the maximum survival rate occurring when environmental manipulations begin after one week 13.

Facilitative Effect of Neurogenesis on Hippocampus-Dependent Learning

Figure 1. (prev. page) Dividing cells in the adult mouse dentate gyrus express early neural markers at 48 h after virus injection. a, Confocal micrograph (a merged image of 15 1-­microm optical sections) of GFP expression in the dentate gyrus in a section that was labelled for the neuronal marker NeuN (red). No co-­labelling of GFP+ cells with NeuN was observed. b, Single confocal plane of a cluster of GFP+ cells labelled with the early neuronal marker Tuj1-­beta (red). c, Single confocal plane of a GFP+ cell with im-­ munoreactivity for the progenitor marker NG2 (red). In b and c, nuclei (DAPI) and GFAP are blue. 7

cells have been found to initially exhibit many of the same distinctive characteristics as neurons generated during development, including an excitatory response to GABA, a lower depolarization threshold, and heightened elicitation of LTP 8. This enhanced excitability suggests that newly generated hippocampal neurons may play a special role in learning-­related plasticity by virtue of their unique electrophysiological properties.

While a larger percentage of these new cells will typically begin to die off about a week after DNA synthesis 9, the survival rate of new hippocampal neurons appears to be critically dependent on the experience and learning that occurs during the new neurons’ maturation.

For example, one month after being injected with BrdU, rats that had lived in an enriched environment retained more BrdU-­labeled neurons than rats that did not 2, indicating that the enriched environment increased the survival rate of proliferating neurons. Adult-­generated Cornell University

Given that new hippocampal neurons can be integrated into existing hippocampal networks, it can be predicted that these neurons may participate in tasks in which the hippocampus plays a functional role. There is a wealth of indirect evidence that supports this view.

For example, conditions such as stress 14, aging 15, and drug use including alcohol 16, nicotine 17, 18, and opiates 19, 20 have all been correlated with both decreases in hippocampal neurogenesis and learning impairments, while enriched environments 2, physical exercise 21 , and estrogen 11, 12 have all been found to increase hippocampal neurogenesis and facilitate learning on hippocampus-­dependent tasks 2.

More direct evidence for a facilitative role of hippocampal neurogenesis in learning comes from experimental procedures in which neurogenesis is partially inhibited, and any resulting decrements in learning are measured. These methods have been used to test the effects of impaired hippocampal neurogenesis on several hippocampus-­dependent and hippocampus– independent tasks.One approach to determine whether neurogenesis is involved in a certain type of learning is to inject the animal with an antimitotic agent which reduces neurogenesis. In one such experiment, Shors and colleagues (2001) administered the antimitotic toxin methylazoxymethanol (MAM) to one group of rats, and compared their performance on a trace eyeblink conditioning task to a control group that did not receive MAM. Trace eyeblink conditioning measures the ability of an animal to create trace memories by learning to associate two stimuli that are separated in time. Unlike other types of associative learning in which there is no temporal separation of stimuli, trace

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Figure  2.  Graphs  of  the  timing  parameters  of  the  CR.  (A)  A  graph  of  the  duration  of  the  response.  (B)  A  graph  of  the  ratio  of  the  percent  of  late  CRs  to  the  percent  of  other  nonalpha  CRs.  The  data  range  from  0.0  to  1.0,  with  1.0  indicating  that  all  responses  LQFOXGHG VRPH VLJQL¿FDQW DFWLYLW\ GXULQJ WKH PV WLPH SHULRG prior  to  US  onset.  h,  Hippocampal  lesion  (complete  and  partial);;  n,  neocortical  lesion;;  s,  sham  lesion. Â

FRQGLWLRQLQJ LV LPSDLUHG E\ KLSSRFDPSDO OHVLRQV ,  and  is  therefore  considereda  hippocampus-­dependent  task.  The  two  groups  did  not  differ  in  their  performance  RQ D KLSSRFDPSXV LQGHSHQGHQW GHOD\ H\HEOLQN FRQGL-­ tioning  task,  suggesting  that  MAM  treatment  did  not  FDXVH D XQLYHUVDO OHDUQLQJ LPSDLUPHQW +RZHYHU WKH JURXS ZLWK LPSDLUHG QHXURJHQHVLV SHUIRUPHG VLJQL¿-­ FDQWO\ ZRUVH WKDQ FRQWUROV RQ WKH WUDFH H\HEOLQN FRQGL-­ tioning  task 7KHVH ¿QGLQJV SURYLGH HYLGHQFH WKDW WKH QHZ FHOOV SURGXFHG E\ KLSSRFDPSDO QHXURJHQHVLV SOD\ an  important  functional  role  in  the  formation  of  trace  PHPRULHV 7KLV LQWHUSUHWDWLRQ LV VWUHQJWKHQHG E\ WKH IDFW WKDW ZKHQ UDWV ZKR KDG UHFHLYHG 0$0 DQG SHU-­ IRUPHG SRRUO\ RQ WUDFH FRQGLWLRQLQJ ZHUH DOORZHG WR UHFRYHU DQG EHJLQ SURGXFLQJ QHZ KLSSRFDPSDO QHXURQV DJDLQ WKHLU SHUIRUPDQFH RQ WKH WUDFH H\HEOLQN FRQGL-­ WLRQLQJ WDVN UHWXUQHG WR WKH OHYHO RI FRQWUROV .  A  similar  effect  has  been  found  during  trace  IHDU FRQGLWLRQLQJ LQ ZKLFK D WRQH &6 LV UHSHDWHGO\ SDLUHG ZLWK D IRRWVKRFN 86 DIWHU D GHOD\ DQG OHDUQLQJ LV PHDVXUHG LQ WHUPV RI IUHH]LQJ EHKDYLRU LQ UHVSRQVH to  the  tone.  MAM-­treated  rats  with  decreased  hip-­ SRFDPSDO QHXURJHQHVLV VKRZHG D VLJQL¿FDQW OHDUQLQJ impairment  on  this  task  compared  to  controls .  How-­ HYHU UDWV WUHDWHG ZLWK 0$0 GLG QRW VKRZ D GHFUHPHQW LQ DOO W\SHV RI KLSSRFDPSXV GHSHQGHQW OHDUQLQJ 7KHLU performance  on  two  other  hippocampus-­dependent  WDVNV VSDWLDO QDYLJDWLRQ LQ WKH 0RUULV ZDWHU PD]H DQG contextual  fear  conditioning,  was  not  impaired  in  rats  WUHDWHG ZLWK 0$0 UHODWLYH WR FRQWUROV .  Another  technique  that  has  been  used  to  inter-­ rupt  adult  neurogenesis  is  focal  or  whole-­brain  irradia-­ WLRQ ZKLFK DYRLGV PDQ\ RI WKH SRWHQWLDO VLGH HIIHFWV RI

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MAM  treatment  while  at  the  same  time  inhibiting  neu-­ URJHQHVLV LQ WKH WUHDWHG DUHD PRUH FRPSOHWHO\ .  Using  WKLV WHFKQLTXH 0DGVHQ DQG FROOHDJXHV IRXQG that  irradiated  rats  performed  worse  than  controls  on  a  hippocampus-­dependent  place-­recognition  task,  but  did  not  show  impairment  on  a  hippocampus-­independ-­ ent  object  recognition  task  .  Likewise,  Winocur  and  FROOHDJXHV IRXQG WKDW LUUDGLDWHG UDWV SHUIRUPHG worse  than  controls  on  a  hippocampus-­dependent  wa-­ ter  maze  non-­match-­to-­sample  (NMTS)  task  with  a  ORQJ GHOD\ EHWZHHQ VDPSOH DQG WHVW WULDOV EXW QRW RQ a  hippocampus-­independent  NMTS  task  with  a  short  GHOD\ .

+RZHYHU D VLPLODU VWXG\ E\ +HUQDQGH] 5DED]D DQG FROOHDJXHV WKDW XVHG D UHJXODU W PD]H LQVWHDG of  a  water  maze  found  no  irradiation-­induced  impair-­ PHQW RQ D 1076 WDVN GXULQJ VKRUW RU ORQJ GHOD\V suggesting  that  the  stressful  nature  of  the  water  maze  WDVN PD\ KDYH FRQWULEXWHG WR WKH LPSDLUPHQW ,QWHUHVW-­ LQJO\ DQRWKHU VWXG\ E\ 6D[H DQG FROOHDJXHV

found  that  inhibition  of  hippocampal  neurogenesis  DFWXDOO\ LPSURYHG SHUIRUPDQFH RQ D 1076 WDVN RQ D radial  arm  maze  in  which  the  animals  were  required  to  GLVUHJDUG KLJKO\ VLPLODU DQG RIWHQ FRQÃ&#x20AC;LFWLQJ LQIRUPD-­ WLRQ IURP SUHYLRXV WULDOV :KLOH FRXQWHULQWXLWLYH WKLV result  is  consistent  with  the  idea  that  new  hippocampal  QHXURQV SOD\ D UROH LQ OLQNLQJ WRJHWKHU PXOWLSOH FRQWH[-­ WXDO HOHPHQWV DFURVV WLPH DQG PD\ WKHUHIRUH LQFUHDVH WKH LQWHUIHUHQFH UHODWHG WR KDYLQJ WR UHPHPEHU GLVWLQFW episodes  within  a  continuous  context.  Also  consistent  with  this  idea,  irradiation  exper-­ LPHQWV KDYH IRXQG VLJQL¿FDQW LPSDLUPHQWV RQ D FRQWH[-­ tual  fear  conditioning  task,  in  which  the  rats  learn  to  as-­ VRFLDWH D VSHFL¿F FRQWH[W ZLWK D IRRWVKRFN HYHQ WKRXJK their  fear  response  itself  was  no  different  from  controls Â

.  This  task  requires  the  subject  to  construct  a  multi-­ dimensional  representation  of  the  context  and  form  an  association  between  the  context  and  a  fear  response,  so  irradiation-­related  impairments  suggest  that  hippocam-­ SDO QHXURJHQHVLV PD\ SOD\ D UROH LQ FRQWH[W SURFHVVLQJ and  generalization  across  learning  instances.

,PSDLUPHQWV KDYH DOVR EHHQ IRXQG LQ LUUDGLDWHG UDWV UHODWLYH WR FRQWUROV IRU VSDWLDO QDYLJDWLRQ RQ WKH Morris  water  maze +RZHYHU WKLV ¿QGLQJ LV IDU IURP FRQFOXVLYH JLYHQ WKDW VHYHUDO RWKHU VWXGLHV KDYH IDLOHG to  show  impairment  on  the  Morris  water  maze  task  in  irradiated  rats .  To  make  matters  more  confusing,  strains  of

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maze  task  than  strains  with  lower  rates  of  neurogenesis  ,  yet  there  was  no  such  correlation  between  strain  and  acquisition  in  a  similar  study  done  with  rats  instead  of  mice  36.  This  suggests  that  the  role  of  hippocampal  neu-­ rogenesis  in  spatial  navigation,  and  probably  other  tasks  as  well,  is  complex  and  can  show  variation  depending  RQ WKH VSHFL¿F SURFHGXUH XVHG DQG VSHFLHV WHVWHG  There  is  still  much  debate  over  how  adult-­ generated  hippocampal  neurons  participate  in  learn-­ ing.  However,  based  on  the  research  to  date,  it  appears  that  adult  hippocampal  neurogenesis  does  play  a  role  in  some,  but  not  all,  forms  of  hippocampus-­dependent  learning.  While  most  results  remain  inconsistent  and  inconclusive,  the  evidence  appears  to  be  strongest  for  tasks  which  require  the  animal  to  retain  information  across  a  delay  (such  as  trace  eyeblink  conditioning  and  delayed  NMTS),  tasks  that  require  context  processing  (such  as  contextual  fear  conditioning),  and  aversive  learning  tasks  that  cause  stress  to  the  animal  (including  fear  conditioning  and  tasks  that  occur  in  a  water  maze).  While  some  authors  have  suggested  that  hippocampal  QHXURJHQHVLV PD\ RQO\ EH QHFHVVDU\ IRU PRUH GLI¿FXOW tasks  28  or  for  more  long-­term  memory  formation  32,  more  research  is  needed  before  any  conclusive  char-­ acterization  of  the  role  of  hippocampal/neurogenesis  in  facilitating  hippocampus-­dependent  learning  can  be  made  23.  35

Evidence for Learning-Induced Survival of New Hippocampal Neurons Â

 While  it  is  clear  that  hippocampal  neurogenesis  can  have  an  effect  on  learning,  it  has  been  suggested  that  the  converse  may  also  be  true,  namely  that  learn-­ ing  can  have  an  effect  on  hippocampal  neurogenesis.  6SHFL¿FDOO\ LW KDV EHHQ K\SRWKHVL]HG WKDW WUDLQLQJ RQ hippocampus-­dependent  tasks  can  facilitate  the  inte-­ gration  of  new  hippocampal  neurons  into  functional  circuits,  thereby  increasing  their  survival  37.  Evidence  for  this  hypothesis  comes  from  a  study  by  Gould  et  al.  (1999)  in  which  rats  injected  with  BrdU  were  trained  on  either  hippocampus-­dependent  tasks  (trace  eye-­ blink  conditioning  or  spatial  navigation  in  the  Morris  water  maze)  or  hippocampus-­independent  tasks  (clas-­ sical  eyeblink  conditioning  or  cue  training  in  the  Mor-­ ris  water  maze).  They  found  that  when  training  began  one  week  after  BrdU  injections,  which  corresponds  to  the  time  when  new  cells  begin  to  either  sprout  ax-­ ons  or  die  off,  a  higher  percentage  of  cells  survived  in  Cornell University

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rats  trained  on  hippocampus-­dependent  tasks  than  on  hippocampus-­independent  tasks.  Furthermore,  they  found  that  if  rats  were  injected  with  BrdU  during  train-­ ing,  rather  than  a  week  before,  there  was  no  increase  in  cell  survival  in  rats  trained  on  hippocampus-­dependent  tasks.  This  suggests  that  hippocampus-­dependent  train-­ ing  does  not  increase  the  rate  at  which  neurogenesis  occurs,  but  instead  increases  the  survival  rate  of  new  neurons  that  have  already  been  produced  37.

6HYHUDO PRUH VWXGLHV KDYH EHHQ GRQH WR FRQÂżUP this  effect  and  clarify  its  mechanisms.  An  experiment  by  Dalla  and  colleagues  (2007)  found  that  it  is  the  pro-­ cess  of  learning  the  task,  rather  than  the  mere  exposure  to  training,  that  increases  new  cell  survival.  Interest-­ LQJO\ WKH\ IRXQG D VLJQLÂżFDQW FRUUHODWLRQ EHWZHHQ WDVN acquisition  and  BrdU-­labeled  cell  number  regardless  of  whether  the  animal  was  trained  on  a  hippocampus-­ dependent  or  â&#x20AC;&#x201C;independent  task  38 7KLV ÂżQGLQJ ZDV expanded  by  Waddell  and  Shors  (2008)  who  found  that  the  cell  survival  rate  was  higher  in  animals  that  took  more  trials  to  learn.  They  also  found  that  acquisition  rate  predicted  cell  survival  more  accurately  than  the  hippocampal  dependence  of  the  task,  suggesting  that  LW PD\ EH WKH GLIÂżFXOW\ RI WKH WDVN ZKLFK LQĂ&#x20AC;XHQFHV the  acquisition  rate)  rather  than  the  hippocampus-­de-­ pendence  of  the  task  that  prompts  the  recruitment  of  new  neurons  into  learning-­related  circuits  and  thereby  increases  their  survival  rate  10.  Further  evidence  for  this  effortful  learning  hypothesis  comes  from  a  study  comparing  male  and  female  rats,  which  found  that  on  average,  female  rats  had  a  slower  acquisition  rate  on  a  trace  eyeblink  conditioning  task,  but  learned  the  task  better  overall,  than  male  rats.  Female  rats  also  retained  a  greater  number  of  BrdU-­labeled  cells,  supporting  the  idea  that  their  survival  rate  is  dependent  on  actually  learning  the  task,  and  is  increased  if  the  task  is  more  GLIÂżFXOW DQG WDNHV PRUH WULDOV WR OHDUQ 39.  While  it  remains  unclear  how  the  hippocampus-­ dependency  of  a  task  is  related  to  new  cell  survival,  it  does  appear  that  training  on  such  tasks  can  increase  the  survival  of  new  neurons.  Furthermore,  the  fact  that  this  effect  is  strongest  when  training  begins  one  week  after  BrdU  administration  when  the  labeled  cells  are  beginning  to  project  axons  and  become  synaptically  in-­ tegrated  10  suggests  that  the  reason  for  this  effect  is  that  the  new  neurons  are  being  recruited  by  the  learning  cir-­ cuits  which  participate  in  the  task,  and  are  spared  from  cell  death.  The  fact  that  survival  rate  can  be  predicted  E\ WKH GLIÂżFXOW\ RI WKH WDVN DQG E\ WKH VXFFHVVIXO

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acquisition  of  the  learned  response  further  suggest  that  JUDQXOH QHXURQV VSHFLÂżF HSLVRGHV FRXOG QRW UHFHLYH it  is  in  these  cases  that  the  new  neurons  are  most  neces-­ unique  neuronal  â&#x20AC;&#x2DC;tagsâ&#x20AC;&#x2122;  that  differentiate  them  from  contextually  similar  episodes,  and  interference  should  sary  and  are  therefore  recruited  most  heavily. therefore  be  more  likely  to  occur  between  distinct  epi-­ A Computational Model of Hippocampal sodic  memories  within  a  given  context.  This  increased  interference  between  episodic  memories  would  also  Neurogenesis PDNH LW PXFK PRUH GLIÂżFXOW WR LGHQWLI\ DQG ELQG WR-­  Taken  together,  the  current  body  of  research  gether  multiple  memories  for  episodes  occurring  in  the  strongly  suggests  that  adult-­generated  hippocampal  same  context,  and  should  therefore  impair  the  ability  neurons  play  an  important  functional  role  in  certain  to  form  rich,  multifaceted  contextual  representations  kinds  of  learning  and  memory,  and  that  their  survival  which  could  later  be  used  to  guide  the  activation  of  ap-­ rates  are  strongly  affected  by  learning  experiences.  SURSULDWH FRJQLWLYH VWUDWHJLHV ZKHQ RQH ÂżQGV RQHVHOI However,  the  fact  that  some  but  not  all  hippocampus-­ in  that  context  again.  Future  research  using  carefully  dependent  tasks  seem  to  be  affected  by  hippocampal  designed  context  discrimination  tasks  will  be  needed  in  neurogenesisbegs  the  question  of  precisely  how  new  order  to  test  these  predictions. hippocampal  neurons  contribute  to  the  overall  function  of  the  hippocampus  and  related  neural  circuits.  Becker  Hippocampal Neurogenesis and and  Wojtowicz  (2007)  recently  proposed  a  computa-­ AďŹ&#x20AC;ective Disorders WLRQDO PRGHO WKDW KHOSV V\QWKHVL]H WKH FXUUHQW ÂżQGLQJV on  hippocampal  neurogenesis  with  the  broader  hip-­  Becker  and  Wojtowiczâ&#x20AC;&#x2122;s  (2007)  model  may  pocampal  literature.  In  their  view,  the  hippocampus  also  help  explain  a  growing  body  of  research  link-­ serves  a  dual  function.  First,  it  is  responsible  for  creat-­ ing  dysfunction  of  the  hippocampus,  and  in  particular  ing  detailed  contextual  representations  that  link  togeth-­ hippocampal  neurogenesis,  to  the  pathophysiology  er  many  aspects  of  the  environment  and  its  associated  of  mood  disorders  such  as  major  depressive  disorder  cognitive  and  behavioral  demands.  Second,  it  acts  as  (MDD).  There  is  strong  evidence  that  stress,  an  impor-­ a  â&#x20AC;&#x2DC;contextual  gateâ&#x20AC;&#x2122;  whereby  it  responds  to  contextual  tant  contributing  factor  to  the  development  of  MDD,  cues  by  activating  the  entire  contextual  representation  has  an  inhibitory  effect  on  hippocampal  neurogenesis  and  priming  other  brain  regions  involved  in  behavior,  14,  and  several  known  effective  treatments  for  MDD  motivation,  cognition,  and  emotion  to  act  accordingly.  have  been  shown  to  enhance  hippocampal  neurogen-­ Newly-­generated  granule  neurons  are  thought  to  play  a  esis,  including  exercise  21,  electroconvulsive  therapy  41,  unique  role  in  this  process  by  virtue  of  their  continuous  and  antidepressant  medications  42.  However,  it  is  not  proliferation,  which  allows  them  to  code  for  separate  yet  clear  how  the  hippocampus,  known  primarily  for  its  episodes  in  time  without  interfering  with  similar  previ-­ role  in  learning  and  memory  functions,  could  contrib-­ ous  episodes,  and  theirdistinctive  maturational  proper-­ ute  to  mood  and  its  dysregulation. ties,  which  allow  for  an  initial  high  level  of  plasticity   Becker  and  Wojtowicz  (2007)  propose  that  the  which  gradually  wanes  over  time  as  they  either  mature  link  resides  in  the  role  of  the  hippocampus  as  a  â&#x20AC;&#x2DC;con-­ or  die  off.  Unique  episodic  memories  encoded  by  pro-­ textual  gateâ&#x20AC;&#x2122;  to  rest  of  the  brain,  including  regions  that  liferating  granule  neurons  can  then  be  integrated  with  process  mood  and  affect  such  as  the  amygdala,  nucleus  other  episodic  memories  that  share  the  same  context  accumbens,  and  prefrontal  cortex.  According  to  their  via  associational  pathways  in  other  hippocampal  re-­ model,  the  hippocampus  is  normally  involved  in  form-­ gions,  resulting  in  the  formation  of  rich  contextual  rep-­ ing  contextual  representations  that  can  then  be  acti-­ resentations  made  up  of  many  distinct  episodic  events  vated  to  modulate  processing  in  these  other  functional  40 . brain  circuits.  However,  if  hippocampal  neurogenesis   This  model  is  especially  useful  because  it  gen-­ is  disrupted,  for  example  by  the  high  levels  of  stress  HUDWHV VSHFLÂżF EHKDYLRUDO SUHGLFWLRQV )RU H[DPSOH LW that  often  precipitate  the  development  of  mood  disor-­ would  predict  an  irradiation-­induced  impairment  in  ders,  the  ability  to  form  contextual  representations  may  performance  on  tasks  that  require  one  to  discriminate  EHFRPH LQWHUUXSWHG UHGXFLQJ WKH DELOLW\ WR Ă&#x20AC;H[LEO\ between  temporally  distinct  episodes  within  the  same  VZLWFK EHWZHHQ WKHP LQ RUGHU WR PDWFK VSHFLÂżF PRGHV context.  Without  the  continuous  proliferation  of  new  of  cognitive  and  emotional  processing  to  their  appro-­ Cornell University

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priate  context.  In  the  case  of  MDD,  this  could  cause  impair-­ ments  in  peopleâ&#x20AC;&#x2122;s  ability  to  generate  appropriately  con-­ textualized  responses  to  emotional  stimuli,  leading  to  a  negative  information-­processing  bias.  Forms  of  such  FRJQLWLYH LQÃ&#x20AC;H[LELOLW\ KDYH EHHQ IRXQG LQ SDWLHQWV ZLWK 0'' ZKR UHJXODUO\ VKRZ D VWURQJ QHJDWLYLW\ ELDV LQ the  face  of  changing  emotional  content  43.  A  greater  XQGHUVWDQGLQJ RI WKH UHODWLRQVKLS EHWZHHQ VWUHVV KLS-­ pocampal  neurogenesis,  context  representations,  and  FRJQLWLYH Ã&#x20AC;H[LELOLW\ VKRXOG WKHUHIRUH EH DQ LPSRUWDQW goal  of  future  research  aimed  at  understanding  both  normal  affective  processing  and  the  neurocognitive  G\VIXQFWLRQV DVVRFLDWHG ZLWK PRRG GLVRUGHUV

Conclusion

:KLOH PXFK PRUH UHVHDUFK PXVW EH GRQH LI ZH are  to  fully  understand  the  purpose  and  functional  im-­ plications  of  adult  hippocampal  neurogenesis,  the  pre-­ VHQW OLWHUDWXUH SRLQWV WR DQ LPSRUWDQW UROH RI QHZO\ JHQ-­ erated  granule  neurons  in  adult  learning  and  memory  processes,  particularly  those  that  rely  on  the  hippocam-­ pus.  This  relationship  appears  to  be  bidirectional,  in  that  learning  experiences  both  require  and  facilitate  WKH SUROLIHUDWLRQ RI QHZ KLSSRFDPSDO QHXURQV 0RUH UHVHDUFK LV QHFHVVDU\ WR KHOS FODULI\ H[DFWO\ KRZ KLS-­ pocampal  neurogenesis  contributes  to  these  processes,  and  may  shed  light  on  the  role  of  the  hippocampus  and  its  unique  neuroplastic  properties  in  learning,  memory,  context  processing,  and  affective  dysregulation.  Future  UHVHDUFK ZLOO XOWLPDWHO\ SURYLGH D JUHDWHU XQGHUVWDQG-­ LQJ RI ZK\ DGXOW KLSSRFDPSDO QHXURJHQHVLV RFFXUV DQG KRZ LW UHODWHV WR WKH IXQFWLRQ RI WKH KLSSRFDPSXV DQG cognitive  functions  it  subserves.

References 1.  Lledo,  P.  M.,  Alonso,  M.,  &  Grubb,  M.  S.  (2006).  Adult  neurogenesis  and  functional  plasticity  inneuronal  circuits.  Nature  5HYLHZV 1HXURVFLHQFH .HPSHUPDQQ * .XKQ + * *DJH ) + 0RUH hippocampal  neurons  in  adult  mice  living  inan  enriched  environ-­ PHQW 1DWXUH 3.  Rakic,  P.  (2002).Adult  neurogenesis  in  mammals,  an  identity  crisis.Journal  of  Neuroscience,  22,  614-­618.

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-RXUQDO RI &RPSDUDWLYH 1HXURORJ\ 1L[RQ . &UHZV ) 7 %LQJH HWKDQRO H[SRVXUH decreases  neurogenesis  in  adult  rathippocampus.  Journal  of  Neu-­ URFKHPLVWU\ 9DQ 3UDDJ + 6FKLQGHU $ ) &KULVWLH % 5 7RQL 1 Palmer,  T.  D.,  &  Gage,  F.  (2002).  Functionalneurogenesis  in  the  DGXOW KLSSRFDPSXV 1DWXUH 'RHWVFK ) +HQ 5 <RXQJ DQG H[FLWDEOH WKH IXQFWLRQ RI QHZ QHXURQV LQ WKH DGXOW PDPPDOLDQ EUDLQ &XUUHQW 2SLQLRQ LQ 1HXURELRORJ\ 'D\HU $ * )RUG $ $ &OHDYHU . 0 <DVVDHH 0 &DPHURQ + $ 6KRUW WHUP DQG ORQJWHUP VXUYLYDO RI QHZ QHXURQV LQ WKH UDW GHQWDWH J\UXV -RXUQDO RI &RPSDUDWLYH 1HXURO-­ RJ\ 10.  Waddell,  J.,  &  Shors,  T.  J.  (2008).Neurogenesis,  learning  DQG DVVRFLDWLYH VWUHQJWK (XURSHDQ -RXUQDO RI1HXURVFLHQFH 3020-­3028.  +DVWLQJV 1 % *RXOG ( 5DSLG H[WHQVLRQ RI D[-­ RQV LQWR WKH &$ UHJLRQ RI DGXOW JHQHUDWHGJUDQXOH FHOOV -RXUQDO RI &RPSDUDWLYH 1HXURORJ\ =KDR & 7HQJ ( 0 6XPPHUV 5 * 0LQJ * *DJH ) (2006).  Distinct  morphologicalstages  of  dentate  granule  neuron  maturation  in  the  adult  mouse  hippocampus.  The  Journal  of  Neu-­ roscience,  26,  3-­11.  7DVKLUR $ 0DNLQR + *DJH ) + ([SHULHQFH VSHFL¿F IXQFWLRQDO PRGL¿FDWLRQ RIWKH GHQWDWH J\UXV WKURXJK DGXOW QHXURJHQHVLV $ FULWLFDO SHULRG GXULQJ DQ LPPDWXUH VWDJH 7KH -RXUQDO RI 1HXURVFLHQFH 0LUHVFX & *RXOG ( 6WUHVV DQG DGXOW QHXURJHQ-­ esis.Hippocampus,  16,  233-­238.

'UDSHDX ( 0D\R : $XURXVVHDX & /H 0RDO 1 3LD]]D 3 9 $EURXV ' 1 6SDWLDOPHPRU\ SHUIRUPDQFHV RI DJHG UDWV LQ WKH ZDWHU PD]H SUHGLFW OHYHOV RI KLSSRFDPSDO QHXUR-­ genesis.  Proceedings  of  the  National  Academy  of  Science,  100, Â

0DWWKHZV ' % 6LOYHUV - 5 7KH XVH RI DFXWH ethanol  administration  as  tool  to  investigatemultiple  memory  V\VWHPV 1HXURELRORJ\ RI /HDUQLQJ DQG 0HPRU\ $EURXV ' 1 $GULDQL : 0RQWDURQ 0 ) $XURXVVHDX & 5RXJRQ * /H 0RDO 0 3LD]]D 3 9 1LFRWLQH self-­administration  impairs  hippocampal  plasticity.  Journal  of  1HXURVFLHQFH 6FHUUL & 6WHZDUW & $ %UHHQ . & %DOIRXU ' -

7KH HIIHFWV RI FKURQLF QLFRWLQH RQ VSDWLDOOHDUQLQJ DQG bromodeoxyuridine  incorporation  into  the  dentate  gyrus  of  the  UDW 3V\FKRSKDUPDFRORJ\ (LVFK $ - %DUURW 0 6FKDG & $ 6HOI ' : 1HVWOHU E.  J.  (2000).Opiates  inhibit  neurogenesis  inthe  adult  rat  hip-­ SRFDPSXV 3URFHHGLQJV RI WKH 1DWLRQDO $FDGHP\ RI 6FLHQFH

6SDLQ - : 1HZVRP * & &KURQLF RSLRLGV LPSDLU DFTXLVLWLRQ RI ERWK UDGLDO PD]H DQG <PD]H FKRLFH HVFDSH 3V\FKRSKDUPDFRORJ\ 9DQ 3UDDJ + &KULVWLH % 5 6HMQRZVNL 7 - *DJH ) +

5XQQLQJ HQKDQFHV QHXURJHQHVLV OHDUQLQJ DQG ORQJ WHUP potentiation  in  mice.  Proceedings  of  the  National  Academy  of  6FLHQFH 22.  Daniel,  J.  M.,  Fader,  A.  J.,  Spencer,  A.L.,  &  Dohanich,  G.  P. Â

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acquisition  of  a  radial  arm  maze.  Hormones  and  Behavior,  32,  217-­225.

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28.  Madsen,  T.  M.,  Kristjansen,  P.  E.  G.,  Bolwig,  T.  G.,  &  Wort-­ wein,  G.  (2003).  Arrested  neuronalproliferation  and  impaired  hippocampal  function  following  fractionated  brain  irradiation  in  the  adult  rat.  Neuroscience,  119,  635-­642.

29.  Winocur,  G.,  Wojtowicz,  J.  M.,  Sekeres,  M.,  Snyder,  J.  S.,  &  Wang,  S.  (2006).  Inhibition  of  neurogenesisinterferes  with  hippocampus-­dependent  memory  function.  Hippocampus,  16,  296-­304.  30.  Hernandez-­Rabaza,  V.,  Llorens-­Martin,  M.,  Velazquez-­ Sanchez,  C.,  Ferragud,  A.,  Arcusa,  A.,  Gumus,  H.  G.,  Gomez-­ Pinedo,  U.,  Perez-­Villalba,  A.,  Rosello,  J.,  Trejo,  J.  L.,  Barcia,  J.  A.,  Canales,  J.  J.  (2009).  Inhibition  of  adult  hippocampal  neuro-­ genesis  disrupts  contextual  learning  but  spares  spatial  working  memory,  long-­term  conditional  rule  retention  and  spatial  reversal.  Neuroscience,  159,  59-­68.

31.  M.D.  Saxe,  G.  Malleret,  S.  Vronskaya,  I.  Mendez,  A.D.  Garcia,  M.V.  Sofroniew,  E.R.  Kandel  and  R.  Hen,  Paradoxical  LQĂ&#x20AC;XHQFH RI KLSSRFDPSDO QHXURJHQHVLV RQ ZRUNLQJ PHPRU\ 3URF Natl  Acad  Sci  U  S  A  104  (2007),  pp.  4642â&#x20AC;&#x201C;4646.  32.  Rola,  R.,  Raber,  J.,  Rizk,  A.,  Otsuka,  S.,  VandenBerg,  S.  R.,  Morhardt,  D.  R.,  &Fike,  J.  R.  (2004).Radiation-­induced  impair-­ ment  of  hippocampal  neurogenesis  is  associated  with  cognitive  GHÂżFLWV LQ \RXQJ PLFH ([SHULPHQWDO 1HXURORJ\ 33.  Raber,  J.,  Rola,  R.,  LeFevour,  A.,  Morhardt,  D.,  Curley,  J.,  Mizumatsu,  S.,  Vandenberg,  S.  R.,  &Fike,J.  R.  (2004).  Radia-­ tion  induced  cognitive  impairments  associated  with  changes  in  indicators  of  hippocampal  neurogenesis.  Radiation  Research,  162,  39-­47.

34.  Snyder,  J.  S.,  Hong,  N.  S.,  McDonald,  R.,  &  Wojtowicz,  J.  M.  (2005).  A  role  for  adult  neurogenesis  inspatial  long-­term  memory.  Neuroscience,  130,  843-­852.

35.  Kempermann,  G.,  &  Gage,  F.  H.  (2002).  Genetic  determi-­ nants  of  adult  hippocampal  neurogenesiscorrelate  with  acquisi-­ tion,  but  not  probe  trial  performance,  in  the  water  maze  task.  European  Journal  of  Neuroscience,  16,  129-­136.

36.  Van  der  Borght,  K.,  Wallinga,  A.  E.,  Lutien,  P.  G.,  Eggen,  B.  J.  L.,  &  Van  der  Zee,  E.  A.  (2005).  Morriswater  maze  learning  in  two  rat  strains  increases  the  expression  of  the  polysialyated  form  of  the  neural  cell  adhesion  molecule  in  the  dentate  gyrus  but  has  no  effect  on  hippocampal  neurogenesis.  Behavioral  Neurosci-­ ence,  119,  926-­932.

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37.  Gould,  E.,  Beylin,  A.,  Tanapat,  P.,  Reeves,  A.,  &  Shors,  T.  J.  (1999).  Learning  enhances  adultneurogenesis  in  the  hippocampal  formation.  Nature  Neuroscience,  2,  260-­265.

38.  Dalla,  C.,  Bangasser,  D.  A.,  Edgecomb,  C.,  &  Shors,  T.  J.  (2007).  Neurogenesis  and  learning:  Acquisitionand  asymptotic  performance  predict  how  many  new  cells  survive  in  the  hip-­ pocampus.  Neurobiology  of  Learning  and  Memory,  88,  143-­148.  39.  Dalla,  C.,  Papachristos,  E.  B.,  Whetstone,  A.  S.,  &  Shors,  T.  J.  (2009).  Female  rats  learn  trace  memoriesbetter  than  male  rats  and  consequently  retain  a  greater  proportion  of  new  neurons  in  their  hippocampi.  Proceedings  of  the  National  Academy  of  Sci-­ ence,  106,  2927-­2932.  40.  Becker,  S.,  &  Wojtowicz,  J.  M.  (2007).  A  model  of  hip-­ pocampal  neurogenesis  in  memory  and  mooddisorders.  Trends  in  Cognitive  Science,  11,  70-­76.  41.  Scott,  B.  W.,  Wojtowicz,  J.  M.,  &  Burnham,  W.  M.  (2000).  Neurogenesis  in  the  dentate  gyrus  of  the  rat  following  electrocon-­ vulsive  shock  seizures.  Experimental  Neurology,  165,  231-­236.

42.  Malberg,  J.,  Eisch,  A.  J.,  Nestler,  E.J.,  &  Duman,  R.  S.  (2000).  Chronic  antidepressant  treatment  increases  neurogenesis  in  adult  rat  hippocampus.  Journal  of  Neuroscience,  20,  9104-­ 9110.

43.  Leppanen,  J.  M.  (2006).  Emotional  information  processing  in  PRRG GLVRUGHUV D UHYLHZ RI EHKDYLRUDO DQG QHXURLPDJLQJ ¿QG-­ ings.  Current  Opinions  in  Psychiatry,  19,  34-­39.

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Omega-3 or Omega-6?

Breastmilk or Formula?

The Controversy Over the EďŹ&#x20AC;ectiveness of Essential Fatty Acids in Infants and Their Mothers on Brain Function and Development Jonathan C. Lin, Human Ecology 14, Human Biology, Health & Society Yoshiko Toyoda, Arts and Sciences 14, Biological Sciences and History Abstract

lation  with  regards  to  dietary  choices  and  the  often  The  chemical  anatomy  of  Omega-­3  and  Omega-­6  controversial  need  for  supplements. differ  only  by  the  placement  of  a  carbon-­carbon  double  bond,  yet  these  two  distinct  macronutrients  Introduction have  drastically  different  effects  on  nutrition  and  cognitive  development  in  pregnant  and  pediatric   With  global  industrialization,  our  diets  have  populations.  Both  macronutrients  can  be  ingested  dramatically  shifted  away  from  traditional  plant-­based  naturally  through  the  diet  or  taken  in  supplements  diets  to  processed  food  diets.  Over  the  past  100-­150  and  formulas.  Today,  our  society  consumes  a  dis-­ years,  consumption  of  n-­6  fatty  acids  has  gone  up  due  proportionate  amount  of  n-­6  fatty  acids  (Omega-­6)  to  the  increased  intake  of  vegetable  oils,  principally  compared  to  Omega-­3.  We  have  attempted  to  make  found  in  corn  and  corn-­based  products1.  N-­6  fatty  acids  XS IRU WKLV GHÂżFLHQF\ E\ FRQVXPLQJ PRUH ÂżVK D or  Omega-­6  fatty  acids  simply  refer  to  the  carbon-­car-­ principal  source  for  Omega-­3,  but  environmental-­ bon  double  bond  in  the  n-­6  position  of  the  polyunsatu-­ ists  warn  us  of  the  potential  dangers  of  mercury  poi-­ rated  fatty  acid  (PUFA).  The  n-­6  counterpart,  n-­3  or  soning.  As  the  consequences  of  poor  nutrition  have  Omega-­3  PUFAs,  which  are  found  most  prominently  in  become  increasingly  more  evident,  some  of  us  have  ¿VK DQG ÂżVK EDVHG SURGXFWV FRPH IURP WKH VDPH IDP-­ KHHGHG WR ZDUQLQJV RI SRWHQWLDO GDQJHUV LQ ÂżVK DQG ily  of  fatty  acids  but  have  their  carbon-­carbon  double  turned  to  supplements.  This  review  seeks  to  explore  bond  located  at  the  n-­3  position.  This  subtle  difference  whether  Omega  3  consumption  will  make  children  affects  our  diet  and  health  and  plays  an  important  role  smarter  by  comparing  the  two  macronutrients.  By  in  our  cognitive  development.  Both  n-­3  and  n-­6  PU-­ weighing  the  strengths  and  weaknesses  of  several  FAs  are  long  chain-­PUFAs  (LC-­PUFAs)  and  are  essen-­ articles  assessing  the  two  fatty  acids  and  their  modes  tial  nutrients2.  We  once  were  a  plant-­based  society  that  of  administration,  we  found  that  despite  the  risk  of  lived  on  diets  with  a  healthy  ratio  of  n-­6  to  n-­3  fatty  WR[LFDQW LQJHVWLRQ LQ ÂżVK PDWHUQDO Q SRO\XQVDW-­ acids.  However,  in  Western  diets  today  the  ratio  of  n-­6  urated  fatty  acid  (n-­3  PUFA/Omega-­3)  consump-­ to  n-­3  fatty  acids  ranges  from  20-­30:1  instead  of  the  tion  increases  a  childâ&#x20AC;&#x2122;s  intelligence  quotient  (IQ)  traditional  range  of  1:1  or  2:11.  It  is  important  to  con-­ at  least  through  four  years  of  life.  After  birth,  long  sider  that  in  the  past  the  life  expectancy  was  also  lower.  chain-­PUFA  supplementation,  which  includes  both  There  are  many  factors  that  might  explain  this  trend  of  Omega-­3  and  Omega-­6,  in  infant  formula  improves  increasing  life  expectancy,  namely  our  advanced  medi-­ neural  development  among  infants  and  the  critical  FDO NQRZOHGJH DQG DELOLW\ WR ÂżJKW GLVHDVH +RZHYHU SHULRG RI VXSSOHPHQWV H[WHQGV EH\RQG WKH ÂżUVW VL[ technology  must  not  overshadow  good  nutritional  care,  weeks  of  life.  Although  only  a  handful  of  articles  are a  necessary  preventative  line  of  defense  against  dis-­ exmined,  there  are  still  insights  drawn  from  this  re-­ ease.

23 search  that  may  be  applied  towards  the  adult  popu-­  Cornell University

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 The  PUFA  docosahexaenoic  acid  (DHA)  is  a  potent  neurobiological  agent  that  affects  neuronal  membrane  structure,  synaptogenesis,  and  myelination.  It  can  be  thought  of  as  a  macronutrient  that  is  important  in  preventing  cognitive  abnormalities.  Studies  in  preterm  KXPDQV LQGLFDWH LPSRUWDQW EHQHÂżWV LQ UHWLQDO DQG cognitive  development  after  DHA  supplementation3.  Today,  PUFAs  such  as  DHA  are  marketed  as  â&#x20AC;&#x2DC;miracle  workersâ&#x20AC;&#x2122;  that  make  people  â&#x20AC;&#x2DC;smarter,â&#x20AC;&#x2122;  and  as  a  result,  GLHWDU\ VXSSOHPHQWV RI ÂżVK RLO KDYH EHFRPH SRSXODU among  health-­conscious  Americans.  As  seen  in  Figure  1,  total  fats,  including  saturated,  trans,  and  n-­6  fats  increased  during  the  transition  to  an  industrial  society  while  n-­3  PUFAs  decreased  moderately.

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Figure  1  Total  fatty  acid  intake  over  time  increased  dramatically  since  the  19th  century.  Intake  of  n-­6  fatty  acids  increased  while  intake  of  n-­3  fatty  acids  decreased  slightly.   The popularity of PUFAs derived from fish oils is backed by scientific data. PUFAs are generally the â&#x20AC;&#x2DC;healthierâ&#x20AC;&#x2122; fats compared to saturated fats and trans fats. They are also important for cognitive functions.

According to Kitajika, et al., PUFAs are essential structural components of the central nervous system2.

Arachidonic acid (AA), a n-6 PUFA, and DHA, a n-3 PUFA, have been shown to be essential for brain growth and cognitive development because they accumulate rapidly in the brain of a developing child in the later part of gestation and early postnatal life4. On the one hand, n-6 PUFA is over-consumed by western society to the point where its excessive consumption may be damaging to our health. On the other hand, DHA (n-3 PUFA) is under-consumed by some populations and its potentially beneficial effects are still being explored.

Mammals obtain DHA either as DHA itself or as the precursor alphe-linolenic acid (ALA), and intermediates between ALA and DHA,

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including eicosapentaenoic acid (EPA)5, DHA protects neural cells from apoptotic death6, induces synaptic growth cones during neuronal development7,8,9, enhances synaptic function10, and regulates nerve growth factor11, among other functions. The effects of PUFAs on brain development have been experimentally studied extensively during the early years of life when the growing infant relies heavily on fatty acids for cognitive development. Normally, an infant acquires enough nutrients from the motherâ&#x20AC;&#x2122;s breastmilk.

However, some mothers are unable to produce milk, others are undernourished, and some mothers prefer not to breastfeed. All of these reasons result in the need for some babies to be fed formula. Formulas are often supplemented to include the essential fatty acids for cognitive development that we have discussed thus far. But are the formulas necessary, sufficient, or even more effective than breastmilk? We reviewed the literature that discusses the potent benefits of n-3 PUFAs compared to n-6 PUFAs and weigh in on these findings. We will focus on the effects of dietary intake of PUFA during pre-natal infancy and infancy, and use the context of PUFAs to examine the effectiveness of supplemented formulas compared to breastmilk.

Omega-3 vs. Omega-6 and Maternal Intake of these PUFAs  Neural  tissues  in  the  brain  show  progressive  enrichment  of  phospholipids  with  LC-­PUFAs,  especially  during  the  last  trimester  of  fetal  development  DQG WKH ÂżUVW PRQWKV DIWHU ELUWK VXJJHVWLQJ WKDW the  availability  of  LC-­PUFAs  is  critical  for  neural  development12.  Animal  studies  have  shown  that  a  GHÂżFLHQF\ RI Q 38)$ PD\ OHDG WR FRJQLWLYH GHÂżFLWV later  in  life13.  This  leads  many  to  believe  that  maternal  VHDIRRG DQG VSHFLÂżFDOO\ Q 38)$ FRQVXPSWLRQ E\ mothers  during  pregnancy  and  lactation  will  make  their  kids  â&#x20AC;&#x2DC;smarter.â&#x20AC;&#x2122;  However,  in  another  study,  infants  who  received  breast  milk  formula  with  n-­3  PUFAs  but  not  n-­6  PUFAs  scored  lower  than  infants  fed  both  kinds  of  PUFAs  on  cognitive  assessments14.  These  seemingly  contradictory  results  reveal  that  a  controversy  exists  over  whether  or  not  maternal  intake  of  seafood  and  n-­3  PUFAs  can  in  fact  increase  a  childâ&#x20AC;&#x2122;s  IQ,  an  indicator  of  cognitive  development.  However,  with  increased  FRQVXPSWLRQ RI SRWHQWLDOO\ EHQHÂżFLDO Q 38)$ risk  and  exposure  of  mothers  and  children  to  toxic  FRQWDPLQDQWV IRXQG LQ ÂżVK VXFK DV PHUFXU\ LQFUHDVHV

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¦ Omega-3 ¦

Furthermore,  comparisons  between  the  effects  of  n-­3  and  n-­6  PUFAs  need  to  be  made  and  the  timing  of  consumption  and  its  effects  assessed.  Upon  discussion  and  interpretation  of  the  following  studies,  we  can  state  ZLWK JUHDWHU FRQ¿GHQFH WKDW GHVSLWH WKH ULVN RI WR[LFDQW ingestion,  maternal  n-­3  PUFA  consumption  during  later  stages  of  pregnancy  increases  a  childâ&#x20AC;&#x2122;s  IQ  at  least  through  four  years.  Much  progress  has  been  made  on  this  controversy  since  the  turn  of  the  century.  In  2003,  +HOODQG HW DO H[DPLQHG WKH HIIHFW RI Q 38)$ supplements  on  mental  development  of  children  of  pregnant  and  lactating  women,  compared  with  that  of  n-­6  PUFA  supplements.  A  control  group  of  pregnant  and  lactating  mothers  on  the  same  diet  without  supplements  was  used.  The  two  supplementsâ&#x20AC;&#x201D;one  enriched  with  n-­3  PUFA  from  cod  liver  oil  and  the  other  with  n-­6  PUFA  from  corn  oilâ&#x20AC;&#x201D;contained  the  same  amount  by  mass  of  either  fatty  acid.  At  four  years  of  age,  90  of  UDQGRPO\ VHOHFWHG FKLOGUHQ WRRN WKH .DXIPDQ $VVHVVPHQW %DWWHU\ IRU &KLOGUHQ . $%& ,Q WKH . $%& D PHQWDO SURFHVVLQJ FRPSRVLWH ZDV XVHG WR assess  intelligence14.  The  notable  inclusion  criteria  for  the  women  in  the  study  included  being  age  19  to  35  and  having  an  intention  to  breastfeed.  Self-­administered  IRRG IUHTXHQF\ TXHVWLRQQDLUHV ZHUH ¿OOHG RXW E\ WKH PRWKHUV IRU GLHWDU\ LQWDNH LQIRUPDWLRQ  At  four  years  of  age,  children  in  the  n-­3  PUFA  JURXS KDG KLJKHU . $%& VFRUHV WKDQ FKLOGUHQ LQ WKH n-­6  PUFA  group  on  the  mental  processing  composite  YV S 7KLV FRPSRVLWH FRUUHODWHG positively  with  DHA  found  in  n-­3  PUFA  supplements(r  S 7KH UHVXOWV RI WKLV VWXG\ VKRZHG WKDW IRXU \HDU ROG FKLOGUHQ ZKRVH PRWKHUV WRRN Q 38)$ supplements  during  pregnancy  had  higher  mental  SURFHVVLQJ VFRUHV WKDQ FKLOGUHQ ZKRVH PRWKHUV WRRN n-­6  PUFA  supplements14 7KXV PDWHUQDO LQWDNH RI DHA  and  n-­3  PUFA  supplementation  during  pregnancy  may  be  important  for  mental  development.  The  study  GLG KDYH ZHDNQHVVHV VXFK DV VHOI DGPLQLVWHUHG IRRG IUHTXHQF\ TXHVWLRQQDLUHV ¿OOHG RXW E\ WKH PRWKHUV IRU their  children  and  a  geographic  scope  limited  to  the  Oslo,  Norway  area.  However,  the  strengths,  such  as  the  double-­blind  nature  of  the  study  and  the  wide  range  of  LQFOXVLRQ H[FOXVLRQ FULWHULD RXWZHLJKHG WKH OLPLWDWLRQV Therefore,  evidence  supports  the  argument  that  n-­3  PUFAs  consumed  by  pregnant  mothers  increases  a  childâ&#x20AC;&#x2122;s  full-­scale  intelligence.  Three  years  later,  Helland  et  al.  conducted  a  Cornell University

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follow-­up  study  from  the  previously  discussed  2003  VWXG\ WR DVVHVV LI Q 38)$ VWLOO KDG EHQH¿FLDO HIIHFWV over  n-­6  PUFA  on  childrenâ&#x20AC;&#x2122;s  intelligence  at  seven  years  of  age.  No  additional  fatty  acid  supplements  were  given;;  143  children,  some  of  which  were  from  the  2003  study,  ZHUH LQYLWHG EDFN DQG UHDVVHVVHG ZLWK WKH . $%&

1R VWDWLVWLFDO GLIIHUHQFHV LQ WKH . $%& VFRUHV at  seven  years  of  age  between  the  two  groups  of  children  ZHUH IRXQG S! 7KH UHVHDUFKHUV DOVR GLG QRW ¿QG DQ\ VLJQL¿FDQW GLIIHUHQFHV EHWZHHQ WKH WZR JURXSV DW HLWKHU IRXU RU VHYHQ \HDUV RI DJH S! +RZHYHU both  groups  did  improve  their  scores  from  four  to  VHYHQ \HDUV RI DJH S 4.  Overall,  the  authors  concluded  that  there  were  no  differences  in  IQ  scores  at  seven  years  of  age  between  children  of  mothers  of  the  two  groups.  Therefore,  Helland  et  al.  argued  that  n-­3  PUFA  consumption  by  pregnant  mothers  does  not  LQFUHDVH D FKLOG¶V IXOO VFDOH LQWHOOLJHQFH DQG VSHFL¿HG WKDW LW GRHV QRW KDYH D EHQH¿FLDO HIIHFW RQ VHYHQ \HDU old  children.  The  same  cognitive  tests  as  those  used  in  2003  were  used  in  the  follow-­up  study,  which  allowed  for  comparisons.  However,  the  validity  of  the  authorâ&#x20AC;&#x2122;s  conclusion  is  reduced  because  of  the  inconsistency  of  VWXG\ VDPSOHV VXEMHFWLYLW\ RI GDWD FROOHFWHG DQG ZHDN control  for  confounding  factors.

,Q D GLIIHUHQW VWXG\ H[DPLQLQJ YDU\LQJ OHYHOV RI PDWHUQDO VHDIRRG LQWDNH GXULQJ SUHJQDQF\ +LEEHOQ et  al.,  in  2007,  used  the  Avon  Longitudinal  Study  of  3DUHQWV DQG &KLOGUHQ $/63$& WR YHULI\ WKH 8 6 Advisory  Boardâ&#x20AC;&#x2122;s  2004  recommendation  that  pregnant  ZRPHQ OLPLW WKHLU VHDIRRG LQWDNH GXULQJ SUHJQDQF\ WR JUDPV SHU ZHHN (LJKW\ ¿YH SHUFHQW RI children  and  their  mothers  elected  to  participate.  To  obtain  data  about  diet  and  other  variable  factors,  mothers  answered  questionnaires  four  times  during  pregnancy  DQG IRXU WLPHV DIWHU ELUWK $W ZHHNV JHVWDWLRQ D VHOI FRPSOHWHG IRRG IUHTXHQF\ TXHVWLRQQDLUH DVNHG IRU WKH QXPEHU RI WLPHV GDLO\ WKDW ZKLWH ¿VK GDUN RU RLO\ ¿VK DQG VKHOO¿VK ZDV FRQVXPHG DQG ZDV XVHG WR REWDLQ Q 38)$ LQWDNH YDOXHV (DFK FKLOG¶V ,4 ZDV PHDVXUHG DW age  eight  using  the  Weschler  Intelligence  Scale  for  Children  III.

7KH HVWLPDWHG Q 38)$ LQWDNH UDQJHG IURP WR J ZHHN PHDQ 6' &KLOGUHQ RI PRWKHUV ZKR UHSRUWHG QR VHDIRRG LQWDNH KDG WKH JUHDWHVW ULVN RI DGYHUVH RU VXERSWLPXP RXWFRPHV :KHQ VHDIRRG LQWDNH ZDV PRGHUDWH J SHU ZHHN WKH ULVN RI VXERSWLPXP RXWFRPH LQ WKH FKLOG UHQ ZDV EHWZHHQ WKH WZR H[WUHPHV RI VHDIRRG

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consumption.  The  results  of  this  studyâ&#x20AC;&#x201D;in  contrast  to  the  U.S.  Advisory  Boardâ&#x20AC;&#x2122;s  recommendationâ&#x20AC;&#x201D;show  that  maternal  consumption  of  more  than  340  g  of  seafood  SHU ZHHN LV DFWXDOO\ EHQHÂżFLDO IRU D FKLOGÂśV FRJQLWLYH development.  The  advice  to  limit  seafood  intake  to  UHGXFH OHYHOV RI PHWK\O PHUFXU\ IRXQG LQ ÂżVK PLJKW reduce  the  intake  of  nutrients  necessary  for  optimum  neurological  development.  Based  on  the  data,  the  authors  concluded  that  the  risk  of  losing  the  potential  LQWHOOHFWXDO EHQHÂżWV RI Q QXWULHQWV H[FHHGV WKH ULVN RI H[SRVXUH WR WUDFH DPRXQWV RI FRQWDPLQDQWV15.  Though  the  observed  cohort  is  studied  with  limited  geographic  samples,  the  researchers  note  the  fact  that  the  U.K.  population  had  a  higher  mean  consumption  of  mercury  Č?J NJ ERG\ZHLJKW WKDQ WKH 8 6 SRSXODWLRQ Č?J NJ ERG\ZHLJKW 15.  This  statistic  lends  more  YDOLGLW\ WR WKH FRQFOXVLRQ WKDW WKHUH DUH EHQHÂżWV RI seafood  consumption.  Thus,  there  is  strong  support  that  maternal  consumption,  and  even  overconsumption,  of  n-­3  PUFAs  during  pregnancy  may  increase  a  childâ&#x20AC;&#x2122;s  intelligence.

,Q RUGHU WR H[DPLQH ÂżVK LQWDNH LQ GLIIHUHQW stages  of  pregnancy  and  its  effects  in  post-­infancy,  *DOH HW DO XVHG WKH :HFKVOHU $EEUHYLDWHG 6FDOH RI ,QWHOOLJHQFH :$6, WR DVVHVV LQWHOOLJHQFH DQG LWV DVVRFLDWLRQ ZLWK PDWHUQDO RLO\ ÂżVK LQWDNH GXULQJ early  and  late  gestation  in  a  sample  of  217  nine-­year-­ old  children.  A  questionnaire  asked  for  the  frequency  RI ZKLWH ÂżVK ÂżVK LQ VDXFHV RLO\ ÂżVK DQG VKHOOÂżVK consumptionâ&#x20AC;&#x201D;which  was  then  recorded  into  eight  categories  representing  different  durations  of  time.  Behavioral  data  was  skewed  so  it  was  dichotomized  to  have  a  reference  category  containing  80-­90%  of  the  data  and  an  upper  tail  representing  adverse  behaviors13.   After  adjusting  for  confounding  variables  such  as  IQ  and  age,  the  infantsâ&#x20AC;&#x2122;  cognitive  function  scores  ZHUH QRW VWDWLVWLFDOO\ VLJQLÂżFDQW EHWZHHQ PRWKHUV ZKR QHYHU DWH ÂżVK DQG WKRVH ZKR DWH ÂżVK WLPHV D ZHHN 7KH DQDO\VHV ZHUH UHSHDWHG ORRNLQJ DW RLO\ ÂżVK LQWDNH RQO\ DQG VLJQLÂżFDQW DVVRFLDWLRQ ZDV QRW IRXQG EHWZHHQ LQWDNH DQG KLJKHU WRWDO GLIÂżFXOWLHV VFRUHV S

7KXV WKH UHVXOWV RI WKLV VWXG\ IRXQG QR VLJQL¿FDQW DVVRFLDWLRQV EHWZHHQ LQWDNH RI RLO\ ¿VK LQ HDUO\ RU ODWH pregnancy  and  intelligence  subcategories13.  It  must  be  QRWHG WKDW WKHUH ZDV QR VLJQL¿FDQW DVVRFLDWLRQ EHWZHHQ IUHTXHQF\ RI HDWLQJ ¿VK LQ HDUO\ SUHJQDQF\ DQG FKLOGUHQœV IXOO VFDOH ,4 EXW ³WKHUH ZDV D VLJQL¿FDQW DVVRFLDWLRQ ZKHQ HDWLQJ ¿VK LQ ODWH SUHJQDQF\´13.  :KLOH WKH LQFOXVLRQ DQG H[FOXVLRQ FULWHULD DUH

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strong,  they  are  overshadowed  by  the  lack  of  an  upper  age  limit  for  eligibility  in  the  study,  which  may  have  led  to  variability.  In  addition,  the  parameters  of  the  results  were  changed  after  collection  of  data,  which  decreases  validity.  Therefore,  their  conclusion  that  n-­3  PUFA  consumption  during  late  pregnancy  does  not  increase  a  childâ&#x20AC;&#x2122;s  full-­scale  intelligence  is  weak.  Considering  the  results  and  weighing  the  strengths  and  weaknesses  of  the  previous  four  articles  GLVFXVVHG LW VHHPV WKDW GHVSLWH WKH ULVN RI WR[LFDQW ingestion,  maternal  n-­3  PUFA  consumption  during  later  stages  of  pregnancy  increases  a  childâ&#x20AC;&#x2122;s  IQ  through  at  least  four  years  of  life.

Breastmilk or Formula? Infant Intake of Long-Chain PUFAs  In  addition  to  maternal  intake  of  PUFAs,  direct  consumption  of  PUFAs  by  infants  may  also  affect  their  EUDLQ GHYHORSPHQW $V DQ H[DPSOH )XMLPRWR HW DO showed  that  dietary  supplementation  of  DHA  improves  learning  skills16,17.  These  â&#x20AC;&#x2DC;essentialâ&#x20AC;&#x2122;  fatty  acids  have  been  added  to  infant  formula18 ZKLFK EHQHÂżWV LQIDQWV who  do  not  have  access  to  naturally  supplemented  breast  milk.  Studies  have  been  conducted  to  assess  the  effects  of  supplemented  formula.  Here,  we  will  review  studies  that  compare  breastmilk,  supplemented  formula,  and  unsupplemented  formula,  and  their  respective  effects  on  neural  development,  in  order  to  determine  which  feeding  pattern  is  best  for  neural  growth  in  infants.  To  assess  the  effects  of  the  three  different  diets  on  neural  development,  Agostoni,  et  al.  studied  WKH LQĂ&#x20AC;XHQFH RI LQFUHDVHG /& 38)$V RQ QHXUDO development  and  fatty  acid  status  in  infants  in  a  randomized  control  trial19.  The  authors  attempted  to  directly  connect  the  psychomotor  performance  of  full-­ WHUP LQIDQWV DW IRXU PRQWKV RI DJH ZLWK /& 38)$ integrated  in  a  formula  regimen.  90  infants  were  fed  RQH RI WKUHH GLIIHUHQW GLHWV EUHDVW IHG /& 38)$ supplemented  formula,  or  standard  formula  lacking  /& 38)$ EXW FRQWDLQLQJ SUHFXUVRUV /$ DQG $/$

for  four  months  after  birth  and  their  blood  DHA  FRQFHQWUDWLRQV DQG SHUIRUPDQFH RQ WKH %UXQHW /H]LQH test,  which  measures  motor  function,  social  reactions,  and  language,  were  recorded19.  The  scores  were  then  XVHG WR FDOFXODWH WKH GHYHORSPHQWDO TXRWLHQW '4 RI the  child.  At  four  months,  infants  who  were  fed  the  HQULFKHG IRUPXOD VFRUHG VLJQL¿FDQWO\ KLJKHU RQ WKH %UXQHW /H]LQH WHVW WKDQ WKRVH IHG WKH VWDQGDUG IRUPXOD

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(DQ=105.3  ±  9.4  vs.  96.5  ±  10.9,  p<0.01).  Breast-­fed  infants  also  scored  higher  than  those  on  standard  formula  (DQ=102.2  ±  11.5).  In  addition,  among  the  infants  who  underwent  blood  sampling,  breast-­fed  infants  (AA=8.5  ±  2.4,  DHA=2.7  ±  0.8)  and  supplemented  formula-­fed  groups  (AA=7.0  ±  1.5,  DHA=2.1  ±  0.6)  had  higher  AA  and  DHA  levels  in  their  blood  than  those  fed  the  standard  formula  (AA=4.4  ±  1.1,  DHA=0.6  ±  0.1).  The  results  of  this  experiment  therefore  support  the  supplementation  of  infant  formula  with  AA  and  DHA,  DV LW FRUUHODWHV VLJQL¿FDQWO\ ZLWK KLJKHU '4 VFRUHV and  higher  blood  levels  of  AA  and  DHA.  A  strength  of  this  experiment  was  that  the  tests  were  carried  out  E\ WKH VDPH PRQLWRU ZKLFK DOORZHG HDFK LQIDQW¶V SHUIRUPDQFH WR EH VWDQGDUGL]HG DQG MXGJHG HTXDOO\ ,Q addition,  quantitative  data  of  blood  lipid  concentrations  VWUHQJWKHQHG WKH SHUIRUPDQFH UHVXOWV¶ YDOLGLW\ &RQVLGHULQJ WKH VWURQJ GHVLJQ DQG KLJK YDOLGLW\ RI WKLV VWXG\ DQG ZHLJKLQJ LW ZLWK WKH LQKHUHQW YDULDELOLW\ RI EUHDVWPLON FRPSRVLWLRQ WKHUH LV VXI¿FLHQW VXSSRUW IRU WKH DXWKRUV¶ FRQFOXVLRQ WKDW /& 38)$ VXSSOHPHQWDWLRQ in  infant  formula  improves  neural  development  in  four-­ month  old  infants.

,Q DQRWKHU VWXG\ KRZHYHU D GLIIHUHQW FRQFOXVLRQ was  reached.  Auestad,  et  al.  conducted  a  double-­blind  VWXG\ LQ ZKLFK UHVHDUFKHUV REVHUYHG WKH FRJQLWLYH GHYHORSPHQW RI LQIDQWV DW PRQWKV 7KLV VWXG\ ZDV D IROORZ XS RI D SUHYLRXV VWXG\ LQ ZKLFK LQIDQWV RI DQG PRQWKV ZHUH H[FOXVLYHO\ EUHDVWIHG for  three  months.  196  infants  were  randomized  within  one  week  after  birth  into  three  groups:  control  formula  containing  no  AA  or  DHA  (n=65),  formula  with  just  DHA  (n=65),  and  formula  with  both  DHA  and  AA  Q 6WDQGDUG WHVWV RI ,4 UHFHSWLYH YRFDEXODU\ H[SUHVVLYH YRFDEXODU\ YLVXDO PRWRU IXQFWLRQ DQG YLVXDO DFXLW\ ZHUH WKHQ DGPLQLVWHUHG  At  12  months,  no  difference  was  found  between  the  three  formula  groups  or  between  the  breastfed  and  formula-­fed  infants  in  terms  of  growth,  visual  DFXLW\ RU PHQWDO DQG PRWRU GHYHORSPHQW +RZHYHU at  14  months,  infants  fed  DHA  but  no  AA  had  lower  YRFDEXODU\ SURGXFWLRQ DQG FRPSUHKHQVLRQ VFRUHV WKDQ infants  who  were  fed  the  unsupplemented  formula  RU ZKR ZHUH EUHDVWIHG ,Q WKH IROORZ XS VWXG\ DW 39  months,  no  difference  was  seen  among  the  three  randomized  formula  groups  or  between  breastfed  and  formula  groups  in  their  neural  developmental  performance,  growth,  or  AA  and  DHA  levels  in  blood.  7KH PRQWK REVHUYDWLRQ RI ORZHU YRFDEXODU\ VFRUH Cornell University

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DV D UHVXOW RI VXSSOHPHQWLQJ IRUPXOD ZLWK RQO\ '+$ DQG QR $$ PD\ KDYH EHHQ D WUDQVLHQW HIIHFW RI '+$ RQ HDUO\ GHYHORSPHQW +RZHYHU WKH ODFN RI GLIIHUHQFHV LQ QHXUDO GHYHORSPHQW JURZWK DQG EORRG /& 38)$ levels  at  39  months  suggests  that  DHA  with  or  without  AA  supports  normal  growth  in  full-­term  infants,  though  no  improved  performance  was  seen.  Thus,  the  authors  concluded  that  adding  both  DHA  and  AA  are  not  GHOHWHULRXV WR LQIDQWV \HW WKHVH PDFURQXWULHQWV GR QRW VHHP WR SURYLGH LQFUHDVHG EHQH¿WV WKURXJK PRQWKV of  age20.

7KLV VWXG\¶V VWUHQJWKV LQFOXGHG D ODUJH VDPSOH group  and  supplementation  of  its  qualitative  data  with  quantitative  data  from  the  growth  and  blood  level  measurements.  Also,  the  follow-­up  design  allowed  the  authors  to  follow  and  compare  data  from  the  same  cohort  RI SDUWLFLSDQWV DW YDU\LQJ WLPH LQWHUYDOV +RZHYHU ZKLOH WKH H[FOXVLRQ FULWHULD ZHUH UHODWLYHO\ VWULFW WKHUH was  no  upper  limit  to  gestational  age.  In  addition,  WKH FRPSDULVRQ JURXS RI EUHDVWIHG LQIDQWV ZDV RQO\ H[FOXVLYHO\ EUHDVWIHG IRU WKUHH PRQWKV DQG FRQVXPHG ZKDWHYHU WKH\ ZLVKHG IRU WKH UHVW RI WKH VWXG\ LQFUHDVLQJ YDULDELOLW\ RI WKH LQIDQWV¶ GLHW DQG SRWHQWLDOO\ UHGXFLQJ WKH HIIHFW RI EUHDVW PLON )LQDOO\ RQO\ RI WKH LQIDQWV IURP WKH VWXG\ SDUWLFLSDWHG LQ WKH IROORZ XS 3HUKDSV ELDV LQ WKH VHOHFWLRQ FULWHULD RFFXUUHG DV LV WKH FDVH ZKHQ SDUHQWV ZKR VHH D SRRU YRFDEXODU\ VFRUH at  12  or  14  months  elect  to  discontinue  their  children  in  WKH VWXG\ ,Q RXU RSLQLRQ WKH ZHDNQHVVHV RI WKLV VWXG\ VHHP WR RXWZHLJK WKH VWUHQJWKV DQG WKXV WKH DXWKRUV¶ FRQFOXVLRQ²WKDW '+$ DQG $$ DUH UHODWLYHO\ QHXWUDO LQ WKHLU HIIHFWV²LV QRW VWURQJO\ XSKHOG

6FLHQWLVWV K\SRWKHVL]H WKDW WKHUH LV D FULWLFDO SHULRG GXULQJ ZKLFK GLHWDU\ VXSSO\ RI /& 38)$V PD\ LQÃ&#x20AC;XHQFH WKH PDWXUDWLRQ RI FRUWLFDO IXQFWLRQ LQ WHUP LQIDQWV 3UHYLRXV UHVHDUFK VXJJHVWV WKDW QHXUDO tissues  in  the  brain  show  progressive  enrichment  of  SKRVSKROLSLGV ZLWK /& 38)$V HVSHFLDOO\ GXULQJ WKH ODVW WULPHVWHU RI IHWDO GHYHORSPHQW DQG WKH ¿UVW months  after  birth21-­25 %LUFK HW DO FRQGXFWHG D VWXG\ WR GHWHUPLQH WKH LPSRUWDQFH RI /& 38)$V DW GLIIHUHQW VWDJHV RI LQIDQF\ IRU WKH PDWXUDWLRQ RI FRUWLFDO IXQFWLRQ 7KH VWXG\ FRPSDUHG UHODWLYH LPSRUWDQFH LQ WKH ¿UVW VL[ ZHHNV RI OLIH ZLWK WKDW RI ZHHN WKURXJK WKH HQG RI \HDU RQH ,I WKH FULWLFDO SHULRG IRU DFFUHWLRQ RI /& 38)$V E\ WKH EUDLQ H[WHQGV EH\RQG ZHHNV RQH ZRXOG H[SHFW WKDW GLHWDU\ /& 38)$ VXSSOHPHQWV LQ LQIDQW IRUPXOD would  improve  cortical  function  in  term  infants  weaned  from  breast-­feeding  at  6  weeks  of  age.  In

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a  randomized  control  trial,  65  healthy  term  infants  were  randomized  into  two  diet  groups,  the  control  group  with  commercially  available  infant  formula  and  the  other  with  the  same  commercial  formula  supplemented  with  0.36%  DHA  and  0.72%  AA.  The  infants  were  fed  their  respective  assigned  diets  from  weeks  7  to  year  one.  Throughout  the  year,  the  infants  were  assessed  for  acuity  (keenness  of  vision),  stereoacuity  (the  ability  to  detect  differences  in  distance),  growth,  and  blood  samples  at  various  time  points.  Despite  a  dietary  supply  of  LC-­PUFAs  from  EUHDVWPLON GXULQJ WKH ÂżUVW ZHHNV RI OLIH LQIDQWV ZKR ZHUH ZHDQHG WR WKH FRQWURO IRUPXOD KDG VLJQLÂżFDQWO\ poorer  visual  acuity  at  17,  26,  and  52  weeks  and  VLJQLÂżFDQWO\ SRRUHU VWHUHRDFXLW\ DW ZHHNV FRPSDUHG to  infants  who  were  weaned  to  LC-­PUFA-­supplemented  formula12.  Better  acuity  and  stereoacuity  at  17  weeks  was  correlated  with  higher  DHA  concentration  in  EORRG VDPSOHV DW ZHHNV ,Q DGGLWLRQ D VLJQLÂżFDQW UHGXFWLRQ LQ WKH XQVDWXUDWLRQ LQGH[ ZKLFK FDQ LQĂ&#x20AC;XHQFH function  of  membrane-­related  enzymes,  receptors,  and  nutrient  transport  systems,  was  found  in  the  control  formula  group  throughout  the  study  period.  The  control  formula  group  also  had  a  higher  Mead  acid  (suggestive  RI HVVHQWLDO IDWW\ DFLG GHÂżFLHQF\ WR $$ UDWLR WKDQ GLG the  supplemented  formula  group.  Overall,  the  average  difference  between  the  LC-­PUFA  supplemented  and  control  formula  groups  was  equivalent  to  one  line  on  an  eye  examination  chart.  The  authors  therefore  concluded  that  the  critical  period  during  which  dietary  /& 38)$ FDQ LQĂ&#x20AC;XHQFH WKH PDWXUDWLRQ RI FRUWLFDO function  extends  beyond  6  weeks  of  age,  and  that  such  VXSSOHPHQWHG IRUPXODV DUH ZHOO WROHUDWHG DQG EHQHÂżFLDO to  the  maturation  of  the  visual  cortex  in  term  infants  weaned  at  6  weeks.  The researchers followed the same infants for a full year (52 weeks) and made assessments at numerous points in time, allowing them to draw comparative data. Because the measurements were quantitative, there was less variability in the data. However, this study drew its cohort from a specific geographic region and obtained infants from two separate hospitals.

While the authors attempted to increase ethnic and socioeconomic variability by taking the infants from two different hospitals, they may have unintentionally subjected their subjects to different qualities of care and thus potentially skewed results. However, the strengths in the design outweigh the weaknesses seen in the variability of the cohort. Therefore, the conclusion

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that the critical period extends beyond 6 weeks and that LC-PUFAs are beneficial to the infantsâ&#x20AC;&#x2122; visual system is likely upheld.

From the previous three studies, which assess the benefits of LC-PUFA supplementation of infant formula and the length of its effects, we have reached the conclusion that LC-PUFA supplementation in infant formulas improve neural development in fourmonth old infants and that the critical period of these supplements and their benefits, specifically on the visual system, extends past the first six weeks of life.

Conclusion  From  the  seven  articles  discussed  above,  we  have  reached  an  overarching  conclusion  that  despite  the  risk  of  mercury  ingestion,  maternal  n-­3  PUFA  consumption  increases  a  childâ&#x20AC;&#x2122;s  IQ  at  least  through  four  years  of  life.  After  birth,  LC-­PUFA  supplementation  in  infant  formula  improves  neural  development  in  infants  and  the  critical  period  of  supplements  extends  EH\RQG WKH ÂżUVW VL[ ZHHNV RI OLIH :KLOH ZH ZHUH RQO\ able  to  analyze  a  handful  of  articles  out  of  the  myriad  DYDLODEOH LQ VFLHQWLÂżF OLWHUDWXUH ZH KDYH DWWHPSWHG WR weigh  the  strengths  and  weaknesses  of  each  articleâ&#x20AC;&#x2122;s  experimental  design  to  better  appreciate  the  reasoning  behind  each  conclusion  and  to  develop  our  own  overall  conclusion.  Based  on  our  conclusion  that  PUFAs,  VSHFLÂżFDOO\ Q 38)$V KDYH SRVLWLYH HIIHFWV RQ human  neural  development,  we  deduced  that  we  should  incorporate  these  healthy  fats  into  our  diet  by  eating  RLO\ ÂżVKHV RU SHUKDSV E\ WDNLQJ VXSSOHPHQWV )XWXUH relevant  work  in  this  area  may  include  analyzing  the  effects  of  n-­3  PUFAs  on  adult  neural  development  and  the  effect  of  supplements  on  adult  diets  compared  to  natural  ingestion.  Supplements  not  only  for  n-­3  PUFAs  but  for  a  variety  of  other  nutrients,  such  as  vitamins  and  minerals,  are  widespread  in  American  health-­conscious  diets,  but  are  these  really  effective?  Further  research  and  analysis  is  required  to  answer  these  integral  questions. Â

References 1.Simopoulos,  AP.  Essential  Fatty  Acids  in  Health  and  Chronic  Disease.  The  American  Journal  of  Clinical  Nutrition.  (1999).  70:560S-­9S. .LWDMLND . 6LQFODLU $ :HLVLQJHU 5 :HLVLQJHU + Mathal,  M.,  Jayasooriya,  A.,  Halver,  J.,  Puskas,  L.  (2004).  Effects  of  Dietary  Omega-­3  Polyunsaturated  Fatty  Acids

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on Brain Gene Expression. Proceedings of the National Academy of Science of the United States of America, 101(30), 10931-­10936.

3.Uauy R., Dangour A. Nutrition in Brain Development and Aging: Role of Essential Fatty Acids. Nutrition Review. (2006). 64:S24-­33.

4.Helland, I., Smith, L., Blomén, B., Saarem, K., Saugstad, O., Drevon, C. (2008). Effect of Supplementing Pregnant and Lactating Mothers with n-­3 Very-­Long-­Chain Fatty Acids on Children’s IQ and Body Mass Index at 7 Years of Age. Pediatrics, 122(2), 472-­479.

5.Innis, SM. (2007). Dietary (n-­3) Fatty Acids and Brain Development. American Society for Nutrition. Journal of Nutrition, 137, 855-­859.

6.Akbar, M. & Kim, H.-­Y. (2002) J. Neurochem. 82, 655– 665.

7.Huettner, J. E. (2003) Prog. Neurobiol. 70, 387–407.

8.Auestad, N. & Innis, S. M. (2000) Am. J. Clin. Nutr. 71, 312S–314S. 9.Bazan, N. G. & Rodriguez de Turco, E. B. (1994) J. Ocul. Pharmacol. 10, 591–604.

10.McGahon, B. M., Martin, D. S. D., Horrobin, D. F. & Lynch, M. A. (1999). Neurocsiences 94, 305–314.

11.Ikemoto, A., Nitta, A., Furukawa., Ohishi, M., Nakamura, A., Fujii, Y. & Okuyama, H. (2000) Neurosci.

Lett. 285, 99–102.

12.Birch, E., Hoffman, D., Castaneda, Y., Fawcett, S., Birch, D., Uauy, R. (2002). A Randomized Controlled Trial of Long-­Chain Polyunsaturated Fatty Acid Supplementation of Formula in Term Infants After Weaning at 6 wk of Age. American Journal of Clinical Nutrition, 75, 570-­580.

13.Gale, C., Robinson, S., Godfrey, K., Law, C., Schlotz, W., O’Callaghan, F.J. (2008). Oily Fish Intake During Pregnancy – Association with Lower Hyperactivity but not with Higher Full-­Scale IQ in Offspring. The Journal of Child Psychology and Psychiatry, 49(10), 1061-­1068.

14.Helland, I., Smith, L., Saarem, K., Saugstad, O., Drevon, C. (2003). Maternal Supplementation With Very-­Long-­Chain n-­3 Fatty Acids During Pregnancy and Lactation Augments Children’s IQ at 4 Years of Age. Pediatrics, 111(1), 39-­44.

15.Hibbeln, J., Davis, J., Steer, C., Emmett, P. (2007).

Maternal Seafood Consumption in Pregnancy and Neurodevelopmental Outcomes in Childhood (ALSPAC study): an Observational Cohort Study. The Lancet, 369(9561), 578-­585.

16.Fujimoto, K., Yao, K., Miyazawa, T., Hirono, H., Nishikawa, M., Kimura, S., Maruyama K., Nonaka, M. (1989). The Effect of Dietary Docosahexaenoate on the Learning Ability of Rats. In: Chandra RK (ed) Health Effects of Fish and Fish Oils. ARTS Biomedical Publishers, St. John’s, newfoundland, pp 275-­284.

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17.Wainwright, PE, Huang YS, Bul-­man-­Fleming, B., Mils DE, Redden P., McCutcheon, D. (1991). The Role of n-­3 Essential Fatty Acids in Brain and Behavioral Development: a Cross-­Fostering Study in the Mouse.

Lipids, 26, 37-­45.

18.Jensen RG. Lipids in Human Milk. (1999). Lipids, 34, 1243-­1271.

19.Agostoni, C., Trojan, S., Bellu, R., Riva, E., Giovannini, M. (1995). Neurodevelopmental Quotient of Healthy Term Infants at 4 Months and Feeding Practice: The Role of Long-­Chain Polyunsaturated Fatty Acids. Pediatric Research, 38(2), 262-­266.

20.Auestad, N., Scott, D.T., Janowsky, J.S., Jacobsen, C., Carroll, R.E., Montalto, M.B., Halter, R., Qiu, W., Jacobs, J.R., Connor, W.E., Connor, S.L., Taylor, J.A., Neuringer, M., Fitzgerald, K.M., Hall, R.T. (2003). Visual, Cognitive, and Language Assessments at 39 Months: A Follow-­up Study of Children Fed Formulas Containing Long-­Chain Polyunsaturated Fatty Acids to 1 Year of Age. Pediatrics, 112(3), 177-­183.

21.Svennerholm L. Distribution and Fatty Acid Composition of Phosphoglycerides in Normal Human Brain. (1968). Journal of Lipid Research, 9, 570-­579.

22.Svennerholm L, Vanier M. The Distribution of Lipids in the Human Nervous System. III. Fatty Acid Composition of Phosphoglycerides of Human Foetal and Infant Brain.

(1973). Brain Research, 50, 341-­351.

23.Martinez M. Tissue Levels of Polyunsaturated Fatty Acids During Early Human Development. (1992). Journal of Pediatrics, 120 (suppl), S129-­138.

24.Martinez M, Conde C, Ballabriga A. Some Chemical Aspects of Human Brain Development. (1974). Pediatric Research, 8, 91-­102.

25.Martinez M, Mougan I. Fatty Acid Composition of Human Brain Phospholipids During Normal Development. (1998). Journal of Neurochemistry, 71, 2528-­2533.

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Parkinson s Disease: A review on both motor and nonmotor symptoms

Diana Hong Arts and Sciences 13, Biological Sciences Abstract Parkinsonâ&#x20AC;&#x2122;s  disease  (PD)  is  considered  a  motor  sys-­ tem  disorder  caused  by  the  degeneration  of  dopa-­ minergic  neurons  in  the  substantia  nigra.  However,  it  is  also  associated  with  a  wide  range  of  non-­motor  symptoms.  Some  of  these  symptoms  include  sensory  dysfunction,  depression,  and  dementia.  Because  the  exact  biochemical  pathways  for  these  non-­motor  symptoms  are  not  yet  completely  understood,  cur-­ rent  treatment  options  for  PD  focus  primarily  on  relieving  motor  symptoms  of  the  disease  and  leave  the  non-­motor  symptoms  inadequately  treated.  It  is  important  to  better  understand  non-­motor  symp-­ toms  because  not  only  are  these  symptoms  associ-­ ated  with  the  rapid  progression  of  PD,  but  many  of  these  symptoms  often  also  precede  the  more  obvi-­ ous  motor  symptoms  such  as  bradykinesia,  tremors,  and  rigidity,  which  could  prove  useful  for  early  di-­ agnosis  of  PD.

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  Parkinsonâ&#x20AC;&#x2122;s  disease  (PD)  is  an  age-­related  neu-­ rodegenerative  disease  that  affects  approximately  2.0%  of  adults  over  the  age  of  651,  and  1  in  300  in  the  gen-­ eral  population2.  PD  is  most  commonly  linked  with  a  degeneration  of  the  dopamine  synthesizing  neurons  in  the  substantia  nigra  that  project  to  the  striatum,  which  causes  an  overall  loss  in  motor  function,  as  presented  by  tremors  and  rigidity  in  movement1,  3-­8  .  Recent  data  SRLQWHG WR WKH SRVVLELOLW\ RI FKURQLF LQĂ&#x20AC;DPPDWLRQ DQG sustained  immune  responses  in  the  brain  in  causing  dopaminergic  cell  death  in  PD4  .  However,  PD  affects  more  than  the  dopaminergic  systems,including  areas  of  the  brain  that  are  not  directly  related  to  motor  control,  such  as  the  amygdala  and  peripheral  autonomic  ner-­

vous  system.1,6  Defects  in  these  areas  lead  to  the  non-­ motor  symptoms  that  affect  many  PD  patients,  such  as  pain,  cognitive  and  sensory  dysfunction6,  as  well  as  depression  and  other  mood  disorders  as  seen  in  20  â&#x20AC;&#x201C;  40%  of  PD  patients.1   Thus,  the  aim  of  this  review  is  to  examine  both  the  motor  and  non-­motor  PD  symptoms  and  review  the  current  understanding  of  the  associated  biochemical  pathways.

Motor Symptoms  Parkinsonâ&#x20AC;&#x2122;s  disease  (PD)  is  often  associated  with  overt  motor  symptoms  that  include  the  asymmet-­ ric  onset  of  bradykinesia,  tremors,  and  rigidity  due  to  the  degeneration  of  dopaminergic  nigrostriatal  neurons  of  the  basal  ganglia1-­8.  Bradykinesia,  or  a  slowness  of  movement,  is  a  trademark  of  basal  ganglia  disorders  DQG LV D FOHDUO\ LGHQWLÂżDEOH V\PSWRP RI 3'8.  With  this  symptom,  PD  patients  experience  a  decrease  in  dexter-­ LW\ DQG ÂżQH PRWRU FRQWURO 2I DOO WKH PRWRU V\PSWRPV the  rest  tremor  is  the  most  well  known  and  is  the  clas-­ sic  motor  dysfunction  associated  with  PD.  This  type  of  tremor  occurs  at  rest  but  decreases  with  voluntary  movement3.  Tremors  can  be  observed  in  the  hands,  lip,  chin,  jaw  and  legs,  but  almost  never  involves  the  neck-­ head  regions  or  the  voice8  .Although  the  tremors  usu-­ ally  remain  asymmetric,  it  may  manifest  into  bilateral  tremors  as  PD  progresses.  The  pathophysiology  of  the  rest  tremors  are  not  fully  understood,  but  it  has  been  generally  accepted  to  be  caused  by  atypical  synchro-­ nous  oscillating  neuronal  activity  within  the  basal  gan-­ glia8.  Tremors  often  accompany  rigidity,  or  the  resis-­ tance  seen  in  the  passive  movement  of  a  limb.  Rigidity  PD\ DOVR SOD\ D UROH LQ WKH UHFXUUHQW SDLQ DIĂ&#x20AC;LFWLQJ 3' patients.  An  early  diagnosis  of  PD  can  be  greatly  sup-­ ported  when  rigidity  increases  with  reinforcing  move-­

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ments  and  is  seen  ipsilateral  to  the  rest  tremor.  At  later  stages  of  PD,  postural  instability  develops  and  can  become  one  of  the  major  devastating  symptoms  and  a  main  cause  of  falls  in  PD  patients.  This  symptom  also  contributes  to  gait  abnormalities  seen  in  patients  with  3' ZKR RIWHQ VKXIĂ&#x20AC;H ZLWK VORZ QDUURZ VWHSV LQ D characteristically  stooped  posture8.

Neuronal Mechanisms Governing Motor Symptoms  Smooth,  well  coordinated  muscle  movement  is  determined  by  the  direct  and  indirect  output  pathways  of  the  basal  ganglia  to  the  globus  pallidus  and  the  substantia  nigra7.  While  the  direct  pathways  disinhibit  the  thalamocortical  neurons,  the  indirect  pathways  LQKLELW WKHVH QHXURQV 7KHVH QHXURQV DUH LQĂ&#x20AC;XHQFHG by  excitatory  inputs  from  the  cortex  and  thalamus  and  by  regulatory  control  of  dopamine  release  from  the  nigrostriatal  neurons.   In  PD,  dopamine  denervation  occurs  with  the  death  of  nigrostriatal  neurons.  Dopamine  denervation  causes  an  imbalance  in  the  activity  of  the  two  basal  ganglion  pathways,  which  is  thought  to  correlate  with  the  motor  symptoms  seen  in  PD2,7.   Neuronal  loss  observed  in  brains  of  PD  patients  may  be  due  to  the  presence  of  Lewy  bodies,  or  a  mass  of  ¿QH ÂżEHUV /HZ\ ERGLHV VHUYH DV GHÂżQLQJ KLVWRORJLFDO characteristics  of  PD  and  have  been  found  in  various  areas  of  the  central  and  peripheral  nervous  systems  in  PD  patients.  The  extensive  distribution  of  these  Lewy  bodies  may  also  be  linked  to  the  wide  range  of  motor  and  non-­motor  symptoms  seen  in  PD  patients9.   In  PD,  Lewy  bodies  are  mainly  comprised  of  a  presynaptic  nerve  terminal  protein  known  as  Ď V\QXFOHLQ9 ,Q D KHDOWK\ EUDLQ ÄŽ V\QXFOHLQ LV IRXQG in  presynaptic  terminals  and  is  absent  in  the  neuronal  F\WRSODVP ,Q WKH QRUPDO DJLQJ SURFHVV ÄŽ V\QXFOHLQ non-­pathologically  accumulates  in  the  substantia  nigra,  but  not  in  other  dopamine  neuronal  nuclei5.  However,  in  PD,  this  protein  develops  inside  nerve  cells  as  pale  and  diffuse  cytoplasmic  inclusions  and  displaces  other  components  of  the  cell2,5.  The  molecular  components  of  Ď V\QXFOHLQ DUH F\WRWR[LF DQG DV ORQJ DV WKHVH WR[LQV DUH made,  Lewy  bodies  expand.  This  causes  an  excessive  build-­up  of  protein  aggregates  in  the  host  cell  and  leads  to  cell  death. Â

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1RQ PRWRU V\PSWRPV DIĂ&#x20AC;LFWLQJ SDWLHQWV ZLWK PD  often  precede  the  more  obvious  motor  symptoms  associated  with  the  disease.  One  recent  hypothesis  suggests  that  the  Lewy  body  pathology  develops  only  after  the  olfactory  system  and  lower  brainstem  areas  have  become  affected.  Recent  data  have  been  found  to  show  a  correlation  between  the  decreased  sensitivity  to  odors  and  the  increased  risk  of  developing  PD.  Olfactory  dysfunction  eventually  effects  up  to  90%  of  patients  with  PD.  Another  common  early  symptom  seen  in  PD  patients  is  constipation.  This  may  be  one  of  the  earliest  symptoms  of  Lewy  body  degeneration  as  seen  in  PD.  Lewy  bodies  found  to  effect  the  peripheral  autonomic  nervous  system  also  affect  the  colonic  sympathetic  denervation  which  has  been  associated  with  a  prolonged  intestinal  passage  time  leading  to  constipation.  Constipation  has  been  reported  as  one  of  the  main  complaints  preceding  the  classic  motor  symptoms  in  about  half  of  PD  patients.  In  one  longitudinal  study  following  the  bowel  habits  of  7,000  men  over  the  course  of  24  years,  those  with  initial  constipation  were  three  times  more  likely  of  developing  PD  over  a  mean  time  period  of  10  years.  Therefore,  the  LGHQWLÂżFDWLRQ RI HDUO\ QRQ PRWRU V\PSWRPV VXFK DV the  decrease  in  function  of  the  olfactory  system  and  the  onset  of  constipation,  could  lead  to  an  earlier  diagnosis  of  PD6,10.

Neuropsychiatric Dysfunctions  PD  patients  are  not  only  affected  by  somatic  non-­motor  symptoms  but  also  neuropsychiatric  non-­ motor  symptoms,  including  depression,  anxiety,  and  apathy.  Studies  have  indicated  that  depression,  FKDUDFWHUL]HG E\ JXLOW ODFN RI FRQ¿GHQFH VDGQHVV and  remorse,  often  occurs  with  anxiety  in  PD  patients.  Depression  or  panic  attacks  have  been  seen  to  antedate  the  onset  of  motor  symptoms  in  up  to  30%  of  patients  with  PD.  Separate  from  the  depression  that  is  usually  seen  in  PD  patients,  apathy  has  also  been  recognized  DV D XQLTXH V\PSWRP RI 3' $SDWK\ LV GH¿QHG DV WKH presence  of  reduced  motivation  that  is  not  related  to  a  decrease  in  conscious  state  or  emotional  distress  and  could  be  caused  by  the  neuronal  degeneration  in  the  reward  centers  of  the  brain,  such  as  the  dopaminergic  projections  between  the  ventral  tegmentum  and  nucleus  accumbens.  Anxiety  and  apathy  are  both  commonly

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ÂŚ Parkinson s ÂŚ

Vol 5 ÂŚ 2011

seen  early  on  in  PD  and  can  be  pre-­clinical  risk   The  underlying  mechanisms  of  dementia  factors1,6,10. in  PD  are  not  yet  fully  understood.  There  have  been  hypotheses  that  Lewy  body  degeneration  is  a  main  driving  factor  for  the  development  of  dementia  in   Neuronal Mechanisms Governing PD6.  There  have  been  studies  linking  the  presence  of  Depression as seen in PD Alzheimer-­type  changes  in  the  brain,  such  as  senile  11  Damage  to  the  limbic  noradrenergic  and  SODTXHV ZLWK WKH ÄŽ V\QXFOHLQ RI /HZ\ ERGLHV .  A  dopaminergic  mechanisms  and  to  the  serotoninergic  decrease  in  hippocampal  volume  has  also  been  seen  neurotransmission  as  seen  in  PD  patients  may  link  in  PD  patients  with  dementia  that  is  comparable  in  LQ LQGLYLGXDOV DIĂ&#x20AC;LFWHG depression  to  a  more  biological  cause  than  to  a  H[WHQW WR WKH GHFUHDVH VHHQ 10 reaction  to  the  disease  itself10.  Neurons  in  the  ventral  with  Alzheimerâ&#x20AC;&#x2122;s  disease .  Connections  have  also  of  motor  symptoms  mesencephalon,  located  near  the  substantia  nigra,  been  made  between  the  severity  12 project  to  limbic  and  cortical  structures  that  control  and  intellectual  impairment .  Using  the  Mini-­Mental  cognition,  emotions,  and  reward-­seeking  behavior.   State  examination  to  assess  intellectual  status,  Huber  There  is  a  greater  degeneration  of  dopaminergic  HW DO IRXQG D VLJQLÂżFDQW QHJDWLYH FRUUHODWLRQ EHWZHHQ neurons  in  this  area  in  PD  patients  who  have  depression  intellectual  impairment  and  the  severity  of  both  than  those  who  do  not.   Depression  associated  with  rigidity  and  bradykinesia.  This  seemed  to  suggest  that  PD  is  also  associated  with  a  decrease  in  serotonin  in  these  motor  symptoms  were  related  to  the  increased  the  dorsal  raphe  nucleus  and  norepinephrine  in  the  intellectual  impairment  seen  in  patients  with  PD.  locus  coeruleus.  The  locus  coeruleus  projects  to  the  anterior  cingulated  gyrus,  the  hippocampus,  the  ventral  striatum,  and  the  amygdala.   The  amygdala,  a  region  of  the  brain  closely  associated  with  motivation  and  emotional  behavior,  is  atrophied  and  consists  of  Lewy  bodies  in  PD  patients  with  depression,  which  may  link  PD  to  depression.  Therefore,  the  relatively  weak  correlation  between  depression  and  the  severity  of  PD  suggests  that  depression  is  not  a  psychological  reaction  to  PD  but  part  of  PD  itself1.

Cognitive Impairment  Some  hypotheses  propose  that  depression  antecedes  dementia1.  Dementia,  another  non-­motor  symptom,  is  seen  in  up  to  40%  of  PD  patients  â&#x20AC;&#x201C;  a  rate  that  is  about  six  times  greater  than  that  of  healthy  individuals.  Dementia  advances  gradually  but  is  associated  with  a  rapid  progression  of  disability,  which  often  puts  many  PD  patients  at  risk  of  nursing  home  placement.  Dementia  is  clinically  characterized  by  impairment  to  visuospatial  abilities,  memory,  and      the  executive  attention  in  the  control  of  thoughts          and  emotions.  Personality  disorders,  hallucinosis,  DQG SV\FKRVLV DUH DOVR VHHQ LQ 3' SDWLHQWV DIĂ&#x20AC;LFWHG with  dementia6,10.

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Discussion  PD,  a  disease  that  is  usually  categorized  as  a  motor  system  disorder,  also  has  many  non-­motor  symptoms.  Neurodegeneration  in  PD  affects  the  central  nervous  system  as  well  as  the  peripheral  nervous  system,  leading  to  a  wide  range  of  classic  motor  symptoms,  such  as  bradykinesia,  tremors,  and  rigidity1,2,4-­8,in  addition  to  non-­motor  symptoms.  Many  non-­motor  symptoms,  such  as  sensory  dysfunction  and  depression,  often  precede  the  more  obvious  motor  symptoms.  Therefore,  it  is  important  to  pay  attention  to  and  correctly  identify  the  early  non-­motor  symptoms,  as  they  could  lead  to  an  earlier  diagnosis  of  PD.   Although  many  drugs  are  currently  prescribed  to  relieve  the  classic  motor  system  malfunctions  seen  in  PD  patients,  these  drugs  often  worsen  non-­motor  symptoms  and  decrease  the  quality  of  life2,7,8.While  newer  treatments  are  beginning  to  treat  both  motor  and  non-­motor  symptoms,  there  are  currently  no  medications  that  stop  the  degeneration  of  dopaminergic  neurons3.  Thus,  future  studies  that  aim  to  gain  a  better  understanding  of  the  relationship  between  the  biochemical  pathways  of  PD  and  the  motor  and  non-­ motor  symptoms  are  warranted.  Furthermore,  being  able  to  pinpoint  the  neurons  that  are  most  susceptible  to  neurodegeneration  could  lead  to  more  effective  treatment  options  for  relieving  both  motor  and  non-­ motor  symptoms  in  PD.

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ÂŚ Parkinson s ÂŚ

References 1.  Lieberman  A.  2006.  Depression  in  Parkinsonâ&#x20AC;&#x2122;s  disease  â&#x20AC;&#x201C;  a  review.  Acta  Neurol  Scand;Íž  113:  1  â&#x20AC;&#x201C;  8.  2.  Schapira  AHC,  Bezard  E,  Brotchie  J,  Frederic  C,  Collingridge  GL,  Ferger  B,  Hengerer  B,  Hirsch  E,  Jenner  P,  Le  Novere  N,  Obeso  JA,  Schwarzschild  MA,  Spampinato  U,  Davidai  G.  2006.  Novel  pharmacological  targets  for  the  treatment  of  Parkinsonâ&#x20AC;&#x2122;s  disease.  Nature  Reviews;Íž  5:  845  â&#x20AC;&#x201C;  854.  3.  Dauer  W,  Przedborski  S.  2003.  Parkinsonâ&#x20AC;&#x2122;s  disease:  mechanisms  and  models.  Neuron;Íž  39:  889  â&#x20AC;&#x201C;  909.

0F*HHU 3/ 0F*HHU (* ,QĂ&#x20AC;DPPDWLRQ DQG neurodegeneration  in  Parkinsonâ&#x20AC;&#x2122;s  disease.  Parkinsonism  and  Related  Disorders;Íž  10:  S3  â&#x20AC;&#x201C;  S7.  5.  Mendez  I,  Vinuela  A,  Astradsson  A,  Mukhida  K,  Hallett  P,  Robertson  H,  Tierney  T,  Holness  R,  Dagher  A,  Trojanowski  JQ,  Isacson  O.  2008.  Dopamine  neurons  implanted  into  people  with  Parkinsonâ&#x20AC;&#x2122;s  disease  survive  without  pathology  for  14  years.  Nature  Medicine;Íž  14(5);Íž  507  â&#x20AC;&#x201C;  509.  6.  Poewe  W.  2008.  Non-­motor  symptoms  in  Parkinsonâ&#x20AC;&#x2122;s  disease.  European  Journal  of  Neurology;Íž  15:  14  â&#x20AC;&#x201C;  20. Â

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7.  Richardson  PJ,  Kase  H,  Jenner  PG.  1997  Adenosine  A2A  receptor  antagonists  as  new  agents  for  the  treatment  of  Parkinsonâ&#x20AC;&#x2122;s  disease.  Trends  in  Pharmacological  Sciences;Íž  18  (4):  338  â&#x20AC;&#x201C;  344.  8.  Shahed  J,  Jankovic  J.  2007.  Motor  symptoms  in  Parkinsonâ&#x20AC;&#x2122;s  disease.  Handbook  of  Clinical  Neurology;Íž  83:  329  â&#x20AC;&#x201C;  342.  9.  Wakabayashi,  K,  Kunikazu  T,  Fumiaki  M,  Takahashi  H.  2007.  The  Lewy  body  in  Parkinsonâ&#x20AC;&#x2122;s  disease:  molecules  implicated  in  the  formation  and  degradation  RI ÄŽ V\QXFOHLQ DJJUHJDWHV 1HXURSDWKRORJ\ Âą 506.

10.  Chaudhuri  KR,  Healy  DG,  Schapira  AHV.  2006.  Non-­motor  symptoms  of  Parkinsonâ&#x20AC;&#x2122;s  disease:  diagnosis  and  management.  Lancet  Neurology;Íž  5:    235  â&#x20AC;&#x201C;  45.  11.  Caballol  N,  Marti  MJ,  Tolosa  E.  2007.  Cognitive  dysfunction  and  dementia  in  Parkinson  Disease.  Movement  Disorders;Íž  22  (S17):  S358  â&#x20AC;&#x201C;  S366.

12.  Huber  SJ,  Paulson  GW,  Shuttleworth  EC.  1988.  Relationship  of  motor  symptoms,  intellectual  impairment,  and  depression  in  Parkinsonâ&#x20AC;&#x2122;s  disease.  Journal  of  Neurology,  Neurosurgery,  and  Psychiatry.  51:  855  â&#x20AC;&#x201C;  858. Â

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