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Excess Manganese Intake During Early Life Alters Brain Catecholaminergic Receptor Expression Mikhail Gadomski, Richard Cathey, Stephane Beaudin, Donald Smith Department of Microbiology and Environmental Toxicology ObjecEve: Determine the developmental window of greatest suscep<bility to excess Mn exposure that alters brain PFC DRD2 and NET expression. Approach: Subjects: 50 male LongEvans rats were used. Mn Exposure: At birth (postnatal day 1), animals were randomly assigned to one of five treatment condi<ons; Control (0 mg Mn/kg/day); (2) 50 mg Mn/kg PND 17; (3) 50 mg Mn/kg/ d PND 815; (4) 50 mg Mn/kg/d PND 1621; and (5) 50 mg Mn/kg/d PND 1–21. Each day rats were weighed, and administered either sucrose vehicle or 50 mg Mn/kg. Tissues were collected PND22. Immunohistochemistry: References: 1 Bouchard et al. 2006 2 Lucchini et al. 2011 3 Arnsten and Rubia 2012 4 Middleton and Strick 2000 5 Kern et al 2010 Summary: Research shows that cell type (neuronal vs. nonneuronal ) can be iden<fied through the use of immunohistochemistry. Iden<fica<on of DRD2 specific to neuronal cells provides vital informa<on on the impact of Mn on the PFC during neonatal life and associated deficits. Over expression of inhibitory signaling proteins in the PFC is a puta<ve mechanism media<ng the a\en<on and behavior deficits seen in children. Results: Our previous study shows that PFC DRD2 is up regulated a]er Mn exposure. We predict that our findings of PND17 will closely resemble these results. 5 In order to determine specificity of DRD2, neuronal markers overlapping proteins can be iden<fied as neurons expressing the protein of interest. RaEonal: Studies in children have associated elevated Mn exposure with behavioral, cogni<ve and motor func<on deficits. 1,2 Elevated exposure may arise from: Contaminated water Industrial sources Diet (soy formula) Cor<cal and basal ganglia structures are densely innervated by dopamine and norepinephrine. 4 The PFC regulates a\en<on, cogni<ve control, mo<va<on and emo<on through connec<ons with the posterior cor<cal and subcor<cal structures. 3 Presynap<c DRD2 regulates dopamine synthesis by inhibi<ng Tyrosine Hydroxylase (TH). Postsynap<cally DRD2 inhibits cyclic adenosine monophosphate(cAMP) inhibi<on of cAMP dependent pathways (second messenger). NET is the main transporter of DA and NE in the prefrontal cortex. We propose that developmental Mn exposure causes permanent up regula<on of DRD2 in PFC. Lifelong up regula<on of DRD2 may underlie a\en<on and cogni<ve deficits in children. DraQ5 (10x) GFAP (10x) NET (10x) 1) Brain perfusion and fixaEon. 0.1MPBS + 4% PFA 2) CryoprotecEon 0.3% sucrose buffer Store at 80 C 3) Develop cryostat secEoning plan ~12mm brain @ 40um = 288 slices Dopamine input to PFC Norepinephrine input to PFC 4) Create secEons slice coronally at 40 um Cryoprotect at 20 C 6) Freefloat stain Wash x 3 Blocking incuba<on Primary incuba<on Wash x 3 Secondary incuba<on Wash x 2 Nuclear stain Wash Mount on slide Tissue 7) Confocal microscopy Exposure Model Shown is the five treatment exposures 5) Design indirect Ab staining protocol 1 Ab:Rabbit an<DRD2 2 Ab:Goat an<Rabbit (conjugated 488) Nuclear stain (DraQ5) DraQ5 + GFAP + NET DRD2 localized in the PFC stained with Alexa Fluor 488 10X 16X 40X Phenotypical descrip<on of PFC circuitry. 3 Future work: Perform immunohistochemistry on <ssues collected. Determine the ability of Mn to program neuronal cells to up regulate the expression of PFC DRD2 during specific developmental windows; PND 17, 715, 1621, 121. NET DRD2 is a G coupled protein receptor whose downstream signal is inhibitory

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Excess  Manganese  Intake  During  Early  Life  Alters  Brain  Catecholaminergic  Receptor  Expression    

Mikhail  Gadomski,  Richard  Cathey,  Stephane  Beaudin,  Donald  Smith  Department  of  Microbiology  and  Environmental  Toxicology  

 ObjecEve:  Determine  the  developmental  window  of  greatest  suscep<bility  to  excess  Mn  exposure  that  alters  brain  PFC  DRD2  and  NET  expression.      

Approach:  Subjects:  50  male  Long-­‐Evans  rats  were  used.    

Mn  Exposure:    At  birth  (postnatal  day  1),  animals  were  randomly  assigned  to  one  of  five  treatment  condi<ons;  Control  (0  mg  Mn/kg/day);  (2)  50  mg  Mn/kg  PND  1-­‐7;  (3)  50  mg  Mn/kg/d  PND  8-­‐15;  (4)  50  mg  Mn/kg/d  PND  16-­‐21;  and  (5)  50  mg  Mn/kg/d  PND  1–21.  Each  day  rats  were  weighed,  and  administered  either  sucrose  vehicle  or  50  mg  Mn/kg.  Tissues  were  collected  PND-­‐22.  

 

 

   

 

Immunohistochemistry:  

References:  1  Bouchard  et  al.  2006  2  Lucchini  et  al.  2011  3  Arnsten  and  Rubia  2012  4  Middleton  and  Strick  2000  5  Kern  et  al  2010    

Summary:  •  Research  shows  that  cell  type  (neuronal  vs.  non-­‐neuronal  )  can  be  

iden<fied  through  the  use  of  immunohistochemistry.  

•  Iden<fica<on  of  DRD2  specific  to  neuronal  cells  provides  vital  informa<on  on  the  impact  of  Mn  on  the  PFC  during  neonatal  life  and  associated  deficits.  

•  Over  expression  of  inhibitory  signaling  proteins    in  the  PFC  is  a  puta<ve  mechanism  media<ng  the  a\en<on  and  behavior  deficits  seen  in  children.  

 

 

Results:    •  Our  previous  study  shows  that  PFC  DRD2  is  up  regulated  a]er  Mn  

exposure.    •  We  predict  that  our  findings  of  PND1-­‐7  will  closely  resemble  these  

results.5          

•  In  order  to  determine  specificity  of  DRD2,  neuronal  markers  overlapping  proteins  can  be  iden<fied  as  neurons  expressing  the  protein  of  interest.  

 

RaEonal:  •  Studies  in  children  have  associated  elevated  Mn  exposure  with  behavioral,  cogni<ve  and  motor  func<on  deficits.  1,2    

 •  Elevated  exposure  may  arise  from:  -­‐  Contaminated  water  

       -­‐  Industrial    sources            -­‐  Diet  (soy  formula)  

   •  Cor<cal  and  basal  ganglia  structures  are  densely  innervated  by  dopamine  and  norepinephrine.4      

                   •  The  PFC  regulates  a\en<on,  cogni<ve  control,  mo<va<on  and  

emo<on  through  connec<ons  with  the  posterior  cor<cal  and  subcor<cal  structures.  3  

                 •  Presynap<c  DRD2  regulates  dopamine  synthesis  by  inhibi<ng  Tyrosine  Hydroxylase  (TH).  Postsynap<cally  DRD2  inhibits  cyclic  adenosine  monophosphate(cAMP)  à  inhibi<on  of  cAMP-­‐dependent    pathways  (second  messenger).  NET  is  the  main  transporter  of  DA  and  NE  in  the  prefrontal  cortex.  

 

 

 

 

 

 

 

•  We  propose  that  developmental  Mn  exposure  causes  permanent  up  regula<on  of  DRD2  in  PFC.  

•  Lifelong  up  regula<on  of  DRD2  may  underlie  a\en<on  and  cogni<ve  deficits  in  children.  

DraQ5  (10x)   GFAP  (10x)   NET  (10x)  

1) Brain  perfusion  and  fixaEon.    

         0.1MPBS  +  4%  PFA    

2)  CryoprotecEon          0.3%  sucrose  buffer            Store  at  -­‐80  C  

3)  Develop  cryostat          secEoning  plan              ~12mm  brain  @  40um                =    288  slices  

Dopamine input to

PFC

Norepinephrine input to PFC

4)  Create  secEons        slice  coronally  at  40  um        Cryoprotect  at  -­‐20  C  

6)  Freefloat  stain            Wash  x  3            Blocking  incuba<on            Primary  incuba<on              Wash  x  3          Secondary  incuba<on            Wash  x  2            Nuclear  stain            Wash            Mount  on  slide      

Tissue  

7)  Confocal  microscopy  

Exposure Model

Shown is the five

treatment exposures

5)  Design  indirect  Ab  staining  protocol        1  Ab:Rabbit  an<-­‐DRD2      2  Ab:Goat  an<-­‐Rabbit                    (conjugated  488)      Nuclear  stain  (DraQ5)  

DraQ5  +  GFAP  +  NET  

DRD2  localized  in  the  PFC  stained  with  Alexa  Fluor  488    

10X   16X   40X  

Phenotypical  descrip<on  of  PFC  circuitry.3  

Future  work:  •  Perform  immunohistochemistry  on  <ssues  collected.  

•  Determine  the  ability  of  Mn  to  program  neuronal  cells  to  up  regulate  the  expression  of  PFC  DRD2  during  specific  developmental  windows;  PND  1-­‐7,  7-­‐15,  16-­‐21,  1-­‐21.  

NET  

DRD2  is  a  G  coupled  protein  receptor  whose  downstream  signal  is  inhibitory