mik symposium poster
TRANSCRIPT
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