This study aims to determine the developmental window of greatest susceptibility to excess manganese (Mn) exposure that alters brain prefrontal cortex (PFC) dopamine receptor D2 (DRD2) and norepinephrine transporter (NET) expression. Male rats were exposed to either a control or 50 mg Mn/kg/day at different developmental periods. Immunohistochemistry will be used to identify DRD2 expression specific to neuronal cells in the PFC. Previous studies have shown that early life Mn exposure can lead to behavioral, cognitive and motor deficits by altering dopamine and norepinephrine signaling in brain regions like the PFC. The results of this study may help explain the neurological effects of Mn exposure during critical periods of brain

1. 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
aen<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
aen<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
aen<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