Rhodes1_Protocol
Project
protocol
—
Contents
Workflow
and
sampling
Equipment
and
supplies
Reagents
and
solutions
Procedures
Definitions
Data
References
Workflow
and
sampling
Workflow
Step |
Procedure
performed |
Age
(wk) |
Frequency
and
duration |
Data
collected |
1 |
8
wk
old
mice
are
divided
equally
by
strain
and
sex
into
exercised
and
non-exercised
conditions
(see
Figure
1
below) |
8 |
- |
|
2 |
The
first
10
days
all
mice
received
daily
injections
of
5-Bromo-2’-deoxyuridine
(BrdU;
50
mg/kg)
to
label
dividing
neurons |
8-9.5 |
daily
for
10
days |
|
3a |
Cohort
1
(controls):
Mice
without
exercise
wheels
are
video-monitored
and
assessed
for
baseline
horizontal
activity
for
13
days,
then
placed
in
shoebox
cages
(without
running
wheels)
for
the
remainder
of
the
experiment |
8-10
|
daily
for
13
days
only |
horizontal
locomotor
activity
|
3b |
Cohort
2
(exercised):Wheel
rotations
are
monitored
continuously
in
1-min
increments
for
mice
with
exercise
wheels
|
8-15 |
daily
for
43
days |
wheel
running
activity |
4 |
After
43
days,
the
two
cohorts
are
anesthetized
with
pentobarbital
(ip)
and
then
perfused
transcardially
with
4%
paraformaldehyde
to
initially
fix
the
brain
|
15 |
- |
- |
5 |
Perfused
brains
are
stored
overnight
in
4%
paraformaldehyde
to
prepare
the
brain
for
sectioning
and
subsequent
staining |
15 |
- |
- |
6 |
Brain
sections
are
immuno-stained
to
visualize
new
neurons
or
neurogenesis |
15 |
- |
number
of
new
neurons,
volume
of
dentate
gyrus |
Figure
1
Experimental
design
and
workflow.
Equipment
and
supplies
•
Custom-made
activity
cages
with
video
tracking
software
(Figure
2)
Cages
(18.5
cm
×
33.5
cm
×
16
cm)
are
constructed
out
of
clear
plastic
with
food
and
water
access
mounted
on
the
side.
Horizontal
distance
traveled
in
the
home
cage
is
recorded
using
TopScan
(Clever
Sys,
Vienna,
VA,
USA)
video
tracking
software.
TopScan
software
is
run
on
a
Dell
Precision
380
workstation
(Dell
Computer,
Round
Rock,
TX,
USA)
which
is
connected
to
a
Nuvico
digital
color
quad
interface
(Nuvico,
Englewood,
NJ,
USA)
and
an
Osprey-2000
(Viewcast,
Dallas,
TX)
or
WinTV
(Hauppauge
Computer
Works,
Hauppauge,
NY,
USA)
capture
card.
Four
Panasonic
WV-CP244
cameras
(Panasonic,
Secaucus,
NJ,USA)
mounted
152
cm
above
the
cages
are
used
to
capture
the
video
used
for
analysis.
•
Standard
polycarbonate
shoebox
home
cages
without
running
wheels
measuring
29
cm
×
19
cm
×
13
cm
(L
×
W
×
H)
(Figure
3B).
•
Standard
polycarbonate
shoebox
cages
with
running
wheels
measuring
36
cm
×
20
cm
×
14
cm
with
a
23
cm
diameter
wheel
(Respironics,
Bend,
OR).
Wheel
rotations
are
monitored
continuously
in
1-min
increments
throughout
the
experiment
via
magnetic
switches
interfaced
to
a
computer
running
VitalView
software
(Respironics,
Bend,
OR)
(Figure
3).
•
Balance
scale
•
Small
rodent
dissecting
kit
•
Peristaltic
pump,
as
part
of
the
perfusion
setup
•
Cryostat
at
-20°C
•
Zeiss
brightfield
light
microscope
•
ImageJ
software
•
AnalySIS
Opti
Version
3.3.776
software
(Soft
Image
System)
•
Leica
SP2
laser
scanning
confocal
microscope
(using
a
40X
oil
objective,
pinhole
size
81.35
µm)
•
Digital
camera
Figure
2.
Customized
housing
cages.
Two
different
types
of
bedding
are
used
depending
on
coat
color.
Corncob
bedding
(Harlan
7097)
is
used
for
dark
coats,
whereas
Sheppard
Paperchip®
bedding
is
used
for
light
coats.
Figure
3.
Panel
A:
A
rack-full
of
cages
with
running
wheels.
Panel
B:
Cages
with
(left)
and
without
(right)
running
wheel.
Panel
C:
Close-up
view
of
a
cage
with
running
wheel.
Reagents
and
solutions
Perfusion
essentials
•
Pentobarbital
sodium
(Lundbeck
Inc./Ovation
Pharmaceuticals
Inc.,
DeerWeld,
IL,
USA)
•
needles
and
syringes
•
4%
paraformaldehyde
in
a
phosphate
buffer
solution
(PBS)
•
30%
sucrose
in
PBS
Common
staining
reagents
•
5-Bromo-2’-deoxyuridine
(BrdU)
•
Tris-buffering
solution
(TBS)
•
0.6%
hydrogen
peroxide
•
50%
de-ionized
formamide
•
10%
20X
saline-sodium
citrate
(SCC)
buffer
•
2N
hydrochloric
acid
(HCl)
•
0.1
M
Boric
acid
•
0.3%
Triton-X
•
3%
goat
serum
in
TBS
(TBS-X
plus)
•
Avidin-Biotin
Complex
(ABC)
staining
system
(Vector,
Burlingame,
CA)
•
Diaminobenzidine
(DAB)
staining
kit
(Sigma,
St.
Louis,
MO)
BrdU-DAB
staining
essentials
for
detection
of
newly
divided
cells
•
primary
antibody
against
BrdU
made
in
rat
(Accurate,
Westbury,
NY)
•
secondary
antibody
against
rat
made
in
goat
at
1:250
in
TBS-X
plus
Double
fluorescent
labeling
for
detection
of
BrdU-positive
(BrdU+)
neuronal
cells
(NeuN+)
•
primary
antibody
cocktail
made
of
rat
anti-BrdU
(1:100;
Accurate,
Westbury,
NY)
and
mouse
anti-NeuN
(1:50;
Chemicon,
Billerica,
MA)
to
detect
neuronal
cells
•
secondary
antibody
cocktail
made
with
goat
conjugated
fluorescent
markers
(1:200;
Cy3
anti-rat,
Cy2
anti-mouse)
•
Superfrost/Plus
microscope
slides
Procedures
1.
Experimental
Design
a.
Mice
are
divided
equally
by
strain
and
sex
into
two
cohorts;
one
cohort
housed
with
running
wheels
and
the
other
cohort
without
(see
Figure
1
above).
b.
The
first
10
days
all
mice
receive
daily
injections
of
BrdU
(50
mg/kg)
for
labeling
dividing
cells.
c.
In
Cohort
1
horizontal
distance
traveled
per
day
in
the
activity
cages
is
recorded
the
first
13
days
using
TopScan
video
tracking
software
(see
Figure
2A
and
2B).
d.
Following
day
13,
non-exercised
mice
are
transferred
to
standard
polycarbonate
shoebox
cages
(no
wheels)
where
they
remain
individually
housed
for
30
more
days
(see
Figure
1).
e.
In
Cohort
2
running
wheel
activities
are
monitored
continuously
in
1-min
increments
throughout
the
experiments
via
magnetic
switches
interfaced
to
a
computer
using
VitalView
software
for
the
entire
43-day
duration
(see
Figure
1
and
Figure
3).
2.
BrdU
administration
a.
Fresh
BrdU
solution
0.5%
is
prepared
a
day
in
advance
by
dissolving
BrdU
in
0.007
N
NaOH
in
0.9%
NaCl.
b.
Each
mouse
is
weighed
to
calculate
the
correct
dose
of
BrdU
(50
mg
per
kg
bw).
c.
Mice
are
injected
with
BrdU
solution
at
about
11:00
am
daily
for
10
days.
d.
Tails
are
marked
to
indicate
test
order,
and
then
returned
in
their
home
cages.
3.
Perfusion
for
immunohistochemistry
a.
After
43
days
of
home
cage
activity
measurement,
the
mice
are
anesthetized
with
(150
mg
per
kg
bw)
sodium
pentobarbital
(ip).
b.
Mice
are
then
perfused
intracardiac
with
4%
paraformaldehyde
in
PBS
using
a
peristaltic
pump.
c.
Brains
are
post-fixed
overnight,
and
transferred
to
30%
sucrose
in
PBS.
d.
Brains
are
then
sectioned
using
a
cryostat
into
40
µm
coronal
sections
(see
Figure
4
below).
Figure
4.
Schematic
illustration
of
the
brain
and
the
three
planes
for
sectioning.
DG
=
dentate
gyrus
(shown
in
green).
e.
Brain
sections
are
stored
in
tissue
cryoprotectant
at
-20°C.
f.
Two
separate
1-in-6
series
of
these
frozen
brain
sections
(i.e.,
series
of
sections
throughout
the
rostro-caudal
extent
of
the
brain
with
240
µm
increments
separating
each
section)
are
immunohistochemically
stained
in
each
of
the
following
ways:
I-
BrdU-DAB
staining
to
detect
the
number
of
BrdU+
newly
divided
cells,
located
adjacent
to
or
in
the
granule
cell
layer
of
the
dentate
gyrus.
a.
Free
floating
sections
are
washed
in
TBS
and
then
treated
with
0.6%
hydrogen
peroxide.
b.
Washed
brain
sections
are
pre-treated
with
50%
de-ionized
formamide,
10%
20XSCC
buffer,
2N
hydrochloric
acid,
0.1
M
Boric
acid
to
denature
DNA
for
BrdU
detection.
c.
Denatured
brain
sections
are
initially
blocked
with
a
solution
of
0.3%
Triton-X
and
3%
goat
serum
in
TBS-X
plus.
d.
Then
the
brain
sections
are
incubated
in
primary
antibody
(anti-BrdU)
for
72
hrs
at
4°C,
washed
with
TBS,
and
then
treated
with
TBS-X
Plus
for
30
min.
e.
Brain
sections
are
then
incubated
in
secondary
antibody
(goat
anti-rat)
for
100
min
at
room
temperature.
f.
Tissue
sections
are
then
treated
using
Avidin-Biotin
Complex
(ABC)
system
for
enzyme-mediated
immunodetection,
commonly
referred
to
as
immunoperoxidase
labeling.
g.
To
generate
a
brown-colored
polymeric
oxidation
product
used
to
visualize
newly
divided
neurons,
the
sections
are
treated
with
diaminobenzidine
(DAB),
which
is
the
substrate
for
the
ABC
system.
h.
After
color
development,
slide-mounted
sections
are
rapidly
dehydrated
and
cover-slipped
for
microscopic
evaluation
and
morphometry.
II-
Double-fluorescent
labeling
to
determine
the
fraction
of
BrdU+
in
the
dentate
gyrus
that
co-label
with
anti-NeuN
(marker
for
neuronal
cells)
(see
Figure
5
below).
a.
The
same
procedure
as
I-a
to
I-h
above
is
used
except
another
cocktail
is
used
for
the
primary
antibody
step:
rat
anti-BrdU
(1:100)
and
mouse
anti-NeuN
(1:50).
b.
The
secondary
antibodies
made
in
goat
are
conjugated
with
fluorescent
markers
(Cy2-anti
mouse
and
Cy3
anti-rat)
at
dilution
1:200,
likewise,
delivered
as
a
cocktail.
Figure
5.
Representative
sections
through
the
dentate
gyrus
of
a
control
non-exercised
and
an
exercised
mouse
stained
for
BrdU
with
DAB
on
the
left.
Double-fluorescent
staining
BrdU
red
and
NeuN
(mature
neuronal
marker)
green
in
the
dentate
gyrus
on
the
right.
4.
Image
analysis,
counts
of
BrdU-labeled
cells,
and
estimating
dentate
gyrus
morphology
I.
BrdU-DAB
a.
The
entire
granule
cell
layer
(bilateral)
of
the
dentate
gyrus
in
the
hippocampus,
represented
in
the
1-in-6
series
is
photographed
by
systematically
advancing
the
field
of
view
of
the
Zeiss
brightfield
light
microscope,
and
taking
multiple
photographs
of
BrdU-DAB
stained
sections,
via
camera
interfaced
to
computer,
under
10X
(total
100X)
magnification.
b.
Positively-labeled
cells
in
these
photographs
are
automatically
counted
using
ImageJ
software.
To
generate
unbiased
estimates
of
total
number
of
BrdU-labeled
cells
in
the
dentate
gyrus,
the
counted
cells
are
first
multiplied
by
0.85
(assuming
15%
of
nuclei
intersect
the
plane
of
the
section,
based
on
the
observation
that
the
average
size
of
nuclei
are
6
microns,
which
is
15%
of
a
40-micron
section).
Secondly,
the
number
of
cells
per
section
is
multiplied
by
96,
the
average
number
of
sections
per
dentate
gyrus.
c.
The
total
volume
of
the
granule
cell
layer
of
the
dentate
gyrus
represented
in
the
series
is
estimated.
II.
Double-fluorescent
labeling
for
BrdU
and
NeuN
a.
For
analyzing
double-labeled
brain
sections,
a
Leica
SP2
laser
scanning
confocal
microscope
(using
a
40X
oil
objective,
pinhole
size
81.35
µm
in
diameter)
is
used
to
determine
the
fraction
of
BrdU+
cells
that
are
double
positive
(BrdU+
NeuN+).
b.
BrdU+
cells
in
the
granule
cell
layer
(represented
in
the
1-in-6
series)
of
the
dentate
gyrus
in
the
hippocampus
are
analyzed
by
focusing
through
the
tissue
in
the
z-axis
to
detect
co-labeling
with
anti-NeuN
(see
Z-plane
stage
encoder
below).
Data
collected
by
investigator
•
Locomotor
activity
•
Beginning
and
ending
body
weight
•
Volume
of
dentate
gyrus
(granule
cell
layer)
•
Number
of
new
dentate
gyrus
neurons
(granule
cell
layer)
•
Density
of
new
dentate
gyrus
neurons
(granule
cell
layer)
Definitions
and
calculations
Brdu:
primarily
used
as
non-radioactive
thymidine
analog
5’bromo-2-deoxyuridine
(BrdU)
to
detect
recently
dividing
cells.
NeuN:
marker
for
neuronal
cells.
z-plane
stage
encoder:
in
studying
hippocampal
cell
proliferation
and
neurogenesis,
the
most
stringent
and
validated
methodology
used
to
quantify
the
number
of
newly
divided
cells
eliminates
many
of
the
inherent
biases
encountered.
This
technique
requires
a
microscope
with
a
z-plane
stage
encoder
and
software
for
optical
dissection
and
cell
counting
analysis.
Neurogenesis
To
quantitate
neurogenesis
(N)
per
volume
of
the
granule
cell
layer
of
the
dentate
gyrus
(DG),
the
following
formula
was
used:
N
=
(t
x
f)
/
v
Where
t
=
total
number
of
BrdU+
cells
in
the
DG
[1]
f
=
fraction
of
BrdU+
cells
that
are
also
positive
for
the
neuronal
cell
marker
(BrdU+
NeuN+)
[2]
v
=
volume
of
DG
(estimated
by
serial
sections)
[1]
[1]
determined
by
BrdU-DAB
staining
[2]
determined
by
double-fluorescent
labeling
(BrdU,
NeuN)
|
|
Test age:
8-15wks
