Crabbe2
Protocol
Project
protocol
—
Contents
Workflow,
sampling,
and
experimental
treatment
Equipment
Reagents,
supplies,
and
solutions
Procedures
Data
References
Workflow,
sampling,
and
experimental
treatment
Workflow
Step |
Procedure
done |
Age
(wks) |
Diazepam
(mg/kg) |
Time
(min) |
Equipment |
Data
collected |
1 |
Mice
are
acclimated |
5-6 |
- |
- |
- |
- |
2 |
Mice
are
given
i.p.
injection
of
treatment
vehicle |
6 |
0 |
- |
- |
- |
3 |
Mice
are
placed
in
the
center
of
activity
monitoring
system |
6 |
0 |
15* |
Omnitech
Activity
monitor |
baseline:
total
horizontal
distance
traveled
(cm),
vertical
(rearing)
activity
(n) |
4 |
Post-activity
rectal
temperature
is
obtained |
6 |
0 |
- |
Digital
thermometer |
baseline
body
temperature
(°C) |
5 |
Mice
are
given
i.p.
injection
of
benzodiazepine
(diazepam) |
6 |
2,
4,
8,
or
16 |
- |
- |
- |
6 |
Mice
are
placed
in
the
center
of
activity
monitoring
system |
6 |
2,
4,
8,
or
16 |
15* |
Omnitech
Activity
monitor |
horizontal
distance
traveled
(cm);
vertical
(rearing)
activity
(n) |
7 |
Post-activity
rectal
temperature
is
obtained |
6 |
2,
4,
8,
or
16 |
<1 |
Digital
thermometer |
30
min
post-treatment
body
temperature
(°C) |
8 |
Mice
are
sacrificed
and
the
brains
are
harvested
and
stored
at
-80°C |
6 |
8
or
16 |
- |
- |
- |
9 |
Mice
are
given
3
test
trials
before
treatment
with
benzodiazepine
(diazepam)
and
then
rested
for
1
hr |
6 |
0 |
>2 |
Rotarod
accelerated
at
1
rpm/s |
(innate
rotarod
ability
is
assessed) |
10 |
Mice
are
given
i.p.
injection
of
high-dose
of
benzodiazepine
(diazepam)
and
then
tested
on
the
rotarod |
7 |
20 |
<2 |
Rotarod
constant
speed
(5
rpm)
|
latency
to
fall
(s) |
11 |
Mice
are
sacrificed
and
the
brains
are
harvested
and
stored
at
-80°C |
7 |
20 |
- |
- |
- |
12 |
Treated
brains
are
processed
and
analyzed |
6-7 |
8,
16,
and
20 |
- |
gas
chromatography |
concentrations
of
benzodiazepines
(DZ,
diazepam)
and
metabolites
(nordazepam
and
oxazepam)
(ng/g) |
*
The
activity
data
(steps
3
&
6)
are
collected
immediately-to-30
min
post
injection.
However,
the
data
reported
are
from
0-15
min
post-injection
because
strain
effects
were
strongest
during
this
period.
Treatment
Low-doses
|
2
or
4
mg
diazepam
per
kg
body
weight
[mg/kg] |
| Intermediate
doses
|
8
or
16
mg
diazepam
per
kg
body
weight
[mg/kg] |
| High-dose
|
20
mg
diazepam
per
kg
body
weight
[mg/kg] |
Delivery
|
intraperitoneal
injection
(i.p.) |
Equipment
- Digital
thermometer,
0.5
mm
diameter
x
2.5
cm,
Sensortek
Thermalert
TH-8
- Animal
monitoring
system,
Omnitech
Activity
Monitoring
Chamber
- Rotarod,
with
constant
and
accelerating
speeds
where
dowels
are
suspended
55-60
cm
above
sawdust
bedding
- Gas
chromatography
-
-80°C
and
-20°C
storage
refrigerators
- Plexiglas
tube
for
brief
restrain
- Decapitating
scissors
- Small
rodent
dissecting
kit
Reagents,
supplies,
solutions
- Benzodiazepine,
diazepam
- Treatment
vehicle:
1
drop
of
Tween
20
+
10
mL
physiological
saline
- Saline
(physiological
saline):
0.9%,
Baxter
Healthcare
Corp,
Deerfield
IL,
USA
- Isopropyl
alcohol:
10%
for
wipe
downs
between
tests
- Syringes
with
needles
Acclimation
to
test
conditions
Mice
are
acclimated
for
1
hr
before
testing
begins.
Procedures
I.
Activity
and
body
temperature
measurement
a.
Each
mouse
is
temporarily
restrained
using
a
Plexiglas
tube
for
at
most
10
s
until
baseline
rectal
(body)
temperature
is
obtained
with
a
pre-lubricated
digital
thermometer
probe.
b.
The
mouse
is
then
injected
i.p.
with
vehicle
or
2,
4,
8,
or
16
mg/kg
dose
of
diazepam,
and
then
placed
in
the
center
of
an
animal
monitoring
system.
c.
Activity
is
automatically
measured
via
infrared
beam
interruptions
every
5
min
for
a
total
duration
of
30
min.
d.
Shortly
after
30
min
in
the
activity
monitoring
chamber,
each
mouse
is
again
restrained
for
a
final
body
temperature
determination.
e.
Following
the
final
body
temperature
measurement,
mice
from
the
8
and
16
mg/kg
dose
groups
are
euthanized
and
brains
dissected
and
stored
at
-80°C
until
assay
is
performed
(see
below).
II.
Measuring
coordination
under
high-dose
diazepam
using
the
rotarod
Because
it
was
not
possible
to
test
all
strains
at
once,
a
2-block
design
was
adopted.
Both
C57BL/6J
and
DBA/2J
strains
were
included
in
blocks
1
and
2
as
internal
(seasonal)
controls.
a.
Initially,
mice
are
tested
for
their
innate
ability
to
perform
on
a
rotarod
that
is
accelerated
at
a
constant
rate
of
1
rpm/s.
b.
After
three
test
trials,
performance
in
Trials
2
and
3
is
averaged
in
revolutions
per
minute
where
in
the
mouse
fell
from
the
rotarod
is
indicative
of
innate
rotarod
ability.
c.
The
mice
are
rested
for
1
hr
subsequently
after
test
trials
and
before
coordination
under
diazepam
treatment
is
measured.
d.
After
being
rested
for
1
hr,
each
mouse
is
injected
i.p.
with
high-dose
of
20
mg/kg
of
diazepam
and
placed
on
a
rotarod
with
a
fixed
speed
of
5
rpm
(rotation
per
min).
e.
Testing
is
terminated
when
the
mouse
falls
off
the
rotarod.
f.
Shortly
after
the
mouse
loses
its
balance,
it
is
decapitated
and
the
brain
is
harvested
for
benzodiazepine
determination.
III.
Determination
of
brain
benzodiazepines
a.
Brain
samples
are
harvested
from
mice
given
8,
16,
and
20
mg/kg
of
diazepam
b.
For
short-term
storage
brain
samples
are
kept
in
-20°C
until
assay
is
performed.
c.
For
long-term
storage
brain
samples
are
kept
in
-80°C
until
assay
is
performed.
d.
Brains
samples
are
processed
and
then
analyzed
with
gas
chromatographic
method.
e.
Brain
concentrations
of
diazepam,
nordazepam,
and
oxazepam
are
determined.
f.
Only
brain
concentrations
of
diazepam
and
nordazepam
are
determined
in
coordination
study.
Data
collected
by
investigator
Total
horizontal
distance
traveled,
total
vertical
(rearing)
activity,
and
body
(rectal)
temperature
with
baseline/vehicle
or
2,
4,
8,
or
16
mg/kg
of
diazepam.
Latency
to
fall
from
the
rotarod
with
high-dose
20
mg/kg
diazepam.
Brain
benzodiazepines
concentrations
in
mice
treated
with
intermediate
and
high-dose
of
diazepam.
|
|
Test age:
6-7wks
