Jones1_Protocol
Project
protocol
—
Contents
Workflow
and
sampling
Equipment
and
consumables
Procedure
Data
Definitions
References
Workflow
and
sampling
Workflow
Step |
Procedure
performed |
Age
(wks) |
Apparatus |
Rx |
Data
collected |
1 |
Mice
are
brought
from
the
vivarium
to
the
test
room
to
acclimate
before
testing
begins |
5-56 |
- |
- |
- |
2 |
Mice
are
weighed
to
determine
anesthetic
dose |
5-56 |
scale |
EquiThesin
4
µL/g
BW |
|
3 |
After
mice
are
completely
sedated,
they
are
prepared
for
surgery,
positioned
and
set-up
for
VsEPs
testing |
5-56 |
surgical
instruments,
VsEPs
testing
system
|
EquiThesin
4
µL/g
BW |
VsEPs
waveforms;
latency
of
peaks
1-3
(P1,
P2,
P3),
amplitudes
of
peaks
1-2
|
4 |
Mice
are
kept
in
thermostatically
controlled
heating
pad
until
they
fully
recover |
5-56 |
- |
- |
|
5 |
VsEPs
testing
system
is
cleaned
before
subsequent
use |
5-56 |
- |
- |
- |
Equipment
and
consumables
•
Balance
scale
•
Disinfectant
or
cleaning
solution
•
Syringe
with
30
gauge
needle
•
Anesthetic:
EquiThesin
(4
µL/g
bw,
(mixture
of
sodium
pentobarbital,
chloral
hydrate,
propylene
glycol,
magnesium
sulfate,
ethanol,
and
water;
see
Jones
et
al.,1997
for
complete
recipe)
•
Small
rodent
surgical
pack
(scissors,
forceps,
scalpels,
retractors,
bone
drill/rongeurs,
etc.)
Figure
1
A
shows
the
mechanical
shaker
and
the
accelerometer
setup.
Panel
B.
Closeup
image
of
the
accelerometer.
Panel
C.
Layout
of
the
mechanical
shaker.
•
Mechanical
shaker
for
delivering
motion
stimuli
(Labworks,
Inc.
Model
ET2-203),
see
Figure
1
A-C
above
•
Accelerometer
(see
Figure
1
B
above)
Figures
2
A
&
B
are
photographs
of
the
electrical
signal
amplifier
setup.
Panel
C.
Display
screen
for
amplified
signals.
Panel
D.
Computer
screen
illustrating
the
signal
output
as
well
as
the
stimulus
generated.
•
Electrical
signal
amplifier:
differential
preamplifier
(Grass
P511
Model
K)
see
Figures
2
A
-
C
above
•
Vestibular
stimulus
generator:
stimuli
are
generated
and
controlled
using
custom
software
and
Tucker
Davis
Technologies
(TDT,
Gainesville,
FL)
modules
(TG6,
DA3-2,
PA4);
response
signals
are
sampled
following
the
onset
of
each
stimulus
(A/D
conversion:
1024
points
at
10
ms/
point,
TDT
AD1
module)
to
produce
a
10.24
ms
response
epoch,
see
Figure
2
D
above
•
Computer
Figure
3
A
depicts
the
layout
for
temperature
control
and
Figure
3
B
is
a
closeup
of
the
temperature
sensor
and
control.
•
Heating
pad,
see
Figure
3
A
above
•
Customized
aluminum
plate
for
coupling
head
to
mechanical
shaker
(optional)
•
Head
mount
or
modified
head
clip
•
Dental
acrylic
•
Thumbscrew,
stainless
steel
screws
•
Stainless
steel
electrodes
Procedures
I.
Pre-surgical
and
surgical
Preparation
a.
Mice
are
weighed
and
then
anesthetized
with
EquiThesin
(4
µl/g
body
weight
injected
intraperitoneally
(i.p.)).
b.
An
incision
is
made
in
the
skin
overlying
the
skull
and
the
skin
is
retracted
to
reveal
the
sagittal
(see
Figure
4
A
below)
and
coronal
skull
sutures.
c.
Two
holes
are
drilled
at
the
vertex
near
the
midline
to
expose
the
dura
and
bare
stainless
steel
(0.127
mm
diameter)
wires
are
coiled
and
inserted
in
the
epidural
space
and
the
holes
are
sealed
with
bone
wax.
These
electrodes
are
used
as
the
noninverting
(G1)
electrodes
(see
Figure
4).
d.
Subcutaneous
electrodes
are
placed
behind
the
left
and
right
ear
pinna
(inverting
leads
(G2)
for
two
channel
recordings),
and
under
the
chin
(common
ground
electrode)
or
ventral
neck
(see
Figure
4
B).
e.
Two
small
pilot
holes
are
drilled
on
the
midline
rostral
and
caudal
to
the
coronal
suture
to
insert
self-tapping
stainless
steel
screws
that
served
as
anchors
for
dental
acrylic.
f.
The
dental
acrylic
is
poured
over
the
area
to
cement
a
thumbscrew
into
place
over
the
vertex.
The
thumbscrew
is
used
to
mechanically
couple
the
mouse's
head
firmly
to
the
mechanical
shaker
platform.
g.
The
animal
is
then
placed
supine-ventro-dorsal
position
on
the
pre-heated
platform,
while
the
head
is
oriented
with
the
nose
pointed
dorsally
(see
Figure
5
below).
h.The
heated
platform
is
adjusted
to
keep
the
core
body
temperature
at
35-39°C.
i.
A
head
mount
is
placed
on
the
cranium
using
methods
published
previously
(Jones
and
Jones,
1999;
Jones
et
al.,
2002).
The
head
mount
is
used
to
couple
the
cranium
securely
to
a
mechanical
shaker
for
precise
and
controlled
stimulation
to
the
head.
Figure
4.
Schematic
illustrations
of
electrode
placements
in
the
head.
Panel
A.
Dorso-ventral
view
of
the
head
showing
the
locations
of
two
(rostral,
caudal)
non-inverting
(G1)
electrodes.
Panel
B.
Left-lateral
view
of
the
head
showing
approximate
locations
of
an
inverting
(G2)
electrode
(red
asterisk)
and
common
ground
electrode
(blue
star).
II.
Measurement
of
VsEPs
Linear
VsEPs
are
compound
action
potentials
from
the
vestibular
portion
of
the
eighth
cranial
nerve
(CN
VIII)
and
central
relays
and
are
generated
in
response
to
motion
stimulus.
These
VsEPs
depend
strictly
upon
the
integrity
of
the
utricle
and
saccule
(gravity
receptors)
of
the
inner
ear.
The
first
response
peak
reflect
activity
of
the
peripheral
vestibular
nerve,
whereas
later
response
peaks
reflect
activity
of
vestibular
relays
within
the
brainstem
and
higher
centers,
which
are
recorded
much
like
the
more
common
auditory
brainstem
response
or
ABR
(see
Figure
6
below,
Jones
et
al.
2005).
a.
After
securing
or
coupling
the
head
to
the
mechanical
shaker
with
anchoring
screws,
motion
stimuli
are
delivered
to
the
head
(Jones,
1992;
Jones
and
Jones,
1996;
Jones
et
al.,
1997).
b.
Linear
up-and-down
jerk
stimulus
pulses
of
2
ms
duration
are
applied
to
the
head
via
naso-occipital
axis
at
a
rate
of
16
linear
jerks/s.
Hence,
head
motion
is
alternated
in
a
downward
and
upward
directions.
c.
A
calibrated
accelerometer
is
then
mounted
on
the
stimulus
platform
and
used
to
monitor
the
acceleration
of
the
stimuli.
The
output
of
the
calibrated
accelerometer
is
then
routed
to
an
electronic
differentiator
to
monitor
the
jerk
component
(i.e.,
the
first
derivative
of
acceleration)
of
the
stimulus.
The
accelerometer
is
calibrated
using
known
jerk
stimuli
that
is
linear
over
ranges
of
amplitudes;
the
stimulus
intensity
ranges
from
-15
dB
to
+6
dB
re:
1.0
g/ms
(where
1.0
g=
9.8
µm/s2)
adjustable
in
3
dB
steps.
Stimulus
amplitude
is
quantified
in
decibels
relative
to
1.0
g/ms
(1.0
g
=
9.8
µm/ms2).
Maximum
stimulus
amplitude
of
+6
dB
re:
1.0
g/ms,
corresponds
to
a
peak
acceleration
of
4
g
and
calculated
peak
platform
displacement
of
26
µm
(Jones
et
al.,
2002).
d.
Two
stimulus
polarities,
normal
and
inverted,
are
used.
Normal
polarity
stimuli
are
defined
as
an
initial
upward
jerk
whereas
inverted
stimuli
as
an
initial
downward
jerk.
e.
Two-channel
signal
averaging
is
used
to
extract
the
vestibular
responses
from
the
background
activity.
f.
Electrophysiological
activity
is
amplified
(200
000X),
filtered
(300-3000
Hz,
-6
dB
points),
and
digitized
(125,000
Hz)
beginning
at
the
onset
of
each
jerk
stimulus
(1024
points,
8
µs
per
point)
to
produce
one
primary
response
trace
or
waveform.
g.
256
primary
responses
are
averaged
to
produce
an
averaged
response
waveform
for
a
given
stimulus
intensity
and
polarity.
h.
Responses
are
replicated
such
that
two
averaged
response
waveforms
are
recorded/collected
for
each
stimulus
intensity
(a
waveform
recorded
for
normal
stimulus
polarity
and
a
waveform
for
inverted
polarity).
Final
individual
response
traces
are
produced
off-line
by
summing
one
averaged
response
to
each
stimulus
polarity
and
dividing
the
result
by
2,
thus
producing
two
response
traces
for
every
stimulus
intensity.
i.
A
threshold
seeking
protocol
is
then
used
where
stimulus
intensity
began
at
+6
dB
and
waveforms
are
collected
in
quiet
(no
sound
stimulus)
and
in
the
presence
of
an
intense
sound
stimulus
or
masker
(50
to
50,000
Hz,
116
dB
SPL).
The
masker
is
used
to
verify
the
absence
of
auditory
responses.
j.
Subsequent
stimulus
level
is
reduced
in
6
dB
steps
until
no
response
could
be
detected
by
visual
inspection
of
the
waveform.
k.
At
that
point,
the
stimulus
intensity
is
reduced
an
additional
3
dB
and
waveforms
are
collected
for
intensity
steps
midway
between
those
already
used.
In
this
manner,
intensity
series
in
3
dB
steps
encompassing
stimulus
levels
above
and
below
thresholds
can
be
collected.
Figure
5
A
shows
a
schematic
illustrations
of
the
vestibular
evoked
potential
(VsEP)
measurement
set-up.
Figure
5
B
depicts
an
image
of
the
actual
set-up.
Note:
the
testing
chamber
is
lined
with
foam
cushions
to
minimized
extraneous
noise
and
electrical
interference.
III.
Quantification
of
VsEP
waveforms
a.
The
first
three
positive
and
negative
response
peaks
are
analyzed
(see
Figure
6
below).
b.
Response
peak
latencies
are
measured
in
milliseconds
(ms)
from
the
onset
of
the
stimulus
to
each
positive
response
peak
(labeled
P1,
P2,
and
P3)
and
negative
(labeled
N1
to
N3)
peaks.
Latencies
provide
a
measure
of
the
timing
of
neural
transmission
and
conduction
through
the
vestibular
neural
pathways.
c.
Peak
to
peak
amplitudes
are
measured
in
microvolts
(µV)
by
subtracting
the
amplitude
of
the
negative
peak
from
its
corresponding
positive
peak
(labeled
P1/
N1,
P2/N2,
P3/N3).
Amplitudes
reflect
the
size
and
general
synchrony
of
the
population
of
neurons
responding
to
the
stimulus.
d.
Thresholds
are
measured
in
decibels
(re:
1.0
g/ms
is
defined
as
the
stimulus
level
midway
between
the
jerk
amplitude
just
producing
a
discernible
response
and
that
stimulus
level
which
did
not.
Thresholds
provide
a
measure
of
the
general
sensitivity
of
the
gravity
receptor
system.
Figure
6.
Graphic
illustrations
of
the
vestibular
evoked
potential
(VsEP)
waveforms.
Panel
A.
Activated
regions
corresponding
to
given
response
peaks.
Panel
B.
Analysis
of
VsEP
waveforms
showing
response
peak
latencies
(red
lines)
and
response
peak
amplitudes
(blue
lines).
Data
collected
by
investigator
-
threshold
stimulus
response
level
- latency
of
positive
evoked
potentials
for
peaks
1,
2,
and
3
- amplitude
of
evoked
potentials
for
peaks
1
and
2
Definitions
and
calculations
Thresholds
are
defined
as
the
intensity
midway
between
the
minimum
intensity
producing
a
response
and
the
maximum
intensity
failing
to
produce
a
response.
Response
peak
latencies
are
measured
in
milliseconds
from
stimulus
onset
to
the
occurrence
of
positive
(labeled
P1
to
P3)
and
negative
(labeled
N1
to
N3)
peaks.
Peak-to-peak
amplitudes
are
measured
in
microvolts
by
subtracting
the
amplitude
of
the
negative
peak
from
its
corresponding
positive
peak
(labeled
P1/
N1,
P2/N2,
P3/N3).
|
|
Test age:
5-56wks