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0022-3565/85/2343-0821$02.0()/O
THE
JOURNAL
Copyright
OF
PHARMACOLOGY
1955 by The
(
AND
American
EXPERIMENTAL
Vol. 214, No. 3
Printed
in U.S.A.
THERAPEUTICS
forPharmacology
Society
and Experimental
Therapeutics
Barbiturate-Induced
Alterations in the Kinetic Parameters
Outward Current in Aplysia Giant Neurons1
JOHN
A.
Epilepsy
HUGUENARD2
Center,
Accepted
and
Veterans
WILKIE
Administration
for publication
of Slow
A. WILSON
Hospital
and Department
of Pharmacology,
Duke University
Medical
Center,
Durham,
North Carolina
June 17. 1985
ABSTRACT
Regulation
of spike frequency
adaptation
has been postulated
to be a neuronal mechanism
of action of the barbiturates
(Zbicz
current
outward
and Wilson,
tion-related,
saturable
and
ponent
of the tail currents,
the amplitude
of the slow phase.
With
moderate
concentrations of barbiturates
there were no changes
in the other
parameters describing
the exponential
decay current.
1981 ). A barbiturate
sensitive
slow outward
current
giant neurons,
and this current
is very
eftective
at regulating
adaptation.
In the current
study a quantitative method
of determining
the effect
of barbiturates
was
is present
in Aplysia
developed
tail current
using
a
analysis,
single-electrode
involved
voltage
clamp.
measuring
the
The
This method,
exponential
A slowly
activating
giant
current
voltage
outward
neurons
is activated
range
of
current
has
been
et al.,
1978).
The
(Cote
by
-40
near-threshold
to -30
mV.
demonstrated
slow
is attained
molluscan
during
neurons,
that
functional
significance
slow
outward
current
of slow
Wilson,
1981)
spike
frequency
important
in the
of drugs.
effects
are
(Zbicz
and
observation
that
models
of epilepsy
associated
individual
1961;
Sawa
affects
the
expected
noted
the
action
of some
thus
This
aspects
well as
with
prolonged
central
neurons
et at.,
periods
(Ward,
Zbicz
clinical
of seizure
human
terrnining
of
may
be
actions
hypothesized
actions
and
process
of barbiturates
hypothesis
in
1978;
of the
of the
been
anticonvulsant
1981).
certain
as
(Cote
that
of
these
of barbiturates
is
based
activity
epileptic
on
the
in animal
cortex
are
of high
frequency
1961;
Kandel
and
firing
of
Spencer,
A,, amplitude
of the fast component
activity
kinetics.
to determine
1) the
the
and
neurons
currents,
cellular
the
on slow
neurons
the
allows
which
and
allows
membrane
and
of tail current decay; A2, amplitude
which
barbiturate
is
action
that
were
slow
be
unique
to
corn1979;
further
of the
the
report
the
analysis
provides
the
current
these
study
outward
of barbiturates
a sensitive
means
effects
in these
neurons
by
of the
of
simultaneous
the
measurement
and
ionic
the
population
soma
activities
of
neurons,
within
of a uniform
accessibility
potentials
of
the
current.
This
preparation
for several
reasons,
including:
outward
of
it was
effects
presents
of slow
effects
to
in de-
current,
kinetics
and
and
important
outward
neurons,
outward
was chosen
of slow
size
which
then
it should
be
is an anticon-
concentration-dependent
identification
which
3)
but rather
by other
anticonvulsant
benzodiazepines
(Zbicz,
study
Kinetic
presence
ready
lipid
current
would
However,
current
a means
of quantitating
outward
current.
This
giant
which
repetitively
mechanism
in enhancing
in Aptysia
the
outward
mechanism,
actions.
outward
shared
and
this
of a voltage-clamp
on the
is a
with
slow
intervention
to fire
properties
develop
on slow
barbiturates
Aplysia
giant
that
1981).
structural
to
current
ganglia
Received
for publication
December
17, 1984.
, This work was supported
by the Veterans
Administration,
Durham,
North
Carolina,
and National
Institutes
of Health
Grant
15212.
2 Present address: Department
of Neurology,
Stanford
University,
School
of
Medicine,
Stanford,
CA 94305.
ABBREVIATIONS:
aminobutyric
acid.
Oosterode,
to understand
desirable
barbiturates
results
it
and is not
as hydantoins
the
correlated
Pharmacological
1963).
to have anticonvulsant
that
if activation
of slow
and
for the enhancement
well
indicating
of neurons
mechanism,
elucidate
not
is not due to a nonspecific
mediated.
et al.,
barbiturates,
pounds
such
de-
of barbiturates
was
drugs,
ability
In order
as anticonvulsants,
voltage-sensitive
enhancement
This
It has
to the
Wilson,
use
the
current
mechanism
related
clinical
potentiate
a resultant
adaptation.
this
group
have
outward
with
in
may be receptor
vulsant
a prolonged
indicating
current
of these
solubility
enhancement
reflected
the magnitude
of slow
were found to cause a concentrastereospecific
increase
in one corn-
of potency
order
outward
Hoyer
control
of firing
rate.
Barbiturates,
which
sedatives
and anesthetics,
velopment
has
outward
depolarizations
in the
This
range
includes
the
average
interspike
potential
that
train
ofaction
potentials
in these
rank
of slow
decay
of the slow outward current at the end of a depolarizing
voltageclamp command.
The tail currents
were made up of two decay
phases with average
half-times
of 7 and 70 sec,
and the tail
in Aplysia
amplitude
accurately
current. Barbiturates
of
2)
neural
of cells
these
giant
of membrane
(both
intra-
extracellular).
of the slow component
of tail current decay; GABA,
‘-
________________________________________________________________________________
821
822
Huguenard
The
kinetic
terizing
analysis
the
of slow
tail
tail
in these
were
were
used
cific
with
were
The
was
slow
not
These
well
data,
acid;
the
together
with
minimal
sedative
Raines
et
suggests
that
by different
at.,
the
1979)
enhancement
(Huguenard
enhancement
with other,
the anticonvulsant
produces
of
slow
outward
nonsedative,
effects.
slow
Wilson,
1985),
current
effects
of the
may
be
barbiturates,
preparation.
Hughes
and
(Marine
Specimens
Tauc,
The
study.
Giant
1961
ganglion
to the
MI)-lined
recording
was
scissors
In the
and
expedite
(millimolar):
70; and
30.
The
492;
in order
were found
mately
outward
of the
ity obtained
highly
drug
A
The
solution
was
volume
of
chamber)
at
was
was
assumed
tion.
(Dye
pH
least
to have
synaptic
under
study,
the
activity
made
control
artificial
sea
ml
immediately
(50
times
to
the
flow
the
any
of
wash
concentrations.
secobarbital
(compliments
them
stereoisomers
0.1
N
Before
in
and
sea
sea
then
change
in
to equilibrium
to
diluent
drug
MO)
over
The
the
were
desired
also
solutions,
concentra-
to this
method,
essentially
current.
First,
forms
final
to
of
the
on
in
of
1 ml
concentration
preparation
parameters
during
Institute
control
was
allowed
obtained
from
to
tail
ling
there
clamp
current
(Wilson
or
was
which
eliminated
ground
circuit.
procedures.
and
voltage
modified
signal
The
Goldner,
of
the
to
provide
drift
reference
(0.1-0.5 megohm)
which
Microelectrodes
were
1975)
giant
due
neuron
an
was
used
under
extracellular
to current
electrode
was placed
pulled
A modified
was
passing
a broken
in the chamber
on a Kopf
vertical
single
for
electrode
either
study.
controlThe
clamp
voltage
reference
through
the
tip
microelectrode
near
the giant
electrode
puller
bath
cell.
from
has
one
during
has
the
been
rates
on
Plotting
the decay
a
obtained
slow
after
of the
phase
sum
of at least
ing”
routine
and
amplitudes
was
tail
current
membrane
a
currents
of
a partial
linear;
approaches
at
least
two
that,
in
inactivation
slow
of a
Therefore,
outward
current,
slow outward
activation
current
in cesium-loaded
cells
et a!., 1982).
Therefore,
by analyzing
a prolonged
voltage
clamp depolarize-
slow
present
outward
the
outward
is not
demonstrated
of measuring
current
outward
current
et a!., 1981).
(Evans
in the
a 60-sec
of slow
consist
reflect
a
develop-
depolarization.
only
a means
(Colmers
depolarization
exists
as
as well.
inward
a measure
to
of
used
two disadvantages
the
have
prolonged
not
currents
current
used
there
current
slow
Second,
( 1982)
a!.
during
does provide
After
as the
appear
et
neurons,
in other
is no tail
time
depolarization
the tail currents
tion,
Eleetrophysiological
circuitry
changes
isolation.
depolarization
Colmers
membrane
One method
in
activation
during
with
a period
be
during
are at least
of 20 to 30 mm.
loaded Aplysia
inward
current
in
prolonged
current
slowly,
appears
current
current
during
in
a period
outward
very
after
of outward
but there
slow
analysis
current
easily
1981)
very
depolarizations
even
cannot
slope
slow
The
similar
for the
final
stabilize,
and
in these
quite
activates
The
7).
level
The
decreases
currents.
changes
but
rise
prolonged
not
measure
especially
over
a
slow
to
gradually
of
cesium
in
solutions.
analysis).
voltage
the slope
maximum
used
fig.
enhancestudy;
clamp
current
of
1981)
neuron
were
current.
current
effects.
and Wilson,
the
does
final
the
problems
voltage
control
Wilson,
intact
repeat
1)
1985),
would be
barbiturate
outward
(see
but
the
and
presented
outward
minutes
of barbiturate
been
slow
(Zbicz
neurons
subthreshold
the
a maximum
(Zbicz
by
For
of many
period
types
current
mV,
Therefore,
has
ment
recording
dissolved
added
the
by the
acid
effects.
superfusate
obtained
(National
NC)
was
(as judged
free
mm.
measure
phenobarbital
were
30
slow
outward
then
was
al.,
et
which
in an
axotomized
quantitating
a period
and
This
reasons:
temporal
the
of
control
soma.
following
and
currents
of study.
of spatial
neurons,
spatial
tail
by axotomy
with
to approach
each
soma
of -30
100
neuropil
(Huguenard
of these
and
axotomy.
range
condito
neuron
degree
the
soma
better
before
in the
the
with
of up
in the
in the
for
the
clamping
obtained
of barbiturate
seen
by voltage-clamping
clamping
study
in
results
(approxi-
of pentobarbital,
and
NY)
pentobarbital
Park,
diluted
The
salts
Louis,
water.
and
Triangle
water.
sodium
St.
Rensselaer,
artificial
Research
NaOH
any
come
Co.,
of secobarbital
Abuse,
artificial
of the
Chemical
of Sterling-Winthrop,
dissolving
Drug
Solutions
(Sigma
exists
obtained
Although
complete
with
a wash
volume
of approximately
20 chamber
volumes.)
The drugs in this study were dissolved
in artificial
sea water at the
desired
voltage
in this
current
of slow
and
on
these
periods
of the
the
the
and
was
regarding
isolate
of
those
processes
then
maneuver
in drug
that
extensive
in somewhat
outward
course
bath,
imposed
Under
soma
of slow
Method
from
A electrical
of the
than
performed
in the
ment
time
circuit
withdrawn
were
over
a time-
performed:
bath.
circuit
obtained.
were
during
error
voltage
the
was
greater
under-
10; MgCl2,
the
study
current
result
control
steps
recorded
relatively
recording
was
return
were
concern
potential
to
to
nA.
of major
two-electrode
due
that
the
in the
Voltage
currents
is some
outward
than
passed
were
work
following
magnitudes
neurons
there
expected
experiment.
chamber
change
indicated
each
giant
ganglia
to those
this
volume
before
after
experiments
before
the
2)
stabil-
1.5
at less
possibility
of
ground
currents
0.1
set
this
microelectrode
the
have
the
cases
water
than
was
artifacts
left submerged
Because
slow
normal
with
of currents
the
vannas
increased
synaptic
the
but
placed
with
suppression
less
the
The experiments
in this
via a single microelectrode
corn-
activity.
a minor
physiological
time-dependent
following
the
filled
therefore
component
currents,
current
the
were
some
within
in
eliminate
voltage-clamped.
that
and
the membrane
potential,
gave similar
results
and 3) in a few cases it was possible
to study
the
twice
placed
limit
recorded
To
experiment,
such
the
tissue
10; CaCl2,
contained
of
largest
constantly
of the
2; KC1,
suppress
equilibrated
dilution
perfused
water
water
to cause
allowed
cell model
or
the
and
magnitude
or less,
were
tail
was
acceptable
the
nA
20 nA.
of study
was
model
a source
R2,
to expose
slow
100
always
were
sea
sea
to 7.8
cell
not
connective
of the
adjusted
model
the
in this
impalement.
currents
by suppressing
desirable.
for
microdissecting
NY)
NaHCO3,
to
used
of an
neuron
of membrane
california
were
the
and
ganglia
artificial
MG
concentration
2x Mg
solutions
10)
cases
artificial
NaCl,
MgSO4,
1967;
from 100- to 300-g animals
(Dow Chemical,
Midland,
Plainview,
the
(204C)
CA)
ganglion
forceps
electrode
chamber,
temperature
position
fine
et a!.,
Apl.vsia
mollusc
some
& Co.,
(Frazier
Palisades,
abdominal
In
with
recording
constant
LP1
marine
(the
chamber.
Tiemann
neurons
and
for LP1) were dissected
bottom
of the Sylgard
dissected
(George
lying
the
Pacific
of study
left pleural
and pinned
sheath
) from
Unlimited,
ganglion
R2
glass
minimized.
that
than
similar
end
the
50
polarization
cell
the
and
were
less
produce
the
sec
neurons
capillary
Generally,
was
currents
dependent
tions,
Methods
The
tail
usually
the giant
marked
and
The
at
(diphenylbarbi-
tested
of current.
polarization
small,
could
an anticonvulsant
fiber-filled
experiments
electrode
activities.
was
nA
in these
stereospe-
diameter
passage
1 mV/100
current
amplitude,
of barbiturates
in
by this method,
that
outside
M KC1. Optimum
microelectrode
resistance
for voltage clamping
Aplysia giant
neurons
with the single electrode
voltage
clamp was found to
be in the range of 0.5 to 1 megohm.
Microelectrode
polarization
due to
current
Barbiturates
and
properties
1975,
the
barbiturates
effect.
fact
the
and
sedative/anesthetic
with
current
associated
perhaps
with
taken
outward
describing
of the tail
The activity
as determined
current,
correlated
barbiturate
turic
outward
activation
established,
barbiturate
1.5 mm
charac-
after
concentration-dependent
increases
in one component
little
effect
on the kinetics.
enhancing
were
induced
of the
to cause
involved
parameters
situation
parameters
study
produced
kinetic
control
as a measure
found
in this
which
current.
in the
Vol. 234
utilized
currents
outward
currents
changes
and Wilson
study
to
elucidate
which
Analysis
the
was
activated
of tail
action
currents
of barbitu-
current.
currents
of slow
two
on
outward
exponential
developed
(zero
currents
decay
could
processes.
order to accurately
intercepts)
of tail
in
time
a semi-logarithmic
graph
revealed
be represented
An
automated
that
by the
“peel-
determine
the kinetics
currents
(Zierler,
1981).
1985
B
could
A
2C
a
in
SECOBARB
also
be reasonably
which
was
the
all
trolled
most
:
constant
U
2tsec
the
intersimulus
for
2400).
TIME
(Seconds)
voltage (V). Of the two current traces the lower, shallower
and
the
upper,
semilog
plots
steeper
tail
of
trace
is in the
currents
from
presence
A.
The
trace is control
of secobarbital.
arrows
mark
the
B,
division
between the slow and fast component
of tail current as determined
the analysis.
In order to obtain the fast component
of tail current,
of tail currents
shown
in figure
Amphtudes
0ev’
2
1
2
squares
6.21
5.94
1 00 MM
2.31
5.51
a Dev, deviation
is an estimate
current analysis and is analogous
of the goodness of fit
to the S.D. It is derived
Deviation
SS
is the
n
number
of points
processes)
Figure
(NC x 2)
-
1 shows
,M
figure
an
example
solution
secobarbital.
The
shows
fit curves
obtained
from
tail
analysis:
by performing
component
and 3) the
repeated
mark
the
of the
NC
until
arrows
were
found
are
were
in the
slight
obtained
curve
ods.
the
from
the
fitting
All
fast
ments
no
more
The
half-times
components
results
of tail
change
kinetics
routine
best
the
were
analysis
described.
whereas
and
the
best
The
from
above
the
to
this
the
component;
as well
were
(e.g.
with
most
7 and
line
obtained
the
right
yielded
70 sec.
However,
cases
2). The
use
it was
two
there
results
meth-
of compart-
obtained
5 to
an
of alternative
fitting
parameters
often
In these
cases
numbers
than
excepwas
most
table
the
for the
were
least-square
equivalent
by
of
barbiturates
by the
nonlinear
fits
, see
left
analysis
in
verified
lb
to the
with
10%.
components
in some
found
with
cases,
that
the
three
tails
was
desensitization.
Voltage
Hz,
conIn
and
was
clamp
-18
kept
currents
dB/octave)
on a chart
using
Some
carefully
12 mm,
to be
(50
digitized
curves
(SAS
Unless
are representative
set
filter
analysis.
program
and
recorder
(Gould
a graphics
pad
(Apple
and the data
were
stored
experiments
also
utilized
direct
fit
were
NLIN)
otherwise
of at least
noted,
three
with
which
a
nonlinear
determined
all
least-
the
observations
and
K
in this
study
experiments.
Results
These
results
neurons
No
seasonal
differences
fire
trains
tail
neurons
chosen
effects
and
further
accurately
first step
with
outward
current
as the conductance
depolarized
four
experiment.
mV
In
current
current
criteria
current
and
in both
depolarization.
Based on the assumption
first
intervals
and
the
10
to
180
was
step
sec.
The
repolarization
of the developing
were
slow
outward
2 shows
curves
that
leak
tail current,
equilibrium
Em
of
upon
a neuron
mV and
with
both
increased
of these
duration
currents
are due
the
membrane,
as shown
in figure
current
was
calculated
of
one
2B.
from
can
For
the
relationship
in the hyperpolarized
region.
Then
was calculated
(after
subtraction
of the leak
on the following
relationship:
g, = IK(EK
Em),
g is the potassium
and
the
pulse
is the same
at the beginning
this
hypothesis
potential
of -50
of potassium
ions
through
the curves
to conductances
current-voltage
the conductance
current)
based
estimates
the results
of a typical
2A is shown
the amplitude
of slow outcurrents
vs. time of depolarization.
There
tail
rise
figure,
analysis
measured
Figure
figure
and
for
amplitudes
cells.
is a correlative
1972)
above
outward
(as
period.
current.
It was hypothesized
that
decay
of slow outward
current
upon
the conductance
underlying
the slow
currents
to the
in
tail
To test
a holding
-32
of tail
current
current
potassium
current
control
all of the
at the end of the depolarizing
underlying
the tail current
to
amplitudes
where
to
reflect
slow outward
current
in this study involved
determining
which
of the
repolarization.
voltage-clamped
to
this
2)
of time
outward
a 2-hr
of slow
mV,
ability
of barbiturates.
accuracy
to flux
convert
slow
elec-
following
current:
1)
-50
periods
83 met
study
than
prolonged
over
giant
initial
the
3) the
for
studied,
for
the
1 megohm,
analysis)
of slow outward
no instantaneous
repolarization;
i.e., that
ward
Upon
4) a stable
current
Aplysia
of 18 months.
which
met
slow
outward
negative
than
potentials
10 sec)
giant
a period
more
greater
of action
by
from
over
observed.
potential
Tail currents
amplitude.
The
the
were
resistance
than
obtained
study
only those
neurons
in the study
of
membrane
input
data
a continuous
impalement,
were
used
cell
the
represent
during
compared
process
in figure
points
experiment
by
slow
subtraction
arrows
points
of the
The
of the tail current
produced
of the
by
i.e. , the
on
including
of approximately
case
1A
contribution
determined.
points.
and the estimates
of the decay
methods
did not differ by more
(two),
the different
current
components)
and/or
of 200 points/cm
magnitude
there
was
lines).
components
was
to be significantly
in the
yielded
currents
(solid
“comparments,”
peeling
techniques
methods
tail
in this
figure
2) the
ofdecay
were
amplitude
changes
with
in
tail
regression,
regression
only
shown
program
Table 1 contains
the results
two curves
in figure
1. The
results
that
and
barbiturate,
of the
analysis
arrows.
increase
(decay
of components
analysis
was similar
to the peeling
used in
1 ) the slowest
component
of decay was deter-
between
a log-linear
in
a
fewest
tail currents
via an analog-to-digital
converter
Scotts Valley, CA) installed
in the computer.
of
effect.
Of the
of the residuals,
current
added
plots
component
there
outward
data
removed
slower
boundary
performing
tional
number
for
and were
of the results of the tail
by the following
formula:
of the squares
is the
with
a log-linear
was
next
was
raw
the
of the
compartmental
slow
and
semi-logarithmic
method
mined
and
of slow
in control
lB
The
in the curve
is the sum
0.092
0.123
the curve.
in
obtained
where
51.7
53.9
of differing
stimuli
experiment.
then
diskettes
measured
6.74
6.00
were
a resolution
fitting
maximal
an
was
( 100 mm) channel
wide
Computer,
(greater
sec
nAmp
In all cases
models
with
between
a low-pass
Concentration-response
resting
Halt-Trnes
1
=
mV
1
-
of
with
collection
trode
criteria
Components
Control
Secobarbital,
are
(that
interval
by
the
tail analysis routine. The results of the analysis for these two curves
shown in table 1 . The stimulus was a 60-sec depolarization
to -34
from a holding potential of -50 mV.
tail.
with
of potentiation
duration
currents
floppy
interval
with
(Mountain
residuals were subtracted from the best fit line of the late time points.
The set of straight lines, as well as the curve fit, were obtained from the
TABLE 1
Results of the analysis
the
Tail
on-line
Fig. 1. Example of the change in tail current produced
by barbiturate.
A,
tracings of slow outward
current obtained
in control and with 1 00 M
secobarbital.
Upper traces are current (I) (up = outward),
lower trace is
a two-component
be obtained
model
effect
on a double
Computer)
on
the
any
conditioned
recorded
1O’V
fit with
fit could
complex
reduce
cases
were
V
least
experiments
to
z
0-
good
823
Current
assumed.
In
4
well
an equally
complexity,
100
of Barbiturate
Kinetics
5
the
conductance
underlying
is the potassium
potential
(-76
mV,
‘K
membrane
potential.
slow outward
current,
EK is the
Russell
and Brown,
After
the
first
10 sec
824
Huguenard
Vol. 234
and Wilson
A
gible
effects
on
tail
kinetics,
i.e.,
the
rate
of
decay
of
each
component.
In order
studying
increments
sponses
4
z
to
tail
determine
the
optimum
holding
holding
potential
around
the resting
obtained
at each
were
an experiment
was depolarized
ED
U
the
currents,
potential,
potential.
potentials
is shown
in figure
4. Here
the giant
to -34 mV from holding
potentials
of -42
A,, whereas
of
decreased
tude
z
0
force.
0
z
U
tion
SflMULUS
DURATION
(Seconds)
upon repolanzation
after increasing
In order to directly compare
tail current,
both
were
for the two
times
84.3
converted
components
of -50
the
rent
is the same
outward
current.
as the
Similar
conductance
Illustration
of the
current.
To determine
calculated
from
calculated
obtained
was added
ofslow
outward
of tail current
on slow
by depolarizing
activation
range
then
measuring
outward
results
are
summarized
outward
plotted
tial,
has
giant
current
which
of tail analysis
vs. the
in figure
of the
fast
current,
has
from
3.
two
and
depolarized
slow
shift
which
the
effect
on
the
for
slow
percentage
of increase
the selective
steep
voltage
increase
dependence
and
the
sea
the
up the tail
Based
to -30
mV),
the amount
components
of tail
components,
the
eliciting
most depolarized
voltage
clamp
Effects
slow component
(-32
mV) than
the
compared
control
level
at each
component
evident.
There
was
between
voltage
control.
that
could
are
tion-dependent,
potential.
negli-
on
analysis
current
and
the
clearly
decreases
are
due
in
to
activated
the
with
mV
the
at the
half-times
different
and
for the
holding
-32
potential
for
the
outward
to
currents.
were
be seen
clearly
sea
water.
This
time of these
experiments
The half-times
of both
were relatively
unaffected
However,
at
was a marked
depicts
several
the tail
voltage
currents
clamp
tail
was
an
(in this case
A2, compared
of quanti-
enhancement
current
in the
phenobarbital
values
have
lower
slow
in the
reversal
of 2 hr
is much
slower
high concentrations
slowing
in the
slower
panel.
than
component
the
is
in
been
it
a concentraof tail
bath
effects
control
turnover
components
concentra-
(greater
than
component.
a semi-logarithmic
A2.
by
Here
of barbiturate
of wash
with
(less than
5 mm).
the fast and slow decay
by changes
in barbiturate
on
ef-
specific
phenobarbital).
to A1, that
increase
currents
barbi-
of slow outward
the major
effect
produces
were produced
depolarization
Several
as a means
of
of
pentobarbital,
The
general
phenobarbital
the slow
a period
or the
loss
component
of tail delay,
course
of effects
produced
of control
selective
Notice
over
used
concentrations
5. These
raw
that
mV,
bar-
without
and then the
be discussed.
barbiturates
slower
the time
increasing
of figure
first,
will
slow
-30
be obtained
tail
in a typical
experiment
marked
increase
in
current
decay.
that
is obtained
tion.
there
magni-
seen
The
and enhancement
In nearly
every
case,
other
the
to percentage
can
in
to
be
holding
will be presented
with each compound
of tail
with
panel
(A1
(A2), and the
were
voltage
amplitude
of
5 demonstrates
obtained
the upper
is best seen
in figure
3B
have been
normalized
can
current
the
at -50
pentobarbital
barbiturate
Notice
the
to the
to increase
examined
in this
study,
including
secobarbital
and amobarbital.
results
artificial
current
cell
mV.
outward
set
slow outward
by barbiturates.
and
of
of tail
fast
-45
findings,
was
of barbiturates
turates
were
phenobarbital,
of the
Figure
poten-
tail
the
depolarizations
slow
above
current
was
the
100 zM pentobarbital
of both components,
but
of the slow
is clearly
A (holding
is marked
This
amplitudes
above
respectively)
in part
solutions,
activation
current
the
experiments
of
current
within
to
in
potassium
current.
on
The
voltage-
-60
are
the
In figure
4B are shown
of tail decay
obtained
fects ofbarbiturates
results
obtained
characterized.
then
these
magnitude
further
steady-state
biturate
tating
current
as
amplitude
mV resulted
As was generally
the case in this study,
the alterathe
tail
current
were
produced
by changes
in the
but not the kinetics
of exponential
decay which
make
normalized
component
in the fast component.
the tail component
Here
cur-
developed
at each
potential.
a representative
experiment
potential
-50 mV).
In control
a higher
threshold
were
current
(-40
to determine
component
(-36
mV). The addition
appears
to shift the voltage
sensitivity
the
tail
from
the slow
in experiments
outward
to potentials
cells
of slow outward
the tail currents
The amplitudes
and A2 are the
the
voltage
dependence
of slow outward
the optimum
parameters
for measuring
of barbiturates
slow
The
±
to the artificial
current
development
development.
dependent
onset
and decay
of this process
The voltage
dependency
of onset
of slow
determined
4.9
to -36
in the
holding
of some slow outward
current
at the holding
potential
3). The
tail currents
will thus
reflect
the difference
the slow outward
current
activated
by the depolariza-
potentials.
tions
of
amplitude
half-
0.3 and
mV from a holding
were
was -32
conductance
results
were
when
100 iM pentobarbital
water,
i.e., the time course
paralleled
the time course
effects
potential
current and
average
mV.
of depolarization
the
in B. The
this experiment
in
of depolarization.
of slow outward
to conductance
4.6 sec. The depolarized
±
potential
durations
the magnitude
of
with
in
neuron
R2
in the range
in decreased
of -40
1985),
for
in small
in amplitude
depolarized
by depolarizing
range
seen
resulted
Inasmuch
increase
holding
potential.
two components
Fig. 2. Onset of slow outward current compared to the amplitude of tail
current. A, 0 are the slow outward current (SOC) with increasing duration
of depolarization.
#{149},total
amplitude
of tail current (A1 plus A2) which is
obtained
voltage
activation
(ci. fig.
between
0
et at.,
This
magnitude
0;
of A2.
be maximized
the
in
4
mV
potentials
(Huguenard
can
driving
U
to -36
holding
amplitude
currents
varied
and tail current
reAn example
of such
of -60
to -36
mV. Both
A, and A2 increase
range
of -60 to -44 mV. However,
further
B
potential
was
200 zM)
Figure
6
plot.
by the same
stimulus,
to -34
mV from a holding
All
a 60-sec
poten-
of
Kinetics
1985
A
of Barbiturate
825
Current
B
25
20
0
CONTROL
.
PENTOBARBITAL
120(
-j
OA1
I-
C
15
#{149}A2
1000
10
0
I-
800
z
5
Ui
Q.
I
0
600
IUi
10
C,,
8
400
Ui
C.)
z
6
200
4
-38
2
-36
-34
DEPOLARIZED
0
-38
-36
DEPOLARIZED
-34
-32
POTENTIAL
-32
POTENTIAL
-30
(mV)
-30
(mV)
Fig. 3. Voltage sensitivity of the onset of two components
of tail current; alterations by 100 MM pentobarbital. A, the amplitude of the fast component
(A1) and the slow component (A2) of tail current are plotted against the depolarized potential. B, the changes produced by pentobarbital
are
normalized
by plotting them as the percentage
of increase
both components,
but the effect on A2 is more pronounced.
±
3.2
sec. Holding potential,
above control levels. Pentobarbital
is shown
Average half-times
of the two components
to alter the voltage dependence
of onset of
in this experiment
were 8.2 ± 0.4 and 69.8
50 mV.
A
w..
4
ED
I-
B
.
z
w
z
600
50
0.
500
4Q
0
400
Qu
300
:::
30
.
20
0
__j
200
100
4
E
‘2
.o0_
I
50
HOLDING
I
I
I
0
1
2
POTENTIAL
Fig. 5. Time course of phenobarbital
off. Control responses
were obtained,
mV)
Fig. 4. Dependence
of tail current
on holding
potential.
The membrane
holding potential was set at various levels between
-60 and -36 mV
of
25 and 50 zM phenobarbital
#{149}
3
TIME
-40
I
#{149}
(B),
I
#{149}
4
I
#{149}
5
I
6
(hours)
effect during wash-on
followed by sequential
and washapplication
and then wash. In the upper panel
and tail currents were obtained after eliciting slow outward current with
a depolarization
to -32 mV. A, amplitudes of the two components of tail
current decay vs. holding potential. B, half-times of the two components
the amplitudes
of the two components
of tail current (A, and A2) during
the course of the experiment
have been plotted. In the lower panel these
values have been normalized
to percentage
of control. The average halftimes of the two components
in this experiment were 8.6 ± 0.4 and 80.9
of tail current
the parameters
± 6.5 sec. The stimulus
was a 60-sec depolarization
holding potential of -50 mV every 12 mm.
decay vs. holding potential.
The open symbols
(amplitude
and half-time) ofthe fast component
represent
of decay,
whereas the closed symbols represent the parameters
of the slow
component of decay. The depolarized potential in this experiment was
-32
mV.
tial
of -50
Table
mV.
Increasing
the
concentration
of pentobarbital
up to 200 tM
results
in a selective
increase
in A2, with
effect
on kinetics
or amplitude
of A,. However,
when
barbiturate
concentration
was increased
to 400 jiM, there
marked
prolongation
of the slow component.
Also,
with
centrations
there
of barbiturates
that
slowed
the
tail
current
the
little
the
is a
con-
kinetics,
was
generally
an
2 is a summary
single
Again,
experiment
it can be
pentobarbital
a slight
are
slowing
outward
of the
shift
results
in
shown
in figure
6.
seen
that
the major
concentration
of the
kinetics
dependent
with
the
of the
higher
to -33 mV from a
holding
tail
current.
analysis
changes
increases
from
induced
by
in A2, and
concentrations
of
pentobarbital.
Most
experiments
involving
barbiturate
enhanced
tail
cur-
826
Huguenard
and Wilson
Vol. 234
ysis
each
for
outward
time
then
4
interval.
These
the alterations
sec).
The
major
increase
With
The
sec
similar
35-day
18
the
± S.D.),
rates
was
However,
was much
A1 in control
two
50%
cases
was
the
In
sponses
major
increase
there
hyperpolarized
was
firing
to
60%
was
a good
the
5 sec)
an
the
of
-60
to
a decrease
rate,
in
in
response
-90
on
leak
mV.
firing
a prolonged
described
decay
tions.
onset
kinetics
Thus,
kinetics
above,
to directly
measure
we devised
experiments
current
TABLE
Results
the
analysis.
depolarizations
barbiturates
had
of slow outward
current
we examined
the effects
of slow outward
current.
of
onset
kinetics
to study
Slow
increasing
very
little
outward
duration,
outward
kinetics
current
and
induced
experiments
analysis
shown
although
range
typical
in which
this
sigmoidal
effect
shape
of effects
curve
for
occurs
within
the concentration
previously,
by
was little
tested.
pentobarbital
The
effects
on
A,
order
of
response
concentration
effect
of 3.2
there
was
until
very
little
across
the
of phenobarbital
concentration
that
there
very
one
little
amounts
secobarbital
every
these
of
for this
effect
on A,.
entire
compared
to control,
two-sided
t
concentration
two compounds
the
stereoisomers
In every
where
were
For
tested
curves
effect
the
in
shown
on the
effects
the R
compared
of
pentobarbital
experiment
on
(+)-
and
S
for activity
and
(n = 5), and
at
(-)-isomers
in enhancing
of
6
Half-limes’
2
1
4.76(0.57)
1.01 (0.16)
3.72(0.38)
6.87(0.44)
6.74 (0.37)
5.72 (0.21)
6.33 (0.22)
2.18(0.17)5*
3.30 (0.32)5*
3.47 (0.20)**
4.88 (0.13)**
4.94(0.47)
6.32 (0.60)5*
6.93 (0.74)’
6.85 (0.40)5*
test.
con-
concen-
were
response
is a saturable
study.
riAmp
#{149}#{149}
P < .001
effect
high
n
a Average and (SE.M.).
is
activated
used.
Also, it should
be noted
that
by pentobarbital
was somewhat
barbiturates.
With
most
com-
effect
In the
be seen
Ampktudes’
Control
Pentobarbital
50MM
100 MM
200 MM
400 pM
was
that
receptor
Components
1
a
tested.
and A2 is somewhat
of a saturable
As mentioned
of A2, with
limited
using
in figure
400
A2, the best fit curve was obtained
with an apparent
Kf) of 115
,M and the maximum
increase
was 3.02 nA (not shown).
Stereoisomers
of barbiturates.
We were able to obtain
was elicited
with
the results
of anal-
from the experiment
as A2.
pentobarbital
of A, shows
2
of tail current
sharp
curve
is rather
steep,
and rather
than
to maximum
effect
within
two orders
of
constant
there
range
amplitude
current,
indirectly
rate
experiments.
in figure
9 it can
at moderate
concentraof barbiturates
on the
Because
it was difficult
of slow
these
shown).
nine
on the
eight
amplitude
of A2 vs. pentobarbital
with a KD of 98 iM and a maximum
Phenobarbital.
as
constant
effect
the
(>200
pounds,
tration
stimulus.
As
from
zM) were
the increase
in A1 produced
atypical
compared
to other
con-
rate,
either
the
a
Barbiturates
plot
the full range
The
best
fit
centrations
conductance
Under
these
initial
the
to
nA (not
effects
effects
on
with
high
in the
slope
shows
of
of depolariza-
in A2.
data
were
obtained
for
in figure
8. The amplitude
a log-linear
curve
of the
was obtained
re-
between
and
increase
by a steeper
magnitude,
magnitude.
that
time
enhancement
of A2. At
there
is approximately
increase,
curve
with
and
of A1 as well
the concentration
between
concentration
process.
However,
going
from
minimum
rather
adaptation
This
obtained
control,
studied.
is a barbiturate
increase
results
response
depicted
saturable
within
The relationship
clearer.
of
of
an enhancement
correlation
first
not
In
A,,
is the
example,
a 1000%
The
is
there
of Individual
slow
corresponding
in
were
with
of development
a concentration-dependent
increase.
current,
frequency
was
region
there
late
±
A2 rises
in A, but
concentration
are graphically
by barbitu-
tails.
There
were
minimal
0 and
10 sec).
However,
there
as
50
of tail
was
(after
current
(between
cell,
a
spike
as evidenced
current
tail
which
adaptation
circumstances
over
a
1.10
nA
pentobarbital
however,
for
of the
the
of such an experiment.
This
changes
in tail component
depolarizations
(up to 740
of
rate
a measure
during
were
obtained
by barbiturate
depolarizations
Effects
as
at which
of the
Pentobarbital.
2. For
The amplitude
maximum
effect
of barbiturates
of the
the
well
was
of barbiturates,
ductance
was
enhancement
component
A2.
obtained,
on slow outward
fast adaptation
in
effect
in
slow
table
conducted
over control.
nA, and the
parameter
of the fast
component,
were
doses
maximum
increase
± 1.61
3.76
this
experiments
enhanced
in
the range
of 400 to 500%
of the control
value.
experiments
(n = 3), the enhancement
of A2
than
average,
and sometimes
the effect
was
on
of amplitude
than
the slow
shown
A2 in control
the
as a 2000%
barbiturates
those
experiments
amplitude
and
within
in few
greater
as striking
to
representative
period,
(average
results
long
major
effect,
of depolarization,
Specific
very
in
used
developed
responses
produced
rate
enhancement
Fig. 6. Semilog plots of tail currents
obtained
in control
and with several
concentrations
of pentobarbital.
The stimulus
was a 60-sec depolarization to -30 from a holding potential
of -50 mV. The results of tail
analysis for the curves in this figure are given in table 2.
example,
was
had
effect
in the
tion.
(Seconds)
TIME
had
current
which
In figure
7 are shown
the results
figure
shows
the time-dependent
amplitude
obtained
with prolonged
U
rent
tail
current
2
sec
57.1
(3.7)
6
69.2(17.9)
67.9 (20.6)
99.9 (14.8)
147
(12)5*
4
6
5
6
1985
Kinetics
of Barbiturate
827
Current
10 r-1tA
4
7,
6
0
5c
0
:o
400
600
T,me of Depolorizoton
4
a
4
2
(Sec)
Fig. 7. Onset kinetics during depolarization;
effects
of pentobarbital
(Pento) compared
to control. The stimulus
was depolarization
to -30
mV from a holding potential of -50 mV. [Pento] = 100 MM. The average
half-times
of the two components
in this experiment
were 7.9 ± 0.3 and
66.3 ± 3.6 sec.
0
0
0
10
1000
100
4
4
0
0
ED
a
4
4
0
C’
100
0
CONC
1000
PHENOBARBITAL
(,tM)
(CONC)-response
relationships for phenobarbital
the amplitude of the two components
of tail current. The
error bars represent ± S.E.M. , n = 9 neurons. Linear regression of the
CONC-response
curves yielded slopes of 0.1 1 (A,) and 2.49 (A2) nA/bFig. 9. Concentration
4
alteration
of
fold change
in phenobarbital
0.0009 (not significant)
CONC.
and 0.40 (P
The correlation
coefficients
were
.001), respectively.
<
4
be
seen
that
the
concentration
4
tude
0
of the
the
0
one
slow
4
analysis
in
these
CONC
PENTOBARB)TAL
(NM)
Fig. 8. Concentration
(CONC)-response
relationships
for alteration
amplitude
of the two components
of tail current by pentobarbital.
error bars represent
± S.E.M.;
n = 8 neurons. Linear regression
of the
The
of the
CONC-response
curves yielded slopes of 2.77 (A1) and 3.23 (A2) nA/i 0fold change in pentobarbital
concentration.
The correlation coefficients
were 0.57 (P < .002) and 0.31 (P < .002), respectively.
current,
the
S (-)-isomer
(+)-isomer.
Figure
which
outward
secobarbital
current
and
in
two
is
stereoisomers
little difference
changes
pears
the
responses
and
tration.
tail
Figure
results
Figure
responses
current
lOB
graded
are
shows
for the same experiment.
can easily
be seen with
seen
the
The
bital
of tail
from
the
components
experiment
than
from
an experiment
1OA shows
obtained
with
in both
each
adaptation
shown
the
100
there
M
stereoisomers
in figure
10. Here
apoutward
obtained
stereoisomer
At each
effective
than
firing.
Figure
for the am-
of secobarit can
easily
at
In a few
the
experiments,
appeared
every
amplisuch
as
to be affects
on
the
elicited
average
spike
interspike
train.
poten-
Stronger
depolari-
zations
resulted
in loss of membrane
incomplete
space clamp,
and therefore
potential
in these
control
due to
cells the slow
outward
in voltage
clamp
current
perfectly
prolonged
of the
the
train.
barbiturate
produced
membrane
With
the
effect
that
minimum
3),
effect
of the
with
it is not
tail
clamp
the
tail
minimum
of amobarbital
of experiments
not determined.
results
and concentration-response
In general,
both of these
more
similar
is, in addition
ment
in
of A,.
one
amplitude
ments
with
of the
with
increase
In three
of two
to control.
to pentobarbital
to the
This
of five
fast
component
compares
pentobarbital,
there
of greater
to increases
zero
of nine
and
a limited
seco-
number
phenobarbital.
there
experiments
amobarbital,
the
relationships
were
compounds
produced
than
in A,,
that
concentra-
in only
were
were
and
effective
tested
currents
be
augmenta-
effects
tail
in
It should
current
barbital
a
result
depolarizations
tion for effects on adaptation.
Other
barbiturates.
The
on
not
that
would
studied,
for
did
during
dependence
surprising
current.
neurons
concentrations
coincided
activated
voltage
1 or 2 mV)
on
voltage
majority
effective
generally
the
sufficient
in the
fig.
(by even
in observing
obtained
currents
very strong
(see
depolarization
difficulty
tion
activation
model
spike
emphasized
in concen-
responses
was more
of sustained
relationship
R
the slow
with
the
slow
increase
Here the effects
of either
each increase
in concentration.
with
the
S (-)-stereoisomer
increases
concentration
tested,
the S (-)-isomer
the R (+)-isomer
in the inhibition
11 shows
the concentration-response
plitude
effective
With
the R (+)-isomer,
10 and 30 tM,
but
markedly.
potent;
more
typical
was tested.
tail current
of secobarbital.
between
control,
to be more
current
10 shows
was
effective
which
had no apparent
effect
on
lack of sensitivity
of tail current
most
likely
due to the inability
to
This
was
the
more
at enhancing
11, there
to simulate
during
insufficient
tail
cases
sufficiently
achieved
(A2).
in figure
at concentrations
parameters.
depolarize
tial
component
depicted
adaptation
tail current
0.
S (-)-isomer
was
the R (+)-isomer
than
was
with
was
some
That
enhance-
secobarbital,
and
an increase
in the
than
50%
in three
experiments
compared
of five
experiwith
di-
828
Huguenard
Vol. 234
and Wilson
(+)
A.
( -)
04
___
J-___
4
0
0
ED
a
4
4
4
0
0
ED
B.
a
4
.-.
.-
-:
‘II
‘‘
0-_.
-I
CONC
SECOBARB
),M)
Fig. I 1. Concentration
(CONC)-response
relationship
for tail component
amplitudes
obtained with stereoisomers
of secobarbital.
Data from same
experiment
in figure 10. (0, S (-)-secobarbital;
#{149},R (+)-secobarbital.)
The error bars represent
± S.E.M.
of each value from within a single
experiment
in which five tail currents were obtained with each CONC.
outward
currents
least
0
cells
-
l
50
f
4
Fig. 10. Effects
of stereoisomers
of secobarbital
on slow outward
current
and adaptation. A, slow outward
current
responses
in control
10, 30 and 100 pM of the R (+)- and S (-)-isomers
of secobarbital.
trace
is voltage,
60-sec
lower
depolarization
trace
is current.
The stimulus
to -30 from a holding
20 mV, 10 nA; horizontal
and
with
Upper
in this case was a
potential
of -50
mV.
Calibration:
vertical =
= 30 sec. B, alterations
in adaptation
responses
produced
by stereoisomers
of secobarbital.
The
change in adaptation
can be seen most readily as the decreased
duration
of firing in response
to the constant
current pulse. The entire series of
responses was performed for (+)-secobarbital,
then a wash was obtained
(0 pM (-)-secobarbital),
and the series was repeated with the different
concentrations
of (-)-secobarbital.
Concentrations
(micromolar)
shown
to the left of the traces. The neuron died before it was possible to obtain
an adaptation
response
in 1 00 MM (-)-secobarbital.
The adaptation
stimulus was 50 nA of depolarizing
current in current clamp after being
held at a -50 mV in voltage clamp. Calibration:
horizontal
=
5 sec;
vertical = 20 mV.
phenylbarbituric
nine
acid
experiments
(Huguenard
with
and
Wilson,
1985)
and
zero
phenobarbital.
outward
number
hippocampal
currents
of vertebrate
pyramidal
have
systems,
cells
been
demonstrated
including
(Brown
and Wilson,
1982),
rat hippocampal
Barker,
1984),
cat spinal
motoneurons
and
mouse
Although
are much
cells
to
of no
regulate
have
and
(Moolenaar
recently
guinea-pig
Griffith,
and
been
effects
tation
of inhibitory
1983;
and
sant
and
Spector,
the slow outward
currents
in these
vertebrate
faster
(usually
less than
1 sec to peak)
than
the
point,
selective
doses
and
which
Barker,
Nevertheless,
we
barbithe augmenreduction
in
exert
1978,
no
1979;
it remains
most
It is
current
would
hypotheses
anticonvulsant
and
at
at
however,
include
responses
are
currents.
Alternative
selective
(Macdonald
1981).
unclear
important
for
GA-
Schulz
which
anticonvul-
action.
In
this
analysis
was
study,
undertaken
frequency
foundly
Using
because
by
means,
it has
this
specific
current,
i.e.,
of
the
measured
of GABA
esthesia
gett,
of
in mice
validated
outward
in neurons,
barbiturates
been
the
slow
slow
increase
is
enhanced
(Huang
This
current
affects
spike
that
barbiturates
stereospecific
in other
responses
in cultured
and
current
current.
result
describing
of the
with
the
secobarbital
spinal
Barker,
1980;
outward
current
S
(-)-
more
stereospecificity
(fig.
systems,
in
the
slow
being
This
adaptation
responses
of tail
in one parameter
in the amplitude
pentobarbital
and
R (+)-compounds.
the
use
outward
and this
process
is so pro(Zbicz
and Wilson,
1981).
shown
alteration
an increase
This
logically
ment
have
of quantitating
adaptation
enhanced
a relatively
tail
we
as a means
rat
cells
slow
for
of barbiturates
that
1979).
At this
phenobarbital)
GABA
Macdonald,
matches
Brag-
forward
actions
slow
sensitive
outward
nervous
system
which
a current.
excitation
BAmimetic
relatively
firing.
(especially
glutamate-mediated
properties
these
barbiturate
central
of such
put
turate
in a
cells in culture
(Segal
(Barrett
et at., 1980)
by
repetitive
reports
stereoisomers
potent
than
don
and
neuroblastoma
affected
know
in this
study
(increasing
over
the firing
behavior
of vertebrate
see
a similar
vertebrate
serve
that
presented
fig. 7),
sec,
that
in the
component.
Discussion
Slow
of
is clearly
possible
exists
nA S,msIohon
---------
00
of
lOs
10)
and
such
physio-
as enhance-
neurons
Waddell
and
and
anBag-
1973).
The
as well
approaches
enhancement
as being
of slow
stereospecific,
a maximum.
by
barbiturates,
is concentration-dependent
Thus,
it appears
that
and
slow
outward
Kinetics
1985
current
enhancement
phenomenon
rather
is a receptor-mediated
steepness
of the
coefficient
there
response.
is the
may
activity
A further
partition
with
anesthetic
values
1.95,
were
1.42,
determined
the
(of
strong
slow
correlation
The
clearly
by all barbiturates
two that are less sedative,
acid.
We
rent
is less
effect
conclude,
sociated
sibly
some
well
and
sedative
anticonvulsant
correlated
are
(Raines
in mice
et al.,
of slow
and
1975,
outward
1979),
current
but
depressant
rather
The
but
reasons
to
for
which
(Huguenard
depressant
shows
potent
and
this
activ(oil-water
general
is an effective
is very
is as-
pos-
enhancing
of barbiturates
related
cur-
barbiturates,
current
acid
rats,
outward
a general
effects.
a property
which
is produced
slow
of the
1) slow outward
2) diphenylbarbituric
action
ment
actions
a
this
studied,
including
diphenylbarbituric
enhanced
with
within
current
been
and
same
not
0.68).
=
actions,
neuronal
coefficient)
action
sant
other
of the
is not
that
to be associated
are 2-fold,
partition
that
have
phenobarbital
order
was
properties
outward
to sedative/anesthetic
with
conclusion
ity
likely
leading
there
(r
(1967)
rank
little
or no
anticonvul-
in the
enhance-
Wilson,
1985).
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Medical
reprint
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requests
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to: W. A. Wilson,
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