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Development of acute tolerance to pentobarbital: Differential effects in mice
Biochcmicai Pharmacology, Vol.28,PP.549-552. OPergamonPress Ltd.1979. Printed in Great Britain
DEVELOPMENT
OF ACUTE TOLERANCE
TO PENTOBARBITAL:
DIFFERENTIAL
EFFECTS
IN MICE
Arthur W.K. Chan and Albert J. Siemens Research
Institute
on Alcoholism,
1021 Main Street,
(Received 26 October
Recently,
[l-3].
These mouse
of ethanol
different
[l-3, 5-71.
rather than water, whereas
shown that the C57BL strain and Ritzman
dose of ethanol higher
from the phenomenon
ethanol
showed qreater portion
impairment
portion
between
by Mellanby
of acute tolerance
the two mouse
[S].
In contrast,
acute tolerance brain ethanol
reflex.
to the
we have [Z].
to an acute
level was
This was taken as
The term acute tolerance
that dogs receiving
was
a single dose of
concentration
during
the
reached
[9]. designed
to ascertain
in C57BL mice was specific
such as pentobarbital.
strains was specific
[4]
solutions
curve than at the same concentration
of the curve
and pento-
It has been demonstrated
to ethanol.
at a given blood ethanol
report deals with studies
occur with other hypnotics
to drink ethanol
In C57BL animals,the
systems
fCNSf sensitivity
than DBA mice to pentobarbital
of acute tolerance observed
as model
intake of alcohol
system
that C57BL mice exhibited
of the blood ethanol
during the descending The present
than DBA animals
14203, U.S.A.
of action of ethanol
central nervous
than at the loss of the righting
derived
development
1978)
in their voluntary
C57BL mice prefer
but DBA mice did not.
for the development
ascending
and mechanisms
is more sensitive
f8] reported
at the recovery
evidence
6 November
DBA mice prefer water over ethanol.
that C57BL mice are less sensitive
Tabakoff
effects
strains differ
and have been shown to possess acute effects
New York
two strains of mice, C57BL and DBA, have been utilized
in studies of the pharmacological barbital
1978: accepted
Buffalo,
the selective
tc ethanol or whether
A demonstration
for ethanol
whether
it would
that the difference
would further
support
the use of
550
Preliminary Communications
these two strains
in studies
Male C57BL/63 Laboratories,
and DBA/ZJ mice
Bar Harbor,
cycle in a controlled Mills, Winfield-
(sleep onset)
weighed
pentobarbital-2-14C
the PROPHET Institutes
Boston,
Mass.)
Procedures
concentrations system,
T&lad
mouse
national
at those
2 LiCi of sodium pento
as described
reflex was
times and brains buffer,
and determination
resource
in saline at
at the time the riyhting
of 0.1 M acetate
and statistical
(Teklad Each
to the experiment.
samples have been described
computer
diet
as a 0.25% w/v solution
for the extraction
and brain
light/dark
at the time the righting
in 2 volumes
were computed
a unique
group
(50 ;il) was collected
and homogenized
in blood
from the Jackson
time and sleep time were determined
and another
Blood
10% w/v NaCl.
Pentobarbital
(22-23O) and received
ii.p.) with approximately
Nuclear,
Sleep onset
(awakening).
contained
room
on ethanol.
singly on a 12/12-hr
A group of mice of each strain was decapitated
[2].
immediately,
They were housed
environmental
(New England
reflex was lost regained
dependence
(11-12 weeks old) were purchased
intraperitoneally
a dose of 50 mg/kg. previously
Maine.
to and physical
Iowa) and tap water -ad lib. for 7 days prior
mouse was injected barbital-2-1'C
of tolerance
pH 5, which
of unchanged
previously
analyses
sponsored
were removed
[Z, lo].
were performed
using
by the National
of Health.
The blood and brain for the two strains
levels of pentobarbital
of mice are shown in Tables
DBA mice awoke at significantly
higher
blood
levels than C57BL mice.
This is in agreement
mice were more sensitive
than the DBA strain
Table
1.
Blood Levels
at the onset of sleep and at awakening 1 and 2
(Table 1) and brain with our previous to pentobarbital
of Pentobarbital
C5?RL/63
onset
*
It is seen that
(Table 2) pentobarbital conclusion
that the C57BL
[Z].
at Sleep Onset and Awakening
Blood Pentobarbital Mouse Strain
respectively.
lig/ml i S.E.M. Awakening
(N = 12)
34.64 _' 1.35
15.32 -c 0.61
DBA/ZJ * (II= 12)
33.03 _c 0.67
20.54 i L 0.64
_.. * Sleep onset times Sleep
time:
+ Siqnifieantly
(min ? S.E.M.):
C57BL,
C57BL,
3.88 F 0.07. DBA, 3.95 ? 0.07,
73.56 + 5.36; DBA, 55.32 t 5.02.
different
(p < c1.001) from CS7EJ, mice.
Preliminary Communications For DBA mice the brain pentobarbital concentrationat awakening (Table 2) was significantlyhigher than at sleep onset, indicating the development of acute tolerance. In contrast, C57BL mice awakened at a significantly lower brain pentobarbital level than that at sleep onset, suggesting no acute tolerance development. This is directly opposite to the reported [8] response to ethanol.
Table 2.
Brain Levels of Pentobarbital at Sleep Onset and Awakening
Brain Pentobarbital uy/y wet wt
* S.E.M.
Mouse Strain
Onset
C57BL/6J (N = 12)
31.44 f 0.75
26.95 * + 1.25
DBA/2J (N = I.21
30.88 _+0.54
37.25 *-I i:1.34
Awakening
* Significantly different (p 5 0.005) from levels at onset of sleep. i Significantly different (p ( 0.001) from C57BL mice.
The lower brain tkan blood levels of pentobarbital in both strains of mice at the time of onset, and, conversely, the higher brain than blood concentrationsof the drug at awakening are probably related to drug distributional factors. It has been reported that C57BL mice, which have a lower brain sensitivity than the DBA strain to the acute effects of ethanol, showed development of acute tolerance to ethanol [S]. We have now shown that with respect to pentobarbital, only the DBA mice, which have a lower brain sensitivity to the acute effects of the drug, developed acute tolerance. Thus, the development of acute tolerance to a drug might be related to the degree of CNS sensitivity to the drug. However, two strains of mice, the long-sleep (LS) and short-sleep (SS), which were selectively bred (not inbred) for their differences in sensitivities to the hypnotic effects of ethanol [ll, 121, both did not develop acute tolerance to ethanol [8]. These two strains of mice showed no difference in sensitivity to the hypnotic effects of pentobarbital [2]. Due to the unavailability of the LS and SS mice, we could not perform the above experiments with these animals. Nevertheless, our present results support the use of C57BL and DBA mice as animal models to determine the relationships between voluntary ethanol intake and neural sensitivity to the acute effects of ethanol, and the rates of development of tolerance to and physical dependence on ethanol. This work is currently in progress.
Acknowledgements: The autkors express their sincere appreciation to Olivia Doyle and Patrick George for excellent technical assistance.
Preliminary Communications
552
REFERENCES C.R. Randall
and D. Lester,
A.J. Siemens
and A.W.K.
A.W.K.
J. Pharmacol.
Chan,
Life SCi. 19, 581 (1976).
Chan, Life Sci. 19, 597
4.
D.A. Rodgers,
5.
I?. Kakihana,
6.
C.W. Schneider, Psych. E,
Psychosomat. D.R. Brown,
466
(1976).
Med. 2&, 498 G.E. McClearn
S.K. Evans,
Exp. Ther. 188, 27 (1974).
(1966). and I.R. Tabershaw,
M.B. Chenoweth
Science
154,
and F.L. Beman, J. Camp. Physiol.
(1966). &
(1973).
7.
C.W. Schneider,
8.
B. Tabakoff
and R.F. Ritzman,
9.
E. Mellanby,
Med. Res. Comm.
P. Trzil and R. D'Andrea,
Phannacol.
Pharmacologist (Gt. Brit.)
20, 193
Biochem.
Behav. 2, 549
Spec. Rep. Ser., NO. 31, London
E. Rubin, H. Gang, P.S. Misra and C.S. Lieber,
Amer. J. Med. 49, 801
11.
G.E. McClearn
2, 409
12.
W.D.W.
and R. Kakihana, V.G. Erwin,
Behav.
Genetics
S.M. Anderson
(1974).
(1978).
10.
Heston,
1574
(1919).
(1970).
(1973).
and H. Robbins,
Life Sci. 14, 365 (1974).
DEVELOPMENT
OF ACUTE TOLERANCE
TO PENTOBARBITAL:
DIFFERENTIAL
EFFECTS
IN MICE
Arthur W.K. Chan and Albert J. Siemens Research
Institute
on Alcoholism,
1021 Main Street,
(Received 26 October
Recently,
[l-3].
These mouse
of ethanol
different
[l-3, 5-71.
rather than water, whereas
shown that the C57BL strain and Ritzman
dose of ethanol higher
from the phenomenon
ethanol
showed qreater portion
impairment
portion
between
by Mellanby
of acute tolerance
the two mouse
[S].
In contrast,
acute tolerance brain ethanol
reflex.
to the
we have [Z].
to an acute
level was
This was taken as
The term acute tolerance
that dogs receiving
was
a single dose of
concentration
during
the
reached
[9]. designed
to ascertain
in C57BL mice was specific
such as pentobarbital.
strains was specific
[4]
solutions
curve than at the same concentration
of the curve
and pento-
It has been demonstrated
to ethanol.
at a given blood ethanol
report deals with studies
occur with other hypnotics
to drink ethanol
In C57BL animals,the
systems
fCNSf sensitivity
than DBA mice to pentobarbital
of acute tolerance observed
as model
intake of alcohol
system
that C57BL mice exhibited
of the blood ethanol
during the descending The present
than DBA animals
14203, U.S.A.
of action of ethanol
central nervous
than at the loss of the righting
derived
development
1978)
in their voluntary
C57BL mice prefer
but DBA mice did not.
for the development
ascending
and mechanisms
is more sensitive
f8] reported
at the recovery
evidence
6 November
DBA mice prefer water over ethanol.
that C57BL mice are less sensitive
Tabakoff
effects
strains differ
and have been shown to possess acute effects
New York
two strains of mice, C57BL and DBA, have been utilized
in studies of the pharmacological barbital
1978: accepted
Buffalo,
the selective
tc ethanol or whether
A demonstration
for ethanol
whether
it would
that the difference
would further
support
the use of
550
Preliminary Communications
these two strains
in studies
Male C57BL/63 Laboratories,
and DBA/ZJ mice
Bar Harbor,
cycle in a controlled Mills, Winfield-
(sleep onset)
weighed
pentobarbital-2-14C
the PROPHET Institutes
Boston,
Mass.)
Procedures
concentrations system,
T&lad
mouse
national
at those
2 LiCi of sodium pento
as described
reflex was
times and brains buffer,
and determination
resource
in saline at
at the time the riyhting
of 0.1 M acetate
and statistical
(Teklad Each
to the experiment.
samples have been described
computer
diet
as a 0.25% w/v solution
for the extraction
and brain
light/dark
at the time the righting
in 2 volumes
were computed
a unique
group
(50 ;il) was collected
and homogenized
in blood
from the Jackson
time and sleep time were determined
and another
Blood
10% w/v NaCl.
Pentobarbital
(22-23O) and received
ii.p.) with approximately
Nuclear,
Sleep onset
(awakening).
contained
room
on ethanol.
singly on a 12/12-hr
A group of mice of each strain was decapitated
[2].
immediately,
They were housed
environmental
(New England
reflex was lost regained
dependence
(11-12 weeks old) were purchased
intraperitoneally
a dose of 50 mg/kg. previously
Maine.
to and physical
Iowa) and tap water -ad lib. for 7 days prior
mouse was injected barbital-2-1'C
of tolerance
pH 5, which
of unchanged
previously
analyses
sponsored
were removed
[Z, lo].
were performed
using
by the National
of Health.
The blood and brain for the two strains
levels of pentobarbital
of mice are shown in Tables
DBA mice awoke at significantly
higher
blood
levels than C57BL mice.
This is in agreement
mice were more sensitive
than the DBA strain
Table
1.
Blood Levels
at the onset of sleep and at awakening 1 and 2
(Table 1) and brain with our previous to pentobarbital
of Pentobarbital
C5?RL/63
onset
*
It is seen that
(Table 2) pentobarbital conclusion
that the C57BL
[Z].
at Sleep Onset and Awakening
Blood Pentobarbital Mouse Strain
respectively.
lig/ml i S.E.M. Awakening
(N = 12)
34.64 _' 1.35
15.32 -c 0.61
DBA/ZJ * (II= 12)
33.03 _c 0.67
20.54 i L 0.64
_.. * Sleep onset times Sleep
time:
+ Siqnifieantly
(min ? S.E.M.):
C57BL,
C57BL,
3.88 F 0.07. DBA, 3.95 ? 0.07,
73.56 + 5.36; DBA, 55.32 t 5.02.
different
(p < c1.001) from CS7EJ, mice.
Preliminary Communications For DBA mice the brain pentobarbital concentrationat awakening (Table 2) was significantlyhigher than at sleep onset, indicating the development of acute tolerance. In contrast, C57BL mice awakened at a significantly lower brain pentobarbital level than that at sleep onset, suggesting no acute tolerance development. This is directly opposite to the reported [8] response to ethanol.
Table 2.
Brain Levels of Pentobarbital at Sleep Onset and Awakening
Brain Pentobarbital uy/y wet wt
* S.E.M.
Mouse Strain
Onset
C57BL/6J (N = 12)
31.44 f 0.75
26.95 * + 1.25
DBA/2J (N = I.21
30.88 _+0.54
37.25 *-I i:1.34
Awakening
* Significantly different (p 5 0.005) from levels at onset of sleep. i Significantly different (p ( 0.001) from C57BL mice.
The lower brain tkan blood levels of pentobarbital in both strains of mice at the time of onset, and, conversely, the higher brain than blood concentrationsof the drug at awakening are probably related to drug distributional factors. It has been reported that C57BL mice, which have a lower brain sensitivity than the DBA strain to the acute effects of ethanol, showed development of acute tolerance to ethanol [S]. We have now shown that with respect to pentobarbital, only the DBA mice, which have a lower brain sensitivity to the acute effects of the drug, developed acute tolerance. Thus, the development of acute tolerance to a drug might be related to the degree of CNS sensitivity to the drug. However, two strains of mice, the long-sleep (LS) and short-sleep (SS), which were selectively bred (not inbred) for their differences in sensitivities to the hypnotic effects of ethanol [ll, 121, both did not develop acute tolerance to ethanol [8]. These two strains of mice showed no difference in sensitivity to the hypnotic effects of pentobarbital [2]. Due to the unavailability of the LS and SS mice, we could not perform the above experiments with these animals. Nevertheless, our present results support the use of C57BL and DBA mice as animal models to determine the relationships between voluntary ethanol intake and neural sensitivity to the acute effects of ethanol, and the rates of development of tolerance to and physical dependence on ethanol. This work is currently in progress.
Acknowledgements: The autkors express their sincere appreciation to Olivia Doyle and Patrick George for excellent technical assistance.
Preliminary Communications
552
REFERENCES C.R. Randall
and D. Lester,
A.J. Siemens
and A.W.K.
A.W.K.
J. Pharmacol.
Chan,
Life SCi. 19, 581 (1976).
Chan, Life Sci. 19, 597
4.
D.A. Rodgers,
5.
I?. Kakihana,
6.
C.W. Schneider, Psych. E,
Psychosomat. D.R. Brown,
466
(1976).
Med. 2&, 498 G.E. McClearn
S.K. Evans,
Exp. Ther. 188, 27 (1974).
(1966). and I.R. Tabershaw,
M.B. Chenoweth
Science
154,
and F.L. Beman, J. Camp. Physiol.
(1966). &
(1973).
7.
C.W. Schneider,
8.
B. Tabakoff
and R.F. Ritzman,
9.
E. Mellanby,
Med. Res. Comm.
P. Trzil and R. D'Andrea,
Phannacol.
Pharmacologist (Gt. Brit.)
20, 193
Biochem.
Behav. 2, 549
Spec. Rep. Ser., NO. 31, London
E. Rubin, H. Gang, P.S. Misra and C.S. Lieber,
Amer. J. Med. 49, 801
11.
G.E. McClearn
2, 409
12.
W.D.W.
and R. Kakihana, V.G. Erwin,
Behav.
Genetics
S.M. Anderson
(1974).
(1978).
10.
Heston,
1574
(1919).
(1970).
(1973).
and H. Robbins,
Life Sci. 14, 365 (1974).