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Alcohol & Alcoholism Vol. 41, No. 1, pp. 24–32, 2006
Advance Access publication 10 October 2005
doi:10.1093/alcalc/agh217
PHARMACOLOGICAL MANIPULATION OF CB1 RECEPTOR FUNCTION ALTERS
DEVELOPMENT OF TOLERANCE TO ALCOHOL
KAREN L. NOWAK1,2, K. YARAGUDRI VINOD1,2 and BASALINGAPPA L. HUNGUND1,2,3*
1
New York State Psychiatric Institute, New York, NY, USA, 2Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, USA and
3
College of Physicians and Surgeons, Colombia University, New York, NY, USA
(Received 11 July 2005; first review notified 18 August 2005; accepted in revised form 30 August 2005; accepted 9 September 2005;
advance access publication 10 October 2005)
INTRODUCTION
resulted in decreased preference. There are also innate
differences in the cannabinoid system in mouse strains
Alcoholism is a psychiatric disorder characterized by impaired
selected for EtOH preference. Significant differences were
control over drinking, leading to tolerance, physical dependfound in the density, binding affinity and function of CB1
ence, uncontrollable craving, and relapse. The effects of alcoreceptors in brain synaptic plasma membranes (SPM) of
hol (EtOH) are mediated via several intracellular signal
EtOH-preferring C57BL/6 and EtOH-avoiding DBA/2 mice
transduction pathways, involving many known classical
(Hungund and Basavarajappa, 2000; Basavarajappa and
neurotransmitters and ion channels. There is now evidence to
Hungund, 2001).
support a role for the endocannabinoid (EC) system in many
Little information exists, however on the role of the
aspects of EtOH-drinking related behaviours.
CB1 receptor function in behavioural tolerance to EtOH.
Manipulation of CB1 receptors results in alterations in
Compelling evidence in favour of a role of the EC system in
EtOH consumption and reward value. Mice lacking CB1
tolerance comes from evidence of neuroadaptive changes
receptors (CB1 KO) have a low preference for EtOH
that occur following chronic EtOH exposure. Studies from our
(Hungund et al., 2003; Poncelet et al., 2003; Wang et al.,
laboratory have previously demonstrated increased levels of
2003; Naassila et al., 2004). Antagonism of CB1 receptors
two naturally occurring cannabimimetic brain constituents,
by SR141716A results in decreased EtOH intake and
arachidonyl ethanolamide (AEA) and 2-arachidonyl glycerol
decreased motivation to consume EtOH (Arnone et al.,
in cultured neuronal cells chronically exposed to EtOH
1997; Colombo et al., 1998; Gallate and McGregor, 1999;
(Basavarajappa and Hungund, 1999a; Basavarajappa
Rodriguez de Fonseca et al., 1999; Freedland et al., 2001;
et al., 2000). Upregulation of AEA and downregulation of
Hungund et al., 2002), while the agonists CP-55,940 and
N-arachidonyl phosphatidyl ethanolamide, a precursor for
WIN 55,212-2 result in dose-dependent increases in consumAEA, have been demonstrated in whole brain plasma memption (Colombo et al., 2002) and increased motivation for
branes of mice chronically exposed to EtOH (Hungund et al.,
EtOH (Gallate et al., 1999). The EtOH deprivation effect is
2002). In addition, downregulation of CB1 receptors and CB1
also blocked by SR141716A treatment in EtOH-preferring
receptor agonist stimulated [35S]GTPgS binding in SPM
of mice chronically exposed to EtOH have been reported
(sP) rats (Serra et al., 2002). Lallemand et al. (2001) have
(Basavarajappa et al., 1998; Basavarajappa and Hungund,
shown, however, that the effects of SR141716A on EtOH
1999b).
consumption depend on the temporal administration of the
The present experiments examined CB1 receptor involveantagonist. Rats given the antagonist following chronic
ment in behavioural tolerance to EtOH through the activation
EtOH administration via inhalation showed a decrease in
and blockade of CB1 receptor function. Because Lallemand
voluntary EtOH consumption, while administration of
et al. (2001) found that alterations in EtOH consumption difSR141716A during chronic alcoholization increased intake.
fered depending on the temporal delivery of the CB1 receptor
In addition, SR141716A treatment in non-alcoholized rats
antagonist used, we used two administration paradigms: (i) coadministration with chronic EtOH inhalation and (ii) adminis*Author to whom correspondence should be addressed at: Nathan Kline
tration following chronic alcoholization. EtOH tolerance was
Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg,
NY 10962, USA. Tel: +1 845 398 5452; Fax: +1 845 398 5451; E-mail: examined using both acute EtOH-induced hypothermia and
sedation as measures.
[email protected]
24
The Author 2005. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved
Downloaded from http://alcalc.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014
Abstract — Aims: The current study investigated the efficacy of CB1 receptor-targeted drugs on the development and expression
of tolerance to alcohol (EtOH). Methods: An EtOH-inhalation model was used to induce tolerance, as measured by EtOH-induced
sedation and hypothermia after a 24 h withdrawal period. Two drug treatment procedures, (i) co-treatment with EtOH and (ii) acute
drug administration following chronic EtOH treatment, were used to test the efficacy of CB1 receptor manipulations on EtOH tolerance.
Results: The effects of the CB1 receptor agonist CP-55,940 varied depending on paradigm and behavioural measure. Chronic CP55,940 co-treatment blocked tolerance to EtOH-induced hypothermia but not to the sedative effect (sleep time) in EtOH-exposed
mice. However, chronic CP-55,940 administration alone resulted in tolerance to the sedative effect of a challenge dose of EtOH in
control mice. Acute CP-55,940 administration after chronic alcoholization blocked the development of tolerance to EtOH-induced sedation compared to the EtOH alone exposed group, but induced tolerance to the hypothermic effects of EtOH in control mice. Chronic
blockade of CB1 receptor function by SR141716A resulted in tolerance to both the sedative and hypothermic effects of EtOH in control
mice, but had no effect on EtOH-exposed mice. Conclusions: The data support a role for the endocannabinoid (EC) system in EtOH
tolerance/dependence and suggest that drugs targeted against EC system could be therapeutically useful in treating alcohol-related
disorders.
CB1 RECEPTOR FUNCTION AND DEVELOPMENT OF TOLERANCE TO ALCOHOL
MATERIALS AND METHODS
Animals
Seven- to eight-week old Swiss-Webster male mice weighing
25–35 g (Taconic Farms, Germantown, NY) were used in the
following experiments (N = 8–12/group). Mice were housed in
groups under standard laboratory conditions (12:12 light/dark
cycle; 23 ± 1 C) with food and water available ad libitum.
All mice were treated in accordance with Institutional and
National Institute of Health guidelines for the use and care
of laboratory mice.
Chronic EtOH inhalation procedure
Mice were made EtOH-tolerant by continuous exposure to
EtOH vapours following the procedure described below.
The infusion chambers were 52 · 34 · 27 cm and made of
Lucite. A syringe pump delivered absolute EtOH at 25.5 mg/
min onto a gauze wick in a flask. An air pump delivered air
at 2–3 l/min through the flask and into the chamber at appropriate flow rates to give nominal EtOH vapour concentrations
of 10–16 mg/l. EtOH-exposed mice were given a priming dose
of EtOH (2 g/kg) in addition to pyrazole before being placed
in the chamber on the first day of the procedure. Pyrazole,
an EtOH dehydrogenase inhibitor, was given to inhibit EtOH
metabolism and stabilize blood EtOH levels. Mice were
exposed to EtOH vapours for 72 h, with daily pyrazole injections. Mice remained in either the EtOH- or air-infusion
chamber constantly throughout this 72 h period, except for
30–40 min each morning for injections. Control mice treated
with a similar dose of pyrazole as the EtOH group, were subjected to identical housing and handling conditions, with the
exception of the EtOH pre-exposure and infusion into the
chamber. Body weights and blood samples via tail blood
extraction were taken daily in the morning between 9 and
10 AM. Blood samples (10 ml) were collected by clipping the
tip of the tail and were mixed 10:90 (v/v) with 3.4% perchloric
acid. The mixture was centrifuged at 12 000 r.p.m. and the
supernatant (50 ml) was used for EtOH estimation by the spectrophotometric enzymatic method as described previously
(Lundquist, 1959; Basavarajappa et al., 1998; Basavarajappa
and Hungund, 1999b). Briefly, 50 ml aliquots of supernatant
were incubated in semicarbazide buffer containing NAD
(2 mM) and alcohol dehydrogenase for 30 min. The resultant
concentration of NADH was determined at 340 nM and corresponding concentrations of EtOH were determined using
a standard curve. Similarly chamber EtOH concentration was
determined by analysing aliquots of air at different ports in
the chamber and expressed as mg/l of air. Delivery of EtOH
was adjusted to give chamber EtOH vapour concentration of
10–12 mg/l of air.
The EtOH inhalation model used in the current studies has
proven to be a valuable paradigm for studying EtOH tolerance
and dependence (Goldstein and Pal, 1971; Blum et al., 1975;
Sprague and Craigmill, 1978; Littleton and Little, 1994;
Kliethermes et al., 2004). In the current studies, 72 h EtOH
exposure resulted in tolerance to the sedative and hypothermic
effects of EtOH.
Behavioural studies
Mice were given a 24 h withdrawal period after 72 h EtOH
vapour exposure prior to behavioural testing.
Acute EtOH-induced sedation
EtOH-induced sedation was measured by determining the
time between loss of the righting reflex (LORR) and time to
regain the righting reflex after the administration of a challenge dose of EtOH (4 g/kg body wt). After the LORR, the
mice were placed on their back in a V-shaped trough and
time was recorded. The mice were deemed to have regained
the righting reflex if they could right themselves three times
within 30 s when again placed on their backs.
Hypothermia
EtOH-induced hypothermia was assessed by monitoring rectal
temperatures, 60 min after a challenge dose of EtOH (2.0 g/kg
body wt). Temperature was determined using a digital rectal
thermometer (Physiotemp model BAT-12, Physiotemp Instruments, Inc., Clifton, NJ). The lubricated rectal probe was
inserted 1.5 cm into the rectum and temperature was allowed
to stabilize before recording. Temperatures were recorded
immediately prior to the EtOH challenge and 60 min thereafter. The difference in body temperature between pre- and
post-injection determined the degree of hypothermia.
The challenge doses of EtOH chosen were based upon
pilot work in the laboratory, determining 2.0 g/kg EtOH was
optimal to reveal the hypothermic effects of EtOH and that
4.0 g/kg produced consistent sedation with which to examine
tolerance.
Effects of co-administration of CP-55,940 with chronic
EtOH exposure, on tolerance to the sedative and
hypothermic effects of EtOH
Tolerance to the sedative and hypothermic effects of EtOH
were assessed following four experimental conditions: (i) EtOH
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Drugs
Solutions (20% v/v) of EtOH were prepared in 0.9% saline.
An aliquot of CP-55,940 (Tocris, Ellisville, MO) was dissolved in 1% dimethyl sulphoxide and 1% Tween 80 and mixed
in saline and was injected at a volume of 1 ml/kg (0.4 mg/kg).
The SR141716A was a kind gift from Sanofi Pharmaceuticals (Montpellier, France) and RBI as part of the Chemical
Synthesis Program of the NIMH, contract #1MH30003RBINIMH. An aliquot of SR141716A was similarly prepared
for a final injection concentration of 3 mg/kg. Pyrazole
(Sigma-Aldrich, St Louis, MO) was dissolved in saline and
injected at a volume of 10 ml/kg (68 mg/kg). The dose of
SR141716A used in the present experiments was chosen based
on substantial findings of the effects of SR141716A on voluntary EtOH consumption and EtOH preference (Arnone et al.,
1997; Gallate and McGregor, 1999; Rodriguez de Fonseca
et al., 1999; Freedland et al., 2001; Lallemand et al., 2001).
In addition, SR141716A has been shown to have a relatively
flat dose–response curve, with 1 and 3 mg/kg having effects
on EtOH and sucrose consumption, while higher doses result
in competing behaviours such as excessive scratching and
grooming (Compton et al., 1996; Darmani et al., 2000; Jarbe
et al., 2002). The dose of CP-55,940 was similarly chosen
based on the literature and pilot studies conducted in this
laboratory (Rubino et al., 1997; Rubino et al., 2000; Biscaia
et al., 2003).
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K. L. NOWAK et al.
inhalation, (ii) co-administration of the CB1 receptor agonist
CP-55,940 and EtOH inhalation, (iii) chronic CP-55,940 treatment, and (iv) vehicle (pyrazole) administration. Groups treated with the CB1 agonist received CP-55,940 (0.4 mg/kg) 24
and 1 h prior to the beginning of the 72-h inhalation protocol
and twice daily for 2.5 days.
Acute effects of CP-55,940 administration following
chronic alcoholization on tolerance to the hypnotic and
hypothermic effects of EtOH
Mice were exposed to chronic EtOH via inhalation and then
given CP-55,940 (0.4 mg/kg) or vehicle 24 h after removal
from the chamber. After 1 h, mice were challenged with 4
or 2 g/kg EtOH and sedation and hypothermia, respectively
were measured.
Effects of acute administration of SR141716A following
chronic alcoholization on tolerance to the hypnotic and
hypothermic effects of EtOH
Mice were exposed to chronic EtOH via inhalation and then
given either SR141716A (3 mg/kg) or vehicle 24 h after
removal from the chamber. After 1 h, mice were challenged
with 4 or 2 g/kg EtOH and sedative effect and hypothermia,
respectively, were measured.
Fig. 1. The blood EtOH levels were estimated in mice following chronic treatment with EtOH, EtOH + CP-55,940, and EtOH + SR141716A. There were
no significant changes (P < 0.99) in the BEL by the co-administration of
either CP-55,940 or SR141716A during alcoholization.
Statistical analyses
Analyses were conducted using two-way ANOVAs (Group:
EtOH or Air · Drug: CP-55,940/SR141716A or Vehicle).
Planned comparisons were done to assess whether: (i) the
drug (CP-55,940 or SR141716A) affected tolerance, (ii) the
drug affected basal levels, and (iii) tolerance developed.
The analyses were performed by the least squares means
method (LSM) using SAS statistical software (Cary, NC)
with Bonferroni adjustments (P < 0.017). One-way ANOVA
test was used to compare the EtOH levels.
RESULTS
The blood EtOH levels
The average blood EtOH levels (BELs) during EtOH exposure
was 1.68 ± 0.05. The BELs were found to be similar in the
EtOH, EtOH ± CP-55,940, and EtOH ± SR141716A groups
(P < 0.99). This indicates no effect of co-treatment of either
CP-55,940 or SR141716A on the EtOH metabolism during
chronic alcoholization (Fig. 1).
Fig. 2. EtOH-induced LORR following chronic treatment with EtOH,
CP-55,940, CP-55,940 + EtOH, or vehicle. Histogram represents the time
(mean ± SEM) between the loss and gain of the righting reflex following
a challenge dose of 4 g/kg EtOH. Tolerance to challenge dose of EtOH was
demonstrated in EtOH-exposed mice compared to control mice given vehicle
(@P < 0.0001). There was a trend towards CP-55,940 reducing the duration of
LORR relative to vehicle-treated controls (#P < 0.0001). Similarly, a significant tolerance to challenge dose of EtOH was evident in the EtOH + CP-55,940
group when compared to control mice ($P < 0.0001).
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Effects of chronic SR141716A administration during
chronic alcoholization on tolerance to the sedative and
hypothermic effects of EtOH
Tolerance to the sedative and hypothermic effects of EtOH
was assessed in four groups of mice, treated with: (i) EtOH
inhalation, (ii) co-administration of the CB1 receptor antagonist SR141716A and EtOH inhalation, (iii) chronic SR141716A
treatment, and (iv) vehicle (pyrazole) administration. Groups
treated with the CB1 antagonist received SR141716A
(3 mg/kg) 24 and 1 h prior to the beginning of the 72-h inhalation protocol and once daily, thereafter. EtOH-induced sedation and hypothermia were measured as stated above.
Effects of co-administration of CP-55,940 during chronic
alcoholization on tolerance to the sedative effects of EtOH
A two-way ANOVA on the amount of time between the loss of
and regaining the righting reflex revealed a significant main
effect for Group (F1,44 = 26.12, P < 0.001) and Drug (F1,44 =
22.28, P < 0.0001), in addition to a significant Group · Drug
interaction (F1,44 = 4.05, P = 0.05). A clear tolerance was
demonstrated between EtOH-exposed mice and control
mice given vehicle, verifying the efficacy of the inhalation
model (mean ± SEM = 30.00 ± 3.47 and 36.73 ± 5.91 vs
75.50 ± 9.51; LSM with Bonferroni adjustment, P < 0.017).
The results also suggest that CP-55,940 differentially affected
EtOH-exposed and control mice. A significant reduction
in the duration of sedation in control mice treated with
CP-55,940 relative to vehicle-treated controls (mean ± SEM =
47.33 ± 4.12 vs 86.5 ± 4.56) was observed but produced no
significant effect in EtOH-tolerant mice (Fig. 2).
CB1 RECEPTOR FUNCTION AND DEVELOPMENT OF TOLERANCE TO ALCOHOL
Effects of co-administration of CP-55,940 during chronic
alcoholization on tolerance to the hypothermic
effects of EtOH
Changes in body temperature 60 min post-injection, analysed
by two-way ANOVA, did not reveal a significant main effect
of Group (F1,37 = 0.80, P > 0.05) nor Drug (F1,37 = 0.124,
P > 0.05). There was, however, a significant Group · Drug
interaction (F1,37 = 11.37, P < 0.01). As above, tolerance
was demonstrated between EtOH-exposed and control
mice given vehicle (mean ± SEM = –1.33 ± 0.39 vs
–2.74 ± 0.31 C; Bonferroni adjusted to P < 0.017). There
was no significant difference between control mice given the
drug vs vehicle. However, CP-55,940 significantly blocked
the development of tolerance to the hypothermic effect of
EtOH (mean ± SEM = –3.0 ± 0.18 vs –1.33 ± 0.39 C), as
there was a significant difference between EtOH-exposed
mice given CP-55,940 vs EtOH and vehicle (Fig. 3).
Effects of acute administration of CP-55,940 after chronic
alcoholization on tolerance to the sedative
effects of EtOH
A two-way ANOVA on the amount of time between the loss
of and regaining the righting reflex revealed a significant
main effect for Group (F1,24 = 35.69, P < 0.001) and Drug
(F1,24 = 8.4, P < 0.007). In addition, there was a significant
Group · Drug interaction (F1,24 = 5.17, P < 0.03). EtOHexposed mice given vehicle had a lower latency of LORR
(57%; P < 0.01) than control mice given vehicle, suggesting
a tolerance to the ataxic effects of EtOH (mean ± SEM =
42.1 ± 10.49 vs 98.8 ± 6.3). There was a trend of decreased
tolerance by the CP-55,940 treatment when compared to the
vehicle-treated group (P < 0.09). However, increased sedative
effect was evident in EtOH-exposed mice treated with
Fig. 4. EtOH-induced LORR following chronic alcoholization and acute
CP-55,940 treatment. Histogram represents the time (mean ± SEM) between
the loss and gain of the righting reflex following a challenge dose of 4 g/kg
EtOH in mice treated with CP-55,940 or vehicle 1 h prior to behavioural
testing. EtOH-exposed mice showed tolerance to the ataxic effects of EtOH
(@P < 0.01). Acute CP-55,940 treatment showed a trend towards blockade
of tolerance when compared to the vehicle-treated group (#P < 0.09).
The CP-55,940 treatment to the EtOH group reduced the development of tolerance when compared to EtOH-exposed mice ($P < 0.001).
CP-55,940 when compared to the EtOH exposed group
(P < 0.001; Fig. 4).
Effects of acute administration of CP-55,940 after chronic
alcoholization on tolerance to the hypothermic
effects of EtOH
Changes in body temperature 60 min post-injection were
analysed using a two-way ANOVA. There was not a significant main effect of Group (F1,38 = 0.54, P > 0.05), but there was
a significant Drug effect (F1,38 = 24.38, P < 0.0001) and a significant Group · Drug interaction (F1,38 = 11.09, P < 0.01). A
clear tolerance was demonstrated between EtOH-exposed vs.
control mice given vehicle, verifying the efficacy of the
inhalation model (mean ± SEM = –0.95 ± 0.30 vs
–2.19 ± 0.20 C; Bonferroni adjusted P < 0.017). The mice
exposed to EtOH + CP-55,940 showed less of a drop in body
temperature compared to control group indicative of tolerance
to hypothermic effect of EtOH (Fig. 5). Control mice treated
with an acute dose of CP-55,940 were also shown to be tolerant to the hypothermic effects of EtOH (mean ± SEM =
0.57 ± 0.39 vs –2.19 ± 0.20 C; CP-55,940 vs vehicle controls,
P < 0.0001), suggestive of rapid cross-tolerance to CP-55,940.
Effects of co-administration of SR141716A during chronic
alcoholization on tolerance to the sedative
effects of EtOH
A two-way ANOVA on the amount of time between the loss of
and regaining the righting reflex revealed a significant main
effect for both Group (F1,42 = 12.33, P < 0.001) and Drug
(F1,42 = 6.94, P < 0.01). There was no significant Group ·
Drug interaction (F3,42 = 2.33, P > 0.05). Tolerance to the sedative effect of EtOH was demonstrated in EtOH-exposed vs
control mice given vehicle as evidenced by a 51% reduction
in sedation (mean ± SEM = 36.73 ± 5.91 vs 75.50 ± 9.51).
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Fig. 3. EtOH-induced hypothermia following chronic treatment with EtOH,
CP-55,940, CP-55,940 + EtOH, or vehicle. Graph represents (Mean ± SEM)
drop in body temperature ( C) at 60 min post-injection following a challenge
dose of 2 g/kg EtOH. There was no significant effect of CP-55,940 on the
development of tolerance to the hypothermic effects of a challenge dose of
EtOH in mice chronically exposed to EtOH. @P < 0.017 compared to control
groups. Co-administration of CP-55,940 with EtOH exposure resulted in
significant decrease in the body temperature compared to the EtOH group
($P < 0.001). Chronic administration of CP-55,940 to EtOH-naı̈ve mice
showed a higher decrease in the body temperature compared to the EtOH
group (xP < 0.038).
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K. L. NOWAK et al.
Fig. 6. EtOH-induced LORR following chronic treatment with EtOH,
SR141716A, SR141716A + EtOH, or vehicle. Histogram represents the time
(mean ± SEM) between the loss and gain of the righting reflex following
a challenge dose of 4 g/kg EtOH. Tolerance to challenge dose of EtOH was
demonstrated in EtOH-exposed mice compared to control mice given vehicle
(@P < 0.009). Chronic SR141716A treatment showed a development of tolerance to the sedative effect of EtOH in alcoholized ($P < 0.0001) and nonalcoholized mice (#P < 0.051) compared to control groups. There was a trend
toward SR141716A reducing the duration of LORR compared to vehicletreated controls.
Planned comparisons of simple main effects using least
square means and Bonferroni correction (P < 0.017) are suggestive, yet not conclusive, of SR141716A reducing the duration of sedation in control mice relative to vehicle-treated
controls (mean ± SEM = 28.72 ± 7.54 and 44.00 ± 7.03 vs
75.50 ± 9.51) without affecting EtOH-exposed mice
(Fig. 6).
Effects of co-administration of SR141716A during chronic
alcoholization on tolerance to the hypothermic
effects of EtOH
Changes in body temperature 60 min post-injection were analysed, revealing significant main effects for both Group
(F1,43 = 7.13, P < 0.01) and Drug (F1,43 = 10.22, P < 0.01),
but no significant Group · Drug interaction (F1,43 = 1.51,
P > 0.05). Tolerance was demonstrated between EtOHexposed and control mice given vehicle, verifying the efficacy
of the inhalation model (mean ± SEM = –2.0 ± 0.39 vs
–3.13 ± 0.33 C; Bonferroni adjusted P < 0.012). As detailed
above, planned comparisons are only suggestive of the fact
that SR141716A attenuated hypothermia in control mice, but
not EtOH-exposed mice, relative to vehicle-treated controls
(mean ± SEM = –1.84 ± 0.26 vs –3.13 ± 0.33 C), suggesting
cross-tolerance to SR141716A (Fig. 7).
Effects of acute administration of SR141716A following
chronic alcoholization on tolerance to the sedative
effects of EtOH
Analysis of sedative effect of challenge dose of EtOH revealed
a significant effect of Group (F1,41 = 22.52, P < 0.0001), but
no significant Drug effect (F1,41 = 0.01, P > 0.05), nor a significant Group · Drug interaction. (F1,41 = 1.17, P > 0.05). The
mice exposed to EtOH alone or EtOH + SR141716A showed
a similar reduction in sedation, indicative of tolerance to the
sedative effects of EtOH, relative to either SR141716Atreated or control mice (Fig. 8).
Effects of acute administration of SR141716A following
chronic alcoholization on tolerance to the hypothermic
effects of EtOH
Analysis of the change in body temperature at 60 min by twoway ANOVA revealed a significant Group effect (F3,39 =
10.02, P < 0.01). There was not a significant effect of Drug
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Fig. 5. EtOH-induced hypothermia following chronic alcoholization and
acute CP-55,940 treatment. Graph represents (mean ± SEM) drop in body
temperature ( C) at 60 min post-injection following a challenge dose of
2 g/kg EtOH in mice treated with CP-55,940 or vehicle 1 h prior to behavioural
testing. Tolerance to challenge dose of EtOH was demonstrated in EtOHexposed mice compared to control mice given vehicle (@P < 0.011). Acute
CP-55,940 treatment showed a rapid cross-tolerance to the hypothermic
effect of EtOH in alcoholized ($P < 0.043) and non-alcoholized mice
(#P < 0.0001).
Fig. 7. EtOH-induced hypothermia following chronic treatment with EtOH,
SR141716A, SR141716A + EtOH, or vehicle. Graph represents (mean ±
SEM) drop in body temperature ( C) following a challenge dose of 2 g/kg
EtOH in mice treated with SR141716A or vehicle during chronic alcoholization and control mice given the antagonist or vehicle. Tolerance to challenge
dose of EtOH was demonstrated in EtOH-exposed mice compared to control
mice given vehicle (@P < 0.012). There was a trend toward SR141716A
attenuating hypothermia compared to alcoholized ($P < 0.0002) and nonalcoholized mice ($P < 0.004) compared to control group.
CB1 RECEPTOR FUNCTION AND DEVELOPMENT OF TOLERANCE TO ALCOHOL
Fig. 9. EtOH-induced hypothermia following chronic alcoholization and
acute SR141716A treatment. Graph represents (mean ± SEM) drop in body
temperature ( C) at 60 min post-injection following a challenge dose of
2 g/kg EtOH in mice treated with SR141716A or vehicle 1 h prior to behavioural testing. Tolerance to challenge dose of EtOH was demonstrated in
EtOH-exposed mice compared to control mice given vehicle (@P < 0.061).
There was significant reduction in the hypothermic effect of EtOH
in acute SR141716A-treated group compared to SR141716A group
(#P < 0.013).
(F1,39 = 2.19, P >0.05), nor a significant Group · Drug interaction (F1,39 = 0.12, P > 0.05). There was no differential
effect of SR141716A on tolerance to the hypothermic effects
of EtOH (Fig. 9).
DISCUSSION
Studies from this laboratory have reported neuroadaptive
changes in the CB1 receptor system that occur following
chronic EtOH exposure, suggesting a role for this system in
EtOH tolerance (Basavarajappa et al., 1998; Basavarajappa
and Hungund, 1999a; Basavarajappa and Hungund, 1999b;
Basavarajappa et al., 2000). The current studies investigated
the efficacy of CB1 receptor-targeted drugs in modulating
the development and expression of tolerance to EtOH in
Swiss-Webster mice. The results indicate that it is possible
to modulate EtOH tolerance with both the CB1 receptor
agonist and the antagonist and that results depend upon
temporal administration of the drugs.
The effects of CP-55,940 varied depending on paradigm
and behavioural measure. When chronic CP-55,940 administration resulted in tolerance to the sedative effects of a challenge dose of EtOH in control mice, but did not differentially
affect tolerance in EtOH-exposed mice. This finding is consistent with reported studies of cross-tolerance between cannabinoids and EtOH (Sprague and Craigmill, 1976; Siemens
and Doyle, 1979; da Silva et al., 2001). When measuring
EtOH-induced hypothermia, tolerance to hypothermic effect
was observed in mice exposed to EtOH chronically. However,
chronic CP-55,940 treatment during chronic alcoholization
blocked tolerance to the hypothermic effects of EtOH. Acute
CP-55,940 administration after chronic alcoholization had
a significant effect on EtOH-induced sedation when compared
to the EtOH-exposed mice, suggesting a blockade of expression of tolerance to sedative effect of EtOH. However, there
was a trend towards reduction in the development of tolerance
in mice treated with CP-55,940 compared to the vehicletreated group. Conversely, administration of the agonist after
chronic alcoholization resulted in tolerance to the hypothermic
effects of EtOH in control mice, and showed a trend toward
attenuating hypothermia in EtOH-exposed mice as well, suggesting that mice displayed rapid cross-tolerance between
the agonist and EtOH.
A possible explanation, though speculative, for the disparate effects for EtOH-induced hypothermia and hypnosis
under both paradigms could be due to differential effects of
the agonist on CB1 receptor function in specific brain regions
controlling these behaviours. The CB1 receptors are widely
distributed throughout the brain, with significantly higher
densities in the basal ganglia and cerebellum, areas known
to be involved in motor coordination and motor function, and
the hypothalamus, which is implicated in thermoregulation (Moldrich and Wenger, 2000). Regarding CP-55,940
co-administration, chronic exposure with either EtOH or
CP-55,940 has been shown to increase endogenous cannabinoid content, specifically in the limbic forebrain, and to desensitize the CB1 receptors (Sim-Selley et al., 2002). Changes in
the degree of endogenous tone could alter the sensitivity of
various neural circuits to both agonists and antagonists.
Alternatively, neurotransmitter systems within different brain
regions that regulate the behavioural effects of EtOH may be
differentially affected by CB1 receptor manipulations. For
example, inhibition of serotonin reuptake and blockade of
dopamine and NMDA receptors are known to potentiate
cannabinoid-evoked hypothermia (Davies and Graham,
1980; Malone and Taylor, 1998; Nava et al., 2000; Rawls
et al., 2002). Thus, differential modulation of one or more
neurotransmitter systems with CP-55,940 may explain the
effects observed.
The behavioural syndromes elicited by EtOH and CB1
receptor agonists only partially overlap (Wiley and Martin,
Downloaded from http://alcalc.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014
Fig. 8. EtOH-induced LORR following chronic alcoholization and acute
SR141716A treatment. Histogram represents the time (mean ± SEM) between
the loss and gain of the righting reflex following a challenge dose of 4 g/kg
EtOH in mice treated with SR141716A or vehicle 1 h prior to behavioural
testing. Mice exposed to EtOH (@P < 0.0011) and EtOH + SR141716A
(#P < 0.0002) had reduced LORRs compared to control and SR141716Atreated groups, respectively, indicative of tolerance to the ataxic effects of
EtOH relative to control mice.
29
30
K. L. NOWAK et al.
signalling system modulates the behavioural and pharmacological effects of EtOH.
Acknowledgements — Supported by NIAAA contract #N01AA22008 and
grant #AA13003. Special thanks to Joe Wanderling and Morris Meisner
from Department of Statistics at NKI, for their assistance with the statistical
analyses and to Sharon Marsico for her help with the manuscript.
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differential effects of both acute and chronic CP-55,940 on
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in control mice relative to vehicle-treated controls. This would
suggest that chronic SR141716A treatment results in crosstolerance to EtOH. When SR141716A was given following
chronic alcoholization, however, there was no effect of the
drug. Mice exposed to EtOH showed clear tolerance to
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treated with the antagonist or vehicle. Although SR141716A
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to cannabinoid receptor agonists in vivo, it is intriguing
that it produced cross-tolerance to EtOH similar to that of
the agonist CP-55,940. Several recent studies have ascribed
different pharmacological properties to SR141716A. It has
been shown to have inverse agonist properties (Bouaboula
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treatment with CP-55,940 resulted in a similar increase in
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has demonstrated that co-administration of SR141716A had
similar effects on the acquisition of morphine sensitization
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2003). This study also demonstrated cross-sensitization
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SR141716A.
Overall, the data support a role for the EC system in EtOH
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Further studies, however, are necessary to fine-tune the
dosage, duration, and timing of the drug treatment in order
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