5-HT3A Receptor Subunit is Required for 5-HT3 Antagonist-Induced Reductions in Alcohol Drinking

Article (PDF Available)inNeuropsychopharmacology 29(10):1807-13 · November 2004with90 Reads
DOI: 10.1038/sj.npp.1300498 · Source: PubMed
Abstract
The ionotropic serotonin subtype-3 (5-HT3) receptor has emerged as a potential therapeutic target in the treatment of alcohol abuse and alcoholism because selective pharmacological antagonists reduce alcohol consumption in preclinical and clinical models. 5-HT binds to the extracellular N-terminus of the 5-HT(3A) receptor subunit but receptor activation is also enhanced by distinct allosteric sites, which indicates the presence of other receptor subunits. It is not known if specific molecular subunits of the 5-HT3 receptor modulate alcohol drinking. To address this issue, we characterized acute locomotor response to alcohol and alcohol consumption in a two-bottle home-cage procedure by congenic C57BL/6J mice with a targeted deletion of the 5-HT(3A) receptor subunit gene. 5-HT(3A)-null mice did not differ from wild-type littermate controls on measures of spontaneous locomotor activity, habituation to a novel environment, or locomotor response to ethanol (0, 0.5, 1, or 2 g/kg). Moreover, null mice did not differ from controls on measures of ethanol (2-10%) intake and preference during or after a two-bottle home-cage sucrose fading procedure. Systemic administration of the 5-HT3 antagonist LY-278,584 (0-10 mg/kg) decreased intake of both sweetened (2% sucrose+10% ethanol) and unsweetened (10% ethanol) ethanol in wild-type mice only. These findings indicate that reduction of alcohol drinking produced by 5-HT3 antagonism is dependent on the presence of 5-HT(3A)-containing receptors.
5-HT
3A
Receptor Subunit Is Required for 5-HT
3
Antagonist-Induced Reductions in Alcohol Drinking
Clyde W Hodge*
,1
, Stephen P Kelley
2
, Alison M Bratt
3
, Kimberly Iller
4
, Jason P Schroeder
1
and
Joyce Besheer
1
1
Department of Psychiatry and Bowles Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA;
2
Neurosciences
Institute, Department of Pharmacology and Neuroscience, University of Dundee, Ninewells Hospital, Dundee, Scotland;
3
Pharmacology
Department, Organon Laboratories Ltd., Newhouse, Lanarkshire, Scotland;
4
Ernest Gallo Clinic and Research Center, Department of Neurology,
University of California San Francisco, Emeryville, California, USA
The ionotropic serotonin subtype-3 (5-HT
3
) receptor has emerged as a potential therapeutic target in the treatment of alcohol abuse
and alcoholism because selective pharmacological antagonists reduce alcohol consumption in preclinical and clinical models. 5-HT binds
to the extracellular N-terminus of the 5-HT
3A
receptor subunit but receptor activation is also enhanced by distinct allosteric sites, which
indicates the presence of other receptor subunits. It is not known if specific molecular subunits of the 5-HT
3
receptor modulate alcohol
drinking. To address this issue, we characterized acute locomotor response to alcohol and alcohol consumption in a two-bottle home-
cage procedure by congenic C57BL/6J mice with a targeted deletion of the 5-HT
3A
receptor subunit gene. 5-HT
3A
-null mice did not
differ from wild-type littermate controls on measures of spontaneous locomotor activity, habituation to a novel environment, or
locomotor response to ethanol (0, 0.5, 1, or 2 g/kg). Moreover, null mice did not differ from controls on measures of ethanol (2–10%)
intake and preference during or after a two-bottle home-cage sucrose fading procedure. Systemic administration of the 5-HT
3
antagonist
LY-278,584 (0–10 mg/kg) decreased intake of both sweetened (2% sucrose þ 10% ethanol) and unsweetened (10% ethanol) ethanol in
wild-type mice only. These findings indicate that reduction of alcohol drinking produced by 5-HT
3
antagonism is dependent on the
presence of 5-HT
3A
-containing receptors.
Neuropsychopharmacology advance online publication, 26 May 2004; doi:10.1038/sj.npp.1300498
Keywords: serotonin; 5-HT
3
; 5-HT
3A
; alcohol drinking; LY-278,584; mice
INTRODUCTION
Serotonin (5-HT) receptors are involved in a variety of
mammalian biochemical, physiological, and behavioral
processes (Barnes and Sharp, 1999). 5-HT receptors are
classified into seven groups (5-HT
1–7
), comprising a total of
at least 14 structurally and pharmacologically distinct
receptor subtypes (Hoyer et al, 1994). The 5-HT
3
receptor
is unique among this G-protein-coupled receptor family as
the only ionotropic receptor (Derkach et al, 1989; Maricq
et al, 1991). Electrophysiologically, 5-HT mediates rapid
excitatory responses through ionotropic 5-HT
3
receptors
(Derkach et al, 1989) and activation of this channel results
in depolarization responses and subsequent desensitization
(Lambert et al, 1989; Yakel and Jackson, 1988).
The 5-HT
3
receptor is comprised of five coassembled
subunits that surround a centrally gated channel (Boess
et al, 1995). The 5-HT
3A
subunit has been cloned (Maricq
et al, 1991) and shown to be distributed peripherally
and centrally in brain regions including the hippocampus,
amygdala, and cortex (Tecott et al, 1993). 5-HT binds to
the extracellular N-terminus of the 5-HT
3A
subunit (Eisele
et al, 1993), but receptor activation can be enhanced
pharmacologically at allosteric sites (Lovinger and
Zhou, 1993). A novel molecular subunit (eg 5-HT
3B
) was
recently found to be coexpressed with the 5-HT
3A
subunit
in human amygdala, caudate, and hippocampus (Davies
et al, 1999) and heteromeric assemblies of human
5-HT
3A
and 5-HT
3B
subunits show channel conductance,
calcium permeability, and current–voltage properties
that closely resemble native neuronal 5-HT
3
channels
(Davies et al, 1999). However, in rats 5-HT
3B
subunit
transcripts are not found in brain (Morales and Wang,
2002), which suggests that rodent neural 5-HT
3
receptors
might be 5-HT
3A
homomeric receptors or heteromeric
receptors containing 5-HT
3A
subunits combined with
subunits other than the 5-HT
3B
subunit (Fletcher and
Barnes, 1998).
Online publication: 28 April 2004 at http://www.acnp.org/citations/
Npp04280403515/default.pdf
Received 5 November 2003; revised 11 March 2004; accepted 17
March 2004
*Correspondence: Dr CW Hodge, Department of Psychiatry, Bowles
Center for Alcohol Studies; CB#7178, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599, USA, Tel: +1 919 843 4823,
Fax: +1 919 966 5679; E-mail: chodge@med.unc.edu
Neuropsychopharmacology (2004), 1–7
&
2004 Nature Publishing Group All rights reserved 0893-133X/04
$
30.00
www.neuropsychopharmacology.org
Ethanol alters the function of 5-HT
3
receptors (Barann
et al, 1995; Jenkins et al, 1996; Lovinger et al, 2000; Lovinger
and Zhou, 1993; Lovinger and Zhou, 1994; Lovinger and
Zhou, 1998) with the most consistent finding being
potentiation of receptor function by ethanol (Lovinger,
1999). Biochemically, infusion of the 5-HT
3
antagonist
tropisetron in the nucleus accumbens decreases elevation
of extracellular dopamine produced by local application of
ethanol (Yoshimoto et al, 1992). Accordingly, a number of
studies have shown that systemic administration of 5-HT
3
antagonists decreases voluntary alcohol drinking by rats
(Fadda et al, 1991; Knapp and Pohorecky, 1992; Kostowski
et al, 1993; Tomkins et al, 1995) and mice (Tomkins et al,
1995), and reduce operant ethanol self-administration by
rats (Hodge et al, 1993). Site-specific injection of 5-HT
3
antagonists in the nucleus accumbens also decreases alcohol
consumption (Jankowska and Kostowski, 1995). In humans,
the 5-HT
3
antagonist ondansetron reduces alcohol-induced
craving in social drinkers (Johnson et al, 1993) and
increases abstinence among alcoholics with a biological
predisposition (Johnson et al, 2000).
Since neural 5-HT
3
receptors might be homomeric or
heteromeric receptors containing 5-HT
3A
subunits and 5-HT
3
antagonists show equal affinity for 5-HT
3A
and 5-HT
3A/B
receptors (Brady et al, 2001), the functional significance of
specific 5-HT
3
receptor subunits in alcohol drinking has not
been determined. To address this issue, we studied voluntary
alcohol drinking both during and after a sucrose fading
procedure in mice with a targeted deletion of the 5-HT
3A
receptor subunit (Kelley et al, 2003; Zeitz et al, 2002b).
Moreover, to determine if the 5-HT
3
antagonists reduce
alcohol drinking by inhibiting 3A containing receptors, we
tested the effects of the 5-HT
3
antagonist LY-278,584 on
alcohol drinking by 5-HT
3A
-null mice and controls.
METHODS
Mice
5-HT
3A
receptor-null mice were derived by homologous
recombination as previously reported (Zeitz et al, 2002a).
F1 hybrid C57BL/6J 129vJ heterozygous progeny were
backcrossed to C57BL/6J mice to produce F9 generation
congenics. Heterozygotes from the F9 generation were bred
to generate male wild-type and 5-HT
3A
-null mutants used in
the present study. Genotyping was conducted in Dr David
Julius’ laboratory at the University of California San
Francisco as described in Zeitz et al (2002a). All experi-
ments were conducted in male mice. The experimenter
conducting behavioral studies was blind to genotype. The
mice were housed in plastic cages lined with Cell Sorb
bedding and provided with food (Harlan, Indianapolis, IN)
and water ad libitum. The vivarium was maintained on a
12 h light/dark cycle (lights on at 06 : 00) at a temperature of
221C. All procedures were carried out in accordance with
the NIH Guide to Care and Use of Laboratory Animals and
institutional guidelines.
Locomotor Activity
Spontaneous locomotor activity and habituation of naive
5-HT
3A
(/, n ¼ 16) and 5-HT
3A
( þ / þ , n ¼ 16) mice was
measured in Plexiglas chambers (43 cm
2
) located in sound-
attenuating cubicles equipped with exhaust fans that
masked external noise (Med Associates, St Albans, VT).
Two sets of 16 pulse-modulated infrared photobeams were
placed on opposite walls at 1-in centers to record xy
ambulatory movements. Activity chambers were computer
interfaced (Med Associates) for data sampling at 100-ms
resolution. Mice were handled and weighed daily for 1 week
before activity testing. Prior to each session, the activity
chamber was wiped clean with 2.5% glacial acetic acid to
limit any confounding odors. The mice were placed in the
corner of the chamber and left to behave freely for 60 min.
At 1 week after baseline locomotor testing, the mice were
used to determine the effects of ethanol on locomotor
activity. The same procedure was used to test ethanol-
induced locomotor activity except that mice were adminis-
tered ethanol (0. 0.5, 1, or 2 g/kg, i.p.) in a Latin-Square
randomized dose order immediately before the start of
activity monitoring. Horizontal distance traveled (cm) was
recorded for 60 min after ethanol injection. Tests sessions
were separated by 3 days.
Alcohol Drinking
Oral alcohol drinking and preference were examined using a
two-bottle choice protocol (Hodge et al, 1999). Eight
experimentally naı
¨
ve male 5-HT
3A
( þ / þ ) and six 5-HT
3A
(/) mice were tested in parallel. Mice were given 1 week
to acclimatize to individual housing conditions and
handling. During this period, water was the only fluid
available. Subsequently, two-bottle drinking sessions were
then conducted 24 h per day, 7 days per week, in the home-
cage. Ethanol and water solutions were in 50-ml plastic
bottles (0.5 ml graduates) equipped with ball-bearing
stoppers to limit spillage. The location (left or right side
of the cage) of ethanol and water solutions was counter-
balanced between animals to control for side preference.
Volume was recorded at the beginning and end of access
periods. No significant spillage was noted throughout the
experiment.
To evaluate potential involvement of the 5-HT
3A
receptor
in the initiation of alcohol drinking, mice were given access
to a series of sweetened ethanol solutions vs water (eg
Samson, 1986). The sucrose (% w/v) and ethanol (% v/v)
content of the solutions were as follows: 10S/2E; 10S/5E;
10S/10E; 5S/10E; 2S/10E; 10E. Mice were exposed to each
sweetened ethanol solution for 3 days and 10% ethanol at
the end for a 6-day period.
After evaluating initiation of ethanol intake, the drinking
solutions were returned to 2S/10E vs water for a 3-day
baseline period. Then, the effects of the 5-HT
3
antagonist
LY-278,584 (0–1 mg/kg) were evaluated on sweetened
ethanol vs water intake. After determining the LY-278,584
dose–response curve, mice were re-exposed to 10E vs water
for a 3-day baseline period. The effects of LY-278,584 were
then redetermined on unsweetened ethanol vs water intake.
Drug dosing was conducted in a randomized Latin-Square
manner. Injections occurred immediately prior to 24-h
drinking sessions no more than twice per week. The day
immediately prior to saline vehicle treatment was used as a
noninjection control in each experiment.
5-HT
3
antagonist-induced reductions in alcohol drinking
CW Hodge et al
2
Neuropsychopharmacology
RESULTS
Locomotor Activity
To determine if any overt motor changes were produced by
the targeted mutation, we first tested 5-HT
3A
-null mice and
wild-type littermates in a novel open-field environment for
1 h. The null mutation produced no effect on spontaneous
locomotor activity (Figure 1a). Moreover, locomotor
activity by both genotypes decreased as a function of time
in the open-field environment (F
time
(5,150) ¼ 13.55,
po0.001), which indicates normal habituation to the
environment. This finding is consistent with our previous
report (Kelley et al, 2003) and supports pharmacological
evidence, which indicates that 5-HT
3
receptor antagonists
have no effects on locomotor behavior (Jones et al, 1988).
The effects of ethanol on locomotor activity of the 5-
HT
3A
-null mice and wild-type littermates are illustrated in
Figure 1b. Ethanol significantly reduced locomotor activity
(F
dose
(3,90) ¼ 5.08, p ¼ 0.003), which was due to reduced
activity at the 2 g/kg ethanol dose as compared to the 1 g/kg
dose (Tukey, p ¼ 0.001). However, the ethanol-induced
activity of the two genotypes did not differ. Further,
planned comparisons revealed that locomotor activity after
ethanol injections did not differ from the saline injection for
the 5-HT
3A
-null mice and wild-type littermates. These
findings are consistent with pharmacological evidence
showing no effect of 5-HT
3
receptor antagonists on
ethanol-induced locomotor activity (Le et al, 1997), and
the absence of ethanol-induced locomotor stimulation in
C57BL/6J mice (Liljequist and Ossowska, 1994).
Alcohol Drinking
When examined through the course of a sucrose fading
procedure (eg Samson, 1986) conducted during daily 24-h
periods in the home cage 5-HT
3A
receptor-null mice did not
differ from wild-type controls on measures of ethanol intake
or preference (Figure 2a and 2b). Repeated measures
ANOVA showed that intake and preference decreased
as a function of reduced sucrose and increased ethanol
concentration in both genotypes (F
dose
(6,72) ¼ 45.57,
po0.001). Although ethanol intake was relatively low for
C57BL/6J mice (Middaugh et al, 1999), no differences were
observed in ethanol, water, or total fluid intake between
genotypes. These data indicate that deletion of the 5-HT
3A
receptor subunit does not influence the acquisition or
Figure 1 Deletion of the 5-HT
3A
receptor subunit produced no
significant effects on motor activity or locomotor response to ethanol. (a)
Spontaneous locomotor activity and habituation to a novel environment.
Both null mice (/, n ¼ 16) and wild-type controls ( þ / þ , n ¼ 16)
showed normal levels of locomotor activity that decreased as a function of
time in the environment. Horizontal distance traveled (cm 7 standard
error) was averaged for each 10 min of a 60 min exposure. (b) Locomotor
response to acute ethanol administration. All data are expressed as mean
(7SEM). * indicates significantly different from initial 10-min period
irrespective of genotype, po0.05, Tukey test.
Figure 2 Initiation and maintenance of alcohol drinking by mice lacking
the 5-HT
3A
receptor gene. (a) Null mice (/, n ¼ 6) consumed the same
amount of sweetened and unsweetened alcohol in a home-cage two-
bottle preference test as wild-type mice ( þ / þ , n ¼ 8). (b) The null
mutation had no effect on ethanol preference expressed as a percentage of
total fluid intake (ethanol mls plus water mls). Each animal’s alcohol intake
(g/kg/24-h) or preference data were averaged over a 3-day exposure
period at each ethanol/sucrose combination and then plotted as a group
mean (7SEM).
5-HT
3
antagonist-induced reductions in alcohol drinking
CW Hodge et al
3
Neuropsychopharmacology
maintenance of alcohol drinking under these experimental
conditions.
Figure 3a shows the effects of the 5-HT
3
antagonist LY-
278,584 on sweetened ethanol (2% sucrose þ 10% EtOH)
intake. LY-278,584 dose dependently decreased intake of
sweetened ethanol (F
dose
(3,33) ¼ 4.6, p ¼ 0.009), which was
primarily due to a significant difference between the no-
injection control and 10 mg/kg dose of LY-278,584 (Tukey,
p ¼ 0.001). There was no genotypic difference or interaction
between LY-278,584 and genotype. Planned comparisons
within the wild-type group showed that ethanol intake
following no-injection differed from the 1 and 10 mg/kg
doses of LY-278,584 (p ¼ 0.009 and 0.01, respectively),
whereas intake after saline differed only from the 10 mg/kg
dose (p ¼ 0.013) (Figure 3a). LY-278,584 produced no
significant effects on alcohol drinking by 5-HT
3A
-null mice.
There was also a main effect of dose of LY-278,584 on
preference for sweetened ethanol relative to water (F
dose
(3,33) ¼ 4.7, p ¼ 0.008), which was attributable to a signifi-
cant reduction in preference only in the wild-type group
(Table 1). There was no effect of genotype, or a genotype by
dose interaction, on sweetened ethanol preference. No
differences were observed in water, or total fluid, intake.
Statistical analysis of unsweetened ethanol intake (g/kg)
found no main effect of genotype or LY-278,584 (Figure 3b).
However, the effects of LY-278,584 on ethanol intake (g/kg)
depended on genotype as evidenced by a significant
interaction (F
dose genotype
(3,33) ¼ 2.9, p ¼ 0.04). Planned
comparisons showed that LY-278,584 (10 mg/kg) signifi-
cantly decreased ethanol intake as compared to no-injection
and saline controls (p ¼ 0.009 and 0.01, respectively) in
wild-type mice only (Figure 3b). Ethanol intake by 5-HT
3A
-
null mice was unchanged by any dose of the antagonist
(Figure 3b). Thus, the significant interaction was due to
antagonist-induced decreases in ethanol intake only within
the wild-type group. RM ANOVA showed a trend toward a
significant decrease in preference for unsweetened ethanol
produced by LY-278,584 (F
dose
(3,33) ¼ 2.5, p ¼ 0.07). There
was no effect of genotype, and no interaction among the
variables. Planned comparisons across genotype and dose
of the 5-HT
3
antagonist showed that the 10 mg/kg dose of
LY-278,584 significantly decreased preference only in the
wild-type group (Table 1). No differences were observed in
water, or total fluid, intake.
DISCUSSION
The 5-HT
3
receptor has emerged as a potential therapeutic
target for the medical management of alcohol abuse and
alcoholism because inhibition of the receptor by selective
antagonists produces positive effects in preclinical (Costall
Figure 3 The 5-HT
3
antagonist LY-278,584 significantly decreased
intake (g/kg/24 h) of sweetened (2S/10E) (a) or unsweetened (10E) (b)
alcohol in a 24-h home-cage two-bottle test in 5-HT
3A
wild-type mice
( þ / þ , n ¼ 8). Data represent mean (7SEM) during a single 24-h period
following i.p. administration of LY-278,584. Variability in ethanol intake by
5-HT
3A
-null mice (/, n ¼ 6) was due to a single outlier (b). Note the
different y-axis scales on the two panels. * indicates significantly different
from no-injection control within genotype; w indicates significantly
different from both no-injection and saline vehicle control within genotype,
po0.05, Tukey test.
Table 1 Effect of LY-278,584 on Preference (mean7SEM) for Sweetened and Unsweetened Ethanol
LY-278,584 (mg/kg, i.p.)
Solution Genotype No injection Saline 1.0 10.0
2S/10E 5-HT
3A
(+/+) 0.71 (0.03) 0.74 (0.09) 0.59 (0.06) 0.46 (0.11)
a
5-HT
3A
(/) 0.74 (0.08) 0.78 (0.06) 0.70 (0.06) 0.69 (0.07)
10E 5-HT
3A
(+/+) 0.21 (0.04) 0.20 (0.04) 0.17 (0.009) 0.10 (0.04)
b
5-HT
3A
(/) 0.27 (0.06) 0.21 (0.06) 0.23 (0.06) 0.23 (0.08)
a
significantly different from no-injection and saline controls within 5-HT
3A
(+/+) group, and from 5-HT
3A
(/) at corresponding dose.
b
significantly different from no-injection control.
5-HT
3
antagonist-induced reductions in alcohol drinking
CW Hodge et al
4
Neuropsychopharmacology
et al, 1993) and clinical (Sellers et al, 1994) studies. A
common finding from preclinical studies is that 5-HT
3
antagonists selectively decrease alcohol drinking and
reinforcement (Fadda et al, 1991; Hodge et al, 1993;
Jankowska and Kostowski, 1995; Knapp and Pohorecky,
1992; McKinzie et al, 1998; Silvestre et al, 1998; Tomkins
et al, 1995), suggesting that alcohol self-administration is at
least partly maintained by 5-HT
3
receptor activity (but see
Beardsley et al, 1994).
Ionotropic 5-HT
3
receptors are thought to consist of five
coassembled subunits that surround a centrally gated
channel (Boess et al, 1995). The 5-HT
3A
subunit (Maricq
et al, 1991) is the only known 5-HT
3
subunit that occurs in
rodent brain (Morales and Wang, 2002). It is not known,
however, if the effects of 5-HT
3
antagonists on alcohol
drinking are mediated by this subunit or other(s), which
may coexpress with the 3A subunit (eg, Fletcher and Barnes,
1998). To address this question, we examined alcohol
drinking by mutant mice lacking the 5-HT
3A
receptor
subunit (Zeitz et al, 2002a). One of the primary findings of
this study is that 5-HT
3A
-null mice did not differ from wild-
type controls on measures of voluntary alcohol drinking
either during or after a sucrose fading initiation procedure.
These data are in contrast with a number of studies showing
that 5-HT
3
antagonists decrease alcohol drinking and
operant self-administration (Hodge et al, 1993; Knapp and
Pohorecky, 1992).
To determine, therefore, if decreases in alcohol drinking
produced by 5-HT
3
antagonists might be dependent on the
5-HT
3A
receptor subunit, we tested the effects of the 5-HT
3
antagonist LY-278,584 on intake (g/kg/24-h) of sweetened
and unsweetened ethanol by null mice and wild types. LY-
278,584 decreased intake of, and preference for, both
ethanol solutions only in wild-type mice. This indicates
that the 5-HT
3A
receptor subunit is required for 5-HT
3
antagonist-induced reductions in alcohol drinking, and
strongly suggests that 5-HT
3
antagonist-induced reductions
in ethanol drinking (Fadda et al, 1991; Hodge et al, 1993;
Jankowska and Kostowski, 1995; Knapp and Pohorecky,
1992; McKinzie et al, 1998; Silvestre et al, 1998; Tomkins
et al, 1995) are likely due to pharmacological blockade of
the 5-HT
3
receptor subunit. Moreover, these data indicate
that other 5-HT
3
receptor subunits, which may be expressed
in brain (Fletcher and Barnes, 1998; Morales and Wang,
2002; Niesler et al, 2003), are not able to form functional
channels that are as sensitive to 5-HT
3
antagonists as native
receptors containing the 3A subunit.
A question that emerges from these data is why deletion
of the 5-HT
3A
receptor had no effect on alcohol drinking
even though the 5-HT
3
antagonist decreased drinking in
wild-type mice. One possible explanation for the lack of
agreement between targeted gene deletion and pharmaco-
logical inhibition of the 5-HT
3
receptor is that develop-
mental compensation occurred in the null mutants, which
may have masked any functional involvement of the
receptor subunit in alcohol drinking. Compensation may
be due to genetic redundancy whereby so-called ‘helper’
genes can take over the function of the targeted gene
(Gerlai, 1996; Gerlai, 2001). Thus, it is possible that the
5-HT
3A
receptor subunit is an important mediator of
alcohol drinking, but compensation by another unknown
pathway concealed this involvement. In this regard, gene
targeting missed the mark of revealing new information
about 5-HT
3
receptor regulation of alcohol drinking.
Whether new information could be gained by investigating
the mechanisms of developmental compensation, such as
uncovering novel interactions between the 5-HT
3A
receptor
subunit and other genes, is a matter of debate (eg Gerlai,
2001; Routtenberg, 1995; Routtenberg, 1996).
Compensation, however, may not occur in all phenotypes
associated with the targeted gene. For instance, we recently
reported that 5-HT
3A
-null mice exhibit less anxiety-
like behavior on three validated animal models of anxiety
(Kelley et al, 2003). This phenotypic profile corresponds
with the known anxiolytic properties of pharmacological
antagonists of 5-HT
3
receptors (eg Costall and Naylor, 1991)
and suggests that the 5-HT
3A
receptor subunit is
an important mediator of anxiety. One might conclude,
therefore, that deletion of the 5-HT
3A
receptor subunit
did not produce compensatory changes in neurobio-
logical pathways associated with anxiety. Moreover, it
seems reasonable to conclude that compensatory changes
that might have masked 5-HT
3A
receptor involvement in
alcohol drinking are not associated with anxiety-like
behavior.
A final remark regarding potential compensatory factors
merits discussion. Genetic background may interact with
the mutated gene in ways that can alter phenotypic
expression. For instance, protein kinase C gamma (PKCg)-
null mutants from a C57BL/6J 129/SvJ mixed genetic
background exhibited reduced ethanol sensitivity and the
absence of ethanol tolerance, however, expression of the
null mutation on a C57BL/6J background eliminated the
phenotypes (Bowers 1999). Mice used in the present study
were congenic C57BL/6J, which are widely known as a
model of high alcohol preference (Belknap, 1993). Thus, it is
plausible that background genes driving alcohol drinking in
this mouse strain might blunt the effects of some null
mutations, such as the 5-HT
3A
receptor.
ACKNOWLEDGEMENTS
This research was supported by Grants AA09981 and
AA011605 (CWH). Portions of the work, including mouse
breeding and genotyping, were supported by funds
provided by the State of California for medical research
on alcohol and substance abuse through the University of
California at San Francisco. We are grateful to Dr David
Julius for initial contribution of the mice.
REFERENCES
Barann M, Ruppert K, Gothert M, Bonisch H (1995). Increasing
effect of ethanol on 5-HT3 receptor-mediated 14C-guanidinium
influx in N1E-115 neuroblastoma cells. Naunyn-Schmiedebergs
Arch Pharmacol 352: 149–156.
Barnes NM, Sharp T (1999). A review of central 5-HT receptors
and their function. Neuropharmacology 38: 1083–1152.
Beardsley PM, Lopez OT, Gullikson G, Flynn D (1994). Serotonin
5-HT3 antagonists fail to affect ethanol self-administration of
rats. Alcohol 11: 389–395.
Belknap JK, Crabbe JC, Young ER (1993). Voluntary consumption
of ethanol in 15 inbred mouse strains. Psychopharmacology 112:
503–510.
5-HT
3
antagonist-induced reductions in alcohol drinking
CW Hodge et al
5
Neuropsychopharmacology
Boess FG, Beroukhim R, Martin IL (1995). Ultrastructure of the
5-hydroxytryptamine3 receptor. J Neurochem 64: 1401–1405.
Bowers BJ, Owen EH, Collins AC, Abeliovich A, Tonegawa S,
Wehner JM (1999). Decreased ethanol sensitivity and tolerance
development in gamma-protein kinase C null mutant mice is
dependent on genetic background. Alcohol: Clin Exp Res 23:
387–397.
Brady CA, Stanford IM, Ali I, Lin L, Williams JM, Dubin AE et al
(2001). Pharmacological comparison of human homomeric
5-HT3A receptors versus heteromeric 5-HT3A/3B receptors.
Neuropharmacology 41: 282–284.
Costall B, Domeney AM, Kelly ME, Naylor RJ (1993). The effects of
5-HT3 receptor antagonists in models of dependency and
withdrawal. Alcohol Alcohol Suppl 2: 269–273.
Costall B, Naylor RJ (1991). Pharmacological properties and
functions of central 5-HT3 receptors. Therapie 46: 437–444.
Davies PA, Pistis M, Hanna MC, Peters JA, Lambert JJ, Hales TG
et al (1999). The 5-HT3B subunit is a major determinant of
serotonin-receptor function. Nature 397: 359–363.
Derkach V, Surprenant A, North RA (1989). 5-HT3 receptors are
membrane ion channels. Nature 339: 706–709.
Eisele JL, Bertrand S, Galzi JL, Devillers-Thiery A, Changeux JP,
Bertrand D (1993). Chimaeric nicotinic-serotonergic receptor
combines distinct ligand binding and channel specificities.
Nature 366: 479–483.
Fadda F, Garau B, Marchei F, Colombo G, Gessa GL (1991). MDL
72222, a selective 5-HT3 receptor antagonist, suppresses
voluntary ethanol consumption in alcohol-preferring rats.
Alcohol Alcohol 26: 107–110.
Fletcher S, Barnes NM (1998). Desperately seeking subunits: are
native 5-HT3 receptors really homomeric complexes? Trends
Pharmacol Sci 19: 212–215.
Gerlai R (1996). Gene-targeting studies of mammalian behavior: is
it the mutation or the background genotype? (comment)
(erratum appears in Trends Neurosci 1996 Jul; 19(7): 271).
Trends Neurosci 19: 177–181.
Gerlai R (2001). Gene targeting: technical confounds and potential
solutions in behavioral brain research. Behav Brain Res 125:
13–21.
Hodge CW, Mehmert KK, Kelley SP, McMahon T, Haywood A,
Olive MF et al (1999). Supersensitivity to allosteric GABA(A)
receptor modulators and alcohol in mice lacking PKCepsilon.
Nat Neurosci 2: 997–1002.
Hodge CW, Samson HH, Lewis RS, Erickson HL (1993). Specific
decreases in ethanol but not water reinforced responding
produced by the 5-HT3 antagonist ICS 205-930. Alcohol 10:
191–196.
Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR,
Mylecharane EJ et al (1994). International Union of Pharmacol-
ogy classification of receptors for 5-hydroxytryptamine (Ser-
otonin). Pharmacol Rev 46: 157–203.
Jankowska E, Kostowski W (1995). The effect of tropisetron
injected into the nucleus accumbens septi on ethanol consump-
tion in rats. Alcohol 12: 195–198.
Jenkins A, Franks NP, Lieb WR (1996). Actions of general
anaesthetics on 5-HT3 receptors in N1E-115 neuroblastoma
cells. Br J Pharmacol 117: 1507–1515.
Johnson BA, Ait-Daoud N, Prihoda TJ (2000). Combining
ondansetron and naltrexone effectively treats biologically pre-
disposed alcoholics: from hypotheses to preliminary clinical
evidence. Alcohol: Clin Exp Res 24: 737–742.
Johnson BA, Campling GM, Griffiths P, Cowen PJ (1993).
Attenuation of some alcohol-induced mood changes and the
desire to drink by 5-HT3 receptor blockade: a preliminary study
in healthy male volunteers. Psychopharmacology 112: 142–144.
Jones BJ, Costall B, Domeney AM, Kelly ME, Naylor RJ, Oakley
NR et al (1988). The potential anxiolytic activity of GR38032F,
a 5-HT3-receptor antagonist. Br J Pharmacol 93: 985–993.
Kelley SP, Bratt AM, Hodge CW (2003). Targeted gene deletion of
the 5-HT3A receptor subunit produces an anxiolytic phenotype
in mice. Eur J Pharmacol 461: 19–25.
Knapp DJ, Pohorecky LA (1992). Zacopride, a 5-HT3 receptor
antagonist, reduces voluntary ethanol consumption in rats.
Pharmacol, Biochem Behav 41: 847–850.
Kostowski W, Dyr W, KrzaScik P (1993). The abilities of
5-HT3 receptor antagonist ICS 205-930 to inhibit alcohol
preference and withdrawal seizures in rats. Alcohol 10: 369–373.
Lambert JJ, Peters JA, Hales TG, Dempster J (1989). The properties
of 5-HT3 receptors in clonal cell lines studied by patch- clamp
techniques. Br J Pharmacol 97: 27–40.
Le AD, Tomkins D, Higgins G, Quan B, Sellers EM (1997).
D1 and D2 receptor antagonists on ethanol- and cocaine-
induced locomotion. Pharmacol Biochem Behav 57: 325–332.
Liljequist S, Ossowska K (1994). Genotypic differences in locomotor
stimulation and dopaminergic activity following acute ethanol
administration. Eur Neuropsychopharmacol 4: 31–38.
Lovinger DM (1999). 5-HT3 receptors and the neural actions of
alcohols: an increasingly exciting topic. Neurochem Int 35:
125–130.
Lovinger DM, Sung KW, Zhou Q (2000). Ethanol and trichloro-
ethanol alter gating of 5-HT3 receptor-channels in NCB-20
neuroblastoma cells. Neuropharmacology 39: 561–570.
Lovinger DM, Zhou Q (1993). Trichloroethanol potentiation of
5-hydroxytryptamine3 receptor-mediated ion current in nodose
ganglion neurons from the adult rat. J Pharmacol Exp Ther 265:
771–776.
Lovinger DM, Zhou Q (1994). Alcohols potentiate ion
current mediated by recombinant 5-HT3RA receptors expressed
in a mammalian cell line. Neuropharmacology 33: 1567–1572.
Lovinger DM, Zhou Q (1998). Alcohol effects on the 5-HT3 ligand-
gated ion channel. Toxicol Lett 100–101: 239–246.
Maricq AV, Peterson AS, Brake AJ, Myers RM, Julius D (1991).
Primary structure and functional expression of the 5HT3
receptor, a serotonin-gated ion channel. Science 254: 432–437.
McKinzie DL, Eha R, Cox R, Stewart RB, Dyr W, Murphy JM et al
(1998). Serotonin3 receptor antagonism of alcohol intake: effects
of drinking conditions. Alcohol 15: 291–298.
Middaugh LD, Kelley BM, Bandy AE, McGroarty KK (1999).
Ethanol consumption by C57BL/6 mice: influence of gender and
procedural variables. Alcohol 17: 175–183.
Morales M, Wang SD (2002). Differential composition of
5-hydroxytryptamine
3
receptors synthesized in the rat CNS
and peripheral nervous system. J Neurosci 22: 6732–6741.
Niesler B, Frank B, Kapeller J, Rappold GA (2003). Cloning,
physical mapping and expression analysis of the human 5-HT3
serotonin receptor-like genes HTR3C, HTR3D and HTR3E. Gene
310: 101–111.
Routtenberg A (1995). Knockout mouse fault lines (comment).
Nature 374: 314–315.
Routtenberg A (1996). Reverse piedpiperase: is the knockout
mouse leading neuroscientists to a watery end? (comment).
Trends Neurosci 19: 471–472.
Samson HH (1986). Initiation of ethanol reinforcement using a
sucrose-substitution procedure in food- and water-sated rats.
Alcohol: Clin Exp Res 10: 436–442.
Sellers EM, Toneatto T, Romach MK, Somer GR, Sobell LC, Sobell
MB (1994). Clinical efficacy of the 5-HT3 antagonist ondanse-
tron in alcohol abuse and dependence. Alcohol: Clin Exp Res 18:
879–885.
Silvestre JS, Palacios JM, Fernandez AG, O’Neill MF (1998).
Comparison of effects of a range of 5-HT receptor modulators on
consumption and preference for a sweetened ethanol solution in
rats. J Psychopharmacol 12: 168–176.
Tecott LH, Maricq AV, Julius D (1993). Nervous system
distribution of the serotonin 5-HT3 receptor mRNA. Proc Natl
Acad Sci USA 90: 1430–1434.
5-HT
3
antagonist-induced reductions in alcohol drinking
CW Hodge et al
6
Neuropsychopharmacology
Tomkins DM, Le AD, Sellers EM (1995). Effect of the 5-HT3
antagonist ondansetron on voluntary ethanol intake in rats and
mice maintained on a limited access procedure. Psychopharma-
cology 117: 479–485.
Yakel JL, Jackson MB (1988). 5-HT3 receptors mediate rapid
responses in cultured hippocampus and a clonal cell line.
Neuron 1: 615–621.
Yoshimoto K, McBride WJ, Lumeng L, Li TK (1992). Alcohol
stimulates the release of dopamine and serotonin in the nucleus
accumbens. Alcohol 9: 17–22.
Zeitz KP, Guy N, Malmberg AB, Dirajlal S, Martin WJ, Sun L
et al (2002a). The 5-HT3 subtype of serotonin receptor
contributes to nociceptive processing via a novel subset of
myelinated and unmyelinated nociceptors. J Neurosci 22:
1010–1019.
Zeitz KP, Guy N, Malmberg AB, Dirajlal S, Martin WJ, Sun L
et al (2002b). The 5-HT3 subtype of serotonin receptor
contributes to nociceptive processing via a novel subset of
myelinated and unmyelinated nociceptors. J Neurosci 22:
1010–1019.
5-HT
3
antagonist-induced reductions in alcohol drinking
CW Hodge et al
7
Neuropsychopharmacology
    • "Thus, USERs in conjunction with current transgenic approaches can be used to increase our understanding of the pharmacological and physiologic effects of ethanol action by establishing precise links between specific receptor subunits and behavioral outcomes. In addition to GlyRs and GABA A Rs, other members of the Cys-loop superfamily of LGICs, including the neuronal nicotinic cholinergic and serotonergic receptors, have been linked to a number of behavioral effects of ethanol administration (Knapp and Pohorecky, 1992; Hodge et al., 2004; Kamens et al., 2010a,b). Since the sequence homology among subunits of Cys-loop receptors is on the order of 30% (Olsen and Sieghart, 2008), this suggests that the applicability of USER technology can be extended to other receptors within the Cys-loop superfamily. "
    [Show abstract] [Hide abstract] ABSTRACT: We recently developed Ultra-Sensitive Ethanol Receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild type (WT) receptors. The current study investigated: 1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and 2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
    Full-text · Article · Mar 2015
    • "All mice were single-housed for this study, and daily fluid intake was recorded. At the start of the 6 weeks of ethanol consumption, the modified sucrose-fading technique (Samson, 1986) was adapted to incentivize the mice to the ethanol solution similar to previous studies (Becker & Lopez, 2004; Camarini & Hodge, 2004; Hodge et al., 2004; Sanna et al., 2011). At all times, an alternative source of untreated water was freely available to all mice. "
    [Show abstract] [Hide abstract] ABSTRACT: Withdrawal from a chronic period of alcohol consumption is commonly associated with the manifestation of depression, potentially exerting a significant influence on treatment prospects and increasing the likelihood of relapse. Better therapeutic strategies need to be developed to assist with rehabilitation. Here, we report the detection of depression-related behaviours in a mouse model of 6-week free-choice ethanol (10%, v/v) consumption followed by 2-week abstinence. Mice abstinent from alcohol showed increased immobility time on the forced-swim test, reduced saccharin consumption and increased latency to feed in the novelty-suppressed feeding test. By comparison, there was no significant effect on anxiety-related behaviours as determined by testing on the light-dark box and elevated plus maze. We found that the provision of running-wheels through the duration of abstinence attenuated depressive behaviour in the forced-swim and novelty-suppressed feeding tests, and increased saccharin consumption. Given the link between withdrawal from addictive substances and depression, this model will be useful for the study of the pathophysiology underlying alcohol-related depression. The findings of this study establish an interaction between physical activity and the development of behavioural changes following cessation of alcohol consumption that could have implications for the development of rehabilitative therapies.
    Full-text · Article · Mar 2013
    • "The differences in experimental findings may be explained by strain or line of rat or the antagonist itself, some of which are not that selective for the 5-HT 3 receptor (Engleman et al., 2008). In a 5-HT 3A receptor knock-out study, the presence of the 5-HT 3A receptor was required for the selective 5-HT 3A receptor antagonist, LY-278-584, to reduce alcohol consumption, underscoring the importance of this receptor in alcohol consumption (Hodge et al., 2004). Transgenic mice overexpressing the 5-HT 3A receptor drank less alcohol than wild-type mice (Engel et al., 1998), but these mice had increased sensitivity to the low dose effects of alcohol (Engel and Allan, 1999). "
    [Show abstract] [Hide abstract] ABSTRACT: The 5-Hydroxytryptamine3 (5-HT3) receptor is a member of the cys-loop family of ligand gated ion channels, of which the nicotinic acetylcholine receptor is the prototype. All other 5-HT receptors identified to date are metabotropic receptors. The 5-HT3 receptor is present in the central and peripheral nervous systems, as well as a number of non-nervous tissues. As an ion channel that is permeable to the cations, Na(+), K(+), and Ca(2+), the 5-HT3 receptor mediates fast depolarizing responses in pre- and post-synaptic neurons. As such, 5-HT3 receptor antagonists that are used clinically block afferent and efferent synaptic transmission. The most well established physiological roles of the 5-HT3 receptor are to coordinate emesis and regulate gastrointestinal motility. Currently marketed 5-HT3 receptor antagonists are indicated for the treatment of chemotherapy, radiation, and anesthesia-induced nausea and vomiting, as well as irritable bowel syndrome. Other therapeutic uses that have been explored include pain and drug addiction. The 5-HT3 receptor is one of a number of receptors that play a role in mediating nausea and vomiting, and as such, 5-HT3 receptor antagonists demonstrate the greatest anti-emetic efficacy when administered in combination with other drug classes.
    Article · Feb 2011
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