The Journal of Neuroscience, July 16, 2003, 23(15):6255-6263
Previous Article | Next Article 
Altered Anxiety-Related Responses in Mutant Mice Lacking the 
4 Subunit of the Nicotinic Receptor
Ramiro Salas,
Fredalina Pieri,
Beryl Fung,
John A. Dani, and
Mariella De Biasi
Division of Neuroscience, Baylor College of Medicine, Houston, Texas
77030
 |
Abstract
|
|---|
Nicotine, acting at nicotinic acetylcholine receptors (nAChRs), is the
primary addictive component of tobacco. Smokers often report an anxiolytic
effect of cigarettes. This relief of anxiety, attributed to nicotine, is an
important contributor to relapse when smokers try to quit. Hence, the study of
the anxiolytic effects of nicotine is important for understanding the
mechanisms underlying nicotine addiction. Mammalian nAChRs are pentameric ion
channels usually composed of
and
subunits. Taking advantage
of4-homozygous-null mice (4-/-), we examined the role of the nAChR
4 subunit in anxiety-related behaviors. The 4-/- mice behaved as
though they were less anxious than wild-type littermates on the elevated-plus
and staircase mazes, tests that measure anxiety-related behaviors. To obtain
an independent, physiological indication of the stress produced by several
tests, we measured changes in heart rate using telemetry. Consistently with
the behavioral phenotype, 4-/- mice had a smaller heart rate increase in
the elevated-plus maze than did wild-type littermates. In contrast, during
social isolation, a separate test for anxiety,4-/- mice exhibited a
greater increase in heart rate than did wild-type littermates. Finally,
4-/- mice were indistinguishable from their wild-type littermates in the
open field, the light/dark box, and the mirrored chamber. The overall results
demonstrate that 4-containing (4*) nAChRs influence behavioral
responses during anxiety-related tests, and that this effect depends on the
type of anxiety-provoking experience. Through their influence on
anxiety-related behavior, 4* nAChRs might influence both tobacco
consumption and smoking relapse.
Key words: nicotinic acetylcholine receptors; 4 subunit; anxiety; elevated-plus maze; open field; light/dark box; telemetry; heart rate
|
Introduction
|
|---|
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric
ligand-gated cation channels that mediate the addictive properties of nicotine
(Dani and De Biasi, 2001
). In
mammals, eight (2 to 7, 9, and 10) and
three (2 to 4) subunits have been cloned
(Sargent, 1993; Elgoyhen et
al., 1994,
2001;
McGehee and Role, 1995;
Lindstrom et al., 1996;
Tassonyi et al., 2002). Most
nAChRs are composed of and subunits. 7 can form either
homopentamers or heteropentamers
(Seguela et al., 1993;
Khiroug et al., 2002), and
9 and 10 are believed to assemble as -only
heteropentamers (Elgoyhen et al.,
2001; Sgard et al.,
2002). Neuronal nAChRs have been implicated in processes such as
memory (Levin and Simon,
1998), anxiety (File et al.,
2000; Ross et al.,
2000), sleep control (Domino
and Yamamoto, 1965;
Salin-Pascual et al., 1999),
antinociception (Marubio et al.,
1999), and autonomic nervous system function (Xu et al.,
1999a,b;
De Biasi, 2002). Nicotinic
receptors may also be involved in neurological disorders such as nocturnal
epilepsy, schizophrenia, Parkinson's disease, and Alzheimer's disease
(Levin and Simon, 1998;
Rusted et al., 2000;
Dani and De Biasi, 2001;
Leonard et al., 2001;
Moulard, 2001).
A number of nicotinic receptor-mutant mice has been generated by various
groups, and their analysis has shed light on the functions of different nAChR
subunits: 3 (Xu et al.,
1999a; Yu et al.,
2000), 4 (Marubio et
al., 1999; Ross et al.,
2000; Labarca et al.,
2001), 5 (N. Wang et
al., 2002; Salas et al.,
2003), 6 (Champtiaux et
al., 2002), 7
(Orr-Urtreger et al., 1997;
Paylor et al., 1998;
Franceschini et al., 2000,
2002;
Broide et al., 2002), 2
(Picciotto, 1995;
Zoli et al., 1999;
Cohen et al., 2002;
Shoaib et al., 2002),
2/4 (Xu et al.,
1999b), and 3 (Booker et
al., 2000). A recurrent behavioral phenotype among nAChR-mutant
mice is a difference in anxiety-like responses on the elevated-plus maze with
little if any effect on other anxiety tests, as revealed by the analysis of
3- and 4-mutant mice (Booker
et al., 2000; Ross et al.,
2000; Labarca et al.,
2001).
Nicotine has been shown to affect anxiety in different ways
(Picciotto et al., 2002). In
rodents, nicotine can be anxiolytic, anxiogenic, or have no effect on anxiety,
depending on the dose used and the route of administration, even when the same
behavioral test is performed (Cheeta et
al., 2000; File et al.,
2000). In humans, according to smokers' accounts, smoking may have
anxiolytic effects, which are likely attributable to the nicotine contained in
cigarettes (Kassel and Unrod,
2000). However, despite the reported anxiolytic effect of
cigarettes, smokers display higher scores in anxiety-related tests than
nonsmokers and smokers who quit (Parrott,
1995).
The 4 subunit of nicotinic receptors is widely expressed in the
peripheral nervous system, but in the rat CNS, it is restricted to a few
regions (Duvoisin et al.,
1989; Dineley-Miller and
Patrick, 1992; Poth et al.,
1997; Xu et al.,
1999b). We show here that, in the mouse, the CNS expression
pattern of 4 is even more restricted than in the rat, with its
expression significantly detected only in the olfactory bulb (Olf), medial
habenula (MHb), pineal gland (Pin), interpeduncular nucleus (IPN), and
inferior colliculus (IC). To date, no specific function for CNS
4-containing (4*) nAChRs has been reported. Our data show that
4-/- mice display behavioral differences when compared with their
wild-type (4+/+) littermates on anxiety-related tests. The 4-/-
mice showed increased exploratory behavior in the elevated-plus maze and
increased climbing activity in the staircase maze, suggesting decreased
anxiety-like behavior. Conversely, social isolation was more anxiogenic in the
4-/- mice. The behavior in the open field, light/dark box, and mirror
chamber was unchanged.
|
Materials and Methods
|
|---|
Animals. 4 -/- mice were
generated as described previously (Xu et
al., 1999b). Experiments were performed on mice backcrossed for
six generations into the C57BL/6J background. All of the tests were performed
on 2- to 7-month-old animals of both sexes. Mice were housed (three to five
per cage) under a 12 hr light/dark cycle with ad libitum access to
food and water. Behavioral experiments were performed during the light phase.
The experimenters were blind to the genotypes until data were gathered.
4 +/+, 4 +/-, and 4 -/- mice were generated by crossing
heterozygous mice. We used all of the 4 +/+ and 4 -/- and some of
the 4 +/- littermates from each litter. As reported previously, 4
-/- mice do not show any obvious physical or neurological deficits
(Xu et al., 1999b). In the
behavioral experiments, a minimum of 15 mice per genotype was used on each
replication, and the experiments were done in two separate batches of mice.
For clarity and space, the data from both replications of the behavioral
experiments were pooled after confirming that both groups showed the same
results (including statistical significance in the elevated-plus and staircase
mazes). Groups of 14–21 mice were used in the telemetry experiments. All
of the procedures were approved by the Baylor College of Medicine Animal
Research Committee and followed the guidelines for animal intramural research
from the National Institutes of Health.
In situ hybridization. Probes for in situ hybridization
were cloned by reverse transcription-PCR using total RNA isolated from a mouse
septal neuroblastoma cell line (SN56 cells)
(Blusztajn et al., 1992). The
primers were designed from the published rat sequences to encompass the third
intracellular loop of each subunit. The template DNA segments and sizes were
as follows: 4 (1046–1789; 743 bp), 4 (1056–1428; 372
bp), and 3 (1119–1566; 447 bp). PCR fragments were subcloned into
pBlue-script and sequenced (Franceschini
et al., 2002). In situ hybridization with
[35S]UTP (DuPont NEN, Boston, MA) riboprobes was performed as
described previously (Broide et al.,
1996). Quantitation of in situ signals was performed as
described previously (Broide et al.,
2002).
Locomotor activity in the open field. Mice were placed in a clear
Plexiglas arena (40 x 40 x 40 cm), and locomotor activity was
measured over a 30 min session using a computer-operated Ethovision system
(Noldus, Wageningen, The Netherlands). This system was also used in all of the
other behavioral experiments. The total distance moved in the arena and the
distance moved in a center square (20 x 20 cm) were recorded. The ratio
of the distance moved in the center to the total distance moved was calculated
and used as a measure of anxiety-related behavior
(Paylor et al., 1998).
Light/dark exploration. The light/dark exploration test, which is
believed to measure anxiety-related behavior
(Crawley, 1980) was performed
by placing the mouse in a cage (44 x 21 x 21 cm) that has two
chambers, one bigger and bright, and the other smaller and dark. The animal
was initially placed in the lighted side, and transitions between sides and
the time spent in each division were recorded for 10 min.
Elevated-plus maze. Mice were placed for 5 min on an elevated-plus
maze consisting of four arms (25 x 7 cm), two with high, black walls (15
cm high), and two without walls. Mice were placed in the intersection between
the arms (7 x 7 cm), and the number of entries into, and the time spent
in, the open and closed arms were recorded. These two parameters were taken as
measures of anxiety-related behavior
(Pellow et al., 1985). For
some animals, the experiment was repeated 1 d later (trial 2).
Mirror chamber. For the mirror chamber test
(Toubas et al., 1990), mice
were placed in a black chamber (30 x 30 x 40 cm) located inside
another chamber (40 x 40 x 40 cm). The interior walls, floor, and
ceiling of the inside chamber are mirrors, and there is also a mirror in the
internal wall of the bigger chamber that is facing the entrance of the
mirrored box. Mice were placed on a corner opposite to the entrance, and their
movement was monitored for 5 min. Entrances into the mirrored chamber, as well
as the time spent in the mirrored chamber, were recorded. Entrances into the
two lateral corridors were recorded and used as control for total
activity.
Staircase maze. The staircase maze test, which is also sensitive
to anxiety-related drug treatments and behaviors, was performed as described
previously (Simiand et al.,
1984; Weizman et al.,
1999). Briefly, mice were placed for 3 min in the staircase, a
rectangular (45 x 10 cm) maze that has six steps (10 x 7.5 cm, 2.5
cm high). We recorded rearing and step climbing (defined as four paws on the
next step), because these behaviors have been shown to be decreased in mice
showing anxious-like behavior (Simiand et
al., 1984; Weizman et al.,
1999).
Telemetry. Radiotelemetry implants (TAEA-F20; Data Sciences, St.
Paul, MN) were used to monitor heart rate (HR) in conscious, freely moving
animals. The body of the implants was inserted into the abdominal cavity of
anesthetized animals, and the leads were positioned in a lead II
electrocardiogram configuration. Animals were allowed a 9 d recovery period
during which weight and food intake were monitored. After recovering from
surgery, an implanted mouse was housed with at least two same-sex littermates
(home cage), and its HR was monitored for 48 hr, taking one measurement every
2 or 10 min. The animal was then transferred to another cage in which it was
kept in isolation for 48 hr (single cage) while HR was monitored. After the
isolation period, the mouse was returned to its home cage, and HR was
monitored until it returned to basal levels (usually within 24 hr).
Light/dark box and elevated-plus tests on telemetry-implanted
mice. Behavioral tests were conducted at least 2 d after the social
isolation experiment was finished, and the heart rate returned to pretest
levels. For the light/dark box test, basal heart rate was measured for 30 min
before the beginning of the experiment. The animals were then allowed to
freely explore the box for 20 min. During this time, the behavior of the
animals was recorded on tape. After the test, mice were returned to their
cage, and HR was monitored for another hour. The average HR for the 20 min the
mouse spent in the light/dark box was compared with the average HR during
baseline. In addition, we compared average HR in the box with the average HR
obtained when the mouse was exploring the lighted chamber. At least 1 d after
the light/dark box experiment, the implanted mice were tested in the
elevated-plus maze. Basal HR was recorded for 30 min before placing the mice
in an elevated-plus maze. The receiver for telemetry was placed above the
maze. Because of spatial constraints of the receiver, the closed arms of the
maze were 7 cm shorter than in the previous experiments. Mice were placed into
the elevated-plus maze and allowed to explore it for 20 min while the HR was
monitored. The animals were subsequently returned to their home cage, and HR
was monitored for 1 additional hr.
Data acquisition and analysis. Behavioral data were analyzed using
Excel (Microsoft; Redmond, WA) or Statistica (StatSoft, Tulsa, OK), and
unpaired Student's t test or ANOVA and Newman–Keuls post
hoc comparisons. Differences were considered significant when p
< 0.05. The HR data were collected using the Dataquest ART version 1.10
system (Data Sciences). The HR parameter files were exported and analyzed
using SigmaPlot 4.1 (SPSS, Chicago, IL) and ANOVA.
|
Results
|
|---|
Expression pattern of the 4 nAChR subunit mRNA in the CNS
The expression pattern of 4 mRNA in the mouse brain is shown in
Figure 1. From rostral to
caudal, the regions of expression were as follows. Relatively high levels of
4 expression were found on the olf
(Fig. 1A). The 4
signal was restricted to mitral cells, which are the main input–output
cells of the olfactory bulb. We found very high levels of 4 mRNA in the
MHb (Fig. 1D), whereas
the IPN showed lower levels of 4 subunit mRNA (G). Along with
the MHb, the pineal gland (Fig.
1J) expressed the highest levels of 4 mRNA, in
agreement with previous results in rat tissue
(Duvoisin et al., 1989;
Dineley-Miller and Patrick,
1992; Zoli et al.,
1995). Detectable, low levels of 4 subunit mRNA were also
found in the inferior colliculus (Fig.
1M), which is the main target for MHb neurons. In
4-/- mice, no 4 mRNA was detected
(Fig. 1B,E,H,K,N).
Sense controls showed no specific signal
(Fig. 1C,F,I,L,O).
Three 4+/+ and three 4-/- mouse brains were used for these
experiments. Representative brain sections are shown for each genotype. The
sense probe was used on 4+/+ brains only.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 1. Expression of4 mRNA in the mouse brain. A, D, G, J, M,
Distribution of4 nAChR mRNA in4 +/+ brain. Arrows indicate the
mRNA signal. B, E, H, K, N, Distribution of 4 nAChR mRNA in
4 -/- brain. C, F, I, L, O, Control experiments performed with
sense probe on 4 +/+ brains. A–C, Olfactory bulb (Olf).
D–F, Medial habenula (MHb). G–I, Interpeduncular
nucleus (IPN). J–L, Pineal gland (Pin). M–O,
Inferior colliculus (IC). Scale bar, 2 mm.
|
|
Several anxiety-related tests show no effect of the 4
genotype
In the open-field test, the total distance traveled by the mouse is a
measure of locomotion and activity, and the ratio of the distance traveled in
the center to the total distance is a measure of exploration and anxiety-like
behavior. In this experiment, 4-/- mice and littermate controls
exhibited similar activity and center distance/total distance ratios.
Therefore, in the open-field test, 4-/- mice revealed normal exploratory
activity and anxiety-related behavior (Fig.
2A,B).
View larger version (21K):
[in this window]
[in a new window]
|
Figure 2. Open-field, light/dark, and mirror chamber tests. A, Open-field
test (4 +/+, n = 39; 4 -/-, n = 31), total
distance traveled. B, Open-field test, distance in the center
(Cent)/total distance (Tot dist) ratio. C, Light/dark test (4
+/+, n = 45;4 -/-, n = 39), number of entries into the
dark chamber. D, Light/dark test, time spent in the lighted chamber.
E, Mirror chamber test (4 +/+, n = 40;4 -/-,
n = 60), entries in to the mirror chamber. F, Mirror chamber
test, time spent in the mirror chamber. Averages (±SEM) are shown for
4 +/+ (filled bars) and 4 -/- (open bars) mice, respectively.
|
|
In the light/dark exploration test, the number of transitions between the
dark and lighted compartments and the time spent in the light are measures of
anxiety-like behavior (Crawley,
1980). The 4-/- mice and their 4+/+ littermates showed
no statistically significant differences in the number of transitions between
the lighted and the dark compartments, or in the time spent in the dark
compartment (Fig.
2C,D).
In the mirror chamber test, the animal chooses between staying in a black
corridor and entering a chamber with mirrored walls, floor, and ceiling
(Toubas et al., 1990). The
number of entries and the time spent in the anxiety-provoking mirror chamber
are recorded. The 4-/- mice and littermate controls showed no
statistically significant differences in entries and time spent in the mirror
chamber (Fig.
2E,F).
4-/- mice show anxiety-related differences in the elevated-plus
maze and the staircase maze
The elevated-plus maze is used to analyze anxiety-related behavior on the
basis of the hypothesis that there is greater stress from being in the open
arms versus the closed arms of an elevated maze. The number of entrances into
the open arms and the time spent in the open arms provide indications of
anxiety-like behavior, and the total number of entrances into all of the arms
is a measure of total activity (Pellow et
al., 1985). 4-/- mice showed a significant increase in both
the time spent and the number of entrances into the open arms
(Fig. 3), with no differences
in total number of entrances. The 4-/- mice not only entered the open
arms more often (Fig.
3A), but also stayed longer after entry
((Fig. 3B). This
result was the first behavioral phenotype found for 4-/- mice, and it
suggests that these animals have lower levels of anxiety-like behavior as
measured in this particular test. We also tested 4+/- mice in the
elevated-plus maze and analyzed the data using ANOVA and Newman–Keuls
post hoc comparisons (open time, F(2,0.05) =
9.35; entry ratio, F(2,0.05) = 4.31). The results on
heterozygous mice were similar to those of the 4-/- animals
(Fig. 3).
Previous research has shown that, when nicotine is injected directly into
the hippocampus, it does not affect performance in the elevated-plus maze on
the first trial, but nicotine has anxiolytic effects on a second trial
performed at least 1 d after the first trial
(Cheeta et al., 2000). On the
basis of that observation, we repeated the elevated-plus experiment 1 d after
the first trial in the maze. The results showed that mice made fewer entries
and stayed for shorter times in the open arms on trial 2, but the 4
genotype still had the same influence as on trial 1. The time spent in the
open arms dropped from 25 ± 5 sec (mean ± SEM) on trial 1 to 6
± 2 sec on trial 2 for 4+/+ mice, and from 66 ± 6 sec to
23 ± 4 sec for 4-/- mice. A similar result was observed in the
percentage of entries in the open arms, which dropped from 41% ± 7 to
14% ± 3 for 4+/+ mice, and dropped from 63% ± 6 to 30%
± 4 for 4-/- animals. These data were analyzed using Student's
t test. The percent change in arm entries and time between days 1 and
2 was not statistically different between 4+/+ and 4-/- mice.
These results indicate that mice explore less the second day, but this
difference is similar in 4+/+ and 4-/- mice. Hence, habituation to
the maze is normal in 4-mutant mice, and the behavior observed is likely
to be attributable to decreased anxiety-like behavior and not to abnormal
learning or habituation to the maze.
The staircase maze (Simiand et al.,
1984) is another test for anxiety-related behavior that has been
validated using different anxiolytics
(Pick et al., 1997;
Weizman et al., 1999). In this
test, mice explore a rectangular maze that has six steps. The number of steps
climbed and the rearing behavior of the mice are recorded as measures of
anxiety-related behavior. The 4-/- mice displayed significantly more
steps up and rearing events than did the 4+/+ littermates
(Fig. 4). The data were
analyzed using ANOVA and Newman–Keuls post hoc comparisons
(steps, F(2,0.05) = 7.41; rearing
F(2,0.05) = 3.69). This result is consistent with our
experiments in the elevated-plus maze.
Heart rate changes confirm the behavioral results
There is a large body of literature in humans that correlates emotions and
anxiety with the activation of the autonomic nervous system
(Berntson et al., 1998;
Friedman and Thayer, 1998;
Johansen-Berg and Walsh, 2001;
O'Connor et al., 2002;
Watkins et al., 2002). Such
activation is reflected by changes in heart rate and blood pressure, among
other factors. To obtain an independent measurement of anxiety-related
effects, we monitored HR changes during the elevated-plus and the
light/dark-box tests. By directly measuring HR without the confounds of
handling or restraint, telemetry gives a quantitative, nonsubjective measure
of a parameter (HR) that is linked to changes in anxiety-related behavior
(Tornatzky and Miczek, 1995;
Southwick et al., 1999;
Bouwknecht et al., 2000). As
shown in Figure 5A,
basal HR was statistically the same for 4+/+ and 4-/- mice, and HR
increases observed when the animals are placed in the light/dark maze were
comparable. We also measured the mean HR of the mice when they were in the
lighted region of the maze, and compared that with the average HR in the maze.
Both 4+/+ and 4-/- mice had increased HR while they explored the
lighted chamber, and these increases were not statistically different. For
4+/+ mice, the average HR in the lighted chamber was 21 ± 7 beats
per minute (bpm) higher than the average HR during the total time the mice
spent in the light/dark box. For the 4-/- mice, the average HR increase
in the lighted chamber was 30 ± 15 bpm. Increases in HR were also
observed in the elevated-plus maze, but the increase in HR was significantly
lower for 4-/- mice than for 4+/+ mice (p < 0.001)
(Fig. 5B). Differences
in basal heart rate across genotypes or experiments were not statistically
significant (4+/+, light/dark box, HRbasal = 485 ± 26
bpm; 4+/+, elevated plus, HRbasal = 517 ± 40
bpm;4-/-, light/dark box, HRbasal = 411 ± 32 bpm;
4-/-, elevated plus, HRbasal = 528 ± 49 bpm;
p > 0.05). These data agree with the behavioral data shown
previously. In the light/dark test, the behavior and HR of 4-/- mice
were normal. In the elevated-plus maze, the behavior and HR of the 4-/-
mice were different from those of the littermate controls.
Stress during social isolation increases heart rate
Single-cage housing has been shown to produce HR increases in rats
(Gardiner, 1977;
Naranjo, 1985), probably
because of changes in anxiety-like responses
(Guidotti et al., 2001). We
examined whether social isolation produces the same effect in mice, and
whether that effect is dependent on the 4 genotype. The HR of 4+/+
and 4-/- mice was measured during normal housing conditions (three to
five littermates per cage) and under social isolation (one mouse per cage).
Animals were monitored for much longer periods of time than in any of the
other behavioral tests: 48 hr in the home cage and the single cage,
respectively. Over these long periods of time, 4+/+ mice (on average)
had statistically the same heart rate as 4-/- animals when housed with
littermates: 453 ± 18 bpm for 4+/+ and 451 ± 21 bpm for
4-/- mice, respectively. Under isolation, the heart rates were 524
± 16 bpm for 4+/+ and 567 ± 17 bpm for 4-/- mice
(p > 0.05). However, although during isolation the HR increased
for both genotypes (Fig.
5D), the HR of 4+/+ mice increased significantly
less (68 + 10 bpm) than the HR of 4-/- mice (115 + 13 bpm; p
< 0.05). The increase in heart rate was calculated by subtracting the HR in
the home cage from the HR in isolation at each hour and averaging the 48
resulting numbers for each mouse. The p value was calculated using
the average for each mouse as an independent value (+/+, n = 12; -/-,
n = 9). This result indicates that the stress of isolation has a
greater impact on 4-/- than on 4+/+ mice.
nAChR subunits that influence anxiety-related tests are coexpressed
in medial habenula and interpeduncular nucleus
The most common phenotype of nAChR-subunit-mutant mice is associated with
anxiety-producing tests, especially the elevated-plus test. 4-/-,
3-/-, 4-/-, and 4 gain-of-function (4
L9'S-heterozygous) mice all have a significant phenotype in the
elevated-plus maze. If there is a specific brain area that influences the
anxiety-related behaviors manifested in the elevated-plus maze, the nAChR
subunit implicated in such behavior shouldbe coexpressed in this area. To test
this hypothesis, we used the in situ hybridization technique to
determine where in the mouse brain 4, 3, and 4 are
coexpressed. Figure 6 shows the
expression of 4 in the same brain areas in which 4 was found
(Fig. 1). No 4 was found
in the mitral cells of the Olf (Fig.
6A, arrows point to the site of expression of 4) or
the Pin (J). However, 4 and 4 are both expressed in the
MHb, the IPN, and the inferior colliculus
(Fig. 6D,G,M). In
4-/- mice, the expression pattern of 4 remains the same, which
argues against the possibility of subunit compensation
(Fig. 6B,E,H,K,N). No
specific signal was observed when sections were probed with sense 4
probe (Fig.
6C,F,I,L,O).
View larger version (84K):
[in this window]
[in a new window]
|
Figure 6. Expression of4 mRNA at the brain levels where4 is expressed.
A, D, G, J, M, Expression of4 mRNA in4 +/+ brains.
Arrows indicate the site of expression of 4. B, E, H, K, N,
Expression of 4 mRNA in 4 -/- brains. C, F, I, L, O,
Sense controls performed on 4 +/+ brains. A–C, Olfactory
bulb (Olf). D–F, Medial habenula (MHb). G–I,
Interpeduncular nucleus (IPN). J–L, Pin. M–O,
IC. Arrows point at sites of expression of 4. Scale bar, 2 mm.
|
|
Figure 7 shows the
expression pattern of 3 mRNA in the brain areas in which 4 was
found (Fig. 1). Arrows point to
the sites of expression of 4. Again, the MHb
(Fig. 7D) and IPN
(G) are sites of coexpression, but no 3 was observed in the
Olf, the Pin, or the IC (A,J,M). No differences were observed between
4+/+ and 4-/- mice (Fig.
7B,E,H,K,N), excluding the occurrence of subunit
compensation in 4 mutant mice, at least at the level of mRNA. Sense
controls showed no signal (Fig.
7C,F,I,L,O).
View larger version (21K):
[in this window]
[in a new window]
|
Figure 7. Expression of3 mRNA at the brain levels where4 is expressed.
A, D, G, J, M, Expression of3 mRNA in4 +/+ brains. Arrows
indicate the site of expression of 4. B, E, H, K, N, Expression
of 3 mRNA in 4 -/- brains. C, F, I, L, O, Sense controls
performed on 4 +/+ brains. A–C, Olfactory bulb (Olf).
D–F, Medial habenula (MHb). G–I, Interpeduncular
nucleus (IPN). J–L, Pineal gland (Pin). M–O,
Inferior colliculus (IC). Arrows point at sites of expression of 4.
Scale bar, 2 mm.
|
|
The results indicate that the only areas in which 4, 3, and
4 are significantly coexpressed are the MHb and the IPN. To verify that
the expression of 3 and 4 was unchanged in 4-/- mice, we
performed quantitative in situ hybridization. Sections from three to
four brains from 4+/+ and 4-/- mice were exposed below saturation
of signal, and specific areas were chosen for densitometry.
Table 1 shows that 4+/+
and 4-/- mice express similar amounts of 3 and 4 mRNA in
the areas of the brain selected for the study.
|
Discussion
|
|---|
Our investigation of the anxiety-related behaviors in 4 nAChR-mutant
mice was motivated by two observations. First, human smokers often mention the
anxiolytic effect of cigarettes, likely arising from nicotine, as a driving
force to continue smoking (Parrott,
1995; Kassel and Shiffman,
1997; Stewart et al.,
1997; Kassel and Unrod,
2000). Second, recent experiments on different nAChR-mutant mice
suggested a link between nAChRs and anxiety-related behavior. It has been
reported that 4-/- mice behave as though they are more anxious than
control mice in the elevated plus, whereas they are normal in a series of
basic behavioral tests (Ross et al.,
2000). In addition, a gain-of-function mutation in the 4
subunit that produces channels with higher conductance also alters
anxiety-like behavior in the mutant mouse. Mice heterozygous for this mutation
display increased anxiety-like behavior in the elevated plus and, to a lesser
extent, in the mirror chamber, but there is no effect in the light/dark box
(Labarca et al., 2001).
Furthermore, mice lacking the 3 nAChR subunit also behave normally in a
standard battery of tests, but display decreased anxiety-related behavior in
the elevated-plus maze, with a smaller effect in the mirror chamber and no
effect in the light/dark box (Booker et
al., 2000). Interestingly, 7-/- mice have been shown to
behave as if less anxious in the open field, with normal behavior in the
light/dark box (Paylor et al.,
1998). That report did not explore the elevated-plus maze. The
fact that anxiety-related phenotypes are consistently found on the
elevated-plus maze and not on open field or light/dark box should not be
surprising. Factor analysis of behaviors on anxiety-related experiments in
animals has shown that different tests reflect different underlying factors
(File, 1992;
Belzung and Le Pape, 1994;
Ramos et al., 1997).
Therefore, the fact that 4-/- and other nicotinic mutants show such a
specific phenotype on the elevated-plus and not on other mazes might simply
reflect the fact that these tests measure different dimensions of anxiety-like
behaviors. Evidence that the measures of anxiety-like behaviors assessed in
different tasks may reflect different aspects of anxiety-like responses comes
from not only the nAChR-mutant mice but also other animal models (Griebel et
al., 1996,
1997,
2000;
Andreatini et al., 2001),
including mice overexpressing the corticotropin-releasing hormone
(van Gaalen et al., 2002).
Another possible explanation is that the effects of 4* nAChRs on anxiety
appear only under the most stressful situations. For example, we demonstrated
that, in wild-type mice, the heart rate increase in the elevated-plus maze is
higher than that in the light/dark box, suggesting that the elevated plus is a
more stressful experience. There could be a threshold of stress beyond which
4* nAChRs become important for the behavioral and physiological
expression of anxiety. The existence of such a threshold would explain why
there are differences in the elevated-plus maze but not in the open field or
light/dark box.
Although the lack of the 4 subunit increases anxiety-like behavior
on the same test in which absence of 3 or 4 decrease anxiety-like
behavior, all three mutations are effective on the same test. This consistent
specificity of the testing suggested that 4*, 3*, and 4*
nAChRs are involved in the neuronal processing associated with the
elevated-plus maze, possibly by influencing the same neuronal areas of the
mouse brain. That reasoning led us to investigate where in the brain 4,
3, and 4 are coexpressed. The 4 subunit was only found (at
the sensitivity levels of in situ hybridization) in the mitral cells
of the Olf, MHb, Pin, IPN, and IC. Within the regions in which 4 was
expressed, 3 and 4 subunits were detected at significant levels
only in MHb and IPN. This finding suggests the possibility that the MHb and
its main target, the IPN, could participate in the anxiety phenotype observed
in 4, 3, and 4 mutant mice. Indeed, nicotine injections in
the dorsal raphe nucleus, which receives innervation from the habenula
(Morris et al., 1999;
Tomizawa et al., 2001), have
been demonstrated to be anxyolitic in the rat
(Cheeta et al., 2001). It has
also been shown that 4* nAChRs dominate function in MHb neurons
(Quick, 1999). In addition,
nicotine produces a shift between the excitatory and the inhibitory actions of
acetylcholine in the MHb–IPN axis: long-term exposure to low levels of
nicotine leaves the inhibitory effect of muscarinic activation unaffected
while decreasing the excitatory effect of nicotinic activation
(Girod and Role, 2001). This
effect is probably mediated by functional inactivation of presynaptic nAChRs.
Therefore, it is possible that 4-/- mice have a constitutive lower
activation of IPN function. Heavy smokers tend to adjust their nicotine intake
to obtain constant levels of circulating nicotine during wake hours
(Russell, 1989). In that
sense, it is tempting to speculate that the lack of 4* receptors in the
MHb could have, by decreasing IPN activity, one of the effects smokers seek: a
decrease in anxiety levels.
There is a body of literature in humans that correlates emotions with the
activation of the autonomic nervous system. This autonomic activation has been
associated with changes in heart rate and blood pressure. There is also
evidence in humans that different natural stressors can provoke unique
configurations of cardiovascular activity. For example, fear produces a
decrease in heart rate (Maschke et al.,
2002), but stress-induced anxiety results in an increase in both
heart rate and blood pressure (Noteboom et
al., 2001; Gonzalez-Bono et
al., 2002). Measuring heart rate responses as an index of
autonomic activation after anxiety-provoking tests in mice was therefore a
natural extension of our studies. If a certain behavioral paradigm provokes
anxiety, the autonomic nervous system will be activated, and the prediction is
that anxiety-like behaviors correlate with increases in heart rate
(Bouwknecht et al., 2000;
De Biasi, 2001;
X. Wang et al., 2002).
Therefore, to have an independent physiological measure of anxiety-related
effects, we implanted 4-/- mice and their control littermates with
telemetry devices. This procedure was used to monitor the heart rate while
mice explored the mazes. We chose the light/dark box test, because no
behavioral phenotype was observed, and we chose the elevated plus, because
that test showed the largest effect. We demonstrated that 4-/- and
4+/+ mice have a similar increase in heart rate when exposed to the
light/dark box. In contrast, 4-/- mice showed significantly lower
increases in heart rate when mice explored the elevated-plus maze. Therefore,
4-/- mice behaved as though they were less anxious in the elevated plus,
and they also showed a decreased physiological reaction to the maze consistent
with less stress. In contrast, the heart rate responses to social isolation
seem to be enhanced in 4-/- mice, which suggests that different
stressors may act through different neuronal systems.
Because the 4 nAChR subunit is highly expressed in the autonomic
nervous system (Rust et al.,
1994), altered autonomic function might also influence the
differences observed between wild-type and mutant mice. It is possible that
the 4-null mutation produces an overall dysregulation of the autonomic
nervous system, and that heart rate responses could be attenuated or enhanced
depending on the stress paradigm used. Additional experimentation will be
needed to address autonomic function in 4-/- mice in basal conditions
and during stress.
In conclusion, we showed that the lack of the nAChR 4 subunit alters
the behavioral responses to certain anxiety-provoking experimental paradigms.
The effect might be dependent on the MHb–IPN expression of the 4
subunit. The data on 4, 3, and 4 coexpression might help to
explain how different nAChR types can exert their influence on anxiety-related
behavioral tests.
A major long-term goal of the nicotine addiction field is to understand how
the specific nAChR subtypes contribute to the addiction process. Identifying
the type and location of the nAChRs involved in anxiety-related behaviors
could lead to therapies that aid in smoking cessation and prevention.
|
Footnotes
|
|---|
Received Aug. 16, 2002;
revised Apr. 23, 2003;
accepted Apr. 30, 2003.
This work was supported by National Institute on Drug Abuse Grants DA12661
and DA09411, National Institute of Neurological Disorders and Stroke Grant
NS21229, American Heart Association of Texas Grant 0150808Y, the Whitaker
Foundation, and the US-Israel Binational Science Foundation.
Correspondence should be addressed to Dr. Mariella De Biasi, Division of
Neuroscience, Baylor College of Medicine, Houston, TX 77030. E-mail:
debiasi{at}bcm.tmc.edu.
Copyright © 2003 Society for Neuroscience
0270-6474/03/236255-09$15.00/0
|
References
|
|---|
Andreatini R, Blanchard C, Blanchard R, Brandao ML, Carobrez AP,
Griebel G, Guimaraes FS, Handley SL, Jenck F, Leite JR, Rodgers J, Schenberg
LC, Da Cunha C, Graeff FG (2001) The brain decade in debate. II.
Panic or anxiety? From animal models to a neurobiological basis. Braz J
Med Biol Res 34:
145–154.[Medline]
Belzung C, Le Pape G (1994) Comparison of different
behavioral test situations used in psychopharmacology for measurement of
anxiety. Physiol Behav 56:
623–628.[Medline]
Berntson GG, Sarter M, Cacioppo JT (1998) Anxiety and
cardiovascular reactivity: the basal forebrain cholinergic link. Behav
Brain Res 94:
225–248.[Web of Science][Medline]
Blusztajn JK, Venturini A, Jackson DA, Lee HJ, Wainer BH
(1992) Acetylcholine synthesis and release is enhanced by
dibutyryl cyclic AMP in a neuronal cell line derived from mouse septum.
J Neurosci 12:
793–799.[Abstract]
Booker TK, Tritto T, Colman J, Cui C, Collins AC, Heinemann SF
(2000) 3 subunits in regulation of anxiety: analysis of
3-null mutant mice. In: The 20th Neuropharmacology
Conference, p 65. New Orleans: Elsevier
Science.
Bouwknecht JA, Hijzen TH, van der Gugten J, Maes RA, Olivier B
(2000) Stress-induced hyperthermia in mice: effects of flesinoxan
on heart rate and body temperature. Eur J Pharmacology
400: 59–66.[Medline]
Broide RS, Robertson RT, Leslie FM (1996) Regulation
of 7 nicotinic acetylcholine receptors in the developing rat
somatosensory cortex by thalamocortical afferents. J Neurosci
16:
2956–2971.[Abstract/Free Full Text]
Broide RS, Salas R, Ji D, Paylor R, Patrick JW, Dani JA, De Biasi M
(2002) Increased sensitivity to nicotine-induced seizures in mice
expressing the L250T 7 nicotinic acetylcholine receptor mutation.
Mol Pharmacol 61:
695–705.[Abstract/Free Full Text]
Champtiaux N, Han ZY, Bessis A, Rossi FM, Zoli M, Marubio L,
McIntosh JM, Changeux JP (2002) Distribution and pharmacology of
6-containing nicotinic acetylcholine receptors analyzed with mutant
mice. J Neurosci 22:
1208–1217.[Abstract/Free Full Text]
Cheeta S, Kenny PJ, File SE (2000) Hippocampal and
septal injections of nicotine and 8-OH-DPAT distinguish among different animal
tests of anxiety. Prog Neuropsychopharmacol Biol Psychiatry
24:
1053–1067.[Medline]
Cheeta S, Irvine EE, Kenny PJ, File SE (2001) The
dorsal raphe nucleus is a crucial structure mediating nicotine's anxiolytic
effects and the development of tolerance and withdrawal responses.
Psychopharmacology (Berl) 155:
78–85.[Medline]
Cohen G, Han ZY, Grailhe R, Gallego J, Gaultier C, Changeux JP,
Lagercrantz H (2002) 2 nicotinic acetylcholine receptor
subunit modulates protective responses to stress: a receptor basis for
sleep-disordered breathing after nicotine exposure. Proc Natl Acad Sci
USA 99:
13272–13277.[Abstract/Free Full Text]
Crawley J (1980) Preliminary report of a simple animal
behavior model for the anxiolytic effects of benzodiazepines. Pharmacol
Biochem Behav 13:
167–170.[Web of Science][Medline]
Dani JA, De Biasi M (2001) Cellular mechanisms of
nicotine addiction. Pharmacol Biochem Behav
70: 439–446.[Web of Science][Medline]
De Biasi M (2001) Heart rate variability studies in
nicotinic mutant mice. Paper presented at 19th Annual Houston
Conference on Biomedical Engineering Research, Houston, Feb 8–9,
2001.
De Biasi M (2002) Nicotinic receptor mutant mice in
the study of autonomic function. Curr Drug Targets
1: 409–414.
Dineley-Miller K, Patrick J (1992) Gene transcripts
for the nicotinic acetylcholine receptor subunit, 4, are distributed in
multiple areas of the rat central nervous system. Brain Res Mol Brain
Res 16:
339–344.[Medline]
Domino EF, Yamamoto KI (1965) Nicotine: effect on the
sleep cycle of the cat. Science 150:
637–638.[Abstract/Free Full Text]
Duvoisin RM, Deneris ES, Patrick J, Heinemann S (1989)
The functional diversity of the neuronal nicotinic acetylcholine receptors is
increased by a novel subunit: 4. Neuron
3: 487–496.[Web of Science][Medline]
Elgoyhen AB, Johnson DS, Boulter J, Vetter DE, Heinemann S
(1994) 9: an acetylcholine receptor with novel
pharmacological properties expressed in rat cochlear hair cells.
Cell 79:
705–715.[Web of Science][Medline]
Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, Boulter J
(2001) 10: a determinant of nicotinic cholinergic receptor
function in mammalian vestibular and cochlear mechanosensory hair cells.
Proc Natl Acad Sci USA 98:
3501–3506.[Abstract/Free Full Text]
File SE (1992) Usefulness of animal models with newer
anxiolytics. Clin Neuropharmacol
[Suppl]15:
525A–526A.
File SE, Cheeta S, Kenny PJ (2000) Neurobiological
mechanisms by which nicotine mediates different types of anxiety. Eur J
Pharmacol 393:
231–236.[Medline]
Franceschini D, Orr-Urtreger A, Yu W, Mackley LY, Bond RA,
Armstrong D, Patrick JW, Beaudet AL, De Biasi M (2000) Altered
baroreflex in 7 deficient mice. Behav Brain Res
113: 3–10.[Web of Science][Medline]
Franceschini D, Paylor R, Broide R, Salas R, Bassetto L, Gotti C,
De Biasi M (2002) Absence of 7-containing neuronal
nicotinic acetylcholine receptors does not prevent nicotine-induced seizures.
Brain Res Mol Brain Res 98:
29–40.[Medline]
Friedman BH, Thayer JF (1998) Anxiety and autonomic
flexibility: a cardiovascular approach. Biol Psychol
49: 303–323.[Web of Science][Medline]
Gardiner SM (1977) The effects of short-term isolation
on systolic blood pressure and heart rate in rats. Med Biol
55: 325–329.[Medline]
Girod R, Role L (2001) Long-lasting enhancement of
glutamatergic synaptic transmission by acetylcholine contrasts with response
adaptation after exposure to low-level nicotine. J Neurosci
21:
5182–5190.[Abstract/Free Full Text]
Gonzalez-Bono E, Moya-Albiol L, Salvador A, Carrillo E, Ricarte J,
Gomez-Amor J (2002) Anticipatory autonomic response to a public
speaking task in women: the role of trait anxiety. Biol Psychol
60: 37–49.[Medline]
Griebel G, Blanchard DC, Blanchard RJ (1996) Evidence
that the behaviors in the Mouse Defense Test Battery relate to different
emotional states: a factor analytic study. Physiol Behav
60:
1255–1260.[Medline]
Griebel G, Perrault G, Sanger DJ (1997) CCK receptor
antagonists in animal models of anxiety: comparison between exploration tests,
conflict procedures and a model based on defensive behaviours. Behav
Pharmacol 8:
549–560.[Web of Science][Medline]
Griebel G, Belzung C, Perrault G, Sanger DJ (2000)
Differences in anxiety-related behaviours and in sensitivity to diazepam in
inbred and outbred strains of mice. Psychopharmacology (Berl)
148:
164–170.[Medline]
Guidotti A, Dong E, Matsumoto K, Pinna G, Rasmusson AM, Costa E
(2001) The socially-isolated mouse: a model to study the putative
role of allopregnanolone and 5-dihydroprogesterone in psychiatric
disorders. Brain Res Brain Res Rev 37:
110–115.[Medline]
Johansen-Berg H, Walsh V (2001) Cognitive
neuroscience: who to play at poker. Curr Biol
11:
R261–R263.[Medline]
Kassel JD, Shiffman S (1997) Attentional mediation of
cigarette smoking's effect on anxiety. Health Psychol
16: 359–368.[Web of Science][Medline]
Kassel JD, Unrod M (2000) Smoking, anxiety, and
attention: support for the role of nicotine in attentionally mediated
anxiolysis. J Abnorm Psychol 109:
161–166.[Medline]
Khiroug SS, Harkness PC, Lamb PW, Sudweeks SN, Khiroug L, Millar
NS, Yakel JL (2002) Rat nicotinic ACh receptor 7 and
2 subunits co-assemble to form functional heteromeric nicotinic receptor
channels. J Physiol (Lond) 540:
425–434.[Abstract/Free Full Text]
Labarca C, Schwarz J, Deshpande P, Schwarz S, Nowak MW, Fonck C,
Nashmi R, Kofuji P, Dang H, Shi W, Fidan M, Khakh BS, Chen Z, Bowers BJ,
Boulter J, Wehner JM, Lester HA (2001) Point mutant mice with
hypersensitive 4 nicotinic receptors show dopaminergic deficits and
increased anxiety. Proc Natl Acad Sci USA
98:
2786–2791.[Abstract/Free Full Text]
Leonard S, Adler LE, Benhammou K, Berger R, Breese CR, Drebing C,
Gault J, Lee MJ, Logel J, Olincy A, Ross RG, Stevens K, Sullivan B, Vianzon R,
Virnich DE, Waldo M, Walton K, Freedman R (2001) Smoking and
mental illness. Pharmacol Biochem Behav
70: 561–570.[Web of Science][Medline]
Levin ED, Simon BB (1998) Nicotinic acetylcholine
involvement in cognitive function in animals. Psychopharmacology
(Berl) 138:
217–230.[Medline]
Lindstrom J, Anand R, Gerzanich V, Peng X, Wang F, Wells G
(1996) Structure and function of neuronal nicotinic acetylcholine
receptors. Prog Brain Res 109:
125–137.[Web of Science][Medline]
Marubio LM, del Mar Arroyo-Jimenez M, Cordero-Erausquin M, Lena C,
Le Novere N, de Kerchove d'Exaerde A, Huchet M, Damaj MI, Changeux JP
(1999) Reduced antinociception in mice lacking neuronal nicotinic
receptor subunits. Nature 398:
805–810.[Medline]
Maschke M, Schugens M, Kindsvater K, Drepper J, Kolb FP, Diener HC,
Daum I, Timmann D (2002) Fear conditioned changes of heart rate
in patients with medial cerebellar lesions. J Neurol Neurosurg
Psychiatry 72:
116–118.[Abstract/Free Full Text]
McGehee DS, Role LW (1995) Physiological diversity of
nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu
Rev Physiol 57:
521–546.[Web of Science][Medline]
Morris JS, Smith KA, Cowen PJ, Friston KJ, Dolan RJ
(1999) Covariation of activity in habenula and dorsal raphe
nuclei following tryptophan depletion. NeuroImage
10: 163–172.[Web of Science][Medline]
Moulard B (2001) Ion channel variation causes
epilepsies. Brain Res Brain Res Rev 36:
275–284.[Medline]
Naranjo JR (1985) Association between hypoalgesia and
hypertension in rats after short-term isolation.
Neuropharmacology 24:
167–171.[Medline]
Noteboom JT, Barnholt KR, Enoka RM (2001) Activation
of the arousal response and impairment of performance increase with anxiety
and stressor intensity. J Appl Physiol
91:
2093–2101.[Abstract/Free Full Text]
O'Connor MF, Allen JJ, Kaszniak AW (2002) Autonomic
and emotion regulation in bereavement and depression. J Psychosom
Res 52:
183–185.[Web of Science][Medline]
Orr-Urtreger A, Goldner FM, Saeki M, Lorenzo I, Goldberg L, De
Biasi M, Dani JA, Patrick JW, Beaudet AL (1997) Mice deficient in
the 7 neuronal nicotinic acetylcholine receptor lack
-bungarotoxin binding sites and hippocampal fast nicotinic currents.
J Neurosci 17:
9165–9171.[Abstract/Free Full Text]
Parrott AC (1995) Stress modulation over the day in
cigarette smokers. Addiction 90:
233–244.[Web of Science][Medline]
Paylor R, Nguyen M, Crawley JN, Patrick J, Beaudet A, Orr-Urtreger
A (1998) 7 nicotinic receptor subunits are not necessary
for hippocampal-dependent learning or sensorimotor gating: a behavioral
characterization of Acra7-deficient mice. Learn Mem
5: 302–316.[Abstract/Free Full Text]
Pellow S, Chopin P, File SE, Briley M (1985)
Validation of open:closed arm entries in an elevated plus-maze as a measure of
anxiety in the rat. J Neurosci Methods
14: 149–167.[Web of Science][Medline]
Picciotto MR (1995) Abnormal avoidance learning in
mice lacking functional high-affinity nicotine receptor in the brain.
Nature 374:
65–67.[Medline]
Picciotto MR, Brunzell DH, Caldarone BJ (2002) Effect
of nicotine and nicotinic receptors on anxiety and depression.
NeuroReport 13:
1097–1106.[Web of Science][Medline]
Pick CG, Peter Y, Paz L, Schreiber S, Gavish M, Weizman R
(1997) Effect of the pregnane-related GABA-active steroid
alphaxalone on mice performance in the staircase test. Brain
Res 765:
129–134.[Medline]
Poth K, Nutter TJ, Cuevas J, Parker MJ, Adams DJ, Luetje CW
(1997) Heterogeneity of nicotinic receptor class and subunit mRNA
expression among individual parasympathetic neurons from rat intracardiac
ganglia. J Neurosci 17:
586–596.[Abstract/Free Full Text]
Quick MW (1999) 34 subunit-containing
nicotinic receptors dominate function in rat medial habenula neurons.
Neuropharmacology 38:
769–783.[Web of Science][Medline]
Ramos A, Berton O, Mormede P, Chaouloff F (1997) A
multiple-test study of anxiety-related behaviours in six inbred rat strains.
Behav Brain Res 85:
57–69.[Web of Science][Medline]
Ross SA, Wong JY, Clifford JJ, Kinsella A, Massalas JS, Horne MK,
Scheffer IE, Kola I, Waddington JL, Berkovic SF, Drago J (2000)
Phenotypic characterization of an 4 neuronal nicotinic acetylcholine
receptor subunit knock-out mouse. J Neurosci
20:
6431–6441.[Abstract/Free Full Text]
Russell MA (1989) Subjective and behavioural effects
of nicotine in humans: some sources of individual variation. Addict
Behav 14:
335–341.[Medline]
Rust G, Burgunder JM, Lauterburg TE, Cachelin AB
(1994) Expression of neuronal nicotinic acetylcholine receptor
subunit genes in the rat autonomic nervous system. Eur J
Neurosci 6:
478–485.[Web of Science][Medline]
Rusted JM, Newhouse PA, Levin ED (2000) Nicotinic
treatment for degenerative neuropsychiatric disorders such as Alzheimer's
disease and Parkinson's disease. Behav Brain Res
113:
121–129.[Web of Science][Medline]
Salas R, Orr-Urtreger A, Broide R, Beaudet AL, Paylor R, De Biasi M
(2003) The nicotinic acetylcholine receptor subunit 5
mediates acute effects of nicotine in vivo. Mol
Pharmacol 63:
1059–1066.[Abstract/Free Full Text]
Salin-Pascual RJ, Moro-Lopez ML, Gonzalez-Sanchez H,
Blanco-Centurion C (1999) Changes in sleep after acute and
repeated administration of nicotine in the rat. Psychopharmacology
(Berl) 145:
133–138.[Medline]
Sargent PB (1993) The diversity of neuronal nicotinic
acetylcholine receptors. Annu Rev Neurosci
16: 403–443.[Web of Science][Medline]
Seguela P, Wadiche J, Dineley-Miller K, Dani JA, Patrick JW
(1993) Molecular cloning, functional properties, and distribution
of rat brain 7: a nicotinic cation channel highly permeable to calcium.
J Neurosci 13:
596–604.[Abstract]
Sgard F, Charpantier E, Bertrand S, Walker N, Caput D, Graham D,
Bertrand D, Besnard F (2002) A novel human nicotinic receptor
subunit, 10, that confers functionality to the 9-subunit.
Mol Pharmacol 61:
150–159.[Abstract/Free Full Text]
Shoaib M, Gommans J, Morley A, Stolerman IP, Grailhe R, Changeux JP
(2002) The role of nicotinic receptor 2 subunits in
nicotine discrimination and conditioned taste aversion.
Neuropharmacology 42:
530–539.[Web of Science][Medline]
Simiand J, Keane PE, Morre M (1984) The staircase test
in mice: a simple and efficient procedure for primary screening of anxiolytic
agents. Psychopharmacology 84:
48–53.[Medline]
Southwick SM, Paige S, Morgan III CA, Bremner JD, Krystal JH,
Charney DS (1999) Neurotransmitter alterations in PTSD:
catecholamines and serotonin. Semin Clin Neuropsychiatry
4: 242–248.[Medline]
Stewart SH, Karp J, Pihl RO, Peterson RA (1997)
Anxiety sensitivity and self-reported reasons for drug use. J Subst
Abuse 9:
223–240.[Web of Science][Medline]
Tassonyi E, Charpantier E, Muller D, Dumont L, Bertrand D
(2002) The role of nicotinic acetylcholine receptors in the
mechanisms of anesthesia. Brain Res Bull
57: 133–150.[Web of Science][Medline]
Tomizawa K, Katayama H, Nakayasu H (2001) A novel
monoclonal antibody recognizes a previously unknown subdivision of the
habenulointerpeduncular system in zebrafish. Brain Res
901:
117–127.[Medline]
Tornatzky W, Miczek KA (1995) Alcohol, anxiolytics and
social stress in rats. Psychopharmacology
121:
135–144.[Medline]
Toubas PL, Abla KA, Cao W, Logan LG, Seale TW (1990)
Latency to enter a mirrored chamber: a novel behavioral assay for anxiolytic
agents. Pharmacol Biochem Behav 35:
121–126.[Web of Science][Medline]
van Gaalen MM, Stenzel-Poore MP, Holsboer F, Steckler T
(2002) Effects of transgenic overproduction of CRH on
anxiety-like behaviour. Eur J Neurosci
15:
2007–2015.[Web of Science][Medline]
Wang N, Orr-Urtreger A, Chapman J, Rabinowitz R, Nachman R, Korczyn
AD (2002) Autonomic function in mice lacking 5 neuronal
nicotinic acetylcholine receptor subunit. J Physiol (Lond)
542:
347–354.[Abstract/Free Full Text]
Wang X, Su H, Copenhagen LD, Vaishnav S, Pieri F, Shope CD,
Brownell WE, De Biasi M, Paylor R, Bradley A (2002)
Urocortin-deficient mice display normal stress-induced anxiety behavior and
autonomic control but an impaired acoustic startle response. Mol Cell
Biol 22:
6605–6610.[Abstract/Free Full Text]
Watkins LL, Blumenthal JA, Carney RM (2002)
Association of anxiety with reduced baroreflex cardiac control in patients
after acute myocardial infarction. Am Heart J
143:
460–466.[Web of Science][Medline]
Weizman R, Paz L, Backer MM, Amiri Z, Modai I, Pick CG
(1999) Mouse strains differ in their sensitivity to alprazolam
effect in the staircase test. Brain Res
839: 58–65.[Medline]
Xu W, Gelber S, Orr-Urtreger A, Armstrong D, Lewis RA, Ou CN,
Patrick J, Role L, De Biasi M, Beaudet AL (1999a) Megacystis,
mydriasis, and ion channel defect in mice lacking the 3 neuronal
nicotinic acetylcholine receptor. Proc Natl Acad Sci USA
96:
5746–5751.[Abstract/Free Full Text]
Xu W, Orr-Urtreger A, Nigro F, Gelber S, Sutcliffe CB, Armstrong D,
Patrick JW, Role LW, Beaudet AL, De Biasi M (1999b) Multiorgan
autonomic dysfunction in mice lacking the 2 and the 4 subunits of
neuronal nicotinic acetylcholine receptors. J Neurosci
19:
9298–9305.[Abstract/Free Full Text]
Yu W, Mackey LY, Xu W, Beaudet AL, De Biasi M (2000)
Bradycardia and ventricular repolarization defects in mice lacking the
3 subunit of neuronal nicotinic acetylcholine receptors. In: The
10th Neuropharmacology Conference, p 113. New
Orleans, Nov 2–4, 2000.
Zoli M, Le Novere N, Hill JA, Changeux JP (1995)
Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat
central and peripheral nervous systems. J Neurosci
15:
1912–1939.[Abstract]
Zoli M, Picciotto MR, Ferrari R, Cocchi D, Changeux JP
(1999) Increased neurodegeneration during ageing in mice lacking
high-affinity nicotine receptors. EMBO J
18:
1235–1244.[Web of Science][Medline]
This article has been cited by other articles:
|
|
|
|
 
Y. S. Mineur, C. Eibl, G. Young, C. Kochevar, R. L. Papke, D. Gundisch, and M. R. Picciotto
Cytisine-Based Nicotinic Partial Agonists as Novel Antidepressant Compounds
J. Pharmacol. Exp. Ther.,
April 1, 2009;
329(1):
377 - 386.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. B. Tekinay, Y. Nong, J. M. Miwa, I. Lieberam, I. Ibanez-Tallon, P. Greengard, and N. Heintz
A role for LYNX2 in anxiety-related behavior
PNAS,
March 17, 2009;
106(11):
4477 - 4482.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. R. Grady, M. Moretti, M. Zoli, M. J. Marks, A. Zanardi, L. Pucci, F. Clementi, and C. Gotti
Rodent Habenulo-Interpeduncular Pathway Expresses a Large Variety of Uncommon nAChR Subtypes, But Only the {alpha}3{beta}4 and {alpha}3{beta}3{beta}4 Subtypes Mediate Acetylcholine Release
J. Neurosci.,
February 18, 2009;
29(7):
2272 - 2282.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Salas, A. Main, D. A. Gangitano, G. Zimmerman, S. Ben-Ari, H. Soreq, and M. De Biasi
Nicotine Relieves Anxiogenic-Like Behavior in Mice that Overexpress the Read-Through Variant of Acetylcholinesterase but Not in Wild-Type Mice
Mol. Pharmacol.,
December 1, 2008;
74(6):
1641 - 1648.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. De Biasi and R. Salas
Influence of Neuronal Nicotinic Receptors over Nicotine Addiction and Withdrawal
Experimental Biology and Medicine,
August 1, 2008;
233(8):
917 - 929.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kedmi and A. Orr-Urtreger
Differential brain transcriptome of {beta}4 nAChR subunit-deficient mice: is it the effect of the null mutation or the background strain?
Physiol Genomics,
January 17, 2007;
28(2):
213 - 222.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. J. Kannankeril, B. M. Mitchell, S. A. Goonasekera, M. G. Chelu, W. Zhang, S. Sood, D. L. Kearney, C. I. Danila, M. De Biasi, X. H. T. Wehrens, et al.
Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy
PNAS,
August 8, 2006;
103(32):
12179 - 12184.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Salas, F. Pieri, and M. De Biasi
Decreased Signs of Nicotine Withdrawal in Mice Null for the {beta}4 Nicotinic Acetylcholine Receptor Subunit
J. Neurosci.,
November 10, 2004;
24(45):
10035 - 10039.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kedmi, A. L. Beaudet, and A. Orr-Urtreger
Mice lacking neuronal nicotinic acetylcholine receptor {beta}4-subunit and mice lacking both {alpha}5- and {beta}4-subunits are highly resistant to nicotine-induced seizures
Physiol Genomics,
April 13, 2004;
17(2):
221 - 229.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
Gene
