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The Journal of Neuroscience, September 1, 2000, 20(17):6431-6441
Phenotypic Characterization of an  4 Neuronal
Nicotinic Acetylcholine Receptor Subunit Knock-Out Mouse
Shelley A.
Ross1,
John
Y. F.
Wong1,
Jeremiah J.
Clifford3,
Anthony
Kinsella4,
Jim S.
Massalas1,
Malcolm K.
Horne1,
Ingrid E.
Scheffer1, 5,
Ismail
Kola2,
John L.
Waddington3,
Samuel F.
Berkovic5, and
John
Drago1
1 Neurosciences Group, Monash University Department of
Medicine and 2 Institute of Reproduction and Development,
Monash Medical Centre, Clayton, Victoria, 3168, Australia,
3 Department of Clinical Pharmacology, Royal College of
Surgeons in Ireland, Dublin 2, Ireland, 4 Department of
Mathematics, Dublin Institute of Technology, Dublin 8, Ireland, and
5 Department of Medicine, University of Melbourne, Austin
and Repatriation Medical Centre, Heidelberg, Victoria, 3084, Australia
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ABSTRACT |
Neuronal nicotinic acetylcholine receptors (nAChR) are present in
high abundance in the nervous system (Decker et al., 1995 ). There are a
large number of subunits expressed in the brain that combine to form
multimeric functional receptors. We have generated an  4
nAChR subunit knock-out line and focus on defining the behavioral role
of this receptor subunit. Homozygous mutant mice (Mt) are normal in
size, fertility, and home-cage behavior. Spontaneous unconditioned
motor behavior revealed an ethogram characterized by significant
increases in several topographies of exploratory behavior in Mt
relative to wild-type mice (Wt) over the course of habituation to a
novel environment. Furthermore, the behavior of Mt in the elevated
plus-maze assay was consistent with increased basal levels of anxiety.
In response to nicotine, Wt exhibited early reductions in a number of
behavioral topographies, under both unhabituated and habituated
conditions; conversely, heightened levels of behavioral topographies in
Mt were reduced by nicotine in the late phase of the unhabituated
condition. Ligand autoradiography confirmed the lack of high-affinity
binding to radiolabeled nicotine, cytisine, and epibatidine in the
thalamus, cortex, and caudate putamen, although binding to a number of
discrete nuclei remained. The study confirms the pivotal role played by
the 4 nAChR subunit in the modulation of a number of
constituents of the normal mouse ethogram and in anxiety as assessed
using the plus-maze. Furthermore, the response of Mt to nicotine
administration suggests that persistent nicotine binding sites in the
habenulo-interpeduncular system are sufficient to modulate motor
activity in actively exploring mice.
Key words:
4; nicotinic receptor; homologous
recombination; anxiety; knock-out; behavioral topography
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INTRODUCTION |
Nicotine is one of the most widely
consumed psychoactive drugs and exerts a number of pharmacological
actions in the CNS and PNS (Decker et al., 1995). Neuronal
nicotinic acetylcholine receptors (nAChR) constitute a heterogenous
family of pentameric oligomers with contributions from 11 subunits (Le
Novere and Changeux, 1995). Five types of subunits
(2-6) and three
types of  subunits (2-4) permit
combinations of and type subunits to form a number of
functional receptors with subunits of two or more different types
(Changeux et al., 1998), whereas subunits
7-9 can form -bungarotoxin-sensitive homopentameric receptors (Corringer et al.,
1995). The topography of nAChR subunits varies (Deneris et al., 1989;
Duvoisin et al., 1989; Wada et al., 1989, 1990; Hill et al., 1993;
Elgoyhen et al., 1994; Court and Perry, 1995; Le Novere et al., 1996;
Brioni et al., 1997; Zoli et al., 1998), with
4 and 2 transcripts
being found in a large number of CNS nuclei, whereas
2, 3,
5, 6,
3, and 4 mRNAs are
restricted to a few cholinergic pathways, which also express
4 and 2.
Despite detailed characterization of nAChR subunits at the molecular
level, not much is known about the in vivo functional role
of individual subunits. Most nAChR ligands show similar patterns of
high-affinity labeling that resembles the distribution of
4/2 subunits. A
number of nAChR agonists that bind to the
4/2 receptor configuration in vitro are known to have an effect on
anxiety (Pomerleau, 1986; Gilbert et al., 1989; Brioni et al., 1993), attention (Brioni et al., 1997), and antinociception (Tripathi et al.,
1982; Damaj et al., 1998), implicating the
4/2 receptor in the
mediation of a number of physiological processes. Analysis of an
independently generated line of 4 nAChR
subunit knock-out mice (Marubio et al., 1999) validated the significant
role played by this receptor subunit in nociception. Loss of nicotinic
binding sites and a decrease of nAChR protein expression has been shown in patients with Alzheimer's disease and patients with Parkinson's disease with cognitive dysfunction (Giacobini, 1991; Brioni et al.,
1997). Furthermore, several mutations in the 4
nAChR subunit have been identified in autosomal dominant nocturnal
frontal lobe epilepsy (Steinlein et al., 1995, 1997). The large number
of subunits suggests a potential for considerable diversity in nAChR
function(s). Defining the specific role played by individual subunits
in determining spontaneous motor behavior and responses to drug
challenge will be aided by ongoing analysis of nAChR subunit gene
knock-out mice (Picciotto et al., 1995; Orr-Urtreger et al., 1997;
Marubio et al., 1999; Xu et al., 1999).
Nicotine is known to reduce anxiety in both chronic smokers
(Gilbert, 1979; Pomerleau, 1986; Gilbert et al., 1989) and
nonsmokers (Hutchinson and Emley, 1973). Anxiolytic-like effects of
nicotine and a select number of neuronal nicotinic receptor agonists
have also been documented in experimental animals (Costall et al., 1989; Brioni et al., 1993; Cao et al., 1993). The differential behavioral profile of neuronal nicotinic agonists implies that the
anxiolytic actions of nicotine may be mediated by a specific subunit configuration of the nAChR. We generated an
4 nAChR knock-out line to test the hypothesis
that mice lacking 4 nAChR subunits would show
behavioral features consistent with heightened basal levels of anxiety.
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MATERIALS AND METHODS |
Animals. All procedures involving the use of live
animals conformed to the National Health and Medical Research Council
(NHMRC) code of practice.
Cloning. A 1.0 kb cDNA probe encoding the putative second
transmembrane domain of the 4 nAChR receptor
subunit was cloned by PCR amplification of embryonic stem (ES)
cell-derived genomic DNA using primers based on conserved regions
between the human (GenBank accession number 135901) and rat (GenBank
accession number 131620) published cDNA sequence. The forward
primer corresponded to nucleotides 484-509, and the reverse primer
corresponded to nucleotides 1472-1500 of the rat sequence. An 11.2 kb
clone was isolated, and homologous flanks were cloned into the pPNT
targeting vector (Tybulewicz et al., 1991). The construct was designed
to create a nonfunctioning allele by removing a 750 bp
BglII/ScaI fragment from the fifth exon (Fig.
1). This fragment contains DNA spanning
from the first hydrophobic transmembrane domain through to the second
intracytoplasmic loop. The 5' flank was a 4.0 kb BglII
fragment, and the 3' flank was a 2.0 kb
ScaI/BglII fragment (Fig. 1).
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Figure 1.
Construction of targeting vector and Southern blot
analysis of ES clones. Representation of the genomic map of the
4 nAChR gene (A), the targeting
vector (B), and the expected allelic disruption
after homologous recombination (C). The
restriction sites shown are abbreviated as follows:
N, NotI; H,
HindIII; Bg, BglII;
Sc, ScaI; X,
XhoI; E, EcoRI;
B, BamHI. NEO
represents the neomycin phosphotransferase resistance gene, and
TK represents the thymidine kinase gene. The origin of
the probes used for homologous recombination screening are also shown.
Probe pE1 is a HindIII-BglII fragment
used to confirm 5' recombination, and probe pE2 is a
HindIII-BamHI fragment used for
confirmation of 3' recombination. D shows the Southern
blot of HindIII-digested ES cell genomic DNA probed with
pE1 (Wt allele is 6 kb, and the recombinant allele is 4.2 kb), showing
correctly targeted clones (C3, C44, C55, and C59); C67 is a randomly
selected nontargeted clone. E shows the Southern blot of
BamHI-digested ES cell genomic DNA probed with pE2; the
Wt allele is 12 kb, and the recombinant allele is 2.5 kb (clones C3,
C44, C55, and C59 are correctly targeted). F shows a
Southern blot of a BamHI-digested probed with a
NEO-specific probe. Clones C3, C44, C55, and C59 have a single band at
the expected size of 8 kb, confirming a single insertion event in all
the clones examined.
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ES cell culture and molecular analysis of transgenic mice.
Linearized targeting construct (50 µg) was electroplated into the J1
line (a gift from Dr. R. Jaenisch, Massachusetts Institute of
Technology, Cambridge, MA) of ES cells using standard techniques (Drago
et al., 1994). A HindIII digest of ES cell clone-derived genomic DNA probed with pE1 (Fig. 1) was used to verify recombination at the 5' end (normal allele is 6.0 kb and the recombinant allele 4.2 kb). A Southern blot of a BamHI digest probed with pE2 was used to verify 3' recombination (normal allele is 12.0 kb and recombinant allele is 2.5 kb). A single incorporation event was confirmed by probing a BamHI Southern blot with a neomycin
phosphotransferase (NEO) gene cDNA probe (Fig. 1). Four recombinants
were identified by Southern blotting (Fig. 1), one of which (C3) was
injected into BALB/C blastocysts, and chimeras were generated. Chimeras were mated with CF1 mice, and a single heterozygous mouse (Hz) was
obtained. The Hz founder was used to generate the entire colony by
subsequent crossing with C57/BL6 mice. Hz progeny, obtained after two
backcrosses with C57/BL6 mice, were mated to establish a number of
mutant mice (Mt) mating pairs and wild-type mice (Wt) mating pairs. All
Mt mice used in this study were therefore derived from Mt intermatings,
and all Wt mice were derived from Wt intermatings. Maximal diversity in
the genetic background was maintained by randomly interchanging
breeders within a given genotype.
Tissue preparation. Adult (Wt, n = 9; Hz,
n = 9; and Mt, n = 11) mice were killed
by decapitation, and the brains were snap-frozen in cold isopentane and
stored at  70°C before use. Twenty micrometer frozen coronal
sections were cut in a cryostat and mounted onto 3-aminopropyl
triethoxysilane (Sigma, St. Louis, MO) -coated slides for in
situ hybridization and gelatin-coated slides for ligand binding studies.
In situ hybridization. In situ hybridization was performed
for 3, 4,
6, 7,
2, 3, and
4 nAChR subunits. The sequences of the
oligonucleotides used are as shown in Table
1. Four oligonucleotides were designed to
identify 4 nAChR-specific transcript. Probes ISACh3 and ISACh4 were designed to hybridize with mRNA encoded in
transcribed gene sequence upstream of the deleted
BglII/ScaI fragment (Fig. 1), ISACh1 and
ISACh2 were designed to hybridize with mRNA derived from this deleted
sequence. This strategy allowed identification of cells that normally
express the 4 nAChR subunit in both Wt and Mt,
as well as verified the knock-out paradigm. All oligonucleotide probes
were 5' end-labeled using a standard kinase protocol (Wong et al.,
1997) with [ -33P]ATP and T4
polynucleotide kinase. Specificity of probes used in this study was
determined by using a 100-fold excess of unlabeled antisense
oligonucleotides added to the in situ hybridization reactions to competitively inhibit probe hybridization. Slides were
exposed to Hyperfilm (Amersham Pharmacia Biotech, Uppsala, Sweden), and the images were scanned. The density of mRNA
expression for 4 (ISACh3/4),
3, 4,
6, 7,
2, 3, and
4 nAChR subunits was quantified using a
microcomputer imaging device (MCID) with software (Image Research Inc.,
Brock University, St. Catherine's, Ontario, Canada). All values are
expressed as counts per minute/square millimeter for mRNA
expression (mean ± SEM). The specific binding for densitometric
purposes was calculated by subtracting background binding (determined
by cold competition) from results using labeled antisense
oligonucleotides alone.
Receptor autoradiography. All nicotinic agonists were
obtained from Sigma.
[3H]nicotine binding.
The slides were preincubated in Krebs'-Ringer's HEPES (in
mM: NaCl 118, KCl 4.8, CaCl2 2.5, MgSO47H2O 1.3, and HEPES
20, pH to 7.5 with NaOH) for 30 min at 4°C. They were then
transferred to Krebs'-Ringer's HEPES containing 5.1 nM
L-[N-methyl-3H]nicotine
(specific activity, 81.5 Ci/mmol; NEN, Boston, MA) and incubated for 90 min at 4°C. After incubation, the slides were washed as follows: two
times for 5 sec each in Kreb's-Ringer's HEPES; two times for
5 sec each in 20 mM HEPES, pH 7.5; and two times
for 5 sec each in distilled water. All washes were performed at 4°C.
The slides were then air dried at room temperature and apposed to
Hyperfilm (Amersham Pharmacia Biotech, Little Chalfont, UK) for 6 weeks
in the presence of tritiated microscales (Amersham Pharmacia Biotech,
Little Chalfont, UK). Binding in the presence of 1 µM unlabeled nicotine did not exceed film background.
[ 3H]epibatidine binding.
Slides were preincubated in Krebs'-Ringer's HEPES for 20 min at room
temperature and then transferred to Krebs'-Ringer's HEPES containing
400 pM
(±)[5,6-bicycloheptyl-3H]epibatidine
(33.8 Ci/mmol; NEN) for 60 min at room temperature. The slides were
then washed as follows: two times for 10 sec each in Krebs'-Ringer's
HEPES; two times for 10 sec each in 0.1× Krebs'-Ringer's HEPES; 10 sec in 5 mM HEPES, pH 7.5, and distilled water
for 5 sec. All washes were performed at 0°C. The slides were then air dried at room temperature and apposed to Hyperfilm for 3 weeks together
with standard tritiated microscales. Nonspecific binding was defined as
the binding in the presence of unlabeled epibatidine (10 µM). Cold competition assays were also
performed with unlabeled 300 µM nicotine and
150 nM cytisine.
[ 3H]cytisine binding.
The slides were incubated at 4°C for 60 min in 50 mM Tris-HCl, pH 7.4, containing (in
mM): 120 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, and 5 [3,5-3H (N)]cytisine
hydrochloride (32Ci/mmol; NEN). The washes were as follows: three times
for 2.5 min each in 50 mM Tris-HCl, pH 7.4, followed by a brief rinse in distilled water, all performed at 4°C.
Nonspecific binding was defined as the binding in the presence of
unlabeled nicotine (10 µM). The film was
exposed for 3 months.
[125I]-bungarotoxin
binding. The slides were preincubated in 50 mM Tris-HCl, pH 7.4, containing 0.1% bovine
serum albumin (BSA) for 30 min at room temperature. They were then
incubated in 50 mM Tris-HCl, pH 7.4, containing
[125I]-bungarotoxin (2000 Ci/mmol; a
gift from Prof. Bevyn Jarrott, Department of Pharmacology, Monash
University, Clayton, Australia) at a concentration of 1.5 nM for 120 min at room temperature. The washes
were as follows: two times for 15 min each in 50 mM Tris-HCl, pH 7.4, and 0.1% BSA; two times for
15 min each in 50 mM Tris-HCl, pH 7.4, followed
by a brief rinse in distilled water, all performed at 4°C.
Nonspecific binding was defined as the binding in the presence of
unlabeled acetylcholine (10 mM). The slides were
exposed to XAR5 film (Eastman Kodak, Rochester, NY) together with
laboratory-prepared standards for 36 hr.
Binding densities were measured using the MCID M4 system under constant
illumination. Standardization was achieved by comparing binding
densities with 3H-microscales and
standards exposed with each film. All values are expressed as
femtomoles per milligram of tissue for receptor binding studies
(mean ± SEM). The specific binding was calculated by subtracting
nonspecific binding determined when labeled ligand was coincubated with
respective unlabeled receptor agonist.
Topography of spontaneous motor behavior. On experimental
days, mice were removed from their home cage and placed individually in
clear glass observation cages (36 × 20 × 20 cm). Behavioral assessments were performed in a manner similar to that used extensively for rats (Clifford and Waddington, 1998) and mice (Clifford et al.,
1998, 1999) using a rapid time-sampling behavioral checklist technique.
For this procedure, each of 10 randomly allocated mice was observed
individually for 5 sec periods at 1 min intervals over 15 consecutive
minutes, using an extended, ethologically based behavioral checklist.
This allowed the presence or absence of the following individual
behaviors (occurring alone or in any combination) to be determined in
each 5 sec period: sniffing; locomotion (coordinated movement of all
four limbs producing a change in location); total rearing (rearing of
any form); rearing from a sitting position (front paws reaching upwards
with hind limbs on floor in sitting position); rearing free (front paws reaching upwards away from any cage wall while standing on hind limbs);
rearing toward a cage wall (front paws reaching upwards on a cage wall
while standing on hind limbs); biting; sifting (sifting movements of
the front paws through cage bedding material); grooming (of any form);
intense grooming (grooming of the face with the forepaws followed by
vigorous grooming of the hind flank or anogenital region with the
snout); vacuous chewing (chewing movements not directed onto any
physical material); chewing (chewing movements directed onto cage
bedding and/or faecal pellets without consumption); eating (chewing
with consumption); climbing (jumping onto cage top with climbing along
grill in inverted or hanging position); and stillness (motionless, with
no behavior evident). This cycle of assessment by behavioral checklist
over a 15 min period (0-15 min) was repeated twice (20-35 and 40-55
min); thereafter, 10 cycles of otherwise identical assessments were
repeated at 80-90, 120-130, 160-170, 200-210, 240-250,
280-290, 340-350, and 360-370 min.
Effect of nicotine on behavior. Examination of the effect of
nicotine was conducted under two conditions: (1) unhabituated, i.e.,
active exploration; and (2) habituated, whereby mice were placed
individually in the clear glass observation cages and left undisturbed
for a period of 3 hr. After injection of drug or vehicle, animals were
assessed using the rapid time-sampling behavioral checklist technique.
The time of injection was taken as the zero time point. For assessment
of spontaneous behavior and effects of nicotine on unhabituated
behavior, animals were used on one occasion only; for the assessment of
effects of nicotine on habituated behavior, animals were used on two
occasions, only separated by a drug-free interval of 1 week.
Rotarod. The rotarod apparatus (Ugo Basile, Milan, Italy)
was used in accelerating mode, gradually increasing from 4 to 40 rpm
over the course of 5 min. Mice were placed on the apparatus at a fixed
speed of 4 rpm for 1 revolution; the apparatus was then set to
accelerating mode, and the stopwatch was started. Latency to fall was
recorded for each mouse in three trials per day, separated by an
intertrial interval of 2 hr. Each mouse was subjected to this schedule
for 3 successive days. Mice that stayed on the rotarod for >360 sec
were considered complete responders; their latencies were recorded as
360 sec. All experiments were performed during the hours of 11:00 A.M.
to 6:00 P.M. to avoid circadian effects.
Elevated plus-maze. Anxiolytic-like activity was evaluated
using the elevated plus-maze, a pharmacologically validated model (Pellow et al., 1985; Brioni et al., 1993), according to procedures described previously in which nicotinic receptor agonists were demonstrated to have an anxiolytic-like effect (Brioni et al., 1993).
The elevated plus-maze was custom-made of black Perspex consisting of
two open arms (5 × 30 cm) and two enclosed arms (5 × 30 × 14 cm) extending from a central platform (5 × 5 cm) mounted on a wooden base raised 57 cm above the floor; thus, the maze
formed a "plus" shape. Overhead light levels on the open and
enclosed arms were similar. At the beginning of the experiment, mice
were placed in the center of the maze facing an enclosed arm, and the
following variables were scored: (1) the time spent on the open and
enclosed arms, reported as time spent on open (or enclosed) arms
expressed as a percentage of total time (300 sec); and (2) the number
of entries into open and closed arms. An arm entry was defined as the
entry of all four feet of the animal into one arm; an arm exit was
defined by the exit of both forelimbs from one arm. Plus-maze behavior
was assessed by direct observation.
Data analysis. For determination of ethograms for
spontaneous behavior over the phase of initial exploratory activity,
the total "counts" for each individual behavior was determined as the number of 5 sec observation windows in which a given behavior was
evident, summed over the initial 3 × 15 min (0-15, 20-35, and
40-55 min) cycle periods and expressed as means ± SEM. Data for
individual behaviors were analyzed using ANOVA following
square-root transformation, to allow examination of interaction effects
in the absence of nonparametric techniques for interaction terms. For
determination of the habituation profiles of these ethograms, total
counts for each individual behavior were summed as above over
each of the following periods: 0-10, 20-30, 40-50, 80-90, 120-130,
160-170, 200-210, 240-250, 280-290, 340-350, and 360-370; these
were expressed also as means ± SEM and analyzed using
repeated-measures ANOVA following square-root transformation.
For determination of the effect of nicotine on both active exploration
and under habituated conditions, the total counts for each individual
behavior were determined as the number of 5 sec observation
"windows" in which a given behavior was evident, summed over the
initial 3 × 15 min (0-15, 20-35, and 40-55 min) cycle periods
and expressed as means ± SEM. Data for individual behaviors were
analyzed using ANOVA following square-root transformation; data were
then analyzed using either Student's t test or
Mann-Whitney U test to identify those particular drug doses
at which responsivity differed by genotype, for a given topography of behavior.
For determination of rotarod performance, latency to fall was
calculated for each mouse on three occasions each day, for 3 successive
days, and expressed as means ± SEM. Data were analyzed using
ANOVA following square-root transformation; data were then analyzed
using either Student's t test or Mann-Whitney U
test to identify those particular time points at which responsivity differed by genotype. For elevated plus-maze performance, the percentage of time spent in and number of entries into open and closed
arms was calculated for each mouse. Data were expressed and analyzed as
above to identify those particular parameters for which responsivity
differed by genotype.
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RESULTS |
Mice homozygous for the 4 nAChR mutation
were born in the expected mendelian proportion, were capable of
reproduction, and were of normal body weight (data not shown).
Hematoxylin and eosin-stained sections of the brain of Mt were examined
histologically, and no differences in the size or location of the brain
nuclei or cortical lamination were apparent (data not shown). In
addition, gross anatomical and histological screening failed to show
any abnormalities in heart, liver, spleen, kidney, lung, muscle, and thymus (data not shown).
In situ hybridization
In situ hybridization performed on a number of animals
(Wt, n = 9; Hz, n = 6; and Mt,
n = 9) identified a strong hybridization signal for
ISACh3 and ISACh4 localized to the thalamus (Th) and cortex (Cx) in Wt
(Fig.
2E,G)
Mt (Fig. 2F,H),
and Hz (data not shown). A moderate hybridization signal was also seen
in the caudate putamen (CPu), hippocampus (Hp), dentate gyrus (DG), and
substantia nigra (SN) (data not shown). ISACh1 and ISACh2 probes, which
hybridize to the deleted sequence, showed no regional-specific signal
throughout the brain of Mt (Fig.
2B,D) and a reduced signal in Hz
(data not shown). The hybridization pattern seen in Wt using ISACh1 and ISACh2 probes was the same as for ISACh3 and ISACh4 (Fig.
2E,G). In situ
hybridization was also performed for 3,
6, 7,
2, 3, and
4 nAChR subunits (Fig.
3). An intense signal for
3 mRNA was detected in the medial habenular
(MHb) (Fig. 3A,B),
6 signal was detected in the SN (Fig.
3C,D), 7 was seen
in the Hp and DG (Fig.
3E,F),
2 showed a strong signal in the MHb and Th,
and moderate hybridization was seen in the Cx, Hp, and DG (Fig.
3G,H), whereas
3 had limited distribution with signal only in
the MHb and the SN (Fig. 3I-L). 4
expression was restricted to the MHb (Fig.
3M,N). There were no
statistically significant differences between Mt and Wt in the relative
abundance of 3, 6,
7, 2, 3, and 4 nAChR
subunits transcripts (Fig. 4).
Furthermore, the level of the 4 nAChR
subunit-specific transcripts detected by probes ISACh3/4 were the same
in Mt and Wt (Fig. 4). These oligonucleotide probes were designed to
hybridize to mRNA transcribed from preserved DNA upstream of deleted
gene sequence and thereby specifically identify
4 nAChR subunit-positive cells.
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Figure 2.
Expression of the 4 neuronal nAChR
subunit in Wt and Mt mouse brain. In situ hybridization
using antisense 4 nAChR-specific cDNA oligonucleotide
probes. A, C, E, and
G represent sections from Wt. B,
D, F, and H were sections
derived from Mt. I and J represent
nonspecific binding (NSB). Sections shown in
A-D were probed simultaneously with probes ISACh1 and
ISACh2, whereas sections shown in E-H were probed with
ISACh3 and ISACh4. No anatomically defined signal was seen in the Mt
with ISACh1/ISACh2, which detects the deleted sequence
(B, D), whereas signal was detected (in
brain regions known to express 4 nAChR) using
ISACh3/ISACh4, which recognizes upstream transcript (F,
H). Sections shown in A,
B, E, and F are taken at
bregma levels 0.8, and C, D,
G, and H are taken at bregma levels
1.82 (Franklin and Paxinos, 1997).
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Figure 3.
Expression of 3,
6, 7,
2, 3, and 4
neuronal nAChR subunits in Wt and Mt mouse brain using in
situ hybridization. No difference was apparent between Wt
(A) and Mt (B) probed with
3-specific probes with both genotypes showing intense
hybridization in the MHb. Hybridization is seen in the SN in Wt
(C) and Mt (D) probed with
6-specific probes, and no difference was seen between Wt
(E) and Mt (F) probed with
7-specific probes with Wt and Mt showing hybridization
in the Hp and DG. Strong hybridization is seen in the MHb and Th, and
moderate hybridization is seen in the Cx, Hp, and DG in Wt
(G) and Mt (H)
probed with 2-specific probes. Hybridization is seen in
the MHb and the SN in Wt (I, K)
and Mt (J, L) probed with
3-specific probes. Hybridization is seen in the MHb in
Wt (M) and Mt (N)
probed with 4-specific probes.
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Figure 4.
Quantitative autoradiography for ISACh3/4,
3, 6,
7, 2,
3, and 4 nAChR subunits in Wt and
Mt. Quantitative analysis of ISACh3/4 (A),
3 (B), 6
(C), 7 (D),
2 (E), 3
(F), and 4
(G) binding in Wt and Mt. The results are
expressed as mean ± SEM (counts per minute/square millimeter).
Statistical analysis was performed using a Student's t
test. There were no statistically significant differences between Mt
and Wt for any of the probes examined. Regions quantitated were Cx,
CPu, Th, MHb, SN, and Hp.
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Receptor autoradiography
Autoradiographic ligand binding experiments were performed on a
number of animals (Wt, n = 9; Hz, n = 11; and Mt, n = 11). Binding experiments conducted in
Wt using tritiated nicotine, cytisine, and epibatidine showed a similar
pattern of high-affinity binding (Fig.
5).
[3H]nicotine labeling was detected at
highest levels in the thalamic nuclei, MHb, interpeduncular nucleus
(IPn), superior colliculus (SC), and presubiculum, and moderate levels
were found in the Cx, CPu, and fasciculus retroflexus (fr) (Fig.
5A,E,I).
[3H]cytisine (Fig. 5M)
binding showed a similar pattern to
[3H]nicotine binding in Wt.
[3H]epibatidine (Fig.
5C,G,K) binding differed
from [3H]nicotine binding in that
[3H]epibatidine binding to the MHb and
fr was more intense as shown by quantitative analysis (Fig.
6).
[3H]nicotine and
[3H]epibatidine binding showed a
qualitatively similar pattern in Mt, with binding for both radioligands
detected in the MHb, IPn, fr, and SC (Fig. 5). The main difference was
that [3H]epibatidine binding was
detected at high levels in MHb, IPn, fr, and SC (Fig.
5D,H,L).
[3H]cytisine binding was only detected
in the IPn of Mt (Fig. 5N). [3H]epibatidine binding in Wt and Mt
with cytisine or nicotine cold competition resulted in loss of SC
signal but preservation of binding in the habenulo-interpeduncular
pathway (i.e., MHb, IPn, and fr) (data not shown).
[125I]-bungarotoxin binding was found
to be unchanged in Mt compared with Wt (Fig.
5O,P). Autoradiographic ligand binding
experiments performed on an independently generated line of
4 nAChR subunit knock-out mice (Marubio et
al., 1999) also demonstrated high level binding to
[3H]epibatidine in a number of nuclei
and reduced [3H]nicotine binding in the
MHb. Quantitative autoradiography, however, demonstrated that
[3H]epibatidine binding was reduced in
Mt compared with Wt in the SC and IPn, whereas there was no difference
in the MHb (Fig. 6). Furthermore, Marubio et al. (1999) showed that
[3H]nicotine binding was found at
reduced levels only in the MHb, whereas we detected
[3H]nicotine binding in both the IPn and
the SC, in addition to the previously described binding sites in the
MHb (Fig. 6). Quantitative analysis confirmed that
[3H]nicotine binding was moderately
reduced in Mt compared with Wt in all three nuclei (Fig. 6).
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Figure 5.
Ligand autoradiography of nicotine receptor
agonists in mouse brain. Autoradiographic mapping using
[3H]nicotine identifying the presence of
high-affinity nicotine receptors in Wt (A,
E, I) and Mt (B,
F, J) mouse brain sections at
bregma levels 2.06 (A, B), 3.08
(E, F), and 3.40
(I, J). A,
E, and I show binding in the Th, Cx,
MHb, fr, SC, and IPn. B, F, and
J show persistent binding in the Mt;
arrows indicate the location of the MHb, fr, SC, and
IPn. Autoradiographic mapping using
[3H]epibatidine in Wt (C,
G, K) and Mt (D,
H, L) mouse brain sections at bregma
levels 2.06 (C, D), 3.08
(G, H), and 3.40
(K, L). Binding is present in Wt in the
Th, Cx, MHb, fr, SC, and IPn. Mt showed binding is in the MHb, fr, SC,
and IPn. Autoradiographic mapping using
[3H]cytisine in Wt
(M) and Mt (N) mouse
brain sections at bregma levels 3.40, showing persistent binding in
the IPn. Autoradiographic mapping using
[I125]-bungarotoxin in Wt
(O) and Mt (P) mouse brain
sections at bregma levels 2.54 (Franklin and Paxinos, 1997). No
difference was apparent between the genotypes.
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Figure 6.
Quantitative autoradiography for nicotinic
agonists in the mouse brain. Quantitative analysis of
[3H]nicotine (A),
[3H]cytisine (B),
[3H]epibatidine (C), and
[125I]-bungarotoxin (D)
binding in Wt, Hz, and Mt. The results are expressed as mean ± SEM (femtomoles per milligram). Statistical analysis was performed
using a one-way ANOVA. *p < 0.05 versus Wt
controls. Regions quantitated were Cx, CPu, Th, MHb, SC, IPn, fr,
retroparafasciculus (RPF), and Hp.
PrS, Presubiculum.
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Topography of spontaneous behavior
General observation
No gross neurological deficits were apparent in 40 Mt (20 females,
weight of 25.97 ± 0.53 gm; 20 males, weight of 31.79 ± 0.87 gm; age of 105 ± 4 d) when compared with 40 Wt controls (20 females, weight of 24.59 ± 59; 20 males, weight of 33.40 ± 0.64 gm; age of 109 ± 6 d); in particular, no epileptic
seizures were observed over prolonged observation.
Exploratory phase
Over an initial 1 hr phase of exploratory activity (Fig.
7A), Mt were characterized by
increased sniffing (+13%; F(1,76) = 6.76, p = 0.01) and decreased grooming (15%;
F(1,76) = 4.98, p = 0.03), for both genders; females groomed less than males for each
genotype.
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Figure 7.
A, Behavioral counts for sniffing
(Sn), locomotion (L), sifting
(S), total rearing (Rt), rearing
from a seated position (Rs), rearing free
(Rf), rearing to wall (Rw),
grooming (Gr), biting (B),
stillness (St), chewing (Ch), vacuous
chewing (VCh), eating (E), and
licking (Li) in Wt (n = 40;
filled columns) versus Mt (n = 40;
open columns). Data are mean ± SEM counts over
a 1 hr phase of initial exploratory activity.
***p < 0.001, **p < 0.01, *p < 0.05 versus wild-types. B,
Behavioral counts for sniffing, eating, grooming, locomotion, rearing
total, stillness, rearing seated, rearing free, and rearing to wall in
Wt (n = 40; squares) versus Mt
(n = 40; diamonds). Data are
mean ± SEM counts per 10 min period at indicated intervals over
habituation phase.
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Habituation phase
Over the subsequent phase of habituation (Fig. 7B),
additional effects were evident. Each of sniffing, total rearing,
rearing seated, rearing to wall, rearing free, and chewing occurred to excess in Mt throughout this phase
(F(1,76) = 17.56, p < 0.001; F(1,76) = 14.95, p < 0.001; F(1,76) = 13.96, p < 0.001;
F(1,76) = 16.15, p < 0.001; F(1,76) = 4.94, p < 0.05; F(1,76) = 5.19, p < 0.05, respectively); for those behaviors
that declined significantly by time bins (i.e., habituation of
sniffing, total rearing, rearing seated, and rearing to wall), this did
not differ by genotype or gender, whereas for those low-frequency
behaviors for which habituation was not apparent (i.e., rearing free
and chewing), males habituated more rapidly than did females for each
genotype. Locomotion also occurred to excess in Mt
(F(1,76) = 11.11, p = 0.001) because of their reduced rate of habituation (time × genotype interaction, F(10,760) = 1.97, p = 0.03) for both genders. Although overall
levels of grooming were comparable, this behavior varied over time bins
in a manner that differed between the genotypes (time × genotype
interaction, F(10,760) = 3.38, p < 0.001) for both genders. Overall levels of
stillness were decreased in Mt (F(1,76) = 24.83, p < 0.001) because of their reduced rate of habituation (time × genotype interaction, F(10,760) = 1.98, p = 0.03) for both genders.
In summary, over an initial exploratory phase, Mt showed increased
sniffing with decreased grooming. Over the subsequent habituation phase, increased sniffing in Mt endured together with the emergence of
increases in most other topographies of behavior; in particular, increased locomotion in Mt was characterized by a reduced rate of
habituation relative to Wt.
Rotarod performance
Among 37 Mt (15 females, weight of 25.01 ± 0.58; 22 males,
weight of 31.32 ± 0.8 gm; age of 97 ± 3 d) and 44 Wt
controls (20 females, weight of 24.45 ± 0.76; 24 male, weight of
33.27 ± 0.51 gm; age of 96 ± 5 d), performance
improved with time (three trials on each of 3 successive test days,
F(8,632) = 18.47, p < 0.001) in a manner that did not differ by genotype or gender (Fig.
8). Thus, in this test of sensorimotor
coordination, Mt evidenced no deficits.
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Figure 8.
Performance on a repeated motor coordination task
in Wt (n = 37; filled columns)
versus Mt (n = 44; open columns).
Mice were evaluated on the rotarod test three times a day for 3 consecutive days. Data are presented as the mean ± SEM of the
daily averages. ***p < 0.001, **p < 0.01 versus same genotype on day 1.
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Elevated plus-maze performance
When compared with 51 Wt controls (21 females, weight of 24.2 ± 0.47; 30 males, weight of 31.44 ± 1.0 gm; age of 96 ± 5 d), 70 Mt (36 females, weight of 25.7. ± 0.47; 34 males, weight
of 32.81 ± 0.71 gm; age of 97 ± 3 d) spent less time
in open arms (F(1,115) = 6.92, p < 0.01) and made fewer open-arm entries
(F(1,115) = 6.9, p < 0.01) in a manner that did not differ by gender (Fig. 9); for each genotype, males entered
fewer arms than females. Thus, in this test of "anxiety-like"
behavior, Mt evidenced heightened levels.
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Figure 9.
The elevated plus-maze assay. Data are presented
as the mean ± SEM of percentage time in (A)
and number of entries into (B) various sectors in
Wt (n = 51; filled columns) and Mt
(n = 70; open columns).
**p < 0.01, *p < 0.05 versus
Wt.
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Effects of nicotine: unhabituated condition
Our results differed from those of Marubio et al. (1999), who
analyzed an independently generated line of 4
nAChR subunit knock-out mice, finding no significant differences from
baseline in nonhabituated locomotor activity in response to 1 or 2 mg/kg nicotine. When comparing 40 Mt (20 females, weight of 25.62 ± 0.46; 20 males, weight of 31.55 ± 0.82 gm; age of 97 ± 3 d) with 40 Wt controls (20 females, weight of 25.16 ± 0.45; 20 males, weight of 32.47 ± 0.55 gm; age 100 ± 2 d), declines in each of sniffing, locomotion, and total rearing over
the three time periods were influenced by dose of nicotine administered
in a genotype-specific manner (genotype × dose × time
interactions, F(8,120) = 2.23, p = 0.03; F(8,120) = 3.98, p < 0.001;
F(8,120) = 2.16, p < 0.05, respectively) (Fig.
10A-C). For sifting,
a generally comparable although blunted profile was apparent (Fig.
10D). For individual topographies of rearing,
grooming, and chewing, less consistent profiles of effect were apparent
(data not shown).
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Figure 10.
Unhabituated mice: response to nicotine
administration. Behavioral counts summed over 0-15, 20-35, and 40-55
min: sniffing (A), locomotion
(B), rearing total (C), and
sifting (D) responses to ()-nicotine (0.1-2.7
mg/kg, s.c.) versus vehicle (V) in
unhabituated Wt (filled columns) and Mt
(open columns). Data are means ± SEM for
n = 8 per group. **p < 0.01, *p < 0.05 versus Wt.
ap < 0.05, bp < 0.01, cp < 0.001 versus vehicle-treated mice
of same genotype.
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In summary, under this unhabituated condition, Mt showed less decline
in sniffing, locomotion, total rearing, and sifting over these three
time periods than was evident in Wt; decline in behavior in Mt was
restored by low to mid doses of nicotine, particularly over the late
(40-55 min) period.
Effects of nicotine: habituated condition
When comparing 20 Mt (10 females, weight of 25.2 ± 0.7; 10 males, weight of 33.2 ± 0.2 gm; age of 95 ± 3 d) with
20 Wt controls (10 females, weight of 26.28 ± 0.6; 10 males,
weight of 32.38 ± 0.38 gm; age of 95 ± 5 d), each of
sniffing, locomotion, and total rearing declined over the three time
periods; for locomotion and total rearing, an action of nicotine to
reduce these behaviors declined with time in a manner that was
influenced by dose of nicotine administered (dose × time
interactions, F(8,120) = 3.14, p = 0.01; F(8,120) = 4.64, p < 0.001, respectively) (Fig.
11A-C). For sifting,
a generally comparable although blunted profile was apparent (Fig.
11D), and for individual topographies of rearing, grooming, and chewing, similar profiles of effect were apparent (data
not shown).
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Figure 11.
Well habituated mice: response to nicotine
administration. Behavioral counts summed over 0-15, 20-35, and 40-55
min: sniffing (A), locomotion
(B), rearing total (C), and
sifting (D) responses to ()-nicotine (0.1-2.7
mg/kg, s.c.) versus vehicle (V) in well
habituated Wt (filled columns) and Hz
(open columns). Data are means ± SEM for
n = 8 per group. **p < 0.01, *p < 0.05 versus Wt.
ap < 0.05, bp < 0.01, cp < 0.001 versus vehicle-treated mice
of same genotype.
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In summary, under this habituated condition, lower levels of behavior
continued to decline with time; nicotine acted mainly to further reduce
behavior, primarily over the early (20-35 min) period, in a manner
that tended to be less prominent in Mt than in Wt. Thus, the above
late-period action of nicotine to restore in Mt a Wt-like behavioral
profile appeared specific to the unhabituated condition.
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DISCUSSION |
The 4/2 subunit
receptor configuration, known to be expressed at high levels in the
thalamus and the habenulo-interpeduncular system (Zoli et al., 1998),
is responsible for the vast majority of agonist binding. In this study,
we have characterized the binding patterns of a number of nicotinic
agonists in normal and 4 nAChR knock-out mice.
These data combined with the results of previous studies (Zoli et al.,
1998) on mice lacking functional 2 nAChRs allows us to further characterize brain nAChRs. Mt maintain high-level [3H]nicotine binding in the MHb and IPn
and low-level binding in the SC and fr, whereas the
2 knock-out mice showed no binding with this
ligand (Picciotto et al., 1995). 2 knock-out
mice had [3H]cytisine binding sites in
the MHb, IPn, and fr (Zoli et al., 1998), whereas Mt had binding in the
IPn but not in the MHb or fr. 2 knock-out mice
differ from Mt in that Mt showed additional [3H]epibatidine binding sites in the SC.
Our [3H]nicotine autoradiography results
differed from ligand binding experiments performed on an independently
generated line of 4 nAChR knock-out mice
(Marubio et al., 1999). In this earlier study, [3H]nicotine binding was found at low
levels only in the MHb, whereas we detected additional low-level
binding sites in the IPn, SC, and fr (Figs. 5, 6).
There are known to be high levels of 3 and
4 (Wada et al., 1989) and low levels of
6 mRNA (Le Novere et al., 1996) in the rodent
MHb, whereas all subunits surveyed are detected in this nucleus
(Deneris et al., 1989; Duvoisin et al., 1989; Wada et al., 1989). We
found no significant differences in the expression profile of a large
number of subunits within the MHb of Mt and Wt. A strong hybridization
signal was detected for 3,
2, 3, and
4, confirming the results of previous studies
(Le Novere et al., 1996). The receptor configuration responsible for
the MHb binding to [3H]nicotine seen in
Mt therefore involves the 2 subunit in
combination with the 3 subunit expressed at
high levels and/or the 6 subunit expressed at
low levels. Nicotine autoradiographic analysis in 2 knock-out mice suggests that the
3 and 4 expression
detected in the MHb is insufficient to mediate
[3H]nicotine binding. The finding that
2 knock-out mice retain high-affinity
[3H]cytisine binding in the MHb and that
Mt lack MHb binding confirms that cytisine binding in the MHb requires
the 4 subunit but does not require the
2 subunit.
[3H]epibatidine binding was detected in
the MHb of both knock-out lines, suggesting that
[3H]epibatidine binding can occur in the
absence of 2 or 4 subunits.
A detailed, topographical analysis of spontaneous and
nicotine-stimulated behavior was undertaken. Over the initial 1 hr
exploratory phase, Mt showed an increase in sniffing and a reduction in
grooming. Over the subsequent phase of habituation, additional effects
were revealed as certain topographies of behavior did not habituate to
the same extent in Mt. For sniffing, both genotypes habituated to
similar extents, whereas for locomotion and topographies of rearing and
chewing, Mt retained a higher level of activity throughout the
habituation phase. Alterations in "motor activity" after nicotine administration have been described previously. Although these studies
have most commonly used techniques that fail to resolve individual
topographies of behavior (Morrison and Stephenson, 1972; Clarke and
Kumar, 1983; Clarke, 1987), state-dependency of effect, e.g., treatment
in a familiar versus novel environment, has been reported (Picciotto et
al., 2000). Using the present ethologically based approach, over the
first 15 min of the exploratory phase, nicotine induced in Wt a
dose-dependent reduction in locomotion, total rearing, sifting, and
less so in sniffing, although there was no such drug effect in Mt;
rather, as above, Mt showed relative preservation of these behaviors
over the exploratory phase, with late decline in behavior restored in
Mt to Wt levels by low to mid doses of nicotine. Under the habituated
condition, however, during which lower levels of behavior continued to
decline with time, nicotine was still able to further reduce
locomotion, total rearing, sifting, and less so in sniffing; this
occurred primarily over the early period in a manner that tended to be
less prominent in Mt than in Wt. Thus, the above late-period action of
nicotine to restore in Mt a Wt-like behavioral profile appeared
specific to the unhabituated condition. The lack of modulation of
locomotion and sniffing behavior in Mt with high doses of nicotine
(Fig. 10) may reflect pharmacological desensitization of remaining
nicotinic receptors (Couturier et al., 1990; Vibat et al., 1995;
Fenster et al., 1997). These data indicate a topographically specific interaction between 4 nAChR knock-out and the
neuronal processes of habituation in determining not only the
regulation of spontaneous behavior but also the effects of nicotine on behavior.
One hypothesis is that the 4 nAChR subunit is
required for activation of inhibitory neural circuits; hence, its
absence would result in an elevated baseline of specific behavioral
topographies. Pharmacological doses of nicotine may nonetheless act
through non-4 nAChR-containing nicotine
binding sites, perhaps those present in the habenulo-interpeduncular
pathway, to reduce inhibitory tone. However, this might only be evident
when such inhibitory neural tone is at a low level, as would be
expected in actively exploring mice; the delayed effect of nicotine
administration in reducing locomotion, sniffing, rearing, and sifting
in this exploratory condition may reflect some temporal inefficiency of this parallel pathway in responding to change. Furthermore, the effect
of an agent that acts primarily to activate inhibitory pathways may be
less apparent in well habituated mice in which inhibitory pathways
(both nAChR-dependent and -independent) might be already prominently
activated. There are a number of studies describing nicotine-induced
release of the inhibitory neurotransmitter GABA either from isolated
synaptosomes or in slice preparations (Lena et al., 1993; Kayadjanian
et al., 1994; McMahon et al., 1994). Of particular relevance to our
findings is the observation of Lena et al. (1993) showing that nicotine
increases the frequency of postsynaptic GABAergic currents in rat
interpeduncular nucleus neurons.
Nicotinic receptor agonists that bind the
4/2 receptor
configuration are known to have an effect on anxiety (Pomerleau, 1986;
Gilbert et al., 1989; Brioni et al., 1993). Nicotine in particular
appears to have anxiolytic-like effects in a number of behavioral
paradigms, including the elevated plus-maze assay (Costall et al.,
1989; Brioni et al., 1993). Our results suggest that the
4 subunit may indeed be intimately involved in
mediating anxiolytic-like effects. The nicotinic receptor agonists
ABT-418 and lobeline share the anxiolytic-like actions of nicotine,
whereas cytisine (Brioni et al., 1993), anabasine, and epibatidine are devoid of anxiolytic-like properties (Decker et al., 1995). The differential behavioral profile of neuronal nicotinic agonists implies
that the anxiolytic-like actions may be mediated by a specific subunit
configuration of the nAChR. If this were indeed the case, we would
expect agonists that have similar effects on anxiety to have comparable
ligand binding profiles with respect to both qualitative (i.e.,
topography of binding) and quantitative (i.e., agonist specific
intensity of binding) parameters. The differential preservation of
binding in Mt may explain the lack of anxiolytic-like effects seen with
cytisine because it implies selectivity for a specific receptor
subpopulation, but it would not readily explain the lack of
anxiolytic-like effect of epibatidine because the binding pattern of
epibatidine is qualitatively similar to nicotine in Mt. Quantitative
autoradiographic analysis demonstrates comparable epibatidine binding
in Wt and Mt in the MHb and fr (although a minor reduction of
epibatidine binding of 16% in the IPn of Mt compared with Wt). In
contrast, nicotine binding shows a substantial reduction of 40% in the
MHb and 45% in the IPn in Mt compared with Wt. The situation is
further complicated by the potential for differential segregation of
nAChR subunits on individual neurons.
The finding of increased exploratory activity evident on assessment of
spontaneous behavior was surprising given the anxiety-like profile
demonstrated by elevated plus-maze analysis. However, elevated anxiety
may not invariably be associated with locomotor hypoactivity.
Withdrawal of benzodiazepine is associated with anxiety-like behavior
and locomotor hyperactivity (Nowakowska et al., 1997). In addition, a
very large number of clinical studies report on the coexistence of
anxiety and motor restlessness after cessation of cigarette
smoking (Hughes and Hatsukami, 1986; Hughes et al., 1991, 1994;
Hilleman et al., 1992; Hughes, 1992; McKenna and Cox, 1992; Jorenby et
al., 1996; Schneider et al., 1996; Shiffman et al., 2000). A putative
state-dependency model in which the behavioral topography of
anxiety-like responses depends on the environmental context may be
useful in understanding the data. In this model, the sustained level of
behavior in mutants over habituation in a "naturalistic" setting
would appear to compliment the finding of reduced open-arm entries in
the "stressful" setting of the plus-maze.
In conclusion, our data suggests that, in a stressful setting, Mt have
a heightened basal level of anxiety-like behavior; furthermore, in a
naturalistic setting, topographies of exploratory behavior are
increased, and these behaviors may nonetheless be modulated by nicotine administration.
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FOOTNOTES |
Received Jan. 10, 2000; revised June 9, 2000; accepted June 14, 2000.
This work is supported in part by the National Health and Medical
Research Council of Australia (NHMRC), the Australian Commonwealth Department of Veteran's Affairs, and an unrestricted educational grant
from Parke-Davis (Australia). S.R. was supported by a Peter Bladin
Scholarship from the Epilepsy Society of Australia. J.D. is a Monash
University Logan Research Fellow. J.J.C. was supported by the NHMRC
Brain Network into Mental Health Diseases. J.L.W. and J.J.C. are
supported by a Galen Fellowship from the Irish Brain Research
Foundation, the Higher Education Authority of Ireland, and the Royal
College of Surgeons in Ireland. We thank Prof. Bevyn Jarrott
(Department of Pharmacology, Monash University) for the gift of
iodinated -bungarotoxin. We also thank Dr. Ian Simpson for his help
with organ histology.
J.Y.F.W. and J.J.C. contributed equally to this work.
Correspondence should be addressed to Dr. J. Drago, Department of
Medicine, 5th Floor E Block, Monash Medical Centre, Clayton Road,
Clayton, Victoria, 3168, Australia. E-mail:
john.drago{at}med.monash.edu.au.
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TOP
ABSTRACT
INTRODUCTION
