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The Journal of Neuroscience, February 15, 2002, 22(4):1208-1217
Distribution and Pharmacology of  6-Containing Nicotinic
Acetylcholine Receptors Analyzed with Mutant Mice
Nicolas
Champtiaux1,
Zhi-Yan
Han1,
Alain
Bessis2,
Francesco Mattia
Rossi1,
Michele
Zoli1, 3,
Lisa
Marubio4,
J. Michael
McIntosh5, and
Jean-Pierre
Changeux1
1 Laboratoire de Neurobiologie Moléculaire,
Centre National de la Recherche Scientifique, Unité de Recherche
Associée 2182, Récepteurs et Cognition, Institut Pasteur
75724 Paris, Cedex 15, France, 2 Laboratoire de Biologie
Cellulaire de la Synapse, Ecole Normale Supérieure, 75005 Paris,
France, 3 Department of Biomedical Sciences, Section of
Physiology, University of Modena and Reggio Emilia, 41100 Modena,
Italy, 4 Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, Texas 77030, and 5 Department
of Biology, University of Utah, Salt Lake City, Utah 84112
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ABSTRACT |
The  6 subunit of the nicotinic acetylcholine receptor (nAChR) is
expressed at very high levels in dopaminergic (DA) neurons. However,
because of the lack of pharmacological tools selective for
6-containing nAChRs, the role of this subunit in the etiology of
nicotine addiction remains unknown. To provide new tools to investigate
this issue, we generated an 6 nAChR knock-out mouse. Homozygous null
mutants (6 /) did not exhibit any gross neurological or
behavioral deficits. A careful anatomic and molecular examination of
6/ mouse brains demonstrated the absence of developmental alterations in these animals, especially in the visual and dopaminergic pathways, where the 6 subunit is normally expressed at the highest levels. On the other hand, receptor autoradiography revealed a decrease
in [3H]nicotine,
[3H]epibatidine, and
[3H]cytisine high-affinity binding in the terminal
fields of retinal ganglion cells of 6/ animals, whereas
high-affinity [125I]-conotoxinMII (CtxMII)
binding completely disappeared in the brain. Moreover, inhibition of
[3H]epibatidine binding on striatal membranes,
using unlabeled CtxMII or cytisine, revealed the absence of
CtxMII-sensitive and cytisine-resistant [3H]epibatidine binding sites in 6/ mice,
although the total amount of binding was unchanged. Because CtxMII,
a toxin formerly thought to be specific for 3 2-containing nAChRs,
is known to partially inhibit nicotine-induced dopamine release, these
results support the conclusion that 6 rather than 3 is the
partner of 2 in the nicotinic modulation of DA neurons. They further
show that 6/ mice might be useful tools to understand the
mechanisms of nicotine addiction, although some developmental
compensation might occur in these mice.
Key words:
nicotinic acetylcholine receptor; knock-out; 6
subunit; -conotoxinMII; dopaminergic system; visual system
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INTRODUCTION |
As many drugs of addiction, nicotine
is thought to exert its addictive properties through stimulation of
dopamine release in the nucleus accumbens (Nac) (Pontieri et al., 1996 ;
Di Chiara, 2000). This effect of nicotine has been attributed to a
direct activation of nAChRs located on dopaminergic (DA) neurons of the ventral tegmental area (VTA). Indeed, nicotine is known to increase the
firing rate of these neurons, both in vitro (Pidoplichko
et al., 1997) and in vivo (Grenhoff et al., 1986). Moreover,
nicotinic antagonists infused into the VTA prevent nicotine elicited
dopamine release in the Nac and disrupt systemic nicotine
self-administration in rats (Corrigall et al., 1994; Nisell et al.,
1994). Therefore, nAChRs expressed by DA neurons represent valuable
targets for the design of new pharmacological agents for the treatment
of nicotine abuse, and much research has been devoted to the
identification of their subunit composition.
Neuronal nAChRs are composed of five subunits belonging to a family of
at least 12 genes exhibiting a discreet expression pattern (Sargent,
1993). DA neurons express 3, 4, 5, 6, 7, 2, and 3
subunits (Le Novère et al., 1996; Charpantier et al., 1998; Klink
et al., 2001). Furthermore, it was recently demonstrated that mice
lacking the 2 subunit are no longer sensitive to nicotine-mediated dopamine release in the Nac and fail to self-administer nicotine (Picciotto et al., 1998). The same study demonstrated that
2-containing nAChRs, present on the soma of DA neurons, mediate a
nicotine-induced increase of their firing rate. Among the other
subunits expressed in VTA, only 3, 4, and 6 are likely to
associate with the 2 subunit to form a ligand binding site [3
and 5 are structural subunits, whereas 7 forms homomeric
pentamers (Le Novère and Changeux, 1995)]. Although
pharmacological evidence suggests 4 and 3 subunits as likely
partners of the 2 subunit (Kulak et al., 1997; Kaiser et al., 1998;
Sharples et al., 2000), the existence of functional 6-containing
nAChRs on DA neurons should not be overlooked. Indeed, the 6 subunit
is specifically and highly expressed in catecholaminergic neurons and
retina (Le Novère et al., 1996; Vailati et al., 1999). In
addition, 6 was recently implicated in the stimulating effect of
nicotine on habituated locomotion (Le Novère et al., 1999), an
effect known to be blocked by lesions of midbrain DA neurons (Boye et
al., 2001). However, the absence of pharmacological information about
6-containing nAChRs has been a deterrent to determining the exact
contribution of these receptors to the nicotinic modulation of DA neurons.
To investigate this issue, we generated a strain of mice lacking the
6 nAChR subunit gene. We describe the generation of these animals
and their anatomical characterization. We first focus on the detection
of possible developmental abnormalities in dopaminergic and visual
pathways, because such alterations might secondarily affect behavior
and have already been detected in 2 nAChR knock-out mice (Rossi et
al., 2001). Then, we perform ligand binding experiments to investigate
the pharmacology and distribution of native 6-containing nAChRs and
evaluate the representation of these receptors in the dopaminergic system.
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MATERIALS AND METHODS |
All animals were used in accordance with the Centre National de
la Recherche Scientifique guidelines for care and use of laboratory animals.
Construction of the targeting vector
We used a PCR-based strategy (Israel, 1993) to screen a mouse
genomic DNA library prepared from the 129Sv/d3 embryonic stem (ES)
cells strain (Stratagene, La Jolla, CA), using primers derived from the
6 cDNA rat sequence. An 18 kb clone was isolated, and homologous
flanking regions were cloned in KS bluescript (Stratagene), on either
side of a Neomycin resistance gene. Ultimately, a diphtheria toxin
expression cassette was introduced outside of the recombination arms.
The construct was designed to generate a nonfunctioning allele by
deleting a 4 kb EcoRI-HindIII fragment including
exons 1 and 2 of the 6 gene. The 5' flanking arm was a 5 kb
EcoRI-EcoRI fragment, and the 3' flanking arm
was a 2.4 kb HindIII-SalI fragment (Fig.
1).
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Figure 1.
Gene targeting of the neuronal
nAChR 6 subunit. a, Construction of the targeting
vector. The homologous recombination event generated a 4 kb genomic
deletion that removes exons 1 and 2. The targeting vector was designed
to obtain a replacement mutation and contains a neomycin resistance
gene (Neor) as a positive selection
marker and the diphtheria toxin gene (DTA) as a negative
selection marker. The expected wild-type and mutant restriction
fragments after PstI digest and hybridization with the
Southern blot probe are shown as dotted lines.
Restriction enzymes are as follows: E,
EcoRI; H, HindIII;
P, PstI; S,
SalI. b, Southern blot analysis of
wild-type (Wt), heterozygous (6+/), and homozygous
null mutant mice (6/). Genomic tail DNA was digested with
PstI and hybridized with an
SalI-PstI probe, external to the
targeting vector. The 4 kb band corresponds to the mutated allele.
c, Detection of 6 mRNA in the brain of Wt and
6/ animals, by in situ hybridization with an
oligonucleotide located in the sixth exon of the 6 gene. In Wt
animals, 6 mRNA is detected in SN-VTA neurons, LC and RGC. No
signal above background levels is detected in 6/ animals.
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Generating the chimeras
The targeting vector was linearized by ScaI and
electroporated in ES cells derived from the 129Sv/Pas mouse strain
(Kress et al., 1998). After 8 d of geneticin selection
(Invitrogen, Gaithersburg, MD), 200 resistant clones were screened by
genomic Southern blot for homologous recombination. Three recombinant
ES cells clones were obtained, one of which was used to generate mouse
chimeras by aggregation (Khillan and Bao, 1997) with morulas obtained
from CD1 mice (Charles River, Wilmington, MA). One single germline transmitter was obtained and bred with CD1 mice.
Maintaining the colony
One F1 heterozygous male and one F1 heterozygous female were
then mated to establish a set of F2 breeder animals (5 males and 10 females) of each genotype (mutant or wild-type). All the animals used
in this study were derived either from the F2 or the F3 generation.
Genetic diversity was maintained by randomly interchanging breeders.
Genomic Southern blot
Genomic Southern blot was performed according to standard
methods (Sambrook et al., 1989). A PstI digest of mouse
tail-derived genomic DNA probed with a SalI-PstI
fragment (Fig. 1) was used to check the recombination at the 3' end
(normal allele fragment is 8.1 kb and recombinant is 4.0 kb). Another
probe (EcoRI-EcoRV fragment of the 6
promoter), internal to the 5' recombination arm, was used to check for
correct recombination on the 5' end, using an XbaI digest of
genomic DNA. (Normal allele fragment is 9.9 kb and recombinant is 12.2 kb). This also excluded the eventuality of an additional random
integration (data not shown).
In situ hybridization
Animals were killed by cervical dislocation, and the brain was
quickly dissected out and frozen in dry ice powder. The tissues were
stored at 80°C until cut on a cryostat (Leica, Deerfield, IL)
(14-µm-thick sections). In situ hybridization was
performed according to Zoli et al. (1995), using synthesized
oligonucleotides (Eurogentec, Brussels, Belgium) according to rat or
mouse sequence, when available (Table 1).
The slides were then exposed for 15 d to Hyperfilm-3H (Amersham
Biosciences, Arlington Heights, IL) together with microscale
radioactive standards (Amersham Biosciences) to correct for
nonlinearity of the film response. The semiquantitative analysis was
performed on autoradiography films. On each section, an averaged
optical density of the background level (obtained by cold competition
with unlabeled oligonucleotide) was subtracted from that of the sampled
area to obtain specific optical density value.
For nAChR subunits (3, 4, 5, 6, 7, 2, 4), the
experiment was performed using 3-6 animals of each genotype. Sections were taken at 17 coronal levels (bregma levels: 3.0, 1.5, 0.9, 0.1, 0.6, 1.0, 1.8, 2.2, 2.7, 2.9, 3.1, 3.5, 3.8, 4.0, 4.8, 5.4, and 7.5 mm according to Franklin and Paxinos
(1997).
For dopamine receptors (D1, D2), tyrosine hydroxylase (TH), and
neuropeptides [cholecystokinin (CCK), preprotachykinin (PPT), and
preproenkephalin (PPE)], the experiment was performed using seven
animals of each genotype. Coronal sections were cut from bregma level
1.0-0.7 mm (striatal level) and from bregma level 3.1 to 3.5 mm
(mesencephalic level). For each level, three adjacent sections per
animal were used for quantitative analysis.
Spontaneous locomotor activity
The open-field apparatus consists of a transparent
31-cm-high × 49-cm-long × 25.4-cm-wide Plexiglas cage.
Movements were detected by a set of 9 × 19 infrared photobeam
detectors, 1.5 cm above the floor of the apparatus. Tests were
performed in a quiet room with ambient fluorescent lighting. For this
experiment we used 15 male 6/ mice and 15 wild-type (Wt)
littermates controls derived from F2 heterozygous mating. Mice naive to
the apparatus were placed in the cage at time point t = 0, and their movements were recorded for a 2 hr period, with time
points taken at intervals of 5 min.
Intraocular injections
Mice were anesthetized with a solution of 1.5% ketamine and
0.05% xylazine in PBS (150 µl/30 gm body weight). Intraocular injections were performed using a glass needle inserted just behind the
corneoscleral margin of the eye. One microliter of a 10 µg/µl solution of cholera toxin subunit B, Alexa Fluor 488 conjugate (Molecular Probes, Eugene, OR) was injected into the vitreous chamber
of one eye and Alexa Fluor 594 into the other. After 24 hr, mice were
anesthetized and perfused with 0.9% saline, followed by 4%
paraformaldehyde in PBS. Cryoprotected brains were cut on a freezing
microtome into 40 µm coronal sections. Sections were mounted on
gelatin-coated slides, air-dried, coverslipped, and then examined under
an epifluorescent microscope.
Receptor autoradiography
Nicotinic receptor ligands. For
[3H]cytisine (5 nM), [3H]nicotine
(5 nM),
[3H]epibatidine (400 pM),
[125I]-bungarotoxin (1.5 nM), and
[125I]-conotoxinMII (CtxMII) (500 pM), binding procedures were performed as
described in Zoli et al. (1998) and Whiteaker et al. (2000). For each
ligand, 17 coronal levels (see in situ hybridization procedures), and one or two sections per level were examined, from
three to five animals of each genotype. Nonspecific binding was
determined by using a 1000× excess of unlabeled ligand.
Cytisine displacement of [3H]epibatidine
binding was performed by adding 50 nM of cold cytisine to
the incubation mix, according to Marks et al. (1998).
Except for [125I]-CtxMII, which was a
generous gift from Dr. J. Michael McIntosh (Departments of Biology and
Psychiatry, University of Utah, Salt Lake City, UT), all radiolabeled
ligands were purchased from Amersham Biosciences. Unlabeled cytisine,
nicotine, epibatidine, and -bungarotoxin were purchased from Sigma
(St. Louis, MO); CtxMII was obtained from Eurogentec.
Dopamine receptor ligands. Levels of D1 and D2 dopamine
receptors were determined by equilibrium binding of
[3H]SCH23390 (NEN, Boston, MA) and
[3H]raclopride (NEN), respectively.
Sections were preincubated for 30 min at room temperature in 50 mM Tris 7.4, 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 2 mM
MgCl2 (SCH23390), or 50 mM
Tris 7.4, 120 mM NaCl, and 5 mM KCl (raclopride). Slides were then incubated for 1 hr at room temperature in preincubation buffer plus 1.5 nM (SCH23390) or 3 nM
(raclopride) of tritiated ligand. Nonspecific binding was determined in
the presence of an excess (100×) of cold ligand (Research
Biochemicals, Natick, MA). After incubation, the sections were washed
three times for 5 min each in ice-cold 50 mM Tris
and briefly rinsed in ice-cold water. Slides were exposed for 20 d
(D1) or 40 d (D2) to Kodak (Eastman Kodak, Rochester, NY) BioMax
MS films (Amersham Biosciences). Eight animals of each genotype were
used for quantification. Measures were made on three adjacent sections
at mesencephalic and striatal levels (bregma 3.4 and 0.9 mm, respectively).
Histological analysis
Immunocytochemistry. Mice were anesthetized with 4%
chloral hydrate (12 µl/gm) and transcardially perfused with 20 ml of
0.9% NaCl (37°C) followed by 100 ml of a 4% paraformaldehyde-PBS
solution. Brains were dissected out and post-fixed overnight in the
same fixative, rinsed in 12% sucrose-PBS at 4°C over 2 d, and
frozen on dry ice powder. The brains were cut from bregma level
1.0-0.7 mm and from bregma level 3.1 to 3.5 mm on a cryostat
(20-µm-thick sections). Sections were then processed according to
standard immunoperoxidase protocols, using the avidin-biotin complex
technique (Pedrazzi et al., 1998). Antibodies against rat tyrosine
hydroxylase (TH) (Calbiochem, La Jolla, CA), and dopamine transporter
(DAT) (MAB369; Chemicon, Temecula, CA) were used at a 1:2000 dilution for TH and 1:1000 for DAT. Briefly, slides were incubated overnight at
4°C, in the presence of the primary antibody diluted in PBS supplemented with 0.2% Triton X-100 and 1% normal goat serum (NGS). After washing with PBS containing 0.1% NGS, sections were incubated for 1 hr at room temperature, with a biotinylated anti-rabbit (TH) or
anti-rat (DAT) secondary antibody (Vector Laboratories, Burlingame, CA)
diluted 1:200 in PBS/1% NGS. Sections were washed in PBS and incubated
with an avidin biotinylated peroxidase complex (ABC) according to
manufacturer's instructions (Vector Laboratories). After peroxidase
detection using diaminobenzidine, sections were washed, dehydrated, and coverslipped.
Nissl staining. Rehydrated, fixed (4% PFA) brain sections
were incubated with toluidine blue (0.1%), washed, dehydrated, and coverslipped.
Analysis. The semiquantitative evaluation of the intensity
of TH and DAT immunostainings as well as automatic cell profile count
of Nissl-stained preparations was performed by means of an automatic
image analyzer (KS300; Zeiss Kontron, Munich, Germany) according to
previously published methods (Zoli et al., 1992). Microdensitometric
analysis of immunostaining was performed with a CCD camera connected to
a microscope using a 2.5× objective. In mesencephalon, a thresholding
procedure was used to determine the integrated optical density value.
TH-immunoreactive cell counting in substantia nigra pars compacta (SNc)
was performed manually on coded slides under a 20× objective using a
calibrated eyepiece grid, at 3.5 mm bregma level. For each staining,
the analysis was performed on two sections per animal. The average
value per animal was used for statistical purposes.
For Nissl-stained preparations, automatic cell profile count was
performed according to Zoli et al. (1993) with a CCD camera connected
to a microscope using a 20× objective (rectangular sampled field,
310 × 235 µm). Briefly, stained profiles were separated from
the background by means of a dynamic thresholding procedure. Then, a
spatial threshold was placed to separate glial (diameter < 7.5 µm) from neuronal (diameter  7.5 µm) profiles, and the number of neuronal profiles per sampled field was measured.
Membrane binding. Membranes from striatum (dorsal and
ventral) and superior colliculus (SC) were prepared according to Marks et al. (1998). Incubations were performed in 6 ml glass tubes, using 50 (Superior colliculus) or 200 µg (striatum) of membrane protein per
tube. Membranes were diluted in 500 µl of binding buffer (NaCl 144 mM; KCl 1.5 mM;
CaCl2 2 mM;
MgSO4 1 mM; HEPES 20 mM, pH 7.5), containing
[3H]epibatidine (500 pM) and the appropriate concentration of
unlabeled ligand (300 pM to 10 µM for CtxMII, and 30 pM to 3 µM for cytisine). For CtxMII displacement, binding buffer was supplemented with BSA
(0.1% w/v), 5 mM EDTA, 2 mM EGTA, and 10 µg/ml each of aprotinin, pepstatin A, and leupeptin (Sigma), to protect the ligand from endogenous proteases. After 2 hr at room temperature, incubations were
terminated by filtration on polyethilenimine-soaked (0.3%, Sigma)
glass fiber filters (type GF/C; Whatman) and washing with 10 ml of
ice-cold binding buffer. Nonspecific binding was determined by adding
an excess (1000×) of unlabeled epibatidine to the incubation mix. All
measures were made in triplicate. The epibatidine concentration and the
volume used for the incubation ensured minimal ligand depletion
(<5%). Results were first fitted to the Hill equation [B = Bo/(1 + (I/IC50)n) ] to determine the likeliness of a monophasic inhibition (B is
the number of binding sites at inhibitor concentration, I, Bo is the binding in the absence of
inhibitor, and n is the Hill coefficient). Depending on
n value, results were fitted using one of the following
equations for one or two binding sites: B = Bo/(1+
(I/IC50)) or B = I B1/(1 + (I/IC50-1)) + B2/(1+ (I/IC50-2)), respectively.
IC50 and Bo
values are expressed as the means ± SEM of six independent
determinations. The corresponding Ki
value was determined assuming competitive inhibition:
Ki = IC50/(1 + L/Kd), where L
is the concentration of [3H]epibatidine
(500 pM), and Kd is the
equilibrium dissociation constant of
[3H]epibatidine in striatum (19.3 µM, according to Marks et al. (1998).
Statistical analysis. Statistical analysis was done with
StatView software (SAS Institute). Data are given as means ± SEM; means were compared using a Student's t test.
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RESULTS |
Generation of 6 knock-out mice
Knock-out mice for the 6 subunit of the nAChRs were generated
by replacing the first two exons of the gene with a Neomycin resistance
(Neor) cassette (Fig. 1a).
These exons encode the ATG initiator codon, the signal peptide, and
part of the N-terminal extracellular domain of the mature subunit.
Southern blot analysis using a flanking genomic probe confirmed that
the expected homologous-recombination had occurred (Fig. 1b). In
situ hybridization using an oligonucleotide probe located in the
sixth exon (downstream to the deletion) detected 6 mRNA in retinal
ganglion cells (RGCs), locus coeruleus (LC), substantia nigra (SN) and
VTA neurons of Wt animals, in agreement with previous observations in
rat or chicken (Le Novère et al., 1996; Vailati et al., 1999). In
heterozygous mice, the intensity of the signal decreased and completely
disappeared in 6/ mice, demonstrating that the deletion of exon
1 and 2, and the insertion of the Neor
cassette, is sufficient to prevent transcription (Fig. 1c).
We then compared the levels of expression of 3, 4, 5, 7,
2, and 4 nAChR subunit mRNA in Wt and 6/ animals.
Quantitative analysis of film autoradiography revealed no alteration in
the relative abundance of the mRNA of these subunits to compensate for
the loss of the 6 subunit (Fig. 2).
[Note that in agreement with Ross et al. (2000), 3 mRNA was not
detected in SN-VTA neurons.]
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Figure 2.
Semiquantitative analysis of 3, 4, 5,
7, 2, and 4 nAChR subunit mRNAs in Wt and Mt mice using
in situ hybridization. For each subunit, optical density
value of the signal was measured and corrected for background and film
linearity. This value was then normalized to 100 in the region where
the signal was maximal. Results are expressed as means ± SEM of
at least three animals. Statistical analysis was performed using an
unpaired Student's t test (p < 0.05). Regions quantified were medial habenula (MHb),
interpeduncular nucleus (IPN), thalamus
(Th), cortex (Cx), VTA, and hippocampus
(Hp). Note the absence of significant differences in the
relative abundance of the mRNA of these subunits, in any brain
region.
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6 knock-out mice are viable and do not exhibit
major defects
Homozygous null mutant mice are viable, present in the expected
Mendelian proportion in litters, grow to normal size, and do not
exhibit major physical or neurological defects. Home cage behavior of
mutant mice appeared normal, and spontaneous locomotor activity in an
open-field observed over a 2 hr period did not reveal significant
differences between Wt and 6/ animals at any time point, either
during the nonhabituated (first hour) or habituated period (second
hour) of the experiment (data not shown). Furthermore, histological
examination of toluidine blue-stained sections of 6/ mice did
not reveal alterations in the size, shape, or location of the major
brain nuclei (data not shown).
Absence of developmental adaptations in the dopaminergic and visual
pathways of 6/ animals
One major drawback of knock-out animal studies is the possible
existence of developmental abnormalities in mutant mice that might
interfere with the interpretation of the results obtained from these
studies. For instance, in mice lacking the 2 nAChR subunit,
impairment of visual acuity derives from abnormal segregation of
retinogeniculate projections during development (Rossi et al., 2001).
To examine whether such alterations exist in 6/ animals, we
performed a detailed examination of the anatomical regions where 6
is known to be expressed.
In the visual system, histological staining of the retina showed that
the cell layering and the RGC number is normal in 6/ mice
compared with their Wt controls (data not shown). Moreover, we studied
the distribution pattern of RGC projections in their thalamic target,
the dorsolateral geniculate nucleus (dLGN), in young (postnatal day 15)
and adult animals. In Wt mice, retinal projections from the two eyes
terminate into separated areas of the dLGN: an outer shell, that
receives afferents exclusively from the contralateral eye and an inner
core that is innervated by the ipsilateral eye. In 6/ mice the
pattern of retinogeniculate labeling was not different from the one
observed in Wt at the same age (data not shown).
The anatomy of the mesostriatal dopaminergic system was studied at both
mesencephalic and striatal levels.
At the striatal level, the semiquantitative analysis of TH and DAT
immunoreactivity failed to detect any alteration in DA nerve terminals
of 6/ mice in the anatomical regions examined [caudate-putamen
(Cpu), Nac, and olfactory tubercle (Tu)]. Intrinsic striatal neurons
also appeared normal in terms of number and morphology, as assessed by
Nissl staining. Using in situ hybridization and receptor
autoradiography, we examined the expression profile of several markers
in these neurons: dopamine receptors, D1 and D2, and neuropeptide
precursors, PPT and PPE. In Wt mice, D1 and D2 mRNA levels were found
to be homogeneously expressed in Cpu, and Tu, and to a lesser extent in
the Nac. The study of [3H]raclopride
(D2) and [3H]SCH23390 (D1) binding
revealed a similar pattern of expression at the protein level. PPT
mRNA, coding for the precursor peptide of substance P, was strongly
expressed in Tu and Nac shell and to a lesser extent in Cpu and Nac
core. Finally, PPE mRNA, coding for the precursor peptide of
methionine-enkephalin, was homogeneously expressed in Cpu, Nac, and Tu.
In 6/ mice, the pattern and level of expression of these markers
were not altered (Table 2).
At the mesencephalic level, the morphology and number of DA neurons did
not differ in Wt and 6/ mice when visualized by TH or DAT
immunoreactivity. We then examined the expression of TH, dopamine
receptors D1, D2, and neuropeptide CCK at the mRNA level. In Wt mice,
TH and D2 mRNA was detected in VTA and substantia nigra pars compacta
(SNc) only, whereas CCK was also expressed in cortex and hippocampus.
D1 mRNA was not detected at the mesencephalic level. Ligand binding
studies further revealed low levels of
[3H]raclopride (D2) binding sites in VTA
and SNc, whereas high levels of
[3H]SCH23390 (D1) binding sites could be
seen in SN reticulata. Statistical analysis did not reveal any
significant difference in the expression of these markers, between Wt
and 6/ animals (Table 2).
Distribution of high-affinity binding sites for nicotinic ligands
in the brain of 6/ mice
We investigated the binding profile of several nicotinic ligands
on coronal sections of 6/ mouse brains. Whereas the distribution of -bungarotoxin binding sites was unchanged in 6/ compared with Wt animals, a decrease in the abundance of high-affinity binding
sites for [3H]nicotine,
[3H]epibatidine, and
[3H]cytisine was found in the terminal
regions of RGC (SC and dLGN) (Fig.
3a-c). Surprisingly, no other
differences were detected, especially in the retina, SN-VTA, and
striatum, brain regions containing cell bodies or terminals of neurons
that express 6. Small decreases, however, might have been masked
because of the relative scarcity of 6-containing nAChRs with respect
to other major nAChR populations (e.g., 42 containing nAChRs)
that also bind these ligands. As shown in a previous paper (Marks et
al., 1998), a subpopulation of
[3H]epibatidine binding sites, enriched
in the mesostriatal and visual pathways, has a relatively low affinity
for cytisine ("cytisine-resistant" sites). We therefore, compared
the distribution of [3H]epibatidine
binding sites after displacement by low cytisine concentration (50 nM), in Wt and 6/ mice. In these
conditions, autoradiography quantification revealed a significant
decrease of the cytisine-resistant fraction of
[3H]epibatidine binding sites in most
regions of the visual and dopaminergic systems of mutant animals
compared with Wt mice (Figs. 3d,
4).
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Figure 3.
Quantitative autoradiography for nicotinic
agonists in the mouse brain. Quantitative analysis of
[3H]cytisine (a),
[3H]nicotine (b),
[3H]epibatidine (c), and
[3H]epibatidine in the presence of 50 nM unlabeled cytisine (d), in Wt and
6/ mice. The results are expressed as the mean specific
optical density ± SEM, normalized to 100 in the dLGN of Wt
animals. AU, Arbitrary unit. Statistical analysis was
performed using an unpaired Student's t test
(*p < 0.05; **p < 0.01)
versus Wt controls. Regions quantified were SC, dLGN, retina
(Ret), VTA, Cpu, IPN, MHb, and Cx.
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Figure 4.
Autoradiograms of cytisine-resistant
[3H]epibatidine binding in the mouse brain.
Sections were incubated with 400 pM
[3H]epibatidine in the presence of 50 nM unlabeled cytisine. See Figure 3 for
quantification.
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Finally, we examined the distribution of
[125I]CtxMII binding sites. In Wt
mice, our results were in agreement with those of Whiteaker et al.
(2000). Briefly, the highest density of
[125I]CtxMII binding sites was found
in the SC (zonal and superficial gray layer), optic tract (opt),
geniculate nucleus, and olivary pretectal nucleus (OPN). Lower levels
were found in the basal ganglia (including Cpu, Tu, and Nac), SN-VTA,
interpeduncular nucleus (IPN), medial habenula (MHb), lateral habenula,
fasciculus retroflexus (fr), and retina. It should be noted that these
regions correspond to 6-expressing neurons (Le Novère et al.,
1996) and to their terminal fields. In 6/ mice, no signal was
detected above background level, indicating a complete disappearance of high-affinity CtxMII binding sites (Fig.
5).
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Figure 5.
Autoradiograms of
[125I]CtxMII binding in the mouse brain.
Sections were incubated with 0.5 nM
[125I]CtxMII, as described in Materials and
Methods. Residual signal seen on the eye section of
6/ animals corresponds to nonspecific binding to pigmented
epithelium. MT, Medial terminal nucleus of the accessory
tract; MVN, medial vestibular nucleus; VLGN,
ventrolateral geniculate nucleus.
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Disappearance of the CtxMII-sensitive and the cytisine-resistant
fractions of [3H]epibatidine binding sites in the
striatum of 6/ mice
Receptor autoradiography experiments showed a high selectivity of
CtxMII for 6-containing nAChRs (see above). Still, previous experiments showed that this ligand is a potent inhibitor of
32-containing nAChRs (Cartier et al., 1996). We, therefore,
examined whether some low-affinity CtxMII binding sites
remained in the striatum of mutant mice. We displaced
[3H]epibatidine binding by unlabeled
CtxMII, in striatal membrane preparations. Consistent with our
observation in autoradiography experiments, total
[3H]epibatidine binding was not
significantly different between Wt and 6/ animals (57.9 ± 2.0 and 53.8 ± 1.7 fmol/mg, respectively). In Wt mice, ~21% of
[3H]epibatidine binding sites could be
displaced by CtxMII, in a monophasic manner (Hill coefficient,
0.96 ± 0.12), and results were fitted to a one site model to
determine the Ki value (Table 3). On the contrary, in 6/
animals, CtxMII had no effect on epibatidine binding at any
concentration (up to 10 µM). Strikingly, the
total number of CtxMII-resistant epibatidine binding sites was
significantly upregulated in 6/ mice (p < 0.01) (Fig. 6). We also performed the
same experiment on SC membrane preparations and found residual
CtxMII sensitivity in 6/ mice, representing 16% of the
CtxMII-sensitive [3H]epibatidine
binding population in Wt animals (Table 3). However, the low amount of
these sites and problems with material availability prevented us from
providing an accurate inhibition curve that could have demonstrated the
low-affinity nature of these sites.
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Figure 6.
Displacement of
[3H]epibatidine binding by CtxMII in mouse
striatal membranes. a, Comparison of total
(gray bars) and CtxMII-resistant (10 µM, white bars)
[3H]epibatidine binding sites in Wt and 6/
animals. Each point represents the mean ± SEM of six separate
determinations, expressed in femtomoles per milligram of protein.
Statistical analysis was performed using an unpaired Student's
t test. A statistically significant fraction (21%;
*p < 0.01) of [3H]epibatidine
binding sites is sensitive to CtxMII displacement in Wt but not in
6/ animals. CtxMII-resistant
[3H]epibatidine binding sites are significantly
(°p < 0.01) upregulated in 6/ animals
compared with their Wt controls, whereas total
[3H]epibatidine binding is unaffected by the
genotype. b, Inhibition curve of
[3H]epibatidine (500 pM) binding by
CtxMII (30 pM to 3 µM) in striatal
membrane preparations of 6/ (filled
symbols) and Wt (open symbols) animals.
Nonspecific binding, determined in the presence of 500 nM
unlabeled epibatidine, was subtracted from each measurement. Results
were then expressed as a percentage of specific binding in the absence
of CtxMII. Each value is the mean ± SEM of six separate
determinations. See also Table 3.
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Because CtxMII-sensitive and cytisine-resistant
[3H]epibatidine binding sites have been
suggested to represent the same receptor population (Whiteaker et al.,
2000), we investigated the displacement of epibatidine binding by
unlabeled cytisine. The resulting inhibition curves are shown in Figure
7. In Wt animals, the Hill coefficient was significantly <1 (n = 0.76 ± 0.03),
suggesting that the inhibition of epibatidine binding by cytisine was
not monophasic. Results were thus fitted using a two sites model that
revealed the existence of cytisine-sensitive and cytisine-resistant
epibatidine binding sites (Table 4), in
agreement with the results of Marks et al. (1998). On the contrary, in
6/ animals, the inhibition curve appeared monophasic (Hill
coefficient, 0.94 ± 0.05), and we could fit the data to a one
site model to determine Ki value
(Table 4). Using a two sites model to fit the data gave similar results because the calculated cytisine-resistant population represented <0.3% of total binding in 6/ animals. Moreover, the measured Ki value for cytisine inhibition of
epibatidine binding in 6/ animals was comparable with the
Ki value of the cytisine-sensitive fraction in Wt mice (0.32 ± 0.03 nM
compared with 0.23 ± 0.03 nM,
respectively).
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Figure 7.
Displacement of
[3H]epibatidine binding by cytisine in mouse
striatal membranes. a, Inhibition curve of
[3H]epibatidine binding by cytisine: membrane
samples were incubated with 500 pM
[3H]epibatidine in the presence of (30 pM to 3 µM) unlabeled cytisine.
Nonspecific binding, determined in the presence of 500 nM
unlabeled epibatidine, was subtracted from each measurement. Results
were then expressed as a percentage of specific binding in the absence
of cytisine. Each point represents the mean ± SEM of five
separate determinations. Data obtained from 6/
(filled symbols) or Wt (open
symbols) striatal membranes preparations were fitted to a one-
(dotted line) or two-site inhibition model (see Material
and Methods), respectively, depending on the value of the Hill number
(see below). Statistical analysis was performed using an unpaired
Student's t test (*p < 0.05).
b, Hill plot representation.
Log(B/(Bo B)) is plotted as a function of log([cytisine]), where
B is the amount of specific
[3H]epibatidine binding and
Bo is the specific binding in the absence of
cytisine. In these conditions, the slope of the curve represents the
Hill number, nH. In Wt animals,
nH is significantly <1
(p < 0.05), suggesting the existence of an
heterogeneous population of binding sites. Conversely, in 6/
animals, nH = 0.94 ± 0.05, which
suggests a homogeneous population of binding sites. See also Table
4.
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DISCUSSION |
We describe the generation and phenotypic characterization of an
6 nAChR subunit knock-out mouse, focusing on the anatomy of neuronal
structures where 6 is normally expressed and on the pharmacological
properties of native 6-containing nAChRs. Deletion of the first two
exons of the 6 gene was sufficient to impair its transcription, as
evidenced by the absence of residual 6 mRNA in homozygous null
mutant mice. These animals exhibit normal general appearance, survival,
growth, anatomy, and locomotor behavior.
The anatomy of the dopaminergic and visual pathways is preserved in
6/ animals
Because 6 mRNA is abundantly expressed in catecholaminergic
neurons and RGCs, we examined whether developmental alterations occurred in the dopaminergic and visual systems of 6/ animals as
a consequence of the loss of the 6 subunit.
In the dopaminergic system, no anatomical alterations could be detected
in either midbrain DA neurons at the somatic and terminal levels, or in
intrinsic striatal neurons. Moreover, the expression of various
molecular markers, including the dopamine biosynthetic enzyme and
transporter (TH and DAT, respectively), dopamine receptors (D1, D2) and
neuropeptides (CCK, PPT, PPE) were normal in 6/ mice, although
the expression of some of these markers has been reported to be
modulated by nicotine or endogenous acetylcholine (Wiener et al., 1989;
Smith et al., 1991; Houdi et al., 1998; Drew et al., 2000). In the
visual system, retinal cell layering as well as the organization of
retinothalamic projections were preserved in 6/ animals. Relying
on our results, we conclude that 6 is not necessary for normal
development of visual and mesostriatal dopaminergic pathways.
Therefore, these mutant animals are likely to be suitable models for
studying the function of 6 in adults.
Moreover, our results contribute to the identification of the subunit
composition of nAChRs responsible for the segregation of retinothalamic
projections. In the visual system of mammals, the projections of the
RGC from each eye are initially intermixed in their thalamic target,
the dLGN, and subsequently segregate into eye-specific layers. This
phenomenon is dependent on spontaneous retinal activity, termed retinal
waves, driven by the activation of nicotinic receptors located on RGC
(Feller et al., 1996; Penn et al., 1998). It was recently demonstrated
that mice lacking the 2 but not the 4 nAChR subunit, display
abnormal segregation of retinogeniculate projections (Rossi et al.,
2001). In this context, our finding that adult 6/ mice exhibit
normal segregation, suggests that nAChRs involved in the segregation
process do not contain 6, although the substitution of 6 by
another subunit cannot be ruled out (see below). These results
further support the hypothesis of Bansal et al. (2000), who proposed
3 as the likely partner of 2 in the genesis of normal retinal waves.
The 6 nAChR subunit is a critical component of native CtxMII
binding sites
Null mutant mice exhibit a complete disappearance of high-affinity
[125I]CtxMII binding sites.
Therefore, all these sites appear to correspond to 6-containing
nAChRs. Three distinct anatomical systems contain detectable levels of
[125I]CtxMII binding. High levels
were shown in the visual system, including retina, RGC fibers (opt),
and their terminal fields (geniculate nucleus, SC, OPN). Lower binding
density was found in two other systems: the mesostriatal dopaminergic
system, which includes SN-VTA neurons and their projection structures
(Tu, Nac, Cpu, lateral habenula), and the habenulo-interpeduncular
system (MHb, fr, and IPN). On the basis of the distribution of 6
mRNA-expressing neurons, we infer that 6-containing nAChRs in the
SN-VTA, MHb, and retina are somatodendritic receptors. In other
regions, these receptors are likely to be transported to a presynaptic
or preterminal localization, as confirmed by the presence of high
levels of [125I]CtxMII binding in
axon systems (optic nerve and tract, fr). Our results expand those of
Goldner et al. (1997), who detected 6 immunoreactivity only in the
SN-VTA. However, it should be noted that CtxMII-insensitive
6-containing nAChRs might also exist, as suggested by the fact that
6 mRNA was detected in areas, such as the LC (Le Novère et
al., 1996; Lena et al., 1999), where no
[125I]CtxMII binding was detected.
Although CtxMII was thought to be a specific inhibitor of
32-containing nAChRs (Cartier et al., 1996), the sensitivity of
6-containing nAChRs to this toxin is not surprising. Indeed, 3
and 6 are highly homologous subunits with 67.3% residue identity in
their mature N-terminal domains, and critical residues for CtxMII
selectivity are conserved in 6 (Harvey et al., 1997). Moreover, it
was recently demonstrated that native 64-containing nAChRs,
purified from chick retina, are blocked by this toxin (Vailati et al.,
1999). Finally, chimeric subunits containing the extracellular domain
of the human 6 subunit fused to the rest of 3 or 4 subunits
can form functional nAChRs, sensitive to CtxMII, when expressed with
2 or 4 in Xenopus oocytes (Kuryatov et al., 2000).
More intriguing, however, is the fact that CtxMII binding sites
completely disappear in 6/ mice, although 3 is still
expressed. It is possible that 32-containing nAChRs are not
detected because of the scarcity of these sites. In the SN-VTA, 3
mRNA is barely detectable using in situ hybridization (Le
Novère et al., 1996; Ross et al., 2000; present study), whereas in regions where 3 mRNA is abundant (e.g., in MHb),
CtxMII-insensitive 34-containing nAChRs are likely to form
(Quick et al., 1999). Another explanation would be that
32-containing nAChRs form low-affinity CtxMII binding sites,
which could not be detected using receptor autoradiography. In the SC,
this hypothesis is supported by our results on CtxMII displacement
of [3H]epibatidine binding on membrane
preparations and the findings of Dougherty et al. (2000), which suggest
that some non-6-containing nAChRs with low affinity for CtxMII
are present in this region. In the striatum however, such sites are
unlikely to exist because in Wt mice, in agreement with Whiteaker et
al., (2000), we report a monophasic of inhibition of
[3H]epibatidine binding by CtxMII,
and because, in 6/ animals, residual
[3H]epibatidine binding is insensitive
to CtxMII displacement (up to 10 µM).
Overall, our results prompt a new interpretation of previous works
describing the pharmacological actions of CtxMII on DA neurons. This
toxin partially inhibits nicotine-induced dopamine release in striatal
synaptosomes (Kulak et al., 1997; Kaiser et al., 1998). Moreover,
patch-clamp recordings revealed CtxMII-sensitive nAChR-mediated
currents in midbrain DA neurons (Klink et al., 2001). Relying on the
supposed selectivity of CtxMII, it was proposed that
32-containing nAChRs mediate these nicotinic effects. We
demonstrate here that this is unlikely to be the case. First, the
selectivity of CtxMII for 32-containing nAChRs is questionable because native 6-containing nAChRs exhibit nanomolar affinity for
this toxin. Moreover the absence of residual CtxMII binding sites in
the striatum of 6/ animals, and the low abundance of 3 mRNA
in the SN/VTA suggest that 32-containing nAChRs are very scarce
in the dopaminergic system. Consequently, the role of the 6 subunit
in the nicotinic modulation of DA neurons and its importance for
nicotine addiction should be reevaluated taking 62-containing
nAChR rather than 32-containing nAChR as the likely candidate for
these effects.
Evidence for the existence compensatory mechanisms in
6/ animals
Pharmacological characterization of native 6-containing nAChRs
was completed using various nicotinic agonists. Cytisine displacement of [3H]epibatidine binding on striatal
membranes strongly suggested the identity of cytisine-resistant and
CtxMII-sensitive epibatidine binding sites in the striatum, becaus |
TOP
ABSTRACT
INTRODUCTION
