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The Journal of Neuroscience, April 1, 2002, 22(7):2522-2529
Involvement of the  3 Subunit in Central Nicotinic Binding
Populations
Paul
Whiteaker1,
Cyrus
G.
Peterson1,
Wei
Xu2,
J. Michael
McIntosh3,
Richard
Paylor2,
Arthur L.
Beaudet2,
Allan C.
Collins1, and
Michael J.
Marks1
1 Institute for Behavioral Genetics, University of
Colorado, Boulder, Colorado 80309, 2 Baylor College of
Medicine, Department of Molecular and Human Genetics, Houston, Texas
77030, and 3 Departments of Biology and Psychiatry,
University of Utah, Salt Lake City, Utah 84132
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ABSTRACT |
The  3 subunit gene was one of the first neuronal
nicotinic acetylcholine receptor (nAChR) subunits to be cloned (Boulter et al., 1986 ), but direct evidence of 3 subunit contributions to
mammalian central nAChR populations has not been presented. The studies
reported here used mice engineered to contain a null mutation in the
3 nAChR subunit gene (Xu et al., 1999) to examine the involvement of
the 3 subunit in central nAChR populations. Heterologously
expressed 3 2 and 34 nAChRs are pharmacologically similar to
native [125I]-conotoxin MII
(-CtxMII)-binding and
3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride
(A85380)-resistant [125I]epibatidine-binding nAChR
subtypes, respectively. The hypothesis that both native sites are
3-subtype nAChRs was tested using quantitative autoradiography in
3-null mutant mice. Somewhat surprisingly, deletion of the 3
nAChR subunit gene did not affect expression of the great majority of
[125I]-CtxMII-binding sites, indicating that
they do not correspond to heterologously expressed 32 nAChRs. The
only exception to this was observed in the habenulointerpeduncular
tract, where 3-dependent [125I]-CtxMII
binding was observed. This finding may suggest the presence of an
additional, minor nicotinic population in this pathway. In contrast,
most A85380-resistant [125I]epibatidine-binding
nAChRs were dependent on 3 gene expression, suggesting that they do
indeed correspond to an 3 nAChR subtype. However, widespread but
lower levels of 3-independent A85380-resistant [125I]epibatidine binding were also seen. Again,
this may indicate the existence of an additional, minor population of
non-3 A85380-resistant sites.
Key words:
nicotinic acetylcholine receptor; 3 subunit; A85380-resistant binding; -conotoxin MII; autoradiography; 3
subunit-null mutant
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INTRODUCTION |
Nicotinic acetylcholine receptors
(nAChRs) are involved in normal CNS functions, including analgesia,
cognition, reward, and motor control (Decker et al., 1995), and have
been implicated in many of the diverse behavioral effects of nicotine
in mammals (Stolerman, 1990). It is generally accepted that nAChRs are
homopentameric or heteropentameric assemblies of homologous subunits
and that different combinations of subunits produce distinct nAChR
subtypes with diverse biophysical and pharmacological properties
(Lindstrom et al., 1996). The potential to form functionally distinct
pentamers, combined with differential expression of nAChR subtypes
across the brain (Wada et al., 1989; Marks et al., 1992; Whiteaker et al., 2000a), underlies the rich variety of effects and roles attributed to nAChRs.
Epibatidine is a nicotinic agonist with high (picomolar) affinity at
many mammalian nAChR subtypes (Badio and Daly, 1994; Houghtling et al.,
1995; Perry and Kellar, 1995; Flores et al., 1996; Davila-Garcia et
al., 1997; Marks et al., 1998; Whiteaker et al., 2000a,b). The majority
of high-affinity epibatidine-binding sites are potently inhibited by
the nicotinic agonist cytisine (cytisine-sensitive sites;
Ki = 0.29 nM)
(Marks et al., 1998). These cytisine-sensitive sites
primarily correspond to the same 42-subtype nAChR that was
identified by other agonist ligands such as
( )-[3H]nicotine and
[3H]cytisine (Whiting and Lindstrom,
1987; Flores et al., 1992; Picciotto et al., 1995; Marubio et al.,
1999). The remaining high-affinity epibatidine-binding sites exhibit
much lower cytisine affinity (cytisine-resistant sites;
Ki > 10 nM)
(Marks et al., 1998; Whiteaker et al., 2000a,b). Some
cytisine-resistant sites are highly sensitive to the nicotinic
antagonist -conotoxin MII (-CtxMII) and may be directly
identified using [125I]-CtxMII
(Whiteaker et al., 2000b). A second population of cytisine-resistant sites that is unaffected by high (3 µM)
concentrations of -CtxMII and is unusually resistant to inhibition
by the nicotinic agonist 3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride
(A85380) (Abreo et al., 1996) has also been identified (Whiteaker et
al., 2000a). Their low A85380 affinity may be used to isolate them pharmacologically from other cytisine-sensitive and -resistant epibatidine-binding sites (A85380-resistant sites). The distribution and pharmacology of both cytisine-resistant populations are suggestive of 3* nAChRs (Whiteaker, 2000a,b), but concrete evidence of
3 involvement has not been provided in either case.
The purpose of this study was to investigate the involvement of the
3 nAChR subunit in the expression of the two cytisine-resistant populations described above using mice engineered to lack the 3
nAChR subunit gene (Xu et al., 1999).
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MATERIALS AND METHODS |
Animals. Mice engineered to contain a null mutation
in the 3 nAChR subunit gene (Xu et al., 1999) were bred at the
Division of Neuroscience, Baylor College of Medicine, in accordance
with procedures approved by the local Animal Care and Utilization committee.
Materials. [125I]Epibatidine
(specific activity, 2200 Ci/mmol) was obtained from DuPont NEN (Boston,
MA). ()-Nicotine bitartrate was bought from BDH Chemicals (Poole,
UK). A85380 was supplied by Research Biochemicals (Natick, MA).
-CtxMII was synthesized as described previously (Cartier et al.,
1996), as was [125I]-conotoxin MII
(Whiteaker et al., 2000a) (specific activity, 2200 Ci/mmol). Hyperfilm
-max and 125I autoradiography
microscales (original activity, 1.2-650 nCi/mg) were purchased from
Amersham Biosciences (Mt. Prospect, IL). All other supplies were
purchased from Sigma (St. Louis, MO).
Quantitative autoradiography of
[125I]-CtxMII and
[125I]epibatidine binding.
Quantitative autoradiography procedures were similar to those described
previously (Pauly et al., 1989; Whiteaker et al., 2000a). Mice (8 d of
age) of each 3 genotype (3+/+,
3+/ , and
3/) were decapitated, and each
brain was removed from the skull and rapidly frozen by immersion in
isopentane (35°C, 10 sec). Tissue was collected from mice from a
single litter (2× 3+/+, 6×
3+/, and 2×
3/). Tissue sections (10 µm
thick) were prepared from frozen, unfixed tissue using a Leica
(Nussloch, Germany) CM1850 cryostat/microtome and were
thaw-mounted onto gelatin/poly-L-lysine-subbed
glass microscope slides (Richard Allen, Richland, MI).
Before exposure to [125I]-CtxMII (0.5 nM), sections were incubated in binding buffer (144 mM NaCl, 1.5 mM KCl; 2 mM
CaCl2, 1 mM
MgSO4, 20 mM HEPES, 0.1% w/v BSA, pH
7.5) plus 1 mM PMSF (to inactivate endogenous serine
proteases) at 22°C for 15 min. For all
[125I]-CtxMII-binding reactions, the
standard binding buffer was supplemented with 5 mM EDTA, 5 mM EGTA, and 10 µg/ml each of aprotinin, leupeptin
trifluoroacetate, and pepstatin A to protect the ligand from endogenous
proteases. A separate series of sections from each mouse was used to
determine nonspecific [125I]-CtxMII
binding (in the presence of 1 µM unlabeled epibatidine). After incubation with [125I]-CtxMII,
the slides were washed as follows: Thirty seconds in binding buffer
plus 0.1% w/v BSA (22°C), 30 sec in binding buffer plus 0.1% w/v
BSA (0°C), 5 sec in 0.1× binding buffer plus 0.01% w/v BSA (twice
at 0°C), and twice at 0°C for 5 sec in 5 mM HEPES, pH
7.5.
Sections for use in [125I]epibatidine
binding were rehydrated in binding buffer at 22°C for 15 min,
followed by incubation with 100 pM
[125I]epibatidine for 2 hr at 22°C.
Three series of adjacent sections were used from each mouse to measure
total [125I]epibatidine binding (no
competing ligand), [125I]epibatidine
binding in the presence of 20 nM cytisine, and
[125I]epibatidine binding in the
presence of 10 nM A85380. Concentrations of unlabeled drugs
were chosen on the basis of results obtained in previous studies
(Whiteaker et al., 2000a,b) and confirmed in pilot experiments.
Nonspecific [125I]epibatidine binding
was defined using a separate series of sections in the presence of 1 mM ()-nicotine tartrate. Slides were washed by sequential
incubation in the following buffers (all steps at 0°C): Five seconds
in binding buffer (twice), 5 sec in 0.1× binding buffer (twice), and 5 sec in 5 mM HEPES, pH 7.5 (twice).
Sections were initially dried with a stream of air and subsequently by
overnight storage (22°C) under vacuum. Mounted, desiccated sections
were apposed to Amersham Hyperfilm -Max. Because large variations in
ligand binding, and thus signal intensity, were observed, several film
exposures were made for each binding condition to ensure that all
measurements could be made within the accurate recording range of the
film (3-7 d for
[125I]-CtxMII-labeled sections; 5-96
hr for [125I]epibatidine-labeled
sections). To allow quantification, each film was also exposed to
125I autoradiography microscale standards
of defined specific activity.
After the films had been exposed to the sections for an appropriate
length of time, they were developed and signal intensity in selected
brain regions was measured by digital image analysis. Films were
illuminated using a Northern Light (Ontario, Canada) light box,
and autoradiographic images of the sections and standards were captured
using a CCD imager camera. Signal intensity was determined using NIH
Image 1.61 software. Where possible, six independent
measurements from different tissue sections were made for each brain
region, under each incubation condition, for each mouse. For each
subject, the absorbance measurements from each brain area were used to
calculate the degree of labeling by reference to the relevant standard
curve, and labeling values were used to determine regional labeling in
each mouse.
Data processing. All calculations and graph preparation were
performed using SigmaPlot for Windows, version 5.0 (Jandel Scientific, San Rafael, CA). Statistical analysis (one-way ANOVA) was performed using SPSS PC+ (Jandel Scientific). Duncan's post hoc test
was used to test for within-region differences.
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RESULTS |
Effect of 3-null mutation on
[125I]-CtxMII-binding nAChRs
[125I]-CtxMII (0.5 nM) was used to detect binding sites with a high affinity
for -CtxMII (Whiteaker et al., 2000b). The distribution of
[125I]-CtxMII-binding sites in the
8 d animals was very similar to that observed in adult animals
(Whiteaker et al., 2000b), with the greatest amounts seen in optic
tract-associated nuclei (superior colliculus, olivary pretectal
nucleus, and the ventrolateral and dorsolateral geniculate nuclei) and
the occulomotor nerve and more moderate expression throughout the
dopaminergic tracts (substantia nigra, ventral tegmental region,
striatum, nucleus accumbens, and olfactory tubercles) (Fig.
1, fourth column). Some
developmental differences were evident, however: the amounts of
[125I]-CtxMII binding observed in the
younger animals were generally higher than those seen in more mature
animals (Whiteaker et al., 2000b). Relative amounts also differed
between some regions. (For instance, in mature animals, the
dorsolateral geniculate nucleus contained 5.0 ± 0.2 fmol/mg
protein and the ventrolateral geniculate nucleus contained 5.8 ± 0.2 fmol/mg protein of
[125I]-CtxMII-binding sites, whereas
in the 8 d animals, the specific binding was 10.9 ± 0.4 and
6.7 ± 0.6 fmol/mg protein, respectively.)
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Figure 1.
Illustration of total, cytisine-resistant, and
A85380-resistant [125I]epibatidine-binding and
[125I]-CtxMII-binding patterns in an 8 d
wild-type mouse brain. Sections (10 µM) from
3+/+ mouse brains were incubated in the presence
of 100 pM [125I]epibatidine alone
(first column),
[125I]epibatidine plus 20 nM cytisine
(second column), 100 pM
[125I]epibatidine plus 10 nM A85380
(third column), and 0.5 nM
[125I]-CtxMII (fourth
column). Panels are digital images of
autoradiograms. ac, Anterior commissure;
cc, corpus callosum; DLGN, dorsolateral
geniculate nucleus; fr, fasciculus retroflexus;
IC, inferior colliculus; MG, medial
geniculate nucleus; MVN, medial vestibular
nucleus; NAcc, nucleus accumbens; opt,
optic tract; OT, olfactory tubercle; PHN,
prepositus hypoglossal nucleus; SC, superior colliculus;
SN, substantia nigra; Str, striatum;
VLGN, ventrolateral geniculate nucleus;
VTA, ventral tegmental area.
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Specific [125I]-CtxMII binding was
assessed in 3+/+,
3+/, and
3/ mice. In most regions in which
[125I]-CtxMII binding was observed,
deletion of the 3 nAChR subunit had no effect, as determined by
one-way ANOVA (Fig. 2).
[125I]-CtxMII binding in the medial
habenula (MH) and fasciculus retroflexus was eliminated, however, in
3/ mice
(F(2,3) = 20.60, p = 0.0177 and F(2,3) = 50.58, p = 0.0049, respectively). In addition,
[125I]-CtxMII binding was
approximately halved in the interpeduncular nucleus (IPN) of
3/ mice (from 4.29 ± 0.54 to
1.98 ± 0.74 fmol/mg protein) (Fig. 2), although this effect was
not statistically significant (F(2,3) = 4.89; p = 0.11). Because these regions are small,
represent only 3 of 18 regions with detectable binding, and contain
only modest amounts of [125I]-CtxMII
binding even in the 3+/+ animals, the
great majority of
[125I]-CtxMII-binding nAChRs
apparently do not require expression of the 3 subunit, as
demonstrated by the high correlation between regional
[125I]-CtxMII binding in
3+/+ and
3/ animals (Fig.
2A; y-intercept, 0.25 fmol/mg protein;
slope, 0.98; r = 0.98).
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Figure 2.
Effect of nAChR 3 subunit-null mutation on
mouse brain regional [125I]-CtxMII (0.5 nM) binding. Levels of specific
[125I]-CtxMII (0.5 nM) binding were
determined in the brains of 3+/+,
3+/, and 3/ genotype
mice and compared in 18 different brain regions. A, Only
the medial habenula and fasciculus retroflexus (open
circles) showed significant loss of specific
[125I]-CtxMII binding in
3/ mice compared with their
3+/+ counterparts (as determined by one-way
ANOVA; medial habenula, F(2,3) = 20.60, p = 0.0177; fasciculus retroflexus,
F(2,3) = 50.58, p = 0.0049). Loss of specific [125I]-CtxMII binding
in the IPN (the remaining habenulo-IPN tract region) approached, but
did not attain, significance (F(2,3) = 4.89; p = 0.11). The regression line (solid
line) was fit to all regions that were not significantly
different between genotypes; also shown are 95% confidence intervals
(dotted lines). B,
Within-region differences were tested for in the habenulo-IPN tract
using Duncan's post hoc test. Significant differences
are denoted h (different from heterozygous null-mutant)
or w (different from wild type). fr,
Fasciculus retroflexus.
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Effect of 3-null mutation on A85380-resistant
[125I]epibatidine-binding nAChRs
Resistance to inhibition by the agonist A85380 has been used
previously to isolate a population of
[125I]epibatidine-binding nAChRs with
low affinity for both cytisine and -CtxMII (Whiteaker et al.,
2000a). The highest levels of A85380-resistant
[125I]epibatidine binding were detected
in the medial habenula-fasciculus retroflexus-IPN tract of
3+/+ animals (Figs. 1 and
3). Indeed, the A85380-resistant binding sites in these regions were by far the most densely expressed nAChR
population measured in the present study. Both of these findings are
consistent with those reported for A85380-resistant binding in adult
animals (Whiteaker et al., 2000a), although the accessory olfactory
bulbs of the younger animals used in this study were almost devoid of
A85380-resistant [125I]epibatidine
binding (2.3 ± 0.4 and 1.0 ± 0.2 fmol/mg protein in the
glomerular and mitral layers, respectively), unlike those of adult
animals (82 ± 9 and 38 ± 3 fmol/mg, respectively).
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Figure 3.
Effect of nAChR 3 subunit-null mutation on
mouse brain regional A85380-resistant
[125I]epibatidine (100 pM) binding.
Levels of specific A85380-resistant
[125I]epibatidine binding were compared in 26 different brain regions for 3+/+,
3+/, and 3/ genotype
mice. Top row, Regional distribution of A85380-resistant
[125I]epibatidine binding measured in femtomoles
per milligram of protein. Because binding levels were so high in the
habenulo-IPN tract, this group is displayed on a different scale from
the remaining regions. Bottom row, Regional effects of
3 null-mutation on A85380-resistant
[125I]epibatidine (100 pM) binding,
presented as percentage of change from wild-type binding. Regions
exhibiting significant effects of 3 null-mutation on
[125I]epibatidine binding were determined with
one-way ANOVA; *p < 0.05 (F(2,3) > 9.55);
**p < 0.01 (F(2,3) > 30.8). Within-region differences were tested using Duncan's
post hoc test. Significant differences are denoted
h (different from heterozygous null-mutant) or
w (different from wild type). AOBG,
Accessory olfactory bulb (glomerular layer); AOBM,
accessory olfactory bulb (mitral cell layer); ATN,
anterior thalamic nucleus; bsc, brachium of the superior
colliculus. DLGN, dorsolateral geniculate nucleus;
fr, fasciculus retroflexus; IC-DC,
inferior colliculus (dorsal cortex); MGN, medial
geniculate nucleus; MH, medial habenula;
MVN, medial vestibular nucleus; NAC,
nucleus accumbens (core); NAS, nucleus accumbens
(shell); OBG, olfactory bulb (glomerular layer);
OBI, olfactory bulb (internal plexiform layer);
omn, occulomotor nerve; OPN, olivary
pretectal nucleus; opt, optic tract; OT,
olfactory tubercle; PHN, prepositus hypoglossal nucleus;
SC-ONL, superior colliculus (optic nerve layer);
SC-SG, superior colliculus (superficial gray);
SC-ZL, superior colliculus (zonal layer);
SN, substantia nigra; STR, striatum;
VLGN, ventrolateral geniculate nucleus;
VTA, ventral tegmental area.
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In contrast to [125I]-CtxMII-binding
nAChRs, the A85380-resistant nAChR population displayed notable
sensitivity to the loss of 3 subunit expression, as illustrated in
Figure 3 and detailed in Table 1. As
shown in Figure 3 (first column), deletion of the
3 nAChR subunit decreased the amount of A85380-resistant [125I]epibatidine binding detected in
the inferior colliculus, medial habenula, and fasciculus retroflexus by
at least 90% (F(2,3) = 10.5, p = 0.044; F(2,3) = 176, p = 0.0008; and
F(2,3) = 62.2, p = 0.0036, respectively). A85380-resistant
[125I]epibatidine binding in the IPN of
mutant animals was significantly reduced
(F(2,3) = 15.3; p = 0.0268). Interestingly, however, this binding in IPN was still 24% of
wild-type levels. In contrast, A85380-resistant binding in mutant
animals was only 9.0 and 5.5% of that seen in wild-type mice in the
fasciculus retroflexus and medial habenula, respectively. Outside the
dorsal cortex of the inferior colliculus and the MH-IPN tract, only
the medial vestibular and prepositus hypoglossal nuclei contained
substantial amounts (>10 fmol/mg protein) of A85380-resistant
[125I]epibatidine binding (Fig. 3,
fourth column). Deletion of the 3 subunit also produced a
significant loss of A85380-resistant [125I]epibatidine binding in these
regions. Low (<10 fmol/mg protein) A85380-resistant
[125I]epibatidine-binding signals were
observed in all of the other regions surveyed. In contrast to the more
densely expressed A85380-resistant [125I]epibatidine-binding populations,
the 3-dependence of these sites was generally low, with the only
exceptions being found in the anterior thalamic and medial geniculate
nuclei (Fig. 3). With one exception (glomerular layer of the olfactory
bulb), A85380-resistant [125I]epibatidine binding in
3+/ mice was indistinguishable from
that in 3+/+ mice. No other examples of
binding differences between 3+/ and
3+/+ mice were seen, suggesting that
loss of one copy of the 3 gene has very little effect on nAChR
expression.
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DISCUSSION |
3 was the first mammalian neuronal nAChR subunit cloned
(Boulter et al., 1986) and one of the first to be studied in an
heterologous expression system (Boulter et al., 1987). The
immunochemical study by Flores et al. (1996) established the existence
of 34 nAChRs in the rat trigeminal ganglion, but functional (Zoli
et al., 1998; Quick et al., 1999; Grady et al., 2001),
immunohistochemical (Yeh et al., 2001), and binding (Whiteaker et al.,
2000a) studies have provided only circumstantial evidence for central
expression of 3* nAChRs. The findings of the present study provide
the first direct evidence for 3* nAChR expression in the mammalian CNS.
Expression of [125I]-CtxMII-binding
sites is independent of 3 nAChR subunit expression in 16 of the 18 regions in which these sites were identified (Fig. 2). These data
indicate that [125I]-CtxMII-binding
nAChRs are largely not 3-dependent, despite being found in regions
(the optic tract and its associated nuclei and dopaminergic terminal
regions) in which 3 protein might be expected to be expressed, based
on the detection of 3 mRNA (Wada et al., 1989; Whiteaker et al.,
2000b). This result was very surprising, because -CtxMII was
originally isolated by virtue of its selectivity for heterologously
assembled 32-subtype nAChRs (Cartier et al., 1996), and
both -CtxMII and [125I]-CtxMII
display similar affinities at native
[125I]-CtxMII-binding nAChRs and
artificially expressed 32-subtype nAChRs (Whiteaker et al.,
2000b). The lack of effect of 3 subunit deletion on
[125I]-CtxMII-binding nAChRs argues
strongly against a role for the 3 nAChR subunit in this binding site.
Recently, it has been established that 6-containing nAChRs can bind
-CtxMII with high (nanomolar) affinity (Vailati et al., 1999;
Kuryatov et al., 2000; Klink et al., 2001). In addition, the 6 nAChR
subunit is widely coexpressed with 3 (LeNovère et al., 1996),
and the two subunits exhibit considerable sequence homology
(LeNovère and Changeux, 1995). Together, these findings suggest
that the majority of mouse brain
[125I]-CtxMII-binding nAChRs may in
fact be of an 6-containing rather than 3-containing subtype as
originally suspected. A component of nAChR-stimulated striatal dopamine
release is sensitive to inhibition by -CtxMII (Kulak et al., 1997;
Kaiser et al., 1998; Grady et al., 2001), establishing that
-CtxMII-sensitive nAChRs are a functional mammalian neuronal nAChR
subtype. In addition, striatal -CtxMII-sensitive sites are dependent
on 2 nAChR subunit expression (Grady et al., 2001), indicating that
mouse brain [125I]-CtxMII-binding
nAChRs require 2 subunit expression. Despite their likely
physiological significance,
[125I]-CtxMII-binding nAChRs are
relatively rare. For instance,
[125I]-CtxMII-binding sites are
outnumbered by both cytisine-sensitive and A85380-resistant
[125I]epibatidine-binding sites in the
terminal regions of the substantia nigra/ventral tegmental area
dopaminergic projections. It is likely, however, that their
concentration on dopaminergic termini strengthens their influence over
this important pathway (Kulak et al., 1997; Kaiser et al., 1998; Grady
et al., 2001; Quik et al., 2001).
Although the 3 subunit is not required for the expression of
[125I]-CtxMII-binding sites in most
brain regions, [125I]-CtxMII binding
in the medial habenula and fasciculus retroflexus was dramatically
reduced in 3/ versus
3+/+ mice (Fig. 2), indicating that in
these regions, [125I]-CtxMII-binding
sites may be 32* nAChRs. Alternatively,
[125I]-CtxMII binding in these
regions may result from low occupancy of a large nicotinic binding
population with a lower affinity for -CtxMII. In either case, the
regions containing 3-dependent [125I]-CtxMII-binding receptors are
small, as are the amounts of binding observed in these regions. Thus,
the great majority of nAChRs identified using
[125I]-CtxMII (0.5 nM) do
not appear to require 3 nAChR subunit expression.
The agonist A85380 has been used to isolate the population of
[125I]epibatidine-binding nAChRs with
low affinity for both cytisine and -CtxMII (Whiteaker et al.,
2000a). The distribution of A85380-resistant [125I]epibatidine-binding sites was very
different from that of
[125I]-CtxMII-binding nAChRs,
demonstrating that two different populations of sites were being
identified (Fig. 1). In the regions containing the highest levels of
A-85380-resistant binding (medial habenula-fasciculus retroflexus-IPN
tract, dorsal cortex of the inferior colliculus, and medial vestibular
and prepositus hypoglossal nucleus), binding was dramatically reduced
in 3-null mutant animals. Although most A85380-resistant
[125I]epibatidine-binding sites require
expression of the 3 nAChR subunit, a substantial population of
A85380-resistant
[125I]epibatidine-binding sites was
retained in the IPN of 3/ mice.
Thus, the IPN may express a novel, 3-independent, A85380-resistant nAChR subtype.
An important role for the 3 subunit in A85380-resistant
[125I]epibatidine-binding sites is
consistent with previous findings. First, A85380-resistant
[125I]epibatidine-binding sites have a
distribution and binding pharmacology suggestive of 34* nAChRs
(Whiteaker et al., 2000a). Second, the activation and binding
pharmacology of receptors that persist in the habenulointerpeduncular
tract of 2-null mutant mice also resemble that of 34* nAChRs
(Zoli et al., 1998; Grady et al., 2001). Third, central 3 subunit
immunoreactivity is concentrated in habenulointerpeduncular tract
nuclei (Yeh et al., 2001). Thus, the past and present findings strongly
suggest that A85380-resistant epibatidine-binding sites correspond to a
functional CNS 34 nAChR subtype similar to that previously
identified in the periphery (Flores et al., 1996).
Widely distributed but low-density (<10 fmol/mg protein)
A85380-resistant [125I]epibatidine (100 pM) binding was observed (Figs. 1, 3, Table 1). In
particular, A85380-resistant sites were found in the optic tract and
associated nuclei and in the cell body regions of the dopaminergic
tract. However, these additional
[125I]epibatidine-binding sites
exhibited much lower 3 dependence than those in the more densely
expressing regions. Given the generally lower affinity of A85380 at
4- versus 2-containing nAChR subtypes (Parker et al., 1998), it
is possible that these sites represent a combination of 4 with a
non-3 subunit.
Loss of 3 expression results in a syndrome with features suggestive
of widespread autonomic dysfunction that is lethal within weeks of
birth (Xu et al., 1999). This dysfunction might theoretically induce
global alterations in nAChR expression. However, the minor effects of
3-null mutation on [125I]-CtxMII
and total [125I]epibatidine-binding
sites (Fig. 4) strongly argue against a generalized disruption of nAChR expression. In addition, the early demise of the 3/ mice necessitated
the use of relatively young mice (8 d of age) in this study. It was
anticipated that these mice might display major developmental
differences in the distribution of nAChR subtypes compared with the
more mature 60- to 90-d-old animals used in previous studies from our
laboratory. In fact, each of the subtypes measured previously was found
in the younger subjects used in this study. Regional distribution of
the sites was qualitatively similar to that of adult mice, although
some quantitative differences were seen.
View larger version (118K):
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Figure 4.
Effects of nAChR 3 subunit-null mutation on
nicotinic binding populations illustrated at the level of the
substantia nigra/ventral tegmental region. First row,
The majority of mouse brain
[125I]epibatidine-binding sites are of the
cytisine-sensitive type and do not show global alterations after loss
of 3 subunit gene expression. Second row,
Cytisine-resistant [125I]epibatidine binding has a
more restricted distribution than cytisine-sensitive binding. The
effects of 3 null mutation vary between regions, with loss of
subunit expression dramatically reducing cytisine-resistant binding in
the fasciculus retroflexus and medial geniculate nucleus but having
minimal effect on this measure in the substantia nigra and ventral
tegmental area. Third row, A85380-resistant
[125I]epibatidine binding is a subset of
cytisine-resistant [125I]epibatidine binding and
is strongly affected by 3 null mutation. Fourth row,
[125I]-CtxMII-binding nAChRs are a second
subset of the cytisine-resistant
[125I]epibatidine-binding population and are
largely unaffected by the loss of 3 subunit expression. All
panels are digital images of autoradiograms.
fr, Fasciculus retroflexus; MG, medial
geniculate nucleus; SC, superior colliculus;
SN, substantia nigra; VTA, ventral
tegmental area.
|
|
In summary, this study has determined the 3 dependence of two
previously identified nicotinic binding populations and has provided
evidence for the possible existence of additional minor populations.
Studies performed with transfected oocytes indicated that 32
nAChRs have a high affinity for -CtxMII. Unexpectedly, [125I]-CtxMII-binding nAChRs showed
almost no dependency on 3 expression (Figs. 2, 4, fourth
row), strongly arguing against a role for the 3 subunit in
native [125I]-CtxMII-binding nAChRs.
The 3 dependence of [125I]-CtxMII
binding in the habenulointerpeduncular tract suggests that these sites
may have a different (3-dependent) composition from the majority of
[125I]-CtxMII-binding nAChRs.
A85380-resistant
[125I]epibatidine-binding sites were
suspected be 34 nAChRs (Whiteaker et al., 2000a) and do indeed
show strong 3 dependence (Fig. 4, third row). In this
case, the persistence of A85380-resistant binding in
3/ mice (particularly in the IPN)
may indicate the presence of a novel nicotinic population in this
nucleus. This is currently under investigation.
Note added in proof. During the preparation of this
manuscript, Champtiaux et al. (2002) confirmed the 6 nAChR subunit
dependence of [125I]-conotoxin MII binding sites using
an 6 subunit null mutant mouse model.
| |
FOOTNOTES |
Received Oct. 10, 2001; revised Dec. 18, 2001; accepted Dec. 28, 2001.
This work was supported by National Institutes of Health Grants
DA-12242, DA-12661, DA-00197, GM-48677, and MH-53631. We thank Dr. Mariella DeBiasi for kindly supplying the 3-null mutant tissue used in this study.
Correspondence should be addressed to Dr. Paul Whiteaker, Institute for
Behavioral Genetics, Campus Box 447, University of Colorado, Boulder,
CO 80309. E-mail: wpaul{at}colorado.edu.
| |
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Mol. Pharmacol.,
May 1, 2003;
63(5):
1169 - 1179.
[Abstract]
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J. M. Kulak, J. L. Musachio, J. M. McIntosh, and M. Quik
Declines in Different beta 2* Nicotinic Receptor Populations in Monkey Striatum after Nigrostriatal Damage
J. Pharmacol. Exp. Ther.,
November 1, 2002;
303(2):
633 - 639.
[Abstract]
[Full Text]
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M. Zoli, M. Moretti, A. Zanardi, J. M. McIntosh, F. Clementi, and C. Gotti
Identification of the Nicotinic Receptor Subtypes Expressed on Dopaminergic Terminals in the Rat Striatum
J. Neurosci.,
October 15, 2002;
22(20):
8785 - 8789.
[Abstract]
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A. J. Mogg, P. Whiteaker, J. M. McIntosh, M. Marks, A. C. Collins, and S. Wonnacott
Methyllycaconitine Is a Potent Antagonist of alpha -Conotoxin-MII-Sensitive Presynaptic Nicotinic Acetylcholine Receptors in Rat Striatum
J. Pharmacol. Exp. Ther.,
July 1, 2002;
302(1):
197 - 204.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION
