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The Journal of Neuroscience, June 15, 1998, 18(12):4461-4472
Identification of Four Classes of Brain Nicotinic Receptors Using
 2 Mutant Mice
Michele
Zoli1, 2,
Clément
Léna1,
Marina R.
Picciotto1, 3, and
Jean-Pierre
Changeux1
1 Centre National de la Recherche Scientifique
Unité de Recherche Associée D1284, Neurobiologie
Moléculaire, Institut Pasteur, 75724 Paris Cédex 15, France, 2 Section of Physiology, Department of Biomedical
Sciences, University of Modena, Modena, Italy, and
3 Department of Psychiatry, Yale University School of
Medicine, New Haven, Connecticut 06508
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ABSTRACT |
Although the expression patterns of the neuronal nicotinic
acetylcholine receptor (nAChR) subunits thus far described are known,
the subunit composition of functional receptors in different brain
areas is an ongoing question. Mice lacking the  2 subunit of the
nAChR were used for receptor autoradiography studies and patch-clamp
recording in thin brain slices. Four distinct types of nAChRs were
identified, expanding on an existing classification [Alkondon M,
Albuquerque EX (1993) Diversity of nicotinic acetylcholine receptors in
rat hippocampal neurons. I. Pharmacological and functional evidence for
distinct structural subtypes. J Pharmacol Exp Ther 265:1455-1473.], and tentatively identifying the subunit composition of nAChRs in different brain regions. Type 1 nAChRs bind
 -bungarotoxin, are not altered in 2  / mice, and contain the
7 subunit. Type 2 nAChRs contain the 2 subunit because they are
absent in 2 / mice, bind all nicotinic agonists used with high
affinity (excluding -bungarotoxin), have an order of potency for
nicotine  cytisine in electrophysiological experiments, and are
likely to be composed of 42 in most brain regions, with other subunits contributing in specific areas. Type 3 nAChRs bind epibatidine with high affinity in equilibrium binding experiments and show that
cytisine is as effective as nicotine in electrophysiological experiments; their distribution and persistence in 2 / mice strongly suggest a subunit composition of 34. Type 4 nAChRs bind
cytisine and epibatidine with high affinity in equilibrium binding
experiments and persist in 2 / mice; cytisine = nicotine in
electrophysiological experiments. Type 4 nAChRs also exhibit faster
desensitization than type 3 nAChRs at high doses of nicotine. Knock-out
animals lacking individual subunits should allow a further
dissection of nAChR subclasses.
Key words:
nicotinic receptor; receptor classification; homologous
recombination; patch clamp; receptor autoradiography; mouse; CNS
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INTRODUCTION |
Neuronal nicotinic acetylcholine
receptors (nAChRs) comprise a family of pentameric oligomers made up of
combinations of 10 different subunits (for review, see Le Novère
and Changeux, 1995 ). The subunits (2-8) contain two
cysteines that contribute to acetylcholine (ACh) binding, whereas the
non- or subunits (2-4) lack these residues but contribute
to the complementary component of the binding site and affect the
pharmacological properties of the receptor (Luetje and Patrick, 1991).
On the basis of phylogenetic and pharmacological properties (Le
Novère and Changeux, 1995), the 7 and 8 subunits comprise
an -bungarotoxin-sensitive subfamily of nAChRs that can form
functional homo-oligomers in reconstituted systems (Couturier et al.,
1990). The 2-6 and 2-4 subunits comprise a second
subfamily that can combine to form a number of functionally different
hetero-oligomers (Sargent, 1993; Role and Berg, 1996). 9 is in a
third family and is expressed in non-neuronal cells in the cochlea
(Elgoyhen et al., 1994).
-Bungarotoxin binding is considered to represent the distribution of
7 subunit-containing nAChRs (Orr-Utreger et al., 1997). However,
most nicotinic ligands show similar patterns of high-affinity labeling
(Clarke et al., 1985; Happe et al., 1994; Aubert et al., 1996) that
resembles the distribution of 4/2 in the brain (Wada et al.,
1989; Hill et al., 1993). More recently, epibatidine has been shown to
bind with very high affinity to nAChRs (Houghtling et al., 1995), with
a distribution comparable to that of 3H-nicotine (Perry and
Kellar, 1995), and to exert a number of classical effects of central
nicotinic agonists (Badio and Daly, 1994; Sullivan et al., 1994;
Gerzanich et al., 1995).
In the mammalian brain, patch-clamp recording demonstrates at least two
different nAChRs in the habenulo-interpeduncular system, putatively
3/4 and 2/4, based on subunit distribution and on
comparison with specific isotypes reconstituted in Xenopus oocytes (Mulle et al., 1991; Connolly et al., 1995). In a series of
papers in which electrophysiological recordings were performed along
with in situ hybridization, several nAChR populations have been characterized in the hippocampal formation of the rat containing 7, 42, and 34 (Alkondon and Albuquerque, 1993, 1995;
Alkondon et al., 1994).
Mice lacking the gene for the 2 nAChR subunit have no detectable
high-affinity 3H-nicotine binding in the brain (Picciotto
et al., 1995). Here we show the persistence of high-affinity binding
for a number of other nicotinic ligands in mutant animals. The
distribution of other subunits is not widespread in wild-type
rodents (Dineley-Miller et al., 1992; Le Novère et al., 1996),
and there is no up- or downregulation of expression of other nicotinic
subunits in mutant animals (Picciotto et al., 1995); therefore, nAChR
binding is restricted to a limited number of regions. Regional analysis
of binding was followed by an exploratory survey of residual nAChRs, using patch-clamp recording in brain slices. We interpret the differences of binding and activation properties of the ligands in the
different brain structures as a consequence of differences in the
subunit composition of the nAChRs, and we expand the existing classification of nAChR subtypes (Alkondon and Albuquerque, 1993).
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MATERIALS AND METHODS |
Animals
Mice were generated by mating parents heterozygous for a
mutation in the 2 subunit of the neuronal nAChR (Picciotto et al., 1995). The genotype of the offspring was determined by PCR, using oligonucleotides identifying the mutant and wild-type copies of the
2 gene. In each experiment, mice homozygous for the mutation in the
2 gene were paired with heterozygous and wild-type 2 mice of the
same sex and from the same litter. All experiments were performed on
brains from mice between 4 and 7 months of age.
Materials
3H-Nicotine, 3H-methylcarbamylcholine,
3H-cytisine, 3H-pirenzepine, and
3H-AF-DX384 were obtained from New England Nuclear (Boston,
MA). 3H-Epibatidine and 125I--bungarotoxin
were purchased from Amersham (Arlington Heights, IL).
3H-Acetylcholine iodide was obtained from Isotopchim.
Unlabeled nicotine, cytisine, atropine, and dimethylphenylpiperazinium
(DMPP) were purchased from Sigma (St. Louis, MO), whereas epibatidine, dihydro--erythroidine (DHE), mecamylamine (MCA), and
methyllycaconitine (MLA) were obtained from Research Biochemicals
(Natick, MA).
Receptor autoradiography
Mice were decapitated, and brains were dissected out and frozen
in crushed dry ice. Sections (14 µm) were cut at the cryostat (Reichert-Jung), mounted on gelatinized slides, and stored for not more
than 2 d at 80°C until use. In the analysis of nicotinic ligand binding, sections were taken at 15 coronal levels [bregma levels: 1.2, 1.0, 0.4, 0.2, 0.5, 1.5, 2.0, 2.9, 3.4, 3.9, 4.8, 5.3, 5.8, 7.0, and 7.5 mm, according to Franklin and Paxinos (1997)]. In the analysis of 125I--bungarotoxin
binding, sections were taken at 10 coronal levels: 1.0, 0.4, 0.5,
1.5, 2.0, 3.4, 4.8, 5.3, 5.8, and 7.5 mm. In the analysis
of muscarinic ligands, sections were taken at five coronal levels: 0.4, 0.5, 1.5, 3.4, and 5.3 mm.
On the day of the experiment the slides were thawed and processed
according to established protocols (see below for details) (Wamsley et
al., 1984; Clarke et al., 1985; Cortes et al., 1986; Regenold et al.,
1987; Happe et al., 1994; Perry and Kellar, 1995; Aubert et al., 1996).
The concentration of radioactive ligands used in the experiments was
close to the KD as reported in previous papers,
using receptor autoradiography or homogenate binding. In each
experiment, sections from a 2 +/+, +/, and / mouse were
processed in parallel. For each ligand at least five brains of each
genotype were used. Slides were exposed to 3H-Hyperfilm
(Amersham) for the length of time indicated and developed in Eastman
Kodak (Rochester, NY) D19 film developer (4 min at 22°C).
Quantitative receptor autoradiography was performed by means of
computer-assisted microdensitometry (VIDAS image analyzer, Kontron,
Munich, Germany) and by using appropriate standards (Benfenati et al.,
1986). Statistical analysis was performed according to the
Mann-Whitney U test.
3H-Nicotine (85 Ci/mmol) was used at a concentration of 5 nM. The incubation was performed at room temperature for 30 min in 50 mM Tris-HCl, pH 7.4. It was followed by four
rinses of 30 sec in the same buffer, followed by a brief rinse in
distilled water, all performed at 4°C. Nonspecific binding was
defined as the binding in the presence of cold nicotine (10 µM). The film exposure time was 3 months.
3H-Cytisine (30 Ci/mmol) was used at a concentration of 5 nM. The incubation was performed at 4°C for 60 min in 50 mM Tris-HCl, pH 7.4, containing (in mM) 120 NaCl, 5 KCl, 2.5 CaCl2, and 1 MgCl2. It
was followed by three rinses for 2.5 min 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 cold nicotine (10 µM). The film exposure time was 3 months.
3H-Methylcarbamylcholine (84 Ci/mmol) was used at a
concentration of 5 nM. The incubation was performed at room
temperature for 30 min in 50 mM Tris-HCl, pH 7.4. It was
followed by two rinses of 30 sec in the same buffer, followed by a
brief rinse in distilled water, all performed at 4°C. Nonspecific
binding was defined as the binding in the presence of cold nicotine (10 µM). The film exposure time was 3 months.
3H-Acetylcholine (84 Ci/mmol) was used at a concentration
of 10 nM. Two preincubations were performed at room
temperature for 15 and 10 min in 50 mM Tris-HCl, pH 7.4, containing (in mM) 120 NaCl, 5 KCl, 2 CaCl2, and 1 MgCl2 plus 1.5 µM atropine. The incubation was performed in the same
buffer for 60 min at 4°C. It was followed by two rinses of 2 min 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 cold nicotine (10 µM). The film exposure time was 3 months.
3H-Epibatidine (53 Ci/mmol) was used at a concentration of
200 pM. The incubation was performed at room temperature
for 30 min in 50 mM Tris-HCl, pH 7.4. It was followed by
two rinses of 5 min in the same buffer, followed by a brief rinse in
distilled water, all performed at 4°C. Nonspecific binding was
defined as the binding in the presence of cold nicotine (10 µM). The film exposure time was 2-4 months.
125I--Bungarotoxin (2000 Ci/mmol) was used at a
concentration of 1.5 nM. The preincubation and incubation
were performed at room temperature for 30 and 120 min, respectively, in
50 mM Tris-HCl, pH 7.4, containing 0.1% bovine serum
albumin. They were followed by six rinses of 30 min in 50 mM Tris-HCl, pH 7.4, and a brief rinse in distilled water,
all performed at 4°C. Nonspecific binding was defined as the binding
in the presence of cold nicotine (1 mM). The film exposure
time was 1-2 d.
3H-Pirenzepine and 3H-AF-DX384 (78 and 120 Ci/mmol, respectively) were used at a concentration of 10 nM. The preincubation and incubation were performed at room
temperature for 15 and 60 min, respectively, containing (in
mM) 120 NaCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3,
2.5 CaCl2, 4.7 KCl, and 5.6 glucose, pH 7.4. They
were followed by three rinses for 4 min in 50 mM Tris-HCl,
pH 7.4, and a brief rinse in distilled water, all performed at 4°C.
Nonspecific binding was defined as the binding in the presence of cold
atropine (1.5 µM). The film exposure time was 1 month.
In situ hybridization
After analysis for mRNA secondary structure via GCG Sequence
Analysis Software 7.1, three oligodeoxynucleotide sequences were chosen
in unique regions of the rat 3, 5, and 4 mRNAs and synthesized with a Cyclone (Biosearch) DNA synthesizer. The probe characteristics and specificity controls are reported in Zoli et al. (1995) and Le
Novère et al. (1996). Specificity control included the
demonstration that (1) two or more probes for each mRNA give identical
labeling pattern, (2) the labeling disappears when labeled probes are
incubated with an excess of cold probe, and (3) probes with the same
base composition but different sequence do not give the specific
labeling pattern. The oligonucleotide probes were labeled at the 3'
end, using 35S-dATP (Amersham) and terminal
deoxynucleotidyl transferase (Boehringer Mannheim, Indianap olis, IN)
and following the specifications of the manufacturer to a specific
activity of 100-300 kBq/pmol. The labeled probes were separated from
unincorporated 35S-dATP by using NucTrap push columns
(Stratagene, La Jolla, CA), precipitated in ethanol, and resuspended in
distilled water containing 50 mM dithiothreitol.
Frozen tissues from two rat and two mouse brains were cut at the
cryostat (14-µm-thick sections) at level 13.8 and 7.5 mm, respectively, thaw-mounted on poly-L-lysine-coated slides,
and stored at 80°C for 1-3 d. The procedure was performed
according to Zoli et al. (1995). Probes were applied at a concentration of 2000-3000 Bq per 30 µl/section (corresponding to ~15
fmol/section). The slides were exposed for 7 d to
3H-Hyperfilm (Amersham).
Patch clamp in brain sections
Preparation of the slices and solutions. The 6- to
15-d-old mice were anesthetized with ether and decapitated; brains were removed and placed in ice-cold Krebs' solution containing (in mM) 126 NaCl, 26 NaHCO3, 25 glucose,
1.25 NaH2PO4, 2.5 KCl, 2 CaCl2, and 1 MgCl2 bubbled with 95%
O2/5% CO2. Slices (300 µM thick) were obtained by using a DSK-1000 slicer (Dosaka, Japan) and
were kept submerged on a net in 200 ml of Krebs' solution. Recordings
were performed under an Axioscop microscope (Zeiss, Oberkochen,
Germany). The neurons could be visualized easily without the help of
phase-contrast optics. Drugs were applied either in the bath or with a
broken patch pipette (tip  50 µm diameter) placed at the surface of
the slice; this pipette allowed for either an outward flow of drug or
an inward flow of extracellular medium. This system exchanged solution
close to the cell in the 1-5 sec range. As a result, fast
desensitizing nAChR currents may have been missed. When applied through
the pipette, the drugs were dissolved in (in mM) 150 NaCl,
10 HEPES, 2 CaCl2, 1 MgCl2, and 2.5 KCl, pH 7.3.
Electrophysiological recordings. The patch pipettes were
pulled from thin hard glass tubes (Hilgenberg, Germany) with a P-87 Sutter Instruments puller, wax-coated, and filled with (in
mM) 135 CsCl, 10 BAPTA, 10 HEPES, 5 MgCl2, and 4 NaATP, pH 7.3, yielding a 2-3 M
resistance. Voltage-clamp experiments were performed with an Axopatch
1D amplifier (Axon Instruments, Foster City, CA). Gigaseals were
obtained without cleaning the cells. Currents were acquired on a PC
computer with the pCLAMP6 package (Axon Instruments). Episodes of drug
application were interspaced by 15-45 min to allow the nAChRs to
recover from desensitization.
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RESULTS |
Binding of neuronal nicotinic receptor ligands in normal
adult mice
The distribution of 3H-nicotine,
3H-cytisine, 3H-acetylcholine,
3H-methylcarbamylcholine, and 3H-epibatidine
labeling in equilibrium binding experiments in section through the
mouse brain tallies well with the distribution of these ligands in the
rat brain (Clarke et al., 1985; Happe et al., 1994; Perry and Kellar,
1995; Aubert et al., 1996) as well as with previous studies in the
mouse brain (Marks et al., 1992). The overall pattern of labeling of
all of the neuronal nicotinic ligands used in this study was similar
(Figs.
1-5).
The highest level of binding was detected in thalamic nuclei,
especially anterior nuclei, and the interpeduncular nucleus (IPn),
especially its dorsal portion, whereas moderate levels were found in
several brainstem nuclei. 3H-Epibatidine binding partially
differed from this general pattern, because, in addition to the
labeling common to all of the other ligands, it was present at high
levels in the medial habenula (MHb), fasciculus retroflexus (fr),
ventral IPn, nucleus tractus solitarii (Sol), area postrema (AP), and
dorsal motor nucleus of the vagus nerve (DMnX). Accordingly,
quantitative analysis of the autoradiograms revealed that the ratio of
3H-epibatidine to 3H-cytisine binding, at the
concentration and specific activity of the ligands used in the present
experiment, was between 1.5 and 2 in most brain areas of wild-type
mice, but the ratio reached 10 in the medial part of MHb, 6.5 in the
lateral part of MHb, 4 in the ventral IPn and fr, 6.5 in the Sol/DMnX,
and 19 in the AP (Table 1). Notably, all
areas with a 3H-epibatidine/3H-cytisine binding
>2 displayed residual binding in 2 / mice (see below), with the
exception of the superior colliculus (SC), which had a ratio of 2.5 but
no residual binding. In view of the large number of nAChR subunits
(including 2) expressed in the retina, which sends major afferents
to the SC, it is possible that multiple 2-containing nAChR isotypes
were detected here.
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Figure 1.
Film autoradiograms of 3H-nicotine,
3H-cytisine, and 3H-epibatidine binding at
bregma level 1.5 mm of 2 +/+, +/, and / mice. The
arrow indicates the medial habenula. Both
3H-cytisine and 3H-epibatidine binding persist
in the medial habenula of 2 / mice. However,
3H-epibatidine is distributed homogeneously in the medial
habenula, whereas 3H-cytisine binding is markedly more
concentrated in the lateral than in the medial portion of this nucleus.
CYT, Cytisine; EPI, epibatidine;
NIC, nicotine.
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Figure 2.
Film autoradiograms of 3H-nicotine,
3H-cytisine, and 3H-epibatidine binding at
bregma level 3.4 mm of 2 +/+, +/, and / mice. The
arrow and double arrow indicate the
ventral and dorsal interpeduncular nucleus, respectively. Both
3H-cytisine and 3H-epibatidine binding persist
in the interpeduncular nucleus of 2 / mice. However,
3H-epibatidine is distributed homogeneously in the
interpeduncular nucleus, whereas 3H-cytisine binding is
markedly more concentrated in the dorsal than in the ventral portion of
this nucleus. For abbreviations, see the legend to Figure 1.
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Figure 3.
Film autoradiograms of
3H-methylcarbamylcholine and 3H-acetylcholine
binding at bregma level 3.4 mm of 2 +/+, +/, and / mice. The
arrow and double arrow indicate the
ventral and dorsal interpeduncular nucleus, respectively. Both ligands
show persisting binding in the dorsal portion of the interpeduncular
nucleus of 2 / mice. ACH, Acetylcholine;
MCC, methylcarbamylcholine.
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Figure 4.
Film autoradiograms of 3H-nicotine,
3H-cytisine, and 3H-epibatidine binding at
bregma level 5.3 mm of 2 +/+, +/, and / mice. The
arrow indicates the dorsal cortex of the inferior
colliculus. Both 3H-cytisine and 3H-epibatidine
binding persist in this area. For abbreviations, see the legend to
Figure 1.
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Figure 5.
Film autoradiograms of 3H-nicotine,
3H-cytisine, and 3H-epibatidine binding at
bregma level 7.5 mm of 2 +/+, +/, and / mice. The
arrow and double arrow indicate the
dorsal motor nucleus of the vagus nerve and the area postrema,
respectively. Only 3H-epibatidine binding persists in these
nuclei of 2 / mice. For abbreviations, see the legend to Figure
1.
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Binding of neuronal nicotinic receptor ligands in 2
/ mice
As described previously (Picciotto et al., 1995), high-affinity
binding of 3H-nicotine was no longer detected in the brains
of homozygous mice lacking the 2 subunit of the nAChR and was
diminished by ~50% in the brains of mice heterozygous for this
mutation (data not shown) (see Picciotto et al., 1995). This was also
the case for the other nicotinic ligands tested in most brain areas
containing detectable levels of nicotine binding in wild-type animals
(Figs. 1-5). Binding of 3H-epibatidine,
3H-cytisine, 3H-acetylcholine, and
3H-methylcarbamylcholine persisted in a few well defined
brain areas, however. The areas with residual binding for one or more of these ligands were the dorsal and ventral parts of the IPn, the MHb,
the fr, the dorsal cortex of the inferior colliculus (DCIC), the dorsal
tegmentum of the rostral medulla oblongata (DTgm), mainly corresponding
to the medial and superior vestibular nuclei, the AP, the Sol, and the
DMnX (Figs. 1-5). When the sections were exposed for 4 months, in two
of five brains of 2 / mice faint binding for
3H-epibatidine also was detected in the medial rim of the
amygdala (mAmy), the brachium of the inferior colliculus (bic), and the laterodorsal tegmental nucleus of the pons (LDTg). Binding was absent
or greatly reduced (in the areas with the highest labeling) in all of
these areas when the ligands were incubated in the presence of 10 µM cold nicotine.
In 2 / mice, 3H-epibatidine was detected at high
levels in dorsal and ventral IPn, MHb, fr, AP, and DMnX; at low levels in DCIC, DTgm, and Sol (Figs. 1, 2, 4, 5); and was barely detectable in
mAmy, bic, and LDTg. Quantitative analysis showed that approximately the same amount of 3H-epibatidine binding in IPn, MHb, fr,
AP, Sol, and DMnX was present in wild-type and mutant mice (Fig.
6), demonstrating that the vast majority
of 3H-epibatidine binding in wild-type mice in these areas
results from nAChRs that do not contain the 2 subunit. Instead, in
DCIC and DTgm, 3H-epibatidine binding to nAChRs containing
2 subunit was predominant (60-70% of the total
3H-epibatidine binding) (Fig. 6). Because the affinity of
3H-epibatidine for 2-containing and non-2-containing
nAChR isoforms was not determined in the present experiments, it was
not possible to know whether the amount of 3H-epibatidine
binding that was left in mutants accurately reflected the number of
binding sites (see Discussion).
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Figure 6.
Quantitative analysis of
3H-epibatidine (EPI in A),
3H-cytisine (CYT in B), and
125I--bungarotoxin (BTX in
C) binding in 2 +/ and / mice. The results are
expressed as the mean percentage values ± SEM of the respective
+/+ mean value. Statistical analysis according to Mann-Whitney
U test; *p < 0.01 versus 2 +/+
mean value. For abbreviations, see the legend to Table 1 plus the
following: CA1, CA3, hippocampal fields CA1 and CA3;
DG, dentate gyrus; Hyp, hypothalamus;
IC, inferior colliculus; IO, inferior
olive; IPnl, interpeduncular nucleus, lateral part;
PAG, periaqueductal gray; R, red nucleus;
Sp5, spinal trigeminal nucleus; VCo,
ventral cochlear nucleus.
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3H-Cytisine had a distribution in mutant mice similar to
that of 3H-epibatidine, with the exception of the total
absence of labeling in AP, Sol, and DMnX (Figs. 5, 6). In addition,
contrary to 3H-epibatidine, only faint labeling was
observed in ventral IPn and medial MHb in mutant mice (see Figs. 1, 2,
6).
In mutant mice, 3H-methylcarbamylcholine and
3H-acetylcholine were detected but at a low level in the
dorsal IPn (see Fig. 3).
125I--Bungarotoxin binding in 2 / mice
Overall, the pattern of 125I--bungarotoxin binding
did not differ substantially in 2 +/+, +/, and / mice. A
quantitative analysis of the autoradiographs did not reveal significant
region-specific differences in the level of -bungarotoxin binding in
the brains of 2 mutant mice as compared with their wild-type
siblings (Fig. 6).
Binding of muscarinic ligands in 2 / mice
In agreement with previous reports, in wild-type mice the
3H-pirenzepine binding was concentrated selectively in
telencephalic areas, namely neocortex, hippocampal formation, and
striatum, whereas 3H-AF-DX384 was present at high levels in
most gray matter areas. These patterns of binding persisted in 2
/ mice. The quantitative analysis performed in neocortex (M1 and
M2), cingulate cortex (M1 and M2), caudate-putamen (M1 and M2),
olfactory tubercle (M1 and M2), medial septum (M2), CA1 and CA3
hippocampal fields (M1 and M2), dentate gyrus (M1 and M2), central,
dorsal, and ventral thalamus (M2), and hypothalamus (M2) confirmed that
no significant difference was present in the areas analyzed (data not
shown) for both ligands between 2 / mice and their
wild-type or heterozygous siblings.
Localization of nicotinic subunit mRNAs in the dorsocaudal
medulla oblongata
The autoradiographic study described above shows that high levels
of 3H-epibatidine binding persist in some nuclei of the
dorsocaudal medulla oblongata of mutant mice. Previous in
situ hybridization studies of nAChR subunit distribution in this
area showed the presence of relatively high levels of 3 and 5
mRNAs but only low (4, and 2) to undetectable (2, 6, and
3) levels of other subunits (Wada et al., 1989, 1990; Le
Novère et al., 1996). No information is available on 4 mRNA in
this region. We therefore studied the distribution of 3, 5, and
4 mRNAs in the caudal medulla oblongata of both rat and mouse brains
by using in situ hybridization histochemistry and compared
it with 3H-epibatidine binding in the same region. The
distribution of 3 and 4 mRNAs was the same. In the rat
caudodorsal medulla oblongata the labeling was rather homogeneous over
the AP, Sol, and DMnX (Fig. 7). In the
mouse the labeling appeared more heterogeneous, being high in the DMnX,
moderate in the AP, and low in the Sol (Fig.
8). This pattern closely resembled that
of 3H-epibatidine in the same areas in both animal species.
5 mRNA labeling was fainter than the labeling of 3 and 4 mRNAs
and barely visible in the dorsocaudal medulla oblongata of the
mouse.
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Figure 7.
Film autoradiograms of 3H-cytisine and
3H-epibatidine binding and 3, 5, and 4 mRNA levels
at bregma level 13.8 mm of adult rats. The arrow and
double arrow indicate the dorsal motor nucleus of the
vagus nerve and the area postrema, respectively. For abbreviations, see
the legend to Figure 1.
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Figure 8.
Film autoradiograms of
3H-cytisine and 3H-epibatidine binding and
3, 5, and 4 mRNA levels at bregma level 7.5 mm of adult
mice. The arrow and double arrow indicate
the dorsal motor nucleus of the vagus nerve and the area postrema,
respectively. For abbreviations, see the legend to Figure 1.
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Electrophysiological analysis of nicotinic receptors in the
thalamus, medial habenula, interpeduncular nucleus, and dorsal motor
nucleus of the vagus nerve
We next attempted to correlate the binding activity with the
electrophysiological responses to various nicotinic agonists in a slice
preparation of mouse brain. The slice preparation allowed for the
accurate localization of the neurons but precluded a fast application
of the agonists because of the slow diffusion of the drugs into the
slice. In wild-type mice we tested neurons in the anterior group of the
thalamus that express a very high level of high-affinity nicotine
binding sites in wild-type mice. We found that micromolar
concentrations of nicotine elicited responses larger than those
elicited by DMPP and that cytisine was a poor agonist in the thalamus
(Fig. 9A). Epibatidine was a
potent agonist at concentrations above 10 nM. The responses
to nicotine were blocked by 1 µM DHE and 10 µM MCA, but not by 5 nM MLA (Fig. 10), an antagonist that completely
blocks the response of -bungarotoxin-sensitive nAChRs at a dose of 1 nM in cultures of hippocampal neurons (Alkondon and
Albuquerque, 1993). No nicotinic response persisted in the thalamus of
mutant mice (Picciotto et al., 1995), demonstrating the necessary
contribution of the 2 subunit to functional nAChRs in the thalamus.
Other areas with similar binding features (disappearance of every type
of nicotinic agonist binding in mutant mice), such as the substantia
nigra and ventral tegmental area, also showed a similar pharmacological
spectrum in wild-type mice (nicotine cytisine) and the
disappearance of the response in mutant mice (Picciotto et al.,
1998).
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Figure 9.
Dose-response curve for epibatidine, nicotine,
cytisine, and DMPP in the brain of 2 +/+ and 2 / mice.
A, Antero-ventral (AV) thalamus of 2 +/+ mice.
B-D, Other brain regions of 2 / mice:
ventromedial MHb (B), (antero-) dorsal IPn
(C), and DMnX (D). Each
point is the mean ± SEM of 2-12 measures.
Normalization between different cells was performed with responses to
10 µM DMPP in the thalamus and to 10 µM
nicotine in the other structures. Then the normalized currents were
multiplied by the average amplitude of the responses to 10 µM nicotine and DMPP. Note the smaller amplitude of
responses in the thalamus as compared with other structures. We have
recorded 51 cells in the thalamus, 31 cells in the MHb, 13 cells in the
DMnX, and 40 cells in the IPn. The average number of different
conditions tested per cell is 4.5.
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Figure 10.
Differential sensitivity to the nicotinic
antagonists methyllycaconitine (MLA),
dihydro--erythroidine (DHE), and mecamylamine
(MCA) of the nicotinic responses in the thalamus of 2
+/+ mice and in the MHb and IPn of 2 / mice. The antagonists
were applied 5-10 min before (and during) the application of nicotine.
MLA (5 nM) had little effect on the nicotinic responses,
whereas 10 µM mecamylamine totally abolished the
nicotinic responses to 10 µM nicotine. The responses were
totally inhibited by 1 µM DHE in the wild-type
thalamus, partially inhibited in the mutant interpeduncular nucleus
(IPn), and not inhibited in the mutant medial habenula
(MHb). (**), MCA application and MLA and DHbE
applications were performed in two different thalamic neurons with
similar control response to 10 µM DMPP.
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We then investigated the nicotinic responses in brain areas in which
high levels of binding remained in mice lacking the 2 subunit.
Responses could be recorded in the structures expressing high-affinity
binding sites for epibatidine alone (ventral MHb, DMnX) or for
epibatidine and cytisine (dorsal IPn) in the mutant mice (small
nicotinic responses also were observed in four of nine cells in
the DCIC, but no pharmacological investigation was undertaken in this
structure). In the MHb, the DMnX, and the IPn we found a similar
activity of cytisine, nicotine, and DMPP in the micromolar range (see
Fig. 9B-D). The sensitivity to nicotine was slightly higher
than that reported in the rat DMnX (Bertolino et al., 1997).
Epibatidine elicited large currents at concentrations above 10 nM. When recorded in neurons from the MHb and IPn of wild-type mice, the amplitude of responses to 10 µM
nicotine, DMPP, and cytisine and the relative potency of these agonists were similar to those observed in mutant mice (Picciotto et al., 1995)
(data not shown). Thus, the contribution of 2-containing nAChRs to
the nicotinic responses recorded in our preparations of the IPn and MHb
is minimal in wild-type mice. Nicotinic responses were blocked by 1 µM MCA, but not by 5 nM MLA in both MHb and IPn, whereas 1 µM DHE reduced the nicotinic responses
in the IPn, but not in the MHb, indicating a difference between the
nAChRs in the two structures (Fig. 10) (Mulle et al., 1991). nAChRs
recorded in 2 / MHb and IPn also differed with regard to the
desensitization time constant of the responses to high concentrations
of nicotinic agonists (mean ± SD for 100 µM
nicotine: 4.16 ± 1.13 sec, n = 4, in the IPn;
20.0 ± 7.34 sec, n = 5, in the MHb) (Fig.
11).
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Figure 11.
Desensitization of nicotinic responses in the IPn
and MHb of 2 / mice. The responses to 100 µM
nicotine in the medial habenula and in the interpeduncular nucleus have
been normalized. The decay of the responses was fit by a single
exponential function (dashed line). In the cells shown,
the desensitization time constant of the responses was 30.0 sec
(MHb) and 3.95 sec (IPn).
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DISCUSSION |
Contrary to what is assumed normally, nicotinic agonists bind with
high affinity to different nAChRs in equilibrium binding experiments,
including some nAChRs that do not contain 2. Instead, muscarinic
receptors remained unchanged in the brain of mutant mice, confirming
that the mutation of a nicotinic subunit specifically affects the
nicotinic cholinergic system. The findings in this study, together with
previous experiments on the anatomy and electrophysiology of nAChRs,
allow us to propose an extension of the classification of brain nAChRs
(Alkondon and Albuquerque, 1993) (Table
2).
Different spectra of high-affinity binding and electrophysiological
responses of neuronal nicotinic ligands for nAChR isotypes
At least four groups of nAChR isotypes can be identified on the
basis of the present data.
Type 1 nAChRs are -bungarotoxin-sensitive and have low
affinity for neuronal nicotinic agonists in equilibrium binding
experiments. Their distribution does not change significantly in 2
/ mice, whereas their disappearance in 7 mutant mice
(Orr-Utreger et al., 1997) confirms that they contain the 7 subunit
and that the 2 subunit is not a component.
Type 2 nAChRs contain the 2 subunit and represent the
vast majority of nAChRs in the mouse brain. The order of agonist
potency for this type of nAChR is consistent with in vitro
expression of 4/2 (Luetje and Patrick, 1991). Cytisine is a poor
agonist for 2-containing nAChRs in the dopaminergic system
(Picciotto et al., 1998) and in cultured hippocampal neurons (Alkondon
and Albuquerque, 1993), indicating that this is a characteristic
property of type 2 nAChRs. Type 2 nAChRs are present at high levels in the thalamus and the habenulo-interpeduncular system, especially the
IPn. All nicotinic ligands used in the present study are able to bind
this group of isotypes with nanomolar or subnanomolar (3H-epibatidine) KD in equilibrium
binding experiments. In contrast to the other ligands that were used,
high-affinity 3H-nicotine binding completely disappears in
2 / animals and can be considered a marker of this group of
nAChRs. By comparing the present results with previous localization
studies, it follows that the composition of the major isoform
constituting type 2 binding is 4/2.
Other subunits are likely to coassemble with 2, namely, 2 and
3, 5 as a third subunit (Vernallis et al., 1993; Wang et al.,
1996), and 4 as a fourth subunit (Conroy and Berg, 1995). An
2/2 isoform likely contributes to type 2 binding because 2,
more than 4, is concentrated in the IPn, which contains the highest
level of 3H-nicotine binding in the brain. It is difficult
to ascertain from our results if 3/2 contributes to type 2 binding in some areas of the mouse brain. The highest levels of 3
are distributed homogeneously in the ventral portion of the MHb (Wada
et al., 1989; Le Novère et al., 1996). The subnuclear
distribution of 3H-nicotine in the MHb (higher in the
lateral than in the medial part) suggests that, at least in this
nucleus, 3 does not contribute substantially to type 2 binding. 6
and 3 mRNAs are enriched in the dopaminergic neurons of the ventral
mesencephalon and the noradrenergic neurons of the locus coeruleus (Le
Novère et al., 1996). Electrophysiological experiments did not
reveal any response to nicotinic agonists in the dopaminergic neurons
of the ventral mesencephalon of 2 / mice (Picciotto et al.,
1998), suggesting that in this area the nAChRs, which putatively
contain 6 and 3, also contain the 2 subunit.
Type 3 nAChRs do not contain 2 and bind only
3H-epibatidine with high affinity in equilibrium binding
experiments. This isotype was clearly identified in the dorsocaudal
medulla oblongata (Sol, DMnX, and AP), in the pineal gland (M. Zoli,
unpublished data), and in the habenulo-interpeduncular system, where it
was transported from the MHb to the IPn. 3H-Epibatidine
binding did not decrease significantly in MHb (especially its medial
portion), fr, and ventral IPn in 2 mutant mice. The ratio of
3H-epibatidine to 3H-cytisine in MHb, fr, and
ventral (but not dorsal) IPn in wild-type mice was higher than in other
brain areas, except the dorsocaudal medulla oblongata, implying that
these sites might have higher affinity for epibatidine than type 2 nAChRs. In preliminary experiments both type 2 and type 3 binding
decreased in parallel when assayed with 0.02 and 0.002 nM
3H-epibatidine (M. Zoli unpublished data), suggesting that
their affinity for epibatidine was similar. Electrophysiological
experiments in the MHb and DMnX of mutant mice showed an agonist
potency consistent with 3/4 nAChRs expressed in oocytes (Luetje
and Patrick, 1991) (also see below) and a slow rate of desensitization.
Although in situ hybridization studies (Wada et al., 1989;
Hill et al., 1993), single channel recordings (Connolly et al., 1995),
and binding studies indicate the presence of multiple subtypes of nAChRs in the MHb, present and previous (Picciotto et al., 1995) results point to the existence of a predominant subtype (a type 3 nAChR) in the MHb of both 2 +/+ and / mice.
Type 3 binding corresponds to the distribution of 3/4 subunits
that are coexpressed at high levels in MHb (Wada et al., 1989; Hill et
al., 1993; Zoli et al., 1995), dorsocaudal medulla oblongata, and
pineal gland (Zoli et al., 1995). On the basis of the expression
pattern of 5, this subunit either is not a component of this
receptor or does not change its binding characteristics.
Type 4 nAChRs do not contain 2 and bind
3H-epibatidine and 3H-cytisine with high
affinity in equilibrium binding experiments. The binding of other
nicotinic ligands is very limited (3H-methylcarbamylcholine
and 3H-acetylcholine) or is absent
(3H-nicotine). This isotype or isotypes were found in the
DCIC, DTgm, and the habenulo-interpeduncular system, where they could be detected in the MHb (especially laterally), IPn (especially dorsally), and fr, implying that this isotype or isotypes are, at least
in part, transported from the MHb to the IPn. Electrophysiological experiments in the dorsal IPn showed an order of potency of agonists consistent with a 4-containing receptor (Luetje and Patrick, 1991).
Currents elicited by high concentrations of nicotinic agonists in the
dorsal IPn (type 4 nAChRs) exhibited a substantially faster desensitization than those in the MHb (type 3 nAChRs). The known distribution of nAChR subunits does not allow for the unequivocal identification of the subunit composition of this receptor or receptors. The putative composition of type 4 nAChRs in intrinsic neurons of the IPn may be 2 and/or 4 with 4. The heterogeneous distribution of 3H-cytisine binding in the MHb of 2
/ mice fits well with the distribution of 4 and 5 (Wada et
al., 1989; Le Novère et al., 1996). It therefore is possible that
receptors composed of 4/4 (with or without 5) and/or
3/5/4 account for type 4 binding in the MHb.
Differences between affinity in equilibrium binding and in
electrophysiological experiments
The equilibrium binding constants for cytisine, nicotine, and
epibatidine in binding experiments are below 10 nM, 10 nM, and 500 pM, respectively (Romano and
Goldstein, 1980; Pabreza et al., 1991; Houghtling et al., 1995), but at
these concentrations none of these agonists activates a significant
current in our study. The active state is detected readily in
electrophysiological experiments, whereas the desensitized states have
higher affinities for the agonists, are more stable in the presence of
agonist, and therefore are detected in equilibrium binding experiments.
There is also a lack of correspondence between the order of potency of
agonists in electrophysiological experiments and their affinity in
equilibrium binding experiments. For instance, the type 2 nAChRs have a
very similar KD for nicotine and cytisine,
whereas the order of potency is nicotine cytisine in
electrophysiological experiments. Similar disparities can be seen for
types 3 and 4 nAChRs. This may be attributable to the fact that the
desensitized states (equilibrium binding) and the active states
(electrophysiological experiments) do not share the same affinity for
these ligands, as has been demonstrated already in the case of the
muscle nAChR (Changeux, 1990). Receptor autoradiography only recognizes
high-affinity binding at equilibrium so that some nAChRs isoforms may
not have been detected in binding experiments. In addition,
electrophysiological data are pooled and averaged for many cells so
that the dose-response curves presented account only for the behavior
of the main subtype present in the cells. Minor populations of nAChRs
thus may have been missed by both techniques.
Conclusions
These experiments demonstrate that there are at least four classes
of nAChRs in the brain, which can be distinguished on the basis of
their distribution, high-affinity binding characteristics, and response
to nicotinic agonists in electrophysiological experiments; the
classification provides a tentative identification of the subunit
composition of these subtypes. Future experiments that use knock-out
animals lacking individual subunits should allow for a further
dissection of the subunit composition of these receptors.
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FOOTNOTES |
Received Nov. 13, 1997; revised March 30, 1998; accepted March 30, 1998.
This work was supported by the Collège de France, the Centre
National pour la Recherche Scientifique, the Association Francaise contre la Myopathie, the Council for Tobacco Research, Biomed and
Biotech contracts from the Commission of the European Communities, and
a grant from the Human Frontier Program. C.L. was supported by a Roux
grant from the Institut Pasteur; M.R.P. was supported by grants from
the Donaghue Foundation, National Alliance for Research on
Schizophrenia and Depression, and Grant DA10455 from National
Institutes of Health. We thank Dr. Nicolas Le Novère for his
careful reading of this manuscript and for useful discussions.
Correspondence should be addressed to Dr. Jean-Pierre Changeux,
Neurobiologie Moléculaire, Institut Pasteur, 28 Rue du Dr. Roux,
75724 Paris Cédex 15, France.
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Top
Abstract
Introduction
