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The Journal of Neuroscience, April 15, 2003, 23(8):3176
Differential Desensitization and Distribution of Nicotinic
Acetylcholine Receptor Subtypes in Midbrain Dopamine Areas
Julian R. A.
Wooltorton,
Volodymyr
I.
Pidoplichko,
Ron S.
Broide, and
John A.
Dani
Division of Neuroscience, Baylor College of Medicine, Houston,
Texas 77030-3498
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ABSTRACT |
Although many psychopharmacological factors contribute to nicotine
addiction, midbrain dopaminergic systems have received much attention
because of their roles in reinforcement and associative learning. It is
generally thought that the mesocorticolimbic dopaminergic system is
important for the acquisition of behaviors that are reinforced by the
salient drives of the environment or by the inappropriate stimuli of
addictive drugs. Nicotine, as obtained from tobacco, can activate
nicotinic acetylcholine receptors (nAChRs) and excite midbrain neurons
of the mesocorticolimbic system. Using midbrain slices from rats,
wild-type mice, and genetically engineered mice, we have found
differences in the nAChR currents from the ventral tegmental area (VTA)
and the substantia nigra compacta (SNc). Nicotinic AChRs containing the
7 subunit (7* nAChRs) have a low expression density.
Electrophysiological analysis of nAChR currents, autoradiography of
[125I]--bungarotoxin binding, and in
situ hybridization revealed that 7* nAChRs are more highly
expressed in the VTA than the SNc. In contrast, 
2* nAChRs are move
evenly distributed at a higher density in both the VTA and SNc. At the
concentration of nicotine obtained by tobacco smokers, the slow
components of current (mainly mediated by 2* nAChRs) become
essentially desensitized. However, the minority 7* component
of the current in the VTA/SNc is not significantly desensitized by
nicotine in the range 
100 nM. These results suggest that
nicotine, as obtained from tobacco, can have multiple effects on the
midbrain areas by differentially influencing dopamine neurons of the
VTA and SNc and differentially desensitizing 7* and non-7 nAChRs.
Key words:
ventral tegmental area; substantia nigra; nicotine
addiction; mesolimbic; 7; 2
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Introduction |
The mesotelencephalic dopaminergic
system is heterogeneous and plays many roles (Gardner and Ashby, 2000
).
Although there is significant overlap, mesostriatal dopamine (DA)
neurons originate mainly in the substantia nigra compacta (SNc), and
mesocorticolimbic DA neurons originate mainly in the ventral tegmental
area (VTA). The dopaminergic systems participate during addiction to
amphetamine, cocaine, and nicotine (Clarke, 1990, 1991; Stolerman and
Shoaib, 1991; Corrigall et al., 1992; Di Chiara and North, 1992;
Nestler, 1992, 1993, 1994; Pontieri et al., 1996; Pich et al., 1997; Di Chiara, 1999, 2000; Balfour et al., 2000; Berke and Hyman, 2000; Dani
and De Biasi, 2001; Dani et al., 2001; Hyman and Malenka, 2001).
A role for the midbrain DA system in nicotine addiction is supported by
a number of findings (Di Chiara, 2000). Nicotine can support
self-administration, and DA antagonists or lesions of DA neurons or of
the nucleus accumbens (NAc) reduce self-administration (Corrigall and
Coen, 1989; Corrigall et al., 1992, 1994; Corrigall, 1999). By acting
at nicotinic acetylcholine receptors (nAChRs), nicotine can activate
VTA and SNc neurons (Clarke et al., 1985; Grenhoff et al., 1986;
Calabresi et al., 1989; Pidoplichko et al., 1997; Picciotto et al.,
1998) and cause release of DA in the NAc of rats (Clarke, 1991; Nisell
et al., 1994, 1995; Pontieri et al., 1996). Furthermore,
presynaptically located nAChRs can potently regulate DA release in the
striatum, including the NAc (Wonnacott et al., 2000; Jones et al.,
2001; Zhou et al., 2001).
Neuronal nAChRs are formed from five subunits, and consequently many
compositionally and functionally different nAChRs are possible (McGehee
and Role, 1995; Role and Berg, 1996; Wonnacott, 1997; Jones et al.,
1999). Five subunits (2-6) and three subunits
(2-4) can assemble to produce a large number of
hetero-oligomeric nAChRs that are distinct but share some functional
and pharmacological properties. Most neuronal nAChRs containing the
7 subunit are functionally similar to homo-oligomeric 7 receptors
studied in exogenous expression systems, but 7 also may form
hetero-oligomeric nAChRs (Yu and Role, 1998). The 7 nAChRs have
rapid activation and desensitization kinetics and are specifically
inhibited by -bungarotoxin (-BTX) and methyllycaconitine (MLA)
(Alkondon et al., 1992; Castro and Albuquerque, 1995; Gray et al.,
1996).
The predominant nAChR-mediated currents from VTA and SNc neurons have
relatively slow kinetics and are inhibited by the nonspecific inhibitor
mecamylamine (Pidoplichko et al., 1997; Picciotto et al., 1998; Klink
et al., 2001). Most of these currents are mediated by 2* nAChRs
because there is a dramatic decrease in these currents when examined
from 2-null mice (Picciotto et al., 1998). However, many other
nicotinic subunits are expressed in these areas, particularly 4,
6, and 3 (Wada et al., 1989, 1990; Le Novère et al., 1996; Goldner et al., 1997; Charpantier et al., 1998; Klink et al., 2001).
The 7 subunit also is present, but the rapid kinetics of the 7*
nAChRs make them very difficult to detect.
The purpose of this study was to examine the contribution of 7*
nAChRs in the SNc and the VTA because their high calcium permeability
enables them to serve important roles (Séguéla et al.,
1993; Castro and Albuquerque, 1995). When comparing ACh-induced currents, differences in the density of 7* nAChRs in the SNc and VTA
were found. Detection of those 7* currents was facilitated by
utilization of heterozygous (+/T), gain-of-function 7L250T mutant
mice (Orr-Urtreger et al., 2000). Furthermore, from the study of both
wild-type and 2-null (
/) mice, it was observed that the
concentrations of nicotine that are obtained by smokers desensitized
the slower components of nicotinic current but had much less effect on
the fast 7* component.
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Materials and Methods |
Midbrain slices and electrophysiology. Midbrain
slices containing the VTA and SNc were prepared from 15- to 24-d-old
Sprague Dawley rats, wild-type C57BL/6J mice, or mutant
mice that were anesthetized before decapitation (Xu et al., 1999;
Orr-Urtreger et al., 2000). Slices (300-350 µm thick, rats; 200-250
µm, mice) were prepared following previously published procedures
(Pidoplichko et al., 1997; Zhou et al., 2001). The cutting solution was
either of the following and usually a 50/50% mixture of the two
solutions (in mM): 230 sucrose, 1 KCl, 1.25 NaH2PO4, 30 NaHCO3, 1 CaCl2, 7 MgCl2, 25 D-glucose;
and 144 NMDG, 1.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 2 CaCl2, 2 MgCl2, 25 D-glucose, 30 NaHCO3. The slices were then transferred to a
holding chamber containing the bath solution (in
mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 21 NaHCO3, 2.5 CaCl2, 1 MgCl2, 25 D-glucose. The
experimental chamber (0.5 ml capacity) had a continuously flowing bath
solution (~5 ml/min) at 32-34°C. The external solutions contained
atropine (0.25-1 µM) to block muscarinic ACh
receptors, and in some experiments 0.5 µM
tetrodotoxin was present to inhibit action potentials. Nicotine and the
various nAChR antagonists were applied via the continuously flowing
bath solution. Patch electrodes had resistances of 3-5 M
when
filled with the internal solution (in mM): 60 CsCH3SO3, 60 KCH3SO3, 10 KCl, 10 EGTA,
10 HEPES, 5 Mg-ATP, 0.3 Na3GTP, pH 7.2.
Neurons that were patch clamped were identified as dopaminergic on the
basis of their appearance and the presence of
Ih current (Grace and Onn 1989; Lacey
et al., 1989; Hausser et al., 1995; Mercuri et al., 1995; Pidoplichko
et al., 1997; Bonci and Malenka, 1999). The presence of
Ih was detected by applying
hyperpolarizing steps from the holding potential (see Fig.
1A, top left panel). Non-dopaminergic neurons in the
VTA/SNc are usually GABAergic, and they do not display
Ih (see Fig. 1B, top
left panel).
Nicotinic currents were activated by pressure applying ACh using a
Picospritzer (General Valve, Parker Hannifin
Corporation, Fairfield, NJ) attached to a narrow "puffer"
pipette that was pulled like a patch pipette. The puffer pipette was
placed ~30 µm from the neuron while ACh was applied (1 mM; 28 psi; 30-200 msec duration unless stated otherwise).
Then, the puffer was reversibly moved ~100 µm away between pressure
applications (usually 30-60 sec) by a computer-controlled motorized
manipulator to prevent desensitization caused by agonist leakage.
Consistently reproducible 7* nAChR currents were measured only by
using the above precautions. It should be noted that unless these
precautions are taken, especially the fast component of the current
(mainly 7*) is underestimated or lost in the larger slower
components of the nAChR current (mainly 2*). In mouse slices,
currents are more likely to be underestimated because the neurons are
smaller and the tissue is denser and less amenable to pressure
application of agonist.
Tissue preparation. Tissue for histology was prepared from
Sprague Dawley rats on postnatal day (P) 16-21
(n = 12) and from C57BL/6J mice aged P60
(n = 6). The rats were used at an age to match the
electrophysiology, but because of their smaller size, mice were used at
an optimal young age for the histology. Fixed brain tissue for
immunohistochemistry was prepared by perfusion with 4%
paraformaldehyde in PBS, pH 7.4. Brains were equilibrated in
30% sucrose, frozen, and sectioned (20 µm) on a sliding microtome. Unfixed brains were frozen in prechilled isopentane, sectioned on a
cryostat (20 µm), and mounted onto either gelatin-coated slides for
receptor binding or slides with an additional coating of
poly-L-lysine for in situ
hybridization. Slide-mounted sections for in situ
hybridization were postfixed with 4% paraformaldehyde in 0.1 M PBS for 1 hr at 22°C, washed, and air dried.
All sections were stored desiccated at 20°C until use. Histological
counterstaining was done with cresyl violet acetate.
Immunohistochemistry. Brain sections for
immunohistochemistry were preincubated in PBS containing 0.4% Triton
X-100, 3% goat serum, and 3% bovine serum albumin for 1 hr at 22°C.
Sections were then incubated overnight at 4°C with an
affinity-purified sheep antibody against tyrosine hydroxylase (TH)
(Chemicon, Temecula, CA) diluted (1:100) in the same
buffer. After several washes, tissue sections were incubated in
biotinylated rabbit anti-sheep antibody (1:200) for 2 hr at 22°C.
Staining for TH was then visualized using the avidin-biotin
immunoperoxidase method (Vectastain, Burlingame, CA) with
3-3'-diaminobenzadine tetrahydrochloride (Sigma, St. Louis, MO) as the chromagen.
Receptor binding. Slide-mounted brain sections for
[125I]--BTX (specific activity = 10-20 µCi/µg; PerkinElmer Life Sciences, Boston, MA)
and [125I]-epibatidine (specific
activity = 7383 µCi/µg; PerkinElmer Life Sciences) binding were processed as described previously (Broide et al., 2002). After the procedure, sections were placed against -max film (Amersham Biosciences, Newark, NJ) for either
3-7 d ([125I]--BTX) or 3-12 hr
([125I]-epibatidine).
Autoradiographic images of brain sections showing
[125I]--BTX and
[125I]-epibatidine binding site
expressions were analyzed, and quantification was performed using
computer-assisted densitometry (NIH Image program, developed at the
National Institutes of Health, Bethesda, MD). The VTA, SNc, and
substantia nigra reticulata (SNr) were identified on corresponding
Nissl-stained sections and using rat and mouse brain atlases (Paxinos
and Watson, 1996; Paxinos and Franklin, 2000). Specific binding was
obtained by subtracting the nonspecific values from total binding
values and is presented as mean gray levels. Binding levels in the
nearby hippocampus and interpeduncular nucleus were obtained as
positive controls. These values were always at least 200% higher than
VTA values. Data was statistically analyzed by a two-tailed Student's
t test.
In situ hybridization. Mouse DNA templates encoding the
third intracellular loop of the 7 (279 bp) and 2 (438 bp) nAChR subunits (Broide et al., 2002) were used to synthesize cRNA riboprobes labeled with [35S]-UTP (DuPont
NEN, Boston, MA). Postfixed brain sections for in
situ hybridization were processed as described previously (Broide et al., 1996). After the procedure, brain sections were placed against
-max film for 1-7 d at 4°C.
Chemicals. Salts, acetylcholine chloride (ACh), ATP
(magnesium salt), GTP (sodium salt), mecamylamine (MEC, hydrochloride), N-methyl-D-glucamine (NMDG), BAPTA,
dihydro--erythroidine (DHE), CNQX, AP-5, bicuculline, and
picrotoxin were obtained from Sigma, and MLA (citrate
salt) was obtained from Research Biochemicals International (Natick, MA).
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Results |
Nicotinic AChR currents from neurons of the VTA and SNc
DA neurons were identified by the presence of
Ih currents (Fig.
1A, Ih, DA) (Hausser et
al., 1995; Mercuri et al., 1995; Pidoplichko et al., 1997; Bonci and
Malenka, 1999). Nicotinic currents were activated by pressure puffs of
1 mM ACh, and some neurons expressed more than
one kinetic component (Fig. 1A). The amplitudes of
the ACh-induced currents are given in Table
1, and a component had to be >15 pA to
be accepted and analyzed. A minority of DA neurons from the VTA
displayed nAChR currents with both fast and slow components: 10 of 33 neurons from rats and 11 of 47 neurons from mice. The fast component of
this current was inhibited by MLA (5 nM). Both
the kinetics and pharmacology indicate that the fast component was
mediated by 7* nAChRs. The fast component of the current was seen
only once without the accompanying slower component: 0 of 33 in rat and
1 of 47 in mice. The slower component was usually present in
measurements from VTA DA neurons: 33 of 33 from rats and 42 of 47 from
mice. With rare exceptions, the slow component of the current was
unaffected by 20 nM MLA, but it was rapidly and
completely inhibited by MEC (1-10 µM) (Fig.
1A). This concentration of MEC is rather
nonselective, inhibiting most types of hetero-oligomeric nAChRs. MEC
(especially above 5 µM) applied for longer
times also inhibited the fast currents mediated by 7* nAChRs (data
not shown). In 4 of 47 DA neurons from mice, no nAChR currents were
detected. This result does not guarantee that nAChRs are not present in those neurons; rather, they simply did not mediate significant current
at the position of the ACh puffer pipette.
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Figure 1.
ACh-evoked currents from VTA neurons.
A, The Ih currents indicate a
DA neuron. Puffs of ACh (40 msec; 1 mM ACh) induced
currents with two components in the top row. The fast component is
inhibited by the 7-specific inhibitor MLA (5 nM). The
slow current in the bottom row is not inhibited by MLA, but it is
inhibited by 5 µM MEC. B, The absence of
Ih indicates a non-DA neuron. The
ACh-induced current (100 msec puff of 1 mM ACh) shows two
components in the top row. The fast component is inhibited by 5 nM MLA. The slow current in the bottom row is not inhibited
by MLA, but it is inhibited by 5 µM MEC. The ACh puffs
were applied once every 60 sec. Each trace is an average of three
currents. Calibration: 50 pA, 0.5 sec for the ACh-induced currents; 200 pA, 0.5 sec for the Ih protocols.
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Non-dopaminergic neurons in the VTA (usually considered GABAergic) were
identified by the absence of Ih (Fig.
1B, non-DA). Those neurons displayed nicotinic
currents similar to those of DA neurons (Fig. 1B).
Only a minority displayed both the fast (MLA-sensitive) and the slow
(MEC-sensitive) components of current: 9 of 29 from rats and 8 of 31 from mice. The fast component without the accompanying slower component
was seen in 0 of 29 rat neurons and in 1 of 31 mouse neurons. In
contrast, the slower component was usually present in measurements from
VTA non-DA neurons: 22 of 29 from rats and 27 of 31 from mice. As in DA
neurons, this slow component was completely inhibited by 5 µM MEC (Fig. 1B). In 7 of 29 non-DA neurons from rats and in 3 of 31 non-DA neurons from mice, no
nAChR currents were detected.
All of the neurons that we studied from the SNc possessed an
Ih current, indicating that they were
dopaminergic neurons. In the SNc the fast component of nAChR current
was more rare, and it was detected significantly in only 1 of 23 SNc
neurons from rat and 3 of 37 from mice. In contrast, the slow component
of the current was seen in 22 of 23 rat SNc neurons and in 31 of 37 mouse SNc neurons. No nicotinic currents were observed in 1 of 23 SNc
DA neurons from rats and in 6 of 37 SNc DA neurons from mice.
Usually the ACh pressure applications that we used were 30-200 msec in
duration. Those ACh puffs revealed two pharmacologically distinct
components of the current: a fast component inhibited by 1 nM MLA and a slow component inhibited by 1 µM
DHE. An additional observation was that longer puffs of ACh (2000 msec) revealed an even slower component of the current (data not
shown). That current was inhibited by 1 µM MEC,
indicating that it was arising from another nAChR type, or types, that
was not sensitive to 1 nM MLA or 1 µM DHE.
Thus, the slower components of current are composed of multiple nAChR
types, some of which are sensitive to DHE and others to MEC.
Mice lacking the 2 subunit have predominantly
MLA-sensitive currents
Because the slow component of the current could obscure the
smaller fast component, we examined 2-null mice to eliminate the
vast majority of the slow current. In these experiments, we took
special care to avoid secondary conductances that were indirectly activated by 7 nAChRs. Intracellular
Ca2+ was strongly buffered by BAPTA (20 mM) to prevent Ca2+-dependent
conductances that may have been initiated by 7 activity. GABA and
glutamate channels were inhibited by bicuculline (20 µM),
CNQX (20 µM), and AP-5 (100 µM) to prevent
synaptic currents potentially induced by presynaptic 7 nAChR
activity. Fast small currents (notches in the current trace) were
detected more easily in the 2-null mice. For that reason a higher
percentage of neurons were positive for fast currents, but the
conclusions about the putative 7-mediated currents were similar to
those obtained using the wild-type mice (Table
2). The MLA-sensitive currents were more
common in the VTA than in the SNc neurons. On a few occasions (Table
2), a slow current that was not inhibited by MLA (5 nM) or
DHE (2 µM) was observed. Although other explanations
are possible, this minority current could involve 3* nAChRs (Le
Novère et al., 1996).
Mice with the gain-of-function 7L250T mutation have larger
MLA-sensitive currents
Often the fast, putative 7* component of the ACh-induced
currents was small. Because some 7* currents were below our level of
acceptance in wild-type mice (15 pA), we were underestimating the
percentage of neurons that contained the 7 subunit. To obtain a
better estimate, we took advantage of heterozygote mutant mice (+/T)
having one copy of the 7 subunit with a leucine to threonine mutation (7L250T) (Orr-Urtreger et al., 2000; Ji et al., 2001). In
these mutant mice the 7 subunit is thought to be expressed at lower
concentrations than in wild-type mice (Orr-Urtreger et al., 2000), but
this L250T mutation causes the 7* currents to appear larger and
slower (Fig. 2) (Revah et al., 1991;
Bertrand et al., 1992; Ji et al., 2001). Thus, the 7* nAChRs
currents are much easier to detect. In the +/T mice, larger
MLA-sensitive currents were seen in all of the VTA DA neurons
(n = 10 of 10) (Fig. 2B,F) and
in the majority of SNc DA neurons (n = 14 of 17) (Fig.
2D,F). In both mutant
7L250T mice (+/T) and wild-type mice (+/+), the putative 7*
currents from SNc neurons were significantly smaller
(p < 0.05) than those from VTA DA neurons (Fig.
2E, Table 3).
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Figure 2.
Mice expressing mutant 7L250T subunits display
much larger MLA-sensitive currents. Pressure applications of 1 mM ACh (arrows) elicited currents from a wild-type VTA DA
neuron (A), from a mutant +/T VTA DA neuron
(B), from a wild-type SNc DA neuron
(C), and from a mutant +/T SNc DA neuron
(D). In each case, (a) denotes the
control current and (b) denotes the current recorded in
the presence of MLA. The (a) (b)
subtraction shows the net MLA-sensitive current. As expected, the
7L250T mutation produces larger and slower ACh-induced currents. The
ACh puffs were 30-60 msec in duration for A,
B, and D, but 200 msec in
C. E, The bar graphs compare the
magnitude of the MLA-sensitive currents in the VTA or SNc and from
wild-type or +/T mice. F, The bar graphs compare
the proportion of the sampled neurons with MLA-sensitive current in the
VTA or SNc and from wild-type or +/T mice. Calibration: 200 pA
(A, B), 50 pA (C, D), 0.5 sec.
*p < 0.05 significantly different from corresponding VTA
values by Student's t test.
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To further verify that this MLA-sensitive component of the current was
arising from 7* type nAChRs, we activated the nAChRs with choline
(Table 3), a relatively specific 7* agonist (Papke et al., 1996;
Alkondon et al., 1997; Zwart and Vijverberg, 2000). Pressure
applications of choline (10 mM, 40 msec) induced
reproducible, MLA-sensitive currents in +/T neurons in 6 of 6 VTA DA
neurons (Fig. 3A), in 8 of 9 VTA non-DA neurons (Fig. 3B), and in 17 of 18 SNc DA neurons
(Fig. 3C). The choline-induced 7* currents were
significantly larger (p < 0.001) in the VTA
than in the SNc (Fig. 3D, Table 3). Especially in these
experiments, the puffer pipette was repeatedly positioned for a number
of trials to find an area of the neuron (usually dendrites) that
responded well to the choline puff.
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Figure 3.
Choline-activated currents from +/T mice
(7L250T) are larger, slower, and inhibited by MLA. A,
Typical currents obtained from a VTA DA neuron. At the arrow, 10 mM choline is pressure puffed for 40 msec (once every 2 min). The current was inhibited by 5 nM MLA.
B, Similar MLA-sensitive currents were observed in VTA
non-DA neurons. C, Typical choline-activated,
MLA-inhibited currents are shown from an SNc DA neuron. Three traces
were averaged for each representative current. Calibration: 100 pA, 0.5 sec. D, Bar graphs representing average amplitudes of
choline-evoked currents recorded from VTA DA, VTA non-DA, and SNc DA
neurons of +/T mice. **p < 0.001 significantly different
from VTA values by Student's t test.
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An additional observation in these heterozygous 7L250T (+/T) mice
was that 5 nM MLA added to the bath decreased the holding current necessary to voltage clamp the cells (n = 9;
data not shown). This result is expected because a proportion of the
homomeric 7 nAChRs expressed in the neurons are composed of all
7L250T subunits, which have been reported to be open in the absence
of applied agonist (Revah et al., 1991; Bertrand et al., 1992).
Anatomical confirmation of greater 7 expression in the VTA than
in the SNc
The ACh- and choline-induced currents indicated that the putative
7* component was larger in the VTA than in the SNc. We wanted an
independent method to verify the conclusions drawn from the current
measurements for the following reasons. 7* currents are difficult to
measure and are sometimes not seen (Picciotto et al., 1998), and a
significant component of non-7, MLA-sensitive current has been
reported in the VTA/SNc (Klink et al., 2001). The 7 subunit
participates in (most if not all) the high-affinity binding sites for
-BTX in the brain (Orr-Urtreger et al., 1997; Whiteaker et al.,
2000). Therefore, we used
[125I]-BTX binding in rat and mouse
midbrain sections to semiquantitatively indicate the presence of the
7 subunit.
In the rat, [125I]--BTX binding was
detected in the VTA and the SNc (Fig. 4).
The [125I]--BTX binding was specific
to the 7* nAChRs because it was displaced by the competitive
antagonist, 10 µM -cobratoxin (Cbt) (Fig.
4A,B). The location of the -BTX binding sites was
identified as the VTA/SN by immunohistochemically labeling sections
from the same brain region for tyrosine hydroxylase, an enzyme required for dopamine synthesis (Fig. 4D). Mean levels of
[125I]--BTX binding site density in
the VTA, SNc, and SNr were determined from autoradiographic images of
brain sections (Fig. 4E). Levels of
[125I]--BTX binding were
significantly higher in the VTA than in the SNc, despite the fact that
the density of DA neurons is higher in the SNc (Fig. 4, compare
A, C, E). A caveat in these measures is that the -BTX binding sites could be located on afferent synaptic terminals rather than on the cell bodies. The rats used for these anatomical studies were about the same age (P16-21) as the rats used
for electrophysiology (P15-24). By using the same age, changes in 7
expression during development could be avoided in our comparison of the
anatomical and electrophysiological results.
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Figure 4.
Distribution of -BTX binding sites in the
midbrain dopaminergic region of the rat. A, An
autoradiographic image of a coronal section through the rat midbrain
dopaminergic regions shows the [125I]--BTX
binding-site distribution. The arrow indicates the VTA, and the
arrowhead indicates the SNc. B, An autoradiographic
image of [125I]--BTX binding in an adjacent
coronal section through the same region after specifically blocking the
-BTX sites with -Cbt, indicating nonspecific binding (NS).
C, Higher magnification of the VTA/SNc area shown in
A. D, Staining for tyrosine hydroxylase
(TH) delineates the VTA, SNc, and SNr in the midbrain dopaminergic
region. E, The bar graph depicts the density of
[125I]--BTX binding-site expression in the VTA,
SNc, and SNr. The values represent specific binding in gray levels and
are means ± SEM from seven rat brains. **p < 0.001 significantly different from VTA values by Student's
t test. Hi, Hippocampus; MG, medial geniculate; SC,
superior colliculus; SNc, substantia nigra compacta; SNr, substantia
nigra reticulata; VTA, ventral tegmental area. Scale bars:
A, B, 1000 µm; C,
D, 500 µm.
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Mouse midbrain sections likewise exhibited a significantly higher level
of [125I]--BTX binding in the VTA
than in the SNc (Fig. 5A,E).
To confirm those results and to identify the midbrain DA regions
expressing 7 subunit mRNA, in situ hybridization was
performed on adjacent brain sections (Fig. 5B). Levels of
7 mRNA expression were higher in the VTA than in the SNc,
corresponding to the pattern of
[125I]--BTX binding. The mice used
for these anatomical studies were older (P60) than the mice used for
electrophysiology (P15-24). Potential changes in 7 expression
during development were not examined. Despite the difference in age,
however, both sets of experiments suggest that 7* nAChRs are more
commonly expressed in the mouse VTA than in the SNc.
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Figure 5.
Distributions of 7 and non-7 nAChRs in the
midbrain dopaminergic region of the mouse. Autoradiographic images of
alternate coronal sections through the midbrain dopaminergic region
showing the distributions of [125I]--BTX
binding sites (A), 7 nAChR subunit mRNA
(B), [125I]-epibatidine
binding sites (C), and 2 nAChR subunit mRNA
(D). The arrow indicates the VTA, and the
arrowhead indicates the SNc. E, The bar graph depicts
the density of [125I]--BTX and
[125I]-epibatidine (Epi) binding site expression
in the VTA, SNc, and SNr. The values represent specific binding in gray
levels and are means ± SEM from three mouse brains.
*p < 0.01 significantly different from VTA values
by Student's t test. Hi, Hippocampus; IPn,
interpenduncular nucleus. Scale bar, 500 µm.
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To determine whether this distribution pattern was the same for all
nAChR types, we characterized the distribution of
[125I]-epibatidine binding and 2
nAChR subunit mRNA. Epibatidine binds with various affinities to a
number of non-7 nAChR subtypes, most of which contain 2
(Whiteaker et al., 2000). Both the VTA and SNc displayed comparable and
high levels of [125I]-epibatidine
binding (Fig. 5C,E). In agreement with that result, the
pattern of 2 mRNA expression was similar in the VTA and SNc (Fig.
5D).
Nicotine at the concentrations obtained from tobacco differentially
desensitizes the nAChR subtypes
It has been shown previously that the higher concentrations of
nicotine achieved by smokers (i.e., 100-500 nM) can
desensitize nAChR currents from VTA DA neurons (Pidoplichko et al.,
1997). We extended the examination of desensitization to lower nicotine concentrations (20-80 nM) that are present in smokers for
longer times. Bath application of 80 nM nicotine strongly
desensitized the slow component (mainly 2* nAChRs) of the
ACh-induced current: 82 ± 4%, n = 7 for VTA DA
neurons (Fig. 6A);
80 ± 7%, n = 10 for VTA non-DA neurons (Fig.
6B). The time course of desensitization in the slices
is shown in Figure 6C. Bath application of 20 nM nicotine also was sufficient to cause
substantial desensitization of the slow component of ACh-induced
current: 45 ± 4%, n = 6 from VTA DA neurons
(Fig. 7A). However, 20 nM nicotine did not desensitize the fast
component of the current during a 20 min exposure (n = 3) (Fig. 7B,C). In most cases, the fast and slow components of the current were not easily separable. Measurement of the fast component was often contaminated by the rising phase of the slow component, particularly when the slow component was the predominant current (Fig. 7C).
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Figure 6.
A 20 min exposure to low concentrations of
nicotine desensitizes the slow component of current from VTA DA and
non-DA wild-type neurons. Responses to pressure applications of ACh
(arrows) are shown for a VTA DA neuron (A) and
for a non-DA neuron (B) in the absence
(a) and presence (b) of 80 nM nicotine (Nic). The (a) (b) subtraction shows the net nicotine-desensitized
current. C, The average time course of desensitization
is shown in response to 80 nM nicotine for the slow
component of current from VTA DA neurons ( ; n = 7) and non-DA neurons ( ; n = 10). Calibration:
25 pA, 1 sec.
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Figure 7.
Exposure to low concentrations of nicotine
differentially desensitize fast and slow nicotinic currents from mouse
VTA DA neurons. A, ACh-induced currents (arrow) in the
absence (Control) and presence (Nic) of 20 nM nicotine. The
time course of the nicotine-induced desensitization for this neuron is
shown on the right. B, Nicotine (20 nM)
desensitizes the slow component of current, but the fast component is
not significantly desensitized. The time course of the nicotine-induced
desensitization for this neuron is shown separately for the slow ()
and fast () components of current. C, In this common
example, the fast component of the ACh-induced current is difficult to
separate from the larger slow component of current in the absence of
nicotine (Control). After exposure to 80 nM nicotine (Nic),
the small, fast component is easier to see. The fast component is
inhibited by MLA (Nic + MLA). Calibration: 50 pA, 1 sec.
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To characterize desensitization of the fast component (putative 7*),
we used mutant mice lacking the 2 nAChR subunit. In the absence of
the 2 subunit, the ACh-induced currents were mainly of the fast,
7* variety. The fast component of the current was not significantly
desensitized by bath-applied nicotine in the range experienced by
smokers (80-500 nM; 20 min; n = 4) (Fig. 8). The ACh-induced currents from 2
/ neurons were inhibited by 5 nM MLA (Fig.
8D) (n = 8 of 8) but were not
inhibited by 1 µM DHE, as expected, in one
trial (Fig. 8E). These results further support the
possibility that the fast component of the current that we studied was
mediated by 7* nAChRs.
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Figure 8.
Exposure to low concentrations of nicotine does
not desensitize the fast, MLA-sensitive currents from VTA DA neurons
from 2-null mice. ACh-induced currents (1 mM ACh, 200 msec puff; arrows) are shown from the same neuron. Control currents are
shown in the left panels, and currents obtained in the presence of
nicotine are shown on the right at the concentrations given in
A-C. D, The ACh-induced currents were
inhibited by 5 nM MLA. E, The ACh-induced
currents were not inhibited by 1 µM DHE. Calibration:
25 pA, 0.5 sec.
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Discussion |
Although the 7* nAChRs are expressed at a low density, they are
commonly present in neurons of the midbrain DA areas. Evidence indicates that the VTA/SNc contains 3-7 and 2-4, and 4,
6, and 2 are common participants in nAChRs from this region (Le Novère et al., 1996; Picciotto et al., 1998; Arroyo-Jimenez et al., 1999; Klink et al., 2001; Azam et al., 2002; Champtiaux et al.,
2002). Our evidence indicates that the fast current mediated by the
putative 7* nAChR type is found at low levels in many of the VTA/SNc
neurons. This component of the current is most likely mediated by 7*
nAChRs for the following reasons. (1) When activated by high agonist
concentrations, the current has rapid kinetics. (2) The current is
greatly enhanced by the 7L250T mutation. (3) The current is
activated by the relatively specific agonist, choline. (4) The
relatively specific inhibitor, MLA, inhibits the current. (5) The
current amplitudes are consistent with the distribution of the
[125I]-BTX binding sites. (6) The
current is present in 2-null mice. When current was measured from
wild-type mice, the putative 7* component was difficult to detect,
and we detected this component only 23% (Table 1) of the time from
mouse VTA DA neurons and 8% from mouse SNc DA neurons. However, when
taking advantage of the 2-null mice, we detected this current more
often (Table 2), and in 7L250T gain-of-function mice, it was
detected in the vast majority of the VTA/SNc neurons (Table 3). Thus,
the low copy number for the 7 subunit could be overcome in the
7L250T mice because we could detect the opening of only a few
7L250T* channels. Without the advantages of the 7L250T mutation
and experimental care to have rapid agonist applications that also
avoid desensitization (as described in Materials and Methods), the
presence of fast, putative 7* currents is underestimated in the
VTA/SNc.
There are at least three caveats that limit the weight that should be
placed on the finding that an 7* nAChR is present at low levels in
the majority of VTA/SNc neurons, as estimated from choline-activated
currents from 7L250T mice. One is that there is an 4* nAChR type
in the VTA/SNc that also can be inhibited by MLA (Klink et al., 2001).
However, because that nAChR type cannot be activated by choline (Klink
et al., 2001), the results with choline-activated currents in 7L250T
mice still support the common presence of the 7 subunit. The next
potential problem is one of sampling. In other studies and this one,
only a couple dozen neurons were examined to arrive at the estimates.
By cutting slices slightly differently and choosing cells on the basis
of visual cues, a different sampling process could occur between laboratories. A final point to consider is that these estimates for
7 in this study and others depend strongly on nAChR mutant mice.
Although the general anatomical features are the same as in wild-type
mice, there could be subtle changes arising from circuit differences
during development, particularly when the 7 gene is altered (Broide
et al., 1996; Adams et al., 2001). It can be concluded with certainty,
nonetheless, that 7* nAChRs are a low density but significant
component in the VTA/SNc.
7 more common in VTA than SNc and 7* nAChRs not easily
desensitized by smoker's nicotine
The second interesting result from this study is that the 7*
type is more highly expressed in the rodent VTA than in the SNc. The
[125I]--BTX binding, the in
situ hybridization for 7 mRNA, and the current measurements all
are consistent in indicating that the 7 subunit is present at a
higher concentration in VTA neurons than in SNc neurons.
The third finding of interest is that the 7* nAChR type in the
VTA/SNc is much less susceptible to desensitization by the low
concentrations of nicotine achieved by smokers. This finding is the
most likely to have important implications. Cigarette smoking delivers
~50-300 nM nicotine throughout the brain on a time scale of many seconds to minutes (Russell, 1987; Benowitz et al., 1989; Henningfield et al., 1993; Gourlay and Benowitz, 1997). Significantly lower concentrations of nicotine will linger in the human brain for
hours. We showed that a 20 min exposure to 80 nM nicotine caused ~80% desensitization of the slower components of ACh-induced currents that are mediated predominantly by 2* nAChRs. This result is consistent with others who have estimated that 42(*) nAChRs have an IC50 for nicotine-induced desensitization
of ~1-60 nM (Lippiello et al., 1987; Wonnacott, 1987;
Peng et al., 1994; Rowell, 1995; Fenster et al., 1999; Quick and
Lester, 2002). However, up to 500 nM nicotine caused very
little desensitization of the VTA/SNc 7* nAChR currents. This result
may be surprising to some because high concentrations of agonist
desensitize 7* nAChRs more rapidly than other nAChR types (Alkondon
and Albuquerque, 1991; Bertrand et al., 1992; Dani et al., 2000; Papke
et al., 2000). Our results are consistent with others who have found
that 7* nAChRs from rodent hippocampus or expressed in oocytes are less easily desensitized by low nicotine concentrations. The
IC50 estimates for nicotine-induced
desensitization of 7* nAChRs range from ~0.5 to 7 µM
(Fenster et al., 1997; Frazier et al., 1998; McQuiston and Madison,
1999; Alkondon et al., 2000; Quick and Lester, 2002).
Significance of the differences in distribution and desensitization
of midbrain nAChR subtypes
Although neurons of the VTA and SNc have much in common, a number
of studies have indicated anatomical, pharmacological, and electrophysiological differences (van Domburg and ten Donkelaar, 1991;
Gardner and Ashby, 2000; Zhou et al., 2002). The roles of the VTA and
SNc are not always separable, but there are some simplified distinctions. The SNc provides the main dopaminergic projections to the
neostriatum and is mainly sensorimotor related. The VTA provides the
main dopaminergic projections to the ventral striatum, the prefrontal
cortex, and limbic areas, and the VTA participates in reinforcement and
associative learning processes. Hence, it is mainly the VTA that
projects to the areas in which dopaminergic mechanisms have been
associated with drugs of addiction, including nicotine (Imperato et
al., 1986; Clarke, 1991; Corrigall et al., 1992; Nisell et al., 1994,
1995; Pontieri et al., 1996; Di Chiara, 1999, 2000; Dani and De Biasi,
2001; Dani et al., 2001). Our results suggest another distinction: the
VTA DA neurons have greater 7* nAChR expression than the SNc DA neurons.
The literature supports the fact that nicotine addiction arises via
processes involving - heteromeric nAChRs. 2* nAChRs support
nicotine self-administration (Picciotto et al., 1998), and dopamine
release driven by action potentials in the striatum strongly depends on
2* nAChRs (Zhou et al., 2001, 2002). Furthermore, 42*
receptors are high-affinity sites for nicotine (Picciotto et al., 1995;
Zoli et al., 1998; Marubio et al., 1999). As shown here, however, after
a short period of time, a smoker's level of nicotine will mainly
desensitize 42* nAChRs (Lippiello et al., 1987; Wonnacott, 1987;
Peng et al., 1994; Rowell, 1995; Pidoplichko et al., 1997; Fenster et
al., 1999; Quick and Lester, 2002), but the 7* nAChRs remain
functional at much higher nicotine concentrations (Fenster et al.,
1997; Frazier et al., 1998; McQuiston and Madison, 1999; Alkondon et
al., 2000; Quick and Lester, 2002). Therefore, after the 2*
heteromeric nAChRs are essentially desensitized, 7* nAChRs are still
mainly functional and better able to maintain their usual roles in the
VTA/SNc. That difference in desensitization between 42* nAChRs
and 7* nAChRs on presynaptic afferents in the VTA is an important
factor underlying nicotine-induced synaptic plasticity (Mansvelder and
McGehee, 2000; Mansvelder et al., 2002). It was hypothesized that after
a short exposure to nicotine, 42* nAChRs on GABAergic afferents
are desensitized, decreasing GABA release and decreasing local
inhibition of DA neurons. The 7* nAChRs on glutamatergic afferents
remain active and enhance glutamate excitation of the DA neurons (Dani
et al., 2001). Together, these nicotine-altered mechanisms enhance
long-term potentiation of excitatory inputs to the DA neurons and
enhance the firing of DA neurons.
We should anticipate other complexities in the desensitization process
when nicotine is present in the brain. Nicotine from tobacco will
desensitize many nAChR types, but not in a uniform or invariant manner.
Compositionally identical nAChRs can experience ongoing modifications
that produce functional differences. Furthermore, extremely active
nicotinic, cholinergic synapses will be more susceptible to
desensitization by a smoker's nicotine. Nicotinic receptors at active
synapses repeatedly experience brief exposures to ~1 mM
ACh. Normally, that very brief ACh exposure at a synapse might not
produce desensitization. If the synaptic stimulation is extremely high,
however, even the fast rates of recovery from desensitization may not
allow complete recovery. When those events are occurring in conjunction
with long exposure to low levels of nicotine, then we can expect that
nAChRs located at active synapses are especially susceptible to
desensitization. Evidence indicates that longer exposures to agonist
allow slower rates of desensitization to come into play, such that
nicotinic receptors can enter longer-lasting states of desensitization
(Lester and Dani, 1994; Reitstetter et al., 1999). Furthermore, the
recovery from desensitization can be variable, slow, complex, and
species dependent (Olale et al., 1997; Dani et al., 2000; Quick and
Lester, 2002).
What are the consequences of having many heteromeric nAChRs
desensitized for relatively long periods? What roles did these receptors normally play that are now altered by nicotine-induced desensitization? Although nicotine self-administration and dopamine release depend on 2* heteromeric nAChRs, the maintenance of 7* nAChR activity in the presence of low concentrations of nicotine also
may be important and play presently unappreciated roles in nicotine
addiction and synaptic plasticity linked to associative learning. It is
particularly intriguing that DA and GABA neurons of the VTA, which is
more involved in associative learning and the addiction process,
express higher levels of 7 than the SNc in both rats and mice.
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FOOTNOTES |
Received July 16, 2002; revised Jan. 17, 2003; accepted Jan. 17, 2003.
This work was supported by grants from the National Institute on Drug
Abuse (DA09411, DA12661, DA05947, DA04077), the National Institute of
Neurological Disorders and Stroke (NS21229), and the Wellcome Trust,
UK. We thank M. W. Quick and R. A. J. Lester for
prepublication access to their manuscript.
Correspondence should be addressed to Dr. John A. Dani, Division of
Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX
77030-3498. E-mail: jdani{at}bcm.tmc.edu.
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References |
-
Adams CE,
Stitzel JA,
Collins AC,
Freedman R
(2001)
Alpha 7-nicotinic receptor expression and the anatomical organization of hippocampal interneurons.
Brain Res
922:180-190[ISI][Medline].
-
Alkondon M,
Albuquerque EX
(1991)
Initial characterization of the nicotinic acetylcholine receptors in rat hippocampal neurons.
J Recept Res
11:1001-1021[ISI][Medline].
-
Alkondon M,
Pereira EF,
Wonnacott S,
Albuquerque EX
(1992)
Blockade of nicotinic currents in hippocampal neurons defines methyllycaconitine as a potent and specific receptor antagonist.
Mol Pharmacol
41:802-808[Abstract].
-
Alkondon M,
Pereira EF,
Cortes WS,
Maelicke A,
Albuquerque EX
(1997)
Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons.
Eur J Neurosci
9:2734-2742[ISI][Medline].
-
Alkondon M,
Pereira EF,
Almeida LE,
Randall WR,
Albuquerque EX
(2000)
Nicotine at concentrations found in cigarette smokers activates and desensitizes nicotinic acetylcholine receptors in CA1 interneurons of rat hippocampus.
Neuropharmacology
39:2726-2739[ISI][Medline].
-
Arroyo-Jimenez MM,
Bourgeois JP,
Marubio LM,
Le Sourd AM,
Ottersen OP,
Rinvik E,
Fairen A,
Changeux J-P
(1999)
Ultrastructural localization of the 4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra.
J Neurosci
19:6475-6487[Abstract/Free Full Text].
-
Azam L,
Winzer-Serhan UH,
Chen Y,
Leslie FM
(2002)
Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons.
J Comp Neurol
444:260-274[ISI][Medline].
-
Balfour DJ,
Wright AE,
Benwell ME,
Birrell CE
(2000)
The putative role of extra-synaptic mesolimbic dopamine in the neurobiology of nicotine dependence.
Behav Brain Res
113:73-83[ISI][Medline].
-
Benowitz NL,
Porchet H,
Jacob P
(1989)
Nicotine dependence and tolerance in man: pharmacokinetic and pharmacological investigations.
Prog Brain Res
79:279-287[ISI][Medline].
-
Berke JD,
Hyman SE
(2000)
Addiction, dopamine, and the molecular mechanisms of memory.
Neuron
25:515-532[ISI][Medline].
-
Bertrand D,
Devillers-Thiery A,
Revah F,
Galzi JL,
Hussy N,
Mulle C,
Bertrand S,
Ballivet M,
Changeaux JP
(1992)
Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain.
Proc Natl Acad Sci USA
89:1261-1265[Abstract/Free Full Text].
-
Bonci A,
Malenka RC
(1999)
Properties and plasticity of excitatory synapses on dopaminergic and GABAergic cells in the ventral tegmental area.
J Neurosci
19:3723-3730[Abstract/Free Full Text].
-
Broide RS,
Robertson RT,
Leslie FM
(1996)
Regulation of 7 nicotinic acetylcholine receptors in the developing rat somatosensory cortex by thalamocortical afferents.
J Neurosci
16:2956-2971[Abstr
TOP
ABSTRACT
Introduction
