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The Journal of Neuroscience, March 1, 2001, 21(5):1452-1463
Molecular and Physiological Diversity of Nicotinic Acetylcholine
Receptors in the Midbrain Dopaminergic Nuclei
Ruby
Klink1,
Alban
de Kerchove
d'Exaerde1,
Michele
Zoli1, 2, and
Jean-Pierre
Changeux1
1 Laboratoire de Neurobiologie Moléculaire,
Centre National de la Recherche Scientifique Unité de Recherche
Associée 2182 "Récepteurs et Cognition," Institut
Pasteur, 75724 Paris Cédex 15, France, and
2 Department of Biomedical Sciences, Section of Physiology,
University of Modena and Reggio Emilia, 41100 Modena, Italy
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ABSTRACT |
Nicotinic acetylcholine receptors (nAChRs) on dopaminergic (DA) and
GABAergic (Gaba) projection neurons of the substantia nigra (SN)
and ventral tegmental area (VTA) are characterized by single-cell
RT-PCR and patch-clamp recordings in slices of rat and wild-type,
2
/, 
4/, and 7/ mice. The eight nAChR subunits
expressed in these nuclei, 3-7 and 2-4, contribute to four
different types of nAChR-mediated currents. Most DA neurons in the SN
and VTA express two nAChR subtypes. One is inhibited by
dihydro--erythroidine (2 µM), -conotoxin MII (10 nM), and methyllycaconitine (1 nM) but does not
contain the 7 subunit; it possesses a putative
465(2)2 composition. The other subtype is
inhibited by dihydro--erythroidine (2 µM) and has a
putative 45(2)2 composition. Gaba neurons in the
VTA exhibit a third subtype with a putative
(4)2(2)3 composition, whereas Gaba
neurons in the SN have either the putative
(4)2(2)3 oligomer or the putative
465(2)2 oligomer. The fourth subtype, a
putative (7)5 homomer, is encountered in less than half
of DA and Gaba neurons, in the SN as well as in the VTA. Neurons in the
DA nuclei thus exhibit a diversity of nAChRs that might differentially
modulate reinforcement and motor behavior.
Key words:
addiction; dopamine; GABA; nAChR; nicotinic; Parkinson's
disease; substantia nigra; ventral tegmental area
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INTRODUCTION |
The midbrain dopaminergic (DA)
nuclei comprising the substantia nigra (SN) and ventral tegmental area
(VTA) play key roles in motility, reinforcement, and associative motor
learning (Berke and Hyman, 2000
). Nicotinic acetylcholine receptors
(nAChRs) are densely distributed in the SN and VTA (Fallon and
Loughlin, 1995) and are probably implicated in the regulation of the
dopaminergic circuits in several pathophysiological conditions, such as
nicotine addiction (O'Brien and McLellan, 1996) and the
nicotine-mediated protection against Parkinson's disease (Baron, 1986;
Fuxe et al., 1990). To develop pharmacological agents selectively
targeted to the different midbrain nicotinic functions, identification of the nAChR subtypes becomes imperative.
Distinguishing between the various native nAChR subtypes and
defining their subunit composition is a major challenge, and especially
so in the DA midbrain. Of the nine nAChR subunits identified so far in
the mammalian CNS, seven were found in the SN-VTA; the mRNA for five of
them (4, 5, 6, 2, 3) was expressed at high levels, and
of those, at least 6 and 3 are thought to be present largely in
tyrosine hydroxylase (TH)-containing DA neurons (Le Novère et
al., 1996). In addition, the presence of 7 was detected in the SN
(from mRNA) (Elliott et al., 1998) and inferred in some DA neurons of
the VTA from electrophysiological studies (Pidoplichko et al., 1997).
Binding studies revealed high-affinity nicotinic binding sites in the
SN-VTA as well as in their striatal terminal fields (Clarke and Pert,
1985); further studies performed in 2 (Picciotto et al.,
1995; Zoli et al., 1998) and 4 (Marubio et al., 1999) null mutant
mice demonstrated the critical role of subunits 2 and 4 in this
high-affinity binding. Functional studies have focused on the
pharmacology of dopamine release in striatal synaptosomes and slices
[Sharples et al. (2000) and references therein]. Although hampered by
a lack of subtype-specific tools, they suggested the presence of more
than one nAChR subtype, comprising a putative 32 ligand binding
interface (Kaiser et al., 1998) but exhibiting a subunit combination
distinct from that of any of the oligomers commonly reconstituted in
expression systems (Kulak et al., 1997). Finally, in vivo
approaches have conclusively implicated 2*-nAChR subtype(s) in
nicotine self-administration and nicotine-elicited dopamine release
(Picciotto et al., 1998). Other in vivo studies have
suggested that an 7-homomeric nAChR subtype in the VTA was involved
in nicotine-elicited dopamine release (Schilstrom et al., 1998) and in
the nicotine withdrawal syndrome (Nomikos et al., 1999).
In this work, we further investigate the identification of nAChR
subtypes present in midbrain DA nuclei. From single-cell studies in the
SN compacta (SNc), SN reticulata (SNr), and VTA of rat and
nAChR-subunit null mutant mice, we reveal that projection neurons in
these areas show diverse nAChR subtypes, and that one of them, present
on DA neurons, is endowed with a hitherto undescribed pharmacological profile.
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MATERIALS AND METHODS |
Slice preparation and electrophysiological
recordings. Coronal, horizontal, or sagittal 300 µm slices were
obtained from the midbrain of Sprague Dawley rats (13-25 d) and
wild-type (WT), 2, 4, and 7 null mutant mice (11-16 d),
incubated in oxygenated ACSF containing 1 mM kynurenic acid at ~32°C for 0.5 hr, then left at room temperature for an additional 0.5 hr. ACSF composition was
as follows (in mM): 126 NaCl, 26 NaHCO3, 2.5 KCl, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, 25 glucose. For recording, a single slice was transferred to a chamber superfused with oxygenated ACSF at 35 ± 0.5°C at a rate of 2 ml/min. Whole-cell recordings were obtained from SNc, SNr, and VTA neurons identified using infrared
videomicroscopy with Nomarski optics. Only presumptive projection
neurons of large and medium size were targeted; presumputive
interneurons of small size were avoided. Patch pipettes were filled
with the following (in mM): 144 K-gluconate, 10 HEPES, 3 MgCl2, 0.2 EGTA, pH 7.2, yielding a 3-5
M
resistance. Recordings were done with an Axoclamp-2A (Axon Instruments, Foster City, CA) amplifier operating under current-clamp or continuous voltage-clamp mode, filtered at 3 kHz, and
stored on digital tape for later analysis. After completion of the
experiment, a video image of the field of view was saved to confirm the
location of the neuron recorded from, by comparison with a rat brain
atlas (Paxinos and Watson, 1986). Data were subsequently acquired
(10-50 kHz; InstruNet board, GWI, Sommerville, MA) and analyzed
(IgorPro software, ver 3.1, Wavemetrics, Lake Oswego, OR). Some traces
were digitally filtered for display.
Drug administration. Fast application of agonist was
achieved by pressure-pulse delivery (14 psi) to a two-channel, theta style pipette (diameter 3.5-4 µM) positioned
under visual control at ~30 µM from the
targeted cell. This allowed sequential, rapid application of two
agonists onto the same neuron, and thus a between-agonists comparison
of induced currents on a truly cell-by-cell basis, without drawbacks
associated with slower drug application systems. Mechanical removal of
agonist from the vicinity of the activated receptors, caused by
diffusion into the slice tissue and constant perfusion of ACSF, was
similar for all agonists because the application pipette was always in
the same position relative to the targeted cell. Quantified kinetic
parameters of agonist-elicited current waveforms were compared only for
the same agonist (ACh) so that intrinsic removal of agonist caused by
uptake and degradation would be identical. For all agonists,
response latency (time between beginning of pressure pulse and a
detectable response) was 12-63 msec. Agonists were dissolved in ACSF
to the specified concentration before filling of the drug application
pipette. Agonists were acetylcholine chloride (ACh), choline
chloride (choline), nicotine tartrate (nicotine), and cytisine (all
from Sigma-Aldrich); atropine (1 µM;
Sigma-Aldrich) was present in the ACSF when ACh was applied. Antagonists were bath-perfused for 10 min before pressure-pulse applications of agonists. Additional perfusion did not increase the
extent of inhibition for any of the antagonists tested. Antagonists were dihydro--erythroidine hydrobromide (DHE), methyllycaconitine citrate (MLA) (both from RBI/Sigma-Aldrich), and -conotoxin MII (Eurogentec) stock solution dissolved in 50 mM Tris + 0.1 mg/ml BSA (perfusion of Tris did
not affect agonist-elicited nicotinic currents; n = 2).
Cytoplasm harvest and single cell RT-PCR. When required, at
the end of the recording, the cytoplasm was retrieved by aspiration into the recording pipette under visual control; the contents were
expelled into a test tube in which the RT reaction was performed in a final volume of 10 µl (Lambolez et al., 1992) and left overnight at 37°C. PCR amplification was performed for detection of the two
GABA synthetizing enzymes GAD 65 and GAD 67, tyrosine
hydroxylase (TH), calbindin (CB), calretinin (CR), parvalbumin (PV),
cholecystokinin (CCK), neurotensin (NT), and the nAChR subunits
2-7 and 2-4. The possibility of contamination by nAChR
subunit cDNAs used in the laboratory was ruled out by inclusion of a
template minus negative control (extracellular solution near harvested neurons).
After analyzing results of the single-cell PCR experiments, the 2
band was found absent or difficult to interpret in 32 of 79 neurons.
This result is at odds with previous in situ hybridization (Le Novère et al., 1996) and immunocytochemical (Hill et al., 1993) analysis of the 2 subunit in the midbrain area, which showed 2 mRNA or protein expression in practically all neurons. We
therefore decided to synthesize a second set of oligonucleotides
(2For2 and 2Rev; see below). With the new set of
oligonucleotides, a clear 2 band was detected with single-cell PCR.
The experiment was repeated in a small sample of DA neurons, and 2
mRNA was detected in nine of nine neurons with the new oligonucleotides but only in five of nine with the old oligonucleotides (2For and
24Rev; see below). The frequency distribution reported for the
2 mRNA is most probably underestimated. The other subunits (see
Discussion) apparently were not associated with similar detection problems.
Multiplex PCR. The set of primers used were located in
different exons to rule out genomic DNA amplification by size
criterion. The two steps of multiplex PCR were performed as described
previously (Léna et al., 1999). The resulting cDNAs of nAChR
subunits 2-7, 2-4, GAD65, GAD67, TH, CB, CCK, CR, NT, and PV
contained in 10 µl RT reaction were first amplified simultaneously.
Taq polymerase (2.5 U) (Qiagen, Hilden, Germany) and 10 pmol
of each of the 31 primers were added in the buffer supplied by the
manufacturer (final volume 100 µl), and 20 cycles (94°C, 1 min;
60°C, 1 min; 72°C, 1 min) of PCR were run. Second rounds of PCR
were then performed using 2 µl of the first PCR as template (final
volume 50 µl). In this second PCR, each cDNA was amplified
individually using its specific primer pair (except for GAD65 and
GAD67, which were amplified together) by performing 40 PCR cycles as
described above. Twenty microliters of each individual PCR were then
run on a 2% agarose gel. To confirm the specificity of PCR product,
the last 30 µl were purified on QIAquick spin column (Qiagen) and
restricted with specific endonucleases. The products that were digested
yielded uniquely identifying fragments. The efficiency of this
RT-multiplex PCR protocol was tested on 1 ng of whole-brain total RNA
(Léna et al., 1999; Porter et al., 1999). The following sets of
primers were used (from 5' to 3'; position 1 is the first base of the start codon, and the number between brackets indicates the initial and
final positions of the PCR primers). Specific endonucleases for the
different cDNAs and their cut position are also between brakets: 2
(accession number L10077) 2 For [325-351], 24 Rev
[880-908] (Léna et al., 1999) NdeI [546]; 3
(accession number L31621) 3 For [301-327], 3 Rev [843-870]
(Léna et al., 1999) AvaI [619]; 4 (accession
number L31620) 4 For [337-356], 4 Rev [581-601] (Porter et
al., 1999) AatII [496]; 5 (accession number J05231) 5 For
[1102-1121], 5 Rev [1369-1392] (Porter et al., 1999) AatII
[1187]; 6 (accession number L08227) 6 For [417-441], 6
Rev [1014-1034] (Léna et al., 1999) EcoRV [685]; 7 (accession number L31619) 7 For [264-293], 7 Rev
[746-773] (Léna et al., 1999) HaeII [359]; 2
(accession number L31622) 2 For [308-335], 24 Rev
[859-884] (Poth et al., 1997) HinFI [649], 2 For2 [10-29],
2 Rev [264-283] BspHI [223]; 3 (accession number
J04636) 3 For [334-360], 3 Rev [772-799] (Léna et al., 1999) NcoI [479, 554]; 4 (accession number U42976)
4 For [284-310], 24 Rev [853-878] (Poth et al., 1997)
AflII [547]; GAD 65 (accesion number M72422) GAD65 For
[713-732], GAD65-67 Rev [1085-1103] (Cauli et al., 1997); GAD67
(accession number M76177) GAD67 For [529-547], GAD65-67 Rev
[1109-1127] (Cauli et al., 1997); TH (accession number NM012740) TH
For [36-65], TH Rev [581-610] (Léna et al., 1999)
BanI [408]; CB (accession number M27839) CB For
[130-156], CB Rev [544-570] (Cauli et al., 1997) EcoRI
[304]; CCK (accession number K01259) CCK For [170-196], CCK
Rev[366-392] (Cauli et al., 1997) PstI [247]; CR (accession number X66974) CR For [138-164], CR Rev [425-451] (Cauli et al., 1997) Eco 47 III [325]; NT (accession number J03185) NT For [30-56], NT Rev [375-401] SphI [120]; PV
(accession number) M12725) PV For [115-141], PV Rev [473-502]
(Cauli et al., 1997) EcoRI [304].
Statistical analysis. Statistical analysis was done with
StatView software (SAS Institute). Averages are given as mean ± SEM; means were compared with the Student's t test
(two-tailed, paired or unpaired) or post hoc analysis of
one-way ANOVA, and frequency distributions were compared with the
2 test (significance level 0.05).
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RESULTS |
Physiological and molecular profile of SN and VTA neurons
Whole-cell recordings were obtained from presumptive projection
neurons of large and medium size in rat SNr, SNc, and VTA at all
rostrocaudal levels. A detailed electrophysiological characterization of firing and membrane potential properties, followed by single-cell RT-PCR, was performed on 79 neurons.
Two main electrophysiological classes composed of neurons of similar
morphology and size were distinguished in each of the SNcs, SNrs, and
VTAs (Table 1). One was typical of the
TH-containing, DA neurons already extensively characterized (Grace and
Onn, 1989; Yung et al., 1991). In the present study, only slight
differences were observed between DA neurons of the SNc and SNr and
those of the VTA (Fig. 1); we refer to
neurons from this group as DA neurons. Neurons in the other
electrophysiological class, generally denoted as non-DA, were referred
to as Gaba neurons because their molecular phenotype was confirmed by
single-cell RT-PCR. We have encountered two subclasses of Gaba neurons
in the SNc and SNr and two different ones in the VTA. The major
subclass in the SNc/SNr (12 of 15 Gaba neurons) exhibited a regular
spiking pattern on application of a depolarizing current pulse (Fig.
1); we denote this subclass as Gaba-regular spiking (Gaba-RS). The
major subclass in the VTA (six of nine Gaba neurons) exhibited a
strongly accommodating firing pattern (Fig. 1); we denote this subclass
as Gaba-accommodating (Gaba-Ac). The intrinsic membrane potential
properties of Gaba-Ac neurons were unlike those of neocortical
GABAergic neurons (Cauli et al., 1997) but resembled those of an
infrequent hippocampal subclass (R. Miles, personal
communication). We found it necessary to maintain the
distinction between Gaba neuron subclasses throughout, because the
biophysical and pharmacological properties of their nAChR-mediated
currents differed.
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Figure 1.
Electrophysiological and molecular profile of VTA
and SN neurons. Electrophysiological classes were distinguished on the
basis of intrinsic membrane potential and firing properties in response
to current steps applied from 64 mV (first
panel from left); on action potential waveform
and duration, measured at rheobase current (second
panel); and on firing frequency at the resting membrane
potential for neurons that were spontaneously active (third
panel). DA neurons in the SNc and SNr were
indistinguishable; they exhibited strong Ih
activation and slow potential oscillations in the subthreshold range.
DA neurons in the VTA showed a less pronounced
Ih activation and a slow ramp potential
before firing initiation. Gaba-Ac neurons in the VTA were characterized
by marked spike-frequency accommodation in response to depolarizing
current steps. Gaba-RS neurons in the SNr fired with a regular
discharge pattern, at all amplitudes of depolarizing current steps and
from depolarized as well as hyperpolarized holding potentials. Vertical
calibration bar is 20 mV in all three panels. The right
panel shows agarose gels of the PCR amplification products
corresponding to the illustrated neuron. Only detected products are
labeled (left to right): nicotinic
subunits 2-7 and 2-4, marker, GAD (GAD 65 and GAD 67), TH, CB,
CCK, CR, NT, and PV. The gel for the GABA-Ac neuron
(third from top) shows a faint TH band;
such (rare) TH bands were discounted. When GAD and TH were coexpressed
(see Results) both bands were of equal intensity.
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Single-cell RT-PCR was performed to assay the differential mRNA
expression of the two neurotransmitter synthesizing enzymes GAD and TH,
two neuropeptides CCK and NT, three
Ca2+-binding proteins CB, CR, and PV, and
nine nAChR subunits 2-7 and 2-4 (Fig. 1). The aim was to
determine whether a specific, potential nAChR-forming combination of
subunits was associated with a particular molecular profile, a
particular electrophysiological class or a particular anatomical
location within the SN-VTA area. The cells included in the analysis
expressed at least one of the neurotransmitter synthesizing enzymes
mRNA, plus at least one of either the neuropeptides or the
Ca2+-binding proteins mRNA, plus at least
one nAChR subunit mRNA. Of the neurons characterized
electrophysiologically, 70 of 79 fulfilled these criteria.
All but one electrophysiologically identified DA neuron expressed TH
mRNA (49 of 50; the remaining neuron expressed only GAD). Gaba neurons
expressed GAD (20 of 20); 19 of the 70 cells (27%) coexpressed TH and
GAD, as already reported under similar experimental conditions (Guyon
et al., 1999). This molecular phenotype appeared independent of the
electrophysiological class and location of the cell
(p = 0.3 and p = 0.9, respectively) but age dependent (Fig.
2A). Coexpression of
catecholaminergic and amino acidergic phenotypes appears to be a common
phenomenon in the brain (Kosaka et al., 1987; Kaneko et al., 1990). In
DA midbrain neurons, the DA-glutamate phenotype seems to predominante
over the DA-GABA phenotype in adult animals (Kosaka et al., 1987;
Campbell et al., 1991; Sulzer et al., 1998). However, the present data
showing a peak of GAD/TH coexpression at postnatal day 16-17
suggests that the prevalence of the GABA phenotype is plastic and may
be regulated developmentally.
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Figure 2.
Frequency distribution of mRNAs for
neurotransmitter synthesizing enzymes, neuropeptides, and
Ca2+-binding proteins in SN and VTA.
A, Percentage of neurons coexpressing GAD and TH mRNA
versus age; numbers at the base of histogram bars
indicate the total number of neurons in each age group bin (2 d).
B1, Percentage of neurons expressing the neuropeptides
CCK and NT and the Ca2+-binding proteins CR, PV, and
CB. B2, Percentage of DA and Gaba neurons that are CCK
positive. CCK is preferentially expressed in DA neurons.
B3, Percentage of CB-positive neurons in the SNc, SNr,
and VTA. CB was found preferentially in the VTA
(**p < 0.01).
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Among the neuropeptides, CCK was distributed widely and NT was
distributed very sparsely (Fig. 2B1); CCK mRNA was
found more frequently in DA than in Gaba neurons
(p < 0.01) (Fig. 2B2),
whereas the sparse distribution of NT mRNA was not correlated with the occurrence of a particular Gaba subclass. Among the
Ca2+-binding proteins, CR mRNA was
detected in practically every neuron, whereas PV and CB mRNAs were more
restricted in their distribution (Fig. 2B1). VTA
neurons preferentially expressed CB mRNA (p < 0.01), regardless of neuron class (Fig. 2B3); PV mRNA
was indicative of neither class nor location.
nAChR subunit mRNAs in SN and VTA neurons
Among the mRNAs of the nine nAChR subunits sampled, 2 mRNA was
never detected, and 4 mRNA was found in practically all neurons (69 of 70). The frequency distribution of the remaining nAChR subunit mRNAs
is shown in Figure 3A; no
single subunit was location specific; however, for some, the nAChR
subunit mRNA distribution was strikingly class specific (Figs. 1,
3B). 5, 6, and 3 mRNA were almost exclusively
(p < 0.001) encountered in DA neurons, and in a
high proportion (>72% of DA neurons); 3 mRNA was also prevalent in
DA neurons (60%) but not significantly more (p = 0.06) than in Gaba neurons (35%); 2 mRNA provisionally appeared (see caveat in Materials and Methods) more frequently distributed in
Gaba than in DA neurons; 7 mRNA was equally distributed (40%) between classes, and 4 mRNA was sparsely distributed but more frequently encountered in Gaba (25%) than in DA (12%) neurons. Hence,
the vast majority of DA neurons expressed the 4-5-6-3 mRNAs, to which 3 mRNA could often be added, whereas the vast majority of Gaba neurons expressed the 4-2 pair occasionally associated with 3 mRNA.
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Figure 3.
Frequency distribution of nicotinic subunit mRNAs
in the SN and VTA. A, Nicotinic subunits are sorted by
order of decreasing prevalence: subunit 4 was present in all
neurons, and subunit 2 was present in none (data not shown).
B, The same distribution segregated with respect to
neuron class. 3, 5, and 6 mRNAs are significantly more
prevalent in DA than in Gaba neurons (***p < 0.001).
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DA neurons were remarkably similar in terms of the nAChR subunit mRNAs
they expressed, regardless of anatomical location. In Gaba neurons, the
available sample was too limited to allow meaningful comparisons;
however, VTA Gaba neurons expressed exclusively the 4-2-3
subunits, whereas SN Gaba neuron subunit expression was more diverse.
The 5, 6, or 3 mRNA, when expressed in Gaba neurons, was
encountered exclusively in the Gaba-RS subclass of the SNr (three of
three for 5, two of two for 6, four of four for 3); in
addition, four of five of 4 mRNA-containing Gaba neurons were in the
SNr. Finally, 7 mRNA appeared with equal probability in VTA or SN
neurons regardless of class (p > 0.99).
Expression of the less prevalent 3, 7, and 4 mRNAs was
evaluated for codistribution with the less prevalent molecular species CB, PV, and NT; the occurrence of 3 mRNA was thus found
significantly correlated with that of the
Ca2+-binding protein CB
(p < 0.05).
Correlation of nAChR subunit mRNAs with nAChR currents
In a subset (n = 37) of the 70 neurons on which
single-cell RT-PCR was performed, the pharmacology of nicotinic
agonist-gated currents was assessed rapidly so as not to compromise the
quality of the subsequent RT-PCR. By using a two-channel drug
application pipette, two agonists could be tested on the soma-proximal
dendritic region of each neuron by fast, pressure-pulse applications of variable duration (see Materials and Methods). The drug combinations used were ACh (1 mM; 30 msec) with the putative
7-homomeric subtype-specific agonist choline (10 mM; 30 msec) in one set of neurons, and nicotine (20 µM; 1 sec) with cytisine (20 µM; 1 sec) in another set. Only 2 of the 37 neurons did not respond to any of the agonists applied, and both were
Gaba VTA neurons.
The aim of the ACh/choline combination (n = 16) was
combined with the single-cell RT-PCR results to validate the
identification of an 7-homomeric type of current when gated by ACh
under our experimental conditions. Choline elicited the characteristic
7-homomeric type of current waveform (Alkondon et al., 1997) in 7 of
16 neurons; 7 mRNA was detected in all choline-responsive neurons
(Fig. 4) and absent in the
choline-unresponsive neurons. ACh elicited multiphasic current
waveforms; a component with fast activation kinetics was attributed to
the 7-homomeric type of current and could be identified in five of
seven of the choline-responsive and 7 mRNA-positive neurons (Fig.
4). Rise times of the fast current were similar in ACh (22 ± 4 msec) and choline-gated currents (13 ± 2 msec); current duration
measured in choline (86 ± 11 msec) was therefore considered
indicative of the time course of the ACh-gated 7-homomeric type of
current. This current is essentially over in 100 msec and therefore
cannot contribute to the ACh current waveform occurring later.
Concerning these later ACh-elicited currents, the examples illustrated
in Figure 4 show that, congruent with the single-cell RT-PCR results,
ACh waveforms in DA neurons were clearly different from those in Gaba
neurons. Later on, we present a detailed analysis of the ACh-gated
currents on a larger sample of SN-VTA neurons.
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Figure 4.
Neurons containing subunit 7 mRNA exhibit fast
currents gated by choline and ACh. Choline (10 mM; 30 msec)
elicits identical current waveforms in DA and Gaba neurons (left
panel). In the same neurons, ACh (1 mM; 30 msec) elicits different current waveforms (middle
panel), in both of which a fast component can be
recognized. In this and subsequent figures, currents were evoked from a
holding potential of 70 mV, and the width of the black
box at the beginning of each trace indicates duration of
pressure-pulse application. Agarose gels (right
panel) corresponding to the DA and Gaba neuron show the
presence of subunit 7 mRNA (gels truncated after the nAChR subunit
wells).
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Cytisine is considered a nicotinic agonist with higher affinity for
4-containing nAChRs, in both reconstituted (Luetje and Patrick,
1991) and native receptors (Zoli et al., 1998). Conflicting evidence
for the role of cytisine and hence of 4 subunit in DA release in the
striatum has been presented (Grady et al., 1992; El-Bizri and Clarke,
1994). With the nicotine/cytisine combination (n = 21),
cytisine elicited a larger current than nicotine in only one VTA Gaba
neuron; otherwise, the cytisine response was between 0 and 44% of the
corresponding nicotine response (Fig. 5).
In none of those neurons was 4 mRNA detected.
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Figure 5.
Nicotine elicits larger currents than cytisine in
neurons lacking subunit 4. Nicotine (20 µM;
left panel) and cytisine (20 µM;
middle panel) were pressure-applied for 1 sec.
Agarose gel (right panel; truncated after the nAChR
subunit wells) shows the absence of subunit 4.
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Diversity of nAChR currents in SN and VTA neurons
An extensive biophysical and pharmacological investigation was
performed on another set of SN-VTA neurons with electrophysiological properties identical to those described previously (n = 73). First, we confirmed the postsynaptic location and nicotinic nature
of the recorded currents. In a low Ca2+
(0.1 mM)/high Mg2+
(10 mM) ACSF solution, which prevents sustained
transmitter release, ACh and nicotine responses were identical in time
course to those in regular ACSF (n = 3); their
amplitude, however, appeared slightly reduced, consistent with
Ca2+ modulation of nAChR channels (Mulle
et al., 1992; Liu and Berg, 1999) (data not shown). In another set of
neurons, the nicotinic channel blocker mecamylamine (10 µM) blocked all ACh/nicotine responses
(n = 4) (data not shown).
ACh (1 mM; 30 msec)-elicited and nicotine (20 µM; 300 msec)-elicited currents were characterized in DA
(n = 38) and Gaba (n = 20) neurons of
the SN-VTA area. All neurons responded to ACh, whereas nicotine
responses were undetectable in three Gaba neurons. As noted previously,
ACh current waveforms differed in shape and amplitude according to
neuron class, whereas nicotine waveforms differed only in amplitude. In
what follows, we will not mention the fast, putative 7-homomeric
component when present in the ACh waveform; the later component,
putatively mediated by heteromeric nAChRs, will be referred to as the
slow component. Table 2 lists the
ACh-elicited slow current parameters quantified in DA neurons and the
two major Gaba subclasses of the SN-VTA (15 of 20 Gaba neurons).
ACh currents in DA neurons of the SNc, SNr, and VTA were similar in
shape (Fig. 6A1). Peak
current was reached slowly (114-543 msec), but current decay was
relatively abrupt; the DA ACh waveform was thus characterized by a
round peak and a sigmoid decay component (concave up). The nicotine
current time course was ~10× slower (rise time 1.6-4.9 sec) (Fig.
6B1), although response latency (see Materials and
Methods) was equally short with ACh or nicotine. Both agonists elicited
currents of larger amplitude in the VTA (n = 16) than
in the SN (n = 22); this difference was more pronounced with nicotine (142 ± 17 vs 93 ± 11 pA; p < 0.05) (Fig. 6B2) than with ACh (302 ± 39 vs
211 ± 31 pA; p = 0.07) (Fig.
6A2).
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Figure 6.
Nicotinic currents in DA neurons of the SN and
VTA. A1, ACh (1 mM; 30 msec) elicits
currents of similar waveform in the VTA and SN, characterized by a
round peak and sigmoid decay. B1, In the same neurons,
nicotine (20 µM; 300 msec) elicits much longer lasting
currents. A2, Mean ACh-elicited current amplitude in the
SN and VTA. B2, Mean nicotine-elicited current amplitude
in the SN and VTA. Nicotine induces larger currents in the VTA than in
the SN (*p < 0.05).
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ACh currents in the two major subclasses of Gaba neurons fell into two
distinct categories with respect to activation kinetics and amplitude.
In the Gaba-Ac neurons of the VTA and in a portion of the Gaba-RS
neurons of the SNr, peak current was reached relatively fast (50-150
msec) and decay was much slower (Fig.
7A1). This Gaba ACh waveform
was thus characterized by a sharp peak and an exponential decay
component (convex up); amplitudes were considerably and significantly
smaller than in the other neuronal classes (Table 2). In the remaining
Gaba-RS neurons of the SNr, ACh waveforms resembled those of DA
neurons; they exhibited a large amplitude and a slow rounded peak
(89-328 msec), although the sigmoid decay component was not as
pronounced (Fig. 7A2). By plotting current amplitude versus
corresponding rise time of ACh currents, the two groups segregated, and
it was apparent that many Gaba-RS neurons exhibited current rise times
and amplitudes characteristic of DA neurons (Fig. 7B).
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Figure 7.
ACh-elicited currents in Gaba neurons of the SN
and VTA. A1, In the Gaba-Ac subclass of the VTA, ACh
elicits current waveforms characterized by a sharp peak, exponential
decay, and small amplitude. A2, In the Gaba-RS subclass
of the SNr, the ACh current waveform is often slower and of larger
amplitude. B, ACh elicited current amplitude plotted
versus corresponding rise time in Gaba-Ac ( ) and Gaba-RS neurons
( ); mean ACh-elicited current amplitude versus mean rise time in DA
neurons was also reported (double triangle).
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Pharmacological properties of nAChRs in SN and VTA neurons
MLA, a putative 7-specific nAChR antagonist, was tested for its
ability to inhibit the fast currents elicited by ACh and choline.
Choline pulses (10 mM; 30 msec) 5 min apart did not cause any substantial nAChR desensitization (89-100% of control responses; n = 4). MLA (1 nM), bath-perfused
for 10 min, antagonized the choline-elicited current (91-100% block;
n = 4). After a 15 min washout period, the current
recovered to 23 and 29% of its control value (n = 2).
These properties (Fig.
8A1) are typical of
7 homomeric nAChRs (Wonnacott et al., 1993; Palma et al., 1996).
However, because ACh (1 mM; 30 msec) was applied
to the same neurons after choline (n = 8), in addition
to fast current antagonism (Fig. 8A2), it was noticed
that MLA (1 nM) could antagonize the slow peak of
the ACh waveform (Fig. 8B2), a component that, as
already shown, is independent of the presence of the 7 mRNA.
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Figure 8.
MLA affects two different types of currents.
A1, In a VTA DA neuron, MLA (1 nM) blocks
the choline-gated current, which partially recovers after a 15 min wash
period. A2, In the same neuron, MLA blocks the fast
component of the ACh-gated current without affecting the slower
component. B1, In another VTA DA neuron, MLA (1 nM) blocks the choline-gated current. B2, In
the same neuron, the ACh-gated current does not show a clear fast
component; however, MLA inhibits the slow peak current occurring later.
In the superimposition panels, MLA trace is in
gray.
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This MLA sensitivity of slow nAChR-mediated currents was further
investigated with ACh (1 mM; 30 msec) and nicotine (20 µM; 300 msec) coapplications. MLA (1-10
nM) differentially antagonized ACh-elicited
responses (Table 3). Responses in 8 of 30 neurons tested were unaffected by MLA (<11% inhibition), in 4 of 30 were highly MLA sensitive (>85% inhibition), and in the rest were
partially sensitive (inhibition between 34 and 61%; mean = 46 ± 2%; n = 18). MLA antagonism was already
maximal at a concentration of 1 nM because
switching from 1 nM to 10 nM MLA did not cause a greater inhibition
(responses in 10 nM MLA were 105-116% of those in 1 nM MLA; n = 3). In addition,
MLA (1 nM) was not measurably reversed after a 15 min wash period (n = 15).
Nicotine-elicited responses exhibited the same MLA sensitivity profile
(after verification that a 10 min period between nicotine pulses was
sufficient to recover from nAChR desensitization; n = 4); however, MLA inhibition was only 24 ± 3%, significantly smaller than that of ACh-elicited responses (p < 0.001; n = 12).
Thus, to sum up, in SN-VTA neurons, ACh and nicotine can elicit slow
nAChR-mediated currents that are as sensitive to MLA as the fast type.
These two MLA-sensitive current types can be distinguished by their
kinetics and their differential recovery from MLA blockade.
Table 3 categorizes all MLA-probed neurons (ACh as agonist) in terms of
neuron class and MLA sensitivity. A minority of DA neurons (24%) are
not MLA sensitive; most are only partially MLA sensitive. However, DA
neurons with nAChR-mediated currents totally blocked by MLA (1/25) do
occur; these are referred to as highly MLA sensitive in Table 3. The
available sample of Gaba neurons was small; however, it appears that
VTA Gaba-Ac neurons are not MLA sensitive. Concerning the SNr Gaba-RS
neurons, it should be noted that these exhibited the ACh-elicited
current waveform characteristic of DA neurons and were highly MLA sensitive.
We interpret partial inhibition of the nAChR-mediated currents by MLA,
which could not be overcome by greater antagonist concentrations, as
being caused by the presence of two nAChR subtypes: one is maximally
blocked by 1 nM MLA and the other is not affected. Most DA
neurons possess both subtypes, and their agonist-induced responses are
therefore partially inhibited by MLA. However, it appears that the
biophysical properties of the two subtypes do not differ greatly. The
current waveform in the presence of MLA also exhibited the
characteristic rounded peak and sigmoid decay (Fig.
8B2); thus, rise time of ACh-elicited current was
slightly but not significantly increased in the presence of MLA
(295 ± 31 msec vs 271 ± 31 msec; p = 0.08;
n = 19 DA neurons).
We further investigated the pharmacological properties of these nAChR
subtypes with the antagonist -conotoxin MII, at concentrations inhibiting striatal DA release (Kulak et al., 1997; Kaiser et al.,
1998) and reported to be specific for 32*-nAChRs (10-100 nM). ACh-elicited currents were not affected by
-conotoxin MII (10 nM) in two of eight DA neurons; in
the remaining six neurons, -conotoxin MII (10 nM)
inhibited ACh currents by 40 ± 4% (20-48%) without an
observable effect on activation kinetics (not quantified) (Fig.
9A). This block was maximal
because switching from a 10 to a 100 nM solution
did not increase the degree of -conotoxin MII antagonism
(n = 3) (Fig. 9B). There was an occlusion
between -conotoxin MII and MLA antagonism because addition of MLA (1 nM) to the toxin solution did not increase the
degree of antagonism (MLA + toxin responses were 91-108% of those of
toxin alone; n = 3 including a toxin-insensitive
neuron) (Fig. 9B). These results therefore indicate that the
MLA-sensitive nAChR subtype is also -conotoxin MII sensitive,
because both antagonists induced a comparable degree of inhibition in
the same proportion of neurons, and their effects were occlusive.
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Figure 9.
-conotoxin MII inhibits the MLA-sensitive
component of ACh-gated currents. A, In an SNc DA neuron,
-conotoxin MII (10 nM) inhibits the ACh-gated current.
In the panel labeled superimposition, the
MLA trace is in gray. B, In the same
neuron, increased concentration of -conotoxin MII (100 nM) does not result in greater inhibition (note change in
scale); addition of MLA (1 nM) to the -conotoxin MII
(100 nM) also has no effect (middle). In the
superimposition panel, the -conotoxin MII (100 nM) and MLA traces are in gray.
-Cntx MII, -Conotoxin MII.
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The antagonist DHE at a concentration of 2 µM
discriminates, in functional assays, between two different 2*-nAChR
subtypes (Marks et al., 1999). Ach- and nicotine-elicited currents in
DA and Gaba neurons were blocked by DHE (2 µM)
(83-100% inhibition; n = 7 including two neurons that
were partially inhibited by -conotoxin MII). These results therefore
indicate that both the MLA/-conotoxin MII-sensitive and -insensitive
nAChR subtypes on SN-VTA neurons are DHE sensitive (data not shown).
nAChR currents in nAChR subunit null mutant mice
Further refinement in the identification of the subunit
composition of nAChRs present in the SN and VTA was attempted by
examining nAChR-mediated currents in 2 (Picciotto et al., 1995),
4 (Marubio et al., 1999), and 7 (Orr-Urtreger et al., 1997) null
mutant mice. First, it was ascertained that neuronal classes and their ACh-elicited currents were similar in rat and mouse; in WT mice (n = 15), identical ACh-elicited current waveforms were
segregated to the same neuronal classes as in rat (Fig.
10, first and second panels from left). In 2 null mutant mice
(n = 19), the only ACh-gated current that could be
elicited in DA, Gaba-Ac, and Gaba-RS neurons was the fast, putative
7-homomeric type of current (Fig. 10, right panel). However, in the SNr, in addition to this fast
current, a slow current was still evoked in a subclass of Gaba neurons characterized by burst firing (the minority type in rat); further investigation of this 2-independent current was not undertaken. In
4 null mutants (n = 23), slow currents were evoked
in most DA neurons (15 of 18); however, ACh current amplitude, rise
time, and decay time were all significantly smaller than in WT mice (p < 0.01) (Table
4; Fig. 10, right
panel). No slow currents could be elicited in the Gaba-Ac
or Gaba-RS neuron subclasses (Fig. 10, right panel).
In 7 null mutants, the only noted difference with WT mice was the
absence of the fast, putative 7-homomeric type component from the
ACh waveform (data not shown).
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Figure 10.
Neuron classes and their ACh-elicited currents in
the SN and VTA of WT and nAChR subunit null mutant mice.
Electrophysiological classes (left panel) and
ACh-elicited current waveforms in WT (second panel from
left) were similar to those described in rat. In
2/ mice, only a fast, 7-homomeric type of ACh-gated current
could be recorded in the illustrated neuronal subclasses (third
panel). In 4/ mice, a slow current could be
elicited only in DA neurons (right panel).
Calibration in second row applies also to top
row.
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MLA sensitivity was also investigated. Similarly to rat, in WT mice
ACh-elicited currents in DA neurons were partially inhibited (45 ± 12%; n = 4), and their rise time was increased
slightly but significantly (from 195 ± 53 msec to 246 ± 78 msec; n = 4; p < 0.01) by MLA (1 nM) (Fig. 11,
left). MLA sensitivity was totally abolished in DA neurons
of 4/ mice (112 ± 6% of control currents; n = 5) (Fig. 11, middle) but remained intact
in DA neurons of 7/ mice (43 and 55% inhibition;
n = 2) (Fig. 11, right). As in rat, the
ACh-elicited current in SNr Gaba-RS neurons of WT mice was highly MLA
sensitive (79 and 82% inhibition; n = 2) (data not shown); as already mentioned, this current was abolished in 4 null
mutants.
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Figure 11.
MLA-sensitive currents in WT and nAChR subunit
null mutant mice. Left, DA neurons in WT mice are
partially inhibited by MLA (1 nM); middle,
MLA sensitivity is abolished in 4/ mice; right,
MLA sensitivity is not affected in 7/ mice. MLA traces are in
gray.
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Results obtained in null mutant mice thus confirm the previous
identification of MLA-sensitive and MLA-insensitive nAChR subtypes. One
important question is whether the MLA-insensitive nAChR subtype in WT
mice is identical to the MLA-insensitive nAChR species present in
4/ mice. A kinetic analysis suggests a negative answer: in WT
mice, MLA blockade uncovered an nAChR-mediated current with slow
activation kinetics (rise time ~250 msec) and sigmoid decay, whereas
the nAChR-mediated current in 4 null mutants had fast activation
kinetics (rise time ~100 msec) and exponential decay (Table 4; Fig.
11). The residual nAChR-mediated current in 4/ mice is currently
being investigated in our laboratory.
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DISCUSSION |
The present work shows that somatodendritic nAChRs on DA and Gaba
projection neurons of the SN and VTA fall into four different subtypes,
each possessing a particular complement of nAChR subunits and
exhibiting different ACh-gated current waveforms and pharmacological properties. One subtype present on neurons that lack the 7 gene or
mRNA exhibits nanomolar sensitivity to MLA.
Methodological considerations
An objective of the present study was to relate
electrophysiological recordings and molecular detection of nAChR mRNAs
at the single-cell level. Yet, some caution is required when
interpreting single-cell PCR studies. In general, the distribution of
nAChR subunit mRNAs in the SN-VTA revealed by single-cell RT-PCR
tallies well with in situ hybridization results (Le
Novère et al., 1996), which showed moderate to high levels of
4-6 and 2-3 mRNA. The case of 3, 7, 2, and 4 mRNA
requires a more specific comment. 3 mRNA was detected in ~60% of
DA neurons with single-cell PCR, but its signal was very low in
in situ hybridization experiments. This suggests that 3
mRNA is expressed in a relatively large proportion of DA neurons but at
low level. With regard to 7 mRNA, single-cell PCR shows its presence
in 40% of DA neurons, whereas the 7 mRNA signal in in
situ hybridization experiments, as well as -bungarotoxin
binding in autoradiography experiments, is very low (Zoli et al.,
1998). The tight correspondence between detection of 7 mRNA and
electrophysiological response to choline is strong proof for the
existence of functional 7-containing nAChRs in some SN-VTA neurons.
As described in Materials and Methods, the set of oligomers for 2
used in the single-cell PCR experiments was likely to detect a large
number of false negative cells. The use of another set of oligomers in
a restricted sample showed that 2 mRNA was present in all DA neurons
analyzed. This latter evidence together with previous in
situ hybridization (Le Novère et al., 1996) and
immunocytochemistry (Hill et al., 1993) findings strongly suggests that
2 mRNA and protein are present in all neurons. This notion is also
supported by the evidence that slow nAChR-mediated currents disappear
from all DA neurons in 2 null mice (Picciotto et al., 1998; and
present results). 4 mRNA was detected in ~10% of DA neurons and
25% of Gaba neurons. 4 mRNA was not detected previously in DA
neurons by using PCR technique on SNc homogenates (Elliott et al.,
1998) or in situ hybridization experiments (Le Novère
et al., 1996). Overall, these techniques converge on the concept that
4 is rarely expressed in DA or Gaba neurons of the SN-VTA. Its
expression and participation in functional nAChRs, however, is
indirectly suggested by the finding that some Gaba neurons in the SNr
still show slow nAChR currents in 2 null mice.
nAChR subtypes as signatures of neuron class diversity
In the present study, in addition to nAChR subunit mRNA
distribution, we investigated that of various molecular markers to disclose any potential association between them. The only significant correlation was that of the Ca2+-binding
protein CB with 3 mRNA, and hence presumably with 3*-nAChRs. In
contrast, neuron class was highly correlated with a specific distribution pattern of nAChR subunit mRNA and nAChR-mediated current
waveform. Thus, nAChR subunit expression and nicotinic agonist-gated
currents were remarkably similar in DA neurons of the SNc, SNr, and VTA
and different from those of Gaba neurons in the same area; moreover,
Gaba neurons exhibited diverse electrophysiological properties
warranting their classification into subclasses as elsewhere in the CNS
(Cauli et al., 1997; Parra et al., 1998), and nAChRs, in general,
followed this diversity. In this regard, because it was not
investigated further, we mention again here the occurrence, in
2/ mice, of a non-2*-nAChR slow current restricted to a
particular Gaba neuron subclass in the SNr.
nAChR subtypes in SN and VTA neurons
To uncover the various nAChR functional subtypes, we used kinetic
and pharmacological analysis in the same neuronal populations in which
single-cell RT-PCR was performed. These revealed the presence of four
different nAChR subtypes on the somatodendritic region of SN-VTA
neurons (discussed below). Similar studies in null mutant mice
confirmed the identification of these four subtypes and added some
useful constraints on their subunit composition. In the following, we
evaluate the data presented in this work, as well as previous relevant
findings, to propose a hypothesis about the molecular composition of
the different nAChR subtypes. We follow the rules currently accepted
for assembly of functional nAChR oligomers (for review, see Corringer
et al., 2000) and the recommended nomenclature (Lukas et al.,
1999).
The 7-homomeric subtype
7 mRNA was detected in less than half of neurons in the SN-VTA.
In all 7-expressing neurons, a choline, and in most cases an
ACh-elicited current with fast kinetics, was observed that was blocked
by MLA (1 nM) and measurably recovered within 15 min of
drug removal. These properties are typical of the 7-homomeric subtype identified in other anatomical locations and experimental systems (Wonnacott et al., 1993; Alkondon et al., 1997; Papke et al.,
2000). Characterization of 7 null mutant mice (Orr-Urtreger et al.,
1997) confirms that with deletion of the 7 subunit, the identified
7-homomeric component disappears, whereas ACh-gated waveforms beyond
100 msec are not affected. Therefore, present and previous evidence
converges to the identification of choline and ACh-gated fast currents
with an (7)5-nAChR oligomer. On the basis of
mRNA expression of the 7 subunit, this subtype is encountered with
equal probability in the SN or VTA and in DA as well as Gaba neurons.
The GABA-Ac subtype
The ACh-elicited current waveform of this nAChR subtype is
identical to that of Type IB [when the
(7)5-nAChR is also present] and Type II
nAChRs, suggested to comprise 4 and 2 subunits (Alkondon and
Albuquerque, 1993). The present work supports this contention because
Gaba-Ac neurons express almost exclusively mRNA for 4 and 2, and
no current could be evoked in 2 (Picciotto et al., 1995) or 4
(Marubio et al., 1999) null mutant mice. The only other subunit
detected in Gaba-Ac neurons was 3, which could be transported to
nerve terminals or form somatodendritic nAChRs together with 4 and
2. If the
TOP
ABSTRACT
INTRODUCTION
