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Vol. 302, Issue 3, 1246-1252, September 2002
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida (M.A., M.J.P., F.N.M, C.W.L.); and Chemistry and Life Sciences, Research Triangle Institute, Research Triangle Park, North Carolina (F.I.C.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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2'-Pyridine ring substituted analogs of epibatidine were assessed for
equilibrium binding affinity, functional potency, and efficacy at rat
neuronal nicotinic receptors expressed in Xenopus oocytes. Binding affinities were determined in membrane homogenates from oocytes expressing 
2
2, 24, 32, 34,
42, or 44. Efficacy (relative to acetylcholine) and
potency were measured electrophysiologically with oocytes expressing
34, 42, and 44. Hydroxy, dimethylamino, and
trifluoromethanesulfonate analogs had affinities too low for accurate
measurement. The bromo analog had affinities 4- to 55-fold greater at
2 than at 4-containing receptors, modestly greater efficacy at
44 than at 42, and 5- to 10-fold greater potency at a44
than at 34 or 42. The fluoro analog displayed affinities
52- to 875-fold greater at 2- than at 4-containing receptors,
efficacy at 44 receptors 3-fold greater than at 42 and
34, and was equipotent at all receptors tested. The norchloro
analog showed affinities 114- to 3500-fold greater at 2- than at
4-containing receptors, 2-fold greater efficacy at 42 and
44 than at 34, and 4- to 5-fold greater potency at 44
and 34 than at 42. The amino analog displayed affinities
10- to 115-fold greater at 2- than at 4-containing receptors,
3-fold greater efficacy at 34 than at 42, and 2- to 4-fold
greater potency at 34 and 44 than at 42. Although these compounds displayed a variety of differences in affinity, efficacy, and potency, with one exception (binding affinity and functional potency at 44 receptors) there were no significant correlations among these properties.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Neuronal
nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion
channels assembled as pentamers of and subunits, forming a
variety of different receptor subtypes. There are nine known subunits (2-10) and three known subunits (2-4) for neuronal nAChRs (Corringer et al., 2000
; Elgoyhen et al., 2001). These
subunits assemble in a variety of homomeric and heteromeric combinations to form receptors with different biophysical and pharmacological properties and with distinct distributions in the
central and peripheral nervous systems (Corringer et al., 2000). A
predominant CNS nAChR is the 42 subtype, whereas a predominant
ganglionic nAChR is the 34* subtype (Lucas et al., 1999; Xu et
al., 1999; Picciotto et al., 2000; Quik et al., 2000). Neuronal nAChR
ligands with potential subtype selectivity are under investigation as
analgesics (Qian et al., 1993; Rogers and Iwamoto, 1993; Badio and
Daly, 1994), anxiolytics (Decker et al., 1995; Brioni et al., 1997),
and as therapeutics for CNS disorders, including Alzheimer's Disease,
Parkinson's Disease, and schizophrenia (Qian et al., 1993; Rogers and
Iwamoto, 1993; Vidal, 1996; Brioni et al., 1997; Hellstrom-Lindahl et
al., 1999).
Epibatidine is a potent agonist at neuronal nAChRs (Badio and Daly,
1994; Gerzanich et al., 1995; Alkondon et al., 1998). Epibatidine is
reported to exhibit high-potency analgesic activity with a longer
duration of action than nicotine (Qian et al., 1993; Rogers and
Iwamoto, 1993; Badio and Daly, 1994), and epibatidine analogs are
currently under investigation as nonopioid analgesics. However,
epibatidine is reported to produce various toxicities in rodents,
including increased heart rate, motor incoordination, and seizure
(Sullivan et al., 1994; Bonhaus et al., 1995; Horti et al., 1998). The
toxicity of epibatidine may arise from its capacity to activate many
different neuronal nAChR subtypes. Separation of the analgesic effects
from the toxicities may be possible if subtype-selective analogs of
epibatidine can be developed.
In this study, we examine the effects of modifying the epibatidine
molecule on equilibrium binding affinity, efficacy, and functional
potency at simple heteromeric neuronal nAChRs. In particular, we focus
on the effect that 2'-pyridine ring substitutions have on selectivity
for subunit combinations (42 and 34) representative of
neuronal nAChRs found in the central and peripheral nervous systems, respectively.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials. Xenopus laevis frogs were purchased from Nasco (Ft. Atkinson, WI). Care and use of Xenopus frogs in this study have been approved by the University of Miami Animal Research Committee and meets the guidelines of the National Institutes of Health. RNA transcription kits were from Ambion (Austin, TX). [3H]Epibatidine was from PerkinElmer Life Sciences (Boston, MA). Acetylcholine, (+)-epibatidine, gentamicin, HEPES, polyethylenimine, and 3-aminobenzoic acid ethyl ester were from Sigma-Aldrich (St. Louis, MO). Collagenase B was from Roche Molecular Biochemicals (Indianapolis, IN). 934-AH glass microfiber filters were from Whatman (Clifton NJ).
Epibatidine Analogs.
Norchloro-epibatidine (NEP),
fluoro-norchloro-epibatidine (NFEP), bromo-norchloro-epibatidine
(NBEP), amino-norchloro-epibatidine (NNEP),
hydroxy-norchloro-epibatidine (NOHEP),
dimethylamino-norchloro-epibatidine (NDMNEP), and
trifluoromethanesulfonate-norchloro-epibatidine (NTEP) were synthesized
as described previously (Carroll et al., 2001). All analogs are
racemic. Structures are shown in Fig. 1.
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Expression of Neuronal nAChRs in Xenopus
Oocytes.
cDNA clones encoding rat 2, 3, 4, 2, and 4
subunits in the pGEMHE high-expression vector (Liman et al., 1992) were
used for cRNA transcription.
m7G(5')ppp(5')G-capped cRNA was synthesized in
vitro from linearized template cDNA using an mMessage mMachine
kit (Ambion). Mature X. laevis frogs were
anesthetized by submersion in 0.1% 3-aminobenzoic acid ethyl ester and
oocytes were surgically removed. Follicle cells were removed by
treatment with collagenase B for 2 h at room temperature. Oocytes
were injected with 8 to 18 ng of cRNA in 23 to 50 nl of water and
incubated at 19°C in modified Barth's saline [88 mM NaCl, 1 mM KCl,
2.4 mM NaHCO3, 0.3 mM
Ca(NO3)2, 0.41 mM
CaCl2, 0.82 mM MgSO4, 100 µg/ml gentamicin, 15 mM HEPES, pH 7.6] for 2 to 10 days. RNA
transcripts encoding each subunit were injected into oocytes at a molar
ratio of 1:1.
Competition Binding Assays.
Crude membrane homogenates were
prepared from Xenopus oocytes expressing various neuronal
nAChR subunit combinations as described previously (Parker et al.,
1998). Briefly, up to 15 oocytes (depending on expression levels) were
homogenized per milliliter of buffer containing 140 mM NaCl, 1.5 mM
KCl, 2 mM CaCl2, 1 mM
MgSO4, and 25 mM HEPES, pH 7.5, with 0.1 mM
phenylmethylsulfonyl fluoride added immediately before the
experiment, using a model PT 10/35 homogenizer (Brinkman, Atlanta, GA).
Homogenates were centrifuged at 4°C at 2000g for 10 min.
The supernatant was removed for use in experiments, avoiding both the
surface lipid layer and the pellet. Receptor expression levels averaged
480 fmol/mg of total protein (16 fmol/oocyte).
, 2001).
Because of the variation in receptor expression level from day to day
after injection of the oocytes and among oocyte batches, all results
were normalized as the percentage of maximum specific binding.
In Table 1 and Fig. 2,
Kd values for (±)-epibatidine,
obtained by saturation analysis in Parker et al. (1998), are compared with Ki values derived from
competition analysis (as described above). To ensure the validity of
this comparison, we performed competition analysis for (±)-epibatidine
and 42 receptors. We obtained a
Ki of 42 ± 2 pM (mean ± S.E.M. of three experiments, each in triplicate), similar to the
Kd of 30 ± 4 pM obtained in saturation analysis.
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). Kd values shown in Table 1 were
used in the calculation of Ki.
Electrophysiological Methods.
Agonist-induced current
responses were measured under two-electrode voltage clamp, using a
TEV-200A voltage-clamp unit (Dagan, Minneapolis, MN). Micropipettes
were filled with 3 M KCl and were used at resistances of 0.3 to 2.0 M
. Current responses were recorded at a holding potential of 
40 mV
to minimize the contribution of calcium-activated chloride channels.
Recordings were sampled at 100 Hz and filtered at 20 Hz (3 db). Data
was acquired, stored, and analyzed on a Macintosh G3 computer using
AxoGraph 4.6 software (Axon Instruments, Union City, CA).
42, 20 µM;
34, 110 µM; and 44, 5 µM). Current responses to
epibatidine and epibatidine analogs were normalized to the preceding
acetylcholine-induced response, allowing determination of each test
drug response relative to the maximum response to acetylcholine at each
receptor. Initial experiments with epibatidine determined with and
without this normalization resulted in different efficacies (Fig.
3A). All subsequent experiments were
normalized as described above.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Epibatidine Analogs.
Seven racemic analogs of epibatidine,
with substitutions at the 2' position of the pyridine ring, were
synthesized as described previously (Carroll et al., 2001). The
structures of epibatidine and the analogs are shown in Fig. 1.
Competition for [3H]Epibatidine Binding by Novel
Epibatidine Analogs.
We determined the equilibrium binding
affinities of the epibatidine analogs for several neuronal nAChR
subunit combinations (22, 32, 42, 24, 34,
and 44) by competition for
[3H]epibatidine binding to Xenopus
oocyte homogenates. The calculated Ki
values and Hill coefficients derived from the competition analyses are
shown in Table 1. Several analogs (NOHEP, NDMNEP, and NTEP) had
affinities that were too low to accurately measure. Rough estimates of
the affinities of these analogs for 2-containing receptors are
provided in Table 1. No estimates of affinity were obtained for
4-containing receptors. These three epibatidine analogs were not
pursued further. All subsequent work in this study is focused on the
four analogs with measurable affinities (NEP, NFEP, NBEP, and NNEP).
), the 2-containing receptors displayed
higher affinities for the epibatidine analogs than did the
4-containing receptors. However, some of the analogs displayed much
greater selectivity for 2-containing receptors over 4-containing receptors than we have observed with other compounds. Differences in
affinity for different receptors based on the subunit were also
observed, but the differences were modest. The binding affinities of
epibatidine and the four analogs for the various neuronal nAChR subunit
combinations are compared in Fig. 2.
The bromo-substitution in NBEP resulted in an analog with affinities
for the various subunit combinations that were very similar to those of
epibatidine itself. The fluoro-substitution (NFEP) resulted in an
analog with increased affinity for 2-containing receptors and
decreased affinity for 4-containing receptors. The largest
difference was seen for 3-containing receptors, with the 32
receptor having a 550-fold higher affinity than the 34 receptor.
The norchloro analog (NEP) was also highly selective for
2-containing receptors. Again, the greatest difference in affinity
was seen with 32 and 34 (a 3500-fold difference). The amino
substitution (NNEP) caused a loss of affinity for both 2- and
4-containing receptors; however, NNEP remained selective for 2
receptors over 4 receptors.
Electrophysiological Analysis.
We examined the functional
potency and efficacy of (+)-epibatidine and the four analogs that
displayed measurable equilibrium binding affinities (NBEP, NFEP, NEP,
and NNEP) for activation of three neuronal nAChR subunit combinations.
42 is a major subtype in the CNS, whereas 34 represents a
subtype (34*) found in the periphery (for explanation of
nomenclature, see Lucas et al., 1999). The 44 subunit combination
was also examined as a point of comparison between 42 and
34.
42 receptor reported in the literature (see
Discussion), we initially constructed concentration-response
curves for epibatidine at 42 receptors using two different
protocols (Fig. 3A). In both protocols, three applications of
acetylcholine (20 µM, the EC20 for 42
receptors) were given before epibatidine applications to ensure
stability of responses. In the first protocol, the epibatidine dose-response curve was determined by measuring epibatidine responses in succession and normalizing to the ACh response immediately preceding
the first epibatidine response. In the second protocol, a normalizing
ACh application (20 µM) was added before each epibatidine application
to correct for possible receptor desensitization or inactivation after
repeated agonist application. When dose-response curves for epibatidine
were determined by the first method, an efficacy of 25 ± 3%
(relative to the maximum ACh response) was obtained. When dose-response
curves were determined by the second method, an efficacy of 58 ± 6% was obtained. Although the efficacy values determined by the two
methods were quite different (p < 0.01), the
EC50 values were similar (11 ± 5 nM by the
first method, 14 ± 5 nM by the second method).
Our observation that the measured efficacy of epibatidine varies
depending on the normalization method (Fig. 3A) suggested that
epibatidine may be causing a long-lasting inactivation of the
receptors. To test this idea, we examined the effect of high concentrations of ACh or epibatidine on subsequent responses to ACh
(Fig. 3B). Agonists were applied to 42-expressing oocytes for
10 s at 5-min intervals. Three applications of 20 µM ACh were followed by an application of 75 µM ACh, or a concentration of epibatidine (45 nM), yielding a similar degree of receptor activation. Three more applications of 20 µM ACh followed. These last three applications of 20 µM ACh are plotted in Fig. 3B as a percentage of
the 20 µM ACh application immediately preceding application of the
high concentration of ACh or epibatidine. Although application of the
high concentration of ACh had no effect on subsequent ACh responses,
application of the high concentration of epibatidine resulted in a
decrease in subsequent ACh responses. This effect of epibatidine did
not reverse after 15 min of washing. In light of this apparent
inactivation of a portion of the receptors upon epibatidine exposure,
we decided to use the second method (renormalizing after each
epibatidine application) in all subsequent work.
In addition to 42, we also examined epibatidine activation of the
34 and 44 receptors. Figure 3C compares the epibatidine dose-response curves for 42, 34, and 44 receptor
subtypes. At 44 receptors, epibatidine exhibited an efficacy of
71 ± 7%, similar to the efficacy at 42 receptors. At the
34 receptor, epibatidine exhibited an efficacy of 39 ± 3%,
significantly lower than at either the 42 or 44 receptor.
The fluoro-substituted analog (NFEP) displayed the greatest improvement
in subtype selectivity (in terms of efficacy). Dose-response curves for
NFEP at each receptor subtype are shown in Fig. 3D. Although NFEP
exhibited high efficacy at 44 receptors (131 ± 9%),
efficacies were significantly lower at 42 and 34 receptors (41 ± 10 and 40 ± 3%, respectively). Although NFEP
produced differences in maximum response, the
EC50 values were similar for the three receptor
subtypes (Table 2). Dose-response curves
were also constructed for NBEP, NEP, and NNEP activation of 42,
34, and 44 receptors. Efficacies relative to ACh for
epibatidine and the four analogs are compared in Fig.
4. Efficacy, EC50,
and nH values are provided in Table 2.
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42, 34, and
44 receptors (65 ± 8, 82 ± 20, and 102 ± 10%,
respectively). The efficacy at 42 receptors was significantly
less than at 44 receptors. NBEP was significantly less potent at
42 (EC50 = 94 ± 6 nM) and 34
(EC50 = 189 ± 51 nM) receptors relative to
44 (EC50 = 20 ± 8 nM).
The amino-substituted analog (NNEP) was the only analog to display
selectivity (in terms of efficacy) for the 34 receptor over the
42 receptor. NNEP displayed an efficacy at the 34 receptor
(82 ± 20%) that was similar to ACh, whereas the efficacy of NNEP
at the 42 receptor was significantly lower (29 ± 4%). NNEP
was less potent at 42 (EC50 = 7600 ± 627 nM) than at 34 (EC50 = 3240 ± 449 nM) and at 44 (EC50 = 1840 ± 622 nM).
NEP exhibited high efficacy at both 42 and 44 receptors
(112 ± 19 and 147 ± 34%, respectively). This analog
exhibited a significantly lower efficacy of 61 ± 10% at 34
receptors. NEP was less potent at 42 receptors
(EC50 = 3630 ± 646 nM) than at 34
(EC50 = 702 ± 57 nM) and 44
(EC50 = 982 ± 54 nM).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we have examined the equilibrium binding affinities
and functional activities of several epibatidine analogs at neuronal
nAChRs expressed in Xenopus oocytes. We found that the
epibatidine analogs with measurable binding affinities were selective
for 2-containing receptors over 4-containing receptors. This is
similar to what we have previously reported using epibatidine and
several common agonists (Parker et al., 1998). However, the degree of
selectivity was much greater than in our previous work. In particular,
the fluoro-substituted (NFEB) and norchloro (NEP) analogs of
epibatidine showed several hundred- to several thousand-fold selectivity for 2-containing receptors. In general, substitution of
the 2' chlorine of epibatidine with electron-withdrawing groups, such
as the fluoro and bromo groups, or the neutral hydrogen, resulted in
analogs with high affinity for 2-containing receptors and low, but
measurable affinities for 4-containing receptors. Substitution with
electron-donating groups (hydroxy and dimethylamino) resulted in
affinities for all subunit combinations that were too low to accurately
measure. However, effects on affinity could not be attributed solely to
electron-donating or -withdrawing properties of the substituents.
Substitution with the electron-withdrawing trifluoromethanesulfonate
group produced an analog with a very low affinity for all subunit
combinations, perhaps due to the large increase in steric bulk. Also,
substitution with the electron donating amino group resulted in an
analog with moderate affinities for some subunit combinations.
Although the great increase in selectivity seen in the equilibrium
binding assays was promising, the affinity values for the analogs
derived from the binding assay were not generally predictive of the
relative potencies determined in the functional assay (Tables 1 and 2).
For example, the equilibrium binding of NFEP for the 42 receptor
was 228-fold greater than the affinity for the 34 receptor.
However, NFEP displayed equal functional potency at the 42 and
34 receptors. NEP provides an even more striking example.
Although the binding affinity of NEP was 1300-fold greater for 42
than for 34, the functional potency of NEP was 5-fold greater at
the 34 receptor than at the 42 receptor. For both 42
and 34, the functional potency of epibatidine and the four analogs failed to correlate with the equilibrium binding affinity (Table 3). However, for 44 there
was a significant correlation between functional potency and
equilibrium binding affinity.
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The affinity measured in our binding assay reflects a distribution of
receptor states. Because the receptors are in equilibrium between
closed, open, and desensitized states, the binding affinity measured is
dependent on agonist affinities for the individual states as well as
the equilibrium constants for transitions among the different states.
The desensitized state of the receptor has an exceptionally high
affinity for agonists. Furthermore, the open activated state of the
receptor is transient, and in our binding assay, the agonist
concentration is too low for significant binding to the closed state of
the receptor. This suggests that the equilibrium binding affinity
predominantly reflects the affinity of agonists for the desensitized
state. Because of the higher affinity of agonists for this state,
equilibrium binding affinities are 2 to 4 orders of magnitude higher
than that for the closed activateable state (estimated from
EC50 values for activation) measured in
functional assays (Harvey and Luetje, 1996; Parker et al., 1998). The
affinity of epibatidine for the closed activateable state and the
desensitized state has been reported to differ by ~3 orders of
magnitude, depending on receptor subunit combination (Gerzanich et al.,
1995; Gopalakrishnan et al., 1996). Thus, independent variation in
ligand affinity for different states of the receptors may underlie the
differences in selectivity measured in equilibrium binding assays and
functional potency assays of the epibatidine analogs studied herein.
The large effect of 2'-pyridine ring substitutions on subunit
selectivity in equilibrium binding assays that we have observed is
interesting in light of the recently reported computational docking of
epibatidine into the binding site of a homology model of the
extracellular domain of an 7 homomeric neuronal nAChR (Le Novere et
al., 2002). In this model, which is suggested to represent the
desensitized form of the receptor, the 2'-pyridine chlorine of
epibatidine interacts with residues of the "complementary
component" of the binding pocket. In heteromeric neuronal nAChRs,
such as 42 and 34, the complementary component is supplied
by the subunit. Although the selectivity in equilibrium binding
assays will be of use in experimental identification of receptor
subunit composition, identification of analogs of potential therapeutic
usefulness requires assessment of functional potency and efficacy.
The reported efficacy of epibatidine for activation of 42
receptors varies from ~20 to ~100% of the maximum ACh response (Gerzanich et al., 1995; Buisson et al., 2000; Spang et al., 2000). We
have found that the measured efficacy of epibatidine is dependent on
the method used to normalize the data (Fig. 3A). If the responses to
epibatidine are recorded in succession and normalized to an ACh
response that precedes the entire series of epibatidine responses, the
measured efficacy is quite low (25 ± 3%). If normalizing ACh responses are interleaved between the epibatidine responses then the
measured efficacy is higher (58 ± 6%). This difference seems to
be due to exposure to epibatidine causing an inactivation of a portion
of the receptors. This inactivated state seems to be relatively
long-lived, because the receptors do not reappear even after extensive
washing (Fig. 3B). Buisson et al. (2000) also observed this effect of
epibatidine, proposing that epibatidine remains tightly bound,
maintaining the receptor in a nonactivatable state. The remaining
functional receptors did not seem to be affected, because
EC50 values, Hill coefficients, and
desensitization rates before and after epibatidine exposure were the
same (Buisson et al., 2000). This effect of epibatidine supports the
necessity of repeated ACh normalization during epibatidine and analog
efficacy measurements. We also observed decreases in the amplitude of
the normalizing ACh applications after application of the various epibatidine analogs (data not shown).
Similar to what we observed for functional potencies, equilibrium
binding affinity values for the epibatidine analogs were not generally
predictive of the relative efficacies determined in the functional
assay (Tables 1 and 2). For example, although NEP was selective for
42 over 34 in the binding assay (1300-fold) and the
efficacy assay (2-fold), NFEP was selective for 42 over 34
(228-fold) and 44 (52-fold) in the binding assay, but was selective for 44 over 42 and 34 (3-fold) in the
efficacy assay. Another striking example is provided by NNEP, which had a 20-fold greater affinity for 42 receptors than for 34
receptors, but was 3-fold more efficacious at 34 receptors than
at 42 receptors. There were no significant correlations between
equilibrium binding affinity and efficacy for 42, 34, or
44 (Table 3).
The large increases in neuronal nAChR subtype selectivity that we
observed in equilibrium binding assays with several epibatidine analogs
suggested that these analogs might have useful functional selectivity
for CNS neuronal nAChRs (typified by 42) over peripheral neuronal
nAChRs (typified by 34). However, differences in functional potency and efficacy were modest, suggesting that these analogs are
unlikely to be selective enough for CNS neuronal nAChRs over peripheral
nAChRs to avoid peripheral toxicity. This has been a problem with other
epibatidine analogs. Halogen-substituted and methylated analogs of
epibatidine have been reported to produce fewer toxicities in rodents
(Badio et al., 1997; Horti et al., 1998); however, the reduction in
toxic effects seen in these studies was not sufficient to ensure safe
therapeutic/toxicity ratios. Epiboxidine, a methylisoxazole analog of
epibatidine has also been reported to produce fewer toxicities than
epibatidine in mice (Badio et al., 1997). However, epiboxidine was less
potent than epibatidine as an antinociceptive agent and, in sodium flux assays, was not significantly less potent than epibatidine at ganglionic nAChRs. Thus, the development of CNS-selective epibatidine analogs will require continued effort. The general failure of efficacy
and functional potency to correlate with binding affinity (Table 3)
indicates that a combined physiological and pharmacological approach
for determination of binding affinity, efficacy, and functional potency
will be required for the development of epibatidine analogs with
improved functional subtype selectivity.
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Acknowledgments |
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We thank Ana Mederos for excellent technical assistance.
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Footnotes |
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Accepted for publication May 6, 2002.
Received for publication March 6, 2002.
1 Present address: Department of Neuroscience, Duke University, Durham, NC 27710.
This work was supported by Grants DA08102 (to C.W.L.) and DA12001 (to F.I.C.) from the National Institute on Drug Abuse. M.J.P. was supported in part by T32-HL07188.
DOI: 10.1124/jpet.102.035899
Address correspondence to: Dr. Charles W. Luetje, Department of Molecular and Cellular Pharmacology (R-189), University of Miami School of Medicine, P.O. Box 016189, Miami, FL 33101. E-mail: cluetje{at}chroma.med.miami.edu
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Abbreviations |
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nAChR, nicotinic acetylcholine receptor; CNS, central nervous system; NEP, norchloro-epibatidine; NFEP, fluoro-norchloro-epibatidine; NBEP, bromo-norchloro-epibatidine; NNEP, amino-norchloro-epibatidine; NOHEP, hydroxy-norchloro-epibatidine; NDMNEP, dimethylamino-norchloro-epibatidine; NTEP, trifluoromethanesulfonate-norchloro-epibatidine; ACh, acetylcholine.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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, p < 0.05; 