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Vol. 55, Issue 1, 39-49, January 1999
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois
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Summary |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Ethanol, at physiologically relevant concentrations, significantly
enhanced high-affinity neuronal nicotinic acetylcholine receptor
(NnAChR) currents insensitive to 
-bungarotoxin (-BuTX-ICs) in
cultured rat cortical neurons in a fast and reversible manner, as
determined by standard whole-cell patch-clamp recording techniques. The
enhancement was (mean ± S.D.) 7.7 ± 5% to 192 ± 52%
upon coapplication of 3 to 300 mM ethanol with 1 to 3 µM ACh. No
plateau for this ethanol-induced enhancement of -BuTX-ICs was
reached. The maximal -BuTX-IC evoked by very high concentrations of
ACh also was increased upon coapplication of ethanol. In contrast,
ethanol weakly inhibited low-affinity NnAChR currents sensitive to
-BuTX (-BuTX-SCs) (5 ± 4% to 29 ± 6% inhibition by
10 to 300 mM ethanol at 300 to 1000 µM ACh). This neuronal
preparation also enabled comparison of ethanol action on NnAChRs with
its action on N-methyl-D-aspartate receptor
currents and 
-aminobutyric acid receptor currents within the same
neurons. Ethanol (100 mM) was more potent at enhancing NnAChR
-BuTX-ICs (61 ± 9% enhancement) than it was at enhancing -aminobutyric acid receptor current (3 ± 3% enhancement
not
statistically significant) or at inhibiting
N-methyl-D-aspartate receptor currents (~35 ± 7% inhibition). Thus, NnAChRs, particularly those
insensitive to -BuTX, may be sensitive conduits through which
ethanol can mediate some of its actions in the brain.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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In
animals, tolerance to some of the reinforcing effects of ethanol can be
induced by chronic nicotine administration, and tolerance to some of
the effects of nicotine can be induced by chronic ethanol
administration (Zacny, 1990
; Collins, 1996). Such cross-tolerance
infers the involvement of central nervous system (CNS) nicotinic
acetylcholine receptors (NnAChRs) in the mediation of ethanol
action in the brain. Ethanol is known to modulate the function of
numerous neurotransmitter-gated receptor-ion channels important in the
CNS, including inhibition of
N-methyl-D-aspartate receptors
(NMDARs), enhancement of -aminobutyric acid receptors (GABAARs), enhancement of type III
5-hydroxytryamine (or serotonin) receptors
(5-HT3Rs), as well as inhibition of
voltage-activated calcium channels (Mullikin-Killpatrick and
Treistman, 1993; Crews et al., 1996; Lovinger, 1997). Although ethanol
modulation of the nicotinic receptors at the neuromuscular junction is
well documented (Miller et al., 1991), the very limited reports
concerning the direct action of acute application of ethanol on NnAChRs
have been restricted to our previous study of ganglionic-type NnAChRs natively expressed by PC12 cells (Nagata et al., 1996) and a recent study by Covernton and Connolly (1997) using cloned NnAChRs
recombinantly expressed in Xenopus oocytes.
Our previous study (Nagata et al., 1996) indicated variable effects on
NnAChR current amplitude upon application of low concentrations of
ethanol, whereas Covernton and Connolly (1997) reported enhancement of
NnAChR currents upon application of high concentrations of ethanol.
Covernton and Connolly (1997) also reported variable effects of
enhancement and inhibition at the lower ethanol concentrations for the
3
4 subtype NnAChR, which is considered to be a major NnAChR
subtype in PC12 cells (Rogers et al., 1992). Considering that ethanol
and nicotine are the two most used and abused drugs, and that their
effects are most certainly manifest in the brain, more effort to
delineate the molecular and cellular mechanisms of their effects on
NnAChRs expressed in central neurons is of crucial importance.
However, because of diffuse expression of NnAChRs in the brain and the
resultant difficulty of obtaining NnAChR responses in primary neuronal
cultures, the effects of ethanol on the primary NnAChRs expressed in
central neurons (generally thought to be the 42 and 7
subtypes) have not been forthcoming. This has led to attempts to
establish and characterize cloned NnAChRs in recombinant expression
systemsan approach also seemingly plagued with difficulties. Even
when achieved it has been indicated that cloned NnAChRs expressed
either transiently in Xenopus oocytes or stably in mammalian
cell lines may not accurately represent native NnAChRs in neurons
(Cooper and Millar, 1997; Lewis et al., 1997; Sivilotti et al., 1997).
In the present study, long-term cultures of rat cortical neurons were
established from which a tangible number of neurons were found to
exhibit nicotinic acetylcholine currents. These ACh-evoked NnAChR
currents had sufficient amplitude to enable apt investigation of
ethanol action on central NnAChR-mediated ion channel
activity. The primary findings were that ACh-evoked NnAChR currents
sensitive to -bungarotoxin (-BuTX-SCs) were modestly inhibited by
ethanol, whereas ACh-evoked NnAChR currents insensitive to
-bungarotoxin (-BuTX-ICs) were significantly enhanced by low
concentrations of ethanol. The neuronal preparation also made it
possible to compare ethanol sensitivities of NnAChRs, NMDARs, and
GABAARs within the same neuron. Such comparisons
demonstrated that the potency of ethanol in enhancing NnAChR
-BuTX-ICs was greater than that for the enhancement of
GABAAR currents and that for the inhibition of
NMDAR currents exemplifying the diversity of outcomes that ethanol
could produce even at the single neuron level.
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Materials and Methods |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Cell Preparation.
Rat cortical neurons were isolated and
cultured by a procedure slightly modified from that described elsewhere
(Marszalec and Narahashi, 1993). In brief, rat embryos were removed
from 17-day pregnant Sprague-Dawley rats under methoxyflurane
anesthesia. Small wedges of frontal cortex were excised and
subsequently incubated in phosphate-buffered saline solution containing
0.25% (w/v) trypsin (Type XI; Sigma-Aldrich, St. Louis, MO) for 20 min
at 37°C. The digested tissue was then mechanically triturated by
repeated passages through a Pasteur pipette, and the dissociated cells
were suspended in neurobasal medium with B-27 supplement (GiBCO,
Gaithersburg, MD) and 2 mM glutamine. The cells were added to 35-mm
culture wells at a concentration of 100,000 cells/ml. Each well
contained five 12-mm coverslips (previously coated with
poly-L-lysine) overlaid with confluent glia that had been
plated 2 to 4 weeks earlier. The cortical neuron/glia cocultures were
maintained in a humidified atmosphere of 90% air/10% CO2
at 37°C. Cells used for electrophysiological experiments were
cultured for 1 to 9 weeks.
Electrophysiological Recordings.
All chemicals were reagent
grade or higher from Sigma-Aldrich unless otherwise specified. The
standard external solution consisted of: 140 mM NaCl, 5 mM KCl, 1.5 mM
CaCl2, 1 mM MgCl2, 15 mM acid-HEPES, 10 mM
Na-HEPES, with 3 µM LaCl3, 0.2 µM TTX, and 0.3 µM
atropine sulfate, pH 7.3. Two different pipette solutions were used:
one with high calcium-buffering capacity, and a second with 5 mM Mg-ATP added. The two different solutions were used to evaluate the possible effects of internal Ca++ or energy processes on NnAChR
activity. The pipette solution with high calcium-buffering capacity
consisted of: 140 mM cesium-gluconate, 15 mM NaCl, 5 mM
potassium-gluconate, 15 mM acid-HEPES, 10 mM sodium-HEPES, 35 mM
cesium-BAPTA
[1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; free BAPTA 
20 mM], 12 mM calcium-gluconate (free Ca++
100 nm), 4 mM magnesium-gluconate (free Mg++ 1 mM).
The ATP-containing pipette solution consisted of: 140 mM
cesium-gluconate, 15 mM NaCl, 5 mM potassium-gluconate, 1 mM MgCl2, 15 mM acid-HEPES, 10 mM sodium-HEPES, 11 mM EGTA, 1 mM CaCl2, 5 mM magnesium-ATP, and 0.2 mM sodium-GTP.
Pipettes were pulled from borosilicate glass capillaries (Kimax-51; 1.8 mm o.d., 1.5 i.d.; Kimble Glass Co., Vineland, NJ) or Clark Patch
Glass (PG120T; 1.2 o.d., 0.93 i.d.; Warner Instruments,
Hamden, CT) and lightly fire-polished to a final resistance of 2 to 4 M
when filled with the internal solution.
3 dB) via either an Axopatch
200 amplifier or an Axopatch 1-C amplifier (Axon Instruments, Foster
City, CA). The holding potential was -70 mV, unless otherwise specified. Recorded currents were directly digitized at 1 to 10 kHz,
acquired to the hard disk of the microcomputer via a Digidata 1200 ADC/DAC interfaced to a microcomputer under control of the ClampEx
module of the Pclamp6 software package (Axon Instruments). Agonists and
test compounds were applied to cells via a modified U-tube system
(Marszalec and Narahashi, 1993) with a solution-exchange time of 10 to
15 ms as measured by change in junction potential. Dihydro--erythroidine·HBr (DHE) and mecamylamine·HCl were
obtained from Research Biochemicals International, Natick, MA. In the
present study, the term "coapplication" refers to the short (0.5-3
s), simultaneous application of effector(s) with agonist via the U-tube only (the application solution contains both the effector(s) and agonist), not via perfusion through the external bathing solution. The
term "pre-exposure" refers to the application of effector(s) via
perfusion through the external bathing solution. The ethanol used in
these experiments was nondenatured, absolute (200 proof) ethyl alcohol
USP (Midwest Grain Products, Weston, MO) bought and stored in glass containers.
Analysis. Current records initially were analyzed via the ClampFit module of the Pclamp6 to assess whole-cell current amplitudes and decay kinetics. Cumulative concentration-response results subsequently were compiled for graphical analysis in SigmaPlot. Analysis of variance and/or Student's t tests were performed to assess significance of differences between test and control measurements.
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Description of Cortical Neurons in Culture.
The neuronal
cultures consisted of a variety of differently shaped neurons appearing
as pyramidal, irregularly rounded, ovoid, and rectangular. After about
1 week in culture, most neurons projected from two to as many as five
or more neurites. The ovoid cells generally projected two major
neurites, the pyramidal cells projected three to four, and the
irregularly rounded and rectangular cells projected four or more. After
about 2 weeks, a dense and complex network of neuronal processes was
established. At this time and later, it became apparent that synapses
between these cultured neurons had "reformed." This was evidenced
by spontaneous activity that was recorded as excitatory and/or
inhibitory postsynaptic currents, primarily mediated by NMDA and GABA
receptors (Marszalec et al., 1998). After about 4 to 5 weeks, many of
the pyramidal cells began to show signs of degeneration (blebbing and
neurite degradation), while the irregularly rounded, ovoid, and
rectangular cells remained relatively healthy looking until about 8 to
9 weeks in culture. Also more apparent after 4 to 5 weeks in culture
were persistent, miniature spontaneous excitatory and inhibitory
currents, which were not blocked by tetrodotoxin. Because such activity potentially could cause interference in assessing evoked NnAChR currents, 6-cyano-7-nitroquinoxaline-2,3-dione (1-3 µM),
2-amino-5-phosphonopentanoic acid (30 µM), and bicuculline (30 µM)
or picrotoxin (30 µM) often were added to the external solution to
reduce these miniature spontaneous excitatory and inhibitory currents.
Inclusion of these agents in the extracellular solution reduced only
the background activity and had no observable effect on the ACh- or
nicotine-evoked currents.
Properties of NnAChR Currents.
ACh in the presence of 0.1 to
0.3 µM atropine was routinely used to assess NnAChR activity in
cultured cortical neurons. In general, in the absence of
-BuTX all neurons that responded to short applications of ACh
(at concentrations 
300 µM) always exhibited a quickly decaying
current. In many neurons (pyramidal-shaped neurons in particular,
although not restricted to this type of neuron) this quickly decaying
current was the only type of current observed (Fig
1A). Some neurons exhibited a slowly
decaying current component along with the quickly decaying current (Fig
1B). However, in general, neurons did not exhibit the slowly decaying
current by itself.
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-BuTX through the external bathing solution for (Fig. 1A). These -BuTX-sensitive currents (-BuTX-SCs) displayed low ACh affinity (EC50 = 330 ± 30 µM ACh
with a Hill coefficient of 1.3; n = 7) (Fig. 1F) and
showed some rundown that could be attenuated by inclusion of Mg-ATP and
Na-GTP in the pipette solution. No difference in the character of these
currents was observed when high or low
Ca++-buffering-capacity pipette solutions was
used, indicating no interference by calcium-dependent currents in these
assessments. Such properties generally are attributed to homomeric 7
subtype NnAChRs, and, in all likelihood, the -BuTX-SCs are mediated
by 7-type NnAChRs.
The current characterized by slow decay showed no sensitivity to
-BuTX and persisted well after (>10 min) exposure to the toxin (remaining current in Fig. 1B). In the constant presence of 30 to
100 nM -BuTX both perfused through the bath and included in the
application solutions, the -BuTX-ICs could be inhibited by
mecamylamine (Fig. 1C) or DHE (Fig. 1D). These -BuTX-ICs displayed a relatively high ACh affinity (EC50 = 2.7 ± 0.2 µM ACh with a Hill coefficient of 0.8;
n = 23) (Fig. 1F), although considerable cell-to-cell
variance in the calculated ACh EC50 was indicated
by the individual ACh concentration-response relationships, which
ranged from 1.3 to 14 µM ACh. The -BuTX-ICs exhibited no significant rundown whether or not Mg-ATP or Na-GTP was included in the
pipette solution. No significant difference in current character was
observed when using either the high or the low
Ca++-buffering capacity pipette solution.
-BuTX-ICs could also be activated by nicotine,
dimethylphenylpiperazinium (DMPP), and cytisine. At a concentration of
3 µM for all agonists (a concentration chosen so as not to introduce complexities due to desensitization) and in the presence of 30 to 100 nM -BuTX, the -BuTX-ICs were most effectively activated by ACh,
followed closely by nicotine (88 ± 16% that of ACh;
n = 5), with DMPP being modestly effective (40 ± 6% that of ACh; n = 5), and cytisine being least
effective (13 ± 3% that of ACh; n = 4) (Fig.
1E). Perhaps noteworthy is the very apparent slow inactivation of
current evoked by either nicotine or cytisine. This was not an
incidental application/removal methodological effect and was always
observed when currents were evoked with these two agonists, possibly
suggesting differences in affinity or mechanisms of
activation/inactivation for nicotine and cytisine versus ACh and DMPP.
The properties of high ACh affinity, nanomolar to low micromolar
potency of the DHE inhibition, and poor cytisine activation exhibited by the -BuTX-ICs indicates that they most closely resemble 42-subtype NnAChRs (Luetje and Patrick, 1991; Buisson et
al., 1996; Chavez-Noriega et al., 1997; Fenster et al., 1997). However, contributions by other NnAChR subunits, such as 4 and 5, or even
2 or 3 contribution cannot be ruled out at present in the absence
of immunological data and considering the variance in the ACh
sensitivity indicated by the relatively large range in the individual
cell-to-cell ACh EC50 values.
Ethanol on -BuTX-SCs.
To assess the effects of ethanol on
-BuTX-SCs separately from -BuTX-ICs, only those ACh currents
exhibiting low affinity (little or no response at ACh concentrations
30 µM) that quickly decayed back to the baseline (very fast
desensitization) within 1 to 3 s of ACh application were deemed
-BuTX-SCs and used to further assay effects of ethanol.
-BuTX-SCs evoked by concentrations of 300 µM ACh (near its
EC50 for this type of current) showed weak
inhibition of the peak amplitude upon coapplication of ethanol at
concentrations of 10 to 300 mM. To verify that more prominent effects
of ethanol were not missed as a consequence of the very fast onset and
decay of the -BuTX-SCs, triplet comparisons of control (Fig
2A, first set) were compared with that
upon coapplication of 100 mM ethanol (Fig. 2A, second set), with that
upon pre-exposure to 100 mM ethanol via perfusion through the external
bath solution for 3 to 6 min (Fig. 2A, third set), and with that upon
washout of the ethanol (Fig. 2A, fourth set). The pre-exposure protocol
did result in slightly more inhibition. However, pre-exposure likely
sacrifices some specificity in terms of direct action of ethanol on
-BuTX-SC, because ethanol is known to affect many intracellular
regulatory systems (Diamond and Gordon, 1997). Thus, the more prolonged
presence of ethanol increases the possibility of inclusion of indirect effects of ethanol. Still, the detectable effect of ethanol on -BuTX-SCs was quite modest. Yet, although only modest, the
inhibition observed upon coapplication of ethanol was statistically
significant at concentrations of ethanol 30 mM (Table
1). However, over the entire 3 to 300 mM
ethanol concentration range, a half-maximal inhibition was never
attained using the coapplication protocol (Fig. 2B; Table 1).
Furthermore, although a small decrease in the
Imax and a small increase in the ACh
EC50 upon coapplication of 100 mM ethanol were
indicated, as illustrated in Fig. 2C, these changes did not pass tests
for statistical significance. No effects on the kinetics of current
onset or decay were detectable.
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Ethanol on -BuTX-ICs.
The assessment of the effects of
ethanol on -BuTX-ICs separately from -BuTX-SCs was technically
more straightforward. Only those ACh-evoked currents remaining after
constant perfusion of 30 to 100 nm -BuTX exhibiting high affinity
(considerable response at ACh concentrations 30 µM) that never
decayed back to the baseline (slow desensitization) within 1 to 3 s of ACh application were deemed -BuTX-ICs and used to further assay
effects of ethanol.
-BuTX-ICs evoked by concentrations of 3 µM ACh (near its
EC50 for this type of current) exhibited
significant amplitude enhancement upon coapplication of ethanol at
concentrations of 3 to 300 mM (Table 2).
No consistent effects on the kinetics of current decay were found.
Shown in Fig. 3A is enhancement by coapplication of 10, 30, and 100 mM ethanol, which are at the low,
medium, and high range of physiological relevance. Pre-exposure to
ethanol was not necessary for this effect of ethanol on -BuTX-ICs to
be observed, and only coapplication of ethanol with ACh was used to
assay this effect further so as to minimize the inclusion of possible
indirect effects of ethanol. However, for comparative purposes, a few
experiments were conducted whereby ethanol (100 mM) was perfused
through the external bath solution for 3 to 6 min (Fig. 3B). Using this
pre-exposure protocol, enhancement of the -BuTX-ICs similar to that
upon simultaneous coapplication was observed. Although, after washout
of bath-perfused ethanol, the current often did not fully recover,
which again may be indicative of additional, less direct actions of
ethanol on -BuTX-insensitive NnAChRs.
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-BuTX-ICs that were statistically significant down to
3 mM ethanol (Table 2). Very small enhancements of -BuTX-ICs were
sometimes observed upon coapplications of 1 mM ethanol, but were not
statistically significant. The marked enhancement at 300 mM ethanol
showed no apparent trend toward a maximum. This notion was corroborated
by even greater enhancement upon coapplication of 1 M ethanol (Fig.
3D). However, note that the coapplication of 1 M ethanol appears to
induce changes in the kinetics of current decay, which may suggest
additional mechanisms of ethanol action at such high ethanol concentrations.
Ethanol was able to enhance -BuTX-ICs at both low and high
concentrations of ACh (Fig. 4A). The
ethanol-induced enhancements were greater at lower than at higher
concentrations of ACh (Fig. 4B, inset), yet coapplication of ethanol
still resulted in a somewhat substantial increase in the calculated
Imax (32 ± 11% increase; P < .004) (Fig. 4B). Coapplication of ethanol also caused a small but
significant decrease in the calculated ACh EC50
(1.3 ± 0.4 µM decrease; P < .01) without
significantly changing the Hill coefficient (0.02 ± 0.1 decrease;
P < .9). The higher variance in ethanol enhancement at
low concentrations of ethanol (Fig. 4B, inset) likely
reflects the variation in cell-to-cell ACh sensitivity noted above.
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Specificity of Ethanol Enhancement of -BuTX-ICs.
When
applied in the absence of ACh, ethanol at concentrations >300 mM
sometimes generated small currents, again perhaps insinuating that
ethanol may exert additional mechanisms of action at very high
concentrations. However, it generally was found that little or no
current could be observed upon applications of ethanol 300 mM (Fig.
5A, first record). Such currents, when
observed, were very small (<10% of control) and could not explain the
large enhancement of -BuTX-ICs when ethanol was coapplied with ACh.
Furthermore, the enhancements of -BuTX-ICs at 300 mM ethanol were
specific to the ACh-evoked -BuTX-ICs as demonstrated by the
enhancement of ACh-evoked -BuTX-ICs induced by 300 mM ethanol being
inhibited by coapplication of DHE (Fig. 5A).
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Comparison of Ethanol on -BuTX-ICs with Those Evoked by NMDA and
GABA.
As described in the introductory remarks, ethanol can affect
many different types of receptor channels. Much importance of ethanol
action in the brain has been attributed to its effects on NMDARs and
GABAARs. Therefore, experiments were conducted to compare
the effect of ethanol on ACh-, GABA-, and NMDA-evoked currents in the
cortical neuron preparation. To avoid cell-to-cell variations and to
provide inherent controls for ethanol action on each receptor type, the
effects of ethanol were compared between GABA and ACh responses or
between NMDA and ACh responses that were evoked from the same neuron.
As depicted in Fig. 5B, GABA-evoked currents were not significantly
enhanced by coapplication of 100 mM ethanol, but could be modestly
enhanced by coapplications of 300 mM ethanol. ACh-evoked -BuTX-ICs
in the same neurons were markedly enhanced by the same concentrations
of ethanol. As shown in Fig. 5C, NMDA-evoked currents were inhibited by
coapplications of 100 mM ethanol, whereas ACh-evoked -BuTX-ICs in
the same neuron were enhanced by coapplications of 100 mM ethanol. Such
actions of ethanol on GABAAR and NMDAR function depicted in
these results are not new findings. However, coupling these effects of
ethanol with that on NnAChRs at the intraneuron level illustrates that the overall effect ethanol exerts on neuronal activity would be quite
dependent on the receptor constituency of each individual neuron.
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The results in the present study indicated that ethanol, at
physiologically relevant concentrations, weakly inhibited ACh-evoked -BuTX-SCs, whereas it significantly enhanced -BuTX-ICs. These results exemplify the diverse effects ethanol can have on two types of
NnAChRs and involve complexities that are best discussed by considering
the effects on each separately.
Ethanol on -BuTX-SCs.
The results in the present study
indicated statistically significant inhibition of -BuTX-SCs upon
coapplication of concentrations of ethanol 30 mM with 300 µM
ACh (near its EC50). However, the findings that an actual
IC50 for ethanol inhibition was never observed, and that
changes in the Imax and ACh EC50 for the
response were not statistically significant indicate that the
inhibition should not be interpreted without careful consideration of
the limitations of the experimental methods used in this assessment. That is, the 10- to 15-ms solution exchange time estimated for the
-tube application system utilized was longer than the 3- to 4-ms
rise time to reach 10 to 90% of the peak -BuTX-SCs, and was merely
about equal to the 18- to 22-ms decay for this current. Thus, accurate
resolutions of the peak amplitude and/or any small change in current
onset or decay, particularly at higher ACh concentrations, were in all
likelihood not achieved. Even if the measured peak current appears
consistent in controls, problems with interpreting changes still
remain. For example, if ethanol actually enhances the amplitude of
-BuTX-sensitive NnAChR current, but also enhances the rate of
current fast decay (often interpreted as acute desensitization), the
combined effect causes the peak current to be even shorter lived. This
shorter-lived peak, although greater in amplitude than the control,
would actually appear to be lesser in amplitude due to it being even
more inadequately resolved than the control. Therefore, the inhibition
observed in the present can only be characterized as "apparent
inhibition" and awaits employment of a faster effector application
system that can accurately and consistently resolve the -BuTX-SC
response. Further interpretations of the effects of ethanol on the
-BuTX-SCs would be premature at this time.
-BuTX-SCs can be considered as mediated by the 7-subtype
NnAChR, and because similar activation (6-7 ms time-to-peak) and
desensitization (12-20-ms decay) kinetics for the cloned 7 subtype
NnAChRs have been reported in other studies (Puchacz et al., 1994;
Gopalakrishnan et al., 1995), inconsistencies in results obtained by different investigators studying this type of receptor might be expected considering the problem of accurately resolving the
response. For instance, the lack of obtaining a relevant ethanol IC50 for observed inhibition of -BuTX-SCs is
in contrast to the reported 33 mM IC50 for
ethanol inhibition of cloned 7 NnAChRs expressed in
Xenopus oocytes (Yu et al., 1996). This could be an
indication of substantial differences between recombinant and native
NnAChRs; however, it may simply be due to differences in effector
application methodology, because the solution exchange time is
generally on the order of seconds to even minutes in oocyte experiments, which is even less than the 10- to 15-ms solution exchange
time in the present study. This comparison actually may suggest that
the observed inhibition of -BuTX-SCs or 7-mediated currents by
ethanol is only apparent inhibition and that the true effects remain to
be determined.
Ethanol on -BuTX-ICs.
Over the 3 to 300 mM ethanol
concentration range tested, ethanol's consistent effect was only to
enhance the amplitude of the -BuTX-ICs, without significantly
affecting the decay kinetics, with the threshold concentration for
observing this effect at concentrations of 1 to 3 mM ethanol. This is
in contrast to our previous results whereby the more consistent effect
of ethanol at low millimolar to high micromolar concentrations was to
increase the rate of decay of NnAChR whole-cell currents in PC12 cells while causing varied inhibition and enhancement of the current amplitude (Nagata et al., 1996). Although this contrast certainly deserves further attention, it may yet again exemplify the diversity of
ethanol action on different subtypes of NnAChRs, because the primary
functional NnAChRs in PC12 cells are most likely 3-containing NnAChRs in combination with 4 and/or probably 2 and 5, whereas the -BuTX-ICs in cortical neurons of the present study, according to
their pharmacological profile, appear most likely to be mediated by
4-containing NnAChRs in combination with 2. Perhaps recent studies on known combinations of cloned NnAChRs provide more insight into such a contention. That is, Covernton and Connolly (1997) reported
that the current amplitude of cloned 34 NnAChR subtypes expressed
in Xenopus oocytes was the most sensitive of all of the
cloned NnAChR subtypes they tested, being affected by ethanol at
concentrations of as low as 1 mM. Also in that study, the effect of
ethanol at very low concentrations sometimes was seen as enhancement and at other times was seen as inhibition. No indication of changes in
kinetics were mentioned in that study, but the effector application exchange rate in Xenopus oocyte experiments generally
limits accurate assessment of such kinetic parameters. However, Cardoso
et al. (1998) reported the current amplitude of cloned 34 NnAChRs
expressed in Xenopus oocytes to be enhanced by ethanol,
but it was found to be the among the least sensitive of the cloned
NnAChRs they tested. Thus, it appears that ethanol action on 34
NnAChRs is in need of more detailed investigation and may be the
culprit in terms of the contrast between the present results of ethanol action on NnAChRs in cortical neurons and our previous results on
NnAChRs in PC12 cells.
-BuTX-ICs in
cortical neurons, it was found that no apparent plateau for ethanol
enhancement of -BuTX-ICs could be reached at concentrations of
ethanol as high as 1000 mM, and -BuTX-ICs were enhanced even at very
high concentrations of ACh, which were maximal in controls. It is also
noteworthy to point out that ethanol, at high concentrations, is known
to produce anesthetic action. Yet, at all concentrations of ethanol
tested in the present study, only enhancement was observed, which is in
contrast to the inhibitory effects of general anesthetics on cloned
-BuTX-insensitive NnAChRs expressed in oocytes reported recently
(Flood et al., 1997; Violet et al., 1997). Preliminary results obtained
thus far indicate only inhibition of -BuTX-ICs in cortical neurons
by the volatile general anesthetic halothane, as well as the long-chain
alcohol, n-octanol. Thus, apparently ethanol does not have
the same mechanism(s) of action on -BuTX-ICs as these general anesthetics.
Pertaining to mechanism of action, these aspects of the ethanol
enhancement of -BuTX-ICs could be interpreted in several different
ways. One plausible way of interpreting the ethanol-induced enhancement
of -BuTX-insensitive currents is that ethanol increases -BuTX-insensitive NnAChR channel conductance. This would be
consistent with the result that ethanol caused enhancements of the
responses evoked by concentrations of ACh that were maximal in
controls. However, the enhancements induced by higher concentrations of ethanol are difficult to reconcile by a mechanism involving increases in channel conductance alone. If, for example, 25 pS is taken as a
plausible single-channel conductance for -BuTX-insensitive NnAChRs,
and ethanol acts only to increase the open-channel conductance, the
400% enhancement over control induced by 1000 mM ethanol would correspond to a 75 pS increase in single-channel conductance. Such a
large drug-induced conductance increase has never been reported, and
changes in structure and function of a receptor channel sufficient to
cause such an increase in conductance would be difficult to conceive.
Nonetheless, such an action by ethanol cannot be completely ruled out
at this time, and single-channel studies will be necessary to resolve
this issue.
A more plausible interpretation of the results would be for ethanol to
stabilize the open state of -BuTX-insensitive NnAChRs. This has been
suggested as a mechanism for ethanol action on Torpedo nAChRs by Wu et al. (1994), who asserted that the major effect of ethanol is to increase the open/closed equilibrium of nAChRs while
causing only a minor change in their affinity for agonist. Such a
mechanism is consistent with increased enhancement of
-BuTX-insensitive currents upon increasing ethanol concentration and
with the small decrease in the EC50 for ACh
activation exhibited by -BuTX-insensitive responses in the presence
of ethanol. It is also sufficient to explain the ethanol-induced
enhancement of -BuTX-insensitive maximal response if the open
probability for NnAChRs is considerably low when using ACh as an
agonist. Very low channel open probability (Popen = .006-.1) has been reported for
-BuTX-insensitive NnAChRs in chick lateral spiriform nucleus when
using ACh as an agonist (Weaver and Chiappinelli, 1996). Thus, the
results obtained in the present study in which ethanol enhances both
submaximal and maximal -BuTX-insensitive NnAChR responses while
causing only small decreases in the ACh EC50 are
consistent with a mechanism whereby ethanol increases the open/closed
equilibrium of -BuTX-insensitive NnAChRs mainly by increasing the
probability of channel opening.
However, intricacies in the activity of -BuTX-insensitive NnAChRs,
such as the variation in the ACh dose-response evident in the present
study, as well as evidence from other studies indicating that NnAChRs
can enter multiple, long-lived desensitized states (Lester and Dani,
1994) and/or NnAChRs can exist on the surface of neurons as
nonfunctional receptors (Margiotta et al., 1987) unquestionably make
interpreting the action of any effector complicated. Therefore, further
investigation into such details of NnAChR activity is absolutely
necessary to elucidate more fully the actions of ethanol on NnAChRs.
Physiological Relevance.
Many studies (Diamond, 1990) have
established that in naïve subjects, the evident behavioral
changes associated with increases in blood alcohol concentrations are
as follows (behaviorassociated blood alcohol concentration): altered
mood, impaired attention6 to 20 mM; impaired cognition and
coordination, and sedation20 to 40 mM; intoxication, ataxia40 to 65 mM; severe stupor, coma, death65 to 110 mM. In addition, subjects can
achieve what is referred to as acute tolerance, whereby they exhibit
intoxication at a blood alcohol concentration of 40 mM after 1.5 h
of consuming ethanol, yet later appear sober with a 60 mM blood alcohol
concentration after 4.5 h of continued consumption of ethanol.
Heavy drinkers and alcoholics can achieve what is referred to as
chronic tolerance whereby subjects can maintain blood alcohol
concentrations of 30 to 120 mM ethanol after consuming ethanol for
6 h and still appear sober. According to such studies, severe
alcoholics can attain blood alcohol concentrations as high as 330 mM
and still remain conscious and alert.
-BuTX-insensitive NnAChR
function is likely to be an important factor during acute intoxication,
because the enhancement of -BuTX-insensitive NnAChR function over a
10 to 30 mM ethanol concentration range was a significant 10 to 30% at
EC50 ACh. The present study also revealed that at
EC50 ACh, over a 100 to 300 mM ethanol
concentration range, which may be reached in the brain under conditions
of tolerance, 52 to 164% enhancement of -BuTX-insensitive NnAChRs
was evident (although the present study did not focus on whether
NnAChRs might become tolerant). Moreover, the significant ethanol
enhancement of -BuTX-insensitive NnAChR function (30% increase in
Imax) when activated by high concentrations of
ACh is of considerable relevance taken in context of the possible
ranges of neurotransmitter concentrations at synapses.
The enhancement of -BuTX-insensitive NnAChR function perhaps becomes
even more pertinent when considering the mounting evidence indicating
that NnAChRs in the brain may be primarily presynaptic or
perisynaptic and can modulate the release of many other
neurotransmitters. These include dopamine, norepinephrine, GABA, and
glutamate (Role and Berg, 1996; Colquhoun and Patrick, 1997; Lindstrom,
1997; Wonnacott, 1997). Thus, although many reports suggest that
ethanol exerts its effects in the brain mainly via direct modulation of GABAARs and NMDARs as well as voltage-gated
calcium channels, the results obtained in the present study suggest
reevaluation of this tenet so as to include central NnAChRs. This
becomes especially apparent when considering the results of the present
study, which indicate that within a single neuron, multiple
ethanol-sensitive receptors can mutually exist, each with extremely
different ethanol-induced modulation. Thus, the overall effect(s) of
ethanol on such an immense neuronal network as the brain, which is
ultimately determined by the complex summation of ethanol action at
single neurons, would be extremely diverse. The findings that
mecamylamine, a NnAChR antagonist, can counter ethanol-induced dopamine
release in the nucleus accumbens (Blomqvist et al., 1997) in addition to countering ethanol-induced depression of cerebellar Purkinje neuron
firing (Freund and Palmer, 1997) are recent examples indicating NnAChR
involvement in the complexity and diversity of ethanol mediating its
action in the brain.
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Acknowledgments |
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We thank Nayla Hasan for maintenance of cell cultures and Dr. Dennis Twombly for his comments on the manuscript.
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Footnotes |
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Received July 2, 1998; Accepted September 24, 1998
This work was supported by National Institutes of Health Grants R01 AA07836, R01 NS14144, and F32 AA05447.
Send reprint requests to: Dr. Toshio Narahashi, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611-3008. E-mail: tna597{at}nwu.edu
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Abbreviations |
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NnAChR, neuronal nicotinic acetylcholine
receptor;
-BuTX, -bungarotoxin;
-BuTX-SC, -BuTX-sensitive
current;
-BuTX-IC, -BuTX-insensitive current;
DHE, dihydro--erythroidine, DMPP, dimethylphenylpiperazinium;
GABA, -aminobutyric acid;
GABAAR, GABAA receptor;
NMDA, N-methyl-D-aspartate;
NMDAR, NMDA
receptor.
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References |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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  J. Liu, T. Vaithianathan, K. Manivannan, A. Parrill, and A. M. Dopico Ethanol Modulates BKCa Channels by Acting as an Adjuvant of Calcium Mol. Pharmacol., September 1, 2008; 74(3): 628 - 640. [Abstract] [Full Text] [PDF]
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 P. Steensland, J. A. Simms, J. Holgate, J. K. Richards, and S. E. Bartlett Varenicline, an {alpha}4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking PNAS, July 24, 2007; 104(30): 12518 - 12523. [Abstract] [Full Text] [PDF]
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) and 
). The data
points are the mean peak current amplitudes with S.E.M. expressed as a
fraction of the maximal current obtained at 10,000 µM ACh for
). The data points are the mean peak current amplitudes
with S.E.M. expressed as a fraction of the maximal control current
(3000 µM ACh in the absence of ethanol). Data are from at least 5 neurons (different from those used for B). The ACh EC50 in
controls was 300 ± 34 µM ACh with a Hill coefficient of 1.3. The ACh EC50 upon coapplications of 100 mM ethanol was
322 ± 15 µM ACh with a Hill coefficient of 1.4.
). The data points are the mean
peak current amplitudes with S.E.M. expressed as a fraction of the
maximal control current (300-1000 µM ACh). Data are from at least
five neurons (different from those used for Fig. 
flux) are
outward (positive: up) and ACh-evoked