Department of Pharmacology, The George Washington University School of Medicine and Health Sciences, Washington, DC 20037
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ABSTRACT |
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Zhu, Ping Jun and
Vincent A. Chiappinelli.
Nicotine Modulates Evoked GABAergic Transmission in the Brain.
J. Neurophysiol. 82: 3041-3045, 1999.
The effects of nicotine on evoked GABAergic synaptic transmission were
examined using whole cell recordings from neurons of the lateral
spiriform nucleus in embryonic chick brain slices. All synaptic
activities were abolished by the GABAA receptor antagonist, bicuculline (20 µM). Under voltage-clamp with KCl-filled pipettes (holding potential 70 mV), nicotine (0.1-1.0 µM) increased the frequency of spontaneous GABAergic currents in a dose-dependent manner.
Nicotine enhanced electrically evoked GABAergic transmission only at
relatively low concentrations of 50-100 nM (but not 25 nM), which
approximate the concentrations of nicotine in the blood produced by
cigarette smoking. At higher concentrations nicotine had either no
effect (0.25 µM) or diminished (0.5-1.0 µM) evoked GABAergic
neurotransmission. Nicotine had no significant effect on the
postsynaptic current induced by exogenous GABA (30-50 µM). These
data imply that nicotine levels attained in smokers are sufficient to
enhance evoked GABAergic transmission in the brain, and that this
effect is most likely mediated through activation of presynaptic
nicotinic receptors.
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INTRODUCTION |
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In the CNS, nicotinic acetylcholine receptors
(nAChRs) play an important role in short-term memory, attention, and
anxiety (Levin 1992; Ohno et al. 1993
).
The central nAChRs have a modulatory role in transmitter release.
Nicotine, a nicotinic receptor agonist and a major constituent of
tobacco, increases release of several neurotransmitters (Role
and Berg 1996
) and enhances the frequency of spontaneous
postsynaptic currents mediated by glutamate and GABA (Alkondon
et al. 1999
; Gray et al. 1996
; McGehee et
al. 1995
; McMahon et al. 1994
). Although it has
been reported that activation of nicotinic receptors enhances evoked
glutamatergic transmission (McGehee et al. 1995
), the
effects of nicotine on electrically evoked GABAergic transmission have
not been examined. Furthermore, the effects of low concentrations,
close to the steady-state level of nicotine in blood provided by
cigarette smoking (Benowitz et al. 1989
), on GABAergic
synaptic function need to be tested.
In the present study, we examined the effects of nicotine on GABAergic
transmission in the chick lateral spiriform nucleus (SPL), a thalamic
pretectal nucleus containing a high-density of
3H-nicotine binding sites (Sorenson and
Chiappinelli 1990) and GABAA receptors
(Veenman et al. 1994
). Our data indicate that unlike its
effect on spontaneous GABA currents, nicotine enhanced evoked GABAergic
transmission only at low concentrations (50-100 nM) and caused a
reduction in evoked responses at 0.5-1.0 µM.
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METHODS |
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Brain slices (400 µm) containing the SPL were prepared from
chick embryos (18 days of incubation). Slices were cut with a Vibroslice (Campden Instruments, Sileby, UK) in cold buffer
bubbled with 95% O2-5% CO2. The composition
of the external recording buffer was (in mM) 126 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.2 Na2HPO4, 25 NaCO3, and 10 glucose.
The slice was continuously perfused (~4 ml/min) at room temperature
(23°C) in a recording chamber as previously described (Guo et
al. 1998). Drugs were applied by bath perfusion except GABA,
which was given by pressure ejection through a fast perfusion system
(ALA Scientific Instruments, Westbury, NY).
The SPL neurons were visualized under an Axioskop fixed-stage
microscope (Zeiss, Oberkochen, Germany) at ×400 for voltage-clamp recording. Patch pipettes were made by a two-stage microelectrode puller and had resistances of 4-7 M after filling with internal solution containing (in mM) 140 KCl, 10 HEPES, 5.5 EGTA, 2 Mg-ATP, 2 MgCl2, and 5 QX314 (added to block sodium spikes).
Conventional patch-clamp techniques were used with an Axoclamp-2A
amplifier (Axon Instruments, Burlingame, CA). Holding potential was
60 to
70 mV. Approximately half-maximal postsynaptic current (PSC) was electrically evoked through a bipolar electrode placed adjacent to
the medial side of the SPL at 0.05 Hz. The evoked PSC was recorded with
a video cassette recorder. The data were sampled by pClamp7 software
through a Digidata 1200 interface. The figures were plotted with SigmaPlot.
Drugs were obtained as follows: ()-nicotine bitartrate, bicuculline
methiodide (BIC), and
-amino-n-butyric acid (GABA) from Sigma (St.
Louis, MO); lidocaine N-ethyl bromide (QX314) from RBI
(Natick, MA); tetrodotoxin (TTX) from Calbiochem (San Diego, CA); and
dihydro-
-erythroidine (DH
E) was a gift from Merck, Sharp & Dohme
Research Labs (Rahway, NJ). The data were expressed as means ± SE; differences between means were examined by the paired
t-test unless otherwise indicated.
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RESULTS |
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Whole cell recordings demonstrated that in SPL neurons, spontaneous events were fully blocked (Fig. 1A) and electrically evoked responses (medial placement of stimulating electrode) were suppressed by 98 ± 1% (mean ± SE, n = 8, Fig. 1B) in the presence of the GABAA receptor antagonist bicuculline (20 µM), indicating that these synaptic activities were primarily GABAergic. These GABAergic events were inward currents due to the presence of KCl-containing internal solution in the patch pipettes.
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Bath application of nicotine to slices containing the SPL produced a
sustained postsynaptic inward current and an increase in spontaneous
GABAergic synaptic activity as previously described (McMahon et
al. 1994). Nicotine increased the frequency of spontaneous GABA
currents in a dose-dependent manner (Fig.
2A). Overall, there were
42 ± 11% (n = 12), 154 ± 25%
(n = 8), and 465 ± 76%
(n = 7) increases in spontaneous events in the
presence of 0.1, 0.25, and 1.0 µM nicotine, respectively. Nicotine
also caused a sustained inward current of 20 ± 4, 63 ± 12, and 184 ± 42 (pA) at 0.1 µM (n = 12), 0.25 µM (n = 8), and 1.0 µM (n = 7), respectively.
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A different pattern was observed for nicotine effects on electrically evoked GABAergic transmission. Whereas nicotine dose-dependently increased the frequency of spontaneous currents, evoked GABAergic transmission was only enhanced at concentrations of 50-100 nM (Fig. 2B). At 50 nM, nicotine produced a significant 9 ± 2.3% increase in evoked GABAergic current (834 ± 190 pA in nicotine, 743 ± 168 pA in control, n = 5, P < 0.05). In 14 cells tested, 100 nM nicotine significantly increased evoked postsynaptic GABAergic currents by 19 ± 3.4% (954 ± 159 pA in nicotine, 804 ± 138 pA in control, P < 0.001). In contrast, nicotine at high concentrations (0.5-1.0 µM) caused a significant reduction in the evoked GABAergic transmission (Fig. 2B). In seven cells tested, nicotine (0.5-1.0 µM) caused a 114 ± 16 pA reduction in the evoked GABAergic current from the control level of 918 ± 184 pA (P < 0.001). At an intermediate concentration (0.25 µM), nicotine had no significant effect on evoked postsynaptic GABAergic current (712 ± 42 pA in 0.25 µM nicotine compared with control level of 746 ± 123 pA, n = 8). At a very low concentration (25 nM), nicotine also had no significant effect on evoked GABAergic currents (760 ± 79 pA in nicotine, 687 ± 44 pA in control, P > 0.3, n = 5). Further, nicotine (100 nM) enhanced the evoked GABAergic current without having any significant effect on the mean amplitude of GABAergic spontaneous events. In nine cells tested, the mean amplitude of spontaneous GABAergic events was 217 ± 13 pA in control and 212 ± 13 pA in the presence of 100 nM nicotine (P > 0.6).
Dihydro--erythroidine (DH
E), a nicotinic receptor antagonist,
blocked the effects of nicotine on electrically evoked GABAergic transmission (Fig. 3). In three cells
tested, nicotine alone (100 nM, 1.0 µM) caused a 207 ± 37 pA
increase (P < 0.05) and a 296 ± 78 pA
reduction in evoked GABAergic current, respectively, from control
values of 959 ± 112 and 1,186 ± 109 pA. However, in the presence of 60 µM DH
E, nicotine (0.1, 1.0 µM) only altered
evoked GABAergic current by 65 ± 79 (P > 0.6) and 55 ± 43 pA (P > 0.5), respectively.
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The effect of nicotine on the postsynaptic current induced by exogenous application of GABA was examined in the presence of 0.5 µM TTX. Nicotine had no significant effect on the postsynaptic current produced by pressure ejection of GABA onto SPL neurons. In five cells, the control inward current elicited by GABA (30-50 µM) was 3.5 ± 0.36 nA and 98 ± 2.3% of control in the presence of 0.5-1.0 µM nicotine. At 100 nM nicotine, there was also no significant change from control (only 2 ± 2.6% decrease, P > 0.4, n = 3).
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DISCUSSION |
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Nicotine, a potent neuromodulator in the CNS, enhances the release
of a variety of transmitters and increases synaptic transmission mediated by glutamate (McGehee et al. 1995; Role
and Berg 1996
). In the present study, we demonstrate that low
concentrations of nicotine (50-100 nM) enhance evoked GABAergic
currents in chick SPL neurons. Higher concentrations of nicotine
(0.5-1.0 µM) reversibly decrease evoked GABAergic currents in SPL
neurons. This is in contrast to the dose-dependent enhancement of the
frequency of spontaneous GABAergic currents produced by nicotine.
Although nicotine in blood can reach a peak concentration of 0.5 µM
after cigarette consumption (Henningfield et al. 1993), steady-state nicotine levels are generally ~100 nM (Benowitz
et al. 1989
; Russell 1989
), and those in
brain are equivalent to or even greater than those in blood. The
present study indicates that concentrations of nicotine similar to
steady-state levels found in smokers significantly increase both
spontaneous and evoked GABAergic synaptic activities. Considerable
heterogeneity exists in the subunit compositions and pharmacological
properties of central nAChRs, including those located on presynaptic
terminals (Alkondon et al. 1999
; Guo et al.
1998
). Particularly relevant to the present investigation is
that nicotine rapidly desensitizes (within ms) some nAChRs, including
most
7-containing receptors. The bath application method used to
apply nicotine in the present study is relatively slow, and thus it is
unlikely that the effects we observe, which persisted throughout 1- to
2-min exposures to nicotine, were due to activation of such rapidly
desensitizing receptors. Likewise, in smokers exposed during the day to
comparable steady-state levels of nicotine, most of these receptors
would presumably be desensitized.
Both pre- and postsynaptic mechanisms could contribute to the nicotinic
effect on evoked GABAergic transmission. Because nicotine had no
significant effect on the postsynaptic currents elicited by exogenous
GABA (30-50 µM), the postsynaptic sensitivity to GABA was unchanged
by activation of nicotinic receptors. In these experiments GABA was
applied using a fast perfusion system to mimic evoked release of
transmitter. It should be noted that there remain some differences
between this approach and electrically evoking GABA release, especially
in the magnitude of the currents generated by the two techniques, with
approximately fourfold higher currents produced by exogenous GABA.
Because no evidence was obtained for nicotine altering postsynaptic
GABA currents, a presynaptic mechanism most likely accounts for the
observed effects of nicotine. Nicotine also enhances glutamatergic
transmission in the CNS by a presynaptic mechanism involving an
increase in presynaptic [Ca2+]i (Gray
et al. 1996; McGehee et al. 1995
).
There are several possible explanations for the decrease in
evoked transmission observed at higher concentrations of nicotine. The
first hypothesis requires two subtypes of nicotinic receptors on the
GABAergic afferents. The first subtype enhances spontaneous GABA
release, and the second subtype, located nearby GABA release sites,
enhances evoked GABA release at low concentrations of nicotine, but
desensitizes at higher concentrations of the agonist. This hypothesis
would be feasible if there was ongoing acetylcholine (ACh) release that
up-regulated GABAergic transmission during control stimulation.
However, because DHE, a nicotinic receptor antagonist, did not alter
evoked GABAergic transmission elicited by stimulating the medial side
of the SPL (see Fig. 3), we have no evidence for ongoing ACh release at
this site. Furthermore, fibers containing choline acetyltransferase are
not present on the medial side of the SPL. [Cholinergic fibers are
present on the lateral side of the nucleus, and electrical stimulation
of these lateral fibers does evoke postsynaptic nicotinic responses in
SPL neurons (Nong et al. 1999
)]. The second hypothesis
is that high concentrations of nicotine may cause the release of other inhibitory transmitters (Reiner et al. 1982a
,b
) which in
turn reduce evoked GABAergic responses. Alternatively, the reduction in
evoked GABAergic transmission induced by higher concentrations of
nicotine may be mediated through presynaptic inhibition
(Clements et al. 1987
; Ryall 1978
)
resulting from depolarization of GABAergic terminals by nicotine.
Enhanced spontaneous GABA release induced by nicotine could also
contribute to the reduction of evoked GABAergic transmission at high
concentrations of the agonist. The released GABA could act on
presynaptic GABAB receptors to reduce GABAergic
transmission (Davies and Collingridge 1993). Increased
spontaneous release of GABA might also lead to a decrease in the amount
of GABA released in response to electrical stimulation.
The significance of nicotinic enhancement of evoked GABAergic transmission in the brain is potentially far-reaching, because GABA is the principal inhibitory neurotransmitter in the brain. It remains to be seen whether the effects we observe here are representative of the relationship between nAChRs and GABAergic transmission in other regions of the brain.
In summary, nicotine increased spontaneous GABAergic synaptic currents recorded from chick SPL neurons in a dose-dependent manner. At low concentrations (50-100 nM), nicotine enhanced evoked GABAergic transmission, whereas higher concentrations of nicotine reduced the evoked GABAergic current. Moreover, the nicotinic effect on GABAergic transmission appears to be through a presynaptic mechanism.
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ACKNOWLEDGMENTS |
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This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-17574 to V. A. Chiappinelli.
Present address of P. J. Zhu: Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Park Bldg., Rm. 118, 12420 Parklawn Dr., Rockville, MD 20852.
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FOOTNOTES |
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Address for reprint requests: V. A. Chiappinelli, Dept. of Pharmacology, The George Washington University School of Medicine and Health Sciences, 2300 Eye St. N.W., Washington, DC 20037.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 April 1999; accepted in final form 17 August 1999.
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REFERENCES |
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