1Departments of Anesthesiology and Pharmacology and Physiology, New Jersey Medical School, Newark, New Jersey 07103-2714; and 2Anaesthesia Research Department, McGill University, Montreal, Quebec H3G 1Y6, Canada
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ABSTRACT |
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Ye, Jiang Hong,
Liang Tao,
Li Zhu,
Kreimir Krnjevi
, and
Joseph J. McArdle.
Ethanol Inhibition of Glycine-Activated Responses in Neurons
of Ventral Tegmental Area of Neonatal Rats.
J. Neurophysiol. 86: 2426-2434, 2001.
The brain is particularly
sensitive to alcohol during the period of its rapid growth. To better
understand the mechanism(s) involved, we studied ethanol effects on
glycine-activated responses of ventral tegmental area (VTA) neurons
isolated from the newborn rat, using whole cell and gramicidin
perforated patch-clamp techniques. Previously we reported that 0.1-40
mM ethanol enhances glycine-induced responses of 35% of VTA neurons
(Ye et al. 2001
). We now direct our attention to the
inhibitory effects of ethanol observed in 45% (312 of 694) of neonatal
VTA neurons. Under current-clamp conditions, 1 mM ethanol had no effect
on the membrane potential of these cells, but it decreased
glycine-induced membrane depolarization and the frequency of
spontaneous action potentials. Under voltage-clamp conditions, 0.1-10
mM ethanol did not elicit a current but depressed the glycine-induced
currents. The ethanol-induced inhibition of glycine current was
independent of membrane potential (between
60 and +60 mV). Likewise,
ethanol did not alter the reversal potential of the glycine-activated
currents. Ethanol-mediated inhibition of glycine current depended on
the glycine concentration. While ethanol strongly depressed currents
activated by 30 µM glycine, it had no appreciable effect on maximal
currents activated by 1 mM glycine. In the presence of ethanol (1 mM),
the EC50 for glycine increased from 32 ± 5 to 60 ± 3 µM. Thus ethanol may decrease the agonist affinity of
glycine receptors. A kinetic analysis indicated that ethanol shortens
the time constant of glycine current deactivation but has no effect on
activation. In conclusion, by altering VTA neuronal function,
ethanol-induced changes in glycine receptors may contribute to
neurobehavioral manifestations of the fetal alcohol syndrome.
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INTRODUCTION |
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Ethanol, a brain
depressant and an addictive drug, is strongly teratogenic. As the brain
is particularly sensitive to neurotoxic effects during its rapid
growth, fetal exposure to alcohol could lead to learning and memory
deficits associated with Fetal Alcohol Effects and/or Fetal Alcohol
Syndrome (Clarren and Smith 1978). The mechanisms
underlying ethanol's effects on the developing human brain, however,
are poorly understood.
According to Ikonomidou et al. (2000), potentiation of
GABAA receptors by ethanol contributes to
widespread apoptotic neurodegeneration in the developing rat forebrain.
A role for glycine receptors (GlyR) in this process is not known. Like
GABA (Krnjevi
1997
), glycine activates
Cl
selective channels (Werman et al.
1968
) and is a major inhibitory neurotransmitter in the mature
CNS. The GlyR consists of
and
subunits, which are heterogeneous
and undergo developmental changes. For example, the
2 subunit is
present in the fetus until 2-3 wk after birth. Afterward the
1
subunit becomes dominant (Becker et al. 1988
).
The physiological and pharmacological properties of GlyRs, including
ethanol sensitivity, depend on their subunit composition (Akagi
and Miledi 1988
; Eggers et al. 2000
; Ye
2000
). These properties are likely different for adult and
immature types of receptors. In addition, whereas activation of GlyRs
of adult mammalian CNS results in neuronal hyperpolarization,
activation of GlyRs of the developing CNS results in membrane
depolarization and neuronal excitation, owing to a more positive
chloride equilibrium potential (Cherubini et al. 1991
;
Ye 2000
).
Several studies have shown that ethanol enhances glycine-activated
currents in different preparations: e.g., synaptoneurosomes (Engblom and Akerman 1991), cultured neurons from chicks
(Celentano and Wong 1994
) and mice
(Aguayo and Pancetti 1994
; Aguayo et al. 1996
), Xenopus oocytes, as well as mammalian cell
lines expressing homomeric GlyRs (Mascia et al. 1996a
,b
;
Valenzuela et al. 1998
; Q. Ye et al.
1998
) and freshly isolated hypoglossal motoneurons (Eggers et al. 2000
). However, data from upper brain
stem neurons of neonatal animals are lacking.
The ventral tegmental area (VTA) contains the cells of origin of the
mesolimbic system. It plays a pivotal role in the mediation of the
rewarding effects of drugs of abuse, including ethanol (Gatto et
al. 1994; Wise 1996
). In our recent experiments,
glycine elicited responses from most (82%) VTA neurons (Ye
2000
; J. H. Ye et al. 1998
). Ethanol
enhanced the glycine-activated current in 35% (173/494) of these cells
(Ye et al. 2001
). In the present article, we report that
ethanol (0.1-10 mM) also reduces responses to glycine in 45%
(312/694) of neonatal VTA neurons. Thus ethanol can depress their excitability.
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METHODS |
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Isolation of neurons and electrophysiological recording
The care and use of animals and the experimental protocol of
this study were approved by the Institutional Animal Care and Use
Committee of the University of Medicine and Dentistry of New Jersey
(protocol No. 0752). Sprague-Dawley rats (5-14 days old) were
decapitated as described earlier (Ye 2000). The brain
was quickly excised, placed into ice-cold saline saturated with 95% O2-5% CO2, glued to the
chilled stage of a vibratome (Campden Instruments), and sliced to a
thickness of 300-400 µm. Slices were transferred to the standard
external solution
containing 1 mg pronase/6 ml and saturated with
O2
and incubated at 31°C for 20 min. After an
additional 20 min incubation in 1 mg thermolysin/6 ml, the VTA was
identified medial to the accessory optic tract and lateral to the
fasciculus retroflexus under a dissecting microscope. Micro-punches of
the VTA were isolated and transferred to a 35-mm culture dish. Mild
trituration through heat-polished pipettes of progressively smaller tip
diameters dissociated single neurons. Within 20 min of trituration,
isolated neurons attached to the bottom of the culture dish and were
ready for electrophysiological experiments. Based on morphology under
the light microscope, the cells acutely isolated from VTA were of two
types: bipolar and multipolar. The majority was bipolar with one to
three dendritic processes emerging from each end of the fusiform soma
(20-40 µm in length and 15-25 µm in diameter). The multipolar
neurons were larger with a diameter of 35-60 µm and four to five
major dendrites. Most of the cells were tyrosine hydroxylase-positive,
which is in good agreement with the recent report of Brodie et
al. (1999)
. There were no appreciable differences in the
response of these two groups of neurons to ethanol.
The saline in which the brain was dissected contained (in mM) 128 NaCl,
5 KCl, 1.2 NaH2PO4, 26 NaHCO3, 9 MgCl2, 0.3 CaCl2, and 2.5 glucose. The pH was adjusted to
7.4 with HCl. The standard external solution contained (mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES. The pH was adjusted to 7.4 with Tris base and
the osmolarity to 320 mM with sucrose. With 100 mM ethanol, the pH and
the osmolarity of the solution were unchanged. Patch pipette solutions
contained (mM) 150 KCl and 10 HEPES for gramicidin-perforated patch
recording and 120 CsCl, 21 TEA-Cl, 4 MgCl2, 11 ethyleneglycol bis-(-aminoethylether)-N,N,N'N'-tetraacetic
acid (EGTA), 10 CaCl2, 10 HEPES, and 2 Mg-ATP for
conventional whole cell recording. The pH was adjusted to 7.2 with Tris
base, and the osmolarity to 280 mM with sucrose. The patch electrodes
had a resistance between 3 and 5 M when filled with the preceding
solutions. The gramicidin-perforated-patch technique (Abe et al.
1994
) was used to record glycine-induced whole cell responses.
Gramicidin enters the membrane lipid bilayer to form transmembrane
pores which are impermeable to chloride; this preserves the
normal internal chloride concentration and is important for the study
of chloride channels. The gramicidin stock solution of 10 mg/ml was
prepared in methanol (J. T. Baker, Phillipsburg, NJ). It was
diluted in the pipette solution to a final concentration of 50-100
µg/ml just before the experiment. The pipette tip was filled with
gramicidin-free solution by brief immersion prior to back filling.
After establishing the giga-seal in the cell-attached configuration by
gentle suction, no further negative pressure was applied. The progress
of perforation was monitored by measuring the decrease in membrane
resistance with repeated 10-mV hyperpolarizing voltage steps from a
holding potential (VH) of
50 mV. The
entry into the perforated-patch mode was signaled by an increase in the
amplitude of the capacitive transient. The access resistance reached a
steady level of 20 M
within 30 min after making the giga-seal. At
this time, whole cell recording began. Throughout all experimental
procedures the bath was continually perfused with the standard external
solution. All glycine-induced responses were elicited in this solution
at an ambient temperature of 20-23°C.
Currents were recorded under voltage-clamp with an Axopatch 1D or 200 B
amplifier (Axon Instruments, Foster City, CA) interfaced to a Digidata
1200 or a Digidata 1320A (Axon Instruments) and directly digitized with
pCLAMP 8 software for further off-line analysis. The junction potential
between the patch pipette and the bath solutions was nulled just before
forming the giga-seal. The liquid junction potential between the bath
and the electrode was 3.3 mV, as calculated from the generalized
Henderson equation using the Axoscope junction potential calculator
(Barry 1996). This value was corrected off-line when
estimating the reversal potential of glycine-activated currents. In
most experiments, the series resistance before compensation was 15-25
M
. Routinely, 80% of the series resistance was compensated; hence,
there was a 3 mV error for 1 nA of current.
Chemical applications
Solutions of agonist, antagonists, alcohol, and gramicidin were
prepared on the day of experimentation. Glycine, strychnine, gramicidin, and
N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) were obtained from Sigma Chemical (St Louis, MO). TPEN was
prepared in dimethyl sulfoxide (DMSO) and diluted to its final concentration in standard external solution. The final concentration of
DMSO was always <0.1%; it did not induce any ionic current and had no
effect on the glycine response at the concentrations used. Ethanol
(100% and 95%, prepared from grain) was obtained from Pharmco
(Brookfield, CT, or Bayonne, NJ), or from U.S. Industrial Chemicals,
Division of National Distillers of Chemical (New York, NY), and was
stored in glass bottles. We found no significant difference between the
data collected using ethanol from different manufactures. Solutions
were applied to a dissociated neuron with a superfusion system via a
multi-barreled pipette as described previously (Ye et al.
2001). The tip of the superfusion pipette was usually placed
50-100 µm away from the cell, a position that allowed rapid as well
as uniform drug application while preserving the neuron's mechanical
stability. This system allows complete exchange of solutions in the
vicinity of the neuron within 20 ms. The speed of solution exchange was
measured by reducing the external Na+
concentration from 140 to 10 mM (plus 130 mM
N-methyl-D-glucamine, NMDG) during a kainate
application. Since kainate currents do not desensitize, the rate of
decrease of kainate responses reflects the rate of solution change
(Ye et al. 2001
). In the later half of the study, we
replaced all plastic in the system with Teflon and glass and eliminated
all metals.
Data analyses
Whole cell current decays were fitted by a Chebychev algorithm (pClamp). Concentration-response data were analyzed with a nonlinear curve-fitting program (Sigma Plot, Jandel Scientific). Data were statistically compared using Student's t-test at a significance level of P < 0.05, otherwise as indicated. For all experiments, average values are expressed as mean ± SE with the number of neurons indicated in parentheses. To obtain a concentration-response relationship for VTA glycine receptors, all neurons were exposed to three or four concentrations of glycine, in the range of 0.003-1 mM. For each concentration, four to six responses from a given neuron were normalized to the peak current evoked by 30 µM glycine. The normalized values from three to five neurons at each concentration of glycine were averaged. Using a Simplex algorithm (Sigma plot, Jandel Scientific), these averages were then fitted to the Hill equation: I = Imax/[1 + (EC50/C)n], where I, Imax, C, EC50 and n are glycine-activated current, maximal glycine-activated current, glycine concentration, the concentration for 50% of maximum response, and the Hill coefficient, respectively.
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RESULTS |
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Ethanol can decrease glycine-induced depolarization and neuronal excitability
We first studied ethanol's effects under current-clamp conditions
with the gramicidin-perforated patch technique. In agreement with our
earlier report (Ye 2000), glycine elicited
depolarization and, occasionally, action potentials in VTA neurons from
neonatal rats (Fig. 1). This
depolarization is explained by a reversal potential for glycine's
action on neonatal neurons that is much more positive (near
25 mV)
than the resting potential (
68 ± 2.5 mV, n = 5). In accord with our recent reports (Ye 2000
;
J. H. Ye et al. 1998
), all these effects of glycine were
antagonized by 0.1 µM strychnine (data not shown).
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To determine whether ethanol has a direct effect, we routinely applied ethanol alone to the cells before it was co-applied with the agonist (as shown in Fig. 1A). When a direct effect of ethanol was detected, we preequilibrated the cell with ethanol before it was co-applied with glycine, using the protocol illustrated in Fig. 6A. As demonstrated in Fig. 1, ethanol alone did not alter the membrane potential. However, when co-applied with 10 µM glycine, ethanol reduced both the amplitude of the depolarization and the number of action potentials. On average, the steady depolarization induced by 10 µM glycine was 13 ± 3 mV in the absence and 3 ± 3 mV in the presence of 1 mM ethanol; these values are significantly different (P < 0.01; n = 5). Moreover, 10 µM glycine evoked a significantly greater number of action potentials in the absence of 1 mM ethanol (2 ± 1) than in its presence (0 ± 1; for n = 6, P < 0.01).
Ethanol inhibits glycine-activated currents
Glycine elicits responses from most VTA neurons (82%) of neonatal
rats (Ye et al. 2001). We also reported that acute
applications of 0.1-40 mM ethanol enhanced glycine responses in 35%
of VTA neurons (Ye et al. 2001
). In the present
experiments, we found that 0.1-10 mM ethanol inhibited glycine-induced
currents of many neurons. Figure
2A shows typical examples
of currents activated by 30 µM glycine alone (A, a) and in
the presence of 0.1, 1, and 10 mM ethanol (A, b-d,
respectively); the currents recovered to the control level after
washout of ethanol (A, e). At concentrations between 0.1 and
3 mM, ethanol reduced glycine currents in a concentration-dependent manner; but 10 mM or higher concentrations were equally or less effective (see following text). Similar results were obtained when
ethanol was applied in the reversed order of concentration that is from
higher to lower. Such inhibition of glycine-activated current by
ethanol occurred in 45% (312/694) of the neurons tested. Figure
2B is a plot of the means of normalized ethanol inhibition as a function of ethanol concentration. On average, 3 and 10 mM ethanol
decreased the peak current induced by 30 µM glycine to 67 ± 6%
(n = 18) and 69 ± 5% (n = 48) of
control, respectively. At even higher ethanol concentrations (100 mM),
there was no inhibitory effect on glycine-activated currents. The
mechanisms underlying the disappearance of the inhibitory effect at
higher ethanol concentrations are unclear and currently under study.
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Age-dependence of ethanol's action
The preceding experiments were performed on neurons from neonatal rats (<14 days). GlyRs are known to be heterogeneous and to change during development. To determine whether developmental changes of GlyRs affect their response to ethanol, we tested ethanol's effects on VTA neurons isolated from more mature (27- to 34-day-old) rats prepared in an identical manner. Ethanol (1 mM) potentiated, had no effect and inhibited GlyRs in 72% (28/39), 23% (9/39), and 5% (2/39) of the neurons, respectively. This is in sharp contrast to the neonatal rats, where ethanol potentiated, had no effect and inhibited the GlyRs in 35, 20, and 45% of the neurons, respectively. Thus GlyRs of neonatal neurons are more sensitive to ethanol inhibition.
Additional control experiments
The inhibitory effect of ethanol on glycine response was
unexpected. It is possible that the inhibition resulted from a plastic product, bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (BTMPS; Tinuvin 770), a sterically hindered amine light and radiation stabilizer manufactured by Ciba-Geigy (Summit, NJ). This chemical inhibits nicotinic acetylcholine receptors expressed in
Xenopus oocytes in a use-dependent manner (Papke et
al. 1994). To eliminate this possibility, we replaced all the
plastic syringes with glass ones and installed metal-free Teflon tubing
in the perfusion system. In these experimental conditions, ethanol
caused a similar depression of glycine currents recorded in 260 neurons.
The initial experiments on 494 neurons were performed with 100% ethanol prepared from grain (U.S. Industrial Chemicals, Division of National Distillers of Chemical). Complete dehydration of ethanol is known to introduce contaminants. So we repeated the experiments on 200 neurons with 95% ethanol prepared from grain (Pharmco). To ensure that the measurement and other conditions were the same, in some of the experiments we examined the inhibitory effect of ethanol from both sources in the same neurons. There were no significant differences: 100 and 95% ethanol (both at 10 mM) inhibited currents activated by 30 µM glycine to 68 ± 5 and 69 ± 5%, respectively (n = 7, P > 0.05). Thus the inhibition of GlyR cannot be attributed to contaminants introduced during complete dehydration.
Ethanol inhibition of GlyRs is not due to zinc contamination
High levels of contaminating zinc may be present in ethanol from
commercial sources. Because zinc is a potent modulator of glycine
receptors (Laube et al. 1995, 2000
), we compared the
effects of zinc with those of ethanol. As illustrated in Fig.
3, zinc had biphasic
effects: at concentrations of 0.5-10 µM, zinc potentiated, but at
concentrations >50 µM, it depressed the glycine-activated current.
These results agree with a recent report of biphasic effects of zinc on
human GlyRs (Laube et al. 2000
). By contrast, glycine-induced responses are depressed by low (0.1-10 mM) and enhanced by high ethanol (1-40 mM) concentrations. In view of the
opposite concentration dependence of the effects of zinc and ethanol,
contamination by zinc cannot explain our observations.
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In addition, ethanol was tested in the presence of TPEN, a chelator of
zinc (and other heavy metals). In agreement with a recent study on
Zebrafish hindbrain (Suwa et al. 2001), applications of
TPEN (100 µM) alone reduced the glycine-activated current, indicating
that traces of heavy metals potentiate glycine in control conditions.
On average, 100 µM TPEN, 1 mM ethanol, and TPEN + ethanol depressed
currents activated by 30 µM glycine by 17 ± 5, 13 ± 5, and 31 ± 6% (n = 3), respectively. After
subtracting the effect of TPEN, ethanol depressed glycine currents to
14 and 13%, respectively, in the presence or absence of TPEN
(P > 0.5, Fig. 3, C and D).
These results are further evidence that ethanol's actions on the GlyRs
cannot be attributed to zinc contamination.
Ethanol-mediated inhibition of glycine-activated current is independent of membrane voltage
In view of recent evidence that the GlyR-channel is voltage
dependent (Legendre 1999) and may include an
alcohol-receptor site (Wick et al. 1998
), we examined
the voltage dependence of the depressant action of ethanol on
glycine-activated currents. As illustrated in Fig.
4 (A and B), the
current-voltage relations obtained with a voltage ramp were linear and
ethanol decreased glycine-activated current to a similar extent at all
voltages between +60 and
60 mV. Thus ethanol's effect on
glycine-activated current is independent of membrane potential.
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Furthermore, in the presence of ethanol, glycine-activated currents
remained selectively permeable to Cl since
their reversal potential remained close to the calculated Nernst
potential for Cl
(
1 mV in our experimental
conditions). As a control for the presence of a time-dependent
component of current that is slower than the voltage ramp, we also
studied the effect of ethanol while holding the membrane potential
constant for >3 min. As illustrated in Fig. 4C, ethanol
inhibited the glycine-activated current equally when the membrane was
held at
30 and +30 mV.
Ethanol inhibition of glycine-activated current depends on glycine concentration
Ethanol might inhibit glycine-activated current by decreasing the affinity of the receptor for glycine, and/or by decreasing the efficacy of glycine at the receptor. To explore these possibilities, we tested ethanol on currents induced by a wide range of glycine concentration (3-1,000 µM). Typical glycine-activated current records, obtained in the absence and presence of 1 mM ethanol, are shown in Fig. 5. Ethanol had a greater effect on the current induced by 30 µM glycine (Ab) than on the current induced by 300 µM glycine (Bb). On average, 1 mM ethanol decreased the peak current activated by 10, 30, and 1,000 µM glycine to 66 ± 2 (n = 7), 75 ± 3 (n = 7) and 97 ± 4% (n = 6) of control, respectively. As can be seen from the corresponding concentration-response curves in Fig. 5C, the EC50 of glycine was significantly greater in the presence (60 ± 3 µM, n = 4, P < 0.01) than in the absence (32 ± 5 µM, n = 4) of 1 mM ethanol. Thus ethanol lowered the apparent affinity of glycine for GlyRs.
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Ethanol's effects on the kinetics of glycine-activated current
As changes in either agonist affinity or channel opening
probability can alter the EC50 of agonists (see
Colquhoun 1998), we analyzed the kinetics of
glycine-activated current. To allow accurate measurement of time
constants within the limits of the fast perfusion system (time constant
of ~10 ms), glycine was applied at concentrations <30 µM. To
ensure that the measurement of the rate of activation and deactivation
was not influenced by the rates of onset and offset of ethanol's
action, we applied ethanol for 2 s before and after the
application of glycine.
In agreement with previous observations (Akaike and Kaneda
1989; Harty and Manis 1998
), after a brief
latent period, the onset of the inward current following glycine
concentration jumps could be fitted by a single exponential function
(Fig. 6A). The approximately linear relation between 1/
on and the
concentration of glycine (Fig. 6B) is consistent with a
one-binding site model,
on = 1/(Ckon+ koff) and
off = 1/koff (where C is the
concentration of glycine). The slope of these curves gave an estimated
value for the rate of association of glycine
(kon) that was not changed
significantly by ethanol: kon was
close to 9.9 × 104
mol
1s
1 in 0, 0.1, and 1 mM ethanol.
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We also measured the deactivation time constant
(off) for glycine-activated channels from the
time course of responses when glycine was rapidly washed from the
external medium. The values of
off obtained by
fitting single exponentials to the current decay after glycine
concentration jumps did not change significantly with glycine
concentration (Fig. 6C). In contrast, ethanol decreased the
off of current activated by 5 µM glycine
from a control value of 340 ± 25 ms in the absence of ethanol to
290 ± 22 ms in 0.1 mM ethanol and 230 ± 25 ms in 1 mM
ethanol. Thus the deactivation time constant was highly dependent on
ethanol's concentration (ANOVA, P < 0.01;
n = 6) but was independent of glycine's concentration (ANOVA, P > 0.5; n = 6). Furthermore,
the y intercept of these plots was used to estimate the
dissociation rate (koff), which was
substantially increased by ethanol (Fig. 6C): 2.9, 3.7, and 4.4 s
1, for 0, 0.1, and 1 mM ethanol,
respectively (ANOVA, P < 0.05; n = 6).
Hence the apparent dissociation constant
(KD) for glycine increased from ~29
to 45 µM. These values are close to those obtained directly from the
glycine dose-response curves.
Ethanol does not increase receptor desensitization
The depression of glycine-activated currents by ethanol could
result from increased receptor desensitization. Indeed, previous authors suggested that faster desensitization contributes to alcohol modulation of the nicotinic acetylcholine (nACh) receptor
(Nagata et al. 1996). To test this hypothesis, we
studied the desensitization of glycine-activated current in the absence
and presence of ethanol. As shown in Fig.
7, the decay rate of current activated by
10 µM glycine was reduced rather than increased by 1 mM ethanol. The
decay of glycine-activated currents both in the absence and presence of
ethanol could be fitted with single exponentials. The ratio of the
decay time constants (
EtOH:
control) in Fig. 7 is 1.75. For four neurons,
1 mM ethanol significantly increased the time constant of
desensitization (P < 0.05).
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DISCUSSION |
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Our principal finding is that 0.1-10 mM ethanol decreased
glycine-induced responses in VTA neurons of neonatal rats. This is the
first report that ethanol can inhibit GlyRs of native neonatal central
neurons. Our study confirms and extends previous findings on
interactions between ethanol and GlyRs obtained from recombinant expression systems or native preparations by electrophysiological recording or neurochemical methods (Aguayo and Pancetti
1994; Aguayo et al. 1994
; Celentano et
al. 1988
; Engblow and Akerman 1991
; Harty
and Manis 1998
; Mascia et al. 1998
; Mihic
et al. 1997
; Wick et al. 1998
; Ye et al.
2001
). In addition, our results suggest that ethanol binds to a
site on the GlyR that can allosterically reduce the affinity of this
receptor for glycine.
Ethanol inhibition of the glycine-activated current of VTA neurons is genuine
Because this finding differs radically from previous results (see following text), and there is a strong historical precedent for artifacts in alcohol studies, it was important to rule out spurious effects due to the presence of contaminants and other possible artifacts. We therefore took the following additional precautions: we 1) replaced all plastic tubing with Teflon and glass and ensured there was no metal in the perfusion system; 2) used 95% ethanol prepared from grain, stored in glass bottles, and did control experiments with ethanol from different sources; 3) tested ethanol in the presence of TPEN, a zinc chelator; 4) demonstrated that the inhibitory effect of low (1 mM) ethanol does not occur in VTA neurons from older animals, prepared in an identical manner; and 5) tested a substantial number of neurons at each concentration of ethanol to ensure that the effects of glycine were reproducible.
Glycine concentration and ethanol's effects
Ethanol potentiations are more easily observed at low concentrations of agonist, typically EC20. With glycine concentrations near EC50, potentiations are harder to record because they are smaller and receptors are more prone to desensitization. One may argue that if two effects occur by actions at distinct sites, agonist concentrations near EC50 may bias the measurements in the direction of negative receptor modulation (inhibition). However, in our previous experiments, we clearly found ethanol potentiation of glycine responses in neurons exposed to 30 µM glycine. This suggests that applying glycine at concentrations near the EC50 does not preclude detection of potentiation by ethanol. The inhibitory effect of ethanol observed in the current study was therefore probably not due to masking of a potentiating action by receptor desensitization.
Comparison with previously reported interactions between ethanol and glycine
Many studies on several preparations have shown that ethanol and
other anesthetics enhance neuronal responses to glycine (Aguayo and Pancetti 1994; Aguayo et al. 1996
;
Celentano et al. 1988
; Eggers et al.
2000
; Engblom and Akerman 1991
; Mascia et
al. 1996a
,b
; Mihic 1999
; Mihic et al.
1997
; Tapia et al. 1998
;
Valenzuela et al. 1998
; Ye et al. 2001
).
In our own recent study (Ye et al. 2001
), 0.1-40 mM
ethanol potentiated, depressed, or had no effect on GlyR-mediated
responses of 35, 45, and 20% of neonatal VTA neurons. This is in good
agreement with reports that ethanol potentiates, inhibits, or has no
effect on GABAA receptor-mediated synaptic responses of neurons from different brain regions, as well as within a
single neuronal population (Harris 1999
; Weiner
et al. 1997
).
The factors underlying the variability of the
ethanol-GABAA receptors interaction are unclear.
Several factors, such as the subunit composition of the receptor, its
phosphorylation state, and the methods for GABA and ethanol
application, have been suggested. Similar factors may also be involved
in ethanol-GlyRs interactions. Since most (72%) adult VTA neurons
showed potentiation by ethanol and only 5% (2 neurons) showed an
inhibition, the different in ethanol effects could be due to the known
developmental shift from 2 to
1 subunit-containing receptors.
This possibility is currently under investigation using compounds, such
as cyanotriphenylborate, which distinguish between
1 and
2
subunit-containing receptors (Rundstrom et al.
1994
).
Alternatively, the various responses to ethanol could indicate
regulation of the channel protein by phosphorylation (Mascia et
al. 1998; Swope et al. 1999
). This possibility
is supported by an earlier finding that ethanol's effect on
glycine-evoked responses seems to depend in part on the phosphorylation
state of GlyRs (Mascia et al. 1998
). Our preliminary
results indicate that protein phosphorylation is indeed involved in
ethanol-GlyR interactions in VTA neurons (unpublished data). In
addition, we previously found that the equilibrium potential of
chloride shifts from
29 mV in neonatal to
50 mV in adult VTA
neurons (Ye 2000
). This will certainly affect the
driving force of Cl
. The impact of this on the
results needs further study.
Mechanism of ethanol actions
Ethanol may alter the permeability of Cl
channels. However, the fact that the reversal potential of glycine
currents remained unchanged indicates that ethanol does not alter the
ion selectivity of the channel. A related question is whether
ethanol's effects are voltage dependent. As ethanol is not charged at
physiological pH, any voltage dependence would result from an
ethanol-induced conformational change of the glycine receptor channel
that affected its voltage sensitivity. This is unlikely because
ethanol's inhibition of glycine-activated current was independent of
membrane voltage.
More significant is the fact that in the presence of ethanol, the glycine concentration response curve shifted to the right in a parallel manner without a change in the maximal value. A simple explanation would be that ethanol binds to a site on the GlyR that can allosterically reduce the affinity of the receptor for glycine. This possibility is supported by two findings: that ethanol depressed glycine-activated currents by shifting the dose-response curves and that it enhanced the rate of offset of glycine responses. Thus the effects of ethanol may be attributed to faster dissociation of glycine from its binding site, even though other possibilities are not excluded.
The absence of ethanol effects on on can
result from either no change in the rate constants of binding and
unbinding (in the standard equation for binding described earlier) or
in a more complex kinetic modification with no changes in the balance
between these rate constants. Furthermore a decrease in the
deactivation time constant could also come about in several different
ways. For example, ethanol could increase the agonist dissociation rate constant, as in the ethanol-mediated inhibition of P2X purinoceptor function in bullfrog dorsal root ganglion neurons (Li et al.
1998
).
Physiological consequences of ethanol inhibition of GlyR responses
Recent studies have revealed dramatically different effects of
both GABAA and glycine during early development.
Neonatal cells have a relatively high intracellular
[Cl]. Therefore in contrast to their
inhibitory effects in adult neurons, both glycine and GABA induce an
outward flux of Cl, resulting in neuronal depolarization and excitation
(Cherubini et al. 1991
; Ye 2000
).
Glycine-induced membrane depolarization could result in the activation
of voltage-gated Ca2+ channels and NMDA receptor
channels, thus raising intracellular Ca2+. As an
important second messenger, cytoplasmic Ca2+
plays a critical role in many neuronal functions, including a trophic
function at early stages of neuronal development (Cherubini et
al. 1991
; Reichling et al. 1994
). In addition,
recent evidence indicates that Ca2+ influx,
triggered by the activation of embryonic GlyRs, is required for the
synaptic localization of GlyR and gephyrin, its anchoring protein
(Betz et al. 1999
). This Ca2+
influx is crucial for the formation of gephyrin and GlyR clusters at
developing postsynaptic sites (Kirsch and Betz 1998
).
Thus GlyR-mediated excitatory responses during embryonic development play an important role in synaptogenesis and functional
verification
an essential step in the proper targeting of postsynaptic
receptors to developing synaptic connections (Betz et al.
1999
).
By dampening growth-promoting increases in cytoplasmic
[Ca2+], ethanol-mediated inhibition of glycine
may be responsible for abnormal CNS development. Currently, the
mechanisms underlying Fetal Alcohol Syndrome and/or Fetal Alcohol
Effect remain obscure. It is thought that the brain is particularly
sensitive to the neurotoxic effects of ethanol during the period of
rapid growth and synaptogenesis, which occurs postnatally in rats but
prenatally (during the last trimester of gestation) in humans. During
this period, transient ethanol exposure can delete millions of neurons from the developing brain (Ikonomidou et al. 2000).
Alcohol has other negative effects, such as causing neurons to grow
incorrectly. Because even very low concentrations of ethanol can
inhibit GlyR-mediated excitatory responses, this effect may be
particularly significant for such neurotoxic effects of ethanol.
In conclusion, 0.1-10 mM ethanol depressed glycine-induced excitatory responses in 45% of neurons freshly dissociated from the VTA of neonatal rats. This effect may be due to an ethanol-induced decrease of glycine affinity for its receptor. This finding may shed light on the role of the GlyR in the neurotoxic effects of alcohol observed in Fetal Alcohol Syndrome and/or Fetal Alcohol Effect.
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ACKNOWLEDGMENTS |
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The authors thank E. Sceusi for editing the manuscript.
This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA-11989 to J. H. Ye.
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FOOTNOTES |
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Address for reprint requests: J. H. Ye, Dept. of Anesthesiology, New Jersey Medical School (UMDNJ), 185 S. Orange Ave., Newark, NJ 07103-2714 (E-mail: ye{at}umdnj.edu).
Received 14 March 2001; accepted in final form 3 August 2001.
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REFERENCES |
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