![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 296, Issue 1, 77-83, January 2001
,Departments of Anesthesiology (J.H.Y., L.T., J.R., R.S., P.L.L., J.J.M.) and Pharmacology and Physiology (J.H.Y., P.L.L., D.A.S., J.J.M.), New Jersey Medical School, Newark, New Jersey; and Anaesthesia Research Department, McGill University, Montréal, Canada (K.K.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The potentiation of glycine-induced responses by ethanol (EtOH) was
studied in neurons freshly dissociated from the ventral tegmental area
(VTA) of 5- to 14-day-old postnatal rats using whole-cell and
gramicidin-perforated patch-clamp techniques. Under current-clamp
conditions, EtOH increased glycine-induced membrane depolarization and
action potential firing. Under voltage-clamp conditions, EtOH (0.1-40
mM) alone did not elicit a current. When coapplied with glycine, EtOH
enhanced the glycine-induced current in 35% (180 of 474) of the
neurons. The EtOH-induced enhancement of glycine current was
independent of membrane potential (between 
60 and +60 mV); the
reversal potential was not changed. Concentration-response analysis
showed that in the presence of EtOH (10 mM), the EC50 for
glycine decreased from 25 ± 4 to 14 ± 3 µM; the Hill
coefficient increased from 1.5 ± 0.2 to 1.9 ± 0.3. Kinetic
analysis of glycine currents indicated that EtOH decreased the time
constant of activation and increased the time constant of deactivation
of glycine-gated chloride channels. EtOH may accelerate glycine
association with its receptor at the agonist binding site and increase
the apparent agonist affinity. Our observations suggest that, at
pharmacologically relevant concentrations, EtOH alters the function of
glycine receptors and thus the excitability of neonatal VTA neurons.
This action of EtOH may contribute to the neurobehavioral disturbances
associated with fetal alcohol syndrome.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The
fetal central nervous system (CNS) is one of the most sensitive targets
of ethanol (EtOH). Exposure of the human fetus to EtOH results in a
combination of abnormalities termed fetal alcohol syndrome or fetal
alcohol effects (Clarren and Smith, 1978
). The most common
manifestations of fetal alcohol syndrome/fetal alcohol effects are
neurobehavioral disturbances, such as hyperactivity, learning
disabilities, depression, and psychosis (Clarren and Smith, 1978). The
mechanisms underlying EtOH effects on the developing human brain,
however, are poorly understood.
According to a very recent report (Ikonomidou et al., 2000), EtOH
not only blocks
N-methyl-D-aspartate-type
glutamate receptors but also strongly activates
GABAA receptors, thus triggering widespread apoptotic neurodegeneration in the developing rat forebrain. Like GABA
(Krnjevi, 1997), glycine is a major inhibitory neurotransmitter in the mature CNS, which activates Cl
-selective
channels. In the adult mammalian CNS, activation of these channels
results in neuronal hyperpolarization. Recent studies have revealed
dramatically different effects of these transmitters in early
development. Neonatal cells feature relatively high intracellular [Cl], owing to the active inward transport of
Cl. Therefore, in contrast to their effects in
adult cells, both glycine and GABA induce an outward flux of
Cl, resulting in neuronal depolarization and
excitation (Cherubini et al., 1991; Ye, 2000).
The glycine receptor/channel (GlyR) consists of 
- and 
-subunits
that combine to form a pentameric receptor complex mediating transmembrane flux of Cl (Betz,
1991). Molecular cloning studies have revealed a developmental heterogeneity of GlyR subunits. For example, the
2-subunit appears only during an early
developmental stage (from fetus to 2-3 weeks after birth), and is
subsequently replaced by the 1-subunit (Betz, 1991). According to other studies (Akagi and Miledi, 1988; Ye, 2000),
the physiological and pharmacological properties of the GlyRs depend on
their subunit composition and probably differ in adult and immature types.
In contrast to the numerous studies on GABA receptors, studies of the
effects of EtOH on GlyRs are fewer and more limited in scope. Engblom
and Akerman (1991) reported that EtOH potentiates glycine-activated
Cl uptake into synaptoneurosomes of whole-rat
brain. In addition, central depressant effects of EtOH were shown to be
enhanced by glycine and the glycine precursor serine (Williams et al.,
1995); the specific antagonist strychnine (STR) blocked this action, indicating that glycine enhances EtOH effects via STR-sensitive GlyRs
(Schiller et al., 1995). More recently, EtOH's positive modulatory
effect on recombinant GlyRs was shown to be determined by a single
amino acid in the subunit of the STR-sensitive GlyR (Mascia et al.,
1996; Mihic et al., 1997). Electrophysiological studies are supportive,
revealing a positive modulation of glycine-activated current
(IGly) by EtOH in cultured neurons from chicks
(Celentano and Wong, 1994), mice (Aguayo and Pancetti, 1994; Aguayo et
al., 1996), and Xenopus oocytes and mammalian cell lines
expressing homomeric GlyRs (Mascia et al., 1996; Valenzuela et al.,
1998; Ye et al., 1998). 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 EtOH (Gatto et al.,
1994; Wise, 1996). Recent experiments in this laboratory have shown
that glycine-mediated responses can be recorded in the majority of VTA
neurons (Ye, 1999, 2000). Despite the importance of the VTA in the
reinforcement of drug abuse, EtOH effects on the GlyRs of VTA have not
been studied. In the present article, we show that pharmacologically
relevant concentrations of EtOH (0.1-40 mM) greatly potentiate the
glycine-activated responses of neonatal VTA neurons and thus enhance
their excitability.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
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 University of Medicine and Dentistry of New Jersey (protocol
number 0752). We performed our experiments on VTA neurons prepared as
described in Ye (2000). Briefly, 5- to 14-day-old Sprague-Dawley rats
were decapitated. The brain was quickly excised, placed into ice-cold
saline saturated with 95% O2 and 5%
CO2, glued to the chilled stage of a vibratome
(Campden Instruments, Leics, UK), and sliced to a thickness of
300 to 400 µm. Slices were transferred to the standard external
solution saturated with O2, containing 1 mg of
pronase/6 ml and incubated (31°C) for 20 min. After an additional
20-min incubation in 1 mg of 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
of these tissue punches 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.
when
filled with the above-mentioned solutions. The gramicidin-perforated patch technique (Abe et al., 1994) was used to record the
glycine-induced whole-cell responses. The gramicidin stock solution of
10 mg/ml was prepared in methanol (J.T. Baker, Inc., Phillipsburg, NJ). It was diluted in the pipette solution to a final concentration of 50 to 100 µg/ml just before the experiment. The pipette tip was filled
with gramicidin-free solution by brief immersion before 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. When
conditions stabilized, whole-cell recording began. Throughout all
experimental procedures the bath was continuously 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
amplifier (Axon Instruments, Foster City, CA) interfaced to a Digidata
1200 (Axon Instruments) and directly digitized with pCLAMP 6 software
for further off-line analysis. The junction potential between the patch
pipette and the bath solutions was nulled immediately 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 to accurately estimate the
reversal potential of IGly. In most experiments,
the series resistance before compensation was 15 to 25 M. Routinely,
80% of the series resistance was compensated; hence, there was a 3-mV
error for 1 nA of current.
Chemical Application.
Solutions of glycine,
strychnine, gramicidin (Sigma Chemical Co., St. Louis, MO) and EtOH
(Pharmco, Bayonne, NJ) were prepared on the day of experimentation.
Solutions were applied to a dissociated neuron with a superfusion
system via a multibarreled pipette (as described previously; Ye, 2000).
The tip of the superfusion pipette was usually placed 50 to 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 change was
determined by reducing the external Na+
concentration from 140 to 10 mM (plus 130 mM
N-methyl-D-glucamine) during a kainate
application. Because the kainate currents do no desensitize, the rate
of decrease of kainate responses reflected the rate of solution change
(Ye et al., 1999).
0.05, otherwise as indicated. For all experiments,
average values are expressed as mean ± S.E.M. with the number of
neurons indicated in parentheses. To generate a concentration-response
relationship for VTA glycine receptors, all neurons were exposed to 1 mM glycine and two to three lower concentrations (0.003-0.3 mM). For
each concentration, four to six responses for a given neuron were
normalized to the peak current amplitude in response to 1 mM glycine.
The normalized values from three to five neurons at each concentration were normalized. These averages were then fit using a Simplex algorithm
(Sigma Plot; Jandel Scientific) with the Hill equation: I = (Imax × Cn)/(Cn + EC50n), where I,
Imax, C,
EC50, and n are
IGly, maximal IGly, glycine concentration, the concentration for 50% of maximum, and the Hill coefficient, respectively.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
EtOH Enhances Glycine-Induced Depolarization and Neuronal
Excitability.
EtOH's effect on glycine response was first studied
under current-clamp conditions with the gramicidin-perforated patch
technique. In agreement with our previous report (Ye, 2000), glycine
elicited depolarization and, in some cases, action potentials in VTA
neurons from neonatal rats (Fig. 1). This
depolarization is explained by a reversal potential for glycine's
action (EGly) that is much more positive (near
25 mV in neonatal neurons) than the resting potential (68 ± 2.5 mV, n = 5).
|
);
but even when it did not alter the membrane potential, its
coapplication with glycine enhanced both the amplitude of the
depolarization and the number of action potentials elicited by 10 µM
glycine (Fig. 1). On average, the depolarization induced by 10 µM
glycine was 13 ± 3 mV in the absence and 23 ± 3 mV in the
presence of 10 mM EtOH; these values are significantly different (P < 0.01, n = 8). Moreover, 10 µM
glycine evoked a significantly greater number of spikes in the presence
of 10 mM EtOH (5 ± 1) than in its absence (2 ± 1, n = 7, P < 0.01). In the absence of glycine, 10 mM EtOH did not increase firing elicited by a depolarizing stimulus (2 ± 1 versus 2 ± 1 spikes in the absence and presence of 10 mM EtOH; P > 0.05, n = 4).
EtOH Potentiates IGly.
Our previous findings on
neonatal VTA neurons recorded under voltage clamp (Ye, 2000) can be
summarized as follows. Most VTA neurons (82%) are sensitive to
glycine. When elicited by a near-threshold concentration of glycine
(3-5 µM), in the majority of cases (64%) IGly
decays; with higher glycine concentrations, IGly
always decays to a lower steady-state value. Peak
IGly increases sigmoidally with the concentration
of glycine, with an EC50 of 37 ± 8 µM and a Hill coefficient of 1.5. When internal and external
[Cl] are equal, IGly
reverses near 0 mV. In recordings made with the gramicidin-perforated
patch technique, EGly was 29 mV.
|
; but see Aguayo et al., 1996).
Responses to glycine in the absence and presence of 40 mM EtOH were
completely blocked by 500 nM STR. This suggests the involvement of an
immature form of the GlyR because the STR IC50
for the adult GlyR is only 12 nM compared with 110 nM for the neonatal
GlyR (Ye, 2000). This observation also indicates that potentiated
current resulted from an increase in GlyR function and not from an
additional effect of the agonist in combination with EtOH.
EtOH-Mediated Enhancement of IGly 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 looked for evidence regarding the possibility that EtOH's potentiation of IGly is
also voltage-dependent. As illustrated in Fig.
3, A and B, the current-voltage curves obtained with a voltage-ramp were linear and EtOH enhanced
IGly to a similar extent at all voltages between
+60 and 60 mV. Thus, EtOH's effect on IGly is
independent of voltage.
|
because
EGly remained close to the calculated Nernst
potential for Cl (1 mV in our experimental
conditions). Because voltage dependence could have a time-dependent
component that is slower than the slew rate of the voltage ramp, we
also studied the effect of EtOH while holding the membrane potential
constant for >3 min. As illustrated in Fig. 3C,
IGly decays during a continuous application of
glycine. The decay of IGly could be fit with a
single exponential function (Fig. 3C), and appeared to be
voltage-dependent. The decay time constant increases with
depolarization: it was 23 ± 5 s for 30 mV and 55 ± 5 s for +30 mV, respectively (n = 4, P < 0.01).
When a brief pulse of 10 mM EtOH was applied during a longer lasting
pulse of 10 µM glycine, IGly was immediately
enhanced. After the EtOH pulse ended, IGly
returned to the control level. The percentage potentiation of
IGly was not significantly different at membrane
potentials of 30 and +30 mV (126 ± 8 and 124 ± 6%, respectively, P > 0.1). The onset of the EtOH effect
could be fit by a single exponential (smooth line, Fig. 3D, right). The time constant of onset of EtOH's action was 305 ± 10 ms
(n = 5). However, the offset time constant could not be
readily estimated because the effect of a sustained application of EtOH
decreased with time (Fig. 3D).
EtOH-Mediated Enhancement of IGly Depends on Glycine
Concentration.
EtOH might augment IGly by
increasing the affinity of the receptor for glycine, by increasing the
efficacy of glycine at the receptor, or both. In an attempt to explore
these possibilities, EtOH was tested on currents induced by 3 to 1000 µM glycine. Typical IGly records, obtained in
the absence and presence of 40 mM EtOH, are shown in Fig.
4A. Although EtOH strongly potentiated
IGly induced by 10 µM glycine (a-c), it had no
appreciable effect on IGly induced by 1 mM
glycine (d-f). On average, 40 mM EtOH potentiated the peak
IGly activated by 10, 30, and 1000 µM glycine
to 160 ± 2% (n = 6), 133 ± 3%
(n = 7), and 98 ± 4% (n = 5) of
control, respectively. Figure 4B presents the glycine
concentration-response curves for data obtained from neurons in control
solution and in the presence of 40 mM EtOH. The
EC50 and Hill coefficient were 25 ± 4 µM
and 1.5 ± 0.2, respectively, in the absence of EtOH and 14 ± 3 µM and 1.9 ± 0.3 in the presence of 40 mM EtOH. In contrast, the maximum response was not affected by 40 mM EtOH, being
1.0 in its absence and 0.98 in its presence (P > 0.5).
|
EtOH Effects on the Kinetics of IGly.
Because
changes in either agonist affinity or channel opening efficacy can
alter the EC50 values of agonists (Colquhoun,
1998), we analyzed the channel kinetics, including activation,
deactivation, and desensitization of GlyR in the absence and presence
of EtOH. To allow accurate measurement of time constants within the
limits of the fast perfusion system (time constant of around 10 ms), we
applied glycine at concentrations of 30 µM or lower. To ensure that
the measurement of the rates of activation and deactivation was not
influenced by the rates of onset and offset of EtOH's action, we
applied EtOH for 5 s before and after the application of glycine.
|
(1) |
on) of a response is as follows: 1/on = + × ([agonist]n/([agonist]n + EC50n), where is the
closing rate constant, is the opening rate constant, [agonist] is
the agonist concentration, n is the number of binding sites,
and EC50 is the concentration of agonist that gave 50% of the maximum possible .
In agreement with previous observations (Akaike and Kaneda, 1989; Harty
and Manis, 1998), after a brief latent period, the onset of the inward
current after glycine concentration jumps could be fit by a single
exponential function (Fig. 5A). The
relationship between 1/on and the
concentration of glycine was approximately linear (Fig. 5B). The slope
of these curves gave an estimated value for the rate of association of
glycine (kon). They are 1.1 × 107 mol1
s1, 1.75 × 107
mol1 s1, and 1.95 × 107 mol1
s1 for 0, 10, and 40 mM EtOH, respectively. The
y-intercept of these plots was used to estimate the
dissociation rate (koff), which was
substantially increased by EtOH. The estimated value of this rate was
1.36, 3.25, and 4.14 s1 in the presence of 0, 10, and 40 mM EtOH, respectively.
|
off) for glycine from the time course of
responses when the added glycine was rapidly washed from the external
medium. In agreement with previous work (Harty and Manis, 1998), the
decreases in IGly after glycine concentration
jumps could also be fit by single exponential functions. The
off did not change significantly with glycine concentration (Fig. 5C). In contrast, EtOH increased the
off of IGly evoked by 10 µM glycine from a control value of 140 ±25 ms in the absence of EtOH
to 210 ± 20 ms in 10 mM EtOH and 300 ± 30 ms in 40 mM EtOH.
Thus, the deactivation time constant was highly dependent upon EtOH's
concentration (ANOVA, P < 0.01, n = 6), but was independent of glycine's concentration (ANOVA,
P > 0.25, n = 5).
Effects of EtOH on Receptor Desensitization.
EtOH potentiation
of IGly could result from a decrease in the rate
of receptor desensitization. Indeed, previous experiments suggested
that slowing of desensitization contributes to alcohol potentiation of
the 5-hydroxytryptamine3 receptor (Zhou et al., 1998). To test this possibility, we studied the desensitization of
IGly in the absence and presence of EtOH. As
shown in Fig. 6, the differences between
peak and late currents, the decay rate of current activated by 3 µM
glycine increased, rather than decreased, by application of 40 mM EtOH.
The ratio of the decay time constants (EtOH:control) in
Fig. 6 is 0.45. For six neurons, 40 mM EtOH significantly decreased the
time constant of desensitization (Student's t test,
P < 0.05). This is in agreement with EtOH acceleration of current decay induced by extended application of GABA (Nakahiro et
al., 1991). Desensitization has previously been studied with whole-cell
recording (Akaike and Kaneda, 1989; Harty and Manis, 1998) and the
desensitization time constant decreased when glycine concentration was
increased, whereas the amount of desensitized current increased (Akaike
and Kaneda, 1989). Therefore, the increase in response amplitude
associated with an increased desensitization evoked by EtOH (Fig. 6)
might result from multiple distinct changes in GlyRs kinetics. Figure
7 illustrates that a further increase in
glycine concentration resulted in a response similar to that obtained
with a lower glycine concentration in the presence of EtOH, indicating
that EtOH might decrease the microscopic
kd for glycine.
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The observations reported here indicate that at pharmacologically relevant concentrations EtOH potentiates the excitatory action of glycine on neonatal mammalian VTA neurons. This is the first report of EtOH effects on the GlyRs of native neonatal central neurons. Our study confirms and expands upon previous findings obtained from recombinant expression systems or native preparations via electrophysiological recording and neurochemical methods.
For the rats studied, the VTA cells are clearly immature and glycine
induces neuronal depolarization and excitation. Glycine receptor/channels, together with other ligand-gated channels, mediate
fast excitatory and inhibitory synaptic transmission in the developing
CNS. These receptors/channels have been shown to have roles in neuronal
proliferation, differentiation, and programmed cell death (Costa et
al., 2000). Accumulating evidence indicates that prenatal and/or early
postnatal EtOH exposure affects neurotransmitter-gated ion channels.
There are several possible mechanisms by which EtOH might act on glycine-gated ion channels. For example, EtOH may alter their ionic permeability. However, EGly was not significantly altered by EtOH, indicating that EtOH does not alter the ion selectivity of the channel. A related question concerned whether EtOH's effects are voltage-dependent. Because EtOH is not charged at physiological pH, any voltage dependence would result from an EtOH-induced conformational change in the glycine receptor channel that affected its voltage sensitivity. More significant is the fact that in the presence of EtOH, the glycine concentration-response curve shifted to the left in a parallel manner without appearing to alter the maximal value. Thus, the ability of EtOH to enhance IGly may be at least in part attributable to a slowing of agonist dissociation.
In addition, we demonstrated that EtOH increased the activation rate.
This observation suggests that EtOH enhances glycine response by
accelerating glycine association to its receptor at the binding site, a
mechanism that has been demonstrated for potentiation of
GABAA receptor function by diazepam in expressed
21
2 receptors (Lavoie and Twyman, 1996). However, caution
must be taken in interpreting the present data. In the proposed model,
the rate of offset of the response is simply 1/, or the reciprocal
of the closing rate constant, i.e., the mean open time of the channel.
This shows that we cannot ignore the gating constants in the
interpretation of a deactivation rate, even in the simplest case. In
addition, we must consider the contribution of EtOH-induced change of
agonist efficacy, as demonstrated for potentiation of
5-hydroxytryptamine3 receptor channel function by
EtOH in NCB-20 neuroblstoma cells (Lovinger et al., 2000).
Theoretically, for agonists with high intrinsic activity at
ligand-gated ion channels, alteration of apparent agonist affinity may
result from changes in agonist efficacy (Colquhoun, 1998). Although we
did not observe an increase in the maximum response to glycine in the
presence of EtOH, we cannot exclude an effect of EtOH on efficacy for
the following reason. In a variety of mechanistic models for receptor
activation, the maximum response approximates to
Imax = E/(E + 1), where E is the efficacy of the agonist (/). At
high values of E, increases in E do not result in
marked increases in maximum response (which would be supported by the
increase in Hill slope; Colquhoun, 1998). However, whether this
involves a change in the true agonist binding affinity, or a change in
gating properties of the channel (or a combination of both), remains to
be seen. Single-channel analysis is necessary because changes in
affinity can be separated from alterations in channel gating by
examination of burst patterns of single ligand-gated ion channels
(Colquhoun and Hawkes, 1995).
Our observation that the glycine-activated current was
heterogeneous with respect to modulation by EtOH in different neurons is consistent with studies from other laboratories (Aguayo and Pancetti, 1994; Aguayo et al., 1996). This may reflect different subunit compositions of glycine receptor/channels with different functional properties. Alternatively, the insensitivity of a subset of
the glycine-gated channels to EtOH could indicate regulation of the
channel protein by a process such as phosphorylation (Mascia et al.,
1998; Swope et al., 1999). This explanation agrees with a previous
finding that EtOH's effect on glycine-evoked responses seems to depend
in part on the phosphorylation state of GlyRs (Mascia et al., 1998). It
is also supported by our observation that EtOH failed to potentiate
glycine in all six neurons recorded with pipettes containing no added
ATP (J. H. Ye and L. Tao, unpublished data).
Glycine-induced membrane depolarization could result in the activation
of voltage-gated Ca2+ channels and
N-methyl-D-aspartate receptor
channels, thus increasing intracellular Ca2+.
Cytoplasmic Ca2+ is an important second messenger
and plays critical roles in many neuronal functions, including the
regulation of development. Glycine-induced increases of intracellular
Ca2+ may, therefore, mediate the trophic function
of GlyRs at early stages of neuronal development (Cherubini et al.,
1991; Reichling et al., 1994). On the other hand, high levels of
intracellular Ca2+ are toxic. In the
present study, pharmacologically relevant concentrations (0.1-40 mM)
of EtOH potentiated glycine-induced depolarization in VTA neurons of
neonatal rats. If glycine does in fact increase intracellular
Ca2+, EtOH's potentiating effect on glycine
action may raise cytoplasmic Ca2+ concentrations
to neurotoxic levels. Hence, the observed change in GlyRs activity may
be responsible for abnormal CNS development, as in fetal alcohol
syndrome. The relevance of our current finding with fetal alcohol
effects and/or fetal alcohol syndrome is clear. Currently, the
mechanisms underlying fetal alcohol effects/fetal alcohol syndrome are
not well understood. It is thought that the brain is particularly
sensitive to the neurotoxic effects of EtOH during the period of
synaptogenesis, or the brain growth spurt period, which occurs
postnatally in rats but prenatally (during the last trimester of
gestation) in humans. Very recently Olney and colleagues (Ikonomidou et
al., 2000) demonstrated that during this period, transient EtOH
exposure can delete millions of neurons from the developing brain.
However, how EtOH kills the neurons is still not well understood. EtOH
potentiation of glycine-induced responses in neurons of neonatal rats
may contribute to the neurotoxic effects of EtOH.
In summary, our experiments on neonatal VTA neurons show that EtOH enhances both the depolarization and the firing elicited by glycine. The actions of EtOH may be due to EtOH-induced acceleration of glycine binding to its receptor and increases of the apparent agonist affinity. This study may shed light on the role of the GlyR in the neurotoxic effects of alcohol observed in fetal alcohol syndrome.
| |
Acknowledgments |
|---|
We thank Dr. Parul Metha and Julius Potian for assistance in analyzing the data.
| |
Footnotes |
|---|
Accepted for publication September 15, 2000.
Received for publication August 1, 2000.
This study was supported by National Institute of Alcohol Abuse and Alcoholism, National Institute of Health Grant AA-11989 (to J.H.Y.).
Send reprint requests to: Jiang Hong Ye, Department of Anesthesiology, New Jersey Medical School (UMDNJ), 185 South Orange Ave., Newark, NJ 07103-2714. E-mail: ye{at}umdnj.edu
| |
Abbreviations |
|---|
CNS, central nervous system;
EtOH, ethanol;
GABA, -aminobutyric acid;
GlyR, glycine receptor/channel;
STR, strychnine;
IGly, glycine-activated current;
VTA, ventral
tegmental area;
VH, holding potential;
EGly, reversal potential of glycine current;
d, time constant
of decay;
on, activation time constant;
off, deactivation time constant.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
current by ethanol in cultured mouse spinal neurons.
J Pharmacol Exp Ther
279:
1116-1122-aminobutyric acid and glycine receptor-coupled Cl flux in rat brain synaptoneurosomes.
J Neurochem
57:
384-390[Medline].This article has been cited by other articles:
|
|
 |
|
  C. Dubois, H. Houchi, M. Naassila, M. Daoust, and O. Pierrefiche Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control J. Physiol., March 1, 2008; 586(5): 1413 - 1427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Koyama, M. S. Brodie, and S. B. Appel Ethanol Inhibition of M-Current and Ethanol-Induced Direct Excitation of Ventral Tegmental Area Dopamine Neurons J Neurophysiol, March 1, 2007; 97(3): 1977 - 1985. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Song, S. C. Chattipakorn, and L. L. McMahon Glycine-Gated Chloride Channels Depress Synaptic Transmission in Rat Hippocampus J Neurophysiol, April 1, 2006; 95(4): 2366 - 2379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Hejazi, C. Zhou, M. Oz, H. Sun, J. H. Ye, and L. Zhang {Delta}9-Tetrahydrocannabinol and Endogenous Cannabinoid Anandamide Directly Potentiate the Function of Glycine Receptors Mol. Pharmacol., March 1, 2006; 69(3): 991 - 997. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Wang, C. Xiao, and J. H. Ye Taurine activates excitatory non-synaptic glycine receptors on dopamine neurones in ventral tegmental area of young rats J. Physiol., June 1, 2005; 565(2): 503 - 516. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-H. Ye, F. Wang, K. Krnjevic, W. Wang, Z.-G. Xiong, and J. Zhang Presynaptic Glycine Receptors on GABAergic Terminals Facilitate Discharge of Dopaminergic Neurons in Ventral Tegmental Area J. Neurosci., October 13, 2004; 24(41): 8961 - 8974. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Lynch Molecular Structure and Function of the Glycine Receptor Chloride Channel Physiol Rev, October 1, 2004; 84(4): 1051 - 1095. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. A. Lobo, M. P. Mascia, J. R. Trudell, and R. A. Harris Channel Gating of the Glycine Receptor Changes Accessibility to Residues Implicated in Receptor Potentiation by Alcohols and Anesthetics J. Biol. Chem., August 6, 2004; 279(32): 33919 - 33927. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. D. Eggers and A. J. Berger Mechanisms for the Modulation of Native Glycine Receptor Channels by Ethanol J Neurophysiol, June 1, 2004; 91(6): 2685 - 2695. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Jiang, K. Krnjevic, F. Wang, and J. H. Ye Taurine Activates Strychnine-Sensitive Glycine Receptors in Neurons Freshly Isolated From Nucleus Accumbens of Young Rats J Neurophysiol, January 1, 2004; 91(1): 248 - 257. [Abstract] |
) or glycine
plus 40 mM EtOH (
). Before pooling, amplitudes of the peak
IGly were first normalized to a near maximum response
elicited by a concentration of glycine (1 mM) that was common to all
sets. For individual neurons, three-point concentration-response curves
to glycine were determined first; similar responses were then obtained
in the presence of EtOH (see Materials and Methods for
details). The continuous line is the fit of the Hill equation.
) EtOH. The data demonstrated an approximately linear relationship
between 1/
