Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington 98195-7290
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ABSTRACT |
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Eggers, Erika D.,
Jennifer A. O'Brien, and
Albert J. Berger.
Developmental Changes in the Modulation of Synaptic Glycine
Receptors by Ethanol.
J. Neurophysiol. 84: 2409-2416, 2000.
During postnatal motoneuron development, the
glycine receptor (GlyR) subunit changes from
2 (fetal) to
1
(adult). To study the effect this change has on ethanol potentiation of
GlyR currents in hypoglossal motoneurons (HMs), we placed neurons into
two groups: neonate [postnatal day 1 to 3 (P1-3)], primarily expressing
2, and juvenile
(P9-13), primarily expressing
1. We found that
glycinergic spontaneous miniature inhibitory postsynaptic currents
(mIPSCs) in neonate HMs are less sensitive to ethanol than in
juveniles. Thirty millimolar ethanol increased the amplitude of
juvenile mIPSCs but did not significantly change neonatal mIPSCs.
However, 100 mM ethanol increased the amplitudes of both neonate and
juvenile mIPSCs. There was a significant difference between age groups in the average ethanol-induced increase in mIPSC amplitude for 10, 30, 50, and 100 mM ethanol. In both age groups ethanol increased the
frequency of glycinergic mIPSCs, but there was no difference in the
amount of frequency increase between age groups. Ethanol (100 mM) also
potentiated evoked IPSCs (eIPSCs) in both neonate and juvenile HMs. As
we observed for mIPSCs, 30 mM ethanol increased the amplitude of
juvenile eIPSCs, but had no significant effect on eIPSCs in neonate
HMs. Ethanol also potentiated currents induced by exogenously applied
glycine in both neonate and juvenile HMs. These results suggest that
ethanol directly modulates the GlyR. To investigate possible mechanisms
for this, we analyzed the time course of mIPSCs and single-channel
conductance of the GlyR in the presence and absence of ethanol. We
found that ethanol did not significantly change the time course of
mIPSCs. We also determined that ethanol did not significantly change
the single-channel conductance of synaptic GlyRs, as estimated by
nonstationary noise analysis of mIPSCs. We conclude that the adult form
of the native GlyR is more sensitive to ethanol than the fetal form.
Further, enhancement of GlyR currents involves mechanisms other than an
increase in the single-channel conductance or factors that alter the
decay kinetics.
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INTRODUCTION |
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Ethanol has been shown to
modulate ligand-gated ion channels (for review see Crews et al.
1996). Depending on the receptor type and subunit composition,
ethanol can have either a potentiating or depressing effect on the
response of neurons to neurotransmitters. Several studies have shown
that ethanol potentiates glycine-activated currents in different types
of cultured cells: hippocampal neurons (Aguayo and Pancetti
1994
), synaptoneurosomes (Engblom and Akerman 1991
), chick spinal cord neurons (Celentano et al.
1988
), mouse spinal cord neurons (Aguayo et al.
1996
), and Xenopus oocytes expressing homomeric
glycine receptor (GlyR) channels (Mascia et al. 1996b
).
In the Xenopus expression system, homomeric GlyR channels
composed of
1 subunits (the adult channel isoform) were more
sensitive to ethanol than channels composed of
2 subunits (the
neonatal isoform) (Mascia et al. 1996b
). It is not known whether native adult GlyRs, which contain both
1 and
subunits, are more sensitive to ethanol than native neonatal GlyRs containing both
2 and
subunits.
Previous studies in our laboratory have shown that hypoglossal
motoneurons (HMs) in the medullary slice preparation receive significant glycinergic inhibitory synaptic input (Singer and Berger 1999; Umemiya and Berger 1994
). HMs
control muscles of the tongue and function in mastication, swallowing,
suckling, and speech. They also play an important role in respiration
by regulating airway patency (Lowe 1980
). Studies have
shown that inhibitory input to HMs increases during rapid eye movement
(REM) sleep (Yamuy et al. 1999
), and that excessive
inhibition of HMs can contribute to obstructive sleep apnea (OSA)
(Remmers et al. 1980
; Wiegand et al.
1991
). OSA severity has been shown to be decreased by blocking
GlyRs with strychnine (Remmers et al. 1980
). Adding
strychnine also resulted in augmented oropharyngeal muscle activity
(Remmers et al. 1980
). These results suggest that
glycinergic synaptic transmission has an important physiologic and
pathologic role in this system.
The ethanol modulation of glycinergic input is also potentially
important. In human studies, OSA severity has also been shown to be
increased by ethanol (Issa and Sullivan 1982;
Scrima et al. 1982
; Taasan et al. 1981
).
Ethanol has also been shown to decrease the respiratory-related
activity of the hypoglossal nerve in vivo and in vitro (Bonora
et al. 1984
; Di Pasquale et al. 1995
; Gibson and Berger 2000
). The decrease in HM activity due
to ethanol could lead to reduction of contraction of the tongue
muscles, and thereby promote OSA.
Thus we studied the effect of ethanol on glycinergic synaptic
transmission to HMs in a medullary slice preparation. HMs express heteromeric GlyR channels, containing both and
subunits
(Singer and Berger 1999
; Singer et al.
1998
). A previous study has shown that the
subunit in HMs
undergoes a developmental shift from being primarily
2 at neonate
ages [postnatal day 0 to 3 (P0-3)] to primarily
1 at juvenile ages (P10-18) (Singer
et al. 1998
). We used this developmental change in receptor
subtype to study the differences in ethanol potentiation with changing
GlyR subunit composition. Specifically, we studied glycinergic
spontaneous miniature inhibitory postsynaptic currents (mIPSCs), evoked
IPSCs (eIPSCs), and currents due to focal glycine application, in HMs to determine whether native GlyR channels are sensitive to ethanol, and
whether the sensitivity changes with subunit composition during the
postnatal period. We also investigated several mechanisms whereby
ethanol may potentiate glycinergic mIPSCs. Preliminary reports of some
of these results have appeared as abstracts (Eggers et al.
1999
; O'Brien et al. 1998
).
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METHODS |
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Brain stem slice preparation
These experiments utilized in vitro slice recordings from Sprague-Dawley rat HMs (P1-13). Rats were anesthetized with either a ketamine-xylazine mixture (200 and 14 mg/kg, respectively) or halothane and then decapitated. The medulla was isolated and sliced into 300-µm-thick sections with a vibratome (Pella) while in ice-cold Ringer solution containing (in mM) 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1 NaH2PO4·H2O, 26.2 NaHCO3, 11 glucose, and 2.5 CaCl2, bubbled with carbogen (95% O2-5% CO2). Slices were incubated at 37°C for 1 h in carbogen-bubbled Ringer solution, and then stored at room temperature in the same solution.
Recordings
Whole cell recordings were made on visualized HMs. Slices were
placed in a recording chamber mounted on a fixed-stage microscope (Zeiss) and perfused with carbogen-bubbled Ringer solution. The microscope had DIC optics and was illuminated with near-infrared light.
Neurons were visualized using a ×40 water immersion lens. The output
of the microscope was fed to an infrared-sensitive video camera
(Hamamatsu) and sent to a video monitor (Sony). HMs were identified
based on location in the slice, near the central canal and ventral to
the dorsal motor nucleus of the vagus, and by their size and shape
(Umemiya and Berger 1994).
Whole cell voltage-clamp recordings were made with electrodes of 2-5
M resistance pulled from borosilicate glass capillary tubes (Warner
Instrument). The pipette was filled with (in mM) 140 CsCl, 2 MgCl2, 10 HEPES, 10 EGTA, 2 ATP, and 0.2 GTP.
Pipette solution pH was adjusted to be 7.3 with CsOH. For those
experiments where evoked synaptic responses were studied, 10 mM QX-314
(Sigma) was also added to the pipette solution to block action
potentials in the recorded cell. Series resistances for whole cell
recording were <10 M
. These were measured at the beginning and end
of each whole cell recording and were uncompensated.
Whole cell recordings were made in the Ringer solution described above.
The bath solution also contained 6,7-dinitro-quinoxaline (DNQX, 10 µM, Research Biochemicals) to block
non-N-methyl-D-aspartate (non-NMDA) glutamate
receptors, D()-2-amino-5-phosphonopentanoic acid (APV, 25 µM, Tocris) to block NMDA receptors and bicuculline methiodide (BMI,
5 µM, Sigma) to block GABAA receptors. This
concentration of BMI was shown to block GABAA
responses in HMs (O'Brien and Berger 1999
). When
glycinergic mIPSCs and the responses of cells to short-duration focal
application of glycine were studied, tetrodotoxin (TTX, 0.5 µM,
Alomone) and CdCl2 (100 µM) were also added to
the bath to block voltage-gated Na+ channel and
calcium channel dependent synaptic transmission. For the electrically
evoked response experiments, an electrode filled with 2 M NaCl was
placed lateral to the hypoglossal motor nucleus, and electrical stimuli
were applied. The stimuli were controlled by a stimulator and a
stimulus isolation unit (S88 and SIU5, Grass Instruments). For
experiments employing focal glycine application, glycine was applied
using a glass electrode filled with 200 µM glycine. Brief
applications of glycine were performed using a Picospritzer II (General Valve).
Voltage-clamp recordings were made in the whole cell configuration
(Vhold = 55 mV, unless otherwise
noted) using the Axopatch 200B amplifier (Axon). Clampex and Fetchex
software (Axon, versions 6, 7, 8) were used to acquire data to control
the glycine application and electrical stimulation. Data were filtered
at 2 kHz and acquired at 5 kHz, unless otherwise noted. After recording
control mIPSCs, glycine responses, or eIPSCs for 10 min, various
concentrations of ethanol were added to the bath solution and allowed
to equilibrate for 10 min. Ethanol enhanced currents were then
recorded. A liquid junction potential of 4-5 mV was measured, and the
command potentials were corrected for this potential only for the
calculation of single-channel conductance for nonstationary analysis of mIPSCs.
Data analysis
Data arising from focal glycine applications and electrical
stimulations were analyzed using Clampfit (Axon, versions 6, 8). mIPSCs
were identified using software developed in our laboratory (algorithm
by Cochran 1993), and analyzed with Excel 97 (Microsoft). The distributions of mIPSC amplitudes were compared using
a Kolmogorov-Smirnov (K-S) test. Differences in averages for groups of
cells were tested with a two-tailed t-test. Values reported
are means ± SE. Decay times were computed by aligning mIPSCs
along the rising phase, averaging the mIPSCs whose decay phase did not
contain any other events and fitting the decay phase of the average
mIPSC with a single exponential function (Igor Pro, Wavemetrics).
To estimate the single-channel conductance of GlyRs, nonstationary
noise analysis (Traynelis et al. 1993) was performed on mIPSCs in the presence and absence of 100 mM ethanol for both neonate
and juvenile HMs. The mIPSCs chosen for this analysis had a 10-90%
rising phase of <1 ms and decay phases free of other events. For each
HM, mIPSCs were aligned along their rising phase and averaged. The
average mIPSC was scaled to the peak of each individual mIPSC and then
subtracted to give a difference current. The difference current for
each mIPSC reflects noise due to single-channel closings. The average
mIPSC amplitude was divided into bins equal to 5% of the peak current.
For all mIPSCs, the average variance for each amplitude bin (average
difference current squared) was computed and plotted versus the binned
average current. The first 25% of this graph was fit to a line to
estimate the single-channel current. This procedure has been determined
to sufficiently fit the initial portion of the noise relationship
(Traynelis et al. 1993
).
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RESULTS |
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Ethanol increases the frequency and amplitude of glycinergic mIPSCs
Glycinergic mIPSCs recorded in HMs
(Vh = 55 mV) were isolated by
blocking non-glycinergic currents [NMDA,
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA), GABAA, voltage-gated
Na+ channels, and Ca2+
channels]. As shown in Fig. 1,
bath-applied ethanol (100 mM) increased both the average amplitude and
frequency of glycinergic mIPSCs. In HMs from juvenile rats
(P9-12), 100 mM ethanol increased the amplitude
by an average of 55 ± 8% (mean ± SE) and decreased the
mIPSC interval by 67 ± 7% (n = 10 cells). These
mIPSCs were fully blocked by bath application of strychnine (2 µM,
Fig. 1).
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Neonatal HMs are less sensitive to ethanol than juvenile HMs
Previous work by Mascia et al. (1996b), using the
Xenopus oocyte expression system, showed that homomeric GlyR
channels containing the
2 subunit are less sensitive to ethanol than
those containing the
1 subunit. Native GlyR channels that express
subunits along with
subunits have not been previously studied.
Therefore we studied glycinergic mIPSCs during two developmental
periods: neonate (P1-3) when glycine channels contain
predominantly
2 subunits, and juvenile (P9-13) when
glycine channels contain predominantly
1 subunits (Singer et
al. 1998
). We observed that ethanol increases the frequency and
amplitude of glycinergic mIPSCs in both age groups. These increases
were studied over a range of ethanol concentrations.
We found that 100 mM ethanol increases the amplitude of mIPSCs in both juvenile and neonatal HMs. Shown in Fig. 2A are recordings of glycinergic mIPSCs in a neonatal and a juvenile HM. Adding 100 mM ethanol to the bathing solution increased the amplitude and frequency in both cases (Fig. 2B). The distributions of amplitudes in control and ethanol conditions, shown in Fig. 2C, were significantly different by the K-S test (P < 0.001). On average in the cells tested, 100 mM ethanol increased the amplitude of mIPSCs from neonatal HMs by 30 ± 3% (n = 15 cells, paired t-test P < 0.001) and from juvenile HMs by 55 ± 8% (n = 10 cells, paired t-test P < 0.001).
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In contrast to the results found at 100 mM ethanol, we observed that 30 mM ethanol increases the amplitude of juvenile HMs while leaving the amplitude of neonatal HMs unchanged. Shown in Fig. 3A are glycinergic mIPSCs from a neonatal and a juvenile HM. Adding 30 mM ethanol to the bath solution increased the frequency of mIPSCs in both cases, but significantly increased the amplitude of only the juvenile HM (Fig. 3, A and B). The mIPSC amplitude distributions for the juvenile HM were significantly different between control and ethanol (K-S, P < 0.001), but the amplitude distributions for the neonate HM were not (K-S, P > 0.01). On average, 30 mM ethanol significantly increased the amplitude of juvenile mIPSCs by 33 ± 6% (n = 7, paired t-test P < 0.005) but did not change the amplitude of neonatal mIPSCs (2 ± 6%, n = 7, NS, paired t-test P = 0.6).
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We studied the differential sensitivity of juvenile and neonate HMs to
ethanol at doses from 1-300 mM in juveniles and 10-300 mM in
neonates. The average percent increase in mIPSC amplitude due to
ethanol at each dose is shown in Fig. 4.
At doses of 10-50 mM for neonatal mIPSCs, there was no statistically
significant amplitude increase due to ethanol (P > 0.5). For this age group, a dose of 300 mM was required to achieve a
50% increase in amplitude. In contrast, at doses 10 mM and higher,
juvenile mIPSCs consistently showed an ethanol-induced increase. The
only dose for which an increase was not typically seen was 1 mM.
Juvenile HMs showed an average of 50% increase at about 100 mM, a
third of the dose required by neonates for an equivalent increase.
There was a significant difference between age groups in the average
increase in mIPSCs due to ethanol for 4 doses: 10 (P < 0.05), 30, 50, and 100 mM (P < 0.01). Thus glycinergic
mIPSCs from neonatal HMs, which are mediated by GlyR channels that
contain primarily the 2 subunit, are less sensitive to ethanol than
mIPSCs from juvenile HMs, which are mediated by GlyRs that contain
primarily the
1 subunit.
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We found that at all doses tested (10-300 mM) ethanol decreased the
interval between successive mIPSC onsets. With a dose of 30 mM ethanol,
the mIPSC interval decreased by 42 ± 16% (n = 7)
in neonate HMs and 48 ± 8% (n = 5) in juvenile
HMs. When we increased the dose of ethanol to 100 mM, we found a
75 ± 5% (n = 12) decrease mIPSC interval in
neonates and 67 ± 7% (n = 9) decrease in
juveniles. We found no significant difference (P > 0.1) in the interval decrease at any dose between the age groups studied: neonate (P1-3, n 3 cells/dose)
and juvenile (P9-13, n
4 cells/dose). This was to
be expected, as an increase in mIPSC frequency would likely be due to a
presynaptic effect, and therefore should not have a correlation with
the subunit shift of postsynaptic GlyRs.
Ethanol enhances evoked IPSCs
We have shown that ethanol increases glycinergic mIPSCs in neonate
and juvenile HMs and that the ethanol sensitivity changes with
development. We next tested whether ethanol enhances evoked synaptic
responses that result from action potential-induced release of glycine
from the presynaptic cell. We studied evoked glycinergic IPSCs (eIPSCs)
by applying electrical stimuli lateral to the hypoglossal motor nucleus
and recording the response in a HM. Shown in Fig. 5, A and B, are
averages of at least 10 eIPSCs from HMs in control and ethanol
conditions. In the cells shown, ethanol (100 mM) increased the eIPSCs
of both neonate (31%) and juvenile (63%) HMs (Fig. 5B). On
average, 100 mM ethanol increased eIPSCs by 39 ± 7% in neonate
HMs (n = 4) and 111 ± 38% in juveniles
(n = 4, Fig. 5C). However, as seen with the
mIPSCs, the sensitivity of glycinergic eIPSCs to ethanol changes with
postnatal development. As shown in Fig. 5A, 30 mM ethanol,
while increasing juvenile eIPSCs by 40%, did not increase the response
of the neonate HM (6%). On average 30 mM ethanol increased juvenile
eIPSCs by 59 ± 26% (n = 4) and did not change
neonate HMs (n = 4,
3 ± 4%, Fig.
5C). Thus the sensitivity of glycinergic eIPSCs also changes
with development, as juvenile eIPSCs are more sensitive to ethanol than
neonate eIPSCs.
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Ethanol can act directly on GlyR channels
We have shown a difference in glycinergic mIPSC and eIPSC amplitude enhancement by ethanol that is correlated with changes in glycinergic receptor subunit composition. However, mIPSCs and eIPSCs are synaptic events and, as such, an increase in amplitude could be mediated by presynaptic changes or direct receptor effects. Therefore we studied the effect of ethanol on currents induced by exogenous focal application of glycine to HMs to determine whether ethanol can directly effect the postsynaptic cell. We focally applied glycine to the soma of HMs, measured the current response, and looked for enhancement of this response by ethanol.
We found that both neonate and juvenile HMs responded to focal application of glycine and that this response could be enhanced by ethanol (Fig. 6). Ethanol (100 mM) increased the amplitude of current response to glycine in neonatal HMs by an average of 35 ± 6% (n = 3, data filtered at 500 Hz). Ethanol (50 mM) increased the response of juvenile HMs to glycine by an average of 48 ± 22% (n = 3). Interestingly, these values were similar to the amplitude increases of glycinergic mIPSCs due to ethanol: in neonate HMs, 100 mM increased the amplitude by 30 ± 3% (n = 15); in juvenile HMs, 50 mM increased the amplitude by 45 ± 9% (n = 5). These results, together with our result that changes in efficacy of ethanol correlate with changes in postsynaptic GlyR subunits, strongly suggest that the ethanol-induced mIPSC amplitude increase is due to a direct modulation of the postsynaptic GlyR channel by ethanol.
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Ethanol does not alter the decay time of mIPSCs
We have shown that ethanol can potentiate currents due to exogenous glycine application and that the sensitivity of glycine responses to ethanol correlates with changes in postsynaptic GlyR subunits. Together these suggest that ethanol is modulating the GlyR channel. Next, we wanted to investigate whether this change in the glycine channel response would also be reflected in the time course of mIPSCs. A change in mIPSC decay time course could reflect changes in the kinetics of GlyR channels. We therefore measured the average decay time of mIPSCs with and without 100 mM ethanol. At this dose of ethanol, GlyRs from both neonatal and juvenile HMs are modulated since the amplitudes of mIPSCs are increased. Therefore we would expect an effect on the decay time course if ethanol modulates channel kinetic properties that contribute to this decay phase.
We found that ethanol (100 mM) did not significantly change the decay
kinetics of mIPSCs. We fit a single exponential to the decay course of
the average mIPSC for each cell with and without ethanol and determined
the decay time constant (decay). The neonatal HMs had an average
decay of 10.5 ± 1.5 ms in control and 11.3 ± 1.5 ms in ethanol (n = 13). The juvenile HMs had an average
decay of
7.4 ± 0.5 ms in control and 7.4 ± 0.6 ms in ethanol (n = 9). In both age groups, ethanol did not
significantly alter the decay time (paired t-test,
P > 0.01).
Ethanol does not alter single-channel conductance
Next, we investigated whether an increase in single-channel
conductance of the GlyR was responsible for the potentiation of the
mIPSC due to ethanol. We estimated the single-channel conductance by
performing nonstationary noise analysis of glycinergic mIPSCs with and
without 100 mM ethanol (described in METHODS). For four cells in both the neonate and juvenile age groups, we obtained the
variance-current relationship that describes single-channel noise. The
first 25% of this relationship was fit with a line to estimate the
single-channel current for control and ethanol conditions (Fig.
7A). The average
single-channel current estimated for neonates was as follows: control
i = 1.7 ± 0.2 pA, ethanol i =
1.9 ± 0.2 pA; juveniles: control i =
1.7 ± 0.2 pA, ethanol i =
1.5 ± 0.3 pA. From this
value the average single-channel conductance could be estimated using
the Cl
driving force. Using the concentrations
of Cl
in the pipette and bath solutions, the
ECl was calculated to be +4 mV. The
holding potential in these experiments was
51 mV. The
Cl
driving force was thus calculated to be
55
mV. Using these values we estimated the average single-channel
conductances as follows (Fig. 7B): neonate control
g = 31 ± 4 pS, ethanol g = 34 ± 3 pS; juvenile control g = 31 ± 3 pS,
ethanol g = 27 ± 5 pS. These values were not
significantly different from each other (P > 0.1). We found that ethanol does not alter the single-channel conductance of
GlyRs, therefore this is not a mechanism for the increase in the
amplitude of glycinergic currents.
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DISCUSSION |
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We have demonstrated that glycinergic synaptic transmission to HMs
is augmented by ethanol in two ways. First, ethanol increases the
amplitude of currents activated by glycine, as shown here for mIPSCs,
focal glycine application, and eIPSCs. Second, ethanol increases the
frequency of mIPSCs, thus increasing the glycinergic input to HMs. HMs
from both neonates (expressing the 2 GlyR subunit) and juveniles
(expressing the
1 GlyR subunit) are sensitive to ethanol. However,
neonate HMs are less sensitive to ethanol than juvenile HMs.
Comparison with previously observed glycine and ethanol interactions
These results agree with and extend previous studies of the
effects of ethanol on glycinergic neurotransmission. It has been observed previously in several systems that the current response to
exogenous glycine application is increased due to ethanol
(Aguayo and Pancetti 1994; Aguayo et al.
1996
; Celentano et al. 1988
; Engblom and
Akerman 1991
; Mascia et al. 1996a
,b
;
Tapia et al. 1998
). Our results concur that ethanol
increases the amplitude of glycinergic current responses and extend the
results of focal glycine application to synaptically mediated
glycinergic mIPSCs and eIPSCs. It has also been observed in spinal
motoneurons that the frequency of combined GABAergic and glycinergic
mIPSCs is increased by ethanol (Cheng et al. 1999
). We
observed that the frequency of glycinergic mIPSCs, isolated from
GABAergic mIPSCs, is also increased in HMs. However, Cheng et
al. (1999)
did not observe an increase in the amplitude of
inhibitory mIPSCs with the application of 70 mM ethanol. Two effects
may contribute to this difference. First, they did not isolate
GABAergic mIPSCs from glycinergic mIPSCs, so it is unclear how much of
the current is due to glycine. Also, they studied spinal motoneurons
from neonatal animals, which presumably contain primarily the
2
subunit of the GlyR. Thus their dose of 70 mM ethanol may not have had a significant effect on the amplitude of GlyR currents.
It has also been previously observed, using the Xenopus
oocyte expression system, that homomeric 2 GlyRs are less sensitive to ethanol than homomeric
1 GlyRs (Mascia et al.
1996b
). We extended this study to look at native synaptic GlyRs
that are heteromeric, containing
subunits as well as
subunits.
We have shown that the difference in ethanol sensitivity shown for
expressed homomeric channels is also present for native heteromeric channels.
Mechanism of ethanol actions
We have shown that ethanol increases the frequency and amplitude of glycinergic mIPSCs. An increase in spontaneous release frequency is most likely a presynaptic effect. The increase in amplitude of mIPSCs could be either pre- or postsynaptic. However, we found that the amplitude of currents due to focal application of glycine also was increased by ethanol, independent of any presynaptic effects and that the efficacy of ethanol was correlated with changes in postsynaptic GlyR subunits. Thus we conclude that ethanol has both a presynaptic and postsynaptic effect on glycinergic neurotransmission.
Previous studies have shown that ethanol can change the frequency of
mIPSCs in spinal motoneurons (Cheng et al. 1999) and at
the frog neuromuscular junction (Kriebel and Bridy
1996
). The mechanism of this effect was not determined. Ethanol
could be affecting presynaptic release by several mechanisms. The
potentiation of glycinergic currents by ethanol has been shown to be
decreased by inhibiting protein kinase C in Xenopus oocytes
(Mascia et al. 1998
) and hippocampal neurons
(Aguayo and Pancetti 1994
). Ethanol's potentiation has
also been shown to decrease with inhibition of G-protein systems
(Aguayo et al. 1996
). Ethanol has been shown to increase
the production of phospholipase C in hepatocytes
(Higashi et al. 1994
) and to modulate intracellular
Ca2+ levels (Rabe and Weight
1988
). All of these mechanisms could potentially contribute to
an increased frequency of spontaneous release.
The postsynaptic effect of ethanol is thought to be a direct effect on
the GlyR channel. It has been previously shown in cultured mouse spinal
cord neurons that ethanol can enhance the response to glycine without
changing lipid fluidity of the membrane (Tapia et al.
1998). Further evidence for a direct effect on the receptor is
that ethanol enhancement can be diminished by specific differences in
protein sequences. For homomeric GlyRs expressed in Xenopus, a mutation of one amino acid significantly reduces the potentiation by
ethanol of the
1 receptor (Mascia et al. 1998
).
Another piece of evidence for a direct protein effect, shown in the
present study, is that developmental changes in sensitivity to ethanol correlate with the developmental change in
subunit composition of
the GlyR.
The increased response of GlyRs due to ethanol could be accomplished by
several changes in single-channel properties. First, ethanol could
increase the conductance of GlyR channels to
Cl. However, we did not see an increase in
single-channel conductance when estimated using nonstationary noise
analysis. Ethanol could also be altering the decay time of mIPSCs, but
we did not observe this. Experiments analyzing the full set of kinetic
properties of isolated GlyRs are needed to determine the precise
mechanism of ethanol's action. It is possible that ethanol is
increasing the number of effective GlyRs by altering neurotransmitter
affinity or open probability. Ethanol has also been shown to increase
the frequency of channel openings for excised
GABAA channels (Tatebayashi et al.
1998
). Further studies are necessary to determine whether this
mechanism could also apply to glycine channels.
Physiological consequences of ethanol potentiation of GlyR responses
We found in this study that neonate (2 containing) GlyRs are
less sensitive to ethanol than juvenile (
1 containing) GlyRs. This
suggests that the glycine neurotransmission system is partially protected from the effects of ethanol at young ages. In addition to
undergoing a developmental subunit shift, the GlyR also shows functional developmental changes. Because of high internal
Cl
concentrations early in development, it is
possible that activating GlyRs depolarizes neurons at young ages. This
depolarization has been shown to increase intracellular
Ca2+ (Flint et al. 1998
; Lo
et al. 1998
; Reichling et al. 1994
), and therefore could be acting as a neurotrophic agent. In HMs this shift in
the Cl
gradient has been shown to occur between
the neonate and juvenile age groups that we studied (Singer et
al. 1998
).
Our results demonstrate that ethanol increases the glycinergic
input to HMs. Excessive inhibition to the hypoglossal motor nucleus is
thought to be a partial cause of OSA (Remmers et al. 1980; Wiegand et al. 1991
). Recently, using an
animal model of REM sleep, a sleep state when OSA is most prevalent,
HMs were shown to have large glycinergic IPSPs (Yamuy et al.
1999
). The prevalence of OSA is also increased by ethanol
administration in humans (Issa and Sullivan 1982
;
Scrima et al. 1982
; Taasan et al. 1981
).
Since we have demonstrated that ethanol increases glycinergic IPSCs in
HMs, this could be a mechanism for ethanol's potentiation of OSA.
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ACKNOWLEDGMENTS |
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We thank W. Satterthwaite for analysis software and P. Huynh for technical assistance.
E. D. Eggers was supported by National Institutes of Health (NIH) National Research Service Award T32 GM-07270. J. A. O'Brien was supported by NIH Training Grant 5T32GM-07108. This research was also supported by NIH Grants HL-49657 and NS-14857 to A. J. Berger.
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FOOTNOTES |
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Address for reprint requests: E. D. Eggers, Dept. of Physiology and Biophysics, School of Medicine, University of Washington, Box 357290, Seattle, WA 98195-7290 (E-mail: eeggers{at}u.washington.edu).
Received 24 April 2000; accepted in final form 27 July 2000.
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