Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195-7290
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ABSTRACT |
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Gibson, Ian C. and
Albert J. Berger.
Effect of Ethanol Upon Respiratory-Related Hypoglossal Nerve
Output of Neonatal Rat Brain Stem Slices.
J. Neurophysiol. 83: 333-342, 2000.
The actions of ethanol (EtOH)
on the respiratory output of the neonatal rat brain stem slice
preparation in vitro are described. Ethanol inhibited
respiratory-related hypoglossal nerve activity in a dose-dependent
manner. The effect of EtOH was evident within 5 min and was reversible
on EtOH washout. The actions of EtOH were qualitatively similar to
those of two other alcohols, methanol and octanol. We investigated the
dose-response relationship for each alcohol and determined that the
order of potency was methanol < EtOH octanol, with
EC50 values of 291 mM, 39.7 mM, and 49.2 µM respectively.
Application of either strychnine (5 µM) or bicuculline (5 µM)
alone, partially but not significantly, reversed the inhibition of
respiratory-related hypoglossal nerve activity produced by 50 mM EtOH.
Preincubation of rhythmic slices with a combination of both strychnine
and bicuculline (both 5 µM) partially, but significantly, blocked the
inhibitory actions of EtOH, suggesting that other mechanisms also play
a role in the action of EtOH. Preincubation of the slices with 25 µM
APV reduced the relative degree of inhibition caused by EtOH suggesting
that N-methyl-D-aspartate (NMDA)-receptor-mediated events can be affected by EtOH. Furthermore inhibition of protein kinase C by incubation with 100 nM staurosporine also reduced the efficacy of EtOH. These results suggest that the
actions of EtOH may be mediated via glycine, GABAA, and
NMDA receptors and that activation of protein kinase C is involved in
the EtOH-induced inhibition of respiratory-related hypoglossal nerve activity.
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INTRODUCTION |
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Ethanol (EtOH) can modify the activity of neurons
in the CNS including the hippocampus (Peoples et al.
1997; Proctor et al. 1992
), neocortex
(Soldo et al. 1998
), and locus coeruleus (Nieber et al. 1998
). Furthermore EtOH is also known to have a
depressant effect on respiration-related hypoglossal nerve output in
humans as well as other mammals (Dawson et al. 1997
;
Di Pasquale et al. 1995
; Krol et al.
1984
; Scrima et al. 1982
). With such a broad spectrum of targets, it is likely that EtOH acts on neurons other than
hypoglossal motoneurons (HMs) within the brain stem to reduce hypoglossal drive.
The depressant activity of EtOH on hypoglossal nerve output can have
serious consequences in humans with obstructive sleep apnoea (OSA),
which is characterized by a collapse of the upper airway during
respiration in sleep. The patency of the airway is determined by the
balance between the activity of neurons that maintain upper airway
muscle tone, which tends to promote airway patency, and the collapsing
influence arising from negative intrathoracic pressure caused by the
inspiratory activity of the diaphragm. The genioglossus is an important
tongue protruder muscle the activity of which promotes upper airway
patency. Thus EtOH, through its action on genioglossal output, can
markedly exacerbate OSA. It has been demonstrated that EtOH has a
marked depressing effect on genioglossal nerve output but spares
phrenic nerve output (Krol et al. 1984), resulting in a
reduced upper airway patency in the on-going presence of diaphragmatic
effort. Other studies have concluded that EtOH acts to increase upper
airway resistance in asymptomatic snorers (Issa and Sullivan
1982
; Mitler et al. 1988
).
Hypoglossal motoneurons receive excitatory, respiratory-related drive
from a region of the ventrolateral brain stem known as the
pre-Bötzinger complex, which now generally is accepted to be the
locus of respiratory rhythm generation (Schwarzacher et al.
1995; Smith et al. 1991
). The interaction of
neurons in this region has been studied, and it is known that in adult
animals, inhibitory neurotransmission is critical for the maintenance
and generation of respiratory rhythm (Hyashi and Lipski
1992
; Pierrefiche et al. 1998
; Ramirez et
al. 1997b
). Application of glycine and/or GABAA receptor antagonists (strychnine and
bicuculline, respectively) markedly affect respiratory output. However,
in neonatal animals, the role of inhibitory amino acids appears to be
less critical as strychnine and bicuculline have lesser effects. This
finding has led to the suggestion that rhythm generation in the newborn rat may be more dependent on pacemaker driven circuits rather than
postsynaptic inhibitory mechanisms (Onimaru et al. 1990
; Smith et al. 1991
). However, both glycine and
GABAA receptor activation still play a role in
the modulation of excitability and output pattern of such rhythmogenic
respiratory neurons (Ramirez et al. 1997b
; Shao
and Feldman 1997
).
When applied to the in vitro rhythmically active slice, both glycine
and GABA produce marked blockade of the respiratory output (Brockhaus and Ballanyi 1998). Conversely, application
of strychnine and bicuculline enhanced the respiratory output (see
Brockhaus and Ballanyi 1998
; Hyashi and Lipski
1992
; Paton and Richter 1995a
; Pierrefiche et al. 1998
; Ramirez et al.
1997b
). In other systems, responses to both glycine
(Aguayo and Pancetti 1994
; Engblom and Åkerman
1991
; Mascia et al. 1998
) and GABA
(Aguayo and Pancetti 1994
) have been shown to be
affected markedly by EtOH. Because of the important role of glycinergic
and GABAergic input in modulation of the respiratory rhythm, EtOH may
have a profound effect on respiratory output if it acts to enhance the
actions of one or both of these inhibitory neurotransmitters. Thus one
of the goals of the present study was to determine if the depression of
rhythmic hypoglossal nerve activity by EtOH is due to its effect on
glycinergic and GABAergic transmission.
Another possible target for EtOH could be excitatory neurotransmission.
Indeed, diminished responses to NMDA receptor activation in the
presence of EtOH have been reported in a number of brain regions
including the nucleus accumbens (Nie et al. 1994),
cortical neurons (Marszalec et al. 1998
), and the
amygdala (Gean 1992
). The effect of NMDA receptor
activation or inhibition has been investigated in the rhythmically
active brain stem preparation (see Funk et al. 1993
;
Ge and Feldman 1998
; Greer et al. 1991
; Kashiwagi et al. 1993
; Takeda and Matsumoto
1998
). We also investigated whether EtOH blocks NMDA-mediated
neurotransmission to reduce respiratory output.
We have used the in vitro brain stem slice preparation (Smith et
al. 1991) that generates stable, robust rhythmic activity that
can be assessed by recording electrical activity from the cut ends of
the hypoglossal rootlets. We found that EtOH reduces respiratory output
from the slice at physiologically relevant concentrations and that this
effect is mimicked qualitatively by other alcohols namely octanol and
methanol. Mechanisms underlying the actions of EtOH were investigated
by bath application of specific receptor antagonists, and we found that
EtOH appears to have a multitude of effects on the transmitter systems
in the rhythmically active slice.
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METHODS |
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Slice preparation
The experiments described in this study were performed on rhythmically active slices taken from the brain stem of neonatal Sprague-Dawley rats aged between 1 and 5 days. Briefly, the rat was anesthetized deeply with halothane and then transected below the forelimbs. The head and forelimbs were then pinned out onto a silicone elastomer (Sylgard) block and submerged in an artificial cerebrospinal fluid (ACSF) containing (in mM) 115 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 24 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose at room temperature. The solution was oxygenated with 95% O2-5% CO2. In some experiments, 10 mM HEPES was included and NaOH was added to adjust the pH to 7.4. The brain stem and spinal cord were exposed, and a transverse cut was made at the level of the superior colliculus. The cerebellum, choroid plexus, and any dura remaining on the brain stem were removed. The brain stem spinal cord was freed and pinned into a separate dish filled with oxygenated ACSF. The pia mater/arachnoid tissue was cut along the midline (above the basilar artery), and the hypoglossal rootlets were teased free using a blunt dissection pin. Finally the basilar artery was pulled carefully from the tissue. The brain stem spinal cord then was pinned onto a molded plasticine bed and locked into the vice of a Vibratome with the rostral end uppermost and bathed in gassed ACSF. If necessary, the plasticine bed could be deformed gently and/or maneuvered such that the dorsal surface of the tissue was flush with the plasticine. Initially, slices of between 50 and 150 µm were cut from the rostral end of the tissue until the compact nucleus ambiguus was evident but the facial nucleus was absent. At this point, a 400- to 700-µm slice was taken that included the most rostral hypoglossal rootlets. This slice then was placed into the recording chamber and superfused with a high K+ ACSF solution containing (in mM) 110 NaCl, 9 KCl, 2 CaCl2, 1 MgCl2, 24 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose gassed with 95% O2-5% CO2. The temperature was maintained between 27 and 28°C.
Electrophysiological recordings
Glass suction electrodes filled with ACSF were used to record
from the cut ends of the hypoglossal rootlets. Signals were amplified
using a CyberAmp 320 (Axon Instruments) at a gain of 500-2,000 and
filtered at 10 kHz. The raw electrical signal also was rectified and
integrated using a custom built "leaky" integrator with a time
constant of 50 ms and gain of 10. Both raw and integrated signals were
digitized (Neuro-Corder, Neurodata Instruments), stored on videotape
and also displayed on a chart recorder (Gould TA-2000). The slice was
left to equilibrate for 30 min before any recordings were made. The
stability of the recording was assessed by sampling
30
rectified-integrated bursts using pClamp 6 or pClamp 7 (Axon
Instruments) and measuring the constancy of the area underneath each burst.
In a separate set of experiments to assess the effect of EtOH and octanol on passive membrane properties, we made whole cell recordings from HMs in thin (300 µm) slices of the brain stem. These experiments were conducted under conditions where fast synaptic transmission was abolished by bath application of TTX (1 µM), 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 µM), APV (20 µM), strychnine (10 µM), and bicuculline (10 µM).
Drug application
All drugs used in this study were diluted from stock solutions to the required concentration in high K+ ACSF and superfused over the slice at a rate of 3-4 ml/min. The solution was removed either from the bath by suction outlet or recirculated from the bath back to the reservoir. As 1-octanol is insoluble in water it first was mixed with methanol and then added dropwise to the ACSF, which was kept stirred constantly to give a final octanol stock concentration of 1 mM (methanol concentration = 100 mM). Subsequent dilution of this stock solution to give submillimolar concentrations of octanol resulted in methanol concentrations of between 1 and 30 mM. These concentrations of methanol alone were not great enough to noticeably affect the respiratory output of the slice (see Fig. 2). All alcohols were added to the ACSF just before they were applied to the slice in an effort to reduce loss by evaporation.
All agents were applied until a steady-state effect on hypoglossal activity had been reached, usually between 10 and 15 min. Values in the text are derived from measurements of the area under the respiratory-related hypoglossal burst and are quoted as means ± SE unless otherwise stated. The area under the burst proved to be more indicative of the effect of the various agents used. Indeed, in some experiments the peak value of the hypoglossal burst was barely affected yet the area was reduced. Averaged burst envelopes were generated from 20 to 30 events aligned on their rising phase.
Unless otherwise stated, all values are given as means ± SE. Statistical analysis was performed with a two-tailed Students t-test, and significance was assumed if P < 0.05.
Chemicals used
Salts for the ACSF were obtained from ICN Pharmaceuticals. Ethanol was obtained from Midwest Grain Products of Illinois, methanol from Baker, octanol, strychnine, bicuculline methiodide and staurosporine from Sigma. D-2-amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d] cyclohepten-5,10-imin-H-maleate (MK-801) were purchased from Research Biochemicals International. Tetrodotoxin (TTX) was obtained from Alomone Labs.
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RESULTS |
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Basic activity
Each burst of hypoglossal nerve activity had a rapid rising phase
that was followed by a slowly decrementing period of activity (Fig.
1B). Analysis of the
rectified, integrated signal from rhythmic slices revealed that the
mean burst frequency was 8.4 ± 2.9 per min (n = 14). The duration of the burst had a mean value of 1,237 ± 87 ms
(range 768-2,040 ms; n = 17). These values are similar to those published elsewhere (Johnson et al. 1994;
McLean and Remmers 1994
; Smith et al.
1991
). Rhythmic activity was stable, and in many instances
still could be recorded up to 18 h later.
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Effect of ethanol
Figure 1A displays a representative experiment in which
EtOH was applied to the slice at concentrations between 10 and 100 mM.
The effect of EtOH was evident within 5 min and induced a dose-dependent reduction in burst frequency (Fig. 1, A and
C). In seven experiments where EtOH was applied sequentially
in increasing concentrations from 10 to 100 mM, the burst frequency
fell by 19.1 ± 7.6%, 39.7 ± 9.0%, and 92.3 ± 5.5%,
respectively. The burst frequency-dose data in Fig. 1C were
fitted to the following logistic function
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Increasing doses of EtOH also reduced the magnitude (assessed as the area under the burst) of the rhythmic hypoglossal discharge (see Fig. 1B). Pooling data from five experiments in which EtOH was applied sequentially revealed that the burst area fell to 86 ± 8%, 67 ± 11%, and 6 ± 6% of control values in 10, 30, and 100 mM EtOH, respectively. Figure 2 shows the dose-response relationship for the actions of EtOH, methanol, and octanol on burst area. Fitting the same logistic function as defined in the preceding text, where E is now the percentage inhibition of burst area, to the data points the EC50 value for EtOH with respect to burst area was determined to be 39.7 mM with a b value of 2.18. The number of observations at each alcohol concentration is given beside the appropriate datum point in Fig. 2.
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In subsequent experiments, single 50 mM EtOH doses routinely were used to inhibit rhythmic hypoglossal discharge as this concentration was close to the burst area EC50 yet still on the linear part of the dose-response curve for EtOH (see Fig. 2). During the course of these subsequent experiments, we noticed that on average the single application of 50 mM EtOH barely affected the hypoglossal interburst interval (reduced by 96.8 ± 5.7% of control, n = 22) even though the effect on burst area, approximately a 50% decrease, was almost the same as that predicted by the dose-response curve for EtOH (Fig. 2) obtained with increasing doses of EtOH. We have not investigated further why the 50 mM dose is so much less effective with respect to the burst rate than the 30 mM dose. However, one possibility is that successive doses of EtOH (i.e., the protocol used to generate the data shown in Fig. 1) may "precondition" the slice such that higher concentrations of EtOH become more effective.
Effect of other alcohols
A previous study showed that the potency of various alcohols in
modulating an ATP-gated ion channel was dependent to some extent on
molecular volume (Li et al. 1994). Accordingly, to see if this phenomenon was preserved in this in vitro preparation, we also
studied the effects of octanol and methanol using the same protocol as
for EtOH. Methanol had little effect at equimolar concentrations to
EtOH, and it was only at the rather high concentration of 300 mM that
inhibitory effects on the bursting rhythm became evident (Fig. 2). In
contrast, octanol had the opposite effect. Inhibitory effects were
observed at low concentrations (10 µM) with 300 µM producing a
total block of respiratory activity. Figure 2 compares the
dose-response relationships for each of the three alcohols. The shift
in EC50 values is clear with the respective EC50 values for octanol, EtOH, and methanol being
49.2 µM, 39.7 mM, and 291 mM, respectively. The b values
were 1.37, 2.18, and 1.21, respectively.
Is glycine involved in EtOH-induced inhibition?
Because EtOH exerted an inhibitory effect on rhythmic
bursting, and it is known from other studies that EtOH can enhance
glycine receptor-mediated currents (Aguayo and Pancetti
1994; Mascia et al. 1996
, 1998
), we examined the
effect of strychnine, a selective antagonist of glycine receptors, on
the rhythmic bursting activity in the presence of EtOH. Application of
50 mM EtOH to rhythmically active slices produced a significant
(P < 0.001) depression of the burst area to 49.0 ± 3.9% (n = 8) of control. Subsequent coapplication of strychnine at 5 µM induced recovery of the burst activity such that the burst area was 60.1 ± 8.7% of the control burst area (Fig. 3,
; P < 0.01 compared with control, n = 8). The burst area in the
presence of EtOH and strychnine was not significantly different from
that in the presence of strychnine alone (P = 0.272). After
15 min of washout of both the EtOH and strychnine there was a
recovery to control levels (105.7 ± 6.4%).
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Is GABA involved in EtOH-induced inhibition?
The other major inhibitory neurotransmitter in the respiratory
network of the neonate rat is GABA (Brockhaus and Ballanyi 1998). Could EtOH also be acting to enhance the actions of GABA and thereby depressing the respiratory drive? This possibility was
investigated using the same protocol as described in the preceding text
for the strychnine experiments. Pooling together data from six
experiments revealed that application of 50 mM EtOH significantly reduced (P < 0.01) the burst area to 55.6 ± 8.6% of control. On coperfusion of EtOH and 5 µM bicuculline, a
specific GABAA receptor antagonist, there was a
recovery of the burst area to a level of 71.4 ± 12.6% compared
with control (P = 0.071). The difference between the
burst in the presence of EtOH alone and EtOH and bicuculline was not
significant (P = 0.326). After washout of the drugs,
the burst area recovered to 103.6 ± 16.9% of control levels
(P = 0.84). These data are shown in Fig. 3 (
).
Effect of preincubation of both strychnine and bicuculline
We studied the effect of preincubating the slice with both 5 µM strychnine and 5 µM bicuculline to determine if EtOH was acting solely through glycine and GABAA receptor-mediated mechanisms. Figure 4 displays the pooled results from five experiments where a 15-min preincubation of both antagonists significantly increased the burst area to 143.4 ± 14.2% of control levels (P < 0.05). After co-application of EtOH (50 mM), the burst area was reduced to 81.6 ± 5.8% of control (P < 0.05). This represented a reduction in burst area of 41.4% compared with that in the presence of the antagonists alone (P < 0.01). Pooling all of the data for the actions of EtOH before the application of either strychnine or bicuculline (see preceding text) gave an average inhibition of burst area of 51.2 ± 4.2% (n = 14). This value is significantly different from that for EtOH-induced burst area inhibition after application of both antagonists (81.6 ± 5.8%, P < 0.01).
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Washing off all drugs resulted in the burst area returning to control levels (103.8 ± 11.8% of control).
Effect of EtOH on NMDA receptor-mediated transmission
Excitatory mechanisms contribute to the generation of respiratory
rhythm (Funk et al. 1993; Greer et al.
1991
; Smith et al. 1991
). As EtOH has been shown
previously to modify responses mediated via NMDA glutamate receptors in
hippocampal (Peoples et al. 1997
) and locus coeruleus
(Nieber et al. 1998
) neurons, we next investigated the
possible effect of EtOH on NMDA transmission in the rhythmic slice. In
five experiments the NMDA receptor antagonist APV (25 µM) was
perfused onto a rhythmic slice for 20 min, causing a significant reduction in burst area to 51.0 ± 4.8% of control values
(P < 0.001, n = 5). Subsequent
application of 50 mM EtOH in the presence of APV further reduced the
burst area to 27.0 ± 4.4% of the control burst area
(P < 0.01). This reduction took the area in the
presence of EtOH and APV to 52.1 ± 5.1% of that burst area
observed in the presence of APV alone (P < 0.01).
Compared with the effect of EtOH alone, the relative effect of EtOH in
the presence of APV was reduced, suggesting that EtOH inhibits NMDA
receptors under our experimental conditions. The effects of APV were
partially reversible on washout with drug-free ACSF (to 69.6 ± 6.1% of control). These data are summarized in Fig.
5, which also demonstrates the time
course of action of the agents applied under our experimental conditions.
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Effect of inhibition of protein kinase C
Protein kinase C (PKC) is known to play a role in the transduction
mechanism of several neurotransmitters including those of glycine
(Mascia et al. 1998), GABA (Krishek et al.
1994
), and glutamate (Ragozzino and Eusebi
1993
). We investigated the possibility that EtOH exerts its
action via a pathway that involves PKC. In five experiments
staurosporine, a PKC inhibitor, was perfused over the slice for 60 min
at a concentration of 100 nM during which time the burst area declined
to 68.3 ± 8.7% of control (Fig. 6,
P < 0.05). After this period 50 mM EtOH was coapplied
with the staurosporine, resulting in a further reduction in burst area of 41.7 ± 5.0% compared with the burst area in staurosporine
(P < 0.05). This compares to an EtOH-induced
inhibition of the burst area of 59.6 ± 8.0% (n = 6) in the absence of staurosporine. The net effect of both the
staurosporine and EtOH was to reduce the burst area to 40.5 ± 7.3% of control values (P < 0.002). The effects of
the staurosporine were at least partially reversible after 15 washes.
These data suggest that activation of PKC may occur in one or more of
the pathways affected by EtOH.
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Effects of the alcohols on passive membrane properties
It has been suggested that the acute effect of EtOH may be
mediated via nonspecific mechanisms such as perturbations of membrane fluidity (Bunting and Scott 1989). To assess if this
occurred, we investigated whether bath application EtOH (100 mM) caused changes in membrane properties of HMs. To do this we used whole cell
recording in current-clamp mode from visualized HMs in thin (200-300
µm), nonrhythmic slices of the brain stem. We measured the resting
membrane potential and determined both the input resistance and
membrane time constant using injections of negative current (
10 to
60 pA, 400-800 ms in duration). We found that neither octanol
(n = 5) nor EtOH (n = 4) significantly
changed these parameters from their control values. The resting
membrane potential was unchanged from a control value of
38.9 ± 3.6 to
38.8 ± 4.3 mV in EtOH. Presumably these relatively
depolarized resting membrane potential values reflect the high
extracellular K+ concentration used in these
studies. The input resistance was unaffected being 94.7 ± 14.2 M
in control conditions and 92.4 ± 10.9 M
in EtOH. The
membrane time constant of 15.3 ± 3.0 ms under control conditions
was unaffected in the presence of EtOH (16.7 ± 2.2 ms). None of
these changes were significant (P > 0.05). These
results suggest that the inhibitory effects of neither octanol nor EtOH
were due to direct effects on HM membrane properties.
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DISCUSSION |
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This study has demonstrated that physiologically relevant doses of EtOH have a marked effect on the respiratory discharge of the neonatal rat brain stem slice. This inhibition is manifest as a reduction of the overall magnitude and frequency of the rhythmic bursts of hypoglossal nerve activity. The inhibition appears to be mediated in part by enhancement of glycinergic and GABAergic transmission based on the sensitivity of the inhibition to strychnine and bicuculline, respectively. A role for EtOH-induced inhibition of NMDA receptor-mediated transmission also can be inferred. Possible mechanisms of action will be discussed in the following text.
Actions of EtOH on respiratory output
Ethanol inhibited the respiratory output from rhythmically active
brain stem slices. Inhibitory actions of EtOH also have been reported
in many tissues including neocortical neurons (Soldo et al.
1998), hippocampus (Peoples et al. 1997
;
Proctor et al. 1992
; Siggins et al.
1987
), and locus coeruleus (Nieber et al. 1998
).
Each of these latter studies used relatively high concentrations of
EtOH (50-100 mM); this only produced modest but significant changes.
In the present study, such concentrations could produce quite marked
degrees of inhibition of the hypoglossal burst envelope. This raises
the possibility that there may be a differential sensitivity to ethanol
between neurons of the brain stem and those of other regions of the
CNS. Indeed a recent study showed that very low concentrations (<10
mM) of EtOH could produce a profound non-dose-dependent inhibition of
hypoglossal nerve discharge and at the doses used had no effect on
phrenic nerve discharge (Di Pasquale et al. 1995
). This
is in clear contrast to the results presented in this study where 10 mM
EtOH had only a small effect on the burst discharge and dose-dependent
effects were seen. One possible reason for the discrepant actions of
EtOH may be that the present study used a reduced preparation compared
with that of Di Pasquale et al. (1995)
, who used the en
bloc brain stem spinal cord preparation. Selective modulation of the
hypoglossal output compared with phrenic discharge also has been
observed in man (Krol et al. 1984
) and cat
(Bonora et al. 1984
). The exact reason for this
selectivity has yet to be resolved.
We also demonstrated in this study that alcohols with different chain
lengths have different EC50s. Methanol had a
greater EC50 compared with EtOH. In marked
contrast, the EC50 for octanol was very much
smaller. Although we did not test any alcohols of intermediate chain
length between EtOH and octanol, the effect we observed was very
similar to the action of EtOH and octanol both on
GABAA receptor-channel kinetics
(Tatebayashi et al. 1998) and on enhancement of
GABAA receptor currents and inhibition of NMDA,
AMPA, and kainate receptor-mediated currents (Dildy-Mayfield et
al. 1996
).
Ethanol reduced the burst rate in a dose-dependent manner with an
EC50 value very close to that for inhibition of
the burst area. It is likely that the EtOH is acting on neurons of the
pre-Bötzinger complex to bring about the depression of bursting
as the hypoglossal nucleus has been shown to have no role in
respiratory rhythm generation (Paton et al. 1994).
Further studies are needed to elucidate the effects of EtOH on those
specific subtypes of neuron of the pre-Bötzinger complex that are
responsible for rhythm generation in the neonatal rat brain stem slice.
Interestingly the effect of a single dose of 50 mM EtOH was greatly
reduced compared with the effect expected from the burst-frequency dose
curve shown in Fig. 1C. A 3% inhibition was observed
compared with an expected value of ~65%. This effect may be due to a
preconditioning effect of EtOH where previous applications of EtOH may
augment the effect of subsequent doses of EtOH. This may reflect a
differential sensitivity to EtOH of the respiratory pattern generating
neurons. A similar augmentation due to EtOH preincubation has been seen recently with respect to EtOH-induced inhibition of NMDA receptors in
cerebellar granule cells (Popp et al. 1999).
Interaction of EtOH with glycinergic transmission
In the neonatal rhythmic slice preparation, the role of
glycinergic transmission has been studied previously (Brockhaus
and Ballanyi 1998; Hyashi and Lipski 1992
;
Paton and Richter 1995a
; Paton et al.
1994
; Pierrefiche et al. 1998
). In this study,
we have shown that the inhibitory effects of EtOH can be reversed partially by coapplication of strychnine. This suggests that EtOH enhances the postsynaptic efficacy of released glycine and/or the
presynaptic release of glycine.
The other question that remains unanswered is whether EtOH acts
selectively at one site or a number of sites. The main targets would be
HMs and the pre-Bötzinger complex, but this does not exclude an
action at other locations within the rhythmically active slice. We have
shown that glycinergic transmission to HMs is enhanced by EtOH
(O'Brien et al. 1998) and further that this effect
appears to be mediated via both pre- and postsynaptic mechanisms.
However, glycinergic transmission to HMs in neonatal rats (0-5 days)
is depolarizing in nature (Singer et al. 1998
), raising
the question of how EtOH could inhibit output by enhancing glycinergic
transmission at this site. One possibility is that EtOH induces a
persistent, glycine receptor activation-mediated depolarizing block of
discharge from HMs. This would result in the observed reduction in
magnitude of respiratory output recorded from the hypoglossal rootlets. An alternative mechanism is that increased glycine receptor activity may function to shunt excitatory synaptic inputs with a similar net
effect on hypoglossal output.
Although EtOH undoubtedly has an action at the level of the HM, it is
unlikely that the critical action on rhythm generation is at this
locus. A number of studies have demonstrated that transections of the
medulla that isolate the dorsal half of the brain stem from the ventral
half results in the block of respiratory output from the hypoglossal
rootlets (McLean and Remmers 1994; Paton et al.
1994
). Therefore HMs are "follower" neurons that are
critically dependent on excitatory and inhibitory synaptic inputs.
Could EtOH be acting on neurons of the pre-Bötzinger complex to
produce its respiratory depression? This region of the brain stem is
accepted as the site of rhythm generation (Schwarzacher et al.
1995; Smith et al. 1991
). Studies in neonatal
animals have demonstrated that neither glycinergic nor GABAergic
synaptic transmission is involved in respiratory rhythm generation at
perinatal ages (Shao and Feldman 1997
). However these
systems do have strong modulatory effects on the rhythm itself. We and
others have found that application of glycine and
GABAA receptor antagonists causes an enhancement
of the respiratory burst observed in the hypoglossal nerve
(Hyashi and Lipski 1992
; Shao and Feldman
1997
). Late-inspiratory neurons of the pre-Bötzinger
complex are themselves subject to inhibition during the inspiratory
phase (Lawson et al. 1989
; Rybak et al.
1997
) presumably by glycinergic and/or GABAergic input. Were
EtOH to enhance this input then the net result could be a depression of
late-inspiratory neurons and therefore a reduced respiratory output. If
this was the case, then the reversal caused by strychnine could be
explained by a blockade of the enhanced glycine activity caused by the
EtOH. The same mechanism cannot be applied to neonatal HMs as they are
subject to depolarizing glycinergic input (Singer et al.
1998
) and thus EtOH-induced enhancement of glycinergic
transmission would augment HM discharge (but see preceding text).
Chemical activation of augmenting expiratory neurons of the
Bötzinger complex in the rabbit results in a depression of both inspiration and expiratory neurons of the caudal ventral respiratory group (Bongianni et al. 1988, 1997
). Thus enhancement of
these Bötzinger complex neurons, because of EtOH-induced
disinhibition, also could explain the observed effects of EtOH. A
similar effect to that of EtOH was observed after perfusion of the
selective glycine uptake blocker sarcosine, which abolished respiratory output from both the slice as a whole and inspiratory neurons (Brockhaus and Ballanyi 1998
).
Role of GABA receptors in the actions of EtOH
Similar effects to those seen with strychnine also were observed
with the application of the selective
GABAA-receptor antagonist bicuculline.
Essentially the same arguments as those given for the action of
strychnine could be applied to the effects of bicuculline. In the
developing respiratory network, it has been proposed that GABA can
modulate respiratory output (Shao and Feldman 1997). Application of EtOH could enhance the inhibitory actions of GABA on
respiratory neurons, an effect reversed by bicuculline. Application of
the GABA uptake blocker nipecotic acid, similar to sarcosine, resulted
in a depression of inspiratory activity (Brockhaus and Ballanyi
1998
). Ethanol has been shown to enhance the actions of GABA in
neocortical neurons (Soldo et al. 1998
), cultured mouse hippocampal neurons (Aguayo and Pancetti 1994
) and
spinal cord neurons (Celentano et al. 1988
). The effects
of EtOH on the GABA receptor complex are only thought to be possible
when the
2L subunit is present
(Wafford et al. 1991
). However, the expression of this
subunit is thought to be dependent on development (Bovolin et
al. 1992
; Wang and Burt 1991
), raising the
possibility that the actions of EtOH could be greater in older animals.
Under the conditions used in our experiments, we did not see any net
significant reversal of EtOH-induced hypoglossal burst depression with
either strychnine or bicuculline. It is possible that significant
recovery could have been achieved using higher doses of the
antagonists. We did not test higher antagonist concentrations as we
were concerned about loss of selectivity of these agents at higher
concentrations (see O'Brien and Berger 1999).
In contrast when strychnine and bicuculline were coapplied, there was an initial significant increase in the burst area. On coperfusion of the antagonists and EtOH, there was a significant fall in burst area (compared with both the control area and that in the presence of the antagonists). Thus we can conclude that at concentrations that are relatively selective for their target receptor neither bicuculline nor strychnine alone can significantly reverse EtOH-induced inhibition of hypoglossal nerve discharge, but in combination they can significantly block the inhibitory effects of EtOH alone.
Role of glutamatergic mechanisms in the actions of EtOH
Preincubation with both strychnine and bicuculline did not
completely abolish the inhibitory effect of EtOH, suggesting that there
are additional mechanisms by which EtOH exerts its depressant action. A
clear target for EtOH would be a reduction of an excitatory drive
within the respiratory rhythm generator. That non-NMDA receptor activation is an essential step in rhythm generation is demonstrated by
the sensitivity of the output to antagonists of non-NMDA receptors, e.g., CNQX (Funk et al. 1993; Greer et al.
1991
; Smith et al. 1991
). However, EtOH has been
shown to affect the actions of NMDA in hippocampal neurons
(Lovinger et al. 1990
; Peoples et al.
1997
), locus coeruleus neurons (Nieber et al.
1998
), and dorsal root ganglion cells (White et al.
1990
). Furthermore enhanced excitatory synaptic input
(presumably glutamatergic in nature) to inspiratory neurons has been
observed after inhibition of both glycinergic and GABAergic input
(Ramirez et al. 1997b
). Under the experimental conditions used in this study where neurons are likely to be more depolarized due to the high K+ concentration, the
Mg2+ block of the NMDA channel may be relieved,
and thus EtOH could function to inhibit excitatory NMDA current. We
have shown here that blockade of NMDA receptors with APV did not affect
the ability of EtOH to subsequently inhibit respiratory discharge.
However, the relative effect of EtOH was reduced in the presence of APV compared with the effect observed with EtOH alone. This suggests that
APV partly occluded the actions of EtOH, implying that EtOH can inhibit
those NMDA receptors that are involved in respiratory rhythm
generation. It has been shown that application of NMDA to
preinspiratory neurons under Ca2+-free conditions
can induce an increase of intrinsic burst frequency or even an
induction of bursting activity (Kashiwagi et al. 1993
). If these neurons that burst under Ca2+-free
conditions are those that underlie respiratory rhythm generation in
neonatal animals, then it may be that the NMDA receptors on these
neurons represent the target for EtOH.
As the APV experiments were done in the absence of strychnine or
bicuculline, the observed reduction was probably due, at least in part,
to EtOH-mediated modulation of inhibitory transmission. The possibility
that the EtOH was affecting non-NMDA receptors was not investigated in
this study because inhibition of ionotropic glutamate receptors
abolishes respiratory rhythm thus making it impossible to determine the
actions of EtOH under these conditions (Funk et al.
1993; Ge and Feldman 1998
; Greer et al.
1991
; Smith et al. 1991
). A recent study has
shown that EtOH can inhibit both AMPA and kainate responses in the
hippocampus (Martin et al. 1995
) and also recombinant
AMPA receptors expressed in HEK-293 cells (Lovinger
1993
).
Role of protein phosphorylation
We noted that perfusion of the rhythmic slice with staurosporine,
an antagonist of PKC, caused a reduction in the burst per se. This
implies that either PKC is activated in one of the neuronal elements
generating the rhythm or alternatively a tonic input (see Lawson
et al. 1989) at some point in the cycle uses PKC. We did not
determine if staurosporine occluded the actions of glycine, GABA,
and/or glutamate. Previous studies have demonstrated that protein
kinase C inhibition with staurosporine causes a reduction in the
EtOH-induced potentiation of homomeric glycine-receptor mediated
currents (Mascia et al. 1998
). Staurosporine also has been shown to block the glutamate-receptor mediated attenuation of
GABAA receptor-mediated currents in hippocampal
CA1 pyramidal neurons (Ragozzino and Eusebi 1993
).
Developmental influences on the action of EtOH
The experiments described here were performed on rats aged between
1 and 5 days. Studies have shown that over the first 2 wk after birth
dramatic changes occur in the synaptic organization and efficacy in the
mammalian respiratory system (Paton and Richter 1995b;
Ramirez et al. 1997a
). One of the greatest changes
occurs in the role of inhibitory transmission, as strychnine (and
bicuculline) display a much greater efficacy in the adult animal
compared with the neonate. Accordingly the effects of EtOH also may
become relatively more potent in the mature animal. However, no studies
so far have directly investigated the actions of EtOH on the
respiratory output from more mature rhythmic slices so comparison with
the present study cannot be made.
Physiological relevance of the action of EtOH
Numerous studies have shown that EtOH markedly exacerbates sleep
disorders of patients with OSA by increasing the atonia of the upper
airways (Remmers et al. 1980). This can result in a number of secondary effects that could prove to be life-threatening, e.g., low arterial oxygen saturation and cardiac arrhythmia. Although the results from this study are in agreement with other studies demonstrating an action of EtOH on hypoglossal output (Bonora et
al. 1984
; Di Pasquale et al. 1995
; Krol
et al. 1984
; St. John et al. 1986
), we have
raised the possibility that EtOH does not selectively affect HMs.
Remmers et al. (1980)
showed that injection of
strychnine into patients with OSA could "normalize" respiratory neuromuscular activity in humans. The strychnine could be acting at a
number of locations within the brain stem and elsewhere with the net
result being an increased hypoglossal output. Several studies,
including the current one, have shown that application of either
strychnine or bicuculline can augment the output of the hypoglossal
nerve (Kimura et al. 1997
; Shao and Feldman
1997
). However, the present study is the first to investigate
the interaction of EtOH and strychnine on respiratory output. The
counteracting effects of strychnine and EtOH have been observed in
humans in cases of concomitant EtOH intoxication and strychnine
poisoning (Spapen et al. 1990
). In the latter instance,
the EtOH intoxication obscured the known effects of strychnine
emphasizing the strong interaction of these two agents.
In conclusion, EtOH clearly acts to inhibit respiratory output recorded in hypoglossal nerves. This action appears to be mediated via a number of mechanisms. Inhibitory neurotransmission appears to be a primary target although actions on NMDA neurotransmission are also likely. Because of its varied neuronal targets, EtOH is likely to affect not only HMs but also to have a predominant action in neonates on neurons of the pre-Bötzinger complex. Further studies are necessary to determine the actions of EtOH on specific neuronal subtypes associated with respiratory rhythm generation.
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ACKNOWLEDGMENTS |
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We acknowledge Dr. J. C. Smith and S. Johnson for invaluable help in the development of the rhythmic slice technique and W. Satterthwaite and P. Huynh for providing technical support.
This study was made possible by Javits Neuroscience Award National Institute of Neurological Disorders and Stroke Grant NS-14857.
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FOOTNOTES |
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Address for reprint requests: A. J. Berger, Dept. of Physiology and Biophysics, School of Medicine, University of Washington, Box 357290, Seattle, WA 98195-7290.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 June 1999; accepted in final form 20 August 1999.
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REFERENCES |
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