Effect of Ethanol Upon Respiratory-Related Hypoglossal Nerve Output of Neonatal Rat Brain Stem Slices

Ian C. Gibson and Albert J. Berger

Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195-7290


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INTRODUCTION
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DISCUSSION
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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.


    INTRODUCTION
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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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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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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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Fig. 1. Actions of ethanol (EtOH) on rhythmic hypoglossal nerve activity. A: rhythmic activity in the medullary slice displayed by the rectified, integrated activity of hypoglossal nerve discharge. Application of EtOH in increasing concentrations causes a progressive decline in both the magnitude of the burst and burst frequency. Complete inhibition of bursting was attained with 100 mM EtOH. B: examples of the burst envelope. Each trace is the average of 30 bursts aligned on their rising phase. All data derived from an experiment performed in a 3-day-old neonatal rat. C: graph showing the percent reduction of the burst rate compared with control (mean ± SE) with increasing doses of EtOH. Data was taken from 7 experiments where EtOH was applied sequentially from 10 to 100 mM. See text for the logistic function used to fit the data points in C.

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
<IT>E</IT><IT>=</IT>((<IT>E</IT><SUB><IT>min</IT></SUB><IT>−</IT><IT>E</IT><SUB><IT>max</IT></SUB>)<IT>/</IT>(<IT>1+</IT>([<IT>D</IT>]<IT>/EC<SUB>50</SUB></IT>)<SUP><IT>b</IT></SUP>)<IT>+</IT><IT>E</IT><SUB><IT>max</IT></SUB>)
where E is effect (% reduction of burst rate), [D] is the concentration of EtOH, EC50 is the concentration of EtOH that produces a 50% reduction of the burst rate and b is a slope factor. Using this function to fit the data points, an EC50 value of 33.3 mM and a b value of 1.68 were determined for EtOH. The effects of EtOH were reversible on washout.

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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Fig. 2. Comparison of the log-dose response curves for the 3 alcohols tested. Each point represents the mean ± SE of the burst area from the last 5 min of a 15-min perfusion of that concentration of either octanol (black-diamond ), ethanol (black-square), or methanol (black-triangle). Data were fit to the logistic function given in the text. Note the clear dose-dependency of each alcohol and the apparent graded effect dependent on chain length of the alcohol. Number of observations for each dose of a given alcohol is indicated next to each datum point.

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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Fig. 3. Actions of EtOH are partially reversed by application of either strychnine or bicuculline. Burst area was reduced significantly by 50 mM EtOH (P < 0.001). Subsequent perfusion of either 5 µM strychnine () or 5 µM bicuculline () resulted in a partial recovery of the burst area back toward control values. Washout of all drugs resulted in a return to control levels. Values are means + SE of 8 and 6 experiments for strychnine and bicuculline, respectively.

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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Fig. 4. Effect of preincubation with strychnine and bicuculline. Both antagonists were perfused at a concentration of 5 µM for >= 15 min resulting in a significant increase in the burst area. Application of 50 mM EtOH produced a very significant reduction in the burst area. On washing there was a return to control levels observed before the application of the antagonists. Each bar represents the normalized mean burst area + SE from 5 experiments. *P < 0.05, **P < 0.001.

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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Fig. 5. Action of APV on the EtOH-induced inhibition of hypoglossal burst area. Results of 5 experiments are summarized here. Each point displays the mean (± SE) hypoglossal burst area for a 1-min period. Superfusion of 25 µM APV (top bar) significantly reduced the burst area (P < 0.001). Subsequent coapplication of 50 mM EtOH (bottom bar) caused a further significant reduction (P < 0.001) in the burst that was partially reversible on washout of APV and EtOH. Inset: effect of the treatments on the hypoglossal burst from 1 experiment. Each trace is the average of 30 bursts aligned on their rising phase.

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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Fig. 6. Staurosporine modulates the action of EtOH. Rhythmic slices were preincubated with 100 nM staurosporine for 60 min during which time the burst area was significantly reduced. There was a further significant reduction following perfusion of EtOH. Burst area partially recovered after washout of EtOH and staurosporine. Number of observations for each condition is indicated in parentheses (*P < 0.05).

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 MOmega in control conditions and 92.4 ± 10.9 MOmega 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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 gamma 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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