Developmental Changes in the Modulation of Synaptic Glycine Receptors by Ethanol

Erika D. Eggers, Jennifer A. O'Brien, and Albert J. Berger

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) alpha  subunit changes from alpha 2 (fetal) to alpha 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 alpha 2, and juvenile (P9-13), primarily expressing alpha 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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1 subunits (the adult channel isoform) were more sensitive to ethanol than channels composed of alpha 2 subunits (the neonatal isoform) (Mascia et al. 1996b). It is not known whether native adult GlyRs, which contain both alpha 1 and beta  subunits, are more sensitive to ethanol than native neonatal GlyRs containing both alpha 2 and beta  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 alpha  and beta  subunits (Singer and Berger 1999; Singer et al. 1998). A previous study has shown that the alpha  subunit in HMs undergoes a developmental shift from being primarily alpha 2 at neonate ages [postnatal day 0 to 3 (P0-3)] to primarily alpha 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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

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 MOmega 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 MOmega . 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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha -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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Fig. 1. Glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in hypoglossal motoneurons (HMs) are enhanced by ethanol and blocked by strychnine. Representative traces from a voltage-clamp recording of glycinergic mIPSCs in a HM from a juvenile rat [postnatal day 9 (P9)] are shown. Action potentials and nonglycinergic currents were blocked by tetrodotoxin (TTX), Cd2+, 6,7-dinitro-quinoxaline (DNQX), D(-)-2-amino-5-phosphonopentanoic acid (APV), and bicuculline methiodide (BMI). In this cell, adding 100 mM ethanol to the bath increased the average mIPSC amplitude 32% and decreased the interval between successive mIPSCs by 58% [distributions significantly different by Kolmogorov-Smirnov (K-S) test, P < 0.001]. Glycinergic mIPSCs were blocked by strychnine (2 µM).

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 alpha 2 subunit are less sensitive to ethanol than those containing the alpha 1 subunit. Native GlyR channels that express beta  subunits along with alpha  subunits have not been previously studied. Therefore we studied glycinergic mIPSCs during two developmental periods: neonate (P1-3) when glycine channels contain predominantly alpha 2 subunits, and juvenile (P9-13) when glycine channels contain predominantly alpha 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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Fig. 2. The average amplitudes of glycinergic mIPSCs from both neonatal and juvenile HMs are increased by 100 mM ethanol. A1 and A2: glycinergic mIPSCs recorded from HMs at neonate (P2) and juvenile (P12) ages. The average mIPSC amplitudes for the cells shown were -39 ± 1 pA (neonate) and -45 ± 2 pA (juvenile). The average mIPSC intervals were 550 ± 23 ms (neonate) and 1,796 ± 110 ms (juvenile). B1 and B2: bath application of 100 mM ethanol increased the average mIPSC amplitude and decreased the average interval between mIPSCs in the cells shown in A. The average mIPSC amplitude with ethanol application was increased to -51 ± 1 pA (neonate) and -74 ± 1 pA (juvenile). The average mIPSC interval decreased to 255 ± 8 ms (neonate) and 287 ± 7 ms (juvenile). C1 and C2: distribution of amplitudes of mIPSCs for cells shown in A and B, with and without 100 mM ethanol. At each age, both the control and ethanol distributions were significantly different from each other (K-S test, P < 0.001). Average mIPSC amplitudes are indicated by the black (control) and white (100 mM ethanol) arrows. The insets show average mIPSCs (>100 mIPSCs averaged/cell) with and without ethanol. One hundred mM ethanol increased the average amplitude by 30% (neonate) and 64% (juvenile).

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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Fig. 3. The average amplitudes of glycinergic mIPSCs from juvenile HMs are increased by 30 mM ethanol, while the amplitudes of mIPSCs from neonatal HMs remain unchanged. A1 and A2: glycinergic mIPSCs recorded from HMs at neonate (P2) and juvenile (P12) ages. The average mIPSC amplitudes were -38 ± 2 pA (neonate) and -79 ± 1 pA (juvenile). The average mIPSC intervals were 3,347 ± 345 ms (neonate) and 247 ± 5 ms (juvenile). B1 and B2: bath application of 30 mM ethanol to the cells shown in A. The average mIPSC amplitude with ethanol application was -39 ± 1 pA (neonate) and -98 ± 1 pA (juvenile). The average mIPSC interval decreased to 1,762 ± 91 ms (neonate) and 80 ± 1 ms (juvenile). C1 and C2: plotted are average mIPSCs with and without ethanol. Thirty mM ethanol increases the amplitude by 25% in the juvenile HM (P < 0.001, K-S) but does not significantly change the amplitude in the neonate HM (3%) (P > 0.01, K-S).

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 alpha 2 subunit, are less sensitive to ethanol than mIPSCs from juvenile HMs, which are mediated by GlyRs that contain primarily the alpha 1 subunit.



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Fig. 4. The potentiating effect of ethanol on glycinergic mIPSC amplitude changes with development. Plotted are the average (n >=  4 cells/dose) %increases of mIPSC amplitudes due to ethanol in juvenile HMs (1-300 mM ethanol) and neonate HMs (10-300 mM ethanol). Glycinergic mIPSCs from juvenile rats were much more sensitive to ethanol (, 50% increase approx 100 mM) than neonatal rats (, 50% increase approx 300 mM). The increase due to ethanol in juvenile HMs was significantly greater than neonate HMs at 4 doses: 10 (**, unpaired t-test, P < 0.05), 30, 50, and 100 mM (*, unpaired t-test, P < 0.01). Error bars are SE.

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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Fig. 5. Evoked glycinergic IPSCs in juvenile HMs are more sensitive to ethanol than those in neonatal HMs. A1 and A2: average eIPSCs from HMs in response to electrical stimuli applied lateral to the hypoglossal motor nucleus. The arrow indicates the time of stimulation, and the stimulus artifact has been deleted. For the cells shown, 30 mM ethanol increased these responses in a juvenile (40%) but not a neonate (-6%) HM. B1 and B2: average responses of a neonate and a juvenile HM electrical stimuli as in A. For the cells shown here, 100 mM ethanol increased these responses in both a neonate (31%) and a juvenile (63%) HM. C: plotted are average percent increases of eIPSCs in response to ethanol. Thirty mM ethanol increased eIPSCs in juvenile HMs by an average of 59 ± 26% (n = 4) but had no effect on neonate eIPSCs (-3 ± 4%, n = 4, unpaired t-test difference P = 0.06). One hundred mM ethanol increased both neonate (39 ± 7%, n = 4) and juvenile (111 ± 38%, n = 4) eIPSCs. The change in peak current amplitude due to 100 mM ethanol was significantly greater than the change due to 30 mM ethanol for neonate HMs (*, unpaired t-test, P < 0.01). Error bars are SE.

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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Fig. 6. Ethanol increases the current responses of neonatal and juvenile HMs due to focal application of glycine onto the cell soma. A and B: plotted are average responses of whole cell voltage-clamp recordings to the application of 200 µM glycine with and without ethanol. In neonatal HMs, ethanol (100 mM) increases the response to glycine by an average of 35 ± 6% (Vh = -60 mV, n = 3). In juvenile HMs ethanol (50 mM) also increased the response to glycine by an average of 48 ± 22% (Vh = -55 mV, n = 3).

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 (tau decay). The neonatal HMs had an average tau 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 tau 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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Fig. 7. Ethanol (100 mM) does not change the single-channel conductance as estimated by nonstationary noise analysis. A1 and A2: examples of nonstationary noise analysis from neonate and juvenile HMs in control () and 100 mM ethanol (open circle ). Plotted are the variance-current relationships for 2 cells. The first 25% of the curve was fit to a line, and the single-channel current and conductance were estimated (driving force is -55 mV): neonate control i = -1.4 pA, g = 26 pS, ethanol i = -1.8 pA, g = 33 pS; juvenile control i = -2.05 pA, g = 38 pS, ethanol i = -1.6 pA, g = 28 pS. B: average values of single-channel conductance from neonate (n = 4) and juvenile (n = 4) HMs with and without ethanol. There was no significant difference in the conductance of control and ethanol groups or neonate and juvenile age groups (P > 0.1). Error bars are SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 2 GlyR subunit) and juveniles (expressing the alpha 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 alpha 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 alpha 2 GlyRs are less sensitive to ethanol than homomeric alpha 1 GlyRs (Mascia et al. 1996b). We extended this study to look at native synaptic GlyRs that are heteromeric, containing beta  subunits as well as alpha  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 alpha 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 alpha  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 (alpha 2 containing) GlyRs are less sensitive to ethanol than juvenile (alpha 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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