Ž .Brain Research 845 1999 224–231www.elsevier.comrlocaterbres

Research report

Ethanol reduces neuronal excitability and excitatory synaptic transmission inthe developing rat spinal cord

Gong Cheng a, Bao-Xi Gao b, Yakov Verbny b, Lea Ziskind-Conhaim b,)

a Department of Anesthesia, Stanford UniÕersity Medical Center, Palo Alto, CA 94305-5117, USAb Department of Physiology and Center for Neuroscience, UniÕersity of Wisconsin Medical School, 1300 UniÕersity AÕenue, Madison, WI 53706, USA

Accepted 10 August 1999

Abstract

Ž .Effects of acute ethanol EtOH exposure on motoneuron excitability and properties of synaptic transmission were examined in spinalcords of postnatal rats. Whole-cell patch clamp recordings and intracellular recordings with high-resistance electrodes were carried out inmotoneurons of 1- to 4-day-old postnatal rats. To determine the effects of extracellular EtOH on action potential waveform, properties ofcurrent-evoked soma action potentials and motoneuron ability to generate repetitive action potential firing were examined. During a brief

Ž .EtOH 70 mM exposure, larger depolarizing current was required for action potential generation, the duration of the after hyperpolarizingpotential increased, and fewer action potentials were produced during a prolonged intracellular current injection. These effects werereversed within 20 min of EtOH removal from the extracellular solution. To determine whether the reduced probability of action potentialgeneration was associated with changes in synaptic transmission, properties of evoked synaptic potentials and spontaneous synapticcurrents were investigated. In the presence of EtOH, the amplitude of dorsal root-evoked synaptic potentials was reduced, the frequencyof spontaneous excitatory postsynaptic currents decreased, while the frequency of inhibitory postsynaptic currents increased. Our datasuggested that acute EtOH exposure suppressed motoneuron electrical activity by decreasing motoneuron excitability and shifting thebalance between excitatory and inhibitory synaptic transmission toward inhibition. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Ethanol; Motoneuron excitability; Action potential waveform; Excitatory synaptic current; Inhibitory synaptic current; Developing spinal cord

1. Introduction

Ž .Ethanol EtOH consumption impairs motor coordina-tion and control, behavioral activities that are regulated byfunctional integration of neuronal activity in various re-gions of the brain and the spinal cord. To explore themechanisms underlying EtOH-induced changes in motoractivities, numerous studies have examined its effects onaction potential firing rate and synaptic transmission inareas of the brain that are known to control motor activityŽ w x.for review, see Refs. 34,38 , but much less is knownabout EtOH actions on neuronal functions in the spinalcord. In decerebrated cats, intravenous administration of

ŽEtOH at concentrations that cause moderate ataxia 100–.600 mgrkg , reduces spontaneous action potential firing in

dorsal horn spinal interneurons, and at higher concentra-

) Corresponding author. Fax: q1-608-265-5512; e-mail:lconhaim@physiology.wisc.edu

Ž .tions 600–900 mgrkg it attenuates the amplitude ofantidromic potentials below the threshold for action poten-

w xtial generation 12 . Based on their data, the authors in-ferred that the reduced firing rate results from changes inNaq and Kq conductances. Similar to the findings in theintact spinal cord, acute exposure to low EtOH concentra-

Ž .tion 20–100 mM depresses the rate of spontaneous ac-tion potential firing and reduces the frequency of sponta-neous excitatory and inhibitory synaptic potentials in dis-

w xsociated spinal neurons 17 . The suppressed excitatoryactivity in those neurons is not associated with a signifi-cant change in membrane permeability.

EtOH further impairs electrical activity in neural net-works by depressing orthodromic excitatory synaptic trans-

w xmission in spinal motoneurons of the cat 12 , and atconcentrations of 65–130 mM it decreases glutamate-mediated monosynaptic reflex in the isolated neonatal rat

w xspinal cord 41 . In contrast to EtOH inhibitory effect onglutamate-mediated synaptic activity, it facilitates the ac-tivity of cholinergic synapses that converge onto Renshaw

w xcells 29 , which in turn increases the activity of the latter

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01968-X

( )G. Cheng et al.rBrain Research 845 1999 224–231 225

neurons that form recurrent inhibitory synapses on mo-toneurons. The opposite actions of acute EtOH exposureon excitatory and inhibitory neural pathways contribute toits depressant effect on excitatory synaptic transmission inthe mammalian spinal cord.

EtOH-induced changes in the functions of diverse cellu-lar activities result from its interactions with specific mem-brane proteins that initiate cellular signal transduction pro-

Ž w x.cesses for review, see Ref. 9 . It has been well docu-mented that EtOH interaction with neurotransmitter recep-tors changes their physiological functions, thus substan-tially altering the level of excitatory and inhibitory synap-

Žtic transmission in various regions of the CNS for review,w xsee Refs. 13,20,38 . In general, EtOH reduces the function

Ž .of excitatory neurotransmitters e.g., glutamate and en-Žhances the activity of inhibitory neurotransmitters e.g.,

.GABA, glycine, adenosine , resulting in a shift towardw xinhibition of synaptic activity 7,32 . One such example is

the interaction of EtOH with N-methyl-D-aspartateŽ .NMDA receptor. Electrophysiological and biochemicalstudies have demonstrated that among the different typesof ionophoric glutamate receptors, the NMDA receptor isthe most sensitive to the inhibitory action of EtOHw x Ž w x.22,24,40 for review, see Refs. 9,13,35 . NMDA recep-tor sensitivity to EtOH depends on its subunit compositionw x42 , therefore EtOH affects NMDA receptors differentlyin various regions of the brain. Non-NMDA receptors areless sensitive to EtOH than NMDA receptors, but they are

w xalso suppressed by EtOH 23,27,31 . In addition to EtOHdepressant action on glutamate receptor function, there issome evidence that it further reduces excitatory neurotrans-mission by inhibiting glutamate release from presynaptic

Ž .terminals. It has been shown that acute EtOH 2 grkgdecreases the extracellular concentrations of glutamate in

w xthe striatum of awake rats 3 , and at concentrations of25–100 mM it inhibits Kq-induced release of glutamate in

w xhippocampal slices 26 .In contrast to EtOH inhibitory action on glutamate-

mediated synaptic transmission, it potentiates glycine- andw xGABA receptor-mediated currents 2,8,33 . Glycine andA

GABA receptors sensitivity to EtOH depends on theirA

specific receptor subunit composition. Therefore, EtOHfunctional interaction with GABA receptors varies con-A

siderably not only in different brain regions but also withinw xa single neuronal population 39 .

To increase our understanding of the effects of acuteEtOH exposure on neuronal activity in the developingmammalian spinal cord, experiments were carried out todetermine the actions of EtOH on neural network activityin the developing rat spinal cord. The primary objectivewas to investigate the actions of a brief EtOH exposure onaction potential properties and motoneuron ability to gen-erate repetitive firing, and on properties of evoked andspontaneous synaptic transmission.

A preliminary description of these results have beenw xpresented in an abstract form 5 .

2. Materials and methods

2.1. Spinal cord preparation

Ž .Postnatal Sprague–Dawley rats 1- to 4-day-old, P1–4were used in this study. Rats were anesthetized by hy-pothermia, and spinal cords were dissected out and placed

Ž .into ice-cold dissecting solution containing in mM : 140.0NaCl, 5.0 KCl, 4.0 CaCl , 1.1 MgCl , 4.2 HEPES and2 2

Ž w x.11.0 glucose pH 7.2 43 . Experiments were performedŽ .using either transverse thick slices 350 mm of the lumbar

spinal cord or hemisected spinal cords. Slices were cutw xfollowing the procedure described previously 11,16 .

Hemisected spinal cords were dissected with ventral andw xdorsal root attached 44 . Slices and hemisected spinal

Žcords were incubated in extracellular solution aerated with. Ž .95% O –5% CO at room temperature 21–238C for 1 h2 2

before electrophysiological recordings were carried out.Ž .The extracellular solution contained in mM : 113.0 NaCl,

3.0 KCl, 2.0 CaCl , 1.0 MgCl , 25.0 NaHCO , 1.02 2 3Ž .NaH PO , and 11.0 glucose pH 7.2 .2 4

The following substances were added to the extracellu-Žlar solution at known concentrations: tetrodotoxin TTX,

. Ž .Sigma , D-2-amino-5-phosphonovaleric acid D-APV , 6-Ž .cyano-7-nitroquinoxaline-2,3-dione CNQX , strychnine

Ž .and bicuculline methchloride Research Biochemicals .

2.2. Electrophysiological recordings

The procedures for whole-cell patch clamp recordingswere identical to those described in our recent studiesw x w x14,15 . Using infrared DIC-videomicroscopy 14,25 ,recordings were performed in visually identified large

Ž .multipolar or round cells 15–25 mm diameter in thelateral and medial ventral horn, which were assumed to be

w xmotoneurons 16 . Patch pipettes with resistances of 4–7MV were fabricated using a Flaming–Brown P-97 pullerŽ . ŽSutter Instruments . The pipette solution contained in

.mM : 140.0 Cs-gluconate, 9.0 CsCl, 1.0 Mg–ATP, 0.1ŽGTP, 10.0 HEPES, and 0.2 EGTA buffered to pH 7.2

.with CsOH . Recordings were carried out at room tempera-Ž .ture 21–238C . Following the formation of a giga seal and

rupture of the membrane, the cell capacitance was com-pensated by adjusting the compensation dial. The 8–12MV series resistance was compensated )60%. Allrecordings were corrected for the liquid junction potentialŽ w x.13 mV 16 .

The procedures for intracellular recording with high-re-sistance electrodes, and electrical stimulation of ventraland dorsal roots in the hemisected spinal cord were identi-

w xcal to those described previously 43,45 . Briefly, mo-Žtoneurons were impaled with high-resistance quartz 120–

. Ž .160 MV or glass 80–120 MV microelectrodes filledwith 3 M potassium acetate. Microelectrodes were fabri-

Ž .cated using the P-2000 puller Sutter Instrument . Mo-Ž .toneurons were identified by brief 300 ms ventral root

( )G. Cheng et al.rBrain Research 845 1999 224–231226

electrical stimuli using tight-fit glass suction electrodesfilled with extracellular solution. Postsynaptic potentials

Ž .were produced by brief 300 ms dorsal root stimuli usingsuction electrodes similar to those used for ventral rootstimulation. Recordings were performed at a regulatedtemperature range of 28–308C.

2.3. Data acquisition and analysis

Membrane resting potential was recorded using high-re-sistance microelectrodes. Membrane resistance was esti-mated from the voltage change produced by intracellular

Ž .injection of hyperpolarizing currents 100–200 pA . Singleand repetitive action potentials were generated from a

Ž .holding potential HP of y60 mV by intracellular injec-tion of depolarizing currents using the whole-cell patchclamp technique. Properties of the first action potential inthe train of a sustained firing were analyzed to determinethe effects of EtOH on action potential waveform. Peakaction potential amplitude was measured from y60 mV,and its duration was measured at threshold potential. Am-plitude and duration of the afterhyperpolarizing potentialŽ .AHP were measured as the peak and duration of therepolarization of the first action potential below the thresh-old potential of the second action potential. Measurements

Žwere performed using pClamp software Axon Instru-.ments .

To simultaneously record both excitatory and inhibitoryspontaneous synaptic currents, whole-cell voltage-clamp

w xrecordings were performed at an HP of y40 mV 14 .Synaptic currents were continuously recorded using either

ŽAxopatch-1D or Axopatch 200A amplifiers Axon Instru-.ments . Currents were digitized at 5 kHz, filtered at 1 kHz,

and stored on an optical disk. Recordings in a givenmotoneuron were carried out for 10 min or until )250events were recorded.

Data are presented as means"S.E.M. Student’s t-testwas used to determine the statistical significance of theresults. The level of statistical significance was 5%.

3. Results

3.1. EtOH reduced motoneuron ability to generate actionpotentials

To determine whether acute EtOH exposure alteredmotoneuron excitability, action potential properties wereexamined in motoneurons in thick spinal cord slices usingwhole-cell current-clamp technique. Single action poten-

Ž .tials were generated by brief 30 ms intracellular injec-Ž .tions of depolarizing currents 20–30 pA, Fig. 1a , while a

sustained action potential firing was produced by a pro-Ž .longed 150 ms intracellular injection of higher intensity

Ž .depolarizing current )30 pA, Fig. 1b . To determine thelowest EtOH concentration that invariably suppressed mo-toneuron excitability, EtOH effect on action potentialwaveform was examined at concentrations ranging from30 to 100 mM. Our findings showed that 70 mM EtOHeffectively reduced the number of action potentials in the

Ž .train of action potentials Fig. 1b , therefore this concentra-tion was used throughout the study to investigate EtOHeffects on action potential properties.

Intracellular recordings with high-resistance microelec-trodes demonstrated that resting membrane potential andresistance did not change in response to acute EtOHexposure. An average resting membrane potential of y62.6

Ž .mV "2.1 S.E.M., ns21 was recorded before EtOHŽ .application, and an average of y63.1 mV "1.8 S.E.M.

was recorded in the presence of EtOH. Membrane resis-tance slightly decreased in the presence of EtOH from 53.4

Ž . Ž .MV "3.7 S.E.M. to 51.8 MV "4.9 S.E.M. . One ofthe apparent changes induced by EtOH exposure was a10-pA increase in the rheobase, the depolarizing currentthat was necessary to generate a soma action potentialŽ .30–50 pA . The larger rheobase did not result from adepolarizing shift in action potential threshold, which re-

Ž .mained at about y40 mV Table 1 . EtOH did not affectthe action potential amplitude and duration, but it signifi-cantly prolonged the duration of the AHP from 33.4 ms

Žbefore EtOH application to 44.6 ms in its presence Fig. 1b.and Table 1 .

The most prominent action of EtOH on motoneuronexcitability was the reduced probability of action potentialgeneration, as evident by the decrease in the number ofaction potentials evoked during a prolonged depolarizing

Ž .current Fig. 1b . The average number of action potentialsgenerated during a depolarizing current of 100 pA and 150

Ž .ms duration was 4.1 "1.4 S.E.M., ns9 , and it wasŽ .reduced to 2.6 "1.4 S.E.M., p-0.05 in the presence of

EtOH. This effect was partially or completely reversedwithin 20 min of EtOH removal from the extracellularsolution.

3.2. EtOH suppressed dorsal root-eÕoked synaptic poten-tials

Dorsal root-evoked postsynaptic potentials consisted oftwo components: short-latency monosynaptic potential and

w xlong-latency polysynaptic potentials 44 . At extracellularŽconcentration F50 mM, EtOH only slightly reduced -

.15% the peak amplitude of the evoked potentials, but atconcentrations G70 mM it invariably depressed the

Ž .synaptic potentials Fig. 2 . In the presence of 70 mMEtOH, peak amplitude was reduced by about 43%: from an

Ž . Žaverage of 9.6 mV "1.1 S.E.M., ns7 to 5.5 mV "1.0.S.E.M. . EtOH had similar inhibitory action on both mono-

and polysynaptic potentials. EtOH did not change thesynaptic latency suggesting that at this concentration it did

( )G. Cheng et al.rBrain Research 845 1999 224–231 227

Fig. 1. A brief EtOH exposure increased the rheobase and reduced the number of action potentials generated during depolarizing current injection in a P3Ž . Ž .motoneuron. a Before EtOH application, intracellular injection of 20 pA depolarizing current was sufficient to produce an action potential control , butŽ . Ž .higher current 30 pA was necessary to generate an action potential in the presence of 70 mM EtOH. b EtOH exposure reduced the probability of

generating a train of repetitive action potentials in response to a prolonged current injection. Depolarizing current of 100 pA produced a sustained burst offive action potentials during the 150-ms current pulse, but during EtOH exposure, only two action potentials were generated by the same current. Actionpotentials were generated from an HP of y60 mV using whole-cell current-clamp recordings.

not affect the conduction velocity of primary afferentprojections. EtOH action was reversible, as evident by the

recovery within 20 min of EtOH removal from the extra-Ž .cellular solution Fig. 2, wash .

Table 1The duration of the AHP increased during EtOH exposure

Ž .Acute EtOH exposure 70 mM, for 10–12 min significantly increased the duration of the AHP, but it did not change action potential threshold, amplitudeand duration as well as AHP amplitude.

Action potential Action potential Action potential AHP amplitude AHP durationŽ . Ž . Ž . Ž . Ž .threshold mV amplitude mV duration ms mV ms

Ž .Control ns13 y41.9"1.2 97.4"4.5 3.3"0.3 23.0"2.1 33.4"4.5aŽ .EtOH ns13 y41.2"1.2 93.9"4.1 3.3"0.3 24.0"3.6 44.6"6.1

aSignificantly longer than in the absence of EtOH, p-0.01.

( )G. Cheng et al.rBrain Research 845 1999 224–231228

Fig. 2. Amplitude of dorsal root-evoked synaptic potential was reduced by EtOH in a P2 motoneuron. Peak amplitude of the evoked potential was reducedŽ .from 12 mV control to 9 mV in the presence of EtOH. EtOH action was reversible, and an amplitude of 12 mV was recorded within 15 min after EtOH

Ž .removal from the extracellular solution wash . Resting membrane potential was y62 mV.

3.3. EtOH increased the ratio of inhibitory-to-excitatoryspontaneous synaptic currents

The EtOH-induced suppression of dorsal root-evokedsynaptic potentials might be attributed to several factors,including a decrease in the excitability of presynapticneurons, a reduced excitatory synaptic transmission andincreased inhibitory synaptic transmission. To determinewhether EtOH shifted the balance between excitatory andinhibitory synaptic transmission, the frequencies of sponta-neous inhibitory and excitatory postsynaptic currentsŽ .sIPSCs and sEPSCs were analyzed in spinal cord slicesusing whole-cell voltage-clamp recordings. sEPSCs andsIPSCs were recorded simultaneously at an HP of y40mV. At that potential, sEPSCs appeared as inward currents

w xand sIPSCs were recorded as outward currents 14 . Weconfirmed our recent findings that the sEPSCs were medi-ated via glutamatergic synapses by blocking these currents

Ž . Ž .by CNQX 10 mM and D-APV 20 mM , non-NMDA andŽ .NMDA receptors antagonists, respectively not shown .

Ž .The outward currents were blocked by strychnine 5 mMŽ .and bicuculline 10 mM , glycine and GABA receptorsA

Ž .antagonists not shown , indicating that they were medi-w xated via glycinergic and GABAergic synapses 14 .

Ž .Acute EtOH exposure 70 mM for 10–12 min signifi-Žcantly decreased sEPSC frequency from 1.1 Hz "0.1

. Ž .S.E.M., ns13 to 0.7 Hz "0.1, p-0.01, Fig. 3 .However, EtOH did not change sEPSC amplitude, which

Ž .was 12.2 pA "1.5 S.E.M., ns13 before EtOH expo-Ž .sure and 10.5 "1.5 S.E.M. in its presence. Although

Fig. 3. EtOH significantly decreased the frequency of sEPSCs and increased the frequency of sIPSCs in a P2 motoneuron. In this motoneuron, EtOHexposure decreased the frequency of sEPSCs from 1.1 to 0.6 Hz, and increased sIPSC frequency from 0.4 to 1.8 Hz. Traces are continuous recordings at anHP of y40 mV.

( )G. Cheng et al.rBrain Research 845 1999 224–231 229

both NMDA and non-NMDA glutamate receptor subtypescontribute to excitatory synaptic transmission in postnatal

w xmotoneurons 19,44 , only fast-rising, fast-decaying sEP-Ž .SCs were recorded in our study Fig. 3 , and those were

Ž .blocked by CNQX not shown , indicating that they werew xmediated solely via non-NMDA receptors 14 . Slow-ris-

ing, slow-decaying NMDA receptor-mediated currentswere not apparent at an HP of y40 mV, probably becauseof the voltage-dependent Mg2q block of the NMDA chan-nel. Our data implied that in the developing rat spinal cord,EtOH-induced suppression of sEPSC frequency resultedfrom a reduction in the frequency of non-NMDAreceptor-mediated sEPSCs.

In contrast to EtOH effect on sEPSCs, it significantlyŽincreased sIPSC frequency from 0.8 Hz "0.2 S.E.M.,

. Ž .ns13 to 1.2 Hz "0.2, p-0.01, Fig. 3 . EtOH did notŽaffect sIPSC amplitude, which was 20.8 pA "3.1 S.E.M.,

. Žns13 before EtOH application and 17.2 pA "2.2.S.E.M. during EtOH exposure. The opposite effects of

EtOH on sEPSC and sIPSC frequencies resulted in asignificantly higher ratio of sIPSC-to-sEPSC frequency,increasing it from 0.7 before EtOH application to 1.7during EtOH exposure.

4. Discussion

In this study we examined the effects of acute EtOHexposure on motoneuron excitability and synaptic trans-mission in the postnatal rat spinal cord. The findingsdemonstrated that EtOH reduced motoneuron electricalactivity by suppressing motoneuron ability to sustain repet-itive action potential firing, and tilting the balance betweenexcitatory and inhibitory synaptic currents toward inhibi-tion. Numerous studies have shown that at intoxicatingconcentrations, acute EtOH exposure depresses neuronalfunctions in various regions of the CNS, but because of itsselective interaction with specific neurotransmitter- and

Žvoltage-gated ionic channels for review, see Refs.w x.7,20,38 , EtOH effects on the pattern of neuronal firingvaries considerably in different areas of the CNS. There-fore, in comparing our findings with the existing literature,we focus the discussion primarily on data describingEtOH-induced dysfunction of neuronal electrical activityin the mammalian spinal cord.

4.1. EtOH exposure reduced motoneuron excitability

Acute EtOH exposure decreased motoneuron ability togenerate repetitive action potential firing, without signifi-cantly changing motoneuron resting membrane potentialand resistance, or soma action potential threshold, ampli-tude or duration. These findings supported previous datashowing that at concentrations )30 mM, EtOH reducedthe rate of spontaneous firing in dissociated mouse spinal

neurons, without significantly changing membrane restingpotential and permeability and action potential amplitudew x17 . Similarly, at concentration -100 mM, EtOH did notalter membrane conductances of central neurons such as

w xdissociated chick spinal neurons 4 and hippocampal neu-w xrons 21 .

The decreased motoneuron ability to sustain repetitiveaction potential firing was associated with larger rheobaseand an increase in AHP duration. The ionic mechanismsunderlying the changes in action potential waveform areunknown, but it is conceivable that EtOH-induced activa-

q Ž .tion of voltage-gated outward K current s , at least par-tially contributed to the suppressed firing rate. Recently wehave demonstrated that various Kq channel subtypes playdifferent roles in modulating action potential firing rate in

w xpostnatal motoneurons 15 . Blocking the big and smallconductance Ca2q-dependent Kq channels reduced AHP

w xduration and increased action potential firing rate 15 ,while inhibition of the delayed rectifier and the transientA-type Kq currents reduced action potential duration anddecreased the frequency of action potential firing, but didnot change AHP duration. Therefore, it is reasonable toassume that EtOH-induced depression of action potentialfiring in postnatal motoneurons resulted from its activationof Ca2q-dependent Kq channels. Indeed, pervious studieshave shown that EtOH activated the big conductanceCa2q-dependent Kq channels in rat neurohypophysial ter-

w x w xminals 10 , in pituitary tumor cells 18 and in planar lipidw xbilayer membranes 6 .

4.2. EtOH exposure reduced synaptic excitation

EtOH depressed motoneuron electrical activity not onlyby reducing its ability to sustain repetitive firing, but alsoby attenuating evoked and spontaneous excitatory synaptictransmission and facilitating spontaneous inhibitory trans-mission. EtOH decreased the amplitude of dorsal root-evoked synaptic potentials by about 40%, similar to itseffect on excitatory postsynaptic potentials recorded extra-cellularly in ventral roots of isolated postnatal spinal cordsw x41 . The suppressed evoked synaptic potentials might beattributed to EtOH interactions with pre- andror post-synaptic cellular mechanisms. It is conceivable that adecrease in presynaptic neuronal excitability resulted inreduced action potential-dependent neurotransmitter re-lease and depressed synaptic responses. An alternativepresynaptic mechanism might be related to EtOH modifi-cation of molecular processes underlying action potential-independent vesicular release. A third possible mechanismthat might be responsible for the smaller dorsal root-evokedpotentials is EtOH interaction with specific postsynapticreceptors, resulting in attenuated glutamate-mediated exci-tatory postsynaptic currents and possibly larger glycine-and GABA-mediated inhibitory currents.

Our data showed that EtOH decreased the frequency ofsEPSCs and increased the frequency of sIPSCs, but it did

( )G. Cheng et al.rBrain Research 845 1999 224–231230

not affect their amplitudes. Based on the assumption thatchanges in presynaptic cellular functions underlie thechanges in the frequency of spontaneous synaptic currents,we hypothesized that EtOH suppressed glutamate releaseand increased glycine andror GABA release. To deter-mine whether EtOH action on glutamate release was inde-pendent of action potential generation in presynaptic termi-nals, experiments have been carried out to investigate theeffects of EtOH on TTX-resistant miniature excitatory

Ž .postsynaptic currents mEPSCs . Our preliminary datademonstrated that similar to its action on sEPSCs, EtOH

Ž .reduced the frequency of mEPSCs not shown , indicatingthat it affected action potential-independent presynapticrelease. These findings supported previous studies showingthat EtOH reduced extracellular glutamate concentrations

w x qin the striatum of awake rats 3 , and depressed K -in-w xduced glutamate release in hippocampal slices 26 .

The findings that EtOH did not affect sEPSC and sIPSCamplitudes might indicate that at the concentration used inour study, EtOH did not interact with postsynaptic excita-tory and inhibitory neurotransmitters receptors. It is likelythat the sEPSCs recorded in our study consisted mostly of

Ž .non-NMDA receptor-mediated currents see Section 3.3 .Based on electrophysiological and biochemical studies, thenon-NMDA receptor is less sensitive to the inhibitory

Žaction of EtOH than the NMDA receptor for review, seew x.Refs. 9,13 , which might explain our observation that

EtOH did not change the amplitude of sEPSCs.Little is known about the specific sensitivity of glycin-

ergic and GABAergic synapses to EtOH. Previous studieshave shown that EtOH exposure enhances glycine recep-tor-mediated responses in various preparations including

w x w xcultured mouse 2 and chick spinal neurons 4 . EtOHactions on GABA receptors vary considerably in differentregions of the brain, and appear to be dependent on the

w x Žcomposition of GABA receptor subunits 1 for review,w x.see Ref. 30 . In fact, it has been proposed that the

sensitivity of both GABA and glycine receptors to EtOHA

depends primarily on the expression of extracellular a1w xreceptor domain 28,36 . Some selectivity of EtOH effects

have been shown in dissociated chick spinal cord neurons,in which EtOH potentiated more the Cly currents gener-ated by exogenous glycine than those produced by GABAw x4 .

Our findings showed that acute EtOH exposure effec-tively suppressed motoneuron electrical activity via differ-ent cellular mechanisms. EtOH decreased motoneuron abil-ity to sustain repetitive action potential firing, and itsopposite actions on excitatory and inhibitory synapsesresulted in a higher ratio of inhibitory-to-excitatory sponta-neous synaptic transmission in the postnatal spinal cord.To increase our understanding of the diverse actions ofEtOH on synaptic transmission in newly formed neuralpathways, we will study the specific interactions of EtOHwith neurotransmitter- and voltage-gated channels in thedeveloping spinal cord.

Acknowledgements

We thank Dr. Peter Lipton for helpful comments on themanuscript. This work was supported by the NationalInstitute of Neurological Disorders and Stroke Grant NS-23808 to Lea Ziskind-Conhaim.

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