Alcohol 30 (2003) 167–174

High-priority communication

Histiotypic electrophysiological responses of culturedneuronal networks to ethanol

Yun Xia1, Guenter W. Gross*Department of Biological Sciences and Center for Network Neuroscience, P.O. Box 305220, University of North Texas, Denton, TX 76203, USA

Received 5 June 2003; received in revised form 19 July 2003; accepted 22 July 2003

Abstract

Embryonic murine neuronal networks cultured on substrate-integrated microelectrode arrays were used to quantify acuteelectrophysiological effects of ethanol by using extracellular, multichannel recording of action potentials. Spontaneously active frontalcortex cultures showed repeatable, concentration-dependent sensitivities to ethanol, with initial inhibition at 20 mM and a spike rate 50%effective concentration (EC50) of 48.8 5.4 mM. Ethanol concentrations of greater than 100 mM led to cessation of activity. The ethanolinhibitions up to the maximum tested 160 mM were reversible. Although ethanol did not change the shape of action potentials, unit-specific spike pattern effects were found. At 40 mM, ethanol decreased neuronal firing in 71%, increased firing in 20%, and generated noeffect in 9% of all units observed (14 cultures, 200 discriminated units). The effects of combined application of ethanol and fluoxetinewere additive. Excellent agreement with findings obtained from experimental studies with animals validates the use of these in vitro systemsfor alcohol research. 2003 Elsevier Inc. All rights reserved.

Keywords: Neuronal networks; Microelectrode arrays; Ethanol; Frontal cortex; Cell culture

1. Introduction

Results from recent studies support the suggestion thatneuronal networks, grown on microelectrode arrays (MEAs)in vitro, are uniquely suited for use as broadband biosensors(Gross et al., 1992, 1995, 1997a, 1997b; Pancrazio et al.,2001; Stenger, et al., 2001). On the basis of pharmacologi-cal data, it seems that receptors, synapses, and cellularmechanisms responsible for pattern generation in speci-fic CNS tissues are retained and represented in culture(Gramowski et al., 2000; Keefer et al., 2001a, 2001b, 2001c;Morefield et al., 2000). Consequently, any compound capa-ble of altering these mechanisms to a degree that influence theperformance and life support of an animal should be reflectedby changes in the spontaneous activity of networks. Suchchanges are considered “cell culture correlate responses” tothe altered behavioral or life-threatening responses that occur

A paper published as a high-priority communication is one that review-ers have identified as being of high scientific significance and have recom-mended that the study findings should be communicated to the scientificcommunity as soon as possible.

* Corresponding author. Tel.: 1-940-565-3615; fax: 1-940-565-4136.E-mail address: gross@nervous.cnns.unt.edu (G. Gross).1 Present address: Pioneer Hi-Bred International, Inc., 7300 NW 62nd

Avenue, P.O. Box 1004, Johnston, IA 50131, USA.Editor: T.R. Jerrells

0741-8329/03/$ – see front matter 2003 Elsevier Inc. All rights reserved.doi: 10.1016/S0741-8329(03)00135-6

in animals. Therefore, the networks are physiological sensorsand support biochemical and pharmacological assays as theyare capable of responding to neuroactive or neurotoxic com-pounds in approximately the same concentration ranges thatalter the functions of an intact mammalian nervous system.To continue the validation of network responses in culture,a frequently used substance, ethanol, has been investigated,and its effects have been compared with findings of invivo studies.

The primary physiological target of ethanol is the CNS.In human beings, acute ethanol intoxication causes impairedjudgment, inappropriate behavior, slurred speech, and inco-ordination. These changes reflect the influence of ethanol onthe brain, especially the prefrontal area, temporal area, andcerebellum. Ethanol intoxication is usually defined as ablood alcohol level (BAL) of 0.1 g/dl (22 mM). A BALof 0.05–0.10 g/dl (11–22 mM) causes slight impairment ofbalance, speech, reasoning, and judgment. A BAL of greaterthan 0.5 g/dl (110 mM) leads to coma and death (Little,1999).

In this study, we investigated the responses of culturedfrontal cortex networks to ethanol and compared the resultswith those obtained in other investigations, especially invivo behavioral and performance studies. Although networksin culture cannot display “behavior,” they can show remark-ably complex changes in their firing patterns. These changesare concentration-correlated with those observed for in vivo

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studies and seem to indicate that such networks can functionas physiological sensors. Cell culture correlate responses alsoseem to be useful for rapid screening of compounds in thedomains of toxicology and drug development.

2. Materials and methods

2.1. Culturing of neuronal networks on microelectrodearrays

Microelectrode arrays were fabricated in-house and pre-pared according to methods described previously (Gross,1979, 1994; Gross et al., 1985; Gross & Kowalski, 1991;Gross & Lucas, 1982). Briefly, indium–tin oxide (ITO)-sputtered plates were photoetched, spin-insulated with poly-siloxane, cured, deinsulated at the electrode tips with lasershots, and electrolytically gold-plated to adjust the interfaceimpedance to 1–2 MΩ at 1 K Hz (Gross et al., 1985).The MEA insulation material is hydrophobic, and butaneflaming was used to activate the surface and generate ahydrophilic adhesion island (3 mm in diameter) centered onthe MEA (Gross & Kowalski, 1991).

Frontal cortex tissues were dissociated from 15- to 16-day-old BALB/c/Icr murine embryos and cultured accordingto the methods of Ransom et al. (1977), with minor modifi-cation that included the use of DNAse during tissue dissocia-tion (Gross, 1979). The cells were seeded on the MEAs as0.1-ml droplets with subsequent addition of 2 ml of mediumconfined to a 4-cm2 area by a silicone gasket. The care anduse of, as well as all procedures involving, animals in thestudy were approved by the institutional animal care anduse committee of the University of North Texas and are inaccordance with the guidelines of the Institutional Care andUse Committee of the National Institute on Drug Abuse,National Institutes of Health, and the Guide for the Care andUse of Laboratory Animals (Institute of Laboratory AnimalResources, Commission on Life Sciences, National ResearchCouncil, 1996). Mice were euthanized by means of CO2

narcosis, followed by cervical dislocation.The cells were incubated in Dulbecco’s modified Eagle’s

medium (DMEM) and supplemented with 5% horse serumand 5% fetal bovine serum for 1 week. Thereafter, the cellswere fed twice weekly with DMEM containing 5% fetalbovine serum. Cells were maintained at 37ºC in an atmos-phere of 10% CO2 and 90% air until ready for use, usually3 weeks after cell seeding (Gross, 1994; Gross & Kowalski,1991). Fig. 1 shows a 5-week-old culture on the 64-elec-trode MEA.

2.2. Recording and data analyses

Microelectrode arrays were placed into recording cham-bers and sustained at 37ºC on a microscope stage. The pHwas maintained at 7.4 with a continuous stream of humidified10% CO2 and 90% air at 5–10 ml/min into a special capfitted with a heated ITO window to prevent condensation.

Fig. 1. Neurofilament-stained network cultured on a 64-electrode recordingmatrix. Transparent indium–tin oxide conductors are 8 µm wide and termi-nate in 4 rows and 16 columns. The right panel shows neuronal circuitsin greater detail. Age: 43 days in vitro; bar: 50 µm.

After assembly, neuronal activities were monitored on oscil-loscopes in real time to provide spike information. Recordingwas performed with the commercially available multichan-nel processor system, a computer-controlled, 64-channel am-plifier system (Plexon Inc., Dallas, TX). Preamplifiers wereplaced on the microscope stage to both sides of the recordingchamber and connected to the MEA by means of zebra strips(Fujipoly America Corporation, Carteret, NJ). Total systemgain was set to approximately 10 K. The amplifier groundwas connected to the stainless steel chamber confining theculture medium.

An important feature of network activity is the organiza-tion of action potentials (spikes) into high-frequency clusters(bursts). Burst patterns represent a simplified level of activityand often reveal the major modes of network activity (Gross,1994). Integration was used to simplify the identificationand quantification of bursts. All analyses were done withbinned data (bin size of 60 s). Single-unit activity was aver-aged across the network to yield mean spike rate, burst rate,burst duration, and integrated burst amplitude per minute. Toavoid excessive network responses to medium changes re-quired for the washout of substances, the native mediumwas exchanged for the wash medium (fresh DMEM con-taining 5% horse serum) at the beginning of the experiment,and the cultures were allowed to stabilize. This state wastermed reference activity. The percent change in activityfor each variable at each ethanol concentration was alwayscalculated relative to this 30- to 60-min reference spontane-ous activity. To follow the changes in network activity with

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time, spike rates and burst rates, averaged across all activeunits selected, were plotted as “minute means.”

2.3. Pharmacological manipulations

Ethanol (CH3CH2OH, 100%) was obtained from SigmaAldrich, Inc. (Milwaukee, WI), and fluoxetine (C17H18F3NO,HCl) was obtained from Sigma Aldrich, Inc. (St. Louis,MO). To obtain relatively even distributions, the drugs weremicropipetted into the culture medium near the edge ofthe circular chamber at four cardinal positions (separatedby 90º). Concentrations were increased sequentially and ac-tivity was recorded continuously during the experimentalepisodes. To remove the test drug, syringes were used toextract the medium and to add fresh wash medium, con-sisting of fresh DMEM with 5% horse serum. Because ofthe high ethanol volatility, the CO2/air flow used to controlpH accelerated the change of ethanol concentrations in thechamber. Therefore, ethanol was added to the wash bottle thathumidified the CO2/air mixture at the same concentrationas that in the recording chamber. Ethanol concentrations weredetermined with a diagnostic kit (Sigma Diagnostics Inc.,St. Louis, MO). Such measurements showed that ethanolconcentrations were stabilized with this method.

A one-way analysis of variance (ANOVA), followed byDunnett test, was used to detect significant difference be-tween the control and experimental episodes. An alpha levelof .05 was considered statistically significant.

3. Results

Frontal cortex cultures ranged in age from 21 to 61 daysin vitro. The cultures tested had an average electrode yieldof 33% (20 channels), with a mean signal-to-noise ratioof 3:1. Frontal cortex cultures often displayed spontaneousactivity with phasic and tonic spiking. Bursts occurred almostsimultaneously among the discriminated units, with some“random” spiking among several units between bursts. Ingeneral, mean network spike rates ranged from 300/min to1,000/min, and burst rates ranged from 10/min to 50/min.The age of the culture was not correlated with initial spikerates and had no overt influence on the activity variablesmeasured. No obvious changes in waveform shapes wereobserved under exposure to ethanol before total activitytermination. Fig. 2 depicts waveforms of the same units

Fig. 2. Multiple, superimposed traces of representative action-potentialwaveforms from four recorded units in the reference state (A) and underexposure to 40 mM ethanol (B). Ethanol did not cause noticeable changesin the shape of action potential.

during reference activity (Fig. 2A) and after 20 min underexposure to 40 mM ethanol (Fig. 2B).

It was observed that ethanol produced unit-specific ef-fects. Fig. 3 displays spike rates from each discriminatedunit of one frontal cortex culture. The horizontal axis repre-sents time (minutes), and the vertical axis represents spikesper minute. Of the 36 units, 40 mM ethanol decreased neu-ronal firing in 72%, increased firing in 17%, and generatedno effect in 11%. For all the ethanol experiments (n 14cultures), 40 mM ethanol decreased neuronal firing in 71%,increased firing in 20%, and generated no effect in 9% (totalnumber of neurons 200).

Fig. 4 shows the typical activity change in response tosequential additions of ethanol. The mean spikes per minute(left ordinate) and mean bursts per minute (right ordinate)averaged across selected active units were plotted againsttime. Ethanol caused progressive inhibition at increasingconcentrations and terminated the network activity at 100mM. For each concentration, the network activity reacheda minimum with partial recovery. Because ethanol wasadded to the gas wash bottle that humidified the CO2/airmixture and ethanol concentrations were monitored with aSigma diagnostic kit, the partial recovery cannot be attrib-uted to changes in ethanol concentration.

All inhibitions caused by ethanol, up to a maximum con-centration of 160 mM, were found to be reversible. Fig. 5shows a network response to two applications of 160 mMethanol. Although burst rates recovered to near normal, spikerate recovery was not 100%. Complete medium changes aftertotal activity cessation reversed spike rates to 78.8% 9.3%(mean S.E.) of reference level (n 6 cultures).

The ethanol concentration-response summary curve, withthe use of all data, including single applications, is shownin Fig. 6A. The 50% effective concentration (EC50)mean S.E. for spike rate was 38.6 3.7 mM (n 14 cul-tures). To display the interculture repeatability, we summa-rized all experiments that were subjected to a complete,multistep concentration-response sequence in Fig. 6B. Thisgenerated a spike rate EC50 of 48.8 5.4 mM (n 5cultures).

Fig. 7A depicts the effects of ethanol on spike and burstproduction. Although some individual units showed slightexcitation or no activity change (as shown in Fig. 3), theoverall response was inhibitory. Both spike and burst produc-tion decreased progressively under increasing ethanol con-centrations. A one-way ANOVA, followed by Dunnett test,indicated that initial concentrations causing significantchanges from reference levels were 20 mM for spike rates(P .03) and 40 mM for burst rates (P .03). The effects ofethanol on burst duration and integrated burst amplitudewere also investigated (Fig. 7B). Ethanol led to concentra-tion-dependent inhibition of burst duration. The initial con-centration that caused significant change was 40 mM (oneway ANOVA, followed by Dunnett test, P .04). Burstamplitude, which reflects the spike frequency in bursts,

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Fig. 3. Individual responses of discriminated units to ethanol. The graphs show the evolution of spike rates (spikes per minute, Y-axis) over time (X-axis).Among the 36 units, 40 mM ethanol (vertical line) decreased neuronal spiking in 72%, increased such firing in 17%, and generated no effect in 11%. Themajor effect was a reduction in activity.

showed a small decrease in response to ethanol. This de-crease was not significantly different from reference activity.

To obtain preliminary information on the responses tomultiple inhibitory compounds, the combined effects of flu-oxetine and ethanol were examined. Fig. 8 displays dose-response curves of only fluoxetine (n 3 cultures) compared

with those obtained from a mixture of fluoxetine plus 20mM ethanol (n 3 cultures). The resulting EC50 was4.3 0.2 µM and 2.7 0.2 µM, respectively. An indepen-dent t test (one tail) indicated that the EC50 of the mixture(fluoxetine 20 mM ethanol) was significantly lower thanthat of fluoxetine alone (P .02). The combined application

Fig. 4. Typical effect of ethanol on frontal cortex culture spontaneous activity. Data points represent average spike and burst rates across all the selectedunits (time bin, 1 min). Both spike and burst rate decreased under exposure to 20 mM ethanol, with progressive reductions associated with increasingconcentrations. Ethanol at 100 mM stopped all activity.

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Fig. 5. Reversibility of frontal cortex network activity after exposure to two additions of high concentrations of ethanol. The total cessation of activity at160 mM was reversed by a complete medium change. The second manipulation displays the same effect.

shifted the fluoxetine EC50 from 4.3 µM to 2.7 µM, dem-onstrating that ethanol potentiates the inhibitory effects offluoxetine.

4. Discussion

Among all the organs affected by ethanol, the CNS mani-fests the most obvious effects, with rapid motor and emo-tional changes. The actions of ethanol are very complex and,despite considerable research effort, the mechanisms are notwell understood. The findings obtained in this study showedthat acute exposure to ethanol decreased the activity offrontal cortex cultures for concentrations ranging from 10to 160 mM. The spike rate EC50 was 48.8 5.4 mM. Atconcentrations of 100–140 mM, ethanol abolished all cellspiking. Complete medium changes after application of 160mM ethanol returned the activity, suggesting no overt cyto-toxicity over time periods up to 100 min at this concentration.Further, the action-potential shapes did not show obviouschanges before total cessation of activity at high ethanolconcentrations. If subtle changes exist, such analyses must

await the completion of new multichannel waveshape-analy-sis programs.

Ethanol caused minor change in integrated burst ampli-tude and significant decreases in burst duration. Integratedburst amplitude reflects the spike frequency in bursts. Thegreater the maximum frequency, the higher the integratedamplitude (Gross, 1994; Morefield et al., 2000). Burst dura-tion reflects the length of such characteristic high-frequencyspike clusters. Although bursting is a ubiquitous featureof spontaneous and evoked activity in vivo (Lisman, 1997)and in vitro (Corner et al., 2002), the mechanisms underlyingbursting are not clearly understood. Considering the com-plexity of the temporal patterns and the great variability ofthe spike patterns within bursts, present methods of analyz-ing this important aspect of neuronal firing are still crude.Nevertheless, these methods already allow powerful fea-ture extraction and have shown highly repeatable sensitivi-ties to pharmacological compounds (Keefer et al., 2001a,2001b, 2001c). In networks derived from cortical tissue,spiking is first seen at approximately 4 days. At 6 days,weak, random bursting appears that develops into stronger,coordinated bursting at 9–12 days after cell seeding. In the

Fig. 6. Concentration-response summary for ethanol with the use of network spike rates. Vertical bar represents mean and S.E. of activity change. A.Summary of all data, including single-concentration exposures. Spike rate 50% effective concentration (EC50): 38.6 3.7 mM. B. Complete concentration-response curves, each derived from five sequential exposures (five different cultures). EC50: 48.8 5.4 mM.

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Fig. 7. A. Spiking and bursting (mean S.E.) in frontal cortex culture under ethanol. Both variables decreased progressively. B. Quantification of burstduration and integrated burst amplitude (mean S.E.) from 14 frontal cortex cultures. Burst duration was more sensitive than burst amplitude.

native state, not modified by pharmacological compounds,cortical networks generally produce single coordinated burstpatterns with characteristic short burst durations of 0.3 to0.8 s (Gopal & Gross, in press).

The concentration-dependent effects of ethanol, in thecurrent study, were consistent with those observed in otherstudies that have addressed the association of blood concen-trations of ethanol with certain behavioral effects. In mice,40 mM ethanol led to a loss of the righting reflex, and 122mM led to sleep and hypothermia. In rats, 20 mM ethanolcaused sedation. In human beings, ethanol, at 5–15 mM,caused slight impairment of attention; 15–30 mM resultedin significant sedation; 30–50 mM induced total mental con-fusion; 50–100 mM led to loss of consciousness and centraldepression; and greater than 100 mM rendered coma andrespiratory arrest (Charness et al., 1989; Little, 1991). Allthese observations were made in the concentration range of10–122 mM, which was also effective in our cell cultures.

More specific effects of ethanol on neuronal firing havebeen investigated in other laboratories. Intraperitoneal injec-tion of ethanol, in a dose range of 1–4 g/kg, caused a decreasein the spontaneous activity of Purkinje cells (Sorensen etal., 1981). This represents a blood concentration of approxi-mately 27–108 mM, assuming uniform distribution. Givens

Fig. 8. Comparison of concentration-response curves from fluoxetine and20 mM ethanol (dashed curves) and fluoxetine only (solid curves). Themixture elicited a statistically significant shift of the fluoxetine 50%effective concentration (EC50) from 4.3 0.2 µM to 2.7 0.2 µM.

and Breese (1990) observed that intraperitoneal injection ofethanol, in a dose range of 0.75–3.0 g/kg (approximate bloodconcentration at 20–80 mM), reduced firing of medial septalneurons. Grupp (1980) reported that a low concentration ofethanol caused an increase in the dorsal hippocampus neu-ronal firing frequency, followed by some reduction in firingrates. Increasing concentrations produced augmented inhibi-tion in firing. The changes in unit firing appeared to becorrelated with the pattern of frontocortical EEG activity.Ethanol inhibited spontaneous central nucleus of the amyg-dala firing rates in rats, when administered in the dose rangeof 0.75–2.5 g/kg (approximate blood concentration at 20–67 mM). Ethanol partially inhibited central nucleus of theamygdala activity at low doses and produced nearly com-plete suppression at high doses (Naylor et al., 2001).

This multichannel study of network spike activity alsorevealed that ethanol caused neuron-specific effects. Sigginset al. (1987) found that, of spontaneously firing CA1 pyrami-dal neurons, 10–350 mM ethanol decreased the firing ratesin 50%, had no effect in 29%, generated biphasic effect in12%, and increased firing in 9%. They also observed thatthe application of 50–200 mM ethanol in hippocampal slicesincreased firing in 32% of the neurons, decreased firing in24%, had biphasic effect in 32%, and generated no obviouseffect in 12%. Intraperitoneal administration of ethanol de-creased the spontaneous firing of noradrenergic neurons inthe locus coeruleus in 62%, increased spiking in 22%, andhad no effect in 16% (Pohorecky & Brick, 1977). Thesefindings agree well with our observations in cell culture inwhich ethanol caused unit-specific effects, with the majoreffect being inhibitory. Summarized from 200 units in 14cultures, 40 mM ethanol decreased neuronal firing in 71%,increased firing in 20%, and generated no effect in 9%.

Results from several studies have demonstrated that etha-nol does not change the action-potential shape, although itreduces the neuronal activity. Benson et al. (1989) reportedthat 50–100 mM ethanol suppressed the firing of CA1 pyra-midal neurons in hippocampal slices, and this effect wasnot attributed to changes in either membrane potential ormembrane conductance. Gruol (1982) found that, in culturedspinal cord neurons, ethanol, at 20–30 mM, caused a reduc-tion in the rates of spontaneous neuronal firing. However, the

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amplitude of these potentials remained the same. In light ofthe stable waveforms seen in our experiments, we contendthat ethanol inhibits the initiation of neuronal firing butnot the propagation of action potentials.

To test whether ethanol potentiates the effects of otherCNS-inhibitory compounds, we studied the effects of fluox-etine and ethanol mixtures on neuronal networks. Fluoxetine,a selective serotonin reuptake inhibitor, is one of the mostcommonly prescribed antidepressants. It is generally sug-gested that individuals who are receiving fluoxetine shouldavoid ethanol because it may increase the sedative effectsof fluoxetine. Although fluoxetine was thought to have agood overdose safety, in high doses it can cause serious sideeffects (Braitberg & Curry, 1995; Neely, 1998). This toxicitywas usually increased by the additional intake of ethanol(Barbey & Roose, 1998), and several fatalities due to acombined use of fluoxetine and ethanol have been reported(Kincaid et al., 1990; Rohrig & Prouty, 1989). However,only limited studies were performed on the combinationeffects of these two compounds. Our current study findingsshow that co-administration of fluoxetine and 20 mM ethanolshifted the fluoxetine concentration-response curve EC50

from 4.3 0.2 µM to 2.7 0.2 µM, indicating that thiscombined application produces a higher inhibitory effect.

The findings obtained from this research demonstrate thatthe cultured neuronal networks report changes in the mediumenvironment at physiologically relevant concentrations.Hence, these findings help validate the use of such net-works as biosensors and as pharmacological assay platforms.Moreover, applications for rapid screening of new com-pounds for toxicity, including additive effects of mixtures,become increasingly feasible as results of more studies con-firm the histiotypic responses of small networks in culture.

Acknowledgments

This study was supported by the DARPA Activity De-tection Technologies Program (program manager, Dr. AlanRudolph). We gratefully acknowledge the technical contri-butions of Anthony Curran, Todd Hall, and Tracy Howard.We also thank Jason Brauner for the critical reading ofthis manuscript.

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