Direct effects of ethanol on central nervous system cultures: An electrophysiological and morphological study

EXPERIMENTAL

Direct

NEL’KOLCGY

55, 390-404

(1977)

Effects of Ethanol on Central Cultures: An Electrophysiological Morphological Study

Nervous and

System

FREDRICK J. SEIL, ARNOLD L. LEIMAN, MARY M. HERMAN, ROBERT A. FISK 1

AND

Department of Neurology, Vetcram Admi)&ratior~ Hospital, Palo Alto. California 94304; Dcpartmm t of Psycltolog~, Uttizwsity of California, Berkelry, California 94720; and Dcpartmrrlt of Pathology (Neuropathology), Stanford University, School of Mcdiciw. Stanford, California 94305 Recrivcd

November

8,1976

Ethanol was applied directly to cultures of mouse cerebellum and cerebral neocortex while spontaneous electrical activity was monitored in the former and stimulus-elicited responses were monitored in the latter. Ethanol characteristically

demonstrated

an initial

excitatory

effect

on cerebellar

cultures,

manifested by an increase in spontaneous discharge rate, followed by a depression of spontaneous activity. Only depressant effects were noted in ethanol-treated cerebral neocortex cultures. In both instances, removal of ethanol by washing with normal medium resulted in a return to baseline electrical activity. The rapid reversibility of the depressant effects was thought to favor a membrane-altering mechanism which impairs ionic exchange. Light and electron microscopic studies of cerebellar cultures treated with 0.21 to 1.55 g ethanol/100 ml medium for 6 to 15 days demonstrated only nonspecific changes, i.e., an increase in lysosomes and autophagic vacuoles, particularly in astrocytes. Both electrophysiological and morphological studies revealed that the effects of ethanol in vitro were generally manifested at concentrations well above levels producing effects ilz z~ivo. 1 Ethanol determinations were performed in the Drug Assay Laboratory, Stanford University, through the courtesy of Drs. Dennis R. Clark and Sumner M. Kalman. Stephan R. Montague provided technical assistance. This study was supported by the Medical Research Service of the Veterans Administration and by U.S. Public Health Service A. A. Grant No. 5 ROI AA00322. Dr. Seil’s present address is Research Service (151), Veterans Administration Hospital, Portland, Oregon 97207, and the Department of Neurology, University of Oregon Health Sciences Center, Portland, Oregon 97201; send correspondence to Dr. Seil at the former address. 390 Cooyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0014

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INTRODUCTION The direct effects of ethanol on the central nervous system (CNS)” have been difficult to assess in animal studies because of the presence of numerous other modulating factors. Such factors, as reviewed by Kalant (13), include effects of ethanol metabolites, influences of portions of the nervous system other than that being studied, vagaries of responses imposed by close, route, and rate of administration of ethanol, and systemic factors such as changes in circulation, respiration and concentrations of various hormones. In some attempts to circumvent these difficulties by use of in v&o preparations, mammalian brain slices or isolated amphibian spinal cords have been employed (6, 8, 14. 23). Tissue culture offers an alternative in zritro preparation. Explants of rodent cerebellum and cerebral neocortex maintained in Maximow chambers developed histiotypic structural characteristics (1, 3, 15, 24-28, 31, 32) and complex electrophysiologic activity (7, 18, 19). Metabolic studies of such cultures have demonstrated high metabolic rates, more like the in z~izro situation than is evident in brain slices (10, 16, 17, 20), while retaining the advantages of the isolated preparation. The purpose of the present paper is to examine the acute effects of ethanol application on spontaneous electrical activity in cerebellar cultures and on stimulus-elicited electrical responses in cultures of cerebral neocortex and to study the structural effects of chronic exposure of cerebellar explants to ethanol. METHODS Cerebellum and cerebral neocortex cultures derived from newborn Swiss-Webster mice were prepared as described previously (2.5, 28). Parasagittally oriented explants of either cerebellar or cerebral neocortical tissue were placed on collagen-coated coverslips with a drop of nutrient medium, sealed in Maximow double coverslip assemblies, and incubated in the lying-drop position at 35.5 to 36°C. The nutrient medium, which was changed twice weekly, consisted of two parts 3 IU/ml low-zinc insulin (supplied by th e S quibb Institute for hZedica1 Research), one part 20% dextrose, four parts bovine serum ultrafiltrate, four parts Eagle’s minimum essential medium with Hanks’ base and added L-glutamine, seven parts Simms’ X-7 balanced salt solution (BSS) with sufficient added HEPES buffer to make its concentration 0.01 M in the fully consituted medium, 2 Abbreviations : CNS, central nervous system ; HEPES, zinc-IV-Z-ethanesulfonic acid ; BSS, balanced salt solution.

N-Z-hydroxyethylpipera-

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and 12 parts serum. Fetal calf serum was used for cerebellar explants, and human placental serum was applied to cultures of cerebral neocortex. Electrophysiologic recording techniques were similar to those previously established in our laboratory (18, 19). Cerebellar cultures selected for functional studies varied in age from 13 to 2.5 days in vitro, and cerebral neocortex explants were recorded at 20 days in vitro. Cultures within those age ranges demonstrated mature patterns of electrical activity (18, 19). Collagen-coated coverslips containing the cultures were placed in a chamber open at the top and mounted on the stage of an inverted microscope. A dc-powered Nichrome coil surrounding the coverslip served to warm the preparation. Temperature was monitored by a thermistor probe inserted into the medium. Etched tungsten stimulating and recording electrodes, the latter with tip diameters of less than 1 pm, were placed under direct visual control. Silver reference electrodes were inserted into the medium bathing the cultures. For recording purposes, this medium consisted of 9 to 10 drops BSS additionally buffered with 0.015 M HEPES. Ethanol diluted 5 to 307 0 in BSS w.as added to the medium in drop increments by pipet. Electrical stimuli were delivered to cerebral neocortex explants by means of a Grass S88 stimulator, and responses were recorded from both cerebellar and cerebral neocortical cultures by a Grass P15 preamplifier and stored on an Ampex 300 tape recorder or photographed from the oscilloscopic display with a Polaroid camera. Precise measurements of ethanol concentrations were determined in some cultures at various levels of electrical activity. Twenty-microliter samples of recording medium with incorporated ethanol were withdrawn by a microliter pipet and placed in tightly stoppered glass test tubes. Ethanol concentrations were determined by head-space gas chromatography using a Porapak Q column and an n-propanol internal standard (D. B. Goldstein, personal communication). Ten such determina.tions were made in three cerebellar cultures, and four determinations in two cerebral neocortex explants. Electrophysiological studies were performed on a total of six cerebellar and two cerebral neocortical cultures. A total of 118 test and 76 control cerebellar cultures selected from 16 different groups of explants were used for morphological studies. Of these, 30 test and 17 control cultures were fixed for electron microscopy, and the remainder were stained as whole-mount preparations with either thionine (26) or a modified Holmes procedure (31) for light microscopic examination. All cultures were also observed in the living state during periods of exposure to ethanol. For electron microscopy, the cultures were rinsed briefly in Simms’ X-7 BSS at room temperature, then fixed 1.5 h in 3% glutaraldehyde-1% paraformaldehyde in 0.1 M phosphate buffer at 37°C (pH 7.2), and rinsed briefly in a wash solution. Methods of postfixation in

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osmium tetroxide and processing and examination by electron microscopy were similar to those previously used in our laboratory (29). For embedding in Epon, the coverslips were inverted over the top of a modified Beem capsule; after polymerization, the coverslips were separataed from the plastic-embeddedexplants. Test cultures were exposed to ethanol incorporated into the nutrient medium in varying concentrations by volume and at varying age ranges as follows: 2.2% (O-14, 11-21, 15-21, 15-22, and 18-33 days in vitro) ; 3.3% (15-22 and 22-29 days i~t GM) ; 4.4% (11-20, 11-21, and l5-22 days ill zritvo) ; and 1370 (l&22 and 25-29 days in z6fr.o). Twenty-four quantitative measurements of ethanol, as described above, were made of medium containing 2.2 and 4.4% ethanol by volume immediately after application to cultures. Values for 2.2% solutions ranged from 0.56 g ethanol/100 ml medium to 0.88 g/100 ml, and values for 4.4% solutions varied from 1.26 to 1.55 g/100 ml. By extrapolation, values for 3.3 and 13% solutions would correspond with levels of at least 0.8 and 3.5 g ethanol,/100 ml medium respectively. Twenty-four additional quantitative measurements were made of medium to which cultures had been exposed for 3 or 4 days, i.e., the interval between feedings. Those determinations revealed a drop in ethanol concentration betlveen one-half and one-third of the original concentration, the bulk of which was estimated to be due to evaporative loss into the culture chamber during the 3 or 4 days of exposure. In spite of such losses,the ethanol concentration in the medium of cultures after exposure to 2.2% solutions was from 0.21 to 0.44 g/100 ml, and from 0.45 to 0.87 g/100 ml in medium containing 4.4% ethanol by volume. RESUI,TS Effects of EfhnoZ O/L Sponfanrons Elrcfvicul ,4rfizify. Ethanol added to cerebellar cultures in small increments produced an increase in rate and usually a regularization of spontaneous discharge. Addition of large increments resulted initially in an increase in discharge rate followed by reversible depression of spontaneous activity. These effects are illustrated in Figs. 1 and 2. Figure 1, recorded from a cerebellar explant 25 clays ill zGh.0, illustrates the typical pattern of adding ethanol in small increments. Phasic spontaneous discharge, characteristic of many cerebellar cortical units (4, IS), is evident in Fig. 1‘4. One minute after addition of ethanol, a mild increase in discharge rate became evident (Fig. 1B). Subsequently there was further increase in rate of discharge and loss of its phasic quality, as well as an apparent reduction of some of the higher-amplitude units (Fig. 1C). This was followed by a decreasein rate of discharge and in amplitude of the remaining units (Fig. 1D). After addition of another sma11increment of ethanol, a further decrease in amplitude of the re-

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to a cerebellar culture 25 days i~t vitro, reFIG. 1. Effects of ethanol application corded extracellularly at 25°C. A-Baseline spontaneous activity with characteristic phasic discharges. B-One minute after addtion of an increment of ethanol, there was an increase in rate of discharge. C-Two minutes later the discharge rate has increased further and become more regular, and there has been a reduction of some of the higher amplitude units. D-After another 2 min, the discharge rate has decreased and the amplitude of the remaining units has become lower. E-One minute after addition of another increment of ethanol, the amplitude of the remaining units has become further reduced. F-Two minutes later the last discharges are evident on the left side of the figure, followed by electrical silence. G-After washing with buffered balanced salt solution, spontaneous electrical activity has returned to approximately baseline rate. The time-base indicator at the bottom of the figure indicates 1 s. maining discharging units became evident (Fig. lE), followed by a progressive decrease in amplitude of cerebellar cortical units until discharge ceased altogether (Fig. 1F). After 1.5 min electrical silence, the culture

ETHANOL

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KEITRAL

CULTC’RES

395

was washed with BSS, with the subsequent return of phasic spontaneous activity (Fig. 1G) within minutes after washing. Some cultures demonstrated such reversibility even after 15 min electrical silence. Cerebellar

FIG. 2. Effects of a single application of ethanol to a 13-day irb V~~YO cerebellar culture recorded at 25°C. A-Baseline spontaneous activity. B-Increase and regularization of spontaneous discharge after addition of ethanol to 2.84 g/100 ml. CWithout further addition of ethanol, the discharge rate and amplitude became progressively lower, followed by electrical silence. II-The culture was washed with balanced salt solution after a 5-min electrical silence, with subsequent return of spontaneous activity. The time-base indiactor at the bottom of the figure indicates 500 ms.

396

SEIL

o Excdatory . Depressant

CEREBELLUM

(D

0

0 I 1.0

0.5 .

AL.

Effects Effects

grams ethanol per IOOcc medium CEREBRUM

ET

.

.O

. 1 2.0

1.5 l

. .

2.5

. I 3.0

, 3.5

l

FIG. 3. The ethanol concentrations at which effects were obtained on electrophysiologic activity in cerebellar and cerebral neocortical cultures after addition of very small or large increments of ethanol. Closed circles represent depressant effects of ethanol, which were the only effects found in cerebral neocortex cultures, and open circles represent excitatory effects. Excitatory effects were operationally defined as an increase in spontaneous discharge rate, not followed by a decrease unless more ethanol was added. Ethanol concentrations were not measured until an effect on electrophysiologic activity was noted, and therefore the lowest concentrations on the graph represent the lowest concentrations at which any effect was produced.

cultures were recorded at temperatures of 25 to 33.5”C. No notable differences attributable to temperature effects were observed within this range. A similar sequence is illustrated in a cerebellar culture 13 days in vitro (Fig. 2), during which ethanol concentrations were measured. After addition of ethanol to 2.84 g/100 ml an excitatory effect became evident (Fig. 2B), followed, without further addition of ethanol, by total depression of

FIG. 4. Effects of ethanol application on stimulus-elicited responses in a cerebral neocortex culture, 20 days in zifro, recorded at 31 to 32°C. A-Baseline response, demonstrating polyphasic spikes followed by a slow wave, which has superimposed spikes. B-After addition of ethanol to 2.17 g/100 ml medium, the amplitude of the slow wave was markedly reduced and the superimposed spikes were no longer present. C-After washing the culture with balanced salt solution, the stimulus-elicited response had returned to approximately baseline level. The time-base indicator at the bottom of the figure denotes 50 ms.

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spontaneous activity (Fig. 2C). This depression was reversed after 5 min by washing with BSS (Fig. 2D). Ethanol was again added to this culture, and depression of spontaneous activity was attained at a concentration of 3.64 g/l00 ml, again reversed by washing with BSS. Measurement of ethanol concentration after washing with BSS demonstrated that all the ethanol within the fluid environment could be removed. The results of quantitative ethanol studies are summarized in Fig. 3. The lowest concentration at which any change was noted ill cerebellar cultures was 0.51 g/100 ml, which produced an increase in spontaneous discharge rate. A dividing line between excitatory and depressant effects occurred at approximately 1.7 g/100 ml. As stated above, only excitatory effects were usually noted at smaller concentrations, and addition of ethanol in increments greater than this concentration generally resulted initially in an excitatory phase, followed by depression, as shown particularly well in Fig. 2. EfJrcts of Ethanol OH Stil,/lfllls-EZic.itrd Rrsjw~~s. In cereljral neocortes cultures, ethanol produced only depressant effects (Fig. 3). As illustrated in Fig. 4A, baseline activity consisted of electrically evoked responses characterized by polyphasic spikes followed by a slow wave, which in some cases had superimposed spikes, as described previously (7, 19). The earliest change, seen at ethanol concentrations as low as 0.27 g/100 ml, cousisted of a change in the slope of the slon wave. This was followed at higher concentrations of ethanol by a decrease iu amplitude and complexity of the slow wave, and. in the case of the response illustrated in Fig. -I, by a re-

FIG. 5. Normal neurons and cerebellar cortical architecture in ethanol-treated cultures. Left-Structurally unaffected Purkinje cells in a cerebellar explant at 14 days is z~itro exposed to starting concentrations of 0.56 to 0.70 g ethanol/100 ml medium from 0 to 14 days. Thionine stain, X475. Right-A cortical laminar arrangement which is normal for a cerebellar explant [see (25) ] in a 22-day i~t n&o culture exposed to starting concentrations of ethanol equivalent to 1.26 to 1.55 g/100 ml from Day 15 to Day 22. The large neurons near the center of the figure are Purkinje cells. Holmes stain, X285.

398

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ET

AL.

duction in the firing rate of the superimposed spikes, At higher concentrations of ethanol, 2.17 g/100 ml in the response illustrated in Fig. 4B, there was a marked reduction in the amplitude of the slow wave. Washing with BSS again resulted in rapid return of the response to approximately baseline level, as illustrated in Fig. 4C. Recordings were made at 25°C in one cerebral neocortex culture and at 31 to 32°C in the other cerebral neocortex explant. No significant differences due to temperature effects were noted within this range. As with the cerebellar cultures, each cerebral neocortex explant was subjected to several trials of ethanol exposure followed by washing with buffered BSS. Light Microscopic Results. Cerebellar cultures exposed 4 days to ethanol in excess of 3.5 g/100 ml demonstrated severe toxic degeneration of all cellular elements. In none of the other groups of cultures exposed to 1.55 to 0.45 g ethanol/100 ml medium (values include the maximum by quantitative determination immediately after application to cultures and the minimum remaining ethanol after 3 or 4 days of exposure) for up to 10 days, or from 0.88 to 0.21 g/l00 ml for LIP to 15 days, were changes evident by light microscopic examination of living or stained preparations. The latter category of cultures included one group exposed from explantation to 14 days in vitro. Specifically, no changes were evident in glia or neurons, including granule and Purkinje cells (Fig. 5A), in myelin, or in overall cerebellar cortical architecture (Fig. 5B). Normal migration and alignment of cells were evident in the group of cultures exposed to ethanol from 0 to 14 days in vitro. Electron Microscopic Results. Ultrastructural examination of cultures which demonstrated no changes by light microscopy upon exposure to ethanol concentrations and durations, as described above, revealed only nonspecific changes of increased dense bodies (lysosomes) and autophagic vacuoles, particularly in astrocytes (Fig. 6). No other cytologic changes were found. Synapses and mitochondria (Fig. 6 and inset), granular neurons, Purkinje cells, and nerve cell processes were similar to those in agematched control cultures (Fig. 7). The presence of myelinated axons in ethanol-treated explants was comparable to controls. DISCUSSION Except for those cultures exposed to extremely high concentrations of ethanol (more than 3.5 g/100 ml) for several days, no specific pathologic changes were evident by both light and electron microscopic examination of cerebellar explants exposed to concentrations of ethanl closer to, but in many instances still well above, those which produce profound functional changes in v&o. In view of the abnormalities of neuronal migration noted in the human fetal alcohol syndrome (11, 12). it is of particular interest

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FIG. 6. Treated cerebellar culture at 21 days i)t zjifro, exposed 10 days to starting concentrations of 0.67 to 0.88 g ethanol/100 ml medium from Day 11. Illustrated is an astrocyte containing numerous dense bodies (lysosomes) and packets of glial filaments. Note the normal mitochondria. X 18,200. Inset : Neuropil of a cerebellar culture at 21 days irz z*itvr~, exposed 6 days to 0.56 g ethanol/100 ml medium at 15 days ira zjitvo. Note the numerous well-preserved synapses. X 12,300.

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AL.

that norma architectural and cellular relationships were present in ethanoltreated cultures. In the only other available study of ethanol effects on neural cultures, no morphologic changes were noted by phase microscopy in either monolayer cultures of clones NN (hamster astroblast line) or the rat C6 glioma exposed 20 days to 100 mM ethanol (21). However, chronic exposure to ethanol led to an increase in choline uptake, suggesting that ethanol alters plasma membrane characteristics, a view further supported by the increased activity of Mg?+-adenosine triphosphatase in similarly treated neural cultures (30). The acute effects of ethanol on spontaneous discharges in cerebellar explants included an increase in overall discharge rate either at relatively

FIG. 7. Control cell (upper field), x 500.

cerebellar culture, 24 days irl zfitro. Note well-developed two granular neurons (lower field), and the adjacent

Purkinje neuropil.

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low (but above ill viz10 range) doses or early in the time course after application of a larger dose, followed by depression of spontaneous activity when larger doses of ethanol lvere applied. Intravenous injection of ethanol in cats has been reported to cause a decrease in the spontaneous discharge frequency of Purkinje cells and an increased firing rate of the majority of cerebellar interneurons (9). The results of the present study can be interpreted to suggest that ethanol may l~lucl~ synaptic transmission, thus isolating Purkinje cells from the inhibitory effects of cerebellar interneurons. Alternatively, ethanol may have an earlier depressant effect on the smaller inhibitory interueurons than on the larger Purkinje cells. Either mechanism could result in an increased discharge rate of the Purkinje cells, the most likely source of the higher-amplitude units recorded in &fro (4, 18). The regularization of discharge rate which accompanies the increase may also reflect the effects of release of Purkinje cells from the influence of cerebellar interneurons, because such regularization of discharge rate has been reported in low cell density areas of cerebellar cultures where Purkinje cells may be relatively isolated, in contrast to more densely populated areas which characteristically display phasic spontaneous discharges (4, 5). The progressive decrease in amplitude of discharging units prior to cessation of electrical activity suggestsa direct depressant effect of ethanol on the membranes of the remaining discharging neurons. Comparison of the impact of ethanol on cultures of cerebellum and cerebral neocortex may provide clues to the anatomic specificity of ethanol effects on the CNS. Effects on cerebral neocortical explants were distinguished from those characteristic of cerebellum by being exclusively depressant. One possible explanation for this difference is that ethanol may block all forms of neural membrane electrogenesis,and the effects on either spontaneous single-unit discharges or stimulus-elicited extracellular field potentials may be determined by anatomic and electrophysiologic specializations of particular regions. In the present study, for example, the initial increase in cerebellar Purkinje cell discharge rate after addition of ethanol may derive from a lower threshold of inhibitory circuitry (finer neurites ?) to the depressant effect of ethanol, whereas in cerebral neocortex cultures inhibitory and excitatory elements may demonstrate no threshold differences to nonselective membrane depressants. The electrical sign of the effect may be a property of the differential safety factor of various rnembranes, e.g., excitatory or inhibitory synapses, or the magnitude of .synaptic activity required for some postsynaptic effect. An alternative possibility is that differences between cerebellar and cerebral neocortical cultures may reflect on regional specialization in terms of neurochemical distinctions, especially those related to synaptic transmission. With regard to the latter, the rapid reversibility of the depressant manifestations of ethanol

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in 7sitro by washing the explants with normal medium favors a membranealtering mechanism which impairs ionic exchange rather than a neurotransmitter-media,ted effect. Other evidence which suggests that ethanol alters cell membrane properties has been obtained in squid giant axon (2, 22) and in slices of rat cerebral cortex (23). The present study demonstrates that ethanol applied directly to the isolated CNS produces functional changes at concentrations well above those which produce effects in vivo, in agreement with other in vitro studies (13). One possible explanation for this phenomenon is that ethanol itself is not the major factor which produces electrical changes in the CNS, but that some ethanol metabolite not produced in functionally significant quantities in the CNS is the key factor which elicits electrophysiologic changes. Other possibilities include the effects of ethanol on the CNS being largely secondary to a variety of systemic effects. Indeed, as Kalant has cautioned, there may be no specific set of direct effects of ethanol on the CNS (13). Some of those possibilities, however, particularly those involving ethanol metabolites, can be evaluated by further experiments with tissue cultures. The tissue culture system, with its metabolic properties more like those of the in viva situation than other in vitro preparations, offers a promising approach to possible mechanismsof ethanol action on the CNS. REFERENCES I. ALLERAND, C. D. 1971. Patterns of neuronal differentiation in developing cultures of neonatal mouse cerebellum: A living and silver impregnation study. 1. Co~lp. Nerwol. 142 : 167-204. 2. ARMSTRONG, C. L., AND L. BINSTOCK. 1964. The effects of severalalcoholson the properties of the squid giant axon. J. Ccn. Phwiol. 48 : 265-277. 3. BORNSTEIN, M. B. 1964. Morphological development of neonatal mouse cerebral cortex in tissue culture. Pages l-11 ilz P. KELLAWAY AND I. PETERSON, Eds., Neurological and Elcctrographic Correlative Studies in Irtfancy. Grune and Stratton, New York. 4. CALVET, M. C. 1974. Patterns of spontaneous electrical activity in tissue cultures of mammalian cortex vs. cerebellum. Brain Res. 69 : 281-295. 5. CALVET, M. C., M. J. DRIAN, AND A. PRIVAT. 1974. Spontaneous electrical patterns in cultured Purkinje cells grown with an antimitotic agent. Brabz Res. 79 : 285-290. 6. CARMICHAEL, F. J., AND Y. ISRAEL. 1975. Effects of ethanol on neurotransmitter release by rat brain cortical slices. J. Pharmacol. E.@. Ther. 193: 824-834. 7. CRAIN, S. M., AND M. B. BORNSTEIN. 1964. Bioelectric activity of mouse cerebral cortex during growth and differentiation in tissue culture. Exp. Newel. 10: 425-450. 8. DAVIDOFF, R. A. 1973. Alcohol and presynaptic inhibition in an isolated spinal cord preparation. Arch. Ncwol. 28 : 60-63. 9. EIDELBERG, E., M. L. BOND, AND A. KELTER. 1971.. Effects of alcohol on cercbellar and vestibular neurones. drch. Int. Pharmacodyw. Ther. 192: 213-219.

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10. HOSKIN, F. C. G., AND C. D. ALLERAND. 1968. Metabolism of specifically labelled glucose by explants of newborn mouse cerebellum. J. NEIITOCIICIII. 15: 427-432. 11. JONES, I(. L., AND D. W. S~IITH. 1973. Recognition of the fetal alcohol syndrome in early infancy. Latlcct 2 : 999-1001. 12. JONES, K. L., D. W. SMITH, AND J. W. HANSON. 1976. The fetal alcohol syndrome: Clinical delineation. .4ntf. N.I-. -4cod. Sci. Sci. 273 : 130-137. 13. KAI,ANT, H. 1975. Direct effects of ethanol on the nervous system. Frd. Proc. 34: 1930-1941. 14. KAI.ANT, H., AND W. GHOSE. 1967. Effects of ethanol and pentobarbital on release of acetylcholine from cerebral cortex slices. J. Pharmcol. Erj. Thu. 158 : 386-393. 15. KIM, S. U. 1972. Light and electron microscopic study of mouse cerebral neocortex in tissue culture. E.rp. Ncurol. 35 : 305-321. 16. LEIIRER, G. M., M. B. BORNSTEIN, C. WEISS, M. FVRMAX, AXD C. LICHTMAN. 1970. Enzymes of carbohydrate metabolism in the rat cerebellum developing in sit~c and ill vitro. Exp. Nrwol. 27 : 410-425. 17. LEHRER, G. M., M. B. BORNSTEIN, C. WEISS, AND D. J. SILIDES. 1970. Enzymatic maturation of mouse cerebral neocortex in zfifro and irk ~itzl. Exp. Nrwol. 26 : 595-606. 18. LEIMAN, A. L., AND F. J. SEIL. 1973. Spontaneous and evoked bioelectric activity in organized cerebellar tissue cultures. E.rp. Ncwol. 40: 748-759. 19. LEIMAN, A. L.. F. J. SEIL, AND J. M. KELLY. 1975. Maturation of electrical activity of cerebral neocortex in tissue culture. Exp. Newel. 48: 275-291. 20. MAKER, H. S., G. M. LEHRER, S. WEISSBARTH, AND M. B. BORNSTEIN. 1972. Changes in LDH isoenzyme of brain developing irl siftr and ill TGfvo. Brain Rcs. 44: 189-196. 21. MASSARELLI, R., P. J. SYAPIN, AND E. P. NOBLE. 1976. Increased uptake of choline by neural cell cultures chronically exposed to ethanol. Life Sri. 18: 397-404. 22. MOORE, J. W., W. ULBRICHT, AND M. TAKATA. 1964. Effect of ethanol on sodium and potassium conductances of the squid axon membrane. J. Gcn. PIzssiol. 48: 279-295. 23. NIICANDER, P., I’. VON BOGUSLAA’SKY, API’D H. WALLGREN. 1971. Actions of ethanol and tertiary butanol on net movements of sodium and potassium in electrically stimulated rat brain tissue ill vitro. Scuud. J. Clin. Lab. Invest. Snppl. 27 (116) : 67. 24. PRITAT, A., AND M. J. DRIAN. 1976. Postnatal maturation of rat Purkinje cells cultivated in the absence of two afferent systems: An ultrastructural study. J. Comfi. Nrrwol. 166: 201-243. 25. SEIL, F. J. 1972. Neuronal groups and fiber patterns in cerebellar tissue cultures. Brain Res. 42: 33-51. 26. SEIL, F. J., AND R. M. HERNDON. 1970. Cerebellar granule cells it1 vitro. A light and electron microscopic study. J. Ccl/ Biol. 45 : 212-220. 27. SEIL, F. J., AND A. L. LEIMAN. 1976. Neural subsystems and learning: Tissue culture approaches. Pages 390-398 in M. R. ROSENZWEIC AND E. L. BENNETT, Eds., Nrural ;~~CC~CIII~SVZS of Lconii)z~/ aMd ilfrmory. AfIT Press, Cambridge, Massachusetts. 28. SEIL, F. J., J. M. KELLY, AND A. L. LEIMAIL.. 1974. Anatomical cerebral neocortex in tissue culture. I?.@. Nrwol. 45 : 435-450.

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J. C., M. M. HERMAN, AND L. J. RUBINSTEIN. 1973. Electron microscopic observation on human glioblastomas and astrocytomas maintained in organ culture systems. Anwv. J. Patlzol. 73 : 589-606. 30. SYAPIN, P. J., V. STEFANOVIC, P. MANDEL, AND E. P. NOBLE. 1976. The chronic and acute effects of ethanol on adenosine triphosphatase activity in cultured astroblast and neuroblastoma cells. J. Neurosci. Res. 2: 147-155. 31. WOLF, M. K. 1964. Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum. J. Cell Biol. 22 : 2X&279. 32. WOLF, M. K., AND M. DUBOIS-DALCQ. 1970. Anatomy of cultured mouse cerebellum. I. Golgi and electron microscopic demonstration of granule cells, their afferent and efferent synapses. J. Comp. Ncttrol. 140 : 261-280. SIPE,