developmental changes in the inhibitory actions of ethanol on glutamate-induced translocation of...

ELSEVIER PII S0741-8329(97)00050-5

Alcohol, Vol. 14, No. 6, pp. 575-580, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved

0741-8329/97 $17.00 + .00

Developmental Changes in the Inhibitory Actions of Ethanol on Glutamate-Induced

Translocation of Protein Kinase C in Cerebellar Granule Neurons

M A N U E L B A R R I O S A N D S T U R E L I L J E Q U I S T

Department o f Clinical Neuroscience, Division o f Drug Dependence Research, Karolinska Hospital, S-1 7176 Stockholm, Sweden

Received 2 D e c e m b e r 1996; Accep ted 21 Feb rua ry 1997

BARRIOS, M. AND S. LILJEQUIST. Developmental changes in the inhibitory actions of ethanol on glutamate-induced translocation of protein kinase C in cerebellar granule neurons. ALCOHOL 14(6) 575-580, 1997.--The effects of increasing concentrations of ethanol (25-200 mM) on the enhancement of [3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding produced by different glutamate receptor agonists, indicative of a translocation of the intracellular enzyme protein kinase C (PKC), were studied in rat cerebellar granule cells at 2, 4, 8, and 12 days in vitro (DIV). Glutamate-produced stimulation of [3H]PDBu binding was inhibited by 50 mM ethanol at 2 DIV, whereas higher ethanol concentrations (> 100 mM) were needed to reduce the increase of [3H]PDBu binding in cells grown for 4, 8, and 12 DIV. Ethanol significantly inhibited NMDA-stimulated [3H]PDBu binding in a concentration-dependent fashion in cells maintained in culture for 4 and 8 days, respectively, with a slightly less pronounced inhibition by ethanol (50 mM) seen in cells kept for 2 and 12 DIV. Application of higher ethanol concentrations (> 100 raM), inhibited the NMDA-induced stimulation in all cell preparations. Following kainic acid-induced enhancement of [3H]PDBu binding, ethanol (100 raM) reduced the binding only in cells maintained for 2 DIV. Even higher ethanol concentrations (200 raM) inhibited the effects of kainic acid only in cells maintained for 2 and 4 DIV, respectively. Our data suggest that various subclasses of glutamate receptors display a developmentally determined differential sensitivity to ethanol at least in cerebellar granule cells in vitro. © 1997 Elsevier Science Inc.

Ethanol Phorbol ester binding Cerebellar granule cells

PKC translocation Glutamate receptors NMDA Development

EXCITATORY neurotransmission in the brain is to a large extent mediated through activation of various subclasses of ionotropic glutamate receptors, named N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole pro- pionic acid (AMPA) receptors, which are known to influence the activity of about 50% of all synapses in the brain (22). Rapidly growing evidence suggests that glutamate not only regulates brain excitability in the adult brain but also acts as a potent modulator of various functions in the developing brain, thereby influencing the regulation of neuronal survival, dendritic and axonal growth, synaptogenesis, and activity- dependent synaptic plasticity (29,30). By using cultured cere-

bellar granule cells as an experimental model system (7) for the study of developmental characteristics of glutamatergic functions, it has been found that glutamate-produced stimulation of intracellular Ca 2+ levels ([Ca2+]i shows a typical developmental profile, that is, an enhanced ability of glutamate to increase Ca 2+ fluxes with increasing postnatal age (16). The developmentally determined effects of glutamate on Ca 2+ fluxes appears to coincide with the occurrence of an increased sensitivity to neurotoxic actions of glutamate (17). Moreover, activation of glutamate receptors not only causes Ca 2+ fluxes and neurotoxicity, but results also in translocation of the intracellular enzyme, protein kinase C (PKC) (28,41,42). In analogy

Requests for reprints should be addressed to Sture Liljequist, Ph.D., Department of Clinical Neuroscience, Division of Drug Dependence Research, Bldg. L1:01, Karolinska Hospital, S-17176 Stockholm, Sweden. Tel: +46-8-5177-5742; Fax: +46-8-5177-523l; E-mail: [email protected]

575

576 BARRIOS AND LILJEQUIST

to findings with Ca 2+ and neurotoxicity, the PKC translocation stimulated by glutamate was found to undergo pronounced changes during early postnatal development of cerebellar granule cells (3). The exact mechanisms behind these dynamic patterns of postnatal glutamate-induced functional responses are currently not known but recent observations suggest that developmentally determined differences in the molecular composition of brain glutamate receptors may play a pivotal role in these events (1,20,32,33,35,44,46).

Recently accumulated knowledge indicates that ethanol, like glutamate, influences the survival, dendritic and axonal growth, synaptogenesis, and activity-dependent synaptic plasticity in developing brain neurons and it is increasingly ac- cepted that these phenomena may be targets for the teratogenic actions of ethanol [for discussion, see (34)]. Moreover, there is convincing evidence that acute administration of moderate to intoxicating concentrations of ethanol are able to inhibit many actions of the excitatory neurotransmitter glutamate in the brain, including NMDA/glutamate-induced electrophysiological activity, functional Ca 2+ responses, activation of transcription factors, release of various neurotrans- mitters, and neurotoxicity [see (9,21,23)]. Additional findings indicate that chronic ethanol exposure results in an enhancement of NMDA receptor-mediated receptor functional responses as well as causes changes in the molecular composition of brain NMDA receptors (15,38,40). Furthermore, recent evidence supports the notion that continuous exposure to ethanol blocks the trophic and neurotoxic actions of NMDA during the early development of cerebellar granule cells in vitro (45).

Intracellular PKC activity has been suggested to be involved in processes associated with the normal development of brain neurons (8,11,29,43). Stimulation of glutamate receptors results in a pronounced enhancement of PKC translocation in cerebellar granule neurons (28,41), and we have recently shown that stimulation of various subclasses of glutamate receptors induce differential patterns of PKC activation during the early development of cerebellar granule cells (3). Based upon those observations and the fact that ethanol inhibits the translocation of PKC (27), we decided to ex- amine these phenomena during early postnatal development of rat cerebellar granule cells. Thus, we have in the present study investigated the effects of ethanol on glutamate-induced stimulation of [3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding, considered to be an indirect measure of PKC translocation from the cytosol to the neuronal membrane, to monolayers of intact cerebellar granule cells from newborn rats at 2, 4, 8, and 12 days in vitro (DIV).

M E T H O D

Cell Cultures

Primary cultures of cerebellar granule cells were obtained from 8-day-old Sprague-Dawley rat cerebellum as previously described by Levi et al. (26). Briefly, after dissection, eight cere- belli were pooled, and sliced with a Mcllwain tissue chopper in two orthogonal directions (slices were 0.4 mm thick), incubated in 0.25% trypsin solution, and dispersed by trituration in a DNase and soybean trypsin inhibitor-containing solution (0.01% and 0.05%, respectively). Cells were plated (106 cells/2 ml/dish) onto 35-mm Nunc dishes (Nunc AC, Denmark) coated with 5 txg/ml of poly-L-lysine (Mw = 70,000-150,000), and cultured for 8 days at 37°C in an atmosphere of 5% COJ95% air in a Basal Eagle's Medium (GIBCO; Taby, Sweden) supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 25 mM

KC1, 2 mM glutamine, and 100 ixg/ml gentamicin (GIBCO). The addition of cytosine-[3-arabinofuranoside (10 IxM; Sigma) 24 h after plating limited the number of glia cells to less than 5% (41). The medium was not changed until the cultures were used in the binding experiments.

[3H]Phorbol Ester Binding to Intact Cells

A detailed description of assay conditions and binding characteristics of 4-[3-[3H]phorbol 12,13-dibutyrate ([3H]PDBu) and its developmental characteristics can be found in our previous studies (3,41,42) in which evidence also was presented in- dicating that [3H]PDBu binding can be used as a measure of PKC translocation in cerebellar granule cells. Briefly, mono- layer cultures were grown for 2, 4, 8, and 12 DIV, respectively. To start the experiments the cultures were washed once with 2 ml of MgZ+-free Locke's solution (154 mM NaC1, 5.5 mM KCI, 3.6 mM CaC12, 3.6 mM NaHCO3, 5.6 mM glucose, 5 mM HEPES, pH 7.4) and incubated in 1 ml of Locke's solution containing 10 nM [3H]PDBu (spec.act. 20 Ci/mmol; Du Pont Scan- dinavia AB, Stockholm, Sweden) in 0.5% fatty acid-free bo- vine serum albumin (Sigma). Monolayers were incubated at 22°C for 15 min, after which they were rapidly washed three times with ice-cold, MgZ+-free, Locke's solution and sus- pended over night with 0.1 M NaOH. Aliquots of the suspen- sion were then used for protein determination (100 txl) and determination of radioactivity (500 Ixl) by liquid scintillation counting (LKB Rackbeta 1217, Uppsala, Sweden). Nonspe- cific binding was determined in the presence of 10 IxM 12- O-tetradecanoylphorbol 13-acetate (TPA, Sigma) and repre- sented about 20% of the total binding. TPA was added to the incubation medium as a 100-fold concentrated solution in 10% dimethyl sulfoxide (DMSO). Other drugs of interest were dissolved either in Locke's solution, slightly warmed if necessary, or in 10% DMSO and premixed to the incubation medium before the start of the cell incubation. These concentrations of DMSO had no effects by themselves on basal [3H]PDBu binding.

Drugs

The glutamate receptor agonists, glutamic acid, NMDA, and kainic acid were all purchased from Sigma, whereas AMPA was purchased from Tocris Neuramin.

Analysis of Data

All binding data were expressed as fmol of [3H]PDBu bound per mg of protein and transformed to percent of baseline binding in untreated cerebellar granule cells. One-way analysis of variance (ANOVA) followed by Bonferroni's test for multiple comparisons with p < 0.05 considered to be sta- tistically significant.

RESULTS

Glutamate-Induced Enhancement of [3 H] P D Bu Binding

Figure 1 shows the stimulation of [3H]PDBu binding induced by 5 txM glutamate to monolayers of intact cerebellar granule cells after 2, 4, 8, and 12 DIV and its inhibition by increasing concentrations of ethanol. As shown, ethanol caused a concentration-dependent reduction of [3H]PDBu binding in all cell preparations. The glutamate-induced stimulation of [3H]PDBu binding was most sensitive to the inhibitory actions of ethanol in cells maintained for 2 DIV evidenced by the fact that already 50 mM ethanol produced a significant blockade of

E T H A N O L AND PKC T R A N S L O C A T I O N 577

[3H]PDBu binding in these neurons, whereas 100 mM ethanol was required to produced a significant inhibition of the glutamate response in all the other cell preparations. Because the magnitude of the glutamate-produced stimulation of PKC differed at various developmental stages (see Fig. 1), the effects of glutamate plus ethanol are presented as percentage of the glutamate-induced maximal enhancement at different points of maturation. In accordance with our previous findings (3), glutamate induced a maximal enhancement of [3H]PDBu binding (260.4 + 16.1% above basal binding) in cells at 2 DIV after which the stimulatory effects of glutamate gradually decreased, reaching its lowest level (about 50% stimulation) at 12 DIV. As noted before (3), there was no significant change in the level of basal [3H]PDBu binding (rang- ing from 371.1 _ 21.4 fmol/mg protein at 4 DIV to 443.1 _ 33.5 fmol/mg protein at 12 DIV) between 2 and 12 DIV and the glutamate-induced enhancement of binding was largely blocked by both competitive and noncompetitive NMDA receptor antagonists, but not by specific AMPA receptor block- ers. For further details of [×H]PDBu binding characteristics in the currently used cerebellar granule cell preparations, see Barrios and Liljequist (3).

NMDA-Induced Enhancement of [3H]PDBu Binding

Results depicted in Fig. 2 demonstrate that increasing concentrations of ethanol inhibited the enhancement of [3H] PDBu binding produced by 50 IxM N M D A in a concentra- t ion-dependent fashion. As shown before (3), the maximally enhanced stimulation of [3H]PDBu binding caused by NMDA (190.8 -+ 11.9% above basal binding) was seen at 2 DIV after which it gradually decreased with increasing number of DIVs. In contrast to the findings obtained with glutamate itself (see above), 50 mM ethanol caused a significant inhibition of NMDA-enhanced [zH]PDBu binding in cells kept for 4 and 8 DIV, respectively, whereas the same concentration of ethanol caused a smaller inhibition of the NMDA-induced enhancement in cells grown for 2 and 12 DIV. Higher concentrations

NMDA

200-

IO0-

==. o-

-"=- 2 DIV • -0-4 DIV "-~-8 DIV --o- 12 DIV

; 5'0 1;0 1;0 2 ; 0

Ethanol, [mM]

FIG. 2. Effects of increasing ethanol concentrations (25-200 raM) on [3H]PDBu binding, expressed as percentage above the baseline, induced by NMDA (50 IxM) in cerebellar granule cells at different stages of development (2, 4, 8, and 12 DIV). Statistical differences with respect to control (ANOVA followed by Bonferroni test): *p < 0.05, **p < 0.01. Data represent the mean ± SEM of 4-6 different cell culture preparations.

of ethanol (->100 raM) inhibited the NMDA response in all cell preparations.

Kainate-Induced Enhancement of [~H]PDBu Binding

Application of ethanol to cells following the stimulation of [3H]PDBu binding by kainic acid (147.4 _+ 17.8% above basal binding) caused inhibition (from > 100 mM ethanol) only in cells maintained for 2 DIV, whereas all other cell preparations remained unaffected at intoxicating concentrations (< 100 mM) of ethanol (Fig. 3). However, very high concentrations of ethanol (200 mM) caused a significant inhibition of

GLUTAMATE

3°°I

~ * .-~-2 DIV 2001 * + o,v

- - 8 DIV ~ ] * --o- 12 DIV

i '°°1 i 0 n ~ ~*

; 5'0 1;0 1;0 2;0 Ethanol, [mM]

FIG. 1. Effect of increasing concentrations of ethanol (25-200 mM) on [3H]PDBu binding, expressed as percentage above basal binding, induced by glutamate (5 txM) in cerebellar granule cells at different stages of development (2, 4, 8, and 12 DIV). Statistical differences with respect to control (ANOVA followed by Bonferroni test): *p < 0.05, **p < 0.01. Data represent the mean ± SEM of 3-5 different cell culture preparations. Statistical differences with respect to non- ethanol-treated control (ANOVA followed by Bonferroni test): *p < 0.05, **p < 0.01.

KAINATE

200- c O & E

,~ 100 ¢i _=

m

a 0- el

--,-8 DIV --o- 12 DIV

; 5'0 1;0 1;0 2;0

Ethanol, [mM]

FIG. 3. Effects of increasing ethanol concentrations (25-200 raM) on [3H]PDBu binding, expressed as percentage above the baseline, induced by kainate (25 ~M) in cerebellar granule cells at different stages of development (2, 4, 8, and 12 DIV). Statistical differences with respect to control (ANOVA followed by Bonferroni test): *p < 0.05, **p < 0.01. Data represent the mean ± SEM of 4--6 different cell culture preparations. Statistical differences with respect to immature cells (2 DIV) (ANOVA followed by Bonferroni test): *p < 0.05, **p < 0.01.

578 BARRIOS AND LILJEQUIST

the kainic acid-produced enhancement of [3H]PDBu binding in cells grown for 2 and 4 DIV, respectively.

DISCUSSION

The most interesting observations in the present study were, first, that the enhancement of [3H]PDBu binding induced by NMDA was significantly blocked by 50 mM ethanol at 4 and 8 DIV, respectively, whereas a slightly weaker inhibition was seen after 2 and 12 DIV, and second, that the glutamate-induced stimulation of [3H]PDBu binding was the only glutamate receptor agonist response inhibited by a moderate concentration of ethanol (50 mM) at 2 DIV.

A prominent feature associated with glutamate receptor activation is a marked increase of [Ca2+]i which, among other things, has been shown to result in a redistribution of Ca2+/ phosphatidylserine-dependent PKC from the cytosol to the neuronal membrane. This translocation of PKC can be de- tected by phorbol ester binding or phoshorylation assays in cerebellar granule cells (28,41). We have recently reported that the magnitude of the enhancement of [3H]PDBu binding produced by various glutamate receptor agonists appears to be developmentally regulated in that the most pronounced stimulation was seen in cells cultured for 2 DIV followed by a gradually decrease of [3H]PDBu binding up to 12 DIV (3). Because, as already pointed out, the PKC translocation is considered to be a CaZ+dependent event, we discussed the puz- zling observation that available knowledge suggests that in particular the strength of NMDA receptor-mediated activation of functional Ca 2+ responses and neurotoxicity appear to develop in the opposite direction than the glutamate-induced stimulation of PKC translocation, that is, NMDA causes gradually increased Ca 2+ fluxes and neurotoxicity with increasing age (increased number of DIV) during early postnatal development of various types of cultured brain neurons (see also Introduction). As suggested by others, we considered the pos- sibility that the observed changes of glutamate-induced functional responses could be associated with developmentally determined variations in the molecular composition of various subclasses of glutamate receptors (5,20,32,33,35).

Accumulated evidence suggests that acute exposure to low and moderate concentrations of ethanol produces inhibition of many NMDA receptor functions in the brain. For example, using cerebellar granule cells it has been found that ethanol reduces NMDA receptor-induced Ca2 + fluxes, transcription factor activity, and neurotoxicity. Because accumulation of [Ca2+]i activates the intracellular enzyme PKC it is of interest to note that results from many studies indicate that ethanol and other alcohols may influence the activity and/or translocation of PKC (14,27,31,36,37,39). In this laboratory we have previously found that ethanol, in concentrations readily at- tainable during intoxication, inhibits NMDA-induced translocation of PKC in cerebellar granule cells after 8 DIV (27). We now extend those results by showing that the inhibitory effects of ethanol on PKC translocation vary during different developmental stages depending on both the subclass of glutamate receptors activated and/or the number of days in culture. Thus, we found that the NMDA-induced enhancement of [3H]PDBu binding was significantly inhibited by 50 mM ethanol at 4 and 8 DIV, with a smaller attenuation seen at 2 and 12 DIV, respectively. Furthermore, we noted that the only glutamate receptor agonist-induced response inhibited by ethanol (50 mM) at 2 DIV was the enhancement of [3H]PDBu binding caused by glutamate itself, whereas no cor- responding inhibition was seen at 2 DIV with any of the other

glutamate receptor agonists used in this study. These findings may imply that the ethanol-produced inhibition of the glutamate response at 2 DIV was mediated through a less dif- ferentiated glutamate receptor complex compared to the inhibitory actions of ethanol seen at 4, 8, and 12 DIV at which times also the effects of NMDA- and KA-induced PKC translocation were inhibited by ethanol. It should be mentioned that, in accordance with several previous reports, the inhibitory effects of ethanol on KA-induced responses were less pronounced than with the other glutamate receptor agonists.

Further studies will be needed to clarify to which extent the observed differences in the inhibitory effects of ethanol on glutamate-enhanced [3H]PDBu binding are related to developmentally determined differences in the molecular configu- ration of glutamate, and in particular NMDA, receptors. Our current observations may, however, be of some significance for the interpretation of earlier data concerning the effects of ethanol on developmental characteristics of cerebellar granule cells. For example, it was recently shown by Wegelius and Korpi (45) that continuous exposure to ethanol during the en- tire early phase of development (1-7 DIV) had no effects on NMDA receptor functions in cells grown under depolarized conditions, whereas ethanol inhibited the trophic actions of NMDA in nondepolarized cells. In contrast to the findings by Wegelius and Korpi, we and others have shown that chronic exposure to similar concentrations of ethanol, but starting after an initial period of 3-5 DIV, results in an increase of NMDA-induced neurotoxicity (10,13,25). Some findings suggest that this enhancement of NMDA neurotoxicity may be due to an enhancement of NMDA-produced accumulation of [Ca2+]i (6,24), although these data have not been generally confirmed (19). Because we now show that the sensitivity of NMDA/glutamate receptors varies during different developmental stages, it is clearly suggested that the pharmacological actions of ethanol may vary with the start and the duration of exposure during this critical period of development. In this context it is of interest to note that developmental changes in glutamate agonist-induced calcium signals have been observed also in cultured Purkinje neurons (18). Furthermore, because chronic ethanol exposure most likely alters the molecular composition of NMDA receptors, it seems reasonable to assume that postnatal exposure to ethanol could cause de- layed maturation and/or altered differentiation of certain glutamate receptor subunits, as, for example, seen in cells grown in depolarized vs. nondepolarized conditions (2,12,33). An altered composition of glutamate receptor subunits produced by chronic ethanol could certainly contribute to changes in glutamate-induced functional responses, that is, Ca ~+ fluxes, transcription factor activity, and/or neurotoxicity. However, this issue clearly needs further elucidation.

The teratogenic actions of ethanol are well established. Stimulation of protein kinase C activity has been considered to be crucially involved in processes associated with synaptogenesis, dendritic and axonal growth, activity-dependent plasticity, and long-term potentiation (4,8,11,43). The fact that ethanol concentrations that can be achieved in brain during intoxication produce significantly inhibitory actions on the translocation of PKC in brain neurons suggests that this mechanism may be of significance for ethanol-produced alter- ations of developmental processes.

ACKNOWLEDGEMENTS

This study was supported by the Swedish Medical Research Council (project No. 7688), The Swedish Foundation [or Alcohol Re-

E T H A N O L A N D PKC T R A N S L O C A T I O N 579

search, Sven and Ebba-Christina Hagbergs Foundation, Orion Pharma, Hans and Loo Ostermans Foundation, Ake Wibergs Foun- dation, and funds from the Karolinska Institute. M. B. was a Visiting Fellow from the Department of Pharmacology, University of Gran-

ada, Spain, and was supported by fellowships from the Ministerio de Asuntos Exteriores in Spain, the European Science Foundation, and the Human Frontier Science Program.

REFERENCES

1. Akazawa, C.; Shigemoto, R.; Bessho, Y.; Nakanishi, S.; Mizuno, N.: Differential expression of five N-methyl-D-aspartate receptor subunits mRNAs in the cerebellum of developing and adult rats. J. Comp. Neurol. 347:150--160; 1994.

2. BalMs, R.; Jorgensen, O. S.; Hack, N.: N-methyl-D-aspartate pro- motes the survival and differentiation of cerebellar granule cells in culture. Neuroscience 27:437451; 1988.

3. Barrios, M.; Liljequist, S.: Developmental changes in glutamate receptor-activated translocation of protein kinase C in cerebellar granule neurons. Dev. Brain Res. 94:22-30; 1996.

4. Ben-Ari, Y.; Aniksztejn, L.; Bregestowski, P.: Protein kinase C modulation of NMDA currents: An important link for LTP induction. Trends Neurosci. 15:333-339; 1992.

5. Bessho, Y.; Nawa, H.; Nakanishi, S.: Selective upregulation of an NMDA receptor subunit mRNA in cultured cerebellar granule cells by K÷-induced depolarization and NMDA treatment. Neu- ron 12:87-95; 1994.

6. Blevins, T.: Mirshahi, T.; Woodward, J. J.: Increased agonist and antagonist sensitivity of N-methyl-D-aspartate stimulated calcium flux in cultured neurons following chronic ethanol exposure. Neurosci. Lett. 200:214--218; 1995.

7. Burgoyne, R. D.; Cambray-Deakin, M. A,: The cellular neuro- biology of neuronal development: The cerebellar granule cell. Brain Res. Rev. 13:77-101; 1988.

8. Cambray-Deakin, M. A.; Adu, J.; Burgoyne, R. D.: Neuritogene- sis in cerebellar granule cells in vitro: A role for protein kinase C. Dev. Brain Res. 53:40-46; 1990.

9. Cebers, G.; Cebere, A.; Zharkovsky, A.; Liljequist, S.: Glycine does not reverse the inhibitory actions of ethanol on NMDA receptor functions in cerebellar granule cells. Naunyn Schmiede- bergs Arch. Pharmacol. 354:736-745; 1996.

lt/. Chandler, L. J.; Newsom, H.; Sumners, C.; Crews, F.: Chronic ethanol exposure potentiates NMDA excitotoxicity in cerebral cortical neurons. J. Neurochem. 60:1578-1581; 1993.

11. Clemens, M. J.; Trayner, I.; Menaya, J.: The role of protein kinase C isoenzymes in the regulation of cell proliferation and differentiation. J. Cell Sci. 103:881-887; 1992.

12. Condorelli, D. F.; Dell'Albani, P.; Aronica, E.; Genazzani, A. A.; Casabona, G.; Corsaro, M.; Bal/tzs, R.; Nicoletti, F.: Growth conditions differentially regulate the expression of a-amino-3-hydroxy- 5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured neurons. J. Neurochem. 61:2133-2139; 1993.

13. Davidson, M. D.; Wilce, W.; Shanley, B. C.: Increased sensitivity of the hippocampus in ethanol-dependent rats to toxic effect of N-methyl-D-aspartic acid in vivo. Brain Res. 606:5-9; 1993.

14. Deitrich, R. A.; Bludeau, P, A.; Baker, R. C.: Investigations of the role of protein kinase C in the acute sedative effects of ethanol. Alcohol. Clin. Exp. Res. 13:737-745; 1989.

15. Follesa, P.; Ticku, M. K.: Chronic ethanol treatment differentially regulates NMDA receptor subunit mRNA expression in rat brain. Mol. Brain Res. 29:99-106; 1995.

16. Frandsen, A.; Drejer, J.: Schousboe, A.: Development of excitatory amino acid dependent 45Ca2÷ uptake in cultured cerebral cortex neurons. Ann. NY Acad. Sci. 560:454-455; 1989,

17. Frandsen, A.; Schousboe, A.: Development of excitatory amino acid induced cytotoxicity in cultured neurons. Int. J. Dev. Neuro- sci. 8:209~ 16; 1990.

18. Gruol, D. L.; Curry, J. G.: Calcium signals elicited by quisqualate in cultured Purkinje neurons show developmental changes in sensitivity to acute alcohol. Brain Res. 673:1-12; 1995.

19. Gruol, D. L.; Parsons, K. L.: Chronic ethanol reduces calcium sig- naling elicited by glutamate receptor stimulation in developing cerebellar neurons. Brain Res. 728:166-174; 1996.

20. Hack, N. J.; Sluiter, A. A.; Balhzs, R.: AMPA receptors in cere-

bellar granule cells during development in culture. Dev. Brain Res. 87:55-61; 1995.

21. Hoffman, P. L.: Effects of alcohol on excitatory amino acid receptor function. In: Kranzler, H., ed. Handbook of experimental pharmacology. The pharmacology of alcohol abuse. Heidelberg: Springer-Verlag; 1995:75-102.

22. Hollmann, M.; Heinemann, S.: Cloned glutamate receptors. Annu. Rev. Neurosci. 17:31-108; 1994.

23. Hou, Y.-N.; Cebers, G.; Cebere, A.; Terenius~ L.; Liljequist, S.: N-methyl-D-aspartate-induced enhancement of activator protein- 1 DNA binding activity is blocked by ethanol in cerebellar granule cells. Eur. J. Pharmacol. 309:307-310; 1996.

24. Iorio, K. R.; Reinlib, L.; Tabakoff, B.: Hoffman, P. L.: Chronic exposure of cerebellar granule cells to ethanol results in increased N-methyl-D-aspartate receptor function. Mol. Pharma- col. 41:1142-1148; 1992.

25. Iorio, K. R.; Tabakoff, B.; Hoffman, P. L.: Glutamate-induced neurotoxicity is increased in cerebellar granule cells exposed chronically to ethanol. Eur. J. Pharmacol. Environ. Toxicol. Phar- macol. 248:209-212; 1993.

26. Levi, G.; Aloisi, F.; Ciott, M. T.; Gallo, V.: Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cells. Brain Res. 290: 77-86; 1984.

27. Liljequist, S.: Effect of ethanol on the translocation of PKC produced by glutamate receptor agonists in cerebellar granule cells. J. Neurochem. 61:$257; 1993.

28. Liljequist, S.; Vaccarino, F. M.; Guidotti, A.: Regulation of phorbol ester binding by putative excitatory and inhibitory amino acid transmitters in cerebellar granule cells in cnlture. Soc. Neurosci. Abstr. 12:264.12; 1986.

29. Lipton, S.; Kater, S. B.: Neurotransmitter regulation of neuronal outgrowth, plasticity, and survival. Trends Neurosci. 12: 265-270; 1988.

30. McDonald, J. W.; Johnston, M. V.: Physiological and pathophysi- ological roles of excitatory amino acids during central nervous system development. Brain Res. Rev. 15:41-70; 1990.

31. Messing, R. O.; Sneade, A. B.; Savidge, B.: Protein kinase C par- ticipates in upregulation of dihydropyridine-sensitive calcium channels by ethanol. J. Neurochem. 55:1383-1389; 1990.

32. Monyer, H.; Burnashev, N.; Laurie, D. J.; Sakmann, B.; Seeburg, P. H.: Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12: 529-540; 1994.

33. Resink, A.; Villa, M.; Benke, D.; Hidaka, H.; MOhler, H.; Bal/~zs. R.: Characterization of agonist-induced downregulation of NMDA receptors in cerebellar granule cell cultures. J. Neurochem. 66:369- 377; 1996.

34. Reynolds, J. D.; Brien, J. F.: Ethanol neurobehavioural teratoge- nesis and the role of L-glutamate in the fetal hippocampus. Can. J. Physiol. Pharmacol. 73:120%1223; 1995.

35. Riva, M. A.; Tascedda, F.; Molteni, R.; Racagni, G.: Regulation of NMDA receptor subunit mRNA expression in the rat brain during postnatal development. Mol. Brain Res. 25:209-216; 1994.

36. Skwish, S.; Shain, W.: Ethanol and diolein stimulate PKC translocation in astroglial cells. Life Sci. 47:1037-1042; 1990.

37. Slater, S. J.; Cox, K. J. A.; Lombardi, J. V,; Ho, C.; Kelly, M. B.; Rubin, E.; Stubbs, C. D.: Inhibition of protein kinase C by alcohols and anaesthetics. Nature 364:82-84; 1993.

38. Snell, L. D.; Nunley, K. R.; Lickteig, R. L.; Browning, M. D.; Tabakoff, B.; Hoffman, P. L.: Regional and subunit specific changes in NMDA receptor mRNA and immunoreactivity in mouse brain following chronic ethanol ingestion. Mol. Brain Res. 40:71-78; 1996.

580 B A R R I O S A N D L I L J E Q U I S T

39. Snell, Li D.; Tabakoff, B.; Hoffman, P. L.: Involvement of protein kinase C in ethanol-induced inhibition of NMDA receptor function in cerebellar granule cells. Alcohol. Clin. Exp. Res. 18:81-85; 1994.

40. Trevisan, L.; Fitzgerald, L. W.; Brose, N.; Gasic, G. P.; Heine- mann, S. F.; Duman, R. S.; Nestler, E. J.: Chronic ingestion of ethanol upregulates NMDAR1 receptor subunit immunoreactivity in rat hippocaInpus. J. Neurochem. 62:1635-1638; 1994.

41. Vaccarino, F.; Guidotti, A.; Costa, E.: Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons. Proc. Natl. Acad. Sci. USA 84: 8707-8711; 1987.

42. Vaccarino, F.; Liljequist, S.; Tallman, J. F.: Modulation of protein kinase C translocation by excitatory and inhibitory amino acids in primary cultures of neurons. J. Neurochem. 57:391-396; 1991.

43. Wakade, A. R.; Wakade, T. D.; Malhotra, R. K.; Bhave, S. J.: Excess K + and phorbol ester activate protein kinase C and sup- port the survival of chick sympathetic neurons in culture. J. Neu- rochem. 51:975-983; 1988.

44. Watanabe, M.; Mishina, M.; lnoue, Y.: Distinct spatiotemporal expressions of five NMDA receptor channel subunit mRNAs in the cerebellum. J. Comp. Neurol. 343:513-519; 1994.

45. Wegelius, K.; Korpi, E. R.: Ethanol inhibits NMDA-induced tox- icity and trophism in cultured cerebellar granule cells. Acta Phys- iol. Scan& 154:25-34; 1995.

46. Williams, K.; Russell, S. L.; Shen, Y. M.; Molinoff, P. B.: Devel- opmental switch in the expression of NMDA receptors occurs in vivo and in vitro. Neuron 10:267-278; 1993.

developmental changes in the inhibitory actions of ethanol on glutamate-induced translocation of...

Documents