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An NMDA receptor antagonist reduces ethanol preference in untrained but not trained rats
Brain Research Bulletin, Vol. 36, No. 5, pp. 421-424, 1995 Copyright © 1995 ElsevierScienceLtd Printed in the USA. All rights reserved 0361-9230/95 $9.50 + .00
Pergamon 0361-9230(94)00215-0
An NMDA Receptor Antagonist Reduces Ethanol Preference in Untrained But Not Trained Rats NAILI LIN AND JOHN I. HUBBARD 1
Department of Physiology and Centre for Neuroscience, University of Otago Medical School, Dunedin, New Zealand [Received 3 December 1993; Accepted 16 August 1994] ABSTRACT: Rats were exposed to a potent antagonist of NMDA receptors, 2-amino-5 phosphonovalerate (AP5) by implanting osmotic pumps delivering AP5 (test group) or the vehicle for AP5 (control group) into the cerebrospinal fluid through previously implanted cannulas. A week later they were given the choice of 6 % ethanol or water for I h. The control group had a significantly greater preference for ethanol than the test group after the first 5 days. Another group of rats was trained almost to a 100% criterion in preferring ethanol to water in the hour the choice was available. Then they were implanted with osmotic pumps and cannulas and administered AP5. These trained rats showed no significant difference between control and test groups in preference for ethanol after the first 5 days. The results support the hypothesis that activation of NMDA receptors is involved in learning to drink ethanol.
semipermanent change in synaptic properties manifest as learning a new preference. It is known already that if rats with water and ethanol available are stimulated electrically in the median forebrain bundle for 30 min daily for 30 days, then their preference for ethanol, in previously aversive concentrations, is significantly increased [1]. The increase in preference appears to be permanent [ 1] and its production does not depend on the presence of ethanol during stimulation [2]. LTP is thought to be the neural basis of many forms of learning. In many cases, this LTP is generated by excitation of the N M D A group of glutamate receptors [3,4,7,8,12]. The drug (_+)2-amino-5-phosphonovaleric acid (AP5) is a selective antagonist of N M D A receptors and we report here the effect of AP5, in concentrations known to block LTP in the hippocampus [20], on ethanol preference under two circumstances. First, when a group of rats was learning a new task, i.e., to drink 6% ethanol in the 1 h of the 24 that it was available (untrained group). Second, on a group of rats previously trained to drink 6% ethanol during the hour (trained group).
KEY WORDS: Ethanol, Rat, 2-Amino-5 phosphonovalerate, Preference, Memory.
INTRODUCTION
METHOD
Progress in understanding the problem of alcohol addiction has been slow. In part, we suggest this is because in animal studies there has been a concentration on the search for a model in which the attainment of a high blood alcohol was the sine qua non of the research. It may be more profitable to concentrate on the beginning of the process which, we consider, begins with the development for a preference for ethanol over other fluids. Indeed, there is ample evidence in rat models that ethanol excites the signalling mechanisms in brain pathways concerned with reward and pleasure. The neural basis of psychomotor stimulant and opiate reward in rats has been known for some time to be the ventral tegmental dopamine system (A 10) with cell bodies in the ventral tegmentum and axon terminals passing through the median forebrain bundle to the nucleus accumbens [5,26]. There is evidence in rats that the same system serves the rewarding function of ethanol ingestion [17]. For instance, the A10 group of dopamine containing cells of conscious, but not anesthetised, rats are known to be excited by IV ethanol in a dose-related fashion [11]. Repetitive activity, produced by effects of ethanol or its metabolites, could induce long-term potentiation (LTP) at an as-yet undetermined site which, in turn, would induce the
Animals
Male S p r a g u e - D a w l e y rats weighing 300 g at the beginning of the experiment were used. They were kept in individual cages at a temperature of 24 ± 2°C and fed an ad lib diet. Unless otherwise noted, they were kept on a 12 L:12 D cycle in which the dark cycle began at 0900 h and drinking of water and ethanol was measured after 1 h of the dark cycle, i.e., at 1000 h. Food, water, and ethanol were initially available ad lib. Rats were given the choice of three tubes, one filled with water, one with the concentration of ethanol being tested on that day ( 3 6%), and one empty. The position of the tubes was varied randomly each day to avoid the setting up of a position habit [22]. The ethanol concentration (wt/volume) was 3% for 4 days, then 5% for 4 days, and finally 6% for 5 days. Untrained Rats
The rats were divided randomly into test (n = 10) and control groups (n = 6). During the 5 days with access to 6% ethanol the control group drank 34.87 ___ 1.34 ml/day of ethanol and water including 1.05 _ 0.14 g/kg ethanol and the test group 34.12 ±
Requests for reprints should be addressed to Prof. J. I. Hubbard, Physiology Department, Medical School, P.O. Box 913, Dunedin, New Zealand. 421
422
1.04 ml/day including 1.35 _+ 0.12 g/kg ethanol. Neither the total volumes nor the amounts of ethanol/kg were significantly different between the two groups (volume t = 0.4359, 8 df, p = 0.67; ethanol t = 1.58, 8 df, p = 0.1525). IVT cannulas were then implanted. One week after operation the rats, which postoperatively had been on water and food ad lib, were again given access to 6% ethanol but only for 1 h daily, for 20 days.
Trained Rats The training was initially the same as the untrained rats except that these rats spent 2 weeks with 6% ethanol. Rats were then allotted randomly to test (n = 9) and control groups (n = 8). Food and water were still available for 24 h but 6% ethanol was only provided for 1 h, from the beginning of the dark cycle. This training continued for 24 days. Then, IVT cannulas were implanted. One week after operation the animals were again given access to 6% ethanol for 1 h in the same manner as before, for 15 days. For both groups the water and ethanol consumption in the hour was recorded daily at the same time each day and the rats' weights were also recorded. Preference was measured as the ratio of the volume of ethanol consumed in the hour to the total volume of fluid consumed (ethanol and water) in the hour.
Drug Delivery AP5 was infused IVT from an osmotic pump (Model 2002, mean pumping rate, at 37°C, 0.48 + 0.02 #l/h, Alza Corporation, Palo Alto, CA). Rats were anesthetized with Equithesin (3 ml/ kg) and IVT cannulas were implanted (coordinates P, 0.7; L, 1.6; H, 4.0) and connected to osmotic pumps placed under the skin of the neck. The pumps contained either AP5 dissolved in 0.9% saline to give a final concentration of 40 nmol (test group), or 0.9% saline (control group). These solutions were sterilized by passing them through a m e m b r a n e filter (Millex G V 0.22 # m filter unit). At the conclusion of the experiment all rats were deeply anesthetized with Equithesin IP and the pump and connection with the cannula were exposed and checked for continuity and patency. A 10#1 injection of methylene blue was then given through the connecting tube and cannula to fill the CSF with dye. The rats were then perfused intracardially with normal saline followed by 10% formol saline. Their brains were removed and hardened in formol saline for 2 - 3 days. Coronal sections (50 # m ) were then cut with a freezing microtome for histological verification of the cannula placements.
Statistics The initial training data of the untrained group, when the animals were drinking 6% ethanol (mean daily volume and mean ethanol consumption, corrected for weight of the rats) were compared using a t-test. For later comparisons, the data (water and ethanol consumption and preference) were not normally distributed. Moreover, preference is a ratio and cannot, therefore, be used untransformed in an A N O V A [27]. Zar [27] recommends transformation to the arcsine. Our data after this conversion were still markedly skew so nonparametric methods were used for all data. First, K r u s k a l - W a l l i s tests (H) were used to examine whether there was a significant difference between the measured data in a 5-day period. If the measured data from a 5-day period were not significantly different by days, the data from those days were combined. M a n n - W h i t n e y tests, corrected for ties (U), were then used with the combined data to compare test and control results, p for significance was set at 0.05.
LIN AND H U B B A R D
RESULTS
Preference Untrained rats in the presence of AP5. W h e n the control and test groups were compared with each other, there was no significant difference in the first 5-day period, but in the following three periods preference for ethanol was significantly lower in the test group (Fig. 1). Trained rats. In the pre-AP5 period as a result of training, there was a very high preference for ethanol in the last 3 of the 5-day periods, shown in Fig. 2A, Indeed, in one of these 5-day periods all rats in the control group drank only ethanol during the daily hour exposure (Fig. 2A). There was no significant difference in mean preference between the control and the test group in any of these periods. In the presence of, and immediately after AP5, when the test and control groups were again compared, there was a significant difference in the first 5-day period but there were no significant differences in later periods (Fig. 2B). Total Drunk Untrained group. The total volume consumed/kg on each day of a 5-day period was not significantly different for any group except for day 8 in the second 5-day period. On this day, presumably at the height of the AP5 effect, there was a significant fall in the total volume drunk by the test group compared with the other 4 days (p = 0.03). On the other 4 days there was no difference between test and control groups in the amount drunk. Actual volumes (ml) in successive 5-day periods were control 3.41 _+ 0.34, 4.2 _+ 0.37, 4.34 _+ 0.33, 4.43 + 0.37; test 3.87 _+ 0.27, 3.36 _+ 0.24, 3.36 _+ 0.27, 4.71 _+ 0.30. Trained group. W h e n test and control groups were compared, except for the 11 - 15-day period predrug w h e n the control group d r a n k significantly more (p = 0.013), there was no 1.2" t
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successive 5 day periods FIG. 1. The effect of AP5 on ethanol preference in an untrained group of rats learning to drink 6% ethanol in a l-h period of availability. The height of the columns indicates the mean preference + 1 SE of the control (white) and test group (black dots)• The numbering on the horizontal axis indicates successive 5-day periods• Black stars above the test group columns for the second, third, and fourth periods of 5 days indicate significant differences (p < 0.05) from the control group during those periods•
N M D A A N T A G O N I S T R E D U C E S P R E F E R E N C E FOR E T H A N O L A
DISCUSSION
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FIG. 2. The effect of AP5 on ethanol preference in a group of rats that had previously learned to drink 6% ethanol in a 1-hour period of availability. (A) Preference in the last three 5-day periods of 24 days training preexposure to AP5. (B) Preference in the same rats during and after exposure to AP5. The height of the columns indicates the mean preference + 1 SE of the control (white) and test group (black dots). The numbering on the horizontal axis indicates successive 5-day periods. In B, the duration of AP5 infusion is indicated by the horizontal black bar at the top of the figure. The black star above the test group column for the first period of 5 days in B indicates a significant difference (p < 0.05) from the control group.
significant difference b e t w e e n the m e a n s of the test a n d control groups in any period in the p r e - A P 5 , AP5, or p o s t - A P 5 conditions. A c t u a l v o l u m e s (ml) in s u c c e s s i v e 5-day periods were, pre-AP5: control 3.88 _ 0.33, 3.16 ± 0.27, 4.3 ± 0.3; test 4.27 ___ 0.29, 3.47 ± 0.22, 3.57 ± 0.24; d u r i n g and after AP5: control 2.69 ___ 0.32, 3.84 ± 0.31, 4.5 ± 0.3, test 2.86 ___ 0.36, 4.21 ± 0.93, 3.97 ___ 0.4.
Our results (Fig. 1) show that in the p r e s e n c e of AP5, preference for ethanol, available for 1 h, is depressed. One possible mechanism for the inhibitory action of AP5 on ethanol preference (Fig. 1) is that it disrupts learning of the ability to drink ethanol in the hour of availability. An alternative explanation is that N M D A receptors are concerned with some other function. Our second experiment was designed to answer this question. In this experiment with rats trained to drink ethanol during the hour of availability (Fig. 2A), we found that AP5 no longer produced a prolonged drop in ethanol preference (Fig. 2B). This is easily explained by the lack of any learning to be disrupted by AP5, as learning had already occurred and is difficult to explain in terms of some other essential function. It is interesting to consider the m e c h a n i s m by which AP5 impaired learning in our experiments and the type of learning impaired. In regard to the type of learning impaired, there appears to be agreement that AP5 disrupts working memory, that is memory required for the duration of a session rather than reference memory, defined as information that is needed across all sessions [23,24]. There is also good evidence that working memory requires both the integrity of the hippocampus [15,21,24] and the concurrent expression of long-term potentiation by hippocampal neurons [6,10]. It is, thus, probable that ethanol activated hippocampal neurons in our experiments. The mechanism by which ethanol activates hippocampal neurons is currently unknown. It is unlikely to be a direct effect on N M D A receptors for most investigators [ 13,14,18,19,25], though not all [16] report that in isolated preparations ethanol inhibits the excitation of glutamate receptors by N M D A . It is more likely that an indirect effect, perhaps involving the A10 group of dopamine containing cells, which are k n o w n to be excited by IV ethanol in vivo, in a dose-related fashion [11], and which have axons that travel in the medial forebrain bundle, provides the excitation that finally impinges on the hippocampal N M D A receptors. ACKNOWLEDGEMENTS The work was performed with the aid of grants from the Health Research Council of New Zealand and the Bequest funds of the University of Otago Medical School. We thank Dr. C. Phillips of Vancouver for his helpful discussion of our initial findings and Prof. C. Burns for her careful review of the manuscript.
REFERENCES 1. Amit, Z.; Stern, M. H.; Wise, R. A. Alcohol preference in the laboratory rat induced by hypothalamic stimulation. Psychopharmacology (Berlin) 17:367-377; 1970. 2. Amit, Z.; Stern, M. H. A further examination of alcohol preference in the laboratory rat induced by hypothalamic stimulation. Psychopharmacology (Berlin) 21:317-327; 1971. 3. Bliss, T. V. P.; Gardner-Medwin, A. R. Long-lasting potentiation of synaptic transmission in the dentate area of the unanaesthetised rabbit following stimulation of the perforant path. J. Physiol. 232:357-374; 1973. 4. Bliss, T. V. P.; Lomo, T. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetised rabbit following stimulation of the perforant path. J. Physiol. 232:331-356; 1973. 5. Bozarth, M. A. Neural basis of psychomotor stimulant and opiate reward: Evidence suggesting the involvement of a common dopaminergic system. Behav. Brain Res. 22:107-116; 1986. 6. Butcher, S. P.; Davis, S.; Morris, R. G. M. A dose-related impairment of spatial learning by the NMDA receptor antagonist, 2-amino5-phosphonovalerate (AP5). Eur. Neuropsychopharmacol. 1: 15-20; 1990.
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7. Collingridge, G. L.; Bliss, T. V. P. NMDA receptors--Their role in long-term potentiation. Trends Neurosci. 10:288- 293; 1987. 8. Collingridge, G. L.; Kehl, S. J.; McLennan, H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. (Lond.) 334:33-46; 1984. 9. Davis, H. P.; Tfibuna, J.; Pulsinelli, W. A.; Volpe, B. T. Reference and working memory of rats following hippocampal damage induced by transient forebrain ischemia. Physiol. Behav. 37:387-392; 1986. 10. Davis, S.; Butcher, S. P.; Morris, R. G. M. The NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) impairs spatial learning and LTP in vivo at intmcerebral concentrations comparable to those that block LTP in vitro. J. Neurosci. 12:21-34; 1992. 11. Gessa, G. L.; Muntoni, F.; Collu, M.; Vargiu, L.; Mereu, G. Low doses of ethanol activate dopaminergic neurons in the ventral tegmental area. Brain Res. 348:201-203; 1985. 12. Harris, E. W.; Ganong, A. H.; Cotman, C. W. Long-term potentiation in the hippocampus involves activation of N-methyl-o-aspartate receptors. Brain Res. 323:132-137; 1984. 13. Hoffman, P. L.; Moses, F.; Tabakoff, B. Selective inhibition by ethanol of glutamate-stimulated cyclic GMP production in primary cultures of cerebellar granule cells. Neuropharmacology 28:1239-1243; 1989. 14. Hoffman, P. L.; Rabe, C. S.; Moses, F.; Tabakoff, B. N-methyl-Daspartate receptors and ethanol inhibition of calcium flux and cyclic GMP production. J. Neurochem. 52:1937-1940; 1989. 15. Jarrard, L. E. Selective hippocampal lesions and behaviour, Physiol. Psychol. 8:198-206; 1980. 16. Lima-Landman, M. T. R.; Albuquerque, E. X. Ethanol potentiates and blocks NMDA-activated single currents in rat hippocampal pyramidal cells. FEBS Lett. 247:61-67; 1989. 17. Lin, N.; Hubbard, J. I. The increased ethanol preference in rats induced by choice, darkness or drugs is reduced by ritanserin. Brain Res. Bull. 33:633-638; 1993.
LIN A N D H U B B A R D
18. Lovinger, D. M.; White, G.; Weight, F. F. Ethanol inhibits NMDAactivated ion current in hippocampal neurons. Science 243:17211724; 1989. 19. Lovinger, D. M.; White, G.; Weight, F. F. NMDA receptor-mediated synaptic excitation selectively inhibited by ethanol in hippocampal slice from adult rat. J. Neurosci. 10:1372-1379; 1990. 20. Morris, R. G. M.; Anderson, E.; Lynch, G. S.; Baudry, M. Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 319:774776; 1986. 21. Morris, R. G. M.; Garrud, P.; Rawlins, J. N. P.; O'Keefe, J. Place navigation impaired in rats with hippocampal lesions. Nature 297:681-683; 1982. 22. Myers, R. D. Methods for chemical stimulation of the brain. In: Myers, R. D., ed. Methods in psychobiology, vol 1. London: Academic Press; 1971:247-280. 23. Ohno, M.; Yamamoto, T.; Watanabe, S. Effects of intrahippocampal injections of N-methyl-D-aspartate receptor antagonists and scopolamine on working and reference memory assessed in rats by a three-panel runway task. J. Pharmacol. Exp. Ther. 263(3):943-950; 1992. 24. Olton, D. S.; Papas, B. C. Spatial memory and hippocampal function. Neuropsycbologia 17:669-682; 1979. 25. White, G.; Lovinger, D. M.; Weight, F. F. Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron. Brain Res. 507:332336; 1990. 26. Wise, R. A.; Bozarth, M. A. Brain reward circuitry: Four circuit elements "wired" in apparent series. Brain Res. Bull. 12:203-308; 1984. 27. Zar, J. H. Biostatistical analysis, second ed. Englewood Cliffs, NJ: Prentice Hall; 1984.
Pergamon 0361-9230(94)00215-0
An NMDA Receptor Antagonist Reduces Ethanol Preference in Untrained But Not Trained Rats NAILI LIN AND JOHN I. HUBBARD 1
Department of Physiology and Centre for Neuroscience, University of Otago Medical School, Dunedin, New Zealand [Received 3 December 1993; Accepted 16 August 1994] ABSTRACT: Rats were exposed to a potent antagonist of NMDA receptors, 2-amino-5 phosphonovalerate (AP5) by implanting osmotic pumps delivering AP5 (test group) or the vehicle for AP5 (control group) into the cerebrospinal fluid through previously implanted cannulas. A week later they were given the choice of 6 % ethanol or water for I h. The control group had a significantly greater preference for ethanol than the test group after the first 5 days. Another group of rats was trained almost to a 100% criterion in preferring ethanol to water in the hour the choice was available. Then they were implanted with osmotic pumps and cannulas and administered AP5. These trained rats showed no significant difference between control and test groups in preference for ethanol after the first 5 days. The results support the hypothesis that activation of NMDA receptors is involved in learning to drink ethanol.
semipermanent change in synaptic properties manifest as learning a new preference. It is known already that if rats with water and ethanol available are stimulated electrically in the median forebrain bundle for 30 min daily for 30 days, then their preference for ethanol, in previously aversive concentrations, is significantly increased [1]. The increase in preference appears to be permanent [ 1] and its production does not depend on the presence of ethanol during stimulation [2]. LTP is thought to be the neural basis of many forms of learning. In many cases, this LTP is generated by excitation of the N M D A group of glutamate receptors [3,4,7,8,12]. The drug (_+)2-amino-5-phosphonovaleric acid (AP5) is a selective antagonist of N M D A receptors and we report here the effect of AP5, in concentrations known to block LTP in the hippocampus [20], on ethanol preference under two circumstances. First, when a group of rats was learning a new task, i.e., to drink 6% ethanol in the 1 h of the 24 that it was available (untrained group). Second, on a group of rats previously trained to drink 6% ethanol during the hour (trained group).
KEY WORDS: Ethanol, Rat, 2-Amino-5 phosphonovalerate, Preference, Memory.
INTRODUCTION
METHOD
Progress in understanding the problem of alcohol addiction has been slow. In part, we suggest this is because in animal studies there has been a concentration on the search for a model in which the attainment of a high blood alcohol was the sine qua non of the research. It may be more profitable to concentrate on the beginning of the process which, we consider, begins with the development for a preference for ethanol over other fluids. Indeed, there is ample evidence in rat models that ethanol excites the signalling mechanisms in brain pathways concerned with reward and pleasure. The neural basis of psychomotor stimulant and opiate reward in rats has been known for some time to be the ventral tegmental dopamine system (A 10) with cell bodies in the ventral tegmentum and axon terminals passing through the median forebrain bundle to the nucleus accumbens [5,26]. There is evidence in rats that the same system serves the rewarding function of ethanol ingestion [17]. For instance, the A10 group of dopamine containing cells of conscious, but not anesthetised, rats are known to be excited by IV ethanol in a dose-related fashion [11]. Repetitive activity, produced by effects of ethanol or its metabolites, could induce long-term potentiation (LTP) at an as-yet undetermined site which, in turn, would induce the
Animals
Male S p r a g u e - D a w l e y rats weighing 300 g at the beginning of the experiment were used. They were kept in individual cages at a temperature of 24 ± 2°C and fed an ad lib diet. Unless otherwise noted, they were kept on a 12 L:12 D cycle in which the dark cycle began at 0900 h and drinking of water and ethanol was measured after 1 h of the dark cycle, i.e., at 1000 h. Food, water, and ethanol were initially available ad lib. Rats were given the choice of three tubes, one filled with water, one with the concentration of ethanol being tested on that day ( 3 6%), and one empty. The position of the tubes was varied randomly each day to avoid the setting up of a position habit [22]. The ethanol concentration (wt/volume) was 3% for 4 days, then 5% for 4 days, and finally 6% for 5 days. Untrained Rats
The rats were divided randomly into test (n = 10) and control groups (n = 6). During the 5 days with access to 6% ethanol the control group drank 34.87 ___ 1.34 ml/day of ethanol and water including 1.05 _ 0.14 g/kg ethanol and the test group 34.12 ±
Requests for reprints should be addressed to Prof. J. I. Hubbard, Physiology Department, Medical School, P.O. Box 913, Dunedin, New Zealand. 421
422
1.04 ml/day including 1.35 _+ 0.12 g/kg ethanol. Neither the total volumes nor the amounts of ethanol/kg were significantly different between the two groups (volume t = 0.4359, 8 df, p = 0.67; ethanol t = 1.58, 8 df, p = 0.1525). IVT cannulas were then implanted. One week after operation the rats, which postoperatively had been on water and food ad lib, were again given access to 6% ethanol but only for 1 h daily, for 20 days.
Trained Rats The training was initially the same as the untrained rats except that these rats spent 2 weeks with 6% ethanol. Rats were then allotted randomly to test (n = 9) and control groups (n = 8). Food and water were still available for 24 h but 6% ethanol was only provided for 1 h, from the beginning of the dark cycle. This training continued for 24 days. Then, IVT cannulas were implanted. One week after operation the animals were again given access to 6% ethanol for 1 h in the same manner as before, for 15 days. For both groups the water and ethanol consumption in the hour was recorded daily at the same time each day and the rats' weights were also recorded. Preference was measured as the ratio of the volume of ethanol consumed in the hour to the total volume of fluid consumed (ethanol and water) in the hour.
Drug Delivery AP5 was infused IVT from an osmotic pump (Model 2002, mean pumping rate, at 37°C, 0.48 + 0.02 #l/h, Alza Corporation, Palo Alto, CA). Rats were anesthetized with Equithesin (3 ml/ kg) and IVT cannulas were implanted (coordinates P, 0.7; L, 1.6; H, 4.0) and connected to osmotic pumps placed under the skin of the neck. The pumps contained either AP5 dissolved in 0.9% saline to give a final concentration of 40 nmol (test group), or 0.9% saline (control group). These solutions were sterilized by passing them through a m e m b r a n e filter (Millex G V 0.22 # m filter unit). At the conclusion of the experiment all rats were deeply anesthetized with Equithesin IP and the pump and connection with the cannula were exposed and checked for continuity and patency. A 10#1 injection of methylene blue was then given through the connecting tube and cannula to fill the CSF with dye. The rats were then perfused intracardially with normal saline followed by 10% formol saline. Their brains were removed and hardened in formol saline for 2 - 3 days. Coronal sections (50 # m ) were then cut with a freezing microtome for histological verification of the cannula placements.
Statistics The initial training data of the untrained group, when the animals were drinking 6% ethanol (mean daily volume and mean ethanol consumption, corrected for weight of the rats) were compared using a t-test. For later comparisons, the data (water and ethanol consumption and preference) were not normally distributed. Moreover, preference is a ratio and cannot, therefore, be used untransformed in an A N O V A [27]. Zar [27] recommends transformation to the arcsine. Our data after this conversion were still markedly skew so nonparametric methods were used for all data. First, K r u s k a l - W a l l i s tests (H) were used to examine whether there was a significant difference between the measured data in a 5-day period. If the measured data from a 5-day period were not significantly different by days, the data from those days were combined. M a n n - W h i t n e y tests, corrected for ties (U), were then used with the combined data to compare test and control results, p for significance was set at 0.05.
LIN AND H U B B A R D
RESULTS
Preference Untrained rats in the presence of AP5. W h e n the control and test groups were compared with each other, there was no significant difference in the first 5-day period, but in the following three periods preference for ethanol was significantly lower in the test group (Fig. 1). Trained rats. In the pre-AP5 period as a result of training, there was a very high preference for ethanol in the last 3 of the 5-day periods, shown in Fig. 2A, Indeed, in one of these 5-day periods all rats in the control group drank only ethanol during the daily hour exposure (Fig. 2A). There was no significant difference in mean preference between the control and the test group in any of these periods. In the presence of, and immediately after AP5, when the test and control groups were again compared, there was a significant difference in the first 5-day period but there were no significant differences in later periods (Fig. 2B). Total Drunk Untrained group. The total volume consumed/kg on each day of a 5-day period was not significantly different for any group except for day 8 in the second 5-day period. On this day, presumably at the height of the AP5 effect, there was a significant fall in the total volume drunk by the test group compared with the other 4 days (p = 0.03). On the other 4 days there was no difference between test and control groups in the amount drunk. Actual volumes (ml) in successive 5-day periods were control 3.41 _+ 0.34, 4.2 _+ 0.37, 4.34 _+ 0.33, 4.43 + 0.37; test 3.87 _+ 0.27, 3.36 _+ 0.24, 3.36 _+ 0.27, 4.71 _+ 0.30. Trained group. W h e n test and control groups were compared, except for the 11 - 15-day period predrug w h e n the control group d r a n k significantly more (p = 0.013), there was no 1.2" t
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N M D A A N T A G O N I S T R E D U C E S P R E F E R E N C E FOR E T H A N O L A
DISCUSSION
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FIG. 2. The effect of AP5 on ethanol preference in a group of rats that had previously learned to drink 6% ethanol in a 1-hour period of availability. (A) Preference in the last three 5-day periods of 24 days training preexposure to AP5. (B) Preference in the same rats during and after exposure to AP5. The height of the columns indicates the mean preference + 1 SE of the control (white) and test group (black dots). The numbering on the horizontal axis indicates successive 5-day periods. In B, the duration of AP5 infusion is indicated by the horizontal black bar at the top of the figure. The black star above the test group column for the first period of 5 days in B indicates a significant difference (p < 0.05) from the control group.
significant difference b e t w e e n the m e a n s of the test a n d control groups in any period in the p r e - A P 5 , AP5, or p o s t - A P 5 conditions. A c t u a l v o l u m e s (ml) in s u c c e s s i v e 5-day periods were, pre-AP5: control 3.88 _ 0.33, 3.16 ± 0.27, 4.3 ± 0.3; test 4.27 ___ 0.29, 3.47 ± 0.22, 3.57 ± 0.24; d u r i n g and after AP5: control 2.69 ___ 0.32, 3.84 ± 0.31, 4.5 ± 0.3, test 2.86 ___ 0.36, 4.21 ± 0.93, 3.97 ___ 0.4.
Our results (Fig. 1) show that in the p r e s e n c e of AP5, preference for ethanol, available for 1 h, is depressed. One possible mechanism for the inhibitory action of AP5 on ethanol preference (Fig. 1) is that it disrupts learning of the ability to drink ethanol in the hour of availability. An alternative explanation is that N M D A receptors are concerned with some other function. Our second experiment was designed to answer this question. In this experiment with rats trained to drink ethanol during the hour of availability (Fig. 2A), we found that AP5 no longer produced a prolonged drop in ethanol preference (Fig. 2B). This is easily explained by the lack of any learning to be disrupted by AP5, as learning had already occurred and is difficult to explain in terms of some other essential function. It is interesting to consider the m e c h a n i s m by which AP5 impaired learning in our experiments and the type of learning impaired. In regard to the type of learning impaired, there appears to be agreement that AP5 disrupts working memory, that is memory required for the duration of a session rather than reference memory, defined as information that is needed across all sessions [23,24]. There is also good evidence that working memory requires both the integrity of the hippocampus [15,21,24] and the concurrent expression of long-term potentiation by hippocampal neurons [6,10]. It is, thus, probable that ethanol activated hippocampal neurons in our experiments. The mechanism by which ethanol activates hippocampal neurons is currently unknown. It is unlikely to be a direct effect on N M D A receptors for most investigators [ 13,14,18,19,25], though not all [16] report that in isolated preparations ethanol inhibits the excitation of glutamate receptors by N M D A . It is more likely that an indirect effect, perhaps involving the A10 group of dopamine containing cells, which are k n o w n to be excited by IV ethanol in vivo, in a dose-related fashion [11], and which have axons that travel in the medial forebrain bundle, provides the excitation that finally impinges on the hippocampal N M D A receptors. ACKNOWLEDGEMENTS The work was performed with the aid of grants from the Health Research Council of New Zealand and the Bequest funds of the University of Otago Medical School. We thank Dr. C. Phillips of Vancouver for his helpful discussion of our initial findings and Prof. C. Burns for her careful review of the manuscript.
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