- Email: [email protected]
GABAA receptor-mediated inhibition by ethanol of long-term potentiation in the basolateral amygdala–dentate gyrus pathway in vivo
Neuroscience 125 (2004) 113–117
GABAA RECEPTOR-MEDIATED INHIBITION BY ETHANOL OF LONGTERM POTENTIATION IN THE BASOLATERAL AMYGDALA–DENTATE GYRUS PATHWAY IN VIVO K. ABE,* Y. NIIKURA AND M. MISAWA
DG field potentials (Abe et al., 2003), demonstrating that the BLA-DG pathway displays synaptic plasticity. This novel form of LTP may represent a synaptic mechanism underlying the formation of memory associated with emotional experiences. Acute ethanol intoxication causes impairment of memory and other cognitive functions in humans. Although behavioral studies with experimental animals have demonstrated that acute administration of ethanol impairs hippocampal-dependent working memory (Givens, 1995; Melchior et al., 1993; Zhang et al., 1994), cellular mechanisms are not fully understood. As a possible mechanism underlying ethanol-induced memory impairments, ethanol has been reported to inhibit the induction of LTP in hippocampal CA1 or DG synapses in vitro (Blitzer et al., 1990; Morrisett and Swartzwelder, 1993; Sinclair and Lo, 1986; Sugiura et al., 1995) and in vivo (Abe et al., 1999b; Sugiura et al., 1994). However, very little is known about the effect of ethanol on LTP in other brain regions. Furthermore, acute administration of ethanol has been shown to impair emotion-associated learning and memory, including passive avoidance learning (Zhang et al., 1994; Castellano and Pavone, 1988; Nabeshima et al., 1988) and fear conditioning (Gould, 2003; Melia et al., 1996; Sanger and Joly, 1986). Therefore, in the present study, we investigated the effect of ethanol on the induction of LTP in the BLA-DG pathway by using anesthetized rats. Furthermore, it has been proposed that ethanol impairs the induction of hippocampal LTP by inhibiting N-methyl-D-aspartate (NMDA) type of glutamate receptors or by potentiating GABAA receptor-mediated inhibitory mechanisms (Blitzer et al., 1990; Morrisett and Swartzwelder, 1993; Schummers and Browning, 2001). To explore mechanisms underlying the effect of ethanol on LTP in the BLA-DG pathway, possible involvement of the NMDA receptor or the GABAA receptor was also investigated.
Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
Abstract—Although ethanol has been reported to inhibit the induction of long-term potentiation in hippocampal CA1 and dentate gyrus synapses of rats, very little is known about the effect of ethanol on synaptic plasticity in other brain regions. Therefore, in the present study, we investigated the effect of ethanol on long-term potentiation in synaptic pathway from the basolateral amygdala to the dentate gyrus by using anesthetized rats in vivo. I.v. (20 – 40%ⴛ2 ml/kg) or i.c.v. (30 – 40%ⴛ5 l) administration of ethanol did not affect the basal amplitude of dentate gyrus field potential evoked by basolateral amygdala stimulation, but significantly inhibited the induction of long-term potentiation following application of tetanic stimulation. Since long-term potentiation in this pathway was independent of N-methyl-D-aspartate receptors, the inhibitory effect of ethanol is unlikely to be caused by suppression of N-methyl-D-aspartate receptor function. Alternatively, long-term potentiation in this pathway was significantly suppressed by the benzodiazepine agonist diazepam (2 mg/kg, i.p.), and the inhibitory effect of ethanol was abolished by the GABAA receptor channel blocker picrotoxin (1 mg/kg, i.p.). The present study demonstrates that ethanol inhibits the induction of long-term potentiation in the basolateral amygdala– dentate gyrus pathway by enhancing GABAA receptormediated neurotransmission. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: alcohol, emotional memory, synaptic plasticity, amygdala, hippocampus.
Emotion and memory are closely related. Among brain regions, the amygdala is involved in emotional and motivational aspects of behavior, and the hippocampus is crucially involved in the formation of memory. The interaction between the amygdala and hippocampus may be a key to elucidating neural mechanisms linking emotion and memory. We have previously found that that electrical stimulation of the basolateral amygdala (BLA) evokes field potentials in the dentate gyrus (DG) of the hippocampus in anesthetized rats (Ikegaya et al., 1996), demonstrating that there is neural connection from the BLA to the DG. Furthermore, application of tetanic stimulation to the BLA produced long-term potentiation (LTP) of the BLA-evoked
EXPERIMENTAL PROCEDURES 2-Amino-5-phosphonovalerate (APV) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other drugs were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All drugs were dissolved in saline. Evoked potential in the BLA-DG pathway was recorded as described previously (Ikegaya et al., 1996; Abe et al., 2003). Briefly, male Wistar rats (7–9 weeks old, 200 –300 g) were anesthetized with urethane (1 g/kg, i.p.) and ␣-chloralose (25 mg/kg, i.p.) and fixed in a stereotaxic frame. For i.v. administration of ethanol, a catheter was inserted into the femoral vein. For i.c.v. administration of ethanol or APV, a stainless steel cylindrical cannula (0.5 mm o.d.) was stereotaxically inserted into the con-
*Corresponding author. Tel: ⫹81-3-5498-5785; fax: ⫹81-3-5498-5787. E-mail address: [email protected] (K. Abe). Abbreviations: APV, 2-amino-5-phosphonovalerate; BLA, basolateral amygdala; DG, dentate gyrus; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate.
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tralateral ventricle (0.8 mm posterior to bregma, 1.5 mm lateral to midline, 3.7 mm ventral to dura). Then, a bipolar stimulating electrode was placed in the BLA (2.8 mm posterior to bregma, 5.2 mm lateral to midline, 7.6 mm ventral to dura), and the evoked potential was extracellularly recorded from the granule cell layer of the DG (3.5 mm posterior to bregma, 4.4 mm lateral to midline, 3.0 mm ventral to dura). Test stimulation (0.08 ms duration) was applied at intervals of 30 s. As previously reported (Ikegaya et al., 1996), maximal stimulation of the BLA evoked biphasic field potentials in the DG, with early non-synaptic component and late synaptic component, while stimulation at modest intensity (⬍40 A) evoked only the late component. Thus, intensity of test stimulation was set so that the early component was not elicited and the amplitude of the late component was half of the maximum amplitude. After stable baseline responses were obtained for 20 min, test drug was administered and tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied to the BLA at the same stimulus intensity through the same electrode as that used for test stimulation. To evaluate changes in synaptic transmission, the evoked potential amplitude was measured as shown in Fig. 1A. All efforts were made for the care and use of animals according to the Guideline for Animal Experiments of Hoshi University. The number of animals used and their suffering was minimized.
RESULTS Effect of ethanol Single-pulse test stimulation of the BLA evoked characteristic positive-going field potentials in the DG (Fig. 1A), consistent with previous observations (Ikegaya et al., 1996; Abe et al., 2003). Since ingested ethanol is rapidly absorbed and circulates in the blood, we chose to administer ethanol through a catheter inserted into the femoral vein. In saline-treated control rats, application of tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) produced robust LTP in the BLA-DG pathway (Fig. 1A and B). I.v. administration of ethanol (20 – 40 v/v%, 2 ml/kg) did not affect the basal evoked potentials, but significantly inhibited the induction of LTP following tetanic stimulation (Fig. 1A). The inhibitory effect of ethanol on LTP induction was dose dependent (Fig. 1B). As shown in Fig. 2, there was no change in the basal evoked potential when it was observed for up to 70 min after i.v. administration of ethanol (40 v/v%⫻2 ml/kg) without applying tetanic stimulation. Furthermore, i.v. administration of ethanol (40 v/v%, 2 ml/kg) 30 min after tetanic stimulation did not change the pre-established LTP (Fig. 3). To examine if ethanol has direct effect on the CNS, ethanol was directly injected into the brain. I.c.v. administration of ethanol (30 – 40 v/v%, 5 l) did not affect the basal evoked potentials, but significantly inhibited the induction of LTP following tetanic stimulation (Fig. 4A). The inhibitory effect of ethanol was dose dependent (Fig. 4B). Possible target of ethanol In our previous study, the induction of LTP in the BLA-DG pathway was not affected by i.c.v. administration of 50 nmol APV, an NMDA receptor antagonist (Abe et al., 2003). To further support that LTP in the BLA-DG pathway is independent of NMDA receptors, we investigated again the effect of APV at a higher dose (150 nmol). However,
Fig. 1. Effect of i.v. administration of ethanol on the induction of LTP in the BLA-DG pathway in vivo. (A) Time-course of changes in evoked potentials. Saline (E, n⫽7) or ethanol (F, 20 v/v%, n⫽5; Œ, 30 v/v%, n⫽5; , 40 v/v%, n⫽7) was intravenously (2 ml/kg) administered 10 min before tetanus, and tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied at time 0. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before tetanic stimulation (time 0). Insets are representative records of evoked potentials at the times denoted by the numbers. Test stimulation was delivered at the time indicated by arrowheads. The amplitude of evoked potential was measured as shown. (B) Dosedependency. The data in A were summarized by calculating the average of evoked potential amplitudes 30 – 60 min after tetanic stimulation as an index of LTP magnitude. All data are means⫾S.E.M. ** P⬍0.01 vs. saline group, Dunnett’s test.
i.c.v. administration of 150 nmol APV had no significant effect on the induction of LTP in the BLA-DG pathway. The mean amplitude of evoked potentials 30 – 60 min after tetanic stimulation was 155.7⫾12.6% in the saline-treated group (n⫽7) and 139.5⫾14.8% (n⫽5) in the group treated with APV (150 nmol, i.c.v.). If ethanol inhibition of BLA-DG LTP is caused by potentiation of GABAergic transmission, the effect should be mimicked by other drugs that potentiate GABAergic transmission and should be abolished under blockade of GABAergic transmission. To test this possibility, we investigated the effects of diazepam, one of the benzodiazepines that potentiate GABA-induced activation of the
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Fig. 2. Effect of i.v. administration of ethanol on basal evoked potentials in the BLA-DG pathway. Ethanol (40 v/v%) was intravenously (2 ml/kg) administered at time 0, and evoked potentials were observed for 70 min without applying tetanic stimulation. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before administration of ethanol (time 0). Insets are representative records of evoked potentials at the times denoted by the numbers. Data are means⫾S.E.M. (n⫽5).
GABAA receptor channel complex, and picrotoxin, a noncompetitive GABAA receptor blocker. To examine pure effects on LTP induction, we chose to use these drugs at relatively low doses which alone did not affect basal responses before tetanic stimulation. I.p. administration of diazepam (2 mg/kg) had no significant effect on the basal evoked potentials, but significantly inhibited the induction of LTP in the BLA-DG pathway (Fig. 5). I.p. administration of picrotoxin (1 mg/kg) had no significant effect on the basal evoked potentials, and tended to enhance LTP, though the difference in LTP magnitude between the saline-treated and picrotoxin-treated groups was not statistically significant (Fig. 6B). When 40 v/v% ethanol was administered under treatment with picrotoxin, it did not affect the induction of LTP (Fig. 6A). The magnitude of LTP was not significantly different between the picrotoxintreated control group and the group treated with picrotoxin and ethanol (Fig. 6B).
Fig. 3. Effect of i.v. administration of ethanol on the maintenance phase of LTP in the BLA-DG pathway. LTP was induced by applying tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) at time 0, and 40 v/v% ethanol was intravenously (2 ml/kg) administered 30 min after tetanus. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before tetanic stimulation (time 0). Data are means⫾S.E.M. (n⫽5).
Fig. 4. Effect of i.c.v. administration of ethanol on the induction of LTP in the BLA-DG pathway. (A) Time-course of changes in evoked potentials. Saline (E, n⫽8) or ethanol (F, 30 v/v%, n⫽5; Œ, 40 v/v%, n⫽5) was intracerebroventricularly (5 l/brain) administered 10 min before tetanus, and tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied at time 0. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before tetanic stimulation (time 0). (B) Dose-dependency. The data in A were summarized by calculating the average of evoked potential amplitudes 30 – 60 min after tetanic stimulation as an index of LTP magnitude. All data are means⫾S.E.M. * P⬍0.05 vs. saline group, Dunnett’s test.
DISCUSSION The main finding in the present study was that i.v. administration of ethanol inhibited the induction of LTP in the BLA-DG pathway. Ethanol in the blood easily crosses the blood– brain barrier and exerts various effects on the CNS. I.c.v. administration of ethanol also inhibited the induction of LTP in the BLA-DG pathway, indicating that ethanol has a direct effect on the brain. The i.v. and i.c.v. doses of ethanol effective in inhibiting BLA-DG LTP were very similar to those in inhibiting hippocampal LTP in anesthetized rats in vivo (Abe et al., 1999b; Sugiura et al., 1994). These results demonstrate that ethanol causes impairment of amygdalo-hippocampal interaction as well as intrahippocampal synaptic plasticity. The relation between blood ethanol concentration and behavioral signs of intoxication has been described in the literature (Rall, 1990). In nontolerant humans, blood ethanol concentration of 0.2– 0.3 mg/ml can lead to delayed reaction time and impairment of fine motor control. An increase in blood ethanol concentration to 2.0 –2.5 mg/ml results in increased impairment of mental ability and motor coordination that is generally recognized as intoxication.
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Fig. 5. Effect of diazepam on the induction of LTP in the BLA-DG pathway. (A) Time-course of changes in evoked potentials. Saline (E, n⫽8) or diazepam (F, 2 mg/kg, n⫽5) was intraperitoneally administered 20 min before tetanus, and tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied at time 0 min. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before tetanic stimulation (time 0). Insets are representative records of evoked potentials at the times denoted by the numbers. (B) The data in A were summarized by calculating the average of evoked potential amplitudes 30 – 60 min after tetanic stimulation as an index of LTP magnitude. All data are means⫾S.E.M. ** P⬍0.01 vs. saline group, Student’s t-test.
Excessive increase in blood ethanol concentration over 2.0 –2.5 mg/ml may result in progressive depression of the CNS such as sedation, stupor and coma. On the other hand, it has previously been reported that blood ethanol concentration 10 min after i.v. administration of 0.5–1 g/kg ethanol reaches 0.6 –1.2 mg/ml in rats (Matsumoto et al., 1994; Shimada et al., 1987). In the present study, the induction of LTP in the BLA-DG pathway was inhibited by i.v. administration of ethanol (40 v/v%⫻2 ml/kg), which corresponds to the dose of 0.6 g/kg and is expected to give blood ethanol concentration of approximately 1 mg/ml. Thus, the blood ethanol concentration effective in inhibiting LTP induction is close to the range over which intoxication occurs in humans.
Fig. 6. Effect of ethanol on the induction of LTP under blockade of GABAergic transmission. Saline or picrotoxin (1 mg/kg) was intraperitoneally administered 30 min before tetanus, and saline or 40 v/v% ethanol was intravenously (2 ml/kg) administered 10 min before tetanus. (A) Time-course of changes in evoked potentials in the group treated with picrotoxin alone (E, n⫽5) and the group treated with picrotoxin and ethanol (F, n⫽5). Tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied at time 0 min. Insets are representative records of evoked potentials at the times denoted by the numbers. For clarity, the data in the group treated with saline alone (n⫽9) and the group treated with ethanol alone (n⫽8) are not shown in A. (B) Comparison of LTP magnitude among the four groups. The average of evoked potential amplitudes 30 – 60 min after tetanic stimulation was calculated as an index of LTP magnitude. All data are means⫾S.E.M. * P⬍0.05 vs. saline group, Tukey-Kramer’s test. There was no significant difference between saline and picrotoxin or between picrotoxin and picrotoxin⫹ethanol.
Since ethanol did not affect the basal evoked potentials in the BLA-DG pathway, ethanol is unlikely to change the release of neurotransmitter or neuron excitability in this pathway under normal conditions. In addition, administration of ethanol after tetanic stimulation had no effect on the pre-established LTP. Thus, it is probable that ethanol selectively affects mechanisms involved in the induction, but not maintenance, of LTP. It has been proposed that ethanol inhibits the induction of hippocampal LTP by inhibiting NMDA receptors or by potentiating GABAA receptor-mediated inhibitory mecha-
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nisms (Blitzer et al., 1990; Morrisett and Swartzwelder, 1993; Schummers and Browning, 2001). The induction of LTP in the BLA-DG pathway was not affected by i.c.v. administration of APV even at a relatively high dose (150 nmol). We previously confirmed that i.c.v. administration of APV at 50 –150 nmol are sufficient to completely block the induction of LTP at the perforant path-DG granule cell synapses in anesthetized rats (Abe et al., 1999a; Mizutani et al., 1991). The results suggest that LTP in the BLA-DG pathway is independent of NMDA receptors, and the impairment of BLA-DG LTP by ethanol is unlikely to be caused by inhibition of NMDA receptors. Alternatively, the induction of LTP in the BLA-DG pathway was inhibited by diazepam, and tended to be enhanced by picrotoxin, indicating that the GABAergic system functions to suppress the induction of LTP. Furthermore, ethanol failed to inhibit the induction of LTP in the BLA-DG pathway under blockade of GABAergic neurotransmission with picrotoxin. Therefore, ethanol inhibition of LTP in this pathway is likely to be caused by potentiation of GABAA receptor-mediated inhibitory mechanisms. In conclusion, we have demonstrated for the first time that ethanol inhibits the induction of LTP in the amygdalohippocampal pathway, probably by potentiation of GABAergic transmission. This effect of ethanol may underlie ethanol-induced impairment of emotion-associated learning and memory.
REFERENCES Abe K, Niikura Y, Misawa M (2003) The induction of long-term potentiation at amygdalo-hippocampal synapses in vivo. Biol Pharm Bull 26:1560 –1562. Abe K, Sugiura M, Yamaguchi S, Shoyama Y, Saito H (1999a) Saffron extract prevents acetaldehyde-induced inhibition of long-term potentiation in the rat dentate gyrus in vivo. Brain Res 851:287–289. Abe K, Yamaguchi S, Sugiura M, Saito H (1999b) The ethanol metabolite acetaldehyde inhibits the induction of long-term potentiation in the rat dentate gyrus in vivo. Br J Pharmacol 127:1805–1810. Blitzer RD, Gil O, Landau EM (1990) Long-term potentiation in rat hippocampus is inhibited by low concentrations of ethanol. Brain Res 537:203–208. Castellano C, Pavone F (1988) Effects of ethanol on passive avoidance behavior in the mouse: involvement of GABAergic mechanisms. Pharmacol Biochem Behav 29:321–324. Givens B (1995) Low doses of ethanol impair spatial working memory
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and reduce hippocampal theta activity. Alcohol Clin Exp Res 19: 763–767. Gould TJ (2003) Ethanol disrupts fear conditioning in C57BL/6 mice. J Psychopharmacol 17:77–81. Ikegaya Y, Saito H, Abe K (1996) Dentate gyrus field potentials evoked by stimulation of the basolateral amygdaloid nucleus in anesthetized rats. Brain Res 718:53–60. Matsumoto H, Fujimiya T, Fukui Y (1994) Role of alcohol dehydrogenase in rat ethanol elimination kinetics. Alcohol Alcohol 29:15–20. Melchior CL, Glasky AJ, Ritzmann RF (1993) A low dose of ethanol impairs working memory in mice in a win-shift foraging paradigm. Alcohol 10:491–493. Melia KR, Ryabinin AE, Corodimas KP, Wilson MC, Ledoux JE (1996) Hippocampal-dependent learning and experience-dependent activation of hippocampus are preferentially disrupted by ethanol. Neuroscience 74:313–322. Mizutani A, Saito H, Abe K (1991) Evidence for involvement of endogenous glycine in the induction of long-term potentiation in the dentate gyrus of anesthetized rats. Eur J Pharmacol 205:303–305. Morrisett RA, Swartzwelder HS (1993) Attenuation of hippocampal long-term potentiation by ethanol: a patch-clamp analysis of glutamatergic and GABAergic mechanisms. J Neurosci 13:2264 –2272. Nabeshima T, Tohyama K, Kameyama T (1988) Reversal of alcoholinduced amnesia by the benzodiazepine inverse agonist Ro 15– 4513. Eur J Pharmacol 155:211–217. Rall TW (1990) Hypotonics and sedatives: ethanol. In: The pharmacological basis of therapeutics (Gilman AG, Rall TW, Nies AS, Taylor P, eds), pp 372–373. New York: Pergamon Press. Sanger DJ, Joly D (1986) Zimelidine does not antagonise the effects of alcohol or diazepam on the acquisition of conditioned fear in mice. Neuropsychobiology 15:29 –33. Schummers J, Browning MD (2001) Evidence for a role for GABAA and NMDA receptors in ethanol inhibition of long-term potentiation. Mol Brain Res 94:9 –14. Shimada J, Miyahara T, Otsubo S, Yoshimatsu N, Oguma T, Matsubara T (1987) Effects of alcohol-metabolizing enzyme inhibitors and beta-lactam antibiotics on ethanol elimination in rats. Jpn J Pharmacol 45:533–544. Sinclair JG, Lo GF (1986) Ethanol blocks tetanic and calcium-induced long-term potentiation in the hippocampal slice. Gen Pharmacol 17:231–233. Sugiura M, Shoyama Y, Saito H, Abe K (1994) Crocin (crocetin digentiobiose ester) prevents the inhibitory effect of ethanol on longterm potentiation in the dentate gyrus in vivo. J Pharmacol Exp Ther 271:703–707. Sugiura M, Shoyama Y, Saito H, Abe K (1995) The effects of ethanol and crocin on the induction of long-term potentiation in the CA1 region of rat hippocampal slices. Jpn J Pharmacol 67:395–397. Zhang Y, Sugiura M, Saito H, Shoyama Y (1994) Acute effects of Crocus sativus L. on passive avoidance performance in mice. Biol Pharm Bull 17:217–221.
(Accepted 22 January 2004)
GABAA RECEPTOR-MEDIATED INHIBITION BY ETHANOL OF LONGTERM POTENTIATION IN THE BASOLATERAL AMYGDALA–DENTATE GYRUS PATHWAY IN VIVO K. ABE,* Y. NIIKURA AND M. MISAWA
DG field potentials (Abe et al., 2003), demonstrating that the BLA-DG pathway displays synaptic plasticity. This novel form of LTP may represent a synaptic mechanism underlying the formation of memory associated with emotional experiences. Acute ethanol intoxication causes impairment of memory and other cognitive functions in humans. Although behavioral studies with experimental animals have demonstrated that acute administration of ethanol impairs hippocampal-dependent working memory (Givens, 1995; Melchior et al., 1993; Zhang et al., 1994), cellular mechanisms are not fully understood. As a possible mechanism underlying ethanol-induced memory impairments, ethanol has been reported to inhibit the induction of LTP in hippocampal CA1 or DG synapses in vitro (Blitzer et al., 1990; Morrisett and Swartzwelder, 1993; Sinclair and Lo, 1986; Sugiura et al., 1995) and in vivo (Abe et al., 1999b; Sugiura et al., 1994). However, very little is known about the effect of ethanol on LTP in other brain regions. Furthermore, acute administration of ethanol has been shown to impair emotion-associated learning and memory, including passive avoidance learning (Zhang et al., 1994; Castellano and Pavone, 1988; Nabeshima et al., 1988) and fear conditioning (Gould, 2003; Melia et al., 1996; Sanger and Joly, 1986). Therefore, in the present study, we investigated the effect of ethanol on the induction of LTP in the BLA-DG pathway by using anesthetized rats. Furthermore, it has been proposed that ethanol impairs the induction of hippocampal LTP by inhibiting N-methyl-D-aspartate (NMDA) type of glutamate receptors or by potentiating GABAA receptor-mediated inhibitory mechanisms (Blitzer et al., 1990; Morrisett and Swartzwelder, 1993; Schummers and Browning, 2001). To explore mechanisms underlying the effect of ethanol on LTP in the BLA-DG pathway, possible involvement of the NMDA receptor or the GABAA receptor was also investigated.
Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
Abstract—Although ethanol has been reported to inhibit the induction of long-term potentiation in hippocampal CA1 and dentate gyrus synapses of rats, very little is known about the effect of ethanol on synaptic plasticity in other brain regions. Therefore, in the present study, we investigated the effect of ethanol on long-term potentiation in synaptic pathway from the basolateral amygdala to the dentate gyrus by using anesthetized rats in vivo. I.v. (20 – 40%ⴛ2 ml/kg) or i.c.v. (30 – 40%ⴛ5 l) administration of ethanol did not affect the basal amplitude of dentate gyrus field potential evoked by basolateral amygdala stimulation, but significantly inhibited the induction of long-term potentiation following application of tetanic stimulation. Since long-term potentiation in this pathway was independent of N-methyl-D-aspartate receptors, the inhibitory effect of ethanol is unlikely to be caused by suppression of N-methyl-D-aspartate receptor function. Alternatively, long-term potentiation in this pathway was significantly suppressed by the benzodiazepine agonist diazepam (2 mg/kg, i.p.), and the inhibitory effect of ethanol was abolished by the GABAA receptor channel blocker picrotoxin (1 mg/kg, i.p.). The present study demonstrates that ethanol inhibits the induction of long-term potentiation in the basolateral amygdala– dentate gyrus pathway by enhancing GABAA receptormediated neurotransmission. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: alcohol, emotional memory, synaptic plasticity, amygdala, hippocampus.
Emotion and memory are closely related. Among brain regions, the amygdala is involved in emotional and motivational aspects of behavior, and the hippocampus is crucially involved in the formation of memory. The interaction between the amygdala and hippocampus may be a key to elucidating neural mechanisms linking emotion and memory. We have previously found that that electrical stimulation of the basolateral amygdala (BLA) evokes field potentials in the dentate gyrus (DG) of the hippocampus in anesthetized rats (Ikegaya et al., 1996), demonstrating that there is neural connection from the BLA to the DG. Furthermore, application of tetanic stimulation to the BLA produced long-term potentiation (LTP) of the BLA-evoked
EXPERIMENTAL PROCEDURES 2-Amino-5-phosphonovalerate (APV) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other drugs were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All drugs were dissolved in saline. Evoked potential in the BLA-DG pathway was recorded as described previously (Ikegaya et al., 1996; Abe et al., 2003). Briefly, male Wistar rats (7–9 weeks old, 200 –300 g) were anesthetized with urethane (1 g/kg, i.p.) and ␣-chloralose (25 mg/kg, i.p.) and fixed in a stereotaxic frame. For i.v. administration of ethanol, a catheter was inserted into the femoral vein. For i.c.v. administration of ethanol or APV, a stainless steel cylindrical cannula (0.5 mm o.d.) was stereotaxically inserted into the con-
*Corresponding author. Tel: ⫹81-3-5498-5785; fax: ⫹81-3-5498-5787. E-mail address: [email protected] (K. Abe). Abbreviations: APV, 2-amino-5-phosphonovalerate; BLA, basolateral amygdala; DG, dentate gyrus; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate.
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tralateral ventricle (0.8 mm posterior to bregma, 1.5 mm lateral to midline, 3.7 mm ventral to dura). Then, a bipolar stimulating electrode was placed in the BLA (2.8 mm posterior to bregma, 5.2 mm lateral to midline, 7.6 mm ventral to dura), and the evoked potential was extracellularly recorded from the granule cell layer of the DG (3.5 mm posterior to bregma, 4.4 mm lateral to midline, 3.0 mm ventral to dura). Test stimulation (0.08 ms duration) was applied at intervals of 30 s. As previously reported (Ikegaya et al., 1996), maximal stimulation of the BLA evoked biphasic field potentials in the DG, with early non-synaptic component and late synaptic component, while stimulation at modest intensity (⬍40 A) evoked only the late component. Thus, intensity of test stimulation was set so that the early component was not elicited and the amplitude of the late component was half of the maximum amplitude. After stable baseline responses were obtained for 20 min, test drug was administered and tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied to the BLA at the same stimulus intensity through the same electrode as that used for test stimulation. To evaluate changes in synaptic transmission, the evoked potential amplitude was measured as shown in Fig. 1A. All efforts were made for the care and use of animals according to the Guideline for Animal Experiments of Hoshi University. The number of animals used and their suffering was minimized.
RESULTS Effect of ethanol Single-pulse test stimulation of the BLA evoked characteristic positive-going field potentials in the DG (Fig. 1A), consistent with previous observations (Ikegaya et al., 1996; Abe et al., 2003). Since ingested ethanol is rapidly absorbed and circulates in the blood, we chose to administer ethanol through a catheter inserted into the femoral vein. In saline-treated control rats, application of tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) produced robust LTP in the BLA-DG pathway (Fig. 1A and B). I.v. administration of ethanol (20 – 40 v/v%, 2 ml/kg) did not affect the basal evoked potentials, but significantly inhibited the induction of LTP following tetanic stimulation (Fig. 1A). The inhibitory effect of ethanol on LTP induction was dose dependent (Fig. 1B). As shown in Fig. 2, there was no change in the basal evoked potential when it was observed for up to 70 min after i.v. administration of ethanol (40 v/v%⫻2 ml/kg) without applying tetanic stimulation. Furthermore, i.v. administration of ethanol (40 v/v%, 2 ml/kg) 30 min after tetanic stimulation did not change the pre-established LTP (Fig. 3). To examine if ethanol has direct effect on the CNS, ethanol was directly injected into the brain. I.c.v. administration of ethanol (30 – 40 v/v%, 5 l) did not affect the basal evoked potentials, but significantly inhibited the induction of LTP following tetanic stimulation (Fig. 4A). The inhibitory effect of ethanol was dose dependent (Fig. 4B). Possible target of ethanol In our previous study, the induction of LTP in the BLA-DG pathway was not affected by i.c.v. administration of 50 nmol APV, an NMDA receptor antagonist (Abe et al., 2003). To further support that LTP in the BLA-DG pathway is independent of NMDA receptors, we investigated again the effect of APV at a higher dose (150 nmol). However,
Fig. 1. Effect of i.v. administration of ethanol on the induction of LTP in the BLA-DG pathway in vivo. (A) Time-course of changes in evoked potentials. Saline (E, n⫽7) or ethanol (F, 20 v/v%, n⫽5; Œ, 30 v/v%, n⫽5; , 40 v/v%, n⫽7) was intravenously (2 ml/kg) administered 10 min before tetanus, and tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied at time 0. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before tetanic stimulation (time 0). Insets are representative records of evoked potentials at the times denoted by the numbers. Test stimulation was delivered at the time indicated by arrowheads. The amplitude of evoked potential was measured as shown. (B) Dosedependency. The data in A were summarized by calculating the average of evoked potential amplitudes 30 – 60 min after tetanic stimulation as an index of LTP magnitude. All data are means⫾S.E.M. ** P⬍0.01 vs. saline group, Dunnett’s test.
i.c.v. administration of 150 nmol APV had no significant effect on the induction of LTP in the BLA-DG pathway. The mean amplitude of evoked potentials 30 – 60 min after tetanic stimulation was 155.7⫾12.6% in the saline-treated group (n⫽7) and 139.5⫾14.8% (n⫽5) in the group treated with APV (150 nmol, i.c.v.). If ethanol inhibition of BLA-DG LTP is caused by potentiation of GABAergic transmission, the effect should be mimicked by other drugs that potentiate GABAergic transmission and should be abolished under blockade of GABAergic transmission. To test this possibility, we investigated the effects of diazepam, one of the benzodiazepines that potentiate GABA-induced activation of the
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Fig. 2. Effect of i.v. administration of ethanol on basal evoked potentials in the BLA-DG pathway. Ethanol (40 v/v%) was intravenously (2 ml/kg) administered at time 0, and evoked potentials were observed for 70 min without applying tetanic stimulation. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before administration of ethanol (time 0). Insets are representative records of evoked potentials at the times denoted by the numbers. Data are means⫾S.E.M. (n⫽5).
GABAA receptor channel complex, and picrotoxin, a noncompetitive GABAA receptor blocker. To examine pure effects on LTP induction, we chose to use these drugs at relatively low doses which alone did not affect basal responses before tetanic stimulation. I.p. administration of diazepam (2 mg/kg) had no significant effect on the basal evoked potentials, but significantly inhibited the induction of LTP in the BLA-DG pathway (Fig. 5). I.p. administration of picrotoxin (1 mg/kg) had no significant effect on the basal evoked potentials, and tended to enhance LTP, though the difference in LTP magnitude between the saline-treated and picrotoxin-treated groups was not statistically significant (Fig. 6B). When 40 v/v% ethanol was administered under treatment with picrotoxin, it did not affect the induction of LTP (Fig. 6A). The magnitude of LTP was not significantly different between the picrotoxintreated control group and the group treated with picrotoxin and ethanol (Fig. 6B).
Fig. 3. Effect of i.v. administration of ethanol on the maintenance phase of LTP in the BLA-DG pathway. LTP was induced by applying tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) at time 0, and 40 v/v% ethanol was intravenously (2 ml/kg) administered 30 min after tetanus. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before tetanic stimulation (time 0). Data are means⫾S.E.M. (n⫽5).
Fig. 4. Effect of i.c.v. administration of ethanol on the induction of LTP in the BLA-DG pathway. (A) Time-course of changes in evoked potentials. Saline (E, n⫽8) or ethanol (F, 30 v/v%, n⫽5; Œ, 40 v/v%, n⫽5) was intracerebroventricularly (5 l/brain) administered 10 min before tetanus, and tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied at time 0. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before tetanic stimulation (time 0). (B) Dose-dependency. The data in A were summarized by calculating the average of evoked potential amplitudes 30 – 60 min after tetanic stimulation as an index of LTP magnitude. All data are means⫾S.E.M. * P⬍0.05 vs. saline group, Dunnett’s test.
DISCUSSION The main finding in the present study was that i.v. administration of ethanol inhibited the induction of LTP in the BLA-DG pathway. Ethanol in the blood easily crosses the blood– brain barrier and exerts various effects on the CNS. I.c.v. administration of ethanol also inhibited the induction of LTP in the BLA-DG pathway, indicating that ethanol has a direct effect on the brain. The i.v. and i.c.v. doses of ethanol effective in inhibiting BLA-DG LTP were very similar to those in inhibiting hippocampal LTP in anesthetized rats in vivo (Abe et al., 1999b; Sugiura et al., 1994). These results demonstrate that ethanol causes impairment of amygdalo-hippocampal interaction as well as intrahippocampal synaptic plasticity. The relation between blood ethanol concentration and behavioral signs of intoxication has been described in the literature (Rall, 1990). In nontolerant humans, blood ethanol concentration of 0.2– 0.3 mg/ml can lead to delayed reaction time and impairment of fine motor control. An increase in blood ethanol concentration to 2.0 –2.5 mg/ml results in increased impairment of mental ability and motor coordination that is generally recognized as intoxication.
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Fig. 5. Effect of diazepam on the induction of LTP in the BLA-DG pathway. (A) Time-course of changes in evoked potentials. Saline (E, n⫽8) or diazepam (F, 2 mg/kg, n⫽5) was intraperitoneally administered 20 min before tetanus, and tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied at time 0 min. The amplitude of evoked potential was expressed as the percentage of baseline values immediately before tetanic stimulation (time 0). Insets are representative records of evoked potentials at the times denoted by the numbers. (B) The data in A were summarized by calculating the average of evoked potential amplitudes 30 – 60 min after tetanic stimulation as an index of LTP magnitude. All data are means⫾S.E.M. ** P⬍0.01 vs. saline group, Student’s t-test.
Excessive increase in blood ethanol concentration over 2.0 –2.5 mg/ml may result in progressive depression of the CNS such as sedation, stupor and coma. On the other hand, it has previously been reported that blood ethanol concentration 10 min after i.v. administration of 0.5–1 g/kg ethanol reaches 0.6 –1.2 mg/ml in rats (Matsumoto et al., 1994; Shimada et al., 1987). In the present study, the induction of LTP in the BLA-DG pathway was inhibited by i.v. administration of ethanol (40 v/v%⫻2 ml/kg), which corresponds to the dose of 0.6 g/kg and is expected to give blood ethanol concentration of approximately 1 mg/ml. Thus, the blood ethanol concentration effective in inhibiting LTP induction is close to the range over which intoxication occurs in humans.
Fig. 6. Effect of ethanol on the induction of LTP under blockade of GABAergic transmission. Saline or picrotoxin (1 mg/kg) was intraperitoneally administered 30 min before tetanus, and saline or 40 v/v% ethanol was intravenously (2 ml/kg) administered 10 min before tetanus. (A) Time-course of changes in evoked potentials in the group treated with picrotoxin alone (E, n⫽5) and the group treated with picrotoxin and ethanol (F, n⫽5). Tetanic stimulation (100 pulses at 100 Hz, twice at an interval of 30 s) was applied at time 0 min. Insets are representative records of evoked potentials at the times denoted by the numbers. For clarity, the data in the group treated with saline alone (n⫽9) and the group treated with ethanol alone (n⫽8) are not shown in A. (B) Comparison of LTP magnitude among the four groups. The average of evoked potential amplitudes 30 – 60 min after tetanic stimulation was calculated as an index of LTP magnitude. All data are means⫾S.E.M. * P⬍0.05 vs. saline group, Tukey-Kramer’s test. There was no significant difference between saline and picrotoxin or between picrotoxin and picrotoxin⫹ethanol.
Since ethanol did not affect the basal evoked potentials in the BLA-DG pathway, ethanol is unlikely to change the release of neurotransmitter or neuron excitability in this pathway under normal conditions. In addition, administration of ethanol after tetanic stimulation had no effect on the pre-established LTP. Thus, it is probable that ethanol selectively affects mechanisms involved in the induction, but not maintenance, of LTP. It has been proposed that ethanol inhibits the induction of hippocampal LTP by inhibiting NMDA receptors or by potentiating GABAA receptor-mediated inhibitory mecha-
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nisms (Blitzer et al., 1990; Morrisett and Swartzwelder, 1993; Schummers and Browning, 2001). The induction of LTP in the BLA-DG pathway was not affected by i.c.v. administration of APV even at a relatively high dose (150 nmol). We previously confirmed that i.c.v. administration of APV at 50 –150 nmol are sufficient to completely block the induction of LTP at the perforant path-DG granule cell synapses in anesthetized rats (Abe et al., 1999a; Mizutani et al., 1991). The results suggest that LTP in the BLA-DG pathway is independent of NMDA receptors, and the impairment of BLA-DG LTP by ethanol is unlikely to be caused by inhibition of NMDA receptors. Alternatively, the induction of LTP in the BLA-DG pathway was inhibited by diazepam, and tended to be enhanced by picrotoxin, indicating that the GABAergic system functions to suppress the induction of LTP. Furthermore, ethanol failed to inhibit the induction of LTP in the BLA-DG pathway under blockade of GABAergic neurotransmission with picrotoxin. Therefore, ethanol inhibition of LTP in this pathway is likely to be caused by potentiation of GABAA receptor-mediated inhibitory mechanisms. In conclusion, we have demonstrated for the first time that ethanol inhibits the induction of LTP in the amygdalohippocampal pathway, probably by potentiation of GABAergic transmission. This effect of ethanol may underlie ethanol-induced impairment of emotion-associated learning and memory.
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(Accepted 22 January 2004)