Introduction

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Cholinergic neuronal systems play an important role in the cognitive deficits associated with aging and neurodegenerative diseases (Bartus et al., 1982; Beninger et al., 1989; Coyle et al., 1983; Kameyama et al., 1986; Levin, 1992; Newhouse, 1990; Sarter, 1991). Although investigations of learning and memory have focused primarily on cholinergic neurotransmission, reports of increased kappa opioid receptor density in the brains of patients with Alzheimer's disease (Hiller et al., 1987) and dynorphin A (1-8)-like immunoreactivity in the hippocampus of aged rats (Jiang et al., 1989) suggest that disruption of opioidergic neurotransmission may also play a role in the cognitive deficits associated with Alzheimer's disease and aging. Recent studies have indicated that neuropeptides modulate learning and memory processes in experimental animals. Of particular interest was the observation that an endogenous kappa opioid agonist, dynorphin A (1-13), reverses the scopolamine-induced impairment of spontaneous alternation performance (Itoh et al., 1993a) and CO-induced delayed amnesia in mice (Hiramatsu et al., 1995, 1997b). We have reported recently that a selective kappa opioid receptor agonist, U-50,488H, also improves the impairment of learning and memory induced by CO exposure (Hiramatsu et al., 1996a) and by carbachol (Hiramatsu et al., 1997a, 1998, in press). However, whether kappa opioid agonists improve memory function is controversial, and neurochemical mechanism underlying the memory improvement by kappa opioid agonists is still unknown. For example, post-training administration of dynorphin A (1-13) has no effect on inhibitory avoidance or shuttle avoidance responses (Izquierdo et al., 1985) and impairs retention of inhibitory avoidance but not of Y-maze discrimination (Introini-Collison et al., 1987). Colombo et al. (1992) reported that dynorphin A (1-13) impaired memory in a dose-dependent manner. However, injection of U-50,488H showed a biphasic effect on memory; low doses tended to enhance, albeit not significantly, whereas high doses significantly impaired memory in 2-day-old chicks (Colombo et al., 1992). Therefore, the role of kappa opioid receptors in memory formation may depend biphasically on the dosage of agonist used.

Evidence that high concentrations of dynorphin decrease [¹⁴C]acetylcholine release (Mulder et al., 1984) corresponds with this hypothesis. On the other hand, the activation of kappa opioid receptors by dynorphin had no effect on high potassium or glutamate-evoked acetylcholine release in rat striatal slices (Arenas et al., 1990), and electrical stimulation or high potassium concentration evoked release of acetylcholine output in brain slices (Lapchak et al., 1989; Heijna et al., 1990). Furthermore, recent results from our laboratory indicated that low doses of dynorphin have no effect on acetylcholine release in normal rats as measured by microdialysis (Mori et al., 1995).

Scopolamine, a muscarinic acetylcholine receptor antagonist, is used widely to investigate cholinergic influence on learning ability in experimental animals. Blockade of nicotinic receptors by mecamylamine also induces the impairment of learning ability. Furthermore, in patients with Alzheimer's disease, not only the muscarinic but also the nicotinic receptors were decreased markedly (Nordberg and Winblad, 1986; Quirion et al., 1986; Whitehouse et al., 1986). Therefore, blockade of both receptors may offer a better amnesia model (Levin et al., 1989; Levin, 1992). Because mecamylamine acts partly as an NMDA receptor antagonist and cholinergic systems modulate glutamatergic systems in the hippocampus (Faden, 1992), we also tested the effects of U-50,488H on learning impairment induced by the typical NMDA receptor antagonist, dizocilpine [(+)-MK-801].

The present study, therefore, was designed to test the hypothesis that a kappa opioid receptor agonist ameliorates both scopolamine-, mecamylamine- and (+)-MK-801-induced learning and memory impairment and the disruption of cholinergic neurotransmission via kappa opioid receptors.

	Methods

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Animals. Male Sprague-Dawley rats (Japan SLC Inc., Shizuoka, Japan), weighing between 250 and 350 g, were used. The animals were housed in a room with controlled lighting (12-h light/dark cycle, lights on 8 A.M. to 8 P.M.) and temperature (23 ± 2°C) for at least 5 days before the experiments and were given free access to food and water. Experimental protocols concerning the use of laboratory animals were approved by the committee of Meijo University and followed the guidelines of the Japanese Pharmacological Society [(1992) Guiding Principles for the Care and Use of Laboratory Animals. Folia Pharmacol Jpn 99:35A] and the interministerial decree of May 25, 1987 (the Ministry of Education).

Surgical procedure. Rats were anesthetized with sodium pentobarbital (50 mg/kg) administered intraperitoneally (i.p.). With use of coordinates from the stereotaxic atlas of Paxinos and Watson (1986), a guide cannula for the microdialysis probe was implanted unilaterally into the hippocampus, and the cannula for drug injection was implanted into the lateral ventricle. The tips of the cannulae were positioned just above the hippocampus (A: 4.1, L: 2.0, V: 3.2 mm from the bregma) and the lateral ventricle (A: 1.0, L: 1.2, V: 4.5 mm from the bregma) of each rat. The animals were allowed to recover from the procedure for 3 to 7 days before the experiment. In the experiment, the dialysis probe (CMA/10, Bioanalytical Systems, Inc., Tokyo, Japan) was inserted through the guide cannula and a 3-mm length of dialysis membrane was then advanced into the hippocampus.

Passive avoidance test. One group of rats was trained in a passive avoidance apparatus which consisted of two compartments, one light (25 × 15 × 15 cm high) and one dark, of the same size connected via a guillotine door. On day 1, each rat was placed in the light compartment and then allowed to enter the dark compartment. Rats that had latencies greater than 60 s were discarded as being outside the normal range (preacquisition trial). The acquisition trial was carried out 15 min after the preacquisition trial. Rats were placed in the light compartment and 30 s later the guillotine door was opened. Once the rat entered the dark compartment, the guillotine door was closed and an electric shock (0.5 mA for 3 s) was delivered to the animal via the floor. The animal was then put back into the home cage and the retention trial was carried out 24 h later. The rat was put in the light compartment and the time taken to enter the dark compartment was recorded (step-through latency). A maximum latency of 300 s was set.

Sampling procedure. The other group of rats was used for microdialysis experiments. The dialysis probe was perfused with Ringer's solution (composition in mM: NaCl, 127.6; KCl, 2.5; CaCl₂, 1.3, pH 6.4-6.8, containing 0.01 mM eserine) at a rate of 2 µl/min, connected to a microinfusion pump (Syringe Infusion Pump 22, Harvard Apparatus, South Natick, MA) via a single-channel liquid swivel. The rats were placed in individual acrylic cages (30 × 30 × 35 cm high) and allowed to adapt for at least 60 min before the experiment was started. The dummy cannulae were replaced with dialysis probes and the perfusate was collected in small (250 µl) disposable microcentrifuge tubes secured to the middle of the tether. The total dead volume from the tip of the probe to the collection tube was usually 4 µl. About 3 h after the probe was inserted, samples (40 µl) were collected at 20-min intervals, and when at least three base-line samples were stable, the drugs were administered. Perfusate samples from the brain were taken up to 120 min after treatment with drugs or saline. The locations of dialysis probes were confirmed after the experiments.

Analysis of dialysates. Acetylcholine and choline in the dialysate were quantified by HPLC with an immobilized enzyme column and an ECD (ECD-300, Eicom Corp., Kyoto, Japan). The mobile phase consisted of 0.1 M sodium phosphate buffer (pH 8.5) containing 1.23 mM 1-decanesulfonate sodium salt and 593 µM tetramethylammonium chloride (Fujimori and Yamamoto, 1987) was delivered by a pump (P-300, Eicom Corp., Kyoto, Japan) at a flow rate of 0.6 ml/min. To protect the analytical column from impurities in the mobile phase and samples, a precolumn (Eicom Corp. Kyoto, Japan) was placed between the pump and injector. Aliquots (25 µl) of the perfusate samples were injected into the HPLC system and separated by a column of Eicompak AC-GEL (6.0 × 150 mm). The enzyme column containing acetylcholinesterase and choline oxidase catalyzed the formation of hydrogen peroxide from acetylcholine and choline. The resultant H₂O₂ was detected by ECD with a platinum electrode at +450 mV. The average basal values of acetylcholine and choline (recorded in the presence of 0.01 mM eserine) were 0.22 ± 0.06 and 2.45 ± 0.46 pmol/min in the hippocampus. Although relatively high concentrations of eserine had to be used to measure extracellular acetylcholine levels, acetylcholine release was able to detect in a similar time course when samples were collected during longer periods.

Locomotor activity. Locomotor activity was measured with Scanet SV-10 (Toyo Sangyo, Co. Ltd., Nakashinkawa, Japan). Interruptions of any of the infrared photocells were recorded on an NEC personal computer (PC-9801 RX). Rats were injected subcutaneously with vehicle or drug, injected subcutaneously with U-50,488H 5 min later and then placed individually in polypropylene cages (41.5 × 24.5 × 18 cm). Locomotor activity was then measured for 120 min, with data recorded separately for 12 consecutive periods of 10 min each. Experiments were conducted during the light phase of the light/dark cycle in a quiet room.

Drugs. The following drugs were used: sodium pentobarbital (Tokyo Chemical Industry Co., Ltd., Japan); U-50,488H (Sigma, St. Louis, MO); n-BNI (Research Biochemicals, Inc., Natick, MA); scopolamine hydrobromide (scopolamine, Tokyo Chemical Industry, Co., Ltd., Tokyo, Japan); dizocilpine [(+)-MK-801, a generous gift from Dr. A.K. Cho, UCLA] mecamylamine hydrochloride (Sigma, St. Louis, MO); dynorphin A (1-13) (Peptide Institute, Inc., Osaka, Japan). All doses were calculated as those of the bases. Drugs were dissolved in isotonic saline solution (Otsuka Pharmaceuticals, Inc., Tokyo, Japan).

Data analysis. The behavioral data are expressed in terms of median, interquartile and 10th and 90th percentile ranges. Significant differences were evaluated using the Mann-Whitney U test for comparisons between two groups and Kruskal-Wallis non-parametric one-way analysis of variance followed by Bonferroni's test for multiple comparisons. Dialysis data are shown as means ± S.E.M of the percentage of base-line level obtained from each rat before drug treatment. To compare the effects of drugs, data were analyzed by two-way repeated measures analysis of variance followed by Bonferroni's test. The data for individual time points was analyzed by one-way analysis of variance followed by Bonferroni's test. P < .05 was taken as the criterion for significance.

	Results

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Effects of U-50,488H on scopolamine-induced learning impairment. Scopolamine (3.3 µmol/kg s.c.) significantly impaired the acquisition of learning when administered 30 min before the acquisition trial (fig. 1). U-50,488H (0.51 µmol/kg s.c.) 25 min before the acquisition trial significantly attenuated the impairment of learning and memory induced by scopolamine in rats, whereas a lower dose of U-50,488H (0.17 µmol/kg s.c.) showed no such effect (fig. 1). U-50,488H (0.17 and 0.51 µmol/kg s.c.) itself administered 25 min before acquisition trials had no effect on learning and memory when administered alone (fig. 1).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effects of U-50,488H on the scopolamine-induced impairment of learning and memory in the step-through type passive avoidance test. Rats were treated with scopolamine (3.3 µmol/kg s.c.) and U-50,488H (0.17-0.51 µmol/kg s.c.) 30 and 25 min before the acquisition trial, respectively. The retention trial was carried out 24 h after the acquisition trial. Values show the median (horizontal bar), first and third quartiles (vertical column) and 10th and 90th percentiles (vertical lines). Figures in parentheses show the numbers of mice used. **P < .01 vs. normal control (Mann-Whitney U test), ##P < .01 vs. scopolamine alone (Bonferroni's test).

Effects of U-50,488H on mecamylamine-induced learning impairment. The effects of U-50,488H on passive avoidance performance in mecamylamine-treated rats are shown in figure 2. Mecamylamine (40 µmol/kg s.c.), a nicotinic acetylcholine receptor antagonist, significantly impaired the acquisition of learning when administered 30 min before acquisition trial (fig. 2). In this model, U-50,488H (0.17 µmol/kg s.c.) administered 25 min before the test session significantly attenuated the impairment of learning and memory induced by mecamylamine in rats. The dose-effect function for U-50,488H was bell shaped (fig. 2).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Effects of U-50,488H on the mecamylamine-induced impairment of learning and memory in the step-through type passive avoidance test. Rats were treated with mecamylamine (40 µmol/kg s.c.) and U-50,488H (0.051-1.7 µmol/kg s.c.) 30 and 25 min before the acquisition trial, respectively. The retention trial was carried out 24 h after the acquisition trial. Values show the median (horizontal bar), first and third quartiles (vertical column) and 10th and 90th percentiles (vertical lines). Figures in parentheses show the numbers of mice used. **P < .01 vs. normal control (Mann-Whitney U test), #P < .05 vs. mecamylamine alone (Bonferroni's test).

Effects of U-50,488H on low doses of scopolamine + mecamylamine-induced learning impairment. Scopolamine (0.17 µmol/kg s.c.) and mecamylamine (12 µmol/kg s.c.) itself did not induce impairment of learning and memory in rats (fig. 3). When given together 30 min before the acquisition trial, these drugs significantly impaired the acquisition of learning (fig. 3). U-50,488H (0.17 µmol/kg s.c.) significantly attenuated the impairment of learning and memory induced by scopolamine + mecamylamine in rats when administered 5 min after the injection of these antagonists (fig. 3).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effects of U-50,488H on the scopolamine + mecamylamine-induced impairment of learning and memory in the step-through type passive avoidance test. Rats were treated with scopolamine (0.17 µmol/kg s.c.), mecamylamine (12 µmol/kg s.c.) and U-50,488H (0.17 µmol/kg s.c.) 30, 30 and 25 min before the acquisition trial, respectively. The retention trial was carried out 24 h after the acquisition trial. Values show the median (horizontal bar), first and third quartiles (vertical column) and 10th and 90th percentiles (vertical lines). Figures in parentheses show the numbers of mice used. **P < .01 vs. normal, and vs. mecamylamine or scoplamine alone, ##P < .01 vs. scopolamine + mecamylamine alone (Bonferroni's test).

Effects of U-50,488H on (+)-MK-801-induced learning impairment. As shown in figure 4, administration of (+)-MK-801 (2.9 µmol/kg s.c.) 30 min before the acquisition trial significantly impaired the acquisition of learning (fig. 4). U-50,488H (0.17 and 0.51 µmol/kg s.c.) did not attenuate the impairment of learning and memory induced by (+)-MK-801 in rats when administered 25 min before acquisition.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of U-50,488H on the (+)-MK-801-induced impairment of learning and memory in the step-through type passive avoidance test. Rats were treated with (+)-MK-801 (2.9 µmol/kg s.c.) and U-50,488H (0.17-0.51 µmol/kg s.c.) 30 and 25 min before the acquisition trial, respectively. The retention trial was carried out 24 h after the acquisition trial. Values show the median (horizontal bar), first and third quartiles (vertical column) and 10th and 90th percentiles (vertical lines). Figures in parentheses show the numbers of mice used. **P < .01 vs. control (Bonferroni's test).

Effects of U-50,488H and dynorphin A (1-13) on scopolamine-induced increase in extracellular acetylcholine levels. Scopolamine (3.3 µmol/kg s.c.) significantly increased the synaptic overflow of acetylcholine in the hippocampus (P < .01) by about 1500% of the base-line levels from 20 to 120 min after injection (fig. 5). In behavioral experiments, U-50,488H attenuated scopolamine-induced impairment of learning and memory (fig. 1). To investigate the possible mechanism of this behavioral effect, the extracellular acetylcholine levels were measured after administration of U-50,488H. U-50,488H (0.51 µmol/kg s.c.) partially but significantly suppressed the increase in extracellular acetylcholine level induced by scopolamine 20 to 100 min after injection in the hippocampus (fig. 5).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effects of U-50,488H and dynorphin A (1-13) on the scopolamine-induced increase in extracellular acetylcholine in the hippocampus. Scopolamine (3.3 µmol/kg s.c.), U-50,488H (0.51 µmol/kg s.c.) and/or dynorphin A (1-13) (0.5 nmol/rat i.c.v.) were injected immediately, 5 and/or 5 min after perfusion, respectively. Values represent the means ± S.E.M. for five rats. (A) P < .01 for [Control] vs. [Scopolamine], [Control] vs. [Scopolamine + U-50,488H], [Scopolamine] vs. [Scopolamine + U-50,488H]; (B) P < .01 for [Control] vs. [Scopolamine], [Control] vs. [Scopolamine + dynorphin A (1-13)] (two-way ANOVA), **P < .01 vs. control (Bonferroni's test).

Effects of U-50,488H on mecamylamine-induced decrease in extracellular acetylcholine levels. Mecamylamine (40 µmol/kg s.c.) significantly decreased the extracellular levels of acetylcholine in the hippocampus (P < .01) by about 25% of the base-line levels from 20 to 120 min after injection (fig. 6). This decrease elicited by mecamylamine lasted for at least 120 min. U-50,488H (0.17 µmol/kg s.c.) abolished the decrease in the extracellular acetylcholine level induced by mecamylamine almost completely in the hippocampus (fig. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effects of U-50,488H on the mecamylamine-induced decrease in extracellular acetylcholine and antagonism by n-BNI in the hippocampus. Mecamylamine (40 µmol/kg i.p.), U-50,488H (0.51 µmol/kg s.c.) and/or n-BNI (4.9 nmol/rat i.c.v.) were injected immediately, 5 and/or 10 min after perfusion, respectively. Values represent the means ± S.E.M. for five rats. P < .01 for [Control] vs. [Mecamylamine], [Mecamylamine] vs. [U-50,488H], [Mecamylamine + U-50,488H] vs. [Mecamylamine + U-50,488H + n-BNI] (two-way ANOVA), *P < .05, **P < .01 vs. control, #P < .05, ##P < .01 vs. mecamylamine, $P < .05, $$P < .01 vs. mecamylamine + U-50,488H (Bonferroni's test).

Effects of U-50,488H on scopolamine + mecamylamine-induced increase in extracellular acetylcholine levels. The low dose of scopolamine (0.17 µmol/kg s.c.) also significantly increased the extracellular levels of acetylcholine in the hippocampus (P < .01) by about 230% of the base-line levels at 40 min after injection (fig. 7), although the increment was less than after a higher dose of scopolamine (fig. 5). However, a lower dose of mecamylamine (12 µmol/kg s.c.) did not affect the extracellular levels of acetylcholine. Combined treatment with these two antagonists significantly increased the extracellular levels of acetylcholine in the hippocampus but less than with scopolamine alone. U-50,488H (0.17 µmol/kg s.c.) partially antagonized the increase in acetylcholine level induced by scopolamine + mecamylamine (fig. 7).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effects of U-50,488H on the scopolamine + mecamylamine-induced increase in extracellular acetylcholine in the hippocampus. Scopolamine (0.17 µmol/kg s.c.), mecamylamine (12 µmol/kg i.p.) and U-50,488H (0.17 µmol/kg s.c.) were injected immediately and 5 min after perfusion, respectively. Values represent the means ± S.E.M. for five to six rats. P < .05 for [Scopolamine + Mecamylamine] vs. [Scopolamine + Mecamylamine + U-50,488H], P < .01 for [Control] vs. [Scopolamine], [Control] vs. [Scopolamine + Mecamylamine], [Control] vs. [Scopolamine + Mecamylamine + U-50,488H] (two-way ANOVA), *P < .05 vs. control (Bonferroni's test).

Effects of U-50,488H on (+)-MK-801-induced increase in extracellular acetylcholine levels. (+)-MK-801 (2.9 µmol/kg s.c.) significantly increased the extracellular levels of acetylcholine in the hippocampus (P < .01) by about 300% of the base-line levels from 60 to 120 min after injection (fig. 8). U-50,488H (0.17 µmol/kg s.c.) partially antagonized the increase in acetylcholine levels induced by (+)-MK-801 (fig. 8).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Effects of U-50,488H on the (+)-MK-801-induced increase in extracellular acetylcholine in the hippocampus. (+)-MK-801 (2.9 µmol/kg s.c.) and U-50,488H (0.17 µmol/kg s.c.) were injected immediately and 5 min after perfusion, respectively. Values represent the means ± S.E.M. for five rats. P < .05 for [(+)-MK-801] vs. [(+)-MK-801 + U-50,488H (0.17 µmol/kg s.c.)], P < .01 for [Control] vs. [(+)-MK-801], [Control] vs. [(+)-MK-801 + U-50,488H (0.17 µmol/kg s.c.)], [Control] vs. [(+)-MK-801 + U-50,488H (0.51 µmol/kg s.c.)], [(+)-MK-801] vs. [(+)-MK-801 + U-50,488H (0.51 µmol/kg s.c.)] (two-way ANOVA), *P < .05 vs. control (Bonferroni's test).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Effects of U-50,488H on extracellular acetylcholine in the hippocampus. U-50,488H (0.17 and 0.51 mmol/kg s.c.) was injected immediately after perfusion. Values represent the means ± S.E. for five rats. P < .01 for [Control] vs. [U-50,488H (0.17 µmol/kg s.c.)], [Control] vs. [U-50,488H (0.51 µmol/kg s.c.)] (two-way ANOVA), *P < .05 vs. control (Bonferroni's test).

Effects of U-50,488H on scopolamine- and (+)-MK-801-induced increases in locomotor activities. It has been reported that kappa opioid receptor agonists decreased locomotor activities in mice and rats. To test whether the doses of U-50,488H used in this experiment had such effects, locomotor activities were measured for 120 min. Scopolamine (3.3 µmol/kg s.c.) and (+)-MK-801 (2.9 µmol/kg s.c.) significantly increased the locomotor activities (fig. 10). U-50,488H (0.17 and 0.51 µmol/kg s.c.) did not affect the hyperactivity induced by scopolamine or (+)-MK-801 (fig. 10). U-50,488H itself also did not affect the locomotor activity in normal rats (fig. 10).

View larger version (35K): [in this window] [in a new window]   Fig. 10.   Effects of U-50,488H on scopolamine- or (+)-MK-801-induced hyperactivity in rats. U-50,488H (0.17 and 0.51 mmol/kg s.c.) was treated 5 min after the injection of scopolamine (A, 3.3 µmol/kg s.c.) or (+)-MK-801 (B, 2.9 µmol/kg i.p.). Values represent the means ± S.E. for six to eight rats. **P < .01 vs. control (Bonferroni's test).

	Discussion

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Systemic administration of muscarinic cholinergic antagonists such as scopolamine impairs the performance of experimental animals in a wide variety of learning and memory tasks, including inhibitory (passive) avoidance (Kameyama et al., 1986) and spatial maze tasks (Buresova et al., 1986; Wirsching et al., 1984). However, recent studies have shown that quisqualic acid, an excitatory amino acid, injected into the nucleus basalis of Meynert reduces cholinergic input to the cortex more completely than ibotenic acid but produces only minimal memory deficits (Dunnett et al., 1987). Furthermore, large decreases in many muscarinic binding sites, as well as nicotinic sites, have been reported in the brains of patients with Alzheimer's disease (Nordberg and Winblad, 1986; Quirion et al., 1986; Whitehouse et al., 1986). Thus, independent manipulation of cholinergic receptor subtypes in experimental animals may provide an inadequate model of cognitive dysfunction. Consistent with this conclusion, the cholinergic dysfunction observed in aging and Alzheimer's disease is accompanied by changes in other neurotransmitter systems such as peptidergic (Hiller et al., 1987; Jiang et al., 1989) and noradrenergic (Scarpace and Abrass, 1988) systems, which may be important in memory modulation. For example, Jiang et al. (1989) reported that dynorphin A (1-8)-like immunoreactivity was increased in the aged rat brain, and this elevation was found only in the hippocampus and frontal cortex. The increase in dynorphin A (1-8)-like immunoreactivity in the aged hippocampus was associated with a decline in spatial learning memory (Jiang et al., 1989).

In our recent studies, dynorphin A (1-13) and U-50,488H reversed CO-induced impairment of learning and memory (Hiramatsu et al., 1995, 1996a, 1997b), in agreement with previous findings, which indicates reversal of the scopolamine-, mecamylamine-, galanin- and carbachol-induced impairment of learning and memory in mice and rats (Hiramatsu et al., 1996b, 1997a,b, 1998, in press; Itoh et al., 1993a). These ameliorative effects of dynorphin A (1-13) were antagonized almost completely by n-BNI (Hiramatsu et al., 1996a, b; Itoh et al., 1993a), a selective kappa opioid receptor antagonist. n-BNI itself had no significant effect on locomotor activity or memory process in either memory-impaired or normal animals. Previously, we reported that one of the mechanisms underlying memory dysfunction after CO exposure would be dysfunction of cholinergic neuronal systems (Hiramatsu et al., 1996c). Taken together, these results suggest that kappa opioid receptor agonists can ameliorate cholinergic dysfunction via the kappa opioidergic system.

Although kappa opioid receptor agonists can ameliorate learning and memory impairment, endogenous kappa opioid agonists may not have exerted tonic (inhibitory) control over the regulation of neurotransmission, because n-BNI did not modify the step-through latency in normal rats (Hiramatsu et al., 1996b). We previously reported that a low dose of dynorphin A (1-13), which has no effect on acetylcholine release in normal rats, prevents galanin-induced decreases in acetylcholine release (Hiramatsu et al., 1996b). Therefore, we hypothesized that endogenous kappa opioid agonists such as dynorphins can compensate for dysfunction in the hippocampal formation and that the kappa opioidergic system in the brain plays an important role in modulating learning and memory when the cholinergic system is impaired.

To clarify this hypothesis, in the present study, we investigated the effects of a selective kappa opioid receptor agonist, U-50,488H, on a muscarinic and/or a nicotinic receptor agonist-induced learning and memory impairment by a step-through type passive avoidance task and an in vivo microdialysis technique.

Muscarinic receptor blockade. Although dynorphin A (1-13) was not studied in the present behavioral experiments, our previous results showed that U-50,488H (Hiramatsu et al., 1996a) and dynorphin A (1-13) (Itoh et al., 1993a) ameliorated the impairment of learning and memory induced by scopolamine. Although U-50,488H attenuated the increase in acetylcholine release, dynorphin A (1-13) did not alter this increment (fig. 5). On the other hand, U-50,488H alone decreased the acetylcholine release in normal rats (fig. 9), whereas dynorphin A (1-13) did not (Hiramatsu et al., 1996b). Therefore, suppression of acetylcholine increase may not be important in amelioration of the impairment of learning and memory.

Nicotinic receptor blockade. Nilsson et al. (1987) suggested that endogenous acetylcholine could positively modulate its own release in the brains of patients with Alzheimer's disease, possibly through activation of nicotinic receptors, when its enzymatic cleavage is prevented. Similar results have been obtained in hippocampal slices from rats in which cholinergic neurons had been lesioned with the neurotoxin AF64A (Potter and Nitta, 1993). The importance of presynaptic nicotinic receptors also was highlighted in a recent study by McGehee et al. (1995). Nicotine was found to enhance both glutamatergic and cholinergic transmission evoked at 0.1 Hz through activation of presynaptic nicotinic receptors. Sensitivity of presynaptic nicotinic autoreceptors might increase during degeneration of cholinergic neurons as a compensatory mechanism. In agreement with these findings, we also showed that low doses of nicotine improved CO-induced amnesia (Hiramatsu et al., 1994).

Involvement of dopaminergic systems. Because U-50,488H does not act exclusively on cholinergic synapses, the possible effects on other neurotransmitter systems cannot be excluded. Kappa opioid receptor agonists inhibit dopamine agonist-induced hyperactivity, diminish striatal and mesolimbic dopamine release and reduce the release of [³H]dopamine from cultured neurons (Ronken et al., 1993; Heijna et al., 1990). Moreover, central catecholamines appear to be involved in the acquisition and maintenance of learning associated with aversion (Ichihara et al., 1989; Oei and King, 1980).

Concurrent muscarinic-nicotinic receptor blockade. If a preferential activation of nicotinic versus muscarinic autoreceptors only occurs in the diseased brain, normal brain tissues may not provide appropriate models for the understanding of cholinergic mechanisms operative in the damaged brain in Alzheimer's disease and for testing drugs expected to compensate for altered cholinergic transmission. Therefore, concurrent blockade of these two components of acetylcholine systems may provide a better animal model of cognitive impairment caused by the loss of cholinergic neurons (Levin and Rose, 1991).

Involvement of excitatory amino acid systems. In addition to their potent kappa opioid activities, the prodynorphin-derived peptides, dynorphin A (1-13) and dynorphin (1-17) exert so-called `non-opiate effects' (Walker et al., 1982). These include various motor and behavioral effects as well as inhibition of the spontaneous or glutamate-induced firing of hippocampal pyramidal cells. Administration of dynorphin A (1-17) also caused marked increases in the extracellular levels of glutamate and aspartate (Faden, 1992). Therefore, it seems that kappa opioid receptor agonists improved learning and memory impairment through the activation of excitatory amino acid neurons. However, the increment in the excitatory amino acid release was not modified by the opioid receptor antagonists. Further, dynorphin A (2-17), which is inactive on opioid receptors, produced alterations in excitatory amino acid similar to dynorphin A (1-17) (Faden, 1992). Massardier and Hunt (1989) showed that dynorphin A (1-13) interacted directly with NMDA receptors by a nonopiate mechanism. Dynorphin A (1-13) selectively inhibited the NMDA subtype of excitatory amino acid receptors, but had no effect on the binding of ligands for the kainate or quisqualate subtypes. This activity of dynorphin A (1-13) was not related to an effect at kappa opioid receptors, because U-50,488H did not have such effects. Our present and previous findings demonstrated that n-BNI blocked the antiamnesic effects of kappa opioid receptor agonists and also that kappa opioid receptor agonists, which were sensitive to n-BNI, suppressed the abolishment of decreases in acetylcholine release induced by galanin, carbachol and mecamylamine (Hiramatsu et al., 1996b, 1997a, 1998, in press; fig. 6).

Possible sites of kappa opioid receptor agonists. Both acetylcholine and glutamate are now thought to play important roles in memory (Aigner, 1995), but the nature of the involvement of cholinergic and glutamatergic systems of the hippocampus in learning and memory processes is still unclear. Both systems are involved in learning and memory, which is impaired by mucarinic (Hiramatsu et al., 1996a; Itoh et al., 1993a; Kameyama et al., 1986) and NMDA antagonists (Hiramatsu et al., 1997b; Maurice et al., 1994; Morris et al., 1986). Recent evidence suggests that the interaction of these two neurotransmitters may be important for some forms of memory and that acetylcholine, in particular, may facilitate glutamate activity by coordinating states of acquisition and recall in the cortex and hippocampus (Aigner, 1995). From our present and previous results (Hiramatsu et al., 1997b; fig. 4), kappa opioid receptor agonists could not ameliorate (+)-MK-801-induced learning and memory impairment. On the other hand, these agonists improved galanin-, carbachol- and mecamylamine-induced impairment of learning and memory and abolished the decrease of acetylcholine release induced by those drugs (Hiramatsu et al., 1996a, b, 1998, in press; figs. 2 and 6).

View larger version (22K): [in this window] [in a new window]   Fig. 11.   Possible sites of kappa opioid receptor agonists which may underlie the ameliorative effects on learning and memory. GABA, -aminobutyric acid.