Effects of systemic administration of β-casomorphin-5 on learning and memory in mice
Introduction
β-Casomorphins belong to a family of opioid peptides derived from food protein. Bovine β-casomorphin-7 (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) was first isolated from an enzymatic digest of bovine β-casein (Brantl et al., 1979). Evidence for the liberation of β-casomorphin fragments from casein by gastrointestinal proteases under in vivo (Svedberg et al. 1985; Meisel and Frister 1989) and in vitro (Jinsmaa and Yoshikawa, 1999) conditions has already been obtained. These peptides have been found to act as μ-opioid receptor agonists in isolated organs (Schlimme and Meisel, 1995) and binding experiments (Chang et al., 1981). β-Casomorphins seem to cross different barriers in the body, including brush-border membranes (Mahe et al., 1989) and the blood–brain barrier (Ermisch et al. 1985; Banks and Kastin 1987). Shorter fragments, such as the N-terminal pentapeptide (bovine β-casomorphin-5), were also found to be active, in fact more active than β-casomorphin-7 (Brantl et al., 1981). Intracerebroventricular administration of β-casomorphin-5 is known to have an antinociceptive effect (Chang et al., 1982). In addition, systemic administration of β-casomorphin-5, but not β-casomorphin-4 or β-casomorphin-7, caused hypoalgesia in 10-day-old rats, reportedly through central mechanisms (Blass and Blom, 1996).#
It is now known that opioid neuronal systems play an important role in the learning and memory processes (Messing et al. 1979; Gallagher 1982). In general, endogenous μ- and δ-opioid receptor agonists have been reported to impair learning and memory (Izquierdo and Netto 1985; Rigter et al. 1979), whereas endogenous κ-opioid receptor agonists ameliorate the impairment of memory (Itoh et al., 1994). Apart from its possible role as an inhibitor, the μ-opioid receptor may play a positive role in learning and memory. We previously demonstrated that intracerebroventricular injection of a high dose of β-casomorphin-5 (10 μg/mouse) induces amnesia, whereas a low dose (0.5 μg/mouse) ameliorates scopolamine-induced amnesia using step-down-type passive avoidance tasks in mice (Sakaguchi et al., 2003). In the present paper, we report the ameliorating effects of systemic administration of β-casomorphin-5 on scopolamine-induced impairment of spontaneous alternation behavior (spatial short-term memory) and passive avoidance response (long-term memory) in mice.#
Materials and methods
Animals
Six-week-old male ddY mice (Nihon SLC, Japan) weighing about 28–33 g were kept in a controlled environment (24ą1 °C, 55ą5% humidity), with a 12-h light/12-h dark cycle (lights on, 6:00 a.m.–6:00 p.m.) and given food and tap water freely. The mice were kept at least 7 days in home cages before starting experiments. Experimental protocols concerning the use of laboratory animals were approved by the Experimental Animal Committee of the Osaka University of Pharmaceutical Sciences.#
Drugs and injection procedure
β-Casomorphin-5 (Tyr-Pro-Phe-Pro-Gly), (Peptide Institute, Minoh, Japan), β-funaltrexamine (Tocris, Ellisville, MO, USA), naloxonazine (Sigma, St. Louis, MO, USA), and scopolamine (Wako Pure Chemicals, Osaka, Japan) were dissolved in saline. β-Casomorphin-5 was injected intraperitoneally (i.p.) in a volume of 100 μl/30 g of body weight. The intracerebroventricular (i.c.v.) injections of β-funaltrexamine and naloxonazine were made according to the methods of Haley and McCormick (1957) and Maurice et al. (1998) with slight modifications. In brief, each mouse was anesthetized lightly with ether, and a 30-gauge stainless-steel needle (Nipro, Tokyo, Japan), 4 mm long, was inserted unilaterally 1 mm to the right of the midline point equidistant from each eye, at an equal distance between the eyes and the ears and perpendicular to the plane of the skull. Drug solutions (5 μl) were delivered gradually within approximately 3 s by a 50 μl microsyringe (705LT; Hamilton Co., Reno, NV, USA). The administration site was checked by injecting Indian ink in preliminary experiments. Neither insertion of the needle nor injection of the vehicle had a significant influence on survival, behavioral responses or cognitive functions. Scopolamine was injected subcutaneously (s.c.) in a volume of 100 μl/30 g of body weight.#
Spontaneous alternation behavior test
Spatial short-term memory was assessed by recording spontaneous alternation behavior in a Y-maze according to the method of Itoh et al. (1994). The maze was made of black painted wood. Each arm was 40 cm long, 12 cm high, 3 cm wide at the bottom and 10 cm wide at the top, and they converged at equal angles. Each mouse, new to the maze, was placed in one arm and allowed to explore the Y-maze for a period of 8 min. Arm choices, including returns to the same arm, were recorded visually, and three consecutive choices of three different arms were counted as an alternation. Thus, the percentage of alternation was determined by dividing the total number of alternations by the total number of choices minus 2. Mice that completed only 8 arm entries or less within 8 min were excluded from further analysis (fewer than 5% of the mice were thus excluded). β-Casomorphin-5 and scopolamine were administered 15 and 30 min before the session, respectively.#
Step-down-type passive avoidance test
The effects of β-casomorphin-5 on non-spatial long-term memory were also investigated using step-down-type passive avoidance tasks according to the method of Ukai et al. (2001b) with some modifications. The dark compartment of the step-through passive avoidance apparatus for rats (Cat.7550, Ugo Basile, Comerio, Italy) was substituted for the step-down passive avoidance apparatus. The apparatus consisted of a cage made of black Perspex panels (23×21×22 cm high) with a grid floor, inserted in a semi-soundproof outer box (40×90×100 cm high). The cage was illuminated with a 30 W lamp during the experimental period. A wooden platform (4×4×4 cm) was fixed at the center of the grid floor. Electric shocks (0.3 mA) were delivered to the grid floor. The test consisted of a training session and a retention session done 24 h after the training. During the training session, each mouse was placed on the platform. When it stepped down and placed its four paws on the grid floor, an electric shock was delivered for 9 s. The retention test was performed in a similar manner, except that no electric shock was applied to the grid floor. Each mouse was placed again on the platform, and the step-down latency (SDL) was recorded, with an upper cut-off time of 300 s. Animals that showed an SDL in the criterion range (3–20 s) during the training session were used for the retention test. (More than 90% of the animals met this criterion.) Mice presenting three times or less with both flinches and vocalizations in the training session were considered to be poorly sensitized to the test and were excluded from further analysis (fewer than 5% of mice were thus excluded). Scopolamine and β-casomorphin-5 were administered 30 min before and immediately after the training session, respectively. β-Funaltrexamine and naloxonazine were injected 24 h before training.#
Statistical analysis
Data from spontaneous alternation behavioral tests were expressed as the meanąS.E.M. The SDL was expressed as the median, first, and third quartiles. Statistical comparisons were made by the Kruskal–Wallis non-parametric one-way analysis of variance followed by Bonferroni test. Differences in values were considered significant when P<0.01 or P<0.05.#
Results
Effect of β-casomorphin-5 on spontaneous alternation behavior
The effects of intraperitoneal administration of β-casomorphin-5 on spontaneous alternation behavior were investigated in mice. In the Y-maze test, β-casomorphin-5 (0.1–20 mg/kg) did not have any marked effects on the percent alternation (H=0.95, not significant, Fig. 1A) or total arm entries (H=5.16, not significant, Fig. 1B).#
Effects of β-casomorphin-5 on scopolamine-induced impairment of spontaneous alternation behavior
Scopolamine (1 mg/kg, s.c.) significantly decreased the percent alternation (Fig. 2A). β-Casomorphin-5 (1 mg/kg, i.p.) significantly improved the scopolamine-induced impairment of spontaneous alternation (H=19.39, P<0.01, Fig. 2A). Scopolamine also increased the total number of arm entries (Fig. 2B). β-Casomorphin-5 (0.1–3 mg/kg, i.p.) had no apparent effect on increases in the total number of arm entries induced by scopolamine, though 10 mg/kg of β-casomorphin-5 decreased the number (H=29.05, P<0.01, Fig. 2B).#
Effects of β-casomorphin-5 on passive avoidance response
Next, we investigated the effects of systemic administration of β-casomorphin-5 on passive avoidance response. β-Casomorphin-5 (0.1–20 mg/kg, i.p.) did not have a significant effect on passive avoidance response (H=6.37, not significant, Fig. 3). We previously reported that the intracerebroventricular injection of β-casomorphin-5 impaired the passive avoidance response, which was mediated through μ-opioid receptors (Sakaguchi et al., 2003). In this study systemic administration of β-casomorphin-5 did not have a significant effect.#
Effect of β-casomorphin-5 on scopolamine-induced impairment of passive avoidance response
In order to test whether or not intraperitoneal administration of β-casomorphin-5 also ameliorates amnesia in the passive avoidance response, we further investigated its effect on the scopolamine-induced impairment of the passive avoidance response. Scopolamine (1 mg/kg, s.c.) significantly shortened the SDL. β-Casomorphin-5 (0.05–10 mg/kg, i.p.) administered immediately after training reversed the scopolamine-induced impairment with a bell-shaped curve, and the effect at 1 mg/kg was significant (H=37.43, P<0.01, Fig. 4). To elucidate the mechanism of amelioration by β-casomorphin-5, the effects in combination with β-funaltrexamine (a μ-opioid receptor antagonist) or naloxonazine (a μ1-opioid receptor antagonist) on passive avoidance response were investigated. β-Funaltrexamine has been found to increase retention of passive-avoidance conditioning (Gallagher, 1985). In our experimental condition, a 5 μg dose of β-funaltrexamine (i.c.v.) markedly improved scopolamine-induced impairment (Fig. 5A), whereas a 0.1 μg dose of β-funaltrexamine (i.c.v.) did not. β-Funaltrexamine (0.1 μg, i.c.v.) almost completely prevented the effect of β-casomorphin-5 (1 mg/kg, i.p.) (Fig. 5A). Furthermore, naloxonazine (5 μg, i.c.v.), which did not influence the scopolamine-induced amnesia, also prevented the effect of β-casomorphin-5 (1 mg/kg, i.p.) (Fig. 5B).#
Discussion
The present results suggest that the intraperitoneal administration of β-casomorphin-5 alone had no effect on both spontaneous alternation behavior and passive avoidance response. Interestingly, the results also showed that a low dose of β-casomorphin-5 (1.0 mg/kg, i.p.) alleviated the scopolamine-induced impairment in both tests. These results extend our previous findings that injection of a high dose of β-casomorphin-5 (10 μg/mouse, i.c.v.) into the central nervous system induces amnesia in a β-funaltrexamine (a μ-opioid receptor antagonist)-reversible manner, whereas a low dose (0.5 μg/mouse, i.c.v.) ameliorates scopolamine-induced amnesia using step-down-type passive avoidance tasks in mice (Sakaguchi et al., 2003). When the data is taken together, β-casomorphin-5 was suggested to improve the impairment of both the short and long-term memory resulting from cholinergic dysfunction. The present findings also indicate that circulating β-casomorphin-5 reaches the central nervous system (CNS). These results are in accordance with the reports that β-casomorphins can cross the blood–brain barrier (Ermisch et al. 1985; Banks and Kastin 1987; Blass and Blom 1996). However, the systemic administration of β-casomorphin-5 does not induce amnesia in the present study, indicating that sufficient β-casomorphin-5 might not reach the brain to induce amnesia when administered i.p. The transport of β-casomorphin across the blood–brain barrier is known to be a saturable process (Banks and Kastin, 1987). In the present report the anti-amnesic effect of β-casomorphin-5 in the passive avoidance test was almost completely prevented by i.c.v. pretreatment with β-funaltrexamine and naloxonazine (a μ1-opioid receptor antagonist), indicating that β-casomorphin-5 acts through a central mechanism involving μ1-opioid receptors.#
β-Casomorphins are known to affect the nervous system, including anti-nociception (Brantl et al. 1981; Chang et al. 1982; Blass and Blom 1996) and spontaneous behavior (Maklakova et al., 1993). Therefore, administration of β-casomorphin-5 may alter sensitivity to electric shocks and locomotor activity, and these effects may alter the behavioral test conditions in a nonspecific manner. In the Y-maze test, the total number of arm entries after β-casomorphin-5 injection indicated that this peptide had no effect on locomotor activity (Fig. 1B). The analgesic activity of β-casomorphin-5 was investigated using electrical stimulation of the rat tail and vocalization or the hot water tail-flick test (Brantl et al. 1981; Chang et al. 1982). These reports described the effective doses of β-casomorphin-5 injected i.c.v. under analgesia as ranging from 10 to 200 nmoles per animal. We previously reported that an ameliorating effect of β-casomorphin-5 was seen at just 0.5 μg (0.86 nmoles, i.c.v.) per mouse, though how much β-casomorphin-5 reached the CNS after intraperitoneal administration was unclear in this study. Furthermore, we evaluated the effects of post-training administration of β-casomorphin-5 on a passive avoidance task. Therefore, the possibility that β-casomorphin-5 influences performance during acquisition or retention testing could be excluded.#
The involvement of opioid receptors could be due to their interaction with other systems known to modulate learning and memory (McGaugh and Cahill, 1997). There is a consensus that brain cholinergic neurotransmission plays a critical role in the processes underlying learning and memory (Everitt and Robbins 1997; Sarter and Bruno 1997). It is well-known that interaction between opioids and the cholinergic system affects memory performance. Moreover, it has also been reported that μ- and δ-opioid receptors are located on cholinergic terminals, which are normally tonically inhibited by the opioid system (Heijina et al., 1990). A μ-opioid receptor ligand (DAMGO) affected cholinergic release in the rodent brain (Gazyakan et al., 2000), and a muscarinic agonist, oxotremorine, is known to antagonize morphine-induced memory impairment (Li et al., 2001). Meanwhile the possibility that the μ-opioid receptor plays a positive role in learning and memory is becoming apparent. For example, the intrahippocampal injection of a μ-opioid receptor antagonist β-funaltrexamine in rats (Meilandt et al., 2004) and μ-opioid receptor gene deletions in mice (Jang et al. 2003; Jamot et al. 2003) impair spatial water maze learning, and significant reductions in [3H] DAMGO binding of μ-opioid receptor in the subiculum and hippocampus of Alzheimer's disease brain were reported (Mathieu-Kia et al., 2001). Also, the intracerebroventricular administration of a low dose (0.03 μg) of endomorphin-1 (an endogenous μ-opioid receptor agonist) improved the scopolamine-induced impairment of spontaneous alternation behavior through the mediation of μ1-opioid receptors (Ukai et al., 2001a) whereas a high dose (10 μg) by itself impaired learning and memory in rodents (Ukai et al., 2001b). Taken together, these findings, including those of the present study, suggest that the effect of an exogenously administered μ-opioid receptor agonist on learning and memory depend on the dose.#
The ameliorating effect of the low dose (1 mg/kg, i.p.) of β-casomorphin-5 on scopolamine-induced amnesia cannot be explained by the inhibitory action of the opioid on cholinergic neurotransmission. At present, the mechanisms for the anti-amnesic effects of β-casomorphin-5 are not clear, except for the involvement of the μ1-opioid receptors in the brain. Long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus are widely regarded to be a cellular basis for learning and memory processes. It was demonstrated that a lack of μ-opioid receptors decreased LTP in the dentate gyrus of the hippocampus (Matthies et al., 2000). In addition, some pharmacological studies showed the involvement of the μ-opioid receptor in mossy fiber LTP induction (Bramham 1992; Derrick et al. 1992). Furthermore, μ-opioid agonist-mediated facilitation of LTD in rat hippocampus was reported (Wagner et al., 2001). Therefore, a low dose of β-casomorphin-5 (1 mg/kg, i.p.) may enhance synaptic plasticity in the hippocampus, although at high dose (10 μg/mouse, i.c.v.) the inhibitory action on cholinergic neurotransmission exceeds the positive effect on learning and memory.#
In summary, we demonstrated that systemic administration of a low dose of β-casomorphin-5 (1.0 mg/kg, i.p.) improves the disturbance of learning and memory resulting from cholinergic dysfunction through central mediation involving μ1-opioid receptors. Our results support the possibility that the μ-opioid receptor plays a positive role in learning and memory.#
Figures and Tables
References
10. B.J.EverittT.W.RobbinsCentral cholinergic systems and cognitionAnnu. Rev. Psychol.481997649684
17. I.IzquierdoC.A.NettoRole of β-endorphin in behavioral regulationAnn. N.Y. Acad. Sci.4441985162177