- Email: [email protected]
β-Lactotensin, a neurotensin agonist peptide derived from bovine β-lactoglobulin, enhances memory consolidation in mice
peptides 28 (2007) 1470–1474
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Short communication
b-Lactotensin, a neurotensin agonist peptide derived from bovine b-lactoglobulin, enhances memory consolidation in mice Kousaku Ohinata a,d, Soushi Sonoda a, Natsumi Inoue a, Rena Yamauchi a,b,c,d, Keiji Wada b,d, Masaaki Yoshikawa a,d,* a
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan c Japan Society for Promotion of Science, Chiyoda-ku, Tokyo 104-8471, Japan d CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 322-0012, Japan b
article info
abstract
Article history:
b-Lactotensin (His-Ile-Arg-Leu) is an ileum-contracting tetrapeptide isolated from bovine
Received 2 March 2007
b-lactoglobulin. We previously reported that a neurotensin agonist b-lactotensin shows
Received in revised form
antinociceptive effect through neurotensin NT2 receptor. We found that centrally or orally
1 June 2007
administered b-lactotensin at a dose of 60 nmol/mouse or 300–500 mg/kg, respectively,
Accepted 1 June 2007
increased memory consolidation in the step-through-type inhibitory avoidance test in mice.
Published on line 8 June 2007
The memory-enhancing activity of b-lactotensin was inhibited by the dopamine D2 receptor antagonist raclopride but not the D1 receptor antagonist SCH23390. Taken together, b-lactotensin might improve memory consolidation through activating the dopamine D2 receptor. # 2007 Elsevier Inc. All rights reserved.
1.
Introduction
A number of bioactive peptides have been found from enzymatic digests of various food proteins. It is known that these peptides often show not only various physiological actions on blood pressure, lipid metabolism, pain response and food intake but also higher brain functions including memory and learning in animals and humans [21–25]. b-Lactotensin (His-Ile-Arg-Leu) is also a bioactive tetrapeptide, which we previously isolated from chymotryptic digest of bovine b-lactoglobulin using a guinea pig ileum-contracting assay [23]. We reported that b-lactotensin had antinociceptive
and cholesterol-lowering effects after central and oral administration acting as a neurotensin agonist [21–23]. Neurotensin, an endogenous tridecapeptide widely distributed in the gastrointestinal tract and the central nervous system (CNS), acts as a neuromodulator in the CNS [2,6,18]. Three subtypes of neurotensin receptors are known: NT1, NT2 and NT3. NT1 and NT2 are G-protein-coupled receptors with seven-transmembrane domains, which have high- and lowaffinity neurotensin binding sites, respectively [2,6,18]. NT3 is a receptor with a single-transmembrane domain; however, its function and signaling are unclear at present. b-Lactotensin binds to both NT1 and NT2 receptors, and its affinity for the NT2
* Corresponding author at: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan. Tel.: +81 774 38 3725; fax: +81 774 38 3774. E-mail address: [email protected] (M. Yoshikawa). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.06.002
peptides 28 (2007) 1470–1474
receptor is 50-fold higher than that of the NT1 receptor [23]. b-Lactotensin is the first natural ligand selective for NT2 receptor. The antinociceptive and hypocholesterolemic effects of b-lactotensin were mediated by the NT2 receptor [21]. In this study, we found a novel function of an NT2 agonist b-lactotensin in memory consolidation. b-Lactotensin enhanced memory consolidation after central or oral administration in the step-through-type inhibitory avoidance test in mice. Dopamine, a monoamine neurotransmitter in the brain, is known to modulate memory and learning performance. Functional and anatomical interactions between the neurotensin and dopamine systems in the CNS are well known [2,6,18]. Then, we investigated whether the memory consolidation-enhancing effect of b-lactotensin was mediated through the dopamine system using selective antagonists for dopamine D1 and D2 receptors.
1471
(ACSF; 138.9 mM NaCl, 3.4 mM KCl, 1.3 mM CaCl2, 4.0 mM NaHCO3, 0.6 mM NaH2PO4, 5.6 mM glucose, pH 7.4) was injected into the lateral ventricle. SCH23390 (0.30 mg/mouse) or raclopride (0.15 mg/mouse) was i.c.v. co-administered with b-lactotensin [14,22].
2.4.
Receptor binding assay
2.
Materials and methods
Dopamine receptor binding assay was performed using membranes from recombinant CHO cells expressed with human D1, D2L or D2S receptor, as described previously [4,7,8,26]. In the D1 receptor assay, 1.4 nM [3H] SCH23390 was incubated with the membranes in homogenizing buffer (50 mM Tris–HCl, 1.4 mM ascorbic acid, 0.001% BSA, 150 mM NaCl, pH 7.4) for 2 h at 37 8C. The radioactivity associated membrane-bound [3H] SCH23390 was measured. In the D2L or D2S receptor assay, 0.16 nM [3H] spiperone was incubated with the membranes in homogenizing buffer for 2 h at 25 8C, and the radioactivity associated with membranes was measured.
2.1.
Reagents
2.5.
b-Lactotensin (His-Ile-Arg-Leu) was synthesized by the Fmoc strategy. SCH23390 (dopamine D1 receptor antagonist) and raclopride (D2 receptor antagonist) were obtained from Tocris Bioscience (Bristol, UK) and Sigma–Aldrich Co. (St. Louis, MO), respectively.
2.2.
Data from the inhibitory avoidance test using step-through apparatus were expressed as the median and interquartile ranges. Mann–Whitney’s U-test was used for comparisons between groups and P values less than 0.05 were considered significant.
Animals
Four-week-old male ddY mice (SLC, Shizuoka, Japan) were used. Mice were housed under regulated conditions (22 8C on a 12 h light–dark cycle, lights on 07:00–19:00), and fed food pellets and water ad libitum. All experiments were approved by the University Animal Committee.
2.3.
Statistical analysis
Step-through-type inhibitory avoidance test
The inhibitory avoidance test was performed as described previously [16,25]. Briefly, the step-through apparatus consisted of light and dark compartments with a grid floor made of stainless steel rods. These rooms were connected by a hole. In the training trial, each mouse was placed into the illuminated room. Mice entered the dark compartment because they are nocturnal. Then, an electric shock (0.12– 0.14 mA) was given until the mouse returned to the light room. Just after the training trial, b-lactotensin was intracerebroventricularly (i.c.v.) or orally administrated [16,25]. The test trial, again placing the mouse in the light room, was performed 24 h after training. The latency time passed in the light compartment (step-through latency) was measured. The cutoff was set at 600 s. I.c.v. administration into the lateral ventricle was performed as described previously [16,25]. Briefly, a 28-gauge stainless steel needle attached to a 0.05 ml Hamilton syringe was inserted perpendicularly through the skull into the brain. The site of injection was 2 mm from either side of the midline on a line drawn through the anterior base of the ears [25]. bLactotensin dissolved in 4 ml artificial cerebrospinal fluid
3.
Results
3.1.
b-Lactotensin enhances memory consolidation
We investigated whether b-lactotensin enhanced memory consolidation in step-through-type inhibitory avoidance test, since neurotensin agonists were known to be associated with higher brain function [1,20]. b-Lactotensin increased step-through latency (P < 0.05) after central administration at a dose of 60 nmol/mouse (Fig. 1a). Orally administered b-lactotensin also increased step-through latency at a dose of 300–500 mg/kg (P < 0.05) in a dosedependent manner (Fig. 1b). Thus, b-lactotensin had memory-enhancing activity after oral and central administration in normal mice.
3.2. Memory-enhancing activity of b-lactotensin was mediated by dopamine D2 receptor We tested whether the memory consolidation-enhancing effect of b-lactotensin was mediated through the dopamine system using selective antagonists for dopamine D1 and D2 receptors, since the interaction between neurotensin and dopamine systems had been suggested in the CNS [2,6,18]. Dopamine D2 antagonist raclopride (0.15 mg/mouse, i.c.v.) abolished the memory-enhancing action of b-lactotensin at a dose of 60 nmol/mouse (P < 0.05); however, D1 antagonist SCH23390 (0.3 mg/mouse, i.c.v.) did not attenuate it (Fig. 2). The affinities for b-lactotensin for D1 and D2 receptors were negligible at 100 mM, suggesting that b-lactotensin did not
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peptides 28 (2007) 1470–1474
Fig. 1 – Effect of oral or central administration of blactotensin on memory consolidation. b-lactotensin at a dose of 20–60 nmol/mouse i.c.v. (a) or 100–500 mg/kg p.o. (b) was given immediately after training. Each value represents the median and interquartile ranges (n = 9). * P < 0.05 compared with control group, Mann–Whitney’s U-test.
show affinities for D1 and D2 receptors. Taken together, b-lactotensin enhances memory consolidation through dopamine release followed by activation of the D2 receptor.
4.
Discussion
We found for the first time that an NT2 agonist, b-lactotensin, derived from bovine milk protein b-lactoglobulin enhanced memory consolidation in the inhibitory avoidance test after oral and central administration in mice (Fig. 1a and b). b-Lactotensin centrally administered just after training improved step-through latency. However, it was inactive, when given 2 h after the training (data not shown), suggesting that b-lactotensin enhances memory consolidation. It is known that peripherally administered neurotensin does not cross the blood-brain barrier (BBB); however, orally active b-lactotensin might be able to cross it, because of its small molecular size. Alternatively, peripheral b-lactotensin signal might be neurally transmitted to the CNS. b-Lactotensin acts as an agonist for neurotensin receptor [21,22]. Centrally administered neurotensin alone stimulated
Fig. 2 – Effect of dopamine receptor antagonist on the enhancement of memory consolidation of b-lactotensin. SCH23390 dopamine D1 receptor antagonist (0.3 mg/ mouse, i.c.v.) or raclopride D2 receptor antagonist (0.15 mg/ mouse, i.c.v.) was co-administered with b-lactotensin (60 nmol/mouse, i.c.v.) immediately after training. Each value represents the median and interquartile ranges ((a) n = 14–15, (b) n = 8). *P < 0.05 compared with each group, Mann–Whitney’s U-test.
memory consolidation (unpublished data). It was reported that i.c.v.-administered neurotensin agonist improved scopolamine-induced amnesia though the neurotensin receptor and centrally administered neurotensin antagonist impaired spatial learning [1,20]. Taken together, neurotensin receptors might be associated with memory consolidation and learning performance. b-Lactotensin-induced memory-enhancing activity was blocked by a dopamine D2 receptor antagonist, suggesting that the memory-enhancing effect of b-lactotensin is mediated through dopamine release followed by activation of the D2 receptor. Peripherally administered drugs stimulating the D2 receptor have been reported to improve cognitive functions, and blockade of the D2 receptor impaired them [11,19]. Inhibitory avoidance is dependent on the hippocampus and amygdale [3,5,9]. Dopamine neurons are known to project to the striatum, nucleus accumbens, prefrontal cortex and amygdala [2,6,18]. The hippocampus also receives dopaminergic innervation, and dopamine is also released within the hippocampus [12,13]. Recently, the binding potential for the D2 receptor in the
peptides 28 (2007) 1470–1474
hippocampus was positively correlated with memory function using positron emission tomography (PET) [19]. There is also correlation between memory impairment and reduction of the dopamine D2 receptor binding of the hippocampus in Alzheimer’s disease patients [10]. In the striatum, we observed that b-lactotensin perfusion stimulated extracellular dopamine release in microdialysis study (data not shown). It might be possible for b-lactotensin to activate the dopamine system, in particular, the dopamine D2 receptor, in the hippocampus followed by enhancing memory consolidation. It was reported that peripheral pretraining administration of D1/D2 agonist apomorphine impaired short- and long-term retention of an inhibitory avoidance task in adult female rats [17]. Interestingly, b-lactotensin also promotes the extinction of fear memory in the fear-conditioning test in mice given considerably potent electrical shock [24]. These results might raise the possibility that b-lactotensin might modulate several steps in memory and learning system. Further investigations will elucidate the mechanisms underlying the b-lactotensininduced enhancement of memory consolidation and extinction of fear memory, including sites of actions, mediators such as dopamine and other neurotransmitters, and their receptors. We also demonstrated that anorexigenic activity of neurotensin agonist was mediated by NT1 receptor followed by activating the histamine H1 receptor [15]. Although a high dose was necessary, b-lactotensin may be a candidate for orally effective pharmaceutical drugs to enhance memory consolidation. In conclusion, we found that orally and centrally administered b-lactotensin had memory-enhancing activity, which was mediated by dopamine D2 receptor.
Acknowledgements This work was supported, in part, by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan to MY and KO, and grants from the Japan Science and Technology Agency (CREST), the Bio-oriented Technology Research Advancement Institution (PROBRAIN) and the Food Science Institute Foundation to MY.
references
[1] Azmi N, Norman C, Spicer CH, Bennett GW. Effects of a neurotensin analogue (PD149163) and antagonist (SR142948A) on the scopolamine-induced deficits in a novel object discrimination task. Behav Pharmacol 2006;17(4):357–62. [2] Caceda R, Kinkead B, Nemeroff CB. Neurotensin: role in psychiatric and neurological diseases. Peptides 2006;27(10):2385–404. [3] Cahill L, McGaugh JL. Mechanisms of emotional arousal and lasting declarative memory. Trends Neurosci 1998;21(7):294–9. [4] Dearry A, Gingrich JA, Falardeau P, Fremeau Jr RT, Bates MD, Caron MG. Molecular cloning and expression of the gene for a human D1 dopamine receptor. Nature 1990;347(6288):72–6.
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[5] Elvander-Tottie E, Eriksson TM, Sandin J, Ogren SO. NMethyl-D-aspartate receptors in the medial septal area have a role in spatial and emotional learning in the rat. Neuroscience 2006;142(4):963–78. [6] Geisler S, Berod A, Zahm DS, Rostene W. Brain neurotensin, psychostimulants, and stress—emphasis on neuroanatomical substrates. Peptides 2006;27(10):2364–84. [7] Grandy DK, Marchionni MA, Makam H, Stofko RE, Alfano M, Frothingham L, et al. Cloning of the cDNA and gene for a human D2 dopamine receptor. Proc Natl Acad Sci USA 1989;86(24):9762–6. [8] Hayes G, Biden TJ, Selbie LA, Shine J. Structural subtypes of the dopamine D2 receptor are functionally distinct: expression of the cloned D2A and D2B subtypes in a heterologous cell line. Mol Endocrinol 1992;6(6):920–6. [9] Holland PC, Bouton ME. Hippocampus and context in classical conditioning. Curr Opin Neurobiol 1999;9(2): 195–202. [10] Kemppainen N, Laine M, Laakso MP, Kaasinen V, Nagren K, Vahlberg T, et al. Hippocampal dopamine D2 receptors correlate with memory functions in Alzheimer’s disease. Eur J Neurosci 2003;18(1):149–54. [11] Kimberg DY, D’Esposito M. Cognitive effects of the dopamine receptor agonist pergolide. Neuropsychologia 2003;41(8):1020–7. [12] Li S, Cullen WK, Anwyl R, Rowan MJ. Dopamine-dependent facilitation of LTP induction in hippocampal CA1 by exposure to spatial novelty. Nat Neurosci 2003;6(5):526–31. [13] Lisman JE, Grace AA. The hippocampal-VTA loop: controlling the entry of information into long-term memory. Neuron 2005;46(5):703–13. [14] MacDonald AF, Billington CJ, Levine AS. Alterations in food intake by opioid and dopamine signaling pathways between the ventral tegmental area and the shell of the nucleus accumbens. Brain Res 2004;1018(1):78–85. [15] Ohinata K, Shimano T, Yamauchi R, Sakurada S, Yanai K, Yoshikawa M. The anorectic effect of neurotensin is mediated via a histamine H1 receptor in mice. Peptides 2004;25(12):2135–8. [16] Ohinata K, Sonoda S, Shimano T, Yoshikawa M. Enterostatin (APGPR) enhances memory consolidation in mice. Peptides 2007;28(3):719–21. [17] Picada JN, Schroder N, Izquierdo I, Henriques JA, Roesler R. Differential neurobehavioral deficits induced by apomorphine and its oxidation product, 8-oxoapomorphine-semiquinone, in rats. Eur J Pharmacol 2002;443(1–3):105–11. [18] St-Gelais F, Jomphe C, Trudeau LE. The role of neurotensin in central nervous system pathophysiology: what is the evidence? J Psychiatry Neurosci 2006;31(4):229–45. [19] Takahashi H, Kato M, Hayashi M, Okubo Y, Takano A, Ito H, et al. Memory and frontal lobe functions; possible relations with dopamine D2 receptors in the hippocampus. Neuroimage 2007;34(4):1643–9. [20] Tirado-Santiago G, Lazaro-Munoz G, Rodriguez-Gonzalez V, Maldonado-Vlaar CS. Microinfusions of neurotensin antagonist SR 48692 within the nucleus accumbens core impair spatial learning in rats. Behav Neurosci 2006;120(Oct 5):1093–102. [21] Yamauchi R, Ohinata K, Yoshikawa M. Beta-lactotensin and neurotensin rapidly reduce serum cholesterol via NT2 receptor. Peptides 2003;24(12):1955–61. [22] Yamauchi R, Sonoda S, Jinsmaa Y, Yoshikawa M. Antinociception induced by beta-lactotensin, a neurotensin agonist peptide derived from beta-lactoglobulin, is mediated by NT2 and D1 receptors. Life Sci 2003;73(15):1917–23. [23] Yamauchi R, Usui H, Yunden J, Takenaka Y, Tani F, Yoshikawa M. Characterization of beta-lactotensin, a
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bioactive peptide derived from bovine beta-lactoglobulin, as a neurotensin agonist. Biosci Biotechnol Biochem 2003;67(4):940–3. [24] Yamauchi R, Wada E, Yamada D, Yoshikawa M, Wada K. Effect of beta-lactotensin on acute stress and fear memory. Peptides 2006;27(12):3176–82.
[25] Yang S, Kawamura Y, Yoshikawa M. Effect of rubiscolin, a delta opioid peptide derived from Rubisco, on memory consolidation. Peptides 2003;24(2):325–8. [26] Zhou QY, Grandy DK, Thambi L, Kushner JA, Van Tol HH, Cone R, et al. Cloning and expression of human and rat D1 dopamine receptors. Nature 1990;347(6288):76–80.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Short communication
b-Lactotensin, a neurotensin agonist peptide derived from bovine b-lactoglobulin, enhances memory consolidation in mice Kousaku Ohinata a,d, Soushi Sonoda a, Natsumi Inoue a, Rena Yamauchi a,b,c,d, Keiji Wada b,d, Masaaki Yoshikawa a,d,* a
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan c Japan Society for Promotion of Science, Chiyoda-ku, Tokyo 104-8471, Japan d CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 322-0012, Japan b
article info
abstract
Article history:
b-Lactotensin (His-Ile-Arg-Leu) is an ileum-contracting tetrapeptide isolated from bovine
Received 2 March 2007
b-lactoglobulin. We previously reported that a neurotensin agonist b-lactotensin shows
Received in revised form
antinociceptive effect through neurotensin NT2 receptor. We found that centrally or orally
1 June 2007
administered b-lactotensin at a dose of 60 nmol/mouse or 300–500 mg/kg, respectively,
Accepted 1 June 2007
increased memory consolidation in the step-through-type inhibitory avoidance test in mice.
Published on line 8 June 2007
The memory-enhancing activity of b-lactotensin was inhibited by the dopamine D2 receptor antagonist raclopride but not the D1 receptor antagonist SCH23390. Taken together, b-lactotensin might improve memory consolidation through activating the dopamine D2 receptor. # 2007 Elsevier Inc. All rights reserved.
1.
Introduction
A number of bioactive peptides have been found from enzymatic digests of various food proteins. It is known that these peptides often show not only various physiological actions on blood pressure, lipid metabolism, pain response and food intake but also higher brain functions including memory and learning in animals and humans [21–25]. b-Lactotensin (His-Ile-Arg-Leu) is also a bioactive tetrapeptide, which we previously isolated from chymotryptic digest of bovine b-lactoglobulin using a guinea pig ileum-contracting assay [23]. We reported that b-lactotensin had antinociceptive
and cholesterol-lowering effects after central and oral administration acting as a neurotensin agonist [21–23]. Neurotensin, an endogenous tridecapeptide widely distributed in the gastrointestinal tract and the central nervous system (CNS), acts as a neuromodulator in the CNS [2,6,18]. Three subtypes of neurotensin receptors are known: NT1, NT2 and NT3. NT1 and NT2 are G-protein-coupled receptors with seven-transmembrane domains, which have high- and lowaffinity neurotensin binding sites, respectively [2,6,18]. NT3 is a receptor with a single-transmembrane domain; however, its function and signaling are unclear at present. b-Lactotensin binds to both NT1 and NT2 receptors, and its affinity for the NT2
* Corresponding author at: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan. Tel.: +81 774 38 3725; fax: +81 774 38 3774. E-mail address: [email protected] (M. Yoshikawa). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.06.002
peptides 28 (2007) 1470–1474
receptor is 50-fold higher than that of the NT1 receptor [23]. b-Lactotensin is the first natural ligand selective for NT2 receptor. The antinociceptive and hypocholesterolemic effects of b-lactotensin were mediated by the NT2 receptor [21]. In this study, we found a novel function of an NT2 agonist b-lactotensin in memory consolidation. b-Lactotensin enhanced memory consolidation after central or oral administration in the step-through-type inhibitory avoidance test in mice. Dopamine, a monoamine neurotransmitter in the brain, is known to modulate memory and learning performance. Functional and anatomical interactions between the neurotensin and dopamine systems in the CNS are well known [2,6,18]. Then, we investigated whether the memory consolidation-enhancing effect of b-lactotensin was mediated through the dopamine system using selective antagonists for dopamine D1 and D2 receptors.
1471
(ACSF; 138.9 mM NaCl, 3.4 mM KCl, 1.3 mM CaCl2, 4.0 mM NaHCO3, 0.6 mM NaH2PO4, 5.6 mM glucose, pH 7.4) was injected into the lateral ventricle. SCH23390 (0.30 mg/mouse) or raclopride (0.15 mg/mouse) was i.c.v. co-administered with b-lactotensin [14,22].
2.4.
Receptor binding assay
2.
Materials and methods
Dopamine receptor binding assay was performed using membranes from recombinant CHO cells expressed with human D1, D2L or D2S receptor, as described previously [4,7,8,26]. In the D1 receptor assay, 1.4 nM [3H] SCH23390 was incubated with the membranes in homogenizing buffer (50 mM Tris–HCl, 1.4 mM ascorbic acid, 0.001% BSA, 150 mM NaCl, pH 7.4) for 2 h at 37 8C. The radioactivity associated membrane-bound [3H] SCH23390 was measured. In the D2L or D2S receptor assay, 0.16 nM [3H] spiperone was incubated with the membranes in homogenizing buffer for 2 h at 25 8C, and the radioactivity associated with membranes was measured.
2.1.
Reagents
2.5.
b-Lactotensin (His-Ile-Arg-Leu) was synthesized by the Fmoc strategy. SCH23390 (dopamine D1 receptor antagonist) and raclopride (D2 receptor antagonist) were obtained from Tocris Bioscience (Bristol, UK) and Sigma–Aldrich Co. (St. Louis, MO), respectively.
2.2.
Data from the inhibitory avoidance test using step-through apparatus were expressed as the median and interquartile ranges. Mann–Whitney’s U-test was used for comparisons between groups and P values less than 0.05 were considered significant.
Animals
Four-week-old male ddY mice (SLC, Shizuoka, Japan) were used. Mice were housed under regulated conditions (22 8C on a 12 h light–dark cycle, lights on 07:00–19:00), and fed food pellets and water ad libitum. All experiments were approved by the University Animal Committee.
2.3.
Statistical analysis
Step-through-type inhibitory avoidance test
The inhibitory avoidance test was performed as described previously [16,25]. Briefly, the step-through apparatus consisted of light and dark compartments with a grid floor made of stainless steel rods. These rooms were connected by a hole. In the training trial, each mouse was placed into the illuminated room. Mice entered the dark compartment because they are nocturnal. Then, an electric shock (0.12– 0.14 mA) was given until the mouse returned to the light room. Just after the training trial, b-lactotensin was intracerebroventricularly (i.c.v.) or orally administrated [16,25]. The test trial, again placing the mouse in the light room, was performed 24 h after training. The latency time passed in the light compartment (step-through latency) was measured. The cutoff was set at 600 s. I.c.v. administration into the lateral ventricle was performed as described previously [16,25]. Briefly, a 28-gauge stainless steel needle attached to a 0.05 ml Hamilton syringe was inserted perpendicularly through the skull into the brain. The site of injection was 2 mm from either side of the midline on a line drawn through the anterior base of the ears [25]. bLactotensin dissolved in 4 ml artificial cerebrospinal fluid
3.
Results
3.1.
b-Lactotensin enhances memory consolidation
We investigated whether b-lactotensin enhanced memory consolidation in step-through-type inhibitory avoidance test, since neurotensin agonists were known to be associated with higher brain function [1,20]. b-Lactotensin increased step-through latency (P < 0.05) after central administration at a dose of 60 nmol/mouse (Fig. 1a). Orally administered b-lactotensin also increased step-through latency at a dose of 300–500 mg/kg (P < 0.05) in a dosedependent manner (Fig. 1b). Thus, b-lactotensin had memory-enhancing activity after oral and central administration in normal mice.
3.2. Memory-enhancing activity of b-lactotensin was mediated by dopamine D2 receptor We tested whether the memory consolidation-enhancing effect of b-lactotensin was mediated through the dopamine system using selective antagonists for dopamine D1 and D2 receptors, since the interaction between neurotensin and dopamine systems had been suggested in the CNS [2,6,18]. Dopamine D2 antagonist raclopride (0.15 mg/mouse, i.c.v.) abolished the memory-enhancing action of b-lactotensin at a dose of 60 nmol/mouse (P < 0.05); however, D1 antagonist SCH23390 (0.3 mg/mouse, i.c.v.) did not attenuate it (Fig. 2). The affinities for b-lactotensin for D1 and D2 receptors were negligible at 100 mM, suggesting that b-lactotensin did not
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peptides 28 (2007) 1470–1474
Fig. 1 – Effect of oral or central administration of blactotensin on memory consolidation. b-lactotensin at a dose of 20–60 nmol/mouse i.c.v. (a) or 100–500 mg/kg p.o. (b) was given immediately after training. Each value represents the median and interquartile ranges (n = 9). * P < 0.05 compared with control group, Mann–Whitney’s U-test.
show affinities for D1 and D2 receptors. Taken together, b-lactotensin enhances memory consolidation through dopamine release followed by activation of the D2 receptor.
4.
Discussion
We found for the first time that an NT2 agonist, b-lactotensin, derived from bovine milk protein b-lactoglobulin enhanced memory consolidation in the inhibitory avoidance test after oral and central administration in mice (Fig. 1a and b). b-Lactotensin centrally administered just after training improved step-through latency. However, it was inactive, when given 2 h after the training (data not shown), suggesting that b-lactotensin enhances memory consolidation. It is known that peripherally administered neurotensin does not cross the blood-brain barrier (BBB); however, orally active b-lactotensin might be able to cross it, because of its small molecular size. Alternatively, peripheral b-lactotensin signal might be neurally transmitted to the CNS. b-Lactotensin acts as an agonist for neurotensin receptor [21,22]. Centrally administered neurotensin alone stimulated
Fig. 2 – Effect of dopamine receptor antagonist on the enhancement of memory consolidation of b-lactotensin. SCH23390 dopamine D1 receptor antagonist (0.3 mg/ mouse, i.c.v.) or raclopride D2 receptor antagonist (0.15 mg/ mouse, i.c.v.) was co-administered with b-lactotensin (60 nmol/mouse, i.c.v.) immediately after training. Each value represents the median and interquartile ranges ((a) n = 14–15, (b) n = 8). *P < 0.05 compared with each group, Mann–Whitney’s U-test.
memory consolidation (unpublished data). It was reported that i.c.v.-administered neurotensin agonist improved scopolamine-induced amnesia though the neurotensin receptor and centrally administered neurotensin antagonist impaired spatial learning [1,20]. Taken together, neurotensin receptors might be associated with memory consolidation and learning performance. b-Lactotensin-induced memory-enhancing activity was blocked by a dopamine D2 receptor antagonist, suggesting that the memory-enhancing effect of b-lactotensin is mediated through dopamine release followed by activation of the D2 receptor. Peripherally administered drugs stimulating the D2 receptor have been reported to improve cognitive functions, and blockade of the D2 receptor impaired them [11,19]. Inhibitory avoidance is dependent on the hippocampus and amygdale [3,5,9]. Dopamine neurons are known to project to the striatum, nucleus accumbens, prefrontal cortex and amygdala [2,6,18]. The hippocampus also receives dopaminergic innervation, and dopamine is also released within the hippocampus [12,13]. Recently, the binding potential for the D2 receptor in the
peptides 28 (2007) 1470–1474
hippocampus was positively correlated with memory function using positron emission tomography (PET) [19]. There is also correlation between memory impairment and reduction of the dopamine D2 receptor binding of the hippocampus in Alzheimer’s disease patients [10]. In the striatum, we observed that b-lactotensin perfusion stimulated extracellular dopamine release in microdialysis study (data not shown). It might be possible for b-lactotensin to activate the dopamine system, in particular, the dopamine D2 receptor, in the hippocampus followed by enhancing memory consolidation. It was reported that peripheral pretraining administration of D1/D2 agonist apomorphine impaired short- and long-term retention of an inhibitory avoidance task in adult female rats [17]. Interestingly, b-lactotensin also promotes the extinction of fear memory in the fear-conditioning test in mice given considerably potent electrical shock [24]. These results might raise the possibility that b-lactotensin might modulate several steps in memory and learning system. Further investigations will elucidate the mechanisms underlying the b-lactotensininduced enhancement of memory consolidation and extinction of fear memory, including sites of actions, mediators such as dopamine and other neurotransmitters, and their receptors. We also demonstrated that anorexigenic activity of neurotensin agonist was mediated by NT1 receptor followed by activating the histamine H1 receptor [15]. Although a high dose was necessary, b-lactotensin may be a candidate for orally effective pharmaceutical drugs to enhance memory consolidation. In conclusion, we found that orally and centrally administered b-lactotensin had memory-enhancing activity, which was mediated by dopamine D2 receptor.
Acknowledgements This work was supported, in part, by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan to MY and KO, and grants from the Japan Science and Technology Agency (CREST), the Bio-oriented Technology Research Advancement Institution (PROBRAIN) and the Food Science Institute Foundation to MY.
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