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Ajinomoto Company Incorporated, Institute of Life Sciences, 210-8681 Kawasaki, Japan
1To whom correspondence should be addressed. Email: miroslav_smriga{at}ajinomoto.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • L-lysine • serotonin • stress • anxiety • colon • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and affects norepinephrine activity in the hypothalamus (2
). Of all indispensable amino acids (AA), 2 Lys is the one most strongly conserved, due to its capacity for storage and slower catabolism (3
). In spite of that, Hrupka et al. (4
) showed that rats are sensitive to changes as small as 0.01% in dietary Lys concentration, when adapted to a Lys-deficient diet. The neural modifications that have been detected consistently in Lys-deficient rats (5
) and the participation of Lys and its metabolites in normal brain functions (6
) raise the possibility of psychobehavioral alterations during Lys deficiency. The human parallels of partial Lys deficiency (7
) make this area worthy of detailed consideration.
On the basis of the effects of other indispensable AA deficiencies on the amygdala region of the brain (8
,9
), we hypothesized that Lys deficiency compromises the circadian pattern of amygdala serotonin (5-HT) activity. Thus, we studied the effects of immediate (the first 24 h) and short-term (4 d) Lys deficiency on circadian 5-HT and dopamine releases in the central nucleus of the amygdala (CeA). Because the amygdalo-frontal 5-HT pathway is instrumental in mood, stress and anxiety regulations (10
), we also tested two psychobehavioral parallels of 5-HT modification, stress-induced anxiety and stress-induced colonic transit. The time periods of Lys deficiency were based on differences in food intake. No changes in daily food intake volume were found within 24 h after a Lys-deficient diet was introduced, whereas a significant decline was found during d 4 of deficiency (2
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Wistar rats (body weight, 250–280 g) (Charles River Japan, Tokyo, Japan) were housed individually in conventional hanging cages (12-h light:dark cycle; dark period, 1900–0700 h). Rats consumed ad libitum distilled water and a control diet formulated as described (2
). Rats were implanted with guide cannulae for the microdialysis probes (Eicom, Kyoto, Japan). The cannulae were placed just above the left central nucleus of the central nucleus of amygdala (CeA), as described in detail (11
). Rats were allowed to recover for 1 wk. They were treated in compliance with the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For the behavioral testing, sound-attenuated, ventilated operant boxes were used. Early in the morning on the day of testing (08:00), a dialysis probe (0.22-mm o.d., 50,000 MW cut-off; length, 2 mm) was inserted into the guide cannulae and secured with a screw. Immediately thereafter, rats, which had been divided into three groups of four were placed into the operant boxes and given food and water. Group 1 was given the Lys-deficient diet for the first time at 0800 h on the day of experiment (5 h before the start of recording) and was allowed to consume the food ad libitum. Group 2 consumed the Lys-deficient diet ad libitum starting 3 d before the experiment; thus, the day of experiment was d 4 of deficiency. Group 3 (control rats) was pair-fed the control diet in an amount equaling the mean food intake of the 4-d Lys-deficient group.
Microdialysis recording started at 1300 h (5 h after the probe insertion and diet access). Data were collected every 30 min and averaged over 1-h periods. The dialysis probe was perfused at a rate of 1.0 µL/min with a modified Ringer’s solution (147 mmol/L NaCl, 10 mmol/L KCl, 1.1 mmol/L CaCl2 · 2H2O, 1.1 mmol/L MgCl2 · 6H2O, pH 6.0). The outflow was connected by a teflon tube to a HPLC auto-injector system (Eicom). 5-HT and dopamine were separated on an Eikompak CA-5 ODS column (5 mm, 4.6 x 150 mm; Eicom), using a solution of phosphate buffer, octane-sulfonic acid, EDTA and methanol as the mobile phase (flow rate, 1.0 mL/min). The working electrode was set at +450 mV against a silver-silver chloride reference electrode. At the end of the experiments, the rats were anesthetized with ether, trunk blood was collected for AA analysis and the brains were fixed with a formalin (10%) solution. Brains were kept at 4°C in several changes of 0.44 mol/L sucrose and 0.10 mol/L phosphate buffer for 5 d. Brain sections were cut on a cryotome and stained with toluidine blue. All rats had a probe placed successfully into the CeA. Group differences were evaluated by one-way ANOVA followed by Dunnett’s test. Differences were considered significant at P < 0.05.
Behavioral study (anxiety).
The elevated plus-maze (EPM) was constructed of dark-gray polyacrylic resin, and consisted of 4 arms (each 50 cm long and 10 cm wide) arranged in a shape of a plus sign and elevated 90 cm above the floor. Two opposite arms were open and 40-cm high walls protected the other two. All arms were interconnected with a 10 cm x 10 cm central area. This area was designed as a "protected" area (offering relative security), similar to closed arms. Behavior was recorded via a computerized video camera (NTSC, Sony, Tokyo, Japan) mounted above the center of the maze. Speed, time spent in the open vs. closed arms, the total path length and number of entries into each arm were recorded using analysis software (SMART, Panlab, Barcelona, Spain). Additional measures comprised frequency scores for rearing in the closed arms (vertical movement against the side and/or end of the walls) and stretched-attend exploratory postures in the open arms. An experienced person, unaware of treatment group, evaluated these measures. A curtain surrounded the whole apparatus so that the rats could not see the experimenter, who observed their behavior on a computer screen. To encourage exploration in the open arms, the room was only dimly lit. The maze was cleaned thoroughly with water containing a detergent before a new rat was exposed to it. Exposure started by placing the rat on the central area facing a closed arm. To avoid significant influences of circadian rhythm, all experiments were conducted between 1130 and 1430 h.
Seven-week-old male Wistar rats (Charles River Japan, Tokyo, Japan), different from the rats used in the first experiment, were divided into two groups of 20 and fed the control diet or the Lys-deficient diet for 3 d before testing. Diets were formulated as described (2
). Controls were fed the Lys-sufficient diet in an amount equaling the mean food intake of the Lys-deficient group. On the day of experiment, both groups were further divided into two subgroups of 10 rats. One subgroup of each diet group was subjected to footshock 5 min before testing in the EPM. Footshock consisted of a 1-mA electric shock (duration, 3 s) delivered to the floor of the noise-isolated cage 5 times, with intrastimulus intervals of 10 s.
All rats were tested on the EPM at 10-min intervals. The first minute of testing was not included in the statistical analysis, to discard possible neophobic responses to the EPM. Immediately after the test, rats were returned to their home cages and blood was collected during the following 5-min period, 
25min after footshock exposure. Plasma corticosterone concentration was analyzed by 125I RIA (Diagnostic Products, Los Angeles, CA), as described (11
). Diet and stress-exposure differences were evaluated by two-way ANOVA followed by Duncan’s multiple range test. Differences were considered significant at P < 0.05.
Behavioral study (stress-induced colonic transit).
Male Wistar rats (body weight, 250–280 g) (Charles River Japan) were housed individually in conventional hanging cages (12-h light:dark cycle; dark period, 1900–0700 h). Rats were divided into two groups of 8. One group consumed the Lys-deficient diet for 6 d and the other was pair-fed the control diet as described above. On d 7, rats were subjected to wrap-restraint stress (12
) for 150 min (1300–1530 h). Fecal excretion (weight of faces) was measured in four intervals (0–30, 30–60, 60–120 and 120–150 min). Time and group differences were evaluated by two-way ANOVA followed by Duncan’s multiple range test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) were characterized by the same circadian pattern in CeA 5-HT activity as control rats. Rats fed the Lys-deficient diet for 3 d before the start of experiment (Group 2) (Fig. 1)
had greater CeA 5-HT release at specific time points from 0100 h onward compared with rats fed the control diet. The effect was canceled by refeeding the Lys-deficient rats with control diet for 24 h (n = 4, data not shown). No Lys-dependent differences in circadian CeA dopamine release were recorded (data not shown).
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). The Lys-deficient diet did not affect plasma corticosterone, measured 5 min after EPM exposure (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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); therefore, taste differences or a nonspecific effect of neophobia did not cause the above differences. Although the whole-brain content of Lys has been shown to be lower in Lys-deficient rats, no specific modifications were found in the amygdala (1
,8
). Hence, inhibition of protein synthesis (due to the low content of Lys) is improbable, but direct effects of Lys and/or its metabolites (13
) on 5-HT neurotransmission in the CeA, or in other functionally interconnected brain areas, are possible. It is a limitation of this study that no specific biochemical interaction between Lys dietary status and the CeA 5-HT was established. It remains to be elucidated whether the observed changes were due to a decreased uptake, or an enhanced release of 5-HT.
Ligand binding studies of Jiang and Gietzen (14
) and Terry-Nathan et al. (15
) have suggested that 5-HT is involved in the anorectic response to an AA-deficient diet. Moreover, Meliza et al. (16
) and Wang et al. (9
) found a specific involvement of the CeA in the learned aversion to a threonine-imbalanced diet. Thus, it is possible that the 5-HT system in the CeA is involved in anorectic responses to all indispensable AA deficiencies. Our data support the hypothesis of Gietzen et al. (8
) that the CeA 5-HT is involved in the conditioned taste aversion response to an AA-deficient diet. Presently, the most feasible explanation for the observed results involves a complex activation of neural pathways from the brain’s chemosensor, the anterior piriform cortex, to the amygdala (8
). The amygdala has been implicated in emotion regulation, including food preferences, fear and responses to stress. According to the 5-HT hypothesis, increases in the activity of CeA 5-HT, as we observed in Lys-deficient rats, have anxiogenic effects (17
,18
).
In part two of the study, the above hypothesis was tested using the EPM apparatus (19
). In addition to conventional measures, we measured rearing in the closed arms, which is a normal exploratory behavior directed into the closed arms, and stretched-attend exploratory posture in the open arms, a behavior displayed in stressful situations (20
). The stretched-attend exploratory posture characterizes approach-avoid conflict, which optimizes the stress-adaptive behavior, and is sensitive to anxiolytic drugs (20
). In line with the 5-HT hypothesis (18
) and the increased CeA 5-HT activity in the Lys-deficient rats, footshock triggered an anxiogenic effect in Lys-deficient rats but not in controls.
Stress plays a major role in functional intestinal disorders. Stimulation of colonic transit is the most consistent pattern in the motility response of the intestinal tract to acute stress (21
). A strong positive relationship between stress-induced anxiety and irritable bowel syndrome was reported in humans (22
). On the basis of the results of the previous parts of this study, we hypothesized that Lys-deficient rats are characterized by greater wrap-restraint stress-stimulated fecal excretion, than their normally fed counterparts. The hypothesis was confirmed during h 2 of wrap-restraint stress exposure. This may have been associated with the increased CeA 5-HT due to Lys deficiency, or Lys deficiency may have triggered a direct 5-HT activation in the colon, similar to the changes in the CeA (23
).
In summary, the rats fed a Lys-deficient diet for 4 d, compared with normally fed rats, were characterized by increases in circadian release of CeA 5-HT and stress-induced anxiety and fecal excretion. Chronic intake of a Lys-deficient diet affects the amygdala 5-HT and alters complex behaviors, such as stress perception. The need for further studies is emphasized by human parallels of Lys deficiency (7
).
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FOOTNOTES |
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Manuscript received 22 July 2002. Initial review completed 22 August 2002. Revision accepted 9 September 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Mori, M., Kawada, T., Ono, T. & Torii, K. (1991) Taste preference, protein nutrition and L-amino acid homeostasis in male Sprague-Dawley rats. Physiol. Behav. 49:987-996.[Medline]
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6. Bernasconi, R., Jones, R. S., Bittiger, H., Olpe, H. R., Heid, J., Martin, P., Klein, M., Loo, P., Braunwalder, A. & Schmutz, M. (1986) Does pipecolic acid interact with the central GABA-ergic system?. J. Neural Transm. 67:175-189.
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9. Wang, Y., Cummings, S. L. & Gietzen, D. W. (1996) Temporal-spatial pattern of c-fos expression in the rat brain in response to indispensable amino acid deficiency. Mol. Brain. Res. 40:35-41.[Medline]
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14. Jiang, J. C. & Gietzen, D. W. (1994) Anorectic response to amino acid imbalance: a selective serotonin3 effect?. Pharmacol. Biochem. Behav. 47:59-63.[Medline]
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16. Meliza, L. L., Leung, P.M.B. & Rogers, Q. R. (1981) Effect of anterior prepyriform and medial amygdaloid lesions on acquisition of taste-avoidance and response to dietary amino acid imbalance. Physiol. Behav. 26:1031-1035.[Medline]
17. Chung, K. K., Martinez, M. & Herbert, J. (2000) c-fos expression, behavioural, endocrine and autonomic responses to acute social stress in male rats after chronic restraint: modulation by serotonin. J. Neurosci. 95:453-463.
18. Gardner, C. R. (1986) Recent developments in 5HT-related pharmacology of animal models of anxiety. Pharmacol. Biochem. Behav. 24:1479-1485.[Medline]
19. Hogg, S. (1996) A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacol. Biochem. Behav. 54:21-30.[Medline]
20. Rodgers, R. J., Haller, J., Holmes, A., Halasz, J., Walton, T. J. & Brain, P. F. (1999) Corticosterone response to the plus-maze: high correlation with risk assessment in rats and mice. Physiol. Behav. 68:47-53.[Medline]
21. Monnikes, H., Tebbe, J. J., Hildebrandt, M., Arck, P., Osmanoglou, E., Rose, M., Klapp, B., Wiedenmann, B. & Hyemann-Monnikes, I. (2001) Role of stress in functional gastrointestinal disorders. Dig. Dis. 19:201-211.[Medline]
22. Lydiard, R. B. (1997) Anxiety and the irritable bowel syndrome. J. Clin. Psychiatry 58:51-58.
23. Borman, R. A., Tilford, N. S., Harmer, D. W., Day, N., Ellis, E. S., Sheldrick, R.L.G., Carey, J., Coleman, R. A. & Baxter, G. S. (2002) 5-HT2B receptors play a key role in mediating the excitatory effects of 5-HT in human colon in vitro. Br. J. Pharmacol. 135:1144-1151.[Medline]
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