n-3 Polyunsaturated fatty acid supplementation reverses stress-induced modifications on brain monoamine levels in mice
2007; Elsevier BV; Volume: 49; Issue: 2 Linguagem: Inglês
10.1194/jlr.m700328-jlr200
ISSN1539-7262
AutoresSylvie Vancassel, Samuel Leman, L. Hanonick, Stéphanie Denis, Jérôme E. Roger, Mathieu Nollet, Sylvie Bodard, Isabelle Kousignian, Catherine Belzung, Sylvie Chalon,
Tópico(s)Stress Responses and Cortisol
ResumoThe aim of this study was to examine the effects of supplementation with n-3 polyunsaturated fatty acids (PUFAs) on stress responses in mice subjected to an unpredictable chronic mild stress (UCMS) procedure. Stress-induced modifications in coat and aggressiveness were evaluated, and phospholipid PUFA profiles and monoamine levels were analyzed in the frontal cortex, hippocampus, and striatum. The results showed that repeated exposure to mild stressors induced degradation in the physical state of the coat, lowered body weight gain, and increased aggressiveness, without any effect of n-3 PUFA supplementation. The UCMS induced a significant decrease in the levels of norepinephrine in the frontal cortex and striatum, and a nonsignificant decrease in the hippocampus. The tissue levels of serotonin (5-HT) were 40% to 65% decreased in the three brain regions studied. Interestingly, the n-3 PUFA supplementation reversed this stress-induced reduction in 5-HT levels. These findings showed that supplementation in n-3 long-chain PUFAs might reverse certain effects of UCMS in cerebral structures involved in stress-related behaviors. The aim of this study was to examine the effects of supplementation with n-3 polyunsaturated fatty acids (PUFAs) on stress responses in mice subjected to an unpredictable chronic mild stress (UCMS) procedure. Stress-induced modifications in coat and aggressiveness were evaluated, and phospholipid PUFA profiles and monoamine levels were analyzed in the frontal cortex, hippocampus, and striatum. The results showed that repeated exposure to mild stressors induced degradation in the physical state of the coat, lowered body weight gain, and increased aggressiveness, without any effect of n-3 PUFA supplementation. The UCMS induced a significant decrease in the levels of norepinephrine in the frontal cortex and striatum, and a nonsignificant decrease in the hippocampus. The tissue levels of serotonin (5-HT) were 40% to 65% decreased in the three brain regions studied. Interestingly, the n-3 PUFA supplementation reversed this stress-induced reduction in 5-HT levels. These findings showed that supplementation in n-3 long-chain PUFAs might reverse certain effects of UCMS in cerebral structures involved in stress-related behaviors. The mammalian brain is particularly rich in docosahexaenoic acid (22:6n-3, DHA), the main n-3 polyunsaturated fatty acid (PUFA). DHA is provided directly by the diet from aquatic sources and, after endogenous synthesis in the liver, from its vegetable dietary precursor α-linolenic acid (18:3n-3, ALA) by successive desaturation and elongation. ALA is present in the brain at very low concentrations, whereas DHA can represent half of the total PUFAs inserted into phospholipids that constitute the structure of neuronal membranes. Accumulation of DHA in brain membranes is particularly high during the perinatal period, coinciding with the formation of synapses (1Clandinin M.T. Brain development and assessing the supply of polyunsaturated fatty acid.Lipids. 1999; 34: 131-137Crossref PubMed Scopus (88) Google Scholar). It has been shown that the accumulation of DHA in the human infant brain during the first 6 months of life is half that of the total amount in the body, around 5 mg per day (2Cunnane S.C. Francescutti V. Brenna J.T. Crawford M. Breast-fed infants achieve a higher rate of brain and whole body docosahexaenoate accumulation than formula-fed infants not consuming dietary docosahexaenoate.Lipids. 2000; 35: 105-111Crossref PubMed Scopus (181) Google Scholar). A diet deficient in ALA results in changes in the composition of cells, organelles, and synaptic membranes in the central nervous system and leads to reduced learning ability. 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Reversibility of n-3 fatty acid deficiency-induced changes in dopaminergic neurotransmission in rats: critical role of developmental stage.J. Lipid Res. 2002; 43: 1209-1219Abstract Full Text Full Text PDF PubMed Google Scholar). In light of these findings, it can be suggested that early deficiency in n-3 PUFAs, particularly DHA, may result in a cascade of suboptimal development of neurotransmitter systems, especially in limbic structures, leading to impaired emotional and cognitive responses to subsequent environmental challenges. In addition, it has been suggested that DHA may be involved in the regulation of stress responses in rats, inasmuch as DHA intake completely reversed anxiety-like behavior in the elevated plus-maze caused by an n-3 PUFA-deficient diet and attenuated freezing behavior in conditioned fear-stress responses (20Takeuchi T. Iwanaga M. Harada E. Possible regulatory mechanism of DHA-induced anti-stress reaction in rats.Brain Res. 2003; 964: 136-143Crossref PubMed Scopus (120) Google Scholar). It has also been shown that n-3 fatty acid-deficient mice were more vulnerable to stress, inasmuch as they behaved similarly to mice fed an adequate diet under normal conditions in the elevated-plus maze but were significantly more anxious under stressful conditions (3Fedorova I. Salem Jr., N. Omega-3 fatty acids and rodent behavior.Prostaglandins Leukot. Essent. Fatty Acids. 2006; 75: 271-289Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). In humans, it has previously been reported that administration of fish oil rich in DHA improved resistance to the mental stress of exams in students (21Hamazaki K. Sawazaki S. Nagasawa T. Nagao Y. Kanagawa Y. Yazawa K. Administration of docosahexaenoic acid influences behavior and plasma catecholamine levels at times of psychological stress.Lipids. 1999; 34: S33-S37Crossref PubMed Google Scholar). Moreover, prevention of stress-induced aggression and hostility by DHA supplementation has been demonstrated in clinical trials (22Hamazaki K. Itomura M. Huan M. Nishizawa H. Sawazaki S. Tanouchi M. Watanabe S. Hamazaki T. Terazawa K. Yazawa K. Effect of omega-3 fatty acid-containing phospholipids on blood catecholamine concentrations in healthy volunteers: a randomized, placebo-controlled, double-blind trial.Nutrition. 2005; 21: 705-710Crossref PubMed Scopus (98) Google Scholar, 23Hamazaki T. Itomura M. Sawazaki S. Nagao Y. Anti-stress effects of DHA.Biofactors. 2000; 13: 41-45Crossref PubMed Scopus (42) Google Scholar). The authors concluded that DHA influences a possible adaptive mechanism during stress by lowering norepinephrine levels. There is currently some evidence regarding the involvement of dietary n-3 PUFA in mood disorders, particularly depression. Cross-national epidemiological analyses have suggested that lower n-3 PUFA levels are related to higher prevalence rates of major and postpartum depression (24Hibbeln J.R. Seafood consumption, the DHA content of mothers' milk and prevalence rates of postpartum depression: a cross-national, ecological analysis.J. Affect. Disord. 2002; 69: 15-29Crossref PubMed Scopus (397) Google Scholar, 25Otto S.J. de Groot R.H. Hornstra G. Increased risk of postpartum depressive symptoms is associated with slower normalization after pregnancy of the functional docosahexaenoic acid status.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 69: 237-243Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and that there is a significant negative correlation between fish consumption and the prevalence of depression (26Hibbeln J.R. Fish consumption and major depression.Lancet. 1998; 351: 1213-1216Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). In addition, several clinical studies have described abnormally low levels of DHA in the plasma and/or erythrocytes of depressed patients (27Peet M. Murphy B. Shay J. Horrobin D.F. Depletion of omega-3 fatty acid levels in red blood cell membranes of depressive patients.Biol. Psychiatry. 1998; 43: 315-319Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 28Maes M. Christophe A. Delanghe J. Altamura C. Neels H. Meltzer H.Y. Lowered omega 3 polyunsaturated fatty acids in serum phospholipids and cholesteryl esters of depressed patients.Psychiatry Res. 1999; 85: 275-291Crossref PubMed Scopus (410) Google Scholar). To understand further the potential relationships between n-3 PUFA and depression, we examined the effects of n-3 PUFA supplementation on various responses induced by chronic stress exposure in mice. Animals were subjected to a chronic mild stress procedure that represents a well-known animal model of depression (29Willner P. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation.Psychopharmacology (Berl.). 1997; 134: 319-329Crossref PubMed Scopus (1525) Google Scholar, 30Willner P. Muscat R. Papp M. An animal model of anhedonia.Clin. Neuropharmacol. 1992; 15: 550A-5551Crossref PubMed Scopus (46) Google Scholar). The consequences of n-3 PUFA supplementation on behavioral parameters and on monoaminergic levels and fatty acid profiles in several brain areas (i.e., the frontal cortex, hippocampus, and striatum) were compared in normal and stress conditions. Fifty-five male BALB/[email protected] mice (Centre d'Elevage Janvier; Le Genest Saint Isle, France) were used in this study. The mice were aged 6 weeks on their arrival and were housed in groups of three to four. They were maintained in a temperature- (22°C) and humidity- (40%) controlled room on an inverted light-dark cycle (light from 20:00 to 8:00) with free access to food (regular chow, Ext M20, SDS; Essex, England) and water. Mice were first acclimatized to the laboratory for 1 week before the start of the experiment. Experiments were conducted in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). Mice were initially distributed into four groups. Mice of two groups were subjected to an unpredictable chronic mild stress (UCMS) procedure for 8 weeks, until the end of the behavioral tests. At the start of the experiment, stressed mice were maintained under the same standard conditions, but they were isolated in individual home cages (8.5 × 22 cm) and had no physical contact with the other mice. Animals from the two nonstressed groups were housed in groups of three or four, with a shelter and some tubes placed in their home cages. Several variables were used to assess the stress-induced effects, i.e., condition of coat, body weight, behavior in the novelty suppression of feeding (NSF) test, and behavior in the resident-intruder test. Condition of the coat and body weight were recorded weekly for all mice. The behavioral tests commenced from the seventh week. Mice were euthanized at the end of the behavioral tests (i.e., 8 weeks after the beginning of the UCMS procedure). The stress protocol used was based on the UCMS procedure described by Willner, Muscat, and Papp (30Willner P. Muscat R. Papp M. An animal model of anhedonia.Clin. Neuropharmacol. 1992; 15: 550A-5551Crossref PubMed Scopus (46) Google Scholar), and adapted to mice by our laboratory (31Ducottet C. Griebel G. Belzung C. Effects of the selective nonpeptide corticotropin-releasing factor receptor 1 antagonist antalarmin in the chronic mild stress model of depression in mice.Prog. Neuropsychopharmacol. Biol. Psychiatry. 2003; 27: 625-631Crossref PubMed Scopus (197) Google Scholar, 32Santarelli L. Saxe M. Gross C. Surget A. Battaglia F. Dulawa S. Weisstaub N. Lee J. Duman R. Arancio O. et al.Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants.Science. 2003; 301: 805-809Crossref PubMed Scopus (3612) Google Scholar, 33Mineur Y.S. Prasol D.J. Belzung C. Crusio W.E. Agonistic behavior and unpredictable chronic mild stress in mice.Behav. Genet. 2003; 33: 513-519Crossref PubMed Scopus (127) Google Scholar, 34Ducottet C. Belzung C. Behaviour in the elevated plus-maze predicts coping after subchronic mild stress in mice.Physiol. Behav. 2004; 81: 417-426Crossref PubMed Scopus (119) Google Scholar). This animal model of depression consists of chronic exposure to various mild social and environmental stressors, none of which is sufficient alone to induce long-lasting effects. The stressors used vary, and they were applied in a different sequence each week to avoid any habituation. We excluded nociceptive stressors and food/water deprivation for ethical reasons. The emphasis in this model is on the chronic and variable nature of the stressors. The stressors used consisted of removal of bedding, wetting the bedding, several repeated changes of bedding, tilting cages by 45° for varying times, placing ∼2 cm of water in the home cage (after removing the bedding), exposure to rat bedding for 15 min, social stress 1 (placing a mouse in a cage that had previously belonged to another mouse), social stress 2 (placing a mouse in another animal's cage and then returning it to its own cage, where it would find that the cage had been occupied by another mouse), restrained stress in small tubes for varying times, lights on during the dark phase, lights off during the light phase, a succession of light and dark periods for 30 min, and switching the light/dark cycle for varying durations. Two parameters were measured throughout the UCMS procedure, i.e., condition of the coat and body weight. The condition of the coat was evaluated each week by examining the coat on different parts of the body (head, neck, dorsal area, ventral area, tail, front and hind paws, and genital area). For each area, a score of 0 was applied if the coat was in good condition, and a score of 1 if it was in very poor condition (disordered, piloerection). The total score was the sum of the score for each area; thus a high score indicated that the coat was in poor condition. This method has been validated in a number of recent studies (31Ducottet C. Griebel G. Belzung C. Effects of the selective nonpeptide corticotropin-releasing factor receptor 1 antagonist antalarmin in the chronic mild stress model of depression in mice.Prog. Neuropsychopharmacol. Biol. Psychiatry. 2003; 27: 625-631Crossref PubMed Scopus (197) Google Scholar, 32Santarelli L. Saxe M. Gross C. Surget A. Battaglia F. Dulawa S. Weisstaub N. Lee J. Duman R. Arancio O. et al.Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants.Science. 2003; 301: 805-809Crossref PubMed Scopus (3612) Google Scholar, 35Griebel G. Simiand J. Serradeil-Le Gal C. Wagnon J. Pascal M. Scatton B. Maffrand J.P. Soubrie P. Anxiolytic- and antidepressant-like effects of the non-peptide vasopressin V1b receptor antagonist, SSR149415, suggest an innovative approach for the treatment of stress-related disorders.Proc. Natl. Acad. Sci. USA. 2002; 99: 6370-6375Crossref PubMed Scopus (429) Google Scholar). Body weight was also measured each week until the end of the UCMS procedure. n-3 PUFA (léroDNV, Laboratoire léro; France) or vehicle devoid of n-3 PUFA (Frial oil; Lesieur, France) was administered daily at 1:30 PM by force feeding at a volume of 0.15 ml throughout the UCMS procedure. léroDNV contained 70% n-3 PUFA (w/w). Mice in the supplemented groups were thus receiving a dose of approximately 80 mg/day n-3 PUFA [6.1 mg DHA and 9.2 mg eicosapentaenoic acid (EPA)], corresponding to the dose that has been previously shown to allow maximal DHA incorporation in brain membranes (36Alessandri J.M. Poumès-Ballihaut C. Langelier B. Perruchot M.H. Raguenez G. Lavialle M. Guesnet P. Incorporation of docosahexaenoic acid into nerve membrane phospholipids: bridging the gap between animals and cultured cells.Am. J. Clin. Nutr. 2003; 78: 702-710Crossref PubMed Scopus (38) Google Scholar). Four groups of mice were formed: a nonstressed group receiving the vehicle (NS-V, n = 13), a stressed group receiving the vehicle (S-V, n = 14), a nonstressed group receiving n-3 PUFA (NS-PUFA, n = 14), and a stressed group receiving n-3 PUFA (S-PUFA, n = 14). The NSF test is a modified version of that used by Santarelli et al. (32Santarelli L. Saxe M. Gross C. Surget A. Battaglia F. Dulawa S. Weisstaub N. Lee J. Duman R. Arancio O. et al.Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants.Science. 2003; 301: 805-809Crossref PubMed Scopus (3612) Google Scholar). The testing apparatus consisted of a wooden 30 cm × 30 cm × 20 cm box with an indirect red light. The floor was covered with 2 cm sawdust. Twelve hours before the test, food was removed from the cages. At the time of testing, a pellet of food (regular chow) was placed on a white paper platform positioned in the center of the box. An animal was placed in a corner of the maze. The latency to manifestly chew the pellet was recorded within a 3 min period. This test induced conflicting motivation between the drive to eat the food pellet and the fear of venturing into the center of the arena. This test was performed at 10 AM. The resident-intruder test consisted of the introduction of an unknown animal into the home cage of test mice to measure their aggressiveness. The intruder was a naïve male C57BL/[email protected] mouse, known for its high passivity and lack of aggression. When done in nonstressed mice, the animals were isolated 24 h before the test to become familiar with their novel environment. The bedding of the isolated stressed mice was changed 24 h before the test to standardize conditions between nonstressed and stressed mice. The test started when the intruder was placed in the home cage of the resident animal and lasted for 5 min. Two parameters were measured: the latency of the first attack and the frequency of attacks on the intruder. This test was performed at 3 PM. Mice (n = 6 for each group) were euthanized by decapitation at the end of the behavioral tests. Brains were quickly removed, and the frontal cortex, hippocampus, and striatum were dissected out on ice, weighed, and frozen in liquid nitrogen. Total lipids were extracted by a modification of the method of Folch, Lees, and Sloane Stanley (37Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissue.J. Biol. Chem. 1957; 226: 497-506Abstract Full Text PDF PubMed Google Scholar). Phosphatidylethanolamine (PE) was separated from total lipids on an aminopropyl-bonded silica gel cartridge (BAKERBOND spe™ Amino; Baker, USA) by the method of Alessandri and Goustard-Langelier (38Alessandri J.M. Goustard-Langelier B. Alterations in fatty acid composition of tissue phospholipids in the developing retinal dystrophic rat.Lipids. 2001; 36: 1141-1152Crossref PubMed Scopus (13) Google Scholar). The fatty acids were methylated with BF3, and the fatty acid methyl esters were analyzed by gas liquid chromatography (Carlo Erba) (39Aïd S. Vancassel S. Poumès-Ballihaut C. Chalon S. Guesnet P. Lavialle M. Effect of a diet-induced (n-3) polyunsaturated fatty acid depletion on cholinergic parameters in the rat hippocampus.J. Lipid Res. 2003; 44: 1545-1551Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and identified by comparison with commercial standards of equivalent chain lengths. The results were expressed as mg fatty acids/100 mg total fatty acids (TFAs; wt %). Mice (n = 6–8 for each group) were euthanized by decapitation at the end of the behavioral tests. Brains were quickly removed, and the frontal cortex, hippocampus, and striatum were dissected out on ice and weighed. Each cerebral region was homogenized in 1 ml of a buffer containing 12 mM HClO4, 0.1 mM EDTA, 0.5 mM Na2S2O5, 3 mM octanesulfonic acid, and 3 mM heptanesulfonic acid with an Ultraturrax T25 at 4°C. After centrifugation at 30,000 g for 20 min at 4°C, 100 μl of the supernatant was stored at −80°C until use. Contents of norepinephrine (NE), DA, dihydroxyphenyl acetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) were measured in each supernatant by HPLC, with electrochemical detection on a Concorde apparatus (Waters; St. Quentin-Yvelines, France). Samples were injected using a Rheodyne 7725i injector valve with a 20 μl injection loop. The mobile phase, consisting of 7% acetonitrile, 3% methanol, 90% 20 mM citric acid, 10 mM monobasic phosphate sodium, 3.25 mM octanesulfonic acid, 3 mM heptanesulfonic acid, 0.1 mM EDTA, 2 mM KCl, 6 ml/l o-phosphoric acid, and 2 ml/l diethylamine, pH 3, was pumped at 0.3 ml/min using a Gold 118 system (Beckman; Fullerton, CA). Separation was performed with a 3 μm C18, 3.2 × 100 mm reversed phase column (LC-22C, BAS; West Lafayette, IN). A glassy carbon working electrode set at 610 mV with reference to an in situ Ag/AgCl reference electrode was used to detect compounds. Signals were recorded and quantified with a Beckman Gold 118 integrator. Amounts of NE, DA, DOPAC, HVA, 5-HT, and 5-HIAA were calculated by comparing peak levels from the supernatant samples with those of external standards. Results are expressed as nmol/mg tissue. All data are expressed as mean ± SEM. For monoamine assays and fatty acid analyses, means were compared by two-way ANOVA (PUFA supplementation × stress factors), followed by the posthoc Bonferroni test in case of significance. For behavioral studies and body weight follow-up, results were compared using nonparametric ANOVA from Kruskal-Wallis, followed by the posthoc Mann-Whitney U test in case of significance. Differences with P < 0.05 were considered significant. Statistical analyses were performed using Statistica 7.0 software (StatSoft®, Inc.; Tulsa, OK). Kruskal-Wallis test revealed significant differences in the state of the coat after 1 week of UCMS (P < 0.001) until the end of UCMS (P < 0.001) (Fig. 1A). The coat state of both stressed groups (groups S-V and S-PUFA) continued to deteriorate, and there was a significant difference compared with nonstressed mice after 1 week UCMS (P < 0.001) until the end of UCMS (P < 0.001). No effect of n-3 PUFA treatment was observed in stressed mice (groups S-V and S-PUFA), from week 1 until week 5. Deterioration was greater during weeks 5 and 7 in the stressed supplemented mice (group S-PUFA) than in the stressed unsupplemented mice (group S-V, P = 0.0501 and P = 0.0568, respectively), but the difference only reached statistical significance during week 6 (P < 0.03). No difference in body weight appeared among the four groups throughout the UCMS regimen (Fig. 1B), except during week 7, when the body weights of both nonstressed groups (NS-V and NS-PUFA) were significantly higher than those of the stressed groups (S-V and S-PUFA; P < 0.03 and P < 0.001, respectively). The Kruskal-Wallis test showed significant differences between the four groups in latency to chew the pellet (P < 0.001) (Fig. 2A). n-3 PUFA supplementation resulted in increased latency to chew the pellet (P < 0.02 for S-V vs. S-PUFA and P < 0.005 for NS-V vs. NS-PUFA). UCMS had no effect in either test. The Kruskal-Wallis test showed significant differences between the groups in latency to attack the intruder (P < 0.005) (Fig. 2B). The UCMS procedure resulted in a significant effect, inasmuch as it reduced the latency of agonistic behaviors (P < 0.05 for NS-V vs. S-V). Similar effects were observed for frequency of attack (data not shown). n-3 PUFA supplementation had no effect in this test. The major fatty acids in the PE of frontal cortex, hippocampus, and striatum are shown in Tables 1, 2, and 3, respectively. In the four groups of mice, saturated fatty acid (SFA) and PUFA accounted for the highest levels of fatty acids. The total SFA and total n-6 + n-3 PUFAs thus accounted for nearly 30% and 45% of the TFAs in each cerebral region, respectively.TABLE 1Main fatty acid contents of PE in the frontal cortexFatty AcidaFor each fatty acid, values with different superscripts (a–c) were significantly different between groups (two-way ANOVA; P < 0.05); n = 6 for all groups.mg/100 mg fatty acidsGroupSFAMUFA20:4n-622:5n-6n-6 PUFAbn-6 PUFA = sum of 18:2n-6, 18:3n-6, 20:3n-6, 22:4n-6, and 22:5n-6.20:5n-322:6n-3n-3 PUFAcn-3 PUFA = sum of 18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3, 22:5n-3, and 22:6n-3.n-6+n-3 PUFA22:5n-6/22:6n-3NS-V26.9 ± 0.610.2 ± 0.69.5 ± 0.3aFor each fatty acid, values with different superscripts (a–c) were significantly different between groups (two-way ANOVA; P < 0.05); n = 6 for all groups.0.7 ± 0.0aFor each fatty acid, values with different supers
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