Effect of a diet-induced n-3 PUFA depletion on cholinergic parameters in the rat hippocampus
2003; Elsevier BV; Volume: 44; Issue: 8 Linguagem: Inglês
10.1194/jlr.m300079-jlr200
ISSN1539-7262
AutoresSaba Aïd, Sylvie Vancassel, Carine Poumès‐Ballihaut, Sylvie Chalon, Philippe Guesnet, Monique Lavialle,
Tópico(s)Adipose Tissue and Metabolism
ResumoBecause brain membranes contain large amounts of docosahexaenoic acid (DHA, 22:6n-3), and as (n-3) PUFA dietary deficiency can lead to impaired attention, learning, and memory performance in rodents, we have examined the influence of an (n-3) PUFA-deprived diet on the central cholinergic neurotransmission system. We have focused on several cholinergic neurochemical parameters in the frontal cortex and hippocampus of rats fed an (n-3) PUFA-deficient diet, compared with rats fed a control diet. The (n-3) PUFA deficiency resulted in changes in the membrane phospholipid compositions of both brain regions, with a dramatic loss (62–77%) of DHA. However, the cholinergic pathway was only modified in the hippocampus and not in the frontal cortex. The basal acetylcholine (ACh) release in the hippocampus of deficient rats was significantly (72%) higher than in controls, whereas the KCl-induced release was lower (34%). The (n-3) PUFA deprivation also caused a 10% reduction in muscarinic receptor binding. In contrast, acetylcholinesterase activity and the vesicular ACh transporter in both brain regions were unchanged.Thus, we evidenced that an (n-3) PUFA-deficient diet can affect cholinergic neurotransmission, probably via changes in the phospholipid PUFA composition. Because brain membranes contain large amounts of docosahexaenoic acid (DHA, 22:6n-3), and as (n-3) PUFA dietary deficiency can lead to impaired attention, learning, and memory performance in rodents, we have examined the influence of an (n-3) PUFA-deprived diet on the central cholinergic neurotransmission system. We have focused on several cholinergic neurochemical parameters in the frontal cortex and hippocampus of rats fed an (n-3) PUFA-deficient diet, compared with rats fed a control diet. The (n-3) PUFA deficiency resulted in changes in the membrane phospholipid compositions of both brain regions, with a dramatic loss (62–77%) of DHA. However, the cholinergic pathway was only modified in the hippocampus and not in the frontal cortex. The basal acetylcholine (ACh) release in the hippocampus of deficient rats was significantly (72%) higher than in controls, whereas the KCl-induced release was lower (34%). The (n-3) PUFA deprivation also caused a 10% reduction in muscarinic receptor binding. In contrast, acetylcholinesterase activity and the vesicular ACh transporter in both brain regions were unchanged. Thus, we evidenced that an (n-3) PUFA-deficient diet can affect cholinergic neurotransmission, probably via changes in the phospholipid PUFA composition. Brain membranes are rich in the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA, 20:4n-6), and docosahexaenoic acid (DHA, 22:6n-3). Mammals must obtain the linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) from which they are derived from their diet. These long-chain PUFAs may be important for the structure and function of many membrane proteins, including receptors, enzymes, and active transport molecules (1Spector A.A. Yorek M.A. Membrane lipid composition and cellular function.J. Lipid Res. 1985; 26: 1015-1035Abstract Full Text PDF PubMed Google Scholar, 2Youdim K.A. Martin A. Joseph J.A. Essential fatty acids and the brain: possible health implications.Int. J. Dev. Neurosci. 2000; 18: 383-399Crossref PubMed Scopus (414) Google Scholar). Dietary α-linolenic acid deficiency that reduces the brain DHA contents has been directly linked to impaired central nervous system (CNS) function (3Bourre J.M. François M. Youyou A. Dumont O. Piciotti M. Pascal G. Durand G. 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This lack of data on the effects of a chronic α-linolenic acid-deficient diet on cholinergic neurotransmission prompted us to examine several parameters of cholinergic function to determine the nature and extent of changes in cholinergic synapse function in rats deprived of (n-3) PUFA. We analyzed those brain areas that have prominent cholinergic inputs from the basal forebrain (frontal cortex and hippocampus) (20Lehman J. Nagy J.I. Atmadja S. Fibiger H.C. The nucleus basalis magnocellularis: the origin of a cholinergic projection to the neocortex of the rat.Neuroscience. 1980; 5: 1161-1174Crossref PubMed Scopus (487) Google Scholar, 21Dutar P. Bassant M.H. Senut M.C. Lamour Y. The septohippocampal pathway: structure and function of a central cholinergic system.Physiol. Rev. 1995; 75: 393-427Crossref PubMed Scopus (291) Google Scholar). Microdialysis was used to monitor basal and KCl-stimulated ACh concentrations in (n-3)-PUFA-deficient rats and in rats fed a diet with an adequate (n-3) PUFA supply. Muscarinic receptors were investigated by autoradiography and vesicular acetylcholine transporter (VAChT) binding sites by the in vivo binding of [125I]benzovesamicol (IBVM). Cholinergic catabolism was assessed by measuring acetylcholinesterase (AChE) activity. The data on the cholinergic neurochemical parameters were correlated with the phospholipid fatty acid compositions of the hippocampal and cortical membranes. Two generations of female Wistar rats were fed a diet containing 6% fat in the form of African peanut oil (deficient in α-linolenic acid), giving about 1,200 mg linoleic acid and <11 mg α-linolenic acid per 100 g diet (deficient diet) (Table 1). Two weeks before mating, a second generation of deficient females was divided into two groups. The first group was fed the deficient diet, and the second group was fed a control diet (peanut oil and rapeseed oil) containing about 1,200 mg linoleic acid and 300 mg α-linolenic acid per 100 g diet. At weaning, the males from each litter were housed two per cage with free access to the same diet as their mothers, and under controlled temperature (22 ± 1°C), humidity (50 ± 10%), and light cycles (7 AM to 7 PM). Experiments were performed on 2- to 3-month-old rats. The experimental protocol complied with the European Community guidelines (directive 86/609/EEC).TABLE 1Composition of the experimental dietsControl(n-3) PUFA Deficient(g/100 g diet)Casein (vitamin free)2222dl-methionine0.20.2Cellulose22Mineral mixaAccording to (10).44Vitamin mixaAccording to (10).11Corn starch42.643.2Sucrose21.321.6FatsbTotal dietary lipids: 6 g/100 g diet.(g/100 g diet)African peanut oil3.86.0Rapeseed oil2.2—Fatty acid composition(mg/100 g diet)18:2n-61,1941,20018:3n-329611The diet provided about 16.5 MJ/kg diet. Lipids provided 13.5% of total calories. Oils were kindly supplied by Lesieur-Alimentaire (Coudekerque, France).a According to (10).b Total dietary lipids: 6 g/100 g diet. Open table in a new tab The diet provided about 16.5 MJ/kg diet. Lipids provided 13.5% of total calories. Oils were kindly supplied by Lesieur-Alimentaire (Coudekerque, France). Rats were killed by decapitation. Their brains were quickly removed, and the hippocampus and the frontal cortex were dissected out on ice, weighed, and frozen in liquid nitrogen. Total lipids were extracted by a modification of the Folch method (22Folch 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). Phospholipid classes (phosphatidylcholine, PC; phosphatidylethanolamine, PE; and phosphatidylserine, PS) were separated from total lipids on an aminopropyl-bonded silica gel cartridge (BAKERBOND speTM Amino) by the method of Alessandri and Goustard-Langelier (23Alessandri 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 then transmethylated with 10% boron trifluoride (Fluka, Socolab, France) at 90°C for 20 min (24Morisson W. Smith L. Preparation of fatty acid methyl esters and dimetylacetals from lipids with boron trifluoride-methanol.J. Lipid Res. 1964; 5: 600-608Abstract Full Text PDF PubMed Google Scholar), and the composition of each phospholipid class was determined by gas chromatography (Carlo Erba) (25Guesnet P. Alasnier C. Alessandri J.M. Durand G. Modifying the n-3 content of the maternal diet to determine the requirements of the foetal and sucking rats.Lipids. 1997; 32: 527-534Crossref PubMed Scopus (39) Google Scholar). The fatty acid methyl esters were identified by comparison with commercial standards of equivalent chain lengths and quantified by integration using the Nelson Analytical Program System (SRA, France). Results are expressed as the percentage of total fatty acids. Differences between control and deficient rats were analyzed by one-way ANOVA followed by a post hoc Bonferroni t-test. The significance of differences between cerebral regions within a single dietary group was analyzed by paired Student's t-test. Significance was set at P < 0.01. Rats were anesthetized at 10 AM with urethane (1.5 mg/Kg ip), and commercially supplied probes (4 mm-long membrane, polycarbonate, 35 kDa cut-off; MAB 6, Sweden) were stereotaxically implanted in the left lateral hippocampus [−5.6 mm anterior to the bregma, 4.4 mm lateral, −7.5 mm from the dura (26Paxinos G. Watson C. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York, NY1986Google Scholar)]. Body temperature was maintained at 37°C using a thermostatically controlled heating blanket (CMA 150, CMA, Microdialysis, Sweden). Ringer solution (147 mM NaCl, 4 mM KCl, 3.4 mM CaCl2) containing 0.5 μM neostigmine (Sigma, France) was perfused through the probe at 2 μl/min. All studies included a 90 min washout period prior to collecting 20 min dialysates. Three dialysate samples were then collected and defined as basal samples. Potassium chloride (KCl, 100 mM) was then added to the perfusion buffer for 40 min. Return to basal ACh level was recovered for the next 1 h 40 min (five samples). Samples of dialysate were stored at –80°C. The rats were decapitated immediately after the microdialysis. Their brains were removed and quickly frozen. The probe location was checked on coronal cryosections. Any results obtained with an incorrectly located probe were discarded. With careful handling and storage, a probe could be reused for as many as five acute experiments. The recovery of ACh through the probe was tested in vitro prior to each experiment using Ringer's solution containing 500 nM ACh. Perfusate samples (20 μl) were assayed for ACh by HPLC with electrochemical detection. ACh was separated on a reverse-phase analytical column (C18 Superspher, 100 mm × 2 mm, 4 μm, Macherey-Nagel, France) using a mobile phase (flow rate 0.3 ml/min) of 50 mM KH2PO4, 0.5 mM tetramethylammonium chloride, 2.5 mM heptane-sulfonic acid, and 0.01% (v/v) bactericide (pH 7). ACh was then enzymatically converted to hydrogen peroxide in a postcolumn solid phase reactor containing covalently bound AChE and choline oxidase, and the resulting H2O2 was detected electrochemically using a platinum electrode operating at a potential of +500 mV. The detection limit was 50 fmol/20 μl dialysate. The signal was recorded and the ACh was quantified by comparison with ACh standard solutions and corrected for the in vitro recovery of the probe. The mean basal ACh release for each animal was estimated by averaging the three samples collected during the 1 h preceding KCl perfusion, and expressed as pmol per 20 μl dialysate. ACh release is expressed as a percentage of the baseline value for each animal. Statistical differences between dietary groups for each collection time were tested using Student's t-test and were considered significant when P < 0.05. Animals were killed by decapitation, and their brains were rapidly removed and frozen in isopentane at −35°C. Coronal cryosections (20 μm) were cut, thaw-mounted on gelatin-coated microscope slides, and stored at −80°C. The [3H]scopolamine binding assay was performed according to Albin et al. (27Albin R.L. Howland M.M. Higgins D.S. Frey K.A. Autoradiographic quantification of muscarinic cholinergic synaptic markers in bat, shrew and rat brain.Neurochem. Res. 1994; 19: 581-589Crossref PubMed Scopus (5) Google Scholar). Sections were incubated for 30 min at room temperature with buffer (pH 7.4) containing 137 mM NaCl, 3 mM KCl, 1 mM EDTA, 8 mM NaHPO4, 1.5 mM KH2PO4, and 5 nM [3H]scopolamine (70 Ci/mmol, Amersham, France) with (nonspecific binding) or without (total binding) 20 μM atropine (Sigma). The sections were then rinsed for 10 min in 4°C buffer, and for 30 s in 4°C distilled water. They were dried and exposed to Biomax MS film (Kodak, France) in an autoradiography cassette for 2 weeks at −80°C. The films were developed, fixed, and the autoradiographs analyzed with an imaging system (Biocom, Visioscan, France). Relative optical densities (RODs) are expressed as the ratio of optical density measured in a region of interest over the optical density of a reference region, the corpus callosum. Measurements were performed on six serial sections for each brain region of eight control and nine deficient rats. RODs were compared between diets for each cerebral region, and between cerebral regions for each dietary group, by Student's t-test. Significance was set at P < 0.05. IBVM was prepared at INSERM U316 (Tours, France) (28Van Dort M.E. Jung Y.W. Gildersleeve D.L. Hagen C.A. Kuhl D.E. Wieland D.M. Synthesis of the 123I- and 125I-labeled cholinergic nerve marker (−)-5-iodobenzovesamicol.Nucl. Med. Biol. 1993; 20: 929-937Crossref PubMed Scopus (36) Google Scholar) and purified by HPLC. The resulting labeled compound had a specific activity of 2,200 Ci/mmol. Groups of six deficient and six control rats (380–400 g) were injected with a (50.1–61.8 μCi) bolus of IBVM via the tail vein and killed 2 h later by decapitation. Their brains were quickly removed, and the hippocampus and frontal cortex were dissected out and weighed. The radioactivity in the tissues was measured in a γ counter (LKB1282 Compugamma), calculated per gram of tissue, and referred to the injected dose (ID). Statistical differences were tested using two-way ANOVA (brain region × diet) followed by a Bonferroni t-test, and were considered significant when P < 0.05. Rats were decapitated, their brains were quickly removed, and the frontal cortex and hippocampus were dissected out on ice, weighed, frozen in liquid nitrogen, and stored at −80°C. AChE activity was determined by the method of Ellman et al. (29Ellman G.L. Courtney K.D. Andres V. Featherstone R.M. A new and rapid colorimetric determination of acetylcholinesterase activity.Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21719) Google Scholar). Tissues were homogenized in 0.1 M phosphate buffer (pH 8.0). The reaction mixture consisted of a 0.4 ml aliquot of homogenate, 2.6 ml phosphate buffer, and 0.1 ml 0.01 M dithiobis-nitrobenzoate (Sigma). Substrate [0.02 ml 0.075 M acetylthiocholine iodide (Sigma)] was added to the reaction mixture and the absorbance at 412 nm was recorded for at least 6 min in a UVIKON spectrophotometer (Kontron Instrument, UVK LAB Service, France). Protein content was determined by the Bradford procedure (30Bradford M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). The differences in enzymatic activity between dietary groups and between brain regions were analyzed by two-way ANOVA (brain region × diet), followed by the Bonferroni t-test. Differences were considered significant when P < 0.05. The overall concentrations of saturated and monounsaturated fatty acids in all three phospholipid classes in the rats fed the (n-3) PUFA-deficient diet and the control rats were essentially the same (Table 2). The (n-3) PUFAs in the controls were almost entirely 22:6n-3, and their concentration was greatest in the PE (24.5 ± 1.5% of total fatty acids in the frontal cortex; 21.6 ± 1.2% in the hippocampus) and the PS (23.7 ± 2.7% in the frontal cortex; 11.7 ± 1.0% in the hippocampus). But the 22:6n-3 accounted for less than 6% of the total in the PC. The DHA concentrations in the three phospholipid classes were significantly higher in the frontal cortex than in the hippocampus (P < 0.01). The major (n-6) PUFA was 20:4n-6. Its concentration was greatest in PE (12.7 ± 1.1% in the frontal cortex; 13.7 ± 1.1% in the hippocampus), followed by PC (6.5 ± 0.1% in the frontal cortex; 8.6 ± 0.3% in the hippocampus) and PS (less than 3%).TABLE 2Main fatty acid compositions of phospholipid classesFrontal CortexHippocampusControl Rats(n-3) PUFA- Deficient RatsControl Rats(n-3) PUFA- Deficient Ratsmg/100 mg fatty acidsPC 16:044.2 ± 0.5bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).44.8 ± 0.5bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).42.6 ± 0.7aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).42.6 ± 1.2aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01). 18:012.1 ± 0.211.6 ± 0.1aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).12.3 ± 0.112.3 ± 0.2bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01). 18:1n-921.1 ± 0.3bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).19.9 ± 0.2bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).19.8 ± 0.3aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).18.6 ± 0.3aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01). 20:4n-66.5 ± 0.1aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).7.0 ± 0.2aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).8.6 ± 0.3bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).10.2 ± 0.8bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01). 22:4n-60.8 ± 0.11.1 ± 0.0aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).0.8 ± 0.11.2 ± 0.0bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01). 22:5n-60.1 ± 0.03.1 ± 0.3*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).0.1 ± 0.03.0 ± 0.3*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01). Σ (n-6) PUFA8.3 ± 0.2aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).12.3 ± 0.4aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).10.3 ± 0.3bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).15.6 ± 0.8bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01). 22:6n-35.4 ± 0.3bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).1.3 ± 0.1aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).4.0 ± 0.1bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).1.5 ± 0.1bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01). Σ (n-3) PUFA5.5 ± 0.3bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).1.3 ± 0.1aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).4.4 ± 0.2aFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).1.5 ± 0.1bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).PE 16:04.8 ± 0.15.2 ± 0.2bFor each fatty acid, a significant difference exists between cortical and hippocampal regions in the same dietary group (paired Student's t-test; P < 0.01).,*For each fatty acid, significantly different from control group in the same cerebral region (one-way ANOVA followed by posthoc Bonferroni t-test; P < 0.01).4.5 ± 0.54.5 ± 0.5aFo
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