[1] 20w Neither the density nor function of striatal dopamine transporters were influenced by chronic n-3 polyunsaturated fatty acid deficiency in rodents

[1] 136w We hypothesized that the chronic dietary deficiency of n-3 polyunsaturated fatty acids (n-3 PUFAs) might affect the density and/or function of dopamine transporters (DAT), which have a major role in regulating the synaptic level of dopamine. This hypothesis was tested by investigating DAT in the striatum using three complementary methods in control and deficient rats. The density of DAT was determined by quantitative autoradiography using [ 125 I]PE2I, a specific ligand of this transporter. Functional investigations were performed (i) in vitro by measuring [ 3 H]dopamine uptake on synaptosomes, and (ii) in vivo using intracerebral microdialysis. The results demonstrated that neither the density nor the function of DAT were influenced by n-3 PUFA deficiency in the striatum. This suggests lower sensitivity to n-3 PUFA deficiency in the striatum than that previously observed in the frontal cortex.

[1] 46w Specific in vitro binding of [ 125 I]PE2I in the striatum of control and deficient rats (Fig. 1) revealed no significant difference in DAT density between both groups (5.84 ^0.38 vs. 6.17 ^0.39 fmol/mg equivalent tissue in control and deficient rats, respectively; n ¼ 5=dietary group).

[2] 197w As shown in Table 3, [ 3 H]DA uptake in striatal synaptosome preparations after Eadie-Hofstee analysis did not show any significant difference between the two groups. The amounts of [ 3 H]MPP 1 and [ 14 C]Man present in dialysate fractions are presented in Fig. 2. [ 3 H]MPP 1 is transported through the DAT from the extra-to the intracellular compartment, whereas [ 14 C]Man, the reference substance, stays in the extracellular space. During the first [ 3 H]MPP 1 perfusion (before cocaine administration) 46% of the initial amount of [ 3 H]MPP 1 perfused was collected in the dialysate. During the second perfusion, when cocaine was added, the amount of [ 3 H]MPP 1 collected increased to 70% in control rats and the difference was statistically significant (P , 0:05, Student's t-test). In deficient rats 43% of the [ 3 H]MPP 1 perfused was collected, whereas there was a significant increase in the presence of cocaine (66%). The pattern of [ 3 H]MPP 1 uptake did not therefore differ significantly between control and deficient rats during the first perfusion (4434 ^363 vs. 4343 ^512 dpm) or during the second perfusion (6770 ^393 vs. 6671 ^521 dpm).

[1] 108w High amounts of polyunsaturated fatty acids (PUFAs) of the n-3 series are incorporated in neuronal cell membrane phospholipids. The main n-3 PUFA, docosahexaenoic acid (DHA, 22:6n-3), is derived from alpha-linolenic acid (ALN, 18:3n-3), which cannot be synthesized by mammalians and must therefore be obtained from the diet. These n-3 PUFAs play a key role in maintaining the physical properties of cerebral membranes, and thus influence membrane function [2]. It has been reported in numerous studies that dietary ALN deficiency in rodents dramatically alters the fatty acid composition of cerebral membrane phospholipids [3,8]. Moreover, the performance of n-3 PUFA-deficient rats is impaired in a variety of cognitive tasks [20].

[2] 80w We recently demonstrated that these behavioural disturbances could be mediated through the effects of n-3 PUFAs on the dopaminergic neurotransmission processes. The overall amount of dopamine (DA) is actually decreased in the frontal cortex of n-3 PUFA-deficient rats throughout life [8]. A more precise approach using intracerebral microdialysis revealed (i) abnormalities in the basal release of DA and its metabolites (Dopac, HVA) and (ii) reduced storage of DA in the pre-synaptic vesicles in the frontal cortex and nucleus accumbens [21][22][23][24].

[3] 293w The dopamine transporter (DAT) is located on pre-synaptic dopaminergic nerve terminals and regulates synaptic neurotransmission by controlling DA concentrations available for binding to pre-synaptic and post-synaptic DA receptors [1]. The DAT is anchored in the lipid bilayer and it could be expected that modification of the architecture of neuronal cell membranes induced by n-3 PUFA deficiency could alter its function, as already shown for membrane-associated proteins [2]. We have already reported that there is no alteration in the binding of the ligand [ 3 H]mazindol to DAT on cerebral sections in n-3 PUFA-deficient rats [8]. However, the method used in this study mainly revealed the protein expression of DAT on the membrane, but not the function of the DA uptake process. The aim of the present study was therefore to explore DAT density by autoradiographic experiments and to investigate DAT function using an in vitro approach on synaptosomes and an in vivo approach by microdialysis in rats fed a diet deficient in ALN compared to control rats receiving ALN. As the striatum is particularly affected by DAT dysfunction [16], all these experiments were performed in this cerebral region. DAT density was measured by in vitro autoradiographic experiments using the highly specific iodinated ligand [ 125 I]PE2I [7]. DAT function was studied in vitro by measuring [ 3 H]DA re-uptake on synaptosomes and in vivo by comparing [ 3 H]MPP 1 and [ 14 C]Mannitol ([ 14 C]Man) uptake using intracerebral microdialysis. MPP 1 is a neurotoxin which is actively transported through DAT into dopaminergic nerve terminals [15], whereas mannitol which is not a substrate for DAT, is used as a marker for the extracellular space. This novel microdialysis method has recently been validated as a relevant approach to evaluate the DAT function [25].

[4] 265w Two generations of female Wistar rats originating from the Laboratoire de Nutrition et Se ´curite ´Alimentaire (INRA, Jouy-en-Josas, France) were fed a diet containing 6 g/100 g fat in the form of African peanut oil specifically deficient in ALN, as already described [8]. This deficient diet provided 1200 mg of linoleic acid (LN, 18:2n-6) but less than 6 mg of ALN per 100 g of diet. Two weeks before mating, female rats originating from the second generation of ALN-deficient rats were divided into two groups. The first group received the deficient diet, and the second group received a diet in which peanut oil was replaced by a mixture of 60% peanut oil and 40% rapeseed oil. This diet (control) which provided the same amount of LN as the deficient diet, and in addition, 200 mg of ALN per 100 g of diet (½n 2 6=½n 2 3 ¼ 6), has previously been shown to restore the maternal amount of DHA [13]. Diets were consumed ad libitum by both groups. At weaning, the male progeny of these two groups of female rats received the same diets as their respective dams. The overall composition of diets and the fatty acid composition of dietary lipids are reported in Tables 1 and 2. Experiments were performed on 250-300 g male rats (2-3 months of age) from both dietary groups. A total of eight litters receiving each diet were used; each litter provided a mean of five male rats which were mixed at weaning. The experimental procedures were in compliance with guidelines from the European Communities Council directories 86/609/ EEC.

[5] 134w For in vitro autoradiographic study, rats (n ¼ 5=dietary group) were sacrificed and brains were rapidly removed on ice and frozen at 2358C. Twenty micron sections were cut at 2208C in a cryostat microtome (Reichert-Jung, Cryocut 1800, Lecia, France), thaw-mounted onto gelatinecoated slides and stored at 2808C until use. Each striatal section was incubated for 90 min at 258C with 100 pM of [ 125 I]PE2I in 100 ml of phosphate buffer (pH 7.4) as previously described [6]. Unspecific binding was determined from adjacent sections incubated in the presence of 100 mM cocaine (Cooper, France). After washing and rinsing, sections were dried and exposed to sensitive films (Hyperfilm bmax, Amersham, France) together with [ 125 I]microscales (Amersham) in X-ray cassettes for 3 days. Regional optical densities were measured using an image analyzer (Biocom, France).

[6] 201w The uptake of [ 3 H]DA was studied in vitro on synaptosome preparations (n ¼ 5=dietary group) as already described [18]. Briefly, the striatum was removed (50 mg of tissue per rat), homogenized and centrifuged to give a nuclear pellet. The supernatant was stored at 48C, and the pellet was suspended and centrifuged again. The two supernatants were pooled and centrifuged. The final pellet was suspended in Krebs-Ringer buffer (pH 7.6) to the required tissue concentration (3 mg of original tissue/ml). DA uptake in crude striatal synaptosomes was assayed using 10 nM of [ 3 H]DA (specific activity 48 Ci/mmol, Amersham, France) and concentrations of non-radiolabelled DA (DA hydrochloride, Pierre Fabre, France) ranged from 50 to 700 nM. Uptake was measured at 37 and 48C (total and non-specific DA uptake) and stopped by rapid filtration under vacuum through Whatman GF/C filters. The filters were placed in scintillation vials containing 7.4 ml of Picofluor 15 (Packard) and counted by liquid scintillation spectrometry (Packard Tricarb 2050 CA). The apparent kinetic parameters of specific DA uptake, the constant K m and the maximal uptake velocity (V max ) were calculated by analogy with the Michaelis-Menten equation using a least-square linear regression in Eadie-Hofstee analysis.

[7] 310w For microdialysis studies, rats were anaesthetized (n ¼ 5=dietary group) with urethane (Sigma Aldrich, France) (1.5 g/kg i.p.) and placed in a stereotaxic apparatus (Stoeling, USA). The probe was implanted into the striatum (AP, 1.0 mm; L, 2.7 mm; DV, 27.2 mm; membrane length, 4 mm; MAB, Sweden) according to Paxinos and Watson [19]. It was continuously infused with Dulbecco's modified liquid (ICN, USA) supplemented with 2.2 mM CaCl 2 and 1.1 mM MgCl 2 at 1 ml/min using a microsyringe pump (Harvard Apparatus, USA). [ 3 H]MPP 1 (2 mCi) and [ 14 C]Man (4 mCi) (specific activity 82 Ci/mmol and 51.5 mCi/mmol, respectively; NEN Life Science, France) were mixed. Radioisotopes were subsequently dissolved in 200 ml of perfusion buffer. Half of the solution (100 ml) was mixed with 100 ml of perfusion buffer, and the other half with 100 ml of perfusion buffer containing 10 mM cocaine (Cooper Pharmaceutique, France). The microinjection pump was mounted with three syringes, one containing perfusion buffer alone, one containing perfusion buffer with [ 3 H]MPP 1 and [ 14 C]Man, and one containing perfusion buffer with [ 3 H]MPP 1 , [ 14 C]Man and cocaine. During the first 90 min after insertion into the brain, the dialysis probe was infused with perfusion buffer alone. The perfusate was changed to buffer with isotopes for 10 min by switching probes. Then perfusion was continued with buffer alone for 30 min. A second perfusion containing isotopes and cocaine was performed 40 min after the initial perfusion. During and after isotope perfusion, dialysates were collected every 2 min, yielding 2 ml per fraction. Six millilitres of scintillation liquid (Optiphase Hisafe, Wallac, UK) were added to each dialysate fraction upon collection. Scintillation counting of [ 3 H]-and [ 14 C]-radioisotopes was carried out using a liquid scintillation analyzer (1215 Rackbeta, LKB Wallac, UK) 12 h later.

[8] 76w Chronic dietary ALN deficiency in the rat modifies the phospholipid fatty acid composition of cerebral membranes in several regions such as the frontal cortex, striatum and cerebellum [9]. Furthermore, such deficiency decreases endogenous monoamine concentrations, in particular the DA content of the frontal cortex [8,9]. As dopaminergic neurotransmission is mainly regulated by DAT anchored in the lipid bilayer, we hypothesized that changes in dopaminergic neurotransmission induced by n-3 PUFA deficiency could involve DAT density and/or function.

[9] 246w The dynamic microdialysis method allowed us to measure these changes, showing that the vesicular storage pool of DA was affected whereas the cytoplasmic compartment remained unchanged by n-3 PUFA deficiency [21,22,24]. Reduction in the storage pool of DA could be due to a decrease in the number of vesicles [23]. The decreased binding of [ 3 H]dihydrotetrabenazine observed in the frontal cortex [21] and the nucleus accumbens [22] might reflect changes in the structure and/or function of the vesicular monoamine transporter (VMAT 2 ). As the VMAT 2 shares several properties with the membrane DAT, it could be hypothesized that changes in dopaminergic neurotrans-mission induced by n-3 PUFA deficiency also affect DAT. We therefore used several approaches involving both the in vitro and in vivo study of DAT. In vitro binding experiments with the highly specific DAT ligand PE2I, which reflects both the affinity and density of binding sites, showed no difference between n-3 PUFA-deficient and control rats. In addition, the functional aspect of the transporter assessed by in vitro DA uptake and by in vivo microdialysis studies showed no effect of deficiency on DAT. Although no precise data are available, the fatty acid composition of vesicular membranes (around VMAT 2 ) and neuronal membranes (around DAT) is probably similarly modified in n-3 PUFA-deficient rats. The lack of effect of n-3 PUFA deficiency on DAT might therefore be indirect evidence of a reduced number of storage vesicles in deficient rats rather than impaired VMAT 2 function.

[10] 152w It is known that domains involved in recognition of a ligand and in translocation and regulatory function of a protein can be modulated by the surrounding bilayer lipid [5,17]. More specifically, it has been shown that the level of membrane DHA is able to act on the Na 1 /K 1 -ATPase activity of nerve endings [2]. Another hypothesis in agreement with our results concerning DAT and VMAT 2 is therefore that the physical properties of the membrane might act on VMAT 2 and not on DAT because of differences in the structural/functioning characteristics of these transporters. Although both are 12 transmembrane segment proteins, VMAT 2 use the proton electrochemical gradient across the vesicular membrane generated by a vacuolar H 1 -ATPase [11] while DAT accumulate DA from the synaptic space in a Na 1 /Cl 2 -dependent manner [1]. These distinct processes might therefore have different sensitivities to their membrane environment.

[11] 152w As DAT play a major physiological role in the control of the synaptic level of DA and locomotor activity [12], the lack of modification of this transporter in n-3 PUFA-deficient animals is in agreement with the lack of motor changes already described in these animals [10]. In addition, we previously observed no significant modification in DA D 2 receptors in the striatum of deficient rats, whereas a decrease was measured in the frontal cortex [8,9]. It can therefore be proposed that cerebral regions have different sensitivity to the amount of n-3 PUFA [4,9]. It would therefore be of great value to explore the effects of n-3 PUFA deficiency on DAT function and/or density in a cerebral region rich in DHA such as the frontal cortex. However, implementation of the methods used to explore the DAT would be difficult, as there are fewer DAT in cortical nerve endings than in the striatum [14].

[12] 37w Using three complementary methods, the present findings demonstrate that n-3 PUFA dietary deficiency does not modify DAT density or function in the striatum. However, we cannot exclude the possibility that DAT are affected in other cerebral regions.