Brain lipid composition in postnatal iron-induced motor behavior alterations following chronic neuroleptic administration in mice Giorgis Isaac1*, Anders Fredriksson2, Rolf Danielsson1, Per Eriksson3 and Jonas Bergquist1 Department of Analytical Chemistry, Biomedical Center, Uppsala University, Sweden ˚ker, Uppsala University, Sweden Department of Neuroscience, Psychiatry Ullera Department of Environmental Toxicology, Evolutionary Biology, Uppsala University, Sweden Keywords Neuroleptics; neurodegenerative and psychiatric disorders; phosphatidylcholine; postnatal iron treatment; sphingomyelin Correspondence J Bergquist, Department of Analytical Chemistry, Uppsala University, Box 599, SE-751 24 Uppsala, Sweden Fax: +46 18 4713692 Tel: +46 18 4713675 E-mail: jonas.bergquist@kemi.uu.se *Present address Division of Biology, Kansas State University, Manhattan, KA, USA (Received 22 December 2005, revised 23 Feburary 2006, accepted 20 March 2006) doi:10.1111/j.1742-4658.2006.05236.x Several studies have shown that deficient uptake or excessive break down of membrane phospholipids may be associated with neurodegenerative and psychiatric disorders The purpose of the present study was to examine the effects of postnatal iron administration in lipid composition and behavior and whether or not the established effects may be altered by subchronic administration of the neuroleptic compounds, clozapine and haloperidol In addition to motor activities such as locomotion, rearing and activity, a targeted lipidomics approach has been used to investigated the brains of eight groups of mice (four vehicle groups and four iron groups) containing six individuals in each group treated with vehicle, low dose clozapine, high dose clozapine and haloperidol Lipids were extracted by the Folch method and analyzed using reversed-phase capillary liquid chromatography coupled on-line to electrospray ionization mass spectrometry (LC ⁄ ESI ⁄ MS) Identification of phosphatidylcholine (PC) and sphingomyelin (SM) molecular species was based on their retention time, m ⁄ z ratio, head group specific up-front fragmentation and analysis of the product ions produced upon fragmentation A comparison between the Ve-groups and Fe-groups showed that levels of PC and SM molecular species and motor activities were significantly lower in Fe-Ve compared to Ve-Ve The effects of neuroleptic treatment with and without iron supplementation were studied In conclusion our results support the hypothesis that an association between psychiatric disorders and lipid and behavior abnormalities in the brain exists The physical properties and function of biological membranes are mediated to a large extent by their lipid composition Not only lipid classes such as phosphatidylcholine (PC), cholesterol, phosphatidylethanolamine, sphingomyelin (SM) but also the molecular species composition of the various phospholipids (PLs) and SM have to be determined in the characterization of lipid membranes [1] Phospholipids comprise a major part of the cell membrane, giving the membrane its structure and integrity SM is an important constituent in nervous tissue and plasma membrane of higher animals and is a phosphocholine ester of ceramide [2,3] A typical structure of a PL consists of three parts: a glycerol backbone, a polar head group and two fatty acid chains esterified at the sn-1 and sn-2 positions The Abbreviations AD, Alzheimer’s disease; EFA, essential fatty acid; IS, internal standard; LC ⁄ ESI ⁄ MS, liquid chromatography coupled on-line to electrospray ionization mass spectrometry; PD, Parkinson’s disease; PCA, principal component analysis; PL, phospholipids; PLA2, phospholipase A2 2232 FEBS Journal 273 (2006) 2232–2243 ª 2006 The Authors Journal compilation ª 2006 FEBS G Isaac et al specific hydrophilic head group varies and includes choline, ethanolamine, serine and inositol As the fatty acid chains can vary in length and degree of unsaturation, each natural PL and SM contain numerous molecular species The fatty acid tails can range from 16 to 24 carbons; typically one chain is saturated (sn-1) and the second (sn-2) contains one or more double bonds [4] Brain lipids have many physiological functions The phospholipid structure of neuronal membrane is essential for normal functioning of the nervous system Numerous hypotheses have been proposed to conceptualize the pathophysiology of neurodegenerative and psychiatric disorders, e.g schizophrenia One of these gaining a major research interest is the study of membrane composition and function, the phospholipid membrane hypothesis, originated by Feldberg [5] and Horrobin [6] The phospholipid hypothesis suggests that in some neurodegenerative and psychiatric disorders the metabolism and structure of membrane phospholipids are abnormal, not just in brain but also in other tissues, e.g red blood cells [7–9] Of particular interest in the context of these disorders are the profound effects that membrane structure can exert on receptor function The specific essential fatty acid (EFA) content of synaptic membrane can modify neuronal functions and produce clinical effects through at least two mechanisms: first, changes in EFA content alter the microenvironment and hence structure and function of membrane receptors, ion channels and enzymes Second, EFAs contribute to cell regulation by acting as a source of precursor for second messengers in intra- and intercellular signal transduction [4,10,11] The same membrane protein can behave quite differently when embedded in a membrane composed of saturated fatty acids or one in which unsaturated fatty acids predominate [4,8] A single change in membrane structure could produce changes in the behavior of all the receptor types associated with that membrane [4,7,8,11] and thus alter the behavior and response of any neurotransmitter system In general, membrane hypotheses are appealing because of their apparent ability to account for a range of disparate findings related to various disorders [4] Iron, the most abundant nonalkaline metal in the human body and brain [12], is involved in several metabolic processes and is essential for a normal neurological development [13], and iron deficiency during critical periods of development is associated with disruption of behavioral performance, e.g in learning and memory tasks [13] Nevertheless, there is accumulating evidence that excessive iron deposits in the brain, which may generate cytotoxic free radical formation Brain lipid composition in postnatal iron-induced mice [14,15], and alterations in iron metabolism play an important role in many neurologic diseases [16–18] Iron-overload and ⁄ or defects of the iron-dependent enzyme complex are implicated in the pathogenesis of neurodegenerative disorders, e.g Parkinson’s disease (PD) and Alzheimer,s disease (AD) [19–21] and in the Hallervorden–Spatz syndrome, involving aberrant brain iron metabolism [22] Several studies have demonstrated that elevations of iron are found in the substantia nigra of patients afflicted with PD [23–26] As demonstrated in several previous studies [27–31], postnatal administration of Fe2+ (7.5 mgỈkg)1, on days 10–12) increased the level of iron in the adult mice and rat brain together with alterations in motor behavior performance This postnatal iron administration can be used as a model for oxidative stress induced brain damage and neurodegenerative disorders Haloperidol, a butyrophenone, first developed as an analgesic, has found wide use as an antipsychotic agent due to its neuroleptic action It is known to block the dopamine receptors of the D2 type which is accepted as a mechanism for its neuroleptic action [32] Clozapine is rather novel and unique prototype atypical, dibenzodiazepine derivative, antipsychotic agent It has been proven effective and significantly superior to placebo, as well as to conventional neuroleptics Approximately 30–60% of all schizophrenic patients who fail to respond to typical antipsychotics may respond to clozapine [33] Membrane PL abnormalities have been studied previously in humans and animals using conventional chromatographic methods in brain [11,34–36], platelets and red blood cells [36,37] and using 31P-magnetic resonance spectroscopy (P-MRS) [36,38–41] Conventional analysis methods of PL and SM molecular species require laborious procedures including separation by column, argentation thin-layer chromatography or liquid chromatography (LC) after pre or postcolumn derivatization [42,43] A general drawback of these approaches is that they preclude the analysis of metabolic studies and the derivatization may subject the lipid to the danger of rearrangement of the fatty acyl chains on the glycerol backbone It is also more time consuming to obtain the derivatives compared to analyzing a total lipid extract directly without any derivatization [44] Although the in vivo determination of metabolic processes in the brain by P-MRS is a powerful method, individual molecular species of phospholipid membrane components can not be measured using present technology [37] Therefore brain extracts have been used to study the pattern and abnormalities of individual molecular species in the cellular level in more detail using LC coupled on-line to mass spectrometry (MS) previously developed by FEBS Journal 273 (2006) 2232–2243 ª 2006 The Authors Journal compilation ª 2006 FEBS 2233 Brain lipid composition in postnatal iron-induced mice G Isaac et al Isaac et al [45] The separation power of LC into either different lipid classes or molecular species within a class together with selective MS detection makes the simultaneous determination of protonated molecules and the identification of several structural elements possible [45,46] The purpose of the present study was to examine both the effect of postnatal iron administration in lipid composition and motor activities and whether or not the established effects may be altered by subchronic administration of the neuroleptic compounds, clozapine and haloperidol We investigated the PC and SM molecular species in brains from eight groups of mice treated with vehicle, low dose clozapine, high dose clozapine and haloperidol Results and Discussion Lipids make up a very high proportion of the brain (50–60% of dry weight) [47] A major proportion of these lipids are EFAs mainly bound to PLs [47] The membrane is a complex structure composed primarily of phospholipids and their constituent fatty acids, that provide scaffolding for many key functional systems, including neurotransmitter receptor binding, signal transduction and transmembrane ion channels Thus, the dynamic state of all membranes, including those of neurons and glia, is dependent on their composition, such that small changes in key phospholipids or the polyunsaturated fatty acids that make up phospholipids can lead to a broad range of membrane dysfunction The interest in the analysis of lipids in general and PL and SM in particular is continuously increasing due to the importance of these molecules related to various disorders Depending on the polar head group, fatty acid chain length and number of double bonds numerous individual lipid molecular species are present in a cell These species differ greatly in their chemical and biological properties under pathological conditions and their identification and quantitation are of great interest Recent advances in LC ⁄ MS have greatly enhanced the identification and profiling of individual molecular species These advances have made possible the field of lipidomics, which aims to identify all endogenous lipids [48–50] In this study a targeted lipidomics approach using LC ⁄ ESI ⁄ MS was applied to the different treatment groups in order to examine alteration in PC and SM molecular species (Table 1) LC/ESI/MS of lipid extracts First direct infusion of crude lipid extracts from mouse brain was tested as suggested by Han et al [51] How2234 Table The table shows the eight treatment groups For details in treatment see Experimental procedures Treatment Ve-groups Fe-groups Vehicle Clozapine (1mgkg)1) Clozapine (5mgkg)1) Haloperidol (1mgkg)1) Ve-Ve Ve-C1 Ve-C5 Ve-H1 Fe-Ve Fe-C1 Fe-C5 Fe-H1 ever this approach did not yield acceptable signals probably reflecting signal suppression by other components in the crude lipid extract In order to reduce this suppression effect, the LC ⁄ ESI ⁄ MS method previously developed [45] for the analysis of PC and SM molecular species was applied with minor modifications The LC column was changed to a larger internal diameter (0.5 mm) in order to avoid frequent plugging of the column, variation in retention time from run to run and to accommodate the large number of runs from the crude lipid extracts Identification of PC and SM molecular species was based on their retention time, m ⁄ z ratio, head group specific up-front fragmentation and analysis of the product ions produced upon fragmentation Shown in Fig are representatives of the extracted normalized ion chromatograms of the most abundant protonated molecular ions of PC and SM molecular species from crude mouse brain extract spiked with deuterium labeled 1,2-dipalmitoyl-snglycero-3-phosphocholine (d4–16 : ⁄ 16 : 0) internal standard (IS) The actual peak area of individual molecular species normalized against the IS in the vehicle group and iron group after three weeks of low dose clozapine (1 mgỈkg)1), high dose clozapine (5 mgỈkg)1) and haloperidol (1 mgỈkg)1) treatment are shown in Table Effect of neuroleptics and iron treatment on lipid membrane A principal component analysis (PCA) of all lipid results (11 variables for 90 samples) resulted in loadings for the first two components (explaining 76% and 10%, respectively, of the total variance) according to Fig 2A The axes were scaled in proportion to the square root of the explained variance; hence the influence of a variable is directly related to its distance from the origin It is seen that all the variables have about the same influence on the lipid variations explained by the two components Moreover, the correlation between two variables is the cosine of the angle between their connection lines to the origin It is seen that most of the variables are clustered, indicating FEBS Journal 273 (2006) 2232–2243 ª 2006 The Authors Journal compilation ª 2006 FEBS G Isaac et al Brain lipid composition in postnatal iron-induced mice Normalized Intensity PC6 0.25 PC2 SM2 Fig Extracted normalized ion chromatograms of the most abundant protonated molecular ions of PC and SM molecular species The peak intensities are normalized to the intensity of the deuterium labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (d4–16 : ⁄ 16 : 0) internal standard (IS) The molecular species were resolved and identified based on their retention time, m ⁄ z ratio, head group specific up-front fragmentation and analysis of the product ions produced upon fragmentation The inserted chromatogram shows the low intensity species Peak identification as in Table 10 15 20 25 30 35 40 45 50 PC9 PC8 PC1 SM1 PC3 PC7 IS 10 15 similar variation pattern, although with some deviation for PC5, PC6 and especially PC3 The correlation coefficient between PC1 and PC4 (bracketing the cluster) is 0.71, while that between PC1 and PC3 (the least concordant variables) is only 0.16 According to Fig 2A, the first principal component is a linear combination with positive contributions (loadings) from all variables, but to some lesser degree from PC3 which then dominates the second component The score values for the two components, together accounting for 86% of the variance, were then analyzed by nested anova to separate the contributions from different treatments, individual animals and replicate analyses Only the scores for the first component showed significant difference between treatment groups, which is the systematic variation we are looking for The random variation in the first component (not related to treatment) was mainly due to individual animals with less contribution from the repeated analysis (87% and 13%, respectively) Justified by these findings, the further data analysis was performed on the scores for the first principal components rather than on the separate lipid concentrations For each animal the average score from the two runs was taken as an over-all measure of the lipid concentration In a few cases only one measurement was available, but as the repeatability error was comparatively low the single score value could be used A one-way anova as well as Kruskal–Wallis’ nonparametric test showed highly significant variation between the treatment groups (P < 0.001) The mean values (least squares means) for the different groups are 20 25 30 Time (min) 35 40 45 50 shown in Fig 2B The neuroleptic treated groups are shown with error bars related to the least significant difference The vehicle treated groups, Ve-Ve and Fe-Ve, define reference levels depicted as error bands Hence there is a significant difference between two means at approximately 5% significance level when the error bars or bands are not overlapping The significance is further confirmed with an ordinary two-sided t-test between two means, and the P-value is reported The confirmation of anova and least significant difference (LSD) comparisons with other tests is due to the slightly significant inhomogeniety of variance according to Bartlett’s test (P ¼ 0.034) First, it can be noted that iron supplementation results in a significant decrease in the amount of PC and SM molecular species (nonoverlapping reference bands, P < 0.025) The effect of subsequent neuroleptic treatment for three weeks can be examined by comparing the corresponding error bars with the Fe-Ve band Both low and high dose clozapine (Fe-C1 and Fe-C5) treated mice did not show significant differences in lipid concentration compared to Fe-Ve However haloperidol (Fe-H1) significantly increased the lipid concentration compared to Fe-Ve (P ¼ 0.009) and to both low and high dose clozapine treated mice (P ¼ 0.004 and P < 0.001, respectively) The intrinsic effect of the neuroleptic treatment was studied by comparing Ve-Ve against Ve-C1, Ve-C5 and Ve-H1 As can be seen in Fig 2B there is no significant change of the PC and SM molecular species when low dose clozapine (Ve-C1) is given compared to vehicle (Ve-Ve) When high dose clozapine (Ve-C5) or FEBS Journal 273 (2006) 2232–2243 ª 2006 The Authors Journal compilation ª 2006 FEBS 2235 2236 0.02 0.03 0.08 0.06 0.43 0.87 0.15 0.26 0.44 0.10 0.02 6·2 3.13 ± 0.59 ± 0.80 ± 1.25 ± 9.79 ± 16.52 ± 3.98 ± 5.72 ± 11.11 ± 2.67 ± 0.69 ± 5·2 2.41 ± 0.50 ± 0.59 ± 0.90 ± 8.13 ± 14.63 ± 3.47 ± 4.76 ± 9.37 ± 2.25 ± 0.57 ± 6·2 3.15 ± 0.63 ± 0.77 ± 1.36 ± 9.88 ± 16.49 ± 4.14 ± 6.03 ± 11.62 ± 2.88 ± 0.71 ± 0.03 0.02 0.05 0.05 0.32 0.60 0.11 0.20 0.44 0.09 0.03 6·2 2.70 ± 0.61 ± 0.79 ± 1.13 ± 9.60 ± 16.11 ± 3.72 ± 5.36 ± 10.19 ± 2.46 ± 0.59 ± Number of animals PC1 (16 : ⁄ 22 : 6, m ⁄ z 806.6) PC2 (16 : ⁄ 18 : 2, m ⁄ z 758.6) PC3 (18 : ⁄ 18 : 2, m ⁄ z 782.6) PC4 (18 : ⁄ 20 : 4, m ⁄ z 808.6) PC5 (16 : ⁄ 16 : 0, m ⁄ z 734.6) PC6 (16 : ⁄ 18 : 1, m ⁄ z 760.6) PC7 (18 : ⁄ 18 : 2, m ⁄ z 786.6) PC8 (18 : ⁄ 20 : 4, m ⁄ z 810.6) PC9 (18 : ⁄ 18 : 1, m ⁄ z 788.6) SM1 (18 : 0, m ⁄ z 731.6) SM2 (24 : 1, m ⁄ z 813.6) 0.02 0.01 0.01 0.01 0.10 0.20 0.05 0.07 0.18 0.03 0.02 6·2 2.59 ± 0.52 ± 0.91 ± 1.17 ± 9.23 ± 15.64 ± 3.55 ± 5.19 ± 9.69 ± 2.39 ± 0.57 ± 0.05 0.02 0.04 0.06 0.32 0.60 0.07 0.14 0.37 0.07 0.04 6·2 2.30 ± 0.47 ± 0.76 ± 0.95 ± 8.53 ± 15.01 ± 3.21 ± 4.61 ± 8.67 ± 2.00 ± 0.50 ± 0.08 0.02 0.05 0.05 0.22 0.51 0.04 0.15 0.30 0.06 0.03 0.11 0.02 0.04 0.02 0.18 0.27 0.09 0.14 0.16 0.05 0.01 5·2 2.38 ± 0.42 ± 0.54 ± 0.86 ± 7.19 ± 13.42 ± 2.97 ± 4.18 ± 7.86 ± 1.78 ± 0.43 ± 0.06 0.03 0.01 0.05 0.37 0.81 0.11 0.17 0.35 0.19 0.04 6·2 2.60 ± 0.48 ± 0.72 ± 1.02 ± 8.21 ± 14.49 ± 3.29 ± 4.71 ± 9.01 ± 2.19 ± 0.56 ± 0.10 0.03 0.11 0.08 0.78 1.26 0.16 0.24 0.41 0.14 0.04 Fe-H1 Fe-C5 Fe-C1 Fe-Ve Ve-H1 Ve-C5 Ve-C1 Ve-Ve Treatment Table Data given as peak area of the molecular species normalized against the peak area of the internal standard [IS (mean values ± SEM)] Each treatment group contains animals except treatments Fe-Ve and Fe-C1 which contain animals due to losses during the extraction procedure The duplicate measurements for each animal were averaged, and for each group the mean values and the corresponding standard errors of the mean were calculated Brain lipid composition in postnatal iron-induced mice G Isaac et al haloperidol (Ve-H1) is given there is a significant decrease in lipid concentration compared to Ve-Ve (P ¼ 0.037 and p ¼ 0.008, respectively) It was also shown by Singh et al that haloperidol reduced biosynthesis of various phospholipids in rat brain by following incorporation of 32P into individual classes of phospholipid [32] The traditional neuroleptic haloperidol decreased the PC and SM molecular species concentration in comparison to clozapine A comparison was also made between the vehicle and iron groups to examine the difference between iron and vehicle supplemented groups after subchronic administration of the neuroleptic compounds An interesting phenomenon is that lipid concentrations were lower in Fe-C1 compared to Ve-C1 (P ¼ 0.038), no significant difference with high dose clozapine and actually higher for mice treated with haloperidol (P < 0.001) Effect of neuroleptics and iron treatment on behavior Motor activities such as locomotion (L), rearing (R) and activity (A) were measured for short (20 min) and long (60 min) terms as mentioned under experimental section The motor activities were analyzed by PCA in a similar way as the lipid data The loading plot (Fig 3A) for the first two components, explaining 83% and 9%, respectively, shows that the behavioral variables contribute almost equally to the dominating first component The second component seems to reflect the difference between short-term and long-term behavior The lowest correlation coefficient, 0.58, is found between L60 and R20 As for the lipid data, one way anova (as well as Kruskal–Wallis’ test) showed a highly significant difference between the treatment groups (P < 0.001) for the score values along the first component Barlett’s test indicated slightly inhomogenuos variance (P ¼ 0.025), which gives cause for the confirmation with alternative tests Also for the second component the scores showed a significant variation between the groups (P ¼ 0.012) However, in view of the lower significance and the small proportion of explained variance (9%) the scores for the second component were not further analyzed The scores for the first principal components were taken as a single-valued measure of the behavior (locomotion, rearing and activity) for each animal The mean value for each group is shown in Fig 3B in the same way as for lipid concentrations (cf Fig 2B) The clear gap between the reference bands (Ve-Ve, Fe-Ve) shows the highly significant decrease in motor FEBS Journal 273 (2006) 2232–2243 ª 2006 The Authors Journal compilation ª 2006 FEBS G Isaac et al Brain lipid composition in postnatal iron-induced mice PC3 B + A PC4 PC2 PC8 PC5 PC7 SM2 PC1 Ve-Ve Fe-Ve - 2nd comp PC5 Lipid conc PC6 Ve- SM1 -C1 -C5 -H1 Fe- 1st comp Fig (A) Loading plot for the lipid concentration variables Phosphatidylcholine (PC) and sphingomyelin (SM) molecular species identification as in Table (B) Mean scores for the treatment groups from lipid data The neuroleptic treated groups are shown with error bars, while the vehicle treated groups, Ve-Ve and Fe-Ve, define reference levels depicted as error bands Overlapping error bars (bands) indicate significant differences according to the least significant difference (LSD) A B + L60 Ve-Ve L20 Activities R60 - 2nd comp A60 Fe-Ve A20 1st comp Ve- R20 Fe- -C1 -C5 -H1 Fig (A) Loading plot for the behavioral variables measured for 20 and 60 Locomotion (L), rearing (R) and activity (A) (B) Mean scores for the treatment groups from behavioral data The neuroleptic treated groups are shown with error bars, while the vehicle treated groups, Ve-Ve and Fe-Ve, define reference levels depicted as error bands Overlapping error bars (bands) indicate significant differences according to the LSD activities with iron supplementation (P < 0.001) Subsequent treatment with low dose clozapine (Fe-C1) had no significant effect in the activities compared to Fe-Ve Haloperidol (Fe-H1) and the higher dose of clozapine (Fe-C5) elevated the motor activities significantly compared to Fe-Ve (P < 0.001) The activities even raised to the same level as Ve-Ve, thus the behavioral effect of iron supplementation was abolished by the neuroleptic treatment The intrinsic effect of neuroleptic treatment on motor activities was studied by comparing Ve-Ve against Ve-C1, Ve-C5 and Ve-H1 As can be seen in Fig 3B there is no significant difference for low (VeC1) and high (Ve-C5) dose clozapine treated mice compared to Ve-Ve but a significant increase with haloperidol (Ve-H1, p ¼ 0.035) The difference in motor activities induced by iron supplementation (Ve-Ve compared to Fe-Ve) was about the same after low dose clozapine treatment (Ve-C1 versus Fe-C1) However, there was no significant difference related to iron supplementation for the groups treated with high dose clozapine (Ve-C5 versus Fe-C5) or haloperidol (Ve-H1 versus Fe-H1) The combined effects in motor activities and lipid concentrations are visualized in Fig 4, where the X-axis represents the lipid concentrations and the Y-axis represents activities The ovals represent error regions based on least significant difference similarly to the error FEBS Journal 273 (2006) 2232–2243 ª 2006 The Authors Journal compilation ª 2006 FEBS 2237 Brain lipid composition in postnatal iron-induced mice Ve-H1 G Isaac et al Fe-H1 + Ve-C5 Ve-C1 Ve-Ve - Activities Fe-C5 Fe-Ve Fe-C1 - Lipid conc + Fig Mean scores for the treatment groups from lipid and behavioral data The oval shapes represent confidence regions related to the LSD Two nonoverlapping groups show significant difference (P < 0.05) considering both motor activities and lipid concentrations Filled ovals represent mice supplemented with iron (Fe-) and open ovals those supplemented with vehicle (Ve-) bands and error bars in Figs and Hence, when two ovals are not overlapping there is a significant difference at approximately 5% significance level Clearly, the iron supplemented mice treated with vehicle or low dose clozapine are similar, standing out from the others The immediate postnatal period is critical for establishment of normal iron content in the adult brain and its regional distribution Investigations of cerebral iron uptake indicate that both iron transport and transferrin, the iron mobilization protein, binding sites are maximal during the postnatal period of rapid brain growth, essentially during the second week post partum in rats and mice [52], and maximal brain iron uptake occurs in 15-day-old rats [53] Dwork et al [54] showed that iron acquired by the brain during this period of development is retained in the brain without being returned to plasma sites In many mammalian species, a period of rapid brain growth occurs during perinatal development, termed ‘brain growth spurt’ [55] In humans this period begins during the third trimester of pregnancy and is maintained throughout the first years of life whereas in rats and mice the corresponding period occurs during the first three-to-four weeks of postnatal life During this critical period of neuronal development the essential processes of brain structure and function are established [56] and fundamental sensory-motor faculties are acquired [57,58] Thus, it appears that there may exist a critical neonatal period of brain development associated with the establishment of normal iron content in the adult brain 2238 Several studies by Ben-Shachar et al [14,59,60] have related the effects of chronic administration of neuroleptic compounds to the involvement of iron in the expressions of neuroleptic-induced dopamine (DA) supersensitivity These studies suggest that the mobilization of iron from peripheral tissues into the brain may exert a role in the mechanism of action of neuroleptic compounds Haloperidol has been shown to alter the property of the blood–brain barrier by enhancing, normally restricted, iron transport into the brain whereas clozapine prohibits iron uptake into the brain [59] Furthermore, Ben-Shachar and Youdim [60] examined the possibility that neuroleptic-induced DA D2 receptor supersensitivity involves an alteration in brain iron content whereby liver nonheme iron stores were depleted in rats treated chronically with haloperidol (5 mgỈkg)1 daily for 14 or 21 days) as compared with values in control rats Ben-Shachar et al [14] showed that FeCl2 (5 mgỈkg)1 per day over 21 days) administered to rats treated with chlorpromazine (10 mgỈkg)1 per day over 21 days) prevented the expressions of DA D2 receptor supersensitivity, both biochemically and behaviourally (apomorphineinduced activity) Moreover, neuroleptics may alter brain phospholipid metabolism and composition by regulating phospholipase A2 (PLA2) activity Possible effect of neuroleptics such as haloperidol on membrane phospholipid metabolism and composition are under investigation Several studies have shown that various neuroleptics reduce the break down of phospholipid membrane by inhibiting PLA2 activity [10,38,61–63] Gattaz et al [61] found platelet PLA2 activity to be greater in schizophrenic patients than in healthy controls, and this elevation was reduced by haloperidol treatment Trzeciak et al [63] reported a decrease in PLA2 activity after a single or four week administration of chlorpromazine, trifluoperazine, haloperidol and sulpiride Fluphenazine and thioridazine caused an increase of PLA2 activity in rat brain both after a single dose and long-term administration [63] Clozapine is good antioxidants and decreases membrane lipid peroxidation [64] whereas haloperidol has been reported to have the opposite effect [65] Conclusions In summary, a targeted lipidomics approach has been used to identify the PC and SM molecular species alteration coupled with behavioral data to investigate the effects of postnatal iron supplementation The effect of subsequent subchronic treatment with neuroleptics was also studied A comparison between the Ve-groups and Fe-groups showed that levels of PC FEBS Journal 273 (2006) 2232–2243 ª 2006 The Authors Journal compilation ª 2006 FEBS G Isaac et al and SM molecular species and motor activities were significantly lower in Fe-Ve compared to Ve-Ve A comparison of Fe-Ve with Fe-C1, Fe-C5 and Fe-H1 showed no significant change in PC and SM molecular species for low and high dose clozapine treated mice while haloperidol administration significantly increased the molecular species Low dose clozapine (Fe-C1) has no significant effect in motor activities but Fe-C5 and Fe-H1 elevated the activities significantly compared to Fe-Ve The intrinsic effects of neuroleptic treatment in the Ve-groups (i.e by comparing Ve-Ve with Ve-C1, Ve-C5 and Ve-H1) were demonstrated No significant change in PC and SM molecular species were found when the mice were treated with low dose clozapine while high dose clozapine and haloperidol treated mice showed significant change in PC and SM molecular species There is no significant change in motor activities for Ve-C1 and Ve-C5 treated mice but with haloperidol (Ve-H1) there was a slight increase in activities An interesting phenomenon is that both lipid concentrations and activities were decreased in Fe-C1 compared to Ve-C1 and for high dose clozapine there is no significant difference When the mice were treated with haloperidol the lipid concentration is significantly higher in Fe-H1 compared to Ve-H1 while there was no change in activities Experimental procedures Brain lipid composition in postnatal iron-induced mice study At the age of weeks the mice were weaned and the males were placed and raised in groups of 4–6 animals in a room maintained for male mice only Male mice, postnatal days 10–12, were administered Fe2+ (see below) or saline At 61 days of age and onwards for three weeks, these mice, weighing 20–24 g, were administered subcutaneously either a neuroleptic compound [Clozapine (1 or mgỈkg)1) or Haloperidol (1 mgỈkg)1)] or Vehicle (Tween-80) Free access to food and water was maintained throughout They were housed in groups of animals and tested only during the hours of light (08.00– 15.00 h) Behavioral testing was initiated three weeks following the start of treatment with the neuroleptic compounds or vehicle All testing was performed in a normally lighted room This test room, in which all 12 ADEA activity test chambers, each identical to the home cage, were placed, was well-secluded and used only for this purpose Each test chamber (i.e motor activity test cage) was placed in a sound-proofed wooden box with 12 cm thick walls and front panels and a small double-glass window to allow observation; each box had a dimmed lighting Mice were killed by cervical dislocation within weeks after completion of behavioral testing The brains from each group were dissected and stored in )80 °C until extraction Experiments were carried out in accordance with the European Communities Council Directive of 24th November 1986 (86 ⁄ 609 ⁄ EEC) after approval from the local ethical committee (Uppsala University and Agricultural Research Council), and by the Swedish Committee for Ethical Experiments on Laboratory Animals (license S93 ⁄ 92 and S77 ⁄ 94, Stockholm, Sweden) Chemicals and reagents Lipid standards of l-a-phosphatidylcholine (PC from egg yolk) and N-acyl-d-sphingosine-1-phosphocholine (SM from bovine brain) were purchased from Sigma Chemical Co (St Louis, MO, USA) A deuterium labeled 1,2-dipalmitoylsn-glycero-3-phosphocholine (d4–16 : ⁄ 16 : 0) was purchased from Avanti Polar Lipids (Alabaster, AL, USA) Ultrapure water was prepared by Milli-Q water system (Millipore, Bedford, MA, USA) Other organic solvents and reagents were of the highest purity commercially available Clozapine (Sandoz, Basel, Switzerland) and Haloperidol (Leo, Halsingborg, Sweden) FerromynÒ (Iron succinate: ă 3.7 mg Fe2+ per mL)1, AB Hassle, Goteborg, Sweden) ă ă Animals Pregnant C57 Bl mice were purchased from B & K, Sollentuna, Sweden Each litter adjusted within 48 h to 8–10 mice and to contain offspring of either sex in about equal number, was kept together with its respective mother in a plastic cage in a room at temperature of 22 ± °C and a 12 ⁄ 12 h constant light ⁄ dark cycle (lights on between 06.00 and 18.00 h) The male offspring only were used in this Behavioral measurements and apparatus Activity test chambers An automated device, consisting of macrolon rodent test cages (40 · 25 · 15 cm) each placed within two series of infra-red beams (at two different heights, one low and one high, and cm, respectively, above the surface of the sawdust, cm deep), was used to measure spontaneous motor activity (RAT-O-MATIC, ADEA Elektronik AB, Uppsala, Sweden) The distance between the infra-red beams was as follows: the low level beams were 73 mm apart lengthwise and 58 mm apart breadth wise in relation to the test chamber; the high level beams, placed only along each long side of the test chamber were 28 mm apart According to the procedures described previously [66], the following parameters were measured: locomotion was measured by the low grid of infra-red beams Counts were registered only when the mouse in the horizontal plane, ambulating around the test-cage Rearing was registered throughout the time when at least one high level beam was interrupted, that is the number of counts registered was proportional to the amount of time spent rearing Total activity was measured FEBS Journal 273 (2006) 2232–2243 ª 2006 The Authors Journal compilation ª 2006 FEBS 2239 Brain lipid composition in postnatal iron-induced mice G Isaac et al by a sensor (a pick-up similar to a gramophone needle, mounted on a lever with a counterweight) with which the test cage was constantly in contact The sensor registered all types of vibration received from the test cage, such as those produced both by locomotion and rearing as well as shaking, tremors, scratching and grooming All three behavioral parameters were measured over three consecutive 20-min periods The motor activity test room, in which all 12 ADEA activity test chambers, each identical to the home cage, were placed, was well-secluded and used only for this purpose Each test chamber (that is activity cage) was placed in a sound-proofed wooden box with 12 cm thick walls and front panels, and day-lighting Motor activity parameters were tested on one occasion only, over three consecutive 20-min periods, at the age of 3–4 months Extraction and Analysis Brain lipids were extracted by Folch method and PC and SM molecular species were analyzed with minor modification of the previously developed LC ⁄ ESI ⁄ MS method [45] Design and Treatment Eight treatment groups were derived from four groups having received Fe2+ as pups on postnatal days 10–12 was administered orally via a metallic gastric tube in a volume of 10 mLỈkg)1 (7.5 mgỈkg)1) body weight [Fe-groups] and four groups having received vehicle (saline) at that same period [Ve-groups] Saline was used as vehicle and to prepare the dose of Fe2+ On day 61 after birth and for three weeks following, two Fe-groups and two Ve-groups received clozapine (1 or mgỈkg)1), one Fe-group and one Ve-group received haloperidol (1 mgỈkg)1), one Fe-group and one Ve-group received vehicle administration to provide the groups shown in Table Spontaneous motor behavior was initiated 24 h following the final injection of clozapine, haloperidol or vehicle Locomotion, rearing and total activity counts were registered over 60 Statistical Analysis With principal component analysis (PCA) the concentrations for the different lipid compounds were linearly combined into score values, and the scores rather than single concentrations were investigated with one-way anova to elucidate the variations between the groups The aim of this type of analysis was to enhance the systematic variation, which could be expected to show up in the few first components A loading plot also reveals to what degree the individual variables (lipids) contribute to the variation pattern Variables with similar loading values are more or less indicators of the same phenomena, which can be confirmed by a high correlation coefficient The mean score (one for each 2240 principal component) for a group represents the lipid state of the group, thus condensed into a single value if only the first component needs to be considered If the mean scores show significant variation between the groups, pair-wise comparisons may be made using the least significant difference (LSD) Visually this was accomplished by representing the means as intervals according to mean ± tỈs ⁄ Ö(2n), where s is the pooled standard deviation and n is the number of replicates in the treatment group When the intervals for two groups are nonoverlapping, there is a significant difference between the means according to the LSD test This approach is based on the approximation Ö(1 ⁄ n1 +1 ⁄ n2)  ⁄ Ö(2n1) +1 ⁄ Ö(2n2), which is exact for n1 ¼ n2 and holds within 0.1% for n1 ¼ and n2 ¼ (the only unequal numbers in this study) However, the application of anova and the LSD comparison test requires equal variances as well as normal distribution for the variations within the groups Therefore, when significance was found it was confirmed by Kruskal– Wallis’ nonparametric test as a robust alternative to anova The significant pair-wise group differences were confirmed by application of an ordinary t-test for the means with unequal variances, a test that also is fairly insensitive to deviations from normality distribution Bartlett’s test for equal variances was applied in order to find out whether the confirmation with alternative tests should be performed The same multivariate approach was taken for the six behavioral variables, enabling test of significant differences in total activities between the groups Finally, a plot of lipid scores versus behavioral scores, with each group depicted as a confidence region, was made in order to visualize the overall influence of postnatal iron supplementation as well as neuroleptic treatments All basic calculations were performed with Microsoft excel 2002 SP3, anova with minitab release 14 (Minitab Inc., State College, PA, USA) and PCA with the unscrambler v7.6 SR1 (CAMO Technologies Inc., Woodbridge, NJ, USA) Acknowledgements Financial support from SIDA ⁄ SAREC Foundation and Uppsala University (GI), the Swedish Society for Medical Research (J.B), and the Swedish Research Council (621-2002-5261, 629-2002-6821 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