• α-linolenic acid;
  • arachidonic acid;
  • brain;
  • eicosapentaenoic acid;
  • linoleic acid;
  • phospholipids


  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acid in the brain, has important functions in the hippocampus. To better understand essential fatty acid homeostasis in this region of the brain, we investigated the contributions of n-3 fatty acid precursors in supplying hippocampal neurons with DHA. Primary cultures of rat hippocampal neurons incorporated radiolabeled 18-, 20-, 22-, and 24-carbon n-3 fatty acid and converted some of the uptake to DHA, but the amounts produced from either [1-14C]α-linolenic or [1-14C]eicosapentaenoic acid were considerably less than the amounts incorporated when the cultures were incubated with [1-14C]22:6n-3. Most of the [1-14C]22:6n-3 uptake was incorporated into phospholipids, primarily ethanolamine phosphoglycerides. Additional studies demonstrated that the neurons converted [1-14C]linoleic acid to arachidonic acid, the main n-6 fatty acid in the brain. These findings differ from previous results indicating that cerebral and cerebellar neurons cannot convert polyunsaturated fatty acid precursors to DHA or arachidonic acid. Fatty acid compositional analysis demonstrated that the hippocampal neurons contained only 1.1–2.5 mol% DHA under the usual low-DHA culture conditions. The relatively low-DHA content suggests that some responses obtained with these cultures may not be representative of neuronal function in the brain.

Abbreviations used

linoleic acid


arachidonic acid


docosapentaenoic acid


tetracosapentaenoic acid


tetracosahexaenoic acid

ALA or 18:3n-3

α-linolenic acid


cytosine arabinoside C

DHA or 22:6n-3

docosahexaenoic acid

EPA or 20:5n-3

eicosapentaenoic acid


glial fibrillary acidic protein


microtubule-associated protein

Docosahexaenoic acid (DHA or 22:6n-3), the most abundant n-3 polyunsaturated fatty acid present in the brain, is required for normal development and function of the nervous system (Lauritzen et al. 2001; Salem et al. 2001; Yavin 2006). DHA is contained in membrane phospholipids, primarily in the ethanolamine and serine phosphoglycerides fractions (Spector 1999), and it has a highly flexible structure that affects matrix and elastic properties of the lipid bilayer (Eldho et al. 2003; Gawrisch et al. 2003; Bruno et al. 2007). DHA promotes neuronal differentiation (Kawakita et al. 2006; Kan et al. 2007), prevents apoptosis (Kim et al. 2000; Florent et al. 2006), and activates neuronal gene expression by functioning as a ligand for the retinoid X receptor in the brain (Mata de Urquiza et al. 2000; Goldstein et al. 2003). In addition, DHA activates the Akt signaling pathway, a process that enhances neuronal survival (Akbar et al. 2005), and phospholipids containing DHA facilitate G protein-coupled signaling (Litman et al. 2001). Compounds synthesized from DHA also can affect neuronal function in various ways. For example, neuroprotectin D1, a 10,17S-docosatriene derivative of DHA formed by the lipoxygenase pathway, protects neural cells (Serhan et al. 2006; Bazan 2007), whereas neuroprostanes, which are isoprostane-like compounds produced from DHA during exposure to oxidative stress, damage the cells by altering the physical properties of the neural membranes (Roberts et al. 1998; Fam et al. 2002).

Because DHA is an essential fatty acid for mammalian cells, it must be supplied either directly to the cells or in the form of another n-3 fatty acid like α-linolenic acid (ALA or 18:3n-3) or eicosapentaenoic acid (EPA or 20:5n-3) that can be converted to DHA. The relative importance of these two mechanisms has been studied in some detail in astrocytes (Moore et al. 1991; Spector and Moore 1993; Williard et al. 2001a). However, little detailed information is currently available regarding neurons, and the purpose of the present study was to investigate this process in primary cultures of neurons isolated from the brain. Rat hippocampal neurons were selected as a model system because DHA has an important role in the hippocampus, a region of the brain that is critical for learning. Data from animal models indicate that a decrease in DHA content, which normally comprises 13% of the fatty acids in the hippocampus (Diau et al. 2005), significantly reduces spatial learning in rats as determined by the Morris water maze test (Lim et al. 2005a,b). This reduction is associated with a decrease in the cell body size of the CA1 pyramidal neurons (Ahmad et al. 2002). DHA also promotes neurite outgrowth in hippocampal neurons (Calderon and Kim 2004). Another reason for selecting hippocampal neurons as the model system is that these cultures are being widely used to investigate many important functional processes, including glutamatergic synapse formation (Jelks et al. 2007), endocannabinoid signaling (Ohno-Shosaku et al. 2007), β-amyloid-induced changes in synaptic vesicle recycling (Kelly and Ferreira 2007), the role of proinflammatory cytokines on synaptic network activity (Vereyken et al. 2007), and the role of dendritic contact in the assembly of the functional pre-synaptic apparatus (Krueger et al. 2003). Moreover, the low-density serum-free defined medium allows healthy neuronal maturation with minimal glia cell growth, enabling us to study fatty acid metabolism in highly enriched neuron cultures.

Most of the available in vivo data indicate that the brain primarily utilizes DHA obtained directly from the diet or produced in the liver from dietary n-3 fatty acid precursors (Scott and Bazan 1989; Su et al. 2000; Rapoport et al. 2001b; DeMar et al. 2005; Lefkowitz et al. 2005). This mechanism is supported by previous results demonstrating that neurons cultured from the forebrain gray matter of 2- to 7-day-old rat pups took up DHA but did not synthesize DHA from ALA or EPA (Moore et al. 1991; Spector and Moore 1993). Likewise, cultures composed of 90% glutaminergic granular neurons and 5–7% GABAergic neurons obtained from the cerebellum took up DHA but did not convert ALA or EPA to DHA (Moore et al. 1991). The cerebral and cerebellar neuron cultures also did not convert linoleic acid (18:2n-6) to arachidonic acid (20:4n-6), the main n-6 fatty acid in the brain, providing additional evidence that neurons cannot convert polyunsaturated fatty acid precursors to the highly unsaturated derivatives required by the brain (Moore et al. 1991). As opposed to these previous results, we now find that primary cultures of rat hippocampal neurons express the complete polyunsaturated fatty acid metabolic pathway (Sprecher et al. 1995), and they are able to convert n-3 fatty acid precursors to DHA and linoleic acid to arachidonic acid.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Primary culture of rat hippocampal neurons

Low-density cultures of dissociated rat hippocampal neurons were prepared using previously described methods (Lim et al. 2003). In brief, hippocampi were dissected from 18-day embryonic rats (Harlan Sprague–Dawley, Indianapolis, IN, USA). The pooled tissue was incubated in HEPES-buffered saline solution (Ca2+/Mg2+-free) containing 0.03% trypsin at 37°C for 15 min. After the tissue was washed, it was dissociated with a fire-polished Pasteur pipette and plated at a density of 3–6 × 103/cm on coverslips (Warner Instruments, Hamden, CT, USA) coated with 0.1% (w/v) poly-l-lysine (Peptides International, Louisville, KY, USA). This low-density culture was plated in Neurobasal media containing B-27 supplement (prepared in our laboratory according to a published formulation; Brewer et al. 1993), 0.5 mM glutamine, and 5% horse serum. This medium was replaced with serum-free Neurobasal medium supplemented with B-27 and 0.5 mM glutamine 4 h after plating. Cells were maintained at 37°C in a humidified environment of 95% air and 5% CO2. In some experiments, cultures maintained for 7 days were treated overnight with 2 μM cytosine arabinoside hydrochloride (Ara-C; Sigma, St Louis, MO, USA) to remove astrocytes, and the cultures were then continued in normal growth medium. Radiolabeled DHA formation was compared in cultures treated with Ara-C and corresponding untreated cultures.

Fluorescence microscopy, imaging, and antibodies

The purity of the hippocampal cultures was determined by immunofluorescence microscopy using a Olympus IX-70 inverted epifluorescence microscope (Leeds, Minneapolis MN, USA) equipped with an Olympus (Center Valley, PA, USA) 10× dry objective, a MAC2002 Ludl shutter (BD Biosciences Bioimaging, Rockville, MD, USA), and fluorescence filter sets (Chroma, Brattleboro, VT, USA) for fluorescein (490 nm band-pass excitation and 528 nm long-pass emission) and rhodamine (555 nm band-pass excitation and 617 nm long-pass emission). Images were acquired with an ORCA II CCD camera (Hamamatsu, Bridgewater, NJ, USA) equipped with a frame grabber EDT DV PCI card controlled by Esee software (Inovision, Chapel Hill, NC, USA). Coverslips on which hippocampal cells were grown were rinsed in Dulbecco’s phosphate-buffered saline (Invitrogen, Carlsbad, CA, USA) before fixation. The coverslips were transferred to 4%p-formaldehyde with 4% glucose in the phosphate buffer for 15 min. Following fixation, the cells were permeabilized with 0.1% Triton X-100 for 20 min and incubated in phosphate-buffered blocking solution (2% glycerol, 0.05 M NH4Cl, 5% fetal bovine serum, and 2% donkey serum) for 2 h. Primary antibodies were added, and the cells were incubated for either 1.5 h or overnight at 4°C. The cells were again washed and incubated for 1 h at 23°C with Alexa 488 and 568 conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) diluted 1 : 100. After a final wash, the cells were mounted in Prolong Antifade mounting media before viewing. Antibodies used included a mouse monoclonal anti-microtubule-associated protein (MAP) 2B (1 : 100; Transduction laboratories, Lexington, KY, USA) as a neuronal marker, and a rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (1 : 1000; Dako Cytomation, Glostrup, Denmark) to define astrocytes. For each individual experiment, 8–10 fields were randomly selected from both Ara-C treated and untreated coverslips. MAP2B- and GFAP-positive neurons were manually counted for quantification and statistics.

Incubation and lipid extraction

The growth medium was removed, and after washing the cells once with the phosphate-buffered saline solution, the cultures which contained an average of 200 μg cell protein were incubated in 1.5 mL serum-free Neurobasal medium containing B-27, 0.5 mM glutamine, supplemental fatty acid where indicated, and 0.1 μM bovine serum albumin (essentially fatty acid-free) for 24–72 h at 37°C in an humidified atmosphere containing 5% CO2. Radiolabeled fatty acids were purchased from American Radiolabeled Chemicals Inc. (St Louis, MO, USA); [1-14C]18:2n-6, [1-14C]18:3n-3, and [1-14C]docosapentaenoic acid ([1-14C]22:5n-3), 50–60 mCi/mmol; and [1-14C]20:5n-3 and [1-14C]22:6n-3, 40–60 mCi/mmol. The corresponding unlabeled fatty acids were obtained from Cayman Chemical (Ann Arbor, MI, USA). Dr Howard Sprecher, Ohio State University, kindly provided [3-14C]tetracosapentaenoic acid ([3-14C]24:5n-3), [3-14C]tetracosahexaenoic acid ([3-14C]24:6n-3), and the corresponding unlabeled fatty acids. The radiolabeled fatty acids were purified by HPLC, and the purity of unlabeled fatty acids was checked by GLC.

The incubations were terminated by removing the medium, washing the cells with ice-cold phosphate-buffered saline solution, and extracting the cell lipids with a 2 : 1 chloroform : methanol mixture. Following isolation of the chloroform phase and removing the solvent under a steam of N2, the lipid extract was dissolved in 0.2 mL chloroform and aliquots were taken for the various lipid analytical procedures. Fatty acid methyl esters were prepared from aliquots of the cell lipid extract by incubating the lipid dissolved in 0.5 mL acetonitrile with 0.25 mL 14% BF3 in methanol at 95°C for 45 min. After addition of 4 mL water, the methyl esters were extracted three times with 1 mL n-heptane. The n-heptane extracts were combined, the solvent removed under a stream of N2, and the methyl esters suspended in 50 μL of acetonitrile (Williard et al. 2001b).


The radiolabeled fatty acid methyl esters were separated by reverse phase HPLC using a gradient system consisting of water acidified to pH 3 with formic acid and acetonitrile at a flow rate of 0.7 mL/min (Williard et al. 2001a). The gradient started at 76% acetonitrile and increased to 100% acetonitrile at 50 min. After the column effluent was mixed with three volumes of Budget Solve scintillation solution (Research Products International Corp., Mt. Prospect, IL, USA), radioactivity was measured by passing the mixture through an in-line flow radioactivity detector (IN/US Inc., Tampa, FL, USA). Radiolabeled fatty acid methyl ester standards were included with each set of chromatograms.

Unlabeled fatty acid methyl esters were separated by GLC (Williard et al. 2001b). Margaric acid (17:0) was added as an internal standard prior to extraction. The separation was performed with a Hewlett-Packard 5890 chromatograph (Palo Alto, CA, USA) containing a 1.9 m × 2 mm glass column packed with GP-10% SP2330 on 100–120 mesh Chromosorb WAW (Supelco, Bellefonte, PA, USA). The carrier gas was N2 at a flow rate of 25 mL/min. Fatty acid methyl esters were detected by flame ionization at 250°C, identified by comparison with the retention times of fatty acid methyl ester standards, and quantified by peak area integration.

Cell lipids were separated by TLC using Whatman LK5D plates (Whatman Inc., Florham Park, NJ, USA) and a solvent mixture containing chloroform : methanol : methylamine : water (60 : 36 : 1.5 : 1). After development and drying, the plates were scanned for radioactivity with a Bioscan TLC plate gas proportional flow detector (Bioscan, Washington, DC, USA). Phospholipid standards (Avanti Polar Lipids, Birmingham, AL, USA) were added to each TLC plate. After scanning, the dried TLC plate was sprayed with 1 mM 8-anilino-1-naphthalene and the lipid standards were visualized under ultraviolet light. The radiolabeled lipids were identified by co-migration with the lipid standards (Vartak et al. 1998).

Statistical analysis

All data are presented as mean ± SD. Differences between mean values were analyzed by Student’s t-test, and a value of p < 0.05 were considered to be statistically significant.


  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Comparative incorporation of n-3 fatty acids

The initial studies compared the uptake of [1-14C]18:3n-3, [1-14C]20:5n-3, and [1-14C]22:6n-3 by the cultured hippocampal neurons. Figure 1 shows that the cells took up each of these fatty acids. When the medium containing 4 μM fatty acid (Fig. 1a), 2.6 times more 22:6n-3 was incorporated into the cell lipids than 18:3n-3, but there was no significant difference between the 20:5n-3 and 22:6n-3 uptake. More of each fatty acid was incorporated when the culture medium contained 10 μM fatty acid, but there was still a twofold difference in the amount of 22:6n-3 taken up when compared with 18:3n-3 (Fig. 1b).


Figure 1.  Comparative incorporation of n-3 PUFA. Primary rat hippocampal cultures were incubated with either 4 μM (a) or 10 μM (b) [1-14C]18:3n-3, [1-14C]20:5n-3, or [1-14C]22:6n-3 for 66 h at 37°C. After removing the medium and washing, the radioactivity contained in the cell lipid extract was measured by liquid scintillation counting and converted to nmol amounts based on the specific radioactivity of the fatty acid substrate added to the culture medium. Each bar is the mean ± SD of values obtained from three separate cultures. ap < 0.01, 4 μM 20:5n-3 versus 4 μM 18:3n-3; bp < 0.01, 4 μM 22:6n-3 versus 4 μM 18:3n-3; cp < 0.05, 10 μM 22:6n-3 versus 10 μM 18:3n-3; dp < 0.05, 10 μM 22:6n-3 versus 4 μM 22:6n-3.

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Docosahexaenoic acid utilization

Table 1 contains results of TLC analysis of the lipids extracted from cells incubated for 66 h with either 4 or 10 μM [1-14C]22:6n-3. Between 90% and 95% of the radioactivity taken up was present in phospholipids, and the amount incorporated into each phospholipid fraction was 30–60% greater in the incubation with 10 μM [1-14C]22:6n-3. The ethanolamine phosphoglycerides contained from 50% to 59% of the incorporated radioactivity, choline phosphoglycerides contained 17–28%, and the combined serine and inositol phosphoglyceride fraction contained 15–18% (n = 6 separate cultures). In agreement with these results, recent studies with PC12 neurons also demonstrated that more DHA was incorporated into ethanolamine phosphoglycerides than any of the other phospholipids (Richardson and Wurtman 2007).

Table 1.   Distribution of radiolabeled DHA in rat hippocampal neuron phospholipidsa
Phospholipid fractionRadiolabeled DHA content (nmol/200 μg cell protein ± SD)b
4 μM [1-14C]22:6n-3c10 μM [1-14C]22:6n-3c
  1. aPrimary rat hippocampal cultures were incubated for 66 h at 37°C with either 4 or 10 μM [1-14C]22:6n-3. After removing the medium and washing the cells, the cell lipids were extracted with a chloroform : methanol mixture and the phospholipids separated by TLC. Radioactivity in the phospholipid fractions was determined with a proportional gas flow radioisotope scanner. bMean ± SD of values obtained from three separate cultures. cConcentration of [1-14C]22:6n-3 added to the culture medium at the start of the incubation. dp < 0.05, 10 μM versus 4 μM [1-14C]22:6n-3. PtdSer + PtdInsI, combined serine and inositol phosphoglyceride fraction; PtdCho, choline phosphoglyceride; PtdEtn, ethanolamine phosphoglyceride; DHA or 22:6n-3, docosahexaenoic acid.

PtdSer + PtdIns0.62 ± 0.0350.82 ± 0.076d
PtdCho0.73 ± 0.2101.08 ± 0.028
PtdEtn1.98 ± 0.1893.16 ± 0.133d

Additional studies were performed to determine whether the DHA incorporated by the hippocampal cells underwent structural modification. Aliquots of the cell lipid extract were methylated and the radiolabeled fatty acid methyl esters were analyzed by HPLC. The results, which are shown in Fig. 2, demonstrate that more than 99% of the incorporated radioactivity remained as unmodified 22:6n-3 in cells that were maintained in culture for either 11 days (Fig. 2a) or 18 days (Fig. 2b) prior to the 24 h incubation with [1-14C]22:6n-3.


Figure 2.  DHA utilization: The HPLC analysis was performed with rat hippocampal cultures maintained in growth medium for 11 days (a) or 18 days (b). The growth medium was replaced with fresh medium containing 5 μM [1-14C]22:6n-3, and the incubation was continued for 24 h at 37°C. Following removal of the medium and washing, the cell lipids were extracted with a chloroform : methanol mixture, methylated, and the fatty acid methyl esters were separated by reverse phase HPLC. After combining with scintillator solution, the radioactivity was detected by passing the mixture through an in-line flow scintillation counter. Similar chromatograms were obtained from two additional cultures in each case.

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Conversion of n-3 fatty acid precursors to DHA

Radiolabeled DHA was produced when the hippocampal cultures were incubated with n-3 fatty acid precursors. Figure 3 contains representative HPLC obtained from cultures incubated for 72 h with 5 μM [1-14C]18:3n-3 (Fig. 3a), [1-14C]20:5n-3 (Fig. 3b), [1-14C]22:5n-3 (Fig. 3c), [3-14C]24:5n-3 (Fig. 3d), and [3-14C]24:6n-3 (Fig. 3e). Quantitative results from these and other cultures that were tested are contained in Table 2. In the 72 h incubations with [1-14C]18:3n-3, 27% of the radioactivity in the cells was present in 22:6n-3, 26% in 22:5n-3, 32% in 20:5n-3, 9% in palmitic acid (16:0), and 0.4% in stearic acid (18:0). Only trace quantities of radiolabeled 18:3n-3 remained in the hydrolyzed cell lipid extract. In the single culture incubated for 72 h with [1-14C]20:5n-3, 50% of the radioactivity was present in 22:6n-3 and 26% in 22:5n-3, 8% remained as 20:5n-3, and 1.5% in 16:0. In the 72 h incubations with 5 μM [1-14C]22:5n-3, 36% of the radioactivity was present in 22:6n-3, 21% in 16:0, 5% in 18:0, and 33% remained as unmodified 22:5n-3. Results from 24 h incubations with [1-14C]18:3n-3 and [1-14C]20:5n-3 demonstrate that the conversion of these fatty acids to 22:6n-3 was time dependent. However, the amounts of radiolabeled 22:6n-3 produced from [1-14C]18:3n-3 and [1-14C]20:5n-3 were 75% and 55% less, respectively, than the amount present in the cell lipids after incubation for 24 h with [1-14C]22:6n-3. The standard deviations of the results obtained with these fatty acids are very large, presumably because of differences in the metabolic status of the cultures.


Figure 3.  Conversion of n-3 fatty acids to DHA. Rat hippocampal cultures were incubated for 72 h at 37°C with 5 μM [1-14C]18:3n-3 (a), [1-14C]20:5n-3 (b), [1-14C]22:5n-3 (c), [3-14C]24:5n-3 (d), or [3-14C]24:6n-3 (e). The procedures and HPLC analysis were the same as described Fig. 2.

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Table 2.   Radiolabeled n-3 fatty acids contained in the cell lipids
Radiolabeled fatty acid substrateaTime of incubation (h)nbn-3 fatty acid products (nmol/200 μg cell protein ± SD)c
  1. aThe cultures were incubated with 1.5 mL medium containing 5 μM fatty acid. bNumber of separate cultures tested. cMean ± SD values are given for results from three to nine separate cultures. Average values are given where the results are from two separate cultures. ND, not detected; 22:5n-3, docosapentaenoic acid; 24:5n-3, tetracosapentaenoic acid; 24:6n-3, tetracosahexaenoic acid; 18:3n-3, α-linolenic acid; 22:6n-3, docosahexaenoic acid; 20:5n-3, eicosapentaenoic acid.

[1-14C]18:3n-32450.383 ± 0.2160.492 ± 0.0430.195 ± 0.225
[1-14C]18:3n-37290.549 ± 0.4090.453 ± 0.0880.468 ± 0.397
[1-14C]20:5n-32440.522 ± 0.0500.688 ± 0.1260.399 ± 0.090
[1-14C]22:5n-3728ND1.916 ± 1.2451.936 ± 0.766
[1-14C]22:6n-3246NDND0.857 ± 0.266
[1-14C]22:6n-3723NDND4.450 ± 0.176

In the 72 h incubations with [3-14C]24:5n-3, 53% of the radioactivity was present in 22:6n-3, 31% in 16:0, 5% in 18:0, and 12% remained as unmodified 24:5n-3. The only appreciable radiolabeled product detected in the 72 h incubation with [3-14C]24:6n-3 was 22:6n-3, accounting for 93% of the incorporated radioactivity. This value is only 15% less that the amount of radiolabeled 22:6n-3 present in the cell lipids after a 72 h incubation with [1-14C]22:6n-3.

Fatty acid composition

GLC analysis was performed to determine whether the radiolabeled fatty acid data reflected actual changes in the fatty acid composition of the hippocampal neurons.

Results from two separate experiments are shown in Table 3. Control cultures maintained in media without fatty acid supplementation contained only 1.1–2.5 mol% 22:6n-3 in the cell lipids. Trends toward higher 20:5n-3, 22:5n-3, and 22:6n-3 mol% in the incubation with EPA, and toward higher 22:6n-3 mol% in the incubation with DHA were noted in the first experiment, but the differences were not statistically significant. While none of the differences between the control cultures and those supplemented with ALA were statistically significant in the second experiment, there was a 3.8-fold increase in 22:6n-3 mol% and a 65% decrease in 22:5n-3 mol% in the cultures supplemented with DHA (p < 0.05). Therefore, the amounts of DHA produced in the studies with either [1-14C]18:3n-3 or [1-14C]20:5n-3 were not large enough to appreciably increase in 22:6n-3 content of the cell lipids. However, an increase in 22:6n-3 mol% occurred in the incubations with DHA, and this is consistent with the larger radiolabeled DHA incorporation observed in Table 2 when the cultures were incubated with [1-14C]22:6n-3 when compared with either [1-14C]18:3n-3 or [1-14C]20:5n-3.

Table 3.   Effect of n-3 fatty acid supplements on the fatty acid composition of rat hippocampal neurons in culture
Fatty acidCell fatty acid composition (mol% ± SD)
Experiment 1aExperiment 2b
  1. aCultures maintained for 24 h in media containing 1 μM albumin (Control) or supplemented with either 5 μM EPA or DHA. Each value is the mean ± SD of data obtained from 4 separate cultures. bCultures maintained for 72 h in media containing 1 μM albumin (Control) or supplemented with either 4 μM ALA or DHA. Each value is the mean ± SD of results obtained from three separate cultures. cp < 0.05, DHA versus Control. EPA or 20:5n-3, eicosapentaenoic acid; DHA or 22:6n-3, docosahexaenoic acid; ALA or 18:3n-3, α-linolenic acid; 22:5n-3, docosapentaenoic acid.

Saturated55.9 ± 9.056.2 ± 8.056.0 ± 9.1 44.8 ± 1.947.0 ± 1.245.9 ± 5.4
Monounsaturated23.9 ± 6.821.9 ± 7.323.2 ± 7.729.1 ± 2.826.7 ± 1.031.5 ± 4.3
n-6 polyunsaturated15.2 ± 2.512.8 ± 3.014.1 ± 1.521.1 ± 1.719.1 ± 0.916.9 ± 2.1
 20:4n-66.7 ± 1.54.7 ± 2.86.3 ± 0.67.8 ± 2.18.5 ± 0.76.4 ± 0.9
n-3 polyunsaturated4.8 ± 1.08.9 ± 4.36.0 ± 1.15.0 ± 1.27.2 ± 1.25.7 ± 1.2
 18:3n-31.0 ± 0.60.6 ± 0.40.4 ± 0.21.2 ± 1.40.7 ± 0.20.5 ± 0.2
 20:5n-30.3 ± 0.22.4 ± 3.20.3 ± 0.10.7 ± 0.41.4 ± 0.40.3 ± 0.2
 22:5n-31.0 ± 0.2 2.4 ± 1.20.9 ± 0.2 3.1 ± 0.73.9 ± 0.51.1 ± 0.2c
 22:6n-32.5 ± 1.23.5 ± 1.24.5 ± 1.21.1 ± 0.21.9 ± 1.64.2 ± 0.9c

Conversion of linoleic to arachidonic acid

Previous studies indicated that primary cultures of rat cerebellar and cerebral neurons did not convert linoleic acid to arachidonic acid (Moore et al. 1991; Spector and Moore 1993). However, the finding that radiolabeled ALA was converted to EPA suggested that the hippocampal neurons also might have the ability to synthesize arachidonic acid from linoleic acid. To evaluate this possibility, HPLC analysis of the methylated cell lipid extract was performed following incubations with radiolabeled linoleic acid (Fig. 4). In cultures incubated for 24 h with 5 μM [1-14C]18:2n-6, 84% of the incorporated radioactivity remained as 18:2n-6, 13% was converted to 20:4n-6, and 3% was recovered in 16:0 (Fig. 4a). In 72 h incubations, only 28% of the incorporated radioactivity remained as 18:2n-6, 47% was converted to 20:4n-6, 0.7% to eicosatrienoic acid (20:3n-6), and 6.7% to 16:0 (Fig. 4b). An unidentified component with a retention times of 46.6 min contained 6.8% of the incorporated radioactivity.


Figure 4.  Conversion of 18:2n-6 to arachidonic acid. The medium contained 5 μM [1-14C]18:2n-6, and the cultures were incubated for 24 h (a) or 72 h (b). The procedures for incubation and HPLC analysis were the same as described in Fig. 2. Similar chromatograms were obtained from a second culture in both cases.

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Tests for glial contamination

Previous studies indicated that primary astrocyte cultures prepared from neonatal rat brain converted large quantities of ALA and EPA to DHA (Moore et al. 1991; Williard et al. 2001a). To determine whether the conversions observed in the hippocampal cultures might be because of astrocyte contamination, hippocampal cultures were double stained for neuronal and glial markers. Figure 5 shows typical results obtained with the MAP2B neuronal marker (red fluorescence) and GFAP glial marker (green fluorescence). In some of the fields examined, no cells stained positive for glial markers (Fig. 5a); in others, one cell was positive (Fig. 5b). The results obtained with representative double stained cultures indicate that less than 5% of all cells were positive for glial markers. To further evaluate the possibility that glial contamination contributed to the amount of DHA produced from n-3 fatty acid precursors, studies were performed in which one set of the hippocampal cultures was treated overnight with 2 μM Ara-C to inhibit the growth of astrocytes. Table 4, which summarizes quantitative data obtained from three separate experiments, demonstrates that the glial content was small and that the composition of the cultures was not affected by Ara-C treatment. The modest effect of Ara-C most likely is due to the already very low level of astrocyte contamination of these cultures, making it difficult to achieve any further decrease.


Figure 5.  Immunofluorescence micrographs of rat hippocampal cultures stained for neuronal and glial markers. Cultures were double stained with the neuronal marker MAP2B (red fluorescence) and glial marker GFAP (green fluorescence). As indicated in these representative photomicrographs, most fields contained either no GFAP-positive cells (a) or one GFAP-positive cell (b).

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Table 4.   Effect of Ara-C treatment on composition of hippocampal cultures
CulturesCell typeaPercent glia
Cells/microscopic field (Mean ± SD)Mean % ± SD
  1. aThe cell counts were obtained from three separate experiments. In each experiment, from 8 to 10 randomly selected separate microscopic fields were examined in the control and Ara-C-treated cultures. The data are the values for the combined results. None of the differences are statistically significant (p > 0.1, Control Neurons vs. Ara-C Neurons; Control Glia vs. Ara-C Glia; or glia % in the Control and Ara-C culture). Likewise, none of the differences in mean values in each of the three separate experiments were statistically significant (p > 0.1). bCells stained with mouse monoclonal anti-MAP2B antibody. cCells stained with a rabbit polyclonal anti-GFAP antibody. dCells treated overnight with 2 μM Ara-C between the seventh and eighth day in culture. Ara-C, cytosine arabinoside C.

Control14.77 ± 1.280.49 ± 0.083.13 ± 0.86
Ara-C treatedd14.46 ± 0.730.44 ± 0.062.80 ± 0.34

In additional experiments, the production of radiolabeled fatty acid products was compared in control cultures and those treated with Ara-C. Both sets of cultures were incubated with 2 μM [1-14C]20:5n-3 for 48 h. No appreciable difference in incorporation of radiolabeled 20:5n-3 was observed; 1.63 ± 0.165 nmol/200 μg cell protein in the controls when compared with 1.56 ± 0.027 in the Ara-C-treated cultures (mean ± SD, n = 3). Likewise, as shown in Fig. 6, HPLC analyses of the methylated cell lipid extracts demonstrated that there were no appreciable differences in the amount of [1-14C]20:5n-3 elongated and desaturated by these cultures. The control cultures converted 43% and 30% of the incorporated radioactivity to 22:5n-3 and 22:6n-3, respectively (Fig. 6a), while those treated with Ara-C converted 44% and 29% to 22:6n-3 and 22:5n-3 (Fig. 6b). These findings, together with the immunofluorescence results, indicate that the synthesis of DHA from n-3 fatty acid intermediates most likely is not because of glial contamination of the hippocampal cultures.


Figure 6.  Effect of cytosine arabinoside (Ara-C) treatment on the conversion of 20:5n-3 to DHA. One set of cultures was maintained in regular (control) medium for 11 days (a). A second set was maintained in regular medium for 7 days, transferred to a medium containing 2 μM Ara-C for 18 h, and then maintained for an additional 4 days in Ara-C-free medium (b). Both sets of cultures then were incubated with 2 μM [1-14C]20:5n-3 for 48 h at 37°C, and the radiolabeled fatty acids contained in the methylated cell lipid extracts were analyzed by reverse phase HPLC as described in Fig. 2.

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  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

These results demonstrate that rat hippocampal neuron cultures can synthesize DHA from n-3 fatty acid precursors. DHA is the main n-3 fatty acid in the brain and is essential for optimum neural function. All of the n-3 fatty acids tested were converted to DHA, including ALA, EPA, and the 22- and 24-carbon intermediates, indicating that the hippocampal neurons express the complete pathway for n-3 fatty acid conversion to DHA (Sprecher et al. 1995). Similar amounts of radiolabeled 22:6n-3 were produced in 72 h from 20:5n-3, 22:5n-3, and 24:5n-3, but five times less DHA was produced from ALA. By contrast, almost twice as much radiolabeled 22:6n-3 was produced from 24:6 than from 20:5n-3, 22:5n-3, or 24:5n-3. These results suggest that the Δ6 desaturase, which acts on both the 18:3n-3 and 24:5n-3 desaturation steps in DHA synthesis (Sprecher et al. 1995), most likely is the rate-limiting enzyme of this pathway in hippocampal neurons.

These results differ from our previous findings indicating that cultured rat cerebral and cerebellar neurons cannot convert ALA and EPA to DHA (Moore et al. 1991; Spector and Moore 1993). The reason for this difference is uncertain, but the comparative results from cultures treated with Ara-C demonstrate that it is not because of glial contamination of the neuronal cultures. DHA has many important functions in the hippocampus (Ahmad et al. 2002; Calderon and Kim 2004; Lim et al. 2005a,b; Lukiw et al. 2005), and the ability to synthesize DHA from ALA and EPA may insure that at least some DHA is available for hippocampal function if the external supply is limited, or it may provide a small amount of DHA for intracellular processes that cannot utilize externally available DHA. Alternatively, the disparity between the present findings and our previous results might be because of differences in the culture conditions. In addition to differences in the plating media, the hippocampal neurons were maintained in serum-free Neurobasal medium supplemented with B-27 and 0.5 mM glutamine (Brewer et al. 1993; Lim et al. 2003), while in our previous studies, the cerebral and cerebellar neurons were maintained in Eagle’s minimum essential medium containing 33 mM glucose, 80 μM fluorodeoxyuridine, 2.5% chick embryo extract, and 10% fetal bovine serum (Moore et al. 1991).

Arachidonic acid is the most abundant n-6 essential fatty acid present in the brain and, like DHA, it is necessary for optimum neural function. Our previous studies indicated that cerebral and cerebellar neuron cultures did not synthesize arachidonic acid from linoleic acid and, therefore, required a preformed source of arachidonic acid (Moore et al. 1991; Spector and Moore 1993). As opposed to these results, the present findings demonstrate that hippocampal neurons readily convert linoleic acid to arachidonic acid. This is consistent with the fact that mammalian cells contain a single polyunsaturated fatty acid desaturation and elongation pathway that utilizes both n-3 and n-6 fatty acids (Sprecher et al. 1995).

Humans, including infants, are known to convert ALA to DHA (Salem et al. 1996; Brenna 2002Burdge 2006). Studies in animal models indicate that this conversion occurs primarily in the liver and that the DHA is stored there and in other tissues prior to transfer to the brain (Scott and Bazan 1989; Lefkowitz et al. 2005; Igarashi et al. 2007). However, the conversion of ALA to DHA also can take place in the developing brain (Dhopeshwarkar and Subramanian 1976; Green and Yavin 1993; Su et al. 2000). Like the cerebral and cerebellar neurons, cultured brain microvessel endothelial cells are unable to convert ALA or EPA to DHA (Moore et al. 1990). Therefore, astrocytes were thought to be the cells where this process occurred, but the present findings demonstrate that DHA synthesis from n-3 fatty acid precursors also can occur in cultured neurons. Because of differences in the radiolabeled fatty acid concentrations and incubation conditions in the present and previous studies, it is not possible to estimate the relative contributions of the astrocytes and neurons to this process.

While results in rats and guinea pigs show that ALA can supply DHA to the nervous system, these studies indicate that dietary DHA is necessary for optimal DHA accretion (Woods et al. 1996; Abedin et al. 1999). Likewise, more DHA accumulates in glial cell ethanolamine and serine phosphoglycerides when the cultures are supplemented with DHA (Raffick et al. 2005). Kinetic studies in rats indicate that the daily uptake of [1-14C]22:6n-3 by the adult rat brain is 370–1160 nmol/g, a quantity sufficient to supply the daily DHA requirement (Rapoport et al. 2001b), whereas only 1% of the [1-14C]18:3n-3 taken up by the rat brain was converted to DHA (DeMar et al. 2005; Igarashi et al. 2007). These in vivo studies were performed with adult rats, whereas the present data were obtained with neurons cultured from 18-day embryonic rats. Likewise, the astrocytes studied previously were cultured from 2- to 7-day-old rat pups (Moore et al. 1991; Williard et al. 2001a). A 12-fold decline in fatty acid Δ6-desaturase activity occurs in the brain during the 21-day postnatal period (Bourre et al. 1990). This decrease may account for the larger conversions of ALA to DHA in the cultured astrocytes and neurons than would be predicted from the in vivo studies in the adult rat brain (DeMar et al. 2005; Igarashi et al. 2007).

Notwithstanding these differences, the present results still are consistent with the conclusion that DHA obtained from the plasma is the main source of DHA for the brain (Rapoport et al. 2001b; DeMar et al. 2005, 2006). Rat brain contains 15 300 nmol/g of esterified DHA, and the daily turnover is between 2% and 8%, or 306 and 1224 nmol/g (Rapoport et al. 2001b). From the results in Table 2, the conversion of 5 μM [1-14C]18:3n-3 to DHA by the neurons is 146 nmol/g cells in 24 h. This suggests that the conversion of ALA to DHA in the neurons can account for only 12–48% of the daily DHA requirement of the adult rat brain. Similar calculations indicate that the hippocampal neurons incubated with 5 μM [1-14C]22:6n-3 incorporated 644 nmol/g cells in 24 h, a value within the range of daily DHA turnover in the rat brain (Rapoport et al. 2001b). Furthermore, this value is 4.4 times larger than the calculated conversion of [1-14C]18:3n-3 to DHA under these conditions. This difference probably accounts for the fact that as opposed to ALA, supplementation of the cultured neuron with DHA for 72 h significantly increased the 22:6n-3 mol% of the cell lipids.

Recycling of ALA carbons for fatty acid synthesis through β-oxidation has been observed in suckling rats and rhesus monkeys (Cunnane et al. 1999), and kinetic studies in rats indicate that almost all of the [1-14C]18:2n-6 or [1-14C]18:3n-3 taken up by the brain undergoes β-oxidation (DeMar et al. 2005, 2006). The hippocampal neuron cultures produced radiolabeled palmitic acid, the main product of fatty acid de novo synthesis, from [1-14C]18:2n-6, [1-14C]18:3n-3, [1-14C]22:5n-3, and [3-14C]24:5n-3. A small amount of radiolabeled stearic acid also was produced from [1-14C]22:5n-3 and [3-14C]24:5n-3. This is most likely because of fatty acid β-oxidation, followed by recycling of the resulting acetyl-CoA. The functional significance of this recycling process is unknown. It may be related to the finding that saturated fatty acids do not enter the brain (Noelle Marbois et al. 1992; Edmond et al. 1998; Edmond 2001), suggesting that one function of polyunsaturated fatty acids uptake may be to provide the acetyl-CoA needed by the brain for non-essential fatty acid synthesis and fatty acid chain elongation. However, this explanation seems unlikely because other studies show that the brain can take up saturated fatty acids from the plasma. For example, [9,10-3H]palmitic acid rapidly crosses the blood–brain barrier in rats and is incorporated into brain phospholipids (Grange et al. 1995; Contreras et al. 1999; Rapoport 2001a), and enough [1-11C]palmitic acid injected intravenously enters the brain for positron emission tomography imaging in monkeys (Arai et al. 1995).

Possibly, the most important result of the present work is that hippocampal neurons cultured in Neurobasal medium supplemented with B-27, which are being used extensively as a model system for studies of neuronal function, contain only 1.1–2.5 mol% DHA. By contrast, brain fatty acid compositional analysis indicates that the hippocampus ordinarily contains 13% DHA, and most other regions of the brain contain between 11% and 15% (Diau et al. 2005). The low-DHA content is likely because of the fact that the cultures were plated in horse serum, which contains <0.1% DHA (Stoll and Spector 1984), and then grown in serum-free Neurobasal medium. Although the B-27 supplement contains 3.6 μM ALA, the results in Table 3 show that this amount of ALA is not sufficient to raise the DHA content of the cell lipids to physiological levels. Thus, it is possible that some functional responses obtained with these widely used hippocampal neuron cultures may be suboptimal or even impaired because of their relatively low-DHA content.


  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

These studies were supported by research Grants R01 HL072845 from the National Heart Lung and Blood Institute (AAS) and R01 NS046450 from the National Institute for Neurological Diseases and Stroke (JWH), National Institutes of Health. We thank Dr Howard Sprecher, Ohio State University, for providing the 24-carbon n-3 fatty acids and isotopes.


  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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