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Brain Res. Author manuscript; available in PMC 2008 November 28.
Published in final edited form as:
Brain Res. 2007 November 28; 1182: 50–59.
Oral Supplementation with Docosahexaenoic Acid and Uridine-5’-
Monophosphate Increases Dendritic Spine Density in Adult Gerbil
Hippocampus
Toshimasa Sakamotoa, Mehmet Canseva,b, and Richard J. Wurtmana,*
a Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139,
USA
b Department of Pharmacology and Clinical Pharmacology, Uludag University, Medical School, Bursa
16059, Turkey
Abstract
Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of
membrane phosphatides and has been implicated in cognitive functions. Low levels of circulating
or brain DHA are associated with various neurocognitive disorders including Alzheimer’s disease
(AD), while laboratory animals, including animal models of AD, can exhibit improved cognitive
ability with a diet enriched in DHA. Various cellular mechanisms have been proposed for DHA’s
behavioral effects, including increases in cellular membrane fluidity, promotion of neurite extension,
and inhibition of apoptosis. However, there is little direct evidence that DHA affects synaptic
structure in living animals. Here we show that oral supplementation with DHA substantially increases
the number of dendritic spines in adult gerbil hippocampus, particularly when animals are co-
supplemented with a uridine source, uridine-5’-monophosphate (UMP), which increases brain levels
of the rate-limiting phosphatide precursor CTP. The increase in dendritic spines (> 30%) is
accompanied by parallel increases in membrane phosphatides, and in pre- and post-synaptic proteins
within the hippocampus. Hence oral DHA may promote neuronal membrane synthesis to increase
the number of synapses, particularly when co-administered with UMP. Our findings provide a
possible explanation for the effects of DHA on behavior and also suggest a strategy to treat cognitive
disorders resulting from synapse loss.
Keywords
docosahexaenoic acid; uridine; membrane synthesis; spine formation; synaptogenesis; phosphatides
1. Introduction
Nutritional supplementation with essential omega-3 fatty acids including docosahexaenoic
acid (DHA) is increasingly practiced (Marszalek and Lodish, 2005). DHA, a 22-carbon
compound with six double bonds, is synthesized from its precursor α-linolenic acid, which
*Correspondence should be addressed to: Richard J. Wurtman, M.D., MIT, 43 Vassar St., 46-5023B, Cambridge MA, 02139, Email:
dick@mit.edu, Phone: 617-253-6731, Fax: 617-253-6882.
Scope: Neurophysiology, Neuropharmacology and other forms of Intercellular Communication
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Sakamoto et al. Page 2
cannot be synthesized in mammals (mammals lack the desaturase enzyme that forms the double
bond at the third carbon from its methyl terminal) (Marszalek and Lodish, 2005). Since
conversion from α-linolenic acid to DHA is very slow, DHA must also be obtained from dietary
sources, for example fatty fish (Marszalek and Lodish, 2005). Like other fatty acids, DHA is
a precursor of diacylglycerol (DAG) (Marszalek and Lodish, 2005), an essential and sometimes
rate-limiting precursor for the phosphatides in cellular membranes (Araki and Wurtman,
1997). Clinical studies suggest that DHA can maintain or enhance cellular functions in various
physiological systems including the nervous system (Marszalek and Lodish, 2005).
The mechanisms by which DHA affects brain functions or behaviors are poorly understood.
The fatty acid readily crosses the blood-brain barrier (Spector, 2001; Hashimoto et al., 2002)
and then cellular membranes, whereupon it is acylated and can be incorporated into DAG
(Marszalek and Lodish, 2005). The DHA-containing DAG is preferentially utilized for
membrane phosphatide synthesis (Marszalek and Lodish, 2005). Low levels of circulating or
brain DHA are reportedly associated with neurodegenerative or psychiatric disorders, such as
Alzheimer’s disease (Soderberg et al., 1991; Schaefer et al., 2006), depression (Logan, 2004;
Appleton et al., 2006) and schizophrenia (Assies et al., 2001; Reddy et al., 2004). Behavioral
studies in laboratory rodents suggest that DHA is involved in learning and memory (Yoshida
et al., 1997; Gamoh et al., 1999; Moriguchi et al., 2000; Calon et al., 2004; Hashimoto et al.,
2002, 2006). Animals given a diet deficient in omega-3 fatty acids exhibit learning impairments
(Yoshida et al., 1997; Moriguchi et al., 2000), while those receiving supplemental DHA show
improved learning (Gamoh et al., 1999; Calon et al., 2004; Hashimoto et al., 2002, 2006).
Various cellular mechanisms have been proposed as mediating DHA’s neurobehavioral effects,
for example increasing cell membrane fluidity (Hashimoto et al., 2006), promoting neurite
extension (Ikemoto et al., 1997; Calderon and Kim, 2004; Marszalek et al., 2004, 2005; Darios
and Davletov, 2006), inhibiting apoptosis (Hashimoto et al., 2002; Calon et al., 2004), and
increasing synthesis of the phosphatides in synaptic membranes (Wurtman et al., 2006).
However, there is little direct evidence that DHA affects synaptic structures in intact animals.
Uridine-5’-monophosphate (UMP) is a precursor of circulating uridine (Cansev et al., 2005).
Like DHA, uridine readily crosses the blood-brain barrier and enters brain cells (Cansev,
2006). It is then phosphorylated by uridine-cytidine kinases to form uridine triphosphate (UTP),
which can be further transformed by CTP synthetase to cytidine triphosphate (CTP), the usual
rate-limiting precursor in phosphatide biosynthesis (Ross et al., 1997). CTP, for example, can
combine with phosphocholine to form cytidine-5’-diphosphocholine (CDP-choline) (Kennedy
and Weiss, 1956), which then combines with DAG to yield phosphatidylcholine (PC), the
major phosphatide in neuronal membranes (Sastry, 1985). Uridine promotes neurite outgrowth
in PC-12 cells treated with nerve growth factor (Pooler et al., 2005), and neurotransmitter
release from the rat striatum in vivo (Wang et al., 2005, 2007). Moreover, when given with
DHA, uridine increases membrane phosphatides and synaptic proteins in the adult gerbil brain
(Wurtman et al., 2006). These results suggest that uridine as well as DHA can enhance
phosphatide synthesis in neuronal or synaptic membranes.
Here we examined the effects of supplemental DHA and uridine on the number of dendritic
spines in brains of living animals. Dendritic spines are small membranous protrusions
extending from dendrites, which compartmentalize post-synaptic responses. They mediate
most excitatory connections and are thought to reflect the number of excitatory synapses in the
central nervous system (Matus, 2000; Hering and Sheng, 2001; Nimchinsky et al., 2002; Yuste
and Bonhoeffer, 2004). We show that oral supplementation with DHA increases the number
of dendritic spines in adult gerbil hippocampus, particularly when animals are also
supplemented with UMP, a uridine source. Oral DHA may thus increase the number of brain
synapses, particularly when co-administered with UMP. We have also examined the ability of
arachidonic acid, an omega-6 fatty acid, to increase hippocampal dendritic spines. Unlike
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DHA, this fatty acid has been shown (Cansev and Wurtman, In Press) not to affect brain levels
of phosphatides or synaptic proteins.
2. Results
2.1. Oral supplementation with DHA increases dendritic spine density in adult gerbil
hippocampus
To examine whether DHA affects the number of dendritic spines, we treated adult gerbils with
the fatty acid daily for 4 weeks, in oral doses of 0, 50, 100 or 300 mg/kg. The spine density
increased significantly in the primary apical dendrites of CA1 pyramidal neurons after the
animals received either the 100 or 300 mg/kg/day doses (Fig. 1). The increases were 12 % (p
= 0.04) with the 100 mg/kg/day dose and 18% (p < 0.001) with the 300 mg/kg/day dose.
2.2. DHA-induced dendritic spine formation in adult gerbil hippocampus is enhanced by co-
supplementation with UMP
We next tested the effect on dendritic spine density of providing both DHA (300 mg/kg/day,
by gavage) and a uridine source, UMP (0.5%, via the standard choline-containing diet) for 4
weeks. A recent study from our laboratory had shown that oral UMP, given concurrently,
enhances the DHA-induced increases in membrane phosphatide and synaptic protein levels in
gerbil brain (Wurtman et al., 2006). DHA supplementation alone caused a significant, 19% (p
= 0.004, Fig. 2B) increase in spine density. In contrast, animals that had received both DHA
and UMP exhibited a 36% increase (p < 0.001 vs. Control), or approximately double the
increase produced by DHA alone (p = 0.008 vs. DHA alone, Fig. 2B). Giving UMP alone did
not significantly increase dendritic spine density (Fig. 2B), although it did when all dendritic
protrusions (including filopodias) were included for statistical analysis (data not shown; see
also Methods for the definition of dendritic spines). In another study, we examined the relation
between duration of treatment with DHA and UMP and spine density; animals received the
two compounds daily for 1, 2, 3 or 4 weeks. The effect of the co-supplementation on spine
density was already apparent at 1 week (p = 0.02), and continued throughout the treatment
period (Fig. 2C).
2.3. Supplementation with DHA or DHA-plus-UMP increases major membrane phospholipids
in adult gerbil hippocampus
Treatment with DHA or DHA-plus-UMP daily for 4 weeks, which increased dendritic spine
density (Fig. 2B), also caused major increases in membrane phosphatides within the gerbil
hippocampus (Fig. 3). The increases with DHA alone were: phosphatidylcholine (PC): 8%;
phosphatidylethanolamine (PE): 26%; phosphatidylserine (PS): 75%; and phosphatidylinositol
(PI): 29% (all p < 0.05 except PC); and those with DHA-plus-UMP were: PC: 28%; PE: 59%;
PS: 160%; and PI: 100% (all p < 0.001 vs. their controls). No significant increases were
observed with UMP alone.
2.4. Supplementation with DHA or DHA-plus-UMP elevates expression of pre- and post-
synaptic proteins in adult gerbil hippocampus
We further tested whether the increases in spine density following treatment with DHA and
UMP were associated with increases in hippocampal levels of pre- and post-synaptic proteins.
If the increased number of dendritic spines represented an increase in the number of synapses,
it might be anticipated that there would be comparable increases in such proteins as PSD-95,
GluR-1 (an AMPA receptor subunit) and Synapsin-1 (Greengard et al., 1993; El-Husseini et
al., 2000; Kasai et al., 2003; Matsuzaki et al., 2004; Ziv and Garner, 2004; Matus, 2005).
Expression levels of PSD-95 (El-Husseini et al., 2000) and GluR-1 (Kasai et al., 2003;
Matsuzaki et al., 2004) are highly associated with growth of dendritic spines and levels of post-
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synaptic responses. Synapsin-1, on the other hand, is expressed in pre-synaptic terminals,
apparently anchoring synaptic vesicles to the actin cytoskeleton for exocytosis or
synaptogenesis (Greengard et al., 1993; Ziv and Garner, 2004). Western blot analysis of
aliquots of the hippocampal homogenates used for the phosphatide assays above (i.e.,
hippocampal homogenates obtained from the same animals as those tested for dendritic spines)
demonstrated significantly elevated expression of all of these synaptic proteins after treatment
with DHA alone or, to a greater extent, with DHA-plus-UMP (Fig. 4A-C). The increases in
PSD-95, GluR-1 and Synapsin-1 with DHA alone were 42%, 29% and 37%, respectively (all
p ≤ 0.05); the increases after treatment with DHA-plus-UMP were 44%, 37% and 57%,
respectively (all p < 0.01). We further examined the expression levels of two cytoskeletal
proteins: Actin and β-tubulin. Actin has been demonstrated to directly regulate morphology of
dendritic spines, and this effect can underlie such manifestations of synaptic plasticity as long-
term potentiation (LTP) and depression (LTD) (Matus, 2000, 2005; Hering and Sheng, 2001;
Nimchinsky et al., 2002; Matsuzaki et al., 2004). On the other hand, β-tubulin is a structural
protein not specifically localized within synaptic structures. We found that the expression level
of Actin was substantially elevated with DHA (by 60%, p = 0.04) and with DHA-plus-UMP
(by 88%, p = 0.004), whereas that of β-tubulin was unaffected with the treatments (Fig. 4D
and E).
2.5. Oral supplementation with an omega-6 fatty acid (arachidonic acid) does not affect spine
density in adult gerbil hippocampus
To examine the specificity of DHA’s effects on spine density, we determined whether
arachidonic acid (ARA), an omega-6 fatty acid, also produces these effects. ARA contains 20
carbons with 4 double bonds; the first of these is situated at the sixth carbon from its methyl
terminal (Marszalek and Lodish, 2005; Smith, 2005). Like DHA, ARA cannot be synthesized
de novo in mammalian tissues, and must be obtained from dietary sources (Marszalek and
Lodish, 2005; Smith, 2005). It also is a precursor for DAG (Marszalek and Lodish, 2005).
Moreover, ARA is known to affect neurite outgrowth (Darios and Davletov, 2006) and synaptic
plasticity (Williams et al., 1989; Ramakers and Storm, 2002; Feinmark et al., 2003). But unlike
DHA, ARA administration daily for 4 weeks failed to affect dendritic spine density in the CA1
region of the gerbil hippocampus (Fig. 5A and B). Similarly, ARA failed to affect levels of
hippocampal phosphatides or synaptic proteins (Fig. 5C and D).
2.6. Supplementation with DHA, UMP or ARA did not affect size of dendritic spines in adult
gerbil hippocampus
In addition to dendritic spine number density, we investigated the effects of DHA, UMP or
ARA on spine size. We measured the length and width of dendritic spines used for the spine
density analysis described above. The mean length and the mean width were obtained for each
dendrite and the averages of the means were compared between treatment groups. The results
showed that none of DHA, UMP or ARA affected these parameters (SI Fig. 8 and 9).
3. Discussion
These studies show that oral supplementation with DHA, an omega-3 fatty acid, increases
dendritic spine density in adult gerbil hippocampus (Fig. 1). This effect of DHA is
approximately doubled when animals also receive UMP (Fig. 2), and is accompanied by
parallel increases in membrane phosphatides (Fig. 3) and in specific pre- and post-synaptic
proteins (Fig. 4). Supplementation with arachidonic acid (ARA), an omega-6 fatty acid, fails
to increase spine density (Fig. 5).
Dendritic spines are small membranous protrusions extending from neuronal dendrites (Matus,
2000; Hering and Sheng, 2001; Nimchinsky et al., 2002; Yuste and Bonhoeffer, 2004). They
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mediate most excitatory connections in the CNS, compartmentalizing post-synaptic responses
(Matus, 2000; Hering and Sheng, 2001; Nimchinsky et al., 2002; Yuste and Bonhoeffer,
2004). In the adult mammalian cortex, all of the mature spines are thought to make synapses,
with a typical spine having a single synapse at its head (Matus, 2000; Hering and Sheng,
2001; Nimchinsky et al., 2002; Yuste and Bonhoeffer, 2004). Hence, the increase in dendritic
spine number that we observe may reflect an increased number of hippocampal synapses as
well. DHA administration has been described as promoting cognitive functions (Gamoh et al.,
1999; Hashimoto et al., 2002, 2006; Calon et al., 2004), yet its effects on synapses have been
obscure. Our finding that oral DHA promotes spine formation suggests that one way through
which DHA enhances cognition involves increasing the number of synapses. This hypothesis
is further supported by our finding that the DHA-induced increase in spine density is
accompanied by parallel increases in such pre- and post-synaptic proteins as PSD-95, GluR-1
and Synapsin-1 (Fig. 4).
DHA is a component of DAG, an immediate precursor of such phosphatides as PC, PE, PS
and PI. DHA-containing DAG is formed by acetylating glycerol-3-phosphate with DHA on
its sn-2 position, and with a saturated fatty acid on its sn-1 position (Marszalek and Lodish,
2005). Oral supplementation with DHA has been shown to elevate both plasma and brain DHA
levels in rats (Hashimoto et al., 2002). In PC 12 cells, DHA added to the medium is rapidly
internalized by the cells, and activated by acyl-CoA synthetases (Marszalek et al., 2004,
2005). It then increases cellular phosphatide levels and promotes neurite outgrowth (Marszalek
et al., 2004, 2005). Given that formation of dendritic spines, like neurite outgrowth, requires
a major increase in the amount of cell membrane, our present finding that DHA-induced spine
formation accompanies substantial increases in the phosphatides suggests that DHA promotes
spine formation by enhancing the synthesis of membrane phospholipids.
Formation of dendritic spines can be induced by synaptic inputs that induce LTP in CA1
pyramidal neurons (Engert and Bonhoeffer, 1999; Maletic-Savatic et al., 1999; Toni et al.,
1999). Because the induction of spine formation is blocked by inhibition of NMDA receptors,
this process appears to be mediated by Ca2+ influx into the post-synaptic dendrite (Engert and
Bonhoeffer, 1999; Maletic-Savatic et al., 1999; Toni et al., 1999). In this context, membrane
synthesis that realizes spine formation may be induced by Ca2+ or synaptic activity, as well as
other related processes such as actin polymerization (Matus, 2000; Matsuzaki et al., 2004). In
the present study, we tested the effects of two different classes of polyunsaturated fatty acids
(i.e., DHA and ARA) on spine density. Like DHA, ARA can be acylated to the sn-2 position
of DAG, and is enriched in neuronal membranes (Marszalek and Lodish, 2005). However,
supplementation with ARA failed to affect brain levels of phosphatides or synaptic proteins in
a previous study (Cansev and Wurtman, In Press), and failed to affect dendritic spine number
or phosphatide levels (Fig. 5) in the present study. The mechanism that allows DHA but not
ARA to promote spine formation or phosphatide synthesis awaits discovery, but it could
involve the substrate specificities of the enzymes that regulate the utilization of the fatty acids
for membrane synthesis (Marszalek and Lodish, 2005; Marszalek et al., 2005).
Our finding that oral supplementation with uridine substantially enhances DHA-induced spine
formation provides confirmation that uridine can affect membrane phosphatide synthesis in
neurons (Cansev, 2006; Wurtman et al., 2006; Cansev and Wurtman, In Press). Uridine, a
pyrimidine nucleoside that readily crosses the blood-brain barrier, is a precursor of UTP, and
subsequently of CTP (Cansev et al., 2005; Cansev, 2006), the major rate-limiting precursor in
the synthesis of membrane phosphatides (i.e., PC, PE, PS and PI) (Ross et al., 1997). In brain
cells, CTP reacts with phosphocholine or phosphoethanolamine to form CDP-choline or CDP-
ethanolamine (Kennedy and Weiss, 1956; Sastry, 1985). These two products then react with
DAG to generate PC or PE, respectively (CTP can also react directly with DAG to form CDP-
DAG, which then reacts with inositol to yield PI; PS is mainly synthesized by a base exchange
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mechanism in which free serine is substituted for the nitrogen base of a phosphatide)
(Borkenhagen et al., 1961; Sastry, 1985). These metabolic processes can be limited by the
availability of CTP or the proportion of DAG represented by a particular species (i.e., one
containing a DHA or other omega-3 fatty acid). The effect on phosphatide synthesis of forming
additional DHA-containing DAG would thus be maximized when uridine administration also
increases CTP levels. Consistent with this hypothesis, our present study demonstrated that the
promoting effects of oral DHA on spine formation and membrane synthesis are substantially
potentiated by co-supplementation with UMP (a uridine source). The combined effects were
more than the sums of the individual effects observed when each of the compounds was given
alone (Figs. 2B and 3; see also Fig. 6).
The effects of DHA or uridine on spine formation may also be mediated by pre-synaptic
mechanisms. Results from various model systems suggest that both DHA (Ikemoto et al.,
1997; Calderon and Kim, 2004; Marszalek et al., 2004, 2005; Darios and Davletov, 2006) and
uridine (Pooler et al., 2005; Wang et al., 2005, 2007) promote axonal growth and exocytosis.
A recent study has demonstrated that DHA can activate the SNARE protein Syntaxin-3 (Darios
and Davletov, 2006). Uridine, through UTP, can activate P2Y receptors (Pooler et al., 2005),
which are expressed in hippocampal neurons (Abbrachio et al., 2006) and implicated in pre-
synaptic induction of LTP (Price et al., 2003). Formation of dendritic spines or synaptogenesis
in mammalian brains can be induced or initiated by pre-synaptic neurons (Matus, 2000; Hering
and Sheng, 2001; Nimchinsky et al., 2002; Yuste and Bonhoeffer, 2004). The increases in spine
density with DHA and UMP (Figs. 1 and 2) may be due to their promoting effects on pre- and/
or post-synaptic mechanisms.
Various psychiatric and neurological disorders, for example, schizophrenia, mental retardation
syndromes and Alzheimer’s disease (AD), are accompanied by subnormal numbers of brain
synapses or dendritic spines (Selkoe, 2002; Blanpied and Ehlers, 2004). Oral administration
of DHA and uridine may provide a means to compensate for or prevent synapse loss in these
disorders. Studies on animal models of AD have demonstrated that administration of DHA can
improve their cognitive performance (Calon et al., 2004; Hashimoto et al., 2002, 2006).
Promoting synapse formation may be a primary mechanism underlying the effect of DHA on
behavior.
4. Experimental Procedures
4.1. Animals
Normal adult male gerbils (M. unguiculatus, 60~80g body weight) were given a standard rodent
diet (Harlan Teklad) (Wurtman et al., 2006) and, by gavage, various doses of DHA or its diluent
(5% gum arabic) daily for 4 weeks. Animals receiving UMP were given the above diet
supplemented with 0.5% UMP. Animals supplemented with arachidonic acid (ARA) were
treated in the same way as the DHA-supplemented animals, except that they received ARA in
the place of DHA. All the diets also contained 0.1% choline. The fatty acids were purchased
from Nu-Chek Prep, Inc. We used gerbils because, unlike rats, this species’ pyrimidine
metabolism closely resembles that of humans (Cansev, 2006).
4.2. Tissue preparation
Animals that had been treated for 4 weeks were sacrificed with CO2 gas inhalation and then
decapitated by guillotine. The hippocampi were then extracted quickly from each brain
hemisphere; one was sliced at 300 μm thickness with a tissue chopper for neuronal imaging
and the other was homogenized for membrane and protein assays (see below).
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4.3. Neuroimaging
Immediately after slicing, tissues were fixed overnight with 4% paraformaldehyde at 4°C and
then stained with a lipophilic membrane staining dye, DiI (CellTracker™ CM-DiI, Invitrogen),
using a needle to embed the dye in CA1 regions of the hippocampus. Images of CA1 pyramidal
neurons were obtained using a confocal microscope (Olympus, FV300). Individual slices were
placed in PBS on a coverglass and imaged at 568 nm excitation to capture emission signals
from DiI with a 60x water-immersion objective (Olympus, UPlan Apo). A three-dimensional
image stack was obtained at 0.5 μm intervals in the vertical (Z) - axis, using a FluoView
software (1024 x1024 pixels) with 3x zoom. The total depth of imaging space was mostly 5~10
μm (i.e., 10~20 optical sections). Five to 10 neurons were randomly sampled from 4~5 slices
for individual animals. An individual image stack was analyzed on its two-dimensional
projection using a MetaMorph software (Molecular Devices). Small protrusions extending
from the primary apical dendrite of a CA1 pyramidal neuron (where CA3 axons form excitatory
synapses with CA1 dendrites) were counted by drawing lines to measure the lengths of the
protrusions from the edge of the dendritic shaft. Only one dendritic segment was analyzed per
neuron. Protrusions whose lengths fell between 0.3 to 2 μm were defined as dendritic spines
and included for analysis (Hering and Sheng, 2001; Nimchinsky et al., 2002; Yuste and
Bonhoeffer, 2004). Stacked images were also inspected section by section to ensure that all
the countable spines were counted in a defined length of dendrite (about 30 μm on average).
The data were expressed as “spine density” (i.e., number of spines per unit length of dendrite)
for statistical analysis. To confirm the reliability of the measurement, part of the spine counting
was repeated by a second person, who was blind to the group identity of images. The correlation
coefficient in spine density between the two observers was 0.9 (n = 30 dendrites; see SI Fig.
10). Spine width (see SI Fig. 9) was defined as the apparent diameter of a spine head.
4.4. Biochemical assays
Phosphatides were extracted and measured as described previously (Wurtman et al., 2006).
Briefly, individual hippocampi were weighed and homogenized in 20 volumes of ice-cold
deionized water using a 5 ml Teflon-glass homogenizer (Wheaton) with 10 strokes by hand. 1
ml of each aliquot was mixed with 3 ml of a chloroform/methanol mixture (2:1 v/v) and
vortexed, followed by sequential addition of 3 ml of chloroform/methanol mixture (2:1 v/v)
and 1 ml of ice-cold deionized water. The mixture was allowed to stand overnight at 4°C (18–
20 h). Next day, the organic (lower) and aqueous (upper) phases of the mixture were separated
by centrifugation (10 min at 4 °C; 1000 g). Aliquots (0.1–0.4 ml) of the organic phase were
dried under vacuum for phosphatide analysis. Residues of 0.4-ml aliquots of the organic phase
were reconstituted in 40 μl of methanol and subjected to thin-layer chromatography using silica
G plates (Adsorbosil Plus-1, Alltech) and a system consisting of chloroform/ethanol/
triethylamine/water (30:34:30:8) as the mobile phase. Phospholipid standards were used to
identify the corresponding bands under UV light after the plates were sprayed with 0.1%
diphenylhexatriene in petroleum ether. Bands for individual phosphatide classes (PC, PE, PS
and PI) were scraped off the plates and extracted into 1 ml of methanol, dried under vacuum
and assayed for phosphorus content. Aliquots of the hippocampal homogenates were assayed
for total protein amounts by a bicinchoninic acid reagent (Perkin-Elmer).
Expression of synaptic marker proteins was assayed by Western blot. Aliquots of the
hippocampal homogenates were mixed with equal volumes of KFL loading buffer and boiled
for 5 min prior to gel electrophoresis. Equal amounts of protein (20 μg) were loaded for each
lane and separated using SDS-PAGE (4–20%; Bio-Rad). Proteins were then transferred onto
polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore). The remaining
binding sites were blocked with 4% non-fat dry milk (Carnation) for 30 min in TBST buffer.
Membranes were rinsed 5 times in TBST and immersed in TBST containing the antibody of
interest [mouse anti-PSD-95 (Upstate); rabbit anti-GluR-1 (Chemicon); mouse anti-
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Synapsin-1 (Calbiochem); and mouse anti-F-actin (Abcam). Following overnight incubation
and 5 rinses in TBST, blots were incubated for 1 h with an appropriate peroxidase-linked
secondary antibody. Blots were then rinsed in TBST 5 times, and protein–antibody complexes
were detected and visualized by the ECL system (Amersham Biosciences) on Kodak X-AR
film. Films were digitized using a Supervista S-12 scanner with a transparency adapter (UMAX
Technologies). Immunoreactive bands were compared densitometrically using the Public
Domain NIH Image program, Image J (available at http://rsb.info.nih.gov/ij/). Areas under the
absorbance curves were expressed as arbitrary units, and were normalized as percentage of the
average for controls in the same blot. β-tubulin was used as the loading control for the proteins
tested. For this purpose, PVDF membranes were rinsed in stripping buffer (Pierce; Rockford,
IL) for 30 min and then reincubated with mouse anti-tubulin beta III subunit (Chemicon).
4.5. Statistics
Data were expressed as mean ± standard error of the mean (SEM) for each treatment group.
Unless otherwise noted, one-way analysis of variance (ANOVA, SigmaPlot) was used to
compare group means, with post-hoc analysis as needed. The significance (α) level was set at
p = 0.05.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
We thank Ken-ichi Okamoto for valuable advices on dendritic imaging, and Morgan Sheng and Jing Kang for
comments on the manuscript. We also thank Julia Fong, Vivian Tang, Kaitlin Kamrowski and Peter Fung for their
technical assistance, and Numico Research for UMP. This work was supported by grants from the National Institutes
of Health (Grant MH-28783) and the Center for Brain Sciences and Metabolism Charitable Trust.
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Fig. 1. Oral supplementation with DHA increases dendritic spine density in adult gerbil
hippocampus
Eight animals were randomly divided into 4 groups and were supplemented with 0, 50, 100 or
300 mg/kg of DHA daily for 4 weeks. (A) Primary apical dendrites of CA1 pyramidal neurons.
(B) Animals supplemented with 100 or 300 mg/kg/day showed increased spine density: a 12
% increase after the 100 mg/kg/day dose (*p = 0.04) and an 18 % increase after the 300 mg/
kg/day dose (**p < 0.001 vs. 0 mg/kg/day). n = 16 ~ 20 neurons from 2 animals per group.
One-way ANOVA followed by Tukey’s test.
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Fig. 2. DHA-induced dendritic spine formation in adult gerbil hippocampus is enhanced by co-
supplementation with UMP
Animals received UMP (0.5%), DHA (300 mg/kg) or both daily for 4 weeks; control gerbils
received neither. (A) Apical dendrites of CA1 pyramidal neurons. (B) Animals supplemented
with DHA exhibited a significant increase in spine density (by 19%, *p = 0.004 vs. Control);
those receiving both DHA and UMP exhibited a greater increase (by 36%, **p < 0.001 vs.
Control or by 17%, p = 0.008 vs. DHA). n = 20 ~ 25 neurons from 4 animals per group. One-
way ANOVA followed by Tukey’s test. (C) The effect of DHA-plus-UMP on spine density
was apparent by 1 week after the start of the treatment. The treated groups (total 8 animals)
received both UMP (0.5%) and DHA (300 mg/kg) daily for 1, 2, 3 or 4 weeks; the control
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groups (8 animals) were given only a regular diet. n = 12 ~ 20 neurons from 2 animals per
group. Two-way ANOVA followed by Tukey’s test. *p = 0.02; ** p < 0.001.
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Fig. 3. Supplementation with DHA or DHA-plus-UMP increases major membrane phospholipids
in adult gerbil hippocampus
(A) PC, phosphatidylcholine; (B) PE, phosphatidylethanolamine; (C) PS, phosphatidylserine;
(D) PI, phosphatidylinositol. The increases ranged from 26% (PE by DHA) to 160% (PS by
DHA-plus-UMP) compared with their controls. n = 4 ~ 7 animals per group including all the
animals used for the dendritic spine analysis shown in Fig. 2B. For those animals, the
hippocampus was collected from the contralateral hemisphere. The data are shown as
percentage of the average of Controls. One-way ANOVA followed by Tukey’s test. * p < 0.05;
*** p < 0.001 vs. Controls.
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Fig. 4. Supplementation with DHA or DHA-plus-UMP elevates expression of pre-and post-synaptic
proteins in adult gerbil hippocampus
(A and B) Expression levels of the post-synaptic proteins PSD-95 (A) and GluR-1 (B) were
increased by 42% and 29% with DHA alone, and by 44% and 37% with DHA-plus-UMP,
respectively. (C) Expression of the pre-synaptic protein Synapsin-1 was increased by 37% with
DHA alone and by 57% with DHA-plus-UMP. (D and E) Expression of cytoskeletal proteins.
The level of Actin (D), which can regulate dendritic spine structures, was increased by 60%
with DHA alone and by 88% with DHA-plus-UMP, whereas that of -tubulin (E), which is not
specifically localized within synaptic structures, was unaffected with any of the treatments.
Hippocampi used for this experiment were collected from the contralateral hemisphere in
animals used for the dendritic spine analysis shown in Fig. 2B. n = 4 animals per group. The
data are shown as percentage of the average of Controls. One-way ANOVA followed by
Tukey’s test. * p ≤ 0.05; ** p < 0.01 vs. Controls. See also supplementary information (SI)
Fig. 7.
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Fig. 5. Oral supplementation with arachidonic acid (ARA) does not increase dendritic spine density
in adult gerbil hippocampus
(A) Primary apical dendrites of CA1 pyramidal neurons. (B) Supplementation with ARA did
not change spine density in adult gerbil CA1. n = 18 ~21 neurons from 2 animals per group.
(C) Relative amounts of phosphatides in the hippocampi obtained from animals treated with
300 mg/kg/day of ARA. The data are shown in percentage of the average of controls for each
phosphatide. There was no statistical difference in any phosphatide between the Control and
ARA groups. t-test was performed for each phosphatide (the α level was adjusted to .012 for
multiple comparisons). PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS,
phosphatidylserine; PI, phosphatidylinositol. (D) Western blot analysis for pre- and post-
synaptic marker proteins in the same hippocampal homogenatesas as those used for the
phosphatide assay (β-tubulin was used as a negative control). Supplementation with ARA did
not significantly increase the expression of the marker proteins. Syn-1, Synapsin-1. n = 4
animals for each of Control and ARA groups. Tested by multiple t-tests for individual proteins
(α = 0.01).
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Fig. 6. A model for spine formation by supplementation with DHA and UMP
The diagram shows phosphatidylethanolamine (PE) synthesis as an example. In neurons, DHA
is acylated into the sn-2 position of glycerol-3-phosphate (G3P) to form DAG, together with
a saturated fatty acid at the sn-1 position. UMP, a precursor of uridine, is metabolized to CTP,
the major rate-limiting precursor in membrane phosphatide synthesis. CTP then reacts with
phosphoethanolamine to form CDP-ethanolamine. PE is synthesized by combining DAG and
CDP-ethanolamine to form the phosphatide in dendritic spines. DHA and uridine may also
activate other neuronal mechanisms (e.g., axonal growth and exocytosis) and cell signaling
pathways to induce spine formation (see text).
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