Behavioural Brain Research 204 (2009) 129–132

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Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr

Research report

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine improvescognitive decline by enhancing long-term depression
Takahiro Yaguchi, Tetsu Nagata, Tomoyuki Nishizaki ∗Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya Hyogo 663-8501, Japana r t i c l ei n f oArticle history:
Received 28 April 2009
Received in revised form 20 May 2009
Accepted 25 May 2009
Available online 29 May 2009
Keywords:
Phosphatidylcholine
Long-term depression
AMPA receptor
Water maze test
Cognitive impairmenta b s t r a c t
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPhtCho) (1 ␮M) enhanced long-term depression(LTD), a synaptic plasticity relevant to learning and memory, in the CA1 region of rat hippocampal slices,where expression of the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluR1 on the plasma membrane was decreased. In the water maze test, oral administration withPOPhtCho (5 mg/kg) signiﬁcantly shortened the prolonged retention latency for rats intraperitoneallyinjected with scopolamine (1 mg/kg), while the acquisition latency was not affected. For humans withmild cognitive impairment and dementia (average of Mini Mental State Examination score, 18), oral intake
with POPhtCho (300 mg/day, once after breakfast) everyday raised the score to over 20, corresponding
to normal cognitive functions, throughout 6 months after intake. The results of the present study, thus,
indicate that POPhtCho could ameliorate cognitive disorders, possibly by enhancing LTD.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
Phosphatidylcholine, a component of the plasma membrane,
is hydrolyzed into cis-unsaturated free fatty acid and lysophosphatidylcholine at the ␤ position by phospholipase A2 (PLA2 ) [9].
Phosphatidylcholine, alternatively, is hydrolyzed into choline andphosphatidic acid at the ␥ position by phospholipase D (PLD), andphosphatidic acid is further hydrolyzed into cis-unsaturated freefatty acid and lysophosphatidic acid at the ␤ position by PLA2[9]. Choline serves as a selective agonist of ␣7 acetylcholine (ACh)receptors [1] that are preferentially localized on presynaptic terminals and involve neurotransmitter release. In addition, choline
produces ACh together with acetyl-CoA as catalyzed by choline
acetyltransferase, to activate nicotinic and muscarinic ACh receptors. cis-Unsaturated free fatty acids such as arachidonic, linoleic,
and linolenic acid induce a long-lasting facilitation of hippocampal synaptic transmission by targeting presynaptic nicotinic ACh
receptors under the inﬂuence of protein kinase C (PKC) [5,6]. This,in the light of the fact that presynaptic nicotinic ACh receptors
participate in the expression of long-term potentiation (LTP) [4,7],a cellular model of learning and memory, raises the possibility
that cis-unsaturated free fatty acids enhance cognitive functions.
Lysophosphatidic acid and lysophosphatidylcholine are also shown
to potentiate nicotinic ACh receptor responses [3,8], suggesting acontribution to LTP expression. Phosphatidylcholine, thus, mightserve as an enhancer of cognitive functions. In support of this note,
we earlier found that 1,2-dilynoleoyl-sn-glycero-3-phosphocholine(DLPhtCho) improves scopolamine-induced learning and memory
deﬁcits by facilitating hippocampal synaptic transmission under
the control of ␣7 ACh receptors [11]. The present study was
conducted to assess the effect of the other phosphatidylcholine, 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPhtCho), on
cognitive functions.
We show here that POPhtCho improves scopolamine-induced
impairment of spatial learning and memory for rats, possibly byenhancing long-term depression (LTD) in concert with decreased
expression of the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluR1 on the plasma membrane
and that the lipid ameliorates cognitive decline for humans.
2. Materials and methods
2.1. Animal care
All procedures have been approved by the Animal Care and Use Committee atHyogo College of Medicine and were in compliance with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals.
2.2. Participants
The study for human was approved by Institutional Review Board at Hyogo
College of Medicine and informed consent was obtained from all the participants.
2.3. Field excitatory postsynaptic potential (fEPSP) recording∗ Corresponding author. Tel.: +81 798 45 6397; fax: +81 798 45 6649.
E-mail address: tomoyuki@hyo-med.ac.jp (T. Nishizaki).
0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2009.05.027

The hippocampus was isolated from male Wistar rats (5–7 w) and hippocampal
slices (400 ␮m in thickness) were prepared. fEPSPs were recorded from the CA1 area

130T. Yaguchi et al. / Behavioural Brain Research 204 (2009) 129–132of hippocampal slices by electrically stimulating the Schaffer collateral at 0.033 Hz
in artiﬁcial cerebrospinal ﬂuid (ACSF) (in mM: 117 NaCl, 3.6 KCl, 1.2 NaH2 PO4 , 1.2MgCl2 , 2.5 CaCl2 , 25 NaHCO3 and 11.5 glucose) oxygenated with 95% O2 and 5% CO2
at 34 ◦ C. LTD was induced by applying low frequency stimulation (LFS) (120 pulses
at 2 Hz, ﬁve trains with an inter-train interval of 60 s) in the presence and absenceof POPhtCho (Avanti Polar Lipids, Inc., Alabaster, AL, USA) (1 ␮M).
2.4. Distribution of GluR1 and GluR2 in rat hippocampal slices
Rat hippocampal slices were taken out 10 min before and 60 min after LFS,
that induced LTD, in the absence and presence of POPhtCho (1 ␮M). Then, fractions of the CA1 area around an electrode to record fEPSPs were cut off fromslices and homogenized in 100 ␮l of a lysate solution (210 mM mannitol, 70 mM
sucrose, 1 mM EDTA, 10 mM HEPES, and 1% protease inhibitor cocktail, pH 7.5).
Homogenates were centrifuged at 11,000 rpm for 15 min, and the supernatant was
further super-centrifuged at 100,000 × g for 1 h. The pellet and the supernatantwere used as a plasma membrane-enriched and cytosol-enriched component,respectively. Each sample (20 ␮g protein) was loaded on 10% sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene
diﬂuoride membranes. After blocking with 0.1% Tween-20 containing 5% BSA,
blotting membranes were reacted with an antibody against GluR1 (1:5000)(Upstate, Lake Placid, NY, USA) or GluR2 (1:800) (CHEMICON, Temecula, CA, USA)
followed by an HRP-conjugated anti-rabbit IgG antibody (1:5000) or anti-mouse
IgG antibody (1:5000), respectively. Immunoreactivity was detected with an ECL kit(GE Healthcare Biosciences, Piscataway, NJ, USA) and signal density was measured
with an Image Gauge software (FUJIFILM, Tokyo, Japan).

2.6. Mini Mental State Examination (MMSE) test
MMSE test was performed on 67 subjects (31 males and 36 females) ranging inage from 59 to 93 years (average, 77.1 ± 0.8 years old), who had age-related cognitive
disorders before and after oral intake with POPhtCho (300 mg/day, once after breakfast), that was extracted from egg lecithin, as a POPhtCho group and on 9 subjects(4 males and 5 females) ranging in age from 74 to 90 years (average, 79.4 ± 2.8 yearsold) without taking POPhtCho as a control group. Full marks are 30, and less than 20
corresponds to mild cognitive impairment and dementia.
2.7. Statistical analysis
Statistical analysis was carried out using Fisher’s Protected Least Signiﬁcant
Difference (PLSD) test and t-test.

3. Results
3.1. POPhtCho enhances LTDOur ﬁrst attempt was to see the effect of POPhtCho on synaptic plasticity relevant to learning and memory. We have earlier

2.5. Water maze test
For animal model of learning and memory deﬁcits, male Wistar rats (7 w) wereintraperitoneally injected with scopolamine (1 mg/kg). A circular plastic water tankwith 180 cm in diameter and 45 cm in deep was used for a water maze test. The
entire inside of the pool was painted black, and the pool was ﬁlled up to 25 cm fromthe bottom with muddy water containing India ink at 22 ◦ C. A platform (11 cm indiameter) painted black was placed into water, the top sinking 1 cm below water
surface. The pool was put in a test room, where there were several marks that rats
were able to see from the pool. The position of the marks remained unchanged
throughout testing. A platform was located in the constant position, i.e., in the middle of one quadrant, equidistant from the center and edge of the pool. Rats facing
the wall of the pool were placed into water at one of the ﬁve positions selected
at random, and time from start to escape onto the platform (acquisition latency)
was measured. When succeeded, rats were allowed to stay on the platform for 10 s.
POPhtCho dissolved with polyethylene glycol (PEG) (ﬁnal volume, 0.1 ml) or PEG
alone (ﬁnal volume, 0.1 ml) was orally administered to rats everyday throughout
experiments from 7 days prior to injection with scopolamine or saline. Water mazetask was performed two trials per day, and the second trial began 2 min after theend of the ﬁrst trial. Rats received the task for consecutive 8 days and the acquisition latency (time form the start to arrival onto the plate) was measured. Sevendays later, the platform was removed and the retention latency (time from the start
to arrival to the place where the platform had been set, 30 s in maximum) wasmeasured.

Fig. 1. POPhtCho enhances LTD. fEPSPs were monitored from the CA1 region of rat
hippocampal slices, and LFS, to induce LTD, was applied to slices in the absence(control) and presence of POPhtCho (1 ␮M). In the graph, each point represents themean (±SEM) percentage of basal EPSP slope (0 min) (n = 8).

Fig. 2. POPhtCho reduces expression of AMPA receptors on the plasma membrane
during LTD. Western blotting using an antibody against GluR1 (A) or GluR2 (B) wascarried out in the plasma membrane and cytosolic component from rat hippocampal slices taken out 10 min before and 60 min after LFS, that induced LTD, in theabsence and presence of POPhtCho (1 ␮M). In the graphs, each column represents
the mean (±SEM) ratio of signal intensity for GluR1 or GluR2 in the plasma membrane component/signal intensity for each subunit in total cells (n = 4). P values,
unpaired t-test.

T. Yaguchi et al. / Behavioural Brain Research 204 (2009) 129–132

conﬁrmed that POPhtCho (1 ␮M) does not affect basal fEPSPs
elicited from the CA1 region of rat hippocampal slices and Schaffer
collateral-CA1 LTP [11]. In the present study, POPhtCho (1 ␮M) signiﬁcantly enhanced LTD as compared with control LTD (P < 0.0001,
Fisher’s PLSD test) (Fig. 1).
3.2. POPhtCho decreases GluR1 expression on the plasma
membrane during LTD
To understand the mechanism underlying POPhtCho-induced
enhancement in LTD, we examined distribution of the AMPA receptor subunits GluR1 and GluR2 in rat hippocampal slices. POPhtCho(1 ␮M) had no effect on expression of GluR1 or GluR2 on the plasma
membrane before LTD induction (Fig. 2A and B). POPhtCho (1 ␮M)signiﬁcantly reduced GluR1 expression on the plasma membrane
during LTD as compared with the expression in the absence ofPOPhtCho (Fig. 2A). Likewise, POPhtCho (1 ␮M) decreased GluR2expression on the plasma membrane during LTD, although it was
not signiﬁcant (Fig. 2B). The reduced GluR1 expression on theplasma membrane during LTD could account for POPhtCho-induced
enhancement in LTD.
3.3. POPhtCho improves scopolamine-induced impairment of
spatial learning and memory for rats
We next examined the effect of POPhtCho on impairment of
spatial learning and memory using water maze test. For control rats, oral administration with POPhtCho (1 mg/kg) had noeffect on the acquisition and retention latency (Fig. 3A and B).
Intraperitoneal injection with scopolamine markedly prolonged
both the acquisition and retention latency (Fig. 3A–D), indicat-131

ing scopolamine-induced impairment of learning and memory.
No signiﬁcant improvement against the prolonged acquisition andretention latency was obtained with oral administration with
1 mg/kg of POPhtCho (Fig. 3A and B). In contrast, oral administration
with 5 mg/kg of POPhtCho signiﬁcantly shortened the prolonged
retention latency (P = 0.0276, unpaired t-test as compared with thatfor rats treated with scopolamine without POPhtCho) (Fig. 3D),
while the prolonged acquisition latency was not affected (Fig. 3C).
This implies that POPhtCho is capable of improving spatial learningand memory deﬁcits induced by scopolamine.
3.4. POPhtCho ameliorates cognitive disorders for humans
We ﬁnally examined whether POPhtCho improves cognitive disorders for humans. Of 67 subjects for a POPhtCho group 43 ones
belonged to mild cognitive impairment and dementia, and the average MMSE score before oral intake with POPhtCho was 18.1 ± 0.6(Fig. 4). Oral intake with POPhtCho (300 mg/day, once after breakfast) everyday signiﬁcantly raised MMSE scores, the average scorereaching over 20, i.e., normal cognitive functions, throughout 6months after intake (Fig. 4). In contrast, no increase in MMSE score
was obtained with control subjects, who did not take POPhtCho,
throughout 3 months (Fig. 4). These results indicate that POPhtCho
is still capable of ameliorating cognitive disorders for humans.
4. Discussion
PhtCho is hydrolyzed into cis-unsaturated free fatty acid andlysophosphatidylcholine by PLA2 or into choline and phosphatidic acid by PLD followed by further hydrolysis of phosphatidic
acid into cis-unsaturated free fatty acid and lysophosphatidic acid

Fig. 3. POPhtCho improves scopolamine-induced spatial learning and memory deﬁcits. Water maze task was performed two trials per day for consecutive 8 days and theacquisition latency was measured. Seven days later, the retention latency was measured. Rats were orally administered with PEG or POPhtCho (POPC) (1 and 5 mg/kg)
everyday throughout experiments from 7 days prior to treatment with saline [scopolamine (−)] or scopolamine (1 mg/kg) [scopolamine (+)]. Saline or scopolamine wasintraperitoneally injected 30 min prior to water maze task. (A and C) Each point represents the mean (±SEM) acquisition latency from consecutive 2 days (n = 15). P value,
Fisher’s PLSD test. (B and D) Each column represents the mean retention latency (n = 15). P value, unpaired t-test.

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T. Yaguchi et al. / Behavioural Brain Research 204 (2009) 129–132

retention latency for rats treated with scopolamine, although theprolonged acquisition latency was not affected. This implies that
POPhtCho improves scopolamine-induced learning and memory
deﬁcits, possibly by enhancing LTD. This also suggests that LTD
is related to retention but not acquisition for spatial learning andmemory. The most striking ﬁnding in the present study is that
POPhtCho raised lowered MMSE scores, corresponding to mild cognitive impairment and dementia, to normal levels for humans. This
represents that POPhtCho could serve as an enhancer of cognitivefunctions.
In conclusion, the results of the present study demonstrate that
POPhtCho improves spatial learning and memory deﬁcits for rats,
possibly by enhancing LTD in association with decreased expression of the AMPA receptor subunit GluR1 on the plasma membrane,
and that POPhtCho ameliorates cognitive disorders for humans.
This provides evidence that POPhtCho exerts its beneﬁcial action
on cognitive functions.
References
Fig. 4. POPhtCho ameliorates cognitive disorders. MMSE test was carried out once amonth on 67 subjects before and after oral intake with POPhtCho (300 mg/day, once
after breakfast) everyday [POPhtCho (+)] and 9 subjects without taking POPhtCho[POPhtCho (−)]. In the graph, each point represents the mean (±SEM) MMSE scoreat the periods as indicated. ***P < 0.0001, paired t-test.

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