Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112
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ABSTRACT |
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Chen, Chu, Jeffrey C. Magee, Victor Marcheselli, Mattie Hardy, and Nicolas G. Bazan. Attenuated LTP in Hippocampal Dentate Gyrus Neurons of Mice Deficient in the PAF Receptor. J. Neurophysiol. 85: 384-390, 2001. Platelet-activating factor (PAF), a bioactive lipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) derived from phospholipase A2 and other pathways, has been implicated in neural plasticity and memory formation. Long-term potentiation (LTP) can be induced by the application of PAF and blocked by a PAF receptor (PAF-R) inhibitor in the hippocampal CA1 and dentate gyrus. To further investigate the role of PAF in synaptic plasticity, we compared LTP in dentate granule cells from hippocampal slices of adult mice deficient in the PAF-R and their age-matched wild-type littermates. Whole cell patch-clamp recordings were made in the current-clamp mode. LTP in the perforant path was induced by a high-frequency stimulation (HFS) and defined as >20% increase above baseline of the amplitude of excitatory postsynaptic potentials (EPSPs) from 26 to 30 min after HFS. HFS-induced enhancement of the EPSP amplitude was attenuated in cells from the PAF-R-deficient mice (163 ± 14%, mean ± SE; n = 32) when compared with that in wild-type mice (219 ± 17%, n = 32). The incidence of LTP induction was also lower in the cells from the deficient mice (72%, 23 of 32 cells) than in the wild-type mice (91%, 29 of 32 cells). Using paired-pulse facilitation as a synaptic pathway discrimination, it appeared that there were differences in LTP magnitudes in the lateral perforant path but not in the medial perforant path between the two groups. BN52021 (5 µM), a PAF synaptosomal receptor antagonist, reduced LTP in the lateral path in the wild-type mice. However, neither BN52021, nor BN50730 (5 µM), a microsomal PAF-R antagonist, reduced LTP in the lateral perforant path in the receptor-deficient mice. These data provide evidence that PAF-R-deficient mice are a useful model to study LTP in the dentate gyrus and support the notion that PAF actively participates in hippocampal synaptic plasticity.
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INTRODUCTION |
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Platelet-activating factor
(PAF), a bioactive lipid
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) generated
by phospholipase A2 and other pathways
(Bazan 1995), has been implicated in neural plasticity
and memory formation. These functional implications of PAF are based on
studies showing that tetanic stimulation-induced long-term potentiation
(LTP) is blocked by a PAF receptor (PAF-R) antagonist in rat
hippocampal CA1 (Arai and Lynch 1992
; Kato et al.
1994
), in dentate gyrus (Kato and Zorumski
1996
), and in medial vestibular nuclei (Grassi et al.
1998
). PAF also enhances excitatory postsynaptic responses
(Clark et al. 1992
; Kato and Zorumski
1996
; Kato et al. 1994
; Kornecki et al.
1996
; Wieraszko et al. 1993
) and increases the
frequency of spontaneous miniature excitatory postsynaptic currents
(Clark et al. 1992
; Kato and Zorumski
1996
; Kato et al. 1994
) in hippocampal neurons.
PAF inhibits ionotropic GABA receptor activity in primary cultured
hippocampal neurons (Chen and Bazan 1999
), further
contributing to the notion that accumulation of PAF promotes enhanced
excitatory neurotransmission by decreasing inhibitory synaptic input.
There is evidence that PAF release from hippocampal slices is
dramatically increased during the induction of LTP by a high-frequency
stimulation (Kornecki et al. 1996
). In addition, PAF has
been proposed to be involved in memory formation (Izquierdo et
al. 1995
; Teather et al. 1998
). Furthermore PAF
content in brain is elevated during ischemia or seizures (Kumar
et al. 1988
; Nishida and Markey 1996
),
and PAF receptor antagonists reduce glutamate-induced neurotoxicity and cell death (Mukherjee et al. 1999
; Ogden et al.
1998
). Therefore PAF is of significance both in cell function
as well as in pathological conditions (Bazan and Allan
1998
).
Recent progress in the introduction of targeted mutations into the
mouse genome has made it possible to test specific gene products in
synaptic function. A seven-transmembrane-spanning domain PAF-R has been
cloned (Honda et al. 1991), and a mouse lacking this
PAF-R has been generated (Ishii et al. 1998
). To assess
the usefulness of this mutant for studying synaptic plasticity and to
further investigate the role of PAF in synaptic plasticity, LTP
induction in the perforant path was evaluated in dentate granule cells
from hippocampal slices of adult mice deficient in the PAF-R and their
age-matched wild-type littermates using the whole cell patch-clamp
technique. Our results indicate that LTP is attenuated in lateral
perforant path-dentate granule cell synapses from mice deficient in the
PAF receptor and support the notion that PAF is involved in hippocampal
synaptic plasticity.
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METHODS |
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Generation of deficient mice
PAF-R deficient mice (hybrids between C57BL/6J and 129/Ola
strains) (Ishii et al. 1998) and their wild-type
littermates were kindly provided by Takao Shimizu (Department of
Biochemistry, Faculty of Medicine, University of Tokyo).
PCR analysis
Quick Tail DNA Prep for Genotyping: 1-2 cm mice tail was added to digestion buffer [5 mM EDTA, 200 mM NaCl,100 mM Tris-HCl (pH, 8.0; 0.2% SDS), 0.5 mg/ml proteinase K, and 12.5 µg/ml ribonuclease A], digested overnight in a water bath at 55°C. The DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), washed twice in chloroform/isoamyl alcohol (26:4), and precipitated with 1:1 isopropanol. Pellets were resuspended in Tris-EDTA (pH, 8) and heated for 2 h at 65°C. PCR was as follows: 0.5 µl DNA, 1× PCR buffer, 1.5 mM MgCl, 0.1 mM dNTPs, 200 ng primers (forward: 5' gcc tgc ttg ccg at atc atg gtg gaa aat 3', reverse: 3' gcg atg cgc tgc gaa tcg gga gcg gcg ata 5' for deficient allele and forward: 5' tat ggc tga cct gct ctt cct gat 3', reverse: 3' tat tgg gca cta ggt tgg tgg agt 5' for wild-type allele), water, and Taq DNA Polymerase (1 U) in a total volume of 25 µl. PCR was carried out using a Perkin Elmer GeneAmp PCR System 9600 cycler with the following conditions: 94°C for 0.25 min, 60°C for 0.25 min, 72°C for 0.5 min for 30 cycles. The PCR product was analyzed on a 1.8% agarose gel containing ethidium bromide (Fig. 1).
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Slice preparation
Hippocampal slices were prepared from either sex of
PAF-R-deficient mice (body wt: 26.2 ± 0.8 g,
n = 72) and age-matched wild-type littermates (body wt:
25.1 ± 1 g, n = 69), using standard
procedures as described previously (Magee et al. 1996).
Briefly, the brain was rapidly removed after decapitation and placed in
cold oxygenated (95% O2-5%
CO2)
low-Ca2+/high-Mg2+ slicing
solution composed of (in mM) 2.5 KCl, 7.0 MgCl2,
28.0 NaHCO3, 1.25 NaH2PO4, 0.5 CaCl2, 7.0 glucose, 3 pyruvic acid, 1 ascorbic
acid, and 117 choline Cl or 234 sucrose. Then the brain was trimmed and
glued to the stage of a vibratome (Technical Products International,
St. Louis, MO) and immersed in a bath of cold, oxygenated slicing
solution. Slices were cut at a thickness of 400 µm and transferred to
a holding chamber in an incubator containing oxygenated artificial
cerebrospinal fluid (ACSF) composed of (in mM) 125.0 NaCl, 2.5 KCl, 1.0 MgCl2, 25.0 NaHCO3, 1.25 NaH2PO4, 2.0 CaCl2, 25.0 glucose, 3 pyruvic acid, and 1 ascorbic acid at 36°C. Slices were maintained in an incubator
containing oxygenated ACSF at room temperature (22-24°C) for
1.5 h
before recordings. Slices were then transferred, as needed, to a
recording chamber where they were continuously perfused with the 95%
O2-5% CO2 saturated standard ACSF (without pyruvic acid and ascorbic acid) at 34-35°C. Individual dentate granule cells were visualized with a Zeiss Axioskop
microscope (Oberkochen, Germany).
Electrophysiological recordings
Whole cell patch-clamp recordings were made using an
Axoclamp-2B patch-clamp amplifier in bridge mode. Data were
acquired (25 kHz, filtered at 1 kHz) using a DigiData 1200 interface
and pClamp 7.01 software (Axon Instruments, Foster City, CA). Recording pipettes (6-9 M) were pulled from borosilicate glass with a
micropipette puller (Sutter Instrument) and fire polished on a
microforge (Narishige Scientific Instrument) prior to use. The internal
pipette solution contained (in mM) 120 K gluconate, 20 KCl, 4 NaCl, 10 HEPES, 0.5 EGTA, 0.28 CaCl2, 4 Mg2ATP, 0.3 Tris2GTP, and
14 phosphocreatine (pH, 7.25 with KOH). Series resistance ranged from
15 to 30 M
as estimated directly from the amplifier and was
monitored during recordings by injecting a 20-ms hyperpolarizing
current (50 pA) before delivering a stimulus. The mean input resistance
(estimated from a 50-pA hyperpolarizing current injection) was 138 ± 8 M
(n = 52) for cells from the wild-type mice
and 135 ± 9 M
(n = 68) for cells from the
deficient mice, and the mean resting membrane potential was
74 ± 1 mV (n = 52) for cells from the wild-type mice and
73 ± 1 mV (n = 68) for cells from the deficient
mice. Excitatory postsynaptic potentials (EPSPs) were recorded in
response to stimulation of the perforant path at a frequency of 0.05 Hz. Stimuli were elicited via a bipolar tungsten electrode. The
synaptic input pathway was determined by stimulation electrode
positioning and paired-pulse stimulating (inter-pulse interval, 80-100
ms). As reported (Colino and Malenka 1993
; Min et
al. 1998
; Reid and Clements 1999
; Zucker
1989
), medial perforant path (MPP) stimulation elicited
paired-pulse depression, whereas lateral perforant path (LPP)
stimulation elicited facilitation. Paired-pulse ratio was calculated as
P2/P1 (P1, the amplitude of the first EPSP; P2, the amplitude of the
second EPSP). The amplitude range of the evoked EPSPs was always
adjusted to 2-6 mV (<30% of threshold for generating an action
potential). LTP in the perforant path was induced by a high-frequency
stimulation (HFS) consisting of eight trains, each of eight pulses at
200 Hz with an intertrain interval of 2 s as described by
Wang et al. (1996)
. Postsynaptic depolarization was
induced by injecting a depolarizing current (0.5 nA) during HFS. LTP
was operationally defined as >20% increase above baseline for the
amplitude of EPSPs from 26 to 30 min after HFS.
Drug solutions
All chemicals and drugs were purchased from Sigma except for
bicuculline, which was purchased from RBI (Natick, MA), and BN52021, which was purchased from Biomol (Plymouth Meeting, PA). BN52021 and
BN50730 were dissolved in DMSO at 100 mM and diluted to a desired
concentration with the standard ACSF. In experiments where the PAF-R
antagonists were applied, slices were pretreated with either 5 µM
BN52021 or BN50730 for 1.5 h and then were continuously perfused with
the antagonists during recordings. All the bath perfused solutions,
including drug solutions, contained 10 µM bicuculline to block
ionotropic GABA receptors.
All experiments were performed blind, i.e., the person who carried out the recordings did not know the genotypes of the animals. After finishing the recordings and basic data analyses, the codes were broken from the chips that were previously implanted in the animals for identification. The data were classified into two groups based on the recordings made from the deficient and wild-type mice. Data are presented as means ± SE. Unless stated otherwise, Student's t-test and one-way ANOVA with Fishers PLSD post hoc were used for statistical comparison when appropriate. Differences were considered significant when P < 0.05. The care and use of the animals reported on this study were approved by the Animal Care and Use Committee of Louisiana State University Health Sciences Center.
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RESULTS |
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Induction of LTP is attenuated in the lateral perforant path-dentate gyrus cell synapses of PAF-R-deficient mice
Synaptic transmission and plasticity were examined in dentate granule cells of PAF-R-deficient mice and their age-matched wild-type littermates. There were no abnormalities in basic membrane properties including resting membrane potential, input resistance (see METHODS), current required to evoke EPSPs (1-10 µA), and action potential generation (8-10 spikes per burst) in cells from the receptor deficient slices. As indicated in Fig. 2, however, the potentiation of EPSP amplitude by HFS was significantly reduced in cells from PAF-R-deficient mice (mean potentiation: 163 ± 14% of baseline 26-30 min after HFS, n = 32) when compared with that in the wild-type mice (219 ± 17%, n = 32). The incidence of LTP induction was also lower in the receptor deficient mice (72%, 23 of 32 cells) than in the wild-type mice (91%, 29 of 32 cells, Fig. 2D).
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Using a standard PPF protocol, we attempted to determine if the differences in LTP induction were pathway specific. Input from the medial and lateral perforant pathways can be determined based on ratios of PPF (see METHODS). As shown in Fig. 3A, there were no significant differences in magnitudes of LTP in MPP between the PAF-R-deficient mice (179 ± 23% of baseline 26-30 min after HFS, n = 15) and wild-type mice (175 ± 21%, n = 7). However, significant differences in magnitudes of LTP in LPP were found between the deficient mice (161 ± 18% of baseline 26-30 min after HFS, n = 14) and wild-type mice (235 ± 23%, n = 20). In addition, there were significant differences in rations of the paired-pulse facilitation in LPP before and after HFS in both PAF-R deficient mice (P2/P1: from 1.18 ± 0.04 to 1.02 ± 0.03, n = 14) and wild-type mice (P2/P1: from 1.18 ± 0.04 to 1.06 ± 0.03, n = 20), but there were no differences in changes in the ratios of the paired-pulse facilitation before and after HFS between the receptor-deficient (0.16 ± 0.02, n = 14) and wild-type mice (0.12 ± 0.02, n = 20).
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PAF-R antagonist reduces LTP in wild-type mice but not in PAF-R mutants
Tetanic stimulation-induced LTP can be blocked by the synaptosomal
PAF-R antagonist 52021 in rat hippocampus (Kato and Zorumski 1996; Kato et al. 1994
). To examine whether
HFS-induced LTP in LPP in PAF-R-deficient mice could be blocked by the
PAF-R antagonist, slices were pretreated with 5 µM BN52021 for
1.5 h at 22-24°C, and then continuously perfused with the
antagonist throughout the experiments at 34-35°C. It appears that
LTP in LPP in wild-type mice was significantly reduced in 5 µM
BN52021-treated slices (162 ± 24% of baseline 26-30 min after
HFS, n = 20) when compared with that in nontreated
slices (235 ± 23%, n = 20; Fig.
4). However, there was no difference in
LTP in LPP in PAF-R deficients in 5 µM BN52021-treated slices
(164 ± 12% of baseline 26-30 min after HFS, n = 16) when compared with that in nontreated slices (161 ± 18%,
n = 14; Fig. 5,
B and D). To test whether BN50730, a microsomal PAF-R antagonist, has an effect on LTP in LPP in PAF-R-deficient mice,
slices were pretreated with 5 µM BN50730 in an incubator for 1.5 h, then continuously perfused with it during recordings. As shown in
Fig. 5, A and C, HFS-induced LTP in LPP was not
reduced in BN50730-treated slices (172 ± 12% of baseline 26-30
min after HFS, n = 20) when compared with that in
nontreated slices (161 ± 18%, n = 14). It has
been demonstrated before that BN50730 has no effect on LTP induction in
normal rat hippocampus (Kato and Zorumski 1996
;
Kato et al. 1994
), therefore we did not test the effect
of this microsomal PAF-R antagonist on LTP in wild-type animals.
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DISCUSSION |
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The idea that PAF is involved in hippocampal synaptic plasticity
is based on the observations that tetanic-induced LTP is blocked by the
PAF-R antagonist, and spontaneous miniature postsynaptic current or
evoked excitatory postsynaptic activity is enhanced by application of
PAF (Arai and Lynch 1992; Clark et al.
1992
; Kato and Zorumski 1996
Kato et al.
1994
; Kornecki et al. 1996
; Wieraszko et
al. 1993
). Two types of PAF binding sites in brain, synaptosomal (plasma membrane) and microsomal, have been described (Marcheselli et al. 1990
). But thus far only the
synaptosomal PAF-R has been cloned (Honda et al. 1991
).
To further investigate the role of PAF in hippocampal synaptic
plasticity, we used mice with a targeted deletion of the PAF plasma
membrane receptor gene (Ishii et al. 1998
). These mice
grow and reproduce normally and do not present obvious
behavioral and cellular abnormalities. However, HFS-induced LTP of the
lateral perforant path input to dentate granule cells is attenuated in
the receptor-deficient mice when compared with that in wild-type
controls. This is further evidence that PAF participates in modulating
hippocampal excitatory synaptic transmission.
In addition to reduced LTP induction, the synaptosomal PAF-R antagonist
52021 (5 µM) reduces the magnitude of LTP in wild-type mice but not
in the PAF-R-deficient mice, indicating that the synaptosomal PAF-R has
likely been physically and functionally deleted. This is consistent
with previous reports (Kato and Zorumski 1996;
Kato et al. 1994
) showing that BN50730 (5 µM), a
microsomal PAF-R antagonist, has no effect on the LTP induction in
PAF-R-deficient mice.
The medial and lateral perforant paths represent two separate input
pathways to the dentate gyrus. The medial perforant path synapses on
the central third and lateral perforant path onto the distal third of
the dendritic tree of dentate granule cells (Witter
1993). It has been demonstrated that synaptic transmission in
these pathways displays profound differences in terms of physiology and
pharmacology (Abraham and McNaughton 1984
; Kahle
and Cotman 1989
; McNaughton 1980
; Min et
al. 1998
), and our data support this idea. While the mechanisms
of LTP expression in these two pathways are still not clear, recent
evidence suggests that LTP of the medial perforant path results from an
increased postsynaptic response to neurotransmitter (Reid and
Clements 1999
). Our data indicate that LTP induction in lateral
perforant path is reduced in the PAF-R-deficient mice when compared
with that in wild-type controls, suggesting that the role of PAF in the
expression of LTP in these two pathways may be different. It has been
proposed that PAF may act as a retrograde messenger at a presynaptic
receptor in hippocampal CA1 LTP because of increases in the frequency
of miniature excitatory postsynaptic currents and synaptic evoked responses by postsynaptic application of PAF (Clark et al.
1992
; Kato et al. 1994
; but see Kobayashi
et al. 1999
). Similar phenomena have also been observed in rat
hippocampal dentate region (Kato and Zorumski 1996
). A
PAF-mediated increase in the probability release may be a mechanism of
LTP expression in the lateral perforant path. This may be the reason
why there is a difference in LTP in the lateral perforant, but not in
the medial perforant path between the PAF-R-deficient mice and
wild-type controls. Interestingly, it has been reported recently that
LTP in lateral perforant path, but not in medial perforant, is impaired
in mice deficient in the protein phosphatase inhibitor-1 (Allen
et al. 2000
). This finding together with our results suggest
that there may be heterogeneous molecular signaling regulation of LTP
in the lateral perforant path
and medial perforant path
dentate
granule cell synapses.
In summary, we have found that HFS-induced LTP is reduced in lateral perforant path-dentate granule cell synapses in PAF-R-deficient mice when compared with that of wild-type mice. BN52021 (5 µM), a PAF synaptosomal receptor antagonist, reduced LTP in the lateral perforant path in wild-type mice. However, neither BN52021, nor 50730 (5 µM), a microsomal PAF-R antagonist, reduced LTP in lateral perforant path in the receptor-deficient mice. These data provide evidence to support the notion that PAF participates in hippocampal synaptic plasticity and that the PAF-R-deficient mice are a useful model. In addition, hippocampal dentate gyrus is of significance in leaning and memory as well as for epileptogenesis. Alterations in PAF synthesis or PAF-R function may contribute to synaptic dysfunction and pathogenesis. Thus it would be of interest and significance to employ these receptor deficient mice to perform behavioral studies that will further our understanding of relationships between LTP and learning and memory, and role of PAF in learning and memory as well as in epileptogenesis.
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ACKNOWLEDGMENTS |
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We thank Professor Takao Shimizu for the kind gift of the breeding
mice of the PAF receptor /
and +/+.
This work was supported by National Institute of Neurological Disorders and Stroke Grant R01-NS-23002.
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FOOTNOTES |
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Address for reprint requests: N. G. Bazan, Neuroscience Center, Louisiana State University Health Sciences Center, 2020 Gravier St., New Orleans, LA 70112 (E-mail: nbazan{at}lsuhsc.edu).
Received 30 May 2000; accepted in final form 19 September 2000.
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REFERENCES |
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