Integrins Modulate Fast Excitatory Transmission at
Hippocampal Synapses*
Enikö A.
Kramár
§,
Joie A.
Bernard¶,
Christine M.
Gall¶, and
Gary
Lynch
From the
Department of Psychiatry and Human Behavior,
University of California, Irvine, California 92612-1695 and the
¶ Department of Anatomy and Neurobiology, University of
California, Irvine, California 92697-1275
Received for publication, October 6, 2002, and in revised form, January 8, 2003
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ABSTRACT |
The present study provides the first evidence
that adhesion receptors belonging to the integrin family modulate
excitatory transmission in the adult rat brain. Infusion of an integrin
ligand (the peptide GRGDSP) into rat hippocampal slices reversibly
increased the slope and amplitude of excitatory postsynaptic
potentials. This effect was not accompanied by changes in paired pulse
facilitation, a test for perturbations to transmitter release, or
affected by suppression of inhibitory responses, suggesting by
exclusion that alterations to
-amino-3-hydroxy-5-methyl-4-isoxazole
propionate (AMPA)-type glutamate receptors cause the enhanced
responses. A mixture of function-blocking antibodies to integrin
subunits
3,
5, and
v blocked ligand effects on synaptic responses. The ligand-induced increases were (i) blocked by inhibitors of Src tyrosine
kinase, antagonists of N-methyl-D-aspartate
receptors, and inhibitors of calcium
calmodulin-dependent protein kinase II and (ii) accompanied
by phosphorylation of both the Thr286 site on
calmodulin-dependent protein kinase II and the
Ser831 site on the GluR1 subunit of the AMPA receptor.
N-Methyl-D-aspartate receptor antagonists
blocked the latter two phosphorylation events, but Src kinase
inhibitors did not. These results point to the conclusion that synaptic
integrins regulate glutamatergic transmission and suggest that they do
this by activating two signaling pathways directed at AMPA receptors.
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INTRODUCTION |
Integrins are heterodimeric (
,
), membrane-spanning proteins
that anchor cells to the extracellular matrix and to adhesion proteins
on opposing cells (1, 2). They also organize the elements of the
submembrane actin cytoskeleton (3) and act as initiating receptors for
a diverse array of intracellular signaling cascades (4-6). As might be
expected from these latter roles, integrins have potent interactions
with neighboring membrane-associated proteins, including ion channels
(7-10) and trophic factor receptors (11-15). Whether these lateral
interactions also occur in synaptic junctions in the adult brain, which
are known to have high concentrations of integrins (16-20), has not
been studied. This leaves open the possibility that transmitter
receptors are tonically regulated by co-localized integrins. The
question takes on added significance because of the recent finding that
integrin expression is highly differentiated across brain regions as
well as between the dendritic domains of individual neurons (16,
21-24). If integrin binding does affect transmission, then it is
likely that this influence will vary significantly across synaptic systems.
Previous studies on the contributions of integrins to adult synaptic
physiology have been restricted to analyses of long term potentiation
(LTP).1 Small peptides and
toxins containing the matrix sequence (arginine-glycine-aspartate (RGD)) recognized by most integrins had little effect on the induction or initial expression of LTP but clearly interfered with its
stabilization (25-29) (i.e. in the presence of these
soluble ligands, potentiation slowly decayed to baseline). Whereas
function-blocking antibodies against the
5 (27) or
3 (30) integrin subunits caused partial blockade of
consolidation as did reductions in integrin-associated protein (31),
local applications of a mixture containing neutralizing antibodies
against integrin subunits
3,
5, and
v caused a complete reversal in LTP (25). Integrin
effects on plasticity are not restricted to hippocampus or LTP.
RGD-containing peptides interfere with rapid limbic kindling (32) and
activity-dependent synaptic changes in
Drosophila (33). In the latter case, it was possible to
identify the pertinent
integrin subunit (34, 35).
While connecting integrins to synaptic operations, the above results do
not address the question of whether integrins influence neurotransmitter receptors other than very indirectly through processes
mediating LTP consolidation. The experiments described here tested for
such effects in adult rat hippocampal slices. The results indicate that
integrin binding can increase fast, excitatory synaptic responses and,
in addition, provide evidence that they do so by modifying the
properties of AMPA-type glutamate receptors.
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EXPERIMENTAL PROCEDURES |
Hippocampal Slices--
Hippocampal slices were prepared as
previously described (30). Briefly, male Sprague-Dawley rats (42-60
days old) were anesthetized with halothane (Sigma) and decapitated. The
brain was quickly removed and placed in 0 °C oxygenated dissection
medium containing 124 mM NaCl, 3 mM KCl, 1.25 mM KH2PO4, 5 mM
MgSO4, 3.4 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose.
Transverse hippocampal slices (350 µm thick) through the middle third
of the septo-temporal axis of hippocampus were prepared using a
McIlwain tissue chopper and then transferred to an interface recording
chamber containing preheated aCSF consisting of 124 mM
NaCl, 3 mM KCl, 1.25 mM
KH2PO4, 2.5 mM MgSO4,
3.4 mM CaCl2, 26 mM
NaHCO3, and 10 mM glucose (30) and maintained
at 31 ± 1 °C. Slices were continuously perfused with this
solution at a rate of 75 ml/h while the surface of the slices were
exposed to warm, humidified 95% O2, 5% CO2.
Recordings began following at least 1 h of incubation.
Physiology--
Field excitatory postsynaptic potentials
(fEPSPs) were recorded from stratum radiatum of CA1b using a single
glass pipette filled with 0.15 M NaCl (yielding a
resistance of 2-3 megaohms) in response to orthodromic stimulation
(twisted nichrome wires, 65 µm) of the Schaffer
collateral-commissural projections in CA1 stratum radiatum. Pulses were
delivered to the stimulation electrode at 0.05 Hz with current test
intensity adjusted to obtain 50-60% of the maximum fEPSP. After
establishing a 10-20-min stable baseline, test compounds (see below)
were introduced into the infusion line by switching from control
aCSF to drug-containing aCSF. Measurements from the recorded
evoked potential included fEPSP slope and amplitude that were digitized
by NACGather (Theta Burst Corp.) and stored on a disk. The sample size
for all experiments represents the number of animals used.
Drug Application--
The integrin ligand peptide,
Gly-Arg-Gly-Asp-Ser-Phe (GRGDSP; Calbiochem), the integrin binding
control peptide, Phe-Ser-Asp-Gly-Arg-Gly (SDGRG; Sigma), the NMDA
antagonist D-(
)-aminophosphonopentanoic acid (AP5)
(Sigma), the AMPA type glutamate receptor blocker
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Sigma), and the GABA-A
antagonist picrotoxin (Sigma) were prepared fresh daily in aCSF prior
to being added to the infusion line. The Src tyrosine kinase inhibitor,
PP2 (Calbiochem); the CaMKII inhibitor, KN-93, and its control peptide
KN-92 (Calbiochem); and the protein kinase A inhibitor H-89
(Calbiochem) were dissolved in dimethyl sulfoxide and stored at
20 °C until the day of the experiment. Prior to application of
PP2, KN-93, KN-92, or H-89, stock solutions were diluted with aCSF to a
final working concentration containing 0.05% dimethyl sulfoxide.
Neutralizing Antibody Infusion--
Function-blocking monoclonal
antibodies to human
3,
5, and
v integrin subunits (anti-
3, MAB1952Z;
anti-
5, MAB1956; anti-
v, MAB1953;
Chemicon International, Temecula, CA) were combined and diluted in aCSF
to a working pipette concentration of 0.2 mg/ml each and then loaded
into a glass micropipette (tip diameter 25 µm). Local applications of
the anti-integrin mixture or aCSF (control) were achieved by
positioning the infusion pipette in the bath immediately adjacent and
slightly upstream (relative to aCSF infusion) from the hippocampal
slice (27). Ejection pressure, set at 8-12 p.s.i. with a pulse
duration of 10 ms, was estimated to deliver 3 nl per pulse at 10-s
intervals. The infusion pipette contained red dye, so that the
diffusion of the ejected solution could be monitored using a dissection
microscope. Treatment with the anti-integrin mixture began 20 min
before the application of 0.5 mM GRGDSP and continued for
60 min until the end of dual application with GRGDSP. Control
experiments involved pipette ejection of aCSF by the same technique and
schedule in the presence of GRGDSP.
Western Blots--
The effects of integrin ligands on the
activation state of calcium calmodulin protein kinase II (CaMKII) and
on phosphorylation of the GluR1 subunit of the AMPA class glutamate
receptor was evaluated by Western blot analysis with antibodies
directed to the phosphorylated forms of CaMKII (Thr286) and
GluR1 (Ser831). Hippocampal slices were prepared and placed
into the electrophysiological recording chamber with aCSF perfusion as
described above. After a 1-h incubation period, one of the six slices
was used to monitor baseline field potentials in the absence and
presence of test compounds; control slices were exposed to aCSF during
the same time interval. At the end of treatment, the slice being
monitored for treatment effects was discarded, and the remaining five
co-treated slices within the same recording chamber were pooled and
homogenized in aCSF containing protease inhibitor mixture (PIC
Complete; Amersham Biosciences) and phosphatase inhibitor mixture 1 (P2850; Sigma) as per the kit instructions. Homogenates were processed
for Western blot analysis with visualization of immunoreactive bands
using the enhanced chemiluminesence ECL Plus kit (Amersham Biosciences) as described in detail elsewhere (30). Blots were probed with anti-phospho-CaMKII Thr286 (catalog no. 05-533; Upstate
Biotechnology, Inc., Lake Placid, NY) or anti-phospho-GluR1
Ser831 (catalog no. 06-772; Upstate Biotechnology) at a
1:500 dilution in Tris-buffered saline with 5% bovine serum albumin
overnight at 4 °C. Experiments were run with experimental and
control tissue processed through all procedures simultaneously.
Densities of immunoreactive bands were evaluated by densitometry of ECL
films using the AIS imaging system (Imaging Research, St.
Catherines, Canada). All treatment effects described are for measures
of paired experimental and control samples run on the same Western blot.
All animal treatments were conducted in accordance with protocols
approved by the University of California at Irvine Institutional Animal
Use Committee and with the National Institute of Health Guide for the
Care and Use of Animals.
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RESULTS |
Effects of Integrin Ligands on Synaptic Responses
The effects of 60-min applications of GRGDSP on fEPSPs recorded in
the apical dendrites of hippocampal region CA1 are summarized in Fig.
1. Infusions of 1 mM GRGDSP
(Fig. 1A; n = 5) caused a consistent
increase in the slope and amplitude of the response beginning at 20-30
min after the start of infusion. This effect on fEPSP slope and
amplitude was highly reproducible and produced an average of 17 ± 2.9% (± S.D.) and 20 ± 6.5% increase over baseline by 1 h
after the start of infusion, respectively. Similar results were
obtained from slices treated with 0.5 mM GRGDSP (Fig.
1B, n = 9). At the end of a 60-min infusion
period, mean fEPSP slope and amplitude increased to 18 ± 6.8 and
20 ± 6.9% over baseline, respectively, and were not
statistically different from the field responses recorded from slices
treated with 1 mM GRGDSP (fEPSP slope, t = 0.12, p = 0.91; amplitude, t = 0.11, p = 0.91; between groups t test). Results
using doses of 0.1 (n = 5), 0.01 (n = 6), and 0.001 mM (n = 6) GRGDSP as compared
with the reverse-sequence control peptide SDGRG at 0.5 mM
(n = 6) are also presented in Fig. 1, C-F.
As shown, 0.1 mM GRGDSP produced an observable threshold for RGD-induced increases in fEPSP slope (10 ± 4.6%) and
amplitude (10 ± 5.4%). Lower doses of 0.01 and 0.001 mM GRGDSP and the reverse-sequence control peptide SDGRG at
0.5 mM had no detectable influence on synaptic responses
(mean fEPSP slope: 0.01 mM GRGDSP = 5 ± 2.3%; 0.001 mM GRGDSP =
2 ± 4.3%; 0.5 mM SDGRG =
2 ± 4.7%; mean amplitude: 0.01 mM GRGDSP = 6 ± 3.1%; 0.001 mM
GRGDSP = 2 ± 2.2%; 0.5 mM SDGRG = 3 ± 3.7%). Fig. 1G summarizes the dose-response data. One-way analysis of variance indicated that there were significant differences between groups infused with GRGDSP and SDGRG (fEPSP slope:
F = 18.5, p < 0.0001; amplitude:
F = 15.4, p < 0.0001). Post
hoc comparisons confirmed that slices perfused with 0.1, 0.5, and 1 mM GRGDSP had significantly greater fEPSP slopes
and amplitudes measured 60 min after the start of infusion as compared with slices treated with 0.001 or 0.01 mM GRGDSP or with
0.5 mM SDGRG. The dose range over which GRGDSP was
effective in increasing field responses corresponds to that at which it
blocks integrin adhesive functions in tissues throughout the body. Also
in accord with prior results, GRADSP, a weaker integrin ligand (36),
was substantially less effective than GRGDSP at all concentrations tested (data not shown).

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Fig. 1.
The GRGDSP integrin ligand increases
CA1 Schaffer collateral fEPSP slope and amplitude in a
dose-dependent manner. Representative examples of the
effects of infusing 0.001-1 mM GRGDSP
(A-E) and 0.5 mM SDGRG (F) on
fEPSP slope are plotted as the percentage change from baseline.
For each graph, insets show examples of evoked field
potentials taken at time points just before peptide infusion and
immediately after the 60-min testing period and illustrate treatment
effects on fEPSP amplitude. Calibration was as follows: 0.5 mV, 5 ms.
G, plot of grouped data (mean ± S.E.) showing the
effects of GRGDSP applied at 0.001-1 mM and of the reverse
sequence SDGRG at 0.5 mM on fEPSP slope and amplitude
(plotted as percentage change from baseline) as measured during the
last 5 min of the 60-min peptide infusion period. With GRGDSP applied
at 0.1-1 mM, the fEPSP slope and amplitude were
significantly greater than in slices treated with 0.001 and 0.01 mM GRGDSP and the control peptide SDGRG (*,
p < 0.0001; Neuman-Kuels post
hoc test).
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Slices treated with 0.5 or 1 mM GRGDSP were used to test
for reversibility of the ligand-induced increases in response size. Pooled data for the two concentrations are summarized in Fig. 2. After a 1-h application of the
integrin ligand, fEPSP slope and amplitude continued to increase for
about 25 min after the start of washout, reaching a peak increase of
22 ± 7.1 and 28 ± 6.4%, respectively. The increase was
maintained for an additional 25 min (i.e. ~50 min after
onset of washout), after which a gradual decline became evident.
Washout of competitive agonist/antagonists begins within 5-10 min in
the slice perfusion system used in the present study; the unusually
long washout pattern obtained with GRGDSP is suggestive of a compound
that triggers a second messenger system with activation/deactivation
times on the order of several minutes.

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Fig. 2.
GRGDSP induces a lasting enhancement of field
EPSPs. After establishing a stable 10-min baseline, GRGDSP (0.5 and 1 mM) was infused for 1 h as indicated by the
horizontal bar; plot shows group mean ± S.E. fEPSP slopes and amplitudes expressed as a percentage of baseline
values (n = 5 slices). As shown, both fEPSP slope and
amplitude increased steadily to reach maximal values about 20 min after
washout of the peptide, before beginning a gradual decline toward
baseline, which lasted over 3 h.
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The fast, feed forward inhibitory postsynaptic potential disynaptically
activated by the Schaffer-commissural fibers affects the amplitude and
waveform of the fEPSP. Changes in these potentials could therefore
account for some, although not all, of the GRGDSP-induced increases
described above. To test this possibility, slices (n = 4) were treated with the GABA-A receptor antagonist picrotoxin (5 µM) prior to and during infusion of 1 mM
GRGDSP. As illustrated in Fig.
3A, picrotoxin did not reduce
the effects of the integrin ligand on fEPSP slope (31 ± 8.8%; 30 min post-GRGDSP infusion) and amplitude (41 ± 18.0%; 30 min
post-GRGDSP infusion). These increases are as large as or larger than
those obtained in the absence of the GABA-A receptor antagonist (see
Fig. 2). The traces in Fig. 3, B-D, make the additional
point that GRGDSP did not distort the waveform of the fEPSP. In accord
with earlier work (37, 38), 5 µM picrotoxin prolonged the
decay phase of the fEPSP and eliminated the positive wave that follows
it (Fig. 3B). The waveform of the RGD-enhanced response,
normalized to base-line amplitudes, was identical to responses recorded
during treatment with picrotoxin alone (Fig. 3C).

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Fig. 3.
GRGDSP-induced fEPSP waveform changes are not
influenced by GABA-A receptors. A, plots of mean ± S.E. slopes and amplitudes of fEPSPs recorded from hippocampal
slices (n = 4) perfused with 5 µM
picrotoxin (PTX) for 50 min followed by picrotoxin plus 1 mM GRGDSP for an additional 1 h, after which washout
began. As shown, picrotoxin did not interfere with GRGDSP-induced
increases in fEPSP slope and amplitude. During washout, fEPSP slope and
amplitude reached a maximum of 30 and 40% above baseline values,
respectively, before slowly declining. B-D, representative
fEPSPs collected during picrotoxin and GRGDSP infusion, showing that
waveform changes in GRGDSP-treated slices are not due to GABA-A
receptor activities. Each trace shows the average response
during a 3-min recording period. B, following a stable
baseline, slices were treated with 5 µM picrotoxin for
1 h. The trace on the left is an average
field response during baseline recording. The field response in the
middle is an average trace taken in the presence
of picrotoxin. On the far right, the two
responses are superimposed. Calibration was as follows: 5 ms, 0.25 mV.
C, following 1-h infusion of picrotoxin, 1 mM
GRGDSP was added to the perfusion medium for an additional 1-h
recording period. The trace on the left
represents an average field response in the presence of picrotoxin.
Normalized for amplitude, the trace in the middle
shows an average waveform taken in the presence of picrotoxin plus
GRGDSP. At the right, the two traces are shown superimposed.
Calibration was as follows: 5 ms, 0.25 mV. D, for
comparison, a separate group of slices were treated for 1 h with 1 mM GRGDSP only. The field response on the left
represents an average trace during the baseline recording period. The
middle trace has been normalized for amplitude
and shows an average response in the presence of GRGDSP. The two
responses are superimposed on the far right.
Calibration was as follows: 5 ms, 1 mV.
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Increases in the size of synaptic responses with minimal distortion of
their waveform, as described above, can be produced by enhancing the
probability of transmitter release. Paired pulse facilitation, an
effect known to be sensitive to perturbations in neurotransmitter
release probability, was used to test the possibility that integrin
related changes in response size are due to presynaptic alterations. As
shown in Fig. 4, paired pulse facilitation at intervals of 20, 50, 100, and 200 ms was not detectably changed from baseline values following infusion of 0.5 mM
GRGDSP (p
0.2 at all intervals; paired t
test, two-tailed). These data suggest that the molecular mechanisms
regulating GRGDSP-induced enhancement in synaptic transmission are
postsynaptic.

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Fig. 4.
GRGDSP does not affect paired pulse
facilitation. The graph shows the degree of paired
pulse facilitation (initial slope of response measure) expressed as a
function of the interpulse interval (20, 50, 100, and 200 ms) for
responses recorded before (open circle) and after
(closed circle) application of 0.5 mM
GRGDSP. Each point represents the mean ± S.E. of five separate
experiments. Treatment with GRGDSP did not influence the degree of
paired pulse facilitation at any interpulse interval tested.
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Previous studies established that field EPSPs recorded under conditions
described here do not contain significant contributions from NMDA
receptors (38). In accord with this, as shown in Fig. 5, infusion of the AMPA receptor
antagonist CNQX largely blocked synaptic responses and completely
prevented GRGDSP-induced increases in field potentials (40 min
post-CNQX infusion; mean ± S.D. fEPSP amplitude = 15 ± 1.8% versus 60 min post-GRGDSP + CNQX; mean = 13 ± 0.3%; p = 0.008, paired t test,
two-tailed). Moreover, the remaining synaptic response, presumably
mediated by the NMDA receptor and insensitive to CNQX infusions (39),
was completely eliminated during the application of 50 µM
AP5 (30 min post-AP5 infusion = 2.8 ± 1.5%). This result,
combined with those obtained with the GABA-A receptor antagonists and
paired pulse facilitation, point to a change in AMPA receptors as the
simplest explanation for the GRGDSP-induced increase in fEPSP slope and
amplitude.

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Fig. 5.
The AMPA receptor antagonist CNQX prevents
GRGDSP-induced increases in synaptic responses. After recording
stable baseline responses, 10 µM CNQX infused into the
bath caused a marked reduction in the synaptic response amplitude that
reached a plateau by 40 min postinfusion (group means plotted for
n = 4 experiments). Infusion of 0.5 mM
GRGDSP in the presence of CNQX did not significantly change the
remaining field potentials through a 60-min treatment period. The
fEPSPs remaining in the presence of CNQX were completely blocked by 50 µM AP5. The inset shows representative field
traces recorded (from left to right) (i) during
initial baseline recordings, (ii) 40 min after the start of CNQX
infusion, (iii) 60 min after application of GRGDSP + CNQX, and (iv) 30 min after infusion of AP5 in the presence of CNQX.
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Biochemical Pathways Responsible for Increases in Field
EPSPs
Pharmacological Results--
As noted, integrins stimulate a
number of postsynaptic signaling cascades including those driven by the
Src family kinases (40, 41). The Src inhibitor PP2 was used to test
whether the latter pathways contributed to GRGDSP-induced increases in
Schaffer collateral fEPSPs. PP2 (10 µM, n = 6) applied alone had no detectable effect on fEPSPs (percentage
change in slope after a 60-min PP2 infusion =
5 ± 5.9%,
p > 0.1) but completely blocked the effect of GRGDSP
(n = 6) on fEPSP slope (percentage change after 60 min = 1 ± 4.9%; p > 0.2) (Fig.
6, A and B).
Because Src tyrosine kinases phosphorylate the NMDA receptor NR2A and
NR2B subunits (42-44), the findings with PP2 raise the possibility
that the GRGDSP-induced increase in fEPSPs is secondary to changes in
NMDA receptors. Tests of this were carried using AP5, a competitive
antagonist of NMDA receptors. As presented in Fig.
7A, 50 µM AP5
(n = 9) substantially reduced the effect of GRGDSP on
synaptic responses: the integrin ligand by itself caused an 18 ± 6.6% increase in the slope after 1 hour of infusion but only a 4 ± 11.6% increase in slices pretreated with AP5. Whereas this
difference was statistically robust (t = 7.6;
p < 0.0001, between groups t test,
two-tailed), AP5 did not completely block the GRGDSP-induced increase
in facilitation in three of the nine slices tested.

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Fig. 6.
Integrin ligand binding effects on synaptic
transmission involve Src-tyrosine kinase signaling. A,
graph shows the mean ± S.E. fEPSP slope, plotted as percentage
change from baseline, for six experiments in which slices were treated
with 10 µM PP2 alone. Inset, traces were taken
at the time points indicated by the lowercase
letters. As shown, PP2 had no significant effect on field
responses over the 60-min treatment interval. B, in a
separate set of experiments (n = 6), hippocampal slices
were treated with 10 µM PP2 for 30 min and then with PP2
plus 0.5 mM GRGDSP. Representative field responses
(inset) were taken at the time points indicated by
lowercase letters. As shown, PP2 completely
blocked GRGDSP effects on fEPSP slope and amplitude. Trace calibration
for both panels was as follows: 5 ms, 0.5 mV.
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Fig. 7.
The NMDA receptor antagonist AP5 markedly
attenuates GRGDSP-induced increases in field EPSPs. A,
plot of Schaffer collateral fEPSP slopes (mean ± S.E.) in
hippocampal slices treated with 0.5 M GRGDSP for 1 h
(filled circles; n = 9; group
mean values ± S.E. shown) or with the NMDA receptor antagonist,
AP5 (50 µM) for 20 min followed by AP5 plus 0.5 mM GRGDSP for 1 h (open circles;
n = 9). As shown, AP5 significantly reduced the effects
of GRGDSP on fEPSP slope. B, plot of fEPSP slopes (mean ± S.E.) in hippocampal slices (n = 3) treated with 0.5 mM GRGDSP alone for 1 h followed by AP5 alone for 30 min. As shown, the size of field responses did not decline with the
addition of 50 µM AP5. Theta Burst patterned stimulation
(TBS; 10 bursts at 5 Hz, each composed of four pulses at 100 Hz) was delivered immediately after AP5 infusion to verify that the AP5
dose used was sufficient to block NMDA receptor function and,
consequently, the induction of LTP.
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The above result is as predicted from the hypothesis that activation of
NMDA receptors is needed to induce integrin-related increases in AMPA
receptor-mediated synaptic currents. However, while unlikely given
their voltage dependence, it is also possible that NMDA receptors
contribute directly to the enhanced responses. If so, then infusion of
AP5 should at least partially reverse the effects of GRGDSP. As shown
in Fig. 7B (n = 3), this prediction could
not be confirmed; AP5 applied at concentrations sufficient to block
NMDA receptor-driven operations, as evidenced by the total blockade of
LTP normally induced by high frequency bursts of afferent stimulation,
did not reverse the effects of GRGDSP on fEPSPs. It is known from
several previous studies that GRGDSP does not reduce induction or
expression of consolidated potentiation (27, 45, 46).
The results with AP5 emphasize the point that changes in AMPA receptors
are likely to be the major cause of GRGDSP-induced increases in field
responses. Phosphorylation of AMPA receptors by the
calcium/calmodulin-dependent protein kinase II (CaMKII) is
one route whereby their functioning could be enhanced (47-49). This
was tested by infusing the CaMKII inhibitor KN93 (5 µM)
for 30 min prior to and during the 1-h infusion of 0.5 mM
GRGDSP (n = 6). Fig.
8A shows that KN-93 had no
detectable effect on baseline responses but completely blocked the
increases in fEPSP slope normally produced by the integrin ligand
(change from baseline of
3 ± 4.2%, p > 0.20).
In contrast, Fig. 8B shows that the structural analogue
KN-92 (5 µM, n = 4), which lacks CaMKII
antagonist activity, had no effect on GRGDSP-induced increases in fEPSP
slope (16 ± 5.0%; t = 5.9, p = 0.002, paired t test, two-tailed).

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Fig. 8.
The CaMKII inhibitor, KN-93, blocks
GRGDSP-induced increases in synaptic responses. A, plot
of fEPSP slopes (mean ± S.E.) in hippocampal slices pretreated
with the CaMKII inhibitor peptide, KN-93 (5 µM), for 30 min followed by the infusion of KN-93 plus 0.5 mM GRGDSP
(n = 6). Traces (inset) show field responses
taken at time points indicated by lowercase
letters. As shown, KN-93 completely blocked GRGDSP-induced
enhancement of fEPSPs. B, plot of the mean ± S.E.
fEPSP slope in a separate set of experiments (n = 4) in
which slices were pretreated with 5 µM KN-92 (control
compound) for 30 min followed by treatment with KN-92 plus 0.5 mM GRGDSP. Representative field responses
(inset) are taken at time points indicated by
lowercase letters. At the end of the 1-h
recording period, GRGDSP induced a significant increase in fEPSP slope
as compared with baseline responses. Calibration for both
panels was as follows: 5 ms, 0.5 mV.
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In order to rule out the possibility that GRGDSP is producing
nonspecific effects on kinases that modulate AMPA receptor function, the effects of the protein kinase A inhibitor H-89 on GRGDSP-induced increases in fEPSPs were examined. Hippocampal slices were pretreated with H-89 (5 µM) for 30 min before the application of 0.5 mM GRGDSP (Fig. 9).
Application of H-89 alone had no significant effect on baseline
responses (mean ± S.D. fEPSP slope during baseline = 1 ± 4.7% versus 30 min post H-89 infusion = 0 ± 5.1%; p = 0.6, paired t test, two-tailed),
and H-89 applied prior to and during GRGDSP infusion did not prevent or
attenuate GRGDSP-induced increases in synaptic responses (60 min
post-H-89 + GRGDSP infusion = 16 ± 3.5%). These results
demonstrate that protein kinase A does not make a significant
contribution to integrin ligand effects on AMPA receptor function.

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Fig. 9.
GRGDSP-induced increases in field potentials
do not involve protein kinase A (PKA). The
graph shows the mean ± S.E. fEPSP slope plotted as a
percentage change from baseline in four hippocampal slices pretreated
with the protein kinase A inhibitor H-89 (5 µM) for 30 min before co-application of 0.5 mM GRGDSP plus H-89. As
shown, H-89 did not prevent GRGDSP-induced increases in field
responses.
|
|
In situ hybridization analyses have shown that integrin
receptor expression is regionally differentiated in the hippocampus and
neocortex (21). Together with immunocytochemical findings, these
results indicate that eight different RGD-binding integrins are
expressed by the hippocampal CA1 pyramidal cells including
1
1,
3
1,
5
1,
8
1,
v
1,
v
3,
v
5, and
v
8
(50). To verify that GRGDSP-mediated increases in field potentials are indeed integrin-mediated and to gain information on the specific integrin receptors involved, hippocampal slices were treated with a
mixture of function-blocking antibodies to integrin
subunits 3, 5, and v in the presence of GRGDSP. As illustrated in Fig. 10A, control slices
pretreated with local application of aCSF followed by co-application of
aCSF and 0.5 mM GRGDSP exhibited a significant increase in
synaptic responses relative to baseline by 60 min postinfusion
(mean ± S.D. fEPSP slope during baseline = 0 ± 3.3% versus 60 min post-GRGDSP + aCSF infusion = 16 ± 5.8%; p = 0.03, paired t test, two-tailed).
In contrast, in a separate group of slices pretreatment with the
anti-integrin mixture completely blocked the increase in field
potentials produced by GRGDSP (mean ± S.D. fEPSP slope during
baseline = 0 ± 1.2% versus antibodies + GRGDSP = 3 ± 5.0%; p = 0.36, paired
t test, two-tailed) (Fig. 10B). A significant
difference in mean fEPSP slope between the aCSF and antibody-treated
slices was observed 60 min post-GRGDSP infusion (p = 0.008, between groups t test, two-tailed).

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Fig. 10.
Local applications of an anti-integrin
mixture prevents GRGDSP-induced increases in synaptic responses.
A, an infusion pipette containing either a mixture of
function blocking antibodies to integrin subunits 3,
5, and v, or aCSF (control) was situated
within the bath immediately upstream from the slice. Local application
of pipette contents began 20 min before the introduction of 0.5 mM GRGDSP into the bath. As shown, the increase in synaptic
responses induced by GRGDSP was prevented in slices treated with
integrin function-blocking antibodies. B, representative
field responses collected from experimental and control
(i.e. aCSF-infused) slices during the baseline recording
period (a), 20 min after the start of pipette infusion
(b), and 60 min after the co-application of GRGDSP and
pipette contents (c).
|
|
Biochemical Results--
Pharmacological results presented above
are consistent with the hypothesis that RGD-induced increases in fEPSPs
are dependent upon activation of CaMKII followed by phosphorylation of
sites on the AMPA receptor known to regulate receptor kinetics (51, 52). Western blot analyses with phosphospecific antibodies were used to
test essential predictions of this argument. Slices were exposed to 0.5 mM GRGDSP (experimental) or aCSF (control) for 1 h;
electrophysiological recordings were collected from one of the
GRGDSP-treated slices to verify that increases in fEPSPs did occur as
described above. Fig. 11 shows Western
blots of whole homogenates probed with phosphospecific antibodies to
the CaMKII autophosphorylation site (Thr286). As shown in
the upper blots (both A and B), levels of the
phosphorylated kinase were far greater in GRGDSP-treated as compared
with control slices: in each of nine paired-slice experiments,
immunoreactivity was substantially greater in the RGD-treated slice
(1.3-16-fold; mean increase = 5.5-fold). Samples from seven of
these experiments were probed for levels of GluR1 phosphorylated at the
Ser831 site recognized by CaMKII (51, 52). Phospho-GluR1
was also markedly increased by GRGDSP treatment in each experiment
(2-18-fold; mean increase = 6.4-fold) (Fig. 11, A and
B, bottom panels).

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Fig. 11.
Integrin ligands induce an increase in
CaMKII Thr286 and GluR1 Ser831
phosphorylation. A and B, Western blots of
whole homogenates showing levels of phospho-CaMKII and phospho-GluR1 in
acute hippocampal slices treated with a 1-h infusion of aCSF
(con), 0.5 mM GRGDSP (RGD), and
GRGDSP + 10 µM PP2 (A) and aCSF
(con), GRGDSP (RGD), and GRGDSP + 50 µM AP5 (30 µg of protein/lane) (B).
|
|
Experiments described above demonstrated that GRGDSP-induced increases
in fEPSPs were blocked by antagonists of the NMDA receptor (i.e. AP5) and Src tyrosine kinase (i.e. PP2).
The effects of AP5 and PP2 on phospho-CaMKII Thr286 and
phospho-GluR1 Ser831 were tested to determine whether the
concomitant increases in phosphorylation of CaMKII and GluR1 involve
the same signaling pathways. As shown in Fig. 11, AP5 markedly
attenuated GRGDSP-induced increases in both CaMKII Thr286
and GluR1 Ser831 phosphorylation, whereas 10 µM PP2 did not affect either measure (three experiments
for AP5; four for PP2).
 |
DISCUSSION |
The above results provide the first evidence that integrins can
influence the size of fast, excitatory synaptic responses in the mature
brain. Small peptides with the matrix sequence recognized by the types
of integrins found in the hippocampus (i.e. the RGD-binding integrins) (1, 2) increased the size of fEPSPs over concentration ranges at which they are known to be effective ligands (36, 53).
Moreover, a pool of function-blocking antibodies against the
3,
5, and
v integrin
subunits blocked the RGD-induced increases in fEPSPs. The enhanced
responses were not accompanied by changes in paired pulse facilitation
and accordingly cannot be ascribed to increase probability of glutamate
release. NMDA receptors cannot be the agents for expressing the
increased response size, because, as evidenced by studies with
antagonists, the NMDA receptors do not contribute significantly to the
slope and amplitude of the augmented fEPSPs, and the latter responses
were blocked by the AMPA receptor antagonist CNQX. Reductions in fast
inhibitory postsynaptic potentials would not be expected to affect the
initial components of the response, and, in any event, the GABA-A
receptor antagonist picrotoxin did not reduce the effects of the
integrin ligands. By exclusion, then, it can be concluded that the
expression mechanism for the observed increase in synaptic responses
involves changes in AMPA-type glutamate receptors.
The sequence of events leading to the AMPA response facilitation by
GRGDSP involves both NMDA receptors and tyrosine kinases sensitive to
the inhibitor PP2. Regarding the former, the NMDA receptor antagonist
AP5 caused a marked, although not complete, reduction in the integrin
ligand-induced changes in CA1 field potentials; it appears then that
NMDA receptors are needed to induce the integrin-related increases but
not to express them. Among the PP2-sensitive kinases potentially
involved in the above effects, Src is particularly attractive, because
it is known to be activated by integrins via integrin-related tyrosine
kinases (e.g. focal adhesion kinase and its homologue PYK2)
and to phosphorylate NMDA receptors (42-44). Moreover, Src
phosphorylation results in an increase in the NMDA receptor-mediated
currents (54) needed for the ligand-induced increases in responses.
However, as discussed below, there are reasons to assume that the
PP2-sensitive kinase exerts its integrin-driven effects on synaptic
physiology through links other than the NMDA receptor.
In other circumstances (e.g. LTP), NMDA receptors trigger
changes in AMPA receptor-mediated responses by transiently increasing postsynaptic calcium levels (55). CaMKII is present in high concentrations in the postsynaptic density (56, 57) and thus is well
positioned to respond to such effects. Moreover, AMPA receptors are
substrates for CaMKII, and there is evidence that the pertinent
phosphorylation alters synaptic currents (51). The idea that CaMKII is
involved in the RGD-induced increases in synaptic responses was
supported by experiments showing that antagonists of the enzyme
completely block the increases. Moreover, the integrin ligand caused a
marked and persistent increase in phosphorylation of CaMKII at its
autophosphorylation (activation) site and parallel increases in
phosphorylation of GluR1 at Ser831, its CaMKII site. As
with GRGDSP effects on fEPSPs, both phosphorylation events were
AP5-sensitive.
Taken together, the above results suggest a hypothesis in which 1)
integrin binding influences local Src activity, 2) Src facilitates NMDA
receptor functioning, 3) calcium passing through the NMDA receptor
activates CaMKII, and 4) phosphorylation of AMPA receptors by CaMKII
results in larger synaptic currents. This scenario fits with the
observation that the integrin ligand elicits a slow increase in fEPSP
measures that continues to build for about 25-40 min after its removal
from the tissue bath. This slow buildup and overshoot also accords with
the slow increase in CaMKII and AMPA receptor phosphorylation reported
to occur after high frequency afferent stimulation (51). However,
differences in the effects of antagonists on GRGDSP-induced
phosphorylation events and fEPSP enhancement require some modification
to the hypothesis. In other words, the Src antagonist PP2 blocked the effects of GRGDSP on fEPSP slope but not on CaMKII or GluR1
phosphorylation. This suggests that two pathways beginning with
integrins and ending with AMPA receptors are needed to enhance synaptic
responses: 1) integrin
NMDA
CaMKII
GluR1 Ser831
phosphorylation and 2) integrin
PP2-sensitive kinase (Src?)
AMPA receptor. Both signaling pathways appear to be necessary, but
neither is sufficient for integrin-mediated increases in fEPSP responses. Several variants to the scheme illustrated in Fig. 12 are plausible, but of particular
interest is the possibility that the contribution of the NMDA receptor
is constitutive rather than induced.

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Fig. 12.
Schematic illustration of signaling pathways
likely to underlie integrin receptor mediated increases in synaptic
responses. As described, synaptic responses evaluated here largely
reflect AMPA receptor currents. The present results show that integrin
ligation leads to increases in synaptic responses that depend on
signaling through Src kinase and the NMDA receptor (and are blocked by
PP2 and AP5, respectively). However, evidence that ligand-induced
increases in CaMKII and GluR1 Ser831 phosphorylation are
blocked by antagonism of the NMDA receptor but not Src kinase indicates
that at least two separate pathways are involved: (i) an
Src-independent pathway through which integrin ligation leads
sequentially to increased NMDA receptor currents, activation of CaMKII,
and then phosphorylation of GluR1 Ser831, leading to
increased AMPA receptor function, and (ii) a second pathway through
which integrin binding leads to activation of a Src family kinase,
which then, directly or indirectly, modulates activity of the
AMPA receptor. Results in the literature suggest additional lines of
communication that could contribute to these effects, including the
facilitation of AMPA receptor membrane insertion by activation of
CaMKII (65), integrin activation of Src kinase that in turn
phosphorylates the NMDA receptor and up-regulates NMDA
receptor-mediated calcium influx (54), and integrin-mediated increases
in phospholipase C activity (80) that could further augment activation
of the CaMKII path.
|
|
How phosphorylation of AMPA receptors results in enhanced responses
remains an open question. An LTP-like effect seems unlikely, because
induction and expression of potentiation are not occluded by GRGDSP
(28, 29), and PP2, while blocking RGDSP-induced increases in fEPSPs,
does not prevent LTP (58). Perhaps the simplest alternative would be an
increase in the numbers of receptors in the postsynaptic membrane (see
Fig. 12). A number of investigators have proposed that rapid insertion
and recycling of AMPA receptors occurs under a variety of circumstances
at synapses (59-63), even within the mature hippocampus (64). Possibly
relevant to this idea, CaMKII phosphorylation of AMPA receptors has
been implicated in AMPA receptor insertion (60, 65). It is also
pertinent that integrin signaling increases surface expression of
co-distributed trophic factor receptors in a Src
kinase-dependent fashion (14), perhaps by stabilizing
intracellular associations that prevent internalization. Thus, it is
not unreasonable to propose that changes in integrin functioning affect
the number of receptors available for transmitter binding. In any
event, the paradigm described here seems particularly appropriate for
investigating the broad question of how AMPA receptor phosphorylation
affects synaptic physiology.
Finally, there are the questions of whether and to what degree
integrins regulate the size of synaptic responses in situ. To address these issues, it is necessary to first ask how dynamic the
receptors are in fully mature synapses. Integrins are activated and
inactivated in a matter of minutes in other tissues (66-68), and
integrin activation can be mediated by agents known to be regulated by
neuronal activity (e.g. protein kinase C, CaMKII) (67) (for
review, see Refs. 68 and 69). In addition, in the brain, neuronal
activity increases extracellular proteases (70-73) that could modulate
integrin signaling through the generation or exposure of new ligands
from limited degradation of matrix (74-76) and/or through a turnover
of the adhesion receptors themselves (77-79). If effects of this kind
occur on a routine basis in the brain, then a time-varying integrin
influence over transmission is plausible. An intriguing possibility
related to this is that a loop exists between the transmission and
adhesive functions of the synapse, such that afferent activity affects
levels of integrin binding, which in turn adjust the strength of
postsynaptic currents.
 |
FOOTNOTES |
*
This work was supported by NIMH, National Institutes of
Health (NIH), Grant MH61007 (to G. L.) and NINDS, NIH, Grant NS37799 (to C. M. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: 101 Theory Dr., Suite
250, University Research Park, Irvine, CA 92612-1695. Tel.: 949-824-7001; Fax: 949-824-3559; E-mail: ekramar@uci.edu.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M210225200
 |
ABBREVIATIONS |
The abbreviations used are:
LTP, long term
potentiation;
AMPA,
-amino-3-hydroxy-5-methyl-4-isoxazole
propionate;
fEPSP, field excitatory postsynaptic potential;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
GABA,
-aminobutyric acid;
CaMKII, calcium calmodulin-dependent protein kinase
II;
AP5, D-(
)-aminophosphonopentanoic acid;
PP2, protein
phosphatase 2;
NMDA, N-methyl-D-aspartate;
aCSF, artificial cerebrospinal fluid.
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