Integrins Modulate Fast Excitatory Transmission at Hippocampal Synapses*

Enikö A. KramárDagger §, Joie A. Bernard, Christine M. Gall, and Gary LynchDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha 3, alpha 5, and alpha 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins are heterodimeric (alpha , beta ), 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 alpha 5 (27) or alpha 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 alpha 3, alpha 5, and alpha 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 alpha  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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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha 3, alpha 5, and alpha v integrin subunits (anti-alpha 3, MAB1952Z; anti-alpha 5, MAB1956; anti-alpha 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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).

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.

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.

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.

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.

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.

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.

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 alpha 1beta 1, alpha 3beta 1, alpha 5beta 1, alpha 8beta 1, alpha vbeta 1, alpha vbeta 3, alpha vbeta 5, and alpha vbeta 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 alpha  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 alpha 3, alpha 5, and alpha 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 3, alpha 5, and alpha 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 right-arrow NMDA right-arrow CaMKII right-arrow GluR1 Ser831 phosphorylation and 2) integrin right-arrow PP2-sensitive kinase (Src?) right-arrow 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, alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionate; fEPSP, field excitatory postsynaptic potential; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; GABA, gamma -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Plow, E. F., Haas, T. A., Zhang, L., Loftus, J., and Smith, J. W. (2000) J. Biol. Chem. 275, 21785-21788[Free Full Text]
2. Hynes, R. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
3. Geiger, B., Bershadsky, A., Pankov, R., and Yamada, K. M. (2002) Nat. Cell Biol. 793-805
4. Yamada, K., and Miyamoto, S. (1995) Curr. Opin. Cell Biol. 7, 681-689[CrossRef][Medline] [Order article via Infotrieve]
5. Howe, A., Aplin, A., Alahari, S., and Juliano, R. (1998) Curr. Opin. Cell Biol. 10, 220-231[CrossRef][Medline] [Order article via Infotrieve]
6. Schwartz, M., Schaller, M., and Ginsberg, M. (1995) Annu. Rev. Cell Dev. Biol. 11, 549-599[CrossRef][Medline] [Order article via Infotrieve]
7. Wu, X., Mogford, J. E., Platts, S. H., Davis, G. E., Meininger, G. A., and Davis, M. J. (1998) J. Cell Biol. 143, 241-252[Abstract/Free Full Text]
8. Wu, X., Davis, G. E., Meininger, G. A., Wilson, E., and Davis, M. J. (2001) J. Biol. Chem. 276, 30285-30292[Abstract/Free Full Text]
9. Platts, S. H., Mogford, J. E., Davis, M. J., and Meininger, G. A. (1998) Am. J. Physiol. 275, H1449-H1454[Abstract/Free Full Text]
10. Wildering, W. C., Herman, P. M., and Bulloch, A. G. M. (2002) J. Neurosci. 22, 2419-2426[Abstract/Free Full Text]
11. Porter, J. C., and Hogg, N. (1998) Trends Cell Biol. 8, 390-396[CrossRef][Medline] [Order article via Infotrieve]
12. Miyamoto, S., Teramoto, H., Gutkind, J. S., and Yamada, K. M. (1996) J. Cell Biol. 135, 1633-1642[Abstract]
13. Schneller, Vuori, K., and Ruoslahti, E. (1997) EMBO J. 16, 5600-5607[Abstract/Free Full Text]
14. Moro, L., Dolce, L., Cabodi, S., Bergato, E., Erba, E. B., Smeriglio, M., Turco, E., Retta, S. F., Giufridda, M. G., Venturino, M., Godovac-Zimmermann, J., Conti, A., Schaefer, E., Berguino, L., Tacchetti, C., Gaggini, P., Silengo, L., Tarone, G., and Defilippi, P. (2002) J. Biol. Chem. 277, 9405-9414[Abstract/Free Full Text]
15. Yamada, K. M., and Even-Ram, S. (2002) Nat. Cell Biol. 4, E75-E76[CrossRef][Medline] [Order article via Infotrieve]
16. Schuster, T., Krug, M., Stalder, M., Hackel, N., Gerardy-Schahn, R., and Schachner, M. (2001) J. Neurobiol. 49, 142-158[CrossRef][Medline] [Order article via Infotrieve]
17. Nishimura, S. L., Boylen, K. P., Einheber, S., Milner, T. A., Ramos, D. M., and Pytela, R. (1998) Brain Res. 791, 271-282[CrossRef][Medline] [Order article via Infotrieve]
18. Einheber, S., Schnapp, L., Salzer, J., Cappiello, Z., and Milner, T. A. (1996) J. Comp. Neurol. 370, 105-134[CrossRef][Medline] [Order article via Infotrieve]
19. Rodriguez, M. A., Pesold, C., Liu, W. S., Kriho, V., Guidotti, A., Pappas, G. D., and Costa, E. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3550-3555[Abstract/Free Full Text]
20. Bahr, B., and Lynch, G. (1992) Biochem. J. 281, 137-142[Medline] [Order article via Infotrieve]
21. Pinkstaff, J. K., Detterich, J., Lynch, G., and Gall, C. (1999) J. Neurosci. 19, 1541-1556[Abstract/Free Full Text]
22. Bi, X., Zhou, J., Lynch, G., and Gall, C. M. (2001) J. Comp. Neurol. 435, 184-193[CrossRef][Medline] [Order article via Infotrieve]
23. King, V. R., McBride, A., and Priestly, J. V. (2001) J. Neurocytol. 30, 243-252[CrossRef][Medline] [Order article via Infotrieve]
24. Murase, S.-I., and Hayashi, Y. (1998) J. Comp. Neurol. 395, 161-176[CrossRef][Medline] [Order article via Infotrieve]
25. Kramár, E. A., and Lynch, G. (2003) Neurocience, in press
26. Hernandez, R. M., Garza, J. M., Graves, M. E., Martinez, J. L. J., and LeBaron, R. G. (2001) Biol. Bull. 201, 236-237[Free Full Text]
27. Chun, D., Gall, C. M., Bi, X., and Lynch, G. (2001) Neuroscience 105, 815-829[CrossRef][Medline] [Order article via Infotrieve]
28. Bahr, B. A., Staubli, U., Xiao, P., Chun, D., Ji, Z.-X., Esteban, E. T., and Lynch, G. (1997) J. Neurosci. 17, 1320-1329[Abstract/Free Full Text]
29. Staubli, U., Chun, D., and Lynch, G. (1998) J. Neurosci. 18, 3460-3469[Abstract/Free Full Text]
30. Kramár, E. A., Bernard, J. A., Gall, C. M., and Lynch, G. (2002) Neuroscience 110, 29-39[CrossRef][Medline] [Order article via Infotrieve]
31. Chang, H. P., Lindberg, F. P., Wang, H. L., Huang, A. M., and Lee, E. H. Y. (1999) Learning Memory 6, 448-457[Abstract/Free Full Text]
32. Grooms, S., and Jones, L. (1997) Neurosci. Lett. 231, 139-142[CrossRef][Medline] [Order article via Infotrieve]
33. Rohrbough, J., Grotewiel, M. S., Davis, R. L., and Broadie, K. (2000) J. Neurosci. 20, 6868-6878[Abstract/Free Full Text]
34. Grotewiel, M. S., Beck, C. D., Wu, C. F., and Greenspan, R. J. (1998) Nature 18, 7847-7855
35. Connolly, J. B., and Tully, T. (1998) Curr. Biol. 8, R386-R389[Medline] [Order article via Infotrieve]
36. Pierschbacher, M., and Ruoslahti, E. (1984) Nature 304, 30-33
37. Alger, B. E., and Nicoll, R. A. (1982) J. Physiol. 328, 105-123[Medline] [Order article via Infotrieve]
38. Muller, D., and Lynch, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9346-9350[Abstract]
39. Muller, D., Joly, M., and Lynch, G. (1988) Science 242, 1694-1697[Medline] [Order article via Infotrieve]
40. Duong, L. T., and Rodan, G. A. (2000) Cell Motil. Cytoskeleton 47, 174-188[CrossRef][Medline] [Order article via Infotrieve]
41. Girault, J.-A., Costa, A., Dirkinderen, P., Studler, J.-M., and Toutant, M. (1999) Trends Neurosci. 22, 257-263[CrossRef][Medline] [Order article via Infotrieve]
42. Hisatsune, C., Umemori, H., Mishina, M., and Yamamoto, T. (1999) Genes Cells 4, 657-666[Abstract/Free Full Text]
43. Lu, W. Y., Xiong, Z. G., Lei, S., Orser, B. A., Dudek, E., Browning, M. D., and MacDonald, J. F. (1999) Nat. Neurosci. 2, 331-338[CrossRef][Medline] [Order article via Infotrieve]
44. Grosshans, D. R., Clayton, D. A., Coultrap, S. J., and Browning, M. D. (2001) Nat. Neurosci. 5, 27-33[CrossRef]
45. Staubli, U., Vanderklish, P. W., and Lynch, G. (1990) Behav. Neural Biol. 53, 1-5[Medline] [Order article via Infotrieve]
46. Xiao, P., Bahr, B., Staubli, U., Vanderklish, P., and Lynch, G. (1991) NeuroReport 2, 461-464[Medline] [Order article via Infotrieve]
47. Poncer, J. C., Esteban, J. A., and Malinow, R. (2002) J. Neurosci. 22, 4406-4411[Abstract/Free Full Text]
48. Liao, D., Scannevin, R. H., and R, H. (2001) J. Neurosci. 21, 6008-6017[Abstract/Free Full Text]
49. Nishizaki, T., and Matsumura, T. (2002) Mol. Brain Res. 98, 130-134[CrossRef][Medline] [Order article via Infotrieve]
50. Gall, C. M., and Lynch, G. (2003) in Recent Advances in Epilepsy Research: Molecular Mechanisms of Epileptogenesis (Scharfman, H. , and Binder, D., eds) , Landes Bioscience, Georgetown, TX, in press
51. Barria, A., Muller, D., Derkach, V., Griffith, L. C., and Soderling, T. R. (1997) Science 276, 2042-2045[Abstract/Free Full Text]
52. Mammen, A. L., Kameyama, K., Roche, K. W., and Huganir, R. L. (1997) J. Biol. Chem. 272, 32528-32533[Abstract/Free Full Text]
53. Ruoslahti, E., and Pierschbacher, M. D. (1986) Cell 44, 517-518[Medline] [Order article via Infotrieve]
54. Ali, D. W., and Salter, M. W. (2001) Curr. Opin. Neurobiol. 11, 336-342[CrossRef][Medline] [Order article via Infotrieve]
55. Lynch, G., Larson, J., Kelso, S., Barrionuevo, G., and Schottler, F. (1983) Nature 305, 719-721[Medline] [Order article via Infotrieve]
56. Kennedy, M. B. (2000) Science 290, 750-754[Abstract/Free Full Text]
57. Silva, A. J., Stevens, C. F., Tonegawa, S., and Wang, Y. (1992) Science 257, 201-206[Medline] [Order article via Infotrieve]
58. Bi, F., Broutman, G., Foy, M. R., Thompson, R. F., and Baudry, M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3606-3607
59. Liang, F., and Huganir, R. L. (2001) J. Neurochem. 77, 1626-1631[CrossRef][Medline] [Order article via Infotrieve]
60. Shi, S. H., Hayashi, Y., Esteban, J. A., and Malinow, R. (2001) Cell 105, 331-343[Medline] [Order article via Infotrieve]
61. Lüscher, C., Nicoll, R. A., Malenka, R. C., and Muller, D. (2000) Nat. Neurosci. 3, 545-550[CrossRef][Medline] [Order article via Infotrieve]
62. Shi, S.-H., Hayachi, Y., Petralia, R. S., Zaman, S. H., Wenthold, R. J., Svoboda, K., and Malinow, R. (1999) Science 284, 1811-1816[Abstract/Free Full Text]
63. Iwakura, Y., Nagano, T., Kawamura, M., Horikawa, H., Ibaraki, K., Takei, N., and Nawa, H. (2001) J. Biol. Chem. 276, 40025-40032[Abstract/Free Full Text]
64. Broutman, G., and Baudry, M. (2001) J. Neurosci. 21, 27-34[Abstract/Free Full Text]
65. Hayashi, Y., Shi, S.-H., Piccini, J. A., Poncer, J.-C., and Malinow, R. (2000) Science 287, 2262-2267[Abstract/Free Full Text]
66. Bouvard, D., Molla, A., and Block, M. R. (1998) J. Cell Sci. 111, 657-665[Abstract/Free Full Text]
67. Hughes, P. E., and Pfaff, M. (1998) Trends Cell Biol. 8, 359-364[CrossRef][Medline] [Order article via Infotrieve]
68. Kolanus, W., and Seed, B. (1997) Cur. Opin. Cell Biol. 9, 725-731[CrossRef][Medline] [Order article via Infotrieve]
69. Soderling, T. R., and Derkach, V. A. (2000) Trends Neurosci. 23, 75-80[CrossRef][Medline] [Order article via Infotrieve]
70. Gualandris, A., Jones, T. E., Strickland, S., and Tsirka, S. E. (1996) J. Neurosci. 16, 2220-2225[Abstract]
71. Wu, Y. P., Siao, C. J., Lu, W., Sung, T. C., Frohman, M. A., Milev, P., Bugge, T. H., Degen, J. L., Levine, J. M., Margolis, R. U., and Tsirka, S. E. (2000) J. Cell Biol. 148, 1295-1304[Abstract/Free Full Text]
72. Zhang, J. W., Deb, S., and Gottschall, P. E. (1998) Eur. J. Neurosci. 10, 3358-3368[CrossRef][Medline] [Order article via Infotrieve]
73. Momota, Y., Yoshida, S., Ito, J., Shibata, M., Kato, K., Sakurai, K., Matsumoto, K., and Shiosaka, S. (1998) Eur. J. Neurosci. 10, 760-764[CrossRef][Medline] [Order article via Infotrieve]
74. Davis, G. E., Bayless, K. J., Davis, M. J., and Meininger, G. A. (2000) Am. J. Pathol. 156, 1489-1498[Abstract/Free Full Text]
75. Fukai, F., Ohtaki, M., Fujii, N., Yajima, H., Ishii, T., Nishizawa, Y., Miyazaki, K., and Katayama, T. (1995) Biochemistry 34, 11453-11459[Medline] [Order article via Infotrieve]
76. Sage, E. (1997) Trends Cell Biol. 7, 182-186[CrossRef]
77. Hoffman, K. B., Martinez, J., and Lynch, G. (1998) Brain Res. 811, 29-33[CrossRef][Medline] [Order article via Infotrieve]
78. Hoffman, K. B., Larson, J., Bahr, B. A., and Lynch, G. (1998) Brain Res. 811, 152-155[CrossRef][Medline] [Order article via Infotrieve]
79. Endo, A., Nagai, N., Urano, T., Takada, Y., Hashimoto, K., and Takada, A. (1999) Neurosci. Res. 33, 1-8[CrossRef][Medline] [Order article via Infotrieve]
80. Zhang, X., Chattopadhyay, A., Ji, Q. S., Owen, J. D., Ruest, P. J., Carpenter, G., and Hanks, S. K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9021-9026[Abstract/Free Full Text]


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