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The Journal of Neuroscience, May 1, 1998, 18(9):3460-3469
Time-Dependent Reversal of Long-Term Potentiation by an
Integrin Antagonist
Ursula
Stäubli,
Daniel
Chun, and
Gary
Lynch2
Center for Neural Science, New York University, New York, New York
10003, and 2 Center for the Neurobiology of Learning and
Memory, University of California, Irvine, California 92697
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ABSTRACT |
The integrin antagonist Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) was
applied by local ejection to one of two recording sites in hippocampal slices at various times before and after long-term potentiation (LTP)
was induced at both sites with theta burst stimulation. Applications 10 min before, immediately after, and 10 min after induction caused LTP at
the experimental site to decay steadily relative to that at the
within-slice control site. However, application at 25 min or more after
induction had no detectable effect on potentiation. Similar results
were obtained when the integrin antagonist was perfused into the slice
rather than applied locally. The time period after induction during
which GRGDSP interfered with LTP consolidation corresponds to that
during which LTP is susceptible to reversal by low-frequency afferent
stimulation and newly formed memories are vulnerable to various
disruptive treatments. Comparable experiments using a peptide that
blocks an extracellular binding site of neural cell adhesion molecules (NCAMs) did not yield time-dependent reversal of LTP; i.e., an antagonist that interacts with the fourth immunoglobulin-like domain
reduced LTP when applied before induction but not afterward. Moreover,
LTP formation occurred normally in the presence of an antibody against
the fibronectin repeat domain of NCAM. These results suggest that
integrin activation and signaling occurring over several minutes after
LTP induction are necessary for stabilizing synaptic potentiation and
by inference may be required for the conversion of new memories into a
not readily disrupted state.
Key words:
LTP reversal; adhesion receptors; integrins; NCAMs; consolidation; memory; hippocampus; retrograde amnesia
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INTRODUCTION |
Long-term potentiation (LTP) is
vulnerable to disruption for several minutes after its induction but
then becomes resistant to even extreme treatments. For example,
episodes of hypoxia, too brief to more than transiently affect control
synaptic responses, eliminate LTP if applied within 1-2 min of
induction, but are without effect if administered 30 min later (Arai et
al., 1990 ). Reversal of LTP by afferent stimulation (Barrionuevo et
al., 1980; Stäubli and Lynch, 1990; Larson et al., 1993) is also
time dependent: low-frequency stimulation erases potentiation when
delivered within 1-2 min of theta burst stimulation (TBS) but has
progressively less influence as the time after induction approaches 30 min (Stäubli and Chun, 1996a,b). These and other results indicate
that although the neurochemical processes that consolidate LTP are set
in motion by synaptic events in the millisecond range, they require
many minutes to reach completion. In these features, the substrates of
potentiation resemble those for the encoding of memory (Duncan, 1949;
Riccio et al., 1968; Popik et al., 1994).
Neural cell adhesion molecules (NCAMs) and integrins, two classes of
cell surface receptors involved in the assembly of pericellular matrices (Akiyama et al., 1989; Wu et al., 1995a,b) and maintenance of
contact morphology (Horwitz et al., 1986; Burridge et al., 1988; Hynes,
1992) have been implicated in the formation of LTP (Stäubli et
al., 1990; Xiao et al., 1991; Lüthi et al., 1994; Rønn et al.,
1995; Bahr et al., 1997). Specifically, the processes triggered by the
initiation of LTP are believed to involve activation of adhesion
receptors that control configurational changes of the synaptic
architecture and rearrange the membrane environment. It has been
suggested that the two classes of adhesion receptors contribute to
separate phases of LTP formation, with NCAMs being involved in the
development of LTP (Lüthi et al., 1994; Rønn et al., 1995), a
stage initiated and completed within 30 sec of induction (Gustafsson et
al., 1989), and integrins contributing to later stabilization phases
(Xiao et al., 1991; Bahr et al., 1997). Integrins commonly exist in a
quiescent state, requiring an activation event, often triggered by
other types of transmembrane receptors, for their adhesive properties
to appear (Newton et al., 1997). The conversion from a latent to an
active state can involve several minutes (van Willigen et al., 1996;
Newton et al., 1997), and it is thus possible that the protracted
consolidation period for LTP, and by inference possibly memory,
reflects the time needed to mobilize integrin adhesion receptors.
The present studies tested predictions arising from the hypothesis that
integrin, but not NCAM, chemistries dictate the protracted time period
during which LTP is vulnerable to disruption. A technique was used in
which LTP could be induced simultaneously at two sites in the same
hippocampal slice, only one of which was exposed to adhesion receptor
antagonists or control compounds. The use of within-slice and same
time-frame comparisons reduced several sources of variance typically
present in LTP experiments and thus increased the likelihood of
accurately defining effects arising from the experimental
manipulations. The compounds tested included (1) the peptide
Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) (Pierschbacher and Ruoslahti, 1984),
which competes with the recognition site for a large subclass of
integrins, (2) the control peptide Gly-Arg-Ala-Asp-Ser-Pro (GRADSP),
(3) the peptide MS2, which binds to the fourth Ig-like region in the
extracellular domain of NCAMs, and (4) an antibody selective for the
fibronectin type III repeat region, an extracellular segment of NCAMs
closer to the membrane than the Ig-like domains. The specific question
examined was whether the integrin antagonist, alone of the test
compounds, would reverse already established LTP and do so with
decreasing efficiency during the period beginning immediately after
induction and ending 30 min later; i.e., during the same period that
reversal by afferent activity becomes progressively less effective.
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MATERIALS AND METHODS |
Rat hippocampal slices were prepared from 2- to 3-month-old
Sprague Dawley rats and maintained in an interface chamber using standard conditions, as described in earlier work (Stäubli and Chun, 1996a,b). The rats were decapitated, and their brains were removed rapidly and placed in 0°C oxygenated (95%
O2/5% CO2) artificial CSF (aCSF)
of the following composition (in mM): NaCl 124, KCl 3, KH2PO4 1.25, MgSO4 2.5, CaCl2 3.4, NaHCO3 36, D-glucose 10, and L-ascorbate 2. The hippocampi were quickly dissected
free in ice-cold aCSF, placed on a McIlwain tissue chopper, cut into 400 µm sections, and collected in a petri dish containing ice-cold aCSF. The slices were then immediately placed on a nylon net in an
interface chamber and maintained at a temperature of 31 ± 1°C. They were perfused continuously with preheated aCSF at a rate of 75 ml/hr while their upper surface was exposed to warm humidified 95%
O2/5% CO2.
Recording and stimulating began after an incubation time of at least 1 hr. Experiments using local drug application via pressure ejection
involved two extracellular recording sites (glass micropipettes filled
with 2 mM NaCl) in the apical dendrites of fields
CA1a and CA1c, with one of the two sites randomly selected
for drug application and the other serving as control. Stimulation
pulses were delivered to the Schaffer-commissural axons passing
through stratum radiatum using a bipolar stimulating electrode (twisted nichrome wires, 65 µm) centered between the two recording electrodes, approximately 500 µm apart from each. The stimulus strength was adjusted to produce two field EPSPs with amplitudes that were ~60%
of the maximum spike-free response. After stable recording for at least
20 min, application of the various compounds to be tested for their
effect on LTP began.
The peptide GRGDSP (Calbiochem, San Diego, CA), which blocks integrin
binding to a diverse collection of ligands by competing with the
Arg-Gly-Asp (RGD) consensus binding sequence, was diluted to 0.5 mM with aCSF and applied locally by pressure ejection
(Picospritzer; General Valve, Fairfield, NJ) from a glass micropipette
placed next to (within 150 µm), and at the same depth as (~50-100
µm), the test recording electrode. Pipette ejection pressure was set at 8-12 psi (pulse duration 10 msec) to supply ~3 nl of peptide every 5 sec throughout the experiment, starting at various time points
before or after attempts to induce LTP and continuing throughout the
experiment. A higher concentration of GRGDSP (2 mM) was tested in an additional set of experiments,
with peptide ejection beginning 20 min before LTP induction. Control
experiments involved local application of 0.5 mM GRADSP
(Calbiochem), a peptide lacking the RGD sequence, with drug application
starting 20 min before LTP induction.
The same arrangements were used to compare the effects on physiology of
two compounds that interact with the extracellular domain of neural
cell adhesion molecules (NCAMs), the second class of adhesion
receptors. Specifically, the protein fragment MS2 (2 mg/ml), which
contains the first 19 amino acids of the fourth Ig-like domain of NCAM,
i.e.,
Ac-Glu-Ala-Ser-Gly-Asp-Pro-Ile-Pro-Ser-Ile-Thr-Trp-Arg-Thr-Ser-Thr-Arg-Asn-Ile-NH2 (Horstkorte et al., 1993), and was synthesized by Dr. C. Glabe (University of California at Irvine), as well as an antibody against the fibronectin type III repeat domain of NCAM (4 mg/ml), were used
[generous gift of Drs. E. Bock and M. Olsen (University of Copenhagen)].
LTP was induced by delivering TBS, consisting of ten theta bursts
containing four pulses at 100 Hz each, separated by 200 msec. The
stimulation intensity was not increased during TBS. Within-slice
comparisons between potentiation at the site receiving the injection of
the antagonist ("test response") versus potentiation at the site
that did not ("control response"), were used to test for blockade
of LTP.
In experiments involving bath perfusion rather than local application
of the integrin antagonist, GRGDSP was added directly to the chamber
via perfusion pump, at a final concentration of 0.5 mM.
Drug infusion started at various time points before and after attempts
to induce LTP and lasted 60 min. Care was taken to keep the total rate
of perfusion constant throughout the experiment (25 ml/hr) by adjusting
the flow rate of the primary source of aCSF accordingly. One recording
electrode in stratum radiatum of field CA1b and two stimulating
electrodes (test and control), placed in equidistant positions in
fields CA1a and CA1c to stimulate nonoverlapping Schaffer
collateral/commissural projections, were used in the perfusion
studies.
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RESULTS |
Local pressure ejection of the aCSF carrier vehicle used in these
experiments had no detectable effect on slice physiology or
potentiation. In accord with previous work involving bath perfusion (Stäubli et al., 1990; Xiao et al., 1991), GRGDSP did not alter the shape or size of baseline synaptic responses. Figure
1 summarizes experiments in which local
ejection of the peptide was initiated 10 min before
(A), immediately after (B), 10 min
after (C), 25 min after (D), or 45 min after the induction of LTP (E). As shown, when
the infusion was started 10 min before TBS it resulted in an LTP that,
in marked contrast to the potentiation elicited by the same stimulation
at the control site, decayed steadily throughout the 60 min recording
period after induction. Within-slice comparisons showed that the
difference between control and experimental recording sites for the
last 10 min were statistically significant (T(5) = 13.22; p < 0.001; two-tailed paired t
test). The initial potentiation ("short-term potentiation")
generated by TBS was left intact by GRGDSP (T(5) = 1.89 and 3.53; not significant, for comparisons of control versus
test LTP during the first 5 and 10 min after induction). This finding
is in agreement with earlier results (Bahr et al., 1997) and confirms
that integrin antagonists interfere with neither the physiological
events that induce LTP nor the development phase that occurs within the
first 30 sec of LTP (Gustafsson et al., 1989). Infusions begun
immediately after TBS were equally effective at destabilizing
potentiation (T(3) = 7.46; p < 0.01, for comparison of the last 10 min of the LTP recording period). The infusion at 10 min after TBS, although it had no obvious immediate effect on the potentiated responses, also blocked stabilization to a
significant degree (T(4) = 3.17;
p < 0.05, for the last 10 min). Infusions at 25 and 45 min after TBS (Figs. 1D,E) caused no change in
potentiated response compared with LTP at the within-slice control
sites; i.e., responses recorded 50-60 min (25 min group) and 80-90
min (45 min group) after TBS exhibited virtually the same degree of
potentiation at the GRGDSP and control sites [159.7 ± 9.4% vs
165.4 ± 7.1% (n = 6) for the 25 min group, and
165.2 ± 3.2% vs 166 ± 16.3% (n = 4) for
the 45 min group].
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Figure 1.
Time-dependent reversal of LTP by integrin
antagonist GRGDSP. A-E, Experiments in which LTP in
area CA1 was simultaneously monitored at a test ( ) and control site
( ) within the same slice. Local ejection of the integrin antagonist
GRGDSP (0.5 mM) at the test site was initiated at various
times before and after LTP induction, i.e., 10 min before
(A) (n = 6), immediately
after (B) (n = 4), 10 min
after (C) (n = 5), 25 min
after (D) (n = 6), and 45 min
after TBS (E) (n = 4). Each
data point represents the group mean of one response per animal
(±SEM). A', Superimposed representative responses from
an individual experiment. The potentials were recorded from the control
and within-slice test site at the times indicated by the numbers in
A, i.e., 10-15 min before as well as 5 min and 45 min
after TBS, with the dotted waveform representing the
response recorded at 45 min. C', E', Same as in
A', except that the responses are taken from experiments
included in the groups summarized in C and
E. Calibration: 1 mV, 10 msec.
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Figure 2A combines
within-slice comparisons for all groups of slices and infusion periods.
The percentage potentiation of the experimental response is expressed
as a fraction of that in the paired (same slice) control response.
ANOVA using paired differences at 35-45 min after application of the
inhibitor, or at 35-45 min after TBS for the  10 min group, indicated
that a time-dependent drug effect was present (F = 5.55; p < 0.01). As shown, the magnitude of LTP at
sites exposed to the antagonist before ( ) or immediately after ()
TBS was reduced to 50% of that in control synapses by the end of
testing. Lesser but still substantial impairments were obtained with
infusions begun at 10 min after induction ( ); in contrast, LTP at
sites treated with the antagonist at or beyond the 25 min time point
( ) was not detectably different from the potentiation at the control
sites. The within-slice comparisons for this last group were
statistically different from the within-slice comparisons for the 10 min before TBS group (p < 0.01, Newman-Keuls), the immediate group (p < 0.05), and the 10 min
after TBS group (p < 0.05).
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Figure 2.
GRGDSP, but not the control peptide GRADSP,
interferes with LTP stabilization. A, Same experiments
as those illustrated in Figure 1, except that the data (mean ± SEM) for all groups are united in one graph and expressed as the ratio
of the percent LTP at the test site divided by the percent LTP recorded
simultaneously at the within-slice control site (: start of infusion
10 min before TBS, n = 6; : immediately after
TBS, n = 4; : 10 min after TBS,
n = 5; : >25 min after TBS,
n = 10). B, GRGDSP was applied at a
higher concentration (2 mM) and earlier, i.e., 20 min
before LTP induction at both test and control site in a group of seven
slices. C, Control experiments examining the effect of
the non-RGD-containing peptide GRADSP (0.5 mM) in a group
of seven slices, using the same experimental protocol as in
B.
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The 0.5 mM concentrations used in the above experiments are
sufficient to block integrins (Cardwell and Rome, 1988; Haskel and
Abendschein, 1989; Bahr and Lynch, 1992), but additional experiments (n = 7) were conducted to determine whether higher
concentrations would result in a more rapid decrease in LTP. As shown
in Figure 2B, application of the peptide at 2.0 mM beginning at 20 min before LTP induction resulted in a
continuous and marked decay of potentiation at the test site
(T(6) = 6.32; p < 0.001, for
comparisons of control versus test LTP during the last 10 min). The
average within-slice difference in potentiation between test and
control sites during the last 10 min of recording was not obviously
different for 0.5 mM versus 2 mM, i.e.,
41.4 ± 6.5% for 0.5 mM versus 36.5 ± 6.8% for
2 mM. That GRGDSP, even when administered at 2 mM, did not influence the initial potentiation
(T(6) = 1.13 and 1.85; not significant, for the
first 5 and 10 min after induction), indicates that early LTP events
are mediated by a mechanism other than integrins.
Figure 2C shows the results from experiments using GRADSP, a
non-RGD-containing control peptide that was pressure-ejected at a
concentration of 0.5 mM. This compound gave no evidence of interfering with LTP induction, development, or stabilization (T(6) = 1.30; not significant, for comparisons
of control versus test LTP during the last 10 min of recording after
induction).
Results similar to those collected with local ejection of GRGDSP were
obtained by bath application. As illustrated in Figure 3A, adding GRGDSP before TBS
resulted in LTP that decayed steadily throughout the subsequent
recording period. Infusions beginning immediately or 15 min after
induction (Figs. 3B,C) also interfered with LTP
stabilization, whereas those begun at or after 30 min had no detectable
influence on potentiation (Fig. 3D). ANOVA of the degree of
LTP in place 60-70 min after the start of the peptide infusion
revealed a significant effect of perfusion onset time (F = 7.46; p < 0.001). Post
hoc comparisons indicated that LTP was greater in the long delay
(30/45 min) group (155 ± 6%) than in the 10 min before TBS
(118 ± 5%; p < 0.01, Neuman-Keuls), the 0 min
(126 ± 5%; p < 0.05), or the 15 min after TBS
groups (128 ± 9%; p < 0.01), despite being
assessed at a greater interval after induction. An additional ANOVA
comparing the degree of potentiation measured during the last 10 min of
recording in each time group confirmed the presence of a significant
effect of time (F = 6.6; p < 0.01).
Specific comparisons indicated that the degree of LTP was significantly
larger in the 30/45 min group than in the 10 min before TBS
(p < 0.01; Neuman-Keuls), the 0 min
(p < 0.05), and the 15 min after TBS groups
(p < 0.05).
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Figure 3.
Whole-slice perfusion of integrin antagonist
GRGDSP causes time-dependent reversal of LTP. A-D, Bath
perfusion of the peptide (0.5 mM) was initiated at
different times (horizontal bar) before and after LTP
induction: A, 10 min before TBS (n = 5); B, immediately after TBS (n = 4); C, 10 min after TBS (n = 6); and
D, 30 min (n = 5) and 45 min
(n = 4) after TBS (data pooled for both time
points). Each circle represents the group mean of one
response per animal (±SEM).
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Figures 4 and
5 summarize results obtained with local
application of two compounds that interact with the extracellular
domain of NCAMs, the second class of cell surface adhesion molecules implicated in the production of LTP (Lüthi et al., 1994; Muller et al., 1996). Time-dependent reversal was not obtained with local infusion of either compound. As shown in Figure 4A,
local ejection of MS2 in advance of TBS caused a distinct reduction in
immediate potentiation compared with LTP at the within-slice control
site, an effect that was evident from the first response after TBS. The
potentiation in both pathways stabilized within 15 min, but the initial
gap between control and test LTP persisted throughout the rest of the
experiment. Average responses were 153.7 ± 3.8% (test) versus
174.8 ± 5.9% (control) for the first 10 min after TBS
(T(9) = 6.84; p < 0.001) and
134.7 ± 3.7% (test) versus 157.0 ± 3.4% (control) for
45-55 min after TBS (T(9) = 6.42;
p < 0.001). It thus appears that MS2 reduces the
initial magnitude of LTP but has no impact on its stabilization,
suggesting that the NCAM antagonist interferes with LTP induction and
the LTP development phase, in agreement with findings by others
(Lüthi et al., 1994; Rønn et al., 1995). This mode of operation
distinguishes MS2 from integrin antagonists, which leave immediate
potentiation intact but interfere with a later step in the sequence
leading to LTP stabilization (Stäubli et al., 1990; Bahr et al.,
1997; present study). Microejection of MS2 initiated immediately and 10 min after TBS (Figs. 4B,C) had no initial or delayed
impact on test LTP compared with that at the control site, an
observation that differs from the postinduction time course over which
integrin antagonists were found to be effective at destabilizing LTP in the present study. The lack of effect of MS2 when applied after LTP
induction demonstrates that microejection of bioactive compounds is
readily accomplished without retroactive changes in recently induced
potentiation.
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Figure 4.
MS2, an NCAM antagonist, reduces the amount of
initial LTP when applied before, but not after, induction.
A-C, Experiments in which LTP was induced
simultaneously at control () and test () sites within the same
slice. The polypeptide MS2, which binds to the fourth immunoglobulin
domain of NCAM, was pressure-ejected at 2 µg/µl at the test site,
starting at various times (horizontal bar) before and
after LTP induction: A, 10 min before
(n = 10); B, immediately after
(n = 7); or C, 10 min after TBS
(n = 4). Each data point represents
the group mean of one response per animal (±SEM). Superimposed
waveforms on the right of each graph illustrate
representative recordings from individual experiments taken at the
times indicated by the numbers in the graphs.
Dotted waveform is the response collected 45 min after
TBS (A, B) or start of peptide application
(C). Calibration: 1 mV, 10 msec.
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Figure 5.
NCAM antibody does not affect LTP induction,
development, or stabilization. A, An antibody against
the fibronectin type III repeat domain of NCAM was pressure-ejected at
4 µg/µl, starting 10 min (n = 4) and 20 min
(n = 5) before LTP induction (data pooled for both
time points). B, Same as in A, except
that antibody application was initiated 30 min before LTP induction
(n = 5). C, Representative waveforms
from an individual experiment taken at the times indicated by the
numbers in A. Calibration: 1 mV, 10 msec.
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Finally, as illustrated in Figure 5A, antibodies against the
fibronectin type III repeat region of NCAM had no effect on LTP when
pressure-ejected 10 or 20 min before induction, a result that remained
unchanged when the drug application time before TBS was increased to 30 min (Fig. 5B). A previous study testing NCAM antibodies
against the fourth Ig-like binding domain found a significant LTP
impairment, although the concentration ejected was 80 times lower
(Lüthi et al., 1994), suggesting that our negative observation is
not likely attributable to an insufficient antibody level but rather
that the fibronectin site of NCAM is not essential for LTP.
Because the degree of LTP resulting from TBS is known to be
closely tied to the amount and duration of the postsynaptic
depolarization occurring during each burst (Arai and Lynch, 1992b), it
was of interest to determine whether the reduction in initial LTP
observed with MS2 was caused by the presence of the peptide during TBS, thereby causing interference with induction mechanisms. Comparisons were made of the degree of individual burst facilitation between control and test pathways in experiments involving drug application before TBS. Typically, and as confirmed in Figure
6, bursts 2-10 are markedly facilitated
under control conditions, with the effect being greater in the early
rather than the late segments of the train (Arai and Lynch, 1992b).
There were no obvious differences in burst facilitation between control
and test pathways of slices treated with GRGDSP (Fig.
6A). In contrast, MS2 dramatically reduced the area
of test relative to control burst responses across the entire train
(Fig. 6B). Comparisons of the degree of test burst facilitation obtained in presence of MS2 with that measured during application of GRGDSP, GRADSP, or the NCAM antibody revealed a significant suppressive action of MS2 in all cases (Fig.
6C,D,F). This pattern of results strongly suggests
that the reduction in immediate LTP seen with MS2 reflects an
interaction with the induction rather than the development or
stabilization of LTP.
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Figure 6.
MS2 reduces facilitation of postsynaptic theta
burst responses during LTP-inducing afferent stimulation, whereas
GRGDSP, GRADSP, and the NCAM antibody have no effect. A,
Increase in burst area (mean ± SEM) across a train of 10 bursts
expressed relative to the initial burst response for both control
(n = 5) and test (n = 5)
pathways of slices in which GRGDSP (2 mM) was applied
locally at the test site starting 20 min before LTP induction.
B, Same as in A, but showing comparisons
between control (n = 5) and test
(n = 7) pathways of slices involving local ejection
of MS2 (2 µg/µl) at the test site starting 20 min before TBS.
C, Data adapted from A and
B, comparing results between the two groups of test
pathways. D, Comparisons of the amount of burst
facilitation between test pathways of slices exposed to the control
peptide GRADSP (n = 8) or MS2
(n = 7), with drug application beginning 20 min
before TBS in both cases. E, Comparisons of burst
facilitation between test pathways of slices in which GRGDSP (2 mM; n = 5) or NCAM antibodies (4 µg/µl; n = 5) were applied locally, starting 20 min before TBS. F, Data adapted from C
and E showing comparisons between test pathways treated
with MS2 (n = 7) or NCAM antibody
(n = 5). Significance levels:
*p < 0.05, **p < 0.01;
t test.
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DISCUSSION |
Proteins with integrin epitopes that bind to appropriate
ligands via the consensus RGD sequence are concentrated in forebrain synapses (Bahr and Lynch, 1992; Grooms et al., 1993; Paulus et al.,
1993; Einheber et al., 1996; Bahr et al., 1997). Previous work
implicated the synaptic integrins in LTP consolidation by showing that
diverse peptide antagonists of RGD binding prevent the formation of
stable potentiation without affecting synaptic potentials or the
complex physiological responses to theta bursts (Xiao et al., 1991;
Bahr et al., 1997). The dose dependency of these effects corresponded
to that for peptide suppression of integrin-mediated adhesion in
various tissues (Cardwell and Rome, 1988; Haskel and Abendschein,
1989). Similar sized peptides with no relationship to the RGD site did
not interact with LTP (Xiao et al., 1991; Bahr et al., 1997). The
present experiments significantly extended this control by showing that
a single amino acid substitution in the RGD segment of infused peptide
(i.e., GRADSP) was sufficient to remove any effect on LTP. Assuming
from these observations that integrins contribute to consolidation, it
was expected that the peptide antagonists (1) would reverse long-term
potentiation in a time-dependent manner and (2) be effective over the
same time period as low-frequency synaptic activity, i.e., if
administered within ~15-30 min of induction (Stäubli and Chun,
1996a,b). The results from the present study confirm both predictions
and in addition specify that integrin-binding events, rather than
participating in early consolidation steps, contribute to delayed
stabilization events beginning between 5 and 10 min after TBS.
The LTP blocking effects obtained with agents that interfere with
NCAMs, the second class of adhesion receptors, differed from those
found with integrin antagonists. Previous work by others using
antibodies against the fourth Ig-like domain implicated NCAMs in LTP
induction (Rønn et al., 1995) and early stabilization processes
occurring in the first few minutes after induction (Lüthi et al.,
1994). The present results modify and extend these findings. (1) The
peptide MS2, which binds to the fourth Ig-like domain, reduced the
magnitude of LTP by a constant amount, from the beginning to the end of
the recording period after TBS, but only if it was present during TBS.
Despite this reduction in LTP amount, the potentiation stabilized
normally. A subsequent analysis of burst responses revealed that MS2
significantly suppressed response facilitation across the entire TBS
train, a result consistent with an impairment in induction, but not
excluding an additional deficit in LTP development. (2) LTP induction,
development, and stabilization remained unaffected by the application
of an antibody against the fibronectin type III repeat region of NCAMs,
suggesting that this binding site, in contrast to the fourth Ig-like
domain, does not contribute to LTP.
Burst response facilitation during LTP induction was not affected by
any of the other agents involved in this study, as would be expected
from compounds that selectively interfere with consolidation as opposed
to induction (i.e., the integrin antagonist GRGDSP) or have no impact
on LTP at all (i.e., the fibronectin antibody and the integrin control
peptide GRADSP). In all, this pattern of results suggests that the two
classes of cell surface adhesion receptors, NCAMs and integrins,
participate in distinctly different stages of LTP, with the former
playing a role in induction and perhaps also development, and the
latter contributing to stabilization processes taking place between 5 and 30 min after induction of potentiation.
The extended period over which LTP was found vulnerable to GRGDSP
presumably reflects the time needed to engage latent integrins. Integrins are activated by various bioactive molecules, one of the most
prominent of which, the platelet activating factor (PAF), is rapidly
generated in the brain and has receptors concentrated in synapses
(Marcheselli et al., 1990; Mori et al., 1996). The mechanisms whereby
PAF operates on integrins are not well understood, although recent work
points to kinase activation and Ser-Thr phosphorylation of the  subunit of the integrin dimer as being critical, at least for platelets
(van Willigen et al., 1996). Other studies using endothelial cells
suggest that tyrosine phosphorylation of the focal adhesion kinase
closely associated with the adhesion molecules is involved (Soldi et
al., 1996). In any event, bursts of afferent activity are likely to
generate at least one activation signal (stimulation of PAF receptors)
in the immediate vicinity of latent synaptic integrins. Studies showing
that inhibitors of PAF receptors block LTP are of interest with regard
to this idea (del Cerro et al., 1990; Arai and Lynch, 1992a; Bazan et
al., 1997).
Once activated, integrins can be expected to produce, over time, two
types of changes pertinent to consolidation. First, by cross-linking
the membrane cytoskeleton with extracellular matrix components (Horwitz
et al., 1986), newly functional integrins will shape and stabilize
morphological changes caused by high-frequency stimulation. Numerous
electron microscopic studies have shown that LTP occurs in association
with rapidly appearing, persistent modifications in synaptic anatomy
(Lee et al., 1980; Desmond and Levy, 1983; Chang and Greenough, 1984).
Second, integrin engagement triggers a mitogen-activated protein (MAP)
kinase cascade (Chen et al., 1994) that interacts with other signal
transduction pathways to modify gene expression. Although integrin
effects on adhesion and cell morphology can occur independently of
these events (Clark and Hynes, 1996; Lin et al., 1997), the link to MAP
kinases could account for the gene induction reported to occur with
high-frequency synaptic activity (Isackson et al., 1991; Andreasson and
Worley, 1995; Link et al., 1995) and potentially could add a genomic
contribution to the later stages of LTP consolidation.
Links between integrin activation and the phenomenon of LTP reversal
remain to be explored. An intriguing possibility is suggested by
experiments showing that stimulation of adenosine receptors within
minutes after TBS selectively erases potentiation (Arai et al., 1990)
and that antagonists of the receptors prevent LTP reversal by
repetitive stimulation (Larson et al., 1993; Stäubli and Chun,
1996b; Abraham and Huggett, 1997). Related to these results is the
finding that repetitive stimulation at frequencies well suited for
reversal causes an efflux of adenosine at synaptic sites (Cunha et al.,
1996). These observations are of interest in the present context
because of evidence that adenosine receptors inhibit activation of
integrins and that endogenously formed adenosine regulates adhesion in
leukocytes (Thiel et al., 1996). In all, the adenosine-integrin
connection, if present in the brain, provides a possible route whereby
low-frequency afferent activity could disrupt the stabilization of
recently induced LTP.
A final and critical issue concerns the behavioral relevance of the
present findings. The observation that two very different manipulations, i.e., RGD peptides and low-frequency synaptic activity, are effective at causing depotentiation over the same time frame is
intriguing not only because it supports the notion that the stabilization of LTP requires ~15-30 min to reach completion, but
also because the estimated consolidation time during which newly
acquired memories are susceptible to disruption by temporary inactivation of hippocampal processes (electroconvulsive shock, hypothermia, etc.) is typically on the order of 15 min to <1 hr (Duncan, 1949; Riccio et al., 1968; Popik et al., 1994). Although these
numbers are based on animal models of memory consolidation, studies on
the duration of retrograde amnesia in humans after accidental head
injury (excluding lesions) have provided similar estimates (Russell,
1959). In contrast, permanent memory loss of events that occurred at
longer intervals is associated with lesions or extreme trauma, such as
severe concussion or coma, that caused damage of the medial temporal
lobe (Moscovitch, 1994; Nadel and Moscovitch, 1997).
Establishment of a link between the role of integrins in LTP
stabilization and possible contributions to memory consolidation will
require comparisons of how intracerebral injections of antagonists into
freely moving rats affect recently induced potentiation and recently
encoded memories. Chronic recording studies have established that the
in vivo time course for LTP erasure with low-frequency stimulation is about the same as that observed in slices (U. Stäubli and J. Scafidi, unpublished observations), but this point
remains to be tested for integrin antagonists. With regard to memory, it has been reported that agents that interfere with NCAM interactions disrupt spatial learning in rodents (Arami et al., 1996; Becker et al.,
1996), but behavioral results of any type for RGD peptides are
lacking.
| |
FOOTNOTES |
Received Jan. 15, 1998; accepted Feb. 18, 1998.
This work was supported in part by the Whitehall Foundation Grant
M97R05 (U.S.) and the Air Force Office of Scientific Research (AFOSR
F49620-95-1-0304) (G.L.).
Correspondence should be addressed to Dr. Ursula Stäubli, New
York University, Center for Neural Science, New York, NY
10003.
| |
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Top
Abstract
Introduction
