Keywords: Long-term potentiation, NMDA receptor, Hippocampus

7079 Journal of Physiology (1997), 504.2, pp. 379—385 379

Activation of NMDA receptors is necessary for the induction of

associative long-term potentiation in area CA1 of the rat
hippocampal slice
K. P. S. J. Murphy*, G. P. Reid†, D. R. Trentham† and T. V. P. Bliss
Divisions of Neurophysiology and †Physical Biochemistry, National Institute for Medical
Research, Mill Hill, London NW7 1AA, UK
1. It is commonly assumed that the role of the strongly activated heterosynaptic input during
the induction of associative long-term potentiation (LTP) is to relieve the magnesium blockade
of NMDA receptors located at the weakly stimulated synapses and thereby allow the weak
input to undergo potentiation. We tested this assumption by using a caged form of the
NMDA receptor antagonist, ª_(−)-2-amino-5-phosphonopentanoic acid (ª_AP5) to block the
activation of NMDA receptors at the weak input in a conditioning protocol for the induction
of associative LTP in area CA1 of the rat hippocampal slice.
2. The effect of releasing ª_AP5 by flash photolysis of 100 ìÒ caged ª-AP5 (N-[1-(2-nitro-
phenyl)ethoxycarbonyl]-ª-AP5) on pharmacologically isolated NMDA receptor-mediated
field EPSPs was examined in area CA1. The slope of the EPSP was reduced by 71% within
50 ms of the initiation of the photolytic reaction when the concentration of released ª_AP5
had reached 2·0—2·5 ìÒ and was reduced by 95 % within 1 min (10 ìÒ ª_AP5 released).
3. Associative LTP was induced by pairing a strong tetanus to one input with a weak tetanus
(subthreshold for homosynaptic LTP) to a second input. The strong tetanus preceded the
weak by 50 ms. Rapid application of ª_AP5, by flash photolysis of caged ª_AP5, coincident
with the last shock of the strong tetanus, resulted in the blockade of NMDA receptor
activation during the period of the weak tetanus. Associative LTP was blocked by photolysis
of caged ª_AP5 but was normally expressed in experiments using caged ¬-AP5.
4. We conclude that activation of NMDA receptors at the weakly activated input is an essential
requirement for synaptically induced associative LTP.
Associativity is a characteristic feature of LTP in area CA1 unmasking of NMDA receptor-mediated potentials by
of the hippocampus (for review see Bliss & Collingridge, intracellular injections of depolarizing current (Kelso,
1993; see also Gustafsson & Wigstr‡om, 1986). A synaptic Ganong & Brown, 1986; Gustafsson, Wigstr‡om, Abraham
input subjected to a weak conditioning protocol that is & Huang, 1987) and facilitation of NMDA receptor-
subthreshold for the induction of homosynaptic LTP mediated transmission at one set of synaptic inputs by
undergoes potentiation when the same protocol is delivered temporal association with activity at a second set (Clark &
in temporal association with a second, strongly activated Collingridge, 1996). However, the assumption that NMDA
input. Associativity can be explained by the voltage- receptor activation is essential for the induction of
dependent properties of the NMDA receptor. It has been associative LTP has not hitherto been tested directly.
proposed that electrotonically propagated depolarization We used a form of the NMDA receptor antagonist ª_AP5
produced by the strongly activated input relieves the (ª-(−)-2-amino-5-phosphonopentanoic acid) to test the
voltage-dependent blockade by Mg¥ ions of NMDA hypothesis that activation of NMDA receptors located on
channels at weakly activated synapses. In this way, the the weakly stimulated input is a prerequisite for induction
conditions necessary for opening of the NMDA receptor of associative LTP. To induce associative LTP the weak
channel — release of transmitter and strong depolarization — input must be activated less than 100 ms after the end of
are satisfied at the weak input, leading to associative LTP. the tetanus to the strong input (Gustafsson & Wigstr‡om,
Evidence supporting a role for NMDA receptors in 1986). Our strategy has been to release ª_AP5 photo-
associative LTP is based on indirect approaches such as the chemically during the interval between strong and weak

*To whom correspondence should be addressed at the Department of Pharmacology, Tennis Court Road, Cambridge CB2 1QJ, UK.
This manuscript was accepted as a Short Paper for rapid publication.
380 K. P. S. J. Murphy, G. P. Reid, D. R. Trentham and T. V. P. Bliss
conditioning stimuli, so that NMDA receptors are blocked
only during activation of the second, weakly activated
input (Lester, Clements, Westbrook & Jahr, 1990). Our
results demonstrate that activation of NMDA receptors on
the weakly activated pathway is essential for induction of
associative LTP.
Some of the results presented here have been published in
preliminary form (Murphy, Reid, Chepkova, Trentham &
Bliss, 1997).
METHODS
Preparation of hippocampal slices
Hippocampal slices were prepared from young male Sprague—
Dawley rats (70—140 g). Animals were killed by stunning followed
by cervical dislocation and decapitation. The brain was rapidly
removed and the hippocampus dissected out and placed in cold
(0—4°C) oxygenated (95% Oµ—5% COµ) artificial cerebrospinal fluid
(ACSF) of the following composition (mÒ): NaCl, 120; KCl, 3;
MgSOÚ, 2; CaClµ, 2; NaHµPOÚ, 1·2; NaHCO×, 23; glucose, 11.
Transverse slices (•400 ìm thick) were cut with a McIlwain tissue
chopper, the CA3 subfield was excised, and the resulting minislice
transferred directly to an interface-type recording chamber
(Scientific Systems Design Inc.) maintained at 26—29°C and
perfused (at 100 ìl min¢) with oxygenated ACSF.
Stimulation and recording arrangements
Extracellular field EPSPs were recorded using glass microelectrodes
(filled with 0·5 Ò sodium acetate and 2% Pontamine Blue;
5—12 MÙ) placed in stratum radiatum of area CA1. Responses
were elicited by stimuli delivered alternately to two independent
pathways, at a frequency of 0·033 Hz to each pathway, via
monopolar tungsten electrodes (tip diameter, 0·1 mm; AM Systems,
Everett, WA, USA) placed either side of the recording site (within
100—300 ìm of the recording electrode). The independence of each
input was verified by demonstrating that the response evoked by
each stimulating electrode was not affected by prior activation of
the other (20—60 ms interpulse interval). The test shock (constant
current duration, 40 ìs; current amplitude, 40—200 ìA) for each
pathway was set to elicit a field EPSP with a slope which was
30—40% of maximum.
Protocols for inducing associative LTP
Associative LTP was induced by temporally pairing a strong
tetanus to one pathway (designated ‘strong pathway’) with a
weaker tetanus to the other (‘weak pathway’). Use of caged ª-AP5
gave us precise temporal control over NMDA receptor blockade, but
no control over the spatial distribution of released ª_AP5, since the
photolytic flash illuminated the whole slice. Moreover, blockade of
NMDA receptors by ª_AP5 persisted for many minutes after
photolysis of the caged precursor (see Fig. 3A). These factors
imposed constraints on the design of the protocol for inducing
associative LTP. First, the interval between tetani to the strong
and weak pathways had to be long enough to allow ª_AP5 to be
released from its cage and bind to NMDA receptors, but short
enough so that, in control conditions (that is, in the absence of
ª_AP5), associative LTP would be induced. An interval of 50 ms
satisfied these requirements. Second, because of the persistence of
ª_AP5 once released from its cage, we were unable to give repeated
pairings of strong and weak tetani, but instead had to adopt a one-
trial associative protocol. We found that weak tetani which
produced associative LTP when paired with the strong tetanus,
often produced some degree of LTP when given alone. In the
J. Physiol. 504.2
protocol that we finally adopted, the strong tetanus consisted of
three trains of 100 pulses at 100 Hz, with an intertrain interval of
10 s; the weak tetanus, delivered 50 ms after the end of the last
train of the strong tetanus, consisted of 20 pulses at 33 Hz. (Three
trains to the strong input was considerably more effective at
inducing associative LTP than a single train, presumably because
the preceding two trains greatly enhanced the response generated
by the third train.) We excluded from analysis all those slices in
which the weak tetanus alone produced LTP of 20% or more,
measured 35—40 min after the tetanus, or in which the LTP
produced by the strong tetanus was less than twice that produced
by the weak tetanus.
Synthesis and flash photolysis of caged ª- and ¬_AP5
Caged ª_AP5 was synthesised as its sodium salt in 53% overall
yield as described for a caged glutamate by Corrie et al. (1993)
except that ª_AP5 rather than a protected derivative was used as
starting material in condensation with 1-(2-nitrophenyl)ethyl
chloroformate. The Sephadex LH20 column step was not required.
Caged ¬-AP5 was similarly prepared. The product quantum yield
of caged ª_AP5 was 0·63 and identical to that for caged ATP (P Å_1-
(2-nitrophenyl)ethyl ester of ATP). The rate of caged ª_AP5
photolysis was measured as shown in Fig. 1 based on the protocol
described by Corrie et al. (1993). Depending on temperature
(26—29°C), 20—25% of the total ª_AP5 released upon caged ª_AP5
photolysis was formed 50 ms after the flash, 50% at 85 ms and
greater than 95% at 350 ms (pH 7·4, 29°C and 0·14 Ò ionic
strength). The extent of photolysis of caged AP5 in the recording
chamber was estimated with reference to measured photolysis of
caged ATP (Ogden, Capiod, Walker & Trentham, 1990; Murphy,
Williams, Bettache & Bliss, 1994). Caged compound was applied
via the bathing medium and photolysed by exposure to a 1 ms
70 mJ pulse of near-UV light from a xenon flash lamp transmitted
through a Schott UG11 bandpass filter (300—350 nm), irradiating
the whole slice. The extent of photolysis within the slice and
surrounding medium (Khodakhah & Ogden, 1995) was typically
10% of the caged compound.
To prevent functional activation of NMDA receptors during
application of the weak tetanus, 10 ìÒ ª_AP5 was photolytically
released from bath-applied caged ª_AP5 coincident with the last
pulse of the strong tetanus, and 50 ms before the first pulse of the
weak conditioning tetanus. Effectiveness of released ª_AP5 was
verified by examining its effect on pharmacologically isolated
NMDA receptor-mediated potentials (using nominally magnesium-
free ACSF containing 10 ìÒ 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX); Tocris Cookson).
RESULTS
Associative LTP
The salient features of the associative conditioning protocol
adopted for these experiments is shown in Fig. 2.
Once stability of evoked responses was established, a
subthreshold tetanus was applied to the weak pathway
and responses monitored for 40 min to ensure that any
homosynaptic LTP was below the arbitrary limit of 20%
(see Methods). In control experiments to demonstrate
associative LTP (Fig. 2A, upper panel), the weak tetanus
produced a marginal potentiation of 10·4 ± 2·7 % (n = 8
slices; mean ± s.e.m.). However, when the weak tetanus was
reapplied in temporal association with the tetanus to the
J. Physiol. 504.2 Caged AP5 and associative long-term potentiation 381

strong pathway, a robust potentiation was produced in
both inputs (40·1 ± 10·5 and 42·4 ± 12·0 % in the weak
and strong pathways, respectively, measured 35—40 min
after associative conditioning, n = 8 slices). Associative
potentiation in the weak pathway was not the consequence
of priming by the first weak tetanus (Christie, Stellwagen &
Abraham, 1995); in five additional experiments, the strong
tetanus was omitted and repetition of the weak tetanus
failed to produce further potentiation (2·9 ± 4·4 %, n = 5;
see Fig. 2A, open symbols).
Caged AP5 and NMDA receptor-mediated
transmission
The strategy of using a caged compound to modify NMDA
receptor function in a time-resolved manner requires that
the compound is biologically inert, undergoes rapid
photolysis and that released byproducts are inactive. Data
illustrated in Fig. 3 demonstrate that caged AP5 fulfils these
requirements. Exposure to near-UV light alone or to 100 ìÒ
caged ª_AP5 had no effect on synaptic transmission, but

Figure 1. Rate of ª_AP5 formation after flash photolysis of caged ª_AP5

The schemetic diagram shows a minimal mechanism for caged ª_AP5 photolysis at pH 7·4. The photolysis
kinetics were measured from time courses of the aci-nitro intermediate and proton concentration changes.
Absorption changes at 406 nm (A406nm ) are predominantly associated with formation and decay of the aci-
nitro intermediate (steps 1 and 2), controlled by rate constants kÔ and kµ. Absorption changes at 615 nm
(A615 nm) arise from the proton indicator Bromothymol Blue and are associated with just one proton release
(stepwise decrease in absorption) controlled by k1 and then with two proton uptake (increase in absorption)
controlled predominantly by k2 (Corrie et al. 1993) and to a lesser extent by k×. There was a 0·002
absorption decrease at 615 nm (10% of the total signal) concomitant with the flash due to photobleaching
of the Bromothymol Blue. After correction for this the 1:2 stoichiometry of proton release to uptake is
seen. Reaction conditions (mÒ): caged ª_AP5, 0·55; NaCl, 130; dithiothreitol, 2; sodium phosphate, 1;
Bromothymol Blue (absorbance, 1 at 615 nm) at pH 7·4 and 29°C in a 4 mm path-length cell. The arrows
on the time axes mark the 20 ìs 320 nm laser pulse (hv) that initiates photolysis. From the right-hand trace
(615 nm record) 33% of the released ª_AP5 had been formed at 50 ms after the laser pulse. The pH at the
end of the experiment was checked and had decreased to pH 7·26. The pH changes during the series of
experiments never exceeded ±0·15; the percentage of released ª_AP5 at pH 7·4 needed to be corrected for
this, resulting in a mean corrected value of released ª_AP5 at 50 ms of 25%. Refer to Corrie et al. (1993)
for pH dependence of the reaction and for further experimental details.
382 K. P. S. J. Murphy, G. P. Reid, D. R. Trentham and T. V. P. Bliss J. Physiol. 504.2

photolysis of 100 ìÒ caged ª-AP5, liberating approximately
2·0—2·5 ìÒ ª_AP5 at 50 ms and 10 ìÒ ª_AP5 overall (see
Methods), rapidly depressed NMDA receptor-mediated
transmission. Within 50 ms of the photolytic pulse, the
slope of the NMDA receptor-mediated EPSP was reduced
by 71·2 ± 5·7 % (n = 5 slices), reaching a maximum mean
depression of 94·4 ± 3·9 % (means of 6 consecutive responses
collected 15—165 s after photolysis). Bath application of 2·0
and 2·5 ìÒ ª_AP5 reduced the slope of NMDA receptor-
mediated potentials by 63·9 ± 5·5 % and 75·3 ± 2·0 %,

Figure 2. Induction of associative LTP using a one-trial pairing protocol

A, the upper and lower panels show percentage change in slope of field EPSPs displayed as a function of
time, evoked by stimulation of weak and strong afferent pathways, respectively. Data are expressed as
pooled means; s.e.m. indicated by error bars are plotted for every 5th datum point. In the upper panel,
application of the weak conditioning tetanus to the weak input is denoted by open arrowheads and in the
lower plot, application of the strong tetanus to the strong input is indicated by the filled arrowhead. The
period of associative conditioning is denoted by juxtaposition of both filled and open arrowheads (upper
panel only). In both panels filled symbols represent pooled data from the same eight control experiments. In
the upper panel, open symbols are pooled means from a separate series of five experiments in which the
second weak tetanus was given in the absence of the strong conditioning stimulus. In the upper panel, data
points are normalized with respect to the 5 min period prior to application of the second weak tetanus. In
the lower panel, data points are normalized with respect to the control period prior to the strong tetanus. B,
records a—c are taken from a representative experiment; each is the average of five consecutive responses
evoked in the weak input and collected at the times a—c indicated in the upper panel.
J. Physiol. 504.2 Caged AP5 and associative long-term potentiation 383

respectively (n = 4 slices), and 10 ìÒ ª_AP5 reduced the
slope of the potentials by 97·9 ± 2·6 % (n = 3 slices). The
fact that 2·0—2·5 ìÒ ª_AP5 photoreleased at 50 ms induces
the same depression as 2·0—2·5 ìÒ bath applied ª_AP5
indicates that the rate of ª_AP5 interaction with the
receptor is rapid on the 50 ms time scale.
In further experiments we compared effects of submaximal
concentrations of ª_AP5 delivered by the two methods.
Release of 1 ìÒ ª_AP5 by flash photolysis resulted in a
depression of 37·8 ± 5·9 % (n = 5 slices), which was not
significantly different from the depression caused by bath
application of 1 ìÒ ª_AP5 (43·4 ± 3·7 %, n = 8 slices;
P > 0·1, Student’s two-tailed t test). These results suggest
that the assay of the concentration of photoreleased ª_AP5
described in Methods is satisfactory.
In addition to ª_AP5, photolysis of caged ª-AP5 yields
equimolar concentrations of 2-nitrosoacetophenone, carbon
dioxide and hydroxide ion (see Fig. 1). To exclude the
possibility that the byproducts might impair the function of
NMDA channels (Traynelis & Cull-Candy, 1990), experiments
were performed using a caged compound in which the
biologically inert enantiomer ¬_AP5 was substituted for
ª_AP5. Photolysis of 100 ìÒ caged ¬-AP5 had no effect on
NMDA receptor-mediated transmission (n = 5 slices, Fig. 3).
These experiments demonstrate that antagonism at the
NMDA receptor was due to ª_AP5.
NMDA receptors and associative LTP
NMDA receptor dependency of associative LTP is
illustrated in Fig. 4. Photolytic release of 10 ìÒ ª_AP5,
50 ms before the start of the weak tetanus prevented
expression of LTP in the weak pathway (3·0 ± 0·8 %, n = 6
slices, measured 40 min post tetanus) but did not block
induction of homosynaptic LTP in the strong pathway
(65·2 ± 13·9 %); expression of LTP in the strong pathway
did not differ significantly from that expressed in control
experiments (see Fig. 2, P > 0·2, Student’s two-tailed

Figure 3. Caged ª- and ¬_AP5 and blockade of NMDA receptor-mediated transmission

A, mean percentage change in slope of NMDA receptor-mediated potentials plotted against time; s.e.m.
indicated by error bars for every 5th datum point. In the upper panel (n = 7), a 1 ms pulse of near-UV
light (arrow) was applied in the absence and presence (filled bar) of 100 ìÒ caged ª_AP5. Photolytic release
of 10 ìÒ ª_AP5 had an immediate effect on NMDA receptor-mediated transmission as demonstrated by
the marked reduction of 72% seen for the first datum point collected 50 ms after the flash (n = 5 for this
point only). In the lower panel, caged ¬-AP5 was substituted for caged ª_AP5. Photolytic release of 10 ìÒ
¬_AP5 and associated photoproducts had no effect on NMDA receptor transmission (n = 5). Bath
application of 25 ìÒ ª_AP5 (open bar) abolished the synaptic potential in both series of experiments.
B, superimposed consecutive responses from a representative experiment, collected immediately before and
at 50 ms and 15 s after the flash. The NMDA receptor-mediated potentials were isolated pharmacologically
in magnesium-free ACSF and in the presence of 10 ìÒ CNQX.
384 K. P. S. J. Murphy, G. P. Reid, D. R. Trentham and T. V. P. Bliss J. Physiol. 504.2

t test). Antagonistic effects of photolytically released ª_AP5 DISCUSSION

were reversible: once photoproducts had been washed from
slices, repetition of the conditioning protocol resulted in
successful induction of associative LTP (23·8 ± 2·4 %;
P = 0·17 (Welch t test) compared with associative LTP
expressed in control slices). To exclude the possibility that
apparent block of associative LTP might be attributable to
exposure to near-UV light or action of byproducts,
additional experiments were performed using caged ¬-AP5.
Photolysis of 100 ìÒ caged ¬-AP5, 50 ms prior to application
of the weak tetanus did not prevent induction of associative
LTP (27·5 ± 4·7 %, n = 6 slices). These result establish that
block of associative LTP by photolysis of caged ª_AP5 can
be attributed to ª_AP5.
Explanation for associative LTP has conventionally focused
on the voltage dependency of the NMDA receptor as the
associative mechanism. The strong heterosynaptic input, in
a two pathway protocol, is presumed to provide sufficient
depolarization to relieve Mg¥ blockade of NMDA receptors
at the weakly activated input, thus allowing Ca¥ to enter
the postsynaptic cell and trigger induction of associative
LTP. On this view, selective blockade of NMDA receptors at
the weak input should prevent induction of associative LTP.
However, there are other models which could explain the
induction of associative LTP, and which do not require
involvement of NMDA receptors. Several lines of evidence
suggest that an increase in intracellular calcium in

Figure 4. Blockade of NMDA receptor activation prevents induction of associative LTP

A, upper and lower panels are pooled data from 6 experiments and show mean values of the percentage
change in EPSP slope for weak and strong inputs, respectively. Weak and strong conditioning tetani are
denoted by open and filled arrowheads, respectively; in the upper panel, juxtaposition of these symbols
indicates the periods of associative conditioning. A 1 ms flash of near-UV light (arrow) was applied in the
presence of 100 ìÒ caged ª_AP5 (indicated by the filled bar) coincident with the last shock of the strong
tetanus and 50 ms before the weak conditioning stimulus. Repetition of the associative conditioning
protocol resulted in the induction of associative LTP in the absence of caged ª_AP5. B, pooled data from 6
experiments where 100 ìÒ caged ¬_AP5 was substituted for caged ª_AP5 (only weak pathway is shown).
Photolysis of caged ¬_AP5 did not prevent induction of associative LTP. C, averaged traces (5 consecutive
responses) from a representative experiment showing the EPSP evoked by stimulation of the weak input,
and corresponding to times a—d in A.
J. Physiol. 504.2 Caged AP5 and associative long-term potentiation
conjunction with weak synaptic activity is sufficient to
induce LTP (Turner, Baimbridge & Miller, 1982; Malenka,
Kauer, Zucker & Nicoll, 1988; Aniksztejn & Ben Ari, 1991).
Diffusion of calcium from the strongly stimulated input,
possibly augmented by local entry via voltage-dependent
calcium channels at the weakly activated input, may fulfil
the conditions for induction of associative LTP. Another
possibility is that an increase in intracellular calcium
concentration in conjunction with postsynaptic activation of
metabotropic glutamate receptors at the weakly stimulated
input may be sufficient to induce associative LTP. In either
case, blockade of NMDA receptors at the weak input would
not be expected to prevent induction of associative LTP.
In this study we have set out to test the hypothesis that
NMDA receptor activation at weakly activated synapses is
essential for the induction of associative LTP in a two-
pathway protocol. The most direct approach would be to
block selectively NMDA receptors in the weak pathway
whilst sparing those in the strong pathway. The method of
Scanziani, Malenka & Nicoll (1996), in which one pathway,
but not the other, is subjected to low frequency activation in
the presence of the use-dependent NMDA channel blocker
MK_801, results in the selective blockade of NMDA receptor
function in the stimulated pathway. Our attempts to exploit
this protocol were not successful. We found that, almost
invariably, both stimulated and unstimulated pathways
were blocked by MK_801.
In the experiments reported here we have employed an
alternative strategy, using flash photolysis of caged ª_AP5
to gain precise temporal control over NMDA receptor
function. This has allowed us to initiate a blockade of
NMDA receptors in the interval between the end of the
strong tetanus to one pathway and the beginning of the
weak tetanus to the other, in a two-pathway associative
protocol. Our results demonstrate that NMDA receptor
activation at weakly activated synapses is necessary for the
induction of associative LTP.
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Author’s email address
K.P. S. J. Murphy:kpsjm2@cam.ac.uk
Received 23 June 1997; accepted 15 August 1997.