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Brief O-Burst Stimulation Induces a

Transcription-Dependent Late Phase of LTP
Requiring cAMP in Area CA I of the
M o u s e Hippocampus
P e t e r V. N g u y e n I a n d Eric R. K a n d e l 2
Howard Hughes Medical Institute and Center for Neurobiologyand Behavior
College of Physicians and Surgeons of Columbia University
New York, New York 10032

Prior induction o f O-LTP also occluded the

potentiation elicited b y c h e m i c a l activation
of PKA. Our results s h o w that, like non-O
LTP, O-induced LTP in area CA1 o f the
m o u s e h i p p o c a m p u s also involves
transcription, translation, a n d PKA and
suggest that cAMP-mediated gene
t r a n s c r i p t i o n m a y be a c o m m o n m e c h a n i s m
responsible for the late phases of LTP
induced b y b o t h O and n o n - O patterns of
stimulation.

Abstract
M e m o r y storage in the m a m m a l i a n b r a i n
can be divided into a s h o r t - t e r m phase that
is i n d e p e n d e n t o f n e w p r o t e i n synthesis a n d
a l o n g - t e r m p h a s e that requires synthesis o f
n e w RNA a n d proteins. A cellular m o d e l for
these two phases has e m e r g e d f r o m studies
o f l o n g - t e r m p o t e n t i a t i o n (LTP) in the t h r e e
m a j o r excitatory synaptic p a t h w a y s in the
h i p p o c a m p u s . One especially effective
p r o t o c o l for i n d u c i n g robust a n d persistent
LTP is "O-burst" stimulation, w h i c h is
designed to m i m i c the firing patterns of
hippocampal neurons recorded during
e x p l o r a t o r y b e h a v i o r in intact awake
animals. Unlike LTP induced b y non-O
tetanization regimens, little is k n o w n about
the b i o c h e m i c a l m e c h a n i s m s u n d e r l y i n g
O-burst LTP in the h i p p o c a m p u s . In the
p r e s e n t study, we e x a m i n e d O-burst LTP in
the Schaffer collateral pathway. We f o u n d
that 3 sec o f O-burst stimulation induced a
r o b u s t a n d persistent potentiation (O L-LTP)
in m o u s e h i p p o c a m p a l slices. This O L-LTP
was d e p e n d e n t o n NMDA r e c e p t o r
activation. The initial o r early p h a s e o f
O-LTP did not require either p r o t e i n o r RNA
synthesis a n d was i n d e p e n d e n t of
cAMP-dependent p r o t e i n kinase (PKA)
activation. In contrast, the late p h a s e of
O-LTP r e q u i r e d synthesis o f proteins and
RNA a n d was b l o c k e d b y inhibitors of PKA.

Introduction
Memory storage consists of at least two distinct temporal phases: short-term memory, lasting
minutes to hours, and long-term memory, which
may persist for days, weeks, or even longer (for
reviews, see Polster et al. 1991; Squire 1992). Brief
inhibition of either protein synthesis or transcription selectively blocks induction of long-term
memory without affecting short-term memory
(Davis and Squire 1984; Castellucci et al. 1989;
Crow and Forrester 1990; Tully et al. 1994). In
contrast to its induction, maintenance of long-term
memory is independent of new protein synthesis
and transcription (Davis and Squire 1984).
Cell biological studies of the conversion of
short- to long-term memory in invertebrates have
revealed some of the molecular mechanisms underlying this transition. In the marine snail, Aplysia, studies of memory for sensitization of the gilland siphon-withdrawal reflexes have shown that a
clear distinction exists between the mechanisms
involved in short-term and long-term presynaptic
facilitation (a cellular mechanism contributing to
sensitization). Short-term facilitation involves post-

1Present address: Center for Neuroscience Research, McGill University, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4
ZCorresponding author.

LEARNING & MEMORY 4:230-243 9 1997 by Cold Spring Harbor Laboratory Press ISSN1072-0502/97 $5.00

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O-BURST L TP IN THE MOUSE HIPPOCAMPUS

pression (Frey et al. 1993; Matthies and Reymann

1993; Huang and Kandel 1994; Huang et al. 1994;
Nguyen et al. 1994; Nguyen and Kandel 1996).
Stimulation protocols for LTP induction in the
hippocampus vary considerably, but LTP is typically induced by applying 1-sec trains of high-frequency (100-Hz) stimulation (Bliss and Lomo 1973;
Bliss and Collingridge 1993). It is unclear whether
hippocampal neurons in vivo fire at 100 Hz for a
full second. Pyramidal cells in CA1 commonly fire
short (30- to 40-msec) bursts of three to four spikes
(Kandel and Spencer 1961; Ranck 1973), with the
bursts being repeated at the "O" frequency (Green
et al. 1960). O is a 5- to 12-Hz electroencephalographic wave that appears when animals are engaged in exploratory, attentive behavior (Grastyan
et al. 1959; Vanderwolf 1969; Bland 1986). O
rhythm may initiate LTP, because brief 30-msec
bursts of stimuli (100 Hz) delivered repeatedly at 5
Hz for 1-2 sec ("O-burst" stimulation) effectively
induces LTP in the rat hippocampus in vitro (Larson and Lynch 1986; Larson et al. 1986) and in vivo
(Staubli and Lynch 1987).
Numerous studies have recently explored the
biochemical mechanisms of L-LTP in hippocampal
slices (for review, see Abel et al. 1995; Nguyen et
al. 1995). These experiments have used 1-sec tetani and have shown that L-LTP requires macromolecular synthesis and PKA recruitment. In contrast,
little is known about the biochemical mechanisms
of L-LTP induced by O-burst stimulation.
In the present study, we asked the following
questions: Can L-LTP be induced in area CA1 of
hippocampal slices by applying brief O-burst
stimulation? Is PKA activity essential for induction
of O L-LTP? Are protein synthesis and gene transcription critical for O L-LTP and, if so, when are
these processes engaged?

translation modification of pre-existing proteins

and is mediated by cAMP-dependent protein kinase
A (PKA) and protein kinase C (PKC) (Castellucci et
al. 1980; Montarolo et al. 1986; Ghirardi et al. 1992;
Byrne et al. 1993). Long-term facilitation, in contrast, requires new protein synthesis and cAMPmediated gene expression, through activation of
cAMP response element binding-1 (CREB-1) and relief from repression of CREB-2 (Montarolo et al.
1986; Dash et al. 1990; Alberini et al. 1994; Bartsch
et al. 1995), and also involves growth of new synaptic connections (Glanzman et al. 1990; Nazif et
al. 1991). Similarly, studies on Drosophila suggest
that short-term memory and learning require PKA,
whereas long-term memory requires CREB-initiated
gene expression (Tully et al. 1994; Yin et al. 1994;
for mouse data, see Bourtchouladze et al. 1994).
Mechanisms similar to those revealed in Aplysia and Drosophila may also underlie explicit
memory storage in the mammalian brain (for reviews, see Abel et al. 1995; Nguyen et al. 1995).
Explicit learning involves the acquisition of information about people, places, and things and is critically dependent on structures within the temporal
lobe, including the hippocampus (Scoville and Milner 1957; Hirsh 1974). Within the hippocampus,
there are three major serial excitatory synaptic
pathways: the perforant, mossy fiber, and Schaffer
collateral pathways that connect the entorhinal
cortex to the dentate gyrus, the dentate gyrus to
area CA3, and area CA3 to area CA1, respectively
(Andersen et al. 1977; Amaral 1993). Damage to
any one of these three serial pathways is thought to
be sufficient to severely impair memory in humans
(Zola-Morgan et al. 1986).
Hippocampal neurons can undergo long-lasting increases in synaptic efficacy after brief highfrequency stimulation of any of the three excitatory pathways (Bliss and Lomo 1973). In awake
animals, the activity-dependent increase in synaptic strength can last for hours to days and is called
long-term potentiation (LTP). LTP has been studied
extensively in hippocampal slices (Andersen et al.
1977; for review, see Bliss and Collingridge 1993).
As with behavioral memory, LTP in all three hippocampal regions consists of at least two biochemically distinct temporal phases. There is an
early phase lasting 1-2 hr that is independent of
protein and RNA synthesis and a later, more persistent phase (L-LTP), beginning after 1-2 hr and
lasting up to 8 hr in slices (for review, see Huang et
al. 1996a). This L-LTP requires new protein and
RNA synthesis, and PKA activation for its full ex-

Materials a n d M e t h o d s
Adult (7- to 11-week old) male C57BL/6J mice
(The Jackson Laboratory) were decapitated, and
their brains were rapidly removed and immersed in
cold (4~ artificial cerebrospinal fluid (ACSF). Isolated hippocampi were cut transversely (400-1am
thickness) with a McIlwain tissue chopper, and the
resulting slices were placed on a nylon mesh in an
interface chamber maintained at 28~ Slices were
continuously superfused with ACSF (1 ml/min)
bubbled with a mixture of 95% 02 and 5% CO 2 and
were allowed to recover for 60-90 min before re-

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Nguyen and Kandel

cordings w e r e attempted. The c o m p o s i t i o n of the
ACSF solution was as follows (125 mM NaC1, 1.5
mM MgSO4, 4.5 m i KC1, 26 m i NaHCO3, 2.5 m i
CaC12, 1 mM NaH2PO4, and 10 m i glucose.
For extracellular stimulation of the Schaffer
collateral pathway, a bipolar n i c k e l - c h r o m i u m
electrode (Medwire Corp.) was placed in the strat u m radiatum layer of area CA1. Extracellular field
EPSPs w e r e r e c o r d e d w i t h a glass microelectrode
(5- to 8-M~ resistance) filled w i t h ACSF and positioned in stratum radiatum of area CA1. For all experiments, test stimuli (0.05-msec pulse w i d t h )
w e r e delivered o n c e / m i n , and the stimulus intensity was set to give baseline field EPSP slopes -40%
of m a x i m a l evoked slopes. Slices that s h o w e d
m a x i m a l field EPSP sizes <3 mV w e r e rejected.
LTP was i n d u c e d b y applying 3 sec of continuous O-burst stimulation: 15 bursts of four pulses at
100 Hz, w i t h an interburst interval of 200 msec.
For some e x p e r i m e n t s , LTP was i n d u c e d by giving
an equivalent n u m b e r of pulses w i t h i n 1 sec (i.e., a
single 1-sec train of 60 Hz).
All drugs w e r e m a d e fresh in perfusate, e x c e p t
for actinomycin-D (dissolved in 0.05% ethanol final
c o n c e n t r a t i o n ) and KT-5720 (dissolved in 0.1%
DMSO final concentration).
Student's u n p a i r e d t-test was used for all statistical c o m p a r i s o n s of m e a n field EPSP slopes.

h r after O-burst stimulation w e r e 186_+ 7%,

170 _ 7%, and 174 _+ 6% of baseline, respectively
(n = 12 slices from six animals) (Fig. 1A).
Is this O-induced L-LTP d e p e n d e n t on NMDA
receptor activation? In the p r e s e n c e of 100 ~IMAPV
(2-amino-5-phosphonovalerate), an NMDA receptor antagonist, 3-sec O-burst stimulation failed to
potentiate transmission in area CA1 (Fig. 1B). This
block of LTP induction was reversed w i t h w a s h o u t
of APV (Fig. 1B). Hence, these e x p e r i m e n t s s h o w
that, like earlier studies on rat h i p p o c a m p a l LTP
i n d u c e d by O-bursts (Larson and Lynch 1988),
O-LTP i n d u c t i o n in area CA1 of m o u s e h i p p o c a m pal slices is d e p e n d e n t on NMDA r e c e p t o r activation.
Can the temporal pattern of stimulation critically shape the duration of synaptic potentiation?
W e addressed this question by applying the same
total n u m b e r of stimulus pulses (60) in a temporally c o m p r e s s e d manner. After a 1-sec train of 60Hz stimulation, transmission in the Schaffer collate r a l - c o m m i s s u r a l p a t h w a y was facilitated to a
lesser degree than following O-burst stimulation
(Fig. 1A). Mean fEPSP slopes in 60-Hz slices w e r e
117 _+ 11%, 117 _+ 9%, and 112 + 8% after 1, 2, and
3 hrs, respectively (n = 6). These values w e r e significantly lower than those observed in O-burst
slices ( P < 0 . 0 2 ) . These results indicate that
O-burst stimulation is a very effective protocol for
induction of stable, long-lasting potentiation in
area CA1 of m o u s e h i p p o c a m p a l slices and that the
temporal spacing of stimulus pulses is critical for
p r o d u c i n g such robust synaptic plasticity.

Results
O-BURST STIMULATION INDUCES LONG-LASTING
POTENTIATION IN AREA CA1 OF MOUSE
HIPPOCAMPAL SLICES

TRANSCRIPTION AND TRANSLATION ARE

REQUIRED FOR EXPRESSION OF LATE PHASES OF
O-BURST LTP

Previous studies have s h o w n that O-burst

stimulation can i n d u c e robust and persistent facilitation of transmission in area CA1 of intact animals
and rat h i p p o c a m p a l slices (Larson et al. 1986;
Staubli and Lynch 1987). In contrast, little is
k n o w n about the b i o c h e m i c a l m e c h a n i s m s responsible for O-burst LTP in the m o u s e h i p p o c a m p u s .
As an initial step toward characterizing the bioc h e m i c a l p a t h w a y s that m a y contribute to O-burst
LTP, w e tested for the i n d u c t i o n of L-LTP in m o u s e
h i p p o c a m p a l slices by applying 3 sec of O-burst
stimulation to the Schaffer collateral-commissural
pathway. W e f o u n d that O-burst stimulation
yielded a robust and stable facilitation of synaptic
transmission that persisted for 3 - 6 hr (Fig. 1A; 6-hr
data not shown). Mean values for field excitatory
postsynaptic potential (fEPSP) slopes at 1, 2, and 3

W e t h e n explored the putative roles of RNA

synthesis and translation in the e x p r e s s i o n of O
L-LTP in m o u s e h i p p o c a m p a l slices. In the prese n c e of 40 ILIMactinomycin D (ACT-D; applied for 1
hr b e g i n n i n g 30 m i n before O), a transcriptional
inhibitor, 3 sec of O-burst stimulation i n d u c e d LTP
that gradually decayed to 125 _+ 5% and 114 + 9%
(n = 6) of pre-O baseline after 90 m i n and 3 hr,
respectively (Fig. 2A). W h e n ACT-D was applied
for 1 hr b e g i n n i n g 30 m i n after O, the levels of
potentiation w e r e t h e n 158 _ 7% and 160 _+ 3%
(n=4)
after 90 m i n and 3 hr, respectively
(P < 0.02; Fig. 2A). Hence, there exists a time wind o w of transcription critical for full m a i n t e n a n c e of

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O - B U R S T L TP IN THE M O U S E H I P P O C A M P U S

ls

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Time (min)

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,
20

40

~"-'-'-'-"1
60
80

Time (min)

synthesis and translation for full expression. Furthermore, in the presence of these inhibitors of
transcription and translation, the later phases of
LTP were absent.

O-burst LTP; this time period extends from O

stimulation to 30 min after induction of potentiation.
A requirement for gene transcription suggests
further that protein synthesis may be involved in
the expression of O-LTP. To test this hypothesis,
we applied anisomycin (30 ~M, for 1 hr starting 30
min before O) to mouse hippocampal slices and
observed that potentiation in area CA1, as in the
ACT-D experiments, decayed slowly to near baseline values: Mean fEPSP slopes after 3 hr were
116 +_6% (n = 6) in drug-treated slices and
161 + 8% (n = 6) in drug-free controls (P < 0.05)
(Fig. 2B).
These experiments show that O-LTP in area
CA1 of mouse hippocampal slices requires RNA

Figure 1: O-Burst, but not 60-Hz stimulation, leads to L-LTP of synaptic transmission
in area CA1 of mouse hippocampal slices. (A)
A brief episode (3 sec) of O-pattern stimulation elicited L-LTP that persisted for at least 3
hr, whereas the same number of stimulus
pulses applied in a compressed fashion (60
Hz, 1 sec) produced a gradually decaying
form of synaptic facilitation. O or 60-Hz
stimulation was applied at the time marked
by the arrow. Sample fEPSP traces were recorded 15 min before and 3 hr after O. Scale
bars, 2 mV, 10 msec. (B) LTP produced by
O-burst stimulation is dependent on NMDA
receptor activation. In the presence of 100 ~IM
APV, O-burst stimulation (at arrow) failed to
elicit any potentiation. Washout of APV, followed by a second episode of O-burst stimulation (3 sec) at about 65 min, resulted in
some potentiation (A). The upper curve (r-l)
shows data identical to the O curve of A.

INHIBITORS OF cAMP-DEPENDENT PROTEIN

KINASE BLOCK THE LATE PHASES OF LTP
INDUCED BY O-BURST STIMULATION IN AREA CA1
W h i c h biochemical signal transduction pathways are involved in the induction of O-LTP? One
candidate is the cAMP-PKA pathway, w h i c h has
been shown to be critical for expression of the late
phases of hippocampal LTP induced by non-O patterns of tetanization (Frey et al. 1993; Huang and

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Nguyen and Kandel

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30

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120

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Time (rain)

B
Figure 2:

Transcription and protein syn-

30"" I

30o]

thesis are necessary for e x p r e s s i o n of O - i n duced L-LTP. (A) Application of 40 IJM

ACT-D, a transcriptional inhibitor, blocked

the late stages of LTP induced by 3 sec of
O-burst stimulation, but only when the
drug was given before and after stimulation (lower curve, A). Later application of
ACT-D (beginning 30 min after O) failed to
affect L-LTP (upper curve, R). (B) Anisomycin, a protein synthesis inhibitor,
blocked L-LTP when applied for 1 hr overlapping with O (lower curve, I-1). Sample
traces were measured 15 min before and 3
hr after O in both A and B. Scale bars, 2
mV, 10 msec.

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Kandel 1994; Huang et al. 1994; Nguyen and Kandel 1996).

Using two different inhibitors of PKA, we
tested the hypothesis that PKA activation is necessary for induction of the late phases of O-LTP. A
brief application of KT-5720 (1 laM,bath-applied for
30 min beginning 15 min before O), an inhibitor of
the catalytic subunit of PKA (Kase et al. 1987), did
not affect initial induction of O-LTP (Fig. 3A), but
caused a relatively rapid decay of potentiation to
baseline values: Mean fEPSP slopes measured 60
min and 3 hr after 3 sec of O stimulation were
123 + 5% and 109 -+ 6%, respectively (n = 6).
When KT-5720 was applied slightly later (starting
30 min after O), potentiation was intact: Mean
fEPSP slopes recorded 60 min and 3 hr post-O were

186 + 6% and 174 + 6%, respectively (n = 4;

P < 0.05).
A second inhibitor of PKA, Rp-cAMPS (100 pM,
applied for 30 min starting 15 min before O), also
blocked the later phases of O-LTP (Fig. 3B). Mean
fEPSP slopes recorded 3 hr after O in drug-treated
and drug-free slices were 106 _+ 10% (n - 5) and
161 _+ 9% (n = 5), respectively (P < 0.05). Unlike
KT-5720, Rp-cAMPS is known to act on the regulatory subtmit of PKA, preventing its dissociation
from the catalytic subunits and thereby maintaining PKA in its inactive tetrameric form (Dostmann
1995).
These results show that PKA activation is crucial for full expression of the late phases of O-LTP
in area CA1 of the mouse hippocampus and sug-

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O-BURST L TP IN THE MOUSE HIPPOCAMPUS

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30

60

90

120

150

180

Time (min)

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AMPS, E a r l y

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Late

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fl=5

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n=5

Rp-cAMPS, 1001~M
Rp-cAMPS, 1001aM
/

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30

60

.'

'l

90

120

150

180

Time (min)

tion of PKA. We tested this idea by first applying

Sp-cAMPS (100 ~M, given for 15 min) to mouse
hippocampal slices (Fig. 4A). This activator of PKA
produced an initial transient depression of transmission in area CA1, followed by a gradual facilitation that reached plateau values of 165 _+ 9% and
167 + 3% (n - 5) 90 min and 2 hr after application,
respectively (Fig. 4A).
In a separate group of slices, we next gave 3
sec of O-burst stimulation, decreased the stimulus
intensity to bring transmission back down to baseline levels immediately after O, and applied 100 ~M
Sp-cAMPS for 15 min, beginning 5 min before
O-burst stimulation (Fig. 4B). The level of potentiation measured immediately after O-burst stimulation was 172 + 14% (n - 4). More importantly,

gest that, like some forms of LTP induced by non-O

patterns of high-frequency stimulation, the late
phases of O-LTP also d e p e n d on activity-dependent
recruitment of PKA in hippocampal neurons. Furthermore, a critical period of PKA activation appears to be required for the late phases of O-LTP.

A cAMP ANALOG PRODUCES LONG-LASTING

POTENTIATION THAT IS OCCLUDED BY PRIOR
O-BURST STIMULATION
If the cAMP-PKA signal transduction pathway
is critically involved in O-LTP, then induction of
LTP by O-burst stimulation should occlude subsequent potentiation produced by chemical activa-

Figure 3: Inhibitors of PKA block expression of L-LTP following O-burst stimulation.

(A) KT-5720, an inhibitor of the catalytic subunit of PKA, elicited a gradual decay of O-induced LTP when application overlapped
with O (KT5720 Early, lower curve) but had
no effect on L-LTP when applied 30 min after
O (KT5720 Late, upper curve). (B) RpcAMPS, an inhibitor of PKA that acts on the
regulatory subunit, also prevented expression
of L-LTP following O, but only when application of drug overlapped O-burst stimulation (lower curve). Later administration of
Rp-cAMPS failed to affect L-LTP expression
(upper curve). Traces were recorded 15 min
before and 2 hr after O. Scale bars, 2 mV, 10
msec.

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Nguyen and Kandel

~ l O O p M

absent during Sp-cAMPS application following O

stimulation (Fig. 4B). This may have stemmed from
facilitatory processes induced in CA1 pyramidal
cells by O, which would mask the depression induced by Sp-cAMPS.
These findings complement those obtained
with pharmacological inhibitors of PKA, strongly
supporting the idea that O-burst stimulation activates the cAMP-PKA signal transduction pathway,
which, in turn, triggers molecular events that are
necessary for expression of the late stages of O-LTP
in area CA1.

300
250
.IQ

"6

n=5

200

g
150

o
03
nco
n
uJ
"o
._r

100
50
Sp-cAMPS, 100p.M

LL

-30

3'0
6'0
Time (min)

9'0

1;0 "

SHORT-LASTING POTENTIATION INDUCED BY A

COMPRESSED PATTERN OF STIMULATION
REQUIRES NEITHER TRANSCRIPTION NOR PKA
ACTIVATION

300

b__
.--

250

,,..

200

c_

It is clear that O-burst stimulation induces

long-lasting potentiation that is dependent on transcription and PKA activation, whereas a temporally
compressed pattern of stimulation that uses the
same number of stimulus pulses fails to produce
persistent and robust facilitation. Does such shortlasting potentiation also require gene transcription
and PKA activity?
To address this question, we applied 1 sec of
60-Hz stimulation to the Schaffer collateral-commissural pathway of mouse hippocampal slices in
the presence of either ACT-D (Fig. 5A) or RpcAMPS (Fig. 5B). In the absence of either drug,
levels of transmission were moderately potentiated
for a relatively short period of time: Mean fEPSP
slopes measured 2 hr after 60-Hz stimulation in the
two control groups were 118 _+7% and 116 + 9%
of baseline values (n - 5 for each group). In the
presence of the transcriptional inhibitor ACT-D (40
HMapplied for 1 hr, Fig. 5A), the mean fEPSP slope
recorded 2 hr after 60-Hz stimulation was 110 + 5%
(n - 5), which was not significantly different from
drug-free controls (P > 0.5). Rp-cAMPS (100 ~aM), a
PKA inhibitor, also did not affect potentiation after
2 hr: The mean fEPSP slope measured here was
118 + 8% (n = 5; P > 0.5; Fig. 5B).
The results of these experiments suggest that
the temporal pattern of stimulation leading to synaptic potentiation in the hippocampus can determine the particular biochemical requirements and
duration of facilitation induced. Specifically, a temporally compressed pattern of stimulation (60 Hz,
1 sec) was less effective in eliciting a transcriptionand cAMP-dependent form of long-lasting potentia-

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-30

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60

90

120

150

180

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Figure 4: O-Burst stimulation occludes synaptic potentiation produced by an analog of cAMP. (A) A brief,
15-min application of Sp-cAMPS, an activator of PKA,
elicited a transient depression and then a long-lasting
facilitation of synaptic transmission in area CAl. Sample
traces were recorded 15 min before and 2 hr after onset
of Sp-cAMPS application. (B) O-Burst stimulation (3 ser
at arrow) given 5 min after the onset of application of
Sp-cAMPS prevented subsequent potentiation by SpcAMPS. Note that the initial depression seen in A was
less pronounced here, perhaps because of overlapping
facilitation induced by O-burst stimulation. Sample
traces were recorded at marked times. Trace b was measured immediately after O. Scale bars, 2 mV, 10 msec.

we found that Sp-cAMPS did not produce significant facilitation of transmission in area CA1 following O-burst stimulation: Mean fEPSP slopes measured 1 hr, 90 min, and 2 hr after Sp-cAMPS application were 109_+13%, 91_+6%, and 99-+8%
(n = 4), respectively. These values were not significantly different from pre-O baseline measurements
(P > O.5). A marked depression of transmission was

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O-BURST L IP IN THE MOUSE HIPPOCAMPUS

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Figure 5: A compressed pattern of stimulation elicits

only short-lasting potentiation that is independent of
transcription and PKA. (A) A brief, 1-sec train of 60 Hz
produced short-lasting facilitation in slices treated with
ACT-D (a transcriptional inhibitor; I-1) that was indistinguishable from control slices (A). (B) The same stimulation pattern still elicited a transient facilitation that was
not significantly different between control slices (A) and
slices treated with a PKA inhibitor, Rp-cAMPS (I-1).

tion than was a more temporally spaced, O-burst

pattern of activation.

Discussion
A COMPARISON OF L-LTP INDUCED BY O AND
NON-O PATTERNS OF STIMULATION
Many recent experiments have shown that
multiple trains (three or more) of 100-Hz stimulation are needed to produce L-LTP lasting 3 hr or
more (Frey et al. 1993; Matthies and Reymann
1993; Huang and Kandel 1994; Huang et al. 1994;
Nguyen et al. 1994; Nguyen and Kandel 1996). In
all three hippocampal regions (dentate gyrus, CA3,
CA1), this L-LTP requires protein and RNA synthe-

sis and is mediated by PKA and cAMP (Frey et al.

1993; Matthies and Reymann 1993; Huang et al.
1994; Nguyen et al. 1994; Nguyen and Kandel
1996). In contrast, short-lasting potentiation that
decays to baseline within 2 hr does not require
macromolecular synthesis and is induced with
fewer stimulus trains (Huang and Kandel 1994;
Nguyen and Kandel 1996).
Our results with O-burst L-LTP show (for the
first time in mouse hippocampal slices) that, like
conventional L-LTP, O L-LTP in the CA1 region requires the synthesis of new protein and RNA, as
well as PKA recruitment. The necessity for transcription and PKA activity occurred during a critical time window overlapping with b-burst stimulation, because delayed post-O application of inhibitors of either transciption or PKA failed to
block O L-LTP.
What makes b-burst stimulation an especially
effective protocol for induction of LTP? Stimulation
of hippocampal afferents initiates EPSPs in pyramidal cells but also recruits IPSPs in these cells by
means of feed-forward activation of interneurons
(Alger and Nicoll 1982). These feed-forward IPSPs
become refractory for 200-500 msec thereafter
(McCarren and Alger 1985). Hence, the O interburst interval of 200 msec delineates a period
when IPSPs are difficult to recruit. Repeated application of brief bursts of stimuli, at the 5-Hz O frequency, allows for more effective temporal summation of EPSPs in the absence of strong feed-forward inhibition that would otherwise truncate
excitatory transmission. Modest LTP can be produced by preceding a single brief burst with a
priming pulse at a 200-msec interval (Rose and
Dunwiddie 1986; Diamond et al. 1988), and robust
LTP can be elicited with as few as 10 bursts (40
pulses) delivered at the O frequency (Larson and
Lynch 1986; Larson et al. 1986; Staubli and Lynch
1987). Spaced stimulation at the O frequency of 5
Hz or at much lower interburst frequencies (e.g.,
1-sec duration trains of 100 Hz given every 1-10
min), may favor L-LTP expression because transcription (a requirement for L-LTP expression, for
review, see Huang et al. 1996a) may be more
strongly activated after such protocols. There is
evidence that CREB expression is increased after
repeated spaced stimulation leading to L-LTP in
hippocampal neurons but not after single-train
compressed stimulation (Impey et al. 1996). Brief,
compressed stimulation patterns may fail to induce
L-LTP because such protocols do not elicit sufficient calcium influx through NMDA receptor chan-

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Nguyen and Kandel

nels and through voltage-gated calcium channels

(e.g., L-type Ca 2+ channels) to trigger gene expression (for review, see Gallin and Greenberg 1995;
see also Malenka 1991). Recent work indicates that
L-type Ca 2+ channels may play a role in eliciting
synapse-to-nucleus signaling involving CREB phosphorylation (Deisseroth et al. 1996). These channels have slow inactivation kinetics and high activation thresholds, which would favor stronger,
more prolonged (i.e., temporally spaced) depolarization regimens for eliciting greater Ca 2+ influx
over the longer stimulation regimens required for
effective L-LTP induction (Bito et al. 1996; Deisseroth et al. 1996). Also, recovery from protein
phosphorylation may be slower following spaced
stimulation protocols (Bito et al. 1996), and this
may facilitate expression of more persistent forms
of synaptic potentiation (for review, see Huang et
al. 1996a).
Our present results extend earlier work in rats
(Larson and Lynch 1986, 1988; Larson et al. 1986)
by showing that temporally spaced patterns of
stimulation are not only effective for initiating LTP
but are also very effective for producing long-lasting, robust L-LTP in mouse hippocampal slices. In
contrast, delivering the same total number of
pulses in a temporally compressed fashion (60 Hz,
1 sec) did not elicit long-lasting facilitation in area
CA1. This latter protocol induced only a transient
potentiation that, unlike O-burst L-LTP, was independent of transcription and PKA activation. In retrospect, our present findings are in agreement
with, and further extend, earlier experiments that
have shown effective L-LTP induction in all three
hippocampal regions by repeated application of
1-sec-duration, lO0-Hz trains spaced 1-10 min
apart (Frey et al. 1993; Matthies and Reymann
1993; Huang et al. 1994; Nguyen and Kandel
1996).
Which induction protocol is most suited for
producing L-LTP? The present study does not provide any evidence to suggest that one single protocol is the most appropriate for inducing L-LTP. It
is evident that a number of different stimulation
regimens, varying in strength and duration, can induce robust L-LTP. Our findings do indicate, however, that despite its more subtle temporal characteristics, a O-burst pattern of stimulation can nonetheless invoke subcellular mechanisms and signal
transduction pathways that are, at least superficially, identical to those involved in non-O L-LTP.
Hence, a similar and conserved molecular program
of events, involving NMDA receptor activation,

cAMP, PKA, transcription, and translation, may underlie L-LTP induced by a variety of stimulus patterns.

RELATIONSHIP BETWEEN O-BURST LTP, FIRING

PATTERNS OF HIPPOCAMPAL NEURONS, AND
SOME FORMS OF LEARNING AND MEMORY
The first evidence to link the electrical activity
of CA1 neurons to spatial processes was provided
through chronic single-unit recordings made by
O'Keefe and Dostrovsky (1971). They found that
CA1 pyramidal cells fired selectively when awake,
unrestrained rats were placed in specific locations
in a defined environment during spatial exploration. These "place cells" encoded spatial relationships between distal cues, as well as direction and
speed of movement (Olton et al. 1978; O'Keefe
1979). It is known that CA1 pyramidal cells fire in
short bursts of two to seven spikes ("complex
spike" bursts) lasting 30 msec (Ranck 1973; Fox
and Ranck 1981; Muller et al. 1987; Thompson and
Best 1989). In rats exploring new surroundings,
this firing pattern occurs in conjunction with a
cholinergically regulated, 5-Hz O rhythm of membrane potential oscillations (Vanderwoff 1969;
Bland 1986; Eichenbaum et al. 1987; Muller et al.
1987; Otto et al. 1991; Lee et al. 1994; Ylinen et al.
1995).
What is the relationship between O-burst firing of CA1 pyramidal neurons and LTP induction?
In isolation, a single 30-msec burst of four pulses
does not generally produce robust LTP (but see
Huerta and Lisman 1995), but several bursts repeated with a 200-msec interburst interval (O frequency) induces substantial LTP in vivo and in
vitro (Larson and Lynch 1986; Larson et al. 1986;
Rose and Dunwiddie 1986; Staubli and Lynch
1987; Diamond et al. 1988; Pavlides et al. 1988).
Hippocampal pyramidal cells fire in bursts similar
to the O pattern found to be optimal for LTP induction, when rats are sampling odors in an olfactory learning task (Otto et al. 1991). These collective studies all support the notion that natural patterns of hippocampal neuronal activity, observed
during learning, can induce robust LTP. Hence,
there is ample evidence to believe that the molecular events invoked after O stimulation occur during
learning and lead to long-term synaptic plasticity
that may underlie the consolidation of long-term
memory. Also, there appears to be a strong correlation between O-burst firing of hippocampal CA1

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O-BURST L TP IN THE MOUSE HIPPOCAMPUS

neurons, synaptic LTP induced by this firing in

these same neurons, and some kinds of learning in
the intact animal (Otto et al. 1991; Huerta and Lisman 1995).

WHICH cAMP-INDUCIBLE GENES ARE CRITICAL

FOR O-BURST L-LTP?
L-LTP in all three regions of the rat hippocampus requires transcription and translation and can
be mimicked by pharmacological activation of PKA
and the cAMP signal transduction pathway (Frey et
al. 1993; Huang et al. 1994; Huang and Kandel
1994; Nguyen et al. 1994; Nguyen and Kandel
1996; see also Chavez-Noriega and Stevens 1994).
Our observations that O L-LTP was blocked by inhibitors of either gene transcription or PKA suggest that cAMP-inducible genes are involved in LLTP in area CA1 of the mouse hippocampus. Genetic evidence supporting this idea derives from
the observations that selective ablation of the ot
and 8 isoforms of CREB eliminated LTP and longterm memory in mice, without affecting short-term
memory (Bourtchouladze et al. 1994). Also, a galactosidase reporter gene driven by CRE (_cAMP response _element) is induced during L-LTP (Impey et
al. 1996). These studies thus show that a cAMPmodulated transcription factor, CREB, may activate
downstream effector genes for L-LTP expression in
the hippocampus. It is not yet k n o w n w h e t h e r
O-burst L-LTP depends on CREB-activated gene expression.
Mthough our study has implicated PKA in the
late phase of O-LTP, we cannot rule out a role for
PKA in the early stages (1-2 hr post-O) of potentiation (see Blitzer et al. 1995). The early phase
may be less sensitive to disruption by the modest
concentrations of PKA inhibitors used here and
may in fact be disrupted by higher concentrations
of inhibitors or by more extended application of
the inhibitors. However, at higher drug concentrations, other kinases may be affected and the specificity of action of these inhibitors may be compromised. Also, a recent study using transgenic overexpression of an inhibitory subunit of PKA (Abel et
al. 1997) has shown that short-lasting potentiation
induced by one or two 100-Hz trains is normal,
whereas L-LTP induced by four trains is selectively
disrupted. Hence, the available evidence to date
argues for a critical and apparently selective role
for PKA in the late phase of LTP.
In Aplysia, long-term (but not short-term) fa-

cilitation is mediated by translocation of the catalytic subunit of PKA to the nucleus of sensory neurons (Bacskai et al. 1993), where it may phosphorylate CREB and other transcription factors that
switch on cAMP-inducible immediate-early genes
(Dash et al. 1990; Kaang et al. 1993). One gene that
is induced in sensory neurons by cAMP is the Aplysia homolog of the mammalian transcription factor
C/EBP (ApC/EBP; Alberini et al. 1994). Blocking
the function of ApC/EBP in sensory neurons
blocks long-term but not short-term facilitation (A1berini et al. 1994).
In mice, genetic ablation of either a catalytic
subunit (O13-1) or a regulatory subunit (RI-[3) of
PKA eliminates the late phase of LTP in areas CA1
and CA3 (Huang et al. 1995; Qi et al. 1996). Overexpression of an inhibitory form of a PKA regulatory subunit (RI-00 also eliminates L-LTP in area
CA1 (Abel et al. 1997), suggesting that in the hippocampus, as in Aplysia, cAMP and PKA may play
a role in the recruitment of transcription factors
(e.g., CREB, C/EBP) for L-LTP expression.
Which effector genes are recruited during O
L-LTP? To date, no previous study has examined
the roles of gene induction and protein synthesis in
L-LTP induced by O-burst stimulation. That O L-LTP
was blocked by transcriptional and translational inhibitors suggests that late effector genes and the
proteins encoded by them may be recruited after
O-burst stimulation. Some candidate genes that
may be activated during L-LTP are those encoding
tissue-plasminogen activator (Qian et al. 1993; Frey
et al. 1995; Huang et al. 1996b), cell adhesion molecules (Bailey et al. 1992; Mayford et al. 1992; Cremer et al. 1994), and voltage-dependent K + channels (Kaang et al. 1992). The latter are particularly
noteworthy, because their expression levels can
shape synaptic efficacy (Kaang et al. 1992) and can
be regulated by cAMP and CREB (Mori et al. 1993).
If hippocampal LTP, like long-term facilitation in
Aplysia (Bailey and Kandel 1993), involves an organized repertoire of synaptic growth and differentiation (Desmond and Levy 1986a,b; Lisman and
Harris 1993; Edwards 1995), then these genes may
very well prove to be pivotal for triggering structural changes during L-LTP.

Acknowledgments
We thank Irma Trumpet, Harriet Ayers, and Chuck Lain
for preparing the manuscript and Danny Winder for critical
comments. This work was supported by the Howard Hughes
Medical Institute and grant MH 45923-07 to E.R.K.P.V.N. is

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Nguyen and Kandel

a fellow of the Medical Research Council of Canada. E.R.K.
is Senior Investigator of the Howard Hughes Medical
Institute.
The publication costs of this article were defrayed in
part by payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
USC section 1734 solely to indicate this fact.

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Received February 18, 1997; accepted in revised form April

9, 1997.

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Brief theta-burst stimulation induces a transcription-dependent late

phase of LTP requiring cAMP in area CA1 of the mouse
hippocampus.
P V Nguyen and E R Kandel
Learn. Mem. 1997 4: 230-243
Access the most recent version at doi:10.1101/lm.4.2.230

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