394 
STORING COVARIANCE BY THE ASSOCIATIVE 
LONG-TERM POTENTIATION AND DEPRESSION 
OF SYNAPTIC STRENGTHS IN THE HIPPOCAMPUS 
Pattic K. Stanton* and Terrence J. Sejnowski ? 
Department of Biophysics 
Johns Hopkins University 
Baltimore, MD 21218 
ABSTRACT 
In modeling studies of memory based on neural networks, both the selective 
enhancement and depression of synaptic strengths are required for efficient storage 
of information (Sejnowski, 1977a, b; Kohonen, 1984; Bienenstock et al, 1982; 
Sejnowski and Tesauro, 1989). We have tested this assumption in the hippocampus, 
a cortical structure of the brain that is involved in long-term memory. A brief, 
high-frequency activation of excitatory synapses in the hippocampus produces an 
increase in synaptic strength known as long-term potentiation, or LTP (Bliss and 
Lomo, 1973), that can last for many days. LTP is known to be Hebbian since it 
requires the simultaneous release of neurotransmitter from presynaptic terminals 
coupled with postsynaptic alepolarization (Kelso et al, 1986; Mannow and Miller, 
1986; Gustaffson et al, 1987). However, a mechanism for the persistent reduction of 
synaptic strength that could balance LTP has not yet been demonstrated. We stu- 
died the associative interactions between separate inputs onto the same dendritic 
trees of hippocampal pyramidal cells of field CA1, and found that a low-frequency 
input which, by itself, does not persistently change synaptic strength, can either 
increase (associative LTP) or decrease in strength (associative long.term depression 
or LTD) depending upon whether it is positively or negatively correlated in Ome 
with a second, high-frequency bursting input. LTP of synaptic strength is Hebbian, 
and LTD is anti.Hebbian since it is elicited by pairing presynaptic firing with post- 
synaptic hyperpolarization sufficient to block postsynaptic activity. Thus, associa- 
tive LTP and associative LTD are capable of storing information contained in the 
covariance between separate, converging hippocampal inputs. 
* Present address: Departments of Neuroscience and Neurology, Albert Einstein College 
of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461 USA. 
?Present address: Computational Neurobiology Laboratory, The Salk Institute, P.O. Box 
85800, San Diego, CA 92138 USA. 
Storing Covariance by Synaptic Strengths in the Hippocampus 395 
INTRODUCTION 
Associative LTP can be produced in some hippocampal neurons when low- 
frequency, (Weak) and high-frequency (Strong) inputs to the same cells are simultane- 
ously activated (Levy and Steward, 1979; Levy and Steward, 1983; Barrionuevo and 
Brown, 1983). When stimulated alone, a weak input does not have a long-lasting effect 
on synaptic strength; however, when paired with stimulation of a separate strong input 
sufficient to produce homosynaptic LTP of that pathway, the weak pathway is associa- 
tively potentiated. Neural network modeling studies have predicted that, in addition to 
this Hebbian form of plasticity, synaptic strength should be weakened when weak and 
strong inputs are anti-correlated (Sejnowski, 1977a,b; Kohonen, 1984; Bienenstock et al, 
1982; Sejnowski and Tesauro, 1989). Evidence for heterosynaptic depression in the hip- 
pocampus has been found for inputs that are inactive (Levy and Steward, 1979; Lynch et 
al, 1977) or weakly active (Levy and Steward, 1983) during the stimulation of a strong 
input, but this depression did not depend on any pattern of weak input activity and was 
not typically as long-lasting as LTP. 
Therefore, we searched for conditions under which stimulation of a hippocampal 
pathway, rather than its inactivity, could produce either long-term depression or potentia- 
tion of synaptic strengths, depending on the pattern of stimulation. The stimulus para- 
digm that we used, illustrated in Fig. 1, is based on the finding that bursts of stimuli at 5 
Hz are optimal in eliciting LTP in the hippocampus (Larson and Lynch, 1986). A high- 
frequency burst (STRONG) stimulus was applied to Schaffer collateral axons and a low- 
frequency (WEAK) stimulus given to a separate subicular input coming from the oppo- 
site side of the recording site, but terminating on dendrites of the same population of CA1 
pyramidal neurons. Due to the rhythmic nature of the strong input bursts, each weak 
input shock could be either superimposed on the middle of each burst of the strong input 
(IN PHASE), or placed symmetrically between bursts (OUT OF PHASE). 
RESULTS 
Extracellular evoked field potentials were recorded from the apical dendritic and 
somatic layers of CA1 pyramidal cells. The weak stimulus train was first applied alone 
and did not itself induce long-lasting changes. The strong site was then stimulated alone, 
which elicited homosynaptic LTP of the strong pathway but did not significantly alter 
amplitude of responses to the weak input. When weak and strong inputs were activated 
IN PHASE, there was an associative LTP of the weak input synapses, as shown in Fig. 
2a. Both the synaptic excitatory post-synaptic potential (e.p.s.p.) (Ae.p.s.p. = 4-49.8 :k 
7.8%, n=20) and population action potential (Aspike = +65.4 :k 16.0%, n=14) were 
significantly enhanced for at least 60 rain up to 180 min following stimulation. 
In contrast, when weak and strong inputs were applied OUT OF PHASE, they eli- 
cited an associative long-term depression (LTD) of the weak input synapses, as shown in 
Fig. 2b. There was a marked reduction in the population spike (46.5 :k 11.4%, n=10) 
with smaller decreases in the e.p.s.p. (-13.8 :k 3.5%, n=13). Note that the stimulus pat- 
terns applied to each input were identical in these two experiments, and only the relofive 
396 Stanton and Sejnowski 
phase of the weak and strong stimuli was altered. With these stimulus patterns, synaptic 
strength could be repeatedly enhanced and depressed in a single slice, as illustrated in Fig 
2c. As a control experiment to determine whether information concerning covariance 
between the inputs was actually a determinant of plasticity, we combined the in phase 
and out of phase conditions, giving both the weak input shocks superimposed on the 
bursts plus those between the bursts, for a net frequency of 10 Hz. This pattern, which 
resulted in zero covariance between weak and strong inputs, produced no net change in 
weak input synaptic strength measured by extracellular evoked potentials. Thus, the asso- 
b 
ASSOCIATIVE STIMULUS PARADIGMS 
IN:)ITI VE.LY CORRELATED - "IN PHASE" 
NEGATIVELY CORRELATED - "OUT OF PHASE 
Figure 1. Hippocampal slice preparation and stimulus paradigms. a: The in vitro hippo- 
campal slice showing recording sites in CA1 pyramidal cell somatic (stratum pyrami- 
dale) and dendritic (stratum radiatum) layers, and stimulus sites activating Schaffer col- 
lateral (STRONG) and commissural (WEAK) afferents. Hippocampal slices (400 l.m 
thick) were incubated in an interface slice chamber at 34-35 o C. Extracellular (1-5  
resistance, 2M NaCI filled) and intracellular (70-120 M, 2M K-acetate filled) record- 
ing electrodes, and bipolar glass-insulated platinum wire stimulating electrodes (50 l.m 
tip diameter), were prepared by standard methods (Mody et al, 1988). b: Stimulus para- 
digms used. Strong input stimuli (STRONG INPUT) were four trains of 100 Hz bursts. 
Each burst had 5 stimuli and the interburst interval was 200 msec. Each train lasted 2 
seconds for a total of 50 stimuli. Weak input stimuli (WEAK INPUT) wre four trains of 
shocks at 5 Hz frequency, each train lasting for 2 seconds. When these inputs were IN 
PHASE, the weak single shocks were superimposed on the middle of each burst of the 
strong input. When the weak input was OUT OF PHASE, the single shocks were placed 
symmetrically between the bursts. 
Storing Covariance by Synaptic Strengths in the Hippocampus 397 
ciative LTP and LTD mechanisms appear to be balanced in a manner ideal for the 
storage of temporal covariance relations. 
The simultaneous depolarization of the postsynaptic membrane and activation of 
glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype appears to be neces- 
sary for LTP induction (Coilingridge et al, 1983; Harris etal, 1984; Wigstrom and Gus- 
taftson, 1984). The sread of current from strong to weak synapses in the dendritic tree, 
a ASSOCIATIVE LONG.TERM POTENTIATION 
b A$OCIATIVE LONG.TERM DEPRESSION 
I I 
Pre Pot 
s IIIII IIIII IIIII IIIII    s 
/  
Time 
Figure 2. Illustration of associative long-term potentiation (LTP) and associative long- 
term depression (LTD) using extracellular recordings. a: Associative LTP of evoked 
exitatory postsynapti potentials (e.p.s.p.'s) and population action potential responses in 
the weak input. Test responses are shown before (Pre) and 30 min after (Post) applica- 
tion of weak stimuli in phase with the oactive strong input. b: Associative LTD of 
evoked e.p.s.p.'s and population spike responses in the weak input. Test responses are 
shown before (Pre) and 30 rain after (Post) application of weak stimuli out of phase with 
the coactive strong input c: Time course of the changes in population spike amplitude 
observed at each input for a typical experiment. Test responses from the strong input (S, 
open circles), show that the high-frequency bursts (5 pulses/100 Hz, 200 msec interburst 
interval as in Fig. 1) elicited synapse-specific LTP independent of other input activity. 
Test responses from the weak input (W, filled circles) show that stimulation of the weak 
pathway out of phase with the strong one produced associative LTD (Assoc LTD) of this 
input. Associative LTP (Assoc LTP) of the same pathway was then elicited following in 
phase stimulation. Amplitude and duration of associative LTD or LTP could be increased 
by stimulating input pathways with more trains of shocks. 
398 Stanton and Sejnowski 
coupled with release of glutamate from the weak inputs, could account for the ability of 
the strong pathway to associatively potentiate a weak one (Kelso et al, 1986; Malinow 
and Miller, 1986; Gustaffson et al, 1987). Consistent with this hypothesis, we find that 
the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APS, 101.12vl) blocks 
induction of associative LTP in CA1 pyramidal neurons (data not shown, n=5). In con- 
trast, the application of AP5 to the bathing solution at this same concentration had no 
significant effect on associative LTD (data not shown, n=6). Thus, the induction of LTD 
seems to involve cellular mechanisms different from associative LTP. 
The conditions necessary for LTD induction were explored in another series of 
experiments using intracellular recordings from CA1 pyramidal neurons made using 
standard techniques (Mody et al, 1988). Induction of associative LTP (Fig 3; WEAK 
S+W IN PHASE) produced an increase in amplitude of the single cell evoked e.p.s.p. and 
a lowered action potential threshold in the weak pathway, as reported previously (Bar- 
rionuevo and Brown, 1983). Conversely, the induction of associative LTD (Fig. 3; 
WEAK S+W OUT OF PHASE) was accompanied by a long-lasting reduction of e.p.s.p. 
amplitude and reduced ability to elicit action potential firing. As in control extracellular 
experiments, the weak input alone produced no long-lasting alterations in intracellular 
e.p.s.p.'s or firing properties, while the strong input alone yielded specific increases of 
the suong pathway e.p.s.p. without altering e.p.s.p.'s elicited by weak input stimulation. 
WEAK 
($UBICULUM) 
STRONG 
SCHAFFER) 
PRE 30 rain POST 30 rain POST 
S+W OUT OF PHASE S+W IN PHASE 
4mV 
_.i25 mec 
Figure 3. Demonstration of associative LTP and LTD using intracellular recordings from 
a CA1 pyramidal neuron. Intracellular e.p.s.p.'s prior to repetitive stimulation (Pre), 30 
rain after out of phase stimulation (S+W OUT OF PHASE), and 30 rain after subse- 
quent in phase stimuli (S+W IN PHASE). The strong input (Schaffer collateral side, 
lower traces) exhibited LTP of the evoked e.p.s.p. independent of weak input activity. 
Out of phase stimulation of the weak (Subicular side, upper traces) pathway produced a 
marked, persistent reduction in e.p.s.p. amplitude. In the same cell, subsequent in phase 
stimuli resulted in associative LTP of the weak input that reversed the LTD and enhanced 
amplitude of the e.p.s.p. past the original baseline. (RMP = -62 mV, R N = 30 M,Q) 
Storing Covarianee by Synaptie Strengths in the Hippocampus 399 
A weak stimulus that is out of phase with a strong one arrives when the postsynap- 
tic neuron is hyperpolarized as a consequence of inhibitory postsynaptic potentials and 
afterhyperpolarization from mechanisms intrinsic to pyramidal neurons. This suggests 
that postsynaptic hyperpolarization coupled with presynaptic activation may trigger LTD. 
To test this hypothesis, we injected current with intraceHular microelectrodes to hyperpo- 
larize or alepolarize the cell while stimulating a synapfic input. Pairing the injection of 
alepolarizing current with the weak input led to LTP of those synapses (Fig. 4a; STIM; 
a  rain POST 
PRE STIM ,,,- DEPOL 
$TIM (S Hz) 
25 euec 
CONTROL '"-'' ._,...r I 
STIM (S Hz) 
RE 
CONTROL 
($UIICULUM) 
0 rain POST 
$TIM + HYPERPOL 
Figure 4. Pairing of postsynaptic hyperpolarization with stimulation of synapses on CA1 
hippocampal pyramidal neurons produces LTD specific to the activated pathway, while 
pairing of postsynaptic alepolarization with synaptic stimulation produces synapse- 
specific LTP. a: Intracellular evoked e.p.s.p.'s are shown at stimulated (STIM) and 
unstimulated (CONTROL) pathway synapses before (Pre) and 30 rain after (Post) pair- 
ing a 20 mV alepolarization (constant current +2.0 hA) with 5 Hz synaitic stimulation. 
The stimulated pathway exhibited associative LTP of the e.p.s.p., while the control, 
unstimulated input showed no change in synaptic strength. (RMP = .65 mV; R N = 35 
M) b: Intracellular e.p.s.p.'s are shown evoked at stimulated and control pathway 
synapses before (Pre) and 30 min after (Post) pairing a 20 mV hyperpolarization (con- 
stant current -1.0 hA) with 5 Hz synaptic stimulation. The input (STIM) activated during 
the hyperpolarization showed associative LTD of synaptic evoked e.p.s.p.'s, while 
synaptic strength of the silent input (CONTROL) was unaltered. (RMP = -62 mY; R N = 
38 Mi"i) 
400 Stanton and Sejnowski 
+64.0 -9.7%, n=4), while a control input inactive during the stimulation did not change 
(CONTROL), as reported previously (Kelso et al, 1986; Malinow and Miller, 1986; Gus- 
taffson et al, 1987). Conversely, prolonged hyperpolarizing current injection paired with 
the same low-frequency stimuli led to induction of LTD in the stimulated pathway (Fig. 
4b; STIM; -40.3:1: 6.3%, n=6), but not in the unstimulated pathway (CONTROL). The 
application of either alepolarizing current, hyperpolarizing current, or the weak 5 Hz 
synaptic stimulation alone did not induce long-term alterations in synaptic strengths. 
Thus, hyperpolarization and simultaneous presynaptic activity supply sufficient condi- 
tions for the induction of LTD in CA1 pyramidal neurons. 
CONCLUSIONS 
These experiments identify a novel form of anti-Hebbian synaptic plasticity in the 
hippocampus and confirm predictions made from modeling studies of information storage 
in neural networks. Unlike previous reports of synaptic depression in the hippocampus, 
the plasticity is associative, long-lasting, and is produced when presynaptic activity 
occurs while the postsynaptic membrane is hyperpolarized. In combination with Hebbian 
mechanisms also present at hippocampal synapses, associative LTP and associative LTD 
may allow neurons in the hippocampus to compute and store covariance between inputs 
(Sejnowski, 1977a, b; Stanton and Sejnowski, 1989). These finding make temporal as 
well as spatial context an important feature of memory mechanisms in the hippocampus. 
Elsewhere in the brain, the receptive field properties of cells in cat visual cortex 
can be altered by visual experience ptired with iontophoretic excitation or depression of 
cellular activity (Fregnac et al, 1988; Greuel et al, 1988). In particular, the chronic hyper- 
polarization of neurons in visual cortex coupled with presynaptic mmsmitter release leads 
to a long-term depression of the active, but not inactive, inputs from the lateral geniculate 
nucleus (Reiter and Stryker, 1988). Thus, both Hebbian and anti-Hebbian mechanisms 
found in the hippocampus seem to also be present in other brain areas, and covariance of 
firing patterns between converging inputs a likely key to understanding higher cognitive 
function. 
This research was supported by grants from the National Science Foundation and 
the Office of Naval research to TJS. We thank Drs. Charles Stevens and Richard Morris 
for discussions about related experiments. 
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