Hierarchical order of coexisting pre- and postsynaptic
forms of long-term potentiation at synapses
Ryong-Moon Shina,1, Keith Tullyb, Yan Lib, Jun-Hyeong Chob, Makoto Higuchia, Tetsuya Suharaa, and Vadim Y.
aMolecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555, Japan; andbDepartment of Psychiatry, McLean Hospital, Harvard
Medical School, Belmont, MA 02478
Edited* by Thomas C. Südhof, Stanford University School of Medicine, Palo Alto, CA, and approved September 28, 2010 (received for review July 6, 2010)
Synaptic rules that may determine the interaction between coex-
isting forms of long-term potentiation (LTP) at glutamatergic cen-
tral synapses remain unknown. Here, we show that two mecha-
nistically distinct forms of LTP could be induced in thalamic input to
the lateral nucleus of the amygdala (LA) with an identical pre-
synaptic stimulation protocol, depending on the level of post-
synaptic membrane polarization. One form of LTP, resulting from
pairing of postsynaptic depolarization and low-frequency presyn-
(“post-LTP”). The same stimulation in the absence of postsynaptic
depolarization led to LTP, which was induced and expressed pre-
synaptically (“pre-LTP”). The inducibility of coexisting pre- and
postsynaptic forms of LTP at synapses in thalamic input followed
a well-defined hierarchical order, such that pre-LTP was suppressed
when post-LTP was induced. This interaction was mediated by ac-
tivation of cannabinoid type 1 receptors by endogenous cannabi-
noids released in the lateral nucleus of the amygdala in response to
activation of the type 1 metabotropic glutamate receptor. These
results suggest a previously unknown mechanism by which the
hierarchy of coexisting forms of long-term synaptic plasticity in
the neural circuits of learned fear could be established, possibly
reflecting the hierarchy of memories for the previously experi-
enced fearful events according to their aversiveness level.
associative learning, which results from memorizing the tem-
poralassociation between biologically neutral conditioned stimuli
(CS) and aversive unconditioned stimuli (US) during behavioral
training (1, 2). In the course of auditory fear conditioning, signals
produced by the acoustic conditioned stimulus enter the lateral
nucleus of the amygdala (LA) through projections originating in
the auditory thalamus (thalamic input) and indirect projections
from the auditory cortex (cortical input) (3). The acquisition of
fear memory to auditory stimulation is mediated by long-term
potentiation (LTP)-like synaptic enhancements in the CS path-
ways, including both cortical and thalamic inputs to the LA (4–9).
Different forms of LTP could be observed, however, at synapses
in the amygdala (7, 8, 10–12) as well as in other regions of
the brain (13, 14), depending on the presynaptic activity levels
and degree of postsynaptic depolarization. Thus, conventional
pairing-induced LTP and spike timing-dependent LTP in tha-
lamic projections to the LA are expressed postsynaptically and
may implicate trafficking of AMPA receptors at stimulated syn-
apses (“post-LTP”) (8, 15), whereas LTP in cortical input to the
LA is expressed presynaptically, resulting from an increase in the
probability of neurotransmitter release (“pre-LTP”) (7). Little is
known, however, about whether the coexisting forms of LTP at
glutamatergic synapses interact with each other during the in-
duction process, and if they do, how such interactions could be
mediated. It prompted us to ask which synaptic mechanisms de-
ear conditioning is one of the best experimental models of
termine the order in which the coexisting forms of LTP in the CS
projections to the LA are induced.
Here, we report that the induction of LTP in thalamic input to
the LA, which is both induced and expressed postsynaptically,
suppresses the mechanisms of pre-LTP coexisting at the same
synapses, thus potentially preventing situations where different
forms of synaptic plasticity are simultaneously expressed.
GluR5 Kainate Receptor-Dependent Pre-LTP Is Readily Induced in
Thalamic Input to the LA. To explore the interactions between dif-
ferent forms of LTP in the CS pathways, we recorded excitatory
postsynaptic currents (EPSCs) in LA neurons evoked by stimula-
tion of either cortical or thalamic inputs to the LA (1, 16). Stim-
ulation of thalamic input for 2 min with paired pulses (50-ms
interpulse interval) at 2 Hz frequency and a holding potential
D, and E and Figs. S1A and S2 A–E), whereas the same induction
protocol failed to induce LTP in the cortico-amygdala pathway
specific. The inducibility of LTP under these conditions was in-
sensitive to changes in GABA-mediated inhibition (Fig. S3). Un-
like conventional pairing-induced and spike timing-dependent
LTP (7, 17), this form of synaptic potentiation was not blocked by
traacetic acid (BAPTA, 20 mM) in the recording pipette solution,
and therefore, it did not require postsynaptic Ca2+influx for its
induction (Fig. 1 F and H). Pretreatment of slices for 30 min
with the cell-permeable Ca2+chelator 1,2-Bis(2-aminophenoxy)
ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)
(BAPTA-AM) blocked the induction of LTP (Fig. 1 F and H and
Fig. S4), indicating that presynaptic Ca2+influx might be impli-
cated in the induction process. This form of LTP was insensitive
to both the metabotropic glutamate receptors (mGluR) antago-
the NMDA receptor antagonist D-(-)-2-Amino-5-phosphono-
pentanoic acid (D-APV, 50 μM) (Fig. 1 G and H). LTP was
completely blocked, however, by the selective antagonists of the
GluR5subunit-containing kainate receptors, (RS)-1-(2-Amino-2-
1 μM) (Fig. 1 G and H) or (S)-1-(2-Amino-2-carboxyethyl)-3-
Author contributions: R.-M.S. and V.Y.B. designed research; R.-M.S., K.T., Y.L., and J.-H.C.
performed research; M.H. and T.S. contributed new reagents/analytic tools; R.-M.S., K.T.,
Y.L., J.-H.C., M.H., T.S., and V.Y.B. analyzed data; and V.Y.B. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 2, 2010
| vol. 107
| no. 44
CaCl2, 1.0 MgSO4, 1.25 NaH2PO4, 26.0 NaHCO3, 10 glucose, and 0.1 picro-
toxin (unless noted otherwise) and equilibrated with 95% O2and 5% CO2
(pH 7.3–7.4) at room temperature (22–24 °C). Whole-cell recordings of
compound or unitary EPSCs were obtained from pyramidal neurons in the
lateral amygdala under visual guidance (differential interference contrast/
infrared optics) with an EPC-9 amplifier and Pulse v8.40 software (HEKA
Elektronik). The cells were classified as principal neurons based on their
appearance and their ability to show spike frequency adaptation to the
prolonged depolarizing current injection. Synaptic responses were evoked
by field stimulation of the fibers in either the external capsule (cortical in-
put) or the internal capsule (thalamic input) at 0.05 Hz. The patch electrodes
(3–5 MΩ resistance) contained (in mM) 120 K-methane-sulfonate, 5 NaCl, 1
MgCl2, 0.2 EGTA, 10 Hepes, 2 MgATP, and 0.1 NaGTP (adjusted to pH 7.2
with KOH). Currents were filtered at 1 kHz and digitized at 5 kHz. Unitary
EPSCs were evoked by low-intensity current pulses (20–40 μA; 100 μs dura-
tion) applied through a fine-tipped (∼2 μM), concentric stimulating elec-
trode consisting of a patch pipette that was coated with silver paint. The two
leads of the stimulus isolation unit (ISO-Flex, Master-8 stimulator; AMPI)
were connected to the inside of the pipette and the external silver coat. The
stimulating pipettes were positioned to activate either cortical or thalamic
input to the LA (Fig. 1A). The recording was used if the mean EPSC ampli-
tude showed a steep all-or-none threshold as a function of stimulating
current intensity and if there was no change in potency (the mean size of
responses, excluding failures of synaptic transmission) during double-pulse
stimulation with a 50-ms interpulse interval, indicating stimulation of a sin-
gle presynaptic input. The EPSC amplitude was measured as the difference
between the mean current during a prestimulus baseline and the peak
current over a 1- to 2-ms window. In all LTP experiments, the stimulus in-
tensity was adjusted to produce synaptic responses with an amplitude that
was ∼20–25% of maximum amplitude EPSC. For induction of pre-LTP, 240
paired presynaptic stimuli (with 50-ms interpulse intervals) were delivered at
2 Hz to the presynaptic fibers at a holding potential of −70 mV. For in-
duction of post-LTP, the same stimulation was delivered at a holding po-
tential of +30 mV. Summary LTP graphs were constructed by normalizing
data in 60s epochs to the mean value of the baseline EPSP.
ACKNOWLEDGMENTS. Support was provided by Grant-in-Aid for Scientific
Research 19390309 from the Japan Society for the Promotion of Science (to
R.-M.S.), Grant-in-Aid for the Molecular Imaging Program from the Ministry
of Education, Culture, Sports, Science, and Technology, Japan (to R.-M.S);
National Institutes of Health Grants MH083011 and MH090464 (to V.Y.B.);
United States Army Medical Research Acquisition Activity (USAMRAA) Grant
W81XWH-08-2-0126 (to V.Y.B.); and the Whitehall Foundation (V.Y.B.).
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