Download Long-Term Depression in Identified Stellate Neurons of Juvenile Rat

J Neurophysiol 97: 727–737, 2007.
First published November 29, 2006; doi:10.1152/jn.01089.2006.
Long-Term Depression in Identified Stellate Neurons of Juvenile Rat
Entorhinal Cortex
Pan-Yue Deng and Saobo Lei
Department of Pharmacology, Physiology and Therapeutics, School of Medicine and Health Sciences, University of North Dakota,
Grand Forks, North Dakota
Submitted 11 October 2006; accepted in final form 22 November 2006
INTRODUCTION
Long-term potentiation (LTP) and depression (LTD) are
considered as the cellular models for learning and memory
(Izquierdo 1994). The entorhinal cortex (EC) is part of a
network that aids in the consolidation and recall of memories
(for reviews, see Dolcos et al. 2005; Haist et al. 2001; Squire
et al. 2004; Steffenach et al. 2005). The EC has been regarded
as the gateway to the hippocampus because it mediates the
majority of connections between the hippocampus and other
cortical areas (Witter et al. 1989, 2000). Sensory inputs converge onto the superficial layers (layers I–III) of the EC
(Burwell 2000), which give rise to dense projections to the
hippocampus (Amaral and Witter 1995). On the other hand,
neurons in the deep layers of the EC (layers IV–VI) relay a
large portion of hippocampal output projections back to the
superficial layers of the EC (Dolorfo and Amaral 1998a,b;
Gloveli et al. 2001; Kohler 1986, 1988; van Haeften et al.
2003) and to other cortical areas (Amaral and Witter 1995;
Address for reprint requests and other correspondence: S. Lei, Dept. of
Pharmacology, Physiology and Therapeutics, School of Medicine and Health
Sciences, University of North Dakota, Grand Forks, ND 58203 (E-mail:
[email protected]).
www.jn.org
Witter et al. 1989). Similar to other synapses in the brain, the
EC expresses N-methyl-D-aspartate (NMDA) receptor-dependent LTP (Alonso et al. 1990; de Curtis and Llinas 1993; Yang
et al. 2004) and LTD (Bouras and Chapman 2003; Cheong et
al. 2002; Kourrich and Chapman 2003; Solger et al. 2004;
Yang et al. 2004; Zhou et al. 2005). However, the synapse
specificity and the cellular and molecular mechanisms underlying the long-term plasticity in the EC remain to be determined.
About 70% of the neurons in layer II of the EC are stellate
neurons (Klink and Alonso 1997) the axons of which form the
perforant path that innervates the dentate gyrus granule cells,
CA1 and CA3 pyramidal neurons, and several subtypes of
hippocampal interneurons (Ruth et al. 1982, 1988; Steward and
Scoville 1976). Stellate neurons themselves receive glutamatergic innervations from the pyramidal neurons in the deep
layers of the EC (Witter et al. 2000). The excitability of the
stellate neurons therefore is likely to play a pivotal role in
controlling the functions of the hippocampus. Whereas the
cellular and molecular mechanisms of long-term plasticity at
hippocampal synapses are well studied, those at the EC synapses have not been determined. In the present study, we
studied the long-term plasticity at synapses formed between
layer II stellate neurons and the inputs of the pyramidal
neurons in the deep layers of the EC in juvenile rats. Our
results indicate that stellate neuron synapses display NMDA
receptor-dependent LTD with no expression of LTP. We have
also shown that the expression of LTD was mediated by
AMPA receptor endocytosis that required the functions of
calcineurin and ubiquitin-proteasome system.
METHODS
Hippocampal slice preparation
Horizontal hippocampal slices (400 ␮m) including the EC, subiculum and hippocampus were cut using a Vibratome (Leica VT1000S)
usually from 15- to 22-day-old Sprague Dawley rats as described
previously (Deng and Lei 2006; Deng et al. 2006). After being deeply
anesthetized with isoflurane, rats were decapitated, and their brains
were dissected out in ice-cold saline solution that contained (in mM)
130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 5.0
MgCl2, and 10 glucose, saturated with 95% O2-5% CO2, pH 7.4.
Slices were initially incubated in the preceding solution at 35°C for 40
min for recovery and then kept at room temperature (⬃24°C) until
use. All animal procedures conformed to the guidelines approved by
the University of North Dakota Animal Care and Use Committee.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0022-3077/07 $8.00 Copyright © 2007 The American Physiological Society
727
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
Yue P-Y, Lei S. Long-term depression in identified stellate neurons of
juvenile rat entorhinal cortex. J Neurophysiol 97: 727–737, 2007. First
published November 29, 2006; doi:10.1152/jn.01089.2006. The entorhinal cortex (EC) serves as a gateway to the hippocampus and plays
a pivotal role in memory processing in the brain. Superficial layers of
the EC convey the cortical input projections to the hippocampus,
whereas deep layers of the EC relay hippocampal output projections
back to the superficial layers of the EC or to other cortical regions.
Whereas the EC expresses long-term potentiation (LTP) and depression (LTD), the underlying cellular and molecular mechanisms have
not been determined. Because the axons of the stellate neurons in
layer II of the EC form the perforant path that innervates the dentate
gyrus granule cells of the hippocampus, we studied the mechanisms
underlying the long-term plasticity in identified stellate neurons.
Application of high-frequency stimulation (100 Hz for 1 s, repeated 3
times at an interval of 10 s) or forskolin (50 ␮M) failed to induce
significant changes in synaptic strength, whereas application of pairing (presynaptic stimulation at 0.33 Hz paired with postsynaptic
depolarization from ⫺60 to ⫺10 mV for 5 min) or low-frequency
stimulation (LFS, 1 Hz for 15 min) paradigm-induced LTD. Pairingor LFS-induced LTDs were N-methyl-D-aspartate receptor-dependent
and occluded each other suggesting that they have the similar cellular
mechanism. Pairing-induced LTD required the activity of calcineurin
and involved AMPA receptor endocytosis that required the function
of ubiquitin–proteasome system. Our study provides a cellular mechanism that might in part explain the role of the EC in memory.
728
P.-Y. DENG AND S. LEI
Whole cell and perforated-patch recordings
Recordings of evoked field potentials
FIG. 1. Recording method and identification of stellate neurons in the
entorhinal cortex (EC). A: placement of recording (R) and stimulation (S)
electrodes in the medial EC. The schematic illustration was adapted from
Aicardi et al. (2004). DG, dentate gyrus; SubC, subiculum; PER, perirhinal. B:
stellate neuron identified under an infrared video microscopy. C: voltage
responses (top) generated by current injection from ⫹0.1 to ⫺1 nA at an
interval of ⫺0.1 nA (bottom). Note the “sag” response generated by hyperpolarizing current injection. Usually, rebound action potentials were generated on
the termination of the hyperpolarizing current pulses because on immediate
formation of whole cell recordings, low concentration of QX-314 (0.2 mM) in
the recording pipettes was not effective at blocking Na⫹ channels. D: plot of
percentage of sag response vs. the hyperpolarizing currents injected (n ⫽ 14).
J Neurophysiol • VOL
An extracellular recording pipette (2–5 M⍀) containing 2 M NaCl
was positioned in layer II of the EC and a concentric bipolar stimulation electrode (Frederick Haer) was placed in layer IV to stimulate
the inputs from the deep layers. Constant current pulses (0.1 ms,
100 –300 ␮A) were delivered using a stimulus generator (WPI, Model
A300) and a stimulus isolation unit (Model A360). Evoked field
potentials were amplified 100 times, filtered at 1 Hz, and digitized at
10 kHz. The slope of evoked field potential responses was measured
using the program Clampfit (Axon Instruments).
Peak-scaled nonstationary variance analysis
Peak-scaled nonstationary variance analysis was used to calculate
the conductance and numbers of synaptic AMPA receptors (Benke et
al. 1998; Lei and McBain 2002; Lei et al. 2003; Traynelis and
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
Whole cell patch-clamp recordings using an Axopatch 200B or a
Multiclamp 700B (Axon Instruments, Foster City, CA) in current- or
voltage-clamp mode were made from stellate neurons in layer II of the
EC visually identified with infrared video microscopy (Olympus
BX51WI) and differential interference contrast optics. Unless stated
otherwise, recording electrodes were filled with the following (in
mM): 100 K-gluconate, 0.6 EGTA, 5 MgCl2, 8 NaCl, 2 ATP2Na, 0.3
GTPNa, 40 HEPES, and 0.2 QX-314, pH 7.4. For perforated-patch
recordings, recording pipettes were tip-filled with the above intracellular solution and then back-filled with freshly prepared intracellular
solution containing amphotericin B (200 ␮g/ml, Calbiochem, San
Diego, CA) (Rae et al. 1991). Patch pipettes had resistances of 4 – 6
M⍀ when filled with the preceding solution. Stable series resistances
(50 –70 M⍀) were usually obtained ⬃30 min after forming a gigaohm
seal. The extracellular solution comprised (in mM) 130 NaCl, 24
NaHCO3, 3.5 KCl, 1.25 NaHPO4, 2.5 CaCl2, 1.5 MgCl2, 10 glucose,
and 0.01 bicuculline methobromide, saturated with 95% O2-5% CO2,
pH 7.4. Stellate neurons were identified by their location, shape, and
electrophysiological properties. Stellate neurons are located in layer II
or the border of layer II and III, and they have larger and polygonal
soma with variable number of main dendrites radiating out from the
cell body but are devoid of a clearly dominant dendrite (van der
Linden and Lopes da Silva 1998) (Fig. 1, A and B). These neurons
have unique electrophysiological properties, i.e., hyperpolarizing current pulse injection always caused the membrane potential to attain an
early peak and then “sag” to a steady-state level (Alonso and Klink
1993) (Fig. 1C). The generation of sag response is due to the selective
expression of hyperpolarization-activated cation channels (H-channels) in stellate neurons (Dickson et al. 2000). Because QX-314 is a
potential H-channel blocker (Bischoff et al. 2003) and its inclusion in
the recording pipettes would reduce the sag response, we initially tried
to use intracellular solution that did not contain QX-314. However,
the contamination of the action potential prevented reliable recordings
of excitatory postsynaptic currents (EPSCs). We therefore included a
low concentration (0.2 mM) of QX-314 in the recording pipettes.
After formation of whole cell recordings, we quickly (usually in ⬍2
min) recorded the voltage responses by injecting currents from ⫹0.1
to ⫺1 nA at an interval of ⫺0.1 nA (Fig. 1C) in current clamp. In this
condition, we can still observe nice initial sag response but prevented
the contamination of action potentials on the recordings of EPSCs
when QX-314 was dialyzed into the soma and dendrites later. The
effect of QX-314 on sag response was likely to be negligible because
action potentials were still observed in this period (Fig. 1C). We used
the method developed by van der Linden and Lopes da Silva (1998)
and Dorval and White (2005) by calculating the percentage of the sag
according to the equation [(peak – steady-state)/peak ⫻ 100%]. Plot
of the percentage of the sag responses versus the currents injected
showed a linear relationship (Fig. 1D). Because Dorval and White
(2005) defined stellate neurons as cells displaying ⬎20% sag response
and our cells surpassed this criterion at every current injected (Fig.
1D), we defined cells exhibiting ⬎20% sag in response to ⫺1 nA
hyperpolarizing current injection as stellate neurons. After identifying
the stellate neurons in current clamp, we switched to voltage clamp to
record AMPA receptor-mediated EPSCs evoked by placing a stimulation electrode in layer IV to stimulate the inputs from the deep layers
(Fig. 1A). The holding potential was at ⫺60 mV. In this condition, the
recorded responses were completely blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) (10 ␮M), indicating that they were mediated
by AMPA/kainate receptors. Series resistance was rigorously monitored by the delivery of 5-mV voltage steps after each evoked current.
Experiments were discontinued if the series resistance changed by
⬎10%. Synaptic responses were evoked at 0.33 Hz by low-intensity
stimulation (80- to 100-␮s duration; 40- to 80-␮A intensity) via a
constant-current isolation unit (A360; World Precision Instrument,
Sarasota, FL) connected to a patch electrode filled with oxygenated
extracellular solution. Data were filtered at 2 kHz, digitized at 10 kHz,
acquired on-line, and analyzed off-line with pClamp9 software (Axon
Instruments). Synaptic responses were included in analysis if the rise
time and decay time constants were monotonic and possessed no
apparent multiple or polysynaptic waveforms. Three paradigms were
used to induce long-term plasticity at the stellate neuron synapses:
high-frequency stimulation (HFS, 100 Hz for 1 s, repeated 3 times at
an interval of 10 s, holding potential: ⫺60 mV for whole cell and
perforated-patch recordings), pairing (presynaptic stimulation at 0.33
Hz paired with postsynaptic depolarization from ⫺60 to ⫺10 mV for
5 min) and low-frequency stimulation (LFS, 1 Hz for 15 min, holding
potential: ⫺60 mV). HFS was conducted with PluseMaster A300
(World Precision Instrument).
LTD IN ENTORHINAL CORTEX STELLATE NEURONS
Data analysis
Data are presented as the means ⫾ SE. Student’s paired or unpaired
t-test or ANOVA was used for statistical analysis as appropriate; P
values are reported throughout the text and significance was set as
P ⬍ 0.05.
Chemicals
AM251, (⫾)-amino-4-carboxy-methyl-phenylacetic acid (MCPG),
lactacystin, pep2m peptide (KRMKVAKNAQ), and (2S,2⬘R,3⬘R)-2(2⬘,3⬘-dicarboxycyclopropyl)glycine (DCG-IV) were products of Tocris (Ellisville, MO). Epoxomicin and forskolin were purchased from
BIOMOL (Plymouth Meeting, PA). All other compounds were products of Sigma-Aldrich (St. Louis, MO).
RESULTS
Stellate neuron synapses are devoid of HFS-induced LTP but
exhibit LFS- and pairing-induced LTD
Stellate neurons in layer II of the EC receive glutamatergic
innervation from the pyramidal neurons in the deep layers and
the axons of stellate neurons form the perforant path that
innervates the dentate gyrus granule cells. We initially examined the long-term plasticity at the stellate neuron synapses.
We recorded from the stellate neurons in layer II of the EC and
stimulated the inputs from the deep layers by placing a stimulation electrode in layer IV (Fig. 1A). After recording sag
response in current clamp to identify stellate neurons, we
switched to voltage clamp to record AMPA receptor-mediated
EPSCs. GABAA responses were blocked by including bicuculline (10 ␮M) in the extracellular solution. In this condition,
HFS (100 Hz for 1 s, repeated 3 times at an interval of 10 s),
a protocol used widely to induce LTP, failed to induce significant changes in AMPA EPSCs (99 ⫾ 7% of control, n ⫽ 8,
P ⫽ 0.87, Fig. 2A). We tested the efficacy of the induction
protocol by recording from CA1 pyramidal neurons of the
hippocampus and placing the stimulation electrode in the
stratum radiatum to stimulate Schaffer collateral fibers. At this
synapse type, application of the same HFS paradigm induced
robust posttetanic potentiation and LTP (167 ⫾ 19% of control, n ⫽ 5, P ⫽ 0.02, Fig. 2B), excluding the inefficiency of
the induction protocol. To test the possibility that intracellular
constituents required for LTP induction in stellate neurons of
the EC were washed out during the period of whole cell
FIG. 2. Stellate neuron synapses do not express high-frequency stimulation (HFS)-induced long-term potentiation (LTP). A: HFS
(100 Hz for 1 s, repeated 3 times at an interval
of 10 s) failed to change synaptic strength (n ⫽
8). Top: current traces averaged from 20 excitatory postsynaptic currents (EPSCs) before
and after the application of HFS. B: application of the same HFS-induction paradigm induced LTP at the Schaffer collateral-CA1 pyramidal neuron synapses of the hippocampus
(n ⫽ 5). C: HFS failed to induce LTP when
applied at the stellate neuron synapses using
perforated-patch recordings (n ⫽ 6). D and E:
application of HFS in normal (2.5 mM, n ⫽ 5,
D) or higher (5 mM, n ⫽ 5, E) extracellular
Ca2⫹ did not induce LTP when field potentials
were recorded from layer II of the EC and the
inputs from the deep layers were stimulated.
Top left: traces averaged from 10 field excitatory postsynaptic potentials (fEPSPs) recorded
at the time indicated. Top right: 2 averaged
fEPSPs in the left were overlaid in an enlarged
scale to show that there was no change in the
slope and amplitude of the fEPSPs. F: HFS
failed to induce LTP in slices cut from adult
rats (n ⫽ 7). The figure was arranged in the
same way as D and E.
J Neurophysiol • VOL
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
Jaramillo 1998; Traynelis et al. 1993) before and after the induction of
LTD. The recorded AMPA receptor EPSCs were initially inspected
visually to exclude those responses contaminated with spontaneous
synaptic activity. Only those traces showing fast rise time (20 – 80%
rise time ⬍0.8 ms) and smooth decay (successful fitting of a period of
87 ms from the peak by two exponential functions) were selected for
analysis. The selected EPSCs were aligned and averaged. The average
response was scaled to the peak and subtracted from individual
responses to compute the variance. A period of 0 – 87 ms commencing
at the EPSC peak was selected for analysis. The average EPSC
response was then divided into 100 equally sized bins and the
corresponding variances pooled. The binned variance was plotted
against the mean current amplitude, and the single-channel current
and the number of AMPA receptors were calculated by fitting the data
according to the following equation: ␴2 ⫽ iI – I2/N ⫹ ␴base, where ␴2
is the variance, I is the mean current, N is the number of open
channels, i is the single-channel current, and ␴base is the background
variance. The single-channel conductance was measured by ␥ ⫽
i/(E – Erev), where E is the holding potential, and Erev is the
reversal potential that was measured to be close to 0 mV under our
recording conditions.
729
730
P.-Y. DENG AND S. LEI
⫺60 to ⫺10 mV for 5 min without presynaptic stimulation did
not significantly change AMPA receptor-mediated EPSCs
(98 ⫾ 7% of control, n ⫽ 6, P ⫽ 0.76, Fig. 3A). To test
whether LTP could be induced after preconditioning the slices
with the pairing-induced LTD, we applied HFS induction
protocol after the expression of the pairing-induced LTD. In
five experiments, application of the pairing protocol induced
the expression of LTD (56 ⫾ 9% of control, n ⫽ 5), but
subsequent application of HFS did not significantly change the
synaptic strength (97 ⫾ 2% of the value prior to HFS paradigm, n ⫽ 5, P ⫽ 0.11, Student’s paired t-test, data not shown),
suggesting that there is no bidirectionality of synaptic plasticity. Furthermore, application of the pairing paradigm induced
robust LTP at the Schaffer collateral-CA1 pyramidal neuron
synapses of the hippocampus (213 ⫾ 28% of control, n ⫽ 6,
P ⫽ 0.009, Fig. 3B) excluding the inefficacy of the induction
paradigm. We also used perforated-patch recordings to test
whether intracellular molecules required for LTP induction
were washed out during the processes of whole cell recordings.
In this recording configuration, application of the pairing
paradigm still induced LTD (57 ⫾ 5% of control, n ⫽ 9, P ⬍
0.0001, Fig. 3C). We also performed the experiments in high
concentration of extracellular Ca2⫹ (5 mM). Application of the
pairing paradigm in 5 mM Ca2⫹ still induced LTD (47 ⫾ 8%
of control, n ⫽ 7, P ⫽ 0.0005, Fig. 3D).
We next examined whether the stellate neuron synapses
express chemical LTP by applying forskolin, an adenylyl
cyclase activator that increases the generation of cyclic AMP
and induces LTP (Huang et al. 1994; Maccaferri et al. 1998;
Weisskopf et al. 1994). Application of forskolin (50 ␮M) did
not induce LTP at the stellate neuron synapses (95 ⫾ 6% of
control, n ⫽ 7, P ⫽ 0.44, Fig. 3E), whereas application of the
same concentration of forskolin induced robust LTP at the
FIG. 3. Stellate neuron synapses express
pairing- and low-frequency stimulation
(LFS)-induced long-term depression (LTD)
but not forskolin-induced LTP. A: application of pairing (presynaptic stimulation at
0.33 Hz paired with postsynaptic depolarization from ⫺60 to ⫺10 mV for 5 min) paradigm induced LTD (n ⫽ 20), whereas
postsynaptic depolarization from ⫺60 to
⫺10 mV for 5 min without presynaptic stimulation did not change synaptic strength
(n ⫽ 6). Top: traces averaged from 10
AMPA EPSCs as indicated in the figure. The
rest of the figure was arranged in the same
way. B: application of the same pairing paradigm induced LTP at the Schaffer collateralCA1 pyramidal neuron synapses (n ⫽ 6). C:
pairing-induced LTD at the stellate neuron
synapses recorded by perforated patch recordings (n ⫽ 9). D: pairing still induced LTD
when extracellular Ca2⫹ concentration was elevated to 5 mM (n ⫽ 7). E: application of
forskolin (50 ␮M) did not induce LTP at the
stellate neuron synapses (n ⫽ 7). F: stellate
neuron synapses expressed LFS (1 Hz, for 15
min)-induced LTD (n ⫽ 6).
J Neurophysiol • VOL
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
recordings, we utilized perforated-patch recordings to perform
the experiments. Application of the HFS induction protocol
still failed to induce LTP (101 ⫾ 8% of control, n ⫽ 6, P ⫽
0.92, Fig. 2C) in this recording configuration.
The preceding experiments were performed in the presence
of bicuculline to block GABAA receptors. To test whether
inclusion of bicuculline prevented the induction of LTP, we
recorded the evoked field potentials in the absence of bicuculline from layer II of the EC by positioning the stimulation
electrode in layer IV to stimulate the inputs from the deep
layers. In the presence of normal Ca2⫹ concentration (2.5
mM), application of the HFS induction paradigm failed to
induce LTP (105 ⫾ 14% of control, n ⫽ 5, P ⫽ 0.72, Fig. 2D).
Furthermore, increasing the extracellular Ca2⫹ concentration
to 5 mM failed to induce LTP (99 ⫾ 5% of control, n ⫽ 5, P ⫽
0.91, Fig. 2E). Because all the preceding experiments were
conducted in slices from juvenile rats, we then extended our
experiments to adult rats to test whether the expression of LTP
was developmentally regulated. Application of the HFS paradigm did not induce LTP in slices cut from adult rats (94 ⫾ 6%
of control, n ⫽ 7, P ⫽ 0.35, Fig. 2F). Taken together, we
concluded that the stellate neuron synapses do not express
HFS-induced LTP.
We then used the pairing paradigm (presynaptic stimulation
at 0.33 Hz together with postsynaptic depolarization from ⫺60
to ⫺10 mV for 5 min) to test whether stellate neuron synapses
exhibit other forms of LTP. Application of the pairing paradigm did not induce LTP but instead induced LTD (60 ⫾ 5%
of control, n ⫽ 20, P ⬍ 0.00001, 20 min after the induction
protocol, Fig. 3A). Although the pairing-induced LTD in some
cells developed slowly and stabilized ⬃15 min after the induction protocol (Fig. 3A), it was not due to the run-down of
synaptic strength because postsynaptic depolarization from
LTD IN ENTORHINAL CORTEX STELLATE NEURONS
731
mossy fiber-CA3 pyramidal neuron synapses of the hippocampus (188 ⫾ 17% of control, n ⫽ 5, P ⫽ 0.006, data not shown),
suggesting that the stellate neuron synapses do not express
forskolin-induced chemical LTP.
Because application of LFS using field potential recordings
induced LTD in the superficial layers of EC (Cheong et al.
2002; Kourrich and Chapman 2003; Solger et al. 2004), we
then tested whether stellate neuron synapses express this type
of LTD. Application of LFS (1 Hz for 15 min) produced robust
LTD (59 ⫾ 11% of control, n ⫽ 6, P ⫽ 0.01, Fig. 3F),
suggesting that stellate neuron synapses express LFS-induced
LTD.
Among all the paradigms used to induce long-term synaptic
plasticity, we observed the pairing- and LFS-induced LTD. We
next examined whether increases in intracellular Ca2⫹ were
required for these two forms of LTD. Inclusion of bis-(oaminophenoxy)-N,N,N’,N’-tetraacetic acid (BAPTA; 30 mM)
in the recording pipettes blocked pairing-induced LTD (95 ⫾
4% of control, n ⫽ 7, P ⫽ 0.22, Fig. 4A), indicating that an
increase in intracellular Ca2⫹ is required for pairing-induced
LTD. Similarly, inclusion of BAPTA (30 mM) in the recording
pipettes blocked LFS-induced LTD (96 ⫾ 2% of control, n ⫽
6, P ⫽ 0.16, Fig. 4B). These results suggest that both pairingand LFS-induced LTDs require an increase in postsynaptic
Ca2⫹.
We then tested whether NMDA receptors were required for
pairing- or LFS-induced LTDs. Application of DL-APV (100
␮M) blocked both pairing-induced LTD (99 ⫾ 9% of control,
n ⫽ 11, P ⫽ 0.96, Fig. 4C) and LFS-induced LTD (98 ⫾ 3%
of control, n ⫽ 5, P ⫽ 0.55, Fig. 4D), suggesting that NMDA
receptors are required for both pairing- and LFS-induced LTDs
consistent with previous results (Cheong et al. 2002; Kourrich
and Chapman 2003; Solger et al. 2004).
If NMDA receptors are required for both the pairing- and
LFS-induced LTDs, it is possible that these two forms of LTD
share the same mechanism. If so, expression of one form of
LTD would occlude the expression of the other and vice versa.
We applied the induction protocol for the first form of LTD
three times to saturate its expression before applying the
induction protocol for the second form of LTD. Application of
the pairing paradigm the second time reduced AMPA EPSCs to
61 ⫾ 7% of control (n ⫽ 6); this was not significantly different
from the EPSCs after the third application of the induction
protocol (53 ⫾ 10% of control, n ⫽ 6, P ⫽ 0.28, Fig. 4E),
suggesting that the expression of LTD was saturated by the
pairing paradigm. Subsequent application of LFS protocol (1
Hz for 15 min) did not induce LTD (96 ⫾ 17% of the value
before application of 1-Hz protocol, n ⫽ 6, P ⫽ 0.76, Fig. 4E).
Similarly, application of LFS protocol (1 Hz for 15 min) twice
reduced AMPA EPSCs to 39 ⫾ 8% of control (n ⫽ 5); this was
not significantly different from the EPSCs after the third
application of the induction protocol (31 ⫾ 5% of control, n ⫽
5, P ⫽ 0.38, Fig. 4F), suggesting that LFS-induced LTD was
saturated. In this condition, application of the pairing paradigm
failed to induce LTD (95 ⫾ 9% of the value prior to the
application of pairing protocol, n ⫽ 5, P ⫽ 0.44, Fig. 4F).
J Neurophysiol • VOL
FIG. 4. Pairing- and LFS-induced LTDs share the same mechanism. A:
pairing-induced LTD was blocked by intracellular application of bis-(oaminophenoxy)-N,N,N’,N’-tetraacetic acid (BAPTA, 30 mM, n ⫽ 7). Top:
current traces averaged from 10 AMPA EPSCs as indicated in the figure. The
rest of the figure was arranged in the same way. B: intracellular application of
BAPTA (30 mM) blocked LFS-induced LTD (n ⫽ 6). C: bath application of
DL-APV (100 ␮M) blocked pairing-induced LTD (n ⫽ 11). D: bath application
of DL-APV (100 ␮M) blocked LFS-induced LTD (n ⫽ 5). E: pairing-induced
LTD occluded the LFS-induced LTD at the stellate neuron synapses (n ⫽ 6).
F: expression of LFS-induced LTD occluded pairing-induced LTD at the
stellate neuron synapses (n ⫽ 5).
These results suggest that pairing- and LFS-induced LTDs
share the similar mechanism at stellate neuron synapses.
Stellate neuron synapses express lower NMDA/AMPA
EPSC ratio
Activation of NMDA receptors can produce either LTP or
LTD. It is generally believed that high and fast increases in
intracellular Ca2⫹ are supposed to induce LTP, whereas low
and slow increases in intracellular Ca2⫹ generate LTD. The
density of NMDA receptors at individual synapses is likely to
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
Stellate neuron LTDs are mediated by an increase in
postsynaptic Ca2⫹ and are NMDA receptor-dependent
732
P.-Y. DENG AND S. LEI
FIG. 5. Pairing-induced LTD is associated with a low expression of Nmethyl-D-aspartate (NMDA) receptors at the stellate neuron synapses but
independent of L-type Ca2⫹ channels, metabotropic glutamate receptors and
cannabinoids receptors. A: ratio of NMDA/AMPA EPSCs measured at the
hippocampal CA1 pyramidal neuron synapses (n ⫽ 7) and at the EC stellate
neuron synapses (n ⫽ 6). Top: current traces averaged from 20 EPSCs
recorded at ⫺60 mV (bottom) and ⫹40 mV (top). 䡠 䡠 䡠 , position where NMDA
EPSCs were measured. Note that the ratio of NMDA/AMPA EPSCs was lower
at the stellate neuron synapses (** P ⫽ 0.005). B–D: application of nimodipine
(10 ␮M, n ⫽ 7, B), (⫾)-amino-4-carboxy-methyl-phenylacetic acid (MCPG; 1
mM, n ⫽ 5, C) or AM251 (10 ␮M, n ⫽ 6, D) failed to block pairing-induced
LTD. Top: current traces averaged from 10 AMPA EPSCs as indicated in the
figure.
J Neurophysiol • VOL
Pairing-induced LTD at the stellate neuron synapses is
independent of L-type Ca2⫹ channels, metabotropic
glutamate receptors, or cannabinoids receptors
Because the pairing-induced LTD has not been reported
previously in the EC, we next studied the mechanisms underlying this form of LTD. Pairing-induced LTD was not blocked
by application of nimodipine (10 ␮M, 50 ⫾ 3% of control, n ⫽
5, P ⬍ 0.0001, Fig. 5B), suggesting that L-type Ca2⫹ channels
are not required. To ensure that the used nimodipine was
effective, we induced LTD at the mossy fiber-CA3 pyramidal
neuron synapses in the presence of nimodipine because membrane depolarization (from ⫺60 to ⫺10 mV for 5 min) induces
L-type Ca2⫹ channel-dependent LTD at this synapse type (Lei
et al. 2003). Application of nimodipine (10 ␮M) blocked
depolarization-induced LTD at the mossy fiber-CA3 pyramidal
neuron synapses (93 ⫾ 8% of control, n ⫽ 6, P ⫽ 0.4),
whereas depolarization still induced LTD in the absence of
nimodipine at this synapse type (56 ⫾ 3% of control, n ⫽ 6,
P ⬍ 0.001, data not shown). Because metabotropic glutamate
receptor (mGluR)-mediated LTD has been observed in the
perirhinal cortex (McCaffery et al. 1999), we tested whether
pairing-induced LTD was mediated via activation of mGluRs.
Application of the broad spectrum, nonselective group I/II
mGluR antagonist MCPG (1 mM) failed to block pairinginduced LTD (47 ⫾ 6% of control, n ⫽ 5, P ⫽ 0.001, Fig. 5C),
suggesting that the function of mGluRs is not required for
pairing-induced LTD at the stellate neuron synapses. We also
performed a positive control experiment to ensure that MCPG
was effective. Because mossy fiber terminals of the hippocampus express group II mGluRs and application of DCG-IV
activates these receptors and inhibits glutamate release (Lei et
al. 2003), we recorded AMPA EPSCs from the CA3 pyramidal
neurons and stimulated mossy fibers by placing the stimulation
electrode in the stratum lucidum. Application of DCG-IV (1
␮M) inhibited AMPA EPSCs to 76 ⫾ 3% of control (n ⫽ 6)
in the presence of MCPG (1 mM), whereas application of the
same concentration of DCG-IV alone inhibited AMPA EPSCs
to 18 ⫾ 2% of control (n ⫽ 6, P ⫽ 0.0001, Student’s unpaired
t-test), suggesting that MCPG was effective at blocking
mGluRs. Together, these results indicate that group I/II
mGluRs are not required for the induction of LTD at the
stellate neuron synapses.
In many cells, postsynaptic depolarization can potentially
release endogenous cannabinoids, which translocate to the
presynaptic terminals to transiently inhibit transmitter release
(for review, see Alger 2002), and cannabinoids are involved in
LTP (Carlson et al. 2002; Misner and Sullivan 1999) as well as
LTD (Chevaleyre and Castillo 2003; Gerdeman et al. 2002).
We therefore tested whether pairing-induced LTD requires the
function of cannabinoids. In slices pretreated and perfused with
the CB1 receptor antagonist AM-251 (10 ␮M), pairing still
induced robust LTD (62 ⫾ 10% of control, n ⫽ 6, P ⫽ 0.01,
Fig. 5D). To ensure that AM-251 was effective at blocking
CB1 receptors, we recorded GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) from CA1 pyramidal neurons of the hippocampus by placing the stimulation electrode in
the stratum radiatum. We replaced the intracelulluar K⫹gluconate with the same concentration of CsCl and the extracellular solution contained DNQX (10 ␮M) to block AMPA
responses. The cells were held at ⫺60 mV. In this condition,
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
be a determinant that controls the amount and velocity of Ca2⫹
influx when NMDA receptors are activated. Because application of the pairing-paradigm induced LTP at CA1 pyramidal
neuron synapses but LTD at stellate neuron synapses, we
reasoned that the distinct effects might be due to differences in
NMDA receptor density at those two synapses, i.e., the density
of NMDA receptors at the stellate neuron synapses is lower
than that at the CA1 pyramidal neuron synapses. We tested this
possibility by comparing NMDA/AMPA EPSC ratio at these
two synapse types. We initially recorded AMPA EPSCs at
⫺60 mV and then recorded the EPSCs mediated by both
NMDA and AMPA receptors at ⫹40 mV. NMDA receptormediated current was measured 50 ms after the stimulus
artifact at ⫹40 mV (Lei and McBain 2004). NMDA/AMPA
EPSC ratio at the stellate neuron synapses (0.81 ⫾ 0.13, n ⫽
6) was lower than that measured at CA1 pyramidal neuron
synapses of the hippocampus (1.37 ⫾ 0.10, n ⫽ 7, P ⫽ 0.005,
Fig. 5A), suggesting that the low density of NMDA receptors
at the stellate neuron synapses might be the reason that application of the pairing paradigm at this synapse type induced
LTD instead of LTP.
LTD IN ENTORHINAL CORTEX STELLATE NEURONS
733
application of WIN 55212–2 (2 ␮M), an agonist for cannabinoid receptors, inhibited IPSCs to 26 ⫾ 4% of control (n ⫽ 5,
P ⬍ 0.001). This effect was blocked by application of AM-251
(2 ␮M, 98 ⫾ 6% of control, n ⫽ 6, P ⫽ 0.8, data not shown),
suggesting that AM-251 was effective at blocking CB1 receptors, consistent with previous results (Hájos and Freund 2002).
Together, these results indicate that pairing-induced LTD is
unlikely to be mediated by cannabinoids.
Postsynaptic expression of pairing-induced LTD at stellate
neuron synapses
Stellate neuron LTD involves a reduction in postsynaptic
AMPA receptor number
LTD at the stellate neuron synapses could result from a
decrease in single-channel conductance (␥) or open probability
of AMPA receptors or a reduction in the number of available
postsynaptic AMPA receptors. To differentiate these possibilities, we used peak-scaled nonstationary variance analysis
(Benke et al. 1998; Lei and McBain 2002; Lei et al. 2003;
Traynelis and Jaramillo 1998; Traynelis et al. 1993). The
synaptic current amplitude varies not only as a result of random
channel gating but also with variations in the transmitter
release, the temporal and spatial transmitter concentration
profile and the numbers of channels in the postsynaptic membranes (when synaptic currents arise from different release
FIG. 6. Postsynaptic expression of pairing-induced LTD. A: lack of change
in CV (SD/mean) before and after LTD induction (n ⫽ 20). Top: consecutive
15 EPSCs before (left) and after (right) LTD induction. Empty circles:
individual experiments; black circles, mean data. B: paired-pulse ratio (PPR)
evoked by 2 stimuli at an interval of 40 ms was not altered after the induction
of LTD (n ⫽ 10). Top left: EPSCs evoked by 2 stimuli before (thin) and after
(thick) LTD induction. Top right: EPSCs before and after LTD induction were
scaled to show the lack of difference in PPR. Empty circles: individual
experiments; black circles, mean data.
J Neurophysiol • VOL
FIG. 7. Peak-scaled nonstationary variance analysis shows that pairinginduced LTD is related to a reduction in AMPA receptor number with no
changes in AMPA receptor conductance. A, top: average of 30 EPSCs before
the induction of LTD was scaled to the peak of a single EPSC to calculate the
variance. Bottom: plot of variance vs. current to calculate the conductance (␥ ⫽
36 pS) and number (n ⫽ 120) of postsynaptic AMPA receptors. B, top: average
of 30 EPSCs from the same cell after the induction of LTD was scaled to the
peak of a single EPSC to calculate the variance. Bottom: variance and current
relationship from the same cell after the induction of LTD. Note that the
conductance was unchanged (␥ ⫽ 34 pS) but that the number of AMPA
receptors was reduced (N ⫽ 82). C: summarized data for single-channel
conductance from 7 neurons before and after the induction of LTD. Individual
experiments and mean data are indicated (E and F, respectively). D: summarized data for the number of the AMPA receptors before and after LTD
induction. Note that the number of AMPA receptors was significantly reduced
after the induction of LTD (n ⫽ 7; P ⫽ 0.0007).
sites). Because variations in amplitude will contaminate the
calculation of variance attributable to channel gating, the
individual events have to be scaled such that their peak amplitude equals the average peak amplitude. Therefore peakscaled variance analysis yields estimates of the single-channel
conductance and the number of open channels without being
able to resolve the open probability. Using this analysis, we
probed whether LTD was related to a reduction in singlechannel conductance or postsynaptic AMPA receptor number
or both.
The single-channel conductance of the AMPA receptors
calculated by the peak-scale variance analysis was not significantly changed after the induction of LTD (control, 29.1 ⫾ 3.0
pS; LTD, 28.1 ⫾ 3.6 pS; n ⫽ 7; P ⫽ 0.59, Fig. 7). In contrast,
the number of AMPA receptors on the postsynaptic membrane
was significantly reduced after LTD induction (control, 168 ⫾
12; LTD, 100 ⫾ 8; n ⫽ 7; P ⫽ 0.0007, Fig. 7). These results
suggest that stellate neuron LTD is associated with a reduction
in the number of postsynaptic AMPA receptors, consistent
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
We then examined whether the expression of pairing-induced LTD at stellate neuron synapses was pre- or postsynaptic
in origin using the following two approaches. First, we calculated the coefficient of variation (CV ⫽ SD/mean) before and
after the induction of LTD (Fig. 6A). The values of CV were
not changed after LTD induction (control: 0.141 ⫾ 0.017,
LTD: 0.146 ⫾ 0.018, n ⫽ 20, P ⫽ 0.39, Fig. 6A). Second, we
calculated the paired-pulse ratio (PPR) before and after LTD
induction. PPR was not significantly changed after the induction of LTD (control: 1.09 ⫾ 0.05, LTD: 1.11 ⫾ 0.07, n ⫽ 10,
P ⫽ 0.76, Fig. 6B). These data suggest that the expression of
LTD is postsynaptic.
734
P.-Y. DENG AND S. LEI
with the mechanisms observed at other synapses (Linden
2001).
Stellate neuron LTD involves AMPA receptor internalization
Stellate neuron LTD is ubiquitination-dependent
The endocytosis of AMPA receptors has recently been
shown to require the activity of the ubiquitin-proteasome
system (Bingol and Schuman 2004; Burbea et al. 2002; Colledge et al. 2003; Turrigiano 2002). Ubiquitination is conducted in a stepwise manner by E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin
ligases, which recognize target proteins and catalyze the covalent attachment of ubiquitin to these target substrates. Ubiquitinated membrane proteins are substrates for endocytosis and
eventually degraded by proteasome. Inhibition of the proteasome activity has been shown to block NMDA receptormediated LTD in hippocampal CA1 pyramidal neuron synapses (Colledge et al. 2003). We next tested whether the
function of proteasome was required for stellate neuron LTD
using two proteasome inhibitors of different structures: lactacystin and epoxomicin. Dialysis of lactacystin (10 ␮M) into the
cells via the recording pipettes blocked pairing-induced LTD
(94 ⫾ 7% of control, n ⫽ 6, P ⫽ 0.46, Fig. 8B), whereas
inclusion of the vehicle (0.05% DMSO) in the recording
pipettes failed to block pairing-induced LTD (54 ⫾ 2% of
control, n ⫽ 5, P ⬍ 0.0001, Fig. 8B). Pretreatment of slices
with epoxomicin (100 nM), a cell-permeable proteasome inhibitor, also blocked pairing-induced LTD at the stellate neuron synapses (90 ⫾ 6% of control, n ⫽ 6, P ⫽ 0.63, Fig. 8C).
Together, these results indicate that the function of proteasome
J Neurophysiol • VOL
FIG. 8. Pairing-induced LTD is associated with endocytosis of AMPA
receptors. A: intracellular perfusion of NSF-inhibitory peptide (pep2m, 0.5
mM) reduced AMPA EPSCs and blocked pairing-induced LTD (n ⫽ 7). Top:
current traces averaged from 10 AMPA EPSCs as indicated in the figure. The
rest of the figure was arranged in the same way. B: intracellular application of
a proteasome inhibitor, lactacystin (10 ␮M), via the recording pipettes blocked
pairing-induced LTD (n ⫽ 6), whereas inclusion of the vehicle (0.05% DMSO)
in the recording pipettes was without effect (n ⫽ 5). C: pretreatment and
perfusion of the slices with epoxomicin (100 nM) blocked pairing-induced
LTD (n ⫽ 6). D: application of the calcineurin inhibitor, FK506 (50 ␮M)
blocked pairing-induced LTD (n ⫽ 6), whereas application of the inactive
analog rapamycin (50 ␮M) was without effect (n ⫽ 6).
is required for the endocytosis of AMPA receptors and LTD at
the stellate neuron synapses.
Calcineurin is required for stellate neuron LTD
In hippocampal CA1 pyramidal neurons, NMDA receptormediated modest increase in postsynaptic Ca2⫹ preferentially
activates phosphatase 2B, calcineurin, to induce AMPA receptor endocytosis (Beattie et al. 2000; Morishita et al. 2005) and
LTD (Mulkey et al. 1993, 1994). We next tested whether
calcineurin was required for pairing-induced LTD at the stellate neuron synapses. We pretreated the slices with the calcineurin inhibitor, FK506 (50 ␮M), which was also continuously bath applied during electrophysiological recordings. In
this condition, application of the pairing paradigm failed to
induce LTD (94 ⫾ 3% of control, n ⫽ 6, P ⫽ 0.12, Fig. 8D),
whereas pretreatment with and bath application of rapamycin
(50 ␮M), a compound with a structure similar to FK506 but
lacking any calcineurin inhibitory activity (Kunz and Hall
1993), had no effect on pairing-induced LTD (55 ⫾ 2% of
control, n ⫽ 6, P ⬍ 0.0001, Fig. 8D). These results indicate
that the calcium-dependent phosphatase, calcineurin is required for LTD at the stellate neuron synapses of the EC.
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
Postsynaptic AMPA receptor translocation is an important
mechanism in the expression of NMDA receptor-dependent
LTD (Carroll et al. 1999; Lüscher et al. 1999; Lüthi et al. 1999;
Man et al. 2000; Matsuda et al. 2000; Sheng and Kim 2002;
Song and Huganir 2002; Wang and Linden 2000). NMDA
receptor-dependent LTD expression involves a pool of AMPA
receptors regulated by NSF-GluR2 interactions at Schaffer
collateral-CA1 pyramidal neuron (Lüscher et al. 1999; Lee et
al. 2002; Lüthi et al. 1999; Noel et al. 1999) and cerebellar
(Steinberg et al. 2004) synapses. NMDA receptor-induced
endocytosis of AMPA receptors is dependent on both Ca2⫹ and
the activity of protein phosphatase (Beattie et al. 2000; Ehlers
2000; Morishita et al. 2005). We next examined whether
expression of LTD at stellate neuron synapses similarly involved a translocation of AMPA receptors using the broadspectrum NSF inhibitory peptide, commonly referred to as
pep2m (Lüscher et al. 1999; Lüthi et al. 1999; Lee et al. 2002;
Shi et al. 2001). Infusion of pep2m (0.5 mM) into the cells via
the recording pipette led to a reduction in AMPA receptor
EPSCs (51 ⫾ 8% of control, n ⫽ 7, P ⫽ 0.0009, Fig. 8A),
suggesting that translocation of AMPA receptors at stellate
neuron synapses is dependent on NSF-GluR2 interactions. In
the continuous presence of pep2m, application of the pairing
paradigm failed to induce LTD (86 ⫾ 7% of the value before
pairing, n ⫽ 7, P ⫽ 0.1, Fig. 8A), suggesting that LTD at
stellate neuron synapses involves the endocytosis of a pool of
AMPA receptors that require the function of NSF.
LTD IN ENTORHINAL CORTEX STELLATE NEURONS
DISCUSSION
J Neurophysiol • VOL
receive inputs from other cortical regions such as olfactory
cortices. Actually NMDA receptor-dependent LTP has been
observed in the superficial layers using field recordings when a
stimulation electrode was placed in the molecular layer of the
EC (layer I) (Alonso et al. 1990). Therefore the expression of
LTP at the stellate neuron synapses formed with other inputs
remains to be determined.
Whereas the stellate neuron synapses do not express LTP,
application of either pairing- or LFS-induction paradigm induced LTD at this synapse type. The expression of these two
forms of LTD is dependent on NMDA receptors and requires
an increase in postsynaptic Ca2⫹. The expression of one type
of LTD occluded that of the other suggesting that the involved
mechanisms are the same. We further studied the signaling
mechanisms downstream of Ca2⫹ influx via NMDA receptors.
Our results suggest that the Ca2⫹-dependent phosphatase calcineurin is the target of Ca2⫹. Activation of calcineurin leads to
the endocytosis of AMPA receptors, which are then degraded
by proteasome resulting in a reduction in synaptic strength.
The superficial layers (II and III) of the EC relay most of the
inputs from cortical associational areas to the hippocampus,
whereas the deep layers (V and VI) receive inputs from the
cingular cortex, are the main target of hippocampal output, and
send extensive projections back to the superficial layers of the
EC and to the neocortex (Witter et al. 2000). Individual
neurons in the deep layers (layer V) respond to consecutive
stimuli with persistent firing (Egorov et al. 2002). The persistent activity from deep layers of the EC is likely to generate
continuous depolarization of the neurons such as stellate neurons in the superficial layers. Similar to the pairing protocol,
persistent depolarization of stellate neurons is likely to activate
NMDA receptors and induce LTD. Because the axons of the
stellate neurons are the major component of the perforant path
to control the function of the hippocampus, stellate neuron
LTD is likely to produce a long-term inhibition of the hippocampus. Therefore the physiological significance of the
pairing- or LFS-induced LTD is to provide a feedback modulation of the hippocampal-parahippocampal functions. Because
the EC in the parahippocampal region is crucially involved in
the acquisition, consolidation, and retrieval of long-term memory traces for which working memory operations are essential,
the feedback modulation from the stellate neuron synapses is
likely to influence the memory processes.
GRANTS
This work was supported by Division of Research Resources Grant
5P20RR-017699-02.
REFERENCES
Aicardi G, Argilli E, Cappello S, Santi S, Riccio M, Thoenen H, Canossa
M. Induction of long-term potentiation and depression is reflected by
corresponding changes in secretion of endogenous brain-derived neurotrophic factor. Proc Natl Acad Sci USA 101: 15788 –15792, 2004.
Alger BE. Retrograde signaling in the regulation of synaptic transmission:
focus on endocannabinoids. Prog Neurobiol 68: 247–286, 2002.
Alonso A, de Curtis M, Llinas R. Postsynaptic Hebbian and non-Hebbian
long-term potentiation of synaptic efficacy in the entorhinal cortex in slices
and in the isolated adult guinea pig brain. Proc Natl Acad Sci USA 87:
9280 –9284, 1990.
Alonso A, Klink R. Differential electroresponsiveness of stellate and pyramidal-like cells of medial entorhinal cortex layer II. J Neurophysiol 70:
128 –143, 1993.
Amaral DG, Witter MO. Hippocampal formation. In: The Rat Nervous
System, edited by Paxinos G. San Diego, CA: Academic, 1995, p. 443– 493.
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
Our study is the first one to investigate the cellular and
molecular mechanisms underlying LTD in the EC. Our results
demonstrate that the EC stellate neuron synapses express
NMDA receptor-dependent LTD. The expression of stellate
neuron LTD is postsynaptic and requires an increase in intracellular Ca2⫹ and the activity of Ca2⫹-dependent phosphatase
2B, calcineurin. Stellate neuron LTD involves a reduction in
the number of postsynaptic AMPA receptors. The endocytosis
of AMPA receptors requires the function of ubiquitin-proteasome system.
Whereas LFS-induced NMDA receptor-dependent LTD was
detected in the superficial layers of the EC using extracellular
field recordings (Cheong et al. 2002; Kourrich and Chapman
2003; Solger et al. 2004; Yang et al. 2004), the following
questions still remain to be answered. At which synapse(s) in
the superficial layers of the EC does LTD occur?. How does
NMDA receptor activation lead to a reduction in synaptic
strength in the superficial layers of the EC? Are there any other
forms of synaptic plasticity in this brain region? Our study
addressed these questions.
Because ⬃70% of the cells in layer II of the superficial
layers are stellate neurons (Klink and Alonso 1997) and the
axons of these neurons form the major components of the
perforant path that provides the major inputs to the hippocampus, we chose to study the long-term plasticity at the stellate
neuron synapses. We initially identified the stellate neurons by
their location, morphology, and electrophysiological properties. We then combined whole cell, perforated-patch, and field
recordings and studied the cellular mechanisms of long-term
plasticity at the stellate neuron synapses. Our results suggest
that the stellate neuron synapses are devoid of HFS- or forskolin-induced LTP. Whereas the reason for the incompetence
of stellate neuron synapses to express LTP is not absolutely
clear, our results suggest that it is due to the low expression of
NMDA receptors at this synapse type because the ratio of
NMDA/AMPA EPSCs is lower at this synapse type. Because
HFS induces a fast and large increase in intracellular Ca2⫹ to
generate LTP, application of this induction paradigm at the
stellate neuron synapses where the expression of NMDA receptors is lower may not elevate the intracellular Ca2⫹ to a
level to induce LTP. This might be the same reason that
application of the pairing-paradigm induces LTP at the Schaffer collateral-CA1 pyramidal neuron synapses of the hippocampus but LTD at the stellate neuron synapses of the EC.
Application of forskolin induces LTP at hippocampal mossy
fiber-CA3 pyramidal neuron synapses (Huang et al. 1994;
Weisskopf et al. 1994) but not at mossy fiber-CA3 interneuron
synapses (Maccaferri et al. 1998), suggesting that the intrinsic
properties of the synapses determine the expression of this
chemical-induced LTP. Indeed, forskolin-induced LTP is presynaptically expressed and requires signaling cascades including adenylyl cyclase, cyclic AMP, protein kinase A, and
Rab3A (Huang et al. 1994, 1995; Lonart et al. 1998; Villacres
et al. 1998). The inability of the stellate neuron synapses to
express this form of LTP suggests that one or more of these
signaling molecules are missing at this synapse type. However,
in the present study, we have only examined the induction of
LTP at the synapses formed between stellate neurons and the
inputs from the deep layers of the EC. Stellate neurons also
735
736
P.-Y. DENG AND S. LEI
J Neurophysiol • VOL
Huang YY, Li XC, Kandel ER. cAMP contributes to mossy fiber LTP by
initiating both a covalently mediated early phase and macromolecular
synthesis-dependent late phase. Cell 79: 69 –79, 1994.
Izquierdo I. Pharmacological evidence for a role of long-term potentiation in
memory. FASEB J 8: 1139 –145, 1994.
Klink R, Alonso A. Morphological characteristics of layer II projection
neurons in the rat medial entorhinal cortex. Hippocampus 7: 571–583, 1997.
Kohler C. Intrinsic connections of the retrohippocampal region in the rat
brain. II. The medial entorhinal area. J Comp Neurol 246: 149 –169, 1986.
Kohler C. Intrinsic connections of the retrohippocampal region in the rat
brain. III. The lateral entorhinal area. J Comp Neurol 271: 208 –228, 1988.
Kourrich S, Chapman CA. NMDA receptor-dependent long-term synaptic
depression in the entorhinal cortex in vitro. J Neurophysiol 89: 2112–2119,
2003.
Kunz J, Hall MN. Cyclosporin A, FK506 and rapamycin: more than just
immunosuppression. Trends Biochem Sci 18: 334 – 8, 1993.
Lee SH, Liu L, Wang YT, Sheng M. Clathrin adaptor AP2 and NSF interact
with overlapping sites of GluR2 and play distinct roles in AMPA receptor
trafficking and hippocampal LTD. Neuron 36: 661– 674, 2002.
Lei S, McBain CJ. Distinct NMDA receptors provide differential modes of
transmission at mossy fiber-interneuron synapses. Neuron 33: 921–933,
2002.
Lei S, McBain CJ. Two Loci of expression for long-term depression at
hippocampal mossy fiber-interneuron synapses. J Neurosci 24: 2112–2121,
2004.
Lei S, Pelkey KA, Topolnik L, Congar P, Lacaille JC, McBain CJ.
Depolarization-induced long-term depression at hippocampal mossy fiberCA3 pyramidal neuron synapses. J Neurosci 23: 9786 –9795, 2003.
Linden DJ. The expression of cerebellar LTD in culture is not associated with
changes in AMPA-receptor kinetics, agonist affinity, or unitary conductance. Proc Natl Acad Sci USA 98: 14066 –14071, 2001.
Lonart G, Janz R, Johnson KM, Sudhof TC. Mechanism of action of rab3A
in mossy fiber LTP. Neuron 21: 1141–1150, 1998.
Lüscher C, Xia H, Beattie EC, Carroll RC, von Zastrow M, Malenka RC,
Nicoll RA. Role of AMPA receptor cycling in synaptic transmission and
plasticity. Neuron 24: 649 – 658, 1999.
Lüthi A, Chittajallu R, Duprat F, Palmer MJ, Benke TA, Kidd FL, Henley
JM, Isaac JT, Collingridge GL. Hippocampal LTD expression involves a
pool of AMPARs regulated by the NSF-GluR2 interaction. Neuron 24:
389 –399, 1999.
Maccaferri G, Toth K, McBain CJ. Target-specific expression of presynaptic
mossy fiber plasticity. Science 279: 1368 –1370, 1998.
Man HY, Lin JW, Ju WH, Ahmadian G, Liu L, Becker LE, Sheng M,
Wang YT. Regulation of AMPA receptor-mediated synaptic transmission
by clathrin-dependent receptor internalization. Neuron 25: 649 – 662, 2000.
Matsuda S, Launey T, Mikawa S, Hirai H. Disruption of AMPA receptor
GluR2 clusters following long-term depression induction in cerebellar
Purkinje neurons. EMBO J 19: 2765–2774, 2000.
McCaffery B, Cho K, Bortolotto ZA, Aggleton JP, Brown MW, Conquet
F, Collingridge GL, Bashir ZI. Synaptic depression induced by pharmacological activation of metabotropic glutamate receptors in the perirhinal
cortex in vitro. Neuroscience 93: 977–984, 1999.
Misner DL, Sullivan JM. Mechanism of cannabinoid effects on long-term
potentiation and depression in hippocampal CA1 neurons. J Neurosci 19:
6795– 6805, 1999.
Morishita W, Marie H, Malenka RC. Distinct triggering and expression
mechanisms underlie LTD of AMPA and NMDA synaptic responses. Nat
Neurosci 8: 1043–1050, 2005.
Mulkey RM, Herron CE, Malenka RC. An essential role for protein
phosphatases in hippocampal long-term depression. Science 261: 1051–
1055, 1993.
Mulkey RM, Endo S, Shenolikar S, Malenka RC. Involvement of a
calcineurin/inhibitor-1 phosphatase cascade in hippocampal long-term depression. Nature 369: 486 – 488, 1994.
Noel J, Ralph GS, Pickard L, Williams J, Molnar E, Uney JB, Collingridge GL, Henley JM. Surface expression of AMPA receptors in
hippocampal neurons is regulated by an NSF-dependent mechanism. Neuron
23: 365–376, 1999.
Rae J, Cooper K, Gates P, Watsky M. Low access resistance perforated
patch recordings using amphotericin B. J Neurosci Methods 37: 15–26,
1991.
Ruth RE, Collier TJ, Routtemberg A. Topography between the entorhinal
cortex and the dentate septotemporal axis in rats. I. Medial and intermediate
entorhinal projecting cells. J Comp Neurol 209: 69 –78, 1982.
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
Beattie EC, Carroll RC, Yu X, Morishita W, Yasuda H, von Zastrow M,
Malenka RC. Regulation of AMPA receptor endocytosis by a signaling
mechanism shared with LTD. Nat Neurosci 3: 1291–1300, 2000.
Benke TA, Luthi A, Isaac JT, Collingridge GL. Modulation of AMPA
receptor unitary conductance by synaptic activity. Nature 393: 793–797,
1998.
Bingol B, Schuman EM. A proteasome-sensitive connection between PSD-95
and GluR1 endocytosis. Neuropharmacology 47: 755–763, 2004.
Bischoff U, Brau ME, Vogel W, Hempelmann G, Olschewski A. Local
anaesthetics block hyperpolarization-activated inward current in rat small
dorsal root ganglion neurones. Br J Pharmacol 139: 1273–1280, 2003.
Bouras R, Chapman CA. Long-term synaptic depression in the adult entorhinal cortex in vivo. Hippocampus 13: 780 –790, 2003.
Burbea M, Dreier L, Dittman JS, Grunwald ME, Kaplan JM. Ubiquitin
and AP180 regulate the abundance of GLR-1 glutamate receptors at
postsynaptic elements in C. elegans. Neuron 35: 107–120, 2002.
Burwell RD. The parahippocampal region: corticocortical connectivity. Ann
NY Acad Sci 911: 25– 42, 2000.
Carlson G, Wang Y, Alger BE. Endocannabinoids facilitate the induction of
LTP in the hippocampus. Nat Neurosci 5: 723–724, 2002.
Carroll RC, Lissin DV, von Zastrow M, Nicoll RA, Malenka RC. Rapid
redistribution of glutamate receptors contributes to long-term depression in
hippocampal cultures. Nat Neurosci 2: 454 – 460, 1999.
Cheong MY, Yun SH, Mook-Jung I, Kang Y, Jung MW. Induction of
homosynaptic long-term depression in entorhinal cortex. Brain Res 954:
308 –310, 2002.
Chevaleyre V, Castillo PE. Heterosynaptic LTD of hippocampal GABAergic
synapses: a novel role of endocannabinoids in regulating excitability. Neuron 38: 461– 472, 2003.
Colledge M, Snyder EM, Crozier RA, Soderling JA, Jin Y, Langeberg LK,
Lu H, Bear MF, Scott JD. Ubiquitination regulates PSD-95 degradation
and AMPA receptor surface expression. Neuron 40: 595– 607, 2003.
de Curtis M, Llinas RR. Entorhinal cortex long-term potentiation evoked by
theta-patterned stimulation of associative fibers in the isolated in vitro
guinea pig brain. Brain Res 600: 327–330, 1993.
Deng PY, Lei S. Bidirectional modulation of GABAergic transmission by
cholecystokinin in hippocampal dentate gyrus granule cells of juvenile rats.
J Physiol 572: 425– 442, 2006.
Deng PY, Porter JE, Shin HS, and Lei S. Thyrotropin-releasing hormone
increases GABA release in rat hippocampus. J Physiol 577: 497–511, 2006.
Dickson CT, Magistretti J, Shalinsky MH, Fransen E, Hasselmo ME,
Alonso A. Properties and role of Ih in the pacing of subthreshold oscillations
in entorhinal cortex layer II neurons. J Neurophysiol 83: 2562–2579, 2000.
Dolcos F, LaBar KS, Cabeza R. Remembering one year later: role of the
amygdala and the medial temporal lobe memory system in retrieving
emotional memories. Proc Natl Acad Sci USA 102: 2626 –2631, 2005.
Dolorfo CL, Amaral DG. Entorhinal cortex of the rat: organization of
intrinsic connections. J Comp Neurol 398: 49 – 82, 1998a.
Dolorfo CL, Amaral DG. Entorhinal cortex of the rat: topographic organization of the cells of origin of the perforant path projection to the dentate
gyrus. J Comp Neurol 398: 25– 48, 1998b.
Dorval AD Jr, White JA. Channel noise is essential for perithreshold
oscillations in entorhinal stellate neurons. J Neurosci 25: 10025–10028,
2005.
Egorov AV, Hamam BN, Fransen E, Hasselmo ME, Alonso AA. Graded
persistent activity in entorhinal cortex neurons. Nature 420: 173–178, 2002.
Ehlers MD. Reinsertion or degradation of AMPA receptors determined by
activity-dependent endocytic sorting. Neuron 28: 511–525, 2000.
Gerdeman GL, Ronesi J, Lovinger DM. Postsynaptic endocannabinoid
release is critical to long-term depression in the striatum. Nat Neurosci 5:
446 – 451, 2002.
Gloveli T, Dugladze T, Schmitz D, Heinemann U. Properties of entorhinal
cortex deep layer neurons projecting to the rat dentate gyrus. Eur J Neurosci
13: 413– 420, 2001.
Haist F, Bowden Gore J, Mao H. Consolidation of human memory over
decades revealed by functional magnetic resonance imaging. Nat Neurosci
4: 1139 –1145, 2001.
Hájos N, Freund TF. Pharmacological separation of cannabinoid sensitive
receptors on hippocampal excitatory and inhibitory fibers. Neuropharmacology 43: 503–510, 2002.
Huang YY, Kandel ER, Varshavsky L, Brandon EP, Qi M, Idzerda RL,
McKnight GS, Bourtchouladze R. A genetic test of the effects of mutations in PKA on mossy fiber LTP and its relation to spatial and contextual
learning. Cell 83: 1211–1222, 1995.
LTD IN ENTORHINAL CORTEX STELLATE NEURONS
J Neurophysiol • VOL
Turrigiano GG. A recipe for ridding synapses of the ubiquitous AMPA
receptor. Trends Neurosci 25: 597–598, 2002.
van der Linden S, Lopes da Silva FH. Comparison of the electrophysiology
and morphology of layers III and II neurons of the rat medial entorhinal
cortex in vitro. Eur J Neurosci 10: 1479 –1489, 1998.
van Haeften T, Baks-te-Bulte L, Goede PH, Wouterlood FG, Witter MP.
Morphological and numerical analysis of synaptic interactions between
neurons in deep and superficial layers of the entorhinal cortex of the rat.
Hippocampus 13: 943–952, 2003.
Villacres EC, Wong ST, Chavkin C, Storm DR. Type I adenylyl cyclase
mutant mice have impaired mossy fiber long-term potentiation. J Neurosci
18: 3186 –3194, 1998.
Wang YT, Linden DJ. Expression of cerebellar long-term depression requires
postsynaptic clathrin-mediated endocytosis. Neuron 25: 635– 647, 2000.
Weisskopf MG, Castillo PE, Zalutsky RA, Nicoll RA. Mediation of hippocampal mossy fiber long-term potentiation by cyclic AMP. Science 265:
1878 –1882, 1994.
Witter MP, Groenewegen HJ, Lopes da Silva FH, Lohman AH. Functional
organization of the extrinsic and intrinsic circuitry of the parahippocampal
region. Prog Neurobiol 33: 161–253, 1989.
Witter MP, Naber PA, van Haeften T, Machielsen WC, Rombouts SA,
Barkhof F, Scheltens P, Lopes da Silva FH. Cortico-hippocampal communication by way of parallel parahippocampal-subicular pathways. Hippocampus 10: 398 – 410, 2000.
Yang S, Lee DS, Chung CH, Cheong MY, Lee CJ, Jung MW. Long-term
synaptic plasticity in deep layer-originated associational projections to
superficial layers of rat entorhinal cortex. Neuroscience 127: 805– 812,
2004.
Zhou YD, Acker CD, Netoff TI, Sen K, White JA. Increasing Ca2⫹
transients by broadening postsynaptic action potentials enhances timingdependent synaptic depression. Proc Natl Acad Sci USA 102: 19121–19125,
2005.
97 • JANUARY 2007 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on April 29, 2017
Ruth RE, Collier TJ, Routtemberg A. Topographical relationship between
the entorhinal cortex and the septotemporal axis of the dentate gyrus in rats.
II. Cells projecting from lateral entorhinal subdivisions. J Comp Neurol 270:
506 –516, 1988.
Sheng M, Kim MY. Postsynaptic signaling and plasticity mechanisms. Science 298: 776 –780, 2002.
Shi S, Hayashi Y, Esteban JA, Malinow R. Subunit-specific rules governing
AMPA receptor trafficking to synapses in hippocampal pyramidal neurons.
Cell 105: 331–343, 2001.
Solger J, Wozny C, Manahan-Vaughan D, Behr J. Distinct mechanisms of
bidirectional activity-dependent synaptic plasticity in superficial and deep
layers of rat entorhinal cortex. Eur J Neurosci 19: 2003–2007, 2004.
Song I, Huganir RL. Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci 25: 578 –588, 2002.
Squire LR, Stark CE, Clark RE. The medial temporal lobe. Annu Rev
Neurosci 27: 279 –306, 2004.
Steffenach HA, Witter M, Moser MB, Moser EI. Spatial memory in the rat
requires the dorsolateral band of the entorhinal cortex. Neuron 45: 301–313,
2005.
Steinberg JP, Huganir RL, Linden DJ. N-ethylmaleimide-sensitive factor is
required for the synaptic incorporation and removal of AMPA receptors
during cerebellar long-term depression. Proc Natl Acad Sci USA 101:
18212–18216, 2004.
Steward O, Scoville SA. The cells of origin of entorhinal afferents to the
hippocampus and fascia dentata of the rat. J Comp Neurol 169: 347–370,
1976.
Traynelis SF, Jaramillo F. Getting the most out of noise in the central
nervous system. Trends Neurosci 21: 137–145, 1998.
Traynelis SF, Silver RA, Cull-Candy SG. Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy
fiber-granule cell synapse. Neuron 279 –289, 1993.
737