# Postsynaptic Signals Mediating Induction of Long-Term Synaptic Depression in the Entorhinal Cortex

Authors: Saïd Kourrich; Stephen D. Glasgow; Douglas A. Caruana; C. Andrew Chapman
Journal: Neural Plasticity
 (2008)

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## Abstract

The entorhinal cortex receives a large projection from the piriform cortex, and synaptic plasticity in this pathway may affect olfactory processing. In vitro whole cell recordings have been used here to investigate postsynaptic signalling mechanisms that mediate the induction of long-term synaptic depression (LTD) in layer II entorhinal cortex cells.  To induce LTD, pairs of pulses, using a 30-millisecond interval, were delivered at 1 Hz for 15 minutes. Induction of LTD was blocked by the NMDA receptor antagonist APV and by the calcium chelator BAPTA, consistent with a requirement for calcium influx via NMDA receptors. Induction of LTD was blocked when the FK506 was included in the intracellular solution to block the phosphatase calcineurin. Okadaic acid, which blocks activation of protein phosphatases 1 and 2a, also prevented LTD. Activation of protein phosphatases following calcium influx therefore contributes to induction of LTD in layer II of the entorhinal cortex.


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## Body

## 1. Introduction

The mechanisms that mediate the
induction of long-term synaptic potentiation (LTP) [1, 2] and depression (LTD) [3–5]
have been studied intensively within the hippocampus, but less is known about the
signalling mechanisms for LTP and LTD in the entorhinal cortex. Because the
entorhinal cortex receives highly processed inputs from sensory and association
cortices and also provides the hippocampal region with much of its sensory
input [6, 7], lasting changes in the strength of synaptic inputs to the entorhinal
cortex could alter the manner in which multimodal cortical inputs are
integrated, modulate the strength of transmission of specific patterns of sensory
input within the hippocampal formation, and contribute to mnemonic function [8–11]. Determining the effective stimulation
parameters and the intracellular signals that mediate synaptic plasticity in
the entorhinal cortex should allow insight into basic mechanisms that contribute
to the cognitive functions of the parahippocampal region.Long-term potentiation of cortical inputs to
the superficial layers of the entorhinal cortex has been described in vivo [11–14] and in vitro [15, 16]. Stimulation
patterns required to induce LTP tend to be more intense in the entorhinal
cortex than in the hippocampus [12, 14], and we have also found that induction
of LTD in the entorhinal cortex requires intense low-frequency stimulation [17, 18]. In the hippocampus, conventional
1 Hz stimulation trains have been most
effective in slices taken from juvenile animals [19, 20] but are generally
ineffective in adult slices [21–23] and in intact animals ([31, 32], see also [33]).
Similarly, 1 Hz stimulation induces entorhinal LTD in slices from young animals
[28, 29] but is not effective in vivo [17] or in slices from older animals [18].
Repeated stimulation using pairs of pulses separated by a short 25- to 50-millisecond
interval can induce LTD more effectively in both the CA1 ([24–26], but see [27]) and
entorhinal cortex [17, 18, 33, 34]. In the CA1, the LTD induced by this stimulation
pattern is NMDA receptor-dependent, but it also depends upon activation of
local inhibitory mechanisms by the pulse-pairs [30, 31]. In the entorhinal cortex, however, repeated
paired-pulse stimulation using a 10-millisecond interval that evokes maximal paired-pulse
inhibition does not induce LTD, and LTD is induced when a 30-millisecond
interval is used that evokes maximal paired-pulse facilitation [17]. The LTD can also be enhanced when GABAA transmission is reduced with bicuculline [18]. This further suggests that LTD
in the entorhinal cortex does not require activation of local inhibitory
mechanisms but rather requires prolonged stimulation patterns that are strong
enough to overcome local inhibition and lead to NMDA receptor activation. Strong
local inhibition in the entorhinal cortex [8, 35] may thus place a restraint on
activity-dependent synaptic modification. Consistent with this idea is the
finding that the same pairing stimulation protocol that induces LTP in
hippocampus leads to LTD in entorhinal cortex [28].Signalling mechanisms that mediate LTD in the
superficial layers of the entorhinal cortex share some similarities with NMDA
receptor-dependent LTD in the hippocampus. Long-term depression of superficial
layer inputs to layer II is dependent on NMDA receptor activation both in vivo
and in vitro [17, 18, 28, 33] but does not require activation of group I/II
metabotropic glutamate receptors ([18, 28], see [36, 37]). In the hippocampus, moderate
and prolonged influx of calcium via NMDA receptors activates calmodulin which leads
to LTD via activation of the protein phosphatase calcineurin (PP2b). Calcineurin
increases the activity of protein phosphatase 1 by reducing the activity of
inhibitor 1, and this can cause rapid reductions in AMPA-mediated responses [2, 38, 39]. Hippocampal LTD is expressed partly through the reduced conductance of
AMPA receptors caused by dephosphorylation of the GluR1 subunit by PP1 [2, 4], but
careful study has shown that calcineurin-dependent LTD in deep layer inputs to
layer II neurons in the young entorhinal cortex is not associated with a
reduced AMPA conductance, but rather involves internalization of AMPA receptors
and their proteosome-mediated degradation [28].In the present study, the early postsynaptic
signalling mechanisms that mediate LTD in layer I inputs to layer II neurons of
the medial entorhinal cortex have been investigated using recordings of whole
cell excitatory postsynaptic potentials. Long-term depression was induced using
a prolonged paired-pulse stimulation pattern that was previously found to be
effective for induction of NMDA-receptor-dependent LTD [18]. Pharmacological agents applied to the
bathing medium or intracellular solution were used to assess the dependence of
LTD on calcium-dependent signalling mechanisms including the phosphatases calcineurin
and PP1/PP2a.


## 2. Experimental Procedures

### 2.1. Slices and Whole Cell Recordings

Experiments were performed on slices from male
Long-Evans rats (4 to 8 weeks old). Animals were anesthetized with halothane and brains were rapidly removed
and cooled (4°C) in oxygenated artificial cerebrospinal fluid (ACSF). ACSF
consisted of (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2
MgSO4, 2 CaCl2, 26 NaHCO3, and 10 dextrose and
was saturated with 95% O2–5% CO2. All chemicals were obtained
from Sigma (St. Louis, Mo, USA) unless otherwise indicated. Horizontal slices (300μm) were cut with a
vibratome (WPI, Vibroslice
NVSL, Sarasota, Fla, USA) and were allowed to recover for at least one hour before
recordings. Slices were maintained in a recording chamber with oxygenated ACSF
at a rate of 2.0 mL/min, and a temperature from 22 to 24°C was used to
minimize metabolic demands on slices [18, 28]. Neurons were viewed with an
upright microscope (Leica
DML-FS, Wetzlar, Germany) equipped with a 40x objective, differential interference
contrast optics, and an infrared video camera (Cohu, 4990 series, San Diego, Calif, USA).Whole-cell current clamp recordings were
obtained using patch pipettes pulled from borosilicate glass (1.0 mm OD, 4–7 MΩ) using a horizontal puller (Sutter Instr., P-97, Novato, Calif, USA)  and filled with a solution containing (in
mM) 140 K-gluconate, 5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2
ATP-tris, 0.4 GTP-tris (pH adjusted to 7.25 with KOH). Tight seals (>1 GΩ)
between the pipette and soma of cells in layer II of the medial entorhinal
cortex were obtained in voltage-clamp, and whole-cell configuration was
obtained using suction. Synaptic responses were evoked with a bipolar
stimulating electrode constructed from two fine tungsten electrodes (1 MΩ; Frederick Haer & Co., Bowdoin, Me, USA)
placed in layer I of the medial entorhinal cortex, 0.4 to 0.8 mm rostral to the
recording electrode. Constant current pulses (0.1 millisecond, 60–250μA) were
delivered using a pulse generator (WPI, Model A300) and stimulus isolator (WPI,
A360). Responses to synaptic activation and intracellular current injection
were obtained with an Axopatch 200B amplifier (Axon Instr., Sunnyvale, Calif, USA), filtered (10 kHz low-pass),
displayed on a digital oscilloscope (Gould 1602), and digitized at 20 kHz (Axon
Instr., Digidata 1322A) for storage on computer hard disc using the software
package Clampex 8.2 (Axon Instr.). Series and input resistances were monitored
regularly using −100 pA pulses. Recordings were accepted if the series
resistance was <35 MΩ, and if input resistance and resting membrane
potential were stable.


### 2.2. LTD Induction and Pharmacology

Whole-cell current clamp recordings of EPSPs
were monitored 10 minutes before and 30 minutes after LTD induction by
delivering test-pulses every 20 seconds. Intensity was adjusted to evoke EPSPs
that were approximately 3 to 4 mV in amplitude, and cells were held 5 mV below
threshold when necessary to prevent the occurrence of spikes in response to
EPSPs. Stimulus parameters for LTD
induction were based on those used previously in vivo and in vitro
[17, 18]. The induction of LTD was tested using pairs of stimulation pulses (30-millisecond
interpulse interval) delivered at a frequency of 1 Hz for either 7.5 or 15 minutes
[18]. Control cells received test-pulses throughout the recording period and
did not receive conditioning stimulation.Signalling mechanisms mediating the induction
of LTD were tested using stock solutions of pharmacological agents that were
stored frozen and diluted on the day of use. NMDA glutamate receptors were
blocked by constant bath application of 50μM DL-2-amino-5-phosphonovalerate
(APV). The calcium chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N′N′-tetraacetic
acid (BAPTA, 10 mM) was included in the recording electrode solution to block
increases in intracellular calcium. To block activation of the
calmodulin-dependent protein phosphatase calcineurin (PP2b) slices were pre-exposed
to 250μM cyclosporin A (Toronto Research Chemicals Inc., North York, Ontario, Canada) for 1.5 to 3 hours [39]. In other
experiments, FK506 (50μM) was included in the recording electrode solution to
block calcineurin [39, 40]. In other experiments, okadaic acid (0.1 or 1.0μM)
was included in the recording solution to block activation of protein phosphatases
1 and 2a [40, 41]. Control recordings without paired-pulse stimulation were
used to verify the stability of recordings in cells filled with FK506 and 1.0μM
okadaic acid.


### 2.3. Data Analysis

Synaptic
responses and electrophysiological properties of layer II neurons were analyzed
using the program Clampfit 8.2 (Axon Instr.). Data were standardized to the mean of baseline responses for plotting
and were expressed as the mean ±SEM. Changes in EPSP amplitude were assessed
using mixed-design ANOVAs and Neuman-Keuls tests that compared the average responses
during the baseline period, 5 minutes after conditioning stimulation, and during
the last 5 minutes of the recording period.Layer II neurons
were classified as putative stellate
or nonstellate neurons based on electrophysiological characteristics described
by Alonso and Klink [42]. Stellate neurons were characterized by the presence
of low-frequency subthreshold membrane potential oscillations, a depolarizing
afterpotential following spikes, and prominent inward rectification in response
to hyperpolarizing current pulses. Both
pyramidal and stellate neurons in layer II can show inward rectifying sag
responses [43]. Here, neurons recorded were clearly in layer II, usually near
the border with layer I, and a proportion of these neurons did not show clear
sag and were classified as pyramidal neurons. Input resistance was
determined from the peak voltage response to −100 pA current pulses (500-millisecond
duration), and rectification ratio was quantified by expressing peak input
resistance as a proportion of the steady-state resistance at the end of the current
pulse.


## 2.1. Slices and Whole Cell Recordings

Experiments were performed on slices from male
Long-Evans rats (4 to 8 weeks old). Animals were anesthetized with halothane and brains were rapidly removed
and cooled (4°C) in oxygenated artificial cerebrospinal fluid (ACSF). ACSF
consisted of (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2
MgSO4, 2 CaCl2, 26 NaHCO3, and 10 dextrose and
was saturated with 95% O2–5% CO2. All chemicals were obtained
from Sigma (St. Louis, Mo, USA) unless otherwise indicated. Horizontal slices (300μm) were cut with a
vibratome (WPI, Vibroslice
NVSL, Sarasota, Fla, USA) and were allowed to recover for at least one hour before
recordings. Slices were maintained in a recording chamber with oxygenated ACSF
at a rate of 2.0 mL/min, and a temperature from 22 to 24°C was used to
minimize metabolic demands on slices [18, 28]. Neurons were viewed with an
upright microscope (Leica
DML-FS, Wetzlar, Germany) equipped with a 40x objective, differential interference
contrast optics, and an infrared video camera (Cohu, 4990 series, San Diego, Calif, USA).Whole-cell current clamp recordings were
obtained using patch pipettes pulled from borosilicate glass (1.0 mm OD, 4–7 MΩ) using a horizontal puller (Sutter Instr., P-97, Novato, Calif, USA)  and filled with a solution containing (in
mM) 140 K-gluconate, 5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2
ATP-tris, 0.4 GTP-tris (pH adjusted to 7.25 with KOH). Tight seals (>1 GΩ)
between the pipette and soma of cells in layer II of the medial entorhinal
cortex were obtained in voltage-clamp, and whole-cell configuration was
obtained using suction. Synaptic responses were evoked with a bipolar
stimulating electrode constructed from two fine tungsten electrodes (1 MΩ; Frederick Haer & Co., Bowdoin, Me, USA)
placed in layer I of the medial entorhinal cortex, 0.4 to 0.8 mm rostral to the
recording electrode. Constant current pulses (0.1 millisecond, 60–250μA) were
delivered using a pulse generator (WPI, Model A300) and stimulus isolator (WPI,
A360). Responses to synaptic activation and intracellular current injection
were obtained with an Axopatch 200B amplifier (Axon Instr., Sunnyvale, Calif, USA), filtered (10 kHz low-pass),
displayed on a digital oscilloscope (Gould 1602), and digitized at 20 kHz (Axon
Instr., Digidata 1322A) for storage on computer hard disc using the software
package Clampex 8.2 (Axon Instr.). Series and input resistances were monitored
regularly using −100 pA pulses. Recordings were accepted if the series
resistance was <35 MΩ, and if input resistance and resting membrane
potential were stable.


## 2.2. LTD Induction and Pharmacology

Whole-cell current clamp recordings of EPSPs
were monitored 10 minutes before and 30 minutes after LTD induction by
delivering test-pulses every 20 seconds. Intensity was adjusted to evoke EPSPs
that were approximately 3 to 4 mV in amplitude, and cells were held 5 mV below
threshold when necessary to prevent the occurrence of spikes in response to
EPSPs. Stimulus parameters for LTD
induction were based on those used previously in vivo and in vitro
[17, 18]. The induction of LTD was tested using pairs of stimulation pulses (30-millisecond
interpulse interval) delivered at a frequency of 1 Hz for either 7.5 or 15 minutes
[18]. Control cells received test-pulses throughout the recording period and
did not receive conditioning stimulation.Signalling mechanisms mediating the induction
of LTD were tested using stock solutions of pharmacological agents that were
stored frozen and diluted on the day of use. NMDA glutamate receptors were
blocked by constant bath application of 50μM DL-2-amino-5-phosphonovalerate
(APV). The calcium chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N′N′-tetraacetic
acid (BAPTA, 10 mM) was included in the recording electrode solution to block
increases in intracellular calcium. To block activation of the
calmodulin-dependent protein phosphatase calcineurin (PP2b) slices were pre-exposed
to 250μM cyclosporin A (Toronto Research Chemicals Inc., North York, Ontario, Canada) for 1.5 to 3 hours [39]. In other
experiments, FK506 (50μM) was included in the recording electrode solution to
block calcineurin [39, 40]. In other experiments, okadaic acid (0.1 or 1.0μM)
was included in the recording solution to block activation of protein phosphatases
1 and 2a [40, 41]. Control recordings without paired-pulse stimulation were
used to verify the stability of recordings in cells filled with FK506 and 1.0μM
okadaic acid.


## 2.3. Data Analysis

Synaptic
responses and electrophysiological properties of layer II neurons were analyzed
using the program Clampfit 8.2 (Axon Instr.). Data were standardized to the mean of baseline responses for plotting
and were expressed as the mean ±SEM. Changes in EPSP amplitude were assessed
using mixed-design ANOVAs and Neuman-Keuls tests that compared the average responses
during the baseline period, 5 minutes after conditioning stimulation, and during
the last 5 minutes of the recording period.Layer II neurons
were classified as putative stellate
or nonstellate neurons based on electrophysiological characteristics described
by Alonso and Klink [42]. Stellate neurons were characterized by the presence
of low-frequency subthreshold membrane potential oscillations, a depolarizing
afterpotential following spikes, and prominent inward rectification in response
to hyperpolarizing current pulses. Both
pyramidal and stellate neurons in layer II can show inward rectifying sag
responses [43]. Here, neurons recorded were clearly in layer II, usually near
the border with layer I, and a proportion of these neurons did not show clear
sag and were classified as pyramidal neurons. Input resistance was
determined from the peak voltage response to −100 pA current pulses (500-millisecond
duration), and rectification ratio was quantified by expressing peak input
resistance as a proportion of the steady-state resistance at the end of the current
pulse.


## 3. Results

Stable recordings were obtained from 57 putative
stellate neurons and 21 putative nonstellate cells. Peak input resistance was
similar in stellate and pyramidal neurons (stellate, 95 ± 6 MΩ; pyramidal, 96
± 10 MΩ) but there was a much larger sag in voltage responses to hyperpolarizing
current injection in stellate cells (rectification ratio 1.37±0.04 in stellate
cells versus 1.06±0.01 in pyramidal cells). The amplitude of baseline synaptic
responses evoked by layer I stimulation was similar in stellate (3.9±0.2 mV)
and pyramidal cells (3.7±0.4 mV), and the amount of depression induced was
also similar for recording conditions in which significant LTD was obtained (71.2±5.6% in 14 stellate and 76.8±7.6% in 6 pyramidal cells).


### 3.1. LTD Induction

To determine if a relatively brief LTD
induction protocol could be used to induce LTD in whole-cell recordings, the
first tests attempted to induce LTD using paired-pulse delivery at 1 Hz for 7.5
minutes (n=10) which can induce
moderate LTD of field potentials in a gas-fluid interface recording chamber  [18].
Paired-pulse stimulation for 7.5 minutes did not induce depression of EPSPs
relative to control cells (93.0±10.0% of baseline after 30 minutes; F2,28=0.09,P=.92). We previously observed stronger LTD of field potentials in the
interface recording chamber after 15 minutes versus 7.5 minutes of paired-pulse
stimulation [18], and prolonged
paired-pulse stimulation for 15 minutes also reliably induced LTD of whole-cell
EPSPs (n=7, Figure 1). EPSP amplitude was reduced to 56.3±9.5% of
baseline levels 5 minutes after the conditioning stimulation, and remained at
58.6±6.1% of baseline levels at the end of the 30 minutes follow-up
period (F2,22=14.2,P<.001).
Responses in control cells were stable (n=6),
and remained at 99.6±2.6% of baseline levels at the end of the recording period (Figures 1(b2), 1(c)).Prolonged, low-frequency stimulation induces long-term depression of
EPSPs in neurons in layer II of the entorhinal cortex. (a) The location of stimulating and recording
electrodes in acute slices containing the entorhinal cortex. (b) and (c) Long-term
depression was induced by repetitive delivery of pairs of stimulation pulses at a rate of 1 Hz for 15 minutes (PP-LFS). The amplitude
of synaptic responses remained stable in control cells that did not receive conditioning
stimulation. Traces in (b) compare responses
recorded during the baseline period (1) and during the follow-up period (2) in a
neuron that received low-frequency stimulation (b1) and in a control cell (b2). Responses were obtained at the times indicated in (c).
Averaged points in (b) indicate the mean ±1 SEM in this
and subsequent figures. (d) Long-term depression was not
reliably induced when low-frequency stimulation was delivered for only 7.5 minutes
rather than 15 minutes, indicating that induction of LTD requires prolonged
stimulation.


### 3.2. NMDA Receptors and Postsynaptic Calcium

The NMDA receptor antagonist MK-801 blocks
induction of LTD in the entorhinal cortex in vivo [17] and the NMDA receptor blocker APV has been shown to prevent LTD
of field potentials and EPSPs in entorhinal cortex slices [18, 28, 33]. We therefore tested for
the NMDA receptor-dependence of LTD of EPSPs in the current preparation using
constant bath application of APV (50μM). Induction of LTD by 15 minutes of
paired-pulse stimulation was blocked by APV (n=6, Figure 2(a)). There was a tendency for responses to be
potentiated immediately following conditioning stimulation, but this variable effect
was not statistically significant, and responses were close to baseline levels
at the end of the recording period (96.7±13.2% of baseline;
F2,10=2.99,P=.09).The induction of long-term
depression is dependent on activation of NMDA glutamate receptors and on
increases in postsynaptic calcium. (a) Constant bath application of the
NMDA receptor antagonist APV (50μM) blocked the induction of long-term
depression by 15 minutes of paired-pulse low-frequency stimulation (PP LFS). (b) Blocking increases in
postsynaptic calcium by including the calcium chelator BAPTA (10 mM) in the recording
electrode solution also blocked the induction of LTD. The transient facilitation of EPSPs immediately
following stimulation was significant for the BAPTA condition but not the APV
condition, and responses were at baseline levels at the end of the recording
periods. The block of lasting depression suggests that calcium influx via NMDA
receptors is required for induction of LTD.The role of postsynaptic calcium in LTD
induction was tested by recording from cells in which the calcium chelator
BAPTA (10 mM) was included in the recording electrode solution (10 mM,n=6, Figure 2(b)). Cells filled with
BAPTA had longer-duration action potentials than control cells (6.1±0.7 versus
3.3±0.1 milliseconds measured at the base; t1,9=3,57,P<.01) consistent with a reduction in
calcium-dependent potassium conductances. The induction of LTD was blocked in
cells loaded with BAPTA. There was a significant increase in the amplitude of
EPSPs immediately following paired-pulse stimulation (to 122.3±6.0% of baseline; F2,10=5.46,P<.05; N–K, P<.05), but responses returned to
baseline levels within 10 minutes and were at 94.8±7.1% of baseline levels
after 30 minutes (N–K, P=0.50, Figure
2(b)). An increase in postsynaptic calcium is therefore required for induction
of LTD in layer II neurons of the entorhinal cortex.


### 3.3. Protein Phosphatases

The  role of the calmodulin-dependent protein phosphatase
calcineurin (PP2b) in LTD in layer II neurons was tested using either
pre-exposure to 250μM cyclosporin A in the bathing medium [39], or by
including 50μM FK506 postsynaptically in the recording electrode solution.  In cells pre-exposed to cyclosporin A, paired-pulse stimulation was followed by a depression in EPSP amplitude that
reached
82.4±7.5% of baseline levels after 30 minutes (Figure 3(a)).
Although the depression in the cyclosporin group was not statistically
significant (F2,10=3.51,P=0.07,n=6), the depression obtained was also not significantly less than that
observed in control ACSF (F1,11=3.79,P=.08). The result was therefore ambiguous with respect to the role
of calcineurin in LTD. To test the involvement of calcineurin more definitively
and to avoid potential presynaptic effects, the calcineurin blocker FK506 was
included in the recording electrode solution for additional groups of cells [40].
Responses in cells filled with FK506 showed a significant potentiation
immediately following paired-pulse stimulation (n=8), but there was no lasting
change in response amplitudes in comparison to control cells filled with FK506
that did not receive conditioning  stimulation (n=7). Responses were increased
to 134.9±10.5% of baseline levels immediately following  paired-pulse
stimulation, (F2,26=7.71,P<.01; N–K, P<.001;n=8) but returned to 102.2±6.1% of baseline levels after 30 minutes  (Figure 3(b)).Long-term depression is dependent
on activation of the calmodulin-dependent protein phosphatase calcineurin.
Although LTD was only partially inhibited by pre-exposure to cyclosporin A, it
was completely blocked when FK506 was included in the recording electrode
solution. (a) Pre-exposure of slices to the calcineurin inhibitor cyclosporin
A (250μM) for 1.5 to 3 hours resulted in a partial block of LTD by repeated
paired-pulse stimulation. The amount of LTD induced was smaller than in control
ACSF and was close to statistical significance (n=6,P=.07). (b) Including the FK506 in the recording electrode solution to directly block
postsynaptic calcineurin prevented the induction of LTD. Analysis of group responses showed a
significant increase in responses during the baseline period, but responses in
control cells indicate that this increase is transient and unlikely to have
affected measurement of LTD. Inhibition of postsynaptic calcineurin therefore
prevents induction of LTD in layer II cells of the entorhinal cortex.Inspection of averaged responses suggested that
there was an initial increase in responses during the baseline period among
cells filled with FK506, and comparison of responses recorded during the first
and last minutes of the baseline period showed that the increase was
significant (t14=3.09,P<.01).
Interestingly, then, interfering with calcineurin function can lead to enhanced
basal synaptic transmission in entorhinal neurons. This increase is not likely
to have affected measures of LTD in conditioned cells, however, because control
responses showed only a transient increase after which responses remained stable.Protein phosphatase 1 is thought to contribute directly
to suppression of hippocampal EPSPs during LTD by dephosphorylation of the GluR1
AMPA receptor subunit. The involvement of PP1 to LTD in the entorhinal cortex
was therefore tested by including okadaic acid in the recording electrode
solution. In early experiments, a low concentration of 0.1μM okadaic acid [41] did not block LTD
induction, and responses were depressed to 72.7±8.7% of baseline levels at the
end of the recording period (F2,24=4.65,P<.05; N–K, P<.001;n=8). However, increasing the concentration of okadaic
acid to 1.0μM [40] blocked the
induction of LTD. There was a variable and nonsignificant reduction in
responses immediately following conditioning stimulation (to 89.0±14.9% of
baseline) and responses were also near baseline levels after 30 minutes (96.0±6.6% of baseline 30; F2,22=0.18,P=.84;n=7; Figure 4). Activation of PP1 is therefore likely to contribute
to mechanisms of LTD in the entorhinal cortex.The induction of LTD was blocked in
a dose-dependent manner by including okadaic acid in the recording electrode
solution to block activation of protein phosphatase 1 (PP1). (a)
and (b) A low concentration of 0.1μM okadaic acid failed to block LTD
induction, but raising the concentration to 1.0μM resulted in a block of LTD
induction (compare traces in A1 versus A2). Responses in
control cells filled with 1.0μM okadaic acid that did not receive conditioning
stimulation remained stable. The block of LTD by okadaic acid suggests that activation
of PP1 mediates LTD in the entorhinal cortex.


## 3.1. LTD Induction

To determine if a relatively brief LTD
induction protocol could be used to induce LTD in whole-cell recordings, the
first tests attempted to induce LTD using paired-pulse delivery at 1 Hz for 7.5
minutes (n=10) which can induce
moderate LTD of field potentials in a gas-fluid interface recording chamber  [18].
Paired-pulse stimulation for 7.5 minutes did not induce depression of EPSPs
relative to control cells (93.0±10.0% of baseline after 30 minutes; F2,28=0.09,P=.92). We previously observed stronger LTD of field potentials in the
interface recording chamber after 15 minutes versus 7.5 minutes of paired-pulse
stimulation [18], and prolonged
paired-pulse stimulation for 15 minutes also reliably induced LTD of whole-cell
EPSPs (n=7, Figure 1). EPSP amplitude was reduced to 56.3±9.5% of
baseline levels 5 minutes after the conditioning stimulation, and remained at
58.6±6.1% of baseline levels at the end of the 30 minutes follow-up
period (F2,22=14.2,P<.001).
Responses in control cells were stable (n=6),
and remained at 99.6±2.6% of baseline levels at the end of the recording period (Figures 1(b2), 1(c)).Prolonged, low-frequency stimulation induces long-term depression of
EPSPs in neurons in layer II of the entorhinal cortex. (a) The location of stimulating and recording
electrodes in acute slices containing the entorhinal cortex. (b) and (c) Long-term
depression was induced by repetitive delivery of pairs of stimulation pulses at a rate of 1 Hz for 15 minutes (PP-LFS). The amplitude
of synaptic responses remained stable in control cells that did not receive conditioning
stimulation. Traces in (b) compare responses
recorded during the baseline period (1) and during the follow-up period (2) in a
neuron that received low-frequency stimulation (b1) and in a control cell (b2). Responses were obtained at the times indicated in (c).
Averaged points in (b) indicate the mean ±1 SEM in this
and subsequent figures. (d) Long-term depression was not
reliably induced when low-frequency stimulation was delivered for only 7.5 minutes
rather than 15 minutes, indicating that induction of LTD requires prolonged
stimulation.


## 3.2. NMDA Receptors and Postsynaptic Calcium

The NMDA receptor antagonist MK-801 blocks
induction of LTD in the entorhinal cortex in vivo [17] and the NMDA receptor blocker APV has been shown to prevent LTD
of field potentials and EPSPs in entorhinal cortex slices [18, 28, 33]. We therefore tested for
the NMDA receptor-dependence of LTD of EPSPs in the current preparation using
constant bath application of APV (50μM). Induction of LTD by 15 minutes of
paired-pulse stimulation was blocked by APV (n=6, Figure 2(a)). There was a tendency for responses to be
potentiated immediately following conditioning stimulation, but this variable effect
was not statistically significant, and responses were close to baseline levels
at the end of the recording period (96.7±13.2% of baseline;
F2,10=2.99,P=.09).The induction of long-term
depression is dependent on activation of NMDA glutamate receptors and on
increases in postsynaptic calcium. (a) Constant bath application of the
NMDA receptor antagonist APV (50μM) blocked the induction of long-term
depression by 15 minutes of paired-pulse low-frequency stimulation (PP LFS). (b) Blocking increases in
postsynaptic calcium by including the calcium chelator BAPTA (10 mM) in the recording
electrode solution also blocked the induction of LTD. The transient facilitation of EPSPs immediately
following stimulation was significant for the BAPTA condition but not the APV
condition, and responses were at baseline levels at the end of the recording
periods. The block of lasting depression suggests that calcium influx via NMDA
receptors is required for induction of LTD.The role of postsynaptic calcium in LTD
induction was tested by recording from cells in which the calcium chelator
BAPTA (10 mM) was included in the recording electrode solution (10 mM,n=6, Figure 2(b)). Cells filled with
BAPTA had longer-duration action potentials than control cells (6.1±0.7 versus
3.3±0.1 milliseconds measured at the base; t1,9=3,57,P<.01) consistent with a reduction in
calcium-dependent potassium conductances. The induction of LTD was blocked in
cells loaded with BAPTA. There was a significant increase in the amplitude of
EPSPs immediately following paired-pulse stimulation (to 122.3±6.0% of baseline; F2,10=5.46,P<.05; N–K, P<.05), but responses returned to
baseline levels within 10 minutes and were at 94.8±7.1% of baseline levels
after 30 minutes (N–K, P=0.50, Figure
2(b)). An increase in postsynaptic calcium is therefore required for induction
of LTD in layer II neurons of the entorhinal cortex.


## 3.3. Protein Phosphatases

The  role of the calmodulin-dependent protein phosphatase
calcineurin (PP2b) in LTD in layer II neurons was tested using either
pre-exposure to 250μM cyclosporin A in the bathing medium [39], or by
including 50μM FK506 postsynaptically in the recording electrode solution.  In cells pre-exposed to cyclosporin A, paired-pulse stimulation was followed by a depression in EPSP amplitude that
reached
82.4±7.5% of baseline levels after 30 minutes (Figure 3(a)).
Although the depression in the cyclosporin group was not statistically
significant (F2,10=3.51,P=0.07,n=6), the depression obtained was also not significantly less than that
observed in control ACSF (F1,11=3.79,P=.08). The result was therefore ambiguous with respect to the role
of calcineurin in LTD. To test the involvement of calcineurin more definitively
and to avoid potential presynaptic effects, the calcineurin blocker FK506 was
included in the recording electrode solution for additional groups of cells [40].
Responses in cells filled with FK506 showed a significant potentiation
immediately following paired-pulse stimulation (n=8), but there was no lasting
change in response amplitudes in comparison to control cells filled with FK506
that did not receive conditioning  stimulation (n=7). Responses were increased
to 134.9±10.5% of baseline levels immediately following  paired-pulse
stimulation, (F2,26=7.71,P<.01; N–K, P<.001;n=8) but returned to 102.2±6.1% of baseline levels after 30 minutes  (Figure 3(b)).Long-term depression is dependent
on activation of the calmodulin-dependent protein phosphatase calcineurin.
Although LTD was only partially inhibited by pre-exposure to cyclosporin A, it
was completely blocked when FK506 was included in the recording electrode
solution. (a) Pre-exposure of slices to the calcineurin inhibitor cyclosporin
A (250μM) for 1.5 to 3 hours resulted in a partial block of LTD by repeated
paired-pulse stimulation. The amount of LTD induced was smaller than in control
ACSF and was close to statistical significance (n=6,P=.07). (b) Including the FK506 in the recording electrode solution to directly block
postsynaptic calcineurin prevented the induction of LTD. Analysis of group responses showed a
significant increase in responses during the baseline period, but responses in
control cells indicate that this increase is transient and unlikely to have
affected measurement of LTD. Inhibition of postsynaptic calcineurin therefore
prevents induction of LTD in layer II cells of the entorhinal cortex.Inspection of averaged responses suggested that
there was an initial increase in responses during the baseline period among
cells filled with FK506, and comparison of responses recorded during the first
and last minutes of the baseline period showed that the increase was
significant (t14=3.09,P<.01).
Interestingly, then, interfering with calcineurin function can lead to enhanced
basal synaptic transmission in entorhinal neurons. This increase is not likely
to have affected measures of LTD in conditioned cells, however, because control
responses showed only a transient increase after which responses remained stable.Protein phosphatase 1 is thought to contribute directly
to suppression of hippocampal EPSPs during LTD by dephosphorylation of the GluR1
AMPA receptor subunit. The involvement of PP1 to LTD in the entorhinal cortex
was therefore tested by including okadaic acid in the recording electrode
solution. In early experiments, a low concentration of 0.1μM okadaic acid [41] did not block LTD
induction, and responses were depressed to 72.7±8.7% of baseline levels at the
end of the recording period (F2,24=4.65,P<.05; N–K, P<.001;n=8). However, increasing the concentration of okadaic
acid to 1.0μM [40] blocked the
induction of LTD. There was a variable and nonsignificant reduction in
responses immediately following conditioning stimulation (to 89.0±14.9% of
baseline) and responses were also near baseline levels after 30 minutes (96.0±6.6% of baseline 30; F2,22=0.18,P=.84;n=7; Figure 4). Activation of PP1 is therefore likely to contribute
to mechanisms of LTD in the entorhinal cortex.The induction of LTD was blocked in
a dose-dependent manner by including okadaic acid in the recording electrode
solution to block activation of protein phosphatase 1 (PP1). (a)
and (b) A low concentration of 0.1μM okadaic acid failed to block LTD
induction, but raising the concentration to 1.0μM resulted in a block of LTD
induction (compare traces in A1 versus A2). Responses in
control cells filled with 1.0μM okadaic acid that did not receive conditioning
stimulation remained stable. The block of LTD by okadaic acid suggests that activation
of PP1 mediates LTD in the entorhinal cortex.


## 4. Discussion

The current paper has used prolonged repetitive
paired-pulse stimulation to induce LTD in layer I inputs to layer II neurons of
the medial entorhinal cortex and has determined the early postsynaptic signals
that mediate LTD in these cells. Consistent with previous observations, the LTD
observed here was obtained in both putatively identified stellate [28] and
pyramidal [44] cells. The induction of LTD was blocked by the NMDA glutamate
receptor antagonist APV, and by the calcium chelator BAPTA, indicating that
calcium influx via NMDA receptors is required for LTD. The induction of LTD was
also blocked by the calcineurin inhibitor FK506, and by okadaic acid which
blocks activation of protein phosphatases 1 and 2a. Calcineurin is required for
LTD of deep layer inputs to layer II stellate cells [28], and
calcineurin-dependent activation of PP1 contributes to NMDA receptor-dependent
LTD of AMPA responses in the hippocampus [2, 4].The dependence of LTD in the entorhinal cortex
on activation of NMDA receptors has been a consistent finding in vivo and in
slices. It has been observed following stimulation protocols including 1 Hz trains,
pairing of presynaptic stimulation at 0.33 Hz with postsynaptic depolarization
[28], repeated paired-pulse stimulation [18, 33], and spike-timing-dependent
induction of LTD [44]. Long-term depression was blocked by including the calcium
chelator BAPTA in the recording electrode solution (Figure 2) [28], and this is
consistent with calcium influx via NMDA receptors as a critical trigger for
entorhinal LTD. Metabotropic glutamate receptor activation and release of
calcium from intracellular stores can contribute to LTD in the hippocampus [2, 36, 37, 45], but activation of metabotropic glutamate receptors is not required
for entorhinal LTD [18, 28]. Calcium influx through voltage-gated calcium
channels can contribute to spike-timing-dependent LTD in the entorhinal cortex,
however. Cells with broadened action potentials that result in larger calcium transients show greater NMDA
receptor-dependent spike-timing-dependent LTD in layer II-III cells [44]. Calcium influx through voltage-gated
channels also mediates bidirectional spike-timing-dependent plasticity of
inhibitory synapses in entorhinal cortex [46]. A form of long-term depression
on layer V-VI neurons, expressed presynaptically through reduced transmitter
release, is also dependent on activation of voltage-dependent calcium channels
[33]. Calcium signalling mediated by voltage-gated channels therefore plays a
number of roles in modulating synaptic plasticity in the entorhinal cortex.The contribution of the
calmodulin-dependent protein phosphatase calcineurin to LTD was tested by
incubating slices in cyclosporin A or by including FK506 in the recording
electrode solution. Cyclosporin A appeared to cause a partial block of LTD, and
responses were reduced to 82.4% of baseline as compared to 58.6%
in untreated cells (compare Figures1(c)
and 3(a)), but the sizes of these LTD effects were not statistically different.
We obtained a more conclusive result with FK506, however, and LTD was
completely blocked by including FK506 in the recording electrode solution. Including
FK506 in the bathing medium has been used to block calcineurin-dependent
depression effects in entorhinal cortex [28], and in excitatory [47] and
inhibitory [48] synapses of the CA1 region. Here, we have loaded FK506 into the
recording electrode solution to avoid possible presynaptic effects of the drug
and to ensure that FK506 could act on calcineurin [39, 40, 49, 50]. The
block of LTD by FK506 indicates that LTD is dependent on calcineurin, and this
suggests that cyclosporin A resulted in only a partial block of calcineurin
activity.Calcineurin is thought
to mediate expression of LTD in part by dephosphorylating inhibitor 1 and
thereby increasing the activity of PP1 [2, 4, 39]. The PP1/PP2a inhibitor okadaic
acid blocks LTD in the CA1 region [38, 40], and we have shown here that the induction
of LTD in the entorhinal cortex was blocked by including okadaic acid in the
recording electrode solution. This is the first report of LTD in the entorhinal
cortex dependent on PP1/PP2a. Protein phosphatases can regulate synaptic
function through a variety of mechanisms [51] that include dephosphorylation of
the ser-845 residue on the AMPA GluR1 subunit, and LTD in the entorhinal cortex
may be expressed partly through this mechanism. In addition, the work of Deng
and Lei [28] has found entorhinal LTD to be associated with a reduction in the
number of postsynaptic AMPA receptors, with no change in AMPA receptor
conductance, and has shown that this effect is dependent on proteosomes that degrade
AMPA receptors internalized through ubiquitinization. As in the hippocampus,
therefore, entorhinal LTD can be expressed through mechanisms involving
trafficking of AMPA receptors [52].Long-term depression was induced here using
strong repetitive paired-pulse stimulation which we have used previously to
induce LTD in the entorhinal cortex in vivo and in slices ([17, 18], see also [33, 34]). LTD was induced following 15 minutes, but not 7.5 minutes of paired-pulse
stimulation; this is consistent with a requirement for prolonged activation of
calcium-dependent signalling mechanisms, and is also consistent with the
possibility that NMDA receptor-dependent metaplastic changes early in the train
may promote LTD induced by stimuli that occurred later in the 15-minute
duration trains [53]. We previously found 1 Hz stimulation to be ineffective in
vivo and in slices from Long-Evans rats [17, 18], but deep layer inputs to
stellate neurons in slices from 2 to 3 week-old Sprague-Dawley rats express
NMDA receptor-dependent LTD following 15 minutes of 1 Hz stimulation, or
following low-frequency stimulation paired with postsynaptic depolarization [28].
Thus, there may be developmental, strain-related, or pathway-specific factors
that affect the ability of 1 Hz stimulation to activate these signalling mechanisms.The entorhinal cortex
is embedded within the temporal lobe through an extensive array of anatomical
connections [7] and has been linked behaviorally to a variety of sensory and
cognitive functions (e.g., [9, 10]). Lasting synaptic plasticity in the
entorhinal cortex is therefore likely to serve a variety of functions depending
on the synaptic pathways involved. Synaptic depression effects are generally thought
to complement synaptic potentiation during the formation of memory [45, 54–56], and it is possible that depression effects contribute to short and/or
long-term memory processing. However, the laminar architecture of the
entorhinal cortex, with superficial layers mediating much of the cortical input
to the hippocampal formation, suggests that long-term depression of synaptic
transmission in layer II may lead to long-term reductions in the salience of
particular elements or patterns of cortical input and may thus lead to lasting
changes in the multimodal inputs processed by the hippocampal formation.  Similarly, the general resistance of the
entorhinal cortex to induction of LTD could serve to maintain relatively stable
information processing and integration of multimodal sensory inputs within the
medial entorhinal cortex.



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*Source: 840374-2008-07-22.xml*