# Differential Induction of Long-Term Potentiation in the Horizontal versus Columnar Superficial Connections to Layer II Cells of the Entorhinal Cortex

Authors: Li Ma; Angel Alonso; Clayton T. Dickson
Journal: Neural Plasticity
 (2008)

---

## Abstract

The entorhinal cortex (EC) is a nodal and independent mnemonic element of the medial temporal lobe memory circuit as it forms a bidirectional interface between the neocortex and hippocampus. Within the EC, intra- and inter-lamellar associational connections occur via horizontal and columnar projections, respectively. We undertook a comparative study of these two inputs as they converge upon EC layer II cells using whole-cell patch techniques in an adult rat EC horizontal slice preparation in which the deepest layers (V-VI) had been dissected out. Electrical stimulation of layers I and III during GABA blockade allowed us to study excitatory synaptic properties and plasticity in the horizontal and columnar fibre systems, respectively. Both pathways exhibited AMPA- and NMDA-receptor mediated transmission and both exhibited long-term potentiation (LTP) after high-frequency (tetanic) stimulation. LTP in the horizontal, but not in the columnar pathway, was blocked by NMDA receptor antagonism. Intriguingly, LTP in both appeared to be mediated by post synaptic increases inCa2+  that may be coupled to differing second messenger pathways. Thus, the superficial excitatory horizontal and columnar associative pathways to layer II have divergent mechanisms for LTP which may endow the EC with even more complex and dynamic processing characteristics than previously thought.


---

## Body

## 1. Introduction

The entorhinal cortex (EC) is a prominent
component of the medial temporal lobe memory system. The superficial layers (II
and III) of the EC receive an extensive input from multimodal sensory
associational areas of neocortex and, in turn, project to all subregions of the
hippocampal formation. Output stations of the hippocampus, including CA1 and
subiculum, project back to the deep layers of entorhinal cortex (VI & V), reciprocating
the input channels [1, 2]. Therefore, the EC serves as a
bidirectional interface between the neocortex and hippocampal formation and as
such forms a nodal part of the cortico-hippocampo-cortical loop that is the
brain’s hardware for the formation of declarative memories.The importance of the EC in memory
processing, however, is thought to go beyond just its interconnections with the
hippocampus. One suggestion is that the EC and other parahippocampal regions
serve as a temporary memory store that is critical to normal hippocampal-dependent
memory processing [3]. Behavioral studies have
suggested that lesions involving the EC are followed by learning and memory
deficits in mammals (reviewed in [4]). Indeed, early stage tissue
from Alzheimer’s patients in which memory impairments are just subclinical demonstrates neurodegeneration in the superficial layers of the EC specifically [5, 6]. Still other work has shown that
embryonic entorhinal transplants partially ameliorate the deficits in spatial
memory in adult rats with EC lesions [7]. Perhaps the strongest evidence for
an independent role of the EC in memory is that a number of entorhinal cells
demonstrate persistent activity in the delay phase of memory tasks which
correlates to the retention of information necessary to perform during a
subsequent go phase [8, 9]. Indeed, entorhinal cells also demonstrate intrinsic “memory-like”
persistent firing properties dependent upon associative convergence of
excitatory inputs and cholinergic neuromodulation [10].Synaptic plasticity of excitatory
glutamatergic responses, via long-term potentiation (LTP), has been proposed as
a mechanism underlying learning and memory. The most commonly studied form
involves an NMDA-receptor dependent process whereby postsynaptic Ca2+ influx through this ligand-gated channel induces changes via a series of
intracellular second messengers (typically beginning with the calcium/calmodulin-dependent
kinase: CaMKII) that result in the enhancement of neurotransmission [11].It has also been shown that LTP can occur through non-NMDA dependent
triggers such as activation of either voltage-dependent calcium channels or
metabotropic glutamatergic receptors which can also lead to increases in Ca2+ influx and LTP via potentially overlapping intracellular mechanisms [12–14]. In addition, non-CaMKII-dependent processes
have also been elucidated [11, 15, 16]. Although perhaps differing in
their cellular induction mechanisms, all of the above are thought to express
their effects mainly through postsynaptic changes to AMPA-type receptors that result
in an enhancement of glutamate responsiveness. Changes to presynaptic release (due to growth of new contacts and
enhancement of release machinery) have also been suggested to play a role (reviewed
in [11, 15]).A different form of LTP exists that appears
to involve a completely different induction and expression mechanisms. This form, first elucidated in the mossy
fibre input to CA3 pyramidal neurons in the hippocampus, does not require
either NMDA receptor activation or an increase in postsynaptic Ca2+ [17]. The locus of induction and
expression of this form of LTP is presynaptic [18–20], involving no changes in
postsynaptic receptivity. This presumed increase in neurotransmitter release is
accompanied by a marked and long-lasting decrease in the paired-pulse
facilitation ratio [18].The cellular mechanisms underlying LTP in
the EC remain understudied. Field recordings of LTP phenomenon have been
reported in layer II and some interesting differences between deep and
superficial associational pathways have been reported [21, 22]. To date, no studies have assessed, in the same superficial layer II
cells, the differential properties of LTP in these two important pathways. Here,
using whole-cell-recording technique, we investigated the physiology,
pharmacology, and plasticity of both horizontal and columnar associative inputs
to layer II cells using whole cell techniques in an in vitro slice preparation.
These results have been previously published in abstract form [23].


## 2. Materials and Methods

### 2.1. General

Brain
slices were prepared from male Long-Evans rats (100–200 g) using
standard procedures [24]. All methods used conformed to the guidelines established by the
Canadian Council on Animal Care and the Society for Neuroscience. Animals were quickly
decapitated, and the brain was rapidly removed, blocked, and placed in a cold
(4–6°C) oxygenated
normal Ringer’s solution containing (in mM): 124 NaCl, 5 KCl. 1.3 NaH2PO4,
2.0 CaCl2, 2.0 MgSO4, 26 NaHCO3, and 10
glucose (pH 7.4, by gaseous saturation with 95% O2 & 5% CO2).
Horizontal slices from the retrohippocampal region were cut at a thickness of
400μm using a vibratome (Pelco, Redding,
Calif, USA).
After at least an hour recovery period during which they were submerged in
normal Ringer’s at room temperature (23°C–25°C), individual
slices were transferred to a recording chamber located on the stage of an
upright, fixed-stage microscope (Axioskop, Zeiss). Slices were submerged and perfused
continuously with a saturated (95% O2 + 5% CO2) solution containing
(in mM) 126 NaCl, 2.5 KCl, 1.3 NaH2PO4, 2.4 CaCl2,
1.4 MgSO4, 26 NaHCO3, 10 glucose, and 0.05 picrotoxin (a
GABAA receptor antagonist) at room temperature. Before being transferred, a cut was made between
layer III and layer V regions of entorhinal cortex to prevent the propagation
of epileptiform activity. The microscope
was equipped with a water immersion objective (40–63X: long-working
distance), Nomarski optics, and a near-infrared charge-coupled device (CCD)
camera (Sony XC-75). With this equipment,
individual cells in layer II could be visualized via video microscopy and
targeted for recording [25].


### 2.2. Recording

Somatic
whole-cell voltage-clamp recordings [26] were made under visual control using 4–7 MΩ electrodes. The
whole-cell solution contained (in mM): 130 K-Gluconate, 5 NaCl, 2 MgCl2,
10 Hepes, 0.5 EGTA, 2 ATP, 0.4 GTP (pH 7.2–7.4 with 1 M KOH; osmolarity: 290–300 mOsm). In experiments designed to chelate intracellular Ca2+, 10 mM
BAPTA was substituted for equimolar K-Gluconate. To investigate properties of
evoked EPSCs at varying membrane potentials, 130 mM Cs-Gluconate and 0.8 mM QX314-Cl
were used in the whole-cell solution in place of K-Gluconate. The liquid
junction potential was estimated using the method of Neher (1992) and was found
to be 8 mV. No correction of transmembrane potential was applied for the liquid
junction potential unless indicated otherwise.Whole
cell voltage clamp recordings were made using an Axoclamp2A or Axopatch200B amplifier
(Axon Instruments, Foster City,
Calif, USA). The low-pass filter (−3 dB) was set to 1 kHz,
and the resulting current trace was digitized to computer at 2 kHz using a
Digidata 1320 A-D converter driven by Pclamp7 (Axon Instruments).Cells
were held at −60 to −70 mV during recordings unless indicated otherwise. Series
resistance was measured automatically using Pclamp7 software and those with values higher than
30 MΩ in whole-cell configuration were discarded. In
the remainder, series resistance was compensated >40% with the amplifier’s
built-in compensation circuitry. Input resistance of
cells (averaging169.67±5.67 MΩ, N=171) was calculated by measuring current
deflections in response to 5 mV hyperpolarizing voltage pulses from the holding
potential and was monitored through each recording. A short-duration
hyperpolarizing voltage step (−5 mV, 250 millisecods) was always applied to each
sweep just prior to stimulation to monitor any change in input resistance and
series resistance. Cells exhibiting detectible changes in the elicited current process
to the hyperpolarization during recording were discarded.


### 2.3. Stimulation and Experimental Protocol

Electrical stimulation of afferent pathways to
layer II was conducted through bipolar, stainless steel electrodes insulated
except at their tips. These were
positioned on the surface of the slice in layers I/II or III (see diagram in
Figure1(a)). Afferent inputs were activated in this way by passing short
(100μs) current pulses of 0.5–4.0 mA using a pulse
generator (PG-4000 Cygnus Tech, NY) coupled to a stimulus isolation/constant
current generator (Model A395 WPI). EPSCs were evoked once every 30 seconds,
and the basal amplitude of EPSCs was set to 100–200 pA by adjusting the
stimulation intensity. EPSCs were accepted as being monosynaptic if they
exhibited short and consistent latencies that did not change with increasing
stimulus intensity.Figure 1
Properties of evoked EPSCs in EC layer II
neurons. (a) Schematic diagram of
the ascending columnar (arrow a) and the horizontal (arrow b) inputs converging
on a layer II cell in entorhinal cortex (I, II, and III refer to the
superficial layers of the EC). (b) Stimulation
of layer III evoked excitatory postsynaptic currents that had different
profiles at hyperpolarized (−70 mV) versus depolarized (+30 mV) potentials and
sensitivity to APV. For these experiments, 130 mM CsGluconate and 0.8 mM
QX314-Cl were substituted for KGluconate in the recording pipette. Corrections were made for the liquid junction
potential. In control conditions (leftmost panel), EPSCs demonstrated only a fast
decay component at −70 mV, but showed a slower decay component at +30 mV. In the presence of 50μM DL-APV (middle panel),
only the fast component was observed at both holding potentials (a direct
comparison is shown in the superimposed traces in the rightmost panel). (c) I-V relationship for EPSC components
measured at peak and 30 milliseconds post peak for the same cell (see dotted
lines in (b)). The peak amplitude (closed square plots)
showed a linear relationship across the full range of membrane potentials
tested (−70 to +30 mV). This was unchanged following application of APV (open
square plots). In control conditions, the amplitude measured 30 milliseconds following
the peak (filled circles) was lower and showed a negative slope region at
depolarized membrane potentials levels (−60 to −20 mV) but became nearly linear
thereafter. In the presence of APV (open circles), the negative slope region
was abolished and the resultant plot was linear across the full range of
membrane potentials.Following
at least 5 minutes of stable baseline recording, LTP was elicited using a high-frequency
(tetanic) stimulation train (HFS: 100 Hz; 1 second) at the same intensity used
for baseline recording. Typically, HFS
was delivered no more than approximately 15 minutes following the formation of whole
cell configuration. Little to no potentiation was observed if HFS was applied
at times greater than 30 minutes after achieving whole cell mode. Successful
LTP was evaluated statistically in comparison to baseline values at a time
point 30 minutes following HFS for each experiment.Paired
pulse facilitation protocols were used in order to assess any changes in
presynaptic release mechanisms. The two pathways were stimulated either singly
or alternatively at short intervals (50–70 milliseconds).
PPF ratio was computed by dividing the peak amplitude of the second EPSC by the
peak amplitude of the first in each experiment.Application
of drugs was conducted by adding stock solutions directly to the superfusion medium
to make appropriate final concentrations. DL-APV (Tocris) and CNQX (Tocris)
were made up as a 10 mM stock solution in an equivalent volume mix of 1M NaOH
and dimethyl sulfoxide. These were pipetted at smaller volumes into centrifuge
tubes and stored frozen (−20°) until used. Picrotoxin and BAPTA were purchased
from “Sigma (St. Louis, Mo).”


### 2.4. Data Analysis

The
amplitude of EPSCs was measured offline using Clampfit 6 (Axon Instruments), by
calculating the difference between the peak deflections relative to the average holding
current level computed for the 10 milliseconds preceding the stimulus. Average traces as shown in figure insets
represented means of 5–10 individual EPSC
sweeps. Peak amplitudes or paired-pulse facilitation (PPF) ratios were computed
from these averages. Potentiation of EPSC amplitudes or PPF ratios following
the application of HFS was expressed by normalizing amplitudes to the baseline values
pre-HFS. Reported values reflect arithmetic means ± standard error of the mean
(SEM), and statistical significance was determined by paired and unpaired Student’st-tests.


## 2.1. General

Brain
slices were prepared from male Long-Evans rats (100–200 g) using
standard procedures [24]. All methods used conformed to the guidelines established by the
Canadian Council on Animal Care and the Society for Neuroscience. Animals were quickly
decapitated, and the brain was rapidly removed, blocked, and placed in a cold
(4–6°C) oxygenated
normal Ringer’s solution containing (in mM): 124 NaCl, 5 KCl. 1.3 NaH2PO4,
2.0 CaCl2, 2.0 MgSO4, 26 NaHCO3, and 10
glucose (pH 7.4, by gaseous saturation with 95% O2 & 5% CO2).
Horizontal slices from the retrohippocampal region were cut at a thickness of
400μm using a vibratome (Pelco, Redding,
Calif, USA).
After at least an hour recovery period during which they were submerged in
normal Ringer’s at room temperature (23°C–25°C), individual
slices were transferred to a recording chamber located on the stage of an
upright, fixed-stage microscope (Axioskop, Zeiss). Slices were submerged and perfused
continuously with a saturated (95% O2 + 5% CO2) solution containing
(in mM) 126 NaCl, 2.5 KCl, 1.3 NaH2PO4, 2.4 CaCl2,
1.4 MgSO4, 26 NaHCO3, 10 glucose, and 0.05 picrotoxin (a
GABAA receptor antagonist) at room temperature. Before being transferred, a cut was made between
layer III and layer V regions of entorhinal cortex to prevent the propagation
of epileptiform activity. The microscope
was equipped with a water immersion objective (40–63X: long-working
distance), Nomarski optics, and a near-infrared charge-coupled device (CCD)
camera (Sony XC-75). With this equipment,
individual cells in layer II could be visualized via video microscopy and
targeted for recording [25].


## 2.2. Recording

Somatic
whole-cell voltage-clamp recordings [26] were made under visual control using 4–7 MΩ electrodes. The
whole-cell solution contained (in mM): 130 K-Gluconate, 5 NaCl, 2 MgCl2,
10 Hepes, 0.5 EGTA, 2 ATP, 0.4 GTP (pH 7.2–7.4 with 1 M KOH; osmolarity: 290–300 mOsm). In experiments designed to chelate intracellular Ca2+, 10 mM
BAPTA was substituted for equimolar K-Gluconate. To investigate properties of
evoked EPSCs at varying membrane potentials, 130 mM Cs-Gluconate and 0.8 mM QX314-Cl
were used in the whole-cell solution in place of K-Gluconate. The liquid
junction potential was estimated using the method of Neher (1992) and was found
to be 8 mV. No correction of transmembrane potential was applied for the liquid
junction potential unless indicated otherwise.Whole
cell voltage clamp recordings were made using an Axoclamp2A or Axopatch200B amplifier
(Axon Instruments, Foster City,
Calif, USA). The low-pass filter (−3 dB) was set to 1 kHz,
and the resulting current trace was digitized to computer at 2 kHz using a
Digidata 1320 A-D converter driven by Pclamp7 (Axon Instruments).Cells
were held at −60 to −70 mV during recordings unless indicated otherwise. Series
resistance was measured automatically using Pclamp7 software and those with values higher than
30 MΩ in whole-cell configuration were discarded. In
the remainder, series resistance was compensated >40% with the amplifier’s
built-in compensation circuitry. Input resistance of
cells (averaging169.67±5.67 MΩ, N=171) was calculated by measuring current
deflections in response to 5 mV hyperpolarizing voltage pulses from the holding
potential and was monitored through each recording. A short-duration
hyperpolarizing voltage step (−5 mV, 250 millisecods) was always applied to each
sweep just prior to stimulation to monitor any change in input resistance and
series resistance. Cells exhibiting detectible changes in the elicited current process
to the hyperpolarization during recording were discarded.


## 2.3. Stimulation and Experimental Protocol

Electrical stimulation of afferent pathways to
layer II was conducted through bipolar, stainless steel electrodes insulated
except at their tips. These were
positioned on the surface of the slice in layers I/II or III (see diagram in
Figure1(a)). Afferent inputs were activated in this way by passing short
(100μs) current pulses of 0.5–4.0 mA using a pulse
generator (PG-4000 Cygnus Tech, NY) coupled to a stimulus isolation/constant
current generator (Model A395 WPI). EPSCs were evoked once every 30 seconds,
and the basal amplitude of EPSCs was set to 100–200 pA by adjusting the
stimulation intensity. EPSCs were accepted as being monosynaptic if they
exhibited short and consistent latencies that did not change with increasing
stimulus intensity.Figure 1
Properties of evoked EPSCs in EC layer II
neurons. (a) Schematic diagram of
the ascending columnar (arrow a) and the horizontal (arrow b) inputs converging
on a layer II cell in entorhinal cortex (I, II, and III refer to the
superficial layers of the EC). (b) Stimulation
of layer III evoked excitatory postsynaptic currents that had different
profiles at hyperpolarized (−70 mV) versus depolarized (+30 mV) potentials and
sensitivity to APV. For these experiments, 130 mM CsGluconate and 0.8 mM
QX314-Cl were substituted for KGluconate in the recording pipette. Corrections were made for the liquid junction
potential. In control conditions (leftmost panel), EPSCs demonstrated only a fast
decay component at −70 mV, but showed a slower decay component at +30 mV. In the presence of 50μM DL-APV (middle panel),
only the fast component was observed at both holding potentials (a direct
comparison is shown in the superimposed traces in the rightmost panel). (c) I-V relationship for EPSC components
measured at peak and 30 milliseconds post peak for the same cell (see dotted
lines in (b)). The peak amplitude (closed square plots)
showed a linear relationship across the full range of membrane potentials
tested (−70 to +30 mV). This was unchanged following application of APV (open
square plots). In control conditions, the amplitude measured 30 milliseconds following
the peak (filled circles) was lower and showed a negative slope region at
depolarized membrane potentials levels (−60 to −20 mV) but became nearly linear
thereafter. In the presence of APV (open circles), the negative slope region
was abolished and the resultant plot was linear across the full range of
membrane potentials.Following
at least 5 minutes of stable baseline recording, LTP was elicited using a high-frequency
(tetanic) stimulation train (HFS: 100 Hz; 1 second) at the same intensity used
for baseline recording. Typically, HFS
was delivered no more than approximately 15 minutes following the formation of whole
cell configuration. Little to no potentiation was observed if HFS was applied
at times greater than 30 minutes after achieving whole cell mode. Successful
LTP was evaluated statistically in comparison to baseline values at a time
point 30 minutes following HFS for each experiment.Paired
pulse facilitation protocols were used in order to assess any changes in
presynaptic release mechanisms. The two pathways were stimulated either singly
or alternatively at short intervals (50–70 milliseconds).
PPF ratio was computed by dividing the peak amplitude of the second EPSC by the
peak amplitude of the first in each experiment.Application
of drugs was conducted by adding stock solutions directly to the superfusion medium
to make appropriate final concentrations. DL-APV (Tocris) and CNQX (Tocris)
were made up as a 10 mM stock solution in an equivalent volume mix of 1M NaOH
and dimethyl sulfoxide. These were pipetted at smaller volumes into centrifuge
tubes and stored frozen (−20°) until used. Picrotoxin and BAPTA were purchased
from “Sigma (St. Louis, Mo).”


## 2.4. Data Analysis

The
amplitude of EPSCs was measured offline using Clampfit 6 (Axon Instruments), by
calculating the difference between the peak deflections relative to the average holding
current level computed for the 10 milliseconds preceding the stimulus. Average traces as shown in figure insets
represented means of 5–10 individual EPSC
sweeps. Peak amplitudes or paired-pulse facilitation (PPF) ratios were computed
from these averages. Potentiation of EPSC amplitudes or PPF ratios following
the application of HFS was expressed by normalizing amplitudes to the baseline values
pre-HFS. Reported values reflect arithmetic means ± standard error of the mean
(SEM), and statistical significance was determined by paired and unpaired Student’st-tests.


## 3. Results

### 3.1. Characterization of EPSCs in Layer II Cells

Application of
electrical stimulation to either lateral positions in layers I/II or deep
positions in layer III evoked pure excitatory postsynaptic currents (EPSCs) in
layer II cells under our recording and superfusion conditions (with added 50μM
picrotoxin). These EPSCs were routinely characterized as monosynaptic but
occasionally polysynaptic or even antidromic responses were observed. In these latter cases, the stimulation
electrode was physically adjusted or even moved on the surface of the slice
until it evoked a pure monosynaptic response. The latency of responses was 4.01±0.13 milliseconds (layer I stimulation) and 3.85±0.11 milliseconds (layer III
stimulation). These responses were completely blocked by TTX (1μM), cadmium
(200μM), and the nonspecific glutamate antagonist, kynurenic acid (0.5 mM) (not
shown).To characterize the basic properties of the
evoked EPSCs, we recorded the response over a broad range of membrane
potentials (see Figures1(b), 1(c)—layer III stimulation in this case). EPSCs
displayed only a fast decay component when the membrane potential was held at −70 mV.
A slow decay component became apparent as the holding potential was depolarized
(see Figure 1(b), n=6). These fast and slow decay components of EPSC appeared
similar to AMPA- and NMDA-receptor mediated currents in other parts of the central
nervous system. In order to examine this possibility in further detail, we
constructed the current-voltage (I-V) relation of the EPSC response measured at
the peak and 30 milliseconds after the peak (see dashed lines in Figure
1(b)).
The peak I-V relation (filled squares in Figure 1(c)) was linear over the
entire voltage range with a reversal potential of 0 mV whereas the I-V relation
measured 30 milliseconds after the peak (filled circles in Figure 1(c)) was
nonlinear with a region of negative slope resistance in the range of −60 to −20 mV. This nonlinear I-V behavior is typical for NMDA-receptor mediated EPSCs.
Indeed, bath application of the NMDA receptor antagonist DL-APV (50μM) abolished the slow decaying component of the
EPSC at depolarized potentials without any significant effect on the early
component (see Figures 1(b), 1(c)). In consequence, in the presence of DL-APV
the peak I-V relation remained unchanged (open squares in Figure 1(c)). A small
synaptic current could still be measured at 30 milliseconds but this displayed
a completely linear I-V relation (open circles in Figure 1(c)) and was thus due
to the late phase of the fast EPSC. In contrast to DL-APV, the selective
non-NMDA receptor blocker CNQX (10μM) completely abolished the EPSC at
hyperpolarized potentials (not shown). These data suggest that the properties
of EPSCs evoked by stimulation of both horizontal and columnar afferents in EC
layer II cells are similar to their glutamatergic counterparts in the
hippocampus, or indeed, in other parts of the central nervous system.


### 3.2. Independence of the Horizontal and Vertical Associational Pathways

In order to assure
that our stimulation conditions evoked activity in independent sets of inputs
converging in layer II, we used homosynaptic and heterosynaptic paired pulse facilitation
(PPF) protocols. The two pathways were stimulated independently or alternatively
at short intervals (50–70 milliseconds)
and we measured whether the amplitude of EPSCs evoked by the second stimulation
pulse was increased following the first. Since PPF reflects an increase in
neurotransmitter release based on prior activity in the same synapse, any facilitation
observed across different site stimulation would reflect the activation of
overlapping synapses. Consistent with this idea, each pathway could
independently demonstrate PPF (see Figures2(a), 2(b): left panels). In
contrast, heterosynaptic stimulation protocols (either layer I followed by
layer III or vice versa) failed to show any PPF (see Figures 2(a), 2(b): right
panels). Using the same protocol in a total of three cells, we obtained similar
results which indicated that the two converging pathways were indeed
independent.Afferent independence between the
ascending columnar and horizontal associational pathways. The two pathways were
stimulated repetitively (left panels) or alternatively (right panels) using a short
interpulse interval (60 milliseconds). Each trace represents an average of 16
sweeps. Although paired-pulse facilitation (PPF) was observed in both (a) the ascending and (b) horizontal pathways when stimulated
independently (right panels), no facilitation was observed when stimulating
each alternatively (left panels). Dotted lines indicate the maximum amplitude
to the first pulse in each case.
(a)(b)


### 3.3. LTP of the Horizontal Associational Pathway

Previous work
using both field and intracellular methods have demonstrated that the inputs to
layer II activated by stimulation of layer I (which include horizontal
association fibres from layer II and extraentorhinal afferent fibre systems) exhibit
homosynaptic LTP [21, 27]. We confirmed this in our own whole cell recording
conditions using HFS protocols. Posttetanic potentiation (PTP) was observed in
all recorded cells following the application of high-frequency stimulation
(100 Hz, 1 second) to layer I at sites lateral to the recording electrode
position (see Figure 3(a)). LTP of EPSCs
following PTP was observed in 65% of cells tested (15 of 23) and lasted for
more than 30 minutes following HFS. The average normalized value of peak EPSCs
in these cells increased by a factor of 1.401±0.087 of baseline values at 30 minutes following HFS (see Figure 3(b)). This value was significantly different from
baseline (n=15,P<.01).LTP in the horizontal associative
pathway of the superficial entorhinal cortex. The stimulation electrode was
positioned on the surface of layer I, lateral to the recording electrode. (a) Normalized scaled amplitude of peak
EPSCs evoked by test pulses in a layer II cell is plotted against time. HFS at
time zero led to an immediate and long-lasting potentiation of EPSCs. The inset
shows two traces reflecting averaged EPSCs at baseline (pre-HFS) and at 30
minutes post-HFS. (b) Average
normalized scaled amplitude of peak EPSCs across 15 cells. The value at 30
minutes post-HFS (1.401±0.087) is significantly different from baseline (P<.01).
(a)(b)To determine
whether induction of LTP in this pathway required NMDA receptor activation, we
tested slices treated with the NMDA receptor antagonist DL-APV (50μM). Under these conditions, HFS to lateral sites in layer I only produced a short
term (PTP) facilitation of EPSCs in all cells tested. These values returned to baseline levels within 10
minutes following HFS (see Figure 4(a)). The average normalized value of peak EPSCs
was 0.979±0.095 at 30 minutes after HFS
in the presence of APV which was not significantly different from baseline
values (n=6,P>.01, see Figure
4(b)).Induction of LTP in the horizontal
pathway required activation of NMDA-receptors. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell is plotted against time. Time point of application of 50μM
DL-APV is indicated by the line. HFS at time zero led to short-term (PTP) but
not long-term potentiation. The inset shows two traces reflecting averaged EPSCs
at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude of peak EPSCs across 6 cells
in the presence of APV as indicated by the line. The value at 30 minutes post-HFS
(0.979±0.095) was not significantly
different from baseline (P>.05).
(a)(b)To determine if HFS-induced LTP was also dependent
upon a rise in postsynaptic Ca2+, we tested the effects of dialysing
the fast chelator BAPTA (10 mM) into the
postsynaptic cell. As in NMDA blockade conditions, HFS of lateral layer I led to
a short term facilitation of EPSCs only. This facilitation returned to baseline
levels in slightly less than 10 minutes following the application of HFS (see Figure 5(a)).
The average increase in the amplitude of EPSCs at 30 minutes post-HFS (1.008±0.068) was not significantly different from
baseline (n=5,P>.01, see Figure 5(b)).Induction of LTP in the horizontal
pathway required an increase in postsynaptic calcium. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell loaded with 10 mM BAPTA is plotted against time. HFS was
routinely applied about 15 minutes after formation of whole-cell mode to ensure
adequate BAPTA diffusion. The inset shows two traces reflecting averaged EPSCs
at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude of peak EPSCs across 5
BAPTA-loaded cells. HFS at time zero induced only short-term (PTP) but not
long-term potentiation. The value at 30
minutes post-HFS (1.008±0.068) was not significantly different
from baseline (P>.05).
(a)(b)In order to assess
if the expression of LTP in the horizontal associative pathway was solely
dependent on postsynaptic mechanisms, we investigated any changes in PPF ratio.
HFS to lateral layer I led to an immediate and obvious decrease in PPF which
soon returned to stable (and near-baseline) levels within 5 minutes (see Figure6(a)). PPF ratio at 30 and 50 minutes post-HFS was 0.917±0.084 and 0.938±0.062, respectively, which were both slightly, but significantly, less than
baseline (n=8,P<.01, see Figure 6(b)).
Thus, while the primary component of LTP in this pathway appeared to be postsynaptic
in expression, a small component also appeared to involve a presynaptic locus.Expression of LTP in the horizontal
pathway was associated with a slight change in paired-pulse facilitation (PPF) ratio.
(a) Normalized scaled amplitude of
the peak EPSCs evoked by the first of a pair of test pulses separated by 60 milliseconds
(filled circles) and the normalized paired pulse facilitation ratio (open
circles) in a layer II cell is plotted against time. The inset shows two traces
reflecting averaged paired EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS.
PPF ratio was calculated as the amplitude of the peak of the second EPSC
divided by the first. (b) Average
normalized scaled amplitude of PPF ratio in 10 cells. HFS at time zero induced
a short-term decrease which decayed back to near baseline levels. The values at
30 and 50 minutes after HFS, however, (0.944±0.065 and 0.966±0.068, resp.) were significantly different
from baseline (P<.01).
(a)(b)


### 3.4. LTP of the Ascending Columnar Associational Pathway

Application of HFS
to afferent fibres in layer III caused an immediate and long-lasting
potentiation of EPSCs in 79% of cells tested (23 of 29) which lasted for the
duration of the recording situation (>30 minutes post-HFS). Short-term posttetanic
potentiation (PTP) was a consistent feature of this facilitation and lasted for
just less than 5 minutes following HFS (see Figure7(a)). The average
normalized value of peak EPSCs increased by a factor of 1.65±0.102 and 1.68±0.128 of baseline (pre-HFS
values) at 30 and 50 minutes, respectively, following HFS (see Figure 7(b)). Both values
were significantly different from baseline (n=23,P<.01).LTP in the columnar associative (ascending)
pathway of superficial entorhinal cortex. The stimulation electrode was
positioned on the surface of layer III, just deep to the recording electrode. (a) Normalized scaled amplitude of peak
EPSCs evoked by test pulses in a layer II cell is plotted against time. Application
of HFS delivered at time zero led to an immediate and long-lasting increase in the
amplitude of EPSCs. This posttetanic potentiation lasted less than 5 minutes
before it reached a stable and potentiated level. The inset shows two traces reflecting
averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 11 cells. The values at 30 and 50 minutes post-HFS (1.65±0.102 and 1.68±0.128, resp.) were significantly
different from baseline (P<.01).
(a)(b)To determine
whether induction of LTP in this pathway required NMDA receptor activation, we
tested slices treated with the NMDA receptor antagonist DL-APV (50μM). Surprisingly, both the induction and
maintenance of LTP appeared unaffected by this manipulation even though slices
were superfused for at least 10 minutes prior to HFS and in some cases,
constantly during the entire experimental protocol. Results for both washout
(10 to 20 minutes following high-frequency stimulation) and constant perfusion
conditions were pooled together for statistic analysis since no difference was
observed between these conditions. As in control conditions, enhancement of
EPSCs in APV-treated slices became stable and long lasting after short-term PTP
(see Figure 8(a)). The average normalized value of peak EPSCs in the presence of APV increased by a factor of
1.695±0.145 and 1.707±0.19 of baseline (pre-HFS) values at 30 and 50 minutes,
respectively, values that were 1 significantly different from baseline values (n=10,P<.01) and 2 not significantly different from increases found in
control conditions (P>.05, see Figure
8(b)). In an additional two experiments, we also tested the effects of a higher
concentration of APV (100μM), but both the induction and expression of LTP were
still unaffected (data not shown).Induction of LTP in the ascending
pathway did not require activation of NMDA-receptors. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell is plotted against time. Time point of application of 50μM
DL-APV is indicated by the line (note, all experiments involved superfusions of
APV for more than 10 minutes before application of HFS at time zero). HFS led
to both short-term (PTP) and long-term potentiation. The inset shows two traces
reflecting averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 10 cells in the presence of APV as indicated by the line. The
value 30 and 50 minutes after was 1.695±0.145 and 1.707±0.19, respectively,
which were both significantly different from baseline values (P<.01).
(a)(b)Given that
calcium flux arising from activation of either postsynaptic calcium channels or
metabotropic glutamate receptors can also induce synaptic plasticity
independently of NMDA receptor activation, we tested whether fast chelation of
free intracellular calcium with BAPTA would block LTP in this pathway. High-frequency stimulation was applied 15
minutes after whole cell configuration to allow for full intracellular dialysis
of BAPTA to occur. With 10 mM BAPTA in the
pipette solution, the amplitude of LTP was markedly reduced although PTP was
consistently induced. Under this condition (see Figure9(a)), the average
increase in the normalized peak value of EPSCs was only 1.427±0.092 30 minutes post-HFS, a value reflecting a significant reduction (to
86%) of synaptic enhancement from control experiments (see Figure 9(b);
n=5). When we included 20 mM BAPTA in the
recording pipette we could completely abolish LTP (but not PTP: n=7). However, this manipulation also significantly
decreased the input resistance of the postsynaptic cell by an average of 20% (data
not shown).Induction of LTP in the ascending
pathway was not blocked by sequestering of free postsynaptic calcium. (a) Normalized scaled amplitude of peak
EPSCs evoked by test pulses in a layer II cell loaded with 10 mM BAPTA is
plotted against time. HFS was routinely applied about 15 minutes after
formation of whole-cell mode to ensure adequate BAPTA diffusion. The inset
shows two traces reflecting averaged EPSCs at baseline (pre-HFS) and at 30
minutes post-HFS. HFS-induced enhancement of EPSCs was still observed. (b) Average normalized scaled amplitude
of peak EPSCs across 5 BAPTA-loaded cells. HFS at time zero induced both
short-term (PTP) and long-term potentiation. The value at 30 minutes post-HFS (1.427±0.092) was slightly reduced compared
to control conditions but significantly different from baseline (P<.05).
(a)(b)The
above data, though not completely conclusive, suggest that postsynaptic calcium
entry may partially play a role in LTP induction in the layer II-II pathway.
Since NMDA receptor-channels were obviously not the source of this calcium, we
then explored two other possibilities: (1) calcium entry via voltage-gated Ca2+ channels and (2) calcium entry via metabotropic glutamate receptors (mGluRs). To
assess the first possibility we attempted to induce LTP by pairing
low-frequency presynaptic stimulation (0.05–01 HZ) with large
amplitude postsynaptic depolarizing voltage-clamp steps (200 milliseconds at −20 mV)
which have been shown to elicit LTP in other systems. However, this protocol
did not cause any significant change in the amplitude of the baseline EPSC (not
shown, P>.05,n=5). To assess the second possibility, we blocked mGluRs with the broad
spectrum antagonist MCPG (100μM).
However, in 5 neurons tested, HFS continued to induce LTP to a similar extent
as in control conditions (not shown).Previous
findings using field potential recordings [22] have shown that LTP in
the ascending layer V to layer associative pathway of the EC is partially NMDA-independent.
By measuring changes in paired-pulse facilitation (PPF), these researchers suggested
that this non-NMDA component of LTP reflected an increase in presynaptic
transmitter release. To assess whether LTP in the layer III input to layer II cells
was expressed presynaptically, we computed changes in the PPF ratio before and
after expression of LTP. PPF from the intrinsic ascending layer III pathway was
observed in 77% cells (17 of 23 cells) during control (pre-HFS) conditions in
the presence of 2.4 mM extracellular Ca2+. The range of ratios observed
was between 1.08 and 1.96, with an average value of 1.36±0.081. Only cells displaying both PPF and LTP were examined
(see Figure 10(a)). Directly following (1 minute post) HFS (i.e., during the
expression of PTP) the normalized PPF ratio was reduced by a factor of 0.64±0.048 compared to baseline values (n=10, see Figure
10(b)). This decrease, however, was only transient since PPF values showed an
initial quick and later slow increase which brought them back to near baseline
(unity) values (see Figure 10(b)). The average normalized PPF ratio measured 30 and 50
minutes after application of HFS was 0.944±0.065 and 0.966±0.068, respectively, which were only slightly, but significantly,
different from that during baseline period (P<.01). Thus, it would appear that only a small component
of the LTP expressed in this pathway reflected an increase in neurotransmitter
release.Expression of LTP in the
ascending pathway was associated with a slight change in PPF ratio. (a) Normalized scaled amplitude of the peak
EPSCs evoked by the first of a pair of test pulses separated by 60 milliseconds
(filled circles), and the normalized PPF ratio (open circles) in a layer II
cell is plotted against time. The inset shows two traces reflecting averaged
paired EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. PPF ratio was
calculated as the amplitude of the peak of the second EPSC divided by the
first. (b) Average normalized scaled
amplitude of PPF ratio in 8 cells. HFS at time zero induced a short-term
decrease of PPF which decayed back to near baseline levels. The values at 30
and 50 minutes after HFS, however, (0.917±0.084 and 0.938±0.062, resp.)
were significantly different from baseline (P<.01).
(a)(b)


### 3.5. Molecular Mechanistic Differences in LTP of the Horizontal and Columnar Associative
Pathways

Based on the differences in induction properties of
LTP in the two pathways examined, we sought to evaluate their potential
differential dependence on independent second-messenger pathways. We first
tested the effect of the widely-used CamKII specific inhibitor, KN62. KN62 (3.8μM) was applied to slices though
bath perfusion for more than 10 minutes before HFS was delivered. This compound
had differential effects on LTP expressed in the horizontal versus the columnar
associative pathways. HFS of layer I in the presence of KN62 failed to elicit
LTP in layer II cells (see Figure 11). The normalized average of EPSC amplitude at
30 minutes post-HFS was 0.8998±0.0545, which was actually significantly reduced
with respect to baseline values (n=6,P<.05). In contrast, HFS of layer III in the
presence of KN62 was still able to produce LTP in all layer II cells tested (see Figure 12),
although in two cells it did suppress the expression of PTP. The normalized average
of EPSC amplitude at 30 minutes after HFS was 1.656±0.124, a value which
reflected a significant facilitation (n=11,P<.01) and which was not significantly different
from the degree of potentiation expressed in control conditions (unpaired t-test, P>.05).Induction of LTP in the horizontal
pathway required intact CaMKII signalling. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell is plotted against time. Time point of application of 3.8μM
KN62 is indicated by the line. HFS at time zero led to a slight short-term potentiation
(PTP) but no long-term potentiation. The inset shows two traces reflecting
averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 6 cells in the presence of KN62 as indicated by the line.
The value at 30 minutes post-HFS (0.8998±0.0545) was significantly reduced from
baseline (P<.05).
(a)(b)Induction of LTP in the ascending
pathway was not dependent upon intact CaMKII signalling. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell is plotted against time. Time point of application of 3.8μM
KN62 is indicated by the line. HFS led to only a slight short-term potentiation
(PTP) but a prominent long-term potentiation. The inset shows two traces reflecting
averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 10 cells in the presence of KN62 as indicated by the line.
The value 30 minutes after HFS was 1.656±0.124 which was significantly
different from baseline values (P<.01) and no different from LTP in
control conditions (P>.05).
(a)(b)Since LTP in the ascending pathway did not appear
to be dependent on CaMKII, we next tested its dependence on PKA signalling. To
inhibit PKA activity, we used bath applications of the catalytic subunit
inhibitor KT5720 (250 nM).
This manipulation, applied for 10 minutes before HFS, did not completely block LTP
in layer II cells but did significantly suppress it (see Figure13). The normalized average of EPSC amplitude at
30 minutes post-HFS was 1.338±1.016 (n=8) which was significantly smaller
than that shown in control conditions (unpaired t-test, P<.01). Thus, at least part of the LTP expressed in this pathway
appears to depend upon PKA signalling.Induction of LTP in the ascending
pathway was partially dependent upon intact PKA signalling. (a) Normalized scaled amplitude of peak
EPSCs evoked by test pulses in a layer II cell is plotted against time. Time
point of application of 250 nM KT5720 is indicated by the line. HFS led to intact
short-term potentiation (PTP) but the degree of long-term potentiation was
slightly decreased although still intact. The inset shows two traces reflecting
averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 10 cells in the presence of KT5720 as indicated by the
line. The value 30 minutes after HFS was1.338±1.016 which was significantly
different from baseline values (P<.01) but was also significantly
reduced in comparison to control conditions (P<.01).
(a)(b)


## 3.1. Characterization of EPSCs in Layer II Cells

Application of
electrical stimulation to either lateral positions in layers I/II or deep
positions in layer III evoked pure excitatory postsynaptic currents (EPSCs) in
layer II cells under our recording and superfusion conditions (with added 50μM
picrotoxin). These EPSCs were routinely characterized as monosynaptic but
occasionally polysynaptic or even antidromic responses were observed. In these latter cases, the stimulation
electrode was physically adjusted or even moved on the surface of the slice
until it evoked a pure monosynaptic response. The latency of responses was 4.01±0.13 milliseconds (layer I stimulation) and 3.85±0.11 milliseconds (layer III
stimulation). These responses were completely blocked by TTX (1μM), cadmium
(200μM), and the nonspecific glutamate antagonist, kynurenic acid (0.5 mM) (not
shown).To characterize the basic properties of the
evoked EPSCs, we recorded the response over a broad range of membrane
potentials (see Figures1(b), 1(c)—layer III stimulation in this case). EPSCs
displayed only a fast decay component when the membrane potential was held at −70 mV.
A slow decay component became apparent as the holding potential was depolarized
(see Figure 1(b), n=6). These fast and slow decay components of EPSC appeared
similar to AMPA- and NMDA-receptor mediated currents in other parts of the central
nervous system. In order to examine this possibility in further detail, we
constructed the current-voltage (I-V) relation of the EPSC response measured at
the peak and 30 milliseconds after the peak (see dashed lines in Figure
1(b)).
The peak I-V relation (filled squares in Figure 1(c)) was linear over the
entire voltage range with a reversal potential of 0 mV whereas the I-V relation
measured 30 milliseconds after the peak (filled circles in Figure 1(c)) was
nonlinear with a region of negative slope resistance in the range of −60 to −20 mV. This nonlinear I-V behavior is typical for NMDA-receptor mediated EPSCs.
Indeed, bath application of the NMDA receptor antagonist DL-APV (50μM) abolished the slow decaying component of the
EPSC at depolarized potentials without any significant effect on the early
component (see Figures 1(b), 1(c)). In consequence, in the presence of DL-APV
the peak I-V relation remained unchanged (open squares in Figure 1(c)). A small
synaptic current could still be measured at 30 milliseconds but this displayed
a completely linear I-V relation (open circles in Figure 1(c)) and was thus due
to the late phase of the fast EPSC. In contrast to DL-APV, the selective
non-NMDA receptor blocker CNQX (10μM) completely abolished the EPSC at
hyperpolarized potentials (not shown). These data suggest that the properties
of EPSCs evoked by stimulation of both horizontal and columnar afferents in EC
layer II cells are similar to their glutamatergic counterparts in the
hippocampus, or indeed, in other parts of the central nervous system.


## 3.2. Independence of the Horizontal and Vertical Associational Pathways

In order to assure
that our stimulation conditions evoked activity in independent sets of inputs
converging in layer II, we used homosynaptic and heterosynaptic paired pulse facilitation
(PPF) protocols. The two pathways were stimulated independently or alternatively
at short intervals (50–70 milliseconds)
and we measured whether the amplitude of EPSCs evoked by the second stimulation
pulse was increased following the first. Since PPF reflects an increase in
neurotransmitter release based on prior activity in the same synapse, any facilitation
observed across different site stimulation would reflect the activation of
overlapping synapses. Consistent with this idea, each pathway could
independently demonstrate PPF (see Figures2(a), 2(b): left panels). In
contrast, heterosynaptic stimulation protocols (either layer I followed by
layer III or vice versa) failed to show any PPF (see Figures 2(a), 2(b): right
panels). Using the same protocol in a total of three cells, we obtained similar
results which indicated that the two converging pathways were indeed
independent.Afferent independence between the
ascending columnar and horizontal associational pathways. The two pathways were
stimulated repetitively (left panels) or alternatively (right panels) using a short
interpulse interval (60 milliseconds). Each trace represents an average of 16
sweeps. Although paired-pulse facilitation (PPF) was observed in both (a) the ascending and (b) horizontal pathways when stimulated
independently (right panels), no facilitation was observed when stimulating
each alternatively (left panels). Dotted lines indicate the maximum amplitude
to the first pulse in each case.
(a)(b)


## 3.3. LTP of the Horizontal Associational Pathway

Previous work
using both field and intracellular methods have demonstrated that the inputs to
layer II activated by stimulation of layer I (which include horizontal
association fibres from layer II and extraentorhinal afferent fibre systems) exhibit
homosynaptic LTP [21, 27]. We confirmed this in our own whole cell recording
conditions using HFS protocols. Posttetanic potentiation (PTP) was observed in
all recorded cells following the application of high-frequency stimulation
(100 Hz, 1 second) to layer I at sites lateral to the recording electrode
position (see Figure 3(a)). LTP of EPSCs
following PTP was observed in 65% of cells tested (15 of 23) and lasted for
more than 30 minutes following HFS. The average normalized value of peak EPSCs
in these cells increased by a factor of 1.401±0.087 of baseline values at 30 minutes following HFS (see Figure 3(b)). This value was significantly different from
baseline (n=15,P<.01).LTP in the horizontal associative
pathway of the superficial entorhinal cortex. The stimulation electrode was
positioned on the surface of layer I, lateral to the recording electrode. (a) Normalized scaled amplitude of peak
EPSCs evoked by test pulses in a layer II cell is plotted against time. HFS at
time zero led to an immediate and long-lasting potentiation of EPSCs. The inset
shows two traces reflecting averaged EPSCs at baseline (pre-HFS) and at 30
minutes post-HFS. (b) Average
normalized scaled amplitude of peak EPSCs across 15 cells. The value at 30
minutes post-HFS (1.401±0.087) is significantly different from baseline (P<.01).
(a)(b)To determine
whether induction of LTP in this pathway required NMDA receptor activation, we
tested slices treated with the NMDA receptor antagonist DL-APV (50μM). Under these conditions, HFS to lateral sites in layer I only produced a short
term (PTP) facilitation of EPSCs in all cells tested. These values returned to baseline levels within 10
minutes following HFS (see Figure 4(a)). The average normalized value of peak EPSCs
was 0.979±0.095 at 30 minutes after HFS
in the presence of APV which was not significantly different from baseline
values (n=6,P>.01, see Figure
4(b)).Induction of LTP in the horizontal
pathway required activation of NMDA-receptors. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell is plotted against time. Time point of application of 50μM
DL-APV is indicated by the line. HFS at time zero led to short-term (PTP) but
not long-term potentiation. The inset shows two traces reflecting averaged EPSCs
at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude of peak EPSCs across 6 cells
in the presence of APV as indicated by the line. The value at 30 minutes post-HFS
(0.979±0.095) was not significantly
different from baseline (P>.05).
(a)(b)To determine if HFS-induced LTP was also dependent
upon a rise in postsynaptic Ca2+, we tested the effects of dialysing
the fast chelator BAPTA (10 mM) into the
postsynaptic cell. As in NMDA blockade conditions, HFS of lateral layer I led to
a short term facilitation of EPSCs only. This facilitation returned to baseline
levels in slightly less than 10 minutes following the application of HFS (see Figure 5(a)).
The average increase in the amplitude of EPSCs at 30 minutes post-HFS (1.008±0.068) was not significantly different from
baseline (n=5,P>.01, see Figure 5(b)).Induction of LTP in the horizontal
pathway required an increase in postsynaptic calcium. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell loaded with 10 mM BAPTA is plotted against time. HFS was
routinely applied about 15 minutes after formation of whole-cell mode to ensure
adequate BAPTA diffusion. The inset shows two traces reflecting averaged EPSCs
at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude of peak EPSCs across 5
BAPTA-loaded cells. HFS at time zero induced only short-term (PTP) but not
long-term potentiation. The value at 30
minutes post-HFS (1.008±0.068) was not significantly different
from baseline (P>.05).
(a)(b)In order to assess
if the expression of LTP in the horizontal associative pathway was solely
dependent on postsynaptic mechanisms, we investigated any changes in PPF ratio.
HFS to lateral layer I led to an immediate and obvious decrease in PPF which
soon returned to stable (and near-baseline) levels within 5 minutes (see Figure6(a)). PPF ratio at 30 and 50 minutes post-HFS was 0.917±0.084 and 0.938±0.062, respectively, which were both slightly, but significantly, less than
baseline (n=8,P<.01, see Figure 6(b)).
Thus, while the primary component of LTP in this pathway appeared to be postsynaptic
in expression, a small component also appeared to involve a presynaptic locus.Expression of LTP in the horizontal
pathway was associated with a slight change in paired-pulse facilitation (PPF) ratio.
(a) Normalized scaled amplitude of
the peak EPSCs evoked by the first of a pair of test pulses separated by 60 milliseconds
(filled circles) and the normalized paired pulse facilitation ratio (open
circles) in a layer II cell is plotted against time. The inset shows two traces
reflecting averaged paired EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS.
PPF ratio was calculated as the amplitude of the peak of the second EPSC
divided by the first. (b) Average
normalized scaled amplitude of PPF ratio in 10 cells. HFS at time zero induced
a short-term decrease which decayed back to near baseline levels. The values at
30 and 50 minutes after HFS, however, (0.944±0.065 and 0.966±0.068, resp.) were significantly different
from baseline (P<.01).
(a)(b)


## 3.4. LTP of the Ascending Columnar Associational Pathway

Application of HFS
to afferent fibres in layer III caused an immediate and long-lasting
potentiation of EPSCs in 79% of cells tested (23 of 29) which lasted for the
duration of the recording situation (>30 minutes post-HFS). Short-term posttetanic
potentiation (PTP) was a consistent feature of this facilitation and lasted for
just less than 5 minutes following HFS (see Figure7(a)). The average
normalized value of peak EPSCs increased by a factor of 1.65±0.102 and 1.68±0.128 of baseline (pre-HFS
values) at 30 and 50 minutes, respectively, following HFS (see Figure 7(b)). Both values
were significantly different from baseline (n=23,P<.01).LTP in the columnar associative (ascending)
pathway of superficial entorhinal cortex. The stimulation electrode was
positioned on the surface of layer III, just deep to the recording electrode. (a) Normalized scaled amplitude of peak
EPSCs evoked by test pulses in a layer II cell is plotted against time. Application
of HFS delivered at time zero led to an immediate and long-lasting increase in the
amplitude of EPSCs. This posttetanic potentiation lasted less than 5 minutes
before it reached a stable and potentiated level. The inset shows two traces reflecting
averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 11 cells. The values at 30 and 50 minutes post-HFS (1.65±0.102 and 1.68±0.128, resp.) were significantly
different from baseline (P<.01).
(a)(b)To determine
whether induction of LTP in this pathway required NMDA receptor activation, we
tested slices treated with the NMDA receptor antagonist DL-APV (50μM). Surprisingly, both the induction and
maintenance of LTP appeared unaffected by this manipulation even though slices
were superfused for at least 10 minutes prior to HFS and in some cases,
constantly during the entire experimental protocol. Results for both washout
(10 to 20 minutes following high-frequency stimulation) and constant perfusion
conditions were pooled together for statistic analysis since no difference was
observed between these conditions. As in control conditions, enhancement of
EPSCs in APV-treated slices became stable and long lasting after short-term PTP
(see Figure 8(a)). The average normalized value of peak EPSCs in the presence of APV increased by a factor of
1.695±0.145 and 1.707±0.19 of baseline (pre-HFS) values at 30 and 50 minutes,
respectively, values that were 1 significantly different from baseline values (n=10,P<.01) and 2 not significantly different from increases found in
control conditions (P>.05, see Figure
8(b)). In an additional two experiments, we also tested the effects of a higher
concentration of APV (100μM), but both the induction and expression of LTP were
still unaffected (data not shown).Induction of LTP in the ascending
pathway did not require activation of NMDA-receptors. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell is plotted against time. Time point of application of 50μM
DL-APV is indicated by the line (note, all experiments involved superfusions of
APV for more than 10 minutes before application of HFS at time zero). HFS led
to both short-term (PTP) and long-term potentiation. The inset shows two traces
reflecting averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 10 cells in the presence of APV as indicated by the line. The
value 30 and 50 minutes after was 1.695±0.145 and 1.707±0.19, respectively,
which were both significantly different from baseline values (P<.01).
(a)(b)Given that
calcium flux arising from activation of either postsynaptic calcium channels or
metabotropic glutamate receptors can also induce synaptic plasticity
independently of NMDA receptor activation, we tested whether fast chelation of
free intracellular calcium with BAPTA would block LTP in this pathway. High-frequency stimulation was applied 15
minutes after whole cell configuration to allow for full intracellular dialysis
of BAPTA to occur. With 10 mM BAPTA in the
pipette solution, the amplitude of LTP was markedly reduced although PTP was
consistently induced. Under this condition (see Figure9(a)), the average
increase in the normalized peak value of EPSCs was only 1.427±0.092 30 minutes post-HFS, a value reflecting a significant reduction (to
86%) of synaptic enhancement from control experiments (see Figure 9(b);
n=5). When we included 20 mM BAPTA in the
recording pipette we could completely abolish LTP (but not PTP: n=7). However, this manipulation also significantly
decreased the input resistance of the postsynaptic cell by an average of 20% (data
not shown).Induction of LTP in the ascending
pathway was not blocked by sequestering of free postsynaptic calcium. (a) Normalized scaled amplitude of peak
EPSCs evoked by test pulses in a layer II cell loaded with 10 mM BAPTA is
plotted against time. HFS was routinely applied about 15 minutes after
formation of whole-cell mode to ensure adequate BAPTA diffusion. The inset
shows two traces reflecting averaged EPSCs at baseline (pre-HFS) and at 30
minutes post-HFS. HFS-induced enhancement of EPSCs was still observed. (b) Average normalized scaled amplitude
of peak EPSCs across 5 BAPTA-loaded cells. HFS at time zero induced both
short-term (PTP) and long-term potentiation. The value at 30 minutes post-HFS (1.427±0.092) was slightly reduced compared
to control conditions but significantly different from baseline (P<.05).
(a)(b)The
above data, though not completely conclusive, suggest that postsynaptic calcium
entry may partially play a role in LTP induction in the layer II-II pathway.
Since NMDA receptor-channels were obviously not the source of this calcium, we
then explored two other possibilities: (1) calcium entry via voltage-gated Ca2+ channels and (2) calcium entry via metabotropic glutamate receptors (mGluRs). To
assess the first possibility we attempted to induce LTP by pairing
low-frequency presynaptic stimulation (0.05–01 HZ) with large
amplitude postsynaptic depolarizing voltage-clamp steps (200 milliseconds at −20 mV)
which have been shown to elicit LTP in other systems. However, this protocol
did not cause any significant change in the amplitude of the baseline EPSC (not
shown, P>.05,n=5). To assess the second possibility, we blocked mGluRs with the broad
spectrum antagonist MCPG (100μM).
However, in 5 neurons tested, HFS continued to induce LTP to a similar extent
as in control conditions (not shown).Previous
findings using field potential recordings [22] have shown that LTP in
the ascending layer V to layer associative pathway of the EC is partially NMDA-independent.
By measuring changes in paired-pulse facilitation (PPF), these researchers suggested
that this non-NMDA component of LTP reflected an increase in presynaptic
transmitter release. To assess whether LTP in the layer III input to layer II cells
was expressed presynaptically, we computed changes in the PPF ratio before and
after expression of LTP. PPF from the intrinsic ascending layer III pathway was
observed in 77% cells (17 of 23 cells) during control (pre-HFS) conditions in
the presence of 2.4 mM extracellular Ca2+. The range of ratios observed
was between 1.08 and 1.96, with an average value of 1.36±0.081. Only cells displaying both PPF and LTP were examined
(see Figure 10(a)). Directly following (1 minute post) HFS (i.e., during the
expression of PTP) the normalized PPF ratio was reduced by a factor of 0.64±0.048 compared to baseline values (n=10, see Figure
10(b)). This decrease, however, was only transient since PPF values showed an
initial quick and later slow increase which brought them back to near baseline
(unity) values (see Figure 10(b)). The average normalized PPF ratio measured 30 and 50
minutes after application of HFS was 0.944±0.065 and 0.966±0.068, respectively, which were only slightly, but significantly,
different from that during baseline period (P<.01). Thus, it would appear that only a small component
of the LTP expressed in this pathway reflected an increase in neurotransmitter
release.Expression of LTP in the
ascending pathway was associated with a slight change in PPF ratio. (a) Normalized scaled amplitude of the peak
EPSCs evoked by the first of a pair of test pulses separated by 60 milliseconds
(filled circles), and the normalized PPF ratio (open circles) in a layer II
cell is plotted against time. The inset shows two traces reflecting averaged
paired EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. PPF ratio was
calculated as the amplitude of the peak of the second EPSC divided by the
first. (b) Average normalized scaled
amplitude of PPF ratio in 8 cells. HFS at time zero induced a short-term
decrease of PPF which decayed back to near baseline levels. The values at 30
and 50 minutes after HFS, however, (0.917±0.084 and 0.938±0.062, resp.)
were significantly different from baseline (P<.01).
(a)(b)


## 3.5. Molecular Mechanistic Differences in LTP of the Horizontal and Columnar Associative
Pathways

Based on the differences in induction properties of
LTP in the two pathways examined, we sought to evaluate their potential
differential dependence on independent second-messenger pathways. We first
tested the effect of the widely-used CamKII specific inhibitor, KN62. KN62 (3.8μM) was applied to slices though
bath perfusion for more than 10 minutes before HFS was delivered. This compound
had differential effects on LTP expressed in the horizontal versus the columnar
associative pathways. HFS of layer I in the presence of KN62 failed to elicit
LTP in layer II cells (see Figure 11). The normalized average of EPSC amplitude at
30 minutes post-HFS was 0.8998±0.0545, which was actually significantly reduced
with respect to baseline values (n=6,P<.05). In contrast, HFS of layer III in the
presence of KN62 was still able to produce LTP in all layer II cells tested (see Figure 12),
although in two cells it did suppress the expression of PTP. The normalized average
of EPSC amplitude at 30 minutes after HFS was 1.656±0.124, a value which
reflected a significant facilitation (n=11,P<.01) and which was not significantly different
from the degree of potentiation expressed in control conditions (unpaired t-test, P>.05).Induction of LTP in the horizontal
pathway required intact CaMKII signalling. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell is plotted against time. Time point of application of 3.8μM
KN62 is indicated by the line. HFS at time zero led to a slight short-term potentiation
(PTP) but no long-term potentiation. The inset shows two traces reflecting
averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 6 cells in the presence of KN62 as indicated by the line.
The value at 30 minutes post-HFS (0.8998±0.0545) was significantly reduced from
baseline (P<.05).
(a)(b)Induction of LTP in the ascending
pathway was not dependent upon intact CaMKII signalling. (a) Normalized scaled amplitude of peak EPSCs evoked by test pulses
in a layer II cell is plotted against time. Time point of application of 3.8μM
KN62 is indicated by the line. HFS led to only a slight short-term potentiation
(PTP) but a prominent long-term potentiation. The inset shows two traces reflecting
averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 10 cells in the presence of KN62 as indicated by the line.
The value 30 minutes after HFS was 1.656±0.124 which was significantly
different from baseline values (P<.01) and no different from LTP in
control conditions (P>.05).
(a)(b)Since LTP in the ascending pathway did not appear
to be dependent on CaMKII, we next tested its dependence on PKA signalling. To
inhibit PKA activity, we used bath applications of the catalytic subunit
inhibitor KT5720 (250 nM).
This manipulation, applied for 10 minutes before HFS, did not completely block LTP
in layer II cells but did significantly suppress it (see Figure13). The normalized average of EPSC amplitude at
30 minutes post-HFS was 1.338±1.016 (n=8) which was significantly smaller
than that shown in control conditions (unpaired t-test, P<.01). Thus, at least part of the LTP expressed in this pathway
appears to depend upon PKA signalling.Induction of LTP in the ascending
pathway was partially dependent upon intact PKA signalling. (a) Normalized scaled amplitude of peak
EPSCs evoked by test pulses in a layer II cell is plotted against time. Time
point of application of 250 nM KT5720 is indicated by the line. HFS led to intact
short-term potentiation (PTP) but the degree of long-term potentiation was
slightly decreased although still intact. The inset shows two traces reflecting
averaged EPSCs at baseline (pre-HFS) and at 30 minutes post-HFS. (b) Average normalized scaled amplitude
of peak EPSCs across 10 cells in the presence of KT5720 as indicated by the
line. The value 30 minutes after HFS was1.338±1.016 which was significantly
different from baseline values (P<.01) but was also significantly
reduced in comparison to control conditions (P<.01).
(a)(b)


## 4. Discussion

In this
study, we investigated the basic characteristics of synaptic communication and
plasticity in two independent pathways converging on EC layer II principal
neurons, the horizontal input (containing cortical afferents and intralaminar associative
connections between cells of layer II), and the ascending columnar
(interlaminar) layer III input, respectively. Although prior work has been
conducted on the properties of LTP in the superficial layers of the EC, few
have focussed on cellular mechanisms and none has compared the superficial
associative pathways that converge upon layer II. Indeed, a recent whole-cell
investigation of plasticity in EC layer II stellates concluded that only LTD is
present in conditions similar to those reported here [28].Our results demonstrated that both associational pathways within the
EC were independent and had basic physiological and pharmacological characteristics
similar to other glutamatergic synapses in the central nervous system. In
addition, both displayed robust LTP evoked by HFS that was accompanied by small
decreases in paired pulse facilitation ratio. Importantly, we confirmed that
the induction mechanisms of LTP in these two convergent pathways demonstrated
some important differences in terms of their dependence upon NMDA receptors and
on their dependence upon postsynaptic calcium. This difference may reflect a
differential dependence on second-messenger systems. While this is not the
first direct demonstration of differential plasticity in independent pathways
that terminate in the superficial layers of the EC, it is the first cellular
investigation of this type. This
confirms that the intrinsic circuit dynamics of the EC are highly complex.


## 4.1. Physiology of the Horizontal and Ascending Excitatory Inputs to EC Layer II

The projection
cells of EC layer II are important components of the medial temporal lobe
memory circuit since they receive highly processed neocortical information and
provide the hippocampus with its most massive input via the perforant path.
Through feedforward excitatory (and presumably inhibitory) collaterals, these
cells also form part of a rich autoassociative network [29] whereby
neighbouring neurons can influence and entrain each other [30]. As well, the
activity of these cells is influenced by inputs which derive from laminae
located deeper to it [1, 2, 31, 32]. The functional significance of convergent
horizontal and ascending pathways in layer II is unclear although it could be relevant
in order to compare the results of processing in deeper layers to that
conducted in the most superficial.We have demonstrated in the present study that excitatory
feedforward interlaminar communication is not only a feature of the traditional
hippocampal input and output laminae of the EC (i.e., the most deep and the
most superficial) but is also a feature of the independent superficial laminae
(III and II) which comprise the cells of origin of the temporal ammonic and perforant
pathways, respectively. As well, we
demonstrated that both the horizontal and ascending associative pathways have
properties consistent with excitatory glutamatergic synapses at a variety of
locations in the central nervous system. Both demonstrated NMDA and non-NMDA (presumably AMPA) components. Indeed,
pharmacological manipulations using both APV and CNQX completely abolished excitatory
synaptic responses.Also similar to other glutamatergic synapses throughout the nervous
system, both the horizontal and ascending associative pathways exhibited
LTP. These results are in stark contrast
to those of Deng and Lei [28] who failed to
show LTP in either the horizontal or ascending (layer V) inputs to EC layer II
stellate cells using a variety of stimulation paradigms including HFS. The only
apparent differences between our conditions was their inclusion of QX-314
(0.2 mM) in the whole-cell pipette (in order to suppress action potential
generation) and their baseline periods used (>30 minutes) in order to attain
stable series resistances following whole cell dialysis. Although we found that complete whole-cell
dialysis over this time periods greater than 30 minutes appeared to negatively
affect the probability of achieving LTP, Deng and Lei were also unable to
demonstrate LTP using perforated patch methods. Thus, whole-cell washout is presumably not the cause of the
discrepancy. However, we do note that both
field and sharp-electrode intracellular recording methods have consistently
demonstrated LTP in the superficial EC [21, 22, 27, 33–35].
Interestingly, Alonso et al. [27] suggested
that the intrinsic rhythmic properties of EC layer II stellates, brought about
by the interaction of Ih and INap [25], may play a
pivotal role in the induction of plasticity of afferent inputs. Since QX-314 blocks both these currents and
suppresses resonant and oscillatory behavior [36], this
manipulation may indeed be relevant.We did not test if the expression of LTP in the layer I input was
NMDA or non-NMDA dependent [33, 35] although with
continued perfusions of APV in some experiments we continued to observe LTP in
the layer III pathway, showing that it is presumably mediated by non-NMDA glutamate
receptors.


## 4.2. Differential Plasticity of the Horizontal and Ascending Excitatory Inputs to EC Layer II

Although
both pathways demonstrated intact NMDA receptor mediated transmission, only the
induction of LTP in the horizontal pathway required NMDA signalling. As well,
the calcium dependence of LTP in the layer III to II inputs did not appear to
be as critical as that in the horizontal pathway. This suggests that the second-messenger
systems mediating LTP in the two different input pathways are quite different
and potentially independent. This makes layer II cells similar to other CNS
neurons (such as CA3 pyramids in the hippocampus) in which different afferent inputs
demonstrate NMDA- dependent and -independent LTP simultaneously [18]. A major
difference is that the bulk of the LTP in EC layer II neurons in both pathways
appears to be expressed via postsynaptic changes. Similar differences have been observed in LTP
mechanisms in early postnatal (<P9) cortex and hippocampus [37, 38]. However,
this is the first report to our knowledge showing this kind of difference in
two independent pathways to the same sets of neurons in adult (>P27) brain.



---

*Source: 814815-2008-06-30.xml*