# Dopaminergic Suppression of Synaptic Transmission in  the Lateral Entorhinal Cortex

Authors: Douglas A. Caruana; C. Andrew Chapman
Journal: Neural Plasticity
 (2008)

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## Abstract

Dopaminergic projections to the superficial layers of the lateral entorhinal cortex can modulate the strength of olfactory inputs to the region.  We have found that low concentrations of dopamine facilitate field EPSPs in the entorhinal cortex, and that higher concentrations of dopamine suppress synaptic responses.  Here, we have used whole-cell current clamp recordings from layer II neurons to determine the mechanisms of the suppression.  Dopamine (10 to 50μM)
hyperpolarized membrane potential and reversibly suppressed the amplitude of EPSPs evoked by layer I stimulation.  Both AMPA- and NMDA-mediated components were suppressed, and paired-pulse facilitation was also enhanced indicating that the suppression is mediated largely by reduced glutamate release.  Blockade of D2-like receptors greatly reduced the suppression of EPSPs.  Dopamine also lowered input resistance, and reduced the number of action potentials evoked by depolarizing current steps.  The drop in input resistance was mediated by activation of D1-like receptors, and was prevented by blocking K+ channels with TEA.  The dopaminergic suppression of synaptic transmission is therefore mediated by a D2 receptor-dependent reduction in transmitter release, and a D1 receptor-dependent increase in a K+ conductance.  This suppression of EPSPs may dampen the strength of sensory inputs during periods of elevated mesocortical dopamine activity.


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## Body

## 1. Introduction

The entorhinal cortex is an
important interface that links primary sensory and association cortices to the
hippocampal formation, and it is critical for the sensory and mnemonic
functions of the medial temporal lobe [1–4]. In the rat, the lateral division of the
entorhinal cortex receives most of its cortical inputs from the olfactory
cortex and perirhinal cortex, and the medial entorhinal cortex receives visual
and multimodal inputs mainly via the postrhinal cortex [5–7]. This pattern of cortical input to the medial
and lateral divisions of the entorhinal cortex contributes to their different
roles in sensory and cognitive processing [8–10]. In addition, neuromodulatory transmitters
innervate both the medial and lateral entorhinal cortices and can have powerful
effects on sensory and mnemonic function in these regions. Specifically, acetylcholine and serotonin
both modulate synaptic transmission and rhythmic EEG activities in the medial
entorhinal cortex [11–15]. Further, midbrain dopamine neurons send one
of their largest cortical projections to the superficial layers of the lateral
entorhinal cortex where they target principal cell islands [16–18]. Relatively little is known, however,
regarding the neuromodulatory effects of dopamine in the lateral entorhinal
cortex.The large dopaminergic projection
to the prefrontal cortex is known to regulate cellular processes related to
working memory [19–21], and
dopaminergic inputs to the lateral entorhinal cortex are also likely to affect
mechanisms of sensory and mnemonic function. In the prefrontal cortex, activation of D1 receptors can
suppress glutamate release in layer V [22–24] but can
enhance glutamatergic transmission in layer III [25, 26]. Further, the positive effect of D1 receptor activation on working memory follows an inverted U-shaped function
[27], and strong or weak stimulation of D1 receptors can also have
opposite effects on NMDA receptor-mediated synaptic currents [20, 28]. We have also found that dopamine has
dose-dependent bidirectional effects in layer II of the lateral entorhinal
cortex. In awake animals, increasing
levels of dopamine with a selective reuptake inhibitor facilitates synaptic
responses evoked by stimulation of the piriform cortex, and field excitatory
postsynaptic potentials (fEPSPs) are also facilitated by a low concentration of
dopamine in vitro [29]. Higher concentrations of dopamine, however,
suppress fEPSPs, and similar suppression effects have been observed by others
in medial entorhinal cortex layer II [30] and layer III [31]. Dopamine can also reduce the input resistance
of layer II neurons in the medial entorhinal cortex [30] and reduce temporal
summation in layer V neurons of the lateral division through an increase in the Ih current [32]. Dopamine may therefore modulate synaptic
function in the lateral entorhinal cortex through multiple mechanisms.We have used whole-cell current
clamp recordings to investigate the mechanisms of the suppression of EPSPs by
dopamine in electrophysiologically identified “fan” cells in layer II of the
lateral entorhinal cortex. Receptor
blockers were used to determine the dopamine receptors that mediate the
suppression of EPSPs, and paired-pulse tests were used to assess whether the
suppression is expressed pre- or postsynaptically. Changes in the intrinsic excitability of fan
cells were also monitored using responses to hyperpolarizing and depolarizing
current steps. In addition to a D2-like
receptor-mediated suppression of transmitter release, we show evidence that
EPSPs are also reduced by an increased K+ conductance dependent on
activation of D1 receptors.


## 2. Materials and Methods

### 2.1. Tissue Slices

Methods for obtaining whole cell current clamp recordings
were similar to those described previously [13, 29, 33, 34].  Male Long-Evans rats between 4 and 6 weeks old
were anesthetized with halothane, decapitated, and their brains rapidly removed
and transferred into cold (4°C) artificial cerebrospinal fluid (ACSF) saturated
with 95% O2 and 5% CO2 containing (in mM) 124 NaCl, 5
KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2,
26 NaHCO3, and 10 dextrose (pH  ≈7.3; 300–310 mOsm). All chemicals were obtained from Sigma-Aldrich, Mo, USA. Horizontal slices (300 μm thick) were cut
using a vibratome (WPI, Vibroslice,
Fla, USA),
and slices recovered for at least one hour at 22 to 24°C. Slices were transferred individually to a
recording chamber and visualized using an upright microscope (Leica, Richmond
Hill, Canada, DM-LFS)
equipped with differential interference contrast optics, a 40x water immersion
objective, and a near-infrared camera (COHU, Inc., Calif, USA). Submerged slices were superfused with
oxygenated ACSF at a rate of 1.5 to 2.0 mL/min. Slices containing the lateral entorhinal cortex were taken from ventral
sections about 1.9 to 3.4 mm above the interaural line [35]. Layer II was identified based on the presence
of cell “islands” about 150 μm from the cortical surface [36–39].


### 2.2. Stimulation and Recording

Patch recording pipettes for whole cell recordings were
prepared from borosilicate glass (1.0 mm OD, 4 to 8 MΩ) using a horizontal
puller (P-97, Sutter Instr., Calif,
USA) and were filled with a solution containing (in mM) 140 K-gluconate,
5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 ATP-Tris, and 0.4 GTP-Tris
(pH adjusted to 7.24–7.32 with KOH; 270–280 mOsm). Pipettes were placed in contact with somata
of layer II neurons, and gentle suction was applied under voltage clamp to form
a tight seal (1–3 GΩ). Whole cell configuration was achieved by
increased suction, and experiments began after cells stabilized (typically
within 3 to 5 minutes after break-in). Current clamp recordings were obtained using an Axopatch 200B amplifier
(Axon Instr., Calif, USA)
and displayed on a digital oscilloscope (Gould 1604). Recordings were filtered at 10 kHz and
digitized at 20 kHz (Axon Instr., Digidata 1322A) for storage on computer hard
disk. Recordings were accepted if the
series resistance was ≤25 MΩ (mean = 16.9±0.9 MΩ) and if input resistance and
resting potential were stable. A bipolar
stimulating electrode made from two tungsten electrodes (FHC, 1.0 MΩ) was
positioned to span layer I near the border with layer II approximately 0.2 to
0.6 mm rostral to the recording electrode. Synaptic responses were evoked with 0.1 millisecond constant current
pulses delivered using a stimulus timer and isolation unit (WPI, Mass, USA, models A300 and
A360). Stimulation intensity was
adjusted to evoke responses approximately 75% of maximal (75 to 300 μA).All neurons
(n = 118) included for analyses were identified as “fan” cells based on
electrophysiological characteristics described previously [40, 41]. In comparison to stellate cells of the medial
entorhinal cortex, fan cells show modest inward rectification during
hyperpolarizing current steps, a small depolarizing afterpotential following
single spikes, and do not show prominent theta-frequency membrane potential
oscillations at subthreshold voltages [40–42].


### 2.3. Dopaminergic Modulation of Synaptic Responses

The effects of dopamine on glutamate-mediated synaptic
transmission in the lateral entorhinal cortex are largely uncharacterized. We therefore recorded both mixed and isolated
components of excitatory postsynaptic potentials (EPSPs) evoked by stimulation
of layer I before and after 5-minute bath-application of 1, 10, or 50μM
dopamine. Results obtained using high
concentrations of dopamine must be interpreted cautiously because of the
possibility of nonspecific effects. However,
dopamine degrades through oxidization within the slice preparation, and similar
concentrations of dopamine have been used previously, and interpreted in light
of the effects of specific antagonists, in reports examining the effects of
dopamine on synaptic transmission in both the entorhinal [29–32] and
prefrontal [23, 43] cortices. Responses
were evoked once every 20 seconds, and the mean of 10 responses was obtained
for analysis. Baseline responses were
obtained at resting potential and, because dopamine usually hyperpolarizes fan
cells, constant current was often required to return cells to the original
membrane potential for recordings in the presence of dopamine. Sodium metabisulfite (50 μM) was coapplied to
slow the oxidation of dopamine [29, 31, 43], and ambient lighting was also
reduced. Possible effects of sodium metabisulfite
were assessed with a vehicle control group. Drugs were routinely stored at −20°C as
concentrated stock solutions until needed, but dopamine HCl was dissolved just
prior to bath application.Paired-pulse
tests were used to determine whether dopamine modulates EPSPs through a pre- or
postsynaptic mechanism [13]. Pairs of
stimulation pulses separated by an interval of 30 milliseconds were delivered
before and after 5-minute bath-application of 1, 10, or 50 μM dopamine. Stimulation intensity was reduced to evoke
EPSPs approximately 50% of maximal, and ten responses were averaged for
analyses. Paired-pulse facilitation was
quantified by expressing the amplitude of the second response as a percentage
of the first response.Mechanisms mediating the
suppression of EPSPs by high concentrations of dopamine were investigated by
assessing the effects of 50μM dopamine on isolated components of synaptic
responses. After baseline recordings in
normal ACSF, AMPA receptor-mediated responses were isolated with bath
application of 50 μM 2-amino-5-phosphonovaleric acid (APV) and 25 μM
bicuculline methiodide, or NMDA receptor-mediated responses were isolated with
20 μM 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX) and 25 μM
bicuculline. GABA-mediated IPSPs were
isolated with either 1 mM kynurenic acid or 20 μM CNQX with 50 μM APV. Isolated synaptic responses were recorded
before and after 5-minute application of 50 μM dopamine. Isolated AMPA receptor-mediated responses
were also used to determine if dopamine suppresses EPSPs primarily through D1-
or D2-like receptors. Baseline responses were recorded in the presence of either the D1 receptor antagonist SCH23390 (50 μM) or the D2 receptor antagonist
sulpiride (50 μM) [29–31], and 50 μM dopamine was then applied for 5 minutes. Sulpiride was prepared daily in a stock
solution of 6% DMSO in ACSF titrated with 0.1 N HCl, and there was a final
concentration of 0.1% DMSO with sulpiride.The effects of dopamine on the
intrinsic excitability of fan cells were assessed by monitoring responses to
hyperpolarizing and depolarizing current steps. Changes in action potentials, afterhyperpolarizations, input resistance
and inward rectification were examined before and after 5-minute bath
application of 1, 10, or 50μM dopamine. The number of action potentials elicited in response to suprathreshold
current injection can be used to characterize neuronal excitability [32], and
we therefore determined the number of spikes fired in response to a single 500 millisecond-duration
depolarizing current pulse from a constant holding potential (typically rest)
using a pulse amplitude that elicited 3 to 5 action potentials [32]. Receptors that mediate the dopamine-induced
change in input resistance were investigated using SCH23390 or sulpiride, and
the ionic conductances involved were assessed using 0.5 μM tetrodotoxin (TTX)
or 30 mM tetraethylammonium (TEA). Blockers were preapplied for 5–10 minutes prior
to coapplication of dopamine for 5 minutes.


### 2.4. Data Analysis

Electrophysiological
characteristics of fan cells and changes in synaptic responses were analyzed
using the software program Clampfit 8.2 (Axon Instr., Calif,
USA). The amplitudes of averaged EPSPs were
measured relative to the prestimulus baseline, and paired-pulse facilitation
was determined by expressing the amplitude of the second response as a
proportion of the amplitude of the first response. Action potential amplitude was measured from
resting potential, and action potential width and fast and medium
afterhyperpolarizations were measured from threshold. Input resistance was calculated by measuring
peak and steady-state voltage responses to −200 pA current
steps (500 milliseconds), and inward rectification was quantified by expressing
the peak input resistance as a proportion of the steady-state resistance
(rectification ratio). All data were
expressed as the mean ±SEM for plotting, and changes in response properties
were assessed using paired samplest-tests or mixed design ANOVAs.


## 2.1. Tissue Slices

Methods for obtaining whole cell current clamp recordings
were similar to those described previously [13, 29, 33, 34].  Male Long-Evans rats between 4 and 6 weeks old
were anesthetized with halothane, decapitated, and their brains rapidly removed
and transferred into cold (4°C) artificial cerebrospinal fluid (ACSF) saturated
with 95% O2 and 5% CO2 containing (in mM) 124 NaCl, 5
KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2,
26 NaHCO3, and 10 dextrose (pH  ≈7.3; 300–310 mOsm). All chemicals were obtained from Sigma-Aldrich, Mo, USA. Horizontal slices (300 μm thick) were cut
using a vibratome (WPI, Vibroslice,
Fla, USA),
and slices recovered for at least one hour at 22 to 24°C. Slices were transferred individually to a
recording chamber and visualized using an upright microscope (Leica, Richmond
Hill, Canada, DM-LFS)
equipped with differential interference contrast optics, a 40x water immersion
objective, and a near-infrared camera (COHU, Inc., Calif, USA). Submerged slices were superfused with
oxygenated ACSF at a rate of 1.5 to 2.0 mL/min. Slices containing the lateral entorhinal cortex were taken from ventral
sections about 1.9 to 3.4 mm above the interaural line [35]. Layer II was identified based on the presence
of cell “islands” about 150 μm from the cortical surface [36–39].


## 2.2. Stimulation and Recording

Patch recording pipettes for whole cell recordings were
prepared from borosilicate glass (1.0 mm OD, 4 to 8 MΩ) using a horizontal
puller (P-97, Sutter Instr., Calif,
USA) and were filled with a solution containing (in mM) 140 K-gluconate,
5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 ATP-Tris, and 0.4 GTP-Tris
(pH adjusted to 7.24–7.32 with KOH; 270–280 mOsm). Pipettes were placed in contact with somata
of layer II neurons, and gentle suction was applied under voltage clamp to form
a tight seal (1–3 GΩ). Whole cell configuration was achieved by
increased suction, and experiments began after cells stabilized (typically
within 3 to 5 minutes after break-in). Current clamp recordings were obtained using an Axopatch 200B amplifier
(Axon Instr., Calif, USA)
and displayed on a digital oscilloscope (Gould 1604). Recordings were filtered at 10 kHz and
digitized at 20 kHz (Axon Instr., Digidata 1322A) for storage on computer hard
disk. Recordings were accepted if the
series resistance was ≤25 MΩ (mean = 16.9±0.9 MΩ) and if input resistance and
resting potential were stable. A bipolar
stimulating electrode made from two tungsten electrodes (FHC, 1.0 MΩ) was
positioned to span layer I near the border with layer II approximately 0.2 to
0.6 mm rostral to the recording electrode. Synaptic responses were evoked with 0.1 millisecond constant current
pulses delivered using a stimulus timer and isolation unit (WPI, Mass, USA, models A300 and
A360). Stimulation intensity was
adjusted to evoke responses approximately 75% of maximal (75 to 300 μA).All neurons
(n = 118) included for analyses were identified as “fan” cells based on
electrophysiological characteristics described previously [40, 41]. In comparison to stellate cells of the medial
entorhinal cortex, fan cells show modest inward rectification during
hyperpolarizing current steps, a small depolarizing afterpotential following
single spikes, and do not show prominent theta-frequency membrane potential
oscillations at subthreshold voltages [40–42].


## 2.3. Dopaminergic Modulation of Synaptic Responses

The effects of dopamine on glutamate-mediated synaptic
transmission in the lateral entorhinal cortex are largely uncharacterized. We therefore recorded both mixed and isolated
components of excitatory postsynaptic potentials (EPSPs) evoked by stimulation
of layer I before and after 5-minute bath-application of 1, 10, or 50μM
dopamine. Results obtained using high
concentrations of dopamine must be interpreted cautiously because of the
possibility of nonspecific effects. However,
dopamine degrades through oxidization within the slice preparation, and similar
concentrations of dopamine have been used previously, and interpreted in light
of the effects of specific antagonists, in reports examining the effects of
dopamine on synaptic transmission in both the entorhinal [29–32] and
prefrontal [23, 43] cortices. Responses
were evoked once every 20 seconds, and the mean of 10 responses was obtained
for analysis. Baseline responses were
obtained at resting potential and, because dopamine usually hyperpolarizes fan
cells, constant current was often required to return cells to the original
membrane potential for recordings in the presence of dopamine. Sodium metabisulfite (50 μM) was coapplied to
slow the oxidation of dopamine [29, 31, 43], and ambient lighting was also
reduced. Possible effects of sodium metabisulfite
were assessed with a vehicle control group. Drugs were routinely stored at −20°C as
concentrated stock solutions until needed, but dopamine HCl was dissolved just
prior to bath application.Paired-pulse
tests were used to determine whether dopamine modulates EPSPs through a pre- or
postsynaptic mechanism [13]. Pairs of
stimulation pulses separated by an interval of 30 milliseconds were delivered
before and after 5-minute bath-application of 1, 10, or 50 μM dopamine. Stimulation intensity was reduced to evoke
EPSPs approximately 50% of maximal, and ten responses were averaged for
analyses. Paired-pulse facilitation was
quantified by expressing the amplitude of the second response as a percentage
of the first response.Mechanisms mediating the
suppression of EPSPs by high concentrations of dopamine were investigated by
assessing the effects of 50μM dopamine on isolated components of synaptic
responses. After baseline recordings in
normal ACSF, AMPA receptor-mediated responses were isolated with bath
application of 50 μM 2-amino-5-phosphonovaleric acid (APV) and 25 μM
bicuculline methiodide, or NMDA receptor-mediated responses were isolated with
20 μM 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX) and 25 μM
bicuculline. GABA-mediated IPSPs were
isolated with either 1 mM kynurenic acid or 20 μM CNQX with 50 μM APV. Isolated synaptic responses were recorded
before and after 5-minute application of 50 μM dopamine. Isolated AMPA receptor-mediated responses
were also used to determine if dopamine suppresses EPSPs primarily through D1-
or D2-like receptors. Baseline responses were recorded in the presence of either the D1 receptor antagonist SCH23390 (50 μM) or the D2 receptor antagonist
sulpiride (50 μM) [29–31], and 50 μM dopamine was then applied for 5 minutes. Sulpiride was prepared daily in a stock
solution of 6% DMSO in ACSF titrated with 0.1 N HCl, and there was a final
concentration of 0.1% DMSO with sulpiride.The effects of dopamine on the
intrinsic excitability of fan cells were assessed by monitoring responses to
hyperpolarizing and depolarizing current steps. Changes in action potentials, afterhyperpolarizations, input resistance
and inward rectification were examined before and after 5-minute bath
application of 1, 10, or 50μM dopamine. The number of action potentials elicited in response to suprathreshold
current injection can be used to characterize neuronal excitability [32], and
we therefore determined the number of spikes fired in response to a single 500 millisecond-duration
depolarizing current pulse from a constant holding potential (typically rest)
using a pulse amplitude that elicited 3 to 5 action potentials [32]. Receptors that mediate the dopamine-induced
change in input resistance were investigated using SCH23390 or sulpiride, and
the ionic conductances involved were assessed using 0.5 μM tetrodotoxin (TTX)
or 30 mM tetraethylammonium (TEA). Blockers were preapplied for 5–10 minutes prior
to coapplication of dopamine for 5 minutes.


## 2.4. Data Analysis

Electrophysiological
characteristics of fan cells and changes in synaptic responses were analyzed
using the software program Clampfit 8.2 (Axon Instr., Calif,
USA). The amplitudes of averaged EPSPs were
measured relative to the prestimulus baseline, and paired-pulse facilitation
was determined by expressing the amplitude of the second response as a
proportion of the amplitude of the first response. Action potential amplitude was measured from
resting potential, and action potential width and fast and medium
afterhyperpolarizations were measured from threshold. Input resistance was calculated by measuring
peak and steady-state voltage responses to −200 pA current
steps (500 milliseconds), and inward rectification was quantified by expressing
the peak input resistance as a proportion of the steady-state resistance
(rectification ratio). All data were
expressed as the mean ±SEM for plotting, and changes in response properties
were assessed using paired samplest-tests or mixed design ANOVAs.


## 3. Results

### 3.1. Electroresponsiveness of Layer II Fan Cells

A total of 118 fan cells in layer
II of the lateral entorhinal cortex were identified electrophysiologically and
included for analysis, and the characteristics of these cells were similar to
those reported previously [40, 41]. Fan
cells had a mean resting membrane potential of −58.8±0.6 mV, and
a peak input resistance of 99.1±2.1 MΩ. Most cells (108 of 118) demonstrated a small delayed inward
rectification in response to hyperpolarizing current steps (rectification
ratio: 1.10±0.01). Action potentials (amplitude: 128.8±0.7 mV, width: 4.1±0.1 milliseconds, threshold: −44.1±0.8 mV)
were typically followed by fast and medium afterhyperpolarizations (−3.3±0.3 mV and −5.8±0.3 mV) with
a small depolarizing afterpotential. Averaged EPSPs evoked by stimulation of layer I had a mean amplitude of
4.4±0.2 mV. Continuous recordings of
membrane potential were obtained in a subset of 28 cells to assess subthreshold
membrane potential oscillations and, similar to findings of Tahvildari and
Alonso [40], fan cells did not display prominent oscillations (data not
shown).


### 3.2. Dopaminergic Modulation of EPSPs

We previously found
concentration-dependent effects of dopamine on field EPSPs in layer II in vitro, in which 10μM dopamine
facilitated fEPSPs and 50 to 100 μM dopamine suppressed fEPSPs [29]. We obtained similar concentration-dependent
effects in whole cell EPSPs recorded here before and after 5-minute bath
application of dopamine. Application of
50 μM dopamine resulted in a strong suppression of synaptic response to 38.5±5.8% of baseline levels (see Figure 1(a); t8 = 7.75, P<.001; n = 9) that could be reversed by 15 minutes washout in normal ACSF (3
cells). We initially expected 10 μM
dopamine to facilitate EPSPs [29], but found that 10 μM dopamine instead caused
a small synaptic suppression (to 87.0±5.8% of baseline; see Figure 1(b); t15 = 2.31, P<.05; n = 18). However, a lower concentration of 1 μM dopamine significantly enhanced
responses to 132.7±4.4% of baseline levels (see Figure 1(c); t6 =
5.04, P<.01; n = 7). In our previous study using a gas-fluid interface chamber, a larger bath
volume and slower flow-rate may have increased dopamine oxidation and reduced
the effective concentration of dopamine at the slice, and this may account for
why a higher applied concentration facilitated responses in that study
[29]. Bath
application of the antioxidant sodium
metabisulfite alone had no significant effect on the amplitude of whole cell
EPSPs (see Figure 1(d); n = 8).Dopamine has dose-dependent and
bidirectional effects on the amplitude of mixed EPSPs in layer II fan
cells. (a) FiftyμM dopamine
significantly reduces the amplitude of synaptic responses. Traces show averaged EPSPs before (ACSF) and
after 5-minute bath application of dopamine (DA) in a representative cell. Group data indicate the mean amplitude of
EPSPs before and after dopamine (*,P<.001). Bars indicate ±1 SEM in this and subsequent
figures, and * indicates P<.05 unless otherwise indicated. (b) A lower concentration of 10 μM dopamine
causes a smaller suppression of synaptic responses. (c) The low 1 μM concentration of dopamine enhances
the amplitude of synaptic responses (*,P<.01). (d) Bath
application of vehicle (50 μM sodium metabisulfite; Veh) does not significantly
affect synaptic transmission.
(a)(b)(c)(d)Paired-pulse tests were used to
determine if synaptic suppression and facilitation effects were likely
expressed pre- or postsynaptically. Pairs of pulses were delivered before and after 5-minute dopamine
application, and a 30-millisecond interpulse interval was used that results in
optimal paired-pulse facilitation [13, 44–46]. If EPSPs are reduced through a reduction in
transmitter release, then a greater amount of transmitter should be available
for release in response to the second stimulation pulse and paired-pulse
facilitation should be enhanced [47–49]. Changes in EPSPs mediated by alterations in
postsynaptic receptors, however, should not be associated with changes in
paired-pulse ratio. High concentrations
of dopamine that reduced EPSP amplitude were also found to enhance paired-pulse
facilitation (see Figures 2(a), 2(b); t13 = 2.78, P<.05 for 10 μM; t8 = 2.97, P<.05 for 50 μM), suggesting that dopamine reduced EPSPs by
suppressing glutamate release. In
contrast, the low concentration of 1 μM dopamine that facilitated EPSPs had no
significant effect on paired pulse facilitation (see Figure 2(c)), suggesting
that the facilitation of EPSPs was mediated primarily by an increased
postsynaptic response to glutamate. The
dopaminergic facilitation of the conditioning response was smaller during
paired-pulse tests in which stimulus intensity was reduced to avoid spiking (see
Figures 1(c) versus 2(c)) but a similar dopaminergic facilitation of fEPSPs with
no effect on paired-pulse ratio has been observed in the entorhinal cortex in vivo [29].High concentrations of dopamine increase
paired-pulse facilitation. (a) Pairs of stimulation pulses with a 30 millisecond interpulse interval
were delivered before and after 5-minute bath application of 50μM
dopamine. Averaged traces at left show
responses recorded before (ACSF) and after (DA) dopamine from a representative
cell. Note the suppression of the
response to the first pulse and the large facilitation of the second response
following dopamine (dotted line). Traces
at right have been scaled to the amplitude of the first response in normal ACSF
to aid comparison. Group data are shown
on the right. (b) Paired-pulse
facilitation was also enhanced by 10 μM dopamine. (c) In contrast, the low concentration of 1 μM
dopamine does not affect paired-pulse ratio.
(a)(b)(c)


### 3.3. Isolated Synaptic Responses

The suppression of EPSPs by high concentrations of dopamine
was examined more closely using pharmacologically isolated synaptic
responses. Consistent with a suppression
of glutamate release from presynaptic terminals, bath application of 50μM
dopamine significantly attenuated both the isolated AMPA- and NMDA-mediated
responses. The NMDA component was
reduced to 26.0±7.5% of baseline (see Figure 3(b); t7 = 3.32, P<.05; n = 8) and the AMPA component was reduced to 41.7±5.6% of baseline (see
Figure 3(a); t5 = 3.50, P<.05; n = 6).Dopamine suppresses
the amplitude of both AMPA- and NMDA receptor-mediated components of
EPSPs. (a) AMPA-mediated EPSPs
recorded in the presence of APV and bicuculline were suppressed by 50μM
dopamine. Averaged traces show EPSPs
recorded before (BL) and after (DA) dopamine application, and group data are
shown at right. (b) Isolated NMDA
receptor-mediated EPSPs recorded in the presence of CNQX and bicuculline are
also suppressed by a high concentration of dopamine. Group data show a consistent suppression of
the small isolated NMDA response.
(a)(b)Dopamine receptor subtypes
underlying the suppression of AMPA-mediated synaptic responses were
investigated by applying 50μM dopamine in the presence of either the D1 receptor antagonist SCH23390 (50 μM) or the D2 receptor antagonist
sulpiride (50 μM). Similar to previous
reports that have used selective agonists in the medial [30, 31] and lateral
[29] entorhinal cortex, application of either the D1 agonist
SKF38393 (25 to 50 μM; n = 9) or the D2 agonist quinpirole (20 to 40 μM; n = 10) had no effect on EPSPs (data not shown), and we therefore used
receptor blockers known to affect synaptic responses in the lateral entorhinal
cortex [29]. Application of antagonists
alone had no effect on EPSPs, and the D1 antagonist SCH23390 did not
block the suppression of AMPA-mediated EPSPs (see Figure 4(a); t4 =
3.0, P<.05; n = 5), suggesting that D1 receptors do not
mediate the suppression. However, blockade of D2 receptors with
sulpiride significantly reduced the effects of dopamine on AMPA-mediated
EPSPs. Coapplication of dopamine with
sulpiride (n = 5) resulted in a nonsignificant suppression of synaptic
responses, and the size of the suppression was significantly smaller than that
observed with dopamine alone (79.8±7.2% versus 41.7±5.6% of baseline; F1,9 = 18.10, P<.001; see Figure 4(b1)). Sulpiride also prevented the enhancement of
paired-pulse facilitation induced by 50 μM dopamine (see Figure 4(b2)). Although this indicates that the dopaminergic
suppression of EPSPs is largely dependent upon activation of D2-like
receptors, the suppression of responses in the presence of sulpiride was close
to statistical significance (t4 = 2.65, P = .06), suggesting
that a non-D2 receptor-mediated mechanism mediates the residual
suppression.Dopamine suppresses
isolated AMPA-mediated EPSPs via a D2 receptor-dependent
mechanism. (a) Coapplication of the D1 receptor antagonist SCH23390 (50 μM) did not prevent the dopamine-induced
reduction in EPSP amplitude. (b) However, coapplication of the D2 receptor antagonist
sulpiride (50 μM) significantly attenuated the dopaminergic suppression of
EPSPs. Sulpiride also prevented the
enhancement of paired-pulse facilitation induced by dopamine (b2).


### 3.4. Dopaminergic Suppression of IPSPs

Biphasic IPSPs were recorded from
fan cells held near action potential threshold (−51 to −48 mV) and
exposed to either 1 mM kynurenic acid or a combination of 50μM APV and 20 μM
CNQX to block ionotropic glutamate transmission. A concentration of 50 μM dopamine suppressed
both the early GABAA- and late GABAB-mediated components
of the IPSP. The early IPSP was reduced
to 84.5±8.7% of baseline levels, and the late IPSP was reduced to 62.3±11.1%
of baseline levels (see Figure 5(b); early, t8 = 2.41, P<.05, n = 9; late, t7 = 2.46, P<.05, n = 8). The dopaminergic suppression of GABA synapses
indicates that the reduction of EPSPs by dopamine is unlikely to be due to
increased GABAergic inhibition of fan cells.Dopamine suppresses
both the fast and slow components of the mixed monosynaptic IPSP in fan
cells. (a) GABA-mediated IPSPs were
isolated pharmacologically with ionotropic glutamate receptor blockers and
recorded at membrane potentials just below action potential threshold. Both the early (circle) and late (square)
components of the biphasic IPSP were suppressed by 50μM dopamine (DA). (b) Group data reflect a significant suppression
of both the early and late IPSPs.
(a)(b)


### 3.5. Modulation of Intrinsic Excitability

Bath  application of dopamine also
hyperpolarized resting membrane potential and reduced the input resistance of
fan cells. Membrane potential was
increased from −56.1±2.0 to −59.7±1.4 mV (see
Figure 6(a); t8 = 4.73, P<.001; n = 9), and peak input
resistance was reduced from 90.3±7.6 to 68.9±3.1 MΩ by 50 μM dopamine (see Figure
6(b); t7 = 4.27, P<.01; n = 8). Similar changes in membrane potential and
input resistance were observed for 10 μM dopamine (not shown) and have also
been reported following application of high concentrations of dopamine in
whole-cell recordings from medial entorhinal cortex stellate cells [30]. Changes were not due to the vehicle, because
control cells and cells exposed to 1 μM dopamine did not show a drop in input resistance
or hyperpolarization of membrane potential.Dopamine
hyperpolarizes membrane potential and reduces the input resistance of layer II
fan cells. (a) Membrane potential was shifted to more hyperpolarized
potentials by dopamine (*,P<.001). (b) Dopamine also reduced both peak and
steady-state input resistance (*,P<.01). (c) Voltage responses to applied current steps
before (c1) and after (c2) bath application of 50 μM
dopamine in a representative cell. Action potentials are truncated. Circles in (c1) indicate the latencies at which peak and
steady-state input resistance were measured. Inset traces in (c2) compare the initial voltage deflection to
a −200 pA current
step before and after application of dopamine. Arrows indicate voltage responses before and after dopamine that were
similar in amplitude and which allow comparison of the magnitude of the inward
rectification. Note also the reduced input resistance
across the entire range of hyperpolarizing current pulses. (d) Current-voltage plots show peak and
steady-state responses to current steps of increasing size. Arrows indicate points at which a comparable
degree of inward rectification was observed during hyperpolarization to similar
voltages before and after dopamine application.In layer V entorhinal cortex
cells dopamine causes a reduction in excitability and a drop in input
resistance through an increase in the hyperpolarization-activated currentIh [32], and changes in Ih were therefore assessed in
layer II fan cells. However, dopamine
did not significantly affect the amount of inward rectification, and the
rectification ratio remained stable (see Figure 6(d); 1.09±0.02 in ACSF and in
50 μM dopamine, t7 = 0.00, P = 1.00).Dopamine suppressed the
excitability of fan cells, and application of 10 and 50μM dopamine reduced the
number of action potentials evoked by brief 500 milliseconds depolarizing
current pulses (see Figure 7). The
number of spikes was reduced from 4.1±0.1 to 2.8±0.5 spikes by 10 μM dopamine
(see Figure 7(b); t17 = 2.54, P<.05; n = 18). A higher 50 μM concentration of dopamine caused a similar reduction in the number of spikes
(from 3.9±0.2 to 2.8±0.6) that was not statistically significant (t8 = 1.82, P = .11; n = 9). The
reduction in spiking could result in part from reduced input resistance, but it
was not due to membrane hyperpolarization because cells were tested at the same
membrane potential both before and after dopamine application.Figure 7
The number of action
potentials elicited by positive current steps is reduced by dopamine. (a) Traces show action potentials generated in
response to 500 milliseconds duration, 60 pA current steps before and after
application of 50μM dopamine. The
example shown reflects a particularly large reduction to only one action
potential following application of dopamine. Action potentials are truncated. (b) Group data show a reduction in firing for both the 10 and 50 μM
conditions but only the reduction in the 10 μM condition was significant.The drop in
input resistance induced by 50μM dopamine was blocked by coapplication of the
D1 receptor antagonist SCH23390 (and there was actually a very small
but reliable increase in Rin in 4 of 5 cells; t4 = 2.60, P = .06; see Figure 8(a)). The drop in input resistance was not affected
by coapplication of the D2 receptor antagonist sulpiride (t4 = 9.71, P<.001; n = 5; Figure 8(b)). The reduction in input resistance induced by
dopamine is therefore dependent on activation of D1, but not D2,
receptors.Blockade of D1,
but not D2, receptors prevents the dopamine-induced reduction in
input resistance. (a) Bath-application of the
D1 receptor antagonist SCH23390 (50 μM) prevented the reduction in
input resistance induced by 50 μM dopamine. Traces at left show voltage responses to a series of current steps
during baseline recordings in SCH23390 and during subsequent dopamine
application. Traces at right compare the initial voltage responses to −200 pA
steps before and after dopamine application. Note that input resistance is unchanged when D1 receptors are
blocked. (b) The D2 receptor blocker sulpiride (50 μM) does
not prevent changes in input resistance induced by dopamine (*,P<.001).The conductances that mediated
the reduced input resistance were investigated using blockers of Na+ and K+ channels. The Na+ channel blocker TTX was used to verify that reductions in input resistance were
not due to an increase in action potential-dependent synaptic inputs to fan
cells, or due to an altered Na+ conductance. Blockade of Na+ channels with TTX
did not prevent the drop in input resistance induced by dopamine (see Figure 9(a);
peak, t4 = 6.02, P<.01; steady-state, t4 =
8.21, P<.01; n = 5). It has
been suggested that the reduced input resistance induced by dopamine in medial
entorhinal cortex stellate cells might be mediated by an increased K+ conductance [30], and we therefore assessed the effects of dopamine on input
resistance in the presence of the K+ channel blocker TEA (30 mM; n =
5). Coapplication of TEA blocked the
reduction in input resistance induced by dopamine (see Figure 9(b)), indicating
that the D1 receptor-dependent reduction in input resistance
involves an increased K+ conductance. The increased K+ conductance is
likely to contribute to the hyperpolarization of membrane potential induced by
dopamine, and may also account for the reduced excitability of fan cells (see Figure
7). The reduced input resistance may
also contribute to the dopamine-induced suppression of EPSPs; the D2 receptor blocker sulpiride
did not fully prevent the suppression of AMPA-mediated EPSPs (see Figure 4(b1)),
and the D1 receptor-mediated reduction in input resistance could
contribute to part of the EPSP suppression.Blocking potassium channels prevent
the dopamine-induced reduction in input resistance. (a) Blockade of Na+ channels with 0.5 μM TTX does not prevent the reduction of peak or steady-state input resistance
induced by 50 μM dopamine (*,P<.01). Conventions are as in Figure 8. (b) In contrast, coapplication of the K+ channel blocker TEA (30 mM) prevented the dopamine-induced reduction in input
resistance.


## 3.1. Electroresponsiveness of Layer II Fan Cells

A total of 118 fan cells in layer
II of the lateral entorhinal cortex were identified electrophysiologically and
included for analysis, and the characteristics of these cells were similar to
those reported previously [40, 41]. Fan
cells had a mean resting membrane potential of −58.8±0.6 mV, and
a peak input resistance of 99.1±2.1 MΩ. Most cells (108 of 118) demonstrated a small delayed inward
rectification in response to hyperpolarizing current steps (rectification
ratio: 1.10±0.01). Action potentials (amplitude: 128.8±0.7 mV, width: 4.1±0.1 milliseconds, threshold: −44.1±0.8 mV)
were typically followed by fast and medium afterhyperpolarizations (−3.3±0.3 mV and −5.8±0.3 mV) with
a small depolarizing afterpotential. Averaged EPSPs evoked by stimulation of layer I had a mean amplitude of
4.4±0.2 mV. Continuous recordings of
membrane potential were obtained in a subset of 28 cells to assess subthreshold
membrane potential oscillations and, similar to findings of Tahvildari and
Alonso [40], fan cells did not display prominent oscillations (data not
shown).


## 3.2. Dopaminergic Modulation of EPSPs

We previously found
concentration-dependent effects of dopamine on field EPSPs in layer II in vitro, in which 10μM dopamine
facilitated fEPSPs and 50 to 100 μM dopamine suppressed fEPSPs [29]. We obtained similar concentration-dependent
effects in whole cell EPSPs recorded here before and after 5-minute bath
application of dopamine. Application of
50 μM dopamine resulted in a strong suppression of synaptic response to 38.5±5.8% of baseline levels (see Figure 1(a); t8 = 7.75, P<.001; n = 9) that could be reversed by 15 minutes washout in normal ACSF (3
cells). We initially expected 10 μM
dopamine to facilitate EPSPs [29], but found that 10 μM dopamine instead caused
a small synaptic suppression (to 87.0±5.8% of baseline; see Figure 1(b); t15 = 2.31, P<.05; n = 18). However, a lower concentration of 1 μM dopamine significantly enhanced
responses to 132.7±4.4% of baseline levels (see Figure 1(c); t6 =
5.04, P<.01; n = 7). In our previous study using a gas-fluid interface chamber, a larger bath
volume and slower flow-rate may have increased dopamine oxidation and reduced
the effective concentration of dopamine at the slice, and this may account for
why a higher applied concentration facilitated responses in that study
[29]. Bath
application of the antioxidant sodium
metabisulfite alone had no significant effect on the amplitude of whole cell
EPSPs (see Figure 1(d); n = 8).Dopamine has dose-dependent and
bidirectional effects on the amplitude of mixed EPSPs in layer II fan
cells. (a) FiftyμM dopamine
significantly reduces the amplitude of synaptic responses. Traces show averaged EPSPs before (ACSF) and
after 5-minute bath application of dopamine (DA) in a representative cell. Group data indicate the mean amplitude of
EPSPs before and after dopamine (*,P<.001). Bars indicate ±1 SEM in this and subsequent
figures, and * indicates P<.05 unless otherwise indicated. (b) A lower concentration of 10 μM dopamine
causes a smaller suppression of synaptic responses. (c) The low 1 μM concentration of dopamine enhances
the amplitude of synaptic responses (*,P<.01). (d) Bath
application of vehicle (50 μM sodium metabisulfite; Veh) does not significantly
affect synaptic transmission.
(a)(b)(c)(d)Paired-pulse tests were used to
determine if synaptic suppression and facilitation effects were likely
expressed pre- or postsynaptically. Pairs of pulses were delivered before and after 5-minute dopamine
application, and a 30-millisecond interpulse interval was used that results in
optimal paired-pulse facilitation [13, 44–46]. If EPSPs are reduced through a reduction in
transmitter release, then a greater amount of transmitter should be available
for release in response to the second stimulation pulse and paired-pulse
facilitation should be enhanced [47–49]. Changes in EPSPs mediated by alterations in
postsynaptic receptors, however, should not be associated with changes in
paired-pulse ratio. High concentrations
of dopamine that reduced EPSP amplitude were also found to enhance paired-pulse
facilitation (see Figures 2(a), 2(b); t13 = 2.78, P<.05 for 10 μM; t8 = 2.97, P<.05 for 50 μM), suggesting that dopamine reduced EPSPs by
suppressing glutamate release. In
contrast, the low concentration of 1 μM dopamine that facilitated EPSPs had no
significant effect on paired pulse facilitation (see Figure 2(c)), suggesting
that the facilitation of EPSPs was mediated primarily by an increased
postsynaptic response to glutamate. The
dopaminergic facilitation of the conditioning response was smaller during
paired-pulse tests in which stimulus intensity was reduced to avoid spiking (see
Figures 1(c) versus 2(c)) but a similar dopaminergic facilitation of fEPSPs with
no effect on paired-pulse ratio has been observed in the entorhinal cortex in vivo [29].High concentrations of dopamine increase
paired-pulse facilitation. (a) Pairs of stimulation pulses with a 30 millisecond interpulse interval
were delivered before and after 5-minute bath application of 50μM
dopamine. Averaged traces at left show
responses recorded before (ACSF) and after (DA) dopamine from a representative
cell. Note the suppression of the
response to the first pulse and the large facilitation of the second response
following dopamine (dotted line). Traces
at right have been scaled to the amplitude of the first response in normal ACSF
to aid comparison. Group data are shown
on the right. (b) Paired-pulse
facilitation was also enhanced by 10 μM dopamine. (c) In contrast, the low concentration of 1 μM
dopamine does not affect paired-pulse ratio.
(a)(b)(c)


## 3.3. Isolated Synaptic Responses

The suppression of EPSPs by high concentrations of dopamine
was examined more closely using pharmacologically isolated synaptic
responses. Consistent with a suppression
of glutamate release from presynaptic terminals, bath application of 50μM
dopamine significantly attenuated both the isolated AMPA- and NMDA-mediated
responses. The NMDA component was
reduced to 26.0±7.5% of baseline (see Figure 3(b); t7 = 3.32, P<.05; n = 8) and the AMPA component was reduced to 41.7±5.6% of baseline (see
Figure 3(a); t5 = 3.50, P<.05; n = 6).Dopamine suppresses
the amplitude of both AMPA- and NMDA receptor-mediated components of
EPSPs. (a) AMPA-mediated EPSPs
recorded in the presence of APV and bicuculline were suppressed by 50μM
dopamine. Averaged traces show EPSPs
recorded before (BL) and after (DA) dopamine application, and group data are
shown at right. (b) Isolated NMDA
receptor-mediated EPSPs recorded in the presence of CNQX and bicuculline are
also suppressed by a high concentration of dopamine. Group data show a consistent suppression of
the small isolated NMDA response.
(a)(b)Dopamine receptor subtypes
underlying the suppression of AMPA-mediated synaptic responses were
investigated by applying 50μM dopamine in the presence of either the D1 receptor antagonist SCH23390 (50 μM) or the D2 receptor antagonist
sulpiride (50 μM). Similar to previous
reports that have used selective agonists in the medial [30, 31] and lateral
[29] entorhinal cortex, application of either the D1 agonist
SKF38393 (25 to 50 μM; n = 9) or the D2 agonist quinpirole (20 to 40 μM; n = 10) had no effect on EPSPs (data not shown), and we therefore used
receptor blockers known to affect synaptic responses in the lateral entorhinal
cortex [29]. Application of antagonists
alone had no effect on EPSPs, and the D1 antagonist SCH23390 did not
block the suppression of AMPA-mediated EPSPs (see Figure 4(a); t4 =
3.0, P<.05; n = 5), suggesting that D1 receptors do not
mediate the suppression. However, blockade of D2 receptors with
sulpiride significantly reduced the effects of dopamine on AMPA-mediated
EPSPs. Coapplication of dopamine with
sulpiride (n = 5) resulted in a nonsignificant suppression of synaptic
responses, and the size of the suppression was significantly smaller than that
observed with dopamine alone (79.8±7.2% versus 41.7±5.6% of baseline; F1,9 = 18.10, P<.001; see Figure 4(b1)). Sulpiride also prevented the enhancement of
paired-pulse facilitation induced by 50 μM dopamine (see Figure 4(b2)). Although this indicates that the dopaminergic
suppression of EPSPs is largely dependent upon activation of D2-like
receptors, the suppression of responses in the presence of sulpiride was close
to statistical significance (t4 = 2.65, P = .06), suggesting
that a non-D2 receptor-mediated mechanism mediates the residual
suppression.Dopamine suppresses
isolated AMPA-mediated EPSPs via a D2 receptor-dependent
mechanism. (a) Coapplication of the D1 receptor antagonist SCH23390 (50 μM) did not prevent the dopamine-induced
reduction in EPSP amplitude. (b) However, coapplication of the D2 receptor antagonist
sulpiride (50 μM) significantly attenuated the dopaminergic suppression of
EPSPs. Sulpiride also prevented the
enhancement of paired-pulse facilitation induced by dopamine (b2).


## 3.4. Dopaminergic Suppression of IPSPs

Biphasic IPSPs were recorded from
fan cells held near action potential threshold (−51 to −48 mV) and
exposed to either 1 mM kynurenic acid or a combination of 50μM APV and 20 μM
CNQX to block ionotropic glutamate transmission. A concentration of 50 μM dopamine suppressed
both the early GABAA- and late GABAB-mediated components
of the IPSP. The early IPSP was reduced
to 84.5±8.7% of baseline levels, and the late IPSP was reduced to 62.3±11.1%
of baseline levels (see Figure 5(b); early, t8 = 2.41, P<.05, n = 9; late, t7 = 2.46, P<.05, n = 8). The dopaminergic suppression of GABA synapses
indicates that the reduction of EPSPs by dopamine is unlikely to be due to
increased GABAergic inhibition of fan cells.Dopamine suppresses
both the fast and slow components of the mixed monosynaptic IPSP in fan
cells. (a) GABA-mediated IPSPs were
isolated pharmacologically with ionotropic glutamate receptor blockers and
recorded at membrane potentials just below action potential threshold. Both the early (circle) and late (square)
components of the biphasic IPSP were suppressed by 50μM dopamine (DA). (b) Group data reflect a significant suppression
of both the early and late IPSPs.
(a)(b)


## 3.5. Modulation of Intrinsic Excitability

Bath  application of dopamine also
hyperpolarized resting membrane potential and reduced the input resistance of
fan cells. Membrane potential was
increased from −56.1±2.0 to −59.7±1.4 mV (see
Figure 6(a); t8 = 4.73, P<.001; n = 9), and peak input
resistance was reduced from 90.3±7.6 to 68.9±3.1 MΩ by 50 μM dopamine (see Figure
6(b); t7 = 4.27, P<.01; n = 8). Similar changes in membrane potential and
input resistance were observed for 10 μM dopamine (not shown) and have also
been reported following application of high concentrations of dopamine in
whole-cell recordings from medial entorhinal cortex stellate cells [30]. Changes were not due to the vehicle, because
control cells and cells exposed to 1 μM dopamine did not show a drop in input resistance
or hyperpolarization of membrane potential.Dopamine
hyperpolarizes membrane potential and reduces the input resistance of layer II
fan cells. (a) Membrane potential was shifted to more hyperpolarized
potentials by dopamine (*,P<.001). (b) Dopamine also reduced both peak and
steady-state input resistance (*,P<.01). (c) Voltage responses to applied current steps
before (c1) and after (c2) bath application of 50 μM
dopamine in a representative cell. Action potentials are truncated. Circles in (c1) indicate the latencies at which peak and
steady-state input resistance were measured. Inset traces in (c2) compare the initial voltage deflection to
a −200 pA current
step before and after application of dopamine. Arrows indicate voltage responses before and after dopamine that were
similar in amplitude and which allow comparison of the magnitude of the inward
rectification. Note also the reduced input resistance
across the entire range of hyperpolarizing current pulses. (d) Current-voltage plots show peak and
steady-state responses to current steps of increasing size. Arrows indicate points at which a comparable
degree of inward rectification was observed during hyperpolarization to similar
voltages before and after dopamine application.In layer V entorhinal cortex
cells dopamine causes a reduction in excitability and a drop in input
resistance through an increase in the hyperpolarization-activated currentIh [32], and changes in Ih were therefore assessed in
layer II fan cells. However, dopamine
did not significantly affect the amount of inward rectification, and the
rectification ratio remained stable (see Figure 6(d); 1.09±0.02 in ACSF and in
50 μM dopamine, t7 = 0.00, P = 1.00).Dopamine suppressed the
excitability of fan cells, and application of 10 and 50μM dopamine reduced the
number of action potentials evoked by brief 500 milliseconds depolarizing
current pulses (see Figure 7). The
number of spikes was reduced from 4.1±0.1 to 2.8±0.5 spikes by 10 μM dopamine
(see Figure 7(b); t17 = 2.54, P<.05; n = 18). A higher 50 μM concentration of dopamine caused a similar reduction in the number of spikes
(from 3.9±0.2 to 2.8±0.6) that was not statistically significant (t8 = 1.82, P = .11; n = 9). The
reduction in spiking could result in part from reduced input resistance, but it
was not due to membrane hyperpolarization because cells were tested at the same
membrane potential both before and after dopamine application.Figure 7
The number of action
potentials elicited by positive current steps is reduced by dopamine. (a) Traces show action potentials generated in
response to 500 milliseconds duration, 60 pA current steps before and after
application of 50μM dopamine. The
example shown reflects a particularly large reduction to only one action
potential following application of dopamine. Action potentials are truncated. (b) Group data show a reduction in firing for both the 10 and 50 μM
conditions but only the reduction in the 10 μM condition was significant.The drop in
input resistance induced by 50μM dopamine was blocked by coapplication of the
D1 receptor antagonist SCH23390 (and there was actually a very small
but reliable increase in Rin in 4 of 5 cells; t4 = 2.60, P = .06; see Figure 8(a)). The drop in input resistance was not affected
by coapplication of the D2 receptor antagonist sulpiride (t4 = 9.71, P<.001; n = 5; Figure 8(b)). The reduction in input resistance induced by
dopamine is therefore dependent on activation of D1, but not D2,
receptors.Blockade of D1,
but not D2, receptors prevents the dopamine-induced reduction in
input resistance. (a) Bath-application of the
D1 receptor antagonist SCH23390 (50 μM) prevented the reduction in
input resistance induced by 50 μM dopamine. Traces at left show voltage responses to a series of current steps
during baseline recordings in SCH23390 and during subsequent dopamine
application. Traces at right compare the initial voltage responses to −200 pA
steps before and after dopamine application. Note that input resistance is unchanged when D1 receptors are
blocked. (b) The D2 receptor blocker sulpiride (50 μM) does
not prevent changes in input resistance induced by dopamine (*,P<.001).The conductances that mediated
the reduced input resistance were investigated using blockers of Na+ and K+ channels. The Na+ channel blocker TTX was used to verify that reductions in input resistance were
not due to an increase in action potential-dependent synaptic inputs to fan
cells, or due to an altered Na+ conductance. Blockade of Na+ channels with TTX
did not prevent the drop in input resistance induced by dopamine (see Figure 9(a);
peak, t4 = 6.02, P<.01; steady-state, t4 =
8.21, P<.01; n = 5). It has
been suggested that the reduced input resistance induced by dopamine in medial
entorhinal cortex stellate cells might be mediated by an increased K+ conductance [30], and we therefore assessed the effects of dopamine on input
resistance in the presence of the K+ channel blocker TEA (30 mM; n =
5). Coapplication of TEA blocked the
reduction in input resistance induced by dopamine (see Figure 9(b)), indicating
that the D1 receptor-dependent reduction in input resistance
involves an increased K+ conductance. The increased K+ conductance is
likely to contribute to the hyperpolarization of membrane potential induced by
dopamine, and may also account for the reduced excitability of fan cells (see Figure
7). The reduced input resistance may
also contribute to the dopamine-induced suppression of EPSPs; the D2 receptor blocker sulpiride
did not fully prevent the suppression of AMPA-mediated EPSPs (see Figure 4(b1)),
and the D1 receptor-mediated reduction in input resistance could
contribute to part of the EPSP suppression.Blocking potassium channels prevent
the dopamine-induced reduction in input resistance. (a) Blockade of Na+ channels with 0.5 μM TTX does not prevent the reduction of peak or steady-state input resistance
induced by 50 μM dopamine (*,P<.01). Conventions are as in Figure 8. (b) In contrast, coapplication of the K+ channel blocker TEA (30 mM) prevented the dopamine-induced reduction in input
resistance.


## 4. Discussion

We show here that dopamine has
powerful suppressive effects on glutamate-mediated synaptic transmission in
layer II fan cells of the lateral entorhinal cortex. Our findings suggest that the suppression of
EPSPs involves the combined actions of a D2 receptor-mediated
reduction in neurotransmitter release and a D1 receptor-mediated
increase in a K+ conductance that reduces cellular input
resistance. Previously, we found that
field EPSPs were enhanced by low concentrations of dopamine in vitro, and by blocking dopamine
reuptake in awake animals [29]. This
suggested that moderate increases in dopamine release might facilitate synaptic
responses in the entorhinal cortex, and enhance transmission of sensory information
to the rest of the hippocampal formation. Here, we have replicated the synaptic facilitation with a low 1 μM
concentration of dopamine and have also shown that high concentrations of
dopamine induce a strong and reversible suppression of intracellular
EPSPs. Similar suppression effects have
been observed in the medial entorhinal cortex [30, 31] and prefrontal cortex
[22, 23, 50, 51] using comparable doses of dopamine.


### 4.1. Suppression of Glutamate Release

The suppression of EPSPs by high
concentrations of dopamine was found to be largely dependent on D2 receptors since coapplication of the D2 receptor antagonist
sulpiride blocked most of the reduction. Dopamine also enhanced paired-pulse facilitation which suggests that the
suppression of EPSPs resulted from a reduction in presynaptic glutamate release
[47, 49]. The suppression of both AMPA-
and NMDA-mediated components of the synaptic response is also consistent with
reduced transmitter release. Although
similar reductions in EPSPs have been shown in stellate cells of the medial
entorhinal cortex, the suppression was dependent on D1, and not D2,
receptor activation [30]. However,
Stenkamp et al. (1998) showed a reduction in synaptic responses in layer III of
the medial entorhinal cortex through activation of both D1and D2 receptors, and
results of paired-pulse tests in their study suggested that the suppression was
also mediated by reduced glutamate release.Dopamine has been shown to
suppress AMPA-mediated synaptic responses in the prefrontal cortex through a D1 receptor-mediated suppression of transmitter release [22–24]. Strong activation of D1 receptors
can also suppress synaptic responses through a retrograde signaling
cascade. Weak D1 receptor
activation can enhance NMDA responses, but stronger D1 receptor
activation can lead to more intense NMDA receptor activation and the release of
adenosine that suppresses transmitter release by acting on presynaptic A1 receptors that suppress voltage-gated Ca2+ channels [28, 52, 53]. In the striatum, activation of
presynaptic D2 receptors suppresses N-type Ca2+ currents
and inhibits acetylcholine release from striatal cholinergic interneurons
[54]. D2 receptors have also
been linked to a suppression of responses in the parabrachial nucleus [55],
ventral tegmental area [56], and striatum [57, 58] via a D2-mediated
reduction in glutamate release. A
similar D2-mediated mechanism underlies the suppression of GABA
release from striatal inhibitory cells onto cholinergic interneurons [59]. Similar mechanisms may mediate the dopaminergic
suppression of glutamate release in the entorhinal cortex.The dopaminergic suppression of
EPSPs observed here cannot be explained by increased transmission at GABA
synapses because we found that dopamine reduced
monosynaptic GABAA and GABAB IPSPs. The suppression is also unlikely to be due to
increased activation of feedback inhibition [60] because dopamine reduced both
glutamatergic transmission and the number of spikes in fan cells (see Figure 7). The suppression of monosynaptic
IPSPs that we observed may have resulted from a D2-mediated
reduction in GABA release [59, 61] and reduced input resistance in fan cells
could also have contributed. These
possibilities are consistent with the parallel reductions observed in GABAA and GABAB IPSPs. Recordings
of spontaneous and/or miniature IPSCs would be useful to determine the
mechanisms of the reduced IPSPs.


### 4.2. Modulation of Intrinsic Excitability

In addition
to the D2-mediated suppression of transmitter release, high
concentrations of dopamine also appear to suppress synaptic transmission
through a D1-receptor dependent mechanism. Sulpiride did not completely block the
suppression of EPSPs (see Figure 4(b1)), and a D1 receptor-dependent activation of a TEA-sensitive K+ conductance
appears to mediate the residual suppression via a reduction in input
resistance. Blockade of synaptic
transmission and voltage-gated Na+ channels with TTX did not prevent
the drop in input resistance induced by dopamine indicating that it is not due
to increased spontaneous synaptic drive or to an increased Na+ conductance. However, the broadly acting
K+ channel blocker TEA prevented the drop in input resistance,
indicating that dopamine activates a K+ conductance. The drop in input resistance was also
prevented by blockade of D1, but not D2, receptors,
indicating that dopamine activates K+ channels via D1 receptors. High concentrations of
dopamine also hyperpolarize membrane potential and reduce input resistance in
stellate cells of the medial entorhinal cortex, and it was also suggested that
these changes might be mediated by an increased K+ conductance [30].A large number of K+ conductances are affected by TEA, and it is therefore not clear which type(s)
may be responsible for the drop in input resistance observed here. Background leak channels are insensitive to
TEA [62] and are therefore not likely to contribute. Voltage-gated K+ currents are
blocked by TEA, but dopamine in the prefrontal cortex tends to enhance neuronal
excitability by suppressing these
currents (see also [43, 63]). Several
reports in CA1 pyramidal cells have found that dopamine hyperpolarizes membrane
potential, reduces input resistance, and increases afterhyperpolarizations
through a D1-receptor mediated increase in Ca2+-activated
K+ currents ([64, 65], see also [66]), but others have found an increase
in the excitability of CA1 neurons due to a suppression of Ca2+-activated K+ currents (see also [32, 67, 68]). Here, there was no clear increase in
afterhyperpolarizations, suggesting that Ca2+-dependent K+ currents do not mediate the change in input resistance. Activation of D1 receptors can
also have dose-dependent effects on activation of inward rectifying K+ currents (IRKCs). In the prefrontal
cortex, D1 receptor activation typically inhibits IRKC by direct effects of cAMP on IRK channels, but strong
activation can increase IRKC via phosphorylation of the channels through
elevated levels of PKA [69]. This could
explain why a significant reduction in input resistance was observed here only
at the higher concentrations of dopamine. Clearly, however, further experiments will be required to determine the
nature of the D1 receptor-dependent K+ conductance in fan
cells.We observed a decrease in fan
cell firing during depolarizing current steps after dopamine, and the reduced
spiking may reflect the drop in cellular input resistance. A surprising finding was that while the D1 receptor antagonist SCH23390 prevented the dopamine-induced reduction in input
resistance it did not completely eliminate the reduction in the number of
spikes, suggesting that reduced input resistance cannot entirely account for
the reduction in spiking, and that other mechanisms may also contribute. D1 receptor activation can increase spiking in prefrontal neurons
by enhancing the persistent Na+ current (INaP) and suppressing a slowly-inactivating K+ conductance [43, 70], but a suppression of spiking via a reduction in INaP has also been observed
[71]. In layer V entorhinal cortex
neurons, dopamine reduces input resistance and leads to a reduction of spiking
though an increase in Ih [32]. Here, there was no apparent change
in Ih in fan cells, and
action potential threshold and afterhyperpolarizations were not affected,
suggesting that the underlying currents were not modified. Dopaminergic effects on INaP were not directly assessed in the present study,
and the drop in input resistance could mask possible reductions in depolarizing
responses to current injection related to INaP. However, in tests in which SCH23390 prevented
a change in input resistance, we found no reduction in the response to +20 pA
pulses. This argues against a D1-mediated
reduction in INaP, but it
is still possible that dopamine may reduce spiking via a D2 receptor-mediated reduction in INaP [71].


## 4.1. Suppression of Glutamate Release

The suppression of EPSPs by high
concentrations of dopamine was found to be largely dependent on D2 receptors since coapplication of the D2 receptor antagonist
sulpiride blocked most of the reduction. Dopamine also enhanced paired-pulse facilitation which suggests that the
suppression of EPSPs resulted from a reduction in presynaptic glutamate release
[47, 49]. The suppression of both AMPA-
and NMDA-mediated components of the synaptic response is also consistent with
reduced transmitter release. Although
similar reductions in EPSPs have been shown in stellate cells of the medial
entorhinal cortex, the suppression was dependent on D1, and not D2,
receptor activation [30]. However,
Stenkamp et al. (1998) showed a reduction in synaptic responses in layer III of
the medial entorhinal cortex through activation of both D1and D2 receptors, and
results of paired-pulse tests in their study suggested that the suppression was
also mediated by reduced glutamate release.Dopamine has been shown to
suppress AMPA-mediated synaptic responses in the prefrontal cortex through a D1 receptor-mediated suppression of transmitter release [22–24]. Strong activation of D1 receptors
can also suppress synaptic responses through a retrograde signaling
cascade. Weak D1 receptor
activation can enhance NMDA responses, but stronger D1 receptor
activation can lead to more intense NMDA receptor activation and the release of
adenosine that suppresses transmitter release by acting on presynaptic A1 receptors that suppress voltage-gated Ca2+ channels [28, 52, 53]. In the striatum, activation of
presynaptic D2 receptors suppresses N-type Ca2+ currents
and inhibits acetylcholine release from striatal cholinergic interneurons
[54]. D2 receptors have also
been linked to a suppression of responses in the parabrachial nucleus [55],
ventral tegmental area [56], and striatum [57, 58] via a D2-mediated
reduction in glutamate release. A
similar D2-mediated mechanism underlies the suppression of GABA
release from striatal inhibitory cells onto cholinergic interneurons [59]. Similar mechanisms may mediate the dopaminergic
suppression of glutamate release in the entorhinal cortex.The dopaminergic suppression of
EPSPs observed here cannot be explained by increased transmission at GABA
synapses because we found that dopamine reduced
monosynaptic GABAA and GABAB IPSPs. The suppression is also unlikely to be due to
increased activation of feedback inhibition [60] because dopamine reduced both
glutamatergic transmission and the number of spikes in fan cells (see Figure 7). The suppression of monosynaptic
IPSPs that we observed may have resulted from a D2-mediated
reduction in GABA release [59, 61] and reduced input resistance in fan cells
could also have contributed. These
possibilities are consistent with the parallel reductions observed in GABAA and GABAB IPSPs. Recordings
of spontaneous and/or miniature IPSCs would be useful to determine the
mechanisms of the reduced IPSPs.


## 4.2. Modulation of Intrinsic Excitability

In addition
to the D2-mediated suppression of transmitter release, high
concentrations of dopamine also appear to suppress synaptic transmission
through a D1-receptor dependent mechanism. Sulpiride did not completely block the
suppression of EPSPs (see Figure 4(b1)), and a D1 receptor-dependent activation of a TEA-sensitive K+ conductance
appears to mediate the residual suppression via a reduction in input
resistance. Blockade of synaptic
transmission and voltage-gated Na+ channels with TTX did not prevent
the drop in input resistance induced by dopamine indicating that it is not due
to increased spontaneous synaptic drive or to an increased Na+ conductance. However, the broadly acting
K+ channel blocker TEA prevented the drop in input resistance,
indicating that dopamine activates a K+ conductance. The drop in input resistance was also
prevented by blockade of D1, but not D2, receptors,
indicating that dopamine activates K+ channels via D1 receptors. High concentrations of
dopamine also hyperpolarize membrane potential and reduce input resistance in
stellate cells of the medial entorhinal cortex, and it was also suggested that
these changes might be mediated by an increased K+ conductance [30].A large number of K+ conductances are affected by TEA, and it is therefore not clear which type(s)
may be responsible for the drop in input resistance observed here. Background leak channels are insensitive to
TEA [62] and are therefore not likely to contribute. Voltage-gated K+ currents are
blocked by TEA, but dopamine in the prefrontal cortex tends to enhance neuronal
excitability by suppressing these
currents (see also [43, 63]). Several
reports in CA1 pyramidal cells have found that dopamine hyperpolarizes membrane
potential, reduces input resistance, and increases afterhyperpolarizations
through a D1-receptor mediated increase in Ca2+-activated
K+ currents ([64, 65], see also [66]), but others have found an increase
in the excitability of CA1 neurons due to a suppression of Ca2+-activated K+ currents (see also [32, 67, 68]). Here, there was no clear increase in
afterhyperpolarizations, suggesting that Ca2+-dependent K+ currents do not mediate the change in input resistance. Activation of D1 receptors can
also have dose-dependent effects on activation of inward rectifying K+ currents (IRKCs). In the prefrontal
cortex, D1 receptor activation typically inhibits IRKC by direct effects of cAMP on IRK channels, but strong
activation can increase IRKC via phosphorylation of the channels through
elevated levels of PKA [69]. This could
explain why a significant reduction in input resistance was observed here only
at the higher concentrations of dopamine. Clearly, however, further experiments will be required to determine the
nature of the D1 receptor-dependent K+ conductance in fan
cells.We observed a decrease in fan
cell firing during depolarizing current steps after dopamine, and the reduced
spiking may reflect the drop in cellular input resistance. A surprising finding was that while the D1 receptor antagonist SCH23390 prevented the dopamine-induced reduction in input
resistance it did not completely eliminate the reduction in the number of
spikes, suggesting that reduced input resistance cannot entirely account for
the reduction in spiking, and that other mechanisms may also contribute. D1 receptor activation can increase spiking in prefrontal neurons
by enhancing the persistent Na+ current (INaP) and suppressing a slowly-inactivating K+ conductance [43, 70], but a suppression of spiking via a reduction in INaP has also been observed
[71]. In layer V entorhinal cortex
neurons, dopamine reduces input resistance and leads to a reduction of spiking
though an increase in Ih [32]. Here, there was no apparent change
in Ih in fan cells, and
action potential threshold and afterhyperpolarizations were not affected,
suggesting that the underlying currents were not modified. Dopaminergic effects on INaP were not directly assessed in the present study,
and the drop in input resistance could mask possible reductions in depolarizing
responses to current injection related to INaP. However, in tests in which SCH23390 prevented
a change in input resistance, we found no reduction in the response to +20 pA
pulses. This argues against a D1-mediated
reduction in INaP, but it
is still possible that dopamine may reduce spiking via a D2 receptor-mediated reduction in INaP [71].


## 5. Conclusions

We have shown here that dopamine
has concentration-dependent, bidirectional effects on glutamate-mediated
synaptic transmission in principal cells of layer II of the lateral entorhinal
cortex. The lateral entorhinal cortex receives
a major input from the piriform cortex [5–7], and dopaminergic innervation of
the superficial layers is likely to have a strong modulatory effect on
olfactory processing. In the prefrontal
cortex, moderate activation of dopaminergic inputs promotes working memory
function, but excessive dopamine activation leads to a decrement in performance
[20, 27]. In the entorhinal cortex,
moderate increases in dopamine concentration may enhance the salience of
olfactory representations carried to the lateral entorhinal cortex (see Figure 1(c); see also 29), but large increases in dopamine associated with drug
effects or acute stress [27] may dampen synaptic inputs to the superficial
layers and suppress working memory function [72–74] or induction of lasting
synaptic plasticity [75]. The
dopaminergic suppression of synaptic transmission in layer II is also likely to
inhibit the propagation of sensory information to the rest of the hippocampal
formation such that only strong and synchronous inputs to the entorhinal region
may be sufficient to activate entorhinal projection neurons.



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*Source: 203514-2008-08-25.xml*