# The Role ofGLUK5-Containing Kainate Receptors in  Entorhinal Cortex Gamma Frequency Oscillations

Authors: Heather L. Stanger; Rebekah Alford; David E. Jane; Mark O. Cunningham
Journal: Neural Plasticity
 (2008)

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## Abstract

Using in vitro brain slices of hippocampus and cortex, neuronal oscillations in the frequency range of 30–80 Hz (gamma frequency oscillations) can be induced by a number of pharmacological manipulations. The most routinely used is the bath application of the broad-spectrum glutamate receptor agonist, kainic acid. In the hippocampus, work using transgenic kainate receptor knockout mice have revealed information about the specific subunit composition of the kainate receptor implicated in the generation and maintenance of the gamma frequency oscillation. However, there is a paucity of such detail regarding gamma frequency oscillation in the cortex. Using specific pharmacological agonists and antagonists for the kainate receptor, we have set out to examine the contribution of kainate receptor subtypes to gamma frequency oscillation in the entorhinal cortex. The findings presented demonstrate that in contrast to the hippocampus, kainate receptors containing theGLUK5 subunit are critically important for the generation and maintenance of gamma frequency oscillation in the entorhinal cortex. Future work will concentrate on determining the exact nature of the cellular expression of kainate receptors in the entorhinal cortex.


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## Body

## 1. Introduction

KARs are made up of various combinations of five subunits: GLUK5,
GLUK6, GLUK7, KA1, and KA2 [1, 2] which are abundantly expressed in the neocortex [3]. These subunits make up tetramers of either
homomeric or heteromeric assemblies, with GLUK5–7 being able to form
functional homomeric receptors [1, 4]. KA1
and KA2 cannot form functional receptors when expressed alone [5, 6], yet are
able to form functional KARs when expressed heteromerically with other subunits
[1, 7, 8]. Differential patterns
of expression of KARs in the CNS coupled with the existence of splice variants
and mRNA editing suggest complex neurophysiological
roles for the various subunits, and different roles in neuronal networks
depending on their localization [9, 10].Of particular interest is the role of KARs in the generation and
maintenance of network neuronal oscillatory activity in cortical regions [9, 10].
Gamma frequency oscillations occur between 30–80 Hz and have
been observed in many areas of the brain, including the hippocampus [11–13] and cortical regions [14–16]. Cortical gamma oscillations are important in
higher brain functions, such as learning, memory, and cognition [17–19], as well as processing
of sensorimotor information [15, 16, 20]. In carrying out these functions, cortical
gamma oscillations are implicated in various central processes, including
long-term potentiation (LTP) and synaptic plasticity [21], with important roles in temporal regulation of neuronal activity.Gamma frequency oscillations are recordable from the MEC during wakefulness in
humans [22, 23], as well as in vivo in rodents [12], in vitro from
perfused guinea pig brains [24, 25], and isolated rat brain
slices [26, 27]. These gamma oscillations in the MEC play a role in the
formation, processing, storage, and retrieval of memories [17, 18]. Previously it has been
demonstrated in an in vitro
preparation of the MEC
that application of nanomolar concentrations of kainate (200–400 nM) can
induce persistent gamma frequency oscillations [26–28]. Using this in vitro model of MEC gamma frequency
oscillations it has been elucidated that this activity is primarily generated
by inhibitory-based neuronal networks [29–31]. A similar mechanism for
the generation of gamma frequency activity has been demonstrated in both the
hippocampus and neocortex [32, 33].To date our understanding of the role of KARs in
neuronal network activity has been hampered by a paucity of selective
pharmacological agents. The competitive AMPA/KAR antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline
(NBQX), shows little selectivity between AMPA receptors and KARs at high
concentrations, yet at low concentrations (1 μM) can be used to block AMPA receptors, and isolate KAR responses [34, 35]. However, NBQX shows no
selectivity between different KAR subunits.
The role of GLUK5 and GLUK6 subunits in neuronal
oscillatory activity in the hippocampus has been previously investigated using
receptor knockout mice [9, 10]. However, interpretation
of work using transgenic models should be viewed in the light of the knowledge
that compensatory factors may play a role.
The recent development of pharmacological agents with specificity for distinct
subunits has led to the possibility of a detailed pharmacological investigation
of the role of specific KARs in cortical gamma frequency oscillations. (S)-3-(2-Carboxybenzyl)willardiine
(UBP302) is a novel selective GLUK5-containing KAR antagonist, with
activity at both homomeric and heteromeric GLUK5-containing
receptors [36, 37]. The activity of UBP302 on GLUK7 is
controversial, Dolman et al. [37] showed that UBP296 (racemic form of UBP302)
only weakly inhibited [3H]kainate binding to human GLUK7 (Ki value of 374 ± 122 μM). However, in an electrophysiological assay
UBP302 was found to block rat homomeric GLUK7 receptors with an IC50 value of 4 μM but
at a concentration of 100 μM
only very weakly blocked rat GLUK6/GLUK7 receptors
[38]. 5-Carboxy-2,4-di-benzamido-benzoic
acid (NS3763) is another novel glutamate antagonist, which is selective and
noncompetitive for homomeric GLUK5-containing KARs [39, 40]. (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-isoxazol-4-yl)propanoic acid
(ATPA) is a selective GLUK5-containing receptor agonist [41]. ATPA
has been shown to depress excitatory and GABAergic synaptic transmission in the
hippocampus [42, 43]. However, Cossart et al.
[35] demonstrated that lower concentrations of ATPA could directly
depolarise hippocampal GABAergic interneurons leading to increases in the
levels of tonic inhibition onto pyramidal neurons. More recently, similar
concentrations of ATPA to that used in the Cossart et al. [35] study have
been shown to facilitate both evoked and action potential-independent glutamate
release in the neocortex [44].The data presented here demonstrates a role of GLUK5-containing
KARs in the MEC by
examining the contribution of these receptor subtypes to gamma frequency
oscillations. Using a pharmacological approach, we have demonstrated that GLUK5-containing
KARs are important for the maintenance of gamma frequency oscillations in the MEC. Moreover, the
selective activation of GLUK5-containing KARs can induce persistent
gamma frequency oscillations in the MEC. We also demonstrate that it is the specific activation of
homomeric GLUK5-containing KARs that is important for the generation
of gamma frequency oscillations in the MEC.


## 2. Material and Methods

### 2.1. Preparation of EC-Hippocampal Slices

All procedures involving animals were carried out in accordance with
UK Home Office Legislation. Male Wistar
rats, weighing >150 grammes, were first anaesthetised by inhalation of the
volatile anaesthetic isofluorane. This
was immediately followed by intramuscular injection of a terminal dose of
≥100 mg/kg ketamine and ≥10 mg/kg xylazine.
After confirmation of deep anaesthesia, rats were intracardiacally
perfused with~50 mL sucrose-modified
artificial cerebral spinal fluid (aCSF), composed of (in millimolar (mM)): 252
sucrose, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2·2H2O,
10 glucose, and 24 NaHCO3.
All salts were obtained from BDH Laboratory Supplies (Poole, UK), except
MgSO4 which was obtained from Sigma Chemical Co (Mo, USA).The whole brain was rapidly removed and maintained in a bath of cold
sucrose-modified aCSF (4-5°C)
during the dissection procedure.
Horizontal slices (450 μm thick) were cut using a vibroslice (Leica
VT1000S). Transverse EC-hippocampal
slices were then transferred either to a holding chamber or directly to the
recording chamber. They were maintained
at 32 ± 1°C, at the interface between a continuous perfusion (~2-3 mL/min) of
NaCl-based aCSF (containing (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4,
1 MgSO4, 1.2 CaCl2·2H2O, 10 glucose, 24 NaHCO3)
and humidified carbogen gas (95% O2/5% CO2). Slices were allowed to equilibrate for 60
minutes before any recordings were taken.


### 2.2. Electrophysiological Recording and Drug Application

Extracellular recordings were taken using glass electrodes pulled
from borosilicate glass capillaries (GC129 TF-10, 1.2 mm OD/0.94 mm ID) (Harvard
Apparatus, Kent, UK) using a Flaming/Brown micropipette puller, model P-97
(Sutter Instrument Co., Calif, USA). This created electrodes with resistances of
2–4 MΩ. Electrodes were filled with
NaCl-based aCSF and positioned in Layer III of the MEC.
Control readings were taken from slices before drug application to
confirm that any network activity seen following treatment was due to the
presence of drugs.To evoke gamma frequency oscillations, 400 nM kainic acid ((2S,3S,4S)-3-carboxymethyl-4-(prop-1-en-2-yl)pyrrolidine-2-carboxylic
acid; Tocris Cookson, Bristol, UK) was bath applied to EC-hippocampal slices
and left to equilibrate for 2-3 hours or until
gamma oscillations had stabilised. All other drugs were bath applied to slices
at known concentrations: UBP302 ((S)-3-(2-carboxybenzyl)willardiine;
gift from Dr. David Jane, Department of Pharmacology, University of Bristol,
UK) at 10 μM; ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid;
Tocris Cookson, Bristol, UK) at 1–5 μM; NS3763
(5-carboxy-2,4-di-benzamido-benzoic acid; Tocris, Bristol, UK) at 10–15 μM; NBQX
(2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline;
Tocris, Bristol, UK) at 1–10 μM; and Carbachol (Sigma, UK) at 10–20 μM.


### 2.3. Data Acquisition

An AppleMac computer with the Axograph OSX software package
(AxographX, Dr. John Clements, Australia) was used for all data
acquisition. Signals were analogue
filtered at 0.01–0.3 kHz and then digitized at a frequency of 10 kHz. Power spectra were constructed, where power
at a given gamma frequency was defined as the area under the peak between 20
and 80 Hz. Power spectra were generated
from digitized data, using 60 second epochs of recorded activity, and it was
from these spectra that values for gamma oscillation peak frequency, peak
amplitude, and spectral area power in the gamma frequency band were obtained.


### 2.4. Data Analysis

Data analysis was carried out using Excel and Kaleidagraph software
packages. Kaleidagraph software was used
to generate pooled power spectra, and the Excel package was used to calculate
the mean and standard error of mean (SEM) of results, and to draw up histograms
and line graphs. All data is presented as mean ± SEM. SigmaStat (Systat software, USA) was used for
all statistical tests. Normality tests
were carried out, and if data was found to be normally distributed, two-tailed
pairedt-tests were run. However,
if data failed the normality test, the Wilcoxon signed rank test was carried
out. This provided us with P-values for
all data sets, and the significance level was set at 95%; values less than P = .05
were deemed to be statistically significant.


## 2.1. Preparation of EC-Hippocampal Slices

All procedures involving animals were carried out in accordance with
UK Home Office Legislation. Male Wistar
rats, weighing >150 grammes, were first anaesthetised by inhalation of the
volatile anaesthetic isofluorane. This
was immediately followed by intramuscular injection of a terminal dose of
≥100 mg/kg ketamine and ≥10 mg/kg xylazine.
After confirmation of deep anaesthesia, rats were intracardiacally
perfused with~50 mL sucrose-modified
artificial cerebral spinal fluid (aCSF), composed of (in millimolar (mM)): 252
sucrose, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2·2H2O,
10 glucose, and 24 NaHCO3.
All salts were obtained from BDH Laboratory Supplies (Poole, UK), except
MgSO4 which was obtained from Sigma Chemical Co (Mo, USA).The whole brain was rapidly removed and maintained in a bath of cold
sucrose-modified aCSF (4-5°C)
during the dissection procedure.
Horizontal slices (450 μm thick) were cut using a vibroslice (Leica
VT1000S). Transverse EC-hippocampal
slices were then transferred either to a holding chamber or directly to the
recording chamber. They were maintained
at 32 ± 1°C, at the interface between a continuous perfusion (~2-3 mL/min) of
NaCl-based aCSF (containing (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4,
1 MgSO4, 1.2 CaCl2·2H2O, 10 glucose, 24 NaHCO3)
and humidified carbogen gas (95% O2/5% CO2). Slices were allowed to equilibrate for 60
minutes before any recordings were taken.


## 2.2. Electrophysiological Recording and Drug Application

Extracellular recordings were taken using glass electrodes pulled
from borosilicate glass capillaries (GC129 TF-10, 1.2 mm OD/0.94 mm ID) (Harvard
Apparatus, Kent, UK) using a Flaming/Brown micropipette puller, model P-97
(Sutter Instrument Co., Calif, USA). This created electrodes with resistances of
2–4 MΩ. Electrodes were filled with
NaCl-based aCSF and positioned in Layer III of the MEC.
Control readings were taken from slices before drug application to
confirm that any network activity seen following treatment was due to the
presence of drugs.To evoke gamma frequency oscillations, 400 nM kainic acid ((2S,3S,4S)-3-carboxymethyl-4-(prop-1-en-2-yl)pyrrolidine-2-carboxylic
acid; Tocris Cookson, Bristol, UK) was bath applied to EC-hippocampal slices
and left to equilibrate for 2-3 hours or until
gamma oscillations had stabilised. All other drugs were bath applied to slices
at known concentrations: UBP302 ((S)-3-(2-carboxybenzyl)willardiine;
gift from Dr. David Jane, Department of Pharmacology, University of Bristol,
UK) at 10 μM; ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid;
Tocris Cookson, Bristol, UK) at 1–5 μM; NS3763
(5-carboxy-2,4-di-benzamido-benzoic acid; Tocris, Bristol, UK) at 10–15 μM; NBQX
(2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline;
Tocris, Bristol, UK) at 1–10 μM; and Carbachol (Sigma, UK) at 10–20 μM.


## 2.3. Data Acquisition

An AppleMac computer with the Axograph OSX software package
(AxographX, Dr. John Clements, Australia) was used for all data
acquisition. Signals were analogue
filtered at 0.01–0.3 kHz and then digitized at a frequency of 10 kHz. Power spectra were constructed, where power
at a given gamma frequency was defined as the area under the peak between 20
and 80 Hz. Power spectra were generated
from digitized data, using 60 second epochs of recorded activity, and it was
from these spectra that values for gamma oscillation peak frequency, peak
amplitude, and spectral area power in the gamma frequency band were obtained.


## 2.4. Data Analysis

Data analysis was carried out using Excel and Kaleidagraph software
packages. Kaleidagraph software was used
to generate pooled power spectra, and the Excel package was used to calculate
the mean and standard error of mean (SEM) of results, and to draw up histograms
and line graphs. All data is presented as mean ± SEM. SigmaStat (Systat software, USA) was used for
all statistical tests. Normality tests
were carried out, and if data was found to be normally distributed, two-tailed
pairedt-tests were run. However,
if data failed the normality test, the Wilcoxon signed rank test was carried
out. This provided us with P-values for
all data sets, and the significance level was set at 95%; values less than P = .05
were deemed to be statistically significant.


## 3. Results

### 3.1. Induction of Kainate-Driven Gamma Oscillations in the MEC

Previously, it has been demonstrated that low concentrations of
kainic acid (kainate) evoke gamma frequency activity in the rat MEC in vitro [26, 27]. In this investigation, we produced persistent
gamma oscillations in the MEC
by bath application of kainate (400 nM) (Figure 1). Robust gamma frequency oscillations (39.4±1.6 Hz; n = 17) were evoked in layer III of the MEC in all slices to which kainate had been
applied (n = 17). This activity was generated within 15 minutes of kainate
superfusion, a stable baseline was observed after 60–90 minutes. As
previously reported [19], application of the competitive AMPA/KAR antagonist NBQX (10 μM) effectively abolished these kainate-induced gamma oscillations
(n = 3) (Figure 1).Figure 1
Gamma frequency
oscillations can be induced in layer III of the MEC by application of kainate. (a) Extracellular field recordings showing 1
second epochs of activity (i) in control setting, (ii) following application of
400 nM kainate, and (iii) following application of 10μM NBQX in the presence of 400 nM kainate. Scale
bar represents 200 milliseconds and 100 μV.


### 3.2. A role for GLUK5-Containing KARs in
the Maintenance of Kainate-Driven Gamma Oscillations

A possible role for GLUK5-containing KARs in the
maintenance of kainate-driven gamma frequency oscillations in the MEC was investigated by
testing the ability of the GLUK5 selective antagonist, UBP302, to
inhibit preestablished kainate-induced gamma activity. Gamma oscillations were
generated in the MEC
by bath application of kainate (400 nM) and allowed to stabilise (n = 9) (Figure 2(a)). In the presence of UBP302 (10 μM), the amplitude of kainate-induced gamma oscillations was
significantly reduced (control, 116.7±44.1 μV2/Hz; v. UBP302, 70.0±30.3 μV2/Hz; P < .05; n = 9), and area power of
oscillations was also significantly decreased (control, 1586.0±503.3 μV2/Hz.Hz; v. UBP302, 1155.1±441.4 μV2/Hz.Hz; P < .05; n = 9) (Figures 2(a), 2(b)). However, following UBP302 application, the
frequency of oscillations remained unchanged (control, 40.4±2.1 Hz; v. UBP302, 38.9±2.6 Hz; P > .1; n = 9). Washout of the effects of UBP302 on gamma
frequency oscillations could not be achieved (n = 9) (Figures 2(a), 2(b)).Antagonizing GLUK5-containing
KARs with UBP302 inhibits kainate-driven gamma frequency oscillations in the MEC. (a)
Extracellular field recordings showing 1 second epochs of activity (i)
in the presence of 400 nM kainate, (ii) following 10 μM UBP302 application, and (iii) during a
washout period into 400 nM kainate. (b) Pooled power spectra (n = 9) produced from 60
second epochs of extracellular field recorded data, showing a control recording
(black), a recording in the presence of 400 nM kainate (blue), application of 10 μM UBP302 (green), and washout back into 400 nM
kainate (red). Scale bar represents 200 milliseconds
and 100 μV.
(a)(b)


### 3.3. A role for GLUK5-Containing
KARs in the Generation of Gamma Oscillations in the MEC

To investigate the role that GLUK5-containing KARs may
play in the induction of kainate-driven gamma oscillations, we carried out two
experiments, using the selective GLUK5-containing KAR agonist, ATPA,
and antagonist, UBP302.First, we tested the ability of UBP302 to inhibit the generation of
a kainate-driven gamma frequency oscillation in the MEC.
Slices were preincubated in UBP302 (10μM) for 30 minutes. As
expected, UBP302 administration caused no neuronal network activity in slices
(n = 11) (Figure 3(a)). However, when kainate
was applied to slices following preincubation with UBP302, gamma frequency oscillations
were generated in all slices (n = 11) (Figure 3(a)).
On washout into kainate alone (400 nM), although the frequency of
oscillations did not change significantly (in presence of kainate following
preincubation with UBP302, 45.4±2.0 Hz; v. 400 nM kainate alone, 40.3±0.9 Hz; P > .05;
n = 11), oscillations were seen to increase significantly in both peak amplitude
(in presence of kainate following preincubation with UBP302, 39.2±12.1 μV2/Hz; v. 400 nM
kainate alone, 122.9±32.8 μV2/Hz; P < .05; n = 11) and area power (in
presence of kainate following preincubation with UBP302, 545.1±159.6 μV2/Hz.Hz; v. 400 nM kainate alone, 1302.5±241.4 μV2/Hz.Hz; P < .05;
n = 11) (Figures 3(a), 3(b)).Preincubation of slices in
UBP302 inhibits the ability of the MEC network to produce kainate-driven gamma frequency
oscillations. (a) Extracellular field recordings showing 1 second epochs of
activity (i) following preincubation with 10μM UBP302, (ii) following application of 400 nM
kainate onto preincubated slices, and (iii) during a washout period into 400 nM
kainate. (b) Pooled power spectra (n = 11) produced from 60 second epochs of
extracellular field recorded data, showing a control recording (black), a
recording following 10 μM UBP302 preincubation (blue), 400 nM kainate
application following preincubation (green), and washout into 400 nM kainate
(red). Scale bar represents 200 milliseconds and 100 μV.
(a)(b)We next investigated whether gamma frequency oscillations could be
generated in the MEC
by application of the GLUK5 selective agonist, ATPA. ATPA was bath applied to slices at
concentrations of 1 μM, 2 μM, and 5 μM. ATPA induced gamma frequency oscillations in
the MEC in the
majority of slices to which the agonist was applied (n = 18 out of a total n = 26)
(Figure 4(a)). Slices showing gamma
oscillations upon ATPA application were observed to be dorsal MEC slices. Upon increasing the concentration of ATPA in
slices demonstrating gamma oscillations, the mean frequency, peak amplitude,
and area power of oscillations increased (n = 10) (Figures 4(a), 4(b)). The frequency of oscillations increased from
23.0±1.9 Hz, to 34.0±2.4 Hz, and to 44.2±1.5 Hz (n = 10) (Figure 4(b)), the
peak amplitude increased from 0.9±0.5 μV2/Hz, to 4.1±2.3 μV2/Hz, and to 23.3±8.7 μV2/Hz (n = 10) (Figure 4(b)), and the power increased from 23.1±12.6 μV2/Hz.Hz, to
88.4±38.9 μV2/Hz.Hz,
and to 201.8±71.0 μV2/Hz.Hz, at
the respective concentrations of ATPA (1 μM, 2 μM, and 5 μM) (n = 10) (Figure 4(b)). Control
readings, taken before ATPA administration, showed that no network activity was
spontaneously present in slices (n = 26) (Figure 4(a)i). ATPA-induced gamma frequency
oscillations were susceptible to the AMPA/KAR antagonist NBQX (10 μM) (n = 3).Activation of GLUK5-containing
KARs can induce gamma frequency oscillations in the MEC.
(a) Extracellular field
recordings showing 1 second epochs of activity in a control setting (i) following
application of 1 μM,
(ii) 2 μM
(iii), and 5 μM
ATPA (iv). (b) Pooled line graphs (n = 10) demonstrating the
effects of varying ATPA concentration on (i) frequency, (ii) area power, and
(iii) peak amplitude of gamma oscillations in the MEC. Scale
bar represents 200 milliseconds and 100 μV.
(a)(b)We next investigated the effect of UBP302 on ATPA-induced gamma
frequency oscillations in the MEC. Gamma frequency
oscillations were induced in slices by bath application of ATPA (2–5μM) (n = 4) (Figure 5(a)i). UBP302
(10 μM) application caused
no significant change in the frequency of gamma oscillations (control, 42.7±3.9 Hz; v. UBP302, 33.9±7.3 Hz; P > .1; n = 4) and yet had significant
effects on both the peak amplitude (control, 20.8±7.1 μV2/Hz; v. UBP302, 6.6±2.8 μV2/Hz; P < .05; n = 4) and power of oscillations
(control, 359.7±117.9 μV2/Hz.Hz; v. UBP302, 141.7±61.9 μV2/Hz.Hz; P < .05;
n = 4) (Figures 5(a)ii, 5(b)). The effects of
UBP302 on an ATPA-induced gamma frequency oscillations were not reversible on
washout (n = 4).ATPA-generated gamma
frequency oscillations in the MEC are reduced by application of the GLUK5 selective
antagonist, UBP302. (a) Extracellular field recordings showing 1
second epochs of activity (i) in the presence of 5 μM
ATPA and (ii) following application of 10 μM UBP302.
(b) Pooled power spectra (n = 4) produced from 60 second epochs of
extracellular field recorded data, showing a control recording (black),
recording in the presence of 5 μM
ATPA (blue), and application of 10 μM UBP302 (green). Scale bar represents 200 milliseconds and 100 μV.
(a)(b)


### 3.4. A role for Homomeric GLUK5-Containing
KARs in Gamma Frequency Oscillations

The GLUK5 selective KAR antagonist, NS3763, was used to
investigate the contribution of homomeric GLUK5-containing KARs to
gamma activity in the MEC. NS3763 selectively antagonises homomeric GLUK5 KARs [39] and experiments were carried out to determine the role of these
homomeric receptors in both kainate- and ATPA-induced gamma oscillations.Application of NS3763 (10–15μM) caused significant decreases in both peak amplitude (control,
100.2±48.4 μV2/Hz; v. NS3763, 46.0±23.7 μV2/Hz; P < .05;
n = 8) and area power (control, 822.9±273.2 μV2/Hz.Hz; v. NS3763, 449.4±182.3 μV2/Hz.Hz; P < .05; n = 8) of kainate-induced gamma
oscillations in the MEC
(Figures 6(a), 6(b)). However, no effect was
seen on the frequency of kainate-generated oscillations (control, 38.3±2.7
Hz; v. NS3763, 36.0±1.5 Hz; P > .1; n = 8) (Figure 6(b)).Blocking homomeric GLUK5-containing
KARs significantly reduces kainate-driven gamma frequency oscillations in the MEC. (a) Extracellular field recordings showing 1
second epochs of activity (i) in the presence of 400 nM kainate and (ii)
following application of 10 μM NS3763.
(b) Pooled power spectra (n = 8)
produced from 60 second epochs of extracellular field recorded data, showing a
control recording (black), recording in the presence of 400 nM kainate (blue),
application of 10 μM
NS3763 (green), and a washout back into 400 nM kainate (red). Scale bar represents 200 milliseconds and 100 μV.
(a)(b)Application of NS3763 (10–15μM) to slices demonstrating ATPA-induced gamma oscillations caused no
significant change in the frequency of oscillations (control, 46.7±3.8 Hz; v. NS3763, 38.5±6.2 Hz; P > .1; n = 8) (Figures 7(a), 7(b)). However, the presence of NS3763 caused a
significant decrease in both the peak amplitude (control, 191.9±63.1 μV2/Hz; v. NS3763, 28.5±13.6 μV2/Hz; P < .05; n = 8) and area power (control, 1192.9±342.8 μV2/Hz.Hz; v. NS3763, 333.6±120.5 μV2/Hz.Hz; P < .05;
n = 8) of gamma oscillations (Figures 7(a), 7(b)). The effects of NS3763 on either
kainate- or ATPA-induced gamma frequency oscillations were not reversible on
washout (n = 12).Blocking homomeric GLUK5-containing
KARs effectively reduces power and amplitude of ATPA-generated gamma frequency
oscillations in the MEC (a)
Extracellular field recordings showing 1 second epochs of activity (i)
in the presence of 5 μM ATPA and (ii) following application of 15 μM
NS3763. (b) Pooled power spectra (n = 4) produced from 60
second epochs of extracellular field recorded data, showing a control recording
(black), recording in the presence of 5 μM ATPA (blue), and application of 15 μM
NS3763 (green). Scale bar represents 200
milliseconds and 100 μV.
(a)(b)


### 3.5. A role for GLUK5-Containing KARs in
Carbachol-Induced Gamma Oscillations

Cortical gamma frequency oscillations can also be induced by
application of carbachol, an agonist at muscarinic acetylcholine receptors
(mAChRs) [24, 25, 45–49]. It
is unclear as to the role of GLUK5-containing KARs in a
cholinergic-mediated gamma frequency oscillation in the MEC. Carbachol will cause an increase in
the release of glutamate in the form of rhythmic EPSPs [46]. This, in turn, may
lead to activation of KARs [50]. In agreement with previous studies in the MEC [24, 25] bath
application of carbachol (10–20 μM) generated persistent
gamma frequency oscillations (n = 6) (Figure 8(a)i).
Application of UBP302 (10 μM) had no significant effect on the frequency (control, 41.7±1.6
Hz; v. UBP302, 40.3±0.6 Hz; P > .1; n = 6), peak amplitude (control,
5.9±3.1 μV2/Hz; v. UBP302, 5.5±2.5 μV2/Hz; P > .1;
n = 6) or power (control, 155.2±72.7 μV2/Hz.Hz; v. UBP302, 148.7±62.8 μV2/Hz.Hz; P > .1; n = 6) of preestablished
carbachol-driven gamma oscillations (Figures 8(a)ii, 8(b)). This lack of effect was further demonstrated
by washout back into carbachol causing no significant change in observed gamma
frequency oscillations (Figures 8(a)iii, 8(b)).Carbachol-induced gamma
frequency oscillations are not dependent on GLUK5-containing
KARs. (a) Extracellular field recordings showing 1
second epochs of activity (i) in the presence of 20 μM
carbachol, (ii) following 10 μM UBP302 application, and (iii) during a
washout period into 20 μM carbcahol.
(b) Pooled power spectra (n = 6)
produced from 60 second epochs of extracellular field recorded data, showing a
control recording (black), recording in the presence of 20 μM
carbachol (blue), application of 10 μM UBP302 (green), and washout back into 20 μM
carbachol (red). Scale bar represents
200 milliseconds and 100 μV.
(a)(b)


## 3.1. Induction of Kainate-Driven Gamma Oscillations in the MEC

Previously, it has been demonstrated that low concentrations of
kainic acid (kainate) evoke gamma frequency activity in the rat MEC in vitro [26, 27]. In this investigation, we produced persistent
gamma oscillations in the MEC
by bath application of kainate (400 nM) (Figure 1). Robust gamma frequency oscillations (39.4±1.6 Hz; n = 17) were evoked in layer III of the MEC in all slices to which kainate had been
applied (n = 17). This activity was generated within 15 minutes of kainate
superfusion, a stable baseline was observed after 60–90 minutes. As
previously reported [19], application of the competitive AMPA/KAR antagonist NBQX (10 μM) effectively abolished these kainate-induced gamma oscillations
(n = 3) (Figure 1).Figure 1
Gamma frequency
oscillations can be induced in layer III of the MEC by application of kainate. (a) Extracellular field recordings showing 1
second epochs of activity (i) in control setting, (ii) following application of
400 nM kainate, and (iii) following application of 10μM NBQX in the presence of 400 nM kainate. Scale
bar represents 200 milliseconds and 100 μV.


## 3.2. A role for GLUK5-Containing KARs in
the Maintenance of Kainate-Driven Gamma Oscillations

A possible role for GLUK5-containing KARs in the
maintenance of kainate-driven gamma frequency oscillations in the MEC was investigated by
testing the ability of the GLUK5 selective antagonist, UBP302, to
inhibit preestablished kainate-induced gamma activity. Gamma oscillations were
generated in the MEC
by bath application of kainate (400 nM) and allowed to stabilise (n = 9) (Figure 2(a)). In the presence of UBP302 (10 μM), the amplitude of kainate-induced gamma oscillations was
significantly reduced (control, 116.7±44.1 μV2/Hz; v. UBP302, 70.0±30.3 μV2/Hz; P < .05; n = 9), and area power of
oscillations was also significantly decreased (control, 1586.0±503.3 μV2/Hz.Hz; v. UBP302, 1155.1±441.4 μV2/Hz.Hz; P < .05; n = 9) (Figures 2(a), 2(b)). However, following UBP302 application, the
frequency of oscillations remained unchanged (control, 40.4±2.1 Hz; v. UBP302, 38.9±2.6 Hz; P > .1; n = 9). Washout of the effects of UBP302 on gamma
frequency oscillations could not be achieved (n = 9) (Figures 2(a), 2(b)).Antagonizing GLUK5-containing
KARs with UBP302 inhibits kainate-driven gamma frequency oscillations in the MEC. (a)
Extracellular field recordings showing 1 second epochs of activity (i)
in the presence of 400 nM kainate, (ii) following 10 μM UBP302 application, and (iii) during a
washout period into 400 nM kainate. (b) Pooled power spectra (n = 9) produced from 60
second epochs of extracellular field recorded data, showing a control recording
(black), a recording in the presence of 400 nM kainate (blue), application of 10 μM UBP302 (green), and washout back into 400 nM
kainate (red). Scale bar represents 200 milliseconds
and 100 μV.
(a)(b)


## 3.3. A role for GLUK5-Containing
KARs in the Generation of Gamma Oscillations in the MEC

To investigate the role that GLUK5-containing KARs may
play in the induction of kainate-driven gamma oscillations, we carried out two
experiments, using the selective GLUK5-containing KAR agonist, ATPA,
and antagonist, UBP302.First, we tested the ability of UBP302 to inhibit the generation of
a kainate-driven gamma frequency oscillation in the MEC.
Slices were preincubated in UBP302 (10μM) for 30 minutes. As
expected, UBP302 administration caused no neuronal network activity in slices
(n = 11) (Figure 3(a)). However, when kainate
was applied to slices following preincubation with UBP302, gamma frequency oscillations
were generated in all slices (n = 11) (Figure 3(a)).
On washout into kainate alone (400 nM), although the frequency of
oscillations did not change significantly (in presence of kainate following
preincubation with UBP302, 45.4±2.0 Hz; v. 400 nM kainate alone, 40.3±0.9 Hz; P > .05;
n = 11), oscillations were seen to increase significantly in both peak amplitude
(in presence of kainate following preincubation with UBP302, 39.2±12.1 μV2/Hz; v. 400 nM
kainate alone, 122.9±32.8 μV2/Hz; P < .05; n = 11) and area power (in
presence of kainate following preincubation with UBP302, 545.1±159.6 μV2/Hz.Hz; v. 400 nM kainate alone, 1302.5±241.4 μV2/Hz.Hz; P < .05;
n = 11) (Figures 3(a), 3(b)).Preincubation of slices in
UBP302 inhibits the ability of the MEC network to produce kainate-driven gamma frequency
oscillations. (a) Extracellular field recordings showing 1 second epochs of
activity (i) following preincubation with 10μM UBP302, (ii) following application of 400 nM
kainate onto preincubated slices, and (iii) during a washout period into 400 nM
kainate. (b) Pooled power spectra (n = 11) produced from 60 second epochs of
extracellular field recorded data, showing a control recording (black), a
recording following 10 μM UBP302 preincubation (blue), 400 nM kainate
application following preincubation (green), and washout into 400 nM kainate
(red). Scale bar represents 200 milliseconds and 100 μV.
(a)(b)We next investigated whether gamma frequency oscillations could be
generated in the MEC
by application of the GLUK5 selective agonist, ATPA. ATPA was bath applied to slices at
concentrations of 1 μM, 2 μM, and 5 μM. ATPA induced gamma frequency oscillations in
the MEC in the
majority of slices to which the agonist was applied (n = 18 out of a total n = 26)
(Figure 4(a)). Slices showing gamma
oscillations upon ATPA application were observed to be dorsal MEC slices. Upon increasing the concentration of ATPA in
slices demonstrating gamma oscillations, the mean frequency, peak amplitude,
and area power of oscillations increased (n = 10) (Figures 4(a), 4(b)). The frequency of oscillations increased from
23.0±1.9 Hz, to 34.0±2.4 Hz, and to 44.2±1.5 Hz (n = 10) (Figure 4(b)), the
peak amplitude increased from 0.9±0.5 μV2/Hz, to 4.1±2.3 μV2/Hz, and to 23.3±8.7 μV2/Hz (n = 10) (Figure 4(b)), and the power increased from 23.1±12.6 μV2/Hz.Hz, to
88.4±38.9 μV2/Hz.Hz,
and to 201.8±71.0 μV2/Hz.Hz, at
the respective concentrations of ATPA (1 μM, 2 μM, and 5 μM) (n = 10) (Figure 4(b)). Control
readings, taken before ATPA administration, showed that no network activity was
spontaneously present in slices (n = 26) (Figure 4(a)i). ATPA-induced gamma frequency
oscillations were susceptible to the AMPA/KAR antagonist NBQX (10 μM) (n = 3).Activation of GLUK5-containing
KARs can induce gamma frequency oscillations in the MEC.
(a) Extracellular field
recordings showing 1 second epochs of activity in a control setting (i) following
application of 1 μM,
(ii) 2 μM
(iii), and 5 μM
ATPA (iv). (b) Pooled line graphs (n = 10) demonstrating the
effects of varying ATPA concentration on (i) frequency, (ii) area power, and
(iii) peak amplitude of gamma oscillations in the MEC. Scale
bar represents 200 milliseconds and 100 μV.
(a)(b)We next investigated the effect of UBP302 on ATPA-induced gamma
frequency oscillations in the MEC. Gamma frequency
oscillations were induced in slices by bath application of ATPA (2–5μM) (n = 4) (Figure 5(a)i). UBP302
(10 μM) application caused
no significant change in the frequency of gamma oscillations (control, 42.7±3.9 Hz; v. UBP302, 33.9±7.3 Hz; P > .1; n = 4) and yet had significant
effects on both the peak amplitude (control, 20.8±7.1 μV2/Hz; v. UBP302, 6.6±2.8 μV2/Hz; P < .05; n = 4) and power of oscillations
(control, 359.7±117.9 μV2/Hz.Hz; v. UBP302, 141.7±61.9 μV2/Hz.Hz; P < .05;
n = 4) (Figures 5(a)ii, 5(b)). The effects of
UBP302 on an ATPA-induced gamma frequency oscillations were not reversible on
washout (n = 4).ATPA-generated gamma
frequency oscillations in the MEC are reduced by application of the GLUK5 selective
antagonist, UBP302. (a) Extracellular field recordings showing 1
second epochs of activity (i) in the presence of 5 μM
ATPA and (ii) following application of 10 μM UBP302.
(b) Pooled power spectra (n = 4) produced from 60 second epochs of
extracellular field recorded data, showing a control recording (black),
recording in the presence of 5 μM
ATPA (blue), and application of 10 μM UBP302 (green). Scale bar represents 200 milliseconds and 100 μV.
(a)(b)


## 3.4. A role for Homomeric GLUK5-Containing
KARs in Gamma Frequency Oscillations

The GLUK5 selective KAR antagonist, NS3763, was used to
investigate the contribution of homomeric GLUK5-containing KARs to
gamma activity in the MEC. NS3763 selectively antagonises homomeric GLUK5 KARs [39] and experiments were carried out to determine the role of these
homomeric receptors in both kainate- and ATPA-induced gamma oscillations.Application of NS3763 (10–15μM) caused significant decreases in both peak amplitude (control,
100.2±48.4 μV2/Hz; v. NS3763, 46.0±23.7 μV2/Hz; P < .05;
n = 8) and area power (control, 822.9±273.2 μV2/Hz.Hz; v. NS3763, 449.4±182.3 μV2/Hz.Hz; P < .05; n = 8) of kainate-induced gamma
oscillations in the MEC
(Figures 6(a), 6(b)). However, no effect was
seen on the frequency of kainate-generated oscillations (control, 38.3±2.7
Hz; v. NS3763, 36.0±1.5 Hz; P > .1; n = 8) (Figure 6(b)).Blocking homomeric GLUK5-containing
KARs significantly reduces kainate-driven gamma frequency oscillations in the MEC. (a) Extracellular field recordings showing 1
second epochs of activity (i) in the presence of 400 nM kainate and (ii)
following application of 10 μM NS3763.
(b) Pooled power spectra (n = 8)
produced from 60 second epochs of extracellular field recorded data, showing a
control recording (black), recording in the presence of 400 nM kainate (blue),
application of 10 μM
NS3763 (green), and a washout back into 400 nM kainate (red). Scale bar represents 200 milliseconds and 100 μV.
(a)(b)Application of NS3763 (10–15μM) to slices demonstrating ATPA-induced gamma oscillations caused no
significant change in the frequency of oscillations (control, 46.7±3.8 Hz; v. NS3763, 38.5±6.2 Hz; P > .1; n = 8) (Figures 7(a), 7(b)). However, the presence of NS3763 caused a
significant decrease in both the peak amplitude (control, 191.9±63.1 μV2/Hz; v. NS3763, 28.5±13.6 μV2/Hz; P < .05; n = 8) and area power (control, 1192.9±342.8 μV2/Hz.Hz; v. NS3763, 333.6±120.5 μV2/Hz.Hz; P < .05;
n = 8) of gamma oscillations (Figures 7(a), 7(b)). The effects of NS3763 on either
kainate- or ATPA-induced gamma frequency oscillations were not reversible on
washout (n = 12).Blocking homomeric GLUK5-containing
KARs effectively reduces power and amplitude of ATPA-generated gamma frequency
oscillations in the MEC (a)
Extracellular field recordings showing 1 second epochs of activity (i)
in the presence of 5 μM ATPA and (ii) following application of 15 μM
NS3763. (b) Pooled power spectra (n = 4) produced from 60
second epochs of extracellular field recorded data, showing a control recording
(black), recording in the presence of 5 μM ATPA (blue), and application of 15 μM
NS3763 (green). Scale bar represents 200
milliseconds and 100 μV.
(a)(b)


## 3.5. A role for GLUK5-Containing KARs in
Carbachol-Induced Gamma Oscillations

Cortical gamma frequency oscillations can also be induced by
application of carbachol, an agonist at muscarinic acetylcholine receptors
(mAChRs) [24, 25, 45–49]. It
is unclear as to the role of GLUK5-containing KARs in a
cholinergic-mediated gamma frequency oscillation in the MEC. Carbachol will cause an increase in
the release of glutamate in the form of rhythmic EPSPs [46]. This, in turn, may
lead to activation of KARs [50]. In agreement with previous studies in the MEC [24, 25] bath
application of carbachol (10–20 μM) generated persistent
gamma frequency oscillations (n = 6) (Figure 8(a)i).
Application of UBP302 (10 μM) had no significant effect on the frequency (control, 41.7±1.6
Hz; v. UBP302, 40.3±0.6 Hz; P > .1; n = 6), peak amplitude (control,
5.9±3.1 μV2/Hz; v. UBP302, 5.5±2.5 μV2/Hz; P > .1;
n = 6) or power (control, 155.2±72.7 μV2/Hz.Hz; v. UBP302, 148.7±62.8 μV2/Hz.Hz; P > .1; n = 6) of preestablished
carbachol-driven gamma oscillations (Figures 8(a)ii, 8(b)). This lack of effect was further demonstrated
by washout back into carbachol causing no significant change in observed gamma
frequency oscillations (Figures 8(a)iii, 8(b)).Carbachol-induced gamma
frequency oscillations are not dependent on GLUK5-containing
KARs. (a) Extracellular field recordings showing 1
second epochs of activity (i) in the presence of 20 μM
carbachol, (ii) following 10 μM UBP302 application, and (iii) during a
washout period into 20 μM carbcahol.
(b) Pooled power spectra (n = 6)
produced from 60 second epochs of extracellular field recorded data, showing a
control recording (black), recording in the presence of 20 μM
carbachol (blue), application of 10 μM UBP302 (green), and washout back into 20 μM
carbachol (red). Scale bar represents
200 milliseconds and 100 μV.
(a)(b)


## 4. Discussion

A number of studies have examined the contribution of various KAR
subunits to gamma frequency oscillations in the hippocampus in vitro. Fisahn et al. [10] focused on the roles of
GLUK5 and GLUK6 subunits in kainate-induced hippocampal
gamma oscillations, using brain slices from transgenic GLUK5 and GLUK6 receptor knockout mice. Knockout of GLUK5 caused increased
sensitivity of the hippocampal network to the effects of kainate and higher
susceptibility to oscillatory and epileptogenic activity. Slices from GLUK6-knockout mice
could not support kainate-induced gamma oscillations or epileptiform activity,
suggesting distinct roles for GLUK5 and GLUK6 subunits in
the hippocampus. Fisahn et al. [10] concluded that GLUK5-containing
receptors may be expressed on axons of hippocampal interneurons and have a
function in inhibitory tone, and that GLUK6-containing KARs may be
found in the somatodendritic region of pyramidal cells and interneurons, and
provide excitatory drive. Functional receptors of both subtypes must interact
to allow generation of stable gamma oscillations in the hippocampus [9, 10].Subsequently, Brown et al. [51] used pharmacological approaches
to investigate the role of GLUK5-containing receptors in hippocampal
gamma oscillations. This study used the GLUK5-selective agonists
ATPA and iodowillardiine but found that neither could induce gamma network
activity in area CA3 of rat hippocampal slices. The GLUK5 selective
antagonist, UBP296, when preincubated with hippocampal slices, did not prevent
induction of kainate-driven gamma oscillations. However, UBP296 produced an
approximately 50% reduction in the power of preestablished kainate-induced
gamma frequency oscillations. This paper
concluded that GLUK5-containing KARs alone cannot generate gamma
oscillations in the hippocampus but may be involved in maintenance of
hippocampal gamma activity generated through other KAR subtypes.In the present study, we have demonstrated that, similarly to in the
hippocampus [51], GLUK5-containing KARs in the MEC have a role in the maintenance of
kainate-driven oscillations. UBP302, a
GLUK5 selective antagonist, caused reductions in peak amplitude and
spectral power of preestablished kainate-induced gamma frequency oscillations
in the MEC.
Furthermore, pretreatment of slices with UBP302 partially inhibited generation
of kainate-induced gamma frequency oscillations, suggesting that GLUK5-containing
KARs are at least partially responsible for the induction of gamma oscillations
by kainate application. These data
suggest that in the MEC,
differently to in the hippocampus [5, 8, 9], activation of GLUK5-containing
KARs plays a role in the ability of MEC neuronal networks to generate gamma frequency
oscillations. Moreover, in contrast to
hippocampal gamma evoked by kainate, MEC gamma generated with GLUK5 agonists demonstrates
a frequency increment with increased excitatory drive. This may reflect the
manifestation of fundamentally different mechanisms of local circuit gamma
oscillation generation in these two regions.It was not surprising then that application of the GLUK5 subunit selective agonist ATPA [41] successfully evoked gamma frequency oscillations in the MEC. However, care must
be taken with interpretation of this data as ATPA has recently been shown not
to be entirely selective for GLUK5-containing KARs [40]. In fact, ATPA can activate
both homomeric and heteromeric KAR complexes containing GLUK5, and
also GLUK6/KA2 heteromeric KARs [48]. Thus, it cannot initially
be assumed that these gamma oscillations have been generated via GLUK5-containing
receptor complexes, since they could have been induced through GLUK6/KA2
heteromeric receptors. UBP302, however, is an antagonist with selectivity for
GLUK5-containing KARs [36, 37].
Whilst UBP302 has been shown to block GLUK7 with an IC50 value of 4 μM—this makes it
~10-fold selective for GLUK5 versus GLUK7—it does not have activity on GLUK6 or GLUK6/KA2 up to 100 μM.
Indeed, some controversy surrounds the activity of UBP302 on GLUK7 as it has been reported that UBP302 failed to demonstrate any potent activity
in a binding assay on GLUK7 (personal communication, D.E. Jane). In
any case, as UBP302 only blocks homomeric GLUK7 and activation of
GLUK7 requires very high glutamate concentrations (EC50 value 5.9 mM) [52] it may not be relevant to this study. Application of UBP302
onto slices showing ATPA-generated network activity causes reduced peak
amplitude and an approximately 60% reduction in area power of gamma frequency
oscillations. This inhibition of ATPA-generated gamma oscillations by UBP302
suggests that the observed activity must, at least in part, be due to
activation of GLUK5-containing KARs.Moreover, NS3763 application caused a significant reduction in peak
amplitude and spectral power of preestablished kainate-driven gamma
oscillations. This demonstrates that homomeric GLUK5-containing KARs
are at least partially responsible for the maintenance of these kainate-driven
gamma frequency oscillations. Application of NS3763 to preestablished
ATPA-generated oscillations caused an approximately 80% reduction in area power
of gamma frequency oscillations and also a reduction in peak amplitude. This suggests that a large component of an
ATPA-driven gamma oscillation is maintained through homomeric GLUK5 KARs. The activity of the selective homomeric GLUK5-containing KAR
antagonist, NS3763, on both kainate- and ATPA-generated gamma frequency
oscillations, tells us that homomeric GLUK5-containing KARs are
involved in the observed network activity.It has been suggested that carbachol-driven activity could cause
excess glutamate release and that this overspill of glutamate could activate
KARs [50]. The lack of effect of the
GLUK5 selective antagonist, UBP302, on carbachol-induced gamma
oscillations in the MEC
suggests that GLUK5-containing KARs are not involved in the
generation or maintenance of gamma oscillations induced via activation of
muscarinic cholinergic receptor.
However, we cannot rule out the possibility that other KAR subtypes may
be involved in these mAChR-mediated gamma oscillations.We have shown that GLUK5-containing KARs are implicated
in the generation and maintenance of gamma frequency oscillations in the MEC evoked by
kainate. However, we can only speculate
on the cellular localisation of these GLUK5-containing receptors in
the MEC. Research performed by Christensen et al. [40] in the
hippocampus, suggested likely localisations of KAR subtypes in hippocampal CA1
inhibitory interneurons terminating with pyramidal cells, concluding that
heteromeric GLUK6/KA2 receptors are expressed in somatodendritic
compartments of interneurons, and that GLUK5 complexes, with either
GLUK6 or KA2, are found at presynaptic terminals. It seems likely from our results that in the MEC, both homomeric and
heteromeric GLUK5-containing KARs are present.Presynaptic KARs are involved in regulation and modulation of
neurotransmitter release at inhibitory and excitatory synapses in the
hippocampus [50, 53, 54]. In contrast,
postsynaptic KARs mediate excitatory postsynaptic currents (EPSCs) in many
brain regions [35, 55]. KAR activation in the hippocampus modulates
GABA release at terminals of inhibitory interneurons and causes an increase in
spontaneous IPSCs but a reduction in the amplitude of these IPSCs impinging on
to CA1 interneurons [40]. This suggests that KARs may
be present in two distinct populations in hippocampal inhibitory interneurons,
and the same may be true of KARs in the MEC [2, 40]. However, other reports
have demonstrated that kainate can increase the frequency and amplitude of
spontaneous IPSCs, but not action potential-independent miniature IPSCs in
stratum radiatum interneurons [56]. Moreover, these authors also
observed that kainate can directly depolarise the axonal plexus of inhibitory
interneurons producing both increased antidromic and presumably orthodromic
spiking. This effect would explain the ability of KAR activation to increase
spontaneous but not miniature IPSC activity. The presence of KARs at an axonal
loci has been well documented in the hippocampus, most notably in mossy fibres [57, 58].As outlined in the previous paragraph, there is a large corpus of
data on the role of KAR in the hippocampus. However, with respect to the MEC there is a paucity of
such information. In order to put the current results presented in this paper
into context, future work will concentrate on combining intracellular
recordings from individual neurones (pyramidal and interneuron), specific
pharmacological KAR tools, and transgenic KAR subunit knockout animals [9, 10] to
elucidate the exact nature of cellular expression of KARs in the MEC.



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*Source: 401645-2008-11-17.xml*